Good neutrophil inhibitor and pharmaceutical composition for preventing or treating clonal hematopoiesis

Abstract

To develop and provide formulations and pharmaceutical compositions having high efficacy and safety, to specifically inhibit the proliferation of myeloid cells and prevent clonal hematopoiesis (CH) and myeloproliferative neoplasms (MPN) or treat them after the onset, as well as clonal hematopoietic models to develop with cardiovascular diseases as complications.SOLUTION: A neutrophil inhibitor contains an ALK1 inhibitor as an active ingredient, the activation of the neutrophils is based on the activation of ALK1 through JAK-STAT signal pathway.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a neutrophil inhibitor containing an ALK1 inhibitor as an active ingredient, and a pharmaceutical composition for preventing or treating clonal hematopoiesis (CH) or myeloproliferative neoplasm using the same.

Background Art

[0002] Myeloproliferative neoplasms (hereinafter often abbreviated as "MPN" in this specification) are a group of diseases characterized by chronic proliferation of mature myeloid cells, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). MPN often presents with cardiovascular diseases in veins and arteries as complications, and is a refractory disease with the possibility of progression to acute leukemia. Cardiovascular diseases known as complications of MPN include arterial thrombosis such as myocardial infarction and venous thrombosis such as hepatic vein occlusion. One of the causes of these thromboses is considered to be the infiltration and / or proliferation of myeloid cells into the vascular intima, which causes intimal thickening and muscular organization of the blood vessels. In addition, infiltration and proliferation of myeloid cells in the stroma of organs are also assumed for some other complications. However, there are few useful animal models for investigating the pathogenesis of cardiovascular diseases associated with blood diseases, and the detailed mechanism of infiltration of these myeloid cells into tissues has not been clarified.

[0003] The main cause of MPN is the constitutive activation of the JAK-STAT signaling pathway due to driver mutations in JAK2, calreticulin, or thrombopoietin receptor, and the disease state progresses by adding abnormalities such as epigenetics-related gene mutations (Non-Patent Document 1).

[0004] However, in recent years, these mutations have been frequently observed in populations without blood and cardiovascular diseases, and it has been revealed that they are particularly observed in more than 15% of individuals after the age of 70 (Non-Patent Document 2). Similar to MPN, in the stroma of the organs of these patients, proliferation of myeloid cells containing the mutations is frequently observed, and such a pathological condition has been named clonal hematopoiesis (hereinafter abbreviated as "CH"). In this CH, the risk of developing cardiovascular disease increases, and some cases progress to MPN. Therefore, therapeutic intervention for CH individuals is desired even at a point in time before reaching a severe hematological pathology. However, there were also many unclear points regarding the causal relationship between the activation of the JAK-STAT signaling pathway and the onset of clonal hematopoiesis.

[0005] Since the etiology has not been clarified at the molecular level, preventive methods for cardiovascular diseases related to CH and MPN, and therapeutic methods for fundamentally improving them have not yet been established, and symptomatic treatment for each symptom that appears is the mainstream. Among the few treatment methods, hematopoietic stem cell transplantation has become the only treatment method that can remove myeloid cells containing somatic mutations such as the JAK2 gene. However, according to this treatment method, not only does the risk of serious complications increase, but poor prognosis is also often seen, and death cases due to this are also scattered (Non-Patent Document 3). In addition, inhibitory therapy of JAK2 with tyrosine kinase inhibitors has also been carried out, but side effects that are difficult to tolerate are seen, such as myelosuppression and immunosuppression, except for patients with cancer and immune abnormalities, and since it is an application to the signaling pathway in which mutations occur, many individuals show resistance to these inhibitors. Thus, existing treatment methods have large side effects and risks and could not be applied to CH individuals and MPN patients unless they developed severe hematological pathologies. Therefore, there has been a demand for preventive and therapeutic methods for blood and cardiovascular diseases that are applicable to MPN patients who have not yet presented severe hematological pathologies and CH individuals at the pre-stage of blood and cardiovascular diseases, are highly safe, and are effective.

Prior Art Documents

Non-Patent Documents

[0006] [Non-Patent Document 1] Vainchenker W. and Kralovics R. 2017, Blood, 129 (6): p.667-679. [Non-Patent Document 2] Bejar R., 2017, Leukemia, 31 (9): p.1869-1871. [Non-Patent Document 3] Gupta R, et al., 2019, Bone Marrow Transplant, 55: p.877-883. [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] The present invention aims to develop and provide effective and safe formulations and pharmaceutical compositions for specifically suppressing the proliferation of myeloid cells and preventing or treating clonal hematopoiesis (CH) and myeloproliferative neoplasms (MPN), and also to provide a clonal hematopoiesis model that develops cardiovascular disease as a complication. [Means for Solving the Problems]

[0008] To solve the above problems, the present inventors developed a therapeutic agent for clonal hematopoiesis based on the suppression of neutrophils, which are one of the myeloid cells. The present inventors found that when transgenic mice having the V617F mutation of JAK2, which is a driver mutation of MPN (often referred to as the "JAK2 V617F mutation" in this specification), are exposed to a hypoxic environment, neutrophils are strongly activated and their infiltration into the vascular intima is significantly increased compared to wild-type mice. In addition, the systolic pressure increased and the weight ratio increased in the right ventricle, which is the origin of the pulmonary artery. From this, it was found that a model that develops cardiovascular disease as a complication can be created by combining a model mouse for a blood disease and hypoxic exposure. Therefore, the present inventors transplanted JAK2 into wild-type mice in which endogenous hematopoietic stem cells were killed V617FWhen hematopoietic stem cells derived from bone marrow of transgenic mice containing mutations were transplanted, the transplanted mice had a strong induction of neutrophil infiltration into the vascular intima by hypoxia exposure, causing intimal thickening, and furthermore, an increase in systolic pressure and weight ratio in the right ventricle. As a result of analyzing the gene expression level, highly expressed ALK1 was found in infiltrated neutrophils containing the V617F mutation. It was revealed that the production of this ALK1 is directly regulated by STAT3, a transcription factor downstream of the JAK-STAT signaling pathway. The present invention is based on this new finding and provides the following.

[0009] (1) A neutrophil inhibitor containing an ALK1 inhibitor as an active ingredient, wherein the activation of the neutrophils is based on the activation of ALK1 via the JAK-STAT signaling pathway, said inhibitor. (2) The neutrophil inhibitor according to (1), wherein the activation of the ALK1 is caused by any one or more protein mutations in JAK2, calreticulin, or thrombopoietin receptor. (3) The neutrophil inhibitor according to (2), wherein the protein mutation of the JAK2 is a V617F mutation or a mutation in exon 12 of JAK2. (4) The neutrophil inhibitor according to any one of (1) to (3), wherein the ALK1 inhibitor contains at least one selected from the group consisting of K02288, LDN-212854, ML347, San78-130, PF-03446962, and Dalantercept. (5) A pharmaceutical composition for preventing or treating clonal hematopoiesis or myeloproliferative neoplasms, containing the neutrophil inhibitor according to any one of (1) to (4) as an active ingredient. (6) A pharmaceutical composition for preventing or treating intimal thickening caused by activated neutrophils, containing the neutrophil inhibitor according to any one of (1) to (4) as an active ingredient. (7) A pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of leukocytosis, neutrophilia and neutrophilopenia, arteriosclerosis and venous sclerosis, aortic aneurysm, hypertension, pulmonary hypertension, arterial and venous thrombosis, myeloid and lymphocytic leukemia, myelodysplastic syndrome, aplastic anemia, paroxysmal nocturnal hemoglobinuria, malignant lymphoma, and multiple myeloma, which contains as an active ingredient a neutrophil inhibitor according to any one of (1) to (4). (8) A mammal containing blood cells and / or myeloid cells containing a mutation that activates ALK1 via the JAK-STAT signaling pathway and exposed to a hypoxic environment. (9) A mammal that is a disease model, wherein the disease is selected from the group consisting of arteriosclerosis and venous sclerosis, aortic aneurysm, hypertension, pulmonary hypertension, and arterial and venous thrombosis caused by activated neutrophils, and the mammal according to (8). (10) A method for isolating a therapeutic agent for a disease, (8) An administration step of administering a candidate drug to the mammal according to (8) or (9), A detection step of detecting the activation of neutrophils based on the activation of ALK1 in the test animal that has undergone the administration step and the control animal that is the mammal according to (8) or (9) to which the candidate drug has not been administered, and An isolation step of isolating the candidate drug as the therapeutic agent when the activation of the neutrophils is significantly suppressed in the test animal compared to the control animal, The disease is selected from the group consisting of arteriosclerosis and venous sclerosis, aortic aneurysm, hypertension, pulmonary hypertension, and arterial and venous thrombosis caused by activated neutrophils, and the method. (11) A method for isolating a prophylactic agent for a disease, An administration step of administering a candidate drug to a mammal containing cells with a mutation that activates ALK1 via the JAK-STAT signaling pathway, An exposure step of exposing the test animal that has undergone the administration step and the control animal that is a mammal containing cells with a mutation that activates ALK1 via the JAK-STAT signaling pathway to which the candidate drug has not been administered to a hypoxic environment, A detection step of detecting activation of neutrophils based on activation of ALK1 in the test animals and control animals, and including an isolation step of isolating the candidate agent as the preventive agent when the activation of the neutrophils is significantly suppressed in the test animals as compared with the control animals, wherein the disease is selected from the group consisting of arteriosclerosis and venous sclerosis, aortic aneurysm, hypertension, pulmonary hypertension, and arterial and venous thrombosis caused by activated neutrophils, the method.

