A method for preventing and treating liver tissue damage and related conditions

Abstract

The use of plasminogen in the treatment and / or elimination of liver damage provides a new therapeutic approach for the treatment of different types of liver damage.

Description

[0001] This application is a divisional application of Chinese patent application No. 201680073668.6, entitled "A method for preventing and treating liver tissue damage and related diseases", filed on December 16, 2016 (PCT application No. PCT / CN2016 / 110451). Technical Field

[0002] This invention relates to the use of plasminogen or plasmin in the prevention and / or treatment of liver tissue damage from various causes, thereby providing a novel treatment strategy for liver tissue damage and related conditions. background Liver injury, also known as liver tissue damage, refers to parenchymal liver disease caused by various factors. It encompasses a series of pathological changes, including inflammation, degeneration, necrosis, and fibrosis of the liver tissue. Common causes include inflammation, liver congestion, viral infection, poisoning, medications, and radiation. Some diseases also involve damage to liver tissue cells, such as diabetes, hepatitis, hypertension, and atherosclerosis.

[0003] Drugs are a very common cause of liver tissue damage. Commonly used drugs that cause liver tissue damage include: anti-tuberculosis drugs: rifampin, isoniazid, ethambutol, etc.; anti-tumor drugs: cyclophosphamide, methotrexate, 5-fluorouracil, carboplatin, cisplatin, etc.; lipid-lowering drugs: statins (atorvastatin, lovastatin), fenofibrate, clofibrate, niacin, etc.; steroid hormones: estrogen drugs, oral contraceptives, anabolic steroids, etc.; cardiovascular drugs: amiodarone, warfarin, calcium channel blockers, etc.; antirheumatic drugs: indomethacin, fenbufen, aspirin, indomethacin, etc.; antibiotics: chloramphenicol, roxithromycin, ketoconazole, penicillins, sulfonamides, etc.; anti-allergy drugs: promethazine (phenergan), chlorpheniramine (chlorpheniramine), loratadine (loratadine), etc.; anti-ulcer drugs: cimetidine, ranitidine, famotidine, etc.; antifungal drugs such as ribavirin, etc.

[0004] Alcohol poses a significant threat to the liver. Long-term or intermittent heavy drinking can cause liver tissue damage, and the greater the amount of alcohol consumed and the longer the duration of drinking, the more severe the consequences. Alcohol directly poisons liver cells, affecting their structure and function.

[0005] Alcoholic liver injury is a chronic toxic liver injury caused by long-term heavy drinking. It typically begins as fatty liver, progressing to alcoholic hepatitis, liver fibrosis, and cirrhosis. Its main clinical features include nausea, vomiting, jaundice, and may include hepatomegaly and tenderness. Severe alcohol abuse can induce widespread hepatocellular necrosis and even liver failure. Alcoholic liver disease is one of the most common liver diseases in my country, seriously endangering public health. [1] .

[0006] In addition to alcohol-induced toxic liver damage, other "hepatotoxic substances," such as toxic chemicals in the environment and certain drugs, can also cause liver damage. As an important detoxification organ in the human body, the liver has a dual blood supply from the hepatic artery and hepatic vein. Chemical substances can enter the liver through the portal vein or systemic circulation in the gastrointestinal tract for transformation, so the liver is easily damaged by toxic substances in chemicals. There are some substances in nature and human industrial production processes that are toxic to the liver, called "hepatotoxic substances." These toxins are generally susceptible to in the population, have a short incubation period, and the pathological process is directly related to the amount of chemical substances, which can cause different degrees of hepatocyte necrosis, fatty degeneration, and cirrhosis. Pathological manifestations include (1) fatty degeneration. Carbon tetrachloride, yellow phosphorus, etc. can interfere with the synthesis and transport of lipoproteins, forming fatty liver. (2) lipid peroxidation reaction, which is a special manifestation of toxic liver damage. For example, carbon tetrachloride produces a highly oxidizing intermediate product in the body, which leads to lipid peroxidation on biological membranes, destroys membrane phospholipids, and changes the structure and function of cells. (3) Cholestasis reaction is mainly related to damage to hepatocyte membranes and microvilli, which leads to impaired bile acid excretion. [2] .

[0007] Radiation can also cause liver tissue damage. Generally, radiation sources are high-energy electromagnetic waves or high-energy particles generated by natural or artificial energy sources. Both instantaneous exposure to high doses of radiation and prolonged exposure to low doses of radiation can cause tissue damage. The energy of radiation damages cell chromosomes and enzymes, disrupting normal cell function.

[0008] Diabetic liver disease refers to the pathological changes in liver histology and function caused by diabetes. Known liver damage caused by diabetes includes: abnormal liver enzymes, which can lead to carbon dioxide accumulation, acidosis, reduced oxygen supply, and increased oxygen consumption in hepatocytes, resulting in increased hepatic transaminase activity, bilirubin metabolism disorders, and in severe cases, hepatocyte necrosis; fatty liver, which ranks third among all causes of fatty liver, with 21%–78% of diabetic patients also having fatty liver; hepatitis, cirrhosis, and liver cancer, with the prevalence of viral hepatitis in diabetic patients being approximately 2–4 times that of the general population, and the incidence of primary liver cancer being approximately 4 times that of the general population. Diabetic liver disease not only impairs the quality of life of millions of patients but also imposes a huge burden of cost and requires significant care from the healthcare system.

[0009] Viral infections of the liver are also a common cause of liver damage, such as hepatitis B, hepatitis C, and hepatitis E.

[0010] Blood stasis in the liver can also cause liver tissue damage. Blood stasis in the liver is mainly caused by the following factors: hepatic venous occlusive disease, Budd-Kiarli syndrome, chronic right heart failure, and constrictive pericarditis.

[0011] Any disease that obstructs the return of blood from the inferior vena cava to the heart can lead to liver congestion, such as rheumatic heart valve disease, chronic constrictive pericarditis, hypertensive heart disease, ischemic heart disease, pulmonary heart disease, and congenital heart disease.

[0012] Congestive liver injury initially affects the central region of the lobule, with congestion and dilation of the central lobule vein. The degree of dilation of the sinusoids varies depending on their distance from the central lobule vein. Central lobule hepatocytes are compressed, deformed, and atrophied, with granular degeneration in the cytoplasm, nuclear pyknosis, mitosis, cell necrosis, and brown pigmentation. The brown pigmentation is located in the central lobule, possibly due to cholestasis. The liver parenchyma adjacent to the central vein shows the most severe degeneration and necrosis. As congestion worsens, necrotic tissue extends towards the portal region. In patients with severe congestion, only relatively normal liver tissue is present in the portal region. Over time, the reticular fibers around the central vein may collapse, and reticular fibrous tissue and fine fiber bundles can be seen extending from the central vein to another central vein.

[0013] Treatment for liver tissue injury currently mainly includes controlling and treating the underlying cause, as well as supportive care. For a long time, scientists have been searching for drugs with direct and effective repair effects on damaged liver tissue. This invention has also conducted in-depth research in this area. Experiments have found that plasminogen, a naturally occurring protein in the human body, has a good repairing effect on liver tissue damage caused by poisoning, radiation, chemotherapy drugs, and diabetes. Plasminogen shows promise as a new strategy for treating liver tissue injury and related conditions.

[0014] Plasminogen (plg) 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 primarily synthesized in the liver and is abundant in extracellular fluid. The plasma plasminogen concentration is approximately 2 μM. Therefore, plasminogen is a significant potential source of protein hydrolysis activity in tissues and body fluids. [5,6] Plasminogen exists in two molecular forms: glutamate-plasminogen and lysine-plasminogen. The naturally secreted and uncleaved form of plasminogen has a single N-terminal glutamate amino acid, hence the name glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed at Lys76-Lys77 to form lysine-plasminogen. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate. The Arg560-Val561 peptide bond of both forms of plasminogen can be cleaved by uPA or tPA, leading to the formation of the disulfide-linked double-stranded protease plasmin. [7]The N-terminal portion of plasminogen contains five homologous tricyclic rings, known as kringles, while the C-terminal portion contains a protease domain. Some kringles contain lysine binding sites that mediate the specific interaction between plasminogen and fibrin and its inhibitor α2-AP. A recently discovered 38 kDa plasminogen fragment, including kringles 1-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, can be produced by the hydrolysis of plasminogen by several proteases. Invention Summary On one hand, this invention relates to the use of plasminogen or plasmin in the preparation of medicaments, articles, and kits for the prevention and / or treatment of liver tissue injury and related conditions in subjects. This invention also relates to a pharmaceutical method comprising co-preparing plasminogen with a pharmaceutically acceptable carrier into medicaments, articles, and kits for the prevention and / or treatment of liver tissue injury and related conditions in subjects.

[0015] In one embodiment, the liver tissue injury and related symptoms are liver injury and related symptoms caused by radiation or chemicals. In one embodiment, the liver injury and related symptoms caused by radiation or chemicals are radiotherapy and chemotherapy methods and drugs used in cancer treatment. In one embodiment, the radiation is radiation caused by accidents or other events such as the work environment. In one embodiment, the liver tissue injury and related symptoms are toxic liver injury and related symptoms. In one embodiment, the toxic liver injury is toxic liver injury caused by "hepatotoxic substances" including alcohol. In one embodiment, the liver tissue injury and related symptoms are caused by diabetes and are one of the complications of diabetes. In one embodiment, the liver tissue injury and related symptoms are caused by hepatitis due to viral infection of the liver, such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus. In one embodiment, the liver tissue injury and related symptoms are drug-induced liver injury and related symptoms. In one embodiment, the liver tissue injury and related symptoms are caused by intrahepatic blood stasis (hepatic congestion). In one implementation, the liver tissue injury and related conditions include diabetic liver injury and related conditions, toxic liver injury and related conditions, drug-induced liver injury or related conditions, radiation-induced liver injury and related conditions, viral infection-related liver injury and related conditions, and congestive liver injury and related conditions. In another implementation, the liver tissue injury and related conditions include abnormal liver function, abnormal liver enzymes, discomfort and tenderness in the liver area, hepatomegaly, splenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis, and liver cancer caused by liver tissue injury.

