WO2014121481A1 - 长效人重组可溶性肿瘤坏死因子α受体在制备预防和治疗慢性肝病重症肝损伤药物中的用途 - Google Patents

长效人重组可溶性肿瘤坏死因子α受体在制备预防和治疗慢性肝病重症肝损伤药物中的用途 Download PDF

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WO2014121481A1
WO2014121481A1 PCT/CN2013/071497 CN2013071497W WO2014121481A1 WO 2014121481 A1 WO2014121481 A1 WO 2014121481A1 CN 2013071497 W CN2013071497 W CN 2013071497W WO 2014121481 A1 WO2014121481 A1 WO 2014121481A1
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liver
necrosis factor
liver disease
receptor
tumor necrosis
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PCT/CN2013/071497
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English (en)
French (fr)
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李海
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李震义
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Priority to EP13874779.5A priority Critical patent/EP2954904B9/en
Priority to CN201380072579.6A priority patent/CN105163750B/zh
Priority to PCT/CN2013/071497 priority patent/WO2014121481A1/zh
Publication of WO2014121481A1 publication Critical patent/WO2014121481A1/zh
Priority to US14/821,311 priority patent/US20160101153A1/en
Priority to US15/372,011 priority patent/US10071137B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/22—Hormones
    • A61K38/2292—Thymosin; Related peptides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the field of genetic engineering technology and gene function application, and relates to a novel medicinal use of a recombinant soluble tumor necrosis factor alpha receptor (HusTNFR) gene, and particularly relates to a long-acting human recombinant soluble tumor necrosis factor alpha receptor (LHusTNFR) for chronic liver disease. Prevention of severe liver injury on chronic liver disease. Background technique
  • Chronic liver disease also known as chronic liver disease
  • chronic liver disease is a patient with chronic liver disease.
  • the liver has suffered one or more major blows, causing severe liver damage caused by acute and massive hepatocyte necrosis. It can affect important organs outside the liver, causing multiple organ failure including the liver.
  • the disease has a high (40-80%) mortality rate in the short term and is a serious liver disease that threatens the lives of patients.
  • Chronic liver disease is characterized by severe liver injury diseases. 1) There is a chronic liver-based disease.
  • Chronic liver basic diseases refer to chronic hepatitis, liver fibrosis or cirrhosis caused by various causes.
  • Specific diseases may include chronic hepatitis caused by hepatitis virus, liver fibrosis or liver cirrhosis, chronic alcoholic liver disease, non-alcoholic fatty liver disease, autoimmune liver disease or drug-induced liver damage; 2) relative It is clear that the causes of acute hepatic injury may include factors such as intestinal endotoxin, taking liver damage drugs, alcohol abuse, hepatitis virus mutation, recurrence of original hepatitis virus, infection with new hepatitis virus or chemical damage. 3) Short-term (the disease course is usually less than 4 weeks), rapid liver function deterioration, and can progress to multiple organ failure; and 4) Mortality exceeds 40-80% within 3 months.
  • liver injury and severe liver injury belong to two different types of liver disease.
  • Acute hepatocyte necrosis is the key pathogenesis of severe liver injury.
  • Large/sub-heavy liver necrosis is a characteristic pathological change of liver tissue in this disease.
  • the liver injury is only mild liver inflammation and necrosis, and there is no large block/sub-macro liver necrosis in pathology. This is also a key feature distinguishing between liver injury and severe liver injury.
  • the outcome of the disease is different. Liver damage does not progress to a single organ or multiple organ failure, and death does not occur; and patients with severe liver injury often progress to multiple organ failure with a mortality rate of 40-80%. Again, the clinical effects vary widely.
  • Severe liver injury is classified into acute severe liver injury and severe liver disease due to the presence of underlying disease in the liver before onset. Severe liver injury occurring in healthy liver is acute severe liver injury (also known as acute liver failure), and severe liver injury based on chronic liver disease is severe liver disease of chronic liver disease (also known as slow plus acute liver failure).
  • Acute severe liver injury occurs in a healthy liver. After acute and massive hepatocyte necrosis, healthy liver has a strong ability to regenerate. With the help of anti-hepatocyte necrosis drugs, the rapid regeneration of autologous hepatocytes can restore liver function to the minimum level that meets the basic needs of the body, thus improving mortality.
  • Chronic liver disease Severe liver injury occurs in the liver where there are diseases such as chronic hepatitis, liver fibrosis or cirrhosis. Chronic lesions Liver is more likely to develop acute hepatocyte necrosis under the influence of liver damage than healthy liver.
  • the liver of chronic liver disease cannot be rapidly regenerated, resulting in the diseased liver unable to bear the basic body in a short time. The need to operate, resulting in death of the patient.
  • liver regeneration defects and more prone to acute hepatocyte necrosis are two important differences between severe liver disease and acute severe liver injury, and thus clinically serve as two different types of diseases.
  • the methods for diagnosis and treatment are different, and the drugs effective for the treatment of acute severe liver injury are ineffective for severe liver injury caused by chronic liver disease.
  • soluble TNF ci receptors can alleviate liver damage caused by mild hepatocyte necrosis to some extent. However, it is not possible to effectively reduce the mortality of acute severe liver injury or severe liver disease caused by extensive hepatocyte necrosis. And people in the field have not been able to know why the effect is not satisfactory. Therefore, it is currently difficult to practically apply soluble TNF ci receptors to clinical treatment of severe liver injury.
  • a long-acting soluble tumor necrosis factor alpha receptor for the preparation of a medicament for preventing and/or treating hepatic necrosis which occurs on the basis of severe liver disease and chronic liver disease of chronic liver disease.
  • the treatment is an early stage of treating hepatocellular necrosis-related diseases in the early stages of severe liver disease and chronic liver disease.
  • the early stage of severe liver disease of chronic liver disease refers to severe liver disease of chronic liver disease in the presence of systemic inflammatory response syndrome (SIRS).
  • SIRS systemic inflammatory response syndrome
  • systemic inflammatory response syndrome refers to peripheral blood pro-inflammatory cytokines TNFa, IL-6 elevation or proinflammatory cytokines TNFa, IL-6, anti-inflammatory cytokines IL-10 is elevated.
  • the long-acting soluble tumor necrosis factor alpha receptor has a half-life of 12-140 hours.
  • the long-acting soluble tumor necrosis factor a receptor is selected from the group consisting of:
  • a human type I tumor necrosis factor a receptor and human igGl fusion protein of Fc fragment
  • the long-acting soluble tumor necrosis factor a receptor can significantly reduce the mortality of liver disease in chronic liver disease patients
  • the level of caspase 3 activity in the liver decreased by 70-80%;
  • liver NF- ⁇ B levels by 30-50%
  • the long-acting soluble tumor necrosis factor alpha receptor significantly reduces mortality in animals with severe chronic liver disease and early liver injury;
  • the level of caspase 3 activity in the liver decreased by 70-80%;
  • liver NF- ⁇ B levels by 30-50%
  • the chronic liver disease refers to a disease caused by a virus, an immune injury, a poison (or a drug), an alcohol or a high-fat diet, which causes a chronic liver disease.
  • a virus an immune injury
  • a poison or a drug
  • an alcohol or a high-fat diet which causes a chronic liver disease.
  • the liver has suffered a major blow to induce acute, massive hepatocyte necrosis caused by rapid deterioration of liver function, and can progress to multiple organ failure, a type of liver disease with a mortality rate of more than 20% in the short term.
  • the severe liver disease of the chronic liver disease comprises: severe alcoholic hepatitis, hepatitis virus-related hepatitis, liver fibrosis or cirrhosis based on chronic acute liver failure, non-alcoholic fatty liver disease occurs on the basis of Severe liver injury, severe liver injury based on autoimmune liver disease, and drug-induced severe liver injury based on chronic liver disease.
  • the hepatocyte necrosis is acute large-area hepatocyte necrosis (macro/submassive hepatic necrosis is one of the characteristic features of liver pathology).
  • a pharmaceutical composition comprising: (i) an effective amount of a long-acting soluble tumor necrosis factor alpha receptor selected from the group consisting of:
  • a human type I tumor necrosis factor ci receptor and human IgGl fusion protein of Fc fragment
  • the pharmaceutical composition further comprises an effective amount of one or more drugs selected from the group consisting of:
  • a method for preventing hepatocyte necrosis which occurs on the basis of severe liver disease and chronic liver disease caused by chronic liver disease, which comprises administering an effective amount of long-acting soluble tumor necrosis factor a to a person in need of treatment. Receptor.
  • a method for treating an early stage of hepatocellular necrosis-related diseases based on severe liver disease and chronic liver disease based on chronic liver disease wherein the method is effective for administering an effective amount to a person in need of treatment Soluble tumor necrosis factor alpha receptor.
  • the long-acting soluble tumor necrosis factor alpha receptor is selected from the group consisting of: a. human type I tumor necrosis factor ci receptor and human IgGl: fusion protein of Fc fragment,
  • the present invention provides that the TNF ci receptor effectively and rapidly prevents the occurrence of severe liver injury of chronic liver disease, thereby clinically becoming a serious liver injury for chronic liver disease (slow plus acute liver failure). Good medicine.
  • Fig. 1 shows the liver damage pathological score of the model 1 of Example 4; wherein the left column is the chronic liver fibrosis liver, the middle group is the long-acting type II receptor subcutaneous injection prevention group, and the right column is the control group.
  • Figure 2 shows the dynamic changes of the model-peripheral blood and pro-inflammatory cytokines of anti-inflammatory cytokines after the LPS/D-GaIN attack in Example 4; It is a dynamic change of proinflammatory factors (TNFa, IL-6) and anti-inflammatory factor (IL-10) in peripheral blood, and Figure 2B is a pro-inflammatory factor (TNFa, IL-6) and anti-inflammatory factor (IL-10) in liver tissue.
  • Dynamic changes the solid line is the saline-treated control group, and the dotted line is the long-acting type II receptor (type II HusTNFR-IgGl: Fc) prevention group via the subcutaneous injection route.
  • Fig. 3 shows the liver lesion pathological score of the model 1 of Example 5; wherein the left column is the chronic liver fibrosis liver, the middle group is the prevention group for the long-acting type II receptor administered intravenously, and the right column is the control group.
  • Figure 4 shows the dynamic changes in peripheral blood and liver-derived pro-inflammatory cytokines and anti-inflammatory cytokoines after LPS/D-GaIN attack in Figure 5; Peripheral blood proinflammatory factors (TNFa, IL-6) and anti-inflammatory factors (IL-10) are dynamically altered.
  • Figure 4B shows the dynamics of proinflammatory factors (TNFa, IL-6) and anti-inflammatory factors (IL-10) in liver tissue. Change; the solid line is the control group administered with saline, and the dotted line is the long-acting type II receptor (type II HusTNFR-IgGl: Fc) prevention group administered intravenously. detailed description
  • the inventors After long-term and extensive research and experiments, the inventors first discovered that the prevention of acute death of large areas of hepatocytes requires the sustained and stable blocking effect of soluble TNFa receptor on hepatocytes. Therefore, the inventors have modified the TNFa receptor by various methods to prepare a long-acting soluble TNFa receptor that can maintain stable and sustained therapeutic effects in the blood and liver of the body, effectively prolonging the soluble TNFa receptor as an activity. Part of the time in the body.
  • the inventors For chronic liver disease with severe liver disease, the inventors have greatly improved the efficacy of TNFa receptor in the prevention and treatment of acute hepatocyte necrosis based on chronic liver disease, so that for the first time, the improved TNFa receptor can be applied to chronic liver disease based on large area/large amount. Severe liver disease caused by hepatocyte necrosis caused by chronic liver disease. The present invention has been completed based on this.
  • long-acting soluble tumor necrosis factor alpha receptor refers to a tumor necrosis factor ci receptor having a longer half-life (i.e., an effective concentration that can be maintained for a longer period of time in vivo).
  • the "long-acting soluble tumor necrosis factor alpha receptor” has a half-life of more than 12 hours (e.g., 12-140 hours).
  • the half-life of the tumor necrosis factor a receptor can be extended by a variety of methods, including but not limited to: linking the tumor necrosis factor alpha receptor to the human IgGl: Fc fragment, and linking the tumor necrosis factor alpha receptor to the PEG, using PEG - The liposome mixture entraps the tumor necrosis factor alpha receptor.
  • the "long-acting soluble tumor necrosis factor ci receptor” is a "long-acting human recombinant soluble tumor necrosis factor alpha receptor".
  • the object of the present invention is to provide a novel medicinal use of a recombinant soluble tumor necrosis factor alpha receptor (HusTNFR) gene, in particular a long-acting human recombinant soluble form formed by recombinant soluble tumor necrosis factor alpha receptor (HusTNFR) gene or modified HusTNF protein.
  • the invention adopts a long-acting human recombinant soluble tumor necrosis factor alpha receptor through an animal model of chronic liver disease with severe liver injury, and intervenes in the severe liver injury of chronic liver disease in rats, and the results show that the mortality rate of the intervention group and the model group are 0 and 60-90%.
  • tumor necrosis factor is a recombinant long-acting soluble protein that binds tumor necrosis factor alpha to the corresponding cell membrane receptor, including long-acting human recombinant soluble type I tumor necrosis factor a receptor (LHusTNFRI) and long-acting human recombinant soluble type II.
  • Tumor necrosis factor alpha receptor LHusTNFRII
  • HusTNFRI or HusTNFRII carboxy terminus linked to human immunoglobulin IgG: Fc fragment, or (2) PEG attached at the amino or carboxy terminus, or (3) PEG-liposome coated HusTNFRI or HusTNFRII.
  • the LHusTNFRI and LHusTNFRII are more effective in preventing and treating chronic liver disease with severe liver injury than HusTNFRI or HusTNFRII.
  • the animal model of severe liver injury caused by chronic liver disease is as follows: 1) after sensitization of heterologous serum albumin, repeated tail vein injection for 6 weeks induces chronic liver injury and liver fibrosis, and D-galactosamine and endotoxin are administered.
  • Intradermal injection attack method modeling 2) repeated subcutaneous injection of small doses of carbon tetrachloride for 8 weeks to cause liver cirrhosis model, D-galactosamine and endotoxin injection method for modeling; 3) low concentration After alcohol-fed rats for 12 weeks, chronic alcoholic liver injury and liver fibrosis model were induced, then D-galactosamine and endotoxin were administered intradermally to attack the model; 4) rats fed a high-fat diet for 12 weeks resulted in A model of nonalcoholic fatty liver disease, followed by intradermal injection of D-galactosamine and endotoxin to challenge the model.
  • the above four types of chronically added acute animals have a mortality rate of between 60 and 100% due to liver failure.
  • the above model is in the SIRS stage (TNF a, IL-6 and IL-10 are significantly elevated) 0-2 hours after D-galactosamine and endotoxin attack, and 2-24 hours is non-SIRS stage (TNF a, IL- 6 and IL-10 fall back to the pre-onset level).
  • the present invention produced a severe liver disease of chronic liver disease in which ConA was repeatedly stimulated to induce GalS/GLS after CARS induction. Animal model.
  • TNFR human type I TNF alpha receptor
  • IgGl Fc fragment fusion gene expressed recombinant protein
  • human type II TNF a receptor and human IgGl Fc fragment fusion gene expressed recombinant protein, or c human type I TNF a receptor protein amino terminus linked to PEG, or
  • PEG-liposome mixture entraps human type I TNF a receptor protein
  • a PEG-liposome mixture entraps a human type II TNF a receptor protein.
  • Preparation of long-acting human recombinant soluble tumor necrosis factor alpha receptor (LHusTNFR) for prevention and treatment 1) Intradermal injection of D-galactosamine and endotoxin after repeated injection of heterologous serum albumin to induce chronic liver injury and liver fibrosis
  • Rat model of severe liver injury with chronic liver disease Rat model of severe liver injury with chronic liver disease by intradermal injection of D-galactosamine and endotoxin after repeated subcutaneous injection of small dose of carbon tetrachloride for 8 weeks; 3) Rats fed with low concentration of ethanol for 12 weeks caused chronic alcoholic liver injury and liver fibrosis model, and then given D-galactosamine and endotoxin intradermal injection of chronic liver disease in rats with severe liver injury; 4) Rats fed a high-fat diet for 12 weeks resulted in a model of non-alcoholic fatty liver disease, followed
  • a method for producing a type I LHusTNFR gene of SEQ ID NO: 1, comprising the gene encoding a type I sTNFR (human) extracellular amino acid (position of 1-71 of g ⁇ SEQ ID NO: 1)
  • the amino acid encoding the human immunoglobulin Y 1 chain Fc fragment (IgG1 : Fc) (172-403 of SEQ ID NO: 1) is preferentially recombined with the relevant plasmid, and restriction endonuclease digestion by DNA restriction enzyme screening carries type I TNFR- IgGl: a positive clone of the Fc fusion fragment, and nucleotide sequence analysis confirmed whether the gene is correct.
  • a method for producing a type II LHusTNFR gene of SEQ ID NO: 2 comprising encoding a human immunoglobulin encoding a gene encoding a type II sTNFR (human) extracellular amino acid (ie, positions 1-235 of SEQ ID NO: 2)
  • the Y 1 chain Fc fragment (IgG1 : Fc) amino acid (positions 236-467 of S ⁇ SEQ ID NO: 2) was recombined with the relevant plasmid, and restriction endonuclease digestion by DNA restriction enzyme screening carried the type II TNFR-IgG1 : Fc fusion fragment Positive clones, nucleotide sequence analysis verify that the gene is correct.
  • the above-described cDNA fragments of type I and type II TNFR-IgGl: Fc were recombined with an expression vector to form a recombinant expression plasmid.
  • the invention is not limited to a particular expression plasmid.
  • the invention employs a eukaryotic expression vector.
  • the above recombinant expression vector can be introduced into a suitable host cell by a conventional method.
  • the present invention is not limited to any particular host cell as long as it is capable of expressing the recombinant expression vector.
  • the invention uses mammalian CHO cells and the like.
  • the invention is not limited to a particular expression plasmid.
  • the invention employs a eukaryotic expression vector.
  • the above recombinant expression vector can be introduced into a suitable host cell by a conventional method.
  • the present invention is not limited to any particular host cell as long as it is capable of expressing the recombinant expression vector.
  • the invention employs mammalian CHO cells and the like.
  • Long-acting type I HusTNFR was synthesized by coupling active mPEG with a molecular weight (MW) of 20,000 or more to the amino terminus or carboxy terminus of type I HusTNFR.
  • the invention is not limited to a particular mPEG.
  • the invention uses mPEG2-ALD, MW 4,0000 (Shearwater corporation, New Jersey, USA) to link to the amino terminus of type I HusTNFR.
  • the carboxy terminus of type I HusTNFR was ligated using mPEG2-NHS easter, MW 4,0000 (Shearwater corporation, New Jersey, USA).
  • the reaction conditions were pH 7.9 for 12 hours.
  • the invention is not limited to a particular expression plasmid.
  • the invention uses a eukaryotic expression vector.
  • the above recombinant expression vector can be introduced into a suitable host cell by a conventional method.
  • the present invention is not limited to any particular host cell as long as it is capable of expressing the recombinant expression vector.
  • the invention uses mammalian CHO cells and the like.
  • Long-acting type II HusTNFR was synthesized by coupling active mPEG with a molecular weight (MW) of 20,000 or more to the amino terminus or carboxy terminus of type I HusTNFR.
  • the invention is not limited to a particular mPEG.
  • the invention uses mPEG2-ALD, MW 4,0000 (Shearwater corporation, New Jersey, USA) to couple to the amino terminus of Type II HusTNFR.
  • the carboxy terminus of type II HusTNFR was ligated using mPEG2-NHS easter, MW 4,0000 (Shearwater corporation, New Jersey, USA).
  • the reaction conditions were pH 7.9 for 12 hours.
  • Long-acting type I and type II HusTNFR were synthesized by encapsulating long-circulating liposome-polyethylene glycol-derived phospholipids with type I HusTNFR or type II HusTNFR, respectively.
  • NHS-PEG 354 o-AL Nhydroxysulfosuccinimide - polyoxyethylene (MW3540)-maleimide) (molecular weight 3477, American Aanti Polar) Lipids) and TEA was reacted at a molar ratio of 1:1:0.1 at 25 °C for 6 hours, centrifuged, evaporated to dryness and vacuum dried to give DOPE-PEG-MAL (molecular weight: 4108.04).
