WO2017101869A1 - 一种预防和治疗肝组织损伤及其相关病症的方法 - Google Patents

一种预防和治疗肝组织损伤及其相关病症的方法 Download PDF

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WO2017101869A1
WO2017101869A1 PCT/CN2016/110451 CN2016110451W WO2017101869A1 WO 2017101869 A1 WO2017101869 A1 WO 2017101869A1 CN 2016110451 W CN2016110451 W CN 2016110451W WO 2017101869 A1 WO2017101869 A1 WO 2017101869A1
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plasminogen
liver
damage
tissue damage
related conditions
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PCT/CN2016/110451
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English (en)
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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Priority to US16/062,389 priority Critical patent/US20190151421A1/en
Priority to CN201680073668.6A priority patent/CN108463241A/zh
Priority to EP16874926.5A priority patent/EP3391901B1/en
Priority to CA3008475A priority patent/CA3008475C/en
Priority to JP2018550636A priority patent/JP6815413B2/ja
Publication of WO2017101869A1 publication Critical patent/WO2017101869A1/zh

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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
    • 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)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • 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
    • 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)
    • C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA
    • 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)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • 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)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23048—Plasminogen activator Pla (3.4.23.48)
    • 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 the use of plasminogen or plasmin in preventing and/or treating liver tissue damage caused by various causes, thereby providing a novel therapeutic strategy for treating liver tissue damage and related disorders.
  • Liver injury or liver tissue damage is a liver parenchymal lesion caused by various causes, and is a general term for a series of pathological changes such as liver tissue inflammation, liver cell degeneration, necrosis, and liver tissue fibrosis. Common causes are inflammation, liver congestion, viral infections, poisoning, drugs, radiation, and the like. Some diseases are also associated with damage to liver tissue cells, such as diabetes, hepatitis, hypertension, atherosclerosis, and the like.
  • Drugs are a very common cause of liver tissue damage.
  • Common drugs that cause liver tissue damage include: anti-tuberculosis drugs: rifampicin, isoniazid, ethambutol; anti-tumor drugs: cyclophosphamide, methotrexate, 5-fluorouracil, carboplatin, cisplatin, etc.
  • Lowering blood lipids statins (atorvastatin, lovastatin), fenofibrate, clofibrate, niacin, etc.; steroid hormones: estrogens, oral contraceptives, male anabolic hormones; Cardiovascular drugs: amiodarone, warfarin, calcium antagonists, etc.; anti-rheumatic drugs: indomethacin, fenbufen, aspirin, indomethacin, etc.; antibiotics: chloramphenicol, roxithromycin, ketoconazole , penicillins, sulfonamides, etc.; anti-allergic drugs: promethazine (phenazone), chlorpheniramine (chlorpheniramine), loratadine (ceramide), anti-ulcer drugs: cimetidine, Ranitidine, famotidine, etc.; antifungal drugs such as ribavirin and the like.
  • Alcohol is a huge threat to the liver. Long-term or intermittent heavy drinking can cause liver damage. The amount of alcohol consumed and the longer the duration of drinking, the more serious the consequences. Alcohol directly poisons liver cells and affects their structure and function.
  • Alcoholic liver injury is a chronic toxic liver injury caused by liver disease caused by prolonged heavy drinking. In the early stage, it usually manifests as fatty liver, which in turn can develop into alcoholic hepatitis, liver fibrosis and cirrhosis. Its main clinical features are nausea, vomiting, jaundice, liver enlargement and tenderness. In severe alcohol abuse, extensive hepatocyte necrosis and even liver failure can be induced. Alcoholic liver disease is one of the common liver diseases in China, which seriously endangers people's health [1] .
  • pro-hepatic poisons In addition to toxic liver damage caused by alcohol, other "pro-hepatic poisons", such as chemical toxic substances in the environment and certain drugs, can also cause liver damage.
  • the liver As an important detoxification organ of the human body, the liver has a dual blood supply of hepatic artery and hepatic vein. Chemical substances can be transformed into the liver through the portal vein or systemic circulation of the gastrointestinal tract, so the liver is easily damaged by toxic substances in the chemical.
  • pro-hepatic poisons there are some substances that are toxic to the liver, called "pro-hepatic poisons”. These poisons are generally susceptible in the population, and the incubation period is short. The process of the lesion is directly related to the amount of chemical substances.
  • liver hepatocyte necrosis, steatosis, cirrhosis causes liver hepatocyte necrosis, steatosis, cirrhosis to varying degrees.
  • Pathological manifestations include (1) steatosis. Carbon tetrachloride, yellow phosphorus, etc. can interfere with the synthesis and transport of lipoproteins to form fatty liver. (2) Lipid peroxidation, which is a special manifestation of toxic liver injury. For example, carbon tetrachloride is metabolized in the body to produce a highly oxidative intermediate, which leads to lipid peroxidation and destruction on biofilm. Membrane phospholipids alter the structure and function of cells. (3) cholestasis reaction, mainly related to damage of liver cell membrane and microvilli, causing bile acid excretion disorder [2] .
  • Radiation can also cause liver tissue damage.
  • the radiation source is a high-energy electromagnetic wave or high-energy particle produced by natural or artificial energy. Rapid irradiation of large doses of radiation or long-time exposure of low-dose radiation may cause tissue damage.
  • the energy of radiation destroys the chromosomes and enzymes of the cells, causing the normal function of the cells to be disordered.
  • Diabetic liver tissue damage refers to a lesion of liver histology and function changes caused by diabetes. It is known that liver damage caused by diabetes includes: liver enzyme abnormalities, which can cause carbon dioxide accumulation in liver cells, acidosis, reduction of oxygen supply, increase of oxygen consumption, increase of liver transaminase activity, bilirubin metabolism disorder, and severe Cause liver cell necrosis; fatty liver, in all causes of fatty liver, diabetes accounted for the third place, of which 21% to 78% of diabetic patients with fatty liver; hepatitis, cirrhosis and liver cancer, including viral hepatitis in diabetic patients The prevalence rate is about 2 to 4 times that of normal people, and the incidence of primary liver cancer is about 4 times that of normal people. Diabetic liver disease not only damages the quality of life of millions of patients, but also creates a huge burden of care and the care needed for the strength of the healthcare system.
  • Viral infection of the liver is also a common cause of liver damage, such as hepatitis B virus, hepatitis C virus, hepatitis E virus, and the like.
  • Hepatic blood deposits can also cause liver tissue damage.
  • the accumulation of blood in the liver is mainly caused by several factors: hepatic venular occlusive disease, Bad-Kiari syndrome, chronic right heart dysfunction and constrictive pericarditis.
  • Any disease that causes the blood in the inferior vena cava to be blocked can cause liver congestion, such as rheumatic valvular heart disease, chronic constrictive pericarditis, hypertensive heart disease, ischemic heart disease, pulmonary heart disease, congenital heart disease. Wait.
  • Congestive liver injury initially involves the central area of the lobule, central venous congestion, expansion, and the degree of hepatic sinus expansion is different from that of the central venous sinus.
  • the central hepatocytes of the lobule are compressed, deformed and atrophied, and the cytoplasm is present.
  • Particle-like changes, nucleus pyknosis, nuclear fission, cell necrosis, accompanied by brown pigmentation, brown pigment in the center of the lobules may be caused by cholestatic, liver necrosis and necrosis adjacent to the central vein is the most serious, with the increase of congestion, necrosis
  • the tissue extends to the portal area. Patients with severe congestion have normal liver tissue only in the portal area. Over time, the reticular fibers around the central vein can collapse. It can be seen that the reticular fibrous tissue and the fine fiber bundle extend from the central vein to the other. Central vein.
  • liver tissue damage currently mainly includes the control and treatment of the cause, as well as supportive treatment.
  • scientists have long been looking for drugs that have direct, good repair effects on damaged liver tissue.
  • the present invention has also conducted intensive research on this. The experiment found that a protein substance naturally present in the human body, plasminogen has a good repair effect on liver tissue damage caused by poisoning, radiation, chemotherapy drugs and diabetes. Plasmin is expected to be a new strategy for the treatment of liver tissue damage and its related disorders.
  • Plasminogen is an inactive precursor of plasmin. It is a single-chain glycoprotein composed of 791 amino acids with a molecular weight of approximately 92 kDa [3,4] . 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 [5,6] . 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.
  • glutamate-plasminogen is hydrolyzed to Lys-Lysinogen at Lys76-Lys77.
  • lysine-plasminogen has a higher affinity for fibrin and can be activated by PA 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 [7] .
  • plasminogen contains five homologous tricycles, the so-called kringle, which contains a protease domain. Some kringles contain a lysine binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor alpha2-AP.
  • plasminogen fragment of 38 kDa, including kringlel-4, is a potent inhibitor of angiogenesis. This fragment was named angiostatin and can be produced by hydrolysis of plasminogen by several proteases.
  • the invention relates to the use of plasminogen or plasmin in the manufacture of a medicament, article, kit for preventing and/or treating liver tissue damage and related disorders in a subject.
  • the invention further relates to a pharmaceutical method comprising the manufacture of a medicament, article, kit for the prevention and/or treatment of liver tissue damage and related disorders in a subject, together with a pharmaceutically acceptable carrier.
  • the liver tissue damage and related conditions are liver damage caused by radiation or chemicals and related conditions.
  • radiation or chemical-induced liver damage and related conditions are chemoradiotherapy methods and drugs used in cancer treatment.
  • the radiation is radiation due to an event such as an accident or working environment.
  • the liver tissue damage and related conditions are toxic liver damage and related conditions.
  • the toxic liver injury is a toxic liver injury caused by "pro-hepatic poisons" including alcohol.
  • the liver tissue damage and related conditions are caused by diabetes and are one of the complications of diabetes.
  • the liver tissue damage and related conditions are caused by hepatitis caused by viral infection of the liver, such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus Caused by hepatitis.
  • the liver tissue damage and related disorders are drug-induced liver damage and related disorders.
  • the liver tissue damage and related conditions are caused by intrahepatic blood stasis (hepatic congestion).
  • the liver tissue damage and related disorders are diabetic liver injury and related disorders, toxic liver injury and related disorders, drug-induced liver injury or related disorders, radiation-induced liver injury, and related Conditions, viral infectious liver damage and related conditions, congestive liver damage and related conditions.
  • the liver tissue damage and related conditions include liver dysfunction caused by liver tissue damage, liver enzyme abnormalities, Liver discomfort and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis and liver cancer.
  • the plasminogen has a sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with sequence 2, 6, 8, 10 or 12. Identity, and still have 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, plasminogen, microplasminogen, delta-plasminogen or Any combination thereof.
  • the plasminogen or plasmin is administered systemically or locally, including surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, topical, intraarticular or rectal administration.
  • the condition associated with the diabetic liver injury or the condition associated with toxic liver damage includes: liver enzyme abnormalities, liver discomfort and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis , fatty liver, cholangitis, cirrhosis, liver necrosis and liver cancer.