Advantages of the Invention

[0010] According to the neutrophil activation inhibitor of the present invention, infiltration of neutrophils into the vascular intima and intimal thickening caused thereby can be suppressed. Further, the model of the present invention becomes an analysis model for cardiovascular diseases such as pulmonary hypertension due to abnormalities of neutrophils.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] 1. Neutrophil inhibitor 1-1. Overview A first aspect of the present invention is a neutrophil inhibitor. The neutrophil inhibitor of the present invention contains an ALK1 inhibitor as an active ingredient and inhibits the activation of neutrophils based on the activation of ALK via the JAK-STAT signaling pathway. The neutrophil inhibitor of the present invention can be an active ingredient of a pharmaceutical composition for preventing or treating clonal hematopoiesis (CH) or myeloproliferative neoplasm (MPN).

[0013] 1-2. Definitions As used herein, "clonal hematopoiesis (CH)" refers to the clonal proliferation of hematopoietic cells, and includes "clonal hematopoiesis of indeterminate potential (abbreviated as "CHIP" herein) and "age-related clonal hematopoiesis (abbreviated as "ARCH" herein)". CH is found in patients who have not been diagnosed with a blood disease, and is classified into CHIP with a mutant allele ratio of 2% or more and ARCH with a mutant allele ratio of less than 2% based on the ratio of the mutant allele to the normal allele. In fact, cases of developing MPN or cardiovascular diseases have been reported even when the mutant allele ratio is about 0.1% (see, for example, Perricone M, et al., Oncotarget, 2017 and Lippert E, Haematologica, 2014). Other main characteristics of these clonal hematopoiesis include no morphological changes in blood cells and not meeting the diagnostic criteria for current symptoms related to hematological tumors (for example, lymphadenopathy and / or changes in blood cell counts). Patients in this state are considered to have a high possibility of developing blood diseases involving the clonal proliferation of blood cells. The boundaries among clonal hematopoiesis, CHIP, and ARCH are unclear, but in this specification, they are referred to as clonal hematopoiesis or CH without particularly distinguishing them.

[0014] As used herein, "blood disease" is synonymous with hematological disease. It includes diseases caused by abnormalities in the quantity and function of red blood cells and white blood cells, as well as abnormalities in blood coagulation factors and platelets, preferably abnormalities in the number and function of white blood cells. The sites where abnormalities occur widely include the circulatory system, vascular tissue, peripheral blood, and bone marrow, preferably vascular tissue or peripheral blood. Specific examples of blood diseases include leukocytosis, neutrophilia and neutropenia, myeloid and lymphoid leukemia, myeloproliferative neoplasms, myelodysplastic syndromes, aplastic anemia, paroxysmal nocturnal hemoglobinuria, malignant lymphoma, and multiple myeloma, etc.

[0015] "Cardiovascular disease" is a general term for disorders or diseases occurring in the heart or circulatory system. Usually, it means a disease in which a part of the heart or vascular tissue degenerates or blood pressure and blood flow are abnormal due to a thrombus. Specifically, it includes arteriosclerosis and venous sclerosis, aortic aneurysm, hypertension, pulmonary hypertension, venous thrombosis including hepatic vein occlusion, and arterial thrombosis including cerebral infarction and myocardial infarction, etc.

[0016] "Myeloproliferative neoplasms (MPN)" are diseases caused by the tumorigenesis of hematopoietic stem cells, characterized by marked proliferation of myeloid cells. MPN is broadly classified into Philadelphia chromosome-positive chronic myeloid leukemia (CML) and Philadelphia chromosome-negative MPN according to the presence or absence of the Philadelphia chromosome. Furthermore, Philadelphia chromosome-negative MPN is classified into chronic neutrophilic leukemia (CNL), polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome / myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS / MPN-RS-T), and myeloproliferative neoplasms, unclassifiable (MPN-U), etc. In particular, among Philadelphia chromosome-negative MPN, PV, ET, and PMF are relatively frequent and are regarded as classical MPN. In the early stage of onset, it shows hyperplasia of myeloid cells with differentiation ability and an increase in granulocytes, erythrocytes, and platelets in the peripheral blood, but it deteriorates systemically and eventually leads to myelofibrosis, etc. MPN is accompanied by growth factor independence or hypersensitivity, myeloid cell hyperplasia, extramedullary hematopoiesis, splenomegaly and hepatomegaly, and thrombotic and / or hemorrhagic predisposition. Diagnostic methods for these diseases are known in the art (for example, the Revised Edition of the 2018 Edition of the Hematopoietic Tumor Diagnosis and Treatment Guidelines, Japanese Society of Hematology). Also, as described above, it is known that some patients presenting with CH develop MPN as the disease progresses.

[0017] As used herein, the term "myeloid cells" means cells that can generate white blood cells, particularly monocytes and granulocytes (i.e., eosinophils, neutrophils, and basophils). Myeloid cells include normal hematopoietic stem cells, but also abnormal cells that have acquired the ability to differentiate into the above blood cells as a result of mutations or the like. The proliferation of myeloid cells and their differentiation into blood cells occur in the bone marrow in normal individuals, but also include proliferation and differentiation outside the bone marrow, i.e., extramedullary hematopoiesis.

[0018] As used herein, the term "blood cells" means red blood cells, platelets, white blood cells, and myeloid cells contained in the blood. Blood cells do not necessarily have to be purified and may be a mixture of multiple types of blood cells, or may contain other blood components or the like, but are preferably a mixture containing neutrophils or myeloid cells. As used herein, the term "blood" includes whole blood, plasma, and serum. Regardless of the type of whole blood, examples include venous blood, arterial blood, or umbilical cord blood.

[0019] "Neutrophils" account for 50 - 60% of white blood cells and are blood cells that play an important role in the body's defense against pathogenic microorganisms such as bacteria and fungi. For example, when foreign substances invade the body due to a bacterial infection or the like, neutrophils mainly present in the blood are activated by stimuli resulting from the body's inflammatory response or the like, move through the blood to near the infection site, infiltrate from the blood vessel wall to the intima of the blood vessel, and then migrate through the tissue to move to the infection site. At that time, they phagocytize foreign substances and also cause inflammatory effects such as secreting chemokines and neutrophil elastase, and acquire the activity to eliminate foreign substances. Chemokines promote the infiltration of macrophages and the like, and neutrophil elastase, which is a serine protease, sterilizes the inflammatory site. The state of having such activity is referred to herein as "activation of neutrophils". Here, the activation of neutrophils may be caused by stimuli from outside the cell as described above, or may be caused by some abnormality within the cell. Regardless of the number or degree of various actions of neutrophils as a result of activation. If neutrophils are overly activated, they may overly infiltrate the tissue, and the protein in the tissue may be decomposed by the less specific neutrophil elastase, which may have an adverse effect on normal tissue.

[0020] As used herein, "activated neutrophils" means neutrophils that have been activated and are distinguished from normal neutrophils in an inactive state. As long as they are activated, the level of the activated state is not a concern.

[0021] As used herein, "suppression of neutrophils" includes suppression of neutrophil activation, inactivation of activated neutrophils, and partial suppression of the actions of activated neutrophils. This suppression does not need to be complete, and as long as at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% of the increase due to activation is suppressed compared to normal neutrophils.

[0022] "Infiltration" means that inflammatory cells or tumor cells expand the site of activity and spread to adjacent regions or further to the surrounding areas from adjacent regions. In this specification, it particularly refers to the invasion of neutrophils from the blood into the intima of blood vessels. For example, neutrophils moving by rolling in the blood strongly adhere via adhesion molecules on the cell membrane of vascular endothelial cells and infiltrate into the intima of blood vessels by squeezing between vascular endothelial cells. However, the mechanism of infiltration is not limited to this. For example, instead of squeezing between vascular endothelial cells, it may infiltrate by killing vascular endothelial cells. Infiltration may also be induced by chemokines or the like released from inflammatory sites in tissues, or by abnormalities within neutrophils. Neutrophils do not need to remain in the intima of blood vessels after infiltration and may move into the outer membrane of blood vessels or tissues outside the blood vessels.

[0023] "The vascular intima" is one of the layers that make up blood vessels. It is the innermost layer among the three layers consisting of the intima, media, and adventitia, and is the layer that comes into contact with blood. The intima includes a layer of endothelial cells and, depending on the type of blood vessel, connective tissue, smooth muscle, and the internal elastic lamina, etc. Also, different from the media mainly composed of smooth muscle and the adventitia composed of connective tissue and elastic fibers, etc., the intima is included in all blood vessels, large and small. The blood vessels in this specification include arteries, veins, and capillaries, and the sizes of arteries and veins can be large, medium, or small, and arterioles and venules are also included. In this specification, the vascular intima is preferably the intima of arteriovenous vessels of a certain size or larger.

[0024] "Vascular intimal hyperplasia" generally refers to a series of reactions in damaged blood vessels where, as a result of cell proliferation occurring for repair, the blood vessel wall thickens and the lumen narrows. This hyperplasia is caused by the proliferation and migration of medial smooth muscle cells to the lumen side and the proliferation of smooth muscle in the intima, thereby forming a thick intimal layer containing multiple layers of smooth muscle. In this specification, it includes not only cases of blood vessel damage but also cases of abnormalities in blood cells such as myeloid cells or neutrophils. Vascular intimal hyperplasia is considered the initial lesion of arterial and venous sclerosis and arterial and venous thrombosis because it hardens the blood vessel wall and narrows the lumen. The degree of hyperplasia is not particularly limited, but preferably, the blood vessel wall becomes significantly thicker (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more) or significantly thicker compared to normal.

