WO2007041964A1 - Utilisation de recepteur long du facteur de necrose tumorale alpha soluble recombinant humain de demi-vie dans la preparation de medicament pouvant prevenir l'insuffisance hepatique - Google Patents

Utilisation de recepteur long du facteur de necrose tumorale alpha soluble recombinant humain de demi-vie dans la preparation de medicament pouvant prevenir l'insuffisance hepatique Download PDF

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WO2007041964A1
WO2007041964A1 PCT/CN2006/002689 CN2006002689W WO2007041964A1 WO 2007041964 A1 WO2007041964 A1 WO 2007041964A1 CN 2006002689 W CN2006002689 W CN 2006002689W WO 2007041964 A1 WO2007041964 A1 WO 2007041964A1
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Prior art keywords
tumor necrosis
necrosis factor
factor alpha
type
human
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PCT/CN2006/002689
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English (en)
French (fr)
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Hai Li
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Hai Li
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Priority to JP2008534855A priority Critical patent/JP2009511510A/ja
Priority to US12/090,037 priority patent/US8227404B2/en
Publication of WO2007041964A1 publication Critical patent/WO2007041964A1/zh
Priority to US13/529,402 priority patent/US8575088B2/en

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    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of genetic engineering technology and gene function application, and relates to a new medicinal use of recombinant soluble tumor necrosis factor receptor (HusTNFR) gene, specifically relates to long-acting human recombinant soluble tumor necrosis factor alpha receptor (LHusTNFR) for acute and sub- Prophylactic and therapeutic use of liver in acute liver failure.
  • HumanTNFR recombinant soluble tumor necrosis factor receptor
  • LHusTNFR long-acting human recombinant soluble tumor necrosis factor alpha receptor
  • Fulminant hepatic failure refers to the absence of liver disease in patients before the disease and a large number of hepatocyte necrosis or severe liver damage in a short period of time, and hepatic development occurs 8 weeks after the onset of the first symptoms or within 10 days after the onset of jaundice.
  • a syndrome of encephalopathy It is characterized by acute onset, critical illness, lack of effective treatment, and high mortality.
  • Acute inflammation and necrosis of hepatocytes can lead to two different diseases: acute hepatitis and fulminant hepatic failure (also known as acute liver failure).
  • acute hepatitis hepatitis caused by hepatitis virus is called acute viral hepatitis; hepatitis caused by alcohol is called acute alcoholic hepatitis.
  • acute viral hepatitis hepatitis caused by alcohol
  • acute alcoholic hepatitis hepatocyte necrosis is prevalent in the above-mentioned hepatitis, the degree of necrosis is light enough to cause a malfunction of the liver's normal function.
  • TNF ci binding to its receptor is the initial pathway for activating hepatocyte necrosis and plays an important role in the mechanism of hepatocyte injury. If blocking TNF a binds to its receptor, it blocks the originating pathway of hepatocyte necrosis, making it possible to treat FHF.
  • TNF a inhibitors that directly block TNF a binding to its receptor: TNF a monoclonal antibody, soluble TNF a receptor analog. These TNF a inhibitors can bind to TNF a in the body, thereby theoretically preventing the binding of TNF a in the blood or intercellular fluid to the TNF a receptor on the hepatocyte membrane to activate the hepatic cell death pathway.
  • TNF a monoclonal antibody in addition to blocking the binding of TNF a to type I receptor, also activates TNF a on the cell membrane to cause apoptosis of target cells, and thus is not suitable as a drug candidate.
  • a long-acting soluble tumor necrosis factor alpha receptor for the manufacture of a medicament for the prevention or treatment of hepatocyte necrosis or liver failure.
  • the long-acting soluble tumor necrosis factor alpha receptor is a long-acting human recombinant soluble tumor necrosis factor alpha receptor.
  • the long-acting soluble tumor necrosis factor alpha receptor has a half-life of from 12 to 140 hours (more preferably, from 24-72 hours).
  • the long-acting soluble tumor necrosis factor alpha receptor is selected from the group consisting of: a. a fusion protein of human type I tumor necrosis factor alpha receptor and human IgGl: Fc fragment (more preferably, The carboxy terminus of the human type I tumor necrosis factor alpha receptor is linked to the amino terminus of the IgG:Fc fragment),
  • a fusion protein of human type II tumor necrosis factor alpha receptor and human IgG1 : Fc fragment (more preferably, the carboxy terminus of human type II tumor necrosis factor alpha receptor is linked to the amino terminus of IgG: Fc fragment),
  • the long-acting soluble tumor necrosis factor alpha receptor can decrease the IL-6 level of hepatocytes by 40-50%; or
  • the liver failure is acute and/or subacute liver failure.
  • the hepatocyte necrosis is a large area of hepatocyte necrosis.
  • the hepatocyte necrosis is acute large area hepatocyte necrosis.
  • a pharmaceutical composition comprising:
  • an effective amount e.g., 0.0001 to 50% by weight, more preferably 0.0001 to 20% by weight, most preferably 0.001 to 10% by weight
  • a long-acting soluble tumor necrosis factor alpha selected from the group consisting of Body:
  • fusion protein of human type II tumor necrosis factor alpha receptor and human IgG1 Fc fragment
  • k a fusion protein of human type II tumor necrosis factor ct receptor and human serum albumin
  • the pharmaceutical composition further contains an effective amount (such as 0.0001-50wt%, more preferably 0.0001-20wt%, most preferably 0. 001- 10wt3 ⁇ 4) - one or more drugs selected from the group consisting of:
  • hepatocyte growth factor huHGF
  • glutathione reduced glutathione
  • matrine a method for preventing or treating hepatocyte necrosis or liver failure, wherein an effective amount is administered to a person in need of treatment (e.g., 0.0001 to 50% by weight, more preferably 0.0001 to 20wV3 ⁇ 4, most Preferably, it is 0. 001-10 wt%) of a long-acting soluble tumor necrosis factor alpha receptor.
  • the long-acting soluble tumor necrosis factor alpha receptor is selected from the group consisting of: a. a fusion protein of human type I tumor necrosis factor alpha receptor and human IgG1 : Fc fragment,
  • fusion protein of human type II tumor necrosis factor alpha receptor and human IgG1 Fc fragment
  • liver failure is acute and/or subacute liver failure.
  • the present inventors After long-term and extensive research and experiments, the present inventors have found for the first time that the prevention and treatment of acute necrosis of large-area hepatocytes requires a sustained and stable blocking effect of soluble TNF a receptor on hepatocytes. It is necessary to maintain a stable and long-lasting concentration of soluble TNF ci receptor in the blood and liver of the body. For mild hepatocyte necrosis in acute hepatitis, a short-acting, pulse-type conventional TNF ci receptor can achieve results.
  • the inventors have revealed that the conventional soluble TNF ⁇ receptor cannot effectively prevent large-area acute hepatocyte necrosis in acute liver failure because of its short half-life and instability in the body, and the inability to maintain stable and long-lasting anti-hepatocyte necrosis. . Therefore, the present inventors have modified a TNF a receptor by various methods to prepare a long-acting TNF ci receptor which can maintain stable and sustained therapeutic effects in the blood and liver of the body, and effectively prolongs the TNF cc receptor.
  • long-acting soluble tumor necrosis factor a receptor refers to a tumor necrosis factor a 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 a 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 a receptor to the human IgGl:Fc fragment, and linking the tumor necrosis factor a receptor to the PEG, using PEG -
  • the liposome mixture entraps the tumor necrosis factor a receptor or binds the tumor necrosis factor a receptor to human serum albumin.
  • the "long-acting soluble tumor necrosis factor alpha receptor" is a "long-acting human recombinant soluble tumor necrosis factor a receptor".
  • the object of the present invention is to provide a novel medicinal use of the recombinant soluble tumor necrosis factor a receptor (HusTNFR) gene, in particular a long-acting human recombinant formed by recombinant soluble tumor necrosis factor a receptor (HusTNFR) gene or modified HusTNFR protein.
  • the invention adopts a long-acting human recombinant soluble tumor necrosis factor a receptor to interfere with fulminant hepatic failure in mice through a classical animal model of acute and subacute liver failure, and the results show that the mortality rates of the Qianqian group and the model group are respectively It is 0 and 80%.
  • the above tumor necrosis factor is a recombinant long-acting soluble protein that binds tumor necrosis factor a to the corresponding cell membrane receptor, including long-acting human recombinant soluble type I tumor necrosis factor a receptor (LHusTNFRI) and long-acting human weight.
  • LHusTNFRI long-acting human recombinant soluble type I tumor necrosis factor a receptor
  • the soluble half-type tumor necrosis factor alpha receptor has a half-life longer than that of the normal human recombinant soluble type I (HusTNFRI) and type II tumor necrosis factor alpha receptor (HusTNFRII).