  • DOPE-PEG-MAL was reacted with EPC (L-a-Phosphatidylcholine, MW 760.09), cholesterol (MW 386.67) and mPEG-2000-DOPE (MW obtained 2801 ⁇ 51) (Aanti Polar Lipids, USA) in a chloroform reaction system.
  • EPC L-a-Phosphatidylcholine, MW 760.09
  • cholesterol MW 386.67
  • mPEG-2000-DOPE MW obtained 2801 ⁇ 51
  • PBS pH 7.4
  • HusTNFR of SEQ ID NO: 3 or Type II HusTNF of SEQ ID NO: 4 in PBS to the above long-circulating liposome-polyethylene glycol-derived phospholipid in PBS, 4 vortexing for 30 min, passing CL -4B (Pharmacia, USA) Removes free type I HusTNFR or type II HusTNFR to obtain long-acting type I and type II HusTNFR.
  • the half-life of type I and type II HusTNFR-IgG:Fc fusion proteins was determined to be 40-100 hours in vivo; half-life of PEG-HusTNFR and long-circulating liposome-polyethylene glycol-derived phospholipid-embedded HusTNFR in vivo For 3-5.5 days.
  • LHusTNFR prepared by the above method have a half-life of more than 12 hours (12-140 hours), and achieve long-term requirements.
  • the half-life of the general soluble tumor necrosis factor a receptor is only 50 minutes to 2 hours.
  • the invention adopts the genetic engineering method to produce LHusTNFR, and the obtained product has the function of effectively preventing and treating severe liver injury of chronic liver disease. Compared with the conventional HusTNFR properties, the half-life of LHusTNFR was significantly prolonged, and the efficacy of preventing and treating chronic liver disease with severe liver injury and reducing mortality was significantly improved.
  • the invention discloses that the long-acting soluble tumor necrosis factor alpha receptor can effectively prevent and treat chronic liver disease severe liver injury model animals, and significantly reduce the disease mortality. That is, various types of long-acting soluble tumor necrosis factor alpha receptors can reduce the mortality of severe liver disease animals with different pathogenesis from chronic liver disease to 60%-90%.
  • the present invention discloses that long-acting soluble tumor necrosis factor alpha receptor significantly reduces disease in animal models
  • the mechanism of the rate is to prevent hepatocyte apoptosis, protect liver cell survival and promote hepatocyte regeneration.
  • Our study further elucidates the role of long-acting soluble tumor necrosis factor alpha receptor in the prevention and treatment of severe liver disease in chronic liver disease. The following two mechanisms are required: 1) to reduce hepatic apoptosis signal generation by blocking TNF ci signaling, and to exert anti-hepatocyte The role of acute necrosis; 2) Increase the ability of hepatocytes to survive and regenerate by increasing the expression of IL-22 and IL-22 receptors. (It has been reported in the literature that IL-22 has the function of promoting hepatocyte regeneration and increasing hepatocyte survival).
  • the present invention describes effective prevention and treatment of chronic liver disease.
  • chronic liver disease severe liver injury animal which is prevented/treated by long-acting soluble tumor necrosis factor alpha receptor, and the severity of hepatocyte apoptosis is obviously reduced, and the IL-22 signal which protects liver cells and promotes hepatocyte regeneration is significant at the same time. Elevated to play a role in effectively treating the disease and significantly reducing mortality.
  • the present invention also innovatively discovers the different effects of long-acting soluble tumor necrosis factor alpha receptor on different disease stages of chronic liver disease in severe liver disease animals. It can significantly reduce the mortality of animals with early disease, but has no effect on the mortality of the disease in the middle and late stages.
  • Chronic liver disease Severe liver injury is divided into three stages according to the evolution of the immune status of the disease: early stage systemic inflammatory response syndrome (SIRS) stage, mid-term non-SIRS stage and late-compensatory anti-inflammatory reaction synthesis Complicmentery anti-inflammatory response syndrome (CARS) stage.
  • SIRS systemic inflammatory response syndrome
  • CARS Complicmentery anti-inflammatory response syndrome
  • the inventors found that long-acting soluble tumor necrosis factor alpha receptors (including TNFRII-IgG: Fc) can significantly reduce the early systemic inflammatory response syndrome (SIRS) in rats with severe liver disease.
  • SIRS is immunologically characterized by elevated levels of TNF a and IL-6 in peripheral blood (ie, elevated pro-inflammatory factors) or a common rise in blood TNF d, IL-6, and IL-10 (pro-inflammatory and anti-inflammatory factors simultaneously rising) High), occurs in the early stages of severe liver disease with chronic liver disease.
  • TNF a and IL-6 in peripheral blood
  • IL-6 in peripheral blood
  • IL-10 pro-inflammatory and anti-inflammatory factors simultaneously rising
  • TNFRII-IgG Fc (Entarcept, trade name: Enbrel) for the treatment of refractory acute alcoholic hepatitis (a type of severe liver disease with severe liver disease, alcoholic liver disease-based disease)
  • Fc Enterarcept, trade name: Enbrel
  • results were not significantly lower than the results.
  • patients enrolled in advanced patients or who were not treated with glucocorticoid therapy were enrolled. This part of the patient is often in the CARS stage, the immune system is characterized by a significant decrease in the expression of HLA-DR in monocytes, an increase in peripheral blood anti-inflammatory factor IL-10, and an increase in pro-inflammatory factors such as TNF a and IFN Y.
  • SIRS occurs 0-2 hours after acute liver injury induces severe liver injury.
  • Long-acting soluble tumor necrosis factor- ⁇ receptor therapy for 0-2 hours of severe liver injury can reduce mortality by 60-90% to 0.
  • Animals given long-acting soluble tumor necrosis factor ci receptor therapy did not significantly reduce mortality. Therefore, the present invention identifies different therapeutic effects of long-acting soluble tumor necrosis factor alpha receptor on severe chronic liver disease (SIRS) and advanced (non-SIRS or CARS stage), explaining why it is late, in CARS Stages of chronic liver disease are the causes of poor efficacy in animals with severe liver injury.
  • SIRS severe chronic liver disease
  • CARS stages of chronic liver disease are the causes of poor efficacy in animals with severe liver injury.
  • long-acting soluble tumor necrosis factor ci receptor has obvious curative effect on animals with severe chronic liver disease with early stage of SIRS, which can significantly reduce the mortality.
  • the present inventors have also found that the prevention of the use of long-acting soluble tumor necrosis factor alpha receptor can prevent the occurrence of severe liver damage in chronic liver disease by blocking a large number of acute hepatocyte necrosis and preventing the occurrence of SIRS.
  • the four animal models of chronic liver disease severe liver injury selected in the present invention respectively represent, but are not limited to, ethanol (alcohol), high fat diet, immune damage or chemical poisons on the liver of model animals causing chronic liver damage, liver fibrosis or cirrhosis
  • Acute liver cell injury characterized by acute hepatocyte necrosis, liver inflammation, rapid deterioration of liver function, and short-term high mortality after endotoxin, viral nucleic acid, etc.
  • the present invention also provides a pharmaceutical composition for treating hepatocyte necrosis or severe liver disease of chronic liver disease comprising the above-described long-acting soluble tumor necrosis factor alpha receptor, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, andpharmaceutically acceptable aqueous carrier medium, wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for the treatment of hepatocyte necrosis or severe liver injury of chronic liver disease.
  • other related therapeutic agents can be used simultaneously.
  • therapeutic agents include, but are not limited to, alpha-thymosin, human hepatocyte growth factor (huHGF), reduced glutathione, matrine, human serum albumin, polyene phosphatidylcholine, various coenzymes and vitamins. , blood will be net (Chinese medicine).
  • compositions of the present invention comprise a safe and effective amount of the above-described long-acting soluble tumor necrosis factor alpha receptor of the present invention together with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be formulated into an injection form, for example, with physiological saline or containing hydrochloric acid. An aqueous solution of glucose and other adjuvants is prepared by a conventional method. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 0.1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • a safe and effective amount of the long-acting soluble tumor necrosis factor alpha receptor of the invention is administered to a mammal, wherein the safe and effective amount is usually at least about 1 microgram per kilogram of body weight, and in most cases not More than about 8 mg/kg body weight, preferably about 10 micrograms per kilogram body weight - about 1 mg/kg body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention.
  • Model I heterogeneous serum albumin sensitization and repeated injection of chronic liver injury/hepatic fibrosis after administration of D-galactosamine (GaIN) and endotoxin (LPS) injection of chronic liver disease in rats with severe liver injury, divided into three Stage completion
  • a, xenogenic albumin sensitization Wistar rats, females weighing 120-150 g. Human serum albumin was diluted with physiological saline and emulsified with an equal amount of incomplete Freund's adjuvant. Each rat was injected subcutaneously at a dose of 0.5 ml per ml (containing albumin 4 mg) for 4 times. 2 times interval 14 days, 3rd and 4th time interval 10 days. Blood samples were taken 10 days after the last immunization, and antibody-positive rats were taken for subsequent experiments;
  • Immunofluorescence showed that the immune system was deposited in the portal, hepatic sinus and blood vessel wall.
  • the stellate cells were transformed into myofibroblasts by electron microscopy and distributed in the portal area. There is a lot of collagen deposition around it. The deposited collagen forms a broad separation.
  • D-GaIN/LPS acutely attacked the above-mentioned liver fibrosis rats: 2 times a week, a total of 6 weeks of tail vein injection of albumin and liver fibrosis in rats given D-GaIN 400mg / kg plus LPS ⁇ ⁇ g / kg abdominal cavity injection.
  • the mortality of the 24-hour model group was 80%-90%, and the average survival time was about 16 hours.
  • the alanine aminotransferase began to rise 4 hours after D-GaIN plus LPS injection, with a peak of 8-12 hours. The 4 hours after the total bilirubin injection increased until the animal died.
  • Plasma TNFa levels increased significantly after 0.5 hours after D-GaIN plus LPS injection, peaked at 1 hour, decreased at 2 hours, and reached normal levels at 4 hours.
  • Plasma IL-6 increased significantly after 0.5 hours, reached a peak at 2 hours, decreased at 3 hours, and returned to normal levels at 8 hours.
  • Plasma IL-10 levels increased significantly after 1 hour, peaked at 2 hours, and reached normal levels at 4 hours.
  • Gross liver specimens showed severe hyperemia and enlargement of the liver; liver pathology showed focal or flaky necrosis with leukocyte response in 4 hours of regenerative nodules, apoptotic bodies scattered, and mild edema in the portal area.
  • hepatic necrosis increased or flaky fusion in the nodules, including erythrocytosis, interstitial cells and bile duct epithelial hyperplasia.
  • most of the regenerative nodules showed large or sub-heavy liver cells.
  • Residual hepatocytes vesicular lipid or vacuolar degeneration, complete fiber retention.
  • Electron microscopy showed a small amount of lipid droplets in 4 hours of hepatocytes, and mitochondrial mites ruptured and disappeared; hepatocytes showed early apoptosis of microvilli and irregular shape of nuclear shrinkage.
  • the apoptotic cells increased significantly in 8 hours and the exfoliated apoptotic bodies were seen.
  • Kupffer cells activate the protruding foot processes to contact or phagocytose apoptotic bodies.
  • Model 2 repeated subcutaneous injection of small doses of carbon tetrachloride 8 weeks after the liver cirrhosis model was given D-galactosamine and endotoxin injection to prepare a model of severe liver injury in chronic liver disease, completed in two stages:
  • D-GaIN/LPS acutely attacks the above cirrhotic rats: D-GaIN 400mg/kg plus LPS
  • Model 3 rats fed with low concentration of ethanol for 12 weeks caused chronic alcoholic liver injury and liver fibrosis model, then D-galactosamine and endotoxin were injected intradermally to produce a model of severe liver injury with chronic liver disease, which was completed in two stages. ;
  • D-GaIN/LPS acutely attacks the above cirrhotic rats: D-GaIN 400mg/kg plus LPS
  • the mortality of the 24-hour model group was 80%-90%, and the average survival time was about 16 hours.
  • the alanine aminotransferase began to rise 4 hours after D-GaIN plus LPS injection, with a peak of 8-12 hours.
  • the 4 hours after the total bilirubin injection increased until the animal died.
  • Plasma TNFa levels increased significantly after 8 hours, and IL-10 levels increased with time.
  • Pathology showed large hepatic mass and sub-macro necrosis, sinusoidal dilatation, red blood cell filling, kupffer cell activation, and cytoplasmic hypertrophy. Residual hepatocytes vesicular lipids or vacuolar degeneration, complete fiber retention.
  • Electron microscopy showed a small amount of lipid droplets in 4 hours of liver cells, and mitochondrial sputum ruptured and disappeared; hepatocytes showed early apoptosis due to microvilli reduction and irregular shape of nuclear shrinkage.
  • the apoptotic cells increased significantly in 8 hours and the exfoliated apoptotic bodies were seen.
  • Kupffer cells activate extended foot processes to contact with hepatocytes or phagocytose apoptotic bodies.
  • Rats fed a high-fat diet for 12 weeks cause a model of nonalcoholic fatty liver disease, followed by D-galactosamine and endotoxin injection. A model of severe liver injury with chronic liver disease was made.
  • D-GaIN/LPS acutely attacked the above-mentioned cirrhotic rats: D-GaIN 400 mg/kg plus LPS 100 ⁇ g/kg was intraperitoneally injected. The mortality rate of the rats in the 24-hour model group was 80%-90%, and the average survival time was about 16 hours.
  • Alanine aminotransferase began to rise 4 hours after D-GaIN plus LPS injection, with a peak of 8-12 hours. The increase in total bilirubin 4 hours after injection increased until the animal died. Plasma TNFa levels increased significantly after 8 hours, and IL-10 levels increased with time.
  • Pathology showed large hepatic mass and sub-macro necrosis, sinusoidal dilatation, red blood cell filling, kupffer cell activation, and cytoplasmic hypertrophy. Residual liver Cellular vesicles or vacuolar degeneration, complete fiber retention. Electron microscopy showed a small amount of lipid droplets in the liver cells for 4 hours, and the mitochondria ruptured and disappeared; the hepatocytes showed early microapoptosis, and the shape of the nuclear shrinkage was irregular and other early apoptosis. The apoptotic cells increased significantly in 8 hours and the exfoliated apoptotic bodies were seen. Kupffer cells activate the protruding foot processes to contact or phagocytose apoptotic bodies.
  • ConA repeatedly stimulates chronic liver injury caused by GalN/LPS after induction of CARS.
  • NAC N-acetylcysteine
  • a rat model of drug-induced acute severe liver injury was prepared using a general-purpose dose of Acetaminophen (acetaminophen), with a short-term mortality rate of approximately 40%.
  • the rat model of cirrhosis induced by carbon tetrachloride was prepared according to the a step of the first model of the first embodiment, and the same dose of acetaminophen was administered to prepare an animal model of severe liver injury with chronic liver disease, and the short-term mortality was 40-60%.
  • Healthy rat NAC was administered intravenously 6 hours prior to administration of acetaminophen to prepare a model of acute severe liver injury with NAC.
  • NAC neuropeptide acetylase acetylase fungus .
  • the same dose of NAC was administered intravenously to cirrhotic rats 6 hours before acetaminophen administration to prepare for the prevention of severe liver injury in chronic liver disease.
  • the same dose of NAC was given to healthy and cirrhotic rats 0.5 hours after acetaminophen administration.
  • NAC was used to treat acute severe liver injury and severe liver injury model of chronic liver disease.
  • the NAC prevention group and the treatment group reduced the model group mortality from 40% to 10%-15%.
  • the mortality of NAC in the prevention and treatment of acetaminophen-induced chronic liver disease in severe liver injury was 40-60%, which was indistinguishable from the control group. It indicated that NAC could not effectively reduce the mortality of animals with severe liver injury induced by acetaminophen in chronic liver disease.
  • NAC N-glutamylamine
  • TNF tumor necrosis factor
  • LHusTNFR Long-acting human recombinant soluble type II tumor necrosis factor (TNF) ⁇ receptor (type II LHusTNFR) represented by type II HusTNFR-I g Gl: Fc is a subcutaneous injection route to prevent severe liver injury in rats with chronic liver disease.
  • Type II LHusTNFR (Method (2) Form II LHusTNFR-IgGl: Fc is a prophylactic drug), dose 5-50 mg/kg, In a preferred embodiment, the present invention uses a dose of 12.5 mg/kg.
  • the treatment group was named the long-acting type II receptor subcutaneous injection prevention group.
  • Long-acting type II soluble receptor type II LHusTNFR-IgGl: Fc is not limited to D-GalN/LPS specific time before acute attack, as long as it is administered before D-GalN/LPS acute strike, the time is preferably D-GalN/ 36 hours to 0 hours before LPS acute strike. In a preferred embodiment, the present invention uses D-GalN/LPS 8 hours before the acute strike.
  • a conventional HusTNFR subcutaneous injection was administered at a dose of 12.5 mg/kg, which was the conventional type II receptor prevention group; the control group was subcutaneously injected with the same volume of normal saline.
  • Subcutaneous injection of long-acting type II TNF ci receptor based on xenogenic albumin-induced immune liver fibrosis, carbon tetrachloride-induced cirrhosis, alcohol-induced alcoholic liver disease, and high-fat diet-induced nonalcoholic liver disease in rats The mortality rate of the rats in the prevention group at 24 hours after D-GalN/LPS injection was 0, while that in the conventional type II TNF a receptor prevention group was 66%, and no long-acting or conventional type II TNF a receptor treatment was given.
  • control group chronic liver disease severe liver injury
  • gross pathology showed that the characteristics of chronic liver damage, liver fibrosis and cirrhosis of the liver in the long-acting type II receptor prevention group were still preserved, but the liver parenchyma was only slightly hyperemic and swollen, and there was a small amount of focal necrosis, no large or sub-bulk. Necrosis.
  • Liver tissue damage score The injury score of the liver injury (semi-quantitative score based on the degree of liver tissue lesions observed in HE staining of the rat liver tissue section, and the lesion tissue area) was 2 ⁇ 1 point.
  • the pathological score of liver injury is shown in Figure 1.
  • the left column is chronic liver fibrosis liver
  • the middle group is the long-acting type II receptor subcutaneous injection prevention group
  • the right column is the control group.
  • the level of NF- ⁇ in the liver of the prevention group was 30-50% lower than that of the control group.
  • the expression of IL-22 and IL-22 receptors in liver tissue was increased by 2-5 fold.
  • the above results indicate that the long-acting type II receptor subcutaneous injection prevention group 1) reduces hepatocyte massive necrosis and slows liver inflammation by inhibiting apoptosis of hepatocytes, 2) promotes hepatocyte survival and regeneration; 3) inhibits chronic liver disease with severe liver injury Early systemic inflammatory response syndrome (SIRS) in liver failure animals significantly reduces mortality.
  • SIRS Early systemic inflammatory response syndrome
  • Fc Long-acting human recombinant soluble type II tumor necrosis factor (TNF) ci receptor (type II LHusTNFR) represented by type II HusTNFR-I g Gl: Fc is an intravenous administration route to prevent severe liver injury in rats with chronic liver disease.
  • TNF tumor necrosis factor
  • Type II LHusTNFR was injected into the tail vein (Method 2 (2) LHusTNFR-IgGl: Fc is a preventive drug), dose 5-50mg/kg In a preferred embodiment, the present invention uses a dose of 12.5 mg/kg.
  • the treatment group was named the long-acting type II receptor intravenous injection prevention group.
  • Fc is not limited to D-GaIN/LPS specific time before acute attack, as long as it is administered before D-GaIN/LPS acute strike, the time is preferably D-GaIN/ 36 hours to 0 hours before LPS acute strike.
  • the invention uses D-GaIN/LPS for 2 hours before the acute strike.
  • a conventional HusTNFR tail vein injection was also given to rats in the conventional type II receptor prevention group at a dose of 12.5 mg/kg.
  • the control group was subcutaneously injected with the same volume of normal saline.
  • liver damage score The injury score of the liver injury (semi-quantitative score based on the degree of liver tissue lesions observed in HE staining of the rat liver tissue section, and the lesion tissue area) was 2 ⁇ 1 point. Significantly lower than the control group of 5 ⁇ 2 points (p ⁇ 0.01). Taking model 1 as an example, the pathological score of liver injury is shown in Figure 3. The left column is chronic liver fibrosis liver, the middle group is the long-acting type II receptor subcutaneous injection prevention group, and the right column is the control group.
  • the level of NF- ⁇ in the liver of the prevention group was 30-50% lower than that of the control group.
