WO2009127140A1 - 7p及其衍生肽和其应用 - Google Patents

7p及其衍生肽和其应用 Download PDF

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Publication number
WO2009127140A1
WO2009127140A1 PCT/CN2009/071204 CN2009071204W WO2009127140A1 WO 2009127140 A1 WO2009127140 A1 WO 2009127140A1 CN 2009071204 W CN2009071204 W CN 2009071204W WO 2009127140 A1 WO2009127140 A1 WO 2009127140A1
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WIPO (PCT)
Prior art keywords
oligopeptide
group
pharmaceutically acceptable
ester
acceptable salt
Prior art date
Application number
PCT/CN2009/071204
Other languages
English (en)
French (fr)
Inventor
程云
虞瑞鹤
赵万洲
赵军
郭仁锋
Original Assignee
Cheng Yun
Yu Ruihe
Zhao Wanzhou
Zhao Jun
Guo Renfeng
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from CNA2008101044251A external-priority patent/CN101559217A/zh
Priority claimed from CNA2008101046045A external-priority patent/CN101565452A/zh
Application filed by Cheng Yun, Yu Ruihe, Zhao Wanzhou, Zhao Jun, Guo Renfeng filed Critical Cheng Yun
Priority to CN200980101141XA priority Critical patent/CN101883781B/zh
Publication of WO2009127140A1 publication Critical patent/WO2009127140A1/zh

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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
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention is in the field of peptide pharmacology, and in particular, the present invention relates to 7P and its derived oligopeptide or a pharmaceutically acceptable salt or ester thereof, especially a novel 7P-derived oligopeptide. Further, the present invention relates to a pharmaceutical composition, a kit, a preparation method and a pharmaceutical application containing such an oligopeptide, particularly for the prevention and/or treatment of new uses such as nephritis.
  • the peptide having the sequence GQTYTSG (abbreviated as 7P herein) is an immunogenic peptide originally designed according to the hepatitis C virus, and the inventors disclosed the use as the type C in the Chinese specialties ljCN1194986C and CN1216075C, respectively. 7P of hepatitis virus immunogenic peptide and a partial derivative thereof. In addition, in PCT application WO20 07/137456A, the inventors have also discovered that 7P and its partial derivatives can also be used for the prevention and/or treatment of liver damage, in particular for preventing or treating immune liver damage and/or liver. Liver damage caused by toxic chemicals.
  • the routes of administration in the above examples of the examples are all injections, since peptide drugs are subject to low intestinal tract The absorption rate, the degradation caused by peptidase in the digestive system, and the short half-life after absorption in the living body, the development of oral peptide preparations is still in its infancy, no major progress, and currently developed and commercialized peptides Most of the drugs are in the form of injections, and it is inevitable that the patients will receive the pain and cumbersome procedures brought about by injection. Especially for preventive drugs, such an injection route will make it difficult for patients to adhere to prophylactic administration for a long time.
  • One of the objects of the present invention is to provide novel 7P-derived peptides, including 7P truncated peptides and 7P variant peptides, which are not GQTYTSG, GQTYTSGAs and GQTYTSGG. Further, the present invention also provides a pharmaceutical composition, a kit, and the like comprising the novel 7P-derived peptide, and a preparation method thereof, and a prophylactic and therapeutic application thereof for liver damage and nephritis, particularly for liver damage Prevention and treatment.
  • the second object of the present invention is to provide a method for preventing and treating new diseases such as chronic immune liver damage, liver-induced liver damage and nephritis by using 7P and 7P-derived peptides, in particular, 7P for preventing and treating nephritis.
  • the present invention also provides an application for preparing a corresponding drug, and a corresponding pharmaceutical composition, a kit, and the like.
  • the third object of the present invention is to provide a method for digestive administration of 7 ⁇ and 7 ⁇ derived peptides and corresponding preparations
  • the invention provides a 7 ⁇ and 7 ⁇ derived peptide or a pharmaceutically acceptable salt or ester thereof, i.e., the invention provides an oligopeptide of formula I or a pharmaceutically acceptable thereof Salt or ester,
  • Xaa3 is Thr or Ser
  • Xaa4 is Tyr, Phe, Trp or Val
  • Xaa5 is missing, Ser or Thr, [18] Xaa6 is missing, Gly or Ala, and
  • Xaa7 is a deletion, Ala or Gly.
  • the first aspect of the invention preferably provides novel 7P and 7P derived peptides or pharmaceutically acceptable salts or esters thereof, i.e., preferably provides an oligopeptide of formula I or a pharmaceutically acceptable salt or ester thereof, wherein the oligopeptide is not Gly-Gln-Thr-Tyr-Thr-Ser-Gly-Xaa7, wherein X aa 7 is a deletion, Ala or G ly (ie, the oligopeptide is not GQTYTSG, GQTYTSGA, and GQTYTSGG).
  • amino acid or amino acid residues can be defined in Table 1, and these abbreviations may refer to L-type amino acids, and may also refer to D-type amino acids.
  • amino acid or amino acid residue refers to an L-form amino acid or amino acid residue.
  • deletion as used herein means that the deleted amino acid residue is not present in the peptide sequence.
  • Xaa7 when Xaa7 is deleted, Xaa6 in the sequence of Formula I is the C-terminal amino acid of the sequence of Formula I; when Xaa2 is deleted, if Xaal and Xaa3 are present, IJXaal is directly linked to Xaa3.
  • Suitable modifications are prepared as multimers, towards terminal amino groups, carboxyl groups or
  • the side chain group is modified to form a pharmaceutically acceptable ester, a conjugate comprising a sequence of Formula I, a fusion protein comprising a sequence of Formula I, or a combination of such modifications, etc., which may also be encompassed by the present invention.
  • cyclization of a linear peptide 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.
  • ester refers to an ester which is suitable for contact with the tissues of a human or animal without excessive toxicity, irritation or allergies and the like. In general, 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 the amino acid side chain groups include, but are not limited to, the esterification of threonine, 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 fluorenyl, and N-acyl modification.
  • Modifications of the C-terminal carboxyl group include, but are not limited to, amides, lower mercaptoamides, dinonylamides, and lower mercaptoester 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
  • the amino group at the N-terminus of the polypeptide and the carboxyl group at the C-terminus and the amino acid side chain group are modified, that is, the chemical group at the N-terminus is still the a-amino group (-NH 2 ) 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 Polymers of monomethoxy-PEG, monomethoxy-PEG aldehyde, methoxy PEG-succinimidyl propionic acid, polyvinyl alcohol, dextran, cellulose or other saccharides .
  • 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,
  • 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)). E.g,
  • the PEGylation can be carried out by an acylation reaction or by a thiolation reaction using a reactive polyethylene glycol molecule.
  • the conjugate is formed by a condensation-activated PEG in which the hydroxyl or amino group at the end of the PEG is replaced by an activated linker molecule (see, for example, Karasiewicz et al.
  • 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, which comprises the sequence of the formula I.
  • the protein is a human albumin, bovine albumin or Fc of an 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.
  • therapeutic proteins that are genetically engineered to human albumin have a longer half-life.
  • the resulting fusion protein can increase the circulating half-life (see US 5,750,375, US 5,847,725, US Patent No. 6,291,646; Barouch et al, Journal of Immunology, 61: 1875-1882 (1998); Barouch et al, Proc. Natl. Acad. Sci. USA, 97(8): 4192-4197 (April 11, 2000); and Kim et al, Transplant Proc, 30 (8): 4031-4036 (1998))°
  • salt refers to a salt that is suitable for contact with the tissues of a human or animal without excessive toxicity, irritation or allergic reaction, 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, methanesulfonic acid Salt, nicotinate, 2-naphthalenesulfonate, oxalate, 3-phenylpropionate, propionate, succinate, tartrate, phosphate, glutamate, bicarbonate, p-toluene Sulfonate 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 phosphates, 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 oligopeptide has an action of preventing or treating liver damage or nephritis. This can be verified by an animal model of liver injury or nephritis provided in a specific embodiment of the invention.
  • prevention and “treatment” as used herein have the meaning as commonly understood by those skilled in the art that “prevention” refers to the administration of a pharmaceutical composition or active compound prior to onset or before the onset of symptoms to prevent, delay and / or to reduce the onset of symptoms or symptoms of the corresponding disease; “treatment” means, after or after the onset of the disease, or after or after the onset of symptoms, the administration of the pharmaceutical composition or active compound to eliminate the corresponding conditions, reduce the corresponding The severity of the symptoms, or delay the development of the corresponding condition.
  • the oligopeptide according to the first aspect of the present invention comprises a TYT sequence, that is, in Formula I, Xaa3 is Thr, and Xaa4 is Tyr, and Other variable amino acid residues in Formula I can be substituted or deleted.
  • the oligopeptide of the first aspect of the invention is truncated on the basis of a 7P peptide, such as an oligopeptide obtained by removing one or two amino acid residues at the C-terminus of the 7P peptide, and/or a 7P peptide.
  • the oligopeptide according to the first aspect of the present invention is an oligopeptide obtained by centripetal truncation of a 7P peptide, that is, an oligopeptide obtained by removing one or two amino acid residues at the C-terminus and the N-terminal end of the 7P peptide.
  • Peptide an oligopeptide obtained by removing one or two amino acid residues at the C-terminus and the N-terminal end of the 7P peptide.
  • the amino acid sequence ⁇ 1 J of the oligopeptide is selected from the group consisting of GQTYTS, QTYTSGs GQTYT, QTYTS, QTYT, TYTS, and TYT.
  • the oligopeptide of the formula I described in the first aspect of the invention may be an oligopeptide obtained by substituting or deleting one or two amino acid residues in the amino acid sequence of GQTYTSG, and the oligopeptide still has Prevent or treat liver damage or nephritis. More preferably, the oligopeptide is in the amino acid sequence of GQTYTSG An oligopeptide obtained by replacing one amino acid residue, according to the experimental results of the variant peptide of the embodiment of the present invention, such a variant peptide is still mostly capable of preventing or treating liver damage.
  • the oligopeptide of the formula I described in the first aspect of the invention may be an oligopeptide obtained by substituting or deleting one or two amino acid residues in the amino acid sequence of GQTYTSG, and the oligopeptide still has Prevent or treat liver damage or nephritis. More preferably, the oligopeptide is in the amino acid sequence of GQTYTSG An oligo
  • the oligopeptide is an oligopeptide selected from the group consisting of AQTYTSG, GNTYTSGs GQSYTSG, GQTYTTG, GQTYTS As GQTFTSGs GQTWTSGs and GQTVTSG.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the oligopeptide of the first aspect of the invention or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means a non-toxic solid, semi-solid or liquid filler, diluent, adjuvant, encapsulating material or other formulation excipient.
  • the carrier to be used may be adapted to the corresponding administration form, and may be formulated into an injection, (injectable) lyophilized powder, a spray, an oral solution, an oral suspension, a tablet, a capsule, using a carrier known to those skilled in the art.
  • Formulations such as enteric-coated tablets, pills, powders, granules, sustained release or delayed release.
  • the oligopeptide according to the first aspect of the present invention is administered by injection and by the digestive tract, and therefore, the pharmaceutical composition of the present invention is preferably an injection or a preparation for administration via the digestive tract, that is, Carriers suitable for administration by injection and administration by the digestive tract are particularly preferred.
  • digestive administration refers herein to a mode of administration of a pharmaceutical preparation through a patient's digestive tract, including oral, intragastric, and enema administration, preferably oral.
  • the carrier used may be adapted to the corresponding mode of administration, such as when administered by the digestive tract, and may be formulated into oral solutions, oral suspensions, tablets, capsules, using carriers known to those skilled in the art. Enteric-coated tablets, pills, powders, granules, sustained release or delayed release release.
  • liver injury refers to damage or lesions in liver tissue or cells. According to the cause of liver damage, liver damage can be divided into liver damage caused by viral liver damage, immune liver damage and hepatotoxic chemicals.
  • the pharmaceutical composition of the present invention is preferably used for the prevention and/or treatment of liver damage caused by immunological liver damage and/or hepatotoxic chemicals, particularly immune liver damage and alcohol-induced liver damage. According to the speed of liver injury, liver damage can be divided into acute liver injury and chronic liver injury.
