Treatment and prevention of remote ischemia-reperfusion injury

Abstract

To provide methods for treating and / or preventing remote ischemia reperfusion injury (IRI).SOLUTION: A method disclosed herein comprises intravenously, intraarterially or subcutaneously administering a contact activation system inhibitor selected from the group consisting of a C1 esterase inhibitor (C1INH), a kallikrein inhibitor and a FXII inhibitor to a patient who is undergoing or has received surgical intervention prior to and / or during and / or after the surgical intervention, or prior to and / or after start of reperfusion, at a dose of 1-1000 IU / kg body weight.SELECTED DRAWING: None

Description

[Background technology]

[0001] Establishment of a bloodless environment is a prerequisite for reconstructive and orthopedic surgery, which is commonly achieved by the application of a tourniquet. Lack of blood and oxygen, termed ischemia, induces time-dependent molecular and structural changes in the affected tissues. Then, when blood flow is restored, complex inflammatory cascades, such as the complement system, the coagulation system, and the plasma kallikrein-kinin system, are activated, resulting in ischemia-reperfusion injury (IRI). Tourniquet-induced IRI is manifested in edema formation, reduced myocyte viability, and apoptosis, which greatly impacts the outcome of surgical interventions. Moreover, IRI is known to induce, in addition to the local inflammatory response, a systemic inflammatory response that causes distant tissue and organ injury, a phenomenon well known in the clinic. To date, there are no drugs available in the clinic that can treat the systemic inflammatory response in addition to the local inflammatory response after IRI (1).

[0002] The natural complement inhibitor C1 esterase inhibitor (C1INH) has been demonstrated to inhibit all three complement pathways (classical, lectin, and alternative), the coagulation system, and the kallikrein-kinin system (2-4). Plasma-derived C1INH has been successfully used in the clinic to treat C1INH-deficient patients suffering from hereditary angioedema (HAE) (2). Several animal studies have demonstrated the therapeutic potential of plasma-derived C1INH in local IRI, i.e., solid organ transplantation, myocardial infarction, stroke, liver, and intestinal IRI (4). Two different studies have demonstrated the potential therapeutic effect of plasma-derived C1INH on local skeletal muscle IRI through the complement system (5, 6). A study using transgenic mice overexpressing human C1INH with "supraphysiologically" high plasma levels showed reduced distant organ damage in the lung and skeletal muscle in a lower body IRI model (7). However, in this model, C1INH was constitutively expressed, including during embryonic development, potentially resulting in altered physiology and pathophysiological reactivity.To date, no effect of plasma-derived C1INH on IRI-induced distant tissue or organ injury has been demonstrated.

[0003] Surprisingly, the inventors were able to demonstrate the therapeutic effect of plasma-derived C1INH on not only local but also distant tissue and organ damage using a rat animal model of tourniquet-applied hind limb IRI. For example, a significant reduction in pulmonary edema was observed. C1INH plays a major role in the inhibition of the contact activation system, and its effect on distant tissue and organ damage indicates a major pathophysiological role of components of this system not only in local tissue and organ damage but also in the distant damage observed during IRI. The results indicate that substances targeting the contact activation system, such as C1INH, kallikrein inhibitors, or factor XII (FXII) inhibitors, improve or even prevent distant tissue and organ damage. Summary of the Invention [Means for solving the problem]

[0004] The present invention relates, inter alia, to the subject matter defined in the following items [1] to

[32] .

[0005] [1] A contact activation system inhibitor selected from the group consisting of C1 esterase inhibitor (C1INH), a kallikrein inhibitor, and a factor XII (FXII) inhibitor, for use in the treatment and / or prevention of remote ischemia-reperfusion injury (IRI), comprising administering the contact activation system inhibitor to an individual.

[0006] [2] The contact activation system inhibitor for use according to item [1], wherein the contact activation system inhibitor is C1INH, preferably human C1INH, more preferably human plasma-derived C1INH, or human recombinant C1INH.

[0007] [3] A contact activation system inhibitor for use according to item [1], wherein the contact activation system inhibitor is an FXII inhibitor.

[0008] [4] The contact activation system inhibitor for use according to any one of items [1] to [3], wherein the contact activation system inhibitor is administered parenterally to an individual.

[0009] [5] A contact activation system inhibitor for use according to any one of items [1] to [4], wherein the contact activation system inhibitor is administered to an individual intravenously, intraarterially or subcutaneously.

[0010] [6] The contact activation system inhibitor for use according to any one of items [1] to [5], wherein the contact activation system inhibitor is C1INH and the dose of C1INH administered to the individual is 1 to 5000 IU per kg of body weight, preferably 1 to 1000 IU per kg of body weight, more preferably 10 to 500 IU per kg of body weight.

[0011] [7] The contact activation system inhibitor for use according to item [6], wherein the dose of C1INH administered to an individual is 10 to 250 IU per kg of body weight, preferably 20 to 100 IU per kg of body weight.

[0012] [8] The contact activation system inhibitor for use according to item [3] or [4], wherein the contact activation system inhibitor is an FXII inhibitor and the dose of the FXII inhibitor administered to the individual is suitable for completely inhibiting the amidolytic activity of FXIIa. Preferably, when the FXII inhibitor is an antibody, the dose administered to the individual is 0.01 to 50 mg / kg body weight, preferably 0.1 to 10 mg / kg body weight.

[0013] [9] A contact activation system inhibitor for use according to any one of items [1] to [8], wherein the individual is a patient who is about to undergo or is undergoing surgical intervention.

[0014]

[10] The contact activation system inhibitor for use according to item [9], wherein the contact activation system inhibitor, preferably C1INH, is administered to the patient within 7 days, preferably within 6 days, more preferably within 5 days, and even more preferably within 4 days before the start of surgical intervention or the start of reperfusion. More preferably, the contact activation system inhibitor is administered within 72 hours, preferably within 48 hours, more preferably within 24 hours, more preferably within 12 hours, and even more preferably within 6 hours before the start of surgical intervention or the start of reperfusion.

[0015]

[11] A contact activation system inhibitor for use according to item [9] or

[10] , wherein the contact activation system inhibitor, preferably C1INH, is administered to the patient during surgical intervention.

[0016]

[12] A contact activation system inhibitor for use according to any one of items [9] to

[11] , wherein the contact activation system inhibitor, preferably C1INH, is administered to the patient after completion of the surgical intervention or after the start of reperfusion.

[0017]

[13] The contact activation system inhibitor, preferably C1INH, is administered to the patient within 72 hours, preferably within 48 hours, more preferably within 24 hours, more preferably within 12 hours, even more preferably within 6 hours, or most preferably immediately after the end of the surgical intervention or the start of reperfusion, after the end of the surgical intervention or the start of reperfusion. A contact activation system inhibitor for the uses described.

[0018]

[14] The contact activation system inhibitor for use according to any one of items [9] to

[13] , wherein the surgical intervention is reconstructive surgery or trauma surgery.

[0019]

[15] The contact activation system inhibitor for use according to any one of items [9] to

[13] , wherein the surgical intervention is orthopedic surgery.

[0020]

[16] The contact activation system inhibitor for use according to item

[15] , wherein the orthopedic surgery is selected from the group consisting of knee surgery, shoulder surgery, and hand surgery.

[0021]

[17] The contact activation system inhibitor for use according to any one of items [9] to

[13] , wherein the surgical intervention is transplantation.

[0022]

[18] The contact activation system inhibitor for use according to item

[17] , wherein the transplant is an organ, tissue or cell transplant, preferably a kidney, liver, lung, intestine, pancreas, heart, limb (e.g., hand), skin, or pancreatic islet cell transplant.

[0023]

[19] The contact activation system inhibitor for use according to any one of items [9] to

[13] , wherein the surgical intervention is vascular surgery or surgery to treat a collision / crush injury.

[0024]

[20] The contact activation system inhibitor for use according to any one of items [9] to

[13] , wherein the surgical intervention is the insertion of an instrument, preferably a catheter, for delivering a pharmacologically active substance, such as a thrombolytic substance or a vasodilator, or the injection of a pharmacologically active substance, such as a thrombolytic substance or a vasodilator, into the individual, or the insertion of an instrument for mechanical removal of a complete or partial vascular occlusion.

[0025]

[21] The contact activation system inhibitor for use according to item

[20] , wherein the individual is suffering from myocardial infarction, stroke, thrombosis, preferably deep vein thrombosis or a thrombotic event on a foreign surface such as a stent, thromboembolism, pulmonary embolism, chronic ischemia due to atherosclerosis, preferably chronic limb ischemia due to peripheral atherosclerosis, or other complete or partial vascular occlusion.

[0026]

[22] The contact activation system inhibitor for use according to any one of items [1] to [8], wherein the remote IRI is due to improvement of blood flow impairment in the individual, either spontaneously or induced by means other than surgical intervention.

[0027]

[23] A contact activation system inhibitor for use according to item

[22] , wherein administration is, for example, prophylactically, before reperfusion or as soon as possible after reperfusion has occurred.