  • the expression of IL-22 and IL-22 receptors in liver tissue is increased by 2-5 fold.
  • the above results indicate that the long-acting type II receptor subcutaneous injection prevention group 1) reduces hepatocyte massive necrosis and slows liver inflammation by inhibiting apoptosis of hepatocytes, 2) promotes hepatocyte survival and regeneration; 3) inhibits chronic liver disease with severe liver injury Early systemic inflammatory response syndrome (SIRS) in liver failure animals significantly reduces mortality.
  • SIRS Early systemic inflammatory response syndrome
  • TNF tumor necrosis factor
  • ⁇ LHusTNFR Long-acting human recombinant soluble type II tumor necrosis factor (TNF) a receptor ( ⁇ LHusTNFR), which is represented by type II HusTNFR-I g Gl: Fc, is used to treat severe liver disease in rats with chronic liver disease in early stage. experiment
  • the severe liver injury model of four types of chronic liver disease in Example 1 and the severe liver injury model of chronic liver disease in the CARS state in Example 2 were 0, 30 minutes, 1 hour, 1.5 hours after the acute attack of D-GalN/LPS.
  • Intravenous injection of type II LHusTNFR in hours, 3 hours, 4 hours, 6 hours and 8 hours (Method (2) type II LHusTNFR-IgGl: Fc is a therapeutic drug)
  • the dosage range is 5-50 mg/kg, in a preferred
  • the present invention uses a dose of 12.5 mg/kg.
  • Example 1 The four types of chronic liver disease animals in Example 1 were treated with type II LHusTNFR-IgGl: Fc at 0, 30 minutes, 1 hour, and 1.5 hours after D-GaIN/LPS acute challenge, and the 24-hour mortality rate was zero.
  • the mortality rate of the control group of chronic liver disease with severe liver injury was 90%.
  • the CRAS model in Example 2 was given type II LHusTNFR-IgGl: Fc treatment at 0, 30 minutes, 1, 1.5, 2, 3, 4, 6 and 8 hours after D-GaIN/LPS acute shock, with 24 hours of death. There was no difference in mortality between the control group and the control group of chronic liver disease with severe liver injury. The mortality rate was 15-20%.
  • Fc has an effective therapeutic effect on severe liver disease in early chronic liver disease, but is ineffective in animals with severe liver disease of chronic liver disease other than SIRS and CARS.
  • Peripheral plasma pro-inflammatory cytokines TNF, IL-6 and anti-inflammatory cytokine IL-10 peaks significantly lower than chronic liver disease severe liver injury control group; liver TNF a, IL-6 peak Compared with the chronic liver disease, the severe liver injury control group decreased significantly.
  • the level of NF- ⁇ in the liver decreased by 30-50% compared with the control group.
  • the expression of IL-22 and IL-22 receptors in liver tissue was increased by 2-5 fold. In the rats administered at 3 hours, 4 hours, 6 hours and 8 hours after D-GaIN/LPS acute shock, there was a large or sub-macro necrosis in the liver.
  • the pathological liver injury score (injury score) was not significantly different from the saline-treated chronic liver disease severe liver injury control group.
  • the peaks of plasma pro-inflammatory factors TNF a, IL-6 and anti-inflammatory factor IL-10 were not significantly different from those of chronic liver disease with severe liver injury.
  • the above studies show that subcutaneous injection of type II LHusTNFR-IgGl: Fc reduces mortality by inhibiting massive hepatocyte necrosis, promoting hepatocyte survival and regeneration, and reducing systemic inflammatory response syndrome (SIRS) in severe chronic liver disease with severe liver disease.
  • SIRS systemic inflammatory response syndrome
  • the severe liver injury model of four types of chronic liver disease in Example 1 and the severe liver injury model of chronic liver disease in the CARS state in Example 2 were 0, 30 minutes, 1 hour, 1.5 hours after the acute attack of D-GaIN/LPS.
  • Intravenous injection of type II LHusTNFR in hours, 3 hours, 4 hours, 6 hours and 8 hours (Method (2) type II LHusTNFR-IgGl: Fc is a therapeutic drug)
  • the dose range is 5-50mg/kg, in one
  • the dosage of the invention is 12.5 mg/kg.
  • Example 1 The four types of chronic liver disease animals in Example 1 were treated with type II LHusTNFR-IgGl: Fc at 0, 30 minutes, 1 hour, and 1.5 hours after D-GaIN/LPS acute challenge, and the 24-hour mortality rate was zero.
  • the mortality rate of the control group of chronic liver disease with severe liver injury was 80-90%.
  • the CRAS model in Example 2 was 0, 30 minutes, 1, 1.5, 2, 3, 4, 6 and 8 hours after the D-GaIN/LPS acute strike.
  • Type II LHusTNFR-IgGl Fc treatment, its 24-hour mortality, chronic liver disease, severe liver injury, normal saline treatment, mortality, no difference, 15-20%.
  • the above experiments showed that the tail vein injection of type II LHusTNFR-IgGl: Fc has an effective therapeutic effect on early chronic liver disease with severe liver disease, but is ineffective for animals with severe liver disease of chronic liver disease other than SIRS and CARS.
  • IL-22 and IL-22 receptors were increased by 2-5 fold.
  • Peripheral plasma pro-inflammatory cytokines TNFa, IL-6 and anti-inflammatory cytokine IL-10 peaks were significantly lower than chronic liver disease severe liver injury control group (p ⁇ 0.01); liver TNFa, The peak of IL-6 was significantly lower than that of the control group with severe liver disease in chronic liver disease (p ⁇ 0.01).
  • the pathological liver tissue injury score (injury score) was not significantly different from the saline-treated chronic liver disease severe liver injury control group.
  • TNF tumor necrosis factor
  • Fc is a subcutaneous injection route to prevent severe liver injury in rats with chronic liver disease.
  • LHusTNFR-IgGl Fc is a preventive drug
  • dose 5-50mg/kg In a preferred embodiment, the dosage of the invention is 12.5 mg/kg.
  • the treatment group was named as the long-acting type I receptor subcutaneous injection prevention group.
  • Fc is not limited to D-GaIN/LPS specific time before acute strike, as long as it meets the D-GaIN/LPS priority It can be administered before sexual strike, and the time is preferably 36 hours to 0 hours before the acute attack of D-GaIN/LPS. In a preferred embodiment, the invention uses D-GaIN/LPS 8 hours prior to acute challenge.
  • Another type I HusTNFR subcutaneously injected rats, the dose of 12.5mg / kg, is the conventional type I receptor prevention group; the control group is subcutaneous injection of the same volume of normal saline in the same type of rats.
  • Long-acting type I receptor subcutaneous injection prevention group based on heterologous albumin-induced immune liver fibrosis, carbon tetrachloride-induced liver cirrhosis, alcohol-induced alcoholic liver disease, and high-fat diet-induced nonalcoholic liver disease rats
  • the mortality rate of rats at 0 hours after D-GaIN/LPS injection was 0, while that of the conventional type I receptor prevention group was 50%, and that of the control group was 90%.
  • Gross pathology showed that the chronic liver damage, liver fibrosis and cirrhosis of the liver in the long-acting type I receptor prevention group remained, but the liver parenchyma was only slightly hyperemic and swollen, and there was a small amount of focal necrosis, no large or sub-bulk.
  • liver tissue damage score The injury score of the liver injury (semi-quantitative score based on the degree of liver tissue lesions observed in HE staining of the liver tissue of the mouse, and the area of the damaged liver tissue) was 2 ⁇ 1 point. Significantly lower than the control group of 5 ⁇ 2 points (p ⁇ 0.01).
  • Pathological TUNEL staining showed that the apoptotic bodies in the control group were 40-70/high power field, which was significantly higher than the long-acting type I receptor subcutaneous injection prevention group (4-9/high power field, p ⁇ 0.01. Liver caspase3 detection, The CaS p aS e3 activity in the prevention group was 3-4 times lower than that in the control group.
  • Peripheral plasma pro-inflammatory cytokines TNF ct, IL-6 and anti-inflammatory cytokine IL-10 peak significantly lower than chronic liver disease severe liver injury control group; liver TNF ⁇ , IL-6 The peak value was significantly lower than that of the chronic liver disease with severe liver injury.
  • the level of NF- ⁇ in the liver of the prevention group was reduced by 25-50% compared with the control group.
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-6 times.
  • the above results indicate that the long-acting type I receptor subcutaneous injection prevention group 1) reduces hepatocyte massive necrosis and slows liver inflammation by inhibiting apoptosis of hepatocytes, 2) promotes hepatocyte survival and regeneration; 3) inhibits chronic liver disease with severe liver injury Early systemic inflammatory response syndrome (SIRS) in liver failure animals significantly reduces mortality.
  • SIRS Early systemic inflammatory response syndrome
  • Fc Long-acting human recombinant soluble type I tumor necrosis factor (TNF) ci receptor (type I LHusTNFR) represented by type I HusTNFR-I g Gl: Fc is an intravenous administration route to prevent severe liver injury in rats with chronic liver disease.
  • TNF tumor necrosis factor
  • Type I LHusTNFR-IgGl Fc is a preventive drug
  • dose 5-50mg/kg the present invention uses a dose of 12.5 mg/kg.
  • Processing group Named the long-acting type I receptor intravenous injection prevention group.
  • Type I LHusTNFR-IgGl: Fc is not limited to a specific time before D-GaIN/LPS acute strike, as long as it is administered before D-GaIN/LPS acute strike, the time is preferably 36 hours before D-GaIN/LPS acute strike 0 hours.
  • the invention uses D-GaIN/LPS 2 hours prior to acute challenge.
  • Another type I HusTNFR tail vein injection rats were given at a dose of 12.5 mg/kg, which was the conventional type I receptor prevention group; the control group was subcutaneously injected with the same volume of normal saline.
  • Long-acting type I receptor intravenous injection prevention group based on heterologous albumin-induced immune liver fibrosis, carbon tetrachloride-induced liver cirrhosis, alcohol-induced alcoholic liver disease, and high-fat diet-induced nonalcoholic liver disease rats The mortality rate of rats at 0 hours after D-GaIN/LPS injection was 0, while that of the conventional type I receptor prevention group was 60%, and that of the control group was 90%.
  • liver fibrosis and cirrhosis of the liver in the long-acting type I receptor prevention group remained, but the liver parenchyma was only slightly hyperemic and swollen, and there was a small amount of focal necrosis, no large or sub-bulk.
  • necrosis Liver tissue damage score
  • the injury score of the liver injury was 2 ⁇ 1 point. Significantly lower than the control group of 6 ⁇ 2 points (p ⁇ 0.01).
  • Pathological TUNEL staining showed that the apoptotic bodies in the control group were 30-60/high power field, which was significantly higher than the long-acting type I receptor subcutaneous injection prevention group (4-7/high power field, p ⁇ 0.01. Liver caspase3 detection, The CaS p aS e3 activity in the prevention group was 3-4 times lower than that in the control group.
  • Peripheral plasma pro-inflammatory cytokines TNF ct, IL-6 and anti-inflammatory cytokine IL-10 peak significantly lower than chronic liver disease severe liver injury control group; liver TNF ⁇ , IL-6 The peak value was significantly lower than that of the chronic liver disease with severe liver injury.
  • the level of NF- ⁇ in the liver of the prevention group was reduced by 25-50% compared with the control group.
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-5 times.
  • the above results indicate that the long-acting type I receptor subcutaneous injection prevention group 1) reduces hepatocyte massive necrosis and slows liver inflammation by inhibiting apoptosis of hepatocytes, 2) promotes hepatocyte survival and regeneration; 3) inhibits chronic liver disease with severe liver injury Early systemic inflammatory response syndrome (SIRS) in liver failure animals significantly reduces mortality.
  • SIRS Early systemic inflammatory response syndrome
  • TNF tumor necrosis factor
  • the severe liver injury model of four types of chronic liver disease in Example 1 and the CARS state in Example 2 Severe liver injury model of chronic liver disease was subcutaneously injected with type II LHusTNFR at 0, 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours and 8 hours after acute shock of D-GalN/LPS (method ( 1)
  • the prepared type I LHusTNFR-IgGl: Fc is a therapeutic drug), and the dose ranges from 5 to 50 mg/kg.
  • the present invention uses a dose of 12.5 mg/kg.
  • Example 1 Four types of chronic liver disease animals in Example 1 were treated with type II LHusTNFR-IgGl: Fc at 0, 30 minutes, 1 hour, and 1.5 hours after D-GalN/LPS acute challenge, with a 24-hour mortality rate of zero. D-GalN/LPS acutely combats four types of chronic liver disease animals at 2 hours, 3 hours, 4 hours, 6 hours and 8 hours after administration of type I LHusTNFR-IgGl: Fc-treated animals with a mortality rate of 50%, 90%, 90 %, 90%. The mortality rate of the control group of chronic liver disease with severe liver injury was 90%.
  • the CRAS model in Example 2 was given type I LHusTNFR-IgGl: Fc treatment at 0, 30 minutes, 1, 1.5, 2, 3, 4, 6 and 8 hours after D-GalN/LPS acute challenge, with 24 hours of death. There was no difference in mortality between the control group and the control group of chronic liver disease with severe liver injury. The mortality rate was 15-20%.
  • Fc has an effective therapeutic effect on severe liver disease in early chronic liver disease, but is ineffective in animals with severe liver disease of chronic liver disease other than SIRS and CARS.
  • the level of NF- ⁇ B in the liver decreased by 30-50% compared with the control group.
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-5 times. In the rats administered 3 hours, 4 hours, 6 hours and 8 hours after D-GalN/LPS acute shock, there was a large or sub-macro necrosis in the liver.
  • the pathological liver tissue injury score (injury score) was not significantly different from the saline-treated chronic liver disease severe liver injury control group.
  • the peaks of plasma pro-inflammatory factors TNF a, IL-6 and anti-inflammatory factor IL-10 were not significantly different from those of chronic liver disease with severe liver injury.
  • the above studies show that subcutaneous injection of type I LHusTNFR-IgGl: Fc inhibits massive liver cell necrosis and systemic inflammatory response syndrome (SIRS) in severe chronic liver disease with early liver disease, thereby reducing mortality, but for severe liver disease in non-early chronic liver disease Injured rats are unable to effectively inhibit massive hepatocyte necrosis and block SIRS, thereby failing to reduce mortality.
  • SIRS systemic inflammatory response syndrome
  • TNF tumor necrosis factor
  • LHnsTNFR Long-acting human recombinant soluble type I tumor necrosis factor (TNF) ci receptor (type I LHnsTNFR) blue represented by type I HusTNFR-I g Gl : Fc blue ⁇
  • the severe liver injury model of four types of chronic liver disease in Example 1 and the severe liver injury model of chronic liver disease in the CARS state in Example 2 were 0, 30 minutes, 1 hour, 1.5 hours after the acute attack of D-GalN/LPS.
  • Intravenous injection of type II LHusTNFR in hours, 3 hours, 4 hours, 6 hours and 8 hours (method (1) LHusTNFR-IgGl: Fc is a therapeutic drug), the dose range is 5-50mg/kg, in one In a preferred embodiment, the dosage of the invention is 12.5 mg/kg.
  • Example 1 The four types of chronic liver disease animals in Example 1 were treated with type I LHusTNFR-IgGl: Fc at 0, 30 minutes, 1 hour, and 1.5 hours after D-GaIN/LPS acute challenge, and the 24-hour mortality rate was zero.
  • the mortality rate of the control group of chronic liver disease with severe liver injury was 90%.
  • the CRAS model in Example 2 was given type I LHusTNFR-IgGl: Fc treatment at 0, 30 minutes, 1, 1.5, 2, 3, 4, 6 and 8 hours after acute shock of D-GaIN/LPS, with 24 hours of death. There was no difference in mortality between the control group and the control group of chronic liver disease with severe liver injury. The mortality rate was 15-20%.
  • Fc has an effective therapeutic effect on severe liver disease in early chronic liver disease, but is ineffective in animals with severe liver disease of chronic liver disease other than SIRS and CARS.
  • Peripheral plasma pro-inflammatory cytokine s TNFa, IL-6 and anti-inflammatory cytokine IL-10 peaks were significantly lower than chronic liver disease severe liver injury control group (p ⁇ 0.01); liver TNFa The peak of IL-6 was significantly lower than that of the control group with severe liver disease in chronic liver disease (p ⁇ 0.01).
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-5 times. In the rats administered at 2 hours, 3 hours, 4 hours, 6 hours and 8 hours after D-GaIN/LPS acute shock, there was a large or sub-macro necrosis in the liver.
  • the pathological liver tissue injury score (injury score) was not significantly different from the saline-treated chronic liver disease severe liver injury control group. Peaks of plasma pro-inflammatory factors TNF ci, IL-6 and anti-inflammatory factor IL-10 The value was not significantly different from the control group of chronic liver disease with severe liver injury.
  • the above studies show that intravenous injection of type I LHusTNFR-IgGl r Fc reduces mortality by inhibiting massive hepatocyte necrosis and systemic inflammatory response syndrome (SIRS) in severe chronic liver disease with severe liver disease, but for non-early chronic liver disease with severe liver injury in rats It is not effective in inhibiting massive hepatocyte necrosis and preventing the occurrence of SIRS, thereby reducing its mortality.
  • SIRS systemic inflammatory response syndrome
  • the product of human type I tumor necrosis factor alpha receptor protein amino terminus and PEG, and the product of type I tumor necrosis factor alpha receptor protein carboxy terminal and PEG are respectively administered intravenously to prevent and treat early chronic liver disease in rats.
  • the product of the amino terminal of human type I tumor necrosis factor alpha receptor protein and PEG, and the product of the carboxy terminal of type I tumor necrosis factor alpha receptor protein and PEG (method (3) prepared by type I LHusTNFR-PEG) Four types of chronic liver disease models with severe liver injury were given to the tail vein before D-GaIN/LPS acute shock.
  • the dose is 5-50 mg/kg, and in a preferred embodiment, the dosage of the invention is 12.5 mg/kg.
  • the treatment group was named the long-acting type I receptor intravenous injection prevention group.
  • Type I LHusTNFR-PEG is not limited to D-GaIN/LPS for a specific time before acute shock, as long as it is administered before D-GaIN/LPS acute strike, the time is preferably 36 hours to 0 hours before D-GaIN/LPS acute strike. .
  • the invention uses D-GaIN/LPS 2 hours prior to acute challenge.
  • Another conventional HusTNFR tail vein injection rats were given at a dose of 12.5 mg/kg, which was the conventional type I receptor prevention group; the control group was subcutaneously injected with the same volume of normal saline.
  • the liver injury score was 2 ⁇ 1 for the liver injury, which was significantly lower than the 5 ⁇ 2 points of the control group (p ⁇ 0.01).
  • Pathological TUNEL staining showed that the control group had 40-60/high power fields of apoptotic bodies under microscope, which was significantly higher than 5-8/high power field of the long-acting type II receptor subcutaneous injection prevention group, p ⁇ 0.01.
  • Liver caspase3 detection, The caspase3 activity in the prevention group was 3-4 times lower than that in the control group.
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-5 times.
  • Human type II tumor necrosis factor a receptor protein amino terminus and PEG-linked product and type I tumor necrosis factor alpha receptor protein carboxy terminal and PEG-linked product are respectively administered intravenously to prevent and treat early chronic liver disease in rats.
  • the product of the binding of the amino terminus of the human type II tumor necrosis factor alpha receptor protein to the PEG and the carboxy terminal of the type II tumor necrosis factor alpha receptor protein and the PEG (the type I LHusTNFR-PEG prepared by the method (4))
  • Four types of chronic liver disease models with severe liver injury were given to the tail vein before D-GaIN/LPS acute shock.
  • the dose is 5-50 mg/kg, and in a preferred embodiment, the dosage of the invention is 12.5 mg/kg.
  • the treatment group was named the long-acting type II receptor intravenous injection prevention group.
  • Type II LHusTNFR-PEG is not limited to D-GaIN/LPS for a specific time before acute shock, as long as it is administered before D-GaIN/LPS acute strike, the time is preferably 36 hours to 0 hours before D-GaIN/LPS acute strike. .
  • the invention uses D-GaIN/LPS 2 hours prior to acute challenge.
  • Another conventional HusTNFR tail vein injection rats were given at a dose of 12.5 mg/kg, which was the conventional type I receptor prevention group; the control group was subcutaneously injected with the same volume of normal saline.
  • the liver injury score was 2 ⁇ 1 for the liver injury, which was significantly lower than the 5 ⁇ 2 score of the control group (p ⁇ 0.01).
