WO2018001273A1 - 肽或其盐、其制备方法、及其制备预防和/或治疗肝损伤药物的用途 - Google Patents

肽或其盐、其制备方法、及其制备预防和/或治疗肝损伤药物的用途 Download PDF

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WO2018001273A1
WO2018001273A1 PCT/CN2017/090552 CN2017090552W WO2018001273A1 WO 2018001273 A1 WO2018001273 A1 WO 2018001273A1 CN 2017090552 W CN2017090552 W CN 2017090552W WO 2018001273 A1 WO2018001273 A1 WO 2018001273A1
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val
peptide
pro
tyr
fmoc
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PCT/CN2017/090552
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English (en)
French (fr)
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金�一
孙勇兵
王日康
杨世林
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江西本草天工科技有限责任公司
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Priority claimed from CN201610503384.8A external-priority patent/CN107551258B/zh
Priority claimed from CN201610503413.0A external-priority patent/CN107556363B/zh
Application filed by 江西本草天工科技有限责任公司 filed Critical 江西本草天工科技有限责任公司
Publication of WO2018001273A1 publication Critical patent/WO2018001273A1/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
    • A61K38/07—Tetrapeptides
    • 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
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a peptide or a salt thereof, a process for the preparation thereof, and a use thereof for the preparation of a medicament for preventing and/or treating liver damage.
  • a peptide is a compound obtained by dehydration condensation of a plurality of amino acid molecules.
  • a variety of peptide drugs are known, for example, VVYP, which is a tetrapeptide composed of the amino acids L-Val, L-Tyr and L-Pro according to the sequence of L-val-L-val-L-Tyr-L-Pro.
  • the compound which was originally composed of a compound of pig blood protein prepared by the method of enzymatic hydrolysis by Professor Kagawa Kagawa of Japan, see lifescience, 1996, 58(20): 1745-1755, which has a reduced porcine protein mixture and VVYP. The role of cholesterol and blood lipids.
  • U.S. Patent US 9233999 B2
  • U.S. Patent No. 5,958,885 a method for synthesizing VVYP is disclosed in the U.S. Patent No. 5,958,885, but the method has obvious defects, and trifluoroacetic acid must be used in each step, resulting in high production cost; three wastes are used, and hydrogen fluoride is used as a cutting reagent.
  • the peptides are cleaved from the resin, which is a highly toxic acid.
  • Val-Val-Tyr-Pro peptide (ie, VVYP) has been reported to have an effect of inhibiting blood triglyceride concentration, preventing or treating obesity, hyperlipidemia and its associated hypertension and arteries in humans or animals. Circulatory diseases such as sclerosis (JP-A-9-255698). Further, the Val-Val-Tyr-Pro peptide has an effect of lowering the blood sugar level of a subject in a hyperglycemic state, and can be used for preventing or treating a disease caused by hyperglycemia (Special Table 2008-519758).
  • Val-Val-Tyr-Pro peptide has the effect of inhibiting the re-elevation of cholesterol in a patient in a high cholesterol state or in the prior stage, and can be used for preventing or treating a pathological condition caused by a high cholesterol state caused by a rise in cholesterol or The disease, and Val-Val-Tyr-Pro peptide has a blood pressure lowering effect (2007-137816). It has also been reported in Chinese patent literature that Val-Val-Tyr-Pro peptide has anti-stress, uneasiness, and low emergency response (CN101633945A).
  • liver damage caused by factors such as viral infections, drugs, chemical poisons (such as chemical pollutants in the environment), alcohol abuse, etc. is increasingly seriously jeopardizing people's health.
  • Liver injury Induction of hepatitis, cirrhosis and even liver cancer seriously affects the quality of life and health.
  • China is recognized as a major hepatitis country in the world, with about 130 million hepatitis virus carriers.
  • the environmental pollution caused by the rapid development of China's economy, the increasing consumption of alcohol, the unreasonable use and abuse of drugs, and the resulting chemical, alcoholic and drug-induced liver damage have increased year by year in China. Therefore, there is a huge market demand for drugs for treating liver damage.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • the clinical first-line drug bicyclol which reduces transaminase is a biphenyl diester structural analog, which has good liver-protecting effect and certain anti-hepatitis B virus activity. It is suitable for carbon tetrachloride (CCl 4 ) and D-galactosamine. Liver damage caused by acetaminophen and mouse immunological hepatitis caused by BCG plus lipopolysaccharide have the effect of reducing transaminase. However, clinical application found that bicyclol has a much lower effect on ALT levels than AST.
  • the ALT level is significantly reduced, the AST level is not lowered or even increased, and the ratio of alanine aminotransferase/aspartate aminotransferase is increased. .
  • many doctors only use bicyclol for patients with mild liver injury, mainly to treat elevated ALT levels.
  • Bicyclol has very poor water solubility, low oral bioavailability and large fluctuations, and cannot be made into an injection, and the virus strain is resistant. The main reduction in ALT levels and poor water solubility limits the clinical use of bicyclol, and clinically targeted drugs to protect liver mitochondria and thereby reduce AST levels are yet to be developed.
  • the drugs used for the treatment of severe liver injury are commonly used such as intravenous administration of glycyrrhizin, glycyrrhizic acid preparation, cell membrane protective agent, and other phosphatidylcholine.
  • These chemical drugs have certain side effects and have long-term therapeutic effects.
  • the liver's ability to clear many drugs is reduced, and excessive use of drugs may cause liver damage. Therefore, it is urgent to find a liver repairing drug with better enzyme-lowering effect and low toxicity.
  • the present inventors conducted a long-term and intensive study and found that a resin as a starting material, an amino acid having a protective group is attached to a resin, and then a protective group is removed, and each amino acid is sequentially linked. Then, the peptide is cut and the side chain protecting group is removed to obtain a crude product, and after purification, the desired peptide or a salt thereof is obtained, thereby providing preparation of a peptide compound with simple process, low toxicity, high yield and high purity.
  • new peptide compounds such as VVYP; AVLP; IGFP; LLIIVP and STAP and salts thereof can also be provided.
  • the polypeptide compound VVYP (peptide represented by the sequence Val-Val-Tyr-Pro) has an excellent liver-protecting effect, lowering transaminase, especially lowering AST level, reducing liver tissue pathological changes and The effect of repairing liver damage has an excellent effect on liver damage, such as chemical, viral, pharmaceutical, or alcoholic (e.g., acute and chronic) liver damage, and prevention and treatment of acute alcoholic poisoning.
  • the present inventors completed the present invention based on the above studies.
  • the present invention relates to the following aspects.
  • a method for producing a peptide or a salt thereof comprising:
  • Step A using a resin as a starting material, linking the C-terminal amino acid of the constituent peptide having an ⁇ -amino protecting group to the resin in the presence of an alkaline reagent,
  • Step B removing the protecting group with a capping reagent
  • Step C in the presence of an alkaline reagent, the respective amino acids constituting the peptide are sequentially connected in the order from the C-terminus to the N-terminus of the peptide to form a peptide-ligating resin, wherein each amino acid linkage is first linked to have ⁇ .
  • the amino acid of the amino protecting group is subjected to peptide extraction, and then the protecting group is removed by a capping reagent to perform capping,
  • Step D performing peptide digestion to obtain a crude product of a peptide or a salt thereof
  • Step E Purification of the crude product to obtain the peptide or a salt thereof.
  • step C The production method of the above [1] to [5], wherein the alkaline agent in the step C is selected from the group consisting of: N,N-diisopropylethylamine (DIEA) and N-methylmorpholine (NMM). .
  • the coupling reagent in step C is selected from the group consisting of O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-tetramethylurea Hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yl-oxytripyrrolidinylphosphonate (PyBop).
  • step A and step C the amino acid having an ⁇ -amino protecting group is fed in an amount of from 1.5 to 6 times the number of moles of the resin to be attached;
  • the alkaline reagent is charged in an amount of 2 to 10 times the number of moles of the resin to be connected;
  • step C in the peptide reaction for linking each amino acid, the amount of the coupling reagent HBTU is equal to the amount of the amino acid having an ⁇ -amino protecting group; the amount of HOBT charged is, in terms of moles, 1.1 to 5 times the amino acid of the ⁇ -amino protecting group; DIEA is fed in an amount of 2 to 10 times the number of moles of the amino acid having an ⁇ -amino protecting group
  • the protecting group of the amino acid is Fmoc;
  • the peptide reagent is: selected from the group consisting of dichloromethane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, tetrahydrofuran and One or more of ethyl acetate.
  • the capping reagent is piperidine (PIP): DMF is equal to 1:2 to 10 (volume ratio); the amount of the capping agent added and the weight of the resin to be uncapped are 5 to 25 ml of capping reagent / g of resin.
  • step E the crude product is dissolved in a dilute acid solution for separation and purification.
  • the acid is selected from the group consisting of sulfuric acid, hydrochloric acid, hydrobromic acid, methanesulfonic acid, naphthalenesulfonic acid, acetic acid, fumaric acid, maleic acid, tartaric acid, 2,5-dihydroxybenzoic acid, methanesulfonic acid, ethylsulfonate Acid, benzenesulfonic acid and p-toluenesulfonic acid.
