WO2007137456A1 - Peptide pour la prévention ou le traitement d'atteinte hépatique et son dérivé ainsi que son utilisation - Google Patents

Peptide pour la prévention ou le traitement d'atteinte hépatique et son dérivé ainsi que son utilisation Download PDF

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Publication number
WO2007137456A1
WO2007137456A1 PCT/CN2006/001176 CN2006001176W WO2007137456A1 WO 2007137456 A1 WO2007137456 A1 WO 2007137456A1 CN 2006001176 W CN2006001176 W CN 2006001176W WO 2007137456 A1 WO2007137456 A1 WO 2007137456A1
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WIPO (PCT)
Prior art keywords
peptide
gly
group
xaa4
formula
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PCT/CN2006/001176
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English (en)
French (fr)
Inventor
Yun Cheng
Ruihe Yu
Wanzhou Zhao
Jun Zhao
Jing Li
Original Assignee
Yun Cheng
Ruihe Yu
Wanzhou Zhao
Jun Zhao
Jing Li
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Filing date
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Application filed by Yun Cheng, Ruihe Yu, Wanzhou Zhao, Jun Zhao, Jing Li filed Critical Yun Cheng
Priority to CN200680051751XA priority Critical patent/CN101336110B/zh
Priority to JP2009512388A priority patent/JP5031827B2/ja
Priority to US12/066,636 priority patent/US7985734B2/en
Priority to PCT/CN2006/001176 priority patent/WO2007137456A1/zh
Priority to EP06742063A priority patent/EP2030628B1/en
Publication of WO2007137456A1 publication Critical patent/WO2007137456A1/zh
Priority to US13/162,981 priority patent/US8263562B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention relates to the use of hepatitis C virus immunogenic peptides and derivatives thereof for preventing or treating liver damage, and in particular to the peptide or derivative thereof The use of preventing or treating liver damage caused by immune liver damage and hepatotoxic chemicals, and its use in the prevention or treatment of hepatitis C.
  • the invention also relates to a pharmaceutical composition comprising the peptide or a derivative thereof, a process for the preparation thereof, and a polynucleotide encoding the peptide.
  • Background Art Viral hepatitis is a serious disease that is harmful to human health, and its pathogen is a heterogeneous hepadnavirus.
  • hepatitis C virus is the causative agent of hepatitis C.
  • HAV hepatitis C virus
  • HCV hepatitis C virus
  • Hepatitis C was originally called non-A, non-B hepatitis caused by blood transfusion, but future studies have shown that hepatitis C virus is not only transmitted by blood transfusion, but other ways such as digestive tract, sexual contact, etc. can cause it. propagation.
  • HAV hepatitis C virus
  • HCV hepatitis C virus
  • HCV is an RNA virus belonging to the Flaviviridae family. Studies (see, for example, Choo et al, Science 244:359-362 (1989); Choo et al, Proc. Natl. Acad. Sci. USA 88:2451-2455 (1991); Han et al, Proc. Natl. Acad. Sci. US A 88: 1711-1715 (1991) shows that the genome of HCV is a single-stranded positive-stranded RNA of about 9.4 kb and has an open reading frame (ORF) that spans the entire genome. This ORF encodes a 3011 or 3010 amino acid virion precursor.
  • ORF open reading frame
  • the HCV genome encodes a nucleocapsid core protein (C), two envelope glycoproteins (E1 and E2) and five non-structural proteins (NS1 NS5) regions.
  • C nucleocapsid core protein
  • E1 and E2 envelope glycoproteins
  • NS1 NS5 non-structural proteins
  • HVR1 region hypervariable region 1 of the hepatitis C virus E2 region contains an important neutralizing epitope, which can induce the host to produce neutralizing antibodies (see, Shirai et al, J. Immunol, 162). : 568-576 (1999)).
  • the HVR1 region's genes are highly variable, making HCV possible to escape the body's immune recognition. This may also be the main cause of chronic hepatitis caused by HCV.
  • Interferon Interferon
  • RNaseL Rease C
  • Manns et al The Lancet, 358: 958-965 (2001)
  • PEGylated interferon and ribavirin used a combination of PEGylated interferon and ribavirin to treat hepatitis C.
  • this improved therapy only has a sense of type 2 and type 3 viruses. Dyed patients are particularly effective, but have limited efficacy in patients infected with the la, lb, and 4 genotype strains. To this end, people have actively tried various means to develop and develop vaccines that reduce the rate of HCV infection and drugs for treating hepatitis.
  • HCV and other viruses cause liver damage through viral cytopathic effects
  • studies have shown that the immune response against HCV is the main cause of liver damage.
  • lymphocytes especially cytotoxic T lymphocytes
  • lymphocytes can not completely eliminate HCV, but cause liver cell immune damage during hepatocyte clearance of HCV-infected hepatocytes.
  • Apoptosis which can even lead to cirrhosis and hepatocellular carcinoma (see, for example, Nelson et al, J. Immunol., 158: 1473-1481 (1997); Wong et al, J. Immunol., 160: 1479-1488).
  • HCV immunogenic peptide triggering an immune response against HCV as an epidemic may cause immunological liver damage.
  • Such a mechanism poses great difficulties in the development of prophylactic vaccines or therapeutics that are actually available for hepatitis C (especially chronic hepatitis C) using immunogenic peptides against HCV.
  • liver damage In addition to immune liver damage and pathogenic liver damage, it is well known that hepatotoxic chemicals can also cause liver damage. Some drugs are known to cause liver damage, resulting in liver cell lysis and liver necrosis.
  • the analgesic acetaminophen that is, paracetamol, chemically known as 4-(N-acetylamino)phenol
  • the analgesic acetaminophen is a substance with liver damage that causes liver necrosis in humans.
  • long-term use of antibiotics such as rifampicin, pyrazinamide, isoniazid
  • long-term use of estrogen in menopause can also cause severe hepatocyte necrosis, leading to liver damage such as acute or chronic hepatitis, jaundice and liver fibrosis.
  • Chemicals that cause liver damage include those that produce large amounts of active free radicals, especially those that produce oxygen free radicals, which usually cause hepatotoxicity through oxidation.
  • the present inventors have obtained an HCV immunogenic peptide and a derivative thereof which, surprisingly, are capable of preventing or treating liver damage, which is not limited to the immunity of HCV-infected hepatocytes. damage.
  • the peptides of the present invention and derivatives thereof can be used for the prevention or treatment of liver damage caused by immunological liver damage, pathogenic liver damage, and hepatotoxic chemicals.
  • Summary of the invention The present invention relates to a peptide and a derivative thereof which, in addition to inducing an immune response against HCV, are capable of preventing or treating liver damage, particularly for preventing or treating immune liver damage, and the liver damage is not limited to HCV infection. Immune damage to liver cells.
  • the present invention provides the use of a peptide comprising the sequence of Formula I, or a pharmaceutically acceptable salt or ester thereof,
  • Xaal is missing, Ala, Gly, Val, Leu or lie,
  • Xaa2 is Thr or Ser
  • Xaa3 is Tyr, Phe or Trp
  • Xaa4 is missing, Ala, Gly, Val, Leu, lie or Pro,
  • the use is for the prevention or treatment of liver damage or for the preparation of a medicament for preventing or treating liver damage. That is, the present invention provides a peptide comprising the sequence of Formula I or a pharmaceutically acceptable salt or ester thereof for use in preventing or treating liver damage, and a peptide comprising the sequence of Formula I or a pharmaceutical thereof Use of an acceptable salt or ester for the manufacture of a medicament for the prevention or treatment of liver damage.
  • liver injury refers to damage or lesions in liver tissue or cells.
  • liver damage mainly manifests as hepatocyte degeneration, intrahepatic phlebitis, spotted necrosis or focal necrosis in the liver, inflammatory cell infiltration in the liver and portal area, or fibroblast proliferation, or hepatomegaly. In severe cases, it can lead to cirrhosis, liver cancer and so on.
  • pathological phenomenon to evaluate liver damage when hepatocytes undergo cytosolic damage, the amount of transaminase released from the damaged hepatocytes to the circulating blood is increased, and the activity of these transaminase in serum can be measured.
  • liver damage can be assessed by measuring alanine aminotransferase or aspartate aminotransferase levels.
  • the liver damage in the present invention is liver damage reflected by an increase in serum alanine aminotransferase or aspartate aminotransferase levels, and it is preferred that the therapeutic indicator for preventing or treating liver damage in the present invention is a degree of reduction in alanine aminotransferase or aspartate aminotransferase levels.
  • the effect of the peptide of the present invention having the sequence of the formula I or a pharmaceutically acceptable salt or ester thereof on liver damage can be determined by the degree of reduction of the above pathological phenomenon or the degree of decrease in the level of alanine aminotransferase or aspartate aminotransferase.
  • the liver damage according to the present invention is liver damage caused by immune liver damage or hepatotoxic chemicals, that is, the present invention preferably provides a peptide comprising the sequence of formula I or a pharmaceutically acceptable salt or ester thereof in prevention or
  • the present invention preferably provides a peptide comprising the sequence of formula I or a pharmaceutically acceptable salt or ester thereof for the preparation of a prophylactic or therapeutic immunological liver injury or Application in drugs for liver damage caused by hepatotoxic chemicals.
  • the peptides of the invention can be used to alleviate immune liver damage induced by BCG and lipopolysaccharide. Further, in the liver damage model caused by the specific hepatotoxic chemical of the present invention, the peptide of the present invention can be used for preventing liver damage caused by D-galactosamine, and the peptide of the present invention can also be used for treatment by four. Liver damage caused by carbon chloride.
  • the "peptide of the sequence of the formula I" of the present invention refers to a peptide of the sequence of the formula I or a modified functional equivalent thereof, that is, the N-terminal amino group and the C-terminal of the peptide of the sequence of the formula I.
  • the carboxyl group and the amino acid side chain group may be modified without being modified, and the activity for preventing or treating liver damage is not substantially reduced.
  • the “functional equivalent” herein refers to a modified product containing the sequence of the formula I, which does not substantially reduce the activity for preventing or treating liver damage, that is, does not reduce the activity of preventing or treating liver damage by 50%, preferably not by 30%. More preferably, the activity is not reduced by 10%, and most preferably, the activity is not lowered.
  • Certain pathological phenomena or levels of certain transaminase in serum can be used to determine the activity of a functional equivalent to prevent or treat liver damage, preferably using the pathological scoring criteria described in the specific embodiments of the invention or serum alanine aminotransferase or aspartate aminotransferase Level to determine activity.
  • suitable modifications are, for example, cyclization, preparation into multimers, modification of terminal amino, carboxy or side chain groups to form pharmaceutically acceptable esters, conjugates comprising the sequence of formula I , a fusion protein comprising the sequence of formula I, or a combination of these modifications, and the like.
  • Cyclization of linear peptides such as condensation of the amino group at the N-terminus of the peptide with the carboxyl group at the C-terminus, can generally extend the half-life of the peptide in a physiological environment.
  • “Pharmaceutically acceptable ester” refers to an ester suitable for contact with the tissues of a human or animal without excessive toxicity, irritation or allergies and the like.
  • esterification can reduce the hydrolysis of peptides by proteases in the body.
  • Modification of the terminal amino, carboxyl or side chain groups of the peptides of the invention can form pharmaceutically acceptable esters.
  • Modifications to amino acid side chain groups include, but are not limited to, threonine, esterification of a serine side chain hydroxyl group with a carboxylic acid.
  • Modifications of the N-terminal amino group include, but are not limited to, de-amino, N-lower fluorenyl, N-di-lower alkyl, and N-acyl modifications.
  • Modifications of the C-terminal carboxyl group include, but are not limited to, amides, lower alkyl amides, dialkyl amides, and lower alkyl ester modifications.
  • the terminal group is protected with a protecting group known to those skilled in the art of protein chemistry, such as acetyl, trifluoroacetyl, Fmoc (9-fluorenyl-methoxycarbonyl), Boc (tert-butoxycarbonyl), Alloc (allyloxycarbonyl), C 1-6 alkyl, C 2 .8 alkenyl, C 7-9 aryl fluorenyl and the like.
  • the chemical group at the N-terminus is still the ⁇ -amino group on the first amino acid.
  • the chemical group at the C-terminus is the carboxyl group (-COOH) of the C-terminal amino acid.
  • the present invention also preferably acylates the carboxyl group at the C-terminus, that is, the chemical group at the C-terminus is -CO NH 2 .
  • the conjugate comprising the sequence of formula I comprises a pharmaceutically acceptable water-soluble polymer moiety using methods known in the art. Typically, the conjugate exhibits a circulating half-life of a peptide that extends the sequence of Formula I.
  • Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(dJ-oxy-PEG, aryloxy-PEG, poly-(N-vinylpyrene) Pyrrolidone) PEG, trimethoxy PEG, monomethoxy-PEG propionaldehyde, PEG propionaldehyde, bis-succinimide carbonate PEG, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymerization , polyoxyethylene polyol (eg, glycerol), monomethoxy-PEG butyraldehyde, PEG butyraldehyde, monomethoxy-PEG acetaldehyde, PEG ace
  • Suitable PEGs can have a molecular weight of from about 600 to about 60,000, including, for example, 5,000 Daltons, 12,000 Daltons, 20,000 Daltons, 30,000 Daltons, and 4,000,000 Dalton, which can be straight or branched. Conjugates of peptides comprising sequences of formula I may also include mixtures of such water soluble polymers. PEGylation can be carried out by PEGylation reactions known in the art (see, for example, Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27: 290 (1994) ), and Francis et al., Int J Hematol 68: 1 (1998)).
