US20160101153A1 - Use of long-acting human recombinant soluble tumor necrosis factor a receptor in the preparation of drugs for preventing and treating severe liver injury on chronic liver disease - Google Patents

Use of long-acting human recombinant soluble tumor necrosis factor a receptor in the preparation of drugs for preventing and treating severe liver injury on chronic liver disease Download PDF

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US20160101153A1
US20160101153A1 US14/821,311 US201514821311A US2016101153A1 US 20160101153 A1 US20160101153 A1 US 20160101153A1 US 201514821311 A US201514821311 A US 201514821311A US 2016101153 A1 US2016101153 A1 US 2016101153A1
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Hai Li
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    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
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    • 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
    • A61K38/38—Albumins
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    • 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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • 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

  • This invention which involves the use of new drugs that include the recombinant soluble tumor necrosis ⁇ receptor (HusTNFR), belongs to the gene engineering technology and gene function application field. This invention specifically involves the prevention and treatment of severe liver injury on chronic liver disease by long-acting HusTNFR (LHusTNFR).
  • LHusTNFR long-acting HusTNFR
  • Severe liver injury on chronic liver disease refers to patients with underlying chronic liver diseases who have recently suffered one or more major injuries in the liver, thus causing severe liver injury induced by acute and massive hepatocyte necrosis, which can involve important extrahepatic organs and cause multiple organ failure, including liver failure.
  • This disease has a very high short-term mortality (40-80%) and is a severe liver disease that threatens the lives of patients.
  • the characteristics of chronic liver disease with severe liver injury are the following: 1) the presence of chronic underlying liver disease.
  • Chronic underlying liver disease refers to chronic hepatitis, liver fibrosis or cirrhosis induced by a variety of causes.
  • liver diseases include hepatitis virus-induced chronic hepatitis, liver fibrosis or cirrhosis, chronic alcoholic liver disease, non-alcoholic fatty liver disease, autoimmune liver disease, or drug-induced liver injury.
  • liver injury medications including intestinal endotoxins, taking liver injury medications, alcoholism, hepatitis virus mutations, recurrence of the original hepatitis virus, infection with a new hepatitis virus, or chemical injury.
  • liver injury medications including intestinal endotoxins, taking liver injury medications, alcoholism, hepatitis virus mutations, recurrence of the original hepatitis virus, infection with a new hepatitis virus, or chemical injury.
  • liver injury duration usually within 4 weeks
  • the 3-month mortality rate is greater than 40-80%.
  • liver injury and severe liver injury are classified as different liver diseases.
  • the pathogenesis of these 2 diseases is different.
  • the development of acute hepatic necrosis is the key pathogenic characteristic of severe liver injury, and massive/submassive hepatic necrosis is the characteristic pathological change in liver tissues during this disease.
  • liver injury involves only mild hepatic inflammation and necrosis, and there is no massive/submassive hepatic necrosis in its pathology. This is the key feature distinguishing liver injury from severe liver injury.
  • the disease outcomes differ. Liver injury will not develop into single organ or multiple organ failure and will not result in death, whereas severe liver injury patients usually progress to multiple organ failure and have a mortality rate of 40-80%.
  • the clinical treatment results differ significantly.
  • liver injury is divided into acute severe liver injury and severe liver injury on chronic liver disease. Severe liver injury that occurs in a healthy liver is called acute severe liver injury (also known as acute liver failure) whereas acute liver injury that occurs during chronic liver disease is called severe liver injury on chronic liver disease (also known as acute-on-chronic liver failure).
  • Acute severe liver injury occurs in healthy livers. After acute and massive hepatocyte necrosis, healthy livers have a very strong regenerative ability. With the help of anti-hepatic necrosis drugs, liver cells can rapidly regenerate to restore the liver function to the lowest level necessary for the basic requirements of body operation, thus improving the mortality rate. Severe liver injury on chronic liver disease occurs in livers with chronic hepatitis, fibrosis, or cirrhosis. Livers with chronic lesions are more susceptible to acute hepatocyte necrosis after liver injury than are healthy livers.
  • livers with chronic liver disease cannot rapidly regenerate after acute and massive hepatic necrosis; thus, the diseased liver cannot bear the basic operational demands of the body within a short time period, resulting in patient death.
  • liver degeneration defects and being prone to acute hepatic necrosis are two important differences between severe liver injury on chronic liver disease and acute severe liver injury; consequently, they are classified as two different groups of diseases in clinical situations. They have different diagnostic and therapeutic methods.
  • drugs that are effective for acute severe liver injury are ineffective for severe liver injury on chronic liver disease.
  • the conventional soluble TNF ⁇ receptor can relieve liver injury caused by mild hepatocyte necrosis to some extent. However, it cannot effectively reduce acute severe liver injury or severe liver injury on chronic liver disease caused by large areas of hepatocyte necrosis. In addition, researchers in this field still cannot understand the reason for the unsatisfactory results. Therefore, it is currently difficult to practically apply the soluble TNF ⁇ receptor in the clinical treatment of severe liver injury.
  • the TNF ⁇ receptor shows poor efficacy for the treatment of severe liver injury on chronic liver disease to further improve TNF ⁇ receptor treatment and to use this agent to effectively and rapidly prevent the development of severe liver injury on chronic liver disease. Therefore, the TNF ⁇ receptor can become an excellent drug for the prevention and treatment of severe liver injury on chronic liver disease (acute-on-chronic liver failure) in clinical situations.
  • the first aspect of the invention involves the use of LHusTNFR in the preparation of drugs for preventing and/or treating severe liver injury on chronic liver disease or hepatocyte necrosis based on chronic liver diseases.
  • the mentioned treatment is the treatment of the early-stage severe liver injury on chronic liver disease and the early-stage hepatic necrosis related disease based on chronic liver disease.
  • the mentioned early-stage severe liver injury on chronic liver disease refers to the severe liver injury on chronic diseases with the status of SIRS.
  • the mentioned SIRS refers to the increase of peripheral blood pro-inflammatory cytokines TNF ⁇ and IL-6 or the increase of the pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 together.
  • the mentioned LHusTNFR has a half-life of 12-140 hours.
  • the mentioned LHusTNFR is selected from:
  • the mentioned LHusTNFR can significantly reduce the mortality rate of animals with severe liver injury on chronic liver disease
  • the mentioned LHusTNFR can significantly decrease the mortality rate of animals with early-stage severe liver injury on chronic liver disease
  • chronic liver disease with severe liver injury refers to diseases that occur in the presence of chronic liver diseases and that are caused by viruses, immune injury, toxins (or drugs), alcohol, or a high-fat diet; this class of liver diseases occurs as a result of the rapid deterioration of liver functions caused by a recently suffered major blow to the liver that induces acute and massive liver cell necrosis that can develop into multiple organ failure with a short-term mortality rate greater than 20%.