  • 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, or 4) Human serum albumin is linked at the amino or carboxy terminus.
  • the LHusTNFRI and LHusTNFRII are more effective in preventing and treating acute and subacute liver failure than HusTNFRI or HusTNFRII.
  • the animal model of acute, subacute liver failure is prepared by the classical D-galactosamine and endotoxin intradermal injection of large (small) mice.
  • the animal model has a mortality rate of 60-80% due to liver failure within 48 hours.
  • TNFR human type I TNF a receptor
  • human IgG1 Fc fragment fusion gene
  • human type II TNF ci receptor and human IgGl Fc fragment fusion gene expressed recombinant protein
  • PEG-liposome mixture entraps human type I TNF a receptor protein
  • PEG-liposome mixture entraps human type II TNF a receptor protein
  • a recombinant protein expressed by human type I TNF a receptor and human serum albumin fusion gene or
  • LHusTNFR human recombinant soluble tumor necrosis factor- ⁇ receptor
  • a method for producing a type I LHusTNFR gene of SEQ ID NO: 1, comprising encoding a gene encoding a type I sTNFR (human) extracellular amino acid (ie, positions 1-171 of SEQ ID NO: 1) Human immunoglobulin Y 1 chain Fc fragment (IgG1 : Fc) amino acid (ie 172-403 of SEQ ID NO: 1 (recommended preferentially with the relevant plasmid, DNA restriction endonuclease digestion screening for carrying 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 (g236 236-467 of 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-IgG1: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 uses a prokaryotic expression vector, such as PET28 or the like.
  • 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 present invention uses Saccharomyces cerevisiae BL21 and the like.
  • the expression product of the present invention is present in the cell body of the host cell as an inclusion body, and the inclusion body is isolated by bacteria, and the inclusion body is dissolved in a high concentration of urea or guanidine hydrochloride, and the LHusTNFR is isolated and purified, and an active type I is obtained after appropriate renaturation.
  • a cDNA encoding a type I sTNFR (human) extracellular amino acid (i.e., position 1-171 of SEQ ID NO: 1) was recombined with an expression vector to form a recombinant expression plasmid (SEQ ID NO: 3).
  • the invention is not limited to a particular expression plasmid.
  • the invention uses a prokaryotic expression vector, such as PET28 or the like.
  • 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 present invention uses Escherichia coli BL21 or the like.
  • the above expression product is present in the cell body of the host cell as an inclusion body, and the inclusion body is isolated by bacteria, and the inclusion body is dissolved by high concentration urea or guanidine hydrochloride, and the type I HusTNFR is isolated and purified, and the active type I HusTNFR is obtained after appropriate renaturation. .
  • Long-acting type I HusTNFR was synthesized by coupling active raPEG having a molecular weight (MW) of 2, 0000 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, MN 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 f3 ⁇ 4 7. 9 and the time was 12 hours.
  • the cDNA encoding the type II sTNFR (human) extracellular amino acid 1-235 was recombined with an expression vector to form a recombinant expression plasmid (SEQ ID NO: 4).
  • the invention is not limited to a particular expression plasmid.
  • the invention uses a prokaryotic expression vector, such as pET28 and the like.
  • 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 present invention uses Escherichia coli BL21 or the like.
  • the above expression product is present in the cell body of the host cell as an inclusion body, and the inclusion body is isolated by bacteria, and the inclusion body is dissolved by high concentration urea or guanidine hydrochloride, and the type II HusTNFR is isolated and purified, and the active scorpion HusTNFR is obtained after appropriate renaturation. .
  • the long-acting type II HusTNFR was synthesized by coupling an active mPEG having a molecular weight of 2, 0000 or more to the amino terminus or the carboxy terminus of the 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 II HusTNFR.
  • the carboxy terminus of type II HusTNFR was coupled using mPEG2-NHS easter, MW 4, 0000 (Shearwater corporation, New Jersey, USA).
  • the reaction conditions were pH 7.9 and the time was 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.
  • DOPE Dioleoylphosphatidylethanolamine
  • NHS-PEG 354 under triethylamine catalyzed conditions.
  • -MAL Nhydroxysulfosuccinimide
  • DOPE-PEG- MAL prepared above and EPC (L-a-Phosphatidylcholine, MW 760. 09), cholesterol (MW 386. 67) and mPEG-2000-DOPE (MW obtained 2801. 51) (Aanti Polar Lipids, USA)
  • EPC Lipid Precipients
  • mPEG-2000-DOPE MW obtained 2801. 51
  • PBS pH 7.4
  • a type II TNF human extracellular amino acid
  • G(n)S - linker ie, 236-245
  • the fusion cDNA fragment of type I and type II TNF ⁇ receptor-human serum albumin described above was reconstituted with an expression vector to form a recombinant expression plasmid.
  • the invention is not limited to a particular expression plasmid.
  • the present invention uses a yeast eukaryotic expression vector, such as Saccharomyces cerevisiae or pichia yeast.
  • 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 present invention uses Saccharomyces cerevisiae BL21 and the like.
  • the expression product of the present invention is present in the cell body of the host cell in the form of inclusion bodies, and the inclusion body is isolated by bacteria.
  • the high concentration urea or guanidine hydrochloride dissolves the inclusion body, and the LHusTNFR is isolated and purified, and the active LHusTNFR is obtained after appropriate renaturation.
  • 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 alpha receptor is only 50 minutes to 2 hours.
  • the invention adopts the genetic engineering method to produce LHusTNFR, and the obtained product has anti-acute and acute effects.
  • the function of acute liver failure Compared with the conventional HusTNFR properties, the half-life of LHusTNFR was significantly prolonged, and the efficacy of preventing and treating acute and subacute liver failure and reducing mortality was significantly improved.
  • the present invention also provides a pharmaceutical composition for treating hepatocyte necrosis or liver failure 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, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually about 5-8, preferably at a pH of about 6-8, although the ⁇ value can vary with The nature of the substance being formulated 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 liver failure.
  • other related therapeutic agents can be used simultaneously.
  • therapeutic agents include, but are not limited to, human hepatocyte growth factor (huHGF), reduced glutathione, and matrine.
  • 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 prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 0.1 microgram per kilogram of body weight per day to about 5 milligrams per kilogram of body weight.
  • 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 milligram per kilogram body weight.
  • specific doses should also take into account 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 merely illustrative of the invention and are not intended to limit the scope of the invention.
  • Example 1 Long-acting human recombinant soluble type I tumor necrosis factor (TNF) alpha receptor (type I LHusTNFR) represented by type I HusTNFR-IgGl: Fc was used to prevent acute (explosive) liver failure in mice.
  • TNF tumor necrosis factor
  • the mouse model of fulminant hepatic failure was prepared by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) or Con-A (T cell mitogens concanavalin A), respectively, with a 48-hour mortality rate of 80% and 50%, respectively.
  • Gross liver specimens showed severe hyperemia and enlargement of the liver; pathological sections stained with HE showed large-area, severe hepatocyte necrosis.
  • the type I LHusTNFR prepared as described above (type I LHusTNFR-IgGl: Fc prepared by method (1) is a prophylactic drug) subcutaneously injected into C57BL/6 mice (GalN/LPS group) or BALB/c mice (Con-A group) , the dose is 12.
  • 5mg / kg is the long-acting type I receptor prevention group; conventional HusTNFR subcutaneous injection of C57BL / 6 mice (GalN / LPS group) or BALB / c mice (Con-A group), dose 12 5 mg/kg, which is a conventional type I receptor prevention group; the control group is a subcutaneous injection of the same volume of normal saline. After 16 hours, the prevention group and the control group were observed by subcutaneous injection of GalN/LPS or Con-A for 48 hours. After 48 hours, the mortality of the three groups was 80% in the GalN/LPS model control group (50% in the Con-A model control group) and 50% in the conventional type I receptor prevention group (30% in the Con-A group).
  • the long-acting type I receptor prevention group was 0% (0 in the Con-A group). Gross pathology showed that the liver of the long-acting type I receptor prevention group was only mildly hyperemic and swollen. HE staining showed mild hepatic cells and focal necrosis. No large area of hepatocyte necrosis was observed in the control group. Total RNA and nuclear protein were extracted from the liver. Real-time PCR was used to detect IL-6, MIP-2 and bcl-xL mRNA levels, and EMSA was used to detect NF-kB levels.
  • IL-6, MIP-2 and bcl-xL mRNA levels decreased by 82.3% in the long-acting type I receptors in the GalN/LPS acute liver failure prevention group compared with the conventional type I receptor prevention group and the control group, respectively.
  • 4% and 37.5%, 78. 1%, 52. 2%; 84.3%, 37.8%, NF-kB levels decreased by 87. 4% and 37.5%, respectively.