[0016] In one embodiment, the plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequences 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, the plasminogen is a protein containing a plasminogen active fragment and still retaining plasminogen activity.

[0017] In one embodiment, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen or plasmin is administered systemically or locally, including topically, intravenously, intramuscularly, subcutaneously, by inhalation, intrathecally, locally, intra-articularly, or rectally. In one embodiment, the related symptoms of diabetic liver injury or toxic liver injury include: abnormal liver enzymes, discomfort and tenderness in the liver area, hepatomegaly, splenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis, and liver cancer. In one embodiment, the diabetic liver injury and its related symptoms are caused by macrovascular, microvascular, and microvascular complications resulting from diabetes. In one embodiment, the plasminogen may be administered in combination with one or more other drugs. In one embodiment, the other drugs include: hepatoprotective drugs, antidiabetic drugs, antithrombotic drugs, anticoagulants, lipid-lowering drugs, anticardiovascular drugs, and anti-infective drugs.

[0018] In one implementation, the subject is a mammal, preferably a human.

[0019] In one implementation, the liver damage caused by diabetes is due to macrovascular, microvascular, and microvascular lesions caused by diabetes.

[0020] In one implementation, the subject has a deficiency of plasminogen or plasminogen. Specifically, the deficiency is congenital, secondary, and / or localized.

[0021] In one embodiment, plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, plasminogen is a protein with 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, or 1 amino acid additions, deletions, and / or substitutions based on sequence 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, plasminogen is a protein containing a plasminogen active fragment and still retaining plasminogen activity. In one embodiment, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen is selected from conserved substitution variants of Glu-plasminogen, Lys-plasminogen, small plasminogen, δ-plasminogen, or microplasminogen. In one embodiment, the plasminogen is a natural human plasminogen, such as a direct homologue of the plasminogen shown in Sequence 2, for example, a direct homologue of plasminogen from primates or rodents, such as a direct homologue of plasminogen from gorillas, rhesus monkeys, mice, cattle, horses, and dogs. Most preferably, the amino acid sequence of the plasminogen of the present invention is as shown in Sequence 2, 6, 8, 10, or 12.

[0022] In one embodiment, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In one embodiment, the plasminogen is administered at a dose of 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, or 10-100 mg / kg (based on body weight) or 0.0001-2000 mg / cm³. 2 0.001-800 mg / cm 2 0.01-600 mg / cm 2 0.1-400 mg / cm 2 1-200 mg / cm 2 1-100mg / cm 2 10-100 mg / cm 2The dosage (calculated per square centimeter of body surface area) is preferably applied at least once, and preferably at least daily. In the case of topical application, the above dosage may be further adjusted as needed.

[0023] In one embodiment, the plasminogen is administered systemically or locally, preferably via the following routes: surface, intravenous, intramuscular, subcutaneous, inhalation, spinal, local injection, intra-articular injection, or rectal administration. In one embodiment, the local administration is performed via a catheter containing plasminogen in the liver region.

[0024] On one hand, this invention relates to a method for preventing and / or treating liver tissue injury and related conditions in a subject, comprising administering an effective amount of plasminogen or plasmin to the subject. This invention also relates to the use of plasminogen or plasmin for the prevention and / or treatment of liver tissue injury and related conditions in a subject.

[0025] In one embodiment, the liver tissue injury and related symptoms are liver injury and related symptoms caused by radiation or chemicals. In one embodiment, the liver injury and related symptoms caused by radiation or chemicals are radiotherapy and chemotherapy methods and drugs used in cancer treatment. In one embodiment, the radiation is radiation caused by an accidental event. In one embodiment, the liver tissue injury and related symptoms are toxic liver injury and related symptoms. In one embodiment, the toxic liver injury is toxic liver injury caused by "hepatotoxic substances" including alcohol. In one embodiment, the liver tissue injury and related symptoms are caused by diabetes and are one of the complications of diabetes. In one embodiment, the liver tissue injury and related symptoms are caused by hepatitis due to viral infection of the liver, such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus. In one embodiment, the liver tissue injury and related symptoms are drug-induced liver injury and related symptoms. In one embodiment, the liver tissue injury and related symptoms are caused by intrahepatic blood stasis (hepatic congestion). In one implementation, the liver tissue injury and related conditions include diabetic liver injury and related conditions, toxic liver injury and related conditions, drug-induced liver injury or related conditions, radiation-induced liver injury and related conditions, viral infection-related liver injury and related conditions, and congestive liver injury and related conditions. In another implementation, the liver tissue injury and related conditions include abnormal liver function, abnormal liver enzymes, discomfort and tenderness in the liver area, hepatomegaly, splenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis, and liver cancer caused by liver tissue injury.

[0026] In one embodiment, the plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequences 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, the plasminogen is a protein containing a plasminogen active fragment and still retaining plasminogen activity. In one embodiment, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, micro-plasminogen, δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen or plasmin is administered systemically or locally, including topically, intravenously, intramuscularly, subcutaneously, by inhalation, intraspinally, locally, intra-articularly, or rectally. In one embodiment, the related symptoms of diabetic liver injury or toxic liver injury include: abnormal liver enzymes, discomfort and tenderness in the liver area, hepatomegaly, splenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis, and liver cancer. In one embodiment, the diabetic liver injury and its related symptoms are caused by macrovascular, microvascular, and microvascular complications resulting from diabetes. In one embodiment, the plasminogen may be administered in combination with one or more other drugs. In one embodiment, the other drugs include: hepatoprotective drugs, antidiabetic drugs, antithrombotic drugs, anticoagulants, lipid-lowering drugs, anticardiovascular drugs, and anti-infective drugs.

[0027] In one implementation, the subject is a mammal, preferably a human.

[0028] In one implementation, the liver damage caused by diabetes is due to macrovascular, microvascular, and microvascular lesions caused by diabetes.

[0029] In one implementation, the subject has a deficiency of plasminogen or plasminogen. Specifically, the deficiency is congenital, secondary, and / or localized.

[0030] In one embodiment, plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, plasminogen is a protein with 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, or 1 amino acid additions, deletions, and / or substitutions based on sequence 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, plasminogen is a protein containing a plasminogen active fragment and still retaining plasminogen activity. In one embodiment, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen is selected from conserved substitution variants of Glu-plasminogen, Lys-plasminogen, small plasminogen, δ-plasminogen, or microplasminogen. In one embodiment, the plasminogen is a natural human plasminogen, such as a direct homologue of the plasminogen shown in Sequence 2, for example, a direct homologue of plasminogen from primates or rodents, such as a direct homologue of plasminogen from gorillas, rhesus monkeys, mice, cattle, horses, and dogs. Most preferably, the amino acid sequence of the plasminogen of the present invention is as shown in Sequence 2, 6, 8, 10, or 12.

[0031] In one embodiment, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In one embodiment, the plasminogen is administered at a dose of 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, or 10-100 mg / kg (based on body weight) or 0.0001-2000 mg / cm³. 2 0.001-800 mg / cm 2 0.01-600 mg / cm 2 0.1-400 mg / cm 2 1-200 mg / cm 2 1-100mg / cm 2 10-100 mg / cm 2The dosage (calculated per square centimeter of body surface area) is preferably applied at least once, and preferably at least daily. In the case of topical application, the above dosage may be further adjusted as needed.

[0032] In one embodiment, the plasminogen is administered systemically or locally, preferably via the following routes: surface, intravenous, intramuscular, subcutaneous, inhalation, spinal, local injection, intra-articular injection, or rectal administration. In one embodiment, the local administration is performed via a catheter containing plasminogen in the liver region.

[0033] On one hand, the present invention relates to a plasminogen or plasminogen for the prevention and / or treatment of liver tissue damage and related conditions in subjects, a pharmaceutical composition comprising said plasminogen or plasminogen, or an article or kit comprising said plasminogen or plasminogen.

[0034] In one embodiment, the liver tissue injury and related symptoms are liver injury and related symptoms caused by radiation or chemicals. In one embodiment, the liver injury and related symptoms caused by radiation or chemicals are radiotherapy and chemotherapy methods and drugs used in cancer treatment. In one embodiment, the radiation is radiation caused by an accidental event. In one embodiment, the liver tissue injury and related symptoms are toxic liver injury and related symptoms. In one embodiment, the toxic liver injury is toxic liver injury caused by "hepatotoxic substances" including alcohol. In one embodiment, the liver tissue injury and related symptoms are caused by diabetes and are one of the complications of diabetes. In one embodiment, the liver tissue injury and related symptoms are caused by hepatitis due to viral infection of the liver, such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus. In one embodiment, the liver tissue injury and related symptoms are drug-induced liver injury and related symptoms. In one embodiment, the liver tissue injury and related symptoms are caused by intrahepatic blood stasis (hepatic congestion). In one implementation, the liver tissue injury and related conditions include diabetic liver injury and related conditions, toxic liver injury and related conditions, drug-induced liver injury or related conditions, radiation-induced liver injury and related conditions, viral infection-related liver injury and related conditions, and congestive liver injury and related conditions. In another implementation, the liver tissue injury and related conditions include abnormal liver function, abnormal liver enzymes, discomfort and tenderness in the liver area, hepatomegaly, splenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis, and liver cancer caused by liver tissue injury.

