WO2001015723A1 - Preventives or remedies for ischemic injury or ischemic reperfusion injury - Google Patents

Abstract

Novel preventives or remedies for ischemic injury or ischemic reperfusion injury which contain heparin cofactor II. These preventives or remedies exert a myocardial protective effect in model dogs with myocardial ischemic reperfusion, a myocardial protective effect in model rats with myocardial ischemic reperfusion, etc., which indicates that these drugs are useful in preventing or treating ischemic injury or ischemic reperfusion injury, in particular, ischemic injury or ischemic reperfusion injury in association with ischemic heart diseases.

Description

 Specification

 Agent for preventing or treating ischemic injury or ischemia-reperfusion injury

 Technical field

 The present invention relates to a prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury, comprising heparin cofactor II (hereinafter referred to as “H C II”) as an active ingredient.

 Background art

 In the heart, in the early stage of a myocardial infarction attack caused by occlusion of the coronary artery, serious conditions such as cardiogenic shock and fatal arrhythmia may occur. (Thrombolysis, percutaneous transluminal coronary angioplasty (PTCA), coronary artery bypass surgery (CABG), etc.) are performed. Recently, however, attention has been paid to a condition called ischemia-reperfusion injury in which the myocardial fiber damage is exacerbated by the resumption of blood flow.

 This ischemia-reperfusion injury occurs not only in the heart but also after reperfusion of organ tissues that have been subjected to ischemia in various organs, such as when blood flow is stopped during transplantation of organs including blood vessels and resumed. Is also a problem. Therefore, it is known to occur not only in the heart but also in many tissues such as the brain, kidney, liver, lung, knee, and digestive tract.

 At present, it is thought that one of the causes of the ischemia-reperfusion injury is energy metabolism increase due to rapid reoxygenation, increase in reactive oxygen species, lipid peroxide, etc. The details of the actual condition are unknown.

 Under such circumstances, various studies have been made on the use of active oxygen scavengers or antioxidants as preventive or therapeutic methods for ischemic injury or ischemia-reperfusion injury. However, the effect of preventing or treating the disease is not sufficient. Therefore, development of a preventive or therapeutic agent for such ischemic injury or ischemia-reperfusion injury is demanded.

HC II, like antithrombin III (hereinafter referred to as "ΑΤΙΠ"), is an important anticoagulant protein existing in the body (Matsuo et al .: Biomedical Perspectives, 2, 269-274 (1993)). According to acrylamide gel electrophoresis (SDS-PAGE) analysis, it is a single-chain plasma glycoprotein with a molecular weight of 72 kDa (Dougls M. Tollfsen et al .: J. Biol. Chemistry, 257, 2167-2169 (1982)). . As the physiological function of HCII, it is known that it inhibits thrombin and other proteases. However, it has a different site of action from と し て and seems to function as a thrombin inhibitor outside the blood vessels compared to blood vessels 內. Therefore, since Hen exhibits an anticoagulant effect in a specific tissue, tissue or cell, the present inventors have suggested that systemic administration of HCII to veins, arteries, etc. may cause a disease that causes sepsis or abnormal blood flow. It has been found that it is effective in preventing or treating diseases such as organ disorders such as liver, lung, kidney, and brain (Japanese Patent Application Laid-Open No. 9-176040).

 In addition, the present inventors have found that HCII is also effective for the treatment of a disease caused by enhanced dysfunction of in vivo cells such as macrophages even when administered locally for the disease (Japanese Patent Application No. No. 191977).

 Furthermore, the present inventors have found that HCII has a blood pressure lowering inhibitory action, and in particular, is useful as a preventive or therapeutic agent for blood pressure lowering because it suppresses blood pressure lowering caused by endotoxin shock. Japanese Patent Application No. 11-93447).

 Thus, when HCII is formulated as an active ingredient, its usefulness as a drug is considered.

 However, there is no known effect of HCII on the prevention or treatment of ischemic injury or ischemia-reperfusion injury.

 Disclosure of the invention

 An object of the present invention is to provide a novel prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury, and to provide a new pharmaceutical use of HCII.