  • PEGylation can be carried out by an acylation reaction or by an alkylation reaction using a reactive polyethylene glycol molecule.
  • the conjugate is formed from a condensation activated PEG wherein the hydroxyl or amino group at the PEG terminus is replaced by an activated linker molecule (see, eg, Karasiewicz et al, US 5382657 A).
  • the conjugate comprising the sequence of formula I may also be a conjugate of a peptide of the sequence of formula I which is crosslinked with other proteins.
  • the other protein is preferably the Fc portion of a human albumin, bovine albumin or IgG molecule.
  • the peptide of the invention is crosslinked with bovine serum albumin to form a peptide conjugate.
  • the peptide of the present invention having the sequence of the formula I may also be a fusion peptide or fusion protein comprising a sequence of the formula I and a peptide or a protein of the formula I.
  • the protein is the Fc portion of a human albumin, bovine albumin or IgG molecule.
  • Albumin can be genetically engineered to the peptide of the present invention containing the sequence of formula I to extend the half-life.
  • human albumin is the most common natural blood protein in the human circulatory system, which can maintain circulation in the body for more than 20 days. Studies have shown that therapeutic proteins that are genetically engineered to human albumin have a longer half-life.
  • Xaal is Gly
  • Xaa2 is Thr
  • Xaa3 is Tyr
  • Xaa4 is a deletion
  • Ala or Gly and more preferably: Xaa4 is a deletion.
  • deletion means that the deleted amino acid residue is not present in the peptide sequence, for example, when Xaa4 is deleted, the Gly in the sequence of Formula I is the C-terminal amino acid of the sequence of Formula I.
  • amino acid or amino acid residues can be defined in Table 1, and these abbreviations can refer to L-type amino groups. Acid can also be referred to as a D-type amino acid. Preferred amino acids refer to amino acids of the L-form.
  • amino acid or amino acid residues can be divided into the following groups according to the similarity of their side chain properties: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R , D, N, C, E, Q, G, H, K, S, T), amino acids of the aliphatic side chain (G, A, V, L, I, P), amino acids with hydroxyl side chains (S , T, Y), amino acids with side chains of sulfur atoms (C, M), amino acids with carboxylic acid and amide side chains (D, N, E, Q), amino acids with basic group side chains (R, K, ⁇ ), amino acids containing aromatic side chains (H, F, Y, W).
  • amino acids or amino acid residues in the same group have similar properties.
  • “Pharmaceutically acceptable salt” refers to a salt suitable for contact with the tissues of a human or animal without excessive toxicity, irritation or allergies and the like. Pharmaceutically acceptable salts are well known in the art. Such salts may be prepared during the final isolation and purification of the polypeptides of the invention, or may be prepared separately by reacting the peptide with a suitable organic or inorganic acid or base.
  • Representative acid addition salts include, but are not limited to, acetate, dihexanoate, alginate, citrate, aspartate, benzoate, besylate, hydrogen sulfate, butyrate , camphorate, camphor sulfonate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate Acid salt, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, 3-phenylpropionate, propionate, succinate, tartrate , phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate.
  • Preferred acids which can be used to form pharmaceutically acceptable salts are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, oxalic acid, maleic acid, succinic acid and citric acid.
  • the cations in the pharmaceutically acceptable base addition salts include, but are not limited to, alkali metal or alkaline earth metal ions such as lithium, sodium, potassium, calcium, magnesium, and aluminum, and non-toxic quaternary ammonium cations such as ammonium, tetramethylammonium, Tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, diethylamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.
  • Preferred base addition salts include phosphorus Acid salts, tris and acetates. These salts are generally capable of increasing the solubility of the polypeptide, and the salt formed does not substantially alter the activity of the polypeptide.
  • the polypeptide of the present invention may be used singly or in the form of a pharmaceutically acceptable salt.
  • the use of the first aspect of the invention also includes further use in the treatment and/or prevention of hepatitis C. That is, in the treatment or prevention of liver damage, the present invention also provides a peptide comprising the sequence of Formula I or a pharmaceutically acceptable salt or ester thereof for preventing or treating liver damage and hepatitis C. And a use of a peptide comprising the sequence of formula I or a pharmaceutically acceptable salt or ester thereof for the manufacture of a medicament for preventing or treating liver damage and hepatitis C.
  • the peptide of the present invention has an effect of inducing the production of cytokines Y-IFN, IL-4, IL-10 and antibodies.
  • Y-IFN is a major cytokine secreted by type 1 T helper cells (Thl) and is one of the main cytokines of the human immune system against viral infection. It can be used to clear HCV by cellular immunity against HCV, and IFN is also currently A mature therapy for the treatment of HCV.
  • the present invention provides a pharmaceutical composition for use in the application of the first aspect of the invention, comprising a peptide comprising the sequence of Formula I, or a pharmaceutically acceptable salt or ester thereof, and pharmaceutically acceptable a,
  • Xaal is missing, Ala, Gly, VaK Leu or lie,
  • Xaa2 is Thr or Ser
  • Xaa3 is Tyr, Phe or Trp
  • Xaa4 is missing, Ala, Gly, Val, Leu, He or Pro.
  • the pharmaceutical composition of the present invention is useful for preventing or treating liver damage.
  • the pharmaceutical composition of the present invention can reduce certain pathological phenomena caused by liver damage and/or reduce the level of certain transaminase in serum, preferably reducing the pathological scoring standard described in the specific embodiment of the present invention or lowering the serum in the valley Alanine aminotransferase or aspartate aminotransferase levels.
  • the pharmaceutical composition of the present invention has an effect of inducing the production of the cytokines Y-IFN, IL-4, IL-10 and antibodies. Therefore, the pharmaceutical composition of the present invention is preferably further useful for the treatment and/or prevention of hepatitis C.
  • pharmaceutically acceptable carrier refers to a non-toxic solid, semi-solid or liquid filler, diluent, adjuvant, encapsulating material or other formulation excipient.
  • the pharmaceutical compositions may be formulated into various dosage forms, such as tablets, films, pills, capsules (including sustained release), depending on the purpose of the treatment, the route of administration, in accordance with the teachings in the art. Or a delayed release form), a powder, a granule, an elixir, a syrup and an emulsion, a sterile solution or suspension, an aerosol or liquid spray, a drop, an injection, an automatic injection device or a suppository.
  • the above active pharmaceutical ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, isotonic glucose solution, glycerol, physiological saline. Or a combination thereof.
  • an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, isotonic glucose solution, glycerol, physiological saline.
  • Excipients such as human serum albumin, low molecular weight peptides, amino acid and metal cations may also be added to the composition, and may also be added.
  • Agents such as Freund's complete adjuvant, Freund's incomplete adjuvant, poly CpG and the like.
  • the pharmaceutical composition of the present invention comprising a peptide comprising the sequence of formula I or a pharmaceutically acceptable salt or ester thereof can also be prevented or treated alone without the addition of an adjuvant.
  • the peptide of the present invention can also significantly increase the amount of interferon secreted by the body without the addition of an adjuvant.
  • the peptide of the present invention can also significantly reduce liver damage without the addition of an adjuvant. This suggests that the mechanism for preventing or treating liver damage and hepatitis C of the peptide of the present invention is not limited to the induction of an immune response against HCV. While inhibiting viral replication and eliminating viruses, the peptide of the present invention may also inhibit excessive immune inflammatory response of the body, thereby achieving the purpose of reducing liver tissue damage. Accordingly, the composition of the second aspect of the invention is preferably free of adjuvant.
  • Xaal is Gly
  • Xaa2 is Thr
  • Xaa3 is Tyr
  • Xaa4 is a deletion
  • Ala or Gly and more preferably Xaa4 is a deletion.
  • the present invention is also directed to the use of the pharmaceutical composition of the second aspect of the present invention for the preparation of a medicament for preventing or treating liver damage; the pharmaceutical composition of the second aspect of the invention also relates to the prevention or treatment of liver Application in injury.
  • the above application further comprises further treatment and/or prevention of hepatitis C.
  • compositions of this invention may be administered by methods of administration well known to those skilled in the art, such as oral, rectal, sublingual, pulmonary, transdermal, iontophoretic, vaginal and intranasal administration.
  • the pharmaceutical compositions of the invention are preferably administered parenterally, such as subcutaneously, intramuscularly or intravenously.
  • the dose to be administered varies depending on the form of the preparation and the desired time of action and the condition of the subject to be treated, and the amount required for the actual treatment can be conveniently determined by the physician based on actual conditions (e.g., the patient's condition, body weight, etc.).
  • the dosage of the pharmaceutical composition of the present invention may be from 1 ng to 10 g per kg of adult body weight.
  • the preferred dosage is from 100 ng to 10 mg per kg body weight, more preferably from 1 to 1 mg per kg, most preferably every kg ( ⁇ g - 10 ( ⁇ g. for oral intake)
  • the dose may be 1 ⁇ 8 - 10 g per kg body weight per day, preferably 10 g - lg per kg body weight per day, more preferably 10 ( ⁇ g - 10 mg per day).
  • the present invention provides a peptide, or a pharmaceutically acceptable salt or ester thereof, for use in the use of the first aspect of the invention or in the pharmaceutical composition of the second aspect of the invention, wherein
  • the peptide contains the sequence of formula I,
  • Xaal is missing, Ala, Gly, Val, Leu or lie,
  • Xaa2 is Thr or Ser
  • Xaa3 is Tyr, Phe or Trp, and Xaa4 is missing, Ala, Gly, Val, Leu, He or Pro.
  • Xaal is preferably Gly
  • Xaa2 is Thr
  • Xaa3 is Tyr
  • Xaa4 is a deletion, Ala or Gly, and more preferably Xaa4 is a deletion.
  • the peptide of the present invention is a purified peptide, i.e., purified to 80% purity, preferably 90% purity, more preferably 95% purity, particularly preferably in a state of drug purity, i.e., a purity of 98% or more, and no source of infection. And heat source.
  • the peptides of the invention are substantially free of other polypeptides or proteins, especially those derived from animal sources.
  • the present invention provides a polynucleotide encoding the peptide of the third aspect of the present invention.
  • polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5' to the 3' end, including RA and DNA, which can be derived from natural Prepared by isolation, in vitro synthesis or recombinant expression from the source.
  • the present invention provides a process for the preparation of the peptide of the third aspect of the invention, or a pharmaceutically acceptable salt or ester thereof, the process comprising the step of chemically synthesizing the peptide or fusion expressing the peptide.
  • the step of further reacting the peptide to form the salt or ester may also be included in the preparation method of the present invention, depending on the desired pharmaceutically acceptable salt or ester structure.
  • the polypeptide of the invention is preferably synthesized by a solid phase method.
  • the polynucleotide of the fifth aspect of the present invention is introduced into an expression vector and expressed in a host cell, thereby preparing the peptide.
  • Suitable expression vectors include plasmids, cosmids, phages or viruses, and the like; suitable host cells include bacteria, fungi, and eukaryotic cells.
  • Figure 2 Y-IFN secreted by rat spleen cells inoculated with the peptide of the present invention with and without the addition of an adjuvant The amount.
  • Figure 3 shows the effect of the peptide of the present invention on serum biochemical parameters in a rat model of immunological liver injury induced by BCG and lipopolysaccharide, wherein * indicates that the group shown can significantly reduce transaminase levels relative to the model group.
  • Figure 4 is a graph showing the results of immunizing the peptide of the present invention against the degree of liver lesions in a rat model of immunological liver injury induced by BCG and lipopolysaccharide, wherein * indicates that the group shown can significantly reduce the degree of liver lesions relative to the model group.
  • Figure 5 is an illustration of the effect of the peptide of the present invention on serum biochemical parameters in a rat model of acute liver injury induced by D-galactosamine, wherein * indicates that the group shown can significantly reduce transaminase levels relative to the model group.
  • Figure 6 shows the effect of the peptide of the present invention on serum biochemical parameters in a mouse model of liver injury induced by carbon tetrachloride, wherein * indicates that the group shown can significantly reduce transaminase levels relative to the model group.
  • Figure 7 is a graph showing the results of immunizing the peptide of the present invention on the degree of liver lesions in a mouse model of liver injury induced by carbon tetrachloride, wherein * indicates that the group shown can significantly reduce the degree of liver lesions relative to the model group.
  • peptide A Three peptides as shown in the following sequence were synthesized by a solid phase peptide synthesis method using an automatic peptide synthesizer Model 413A (available from Perkin Elmer Co., Ltd.): GQTYTSG (hereinafter referred to as “peptide A”), GQTYTSGA (hereinafter referred to as “ Peptide B”) and GQTYTSGG (hereinafter referred to as “peptide C”).
  • the amino acid residues in these three peptides are all L-type amino acids.
  • the specific procedure for the synthesis is as follows: First, the reactive group on the amino acid monomer is protected: the alpha amino group of the amino acid is protected with 9-fluorenylmethoxycarbonyl (Fmoc); and the following specific amino acids are side-chain protected: Ser and Thr
  • the side chain protecting group is a tert-butyl group and the Gin is a trityl group (Trt).