[0025] As used herein, "significant" means statistically significant. Statistically significant means that there is a significant difference between the measured value of the test subject and the control value when the difference is statistically processed. For example, when the p-value (significance level) of the obtained value is small, specifically less than 5% (p < 0.05), less than 1% (p < 0.01), or less than 0.1% (p < 0.001). The "p (value)" shown here indicates the probability that the test statistic accidentally takes that value in the distribution based on the null hypothesis in a statistical test. Therefore, the smaller the "p", the lower the probability that the test statistic takes that value, meaning that the null hypothesis is more likely to be rejected. For the test method of statistical processing, any known test method capable of determining the presence or absence of significance can be appropriately used without particular limitation. For example, Student's t-test, paired Student's t-test, Welch's t-test, Wilcoxon rank sum test, analysis of variance, Tukey's post hoc test, etc. can be used, but there is no particular limitation.

[0026] The "JAK-STAT signaling pathway" is a signaling pathway that starts with the binding of a ligand to a tyrosine kinase-coupled receptor to which JAK, a tyrosine kinase, is bound on the cytoplasmic side. Usually, the tyrosine residues of JAK itself and the receptor are phosphorylated by the binding of the ligand. Subsequently, the transcription activator (STAT) bound to the phosphorylated tyrosine is phosphorylated by JAK to form a dimer, which translocates into the nucleus to regulate the transcription of various genes. In mammalian cells, the JAK family consists of 4 members (JAK1, JAK2, JAK3, and Tyk2), and the STAT family is known to have 7 members (STAT1, 2, 3, 4, 5a, 5b, 6). In this specification, it is preferably the JAK2-STAT3 signaling pathway.

[0027] As used herein, "mutation" refers to a mutation in a nucleotide sequence or an amino acid sequence that can cause the etiology of MPN unless otherwise specified, preferably a mutation that activates the JAK-STAT signaling pathway, a mutation that activates the JAK2-STAT3 signaling pathway. This mutation activates ALK1. Therefore, this mutation is specifically referred to as "mutation that activates ALK1 via the JAK-STAT signaling pathway" in this specification. The mutation that activates ALK1 via the JAK-STAT signaling pathway is not limited as long as it is a mutation that activates the JAK-STAT signaling pathway, and any mutation is included. In this specification, the mutation may be present systemically or in some organs, tissues, or cells. Also, the mutation may be transiently present intracellularly or may be stably and continuously present in a state incorporated into a chromosome or the like.

[0028] "JAK2" refers to the Janus Kinase2 protein. The amino acid sequence of wild-type human JAK2 is represented by SEQ ID NO: 1. As used herein, "mutation of JAK2" includes "JAK2 V617F mutation" in which valine at position 617 is substituted with phenylalanine, "mutation in exon 12 (including substitution, deletion, insertion, and duplication mutations)" present within a 44-nucleotide region of the JAK2 gene encompassing amino acids 533-547, and mutations occurring outside this region. Preferably, it is a V617F JAK2 mutation or a small in-frame deletion mutation of 3 to 12 nucleotides in exon 12, and more preferably, it is a V617F JAK2 mutation.

[0029] "Calreticulin (CALR)" refers to the major Ca present in the endoplasmic reticulum 2+It is an associative molecular chaperone. The amino acid sequence of wild-type human CALR is represented by SEQ ID NO: 2. The "mutation of calreticulin (CALR)" as used herein is an insertion and / or deletion (indel) mutation, which is a frameshift mutation occurring in exon 9. As a result, a mutant protein with a novel C-terminus lacking the amino acid sequence (KDEL sequence) important for localization to the endoplasmic reticulum is generated. Specific examples include c.1092_1143del (L367 fs*46) which is a CALRdel52 / I-type mutation, or c1154_1155insTTGTC (K385 fs*47) which is a 5-bp insertion CALRins5 / II-type mutation, and the like.

[0030] "Thrombopoietin receptor (MPL)" refers to the thrombopoietin receptor protein which is the product of the oncogene of myeloid leukemia, and is called MPL from the abbreviation of its disease name. The amino acid sequence of wild-type human MPL is represented by SEQ ID NO: 3. This protein is a membrane protein that is activated by the binding of thrombopoietin, a natural ligand. When the ligand binds, dimerization of MPL is induced, a conformational change occurs, and activation of the JAK2 kinase signaling pathway is induced. The "mutation of thrombopoietin receptor (MPL)" as used herein includes the MPLS505N mutation, as well as the MPLW515L, MPLW515K, MPLW515A, PMLW515R, and MPLW515S mutations, and these mutations change MPL into a constitutively activated type.

[0031] 1-3. Composition The neutrophil inhibitor of the present invention contains an ALK1 inhibitor as an essential component. This will be specifically described below. <ALK1 inhibitor> "Activin receptor-like kinase 1 (ALK1)" is a receptor-type kinase belonging to the TGF-β superfamily of receptors encoded by the Acvrl1 gene. The nucleotide sequence of the wild-type human Acvrl1 gene is represented by SEQ ID NO: 4, and the amino acid sequence of wild-type human ALK1 is represented by SEQ ID NO: 5. ALK1 is a type I serine / threonine kinase receptor mainly expressed in vascular endothelial cells and associates with type II receptors upon ligand binding. Activated ALK1 phosphorylates Smad1 / 5 / 8, and phosphorylated Smad1 / 5 / 8 translocates into the nucleus to regulate the transcription of various genes.

[0032] In the present invention, an "ALK1 inhibitor" is an agent that inhibits the activity of the ALK1-Smad1 / 5 / 8 signaling pathway. It is not particularly limited and includes any substance that inhibits the expression or function of ALK1, as well as any substance that inhibits the action of ALK1. Preferably, it refers to a substance that specifically inhibits ALK1.

[0033] The ALK1 inhibitors in the present invention include, but are not limited to, known small-molecule compounds and their salts, antibodies and their active fragments, and nucleic acid drugs, etc. Furthermore, these may be in the form of prodrugs. The "prodrug" referred to here is a small-molecule compound that undergoes a chemical change under physiological conditions and consequently changes into an inhibitor in the active form.

[0034] Specific examples of ALK1 inhibitors include, if they are small-molecule compounds, K02288 (3-[6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl]phenol), LDN-212854 (5-[6-[4-(piperazin-1-yl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), ML347 (5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), or San78-130 ((3S,8S,9S)-8,9,16-trihydroxy-14-methoxy-3-methyl-3,4,5,6,9,10,11,12-octahydro-1H-benzo[c][1]oxacyclotetradecine-1,7(8H)-dione), and their salts, etc.

[0035] As used herein, "its salt" refers to a salt of the low molecular weight compound. Any pharmaceutically acceptable salt may be used, and there is no particular limitation. Examples include alkali metal salts (e.g., sodium salt, potassium salt, etc.), alkaline earth metal salts (e.g., calcium salt, magnesium salt, etc.), and amine salts (e.g., tri(n-butyl)amine salt, triethylamine salt, pyridine salt, amino acid salt, etc.). In the present invention, one or more selected from these can be used as the "ALK1 inhibitor or its salt".

[0036] In addition, examples of the antibody include anti-ALK1 antibodies that specifically recognize and bind to ALK1. Specific examples of the anti-ALK1 antibody include PF-03446962 and Dalantercept.

[0037] As used herein, "antibody" refers to an immunoglobulin, chimeric antibody, humanized antibody, or synthetic antibody.

[0038] When the antibody is an immunoglobulin, it may be either a polyclonal antibody or a monoclonal antibody. The immunoglobulin can be of any class, e.g., IgG, IgE, IgM, IgA, IgD, and IgY, or any subclass, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.

[0039] A "chimeric antibody" is an antibody in which the constant region of one antibody is replaced with the constant region of another antibody. In the present invention, it means an antibody in which the constant region of an anti-human ALK1 antibody derived from an animal other than human is replaced with an appropriate constant region derived from human. For example, an antibody in which the constant region of an anti-human ALK1 mouse monoclonal antibody is replaced with the constant region of a human antibody is applicable.

[0040] A "humanized antibody" is a mosaic antibody that is artificially constructed by combining the CDR groups (i.e., CDR1, CDR2, and CDR3) derived from a certain antibody (usually a non-human antibody, such as a mouse antibody), the FR groups (i.e., FR1, FR2, FR3, and FR4) of a human antibody, and the constant region. Such a humanized antibody is also referred to as a CDR-grafted antibody (Nature (1986) Vol. 321, 522).

[0041] A "synthetic antibody" refers to, for example, an antibody or antibody fragment newly synthesized using recombinant DNA techniques. Specifically, but not limited thereto, a monomeric polypeptide molecule in which one or more VLs and one or more VHs of the antibody of the present invention are artificially linked via a linker peptide or the like having an appropriate length and sequence, or a multimeric polypeptide thereof is applicable. Examples of the monomeric polypeptide molecule include a single-chain Fv (scFv: single chain Fragment of variable region) (see Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL) and an Fc fusion protein. An Fc fusion protein is a recombinant protein containing the high-affinity IgE receptor α chain and the Fc fragment of an immunoglobulin. Examples of the multimeric polypeptide include a diabody, a triabody, or a tetrabody. A diabody is a molecule having a structure based on the dimeric structure of a single-chain Fv (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448). In a diabody, which is a bivalent antibody fragment, each antigen-binding site does not necessarily bind to the same epitope, and each may have bispecificity for recognizing and binding to different epitopes. Triabody and tetrabody have trimeric and tetrameric structures based on the single-chain Fv structure, similar to diabody. They are trivalent and tetravalent antibody fragments, respectively, and may be multispecific antibodies.