  • Liver damage can be assessed by pathological phenomena (such as pathological scores of tissue samples from animal models), or by changes in serum levels of alanine aminotransferase or aspartate aminotransferase, or by malondialdehyde in liver tissue homogenate or Changes in levels such as glutathione are reflected.
  • the pharmaceutical composition of the present invention is useful for prevention and/or treatment of liver damage which is acute Immunological liver injury and chronic immune liver injury, such as D-galactosamine-induced acute liver injury, galactosamine and Freund's complete adjuvant-induced chronic immune liver injury and serum-induced chronic immune liver injury.
  • a pharmaceutical composition comprising a novel 7P-derived peptide has been shown to be useful for the prevention and/or treatment of acute immune liver injury.
  • a pharmaceutical composition comprising 7P is shown to be useful for the prevention and/or treatment of chronic immune liver damage and alcohol-induced liver damage, and the pharmaceutical composition may be administered via the digestive tract. The preparation greatly reduces the pain of the patient's administration.
  • the present invention provides the use of the oligopeptide of the first aspect of the present invention or a pharmaceutically acceptable salt or ester thereof for the preparation of a medicament for preventing and/or treating liver damage.
  • the oligopeptide is preferably a novel 7P-derived peptide, ie the oligopeptide is an oligopeptide of formula I, and is not Gly-Gln-Thr-Tyr-Thr-Ser-Gly-Xaa7, wherein X aa 7 is deleted , Ala or Gly.
  • the liver damage is an immune liver injury such as chronic immune liver damage and acute immune liver damage; it is also preferable that the liver damage is liver damage caused by hepatotoxic chemicals, particularly alcohol-induced liver damage.
  • the drug is a pharmaceutical preparation for injection or administration via the digestive tract. According to a specific embodiment of the present invention, it is also preferred that 7P or a pharmaceutically acceptable salt or ester thereof is used for the preparation of a medicament for preventing and/or treating chronic immunological liver injury or alcohol-induced liver damage, more preferably wherein The drug is a pharmaceutical preparation for administration via the digestive tract.
  • the process for preparing a medicament comprises the step of mixing the oligopeptide of the first aspect of the invention or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable carrier, for formulation into a liver for preventing and/or treating Injured pharmaceutical preparation.
  • the manner in which the drug is prepared can be prepared into a suitable formulation by a method familiar to those skilled in the art to enable administration of the corresponding formulation.
  • the present invention provides a method of preventing and/or treating liver damage comprising administering to a subject an effective amount of the oligopeptide of the first aspect of the invention or a pharmaceutically acceptable thereof Accepted salt or ester.
  • subject refers to a mammal, especially a human. According to the equivalent dose conversion relationship between experimental animals and humans known to those skilled in the art (see generally the guidance of FDA, SFDA and other drug regulatory agencies, see also "Huang Jihan et al. Pharmacological tests in animals and animals and Equivalent dose conversion between humans. Chinese Journal of Clinical Pharmacology and Therapeutics, 2004 Sep;9(9)
  • the effective dose of a human can be derived from the dose of the experimental animal.
  • the "effective amount” herein refers to an effective dose of a human, unless otherwise specified.
  • the effective amount of human is 0.1. ⁇ 100 ⁇ ⁇ / Kg body weight, preferably 0.5 to 5 ( ⁇ g/Kg body weight, more preferably 1 to 3 ( ⁇ g/Kg body weight, more preferably 1.5 to 25 g)
  • the effective amount of human is 1 to 100 ( ⁇ g/Kg body weight) Preferably, it is 10 to 300 ⁇ ⁇ / ⁇ g body weight, more preferably 30-12 ( ⁇ g/Kg body weight, most preferably an effective amount is converted according to a specific embodiment of the present invention.
  • the oligopeptide is New 7P-derived peptides; moreover, it is preferred that liver damage is immune liver damage, including chronic immune liver damage and acute immune liver damage; it is also preferred that liver damage is liver damage caused by hepatotoxic chemicals, especially alcohol-induced liver Further, it is preferred that the administration mode is injection or transgastric administration. According to a specific embodiment of the present invention, a method of preventing and/or treating chronic immunological liver injury, which comprises administering an effective amount to a subject, is also preferred.
  • the tester is administered an effective amount of 7P or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutically acceptable salt or ester thereof, more preferably wherein the mode of administration is by the digestive tract.
  • the mode of administration may be a single administration.
  • the drug may also be administered multiple times, such as administering an effective amount of the above oligopeptide multiple times at intervals of one week.
  • the pharmaceutical composition of the second aspect of the invention may also be used for the prevention and/or treatment of nephritis.
  • nephritis refers to inflammation of kidney tissue or cells.
  • nephritis is serum albumin-induced nephritis and active Heymann's nephritis.
  • Nephritis can be assessed by pathological phenomena (such as pathological scores of tissue samples from animal models) or by physiological and biochemical levels such as urea nitrogen levels, creatinine levels, and average nucleated cell counts in the glomeruli.
  • a pharmaceutical composition comprising 7P is shown to be useful for the prevention and/or treatment of nephritis.
  • the present invention provides the use of the oligopeptide of the first aspect of the present invention or a pharmaceutically acceptable salt or ester thereof for the preparation of a medicament for preventing and/or treating nephritis.
  • the oligopeptide is 7P; moreover, nephritis is serum albumin-induced nephritis and active Heymann's nephritis.
  • the drug is a pharmaceutical preparation for injection or administration via the digestive tract, and particularly preferably an injectable pharmaceutical preparation.
  • the process for preparing a medicament comprises the step of mixing the oligopeptide of the first aspect of the invention, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, for formulation and for use in the prevention and/or treatment of nephritis Pharmaceutical preparations.
  • the manner in which the drug is prepared can be prepared into a suitable formulation by a method familiar to those skilled in the art to enable administration of the corresponding formulation.
  • the present invention provides a method of preventing and/or treating nephritis comprising administering to a subject an effective amount of the oligopeptide of the first aspect of the invention or a pharmaceutically acceptable thereof Salt or ester.
  • the "subject” means a mammal, preferably a human; preferably, the administration is by injection or by the digestive tract, and particularly preferably by injection; in addition, the "effective amount” generally means an effective dose for human.
  • the effective amount of human is 0.1 to 10 ( ⁇ g/kg body weight, preferably 0.5 to 5 ( ⁇ g/kg body weight, more preferably 1 to 3 ( ⁇ g/kg body weight, more preferably 1.5 to 25 g/kg body weight, more preferably 10 to 20 ⁇ ⁇ /13 ⁇ 4 body weight, most preferably an effective amount is converted according to a specific embodiment of the present invention; for oral administration, the effective amount of human is 1 to 1000 ⁇ ⁇ /] 3 ⁇ 4
  • the body weight is preferably 10 to 30 ( ⁇ g/Kg body weight, more preferably 30-12 ( ⁇ g/Kg body weight).
  • the oligopeptide is preferably 7P; moreover, nephritis is preferably serum Albumin-induced nephritis and active Heymann's nephritis.
  • the administration may be a single administration or multiple administrations, such as administering an effective amount of the above oligopeptide multiple times at intervals of one week.
  • the present invention provides a kit comprising
  • a container comprising the oligopeptide of the first aspect of the invention or a pharmaceutically acceptable salt or ester thereof;
  • the kit is a packaged pharmaceutical form well known to the public, and includes a container containing a medicament, such as a bottle, a tube, etc., and includes instructions, which can be contained in a separate form in the kit, also It can be printed on the outer wall of the cartridge or container.
  • the specification provides the oligopeptide or the pharmaceutically acceptable salt or ester thereof according to the first aspect of the present invention, which is administered according to the fourth aspect or the sixth aspect of the present invention. And/or treatment of liver damage or nephritis.
  • the content may include one or more of indications, dosages, and modes of administration, for example, the content may be prevention or treatment of chronic immunological liver injury and/or prevention by administering an effective amount of 7P via the digestive tract. Or treat nephritis.
  • the present invention provides a method of synthesizing the oligopeptide of the first aspect of the invention.
  • the oligopeptide of the present invention can be synthesized by a chemical method such as a solid phase method or a liquid phase method which is commonly used in peptide synthesis, and can be efficiently synthesized by functional group protection and deprotection as needed.
  • a protecting group such as an amino group used in peptide synthesis, a condensing agent for a condensation reaction, and the like are available in the prior art, and are commercially available synthetic devices and reagents, such as various commercially available ones in the solid phase method.
  • Peptide synthesis device The synthesis of peptides of known structure by chemical methods will be apparent to those skilled in the art.
  • the polypeptide of the invention is preferably synthesized by a solid phase method.
  • the oligopeptide precursor of the first aspect of the present invention can also be produced by a biorecombination technique, and the oligopeptide according to the first aspect of the present invention can be chemically or enzymatically cleaved.
  • a DNA sequence encoding a peptide containing the oligopeptide is prepared, introduced into a recombinant vector, transformed into a host, and the desired oligopeptide precursor is isolated and purified by culturing the expression host, and then the oligopeptide is chemically or enzymatically cleaved.
  • FIG. 1 Effect of different 7P truncated peptides on serum alanine aminotransferase activity in rat galactosamine-induced liver injury, where ** indicates that the group shown can significantly reduce transaminase levels relative to the model group, * indicates the indicated group The level of transaminase can be significantly reduced relative to the model group.
  • Figure 2 Effect of different 7P truncated peptides on serum aspartate aminotransferase activity in rat galactosamine-induced liver injury, where ** indicates that the group shown can significantly reduce transaminase levels relative to the model group, * indicates that the indicated group is relatively The model group was able to significantly reduce transaminase levels.
  • FIG. 1 Effect of different 7P truncated peptides on the pathological score of galactosamine liver injury in rats, where ** indicates that the group shown can significantly reduce the pathology score relative to the model group, * indicates that the indicated group is relatively The model group was able to significantly reduce the pathology score.
  • FIG. Administration of 7P via digestive tract to BCG and lipopolysaccharide-induced acute immune liver injury in mice The effect of enzyme levels, where ** indicates that the group shown can significantly reduce transaminase levels relative to the model group.
  • * indicates that the indicated group can significantly reduce transaminase levels relative to the model group.
  • * indicates that the indicated group can significantly reduce transaminase levels relative to the model group.
  • FIG. Effect of 7P administered by the digestive tract on pathological scores of acute immune liver injury induced by BCG and lipopolysaccharide in mice, where ** indicates that the group shown can significantly reduce liver damage compared to the model group.
  • Figure 7 Effect of 7P administered by the digestive tract on the level of alanine aminotransferase in chronic immunological liver injury induced by galactosamine and Freund's complete adjuvant, where ** indicates that the group shown can be relative to the model group. The level of transaminase was significantly reduced, and * indicates that the indicated group was able to significantly reduce transaminase levels relative to the model group.
  • Figure 8 Effect of 7P administered by the digestive tract on the level of aspartate aminotransferase in chronic immunological liver injury induced by galactosamine and Freund's complete adjuvant, wherein ** indicates that the group shown can be very Significantly reduced transaminase levels, * indicates that the indicated groups were able to significantly reduce transaminase levels relative to the model group.
  • FIG. 9 Effect of 7P administered by the digestive tract on pathological scores of chronic immunological liver injury induced by galactosamine and Freund's complete adjuvant, wherein ** indicates that the group shown can be very Significantly reduce the pathological score of liver injury, * indicates that the indicated group can significantly reduce the pathological score of liver injury relative to the model group.
  • Figure 10 Effect of peptide 7P administered by the digestive tract on serum alanine aminotransferase levels in rats with hepatic injury induced by xenogeneic serum, wherein ** indicates that the group shown can significantly reduce transaminase levels relative to the model group, * indicates The indicated group was able to significantly reduce transaminase levels relative to the model group.
  • FIG. 11 Effect of peptide 7P administered by the digestive tract on serum aspartate aminotransferase levels in rats with hepatic injury induced by xenogeneic serum, where ** indicates that the group shown can significantly reduce transaminase levels relative to the model group, * indicates The group was able to significantly reduce transaminase levels relative to the model group.
  • FIG. 12 Effect of peptide 7P administered by the digestive tract on hepatic pathology scores in rats with hepatic injury induced by xenogeneic serum, wherein ** indicates that the group shown can significantly reduce the pathological score of liver injury relative to the model group, * It is indicated that the indicated group can significantly reduce the pathological score of liver injury relative to the model group.