[0035] In one embodiment, the plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequences 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, the plasminogen is a protein containing a plasminogen active fragment and still retaining plasminogen activity. In one embodiment, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, micro-plasminogen, δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen or plasmin is administered systemically or locally, including topically, intravenously, intramuscularly, subcutaneously, by inhalation, intraspinally, locally, intra-articularly, or rectally. In one embodiment, the related symptoms of diabetic liver injury or toxic liver injury include: abnormal liver enzymes, discomfort and tenderness in the liver area, hepatomegaly, splenomegaly, hepatitis, fatty liver, cholangitis, cirrhosis, liver necrosis, and liver cancer. In one embodiment, the diabetic liver injury and its related symptoms are caused by macrovascular, microvascular, and microvascular complications resulting from diabetes. In one embodiment, the plasminogen may be administered in combination with one or more other drugs. In one embodiment, the other drugs include: hepatoprotective drugs, antidiabetic drugs, antithrombotic drugs, anticoagulants, lipid-lowering drugs, anticardiovascular drugs, and anti-infective drugs.

[0036] In one implementation, the subject is a mammal, preferably a human.

[0037] In one implementation, the liver damage caused by diabetes is due to macrovascular, microvascular, and microvascular lesions caused by diabetes.

[0038] In one implementation, the subject has a deficiency of plasminogen or plasminogen. Specifically, the deficiency is congenital, secondary, and / or localized.

[0039] In one embodiment, plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, plasminogen is a protein with 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, or 1 amino acid additions, deletions, and / or substitutions based on sequence 2, 6, 8, 10, or 12, and still retains plasminogen activity. In one embodiment, plasminogen is a protein containing a plasminogen active fragment and still retaining plasminogen activity. In one embodiment, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen is selected from conserved substitution variants of Glu-plasminogen, Lys-plasminogen, small plasminogen, δ-plasminogen, or microplasminogen. In one embodiment, the plasminogen is a natural human plasminogen, such as a direct homologue of the plasminogen shown in Sequence 2, for example, a direct homologue of plasminogen from primates or rodents, such as a direct homologue of plasminogen from gorillas, rhesus monkeys, mice, cattle, horses, and dogs. Most preferably, the amino acid sequence of the plasminogen of the present invention is as shown in Sequence 2, 6, 8, 10, or 12.

[0040] In one embodiment, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In one embodiment, the plasminogen is administered at a dose of 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, or 10-100 mg / kg (based on body weight) or 0.0001-2000 mg / cm³. 2 0.001-800 mg / cm 2 0.01-600 mg / cm 2 0.1-400 mg / cm 2 1-200 mg / cm 2 1-100mg / cm 2 10-100 mg / cm 2The dosage (calculated per square centimeter of body surface area) is preferably applied at least once, and preferably at least daily. In the case of topical application, the above dosage may be further adjusted as needed.

[0041] In one embodiment, the plasminogen is administered systemically or locally, preferably via the following routes: surface, intravenous, intramuscular, subcutaneous, inhalation, spinal, local injection, intra-articular injection, or rectal administration. In one embodiment, the local administration is performed via a catheter containing plasminogen in the liver region.

[0042] In one embodiment, the plasminogen or plasmin is dispensed into a container. Preferably, the product or kit also contains other medications dispensed into other containers within the kit. The kit may also include instructions for use stating that the plasminogen can be used to treat liver tissue damage and related conditions, specifically, for example, diabetic liver injury and related conditions caused by diabetes, toxic liver injury and related conditions, drug-induced liver injury or related conditions, radiation-induced liver injury and related conditions, viral infection-induced liver injury and related conditions, and congestion-induced liver injury and related conditions. Furthermore, it may state that the plasminogen or plasmin can be administered before, simultaneously with, and / or after other medications or therapies.

[0043] This invention explicitly covers all combinations of technical features belonging to the embodiments of this invention, and these combined technical solutions have been explicitly disclosed in this application as if each of the above-described technical solutions had been individually and explicitly disclosed herein. Furthermore, this invention also explicitly covers all sub-combinations of the various embodiments and their elements, and these are disclosed herein as if each such sub-combination had been individually and explicitly disclosed herein.

[0044] Specifically, this application relates to the following: 1. Use of plasminogen in the preparation of medicaments for the prevention and / or treatment of liver tissue damage and related conditions in subjects.

[0045] 2. Use of item 1, wherein the liver tissue injury and related conditions are liver injury and related conditions caused by radiation or chemical substances.

[0046] 3. Use of item 2, wherein the liver damage and related symptoms caused by the radiation or chemical substance are radiotherapy and chemotherapy methods and drugs used in cancer treatment.

[0047] 4. The use of item 2 or 3, wherein the radiation is due to an accident or work environment.

[0048] 5. The use of item 1, wherein the liver tissue injury and related conditions are toxic liver injury and related conditions.

[0049] 6. Use of item 5, wherein the toxic liver injury is alcohol-induced toxic liver injury.

[0050] 7. The use of item 1, wherein the liver tissue damage and related conditions are caused by hepatitis resulting from viral infection of the liver.

[0051] 8. The use of item 1, wherein the liver tissue injury and related conditions are drug-induced liver injury and related conditions.

[0052] 9. The use of item 1, wherein the liver tissue damage and related conditions are caused by intrahepatic blood stasis.

[0053] 10. Use of item 1, wherein the liver tissue injury and related conditions are diabetic liver injury and related conditions.

[0054] 11. According to the use of any one of items 1-10, the diabetic liver injury and related conditions include abnormal liver function.

[0055] 12. Use of any one of items 1-11, wherein the liver tissue injury and related conditions include abnormal liver enzymes.

[0056] 13. According to the use of any one of items 1-12, wherein the plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and still has plasminogen activity.

[0057] 14. According to the use of any one of items 1-12, the plasminogen is a protein containing a plasminogen active fragment and still having plasminogen activity.

[0058] 15. According to the use of any one of items 1-12, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, micro-plasminogen, δ(delta)-plasminogen or any combination thereof.

[0059] 16. According to the use of any one of items 1-12, the plasminogen is selected from the conserved substitution variants of Glu-plasminogen, Lys-plasminogen, small plasminogen, δ-plasminogen or microplasminogen.

[0060] 17. The use of any one of items 1-16, wherein the plasminogen is a human plasminogen.

[0061] 18. According to any of the uses in items 1-17, the plasminogen may be administered in combination with one or more other drugs.

[0062] 19. According to the use of any one of items 1-18, the other drugs are selected from: hepatoprotective drugs, antidiabetic drugs, antithrombotic drugs, anticoagulant drugs, lipid-lowering drugs, anticardiovascular and cerebrovascular disease drugs, and anti-infective drugs.

[0063] 20. Use of any one of items 1-19, wherein the subject is a human being.

[0064] 21. Use of items 1-20, wherein the subject is deficient in or lacks plasminogen. Invention Details Diabetic liver injury refers to the histological and functional changes in the liver caused by diabetes. It is mainly caused by macrovascular, microvascular, and microvascular complications resulting from diabetes. Known liver damage caused by diabetes includes: abnormal liver enzymes, which can lead to carbon dioxide accumulation, acidosis, reduced oxygen supply, and increased oxygen consumption in hepatocytes, increasing hepatic transaminase activity, and disrupting bilirubin metabolism; in severe cases, it can cause hepatocyte necrosis; fatty liver, with diabetes ranking third among all causes of fatty liver, accounting for 21%–78% of diabetic patients; hepatitis, cirrhosis, and liver cancer. The incidence of viral hepatitis in diabetic patients is approximately 2–4 times that of normal individuals, and the incidence of primary liver cancer is approximately 4 times that of normal individuals.

[0065] "Chemical liver injury" or "toxic liver injury" refers to liver damage caused by chemically hepatotoxic substances. These chemicals include alcohol, toxic chemicals in the environment, and certain medications. Both nature and human industrial production processes contain substances toxic to the liver, known as "hepatotoxic agents." These toxins are generally contagious, have short incubation periods, and the pathological process is directly related to the amount of the chemical substance, potentially causing varying degrees of hepatocellular necrosis, fatty degeneration, and cirrhosis.

[0066] "Hepatotoxic substances" refers to the general term for substances that are toxic to the liver. Alcohol is the most common "hepatotoxic substance" 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 from the hepatic artery and hepatic vein. Chemical substances can enter the liver through the portal vein or systemic circulation of the gastrointestinal tract for transformation, so the liver is easily damaged by toxic substances in chemicals. There are some substances that are toxic to the liver in nature and human industrial production processes, which are called "hepatotoxic substances". When they enter the liver, they can cause different degrees of hepatocyte necrosis, fatty degeneration, and cirrhosis. Pathological manifestations include (1) fatty degeneration. Carbon tetrachloride, yellow phosphorus, etc. can interfere with the synthesis and transport of lipoproteins, forming fatty liver. (2) lipid peroxidation reaction, which is a special manifestation of toxic liver damage. For example, carbon tetrachloride produces a highly oxidizing intermediate product in the body, which leads to lipid peroxidation on biological membranes, destroys the phospholipids of the membrane, and changes the structure and function of cells. (3) Cholestasis reaction is mainly related to damage to the hepatocyte membrane and microvilli, which leads to bile acid excretion disorder.