 The present inventors have conducted various studies in order to solve the above-mentioned problems, and as a result, have found that HCII has an effect of preventing or treating ischemia injury or ischemia-reperfusion injury, especially for ischemic heart disease. The present inventors have found that the present invention is useful for prevention or treatment of ischemic injury or ischemia-reperfusion injury, and have completed the present invention. That is, the present invention is as follows.

 (1) A prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury comprising heparin cofactor II as an active ingredient.

 (2) The prophylactic or therapeutic agent according to the above (1), which is a preventive or therapeutic agent for ischemia-reperfusion injury.

(3) Prevention or cure of ischemia-reperfusion injury by reperfusion after ischemia caused by ischemic heart disease Remedies.

 (4) The prophylactic or therapeutic agent according to any of (1) to (3) above, which is administered after ischemia and before reperfusion.

 (5) A method for preventing or treating ischemic injury or ischemia reperfusion injury, which comprises the step of administering heparin cofactor-1 II to a patient.

 (6) A method for preventing or treating ischemia-reperfusion injury, which comprises a step of administering heparin cofactor-1 II to a patient.

 (7) The method according to (6), wherein the ischemia-reperfusion injury is ischemia-reperfusion injury due to reperfusion after ischemia caused by ischemic heart disease.

 (8) A method for preventing or treating ischemia-reperfusion injury, which comprises a step of administering heparin cofactor-1 II after ischemia and before reperfusion.

 (9) Use of heparin cofactor II for the manufacture of a prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury.

 (10) Use of heparin cofactor 製造 for the manufacture of a prophylactic or therapeutic agent for ischemia-reperfusion injury.

 (11) The use of heparin cofactor-II according to the above (10), wherein the ischemia-reperfusion injury is ischemia-reperfusion injury due to reperfusion after ischemia caused by ischemic heart disease.

 (12) Use of heparin cofactor-II for the manufacture of a preventive or therapeutic agent for ischemia-reperfusion injury to be administered after ischemia and before reperfusion.

 (13) A prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury according to any of (1) to (4) above, and the prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury A commercial package containing documentation stating that it can or should be used for prevention or treatment.

 Detailed description of the invention

The origin of the HCII used in the present invention is not particularly limited as long as it is purified to such an extent that it can be used as a medicament. Naturally-derived HCII (for example, human, monkey, pest, puma, Dogs, gray herons, mice, rats and other mammals). (Eg, those derived from a recombinant host genetically manipulated to produce HCII, those synthesized by chemical synthesis, etc.). In addition, one or more amino acids in the amino acid sequence of HCII have mutations such as deletion, substitution, addition or induction, and those in which such mutations have been made by known means. Also, those having a preventive or therapeutic effect on ischemic injury or ischemia-reperfusion injury are encompassed by HC II in the present invention. Naturally derived HCII can be obtained, for example, by purifying human whole blood, plasma, serum, or serum expressed from coagulated blood. For example, as a method for purifying whole plasma, fractionated plasma, corn by fractionation method I, supernatant Π + III or fractionation IV or decryoplasm, anion exchange chromatography, cation exchange chromatography An example is a method of performing a parin treatment on a lithography treatment or a solid-phase treatment (JP-A-9-286797). Furthermore, in order to remove contaminating proteins as much as possible and obtain highly purified HCII, use hydrophobic chromatography, fractionation with a water-soluble polymer, salting out, or use basic amino acid as a ligand. Processing by affinity chromatography can be performed. These processes may be used alone or in combination. The hydrophobic chromatographic treatment is carried out, for example, by bringing a solution containing HC II and a depolymerizing factor into contact with a carrier for hydrophobic chromatography under conditions of pH 6 to 9. The term “molecular-weight-decreasing factor” as used herein refers to a substance that degrades HC II to a molecular weight.