  • the protected amino acid was sequentially coupled with hydrazine, hydrazine-diisopropylcarbodiimide/1-hydroxybenzotriazole as an activating reagent, and coupled for 40 minutes each time.
  • the peptide was reacted with trifluoroacetic acid (85%) at room temperature for 120 minutes to support the polymer. The object is cut and the protecting group is removed. The peptide was then precipitated with anhydrous diethyl ether and then washed several times with anhydrous diethyl ether to sufficiently remove the mercaptan. Precipitated in water/tert-butanol (1:1) and lyophilized to give the crude peptide. The crude peptide was purified by reverse phase HPLC in 30 min, with a gradient of 37-42% EtOAc / 0.9% TFA.
  • Peptide A and bovine serum albumin are crosslinked by glutaraldehyde to form a conjugate.
  • the specific conjugation process was as follows: 1 mg of the peptide A synthesized in Example 1 was taken and dissolved in 0.5 ml of PBS (pH 7.4, 0.02 mol/L); 4.5 m g of BSA was dissolved in 4.5 ml of PBS (pH 7.4, 0.02mol/L). The above peptide A and BSA solutions were mixed, and then 0.1 ml of 0.1% glutaraldehyde was slowly added, and the crosslinking reaction was allowed to proceed for 12 hours at room temperature in the dark.
  • Anti-HCV antibody-positive serum Randomly obtained from hospitalized patients with HCV infection in the 302 Hospital of the People's Liberation Army from 2000 to 2001.
  • Control serum taken from healthy blood donors, the results of laboratory tests reflect that all indicators are normal.
  • Hepatitis B (HBV) patient serum selected from hospitalized patients with hepatitis B in the 302 Hospital of the People's Liberation Army. All tests were positive for hepatitis B virus surface antigen.
  • the peptides (peptide A, peptide 8, peptide C, and peptide conjugate A) were negative for binding to HBV patient serum (10 parts) and healthy human serum (10 parts).
  • the results of peptide-conjugated anti-HCV antibody-positive serum (30 parts) are shown in Table 2.
  • the chi-square test by SATA software shows that peptide A and peptide of the present invention are reactive with serum of HBV patients and healthy human serum.
  • B, Peptide C and Peptide Conjugate A were all able to react significantly with the serum of HCV patients, and the conjugate formed by cross-linking of the peptide with bovine serum albumin increased the reactivity of the peptide of the present invention with the serum of HCV patients.
  • mice both male, 6 weeks old, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences, Beijing
  • groups 5 in each group: (1) control group; (2) peptide group A ( Immunization with peptide A); (3) peptide group B (immunized with peptide B); (4) peptide group C (immunized with peptide C).
  • the peptides (100 ⁇ g/ ⁇ 1 ) used in each group were mixed with 50 ⁇ l of each volume of Freund's complete adjuvant (GIBCOBRL), mixed well with a micro-agitator, and the mouse footpad was injected, thereby completing the pair.
  • booster immunization was performed, i.e., 50 ⁇ l each of the peptides used in each group (100 g/ ⁇ ⁇ ) and Freund's incomplete adjuvant (GIBCOBRL) were immunized in the same manner as the first immunization.
  • a booster immunization was performed in the same manner after 14 days.
  • the antibodies in the serum of the immunized mice were detected according to a conventional indirect ELISA technique.
  • the peptides were coated with peptide VIII, peptide B and peptide C as antigens, respectively, and 10 ⁇ g of the above peptide was added to each well of the 100 ⁇ l alkaline coating solution on the coated plate at 4 ° C. overnight. Block with 120 1 PBS and incubate for 2 hours at 37 °C. Serum from each group of mice (1:100 dilution) was then added to each well and incubated for 1 hour at 37 °C.
  • Sheep anti-mouse IgG 100 ⁇ l (purchased from Huamei Bioproducts Co., Ltd., Beijing) (1:1000 dilution) was added to each well and incubated at 37 ° C for 1 hour. Then, add A and B solution (purchased from Beijing Kewei Reagent Factory) to each well for 50 ⁇ l, and place it in the dark for 5 minutes to measure the optical density (OD) at 450 nm. The results are shown in Figure 1.
  • mice with the above peptides can induce the body to produce a corresponding humoral immune response.
  • the IL-4 ELASA test kit, the IL-10 ELASA test kit, and the Y-IFN ELASA test kit were used to detect the serum of the mice after immunization according to the manufacturer's kit instructions.
  • Cytokine i.e., IL-4, IL-10, ⁇ -IFN
  • Table 3 Cytokine (i.e., IL-4, IL-10, ⁇ -IFN) levels, the results are shown in Table 3.
  • mice immunized with the above peptides had a significant increase in the serum levels of Y-IFN, IL-4, and IL-10.
  • Y-IFN is the main cytokine secreted by Th1, it is also one of the main cytokines of the human immune system against viral infection. It inhibits the synthesis of viral proteins by inducing 2-5A synthetase, thereby activating RNaseL and degrading viral RA. Therefore, the increase in ⁇ -IFN levels indicates that the above peptides can be used to remove HCV.
  • Example 5 Effect of peptide on Y-IFN in rats
  • SD rats (body weight 180g ⁇ 220g, male and female, purchased from the Institute of Animal Science of the Academy of Military Medical Sciences) were randomly divided into the following 5 groups, 10 in each group: (1) blank control group; (2) adjuvant control group; High dose group (immunized with peptide A and adjuvant); (4) low dose group (immunized with peptide A and adjuvant); (5) peptide immunized group (immunized with peptide A).
  • the rats were immunized as follows: From day 0, the first immunization was first performed, that is, peptide A was mixed with Freund's complete adjuvant (the appropriate amount of peptide A was dissolved in physiological saline and the same volume of Fuchs was completely Adjuvant mixing) A water-in-oil chyle-like liquid was injected subcutaneously into rats, wherein the administration volume was 0.1 ml/rat, and the dose was 50 g/kg rat and P, respectively. 25 wg/kg rat.
  • a second immunization dose and immunization was performed with the first immunization, but in which Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • a third immunization was performed, the dose and immunization method were the same as the first immunization, but in which the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • the fourth immunization was carried out, that is, without adjuvant, the rats were intraperitoneally injected with the appropriate amount of peptide A in physiological saline, wherein the dose was 0.1 ml/rat, and the dose was 50 in terms of peptide A. Wg/kg rats and 25 g/kg rats.
  • the rats in the adjuvant control group were injected with the corresponding adjuvant and physiological saline without peptide A at the corresponding time; the rats in the blank control group were injected with physiological saline at the corresponding time.
  • the rats were subcutaneously injected with physiologically dissolved peptide A (with no adjuvant added) to immunize the rats. Rats were immunized four times in total. Here, the volume of each administration was 0.1 ml/rat, and the dose per dose was 50 w g/kg of the rat in terms of peptide A.
  • mice Each group of mice was sacrificed one day after the fourth immunization, and spleen cells were collected.
  • the spleen cells were added to the culture plate at a concentration of 1 ⁇ 10 6 /well, and incubated at 37 ° C for 3 days. Then, the culture supernatant was taken, and the ⁇ -IFN level in the serum of the above-mentioned immunized rat was measured using a Y-IFN ELASA test kit (purchased from R&D Co., Ltd.) according to the manufacturer's kit instructions.
  • Wistar rats (body weight 180g ⁇ 220g, male and female, purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were randomly divided into the following 8 groups, 10 in each group: (1) diammonium glycyrrhizinate injection group (injection of glycyrrhizic acid II) Ammonium, diammonium glycyrrhizinate injection was purchased from Jiangsu Zhengda Tianqing Pharmaceutical Co., Ltd.); (2) Interferon group (injected recombinant human interferon a 2a, recombinant human interferon a 2a was purchased from Shenyang Sansheng Pharmaceutical Co., Ltd.) (3) high dose group (immunized with peptide A); (4) medium dose group (immunized with peptide A); (5) low dose group (immunized with peptide A); (6) low dose adjuvant group (Immune with peptide A and adjuvant); (7) blank control group; (8) model group
  • the low-dose adjuvant group was immunized to the rats as follows: Starting from day 0, the initial immunization was first performed, that is, peptide A was mixed with Freund's complete adjuvant (the amount of peptide A was dissolved in physiological saline and completely mixed with 50 ⁇ l of F. The vehicle was mixed in an equal volume. The oil-in-water emulsion was subcutaneously injected into the rats, wherein the administration volume was 0.1 ml/rat, and the dose was 43.5 g/kg of the rats based on the peptide A. After 4 days, a second immunization, dose and immunization was performed with the first immunization, but in which Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • the dose and immunization method were the same as the first immunization, but in which the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • the fourth immunization was carried out, that is, without adjuvant, the rats were intraperitoneally injected with a suitable amount of peptide A in physiological saline, wherein the dose was 0.1 ml/rat, and the dose was administered.
  • the peptide A was 43.5 wg/kg rat.
  • the rats were subcutaneously injected with the peptide A dissolved in physiological saline (wherein Rats were immunized by adding adjuvant) and a total of four immunizations were performed.
  • the volume of each administration was 0.1 ml/rat, and the dose per dose was based on peptide A, and the high dose group, the middle dose group, and the low dose group were 174, 87, and 43.5 g/kg rats, respectively.
  • the rats in the model group and the blank control group were also injected from the 0th day, a total of four times, each injection was physiological saline, each administration The volume was 0.1 ml per rat.
  • BCG polysaccharide nucleic acid injection was injected into the tail vein of the other 7 groups except the blank control group (by Jiuzhitangsi, Hunan) Produced by Biopharmaceutical Co., Ltd., the specification is 0.35mg of BCG-containing polysaccharide per ml, diluted with physiological saline before injection, and the dosage is 126 ug/kg based on BCG polysaccharide; at the same time, blank control group injection, etc. Volume of normal saline.
  • LPS lipopolysaccharide
  • rats were intraperitoneally injected with diammonium glycyrrhizinate daily, and the dose was 13.5 mg/kg rat based on diammonium glycyrrhizinate.
  • SGPT serum alanine aminotransferase activity was determined according to the instructions of the alanine aminotransferase test kit (purchased from Nanjing Jiancheng Bioengineering Research Institute) and the aspartate aminotransferase test kit (purchased from Nanjing Jiancheng Bioengineering Research Institute). Aspartate aminotransferase (SGOT) activity.
  • the rats were taken from the liver, fixed in 10% formalin solution, dehydrated, embedded in paraffin, prepared (4 ⁇ ⁇ thick), stained with HE, and examined by pathologists under the light microscope for the following lesions.
  • the lesion score (1) with or without hepatocyte degeneration (lipid, edema, acidophilic degeneration, etc.); (2) with or without hepatocyte necrosis (point necrosis, focal necrosis, etc.); (3) with or without central Venous and hepatic sinus dilatation, congestion, intrahepatic vein inflammation; (4) There is no connective tissue hyperplasia and inflammatory cell infiltration in the liver or portal area.
  • the lesion scoring criteria were: 0 (normal), 0.5 (very light), 1 (mild), 2 (moderate), 3 (severe) according to the degree of light to heavy of each lesion. ), 4 points (very severe), focal necrosis doubled scores. The average score of the lesion scores for each group of animals was calculated. The higher the score, the more severe the disease.
  • peptide A can significantly reduce the levels of alanine aminotransferase and aspartate aminotransferase in rats with immune liver injury, while the diammonium glycyrrhizinate injection on rat liver Damage does not have significant protection.
  • liver tissue lesions showed hepatic cell necrosis, with a few focal necrosis; the number of neutrophils in the liver increased, usually around the central vein; mild inflammatory cell infiltration in the portal area.
  • the results of liver tissue lesions showed that the degree of lesions in the sputum dose group, the middle dose group, the low dose adjuvant group and the interferon group was significantly lower than that of the model group (P ⁇ 0.05).
  • Wistar rats (body weight 180g ⁇ 220g, male and female, purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were randomly divided into the following 6 groups, 10 in each group: (1) diammonium glycyrrhizinate injection group (injection of glycyrrhizic acid II) (2) high dose group (immunized with peptide A and adjuvant); (3) medium dose group (immunized with peptide A and adjuvant); (4) low dose group (immunized with peptide A and adjuvant); ) blank control group; (6) model group.
  • the high-dose, middle-dose, and low-dose groups were immunized as follows: From day 0, the initial immunization was first performed, that is, peptide A was mixed with Freund's complete adjuvant (after dissolving the appropriate amount of peptide A with physiological saline) ⁇ 1 Fully's complete adjuvant is mixed in an equal volume to form a water-in-oil chyle-like solution for subcutaneous injection in rats, wherein the dose is 0.2 ml/rat, and the dose is based on peptide A, high.
  • the dose group, the middle dose group, and the low dose group were 174, 87, and 43.5 ug/kg rats, respectively.
  • a second immunization dose and immunization was performed with the first immunization, but in which Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • a third immunization was performed, the dose and immunization method were the same as the first immunization, but in which the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • the fourth immunization was carried out, that is, without adjuvant, the rats were intraperitoneally injected with the appropriate amount of peptide A in physiological saline, wherein the dose was 0.1 ml/rat, and the dose was measured by peptide A, high dose.