  • VVYP VVYP
  • AVLP AVLP
  • IGFP IGFP
  • LLIIVP LLIIVP
  • STAP STAP
  • Y represents: L-tyrosine
  • V represents: L-valine
  • P L-valine
  • A represents: L-alanine
  • L represents: L-leucine
  • I represents: L-isoleucine
  • G stands for: L-glycine
  • F represents: L-phenylalanine
  • S represents: L-serine
  • T L-threonine
  • the salt of the peptide is selected from the group consisting of: a sulfate, a hydrochloride, a hydrobromide, a trifluoroacetate, a methanesulfonate, a naphthalenesulfonic acid, an acetate, a fumarate, a maleate, Tartrate, 2,5-dihydroxybenzoate, methanesulfonate, ethanesulfonate, besylate and p-toluenesulfonate.
  • Step (2) Preparation of Fmoc-L-Tyr(tBu)-L-Pro-2-CTC resin:
  • a capping agent is added, and the amount of the capping agent added and the weight ratio of the Fmoc-L-Pro-2-CTC resin are 5 to 25 ml/g. , draining, washing; adding a mixture of Fmoc-L-Tyr(tBu)-OH, HBTU, HOBT and N,N-diisopropylethylamine (DIEA) dissolved in the solvent of the peptide to carry out the reaction, draining, washing, Drying to obtain Fmoc-L-Tyr(tBu)-L-Pro-2-CTC resin;
  • DIEA N,N-diisopropylethylamine
  • Step (3) Preparation of Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC resin:
  • a decapping reagent is added for the reaction, dried, washed; and Fmoc-L-val- dissolved in the peptide solvent is added.
  • the mixture of OH, HBTU, HOBT and N,N-diisopropylethylamine (DIEA) is reacted, drained, washed and dried to obtain Fmoc-L-val-L-Tyr(tBu)-L-Pro-2- CTC resin;
  • a decapping reagent is added to carry out the reaction, and it is dried and washed; and Fmoc-L dissolved in the peptide solvent is added.
  • -val-OH, HBTU, HOBT and N,N-diisopropylethylamine (DIEA) mixture are reacted, drained, washed and dried to obtain Fmoc-L-val-L-val-L-Tyr(tBu) -L-Pro-2-CTC resin;
  • a decapping reagent is added to carry out a reaction to obtain L-val-L-val- L-Tyr(tBu)-L-Pro-2-CTC resin.
  • a peptide or a salt thereof selected from the group consisting of VVYP; AVLP; IGFP; LLIIVP and STAP and salts thereof.
  • liver damage is viral liver damage.
  • liver injury is an acute or chronic alcoholic liver injury.
  • transaminase is AST (aspartate aminotransferase) and also comprises ALT (alanine aminotransferase).
  • a pharmaceutical composition for preventing or treating liver damage comprising a peptide represented by the sequence Val-Val-Tyr-Pro.
  • composition according to [31] which is a tablet, a capsule, a pill, an injection, a sustained release preparation, a controlled release preparation or a microparticle delivery system.
  • liver damage is viral liver damage.
  • liver damage is an acute or chronic alcoholic liver injury.
  • a method of preventing and/or treating liver damage which comprises administering an effective amount of the peptide shown by the sequence Val-Val-Tyr-Pro to a mammal or a patient in need thereof.
  • liver damage is viral liver damage.
  • liver damage is a drug-induced liver injury.
  • liver injury is an acute or chronic alcoholic liver injury.
  • a method of lowering a transaminase comprising administering to a mammal in need or in a patient an effective amount of the peptide shown by the sequence Val-Val-Tyr-Pro.
  • a pharmaceutical composition for preventing or treating acute alcoholism comprising a peptide represented by the sequence Val-Val-Tyr-Pro.
  • the invention adopts a solid phase synthesis method, and couples the first amino acid of the C-terminal of the peptide to the resin, and then activates the carboxyl group of the second amino acid residue of the C-terminal of the peptide sequence with an activator, and is coupled to the former amino acid.
  • a peptide bond is formed on the amino group, and so on until the entire peptide of interest is synthesized.
  • the unreacted amino acid derivative starting material can be washed away with a solvent.
  • the method for preparing the peptide of the present invention or a salt thereof comprises the following steps:
  • Step A using a resin as a starting material, linking the C-terminal amino acid of the constituent peptide having an ⁇ -amino protecting group to the resin in the presence of an alkaline reagent,
  • Step B removing the protecting group with a capping reagent
  • Step C in the presence of an alkaline reagent, the respective amino acids constituting the peptide are sequentially connected in the order from the C terminal to the N terminal of the peptide to form a peptide linking resin, wherein each amino group is formed.
  • the acid linkage is carried out by first attaching an amino acid having an ⁇ -amino protecting group to the peptide, and then removing the protecting group by a capping reagent to perform capping.
  • Step D performing peptide digestion to obtain a crude product of a peptide or a salt thereof
  • Step E Purification of the crude product to obtain the peptide or a salt thereof.
  • 2-chlorotrityl chloride resin i.e., 2-CTC resin
  • 2-CTC resin 2-chlorotrityl chloride resin
  • the protective group of the ⁇ -amino group of the amino acid it may be an Fmoc group or a Boc group, and an ⁇ -amino group of each amino acid or a derivative thereof is preferably protected with an Fmoc group.
  • the amino acid of the present invention may be a side chain protected amino acid, and as a side chain protecting group, a trifluoroacetic acid such as Boc (tert-butoxycarbonyl), tBu (tert-butyl), Trt (trityl) or the like may be used.
  • TFA a releasable group, preferably Boc (tert-butoxycarbonyl).
  • step A after the amino acid is attached to the resin, an alcohol is further added to further react to block the unreacted site.
  • the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol and octanol, preferably methanol.
  • the alcohol may be added in the form of a mixture with an alkaline agent, for example, in a mixing ratio of DIEA of 1:5 to 10, preferably 1:9.
  • step A the loading rate of the amino acid can be determined using Fmoc-OSu reagent as a control.
  • a reagent for removing the Fmoc group for example, a piperidine solution such as a piperidine (PIP) solution in dimethylformamide (DMF) may be used, wherein the PIP:DMF may be 1:2 to 10 ( The volume ratio) is preferably 1:2 to 5 (volume ratio).
  • PIP piperidine
  • DMF dimethylformamide
  • the reaction temperature of the peptide may be, for example, 35 ° C or lower, preferably 10 to 35 ° C, and more preferably 10 to 25 ° C.
  • the reaction time may be 0.5 to 8 hours.
  • N,N-diisopropylethylamine DIEA
  • NMM N-methylmorpholine
  • DIEA N,N-diisopropylethylamine
  • the solvent may be selected, for example, from dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, ethyl acetate, preferably dichloromethane.
  • the peptide-cleaving reagent for cleaving the peptide from the resin for example, a solvent containing trifluoroacetic acid (TFA) such as a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water, TFA can be used.
  • TFA trifluoroacetic acid
  • TIS trifluoroacetic acid
  • TIS triisopropylsilane
  • water water
  • TFA trifluoroacetic acid
  • TFA trifluoroacetic acid
  • the ratio is 90 to 95%
  • the ratio of TIS is 0 to 5%
  • the ratio of water is 0 to 5%
  • the peptide digestion reaction can be carried out at 20 to 35 ° C for 1 to 5 hours, preferably at 25 ° C for 4 hours.
  • the side chain protecting group can be removed while the peptide blocking reaction proceeds.
  • a method of concentrating the crude peptide for example, a method of filtering the resin, drying the solvent with nitrogen, or the like can be employed.
  • a nanofilter can be employed as a method of purifying the peptide.
  • the crude product is dissolved in dilute acid, stirred, filtered, and the filtrate is purified by a nanofilter.
  • the acid is selected, for example, from sulfuric acid, hydrochloric acid, hydrobromic acid, methylalkylsulfonic acid, naphthalenesulfonic acid, acetic acid, fumaric acid, maleic acid, tartaric acid, 2,5-dihydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid. , benzenesulfonic acid and p-toluenesulfonic acid, and the like. Separation can also be carried out using a C18 column.
  • the amount of the amino acid having an ⁇ -amino protecting group is 1.5 to 6 times, preferably 4 times, the number of moles of the resin to be attached.
  • the amount of the alkaline agent to be charged is 2 to 10 times, preferably 8 times, in terms of the number of moles of the resin to be joined.
  • step C the amount of the alkaline reagent HBTU is equal to the amount of the amino acid having an ⁇ -amino protecting group in the peptide reaction for linking each amino acid.
  • the amount of HOBT charged is 1.1 to 5 times, preferably 1.1 times, of the amino acid having an ⁇ -amino protecting group in terms of moles; and the amount of DIEA is, in terms of moles, an amino acid having a protecting group. 2 to 10 times, preferably 8 times
  • reaction peptides In each of the above reaction peptides, the following method can be used to examine whether the reaction is completed, that is, a little resin is used for the detection of ninhydrin, and if it is negative, the reaction is completed.