  • Figure 13.7P Effect on bovine serum albumin-induced urea nitrogen levels in rat nephritis, where ** indicates that the group shown is significantly different from the model group, and * indicates that the group shown is significantly different from the model group.
  • Figure 14.7P Effect on bovine serum albumin-induced creatinine levels in rat nephritis, where ** indicates that the group shown is significantly different from the model group, and * indicates that the group shown is significantly different from the model group.
  • Figure 16.7 shows the effect of bovine serum albumin on the average number of nucleated cells in the glomeruli of rat nephritis, where ** indicates that the group shown is significantly different from the model group, and * indicates that the group shown is relative to The model group is significantly different.
  • Figure 17.7 shows the effect of the same rat kidney cortex homogenate plus Freund's complete adjuvant on the urea nitrogen level in rats with active Heymann's nephritis AHN), where ** indicates that the group shown is very different from the model group, * Indicates that the group shown is significantly different from the model group.
  • Figure 18.7 shows the effect of the same rat kidney cortex homogenate plus Freund's complete adjuvant on the creatinine level of active Heymann's nephritis (AHN) rats, where ** indicates that the group shown is very different from the model group, * Indicates that the group shown is significantly different from the model group.
  • Figure 19.7P Effect on pathological scores of rats with active Heymann's nephritis AHN) established by homologous rat renal cortex homogenate plus Freund's complete adjuvant, where ** indicates that the indicated group is very significant relative to the model group, * Indicates that the group shown is significantly different from the model group.
  • Figure 21.7 Effect of P variant peptide on D-galactosamine-induced serum biochemical ALT in rats with acute liver injury. Where ** indicates that the group shown is very different from the model group, and * indicates that the group shown is significantly different from the model group.
  • Figure 23.7 Effect of P variant peptide on D-galactosamine-induced pathological scoring of acute liver injury in rats, where ** indicates that the group shown is significantly different from the model group, * indicates that the group shown is relative to The model group is significantly different.
  • Figure 24. Effect of peptide 7P administered by the digestive tract on MDA levels in rats with alcohol-induced liver injury, where ** indicates that the group shown can significantly reduce MDA levels relative to the model group, * indicates that the group shown is relative The model group can significantly reduce MDA levels.
  • FIG. 25 Effect of peptide 7P administered by the digestive tract on GSH levels in rats with alcohol-induced liver injury, where ** indicates that the group shown can significantly increase GSH levels relative to the model group, * indicates that the group shown is relatively The model group can significantly increase the GSH level.
  • FIG. 26 Effect of peptide 7P administered by the digestive tract on the pathological score of rats with alcohol-induced liver injury, where ** indicates that the group shown is significantly different from the model group, * indicates that the group shown is relative to the model Group differences are significant.
  • a peptide as shown in the following sequence was synthesized by a solid phase peptide synthesis method using an automatic peptide synthesizer Model 413A (available from PerkinElmer Co., Ltd.): GQTYTSG (abbreviated as 7P); 7P truncated peptide:
  • GQTYTS (referred to as PI), QTYTSG (referred to as P2), GQTYT (referred to as P3), QTY TS (referred to as P4), QTYT (referred to as P5), TYTS (referred to as P6), TYT (referred to as P7)
  • the 7P synthesized in Example 1 was crosslinked with bovine serum albumin (BSA) by a glutaraldehyde method to form a conjugate.
  • BSA bovine serum albumin
  • the specific conjugation process is as follows: 1 mg of the 7P synthesized in Example 1 is dissolved in 0.5 ml of PBS (pH 7.4, 0.02 mol/L); 4.5 mg is taken.
  • Drug dosage and grouping Experimental animals were randomly divided into 11 groups, namely, blank control group, model group, and positive drug (Ganlixin injection) group (the dose was 13.5 mg/kg. day in terms of diammonium glycyrrhizinate). ), 7P group (87 g/kg « day), 7 truncated peptide groups (ie, Pl, P2, P3, P4, P5, P6, P7 groups, respectively, doses of 87 g / k days), each group 10 SPF grade SD rats (body weight 180g ⁇ 220g, male and female).
  • the model group, the positive drug group, the 7P group and the P1 ⁇ P7 group were intraperitoneally injected with D-aminogalactosamine 600 mg/kg rat body weight, and the injection volume was 1 ml/100 g rat.
  • the blank group was injected with the same amount of normal saline.
  • the rats were weighed first, and the blood was separated. The serum was separated and the alanine aminotransferase and aspartate aminotransferase activities were determined using alanine aminotransferase and aspartate aminotransferase assay kits.
  • the rats were sacrificed by cervical dislocation and histological examination of liver tissue was performed. According to the degree of light to heavy lesions marked as 0, 0.5,
  • the liver injury model replicated in this experiment is mainly characterized by punctate or small focal necrosis of multifocal hepatocytes and inflammatory cell infiltration at the necrosis. Eosinophils appeared in the liver cells, and a small amount of inflammatory cells infiltrated around the central vein and in the portal area. Fibroplasia was not observed in the portal area. Mild lipid changes are seen in most rat liver cells. It is distributed in a small stove. For D-galactosamine-induced acute liver injury in rats, different peptide sequences can reduce the degree of liver damage after application. The results are in order of high to low: 7P group, Pl, P2, P3,
  • the dose was 175 g/kg based on BCG polysaccharide
  • the blank group was injected with the same amount of normal saline.
  • the model group, the positive drug group and the 7P group were injected with 10 ⁇ - lipopolysaccharide (LPS) in the tail vein, and the blank group was injected with the same amount of normal saline.
  • LPS ⁇ - lipopolysaccharide
  • alanine aminotransferase and aspartate aminotransferase activities were determined by alanine aminotransferase and aspartate aminotransferase assay kits, respectively. After the mice were sacrificed by cervical dislocation, an autopsy was performed to perform histopathological examination of the liver tissue.
  • the histopathological study of 7P on BCG and lipopolysaccharide-induced immunological liver injury in mice showed that the mouse model of acute immune liver injury replicated in this study was mainly characterized by subcapsular hepatocyte degeneration and hepatocyte pyknosis. .
  • a small number of hepatocytes have mild necrosis, and a small amount of inflammatory cells infiltrate around the central vein. No fibrous tissue hyperplasia was seen in the portal area.
  • the 7P high- and medium-dose groups could significantly reduce the degree of hepatocyte injury after intragastric administration (P ⁇ 0.01).
  • the low-dose 7P group also significantly reduced the degree of hepatocyte injury after intragastric administration ( ⁇ 0 ⁇ 05) (See Figure 6).
  • Model preparation BALB/, rats were injected intraperitoneally with galactosamine 300mg/kg, and the same abdominal cavity was injected subcutaneously with Freund's complete adjuvant 0.05ml/20g once a week for four weeks; then, abdominal cavity Injection of galactosamine 400mg/kg, subcutaneous injection of Freund's complete adjuvant 0.05ml/20g, once a week, Eight weeks in a row; then, galactosamine 500 mg/kg was intraperitoneally injected into the abdomen with a subcutaneous injection of Freund's complete adjuvant 0.1 ml/20 g of mouse body weight once a week for six weeks.
  • mice 100 samples of chronic immunological liver injury in mice were successfully prepared by serum samples, and randomly divided into 5 groups, P model group and positive drug (Ganlixin injection) group (the dose was measured by diammonium glycyrrhizinate). 19.5mg/kg.day), 7P high dose group (1250 ⁇ ⁇ / kg. day), 7P medium dose group (625 ⁇ ⁇ /
  • mice Kg. days
  • 7P low-dose group 312.5 g/kg. day
  • the same batch of healthy mice into a blank control group, a total of 6 groups, 20 in each group.
  • mice were administered once a day, and administered continuously for 1 month (15 times in total).
  • the drugs were all prepared to the required concentration with physiological saline, and the 7P group (administered by gavage) was administered.
  • 0.2 ml/20 g, positive drug (Ganlixin injection) group was intravenously injected with glycyrrhizin, and the blank control group and model group were given an equal volume of normal saline.
  • the mice were sacrificed, weighed, and the blood was separated. Serum was separated using OLYMPUS.
  • Model preparation Wistar rats were intraperitoneally injected with 0.3ml/200g of pig serum (containing 27mg protein), one to two times a week, for three and a half months (20 times in total), and the liver function was detected by serum biochemistry. Sixty rats with chronic immunological liver injury model were prepared. The experimental animals were randomly divided into 5 groups, 12 rats in each group, namely the model group and the Ganlixin injection group (the dosage was 13.5 mg/kg.day for diammonium glycyrrhizinate), and the 7P high dose group (870 ⁇ ). ⁇ / 13 ⁇ 4 ⁇ day), 7 ⁇ medium dose group (435 g/kg. day), 7P low dose group (217.5 ⁇ ⁇ / kg. day); another 12 healthy rats were used as blank control group.
  • Group 7P was prepared with physiological saline to the required concentration, the dosage volume was 0.2 ml/200 g, administered by intragastric administration; the Ganlixin injection group was administered by tail vein injection; the blank control group and the model group were given. Equal volume of saline. Rats were administered once a day for 1 month (15 times in total), during which rats were intraperitoneally injected with porcine serum once a week (0.3 ml/200 g). After the end of the administration, the body weight was weighed, the blood was separated, and the serum was separated. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were measured by an automatic biochemical analyzer.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • Alkaline phosphatase (AKP), total protein (TP), albumin (ALB), globulin (G), white ball ratio (A/G), and blood glucose (GLU).
  • TP total protein
  • ALB albumin
  • G globulin
  • GLU white ball ratio
  • X average score
  • liver injury model replicated in this experiment mainly showed fibrotic tissue proliferation in the hepatic lobules and in the portal area, and the fibrous tissues of the hyperplasia were connected to each other. However, it has not been wrapped, and the hepatic lobules are separated to form false leaflets. Hepatocyte degeneration is not obvious, and most rats only see a small amount of lipid droplet vacuoles in individual liver cells.
  • the high-dose group of 7P gavage administration reduced the degree of liver fibrosis, and there was a significant difference compared with the model group (P ⁇ 0.05) (see Figure 12).
  • Example 8 Protective effect of 7P on bovine serum albumin (BSA)-induced rat nephritis
  • SD rats were randomly divided into 6 groups, namely, the blank control group, the model group, and the positive drug (dexamethasone injection) group (0.9 mg/kg.day as dexamethasone).
  • the 7P high dose group (174 ⁇ /13 ⁇ 4* days), the 7P medium dose group (87 ⁇ /13 ⁇ 4* days), and the 7P low dose group (43.5 ⁇ /13 ⁇ 4* days), 10 rats in each group.
  • Each of the 7P dose groups was administered subcutaneously in a dose of 0.1 ml/100 g body weight; the dexamethasone injection group was intraperitoneally administered at a dose of 0.1 ml/100 g body weight.
  • Bovine serum albumin (BSA) was administered to the model group, the positive drug (dexamethasone injection) group, the 7P high dose group, the 7P medium dose group, and the 7P group at a dose of 150 mg Zkg body weight 12 days before the experiment.
  • Rats in the low-dose group were pre-immunized by intraperitoneal injection once, and were intraperitoneally injected once every 300 mg Zkg body weight on the first day and the 12th day of the experiment.
  • the blank control rats were replaced with an equal volume of normal saline.
  • the positive drug (dexamethasone injection) group, the 7P high-dose group, the 7P medium-dose group, and the 7P low-dose group were administered subcutaneously for dexamethasone or 7P-subjects every other day.
  • the blank control group and the model group were subcutaneously injected with an equal volume of normal saline one day at a time, and a total of 15 times were administered.
  • the rats were weighed first, the blood was separated, the serum was separated, and the creatinine (Cre-S) sarcosine oxidase assay kit and the urea nitrogen (BUN) UV-GLDH assay kit were isolated. The instructions determine creatinine and urea nitrogen.
  • an autopsy was performed. The kidney tissues were fixed with 10% formaldehyde, embedded in paraffin, sectioned, and stained with HE for histopathological examination.
  • the 7P high- and medium-dose group significantly reduced urea nitrogen (BUN) and creatinine (Cre-S) ⁇ ( ⁇ ( ⁇ or ! ⁇ ..) in experimental nephritis rats (see Figures 13, 14).