  • Pathological TUNEL Staining showed that the control group had 40-60/high power field of apoptotic bodies under microscope, which was significantly higher than 5-8/high power field of the long-acting type II receptor subcutaneous injection prevention group, p ⁇ 0.01.
  • Liver caspase3 detection, prevention group The caspase3 activity was 3-4 times lower than that of the control group.
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-5 times.
  • the product of the binding of the amino terminus of the human type II tumor necrosis factor alpha receptor protein to the PEG and the carboxy terminal of the type II tumor necrosis factor ci receptor protein (the type I LHusTNFR-PEG prepared by the method (3))
  • Four types of chronic liver disease models with severe liver injury were given a tail vein injection 0.5, 1, 2, 3, 4, 6 and 8 hours after acute shock of D-GaIN/LPS. 0.5, 1, 2 hours chronic liver disease, severe liver injury, animal mortality rate of 0, 4 hours and subsequent rat mortality rate of 80-90%. It is suggested that the type I LHusTNFR-PEG linked by the above two different end groups can effectively treat severe liver injury and reduce mortality in early chronic liver disease.
  • LHusTNFR was administered to the tail vein before the acute shock of D-GaIN/LPS in the above four types of chronic liver disease models.
  • the dose is 5-50 mg/kg, and in a preferred embodiment, the dosage of the invention is 12.5 mg/kg.
  • the treatment group was named the long-acting type I receptor intravenous injection prevention group.
  • Long-acting type I LHusTNF is not limited to D-GaIN/LPS for a specific time before acute shock, as long as it is administered before D-GaIN/LPS acute strike, the time is preferably 36 hours to 0 hours before D-GaIN/LPS acute strike. .
  • the invention uses D-GaIN/LPS 2 hours prior to acute challenge.
  • Another type I HusTNFR tail vein injection rats were administered at a dose of 12.5 mg/kg, which was the conventional type I receptor prevention group; the control group was subcutaneously injected with the same volume of normal saline.
  • Long-acting type I LHusTNFR-PEG intravenous injection prevention group based on xenogenic albumin-induced immune liver fibrosis, carbon tetrachloride-induced liver cirrhosis, alcohol-induced alcoholic liver disease, and high-fat diet-induced nonalcoholic liver disease in rats
  • Rat mortality was 0 at 24 hours after D-GaIN/LPS injection, whereas the death of the conventional type I receptor prevention group The mortality rate was 60%, and the control group was 90%.
  • liver injury score was 2 ⁇ 1 in the liver injury score, which was significantly lower than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • Pathological TUNEL staining showed that the apoptotic bodies in the control group were 40-60/high power field, which was significantly higher than the long-acting type II receptor subcutaneous injection prevention group (6-8/high power field, p ⁇ 0.01.
  • Liver caspase3 detection, The caspase3 activity in the prevention group was 2-4 times lower than that in the control group.
  • Peripheral plasma pro-inflammatory cytokines TNF ⁇ , IL-6 and anti-inflammatory cytokine IL-10 peaks significantly lower than chronic liver disease severe liver injury control group; liver TNF ci, IL-6 The peak value was significantly lower than that of the chronic liver disease with severe liver injury.
  • the level of NF- ⁇ in the liver of the prevention group was 20-40% lower than that of the control group.
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-5 times.
  • PEG liposome-embedded human type I tumor necrosis factor- ⁇ receptor protein (type I LHusTNFR-PEG prepared by method (5)) was given to D-GaIN/LPS acute shock in the above four types of chronic liver disease severe liver injury model. 1, 2, 3, 4, 6 , 8 hours were given to the tail vein. 0.5, 1, 2 hours chronic liver disease The mortality rate of severe liver injury animals was 0, 4 hours and the mortality rate of rats was 80-90%. It is indicated that PEG liposome-type I LHusTNFR-PEG can effectively treat severe liver disease and reduce mortality in early chronic liver disease.
  • PEG liposome-embedded human type II tumor necrosis factor a receptor intravenous administration to prevent severe liver injury in chronic liver disease and treatment of severe liver injury in early rat chronic liver disease
  • PEG liposome-embedded human type II tumor necrosis factor- ⁇ receptor (long-acting type II LHusTNFR prepared by method (5)) was given to the tail before the acute shock of D-GaIN/LPS was given to the four types of chronic liver disease models.
  • Intravenous injection The dose is 5-50 mg/kg, and in a preferred embodiment, the dosage of the invention is 12.5 mg/kg.
  • the treatment group was named the long-acting type I receptor intravenous injection prevention group.
  • Long-acting type I LHusTNF is not limited to D-GaIN/LPS for a specific time before acute shock, as long as it is administered before D-GaIN/LPS acute strike, the time is preferably 36 hours to 0 hours before D-GaIN/LPS acute strike. .
  • the invention uses D-GaIN/LPS 2 hours prior to acute challenge.
  • Another type I HusTNFR tail vein injection rats were administered at a dose of 12.5 mg/kg, which was the conventional type I receptor prevention group; the control group was subcutaneously injected with the same volume of normal saline.
  • the liver injury score was 2 ⁇ 1 in the liver injury score, which was significantly lower than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • Pathological TUNEL staining showed that the apoptotic bodies in the control group were 40-60/high power field, which was significantly higher than the long-acting type II receptor subcutaneous injection prevention group (6-8/high power field, p ⁇ 0.01.
  • Liver caspase3 detection, The caspase3 activity in the prevention group was 2-4 times lower than that in the control group.
  • Peripheral plasma pro-inflammatory cytokines TNF ⁇ , IL-6 and anti-inflammatory cytokine IL-10 peaks significantly lower than chronic liver disease severe liver injury control group; liver TNF ci, IL-6 The peak value was significantly lower than that of the chronic liver disease with severe liver injury.
  • the level of NF- ⁇ in the liver of the prevention group was 20-40% lower than that of the control group.
  • the expression levels of IL-22 and IL-22 receptors in the liver increased by 2-5 times.
  • PEG liposome-embedded human type II tumor necrosis factor- ⁇ receptor protein (type II LHusTNFR-PEG prepared by method (5)) was given to D-GaIN/LPS acute shock in the above four types of chronic liver disease severe liver injury model. 1, 2, 3, 4, 6, 8 hours were given to the tail vein. 0.5, 1, 2 hours chronic liver disease The mortality rate of severe liver injury animals was 0, 4 hours and the mortality rate of rats was 80-90%. It is indicated that PEG liposome-type II LHusTNFR-PEG can effectively treat severe liver disease and reduce mortality in early chronic liver disease.

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Abstract

本发明公开了一种重组可溶性肿瘤坏死因子α受体（HusTNFR）基因的新药用途。本发明采用I型或II型长效人重组可溶性肿瘤坏死因子α受体，通过5种动物模型，对慢性肝病重症肝损伤的大鼠进行干预，结果显示，半衰期12-140小时的长效人重组可溶性肿瘤坏死因子α受体具有预防慢性肝病重症肝损伤的发生，以及治疗早期慢性肝病重症肝损伤的良好疗效，其疗效较非长效HusTNFR有明显提高。

Description

长效人重组可溶性肿瘤坏死因子 α受体在制备预防和治疗慢性肝病重症 肝损伤药物中的用途
技术领域
本发明属基因工程技术、 基因功能应用领域， 涉及重组可溶性肿瘤坏死因 子 α受体 (HusTNFR)基因的新药用用途， 具体涉及长效人重组可溶性肿瘤坏死 因子 α受体 (LHusTNFR)对慢性肝病基础的重症肝损伤 （severe liver injury on chronic liver disease ) 的预防禾口治疗。 背景技术
慢性肝病基础的重症肝损伤 （又称慢性肝病重症肝损伤） 是指有慢性肝脏 基础疾病的患者， 近期肝脏又遭受一次或多次重大打击， 诱发急性、 大量肝细 胞坏死导致的严重肝脏损伤， 可累及肝外重要脏器， 造成包括肝脏在内的多脏 器功能衰竭。 该病短期有很高的 （40-80% ) 死亡率， 是一种威胁患者生命的严 重肝脏疾病。 慢性肝病重症肝损伤疾病的特点是 1 )存在慢性肝脏基础性疾病。 慢性肝脏基础性疾病是指各种病因引起的慢性肝炎 （chronic hepatitis ) 、 肝纤 维化 (liver fibrosis)或肝硬化 (cirrhosis)。 具体病种上， 可包括肝炎病毒导致的慢 性肝炎、 肝纤维化或肝硬化、 慢性酒精性肝病、 非酒精脂肪性肝病、 自身免疫 性肝病或药物性肝损等肝脏基础疾病； 2 ) 有相对明确， 重大肝脏打击 （acute hepatic injury) 的诱因， 可包括肠源性内毒素、 服用肝损伤药物、 酗酒、 肝炎 病毒变异、原有肝炎病毒复发、感染新肝炎病毒或化学物损伤等肝脏损伤因素； 3 ) 短期内 （病程一般在 4 周以内） 出现快速肝功能恶化， 并可发展至多脏器 功能衰竭； 以及 4 ) 3个月内死亡率超过 40-80%。
临床上， 肝损伤 ( liver injury) 禾口重症肝损伤 ( severe liver injury) 属于两 类不同的肝脏疾病。 首先， 两者发病机制不同。 发生急性肝细胞坏死是重症肝 损伤的关键发病机制， 大块 /亚大块肝坏死是该病肝组织的特征性病理改变。 而 肝损伤仅为轻度的肝脏炎症和坏死， 病理上不存在大块 /亚大块肝坏死。 这也是 区别于肝损伤与重症肝损伤的的关键特征。 其次， 疾病转归结局不同。 肝损伤 不会发展至单个脏器或多脏器功能衰竭， 也不会发生死亡； 而重症肝损伤患者 往往进展为多脏器功能衰竭， 死亡率高达 40-80%。 再次， 临床治疗效果差别巨 大。 在临床实践中， 多种药物可有效预防和治疗普通的肝损伤。 例如有文献报 道， 短半衰期的常规 TNF ci受体可有效治疗或预防肝损伤。 但对重症肝损伤基 本无效。 目前尚无任何化合物、 生物技术药物或天然药物有效预防或治疗慢性 肝病基础上的重症肝损伤。 这是导致该类疾病死亡率居高不下， 短期的疾病死 亡率在 40-80%左右的根本原因。
重症肝损伤根据发病前肝脏是否存在基础疾病分为急性重症肝损伤和慢 性肝病重症肝损伤。 发生于健康肝脏的重症肝损伤为急性重症肝损伤 （又称急 性肝衰竭），而发生于慢性肝病基础上的重症肝损伤为慢性肝病重症肝损伤（又 称慢加急性肝衰竭） 。
急性重症肝损伤发生于健康肝脏。 在急性、 大量肝细胞坏死后， 健康肝脏 具有强大的再生能力， 在抗肝细胞坏死药物帮助下自身肝细胞可快速再生使肝 功能恢复至满足机体运行基本需求的最低水平， 从而改善死亡率。 慢性肝病重 症肝损伤发生在存在慢性肝炎、 肝纤维化或肝硬化等疾病的肝脏。 慢性病变肝 脏较健康肝脏更容易在肝损诱因打击下出现急性肝细胞坏死。 同时因基础疾病 造成的肝组织局部血液循环障碍、 残存肝细胞质量差等原因， 在急性、 大量肝 细胞坏死发生后， 慢性肝病肝脏无法进行快速再生， 导致病变肝脏在短时间内 无法承担机体基本运转需求， 从而导致患者死亡。
因此， 显著的肝脏再生缺陷及更容易发生急性肝细胞坏死是慢性肝病重症 肝损伤与急性重症肝损伤的两个重要区别， 从而在临床上作为两类不同的疾 病。 其诊断和治疗的方法均有不同， 同时对急性重症肝损伤治疗有效的药物对 慢性肝病重症肝损伤无效。
常规可溶性 TNF ci受体在一定程度上可缓解轻度肝细胞坏死导致的肝损 伤。 但是， 无法有效降低因大面积肝细胞坏死导致的急性重症肝损伤或慢性肝 病重症肝损伤的死亡率。 并且本领域人员至今无法得知其效果不理想的原因。 因此， 目前还难以在实践上将可溶性 TNF ci受体应用于重症肝损伤的临床治 疗。
综上， 本领域迫切需要找到 TNF ci受体对于慢性肝病重症肝损伤疗效差的 关键所在， 以进一步对 TNF ci受体进行改良， 使之能有效、 快速阻止慢性肝病 重症肝损伤的发生。 从而在临床上真正成为用于防治慢性肝病重症肝损伤 （慢 加急性肝衰竭） 的良药。 发明内容
在本发明的第一方面， 提供了长效可溶性肿瘤坏死因子 α受体在制备预防 和 /或治疗慢性肝病重症肝损伤及慢性肝病基础上发生的肝细胞坏死药物中的 用途。
