WO2010126169A1 - Composition pharmaceutique utilisée en prévention des maladies vasculaires et comprenant un inhibiteur d'alk1 comme principe actif - Google Patents

Composition pharmaceutique utilisée en prévention des maladies vasculaires et comprenant un inhibiteur d'alk1 comme principe actif Download PDF

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WO2010126169A1
WO2010126169A1 PCT/JP2010/057924 JP2010057924W WO2010126169A1 WO 2010126169 A1 WO2010126169 A1 WO 2010126169A1 JP 2010057924 W JP2010057924 W JP 2010057924W WO 2010126169 A1 WO2010126169 A1 WO 2010126169A1
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bmp9
alk1
antibody
pharmaceutical composition
protein
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冨塚一磨
清水清
柿谷誠
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協和発酵キリン株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a pharmaceutical composition containing an activin receptor-like kinase 1 (Activin receptor-like kinase 1; hereinafter referred to as ALK1) inhibitor as an active ingredient, and a vascular disorder treatment using the same. It is about the method.
  • ALK1 activin receptor-like kinase 1
  • a vascular endothelial cell refers to a flat and thin layer of cells constituting the inner surface of a blood vessel.
  • Vascular endothelial cells produce and secrete various physiologically active substances, and are involved in antithrombotic action, regulation of vascular tonus, and inflammatory reaction (white blood cell adhesion, migration).
  • Healthy endothelial cells express various antithrombotic molecules, function in platelet aggregation inhibition, blood coagulation inhibition, fibrinolytic activity, and vascular relaxation, and function in antithrombotic properties (Non-patent Document 1).
  • vascular endothelial cells constantly express extracellular nucleotide-degrading enzymes on the cell membrane, degrade ADP released from platelets, and suppress platelet aggregation.
  • Non-patent Document 1 vascular endothelial cells are damaged, specifically, a state in which vasodilatability to a specific stimulating substance such as acetylcholine and bradykinin is reduced, an inflammatory state, a thrombotic state (thrombus tends to occur) (Non-patent documents 2 and 3).
  • vascular endothelial cells refers to inflammatory substances such as oxidized LDL, glycated protein, inflammatory cytokines (IL-1, TNF- ⁇ , etc.), endotoxin, etc. acting on vascular endothelial cells, and E-selectin, VCAM- 1, refers to the state in which ICAM-1 and NF-kB are expressed (Non-patent Document 2).
  • E-selectin and VCAM-1 are molecules that are specifically expressed in vascular endothelial cells, and are regarded as vascular disorder markers because they are induced during inflammation.
  • the E-selectin, VCAM-1 is considered to be involved in inflammation through the following mechanism.
  • E-selectin and VCAM-1 which are adhesion molecules, are expressed on vascular endothelial cells.
  • E- selectin on vascular endothelial cells express, leukocytes such as monocytes and neutrophils, via the selectin ligand (sugar (such as sialyl Le x antigens) expressed on the cell surface, weak vascular endothelial cells
  • leukocytes are integrin receptors such as LFA-1 or VLA4 expressed on the surface of leukocytes and VCAM-1 or ICAM-I expressed on the surface of vascular endothelial cells by inflammatory substances.
  • Non-patent Document 4 vascular endothelial injury and thrombotic state
  • normal vascular endothelial cells are in an antithrombotic state, but once inflammation is caused by injury or infection, antithrombosis in endothelial cells is part of the physiological defense reaction.
  • VWF von Willebrand factor
  • TF tissue factor
  • PAI- plasminogen activator inhibitor-1
  • thrombus formation is likely to be induced (Non-patent Document 1).
  • arteriosclerosis, hypertension, chronic kidney disease (CKD), diabetes, heart disease, lipid which are known to be caused by vascular endothelial disorder
  • lifestyle-related diseases such as abnormalities, acute renal failure, and thrombosis is particularly well known.
  • vascular endothelial damage is considered the first stage of atherosclerosis progression, and recent studies have demonstrated that vascular endothelial damage is a useful prognostic indicator of atherosclerotic disease, etc.
  • the importance of vascular endothelial injury in the development of atherosclerosis is unquestionable (Non-Patent Documents 3 and 5).
  • vascular endothelial damage is observed not only in hemodialysis patients but also in patients in the preservation period, and it has been reported that vascular endothelial damage is related to pathological progression (Non-Patent Documents 6-8).
  • Vascular endothelial injury due to renal disease is said to occur because the imported renal arterioles fail to contract due to abnormalities in the paraglomerular device caused by renal disease, and systemic blood pressure is transmitted directly to the glomeruli (non-patented). Reference 8).
  • pathological progression in hypertension, type I diabetes, type II diabetes, heart disease, dyslipidemia, etc., and high cardiovascular disease (CVD) risk in these diseases are also regarded as vascular endothelial disorder.
  • Non-Patent Documents 9-15 In addition, in type II diabetes and hypertension, the presence of vascular endothelial damage has been recognized before the onset of the symptom, and a relationship with the onset of the disease has also been suggested (Non-patent Documents 16 and 17).
  • Non-patent Document 18 vascular endothelial injury in the local region of the kidney is involved in pathological progression such as acute renal failure.
  • Vascular endothelial injury is also known to be deeply related to insulin resistance, which is considered to be the background of metabolic syndrome.
  • Vascular endothelial cells receive the action of insulin and produce nitric oxide (NO) via phosphatidylinositol 3-kinase (PI3K) to control vasodilation, but are considered to act on vascular endothelial damage, high blood sugar, lipid
  • NO nitric oxide
  • PI3K phosphatidylinositol 3-kinase
  • vascular endothelial disorders are closely related to insulin resistance, and lifestyle habits such as arteriosclerotic disease, hypertension, chronic kidney disease, acute renal failure, diabetes, heart disease, and thrombosis are observed. Perceived as the cause of the disease.
  • lifestyle habits such as arteriosclerotic disease, hypertension, chronic kidney disease, acute renal failure, diabetes, heart disease, and thrombosis.
  • Perceived as the cause of the disease.
  • many factors aging, smoking, inflammation, trauma, dyslipidemia, hyperglycemia, etc.
  • the true mechanism is not elucidated (Non-patent Document 22).
  • ALK1 belongs to the type I receptor group of the TGF-beta superfamily and is specifically expressed on the vascular endothelium, and is considered to be a causative gene of hereditary hemorrhagic telangiectasia (HHT) ( Non-patent document 23).
  • ALK1 phosphorylates intracellular signaling molecules Smad1 and Smad5 and transmits signals into the cell. From the analysis using ALK1-deficient mice, it is also known that ALK1 is an important molecule for blood vessel construction (Non-patent Documents 24 and 25).
  • BMP9 belonging to the bone morphogenetic protein (hereinafter referred to as BMP) family is known (Non-patent Documents 26 and 27).
  • BMP belongs to the TGF-beta superfamily and has been identified as a molecule having the ability to induce ectopic bone growth and cartilage formation (Non-patent Documents 28 and 29). Recently, BMP family molecules have generally been found to be involved in the growth, differentiation, and apoptosis of various cells and are important for tissue and organ morphogenesis (Non-patent Documents 30 and 31). BMP9 belonging to the BMP family molecule has been reported to promote the formation of hypertrophic chondrocytes and differentiation from mesenchymal cells to cartilage (Non-patent Document 32), as well as other BMP family molecules.
  • Non-patent Document 33 hepcidin expression regulation
  • Non-Patent Document 34 sugar metabolism involved in maintaining the homeostasis of the drug
  • the expression organ it has been reported that it is mainly expressed in the spinal cord and inter-segmental membrane in the fetal period and in the liver in the adult period (Non-patent Documents 34 to 36).
  • BMP9 is a circulating factor in blood (Non-patent Document 37).
  • the protein structure of BMP9 as with other BMP families, after synthesis as a single-chain precursor protein (pre-pro body), the signal peptide region is cleaved, and the C-terminal side is cleaved in the cell.
  • the cysteine residue present in the dimer forms a dimer (prodimer) via a disulfide bond. Then, it is cleaved by furin-like protease into the C-terminal side (matter dimer), which is the active body, and the N-terminal propeptide region (pro-region) having no disulfide bond. Two molecules of the cleaved N-terminal propeptide region form a complex with one molecule of the C-terminal nature dimer via a non-covalent bond, and are secreted from the cell in the form of the complex. Is known (Non-patent Document 38).
  • BMP signaling generally requires two receptors of type I and type II belonging to the TGFbeta superfamily having a serine / threonine kinase domain in the intracellular region.
  • ALK1 is known as a type I receptor for BMP9, and BMP type II receptor (BMPRII), activin type IIa receptor (ActRIIa), and activin type IIb receptor (ActRIIb) are known as type II receptors (Non-patent Document 27). .
  • ALK1 extracellular region polypeptide-Fc fusion protein or ALK1 extracellular region polypeptide antibody as a therapeutic agent for angiogenesis-related diseases such as cancer and rheumatoid arthritis
  • BMP9 protein The possibility as a therapeutic agent in breast cancer or prostate cancer (Patent Document 3) has been reported.
  • an ALK1 inhibitor comprising a compound having a pyrazolopyrimidine skeleton as an active ingredient as a therapeutic agent in angiogenesis-related diseases has been reported (Patent Document 4).
  • an ALK1 inhibitor has a vascular disorder inhibitory activity. That is, the present inventors have found that when BMP9, which is a ligand of ALK1, is allowed to act on vascular endothelial cells, E-selectin and VCAM-1 that are vascular endothelial injury markers are remarkably induced. Furthermore, when a mouse overexpressing BMP9 was produced, it was found that blood was accumulated in the abdominal cavity / thoracic cavity due to the excessive action of BMP9, and redness due to blood leakage was caused in lymph nodes. We have succeeded in confirming the action in vivo.
  • anti-BMP9 therapy that inhibits has anti-inflammatory effects including suppression of vascular injury.
  • anti-BMP9 therapy is effective for diseases such as heart disease, arteriosclerosis, renal disease, and dyslipidemia. The possibility that it could be an effective treatment was confirmed.
  • the present inventor considered that the present invention can be provided as a pharmaceutical composition for suppressing vascular injury using an ALK1 inhibitor such as a BMP9 neutralizing antibody as an active ingredient, and has completed the present invention. That is, the present invention is as follows. [1] A pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient. [2] The pharmaceutical composition of [1], wherein the ALK1 inhibitor is selected from any of the following.
  • the antibody that binds to BMP9 or the antibody fragment thereof (2) The antibody that binds to the ALK1 extracellular region polypeptide or the antibody fragment is a polyclonal antibody, a peptide antibody, a monoclonal antibody or a fragment thereof.
  • the pharmaceutical composition is a polyclonal antibody, a peptide antibody, a monoclonal antibody or a fragment thereof.
  • [5] Peptide in which the antibody fragment contains Fab, Fab ′, F (ab ′) 2, single chain antibody (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) and CDR The pharmaceutical composition of [3] or [4], which is an antibody fragment selected from [6]
  • [8] Diseases associated with vascular disorders include renal diseases, arteriosclerotic diseases, hypertension, pulmonary hypertension, heart disease, diabetes, diabetic complications, thrombosis, dyslipidemia and other lifestyle-related diseases, vasculitis
  • the pharmaceutical composition according to [7] which is selected from a disease accompanied by the disease or a disease shown to be possibly involved in BMP9.
  • the pharmaceutical composition of [8], wherein the renal disease is chronic glomerulonephritis including IgA nephropathy, diabetic nephropathy, lupus nephritis, nephrosclerosis or rapidly progressive glomerulonephritis.
  • Arteriosclerotic diseases include cerebrovascular disorders (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc.), ischemic heart disease (myocardial infarction, angina pectoris, etc.), aortic aneurysm, [8] The pharmaceutical composition according to [8], which is aortic dissection, nephrosclerosis or obstructive arteriosclerosis. [11] The pharmaceutical composition according to [8], wherein the heart disease is valvular heart disease, myocardial infarction, angina pectoris, cardiomyopathy.
  • the pharmaceutical composition according to [8], wherein the diabetic complication is arteriosclerosis, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic gangrene, chronic infection or cataract.
  • the thrombosis is pulmonary thromboembolism, cerebral infarction, myocardial infarction, acute limb thrombosis, intestinal necrosis (upper mesenteric artery thrombosis).
