WO2018107693A1 - 一种预防和治疗系统性硬化症的方法 - Google Patents

一种预防和治疗系统性硬化症的方法 Download PDF

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WO2018107693A1
WO2018107693A1 PCT/CN2017/089053 CN2017089053W WO2018107693A1 WO 2018107693 A1 WO2018107693 A1 WO 2018107693A1 CN 2017089053 W CN2017089053 W CN 2017089053W WO 2018107693 A1 WO2018107693 A1 WO 2018107693A1
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fibrosis
plasminogen
tissue
drugs
tissue organ
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French (fr)
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李季男
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深圳瑞健生命科学研究院有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method of preventing and treating systemic sclerosis comprising administering to a subject an effective amount of plasminogen.
  • Fibrosis is a pathological change characterized by fibroblast activation and proliferation, increased fibrous connective tissue in tissues and organs, and decreased parenchymal cells. Progression can lead to tissue and organ structural damage and loss of function. Fibrosis of vital organs seriously affects the quality of life of patients and even life-threatening. Worldwide, tissue fibrosis is the leading cause of disability and death in many diseases. According to statistics from the United States, about 45% of the country's deaths from various diseases can be attributed to tissue fibroproliferative diseases.
  • Fibrotic diseases include diseases involving multiple systems, such as systemic sclerosis, multifocal fibrosis, scleroderma, renal multisystemic fibrosis, and organ-specific diseases such as skin, heart, lungs, Liver, kidney fibrosis, etc.
  • the causes of different fibrotic diseases are different, such as tissue and organ damage, infection, immune response, chronic inflammation, etc., but their common feature is excessive deposition of extracellular matrix (ECM) in tissues and organ tissue remodeling [1] -3] .
  • ECM extracellular matrix
  • Cardiac fibrosis occurs in the development and progression of a variety of heart diseases, such as viral myocarditis, myocardial infarction, and hypertensive heart disease. Excessive cardiac fibrosis leads to impaired cardiac function, which is the main cause of heart disease in various diseases. Inhibition and reversal of myocardial fibrosis has become an important link in the treatment of cardiovascular diseases.
  • Hepatic fibrosis refers to the pathological process of abnormal proliferation of connective tissue in the liver caused by various pathogenic factors and excessive precipitation of diffuse extracellular matrix in the liver.
  • Various factors can cause liver fibrosis, such as viral infection, inflammatory response, oxidative stress and alcohol abuse.
  • the pathological features of liver fibrosis are a large number of fibrous tissue hyperplasia and deposition in the portal area and hepatic lobules, but no interlobular septa have been formed.
  • the cirrhosis has pseudolobule formation, the central venous area and the portal area are separated, and the normal structure of the liver is affected. To the destruction, liver fibrosis is further developed as cirrhosis.
  • Viral hepatitis is the main cause of chronic liver disease in China.
  • Liver tissue fibrosis of chronic viral hepatitis is related to intrahepatic inflammation, necrosis, viral replication, etc., and is reversible in the early stage.
  • antiviral therapy, adjustment of immune function and protection of liver cells and anti-fibrosis are combined to prevent active liver fibrosis.
  • Pulmonary fibrosis diseases include idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, pneumoconiosis, fibrosis caused by drugs and radiation, and fibrotic alveolitis associated with collagen vascular disease, and a wide range of causes Disease spectrum.
  • the main pathological features include lung tissue mesenchymal cell proliferation, extracellular matrix hyperplasia and remodeling of lung parenchyma.
  • anti-inflammatory, anti-oxidation, anti-fibroblast proliferation and collagen deposition and lung transplantation are mainly used to treat pulmonary fibrosis.
  • Renal fibrosis is a pathological process in which extracellular matrix and inappropriate connective tissue accumulate in the kidney, leading to structural changes and impaired function of the kidney, and is a common pathway for almost all kidney diseases to progress to end-stage renal failure.
  • the process of renal fibrosis involves inflammatory reaction, apoptosis of innate cells and immune cells, and imbalance of various regulatory fibrotic factors, so it can resist renal fibrosis through anti-inflammatory, anti-apoptotic and treatment against fibrotic factors.
  • Chronic lesions of tissues and organs are often accompanied by fibrosis, such as chronic inflammation of the lungs, chronic lesions, and pulmonary fibrosis.
  • liver fibrosis such as hepatitis B, hepatitis C, alcoholic liver, fatty liver, schistosomiasis, etc. are associated with early liver fibrosis.
  • the compensatory function of the liver is very strong.
  • Fibrotic lesions have been lurking in various chronic liver diseases. It is usually discovered when it develops into cirrhosis. In fact, cirrhosis is already a serious stage of liver fibrosis.
  • Others such as chronic nephritis, glomerulonephritis, and renal tubular inflammation have renal fibrosis; vascular fibrosis occurs in cardiovascular, cerebrovascular, lower extremity arteriosclerosis, or narrowing, or obstruction.
  • the scar tissue is the fibrous connective tissue of the aging stage formed by the morphological transformation of the granulation tissue.
  • fibroblasts divide, proliferate, migrate to the damaged site, produce extracellular matrix, form scar tissue, and repair wounds.
  • the formation of scars is a process of progressive fibrosis of granulation tissue.
  • the reticular fibers are collagenized, the collagen fibers are thickened, and at the same time, the fibroblasts are less and less, and a small amount of the remaining fibers are converted into fibroblasts; Absorption, neutrophils, macrophages, lymphocytes and plasma cells disappeared; capillaries closed, degenerated, disappeared, leaving few small arteries and venules.
  • the granulation tissue is transformed into scar tissue which is mainly composed of collagen fibers and has scarce blood vessels, and the human eye is white and the texture is tough.
  • the scar is tough and inelastic, and the scar contraction can cause organ deformation and dysfunction, it occurs in the vicinity of the joint and scars of important organs, often causing joint spasm or limited mobility, such as in the digestive tract, urinary tract and other chambers.
  • Organs cause stenosis of the lumen and cause movement disorders near the joint.
  • Scarring adhesions between organs or between organs and body walls often different Affect its function. If extensive fibrosis or glassy changes occur after extensive damage in an organ, organ hardening is caused.
  • SSc Systemic sclerosis
  • scleroderma is a systemic autoimmune disease characterized by localized or diffuse skin thickening and fibrosis.
  • the lesions are characterized by skin fibrosis and vascular onion skin-like changes, which eventually lead to skin sclerosis and vascular ischemia.
  • the disease is characterized by localized or diffuse skin thickening and fibrosis. In addition to skin involvement, it can also affect the internal organs (heart, lung and digestive tract).
  • Atherosclerosis often leads to ischemic damage to tissues and organs, which in turn causes fibrotic lesions in tissues and organs.
  • Atherosclerosis is a chronic, progressive arterial disease in which some or all of the fat deposited in the arteries blocks blood flow. Atherosclerosis occurs when the smooth, firm arterial intima is roughened, thickened, and blocked by fat, fibrin, calcium, and cellular debris.
  • Atherosclerosis is a chronic inflammatory hyperplasia of the arterial intima, leading to stenosis or occlusion of large and medium-sized arteries, causing ischemic injury, fibrosis, and even necrosis of the corresponding organs.
  • Atherosclerosis is closely related to diabetes, which is characterized by early, severe and poor prognosis of atherosclerosis in diabetic patients, and atherosclerosis is the leading cause of death in diabetic patients. It is found that the pathological changes of coronary artery in diabetic patients are mainly characterized by more blood vessels involved in the lesions, severe coronary stenosis, and more serious lesions. The mechanism is considered to be atherosclerosis caused by abnormal blood glucose metabolism.
  • plasminogen can improve the fibrosis of tissues and organs, improve the function of tissues and organs, and thus open up a brand new field for the prevention and treatment of tissue and organ fibrosis and related diseases.
  • the invention includes the following items:
  • WHAT IS CLAIMED IS 1. A method of preventing and/or treating fibrosis and a related disorder thereof in a systemic sclerosis subject comprising administering to the subject an effective amount of plasminogen.
  • a method of preventing and/or treating collagen deposition or fibrosis of a tissue of a subject and a related condition thereof comprising administering to the subject an effective amount of plasminogen, wherein the subject is susceptible to tissue Fibrosis of organs, tendency to fibrosis of tissues, or other diseases accompanied by fibrosis of tissues and organs.
  • tissue organ collagen deposition or fibrosis comprises cutaneous fibrosis, vascular fibrosis, cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis.
  • tissue organ collagen deposition or fibrosis comprises infection, inflammation, hypersensitivity, tumor, tissue ischemia, tissue organ congestion, chemical, radiation or environmental pollution caused by damage Or accompanying.
  • tissue organ collagen deposition or fibrosis comprises tissue organ collagen deposition or fibrosis resulting from a tissue organ lesion caused by a bacterial, viral or parasitic infection.
  • tissue organ collagen deposition or fibrosis comprises pulmonary fibrosis caused by Mycobacterium tuberculosis infection.
  • tissue organ collagen deposition or fibrosis is liver fibrosis caused by hepatitis B virus, hepatitis C virus or hepatitis E virus infection.
  • tissue organ collagen deposition or fibrosis is liver fibrosis caused by schistosome infection.
  • tissue organ collagen deposition or fibrosis is renal fibrosis caused by chronic glomerulonephritis, pyelonephritis, nephrotic syndrome, renal insufficiency, and uremia.
  • tissue organ collagen deposition or fibrosis is caused by fat causing damage to tissues and organs.
  • tissue organ collagen deposition or fibrosis is lung fibrosis caused by lung cancer, liver fibrosis caused by liver cancer, or renal fibrosis caused by kidney cancer.
  • tissue organ collagen deposition or fibrosis is coronary ischemic fibrosis caused by coronary atherosclerosis or coronary heart disease.
  • tissue organ collagen deposition or fibrosis is renal fibrosis caused by chronic ischemic renal injury.
  • tissue organ collagen deposition or fibrosis is drug-induced liver fibrosis or renal fibrosis.
  • tissue organ collagen deposition or fibrosis is caused by a systemic immune disease such as systemic lupus erythematosus, systemic sclerosis, ankylosing spondylitis.
  • tissue organ fibrosis-related disorder comprises a condition caused by a weakened, impaired or lost function of a tissue organ due to a fibrotic lesion.
  • tissue organ fibrosis-related disorder comprises atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, cerebral ischemia, cerebral infarction, renal dysfunction Total, uremia, liver dysfunction, cirrhosis, hepatic coma, dyspnea, emphysema, pulmonary heart disease, pulmonary fibrosis, ankylosing spondylitis.
  • tissue organ fibrosis or a related disorder thereof reduces fibrosis of the tissue organ, relieves tissue organ fibrosis-related disorders or improves by reducing collagen deposition in tissues and organs The function of the tissue organ.
  • the other drug comprises: a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a liver protection drug, and an anti-fiber Chemical drugs, antiarrhythmic drugs, cardiotonic drugs, diuretic drugs, anti-tumor drugs, radiotherapy and chemotherapy drugs, inflammation regulating drugs, immunomodulatory drugs, antiviral drugs, antibiotics.
  • the other drug comprises: a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a liver protection drug, and an anti-fiber Chemical drugs, antiarrhythmic drugs, cardiotonic drugs, diuretic drugs, anti-tumor drugs, radiotherapy and chemotherapy drugs, inflammation regulating drugs, immunomodulatory drugs, antiviral drugs, antibiotics.
  • plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, and delta-fibrinolytic Proenzymes or their variants that retain plasminogen activity.
  • plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant thereof that still retains plasminogen activity Or a fragment.
  • a plasminogen for use in the method of any of items 1-43.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the method of any of items 1-43.
  • a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of any of items 1-43 and (ii) for delivery of said plasminogen to The subject of the subject.
  • the kit of item 46 wherein the member is a syringe or vial.
  • kit of item 46 or 47 further comprising a label or instructions for use, the label or instructions for use instructing administration of the plasminogen to the subject to perform any of the items 1-43 method.
  • a pharmaceutical composition comprising (i) plasminogen or a plasminogen-containing composition for use in the method of any one of items 1-43, wherein said label indicates administration of said plasminogen or composition
  • the subject is the method of any one of the preceding items 1-43.
  • kits or article of item 50 wherein the other drug is selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, and a drug.
  • a hypolipidemic drug an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, and a drug.
  • the invention relates to a method of preventing and/or treating collagen deposition or fibrosis and associated disorders in a tissue of a subject, comprising administering to the subject an effective amount of plasminogen, wherein said subject Susceptible to tissue fibrosis, tissue organ fibrosis or other diseases And accompanied by tissue and organ fibrosis.
  • the invention further relates to the use of plasminogen for preventing and/or treating collagen deposition or fibrosis in tissues and organs of a subject and related disorders.
  • the invention further relates to the use of plasminogen for the manufacture of a medicament for the prevention and/or treatment of collagen deposition or fibrosis of tissue and organs in a subject and related disorders thereof.
  • the present invention relates to plasminogen for preventing and/or treating collagen deposition or fibrosis of tissues and tissues of a subject and related disorders thereof.
  • the tissue organ collagen deposition or fibrosis comprises cutaneous fibrosis, vascular fibrosis, cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis.
  • the tissue organ collagen deposition or fibrosis comprises or is accompanied by an infection, inflammation, hypersensitivity reaction, tumor, tissue ischemia, tissue organ congestion, chemical, radiation, or environmental pollution. of.
  • the tissue organ collagen deposition or fibrosis comprises tissue organ collagen deposition or fibrosis caused by bacterial or viral or parasitic infection caused by tissue and organ diseases, wherein the tissue organ collagen deposition or fibrosis comprises Liver fibrosis caused by tuberculosis infection, liver fibrosis caused by hepatitis B virus, hepatitis C virus or hepatitis E virus infection, and liver fibrosis caused by schistosomiasis infection.
  • the tissue organ collagen deposition or fibrosis is caused by a sterile inflammation or an autoimmune response.
  • the tissue organ collagen deposition or fibrosis is renal fibrosis caused by chronic glomerulonephritis, pyelonephritis, nephrotic syndrome, renal insufficiency, and uremia.
  • the tissue organ collagen deposition or fibrosis is caused by cancer causing tissue and organ damage.
  • the tissue organ collagen deposition or fibrosis is lung fibrosis caused by lung cancer, liver fibrosis caused by liver cancer, or renal fibrosis caused by kidney cancer.
  • the tissue organ collagen deposition or fibrosis is caused by chronic ischemic tissue damage.
  • the tissue organ collagen deposition or fibrosis is renal fibrosis caused by coronary atherosclerosis, cardiac ischemic fibrosis caused by coronary heart disease, and/or chronic ischemic renal injury.
  • the tissue organ collagen deposition or fibrosis is caused by tissue organ congestion caused by cardiovascular disease.
  • the tissue organ collagen deposition or fibrosis is hepatic congestion or pulmonary congestion.
  • the tissue organ collagen deposition or fibrosis is caused by a drug.
  • the tissue organ collagen deposition or fibrosis is drug-induced liver fibrosis or renal fibrosis.
  • the tissue organ collagen deposition or fibrosis is pulmonary fibrosis caused by inhalation chemicals or environmental contaminants.
  • the tissue organ collagen deposition or fibrosis is, for example, systemic lupus erythematosus, systemic sclerosis Syndrome, ankylosing spondylitis caused by systemic immune disease.
  • the tissue organ fibrosis is idiopathic pulmonary fibrosis.
  • the tissue organ fibrosis-related disorder includes a disorder caused by a weakened, impaired or lost function of a tissue organ due to a fibrotic lesion.
  • the tissue organ fibrosis-related disorders include atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, cerebral ischemia, cerebral infarction, renal insufficiency, uremia, liver dysfunction, cirrhosis, liver Coma, difficulty breathing, emphysema, pulmonary heart disease, pulmonary fibrosis, ankylosing spondylitis.
  • the plasminogen may be used in combination with one or more other drugs or treatments.