  • type I soluble TNF cx receptors block the binding of TNF ct to type I receptors in acute liver failure, inhibiting TNF ⁇ from causing hepatocyte necrosis by transmitting signals to hepatocyte membrane type I receptors to the nucleus. Acute liver failure.
  • Example 2 Long-acting human recombinant soluble type II tumor necrosis factor (TNF) ⁇ receptor ( ⁇ LHusTNFR) represented by type II HusTNFR-IgGl: Fc was used to prevent acute (explosive) liver failure in mice.
  • TNF tumor necrosis factor
  • ⁇ LHusTNFR type II HusTNFR-IgGl
  • mice model of fulminant hepatic failure was prepared by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) or Con-A (T cell mitogens concanavalin A), with a 48-hour mortality rate of 80% and 50%, respectively.
  • Gross liver specimens showed severe hyperemia and enlargement of the liver; pathological sections stained with HE showed extensive and severe hepatocyte necrosis.
  • the type II LHusTNFR produced by the present invention (type II LHusTNFR-IgGl prepared by the method (2): Fc is a prophylactic drug) is subcutaneously injected into C57BL/6 mice (GalN/LPS group) or BALB/c mice (Con-A). Group), the dose is 12. 5mg/kg, which is the long-acting type II receptor prevention group; conventional HusTNFR is injected subcutaneously into C57BL/6 mice (GalN/LPS group) or BALB/c mice (Con-A group), The dose was 12. 5 mg/kg, which was the conventional type II receptor prevention group; the control group was the same type of mice injected subcutaneously with the same volume of physiological saline.
  • the prevention group and the control group were observed by subcutaneous injection of GalN/LPS or Con-A for 48 hours.
  • the mortality of the three groups was 80% in the GalN/LPS model control group (50% in the Con-A model control group) and 50% in the conventional type I receptor prevention group (Con-A group) 30%), 0% of the long-acting type I receptor prevention group (0 in the Con-A group).
  • Gross pathology showed that the liver of the long-acting type I receptor prevention group was only mildly hyperemic and swollen.
  • HE staining showed mild hepatic cells and focal necrosis. No large area of hepatocyte necrosis was observed in the control group.
  • RNA and nuclear protein were extracted from the liver.
  • Real-time PCR was used to detect IL-6, MIP-2 and bcl-xL mRNA levels
  • EMSA was used to detect NF-kB levels.
  • the result shows that long-acting type II The receptors in the GalN/LPS acute liver failure prevention group and the conventional type II receptor prevention group compared with the control group, IL-6, MIP-2 and bcl-xL mRNA levels decreased by 78. 3 ° /. , 443 ⁇ 4); 72. 1%, 52.2%; 77.3%, 37.8%, NF-kB levels decreased by 78.4% and 37.5%, respectively.
  • Type II soluble TNF ⁇ receptor inhibits the binding of TNF a to type II receptor in acute liver failure, and inhibits TNF a from causing hepatocyte necrosis by transmitting signals to hepatocyte membrane type II receptor to the nucleus. Acute liver failure. The results also showed that the effect of long-acting sputum soluble TNF a receptor on acute liver failure was significantly higher than that of conventional type II soluble TNF a receptor.
  • TNF tumor necrosis factor
  • ⁇ LHusTNFR Long-acting human recombinant soluble type II tumor necrosis factor (TNF) a receptor ( ⁇ LHusTNFR) represented by type II HusTNFR-human serum albumin was used to prevent acute (explosive) liver failure in mice.
  • the mouse model of fulminant hepatic failure was prepared by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) or Con_A (T cell mitogens concanavalin A), with a 48-hour mortality rate of 80% and 50%, respectively.
  • Gross liver specimens showed severe hyperemia and enlargement of the liver; pathological sections stained with HE showed extensive and severe hepatocyte necrosis.
  • Type II LHusTNFR prepared by the present invention is a prophylactic drug subcutaneously injected into C57BL/6 mice (GalN/LPS group) or BALB/c mice (Con-A) Group), dose between 5-30 mg/kg, for long-acting type II receptor prevention group; conventional HusTNFR subcutaneous injection of C57BL/6 mice (GalN/LPS group) or BALB/c mice (Con-A group) The dose was 12. 5 mg/kg, which was the conventional type II receptor prevention group; the control group was the same type of mice injected subcutaneously with the same volume of physiological saline.
  • the prevention group and the control group were observed 48 hours after subcutaneous injection of GalN/LPS or Con-A.
  • the mortality of the three groups was 80% in the GalN/LPS model control group (50% in the Con-A model control group) and 50% in the conventional type I receptor prevention group (Con-A group) 30%), 0% of the long-acting type I receptor prevention group (0 in the Con-A group).
  • Gross pathology showed that the long-acting type I receptor prevention group had only mild hyperemia and swelling in the liver.
  • HE staining showed that the liver cells were mild and spotted, and no large area of hepatocyte necrosis was observed in the control group.
  • Total RNA and nuclear protein were extracted from the liver.
  • Type II TNFR-human serum albumin inhibits the binding of TNF ⁇ to type I receptors in acute liver failure, and inhibits TNF ⁇ from causing hepatocyte necrosis by transmitting signals to hepatocyte membrane type I receptors to the nucleus. Acute liver failure.
  • the results also showed that the long-acting type II soluble TNF a receptor significantly improved the acute liver failure compared with the conventional type II soluble TNF a receptor.
  • Example 4 Long-acting human recombinant soluble type I tumor necrosis factor (TNF) a receptor (type I LHusTNFR) represented by type I HusTNFR-IgGl: Fc was used to prevent subacute liver failure in rats.
  • Rat subacute liver failure model was prepared by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) with a weekly mortality rate of 60%. Gross liver specimens showed severe congestion and swelling of the liver; pathological sections stained with HE showed large areas, Severe hepatocyte necrosis.
  • GalN/LPS D-galactosamine/endotoxin
  • the type I LHusTNFR prepared by the present invention (the type I LHusTNFR-IgGl: Fc prepared by the method (1) is a prophylactic drug) is administered subcutaneously to a Spraque-Dawley rat at a dose of 12. 5rag/kg, which is a long-acting type I receptor prevention group.
  • Conventional HusTNFR was injected subcutaneously with Spraque-Dawley rats 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. After 16 hours, the prevention group and the control group were observed for one week after subcutaneous injection of GalN/LPS.
  • RNA and nuclear protein were extracted from the liver. Real-time PCR was used to detect IL-6, MIP-2 and bcl-xL mRNA levels, and EMSA was used to detect NF-kB levels.
  • Example 5 Long-acting human recombinant soluble type II tumor necrosis factor (TNF) alpha receptor (type II LHusTNFR) represented by type II HusTNFR-IgGl: Fc to prevent subacute liver failure in rats
  • TNF tumor necrosis factor
  • Rat subacute liver failure model was prepared by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) with a weekly mortality rate of 60%. Gross liver specimens showed severe hyperemia and enlargement of the liver; pathological sections stained with HE showed extensive and severe hepatocyte necrosis.
  • GalN/LPS D-galactosamine/endotoxin
  • the preparation type LHusTNFR prepared by the present invention (type II LHusTNFR-IgGl prepared by the method (2): Fc is a prophylactic drug) is administered subcutaneously to Spraque-Dawley rats at a dose of 12.5 mg/kg, which is a long-acting type II receptor prevention group.
  • Conventional HusTNFR was injected subcutaneously into Spraque-Dawley rats 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. After 16 hours, the prevention group and the control group were observed for one week after subcutaneous injection of GalN/LPS.
  • RNA and nuclear protein were extracted from the liver. Real-time PCR was used to detect IL-6, MIP-2 and bcl-xL mRNA levels, and EMSA was used to detect NF-kB levels.
  • IL-6, MIP-2 and bcl-xL mRNA levels decreased by 88.5%, 46.7%, respectively, in the long-acting type II receptor prophylaxis group compared with the conventional type II receptor prophylaxis group and the control group, respectively.
  • 1%, 52. 2%; 78.3%, 37.8%, NF-kB levels decreased by 92.1% and 37.5%, respectively.
  • type II soluble TNF t receptor blocks TNF cc and type I in acute liver failure.
  • the combination of the body inhibits acute liver failure in which TNFa occurs by causing hepatocyte necrosis by transmitting signals to the nucleus of the hepatocyte membrane type I receptor.
  • Example 6 Long-acting human recombinant soluble type II tumor necrosis factor (TNF) a receptor (type II LHusTNFR) represented by type II HusTNFR-human serum albumin to prevent subacute liver failure in rats
  • Rat subacute liver failure model was induced by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) with a weekly mortality rate of 60%.
  • Gross liver specimens showed severe hyperemia and enlargement of the liver; pathological sections stained with HE showed extensive and severe hepatocyte necrosis.