  • the peptide compound or a salt thereof can be prepared by a simple process, low toxicity, high yield, high purity, such as VVYP; YPYP; YPVYP; VYPVYP; YPYPVYP and YPVYPVYP, wherein Y represents: L-tyramine Acid; V represents: L-valine; P represents: L-valine.
  • new peptide compounds such as VVYP; AVLP; IGFP; LLIIVP and STAP can also be provided.
  • the salt of the above peptide is selected from the group consisting of: sulfate, hydrochloride, hydrobromide, trifluoroacetate, methanesulfonate, naphthalenesulfonic acid, acetate, fumarate, maleate, Tartrate, 2,5-dihydroxybenzoate, methanesulfonate, ethanesulfonate, besylate and p-toluenesulfonate.
  • the method comprises the following steps:
  • the 2-CTC resin was taken, soaked in dry dichloromethane to fully swell the resin, and then a mixture of Fmoc-L-Pro-OH and N,N-diisopropylethylammonium (DIEA) dissolved in the peptide solvent was added.
  • the reaction was carried out at 25 ° C for 4 hours, then a mixed solvent of methanol and DIEA (9:1, volume ratio) was added to continue the reaction for 1 h, the unreacted sites were blocked, drained, washed with DMF and dichloromethane, respectively, and dried under vacuum to obtain Fmoc. -L-Pro-2-CTC resin.
  • the substitution value of the 2-CTC resin is 0.2 to 1.6 mmol/g
  • the peptide solvent is DMF
  • 2-CTC resin has a weight-volume concentration of 5 to 25 ml/g
  • the amount of the Fmoc-L-Pro-OH (molar number) is 1.5 to 6 times that of the resin;
  • the amount of DIEA charged (molar number) is 2 to 10 times that of the resin.
  • a capping reagent is added, and the reaction is carried out at 20 to 35 ° C for 5 to 60 minutes, dried, and then washed with DMF; and Fmoc dissolved in the solvent of the peptide is added.
  • the capping reagent is PIP:DMF equal to 1:2-10, volume ratio, the same as below;
  • the peptide solvent is DMF
  • the weight concentration of the Fmoc-L-Pro-2-CTC resin is 5 to 25 ml / g;
  • the dosage (molar number) of Fmoc-L-Tyr(tBu)-OH is 1.5 to 6 times that of the resin
  • the feed amount (molar number) of HBTU is equal to Fmoc-L-Tyr(tBu)-OH;
  • the feed amount (molar number) of HOBT is 1.1 to 5 times that of Fmoc-L-Tyr(tBu)-OH;
  • the amount of DIEA charged (molar number) is 2 to 10 times that of Fmoc-L-Tyr(tBu)-OH;
  • the amount of the capping agent added and the weight ratio of the Fmoc-L-Pro-2-CTC resin were 5 to 25 ml/g.
  • a decapping reagent is added, and the reaction time is 5 to 60 minutes at 20 to 35 ° C, drained, and then DMF is used.
  • the weight concentration of the Fmoc-L-Tyr(tBu)-L-Pro-2-CTC resin is 5-25 ml/g;
  • the amount of the Fmoc-L-val-OH (molar number) is 1.5 to 6 times that of the resin;
  • the amount of HBTU charged (molar number) is equal to Fmoc-L-val-OH;
  • the amount of HOBT charged (molar number) is 1.1 to 5 times that of Fmoc-L-val-OH;
  • the amount of DIEA charged (molar number) is 2 to 10 times that of Fmoc-L-val-OH;
  • the amount of the capping agent added and the weight ratio of the Fmoc-L-Tyr(tBu)-L-Pro-2-CTC resin were 5 to 25 ml/g.
  • a capping reagent is added, and the reaction is carried out at 20 to 35 ° C for 5 to 60 minutes, drained, and then used.
  • the weight concentration of the Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC resin is 5-25 ml/g;
  • the amount of the Fmoc-L-val-OH (molar number) is 1.5 to 6 times that of the resin;
  • the amount of HBTU charged (molar number) is equal to Fmoc-L-val-OH;
  • the amount of HOBT charged (molar number) is 1.1 to 5 times that of Fmoc-L-val-OH;
  • the amount of DIEA charged (molar number) is 2 to 10 times that of Fmoc-L-val-OH;
  • the amount of the capping agent added and the weight ratio of Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC resin were 5 to 25 ml/g.
  • the L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC resin obtained in the above step (5) is added to the peptide-cutting reagent;
  • the peptide-cutting reagent is TFA, triisopropylsilane (TIS) and water mixture, TFA
  • TFA triisopropylsilane
  • the ratio is 90 to 95%, the ratio of TIS is 0 to 5%, the ratio of water is 0 to 5%; the reaction is carried out at 20 to 35 ° C for 1 to 5 hours, and the solvent is distilled off under reduced pressure to obtain VVYP ⁇ after peptide digestion.
  • TFA crude TFA crude.
  • the reaction was carried out at 100 to 35 ° C for 1 to 5 hours, and the solvent was dried with nitrogen to obtain a crude product after peptide digestion.
  • the crude VVYP can be dissolved in dilute acid, stirred, filtered, and the filtrate is purified by a nanofilter.
  • the crude product can also be separated and purified by a C18 column.
  • the crude product of VVYP is dissolved in 5% dilute acetic acid, stirred for 5 hours, filtered, and the filtrate is separated and purified by a C18 column.
  • the crude product is dissolved in different dilute acids, such as sulfuric acid, hydrochloric acid, trifluoroacetic acid, hydrobromic acid, methanesulfonic acid, naphthalenesulfonic acid, acetic acid, fumaric acid, maleic acid, tartaric acid, 2,5-dihydroxybenzene
  • acids such as sulfuric acid, hydrochloric acid, trifluoroacetic acid, hydrobromic acid, methanesulfonic acid, naphthalenesulfonic acid, acetic acid, fumaric acid, maleic acid, tartaric acid, 2,5-dihydroxybenzene
  • methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or the like a pure VVYP salt can be obtained.
  • the pure product of VVYP may exist in the form of a salt, including a sulfate, a hydrochloride, a hydrobromide, a trifluoroacetate, a methanesulfonate, a naphthalenesulfonic acid, an acetate, a fumarate, a horse.
  • the present invention also established a model of acute liver injury of carbon tetrachloride, acetaminophen and alcohol, and observed the Val-Val-Tyr-Pro peptide against carbon tetrachloride, acetaminophen and alcoholic liver injury.
  • the protective effect and its mechanism of action, pharmacological experiments show that Val-Val-Tyr-Pro peptide has very good liver protection and liver protection, lower transaminase, especially lower AST level, and reduce liver tissue pathology in the above three models of liver injury. Sexual changes and the role of repairing liver damage.
  • the VVYP of the present invention can be used for the preparation of an anti-hepatocarcinoma active drug.
  • the polypeptide drug VVYP (i.e., the peptide represented by Val-Val-Tyr-Pro) can be produced according to the aforementioned method of the present invention, can also be produced according to a known production method, and is also commercially available. This compound has good water solubility.
  • One aspect of the present invention relates to the effect of the compound VVYP on liver damage (chemical liver damage) caused by chemical substances.
  • ALT serum propylaminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • CHO1 total cholesterol
  • SOD superoxide dismutase
  • GSH-Px glutathione peroxidase
  • Val-Val-Tyr-Pro peptide significantly reduced the levels of ALT, AST and total bilirubin (TBIL), and the effects of reducing ALT, AST and TBIL were significantly better than the positive drug bicyclol, while bicyclol was applied to AST and The effect of reducing TBIL levels is not obvious.
  • Val-Val -Tyr-Pro peptide protection group (8mg/kg, 16mg/kg, 32mg/kg) can significantly increase the activity of SOD and GSH-Px, and the activity of SOD recovered to 99%, 50.89% and 44.54% of the control group, respectively.
  • Px activity returned to the control group of 96.8%, 94.9% and 111.2%, respectively. Therefore, the protective mechanism of Val-Val-Tyr-Pro peptide is related to the reduction of antioxidant enzyme activity and inhibition of oxidative stress.
  • Another aspect of the invention relates to the effect of the compound VVYP of the invention on alcoholic liver injury.
  • the present inventors induced acute alcoholic liver injury by intragastric administration of 56% red star Erguotou in a mouse model of liver injury caused by alcohol, and detected serum biochemical indicators of liver function (AST, ALT, TBIL, CHO1 and TG). Changes in the protective effect of Val-Val-Tyr-Pro peptide on alcoholic liver injury.
  • Val-Val-Tyr-Pro peptide can significantly reduce the increase of AST, ALT and TG in serum of mice given alcohol group, showing good effect, in which Val-Val-Tyr-Pro peptide reduces TG effect and is equivalent to bicyclol Val-Val-Tyr-Pro peptide reduced ALT and AST levels better than bicyclol, while bicyclol reduced AST levels in mice with alcoholic liver injury without significant differences.
  • a further aspect of the invention relates to the effect of the compound VVYP of the invention on viral and/or drug-induced liver damage.