  • the plasminogen may be combined with one or more drugs selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, and a solution.
  • Thrombosis drugs liver protection drugs, anti-fibrotic drugs, anti-arrhythmia drugs, cardiotonic drugs, diuretic drugs, anti-tumor drugs, radiotherapy and chemotherapy drugs, inflammation regulating drugs, immunomodulatory drugs, antiviral drugs, antibiotics.
  • the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 with the sequence 2, 6, 8, 10 or 12. % sequence identity and still have plasminogen activity.
  • the amino acid of the plasminogen is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen is added, deleted, and/or substituted on the basis of sequence 2, 6, 8, 10, or 12, 1-100, 1-90, 1-80, 1-70 , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and still a protein with plasminogen activity.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen or their retained plasmin A variant of the original activity.
  • the plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant or fragment thereof that still retains plasminogen activity.
  • plasminogen from a primate or rodent is a homologue, such as a plasminogen homolog from gorilla, rhesus, mouse, cow, horse, dog.
  • the amino acid sequence of the plasminogen of the present invention is as ordered Columns 2, 6, 8, 10 or 12 are shown.
  • the subject is a human. In some embodiments, wherein the subject lacks or lacks plasminogen. In particular, the deficiency or deficiency is innate, secondary and/or local.
  • the plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or passage. rectum.
  • the topical administration is by direct administration to an osteoporotic region, such as by dressings, catheters, and the like.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- 100mg / cm 2, 10-100mg / cm 2 ( calculated per square centimeter of body surface area) of the dose administered, preferably repeated at least once, preferably at least daily administration.
  • the above dosages may be further adjusted as appropriate.
  • the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the methods of the invention.
  • the present invention relates to a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of the invention and (ii) for delivery of said plasminogen to The subject of the subject, in particular, the member is a syringe or vial.
  • the kit further comprises a label or instructions for use, the label or instructions for use indicating administration of the plasminogen to the subject to perform the methods of the invention.
  • the present invention is also an article comprising: a container containing a label; and (i) a plasminogen or a pharmaceutical composition comprising plasminogen for use in the method of the present invention, wherein The label indicates administration of the plasminogen or composition to the subject to practice the methods of the invention.
  • the kit or article further comprises an additional one or more components or containers containing other drugs.
  • the other drug is selected from the group consisting of a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a hepatoprotective drug, and an anti-fiber Drug, anti-heart Abnormal drugs, cardiotonic drugs, diuretic drugs, anti-tumor drugs, radiotherapy and chemotherapy drugs, inflammation regulating drugs, immunomodulatory drugs, antiviral drugs, antibiotics.
  • Fibrosis is the activation of fibroblasts after continuous damage to tissues, organs such as lung, liver, kidney, blood vessels, peritoneum, pancreas, skin, etc. caused by inflammation, infection, immune response, ischemia, chemicals, radiation, etc. Proliferation, increased fibrous connective tissue in tissues and organs, decreased parenchymal cells, destruction of tissues, organ structure and loss of function. This term is used interchangeably with "fibrotic lesions.”
  • the term fibrotic lesions covers various stages of cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, vascular fibrosis, skin fibrosis and other tissue and organ fibrotic lesions, as well as various disease occurrence and development processes. Cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, vascular fibrosis, skin fibrosis and other tissue and fibrosis lesions.
  • organ fibrosis-related disorders After fibrotic lesions of tissues and organs, their normal structures change, and the corresponding functions are weakened or lost. The related diseases caused by this are called "organic organ fibrosis-related disorders".
  • Heart fibrosis refers to the occurrence and development of heart disease caused by various causes (such as inflammation, infection, immune response, ischemia, chemicals, radiation) or accompanying heart tissue damage or various causes. Fibrotic lesions. Cardiac fibrosis causes impaired cardiac function, and the resulting condition is called “cardiac fibrosis-related disorder", including but not limited to symptoms of various organs, tissue ischemia, and conditions caused by impaired cardiac function, such as coronary heart disease , angina pectoris, myocardial infarction, arrhythmia, cerebral ischemia, dyspnea, renal insufficiency, etc.
  • Liver fibrosis refers to abnormal proliferation of connective tissue in the liver caused by or associated with various causes such as inflammation, infection (eg, viral infection), immune response, ischemia, chemicals, radiation, oxidative stress, and alcohol abuse. Pathological changes (lesions) in which the diffuse extracellular matrix of the liver is excessively precipitated and the normal structure of the liver is destroyed. Further development of liver fibrosis is cirrhosis, and is also encompassed within the scope of the term “liver fibrosis” of the present invention. Liver fibrotic lesions result in impaired liver function, and the resulting condition is referred to as "hepatic fibrosis-related disorder.”
  • Pulmonary fibrosis refers to the proliferation of lung tissue mesenchymal cells, the deposition of extracellular matrix, and the weight of lung parenchyma caused by or associated with various causes (such as inflammation, infection, immune response, ischemia, chemicals, radiation). The pathological process caused by the structure. Pulmonary fibrotic lesions result in impaired lung function, and the resulting condition is referred to as "pulmonary fibrosis-related disorder.”
  • Kidney fibrosis refers to the pathology of connective tissue in the kidney caused by various causes (such as inflammation, infection, immune response, ischemia, chemicals, radiation), or the pathological changes and functional impairment of the kidney. process. Renal fibrotic lesions are a common pathway in which almost all kidney diseases progress to the later stages.
  • Kiddney fibrosis-related disorder for example, renal insufficiency, renal failure, uremia, and the like.
  • Chronic lesions of tissues and organs are often accompanied by fibrosis, such as chronic inflammation of the lungs, chronic lesions, and pulmonary fibrosis.
  • liver fibrosis such as hepatitis B, hepatitis C, alcoholic liver, fatty liver, schistosomiasis, etc., all associated with early liver fibrosis.
  • Others such as chronic nephritis, glomerulonephritis, and renal tubular inflammation have renal fibrosis; vascular fibrosis occurs in cardiovascular, cerebrovascular, lower extremity arteriosclerosis, or narrowing, or obstruction.
  • fibrosis or "fibrotic lesion" of the present invention encompasses fibrotic lesions often associated with chronic lesions of various tissues and organs of the body.
  • Systemic sclerosis or “scleroderma” is a systemic autoimmune disease characterized by localized or diffuse skin thickening and fibrosis.
  • the lesions are characterized by skin fibrosis and vascular onion skin-like changes, which eventually lead to skin sclerosis and vascular ischemia.
  • the disease is characterized by localized or diffuse skin thickening and fibrosis. In addition to skin involvement, it can also affect the internal organs (heart, lung and digestive tract).
  • Atherosclerosis is a chronic, progressive arterial disease in which the fat deposited in the arteries partially or completely blocks blood flow. Atherosclerosis is a gradual process. When the concentration of lipids in the blood is greatly increased, fat streaks are formed along the walls of the artery. These streaks cause fat and cholesterol deposits that attach to the originally smooth arterial intima, forming a nodule. These nodules then grow fibrotic scar tissue, resulting in calcium deposition. The deposited calcium gradually evolves into a chalky hard film (called an atheroma) that cannot be removed.
  • the ischemic injury of the tissue or organ caused by the obstruction of the artery in the tissue or the organ may cause fibrotic lesions of the tissue or organ, such as the heart and the lung. , liver, kidney, blood vessels, peritoneum, pancreas, skin fibrosis.
  • Atherosclerosis is often accompanied by the occurrence of atherosclerosis, and its mechanism is considered to be atherosclerosis caused by abnormal blood glucose metabolism.
  • atherosclerosis caused by diabetes is not a single factor. Instead, it induces and promotes the development and progression of atherosclerosis through multiple pathways and more complex mechanisms [4] .
  • Diabetes and its accompanying atherosclerosis can lead to tissue, organ damage and fibrosis, such as fibrosis of tissues, organs such as heart, lung, liver, kidney, blood vessels, peritoneum, pancreas, skin, and the like.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that hydrolyzes several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans [5] . In addition, plasmin activates some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis [6,7] . Plasmin is formed by proteolytic plasminogen by two physiological PAs: tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA).
  • tPA tissue plasminogen activator
  • uPA urokinase-type plasminogen activator
  • PAI-1 plasminogen activator inhibitor-1
  • PAI-2 lysogen activator inhibitor-2
  • Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa [10,11] . Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. The plasma plasminogen content is approximately 2 ⁇ M. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids [12,13] . Plasminogen exists in two molecular forms: glutamate-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved forms of plasminogen have an amino terminal (N-terminal) glutamate and are therefore referred to as glutamate-plasminogen.
  • plasminogen glutamate-plasminogen is hydrolyzed to Lys-Lysinogen at Lys76-Lys77.
  • lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide-linked double-chain protease plasmin [14] .
  • the amino terminal portion of plasminogen contains five homologous tricycles, the so-called kringles, which contain a protease domain.
  • Some kringles contain a lysine binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor ⁇ 2-AP.
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycans and gelatin, suggesting that plasmin also plays an important role in ECM reconstruction [11,16, 17] .
  • plasmin can also degrade other components of ECM, including MMP-1, MMP-2, MMP-3 and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis [18] .
  • plasmin has the ability to activate certain potential forms of growth factors [19-21] . In vitro, plasmin also hydrolyzes components of the complement system and releases chemotactic complement fragments.
  • Plasmid is a very important enzyme found in the blood that hydrolyzes fibrin clots into fibrin degradation products and D-dimers.
  • Plasinogen is a zymogen form of plasmin, which is composed of 810 amino acids, based on the sequence in swiss prot, based on the native human plasminogen amino acid sequence (sequence 4) containing the signal peptide. 90 kD, a glycoprotein synthesized mainly in the liver and capable of circulating in the blood, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO:3.
  • Full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle 1-5).
  • the signal peptide includes the residue Met1-Gly19
  • PAp includes the residue Glu20-Val98
  • Kringle1 includes the residue Cys103-Cys181
  • Kringle2 includes the residue Glu184-Cys262
  • Kringle3 includes the residue Cys275-Cys352
  • Kringle4 Including the residue Cys377-Cys454
  • Kringle5 includes the residue Cys481-Cys560.
  • the serine protease domain includes the residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing a 19 amino acid signal peptide), and the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is sequence 2. Shown. In vivo, there is also a Lys-plasminogen which is hydrolyzed from amino acids 76-77 of Glu-plasminogen, and as shown in SEQ ID NO: 6, the cDNA sequence encoding the amino acid sequence is as shown in SEQ ID NO: 5 Shown. Delta-plasminogen is a fragment of full-length plasminogen lacking the Kringle2-Kringle5 structure, containing only the Kringle1 and serine protease domains [22,23].
  • the delta-plasminogen has been reported in the literature.
  • the amino acid sequence (SEQ ID NO: 8) [23] and the cDNA sequence encoding the amino acid sequence is shown in Sequence 7.
  • Mini-plasminogen consists of Kringle5 and a serine protease domain, which has been reported in the literature to include the residue Val443-Asn791 (starting amino acid with a Glu residue of Glu-plasminogen sequence not containing a signal peptide) [24] , the amino acid sequence thereof is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9.
  • Micro-plasminogen contains only the serine protease domain, and its amino acid sequence has been reported to include the residue Ala543-Asn791 (from the Glu residue of the Glu-plasminogen sequence containing no signal peptide). [ amino acid] [25] , there is also a patent document CN102154253A reported that its sequence includes the residue Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence containing no signal peptide is the starting amino acid), and the patent sequence refers to the patent document. CN102154253A, the amino acid sequence of which is shown in SEQ ID NO: 12, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 11.
  • Plasin of the present invention is used interchangeably with “fibrinolytic enzyme” and “fibrinolytic enzyme”, and has the same meaning; “plasminogen” and “plasminogen”, “fibrinolytic enzyme” "Original” is used interchangeably and has the same meaning.
  • the term "deficiency" of plasminogen means that the content or activity of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject;
  • the meaning of "deficient" of plasminogen is that the content or activity of plasminogen in the subject is significantly lower than that of normal people, and even the activity or expression is minimal, and only by external supply can maintain normal physiological functions.
  • plasminogen adopts a closed inactive conformation, but when bound to the surface of a thrombus or cell, it is converted to openness mediated by plasminogen activator (PA).
  • PA plasminogen activator
  • Conformational active plasmin The active plasmin further hydrolyzes the fibrin clot into a fibrin degradation product and a D-dimer, thereby dissolving the thrombus.
  • the PAp domain of plasminogen contains an important determinant that maintains plasminogen in an inactive blocking conformation, while the KR domain is capable of binding to lysine residues present on the receptor and substrate.
  • plasminogen activators include tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hag Mann factor) and so on.
  • a "plasminogen active fragment” refers to an active fragment that binds to a target sequence in a substrate and exerts a proteolytic function in a plasminogen protein.
  • the technical solution of the present invention relating to plasminogen covers the technical solution of replacing plasminogen with a plasminogen active fragment.
  • Plasmin The original active fragment is a protein comprising a serine protease domain of plasminogen.
  • the plasminogen active fragment of the present invention comprises the sequence 14, and the sequence 14 has at least 80%, 90%, 95%, 96%, A protein of 97%, 98%, 99% homologous amino acid sequence.
  • the plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still retaining the plasminogen activity.
  • blood plasminogen and its activity assays include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, inhibition of plasma tissue plasminogen activator Detection of antigens, plasma plasmin-anti-plasmin complex assay (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg detection of plasma tissue plasminogen activator antigen
  • plgA Detection of plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma plasmin-anti-plasmin complex assay
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the plasma to be tested, and the PLG in the tested plasma is converted into PLM under the action of SK, and the latter acts on The chromogenic substrate is then measured spectrophotometrically and the increase in absorbance is directly proportional to the plasminogen activity.
  • plasminogen activity in blood can also be measured by immunochemical method, gel electrophoresis, immunoturbidimetry, or radioimmunoassay.
  • ortholog or ortholog refers to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs, orthologs. It specifically refers to a protein or gene that has evolved from the same ancestral gene in different species.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs of plasminogen activity derived from different species.
  • Constant substitution variant refers to a change in one of the given amino acid residues without altering the overall conformation and function of the protein or enzyme, including but not limited to similar properties (eg, acidic, basic, hydrophobic, etc.)
  • the amino acid replaces the amino acid in the amino acid sequence of the parent protein.
  • Amino acids having similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and are interchangeable.
  • isoleucine is a hydrophobic amino acid that can be replaced by leucine, methionine or valine. Therefore, the similarity of two protein or amino acid sequences of similar function may be different.
  • Constant substitution variants also includes determining polypeptides or enzymes having more than 60% amino acid identity by BLAST or FASTA algorithm. If it is more than 75%, preferably more than 85%, or even more than 90%. Optimal and have the same or substantially similar properties or functions as the native or parent protein or enzyme.
  • Isolated plasminogen refers to a plasminogen protein that is isolated and/or recovered from its natural environment.
  • the plasminogen will purify (1) to a purity greater than 90%, greater than 95%, or greater than 98% by weight, as determined by the Lowry method, eg, over 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is by use Coomassie blue or silver staining was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide peptide
  • protein protein
  • fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue);
  • percent amino acid sequence identity with respect to a reference polypeptide sequence is defined as the introduction of a gap as necessary to achieve maximum percent sequence identity, and without any conservative substitution being considered as part of sequence identity, in the candidate sequence The percentage of amino acid residues that are identical in amino acid residues in the reference polypeptide sequence. Comparisons for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum contrast over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity percent values are generated using the sequence comparison computer program ALIGN-2.
  • amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as having or comprising relative to, and, or for a given amino acid sequence)
  • a given amino acid sequence A of a certain % amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and wherein Y is the total number of amino acid residues in B.
  • % amino acid sequence identity of A with respect to B may not be equal to the % amino acid sequence of B relative to A.
  • All % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless explicitly stated otherwise.