  • Type II LHusTNFR prepared by the present invention (Method (6> Preparation of Type II LHusTNFR - human serum albumin as a prophylactic drug) is administered subcutaneously to Spraque-Dawley rats at a dose of 5-30 m g / kg, which is a long-acting type II receptor.
  • the prevention group conventional HusTNFR subcutaneous injection of Spraque-Dawley rats, the dose of 12. 5rag / kg, is the conventional type II receptor prevention group;
  • the control group is subcutaneous injection of the same volume of saline in the same type of mice. 16 hours after the prevention group and control The group was observed by subcutaneous injection of GalN/LPS for one week.
  • RNA and nucleus were extracted from the liver. Protein, real-time PCR was used to detect IL-6, MIP-2 and bcl-xL mRNA levels, and EMSA was used to detect NF-kB levels.
  • results also indicate long-acting type II soluble TNF The effect of a receptor on prevention of acute liver failure is significantly higher than that of conventional type II soluble TNFa receptor.
  • TNF tumor necrosis factor
  • Example 7 Long-acting human recombinant soluble type I tumor necrosis factor (TNF) represented by type II HusTNFR-IgGl: Fc A receptor (type I LHusTNFR) in the treatment of subacute liver failure in rats
  • Rat subacute liver failure model was prepared by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) with a weekly mortality rate of 60%. Gross liver specimens showed severe hyperemia and enlargement of the liver; pathological sections stained with HE showed extensive and severe hepatocyte necrosis.
  • GalN/LPS D-galactosamine/endotoxin
  • a rat model of subacute liver failure was prepared by subcutaneous injection of GalN/LPS. After 8 hours, subcutaneous injection of type I LHusTNFR (method (1) type I LHusTNFR-IgGl: Fc is a prophylactic agent), dose 12. 5 mg / kg, for long-acting type I receptor treatment group; conventional HusTNFR subcutaneous injection of Spraque- Dawley rats, dose 12. 5rag / kg, for the conventional type I receptor treatment group; control group for subcutaneous injection of the same volume of normal saline Rat. Observe for a week. One week after GalN/LPS injection, the three groups of mortality were: 60% in the control group, 30% in the conventional type I receptor prevention group, and 0% in the long-acting type I receptor prevention group.
  • RNA and nuclear protein were extracted from the liver.
  • Real-time PCR was used to detect IL-6, MIP-2 and bcl-xL mRNA levels, and EMSA was used to detect NF-kB levels.
  • the results showed that IL-6, MIP-2 and bcl-xL mRM levels decreased by 90.5%, 46.7%, respectively, in the long-acting type I receptor prevention group compared with the conventional type I receptor prevention group and the control group, respectively. 1%, 52.
  • Rat subacute liver failure model was induced by subcutaneous injection of D-galactosamine/endotoxin (GalN/LPS) with a weekly mortality rate of 60%.
  • Gross liver specimens showed severe hyperemia and enlargement of the liver; pathological sections stained with HE showed extensive and severe hepatocyte necrosis.
  • a rat model of subacute liver failure was prepared by subcutaneous injection of GalN/LPS. After 8 hours, subcutaneous injection of type II LHusTNFR (Method (1) type II LHusTNFR-IgGl: Fc is a prophylactic drug), dose 12. 5 mg / kg, for long-acting type II receptor treatment group; conventional HusTNFR subcutaneous injection of Spraque- Dawley rats, dose 12. 5 mg / kg, were the conventional type II receptor treatment group; the control group was the same type of mice injected subcutaneously with the same volume of normal saline. Observe for a week.
  • RNA and nuclear protein were extracted from the liver. Real-time PCR was used to detect IL-6, MIP-2 and bcl-xL mRNA levels, and EMSA was used to detect NF-kB levels.
  • IL-6, MIP-2 and bcl-xL mRNA levels decreased by 89%, 44.3%, 76%, 49, respectively, in the long-acting type II receptor prophylaxis group compared with the conventional type II receptor prophylaxis group and the control group.
  • % and 83%, 35.2%, NF-kB levels decreased by 79.6% and 36%, respectively.
  • type II soluble TNF ⁇ receptor inhibits TNF a binding to hepatocyte membrane by blocking the binding of TNF a to type II receptor in acute liver failure; Subacute receptors transmit signals to the nucleus leading to subacute liver failure that occurs in hepatocyte necrosis.
  • Example 9 Long-acting human recombinant soluble type II tumor represented by type II HusTNFR-human serum albumin Necrosis factor (TNF) ⁇ receptor (type II LHusTNFR) in rats with subacute liver failure.
  • TNF human recombinant soluble type II tumor represented by type II HusTNFR-human serum albumin Necrosis factor (TNF) ⁇ receptor (type II LHusTNFR) in rats with subacute liver failure.
  • TNF human recombinant soluble type II tumor represented by type II HusTNFR-human serum albumin Necrosis factor (TNF) ⁇ receptor (type II LHusTNFR) in rats with subacute liver failure.
  • TNF human recombinant soluble type II tumor represented by type II HusTNFR-human serum albumin Necrosis factor (TNF) ⁇ receptor (type II LHusTNFR) in rats with subacute liver failure.
  • TNF human recombinant soluble type II tumor represented by type II HusTNFR-human
