Composition for treating blood coagulation disorders comprising mirna inhibitor

Abstract

The present invention relates to a use of an miRNA inhibitor for treating blood coagulation disorders. The present inventors have derived miRNA, which is a therapeutic target for blood coagulation disorders, from a thrombosis mouse model. In addition, an inhibitor of the derived miRNA was experimentally determined to inhibit thrombus formation and reduce plasma D-dimer levels. Thus, the miRNA inhibitor according to the present invention can be variously used in the fields of blood coagulation disorder prevention, alleviation, and treatment.

Description

Composition for treating blood coagulation disorders comprising a miRNA inhibitor

[0001] The present invention relates to the use of a miRNA inhibitor for treating blood coagulation disorders.

[0002] In addition to death due to the cancer itself, the main causes of death and disability in patients with malignant tumors include death or disability due to cerebral infarction or pulmonary embolism, which are caused by various procoagulants secreted by cancer cells and which cause thrombosis.

[0003] While thrombi are ideally formed within bleeding vessels, thrombi in intact vessels are pathogenic. Thrombi in intact vessels are caused by injuries that result in small changes in the surface of endothelial cells or disruption of the endothelium. Even relatively small changes can initiate this process by causing platelets to contact collagen, and these small changes can arise from a variety of causes. These causes include stasis (i.e., reduced blood flow within the atrium or blood vessels), which causes damage due to oxygen deprivation, reducing shear forces and thus inhibiting platelet interactions. Another cause is damage to the endothelium's inner layer during atherosclerosis, which is known to be destroyed at the site of atherosclerotic lesions.

[0004] There are two main methods proposed to remove blood clots. One method involves dissolving, compressing, or removing fibrin. This method is quite popular because fibrin is the main component of blood clots. Currently, various thrombolytic agents developed to treat vascular diseases include plasminogen activators that activate plasminogen and convert insoluble fibrin into soluble fibrin degradation products. Another method uses thrombin-like enzymes to reduce the amount of fibrinogen, a precursor of fibrin. Fibrin formed by thrombin-like enzymes and thrombin both produce fibrin peptide A from the Aa chain of fibrinogen. However, fibrin formed by thrombin-like enzymes forms an unstable form, des-A-fibrin. This form is more easily degraded by plasmin than fibrin formed by thrombin, and it has been reported that des-A-fibrin activates plasminogen.

[0005] Accordingly, the inventors of the present invention have completed the present invention by deducing that miR-205-5p is a therapeutic target for blood coagulation disorders in a mouse model of thrombosis and confirming the therapeutic effect of a miR-205-5p inhibitor on blood coagulation disorders.

[0006] Accordingly, the purpose of the present invention is to provide a composition comprising an inhibitor of miR-205-5p represented by the base sequence of SEQ ID NO: 1.

[0007] Another object of the present invention is to provide a method for treating a blood coagulation disorder, comprising administering a miR-205-5p inhibitor represented by the base sequence of SEQ ID NO: 1 to a subject in need thereof.

[0008] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating blood coagulation disorders, comprising an inhibitor of miR-205-5p represented by the base sequence of SEQ ID NO: 1.

[0009] In addition, the present invention provides an antithrombotic composition comprising an inhibitor of miR-205-5p represented by the base sequence of SEQ ID NO: 1.

[0010] In addition, the present invention provides a composition for improving blood circulation comprising an inhibitor of miR-205-5p represented by the base sequence of SEQ ID NO: 1.

[0011] In addition, the present invention provides a method for treating a blood coagulation disorder, comprising administering a miR-205-5p inhibitor represented by the base sequence of SEQ ID NO: 1 to a subject in need thereof.

[0012] The present inventors have identified miRNAs as therapeutic targets for blood coagulation disorders in a mouse model of thrombosis. Furthermore, we experimentally confirmed that inhibitors of the identified miRNAs suppress thrombosis and reduce plasma D-dimer levels. Therefore, the miRNA inhibitors of the present invention can be utilized in various fields, including the prevention, improvement, and treatment of blood coagulation disorders.