  • the mentioned chronic liver diseases with severe liver injury include the following: severe alcoholic hepatitis, hepatitis virus associated hepatitis, acute-on-chronic liver failure caused by liver fibrosis or cirrhosis, severe liver injury caused by nonalcoholic fatty liver disease, severe liver injury caused by autoimmune liver disease, and drug-induced severe liver injury caused by chronic liver disease.
  • hepatic necrosis refers to acute and large area hepatic necrosis (massive/submassive hepatic necrosis), which is one of the characteristic manifestations in liver pathology.
  • the second aspect of the invention provides a pharmacological composition wherein the said pharmacological composition contains the following:
  • the mentioned pharmacological composition also contains an effective amount of one or more drugs selected from the drugs listed below:
  • the third aspect of the invention provides a method for the prevention of severe liver injury on chronic liver disease and hepatocyte necrosis on the basis of chronic liver diseases; thus, people who require treatment are given LHusTNFR.
  • the fourth aspect of this invention provides a method for the treatment of early-stage severe liver injury on chronic liver disease and early-stage hepatocyte necrosis on the basis of chronic liver diseases; thus, people who require treatment are given LHusTNFR.
  • the mentioned LHusTNFR is selected from the following group:
  • this invention provides TNF ⁇ that can effectively and rapidly prevent the development of severe liver injury on chronic liver disease; therefore, it can be an excellent drug for the prevention and treatment of severe liver injury on chronic liver disease (acute-on-chronic liver failure) in clinical situations.
  • FIG. 1 shows the liver injury pathological scores in model 1 of embodiment 4.
  • the left column is the liver with chronic liver fibrosis.
  • the middle column is the long-acting type II receptor subcutaneous injection prevention group, and the right column is the control group.
  • FIG. 2A and FIG. 2B respectively, show the dynamic changes of the peripheral blood and liver tissue pro-inflammatory cytokines and anti-inflammatory cytokines after the LPS/D-GaIN challenge in model 1 of embodiment 4.
  • FIG. 2A shows the dynamic changes of the peripheral blood pro-inflammatory cytokines (TNF ⁇ and IL-6) and the anti-inflammatory cytokine (IL-10).
  • FIG. 2B shows the dynamic changes of the liver tissue pro-inflammatory cytokines (TNF ⁇ and IL-6) and the anti-inflammatory cytokine (IL-10).
  • the solid line is the control group receiving normal saline treatment.
  • the dotted line is the prevention group that was treated with the long-acting type II receptor (type II HusTNFR-IgG1:Fc) through the subcutaneous injection route.
  • FIG. 3 shows the liver injury pathological scores in model 1 of embodiment 5.
  • the left column is the liver with chronic liver fibrosis.
  • the middle column is the prevention group receiving the long-acting type II receptor through the intravenous route.
  • the right column is the control group.
  • FIG. 4A and FIG. 4B respectively, show the dynamic changes of the peripheral blood and liver tissue pro-inflammatory cytokines and anti-inflammatory cytokines after the LPS/D-GaIN challenge in model 1 of embodiment 5.
  • FIG. 4A shows the dynamic changes of the peripheral blood pro-inflammatory cytokines (TNF ⁇ and IL-6) and the anti-inflammatory cytokine (IL-10).
  • FIG. 4B shows the dynamic changes of the liver tissue pro-inflammatory cytokines (TNF ⁇ and IL-6) and the anti-inflammatory cytokine (IL-10).
  • the solid line is the control group receiving normal saline treatment.
  • the dotted line is the prevention group that was treated with the long-acting type II receptor (type II HusTNFR-IgG1:Fc) through the intravenous route.
  • inventors for the first time showed that the prevention and treatment of larger area acute hepatocyte necrosis requires the persistent and stable inhibitory function of soluble TNF ⁇ receptors. Therefore, inventors modified TNF ⁇ receptors through a variety of methods to prepare one group of long-acting soluble TNF ⁇ receptors that can maintain stable and persistent treatment functions in the blood and liver of the body, which can effectively prolong the action time of soluble TNF ⁇ receptors as an active component in the body.
  • inventors greatly increased the efficacy of TNF ⁇ for chronic liver disease. Therefore, for the first time, the modified TNF ⁇ receptors can be applied to large-area/massive hepatic necrosis-induced chronic liver disease with severe liver injury on chronic liver disease. Thus, this invention was completed accordingly.
  • LHusTNFR which is formed by different protein structures and has an excellent efficacy for the prevention and treatment of chronic liver disease with severe liver injury in different types of model animals through embodiments.
  • long-acting soluble tumor necrosis factor ⁇ receptor refers to tumor necrosis factor ⁇ receptor with an extended half-life (i.e., it can last for a longer time in the body at an effective concentration). Generally, the half-life of “long-acting soluble tumor necrosis factor ⁇ receptor” is greater than 12 hours (e.g., 12-140 hours). The half-life of tumor necrosis factor ⁇ receptor can be extended by a variety of methods, which include but are not limited to manipulating the connection between tumor necrosis factor ⁇ receptor and the human IgG1:Fc fragment, the connection between tumor necrosis factor ⁇ receptor and PEG, and embedding tumor necrosis factor ⁇ receptor in the PEG-liposome mixture. The preferred “long-acting soluble tumor necrosis factor ⁇ receptor” is the “long-acting human recombinant soluble tumor necrosis factor ⁇ receptor”.
  • the purpose of this invention was to provide new medical uses of the recombinant soluble tumor necrosis factor ⁇ receptor gene (HusTNFR), particularly new uses for the prevention of chronic liver disease with severe liver injury and the treatment of early-stage chronic liver disease with severe liver injury using long-acting human recombinant tumor necrosis factor ⁇ receptor (LHusTNFR), which is formed by the HusTNFR gene or the modified HusTNFR protein.
  • Human soluble tumor necrosis factor ⁇ receptor gene LHusTNFR
  • LHusTNFR long-acting human recombinant tumor necrosis factor ⁇ receptor
  • An additional purpose of this invention was to provide a drug to further greatly reduce the mortality of chronic liver disease with severe liver injury compared to the conventional soluble tumor necrosis factor ⁇ receptor. It can increase the clinical treatment efficacy mainly through the extension of the half-life of the soluble TNF ⁇ receptor to prolong the action time of the drugs.
  • This invention used LHusTNFR to perform an intervention on rats with severe liver injury on chronic liver disease in severe liver injury on chronic liver disease animal models.
  • the results showed that the mortality rates of the intervention group and the model group were 0 and 60-90%, respectively.
  • the mentioned tumor necrosis factor is the recombinant long-acting soluble protein that results from the binding between TNF ⁇ and the corresponding cell membrane receptor, including the long-acting human soluble type I tumor necrosis factor ⁇ receptor (LHusTNFRI) and the long-acting human recombinant soluble type II tumor necrosis factor ⁇ receptor (LHusTNFRII).
  • LHusTNFRI long-acting human soluble type I tumor necrosis factor ⁇ receptor
  • LHusTNFRIII long-acting human recombinant soluble type II tumor necrosis factor ⁇ receptor
  • Their half-life is extended more than 10-fold compared to that of the general human recombinant soluble type I (HusTNFRI) and type II (HusTNFRII) tumor necrosis factor ⁇ receptors.