[0028]

[24] A contact activation system inhibitor for use according to any one of items [1] to

[23] , wherein the contact activation system inhibitor is human C1INH isolated from human plasma.

[0029]

[25] A method for treating and / or preventing remote ischemia-reperfusion injury (IRI), comprising administering to an individual an effective dose of a contact activation system inhibitor.

[0030]

[26] A method for preventing ischemia-reperfusion injury (IRI) in a patient about to undergo or undergoing a surgical intervention, comprising administering an effective dose of a contact active agent to a patient prior to and / or during and / or after the surgical intervention, or prior to and / or after the initiation of reperfusion. The method comprises administering to the patient an inhibitor of the ischemia-reperfusion injury (IRI), wherein the ischemia-reperfusion injury (IRI) affects an organ, limb, or tissue remote from the surgical site and / or the initial site of ischemia.

[0031]

[27] The method according to item

[25] or

[26] , wherein the contact activation system inhibitor is selected from the group consisting of C1INH, FXII inhibitors, and kallikrein inhibitors.

[0032]

[28] The method according to item

[27] , wherein the contact activation system inhibitor is C1INH, preferably human C1INH, more preferably plasma-derived human C1INH.

[0033]

[29] The method according to item

[27] , wherein the contact activation system inhibitor is an FXII inhibitor.

[0034]

[30] The method according to any one of items

[25] to

[29] , wherein remote ischemia-reperfusion injury (IRI) affects the lungs, kidneys, brain, liver, heart, intestines, pancreas, or other organs, and limbs.

[0035]

[31] The method according to any one of items

[25] to

[30] , wherein ischemia-reperfusion injury (IRI) includes multiple organ dysfunction syndrome or systemic inflammatory response syndrome.

[0036]

[32] The contact activation system inhibitor for use according to item [2], wherein the contact activation system inhibitor is C1INH, preferably human C1INH, and is combined with synthetic or natural glycosaminoglycans, such as heparin, N-acetylheparin, heparan sulfate, dextran sulfate, dermatan sulfate, and chondroitin sulfate, and / or with other substances, such as intravenous immunoglobulin, antithrombin III, α1-antitrypsin, or FXII inhibitor, to enhance the therapeutic effect. These substances can be administered as combination therapy or polytherapy. [Brief description of the drawings]

[0037] [Figure 1] Figure 1: C1INH significantly prevents local skeletal muscle edema as assessed by gastrocnemius wet:dry weight ratio. A: Wet:dry weight ratio analysis. B: Representative pictures of C1INH untreated (left) and C1INH treated (right) limbs. Each point represents the value of one animal. Horizontal bars represent the mean values ​​of different animals. Data are shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test using GraphPad Prism5 software. Statistical significance between samples was indicated as *p<0.05; **p<0.01; ***p<0.001. [Diagram 2] Figure 1 shows that C1INH significantly prevents remote pulmonary edema. Each point represents the value of one animal. Horizontal bars represent the mean value of different animals. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test using GraphPad Prism5 software. Statistical significance between samples was indicated as *p<0.05; **p<0.01; ***p<0.001. [Diagram 3]Figure 1 shows the effect of C1INH on the expression of bradykinin receptors B1R and B2R in the lungs, analyzed by IF. A: Expression of B1R. B: Expression of B2R. Each point represents the value of one animal. Horizontal bars represent the mean value of different animals. Data are shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test using GraphPad Prism5 software. Statistical significance between samples was indicated as *p<0.05; **p<0.01; ***p<0.001. [Figure 4] Figure 1: C1INH prevents fibrin deposition in the lung. Each point represents the value of one animal. Horizontal bars represent the mean value of different animals. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test using GraphPad Prism5 software. Data are shown as mean ± SD. Statistical significance between samples was indicated as *p<0.05; **p<0.01; ***p<0.001. [Diagram 5] Figure 1: C1INH reduces fibrin deposition in the contralateral lower limb. Each point represents the value of one animal. Horizontal bars represent the mean value of different animals. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test using GraphPad Prism5 software. Statistical significance between samples was indicated as *p<0.05; **p<0.01; ***p<0.001. [Figure 6] Figure 1: C1INH inhibits C3b and factor B deposition in the contralateral hindlimb. Each point represents the value of one animal. A: C3b deposition. B: Factor B deposition. Horizontal bars represent the mean values ​​of different animals. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test using GraphPad Prism5 software. Statistical significance between samples was indicated as *p<0.05; **p<0.01; ***p<0.001. [Figure 7]Figure 1 shows that heparan sulfate release is attenuated by C1INH in the contralateral hindlimb. Each point represents the value of one animal. Horizontal bars represent the mean value of different animals. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett's post-hoc test using GraphPad Prism5 software. Statistical significance between samples was indicated as *p<0.05; **p<0.01; ***p<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] In one aspect, the present invention relates to a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, for use in the treatment and / or prevention of remote ischemia-reperfusion injury (IRI) comprising administering a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, to an individual.

[0039] The term "treatment" or "treating" shall be understood to include the complete cure of a pathological condition, as well as the amelioration or alleviation of said condition.

[0040] The term "prevention" is understood to include the complete prevention, protection, or reduction of the severity of a pathological condition, as well as the reduction of the risk of an individual becoming ill with said condition. The term is also understood to include the pre-preparation of tissues by administering a contact activation system inhibitor as described herein, preferably a C1INH or FXII inhibitor, at a very early stage (e.g., before surgical intervention, before complete diagnosis of distant IRI) to prevent tissue damage.

[0041] The term "individual" refers to a human or animal subject.

[0042] The term "effective amount" is specifically intended to encompass any amount of an agent according to the present disclosure that is sufficient to effect the desired therapeutic or protective result upon administration to an individual.

[0043] Remote or distal IRI As used herein, the term "ischemia-reperfusion injury" (IRI) refers to injury resulting from the restoration of blood flow to parts of a tissue or organ that were previously deprived of blood flow due to an ischemic event.

[0044] IRI can be caused, for example, by a natural event (e.g., restoration of blood flow after a myocardial infarction), trauma, or one or more surgical procedures or other therapeutic interventions that restore blood flow to a tissue or organ that has had a reduced blood supply. Examples of such procedures include coronary artery bypass surgery, coronary angioplasty, and organ transplant surgery.

[0045] Reperfusion of ischemic tissue produces both local and systemic or distant responses that can result in widespread microvascular dysfunction and alterations in tissue barrier function. Inflammatory responses can even lead to systemic inflammatory response syndrome (SIRS) or multiple organ dysfunction syndrome (MODS).

[0046] As used herein, the phrases "remote IRI" or "distal IRI" or "remote or distal IRI" are used interchangeably herein and refer to pathophysiological processes, such as thrombotic, thromboembolic and / or inflammatory processes, including cell injury, complement activation and / or edema formation, that affect organs or tissues distinct from the reperfused local ischemic organs and tissues.

[0047] In one embodiment, the remote or distal IRI affects the lungs. Remote or distal IRI in this embodiment can include pulmonary edema, thrombotic processes in the lungs, pulmonary embolism, and / or inflammation of lung tissue.

[0048] In another embodiment, the distant IRI affects the kidney. Distant IRI in this embodiment can include renal failure, edema formation, thrombosis, thromboembolism, and / or inflammation of renal tissue.

[0049] In another embodiment, remote IRI affects the cardiovascular system. Remote IRI in this embodiment may include myocardial stunning (myocardial dysfunction that persists after reperfusion despite the absence of irreversible damage), thrombosis, reperfusion arrhythmias, and / or inflammation / infarction of myocardial tissue.

[0050] In another embodiment, the distant IRI affects the gastrointestinal system, preferably the intestine. Distant IRI in this embodiment can include impaired intestinal barrier function, impaired gastrointestinal motility and absorption, thrombosis, thromboembolism, and / or inflammation of gastrointestinal tissue.

[0051] In another embodiment, the remote IRI affects the central nervous system. Remote IRI in this embodiment can include blood-brain barrier disruption, silent cerebral ischemia, stroke, cerebral edema, elevated intracranial pressure, and / or inflammation of neural tissue.

[0052] Distant IRI can be characterized by inflammation, e.g., systemic inflammation, which can affect, for example, the lungs, gastrointestinal system, cardiovascular system, other limbs, and / or the central nervous system.

[0053] Distant IRI may include systemic inflammatory response syndrome (SIRS) and / or multiple organ dysfunction syndrome (MODS).

[0054] The ischemic event preceding remote IRI may be due to surgery or to a non-surgical vascular occlusion.

[0055] surgical intervention In one embodiment, the remote IRI is due to reperfusion of an ischemic tissue and / or organ following surgical intervention. Surgical intervention includes any surgical procedure.