  • vasculitis Diseases associated with vasculitis are Takayasu's arteritis, giant cell arteritis (temporal arteritis), polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, Kawasaki disease, Henoch-Schönlein purpura
  • the pharmaceutical composition according to [8] which is hypersensitivity vasculitis, systemic lupus erythematosus, or rheumatoid arthritis.
  • liver disease acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, etc.
  • cancer with cancerous ascites / pleural effusion chronic Pancreatitis
  • allergic diseases allergic rhinitis, asthma, airway hypersensitivity, atopic dermatitis, etc.
  • inflammatory diseases delayed allergy, rheumatoid arthritis, arthritis, lung disease, hepatitis, ulcerative colitis, etc.
  • Alzheimer The pharmaceutical composition according to [8], which is multiple sclerosis, diabetic retinopathy, Raynaud's syndrome, Crohn's disease or cancer.
  • a method for treating a disease associated with a vascular disorder using the pharmaceutical composition according to any one of [1] to [15].
  • FIG. 1 is a diagram showing the neutralizing action of purified hsALK1Fc protein on BMP9.
  • FIG. 2 shows gene expression analysis using normal human vascular endothelial cells supplemented with BMP9.
  • a vascular endothelial disorder marker gene and lifestyle diseases hypertension, various renal diseases, diabetes, diabetic complications, (Dyslipidemia, arteriosclerotic disease, thrombosis) related gene is a diagram showing that the expression changes.
  • FIG. 3 is a diagram showing the structure of an N-terminal His-tagged mBMP9 complex recombinant expression vector.
  • FIG. 4 is a diagram showing an SDS-PAGE electrophoretic image of a purified N-terminal His-type mBMP9 complex purified sample by CBB staining.
  • FIG. 4 is a diagram showing an SDS-PAGE electrophoretic image of a purified N-terminal His-type mBMP9 complex purified sample by CBB staining.
  • FIG. 5 is a diagram showing analysis results of systolic blood pressure, diastolic blood pressure, mean blood pressure, and heart rate in rats administered with N-terminal His-type mBMP9 complex protein in a rat telemetry test.
  • FIG. 6 shows the results of blood biochemical analysis using sera of 6 control chimeric mice and 12 UShBMP9 KI chimeric mice reaching 5 weeks of age.
  • FIG. 7 is a diagram showing the structure of an N-terminal His-tagged hBMP9 complex recombinant expression vector.
  • FIG. 8 is a diagram showing an SDS-PAGE electrophoretic image of the N-terminal His-type hBMP9 complex purified preparation by silver staining.
  • FIG. 8 is a diagram showing an SDS-PAGE electrophoretic image of the N-terminal His-type hBMP9 complex purified preparation by silver staining.
  • FIG. 9 shows serum biochemical data of individual mice administered with hBMP9 complex at 100 ⁇ g / head or 360 ⁇ g / head.
  • FIG. 10 shows the results of conducting a biomarker survey (RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen)) using mouse serum administered with hBMP9 complex. Each value (times) at 100 ⁇ g / head or 360 ⁇ g / head indicates a variation ratio with respect to each control group.
  • FIG. 11 is a diagram showing the platelet number lowering action of the BMP9 neutralizing antibody.
  • FIG. 12 shows data obtained when a biomarker survey (RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen)) was performed using mouse serum administered with a BMP9 neutralizing antibody. Each value (times) is calculated by setting the variation ratio in the neutralizing antibody administration group to 1 in the control group.
  • FIG. 13 is a diagram showing an SDS-PAGE electrophoretic image of the purified hsALK1mFc preparation by CBB staining.
  • FIG. 14 is a diagram showing the lowering effect on neutral fat (triglycerol) and total cholesterol of the BMP9 neutralizing antibody found in an in vivo test using SHR-ND rats.
  • FIG. 15 is a diagram showing an inhibitory action on cardiac hypertrophy possessed by a BMP9 neutralizing antibody found from an in vivo test using SHR-ND rats. The heart weight value was corrected using the body weight value of each individual.
  • FIG. 16 is a view showing an inhibitory action of an ALK1 antagonist containing a BMP9 neutralizing antibody on plaque formation, which is a main lesion of arteriosclerosis. The figure shows the plaque area ratio in the aorta of APOE-deficient mice.
  • FIG. 17 shows the antihypertensive action of ALK1 extracellular region polypeptide-Fc fusion protein. The figure shows the systolic blood pressure of SHR rats implanted with telemetry equipment.
  • FIG. 18 is a diagram showing the inhibitory effect of BMP9 neutralizing antibody on the progression of nephropathy found from an in vivo test using a WKY anti-GBM nephritis model. The figure shows the change in serum BUN over time.
  • the present invention provides a pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient.
  • the ALK1 inhibitor contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is a substance that can suppress ALK1 activation, that is, activation of ALK1 kinase activity and suppress ALK1 signal.
  • the ALK1 inhibitor used in the present invention is not particularly limited, and examples thereof include the following (1) to (10): (1) an antibody that binds to BMP9 or an antibody fragment thereof; (2) ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof; (3) A nucleic acid encoding an ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof; (4) an expression vector comprising a nucleic acid encoding an ALK1 extracellular region polypeptide or a variant thereof, or a protein comprising the ALK1 extracellular region polypeptide or a variant thereof; (5) an antibody or an antibody fragment thereof that binds to an ALK1 extracellular region polypeptide; (6) an aptamer for BMP9 or an aptamer for ALK1 extracellular region polypeptide; (7) RNAi molecule targeting ALK1 gene or RNAi molecule targeting
  • the ALK1 inhibitor used in the present invention is particularly preferably an antibody that binds to BMP9 or an antibody fragment thereof that has neutralizing activity.
  • the ALK1 inhibitor that can be included in the pharmaceutical composition of the present invention is described below.
  • BMP9 antibody The antibody that binds to BMP9 is preferably a neutralizing antibody and can be prepared by the following method.
  • BMP9 is known and isolated from humans, mice, Japanese zelkova, etc., and sequence information is disclosed in databases such as GenBank.
  • human BMP9 is registered with GenBank as accession numbers Q9UK05, AAD56960, etc.
  • mouse BMP9 is registered with GenBank as accession numbers Q9WV56, NP_062379, etc., and these can be used.
  • the BMP9 protein a BMP9 protein having a known amino acid sequence registered in GenBank or the like can be used.
  • the BMP9 protein has an amino acid sequence represented by SEQ ID NO: 10 (mouse BMP9) or SEQ ID NO: 24 (human BMP9).
  • BMP9 protein particularly preferably BMP9 protein having the amino acid sequence shown in SEQ ID NO: 24 is used.
  • sequences represented by SEQ ID NO: 10 and SEQ ID NO: 24 include a signal sequence and pro-region.
  • SEQ ID NO: 10 the 319th to 428th sequences correspond to mature parts, and in SEQ ID NO: 24, The 320th to 429th arrays correspond to mature parts.
  • BMP9 includes a variant of BMP9. Such mutants include both natural mutants and artificial mutants.
  • amino acid sequence of BMP9 at least 319 to 428 of SEQ ID NO: 10, or at least 320 to 429 of SEQ ID NO: 24 1 to several amino acid deletions, substitutions, additions or insertions are included in the amino acid sequence having the sequence shown in the second sequence, or 80% or more, preferably 85% or more, more preferably It has an amino acid sequence having 90% or more, for example, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity.
  • the range of “1 to several” is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 1. Three, or one or two.
  • Antigen epitopes for obtaining the target antibody include regions having high antigenicity in the amino acid sequence of BMP9, regions having superficiality, regions that may not have secondary structure, homology with other proteins, or A low region can be selected.
  • the region having high antigenicity can be estimated by the method of Parker et al. [Biochemistry, 25, 5425-5432 (1986)].
  • the superficial region can be estimated by calculating and plotting a hydropathic index, for example. Regions that may not have secondary structure are described, for example, by the method of Chou and Fasman [Adv Enzymol Relat Areas Mol Biol. 47, 45-148 (1978)].
  • a peptide comprising the amino acid sequence can be synthesized by using a peptide synthesis method.
  • peptides of interest include R.I. B. Synthesized using a commercially available peptide synthesizer based on solid phase peptide synthesis developed by Merrifield [Science, 232, 341-347 (1986)], and after removing the protecting group, ion exchange chromatography , Purification by gel filtration chromatography, reverse phase chromatography, etc., alone or in combination.
  • the obtained purified peptide can be used as an immunogen by binding to a carrier protein such as keyhole limpet hemocyanin (KLH) or albumin.
  • KLH keyhole limpet hemocyanin
  • a polyclonal antibody or a monoclonal antibody that binds to BMP9 using BMP9 as an immunogen can also be prepared by a known technique.
  • the term “recombinant” used for BMP9 or a monoclonal antibody, a polyclonal antibody, or other proteins means that these proteins are produced by recombinant DNA in a host cell. Means.
  • a host cell any of prokaryotes (for example, bacteria such as E. coli) and eukaryotes (for example, yeast, CHO cells, insect cells, etc.) can be used.
  • the “antibody” of the present invention may be a peptide antibody, a polyclonal antibody, or a monoclonal antibody.
  • Antibodies produce antibodies that will specifically bind to proteins used for immunization of mice or other suitable host animals (eg, rabbits, cows, horses, sheep, pigs, rodents, etc.). Alternatively, it can be obtained by immunizing an antigen or antigen-expressing cell by subcutaneous, intraperitoneal, or intramuscular route to extract lymphocytes that would be produced. Further, as a host animal, a desired humanized antibody may be obtained by administering an antigen or an antigen-expressing cell to a transgenic animal having a repertoire of human antibody genes [Proc. Natl. Acad. Sci.
  • antibodies can be obtained by immunizing lymphocytes in vitro.
  • a polyclonal antibody can be obtained by collecting and purifying fractions that bind to the antigen from serum obtained from the host animal.
  • monoclonal antibodies can be prepared by fusing lymphocytes with myeloma cells using a suitable fusion reagent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principals. and Practice, 59-103, Academic press, (1986)].
  • the monoclonal antibody of the present invention can be prepared using the hybridoma method [Nature, 256, 495 (1975)] or using the recombinant DNA method (Cabilly et al., US Pat. No. 4,816,567). it can.
  • the antigen protein can be prepared by expressing DNA encoding all or a partial sequence of the BMP9 protein in E. coli, yeast, insect cells, animal cells and the like.
  • Recombinant BMP9 is purified by a method such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography or the like alone or in combination, and this purified preparation is used as an immunogen.
  • the antibody of the present invention may be an intact antibody or an antibody fragment such as (Fab ′) 2 or Fab.
  • chimeric antibodies or mosaic antibodies in which the constant region is replaced with a human constant region eg, mouse-human chimeric antibody; Cabilly et al., US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 89, 6851 (1984)
  • Fully human antibodies are also included in the antibodies of the present invention.
  • the use of chimeric antibodies, mosaic antibodies or humanized antibodies is preferred in order to prevent the appearance of antibodies against heterologous antigens, such as human anti-mouse antibodies (HAMA), Most preferred is a fully human antibody.
  • BMP9 antibody fragment In the present invention, examples of the antibody fragment include Fab, F (ab ′) 2, Fab ′, scFv, diabody, dsFv, and a peptide containing CDR.
  • Fab is a fragment obtained by treating IgG with papain, a proteolytic enzyme (cleaved at the 224th amino acid residue of the H chain), about half of the N chain side of the H chain and the entire L chain are disulfides. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000 bound by binding.
  • the Fab of the present invention can be obtained by treating a monoclonal antibody that binds to BMP9 with papain.
  • a Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. it can.
  • F (ab ′) 2 was obtained by decomposing the lower part of two disulfide bonds in the hinge region of IgG with pepsin, a proteolytic enzyme, and was constructed by binding two Fab regions at the hinge portion. It is a fragment having an antigen binding activity with a molecular weight of about 100,000.
  • F (ab ′) 2 of the present invention can be obtained by treating a monoclonal antibody that binds to BMP9 with pepsin.
  • Fab ′ described below can be produced by thioether bond or disulfide bond.
  • Fab ′ is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2.
  • the Fab ′ of the present invention can be obtained by treating F (ab ′) 2 binding to BMP9 with a reducing agent such as dithiothreitol.
  • DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the vector is introduced into prokaryotic or eukaryotic cells to express Fab ′.
  • VH-P-VL or VL-P-VH polypeptide which is an antibody fragment having antigen-binding activity.
  • the scFv of the present invention obtains cDNAs encoding monoclonal antibodies VH and VL that bind to BMP9, constructs a DNA encoding scFv, and inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector.