[0067] Drug-induced liver injury (DILI) refers to liver damage caused by the drug itself or its metabolites, or by hypersensitivity or decreased tolerance to the drug due to specific individual conditions. It is also known as drug-induced liver disease and can clinically manifest as various acute and chronic liver conditions. Mild cases may resolve spontaneously after discontinuation of the drug, while severe cases can be life-threatening and require aggressive treatment and emergency care. DILI can occur in healthy individuals with no prior history of liver disease or in patients with pre-existing serious conditions; it can occur with overdose or under normal dosage.

[0068] "Radiation-induced liver injury" refers to a type of radiation damage caused by high-energy ionizing radiation, including alpha, beta particles, gamma rays, x-rays, and neutron rays. Both instantaneous exposure to high doses of radiation and prolonged exposure to low doses of radiation can cause tissue damage. The energy of radiation damages chromosomes and enzymes in cells, disrupting normal cellular function.

[0069] "Viral-induced liver injury" is a general term for liver damage caused by viral infections. Common viral infections include those caused by hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus.

[0070] "Congestive liver injury" refers to liver tissue damage caused by blood pooling in the liver. Any disease that obstructs the return of blood from the inferior vena cava to the heart can lead to liver congestion, such as rheumatic heart valve disease, chronic constrictive pericarditis, hypertensive heart disease, ischemic heart disease, pulmonary heart disease, and congenital heart disease.

[0071] Plasminogen is the progenitor form of plasmin. Based on the sequence in Swiss prot, the amino acid sequence of the natural human plasminogen containing the signal peptide (Sequence 4) consists of 810 amino acids, with a molecular weight of approximately 92 kDa. It is a glycoprotein mainly synthesized in the liver and capable of circulating in the blood. The cDNA sequence encoding this amino acid sequence is shown in Sequence 3. The full-length plasminogen contains seven domains: a C-terminal serine protease domain, an N-terminal Pan-Apple (PAp) domain, and five Kringle domains (Kringle 1-5). Referring to the sequence in the Swiss prot, its signal peptide includes residues Met1-Gly19, PAp includes residues Glu20-Val98, Kringle1 includes residues Cys103-Cys181, Kringle2 includes residues Glu184-Cys262, Kringle3 includes residues Cys275-Cys352, Kringle4 includes residues Cys377-Cys454, and Kringle5 includes residues Cys481-Cys560. According to NCBI data, the serine protease domain includes residues Val581-Arg804.

[0072] Glu-plasminogen is a natural, full-length plasminogen composed of 791 amino acids (excluding the 19-amino acid signal peptide). The cDNA sequence encoding this sequence is shown in Sequence 1, and its amino acid sequence is shown in Sequence 2. In vivo, Lys-plasminogen, formed by the hydrolysis of amino acids 76-77 of Glu-plasminogen, also exists, as shown in Sequence 6, and its cDNA sequence encoding this amino acid sequence is shown in Sequence 5. Δ-plasminogen is a full-length plasminogen fragment lacking the Kringle2-Kringle5 structure, containing only Kringle1 and the serine protease domain. [8,9] The amino acid sequence of δ-plasminogen has been reported in the literature (Sequence 8). [9] The cDNA sequence encoding this amino acid sequence is shown in Sequence 7. Mini-plasminogen consists of Kringle5 and a serine protease domain, and some literature reports that it includes residues Val443-Asn791 (starting with Glu residues in the Glu-plasminogen sequence that do not contain a signal peptide as the starting amino acids).

[10] Its amino acid sequence is shown in Sequence 10, and the cDNA sequence encoding this amino acid sequence is shown in Sequence 9. Micro-plasminogen contains only a serine protease domain, and its amino acid sequence has been reported to include residues Ala543-Asn791 (starting with Glu residues in the Glu-plasminogen sequence, which does not contain a signal peptide).

[11] Patent document CN102154253A also reports that its sequence includes residues Lys531-Asn791 (starting with Glu residues in the Glu-plasminogen sequence that do not contain a signal peptide as the starting amino acid). The sequence of this patent is referenced from patent document CN102154253A, and its amino acid sequence is shown in sequence 12. The cDNA sequence encoding this amino acid sequence is shown in sequence 11.

[0073] In this invention, "fibrinolytic enzyme" and "fibrinolytic enzyme" are used interchangeably and have the same meaning; "plasminogen" and "fibrinolytic proenzyme" are used interchangeably and have the same meaning.

[0074] Those skilled in the art will understand that all technical solutions of the present invention for plasminogen are applicable to plasmin. Therefore, the technical solutions described in the present invention cover plasminogen and plasmin.

[0075] During circulation, plasminogen activator (PA) exists in a closed, inactive conformation. However, upon binding to a thrombus or cell surface, it transforms into an open, active plasminogen activator. This active plasminogen further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. The PAp domain of plasminogen contains crucial determinants maintaining its inactive, closed conformation, while the KR domain binds to lysine residues present on receptors and substrates. Several enzymes are known to act as plasminogen activators, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hagmann factor).

[0076] "Plasminogen active fragment" refers to an active fragment in plasminogen protein that can bind to a target sequence in a substrate and perform proteolytic function. This invention relates to plasminogen solutions that utilize plasminogen active fragments instead of plasminogen itself. The plasminogen active fragment described in this invention is a protein containing the serine protease domain of plasminogen. Preferably, the plasminogen active fragment described in this invention comprises sequence 14 and a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to sequence 14. Therefore, the plasminogen described in this invention includes a protein containing this plasminogen active fragment and still retaining the plasminogen activity.

[0077] Currently, methods for measuring plasminogen and its activity in blood 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, detection of plasma tissue plasminogen activator inhibitor antigen, and detection of plasma plasmin-anti-plasmin complex (PAP). The most commonly used method is the chromogenic substrate method: streptokinase (SK) and a chromogenic substrate are added to the test plasma. Under the action of SK, PLG in the test plasma is converted to PLM, which then acts on the chromogenic substrate. The absorbance is then measured using a spectrophotometer, and the increase in absorbance is directly proportional to the plasminogen activity. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, and radioimmunodiffusion methods can also be used to measure plasminogen activity in blood.

[0078] "Orthologs" refer to homologs between different species, including both protein and DNA homologs, also known as orthogonal or vertical homologs. Specifically, they refer to proteins or genes that evolved from a common ancestral gene in different species. The plasminogen of this invention includes natural human plasminogen, as well as plasminogen orthologs or ortholines derived from different species that possess plasminogen activity.

[0079] "Conservative substitution variants" refer to proteins or enzymes in which one of a given amino acid residues is altered without changing the overall conformation and function. This includes, but is not limited to, replacing an amino acid in the parent protein's amino acid sequence with an amino acid of similar properties (e.g., acidity, basicity, hydrophobicity, etc.). Amino acids with similar properties are well-known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine, or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may vary. For example, similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" also include peptides or enzymes that, as determined by the BLAST or FASTA algorithm, have more than 60% amino acid identity, preferably more than 75%, ideally more than 85%, and even better if more than 90%, and have the same or substantially similar properties or functions compared to the native or parent protein or enzyme.

[0080] "Isolated" plasminogen refers to plasminogen protein isolated and / or recovered from its natural environment. In some embodiments, the plasminogen is purified to (1) a purity greater than 90%, greater than 95%, or greater than 98% (by weight), as determined by the Lowry method, for example, greater than 99% (by weight); (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotating cup sequencer; or (3) to homogeneity determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated plasminogen also includes plasminogen prepared from recombinant cells using bioengineering techniques and isolated through at least one purification step.

[0081] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and so on.

[0082] The "percentage of amino acid sequence identity (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence, after introducing gaps where necessary to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparisons for determining percentage amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this invention, the percentage of amino acid sequence identity is generated using the sequence comparison computer program ALIGN-2.

[0083] When using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or can be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity relative to, with, or with respect to a given amino acid sequence B) is calculated as follows: Fraction X / Y multiplied by 100 Where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the alignments of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the lengths of amino acid sequences A and B are not equal, 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. Unless otherwise explicitly stated, all % amino acid sequence identity values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0084] As used herein, the terms “treatment” and “treatment” refer to achieving the desired pharmacological and / or physiological effect. The effect may be complete or partial prevention of the disease or its symptoms, and / or partial or complete cure of the disease and / or its symptoms, and includes: (a) preventing the disease from occurring in a subject who may have a causal factor for the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., blocking its formation; and (c) alleviating the disease and / or its symptoms, i.e., causing the disease and / or its symptoms to subside.

[0085] The terms “individual,” “subject,” and “patient” are used interchangeably in this document to refer to mammals, including but not limited to rats (rat, mouse), non-human primates, humans, dogs, cats, and ungulates (e.g., horses, cattle, sheep, pigs, goats).

[0086] "Therapeutic effective dose" or "effective dose" refers to the amount of plasminogen sufficient to achieve the prevention and / or treatment of a disease when administered to a mammal or other subject. Therapeutic effective doses vary depending on the plasminogen used, the severity of the disease and / or symptoms of the subject to be treated, and factors such as age and weight.

[0087] Preparation of plasminogen according to the present invention Plasminogen can be isolated and purified from nature for further therapeutic uses, or it can be synthesized using standard chemical peptide synthesis techniques. When synthesizing peptides chemically, the process can be carried out in a liquid or solid phase. Solid-phase peptide synthesis (SPPS), in which the C-terminal amino acid of a sequence is attached to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence, is a suitable method for the chemical synthesis of plasminogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. The technique for solid-phase synthesis is described in Barany and Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8. In short, small, insoluble porous beads are treated with functional units on which peptide chains are constructed. After repeated cycles of coupling / deprotection, the attached solid-phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected, exposing a new N-terminal amine that can attach to other amino acids. The peptide remains immobilized on the solid phase and is subsequently cleaved.