[0056] Possible applications according to the present invention include the use of contact activation system inhibitors, preferably C1INH or FXII inhibitors, in conjunction with surgical repair of the thoracic, suprarenal or abdominal aorta due to aneurysmal disease, as well as in the induction of temporary occlusion or bypass of the blood supply to internal organs during and / or after major organ transplantation, including liver, kidney, small intestine, limbs and pancreas. This includes preventing remote IRI by administering in combination with surgical procedures that cause or require it. This also includes preventing remote IRI in combination with surgical procedures that temporarily reduce or block blood flow, including surgical resection of the liver and bile duct, total or partial pancreatectomy, total and partial gastrectomy, esophagectomy, colon surgery, vascular surgery for mesenteric vascular disease, or abdominal insufflation during laparoscopic surgery. Further applications include blunt or penetrating trauma that results in the interruption of blood flow to internal organs, including those resulting from stab wounds or penetrating wounds, or blunt abdominal trauma from automobile accidents. Further applications include crush injury, for example after natural disasters. Further applications include insertion of devices for delivery of pharmacologically active agents such as thrombolytic agents or vasodilators and / or for mechanical removal of complete or partial occlusions, and injection of pharmacologically active agents such as thrombolytic agents or vasodilators, following the onset of an initial thrombotic, or thromboembolic, or another ischemia-induced disorder, including, but not limited to, stroke, myocardial infarction, deep vein thrombosis, atherosclerosis, or thrombotic events on foreign body surfaces.

[0057] Preferably, the surgical intervention is selected from the group consisting of orthopedic surgery, vascular surgery, cardiac surgery, transcatheter procedures, cancer surgery, and trauma surgery. The orthopedic surgery is preferably selected from the group consisting of knee surgery, hand surgery, shoulder surgery, fractured long bones in trauma, hip replacement, and back surgery.

[0058] Vascular surgery may be for repair and / or accidental reasons, such as an aortic aneurysm.

[0059] In one embodiment, the surgery is trauma surgery, for example due to car accidents, general impact injuries and crush injuries (including major trauma with reduced blood volume).

[0060] In a particular embodiment, the surgical intervention is a transplant, preferably an organ transplant.

[0061] Non-surgical vascular obstruction Other preferred applications include diseases or treatments that result in systemic hypotension that disrupts or reduces blood flow to internal organs, including hemorrhagic shock due to blood loss, cardiogenic shock due to myocardial infarction or heart failure, neurogenic shock, nephrogenic shock, or anaphylaxis.

[0062] Remote IRI may be due to improved blood flow following ischemia due to hypoperfusion resulting in peripheral ischemia with or without organ ischemia.

[0063] Distant IRI may also be due to improved blood flow following initial thrombus formation or thromboembolic processes within major organs or tissues, such as stroke and myocardial infarction.

[0064] In one embodiment, remote IRI is due to improved blood flow following a chronic ischemic event, such as chronic limb ischemia due to peripheral atherosclerosis.

[0065] Contact activation system inhibitors The term "contact activation system inhibitor" as used herein refers to any compound capable of inhibiting the contact activation system. Contact activation system inhibitors are selected from the group consisting of C1 esterase inhibitors (C1INH), factor XII (FXII) inhibitors, and kallikrein inhibitors. These inhibitors have the advantage of acting early in the contact activation system pathway and interacting at multiple points in the contact activation system. In contrast, bradykinin inhibitors, such as bradykinin receptor antagonists, act distally in the system, with the main intervention point being at the stage of edema formation (Souza et al., 2003). (See (10)).

[0066] C1 esterase inhibitor C1 esterase inhibitor (C1INH) from human plasma is a glycosylated single-chain polypeptide of 478 amino acid residues. The amino acid sequence of human C1INH is shown in SEQ ID NO: 1. The mean plasma concentration is about 240 μg / mL. Synonyms for C1INH are C1-esterase inhibitor, α2-neuramino-glycoprotein, C1s-inhibitor, and C1-inactivator.

[0067] One International Unit (IU) of C1INH is defined by comparison with the WHO International Standard C1-Inhibitor (plasma). The current primary international standard is NIBSC code: 08 / 262 and the assigned potency of this preparation is 0.89 IU / ampule.

[0068] As used herein, the term "C1INH" refers to a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:1. For the purposes of the present invention, the degree of identity between two amino acid sequences refers to the percentage of amino acids that are identical between the two sequences. The degree of identity of an amino acid sequence to SEQ ID NO:1 is determined by comparing the amino acid sequence to SEQ ID NO:1 using the "BLAST 2 SEQUENCES (blastp)" program (Tatusova et al. (1999) FEMS Microbiol. Lett. 174, pp. 247-250) with the following parameters: Matrix BLOSUM62; open gap 11 and extension gap 1 penalty; gap x_dropoff 50; word size 3 with expectation 10.0; filter: none. According to the present invention, the sequence comparison covers at least 400 amino acids, preferably at least 425 amino acids, more preferably at least 450 amino acids, and most preferably at least 475 amino acids.

[0069] C1INH preferably has C1-inhibitory activity, which can be assayed as described in Drouet et al. (1988, Clin Chim Acta. 174:121-30). More preferably, C1INH is human C1INH having the amino acid sequence shown in SEQ ID NO: 1 (see also UniProt P05155).

[0070] In one embodiment, the C1INH is isolated from human or animal plasma, preferably from human plasma. According to this embodiment, the C1INH is preferably glycosylated like native human C1INH.

[0071] In another embodiment, human or animal C1INH is obtained from a transfected host cell expressing C1INH. Such "recombinantly produced" C1INH can be prepared by any suitable method. For example, it can be prepared in a recombinant host cell or organism transfected with DNA encoding C1INH.

[0072] C1INH suitable for use in the present invention can be obtained from culture medium prepared by engineered cells that secrete the protein and purified by standard methods.

[0073] The protein or polypeptide can be obtained by recombinant techniques using isolated nucleic acid encoding the C1INH polypeptide. General methods of molecular biology are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, NY, 2nd Edition, 1989, and Ausubel et al. (eds.) Current Protocols in Molecular Biology. The sequences are described by Epstein-Barr, Olecular Biology, New York (1987). Suitable sequences can be obtained from genomic or cDNA libraries using standard techniques. Polymerase chain reaction (PCR) techniques can be used. See, for example, PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al. (eds.), Academic Press, New York, NY. Libraries are constructed from nucleic acids extracted from suitable cells. Useful gene sequences can be found, for example, in various sequence databases, such as GenBank for nucleic acids and Swiss-Prot for proteins.

[0074] Standard methods can be used to generate transformed prokaryotic, mammalian, yeast or insect cell lines that express large amounts of polypeptide.Exemplary mammalian cell lines include COS-7 cells, mouse L cells, and CHO cells.See Sambrook (1989) supra, and Ausubel et al., 1987, supra.

[0075] A variety of expression vectors can be used to express DNA encoding C1INH. Conventional vectors used for the expression of recombinant proteins in prokaryotic or eukaryotic cells can be used.

[0076] C1INH can be produced in soluble form, such as a secretion product of transformed or transfected yeast cells, insect cells or mammalian cells. The peptide can then be purified by standard techniques known in the art. For example, purification steps can include ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, etc. See Methods in Enzymology Purification Principles and Practices (Springer-Verlag, NY, 1982).

[0077] Alternatively, C1INH can be produced in insoluble form, such as aggregate or inclusion body. Such form of C1INH is purified by standard techniques well known in the art. An example of purification process includes separating inclusion body from disrupted host cells by centrifugation, then solubilizing inclusion body with chaotropic agent and reducing agent so that peptide adopts biologically active conformation.

[0078] To generate C1INH or fragments thereof with various desired properties, the nucleotide sequence used to transfect host cells can be modified using standard techniques. Such modified C1INH can be altered from the naturally occurring sequence at the primary structure level, for example, by amino acid insertion, substitution, deletion and fusion. These modifications can be used in several combinations to produce the final modified protein chain. Amino acid sequence variants can be prepared with various purposes in mind, including extending or shortening serum half-life, facilitating purification or preparation, enhancing therapeutic efficacy, and reducing the severity or occurrence of side effects during therapeutic use. Amino acid sequence variants are usually predetermined variants that do not occur in nature, but others may be post-translational variants. Such variants can be used in the present invention as long as they retain the biological activity of C1INH. C1INH according to the present invention includes the C1INH molecules disclosed in WO2007 / 073186 and US8,071,532B2.

[0079] Expression in host cells results in a modified glycan structure (compared to plasma-derived C1INH). The C1INH may be modified in one or more of the following ways compared to plasma-derived C1INH: removal of carbohydrate moieties (from naturally occurring or recombinantly expressed variants of the glycoprotein), preferably removal of sialic acid and / or galactose from N-linked glycans, and / or removal of glycans exposing mannose, galactose, N-acetylglucosamine and / or fucose residues. Furthermore, modification of C1INH to extend plasma half-life is envisaged, for example, fusion with half-life-extending moieties, for example, albumin, immunoglobulin Fc regions, or chemical modifications such as pegylation or hesylation of C1INH.

[0080] Factor XII inhibitors The abbreviation "FXII" as used in this application refers to either or both of factor XII (FXII) and activated factor XII (FXIIa). Thus, the term "FXII inhibitor" includes either or both of FXII and FXIIa inhibitors. Furthermore, anti-FXII antibodies include antibodies that bind to either or both of FXII and FXIIa and inhibit either or both of FXII and FXIIa. The term "FXII inhibitor" is also intended to include FXII inhibitors linked to a half-life enhancing polypeptide. The FXII inhibitor can be linked directly to the half-life enhancing polypeptide or can be linked via a linker, preferably a cleavable linker.