  • the diabetic liver injury and related conditions are caused by large blood vessels, small blood vessels, microvascular lesions caused by diabetes.
  • the plasminogen can be administered in combination with one or more other drugs.
  • the other drugs include: a liver-protecting drug, an anti-diabetic drug, an anti-thrombotic drug, an anticoagulant drug, a hypolipidemic drug, an anti-cardiovascular disease drug, and an anti-infective drug.
  • the subject is a mammal, preferably a human.
  • the liver damage caused by diabetes is caused by large blood vessels, small blood vessels, microvascular lesions caused by diabetes.
  • the subject has a low plasmin or plasminogen.
  • the low is congenital, secondary and/or local.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 sexual, and still have plasminogen activity.
  • 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.
  • the plasminogen comprises a plasminogen active fragment, and A protein still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, microplasminogen, delta-plasminogen or random combination. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, delta-plasminogen or Microplasminogen.
  • the plasminogen is a human native plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, fibrinolysis from a primate or rodent
  • the zymogen is a straight homologue, such as a troponin-directed homologue from gorillas, rhesus monkeys, rats, cows, horses, and dogs.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • 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 plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum .
  • the topical administration is by applying a catheter containing plasminogen in the liver region.
  • the invention relates to a method of preventing and/or treating liver tissue damage and related disorders in a subject, comprising administering to the subject an effective amount of plasminogen or plasmin.
  • the invention further relates to the use of plasminogen or plasmin for preventing and/or treating liver tissue damage and related disorders in a subject.
  • the liver tissue damage and related conditions are liver damage caused by radiation or chemicals and related conditions.
  • radiation or chemical-induced liver damage and related conditions are chemoradiotherapy methods and drugs used in cancer treatment.
  • the radiation is radiation due to an contingency event.
  • the Liver tissue damage and related conditions are toxic liver damage and related conditions.
  • the toxic liver injury is a toxic liver injury caused by "pro-hepatic poisons" including alcohol.
  • the liver tissue damage and related conditions are caused by diabetes and are one of the complications of diabetes.
  • the liver tissue damage and related conditions are caused by hepatitis caused by viral infection of the liver, such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus Caused by hepatitis.
  • the liver tissue damage and related disorders are drug-induced liver damage and related disorders.
  • the liver tissue damage and related conditions are caused by intrahepatic blood stasis (hepatic congestion).
  • the liver tissue damage and related disorders are diabetic liver injury and related disorders, toxic liver injury and related disorders, drug-induced liver injury or related disorders, radiation-induced liver injury, and related Conditions, viral infectious liver damage and related conditions, congestive liver damage and related conditions.
  • the liver tissue damage and related conditions include liver dysfunction caused by liver tissue damage, liver enzyme abnormalities, liver discomfort and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, Hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis and liver cancer.
  • the plasminogen has a sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with sequence 2, 6, 8, 10 or 12. Identity, and still have 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, plasminogen, microplasminogen, delta-plasminogen or Any combination thereof.
  • the plasminogen or plasmin is administered systemically or locally, including surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, topical, intraarticular or rectal administration.
  • the condition associated with the diabetic liver injury or the condition associated with toxic liver damage includes: liver enzyme abnormalities, liver discomfort and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis , fatty liver, cholangitis, cirrhosis, liver necrosis and liver cancer.
  • the diabetic liver injury and related conditions are caused by large blood vessels, small blood vessels, microvascular lesions caused by diabetes.
  • the plasminogen can be administered in combination with one or more other drugs.
  • the other drug comprises: a liver-protecting drug, an anti-sugar Urinary drugs, antithrombotic drugs, anticoagulant drugs, hypolipidemic drugs, anti-cardiovascular disease drugs, anti-infective drugs.
  • the subject is a mammal, preferably a human.
  • the liver damage caused by diabetes is caused by large blood vessels, small blood vessels, microvascular lesions caused by diabetes.
  • the subject has a low plasmin or plasminogen.
  • the low is congenital, secondary and/or local.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 sexual, and still have plasminogen activity.
  • 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.
  • 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, plasminogen, microplasminogen, delta-plasminogen or random combination. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, plasminogen, delta-plasminogen or Microplasminogen.
  • the plasminogen is a human native plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, fibrinolysis from a primate or rodent
  • the zymogen is a straight homologue, such as a troponogen-directed homologue from gorillas, rhesus monkeys, rats, cows, horses, and dogs.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • 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 plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum .
  • the topical administration is by applying a catheter containing plasminogen in the liver region.
  • the present invention relates to a plasminogen or plasmin, a pharmaceutical composition comprising the plasminogen or plasmin, or a pharmaceutical composition for preventing and/or treating liver tissue damage and related disorders in a subject, or An article or kit comprising the plasminogen or plasmin.
  • the liver tissue damage and related conditions are liver damage caused by radiation or chemicals and related conditions.
  • radiation or chemical-induced liver damage and related conditions are chemoradiotherapy methods and drugs used in cancer treatment.
  • the radiation is radiation due to an contingency event.
  • the liver tissue damage and related conditions are toxic liver damage and related conditions.
  • the toxic liver injury is a toxic liver injury caused by "pro-hepatic poisons" including alcohol.
  • the liver tissue damage and related conditions are caused by diabetes and are one of the complications of diabetes.
  • the liver tissue damage and related conditions are caused by hepatitis caused by viral infection of the liver, such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus Caused by hepatitis.
  • the liver tissue damage and related disorders are drug-induced liver damage and related disorders.
  • the liver tissue damage and related conditions are caused by intrahepatic blood stasis (hepatic congestion).
  • the liver tissue damage and related disorders are diabetic liver injury and related disorders, toxic liver injury and related disorders, drug-induced liver injury or related disorders, radiation-induced liver injury, and related Conditions, viral infectious liver damage and related conditions, congestive liver damage and related conditions.
  • the liver tissue damage and related conditions include liver dysfunction caused by liver tissue damage, liver enzyme abnormalities, liver discomfort and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, Hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis and liver cancer.
  • the plasminogen has a sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with sequence 2, 6, 8, 10 or 12. Identity, and And still have 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, plasminogen, microplasminogen, delta-plasminogen or Any combination thereof.
  • the plasminogen or plasmin is administered systemically or locally, including surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, topical, intraarticular or rectal administration.
  • the condition associated with the diabetic liver injury or the condition associated with toxic liver damage includes: liver enzyme abnormalities, liver discomfort and tenderness, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis , fatty liver, cholangitis, cirrhosis, liver necrosis and liver cancer.
  • the diabetic liver injury and related conditions are caused by large blood vessels, small blood vessels, microvascular lesions caused by diabetes.
  • the plasminogen can be administered in combination with one or more other drugs.
  • the other drugs include: a liver-protecting drug, an anti-diabetic drug, an anti-thrombotic drug, an anticoagulant drug, a hypolipidemic drug, an anti-cardiovascular disease drug, and an anti-infective drug.
  • the subject is a mammal, preferably a human.
  • the liver damage caused by diabetes is caused by large blood vessels, small blood vessels, microvascular lesions caused by diabetes.
  • the subject has a low plasmin or plasminogen.
  • the low is congenital, secondary and/or local.
  • plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 sexual, and still have plasminogen activity.
  • 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.
  • 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, plasminogen, microplasminogen, delta-plasminogen or random combination. In one embodiment, the plasminogen is a conservative substitution variant selected from the group consisting of Glu-plasminogen, Lys-plasminogen, Small plasminogen, delta-plasminogen or microplasminogen.
  • the plasminogen is a human native plasminogen, such as an ortholog of plasminogen as shown in SEQ ID NO: 2, for example, fibrinolysis from a primate or rodent
  • the zymogen is a straight homologue, such as a troponogen-directed homologue from gorillas, rhesus monkeys, rats, cows, horses, and dogs.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • 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 plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum .
  • the topical administration is by applying a catheter containing plasminogen in the liver region.
  • the plasminogen or plasmin is dispensed in a container.
  • the article or kit further comprises other drugs dispensed in other containers of the kit.
  • the kit may further comprise instructions for use, wherein the plasminogen may be used to treat liver tissue damage and related disorders, in particular, diabetes-induced liver damage and related disorders, toxic liver damage and related a condition, a drug-induced liver injury or a related condition thereof, a liver injury caused by radiation and a related condition thereof, a liver injury caused by a viral infection and a related condition thereof, a liver injury caused by blood stasis, and a related condition thereof, and may further explain that said fiber Lysozyme or plasmin may be administered prior to, concurrently with, and/or after administration of other drugs or therapies.
  • the present invention expressly covers all combinations of the technical features between the embodiments of the present invention, and these combined technical solutions are explicitly disclosed in the present application, just as the above technical solutions have been separately and explicitly disclosed.
  • the present invention also explicitly covers various embodiments and their All subcombinations of the primes are disclosed herein, just as each such subcombination is separately and explicitly disclosed herein.
  • liver damage caused by diabetes includes: liver enzyme abnormalities, which can cause carbon dioxide accumulation in liver cells, acidosis, reduction of oxygen supply, increase of oxygen consumption, increase of liver transaminase activity, bilirubin metabolism disorder, and severe Cause liver cell necrosis; fatty liver, in all causes of fatty liver, diabetes accounted for the third place, of which 21% to 78% of diabetic patients with fatty liver; hepatitis, cirrhosis and liver cancer, including viral hepatitis in diabetic patients The prevalence rate is about 2 to 4 times that of normal people, and the incidence of primary liver cancer is about 4 times that of normal people.
  • “Chemical liver injury” or “toxic liver injury” refers to liver damage caused by chemical hepatotoxic substances. These chemicals include alcohol, chemical toxic substances in the environment, and certain drugs. In nature and human industrial production, there are some substances that are toxic to the liver, called “pro-hepatic poisons”. These poisons are generally susceptible in the population, and the incubation period is short. The process of the lesion is directly related to the amount of chemical substances. Cause liver hepatocyte necrosis, steatosis, cirrhosis to varying degrees.
  • Pro-hepatic poison refers to a general term for substances that are toxic to the liver. Alcohol is the most common "pro-hepatic poison” in life. In addition to alcohol, chemical toxic substances in the environment and certain drugs can also cause liver damage. As an important detoxification organ of the human body, the liver has a dual blood supply of hepatic artery and hepatic vein. Chemical substances can be transformed into the liver through the portal vein or systemic circulation of the gastrointestinal tract, so the liver is easily damaged by toxic substances in the chemical. There are some substances that are toxic to the liver during the production process of nature and human industry, called “pro-hepatic poisons". They enter the liver and can cause liver hepatocyte necrosis, steatosis, and cirrhosis to varying degrees.