  • the expression vector can be expressed and produced by introducing the expression vector into a prokaryotic or eukaryotic organism.
  • Diabody is an antibody fragment in which scFv is dimerized and is an antibody fragment having a bivalent antigen-binding activity.
  • the bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.
  • the diabody of the present invention obtains cDNA encoding VH and VL of the monoclonal antibody that binds to BMP9, constructs the DNA encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less,
  • the DNA can be expressed and produced by inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are bonded via a disulfide bond between the cysteine residues.
  • the amino acid residue to be substituted with the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method [Protein Engineering, 7, 697 (1994)].
  • the dsFv of the present invention obtains cDNAs encoding the monoclonal antibodies VH and VL that bind to the BMP9 of the present invention, constructs a DNA encoding the dsFv, and the DNA is expressed in a prokaryotic expression vector or eukaryotic expression. It can be expressed and produced by inserting into a vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR is composed of at least one region of CDR of VH or VL.
  • Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • the peptide containing the CDR of the present invention constructs DNA encoding the VH and VL CDRs of the monoclonal antibody that binds to BMP9 of the present invention, and inserts the DNA into a prokaryotic expression vector or a eukaryotic expression vector. It can be expressed and produced by introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc t method.
  • ALK1 is a type I cell surface receptor for transforming growth factor ⁇ receptor type 1 (TGF- ⁇ -1).
  • TGF- ⁇ -1 transforming growth factor ⁇ receptor type 1
  • Human ALK1 is a polypeptide of 503 amino acids, which includes a signal sequence (amino acids: 1 to 21), an N-terminal extracellular TGF- ⁇ -1 ligand binding domain, or an extracellular domain (amino acids 22 to 22).
  • Attisano et al. Cell, 1993, vol. 75, pp. 671-680 includes human ALK1 amino acid sequence containing serine at position 172 (Genbank accession number L17075), and US Pat. No. 6,316,217 discloses human ALK1 amino acid sequence having threonine at position 172. (Genbank accession number NM — 000020) is disclosed.
  • the “ALK1 extracellular region polypeptide” is a protein consisting of all or part of the amino acid sequence of the ALK1 extracellular region polypeptide derived from a mammal.
  • the amino acid sequence and cDNA sequence of human ALK1 extracellular region polypeptide are as follows. Amino acid sequence of human ALK1 extracellular region polypeptide (SEQ ID NO: 29): (Genbank ACCESSION No.
  • the underline indicates the signal sequence, and the encircled line indicates the extracellular region polypeptide after cleavage of the signal sequence.
  • CDNA sequence of human ALK1 extracellular region polypeptide SEQ ID NO: 30: In the present invention, the ALK1 extracellular region polypeptide also encompasses variants of the above ALK1 extracellular region polypeptide.
  • Such mutants include both natural mutants and artificial mutants, and substitution of one or more (preferably one or several) amino acids in the amino acid sequence of the ALK1 extracellular region polypeptide, Deletion or addition, or 80% or more, preferably 85% or more, more preferably 90% or more, such as 93% or more, 95% or more, 97% or more, 98% or more, or 99% with the amino acid sequence It contains an amino acid sequence having the above identity and has vascular disorder inhibitory activity.
  • the variant includes one or more (preferably one or several) amino acid substitutions, deletions or additions in the amino acid sequence of SEQ ID NO: 29, or 80% or more of the amino acid sequence.
  • vascular disorder inhibitory activity Preferably 85% or more, more preferably 90% or more, for example, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, and an vascular disorder inhibitory activity. It is what has.
  • the term “several” as used herein generally refers to any integer from 2 to 10. Preferably it is any integer from 2 to 5.
  • the term “identity” refers to the alignment of two amino acid sequences (or nucleotide sequences) so that the number of identical amino acid residues (or nucleotides) is maximized.
  • a protein having a sequence identity of 80% or more, preferably 85% or more accesses a sequence database such as NCBI (US) or EMBL (Europe) and uses a sequence homology search program such as BLAST or FASTA. (Altschul, SF et al. (1990) J. Mol. Biol.
  • BLAST breaks the sequence into fixed-length words, searches for similar fragments in word units, stretches them in both directions until the degree of similarity is maximized, performs local alignment, and finally combines them into the final It is a method of performing a general alignment.
  • FASTA searches for fragments of sequences that match continuously at high speed, performs local alignment by focusing on those fragments that have high similarity, and finally considers these gaps. Is an alignment method.
  • a site-directed mutagenesis method using a PCR method using a primer (including a complementary mutant sequence) synthesized based on the sequence of the ALK1 extracellular region polypeptide is preferable (Kunkel et al., Proc Natl.Acad.Sci.USA, 1985, 82: 488-492; F.M. Ausubel et al., Short Protocols in Molecular Biology, 1995, John Wiley & Clones, J. Sambrook et al., Molecular A: Mol. ed., 1989, Cold Spring Harbor Laboratory Press).
  • Mutation introduction kits for example, manufactured by Takara Shuzo Co., Ltd. are also commercially available, and mutations can be introduced according to the instructions.
  • the template is annealed to the template, DNA synthesis is performed, and the ends are ligated with T4 DNA ligase to purify the DNA containing the target mutation.
  • the mutation includes substitution, deletion, addition, insertion, or a combination thereof.
  • substitution may be either a conservative substitution or a non-conservative substitution, but a conservative substitution is preferred so as not to substantially change the conformation of the ALK1 extracellular region polypeptide.
  • Conservative substitutions can be made between amino acids with similar chemical and physical properties such as structural (eg, branched, aromatic, etc.), electrical (eg, acidic, basic, etc.), polar or hydrophobic, etc. Refers to replacement.
  • Branched amino acids include valine, leucine and isoleucine.
  • Aromatic amino acids include tyrosine, tryptophan, phenylalanine, histidine.
  • Acidic amino acids include glutamic acid and aspartic acid.
  • Basic amino acids include lysine, arginine, and histidine.
  • Polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, cysteine, glycine, proline and the like.
  • Hydrophobic amino acids include alanine, valine, leucine, isoleucine, methionine and the like.
  • Deletion is the loss of one or more amino acid residues.
  • Addition is the attachment of one or more amino acid residues to the N-terminus or C-terminus of the protein.
  • Insertion is the joining of one or more amino acid residues inside a protein.
  • deletion and insertion can be performed on the assumption that the conformation of the ALK1 extracellular region polypeptide is not substantially changed. Therefore, it is preferably limited to deletion or insertion of about 1 to 5 amino acid residues.
  • the protein containing the ALK1 extracellular region polypeptide or a variant thereof is a protein containing the above ALK1 extracellular region polypeptide or a variant thereof, and is not limited to the ALK1 extracellular region polypeptide or a variant thereof.
  • a fusion protein of a mammal and a mammal-derived immunoglobulin Fc protein or a variant thereof for example, a fusion protein described in WO 2008/057461.
  • the expression “comprising” is necessary for the ALK1 extracellular region polypeptide or variant thereof and the heterologous peptide, polypeptide or protein on the N-terminal or C-terminal side of the domain or variant thereof.
  • a heterologous protein is a mammal-derived immunoglobulin Fc protein or a variant thereof.
  • a preferred Fc protein is a human immunoglobulin Fc protein for use in humans.
  • the class and subclass of the immunoglobulin are not limited to the following, but any of IgG, IgD, IgE, IgM, IgA, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgA1, IgA2, etc. Can be used. However, for use in humans, it is desirable to use human immunoglobulin classes and subclasses.
  • the Fc protein can improve the in vivo stability of the ALK1 extracellular domain or a variant thereof. In this case, however, the Fc protein exhibits biological activities such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities in order to avoid in vivo effects due to its biological activity.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • a preferred example of the Fc protein is a human IgG1 Fc variant represented by the amino acid sequence of SEQ ID NO: 31 below.
  • SEQ ID NO: 31 The binding position of the Fc protein may be either the N-terminal side or the C-terminal side of the ALK1 extracellular region polypeptide or a variant thereof, but the C-terminal side is preferred.
  • a specific example of the fusion protein of the ALK1 extracellular region polypeptide and the Fc protein is a protein represented by the amino acid sequence of SEQ ID NO: 32 below, for example.
  • the underlined portion indicates the ALK1 extracellular region polypeptide (SEQ ID NO: 29), and the non-underlined portion indicates the human IgG1 Fc variant protein (SEQ ID NO: 31).
  • a protein containing an ALK1 extracellular region polypeptide or a variant thereof does not necessarily have to be bound or fused with a heterologous peptide, polypeptide or protein.
  • the protein containing the ALK1 extracellular region polypeptide or a variant thereof in the present invention can be prepared by a gene recombination technique commonly used in this industry.
  • the protein is prepared by preparing a DNA encoding the protein of the present invention, constructing an expression vector containing the DNA, transforming or transfecting prokaryotic or eukaryotic cells with the vector, Recovering the desired recombinant protein from the cultured cells.
  • Protein purification is performed by appropriately combining conventional protein purification methods such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and HPLC. Is possible.
  • ⁇ ALK1 extracellular region polypeptide or variant thereof or nucleic acid encoding ALK1 extracellular region polypeptide or protein containing the variant> and ⁇ ALK1 extracellular region polypeptide or its Since it is described in the section, Examples, etc. of the expression vector> containing the nucleic acid encoding the mutant or the ALK1 extracellular region polypeptide or the protein containing the mutant, it can be referred to.
  • gene recombination techniques are described in Ausubel et al. (Above), Sambrook et al. (Above), and can be used for the present invention.
  • the protein containing the ALK1 extracellular region polypeptide or variant thereof in the present invention may be chemically modified.
  • Chemical modifications include, but are not limited to, for example, glycosylation, PEGylation (PEG), acetylation, amidation, phosphorylation and the like. Particularly preferred chemical modifications that can be utilized are glycosylation and pegylation.
  • Pegylation is the attachment of one or more polyethylene glycol (PEG) molecules to amino acid residues such as the N-terminal amino group of proteins and the ⁇ -amino group of lysine (Lys).
  • PEG polyethylene glycol
  • Lys ⁇ -amino group of lysine
  • a PEG molecule is attached to the free amino group of an amino acid.
  • the average molecular weight of PEG is not limited to the following, but can be used in the range of about 3,000 to about 50,000.
  • the PEG end can be, for example, carboxylated, formylated, succinimidylated, or maleimidylated and reacted with the free amino group of the protein.
  • Glycosylation is the attachment of a carbohydrate chain (ie, sugar chain) to an asparagine, serine or threonine residue of a protein.
  • sugar chain binding occurs by recognizing the sequence of Asn-X-Thr / Ser (where X is any amino acid residue other than Pro).
  • a sugar chain can be introduced at a position different from the natural type.
  • a recombinant protein can be glycosylated by expressing a nucleic acid encoding the recombinant protein in a eukaryotic cell (yeast cell, animal cell, plant cell, etc.) by a genetic recombination technique.
  • a eukaryotic cell yeast cell, animal cell, plant cell, etc.
  • the sugar chain structure is not particularly limited, and it is considered that the sugar chain structure varies depending on the cell type selected for expression.
  • human-derived cells, yeast cells capable of synthesizing human sugar chains, Chinese hamster ovary (CHO) cells, and the like can be used. Acetylation and amidation are preferably performed mainly at the N-terminus or C-terminus of the protein.
  • nucleic acid includes both DNA and RNA.
  • DNA includes genomic DNA and cDNA
  • RNA includes mRNA.
  • the nucleic acid in the present invention includes the nucleic acid encoding the ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof, as explained and specifically exemplified above. .
  • the nucleic acid includes a nucleic acid encoding an amino acid sequence containing at least the 22nd to 118th amino acids in the amino acid sequence of the extracellular region polypeptide (SEQ ID NO: 29) of human ALK1.
  • the nucleic acid may further include a nucleotide sequence encoding a signal sequence. Examples of signal sequences are BMP9 signal sequence, Ig ⁇ and the like.
  • the nucleotide sequence encoding the precursor of the human-derived ALK1 extracellular region polypeptide is exemplified below.
  • the underlined site indicates a nucleotide sequence encoding a signal sequence
  • the non-underlined site indicates a nucleotide sequence encoding a mature sequence of an extracellular domain protein.
  • the nucleic acid in the present invention also includes a nucleic acid encoding a fusion protein of a protein containing an ALK1 extracellular region polypeptide or a variant thereof and a heterologous protein as defined above.