[0081] In some embodiments, the FXII inhibitor is a direct inhibitor of FXII. The term "direct" inhibitor refers to an inhibitor that acts by contacting (e.g., binding) with FXII (or FXIIa). In contrast, an indirect inhibitor can act without contacting FXII (or FXIIa) protein; for example, antisense RNA can be used to reduce the expression of the FXII gene, or a molecule can inhibit the effect of FXIIa by directly interacting with a downstream reaction partner of direct FXIIa, such as factor XI, but it does not directly interact with FXII protein. Thus, indirect inhibitors act upstream or downstream of FXII protein, unlike direct inhibitors. Some examples of direct inhibitors are given below. FXII inhibitors are generally non-endogenous inhibitors; i.e., they are not inhibitors that naturally occur in the human or animal body.

[0082] A. Infestin-4 In one embodiment, the application provides an FXII inhibitor comprising the infestin domain 4, infestin-4. In one embodiment, the FXII inhibitor comprises a variant of infestin-4. In another embodiment, the FXII inhibitor comprises infestin domain 4 and optionally infestin domains 1, 2 and / or 3; these proteins are known to be potent inhibitors of FXII (see WO2008 / 098720; see also FEBS Lett. pp. 512-516, 2004). The wild-type polypeptide sequence of infestin-4 is shown (SEQ ID NO: 2). As used herein, the term "variant" refers to a polypeptide having an amino acid mutation, where "mutation" is defined as a substitution, deletion or addition to the sequence of wild-type infestin-4, such changes do not alter the functional ability of the polypeptide to inhibit FXII. The term "variant" includes fragments of wild-type or mutant infestin-4 sequences. Further examples of such variants are provided below.

[0083] In one embodiment, the Infestin-4 variant comprises amino acids 2-13 of the N-terminus of the wild-type Infestin-4 sequence, and no differences are present between the wild-type Infestin-4 sequence and the N-terminus of the wild-type Infestin-4 sequence. The variant comprises at least one to five amino acid mutations outside the N-terminal amino acid or six conserved cysteine ​​residues, and at least 70% homology to the wild-type Infestin-4 sequence. Analysis of structural data from the related inhibitor Rhodnius prolixus (PDB: 1TSO), which binds thrombin, as well as analysis of SPINK-1, which binds chymotrypsin, suggests that the N-terminal amino acids 2-13 of the Infestin-4 sequence may be important for FXII binding, since both share the common feature of an accumulation of contact sites in the N-terminal region. Thus, in one embodiment, the variant of Infestin-4 comprises the conserved N-terminal region of amino acids 2-13 of the wild-type Infestin-4 sequence and at least one to five amino acid mutations outside these conserved N-terminal amino acids that result in a difference from the wild-type Infestin-4 sequence. The mutations may be substitutions, deletions, or additions. As used herein, the term "outside the N-terminal amino acid" refers to any amino acid along the polypeptide chain of the variant other than the sequence VRNPCACFRNYV, i.e., the contiguous stretch of amino acids including amino acids 2-13 from the wild-type Infestin-4 sequence. In another embodiment, the Infestin-4 variant contains six conserved cysteine ​​residues and has at least 70% homology to the wild-type Infestin-4 sequence. In one embodiment, the six conserved cysteine ​​residues are amino acids at positions 6, 8, 16, 27, 31, and 48 of the wild-type Infestin-4 sequence. In one embodiment, the variant contains a final conserved cysteine. In other embodiments, the exact positions of the cysteine ​​residues, and their relative positions to each other, may vary from positions 6, 8, 16, 27, 31, and 48 of the wild-type Infestin-4 sequence due to insertions or deletions in the Infestin-4 variant.Nevertheless, in these embodiments, the Infestin-4 variant may contain all six cysteines and share 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the wild-type Infestin-4 sequence.

[0084] In an embodiment, the variant of Infestin-4 is characterized by inhibiting FXII. The functional activity of inhibiting FXII may be evaluated, for example, by in vitro and / or in vivo characterization, including direct assays to test inhibition of FXII enzyme activity, prolonged clotting time, i.e., activated partial thromboplastin time (aPTT), or in vivo methods to evaluate clotting. Further examples of variants of Infestin-4 are SPINK-1 mutants, which are described below.

[0085] B. SPINK-1 mutants One embodiment includes FXII inhibitors for therapeutic use in humans. Human proteins with high similarity to Infestin-4 can be used. For example, the human protein with the highest similarity to Infestin-4 is SPINK-1 (also known as pancreatic secretory trypsin inhibitor, PSTI), a Kazal-type serine protease inhibitor expressed in the pancreas. The family of Kazal-type serine protease inhibitors is one of many families of serine protease inhibitors. Many proteins from different species have been described (Laskowski M and (Kato I, 49 Ann.Rev.Biochem. pp.593-626, 1980). Different variants can be created based on the wild-type SPINK-1 sequence to increase the homology of the SPINK-1 sequence to Infestin-4. The phrase "increasing homology to Infestin-4" refers to the process of making amino acid mutations to SPINK-1 to make the SPINK-1 sequence closer to the Infestin-4 sequence.

[0086] In one embodiment, SPINK-1 is an N-terminal amine of the wild-type Infestin-4 sequence. The N-terminal portion of the Infestin-4 sequence was mutated to include amino acids 2-13, resulting in a polypeptide sequence designated K1. As noted above, the N-terminal portion of the Infestin-4 sequence is believed to be important for FXII inhibitory function.

[0087] Thus, in one embodiment, the mutant SPINK-1 variant also comprises the N-terminal amino acids 2-13 of the wild-type Infestin-4 sequence and at least one to five amino acid mutations outside said N-terminal amino acids that result in a difference from the wild-type SPINK-1 sequence and increase the homology of the variant to the wild-type Infestin-4 sequence. In another embodiment, the mutant SPINK-1 variant comprises the six conserved cysteine ​​residues and has at least 70% homology to the wild-type SPINK-1 sequence. The mutations may be substitutions, deletions or additions. As defined above, the term "outside said N-terminal amino acids" refers to any amino acid along the polypeptide chain of the variant other than the sequence VRNPCACFRNYV, i.e., the contiguous stretch of amino acids including amino acids 2-13 from the wild-type Infestin-4 sequence. The term "variant" includes fragments of said mutant SPINK-1 sequence. In one embodiment, the six conserved cysteine ​​residues may be the amino acids at positions 9, 16, 24, 35, 38, and 56 of the wild-type SPINK-1 sequence. In one embodiment, the variant includes the final conserved cysteine. In another embodiment, the exact positions of the cysteines, and their relative positions to one another, may vary from positions 9, 16, 24, 35, 38, and 56 of the wild-type SPINK-1 sequence due to insertions or deletions in the SPINK-1 variant. Nevertheless, in these embodiments, the SPINK-1 variant includes all six cysteines. In an embodiment, the SPINK-1 variant is also characterized in that it inhibits FXII. Further SPINK variants are disclosed in EP2497489A1, the disclosure of which is incorporated herein in its entirety.

[0088] C. Other FXII inhibitors In one embodiment, other FXII inhibitors are administered to the patient undergoing medical treatment. As discussed above, the term FXII inhibitor includes both FXII and FXIIa inhibitors. In WO2006 / 066878, the use of antibodies against FXII or the use of FXII inhibitors is proposed. In particular, inhibitors against FXII include antithrombin III (ATIII), angiotensin converting enzyme inhibitors, aprotinin, alpha-1 protease inhibitors, antipain ([(S)-1-carboxy-2-phenylethyl]-carbamoyl-L-Arg-L-Val-Arginal), Z-Pro-Pro aldehyde-dimethyl acetate, DX88 (Dyax Inc., 300 Technology Square, Cambridge, MA 02139, USA; Williams A and Baird LG, 29 Transfus Apheresis Sci. 255-258, 2003), leupeptin, prolyl oligopeptidase inhibitors such as Fmoc-Ala-Pyr-CN, corn trypsin inhibitor, mutants of bovine pancreatic trypsin inhibitor, ecotin, platycorrhizal anticoagulant protein, Cucurbita maxima trypsin inhibitor V (including Cucurbita maxima isoinhibitors), and Hamadarin (disclosed in Isawa H et al., 277 J. Biol. Chem. 27651-27658, 2002).

[0089] In yet other embodiments, the FXII inhibitor is HD-Pro-Phe-Arg-chloromethylketone (PCK) (Tans et al., Eur. J. Biochem. 1987;164:637-42; Kleinschnitz et al., J Exp Med. 2006;203:513-8).

[0090] The FXII inhibitor may be, for example, an analog of the Kunitz-type protease inhibitor domain of the amyloid precursor protein, as disclosed in US Pat. No. 6,613,890, columns 4-8.

[0091] In another embodiment, the FXII inhibitor may be an anti-FXII antibody that binds to FXII and inhibits the activation and / or activity of FXII. Such antibodies are described, for example, in WO2006 / 066878 and Ravon et al., 1 Blood 4134-43, 1995.