在另一优选例中， 所述治疗是治疗慢性肝病重症肝损伤早期阶段及慢性肝 病基础上发生肝细胞坏死相关疾病的早期阶段。
在另一优选例中， 所述慢性肝病重症肝损伤早期阶段是指存在全身炎症反 应综合症 （SIRS ) 状态的慢性肝病重症肝损伤。
在另一优选例中， 所述的全身炎症反应综合症 （SIRS) 是指外周血促炎细 胞因子 TNF a 、 IL-6升高或促炎细胞因子 TNF a 、 IL-6, 抑炎细胞因子 IL-10 均升高。
在另一优选例中， 所述的长效可溶性肿瘤坏死因子 α受体的半衰期为 12-140小时。
在另一优选例中， 所述的长效可溶性肿瘤坏死因子 a受体选自：
a.人 I型肿瘤坏死因子 a受体与人 igGl : Fc片段的融合蛋白，
b.人 II型肿瘤坏死因子 a受体与人 IgGl： Fc片段的融合蛋白，
c.人 I型肿瘤坏死因子 a受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 a受体蛋白羧基端与 PEG联接的产物，
e.人 II型肿瘤坏死因子 a受体蛋白氨基端与 PEG联接的产物，
f.人 II型肿瘤坏死因子 a受体蛋白羧基端与 PEG联接的产物，
h.用 PEG-脂质体混合物包埋人 I型肿瘤坏死因子 a受体蛋白的产物， 或 i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 a受体蛋白的产物。
在另一优选例中， 所述的长效可溶性肿瘤坏死因子 a受体可使慢性肝病重 症肝损伤动物死亡率明显降低；
阻止肝脏组织发生大块 /亚大块坏死；
肝脏的 caspase 3活性水平下降 70-80%;
使肝脏病理 TUNEL阳性细胞计数 /高倍视野下降超过 80%;
使肝组织损伤评分下降 40-70%;
使肝脏大块及亚大块坏死病理特征基本消失；
使肝脏的 NF- κ B水平下降 30-50%;
使外周血 TNF α、 IL-6, IL-10的峰值下降 40-95%； 使肝脏 TNF α 、 IL-6的峰值下降 40-80%; 和 /或
使肝脏 IL-22及 IL-22受体分别升高 2-5倍。
在另一优选例中， 所述的长效可溶性肿瘤坏死因子 α受体可使早期慢性肝 病重症肝损伤动物死亡率明显降低；
阻止肝脏组织发生大块 /亚大块坏死；
肝脏的 caspase 3活性水平下降 70-80%;
使肝脏病理 TUNEL阳性细胞计数 /高倍视野下降超过 80%;
使肝组织损伤评分下降 40-70%;
使肝脏大块及亚大块坏死病理特征基本消失；
使肝脏的 NF- κ B水平下降 30-50%;
使外周血 TNF α、 IL-6, IL-10的峰值下降 40-95%；
使肝脏 TNF α 、 IL-6的峰值下降 40-80%; 和 /或
使肝脏 IL-22及 IL-22受体分别升高 2-5倍。
在另一优选例中， 所述慢性肝病重症肝损伤 （或称慢性肝病基础的重症肝 损伤） 指因病毒、 免疫损伤、 毒物 （或药物） 、 酒精或高脂饮食等导致慢性肝 病基础的疾病， 近期肝脏遭受重大打击诱发急性、 大量肝细胞坏死导致的肝脏 功能快速恶化， 并可进展至多脏器功能衰竭、 短期有超过 20%死亡率的一类肝 脏疾病。
在另一优选例中， 所述慢性肝病重症肝损伤包括： 重度酒精性肝炎， 肝炎 病毒相关肝炎、 肝纤维化或肝硬化基础上的慢加急性肝衰竭， 非酒精脂肪性肝 病基础上发生的重症肝损伤， 自身免疫性肝病基础上发生的重症肝损伤， 和慢 性肝病基础上的药物源性重症肝损伤。
在另一优选例中， 所述的肝细胞坏死是急性大面积肝细胞坏死 （大块或亚 大块坏死 ( massive/submassive hepatic necrosis ) 是其在肝脏病理上的特征表现 之一） 。 在本发明的第二方面， 提供了一种药物组合物， 所述的药物组合物含有： (i) 有效量的选自下组的长效可溶性肿瘤坏死因子 α受体：
a.人 I型肿瘤坏死因子 ci受体与人 IgGl : Fc片段的融合蛋白，
b.人 II型肿瘤坏死因子 α受体与人 igGl : Fc片段的融合蛋白，
c.人 I型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物， d.人 I型肿瘤坏死因子 ci受体蛋白羧基端与 PEG联接的产物， e.人 II型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
f.人 II型肿瘤坏死因子 α受体蛋白羧基端与 PEG联接的产物，
h.用 PEG-脂质体混合物包埋人 I型肿瘤坏死因子 ci受体蛋白的产物， 或 i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 ci受体蛋白的产物， 以及
(ϋ) 药学上可接受的载体。
在另一优选例中， 所述药物组合物还含有有效量的一种或多种选自以下的 药物：
(iii) ci -胸腺肽， 人肝细胞生长因子 （huHGF) 、 还原型谷胱甘肽、 苦参碱、 人体血清白蛋白、 多烯磷脂酸胆碱、 各种辅酶维生素， 血必净 （中成药） 。 在本发明的第三方面， 提供了一种预防慢性肝病重症肝损伤及慢性肝病基 础上发生的肝细胞坏死的方法， 所述方法是给予需要治疗的人员有效量的长效 可溶性肿瘤坏死因子 a受体。 在本发明的第四方面， 提供了一种治疗早期的慢性肝病重症肝损伤及慢性 肝病基础上发生肝细胞坏死相关疾病早期阶段的方法， 所述方法是给予需要治 疗的人员有效量的长效可溶性肿瘤坏死因子 α受体。
在另一优选例中， 所述的长效可溶性肿瘤坏死因子 α受体选自下组： a.人 I型肿瘤坏死因子 ci受体与人 IgGl : Fc片段的融合蛋白，
b.人 II型肿瘤坏死因子 a受体与人 IgGl： Fc片段的融合蛋白，
c.人 I型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 ci受体蛋白羧基端与 PEG联接的产物，
e.人 II型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
f.人 II型肿瘤坏死因子 α受体蛋白羧基端与 PEG联接的产物，
h.用 PEG-脂质体混合物包埋人 I型肿瘤坏死因子 ci受体蛋白的产物， 或 i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 ci受体蛋白的产物。 据此， 本发明提供了 TNF ci受体有效、 快速阻止慢性肝病重症肝损伤的发 生， 从而在临床上真正成为用于防治慢性肝病重症肝损伤 （慢加急性肝衰竭） 的良药。 附图说明
图 1显示了实施例 4、 的模型一的肝脏损伤病理评分； 其中左列为慢性肝 纤维化肝脏， 中列为长效 II型受体皮下注射预防组， 右列为对照组。
图 2显示了实施例 4的模型一外周血及肝组织促炎因子（pro-inflammatory cytokines) 禾卩抑炎因子 ( anti-inflammatory cytokoines) 在 LPS/D-GaIN打击后 的动态改变；其中图 2A是外周血的促炎因子（TNFa、IL-6)及抑炎因子（IL-10) 动态改变， 图 2B 是肝组织促炎因子 （TNFa、 IL-6) 及抑炎因子 （IL-10) 动 态改变； 实线为给予生理盐水处理的对照组， 虚线为经皮下注射途径的长效 II 型受体 （II型 HusTNFR-IgGl: Fc) 预防组。
图 3显示了实施例 5的模型一的肝脏损伤病理评分； 其中左列为慢性肝纤 维化肝脏， 中列为长效 II型受体经静脉途径给药的预防组， 右列为对照组。
图 4 显示了实施 5 的模型一外周血及肝组织促炎因子 （pro-inflammatory cytokines) 禾卩抑炎因子 ( anti-inflammatory cytokoines) 在 LPS/D-GaIN打击后 的动态改变；其中图 4A是外周血的促炎因子（TNFa、IL-6)及抑炎因子（IL-10) 动态改变， 图 4B 是肝组织促炎因子 （TNFa、 IL-6) 及抑炎因子 （IL-10) 动 态改变； 实线为给予生理盐水处理的对照组， 虚线为经静脉途径给药的长效 II 型受体 （II型 HusTNFR-IgGl: Fc) 预防组。 具体实施方式
发明人经过长期而广泛的研究和试验， 首次发现防治大面积肝细胞急性坏 死需要可溶性 TNFa受体对肝细胞持续、 稳定的阻断作用。 因此， 发明人通过 多种方法对 TNFa受体进行改造， 制备了一类可在机体血液及肝脏中维持稳 定、 持续治疗作用的长效的可溶性 TNFa受体， 有效延长了可溶性 TNFa受体 作为活性部分在体内的作用时间。 对于慢性肝病重症肝损伤疾病， 发明人大幅 提高了 TNFa受体防治慢性肝病基础上发生急性肝细胞坏死的疗效， 从而首次 使改良后的 TNFa受体可应用于慢性肝病基础上出现大面积 /大量肝细胞坏死 引起的慢性肝病重症肝损伤。 基于此完成了本发明。
发明人研究发现人长效可溶性肿瘤坏死因子 a受体对慢性肝病基础的重 症肝损伤有良好预防和治疗效果的新用途。 本发明通过实施例揭示了不同蛋白 质结构形成的人长效可溶性肿瘤坏死因子 α受体具备预防和治疗不同类型的 慢性肝病重症肝损伤模型动物的良好疗效。 如本文所用， 所述的 "长效可溶性肿瘤坏死因子 α受体" 是指具有较长的 半衰期 (即在体内能够维持较长时间的有效作用浓度)的肿瘤坏死因子 ci受体。 一般的， 所述的 "长效可溶性肿瘤坏死因子 α受体" 的半衰期超过 12小时 (如 12— 140小时)。 可通过多种方法来延长肿瘤坏死因子 a受体的半衰期， 包括但 不限于： 将肿瘤坏死因子 α受体与人 IgGl : Fc 片段相连接， 将肿瘤坏死因子 α受体与 PEG联接， 用 PEG-脂质体混合物包埋肿瘤坏死因子 α受体。优选的， 所述的 "长效可溶性肿瘤坏死因子 ci受体是 "长效人重组可溶性肿瘤坏死因子 α受体" 。
本发明的目的是提供重组可溶性肿瘤坏死因子 α受体 (HusTNFR)基因新的 药用用途， 尤其是重组可溶性肿瘤坏死因子 α受体 (HusTNFR)基因或修饰 HusTNF 蛋白质后形成的长效人重组可溶性肿瘤坏死因子 a受体 (LHusTNFR) 的预防慢性肝病重症肝损伤和治疗早期慢性肝病重症肝损伤的新用途。
本发明的进一步目的在于提供一种可较常规可溶性 TNF α受体进一步大 幅降低慢性肝病重症肝损伤死亡率的药物。 主要是通过延长可溶性 TNF a受体 药物的半衰期， 延长药物的作用时间， 以提高临床治疗疗效。
本发明采用长效人重组可溶性肿瘤坏死因子 α受体通过慢性肝病重症肝 损伤的动物模型， 对大鼠慢性肝病重症肝损伤进行干预， 结果显示， 干预组与 模型组的病死率分别为 0和 60-90%。
上述肿瘤坏死因子 (TNF)为肿瘤坏死因子 α与相应细胞膜受体结合的重组 长效可溶性蛋白，包括长效人重组可溶性 I型肿瘤坏死因子 a受体 (LHusTNFRI) 及长效人重组可溶性 II型肿瘤坏死因子 α受体 (LHusTNFRII),其半衰期较普通 人重组可溶性 I型 (HusTNFRI)及 II型肿瘤坏死因子 α受体 (HusTNFRII) 延长 10 倍以上。其存在形式是 (l)HusTNFRI或 HusTNFRII羧基末端连接人免疫球蛋白 IgG: Fc片段，或 (2)在氨基或羧基末端连接 PEG，或 (3)PEG-脂质体包裹 HusTNFRI 或 HusTNFRII。 所述的 LHusTNFRI及 LHusTNFRII, 其预防及治疗慢性肝病 重症肝损伤的疗效明显好于 HusTNFRI或 HusTNFRII。
所述的慢性肝病重症肝损伤的动物模型采用 1 ) 异种血清白蛋白致敏后反 复尾静脉注射 6周诱导慢性肝损伤和肝纤维化后，给予 D-氨基半乳糖与内毒素 皮内注射攻击方法造模； 2) 反复小剂量四氯化碳皮下注射 8 周造成肝肝硬化 模型后， 给予 D-氨基半乳糖与内毒素皮内注射攻击方法造模； 3 ) 给予低浓度 乙醇喂养大鼠 12周后造成酒精性肝慢性损伤及肝纤维化模型后， 然后给予 D- 氨基半乳糖与内毒素皮内注射攻击造模； 4) 给予高脂饮食喂养大鼠 12周后造 成非酒精性脂肪肝病模型， 然后给予 D-氨基半乳糖与内毒素皮内注射攻击造 模。 上述四类慢加急性动物其因肝衰竭而导致的死亡率在 60-100%之间。 上述 模型在 D-氨基半乳糖与内毒素打击后 0-2小时处于 SIRS阶段 （TNF a， IL-6 及 IL-10明显升高） ， 2-24小时为非 SIRS阶段 （TNF a , IL-6及 IL-10回落至 发病前水平） 。
为了比较长效人重组可溶性肿瘤坏死因子 α受体对不同病程阶段的慢性 肝病重症肝损伤的疗效研究， 本发明制作了 ConA多次剌激诱导 CARS后给予 GalN/LPS打击的慢性肝病重症肝损伤动物模型。
本发明的长效人重组可溶性肿瘤坏死因子 α受体通过
a. 人 I型 TNF α受体 (TNFR)与人 IgGl： Fc片段融合基因表达的重组蛋白， 或
b. 人 II型 TNF a受体与人 IgGl : Fc片段融合基因表达的重组蛋白， 或 c 人 I型 TNF a受体蛋白氨基端与 PEG联接， 或
d. 人 I型 TNF a受体蛋白羧基端与 PEG联接， 或
e. 人 II型 TNF a受体蛋白氨基端与 PEG联接， 或
f. 人 II型 TNF a受体蛋白羧基端与 PEG联接， 或
h. PEG-脂质体混合物包埋人 I型 TNF a受体蛋白， 或
i. PEG-脂质体混合物包埋人 II型 TNF a受体蛋白。 制备长效人重组可溶性肿瘤坏死因子 α受体 (LHusTNFR)，用于预防和治疗 1 ) 异种血清白蛋白反复注射诱导慢性肝损伤和肝纤维化后给予 D-氨基半乳糖 与内毒素皮内注射的慢性肝病重症肝损伤大鼠模型 2 ) 反复小剂量四氯化碳皮 下注射 8周造成肝肝硬化模型后给予 D-氨基半乳糖与内毒素皮内注射的慢性肝 病重症肝损伤大鼠模型； 3 ) 给予低浓度乙醇喂养大鼠 12周后造成酒精性肝慢 性损伤及肝纤维化模型，然后给予 D-氨基半乳糖与内毒素皮内注射的慢性肝病 重症肝损伤大鼠模型； 4 ) 给予高脂饮食喂养大鼠 12周后造成非酒精性脂肪肝 病模型，然后给予 D-氨基半乳糖与内毒素皮内注射的慢性肝病重症肝损伤大鼠 模型。 结果显示， 长半衰期的 I型或 /和 II型长效人重组可溶性肿瘤坏死因子 α 受体， 具有良好预防和治疗模型动物慢性肝病重症肝损伤的疗效， 明显降低模 型动物死亡率。 表明长效人重组可溶性肿瘤坏死因子 α受体对慢性肝病重症肝 损伤肝具有预防和治疗用途。 可进一步制备大幅降低慢性肝病重症肝损伤死亡 率的药物。 本发明中， （l)SEQ ID NO: 1的 I型 LHusTNFR基因的制备方法， 包括将 编码 I型 sTNFR (人)膜外氨基酸 (g卩 SEQ ID NO: 1的 1-171位）的基因与编码人 免疫球蛋白 Y 1链 Fc段 (IgGl : Fc)氨基酸（S卩 SEQ ID NO: 1的 172-403)优先与 相关质粒重组， DNA限制性内切酶酶切鉴定筛选携带 I型 TNFR-IgGl : Fc融 合片段的阳性克隆，核苷酸序列分析验证基因是否正确。在编码 I型 sTNFR (人) 膜外氨基酸 (即 SEQ ID NO: 1的 1-171位)的基因与人免疫球蛋白 Y 1链 Fc段 (IgGl : Fc)氨基酸（g卩 SEQ ID NO: 1的 172-403)之间可加入 0-6个编码氨基酸 G(n)S的核苷酸作为链接段。
(2) SEQ ID NO: 2的 II型 LHusTNFR基因的制备方法， 包括将编码 II型 sTNFR (人)膜外氨基酸 (即 SEQ ID NO: 2的 1-235位)的基因与编码人免疫球蛋 白 Y 1链 Fc段 (IgGl : Fc)氨基酸（S卩 SEQ ID NO: 2的 236-467位)与相关质粒重 组， DNA限制性内切酶酶切鉴定筛选携带 II型 TNFR-IgGl : Fc融合片段的阳 性克隆， 核苷酸序列分析验证基因是否正确。
将上述的 I型及 II型 TNFR-IgGl : Fc的 cDNA片段与表达载体重组， 形 成重组表达质粒。 本发明不限于特定的表达质粒。 在一优选实施方案中， 本发 明使用真核表达载体。
上述重组表达载体可按常规方法导入适宜宿主细胞。 本发明不局限于任何 特定的宿主细胞， 只要它能够表达所述重组表达载体。 在一优选实施方案中， 本发明使用哺乳动物 CHO细胞等。
以上技术方案中所有基本分子生物学操作均参照 <分子克隆实验指南 >(J. 萨姆布鲁克， D.W.拉塞尔著， 纽约： 冷泉港实验室出版社)。
(3)将编码 I型 sTNFR (人)膜外氨基酸 (S卩 SEQ ID NO: 1 的 1-171 位）的 cDNA与表达载体重组， 形成重组表达质粒 (SEQ ID NO: 3)。
本发明不限于特定的表达质粒。 在一优选实施方案中， 本发明使用真核表 达载体。 上述重组表达载体可按常规方法导入适宜宿主细胞。 本发明不局限于任何 特定的宿主细胞， 只要它能够表达所述重组表达载体。 在一优选实施方案中， 本发明使用哺乳动物 CHO细胞等。
将分子量 (molecular weight, MW)2,0000以上的有活性的 mPEG联接至 I 型 HusTNFR的氨基端或羧基端， 合成长效 I型 HusTNFR。 本发明不限于特定 的 mPEG。 在一优选实施方案中， 本发明使用 mPEG2-ALD，MW 4,0000 (Shearwater corporation, New Jersey, USA) 与 I型 HusTNFR的氨基端联接。使用 mPEG2-NHS easter,MW 4,0000 (Shearwater corporation, New Jersey, USA) 与 I型 HusTNFR的羧基端联接。
反应式： mPEG-CHO + NH2 + NaCNBH3 ~ ^ mPEG-CH2-NH
反应条件为 PH 7.9 时间为 12小时。
(4)将编码 II型 sTNFR (人)膜外氨基酸 1-235 的 cDNA与表达载体重组， 形成重组表达质粒 (SEQ ID NO: 4)。
本发明不限于特定的表达质粒。 在一优选实施方案中， 本发明使用真核表 达载体。
上述重组表达载体可按常规方法导入适宜宿主细胞。 本发明不局限于任何 特定的宿主细胞， 只要它能够表达所述重组表达载体。 在一优选实施方案中， 本发明使用哺乳动物 CHO细胞等。
将分子量 (molecular weight, MW)2,0000以上的有活性的 mPEG联接至 I 型 HusTNFR的氨基端或羧基端， 合成长效 II型 HusTNFR。 本发明不限于特定 的 mPEG。 在一优选实施方案中， 本发明使用 mPEG2-ALD，MW 4,0000 (Shearwater corporation, New Jersey, USA) 与 II型 HusTNFR的氨基端联接。 使 用 mPEG2-NHS easter,MW 4,0000 (Shearwater corporation, New Jersey, USA) 与 II型 HusTNFR的羧基端联接。
反应式： mPEG-CHO + NH2 + NaCNBH3 ~ ^ mPEG-CH2-NH
反应条件为 PH 7.9 时间为 12小时。
(5)将长循环脂质体-聚乙二醇衍生磷脂分别包封 I 型 HusTNFR 或 II 型 HusTNFR, 合成长效 I型及 II型 HusTNFR。
在三乙胺催化的条件下， 二油酰磷脂酰乙醇胺 (DOPEX分子量 744.04，美国 Aanti Polar Lipids) 与 NHS-PEG354o- AL (Nhydroxysulfosuccinimide -polyoxyethylene(MW3540)-maleimide) (分子量 3477， 美国 Aanti Polar Lipids)及 TEA 以摩尔比 1 : 1 :0.1在 25 °C条件下，反应 6小时，经离心、 蒸干及真空干燥得 到 DOPE-PEG-MAL (分子量为 4108.04)
将上述制备的 DOPE-PEG-MAL 与 EPC(L- a—Phosphatidylcholine, MW 760.09)、 cholesterol(MW 386.67)及 mPEG—2000-DOPE(MW 得到 2801 ·51)( 美 国 Aanti Polar Lipids)在氯仿反应体系中以摩尔比 200:20: 10: 1,40 V水 浴， 100-150r/min旋转蒸发。 在蒸干有机溶剂后加入 PBS(PH 7.4)，室温下充分水 化。 用 Mini Extruder反复挤压， 过 100nm滤膜 15次， 即得到长循环脂质体- 聚乙二醇衍生磷脂。
将溶于 PBS的 SEQ ID NO:3的 I型 HusTNFR或 SEQ ID NO: 4的 II型 HusTNF 加入上述长循环脂质体-聚乙二醇衍生磷脂的 PBS溶液中， 4 旋涡 振荡 30min， 通过 CL-4B(Pharmacia， USA) 去除游离的 I型 HusTNFR或 II型 HusTNFR, 得到长效 I型及 II型 HusTNFR。 经测定， I型和 II型 HusTNFR-IgG:Fc融合蛋白在体内的半衰期为 40-100 小时左右； PEG-HusTNFR及长循环脂质体 -聚乙二醇衍生磷脂包埋的 HusTNFR 在体内的半衰期为 3-5.5天。
因此可见， 上述方法制备的不同形式的 LHusTNFR, 其半衰期均大于 12 小时 (为 12-140小时)， 达到长效的要求。 而一般的可溶性肿瘤坏死因子 a受体 的半衰期仅为 50分钟至 2小时。