  • the terms “treating” and “treating” refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the disease or its symptoms, and/or to partially or completely cure the disease and/or its symptoms, and includes: (a) preventing the disease from occurring in the subject, the subject may have The cause of the disease, but not yet diagnosed as having a disease; (b) inhibiting the disease, ie, retarding its formation; and (c) reducing the disease and/or its symptoms, ie causing the disease and/or its symptoms to subside.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to a mammal, including but not limited to a mouse (rat, mouse), a non-human primate, a human, a dog, a cat. Hoofed animals (such as horses, cattle, sheep, pigs, goats).
  • “Therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease.
  • the “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, and the like.
  • Plasminogen can be isolated and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, it can be synthesized in a liquid phase or a solid phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase and then cut off.
  • the plasminogen of the present invention can be produced using standard recombinant methods.
  • a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to a regulatory sequence in an expression vector.
  • Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting eukaryotic host cells (eg, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are typically replicated as an episome in the host organism or as an integral part of the host chromosomal DNA.
  • expression vectors typically contain a selection marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the desired DNA sequence with foreign sources. Those cells are tested.
  • a selection marker eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a subject antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Genus (Pseudomonas) species.
  • expression vectors can also be generated which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell.
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically control expression, optionally in the context of manipulating a gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast e.g., S. cerevisiae
  • Pichia are examples of suitable yeast host cells in which a suitable vector has expression control sequences (e.g., a promoter), an origin of replication, a termination sequence, and the like, as desired.
  • a typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes.
  • Inducible yeast is initiated by a promoter specifically comprising an alcohol dehydrogenase, an isocytochrome C, and an enzyme responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • an anti-Tau antibody of the invention e.g., a polynucleotide encoding a subject anti-Tau antibody.
  • Suitable mammalian host cells include CHO cell lines, various Cos Cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may contain expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.
  • RNA splice sites sites that are ribosome binding.
  • RNA splice sites sites that are ribosome binding.
  • polyadenylation sites sites that are ribosome binding sites.
  • transcription terminator sequences sites that are ribosome binding sites.
  • suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).
  • the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like.
  • Plasminogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure. Or more pure, for example, free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.
  • the jelly can be formed by mixing plasminogen of the desired purity with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)).
  • the therapeutic formulation is prepared as a dry formulation or as an aqueous solution.
  • Acceptable carriers, excipients, and stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as Octadecyldimethylbenzylammonium chloride; chlorinated hexane diamine; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptide (less than about 10 residues) Protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the formulations of the invention may also contain more than one active compound as required for the particular condition being treated, preferably those having complementary activities and no side effects to each other.
  • active compound for example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, and the like.
  • the plasminogen of the present invention may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, may be placed in a glial drug delivery system (eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • glial drug delivery system eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • the plasminogen of the invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can prepare a sustained release preparation.
  • sustained release formulations include solid hydrophobic polymeric semi-permeable matrices having a shape and containing glycoproteins, such as films or microcapsules.
  • sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem .Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S.
  • Patent 3,739,919, EP 58,481 L-glutamic acid and ⁇ -ethyl-L-glutamic acid Copolymer (Sidman, et al, Biopolymers 22: 547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al, supra), or degradable lactic acid-glycolic acid copolymer Such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyric acid.
  • Lupron DepotTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly D-(-)-3-hydroxybutyric acid poly D-(-)-3-hydroxybutyric acid.
  • Ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, while some hydrogels release proteins for a short time.
  • a reasonable strategy for protein stabilization can be designed according to the relevant mechanism. For example, if agglomeration is found The mechanism is to form intermolecular SS bonds by thiodisulfide bond exchange, which can be modified by thiol residues, lyophilized from acidic solution, and controlled. Degree, using appropriate additives, and developing specific polymer matrix compositions stable.
  • compositions of this invention may be effected intramuscularly in different ways, such as by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery).
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectables.
  • Organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffering media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles contain liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.
  • the medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical arts, the dosage of any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, number and route of administration, overall health, and other medications administered simultaneously. .
  • the pharmaceutical composition of the present invention comprising plasminogen may be, for example, in the range of about 0.0001 to 2000 mg/kg per day, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) Subject weight.
  • the dose can be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses in the above ranges are also included in the scope of the present invention.
  • the subject can administer such doses daily, every other day, every week, or according to any other schedule determined by empirical analysis.
  • An exemplary dosage schedule includes 1-10 mg/kg for several days. The therapeutic effect and safety need to be evaluated in real time during the administration of the drug of the present invention.
  • One embodiment of the invention relates to an article or kit comprising a plasminogen or plasmin of the invention useful for treating a cardiovascular disease caused by diabetes and a related disorder thereof.
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, and the like.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that is effective to treat a disease or condition of the invention and has a sterile access port (eg, the container can be an intravenous solution or vial containing a stopper that can be penetrated by a hypodermic needle) of).
  • At least one active agent in the composition is plasminogen/plasmin.
  • the label on or attached to the container indicates that the composition is used to treat the cardiovascular disease caused by diabetes and its related conditions of the present invention.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article comprises a package insert with instructions for use comprising, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease to the patient.
  • FIG. 1 Representative picture of kidney Sirius red staining after 21 days of bleomycin-induced systemic sclerosis in mice given plasminogen.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that in the mouse model of systemic sclerosis induced by bleomycin, the degree of collagen fibrosis (arrow mark) in the vehicle PBS control group was higher than that in the plasminogen group. This indicates that plasminogen can effectively reduce bleomycin-induced renal fibrosis.
  • FIG. 1 Representative picture of Sirius red staining after 21 days of bleomycin-induced systemic sclerosis in mice given plasminogen.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • B is the plasminogen group.
  • the study found that in the bleomycin-induced systemic sclerosis mouse model, the concentration of collagen deposition (arrow mark) in the vehicle control group was higher than that in the plasminogen group. This indicates that plasminogen can effectively reduce bleomycin-induced cardiac fibrosis.
  • FIG. 3 Representative picture of Sirius red staining after 21 days of bleomycin-induced systemic sclerosis in mice given plasminogen.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that in the mouse model of systemic sclerosis induced by bleomycin, the degree of pulmonary fibrosis (arrow mark) in the vehicle PBS group was higher than that in the plasminogen group; the mice in the plasminogen group were given.
  • the alveolar wall morphology of the lungs was close to normal, the cells of inflammation level were significantly reduced, and the degree of fibrosis was significantly lower than that of the vehicle-treated PBS group, and the statistical difference was significant (* indicates P ⁇ 0.05).
  • FIG. 4 Representative pictures of Sirius red staining after zebramycin-induced systemic sclerosis in mice for 21 days after administration of plasminogen.
  • A is a blank control group
  • B is a vehicle control group
  • C is a plasminogen group
  • D is a PLG activity-impaired group.
  • the results showed that in the bleomycin-induced systemic sclerosis mouse model, the dermal PBS group and the PLG activity-impaired group showed a significant increase in the collagen fibers in the upper dermis, the collagen fibers were coarse, the arrangement was dense, and the dermis layer was thickened.
  • the fibroblasts in the dermal layer of the plasminogen group were significantly less than those in the vehicle PBS group, and the thickness of the dermis layer was close to normal.
  • Figure 5 shows the results of immunostaining of type IV collagen in kidneys after administration of plasminogen for 23 days in 24-25-week-old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the positive staining of IV collagen in the plasminogen group (arrow mark) was significantly more than that in the vehicle PBS control group, indicating that plasminogen can improve the fibrosis of the kidney of diabetic mice.
  • FIG. 6 Results of renal masson staining after 35 days of plasminogen administration in 26-week-old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that in the vehicle PBS control group, glomerular mesangial hyperplasia, mesangial matrix increased, renal interstitial fibrosis (arrow mark), hyperplastic fibrosis was blue.
  • the glomerular mesangial cells and matrix in the plasminogen group were significantly less than those in the control group, and renal interstitial fibrosis was significantly reduced. This indicates that plasminogen can improve the fibrotic lesions in the kidneys of diabetic mice.
  • Figure 7 Results of cardiac masson staining after 31 days of administration of plasminogen in 24-25-week-old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that blue hyperplastic collagen fibers (arrow marks) were observed between the myocardial fibers of the vehicle control group and showed mild myocardial fibrosis; a small amount of light blue was observed between the myocardial fibers in the plasminogen group.
  • myocardial fibrosis was significantly reduced in hyperplastic collagen fibers. This indicates that plasminogen can improve the fibrosis of the heart of diabetic mice.
  • FIG 8 Representative pictures of Sirius red staining after 17 days of administration of plasminogen in 17-18 week old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the deposition of collagen fibers (arrow marks) in the plasminogen group was significantly less than that in the vehicle PBS control group. This indicates that plasminogen can reduce fibrosis in the heart of relatively young (17-18 weeks old) diabetic mice.
  • FIG. 9 Representative pictures of Sirius red staining after 26 days of administration of plasminogen in 26-27 week old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the deposition of collagen (arrow mark) in the plasminogen group was significantly less than that in the vehicle PBS control group. This indicates that plasminogen can attenuate the fibrosis of the heart of relatively old (26-27 weeks old) diabetic mice.
  • Figure 10 shows the results of immunostaining of type IV collagen in kidneys after 7 days of administration of plasminogen in cisplatin-induced renal fibrosis model mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the positive expression of type IV collagen (arrow mark) in the control vehicle PBS control group was significantly higher than that in the plasminogen group. This indicates that plasminogen can improve the fibrosis of kidney in cisplatin-induced renal fibrosis model mice.
  • FIG 11 Representative images of Sirius red staining after 30 days of plasminogen administration in ApoE atherosclerotic model mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the deposition of collagen in the plasminogen group (arrow mark) was significantly less than that in the vehicle PBS control group, indicating that plasminogen can attenuate cardiac fibrosis in ApoE atherosclerotic model mice.
  • Figure 12 is a representative picture of Sirius red staining after 30 days of administration of plasminogen in C57 hyperlipidemic model mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the result shows that the fiber The deposition of collagen in the lysozyme group (arrow mark) was significantly less than that of the vehicle PBS control group, indicating that plasminogen can alleviate cardiac fibrosis in hyperlipidemic mice.
  • Fig. 13 shows the results of Sirius red staining of mice exposed to plasminogen for 10 days after induction of chronic kidney injury.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the PLG activity-impaired group
  • D is the quantitative analysis result.
  • the deposition of collagen in the plasminogen group (arrow mark) was significantly less than that in the vehicle PBS control group and the PLG activity-impaired group, and the quantitative analysis of the plasminogen group and the PLG activity-impaired group was statistically significant (* indicates P ⁇ 0.05). It indicates that plasminogen can alleviate renal fibrosis caused by chronic kidney injury and promote the repair of kidney injury.
  • Figure 14 shows the results of islet Sirius red staining after administration of plasminogen for 23 days in 24-25-week-old diabetic mice.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that the islet collagen deposition (arrow mark) in the plasminogen group was significantly less than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can improve islet damage and fibrosis caused by diabetes.
  • Figure 15 Representative images of aortic sinus Sirius red staining 30 days after administration of plasminogen in ApoE atherosclerotic model mice. A and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group. The results showed that the area of collagen deposition (arrow mark) in the plasminogen group was significantly smaller than that in the vehicle PBS control group, indicating that plasminogen can attenuate the level of aortic sinus fibrosis in atherosclerotic model mice.
  • Figure 16 is a representative picture of liver Sirius red staining in mice with liver fibrosis induced by carbon tetrachloride after 14 days of plasminogen administration.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the collagen deposition in the plasminogen group was significantly less than that in the vehicle control group, and the collagen deposition level was similar to that in the blank control mice. This indicates that plasminogen can reduce the deposition of liver collagen and improve liver fibrosis in mice with liver fibrosis.
  • FIG 17 Representative pictures of aortic sinus Sirius red staining after administration of plasminogen for 30 days in a 16-week hyperlipidemia model mouse. A and C were given to the vehicle PBS control group, and B and D were given to the plasminogen group. The results showed that the area of collagen deposition (arrow mark) in the aortic sinus wall of the plasminogen group was significantly smaller than that of the vehicle PBS control group, indicating that plasminogen can attenuate the aortic sinus of hyperlipidemia model mice. Endometrial fibrosis level of the wall.
  • Figure 18 shows the results of Sirius red staining of 3% cholesterol hyperlipidemia model mice after 30 days of plasminogen administration.
  • A is a blank control group
  • B is a vehicle control group
  • C is a plasminogen group
  • D is a quantitative analysis result.
  • the results showed that the collagen deposition in the plasminogen group was given. (arrow mark) was significantly less than the vehicle control group, and the statistical difference was significant; the fibrosis to the plasminogen group was basically restored to normal levels. This indicates that plasminogen can effectively reduce renal fibrosis in 3% cholesterol hyperlipidemia model mice.
  • Figure 19 shows the results of liver staining of Sirius red staining in mice modeled with plasminogen for 28 days of carbon tetrachloride-induced liver fibrosis.
  • A is a blank control group
  • B is a vehicle control group
  • C is a plasminogen group
  • D is a quantitative analysis result.
  • the results showed that the collagen deposition (arrow mark) in the plasminogen group was significantly less than that in the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05); compared with the vehicle PBS control group, the plasmin was given.
  • the original group of collagen deposition levels were closer to the blank control mice. This indicates that plasminogen can reduce the deposition of liver collagen and improve liver fibrosis in mice with liver fibrosis.
  • Example 1 Plasminogen reduces renal fibrosis in mice with systemic sclerosis
  • mice Ten C57 male rats of 12 weeks old were randomly divided into two groups, and the vehicle PBS control group and the plasminogen group were each given 5 rats. The day of the experiment was recorded as day 0 and weighed into groups. On the first day, the model was administered. All mice were injected subcutaneously with bleomycin at 0.1 mg/0.1 ml/day/day to induce systemic sclerosis. Plasminogen or PBS was administered continuously for 21 days. The plasminogen group was injected with plasminogen at a dose of 1 mg/0.1 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Mice were sacrificed on day 22 and kidneys were fixed in 4% paraformaldehyde fixative for 24 hours.
  • the fixed kidney was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water and washed once with water. After staining with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, hematoxylin staining for 1 minute, running water rinse, 1% hydrochloric acid alcohol differentiation, ammonia water returning to blue Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • Ten 12-week-old C57 male rats were randomly divided into two groups, one for the vehicle PBS control group and five for the plasminogen group. On the day of the start of the experiment, the day 0 was recorded and the group was weighed. On the first day, the model was administered. The subcutaneous injection of bleomycin at 0.1 mg/0.1 ml/day/day induced systemic sclerosis [26] and began to give Plasminogen or PBS was administered continuously for 21 days. The plasminogen group was injected with plasminogen at a dose of 1 mg/0.1 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • mice were sacrificed on the 22nd day and the heart was taken at 4 Fix in % paraformaldehyde fixative for 24 hours.
  • the fixed heart was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water and washed once with water. After staining with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, hematoxylin staining for 1 minute, running water rinse, 1% hydrochloric acid alcohol differentiation, ammonia water returning to blue Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • mice Seventeen C57 male rats of 12 weeks old were randomly divided into two groups, 11 rats in the vehicle PBS control group and 6 rats in the plasminogen group. The day of the experiment was recorded as day 0 and weighed into groups. On the first day, the model was administered. The two groups of mice were injected subcutaneously with bleomycin at 0.1 mg/0.1 ml/day/day to induce systemic sclerosis [26]. And started to give plasminogen or PBS for continuous administration for 21 days. The plasminogen group mice were injected with plasminogen at a dose of 1 mg/0.1 ml/day/day, and the same volume of PBS was administered to the vehicle PBS control group in the same manner.
  • mice were sacrificed on day 22 and lung tissue was fixed in 4% paraformaldehyde fixative for 24 hours.