[0092] Other monoclonal antibodies (mAbs) against human FXII include the B7C9 mAb described by Pixley et al. (J Biol Chem 1987;262, 10140-45), the mAb described by Small et al. (Blood 1985;65:202-10); mAbs F1 and F3 described by Nuijens et al. (J. Biol. Chem. 1989;264:12941-49); the B6F5, C6B7, and D2E10 mAbs against the light chain of FXII described in WO89 / 11865; a mAb that selectively binds FXIIa-β over FXII described in WO90 / 08835, and the anti-FXII antibody OT-2 described in WO91 / 17258.

[0093] Additional preferred anti-FXII / FXIIa monoclonal antibodies, and antigen-binding fragments thereof, are described in WO2013 / 014092, which is incorporated herein by reference. These antibodies have a binding affinity for human FXIIa-β that is more than two-fold higher than for human FXII, and can inhibit the amidolytic activity of human FXIIa. In some embodiments, the antibody or antigen-binding fragment (a) binds to mouse FXII / FXIIa; (b) comprises a heavy chain variable (VH) region that is more than 85% identical to the sequence of SEQ ID NO:7; (c) comprises a light chain variable (vL) region that is more than 85% identical to the sequence of SEQ ID NO:8; (d) comprises a heavy chain CDR1 that is at least 80% identical to the sequence of SEQ ID NO:9, and / or a heavy chain CDR2 that is at least 60% identical to SEQ ID NO:10, and / or a heavy chain CDR3 that is at least 80% identical to the sequence of SEQ ID NO:12; (e) comprises a light chain CDR1 that is at least 50% identical to SEQ ID NO:14, and / or a light chain CDR2 of SEQ ID NO:15, and / or a light chain CDR3 of the sequence A-X1-W-X2-X3-X4-X5-R-X6-X7, where X1 can be A or S, X5 can be L or V, and any other X can be any other CDR. n (f) a light chain CDR3 having a CDR1 (SEQ ID NO: 17), wherein the CDR1 is a CDR2 sequence, and the CDR2 sequence may be any amino acid; -8 K is higher than M D (g) binds to human FXIIa-β having the following characteristics: (i) competes with Infestin-4 for binding to human FXIIa-β; or (h) has one or more of the following characteristics: human IgG or a variant thereof, preferably human IgG4 or a variant thereof.

[0094] In another embodiment, the anti-FXII antibody is an IgG antibody that binds human FXII and comprises (a) a VH region comprising a heavy chain CDR1 set forth in SEQ ID NO: 9, a heavy chain CDR2 set forth in SEQ ID NO: 11, and a heavy chain CDR3 set forth in SEQ ID NO: 13; and / or (b) a VL region comprising a light chain CDR1 set forth in SEQ ID NO: 14, a light chain CDR2 set forth in SEQ ID NO: 15, and a light chain CDR3 set forth in SEQ ID NO: 17. The heavy chain CDR2 comprising SEQ ID NO: 11 comprises the sequence GIX1X2X3X4X5X6TVYADSVKG, where X1 is R, N or D, X2 is P, V, I or M; X3 is S, P or A; X4 is G, L, V or T; X5 can be any amino acid, preferably X5 is G, Y, Q, K, R, N or M; and X6 is T, G or S. The heavy chain CDR3 comprising SEQ ID NO:13 comprises the sequence ALPRSGYLX1X2X3X4YYYYALDV, where X1 is I, M or V; X2 is S or K; X3 is P, K, T or H; and X4 is H, N, G or Q. SEQ ID NO:17 and the light chain CDR3 comprises the sequence AX1WX2X3X4X5RX6X7, where X1 is A or S; X2 is D, Y, E, T, W, E or S; X3 is A, N, I, L, V, P, Q or E; X4 is S, D, P, E, Q or R; X5 is L or V; X6 is G, L or K; and X7 is V, A, D, T, M or G.

[0095] In other embodiments, the antigen-binding fragment of an anti-FXII antibody is a fragment of an IgG antibody that binds to human FXII and comprises (a) a VH region comprising a heavy chain CDR1 set forth in SEQ ID NO: 9, a heavy chain CDR2 set forth in SEQ ID NO: 10, and a heavy chain CDR3 set forth in SEQ ID NO: 12; and / or (b) a VL region comprising a light chain CDR1 set forth in SEQ ID NO: 14, a light chain CDR2 set forth in SEQ ID NO: 15, and a light chain CDR3 set forth in SEQ ID NO: 16.

[0096] In one embodiment, the anti-FXII antibody or antigen-binding fragment thereof is the antibody "3F7" used in Example 3. The sequences of the variable regions and CDRs of 3F7 are shown in Table 1.

[0097] [Table 1]

[0098] In yet another embodiment, the anti-FXII antibody or antigen-binding fragment has the structure F7), are selected from affinity matured antibodies VR115, VR112, VR24, VR110, VR119.

[0099] [Table 2]

[0100] As mentioned above, SEQ ID NO: 11 is a degenerate sequence. VR119 comprises SEQ ID NO: 11, where X1 is N, X2 is V, X3 is P; X4 is L, X5 is Y; X6 is G. VR112 comprises SEQ ID NO: 11, where X1 is N, X2 is V, X3 is P, X4 is V, X5 is Q, and X6 is G. VR115 comprises SEQ ID NO: 11, where X1 is D, X2 is I, X3 is P, X4 is T, X5 is K, and X6 is G. VR110 comprises SEQ ID NO: 11, where X1 is D, X2 is M, X3 is P, X4 is T, X5 is K, and X6 is G. VR24 contains a unique LCDR1: SGSSEMTVHHYVY (SEQ ID NO: 18).

[0101] In embodiments involving antibody CDRs, the CDRs are defined according to the KABAT numbering system (Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C (1991) Sequences of proteins of immunological interest, 5th ed., USDepartment of Health and Human services, NIH, Bethesda, MD).

[0102] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-FXII / FXIIa monoclonal antibody or antigen-binding fragment thereof that inhibits FXIIa-α by more than 40%, more than 50%, or more than 60% when used at a molar ratio of FXIIa-α to antibody of 1:0.2. In some embodiments, the antibody or antigen-binding fragment thereof inhibits FXIIa-α by more than 80%, more than 85%, or more than 90% at a molar ratio of FXIIa-α to antibody of 1:0.5. In one embodiment, the antibody achieves complete inhibition of FXIIa-α at a molar ratio of 1:0.5. In one embodiment, the FXIIa-α is human FXIIa-α. In one embodiment, the antibody or antigen-binding fragment thereof has an affinity for human FXIIa at least comparable to that of antibody 3F7.

[0103] As discussed above, "anti-FXII antibodies" include antibodies that bind to and inhibit either or both of FXII and FXIIa. In one embodiment, the antibodies include full-length Ig, Fab, F(ab)2, Fv, scFv, etc. The antibody may be of one or another form or variant thereof. The antibody may be monoclonal or polyclonal. The antibody may be characterized by an isotype of IgM, IgD, IgA, IgG, or IgE, or any subclass thereof, e.g., IgG1, or variants thereof. The antibody may be from a mammalian species, including, but not limited to, human, mouse, rat, rabbit, goat, hamster, or monkey. The antibody may be humanized or CDR-grafted. The antibody may be mutated or modified to alter immunogenicity, half-life, or to confer other advantageous properties associated with therapeutic antibodies. In one embodiment, the antibody is an anti-FXII antibody that binds to an epitope, e.g., a neutralizing epitope, on the heavy or light chain of FXII (where "FXII" includes FXII and FXIIa). The antibody may be of high affinity and / or high avidity for FXII binding. The antibody may be conjugated to a polypeptide, nucleic acid, or small molecule.

[0104] D. FXII inhibitors linked to half-life extending moieties Polypeptides Another aspect of the present application provides an FXII inhibitor linked to a half-life enhancing polypeptide (HLEP). For example, in one embodiment, the FXII inhibitor is linked to a protein that extends the half-life. A "half-life enhancing polypeptide" increases the half-life of the FXII inhibitor in a patient or animal in vivo. For example, albumin and immunoglobulins, as well as fragments or derivatives thereof, have been described as half-life enhancing polypeptides (HLEPs). Ballance et al. (WO2001 / 79271) describe fusion polypeptides of a number of different therapeutic polypeptides that, when fused to human serum albumin, are expected to have a longer functional half-life in vivo and a longer shelf life. The embodiments related to the FXII inhibitor linked to a half-life enhancing polypeptide disclosed in EP2497489A1 can be used according to the present invention and are incorporated herein by reference.

[0105] E. Linker In one embodiment, an intervening peptide linker can be introduced between the therapeutic polypeptide and the HLEP. In one embodiment, a cleavable linker is introduced, particularly when the HLEP interferes with the specific activity of the therapeutic polypeptide, for example, by steric hindrance. In certain embodiments, the linker is cleaved by an enzyme such as a coagulation protease of the intrinsic, extrinsic, or common coagulation pathway. Coagulation proteases of the intrinsic pathway are proteases in the contact activation pathway, including, for example, FXIIa, FXIa, or FIXa. In one embodiment, the linker is cleaved by FXIIa. Proteases of the extrinsic pathway include proteases in the tissue factor pathway, for example, FVIIa. Proteases of the common pathway include proteases involved in the conversion of fibrinogen to fibrin, for example, FXa, FIIa, and FXIIIa.