  • the rats in the group, middle dose group and low dose group were 174, 87 and 43.5 g/kg rats, respectively.
  • the model group and Rats in the blank control group were also injected for a total of four times, each time in a corresponding volume of physiological saline.
  • rats were intraperitoneally injected with D-galactosamine (product of SIGMA) 600 mg/kg rats in 5 groups other than the blank control group, and 0.2 rats per mouse were injected.
  • the blank control group was injected with an equal volume of normal saline. The rats were sacrificed 48 hours after intraperitoneal injection of D-galactosamine in rats.
  • the rats were intraperitoneally injected with 13.5 mg/kg of diammonium glycyrrhizinate daily on the 7th day before the rats were sacrificed, and the rats were intrauterine.
  • the rats 48 hours after intraperitoneal injection of D-galactosamine in rats, the rats were sacrificed by cervical dislocation and blood was collected. After separating the serum, the serum alanine aminotransferase (SGPT) activity was determined according to the instructions of the alanine aminotransferase test kit (purchased from Nanjing Jiancheng Bioengineering Research Institute) and the aspartate aminotransferase test kit (purchased from Nanjing Jiancheng Bioengineering Research Institute). Aspartate aminotransferase (SGOT) activity.
  • peptide A in D-galactosamine-induced acute liver injury model in rats, peptide A can significantly reduce the levels of alanine aminotransferase and aspartate aminotransferase in rats with acute experimental liver injury, and the diammonium glycyrrhizinate injection on acute liver in rats. Damage also has a significant protective effect.
  • mice (body weight 18g ⁇ 22g, purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were randomly divided into the following mice
  • diammonium glycyrrhizinate injection group injection of diammonium glycyrrhizinate
  • interferon group injected recombinant human interferon a 2a
  • high dose group (B) immunized with peptide A and adjuvant); (4) medium dose group (immunized with peptide A and adjuvant); (5) low dose group (immunized with peptide A and adjuvant); (6) adjuvant control group (adjuvant and adjuvant only) Immunization>; (7) blank control group; (8) model group.
  • mice except the blank control group were subcutaneously injected with 0.05 ml of CC1 4 (manufactured by Shanghai Lingfeng Chemical Reagent Co., Ltd.); the blank control group was injected with an equal volume of physiological saline.
  • the rats were immunized as follows: First, the primary immunization was performed, that is, the peptide A was mixed with Freund's complete adjuvant (the appropriate amount of peptide A was dissolved in physiological saline, and ⁇ ⁇ completely The mixture was mixed in an equal volume to form a water-in-oil chyle-like liquid for subcutaneous injection in rats, wherein the administration volume was 0.2 ml/mouse, and the dose was peptide A.
  • the high-dose, middle-dose, and low-dose groups were 250, 125, and 62.5 g/k g mice, respectively.
  • a second immunization, dose and immunization was performed with the first immunization, but in which the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • a third immunization was performed, the dose and immunization method were the same as the first immunization, but in which the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant.
  • the fourth immunization was carried out, that is, without adjuvant, the rats were intraperitoneally injected with the appropriate amount of peptide A in physiological saline, wherein the administration volume was 0.1 ml/mouse, and the dose was measured by peptide A, high dose.
  • the group, the middle dose group, and the low dose group were 250, 125, and 62.5 g/kg mice, respectively.
  • mice in the model group and the blank control group were also injected for a total of four times, each time in a corresponding volume of normal saline;
  • the adjuvant group only the corresponding adjuvant and physiological saline were injected for a total of four times.
  • mice except the blank control group were injected subcutaneously with 0.05 ml.
  • mice were sacrificed.
  • mice were intraperitoneally injected with diammonium glycyrrhizinate daily, and the dose was 19.5 mg / kg of mice based on diammonium glycyrrhizinate. 7 days; On the 7th day before sacrifice, the mice were intraperitoneally injected with interferon daily in the interferon group at a dose of 750,000 units/kg of mice for 7 days.
  • the liver was taken from the rats, fixed in 10% formalin solution, dehydrated, embedded in paraffin, prepared (4 ⁇ ⁇ thick), stained with HE, and examined by pathologists under the light microscope for the presence or absence of lesions.
  • the lesion score, lesion examination item, and scoring criteria were the same as in Example 6.
  • SGPT alanine aminotransferase
  • SGOT aspartate aminotransferase
  • liver tissue lesions mainly showed hepatocyte degeneration, punctate necrosis or focal necrosis in the liver, intrahepatic phlebitis, and a small amount of inflammatory cell infiltration and fibroblast proliferation in the liver and portal area. Liver tissue lesion degree score results indicate high dose group and model Compared with the group, the degree of liver lesions was significantly reduced.
  • mice were significantly enlarged relative to the mouse liver of the control group.
  • the peptide A was administered at a dose of 250 g/kg of mice, the hepatomegaly caused by carbon tetrachloride was significantly reduced. Interferon did not significantly reduce the hepatomegaly induced by carbon tetrachloride in mice.

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Description

预防或治疗肝损伤的肽及其衍生物及其应用 发明领域 本发明涉及丙型肝炎病毒免疫原性肽及其衍生物用于预防或治疗肝损伤的应用，尤其涉 及该肽或其衍生物在预防或治疗免疫性肝损伤和肝毒性化学物质引起的肝损伤中的应用， 以及其在预防或治疗丙型肝炎中的应用。本发明还涉及含有该肽或其衍生物的药物组合物、 制备方法以及编码该肽的多核苷酸。 背景技术 病毒性肝炎是危害人类健康的一类重症疾病， 其病原体是一类结构各异的嗜肝性病毒。 迄今为止， 已经发现的肝炎病毒共有 7个类型，它们分别是 HAV、 HBV、 HCV、 HDV、 HEV 和可能的 TTV及 HGV。 其中， 丙型肝炎病毒（HCV)是引起丙型肝炎的病原体。 丙型肝 炎最初曾被称为由输血引起的非甲非乙型肝炎， 但以后的研究证明， 丙型肝炎病毒不仅由 输血方式传播， 而其它方式如消化道、 性接触等， 均可导致其传播。 目前， 全世界大约有 超过 1亿的感染者， 其中大约 50%-90%的患者病程转为慢性， 在这些慢性感染中， 分别为 8-46%和 11-19%发展为肝硬化和肝细胞性肝癌。