[0042] As used herein, the "active fragment" refers to a partial region of the anti-ALK1 antibody described above, and means a polypeptide chain or a complex thereof having an activity substantially equivalent to the antigen-specific binding activity of the antibody. For example, a polypeptide chain having at least one light chain variable region (VL) and at least one heavy chain variable region (VH) or a complex thereof is applicable. Specific examples include antibody fragments generated by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F(ab')2, Fab', etc.

[0043] Antibodies and the like may be modified by glycosylation, acetylation, formylation, amidation, phosphorylation, or pegylation (PEGylation), etc. Further, as described in the complex quantification step to be described later, antibodies and the like may be labeled.

[0044] The antibodies and the like of the present invention can be derived from any animal including mammals and birds. For example, mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens, or humans, etc. may be mentioned.

[0045] Furthermore, in the case of nucleic acid drugs, examples include nucleic acid aptamers or RNA interference molecules that specifically recognize and bind to the ALK1 or Acvrl1 gene or mRNA.

[0046] As used herein, the "nucleic acid aptamer" refers to a DNA aptamer or an RNA aptamer that specifically binds to ALK1. The nucleic acid aptamer refers to a ligand molecule that binds strongly and specifically to ALK1, etc. through a three-dimensional structure formed based on the secondary structure, and further the tertiary structure of a single-stranded nucleic acid molecule via hydrogen bonds, etc. When the nucleic acid aptamer has the ability to specifically inhibit or suppress the functions such as the physiological activity of ALK1, the nucleic acid aptamer can be a functional inhibitor of ALK1.

[0047] The term "RNA interference molecule" refers to a substance that can induce RNA interference (RNAi) in vivo and suppress (silence) the expression of a target Acvrl1 gene through degradation of the transcription product of the gene. Examples include miRNA (micro RNA) (including pri-miRNA and pre-miRNA), shRNA (short hairpin RNA), or siRNA (small interference RNA).

[0048] In this specification, the term "miRNA" refers to a single-stranded non-coding RNA that is 18 to 25 bases in length and exists in vivo and regulates the expression of the Acvrl1 gene. This RNA is known to bind to the mRNA of the Acvrl1 gene and ALK1 to form a complex and inhibit the translation of the Acvrl1 gene. After being transcribed from the genome in a single-stranded precursor state called pri-miRNA, miRNA is processed in the nucleus by an endonuclease called Drosha into a further single-stranded precursor state called the above pre-miRNA, and then, by the action of an endonuclease called Dicer outside the nucleus, it becomes a mature double-stranded miRNA consisting of a miRNA strand and a miRNA star strand. Among them, the miRNA strand is incorporated into the RISC (RNA-induced silencing complex) complex to become a mature single-stranded miRNA and suppress Acvrl1 gene expression (David P. Bartel, Cell, Vol. 116, 281-297, January 23, 2004).

[0049] In this specification, the term "shRNA" refers to a single-stranded RNA in which the sense strand and the antisense strand of the following siRNA or mature double-stranded miRNA are linked by a short spacer sequence having an appropriate sequence. That is, in shRNA, the sense region and the antisense region base-pair with each other within a molecule to form a stem structure, and at the same time, the spacer sequence forms a loop structure, so that the whole molecule forms a hairpin-type stem-loop structure.

[0050] As used herein, "siRNA" refers to a small double-stranded RNA consisting of a sense strand (passenger strand) having a nucleotide sequence corresponding to a part of the Acvrl1 gene and its antisense strand (guide strand).

[0051] The neutrophil inhibitor of the present invention is characterized in that it can suppress the activation of neutrophils based on the activation of ALK1 via the JAK-STAT signaling pathway. The activation of neutrophils based on the activation of ALK1 via the JAK-STAT signaling pathway generally means that a mutation that activates the JAK-STAT signaling pathway or a phenomenon known to be caused by the activation of this pathway (for example, an increase in the number of blood cells in the blood, etc.) and the activation of neutrophils are recognized. In fact, it is not necessary to confirm the activation of the JAK-STAT signaling pathway and ALK1.

[0052] 2. Pharmaceutical composition for preventing or treating clonal hematopoiesis (CH) or myeloproliferative neoplasm (MPN) 2-1. Overview The second aspect of the present invention is a pharmaceutical composition for preventing or treating clonal hematopoiesis (CH) or myeloproliferative neoplasm (MPN). The pharmaceutical composition of the present invention contains an active ingredient as an essential constituent and a solvent and a pharmaceutically acceptable carrier as optional constituents. According to the composition of the present invention, it is possible to suppress the infiltration of neutrophils into the vascular endothelium and the resulting intimal hyperplasia.

[0053] 2-2. Composition 2-2-1. Constituents The constituents of the pharmaceutical composition for preventing or treating clonal hematopoiesis (CH) or myeloproliferative neoplasm (MPN) of the present invention will be described. The pharmaceutical composition of the present invention contains one or more active ingredients as essential constituents and a solvent and / or a carrier as optional constituents. Hereinafter, each constituent will be specifically described.

[0054] (1) Active ingredient The pharmaceutical composition of the present invention includes, as an essential active ingredient, the neutrophil inhibitor described in the first aspect. Optionally, it may also include one or more therapeutic agents for blood diseases.

[0055] Since the configuration of the neutrophil inhibitor is described in detail in the first aspect, specific description here is omitted. The pharmaceutical composition of the present invention can contain one or more neutrophil inhibitors.

[0056] The content of the active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited. Generally, the content varies depending on the type of the active ingredient, dosage form, and the types of other constituent components such as solvents and carriers described later. Therefore, it may be appropriately determined in consideration of each condition. It is only necessary that the pharmaceutical composition of a single application dose contains an effective amount of the active ingredient. However, when it is necessary to administer a large amount of the pharmaceutical composition to the subject in order to obtain the pharmacological effect of the active ingredient, it can also be administered in several divided doses to reduce the burden on the subject. In this case, the amount of the active ingredient only needs to include an effective amount in the total amount. The "effective amount" refers to the amount necessary to exert the function as an active ingredient and that hardly or does not impart any harmful side effects to the subject to which it is applied. This effective amount can vary depending on various conditions such as information of the subject, application route, and number of applications. Therefore, when the pharmaceutical composition of the present invention is used as a medicine, the content of the active ingredient is ultimately determined by the judgment of a doctor or pharmacist, etc.

[0057] In this specification, the "subject" refers to the application target of the neutrophil inhibitor described in the first aspect or the pharmaceutical composition of this aspect. For example, it is a cell (including cultured cells), tissue, organ, or individual. In the case of an individual, it is preferably a human individual. In the present invention, the subject may be healthy or suffering from some disease, and for example, includes an individual in which the JAK-STAT signaling pathway is activated in any cell, or an individual in which activation of this pathway is expected in the future.

[0058] Activation of the JAK-STAT signaling pathway may be determined by either carrying a mutation that activates this pathway or observing a phenomenon known to be caused by activation of this pathway, such as an increase in the number of blood cells in the blood. In addition, when activation of neutrophils is observed as exemplified in the following examples, it may be determined that this pathway is activated. On the other hand, as an example of an individual in whom activation of this pathway is expected in the future, there may be a case where there is an individual in the family who has the above mutation, or an individual belongs to an age group in which the proportion of individuals with CH is recognized to be a certain level or more (for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more).

[0059] In this specification, "information of the subject" refers to various information regarding the characteristics and conditions of the subject. For example, when the subject is a human individual, examples include age, weight, gender, overall health status, presence or absence of disease, degree of progression or severity of the disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment, etc.

[0060] (2) Solvent The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable solvent as necessary. "Pharmaceutically acceptable solvent" refers to a solvent commonly used in the pharmaceutical technology field. For example, water or an aqueous solution, or an organic solvent may be mentioned. Examples of the aqueous solution include physiological saline, glucose or other isotonic solutions containing adjuvants, phosphate buffer solution, and sodium acetate buffer solution. Examples of the adjuvant include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. Ethanol may be mentioned as the organic solvent.

[0061] (3) Carrier The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier as necessary. "Pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. For example, excipients, binders, disintegrants, fillers, emulsifiers, flow additive regulators, lubricants, human serum albumin, etc. may be mentioned.

[0062] Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, poloxamer, kaolin, silicic acid, or combinations thereof.

[0063] Examples of binders include starch paste using plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

[0064] Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate or salts thereof.

[0065] Examples of fillers include petrolatum, the aforementioned sugars and / or calcium phosphate.

[0066] Examples of emulsifiers include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, propylene glycol fatty acid ester.

[0067] Examples of flow regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

[0068] In addition to the above, if necessary, solubilizers, suspending agents, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, humectants, moisturizers, wetting agents, adsorbents, flavoring agents, disintegration inhibitors, coating agents, coloring agents, preservatives, antiseptics, antioxidants, fragrances, flavoring agents, sweeteners, buffers, isotonic agents, etc. that are commonly used in pharmaceutical compositions and the like can also be appropriately included.