  • heterologous protein is an immunoglobulin Fc protein derived from a mammal, and a human Fc protein is particularly preferable, but it is desirable to introduce a mutation so as to reduce or lose its biological activity (particularly ADCC and CDC).
  • the nucleotide sequence encoding the mutant human IgG1-derived Fc protein is shown in SEQ ID NO: 33.
  • SEQ ID NO: 34 a nucleotide sequence (SEQ ID NO: 34) encoding a fusion protein of this mutant human IgG1-derived Fc protein (underlined portion) and a protein containing a human-derived ALK1 extracellular region polypeptide (non-underlined portion) is as follows: This is illustrated in SEQ ID NO: 34:
  • the nucleotide sequence encoding the fusion protein can further include a nucleotide sequence encoding a signal sequence. Examples of signal sequences are human protein-derived signal sequences such as BMP9 and Ig ⁇ .
  • Nucleic acid homologs encoding the above proteins can be obtained by using well-known techniques using primers and probes prepared based on cDNA synthesized from mRNA encoding ALK1 extracellular region polypeptide gene derived from human or mouse. It can be obtained from cDNA libraries prepared from other mammals and prepared from cells or tissues known to express the gene. Such techniques include PCR methods, hybridization methods (Southern method, Northern method, etc.) and the like. The PCR method is a polymerase chain reaction, which is a denaturing step (about 94 to 96 ° C., about 30 seconds to 1 minute) for dissociating double-stranded DNA into single strands, using a primer as a template.
  • Annealing step for binding to single-stranded DNA (about 55 to 68 ° C., about 30 seconds to 1 minute), extension step for extending DNA strand (about 72 ° C., about 30 seconds to about 30 seconds to 1 minute)
  • a cycle consisting of 1 minute) is regarded as one cycle, and about 25 to 40 cycles are carried out.
  • a preheating treatment is performed at about 94 to 95 ° C. for about 5 to 12 minutes, and after the final cycle of the extension step, an extension reaction can be further performed at 72 ° C. for about 7 to 15 minutes. it can.
  • PCR is performed with a commercially available thermal cycler using a thermostable DNA polymerase (for example, AmpliTaq Gold (registered trademark) (Applied Biosystems)), MgCl 2 , In a PCR buffer containing dNTP (dATP, dGTP, dCTP, dTTP), etc., in the presence of sense and antisense primers (size: about 17-30b, preferably 20-25b) and template DNA.
  • dNTP dATP, dGTP, dCTP, dTTP
  • sense and antisense primers size: about 17-30b, preferably 20-25b
  • template DNA size: about 17-30b, preferably 20-25b
  • the amplified DNA can be separated and purified (ethidium bromide staining) by agarose gel electrophoresis.
  • Hybridization is a technique for detecting a target nucleic acid by forming a double strand with a labeled probe having a length of about 20 to 100 b or more.
  • hybridization can generally be performed under stringent conditions.
  • the stringent conditions are, for example, about 1 to 5 ⁇ SSC, hybridization at room temperature to about 40 ° C., and then about 0.1 to 1 ⁇ SSC, 0.1% SDS, about 45 to 65 ° C. Consists of washing.
  • 1 ⁇ SSC refers to a solution of 150 mM NaCl, 15 mM Na-citric acid, pH 7.0.
  • nucleic acids with a sequence identity of about 80% or more, preferably 85% or more.
  • expression vector containing nucleic acid encoding ALK1 extracellular region polypeptide or variant thereof, or protein containing ALK1 extracellular region polypeptide or variant thereof The nucleic acid can be inserted into a vector and used to produce a protein that is an active ingredient of the pharmaceutical composition of the present invention, or the vector itself can be formulated and used as a pharmaceutical composition.
  • Vectors include, for example, plasmids, phages, viruses and the like. Examples of plasmids include, but are not limited to, E.
  • coli-derived plasmids eg, pRSET, pTZ19R, pBR322, pBR325, pUC118, pUC119, etc.
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, etc.
  • yeast-derived plasmids eg, YEp13, YEp24, YCp50, etc.
  • Ti plasmids etc.
  • examples of phages include ⁇ phages
  • viral vectors include animal virus vectors such as retroviruses, vaccinia viruses, lentiviruses, adenoviruses, adeno-associated viruses, etc.
  • the vector may contain a polylinker or multicloning site for integrating the DNA of interest, and may contain several control elements to express the DNA.
  • the control element includes, for example, a promoter, an enhancer, a poly A addition signal, a replication origin, a selection marker, a ribosome binding sequence, a terminator and the like.
  • selectable markers include drug resistance genes (eg, neomycin resistance gene, ampicillin resistance gene, kanamycin resistance gene, puromycin resistance gene, etc.), auxotrophic complementary genes (eg, dihydrofolate reductase (DHFR) gene, HIS3 gene, LEU2 gene) URA3 gene, etc.).
  • the promoter may vary depending on the host cell.
  • host cells include, but are not limited to, bacteria such as Escherichia such as E. coli, Bacillus such as Bacillus subtilis, Pseudomonas such as Pseudomonas putida, Saccharomyces cerevisiae, Saccharomyces such as Schizosaccharomyces pombe And yeast such as Candida and Pichia, animal cells such as CHO, COS, HEK293, and NIH3T3, insect cells such as Sf9 and Sf21, and plant cells.
  • examples of the promoter include trp promoter, lac promoter, PL or PR promoter.
  • examples of the promoter include gal1 promoter, gal10 promoter, heat shock protein promoter, MF ⁇ 1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like.
  • examples of promoters include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, human CMV early gene promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, metallothionein promoter, polyhedron promoter, etc. Is done.
  • examples of promoters include CaMV promoter and TMV promoter.
  • Examples of transformation or transfection of host cells using expression vectors include electroporation, spheroplast, lithium acetate, calcium phosphate, Agrobacterium, virus infection, liposome, microinjection, Examples include gene gun method and lipofection method.
  • the transformed host is cultured under culture conditions according to the types of bacteria, yeast, animal cells, and plant cells, and the target protein is recovered from the cells or from the culture solution.
  • a medium containing a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by microorganisms is used.
  • carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, as nitrogen sources, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc.
  • Inorganic acids, ammonium salts of organic acids, peptone, meat extract, corn steep liquor, etc., as inorganic substances, potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, Manganese sulfate, copper sulfate, calcium carbonate and the like are used.
  • a medium in which DMEM medium, RPMI 1640 medium, or the like is used as a basic medium and fetal calf serum (FCS) or the like is added thereto is used.
  • FCS fetal calf serum
  • the target protein can be recovered by conventional methods for protein purification, such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, It can be carried out by HPLC or the like.
  • the vector is used for therapy, it is preferably a vector that is not integrated into the subject's genome and that infects cells but is unable to replicate, such as a non-viral vector.
  • Such vectors include, for example, adeno-associated virus vectors, adenovirus vectors and the like. These vectors can include promoters, enhancers, polyadenylation sites, selectable markers, reporter genes, and the like. Examples of viral vectors are described in J. Virol. 67: 5911-5921 (1993), Human Gene Therapy 5: 717-729 (1994), Gene Therapy 1: 51-58 (1994), Human Gene Therapy 5: 793-801 (1994), Gene Therapy 1: 165. 169 (1994) and the like, or improved vectors thereof.
  • an example of a non-viral vector is a human artificial chromosome vector, which is a vector composed of chromosome fragments containing human chromosome-derived centromeres and telomeres.
  • the human chromosome fragment is not particularly limited, but includes, for example, human chromosome 14 fragment, human chromosome 21 fragment (re-listed 2004/031385, Japanese Patent Application Laid-Open No. 2007-295860, etc.).
  • the nucleic acid as defined above is inserted into the vector and administered to the subject, or the vector is administered to the subject by introducing the vector into a tissue or cell collected from the subject and then returning to the subject. can do.
  • the antibody that binds to the ALK1 extracellular region polypeptide in the present invention is an antibody that can bind to the ALK1 extracellular region polypeptide, and is preferably a neutralizing antibody.
  • An antibody that inhibits the binding between ALK1 and ALK1 is preferred.
  • the antibody that binds to the ALK1 extracellular region polypeptide in the present invention includes, for example, an antibody described in WO 2007/40912.
  • An antibody that binds to the ALK1 extracellular region polypeptide or the antibody fragment can be prepared by using the ALK1 extracellular region polypeptide as an antigen protein by the technique specifically shown in the above section (BMP9 antibody). it can.
  • Aptamers are nucleic acid ligands that bind to specific molecules of proteins.
  • the aptamer in the present invention binds to the BMP9 protein or ALK1 extracellular region and can inhibit the function of the BMP9 protein or ALK1.
  • Aptamers can be obtained by preparing a library composed of various nucleic acid chains and selecting a nucleic acid chain that can bind to the target protein from the library. Suitable methods for identifying aptamers include, for example, Systematic Evolution of Ligands by Exponential Enrichment (SELEX). TM ) Method (US Pat. No. 5,270,163).
  • RNAi molecule targeting ALK1 gene or RNAi molecule targeting BMP9 gene An RNAi molecule is a single-stranded or double-stranded nucleic acid molecule comprising an antisense strand having a base sequence complementary to mRNA encoding a target protein. In the RNAi molecule, the antisense strand specifically binds to mRNA encoding the target protein and inhibits expression of the target protein (protein synthesis).
  • target means that the antisense strand contained in the RNAi molecule hybridizes with the target mRNA under stringent conditions.
  • the stringent conditions are, for example, about 1 to 5 ⁇ SSC, hybridization at room temperature to about 40 ° C., and then about 0.1 to 1 ⁇ SSC, 0.1% SDS, about 45 to 65 ° C. Consists of washing.
  • 1 ⁇ SSC refers to a solution of 150 mM NaCl, 15 mM Na-citric acid, pH 7.0.
  • the RNAi molecule in the present invention specifically binds to mRNA encoding BMP9 or ALK1, and inhibits its protein synthesis.
  • the RNAi molecule in the present invention includes antisense RNA, antisense DNA, siRNA, and shRNA.
  • siRNA is a low molecular double-stranded RNA obtained by hybridizing an antisense strand having a base sequence complementary to mRNA encoding a target protein and a sense strand complementary to the antisense strand.
  • shRNA is a single-stranded RNA in which the antisense strand and the sense strand are linked via a linker portion, and the linker portion is folded by forming a loop, and the antisense strand and the sense strand are Hybridizes to form a double stranded portion.
  • the RNAi molecule of the present invention can be designed based on the above-described BMP9 and ALK1 gene sequences, for example, using a siRNA Design Support System (Takara Bio Inc.).
  • a siRNA Design Support System TiRNA Design Support System (Takara Bio Inc.).
  • the low molecular weight compound having an inhibitory activity on the kinase activity of ALK1 in the present invention is not particularly limited as long as it is a compound known to inhibit ALK1 kinase activity.
  • WO 2007 / 147647 N- (2-dimethylamino-ethyl) -3- [5- (4-isopropyl-phenylamino) -pyrazolo [1,5-a] pyrimidine-3-yl] -benzamide Phenyl- [3- (3,4,5-trimethoxy-phenyl) -pyrazolo [1,5-a] pyrimidine-5-yl] -amine; (4-fluoro-phenyl)-[3- (3,4 , 5-trimethoxy-phenyl) -pyrazolo [1,5-a] pyrimidin-5-yl] -amine; N ′-[3- (3-chloro-phenyl) -pyrazolo [1 5-a] pyrimidin -5-yl] -N, N- diethyl - propane -e-1,3-diamine, but not limited to) can be mentioned.
  • the low molecular weight compound having an activity of inhibiting the binding between BMP9 and ALK1 or the binding between BMP9 and type II receptor is the inhibition of the binding between BMP9 and ALK1 or between BMP9 and type II receptor. It refers to a compound that can be produced and is not particularly limited.
  • Type II receptor includes BMP type II receptor (BMPRII), activin type IIa receptor (ActRIIa), and activin type IIb receptor (ActRIIb).
  • the low molecular weight compound included in the present invention may have an activity of inhibiting any one of the binding between BMP9 and BMPRII, the binding between BMP9 and ActRIIa, and the binding between BMP9 and ActRIIb. In addition, it may have an activity of inhibiting any two bonds, or may have an activity of inhibiting these three bonds. Preferable examples include low molecular weight compounds having an activity of inhibiting the binding between BMP9 and BMPRII.