[0013] Figure 1 is a diagram showing the results of measuring the occlusion time and plasma D-dimer level in a mouse model of thrombosis injected with miR-205-5p.

[0014] Figure 2 is a diagram showing the results of real-time blood flow measurement in a mouse model of thrombosis injected with miR-205-5p.

[0015] Figure 3 is a diagram showing the results of H&E staining of vascular tissue of a thrombosis mouse model injected with miR-205-5p.

[0016] Figure 4 is a diagram showing an experimental group designed to evaluate the thrombosis inhibition effect of a miR-205-5p inhibitor.

[0017] Figure 5 is a diagram showing the results of measuring the occlusion time of a mouse model of thrombosis administered with a miR-205-5p inhibitor.

[0018] Figure 6 is a diagram showing the results of real-time blood flow measurement in a mouse model of thrombosis administered with a miR-205-5p inhibitor.

[0019] Figure 7 is a diagram showing the results of measuring the plasma D-dimer level in a mouse model of thrombosis administered with a miR-205-5p inhibitor.

[0020] Hereinafter, the present invention will be described in detail.

[0021] According to an aspect of the present invention, the present invention provides a composition comprising an inhibitor of miR-205-5p represented by the base sequence of SEQ ID NO: 1. The composition may be a pharmaceutical composition for preventing or treating a blood coagulation disorder; an antithrombotic composition; or a composition for improving blood circulation.

[0022] In the present invention, miRNA (microRNA) refers to a non-translated RNA of about 22 nt that acts as a post-transcriptional repressor through base binding to the 3' untranslated region (UTR) of mRNA.

[0023] In a specific embodiment of the present invention, the miR-205-5p inhibitor may be at least one selected from the group consisting of an antisense nucleotide, a locked nucleic acid (LNA), a short interfering RNA (siRNA), and a short hairpin RNA (shRNA) that complementarily bind to miR-205-5p. More preferably, the miR-205-5p inhibitor may be represented by the base sequence of SEQ ID NO: 2.

[0024] The composition of the present invention may be a pharmaceutical composition for preventing or treating blood coagulation disorders.

[0025] In the present invention, a coagulation disorder refers to a disease characterized by abnormal blood coagulation function. This abnormal coagulation function occurs when platelets or coagulation factors malfunction. Specifically, a coagulation disorder refers to a condition in which blood coagulation does not proceed smoothly, preventing clots from forming easily or causing excessive clots.

[0026] In a specific example of the present invention, the blood coagulation disorder may be a thrombosis-related disease, more specifically, a disease caused by thrombus formation. The blood coagulation disorder may be selected from the group consisting of myocardial infarction, angina pectoris, thrombophlebitis, arterial embolism, coronary artery thrombosis, cerebral arterial thrombosis, peripheral vascular thrombosis, deep vein thrombosis, arterial thrombosis, venous thrombosis, ischemic cerebral infarction, arteriosclerosis, hypertension, pulmonary hypertension, cerebral infarction, stroke, chronic arterial occlusive disease, pulmonary infarction, cerebral embolism, renal embolism, and thromboembolism, but is not limited thereto.

[0027] In a specific embodiment of the present invention, the blood coagulation disorder is preferably a cancer-related blood coagulation disorder, and more preferably, the cancer-related blood coagulation disorder may be, but is not limited to, cancer-related stroke. The cancer-related blood coagulation disorder refers to a blood coagulation disorder caused by various pro-coagulant substances secreted from cancer cells, and a representative example of a cancer-related blood coagulation disorder is stroke. In a previous study, the present inventors confirmed that miR-205-5p in microvesicles was significantly increased in a group of patients with cerebral infarction due to cancer-related coagulation disorder, and demonstrated that increased miRNA in blood microvesicles can diagnose the occurrence of cerebral infarction due to coagulation disorder in cancer patients. Therefore, the miR-205-5p inhibitor of the present invention is also deeply related to cancer-related blood coagulation factors, such as cancer-related stroke.