  • BUN urea nitrogen
  • Cre-S creatinine
  • the liver injury model replicated in this experiment is mainly characterized by an increase in glomerular volume, an increase in the number of nucleated cells, a lobulated glomerulus, a widening of the mesangial area, and an increase in the matrix.
  • Model animals were randomly divided into 5 groups, namely the model group and the positive drug (dexamethasone injection) group (0.9 mg/kg.day as dexamethasone).
  • 7P high dose group (174 ⁇ /13 ⁇ 4* days), 7P medium dose group (87 ⁇ /13 ⁇ 4* days), 7P low dose group (43 ⁇ 5 ⁇ ⁇
  • the 7P high-dose group, the 7P medium-dose group, and the 7P low-dose group were administered subcutaneously every other day for 15 times.
  • the blank group and the model group were subcutaneously injected with an equal volume of physiological saline one day at intervals of 15 times.
  • the rats were first weighed, the blood was separated, and the serum was separated.
  • Cre-S creatinine oxidase assay kit and urea nitrogen (BUN) UV-GLDH assay reagent The cartridge instructions determine creatinine and urea nitrogen.
  • an autopsy was performed.
  • the kidney tissues were fixed with 10% formaldehyde, embedded in paraffin, sectioned, and stained with HE for histopathological examination.
  • BUN urea nitrogen
  • Cre-S creatinine
  • renal tubular epithelial cells are severely turbid and swollen, and some renal tubular epithelium is almost eosinophilic necrosis, the lumen is obviously narrow or even occluded, and different types of tubular types can be seen in the renal tubule lumen.
  • the high-dose group of the drug showed a significant improvement in the lesion glomerulus, the glomerular volume was only slightly enlarged, the endothelial cell proliferation was not obvious, and the renal tubular epithelial cells were not.
  • Example 10 Administration of 7P in the digestive tract protects against alcoholic liver injury in mice
  • SPF-class ICR mice (body weight 18-22 g, male and female, purchased from Nantong University Experimental Animal Center) were randomly divided into nine groups of 10 animals each, including: blank control group (distilled water) Acute alcoholic liver injury model group (administered 50% ethanol solution), Tianqing Ganping positive drug group (administered diammonium glycyrrhizinate enteric-coated capsule, which was purchased from Jiangsu Zhengda Tianqing Pharmaceutical Co., Ltd., administered The dosage is 58.5 mg/kg ⁇ +50% ethanol solution with diammonium glycyrrhizinate enteric solution), and the high dose group of SP drug (administer 7P)
  • mice were administered by intragastric administration once a day for 30 consecutive days. On the 30th day, the mice were intragastrically administered with 50% absolute ethanol solution, and the mice were sacrificed after 16 hours. After weighing the mice in each group, they were sacrificed. About 0.19 ⁇ 0.25 g of fresh liver was added, and 2 ml of 0.9% normal saline was added to prepare 10% liver tissue homogenate.
  • MDA malondialdehyde
  • GSH reduced glutathione

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Description

7P及其衍生肽和其应用
Figure imgf000003_0001
[1] 本发明属于肽药物学领域， 具体而言， 本发明涉及 7P及其衍生寡肽或其药学上 可接受的盐或酯， 尤其是新的 7P衍生寡肽。 另外， 本发明还涉及含有这类寡肽 的药物组合物、 药剂盒、 制备方法以及药物应用， 尤其是用于预防和 /或治疗肾 炎等新的用途。
[2] 技术背景
[3] 序列为 GQTYTSG的肽 （本文中简称为 7P) 是一种最初根据丙型肝炎病毒而设 计的免疫原性肽， 本发明人在中国专禾 ljCN1194986C和 CN1216075C中分别披露 了用作丙型肝炎病毒免疫原性肽的 7P及其部分衍生物。 另外， 在 PCT申请 WO20 07/137456A中， 本发明人也发现了 7P及其部分衍生物还能用于预防和 /或治疗肝 损伤的应用， 尤其能预防或治疗免疫性肝损伤和 /或肝毒性化学物质引起的肝损 伤。 这些文献以切实的证据公开了序列为 GQTYTSG的 7P肽及其两个衍生肽 GQT YTSGA和 GQTYTSGG具备针对丙型肝炎病毒的免疫原性肽， 能作为丙型肝炎疫 苗， 而且这些肽能用于预防和 /或治疗肝损伤， 尤其是某些非丙型肝炎病毒引起 的肝损伤。
[4] 然而， 这些文献没有以非常切实的证据公开 7P肽中对肝损伤起预防和治疗作用 的关键结构部分， 另外也没有揭示 7P对除丙型肝炎和肝损伤之外的疾病有治疗 或预防效果。 本发明人对 7P肽结构上的氨基酸残基进行了大量缺失和 /或取代的 筛选工作， 发现了对保持用于预防和 /或治疗肝损伤的能力起比较关键作用的部 分， 由此开发出了一系列 7P衍生肽， 从而提供了更多的药物选择， 尤其是开发 出的 7P截短肽， 不但具有预防和治疗肝损伤的作用， 而且由于多肽分子变小， 降低了生产成本。 尤其是， 本发明的肽除了对于上述文献没有明确提及的慢性 免疫性肝损伤和酒精引起的肝损伤有疗效之外， 更令人惊讶的是， 还对肾炎有 疗效。
[5] 另外， 上述文献实施例中的给药途径都为注射， 由于肽类药物受制于肠道的低 吸收率、 消化系统中肽酶导致的降解、 以及生物体内吸收后半衰期短等影响， 口服的肽类药物制剂的开发还处于初级阶段， 没有重大的进展， 而目前开发出 并商业化的肽类药物大都为注射剂的形式， 不可避免地要使患者接受注射带来 的痛苦和繁琐流程， 尤其对于预防性药物来说， 如此的注射给药途径将使患者 难以长期坚持接受预防性给药。 然而， 令人惊讶的是， 本发明人发现， 无需复 杂的口服制剂配方， 一定量的本发明的肽即使仅溶于生理盐水中， 也能够进行 有效的预防和治疗， 因此可以提供一种简便、 有效的经消化道给药 （如， 口服 给药） 方式。
[6] 发明内容
[7] 本发明的目的之一在于提供新的 7P衍生肽， 包括 7P截短肽和 7P变异肽， 所述新 的 7P衍生肽不是 GQTYTSG、 GQTYTSGAs 和 GQTYTSGG。 另夕卜' 本发明还提 供了包含所述新的 7P衍生肽的药物组合物、 药剂盒等产品， 和其制备方法、 以 及其对肝损伤和肾炎的预防和治疗应用， 尤其是对于肝损伤的预防和治疗。
[8] 本发明的目的之二在于提供 7P及 7P衍生肽对于慢性免疫性肝损伤、 酒精引起的 肝损伤和肾炎等新疾病的预防和治疗方法， 尤其是 7P对肾炎的预防和治疗方法 。 另外， 本发明还提供了制备相应药物的应用以及相应药物组合物、 药剂盒等
^口
厂 ππ。
[9] 本发明的目的之三在于提供 7Ρ及 7Ρ衍生肽的经消化道给药的方式以及相应制剂
[10] 根据以上发明目的， 在本发明的第一方面， 本发明提供 7Ρ及 7Ρ衍生肽或其药学 上可接受的盐或酯， 即本发明提供式 I的寡肽或其药学上可接受的盐或酯，
[11] Xaal-Xaa2-Xaa3-Xaa4-Thr-Xaa5-Xaa6-Xaa7 (式 I)
[12] 其中，
[13] Xaal为缺失、 Gly或 Ala,
[14] Xaa2为缺失、 Gin或 Asn，
[15] Xaa3为 Thr或 Ser，
[16] Xaa4为 Tyr、 Phe、 Trp或 Val，
[17] Xaa5为缺失、 Ser或 Thr, [18] Xaa6为缺失、 Gly或 Ala, 而且
[19] Xaa7为缺失、 Ala或 Gly。
[20] 除了现有的 7P等肽之外， 本发明的第一方面优选提供新的 7P及 7P衍生肽或其药 学上可接受的盐或酯， 即优选提供的是式 I的寡肽或其药学上可接受的盐或酯， 其中所述寡肽不是 Gly-Gln-Thr-Tyr-Thr-Ser-Gly-Xaa7， 其中 Xaa7为缺失、 Ala或 G ly (即所述寡肽不是 GQTYTSG、 GQTYTSGA、 和 GQTYTSGG) 。