  • ⁇ BMP9 antagonist protein having BMP9 inhibitory activity> Although no BMP9 antagonist protein having BMP9 inhibitory activity has been reported so far, various biologically derived BMP antagonists having BMP inhibitory activity have been reported for BMP, and such BMP9 antagonist is also present in BMP9. May exist.
  • Such a BMP9 antagonist can be used as an active ingredient of the pharmaceutical composition of the present invention.
  • the pharmaceutical composition of the present invention can be used for diseases associated with vascular disorders, such as renal diseases, arteriosclerotic diseases, hypertension, heart diseases, diabetes, diabetic complications, thrombosis, dyslipidemia, etc. Lifestyle-related diseases, diseases associated with vasculitis, and diseases in which BMP9 has been shown to be involved.
  • the renal disease is not particularly limited, and includes chronic glomerulonephritis including IgA nephropathy, diabetic nephropathy, lupus nephritis, nephrosclerosis or rapidly progressive glomerulonephritis.
  • Arteriosclerotic disease is not particularly limited, but ischemic heart such as cerebrovascular disorders (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc.), myocardial infarction and angina Diseases, aortic aneurysm, aortic dissection, nephrosclerosis, obstructive arteriosclerosis and the like can be mentioned.
  • the heart disease is not particularly limited, and examples thereof include valvular heart disease, myocardial infarction, angina pectoris, and cardiomyopathy.
  • Diabetic complications include arteriosclerosis, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic gangrene, chronic infection, cataract and the like.
  • the thrombosis is not particularly limited, and examples thereof include pulmonary thromboembolism, cerebral infarction, myocardial infarction, lower limb acute arterial thrombosis, intestinal necrosis (upper mesenteric artery thrombosis) and the like.
  • vasculitis Diseases associated with vasculitis are not particularly limited, but Takayasu arteritis, giant cell arteritis (temporal arteritis), polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, Kawasaki disease, Examples include Henoch-Schönlein purpura, hypersensitivity vasculitis, systemic lupus erythematosus, and rheumatoid arthritis.
  • Diseases that may be involved in BMP9 are liver disease, cancer with cancerous ascites / pleural effusion, chronic pancreatitis, allergic disease, inflammatory disease, Alzheimer, multiple sclerosis, diabetic retinopathy, Raynaud Syndrome, Crohn's disease, cancer, etc.
  • the allergic disease is not particularly limited, and examples include allergic rhinitis, asthma, airway hypersensitivity, and atopic dermatitis.
  • the inflammatory disease is not particularly limited, and examples thereof include delayed allergy, rheumatoid arthritis, arthritis, pulmonary disease, hepatitis, ulcerative colitis and the like.
  • the liver disease is not particularly limited, and examples include acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, and metastatic liver cancer.
  • the pharmaceutical composition of the present invention is preferably a renal disease, arteriosclerotic disease, hypertension, heart disease, diabetes, diabetic complications, thrombosis, dyslipidemia and other lifestyle diseases, particularly preferably renal disease, arteriosclerosis It can be applied to sex diseases, hypertension and thrombosis.
  • Chronic kidney disease (CKD) is considered to be the largest independent risk factor for cardiovascular complications (CVD), but the presence of arteriosclerosis is also recognized in the early stages of CKD.
  • CVD cardiovascular complications
  • the pharmaceutical composition of the present invention contains one or a combination of the above active ingredients.
  • the pharmaceutical composition of the present invention contains an antibody that binds to BMP9 or the antibody fragment as an active ingredient.
  • the amount of the active ingredient contained in the pharmaceutical composition of the present invention should be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the patient to be administered, and is not limited to the following, It can be appropriately determined within a range of about 0.1 ⁇ g / kg to 100 mg / kg, preferably within a range of about 1 ⁇ g / kg to 10 mg / kg.
  • the form (namely, preparation) of the pharmaceutical composition of the present invention is not limited, and includes both oral preparations and parenteral preparations.
  • a preferred form is a parenteral preparation, which includes, but is not limited to, an intravenous preparation, an intramuscular preparation, an intraperitoneal preparation, a subcutaneous preparation, a topical preparation and the like.
  • the parenteral preparation includes, for example, an injection, an instillation, a suppository, a transdermal absorption agent, a liposome or a nanoparticle-encapsulated preparation.
  • Oral preparations include, for example, tablets, pills, granules, capsules, powders, solutions, suspensions, delayed release preparations, enteric preparations and the like.
  • the pharmaceutical composition of the present invention can contain pharmaceutically acceptable excipients, carriers such as diluents, and additives.
  • Carriers include, for example, saline, glycerol, ethanol, almond oil, vegetable oil, sucrose, starch, lactose and the like.
  • Additives include, for example, binders (eg pregelatinized corn starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone etc.), lubricants (eg magnesium stearate, talc, silica etc.), dispersants (eg polyvinylpyrrolidone, corn starch etc.) ), Suspension (eg talc, gum arabic, etc.), emulsifier (eg lecithin, gum arabic, etc.), disintegrant (potato starch, sodium starch glycolate, crospovidone, etc.), buffer (eg phosphate, acetate) Citrate, tris salt, etc.), antioxidants (eg, ascorbic acid, tocopherol, etc.), preservatives (eg, sorbic acid, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate), isotonic agents (eg Eg sodium chloride) , And the like agents (eg, gly
  • enteric preparations for example, polymers such as hydroxypropyl methylcellulose phthalate, methacrylic acid-methyl methacrylate copolymer, methacrylic acid-ethyl acrylate copolymer, hydroxypropyl acetate succinate are used.
  • the dosage of the pharmaceutical composition of the present invention should be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the patient, and is not limited to the following, but for example, about 0.1 ⁇ g per adult day / Kg to 100 mg / kg, preferably about 1 ⁇ g / kg to 10 mg / kg.
  • Administration of the formulation may be administered daily during treatment or at intervals such as several days, two weeks or one month.
  • the pharmaceutical composition of the present invention can be administered to a patient by an administration method suitable for the type and dosage form of the active ingredient contained therein.
  • the pharmaceutical composition of the present invention is administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intranasally, intravaginally, intrarectally, topically, intramuscularly, orally.
  • Injection, infusion, cell transplantation of transformed cells expressing ALK1 extracellular region polypeptide or the like prepared using the above vector, or any combination of the above.
  • the pharmaceutical composition of the present invention contains any of the nucleic acids, expression vectors, aptamers or RNAi molecules described in detail above or a combination thereof as an active ingredient
  • the pharmaceutical composition is administered by using the active ingredient as a target tissue.
  • it can be performed using a technique or technique generally used in gene therapy that can be introduced into cells.
  • Gene delivery methods that can be used to administer the pharmaceutical composition of the present invention include colloidal dispersion systems, liposome-derived systems, artificial virus envelopes, and the like.
  • the delivery system should use macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, liposomes, calcium phosphate method, DEAE dextran method, electroporation method, lipofection method, etc. Can do.
  • ⁇ Evaluation of therapeutic agents> The effect of the pharmaceutical composition of the present invention can be evaluated by alleviating or eliminating the symptoms of the disease in a patient suffering from the disease that has received administration of the pharmaceutical composition.
  • the effect of the pharmaceutical composition of the present invention can be evaluated by genetic or biochemical analysis of genes, biomarkers and serum biochemical parameters that are known to be related to the above-mentioned diseases.
  • genes, biomarkers and serum biochemical parameters include (but are not limited to) the following: gene Biomarker Serum biochemistry parameters EXAMPLES
  • the present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
  • this application is a contract research and industrial technology related to “Development of biological system control infrastructure technology using compounds, etc.” in 2007, New Energy and Industrial Technology Development Organization. This is a patent application subject to Article 19 of the Strengthening Law.
  • hsALK1Fc expression vector is a PEAK8 expression vector obtained by enzymatic digestion of the extracellular region cDNA fragment of human ALK1 and the hIgG1 Fc region cDNA fragment with EcoRI and NotI (both from Roche Diagnostics, Japan). It was prepared by incorporating into (Edge Biosystems).
  • the cDNA sequence of the incorporated hsALK1Fc is as follows (SEQ ID NO: 1).
  • SEQ ID NO: 1 346 amino acids, SEQ ID NO: 2) is shown below.
  • SEQ ID NO: 2 Each fragment was obtained as follows.
  • the extracellular region cDNA of human ALK1 is hsALK1 FW: agaattccccaccatgaccttgtccccccag (SEQ ID NO: 3), hsALK1 RV: aacttagctgggcccatctgtttccccggctg (SEQ ID NO: 4) was cloned from a human lung cDNA library using the PCR method. The obtained fragment was enzymatically digested with EcoR1 and SpeI, and then electrophoresed on an agarose gel to cut out the band. For extraction of DNA from agarose, QIAquick Gel Extraction Kit (Qiagen, Japan) was used.
  • the FIgG region cDNA of hIgG1 is hIgG1 Fc FW: actagtgacaaaactcacacatgcc (SEQ ID NO: 5)
  • hIgG1 Fc RV gcggccgctcatcatttaccc (SEQ ID NO: 6)
  • the obtained fragment was enzymatically digested with SpeI and NotI (both Roche Diagnostics, Japan), and then electrophoresed on an agarose gel to cut out the band.
  • QIAquick Gel Extraction Kit Qiagen, Japan
  • the hsALK1Fc high expression strain was prepared by linearizing the expression vector prepared above using Sfi-I restriction enzyme and then converting the linearized DNA to Lipofectamine TM LTX. Using a reagent (Invitrogen, Japan), the gene was introduced into CHO Ras clone I cells according to the package insert. Twenty-four hours after gene transfer, drug-selected cells were selected using alpha MEM containing 6 ⁇ g / ml puromycin and 10% serum (FCS), and single clones were isolated by limiting dilution. It was.
  • Human IgG ELISA kit purchased from Japan Cosmo Bio Co., Ltd. was used for selection of high expression strains. Next, the obtained high expression strain was inoculated into a large flask of 225 cm 2 and cultured at 37 ° C. until it became confluent. After confirming that the cells were confluent, the cells were washed with PBS (Dulecco's Phosphate Buffered Saline; SIGMA), and then replaced with EX-Cell 302 (SAFC Biosciences) medium diluted to 25% in D / F medium. did.
  • PBS Dulecco's Phosphate Buffered Saline
  • EX-Cell 302 SAFC Biosciences
  • the pretreated culture supernatant was applied to a column equilibrated with PBS, washed with a PBS solution containing PBS and 1.85 M NaCl, and then eluted with 20 mM sodium citrate, 50 mM NaCl, pH 2.
  • the target protein was eluted using 7).
  • AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used.
  • the eluate was subjected to sample concentration while substituting the solvent with PBS using an ultrafiltration membrane VIVASPIN20 10,000 MWCO PES (Sartorius Stedim Japan, Japan). After concentration, sterilization was performed using a 0.22 ⁇ m filter (Millex GV; Japan Millipore, Japan).
  • hsALK1Fc activity measurement of purified hsALK1Fc protein in vitro For evaluation of hsALK1Fc activity, a cell line into which a reporter plasmid capable of detecting a BMP signal was stably introduced was used. Specifically, a reporter plasmid (p (GCCG) 12-Luc / neo) that can detect a BMP signal is introduced into a human liver cancer cell line HepG2 (available from ATCC), and the reporter plasmid is introduced. A stably transfected strain HepG2 (p (GCCG) 12-Luc / HepG2 (38-5)) was prepared.
  • p (GCCG) 12-Luc / HepG2 available from ATCC
  • the BMP signal detection reporter plasmid (p (GCCG) 12-Luc / neo) was linked in tandem with 12 binding sequences of Smad1 / 5/8, which is a BMP signaling factor, upstream of the luciferase gene.
  • Plasmid (p (GCCG) 12-Luc / neo) was obtained from Mol. Biol. Cell. 2000, 11 (2): 555-65. Were prepared according to the method described by Kusanagi et al. The neutralization activity of purified hsALK1Fc against BMP9 was measured as follows.
  • EBM endothelial basic medium-2
  • EBM containing 10 ng / ml BMP9 protein purchased from R & D Systems
  • registered registered medium containing only EBM (registered trademark) -2
  • n 2.
  • the supernatant was removed, washed carefully twice with PBS, and RNA was obtained according to the package insert of RNeasy mini kit (Qiagen, Japan). The obtained RNA was sent to Moritex Co., Ltd.