[0028] In the present invention, the term "prevention" means any act of inhibiting or delaying a blood coagulation disorder by administering the pharmaceutical composition of the present invention, and the term "treatment" means any act of improving or beneficially changing the symptoms of a blood coagulation disorder by the pharmaceutical composition of the present invention.

[0029] The pharmaceutical composition of the present invention may contain, in addition to the inhibitor of miR-205-5p, other ingredients that can preferably provide a synergistic effect on the main effect, within a range that does not impair the main effect aimed at by the present invention.

[0030] Additionally, the pharmaceutical composition of the present invention may further include a component for preserving the inhibitor of miR-205-5p.

[0031] In addition, the pharmaceutical composition of the present invention may further include pharmaceutically acceptable carriers, excipients, and diluents in addition to the above-described effective ingredients for administration.

[0032] The carrier, excipient and diluent may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0033] The pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms using known methods. Representative parenteral dosage forms include injectable dosage forms, preferably isotonic aqueous solutions or suspensions. Injectable dosage forms can be prepared using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. For example, each component can be dissolved in saline or a buffer solution to prepare an injectable formulation.

[0034] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0035] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0036] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include withepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0037] The effective dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and weight, but may be administered at 0.0001 to 100 mg / kg, and preferably 0.001 to 10 mg / kg.

[0038] The composition of the present invention can be used alone or in combination with methods using surgery, chemical treatment, radiotherapy, hormone therapy, drug therapy, and biological response modifiers for the prevention or treatment of blood coagulation disorders.

[0039]

[0040] The composition of the present invention may be an antithrombotic composition; or a composition for improving blood circulation.

[0041] In the present invention, a thrombus refers to a state in which blood coagulates within a blood vessel of a living body, and a thrombus is caused by damage to the vascular endothelium, stagnation of blood flow, changes in blood components, etc., and a pathological condition caused by such a thrombus is called thrombosis. In addition, when a wound is repaired, thrombin, which is activated by a complex production mechanism within the body, changes fibrinogen present in the blood into fibrin, forming an insoluble polymer, and the fibrin monomers are formed by hydrogen bonds to form a soft clot, and this clot is converted into a hard clot by various factors, thereby forming a thrombus.

[0042] The present inventors confirmed that a miR-205-5p inhibitor exhibited excellent antithrombotic effects in a mouse model of thrombosis. The miR-205-5p inhibitor was found to inhibit thrombosis, thereby increasing occlusion time and reducing plasma D-dimer levels to normal levels. Therefore, the miR-205-5p inhibitor of the present invention can be usefully utilized for antithrombotic purposes.

[0043] In a specific embodiment of the present invention, the improvement in blood circulation can be achieved by inhibiting thrombosis. The miR-205-5p inhibitor of the present invention exhibits significant antithrombotic activity, thereby improving blood circulation in a subject.

[0044] The composition of the present invention may further include a known substance having antithrombotic activity and blood circulation improving effect.

[0045] The antithrombotic composition of the present invention; or the composition for improving blood circulation; may be a pharmaceutical composition, a food composition, a health functional food composition, a quasi-drug composition, or an in vitro / vivo composition, but the scope of the present invention is not limited thereto. The in vitro / vivo composition may be used for experimental purposes, and may be a composition intended for treating an individual (e.g., a cell, an organoid, or an experimental animal) in need of antithrombotic and blood circulation improvement.

[0046]

[0047] According to another aspect of the present invention, the present invention provides a method for treating a blood coagulation disorder, comprising administering to a subject in need thereof a miR-205-5p inhibitor represented by the base sequence of SEQ ID NO: 1.

[0048] In a specific example of the present invention, the subject may be, but is not limited to, a subject expected to develop a blood coagulation disorder; a subject who has developed the disorder; or a subject who has been judged to be cured.