本发明采用基因工程方法对 LHusTNFR进行生产，所得产品具有高效防治 慢性肝病重症肝损伤的功能。 与常规 HusTNFR性质比较表明， LHusTNFR半 衰期明显延长，预防及治疗慢性肝病重症肝损伤， 降低死亡率的疗效明显提高。 本发明揭示长效可溶性肿瘤坏死因子 α受体可有效预防、 治疗慢性肝病重 症肝损伤模型动物， 明显降低疾病死亡率。 即各种类型的长效可溶性肿瘤坏死 因子 α 受体可将不同发病机制的慢性肝病重症肝损伤动物的死亡率由 60%-90%降至 0。 尽管在已报道的各种文献中， 某些化合物、 生物技术药物或 天然药物可通过各种机制在一定程度降低慢性肝病重症肝损伤的死亡率 （由 40-80%降至 30-50% )，但在本领域从未有药物将慢性肝病重症肝损伤 60%-90% 的高死亡率降至零死亡的报道。
本发明揭示长效可溶性肿瘤坏死因子 α受体明显降低疾病模型动物死亡 率的机制在于阻止肝细胞凋亡、 保护肝细胞存活及促进肝细胞再生。 我们研究 进一步阐明发挥长效可溶性肿瘤坏死因子 α受体防治慢性肝病重症肝损伤需 要以下两种机制共同的作用： 1 ) 通过阻断 TNF ci信号传导以降低肝脏凋亡信 号产生， 发挥抗肝细胞急性坏死的作用； 2 ) 通过增加 IL-22及 IL-22受体的表 达， 使肝细胞增加存活和再生的能力。 （已有文献报道 IL-22有促进肝细胞再 生和增加肝细胞存活的功能） 。
因此， 本发明阐述了有效防治慢性肝病重症肝损伤除了需要阻断急性肝细 胞凋亡 /坏死外， 还需要同时促进肝细胞存活及再生。 但对于急性重症肝损伤只 需要阻断急性肝细胞凋亡 /坏死即可有效治疗。本发明揭示经长效可溶性肿瘤坏 死因子 α受体预防 /治疗的慢性肝病重症肝损伤动物，其肝细胞凋亡严重程度明 显减轻和具保护肝细胞、 促进肝细胞再生的 IL-22信号显著同时升高， 从而发 挥了有效治疗该疾病并显著降低死亡率的作用。
本发明还创新性地发现长效可溶性肿瘤坏死因子 α受体对慢性肝病重症 肝损伤动物不同疾病阶段的不同疗效。 可明显降低早期疾病的动物死亡率， 但 对疾病中、 晚期的死亡率无降低作用。
慢性肝病重症肝损伤根据疾病免疫状态的演变分为三个阶段：早期-全身性 炎症反应综合症 ( systemic inflammatory response syndrome, SIRS ) 阶段、 中期 -非 SIRS阶段及晚期-代偿性抑炎反应综合症 （complimentery anti-inflammatory response syndrome, CARS ) 阶段。 发明人研究发现长效可溶性肿瘤坏死因子 α 受体（包括 TNFRII-IgG: Fc )可明显降低慢性肝病重症肝损伤早期存在全身性 炎症反应综合症 ( systemic inflammatory response syndrome , SIRS )的大鼠。 SIRS 在免疫学上表现为外周血 TNF a、 IL-6 的升高 （即促炎因子升高） 或血 TNF d、 IL-6及 IL- 10的共同上升 （促炎、 抑炎因子同时升高） ， 出现在慢性肝病 重症肝损伤的早期阶段。 发明人在动物实验中也发现长效可溶性肿瘤坏死因子 a受体（包括 TNFRII-IgG: Fc )无法降低存在 CARS的慢性肝病重症肝损伤大 鼠的死亡率。这与临床上报道的曾有 TNFRII-IgG： Fc( Entarcept，商品名： Enbrel ) 治疗难治性急性酒精性肝炎 （慢性肝病重症肝损伤的一种类型， 酒精性肝病为 基础疾病） 患者后死亡率无明显降低的结果相似。 在上述文献中， 研究入组为 晚期患者或经糖皮质激素治疗无效的患者。 该部分患者往往处于 CARS阶段， 免疫系统表现为单核细胞 HLA-DR表达明显下降，外周血抑炎因子 IL-10升高， 促炎因子 TNF a、 IFN Y不升高等免疫抑制特征。 在本发明揭示的四类慢性肝病重症肝损伤动物模型中， SIRS出现在急性肝 脏打击诱发重症肝损伤后的 0-2小时。 对于重症肝损伤发生 0-2小时期间给予 长效可溶性肿瘤坏死因子 a受体治疗， 可将死亡率有 60-90%降至 0. 而对于重 症肝损伤发生 2小时后、 已度过 SIRS阶段的动物给予长效可溶性肿瘤坏死因 子 ci受体治疗无明显降低死亡率的疗效。 因此， 通过本发明鉴别出长效可溶性 肿瘤坏死因子 α受体对早期慢性肝病重症肝损伤 （存在 SIRS ) 及中晚期 （非 SIRS或 CARS阶段） 的不同治疗效果， 解释了为何对晚期、 处于 CARS阶段 的慢性肝病重症肝损伤动物疗效不佳的原因。 同时也表明长效可溶性肿瘤坏死 因子 ci受体对存在 SIRS 阶段的早期慢性肝病重症肝损伤动物有明显疗效， 可 大幅降低其死亡率。 本发明又发现预防使用长效可溶性肿瘤坏死因子 α受体可 通过阻断大量急性肝细胞坏死及阻止 SIRS 发生， 以预防慢性肝病重症肝损伤 的发生。
本发明中选用的四种慢性肝病重症肝损伤动物模型分别代表， 但不限于乙 醇 （酒精） 、 高脂饮食、 免疫损伤或化学毒物导致慢性肝损、 肝纤维化或肝硬 化的模型动物肝脏上遭受内毒素、 病毒核酸等打击后出现急性肝细胞坏死、 肝 脏炎症、 肝功能快速恶化及短期高死亡率的慢性肝病重症肝损伤特征。 本发明还提供了一种治疗肝细胞坏死或慢性肝病重症肝损伤的药物组合 物，它含有上述的长效可溶性肿瘤坏死因子 α受体， 以及药学上可接受的载体。 通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中， 其中 pH通常约为 5-8， 较佳地 pH约为 6-8， 尽管 pH值可随被配制物质的性质 以及待治疗的病症而有所变化。 配制好的药物组合物可以通过常规途径进行给 药， 其中包括 (但并不限于)： 腹膜内、 静脉内、 或局部给药。
本发明的药物组合物可直接用于肝细胞坏死或慢性肝病重症肝损伤的治 疗。 此外， 还可同时使用其它相关的治疗剂。 这类治疗剂包括但不限于： α - 胸腺肽， 人肝细胞生长因子 （huHGF ) 、 还原型谷胱甘肽、 苦参碱、 人体血清 白蛋白、 多烯磷脂酸胆碱、 各种辅酶及维生素、 血必净 （中成药） 。
本发明的药物组合物含有安全有效量的本发明上述的长效可溶性肿瘤坏 死因子 α受体以及药学上可接受的载体或赋形剂。这类载体包括 (但并不限于): 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组合。 药物制剂应与给药方式 相匹配。 本发明的药物组合物可以被制成针剂形式， 例如用生理盐水或含有葡 萄糖和其它辅剂的水溶液通过常规方法进行制备。 药物组合物如针剂、 溶液宜 在无菌条件下制造。 活性成分的给药量是治疗有效量， 例如每天约 0.1微克 /千 克体重-约 5毫克 /千克体重。
使用药物组合物时， 是将安全有效量的本发明的长效可溶性肿瘤坏死因子 α受体施用于哺乳动物， 其中该安全有效量通常至少约 1微克 /千克体重， 而且 在大多数情况下不超过约 8毫克 /千克体重， 较佳地该剂量是约 10微克 /千克体 重-约 1 毫克 /千克体重。 当然， 具体剂量还应考虑给药途径、 病人健康状况等 因素， 这些都是熟练医师技能范围之内的。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法， 通常按照常规条件如 <分子克隆实验指南 >(J. 萨姆布鲁克， D.W.拉塞尔著， 纽约： 冷泉港实验室出版社)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1
四种慢性肝病重症肝损伤动物模型方法
模型一、异种血清白蛋白致敏及反复注射诱导慢性肝损伤 /肝纤维化后给予 D-氨基半乳糖 （GaIN ) 与内毒素 （LPS ) 注射制作的慢性肝病重症肝损伤大鼠 模型， 分三阶段完成；
a, 异种白蛋白致敏： Wistar大鼠， 雌性 体重 120- 150g。 将人血清白蛋白 用生理盐水稀释后与等量的不完全福氏佐剂乳化， 每只大鼠皮下多点注射， 每 次注射 0. 5ml (内含白蛋白 4mg)， 共 4次。 2次间隔 14天， 第 3、 4次问隔 10 天。 末次免疫后 10天取血测抗体， 取抗体阳性大鼠进行后续实验；
慢性肝损及肝纤维化： 尾静脉注射白蛋白， 每周 2次 第 1 次 2. 5mg /只， 以后每次攻击增加 0. 5mg， 直至 4. 5mg。 维持此剂量至六周。 制作免 疫性肝损伤及肝纤维化模型。该阶段的动物肝脏病理显示 (1)网状纤维及胶原纤 维于汇管区增生， 且向外延伸。 同时， 于中央静脉周围增生， 弗沿肝窦散在分 布， 互相连结. 纤维结缔组织包绕小叶， 部分动物肝脏有假小叶形成； （2)汇 管区及中央静脉周围有淋巴细胞、 单核细胞及嗜酸性细胞浸润， 可见肝小叶动 脉有纤维素样死， 中央静脉周围炎细胞浸润。 免疫荧光示汇管、 肝窦、 血管壁 均有免疫复合物沉着。 电镜所见星状细胞向肌成纤维细胞转化，分布于汇管区。 其周围有大量胶原沉积。 沉积的胶原形成较宽的分隔。
D-GaIN/LPS急性打击上述肝纤维化大鼠： 每周 2次， 共 6周尾静脉注 射白蛋白且造成肝纤维化的大鼠给予 D-GaIN 400mg/kg加 LPS ΙΟΟ μ g/kg腹腔 注射。 24小时模型组大鼠死亡率为 80%-90%， 平均存活时间为 16小时左右。 D-GaIN加 LPS注射后 4小时谷丙转氨酶开始升高， 峰值为 8-12小时。 总胆红 素注射后 4小时升高一直上升至动物死亡。 血浆 TNF a水平在 D-GaIN加 LPS 注射后 0.5小时后明显升高， 1小时到达高峰， 2小时下降， 4小时到达正常水 平。 血浆 IL-6在 0.5小时后明显升高， 2小时到达高峰， 3小时下降， 8小时恢 复至正常水平。 血浆 IL-10水平时后明显升高， 1小时到达高峰， 2小时下降， 4 小时到达正常水平。 肝脏大体标本显示肝脏严重充血、 肿大； 肝脏病理显示 4 小时见再生结节内灶状或片状坏死伴白细胞反应， 凋亡小体散在， 汇管区轻 度水肿。 8 小时后结节内肝细胞坏死灶增多或片状融合， 其中见红细胞增多、 间质细胞及胆管上皮增生。 12小时大多数再生结节内肝细胞呈大块或亚大块坏 死。 血窦扩张， 红细胞填充， kupffer细胞活化、 胞质肥大。 残存肝细胞小泡性 脂变或空泡变性， 纤维间隔完整保留。 电镜 4小时肝细胞见少量脂滴， 线粒体 嵴断裂、 消失； 肝细胞呈现微绒毛减少， 核皱缩形状不规则等早期凋亡表现。 8小时凋亡细胞明显增多并见脱落的凋亡小体。 Kupffer细胞活化伸出的足突与 肝细胞接触或吞噬凋亡小体。 模型二、反复小剂量四氯化碳皮下注射 8周造成肝硬化模型后给予 D-氨基半 乳糖与内毒素注射大鼠方法制作慢性肝病重症肝损伤模型， 分两个阶段完 成：
a. 四氯化碳诱导肝硬化大鼠： 以文献报道的通用方法反复小剂量四氯化 碳皮下注射 8周， 肝脏病理确认存在肝硬化肝脏特有的假小叶结构。
b. D-GaIN/LPS急性打击上述肝硬化大鼠： 给予 D-GaIN 400mg/kg加 LPS
100 w g/kg腹腔注射。 24小时模型组大鼠死亡率为 80%-90%， 平均存活时间为 16小时左右。 D-GaIN加 LPS注射后 4小时谷丙转氨酶开始升高， 峰值为 8-12 小时。 总胆红素注射后 4小时升高一直上升至动物死亡。 血浆 TNF a水平在 8 小时后明显升高， IL-10 水平随时间延长而升高。 病理显示肝脏存在大块及亚 大块坏死坏死带，同时伴有肝硬化假小叶的存在。血窦扩张，红细胞填充， kupffer 细胞活化、胞质肥大。残存肝细胞小泡性脂变或空泡变性， 纤维间隔完整保留。 电镜 4小时肝细胞见少量脂滴， 线粒体嵴断裂、 消失； 肝细胞呈现微绒毛减少， 核皱缩形状不规则等早期凋亡表现。 8 小时凋亡细胞明显增多并见脱落的凋亡 小体。 Kupffer细胞活化伸出的足突与肝细胞接触或吞噬凋亡小体。 模型三、 给予低浓度乙醇喂养大鼠 12 周后造成酒精性肝慢性损伤及肝纤 维化模型，然后给予 D-氨基半乳糖与内毒素皮内注射制作慢性肝病重症肝损伤 模型， 分两阶段完成；
a. 低浓度乙醇诱导酒精性肝病大鼠： 10%乙醇混合高脂饮食喂养大鼠 3-4 个月， 病理证实肝脂肪变性及肝纤维化形成
b. D-GaIN/LPS急性打击上述肝硬化大鼠： 给予 D-GaIN 400mg/kg加 LPS
100 w g/kg腹腔注射。 24小时模型组大鼠死亡率为 80%-90%， 平均存活时间为 16小时左右。 D-GaIN加 LPS注射后 4小时谷丙转氨酶开始升高， 峰值为 8-12 小时。 总胆红素注射后 4小时升高一直上升至动物死亡。 血浆 TNF a水平在 8 小时后明显升高， IL-10 水平随时间延长而升高。 病理显示肝脏存在大块及亚 大块坏死， 血窦扩张， 红细胞填充， kupffer细胞活化、 胞质肥大。 残存肝细胞 小泡性脂变或空泡变性， 纤维间隔完整保留。 电镜 4小时肝细胞见少量脂滴， 线粒体嵴断裂、 消失； 肝细胞呈现微绒毛减少， 核皱缩形状不规则等早期凋亡 表现。 8小时凋亡细胞明显增多并见脱落的凋亡小体。 Kupffer细胞活化伸出的 足突与肝细胞接触或吞噬凋亡小体 模型四、 给予高脂饮食喂养大鼠 12 周后造成非酒精性脂肪肝病模型， 然 后给予 D-氨基半乳糖与内毒素注射制作慢性肝病重症肝损伤模型。
a. 高脂、 高糖饮食诱导非酒精性脂肪性肝病大鼠： 文献报道通用的高 脂混合高糖饮食喂养大鼠 3-4个月后， 病理证实肝脂肪变性、 脂肪性肝炎及部 分肝纤维化形成
b. D-GaIN/LPS 急性打击上述肝硬化大鼠： 给予 D-GaIN 400mg/kg加 LPS 100 μ g/kg腹腔注射。 24小时模型组大鼠死亡率为 80%-90%， 平均存活时 间为 16小时左右。 D-GaIN加 LPS注射后 4小时谷丙转氨酶开始升高， 峰值为 8-12小时。 总胆红素注射后 4小时升高一直上升至动物死亡。 血浆 TNF a水平 在 8 小时后明显升高， IL-10水平随时间延长而升高。 病理显示肝脏存在大块 及亚大块坏死， 血窦扩张， 红细胞填充， kupffer细胞活化、 胞质肥大。 残存肝 细胞小泡性脂变或空泡变性， 纤维间隔完整保留。 电镜 4小时肝细胞见少量脂 滴， 线粒体嵴断裂、 消失； 肝细胞呈现微绒毛减少， 核皱缩形状不规则等早期 凋亡表现。 8小时凋亡细胞明显增多并见脱落的凋亡小体。 Kupffer细胞活化伸 出的足突与肝细胞接触或吞噬凋亡小体。 实施例 2
ConA多次剌激诱导 CARS后给予 GalN/LPS打击的慢性肝病重症肝损伤 小鼠模型
小剂量 ConA 每 48小时一次皮下注射小鼠， 共 3-5次。 检测外周血 IL-10 明显升高， TNF a及 IFN Y无升高时即为 CARS模型。 给予大剂量的 GalN/LPS 打击后 24小时， 动物死亡率为 15-20%。 肝脏病理显示存在范围较小的亚大块 坏死， 同时存在局灶及片状坏死。 实施例 3
NAC ( N-乙酰半胱氨酸）防治药物源性急性重症肝损伤和慢性肝病重症肝 损伤的不同疗效比较
使用文献通用剂量的 Acetaminophen (醋氨酚） 制作药物源性急性重症肝 损伤大鼠模型， 其短期死亡率为 40%左右。 按照实施例一模型二的 a步骤制作 四氯化碳诱导肝硬化大鼠模型， 并给予相同剂量的醋氨酚制作慢性肝病重症肝 损伤动物模型， 短期死亡率为 40-60%。在给于醋氨酚前 6小时静脉注射给予健 康大鼠 NAC以制作 NAC预防急性重症肝损伤模型。在给于醋氨酚前 6小时静 脉注射给予肝硬化大鼠相同剂量的 NAC 静脉注射以制作预防慢性肝病重症肝 损伤。在醋氨酚给药后 0.5小时分别给予健康及肝硬化大鼠相同剂量 NAC制作 NAC治疗急性重症肝损伤和慢性肝病重症肝损伤模型。
对于急性重症肝损伤模型， NAC预防组和治疗组可使模型组死亡率由 40% 降至 10%-15%。 而 NAC预防和治疗醋氨酚诱导的慢性肝病重症肝损伤动物的 死亡率为 40-60%， 与对照组无区别。 表明 NAC无法有效降低醋氨酚诱导的慢 性肝病重症肝损伤动物死亡率。
结果表明， NAC (N-谷氨酰氨） 对健康肝脏发生的急性重症肝损伤生存有 效， 但对肝硬化基础上的慢性肝病重症肝损伤动物无效。 实施例 4
以 II型 HusTNFR-IgGl: Fc为代表的长效人重组可溶性 II型肿瘤坏死因 子 (TNF) α受体 (II 型 LHusTNFR)皮下注射给药途径预防大鼠慢性肝病重症肝 损伤的实验
上述四类慢性肝病重症肝损伤模型给予 D-GalN/LPS急性打击前皮下注射
II型 LHusTNFR (方法 (2)制备的 II型 LHusTNFR-IgGl : Fc为预防药物）， 剂量 5-50mg/kg, 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 该处理组命 名为长效 II 型受体皮下注射预防组。 长效 II 型可溶性受体 II 型 LHusTNFR-IgGl : Fc不限于 D-GalN/LPS急性打击前特定时间， 只要满足提前 于 D-GalN/LPS急性打击前给药即可， 时间优选 D-GalN/LPS急性打击前 36小 时至 0小时。 在一优选实施方案中， 本发明使用 D-GalN/LPS急性打击前 8小 时。 另设常规 HusTNFR皮下注射大鼠， 剂量 12.5mg/kg， 为常规 II型受体预防 组； 对照组为皮下注射相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的免 疫性肝纤维化、 四氯化碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱导 的非酒精性肝病大鼠基础上， 长效 II 型 TNF ci受体皮下注射预防组在 D-GalN/LPS注射后 24小时的大鼠死亡率为 0， 而常规 II型 TNF a受体预防组 的死亡率为 66%，未给予长效或常规 II型 TNF a受体处理的慢性肝病重症肝损 伤模型对照组 （简称 "对照组" ） 的死亡率为 90%。 大体病理显示长效 II型受 体预防组肝脏原有慢性肝损、 肝纤维化及肝硬化特征仍保留， 但肝实质仅轻度 充血肿胀， 存在少量灶状坏死， 无大块或亚大块坏死。 肝组织损伤评分肝脏损 伤病理评分 （injury score) (依据鼠肝脏组织切片 HE染色中所观察到的肝脏 组织病变程度、 病变重要性及受损肝脏组织面积进行半定量评分） 为 2 ± 1分， 明显低于对照组的 5 ± 2分 （p< 0.01 ) 。 以模型一为例， 肝脏损伤病理评分见 图 1， 左列为慢性肝纤维化肝脏， 中列为长效 II型受体皮下注射预防组， 右列 为对照组。
病理 TUNEL染色显示对照组显微镜下凋亡小体 40-60个 /高倍视野，明显 高于长效 II型受体皮下注射预防组为 5-8个 /高倍视野， p< 0.01.肝脏 caspase3 检测， 预防组 CaSpaSe3 活性低于对照组 3-4 倍。 外周血浆促炎因子 ( pro-inflammatory cytokines ) TNF ct ， IL-6 及抑炎因子 ( anti-inflammatory cytokine) IL-10的峰值较慢性肝病重症肝损伤对照组明显下降； 肝脏 TNF α， IL-6峰值较慢性肝病重症肝损伤对照组明显下降 （见图 2 ) 。 预防组肝脏的 NF- κ Β水平较对照组下降 30-50%。 肝组织 IL-22及 IL-22 受体表达升高 2-5倍。 上述结果表明长效 II型受体皮下注射预防组 1 ) 通过抑 制肝实质细胞的凋亡降低肝细胞大量坏死， 减缓肝脏炎症； 2 ) 促进肝细胞存 活及再生； 3 ) 抑制慢性肝病重症肝损伤肝衰竭动物早期的全身炎症反应综合 症 ( systemic inflammatory response syndrome, SIRS ) 从而大幅降低死亡率。 实施例 5
以 II型 HusTNFR-IgGl: Fc为代表的长效人重组可溶性 II型肿瘤坏死因 子 (TNF) ci受体 (II型 LHusTNFR)静脉注射给药途径预防大鼠慢性肝病重症肝 损伤的实验
上述四类慢性肝病重症肝损伤模型给予 D-GalN/LPS急性打击前经尾静脉 注射 II型 LHusTNFR (方法 (2)制备的 II型 LHusTNFR-IgGl： Fc为预防药物）， 剂量 5-50mg/kg， 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 该处理 组命名为长效 II 型受体静脉注射预防组。 长效 II 型可溶性受体 II 型 LHusTNFR-IgGl : Fc不限于 D-GaIN/LPS急性打击前特定时间， 只要满足提前 于 D-GaIN/LPS急性打击前给药即可， 时间优选 D-GaIN/LPS急性打击前 36小 时至 0小时。 在一优选实施方案中， 本发明使用 D-GaIN/LPS急性打击前 2小 时。 另设常规 HusTNFR尾静脉注射大鼠， 剂量 12.5mg/kg， 为常规 II型受体预 防组； 对照组为皮下注射相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的 免疫性肝纤维化、 四氯化碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱 导的非酒精性肝病大鼠基础上， 长效 Π型受体静脉注射预防组在 D-GaIN/LPS 注射后 24小时的大鼠死亡率为 0， 而常规 II型受体预防组的死亡率为 60%， 对照组为 90%。 大体病理显示长效 II型受体预防组肝脏原有慢性肝损、 肝纤维 化及肝硬化特征仍保留， 但肝实质仅轻度充血肿胀， 存在少量灶状坏死， 无大 块或亚大块坏死。 肝组织损伤评分肝脏损伤病理评分 （injury score ) (依据鼠 肝脏组织切片 HE染色中所观察到的肝脏组织病变程度、 病变重要性及受损肝 脏组织面积进行半定量评分）为 2 ± 1分，明显低于对照组的 5 ± 2分 ( p< 0.01 )。 以模型一为例， 肝脏损伤病理评分见图 3， 左列为慢性肝纤维化肝脏， 中列为 长效 II型受体皮下注射预防组， 右列为对照组。