  • LHusTNFRI and LHusTNFRII have significantly preferable efficacies in the prevention and treatment of chronic liver disease with severe liver injury compared to that of HusTNFRI or HusTNFRII.
  • Animal models of severe liver injury on chronic liver diseases use the following procedure: 1) After being sensitized by heterologous serum albumin, the tail vein was repeated injected for 6 weeks to induce chronic liver injury and liver fibrosis. The model was established by the intradermal injection of D-galactosamine and endotoxin. 2) After the liver cirrhosis model was established through repeated subcutaneous injections of a small dose of carbon tetrachloride for 8 weeks, the model was established by the intradermal injection of D-galactosamine and endotoxin.
  • this invention established a chronic liver disease with severe liver injury animal model in which CARS was induced by ConA several times, followed by a GaIN/LPS strike.
  • the LHusTNFR in this invention was constructed through
  • LHusTNFR was prepared for the prevention and treatment steps. 1) After being sensitized by heterologous serum albumin, the tail vein was repeatedly injected for 6 weeks to induce chronic liver injury and liver fibrosis. The model was established by the intradermal injection of D-galactosamine and endotoxin. 2) After the liver cirrhosis model was established through repeated subcutaneous injections of a small dose of carbon tetrachloride for 8 weeks, the model was established by the intradermal injection of D-galactosamine and endotoxin. 3) After the chronic alcoholic liver injury and liver fibrosis model was established by feeding the rats low concentrations of alcohol for 12 weeks, the model was established by the intradermal injection of D-galactosamine and endotoxin.
  • the method for the preparation of type I LHusTNFR included the preferential recombination of the gene encoding the type I sTNFR (human) outer membrane amino acids (positions 1-171 of SEQ ID NO: 1) and the gene encoding the human immunoglobulin ⁇ 1 chain Fc fragment (IgG1:Fc) (positions 172-403 of SEQ ID NO: 1) with related plasmids.
  • DNA restriction endonuclease was used to identify and screen for positive clones that carried the type I TNFR-IgG1:Fc fusion fragment. Nucleotide sequencing was used to validate whether the gene was correct.
  • 0-6 nucleotides encoding amino acids G(n)S could be added as a linker fragment.
  • the method for the preparation of the type II LHusTNFR gene included the recombination of the gene encoding the type II sTNFR (human) outer membrane amino acids (positions 1-235 of SEQ ID NO: 2) and the gene encoding the human immunoglobulin ⁇ 1 chain Fc fragment (IgG1:Fc) amino acids (positions 236-467 of SEQ ID NO: 2) with related plasmids.
  • DNA restriction endonuclease was used to identify and screen for positive clones that carried the type II TNFR-IgG1:Fc fusion fragment. Nucleotide sequencing was used to validate whether the gene was correct.
  • the above type I and type II TNFR-IgG1:Fc cDNA fragments were recombined with expression vectors to construct the recombinant expressing plasmids.
  • This invention was not limited to specific expression plasmids.
  • prokaryote expression plasmids were used.
  • the above recombinant plasmids can be introduced into proper host cells using conventional methods. This invention is not limited to any specific host cell as long as it can express the said recombinant expression plasmids. In the preferred embodiment, this invention used mammalian cells, such as CHO cells.
  • the present invention is not limited to any specific expression plasmids.
  • a prokaryotic expression vector for example, pET28, is used.
  • the said recombinant expression vector may be introduced into an appropriate host cells as previously taught.
  • the present invention is not limited to any specific host cells. Any host cells, as long as it allows the expression of the recombinant expression vectors can be used.
  • E. coli BL21 is employed.
  • the expression product according to the present invention is secreted as inclusion bodies in the cytoplasm of the host cells.
  • the inclusion bodies may be isolated from the lysate of the host cells, and then lysed with high concentration of urea or guanidine hydrochloride.
  • the LHusTNFR type I is purified from the lysate of the inclusion bodies, and then renaturated to give the active LHusTNFR type I.
  • Active mPEG(s) of molecular weight (MW) no less than 20,000 may be coupled to the amino terminal or the carboxyl terminal of HusTNFR type I.
  • the present invention is not limited to any specific mPEG molecules.
  • mPEG2-ALD of MW 40,000 from Shearwater corporation (New Jersey, USA) was coupled to the amino terminal of HusTNFR type I.
  • an mPEG2-NHS easter of MW 40,000 from Shearwater corporation (New Jersey, USA) was coupled to the carboxyl terminal of HusTNFR type I.
  • the reaction can be expressed by the formula:
  • the reaction condition includes pH 7.9 and a duration of 12 hrs.
  • the present invention is not limited to any specific expression plasmids.
  • a prokaryotic expression vector for example, pET28, was used.
  • the said recombinant expression vector may be introduced into an appropriate host cells as previously taught.
  • the present invention is not limited to any specific host cells. Any host cells, as long as it allows the expression of the recombinant expression vectors can be used. In a preferred embodiment, E. coli BL21 was used.
  • the expression product according to the present invention is secreted as inclusion bodies into the cytoplasm of host cells.
  • the inclusion bodies may be isolated from the lysate of the host cells, and then lysed with high concentration of urea or guanidine hydrochloride.
  • the LHusTNFR type II is purified from the lysate of the inclusion bodies, and then renaturated to give the active LHusTNFR type II.
  • Active mPEG(s) with a molecular weight (MW) of no less than 20,000 may be coupled to the amino or the carboxyl terminal of HusTNFR type II.
  • the present invention is not limited to any specific mPEG molecules.
  • mPEG2-ALD of MW 40,000 from Shearwater corporation (New Jersey, USA) was coupled to the amino terminal of HusTNFR type II.
  • an mPEG2-NHS easter of MW 40,000 Shearwater corporation, New Jersey, USA was coupled to the carboxyl terminal of HusTNFR type II.
  • the reaction may be expressed by the formula:
  • the reaction condition includes pH 7.9 and a duration of 12 hrs.
  • the HusTNFR type I or the HusTNFR type II is encapsulated with the long circulating liposome-polyethylene glycol-derived phospholipid to produce the long-acting HusTNFR type I or type II.
  • DOPE-PEG-MAL (WM 4108.04) is produced by reacting dioleoyl-phosphatidylethanolamine (DOPE) (WM 744.04, Aanti Polar Lipids, U.S.A.), NHS-PEG3540-MAL (Nhydroxysulfosuccinimide-polyoxyethylene (MW 3540)-maleimide) (MW 3477, Aanti Polar Lipids, U.S.A.) and TEA in a molar ratio of 1:1:0.1 in the presence of triethylamine as the catalyst at 25° C. for 6 hrs and then subjecting the resultant to centrifugation, evaporation and vacuum dehydration.
  • DOPE dioleoyl-phosphatidylethanolamine
  • MW 744.04 Aanti Polar Lipids, U.S.A.