[21] 本文中所使用的肽及氨基酸、 氨基酸残基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缩写可参照表 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。 在本发明的具体实施方式中， 氨基酸或 氨基酸残基均指 L-型的氨基酸或氨基酸残基。 另外， 本文所使用的"缺失"指的是 缺失的氨基酸残基不存在于肽序列中。 例如， 当 Xaa7缺失吋， 式 I所示序列中的 Xaa6就是式 I所示序列的 C末端的氨基酸； 当 Xaa2缺失吋， 如果存在 Xaal和 Xaa3 ， 贝 IJXaal直接与 Xaa3通过相连。
[22] 表 1氨基酸缩写表
Figure imgf000005_0001
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， 这些也可以涵盖在本发明的范围内。 例 如， 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。
[23] 本文所使用的 "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、 刺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。
[24] 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 -低级垸基和 N -酰基修饰。 C末端羧基基团的修饰包括但不限于酰胺、 低级垸基酰胺、 二垸基 酰胺和低级垸基酯修饰。 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） 、 d— 6烧基、 C 2 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 a-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、 单甲氧基 -PEG醛基、 甲氧基 PEG-琥珀酰亚胺丙酸、 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 6 0,000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垸基化反应来进行。 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)o 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。
[26] 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 6291646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))°
[27] 本文所使用的 "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。
[28] 在本发明的第一方面， 所述寡肽具有预防或治疗肝损伤或肾炎的作用。 这可以 通过本发明具体实施方式中提供的肝损伤或肾炎动物模型来验证。 其中， 本文 所用的 "预防 "和"治疗"具有本领域技术人员所常规理解的含义， 即"预防"是指在 发病前或症状出现前给药药物组合物或活性化合物， 以防止、 延缓和 /或减轻相 应病症的发病或症状的出现； "治疗"是指， 在发病之吋或之后， 或在症状出现之 吋或之后， 给药药物组合物或活性化合物， 以消除相应病况、 减轻相应症状的 严重程度、 或延缓相应病症的发展。
[29] 进一步地， 根据本发明实施例的截短肽的实验结果， 优选本发明第一方面所述 的寡肽包括 TYT序列， 即在式 I中， Xaa3为 Thr， 而且 Xaa4为 Tyr， 而式 I中其他可 变的氨基酸残基可以被替换或缺失。 更优选本发明第一方面所述的寡肽在 7P肽 的基础上截短， 如在 7P肽的 C端去掉 1个或 2个氨基酸残基后得到的寡肽， 和 /或 在 7P肽的 N端去掉 1个或 2个氨基酸残基后得到的寡肽。 更加优选本发明第一方面 所述的寡肽是向心截短 7P肽后得到的寡肽， 即在 7P肽的 C端和 N端同吋去掉 1个 或 2个氨基酸残基后得到的寡肽。 在本发明的具体实施方式中， 所述寡肽的氨基 酸序歹1 J选自 GQTYTS、 QTYTSGs GQTYT、 QTYTS、 QTYT、 TYTS、 和 TYT。
[30] 另外进一步地， 优选本发明第一方面所述的式 I的寡肽可以是 GQTYTSG的氨基 酸序列中替换或缺失一个或两个氨基酸残基而得的寡肽， 而且该寡肽仍旧具有 预防或治疗肝损伤或肾炎的作用。 更优选所述寡肽是 GQTYTSG的氨基酸序列中 替换一个氨基酸残基而得的寡肽， 根据本发明实施例的变异肽的实验结果， 这 样的变异肽大多仍具备预防或治疗肝损伤的作用。 在本发明的具体实施方式中
， 所述寡肽是选自 AQTYTSG、 GNTYTSGs GQSYTSG、 GQTYTTG、 GQTYTS As GQTFTSGs GQTWTSGs 和 GQTVTSG的寡肽。
[31] 在第二方面， 本发明提供药物组合物， 其包括本发明第一方面所述的寡肽或其 药学上可接受的盐或酯， 以及药学上可接受的载体。 其中， 本文所称的"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。 所用载体可与相应的给药形式相适应， 可使用本领域技术人员 所知晓的载体配成注射剂、 （注射用） 冻干粉、 喷雾剂、 口服溶液、 口服混悬 液、 片剂、 胶囊、 肠溶片、 丸剂、 粉剂、 颗粒剂、 持续释放或延迟释释放等制 剂。 在本发明的具体实施方式中， 本发明第一方面所述的寡肽通过注射和经消 化道方式给药， 因此， 本发明的药物组合物优选为注射剂或经消化道给药的制 剂， 即适于配制成注射和经消化道方式给药的载体特别优选的。 其中， "经消化 道给药 "在本文中指药物制剂通过患者消化道的给药方式， 包括口服、 灌胃给药 和灌肠给药等， 优选是口服。 所用载体可与相应的给药形式相适应， 如当釆用 经消化道给药的方式吋， 可使用本领域技术人员所知晓的载体配成口服溶液、 口服混悬液、 片剂、 胶囊、 肠溶片、 丸剂、 粉剂、 颗粒剂、 持续释放或延迟释 释放等剂型。
[32] 本发明第二方面的药物组合物可用于预防和 /或治疗肝损伤， 尤其是急性免疫 性肝损伤和慢性免疫性肝损伤。 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。 根据导致肝损伤的诱因， 肝损伤可以被分成病毒性肝损伤、 免疫性肝损伤和肝毒性化学物质引起的肝损伤。 本发明的药物组合物优选用于 预防和 /或治疗免疫性肝损伤和 /或肝毒性化学物质弓 I起的肝损伤， 尤其是免疫性 肝损伤和酒精引起的肝损伤。 根据肝损伤发病的速度， 肝损伤可以被分成急性 肝损伤和慢性肝损伤。 肝损伤可通过病理现象 （如动物模型的组织样本的病理 评分） 来评估， 也可通过血清中谷丙转氨酶或谷草转氨酶等水平的变化来反映 ， 或也可通过肝组织均浆中丙二醛或谷胱甘肽等水平的变化来反映。 在本发明 的具体实施方式中， 本发明的药物组合物可用于预防和 /或治疗的肝损伤是急性 免疫性肝损伤和慢性免疫性肝损伤， 如 D-氨基半乳糖胺诱导的急性肝损伤、 半 乳糖胺和福氏完全佐剂诱导的慢性免疫性肝损伤和血清诱导的慢性免疫性肝损 伤。 在本发明的一些具体实施方式中， 包含新的 7P衍生肽的药物组合物被证实 可用于预防和 /或治疗急性免疫性肝损伤。 在本发明的另一些具体实施方式中， 包含 7P的药物组合物被证实可用于预防和 /或治疗慢性免疫性肝损伤和酒精引起 的肝损伤， 而且该药物组合物可以是经消化道给药的制剂， 大大减轻了患者给 药吋的痛苦。
[33] 因此， 在第三方面， 本发明提供本发明第一方面所述的寡肽或其药学上可接受 的盐或酯在制备用于预防和 /或治疗肝损伤的药物中的应用。 其中， 优选所述寡 肽是新的 7P衍生肽， 即所述寡肽是式 I的寡肽， 而且不是 Gly-Gln-Thr-Tyr-Thr-Ser -Gly-Xaa7 , 其中 Xaa7为缺失、 Ala或 Gly。 另外， 优选肝损伤是免疫性肝损伤， 如慢性免疫性肝损伤和急性免疫性肝损伤； 也优选肝损伤是肝毒性化学物质弓 I 起的肝损伤， 尤其是酒精引起的肝损伤。 另外， 优选药物是注射或经消化道给 药的药物制剂。 根据本发明的具体实施方式， 也优选 7P或其药学上可接受的盐 或酯在制备用于预防和 /或治疗慢性免疫性肝损伤或酒精引起的肝损伤的药物中 的应用， 更优选其中药物是经消化道给药的药物制剂。 制备药物的过程包括将 本发明第一方面所述的寡肽或其药学上可接受的盐或酯和药学上可接受的载体 混合的步骤， 用以配制成能用于预防和 /或治疗肝损伤的药物制剂。 药物的制备 方式可以釆用本领域普通技术人员所熟悉的方法制备成合适的制剂， 从而能进 行相应制剂的给药。
[34] 相应地， 在第四方面， 本发明提供预防和 /或治疗肝损伤的方法， 其包括向受 试者给药有效量的本发明第一方面所述的寡肽或其药学上可接受的盐或酯。 本 文所称的"受试者"指哺乳动物， 尤其是人。 根据的本领域普通技术人员所公知的 实验动物与人的等效剂量换算关系 （通常可参见 FDA、 SFDA等药品管理机构的 指导意见， 也可参见"黄继汉等.药理试验中动物间和动物与人体间的等效剂量换 算.中国临床药理学与治疗学， 2004 Sep ;9(9)
:1069-1072") 可从实验动物的剂量推导出人的有效剂量。 如未特别指出， 本文 中的"有效量 "指人的有效剂量。 例如， 对于注射给药， 人的有效量为 0.1~100μβ/ Kg体重， 优选为 0.5~5(^g/Kg体重， 更优选为 l-3(^g/Kg体重， 更优选为 1.5~25 g
/Kg体重， 更加优选为 10~2(^g/Kg体重， 最优选根据本发明的具体实施方式换算 出有效量； 对于口服给药， 人的有效量为 l~100(^g/Kg体重， 优选为 10~300μ§/Κ g体重， 更优选为 30-12(^g/Kg体重， 最优选根据本发明的具体实施方式换算出有 效量。 在第四方面， 优选所述寡肽是新的 7P衍生肽； 而且， 优选肝损伤是免疫 性肝损伤， 包括慢性免疫性肝损伤和急性免疫性肝损伤； 也优选肝损伤是肝毒 性化学物质引起的肝损伤， 尤其是酒精引起的肝损伤。 另外， 优选给药方式是 注射或经消化道给药。 根据本发明的具体实施方式， 也优选预防和 /或治疗慢性 免疫性肝损伤的方法， 其包括向受试者给药有效量的 7P或其药学上可接受的盐 或酯或其药学上可接受的盐或酯， 更优选其中给药方式是经消化道给药； 也优 选预防和 /或治疗酒精弓 I起的肝损伤的方法， 其包括向受试者给药有效量的 7P或 其药学上可接受的盐或酯或其药学上可接受的盐或酯， 更优选其中给药方式是 经消化道给药。 给药方式可以是单次给药， 也可以多次给药， 如间隔 1周多次给 药有效量的上述寡肽。
[35] 令人惊讶地， 本发明第二方面的药物组合物还可以用于预防和 /或治疗肾炎。
本文中所用的 "肾炎 "指的是肾脏组织或细胞出现的炎症。 在本发明的具体实施方 式中， 肾炎是血清白蛋白诱导的肾炎和主动型 Heymann's肾炎。 肾炎可通过病理 现象 （如动物模型的组织样本的病理评分） 来评估，也可通过尿素氮水平、 肌酐 水平、 肾小球内平均有核细胞数水平等生理生化水平来反映。 在本发明的另一 些具体实施方式中， 包含 7P的药物组合物被证实可用于预防和 /或治疗肾炎。