  • a rat model of subacute liver failure was prepared by subcutaneous injection of GalN/LPS. After 8 hours, subcutaneous injection of type II LHusTNFR (Method (6) type II LHusTNFR-human serum albumin as a prophylactic drug), dose 5-30 mg / kg, for the long-acting type II receptor treatment group; conventional HusTNFR subcutaneous injection of Spraque - Dawley rats, dose 12. 5 mg / kg, for the conventional sputum receptor treatment group; the control group was the same type of mice injected subcutaneously with the same volume of normal saline. Observe for a week. One week after GalN/LPS injection, the three groups of mortality were: control group 60. /.
  • RNA and nuclear protein were extracted from the liver. Real-time PCR was used to detect IL-6, MIP-2 and bcl xL mRNA levels, and EMSA was used to detect NF-kB levels.

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Description

长效人重组可溶性肿瘤坏死因子 α受体在制备防治肝衰竭药物中的用途 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《 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)对急性及亚急性肝衰竭肝的预防和治疗用途。 背景技术
暴发性肝功能衰竭（fulminant hepatic failure, FHF)是指病前患者无肝病而短 期内出现大量肝细胞坏死或肝功能严重损害，并在首发症状出现后 8周或黄疸出现后 10 天内发生肝性脑病的一种综合征。 其特点为起病急， 病情危重， 缺乏有效治疗手 段， 死亡率高。
肝细胞的急性炎症和坏死可导致两种不同的疾病：急性肝炎和暴发性肝功能衰竭 (又称急性肝功能衰竭） 。 在急性肝炎中， 由肝炎病毒造成的肝炎称为急性病毒性肝 炎；由酒精引起的肝炎称为急性酒精性肝炎。虽然肝细胞坏死普遍存在于上述肝炎中， 但坏死程度轻，不足以造成肝脏正常功能的运转障碍。 FHF 与各种类型的急性肝炎区 别在于前者发生十分严重的肝细胞快速坏死，导致残存的正常肝细胞不能维持肝脏功 能的正常运转而发生肝功能衰竭， 由此引发急遽增高的死亡率。
在我国引起 FHF的主要原因是肝炎病毒感染，其次为药物及毒物中毒，缺血缺氧， 代谢紊乱， 自身免疫性肝炎等等。 目前尚没有特异性针对阻断肝细胞急性坏死发生的 药物， 因此无法有效防止因大面积肝细胞坏死所造成的肝功能衰竭， FHF死亡率一直 难以降低。临床上迫切需要具有特异性快速阻止肝细胞急性坏死的药物以治疗肝功能 衰竭。
近年来的研究发现， TNF ci与其受体结合是激活肝细胞坏死的始发通路，在肝细胞 损伤机制中占有重要地位。 如果阻断 TNF a与其受体结合， 也就阻断了肝细胞坏死的 始发通路， 从而使药物治疗 FHF成为可能。
目前直接阻断 TNF a与其受体结合的 TNF a抑制剂主要有两类： TNF a单克隆抗 体， 可溶性 TNF a受体类似物。 这些 TNF a抑制剂进入体内可以与 TNF a结合， 从而 从理论上阻止血液或细胞间液中 TNF a与肝细胞膜上 TNF a受体结合而激活肝细胞坏 死通路。
近来有研究发现， TNF a单克隆抗体除阻断 TNF a与 I型受体结合外， 还同时激 活细胞膜上 TNF a导致靶细胞凋亡的特点， 因此不适合作为候选药物。
常规可溶性 TNF a受体在一定程度上可降低绝大多数轻度肝细胞坏死、 急性肝炎 动物模型死亡率。但是， 无法有效降低因大面积肝细胞坏死导致的急性或亚急性肝衰
1
确认本 竭的死亡率。 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 a受体应用于临床治疗大面积肝细胞坏死或肝衰竭。
综上， 本领域迫切需要找到 TNF a受体对于肝细胞坏死疗效差的关键所在， 以进 一步对 TNF ci受体进行改良， 使之能有效、 快速阻止大面积肝细胞急性坏死的发生。 从而在临床上真正成为用于防治急性、 亚急性肝衰竭的良药。 发明内容
在本发明的第一方面， 提供长效可溶性肿瘤坏死因子 α受体在制备防治肝细胞 坏死或肝衰竭药物中的用途。
在本发明的另一优选例中， 所述的长效可溶性肿瘤坏死因子 α受体为长效人重 组可溶性肿瘤坏死因子 α受体。
在本发明的另一优选例中 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（更优选的， 为 24-72小时）。
在本发明的另一优选例中， 所述的长效可溶性肿瘤坏死因子 α受体选自： a.人 I 型肿瘤坏死因子 α受体与人 IgGl : Fc片段的融合蛋白（更优选的， 为人 I 型肿瘤坏死因子 α受体的羧基末端与 IgG: Fc片段的氨基末端相连接），
b.人 II型肿瘤坏死因子 α受体与人 IgGl : Fc片段的融合蛋白（更优选的， 为人 II 型肿瘤坏死因子 α受体的羧基末端与 IgG: Fc片段的氨基末端相连接），
c人 I型肿瘤坏死因子 α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 α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 α受体蛋白的产物，
i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 α受体蛋白的产物，
j.人 I型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白， 或
k.人 II型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白。
在本发明的另一优选例中， 所述的长效可溶性肿瘤坏死因子 α受体可使肝细胞 的 IL- 6水平下降 40-50%; 或
使肝细胞的 ΜΙΡ-2水平下降 50- 60%; 或
使肝细胞的 bcl- xl水平下降 30-45%; 或
使肝细胞的 NF- B水平下降 30-45%。
在本发明的另一优选例中， 所述的肝衰竭是急性和 /或亚急性肝衰竭。
在本发明的另一优选例中， 所述的肝细胞坏死为大面积肝细胞坏死。
在本发明的另一优选例中， 所述的肝细胞坏死是急性大面积肝细胞坏死。 在本发明的第二方面， 提供一种药物组合物， 所述的药物组合物含有：
(i) 有效量（如 0. 00001- 50wt%， 更优选的为 0. 0001- 20wt%， 最优选的为 0. 001-10wt%)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 α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 α受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 α受体蛋白羧基端与 PEG联接的产物，
e.人 II型肿瘤坏死因子 α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 ct受体蛋白的产物，
i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 α受体蛋白的产物，
j.人 I型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白， 或
k.人 II型肿瘤坏死因子 ct受体与人血清白蛋白的融合蛋白； 以及
(ii) 药学上可接受的载体。
在本发明的另一优选例中， 所述的药物组合物中还含有有效量的（如 0. 00001-50wt%, 更优选的为 0. 0001-20wt%, 最优选的为 0. 001- 10wt¾)—种或多种选 自以下的药物：
(i i i) 人肝细胞生长因子 （huHGF) 、 还原型谷胱甘肽、 或苦参碱。 在本发明的第三方面， 提供一种预防或治疗肝细胞坏死或肝衰竭的方法， 给予 需要治疗的人员有效量（如 0. 00001- 50wt%， 更优选的为 0. 0001- 20wV¾， 最优选的为 0. 001-10wt%)的长效可溶性肿瘤坏死因子 α受体。
在本发明的另一优选例中， 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 α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 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 α受体蛋白羧基端与 PEG联接的产物，
e.人 II型肿瘤坏死因子 α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 α受体蛋白的产物，
i .用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 α受体蛋 ¾的产物，
j.人 I型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白， 或
k.人 II型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白。 在本发明的另一优选例中， 所述的肝衰竭是急性和 /或亚急性肝衰竭。 具体实施方式
本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 TNF a受体对肝细胞持续、 稳定的阻断作用。 即需在机体的血液及肝脏中维 持稳定、 持久的可溶性 TNF ci受体浓度。 而对于急性肝炎中的轻度肝细胞坏死， 作用 时间短、脉冲型的常规 TNF ci受体即可取得效果。因此本发明人揭示了常规可溶性 TNF α受体无法有效阻止急性肝衰竭中大面积急性肝细胞坏死的原因在于其在机体内半 衰期短， 不稳定， 而无法维持稳定、 持久抗肝细胞坏死的作用。 因此， 本发明人通过 多种方法对 TNF a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 TNF ci受体， 有效延长了 TNF cc受体作为活性部分在体内的作用时 间， 大幅提髙了 TNF ct受体防治急性肝细胞坏死的疗效， 从而首次使改良后的 TNF a 受体可应用于大面积肝细胞坏死引起的急性、 亚急性肝衰竭。 基于此完成了本发明。 如本文所用， 所述的 "长效可溶性肿瘤坏死因子 a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 a受体。一般的， 所述 的 "长效可溶性肿瘤坏死因子 a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 a受体与人 IgGl : Fc片段相连接， 将肿瘤坏死因子 a受体与 PEG联接， 用 PEG-脂质 体混合物包埋肿瘤坏死因子 a受体， 或将肿瘤坏死因子 a受体与人血清白蛋白相连 接。 优选的， 所述的 "长效可溶性肿瘤坏死因子 α受体是 "长效人重组可溶性肿瘤坏 死因子 a受体" 。
本发明的目的是提供重组可溶性肿瘤坏死因子 a受体 (HusTNFR)基因的新的药用 用途， 尤其是重组可溶性肿瘤坏死因子 a受体（HusTNFR)基因或修饰 HusTNFR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)的预防和治疗急性及亚 急性肝衰竭的新用途。