 Examples of the carrier for hydrophobic chromatography include an alkyl group type having 4 to 18 carbon atoms (for example, a butyl group type, an octyl group type, an octyl decyl group type), and a fuunyl group type. Examples of the butyl group type include butyl monoagarose and butyl polyvinyl (trade name: butyl toyopearl, manufactured by Tosoh Corporation). Examples of the octyl group type include octyl agarose, and examples of the octyl decyl group type include: Octyldecyl-garose and the like. Examples of the phenyl group type include phenyl monocellulose (trade name: Phenylseguchi Fine, manufactured by Seikagaku Corporation). By this treatment, HC II is recovered in the unadsorbed fraction, and the low-molecular-weight factor is adsorbed on the carrier.

The fractionation treatment with the water-soluble polymer is preferably performed at 1 to 30 ° / _o (w / v) of the water-soluble polymer. Is performed by adding to a solution containing HCII and a depolymerizing factor so as to have a concentration of 3 to 20% (w / v), more preferably 6 to 12% (w / v). By this treatment, HCII remains in the solution, and the low molecular weight factor precipitates. By centrifuging or filtering the treated solution and collecting the supernatant (filtrate), the low molecular weight factor can be separated from HCII. Here, the water-soluble polymer refers to polyethylene glycol (PEG), a nonionic surfactant or the like. PEG preferably has an average molecular weight of 4000 to 6000. Examples of the surfactant include polyoxyethylene (160) polyoxypropylene (30) glycol, and polyoxyethylene (20) sorbitan monooleate.

 The salting-out treatment is performed by adding a salt such as sodium, potassium, barium, ammonium, etc., for example, chloride, sulfate, phosphate, etc. to a solution containing HCII and a molecular weight reducing agent at a concentration of 0.05 to 0.25 M. Is carried out. Preferably, a method in which barium chloride is added to a concentration of 0.05 to 0.2 M is exemplified. By this treatment, HCII remains in the solution, and the depolymerizing factor precipitates. By centrifuging or filtering the treatment solution and collecting the supernatant or solution, the low molecular weight factor can be separated from HCII.

 In affinity chromatography using a basic amino acid as a ligand, a solution containing HCII and a molecular weight-decreasing factor can be treated with a basic amino acid such as lysine or arginine (preferably lysine) as a ligand under a pH condition of 6 to 9. This is performed by bringing the carrier into contact with a chromatographic carrier for immobilization. Examples of the carrier include insoluble supports such as polysaccharides, silica gel, and fibers. More specifically, it is agar, agarose, cross-linked agarose, cellulose, silica, nylon, hydrophilic vinyl polymer and the like. By this treatment, HCII is recovered in the unadsorbed fraction, and the depolymerizing factor is adsorbed on the carrier. In order to minimize the production of low molecular weight HCII, facilitate subsequent purification, and increase the recovery rate of HCII, the step of separating HCII from the low molecular weight factor should be performed at an early stage of purification. Is more preferred.

It is preferable that the solution containing HCII and the molecular weight-decreasing factor is contacted with immobilized heparin to bind HCII to heparin. The immobilized heparin is, for example, monomeric heparin bound to a solid phase such as an insoluble support such as polysaccharide, silica gel, or fiber. Its support Examples of carriers include agar, agarose, cross-linked agarose, cellulose, silica, nylon, hydrophilic vinyl polymers, and others known in the art. Such heparin immobilization on the solid phase may be performed not only before the above-described step of separating HCII and the molecular weight-decreasing factor but also after it. By this treatment, a part of the polymerized HCII and the low molecular weight HCII is separated into a fraction different from the monomeric HCII (effective HCII) fraction and removed. Here, the polymerized HCII means a polymer of HCII molecules, which has no physiological activity of HCII and low affinity for heparin. More specifically, it is a dimer or higher polymer.

 HCII is washed with a buffer solution having a salt concentration of about 0.05 to 0.2 M or the like, preferably after stepwise increasing the salt concentration and repeating the washing operation, and then washed with a salt concentration of about 0.05 to 1.0 M, preferably 0.1 It is eluted from the immobilized heparin by a method such as contacting with a buffer solution of about 0.5 M, more preferably about 0.1 to 0.2 M. Examples of the salt component for adjusting the ionic strength include sodium chloride, sodium chloride, ammonium sulfate, sodium thiocyanate, and sodium sulfate. Preferably it is sodium chloride. Further, the pH of the buffer is preferably set to 6 to 9.