  • FIG. 2 The values in FIG. 2 are obtained by calculating the gene expression change (fold) due to the addition of BMP9 with the value of the BMP9 unstimulated sample as 1.
  • FIG. 2 it was revealed that the addition of BMP9 to vascular endothelial cells strongly induced expression of E-selectin and VCAM-1 which are markers for vascular endothelial injury (FIG. 2).
  • E-selectin and VCAM-1 which are markers for vascular endothelial injury
  • FIG. 2 strong expression induction of genes such as IL-8 and COX-2, which are considered to be involved in vascular disorders and inflammation, was also observed (FIG. 2).
  • SEQ ID NO: 8 pUSmBMP9 KI vector
  • the polynucleotide sequence from the start codon to the stop codon of the mBMP9 expression unit (the mouse BMP9 signal sequence was replaced with the mouse Ig ⁇ signal sequence [underlined portion] containing the intron region, and the mouse BMP9 pro body sequence was downstream of it.
  • Including 1522 bp, SEQ ID NO: 9) and the amino acid sequence encoded by the cDNA (426 amino acids, the boxed portion indicates the mouse Ig ⁇ signal sequence, SEQ ID NO: 10) are shown below.
  • Mouse Ig ⁇ signal sequence information including an intron region was obtained from the UCSC mouse genome database based on MUSIGKVR1 (accession number K02159) obtained from GenBank.
  • SEQ ID NO: 9 SEQ ID NO: 10
  • a USmBMP9 KI chimeric mouse that expresses mouse BMP9 in a B cell specific manner was prepared according to the examples of the pamphlet of International Publication No. WO 2006/78072. Further, mouse control individuals (control chimeric mice) used in Examples 5 and 6 below were prepared according to the method described in Example 11 of International Publication No. WO 2006/78072.
  • red coloration of mesenteric lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age. 5-1-4.
  • Reddish submandibular lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied.
  • reddening of the submandibular lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age. 5-1-5.
  • Reddish elbow lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied.
  • pancreas reddening 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, 2 individuals at 3 weeks of age, 5 individuals at 4 weeks of age, and 4 individuals at 5 weeks of age, redness was observed on a part of the pancreas surface. From the above results, it is clarified that the excessive action of BMP9 causes reddening of ascites, pleural effusion, lymphoid tissue, small intestine and pancreas, which is thought to be caused by hemorrhagic changes. It was confirmed not only in vitro but also in vivo. 5-2.
  • Vascular permeability test of USmBMP9 KI chimeric mouse Evans Blue (L.S., Inc., Japan) was prepared according to the method described in Example 4 and the control chimeric mouse and USmBMP9 KI chimeric mouse that reached 5 weeks of age. I. At a dose of 30 mg / kg. v. Administered. Sixty minutes later, the administered individuals were necropsied and vascular permeability was evaluated using ascites, pleural effusion and organ properties / changes as indicators. As a result, blue-colored ascites and pleural effusion were observed only in the USmBMP9 KI chimeric mice administered with Evans Blue.
  • N-terminal His-type mBMP9 complex recombinant 7-1 Construction of N-terminal His-type mBMP9 complex recombinant expression vector 7-1-1. Construction of pLN1V5 vector Sense oligo DNA having a BamHI / NheI / SalI site at the 5 ′ end and an XhoI site at the 3 ′ end (V5 tag + Stop codon) and its antisense oligo DNA: V5S and V5AS (Hokkaido System Science Co., Ltd., Japan) Company).
  • V5S GATCCGCTAGCGTCGACGGGTAAGCCCTCCTCAACCCTCTCCTGCGTCTCGATCTCGTGGAC (SEQ ID NO: 11)
  • V5AS TCGAGTCACGTAGAGATCGAGACCGAGGAGAGGGTTTAGGATAGGGCTTACCGTCGACGCTAGCG (SEQ ID NO: 12)
  • the synthetic oligo DNA was introduced into the BamHI-XhoI site on the pLN1 vector described in a report by Kakeda et al. (Gene Ther. 12: 852-856, 2005) to construct a pLN1V5 vector. 7-1-2.
  • Synthesis of mBMP9 DNA fragment having His tag sequence at N terminus PCR primer for adding His tag to N terminus of mouse full length sequence of BMP9 (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16)
  • a reaction solution was prepared according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan), SEQ ID NOS: 13 and 14, 10 pmol of each of two primers in a 50 ⁇ l reaction solution, mouse BMP9-cDNA (as a template) SEQ ID NO: 7) was added and incubated at 94 ° C. for 5 minutes, followed by 25 cycles of amplification at 98 ° C.
  • the resulting 101 bp amplified fragment was amplified Separated and recovered with 0.8% gel.
  • An amplified fragment (NheI His mBMP9) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • a reaction solution was prepared according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan), SEQ ID NOS: 15 and 16, 10 pmol of each of the two primers in a 50 ⁇ l reaction solution, mouse BMP9-cDNA (as a template) SEQ ID NO: 7) was added and incubated at 94 ° C. for 5 minutes, followed by 25 cycles of amplification at 98 ° C. for 10 seconds, 57 ° C. for 5 seconds, and 72 ° C. for 1 minute 20 seconds. The resulting 1249 bp amplified fragment was amplified Separated and recovered with 0.8% gel.
  • the amplified fragment (His mBMP9 SalI) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the DNA amplified fragments (NheI His mBMP9) and (His mBMP9 SalI) obtained by the above two PCRs were added to PrimeSTAR buffer to a total volume of 100 ⁇ l, heated to 100 ° C. for 10 minutes, returned to room temperature, and the His tag region was removed. Annealed. Thereafter, SEQ ID NOs: 13 and 16, 2 ps of each primer, 10 pmol each, Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan) were added, an extension reaction was performed at 72 ° C.
  • the enzyme-treated fragment was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the obtained enzyme-treated fragment was introduced into the NheI / SalI site of the pLN1V5 vector prepared in Example 7-1-1 to construct an N-terminal His-type mBMP9 complex recombinant expression vector (FIG. 3).
  • SEQ ID NO: 17 The following is a polynucleotide sequence (1305 bp, SEQ ID NO: 17) from the start codon to the stop codon of the N-terminal His-type mBMP9 complex recombinant cDNA, and an amino acid sequence (434 amino acids, including the signal sequence of mBMP9 encoded by the cDNA) SEQ ID NO: 18) is shown.
  • the underlined portion represents the signal sequence portion of mouse BMP9
  • the boxed portion represents the histidine tag portion
  • italic represents the mouse BMP9 pro body portion.
  • SEQ ID NO: 17 SEQ ID NO: 18 (including mBMP9 signal sequence (underlined portion)) 7-2.
  • N-terminal His type mBMP9 complex using N-terminal His type mBMP9 complex recombinant expression vector 7-2-1.
  • the N-terminal His-type mBMP9 complex recombinant expression vector obtained in 1) was introduced into E. coli DH5 ⁇ , and DNA was prepared from the resulting transformant using a plasmid purification kit (Qiagen plasmid Maxi kit; Qiagen, Japan). . 7-2-2.
  • Free style 293F cells (Invitrogen, Japan) were used with Free style 293 Expression Medium (Invitrogen, Japan) at 37 ° C, 5% CO 2 , 125 rpm, cell density Were cultured in the range of 1 ⁇ 10 5 to 3 ⁇ 10 6 cells / ml.
  • 35 ml of Opti-MEM I Reduced Serum Medium (Invitrogen, Japan) was added to 1 mg of the expression vector, and 33 ml of 1.3 ml of 293fectin Transfection Reagent (Invitrogen, Japan) was added.
  • Opti-MEM I Reduced Serum Medium 7 ml of Opti-MEM I Reduced Serum Medium was added and incubated at room temperature for 5 minutes each. After incubation, the two solutions were mixed and incubated for another 20-30 minutes at room temperature. Thereafter, the expression vector treated by the above method was added to a medium containing 1 ⁇ 10 9 cells / L Free style 293F cells and cultured for 3 days. 7-3. Purification and preparation of N-terminal His-type mBMP9 complex 7-3-1. Pretreatment of culture supernatant After culture, the supernatant was collected, filtered through a 0.22 ⁇ m filter (0.22 ⁇ m GP Express Membrane 500 ml; Japan Millipore, Japan), and then cooled at 4 ° C. (cold room).
  • Example 7-3 The operation was performed in a clean bench as much as possible. All the processes performed in Example 7-3 were performed in a low temperature room (+ 4 ° C.) or on ice, except for work on a clean bench. SDS-PAGE (CBB staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and a small amount under reducing conditions. Pro monomer was detected (FIG. 4). From this result, it was considered that the purified preparation prepared by the above operation mainly contains a complex (complex body) in which two molecules of pro-region and one molecule of molecule dimer are bound.
  • CBB staining SDS-PAGE (CBB staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and
  • Endotoxin-containing PBS was intravenously administered as a control solution to two rats implanted with a transmitter and reached 11 weeks of age, and the telemetry system Dataquest A.
  • R. T. T. was used to measure systolic blood pressure, diastolic blood pressure, mean blood pressure and heart rate, and it was confirmed that there was no blood pressure fluctuation by administration of endotoxin-containing PBS.
  • the N-terminal His-type mBMP9 complex body prepared by the method of Example 7 was intravenously administered so as to have a dosage of 0.5 mg / kg. Minimum blood pressure, mean blood pressure and heart rate were measured.
  • SEQ ID NO: 20 pUShBMP9 KI vector
  • the polynucleotide sequence from the start codon to the stop codon of the hBMP9 expression unit (the human BMP9 signal sequence was replaced with the mouse Ig ⁇ signal sequence [underlined portion] containing the intron region, and the human BMP9 pro body sequence was downstream of it.
  • Including 1525 bp, SEQ ID NO: 21) and the amino acid sequence encoded by the cDNA (427 amino acids, the boxed portion indicates the mouse Ig ⁇ signal sequence, SEQ ID NO: 22) are shown below.
  • Mouse Ig ⁇ signal sequence information including an intron region was obtained from the UCSC mouse genome database based on MUSIGKVR1 (accession number K02159) obtained from GenBank.
  • Serum biochemical test in UShBMP9 KI chimeric mice Using the serum collected from the posterior vena cava of control chimera mice and UShBMP9 KI chimeric mice, which were prepared according to the method described in Example 9 above, Blood biochemistry analysis was performed with an analyzer 7180 (manufactured by HITACHI). As a result, it was found that the serum LDH, BUN, LDL cholesterol, GOT, and sodium values of the UShBMP9 KI chimeric mice were significantly higher than those of the control chimeric mice (FIG. 6). In addition, a significant decrease in serum albumin and serum total protein was also observed (FIG. 6).
  • BUN is a diagnostic marker for kidney disease and is associated with chronic nephritis, edema, obstructive urinary tract disease, LDL cholesterol is dyslipidemia and arteriosclerotic disease (angina, acute coronary syndrome, myocardial infarction, Serum LDH and GOT are associated with acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, acute myocardial infarction, heart failure, serum sodium It is known that an increase is associated with hypertension, and a decrease in serum albumin and serum total protein is known to be associated with liver dysfunction and nephrotic syndrome. It was suggested.
  • anti-BMP9 therapy is chronic nephritis, edema, obstructive urinary tract disease, dyslipidemia, arteriosclerotic disease, acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, acute myocardial infarction, heart failure It has been shown that it may be an effective treatment for diseases such as nephrotic syndrome and hypertension.
  • N-terminal His-type hBMP9 complex recombinant 12-1 Construction of N-terminal His-type hBMP9 complex recombinant expression vector 12-1-1. Synthesis of hBMP9 DNA fragment having His tag sequence at N-terminus PCR primer for adding His tag to N-terminus of full-length human BMP9 sequence (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26) Prepare a reaction solution according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan).
  • An amplified fragment (His hBMP9 XhoI) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the DNA amplified fragments (NheI His hBMP9) and (HisBMP9 XhoI) obtained by the above two PCRs were added to PrimeSTAR buffer to a total volume of 100 ⁇ l, heated to 100 ° C. for 10 minutes, returned to room temperature, and the His tag region was removed. Annealed. Thereafter, SEQ ID NOs: 23 and 26, 10 pmol each of two kinds of primers, Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan) were added, an extension reaction was performed at 72 ° C.
  • the enzyme-treated fragment was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the obtained enzyme-treated fragment was introduced into the NheI / XhoI site of the pLN1V5 vector to construct an N-terminal His-type hBMP9 complex recombinant expression vector (FIG. 7).