  • Pathological manifestations include (1) steatosis. Carbon tetrachloride, yellow phosphorus, etc. can interfere with the synthesis and transport of lipoproteins to form fatty liver. (2) Lipid peroxidation, which is a special manifestation of toxic liver injury. For example, carbon tetrachloride is metabolized in the body to produce a highly oxidative intermediate, which leads to lipid peroxidation and destruction on biofilm. Membrane phospholipids alter the structure and function of cells. (3) cholestasis reaction, mainly related to damage of liver cell membrane and microvilli, causing bile acid excretion disorder.
  • Drug-induced liver injury is a drug-induced liver injury caused by a drug itself or/and its metabolites or a decrease in hypersensitivity or tolerance to a drug due to a specific constitution. Drug-induced liver disease can be manifested as various acute and chronic liver diseases in the clinic. It can be recovered spontaneously after stopping the drug. The severe one may be life-threatening and require active treatment and rescue. "Drug-induced liver damage” can occur in healthy people who have no previous history of liver disease or in patients who have had serious illnesses; they can occur when the amount of medication is excessive, or in the case of normal dosage.
  • Radioactive liver damage refers to a type of radioactive damage caused by high-energy ionizing radiation including alpha, beta particles, gamma rays, xenon rays, and neutron rays. Rapid irradiation of large doses of radiation or long-time exposure of low-dose radiation may cause tissue damage. The energy of radiation destroys the chromosomes and enzymes of the cells, causing the normal function of the cells to be disordered.
  • “Viral-infected liver injury” is a general term for liver damage caused by viral infection.
  • the virus infection is usually caused by hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus.
  • Consgestive liver injury is a disease of liver tissue damage caused by blood accumulation in the liver. Any disease that causes the blood in the inferior vena cava to be blocked can cause liver congestion, such as rheumatic valvular heart disease, chronic constrictive pericarditis, hypertensive heart disease, ischemic heart disease, pulmonary heart disease, congenital heart disease. Wait.
  • Plasmidogen 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.
  • sequence 4 the native human plasminogen amino acid sequence
  • 92 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.
  • ⁇ -plasminogen is a fragment of full-length plasminogen deficient in Kringle2-Kringle5 structure, containing only Kringle1 and serine protease domains [8,9] .
  • ⁇ -plasminogen has been reported in the literature. amino acid sequence (SEQ ID 8) [9], cDNA sequences encoding the amino acid sequence of the 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) [10] , 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).
  • 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.
  • 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 invention relates to a technical scheme of plasminogen A technical solution for replacing plasminogen with a plasminogen active fragment is covered.
  • the plasminogen active fragment of the present invention 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.
  • 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, plasma tissue fibrinolysis Detection of zymogen activator inhibitor antigen, plasma plasmin-anti-plasmin complex assay (PAP).
  • t-PAA tissue plasminogen activator activity
  • t- PAAg detection of plasma tissue plasminogen activator antigen
  • plgA plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma tissue fibrinolysis Detection of zymogen activator inhibitor antigen, 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 methods, gel electrophoresis, immunoturbidimetry, and 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 the determination of more than 60% ammonia by the BLAST or FASTA algorithm If the polypeptide or the enzyme having the same acidity is more than 75%, preferably more than 85%, even more than 90% is optimal, and the same or substantially similar to the natural or parent protein or enzyme. Nature or function.
  • 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. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity 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
  • plasminogen 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.
  • Frozen 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.
  • Aerosol formulations such as nasal spray formulations comprise purified aqueous or other solutions of the active agents and preservatives and isotonic agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.
  • 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 injectable 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 have a dose ranging, for example, from about 0.0001 to 2000 mg/kg per day, or from 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 of diabetic liver disease and its related conditions need to be evaluated and periodically evaluated in the drug administration process of the present invention.
  • One embodiment of the invention relates to the determination of therapeutic efficacy and therapeutic safety following treatment of a subject with plasminogen.
  • the method for judging the efficacy of the treatment includes, but is not limited to, 1) examination of liver function of the subject, for example, enzymatic levels in the patient such as serum aspartate aminotransferase (ALT), alanine aminotransferase (AST) Whether the levels of total bilirubin, direct bilirubin, indirect bilirubin, albumin, globulin, cholinesterase, alkaline phosphatase, transpeptidase are in a normal range, and are used in the present invention.
  • ALT serum aspartate aminotransferase
  • AST alanine aminotransferase
  • alanine aminotransferase 0 ⁇ 40 ⁇ / L
  • aspartate aminotransferase AST
  • GTT glutamyl transferase
  • Total bilirubin 3.4 ⁇ 20.5 ⁇ mol/L
  • PT prothrombin time
  • PTA activity
  • the progressive reduction of PTA to less than 40% is one of the important diagnostic criteria for liver failure, and ⁇ 20% indicates liver dysfunction.
  • the decrease in PTA in patients is expected to be significantly improved; 3) imaging examination: including abdominal hepatobiliary spleen ultrasound, CT or nuclear magnetic to understand the degree of liver damage recovery 4) tumor marker examination, such as alpha fetoprotein AFP, CA199, AFU, etc.; 5) liver biopsy to determine the degree of recovery of fibrosis and other injuries.
  • the present invention also relates to the determination of the safety of the treatment regimen during and after treatment with a plasminogen and variants thereof, including but not limited to serum half-life, treatment of the subject Half-life, half-toxicity (TD50), and median lethal dose (LD50) were counted, or various adverse events such as sensitization reactions occurred during or after treatment were observed.
  • a plasminogen and variants thereof including but not limited to serum half-life, treatment of the subject Half-life, half-toxicity (TD50), and median lethal dose (LD50) were counted, or various adverse events such as sensitization reactions occurred during or after treatment were observed.
  • One embodiment of the invention relates to an article or kit comprising a plasminogen of the invention useful for treating liver damage caused by diabetes and related conditions 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 liver damage 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, including, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease.
  • Figure 1 shows the change in body weight of 24-25 week old diabetic mice after administration of plasminogen.
  • Figure 2 shows the results of liver HE staining of 24-25 week old diabetic mice after administration of plasminogen for 15 consecutive days.
  • Figure 3 shows the results of microscopic examination of liver fibrin immunostaining in 24-25 week old diabetic mice after administration of plasminogen for 15 consecutive days.
  • Figure 4 shows changes in body weight of 24-25 week old diabetic mice after administration of plasminogen for 31 consecutive days.
  • Figure 5 shows the results of liver HE staining of 24-25 week old diabetic mice after administration of plasminogen for 31 consecutive days.
  • Figure 6 shows the results of liver fibrin immunostaining after administration of plasminogen for 23 consecutive days in diabetic mice of 24-25 weeks old.
  • Figure 7 shows the results of immunostaining of liver F4/80 after administration of plasminogen for 23 consecutive 25-day-old diabetic mice.
  • FIG 8 shows the results of serum alanine aminotransferase (ALT) assay after 31 days of administration of PBS or plasminogen in 24-25 week diabetic mice.
  • Figure 9 shows the results of HE staining in liver of mice with acute liver injury induced by carbon tetrachloride on days 0, 2, and 7 of plasminogen administration.
  • Figure 10 shows the results of liver HE staining after 18, 24, and 48 hours of administration of plasminogen in mice with acute liver injury induced by plg -/- carbon tetrachloride.
  • Figure 11 shows the results of immunostaining of liver fibrin after 18, 24, and 48 hours of administration of plasminogen in mice with acute liver injury induced by plg -/- carbon tetrachloride.
  • Figure 12 shows the results of liver F4/80 immunostaining after 10 days of 5.0 Gy X-ray irradiation of mouse plasminogen.
  • Figure 13 shows the results of liver fibrin immunostaining after 7 days of plasminogen in 10 mg/Kg cisplatin chemotherapy-injured mice.
  • Figure 14 shows the results of liver HE staining after 18, 24, 48 and 7 days of administration of plasminogen in mice with acute liver injury induced by plg -/- carbon tetrachloride.
  • mice Ten male db/db mice, 24-25 weeks old, were randomly divided into two groups, given vehicle PBS control group and plasminogen group, 5 rats each. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the administration was continued for 15 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.
  • Body weights were weighed on days 0, 4, 7, 11, and 16 of plasminogen, respectively. The results showed no significant difference in body weight (Fig. 1) on days 0, 4, 7, 11, and 16 in the plasminogen group and the vehicle PBS control group, indicating that plasminogen had little effect on animal body weight.
  • mice Ten male db/db mice, 24-25 weeks old, were randomly divided into two groups, given vehicle PBS control group and plasminogen group, 5 rats each. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the administration was continued for 15 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. On the 16th day, the mice were sacrificed and the liver tissues were fixed in 10% neutral formalin fixative for 24-48 hours.
  • liver tissues fixed were dehydrated by alcohol gradient and transparent with xylene, and then embedded in paraffin.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol was differentiated, ammonia water returned to blue, and the alcohol gradient was dehydrated and sealed.
  • HE staining hematoxylin and eosin
  • HE staining showed that in the control group of PBS, the hepatocytes showed severe steatosis, lipid deposition, nucleus was squeezed to the edge, cells were mildly water-like degeneration, and hepatic cord disorder; the plasminogen group was given to the vehicle.
  • hepatic steatosis was alleviated, with mild fatty changes, with moderate watery degeneration. This indicates that plasminogen can promote the repair of diabetic liver injury.
  • mice Ten male db/db mice, 24-25 weeks old, were randomly divided into two groups, given vehicle PBS control group and plasminogen group, 5 rats each. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the administration was continued for 15 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 16 and liver tissue was fixed in 10% neutral formalin fixative for 24-48 hours.
  • the fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 10% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after the time was over, the sheep serum was discarded and the PAP circle was used. Out of the organization. Rabbit anti-mouse fibrin (pro) antibody (Abeam) was incubated overnight at 4 ° C and washed twice with TBS for 5 minutes each time.
  • pro normal sheep serum
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and twice with TBS for 5 minutes each time.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and then counterstained for 30 seconds with hematoxylin, rinsed with running water for 5 minutes, and then washed once with TBS.
  • the gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • Fibrinogen is a precursor of fibrin.
  • fibrinogen is hydrolyzed to fibrin [12-14] . Therefore, fibrinogen levels can be used as a marker of the degree of damage.
  • Twenty-two male db/db rats aged 24-25 weeks were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, with 10 rats in each group.
  • the group On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously 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. Body weights were weighed on days 0, 4, 7, 11, 16, 21, 26, and 31, respectively.
  • mice Ten male db/db mice, 24-25 weeks old, were randomly divided into two groups, given vehicle PBS control group and plasminogen group, 5 rats each. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously 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. On the 32nd day, the mice were sacrificed and the liver tissues were fixed in 10% neutral formalin fixative for 24-48 hours.
  • liver tissues fixed were dehydrated by alcohol gradient and transparent with xylene, and then embedded in paraffin.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol was differentiated, ammonia water returned to blue, and the alcohol gradient was dehydrated and sealed.