 The HCII-containing solution can be further subjected to anion exchange chromatography. Examples of anion exchangers used include DEAE type [DEAE-Sephadex, DEAE-Cellulose, DEAE-Sepharose, DEAE-Polybininole (eg: DEAE-Toyopearl), etc.], QAE type [QAE-Sephadex, QAE-Cenorelose , QAE-Sepharose, QAE-Polyvinyl (eg ··· QAE—Toyopearl)].

 Equilibration and washing of the anion exchanger in one treatment of anion exchange chromatography are performed using a 0.005 to 0.1 M citrate buffer (about pH 6 to 8) and a 0.005 to 0.1 M phosphate buffer (about ρΗ6 to 8). ) Or by using 0.005 to 0.1 M Tris-HCl buffer (pH 7 to 9). The elution of HCII from the anion exchanger is a buffer used for equilibration and washing, and contains 0Ο5-0.5Ο, preferably 0.2-0.25Μ sodium chloride. Is used.

Treatment for separating HCII from low molecular weight factor, immobilized heparin treatment and The order of one treatment of ion exchange chromatography is not particularly limited, and can be arbitrarily selected. In addition, from the viewpoint of minimizing polymerization and depolymerization of HCII in the purification process, it is preferable to perform each of the above processes under the conditions of ρΗ6 ~ 9 and 4 ~ 20 ^, unless otherwise specified. . All buffers used in each of the above steps should contain 0.001% to 1.0% (w / v) of a water-soluble polymer (eg, PEG4000) to prevent polymer formation. preferable.

 The HC II active fraction obtained as a result of each of the above purification processes may be contaminated with low molecular weight HC II molecules. HCII means a fragmented HCII molecule that gives a peak (shoulder) that can be distinguished from an effective HCII peak by high-performance liquid chromatography (hereinafter referred to as HPLC). Conditions will be described later). Therefore, in order to separate and remove the low molecular weight HCII, it is preferable that the HCII active fraction is further subjected to a separation step based on a difference in molecular weight such as ultrafiltration and Z or gel filtration chromatography.

 The ultrafiltration membrane used for ultrafiltration is not particularly limited as long as it has a pore size that allows only low molecular weight HCII to pass without passing effective HCII. Membrane of acrylonitrile copolymer, aromatic nylon, polysulfone, polyvinylidene polyfluoride, polyimide resin, etc. is usually used, and various forms such as tubular membrane, flat membrane, spiral module and hollow fiber module are selected and used it can. Since the pore size of the ultrafiltration membrane is not constant, if the difference in molecular weight between the low molecular weight HCII to be removed and the effective HCII is small, first concentrate the effective HCII by ultrafiltration, then It is preferable to carry out filtration chromatography.

Gels used for gel filtration chromatography include cross-linked dextran beads (eg, Sephadex), Futachika polyacrylamide beads (eg, Biogel), agarose gel (eg, Sepharose), and dextran monoacrylamide (eg, Sephacryl). ) And the like. For gel filtration, a 0.01 to 0.05% citrate buffer or a phosphate buffer (about 6 to 9) containing O to 0.5M, preferably 0.1 to 0.2M sodium chloride is used. In order to prevent the polymerization and depolymerization of HCTI as much as possible, it is desirable that the above-mentioned ultrafiltration and gel filtration chromatography be performed under conditions of pH 6 to 9 and 4 to 20. All buffers used in each of the above steps contain 0.001% to 1.0% (w / v) of a water-soluble polymer (eg, PEG4000, etc.) to prevent polymer formation. Is more preferable.

 As a result of each of the above treatments, the purity was 98% or more, preferably 99.9 ° /. The above HC II-containing composition can be obtained (PCT / JP98 / 04939).