[0069] The carrier is used to avoid or suppress the decomposition of the active ingredient by enzymes or the like in the body of the subject, facilitate formulation and administration methods, and maintain the dosage form and drug efficacy, and may be appropriately used as needed.

[0070] (4) Drug delivery system particles (DDS particles) The pharmaceutical composition of the present invention can contain DDS particles as needed. DDS particles refer to particles that contain an active ingredient, other carriers, etc. inside or the like, deliver the contents, particularly the active ingredient, to the target site without decomposing it, and can control the drug distribution in the living body temporally and quantitatively. Since the active ingredient of the pharmaceutical composition of the present invention is a peptide or nucleic acid, the use of DDS particles is also suitable for protecting it from degradation by proteases and nucleases in the living body after administration. The type of DDS particles is not limited. For example, liposomes, polymer micelles, virus particles, etc. can be mentioned.

[0071] 2-2-2. Dosage form The dosage form of the pharmaceutical composition of the present invention is not particularly limited. Any form that can deliver the active ingredient to the target site without inactivating it in the body of the subject may be used.

[0072] Specific dosage forms vary depending on the application method described later. Since the application method can be broadly classified into parenteral administration and oral administration, a dosage form suitable for each administration method may be used.

[0073] For example, if the administration method is parenteral administration, a preferred dosage form is a liquid preparation that enables direct administration to the target site or systemic administration via the circulatory system. Preferred examples of liquid preparations include injections. Injections can be formulated by appropriately combining with solvents, the excipients, emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing in a unit dosage form required for generally recognized pharmaceutical practice.

[0074] If the administration method is oral administration, preferred dosage forms include solid preparations (including tablets, capsules, drops, and troches), granules, powders, powders for external use, and liquid preparations (including oral solutions, emulsions, and syrups). In the case of solid preparations, if necessary, dosage forms with a dosage form known in the art can be used, for example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

[0075] In addition, regarding the specific shape and size of each of the above dosage forms, any of them may be within the range of dosage forms known in the art for each dosage form, and there is no particular limitation. Regarding the production method of the pharmaceutical composition of the present invention, it may be formulated according to a conventional method in the art.

[0076] 2-3. Application method The application method of the pharmaceutical composition of the present invention may be oral administration or parenteral administration. Generally, oral administration is systemic administration, while parenteral administration can be further subdivided into systemic administration and local administration. Examples of local administration include intramuscular administration, subcutaneous administration, tissue administration, and organ administration. Examples of systemic administration by parenteral administration include intravascular administration, such as intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration. When the pharmaceutical composition of the present invention is administered locally, it may be directly administered to the liver by injection or the like. When administered systemically, it may be administered into the circulatory system such as by intravenous injection. The dosage may be any amount effective for the active ingredient to exert its effect. The effective amount is appropriately selected according to the information of the subject as described above.

[0077] In addition, the pharmaceutical composition of the present invention can also be separately combined with one or more known prophylactic or therapeutic agents for other blood diseases. Blood disease therapeutic agents that can be considered for combination include tyrosine kinase inhibitors, interferons, anticancer agents, antiplatelet agents, anticoagulants, thrombolytic agents, and various other molecular target drugs.

[0078] 3. Mammals 3-1. Overview The third aspect of the present invention pertains to a mammal. The mammal of the present invention is characterized by comprising blood cells and / or myeloid cells containing a mutation that activates ALK1 via the JAK-STAT signaling pathway and being exposed to a hypoxic environment. The mammal of the present invention can be used, for example, as a model animal for various blood diseases and cardiovascular diseases associated therewith.

[0079] 3-2. Configuration A mammal comprising blood cells and / or myeloid cells containing a mutation that activates ALK1 via the JAK-STAT signaling pathway The mammal of the present invention comprises blood cells and / or myeloid cells (often referred to herein as "mutant cells") containing a mutation that activates ALK1 via the JAK-STAT signaling pathway. Regarding the mutation, blood cells, and myeloid cells that activate ALK1 via the JAK-STAT signaling pathway, since they have been described in detail in the first aspect, the description thereof is omitted, and here, the configuration specific to the mammal of this aspect will be described.

[0080] The mutant cells in this aspect are not particularly limited, but may be blood cells or myeloid cells derived from tissue stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. Also, the type of mutant cells may not be one kind, but may be a mixture of multiple types of cells. For example, a mixture containing myeloid cells or blood cells, specifically, a mixture containing neutrophils, etc. can be mentioned.

[0081] The mammalian animals of the present invention may have mutant cells throughout the body or may have them in some organs, tissues or cells. Also, the mammalian animals of the present invention may be individuals transplanted with mutant cells derived from other individuals. In that case, it is not necessary for all of the cells to be transplanted to be mutant cells. The period until use in experiments after transplantation is also not limited. When used in experiments, for example, if the transplanted cells are myeloid cells or blood cells, in the blood, the mutant allele ratio may be 0.09% or more, 0.1% or more, 0.15% or more, 0.2% or more, 0.25% or more, 0.5% or more, 1.0% or more, 2.0% or more, 5% or more, 10% or more, 20% or more, 23.5% or more, 24.0% or more, 24.5% or more, 25.0% or more, or 25.5% or more.

[0082] As used herein, the term "mammalian animal" means any animal classified as a mammalian animal, including domestic livestock and poultry and pet animals (e.g., dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc.), as well as experimental animals such as rodents and primates (e.g., guinea pigs, mice, rats, chimpanzees, marmosets, etc.). Therefore, each gene or its mutant gene, or protein or its mutant protein described in this specification may be an endogenous gene or its mutant gene, or protein or its mutant protein, and some of its nucleotides or amino acids may be genes (humanized genes) or proteins (humanized proteins) containing orthologous genes or orthologous proteins derived from humans, respectively.

[0083] 3-2-2. Exposure to a Hypoxic Environment The mammalian animals of the present invention are characterized by being exposed to a hypoxic environment. As used herein, the term "hypoxic environment" means an environment in which a state where the oxygen level is lower than the oxygen concentration in the atmosphere (about 21%) is maintained for a certain period. Examples of a hypoxic environment include cases where a predetermined space such as an airtight room or an airtight container (including a case or a bag) is made hypoxic. The hypoxic state may be adjusted by existing methods for reducing oxygen in a predetermined space. For example, gas replacement methods, deoxygenation methods, respiratory consumption methods, combustion methods, and combinations thereof can be mentioned.

[0084] The "gas replacement method" is a method of replacing the gas in a sealed space with a gas having a low oxygen concentration. This method is excellent in that it can expose mammals to a predetermined low-oxygen environment in a short time. As the gas used for replacement (replacement gas), a gas close to the atmospheric components is preferable. For example, a mixed gas of nitrogen and oxygen, a mixed gas of nitrogen and air, etc. can be mentioned. Gas replacement can be performed, for example, in a sealed space equipped with an exhaust port and an intake port having valves. By opening both valves, the replacement gas is taken in from the intake port and the gas in the container is discharged from the exhaust port.

[0085] The "oxygen removal method" is a method of introducing an oxygen scavenger into a sealed space. It is convenient in that the amount of oxygen in the sealed space can be adjusted by the amount of the oxygen scavenger introduced. As the oxygen scavenger, a reducing agent that absorbs oxygen by utilizing an oxidation reaction, etc. is used. As the reducing agent, for example, iron powder, iron compounds such as iron sulfide, copper powder, etc. can be used.

[0086] The "respiration consumption method" is a method of consuming the oxygen in a sealed space by biological respiration. The type of organism used for oxygen consumption is not limited. Microorganisms that are not directly harmful to the mammals to be used, such as yeast, etc. are convenient, but even if other organisms are not used, the mammals to be used may be enclosed in a sealed space at a relatively high density.

[0087] The "combustion method" is a method of consuming the oxygen in a sealed container by burning a substance.

[0088] Any of these methods is adjusted so that the oxygen concentration in the container or room is in the range of 8 - 19%, 9 - 15%, 9 - 12%, 9 - 11%, or 9.5% - 10.5%. The period is not particularly limited, but it is preferable to expose to a long-term low-oxygen environment for 5 days or more, 1 week or more, 10 days or more, 12 days or more, 2 weeks or more, 15 days or more, 20 days or more, 3 weeks or more, 4 weeks or more, 30 days or more, 31 days or more, 40 days or more, 50 days or more, or 60 days or more.

[0089] 4. Method for Isolating Therapeutic Agent for Blood Diseases 4-1. Overview The fourth aspect of the present invention is a method for isolating a therapeutic agent for blood diseases. The method of this aspect includes an administration step, a detection step, and an isolation step as essential steps, and includes a pretreatment step as an optional step. According to the method of this aspect, a therapeutic agent for blood diseases that can actually be expected to have a therapeutic effect can be isolated from candidate drugs for therapeutic agents for various blood diseases.

[0090] 4-2. Configuration 4-2-1. Pretreatment step Although the specific content of the essential steps will be described later, first, the pretreatment step, which is an optional step, will be explained. In this method, pretreatment can be performed before the administration step. The specific content of the pretreatment is not particularly limited. Depending on the purpose, an appropriate pretreatment known in the art can be adopted. Specific examples of the "pretreatment" include, for example, applying a stimulus for the purpose of acclimation (for example, an administration stimulus, a stimulus accompanied by an environmental change such as a cage change, etc.) to the subject individual, or for calculating the amount of change in activation in neutrophils, for the purpose of obtaining the value of the activation amount before the administration step, the activation of neutrophils similar to that in the subsequent detection step may be detected. When performing pretreatment, it is preferable that the same treatment is performed on all individuals to be compared in the isolation step.