  • HE staining hematoxylin and eosin
  • HE staining showed that the liver was severely steatotic in the vehicle PBS control group (Fig. 5A), lipid deposition, fusion into large fat vacuoles, and the nucleus was squeezed to the edge.
  • Hepatic cord disorder hepatic sinus narrowing, and there are a number of inflammatory lesions ( ⁇ ) in the hepatic cord; in the plasminogen group (Fig. 5B), the liver is mildly steatotic, and the damage is mild water-like degeneration.
  • Master, cytosol dissolution It is mainly distributed in the area between the portal area and the central vein. The area around the portal area and the central vein is relatively light, and mild infiltration of the inflammatory cells at the hepatic cord is seen. It shows that the damage to the liver after plasminogen is obviously repaired.
  • 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 group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously 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 on day 32 and liver tissues were fixed in 10% neutral formalin fixative for 24 hours.
  • the fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 10% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after the time was over, the sheep serum was discarded and the tissue was circled with a PAP pen.
  • Rabbit anti-mouse fibrin (pro) antibody (Abeam) was incubated overnight at 4 ° C and washed twice with TBS 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 for 5 minutes each time.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • Fibrinogen is a precursor of fibrin.
  • fibrinogen is hydrolyzed to fibrin [12-14] . Therefore, fibrinogen levels can be used as a marker of the degree of damage.
  • 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 group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously 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 liver tissues were fixed in 10% neutral formalin fixative for 24 hours.
  • the fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 10% normal sheep serum was blocked for 1 hour. After the time, the serum was removed and the tissue was circled with a PAP pen.
  • Rabbit polyclonal antibody (Abeam) against F4/80 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.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and then counterstained for 30 seconds with hematoxylin, rinsed with running water for 5 minutes, dried by gradient dehydration and sealed, and the sections were observed under a microscope at 400 times.
  • the F4/80 macrophage marker can indicate the extent and stage of the inflammatory response.
  • the results showed that the F4/80 positive level in the plasminogen group was significantly lower in the plasminogen group (Fig. 7B) compared to the vehicle PBS control group (Fig. 7A), indicating that the plasminogen was administered. Liver tissue inflammation is reduced.
  • Figure 7C shows the quantitative analysis of the positive expression of F4/80 immunohistochemistry. The expression of F4/80 in the plasminogen group was significantly reduced, and there was a statistical difference, indicating that injection of plasminogen can significantly promote liver inflammation in diabetic mice. Repair.
  • mice Nine male db/db mice aged 25-28 weeks were randomly divided into two groups, three in the vehicle control group and six in the plasminogen group. On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day, and the drug was administered continuously 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. After 31 days of plasminogen extraction, whole blood was collected from the eyeballs.
  • alanine aminotransferase test kit (Nanjing Institute of Bioengineering, item number C009-2) was used to detect the content of alanine aminotransferase (ALT) in serum by Reitman-Frankel method.
  • Alanine aminotransferase is an important indicator of liver health status [15,16] , and the normal reference value range of alanine aminotransferase is 9-50 U/L.
  • the results showed that the serum ALT level in the control group was significantly higher than the normal physiological index, but the plasminogen group had returned to the normal level in the body, and was significantly lower in the plasminogen group than in the vehicle PBS.
  • the control group was statistically significant (Figure 8). It is indicated that injection of plasminogen can effectively repair liver injury in late diabetic model mice.
  • mice Eighteen 7-8 week old plg +/+ mice, male or female, were randomly divided into two groups, respectively, the vehicle PBS control group and the plasminogen administration group, 9 rats in each group.
  • Two groups of mice were given intraperitoneal injection of carbon tetrachloride at 0.5 mL/kg body weight for two consecutive days to establish an acute liver injury model [17,18] .
  • Carbon tetrachloride should be diluted with corn oil before use. The volume ratio of the former to the latter is 1:7.
  • the day of modeling was day 0, and the first day was given to plasminogen or PBS.
  • the plasminogen group was administered 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 for 7 days. On day 0, 2, and 7, the mice in each group were sacrificed, and the liver was observed by dissection. The liver tissue was fixed in 10% neutral formalin fixative for 24-48 hours. The fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol was differentiated, ammonia water was returned to blue, and the mixture was dehydrated with an alcohol gradient, and observed under a microscope at 200 times.
  • HE staining hematoxylin and eosin
  • HE staining showed that the liver of the vehicle PBS control group (Fig. 9A-C) and the plasminogen group (Fig. 9D-F) on day 0 mainly consisted of fragmental necrosis around the central vein, necrotic regional cells. Nuclear fragmentation, cytoplasmic staining, moderate watery degeneration, cell edema in other non-necrotic areas; central venous dilatation on day 2, hepatocyte structure disorder, a small amount of inflammatory cell infiltration, no significant difference between the two groups.
  • hepatocyte degeneration was observed in the vehicle PBS control group, the cells were mildly edematous, hepatic cord disorder, hepatic sinusoids narrowed, and mild inflammatory cells infiltrated around the portal area, while plasmin was given.
  • the liver of the original group basically returned to normal cytoplasm red staining, hepatic cord rule, and the liver sinus was clear. This indicates that plasminogen can promote the repair of liver damage.
  • mice 7-11 week old plg - / - 18 male mice were randomly divided into two groups, respectively, to the vehicle control group and the PBS-administered group plasminogen, 9 per group.
  • Two groups of mice were given intraperitoneal injection of carbon tetrachloride at a dose of 0.5 mL/kg body weight for a single treatment to establish an acute liver injury model [17,18] .
  • Carbon tetrachloride should be diluted with corn oil before use. The volume ratio of the former to the latter is 1:7. Plasminogen or vehicle PBS was administered within half an hour after modeling.
  • the plasminogen group mice were administered with plasminogen at a dose of 1 mg/0.1 mL/day/day, and the vehicle PBS control group was administered with the same volume of PBS for 2 consecutive days. Three mice in each group were sacrificed at 18, 24, and 48 hours after administration, and liver conditions were recorded by dissection. The liver tissues were then fixed in 10% neutral formalin fixative for 24-48 hours. The fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol was differentiated, ammonia water was returned to blue, and the mixture was dehydrated with an alcohol gradient, and observed under a microscope at 200 times.
  • HE staining hematoxylin and eosin
  • the damage was mainly caused by mild watery degeneration, distributed around the portal area, and the central vein
  • the surrounding hepatocytes were not affected, but improved at 24h compared with 18h, watery degeneration was relieved, liver cells around the central vein were mildly steatotic, and cytoplasm was lightly stained, all accompanied by mild inflammatory cell infiltration. This indicates that plasminogen can promote the repair of liver injury in mice with plg -/- acute liver injury.
  • mice Eighteen 7-week-old plg -/- male mice were randomly divided into two groups, the vehicle-administered PBS control group and the plasminogen-administered group, with 9 rats in each group.
  • Two groups of mice were intraperitoneally injected with carbon tetrachloride at a dose of 0.5 mL/kg body weight to establish an acute liver injury model [17,18] .
  • Carbon tetrachloride should be diluted with corn oil before use. The volume ratio of the former to the latter is 1:7. Plasminogen or vehicle PBS was administered within half an hour after modeling.
  • the plasminogen group was administered 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 for 2 consecutive days.
  • Three mice in each group were sacrificed at 18, 24, and 48 hours after administration, and liver conditions were recorded by dissection.
  • the liver tissues were then fixed in 10% neutral formalin fixative for 24-48 hours.
  • the fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding. The thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time.
  • Fibrinogen is a precursor of fibrin.
  • fibrinogen is hydrolyzed to fibrin [12-14] . Therefore, fibrinogen levels can be used as a marker of the degree of damage.
  • Example 12 Plasminogen promotes repair of liver inflammation in mice by 5.0 Gy X-ray irradiation
  • mice 10 male C57 mice, 6-8 weeks old, were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, with 5 rats in each group.
  • a radiation damage model was established.
  • the mice were uniformly irradiated with a single linear force of 5.0 Gy using a linear accelerator 6 MV X-ray.
  • the absorbed dose rate was 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes).
  • plasminogen was administered within 3 hours.
  • the body weight was weighed and grouped on the 0th day.
  • the radiation treatment was started and plasminogen or vehicle PBS was administered.
  • the administration period was 10 days.
  • the mice were sacrificed on day 21 and the liver was fixed in 10% neutral formalin fixative for 24-48 hours.
  • the fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the Tris-EDTA was repaired for 30 minutes, and after 20 minutes of cooling at room temperature, the water was gently rinsed.
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • the F4/80 immunohistochemistry results showed that the expression of mouse macrophage markers in the vehicle PBS control group (Fig. 12A) after 5.0 Gy X-ray irradiation was higher than that in the plasminogen group (Fig. 12B). After the administration of plasminogen, the inflammation of the liver tissue of the animal was significantly alleviated.
  • Example 13 Plasminogen Reduces Fibrin deposition in liver tissue of cisplatin-induced chemotherapy-injured mice
  • 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 chemotherapy injury model was established, and cisplatin was intraperitoneally injected at a dose of 10 mg/kg. After the model was established, plasminogen was administered to the plasminogen group by tail vein injection at 1 mg/day/day, and the same volume of PBS was administered to the vehicle PBS control group. On the first day of the experiment, the body weight was weighed and grouped on the 0th day.
  • plasminogen or vehicle PBS was administered within 3 hours after the model establishment, and the administration period was 7 days.
  • Mice were sacrificed on day 8 and livers were fixed in 10% neutral formalin fixative for 24-48 hours.
  • the fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the citric acid was repaired for 30 minutes, and after cooling at room temperature for 10 minutes, the water was gently rinsed. Incubate for 15 minutes in 3% hydrogen peroxide and circle the tissue with a PAP pen.
  • Fibrinogen is a precursor of fibrin.
  • fibrinogen is hydrolyzed to fibrin [12-14] . Therefore, fibrinogen levels can be used as a marker of the degree of damage.
  • mice Six 7-week-old plg-/- male mice were randomly divided into two groups, the vehicle-administered PBS control group and the plasminogen-administered group, with 3 rats in each group.
  • Two groups of mice were given intraperitoneal injection of carbon tetrachloride at a dose of 0.5 mL/kg body weight for a single treatment to establish an acute liver injury model [17,18] .
  • Carbon tetrachloride should be diluted with corn oil before use. The volume ratio of the former to the latter is 1:7. Plasminogen or vehicle PBS was administered within half an hour after modeling.
  • the plasminogen group was administered 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 for 7 days. Mice were sacrificed on day 8, liver conditions were dissected and observed, and liver tissue was fixed in 10% neutral formalin fixative for 24-48 hours. The fixed liver tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 5 ⁇ m, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), 1% hydrochloric acid alcohol was differentiated, ammonia water was returned to blue, and the mixture was dehydrated with an alcohol gradient, and observed under a microscope at 200 times.