  • SEQ ID NO: 27 The following is a polynucleotide sequence (1308 bp, SEQ ID NO: 27) from the start codon to the stop codon of the N-terminal His-type hBMP9 complex recombinant cDNA, and an amino acid sequence (435 amino acids, including the signal sequence of hBMP9 encoded by the cDNA) SEQ ID NO: 28).
  • the underlined portion indicates the signal sequence portion of human BMP9
  • the surrounded line indicates the histidine tag portion
  • the italic type indicates the human BMP9 pro body portion.
  • SEQ ID NO: 27 SEQ ID NO: 28 (including hBMP9 signal sequence [ underlined portion ]): 12-2.
  • N-terminal His-type hBMP9 complex using N-terminal His-type hBMP9 complex recombinant expression vector 12-2-1.
  • the N-terminal His-type hBMP9 complex recombinant expression vector obtained in (1) was introduced into Escherichia coli DH5 ⁇ , and DNA was prepared from the obtained transformant using a plasmid purification kit (Qiagen plasmid Maxi kit; Qiagen, Japan). . 12-2-2.
  • Free style 293F cells (Invitrogen, Japan) were used with Free style 293 Expression Medium (Invitrogen, Japan) at 37 ° C, 5% CO 2 , 125 rpm, cell density Is cultured in the range of 1 ⁇ 10 5 to 3 ⁇ 10 6 cells / ml.
  • Opti-MEM I Reduced Serum Medium (Invitrogen, Japan) was added to 1 mg of the expression vector, and 33.7 ml in 1.3 ml of 293fectin Transfection Reagent (Invitrogen, Japan). Of Opti-MEM I Reduced Serum Medium was added and incubated at room temperature for 5 minutes each.
  • PBS Dulecco's Phosphate Buffered Saline; SIGMA
  • a buffer PBS containing 0.5 M Imidazole
  • B buffer PBS containing 0.5 M Imidazole
  • the pretreated 1 L culture supernatant was applied to a Ni Sepharose column (His Trap HP 5 ml; GE Healthcare Biosciences, Japan) equilibrated with PBS. Thereafter, the column was washed in the order of 25 ml of 0% B buffer, 25 ml of buffer solution in which NaCl was added to PBS to adjust the NaCl concentration to 1.85 M, 25 ml of 0% B buffer, 30 ml of 9% B buffer, and 40 ml of 11% B buffer.
  • Example 12-3-2 Ion Exchange Chromatography A buffer was prepared by adding NaCl to PBS as A buffer and adjusting NaCl concentration to 1.85 M by adding NaCl to PBS as B buffer. The purpose obtained in Example 12-3-2 on a strong anion exchange column equilibrated with PBS (Hi Trap Q HP 1 mL; GE Healthcare Biosciences, Japan) under a flow rate of 1 mL / min Protein was added.
  • the column was washed sequentially with 20 ml of 0% B buffer and 10 ml of 2% B buffer. After the washing operation, 20 ml of 7% B buffer was applied to the column, and the target protein was recovered.
  • AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used. Endotoxin removal treatment was performed before use. 12-3-4.
  • Purified sample preparation After replacing the solvent in the purified sample obtained in Example 12-3-3 with PBS using an ultrafiltration membrane VIVASPIN20 10,000 MWCO PES (Sartorius Stedim Japan, Japan), The sample was concentrated. After the concentration operation, filtration was performed with a 0.22 ⁇ m filter (Millex GV; Japan Millipore Corporation, Japan).
  • Example 12-3 The operation was performed in a clean bench as much as possible. All steps performed in Example 12-3 were performed at 4 ° C. (cold room or on ice) except for work on a clean bench. SDS-PAGE (silver staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and a small amount under reducing conditions. Pro monomer was detected (FIG. 8). From this result, it was considered that the purified preparation prepared by the above operation mainly contains a complex (complex body) in which two molecules of pro-region and one molecule of molecule dimer are bound.
  • SDS-PAGE silver staining
  • BMP9 activity was evaluated according to the method described in Example 2.
  • the purified hBMP9 complex body increased Luciferase activity in a dose-dependent manner, and was confirmed to have BMP ligand activity.
  • mice N-terminal His-type hBMP9 complex administration test to mice (in vivo test)
  • Each group composition is as follows.
  • each administration solution was administered into the tail vein at Day 1, Day 3, Day 5, and Day 8, and whole blood was collected from the posterior vena cava at Day 9 and prepared as serum.
  • blood biochemistry analysis was performed using the obtained serum with an automatic analyzer 7180 (manufactured by HITACHI), and a biomarker survey was conducted by entrusting it to Charles River Japan Co., Ltd.
  • RODENTS MULTI-ANALYTE PROFILES Rost MAP V2.0 Plasma Antigen
  • LMP cholesterol increased, pancreatic lipase (LIP) decreased, and serum albumin decreased by BMP9 administration in the 100 ⁇ g / head administered group (3), and all of these fluctuations were controlled by (5) It was significant compared to the group (FIG. 9).
  • Increased LDL cholesterol was associated with dyslipidemia and arteriosclerosis, and decreased serum albumin was associated with nephrotic syndrome, severe liver disease, malnutrition, protein-losing gastroenteropathy, and inflammation.
  • LIP Reduced lipase
  • the biomarkers are known to be associated with the following diseases.
  • anti-BMP9 therapy includes thrombosis, arteriosclerotic disease (acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.), various renal diseases (chronic kidney disease, acute kidney injury, glomerulus).
  • arteriosclerotic disease acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.
  • various renal diseases chronic kidney disease, acute kidney injury, glomerulus.
  • Nephritis hypertension
  • vascular disorder diseases autoimmune diseases such as systemic lupus erythematosus (SLE), Kawasaki disease, vasculitis syndrome, sepsis, etc.
  • allergic diseases allergic rhinitis, asthma, atopic dermatitis, etc.
  • Various inflammatory diseases delayed allergy, rheumatoid arthritis, arthritis, lung disease, hepatitis, acute neutrophilic inflammation, etc.
  • heart diseases hypertensive heart hypertrophy, cardiomyopathy, viral cardiomyopathy, etc.
  • Potential for treatment of cancer Alzheimer's, multiple sclerosis, abnormal angiogenesis in Retinopathy of diabetics, Raynaud's syndrome, and Crohn's disease.
  • the dose was administered intraperitoneally to achieve an amount.
  • administration was carried out 6 times in total including Day1, Day3, Day5, Day8, Day10, and Day13.
  • the blood collected from the orbit was subjected to hematological examination using Bayer ADVIA (registered trademark) 120 Hematology System Analyzer and biomarker investigation was performed using serum collected from the posterior vena cava.
  • biomarker survey RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen) was commissioned to Charles River Japan Co., Ltd. and conducted.
  • hematological examination revealed that the administration of BMP9 neutralizing antibody significantly decreased the number of platelets related to thrombus formation and inflammation by about 25% (FIG. 11).
  • CD40 ligand, CRP, EGF, Endothelin-1, Factor VII, Haptoglobin, IL-1alpha, IL-5, MCP-1, MCP-3, MCP-5, and BMP9 neutralizing antibody were administered.
  • Biomarkers such as MIP-1 beta, MIP-1 gamma, MMP-9, Tissue Factor, TNF-a, TIMP-1, VEGF, RANTES, Lymphoactin, Eotaxin, GCP-2 (Granulocyte Chemical Protein-2) (Fig. 12).
  • biomarkers shown in Example 14 that were confirmed to be increased by administration of recombinant BMP9 were decreased by anti-BMP9 therapy using a BMP9 neutralizing antibody.
  • the biomarkers are known to be associated with the following diseases.
  • anti-BMP9 therapy includes various thrombosis, arteriosclerotic diseases (acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.), various renal diseases (chronic kidney disease, acute kidney injury, thread) Sphere nephritis), hypertension, vascular disorder diseases (autoimmune diseases such as systemic lupus erythematosus (SLE), Kawasaki disease, vasculitis syndrome, sepsis), allergic diseases (allergic rhinitis, airway hypersensitivity, asthma, atopy) Dermatitis, etc.), various inflammatory diseases (delayed allergy, rheumatoid arthritis, arthropathy, lung disease, hepatitis, acute neutrophilic inflammation, ulcerative colitis, etc.), heart disease (hypertensive cardiac hypertrophy, cardiomyopathy) , Chronic heart failure such as viral cardiomyopathy), cancer, diabetic retinopathy, Raynaud's syndrome, and the
  • hsALK1mFc expression vector is a PEAK8 expression vector obtained by enzymatic digestion of the extracellular region cDNA fragment of human ALK1 and the Fc region cDNA fragment of mIgG1 with EcoRI and NotI (both from Roche Diagnostics, Japan). It was prepared by incorporating into (Edge Biosystems).
  • the cDNA sequence of the incorporated hsALK1mFc is as follows (SEQ ID NO: 35).
  • the amino acid sequence encoded by SEQ ID NO: 35 (346 amino acids, SEQ ID NO: 36) is shown below.
  • SEQ ID NO: 36 Each fragment was obtained as follows.
  • the extracellular region cDNA of human ALK1 is hsALK1 FW: agaattccccaccatgaccttgtccccccag (SEQ ID NO: 3), hsALK1 RV: aacttagctgggcccatctgtttccccggctg (SEQ ID NO: 4) was cloned from a human lung cDNA library using the PCR method. The obtained fragment was enzymatically digested with EcoRI and SpeI, and then electrophoresed on an agarose gel to cut out the band.
  • the Fc region cDNA of mIgG1 is mIgG1 FP: acttagcctaaggtcacgtgtgtgt (SEQ ID NO: 37)
  • mIgG1 RP gcggccgcttattattattaccagaggaggggg (SEQ ID NO: 38)
  • the obtained fragment was enzymatically digested with SpeI and NotI (both Roche Diagnostics, Japan), and then electrophoresed on an agarose gel to cut out the band.
  • hsALK1mFc high expression strain was prepared by linearizing the expression vector prepared above using Sfi-I restriction enzyme and then converting the linearized DNA to Lipofectamine TM LTX. Using a reagent (Invitrogen, Japan), the gene was introduced into CHO Ras clone I cells according to the package insert.
  • the cells were washed with PBS (Dulecco's Phosphate Buffered Saline; SIGMA), and then the EX-Cell 302 (SAFC Biosciences) medium was diluted to 25% with the DMEM / F-12 mixed medium. The medium was replaced. After culturing for 5 to 7 days, the culture supernatant was collected, filtered through a 0.22 ⁇ m filter (0.22 ⁇ m GP Express Membrane 500 ml; Japan Millipore, Japan), and then at 4 ° C. (cold room). Cooled down. When stored frozen, it was re-filtered through a 0.22 ⁇ m filter after thawing. 16-3.
  • PBS Dulecco's Phosphate Buffered Saline
  • SIGMA Dulecco's Phosphate Buffered Saline
  • SAFC Biosciences SAFC Biosciences
  • a column of RESOURCE S 6 ml (manufactured by GE Healthcare) is connected to AKTA Explorer, 20 mM MES (pH 5.8) is used as the A1 solution, and 20 mM MES (pH 5.8) containing 1 M NaCl is used as the B1 solution.
  • the ProSep eluate diluted 4 to 10 times with the A1 solution was filtered through a 0.22 ⁇ m filter and applied to the column. After the application, A1 was fed, and after confirming that OD280nm returned to the baseline, the elution program was started.
  • 0.1M sodium phosphate (pH 7.2) containing 2M (NH 4 ) 2 SO 4 was used for the A2 solution
  • 0.1M sodium phosphate (pH 7.2) was used for the B2 solution.
  • the B2 concentration was increased from 50 to 90% and eluted in 20 columns. Each eluted fraction was subjected to SDS-PAGE under non-reducing conditions and then subjected to silver staining, and the fraction containing only dimer was selected and mixed.
  • the ammonium sulfate concentration in the mixture of the 3rd chromatographic fraction was estimated on the same column (RESOURCE PHE) from the electrical conductivity, and the final concentration of ammonium sulfate exceeded 1M.
  • A2 was added and then applied to the column. After the sample was applied, the ammonium sulfate concentration was lowered to 100 to 200 mM at once, stepwise elution was performed, and the sample was collected at 0.5 mL / tube.
  • the fraction containing the purified protein was selected and mixed based on the absorbance at OD 280 nM, and then the buffer was replaced with PBS using NAP-25 (manufactured by GE Healthcare), followed by filter sterilization.