 Among the artificial HCII, those derived from the recombinant host include, for example, native HCII (see, for example, Blinder et al., Biochemistry, 27, 752-759 (1988)), or antithrombin activity is retained or improved. A culture solution or cell extract obtained by culturing a host cell transformed with a gene encoding the mutated HC II (see, for example, JP-A-3-139280), or encoding HC II It can also be obtained from bodily fluids or milk of transgenic animals incorporating the gene.

 The preventive or therapeutic agent for ischemic injury or ischemia-reperfusion injury of the present invention includes an agent for suppressing ischemia injury occurring in a living tissue during ischemia and reperfusion injury occurring during reperfusion after ischemia. I do. Examples of ischemic injury or ischemia-reperfusion injury include heart disorders (eg, ischemic heart disease, myocardial infarction, etc.), cerebrovascular disorders (eg, cerebral thrombosis, cerebral infarction, etc.), kidney disorders (eg, nephritis, UX insufficiency) Etc.), liver disorders (eg, fulminant hepatitis, etc.), lung disorders (eg, acute lung injury, adult respiratory distress syndrome (ARDS), etc.), and knee disorders (eg, knee inflammation, etc.). Examples of ischemic injury or ischemia reperfusion injury include ischemia injury due to vascular occlusion during organ transplantation including blood vessels or surgery involving ischemia, and ischemia reperfusion due to reperfusion after ischemia due to vascular occlusion. Obstacles are a specific example. In addition, the disorders also include new myocardial * a tissue disorders that occur after reperfusion in ischemic heart disease.

The agent for preventing or treating ischemic injury or ischemia-reperfusion injury of the present invention can be formulated by a method known per se. At this time, if necessary, treatments such as heating, sterilization, sterilization filtration, freeze-drying, etc. are performed, and pharmaceutically acceptable additives (eg, carriers, excipients, buffers, isotonicity) in each treatment step. Agents, stabilizers, diluents, solubilizers, etc.) Freeze-dried; ^ form of liquid, liquid, etc. is obtained. In particular, a freeze-dried preparation in which HC II is prepared as a freeze-dried product together with a pharmaceutically acceptable excipient and then dissolved in a suitable vehicle (eg, distilled water, sterile purified water, physiological saline, etc.) at the time of use. It is preferable that The lyophilized preparation is preferably diluted with, for example, distilled water for injection so that the HCII concentration becomes about 0.1 to 100 mg / ml.

 Pharmaceutically acceptable additives that can be incorporated into the prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury of the present invention include, for example, buffers (those that maintain a physiologically preferable pH value of the preparation) , Isotonic agent (the salt is not particularly limited as long as it maintains the physiologically isotonic salt concentration of the preparation), stabilizing agent (eg, sugars such as mannitol, sorbitol, etc.) ) And the like, but not particularly limited thereto.

 The route of administration of the agent for preventing or treating ischemic injury or ischemia-reperfusion injury according to the present invention includes parenteral administration. Examples of the administration form include injections, infusions, or mucous membranes or skin Absorbents and the like. Also, the prophylactic or therapeutic agent of the present invention may be administered locally.

 The preventive or therapeutic agent for ischemic injury or ischemia-reperfusion injury of the present invention can be administered at any time before, during, or after ischemia, and ischemic injury or ischemia-reperfusion injury is predicted. Can be administered when ischemic injury or ischemia-reperfusion injury occurs. This dose can be appropriately increased or decreased according to the patient's symptoms, weight, sex, etc., but is usually 0.1 to 300 mg / kg, preferably; Administer 100 mg / kg once to several times per B. The administration is preferably performed, for example, after ischemia and before reperfusion.

 In the present specification, the amount of HC II is calculated from the numerical value (A280) obtained by measuring the absorbance at a wavelength of 280 nm for 1 mL of an HC II-containing solution having a purity of 98% or more in HPLC analysis under the following conditions. The extinction coefficient was calculated as 5.93.

 HPLC analysis conditions

 Column: G3000SWXL (φ8 mm X 30cm; manufactured by Tosoh Corporation)

Eluent: 0.1 M sodium acetate, 0.3 M sodium chloride, 0, 1% sodium azide (pH 6.5)

 Flow rate: 1. OiiiL / min Detection wavelength: 280nm

 Sample injection volume: 50 / L (A280 = approx. 1 (Min. 0.2 to max. 2))

 The present invention will be described in detail with reference to the following production examples, experimental examples and preparation examples, but the present invention is not limited thereto.