病理 TUNEL染色显示对照组显微镜下凋亡小体 40-60个 /高倍视野，明显 高于长效 II型受体皮下注射预防组为 5-8个 /高倍视野， p< 0.01.肝脏 caspase3 检测， 预防组 CaSpaSe3 活性低于对照组 3-4 倍。 外周血浆促炎因子 ( pro-inflammatory cytokines ) TNF ct， IL-6 及抑炎因子 ( anti-inflammatory cytokine ) IL-10的峰值较慢性肝病重症肝损伤对照组明显下降； 肝脏 TNF α， IL-6峰值较慢性肝病重症肝损伤对照组明显下降 （见图 4 ) 。
预防组肝脏的 NF- κ Β水平较对照组下降 30-50%。 肝组织 IL-22及 IL-22 受体表达升高 2-5倍。 上述结果表明长效 II型受体皮下注射预防组 1 ) 通过抑 制肝实质细胞的凋亡降低肝细胞大量坏死， 减缓肝脏炎症； 2 ) 促进肝细胞存 活及再生； 3 ) 抑制慢性肝病重症肝损伤肝衰竭动物早期的全身炎症反应综合 症 ( systemic inflammatory response syndrome, SIRS ) 从而大幅降低死亡率。 实施例 6
以 II型 HusTNFR-IgGl: Fc为代表的长效人重组可溶性 II型肿瘤坏死因 子 (TNF) a受体 (Π型 LHusTNFR)皮 ^ ϋ给药途径治疗早期大鼠慢性肝病重 症肝损伤的实验
实施例 1中四类慢性肝病重症肝损伤模型及实施例 2中的 CARS状态下的 慢性肝病重症肝损伤模型在给予 D-GalN/LPS急性打击后 0， 30分钟， 1小时， 1.5小时， 2小时， 3小时， 4小时， 6小时及 8小时分别皮下注射 II型 LHusTNFR (方 法 (2)制备的 II型 LHusTNFR-IgGl : Fc为治疗药物）， 剂量范围为 5-50mg/kg， 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 实施例一中的四类慢性 肝病动物在 D-GaIN/LPS急性打击后 0， 30分钟， 1 小时， 1.5小时给予 II型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率为 0。 D-GaIN/LPS急性打击四类 慢性肝病动物后 2 小时， 3 小时， 4 小时， 6 小时及 8 小时给予 II 型 LHusTNFR-IgGl : Fc治疗的动物， 其死亡率为 50%， 90% , 90%, 90%。 慢性 肝病重症肝损伤生理盐水治疗对照组死亡率为 90%。 实施例二中的 CRAS模型 在在 D-GaIN/LPS急性打击后 0， 30分钟， 1， 1.5， 2， 3， 4， 6及 8小时给予 II型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率慢性肝病重症肝损伤生理盐 水治疗对照组死亡率无差别， 为 15-20%。 与上述实验表明皮下注射 II 型 LHusTNFR-IgGl : Fc对早期的慢性肝病重症肝损伤有有效的治疗作用， 而对非 SIRS及 CARS状态的慢性肝病重症肝损伤动物无效。大体病理显示 D-GaIN/LPS 急性打击实施例一中的四类慢性肝病动物后 0， 30分钟， 1小时， 1.5小时给予 II型 LHusTNFR-IgGl : Fc治疗的动物肝脏原有慢性肝损、 肝纤维化及肝硬化 特征仍保留， 但肝实质仅轻度充血肿胀， 存在少量灶状坏死， 无大块或亚大块 坏死。 肝组织损伤评分肝脏损伤病理评分 （injury score ) 为 2±1分， 明显低于 对照组的 5±2分 (p< 0.01 ) 。 外周血浆促炎因子 (pro-inflammatory cytokines ) TNF ， IL-6及抑炎因子 ( anti-inflammatory cytokine) IL-10 的峰值较慢性肝 病重症肝损伤对照组明显下降； 肝脏 TNF a , IL-6峰值较慢性肝病重症肝损伤 对照组明显下降。 肝脏的 NF- κ Β水平较对照组下降 30-50%。 肝组织 IL-22及 IL-22受体表达升高 2-5倍。 而 D-GaIN/LPS急性打击后 3小时， 4小时， 6小 时及 8 小时给药的大鼠， 肝脏存在大块或亚大块坏死。 病理肝组织损伤评分 ( injury score ) 与生理盐水处理的慢性肝病重症肝损伤对照组无明显差异。 血 浆促炎因子 TNF a， IL-6及抑炎因子 IL-10的峰值较慢性肝病重症肝损伤对照 组无明显改变。 上述研究显示皮下注射 II型 LHusTNFR-IgGl : Fc通过对早期 慢性肝病重症肝损伤的大量肝细胞坏死、 促进肝细胞存活及再生以及降低全身 炎症反应综合症 （SIRS ) 有抑制作用从而降低死亡率， 但对非早期慢性肝病重 症肝损伤大鼠无法有效抑制大量肝细胞坏死及阻止 SIRS,从而无法降低其死亡 率。 实施例 7
以 II型 HusTNFR-IgGl: Fc为代表的长效人重组可溶性 II型肿瘤坏死因 子 (TNF) ci受体 (II型 LHnsTNFR)M^|i给药途径治疗早期大鼠慢性肝病重 症肝损伤的实验
实施例 1中四类慢性肝病重症肝损伤模型及实施例 2中的 CARS状态下的 慢性肝病重症肝损伤模型在给予 D-GaIN/LPS急性打击后 0， 30分钟， 1小时， 1.5 小时， 2 小时， 3 小时， 4 小时， 6 小时及 8 小时分别尾静脉注射 II 型 LHusTNFR (方法 (2)制备的 II型 LHusTNFR-IgGl : Fc为治疗药物）， 剂量范围为 5-50mg/kg, 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 实施例一中 的四类慢性肝病动物在 D-GaIN/LPS急性打击后 0， 30分钟， 1小时， 1.5小时 给予 II型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率为 0。 D-GaIN/LPS急 性打击四类慢性肝病动物后 2小时， 3小时， 4小时， 6小时及 8小时给予 II 型 LHusTNFR-IgGl : Fc治疗的动物， 其死亡率为 50%， 90%, 90%, 90%。 慢 性肝病重症肝损伤生理盐水治疗对照组死亡率为 80-90%。实施例二中的 CRAS 模型在在 D-GaIN/LPS急性打击后 0， 30分钟， 1， 1.5， 2， 3， 4， 6及 8小时 给予 II型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率慢性肝病重症肝损伤生 理盐水治疗对照组死亡率无差别， 为 15-20%。 与上述实验表明尾静脉注射 II 型 LHusTNFR-IgGl : Fc对早期的慢性肝病重症肝损伤有有效的治疗作用， 而 对非 SIRS 及 CARS 状态的慢性肝病重症肝损伤动物无效。 大体病理显示 D-GaIN/LPS急性打击实施例一中的四类慢性肝病动物后 0， 30分钟， 1小时， 1.5小时给予 II型 LHusTNFR-IgGl : Fc治疗的动物肝脏原有慢性肝损、 肝纤维 化及肝硬化特征仍保留但肝实质仅轻度充血肿胀， 存在少量灶状坏死， 无大块 或亚大块坏死。 肝组织损伤评分肝脏损伤病理评分 （injury score ) 为 2±1分， 明显低于对照组的 5±2 分 （p< 0.01 ) 。 肝脏的 NF- κ Β 水平较对照组下降 30-50%。 肝组织 IL-22 及 IL-22 受体表达升高 2-5 倍。 外周血浆促炎因子 ( pro- inflammatory cytokines ) TNFa， IL-6 及抑炎因子 ( anti-inflammatory cytokine) IL-10 的峰值较慢性肝病重症肝损伤对照组明显下降 （p< 0.01 ) ； 肝脏 TNFa， IL-6峰值较慢性肝病重症肝损伤对照组明显下降（p< 0.01 ) 。 而 D-GaIN/LPS急性打击后 2小时， 3小时， 4小时， 6小时及 8小时给药的大鼠， 肝脏存在大块或亚大块坏死。 病理肝组织损伤评分 （injury score) 与生理盐水 处理的慢性肝病重症肝损伤对照组无明显差异。 血浆促炎因子 TNF a , IL-6及 抑炎因子 IL-10的峰值较慢性肝病重症肝损伤对照组无明显改变。 上述研究显 示静脉注射 II型 LHusTNFR-IgGl : Fc通过抑制早期慢性肝病重症肝损伤的大 量肝细胞坏死、 促进肝细胞存活及再生以及降低全身炎症反应综合症 （SIRS) 进而降低死亡率， 但对非早期慢性肝病重症肝损伤大鼠无法有效抑制大量肝细 胞坏死及阻止 SIRS的发生， 从而无法降低其死亡率。 实施例 8
以 I型 HusTNFR-IgGl: Fc为代表的长效人重组可溶性 II型肿瘤坏死因 子 (TNF) ci受体 (I 型 LHusTNFR)皮下注射给药途径预防大鼠慢性肝病重症肝 损伤的实验
上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急性打击前皮下注射 I型 LHusTNFR (方法（1)制备的 I型 LHusTNFR-IgGl： Fc 为预防药物）， 剂量 5-50mg/kg, 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 该处理组命 名为长效 I型受体皮下注射预防组。长效 I型可溶性受体 II型 LHusTNFR-IgGl : Fc不限于 D-GaIN/LPS急性打击前特定时间， 只要满足提前于 D-GaIN/LPS急 性打击前给药即可， 时间优选 D-GaIN/LPS急性打击前 36小时至 0小时。 在一 优选实施方案中， 本发明使用 D-GaIN/LPS急性打击前 8小时。 另设常规 I型 HusTNFR皮下注射大鼠， 剂量 12.5mg/kg， 为常规 I型受体预防组； 对照组为 皮下注射相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的免疫性肝纤维 化、 四氯化碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱导的非酒精性 肝病大鼠基础上， 长效 I型受体皮下注射预防组在 D-GaIN/LPS注射后 24小时 的大鼠死亡率为 0， 而常规 I型受体预防组的死亡率为 50%， 对照组为 90%。 大体病理显示长效 I型受体预防组肝脏原有慢性肝损、 肝纤维化及肝硬化特征 仍保留， 但肝实质仅轻度充血肿胀， 存在少量灶状坏死， 无大块或亚大块坏死。 肝组织损伤评分肝脏损伤病理评分 （injury score ) (依据鼠肝脏组织切片 HE 染色中所观察到的肝脏组织病变程度、 病变重要性及受损肝脏组织面积进行半 定量评分） 为 2 ± 1分， 明显低于对照组的 5 ±2分 （p< 0.01 ) 。
病理 TUNEL染色显示对照组显微镜下凋亡小体 40-70个 /高倍视野，明显 高于长效 I型受体皮下注射预防组为 4-9个 /高倍视野， p< 0.01.肝脏 caspase3 检测， 预防组 CaSpaSe3 活性低于对照组 3-4 倍。 外周血浆促炎因子 ( pro-inflammatory cytokines ) TNF ct ， IL-6 及抑炎因子 ( anti-inflammatory cytokine) IL-10的峰值较慢性肝病重症肝损伤对照组明显下降； 肝脏 TNF α， IL-6峰值较慢性肝病重症肝损伤对照组明显下降。 预防组肝脏的 NF- κ Β水平 较对照组下降 25-50%。同时肝脏 IL-22及 IL-22受体表达水平分别升高 2-6倍。 上述结果表明长效 I型受体皮下注射预防组 1 ) 通过抑制肝实质细胞的凋亡降 低肝细胞大量坏死， 减缓肝脏炎症； 2 ) 促进肝细胞存活及再生； 3 ) 抑制慢性 肝病重症肝损伤肝衰竭动物早期的全身炎症反应综合症（systemic inflammatory response syndrome, SIRS ) 从而大幅降低死亡率。 实施例 9
以 I型 HusTNFR-IgGl: Fc为代表的长效人重组可溶性 I型肿瘤坏死因子 (TNF) ci受体 (I 型 LHusTNFR)静脉注射给药途径预防大鼠慢性肝病重症肝损 伤的实验
上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急性打击前经尾静脉 注射 I型 LHusTNFR (方法（1)制备的 I型 LHusTNFR-IgGl： Fc为预防药物）， 剂 量 5-50mg/kg， 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 该处理组 命名为长效 I 型受体静脉注射预防组。 I 型 LHusTNFR-IgGl： Fc 不限于 D-GaIN/LPS急性打击前特定时间，只要满足提前于 D-GaIN/LPS急性打击前给 药即可， 时间优选 D-GaIN/LPS急性打击前 36小时至 0小时。 在一优选实施方 案中， 本发明使用 D-GaIN/LPS急性打击前 2小时。 另设常规 I 型 HusTNFR 尾静脉注射大鼠， 剂量 12.5mg/kg， 为常规 I型受体预防组； 对照组为皮下注射 相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的免疫性肝纤维化、 四氯化 碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱导的非酒精性肝病大鼠基 础上， 长效 I型受体静脉注射预防组在 D-GaIN/LPS注射后 24小时的大鼠死亡 率为 0， 而常规 I型受体预防组的死亡率为 60%， 对照组为 90%。 大体病理显 示长效 I型受体预防组肝脏原有慢性肝损、 肝纤维化及肝硬化特征仍保留， 但 肝实质仅轻度充血肿胀， 存在少量灶状坏死， 无大块或亚大块坏死。 肝组织损 伤评分肝脏损伤病理评分 （injury score) (依据鼠肝脏组织切片 HE染色中所 观察到的肝脏组织病变程度、 病变重要性及受损肝脏组织面积进行半定量评 分） 为 2 ± 1分， 明显低于对照组的 6 ±2分 （p< 0.01 ) 。
病理 TUNEL染色显示对照组显微镜下凋亡小体 30-60个 /高倍视野，明显 高于长效 I型受体皮下注射预防组为 4-7个 /高倍视野， p< 0.01.肝脏 caspase3 检测， 预防组 CaSpaSe3 活性低于对照组 3-4 倍。 外周血浆促炎因子 ( pro-inflammatory cytokines ) TNF ct ， IL-6 及抑炎因子 ( anti-inflammatory cytokine) IL-10的峰值较慢性肝病重症肝损伤对照组明显下降； 肝脏 TNF α， IL-6峰值较慢性肝病重症肝损伤对照组明显下降。 预防组肝脏的 NF- κ Β水平 较对照组下降 25-50%。同时肝脏 IL-22及 IL-22受体表达水平分别升高 2-5倍。 上述结果表明长效 I型受体皮下注射预防组 1 ) 通过抑制肝实质细胞的凋亡降 低肝细胞大量坏死， 减缓肝脏炎症； 2 ) 促进肝细胞存活及再生； 3 ) 抑制慢性 肝病重症肝损伤肝衰竭动物早期的全身炎症反应综合症（systemic inflammatory response syndrome, SIRS ) 从而大幅降低死亡率。 实施例 10
以 I型 HusTNFR-IgGl: Fc为代表的长效人重组可溶性 I型肿瘤坏死因子 (TNF) a受体 (I 型 LHusTNFR)皮^ 给药途径治疗早期大鼠慢性肝病重症 肝损伤的实验
实施例 1中四类慢性肝病重症肝损伤模型及实施例 2中的 CARS状态下的 慢性肝病重症肝损伤模型在给予 D-GalN/LPS急性打击后 0， 30分钟， 1小时， 1.5小时， 2小时， 3小时， 4小时， 6小时及 8小时分别皮下注射 II型 LHusTNFR (方 法 (1)制备的 I型 LHusTNFR-IgGl : Fc为治疗药物）， 剂量范围为 5-50mg/kg， 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 实施例一中的四类慢性 肝病动物在 D-GalN/LPS急性打击后 0， 30分钟， 1 小时， 1.5小时给予 II型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率为 0。 D-GalN/LPS急性打击四类 慢性肝病动物后 2 小时， 3 小时， 4 小时， 6 小时及 8 小时给予 I 型 LHusTNFR-IgGl : Fc治疗的动物， 其死亡率为 50%， 90%, 90%, 90%。 慢性 肝病重症肝损伤生理盐水治疗对照组死亡率为 90%。 实施例二中的 CRAS模型 在在 D-GalN/LPS急性打击后 0， 30分钟， 1， 1.5， 2， 3， 4， 6及 8小时给予 I型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率慢性肝病重症肝损伤生理盐 水治疗对照组死亡率无差别， 为 15-20%。 与上述实验表明皮下注射 I 型 LHusTNFR-IgGl : Fc对早期的慢性肝病重症肝损伤有有效的治疗作用， 而对非 SIRS及 CARS状态的慢性肝病重症肝损伤动物无效。大体病理显示 D-GalN/LPS 急性打击实施例一中的四类慢性肝病动物后 0， 30分钟， 1小时， 1.5小时给予 II型 LHusTNFR-IgGl : Fc治疗的动物肝脏原有慢性肝损、 肝纤维化及肝硬化 特征仍保留， 但肝实质仅轻度充血肿胀， 存在少量灶状坏死， 无大块或亚大块 坏死。 肝组织损伤评分肝脏损伤病理评分 （injury score ) 为 2±1分， 明显低于 对照组的 5±2分 (p< 0.01 ) 。 外周血浆促炎因子 (pro-inflammatory cytokines ) TNF ， IL-6及抑炎因子 （anti-inflammatory cytokine) IL-10 的峰值较慢性肝 病重症肝损伤对照组明显下降； 肝脏 TNF a , IL-6峰值较慢性肝病重症肝损伤 对照组明显下降。 肝脏的 NF- κ B水平较对照组下降 30-50%。 同时肝脏 IL-22 及 IL-22受体表达水平分别升高 2-5倍。 而 D-GalN/LPS急性打击后 3小时， 4 小时， 6小时及 8小时给药的大鼠， 肝脏存在大块或亚大块坏死。 病理肝组织 损伤评分 （injury score) 与生理盐水处理的慢性肝病重症肝损伤对照组无明显 差异。 血浆促炎因子 TNF a， IL-6及抑炎因子 IL-10的峰值较慢性肝病重症肝 损伤对照组无明显改变。上述研究显示皮下注射 I型 LHusTNFR-IgGl : Fc通过 对早期慢性肝病重症肝损伤的大量肝细胞坏死及全身炎症反应综合症 （SIRS) 有抑制作用从而降低死亡率， 但对非早期慢性肝病重症肝损伤大鼠无法有效抑 制大量肝细胞坏死及阻止 SIRS, 从而无法降低其死亡率。 实施例 11
以 I型 HusTNFR-IgGl : Fc为代表的长效人重组可溶性 I型肿瘤坏死因子 (TNF) ci受体 (I 型 LHnsTNFR)藍^ |i给药途径治疗早期大鼠慢性肝病重症 肝损伤的实验
实施例 1中四类慢性肝病重症肝损伤模型及实施例 2中的 CARS状态下的 慢性肝病重症肝损伤模型在给予 D-GalN/LPS急性打击后 0， 30分钟， 1小时， 1.5 小时， 2 小时， 3 小时， 4 小时， 6 小时及 8 小时分别尾静脉注射 II 型 LHusTNFR (方法（1)制备的 I型 LHusTNFR-IgGl : Fc为治疗药物）， 剂量范围为 5-50mg/kg, 在一优选实施方案中， 本发明使用剂量为 12.5mg/kg。 实施例一中 的四类慢性肝病动物在 D-GaIN/LPS急性打击后 0， 30分钟， 1小时， 1.5小时 给予 I型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率为 0。 D-GaIN/LPS急性 打击四类慢性肝病动物后 2小时， 3小时， 4小时， 6小时及 8小时给予 I型 LHusTNFR-IgGl : Fc治疗的动物， 其死亡率为 50%， 90%, 90%, 90%。 慢性 肝病重症肝损伤生理盐水治疗对照组死亡率为 90%。 实施例二中的 CRAS模型 在在 D-GaIN/LPS急性打击后 0， 30分钟， 1， 1.5， 2， 3， 4， 6及 8小时给予 I型 LHusTNFR-IgGl : Fc治疗， 其 24小时死亡率慢性肝病重症肝损伤生理盐 水治疗对照组死亡率无差别， 为 15-20%。 与上述实验表明尾静脉注射 I 型 LHusTNFR-IgGl : Fc对早期的慢性肝病重症肝损伤有有效的治疗作用， 而对非 SIRS及 CARS状态的慢性肝病重症肝损伤动物无效。大体病理显示 D-GaIN/LPS 急性打击实施例一中的四类慢性肝病动物后 0， 30分钟， 1小时， 1.5小时给予 I型 LHusTNFR-IgGl : Fc治疗的动物肝脏原有慢性肝损、 肝纤维化及肝硬化特 征仍保留但肝实质仅轻度充血肿胀，存在少量灶状坏死，无大块或亚大块坏死。 肝组织损伤评分肝脏损伤病理评分 （injury score ) 为 2±1分， 明显低于对照组 的 5±2分 （p< 0.01 ) 。 肝脏的 NF- κ B水平较对照组下降 30-50%。 外周血浆 促炎因子 (pro-inflammatory cytokine s)TNFa， IL-6及抑炎因子 ( anti-inflammatory cytokine) IL-10 的峰值较慢性肝病重症肝损伤对照组明显下降 （p< 0.01 ) ； 肝脏 TNFa， IL-6峰值较慢性肝病重症肝损伤对照组明显下降（p< 0.01 ) 。 同 时肝脏 IL-22及 IL-22受体表达水平分别升高 2-5倍。而 D-GaIN/LPS急性打击 后 2小时， 3小时， 4小时， 6小时及 8小时给药的大鼠， 肝脏存在大块或亚大 块坏死。 病理肝组织损伤评分 （injury score ) 与生理盐水处理的慢性肝病重症 肝损伤对照组无明显差异。 血浆促炎因子 TNF ci , IL-6及抑炎因子 IL-10的峰 值较慢性肝病重症肝损伤对照组无明显改变。 上述研究显示静脉注射 I 型 LHusTNFR-IgGl r Fc通过抑制早期慢性肝病重症肝损伤的大量肝细胞坏死及全 身炎症反应综合症 （SIRS) 进而降低死亡率， 但对非早期慢性肝病重症肝损伤 大鼠无法有效抑制大量肝细胞坏死及阻止 SIRS 的发生， 从而无法降低其死亡 率。 实施例 12