[0091] 4-2-1. Administration step The "administration step" is a step of administering a candidate drug to the mammal of the third aspect. The "candidate drug" is a drug that can exhibit a therapeutic effect on the target blood disease and has the potential to become an active ingredient of the therapeutic composition. In this method, a drug that has the potential to become a therapeutic agent for blood diseases is applicable. The candidate drug may be any of a low-molecular compound, a nucleic acid, a peptide, a cell, or a combination thereof. The dosage of the candidate drug, the solvent and / or carrier to be used, the dosage form, and the administration method may be carried out according to the description of the second aspect.

[0092] The mammalian of the third aspect used in this process is an individual after exposure to a hypoxic environment. However, this process is not limited to after exposure, and may be performed during exposure, that is, during the adjustment of the mammalian of the third aspect, and the number and duration thereof are not limited. For example, administration may be performed only once during hypoxic exposure, or may be performed multiple times intermittently or continuously from the start of exposure to after exposure. When performing multiple times, it is not necessary to use the same administration method for all, and different administration methods can be performed as needed.

[0093] In this process, as a control in a later process, some animals are not administered the candidate drug. The method is not limited, but for example, a solvent that does not contain or contains insufficient candidate drug can be administered. Hereinafter, in this specification, a mammalian administered the candidate drug is referred to as a "test animal", and a mammalian not administered is referred to as a "control animal".

[0094] 4-2-2. Detection step The "detection step" is a step of detecting the activation of neutrophils based on the activation of ALK1 in test animals and control animals during or after the administration step.

[0095] The activation of neutrophils based on the activation of ALK1 can be detected by a known method. Although not limited thereto, detection of neutrophil infiltration into tissues using tissue samples, detection of chemokines or neutrophil elastase activity released into tissues, and vacuolar degeneration in the cytoplasm of neutrophils, measurement of reactive oxygen species, and detection of markers of activated neutrophils using blood samples, etc., or combinations thereof can be mentioned.

[0096] This step can be performed only once during or after the administration step, or can be performed multiple times intermittently or continuously. When performing it multiple times, it is not necessary to use the same detection method for all, and different detection methods can be used as needed. For example, for detection at a time when the treatment of the animal is not yet complete, such as during the administration step, detection using a less invasive blood sample can be selected, and for detection performed after all treatments are completed, detection using a more reliable tissue sample can be selected, but it is not limited to this.

[0097] 4-2-3. Isolation step The "isolation step" is a step of comparing the amount of neutrophil activation between the control animal and the test animal based on the detection result in the detection step, and isolating the administered candidate drug as a therapeutic agent when it is significantly suppressed in the test animal.

[0098] In this step, any statistical method described in the first aspect can be used. Also, for example, when pretreatment is performed for the purpose of calculating the change amount of neutrophil activation, comparisons according to the purpose can be made, such as comparing the increase amplitude of the neutrophil activation amount between the control animal and the test animal. In this case, for example, when the increase in the neutrophil activation amount in the test animal is significantly suppressed compared to the control animal, it is also possible to isolate the candidate drug as a therapeutic agent.

[0099] This step is performed based on the detection result, but it is not necessary to perform it after all detection steps are completed. For example, when performing multiple detections, this step can be performed at the first time, and detection can be continued thereafter.

[0100] 5. Method for isolating a preventive agent for blood diseases 5-1. Overview The fifth aspect of the present invention is a method for isolating a therapeutic agent for blood diseases. The method of this aspect includes an administration step, an exposure step, a detection step, and an isolation step. According to the method of this aspect, an agent that can actually be expected to have a preventive effect can be isolated from candidate drugs for preventive agents for various blood diseases.

[0101] 5-2. Configuration 5-2-1. Pretreatment The "pretreatment" of this aspect is the same as that of the fourth aspect. Therefore, this step may be carried out according to the pretreatment of the fourth aspect.

[0102] 5-2-2. Administration step The "administration step" of this aspect is the same as that of the fourth aspect except that a prophylactic agent candidate is used as the candidate agent. Therefore, this step may be carried out according to the administration step of the fourth aspect.

[0103] 5-2-3. Exposure step The "exposure step" is a step of exposing the mammal that has undergone the administration step to a hypoxic environment. Basically, it may be carried out according to the exposure to the hypoxic environment of the third aspect. Therefore, only the points different from the exposure to the hypoxic environment of the third aspect will be described here.

[0104] This step can be carried out during or after the administration step. In particular, if the action of the candidate agent has occurred before the activation of neutrophils by the hypoxic environment, this step may be started simultaneously with or before the administration step. Also, either the administration step or this step may end first, but it is preferable that the administration step ends simultaneously with or before this step ends.

[0105] 5-2-4. Detection step The "detection step" of this aspect is the same as that of the fourth aspect except that the immediately preceding step is the exposure step. Therefore, this step may be carried out according to the detection step of the fourth aspect.

[0106] 5-2-5. Isolation step The "isolation step" of this aspect is the same as that of the fourth aspect except that what is isolated is a prophylactic agent. Therefore, this step may be carried out according to the isolation step of the fourth aspect.

Example

[0107] <Example 1: JAK2 V617F Preparation of a vascular intimal hyperplasia model in mice in response to hypoxic exposure> (Objective) Establish a model of intimal hyperplasia in mice with the JAK2 mutation known as the MPN driver mutation. V617F In mice containing the mutation, establish a model of intimal hyperplasia.

[0108] (Method) JAK2 V617F Transgenic JAK2 mice with a C57BL / 6J background were used. Wild-type (WT) littermates were used as controls. All animal experiments were approved by the Fukushima Medical University Animal Research Committee (approval number; 2019084). V617F For exposure to a hypoxic environment, these mice were exposed to a normal oxygen partial pressure (21% O2) or a hypoxic environment (10% O2) in a ventilation chamber for 2 weeks. A mixture of air and nitrogen (Teijin Limited) was used to maintain the hypoxic environment.

[0109] After exposure to a normal oxygen partial pressure or a hypoxic environment, the mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol (0.25 mg / g / body weight). The right ventricular pressure was measured by inserting a 1.2F micromanometer catheter (Transonic Scisense) into the right jugular vein (n = 7 - 11). Furthermore, the right ventricular systolic pressure was blindly analyzed using LabScribe3 software (IWORX), and the values in 10 consecutive beats were averaged. The right ventricle and the left ventricle including the septum were separated, and the ratio calculated by dividing the weight of the right ventricle by the weight of the left ventricle was defined as the right ventricular weight ratio. Here, the larger the value of the right ventricular weight ratio, the more hypertrophied the right ventricle is indicated.

[0110] After exposure to a normal oxygen partial pressure or a hypoxic environment, the mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol (0.25 mg / g / body weight). The right ventricular pressure was measured by inserting a 1.2F micromanometer catheter (Transonic Scisense) into the right jugular vein (n = 7 - 11). Furthermore, the right ventricular systolic pressure was blindly analyzed using LabScribe3 software (IWORX), and the values in 10 consecutive beats were averaged. The right ventricle and the left ventricle including the septum were separated, and the ratio calculated by dividing the weight of the right ventricle by the weight of the left ventricle was defined as the right ventricular weight ratio. Here, the larger the value of the right ventricular weight ratio, the more hypertrophied the right ventricle is indicated.

[0111] For histological analysis, lung samples were collected from mice after exposure to each oxygen condition, fixed in 4% paraformaldehyde solution, and paraffin sections were prepared. Frozen lung tissue was embedded in O.C.T. compound (Tissue-Tek). Paraffin-embedded sections were stained with hematoxylin and eosin (HE) and Elastica-Masson (EM), and then used for immunostaining. For immunostaining, an anti-α-smooth muscle actin antibody (αSMA; Santa Cruz Biotechnology) was used. For immunofluorescence staining, paraffin-embedded tissue sections were incubated with an anti-Ly6G antibody (ab25377, Abcam). Subsequently, the sections were incubated with a secondary antibody solution containing Alexa Fluor 594-labeled rabbit anti-rat IgG antibody (cross-adsorbed) (A-21211, Thermo Fisher Scientific). Finally, the sections were mounted using DAPI-containing mounting media (Fluoro Gel II, Electron Microscopy Science). Then, images were acquired using a microscope (BZ-X700, Keyence) with Keyence BZ II Viewer software (Keyence).

[0112] For the measurement of the thickness of the vessel wall, EM-stained sections were used (n = 6 - 8). The inner wall area of the intima and media of pulmonary arteries with a diameter of less than 100 μm was measured, and the ratio of the wall thickness was calculated by dividing the inner wall area by the vessel area using ImageJ software (National Institutes of Health).

[0113] For the measurement of the ratio of muscle-organized pulmonary vessels, sections stained with αSMA were used (n = 6 - 9). Pulmonary arteries with a diameter of less than 50 μm were classified into three groups: non-muscular, partially muscular, and completely muscular. The total number of partially and completely muscular vessels was divided by the total number of vessels to calculate the ratio.

[0114] For the quantification of cell infiltration into the perivascular region, sections stained with Ly6G were used (n = 3). The number of infiltrated Ly6G-positive cells was counted around distal pulmonary arteries with a diameter of 50 - 100 μm.