[0088] The plasminogen of this invention can be produced using standard recombinant methods. For example, the nucleic acid encoding plasminogen is inserted into an expression vector, making it operatively linked to a regulatory sequence in the expression vector. The expression regulatory sequence includes, but is not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression regulation can be a eukaryotic promoter system in the vector, which is capable of transforming or transfecting eukaryotic host cells (e.g., 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 for the collection and purification of plasminogen.

[0089] Suitable expression vectors typically replicate in the host organism either as an episome or as an integrated portion of the host chromosomal DNA. Often, expression vectors contain selection markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to aid in the detection of those cells transformed with the desired exogenous DNA sequence.

[0090] Escherichia coli ( Escherichia coli This is an example of a prokaryotic host cell that can be used to clone the subject antibody encoding a polynucleotide. Other suitable microbial hosts include bacilli, such as Bacillus subtilis (…). Bacillus subtilis ) and other Enterobacteriaceae ( Enterobacteriaceae ), such as Salmonella ( Salmonella ), Serratia ( Serratia ), and various Pseudomonas species ( Pseudomonas ) species. Expression vectors can also be generated in these prokaryotic hosts, which typically contain expression control sequences (e.g., origin of replication) compatible with the host cell. Additionally, many well-known promoters exist, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or promoter systems derived from bacteriophage λ. Promoters typically control expression, optionally in the case of manipulating gene sequences, and possess ribosome binding site sequences, etc., to initiate and complete transcription and translation.

[0091] Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., *Saccharomyces cerevisiae*) S. cerevisiae )) and Pichia pastoris ( Pichia A suitable yeast host cell is an example where a suitable vector has expression control sequences (e.g., promoters), origin of replication, termination sequences, etc., as needed. Typical promoters include 3-phosphoglycerate kinase and other sugar-degrading enzymes. Inducible yeast is initiated by promoters that specifically include those from alcohol dehydrogenases, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.

[0092] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell cultures) can also be used to express and generate the plasminogen of this invention (e.g., a polynucleotide encoding the subject anti-Tau antibody). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). 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 used for these cells may contain expression control sequences, such as origin of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as essential processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from leukoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0093] Once synthesized (chemically or recombinantly), the plasminogen described herein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, high-performance liquid chromatography (HPLC), gel electrophoresis, etc. The plasminogen is substantially pure, for example 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, and is free from contaminants such as cell debris, macromolecules other than the subject antibody, etc.

[0094] Pharmaceutical preparations Therapeutic formulations can be prepared by mixing plasminogen of the desired purity with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) to form lyophilized or aqueous solutions. Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexanediamine chloride; benzyl alkyl ammonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); and low molecular weight peptides (less than approximately (10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as TWEENTM, PLURONICS™, or polyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibody formulations are described in WO 97 / 04801, which is incorporated herein by reference.

[0095] The formulations of the present invention may also contain one or more active compounds required for the specific condition to be treated, preferably those with complementary activities and no side effects between them. Examples include antihypertensive drugs, antiarrhythmic drugs, and drugs for treating diabetes.

[0096] The plasminogen of the present invention can be encapsulated in microcapsules prepared by means of techniques such as coagulation or interfacial polymerization, for example, in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in coarse drop emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0097] The plasminogen of the present invention intended for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter membrane before or after freeze-drying and reconstitution.

[0098] The plasminogen of the present invention can be used to prepare sustained-release formulations. Suitable examples of sustained-release formulations include solid hydrophobic polymer semi-permeable matrices having a defined shape and containing glycoproteins, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-isobutylene ester) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (US Patent 3773919, EP 58, 481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer et al., ibid.), or degradable lactic acid-glycolic acid copolymers such as Lupron. Depot™ (injectable microspheres composed of lacto-glycolic acid copolymer and leuprolide acetate), and poly(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lacto-glycolic acid can continuously release molecules for more than 100 days, while some hydrogels release proteins for a shorter period. Rational strategies for protein stabilization can be designed based on the relevant mechanisms. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiodisulfide bond exchange, stabilization can be achieved by modifying thiol residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

[0099] Dosage and administration The pharmaceutical compositions of the present invention can be administered via various methods, such as intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, intranasal, topical, or intradermal application, or spinal or brain delivery. Aerosol formulations, such as nasal spray formulations, comprise purified aqueous or other solutions of the active agent, as well as preservatives and isotonic agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

[0100] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of 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 buffer media. Parenteral media contain sodium chloride solutions, Ringer's dextran, dextran and sodium chloride, or fixed oils. Intravenous media contain fluids and nutritional supplements, electrolyte supplements, etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, etc.

[0101] Medical professionals determine dosage regimens based on various clinical factors. As is well known in the medical field, the dosage for any patient depends on a number of factors, including the patient's body size, body surface area, age, the specific compound to be administered, sex, frequency and route of administration, overall health, and other medications administered concurrently. The dosage range of the pharmaceutical compositions comprising plasminogen of this invention can be, for example, from about 0.0001 to 2000 mg / kg daily, 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.) based on the subject's body weight. For example, the dosage 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. Doses higher or lower than this illustrative range are also included, particularly taking into account the factors described above. Intermediate doses within the above range are also included within the scope of this invention. Subjects may administer such doses daily, every other day, weekly, or according to any other schedule determined through empirical analysis. An illustrative dosage schedule includes 1-10 mg / kg over several consecutive days. During the administration of the drug of this invention, real-time and periodic assessments of the therapeutic efficacy and safety in diabetic liver disease and related conditions are necessary.

[0102] Therapeutic efficacy and safety One embodiment of the present invention relates to the assessment of therapeutic efficacy and safety after treating a subject with plasminogen agonist. The methods for assessing the therapeutic efficacy include, but are not limited to: 1) examining the subject's liver function, such as checking whether the levels of enzymes in the patient's body, such as serum aspartate aminotransferase (ALT), alanine aminotransferase (AST), total bilirubin, direct bilirubin, indirect bilirubin, albumin, globulin, cholinesterase, alkaline phosphatase, and transpeptidase, are within the normal range. After treatment with plasminogen agonist of the present invention, it is expected that the above-mentioned liver function indicators will return to normal or improve, for example, ALT: 0–40 μ / L, AST: 0–40 μ / L, GGT: less than 40 units, total bilirubin: 3.4–20.5 μmol / L; 2) assessing the patient's liver function. The subjects underwent prothrombin time (PT) and prothrombin activity (PTA) tests: PT is an important indicator reflecting the liver's ability to synthesize coagulation factors, and PTA is a commonly used method to express PT values. It is of great value in judging the progression and prognosis of liver disease. The progressive decrease of PTA to below 40% is one of the important diagnostic criteria for liver failure, and <20% indicates poor liver function. After treating the subjects with plasminogen and its variants in this invention, the decrease in PTA in the patients' bodies is expected to be significantly improved; 3) Imaging examinations: including abdominal ultrasound of the liver, gallbladder and spleen, CT or MRI, to understand the degree of liver damage recovery; 4) Tumor marker tests, such as alpha-fetoprotein (AFP), CA199, AFU, etc.; 5) Liver tissue biopsy to determine the degree of recovery of fibrosis and other damage. Furthermore, the present invention also relates to assessing the safety of the treatment regimen during and after treatment of subjects with plasminogen and its variants, including but not limited to statistical analysis of the subject's serum half-life, therapeutic half-life, median toxic dose (TD50), and median lethal dose (LD50), or observation of various adverse events such as sensitization reactions that occur during or after treatment.

[0103] Products or medicine boxes One embodiment of the present invention relates to an article or flask containing plasminogen of the present invention for the treatment of liver damage and related conditions caused by diabetes. The article preferably includes a container, label, or packaging insert. Suitable containers include bottles, vials, syringes, etc. The container can be made of various materials such as glass or plastic. The container contains a composition that is effective in treating the disease or condition of the present invention and has a sterile inlet (e.g., the container may be an intravenous solution pack or vial containing a stopper that can be penetrated by a hypodermic needle). 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 liver damage and related conditions caused by diabetes as described in the present invention. The article may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. In addition, the product includes a packaging insert with instructions for use, including, for example, instructions for the user of the composition to administer the plasminogen composition and other medications for treating accompanying diseases to the patient. Attached Figure Description

[0104] Figure 1 This shows the change in body weight in 24-25 week old diabetic mice after administration of plasminogen activator.

[0105] Figure 2 This shows the results of HE staining of the liver of 24-25 week old diabetic mice after 15 consecutive days of administration of plasminogen lysate.

[0106] Figure 3 This shows the results of immunostaining microscopic examination of liver fibrin in diabetic mice aged 24-25 weeks after administration of plasminogen for 15 consecutive days.

[0107] Figure 4 This shows the changes in body weight in 24-25 week old diabetic mice after 31 consecutive days of administration of plasminogen activator.

[0108] Figure 5 This shows the results of HE staining of the liver of 24-25 week old diabetic mice after 31 consecutive days of administration of plasminogen lysate.

[0109] Figure 6 This shows the results of liver fibrin immunostaining in 24-25 week old diabetic mice after 31 consecutive days of administration of plasminogen 24-25 weeks of treatment.

[0110] Figure 7 This shows the results of liver F4 / 80 immunostaining in 24-25 week old diabetic mice after 31 consecutive days of administration of plasminogen lysate.