  • the purified hsALK1mFc protein showed a mobility of slightly over 50 kDa under reduction and 90-95 kDa under non-reduction, and was confirmed to be a highly purified protein (FIG. 13).
  • This SHR / NDmc-cp (cp / cp) is known as a spontaneously hypertensive rat with obesity, and is also known as an animal that causes cardiac hypertrophy with hypertension (Metabolism 47, 1199-1204 (1998)).
  • the breeding feed was changed from FR-2 to a special feed (Nippon Nosan Kogyo Co., Ltd.) based on Lab H Standard containing 20% casein, 20% shoecloth, and 8% lard.
  • urine was collected in a metabolic cage for about 24 hours, and then blood was collected from the tail vein.
  • the group composition includes PBS administration group (vehicle administration group), BMP9 neutralizing antibody 0.04 mg / kg administration group, BMP9 neutralizing antibody 0.2 mg / kg administration group, and SHR / NDmc ⁇ as a negative control.
  • PBS administration group vehicle administration group
  • BMP9 neutralizing antibody 0.04 mg / kg administration group
  • BMP9 neutralizing antibody 0.2 mg / kg administration group
  • SHR / NDmc ⁇ as a negative control.
  • One group of cp (+ / +) was set.
  • human BMP9 neutralizing antibody (R & D System, Inc, Clone NO .: 360107) diluted with PBS to 0.10 mg / mL and 0.020 mg / mL was prepared at a dose of 2 mL / kg.
  • the weight was calculated based on the body weight before administration and administered intraperitoneally.
  • PBS was intraperitoneally administered to SHR / NDmc-cp (+ / +) which is control. Thereafter, the test substance was administered once a week. The body weight was measured once a week or twice a week, and blood was collected from the tail vein every other week from one week after administration of the test substance.
  • BMP9 neutralizing antibody had no effect on kidney weight, but suppressed heart hypertrophy in a dose-dependent and significant manner (test by Kruskal-Wallis test + Steel test) for heart weight.
  • the neutralizing antibody for BMP9 is effective for cardiac hypertrophy, which is a problem in heart disease, that is, it may be a therapeutic agent for heart disease.
  • a physiological saline administration group (vehicle administration group), a BMP9 neutralizing antibody administration group, and an hsALK1mFc administration group
  • one group of C57BL / 6N was set as a negative control.
  • test substances saline, BMP9 neutralizing antibody, hsALK1mFc
  • physiological saline was intraperitoneally administered to C57BL / 6 mice as controls.
  • the doses of the BMP9 neutralizing antibody and hsALK1mFc were 1 mg / kg and 0.1 mg / kg, respectively.
  • the breeding feed was changed to a Western diet (F2WTD) (produced by Oriental Yeast Co., Ltd.), which is known to easily induce arteriosclerosis after grouping.
  • F2WTD Western diet
  • BMP9 neutralizing antibody a product purchased from R & D (Clone: 360107) was used, and both the BMP9 neutralizing antibody and hsALK1mFc were prepared in a normal saline solution. Thereafter, test substances (physiological saline, BMP9 neutralizing antibody, hsALK1mFc) were administered intraperitoneally at a frequency of once a week. Blood was collected over time every 2 weeks or 4 weeks after the start of test substance administration.
  • Blood biochemical analysis was performed with an automatic analyzer (Hitachi 7170) using reagents sold by Kyowa Medex Co., Ltd.
  • the whole blood was collected from the abdominal vena cava under isoflurane anesthesia, and then the aorta in the region from directly under the heart to the lower descending aorta was removed.
  • the aorta is cut longitudinally from the anterior and posterior sides with scissors and then cut into 10% formalin solution. Soaked and fixed overnight.
  • the fixed aorta was washed twice with PBS and once with 60% isopropanol, and then immersed in 1.8 mg / mL oil red O staining solution overnight. The next day, after washing once with 60% isopropanol and twice with PBS, excess fat adhering to the outside of the aorta was removed using tweezers and scissors under an optical microscope. Used for image analysis. In the image analysis, the total area of the extracted aorta and the area stained with Oil Red O were calculated, and the plaque area ratio in each sample was calculated by dividing the area stained with Oil Red O by the total area of the aorta.
  • the plaque formation inhibition rate (%) in the hsALKmFc and neutralizing antibody administration group was calculated by the following formula. ((Plaque area rate in the APOE-deficient mouse physiological saline administration group) ⁇ (Plaque area rate in the APOE-deficient mouse test substance administration group)) ⁇ ((Plaque area rate in the APOE-deficient mouse physiological saline administration group)) — (C57BL / 6 plaque area ratio in saline administration group)) X100
  • plaque formation in APOE-deficient mice was suppressed by about 32% by hsALKmFc administration and by about 15% by BMP9 neutralizing antibody administration (FIG. 16).
  • ALK1 antagonists containing BMP9 neutralizing antibody The possibility of being effective was found.
  • the diastolic blood pressure, systolic blood pressure, and heart rate of a rat implanted with a transmitter and reaching the age of 33 weeks are measured by the telemetry system Dataquest A. R. T. T.
  • the groups were divided into 4 groups so that their values were equal to each group.
  • a BMP9 neutralizing antibody purchased from R & D (Clone: 360107) was used.
  • the antibody dilution medium PBS
  • the test substance such as BMP9 neutralizing antibody and hsALK1mFc was administered intraperitoneally the next day. Blood pressure, systolic blood pressure, and heart rate were measured.
  • BMP9 neutralizing antibody was not observed to have an effect on blood pressure
  • a decrease in diastolic blood pressure and systolic blood pressure was observed from about 14 hours after administration, and the decrease was observed on the 4th day. (FIG. 17). From the above results, it was revealed that hsALK1mFc has a hypotensive action.
  • test substance PBS, BMP9 neutralizing antibody
  • PBS was administered to negative control rats.
  • test substance was administered once a week.
  • a BMP9 neutralizing antibody purchased from R & D (Clone: 360107) was used.
  • the body weight was measured once or twice a week, and blood was collected from the tail vein at Day 15, Day 22, Day 29, Day 35 after administration of the anti-GBM antibody.
  • whole blood was collected from the abdominal aorta under isoflurane anesthesia, and then the kidney was removed and the kidney weight was measured.

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Abstract

Cette invention concerne l'utilisation/l'usage d'un inhibiteur d'ALK1 (activin receptor-like kinase) dans une composition pharmaceutique pour prévenir les maladies vasculaires. L'invention concerne spécifiquement une composition pharmaceutique prévenant les maladies vasculaires et contenant un inhibiteur d'ALK1 comme principe actif ; et une méthode de traitement des maladies vasculaires utilisant cette composition pharmaceutique.
PCT/JP2010/057924 2009-04-30 2010-04-28 Composition pharmaceutique utilisée en prévention des maladies vasculaires et comprenant un inhibiteur d'alk1 comme principe actif WO2010126169A1 (fr)

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CN102444469A (zh) * 2011-12-28 2012-05-09 上海交通大学 涡轮入口面积可调式涡轮增压系统
CN102562270A (zh) * 2011-12-28 2012-07-11 上海交通大学 涡轮入口面积自适应式涡轮增压系统
WO2014007198A1 (fr) 2012-07-02 2014-01-09 協和発酵キリン株式会社 Agent thérapeutique à base d'anticorps anti-bmp9 en tant que principe actif, pour le traitement de l'anémie, notamment une anémie rénale et une anémie liée à un cancer
WO2016193872A3 (fr) * 2015-06-05 2017-03-30 Novartis Ag Anticorps ciblant la protéine morphogénétique osseuse 9 (bmp9) et méthodes associées
WO2018144968A1 (fr) * 2017-02-06 2018-08-09 Acceleron Pharma Inc. Compositions et méthodes de traitement d'une insuffisance cardiaque
WO2019117208A1 (fr) 2017-12-12 2019-06-20 協和発酵キリン株式会社 Anticorps anti-ｂｍｐ１０, et agent thérapeutique contre l'hypertension et les maladies d'hypertension ayant pour principe actif cet anticorps
CN110389231A (zh) * 2018-04-19 2019-10-29 北京市心肺血管疾病研究所 支链氨基酸检测物在制备主动脉夹层患者术后预后风险评估试剂盒中的应用
TWI825072B (zh) * 2019-02-13 2023-12-11 日商協和麒麟股份有限公司 抗bmp10抗體及以該抗體為有效成份之針對高血壓及高血壓性疾病之治療劑
CN117385024A (zh) * 2023-11-29 2024-01-12 梅州市人民医院(梅州市医学科学院) 一种lncRNA标志物及其在制备诊断、筛查或评估急性冠状动脉综合征的产品中的应用

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CN102444469A (zh) * 2011-12-28 2012-05-09 上海交通大学 涡轮入口面积可调式涡轮增压系统
CN102562270A (zh) * 2011-12-28 2012-07-11 上海交通大学 涡轮入口面积自适应式涡轮增压系统
WO2014007198A1 (fr) 2012-07-02 2014-01-09 協和発酵キリン株式会社 Agent thérapeutique à base d'anticorps anti-bmp9 en tant que principe actif, pour le traitement de l'anémie, notamment une anémie rénale et une anémie liée à un cancer
US8969040B2 (en) 2012-07-02 2015-03-03 Kyowa Hakko Kirin Co., Ltd Pharmaceutical agent comprising anti-BMP9 antibody as active ingredient for treatment of anemia such as renal anemia and cancer anemia
JPWO2014007198A1 (ja) * 2012-07-02 2016-06-02 協和発酵キリン株式会社 抗ｂｍｐ９抗体を有効成分とする、腎性貧血、がん性貧血などの貧血に対する治療剤
JP2018522540A (ja) * 2015-06-05 2018-08-16 ノバルティス アーゲー 骨形成タンパク質９（ｂｍｐ９）を標的とする抗体およびそれらのための方法
US10047155B2 (en) 2015-06-05 2018-08-14 Novartis Ag Antibodies targeting bone morphogenetic protein 9 (BMP9) and methods therefor
WO2016193872A3 (fr) * 2015-06-05 2017-03-30 Novartis Ag Anticorps ciblant la protéine morphogénétique osseuse 9 (bmp9) et méthodes associées
WO2018144968A1 (fr) * 2017-02-06 2018-08-09 Acceleron Pharma Inc. Compositions et méthodes de traitement d'une insuffisance cardiaque
JP7323456B2 (ja) 2017-12-12 2023-08-08 協和キリン株式会社 抗ｂｍｐ１０抗体及び該抗体を有効成分とする、高血圧および高血圧性疾患に対する治療剤
WO2019117208A1 (fr) 2017-12-12 2019-06-20 協和発酵キリン株式会社 Anticorps anti-ｂｍｐ１０, et agent thérapeutique contre l'hypertension et les maladies d'hypertension ayant pour principe actif cet anticorps
US11970531B2 (en) 2017-12-12 2024-04-30 Kyowa Kirin Co., Ltd. Methods for treating hypertension using an anti-BMP10 monoclonal antibody or fragment thereof
JPWO2019117208A1 (ja) * 2017-12-12 2020-12-17 協和キリン株式会社 抗ｂｍｐ１０抗体及び該抗体を有効成分とする、高血圧および高血圧性疾患に対する治療剤
US11485779B2 (en) 2017-12-12 2022-11-01 Kyowa Kirin Co., Ltd. Anti-BMP10 monoclonal antibody or fragment thereof
CN110389231B (zh) * 2018-04-19 2022-09-06 北京市心肺血管疾病研究所 支链氨基酸检测物在制备主动脉夹层患者术后预后风险评估试剂盒中的应用
CN110389231A (zh) * 2018-04-19 2019-10-29 北京市心肺血管疾病研究所 支链氨基酸检测物在制备主动脉夹层患者术后预后风险评估试剂盒中的应用
TWI825072B (zh) * 2019-02-13 2023-12-11 日商協和麒麟股份有限公司 抗bmp10抗體及以該抗體為有效成份之針對高血壓及高血壓性疾病之治療劑
CN117385024A (zh) * 2023-11-29 2024-01-12 梅州市人民医院(梅州市医学科学院) 一种lncRNA标志物及其在制备诊断、筛查或评估急性冠状动脉综合征的产品中的应用
CN117385024B (zh) * 2023-11-29 2024-04-19 梅州市人民医院(梅州市医学科学院) 一种lncRNA标志物及其在制备诊断、筛查或评估急性冠状动脉综合征的产品中的应用

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