  • the fixed lung tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water and washed once with water. After staining with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, hematoxylin staining for 1 minute, running water rinse, 1% hydrochloric acid alcohol differentiation, ammonia water returning to blue Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • Fig. 3A collagen fibrosis (arrow mark) is higher than the plasminogen group
  • Fig. 3B The lung alveolar wall morphology of the plasminogen group was close to normal, the inflammatory cells were significantly reduced, and the degree of fibrosis was significantly lower than that of the vehicle-treated PBS group, and the statistical difference was significant (Fig. 3C). This indicates that plasminogen can effectively reduce lung fibrosis in bleomycin-induced systemic sclerosis mice.
  • the blank control group was subcutaneously injected with physiological saline at 0.1 ml/day/day, and plasminogen or PBS was started and recorded as the first day, and continuous administration was carried out for 21 days.
  • the plasminogen group was injected with plasminogen at a dose of 1 mg/0.1 ml/day/day, and the same volume of PBS was administered to the vehicle PBS control group.
  • the normal mouse group and the PLG-infected mice were not given.
  • Drug treatment Mice were sacrificed on day 22 and the dorsal skin tissue was fixed in 4% paraformaldehyde fixative for 24 hours. The fixed skin tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding. The thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water and washed once with water. After staining with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, hematoxylin staining for 1 minute, running water rinse, 1% hydrochloric acid alcohol differentiation, ammonia water returning to blue Rinse with running water, dry and seal the neutral gum, and observe under a 100x optical microscope.
  • Sirius red staining can make collagen staining for a long time. As a special staining method for pathological sections, Sirius red staining can specifically display collagen tissue.
  • mice Ten male db/db rats aged 24-25 weeks were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, with 5 rats in each group. On the day of the start of the experiment, the day 0 was recorded and the group was weighed, and on the first day, plasminogen or PBS was administered for 31 days. The plasminogen group was injected with plasminogen at 2 mg/0.2 ml/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed 31 days after plasminogen and kidney tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 4 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate in 3% hydrogen peroxide for 15 minutes. Wash twice with 0.01 M PBS for 5 minutes each time. 10% normal goat serum (Vector laboratories, Inc., USA) was blocked for 1 hour, after which time the serum was removed and the tissue was circled with a PAP pen. Rabbit anti-mouse polyclonal antibody (Abeam) against IV collagen was incubated overnight at 4 °C, and TBS was washed twice for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS.
  • HRP Goat anti-rabbit IgG
  • the mixture was washed with water for 3 times, then hematoxylin was counterstained for 30 seconds, rinsed with running water for 5 minutes, dehydrated with gradient alcohol, transparent and transparent with xylene, sealed with neutral gum, and sliced at 200 times. Observed under an optical microscope.
  • Diabetic nephropathy is a chronic complication of diabetes, and glomerular sclerosis and renal interstitial fibrosis are typical pathological changes [27] .
  • mice Ten male db/db males of 26 weeks old were randomly divided into two groups, given vehicle PBS control group and plasminogen group, with 5 rats in each group. On the day of the start of the experiment, the day 0 was recorded and the group was weighed, and on the first day, plasminogen or PBS was administered for 35 days. The plasminogen group was injected with plasminogen at 2 mg/0.2 ml/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed on day 36 and kidney tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the tissue section was 4 ⁇ m thick, and the sections were dewaxed and rehydrated, and then placed in a potassium dichromate solution overnight. Iron hematoxylin is dyed for 3 to 5 minutes, and the water is slightly washed. 1% hydrochloric acid alcohol was differentiated, treated with ammonia for 1 second, and washed with water. Lichun red acid magenta solution is dyed for 8 minutes and rinsed quickly in water. The aqueous solution of 1% phosphomolybdic acid was treated for about 2 minutes, and the aniline blue solution was counterstained for 6 minutes. 1% glacial acetic acid rinse for about 1 minute. The dried ethanol was dehydrated and the xylene was transparently sealed, and the sections were observed under a 200-fold optical microscope.
  • Masson staining can show fibrosis of the tissue.
  • the results showed that the glomerular mesangial hyperplasia, increased mesangial matrix, mild interstitial fibrosis (marked by the arrow), and hyperplastic fibrosis were blue in the vehicle PBS control group (Fig. 6A).
  • the glomerular mesangial cells and matrix in the plasminogen group (Fig. 6B) were significantly less than those in the control group, and renal interstitial fibrosis was significantly reduced. This indicates that plasminogen can improve the fibrosis of kidneys in diabetic mice.
  • mice Ten male db/db rats aged 24-25 weeks were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, with 5 rats in each group. On the day of the start of the experiment, the day 0 was recorded and the group was weighed, and on the first day, plasminogen or PBS was administered for 31 days. The plasminogen group was injected with plasminogen at 2 mg/0.2 ml/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed 31 days after plasminogen and cardiac tissue was fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the tissue section was 4 ⁇ m thick, and the sections were dewaxed and rehydrated, and then placed in a potassium dichromate solution overnight. Iron hematoxylin is dyed for 3 to 5 minutes, and the water is slightly washed. 1% hydrochloric acid alcohol was differentiated, treated with ammonia for 1 second, and washed with water. Lichun red acid magenta solution is dyed for 8 minutes and rinsed quickly in water. The aqueous solution of 1% phosphomolybdic acid was treated for about 2 minutes, and the aniline blue solution was counterstained for 6 minutes. 1% glacial acetic acid rinse for about 1 minute. The dried ethanol was dehydrated and the xylene was transparently sealed, and the sections were observed under a 200-fold optical microscope.
  • the most common complication of diabetes is excessive accumulation of connective tissue (pathological fibrosis), which may be a characteristic pathological change in diabetic cardiomyopathy [28-29] .
  • Masson staining can show fibrosis of the tissue.
  • the results showed that blue hyperplastic collagen fibers (arrow marks) were observed between the myocardial fibers in the vehicle PBS control group (Fig. 7A), showing mild myocardial fibrosis; in the plasminogen group (Fig. 7B) A small amount of light blue hyperplastic collagen fibers were seen, and myocardial fibrosis was significantly reduced compared with the control group. This indicates that plasminogen can improve the fibrosis of the heart of diabetic mice.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water and washed once with water. After staining with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, hematoxylin staining for 1 minute, running water rinse, 1% hydrochloric acid alcohol differentiation, ammonia water returning to blue Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • mice Nine male db/db males from 26 to 27 weeks old were randomly divided into two groups, 5 in the vehicle control group and 4 in the plasminogen group. On the day of the start of the experiment, the day 0 was recorded and the group was weighed, and on the first day, plasminogen or PBS was administered for 35 days. The plasminogen group was injected with plasminogen at 2 mg/0.2 ml/day/day in the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. Mice were sacrificed 35 days after plasminogen and cardiac tissue was fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water, washed once with water, stained with 0.1% Sirius red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, 1% hydrochloric acid and ammonia were rehydrated to blue, rinsed with water. After drying, the sheets were mounted and observed under a 200x optical microscope.
  • mice Ten male C57 mice, 8-9 weeks old, were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, with 5 rats in each group. After the grouping was completed, a single intraperitoneal injection of cisplatin at 10 mg/kg body weight was used to establish a model of renal fibrosis [30] . After the model was established, plasminogen was administered to the plasminogen group at a dose of 1 mg/0.1 ml/day/day via the tail vein, and the same volume of PBS was administered to the vehicle PBS control group. On the day of the experiment, the body weight was recorded on the 0th day and grouped.
  • Cisplatin is a broad-spectrum anticancer drug with extensive clinical application and reliable efficacy, but it has severe nephrotoxicity, mainly renal tubular and renal interstitial damage, and eventually develops into renal fibrosis [30] .
  • the results of this experiment showed that the positive expression of type IV collagen (arrow mark) in the vehicle PBS control group (Fig. 10A) was significantly higher than that in the lytic plasminogen group (Fig. 10B). This indicates that plasminogen can improve the fibrosis of the kidney of cisplatin kidney fibrosis model mice.
  • mice Thirteen 8-week-old ApoE male mice were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce atherosclerosis [31,32] .
  • 50 ⁇ L of blood was taken from each mouse to measure the total cholesterol concentration, and the mice were randomly divided into two groups according to the test results, and 7 rats in the vehicle PBS control group and 6 in the plasminogen group.
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the high-fat, high-cholesterol diet was fed. Mice were sacrificed on day 31 and hearts were fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene. The thickness of the tissue section was 3 ⁇ m. The sections were dewaxed and rehydrated, washed once with water, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid, and returned to blue with ammonia. Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • mice 11-year-old C57 male mice were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce hyperlipidemia [33,34] .
  • TP2031 high-fat, high-cholesterol diet
  • mice were randomly divided into two groups, and 6 rats in the vehicle PBS control group and 5 in the plasminogen group were administered.
  • the start of administration was recorded as the first day, and the plasminogen group mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the high-fat, high-cholesterol diet was fed. Mice were sacrificed on day 31 and heart tissue was fixed in 4% paraformaldehyde for 24-48 hours. The fixed tissue was paraffin-embedded by alcohol gradient dehydration and xylene transparency. The thickness of the tissue section was 3 ⁇ m. The sections were dewaxed and rehydrated, washed once with water, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid, and returned to blue with ammonia. Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • mice with impaired PLG activity were randomly divided into two groups, the plasminogen group and the vehicle PBS control group. only.
  • the day of modeling was recorded as the first day, and administration was started at the same time.
  • the plasminogen group was administered with plasminogen at 1 mg/0.1 ml/day/day, and the same volume of PBS was administered to the vehicle PBS control group in the same manner for 10 days, and the PLG-infected mice were not. Do the processing.
  • mice On the 11th day, the mice were sacrificed and the kidneys were fixed in 4% paraformaldehyde for 24 hours.
  • the fixed kidney was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water, washed once with water, stained with 0.1% Sirius red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, 1% hydrochloric acid and ammonia were rehydrated to blue, rinsed with water. After drying, the sheets were mounted and sections were observed under a 200x optical microscope.
  • mice Sixteen male db/db mice aged 24-25 weeks were randomly divided into two groups, 10 in the plasminogen group and 6 in the vehicle PBS control group.
  • the plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail vein of the vehicle PBS control group.
  • the day of the experiment was recorded as the 0th day weighing group, and the first day was given to plasminogen or PBS for 31 days.
  • Mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde. The fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • Tissue section thickness is 3 ⁇ m
  • the slices were dewaxed to water and washed once with water, stained with 0.1% Sirius red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, 1% hydrochloric acid and ammonia were rehydrated to blue, rinsed with water, dried and sealed. Sections were observed under a 200x optical microscope.
  • mice Thirteen of the 6-week-old male ApoE mice were fed a high-fat, high-cholesterol diet (Nantong Trofe, TP2031) for 16 weeks to induce an atherosclerosis model [31,32] .
  • the mice after the modeling were taken 50 ⁇ l of blood for three days before the administration to detect the total cholesterol (T-CHO) content, and were randomly divided into two groups according to the T-CHO content, and the vehicle PBS control group was given 7 cells. 6 lysogen group.
  • the first dose was started on the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • mice were sacrificed for 30 days, and the mice were sacrificed on the 31st day.
  • the hearts were fixed in 4% paraformaldehyde for 24-48 hours, respectively, in a 15%, 30% sucrose solution at 4 ° C overnight, embedded in OCT, and frozen section thickness. 8 ⁇ m, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia water, rinsed with water, dried and sealed with neutral gum, sliced Observed under a 40 (15A, 15B), 200-fold (15C, 15D) optical microscope.
  • Example 16 Plasminogen Improves Carbon Tetrachloride-Induced Liver Fibrosis
  • mice of 9 weeks old were randomly divided into three groups: blank control group, vehicle PBS control group and plasminogen group, with 5 rats in each group.
  • the vehicle PBS control group and the plasminogen group mice were intraperitoneally injected with carbon tetrachloride at a dose of 1 mL/kg body weight three times a week for two weeks to establish a liver fibrosis model [36 , 37] .
  • the corresponding volume of corn oil was injected according to the model mouse injection method.
  • Carbon tetrachloride needs to be diluted with corn oil, and the dilution ratio of carbon tetrachloride to corn oil is 1:3.
  • the drug was started after the model was established, and the day of the administration was recorded as the first day.
  • mice in the plasminogen group were injected with human plasminogen at a dose of 1 mg/0.1 mL/day/day, and the vehicle was administered to the tail of the PBS control group.
  • the same volume of PBS was injected intravenously, and the blank control group was not treated with injection for 14 days.
  • the mice were sacrificed on the 15th day and the liver was taken and fixed in 4% paraformaldehyde for 24 hours.
  • the fixed liver was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water, washed once with water, stained with 0.1% Sirius red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, 1% hydrochloric acid and ammonia were rehydrated to blue, rinsed with water. After drying, the sheets were mounted and sections were observed under a 200x optical microscope.
  • mice 11-year-old male C57 mice were fed a high-fat and high-cholesterol diet (Nantong Trophy, item number TP2031) for 16 weeks to induce a hyperlipidemia model [30,31] .
  • This model was assigned to 16-week hyperlipidemia.
  • Mice after modeling continue to feed high cholesterol feed. 50 ⁇ l of blood was taken from each of the three days before administration to detect the total cholesterol (T-CHO) content, and was randomly divided into two groups according to the T-CHO content, 6 in the vehicle PBS control group, and 5 in the plasminogen group. .
  • the start of administration was recorded as the first day, and the plasminogen group was injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were sacrificed on the 31st day, and the hearts were fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the aortic sinus slice was 3 ⁇ m. The slice was dewaxed and rehydrated and washed once.
  • mice of 9 weeks old were fed with 3% cholesterol and high fat diet (Nantong Trofe) for 4 weeks to induce hyperlipidemia [30 , 31] .
  • This model was classified as a model of 3% cholesterol hyperlipidemia.
  • the modeled mice continued to be fed a 3% cholesterol high fat diet.
  • Another 5 male C57 mice of the same age were used as a blank control group, and normal maintenance feed was fed during the experiment.
  • the model mice were randomly divided into two groups according to the total cholesterol concentration and body weight, and given to the plasminogen group and the vehicle PBS control group, each group was 8 only.
  • mice were injected with human plasminogen 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • Mice were dosed for 30 days after the 30th day of administration, and the mice were sacrificed on the 31st day, and the kidneys were fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed tissue was paraffin-embedded after dehydration by alcohol gradient and transparency of xylene.
  • the thickness of the slice was 3 ⁇ m, the slice was dewaxed and rehydrated, washed once with water, stained with 0.1% Sirius red saturated picric acid for 30 minutes, rinsed with running water for 2 min, stained with hematoxylin for 1 minute, rinsed with water, differentiated with 1% hydrochloric acid alcohol, and returned to blue with ammonia. Rinse with running water, dry and seal with neutral gum, and observe under a 200x optical microscope.
  • Example 19 Plasminogen Reduces Hepatic Collagen Deposition During Hepatic Fibrosis Induced by Carbon Tetrachloride
  • mice Twenty female C57 female mice aged 7-8 weeks were randomly divided into three groups, 5 in the blank control group, 7 in the vehicle control group and 8 in the plasminogen group.
  • the vehicle PBS control group and the plasminogen group mice were intraperitoneally injected with carbon tetrachloride at a dose of 1 mL/kg body weight three times a week for four weeks to establish a liver fibrosis model [36 , 37] .
  • a corresponding volume of corn oil is injected.
  • Carbon tetrachloride needs to be diluted with corn oil, and the dilution ratio of carbon tetrachloride to corn oil is 1:3.
  • the drug was administered on the day of modeling, and it was recorded as the first day.
  • the plasminogen group was injected with human plasminogen 1 mg/0.1 mL/day/day into the tail vein, and the same volume of the PBS control group was injected into the tail vein.
  • the blank control group was not treated with injection, and was administered continuously for 28 days.
  • the mice were sacrificed and the liver was fixed in 4% paraformaldehyde for 24 hours.