HCV是属于黄病毒科的 RNA病毒。研究 (如可参见 Choo等， Science 244:359-362 (1989)； Choo等， Proc.Natl.Acad.Sci.USA 88:2451-2455 (1991); Han等， Proc.Natl.Acad.Sci.US A 88: 1711-1715 (1991) )表明， HCV的基因组为一约 9.4kb的单链正链 RNA， 并具有一个几乎跨 越整个基因组的开放阅读框（ORF)。该 ORF编码 3011个或 3010个氨基酸的病毒聚蛋白前 体。 HCV基因组编码的蛋白质有核壳核心蛋白 （C)、 两个包膜糖蛋白 （E1和 E2)并有 5 个非结构蛋白质（NS1 NS5) 区域。 其中， 在丙型肝炎病毒 E2区的高变区 1(HVR1区)内 含有重要的中和性抗原表位， 能诱导宿主产生中和性抗体（如可参见， Shirai等, J. Immunol, 162:568-576(1999))。 然而， 在免疫选择作用下， HVR1 区的基因会产生高度变异， 从而使 HCV可能逃避机体的免疫识别。 这可能也是 HCV导致慢性肝炎的主要原因。
目前， 对于 HCV致病机制尚不清楚， 临床上还没有确实有效的治疗方法和疫苗来防止 其进一步传播。 现在临床上常使用干扰素（IFN)， 通过活化 RNaseL来降解病毒 RNA, 但 其长期有效率仅为 20%左右。 Manns等（The Lancet, 358: 958-965 (2001))通过使用 PEG化 的干扰素和利巴伟林来联合治疗丙型肝炎。 然而这种改进疗法仅仅对 2型和 3型病毒株感 染的病人特别有效， 而对于 la、 lb和 4基因型病毒株感染的病人的效果却很有限。 为此， 人们已经积极尝试各种手段来研制和开发降低 HCV感染率的疫苗以及治疗肝炎的药物。
通过对 HCV基因组的研究， 人们发现了大量针对 HCV的免疫原性肽， 能诱导机体产 生针对 HCV的免疫应答。例如， Chisari等的 US5709995A披露了一系列肽， 其能够诱导产 生 HCV特异性细胞毒性 T淋巴细胞（CTL)。 WO2003/097677A公开了具有强烈产生免疫 反应能力的 HCV抗原肽及其组合物。本发明人的 CN1194986C和 CN1216075C也披露了一 系列能够诱导抗体产生的 HCV免疫原性肽。 尽管早期曾经认为， HCV和某些病毒（如乙 肝病毒、 EB病毒等） 一样是通过病毒致细胞病变而造成肝损伤的， 然而研究表明， 针对 HCV的免疫反应却是引起肝脏损伤的主要原因。尤其是在慢性 HCV患者中，淋巴细胞（尤 其是细胞毒 T淋巴细胞） 非但不能完全有效地清除 HCV， 反而在清除 HCV感染的肝细胞 的过程中会造成肝细胞的免疫性损伤， 引起肝细胞调亡， 由此甚至可以导致肝硬化和肝细 胞性肝癌（例如可参见， Nelson等， J. Immunol., 158:1473-1481(1997); Wong等， J. Immunol., 160:1479-1488 (1998)； Ruggieri等， Virology,229： 68-76 (1997))。 因此， HCV免疫原性肽 作为疫亩引发针对 HCV的免疫反应可能会导致免疫性肝损伤。这样的机制为利用针对 HCV 的免疫原性肽开发实际可用于丙型肝炎 （尤其是慢性丙型肝炎） 的预防疫苗或治疗药物带 来了极大的困难。
除了免疫性肝损伤和病原性肝损伤以外， 众所周知， 肝毒性化学物质也会引起肝损伤。 已知一些药物可以造成肝损伤， 导致肝脏细胞溶解和肝坏死。 例如在大剂量服用时， 镇痛 药醋氨酚（即扑热息痛， 化学名为 4- (N-乙酰基氨基）酚） 就是一种具有肝损伤作用的物 质， 会引起人的肝坏死。 又如， 长期服用利福平、 吡嗪酰胺、 异烟肼等抗生素， 以及更年 期长期服用雌激素等也会造成严重的肝细胞坏死， 导致急性或慢性肝炎、 黄疸和肝纤维化 等肝损伤。 引起肝损伤的化学物质包括那些能产生大量活泼自由基的物质， 尤其是能产生 氧自由基的物质， 它们通常通过氧化作用导致肝毒性。
经过大量的研究， 本发明人获得了一种 HCV免疫原性肽及其衍生物， 令人惊讶的是， 其能够预防或治疗肝损伤， 所述肝损伤不仅限于 HCV感染的肝细胞的免疫性损伤。 例如， 本发明的肽及其衍生物能用于预防或治疗免疫性肝损伤、 病原性肝损伤以及肝毒性化学物 质引起的肝损伤。 发明内容 本发明涉及一种肽及其衍生物， 除了诱导针对 HCV的免疫应答之外， 其还能够预防或 治疗肝损伤， 尤其是预防或治疗免疫性肝损伤， 而且所述肝损伤不局限于 HCV感染的肝细 胞的免疫性损伤。
在第一个方面， 本发明提供了含式 I所示序列的肽或其药学上可接受的盐或酯的应用，
Xaal-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I)
其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser，
Xaa3为 Tyr、 Phe或 Trp， 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu、 lie或 Pro，
所述应用是用于预防或治疗肝损伤或者用于制备预防或治疗肝损伤的药物。 也就是说， 本发明提供了含式 I所示序列的肽或其药学上可接受的盐或酯在预防或治疗肝损伤中的应 用，并提供了含式 I所示序列的肽或其药学上可接受的盐或酯在制备预防或治疗肝损伤的药 物中的应用。
本文中所用的 "肝损伤"指的是肝脏组织或细胞出现的损伤或病变。 临床上， 肝损伤主 要表现为肝细胞变性、 肝内静脉炎、 肝内出现点状坏死或灶状坏死、 在肝内及门管区出现 炎细胞浸润或出现成纤维细胞增生、 或肝脏肿大等， 严重时可导致肝硬化、 肝癌等。 除了 通过上述病理现象来评估肝损伤以外， 当肝细胞发生细胞溶解性的损伤时， 转氨酶由受损 伤的肝细胞中释放到循环血液的量增加了， 测定这些转氨酶在血清中的活性， 就能够确定 肝脏的损伤和评价受损伤的程度。 在本发明的具体实施方式中， 可通过测定谷丙转氨酶或 谷草转氨酶水平来评估肝损伤。 优选本发明中的肝损伤是血清中谷丙转氨酶或谷草转氨酶 水平升高所反映出的肝损伤， 优选本发明中的预防或治疗肝损伤的疗效指标是谷丙转氨酶 或谷草转氨酶水平的降低程度。本发明的含式 I所示序列的肽或其药学上可接受的盐或酯对 肝损伤的作用大小可通过上述病理现象的减少程度或谷丙转氨酶或谷草转氨酶水平的降低 程度来确定。
目前研究已经发现， 大量肝毒性药物和化学物质、 免疫原或病原体都可以造成肝损伤。 优选本发明所述的肝损伤为免疫性肝损伤或肝毒性化学物质引起的肝损伤， 即本发明优选 提供了含式 I所示序列的肽或其药学上可接受的盐或酯在预防或治疗免疫性肝损伤或肝毒 性化学物质引起的肝损伤中的应用，也优选提供了含式 I所示序列的肽或其药学上可接受的 盐或酯在制备预防或治疗免疫性肝损伤或肝毒性化学物质引起的肝损伤的药物中的应用。 目前已经有大量合适的动物模型可用于评估本发明对肝损伤的作用。在一个具体的例子中， 本发明的肽能用于减轻由卡介苗和脂多糖诱导的免疫性肝损伤。 另外， 在本发明具体的肝 毒性化学物质引起的肝损伤模型中， 本发明的肽能用于预防由 D-氨基半乳糖胺造成的肝损 伤， 而且本发明的肽也能用于治疗由四氯化碳造成的肝损伤。
本发明的"含式 I所示序列的肽"指的是如式 I所示序列的肽或者其修饰的功能等同物， 即如式 I所示序列的肽的 N末端的氨基和 C末端的羧基以及氨基酸侧链基团可以不进行修 饰， 也可以在基本不降低预防或治疗肝损伤的活性的前提下进行修饰。 这里的 "功能等同 物"指含有式 I所示序列的修饰产物， 其基本不降低预防或治疗肝损伤的活性， 即不降低 50%的预防或治疗肝损伤的活性， 优选不降低 30%的活性， 更优选不降低 10%的活性， 最 优选不降低活性。 某些病理现象或血清中某些转氨酶的水平可用于确定功能等同物的预防 或治疗肝损伤的活性， 优选使用本发明的具体实施方式中所述的病理评分标准或者血清中 谷丙转氨酶或谷草转氨酶水平来确定活性。
对于本发明的肽， 合适的修饰如， 环化， 制备成多聚体， 对末端氨基、羧基或侧链基团 修饰以形成药学上可接受的酯， 含式 I所示序列的缀合物， 含式 I所示序列的融合蛋白， 或 这些修饰的组合等。 将线性肽环化， 如将肽 N端的氨基和 C端的羧基缩合形成环肽， 通常 可以延长肽在生理环境中的半衰期。 "药学上可接受的酯"指适于与人或动物的组织接触而 且无过多的毒性、 剌激或变态反应等的酯。 通常， 酯化修饰后能降低机体中的蛋白酶对肽 的水解。 对本发明的肽的末端氨基、 羧基或侧链基团进行修饰可以形成药学上可接受的酯。 对氨基酸侧链基团的修饰包括但不限于苏氨酸、丝氨酸侧链羟基与羧酸发生的酯化反应。 N 末端氨基基团的修饰包括但不限于脱-氨基、 N-低级垸基、 N-二 -低级烷基和 N-酰基修饰。 C 末端羧基基团的修饰包括但不限于酰胺、 低级烷基酰胺、 二烷基酰胺和低级烷基酯修饰。 优选末端基团用蛋白质化学领域的技术人员已知的保护性基团保护起来， 如乙酰基、 三氟 乙酰基、 Fmoc (9-芴基-甲氧羰基)、 Boc (叔丁氧羰基）、 Alloc (烯丙氧羰基）、 C 1-6烷基、 C2.8烯基、 C 7-9芳垸基等。在本发明的具体实施方式中，优选不对式 I多肽 N末端的氨基 和 C末端的羧基以及氨基酸侧链基团进行修饰， 即 N末端的化学基团仍旧为第一个氨基酸 上的 α -氨基（-NH2)， C末端的化学基团是 C末端氨基酸的羧基（-COOH)。本发明也优选 对 C末端的羧基进行酰氨化， 即 C末端的化学基团是 -CO NH2。
使用本领域已知的方法，含式 I所示序列的缀合物包含药学上可接受的水溶性多聚物部 分。 通常， 该缀合物显示出能延长式 I所示序列的肽的循环半衰期。 合适的水溶性多聚物 包括聚乙二醇 （PEG)、 单甲氧基 -PEG、 单 -(d-J垸氧基 - PEG、 芳氧基 - PEG、 聚- (N-乙烯吡 咯烷酮） PEG, 三甲氧基 PEG、 单甲氧基- PEG丙醛、 PEG丙醛、 二-琥珀酰亚胺碳酸 PEG、 丙烯乙二醇同聚物、聚丙烯氧化物 /乙烯氧化物共聚物、聚氧乙烯多羟基化合物 (如，甘油）、 单甲氧基- PEG丁醛、 PEG丁醛、 单甲氧基- PEG乙醛、 PEG 乙醛、 甲氧基 PEG-琥珀酰亚胺 丙酸、 甲氧基 PEG -琥珀酰亚胺丁酸、 聚乙烯醇、 右旋糖苷、 纤维素或其他糖类的多聚物。 合适的 PEG可具有约 600至约 60, 000的分子量， 包括如， 5， 000道尔顿， 12， 000道尔顿， 20， 000道尔顿， 30, 000道尔顿，和 40, 000道尔顿， 其可以是直链的或分支的。 含式 I所示 序列的肽的缀合物还可包括这类水溶性多聚物的混合物。 PEG化可通过现有技术中已知的 PEG化反应来进行（如参见， Delgado 等的 Critical Reviews in Therapeutic Drug Carrier Systems 9: 249 (1992), Duncan和 Spreafico的 Clin. Pharmacokinet. 27: 290 (1994),和 Francis 等的 Int J Hematol 68: 1 (1998))。 例如， PEG化可用反应性聚乙二醇分子由酰化反应或由烷 基化反应来进行。 在可选的方法中， 缀合物由缩合活化的 PEG来形成， 其中 PEG末端的羟 基或氨基被活化的接头分子替代 (如参见， Karasiewicz等， US5382657A)。 含式 I所示序列 的缀合物也可以是式 I所示序列的肽同其它蛋白交联形成的缀合物。 所述其它蛋白优选人 白蛋白、 牛白蛋白或 IgG分子的 Fc部分。 在本发明的一个具体实施方式中， 本发明的肽与 牛血清白蛋白交联形成肽缀合物。
本发明的含式 I所示序列的肽也可以是式 I所示序列的肽与其它肽或蛋白质形成的含式 I所示序列的融合肽或融合蛋白。优选其中所述蛋白质为人白蛋白、 牛白蛋白或 IgG分子的 Fc部分。 白蛋白可以通过遗传工程方式偶连到本发明的含式 I所示序列的肽上以延长半衰 期。 其中， 人白蛋白是最普通的天然产生的人循环系统中的血蛋白， 能在体内维持循环超 过 20天。 研究表明， 通过遗传工程方式融合到人白蛋白上的治疗蛋白具有较长的半衰期。 另外研究表明， 对于 Fc部分， 所得的融合蛋白可增加循环半衰期 （参见 US5750 375A, US5843725,美国专利第 6,291, 646号； Barouch等， Journal of Immunology, 61:1875-1882 (1998); Barouch等， Proc. Natl. Acad. Sci. USA, 97(8)： 4192-4197 (4月 11,2000)；和 Kim等, Transplant Proc, 30 (8): 4031-4036(1998))。
在本发明的式 I所示序列的肽中， 优选 Xaal是 Gly， Xaa2是 Thr， Xaa3是 Tyr， 而且 Xaa4为缺失、 Ala或 Gly， 更优选其中： Xaa4为缺失。 本文中所使用的 "缺失"指的是缺失 的氨基酸残基不存在于肽序列中， 例如当 Xaa4缺失时， 式 I所示序列中的 Gly就是式 I所 示序列的 C末端的氨基酸。