[0106] Combinations / Kits Another aspect of the present invention is the combination of C1INH with drugs. Preferably, C1INH can be administered in combination with synthetic or natural glycosaminoglycans, such as heparin, N-acetylheparin, heparan sulfate, dextran sulfate, dermatan sulfate, and chondroitin sulfate, which are known to enhance the activity of C1INH. In addition, C1INH can be combined with other substances, including but not limited to intravenous immunoglobulin, antithrombin III, α1-antitrypsin, or FXII inhibitors, to enhance the therapeutic effect. These substances can be administered as combination therapy or polytherapy. Thus, in the prevention and / or treatment of remote IRI, A kit comprising C1INH and one or more of the above-mentioned substances, including instructions for administration, for use in the prevention and / or treatment of distant IRI is also an aspect of the invention. Thus, one embodiment of the present invention is a kit of parts comprising C1INH and at least one other substance as listed above, for simultaneous, separate or sequential use in the prevention and / or treatment of distant IRI.

[0107] Administration The contact activation system inhibitor, preferably the C1INH or FXII inhibitor described herein, is preferably administered as part of a pharmaceutical composition comprising the contact activation system inhibitor, preferably the C1INH or FXII inhibitor, and a pharma- ceutical acceptable excipient.The pharmaceutical composition can be easily administered parenterally, for example, by intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, intranasal, intrapulmonary, transdermal, or subcutaneous application.Parenteral administration can be achieved by placing the composition of the present invention in a solution, emulsion, or suspension.

[0108] Preferably, the composition comprising a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered intravenously, intraarterially, or subcutaneously by single or multiple bolus injections and / or by continuous infusion or infusion over a specific period of time.

[0109] Alternatively, the pharmaceutical compositions may be administered enterally, for example, by oral or rectal application.

[0110] The dose of C1INH administered to an individual ranges from 1 to 5000 IU per kg of body weight. The dose is preferably within the range of 1 to 2500 IU per kg of body weight, more preferably 1 to 500 IU per kg of body weight, and even more preferably 10 to 500 IU per kg of body weight. The dose is most preferably within the range of 10 IU to 250 IU per kg of body weight, or even 20 IU to 100 IU per kg of body weight. The skilled artisan will be aware of the need to adjust the dose depending on various factors such as the route of administration.

[0111] The dose range of the FXII inhibitor will be adjusted depending on the type of inhibitor used, the route of administration, and other factors well known to those skilled in the art. The dose and administration interval are preferably selected so that the amidolytic activity of FXIIa is completely inhibited for the desired treatment period. For example, the FXII inhibitor antibody is expected to be administered at a dose ranging from 0.01-50 mg / kg body weight, about 0.01-30 mg / kg, about 0.1-30 mg / kg, about 0.1-10 mg / kg, about 0.1-5 mg / kg, about 1-5 mg / kg, about 0.1-2 mg / kg, or about 0.1-1 mg / kg. The treatment may include single or multiple administrations. If multiple administrations are required, they may be administered daily, every other day, weekly, biweekly, monthly, or bimonthly, or as needed. Depositories that slowly and continuously release the antibody or antigen-binding fragment thereof may also be used. A therapeutically effective dose may be a dose that inhibits FXIIa in a subject by at least 50%, preferably at least 60%, 70%, 80%, 90%, more preferably at least 95%, 99% or even 100%.

[0112] In the case of infestin-based FXII inhibitors, such as albumin-fused infestin-4, the dosage may be between 0.1 and 1000 mg / kg of body weight, preferably between 1 and 1000 mg / kg, more preferably between 1 and 500 mg / kg, and even more preferably between 50 and 500 mg / kg.

[0113] For these and other FXII inhibitors, one of skill in the art will be able to adjust the dosage required for treatment and will take into account factors that affect the required dosage, such as the route of administration, the half-life of the inhibitor, the inhibitory activity of the inhibitor, and the affinity of the inhibitor for FXIIa. It is expected that you know the following:

[0114] In the case of treatment of a remote or distal IRI caused by a therapeutic intervention such as a surgical procedure, a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is preferably administered to the individual to be treated prior to the surgical intervention (e.g., limb surgery, heart surgery, organ transplantation, etc.) In one embodiment, a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is thus administered to the individual prior to the start of the surgical intervention.

[0115] A contact activation system inhibitor, preferably a C1INH or FXII inhibitor, may be administered to the patient within 1 week, 6 days, 5 days, 4 days or 72 hours prior to the start of the surgical intervention, preferably within 48 hours, more preferably within 24 hours, even more preferably within 12 hours, and most preferably within 6 hours prior to the start of the surgical intervention.

[0116] For example, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, can be administered to the individual to be treated, for example, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours before the surgical intervention. The contact activation system inhibitor, preferably C1INH or FXII inhibitor, can also be administered to the individual to be treated, for example, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 45 minutes before the therapeutic intervention.

[0117] Alternatively or additionally, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, can be administered to the individual undergoing treatment at or during surgical intervention. In another embodiment, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, is thus administered to the patient during surgical intervention. For example, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, can be administered once or more at intervals (e.g., 15 minutes apart) during the surgical intervention. Alternatively, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, can be administered continuously throughout the duration of the therapeutic intervention.

[0118] Furthermore, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, can be administered to the individual to be treated after surgical intervention. In yet another embodiment, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, is administered to the patient after the end of surgical intervention, preferably within 72 hours after the end of surgical intervention. For example, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, can be administered to the subject to be treated, for example, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 12 hours, about 24 hours, or about 48 hours after surgical intervention. The contact activation system inhibitor, preferably C1INH or FXII inhibitor, can also be administered to the subject to be treated, for example, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 45 minutes after surgical intervention. It is preferred to administer a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, as soon as possible after the surgical intervention and to maintain treatment for at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours, or longer if necessary, after the surgical intervention.

[0119] In yet another embodiment, a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered to the patient during and after completion of the surgical intervention.

[0120] In a further embodiment, a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered to the patient before the start of the surgical intervention, during the surgical intervention and after completion of the surgical intervention.

[0121] In a further embodiment, a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered to the patient before the start of the surgical intervention and after completion of the surgical intervention, but not during the surgical intervention.

[0122] In a further embodiment, a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered to the patient before the start of and during the surgical intervention, but not after completion of the surgical intervention.

[0123] In a further embodiment, a contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered before the start of reperfusion. Preferably, the contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered up to 1 week before reperfusion, up to 6 days, up to 5 days, up to 4 days, preferably up to 72 hours, more preferably up to 48 hours, even more preferably up to 24 hours, up to 12 hours, and most preferably up to 6 hours before reperfusion.

[0124] In a further embodiment, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, is administered after the onset of reperfusion. Preferably, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, is administered up to 72 hours, more preferably up to 48 hours, up to 12 hours, even more preferably up to 6 hours, and most preferably immediately after the onset of reperfusion.

[0125] In a preferred embodiment, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, is administered before and after the start of reperfusion. Preferably, the contact activation system inhibitor, preferably C1INH or FXII inhibitor, is administered up to 1 week, up to 6 days, up to 5 days, up to 4 days, preferably up to 72 hours, more preferably up to 48 hours, even more preferably up to 24 hours, up to 12 hours, and most preferably up to 6 hours before reperfusion, and then the treatment is maintained for a period of time, preferably at least 72 hours, more preferably at least 4 days, even more preferably at least 1 week, most preferably 2 weeks, or longer if necessary, after the start of reperfusion.

[0126] In certain embodiments, the contact activation system inhibitor, preferably a C1INH or FXII inhibitor, is administered to the patient for less than one month, preferably less than 14 days, more preferably less than 7 days, and most preferably less than 48 hours. EXAMPLES

[0127] material and method Animals and housing conditions All experiments were performed under the conditions of the Swiss Animal Protection Law and were approved by the Animal Ethics Committee of the cantonal veterinary department (Cantone of Bern, Switzerland). Male Wistar rats (wild type, domestic animal facility, University of Bern) were housed three per cage under standard housing conditions with food and water available ad libitum. The animals were housed in a room with a controlled temperature of 20 ± 2 °C and an indoor humidity of 45% to 65%, maintaining a 12 / 12-h circadian rhythm (based on winter time). During the light phase, the animals were exposed to a light intensity of 200 lux.

[0128] reagent C1INH (Berinert®) and vehicle (glycine 10 mg / mL, sodium citrate 2.9 mg / mL, and sodium chloride 8.5 mg / mL) were provided by CSL Behring AG (Bern, Switzerland). C1INH was administered as a bolus via the tail vein using an IV cannula (BD Vasculon Plus) 5 min before induction of ischemia.