Production Example 1

The 11 + II II, which was obtained according to the method of Kohn et al. (J. Am. Chem. So, 72, 465 (1950)), was used for the heparin affinity chromatography-carrier (trade name: he (Parintoyopearl: manufactured by Tosoh I) was passed through a lOOmL column which was washed with 10 mM sodium citrate buffer (pH 6.8) containing 21% ethanol, and then 400 mM at 2-10 ° C. of Kuen acid Natoriumu aqueous lOraM containing chloride Natoriumu _(P H7. 0) to afford the eluate fractions were eluted (hereinafter referred to as "Paris emission eluent to") used.

 The heparin eluate was diluted with 20 mM phosphate buffer (pH 8) and washed with the same buffer. After passing through a column packed with an anion exchanger (trade name: QAE Toyopearl 550C: manufactured by Tosoh Corporation), the eluted fraction was eluted using 20 mM phosphate buffer containing 250 πιΜ sodium chloride. Obtained. The eluate was dialyzed against a 0.02 M buffer of Tris-monohydrochloride (pH 8.5). After passing the above-mentioned anion eluate through a column packed with heparin affinity chromatography carrier (trade name: Heparinto Yopearl: manufactured by Tosoh Corporation) washed with the same buffer, lOOmM sodium chloride was added. The eluted fraction was eluted using a 20 mM Tris-monohydrochloride buffer (pH 8.5) to obtain an eluted fraction.

Production Example 2

After adjusting the HCII-containing composition obtained according to Production Example 1 to have an A280 value of 2.5 and an ammonium sulfate content of 1 M, the composition (5 mL) was added to a hydrophobic chromatography support ( Trade name: Phenylsephalose: Amersham Pharmacia) and equilibrated with 20 mM Tris-hydrochloric acid buffer (developing solution) containing 1 M ammonium sulfate (Φ 6 x 5 cm, column volume lOraL) ). After applying the composition to the column, the column volume 15723

 PCT / JP00 / 05974

6 to 8 times the amount of the developing solution and the ram volume with only the concentration of ammonium sulfate reduced to 0.95M, and 2 times the amount of the developing solution are sequentially flowed at a flow rate of 2 mL / min to wash the low molecular weight HC II. Separated. Subsequently, a developing solution having a column volume of 5 times the amount of ammonium sulfate alone reduced to 0.8 M was flowed through the column at the same speed to obtain an H C II fraction containing no low molecular weight H C II.

The HC II fraction, 10 mM sodium phosphate and 0. 15M sodium chloride _(P H7. 0) dialyzed in to replace the buffer and then stored frozen, dissolved using the following experimental examples at use Was.

Experimental Example 1: Myocardial protective effect of H C II in canine myocardial ischemia-reperfusion model

An adult dog weighing 10 to 15 kg was opened on the left VI. Then, a centron (manufactured by St. Holland in the Netherlands) and a conductance catheter (manufactured by Cordis, EuropaNV, Podeu) for measuring left ventricular pressure and volume are inserted from the apex of the left ventricle into the left anterior descending coronary artery (LAD). occluder was installed. A silicone catheter was placed in the left femoral vein for drug administration. HC II (16. 8mg / kg, in terms of the unit of AT III 100U / k _g) or control as 0. 5 ° /. HSA was administered intravenously, and LAD was occluded with an occluder 5 minutes later. Occlusion was performed for 15 minutes, followed by reperfusion. Before and after drug administration, the left ventricular pressure volume map was recorded with the left ventricular volume measuring device Sigma 5 (manufactured by eycom, The Netherlands) 15 minutes after LAD occlusion (immediately after reperfusion) and 15, 60, and 120 minutes after reperfusion. The ventricular end-systolic elastance (Ees, an index of left ventricular contractility) and the arterial effective elastance (Ea, afterload) were calculated, and the ventricular artery coupling (Ees / Ea) was calculated. Also, blood was collected at each time point, Endothelin- 1 the IBL made Endothelin- 1 measurement kit, N0 ₂ - / N0 ₃ - the Cayman manufactured NO ₂ - / N0 ₃ - was measured using a kit.