  • the fixed liver was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water, washed once with water, stained with 0.1% Sirius red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, 1% hydrochloric acid and ammonia were rehydrated to blue, rinsed with water. After drying, the sheets were mounted and sections were observed under a 200x optical microscope.

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Abstract

一种预防和治疗系统性硬化症的方法，包括给药受试者有效量的纤溶酶原。

Description

一种预防和治疗系统性硬化症的方法 技术领域
本发明涉及一种预防和治疗系统性硬化症的方法，包括给药受试者有效量的纤溶酶原。
背景技术
纤维化是一种病理变化，表现为成纤维细胞激活增殖、组织器官内纤维结缔组织增多，实质细胞减少，持续进展可致组织、器官结构破坏和功能丧失。重要脏器的纤维化严重影响患者生存质量，甚至危及生命。在全世界范围内，组织纤维化是许多疾病致残、致死的主要原因，据美国有关统计资料证明，该国因各种疾病而致死的患者中，约45％可以归于组织纤维增生疾病。
纤维化疾病包括累及多系统的疾病，如系统性硬化症、多灶性纤维化、硬皮病、肾源性的多系统纤维化，也包括器官组织特异性疾病，如皮肤、心、肺、肝、肾纤维化等。不同纤维化疾病的病因各不相同，例如组织器官的损伤、感染、免疫反应、慢性炎症等，但其共同的特征是细胞外基质(ECM)在组织中的过度沉积和器官组织重构[1-3]。
心脏纤维化出现于多种心脏疾病的发生与发展过程中，如病毒性心肌炎、心肌梗死和高血压心脏病等。过度的心脏纤维化导致心脏功能受损，是多种疾病走向心衰的主要原因，其中抑制和逆转心肌纤维化己成为治疗心血管疾病的重要环节。
肝纤维化是指由各种致病因子所致肝内结缔组织异常增生，肝内弥漫性细胞外基质过度沉淀的病理过程。多种因素均可引起肝纤维化，如病毒感染、炎症反应、氧化应激和酗酒等。肝纤维化的病理特点为汇管区和肝小叶内有大量纤维组织增生和沉积，但尚未形成小叶内间隔，肝硬化则有假小叶形成，中心静脉区和汇管区出现间隔，肝的正常结构遭到破坏，肝纤维化进一步发展即为肝硬化。我国的慢性肝病中以病毒性肝炎为主，慢性病毒性肝炎的肝组织纤维化与肝内炎症、坏死、病毒复制等有关，而且在早期是可逆的。为此将抗病毒治疗，调整机体免疫功能及保护肝细胞等疗法与抗纤维化结合起来，是防止肝纤维化的积极措施。
肺纤维化疾病包括特发性肺纤维化、结节病、过敏性肺炎、尘肺、药物和放射线导致的纤维化，以及与胶原血管病有关的致纤维化肺泡炎等病因各异、范围广泛的疾病谱。其主要病理特点包括肺组织间充质细胞增殖、细胞外基质增生沉积及肺实质的重构等画。目前主要采用抗炎、抗氧化、抗成纤维细胞增殖和胶原沉积及肺移植等措施治疗肺纤维化。
肾纤维化表现为细胞外基质和不适当结缔组织在肾聚集，导致肾结构改变和功能受损的病理过程，也是几乎所有肾疾病进展到终末期肾衰的共同通路。肾纤维化过程涉及炎症反应，固有细胞和免疫细胞的凋亡以及多种调控纤维化因子失衡等，故可通过抗炎症、抗凋亡和针对纤维化因子治疗等途径抗御肾纤维化。
组织器官的慢性病变常伴有纤维化，例如肺的慢性炎症，慢性病变，伴有肺纤维化。肝纤维化也是这样，像乙肝、丙肝、酒精肝、脂肪肝、血吸虫病等都伴有早期肝纤维化。肝的代偿功能很强，纤维化病变一直潜伏在各种慢性肝病里，一般发展为肝硬化时才被发现，实际上肝硬化已经是肝纤维化的严重阶段。再像慢性肾炎、肾小球炎、肾小管炎等都有肾纤维化；心血管、脑血管、下肢血管硬化、或变窄、或阻塞情况下都有血管纤维化。
皮肤纤维化形成瘢痕组织。瘢痕组织是肉芽组织经改建成熟形成的老化阶段的纤维结缔组织。创伤等情况下，成纤维细胞分裂、增殖，向受损部位迁移，产生细胞外基质，形成瘢痕组织，修复创伤。
瘢痕的形成是肉芽组织逐渐纤维化的过程。此时网状纤维及胶原纤维越来越多，网状纤维胶原化，胶原纤维变粗，与此同时纤维母细胞越来越少，少量剩下者转变为纤维细胞；间质中液体逐渐被吸收，中性粒细胞、巨噬细胞、淋巴细胞和浆细胞先后消失；毛细血管闭合、退化、消失，留下很少的小动脉及小静脉。这样，肉芽组织乃转变成主要由胶原纤维组成的血管稀少的瘢痕组织，肉眼呈白色，质地坚韧。
由于瘢痕坚韧又缺乏弹性，加上瘢痕收缩可引起器官变形及功能障碍，所以发生在关节附近和重要脏器的瘢痕，常引起关节痉挛或活动受限，如在消化道、泌尿道等腔室器官则引起管腔狭窄，在关节附近则引起运动障碍。发生在器官之间或器官与体腔壁之间的瘢痕性粘连，常不同程 度地影响其功能。如器官内广泛损伤后发生广泛纤维化、玻璃样变，则导致器官硬化。
系统性硬化症(SSc)也称为硬皮病，是一种以局限性或弥漫性皮肤增厚和纤维化为特征的全身性自身免疫病。病变特点为皮肤纤维增生及血管洋葱皮样改变，最终导致皮肤硬化、血管缺血。本病临床上以局限性或弥漫性皮肤增厚和纤维化为特征，除皮肤受累外，它也可影响内脏(心、肺和消化道等器官)。
动脉粥样硬化常常导致组织、器官缺血损伤，继而引发组织器官的纤维化病变。动脉粥样硬化是一种慢性的、渐进性动脉疾病，发病时动脉中沉积的脂肪部分或全部堵塞血流。当原本光滑、坚实的动脉内膜变粗糙、增厚，并被脂肪、纤维蛋白、钙和细胞碎屑堵塞时，便出现动脉粥样硬化。动脉粥样硬化是动脉内膜的慢性炎症性增生病变，导致大、中型动脉管腔狭窄或闭塞，引起相应器官组织缺血损伤、纤维化，甚至坏死。
动脉粥样硬化与糖尿病关系密切，表现为糖尿病患者出现动脉粥样硬化的时间早、程度重和预后差，而动脉粥样硬化又是糖尿病患者的主要死亡原因。临床发现糖尿病患者的冠状动脉血管病理改变的特点主要是病变累及的血管较多、冠状动脉狭窄严重，病变更加弥漫严重，其机制多认为是血糖代谢异常引起动脉粥样硬化，随着更进一步深入的研究，更多的结果表明，糖尿病引起动脉粥样硬化并非单一因素所致，而是通过多种途径以及较为复杂的机制来诱发和促进动脉粥样硬化的发生及发展，例如巨噬细胞极化、巨噬细胞移动抑制因子途径、糖基化终产物途径、清道夫受体上调、胰岛素抵抗、泛素-蛋白酶体系统激活、血小板源性生长因子激活途径等[4]。
目前，各种原因导致的纤维化疾病发病人群众多，患者往往有多个器官组织受累，尚缺乏有效的治疗方法，社会和经济负担较重。本发明研究发现纤溶酶原可以改善组织器官的纤维化，改善组织器官功能，从而为组织器官纤维化及其相关病症的预防和治疗开辟了一个全新的领域。
发明概述
本发明包括下述各项：
1.一种预防和/或治疗系统性硬化症受试者纤维化及其相关病症的方法，包括给药受试者有效量的纤溶酶原。
2.项1的方法，其中所述纤溶酶原降低系统性硬化症受试者皮肤纤维化。
3.项1或2的方法，其中所述纤溶酶原降低系统性硬化症受试者肺纤维化。
4.项1-3任一项的方法，其中所述纤溶酶原降低系统性硬化症受试者肾脏纤维化。
5.项1-4任一项的方法，其中所述纤溶酶原降低系统性硬化症受试者心脏纤维化或肝纤维化。
6.一种预防和/或治疗受试者组织器官胶原蛋白沉积或纤维化及其相关病症的方法，包括给药受试者有效量的纤溶酶原，其中所述受试者易患组织器官纤维化、有组织器官纤维化倾向或罹患其它疾病并伴有组织器官纤维化。
7.项6的方法，其中所述组织器官胶原蛋白沉积或纤维化包括皮肤纤维化、血管纤维化、心脏纤维化、肺纤维化、肝纤维化、肾纤维化。
8.项6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化包括感染、炎症、超敏反应、肿瘤、组织缺血、组织器官淤血、化学物质、辐射或环境污染导致的损伤引发的或伴随的。
9.项8的方法，其中所述组织器官胶原蛋白沉积或纤维化包括细菌、病毒或寄生虫感染引起的组织器官病变导致的组织器官胶原蛋白沉积或纤维化。
10.项9的方法，其中所述组织器官胶原蛋白沉积或纤维化包括结核杆菌感染导致的肺纤维化。
11.项9的方法，其中所述组织器官胶原蛋白沉积或纤维化为乙型肝炎病毒、丙型肝炎病毒或戊型肝炎病毒感染导致的肝脏纤维化。
12.项9的方法，其中所述组织器官胶原蛋白沉积或纤维化为血吸虫感染导致的肝纤维化。
13.项6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为无菌性炎症或自身免疫反应导致的。
14.项13的方法，其中所述组织器官胶原蛋白沉积或纤维化为慢性肾小球肾炎、肾盂肾炎、肾病综合征、肾功能不全、尿毒症导致的肾脏纤维化。
15.项6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为脂肪导致组织器官损伤所致的。
16.项15的方法，所述组织器官胶原蛋白沉积或纤维化为肺癌导致的肺纤维化、肝癌导致的肝纤维化或肾脏癌症导致的肾脏纤维化。
17.项6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为慢性缺血性组织损伤导致的。
18.项17的方法，其中所述组织器官胶原蛋白沉积或纤维化为冠状动脉粥样硬化、冠心病导致的心脏缺血性纤维化。
19.项17的方法，其中所述组织器官胶原蛋白沉积或纤维化为慢性缺血性肾损伤导致的肾脏纤维化。
20.项6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为心血管疾病导致的组织器官淤血导致的。
21.项20的方法，其中所述组织器官胶原蛋白沉积或纤维化为肝淤血或肺淤血。
22.项6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为药物导致的。
23.项22的方法，其中所述组织器官胶原蛋白沉积或纤维化为药物性肝纤维化或肾纤维化。
24.项6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为吸入性化学物质或环境污染物导致的肺纤维化。
25.项13的方法，其中所述组织器官胶原蛋白沉积或纤维化为诸如系统性红斑狼疮、系统性硬化症、强直性脊柱炎全身性免疫性疾病导致的。
26.项13的方法，其中所述组织器官纤维化为特发性肺纤维化。
27.项1-26任一项的方法，其中所述组织器官纤维化相关病症包括组织器官因纤维化病变导致的功能减弱、障碍或丧失而引发的病症。
28.项27的方法，其中所述组织器官纤维化相关病症包括动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、脑缺血、脑梗塞、肾功能不 全、尿毒症、肝功能障碍、肝硬化、肝昏迷、呼吸困难、肺气肿、肺心病、肺纤维化、强直性脊柱炎。
29.项1-28任一项的用途，其中所述组织器官纤维化或其相关病症通过降低胶原蛋白在组织器官的沉积减少所述组织器官的纤维化、缓解组织器官纤维化相关病症或改善所述组织器官的功能。
30.项1-29任一项的用途，其中所述组织器官纤维化或其相关病症通过减弱组织器官的纤维化程度缓解组织器官纤维化相关病症或改善所述组织器官的功能。
31.根据项1-30任一项的用途，其中所述纤溶酶原可与一种或多种其它药物或治疗手段联合施用。
32.根据项31的用途，其中所述其它药物包括：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗纤维化药物、抗心律失常药物，强心药物，利尿药物、抗肿瘤药物、放化疗药物、炎症调节药物、免疫调节药物、抗病毒药物、抗生素。
33.项1-32任一项的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
34.项1-33任一项的方法，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
35.项1-34任一项的方法，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。
36.项1-35任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
37.项1-36任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
38.项1-36任一项的方法，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。
39.项1-38任一项的方法，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。
40.项1-39任一项的方法，其中所述纤溶酶原是人天然纤溶酶原。
41.项1-40任一项的方法，其中所述受试者是人。
42.项1-41任一项的方法，其中所述受试者缺乏或缺失纤溶酶原。
43.项1-42任一项的方法，所述缺乏或缺失是先天的、继发的和/或局部的。
44.一种用于项1-43任一项的方法的纤溶酶原。
45.一种药物组合物，其包含药学上可接受的载剂和用于项1-43中任一项所述方法的纤溶酶原。
46.一种预防性或治疗性试剂盒，其包含：(i)用于项1-43中任一项所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。
47.根据项46所述的试剂盒，其中所述构件为注射器或小瓶。
48.项46或47的试剂盒，其还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施项1-43中任一项所述方法。
49.一种制品，其包含：
含有标签的容器；和
包含(i)用于项1-43中任一项所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施项1-43中任一项所述方法。
50.项46-48中任一项的试剂盒或项49的制品，还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
51.项50的试剂盒或制品，其中所述其他药物选自下组：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗纤维化药物、抗心律失常药物，强心药物，利尿药物、抗肿瘤药物、放化疗药物、炎症调节药物、免疫调节药物、抗病毒药物、抗生素。
一方面，本发明涉及一种预防和/或治疗受试者组织器官胶原蛋白沉积或纤维化及其相关病症的方法，包括给药受试者有效量的纤溶酶原，其中所述受试者易患组织器官纤维化、有组织器官纤维化倾向或罹患其它疾病 并伴有组织器官纤维化。本发明还涉及纤溶酶原用于预防和/或治疗受试者组织器官胶原蛋白沉积或纤维化及其相关病症的用途。本发明还涉及纤溶酶原用于制备预防和/或治疗受试者组织器官胶原蛋白沉积或纤维化及其相关病症的药物的用途。此外，本发明还涉及用于预防和/或治疗受试者组织器官胶原蛋白沉积或纤维化及其相关病症的纤溶酶原。在一些实施方案中，所述组织器官胶原蛋白沉积或纤维化包括皮肤纤维化、血管纤维化、心脏纤维化、肺纤维化、肝纤维化、肾纤维化。在另一些实施方案中，所述组织器官胶原蛋白沉积或纤维化包括感染、炎症、超敏反应、肿瘤、组织缺血、组织器官淤血、化学物质、辐射或环境污染导致的损伤引发的或伴随的。具体地，所述组织器官胶原蛋白沉积或纤维化包括细菌、病毒或寄生虫感染引起的组织器官病变导致的组织器官胶原蛋白沉积或纤维化，其中，所述组织器官胶原蛋白沉积或纤维化包括结核杆菌感染导致的肺纤维化、乙型肝炎病毒、丙型肝炎病毒或戊型肝炎病毒感染导致的肝脏纤维化、血吸虫感染导致的肝纤维化。在一些实施方案中，所述组织器官胶原蛋白沉积或纤维化为无菌性炎症或自身免疫反应导致的。具体地，所述组织器官胶原蛋白沉积或纤维化为慢性肾小球肾炎、肾盂肾炎、肾病综合征、肾功能不全、尿毒症导致的肾脏纤维化。在另一些实施方案中，所述组织器官胶原蛋白沉积或纤维化为癌症导致组织器官损伤所致的。具体地，所述组织器官胶原蛋白沉积或纤维化为肺癌导致的肺纤维化、肝癌导致的肝纤维化或肾脏癌症导致的肾脏纤维化。在另一些实施方案中，所述组织器官胶原蛋白沉积或纤维化为慢性缺血性组织损伤导致的。具体地，所述组织器官胶原蛋白沉积或纤维化为冠状动脉粥样硬化、冠心病导致的心脏缺血性纤维化和/或慢性缺血性肾损伤导致的肾脏纤维化。在另一些实施方案中，所述组织器官胶原蛋白沉积或纤维化为心血管疾病导致的组织器官淤血导致的。具体地，所述组织器官胶原蛋白沉积或纤维化为肝淤血或肺淤血。在一些实施方案中，所述组织器官胶原蛋白沉积或纤维化为药物导致的。具体地，所述组织器官胶原蛋白沉积或纤维化为药物性肝纤维化或肾纤维化。在一些实施方案中，所述组织器官胶原蛋白沉积或纤维化为吸入性化学物质或环境污染物导致的肺纤维化。在上述实施方案中，所述组织器官胶原蛋白沉积或纤维化为诸如系统性红斑狼疮、系统性硬化 症、强直性脊柱炎全身性免疫性疾病导致的。在一些实施方案中，所述组织器官纤维化为特发性肺纤维化。
在上述实施方案中，所述组织器官纤维化相关病症包括组织器官因纤维化病变导致的功能减弱、障碍或丧失而引发的病症。具体地，所述组织器官纤维化相关病症包括动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、脑缺血、脑梗塞、肾功能不全、尿毒症、肝功能障碍、肝硬化、肝昏迷、呼吸困难、肺气肿、肺心病、肺纤维化、强直性脊柱炎。
在上述实施方案中，所述纤溶酶原可与一种或多种其它药物或治疗方法联用。具体地，所述纤溶酶原可与一种或多种选自如下的药物联用：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗纤维化药物、抗心律失常药物，强心药物，利尿药物、抗肿瘤药物、放化疗药物、炎症调节药物、免疫调节药物、抗病毒药物、抗生素。
在上述实施方案中，所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
在上述实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
在上述实施方案中，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。具体地，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
在上述实施方案中，纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。例如，来自灵长类动物或啮齿类动物的纤维蛋白溶酶原直向同系物，例如来自大猩猩，恒河猴、鼠、牛、马，狗的纤维蛋白溶酶原直向同系物。最优选，本发明的纤维蛋白溶酶原的氨基酸序列如序 列2、6、8、10或12所示。