[0129] The FXIIa inhibitor rHA-Infestin-4 (Hagedorn et al., 2010, Circulation 121:1510-1517) was administered as a bolus via the tail vein at a dose of 100 mg / kg 5 min before inducing ischemia.

[0130] Surgical procedure Male Wistar rats weighing 250-350 g were used for the following experiments. Induction of anesthesia was performed with 2.5% isoflurane in oxygen in an anesthesia induction chamber, and was then maintained by inhalation of 1.5% isoflurane via a nose mask. For assessment of limb perfusion, the hair of both hind limbs was completely shaved with an electric shaver. The rats were kept on a heating pad, and body temperature was maintained at 37°C. Approximately 30 min after induction of anesthesia, ischemia of one hind limb was induced for 3 h. Ischemia was induced by clamping the femoral artery. Therefore, an incision was made in the groin and the adipose tissue was carefully removed to expose the femoral vessels. The femoral artery was then separated from the vein.

[0131] Before clamping the artery, a tourniquet (standard weight 450 g) was placed under the femoral vessels as previously described (8). The femoral artery was clamped with two microvascular clamps (B1-V, S&T The limbs were temporarily occluded with a tourniquet (Switzerland). Immediately, a tourniquet was tightly wrapped to block microvascular blood flow. Hemodynamics was monitored throughout the procedure (Mouse ox plus, Starlifesciences). After 3 h of ischemia, the limbs were reperfused for 24 h each. During the 24 h of reperfusion, the rats were awakened and treated with analgesics (buprenorphine 0.05 mg / kg, Temgesic Reckitt Benckiser (Switzerland AG)). At the end of the experiment, the ischemic and contralateral gastrocnemius muscles were harvested in addition to the lungs for analysis.

[0132] Assessment of edema formation For the evaluation of edema formation, two samples of gastrocnemius muscle were taken from both lower limbs and immediately weighed to obtain wet weight. Muscle samples or lungs were dried at 80°C for 24 hours until a constant weight was obtained. Dry weight was obtained in a second weighing step. Wet to dry weight ratios were then calculated and compared to the wet to dry weight ratios of the contralateral control muscles.

[0133] Determination of deposited complement components and other proteins in muscle and lung tissue Immunofluorescence (IF) was used to quantify deposition of various components of the complement system, as well as fibrin / fibrinogen, heparan sulfate, bradykinin receptor B1, and bradykinin receptor B2. Tissue samples from the gastrocnemius muscles of both lower limbs and from the lungs were collected, washed in PBS, blotted dry, and embedded in matrix (OCT, Tissue tek) on dry ice. Samples were immediately stored at -20°C until cryosectioning (Cryostat, Leica CM 3050S). Sections were fixed in acetone and rehydrated in TBS. Primary antibodies were incubated overnight at 4°C, followed by secondary antibodies for 1 h at room temperature. Slides were then mounted and covered. Photographs were taken under a fluorescent microscope (Leica) and analyzed using Image J software.

[0134] Measurement of cytokines / chemokines using multiplexed arrays An array of cytokines and chemokines was detected using a multiplex immunoassay consisting of magnetic beads conjugated with capture antibodies specific for the target proteins (Bioplex Pro™ Rat Cytokine Group I panel, BioRad, Hercules, USA). The assay was performed according to the manufacturer's instructions.

[0135] Briefly, plasma was diluted 1:3 and incubated with antibody-conjugated magnetic beads. After a washing step, incubation with biotinylated detection antibodies was performed. Cytokine / chemokine concentrations were measured after streptavidin-phycoerythrin incubation. Standard curves were established using recombinant cytokines / chemokines. Analyte concentrations were calculated using Bio-Plex Manager 4.0 software (Bio-Rad, Hercules, USA).

[0136] Assessment of apoptosis by TUNEL assay Apoptosis was assessed using the TdT-mediated dUTP nick end-labeling (TUNEL) assay (in situ Cell Death Detection Kit, TMR red, Roche, Mannheim, Germany). Briefly, frozen sections of tissue samples were fixed in acetone for 5 min at room temperature, washed, and permeabilized with 0.1% Triton-X-100 on ice. Sections were incubated with the TUNEL reaction mixture for 1 h at 37°C in the dark and counterstained with DAPI. After a washing step, sections were mounted, coverslipped, and analyzed under a fluorescent microscope. Immunofluorescence images were analyzed with Image J (National Institutes of Health, Bethesda, MD, USA). The area covered by TUNEL-positive nuclei (%) was analyzed and calculated, all in relation to the area covered by DAPI-stained nuclei.

[0137] statistical analysis Data are expressed as mean ± SD. Statistical significance was determined by one-way analysis of variance with Dunnett's post-hoc test using GraphPad Prism5 software. P values ​​< 0.05 were considered statistically significant.

[0138] result i) Effect of C1INH on local IRI tissue damage C1INH prevented local IRI-induced skeletal muscle edema As a first step, the therapeutic effect of C1INH (50 IU / kg) was evaluated by preventing local edema formation in the hind paws. Systemic preloading of animals with human plasma-derived C1INH highly significantly prevented the formation of skeletal muscle edema (see, e.g., Fig. 1A and B). Using FXII inhibitors, e.g., monoclonal anti-FXII antibodies, or rHA-Infestin-4, similar results, i.e., a reduction in muscle edema formation, were expected.

[0139] ii) Effect of C1INH on IRI-induced distant tissue and organ injury C1INH inhibited remote organ injury by reducing pulmonary edema The effect of C1INH on distant tissue and organ injury was analyzed. C1INH is a highly potent inhibitor of the plasma kallikrein-kinin system by interacting with FXIIa and kallikrein. As shown in Figure 2, C1INH significantly prevented edema formation, as analyzed by wet:dry weight ratio, thus preserving the integrity of endothelial cells in the lungs.

[0140] Effect of C1INH on the expression of bradykinin receptors in the lung and heart Activation of the plasma kallikrein-kinin system produces bradykinin, a molecule that It is a highly potent vasoactive peptide that induces vasodilation and edema. The kallikrein-kinin system has been shown to play a crucial role in IRI. Antagonists of the bradykinin 2 receptor (B2R) have been successfully used in the clinic to treat HAE patients (9). The effect of C1INH on the expression of B1R and B2R was analyzed by IF. Surprisingly, C1INH significantly inhibited IRI-induced B1R upregulation in lung tissue (see Figure 3A), whereas no inhibition could be observed on the expression of B2R (see Figure 3B). The same effect was observed in cardiac tissue (data not shown).

[0141] In the lung, C1INH inhibited fibrin deposition In addition to complement inhibition, C1INH acts on the coagulation system through its interaction with FXII and FXI. Activation of the coagulation system results in thrombin, which cleaves fibrinogen to fibrin. Treatment with C1INH significantly inhibited fibrin deposition in lung tissue (see Figure 4).

[0142] C1INH prevented remote tissue injury in the contralateral limb by preventing fibrin deposition As mentioned above, we could not detect edema formation in the contralateral non-ischemic limb.Surprisingly, we could observe fibrin deposition in the contralateral lower limb (see Fig. 5).As observed in the lung (see Fig. 4A), we observed that C1INH significantly inhibited fibrin deposition in the non-ischemic limb (see Fig. 5).

[0143] C1INH inhibited complement deposition in the contralateral limb, kidney and liver The ischemic and contralateral non-ischemic limbs were analyzed for the deposition of complement fragments as a marker of complement activation. As shown in Figure 6A and B, C1INH significantly reduced the deposition of C3b (classical and lectin pathways) and factor B (alternative pathway of complement activation) in the reperfused and contralateral limbs. In addition, treatment with C1INH significantly reduced the deposition of C3b in the kidney and factor B in the liver.

[0144] In the contralateral limb, treatment with C1INH preserved the endothelial glycocalyx Healthy ECs are covered by a layer of glycosaminoglycans, such as heparan sulfate (HS), which is important for the anticoagulant and anti-inflammatory properties of the endothelium. HS is rapidly released under conditions of inflammation and tissue injury (11-14). Surprisingly, C1INH prevented the release of HS in the contralateral limb (see Figure 7).

[0145] Inhibitory effects of C1INH on inflammatory cytokine and chemokine networks Cytokines, growth factors, and chemokines were measured using xMAP technology in EDTA plasma taken at baseline, i.e., before induction of ischemia, and at the endpoint after 24 hours of reperfusion. Analysis revealed that treatment with C1INH significantly reduced the levels of IL-1α, IL-4, IL-7, IL-17A, and IL-18, as well as IFN-γ, MIP-1α, MIP-3α, and TNF-α (see Table 3).

[0146] [Table 3]

[0147] The following cytokines could not be detected: IL-1β, IL-2, IL-6, IL-12p70, IL-13, G-CSF and GM-CSF. Data are presented as mean ± SD. Statistical significance was determined by Student's t-test using GraphPad Prism5 software. P values ​​<0.05 were considered statistically significant.

[0148] Effect of C1INH treatment on apoptosis in lung and renal cortex Apoptosis was assessed using the TUNEL assay. Cells in the lung tissue of vehicle-treated animals showed a high percentage of apoptosis (76±21%). Apoptosis was observed in both renal cortex and proximal tubule epithelial cells in vehicle-treated animals (43±25%). In comparison, animals treated with C1INH showed a significant reduction in apoptotic cells (10±12% in lung (P<0.0001) and 7±8% in renal cortex).