[36] 因此， 在第五方面， 本发明提供本发明第一方面所述的寡肽或其药学上可接受 的盐或酯在制备用于预防和 /或治疗肾炎的药物中的应用。 在本发明的具体实施 方式中， 所述寡肽是 7P; 而且， 肾炎是血清白蛋白诱导的肾炎和主动型 Heymann 's肾炎。 另外优选， 药物是注射或经消化道给药的药物制剂， 尤其优选是注射药 物制剂。 制备药物的过程包括将本发明第一方面所述的寡肽或其药学上可接受 的盐或酯和药学上可接受的载体混合的步骤， 用以配制成能用于预防和 /或治疗 肾炎的药物制剂。 药物的制备方式可以釆用本领域普通技术人员所熟悉的方法 制备成合适的制剂， 从而能进行相应制剂的给药。 [37] 相应地， 在第六方面， 本发明提供预防和 /或治疗肾炎的方法， 其包括向受试 者给药有效量的本发明第一方面所述的寡肽或其药学上可接受的盐或酯。 其中 ， "受试者"指哺乳动物， 优选是人； 优选给药方式是注射或经消化道给药， 尤其 优选是注射给药； 另外， "有效量"通常指人的有效剂量。 例如， 对于注射给药， 人的有效量为 0.1~10(^g/Kg体重， 优选为 0.5~5(^g/Kg体重， 更优选为 l-3(^g/Kg 体重， 更优选为 1.5~25 g/Kg体重， 更加优选为 10~20μ§/1¾体重， 最优选根据本 发明的具体实施方式换算出有效量； 对于口服给药， 人的有效量为 1~1000μβ/]¾ 体重， 优选为 10~30(^g/Kg体重， 更优选为 30-12(^g/Kg体重。 在本发明的具体实 施方式中， 所述寡肽优选是 7P; 而且， 肾炎优选是血清白蛋白诱导的肾炎和主 动型 Heymann's肾炎。 给药方式可以是单次给药， 也可以多次给药， 如间隔 1周 多次给药有效量的上述寡肽。
[38] 在第七方面， 本发明提供药剂盒， 其包括，
[39] (1) 包含本发明第一方面所述的寡肽或其药学上可接受的盐或酯的容器； 和
[40] (2) 指示给药预防和 /或治疗肝损伤或肾炎的说明书。
[41] 药剂盒是大众所熟知的带包装的药剂形式， 其包括有装有药剂的容器， 如瓶、 管等， 并包括有说明书， 所述说明书可以以单独形式装于药剂盒内， 也可以印 刷在药剂盒或容器外壁上。 在本发明第七方面中， 所述说明书记载有指示本发 明第一方面所述的寡肽或其药学上可接受的盐或酯根据本发明第四方面或第六 方面所述而给药预防和 /或治疗肝损伤或肾炎的内容。 所述内容可以包括适应症 、 剂量、 和给药方式等之一或多种， 例如， 所述内容可以是使用有效量的 7P经 消化道给药预防或治疗慢性免疫性肝损伤和 /或预防或治疗肾炎。
[42] 在第七方面， 本发明提供合成本发明第一方面所述的寡肽的方法。 本发明的寡 肽可以用在肽合成中常用的固相法或液相法等化学方法合成， 可根据需要可以 通过官能团的保护以及去保护有效地进行合成。 在肽合成中使用的氨基等保护 基团以及缩合反应的缩合剂等都有现有技术可供选择， 并有市售的合成装置和 试剂， 如在固相法中可以利用各种市售的肽合成装置。 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禾卩 J.D.Young白勺 《Solid Phase Peptide Synthesis》 (第二版， Pierce Chemical Co., Rockford, Illinois(1984))禾口 J. Meienhofer的 《Hormonal Proteins and
Peptides)) (第 2卷， AcademicPress， 纽约 (1973)) 等； 用液相法合成多肽可参考 Ε· Schroder和 Κ· 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。 另外， 尽管不优选， 但是通过生物重组技术也可以制备本发明 第一方面所述的寡肽前体， 并通过化学或酶裂解出本发明第一方面所述的寡肽 。 例如， 制备编码含有所述寡肽的肽的 DNA序列， 导入重组载体， 转化表达宿 主， 通过培养表达宿主来分离和提纯出所希望的寡肽前体， 然后化学或酶裂解 出所述寡肽。
[43] 为了便于理解， 以下将通过具体的附图和实施例对本发明进行详细地描述。 需 要特别指出的是， 具体实例和附图仅是为了说明， 并不构成对本发明范围的限 制。 显然本领域的普通技术人员可以根据本文说明， 在本发明的范围内对本发 明做出各种各样的修正和改变， 这些修正和改变也纳入本发明的范围内。 另外 ， 本发明引用了公幵文献， 这些文献也是为了更清楚地描述本发明， 它们的全 文内容均纳入本发明进行参考， 就好像它们的全文已经在本发明说明书中重复 叙述过一样。
[44] 附图说明
[45] 图 1.不同 7P截短肽对大鼠半乳糖胺肝损伤血清谷丙转氨酶活性的影响， 其中 ** 表示所示组相对于模型组能够非常显著降低转氨酶水平， *表示所示组相对于模 型组能够显著降低转氨酶水平。
[46] 图 2.不同 7P截短肽对大鼠半乳糖胺肝损伤血清谷草转氨酶活性的影响， 其中 ** 表示所示组相对于模型组能够非常显著降低转氨酶水平， *表示所示组相对于模 型组能够显著降低转氨酶水平。
[47] 图 3.不同 7P截短肽对大鼠半乳糖胺肝损伤病理学评分的影响， 其中 **表示所示 组相对于模型组能够非常显著降低病理学评分， *表示所示组相对于模型组能够 显著降低病理学评分。
[48] 图 4.经消化道给药 7P对卡介苗和脂多糖诱导的小鼠急性免疫性肝损伤谷丙转氨 酶水平的影响， 其中 **表示所示组相对于模型组能够非常显著降低转氨酶水平
， *表示所示组相对于模型组能够显著降低转氨酶水平。
[49] 图 5.经消化道给药 7P对卡介苗和脂多糖诱导的小鼠急性免疫性肝损伤谷草转氨 酶水平的影响， 其中 **表示所示组相对于模型组能够非常显著降低转氨酶水平
， *表示所示组相对于模型组能够显著降低转氨酶水平。
[50] 图 6.经消化道给药 7P对卡介苗和脂多糖诱导的小鼠急性免疫性肝损伤病理学评 分的影响， 其中 **表示所示组相对于模型组能够非常显著降低肝损伤病理学评 分， *表示所示组相对于模型组能够显著降低肝损伤病理学评分。
[51] 图 7.经消化道给药 7P对半乳糖胺和福氏完全佐剂诱导的小鼠慢性免疫性肝损伤 谷丙转氨酶水平的影响， 其中 **表示所示组相对于模型组能够非常显著降低转 氨酶水平， *表示所示组相对于模型组能够显著降低转氨酶水平。
[52] 图 8.经消化道给药 7P对半乳糖胺和福氏完全佐剂诱导的小鼠慢性免疫性肝损伤 谷草转氨酶水平的影响， 其中 **表示所示组相对于模型组能够非常显著降低转 氨酶水平， *表示所示组相对于模型组能够显著降低转氨酶水平。
[53] 图 9.经消化道给药 7P对半乳糖胺和福氏完全佐剂诱导的小鼠慢性免疫性肝损伤 病理学评分的影响， 其中 **表示所示组相对于模型组能够非常显著降低肝损伤 病理学评分， *表示所示组相对于模型组能够显著降低肝损伤病理学评分。
[54] 图 10.经消化道给药多肽 7P对异种血清致肝损伤大鼠血清谷丙转氨酶水平的影 响， 其中 **表示所示组相对于模型组能够非常显著降低转氨酶水平， *表示所示 组相对于模型组能够显著降低转氨酶水平。
[55] 图 11.经消化道给药多肽 7P对异种血清致肝损伤大鼠血清谷草转氨酶水平的影 响， 其中 **表示所示组相对于模型组能够非常显著降低转氨酶水平， *表示所示 组相对于模型组能够显著降低转氨酶水平。
[56] 图 12.经消化道给药多肽 7P对异种血清致肝损伤大鼠肝脏病理学评分的影响， 其中 **表示所示组相对于模型组能够非常显著降低肝损伤病理学评分， *表示所 示组相对于模型组能够显著降低肝损伤病理学评分。
[57] 图 13.7P对牛血清白蛋白诱导的大鼠肾炎尿素氮水平的影响， 其中 **表示所示 组相对于模型组差异非常显著， *表示所示组相对于模型组差异显著。 [58] 图 14.7P对牛血清白蛋白诱导的大鼠肾炎肌酐水平的影响， 其中 **表示所示组 相对于模型组差异非常显著， *表示所示组相对于模型组差异显著。
[59] 图 15.7P对牛血清白蛋白诱导的大鼠肾炎病理组织学评分的影响， 其中 **表示 所示组相对于模型组差异非常显著， *表示所示组相对于模型组差异显著。
[60] 图 16.7P对牛血清白蛋白诱导的大鼠肾炎肾小球内平均有核细胞数的影响， 其 中 **表示所示组相对于模型组差异非常显著， *表示所示组相对于模型组差异显 著。
图 17.7P对同种大鼠肾皮质匀浆加弗氏完全佐剂建立的主动型 Heymann's肾炎 AHN) 大鼠尿素氮水平的影响， 其中 **表示所示组相对于模型组差异非常显著 ， *表示所示组相对于模型组差异显著。
图 18.7P对同种大鼠肾皮质匀浆加弗氏完全佐剂建立的主动型 Heymann's肾炎 （ AHN) 大鼠肌酐水平的影响， 其中 **表示所示组相对于模型组差异非常显著， * 表示所示组相对于模型组差异显著。
图 19.7P对同种大鼠肾皮质匀浆加弗氏完全佐剂建立的主动型 Heymann's肾炎 AHN) 大鼠病理学评分的影响， 其中 **表示所示组相对于模型组差异非常显著 ， *表示所示组相对于模型组差异显著。
[64] 图 20.7P对同种大鼠肾皮质匀浆加弗氏完全佐剂建立的主动型 Heymann's肾炎 （ AHN) 大鼠肾小球内平均有核细胞数水平的影响， 其中 **表示所示组相对于模 型组差异非常显著， *表示所示组相对于模型组差异显著。
[65] 图 21.7P变异肽对 D-氨基半乳糖胺诱导的大鼠急性肝损伤血清生化指标 ALT的 影响。 其中 **表示所示组相对于模型组差异非常显著， *表示所示组相对于模型 组差异显著。
[66] 图 22.7P变异肽对 D-氨基半乳糖胺诱导的大鼠急性肝损伤血清生化指标 AST的影 响。 其中 **表示所示组相对于模型组差异非常显著， *表示所示组相对于模型组 差异显者。
[67] 图 23.7P变异肽对 D-氨基半乳糖胺诱导的大鼠急性肝损伤病理学评分的影响， 其中 **表示所示组相对于模型组差异非常显著， *表示所示组相对于模型组差异 显著。 [68] 图 24.经消化道给药多肽 7P对酒精致肝损伤大鼠的 MDA水平的影响， 其中 **表 示所示组相对于模型组能够非常显著降低 MDA水平， *表示所示组相对于模型组 能够显著降低 MDA水平。
[69] 图 25.经消化道给药多肽 7P对酒精致肝损伤大鼠的 GSH水平的影响， 其中 **表 示所示组相对于模型组能够非常显著提高 GSH水平， *表示所示组相对于模型组 能够显著提高 GSH水平。
[70] 图 26.经消化道给药多肽 7P对酒精致肝损伤大鼠的病理学评分的影响， 其中 ** 表示所示组相对于模型组差异非常显著， *表示所示组相对于模型组差异显著。
[71] 具体实施方式
[72] 实施例 1.肽的合成
通过固相肽合成方法， 使用 413A型自动肽合成仪 （购自 PerkinElmer公司） 来 合成如以下序列所示的肽： GQTYTSG (简称为 7P) ； 7P截短肽：
GQTYTS (简称为 PI) 、 QTYTSG (简称为 P2) 、 GQTYT (简称为 P3) 、 QTY TS (简称为 P4) 、 QTYT (简称为 P5) 、 TYTS (简称为 P6) 、 TYT (简称为 P7
) ； 7P变异肽： AQTYTSGs GNTYTSGs GQSYTSG、 GQTQTSG、 GQTYVSG 、 GQTYTTGs GQTYTS As GQTFTSG、 GQTWTSG、 GQTVTSG、 GQTRTSG 、 GQTHTSG。 这些肽中的氨基酸残基均为 L-型的氨基酸。 合成的肽用反向 HPL C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 成各个肽。 合成的肽经 HPLC确认纯度≥90%， 用于以下实施例中以测试其疗效 实施例 2.肽缀合物的制备
取实施例 1中合成的 7P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 7P, 将其溶于 0.5mlPBS (pH7.4, 0. 02mol/L) 中； 取 4.5mg
BSA溶于 4.5mlPBS (pH7.4, 0.02mol/L) 中。 混合上述 7P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小吋。 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， 得到肽缀合物。 [76] 实施例 3. 7P及其截短肽对 D-氨基半乳糖胺诱导的大鼠急性肝损伤的预防作用 [77] 1， 实验方法