在上述实施方案中，所述受试者是人。在一些实施方案中，其中所述受试者缺乏或缺失纤溶酶原。具体地，所述缺乏或缺失是先天的、继发的和/或局部的。
在一个实施方案中，所述纤维蛋白溶酶原通过全身或局部给药，优选通过以下途径施用：表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或通过直肠。在一个实施方案中，所述局部给药为直接向骨质疏松区域给药，例如通过敷料，导管等方式来进行。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。在局部施用的情况下，上述剂量还可以根据情况进一步调整。一方面，本发明涉及一种药物组合物，其包含药学上可接受的载剂和用于本发明所述方法的纤溶酶原。
另一方面，本发明涉及一种预防性或治疗性试剂盒，其包含：(i)用于本发明所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)，具体地，所述构件为注射器或小瓶。在一些实施方案中，所述试剂盒还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施本发明所述的方法。
另一方面，本发明还涉及一种制品，其包含：含有标签的容器；和(i)用于本发明所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施本发明所述方法。
在上述实施方案中，所述试剂盒或制品还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。在一些实施方案中，所述其他药物选自下组：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗纤维化药物、抗心 律失常药物，强心药物，利尿药物、抗肿瘤药物、放化疗药物、炎症调节药物、免疫调节药物、抗病毒药物、抗生素。
定义
“纤维化”是肺、肝、肾、血管、腹膜、胰腺、皮肤等组织、器官受到炎症、感染、免疫反应、缺血、化学物质、辐射等各种原因导致持续损伤后，成纤维细胞激活增殖、组织器官内纤维结缔组织增多，实质细胞减少，组织、器官结构破坏和功能丧失的病变。该术语可与“纤维化病变”互换使用。该纤维化病变术语涵盖各种原因导致的心脏纤维化、肺纤维化、肝纤维化、肾纤维化、血管纤维化、皮肤纤维化等组织器官纤维化病变，还包括各种疾病发生、发展过程中伴随产生的心脏纤维化、肺纤维化、肝纤维化、肾纤维化、血管纤维化、皮肤纤维化等组织器官纤维化病变。
组织、器官发生纤维化病变后，其正常结构发生改变，相应的功能减弱或丧失，由此导致的相关病症称为“组织器官纤维化相关病症”。
“心脏纤维化”是指各种原因(例如炎症、感染、免疫反应、缺血、化学物质、辐射)导致的或伴随的心脏组织损伤或各种原因导致的心脏疾病的发生与发展过程中出现的纤维化病变。心脏纤维化病变导致心脏功能受损，由此导致的相关病症称为“心脏纤维化相关病症”，包括但不限于心脏功能受损引起的各器官、组织缺血的症状和病症，例如冠心病、心绞痛、心肌梗死、心律失常、脑缺血、呼吸困难、肾功能不全等。
“肝纤维化”是指由各种原因(例如炎症、感染(例如病毒感染)、免疫反应、缺血、化学物质、辐射、氧化应激和酗酒)导致的或伴随的肝内结缔组织异常增生，肝内弥漫性细胞外基质过度沉淀、肝的正常结构遭到破坏的病理变化(病变)。肝纤维化进一步发展即为肝硬化，也涵盖在本发明“肝纤维化”术语的范围内。肝脏纤维化病变导致肝脏功能受损，由此导致的相关病症称为“肝脏纤维化相关病症”。
“肺纤维化”是指各种原因(例如炎症、感染、免疫反应、缺血、化学物质、辐射)导致的或伴随的肺组织间充质细胞增殖、细胞外基质增生沉积及肺实质的重构而造成的病理过程。肺脏纤维化病变导致肺脏功能受损，由此导致的相关病症称为“肺脏纤维化相关病症”。
“肾脏纤维化”是指各种原因(例如炎症、感染、免疫反应、缺血、化学物质、辐射)导致的或伴随的结缔组织在肾脏的异常聚集，导致肾脏结构改变和功能受损的病理过程。肾脏纤维化病变是几乎所有肾脏疾病进展到后期的共同通路。
肾脏纤维化病变导致肾脏功能受损，由此导致的相关病症称为“肾脏纤维化相关病症”，例如，肾脏功能不全，肾衰，尿毒症等。
组织器官的慢性病变常伴有纤维化，例如肺的慢性炎症，慢性病变，伴有肺纤维化。肝纤维化也是如此，例如乙肝、丙肝、酒精肝、脂肪肝、血吸虫病等都伴有早期肝纤维化。再像慢性肾炎、肾小球炎、肾小管炎等都有肾纤维化；心血管、脑血管、下肢血管硬化、或变窄、或阻塞情况下都有血管纤维化。本发明“纤维化”或“纤维化病变”术语涵盖机体各组织器官的慢性病变常伴有的纤维化病变。
“系统性硬化症”或称“硬皮病”是一种以局限性或弥漫性皮肤增厚和纤维化为特征的全身性自身免疫病。病变特点为皮肤纤维增生及血管洋葱皮样改变，最终导致皮肤硬化、血管缺血。本病临床上以局限性或弥漫性皮肤增厚和纤维化为特征，除皮肤受累外，它也可影响内脏(心、肺和消化道等器官)。
“动脉粥样硬化”是一种慢性的、渐进性动脉疾病，发病时动脉中沉积的脂肪部分或全部堵塞血流。动脉粥样硬化是个渐进的过程。当血液中的脂类浓度大大增加时，便会沿着动脉壁形成脂肪条纹。这些条纹会导致脂肪和胆固醇沉积，这些沉淀依附在原本平滑的动脉内膜上，从而形成小结。这些小结下面继而长出纤维化的瘢痕组织，导致钙沉积。沉积的钙逐渐演变为无法除去的白垩状坚硬薄膜(称为动脉粥样斑)。当与体内的某个组织、器官相连的动脉被堵塞后，因该组织、器官中的动脉受阻而引起的组织、器官的缺血损伤可导致该组织、器官的纤维化病变，例如心脏、肺脏、肝脏、肾脏、血管、腹膜、胰腺、皮肤的纤维化。
糖尿病常伴有动脉粥样硬化的发生，其机制多认为是血糖代谢异常引起的动脉粥样硬化，随着更进一步深入的研究，更多的结果表明，糖尿病引起动脉粥样硬化并非单一因素所致，而是通过多种途径以及较为复杂的机制来诱发和促进动脉粥样硬化的发生及发展[4]。糖尿病以及其伴随的动脉 粥样硬化可导致组织、器官损伤和纤维化，例如心脏、肺脏、肝脏、肾脏、血管、腹膜、胰腺、皮肤等组织、器官的纤维化。
发明详述
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖[5]。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物[6，7]。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PAs的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)[8，9]。
纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa[10，11]。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的潜在来源[12，13]。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成[14]。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位 点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键[15]。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用[11，16，17]。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器[18]。此外，纤溶酶具有激活某些潜在形式的生长因子的能力[19-21]。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域[22，23]，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)[23]，编码 该氨基酸序列的cDNA序列如序列7。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)[24]，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)[25]，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，本专利序列参考专利文献CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤维蛋白溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义为受试者体内纤溶酶原的含量或活性比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义为受试者体内纤溶酶原的含量或活性显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。
在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”是指在纤溶酶原蛋白中，能够与底物中的靶序列结合并发挥蛋白水解功能的活性片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。本发明所述的纤溶酶 原活性片段为包含纤溶酶原的丝氨酸蛋白酶域的蛋白质，优选，本发明所述的纤溶酶原活性片段包含序列14、与序列14具有至少80％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列的蛋白质。因此，本发明所述的纤溶酶原包括含有该纤溶酶原活性片段、并且仍然保持该纤溶酶原活性的蛋白。
目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同 一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”和“处理”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状，和/或部分或完全治愈疾病和/或其症状，并且包括：(a)预防疾病在受试者体内发生，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(b)抑制疾病，即阻滞其形成；和(c)减轻疾病和/或其症状，即引起疾病和/或其症状消退。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠、小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤溶酶原的量。“治疗有效量”会根据所使用的纤溶酶原、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides：Analysis，Synthesis，Biology.第2卷：Special Methods in Peptide Synthesis，Part A.，Merrifield，等J.Am.Chem.Soc.，85：2149-2156(1963)；Stewart等，Solid Phase Peptide Synthesis，2nd ed.Pierce Chem.Co.，Rockford，Ill.(1984)；和Ganesan A.2006Mini Rev.Med Chem.6：3-10和Camarero JA等2005Protein Pept Lett.12：723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的抗-Tau抗体(例如编码主题抗-Tau抗体的多核苷酸)。参见Winnacker，From Genes to Clones，VCH Publishers，N.Y.，N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos 细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等，Immunol.Rev.89：49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等，J.Immunol.148：1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除主题抗体以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体，赋形剂，或稳定剂(Remington′s Pharmaceutical Sciences，16版，Osol，A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物，抗心律失常的药物，治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington′s Pharmaceutical Sciences 16th edition，Osol，A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.，15：167-277(1981)；Langer，Chem.Tech.，12：98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919，EP 58，481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22：547(1983))，不可降解的乙烯-乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过静脉内，腹膜内，皮下，颅内，鞘内，动脉内(例如经由颈动脉)，肌内来实现本发明药物组合物的施用。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射 有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以例如为例如每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天1-10mg/kg。在本发明的药物施用过程中需要实时评估治疗效果和安全性。
制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含可用于治疗由糖尿病引起的心血管病及其相关病症的本发明纤溶酶原或纤溶酶。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原/纤溶酶。所述容器上或所附的标签说明所述组合物用于治疗本发明所述由糖尿病引起的心血管病及其相关病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外， 所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
附图简述
图1博莱霉素诱导的系统性硬化模型小鼠给予纤溶酶原21天后肾脏天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，在博莱霉素诱导的系统性硬化症小鼠模型中，给溶媒PBS对照组肾脏胶原纤维化(箭头标识)程度比给纤溶酶原组高。说明纤溶酶原能有效降低博莱霉素诱导的肾脏纤维化。
图2博莱霉素诱导的系统性硬化模型小鼠给予纤溶酶原21天后心脏天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。研究发现，在博莱霉素诱导的系统性硬化症小鼠模型中，给溶媒PBS对照组心脏胶原蛋白沉积(箭头标识)程度比给纤溶酶原组高。说明纤溶酶原能有效降低博莱霉素诱导的心脏纤维化。
图3博莱霉素诱导的系统性硬化模型小鼠给予纤溶酶原21天后肺天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，在博莱霉素诱导的系统性硬化症小鼠模型中，给溶媒PBS组小鼠肺纤维化(箭头标识)程度比给纤溶酶原组高；给纤溶酶原组小鼠肺部肺泡壁形态接近正常，炎症水平细胞明显减少，纤维化程度明显低于给溶媒PBS组，且统计差异显著(*表示P＜0.05)。
图4博莱霉素诱导的系统性硬化模型小鼠给予纤溶酶原21天后皮肤天狼星红染色代表性图片。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为PLG活性受损组。结果显示，在博莱霉素诱导的系统性硬化症小鼠模型中，给溶媒PBS组和PLG活性受损组，真皮上部胶原纤维束明显增多，胶原纤维粗大，排列致密，真皮层增厚；给纤溶酶原组真皮层中成纤维细胞明显少于给溶媒PBS组，且皮肤真皮层厚度接近正常水平。
图5 24-25周龄糖尿病小鼠给予纤溶酶原31天后肾脏IV型胶原免疫染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组IV胶原阳性着色(箭头标识)明显多于给溶媒PBS对照组，说明纤溶酶原能改善糖尿病小鼠肾脏的纤维化。
图6 26周龄糖尿病小鼠给予纤溶酶原35天后肾脏masson染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给溶媒PBS对照组，肾小球系膜增生，系膜基质增多，肾间质轻度纤维化(箭头标识)，增生的纤维化呈蓝色。给纤溶酶原组肾小球系膜细胞及基质明显比对照组少，肾间质纤维化明显减少。说明纤溶酶原能够改善糖尿病小鼠肾脏的纤维化病变。
图7 24-25周龄糖尿病小鼠给予纤溶酶原31天后心脏masson染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给溶媒PBS对照组心肌纤维之间可见着蓝色的增生的胶原纤维(箭头标识)，呈轻度心肌纤维化；给纤溶酶原组心肌纤维之间可见少量的浅蓝色的增生的胶原纤维，较之于对照组，心肌纤维化明显减轻。说明纤溶酶原能够改善糖尿病小鼠心脏的纤维化。
图8 17-18周龄糖尿病小鼠给予纤溶酶原35天后心脏天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组小鼠胶原纤维的沉积(箭头标识)明显少于给溶媒PBS对照组。说明纤溶酶原能够减少相对年轻(17-18周龄)糖尿病小鼠心脏的纤维化。
图9 26-27周龄糖尿病小鼠给予纤溶酶原35天后心脏天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组小鼠胶原蛋白的沉积(箭头标识)明显少于给溶媒PBS对照组。说明纤溶酶原能减弱相对年老(26-27周龄)糖尿病小鼠心脏的纤维化。