  • NHS-PEG3540-MAL Nhydroxysulfosuccinimide-polyoxyethylene (MW 3540)-maleimide)
  • TEA
  • the long circulating liposome-polyethylene glycol-derived phospholipid is produced by reacting the obtained DOPE-PEG-MAL, EPC (L- ⁇ -Phosphatidylcholine, MW 760.09, cholesterol (MW 386.67), and mPEG-2000-DOPE (MW 2801.51) (Aanti Polar Lipids, U.S.A.) at a molar ratio of 200:20:10:1 in chloroform in a water bath at 40° C., and then rotary evaporating at 100-150 r/min to remove the organic solvents, adding PBS (PH 7.4) to allow complete hydration at room temperature, extruding repeatedly with Mini Extruder, and passing through 100 nm filter for 15 times.
  • DOPE-PEG-MAL EPC
  • EPC L- ⁇ -Phosphatidylcholine, MW 760.09, cholesterol (MW 386.67)
  • mPEG-2000-DOPE MW 2801.51
  • the long-acting HusTNFR type I or type II is obtained by adding HusTNFR type I (SEQ ID NO:3) or HusTNFR type II (SEQ ID NO:4) in PBS to the PBS solution of the obtained long circulating liposome-polyethylene glycol-derived phospholipid, oscillating at 4° C. for 30 min, and passing through CL-4B (Pharmacia, USA) to remove encapsulated HusTNFR type I or HusTNFR type II.
  • half-life of the HusTNFR-IgG type I: and type II:Fc fusion proteins are maintained at effective levels in vivo was from 40 to 100 hrs.
  • the half-life of both PEG-HusTNFR and the HusTNFR encapsuled within the long circulating liposome-polyethylene glycol-derived phospholipid was from 3 to 5.5 days separately.
  • This invention uses genetic engineering methods to produce LHusTNFR.
  • the obtained product is highly efficient for the prevention and treatment of chronic liver disease with severe liver injury.
  • the half-life of LHusTNFR significantly extended, the efficacy for the prevention and treatment of chronic liver disease with severe liver injury and reduced the mortality.
  • LHusTNFR can effectively prevent and treat model animals with severe liver injury on chronic liver disease and can significantly reduce the mortality rate of these diseases. That is, the different types of LHusTNFR can reduce the mortality rate of animals with different mechanisms of severe liver injury on chronic liver disease from 60-90% to 0%. Although many reported studies have shown that some chemicals, biotech drugs, or natural medicines could reduce the mortality rate of chronic liver disease with severe liver injury to some extent through a variety of mechanisms (reduced from 40-80% to 30-50%), there are no reports in this field showing that a drug can reduce the high mortality rate of chronic liver disease with severe liver injury from 60-90% to 0%.
  • This invention reveals that the mechanisms underlying the significant reduction of the mortality rate of disease model animals by LHusTNFR include the prevention of hepatocyte apoptosis, the protection of hepatocyte survival, and the promotion of hepatocyte regeneration.
  • Our study further elucidated that the prevention and treatment of chronic liver disease with severe liver injury by LHusTNFR requires the combined functions of the following two mechanisms: 1) a reduction in the production of liver apoptosis signals by blocking TNF ⁇ signal transduction to exert the function for anti-acute hepatocyte necrosis and 2) an increase in the expression of the IL-22 and IL-2 receptors to increase the survival and regeneration ability of hepatocytes (some literature has previously reported that IL-22 has the ability to promote hepatocyte regeneration and increase hepatocyte survival).
  • this invention reveals that, in addition to blocking acute hepatocyte apoptosis/necrosis, the effective prevention and treatment of chronic liver disease with severe liver injury also requires the promotion of hepatocyte survival and regeneration.
  • effective treatment only requires the blocking of acute hepatocyte apoptosis/necrosis.
  • This invention elucidates that after the prevention/treatment of animals with severe liver injury on chronic liver disease by LHusTNFR, the severity of hepatocyte apoptosis is significantly relieved, and the IL-22 signal that can protect hepatocytes and promote hepatocyte regeneration also increases to effectively treat this disease and significantly reduce the mortality rate.
  • LHusTNFR has a different efficacy on animals at different stages of severe liver injury on chronic liver disease. It can significantly reduce the mortality of animals at the early stage of the disease; however, it cannot reduce mortality at the advanced and late stages of the disease.
  • SIRS early-systemic inflammatory response syndrome
  • CARS late-compensatory anti-inflammatory response syndrome
  • LHusTNFR including TNFRII-IgG:Fc
  • TNFRII-IgG:Fc LHusTNFR
  • the immune system presentations included immune suppression characteristics such as a significant decrease of HLA-DR expression in monocytes, an increase in the peripheral blood anti-inflammatory factor IL-10, and no increase in the pro-inflammatory factors TNF ⁇ and IFN ⁇ .
  • the four types of chronic liver disease with severe liver injury animal models that were used in this invention revealed that SIRS occurs 0-2 hours after the induction of severe liver injury by acute liver injury.
  • SIRS occurs 0-2 hours after the induction of severe liver injury by acute liver injury.
  • the administration of LHusTNFR can reduce the mortality from 60-90% to 0%.
  • animals that had already passed the SIRS stage were administered LHusTNFR; however, the results showed that there was no efficacy in terms of a significant reduction of the mortality rate.
  • this invention identified that LHusTNFR has different treatment effects on the early stage (the presence of SIRS) compared to the middle and late stages (non-SIRS or CARS stage) of chronic liver disease with severe liver injury and explains why the efficacy for animals with severe liver injury on chronic liver disease at the CARS stage is poor.
  • LHusTNFR has a significant efficacy for animals with early-stage chronic liver disease with severe liver injury at the SIRS stage and can greatly reduce the mortality rate.
  • This invention also showed that preventive use of LHusTNFR can prevent the development of chronic liver disease with severe liver injury through blocking massive acute hepatocyte necrosis and inhibiting the occurrence of SIRS.
  • the four animal models of chronic liver disease with severe liver injury selected in this invention represent but are not limited to model animals that have chronic liver injury, liver fibrosis, or liver cirrhosis that is induced by ethanol (alcohol), a high-fat diet, immune injury, or chemical toxins that are attacked by endotoxin or viral nucleic acid to show the characteristics of chronic liver disease with severe liver injury such as acute hepatocyte necrosis, liver inflammation, rapid deterioration of liver functions, and high mortality within a short time period.
  • ethanol alcohol
  • endotoxin or viral nucleic acid chemical toxins that are attacked by endotoxin or viral nucleic acid to show the characteristics of chronic liver disease with severe liver injury such as acute hepatocyte necrosis, liver inflammation, rapid deterioration of liver functions, and high mortality within a short time period.
  • This invention also provides a pharmacological composition for treating hepatocyte necrosis or chronic liver disease with severe liver injury. It contains the aforementioned LHusTNFR and pharmacologically acceptable carriers. Usually, these substances can be prepared in non-toxic, inert, and pharmacologically acceptable aqueous carrier media. The pH is generally approximately 5-8, and the preferred pH is approximately 6-8; however, pH values can be changed as a result of the properties of the prepared substances and diseases to be treated. The prepared pharmacological composition can be administered through conventional routes including (but not limited to) intraperitoneal, intravenous, or local administration.