[0049]

[0050] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0051] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0052]

[0053]

[0054] Experimental Example 1. Creation of a mouse model of thrombosis

[0055] In this experimental example, a mouse model of thrombosis, characterized by thrombosis in the carotid artery, was created using 8-week-old C57BL6 / N mice. Each miRNA was administered according to the experimental objectives, as follows.

[0056]

[0057] [Experiment to discover blood clot formation targets]

[0058] - Control agomir: Control agomir (10 nmol) (SEQ ID NOs. 5 and 6)

[0059] - miR-205-5p agomir: miR-205-5p agomir (10 nmol) (SEQ ID NO: 3 and 4)

[0060]

[0061] [Experiment to confirm the effect of inhibiting blood clot formation]

[0062] - G1: Control agomir (10 nmol SEQ ID NO: 5 and 6) / control antagomir (20 nmol) (SEQ ID NO: 7)

[0063] - G2: miR-205-5p agomir (10 nmol) (SEQ ID NO: 3 and 4) / control antagomir (20 nmol) (SEQ ID NO: 7)

[0064] - G3: miR-205-5p agomir (10 nmol) (SEQ ID NO: 3 and 4) / miR-205-5p antagomir (20 nmol) (SEQ ID NO: 2)

[0065]

[0066] Twenty-four hours after administration, mice were anesthetized with ketamine and xylazine to induce carotid artery thrombosis. The right common carotid artery and left jugular vein of the anesthetized mice were exposed under an operating microscope. Rose Bengal solution was injected at a concentration of 50 mg / kg through the left jugular vein to create a mouse model of thrombosis. The created mouse model of thrombosis was characterized by carotid artery thrombosis.

[0067]

[0068] Experimental Example 2. Blood Flow Measurement

[0069] Two minutes after injecting the rose bengal solution into the above-mentioned thrombosis mouse model, blood flow was measured in real time using a 2D laser blood flow imager (OZ-2 STD, Omegawave) while irradiating the common carotid artery with a 532 nm green laser. The occlusion time was defined as the 'start time at which blood flow was maintained at 25% or less for at least 1 minute.'

[0070]

[0071] Experimental Example 3. Determination of Plasma D-dimer Levels

[0072] To determine plasma D-dimer levels, blood samples were collected from the hearts of a mouse model of thrombosis. Plasma was isolated from the obtained blood. Plasma D-dimer levels in the isolated plasma samples were determined using a mouse D-dimer ELISA kit (cat#abx258705, abbex).

[0073]

[0074] Experimental Example 4. Histological Analysis

[0075] To histologically analyze blood vessels, a mouse model of thrombosis was irradiated with a laser for 16 minutes and then perfused with a 4% paraformaldehyde solution. After perfusion, the right common carotid artery was isolated, embedded in OCT compound, and cryosectioned longitudinally to obtain 10 μm-thick tissue sections. The tissue sections were mounted on slides and stained with hematoxylin & eosin.

[0076]

[0077] Example 1. Discovery of Thrombosis Targets

[0078] We investigated whether miR-205-5p injection induces thrombosis in an animal model of carotid artery thrombosis. Specifically, the results of measurements of occlusion time and plasma D-dimer levels are shown in Figure 1. Real-time blood flow measurements are shown in Figure 2, and histological analysis of blood vessels is shown in Figure 3.

[0079] As shown in Figures 1 to 3, administration of miR-205-5p was confirmed to promote the development of arterial thrombosis. Specifically, blood flow measurements in the miR-205-5p administration group revealed that occlusion began significantly earlier than in the control group (Figures 1 and 2). Figure 2 (a) shows the common carotid artery, and (b) indicates the occluded state. Furthermore, plasma D-dimer levels were confirmed to significantly increase following miR-205-5p administration (Figure 1). Furthermore, histological analysis confirmed carotid artery thrombosis and occlusion in the miR-205-5p administration group (Figure 3).

[0080] The above results indicate that miR-205-5p administration enhances thrombogenesis, suggesting that miR-205-5p inhibitors are promising candidates for the treatment of blood coagulation disorders.