  • Val-Val-Tyr-Pro peptide can protect acetaminophen-induced liver injury in mice, significantly increasing elevated serum transaminase (ALT and AST) levels and total bilirubin (TBIL), and The Val-Val-Tyr-Pro peptide prevents a decrease in AST levels in the liver mitochondria.
  • ALT and AST serum transaminase
  • TBIL total bilirubin
  • the drug containing the compound of the present invention may be used singly or in the form of a pharmaceutical composition which is prepared as follows: a method known per se as a pharmaceutical preparation A method of mixing the compound with a pharmaceutically acceptable carrier, for example, to obtain a tablet (including a sugar-coated tablet, a suspension, an aerosol, a film, a film-coated tablet, a sublingual tablet, Decomposable tablets in the mouth, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), syrups, liquids, emulsions, pellets, nasal preparations, lung preparations (inhalation) Injectables (eg, subcutaneous, intravenous, intramuscular, intraperitoneal), intravenous drip, transdermal absorption preparations, ointments, lotions, adhesives, suppositories (eg, rectal suppositories, vaginal supposi
  • the drug containing the compound of the present invention can be administered orally or parenterally (for example, intravenously, intramuscularly, subcutaneously, intra-organally, intranasally, intradermally, drip, intracerebral, rectal, vaginal, intraperitoneal, In vivo, the tumor is administered proximally, directly to the lesion, etc.).
  • the content of the compound of the present invention varies depending on the form of the preparation, but the content of the compound of the present invention is usually from about 0.01 to 100% by weight, preferably from 0.1 to 50% by weight, based on the total weight of each preparation. More preferably, it is 0.5 to 20% by weight.
  • the dose varies depending on the route of administration, the symptoms, the age of the patient, and the like, the dose is, for example, about 0.005 to 50 mg, preferably about 0.05 to 10 mg, more preferably, per 100 kg of body weight per day for an adult patient suffering from liver damage.
  • About 0.2 to 4 mg of the compound of the present invention can be administered in 1 to 3 parts.
  • the dose thereof is according to the kind and content of the compound of the present invention, the dosage form, the duration of drug release, and the administration of the target animal (for example, a mammal such as a human, a rat, a mouse) , dogs, rabbits, etc.) are different from the purpose of administration.
  • the target animal for example, a mammal such as a human, a rat, a mouse
  • parenteral administration for example, from about 0.1 to about 100 mg of a compound of the invention is released per week.
  • Examples of the above pharmacologically acceptable carrier include excipients (e.g., starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc.), binders (e.g., starch, gum arabic, carboxymethylcellulose, hydroxypropyl) Cellulose, crystalline cellulose, alginic acid, gel, polyvinylpyrrolidone, etc.), lubricant (example For example, magnesium stearate, calcium stearate, talc, etc.), disintegrants (eg, carboxymethylcellulose calcium, talc, etc.), diluents (eg, water for injection, saline, etc.), additives (eg, stabilizers, preservatives, colorants, flavoring agents, solubilizers, emulsifiers, buffers, isotonic agents, etc.), and the like.
  • excipients e.g., starch, lactose, sucrose, calcium carbonate, calcium
  • the prophylactic or therapeutic drug of the present invention can also be used together with other drugs.
  • the administration time of the compound of the present invention and other drugs is not limited, and the compound of the present invention or a pharmaceutical composition thereof and other drugs or pharmaceutical compositions thereof may be administered to a patient simultaneously, or may be Patients are administered at different times.
  • the dose of the other drug can be determined in accordance with the number of administrations used clinically, and can be appropriately selected depending on the patient to be administered, the route of administration, the disease, the combination drug, and the like.
  • the mode of administration of other drugs is not particularly limited as long as the compound of the present invention and other drugs are combined during administration. Examples of such modes of administration include the following methods:
  • a compound of the present invention or a pharmaceutical composition thereof and other drugs are simultaneously formulated to give a single administration preparation.
  • the compound of the present invention or a pharmaceutical composition thereof and other pharmaceuticals or a pharmaceutical composition thereof are separately formulated, and two preparations are obtained, which are simultaneously administered by the same administration route.
  • the compound of the present invention or a pharmaceutical composition thereof and other pharmaceuticals or pharmaceutical compositions thereof are separately formulated, and two preparations are obtained, which are administered at different times by the same administration route.
  • the compound of the present invention or a pharmaceutical composition thereof and other drugs or a pharmaceutical composition thereof are separately formulated, and two preparations are obtained, which are simultaneously administered by different administration routes.
  • composition and other drugs or pharmaceutical compositions thereof are administered sequentially, or sequentially.
  • the mixing ratio of the compound of the present invention and other drugs can be appropriately determined in accordance with the patient to be administered, the route of administration, the disease, and the like.
  • the content of the compound of the present invention in the combination drug of the present invention varies depending on the form of the preparation, it is usually from about 0.01 to about 100% by weight, preferably from about 0.1 to about 50% by weight, more preferably from about 0.5 to about 20%, based on the entire formulation. %.
  • the content of the other drug in the combination drug of the present invention varies depending on the form of the preparation, but is usually from about 0.01 to about 100% by weight, preferably from about 0.1 to about 50% by weight, more preferably from about 0.5 to about 20% by weight, based on the entire formulation.
  • the amount of the additive (e.g., carrier or the like) in the combination drug of the present invention varies depending on the form of the preparation, but is usually from about 1 to about 99.99% by weight, preferably from about 10 to about 90% by weight based on the entire formulation.
  • the peptide reagent is DMF
  • the amino acid loading rate was determined to be 0.98 mmol/g resin. The yield was 89%.
  • VVYP The crude VVYP was dissolved in 5% acetic acid, stirred for 5 h, purified by a nanofiltration machine, and the concentrate was dried by nitrogen, and lyophilized to obtain a pure VVYP product (in the form of VVYP acetate, VVYP ⁇ CH 3 COOH, see Figure 1), the purity is 99.3% (see Figure 2). Obtained 48.3 g of pure VVYP, the yield is 82%
  • the amino acid loading rate was determined to be 1.26 mmol/g.
  • the crude AVLP was dissolved in 5% acetic acid, stirred for 5 h, filtered, and the crude AVLP was purified by a 300 Da nanofiltration membrane, and the purified product of AVLP was obtained.
  • the solvent was dried with nitrogen and lyophilized to obtain pure AVLP (as AVLP B).
  • the acid salt form, AVLP ⁇ CH 3 COOH, see Figure 3) the purity was 98.5%, and the yield of AVLP was 82.1% (see Figure 4).
  • the measurement rate of the amino acid was determined to be 1.21 mmol/g of the resin.
  • the crude IGFP was dissolved in 5% acetic acid, stirred for 5 h, filtered, and the crude IGFP was purified by a 300 Da nanofiltration membrane.
  • the purified product of IGFP was evaporated.
  • the solvent was dried with nitrogen and lyophilized to obtain pure IGFP (IGFP B)
  • the purity was 99.8% (see Figure 6)
  • the yield of IGFP was 82.6%.
  • Test animals clean grade KM mice, male, 120, 18-22g
  • Val-Val-TVr-Pro peptide 99% purity, provided by Jiangxi Bencao Tiangong Technology Co., Ltd.
  • Silibinin National Pharmaceutical Standard H20040299, Tianjin Tianshili Shengte Pharmaceutical Co., Ltd.
  • Carbon tetrachloride purchased from Tianjin Fuchen Chemical Reagent Factory
  • ALT Alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • CHO1 total cholesterol
  • TG triglyceride
  • SOD Superoxide dismutase
  • Glutathione Peroxidase (GSH-Px) assay kit was purchased from Nanjing Institute of Bioengineering
  • Kunming mice were randomly divided into control group, model group, silybin group (150 mg/kg), bicyclol group (25 mg/kg) Val-Val-Tyr-Pro peptide high dose group, Val -Val-Tyr-Pro peptide mid-dose group, Val-Val-Tyr-Pro peptide low dose group, 12 animals per group.
  • mice in the administration group were given the corresponding drug once a day in a random order, and the administration method was intragastric administration, the administration volume was 2 ml/100 g, and the model group and the blank group were given the same amount of 0.5. %Sodium carboxymethyl cellulose. Continuous administration for 5 days.
  • Liver function measurement The collected blood sample (about 600 ⁇ l) was allowed to stand at room temperature for 2 h, and the serum was separated by centrifugation at 3500 rpm for 10 min, and the activity of ALT, AST and ALP in the serum was measured by a multifunctional biochemical analyzer.
  • Liver biopsy Liver histopathology was performed to take a small piece of liver tissue from the same part of the mouse liver lobe, fixed with 10% formaldehyde solution, embedded in paraffin, sectioned (sheet thickness 5 ⁇ m), hematoxylin-eosin (HE) ) staining, and then observing liver histopathological changes under light microscope.
  • the degree of liver lesions is integrated by semi-quantitative methods and multiplied by different weights according to the importance of various lesions. Weighted numbers: hepatocyte necrosis ⁇ 3, hepatocyte water-like change ⁇ 1, congestion, bleeding ⁇ 1, inflammatory cell infiltration ⁇ 1.
  • the total score of liver lesions in each animal was calculated and then statistically processed.