[0111] Figure 8 This shows the serum alanine aminotransferase (ALT) test results of diabetic mice at 24-25 weeks of age after being given PBS or plasminogen 20 for 31 days.

[0112] Figure 9 This shows the results of HE staining of the livers of mice with acute liver injury induced by carbon tetrachloride on days 0, 2, and 7 after administration of plasminogen 24.

[0113] Figure 10 Display plg - / - Results of HE staining of liver tissue in mice with carbon tetrachloride-induced acute liver injury after administration of plasminogen 18, 24, and 48 hours.

[0114] Figure 11 Display plg - / - Immunostaining results of liver fibrin in mice with carbon tetrachloride-induced acute liver injury after administration of plasminogen 18, 24, and 48 hours.

[0115] Figure 12 This shows the results of F4 / 80 immunostaining of mouse livers 10 days after exposure to 5.0 Gy X-ray radiation.

[0116] Figure 13 This shows the results of liver fibrin immunostaining in a mouse model of plasminogen retinolytic damage 7 days after treatment with 10 mg / Kg cisplatin.

[0117] Figure 14 Display plg - / - Results of HE staining of liver tissue in mice with carbon tetrachloride-induced acute liver injury after 18, 24, 48 hours and 7 days of plasminogen administration.

[0118] Example Example 1: Effect of plasminogen on body weight in diabetic mice with late-stage nerve injury Ten 24-25 week old male db / db mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. The first day of the experiment was recorded as day 0, and the second day of administration of plasminogen or PBS was recorded as day 1, with continuous administration for 15 days. Mice in the plasminogen-treated group received 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. Body weight was measured on days 0, 4, 7, 11, and 16 after plasminogen administration. Results showed that the body weight of the plasminogen-treated group and the PBS-treated control group on days 0, 4, 7, 11, and 16... Figure 1 There was no significant difference, indicating that plasminogen has little effect on animal body weight.

[0119] Example 2: Protective effect of plasminogen against liver tissue damage in the late stage of diabetic liver injury Ten 24-25 week old male db / db mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. The day the experiment began was designated as Day 0, and the groups were weighed and grouped. Plasminogen or PBS administration began on the second day of the experiment and was designated as Day 1, with administration continuing for 15 days. Mice in the plasminogen group received 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. On Day 16, mice were sacrificed, and liver tissue was collected and fixed in 10% neutral formalin for 24-48 hours. The fixed liver tissue was then dehydrated using an alcohol gradient, cleared with xylene, and embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, blued with ammonia, and mounted after an alcohol gradient dehydration.

[0120] HE staining results showed that in the PBS-treated control group, hepatocytes exhibited severe steatosis, lipid deposition, and the nuclei were pushed to the periphery. Mild hydropic degeneration and disordered hepatic cords were also observed. In the plasminogen-treated group, compared to the PBS-treated control group, hepatocyte steatosis was reduced, showing mild steatosis with moderate hydropic degeneration predominating. This indicates that plasminogen can promote the repair of diabetic liver damage.

[0121] Example 3: Plasminogen reduces fibrin levels in liver tissue of diabetic mice Ten 24-25 week old male db / db mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. The day the experiment began was designated as day 0, and the groups were weighed and grouped. The second day of the experiment, when plasminogen or PBS was administered, was designated as day 1, and administration continued for 15 consecutive days. Mice in the plasminogen-treated group received 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. On day 16, mice were sacrificed, and liver tissue was collected and fixed in 10% neutral formalin for 24-48 hours. The fixed liver tissue was dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and washed once with water. The sections were then incubated with 3% hydrogen peroxide for 15 minutes and washed twice with water for 5 minutes each time. Block with 10% normal sheep serum (Vector laboratories, Inc., USA) for 1 hour; after 1 hour, discard the sheep serum and circle the tissue with a PAP pen. Incubate overnight at 4°C with rabbit anti-mouse fibrin(ogen) antibody (Abcam), wash twice with TBS for 5 minutes each time. Incubate with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody at room temperature for 1 hour, wash twice with TBS for 5 minutes each time. Develop color using the DAB kit (Vector laboratories, Inc., USA), wash 3 times with water, counterstain with hematoxylin for 30 seconds, rinse with running water for 5 minutes, and then wash once with TBS. Dehydrate and clear in a gradient manner, mount, and observe the sections under a microscope at 200x magnification.

[0122] Fibrinogen is a precursor to fibrin. In the presence of tissue damage, as a stress response to injury, fibrinogen is hydrolyzed into fibrin. [12-14] Therefore, fibrinogen levels can be used as an indicator of the degree of damage.

[0123] Studies have found that giving plasminogen lysate (Plasminogen lysate) Figure 3 B) compared to the control group given PBS ( Figure 3 A) Compared to the group given plasminogen, the level of fibrin in the liver tissue of mice was reduced, indicating that plasminogen has the function of inhibiting fibrin deposition and the damage was repaired to a certain extent.

[0124] Example 4: Effect of plasminogen on body weight in diabetic mice Twenty 24-25 week old male db / db mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with 10 mice in each group. The first day of the experiment was recorded as day 0, and mice were weighed and grouped. The second day of the experiment, when plasminogen or PBS was administered, was recorded as day 1, and administration continued for 31 consecutive days. Mice in the plasminogen-treated group received 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. Mice were weighed on days 0, 4, 7, 11, 16, 21, 26, and 31.

[0125] The results showed no significant difference in body weight between the plasminogen agonist group and the PBS-treated control group on days 0, 4, 7, 11, 16, 21, 26, and 31. Figure 4 This indicates that plasminogen has little effect on animal weight.

[0126] Example 5: Protective effect of plasminogen against liver tissue damage in the late stage of diabetic liver injury. Ten 24-25 week old male db / db mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. The day the experiment began was recorded as day 0, and the groups were weighed and grouped. Plasminogen or PBS administration began on day 2 and was recorded as day 1, with administration continuing for 31 days. Mice in the plasminogen group received 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. On day 32, mice were sacrificed, and liver tissue was collected and fixed in 10% neutral formalin for 24-48 hours. The fixed liver tissue was then dehydrated using an alcohol gradient, cleared with xylene, and embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, blued with ammonia, and mounted after alcohol gradient dehydration.

[0127] HE staining results showed that the control group given the solvent PBS ( Figure 5A) The liver shows severe fatty degeneration, with lipid deposition that merges into large fat vacuoles. Cell nuclei are squeezed to the periphery (↗), hepatic cords are disordered, hepatic sinusoids are narrowed, and there are varying numbers of inflammatory foci at the hepatic cords (↑); administration of plasminogen agonists ( Figure 5 B) Mild fatty degeneration of the liver, with damage mainly consisting of mild hydropic degeneration and cytoplasmic lysis (◥), primarily distributed in the area between the portals and the central vein. The portals and the area around the central vein are less affected, and mild inflammatory cell infiltration is also visible in the hepatic cords. This indicates that the liver damage was significantly repaired after administration of plasminogen lysate.

[0128] Example 6: Plasminogen reduces fibrin levels in liver tissue of diabetic mice Ten 24-25 week old male db / db mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. The day the experiment began was recorded as day 0 for weighing and grouping. The second day of the experiment, when plasminogen or PBS was administered, was recorded as day 1, and administration continued for 31 consecutive days. Mice in the plasminogen-treated group received 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. On day 32, mice were sacrificed, and liver tissue was collected and fixed in 10% neutral formalin for 24 hours. The fixed liver tissue was dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and washed once with water. The sections were then incubated with 3% hydrogen peroxide for 15 minutes and washed twice with water for 5 minutes each time. Block with 10% normal sheep serum (Vector laboratories, Inc., USA) for 1 hour; after 1 hour, discard the sheep serum and circle the tissue with a PAP pen. Incubate overnight at 4°C with rabbit anti-mouse fibrin(ogen) antibody (Abcam), wash twice with TBS for 5 minutes each time. Incubate with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody at room temperature for 1 hour, wash twice with TBS for 5 minutes each time. Develop color according to the DAB kit (Vector laboratories, Inc., USA), wash 3 times with water, counterstain with hematoxylin for 30 seconds, rinse with running water for 5 minutes. Dehydrate and clear in a gradient manner, mount, and observe the sections under a microscope at 200x magnification.

[0129] Fibrinogen is a precursor to fibrin. In the presence of tissue damage, as a stress response to injury, fibrinogen is hydrolyzed into fibrin. [12-14] Therefore, fibrinogen levels can be used as an indicator of the degree of damage.

[0130] Studies have found that giving plasminogen lysate (Plasminogen lysate) Figure 6 B) compared to the control group given PBS ( Figure 6Compared to the group given plasminogen, the level of fibrin in the liver tissue of mice was significantly reduced, indicating that plasminogen injection can significantly reduce fibrin deposition in diabetic mice, reflecting that plasminogen has a significant repair function on the damage to the body of diabetic mice.

[0131] Example 7: Plasminogen reduces inflammation in liver tissue of diabetic mice Ten 24-25 week old male db / db mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. The day the experiment began was designated as Day 0, and the groups were weighed and grouped. The second day of treatment, either plasminogen or PBS, was designated as Day 1, and administration continued for 31 days. Mice in the plasminogen-treated group received 2 mg / 0.2 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. After 31 days of plasminogen treatment, the mice were sacrificed, and liver tissue was collected and fixed in 10% neutral formalin for 24 hours. The fixed liver tissue was dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and washed once. They were incubated with 3% hydrogen peroxide for 15 minutes, washed twice with water for 5 minutes each time, and blocked with 10% normal sheep serum for 1 hour. After the blocking time, the serum was discarded, and the tissue was circled using a PAP pen. Rabbit polyclonal antibody against F4 / 80 (Abcam) was incubated overnight at 4°C, followed by two washes with TBS for 5 minutes each. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour, followed by two washes with TBS. Color development was performed using a DAB kit (Vector laboratories, Inc., USA). After three washes with water, hematoxylin was counterstained for 30 seconds, rinsed with running water for 5 minutes, graded for clearing, and mounted. Sections were observed under a microscope at 400x magnification.