[0081]

[0082] Example 2. Evaluation of the antithrombotic effect of miRNA inhibitors.

[0083] In Example 1, miR-205-5p was confirmed to be a strong candidate for a treatment of blood coagulation disorders. Therefore, the thrombosis-inhibiting effect of a miR-205-5p inhibitor was evaluated using the thrombosis-inducing mouse model prepared in Experimental Example 1. The experimental groups for evaluating the thrombosis-inhibiting effect are shown in Figure 4, and blood flow (Experimental Example 2) and plasma D-dimer levels (Experimental Example 3) were measured. The results of occlusion time and blood flow measurements are shown in Figures 5 and 6, respectively, and the results of confirming plasma D-dimer levels are shown in Figure 7.

[0084] As shown in Figures 5 and 6, the occlusion time of G2 (miR-205-5p agomir monotherapy group) was significantly shortened to 13.61 minutes compared to G1 (untreated group, 28.06 minutes) (p value 0.0209). The occlusion time of G3 (miR-205-5p agomir and antagomir combination therapy group) was confirmed to be 34.84 minutes. In other words, the shortening of occlusion time by miR-205-5p agomir administration was statistically significantly (p value 0.0015) increased by administration of a miR-205-5p inhibitor (antagomir).

[0085] As shown in Figure 7, the plasma D-dimer levels of G1 and G2 were 844.21 ng / mL and 2233.50 ng / mL, respectively, indicating that mIR-205-5p agomir administration significantly increased the plasma D-dimer levels (p value 0.0054). The plasma D-dimer level of G3 was 1034.75 ng / mL, which was statistically significantly lower than that of G2 (p value 0.0140).

[0086] The above results suggest that miR-205-5p inhibitors can inhibit thrombosis.

[0087]

[0088] In summary, the present inventors confirmed that administration of miR-205-5p enhances thrombosis in a mouse model of thrombosis, suggesting that miR-205-5p is a promising candidate for the treatment of blood coagulation disorders. Further experiments confirmed the thrombosis-inhibiting effect of a miR-205-5p inhibitor. Therefore, miR-205-5p inhibitors have diverse applications in the prevention, improvement, and treatment of blood coagulation disorders.

[0089]

[0090] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating blood coagulation disorders, comprising an inhibitor of miR-205-5p represented by the base sequence of sequence number 1.

2. A composition according to claim 1, wherein the miR-205-5p inhibitor is at least one selected from the group consisting of an antisense nucleotide, LNA (locked nucleic acid), siRNA (short interfering RNA), and shRNA (short hairpin RNA) that complementarily bind to miR-205-5p.

3. A composition according to claim 2, wherein the inhibitor of miR-205-5p is represented by the base sequence of SEQ ID NO:

2.

4. In the first paragraph, the blood coagulation disorder is selected from the group consisting of myocardial infarction, angina pectoris, thrombophlebitis, arterial embolism, coronary artery thrombosis, cerebral artery thrombosis, peripheral vascular thrombosis, deep vein thrombosis, arterial thrombosis, venous thrombosis, ischemic cerebral infarction, arteriosclerosis, hypertension, pulmonary hypertension, cerebral infarction, stroke, chronic arterial occlusive disease, pulmonary infarction, cerebral embolism, renal embolism, and thromboembolism.

5. In the first paragraph, the blood coagulation disorder is a cancer-related blood coagulation disorder, composition.

6. A composition according to claim 5, wherein the cancer-related blood coagulation disorder is cancer-related stroke.

7. An antithrombotic composition comprising an inhibitor of miR-205-5p represented by the base sequence of sequence number 1.

8. A composition for improving blood circulation comprising an inhibitor of miR-205-5p represented by the base sequence of sequence number 1.

9. A composition according to claim 8, wherein the improvement of blood circulation is achieved by improving blood circulation through inhibition of thrombosis.

10. A method for treating a blood coagulation disorder, comprising administering to a subject in need thereof an inhibitor of miR-205-5p represented by the base sequence of sequence number 1.

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