[78] 药物剂量及分组： 实验动物随机分为 11组， 即空白对照组、 模型组、 阳性药 （ 甘利欣注射液） 组 （给药量以甘草酸二铵计为 13.5mg/kg.天） 、 7P组 （87 g/kg« 天） 、 7个截短肽组 （即 Pl、 P2、 P3、 P4、 P5、 P6、 P7组' 给药量分别为 87 g/k 天） ， 每组 10只 SPF级的 SD大鼠 （体重 180g〜220g， 雌雄各半） 。
[79] 给药过程： 于大鼠注射半乳糖胺前 7天， 阳性药组每天腹腔注射甘利欣注射液 ； 于大鼠注射半乳糖胺前 14天， 7P组及 P1~P7组均每隔一天皮下注射给药相应的 肽， 同吋， 空白组及模型组间也皮下注射等体积生理盐水。 各组各给药七次， 给药体积为 0.1ml/100g大鼠体重。 于各组最后一次给药后 24小吋， 模型组、 阳性 药组、 7P组及 P1~P7组各腹腔注射 D-氨基半乳糖胺 600mg/kg大鼠体重， 注射体积 为 lml/100g大鼠体重， 空白组注射等量生理盐水。 大鼠腹腔注射 D-氨基半乳糖胺 后 24小吋， 大鼠先称体重， 眼眶釆血后， 分离血清， 使用谷丙转氨酶、 谷草转 氨酶检测试剂盒测定谷丙转氨酶、 谷草转氨酶活性。 同吋， 颈椎脱臼处死大鼠 ， 对肝脏组织进行病理组织学检査。 根据病变由轻到重的程度标记为 0分， 0.5，
1分， 2分， 3分， 4分， 累加所有分数， 并计算出每组动物的均分 （X土 SD 值越高提示病变程度越严重。
[80] 2， 实验结果
[81] 7P及 P1~P7对 D-氨基半乳糖胺诱导的大鼠急性肝损伤血清生化指标的影响结果 如图 1-2所示。 D-氨基半乳糖胺造模组大鼠血清谷丙转氨酶、 谷草转氨酶显著升 高 （P<0.01) ， 表明 D-氨基半乳糖胺可造成大鼠急性肝损伤。 7P及 P1~P7以 87μ§/ kg给药都能显著地降低急性实验性肝损伤大鼠的谷丙转氨酶水平及谷草转氨酶 水平 （P<0.05) ， 其中 7P及 P1和 P7均能非常显著地降低谷草转氨酶水平 （P<0.01
7P及 P1~P7对 D-氨基半乳糖胺诱导的大鼠急性肝损伤病理组织学检査的影响结 果如图 3所示。 病理组织学研究表明， 本实验复制的肝损伤模型主要表现为多灶 性肝细胞点状或小灶状坏死， 坏死处有炎细胞浸润。 肝细胞内出现嗜酸性小体 ， 中央静脉周围及门管区有少量炎细胞浸润， 门管区未査见纤维组织增生。 多 数大鼠肝细胞可见轻度脂变。 呈小灶状分布。 不同多肽药物应用后都能显著减 轻肝脏损伤程度， 其效果按由高到低的顺序依次为： 7P组， Pl， P2, P3，
P5， P6，P4，和 P7。 各用药组与模型组相比都有统计学差异 （参见图 3) 。
[83] 3， 实验结论
[84] 本实验复制的肝损伤模型主要表现为多灶性肝细胞点状或小灶状坏死， 坏死处 有炎细胞浸润。 肝细胞内出现嗜酸性小体， 中央静脉周围及门管区有少量炎细 胞浸润， 门管区未査见纤维组织增生。 多数大鼠肝细胞可见轻度脂变。 呈小灶 状分布。 对 D-氨基半乳糖胺诱导的大鼠急性肝损伤模型， 不同多肽序列药物应 用后能减轻肝脏损伤程度， 其效果按由高到低的顺序依次为： 7P组， Pl， P2, P3 ,
P5， P6， P4，和 P7。 各用药组与模型组相比都有统计学差异， 因而能用于减轻肝 损伤症状。
[85] 实施例 4 7P变异肽对 D-氨基半乳糖胺诱导的大鼠急性肝损伤的作用
[86] 相似地， 合成 7P变异肽， 参照实施例 3的方法， 对由 D-氨基半乳糖胺诱导的 SD 大鼠的急性肝损伤进行处理。 其鉴定结果如图 21、 22、 和 23所示。 根据药物应 用后能减轻肝脏损伤程度， 综合血清学检测结果和病理评分， 其效果按由高到 低的顺序依次为药 9组 （GQTWTSG) ， 药 8组 （GQTFTSG) ， 药 1组 （AQTYTS G) ， 药 2组 (GNTYTSG) ， 药 3组 （GQSYTSG) ， 药 10组 （GQTVTSG) ， 药 6组 （GQTYTTG) ， 药 7组 （GQTYTSA) 。 上述各用药组与模型组相比， 在血 清学检测或病理评分指标上有统计学差异。
[87] 实施例 5.经消化道给药 7P对卡介苗和脂多糖诱导的小鼠急性免疫性肝损伤的疗 效
[88] 1， 实验方法
[89] 药物剂量及分组： 实验动物随机分为 6组， 即空白组、 模型组、 阳性药 （甘利 欣注射液） 组、 7P高剂量组， 7P中剂量组， 7P低剂量组， 每组 12只 BALB/c小鼠 。 其中有甘利欣注射液组 （给药量以甘草酸二铵计为 19.5mg/kg.天） ， 7P高剂量 组 （125(^g/ kg.天） ， 7P中剂量组 (625 g/ kg.天) ， 7P低剂量组 (312.5μβ/ kg.天） ， 空白对照组 （给予等容积生理盐水， 但不给药卡介苗和脂多糖） ， 模 型组 （给予等容积生理盐水） 。 7P组 （即 7P高剂量组、 7P中剂量组、 和 7P低剂 量组） 灌胃给药， 给药体积为 0.2ml/20g小鼠体重。
[90] 给药过程： 7P高、 中、 低剂量组间隔一天给药， 连续给药七次。 阳性药组于小 鼠注射 LPS前 7天每天尾静脉注射甘利欣注射液 19.5mg/kg.天， 空白组及模型组于 小鼠注射 LPS前 7天每天给予等体积生理盐水， 0.2ml/20g。 模型组、 阳性药组及 7 P组于 7P第一次给药后 1天给小鼠尾静脉注射卡介菌多糖核酸注射液 （用生理盐 水 1 : 20稀释） ， 每只小鼠注射 0.2ml (给药量以卡介菌多糖计为 175 g/kg) ， 空 白组注射等量生理盐水。 于 7P第七次给药后 1天， 模型组、 阳性药组及 7P组小鼠 尾静脉注射 10μβ脂多糖 （LPS) ， 空白组注射等量生理盐水。 小鼠静脉注射脂多 糖后 12小吋， 小鼠先称体重， 釆血后， 分离血清， 用谷丙转氨酶、 谷草转氨酶 检测试剂盒分别测定谷丙转氨酶、 谷草转氨酶活性。 颈椎脱臼处死小鼠后， 进 行尸检， 对肝脏组织进行病理组织学检査。
[91] 2， 实验结果
[92] 实验结果如图 4-6所示。 卡介苗和脂多糖造模组小鼠血清谷丙转氨酶、 谷草转 氨酶显著升高， 表明卡介苗和脂多糖可造成小鼠急性免疫性肝损伤。 7P肽灌胃 给药能显著降低急性免疫性肝损伤小鼠谷丙转氨酶及谷草转氨酶 （参见图 4、 5 ) 。 7P对卡介苗和脂多糖诱导的小鼠免疫性肝损伤的组织病理学研究表明， 本 研究复制的小鼠急性免疫性肝损伤模型， 病变主要为包膜下肝细胞变性， 肝细 胞出现核固缩。 少数肝细胞有轻度坏死， 中央静脉周围局部可见少量炎细胞浸 润。 门管区未见纤维组织增生。 7P高、 中剂量组灌胃给药后均能非常显著地减 轻肝细胞损伤程度 （P<0.01) ， 7P低剂量组灌胃给药后亦能显著地减轻肝细胞损 伤程度 （Ρ<0·05) (参见图 6) 。
[93] 实施例 6.经消化道给药 7Ρ对半乳糖胺和福氏完全佐剂诱导的小鼠慢性免疫性肝 损伤的疗效
[94] 1， 实验方法：
[95] 药物剂量及分组： 模型制备： 向 BALB/ 、鼠腹腔注射半乳糖胺 300mg/kg， 同 吋腹部皮下注射福氏完全佐剂 0.05ml/20g， 每周一次， 连续四周； 然后， 腹腔注 射半乳糖胺 400mg/kg， 同吋腹部皮下注射福氏完全佐剂 0.05ml/20g， 每周一次， 连续八周； 然后， 腹腔注射半乳糖胺 500mg/kg， 同吋腹部皮下注射福氏完全佐 剂 0.1ml/20g小鼠体重， 每周一次， 连续六周。 然后， 经血清样本检测， 成功制 备小鼠慢性免疫性肝损伤模型 100只， 随机分为 5组， P模型组、 阳性药 （甘利 欣注射液） 组 （给药量以甘草酸二铵计为 19.5mg/kg.天） 、 7P高剂量组 （1250μ§/ kg.天) 、 7P中剂量组 (625μβ/
kg.天） 、 7P低剂量组 （312.5 g/kg.天） ， 另外同批次健康小鼠成空白对照组， 共 6组， 每组 20只。
[96] 给药过程： 小鼠间隔一天给药一次， 连续给药 1个月 （共 15次） ， 药物均用生 理盐水配置成所需浓度， 7P组 （灌胃给药） 给药体积为 0.2ml/20g， 阳性药 （甘 利欣注射液） 组静脉注射甘利欣， 空白对照组和模型组给予等容积生理盐水。 给药结束后， 小鼠处死， 称体重， 釆血后， 分离血清， 用 OLYMPUS
2700全自动生化分析仪分析谷丙转氨酶和谷草转氨酶。 颈椎脱臼处死小鼠后， 进行尸检， 对肝脏组织进行病理组织学检査。
[97] 2， 实验结果
[98] 实验结果如图 7-9所示。 半乳糖胺和福氏完全佐剂造模组小鼠血清谷丙转氨酶 、 谷草转氨酶、 球蛋白显著升高， 白蛋白、 碱性磷酸酶、 葡萄糖、 白球比显著 下降， 表明半乳糖胺和福氏完全佐剂联合应用可造成小鼠慢性肝损伤。 多肽 7口 服给药能显著性改善慢性实验性肝损伤大鼠的血清生化指标， 表明其具有明显 的保护慢性免疫性肝损伤的作用 （参见图 7、 8) 。 7P对半乳糖胺和福氏完全佐 剂诱导的小鼠慢性免疫性肝损伤的组织病理学研究表明， 本研究成功地复制了 小鼠慢性免疫性肝损伤模型， 病变主要为肝脏内出现肝细胞坏死 （点状或小灶 性坏死） ， 坏死处炎细胞浸润， 炎细胞类型主要为中性粒细胞及单核巨噬细胞 。 肝细胞变性表现为水肿和脂变， 水肿程度极轻， 主要位于肝包膜下方， 少数 位于中央静脉周围。 脂肪变性的肝细胞常集聚成小灶。 免疫 7肽应用后， 各用药 组肝细胞损伤程度减轻， 并与所用剂量呈正相关， 各用药组与模型组相比均有 统计学意义 ^〈(^(^或！^。.。^ (参见图 9) 。
[99] 实施例 7.经消化道给药 7P对异种血清诱导的大鼠慢性免疫性肝损伤的疗效
[100] 1， 实验方法 [101] 模型制备： 向 Wistar大鼠腹腔注射猪血清 0.3ml/200g (含 27mg蛋白质） ， 每周 1 - 2次， 连续注射三个半月 （共 20次） ， 经血清生化检测肝脏功能， 成功制备大 鼠慢性免疫性肝损伤模型 60只。 将实验动物随机分为 5组， 每组 12只大鼠， 即模 型组、 甘利欣注射液组 （给药量以甘草酸二铵计为 13.5mg/kg.天） ， 7P高剂量组 (870μβ/ 1¾·天) ， 7Ρ中剂量组 (435 g/ kg.天) ， 7P低剂量组 (217.5μβ/ kg.天） ； 另取 12只健康大鼠组成空白对照组。
[102] 给药过程： 7P组用生理盐水配置成所需浓度， 给药体积为 0.2ml/200g， 灌胃给 药； 甘利欣注射液组尾静脉注射给药； 空白对照组和模型组给予等容积生理盐 水。 大鼠间隔一天给药一次， 连续给药 1个月 （共 15次） ， 期间， 每周大鼠腹腔 注射猪血清一次 （0.3ml/200g) 。 给药结束后， 称体重， 眼眶釆血后， 分离血清 ， 全自动生化分析仪测定丙氨酸氨基转换酶 （ALT) 、 天门冬氨酸氨基转换酶 AST) 、 总胆红素 （TBIL) 、 碱性磷酸酶 （AKP) 、 总蛋白 （TP) 、 白蛋白 （ ALB) 、 球蛋白 （G) 、 白球比 （A/G) 、 血糖 （GLU) 。 颈椎脱臼处死大鼠后 ， 进行尸检， 对肝脏组织进行病理组织学检査， 根据病变由轻到重的程度标记 为 0分， 0.5， 1分， 2分， 3分， 4分， 累加所有分数， 并计算出每组动物的均分 ( X±SD) ， 分值越高提示病变程度越严重。
[103] 2, 实验结果
[104] 实验结果如图 10-12所示。 异种血清造模组大鼠血清谷丙转氨酶、 谷草转氨酶 、 碱性磷酸酶、 总胆红素、 总胆汁酸显著升高， 总蛋白、 白蛋白、 白球比显著 下降， 表明异种血清可造成大鼠慢性肝损伤。 多肽 7P灌胃给药高、 中剂量组能 显著性改善慢性实验性肝损伤大鼠的血清生化指标 （参见图 10、 11) 。 多肽 7P 对异种血清引起的大鼠慢性免疫性肝脏损伤的组织病理学检査表明， 本实验复 制的肝损伤模型主要表现为肝小叶内及门管区纤维组织增生， 明显者增生的纤 维组织互相连接， 但尚未包挠、 分隔肝小叶形成假小叶。 肝细胞变性不明显， 多数大鼠仅见个别肝细胞内出现少量脂滴空泡。 7P灌胃给药高剂量组应用后能 减轻肝脏纤维化程度， 与模型组相比均有显著性差异 （P<0.05) (参见图 12) 。