图10顺铂肾纤维化模型小鼠给予纤溶酶原7天后肾脏IV型胶原免疫染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给溶媒PBS对照组肾脏IV型胶原阳性表达(箭头标识)明显高于给纤溶酶原组。说明纤溶酶原能改善顺铂肾纤维化模型小鼠肾脏的纤维化。
图11 ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后心脏天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组胶原的沉积(箭头标识)明显少于给溶媒PBS对照组，说明纤溶酶原能减轻ApoE动脉粥样硬化模型小鼠心脏纤维化。
图12 C57高血脂模型小鼠给予纤溶酶原30天后心脏天狼星红染色代表性图片。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤 溶酶原组胶原的沉积(箭头标识)明显少于给溶媒PBS对照组，说明纤溶酶原能减轻高血脂模型小鼠心脏纤维化。
图13嘌呤诱导的慢性肾损伤模型小鼠给予纤溶酶原10天后肾脏天狼星红染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组，C为PLG活性受损组，D为定量分析结果。给纤溶酶原组胶原的沉积(箭头标识)明显少于给溶媒PBS对照组和PLG活性受损组，且给纤溶酶原组与PLG活性受损组定量分析统计差异显著(*表示P＜0.05)。说明纤溶酶原能减轻慢性肾损伤所致的肾脏纤维化，促进肾损伤的修复。
图14 24-25周龄糖尿病小鼠给予纤溶酶原31天后胰岛天狼星红染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组小鼠胰岛胶原沉积(箭头标识)明显少于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能改善糖尿病所致的胰岛损伤和纤维化。
图15 ApoE动脉粥样硬化模型小鼠给予纤溶酶原30天后主动脉窦天狼星红染色代表性图片。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组。结果显示给纤溶酶原组胶原蛋白沉积(箭头标识)的面积明显小于给溶媒PBS对照组，说明纤溶酶原能够消减动脉粥硬化模型小鼠主动脉窦纤维化水平。
图16给予纤溶酶原14天后四氯化碳诱导的肝纤维化模型小鼠肝脏天狼星红染色代表性图片。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给纤溶酶原组胶原蛋白沉积明显少于给溶媒PBS对照组，且与空白对照小鼠胶原蛋白沉积水平接近。说明纤溶酶原能减少肝脏胶原蛋白的沉积，改善肝纤维化模型小鼠肝脏纤维化。
图17 16周高脂血症模型小鼠给予纤溶酶原30天后主动脉窦天狼星红染色代表性图片。A、C为给溶媒PBS对照组，B、D为给纤溶酶原组。结果显示，给纤溶酶原组主动脉窦管壁内膜胶原蛋白沉积(箭头标识)的面积明显小于给溶媒PBS对照组，说明纤溶酶原能够消减高脂血症模型小鼠主动脉窦管壁内膜纤维化水平。
图18给予纤溶酶原30天后3％胆固醇高脂血症模型小鼠肾脏天狼星红染色观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给纤溶酶原组肾脏胶原蛋白沉积 (箭头标识)明显少于给溶媒PBS对照组，且统计差异显著；给纤溶酶原组纤维化基本恢复到正常水平。说明纤溶酶原能有效的减少3％胆固醇高脂血症模型小鼠肾脏纤维化。
图19给予纤溶酶原28天四氯化碳诱导的肝脏纤维化模型小鼠肝脏天狼星红染色观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给纤溶酶原组胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)；与给溶媒PBS对照组相比，给纤溶酶原组胶原沉积水平更加接近空白对照小鼠。说明纤溶酶原能减少肝纤维化模型小鼠肝脏胶原蛋白的沉积，改善肝脏纤维化。
实施例
实施例1纤溶酶原降低系统性硬化症小鼠肾脏纤维化
取12周龄C57雄鼠10只以，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各5只。实验开始当天记为第0天并称重分组，第1天开始造模给药，所有小鼠按0.1mg/0.1ml/只/天皮下注射博莱霉素诱导系统性硬化症，并开始给纤溶酶原或PBS，连续造模给药21天。给纤溶酶原组小鼠按1mg/0.1ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组尾静脉注射相同体积的PBS。在第22天处死小鼠并取肾脏在4％多聚甲醛固定液中固定24小时。固定后的肾脏经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，在博莱霉素诱导的系统性硬化症小鼠模型中，给溶媒PBS对照组(图1A)肾脏胶原纤维化(箭头标识)程度比给纤溶酶原组(图1B)高。说明纤溶酶原能有效降低博莱霉素诱导的肾脏纤维化。
实施例2纤溶酶原降低系统性硬化症小鼠心脏纤维化
取12周龄C57雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各5只。实验开始当天记为第0天并称重分组，第1天开始造模 给药，按0.1mg/0.1ml/只/天皮下注射博莱霉素诱导系统性硬化症[26]，并开始给纤溶酶原或PBS，连续给药21天。给纤溶酶原组小鼠按1mg/0.1ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组尾静脉注射相同体积的PBS，在第22天处死小鼠并取心脏在4％多聚甲醛固定液中固定24小时。固定后的心脏经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
研究发现，在博莱霉素诱导的系统性硬化症小鼠模型中，镜下观察到给溶媒PBS对照组(图2A)心脏胶原蛋白沉积比给纤溶酶原组(图2B)高。说明纤溶酶原能有效降低博莱霉素诱导的心脏纤维化。
实施例3纤溶酶原降低系统性硬化症小鼠肺纤维化
取12周龄C57雄鼠17只，随机分为两组，给溶媒PBS对照组11只和给纤溶酶原组6只。实验开始当天记为第0天并称重分组，第1天开始造模给药，两组小鼠按0.1mg/0.1ml/只/天皮下注射博莱霉素诱导系统性硬化症[26]，并开始给纤溶酶原或PBS，连续造模给药21天。给纤溶酶原组小鼠按1mg/0.1ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组以相同方式给予相同体积的PBS。第22天处死小鼠并取肺组织在4％多聚甲醛固定液中固定24小时。固定后的肺组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
研究发现，在博莱霉素诱导的系统性硬化症小鼠模型中，镜下观察给溶媒PBS组(图3A)胶原纤维化(箭头标识)程度比给纤溶酶原组(图3B)高；给纤溶酶原组小鼠肺部肺泡壁形态接近正常水平，炎症细胞明显减少，纤维化程度明显低于给溶媒PBS组，且统计差异显著(图3C)。说明纤溶酶原可有效降低博莱霉素诱导的系统性硬化症小鼠肺组织纤维化。
实施例4纤溶酶原降低系统性硬化症小鼠皮肤纤维化
取12周龄C57雄鼠15只，随机分为三组，空白对照组、给溶媒PBS(PBS为磷酸缓冲盐溶液(Phosphate Buffer Saline)，本文中为纤溶酶原的溶媒。)对照组和给纤溶酶原组，每组各5只，并取13周龄PLG活性受损小鼠5只。实验开始当天记为第0天称重分组，第二天开始造模给药，给溶媒PBS对照组、给纤溶酶原组以及PLG活性受损小鼠按0.1mg/0.1ml/只/天皮下注射博莱霉素诱导系统性硬化症[26]。空白对照组按0.1ml/只/天皮下注射生理盐水，同时开始给纤溶酶原或PBS并记为第1天，连续造模给药21天。给纤溶酶原组小鼠按1mg/0.1ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS，正常小鼠组和PLG活性受损小鼠不做给药处理。在第22天处死小鼠并取背部皮肤组织在4％多聚甲醛固定液中固定24小时。固定的皮肤组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在100倍光学显微镜下观察。
天狼星红染色可使胶原持久染色，作为病理切片特殊染色方法，天狼星红染色可以特异显示胶原组织。
结果显示，在博莱霉素诱导的系统性硬化症小鼠模型中，显微镜下观察到给溶媒PBS组(图4B)和PLG活性受损组小鼠(图4D)真皮上部胶原纤维束明显增多，胶原纤维粗大，排列致密，真皮层增厚；给纤溶酶原组(图4C)，真皮层中成纤维细胞明显少于给溶媒PBS对照组，皮肤真皮层厚度基本接近正常水平(图4A)。说明纤溶酶原能有效降低博莱霉素诱导的皮肤纤维化。
实施例5纤溶酶原降低糖尿病小鼠肾脏胶原蛋白沉积
24-25周龄db/db雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各5只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS，连续给药31天。给纤溶酶原组小鼠按2mg/0.2ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。给纤溶酶原31天后处死小鼠并取肾脏组织在4％多聚甲醛固定液中固定24小时。固定后的肾脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为4μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟， 0.01MPBS洗2次，每次5分钟。10％正常羊血清(Vector laboratories，Inc.，USA)封闭1小时，时间到后甩去血清，用PAP笔圈出组织。针对IV胶原的兔抗小鼠多克隆抗体(Abcam)4℃孵育过夜，TBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，TBS洗2次。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟，梯度酒精脱水，二甲苯透明透明，中性树胶封片，切片在200倍光学显微镜下观察。
糖尿病肾病是糖尿病慢性并发症，肾小球硬化以及肾间质纤维化是其典型的病理改变[27]。
结果显示，给纤溶酶原组(图5B)IV胶原阳性着色明显多于给溶媒PBS对照组(图5A)，说明纤溶酶原能降低肾脏组织胶原蛋白的沉积(箭头标识)，提示纤溶酶原有望通过降低肾脏组织胶原蛋白的沉积，阻止糖尿病所致的肾脏组织纤维化。
实施例6纤溶酶原改善糖尿病小鼠肾脏纤维化
26周龄db/db雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各5只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS，连续给药35天。给纤溶酶原组小鼠按2mg/0.2ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。第36天处死小鼠并取肾脏组织在4％多聚甲醛固定液中固定24小时。固定后的肾脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为4μm，切片脱蜡复水后放入重铬酸钾溶液过夜。铁苏木素染3到5分钟，流水稍洗。1％盐酸酒精分化，氨水处理1秒，水洗。丽春红酸性品红液染8分钟，水中快速漂洗。1％磷钼酸水溶液处理约2分钟，苯胺蓝液复染6分钟。1％冰醋酸漂洗1分钟左右。无水乙醇脱水二甲苯透明后封片，切片在200倍光学显微镜下观察。
Masson染色可以显示组织的纤维化。结果显示，给溶媒PBS对照组(图6A)肾小球系膜增生，系膜基质增多，肾间质轻度纤维化(箭头标识)，增生的纤维化呈蓝色。给纤溶酶原组(图6B)肾小球系膜细胞及基质明显比对照组少，肾间质纤维化明显减少。说明纤溶酶原能改善糖尿病小鼠肾脏的纤维化。
实施例7纤溶酶原改善24-25周龄糖尿病小鼠心脏纤维化
24-25周龄db/db雄鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各5只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS，连续给药31天。给纤溶酶原组小鼠按2mg/0.2ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。给纤溶酶原31天后处死小鼠并取心脏组织在4％多聚甲醛固定液中固定24小时。固定后的心脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为4μm，切片脱蜡复水后放入重铬酸钾溶液过夜。铁苏木素染3到5分钟，流水稍洗。1％盐酸酒精分化，氨水处理1秒，水洗。丽春红酸性品红液染8分钟，水中快速漂洗。1％磷钼酸水溶液处理约2分钟，苯胺蓝液复染6分钟。1％冰醋酸漂洗大概1分钟左右。无水乙醇脱水二甲苯透明后封片，切片在200倍光学显微镜下观察。
糖尿病最常见的并发症是结缔组织过度累积(病理性纤维化)，心肌间质纤维化可能是糖尿病心肌病变的特征性病理改变[28-29]。
Masson染色可以显示组织的纤维化。结果显示，给溶媒PBS对照组(图7A)心肌纤维之间可见着蓝色的增生的胶原纤维(箭头标识)，呈轻度心肌纤维化；给纤溶酶原组(图7B)心肌纤维之间可见少量的浅蓝色的增生的胶原纤维，较之于对照组，心肌纤维化明显减轻。说明纤溶酶原能改善糖尿病小鼠心脏的纤维化。
实施例8纤溶酶原降低17-18周龄糖尿病小鼠心脏胶原蛋白沉积
17-18周龄db/db雄鼠8只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各4只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS，连续给药35天。给纤溶酶原组小鼠按2mg/0.2ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。给纤溶酶原35天后处死小鼠并取心脏组织在4％多聚甲醛固定液中固定24小时。固定后的心脏经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图8B)小鼠胶原纤维的沉积(箭头标识)明显少于给溶媒PBS对照组(图8A)。说明纤溶酶原能降低心脏组织胶原 蛋白的沉积，提示纤溶酶原有望通过降低心脏组织胶原蛋白的沉积，减少相对年轻(17-18周龄)糖尿病小鼠心脏组织纤维化。
实施例9纤溶酶原降低26-27周龄糖尿病小鼠心脏胶原蛋白沉积
26-27周龄db/db雄鼠9只，随机分为两组，给溶媒PBS对照组5只和给纤溶酶原组4只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS，连续给药35天。给纤溶酶原组小鼠按2mg/0.2ml/只/天尾静脉注射纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。给纤溶酶原35天后处死小鼠并取心脏组织在4％多聚甲醛固定液中固定24小时。固定后的心脏经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红染色60分钟后，流水冲洗，苏木素染色1分钟，流水冲洗，1％盐酸酒精和氨水分化返蓝，流水冲洗，烘干后封片，在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图9B)小鼠胶原纤维的沉积(箭头标识)明显少于给溶媒PBS对照组(图9A)。说明纤溶酶原能降低心脏组织胶原蛋白的沉积，提示纤溶酶原有望通过降低心脏组织胶原蛋白的沉积，减少相对年老(26-27周龄)糖尿病小鼠心脏组织纤维化。
实施例10纤溶酶原降低顺铂肾脏纤维化模型小鼠肾脏的纤维化
8-9周龄健康的雄性C57小鼠10只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各5只。分组完成后，按10mg/Kg体重单次腹腔注射顺铂，建立肾脏纤维化模型[30]。建立模型后，给纤溶酶原组1mg/0.1ml/只/天经尾静脉注射给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS。实验开始当天记为第0天称体重并分组，第1天开始腹腔注射顺铂造模，造模后3小时内给予纤溶酶原或溶媒PBS，给药周期7天。第8天处死小鼠，取肾脏在4％多聚甲醛固定液中固定24小时。固定后的肾脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为5μm，切片脱蜡复水后水洗1次。柠檬酸修复30分钟，室温冷却10分钟后水轻柔冲洗。以3％双氧水孵育15分钟，用PAP笔圈出组织。10％的羊血清(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液。兔抗小鼠IV胶原抗体(Abcam)4℃孵育过夜，TBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，TBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色， 水洗3次后苏木素复染30秒，流水返蓝5分钟，然后TBS洗1次。梯度脱水透明并封片，切片在200倍光学显微镜下观察。
顺铂是临床上应用广泛、疗效可靠的广谱抗肿瘤药物，但具有严重的肾毒性，主要是肾小管及肾间质损伤，最终发展为肾纤维化[30]。该实验结果显示，给溶媒PBS对照组(图10A)肾脏IV型胶原阳性表达(箭头标识)明显高于给溶纤溶酶原组(图10B)。说明纤溶酶原可改善顺铂肾脏纤维化模型小鼠肾脏的纤维化。
实施例11纤溶酶原改善ApoE动脉粥样硬化小鼠心脏纤维化水平
6周龄ApoE雄性小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周，诱导动脉粥样硬化[31，32]。在给药前三天每只小鼠取血50μL，检测总胆固醇浓度，并根据检测结果将小鼠随机分为两组，给溶媒PBS对照组7只以及给纤溶酶原组6只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，期间一直饲喂高脂高胆固醇饲料。于第31天处死小鼠，取心脏于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图11B)胶原的沉积(箭头标识)明显少于给溶媒PBS对照组(图11A)，提示纤溶酶原能通过减少ApoE动脉粥样硬化模型小鼠心脏组织的胶原蛋白沉积，阻止和降低动脉粥样硬化所致的心脏纤维化。