  • the pharmacological composition in this invention can be directly used in the treatment of hepatocyte necrosis or chronic liver disease with severe liver injury.
  • it can also be used with other related treatment agents.
  • This group of treatment agents includes but is not limited to the following: ⁇ -thymosin, human hepatocyte growth factor (huHGF), glutathione, matrine, human serum albumin, polyene phosphatidylcholine, a variety of coenzyme vitamins, and Xuebijing (traditional Chinese medicine).
  • the pharmacological composition in this invention contains a safe and effective amount of the aforementioned LHusTNFR and pharmacologically acceptable carriers or excipients. These carriers include (but are not limited to) saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. Pharmacological preparations should match the drug administration method.
  • the pharmacological composition in this invention can be prepared in an injection form. For example, it can be prepared with normal saline or a water solution with glucose or other adjuvants using conventional methods. Pharmacological compositions, such as injections or solutions, should be prepared under sterile conditions.
  • the drug dosage of the active components is the effective treatment dose, for example, approximately 0.1 ⁇ g/kg body weight to 5 mg/kg body weight daily.
  • the safe and effective dose of LHusTNFR in the formulation is administered to mammals.
  • the safe and effective dose is usually at least 1 ⁇ g/kg body weight, and in most cases, it is no more than 8 ⁇ g/kg body weight.
  • the preferred dose is between 10 ⁇ g/kg body weight and 1 mg/kg body weight.
  • the specific dose should also consider factors such as the administration routes and health conditions of the patient, considerations that are within the skill range of experienced physicians.
  • Model 1 A chronic liver disease with severe liver injury rat model was established by heterologous serum albumin sensitization and repeated injection to induce chronic liver injury/liver fibrosis, followed by the injection of D-galactosamine (GaIN) and endotoxin (lipopolysaccharide, LPS); this process was completed in 3 stages.
  • GaIN D-galactosamine
  • LPS lipopolysaccharide
  • Heterologous albumin sensitization female Wistar rats with a body weight from 120-150 g were used.
  • Human serum albumin was diluted with normal saline and emulsified with an equal amount of incomplete Freund's adjuvant. Multiple-point subcutaneous injections were performed on each rat at a dose of 0.5 ml (containing 4 mg of albumin) in each injection for a total of 4 treatments. The interval between the first and second injection was 14 days, and the interval between the third and fourth injection was 10 days. Ten days after the last immunization, blood was collected for antibody detection. Rats with positive antibodies were selected for subsequent experiments.
  • liver injury and liver fibrosis albumin was injected into the tail vein twice a week. The first injection was at a dosage of 2.5 mg/animal. Subsequently, each challenge increased the dosage by 0.5 mg until reaching 4.5 mg. This dosage was maintained until week 6. The immune liver injury and liver fibrosis model was thus established. At this stage, the liver pathology of the animals showed (1) the hyperplasia of reticular fibers and collagen fibers in the portal area, which extend outwards. In addition, they occurred around the central vein, were distributed and scattered along the liver sinusoids, and were connected to one another.
  • Lobules were wrapped with connective tissues, and some animals exhibited a pseudolobule formation; (2) lymphocyte, monocyte, and eosinophil infiltration occurred around the portal area and the central vein. The cellulose-like death of the lobular artery and inflammatory cell infiltration around the central vein could be observed. The immunofluorescence results showed that the portal area, hepatic sinusoid, and vessel wall all had immune complex deposition. Electron microscopy showed the transformation of stellate cells into myofibroblasts, which were distributed in the portal area. The surroundings had a large amount of collagen deposition. The deposited collagen formed a wider separation.
  • Acute challenge of D-GaIN/LPS in the above fibrotic rats tail vein injections of albumin were conducted twice a week for a total of 6 weeks to induce liver fibrosis in the rats, followed by the intraperitoneal injection of 400 mg/kg of D-GaIN plus 100 ⁇ g/kg of LPS.
  • the mortality rate of the rats in the model group after 24 h was 80-90%, and the mean survival time was approximately 16 hours.
  • alanine aminotransferase began to increase, and the peak value was reached at 8-12 hours.
  • Total bilirubin continuously increased 4 hours after the injection until the death of the animals.
  • hepatocyte necrotic loci in the nodules increased or showed a patchy fusion, and increases in red blood cells and the hyperplasia of interstitial cells and biliary epithelia were observed.
  • the hepatocytes in the majority of the regenerative nodules showed massive or submassive necrosis. Sinus dilation, red blood cell filling, Kupffer cell activation, and cytoplasm enlargement could be observed.
  • electron microscopy showed a small amount of lipid droplets that were broken or had disappeared into the mitochondrial cristae.
  • the hepatocytes showed a decrease of microvilli and early apoptotic presentation such as shrinkage and irregularly shaped nuclei. After 8 hours, the apoptotic cells significantly increased, and detached apoptotic bodies were observed. The extended foot processes of the activated Kupffer cells were in contact with hepatocytes or engulfed apoptotic bodies.
  • Model 2 Repeated subcutaneous injections of a small dose of carbon tetrachloride were performed for 8 weeks to establish the liver cirrhosis model. D-GaIN/LPS was then injected to establish the chronic liver disease with severe liver injury mode. It was completed in 2 stages.
  • liver cirrhosis in rats using carbon tetrachloride: subcutaneous injections of a small dose of carbon tetrachloride were performed using the common method reported in the literature for 8 weeks. The liver pathological results confirmed the presence of the cirrhosis liver specific pseudolobule structure.
  • the acute D-GaIN/LPS challenge was conducted in the above liver cirrhosis rats: an intraperitoneal injection of 400 mg/kg of D-GaIN plus 100 ⁇ g/kg of LPS was performed.
  • the mortality rate of the rats in the model group after 24 hours was 80-90%, and the mean survival time was approximately 16 hours.
  • alanine aminotransferase began to increase, and the peak value was reached at 8-12 hours.
  • the total bilirubin increased 4 hours after the injection and continuously increased until the death of the animal.
  • the pathological results showed massive and submassive necrosis in the liver combined with the presence of cirrhosis pseudolobule, sinus dilation, red blood cell filling, activation of Kupffer cells, and cytoplasm enlargement.
  • electron microscopy showed a small amount of lipid droplets that were broken or had disappeared into the mitochondrial cristae.
  • the hepatocytes showed a decrease of microvilli and early apoptotic presentation such as shrinkage and irregularly shaped nuclei.
  • the apoptotic cells significantly increased, and detached apoptotic bodies were observed.
  • the extended foot processes of the activated Kupffer cells were in contact with hepatocytes or engulfed the apoptotic bodies.
  • Model 3 The rats were fed low concentrations of ethanol for 12 weeks to establish the alcoholic chronic liver injury and liver fibrosis model. An intradermal injection of D-GaIN/LPS was then performed to establish the chronic liver disease with severe liver injury model. It was completed in 2 stages.