As a result, left ventricular function (Ees / Ea) decreased in the control group immediately after reperfusion. On the other hand, Ees / Ea did not decrease in the group to which HC II was administered, suggesting that HC II has an inhibitory effect on left ventricular dysfunction (Table 1). Further, in the control group, NO ₂ one / N0 shortly after reperfusion ₃ - is, endothelin after 15 minutes reperfusion - 1 is increased, but it was suggested that vascular endothelial disorder occurs, either in the HC II group There was no increase in the parameters (Tables 2 and 3). These results suggest that HC II exerts a cardioprotective effect by suppressing left ventricular dysfunction after ischemia-reperfusion and inhibiting endovascular endothelial damage. Table 1. Effect of Heparin Cofactor-IT on the Reduction of Left Ventricular Contractile Function (Ees / Ea) in Canine Myocardial Ischemia / Reperfusion Model (n = 6, mean ± standard error)

実施例 2 ： ラット心筋虚血再灌流モデルにおける H C IIの心筋保護作用

Example 2: Cardioprotective effect of HC II in rat myocardial ischemia-reperfusion model

Approximately 300 g of male Wistar rats were anesthetized by intraperitoneal administration of pentobarbital—Na 50 mg / kg, then fixed in a dorsal position on a heated mat (set in SMS-200J, Medical System Inc., 38). Then, a force renewal was introduced into the trachea, and controlled respiration (10 mL / kg, 70 cpm) was performed using a mechanical respirator (MODEL 683, Harvard Apparatus). In addition, a mirror microphone orifice tip pressure transducer (SPC-320, MILLAR INSTRUCTIONS, INC.) Was inserted into the left ventricle via the right carotid artery for measurement of left ventricular function. After thoracotomy at the left rib, the heart was exposed from inside the thoracic cavity, and a suture was applied to the myocardium around the origin of the anterior descending left coronary artery for ligation of the left coronary artery, and the heart was returned to the thoracic cavity. HC II (42mg / 7.24raL / _kg AT Administer a solvent (10 mM phosphate buffer, 0.15 NaC1, pH 7.0) from the left femoral artery as a control, and 5 minutes later, ligate the coronary artery for 20 minutes to perform myocardial ischemia. Blood was taken. Thereafter, the ligation was released and reperfusion was performed, and the left ventricular function was measured and the weight of the myocardial necrotic site 120 minutes later was measured. Left ventricular function was measured before administration of the drug and at 30, 60, and 120 minutes after reperfusion, using left ventricular pressure analysis software (Vmax TC Analize ver. 1.1.0, Fujitech). Then, LV dP / dt max was calculated as an index of systolic function, and LV dP / dt min was calculated as an index of diastolic function.The weight of the myocardial necrosis was measured by ligating the coronary artery again 120 minutes after reperfusion, and using the EVANS blue solution ( After administering 2 mL of bolus (20 mg / mL) from the right femoral vein, the heart was excised and the weight of the left ventricle was measured. The necrotic part was stained with the solution (37 minutes, 20 minutes), and the weight of the unstained necrotic part was measured, and the weight of the necrotic part was calculated from these values. Weight is smaller in the HC II group than in the control group, and myocardial necrosis due to ischemia-reperfusion due to administration of HC II In the control group, LV dP / dt max at 120 min after reperfusion was lower than before drug administration, but LV dP / dt max was reduced by HCII administration. The decrease in max was suppressed (Table 5), and LV dP / dt πάη was similarly improved by administration of HC II (Table 6). It was suggested that they suppressed necrosis and decreased left ventricular function In the control group, some animals died after reperfusion, but no animals died in the HC II-treated group. 4. Effect of heparin cofactor II on myocardial necrosis in rat myocardial ischemia-reperfusion model (n = 6, mean soil standard error)