  • ALT and AST in the serum of the model group was significantly higher than that of the normal control group, and there was statistical difference, indicating that the experimental model was successful.
  • High, medium and low doses of Val-Val-Tyr-Pro peptide can reduce the activity of ALT and AST in serum of mice with liver injury, which is statistically different from the model group ( * P ⁇ 0.05, ** P ⁇ 0.01)
  • the effects of high, medium and low doses of transaminase were better than those of the positive drug silybin and bicyclol group, and 25 mg/kg bicyclol did not reduce the AST effect.
  • Val-Val-Tyr-Pro peptide can reduce the increase of TBIL in mice with carbon tetrachloride injury.
  • Bicyclol can reduce the TG content in normal and model mice, while the model group and the drug group have no CHO1 content. Impact (Table 1).
  • liver tissue of the normal control group was normal, and the liver cells had no pathological changes such as degeneration and necrosis (Fig. 1).
  • the liver section of the model group showed that the hepatic cord arrangement was disordered or the cell line was narrowed. Or dissolution, focal hepatocyte degeneration and necrosis centered on the central vein, with inflammatory cell infiltration, nuclear pyknosis and other pathological changes (pathological score is 1.00 ⁇ 0.00).
  • Hepatocyte necrosis, degeneration and inflammatory cell infiltration in the mice of the positive drug and Val-Val-Tyr-Pro peptide groups were alleviated to a large extent.
  • antioxidant enzymes in the body including SOD, GSH-Px, catalase (CAT), glutathione reductase (GR), etc., which can catalyze different redox reactions, so that the body maintains relative Stable redox state.
  • SOD and GSH-Px in carbon tetrachloride modules changed significantly, which was 82.5% and 23% lower than that in normal control animals, respectively.
  • Val-Val-Tyr-Pro peptide administration (8, 16 and 32 mg/kg) significantly inhibited the decrease of SOD and GSH-Px activity, and restored SOD activity to 99%, 50.89% and 44.54% of the control group, respectively.
  • -Px activity was restored to 96.8%, 94.9% and 111.2% of the control group, respectively (Table 3).
  • the liver tissue of the mice showed severe damage, and the structure of the liver and large leaves was obviously damaged. A large number of inflammatory cells infiltrated, and most of the liver cells showed pathological changes such as turbid swelling (pathological score was 1.00). ⁇ 0.00), and serum levels of AST and ALT also increased significantly. The degree of liver cell damage in the Val-Val-Tyr-Pro peptide group was significantly reduced. The levels of ALT and AST in the high and medium dose serum of Val-Val-Tyr-Pro peptide were significantly lower than those in the CCl 4 model group.
  • Val-Val-Tyr-Pro peptide has a protective effect on the liver cell membrane and is significantly resistant to 0.1% CCl 4 induced liver damage.
  • SOD is an important antioxidant enzyme in the body. It is known that in eukaryotic cells, copper-zinc superoxide dismutase (Cu, Zn-SOD) is present in extracellular and cytosol according to the metal ions contained in the SOD active center. Manganese superoxide dismutase (Mn-SOD) is present in mitochondria, and the synthesis of Mn-SOD can be induced when oxygen radicals (O 2 -) are formed too much.
  • SOD can catalyze the reaction of oxygen radicals (O 2 -) to produce hydrogen peroxide, and hydrogen peroxide and GSH generate water under the catalysis of GSH-Px, thereby scavenging free radicals.
  • oxygen radicals O 2 -
  • Alcohol 56% purchased from Beijing Red Star Wine Co., Ltd.
  • mice 70 Kunming mice were randomly divided into blank control group, model group, positive drug silybin group (75 mg/kg), positive drug bicyclol group (25 mg/kg), Val-Val-Tyr-Pro High-dose peptide group (25 mg/kg), Val-Val-Tyr-Pro peptide middle dose group (12.5 mg/kg), Val-Val-Tyr-Pro peptide low-dose group (6.25 mg/kg), 10 mice in each group animal.
  • mice in the administration group were given the corresponding drug once in the morning, afternoon and the next morning on the first day of the random order, and the administration method was intragastric administration, and the administration volume was 2 ml/100 g.
  • Modeling of experimental animals The last administration was 8 hours. Except the normal group, each group was given a red ml Erguotou 20 ml/kg model. The normal group was given an equal volume of distilled water. After fasting for 16 hours, the eyelid vein was collected.
  • Serum liver function test The collected blood sample (about 600 ⁇ l) was allowed to stand at room temperature for 2 h, and the serum was separated by centrifugation at 3 000 rpm for 10 min.
  • the serum biosynthesis analyzer measured alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin. Levels of TBIL, cholesterol (CHO1) and triglycerides (TG).
  • Val-Val-Tyr-Pro peptide on serum ALT/AST/TBIL/CHO 1/TG in mice with acute alcoholic liver injury: Compared with the normal group, the serum levels of ALT, AST and TG in the model group increased significantly. The high, medium and low doses of Val-Val-Tyr-Pro peptide were pre-administered three times to significantly reduce the elevation of serum ALT and TG levels caused by Erguotou, and the high dose of Val-Val-Tyr-Pro peptide Lowering serum AST levels, bicyclol 25 mg/kg and silybin 75 mg/kg significantly reduced ALT levels, but the effect of lowering AST levels was not significant. The results are shown in Table 4.
  • Alcohol 56% purchased from Beijing Red Star Wine Co., Ltd.
  • mice in the administration group were given the corresponding drug once in the morning, afternoon and the next morning on the first day of the random order, and the administration method was intragastric administration, and the administration volume was 2 ml/100 g.
  • Serum and liver mitochondrial transaminase assay The collected blood samples (about 600 ⁇ l) were allowed to stand at room temperature for 2 h, and the serum was separated by centrifugation at 3 000 rpm for 10 min. The levels of ALT, AST, TBIL, CHO1 and TG in the serum were determined by a multi-functional biochemical analyzer.
  • the level of serum transaminase was significantly increased in mice after intraperitoneal injection of AP for 16 h.
  • the high, medium and low doses of Val-Val-Tyr-Pro peptide were pre-administered three times to increase the levels of ALT, AST and TBIL induced by AP.
  • Significantly reduced, bicyclol 25mg / kg can significantly reduce ALT, but the effect of reducing AST is not obvious, the results are shown in Table 5.
  • the AST level in mitochondria was significantly decreased after intraperitoneal injection of AP16h in mice, and the pre-administration of bicyclol 25mg/kg ⁇ 3 times could prevent the decrease of AST in liver mitochondria, when different doses of Val-Val-Tyr-Pro peptide were administered, and the model. Compared with the group, the AST level in the liver mitochondria was significantly increased and increased. The high level was higher than the positive drug bicyclol group, and there was no significant difference between the groups in the drug-administered group (P>0.05). The results are shown in Table 6.
  • ALT and AST levels were significantly increased after intraperitoneal injection of acetaminophen (AP) for 16 h, while the high doses of medium- and low-dose Val-Val-Tyr-Pro peptide were administered to the serum three times.
  • the levels of ALT, AST and TBIL decreased, and remained at a normal level.
  • the mechanism of AP toxicity was mainly caused by the conversion of AP to semi-quinone free radicals (NAPQI). Because mitochondrial oxidative respiration was inhibited by various terpenoids, AP The semi-purine free radicals produced by metabolism can affect the function of liver mitochondria and cause damage to mitochondria.
  • Val-Val-Tyr-Pro peptide has obvious protective effect on AP-induced liver injury, which is marked by a significant decrease in serum AST transaminase.
  • Val The -Val-Tyr-Pro peptide can significantly prevent the decrease of AST in the liver mitochondria caused by AP and prevent the leakage of AST in the mitochondria.
  • Val-Val-Tyr-Pro peptide can not only reduce the damage of whole liver cells caused by AP, but also prevent the damage of mitochondria structure and function through the protection of mitochondrial membrane, thus preventing AST and other enzymes in mitochondria. Release.
  • the invention can provide a preparation method of a peptide compound with simple process, low toxicity, high yield and high purity, and further, by using the preparation method of the invention, a new peptide compound, such as VVYP; AVLP; IGFP; LLIIVP and STAP and their salts.
  • a new peptide compound such as VVYP; AVLP; IGFP; LLIIVP and STAP and their salts.
  • the polypeptide compound VVYP has an excellent function of protecting liver and protecting liver, reducing transaminase, especially lowering AST level, reducing pathological changes of liver tissue and repairing liver damage.
  • the effect of the liver disease such as chemical, viral, drug, or alcoholic (such as acute and chronic) liver damage, as well as the prevention and treatment of acute alcoholism has an excellent effect.