  • Alcoholic liver disease rats were induced by low concentrations of ethanol: the rats were fed with 10% ethanol mixed with a high-fat diet for 3-4 months.
  • the pathological results confirmed the development of hepatic steatosis and liver fibrosis.
  • the acute D-GaIN/LPS challenge was conducted in the above liver cirrhosis rats: an intraperitoneal injection of 400 mg/kg of D-GaIN plus 100 ng/kg of LPS was performed.
  • the mortality rate of the rats in the model group after 24 hours was 80-90%, and the mean survival time was approximately 16 hours.
  • alanine aminotransferase began to increase and peaked at 8-12 hours.
  • the total bilirubin increased 4 hours after injection and continuously increased until animal death.
  • the plasma TNF ⁇ levels significantly increased after 8 hours, and the IL-10 levels increased over time.
  • the pathological results showed massive and submassive necrosis in the liver, sinus dilation, red blood cell filling, activation of Kupffer cells, and enlargement of the cytoplasm.
  • electron microscopy showed a small amount of lipid droplets that were broken or had disappeared into the mitochondrial cristae.
  • the hepatocytes showed a decrease of microvilli and early apoptotic presentations such as shrinkage and irregularly shaped nuclei.
  • the apoptotic cells significantly increased, and detached apoptotic bodies were observed.
  • the extended foot processes by the activated Kupffer cells were in contact with hepatocytes or engulfed the apoptotic bodies.
  • Model 4 The rats were fed with a high-fat diet for 12 weeks to establish the non-alcoholic fatty liver model. D-GaIN/LPS was then injected to establish the chronic liver disease with severe liver injury model.
  • Non-alcoholic fatty liver rats were induced by a high-fat and high-sugar diet: the rats were fed with the common high-fat mixed with high-sugar diet for 3-4 months.
  • the pathological results confirmed the development of hepatic steatosis, steatohepatitis, and partial liver fibrosis.
  • the acute D-GaIN/LPS challenge was performed in the above liver cirrhosis rats: an intraperitoneal injection of 400 mg/kg of D-GaIN plus 100 ⁇ g/kg of LPS was performed.
  • the mortality rate of the rats in the model group after 24 hours was 80-90%, and the mean survival time was approximately 16 hours.
  • alanine aminotransferase began to increase, and the peak value was reached at 8-12 hours.
  • the total bilirubin increased 4 hours after injection and continuously increased until animal death.
  • the plasma TNF ⁇ levels significantly increased after 8 hours, and the IL-10 levels increased over time.
  • the pathological results showed massive and submassive necrosis in the liver, sinus dilation, red blood cell filling, activation of Kupffer cells, and high levels of cytoplasm.
  • electron microscopy showed a small amount of lipid droplets that were broken or had disappeared into the mitochondrial cristae.
  • the hepatocytes showed a decrease of microvilli and early apoptotic presentation such as shrinkage and irregularly shaped nuclei.
  • the apoptotic cells significantly increased, and detached apoptotic bodies were observed.
  • the extended foot processes of the activated Kupffer cells were in contact with the hepatocytes or engulfed the apoptotic bodies.
  • a subcutaneous injection of a small dose of ConA was performed every 48 hours for a total of 3-5 times.
  • the detection of peripheral blood showed that IL-10 significantly increased, whereas TNF ⁇ and IFN ⁇ did not increase, which reflected the CARS immune status model.
  • the mortality rate of the animals was 15-20%.
  • Liver pathology showed submassive necrosis in a smaller range with the simultaneous presence of local and patchy necrosis.
  • NAC N-Acetylcysteine
  • the drug-induced acute severe liver injury rat model was established using the dose of acetaminophen that is commonly used in the literature.
  • the short term mortality rate was approximately 40%.
  • the hepatic cirrhosis rat model was established using carbon tetrachloride.
  • an equal dose of acetaminophen was used to establish the animal model of chronic liver disease with severe liver injury, which has a short term mortality of 40-60%.
  • an intravenous injection of NAC was administered to healthy rats to establish the prevention of chronic liver disease with severe liver injury by the NAC model.
  • the NAC prevention group and the treatment group observed a reduction in the mortality of the model to 10-15%, whereas the mortality rate of chronic liver disease with severe liver injury induced by acetaminophen animals prevented and treated by NAC was 40-60%; this latter value did not differ from that in the control group.
  • NAC is effective for the survival of acute severe liver injury that occurs in healthy livers but is not effective for animals with chronic liver disease with severe liver injury on the basis of liver cirrhosis.
  • the subcutaneous injection of type II LHusTNFR (the type II LHusTNFR-IgG1:Fc prepared in method (2) is the preventative drug) into the above four types of chronic liver disease with severe liver injury models was evaluated before the acute challenge of D-GaIN/LPS at a dose of 5-50 mg/kg. In one preferred embodiment, the dose used in the invention was 12.5 mg/kg.
  • This treatment group was named the long-acting type II receptor subcutaneous injection prevention group.
  • the injection of the long-acting type II soluble receptor type II LHusTNFR-IgG1:Fc is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • conventional HusTNFR subcutaneous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type II receptor prevention group.
  • the control group was the same species of rats with a subcutaneous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type II TNF ⁇ receptor subcutaneous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type II TNF ⁇ receptor prevention group was 66%.
  • the mortality rate of the chronic liver disease with severe liver injury model control group that did not receive long-acting or conventional type II TNF ⁇ treatment was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • model 1 the liver injury scores are shown in FIG. 1 .
  • the left column is the liver with chronic liver fibrosis
  • the middle column is the long-acting type II receptor subcutaneous injection prevention group
  • the right column is the control group.
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type II receptor subcutaneous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.01.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group ( FIG. 2 ).
  • liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • Type II TNF ⁇ Receptor Type II LHusTNFR
  • type II LHusTNFR (the type II LHusTNFR-IgG1:Fc prepared in method (2) is the preventative drug) into the above four types of chronic liver disease with severe liver injury models was evaluated before the acute challenge of D-GaIN/LPS at a dose of 5-50 mg/kg. In one preferred embodiment, the dose used in the invention was 12.5 mg/kg.
  • This treatment group was named the long-acting type II receptor intravenous injection prevention group.
  • the injection of the long-acting type II soluble receptor type II LHusTNFR-IgG1:Fc is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • conventional HusTNFR intravenous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type II receptor prevention group.
  • the control group was the same species of rats with a intravenous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type II TNF ⁇ receptor intravenous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type II TNF ⁇ receptor prevention group was 66%.
  • the mortality rate of the chronic liver disease with severe liver injury model control group that did not receive long-acting or conventional type II TNF ⁇ treatment was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • model 1 the liver injury scores are shown in FIG. 1 .
  • the left column is the liver with chronic liver fibrosis
  • the middle column is the long-acting type II receptor subcutaneous injection prevention group
  • the right column is the control group.
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type II receptor intravenous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.01.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group ( FIG. 2 ).
  • liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • type II LHusTNFR the type II LHusTNFR-IgG1:Fc prepared in method 2
  • a dose of 5-50 mg/kg was treated into the above four types of chronic liver disease (presented in embodiment 1) or multiple ConA stimulations induced CARS model (presented in embodiment 2) after 0, 30 minutes, 1 hour, 1.5 hour for the acute challenge of D-GaIN/LPS.
  • the dose used in the invention was 12.5 mg/kg.
  • the 2nd, 3rd, 4th, 6th and 8th hour's mortality after D-GaIN/LPS attacking for all four types of chronic liver disease (presented in embodiment 1) animals was 50%, 90%, 90% and 90% separately. However, the mortality of all four groups which treated by type II LHusTNFR-IgG1:Fc was 0.
  • the control group was the same species of animals with a subcutaneous injection of an equal volume of normal saline. Mortality of the control group was 90% at the 2nd hour.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the treating group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • type II LHusTNFR-IgG1:Fc subcutaneous injection could decrease the mortality for the early stage of chronic liver disease with severe hepatic injury through the following mechanisms: 1) reduced massive hepatocyte necrosis through the inhibition of hepatic parenchymal cell apoptosis, 2) promoted hepatocyte survival and regeneration, and 3) inhibited SIRS in liver failure animals at the early stage of chronic liver disease with severe liver injury, thus greatly reducing the mortality rate.
  • type II LHusTNFR-IgG1:Fc could not improve the survival rate for advanced stage of the disease.
  • type II LHusTNFR the type II LHusTNFR-IgG1:Fc prepared in method 2
  • a dose of 5-50 mg/kg was treated into the above four types of chronic liver disease (presented in embodiment 1) or multiple ConA stimulations induced CARS model (presented in embodiment 2) after 0, 30 minutes, 1 hour, 1.5 hour for the acute challenge of D-GaIN/LPS.
  • the dose used in the invention was 12.5 mg/kg.
  • the 2nd, 3rd, 4th, 6th and 8th hour's mortality after D-GaIN/LPS attacking for all four types of chronic liver disease (presented in embodiment 1) animals was 50%, 90%, 90% and 90% separately. However, the mortality of all four groups which treated by type II LHusTNFR-IgG1:Fc was 0.
  • the control group was the same species of animals with a intravenous injection of an equal volume of normal saline. Mortality of the control group was 90% at the 2nd hour.
  • type II LHusTNFR-IgG1:Fc There was no significant difference of 24 hour mortality rate between type II LHusTNFR-IgG1:Fc and normal saline treating CARS group at different time point by intravenous injection (0, 1 ⁇ 2, 1, 1.5, 2, 3, 4, 6 and 8 hours after D-GaIN/LPS attacking). The 24 hour mortality rate of both group was 15-20%. These data showed type II LHusTNFR-IgG1:Fc can effectively reducing the mortality rate when was treated animal at the early stage of chronic liver disease with severe liver injury.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the treating group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • type II LHusTNFR-IgG1:Fc intravenous injection could decrease the mortality for the early stage of chronic liver disease with severe hepatic injury through the following mechanisms: 1) reduced massive hepatocyte necrosis through the inhibition of hepatic parenchymal cell apoptosis, 2) promoted hepatocyte survival and regeneration, and 3) inhibited SIRS in liver failure animals at the early stage of chronic liver disease with severe liver injury, thus greatly reducing the mortality rate.
  • type II LHusTNFR-IgG1:Fc could not improve the survival rate for advanced stage of the disease.
  • Type ILHusTNFR Type ITNF ⁇ Receptor
  • the subcutaneous injection of type I LHusTNFR (the type I LHusTNFR-IgG1:Fc prepared in method (2) is the preventative drug) into the above four types of chronic liver disease with severe liver injury models was evaluated before the acute challenge of D-GaIN/LPS at a dose of 5-50 mg/kg. In one preferred embodiment, the dose used in the invention was 12.5 mg/kg.
  • This treatment group was named the long-acting type II receptor subcutaneous injection prevention group.
  • the injection of the long-acting type II soluble receptor type I LHusTNFR-IgG1:Fc is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • conventional HusTNFR subcutaneous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type I receptor prevention group.
  • the control group was the same species of rats with a subcutaneous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type I TNF ⁇ receptor subcutaneous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type I TNF ⁇ receptor prevention group was 66%.
  • the mortality rate of the chronic liver disease with severe liver injury model control group that did not receive long-acting or conventional type I TNF ⁇ treatment was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • model 1 the liver injury scores are shown in FIG. 1 .
  • the left column is the liver with chronic liver fibrosis
  • the middle column is the long-acting type II receptor subcutaneous injection prevention group
  • the right column is the control group.
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type I receptor subcutaneous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.01.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group ( FIG. 2 ).
  • liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • the intravenous injection of type I LHusTNFR (the type I LHusTNFR-IgG1:Fc prepared in method (2) is the preventative drug) into the above four types of chronic liver disease with severe liver injury models was evaluated before the acute challenge of D-GaIN/LPS at a dose of 5-50 mg/kg. In one preferred embodiment, the dose used in the invention was 12.5 mg/kg.
  • This treatment group was named the long-acting type II receptor intravenous injection prevention group.
  • the injection of the long-acting type II soluble receptor type I LHusTNFR-IgG1:Fc is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • conventional HusTNFR intravenous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type I receptor prevention group.
  • the control group was the same species of rats with a intravenous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type I TNF ⁇ receptor intravenous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type I TNF ⁇ receptor prevention group was 66%.
  • the mortality rate of the chronic liver disease with severe liver injury model control group that did not receive long-acting or conventional type I TNF ⁇ treatment was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • model 1 the liver injury scores are shown in FIG. 1 .
  • the left column is the liver with chronic liver fibrosis
  • the middle column is the long-acting type I receptor subcutaneous injection prevention group
  • the right column is the control group.
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type II receptor intravenous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.01.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group ( FIG. 2 ).
  • liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • type I LHusTNFR (the type I LHusTNFR-IgG1:Fc prepared in method 2) at a dose of 5-50 mg/kg was treated into the above four types of chronic liver disease (presented in embodiment 1) or multiple ConA stimulations induced CARS model (presented in embodiment 2) after 0, 30 minutes, 1 hour, 1.5 hour for the acute challenge of D-GaIN/LPS.
  • the dose used in the invention was 12.5 mg/kg.
  • the 2nd, 3rd, 4th, 6th and 8th hour's mortality after D-GaIN/LPS attacking for all four types of chronic liver disease (presented in embodiment 1) animals was 50%, 90%, 90% and 90% separately. However, the mortality of all four groups which treated by type I LHusTNFR-IgG1:Fc was 0.