[0115] The elastase assay was performed in lung tissues using the EnzChek Elastase Kit (Molecular Probes) (n = 3). Frozen lung samples (20 mg) were homogenized, mixed with an extraction buffer containing NaAc and sodium azide, and then centrifuged overnight at 4°C. Subsequently, the pellet was re-extracted by adding (NH4)2SO4 buffer. The pellet obtained after centrifugation following overnight precipitation was resuspended in 50 mM Tris-HCl assay buffer (pH 8.0) to reactivate elastase. Then, elastase activity was measured by adding bovine DQ-elastin as a fluorogenic substrate into the wells. The measurements were performed in duplicate. For the analysis of the measurement results, relative values were used with the average value of WT mice under normoxic conditions set as 1.

[0116] Statistical analysis was performed by one-way ANOVA with Tukey's post hoc test. The significance level was set at p = 0.05.

[0117] (Results) The results are shown in Figs. 1 - 4. JAK2 V617F Mice expressing the mutant protein (JAK2 V617F mice) and wild-type mice (WT mice) were analyzed for the heart. As a result, there were no significant differences in the systolic pressure and weight ratio of the right ventricle, which is the origin of the pulmonary artery, between WT mice and JAK2 V617F mice (Fig. 1). Therefore, when subjected to hypoxia (10% O2) exposure, which is a well-established method for inducing pulmonary hypertension in mice, the right ventricular systolic pressure significantly increased in JAK2 V617F mice in response to exposure to the hypoxic environment (Fig. 1A). Also, the right ventricular weight ratio of JAK2 V617F mice was significantly larger than that of WT mice, indicating that more severe right ventricular hypertrophy was caused due to pulmonary hypertension in hypoxic-exposed JAK2 V617F mice (Fig. 1B). Furthermore, in the pulmonary artery, the vascular wall thickness and the proportion of muscularized vessels were higher in JAK2 V617FSignificantly increased in mice (Figure 2). Also, normal oxygen partial pressure exposure and hypoxic exposure of JAK2 V617F Increased infiltration of cells around the pulmonary artery was observed by HE staining in both types of mice.

[0118] Furthermore, when this infiltration was examined in detail, it was found that in both WT mice and JAK2 V617F mice, the infiltration of Ly6G-positive neutrophils into the perivascular region was significantly increased by exposure to a hypoxic environment. Quantitative analysis showed that the increase was significantly higher in JAK2 V617F mice (Figure 3). Also, when elastase activity, which is one of the indicators of neutrophil activation, was measured in lung tissue, a slight increase was seen even in WT mice upon exposure to a hypoxic environment, but a significant and substantial increase was seen in JAK2 V617F mice (Figure 4).

[0119] From the above data, the JAK2 V617F mutation did not promote the spontaneous thickening of the vascular wall and the onset of pulmonary hypertension under normal oxygen partial pressure, but rather promoted the thickening of the vascular wall accompanied by pulmonary artery structural remodeling and the onset of pulmonary hypertension in response to hypoxic exposure. Also, it was suggested that activated neutrophils infiltrating into the perivascular region may play an important role in this remodeling.

[0120] <Example 2: Vascular intimal thickening model in response to hypoxic exposure in mice containing only JAK2 V617F mutation in bone marrow-derived cells> (Objective) JAK2 V617F To clarify whether hematopoietic cell clones containing the mutation infiltrate into the lung tissue of WT mice rather than that of JAK2 V617F mice and contribute to pulmonary hypertension.

[0121] (Method) Bone marrow transplantation was performed as follows. Wild-type C57BL / 6J female mice (Charles River Japan) at 8 - 10 weeks of age were used as recipients and irradiated with ultraviolet light at a total dose of 9.0 Gy 24 hours before bone marrow transplantation to kill the endogenous bone marrow. WT mice and JAK2 mice obtained in the same manner as in Example 1 were used as donors, and total bone marrow cells were collected from the femurs and tibias. The cells were washed with PBS, and 5.0×10 V617F bone marrow cells were injected into recipient mice via the tail vein. 6

[0122] In addition, exposure to normal oxygen partial pressure or a hypoxic environment was performed in the same manner as in Example 1 for 3 weeks from the 5th week to the 8th week after bone marrow transplantation, and the mutant allele ratios were analyzed at 4 weeks and 8 weeks (at the end of the experiment) after transplantation.

[0123] In JAK2 recipient mice exposed to normal oxygen partial pressure or a hypoxic environment (n = 8 each), the mutant allele ratios were analyzed. DNA was isolated from blood collected from the terminal vein using the QuickGene DNA Whole Blood Kit (KURABO), and quantitative PCR was performed using the THUNDERBIRD SYBR qPCR Mix. At this time, the following primers were used: forward primer for donor and recipient mice, 5'-CTTTCTTCGAAGCAGCAAGCATGA-3' (SEQ ID NO: 6); reverse primer for recipient mice, 5'-CTGGCTTTACTTACTCTCCTCTCCACAGAC-3' (SEQ ID NO: 7); reverse primer for donor mice, 5'-AACCAGAATGTTCTCCTCTCCACAGAA-3' (SEQ ID NO: 8). To estimate the allele ratio containing the JAK2 mutation in recipient mice, ΔCt (Ct V617F - Ct V617F V617F ) was calculated. Statistical analysis was performed by a paired t-test. donor total

[0124] Other analyses were performed in the same manner as in Example 1. The sample sizes were as follows: ​​​​· Measurement of right ventricular systolic pressure, n = 7 - 11; · Measurement of right ventricular weight ratio, n = 7 - 11; · Measurement of the inner wall thickness of blood vessels, n = 6 - 8; · Measurement of the proportion of muscularized distal pulmonary arteries, n = 6; · Counting of Ly6G⁺ cells, n = 3; · Measurement of elastase activity, n = 3.

[0125] (Results) The results are shown in Figs. 5 - 9. JAK2 V617F In the peripheral blood of mice transplanted with bone marrow cells (JAK2 V617F recipient mice), the ratio of JAK2 V617F mutant alleles in white blood cells increased from 25.5 ± 1.1% to 34.9 ± 6.7% at 4 and 8 weeks after transplantation in the normoxia - exposed group, and from 24.5 ± 0.8% to 51.1 ± 5.4% in the hypoxic environment, respectively (Fig. 5, p < 0.01 for both groups). However, V617F unlike JAK2 V617F mice, the blood cell counts of JAK2 V617F recipient mice did not show a significant increase compared with mice transplanted with wild - type bone marrow cells (WT recipient mice) under normoxia. This is consistent with the previous finding that recipient mice transplanted with hematopoietic stem / progenitor cells containing JAK2

[0126] In the analysis of the heart, the systolic pressure and weight ratio of the right ventricle were not different between mice transplanted with wild - type bone marrow cells (WT recipient mice) and JAK2 V617F recipient mice under normoxia. However, in response to 3 - week hypoxic exposure, both the systolic pressure and weight ratio of the right ventricle significantly increased in JAK2 V617F recipient mice compared with WT recipient mice (Fig. 6).

[0127] Similarly, in the analysis of tissue samples, the medial wall thickness of the pulmonary artery and the ratio of muscularized vessels were higher in mice transplanted with wild-type bone marrow cells (WT recipient mice) even under normal oxygen partial pressure than in mice transplanted with JAK2 V617F The medial wall thickness of the pulmonary artery and the proportion of muscularized vessels in the recipient mice tended to increase, and further increased significantly after hypoxia exposure (Figure 7). Also, as in the case of Example 1, both the infiltration of neutrophils into the perivascular region and elastase activity were higher in JAK2 V617F recipient mice exposed to a hypoxic environment than in WT recipient mice (Figures 8 and 9).

[0128] From the above data, it was shown that neutrophil activation similar to that in JAK2 V617F mice also occurred in mice containing only the JAK2 V617F mutation. The above results indicate that the thickening of the pulmonary artery in response to exposure to a hypoxic environment and the development of pulmonary hypertension are caused by the presence of myeloid cells containing the JAK2 V617F mutation.

[0129] <Example 3: Origin of Cells Infiltrating the Perivascular Region> (Objective) To clarify the origin of neutrophils infiltrating the perivascular region in JAK2 V617F recipient mice.

[0130] (Method) Bone marrow transplantation was performed in the same manner as in Example 2. CAG-EGFP reporter mice on a C57BL / 6J background were purchased from Japan SLC. JAK2 V617F mice were mated with CAG-EGFP mice to generate JAK2 V617F / CAG-EGFP double transgenic mice (JAK2 V617F -GFP mice).

[0131] JAK2 V617F-GFP mice and wild-type littermates (WT-GFP mice) as a control were used as donors, and bone marrow transplantation was performed in the same manner as in Example 2. After exposure to a hypoxic environment for 3 weeks, fluorescence immunostaining was performed using lung tissue sections in the same manner as in Example 1. As the primary antibodies, an anti-GFP antibody (NBP2-22111, Novus Biologicals) and an anti-α-smooth muscle actin antibody (αSMA: 19245, Cell Signaling Technology) were used. As the secondary antibodies, an Alexa Fluor 488-labeled donkey anti-mouse IgG antibody (ab150105, Abcam) and an Alexa Fluor 594-labeled donkey anti-rabbit IgG ReadyProbes antibody (R37119, Thermo Fisher Scientific) were used. Statistical analysis was performed by an unpaired t-test.

[0132] (Results) The results are shown in Fig. 10. JAK2 V617F In recipient mice transplanted with bone marrow cells derived from -GFP mice, GFP-positive cells were significantly accumulated in the pulmonary artery region as indicated by the arrow, while in recipient mice transplanted with bone marrow cells derived from WT-GFP mice, there were few GFP-positive cells in the lung.