人 I型肿瘤坏死因子 α受体蛋白氨基端与 PEG联接的产物及 I型肿瘤坏死 因子 α受体蛋白羧基端与 PEG联接的产物分别静脉注射给药途径预防及治疗 早期大鼠慢性肝病重症肝损伤的实验
人 I型肿瘤坏死因子 α受体蛋白氨基端与 PEG联接的产物以及 I型肿瘤坏 死因子 α 受体蛋白羧基端与 PEG 联接的产物（方法（3)制备的 I 型 LHusTNFR-PEG)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急 性打击前给予尾静脉注射。 剂量 5-50mg/kg， 在一优选实施方案中， 本发明使 用剂量为 12.5mg/kg。 该处理组命名为长效 I 型受体静脉注射预防组。 I 型 LHusTNFR-PEG 不限于 D-GaIN/LPS 急性打击前特定时间， 只要满足提前于 D-GaIN/LPS急性打击前给药即可， 时间优选 D-GaIN/LPS急性打击前 36小时 至 0小时。 在一优选实施方案中， 本发明使用 D-GaIN/LPS急性打击前 2小时。 另设常规 HusTNFR尾静脉注射大鼠，剂量 12.5mg/kg，为常规 I型受体预防组； 对照组为皮下注射相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的免疫性 肝纤维化、 四氯化碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱导的非 酒精性肝病大鼠基础上，两种氨基及羧基端联结的 I型 LHusTNFR-PEG静脉注 射预防组在 D-GaIN/LPS注射后 24小时的大鼠死亡率均为 0， 而常规 I型受体 预防组的死亡率为 60%， 对照组为 90%。大体病理显示 I型 LHusTNFR-PEG预 防组肝脏病理无大块或亚大块坏死存在。 肝组织损伤评分肝脏损伤病理评分 ( injury score)为 2 ± 1分，明显低于对照组的 5 ± 2分（p< 0.01 )。 病理 TUNEL 染色显示对照组显微镜下凋亡小体 40-60个 /高倍视野，明显高于长效 II型受体 皮下注射预防组为 5-8 个 /高倍视野， p < 0.01.肝脏 caspase3 检测， 预防组 caspase3 活性低于对照组 3-4 倍。 外周血浆促炎因子 （pro-inflammatory cytokines) TNF ， IL-6及抑炎因子 （ anti-inflammatory cytokine ) IL-10的峰值 较慢性肝病重症肝损伤对照组明显下降； 肝脏 TNF ci , IL-6峰值较慢性肝病重 症肝损伤对照组明显下降。 预防组肝脏的 NF- K B水平较对照组下降 30-50%。 同时肝脏 IL-22及 IL-22受体表达水平分别升高 2-5倍。 人 I型肿瘤坏死因子 α受体蛋白氨基端与 PEG联接的产物以及 I型肿瘤坏 死因子 a 受体蛋白羧基端与 PEG 联接的产物（方法（3)制备的 I 型 LHusTNFR-PEG)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急 性打击后 0.5， 1， 2， 3， 4， 6 , 8小时给予尾静脉注射。 0.5， 1， 2小时慢性肝 病重症肝损伤动物死亡率为 0， 4小时及以后大鼠死亡率为 80-90%。 表明上述 两种不同端基联结的 I型 LHusTNFR-PEG均可有效治疗早期慢性肝病重症肝损 伤、 降低死亡率的作用。 实施例 13
人 II型肿瘤坏死因子 a受体蛋白氨基端与 PEG联接的产物及 I型肿瘤坏 死因子 α受体蛋白羧基端与 PEG联接的产物分别静脉注射给药途径预防及治 疗早期大鼠慢性肝病重症肝损伤的实验
人 II型肿瘤坏死因子 α受体蛋白氨基端与 PEG联接的产物以及 II型肿瘤 坏死因子 α 受体蛋白羧基端与 PEG 联接的产物（方法 (4)制备的 I 型 LHusTNFR-PEG)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急 性打击前给予尾静脉注射。 剂量 5-50mg/kg， 在一优选实施方案中， 本发明使 用剂量为 12.5mg/kg。 该处理组命名为长效 II 型受体静脉注射预防组。 II 型 LHusTNFR-PEG 不限于 D-GaIN/LPS 急性打击前特定时间， 只要满足提前于 D-GaIN/LPS急性打击前给药即可， 时间优选 D-GaIN/LPS急性打击前 36小时 至 0小时。 在一优选实施方案中， 本发明使用 D-GaIN/LPS急性打击前 2小时。 另设常规 HusTNFR尾静脉注射大鼠，剂量 12.5mg/kg，为常规 I型受体预防组； 对照组为皮下注射相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的免疫性 肝纤维化、 四氯化碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱导的非 酒精性肝病大鼠基础上， 两种氨基及羧基端联结的 II型 LHusTNFR-PEG静脉 注射预防组在 D-GaIN/LPS注射后 24小时的大鼠死亡率均为 0， 而常规 II型受 体预防组的死亡率为 60%，对照组为 90%。大体病理显示 II型 LHusTNFR-PEG 预防组肝脏病理无大块或亚大块坏死存在。 肝组织损伤评分肝脏损伤病理评分 ( injury score )为 2±1分，明显低于对照组的 5±2分（p< 0.01 )。 病理 TUNEL 染色显示对照组显微镜下凋亡小体 40-60个 /高倍视野，明显高于长效 II型受体 皮下注射预防组为 5-8 个 /高倍视野， p < 0.01.肝脏 caspase3 检测， 预防组 caspase3 活性低于对照组 3-4 倍。 外周血浆促炎因子 （pro-inflammatory cytokines ) TNFa, IL-6及抑炎因子 （ anti-inflammatory cytokine ) IL-10的峰值 较慢性肝病重症肝损伤对照组明显下降； 肝脏 TNF a , IL-6峰值较慢性肝病重 症肝损伤对照组明显下降。 预防组肝脏的 NF- κ B水平较对照组下降 30-50%。 同时肝脏 IL-22及 IL-22受体表达水平分别升高 2-5倍。 人 II型肿瘤坏死因子 α受体蛋白氨基端与 PEG联接的产物以及 II型肿瘤 坏死因子 ci 受体蛋白羧基端与 PEG 联接的产物（方法（3)制备的 I 型 LHusTNFR-PEG)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急 性打击后 0.5， 1， 2， 3， 4， 6 , 8小时给予尾静脉注射。 0.5， 1， 2小时慢性肝 病重症肝损伤动物死亡率为 0， 4小时及以后大鼠死亡率为 80-90%。 表明上述 两种不同端基联结的 I型 LHusTNFR-PEG均可有效治疗早期慢性肝病重症肝损 伤、 降低死亡率的作用。 实施例 14
PEG脂质体包埋人 I型肿瘤坏死因子 α受体静脉注射给药途径预防大鼠慢 性肝病重症肝损伤及治疗早期大鼠慢性肝病重症肝损伤的实验
PEG 脂质体包埋人 I 型肿瘤坏死因子 α受体 (方法 (5)制备的长效 I 型
LHusTNFR)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急性打 击前给予尾静脉注射。 剂量 5-50mg/kg， 在一优选实施方案中， 本发明使用剂 量为 12.5mg/kg。 该处理组命名为长效 I 型受体静脉注射预防组。 长效 I 型 LHusTNF 不限于 D-GaIN/LPS 急性打击前特定时间， 只要满足提前于 D-GaIN/LPS急性打击前给药即可， 时间优选 D-GaIN/LPS急性打击前 36小时 至 0小时。 在一优选实施方案中， 本发明使用 D-GaIN/LPS急性打击前 2小时。 另设常规 I型 HusTNFR尾静脉注射大鼠， 剂量 12.5mg/kg， 为常规 I型受体预 防组； 对照组为皮下注射相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的 免疫性肝纤维化、 四氯化碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱 导的非酒精性肝病大鼠基础上长效 I 型 LHusTNFR-PEG 静脉注射预防组在 D-GaIN/LPS注射后 24小时的大鼠死亡率均为 0， 而常规 I型受体预防组的死 亡率为 60%， 对照组为 90%。大体病理显示 I型 LHusTNFR-PEG预防组肝脏病 理无大块或亚大块坏死存在。 肝组织损伤评分肝脏损伤病理评分（injury score ) 为 2 ± 1分， 明显低于对照组的 5 ± 2分 （p< 0.01 ) 。 病理 TUNEL染色显示 对照组显微镜下凋亡小体 40-60个 /高倍视野，明显高于长效 II型受体皮下注射 预防组为 6-8个 /高倍视野， p< 0.01.肝脏 caspase3检测， 预防组 caspase3活性 低于对照组 2-4倍。 外周血浆促炎因子 （pro-inflammatory cytokines ) TNF α， IL-6及抑炎因子 （anti-inflammatory cytokine ) IL-10的峰值较慢性肝病重症肝 损伤对照组明显下降； 肝脏 TNF ci , IL-6峰值较慢性肝病重症肝损伤对照组明 显下降。 预防组肝脏的 NF- κ Β水平较对照组下降 20-40%。 同时肝脏 IL-22及 IL-22受体表达水平分别升高 2-5倍。
PEG 脂质体包埋人 I 型肿瘤坏死因子 a受体蛋白 （方法 (5)制备的 I 型 LHusTNFR-PEG)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急 性打击后 0.5， 1， 2， 3， 4， 6 , 8小时给予尾静脉注射。 0.5， 1， 2小时慢性肝 病重症肝损伤动物死亡率为 0， 4小时及以后大鼠死亡率为 80-90%。 表明 PEG 脂质体 -I型 LHusTNFR-PEG均可有效治疗早期慢性肝病重症肝损伤、降低死亡 率的作用。 实施例 15
PEG脂质体包埋人 II型肿瘤坏死因子 a受体静脉注射给药途径预防大鼠 慢性肝病重症肝损伤及治疗早期大鼠慢性肝病重症肝损伤的实验
PEG 脂质体包埋人 II 型肿瘤坏死因子 a受体 (方法 (5)制备的长效 II 型 LHusTNFR)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急性打 击前给予尾静脉注射。 剂量 5-50mg/kg， 在一优选实施方案中， 本发明使用剂 量为 12.5mg/kg。 该处理组命名为长效 I 型受体静脉注射预防组。 长效 I 型 LHusTNF 不限于 D-GaIN/LPS 急性打击前特定时间， 只要满足提前于 D-GaIN/LPS急性打击前给药即可， 时间优选 D-GaIN/LPS急性打击前 36小时 至 0小时。 在一优选实施方案中， 本发明使用 D-GaIN/LPS急性打击前 2小时。 另设常规 I型 HusTNFR尾静脉注射大鼠， 剂量 12.5mg/kg， 为常规 I型受体预 防组； 对照组为皮下注射相同体积生理盐水的同类大鼠。 在异种白蛋白诱导的 免疫性肝纤维化、 四氯化碳诱导肝硬化、 乙醇诱导酒精性肝病以及高脂饮食诱 导的非酒精性肝病大鼠基础上长效 Π 型 LHusTNFR-PEG 静脉注射预防组在 D-GaIN/LPS注射后 24小时的大鼠死亡率均为 0， 而常规 II型受体预防组的死 亡率为 60%， 对照组为 90%。大体病理显示 I型 LHusTNFR-PEG预防组肝脏病 理无大块或亚大块坏死存在。 肝组织损伤评分肝脏损伤病理评分（injury score ) 为 2 ± 1分， 明显低于对照组的 5 ± 2分 （p< 0.01 ) 。 病理 TUNEL染色显示 对照组显微镜下凋亡小体 40-60个 /高倍视野，明显高于长效 II型受体皮下注射 预防组为 6-8个 /高倍视野， p< 0.01.肝脏 caspase3检测， 预防组 caspase3活性 低于对照组 2-4倍。 外周血浆促炎因子 （pro-inflammatory cytokines ) TNF α， IL-6及抑炎因子 （anti-inflammatory cytokine ) IL-10的峰值较慢性肝病重症肝 损伤对照组明显下降； 肝脏 TNF ci , IL-6峰值较慢性肝病重症肝损伤对照组明 显下降。 预防组肝脏的 NF- κ Β水平较对照组下降 20-40%。 同时肝脏 IL-22及 IL-22受体表达水平分别升高 2-5倍。
PEG 脂质体包埋人 II 型肿瘤坏死因子 a受体蛋白 （方法 (5)制备的 II 型 LHusTNFR-PEG)分别在上述四类慢性肝病重症肝损伤模型给予 D-GaIN/LPS急 性打击后 0.5， 1， 2， 3， 4， 6， 8小时给予尾静脉注射。 0.5， 1， 2小时慢性肝 病重症肝损伤动物死亡率为 0， 4小时及以后大鼠死亡率为 80-90%。 表明 PEG 脂质体 -II型 LHusTNFR-PEG均可有效治疗早期慢性肝病重症肝损伤、 降低死 亡率的作用。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献 被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申 请所附权利要求书所限定的范围。

Claims

权 利 要 求
1. 长效可溶性肿瘤坏死因子 α受体在制备预防和 /或治疗慢性肝病重症肝 损伤及慢性肝病基础上发生的肝细胞坏死药物中的用途。
2.如权利要求 1所述的用途， 其特征在于， 所述治疗是治疗慢性肝病重症 肝损伤早期阶段及慢性肝病基础上发生肝细胞坏死相关疾病的早期阶段。
3.如权利要求 2所述的用途， 其特征在于， 所述慢性肝病重症肝损伤早期 阶段是指存在全身炎症反应综合症 （SIRS ) 状态的慢性肝病重症肝损伤。
4.如权利要求 3 所述的用途， 其特征在于， 所述的全身炎症反应综合症 ( SIRS ) 是指外周血促炎细胞因子 TNF a、 IL-6升高或促炎细胞因子 TNF a、
IL-6、 抑炎细胞因子 IL-10均升高。
5.如权利要求 1所述的用途， 其特征在于， 所述的长效可溶性肿瘤坏死因 子 a受体的半衰期为 12-140小时。
6.如权利要求 1所述的用途， 其特征在于， 所述的长效可溶性肿瘤坏死因 子 a受体选自：
a.人 I型肿瘤坏死因子 ci受体与人 IgGl : Fc片段的融合蛋白，
b.人 II型肿瘤坏死因子 a受体与人 IgGl： Fc片段的融合蛋白，
c.人 I型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 ci受体蛋白羧基端与 PEG联接的产物，
e.人 II型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
f.人 II型肿瘤坏死因子 α受体蛋白羧基端与 PEG联接的产物，
h.用 PEG-脂质体混合物包埋人 I型肿瘤坏死因子 ci受体蛋白的产物， 或 i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 ci受体蛋白的产物。
7.如权利要求 1所述的用途， 其特征在于， 所述的长效可溶性肿瘤坏死因 子 a受体可使慢性肝病重症肝损伤动物死亡率明显降低；
阻止肝脏组织发生大块 /亚大块坏死；
肝脏的 caspase 3活性水平下降 70-80%;
使肝脏病理 TUNEL阳性细胞计数 /高倍视野下降超过 80%;
使肝组织损伤评分下降 40-70%;
使肝脏大块及亚大块坏死病理特征基本消失；
使肝脏的 NF- κ B水平下降 30-50%; 使外周血 TNF α、 IL-6、 IL-10的峰值下降 40-95%；
使肝脏 TNF α 、 IL-6的峰值下降 40-80%; 和 /或
使肝脏 IL-22及 IL-22受体分别升高 2-5倍。
8.如权利要求 2所述的用途， 其特征在于， 所述的长效可溶性肿瘤坏死因 子 a受体可使早期慢性肝病重症肝损伤动物死亡率明显降低；
阻止肝脏组织发生大块 /亚大块坏死；
肝脏的 caspase 3活性水平下降 70-80%;
使肝脏病理 TUNEL阳性细胞计数 /高倍视野下降超过 80%;
使肝组织损伤评分下降 40-70%;
使肝脏大块及亚大块坏死病理特征基本消失；
使肝脏的 NF- κ B水平下降 30-50%;
使外周血 TNF α、 IL-6, IL-10的峰值下降 40-95%；
使肝脏 TNF α 、 IL-6的峰值下降 40-80%; 和 /或
使肝脏 IL-22及 IL-22受体分别升高 2-5倍。
9.如权利要求 1所述的用途， 其特征在于， 所述慢性肝病重症肝损伤 （或 称慢性肝病基础的重症肝损伤） 指因病毒、 免疫损伤、 毒物 （或药物） 、 酒精 或高脂饮食等导致慢性肝病基础的疾病， 近期肝脏遭受重大打击诱发急性、 大 量肝细胞坏死导致的肝脏功能快速恶化， 并可进展至多脏器功能衰竭、 短期有 超过 20%死亡率的一类肝脏疾病。
10.如权利要求 9所述的用途，其特征在于，所述慢性肝病重症肝损伤包括： 重度酒精性肝炎， 肝炎病毒相关肝炎、 肝纤维化或肝硬化基础上的慢加急性肝 衰竭， 非酒精脂肪性肝病基础上发生的重症肝损伤， 自身免疫性肝病基础上发 生的重症肝损伤， 和慢性肝病基础上的药物源性重症肝损伤。
11.如权利要求 1所述的用途， 其特征在于， 所述的肝细胞坏死是急性大面 积肝细胞坏死 （大块或亚大块坏死 ( massive/submassive hepatic necrosis) 是其 在肝脏病理上的特征表现之一） 。
12.—种药物组合物， 其特征在于， 所述的药物组合物含有：
(i) 有效量的选自下组的长效可溶性肿瘤坏死因子 α受体：
a.人 I型肿瘤坏死因子 ci受体与人 IgGl : Fc片段的融合蛋白，
b.人 II型肿瘤坏死因子 α受体与人 igGl : Fc片段的融合蛋白，
c.人 I型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物， d.人 I型肿瘤坏死因子 ci受体蛋白羧基端与 PEG联接的产物， e.人 II型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
f.人 II型肿瘤坏死因子 α受体蛋白羧基端与 PEG联接的产物，
h.用 PEG-脂质体混合物包埋人 I型肿瘤坏死因子 ci受体蛋白的产物， 或 i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 ci受体蛋白的产物， 以及
(ϋ) 药学上可接受的载体。
13.如权利要求 12所述的药物组合物， 其特征在于， 所述药物组合物还含 有有效量的一种或多种选自以下的药物：
(iii) ci -胸腺肽， 人肝细胞生长因子 （huHGF) 、 还原型谷胱甘肽、 苦参碱、 人体血清白蛋白、 多烯磷脂酸胆碱、 各种辅酶维生素， 血必净 （中成药） 。
14.一种预防慢性肝病重症肝损伤及慢性肝病基础上发生的肝细胞坏死的 方法， 其特征在于， 给予需要治疗的人员有效量的长效可溶性肿瘤坏死因子 α 受体。
15.—种治疗早期的慢性肝病重症肝损伤及慢性肝病基础上发生肝细胞坏 死相关疾病早期阶段的方法， 其特征在于， 给予需要治疗的人员有效量的长效 可溶性肿瘤坏死因子 a受体。
16. 如权利要求 14或 15所述的方法， 其特征在于， 所述的长效可溶性肿 瘤坏死因子 α受体选自下组：
a.人 I型肿瘤坏死因子 α受体与人 igGl : Fc片段的融合蛋白，
b.人 II型肿瘤坏死因子 a受体与人 IgGl： Fc片段的融合蛋白，
c.人 I型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 ci受体蛋白羧基端与 PEG联接的产物，
e.人 II型肿瘤坏死因子 ci受体蛋白氨基端与 PEG联接的产物，
f.人 II型肿瘤坏死因子 ci受体蛋白羧基端与 PEG联接的产物，
h.用 PEG-脂质体混合物包埋人 I型肿瘤坏死因子 ci受体蛋白的产物， 或 i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 ci受体蛋白的产物。
PCT/CN2013/071497 2013-02-07 2013-02-07 长效人重组可溶性肿瘤坏死因子α受体在制备预防和治疗慢性肝病重症肝损伤药物中的用途 WO2014121481A1 (zh)

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