本发明的进一步目的在于提供一种可较常规可溶性 TNF ct受体进一步大幅降低 急性肝衰竭死亡率的药物。 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 a受体通过急性、亚急性肝衰竭的经 典动物模型， 对小鼠暴发性肝功能衰竭进行干预， 结果显示， 千预组与模型组的病死 率分别为 0和 80%。
上述肿瘤坏死因子（TNF)为肿瘤坏死因子 a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（1) 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， 或（4)在氨基或 羧基末端连接人血清白蛋白。所述的 LHusTNFRI及 LHusTNFRII , 其预防及治疗急性、 亚急性肝衰竭的疗效明显好于 HusTNFRI或 HusTNFRII。
所述的急性、 亚急性肝衰竭的动物模型采用经典的 D-氨基半乳糖与内毒素皮内 注射大（小）鼠方法制做。 所述动物模型 48 小时内其因肝衰竭而导致的死亡率高达 60-80%。
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 a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 ci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 ci受体蛋白氨基端与 PEG联接， 或
d. 人 I型 TNF ct受体蛋白羧基端与 PEG联接， 或
e. 人 II型 TNF ct受体蛋白氨基端与 PEG联接， 或
f. 人 II型 TNF ct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， 或
j . 人 I型 TNF a受体与人血清白蛋白融合基因表达的重组蛋白， 或
k. 人 II型 TNF a受体与人血清白蛋白融合基因表达的重组蛋白
制备长效人重组可溶性肿瘤坏死因子 a受体（LHusTNFR) , 用于预防 D-氨基半乳 糖与内毒素诱导小鼠急性肝衰竭模型动物 (简称急性肝衰竭动物），使急性肝衰竭动物 的死亡率由 80%下降至 0%。 使用 LHusTNFR预防和治疗 D-氨基半乳糖与内毒素诱导大 鼠亚急性肝衰竭模型动物（简称亚急性肝衰竭动物）使亚急性肝衰竭动物的死亡率由 80%下降至 0%。 结果显示， 较长半衰期的 I型或 /和 II型长效人重组可溶性肿瘤坏死 因子 a受体， 具有良好预防和治疗模型动物急性及亚急性肝衰竭的疗效， 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 a受体对急性及亚急性肝衰竭肝 具有预防和治疗用途。 可进一步制备大幅降低急性肝衰竭死亡率的药物。 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（即 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（即 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。 (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（g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， 本发明使用原核表 达载体， 例如 PET28等。
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 啤酒酵母菌 BL21等。
本发明的表达产物以包涵体形式存在于宿主细胞的胞体中， 破菌分离包涵体， 高浓度尿素或盐酸胍溶解包涵体， 分离纯化 LHusTNFR, 经适当复性后即获得有活性 的 I型或 II型 LHusTNFR- IgGl: Fc。
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（即 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， 本发明使用原核表达载 体， 例如 PET28等。
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 大肠杆菌 BL21等。 上述表达产物以包涵体形式存在于宿主细胞的胞体中， 破菌分离 包涵体， 高浓度尿素或盐酸胍溶解包涵体， 分离纯化 I型 HusTNFR, 经适当复性后即 获得有活性的 I型 HusTNFR。
将分子量（molecular weight, MW) 2, 0000 以上的有活性的 raPEG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,丽 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- CH0 + RNH2 + NaCNBH3_- mPEG- CH2- NHR
反应条件为 f¾ 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， 本发明使用原核表达载 体， 例如 pET28等。
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 大肠杆菌 BL21等。 上述表达产物以包涵体形式存在于宿主细胞的胞体中， 破菌分离 包涵体， 高浓度尿素或盐酸胍溶解包涵体， 分离纯化 II型 HusTNFR, 经适当复性后 即获得有活性的 Π型 HusTNFR。
将分子量（molecular weight, 蕭） 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- CH0 + RNH2 + NaCNBH3 --" >mPEG- CH2- NHR
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（DOPE) (分子量 744. 04,美国 Aanti Polar Lipids) 与 NHS-PEG354。-MAL (Nhydroxysulfosuccinimide
-polyoxyethylene ( W3540) -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 D0PE-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°C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 lOOnm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 HusTNFR 加入上述长循环脂质体-聚乙二醇衍生磷脂的 PBS溶液中， 4Ό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 HusTNFRo
(6) SEQ ID NO: 5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（即 SEQ ID NO : 5中 1-171位）的基因、 G (n) S— linker (即 172-181 ) 与编码人血清白蛋白氨基酸（即 SEQ ID NO : 5中 182-790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-人血清白蛋白融合片段的阳性克隆， 核苷酸序 列分析验证基因是否正确。
(7) SEQ ID NO: 6的 II型 LHusTNFR基因的制备方法，包括将编码 II型 TNF (人） 膜外氨基酸（即 SEQ ID NO : 2的 1-235位）的基因、 G ( n) S- linker (即 236— 245 ) 与编码人血清白蛋白氨基酸（即 SEQ ID NO: 6的 246- 854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-人血清白蛋白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 TNF α受体-人血清白蛋白的融合 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 明使用酵母真核表达载体， 例如啤酒酵母或 pichia酵母等。
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 啤酒酵母菌 BL21等。
本发明的表达产物以包涵体形式存在于宿主细胞的胞体中， 破菌分离包涵体， 高浓度尿素或盐酸胍溶解包涵体， 分离纯化 LHusTNFR, 经适当复性后即获得有活性 的 LHusTNFR。
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）。 经测定， I 型和 II型 HusTNFR-IgG : FC 融合蛋白在体内的有效作用浓度可维持 70-90小时（即半衰期 35-45小时）； II型 HusTNFR-人血清白蛋白在体内的有效作用 浓度可维持在 60小时左右（即半衰期 30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在体内的有效作用浓度为 6- 11天（即半衰期 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 α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， 所得产品具有髙效抗急性、 亚 急性肝衰竭的功能。 与常规 HusTNFR性质比较表明， LHusTNFR半衰期明显延长， 预 防及治疗急性、 亚急性肝衰竭， 降低死亡率的疗效明显提髙。 本发明还提供了一种治疗肝细胞坏死或肝衰竭的药物组合物， 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 ΡΗ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肝衰竭的治疗。 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： 人肝细胞生长因子 （huHGF) 、 还原型谷胱甘肽及苦参碱。
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 : 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) α受体（I型 LHusTNFR)预防小鼠急性 (爆发性)肝衰竭的实验
分别以 D-氨基半乳糖 /内毒素（GalN/LPS) 或 Con- A (T cell mitogens concanavalin A)皮下注射制作小鼠爆发性肝衰竭模型， 48小时死亡率分别为 80%与 50%。 肝脏大体 标本显示肝脏严重充血、 肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。 通过上述制备的 I型 LHusTNFR (方法（1)制备的 I型 LHusTNFR- IgGl : Fc为预防 药物）皮下注射 C57BL/6小鼠 ( GalN/LPS组） 或 BALB/c小鼠（Con- A组）， 剂量均为 12. 5mg/kg,为长效 I型受体预防组；常规 HusTNFR皮下注射 C57BL/6小鼠（GalN/LPS 组）或 BALB/c小鼠（Con-A组）， 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小鼠。 16 小时后预防组及对照组同时皮下注射 GalN/LPS或 Con- A后观察 48小时。 48小时后， 三组死亡率分别为： GalN/LPS模型 对照组 80% (Con- A模型对照组为 50%)， 常规 I型受体预防组 50% (Con- A组为 30%)， 长效 I型受体预防组 0% (Con- A组为 0)。 大体病理显示长效 I型受体预防组肝脏仅轻 度充血肿胀， HE染色显示肝细胞呈轻度、 点灶状坏死， 未见对照组大面积肝细胞坏 死。提取肝脏总 RNA及核蛋白，运用 real- time PCR检测 IL-6 , MIP-2及 bcl- xL mRNA 水平， EMSA检测 NF- kB水平。 结果显示， 长效 I型受体对 GalN/LPS急性肝衰预防组 与常规 I 型受体预防组与对照组相比 IL-6， MIP-2 及 bcl- xL mRNA 水平分别下降 82. 3%, 44% ; 78. 1%, 52. 2%； 84. 3%, 37. 8%, NF-kB水平分别下降 87. 4%及 37. 5%。 上述 结果显示 I型可溶性 TNF cx受体通过阻断急性肝衰竭中 TNF ct与 I型受体的结合， 抑 制 TNF α通过对肝细胞膜 I型受体向胞核传导信号导致肝细胞坏死而发生的急性肝衰 竭。 结果同时表明长效 I型可溶性 TNF a受体预防急性肝衰竭的效果较常规 I型可溶 性 TNF a受体有大幅提高。 实施例 2 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 (Π型 LHusTNFR)预防小鼠急性 (爆发性)肝衰竭的实验