  • HE staining hematoxylin and eosin

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Abstract

纤溶酶原在治疗和/或消除肝损伤方面的用途，为治疗不同类型的肝损伤提供一条新的治疗途径。

Description

一种预防和治疗肝组织损伤及其相关病症的方法 技术领域
本发明涉及纤溶酶原或纤溶酶在预防和/或治疗各种原因导致的肝组织损伤方面的用途，进而为治疗肝组织损伤及其相关病症提供全新的治疗策略。
背景
肝损伤或称肝组织损伤是各种原因导致的肝脏实质病变，是肝组织炎症、肝脏细胞变性、坏死、肝组织纤维化等一系列病理变化的总称。常见的原因有炎症、肝脏淤血、病毒感染、中毒、药物、辐射等。一些疾病也伴发肝脏组织细胞的损伤，例如糖尿病、肝炎、高血压、动脉粥样硬化等。
药物是引起肝组织损害的一个非常常见的原因。引起肝脏组织损害的常用药物包括：抗结核药物：利福平、异烟肼、乙胺丁醇等；抗肿瘤药物：环磷酰胺、甲氨叠呤、5-氟尿嘧啶、卡铂、顺铂等；调降血脂类：他汀类(阿托伐他汀、洛伐他汀)、非诺贝特、氯贝丁酯、烟酸等；类固醇激素：雌激素类药物、口服避孕药、雄性同化激素等；心血管药物：胺碘酮、华法令、钙离子拮抗剂等；抗风湿药物：消炎痛、芬布芬、阿司匹林、吲哚美辛等；抗生素：氯霉素、罗红霉素、酮康唑、青霉素类、磺胺类等；抗过敏药物：异丙嗪(非那根)、氯苯那敏(扑尔敏)、氯雷他定(开瑞坦)等；抗溃疡药物：西咪替丁、雷尼替丁、法莫替丁等；抗真菌的药物例如利巴韦林等等。
酒精是肝脏的巨大威胁，长期或间断性大量饮酒可引起肝组织损伤，饮酒量大、持续饮用时间越长，其后果越严重。酒精会直接毒害肝细胞，影响其结构及功能。
酒精性肝损伤是一种慢性中毒性肝损伤，是由于长期大量饮酒导致的肝脏疾病。初期通常表现为脂肪肝，进而可发展成酒精性肝炎、肝纤维化和肝硬化。其主要临床特征是恶心、呕吐、黄疸、可有肝脏肿大和压痛。 严重酗酒时可诱发广泛肝细胞坏死，甚至肝功能衰竭。酒精性肝病是我国常见的肝脏疾病之一，严重危害人民健康[1]。
除了酒精导致的中毒性肝损伤外，其它″亲肝毒物″，例如环境中的化学有毒物质及某些药物也可造成肝损伤。作为人体的重要解毒器官，肝脏，具有肝动脉和肝静脉双重血液供应。化学物质可通过胃肠道门静脉或体循环进入肝脏进行转化，因此肝脏容易受到化学物中的毒性物质损害。大自然和人类工业生产过程中均存在一些对肝脏有毒性的物质，称为″亲肝毒物″，这些毒物在人群中普遍易感，潜伏期短，病变的过程与化学物质的量直接相关，可引起肝脏不同程度的肝细胞坏死、脂肪变性、肝硬化。病理表现包括(1)脂肪变性。四氯化碳、黄磷等可干扰脂蛋白的合成与转运，形成脂肪肝。(2)脂质过氧化反应，这是中毒性肝损伤的特殊表现形式，如四氯化碳在体内代谢产生一种氧化能力很强的中间产物，导致生物膜上的脂质过氧化，破坏膜的磷脂，改变细胞的结构与功能。(3)胆汁郁积反应，主要与肝细胞膜和微绒毛受损，引起胆汁酸排泄障碍有关[2]。
辐射也可以导致肝脏组织损伤。一般来说，辐射源是天然或人工能源产生的高能电磁波或高能粒子。大剂量射线瞬间照射或低剂量射线长时间照射都可能引起组织损伤，辐射的能量破坏细胞的染色体、酶，使细胞的正常功能发生紊乱。
糖尿病性肝组织损伤是指由糖尿病引起的肝脏组织学和功能变化的病变。已知糖尿病可引起的肝损伤包括：肝酶学异常，其可引起肝细胞内二氧化碳蓄积、酸中毒、氧供减少、氧消耗增加，使肝脏转氨酶活性增加，胆红素代谢紊乱，重者可引起肝细胞坏死；脂肪肝，在引起脂肪肝的所有病因中，糖尿病占第三位，其中21％～78％糖尿病患者伴有脂肪肝；肝炎、硬化和肝癌，其中糖尿病患者中病毒性肝炎的患病率约为正常人的2～4倍，原发性肝癌的发生率约为正常人的4倍。糖尿病性肝病不仅损害数以百万计的患者的生活质量，同时也造成一个巨大的负担成本和医疗保健系统强度所需的护理。
肝脏的病毒感染也是导致肝损伤的一个常见原因，例如乙型病毒性肝炎、丙型病毒肝炎、戊型病毒肝炎等。
肝内血液淤积也可以造成肝脏组织损伤。血液在肝内淤积主要由以下几种因素引起：肝小静脉闭塞病、巴德一基亚利综合征、慢性右心功能不全和缩窄性心包炎。
任何导致下腔静脉血回心受阻的疾病都可导致肝脏淤血，如风湿性心脏瓣膜病，慢性缩窄性心包炎，高血压性心脏病，缺血性心脏病，肺心病，先天性心脏病等。
淤血性肝损伤最初累及小叶中央区，小叶中央静脉淤血，扩张，肝窦扩张程度与肝窦距小叶中央静脉的远近而有所不同，小叶中央肝细胞受压，变形和萎缩，细胞浆内呈颗粒样变，有核固缩，核分裂，细胞坏死，伴有棕色色素沉着，棕色色素位于小叶中央，可能因淤胆所致，邻近中央静脉的肝实质变性坏死最严重，随淤血的加重，坏死组织向门区延伸，严重淤血患者仅在门区有较正常的肝组织，随时间延长，中央静脉周围的网状纤维可塌陷，可见网状纤维组织和细纤维束自中央静脉延伸到另一中央静脉。
对于肝组织损伤的治疗目前主要包括病因的控制和治疗，以及支持治疗。长期以来，科学家们一直在寻找对损伤肝组织有直接的、良好修复效果的药物。本发明对此也进行了深入的研究。实验发现，人体内天然存在的一种蛋白质物质，纤溶酶原对中毒、辐射、化疗药物和糖尿病导致的肝组织损伤有良好的修复作用。纤溶酶原有望成为治疗肝组织损伤及其相关病症的一种新策略。
纤溶酶原(plasminogen，plg)是纤溶酶的非活性前体，是一种单链糖蛋白，由791个氨基酸组成，分子量约为92kDa[3，4]。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[5，6]。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PA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[7]。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringle，羧基末端部分包含蛋白酶结构域。一些kringle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。最新发现一个为38kDa的纤维蛋白溶酶原片段，其中包括kringlel-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。
发明简述
一方面，本发明涉及纤溶酶原或纤溶酶在制备预防和/或治疗受试者肝组织损伤及其相关病症的药物、制品、药盒中的用途。本发明还涉及一种制药方法，包括将纤溶酶原与药学可接受载体共同制备成预防和/或治疗受试者肝组织损伤及其相关病症的药物、制品、药盒。
在一个实施方案中，所述肝组织损伤及其相关病症为辐射或化学物质引起的肝损伤及其相关病症。在一个实施方案中，辐射或化学物质引起的肝损伤及其相关病症为癌症治疗所使用的放化疗方法和药物。在一个实施方案中，所述辐射是由于意外事件或工作环境等其他事件导致的辐射。在一个实施方案中，所述肝组织损伤及其相关病症为中毒性肝损伤及其相关病症。在一个实施方案中，所述中毒性肝损伤为包括酒精在内的″亲肝毒物″造成的中毒性肝损伤。在一个实施方案中，所述肝组织损伤及其相关病症为糖尿病引起的，是糖尿病的并发症之一。在一个实施方案中，所述肝组织损伤及其相关病症为病毒感染肝脏导致的肝炎所致，例如甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒、戊型肝炎病毒导致的肝炎。在一个实施方案中，所述肝组织损伤及其相关病症为药物性肝损伤及其相关病症。在一个实施方案中，所述肝组织损伤及其相关病症是肝内血液淤积(肝脏淤血)导致的。在一个实施方案中，所述肝组织损伤及其相关病症为糖尿病性肝损伤及其相关病症、中毒性肝损伤及其相关病症、药物性肝损伤或其相关病症、辐射性肝损伤及其相关病症、病毒感染性肝损伤及其相关病症、淤血性肝损伤及其相关病症。在一个实施方案中，所述肝组织损伤及其相关病症包括肝组织损伤导致的肝功能异常、肝酶学异常、 肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌。
在一个实施方案，所述纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案，所述纤维蛋白溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。
在一个实施方案，所述纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案，所述纤溶酶原或纤溶酶全身或局部施用，包括表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或直肠施用。在一个实施方案，所述糖尿病性肝损伤的相关病症或中毒性肝损伤的相关病症包括：肝酶学异常、肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌。在一个实施方案，所述糖尿病性肝损伤及其相关病症是由糖尿病引起的大血管、小血管、微血管病变导致。在一个实施方案，所述纤溶酶原可与一种或多种其它药物联合施用。在一个实施方案，所述其它药物包括：保肝药物、抗糖尿病药物、抗血栓药物、抗凝药物、降血脂药物、抗心脑血管疾病药物、抗感染药物。
在一个实施方案，所述所述受试者为哺乳动物，优选为人。
在一个实施方案中，所述由糖尿病引起的肝损伤是由糖尿病引起的大血管、小血管、微血管病变导致。
在一个实施方案中，所述受试者纤维蛋白溶酶或者纤溶酶原低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案中，纤维蛋白溶酶原是在序列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，并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且 仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、δ-纤溶酶原或微纤维蛋白溶酶原。在一个实施方案中，纤维蛋白溶酶原为人天然纤维蛋白溶酶原，例如序列2所示的纤溶酶原的直向同系物，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。
在一个实施方案中，所述纤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，所述局部给药通过在肝部区域应用含有纤溶酶原的导管来进行。
一方面，本发明涉及一种预防和/或治疗受试者肝组织损伤及其相关病症的方法，包括给药受试者有效量的纤溶酶原或纤溶酶。本发明还涉及纤溶酶原或纤溶酶用于预防和/或治疗受试者肝组织损伤及其相关病症的用途。
在一个实施方案中，所述肝组织损伤及其相关病症为辐射或化学物质引起的肝损伤及其相关病症。在一个实施方案中，辐射或化学物质引起的肝损伤及其相关病症为癌症治疗所使用的放化疗方法和药物。在一个实施方案中，所述辐射是由于意外事件导致的辐射。在一个实施方案中，所述 肝组织损伤及其相关病症为中毒性肝损伤及其相关病症。在一个实施方案中，所述中毒性肝损伤为包括酒精在内的″亲肝毒物″造成的中毒性肝损伤。在一个实施方案中，所述肝组织损伤及其相关病症为糖尿病引起的，是糖尿病的并发症之一。在一个实施方案中，所述肝组织损伤及其相关病症为病毒感染肝脏导致的肝炎所致，例如甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒、戊型肝炎病毒导致的肝炎。在一个实施方案中，所述肝组织损伤及其相关病症为药物性肝损伤及其相关病症。在一个实施方案中，所述肝组织损伤及其相关病症是肝内血液淤积(肝脏淤血)导致的。在一个实施方案中，所述肝组织损伤及其相关病症为糖尿病性肝损伤及其相关病症、中毒性肝损伤及其相关病症、药物性肝损伤或其相关病症、辐射性肝损伤及其相关病症、病毒感染性肝损伤及其相关病症、淤血性肝损伤及其相关病症。在一个实施方案中，所述肝组织损伤及其相关病症包括肝组织损伤导致的肝功能异常、肝酶学异常、肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌。
在一个实施方案，所述纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案，所述纤维蛋白溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案，所述纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案，所述纤溶酶原或纤溶酶全身或局部施用，包括表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或直肠施用。在一个实施方案，所述糖尿病性肝损伤的相关病症或中毒性肝损伤的相关病症包括：肝酶学异常、肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌。在一个实施方案，所述糖尿病性肝损伤及其相关病症是由糖尿病引起的大血管、小血管、微血管病变导致。在一个实施方案，所述纤溶酶原可与一种或多种其它药物联合施用。在一个实施方案，所述其它药物包括：保肝药物、抗糖 尿病药物、抗血栓药物、抗凝药物、降血脂药物、抗心脑血管疾病药物、抗感染药物。