[0132] The F4 / 80 macrophage marker can indicate the degree and stage of the inflammatory response. Results showed that the plasminogen group (…) Figure 7 B) compared to the control group given PBS ( Figure 7 Compared to A), the F4 / 80 positivity level in mice given plasminogen was significantly lower, indicating that the degree of liver tissue inflammation was reduced after plasminogen administration. Figure 7 C represents the quantitative analysis results of F4 / 80 immunohistochemical positive expression. The expression level of F4 / 80 in the plasminogen group was significantly reduced, and the difference was statistically significant, indicating that plasminogen injection can significantly promote the repair of liver inflammation in diabetic mice.

[0133] Example 8: Plasminogen promotes liver repair in diabetic mice Nine 25-28 week old male db / db mice were randomly divided into two groups: a control group (n=3) given PBS and a plasminogen agonist group (n=6). The day the experiment began was designated as Day 0, and mice were weighed and grouped. The second day of administration of either plasminogen agonist or PBS was designated as Day 1, and administration continued for 31 consecutive days. Mice in the plasminogen agonist group received a tail vein injection of 2 mg / 0.2 mL / mouse / day, while the control group received the same volume of PBS. After 31 days of plasminogen agonist administration, whole blood was collected by enucleation. After serum separation, the mice were centrifuged at 4°C and 3500 rpm for 10 minutes, and the supernatant was used for analysis. This experiment used a Reitman-Frankel assay kit (Nanjing Jiancheng Bioengineering Institute, catalog number C009-2) to detect serum alanine aminotransferase (ALT) levels.

[0134] Alanine aminotransferase (ALT) is an important indicator of liver health. [15,16] The normal reference range for alanine aminotransferase (ALT) is 9–50 U / L. The results showed that the serum ALT level in the PBS-treated control group was significantly higher than normal physiological levels, while the plasminogen-treated group had recovered to normal levels. Furthermore, the plasminogen-treated group had significantly lower ALT levels than the PBS-treated control group, and the difference was statistically significant. Figure 8 This indicates that in a mouse model of advanced diabetes, injection of plasminogen agonist can effectively repair liver damage.

[0135] Example 9: Protective effect of plasminogen on the liver in acute liver poisoning 7-8 week old plg + / + Eighteen mice, of any sex, were randomly divided into two groups: a control group receiving PBS (propanediol) and a plasminogen agonist group, with nine mice in each group. Both groups of mice were administered carbon tetrachloride intraperitoneally at a dose of 0.5 mL / kg body weight for two consecutive days to establish an acute liver injury model. [17,18] Carbon tetrachloride must be diluted with corn oil before use, with a volume ratio of 1:7. Day 0 is the day of model establishment. Plasminogen agonist or PBS is administered starting on day 1. Mice in the plasminogen agonist group are given 1 mg / 0.1 mL / mice / day, while the control group is given the same volume of PBS. This administration is repeated for 7 days. On days 0, 2, and 7, three mice from each group are sacrificed, and the liver condition is recorded after dissection. The liver tissue is then fixed in 10% neutral formalin for 24-48 hours. After fixation, the liver tissue is dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections are 5 μm thick, dewaxed, rehydrated, and stained with hematoxylin and eosin (HE staining). Differentiation is performed with 1% hydrochloric acid alcohol, followed by ammonia blue resurfacing, and then dehydrated and mounted using an alcohol gradient. The sections are observed under a microscope at 200x magnification.

[0136] HE staining results showed that the control group given the solvent PBS ( Figure 9AC) and plasminogen agonists (AC) and plasminogen agonists (AC) Figure 9 On day 0, the livers of DF mice showed predominantly fragmented necrosis around the central vein, with fragmented nuclei and pale cytoplasm in the necrotic areas. Moderate hydropic degeneration and cellular edema also occurred in other non-necrotic areas. On day 2, the central vein dilated, hepatocyte structure became disordered, and a small amount of inflammatory cell infiltration was observed, with no significant difference between the two groups. However, on day 7, the control group treated with PBS still showed a small amount of hepatocyte degeneration, mild cellular edema, disordered hepatic cords, narrowing of hepatic sinusoids, and mild inflammatory cell infiltration around the portal areas. In contrast, the livers of the plasminogen-treated group largely recovered, with red cytoplasmic staining, regular hepatic cords, and clear hepatic sinusoids. This indicates that plasminogen can promote the repair of liver damage.

[0137] Example 10: Protective effect of plasminogen on the liver in acute liver poisoning 7-11 week old plg - / - Eighteen male mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with nine mice in each group. Both groups of mice were administered carbon tetrachloride intraperitoneally at a single dose of 0.5 mL / kg body weight to establish an acute liver injury model. [17,18] Carbon tetrachloride must be diluted with corn oil before use, with a volume ratio of 1:7. Plasminogen or PBS (propagated PBS) should be administered within half an hour after model establishment. Mice in the plasminogen group were given 1 mg / 0.1 mL / mice / day, while the control group received the same volume of PBS, for two consecutive days. Three mice from each group were sacrificed at 18, 24, and 48 hours after administration. The liver was dissected and its condition recorded. The liver tissue was then fixed in 10% neutral formalin for 24-48 hours. After fixation, the liver tissue was dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and stained with hematoxylin and eosin (HE staining). Differentiation was performed with 1% hydrochloric acid alcohol, followed by ammonia blue resurfacing, and then dehydrated and mounted using an alcohol gradient. The sections were observed under a microscope at 200x magnification.

[0138] The results showed that the control group given the solvent PBS ( Figure 10 AC showed varying degrees of necrosis at 18h, 24h, and 48h. At 18h and 24h, patchy necrosis was predominant. By 48h, bridging necrosis, nuclear fragmentation, and pale cytoplasm had occurred, with the damage continuously worsening. It was mainly distributed around the central vein, with moderate inflammatory cell infiltration in the necrotic area (↓). Necrosis around the portal area was milder, mainly characterized by mild watery degeneration, accompanied by mild inflammatory cell infiltration and mild bile duct hyperplasia (◥). The plasminogen group (…) Figure 10Compared to the control group, DF (diplase-free liver) showed no significant necrosis at 18h, 24h, and 48h. Damage was mainly mild hydropic degeneration, distributed around the portal area, while hepatocytes around the central vein were unaffected. At 24h, the condition improved compared to 18h, with reduced hydropic degeneration and mild fatty degeneration of hepatocytes around the central vein, accompanied by pale cytoplasm and mild inflammatory cell infiltration. This indicates that plasminogen can promote plc... - / - Repair of liver injury in a mouse model of acute liver injury.

[0139] Example 11: Plasminogen Reduces Fibrin Deposition in Liver Tissue of a Mouse Model of Acute Liver Injury 7-11 week old plg - / - Eighteen male mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with nine mice in each group. Both groups of mice were treated once intraperitoneally with carbon tetrachloride (0.5 mL / kg body weight) to establish an acute liver injury model. [17,18] Carbon tetrachloride must be diluted with corn oil before use, with a volume ratio of 1:7. Plasminogen or PBS (propagated PBS) should be administered within half an hour after model establishment. Mice in the plasminogen group were given 1 mg / 0.1 mL / mice / day, while the control group was given the same volume of PBS, for two consecutive days. Three mice from each group were sacrificed at 18, 24, and 48 hours after administration, and the liver condition was recorded after dissection. The liver tissue was then fixed in 10% neutral formalin for 24-48 hours. After fixation, the liver tissue was dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and washed once. They were incubated with 3% hydrogen peroxide for 15 minutes, washed twice (5 minutes each time), and blocked with 10% normal sheep serum (Vector laboratories, Inc., USA) for 1 hour; after this time, the sheep serum was discarded, and the tissue was circled with a PAP pen. Rabbit anti-mouse fibrin(ogen) antibody (Abcam) was incubated overnight at 4°C, followed by two washes with TBS for 5 minutes each. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour, followed by two washes with TBS for 5 minutes each. Color development was performed using the DAB kit (Vector laboratories, Inc., USA), followed by three washes with water, counterstaining with hematoxylin for 30 seconds, and rinsing with running water for 5 minutes. The slides were graded for dehydration, clearing, and mounting, and observed under a microscope at 200x magnification.

[0140] Fibrinogen is a precursor to fibrin. In the presence of tissue damage, as a stress response to injury, fibrinogen is hydrolyzed into fibrin. [12-14] Therefore, fibrinogen levels can be used as an indicator of the degree of damage.

[0141] The results showed that plasminogen agonists were administered plasminogen agonists at three time points: 18, 24, and 48 hours. Figure 11 The positive staining of fibrin in DF was significantly lighter than that in the control group treated with PBS. Figure 11 The fibrin staining also tends to lighten over time. This indicates that plasminogen injection can reduce fibrin deposition and promote liver damage repair.