分别以 D -氨基半乳糖 /内毒素（GalN/LPS) 或 Con-A (T cell mitogens concanavalin A)皮下注射制作小鼠爆发性肝衰竭模型， 48小时死亡率分别为 80%与 50%。 肝脏大体 标本显示肝脏严重充血、 肿大; HE染色的病理切片显示大面积、 重度的肝细胞坏死。
通过本发明制各的 II型 LHusTNFR (方法（2)制备的 II型 LHusTNFR-IgGl : Fc为 预防药物）皮下注射 C57BL/6小鼠 ( GalN/LPS组 )或 BALB/c小鼠（Con- A组） , 剂量均 为 12. 5mg/kg， 为长效 II 型受体预防组； 常规 HusTNFR 皮下注射 C57BL/6 小鼠 ( GalN/LPS组） 或 BALB/c小鼠（Con- A组）， 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小鼠。 16 小时后预防组及对照组同 时皮下注射 GalN/LPS或 Con-A后观察 48小时。 皮下注射 GalN/LPS 48小时后， 三组 死亡率分别为： GalN/LPS模型对照组 80% (Con-A模型对照组为 50%)， 常规 I型受体 预防组 50% (Con- A组为 30%)，长效 I型受体预防组 0% (Con- A组为 0)。大体病理显示 长效 I型受体预防组肝脏仅轻度充血肿胀， HE染色显示肝细胞呈轻度、 点灶状坏死， 未见对照组大面积肝细胞坏死。 提取肝脏总 RNA及核蛋白， 运用 real- time PCR 检 测 IL-6, MIP-2及 bcl-xL mRNA水平， EMSA检测 NF-kB水平。 结果显示， 长效 II型 受体对 GalN/LPS急性肝衰竭预防组与常规 II型受体预防组与对照组相比 IL- 6, MIP-2 及 bcl-xL mRNA水平分别下降 78. 3°/。，44¾) ; 72. 1%，52. 2%； 77. 3%, 37. 8%, NF- kB水平 分别下降 78. 4%及 37. 5%。 上述结果显示 II型可溶性 TNF α受体通过阻断急性肝衰竭 中 TNF a与 II型受体的结合， 抑制 TNF a通过对肝细胞膜 II型受体向胞核传导信号 导致肝细胞坏死而发生的急性肝衰竭。 结果同时表明长效 Π型可溶性 TNF a受体预 防急性肝衰竭的效果较常规 II型可溶性 TNF a受体有大幅提高。 实施例 3 以 II型 HusTNFR-人血清白蛋白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)预防小鼠急性（爆发性)肝衰竭的实验
分别以 D-氨基半乳糖 /内毒素（GalN/LPS) 或 Con_A (T cell mitogens concanavalin A)皮下注射制作小鼠爆发性肝衰竭模型， 48小时死亡率分别为 80%与 50%。 肝脏大体 标本显示肝脏严重充血、 肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。
通过本发明制备的 II型 LHusTNFR (方法（6)制备的 II型 LHusTNFR-人血清白蛋 白为预防药物）皮下注射 C57BL/6小鼠 （ GalN/LPS组）或 BALB/c小鼠（Con- A组）， 剂 量介于为 5— 30mg/kg , 为长效 II型受体预防组；.常规 HusTNFR皮下注射 C57BL/6小 鼠 （GalN/LPS组） 或 BALB/c小鼠（Con- A组）， 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小鼠。 16 小时后预防组及对照组 同时皮下注射 GalN/LPS或 Con- A后观察 48小时。 皮下注射 GalN/LPS 48小时后， 三 组死亡率分别为： GalN/LPS模型对照组 80% (Con- A模型对照组为 50%)， 常规 I型受 体预防组 50% (Con- A组为 30%)， 长效 I型受体预防组 0% (Con- A组为 0)。大体病理显 示长效 I型受体预防组肝脏仅轻度充血肿胀， HE染色显示肝细胞呈轻度、 点灶状坏 死， 未见对照组大面积肝细胞坏死。 提取肝脏总 RNA及核蛋白， 运用 real- time PCR 检测 IL- 6， MIP-2及 bcl- xL mRNA水平, EMSA检测 NF- kB水平。 结果显示， 长效 II 型受体对 GalN/LPS急性肝衰竭预防组中 IL- 6， MIP-2及 bcl- xL mRNA水平均比对照 组与常规 II型受体预防组明显下降，下降幅度介于 30— 60 %之间。 ΝΚ- κ Β分别下降 60%及 32%。上述结果显示 II型 TNFR-人血清白蛋白通过阻断急性肝衰竭中 TNF α与 I 型受体的结合， 抑制 TNF α通过对肝细胞膜 I型受体向胞核传导信号导致肝细胞坏死 而发生的急性肝衰竭。 结果同时表明长效 II型可溶性 TNF a受体预防急性肝衰竭的 效果较常规 II型可溶性 TNF a受体有大幅提高。 实施例 4: 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)预防大鼠亚急性肝衰竭的实验
以 D-氨基半乳糖 /内毒素（GalN/LPS)皮下注射制作大鼠亚急性肝衰竭模型，一周 死亡率 60%。肝脏大体标本显示肝脏严重充血、肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。
通过本发明制备的 I型 LHusTNFR (方法（1)制备的 I型 LHusTNFR- IgGl : Fc为预 防药物）皮下注射 Spraque-Dawley大鼠， 剂量 12. 5rag/kg, 为长效 I型受体预防组； 常规 HusTNFR皮下注射 Spraque- Dawley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小鼠。 16 小时后预防组及对照组 同时皮下注射 GalN/LPS后观察一周。 皮下注射 GalN/LPS—周， 三组死亡率分别为： 对照组 60%， 常规 I型受体预防组 44%, 长效 I型受体预防组 0%。 大体病理显示长效 I型受体预防组肝脏仅轻度充血肿胀， HE染色显示肝细胞呈轻度、 点灶状坏死， 未见 对照组大面积肝细胞坏死。提取肝脏总 RNA及核蛋白，运用 real-time PCR检测 IL-6, MIP-2及 bcl- xL mRNA 水平， EMSA检测 NF- kB水平。 结果显示长效 I型受体预防组 与常规 I 型受体预防组与对照组相比 IL- 6， MIP-2 及 bcl-xL mRNA 水平分别下降 90. 5%, 46. 7% ; 78. 1%, 52. 2%； 84. 3%， 37. 8%, NF-kB水平分别下降 87. 4%及 37. 5 0 上 述结果显示 I型可溶性 TNF ci受体通过阻断急性肝衰竭中 TNF ci与 1型受体的结合， 抑制 TNF a通过对肝细胞膜 I型受体向胞核传导信号导致肝细胞坏死而发生的亚急性 肝衰竭。 结果同时表明长效 I型可溶性 TNF cc受体预防亚急性肝衰竭的效果较常规 I 型可溶性 TNF a受体有大幅提高。 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) α受体（II型 LHusTNFR)预防大鼠亚急性肝衰竭的实验
以 D-氨基半乳糖 /内毒素（GalN/LPS)皮下注射制作大鼠亚急性肝衰竭模型，一周 死亡率 60%。肝脏大体标本显示肝脏严重充血、肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。
通过本发明制备的工型 LHusTNFR (方法（2)制备的 II型 LHusTNFR-IgGl： Fc为预 防药物）皮下注射 Spraque- Dawley大鼠， 剂量 12. 5mg/kg， 为长效 II型受体预防组； 常规 HusTNFR皮下注射 Spraque- Dawley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小鼠。 16 小时后预防组及对照组 同时皮下注射 GalN/LPS后观察一周。 皮下注射 GalN/LPS—周， 三组死亡率分别为： 对照组 60%， 常规 II型受体预防组 44%， 长效 I型受体预防组 0%。 大体病理显示长 效 I型受体预防组肝脏仅轻度充血肿胀， HE染色显示肝细胞呈轻度、 点灶状坏死， 未见对照组大面积肝细胞坏死。 提取肝脏总 RNA及核蛋白， 运用 real-time PCR 检 测 IL-6, MIP-2及 bcl-xL mRNA水平， EMSA检测 NF-kB水平。 结果显示长效 II型受 体预防组与常规 II型受体预防组与对照组相比 IL- 6， MIP-2及 bcl- xL mRNA水平分 别下降 88. 5%, 46. 7% ; 68. 1%, 52. 2%； 78. 3%, 37. 8%, NF-kB 水平分别下降 92. 1%及 37. 5%。 上述结果显示 II型可溶性 TNF t受体通过阻断急性肝衰竭中 TNF cc与 I型受 体的结合， 抑制 TNF a通过对肝细胞膜 I型受体向胞核传导信号导致肝细胞坏死而发 生的急性肝衰竭。 结果同时表明长效 II型可溶性 TNF a受体预防急性肝衰竭的效果 较常规 II型可溶性 TNF a受体有大幅提髙。 实施例 6:以 II型 HusTNFR-人血清白蛋白为代表的长效人重组可溶性 II型肿瘤 坏死因子（TNF) a受体（II型 LHusTNFR)预防大鼠亚急性肝衰竭的实验
以 D-氨基半乳糖 /内毒素 (GalN/LPS)皮下注射制作大鼠亚急性肝衰竭模型，一周 死亡率 60%。肝脏大体标本显示肝脏严重充血、肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。
通过本发明制备的 II型 LHusTNFR (方法（6〉制备的 II型 LHusTNFR -人血清白蛋白 为预防药物）皮下注射 Spraque- Dawley大鼠， 剂量 5— 30mg/kg， 为长效 II型受体预 防组； 常规 HusTNFR皮下注射 Spraque- Dawley大鼠， 剂量 12. 5rag/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小鼠。 16 小时后预防组及 对照组同时皮下注射 GalN/LPS后观察一周。 皮下注射 GalN/LPS —周， 三组死亡率 分别为： 对照组 60%， 常规 II型受体预防组 44%， 长效 I型受体预防组 0%。 大体病 理显示长效 I型受体预防组肝脏仅轻度充血肿胀， HE染色显示肝细胞呈轻度、 点灶 状坏死， 未见对照组大面积肝细胞坏死。 提取肝脏总 RNA及核蛋白， 运用 real- time PCR 检测 IL- 6， MIP- 2及 bcl- xL mRNA 水平， EMSA检测 NF- kB水平。 结果显示长效 II型受体对 GalN/LPS急性肝衰竭预防组中 IL- έ， ΜΙΡ- 2及 bcl-xL mRNA水平均比对 照组与常规 II型受体预防组明显下降， 下降幅度介于 25— 55 %之间。 NK- κ Β分别下 降 51%及 27%。 上述结果显示 II型可溶性 TNF a受体通过阻断急性肝衰竭中 TNF a与 I型受体的结合， 抑制 TNF a通过对肝细胞膜 I型受体向胞核传导信号导致肝细胞坏 死而发生的急性肝衰竭。 结果同时表明长效 II型可溶性 TNF a受体预防急性肝衰竭 的效果较常规 II型可溶性 TNF a受体有大幅提高。 实施例 7: 以 II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)治疗大鼠亚急性肝衰竭的实验