[0149] iii) Effect of rHA-Infestin-4, an FXII inhibitor, on IRI-induced distant tissue and organ damage Treatment with rHA-Infestin-4 as described above also significantly reduced pulmonary edema, and potentially reduced edema formation in other tissues as well.

[0150] In addition, a significant reduction in fibrin deposition in the lungs (and potentially in other tissues) is observed.

[0151] Following treatment with rHA-Infestin-4, a significant reduction in apoptosis in lung and other tissues is observed. rHA-Infestin-4 also reduces deposition of IgM and complement components, potentially demonstrating an effect on the inflammatory cytokine and chemokine network.

[0152] References 1. Blaisdell, F. W. 2002. The pathophysiology of skeletal muscle ischemia and the reperfusion syndrome: a review. Cardiovasc Surg 10: 620-630. 2. Zeerleder, S. 2011. C1-inhibitor: more than a serine protease inhibitor. Semin Thromb Hemost. 37: 362-374. 3. Caliezi, C., W. A. Wuillemin, S. Zeerleder, M. Redondo, B. Eisele, and C. E. Hack. 2000. C1-Esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema. Pharmacol Rev 52: 91-112. 4. Davis, A. E., III, P. Mejia, and F. Lu. 2008. Biological activities of C1 inhibitor. Mol Immunol 45: 4057-4063. 5. Nielsen, E. W., T. E. Mollnes, J. M. Harlan, and R. K. Winn. 2002. C1-inhibitor reduces the ischaemia-reperfusion injury of skeletal muscles in mice after aortic cross-clamping. Scand J Immunol 56: 588-592. 6. Toomayan, G. A., L. E. Chen, H. X. Jiang, W. N. Qi, A. V. Seaber, M. M. Frank, and J. R. Urbaniak. 2003. C1-esterase inhibitor and a novel peptide inhibitor improve contractile function in reperfused skeletal muscle. Microsurgery23: 561-567. 7. Inderbitzin, D., G. Beldi, I. Avital, G. Vinci, and D. Candinas. 2004. Local and remote ischemia-reperfusion injury is mitigated in mice overexpressing human C1 inhibitor. Eur Surg Res 36: 142-147. 8. Dick, F., J. Li, M. N. Giraud, C. Kalka, J. Schmidli, and H. Tevaearai. 2008. Basic control of reperfusion effectively protects against reperfusion injury in a realistic rodent model of acute limb ischemia. Circulation 118: 1920-1928. 9. Bouillet, L. 2011. Icatibant in hereditary angioedema: news and challenges. Expert Rev Clin Immunol 7: 267-272. 10. Souza, D. G., E. S. Lomez, V. Pinho, J. B. Pesquero, M. Bader, J. L. Pesquero, and M. M. Teixeira. 2004. Role of bradykinin B2 and B1 receptors in the local, remote, and systemic inflammatory responses that follow intestinal ischemia and reperfusion injury. J Immunol 172: 2542-2548. 11. Johnson, G. B., G. J. Brunn, Y. Kodaira, and J. L. Platt. 2002. Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. J Immunol 168. 12. Platt, J. L., G. M. Vercellotti, B. J. Lindman, T. R. Oegema, Jr., F. H. Bach, and A. P. Dalmasso. 1990. Release of heparan sulfate from endothelial cells. Implications for pathogenesis of hyperacute rejection. J Exp Med 171. 13. Ihrcke, N. S., and J. L. Platt. 1996. Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: evidence for proteolysis of cell-s urface molecules. J Cell Physiol 168. [ PubMed ] 14. Ihrcke, NS, Parker W, Reissner KJ, and Platt JL. 1998. Regulation of platelet heparanase during inflammation: role of pH and proteinases. J Cell Physiol https: / / doi.org / 10.1103 / PhysRevLett.91.111011 , Google Scholar Crossref , CAS 15. D. Liu, F. Lu, G. Qin, SM Fernandes, J. Li, and AE Davis, III. 2007. C1 inhibitor-mediated protection from sepsis. J Immunol 179: 3966–3972. [ PubMed ] 16. Storini, C., E. Rossi, V. Marrella, M. Distaso, R. Veerhuis, C. Vergani, L. Bergamaschini, and MG De Simoni. 2005. C1-inhibitor protects against brain ischemia-reperfusion injury via inhibition of cell recruitment and inflammation. Neurobiol. Dec. 19: 10-17.

Claims

1. 1. A pharmaceutical composition comprising a contact activation system inhibitor that is a FXII inhibitor for use in the treatment and / or prevention of remote ischemia-reperfusion injury (IRI), said treatment and / or prevention comprising administering said pharmaceutical composition to an individual, wherein said FXII inhibitor is: (i) a polypeptide consisting of the wild-type Infestin-4 polypeptide sequence (SEQ ID NO:2), or (a) the sequence of SEQ ID NO:2 modified to include 1 to 5 amino acid mutations outside the C-terminus or outside the N-terminus of amino acids 2 to 13 of the N-terminus of SEQ ID NO:2; and / or (b) a sequence that contains at least 90%, 95%, 98% or 99% homology with SEQ ID NO:2 and retention of the six conserved cysteine ​​residues from SEQ ID NO:2; A polypeptide consisting of and / or (ii) an anti-FXII antibody, which binds to FXIIa, including FXIIa alpha and FXIIa beta, and inhibits the amidolytic activity of FXIIa; The pharmaceutical composition is selected from the group consisting of:

2. The pharmaceutical composition of claim 1 , wherein the contact activation system inhibitor is administered to the individual parenterally.

3. The pharmaceutical composition of claim 2 , wherein the contact activation system inhibitor is administered to the individual intravenously, intraarterially, or subcutaneously.

4. The dose of FXII inhibitor administered to an individual is sufficient to completely inhibit the amidolytic activity of FXIIa. The pharmaceutical composition of claim 1, which is suitable for inhibiting

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the individual is a patient who is about to undergo or has undergone a surgical intervention.

6. The method of claim 5, wherein the contact activation system inhibitor is administered to the patient within 6 hours prior to the initiation of surgical intervention or the initiation of reperfusion.

7. 7. The pharmaceutical composition of claim 5 or claim 6, wherein the contact activation system inhibitor is administered to the patient before and / or during and / or after surgical intervention or before and / or after the initiation of reperfusion.

8. The method of claim 7, wherein the contact activation system inhibitor is administered to the patient within 72 hours after the end of the surgical intervention or the start of reperfusion.

9. The pharmaceutical composition according to any one of claims 5 to 8, wherein the surgical intervention is selected from the group consisting of: (a) elective surgery, reconstructive surgery, vascular surgery, cardiac surgery, trauma surgery, surgery for crash or crush injuries, cancer surgery, orthopedic surgery, transplantation, minimally invasive surgery, or (b) insertion of instruments for delivery of pharmacologically active substances such as thrombolytic agents or vasodilators and / or for mechanical removal of complete or partial occlusion after onset of an initial thrombotic, or thromboembolic, or another ischemia-induced disorder, and injection of medicamentarily active substances such as thrombolytic agents or vasodilators.

10. The pharmaceutical composition according to any one of claims 1 to 4, wherein reperfusion occurs spontaneously or the impaired blood flow is improved by intervention other than surgical intervention.

11. A pharmaceutical composition for preventing ischemia-reperfusion injury (IRI) in a patient about to undergo a surgical intervention, comprising an effective dose of a contact activation system inhibitor, said contact activation system inhibitor being a FXII inhibitor, administered before and / or during and / or after the surgical intervention or before and / or after the initiation of reperfusion, wherein the ischemia-reperfusion injury (IRI) affects an organ or tissue remote from the surgical site, and wherein said FXII inhibitor is: (i) a polypeptide consisting of the wild-type Infestin-4 polypeptide sequence (SEQ ID NO:2), or (a) the sequence of SEQ ID NO:2 modified to include 1 to 5 amino acid mutations outside the C-terminus or outside the N-terminus of amino acids 2 to 13 of the N-terminus of SEQ ID NO:2; and / or (b) a sequence that contains at least 90%, 95%, 98% or 99% homology with SEQ ID NO:2 and retention of the six conserved cysteine ​​residues from SEQ ID NO:2; A polypeptide consisting of and / or (ii) an anti-FXII antibody, which binds to FXIIa, including FXIIa alpha and FXIIa beta, and inhibits the amidolytic activity of FXIIa; The pharmaceutical composition is selected from the group consisting of:

12. 12. The pharmaceutical composition of claim 11, wherein the remote ischemia-reperfusion injury (IRI) affects the lungs, heart, brain, kidneys, intestines, pancreas, liver, or limbs / limbs.

13. Remote ischemia-reperfusion injury (IRI), if untreated, can lead to multiple organ dysfunction syndrome or systemic The pharmaceutical composition of claim 11 or 12, which is effective in treating chronic inflammatory response syndrome.

Images