本文中所使用的肽及氨基酸、 氨基酸残基和化学基团的表示方法均为所属领域公认的表 示方法。 其中氨基酸或氨基酸残基的縮写可参照表 1中定义， 这些缩写可以指 L-型的氨基 酸， 也可以指 D-型的氨基酸。 优选氨基酸指 L-型的氨基酸。 其中， 氨基酸或氨基酸残基可 以根据其侧链性质的相似性而分成以下组： 疏水性氨基酸（A、 I、 L、 M、 F、 P、 W、 Y、 V)、 亲水性氨基酸（R、 D、 N、 C、 E、 Q、 G、 H、 K、 S、 T)、 脂肪族侧链的氨基酸 (G、 A、 V、 L、 I、 P)、 含羟基侧链的氨基酸（S、 T、 Y)、 含硫原子侧链的氨基酸 （C、 M)、 含羧酸和酰胺 侧链的氨基酸（D、 N、 E、 Q)、 含碱性基团侧链的氨基酸（R、 K、 Η)、 含芳香族侧链的氨基 酸（H、 F、 Y、 W)。 通常， 处于同组中的氨基酸或氨基酸残基具有相似的性质。
表 1 氨基酸缩写表
氨基酸 三字母缩写 一字母缩写 氨基酸 ：三字母缩写 一字母缩写
丙氨酸 Ala A 亮氨酸 Leu L
精氨酸 Arg R 赖氨酸 Lys K
天冬酰胺 Asn N 蛋氨酸 Met M
天冬氨酸 Asp D 苯丙氨酸 Phe F
半胱氨酸 Cys C 脯氨酸 Pro P
谷氨酰胺 Gin Q 丝氨酸 Ser S
谷氨酸 Glu E 苏氨酸 Thr T
甘氨酸 Gly G 色氨酸 Trp w
组氨酸 His H 酪氨酸 Tyr Y
异亮氨酸 lie I 缬氨酸 Val V
"药学上可接受的盐"指适于与人或动物的组织接触而且无过多的毒性、刺激或变态反 应等的盐。 药学上可接受的盐是本领域熟知的。 这种盐可以在本发明多肽的最终分离和纯 化的过程中制备， 也可以将肽与适当的有机或无机酸或碱反应单独制备。 代表性酸加成盐 包括但不限于乙酸盐、 二己酸盐、 藻酸盐、 柠檬酸盐、 天冬氨酸盐、 苯甲酸盐、 苯磺酸盐、 硫酸氢盐、 丁酸盐、 樟脑酸盐、 樟脑磺酸盐、 甘油磷酸盐、 半硫酸盐、 庚酸盐、 己酸盐、 富马酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、 2-羟基乙磺酸盐、乳酸盐、马来酸盐、 甲磺酸盐、 烟酸盐、 2-萘磺酸盐、 草酸盐、 3-苯基丙酸盐、 丙酸盐、 琥珀酸盐、 酒石酸盐、 磷酸盐、 谷 氨酸盐、 碳酸氢盐、 对甲苯磺酸盐和十一垸酸盐。 能用于形成药学上可接受盐的优选的酸 是盐酸、 氢溴酸、 硫酸、 磷酸、 草酸、 马来酸、 琥珀酸和柠檬酸。 药学上可接受的碱加成 盐中的阳离子包括但不限于碱金属或碱土金属离子如锂、 钠、 钾、 钙、 镁和铝等， 以及非 毒性季铵阳离子如铵、 四甲基铵、 四乙基铵、 甲基胺、 二甲基胺、 三甲基胺、 三乙基胺、 二乙基胺、 乙基胺、 二乙胺、 乙醇胺、 二乙醇胺、 哌啶、 哌嗪等。 优选的碱加成盐包括磷 酸盐、 tris和乙酸盐。 这些盐一般能够增加多肽的溶解性， 而且所形成的盐基本上不改变多 肽的活性。 本发明的多肽可以单独使用， 也可以以药学上可接受的盐形式使用。
本发明第一方面的应用还包括进一步用于治疗和 /或预防丙型肝炎。 也就是说， 在治疗 或预防肝损伤的基础上，本发明还提供了含式 I所示序列的肽或其药学上可接受的盐或酯在 预防或治疗肝损伤和丙型肝炎中的应用，并提供了含式 I所示序列的肽或其药学上可接受的 盐或酯在制备预防或治疗肝损伤和丙型肝炎的药物中的应用。 本发明的肽具有诱导细胞因 子 Y -IFN、 IL-4、 IL-10和抗体产生的作用。 其中 Y -IFN是 1型 T辅助细胞（Thl )分泌的 主要细胞因子，是人体免疫系统抵抗病毒感染的主要细胞因子之一，能够用于通过针对 HCV 的细胞免疫来清除 HCV， 而且 IFN也是当前治疗 HCV的成熟疗法。
在第二个方面，本发明提供了用于本发明第一方面所述应用的药物组合物，其包括含式 I所示序列的肽或其药学上可接受的盐或酯以及药学上可接受的载体，
Xaal -Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I )
其中，
Xaal为缺失、 Ala、 Gly、 VaK Leu或 lie,
Xaa2为 Thr或 Ser，
Xaa3为 Tyr、 Phe或 Trp， 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu、 He或 Pro。
本发明的药物组合物用于预防或治疗肝损伤。本发明的药物组合物可减少由肝损伤带来 的某些病理现象和 /或降低血清中某些转氨酶的水平， 优选可减少本发明的具体实施方式中 所述的病理评分标准或者降低血清中谷丙转氨酶或谷草转氨酶水平。 本发明的药物组合物 具有诱导细胞因子 Y -IFN、 IL-4、 IL-10和抗体产生的作用。 因此， 本发明的药物组合物优 选还可以进一步用于治疗和 /或预防丙型肝炎。
本文中使用的 "药学上可接受的载体" 指无毒固态、 半固态或液态填充剂、 稀释剂、 佐剂、 包裹材料或其他制剂辅料。 根据本领域的公知技术， 可以根据治疗目的、 给药途径 的需要将药物组合物制成各种剂型， 优选该组合物为单位剂量形式， 如片剂、 膜剂、 丸剂、 胶囊 (包括持续释放或延迟释释设形式)、 粉剂、 颗粒剂、 酊剂、 糖浆剂和乳液剂、 消毒的住 射用溶液或悬浮液、 气雾剂或液体喷剂、 滴剂、 针剂、 自动注射装置或栓剂。 例如， 以片 剂或胶襄的服给药， 上述活性药物组分可以与一种口服的无毒的药物学可接受的惰性载体 组合在一起， 如乙醇、 等渗葡萄糖溶液、 甘油、 生理盐水或其组合。 组合物中还可以添加 辅料， 如人血清白蛋白、 低分子量肽、 氨基酸和金属阳离子等蛋白保护剂， 也可以添加佐 剂， 如福氏完全佐剂、 福氏不完全佐剂、 聚 CpG等。
然而令人惊讶的是， 在不添加佐剂的情况下， 本发明的包括含式 I所示序列的肽或其药 学上可接受的盐或酯的药物组合物也能够单独起到预防或治疗肝损伤和 /或预防或治疗丙型 肝炎的作用。 在一个具体的实施方式中， 在不添加佐剂的情况下， 本发明的肽也能够显著 提高机体分泌干扰素的量。 在另一个具体的实施方式中， 在不添加佐剂的情况下， 本发明 的肽也能显著降低肝损伤。 这提示， 本发明的肽的预防或治疗肝损伤和丙型肝炎的作用机 理并不局限于诱导针对 HCV的免疫应答。在抑制病毒复制、 清除病毒的同时， 本发明的肽 可能还可以抑制机体过度的免疫炎性反应， 从而达到减轻肝组织细胞损伤目的。 因此， 本 发明第二个方面的组合物优选不含佐剂。
在本发明药物组合物中的式 I所示序列的肽中， 优选 Xaal是 Gly， Xaa2是 Thr， Xaa3 是 Tyr， 而且 Xaa4为缺失、 Ala或 Gly， 更优选其中 Xaa4为缺失。
另外，本发明还涉及的本发明第二个方面的药物组合物在制备预防或治疗肝损伤的药物 中的应用； 本发明还涉及的本发明第二个方面的药物组合物在预防或治疗肝损伤中的应用。 优选上述应用还包括进一步用于治疗和 /或预防丙型肝炎。
本发明的药物组合物可通过所属领域技术人员所熟知的给药方式来进行给药， 例如口 服、 直肠、 舌下、 肺部、 透皮、 离子透入、 阴道及鼻内给药。 本发明的药物组合物优选胃 肠道外给药， 如皮下、 肌内或静脉内注射。 给药剂量根据制剂形式和期望的作用时间以及 治疗对象的情况而有所变化， 实际治疗所需的量可以由医师根据实际情况（如， 病人的病 情、体重等）而方便地确定。对于一般的成人， 本发明的药物组合物的剂量， 以式 I所示序 列的肽计， 可以是每 kg成人体重 lng - 10g。 对于注射给药模式来说， 优选的剂量是每 kg 体重 lOOng - 10mg, 更优选的是每 kg - lmg, 最优选的是每 kg l(^g - 10(^g。对于口服 摄入来说， 给药量可以是每天每 kg体重 1μ8 - 10g, 优选是每天每 kg体重 10 g - lg, 更优 选的是每天 10(^g - 10mg。
在第三个方面，本发明提供了用于本发明第一方面所述应用或者用于本发明第二方面所 述药物组合物中的肽或其药学上可接受的盐或酯， 其中所述肽含式 I所示序列，
Xaal -Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I )
其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser，
Xaa3为 Tyr、 Phe或 Trp， 而且 Xaa4为缺失、 Ala、 Gly、 Val、 Leu、 He或 Pro。 其中， 优选 Xaal是 Gly， Xaa2是 Thr， Xaa3是 Tyr， 而且 Xaa4为缺失、 Ala或 Gly， 更优选其中 Xaa4为缺失。
本发明的肽是纯化的肽， 即纯化到 80%纯度， 优选 90%纯度， 更优选 95%纯度， 尤其优选达到药物纯的状态， 即纯度是大于等于 98%的纯度， 而且不含感染源和热源。 优 选本发明的肽实质上不含有其它多肽或蛋白质， 尤其是动物来源的那些肽或蛋白质。
另外， 在第四个方面， 本发明提供了编码本发明第三方面所述的肽的多核苷酸。本文中 使用的 "多核苷酸"是指从 5'向 3'末端阅读的脱氧核糖核苷酸或核糖核苷酸碱基的单链或双 链多聚物， 包括 R A和 DNA， 可以从天然来源中分离、 体外合成或重组表达等方式来制 备。
在第五个方面，本发明提供了本发明第三方面所述的肽或其药学上可接受的盐或酯的制 备方法， 其步骤包括， 化学合成所述肽或者融合表达所述肽。 根据所需的药学上可接受的 盐或酯结构， 本发明的制备方法中还可包括将所述肽进一步反应生成所述盐或酯的步骤。
通过化学方法合成已知结构的肽对于所属领域技术人员来说都是显而易见的。详细的方 案可参照以下文献所述的方法进行， 如用固相法合成多肽可参考 J.M. Steward和 J.D. Young 的 《Solid Phase Peptide Synthesis》（第二版， Pierce Chemical Co., Rockford, Illinois(1984))和 J. Meienhofer的 《 Hormonal Proteins and Peptides》（第 2卷， Academic Press, 纽约（1973)) 等；用液相法合成多肽可参考 E. Schroder和 K. Lubke的《The Peptides)) (第 1卷， Academic Press, 纽约 (1965) )等。 在本发明的一个具体实施方案中， 优选通过固相法合成本发明的 多肽。
通过基因工程融合表达并提纯已知结构的肽对于所属领域技术人员来说都是显而易见 的 (可参见 Sambrook等， Molecular Cloning: A Laboratory Manual,第 2版， Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989； 禾口 Ausubel等， 编， Current Protocols in Molecular Biology, JohnWiley and Sons, Inc.NY, 1987)。 如， 将本发明第五个方面的多核 苷酸导入表达载体中， 并在宿主细胞中表达， 从而制备出所述肽。 适宜的表达载体包括质 粒、 粘粒、 噬菌体或病毒等； 适宜的宿主细胞包括细菌、 真菌和真核细胞。 附图说明 图 1 用本发明的肽免疫的小鼠的血清中的相应抗体水平。
图 2在添加佐剂和不添加佐剂的情况下， 接种本发明的肽的大鼠脾细胞分泌的 Y -IFN 的量。
图 3 免疫本发明的肽对卡介苗和脂多糖诱导的大鼠免疫性肝损伤模型中血清生化指标 的影响， 其中 *表示所示组相对于模型组能够显著降低转氨酶水平。
图 4免疫本发明的肽对卡介苗和脂多糖诱导的大鼠免疫性肝损伤模型中肝脏病变程度 的评分结果， 其中 *表示所示组相对于模型组能够显著减轻肝脏的病变程度。
图 5免疫本发明的肽对 D-氨基半乳糖胺诱导的大鼠急性肝损伤模型中血清生化指标的 影响， 其中 *表示所示组相对于模型组能够显著降低转氨酶水平。
图 6免疫本发明的肽对小鼠四氯化碳致肝损伤模型中血清生化指标的影响， 其中 *表示 所示组相对于模型组能够显著降低转氨酶水平。
图 7免疫本发明的肽对小鼠四氯化碳致肝损伤模型中肝脏病变程度的评分结果，其中 * 表示所示组相对于模型组能够显著减轻肝脏的病变程度。
本发明引用了公开文献，这些文献是为了更清楚地描述本发明， 它们的全文内容均纳入 本文进行参考， 就好像它们的全文已经在本文中重复叙述过一样。
为了便于理解，以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是， 这些描述仅仅是示例性的描述， 并不构成对本发明范围的限制。 依据本说明书的论述， 本 发明的许多变化、 改变对所属领域技术人员来说都是显而易见了。 具体实施方式 ,
实施例 1 肽合成
通过固相肽合成方法， 使用 413A型自动肽合成仪（购自 Perkin Elmer公司） 来合成如 以下序列所示的三个肽： GQTYTSG (以下称为 "肽 A")、 GQTYTSGA (以下称为 "肽 B") 和 GQTYTSGG (以下称为 "肽 C")。这三个肽中的氨基酸残基均为 L-型的氨基酸。合成的 具体过程如下： 首先， 保护氨基酸单体上的反应性基团： 氨基酸的 α氨基用 9-芴基甲氧羰 基 (Fmoc)保护； 并对以下特定氨基酸进行侧链保护： 对 Ser和 Thr的侧链保护基为叔丁基， 对 Gin的为三苯甲基 (Trt)。然后，以 Ν,Ν-二异丙基碳二亚胺 /1-羟基苯并三唑作为活化试剂， 使受保护的氨基酸依次偶联， 偶联每次 40分钟。 在 15%乙二硫醇 /二甲硫醚 /茴香醚 (体积 比为 1 : 1 : 1)存在的情况下，肽与三氟乙酸 (85%)在室温反应 120分钟，从而从聚合体支持 物上切割下来， 同时脱除保护基。 接着用无水乙醚沉淀肽， 然后用无水乙醚多次洗涤， 充 分除去硫醇。在水 /叔丁醇 (1： 1)中沉淀，冷冻干燥，得到粗肽。粗肽在 30分钟内以反向 HPLC 纯化， 以 37-42%乙晴 /0.9%TFA梯度进行。然后进行浓缩、冻干。 由此分别合成肽八、肽 B 和肽 C。 合成的这三种肽均为纯度 98 %的白色固体。 实施例 2肽缀合物的制备