在一个实施方案，所述受试者为哺乳动物，优选为人。
在一个实施方案中，所述由糖尿病引起的肝损伤是由糖尿病引起的大血管、小血管、微血管病变导致。
在一个实施方案中，所述受试者纤维蛋白溶酶或者纤溶酶原低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案中，纤维蛋白溶酶原是在序列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，并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、δ-纤溶酶原或微纤维蛋白溶酶原。在一个实施方案中，纤维蛋白溶酶原为人天然纤维蛋白溶酶原，例如序列2所示的纤溶酶原的直向同系物，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。
在一个实施方案中，所述纤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，所述局部给药通过在肝部区域应用含有纤溶酶原的导管来进行。
一方面，本发明涉及一种预防和/或治疗受试者肝组织损伤及其相关病症的纤溶酶原或纤溶酶、包含所述纤溶酶原或纤溶酶的药物组合物、或包含所述纤溶酶原或纤溶酶的制品或药盒。
在一个实施方案中，所述肝组织损伤及其相关病症为辐射或化学物质引起的肝损伤及其相关病症。在一个实施方案中，辐射或化学物质引起的肝损伤及其相关病症为癌症治疗所使用的放化疗方法和药物。在一个实施方案中，所述辐射是由于意外事件导致的辐射。在一个实施方案中，所述肝组织损伤及其相关病症为中毒性肝损伤及其相关病症。在一个实施方案中，所述中毒性肝损伤为包括酒精在内的″亲肝毒物″造成的中毒性肝损伤。在一个实施方案中，所述肝组织损伤及其相关病症为糖尿病引起的，是糖尿病的并发症之一。在一个实施方案中，所述肝组织损伤及其相关病症为病毒感染肝脏导致的肝炎所致，例如甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒、戊型肝炎病毒导致的肝炎。在一个实施方案中，所述肝组织损伤及其相关病症为药物性肝损伤及其相关病症。在一个实施方案中，所述肝组织损伤及其相关病症是肝内血液淤积(肝脏淤血)导致的。在一个实施方案中，所述肝组织损伤及其相关病症为糖尿病性肝损伤及其相关病症、中毒性肝损伤及其相关病症、药物性肝损伤或其相关病症、辐射性肝损伤及其相关病症、病毒感染性肝损伤及其相关病症、淤血性肝损伤及其相关病症。在一个实施方案中，所述肝组织损伤及其相关病症包括肝组织损伤导致的肝功能异常、肝酶学异常、肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌。
在一个实施方案，所述纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并 且仍然具有纤维蛋白溶酶原活性。在一个实施方案，所述纤维蛋白溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案，所述纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案，所述纤溶酶原或纤溶酶全身或局部施用，包括表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或直肠施用。在一个实施方案，所述糖尿病性肝损伤的相关病症或中毒性肝损伤的相关病症包括：肝酶学异常、肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌。在一个实施方案，所述糖尿病性肝损伤及其相关病症是由糖尿病引起的大血管、小血管、微血管病变导致。在一个实施方案，所述纤溶酶原可与一种或多种其它药物联合施用。在一个实施方案，所述其它药物包括：保肝药物、抗糖尿病药物、抗血栓药物、抗凝药物、降血脂药物、抗心脑血管疾病药物、抗感染药物。
在一个实施方案，所述受试者为哺乳动物，优选为人。
在一个实施方案中，所述由糖尿病引起的肝损伤是由糖尿病引起的大血管、小血管、微血管病变导致。
在一个实施方案中，所述受试者纤维蛋白溶酶或者纤溶酶原低下。具体地，所述低下是先天的、继发的和/或局部的。
在一个实施方案中，纤维蛋白溶酶原与序列2、6、8、10或12具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤维蛋白溶酶原活性。在一个实施方案中，纤维蛋白溶酶原是在序列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，并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤维蛋白溶酶原活性的蛋白质。在一个实施方案中，纤溶酶原选自Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、小纤维蛋白溶酶原、微纤维蛋白溶酶原、δ-纤溶酶原或其任意组合。在一个实施方案中，纤溶酶原是选自如下的保守取代变体：Glu-纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、 小纤维蛋白溶酶原、δ-纤溶酶原或微纤维蛋白溶酶原。在一个实施方案中，纤维蛋白溶酶原为人天然纤维蛋白溶酶原，例如序列2所示的纤溶酶原的直向同系物，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。
在一个实施方案中，所述纤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，所述局部给药通过在肝部区域应用含有纤溶酶原的导管来进行。
在一个实施方案中，所述纤溶酶原或纤溶酶被分装在容器中。优选，该制品或药盒还包含其它的药物，分装在该药盒的其它容器中。该药盒还可包含使用说明书，说明所述纤溶酶原可以用于治疗肝组织损伤及其相关病症，具体的，例如糖尿病引起的糖尿病肝损伤及其相关病症、中毒性肝损伤及其相关病症、药物性肝损伤或其相关病症、辐射导致的肝损伤及其相关病症、病毒感染引起的肝损伤及其相关病症、淤血导致的肝损伤及其相关病症，并且可以进一步说明，所述纤溶酶原或纤溶酶可以在其它药物或疗法施用之前，同时，和/或之后施用。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要 素的所有亚组合，并且在本文中公开，就像每一个此类亚组合单独且明确在本文中公开一样。
发明详述
“糖尿病性肝损伤”是指由糖尿病引起的肝脏组织学和功能变化的病变。其主要由糖尿病引起的大血管、小血管、微血管病变导致。已知糖尿病可引起的肝损伤包括：肝酶学异常，其可引起肝细胞内二氧化碳蓄积、酸中毒、氧供减少、氧消耗增加，使肝脏转氨酶活性增加，胆红素代谢紊乱，重者可引起肝细胞坏死；脂肪肝，在引起脂肪肝的所有病因中，糖尿病占第三位，其中21％～78％糖尿病患者伴有脂肪肝；肝炎、硬化和肝癌，其中糖尿病患者中病毒性肝炎的患病率约为正常人的2～4倍，原发性肝癌的发生率约为正常人的4倍。
“化学性肝损伤”或“中毒性肝损伤”是指由化学性肝毒性物质所造成的肝损害。这些化学物质包括酒精、环境中的化学有毒物质及某些药物。大自然和人类工业生产过程中均存在一些对肝脏有毒性的物质，称为″亲肝毒物″，这些毒物在人群中普遍易感，潜伏期短，病变的过程与化学物质的量直接相关，可引起肝脏不同程度的肝细胞坏死、脂肪变性、肝硬化。
“亲肝毒物”是指对肝脏有毒性的物质的总称。酒精是生活中最常见的“亲肝毒物”。除酒精外，环境中的化学有毒物质及某些药物也可造成肝损伤。作为人体的重要解毒器官，肝脏具有肝动脉和肝静脉双重血液供应。化学物质可通过胃肠道门静脉或体循环进入肝脏进行转化，因此肝脏容易受到化学物中的毒性物质损害。大自然和人类工业生产过程中均存在一些对肝脏有毒性的物质，称为″亲肝毒物″。他们进入肝脏，可引起肝脏不同程度的肝细胞坏死、脂肪变性、肝硬化。病理表现包括(1)脂肪变性。四氯化碳、黄磷等可干扰脂蛋白的合成与转运，形成脂肪肝。(2)脂质过氧化反应，这是中毒性肝损伤的特殊表现形式，如四氯化碳在体内代谢产生一种氧化能力很强的中间产物，导致生物膜上的脂质过氧化，破坏膜的磷脂，改变细胞的结构与功能。(3)胆汁郁积反应，主要与肝细胞膜和微绒毛受损，引起胆汁酸排泄障碍有关。
“药物性肝损伤”为药物使用过程中，因药物本身或/及其代谢产物或由于特殊体质对药物的超敏感性或耐受性降低所导致的肝脏损伤称为药物性肝损伤，亦称药物性肝病，临床上可表现为各种急慢性肝病，轻者停药后可自行恢复，重者可能危及生命、需积极治疗、抢救。“药物性肝损伤”可以发生在以往没有肝病史的健康者或原来就有严重疾病的患者身上；可发生在用药超量时，也可发生在正常用量的情况下。
“辐射性肝损伤”是指高能电离辐射包括α、β粒子、γ射线、χ射线和中子射线导致的一种放射性损伤。大剂量射线瞬间照射或低剂量射线长时间照射都可能引起组织损伤，辐射的能量破坏细胞的染色体、酶，使细胞的正常功能发生紊乱。
“病毒感染性肝损伤”为病毒感染导致的肝损伤总称。所述病毒感染常见有甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒、戊型肝炎病毒导致的感染。
“淤血性肝损伤”为血液在肝内淤积导致的肝脏组织损伤病变。任何导致下腔静脉血回心受阻的疾病都可导致肝脏淤血，如风湿性心脏瓣膜病，慢性缩窄性心包炎，高血压性心脏病，缺血性心脏病，肺心病，先天性心脏病等。
“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92kD，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。Δ-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[8，9]，有文献报道了δ-纤溶酶原的氨基酸序列(序列8)[9]，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)[10]，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)[11]，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。
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，对于血液中纤维蛋白溶酶原及其活性测定方法包括：对组织纤维蛋白溶酶原激活剂活性的检测(t-PAA)、血浆组织纤维蛋白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)、血浆组织纤维蛋白溶酶原激活剂抑制物活性的检测、血浆组织纤维蛋白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，吸光度增加与纤维蛋白溶酶原活性成正比。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维蛋白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，β-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纤溶酶原(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、鼻内、表面或皮内施用或脊髓或脑投递来 实现本发明药物组合物的施用。气溶胶制剂如鼻喷雾制剂包含活性剂的纯化的水性或其它溶液及防腐剂和等渗剂。将此类制剂调节至与鼻粘膜相容的pH和等渗状态。
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、定期评估糖尿病肝病及其相关病症的治疗效果和安全性。
治疗效力和治疗安全性