[0133] From the above, it was suggested that the infiltrated Ly6G-positive neutrophils differentiated from bone marrow cells containing the JAK2 V617F mutation and migrated to the pulmonary artery region. Also, analysis using proliferating cell nuclear antigen (PCNA) suggested that the neutrophils accumulated in the perivascular region were not in a proliferative state, so it was considered that neutrophil infiltration occurred mainly by only differentiated neutrophils.

[0134] <Example 4: Changes in gene profiling during neutrophil differentiation due to JAK2 V617F mutation> (Objective) JAK2 V617F Clarify the reason why differentiated neutrophils containing the JAK2 mutation are prone to infiltration at the protein level.

[0135] (Method) Total RNA was extracted from mouse lungs using Trizol reagent (n = 5-6) according to the manufacturer's protocol (Thermo Fisher Scientific). The extracted RNA was further purified using the RNeasy Fibrous Tissue Mini Kit (Qiagen), and cDNA was synthesized using ReverTra Ace qPCR R Master Mix (Toyobo). The mRNA expression of the Acvrl1 gene was measured by quantitative PCR using the THUNDERBIRD SYBR qPCR Mix (Toyobo) on a CFX Connect real-time PCR System (Bio-Rad). Standard curves were generated by serial dilution of the template, and the data were normalized to the amount of 18s rRNA and expressed as fold change relative to the control.

[0136] Instantaneously frozen JAK2 V617FLung samples from mice and WT mice (n = 3 - 6) were first homogenized in lysis buffer (Cell Signaling Technology) containing protease inhibitor cocktail (BD Biosciences). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein aliquots were subjected to SDS - polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Merck Millipore), and probed with the following primary antibodies: anti - phosphorylated STAT3 antibody (p - STAT3; 9145, Cell Signaling Technology), anti - STAT3 antibody (t - STAT3; 4904, Cell Signaling Technology), anti - phosphorylated Smad1 / Smad5 / Smad8 antibody (p - Smad1 / Smad5 / Smad8; AB3848 - I, Merck Millipore), anti - Smad1 antibody (t - Smad; 9743, Cell Signaling Technology), and anti - GAPDH antibody (loading control; 60004 - 1 - Ig, Proteintech). Subsequently, they were incubated with goat anti - rabbit antibody or mouse horseradish peroxidase - conjugated antibody (Santa Cruz Biotechnology). Immunoreactive bands were visualized using the Amersham ECL system (Amersham Pharmacia Biotech UK), and signals were detected using the ImageQuant LAS - 4000 digital imaging system (GE Healthcare). The optical density of individual bands was analyzed using ImageJ software. Statistical analysis was performed by one - way ANOVA with Tukey's post - hoc test.

[0137] (Results) The results are shown in Figures 11 - 13. As a result of gene enrichment analysis, JAK2 V617F In Ly6G+ neutrophils from mouse - derived peripheral blood and lungs, the gene most up - regulated in the JAK - STAT3 pathway was found to be the Acvrl1 gene encoding ALK1. On the other hand, JAK2 V617FIn Ly6G-positive neutrophils from mouse-derived bone marrow, the expression of the Acvrl1 gene was only slightly elevated.

[0138] Consistent with the results of RNA sequencing, JAK2 V617F The mRNA expression level of the Acvrl1 gene derived from mouse lungs was higher than that of wild-type (WT) mice under normal oxygen partial pressure and further increased in response to exposure to a hypoxic environment (Figure 11). Similarly, after hypoxic exposure, the phosphorylation level of Smad1 / 5 / 8 downstream of ALK1 was significantly increased in the lungs of JAK2 V617F mice compared to the lungs of WT mice (Figure 12). On the other hand, the phosphorylation level of STAT3 was also significantly increased in JAK2 V617F mouse lungs compared to WT mouse lungs under normal oxygen partial pressure. After exposure to a hypoxic environment, JAK2 V617F the phosphorylation level of STAT3 in mouse lungs was further upregulated compared to other groups (Figure 13).

[0139] From the above, it was suggested that phosphorylation of STAT3 by JAK2 was promoted under a hypoxic environment, and as a result of this excessive phosphorylation of STAT3, the expression level of the Acvrl1 gene increased, and the downstream signaling pathway of the produced ALK1 was activated, thereby activating neutrophils.

[0140] <Example 5: Inhibition of Neutrophil Activation by JAK2 V617F Mutation by ALK1 Inhibitor> (Objective) Verify the suppression of neutrophils by the ALK1 inhibitor of the present invention. (Method) The same mice as in Example 1 were used. As the ALK1 inhibitors, K02288 (Selleck Chemicals) and LDN-212854 (Selleck Chemicals) were used. DMSO was used as the solvent, and from the day before exposure to a hypoxic environment for two weeks, WT mice and JAK2 V617FMice were administered by intraperitoneal injection twice a week for 2 weeks at a predetermined dose (K02288: 12 mg / kg body weight; LDN-212854: 9 mg / kg body weight). As a control, only DMSO was administered.

[0141] In addition, the phosphorylation level of Smad1 / 5 / 8 was analyzed using the ALK1 inhibitor LDN-212854. For this analysis, JAK2 V617F knock-in HCT116 cells (Horizon Discovery) were used. Cells were cultured in RPMI 1640 medium (Sigma: containing 2 mM L-glutamine and 25 mM sodium bicarbonate) supplemented with 10% FBS, 100 mg / mL streptomycin, and 100 IU / mL penicillin at 37 °C in the presence of 5% CO2. LDN-212854 was administered into the medium twice a week for 2 weeks at a concentration of 0.01 μM, 0.1 μM, 1 μM, or 10 μM. As a control, only DMSO was administered.

[0142] In addition, the analysis in tissues was performed in the same manner as in Example 1, and the analysis of the phosphorylation level of Smad1 / 5 / 8 was performed in the same manner as in Example 4. The number of samples was as follows: · Measurement of right ventricular systolic pressure, n = 6 - 8; · Measurement of right ventricular weight ratio, n = 6 - 8; · Measurement of the inner wall thickness of blood vessels, n = 6; · Measurement of the ratio of muscularized distal pulmonary arteries, n = 6; · Counting of Ly6G-positive cells, n = 3; · Measurement of elastase activity, n = 3; · Analysis of the phosphorylation level of Smad1 / 5 / 8, n = 2.

[0143] (Results) The results are shown in Figures 14 - 19. Administration of the ALK1 inhibitor K02288 resulted in JAK2 V617F in mice compared to control JAK2 V617FThe systolic pressure and hypertrophy of the right ventricle in mice were significantly decreased. In contrast, in hypoxic-exposed WT mice, even when K02288 was administered, no significant changes occurred in the systolic pressure and weight ratio of the right ventricle (Figure 14).

[0144] JAK2 administered with K02288 as an ALK1 inhibitor V617F In the pulmonary arteries of mice, a significant decrease in the inner wall thickness and muscular organization was observed compared to control mice, and it decreased to the same level as that of wild-type mice (Figure 15). Also, the number of Ly6G-positive neutrophils in the perivascular region was significantly decreased in the lungs of K02288-administered JAK2 V617F mice compared to the lungs of control mice (Figure 16). Furthermore, by administering K02288, JAK2 V617F the elastase activity in the lungs of mice was significantly decreased, and no significant difference was found compared to wild-type mice (Figure 17).

[0145] When LDN-212854 was administered as an ALK1 inhibitor, JAK2 V617F in cells containing the mutation, the phosphorylation level of Smad1 / 5 / 8 downstream of ALK1 was suppressed in a concentration-dependent manner by LDN-212854 (Figure 18). Also, similar to the case when K02288 was administered as an ALK1 inhibitor, the systolic pressure and weight ratio of the right ventricle after hypoxic exposure were significantly suppressed in JAK2 V617F mice (Figure 19).

[0146] From the above, it was shown that ALK1 is suppressed by an ALK1 inhibitor, and thereby, not only is the activation of neutrophils caused by the JAK2 V617F mutation significantly suppressed, but also the thickening of the blood vessel wall after hypoxic exposure and the onset of pulmonary hypertension induced by hypoxic exposure can be completely suppressed.

Claims

A pharmaceutical composition for preventing or treating cardiovascular diseases with intimal hyperplasia in clonal hematopoiesis or myeloproliferative neoplasms of an individual containing a mutation that activates the JAK-STAT signaling pathway, which comprises an inhibitor of neutrophils containing the mutation, and contains an ALK1 inhibitor as an active ingredient. The ALK1 inhibitor comprises at least one selected from the group consisting of K02288, LDN-212854, ML347, San78-130 and salts thereof; anti-ALK1 antibodies; and RNA interference molecules that specifically recognize and bind to the Acvrl1 gene or mRNA. The pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the mutation comprises any one or more protein mutations in JAK2, calreticulin, or thrombopoietin receptor.

3. The protein mutation of the aforementioned JAK2 is JAK2 V617F The pharmaceutical composition according to claim 2, which is a mutation or a mutation in exon 12 of JAK2.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the anti-ALK1 antibody comprises PF-03446962 and / or Dalantercept.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cardiovascular disease is one or more selected from the group consisting of arteriosclerosis and venous sclerosis of arteries and veins, aortic aneurysm, hypertension, pulmonary hypertension, venous thrombosis including hepatic vein occlusion, and arterial thrombosis including cerebral infarction and myocardial infarction.

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