  • the control group was the same species of animals with a subcutaneous injection of an equal volume of normal saline. Mortality of the control group was 90% at the 2nd hour.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the treating group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • type I LHusTNFR-IgG1:Fc subcutaneous injection could decrease the mortality for the early stage of chronic liver disease with severe hepatic injury through the following mechanisms: 1) reduced massive hepatocyte necrosis through the inhibition of hepatic parenchymal cell apoptosis, 2) promoted hepatocyte survival and regeneration, and 3) inhibited SIRS in liver failure animals at the early stage of chronic liver disease with severe liver injury, thus greatly reducing the mortality rate.
  • type I LHusTNFR-IgG1:Fc could not improve the survival rate for advanced stage of the disease.
  • type I LHusTNFR the type I LHusTNFR-IgG1:Fc prepared in method 2
  • a dose of 5-50 mg/kg was treated into the above four types of chronic liver disease (presented in embodiment 1) or multiple ConA stimulations induced CARS model (presented in embodiment 2) after 0, 30 minutes, 1 hour, 1.5 hour for the acute challenge of D-GaIN/LPS.
  • the dose used in the invention was 12.5 mg/kg.
  • the 2nd, 3rd, 4th, 6th and 8th hour's mortality after D-GaIN/LPS attacking for all four types of chronic liver disease (presented in embodiment 1) animals was 50%, 90%, 90% and 90% separately. However, the mortality of all four groups which treated by type I LHusTNFR-IgG1:Fc was 0.
  • the control group was the same species of animals with a intravenous injection of an equal volume of normal saline. Mortality of the control group was 90% at the 2nd hour.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the treating group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • type I LHusTNFR-IgG1:Fc intravenous injection could decrease the mortality for the early stage of chronic liver disease with severe hepatic injury through the following mechanisms: 1) reduced massive hepatocyte necrosis through the inhibition of hepatic parenchymal cell apoptosis, 2) promoted hepatocyte survival and regeneration, and 3) inhibited SIRS in liver failure animals at the early stage of chronic liver disease with severe liver injury, thus greatly reducing the mortality rate.
  • type I LHusTNFR-IgG1:Fc could not improve the survival rate for advanced stage of the disease.
  • the dose used in the invention was 12.5 mg/kg.
  • This treatment group was named the long-acting type I receptor intravenous injection prevention group.
  • the injection of type I LHusTNFR-PEG is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • the time point used in the invention was 2 hours before D-GaIN/LPS challenge.
  • conventional HusTNFR subcutaneous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type I receptor prevention group.
  • the control group was the same species of rats with a subcutaneous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type I TNF ⁇ receptor intravenous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type I TNF ⁇ receptor prevention group was 60%.
  • the mortality rate of the control group was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type I receptor intravenous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.001.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • the tail intravenous injection of type I LHusTNFR-PEG into above four types of chronic liver disease was evaluated after 0.5, 1, 2, 3, 4, 6 or 8 hour after the acute challenge of D-GaIN/LPS.
  • the mortality rate of type I LHusTNFR-PEG injecting at time point at 0.5, 1, 2 hours after D-GaIN/LPS challenging was 0, however
  • Those results showed both of two different linked form of type I LHusTNFR-PEG can effectively improve the mortality rate for early stage of chronic liver disease with severe liver injury.
  • the dose used in the invention was 12.5 mg/kg.
  • This treatment group was named the long-acting type II receptor intravenous injection prevention group.
  • the injection of type II LHusTNFR-PEG is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • the time point used in the invention was 2 hours before D-GaIN/LPS challenge.
  • conventional HusTNFR subcutaneous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type II receptor prevention group.
  • the control group was the same species of rats with a subcutaneous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type II TNF ⁇ receptor intravenous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type II TNF ⁇ receptor prevention group was 60%.
  • the mortality rate of the control group was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type I receptor intravenous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.001.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • the tail intravenous injection of type II LHusTNFR-PEG into above four types of chronic liver disease was evaluated after 0.5, 1, 2, 3, 4, 6 or 8 hour after the acute challenge of D-GaIN/LPS.
  • the mortality rate of type II LHusTNFR-PEG injecting at time point at 0.5, 1, 2 hours after D-GaIN/LPS challenging was 0, however
  • Those results showed both of two different linked form of type II LHusTNFR-PEG can effectively improve the mortality rate for early stage of chronic liver disease with severe liver injury.
  • the injection of type I LHusTNFR-PEG-liposomes is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • the time point used in the invention was 2 hours before D-GaIN/LPS challenge.
  • conventional HusTNFR subcutaneous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type I receptor prevention group.
  • the control group was the same species of rats with a subcutaneous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type I TNF ⁇ receptor intravenous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type I TNF ⁇ receptor prevention group was 60%.
  • the mortality rate of the control group was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type I receptor intravenous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.001.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • Those results showed both of two different linked form of type I LHusTNFR-PEG-liposomes can effectively improve the mortality rate for early stage of chronic liver disease with severe liver injury.
  • the injection of type II LHusTNFR-PEG-liposomes is not limited to a specific time before the acute challenge of D-GaIN/LPS as long as the administration of drugs occurs before the acute challenge of D-GaIN/LPS.
  • the preferred time is 0 to 36 hours before the acute challenge of D-GaIN/LPS.
  • the time point used in the invention was 2 hours before D-GaIN/LPS challenge.
  • conventional HusTNFR subcutaneous injection rats are prepared at a dose of 12.5 mg/kg, which is the conventional type II receptor prevention group.
  • the control group was the same species of rats with a subcutaneous injection of an equal volume of normal saline.
  • the mortality rate of rats in the long-acting type II TNF ⁇ receptor intravenous injection group 24 hours after the D-GaIN/LPS injection was 0, whereas the mortality rate in the conventional type II TNF ⁇ receptor prevention group was 60%.
  • the mortality rate of the control group was 90%.
  • liver injury score (based on the severity of the lesion, the importance of the lesion, and the area of injured liver tissues observed in HE staining of rat liver tissue sections; semi-quantitative scoring is conducted) was 2 ⁇ 1 points, which was significantly less than the 5 ⁇ 2 points in the control group (p ⁇ 0.01).
  • the pathological TUNEL staining results showed that there were 40-60 apoptotic bodies/high power field under a microscope in the control group, which was significantly more than those in the long-acting type II receptor intravenous injection prevention group with 5-8 apoptotic bodies/high power field, p ⁇ 0.001.
  • the detection of caspase 3 in the liver showed that the caspase 3 activity in the prevention group was 3 to 4 times lower than that in the control group.
  • the peak values of the peripheral plasma pro-inflammatory cytokines TNF ⁇ and IL-6 and the anti-inflammatory cytokine IL-10 significantly decreased compared to that in the chronic liver disease with severe liver injury control group.
  • the peak values of liver TNF ⁇ and IL-6 significantly decreased compared to those in the chronic liver disease with severe liver injury control group.
  • the liver NF- ⁇ B levels in the prevention group decreased by 30-50% compared to that in the control group, whereas the expression levels of the IL-22 and IL-22 receptors in the liver tissues increased by 2- to 5-fold.
  • Those results showed both of two different linked form of type II LHusTNFR-PEG-liposomes can effectively improve the mortality rate for early stage of chronic liver disease with severe liver injury.

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