[105] 实施例 8. 7P对牛血清白蛋白 （BSA) 诱导的大鼠肾炎的保护作用
[106] 1， 实验方案 [ 107] 实验分组及剂量：
SD大鼠随机分为 6组， 即空白对照组、 模型组、 阳性药 （地塞米松注射液） 组 （ 以地塞米松计为 0.9mg/kg.天） 、
7P高剂量组 （174 §/1¾*天） 、 7P中剂量组 （87 §/1¾*天） 、 7P低剂量组 （43.5 §/1¾*天） ， 每组 10只大鼠。 7P各剂量组均皮下注射给药， 给药体积为 0.1 ml/100g体重； 地塞米松注射液组腹腔注射给药， 给药体积为 0.1 ml/100g体重。
[ 108] 给药过程： 于实验前 12天将牛血清白蛋白 （BSA) 按 150mgZkg体重给模型组 、 阳性药 （地塞米松注射液） 组、 7P高剂量组、 7P中剂量组、 和 7P低剂量组大 鼠腹腔注射 1次来预免疫， 在实验第 1天、 第 12天再按 300mgZkg体重腹腔注射各 1次。 空白对照组大鼠同吋注射等容积的生理盐水代替。 在实验第 1天， 阳性药 (地塞米松注射液） 组、 7P高剂量组、 7P中剂量组、 和 7P低剂量组隔天皮下注 射给药地塞米松或 7P—次， 共给药 15次； 同吋， 空白对照组及模型组间隔一天 皮下注射等体积生理盐水， 共给药 15次。 末次给药 24小吋， 大鼠先称体重， 眼 眶釆血后， 分离血清， 按肌酐 (Cre-S) 肌氨酸氧化酶法检测试剂盒及尿素氮 （ BUN) UV-GLDH法检测试剂盒说明书测定肌酐及尿素氮。 颈椎脱臼处死大鼠后 ， 进行尸检， 对肾脏组织用 10%甲醛固定， 石蜡包埋， 切片， HE染色进行病理 组织学检査。
[ 109] 2 , 实验结果
[ 1 10] 结果如图 13- 16所示。 对牛血清白蛋白 （BSA) 诱导的大鼠肾炎影响的研究结 果表明牛血清白蛋白造模组大鼠尿素氮 （BUN) 、 肌酐 （Cre-S) 水平显著升高
(P<0.01 ) ， 表明牛血清白蛋白多次给药可造成大鼠肾损伤。 7P高、 中剂量组能 显著性降低实验性肾炎大鼠尿素氮 （BUN) 、 肌酐 （Cre-S) ^〈(^(^或！^。.。 ) (参见图 13、 14)。 病理组织学研究表明， 本实验复制的肝损伤模型主要表现为 肾小球体积增大， 有核细胞数增多， 肾小球呈分叶状， 系膜区增宽， 基质增多 。 其它病变包括间质有少量炎细胞浸润及纤维组织增生， 个别大鼠局部肾小管 轻度萎缩。 7P各剂量组与动物模型组相比， 病变程度有所减轻， 其中高剂量组 肾小球内有核细胞数及其它病变与模型组相比均有显著性差别。 肾脏病变程度 评分结果表明： 7P高剂量组与模型组相比有统计学差异 （P<0.01 ) (参见图 15、 1 [ 1 1 1] 实施例 9.7P对同种大鼠肾皮质匀浆加弗氏完全佐剂建立的主动型 Heymann' s肾 炎 （AHN) 的保护作用
[ 1 12] 1， 实验方案
[ 1 13] 取体重 180 - 220gSD大鼠 40只， 雌雄各半， 腹腔注射戊巴比妥钠麻醉， 在无菌 条件下幵腹， 结扎肾动脉， 自结扎处向肾脏插管， 剪断肾静脉， 经插管用生理 盐水反复冲洗肾脏， 使之成灰白色， 取下肾脏， 取肾皮质用电动匀浆器磨成匀 浆， 每次取皮质匀浆 35g， 加弗氏完全佐剂 70ml。 置乳钵研匀。 缓缓加入生理盐 水 140ml边加边磨， 使乳化均匀完全。
[ 1 14] 取 SD大鼠， 随机抽取 10只作正常对照组 (正常组)外， 随机抽取 10只作佐剂对照 组， 大鼠腹腔注射弗氏不完全佐剂生理盐水乳化液， 其余造模组大鼠腹腔注射 上述同种大鼠肾皮质加弗氏完全佐剂液， 每次 2mlZl00g体重， 每两周注射 1次 ， 共造模 12周， 前后共注射 7次。 于造模第 12周正常对照组及模型对照组各取 5 只大鼠作血浆肌酐、 尿素氮及病理检査。 模型经病理检査发生肾炎后模型动物 幵始分组给药。
[ 1 15] 模型动物随机分为 5组， 即模型组、 阳性药 （地塞米松注射液） 组 （以地塞米 松计为 0.9mg/kg.天） 、
7P高剂量组 （174 §/1¾*天） 、 7P中剂量组 （87 §/1¾*天） 、 7P低剂量组 （43·5μβ
/kg.天) ， 另外健康大鼠组成空白组， 取每组 10只大鼠。 阳性药 （地塞米松注射 液） 组、
7P高剂量组、 7P中剂量组、 和 7P低剂量组隔天皮下注射给药一次， 共给药 15次 。 空白组及模型组间隔一天皮下注射等体积生理盐水， 共给药 15次。 末次给药 2 4小吋， 大鼠先称体重， 眼眶釆血后， 分离血清， 按肌酐 (Cre-S) 肌氨酸氧化酶 法检测试剂盒及尿素氮 （BUN) UV-GLDH法检测试剂盒说明书测定肌酐及尿素 氮。 颈椎脱臼处死大鼠后， 进行尸检， 对肾脏组织用 10%甲醛固定， 石蜡包埋 ， 切片， HE染色进行病理组织学检査。
[ 1 16] 2， 实验结果
[ 1 17] 结果如图 17-20所示。 对同种大鼠肾皮质匀浆加弗氏完全佐剂建立的主动型 Hey mann's肾炎 （AHN) 影响的研究结果表明同种大鼠肾皮质匀浆加弗氏完全佐剂 造模组大鼠尿素氮 （BUN) 、 肌酐 （Cre-S) 水平显著升高 （Ρ<0·01) ， 表明同 种大鼠肾皮质匀浆加弗氏完全佐剂多次给药可造成大鼠肾损伤。 7Ρ药物高、 中 剂量组能显著性降低实验性肾炎大鼠尿素氮 （BUN) 、 肌酐 （Cre-S) (P<0.05 或!><0.01) (参见图 17、 18) 。 病理组织学研究表明， 本实验复制的肝损伤模型 主要表现为多数肾小球明显肿胀， 肾小球毛细血管丛内细胞数明显增多， 以内 皮细胞增多为主， 毛细血管壁明显增厚， 并有纤维素样坏死， 毛细血管扩张充 血， 肾小管上皮细胞严重混浊肿胀， 有的肾小管上皮近乎嗜酸性坏死， 管腔明 显狭窄甚至闭塞， 肾小管管腔可见不同类型管型。 7P用药各剂量组与动物模型 组相比， 病变程度有所减轻， 其中药物高剂量组病变肾小球明显改善， 肾小球 体积仅轻度肿大， 内皮细胞增生不明显， 肾小管上皮细胞混浊肿胀减轻， 无肾 小管管腔狭窄， 病变肾小球数量也明显减少。 肾脏病变程度评分结果表明： 7P 药物高剂量组与模型组相比有统计学差异 （P<0.01) (参见图 19、 20) 。
[118] 实施例 10.经消化道给药 7P对对小鼠酒精性肝损伤的保护作用
[119] 1， 实验方法
[120] 将 SPF级 ICR小鼠 （体重 18— 22g， 雌雄各半， 购自南通大学实验动物中心） 随 机分为九组， 每组 10只动物， 分组包括： 空白对照组 (给药蒸馏水)、 急性酒精性 肝损伤模型组 (给药 50%乙醇溶液)、 天晴甘平阳性药组 （给药甘草酸二铵肠溶胶 囊， 其购自江苏正大天晴药业股份有限公司， 给药剂量以甘草酸二铵肠溶计为 5 8.5mg/kg^+50%乙醇溶液） ， SP药物高剂量组 （给药 7P
125(^¾'天+50%乙醇溶液） ， 中剂量组分别为 （给药 7P
625 g/kg^+50%乙醇溶液） ， 低剂量组 （给药 7P
312.5 §/1¾*天+50%乙醇溶液） 。 以上各组均通过灌胃给药， 每天给药 1次， 连 续 30天。 在第 30天， 用 50%无水乙醇溶液对小鼠进行一次性灌胃， 16小吋后将小 鼠处死。 各组小鼠称重后， 处死， 取约 0.19〜0.25 g新鲜肝脏， 加入 2 ml 0.9% 生理盐水， 配成 10%的肝组织匀浆后， 3
500rpm离心 10分钟， 取上清， 然后根据厂商说明， 用丙二醛 （MDA) 检测试剂 盒及还原型谷胱甘肽 （GSH) 检测试剂盒 （均购自南京建成生物工程研究所） 分别测定 MDA及 GSH; 并对肝脏组织用 10%甲醛固定， 石蜡包埋， 切片， HE染 色进行病理组织学检査。
[121] 2， 实验结果
[122] 结果如图 24-26所示。 模型组的肝组织匀浆中过氧化脂质降解产物丙二醛 (MDA )水平显著升高、 谷胱甘肽 （GSH) 水平显著下降， 表明酒精可引起肝脏组织发 生氧化损伤； 而 SP药物高、 中、 低剂量组能显著性降低过氧化脂质降解产物丙 二醛 (MDA)水平， 并能显著提高升高谷胱甘肽 （GSH) 水平 （参见图 24、 25) 。 病理组织学研究表明， 本实验复制的肝损伤模型主要表现为明显肝脂变， 肝 细胞内出现细小的脂质空泡， 少数肝脏伴有细胞水肿； 而药 SP使用后均有减轻 肝脂变的作用。 药物高剂量、 中剂量及低剂量与模型组相比均有统计学差异 （ 参见图 26) 。 这表明， 7P对酒精引起的肝脏组织氧化损伤有保护作用。

Claims

权利要求书
[1] 式 I的寡肽或其药学上可接受的盐或酯，
Xaal-Xaa2-Xaa3-Xaa4-Thr-Xaa5-Xaa6-Xaa7 (式 I)
其中，
Xaal为缺失、 Gly或 Ala,
Xaa2为缺失、 Gin或 Asn，
Xaa3为 Thr或 Ser，
Xaa4为 Tyr、 Phe、 Trp或 Val，
Xaa5为缺失、 Ser或 Thr,
Xaa6为缺失、 Gly或 Ala, 而且
Xaa7为缺失、 Ala或 Gly。
[2] 权利要求 1所述的寡肽或其药学上可接受的盐或酯， 其中所述寡肽不是 Gly-
Gln-Thr-Tyr-Thr-Ser-Gly-Xaa7 , 其中 Xaa7为缺失、 Ala或 Gly。
[3] 权利要求 1或 2所述的寡肽或其药学上可接受的盐或酯， 其中 Xaa3为 Thr, 而 且 Xaa4为 Tyr。
[4] 权利要求 1或 2所述的寡肽或其药学上可接受的盐或酯， 其中所述寡肽是 GQ
TYTSG的氨基酸序列中替换一个氨基酸残基而得的寡肽。
[5] 权利要求 1-4之任一所述的寡肽或其药学上可接受的盐或酯， 其中所述寡肽 是选自 GQTYTS、 QTYTSG、 GQTYT、 QTYTS、 QTYT、 TYTS、 和 TYT的 寡肽； 或者， 所述寡肽是选自 AQTYTSG、 GNTYTSGs GQSYTSG、 GQT YTTG、 GQTYTSAs GQTFTSG、 GQTWTSG、 和 GQTVTSG的寡肽。
[6] 药物组合物， 其包括权利要求 1-5之任一所述的寡肽或其药学上可接受的盐 或酯， 以及药学上可接受的载体。
[7] 权利要求 6所述的药物组合物， 其用于预防和 /或治疗肝损伤， 尤其是急性 免疫性肝损伤、 慢性免疫性肝损伤和 /或肝毒性化学物质引起的肝损伤， 如 酒精引起的肝损伤。
[8] 权利要求 6所述的药物组合物， 其用于预防和 /或治疗肾炎， 尤其是血清白 蛋白诱导的肾炎和 /或主动型 Heymann's肾炎。
[9] 权利要求 6-8之任一所述的药物组合物， 其中寡肽是 GQTYTSG。
[10] 权利要求 6-9之任一所述的药物组合物， 其为经消化道给药的制剂。
[11] 权利要求 1-5之任一所述的寡肽或其药学上可接受的盐或酯在制备用于预防 和 /或治疗肝损伤的药物中的应用， 优选所述寡肽不是 Gly-Gln-Thr-Tyr-Thr-
Ser-Gly-Xaa7 , 其中 Xaa7为缺失、 Ala或 Gly。
[12] 预防和 /或治疗肝损伤的方法， 其包括向受试者给药有效量的权利要求 1-5 之任一所述的寡肽或其药学上可接受的盐或酯， 优选所述给药是经消化道 给药。
[13] 权利要求 1-5之任一所述的寡肽或其药学上可接受的盐或酯在制备用于预防 和 /或治疗肾炎的药物中的应用。
[14] 预防和 /或治疗肾炎的方法， 其包括向受试者给药有效量的权利要求 1-5之 任一所述的寡肽或其药学上可接受的盐或酯。
[15] 药剂盒， 其包括，
(1) 包含权利要求 1-5之任一所述的寡肽或其药学上可接受的盐或酯的容 器； 和
(2) 指示给药预防和 /或治疗肝损伤或肾炎的说明书。
[16] 合成权利要求 1-5之任一所述的寡肽的方法， 优选其中合成是固相合成。
PCT/CN2009/071204 2008-04-18 2009-04-09 7p及其衍生肽和其应用 WO2009127140A1 (zh)

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