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Abstract

提供了肽或其盐及其制备方法，该肽可选自VVYP、AVLP、IGFP、LLIIVP和STAP，该制备方法包括如下步骤：以树脂作为起始原料，将具有保护基团的氨基酸连接至树脂，然后脱去保护基团，依次这样连接各个氨基酸，然后进行切肽和脱侧链保护基团得到粗品，经过纯化后得到所需的肽或其盐。还提供了VVYP肽在制备预防或治疗肝损伤或急性酒精中毒的药物中以及降低转氨酶的药物中的用途。

Description

肽或其盐、其制备方法、及其制备预防和/或治疗肝损伤药物的用途 技术领域
本发明涉及肽或其盐及其制备方法，以及其制备预防和/或治疗肝损伤的药物的用途。
背景技术
肽是由多个氨基酸分子脱水缩合而成的化合物。作为多肽类药物已知有多种，例如，VVYP，是由氨基酸L-Val，L-Tyr和L-Pro按照L-val-L-val-L-Tyr-L-Pro的序列构成的四肽化合物，其最早是由日本的香川恭一教授利用酶解的方法制备的一种猪血蛋白混合物中的主要成分，见lifescience，1996，58(20)：1745-1755，该猪蛋白混合物和VVYP具有降低胆固醇和血脂的作用。作为多肽类药物的制备方法，已知有生物合成和人工合成两种。例如，美国专利(US9233999B2)采用了一种重组蛋白的方法制备了VVYP。此外，美国专利(US5958885)中公开了一种VVYP的合成方法，但是该方法存在明显的缺陷，每一步都必须使用三氟乙酸，导致生产成本很高；三废比较多，使用氟化氢作为切割试剂将肽类从树脂上切割下来，氟化氢是剧毒的酸。
此外，Val-Val-Tyr-Pro肽(即，VVYP)据报道具有抑制血中甘油三酯浓度作用，可预防或治疗人或动物的肥胖症、高血脂症及其并发的高血压症和动脉硬化等循环器官类疾病(特开平9-255698)。此外，Val-Val-Tyr-Pro肽具有降低处于高血糖状态的受试者血糖水平的作用，可用于预防或治疗由高血糖引起的疾病(特表2008-519758)。除此之外，Val-Val-Tyr-Pro肽具有抑制处于高胆固醇状态或该前阶段的患者的胆固醇再上升的作用，可用于预防或治疗由于胆固醇再上升导致的高胆固醇状态引起的病态或疾患，并且Val-Val-Tyr-Pro肽具有降血压作用(2007-137816)。在中国专利文献中也有报道，Val-Val-Tyr-Pro肽具有抗紧张、不安、应急力低下等功能作用(CN101633945A)。
近年来，病毒感染、药物、化学毒物(如环境中的化学污染物等)、酗酒等因素引起的肝损伤，正越来越严重地危害人们的身体健康。肝损伤会 诱发肝炎、肝硬化甚至肝癌，严重影响生活质量和健康水平。中国是世界公认的肝炎大国，约有1.3亿的肝炎病毒携带者。中国经济快速发展带来的环境污染，饮酒消费的不断增大，药物不合理使用和滥用，由此产生的化学性、酒精性和药物性肝损伤在我国有逐年增加的趋势。因此治疗肝损伤药物有着巨大的市场需求。
肝损伤通常会引起血清谷丙转氨酶(ALT)和谷草转氨酶(AST)的升高，降低转氨酶水平是缓解和治疗肝损伤，修复肝功能的重要手段。ALT以肝脏细胞含量最高，主要分布于细胞浆水溶性部分，少量存在于线粒体中，AST主要分布于心肌，其次为肝脏、骨骼肌和肾脏等组织中，在肝脏细胞中约有80％以上存在于线粒体，在轻、中度肝损伤时，以ALT升高为明显，ALT升高远大于AST升高，当严重肝细胞损伤时，线粒体受损，可导致线粒体内的酶被释放入血，此时以AST升高更明显，血清中AST/ALT比值升高。由酒精和药物代谢过程中产生的自由基可损伤线粒体膜脂质，导致线粒体AST外泄。
目前降低转氨酶的临床一线用药双环醇，是联苯双酯结构类似物，具有很好的保肝作用和一定的抗乙肝病毒活性，它对四氯化碳(CCl4)、D-氨基半乳糖和对乙酰氨基酚引起的肝损伤以及卡介苗加脂多糖引起的小鼠免疫性肝炎均有降低转氨酶的作用。但是临床应用发现，双环醇对ALT水平的降低作用远大于AST，患者服用双环醇后，ALT水平显著降低，AST水平不降甚至出现升高的现象，使得谷丙转氨酶/谷草转氨酶的比值升高。经过大量临床应用之后，很多医生仅将双环醇用于轻度肝损伤患者，主要治疗ALT水平的升高。双环醇的水溶性非常差，口服生物利用度低且波动大，不能制成注射剂，病毒株产生耐药现象等。主要降低ALT水平和差的水溶性限制了双环醇的临床使用，临床上针对保护肝线粒体从而降低AST水平的药物有待开发。目前用于治疗重度肝损伤的药物，较常用的有静脉给药的甘利欣、甘草酸制剂，细胞膜保护剂多稀磷脂酰胆碱等，这些化学药物有一定的副作用，且远期治疗效果差，此外，重度肝损伤时，肝脏对许多药物的清除能力下降，使用药物过多易造成肝损伤，因此寻找更好降酶效果且毒性低的肝修复药物迫在眉睫。
但现有技术中没有关于Val-Val-Tyr-Pro肽对预防和治疗肝损伤保护作用中的记载。
发明内容
为了克服上述现有技术中的问题，本发明人进行了长期锐意地研究，发现以树脂作为起始原料，将具有保护基团的氨基酸连接至树脂，然后脱去保护基团，依次连接各个氨基酸，然后进行切肽和脱侧链保护基团得到粗品，经过纯化后得到所需的肽或其盐，可以实现提供一种工艺简单、毒性低、产率高、纯度高的肽类化合物的制备方法，进一步地，利用本发明的制备方法，还可以提供新的肽化合物，例如VVYP；AVLP；IGFP；LLIIVP和STAP以及它们的盐。
此外，本发明人还发现，多肽类化合物VVYP(序列Val-Val-Tyr-Pro表示的肽)具有优异的保肝护肝的作用，降低转氨酶尤其是降低AST水平，减轻肝脏组织病理性改变和修复肝损伤的作用，对于肝损伤，例如化学性、病毒性、药物性、或酒精性(例如急、慢性)肝损伤、以及急性酒精性中毒的预防和治疗具有优异的效果。
本发明人基于上述研究完成了本发明。
本发明涉及以下方面。
[1].肽或其盐的制备方法，该方法包括：
步骤A：以树脂作为起始原料，在碱性试剂的存在下，将具有α-氨基保护基团的构成肽的C端的氨基酸连接至树脂，
步骤B：用脱帽试剂将所述保护基团脱去，
步骤C：在碱性试剂的存在下，按照肽的从C端到N端的顺序依次连接组成肽的各个氨基酸进行反应，从而形成肽连接树脂，其中，每个氨基酸的连接都是先连接具有α-氨基保护基团的氨基酸进行接肽，然后用脱帽试剂将所述保护基团脱去进行脱帽，
步骤D：进行切肽，得到肽或其盐的粗品，
步骤E：纯化粗品，得到所述的肽或其盐。
[2].上述[1]的制备方法，其中，步骤A中的所述树脂为2-氯三苯甲基氯树脂，
[3]上述[1]-[2]中任一项的制备方法，其中，步骤A中的碱性试剂选自：N，N-二异丙基乙氨(DIEA)和N-甲基吗啉(NMM)；
[4]上述[1]-[3]中任一项的制备方法，其中，步骤A中，在将氨基酸连接至树脂后，还包括加入醇进一步反应。
[5]上述[1]-[4]的制备方法，其中，所述醇选自，甲醇、乙醇、丙醇、丁醇、戊醇、己醇、庚醇和辛醇。
[6]上述[1]-[5]的制备方法，其中，步骤C中的碱性试剂选自：N，N-二异丙基乙氨(DIEA)和N-甲基吗啉(NMM)。；步骤C中偶联试剂选自O-苯并三氮唑-N，N，N′，N′-四甲基脲四氟硼酸(TBTU)，O-苯并三氮唑-四甲基脲六氟磷酸盐(HBTU)、1-羟基苯并三唑(HOBT)、六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop)。
[7].上述[1]-[6]中任一项的制备方法，其中，所述各步骤中接肽反应的温度是10～35℃，反应时间是0.5～8小时，
[8].上述[1]-[7]中任一项的制备方法，其中，脱帽反应的温度是20～35℃，反应时间是5～60分钟；
[9].上述[1]-[8]中任一项的制备方法，其中，所述各步骤中切肽试剂是三氟乙酸(TFA)、三异丙基硅烷(TIS)和水的混合物，TFA的比例是90～95％，TIS的比例是0～5％，水的比例是0～5％，在20～35℃反应1～5小时。
[10].上述[1]-[9]中任一项的制备方法，其中，所述步骤E中的纯化是利用纳滤机进行的。
[11].上述[1]-[10]中任一项的制备方法，其中，所述步骤D中利用氮气吹干的方法得到了浓缩的粗品。
[12].上述[1]-[11]中任一项的制备方法，其中，
在步骤A和步骤C中，具有α-氨基保护基团的氨基酸的投料量为，以摩尔数计，是所连接的树脂的1.5～6倍；
[13].上述[1]-[12]中任一项的制备方法，其中，
在步骤A中，碱性试剂的投料量为，以摩尔数计，是所连接的树脂的2～10倍；
[14].上述[1]-[13]中任一项的制备方法，其中，
在步骤C中，在连接每个氨基酸的接肽反应中，偶联试剂HBTU的投料量与具有α-氨基保护基团的氨基酸的量相等；HOBT的投料量为，以摩尔数计，是具有α-氨基保护基团的氨基酸的1.1～5倍；DIEA的投料量为，以摩尔数计，是具有α-氨基保护基团的氨基酸的2～10倍
[15].上述[1]-[14]中任一项的制备方法，其中，