 Necrosis weight 120 minutes after reperfusion / "Ischemia weight,%

 Control group 47 ± 5

HC II 15 ± 3 Table 5. Effect of heparin cofactor-II on lowering left ventricular contractile function (LV dP / dt max) in rat myocardial ischemia-reperfusion model (n = 4 to 6, standard error of the mean)

表 6 .ラット心筋虚血再灌流モデルにおけるへパリンコファクタ一 IIの左心室拡張機 能 （LV dP/dt niin) 低下抑制効果 （n = 4〜6、 平均士標準誤差）

Table 6. Inhibitory effect of heparin cofactor-II on lowering left ventricular diastolic function (LV dP / dt niin) in rat myocardial ischemia-reperfusion model (n = 4 to 6, standard error of the mean)

以上の結果から、 H C IIは虚血障害もしくは虚血再灌流障害を予防または治療する 優れた作用を有することが示された。

From the above results, it was shown that HC II has an excellent effect of preventing or treating ischemic injury or ischemia-reperfusion injury.

Formulation Example 1

 In the vial,

 H C II 8 mg

 Disodium hydrogen phosphate 19mg

 Sodium dihydrogen phosphate 8mg

 Sodium chloride 丄 ismg

The freeze-dried product was dissolved in 20 mL of distilled water for injection at the time of use to prepare an intravenous formulation.

 Industrial applications

The heparin cofactor II of the present invention has a cardioprotective effect in a canine myocardial ischemia-reperfusion model, a myocardial protective effect in a rat myocardial ischemia-reperfusion model, and ischemic injury or ischemia-reperfusion injury from other pharmacological experiments. Has a prophylactic or therapeutic effect on ischemic injury, particularly an inhibitory effect on myocardial tissue damage associated with coronary ischemia-reperfusion, and is a preventive or therapeutic agent for ischemic injury or ischemia-reperfusion injury, particularly ischemia caused by ischemic heart disease Of ischemia-reperfusion injury due to post-perfusion reperfusion Useful for prevention or treatment. This application is based on U.S. Patent Application No. 248031 filed in Japan, the contents of which are incorporated in full herein.

Claims

The scope of the claims  I. A preventive or therapeutic agent for ischemic injury or ischemia-reperfusion injury comprising heparin cofactor π as an active ingredient.  2. The preventive or therapeutic agent according to claim 1, which is a preventive or therapeutic agent for ischemia-reperfusion injury. 3. An agent for preventing or treating ischemia-reperfusion injury due to reperfusion after ischemia caused by ischemic heart disease.  4. The prophylactic or therapeutic agent according to any one of claims 1 to 3, which is administered after ischemia and before reperfusion.  5. A method for preventing or treating ischemic injury or ischemia-reperfusion injury, which comprises the step of administering heparin cofactor-1 to a patient.  6. A method for preventing or treating ischemia-reperfusion injury, comprising the step of administering heparin cofactor-II to a patient.  7. The method of claim 6, wherein the ischemia-reperfusion injury is ischemia-reperfusion injury due to reperfusion after ischemia due to ischemic heart disease. 8. A method for preventing or treating ischemia-reperfusion injury, comprising administering heparin cofactor II after ischemia and before reperfusion.  9. Use of heparin cofactor II for the manufacture of a prophylactic or therapeutic agent for ischemic injury or ischemia-reperfusion injury.  10. Use of heparin cofactor-I I for the manufacture of a prophylactic or therapeutic agent for ischemia-reperfusion injury. .  I I. The use of heparin cofactor II according to claim 0, wherein the ischemia-reperfusion injury is ischemia-reperfusion injury due to reperfusion after ischemia caused by ischemic heart disease.  12. Use of heparin cofactor II for the manufacture of a prophylactic or therapeutic agent for ischemia-reperfusion injury for administration after ischemia and before reperfusion. 13. A preventive or therapeutic agent for ischemic injury or ischemia-reperfusion injury according to any one of claims 1 to 4, and a preventive or therapeutic agent for the ischemic injury or ischemia-reperfusion injury. Is a commercial product containing documentation stating that it can or should be used for treatment Package.