本发明的一个实施方案涉及使用纤溶酶原治疗受试者后，对治疗效力和治疗安全性的判断。其中对所述治疗效力的判断方法包括但不限于：1)对受试者肝功能的检查，例如对患者体内酶学水平如血清门冬氨酸氨基转移酶(ALT)、谷丙转氨酶(AST)、总胆红素、直接胆红素、间接胆红素、白蛋白、球蛋白、胆碱酯酶、碱性磷酸酶、转肽酶等水平是否处于正常值范围进行检查，使用本发明中的纤溶酶原对受试者进行治疗后，预期上述肝 功能指标将恢复至正常值或得到改善，例如，谷丙转氨酶(ALT)：0～40μ/L、谷草转氨酶(AST)：0～40μ/L、谷氨酰转移酶(GGT)：小于40单位、总胆红素：3.4～20.5μmol/L；2)对受试者凝血酶原时间(PT)和活动度(PTA)进行检查：PT是反映肝脏凝血因子合成功能的重要指标，PTA是PT测定值的常用表示方法，对判断肝病进展和预后有较大价值，其中PTA进行性降至40％以下为肝衰竭的重要诊断标准之一，＜20％者提示肝功能不良，使用本发明中的纤溶酶原及其变体对受试者进行治疗后，患者体内PTA的下降预期将得到明显改善；3)影像学检查：包括腹部肝胆脾彩超、CT或核磁，以了解肝损伤恢复程度；4)肿瘤标志物检查，如甲胎蛋白AFP、CA199、AFU等；5)肝组织活检，以判断纤维化及其他损伤的恢复程度。此外，本发明还涉及使用纤溶酶原及其变体对受试者进行治疗过程中和治疗后，所述该治疗方案安全性的判断，包括但不限于对受试者的血清半衰期、治疗半衰期、半数中毒量(TD50)、半数致死量(LD50)进行统计，或对在治疗过程中或治疗后发生的各种不良事件如致敏反应进行观察。
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肝损伤及其相关病症的的本发明纤溶酶原。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肝损伤及其相关病症。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显示24-25周龄的糖尿病小鼠在给予纤溶酶原后体重的变化。
图2显示24-25周龄的糖尿病小鼠在连续15天给药纤溶酶原后肝脏HE染色观察结果。
图3显示24-25周龄的糖尿病小鼠在连续15天给予纤溶酶原后肝脏纤维蛋白免疫染色镜检观察结果。
图4显示24-25周龄的糖尿病小鼠在连续31天给予纤溶酶原后体重变化。
图5显示24-25周龄的糖尿病小鼠在连续31天给予纤溶酶原后肝脏HE染色观察结果。
图6显示24-25周龄的糖尿病小鼠在连续31天给予纤溶酶原后肝脏纤维蛋白免疫染色观察结果。
图7显示24-25周龄的糖尿病小鼠在连续31天给予纤溶酶原后肝F4/80免疫染色观察结果。
图8显示24-25周糖尿病小鼠给予PBS或纤溶酶原31天后血清谷丙转氨酶(ALT)检测结果。
图9显示给予纤溶酶原的第0、2、7天四氯化碳致急性肝损伤小鼠肝脏HE染色观察结果。
图10显示plg-/-四氯化碳致急性肝损伤小鼠给予纤溶酶原18、24、48小时后肝脏HE染色观察结果。
图11显示plg-/-四氯化碳致急性肝损伤小鼠给予纤溶酶原18、24、48小时后肝脏纤维蛋白免疫染色观察结果。
图12显示5.0Gy X射线辐射的小鼠纤溶酶原10天后肝脏F4/80免疫染色观察结果。
图13显示10mg/Kg顺铂化疗损伤模型小鼠纤溶酶原7天后肝脏纤维蛋白免疫染色观察结果。
图14显示plg-/-四氯化碳致急性肝损伤小鼠给予纤溶酶原18、24、48小时及7天后肝脏HE染色观察结果。
实施例
实施例1纤溶酶原对神经损伤后期糖尿病小鼠体重的影响
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，每各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药15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。在给纤溶酶原第0、4、7、11、16天分别称体重。结果显示给纤溶酶原组和给溶媒PBS对照组在第0、4、7、11、16天体重(图1)无显著差异，说明纤溶酶原对动物体重影响不大。
实施例2纤溶酶原对糖尿病肝损伤晚期肝脏组织损伤的保护作用
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，每各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药15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。在第16天处死小鼠并取肝脏组织在10％中性福尔马林固定液中固定24-48小时固定后的肝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，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片。
HE染色结果显示，给溶媒PBS对照组，肝细胞呈重度脂肪变，脂质沉积，细胞核被挤至边缘，细胞轻度水样变性，肝索紊乱；给纤溶酶原组较之于给溶媒对照组，肝细胞脂肪变性减轻，呈轻度脂肪变，以中度水样变性为主。说明纤溶酶原能促进糖尿病肝损伤的修复。
实施例3纤溶酶原减少糖尿病小鼠肝组织纤维蛋白水平
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，每各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，连续给药15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。在第16天处死小鼠并取肝脏组织在10％中性福尔马林固定液中固定24-48小时。固定后的肝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。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液，用PAP笔圈 出组织。兔抗小鼠纤维蛋白(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，流水冲洗5分钟，然后TBS洗1次。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[12-14]。因此可将纤维蛋白原水平作为损伤程度的一个标志。
研究发现，给纤溶酶原组(图3B)与给溶媒PBS对照组(图3A)相比，给纤溶酶原组的小鼠其肝脏组织纤维蛋白的水平降低，说明纤溶酶原具有抑制纤维蛋白水平沉积的功能，损伤得到一定程度的修复。
实施例4纤溶酶原对糖尿病小鼠体重的影响
24-25周龄db/db雄鼠20只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组，每组各10只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，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。在第0，4，7，11，16，21，26，31天分别称体重。
结果显示，给纤溶酶原组和给溶媒PBS对照组在第0，4，7，11，16，21，26，31天体重无显著差异(图4)，说明纤溶酶原对动物体重影响不大。
实施例5纤溶酶原对糖尿病肝损伤晚期肝脏组织损伤的保护作用
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，每各5只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，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。在第32天处死小鼠并取肝脏组织在10％中性福尔马林固定液中固定24-48小时固定后的肝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，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片。
HE染色结果显示，给溶媒PBS对照组(图5A)肝脏呈重度脂肪变性，脂质沉积，融合成大的脂肪空泡，细胞核被挤至边缘
Figure PCTCN2016110451-appb-000001
肝索紊乱，肝窦变窄，且在肝索处有数量不等的炎性灶(↑)；给纤溶酶原组(图5B)肝脏轻度脂肪变性，损伤以轻度水样变性为主，胞浆溶解
Figure PCTCN2016110451-appb-000002
主要分布在汇管区与中央静脉中间的区域，汇管区及中央静脉周围受累较轻，同时可见肝索处轻度炎细胞浸润。说明给纤溶酶原后肝脏的损伤得到明显修复。
实施例6纤溶酶原减少糖尿病小鼠肝组织纤维蛋白水平
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天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，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。在第32天处死小鼠并取肝脏组织在10％中性福尔马林固定液中固定24小时。固定后的肝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。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠纤维蛋白(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，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[12-14]。因此可将纤维蛋白原水平作为损伤程度的一个标志。
研究发现，给纤溶酶原组(图6B)与给溶媒PBS对照组(图6A)相比，给纤溶酶原组的小鼠其肝脏组织纤维蛋白的水平明显降低，说明注射纤溶酶原能够显著降低糖尿病小鼠的纤维蛋白沉积，反映出纤溶酶原对糖尿病小鼠的机体损伤有显著修复功能。
实施例7纤溶酶原减轻糖尿病小鼠肝脏组织的炎症
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天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，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天后处死小鼠并取肝脏组织在10％中性福尔马林固定液中固定24小时。固定后的肝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。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％正常羊血清封闭1小时，时间到后甩去血清，用PAP笔圈出组织。针对F4/80的兔多克隆抗体(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，梯度脱水透明并封片，切片在显微镜下400倍下观察。
F4/80巨噬细胞标志物，可以表示炎症反应的程度和阶段。结果显示，给纤溶酶原组(图7B)与给溶媒PBS对照组(图7A)相比，给纤溶酶原组小鼠的F4/80阳性水平明显降低，说明给纤溶酶原后肝脏组织炎症程度减轻。图7C为F4/80免疫组化阳性表达数定量分析结果，给纤溶酶原组F4/80表达量显著减少，且具有统计学差异，说明注射纤溶酶原能够显著促进糖尿病小鼠肝脏炎症的修复。
实施例8纤溶酶原促进糖尿病小鼠肝脏损的修复
25-28周龄db/db雄鼠9只，随机分为两组，给溶媒PBS对照组3只，给纤溶酶原组6只。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天，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天后摘眼球采全血，待血清析出后4℃ 3500r/min离心10分钟，取上清液进行检测。本实验使用谷丙转氨酶检测试剂盒(南京建成生物工程研究所，货号C009-2)，运用赖氏比色法(Reitman-Frankel)检测血清中谷丙转氨酶(ALT)的含量。
谷丙转氨酶是肝脏健康状态的一个重要指标[15，16]，谷丙转氨酶的正常参考值区间为9～50U/L。检测结果显示，给溶媒PBS对照组血清中ALT的 含量显著高于正常生理指标，而给纤溶酶原组已经恢复到体内的正常水平，并且给纤溶酶原组要显著低于给溶媒PBS对照组，且具有统计学差异(图8)。说明在糖尿病晚期模型小鼠中，注射纤溶酶原能有效地修复肝损伤。
实施例9纤溶酶原对急性肝中毒肝脏的保护作用
7-8周龄plg+/+小鼠18只，雌雄不限，随机分为两组，分别为给溶媒PBS对照组和纤溶酶原给药组，每组9只。两组小鼠按0.5mL/kg体重经腹腔注射给予四氯化碳，连续给予两天，建立急性肝损伤模型[17，18]。四氯化碳使用前需用玉米油稀释，前者与后者的体积比1∶7。造模当天为第0天，第1天开始给纤溶酶原或PBS。给纤溶酶原组小鼠按每天1mg/0.1mL/只/天给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS，连续给药7天。在第0，2，7天分别取两组小鼠各3只处死，解剖观察记录肝脏情况，然后肝脏组织在10％中性福尔马林固定液中固定24-48小时。固定后的肝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，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片，在显微镜下200倍下观察。