[0142] Example 12: Plasminogen promotes the repair of liver inflammation in mice exposed to 5.0 Gy X-rays. Ten healthy male C57 mice aged 6-8 weeks were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. After grouping, a radiation injury model was established. Mice were uniformly irradiated with 5.0 Gy of 6 MV X-rays from a linear accelerator in a single, whole-body irradiation, with an absorbed dose rate of 2.0 Gy / min and an absorbed dose of 5.0 Gy (irradiated for 2.5 minutes). Plasminogen was administered within 3 hours after model establishment. Body weight was measured and groups were formed on Day 0 of the experiment. Radiation treatment began on Day 1, followed by administration of either plasminogen or PBS. The administration period was 10 days, and animals were observed for 11 days after drug withdrawal, for a total experimental period of 21 days. The plasminogen group received 1 mg / 0.1 mL / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. On Day 21, mice were sacrificed and dissected, and their livers were fixed in 10% neutral formalin for 24-48 hours. Fixed liver tissue was dehydrated in a gradient of alcohol and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and washed once with water. Tris-EDTA repair was performed for 30 minutes, followed by cooling at room temperature for 20 minutes and gentle rinsing with water. The sections were incubated with 3% hydrogen peroxide for 15 minutes and circled with a PAP pen. Blocking with 10% normal goat serum (Vectorlaboratories, Inc., USA) for 1 hour was performed; the goat serum solution was then discarded. Rabbit anti-mouse F4 / 80 antibody (Abcam) was incubated overnight at 4°C, followed by washing twice with TBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour, followed by washing twice with TBS for 5 minutes each time. Developing was performed using a DAB kit (Vector laboratories, Inc., USA), followed by washing three times with water, counterstaining with hematoxylin for 30 seconds, and rinsing with running water for 5 minutes. The sections were dehydrated, cleared, and mounted, and observed under a microscope at 200x magnification.

[0143] Immunohistochemical results of F4 / 80 showed that after 5.0 Gy X-ray irradiation to induce modeling, the control group given PBS as the solvent ( Figure 12 The expression levels of mouse macrophage markers in group A were higher than those in the plasminogen-treated group (A). Figure 12B) indicates that after administration of plasminogen, the inflammation in the animal's liver tissue was significantly reduced.

[0144] Example 13: Plasminogen reduces fibrin deposition in liver tissue of mice with cisplatin-induced chemotherapy injury model. Ten healthy male C57 mice aged 8-9 weeks were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with five mice in each group. After grouping, a chemotherapy-induced injury model was established by a single intraperitoneal injection of cisplatin at 10 mg / kg body weight. After model establishment, the plasminogen-treated group received 1 mg / mouse / day via tail vein injection, while the PBS-treated control group received the same volume of PBS. Body weight was measured and groups were formed on Day 0 of the experiment. Intraperitoneal injection of cisplatin to establish the model began on Day 1. Plasminogen-treated or PBS-treated mice were administered within 3 hours of model establishment, with a treatment period of 7 days. Mice were sacrificed on Day 8, and liver tissue was fixed in 10% neutral formalin for 24-48 hours. The fixed liver tissue was dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and washed once with water. Citric acid was applied for 30 minutes, followed by cooling at room temperature for 10 minutes and gentle rinsing with water. Incubate with 3% hydrogen peroxide for 15 minutes, then circle the tissue with a PAP pen. Block with 10% normal sheep serum (Vectorlaboratories, Inc., USA) for 1 hour; discard the sheep serum solution after 1 hour. Incubate overnight at 4°C with rabbit anti-mouse fibrin antibody (Abcam), wash twice with TBS for 5 minutes each time. Incubate with goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody at room temperature for 1 hour, wash twice with TBS for 5 minutes each time. Develop color using a DAB kit (Vector laboratories, Inc., USA), wash three times with water, counterstain with hematoxylin for 30 seconds, and rinse with running water for 5 minutes. Dehydrate and clear in a gradient manner, then mount the slides. Observe the sections under a microscope at 200x magnification.

[0145] Fibrinogen is a precursor to fibrin. In the presence of tissue damage, as a stress response to injury, fibrinogen is hydrolyzed into fibrin. [12-14] Therefore, fibrinogen levels can be used as an indicator of the degree of damage.

[0146] The results showed that the control group given the solvent PBS ( Figure 13 A) The fibrin-positive staining in liver tissue was significantly deeper than that in the plasminogen-treated group. Figure 13 B). This indicates that plasminogen can significantly reduce the fibrin deposited in damaged liver tissue, suggesting that plasminogen can promote the repair of liver damage induced by the chemotherapy drug cisplatin.

[0147] Example 14: Protective effect of plasminogen on the liver in acute liver poisoning Six 7-11 week old plc- / - male mice were randomly divided into two groups: a PBS-treated control group and a plasminogen-treated group, with three mice in each group. Both groups of mice were administered carbon tetrachloride intraperitoneally at a single dose of 0.5 mL / kg body weight to establish an acute liver injury model. [17,18] Carbon tetrachloride must be diluted with corn oil before use, with a volume ratio of 1:7. Plasminogen or PBS (propagated PBS) should be administered within half an hour after model establishment. Mice in the plasminogen group were given 1 mg / 0.1 mL / mouse / day, while the control group received the same volume of PBS, for 7 consecutive days. Mice were sacrificed on day 8, and the liver was dissected and its condition recorded. The liver tissue was then fixed in 10% neutral formalin for 24-48 hours. After fixation, the liver tissue was dehydrated using an alcohol gradient and cleared with xylene before being embedded in paraffin. Tissue sections were 5 μm thick, dewaxed, rehydrated, and stained with hematoxylin and eosin (HE staining). Differentiation was performed with 1% hydrochloric acid alcohol, followed by ammonia blue resurfacing, and then dehydrated and mounted using an alcohol gradient. The sections were observed under a microscope at 200x magnification.

[0148] The results showed that the control group given the solvent PBS ( Figure 14 A), central hepatic vein dilation, endothelial cell necrosis, and extensive focal necrosis of hepatocytes surrounding the central vein with deeply stained nuclear fragmentation. Other non-necrotic areas showed mild hydropic degeneration, cellular edema, and clear cytoplasm, accompanied by mild inflammatory cell infiltration in the necrotic areas; plasminogen agonist group ( Figure 14 (B) No obvious necrosis occurred in the liver; the damage was mainly mild watery degeneration, with a small number of hepatocytes showing increased eosinophilicity and red staining in the cytoplasm. The damage in the plasminogen-treated group was significantly milder than that in the PBS-treated control group, indicating that plasminogen can promote the repair of liver damage in the plc- / - acute liver injury model mice.

[0149] References [1] Xiao-Lan Lu, Jin-Yan Luo, Ming Tao, Yan Gen, Ping ZHAO, Hong-LiZhao, Xiao-Dong Zhang, Nei Dong, Risk factors for alcoholic liver disease in China. World Journal of Gastroenterology. 2004, 10(16). [2] Tim CMA Schreuder, Bart J Verwer, Carin MJ van Nieuwkerk, ChrisJJ Mulder, Nonalcoholic fatty liver disease: An overview of current insightsin pathogenesis, diagnosis and treatment. World Journal of Gastroenterology.2011, 14(16) [3] Wiman, B. and Wallen, P. (1975). Structural relationship between"glutamic acid" and "lysine" forms of human plasminogen and their interactionwith the NH2-terminal activation peptide as studied by affinitychromatography. Eur. J. Biochem. 50 , 489-494. [4] Saksela, O. and Rifkin, D.B. (1988). Cell-associated plasminogenactivation: regulation and physiological functions. Annu. Rev. Cell Biol. 4 ,93-126 [5] Raum, D., Marcus, D., Alper, C.A., Levey, R., Taylor, P.D., andStarzl, T.E. (1980). Synthesis of human plasminogen by the liver. Science 208 ,1036-1037 [6] Wallén P (1980). Biochemistry of plasminogen. In Fibrinolysis,Kline DL and Reddy KKN, eds. (Florida: CRC. [7] Sottrup-Jensen, L., Zajdel, M., Claeys, H., Petersen, T.E., andMagnusson, S. (1975). Amino-acid sequence of activation cleavage site inplasminogen: homology with "pro" part of prothrombin. Proc. Natl. Acad. Sci.U. S. A 72 , 2577-2581. [8] Marder V J, Novokhatny V. Direct fibrinolytic agents:biochemical attributes, preclinical foundation and clinical potential [J].Journal of Thrombosis and Haemostasis, 2010, 8(3): 433-444. [9]Hunt J A, Petteway Jr S R, Scuderi P, et al. Simplifiedrecombinant plasmin: production and fu-nctional comparison of a novelthrombolytic molecule with plasma-derived plasmin [J]. Thromb Haemost, 2008,100(3): 413-419.

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Claims

1. Use of plasminogen in the preparation of medicines for the prevention and / or treatment of liver tissue damage and related conditions in subjects.

2. The use of claim 1, wherein the liver tissue injury and related conditions are liver injury and related conditions caused by radiation or chemical substances.

3. The use of claim 2, wherein the liver damage and related symptoms caused by the radiation or chemical substance are radiotherapy and chemotherapy methods and drugs used in cancer treatment.

4. The use of claim 2 or 3, wherein the radiation is caused by an accident or the working environment.

5. The use of claim 1, wherein the liver tissue injury and related conditions are toxic liver injury and related conditions.

6. The use of claim 5, wherein the toxic liver injury is alcohol-induced toxic liver injury.

7. The use of claim 1, wherein the liver tissue damage and related conditions are caused by hepatitis resulting from viral infection of the liver.

8. The use of claim 1, wherein the liver tissue injury and related conditions are drug-induced liver injury and related conditions.

9. The use of claim 1, wherein the liver tissue damage and related conditions are caused by intrahepatic blood stasis.

10. The use of claim 1, wherein the liver tissue injury and related conditions are diabetic liver injury and related conditions.

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