肽 A与牛血清白蛋白 （BSA)通过戊二醛法交联形成缀合物。 具体缀合过程如下： 取 lmg实施例 1合成的肽 A， 将其溶于 0.5ml PBS (pH7.4, 0.02mol/L) 中； 取 4.5mg BSA溶 于 4.5ml PBS (pH7.4, 0.02mol/L) 中。 混合上述肽 A和 BSA溶液， 然后缓慢加入 0.1 %的 戊二醛 lml， 在室温下避光让交联反应进行 12小时。 然后缓慢加入甘氨酸溶液（lmol / L) 终止反应， 接着用 PBS (pH7.4, 0.02mol/L)透析过夜， 冷冻干燥。 所得的肽 A与 BSA的 交联产物被命名为肽缀合物 A。 实施例 3 肽同丙型肝炎患者血清的反应性研究
3.1血清来源
抗 HCV抗体阳性血清：随机从 2000-2001年解放军 302医院 HCV感染的住院患者中获 得。
对照血清： 取自健康献血人员， 化验检查结果反映各项指标均正常。
乙型肝炎 (HBV)患者血清： 选自解放军 302医院乙型肝炎住院患者， 化验检査均呈乙型 肝炎病毒表面抗原阳性。
3.2采用间接 ELISA法检测血清反应性的方法
根据常规间接 ELISA技术， 釆用 DG3022型酶联免疫检测仪 (购自 Perkin Elmer公司)来 检测本发明的各种肽与各种血清的反应性。 分别将肽 A、 肽 B、 肽 C和肽缀合物 A各 10 μ g加于碱性包被液（0.1mol/L NaHC03， 35 μ ΐ; 0.1mol/L Na2C03 ， 15 μ ΐ; H20， 50 1) 中， 将包被液加入聚丙乙烯微孔板的孔中进行包被， 放置于 4°C过夜。 于 37 V , 向每孔 中加 120 l FCS-PBS封闭 2小时。 然后， 向每孔中加入血清 (1： 100稀释)， 于 37°C孵育 1 小时。然后向每孔中加入羊抗人 IgG (购自华美生物制品公司， 北京) (1： 1000稀释） ， 于 37 Ό孵育 1小时。 每孔加入 A、 B液 (购自北京科卫试剂厂) 50 μ 1， 置暗处放 5分钟， 然后于 450nm波长下检测光吸收度。
3.3结果
肽（肽 A、 肽8、 肽 C和肽缀合物 A) 同 HBV患者血清 (10份)及健康人血清 (10份)结 合试验结果均为阴性。 肽结合抗 HCV抗体阳性血清 (30份)的结果如表 2所示。 经 SATA软 件进行卡方检验可知， 相对于同 HBV患者血清及健康人血清的反应性， 本发明的肽 A、 肽 B、肽 C和肽缀合物 A均能显著地同 HCV患者血清反应， 而肽同牛血清白蛋白交联而形成 的缀合物能增加本发明的肽同 HCV患者血清的反应性。
表 2本发明的肽同 HCV患者血清的反应性
Figure imgf000014_0001
实施例 4肽在小鼠体内免疫原性和血清中细胞因子的研究
4.1 实验动物
取 BALB/c小鼠 （均为雄性， 6周龄， 购自军事医学科学院实验动物中心， 北京）， 分 成以下 4组， 每组 5只： （1 )对照组； （2)肽 A组（用肽 A免疫）； (3 )肽 B组 （用肽 B 免疫）； (4)肽 C组 （用肽 C免疫)。
4.2免疫方法
将每组所用的肽（100 μ g/μ 1)与福氏完全佐剂 (GIBCOBRL公司)各 50 μ 1等体积混合， 用微量搅拌器充分混匀， 注射小鼠足垫， 由此完成对小鼠的第一次免疫。 14天后进行加强 免疫， 即将每组所用的肽 （100 g/μ ΐ) 与福氏不完全佐剂 (GIBCOBRL公司）各 50 μ 1以 与第一次免疫相同的方式进行免疫。 14天后以相同方式再进行一次加强免疫。 14天后再进 行冲击免疫， 即用 200 1 PBS溶解 lOO g肽， 将其经肌肉注射小鼠。在此期间， 对照组只 注射相应的佐剂和 PBS。 第四次免疫后一天处死小鼠， 收取血清。
4.3采用 ELASA法检测血清中的相应抗体水平
根据常规间接 ELISA技术， 检测免疫后的小鼠血清中的抗体。 分别用肽八、 肽 B和肽 C作为抗原包被聚丙乙烯微孔板， 即将 10 μ g上述肽加入到 100 μ 1碱性包被液中包被板上 的每个孔， 于 4°C过夜。 用 120 1 PBS封闭， 于 37°C孵育 2小时。 然后向每个孔分别加入 各组小鼠的血清（1： 100稀释）， 37°C孵育 1小时。向每个孔加入羊抗鼠 IgG 100 μ 1(购自华 美生物制品公司， 北京）（1： 1000稀释)， 37°C孵育 1小时。 然后每孔加入 A、 B液 (购自北 京科卫试剂厂 )50 μ 1， 置暗处放 5分钟， 检测 450nm波长下的光密度 (OD)值， 结果如图 1 所示。
上述实验结果表明， 使用上述肽免疫小鼠都能够诱导机体产生相应体液免疫应答。 4.4采用 ELASA法检测小鼠血清中的细胞因子水平
参照厂商试剂盒的说明书， 分别使用 IL-4 ELASA检测试剂盒、 IL-10 ELASA检测试剂 盒、 Y -IFN ELASA检测试剂盒（均购自 R&D公司）检测经上述免疫后的小鼠血清中的细 胞因子 （即 IL-4、 IL-10、 γ -IFN)水平， 结果如表 3所示。
表 3 免疫的小鼠血清中的细胞因子水平
Figure imgf000015_0001
上述实验结果表明， 使用上述肽免疫的小鼠的血清中 Y -IFN、 IL-4、 IL-10, 都有着显著 的提高。 由于 Y -IFN是是 Thl分泌的主要细胞因子， 也是人体免疫系统抵抗病毒感染的主 要细胞因子之一， 它通过诱导 2-5A合成酶， 进而活化 RNaseL, 降解病毒 R A， 从而抑制 病毒蛋白的合成， 所以 Υ -IFN水平的升髙说明上述肽能够用于清除 HCV。 实施例 5 肽在大鼠体内对 Y -IFN的影响
5.1 实验动物
将 SD大鼠（体重 180g〜220g， 雌雄各半， 购自军事医学科学院动物所）随机分成以下 5组，每组 10只： （1 )空白对照组； （2)佐剂对照组； （3)高剂量组（用肽 A和佐剂免疫）; (4)低剂量组（用肽 A和佐剂免疫)； (5)肽免疫组（用肽 A免疫)。
5.2实验方法
高剂量组、 低剂量组按如下方法免疫大鼠： 从第 0天开始， 首先进行初次免疫， 即将 肽 A与福氏完全佐剂混合（用生理盐水溶解适量肽 A后与等体积福氏完全佐剂混合）而成 油包水的乳麋状液对大鼠进行皮下注射， 其中给药体积为 0.1ml/只大鼠， 而给药量以肽 A 计分别为 50 g/kg大鼠和 25 w g/kg大鼠。 4天后， 进行第二次免疫， 剂量和免疫方式同第 一次免疫， 但是其中用福氏不完全佐剂代替福氏完全佐剂。 4天后， 进行第三次免疫， 剂量 和免疫方式同第一次免疫， 但是其中用福氏不完全佐剂代替福氏完全佐剂。 4天后， 进行第 四次免疫， 即不加佐剂，用生理盐水溶解适量肽 A后腹腔注射大鼠，其中给药体积为 0.1ml/ 只大鼠， 而给药量以肽 A计分别为 50 w g/kg大鼠和 25 g/kg大鼠。在进行免疫期间， 从第 0天开始， 佐剂对照组的大鼠在相应时间被注射不含肽 A的相应佐剂和生理盐水； 空白对 照组的大鼠在相应时间被注射生理盐水。
在高剂量组和低剂量组进行免疫注射的相同时间， 从第 0天开始， 在肽免疫组中， 分 别对大鼠皮下注射用生理盐水溶解的肽 A (其中不加入佐剂）来免疫大鼠， 共进行四次免 疫。 其中， 每次给药体积为 0.1ml/只大鼠， 而每次给药量以肽 A计为 50 w g/kg大鼠。
第四次免疫后一天处死各组小鼠， 收取脾细胞。 将脾细胞以 1X106个 /孔的浓度加入到 培养板上， 37°C孵育 3天。然后取培养上清液，参照厂商试剂盒的说明书，使用 Y -IFN ELASA 检测试剂盒（购自 R&D公司）检测经上述免疫后的大鼠血清中的 γ -IFN水平。 5.3实验结果
大鼠血清中的 y -IFN水平结果如图 2所示。 上述实验结果表明， 免疫的大鼠的血清中 Y -IFN的量相对于对照组有着显著的提高。 更令人惊讶的是， 在不添加佐剂的情况下， 仅 用肽 A免疫的大鼠血清中的 Y -IFN水平甚至高于用相同剂量肽 A和佐剂免疫的。 实施例 6肽对卡介苗和脂多糖诱导的大鼠免疫性肝损伤的保护作用
6.1 实验动物
将 Wistar大鼠 （体重 180g〜220g， 雌雄各半， 购自上海斯莱克实验动物有限公司） 随 机分成以下 8组， 每组 10只： （1 )甘草酸二铵注射液组 （注射甘草酸二铵， 甘草酸二铵注 射液购自江苏正大天晴药业股份有限公司）； (2)干扰素组 （注射重组人干扰素 a 2a， 重组 人干扰素 a 2a购自沈阳三生制药股份有限公司）； (3) 高剂量组 （用肽 A免疫）； （4) 中剂 量组（用肽 A免疫）； (5)低剂量组（用肽 A免疫）； (6)低剂量佐剂组（用肽 A和佐剂免 疫）； (7) 空白对照组； （8)模型组。
6.2实验方法
低剂量佐剂组按如下方法免疫大鼠： 从第 0天开始， 首先进行初次免疫， 即将肽 A与 福氏完全佐剂混合 （用生理盐水溶解适量肽 A后与 50 μ 1福氏完全佐剂等体积混合） 而成 油包水的乳麋状液对大鼠进行皮下注射， 其中给药体积为 0.1ml/只大鼠， 而给药量以肽 A 计为 43.5 g/kg大鼠。 4天后， 进行第二次免疫， 剂量和免疫方式同第一次免疫， 但是其中 用福氏不完全佐剂代替福氏完全佐剂。 4天后， 进行第三次免疫， 剂量和免疫方式同第一次 免疫， 但是其中用福氏不完全佐剂代替福氏完全佐剂。 4天后， 进行第四次免疫， 即不加佐 剂， 用生理盐水溶解适量肽 A后腹腔注射大鼠， 其中给药体积为 0.1ml/只大鼠， 而给药量 以肽 A计为 43.5 w g/kg大鼠。
在低剂量佐剂组进行免疫注射的相同时间， 从第 0天幵始， 在高剂量组、 中剂量组和 低剂量组中， 分别对大鼠皮下注射用生理盐水溶解的肽 A (其中不加入佐剂）来免疫大鼠， 共进行四次免疫。 其中， 每次给药体积为 0.1ml/只大鼠， 而每次给药量以肽 A计， 高剂量 组、 中剂量组、 低剂量组分别为 174、 87、 43.5 g/kg大鼠。
在低剂量佐剂组进行免疫注射的相同时间， 从第 0天幵始， 模型组和空白对照组的大 鼠也进行注射， 共进行四次， 每次注射的是生理盐水， 每次给药体积为 0.1ml/只大鼠。
在对大鼠进行第四次免疫注射前的第 9天， 在除了空白对照组之外的其他 7组中， 给 大鼠尾静脉注射 0.2ml卡介菌多糖核酸注射液（由湖南九芝堂斯奇生物制药有限公司生产， 规格为每毫升含卡介菌多糖 0.35mg，注射前用生理盐水稀释，给药量以卡介菌多糖计为 126 u g/kg); 与此同时， 空白对照组注射等体积生理盐水。 在对大鼠进行第四次免疫注射后的 第 3天， 在除了空白对照组之外的其他 Ί组中， 给大鼠尾静脉注射 10μ g脂多糖（简称为 LPS, SIGMA公司产品）； 与此同时， 空白组注射等体积生理盐水。
另外， 在注射 LPS前的第 7天开始， 在甘草酸二铵注射液组中， 每天向大鼠腹腔注射 甘草酸二铵， 给药量以甘草酸二铵计为 13.5mg/kg大鼠， 共进行 7天； 在注射 LPS前的第 7 天开始， 在干扰素组中， 每天向大鼠腹腔注射干扰素， 给药量以干扰素计为 54万单位 / kg 大鼠， 共进行 7天。
大鼠注射脂多糖后 12小时， 对大鼠先称体重， 然后颈椎脱臼处死大鼠并采血。 分离血 清后， 按谷丙转氨酶检测试剂盒（购自南京建成生物工程研究所)、 谷草转氨酶检测试剂盒 (购自南京建成生物工程研究所）的说明书分别测定血清中谷丙转氨酶 (SGPT)活性、谷草 转氨酶 (SGOT)活性。 同时， 取大鼠取肝脏， 经 10%福尔马林溶液固定、 脱水、 石蜡包埋、 制片（4 μ ιη厚）、 HE染色后， 由病理专业人员在光学显微镜下检查有无下列病变并进行病 变评分： （1 )有无肝细胞变性（脂变、 水肿， 嗜酸变性等）； (2)有无肝细胞坏死（点状坏 死， 灶状坏死等)； (3)有无中央静脉及肝窦扩张、 淤血， 肝内静脉周围炎； （4)肝内或门 管区有无结缔组织增生及炎细胞浸润。 病变评分标准为： 根据每项病变由轻到重的程度分 别标记为 0分（正常）、 0.5分（极轻度）、 1分（轻度）、 2分（中度)、 3分（重度）、 4分 (极重度）， 出现灶状坏死加倍记分。 计算出每组动物病变评分的平均分， 分值越高表示病 变程度越严重。
6.3实验结果
丙转氨酶 (SGPT)活性和谷草转氨酶 (SGOT)活性的实验结果如图 3所示,病变评分的实 验结果如图 4所示。
在模型组中， 大鼠血清谷丙转氨酶、 谷草转氨酶显著升高， 表明卡介苗和脂多糖可造 成大鼠免疫性肝损伤。 以高、 中、 低剂量给药肽 A均能显著降低肝损伤大鼠的谷丙转氨酶 及谷草转氨酶水平,低剂量佐剂组中亦呈现显著的保护急性免疫性肝损伤的作用。 即， 对卡 介苗和脂多糖诱导的大鼠免疫性肝损伤模型来说， 肽 A可以显著降低免疫性肝损伤大鼠的 谷丙转氨酶及谷草转氨酶水平， 而甘草酸二铵注射液对大鼠肝损伤不具有显著的保护作用。
病理组织学研究表明， 本研究成功地复制了免疫性肝损伤模型。在模型组中， 肝组织病 变表现为肝细胞点状坏死， 少数为灶状坏死； 肝脏内中性粒细胞数量增多， 通常位于中央 静脉周围； 门管区有轻度炎细胞浸润。 肝组织病变程度评分结果表明， 相对于模型组， 在 髙剂量组、 中剂量组、 低剂量佐剂组和干扰素组中， 动物的病变程度显著性下降 (P<0.05)。 即， 以不加佐剂的高剂量和中剂量给药肽 A， 以及以低剂量加佐剂的方式给药肽 A，均能够 显著减轻肝脏的病变程度， 而甘草酸二铵注射液对大鼠肝损伤不具有显著的保护作用。 实施例 7肽对 D-氨基半乳糖胺诱导的大鼠急性肝损伤的保护作用 7.1 实验动物
将 Wistar大鼠 （体重 180g〜220g， 雌雄各半， 购自上海斯莱克实验动物有限公司） 随 机分成以下 6组， 每组 10只： （1 )甘草酸二铵注射液组 （注射甘草酸二铵）； （2) 高剂量 组（用肽 A和佐剂免疫）； (3) 中剂量组（用肽 A和佐剂免疫）； (4)低剂量组（用肽 A和 佐剂免疫）； (5) 空白对照组； （6)模型组。
7.2实验方法