以 D-氨基半乳糖 /内毒素（GalN/LPS)皮下注射制作大鼠亚急性肝衰竭模型，一周 死亡率 60%。肝脏大体标本显示肝脏严重充血、肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。
采用皮下注射 GalN/LPS制作亚急性肝衰竭大鼠模型。 8 小时后皮下注射 I 型 LHusTNFR (方法（1)制备的 I型 LHusTNFR- IgGl : Fc为预防药物）， 剂量 12. 5mg/kg， 为长效 I 型受体治疗组； 常规 HusTNFR 皮下注射 Spraque- Dawley 大鼠， 剂量 12. 5rag/kg, 为常规 I型受体治疗组； 对照组为皮下注射相同体积生理盐水的同类小 鼠。 观察一周。 GalN/LPS注射后一周， 三组死亡率分别为： 对照组 60%, 常规 I型受 体预防组 30%, 长效 I型受体预防组 0%。大体病理显示长效 I型受体预防组肝脏仅轻 度充血肿胀， HE 染色显示肝细胞呈轻度、 点灶状坏死， 未见对照组大面积肝细胞坏 死。提取肝脏总 RNA及核蛋白， 运用 real- time PCR检测 IL- 6， MIP-2及 bcl- xL mRNA 水平， EMSA检测 NF- kB水平。 结果显示长效 I型受体预防组与常规 I型受体预防组 与对照组相比 IL-6, MIP-2及 bcl-xL mRM水平分别下降 90. 5%, 46. 7% ; 78. 1%， 52. 2%; 84. 3%' 37. 8%, NF-kB水平分别下降 87. 4%及 37. 5%。 上述结果显示 I型可溶性 TNF a受体通过阻断急性肝衰竭中 TNF ci与 I型受体的结合， 抑制 TNF a通过对肝细胞膜 I型受体向胞核传导信号导致肝细胞坏死而发生的亚急性肝衰竭。 结果同时表明长效 I型可溶性 TNF a受体治疗亚急性肝衰竭的效果较常规 I型可溶性 TNF a受体有大幅 提高。 实施例 8: 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) 受体（II型 LHusTNPR)治疗大鼠亚急性肝衰竭的实验
以 D-氨基半乳糖 /内毒素 (GalN/LPS)皮下注射制作大鼠亚急性肝衰竭模型，一周 死亡率 60%。肝脏大体标本显示肝脏严重充血、肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。
采用皮下注射 GalN/LPS制作亚急性肝衰竭大鼠模型。 8小时后皮下注射 II型 LHusTNFR (方法（1)制备的 II型 LHusTNFR- IgGl : Fc为预防药物）， 剂量 12. 5mg/kg， 为长效 II 型受体治疗组； 常规 HusTNFR 皮下注射 Spraque- Dawley 大鼠， 剂量 12. 5mg/kg, 为常规 II型受体治疗组； 对照组为皮下注射相同体积生理盐水的同类小 鼠。 观察一周。 GalN/LPS注射后一周， 三组死亡率分别为： 对照组 60%, 常规 II型 受体预防组 30%, 长效 II型受体预防组 0%。 大体病理显示长效 I型受体预防组肝脏 仅轻度充血肿胀， HE 染色显示肝细胞呈轻度、 点灶状坏死， 未见对照组大面积肝细 胞坏死。提取肝脏总 RNA及核蛋白， 运用 real-time PCR检测 IL- 6， MIP-2及 bcl-xL mRNA水平， EMSA检测 NF- kB水平。 结果显示长效 II型受体预防组与常规 II型受体 预防组与对照组相比 IL- 6, MIP-2及 bcl- xL mRNA水平分别下降 89%， 44. 3% ; 76%, 49% 及 83%、 35. 2%, NF- kB水平分别下降 79. 6%及 36%。 上述结果显示 II型可溶性 TNF α 受体通过阻断急性肝衰竭中 TNF a与 II型受体的结合， 抑制 TNF a通过对肝细胞膜；！ 型受体向胞核传导信号导致肝细胞坏死而发生的亚急性肝衰竭。 结果同时表明长效 II型可溶性 TNF a受体治疗亚急性肝衰竭的效果较常规 II型可溶性 TNF a受体有大 幅提高。 实施例 9:以 II型 HusTNFR-人血清白蛋白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)治疗大鼠亚急性肝衰竭的实验 以 D-氨基半乳糖 /内毒素（GalN/LPS)皮下注射制作大鼠亚急性肝衰竭模型，一周 死亡率 60%。肝脏大体标本显示肝脏严重充血、肿大； HE染色的病理切片显示大面积、 重度的肝细胞坏死。
采用皮下注射 GalN/LPS制作亚急性肝衰竭大鼠模型。 8小时后皮下注射 II型 LHusTNFR (方法（6)制备的 II 型 LHusTNFR-人血清白蛋白为预防药物）， 剂量 5-30mg/kg, 为长效 II型受体治疗组； 常规 HusTNFR皮下注射 Spraque- Dawley大鼠， 剂量 12. 5mg/kg, 为常规 Π型受体治疗组； 对照组为皮下注射相同体积生理盐水的 同类小鼠。 观察一周。 GalN/LPS注射后一周， 三组死亡率分别为： 对照组 60。/。， 常规 II型受体预防组 30%， 长效 II型受体预防组 0%。 大体病理显示长效 I型受体预防组 肝脏仅轻度充血肿胀， HE染色显示肝细胞呈轻度、 点灶状坏死， 未见对照组大面积 肝细胞坏死。 提取肝脏总 RNA及核蛋白， 运用 real- time PCR 检测 IL-6, MIP- 2及 bcl xL mRNA 水平， EMSA检测 NF- kB水平。 结果显示长效 II型受体对 GalN/LPS急 性肝衰竭预防组中 IL- 6， MIP- 2及 bcl- xL mRNA水平均比对照组与常规 II型受体预 防组明显下降， 下降幅度介于 20— 50 %之间。 NK- κ Β分别下降 41%及 24%。 上述结果 显示 II型可溶性 TNF ci受体通过阻断急性肝衰竭中 TNF a与 II型受体的结合， 抑制 TNF a通过对肝细胞膜 I型受体向胞核传导信号导致肝细胞坏死而发生的亚急性肝衰 竭。 '结果同时表明长效 II型可溶性 TNF a受体治疗亚急性肝衰竭的效果较常规 II型 可溶性 TNF ct受体有大幅提高。 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

1、 长效可溶性肿瘤坏死因子 α受体在制备防治肝衰竭或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， 所述的长效可溶性肿瘤坏死因子 α 受体的半衰期为 12- 140小时。
3、 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 α 受体选自：
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 Π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 α受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 α受体蛋白羧基端与 PEG联接的产物，
e.人 I I型肿瘤坏死因子 α受体蛋白氨基端与 PEG联接的产物，
f.人 I I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 α受体蛋白的产物，
1.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 α受体蛋白的产物，
j .人 I型肿瘤坏死因子 α受体与人血清白蛋 S的融合蛋白， 或
k.人 I I型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白。
4、 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 α 受体可使肝细胞的 IL- 6水平下降 40- 50%; 或
使肝细胞的 ΜΙΡ-2水平下降 50- 60% ; 或
使肝细胞的 bcl- xl水平下降 30-45% ; 或
使肝细胞的 NF- κ B水平下降 30-45%。
5、 如权利要求 1 所述的用途， 其特征在于， 所述的肝衰竭是急性和 /或亚急性 肝衰竭。
6、 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。
7、 一种药物组合物， 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 α受体与人 I gGl : Fc片段的融合蛋白，
b.人 I I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 α受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 受体蛋白羧基端与 PEG联接的产物，
e.人 I I型胂瘤坏死因子 α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 α受体蛋白的产物，
i.用 PEG-脂质体混合物包埋人 Π型肿瘤坏死因子 α受体蛋白的产物，
j.人 I型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白， 或
k.人 II型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白； 以及
(ϋ) 药学上可接受的载体。
8、 如权利要求 7所述的药物组合物， 其特征在于， 所述的药物组合物中还含有 有效量的一种或多种选自以下的药物：
(iii) 人肝细胞生长因子、 还原型谷胱甘肽、 或苦参碱。
9、 一种预防或治疗肝细胞坏死或肝衰竭的方法， 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。
10、 如权利要求 9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 α受体与人 IgGl : Fc片段的融合蛋白，
c人 I型肿瘤坏死因子 α受体蛋白氨基端与 PEG联接的产物，
d.人 I型肿瘤坏死因子 α受体蛋白羧基端与 PEG联接的产物，
e.人 II型肿瘤坏死因子 α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 α受体蛋白的产物，
i.用 PEG-脂质体混合物包埋人 II型肿瘤坏死因子 α受体蛋白的产物，
j.人 I型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白， 或
k.人 II型肿瘤坏死因子 α受体与人血清白蛋白的融合蛋白。
PCT/CN2006/002689 2005-10-14 2006-10-13 Utilisation de recepteur long du facteur de necrose tumorale alpha soluble recombinant humain de demi-vie dans la preparation de medicament pouvant prevenir l'insuffisance hepatique WO2007041964A1 (fr)

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