在步骤A-E中，氨基酸的保护基团均为Fmoc；接肽试剂为：选自二氯甲烷，N，N-二甲基甲酰胺(DMF)，N，N-二甲基乙酰胺，四氢呋喃和乙酸乙酯中的一种或更多种。
[16].上述[1]-[15]中任一项的制备方法，其中，
脱帽试剂为哌啶(PIP)∶DMF等于1∶2～10(体积比)；脱帽试剂的加入量和待脱帽的树脂的重量比例是5～25ml脱帽试剂/g树脂。
[17].上述[1]-[16]中任一项的制备方法，其中，
在步骤E中，是将粗品溶解在稀酸溶液中进行分离纯化。
[18].上述[1]-[17]中任一项的制备方法，其中，
其中，所述酸选自硫酸、盐酸、氢溴酸、甲烷基磺酸、萘磺、醋酸、富马酸、马来酸、酒石酸、2，5-二羟基苯甲酸、甲磺酸、乙磺酸、苯磺酸和对甲苯磺酸。
[19].上述[1]-[18]中任一项的制备方法，其中，所述肽选自：
VVYP；AVLP；IGFP；LLIIVP和STAP，
其中
Y代表：L-酪氨酸；
V代表：L-缬氨酸；
P代表：L-脯氨酸；
A代表：L-丙氨酸；
L代表：L-亮氨酸；
I代表：L-异亮氨酸；
G代表：L-甘氨酸；
F代表：L-苯丙氨酸；
S代表：L-丝氨酸；
T代表：L-苏氨酸；
所述肽的盐选自：硫酸盐、盐酸盐、氢溴酸盐、三氟乙酸盐、甲烷基磺酸盐、萘磺酸、乙酸盐、富马酸盐、马来酸盐、酒石酸盐、2，5-二羟基苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐和对甲苯磺酸盐。
[20].上述[1]-[19]中任一项的制备方法，其中，所述用于接肽的氨基酸是侧链被保护基团保护的氨基酸，所述的侧链保护基团选自：Boc(叔丁 氧羰基)，tBu(叔丁基)，Trt(三苯甲基)，且在步骤D中同步进行切肽和脱侧链保护基团。
[21].上述[1]-[20]中任一项的制备方法，其中，所述肽为VVYP，该方法包括：
步骤(1)：制备Fmoc-L-Pro-2-CTC树脂：
取2-CTC树脂，用干燥的二氯甲烷浸泡，然后加入用接肽溶剂溶解的Fmoc-L-Pro-OH和N，N-二异丙基乙氨(DIEA)的混合物进行反应，加入醇继续反应，抽干，洗涤，干燥，获得Fmoc-L-Pro-2-CTC树脂；
步骤(2)：制备Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂：
在步骤(1)获得的Fmoc-L-Pro-2-CTC树脂中，加入脱帽试剂，脱帽试剂的加入量和Fmoc-L-Pro-2-CTC树脂的重量比例是5～25ml/g，反应，抽干，洗涤；加入用接肽溶剂溶解的Fmoc-L-Tyr(tBu)-OH、HBTU、HOBT和N，N-二异丙基乙氨(DIEA)混合物进行反应，抽干，洗涤，干燥，获得Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂；其中，
步骤(3)：制备Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
在步骤(2)中获得的Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂进行反应，抽干，洗涤；加入接肽溶剂溶解的Fmoc-L-val-OH、HBTU、HOBT和N，N-二异丙基乙氨(DIEA)混合物进行反应，抽干，洗涤，干燥，获得Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂；
步骤(4)：制备Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
在步骤(3)获得的Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂进行反应，抽干，洗涤；加入接肽溶剂溶解的Fmoc-L-val-OH、HBTU、HOBT和N，N-二异丙基乙氨(DIEA)混合物进行反应，抽干，洗涤，干燥，获得Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂；
步骤(5)：制备L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂
在步骤(4)获得中的Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂进行反应，获得L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂。
[22].根据上述[1]-[20]中任一项的方法制备得到的肽或其盐。
[23].一种肽或其盐，其选自下列：VVYP；AVLP；IGFP；LLIIVP和STAP以及它们的盐。
[24].序列Val-Val-Tyr-Pro所示的肽在制备预防或治疗肝损伤的药物中的用途。
[25].项[24]所述的用途，其中，肝损伤是病毒性肝损伤。
[26].项[24]所述的用途，其中，肝损伤是药物性肝损伤。
[27].项[24]所述的用途，其中，肝损伤是急、慢性酒精性肝损伤。
[28].序列Val-Val-Tyr-Pro所示的肽在制备预防或治疗急性酒精中毒的药物中的用途。
[29].序列Val-Val-Tyr-Pro所示的肽在制备降低转氨酶的药物中的用途。
[30].项[29]的用途，其中，转氨酶是AST(谷草转氨酶)，也包含ALT(谷丙转氨酶)。
[31].用于预防或治疗肝损伤的药物组合物，其包含序列Val-Val-Tyr-Pro所示的肽。
[32].项[31]的药物组合物，其是片剂、胶囊、丸剂、注射剂、缓释制剂、控释制剂或微粒给药系统。
[33]序列Val-Val-Tyr-Pro所示的肽，其用于预防或治疗肝损伤。
[34].项[33]所述的肽，其中，肝损伤是病毒性肝损伤。
[35].项[33]所述的肽，其中，肝损伤是药物性肝损伤。
[36].项[33]所述的肽，其中，肝损伤是急、慢性酒精性肝损伤。
[37].肝损伤的预防和/或治疗方法，该方法包括向需要的哺乳动物中或患者中给于有效量的序列Val-Val-Tyr-Pro所示的肽。
[38].项[37]所述的方法，其中，肝损伤是病毒性肝损伤。
[39].项[37]所述的方法，其中，肝损伤是药物性肝损伤。
[40].项[37]所述的方法，其中，肝损伤是急、慢性酒精性肝损伤。
[41].序列Val-Val-Tyr-Pro所示的肽，其用于降低转氨酶，特别是降低AST(谷草转氨酶)。
[42].降低转氨酶的方法，该方法包括向需要的哺乳动物中或患者中给于有效量的序列Val-Val-Tyr-Pro所示的肽。
[43].用于预防或治疗急性酒精中毒的药物组合物，其包含序列Val-Val-Tyr-Pro所示的肽。
[44].序列Val-Val-Tyr-Pro所示的肽在制备抑制SOD和/或GSH-Px活性的药物中的用途。
[45].序列Val-Val-Tyr-Pro所示的肽在制备抗氧化的药物中的用途。
[46].序列Val-Val-Tyr-Pro所示的肽在制备抑制氧化应激损伤的药物中的用途。
[47].序列Val-Val-Tyr-Pro所示的肽在制备抗肝癌药物中的用途。
[48].序列Val-Val-Tyr-Pro所示的肽在制备预防和/或治疗化学性肝损伤的药物中的用途。
附图说明
图1：VVYP乙酸盐的13C-NMR图
图2：VVYP乙酸盐的HPLC图
图3：AVLP乙酸盐的MS图
图4：AVLP乙酸盐的HPLC图
图5：IGFP乙酸盐的MS图
图6：IGFP乙酸盐的HPLC图
图7：Val-Val-Tyr-Pro肽对0.1％CCl4致急性肝损伤小鼠肝脏病理改变的影响
具体实施方式
本发明采用固相合成法，把目的肽C端第一个氨基酸偶联在树脂上，然后以活化剂活化肽序列C-末端第二个氨基酸残基的羧基，并偶联于前一个氨基酸的氨基上形成肽键，依次类推直到合成整个目的肽。未反应的氨基酸衍生物原料可以用溶剂洗掉。
本发明的肽或其盐的制备方法包括以下步骤：
步骤A：以树脂作为起始原料，在碱性试剂的存在下，将具有α-氨基保护基团的构成肽的C端的氨基酸连接至树脂，
步骤B：用脱帽试剂将所述保护基团脱去，
步骤C：在碱性试剂的存在下，按照肽的从C端到N端的顺序依次连接组成肽的各个氨基酸进行反应，从而形成肽连接树脂，其中，每个氨基 酸的连接都是先连接具有α-氨基保护基团的氨基酸进行接肽，然后用脱帽试剂将所述保护基团脱去进行脱帽，
步骤D：进行切肽，得到肽或其盐的粗品，
步骤E：纯化粗品，得到所述的肽或其盐。
作为树脂，可以采用例如2-氯三苯甲基氯树脂(即，2-CTC树脂)。
作为氨基酸的α-氨基的保护基团，可以是Fmoc基团或Boc基团，优选以Fmoc基团保护每一个氨基酸或其衍生物的α-氨基。
本发明的氨基酸可以是侧链被保护的氨基酸，作为侧链保护基团可以采用如Boc(叔丁氧羰基)，tBu(叔丁基)，Trt(三苯甲基)等采用三氟乙酸(TFA)可脱去的基团，优选Boc(叔丁氧羰基)。
在步骤A中，在将氨基酸连接至树脂后，还包括加入醇进一步反应，封闭未反应的位点。所述醇选自，甲醇、乙醇、丙醇、丁醇、戊醇、己醇、庚醇和辛醇，优选甲醇。所述醇可以是以与碱性试剂的混合物的形式加入，例如与DIEA的混合比例为1∶5～10，优选1∶9