HE染色结果显示，给溶媒PBS对照组(图9A-C)和给纤溶酶原组(图9D-F)小鼠的肝脏第0天主要以中央静脉周围碎片状坏死为主，坏死区域胞核碎裂，胞浆淡染，其他未坏死区也发生了中度水样变性，细胞水肿；第2天中央静脉扩张，肝细胞结构紊乱，少量炎性细胞浸润，两组并无明显差异。但在第7天时，给溶媒PBS对照组仍可见少量的肝细胞变性，细胞轻度水肿，肝索紊乱，肝血窦变窄，且在汇管区周围轻度炎细胞浸润，而给纤溶酶原组的肝脏基本恢复正常胞浆红染，肝索规则，肝窦清晰。说明纤溶酶原能够促进肝损伤的修复。
实施例10纤溶酶原对急性肝中毒肝脏的保护作用
7-11周龄plg-/-雄性小鼠18只，随机分为两组，分别为给溶媒PBS对照组和纤溶酶原给药组，每组9只。两组小鼠按0.5mL/kg体重经腹腔注射给予四氯化碳，单次处理，建立急性肝损伤模型[17，18]。四氯化碳使用前需用玉米油稀释，前者与后者的体积比1∶7。造模给完成后半小时内给予纤溶酶原或溶媒PBS。给纤溶酶原组小鼠按每天1mg/0.1mL/只/天给予纤溶酶原， 给溶媒PBS对照组给予相同体积的PBS，连续给药2天。在给药后第18，24，48小时分别取两组小鼠各3只处死，解剖观察记录肝脏情况，然后肝脏组织在10％中性福尔马林固定液中固定24-48小时。固定后的肝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，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片，在显微镜下200倍下观察。
结果显示，给溶媒PBS对照组(图10A-C)在18h、24h、48h均呈现不同程度的坏死，18h、24h以片状坏死为主，48h时已经发生了桥接坏死，核碎裂，胞浆淡染，而且损伤不断加剧，主要分布在中央静脉周围，坏死区中度炎细胞浸润(↓)，汇管区周围坏死较轻，以轻度水样变性为主，伴以轻度的炎细胞浸润，轻度胆管增生
Figure PCTCN2016110451-appb-000003
给纤溶酶原组(图10D-F)在18h、24h、48h较之于对照组，均未出现明显的坏死，损伤以轻度水样变性为主，分布在汇管区周围，而中央静脉周围肝细胞未受累，而24h时比之18h有好转，水样变性减轻，中央静脉周围肝细胞轻度脂肪变性，胞浆淡染，均伴以轻度炎细胞浸润。说明纤溶酶原能够促进plg-/-急性肝损伤模型小鼠肝脏损伤的修复。
实施例11纤溶酶原减轻急性肝损伤模型小鼠肝脏组织纤维蛋白沉积
7-11周龄plg-/-雄性小鼠18只，随机分为两组，分别为给溶媒PBS对照组和纤溶酶原给药组，每组9只。两组小鼠按0.5mL/kg体重经腹腔注射四氯化碳，单次处理，建立急性肝损伤模型[17，18]。四氯化碳使用前需用玉米油稀释，前者与后者的体积比1∶7。造模给完成后半小时内给予纤溶酶原或溶媒PBS。给纤溶酶原组小鼠按每天1mg/0.1mL/只/天给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS，连续给药2天。在给药后第18，24，48小时分别取两组小鼠各3只处死，解剖观察记录肝脏情况，然后肝脏组织在10％中性福尔马林固定液中固定24-48小时。固定后的肝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。以3％双氧水孵育15分钟，水洗2次，每次5分钟。10％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠纤维蛋白(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，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[12-14]。因此可将纤维蛋白原水平作为损伤程度的一个标志。
结果显示，第18、24、48小时三个时间点给纤溶酶原组(图11D-F)纤维蛋白的阳性着色都显著浅于给溶媒PBS对照组(图11A-C)，并且随着时间的延长纤维蛋白的着色也有逐渐变浅的趋势。说明注射纤溶酶原能够减少纤维蛋白的沉积，促进肝损伤的修复。
实施例12纤溶酶原促进5.0GyX射线辐射小鼠肝脏炎症的修复
本实验使用6-8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，建立放射损伤模型，采用直线加速器6MV X射线按5.0Gy对小鼠全身单次均匀照射，吸收剂量率2.0Gy/min，吸收剂量为5.0Gy(照射2.5分钟)。建立模型后，3个小时内给予纤溶酶原。实验开始当天为第0天称量体重并分组，第1天开始辐射处理并给予纤溶酶原或溶媒PBS，给药期为10天给药完成后对动物进行停药观察11天，整个实验期为21天。给纤溶酶原组按1mg/0.1mL/只/天经尾静脉注射给药，给溶媒PBS对照组给予相同体积的PBS。在第21天处死解剖小鼠并取肝脏在10％中性福尔马林固定液中固定24-48小时。固定后的肝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。Tris-EDTA修复30分钟，室温冷却20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％的正常羊血清(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液。兔抗小鼠F4/80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，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
F4/80免疫组化结果显示，5.0Gy X射线照射造模后给溶媒PBS对照组(图12A)的小鼠巨噬细胞标志物的表达量高于给纤溶酶原组(图12B)，说明给纤溶酶原后，动物肝脏组织的炎症显著减轻。
实施例13纤溶酶原使顺铂化疗损伤模型小鼠肝脏组织纤维蛋白的沉积减少
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，建立化疗损伤模型，按10mg/Kg体重单次腹腔注射顺铂。建立模型后，给纤溶酶原组按1mg/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。实验开始当天为第0天称量体重并分组，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，给药期为7天。第8天处死小鼠，取肝脏在10％中性福尔马林固定液中固定24-48小时。固定后的肝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％的正常羊血清(Vector laboratories，Inc.，USA)封闭1小时；时间到后，弃除羊血清液。兔抗小鼠纤维蛋白抗体(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，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
纤维蛋白原是纤维蛋白的前体，在组织存在损伤的情况下，作为机体对损伤的一种应激反应，纤维蛋白原水解成纤维蛋白[12-14]。因此可将纤维蛋白原水平作为损伤程度的一个标志。
结果显示，给溶媒PBS对照组(图13A)肝脏组织中纤维蛋白阳性着色明显深于纤溶酶原给药组(图13B)。说明纤溶酶原能够使损伤的肝脏组织中沉积的纤维蛋白显著减少，表明纤溶酶原能够促进化疗药顺铂所致的肝脏损伤的修复。
实施例14纤溶酶原对急性肝中毒肝脏的保护作用
7-11周龄plg-/-雄性小鼠6只，随机分为两组，分别为给溶媒PBS对照组和纤溶酶原给药组，每组3只。两组小鼠按0.5mL/kg体重经腹腔注射给予四氯化碳，单次处理，建立急性肝损伤模型[17，18]。四氯化碳使用前需用玉米油稀释，前者与后者的体积比1∶7。造模给完成后半小时内给予纤溶酶原或溶媒PBS。给纤溶酶原组小鼠按每天1mg/0.1mL/只/天给予纤溶酶原，给溶媒PBS对照组给予相同体积的PBS，连续给药7天。在第8天处死小鼠，解剖观察记录肝脏情况，然后肝脏组织在10％中性福尔马林固定液中固定24-48小时。固定后的肝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，切片脱蜡复水并用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片，在显微镜下200倍下观察。
结果显示，给溶媒PBS对照组(图14A)，肝脏中央静脉扩张，内皮细胞坏死，中央静脉的周围肝细胞均发生大面积灶状坏死，核碎裂深染，其他未发生坏死的区域有轻度的水样变性，细胞水肿，胞浆透亮，伴以坏死区轻度炎细胞浸润；给纤溶酶原组(图14B)，肝脏未发生明显的坏死，损伤以轻度的水样变性为主，有少量肝细胞胞浆嗜酸性增强，红染。给纤溶酶原组损伤明显轻于给溶媒PBS对照组，说明纤溶酶原能够促进plg-/-急性肝损伤模型小鼠肝脏损伤的修复。
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Claims (13)

  1. 一种预防和/或治疗受试者肝组织损伤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，其中所述肝组织损伤及其相关病症为辐射或化学物质引起的肝损伤及其相关病症。
  3. 权利要求1的方法，其中所述肝组织损伤及其相关病症为中毒性肝损伤及其相关病症。
  4. 权利要求1的方法，其中所述肝组织损伤及其相关病症为糖尿病性肝损伤及其相关病症。
  5. 根据权利要求4的方法，其中所述糖尿病性肝损伤及其相关病症是由糖尿病引起的大血管、小血管、微血管病变导致。
  6. 权利要求1-5任一项的方法，其中所述肝组织损伤及其相关病症包括肝组织损伤导致的肝功能异常、肝酶学异常、肝区不适和触痛、肝肿大、脾肿大、肝脾肿大、肝炎、脂肪肝、胆管炎、肝硬化、肝坏死和肝癌。
  7. 根据权利要求1-6的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少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。
  8. 根据权利要求1-7任一项的方法，其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。
  9. 根据权利要求1-8任一项的方法，其中所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、δ(delta)-纤溶酶原或其任意组合。
  10. 根据权利要求1-9任一项的方法，其中所述纤溶酶原可与一种或多种其它药物联合施用。
  11. 一种用于预防和/或治疗受试者肝组织损伤及其相关病症的制品，其包含含有有效剂量的纤溶酶原的容器，和指导施用所述制品预防和/或治疗受试者肝组织损伤及其相关病症的说明书。
  12. 权利要求11的制品，进一步包含含有一种或多种其它药物的容器。
  13. 权利要求12的制品，其中所述说明书进一步说明所述纤溶酶原可以在所述其它药物施用之前，同时，和/或之后施用。
PCT/CN2016/110451 2015-12-18 2016-12-16 一种预防和治疗肝组织损伤及其相关病症的方法 WO2017101869A1 (zh)

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EP16874926.5A EP3391901B1 (en) 2015-12-18 2016-12-16 Plasminogen for use in treating liver tissue damage
CA3008475A CA3008475C (en) 2015-12-18 2016-12-16 Method for preventing and treating hepatic tissue injury and related disorders thereof
JP2018550636A JP6815413B2 (ja) 2015-12-18 2016-12-16 肝組織損傷及びその関連疾患を予防及び治療するための方法

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EP3391901B1 (en) 2023-07-05
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EP3391901A4 (en) 2019-12-04
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CA3008475A1 (en) 2017-06-22
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