高剂量组、 中剂量组和低剂量组按如下方法免疫大鼠： 从第 0天开始， 首先进行初次 免疫， 即将肽 A与福氏完全佐剂混合（用生理盐水溶解适量肽 A后与 ΙΟΟ μ 1福氏完全佐剂 等体积混合）而成油包水的乳麋状液对大鼠进行皮下注射， 其中给药体积为 0.2ml/只大鼠， 而给药量以肽 A计， 高剂量组、 中剂量组、 低剂量组分别为 174、 87、 43.5 u g/kg大鼠。 14 天后， 进行第二次免疫， 剂量和免疫方式同第一次免疫， 但是其中用福氏不完全佐剂代替 福氏完全佐剂。 14天后， 进行第三次免疫， 剂量和免疫方式同第一次免疫， 但是其中用福 氏不完全佐剂代替福氏完全佐剂。 14天后， 进行第四次免疫， 即不加佐剂， 用生理盐水溶 解适量肽 A后腹腔注射大鼠， 其中给药体积为 0.1ml/只大鼠， 而给药量以肽 A计， 高剂量 组、 中剂量组、 低剂量组分别为 174、 87、 43.5 g/kg大鼠。
在高剂量组、 中剂量组和低剂量进行免疫注射的相同时间， 从第 0天开始， 模型组和 空白对照组的大鼠也进行注射， 共进行四次， 每次注射的是相应体积的生理盐水。
在第四次免疫后 24小时， 在除了空白对照组之外的其他 5组中， 给大鼠腹腔注射 D- 氨基半乳糖胺（SIGMA公司产品） 600mg/kg大鼠， 每只大鼠注射 0.2ml; 与此同时， 空白 对照组注射等体积生理盐水。 在大鼠腹腔注射 D-氨基半乳糖胺后 48小时， 处死大鼠。
另外在甘草酸二铵注射液组中， 在大鼠被处死前的第 7天幵始， 每天向大鼠腹腔注射 甘草酸二铵 13.5mg/kg大鼠， 共进行 Ί天。
在大鼠腹腔注射 D-氨基半乳糖胺后 48小时， 颈椎脱臼处死大鼠并采血。 分离血清后， 按谷丙转氨酶检测试剂盒（购自南京建成生物工程研究所)、 谷草转氨酶检测试剂盒（购自 南京建成生物工程研究所）的说明书分别测定血清中谷丙转氨酶 (SGPT)活性、 谷草转氨酶 (SGOT)活性。
7.3实验结果
实验结果如图 5所示。 在 D-氨基半乳糖胺模型组中， 大鼠血清谷丙转氨酶、 谷草转氨 酶水平显著升高， 表明 D-氨基半乳糖胺可造成大鼠急性肝损伤。 肽 A以高、 中、 低剂量给 药均能显著性降低急性实验性肝损伤大鼠谷丙转氨酶及谷草转氨酶水平。 因此， 在 D-氨基 半乳糖胺诱导的大鼠急性肝损伤模型中， 肽 A可以显著降低急性实验性肝损伤大鼠谷丙转 氨酶及谷草转氨酶水平， 甘草酸二铵注射液对大鼠急性肝损伤亦具有显著的保护作用。 实施例 8肽对小鼠四氯化碳导致的肝损伤的保护作用
8.1 实验动物
将 BALB/c小鼠， （体重 18g〜22g， 购自上海斯莱克实验动物有限公司） 随机分成以下
8组， 每组 10只： （1 )甘草酸二铵注射液组（注射甘草酸二铵）； (2)千扰素组（注射重组 人干扰素 a 2a); (3 )高剂量组（用肽 A和佐剂免疫）； （4)中剂量组（用肽 A和佐剂免疫)； (5 )低剂量组（用肽 A和佐剂免疫)； (6)佐剂对照组（仅用和佐剂免疫〉； (7)空白对照 组； (8)模型组。
8.2实验方法
在初次免疫前一天， 向除了空白对照组之外的所有小鼠腹部皮下注射 0.05ml CC14 (由 上海凌峰化学试剂有限公司生产）； 空白对照组中注射等体积生理盐水。
髙剂量组、 中剂量组、 低剂量组按如下方法免疫大鼠： 首先进行初次免疫， 即将肽 A 与福氏完全佐剂混合（用生理盐水溶解适量肽 A后与 ΙΟΟ μ Ι福氏完全佐剂等体积混合）而 成油包水的乳麋状液对大鼠进行皮下注射，其中给药体积为 0.2ml/只小鼠，而给药量以肽 A 计， 高剂量组、 中剂量组、 低剂量组分别为 250、 125、 62.5 g/kg小鼠。 14天后， 进行第 二次免疫， 剂量和免疫方式同第一次免疫， 但是其中用福氏不完全佐剂代替福氏完全佐剂。 14天后， 进行第三次免疫， 剂量和免疫方式同第一次免疫， 但是其中用福氏不完全佐剂代 替福氏完全佐剂。 14天后， 进行第四次免疫， 即不加佐剂， 用生理盐水溶解适量肽 A后腹 腔注射大鼠， 其中给药体积为 0.1ml/只小鼠， 而给药量以肽 A计， 高剂量组、 中剂量组、 低剂量组分别为 250、 125、 62.5 g/kg小鼠。
在高剂量组、 中剂量组和低剂量进行免疫注射的相同时间， 模型组和空白对照组的小 鼠也进行注射， 共进行四次， 每次注射的是相应体积的生理盐水； 与此同时， 佐剂组中只 注射相应佐剂和生理盐水， 共进行四次。
在第四次免疫前一天， 再次向除了空白对照组之外的所有小鼠腹部皮下注射 0.05ml
CC14; 空白对照组中注射等体积生理盐水。 在第四次免疫后两天， 处死所有小鼠。
另外， 在处死前的第 7天， 在甘草酸二铵注射液组中， 每天向小鼠腹腔注射甘草酸二 铵， 给药量以甘草酸二铵计为 19.5mg /kg小鼠， 共进行 7天； 在处死前的第 7天， 在干扰 素组中， 每天向小鼠腹腔注射干扰素， 给药量以干扰素计为 75万单位 / kg小鼠， 共进行 7 天。
处死所有小鼠前， 对小鼠先称体重， 然后颈椎脱白处死小鼠并釆血。 分离血清后， 按 谷丙转氨酶检测试剂盒（购自南京建成生物工程研究所）、 谷草转氨酶检测试剂盒（购自南 京建成生物工程研究所） 的说明书分别测定谷丙转氨酶 (SGPT)活性、 谷草转氨酶 (SGOT) 活性。同时，取大鼠取肝脏，经 10%福尔马林溶液固定、脱水、石蜡包埋、制片（4μ ιη厚）、 HE染色后， 由病理专业人员在光学显微镜下检査有无病变并进行病变评分， 病变检査项目 和评分标准与实施例 6的相同。
8.3实验结果
丙转氨酶 (SGPT)活性和谷草转氨酶 (SGOT)活性的实验结果如图 6所示，病变评分的实 验结果如图 7所示。
在模型组中， 小鼠血清谷丙转氨酶、 谷草转氨酶水平显著升高， 表明四氯化碳可造成 小鼠急性肝损伤。 肽 A各剂量组都能显著性降低急性实验性肝损伤小鼠中谷丙转氨酶及谷 草转氨酶水平。 干扰素、 佐剂对转氨酶水平均无显著的降低作用。
病理组织学研究表明， 使用四氯化碳能成功地造成急性肝损伤。在模型组中， 肝组织病 变主要表现为肝细胞变性， 肝内出现点状坏死或灶状坏死， 出现肝内静脉炎， 肝内及门管 区可见少量炎细胞浸润及成纤维细胞增生。 肝组织病变程度评分结果表明高剂量组与模型 组相比， 显著减轻了肝脏的病变程度。
另外，在模型组中，相对于对照组的小鼠肝脏，小鼠肝脏显著肿大。当肽 A以 250 g/kg 小鼠的剂量给药时能显著减少该由四氯化碳引起的小鼠肝肿大。 而干扰素对四氯化碳引起 的小鼠肝肿大无显著减少作用。

Claims

权利要求书
1. 含式 I所示序列的肽或其药学上可接受的盐或酯在制备预防或治疗肝损伤的药物中的应 用，
Xaal-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I)
其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser,
Xaa3为 Tyr、 Phe或 Trp， 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu、 He或 Pro。
2. 含式 I所示序列的肽或其药学上可接受的盐或酯在预防或治疗肝损伤中的应用，
Xaal -Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I )
其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 Ile，
Xaa2为 Thr或 Ser，
Xaa3为 Tyr、 Phe或 Trp， 而且
Xaa4为缺失、 A〖a、 Gty、 Val、 Leu、 lie或 Pro。
3. 权利要求 1或 2的应用， 其中肝损伤为免疫性肝损伤或肝毒性化学物质引起的肝损伤。
4. 权利要求 1或 2的应用，其中式 I所示序列中的 Xaal是 Gly， Xaa2是 Thr， Xaa3是 Tyr， 而且 Xaa4为缺失、 Ala或 Gly。
5. 权利要求 4的应用， 其中 Xaa4为缺失。
6. 权利要求 1或 2的应用， 其中所述应用包括进一步用于治疗和 /或预防丙型肝炎。
7. 用于权利要求 1-6所述应用的药物组合物， 其包括含式 I所示序列的肽或其药学上可接 受的盐或酯以及药学上可接受的载体，
Xaal -Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I )
其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser，
Xaa3为 Tyr、 Phe或 Trp， 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu、 lie或 Pro。
8. 权利要求 8的药物组合物， 其不含佐剂。
9. 权利要求 7或 8的药物组合物，其中式 I所示序列中的 Xaal是 Gly， Xaa2是 Thr， Xaa3 是 Tyr， 而且 Xaa4为缺失、 Ala或 Gly。
10.权利要求 7或 8的药物组合物， 其中 Xaa4为缺失。
11.权利要求 7-10所述的药物组合物在制备预防或治疗肝损伤的药物中的应用。
12.权利要求 7-10所述的药物组合物在预防或治疗肝损伤中的应用。
13.权利要求 11或 12的应用， 其中所述应用包括进一步治疗和 /或预防丙型肝炎。
14.用于权利要求 1-6所述应用的肽或其药学上可接受的盐或酯， 其中所述肽含式 I所示序 列，
Xaal -Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (式 I )
其中，
Xaal为缺失、 Ala、 Gly、 Val、 Leu或 lie,
Xaa2为 Thr或 Ser，
Xaa3为 Tyr、 Phe或 Trp， 而且
Xaa4为缺失、 Ala、 Gly、 Val、 Leu, lie或 Pro。
15.权利要求 14的肽或其药学上可接受的盐或酯，其中式 I所示序列中的 Xaal是 Gly，Xaa2 是 Thr， Xaa3是 Tyr， 而且 Xaa4为缺失、 Ala或 Gly。
16.权利要求 15的肽或其药学上可接受的盐或酯， 其中 Xaa4为缺失。
17.权利要求 14-16所述的肽或其药学上可接受的盐或酯的制备方法，其包括化学合成所述 肽或者融合表达所述肽的步骤。
18. 编码权利要求 14-16所述的肽的多核苷酸。
PCT/CN2006/001176 2006-06-01 2006-06-01 Peptide pour la prévention ou le traitement d'atteinte hépatique et son dérivé ainsi que son utilisation WO2007137456A1 (fr)

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CN200680051751XA CN101336110B (zh) 2006-06-01 2006-06-01 预防或治疗肝损伤的肽及其衍生物及其应用
JP2009512388A JP5031827B2 (ja) 2006-06-01 2006-06-01 予防或いは肝損傷を治療するペプチッド
US12/066,636 US7985734B2 (en) 2006-06-01 2006-06-01 Peptides for preventing or treating liver damage
PCT/CN2006/001176 WO2007137456A1 (fr) 2006-06-01 2006-06-01 Peptide pour la prévention ou le traitement d'atteinte hépatique et son dérivé ainsi que son utilisation
EP06742063A EP2030628B1 (en) 2006-06-01 2006-06-01 A peptide for preventing or treating liver damage and its derivant and the use
US13/162,981 US8263562B2 (en) 2006-06-01 2011-06-17 Peptides for preventing or treating liver damage

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WO2013044499A1 (zh) * 2011-09-30 2013-04-04 Cheng Yun 丙型肝炎病毒免疫原性肽或其衍生物在制备预防或治疗结肠炎的药物中的应用
US20140235545A1 (en) * 2011-09-30 2014-08-21 Yun Cheng The use of HCV immunogenic peptide or a derivative thereof in the prevention or treatment of arthritis
CN104780930A (zh) * 2011-09-30 2015-07-15 程云 丙型肝炎病毒免疫原性肽或其衍生物在预防或治疗关节炎中的应用
US9605023B2 (en) 2012-12-18 2017-03-28 Yun Cheng Application of SP peptide or derivative thereof in preparing medicines for preventing or treating asthma
WO2021098854A1 (zh) * 2019-11-21 2021-05-27 上海医药工业研究院 一种治疗肺部疾病的生物肽及其应用
EP4063378A4 (en) * 2019-11-21 2024-06-19 Shanghai Institute of Pharmaceutical Industry Co., Ltd. BIOLOGICAL PEPTIDE FOR THE TREATMENT OF LUNG DISEASES AND ITS USE

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