实施例12纤溶酶原降低高血脂模型小鼠心脏纤维化
6周龄C57雄性小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周，诱导高血脂[33，34]。在给药前三天每只小鼠取血50μL，检测总胆固醇浓度，并据其将小鼠随机分为两组，给溶媒PBS对照组6只以及给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，期间一直饲喂高脂高胆固醇饲料。于第31天处死小鼠，取心脏组织于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水 和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图12B)胶原沉积(箭头标识)明显少于给溶媒PBS对照组(图12A)，提示纤溶酶原能通过减少高血脂模型小鼠心脏组织胶原蛋白沉积，从而阻止和降低高血脂所致的心脏纤维化。
实施例13纤溶酶原修复慢性肾衰竭模型肾脏的纤维化
8-9周龄的PLG活性正常雄性小鼠12只以及PLG活性受损雄性小鼠6只，PLG活性正常小鼠随机分为两组，给纤溶酶原组和给溶媒PBS对照组各6只。三组小鼠每天饲喂0.25％嘌呤饲料(南通特洛菲)，建立慢性肾衰竭模型[35]。造模当天记为第1天，同时开始给药。给纤溶酶原组按1mg/0.1ml/只/天给予纤溶酶原，给溶媒PBS对照组以相同方式给予相同体积的PBS，连续造模给药10天，PLG活性受损小鼠不做处理。第11天处死小鼠取肾脏于4％多聚甲醛中固定24小时。固定后的肾脏经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红染色60分钟后，流水冲洗，苏木素染色1分钟，流水冲洗，1％盐酸酒精和氨水分化返蓝，流水冲洗，烘干后封片，切片在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图13B)胶原的沉积(箭头标识)要明显少于给溶媒PBS对照组(图13A)和PLG活性受损组(图13C)，且给纤溶酶原组与PLG活性受损组统计差异显著(P＝0.018)(图13D)。说明纤溶酶原能显著减轻慢性肾损伤动物肾脏组织中胶原蛋白的沉积，阻止和减轻慢性肾损伤所致的肾脏纤维化。
实施例14纤溶酶原减少糖尿病小鼠胰岛胶原沉积
24-25周龄db/db雄性小鼠16只，随机分为两组，给纤溶酶原组10只，给溶媒PBS对照组6只。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。实验开始当天记为第0天称重分组，第1天开始给纤溶酶原或PBS，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm， 切片脱蜡至水后水洗1次，以0.1％天狼星红染色60分钟后，流水冲洗，苏木素染色1分钟，流水冲洗，1％盐酸酒精和氨水分化返蓝，流水冲洗，烘干后封片，切片在200倍光学显微镜下观察。
结果显示，给纤溶酶原组小鼠(图14B)胰岛胶原沉积(箭头标识)明显低于给溶媒PBS对照组(图14A)，且统计差异显著(图14C)。说明纤溶酶原能显著减轻糖尿病小鼠胰腺组织中胶原蛋白的沉积，阻止和减轻胰腺的损伤和纤维化。
实施例15纤溶酶原改善ApoE动脉粥样硬化小鼠主动脉窦纤维化
6周龄雄性ApoE小鼠13只饲喂高脂高胆固醇饲料(南通特洛菲，TP2031)16周以诱导动脉粥样硬化模型[31，32]。成模后的小鼠在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组7只，给纤溶酶原组6只。开始给药定为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，并于第31天处死小鼠，取心脏于4％多聚甲醛固定24-48小时，分别于15％、30％蔗糖中4℃过夜沉底，OCT包埋，冰冻切片厚度8μm，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，切片在40(15A、15B)、200倍(15C、15D)光学显微镜下观察。
结果显示，给纤溶酶原组(图15B、D)主动脉窦管壁内膜胶原蛋白沉积(箭头标识)的面积明显小于给溶媒PBS对照组(图15A、C)，说明纤溶酶原能够消减动脉粥硬化模型小鼠主动脉窦纤维化水平。
实施例16纤溶酶原改善四氯化碳诱导的肝脏纤维化
9周龄C57雌性小鼠15只，随机分为三组，空白对照组、给溶媒PBS对照组和给纤溶酶原组，每组各5只。给溶媒PBS对照组和给纤溶酶原组小鼠按照1mL/kg体重腹腔注射四氯化碳，每周三次，连续注射两周，建立肝纤维化模型[36，37]，空白对照小鼠按照模型小鼠注射方式注射相应体积的玉米油。四氯化碳需用玉米油稀释，四氯化碳与玉米油稀释比例为1∶3。模型建立后开始给药，开始给药当天记为第1天，给纤溶酶原组小鼠按照1mg/0.1mL/只/天尾静脉注射人源纤溶酶原，给溶媒PBS对照组尾静脉注射同体积的PBS，空白对照组不做注射处理，连续给药14天。第15天处死 小鼠取肝脏，于4％多聚甲醛中固定24小时。固定后的肝脏经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红染色60分钟后，流水冲洗，苏木素染色1分钟，流水冲洗，1％盐酸酒精和氨水分化返蓝，流水冲洗，烘干后封片，切片在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图16C)胶原蛋白沉积明显少于给溶媒PBS对照组(图16B)，且给纤溶酶原组比给PBS组胶原蛋白沉积水平更接近空白对照小鼠(图16A)。说明纤溶酶原能减少肝脏胶原蛋白的沉积，改善肝纤维化模型小鼠肝脏纤维化。
实施例17纤溶酶原降低16周高脂血症模型小鼠主动脉窦纤维化
6周龄雄性C57小鼠11只饲喂高脂高胆固醇饲料(南通特洛菲，货号TP2031)16周以诱导高脂血症模型[30，31]，此模型定为16周高脂血症模型。成模后的小鼠继续饲喂高胆固醇饲料。在给药前三天每只取血50μl以检测总胆固醇(T-CHO)含量，并根据T-CHO含量随机分为两组，给溶媒PBS对照组6只，给纤溶酶原组5只。开始给药记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。给药30天，于第31天处死小鼠，取材心脏于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。主动脉窦切片厚度为3μm，切片脱蜡复水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在40(17A、17B)、200倍(17C、17D)光学显微镜下观察。
结果显示，给纤溶酶原组(图17B、17D)主动脉窦管壁内膜胶原蛋白沉积(箭头标识)的面积明显小于给溶媒PBS对照组(图17A、17C)，说明纤溶酶原能够消减高脂血症模型小鼠主动脉窦纤维化水平。
实施例18纤溶酶原降低3％胆固醇高脂血症模型小鼠肾脏纤维化
9周龄雄性C57小鼠16只饲喂3％胆固醇高脂饲料(南通特洛菲)4周，诱导高脂血症[30，31]，此模型定为3％胆固醇高脂血症模型，成模后的小鼠继续饲喂3％胆固醇高脂饲料。另取相同周龄的雄性C57小鼠5只作为空白对 照组，实验期间饲喂普通维持饲料。在给药前三天每只小鼠取血50μL，检测总胆固醇，模型小鼠根据总胆固醇浓度和体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各8只。开始给药记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。在第30天给药后小鼠给药30天，于第31天处死小鼠，取材肾脏于4％多聚甲醛固定24-48小时。固定后的组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片厚度为3μm，切片脱蜡复水后水洗1次，以0.1％天狼星红饱和苦味酸染色30分钟后，流水冲洗2min，苏木素染色1分钟，流水冲洗，1％盐酸酒精分化，氨水返蓝，流水冲洗，烘干后中性树胶封片，在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图18C)肾脏胶原蛋白沉积(箭头标识)明显少于给溶媒PBS对照组(图18B)，且统计差异显著(图18D)；给纤溶酶原组纤维化基本恢复到正常水平(图18A)。说明纤溶酶原能有效的减少3％胆固醇高脂血症模型小鼠肾脏纤维化。
实施例19纤溶酶原减少四氯化碳诱导肝脏纤维化过程肝脏胶原蛋白沉积
7-8周龄C57雌性小鼠20只，随机分为三组，空白对照组5只、给溶媒PBS对照组7只和给纤溶酶原组8只。给溶媒PBS对照组和给纤溶酶原组小鼠按照1mL/kg体重腹腔注射四氯化碳，每周三次，连续注射四周，建立肝纤维化模型[36，37]，空白对照小鼠腹腔注射相应体积的玉米油。四氯化碳需用玉米油稀释，四氯化碳与玉米油稀释比例为1∶3。造模当天开始给药，记为第1天，给纤溶酶原组小鼠尾静脉注射人源纤溶酶原1mg/0.1mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，空白对照组不做注射处理，连续给药28天。第29天处死小鼠取肝脏于4％多聚甲醛中固定24小时。固定后的肝脏经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红染色60分钟后，流水冲洗，苏木素染色1分钟，流水冲洗，1％盐酸酒精和氨水分化返蓝，流水冲洗，烘干后封片，切片在200倍光学显微镜下观察。
结果显示，给纤溶酶原组(图19C)胶原蛋白沉积明显少于给溶媒PBS对照组(图19B)，且统计差异显著(图19D)；与给溶媒PBS对照组相比，给纤溶酶原组胶原沉积(箭头标识)水平更加接近空白对照小鼠 (图19A)。说明纤溶酶原能减少肝纤维化模型小鼠肝脏胶原蛋白的沉积，改善肝脏纤维化。
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Claims (51)

  1. 一种预防和/或治疗系统性硬化症受试者纤维化及其相关病症的方法，包括给药受试者有效量的纤溶酶原。
  2. 权利要求1的方法，其中所述纤溶酶原降低系统性硬化症受试者皮肤纤维化。
  3. 权利要求1或2的方法，其中所述纤溶酶原降低系统性硬化症受试者肺纤维化。
  4. 权利要求1-3任一项的方法，其中所述纤溶酶原降低系统性硬化症受试者肾脏纤维化。
  5. 权利要求1-4任一项的方法，其中所述纤溶酶原降低系统性硬化症受试者心脏纤维化或肝纤维化。
  6. 一种预防和/或治疗受试者组织器官胶原蛋白沉积或纤维化及其相关病症的方法，包括给药受试者有效量的纤溶酶原，其中所述受试者易患组织器官纤维化、有组织器官纤维化倾向或罹患其它疾病并伴有组织器官纤维化。
  7. 权利要求6的方法，其中所述组织器官胶原蛋白沉积或纤维化包括皮肤纤维化、血管纤维化、心脏纤维化、肺纤维化、肝纤维化、肾纤维化。
  8. 权利要求6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化包括感染、炎症、超敏反应、肿瘤、组织缺血、组织器官淤血、化学物质、辐射或环境污染导致的损伤引发的或伴随的。
  9. 权利要求8的方法，其中所述组织器官胶原蛋白沉积或纤维化包括细菌、病毒或寄生虫感染引起的组织器官病变导致的组织器官胶原蛋白沉积或纤维化。
  10. 权利要求9的方法，其中所述组织器官胶原蛋白沉积或纤维化包括结核杆菌感染导致的肺纤维化。
  11. 权利要求9的方法，其中所述组织器官胶原蛋白沉积或纤维化为乙型肝炎病毒、丙型肝炎病毒或戊型肝炎病毒感染导致的肝脏纤维化。
  12. 权利要求9的方法，其中所述组织器官胶原蛋白沉积或纤维化为血吸虫感染导致的肝纤维化。
  13. 权利要求6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为无菌性炎症或自身免疫反应导致的。
  14. 权利要求13的方法，其中所述组织器官胶原蛋白沉积或纤维化为慢性肾小球肾炎、肾盂肾炎、肾病综合征、肾功能不全、尿毒症导致的肾脏纤维化。
  15. 权利要求6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为脂肪导致组织器官损伤所致的。
  16. 权利要求15的方法，所述组织器官胶原蛋白沉积或纤维化为肺癌导致的肺纤维化、肝癌导致的肝纤维化或肾脏癌症导致的肾脏纤维化。
  17. 权利要求6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为慢性缺血性组织损伤导致的。
  18. 权利要求17的方法，其中所述组织器官胶原蛋白沉积或纤维化为冠状动脉粥样硬化、冠心病导致的心脏缺血性纤维化。
  19. 权利要求17的方法，其中所述组织器官胶原蛋白沉积或纤维化为慢性缺血性肾损伤导致的肾脏纤维化。
  20. 权利要求6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为心血管疾病导致的组织器官淤血导致的。
  21. 权利要求20的方法，其中所述组织器官胶原蛋白沉积或纤维化为肝淤血或肺淤血。
  22. 权利要求6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为药物导致的。
  23. 权利要求22的方法，其中所述组织器官胶原蛋白沉积或纤维化为药物性肝纤维化或肾纤维化。
  24. 权利要求6或7的方法，其中所述组织器官胶原蛋白沉积或纤维化为吸入性化学物质或环境污染物导致的肺纤维化。
  25. 权利要求13的方法，其中所述组织器官胶原蛋白沉积或纤维化为诸如系统性红斑狼疮、系统性硬化症、强直性脊柱炎全身性免疫性疾病导致的。
  26. 权利要求13的方法，其中所述组织器官纤维化为特发性肺纤维化。
  27. 权利要求1-26任一项的方法，其中所述组织器官纤维化相关病症包括组织器官因纤维化病变导致的功能减弱、障碍或丧失而引发的病症。
  28. 权利要求27的方法，其中所述组织器官纤维化相关病症包括动脉粥样硬化、冠心病、心绞痛、心肌梗死、心律失常、脑缺血、脑梗塞、肾功能不全、尿毒症、肝功能障碍、肝硬化、肝昏迷、呼吸困难、肺气肿、肺心病、肺纤维化、强直性脊柱炎。
  29. 权利要求1-28任一项的用途，其中所述组织器官纤维化或其相关病症通过降低胶原蛋白在组织器官的沉积减少所述组织器官的纤维化、缓解组织器官纤维化相关病症或改善所述组织器官的功能。
  30. 权利要求1-29任一项的用途，其中所述组织器官纤维化或其相关病症通过减弱组织器官的纤维化程度缓解组织器官纤维化相关病症或改善所述组织器官的功能。
  31. 根据权利要求1-30任一项的用途，其中所述纤溶酶原可与一种或多种其它药物或治疗手段联合施用。
  32. 根据权利要求31的用途，其中所述其它药物包括：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗纤维化药物、抗心律失常药物，强心药物，利尿药物、抗肿瘤药物、放化疗药物、炎症调节药物、免疫调节药物、抗病毒药物、抗生素。
  33. 权利要求1-32任一项的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
  34. 权利要求1-33任一项的方法，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
  35. 权利要求1-34任一项的方法，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。
  36. 权利要求1-35任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
  37. 权利要求1-36任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
  38. 权利要求1-36任一项的方法，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。
  39. 权利要求1-38任一项的方法，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。
  40. 权利要求1-39任一项的方法，其中所述纤溶酶原是人天然纤溶酶原。
  41. 权利要求1-40任一项的方法，其中所述受试者是人。
  42. 权利要求1-41任一项的方法，其中所述受试者缺乏或缺失纤溶酶原。
  43. 权利要求1-42任一项的方法，所述缺乏或缺失是先天的、继发的和/或局部的。
  44. 一种用于权利要求1-43任一项的方法的纤溶酶原。
  45. 一种药物组合物，其包含药学上可接受的载剂和用于权利要求1-43中任一项所述方法的纤溶酶原。
  46. 一种预防性或治疗性试剂盒，其包含：(i)用于权利要求1-43中任一项所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。
  47. 根据权利要求46所述的试剂盒，其中所述构件为注射器或小瓶。
  48. 权利要求46或47的试剂盒，其还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施权利要求1-43中任一项所述方法。
  49. 一种制品，其包含：
    含有标签的容器；和
    包含(i)用于权利要求1-43中任一项所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施权利要求1-43中任一项所述方法。
  50. 权利要求46-48中任一项的试剂盒或权利要求49的制品，还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
  51. 权利要求50的试剂盒或制品，其中所述其他药物选自下组：降血脂药物、抗血小板药物、降血压药物、扩张血管药物、降血糖药物、抗凝血药物、溶血栓药物，保肝药物，抗纤维化药物、抗心律失常药物，强心药物，利尿药物、抗肿瘤药物、放化疗药物、炎症调节药物、免疫调节药物、抗病毒药物、抗生素。
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