在步骤A中，可以采用Fmoc-OSu试剂作为对照，测定氨基酸的上载率。
作为脱帽试剂，例如脱去Fmoc基团的试剂，可以采用哌啶溶液，例如，哌啶(PIP)的二甲基甲酰胺(DMF)溶液，其中，PIP∶DMF可以为1∶2～10(体积比)，优选1∶2～5(体积比)。
作为接肽反应温度，可以为例如35℃以下，优选10～35℃，进一步优选10～25℃。作为反应时间，可以为0.5～8h。
作为碱性试剂，可以选自例如N，N-二异丙基乙胺(DIEA)、N-甲基吗啉(NMM)，优选N，N-二异丙基乙胺(DIEA)。
作为溶剂，可以选自例如二氯甲烷、N，N-二甲基甲酰胺、N，N-二甲基乙酰胺、四氢呋喃、乙酸乙酯，优选二氯甲烷。
作为用于将肽从树脂上切割下来的切肽试剂，可采用例如包含三氟乙酸(TFA)的溶剂，例如三氟乙酸(TFA)、三异丙基硅烷(TIS)和水的混合物，TFA的比例是90～95％，TIS的比例是0～5％，水的比例是0～5％，切肽反应可以在20～35℃反应1～5小时，优选在25℃，反应4小时。
当氨基酸的侧链存在保护基团时，可以在切肽反应进行的同时，将所述侧链保护基团脱去。
作为浓缩肽粗品的方法，可以采用例如过滤树脂，用氮气吹干溶剂的方法等。
作为肽的纯化方法，可以采用例如纳滤机。例如，将粗品溶解在稀酸中，搅拌，过滤，滤液经过纳滤机纯化。所述酸选自例如硫酸、盐酸、氢溴酸、甲烷基磺酸、萘磺、醋酸、富马酸、马来酸、酒石酸、2，5-二羟基苯甲酸、甲磺酸、乙磺酸、苯磺酸和对甲苯磺酸等。也可以采用C18柱进行分离。
具有α-氨基保护基团的氨基酸的投料量为，以摩尔数计，是所连接的树脂的1.5～6倍，优选4倍。
在步骤A中，碱性试剂的投料量为，以摩尔数计，是所连接的树脂的2～10倍，优选8倍。
在步骤C中，在连接每个氨基酸的接肽反应中，碱性试剂HBTU的投料量与具有α-氨基保护基团的氨基酸的量相等。
HOBT的投料量为，以摩尔数计，是具有α-氨基保护基团的氨基酸的1.1～5倍，优选1.1倍；DIEA的投料量为，以摩尔数计，是具有保护基团的氨基酸的2～10倍，优选8倍
在上述各反应接肽中，可以采用以下方式来考察反应是否完成，即，取少许树脂做茚三酮检测，呈阴性，则表示反应完成。
根据本发明的制造方法，可以工艺简单、毒性低、产率高、纯度高的制备肽类化合物或其盐，例如VVYP；YPYP；YPVYP；VYPVYP；YPYPVYP和YPVYPVYP，其中Y代表：L-酪氨酸；V代表：L-缬氨酸；P代表：L-脯氨酸。
进一步地，利用本发明的制备方法，还可以提供新的肽化合物，例如VVYP；AVLP；IGFP；LLIIVP和STAP。
上述的肽的盐选自：硫酸盐、盐酸盐、氢溴酸盐、三氟乙酸盐、甲烷基磺酸盐、萘磺酸、乙酸盐、富马酸盐、马来酸盐、酒石酸盐、2，5-二羟基苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐和对甲苯磺酸盐。
以下，以VVYP为例，对本发明的制备方法进行说明。
作为本发明的VVYP的制备方法的一个实施方式，包括如下步骤：
(1)制备Fmoc-L-Pro-2-CTC树脂：
取2-CTC树脂，用干燥的二氯甲烷浸泡，使树脂充分溶胀，然后加入用接肽溶剂溶解的Fmoc-L-Pro-OH和N，N-二异丙基乙氨(DIEA)混合物，25℃反应4小时，然后加入甲醇和DIEA的混合溶剂(9∶1，体积比)继续反应1h，封闭未反应的位点，抽干，分别用DMF和二氯甲烷洗涤，真空干燥，获得Fmoc-L-Pro-2-CTC树脂。
所述的2-CTC树脂取代值为0.2～1.6mmol/g；
所述的接肽溶剂是DMF；
2-CTC树脂的重量体积浓度是5～25ml/g；
Fmoc-L-Pro-OH的投料量(摩尔数)是树脂的1.5～6倍；
DIEA的投料量(摩尔数)是树脂的2～10倍。
(2)制备Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂：
在步骤(1)中的Fmoc-L-Pro-2-CTC树脂中，加入脱帽试剂，在20～35℃反应5～60分钟，抽干，然后用DMF洗涤；加入用接肽溶剂溶解的Fmoc-L-Tyr(tBu)-OH、HBTU、HOBT和N，N-二异丙基乙氨(DIEA)混合物，20～35℃反应1～5小时，取少许树脂做茚三酮检测，呈阴性，抽干，然后用DMF和二氯甲烷洗涤，真空干燥，获得Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂；
所述的脱帽试剂是PIP∶DMF等于1∶2～10，体积比，下同；
接肽溶剂是DMF；
Fmoc-L-Pro-2-CTC树脂的重量体积浓度是5～25ml/g；
Fmoc-L-Tyr(tBu)-OH的投料量(摩尔数)是树脂的1.5～6倍；
HBTU的投料量(摩尔数)和Fmoc-L-Tyr(tBu)-OH相等；
HOBT的投料量(摩尔数)是Fmoc-L-Tyr(tBu)-OH的1.1～5倍；
DIEA的投料量(摩尔数)是Fmoc-L-Tyr(tBu)-OH的2～10倍；
脱帽试剂的加入量和Fmoc-L-Pro-2-CTC树脂的重量比例是5～25ml/g。
(3)制备Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
在步骤(2)中的Fmoc-L-Tyr(tBu)-L-Pro-2-CTC resin树脂中，加入脱帽试剂，在20～35℃反应时间是5～60分钟，抽干，然后用DMF洗涤；加入接肽溶剂溶解的Fmoc-L-val-OH、HBTU、HOBT和N，N-二异丙基乙氨(DIEA)混合物，20～35℃反应1～5小时，取少许树脂做茚三酮检测，呈 阴性，抽干，然后用DMF和二氯甲烷洗涤，真空干燥，获得Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂；
Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂的重量体积浓度是5～25ml/g；
Fmoc-L-val-OH的投料量(摩尔数)是树脂的1.5～6倍；
HBTU的投料量(摩尔数)和Fmoc-L-val-OH相等；
HOBT的投料量(摩尔数)是Fmoc-L-val-OH的1.1～5倍；
DIEA的投料量(摩尔数)是Fmoc-L-val-OH的2～10倍；
脱帽试剂的加入量和Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂的重量比例是5～25ml/g。
(4)制备Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
在步骤(3)中的Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂，在20～35℃反应5～60分钟，抽干，然后用DMF洗涤；加入接肽溶剂溶解的Fmoc-L-val-OH、HBTU、HOBT和N，N-二异丙基乙氨(DIEA)混合物，20～35℃反应1～5小时，取少许树脂做茚三酮检测，呈阴性，抽干，然后用DMF和二氯甲烷洗涤，真空干燥，获得Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂；
Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂的重量体积浓度是5～25ml/g；
Fmoc-L-val-OH的投料量(摩尔数)是树脂的1.5～6倍；
HBTU的投料量(摩尔数)和Fmoc-L-val-OH相等；
HOBT的投料量(摩尔数)是Fmoc-L-val-OH的1.1～5倍；
DIEA的投料量(摩尔数)是Fmoc-L-val-OH的2～10倍；
脱帽试剂的加入量和Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂的重量比例是5～25ml/g。
(5)制备制备L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂
在步骤(4)中的Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂，在20～35℃反应5～60分钟，抽干，然后用DMF洗涤，真空干燥，获得L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂。
(6)切肽
将上述步骤(5)所获得的L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂加入到切肽试剂中；切肽试剂是TFA、三异丙基硅烷(TIS)和水的混合物，TFA 的比例是90～95％，TIS的比例是0～5％，水的比例是0～5％；在20～35℃反应1～5小时，减压蒸去溶剂，得到切肽后的VVYP·TFA粗品。
(7)浓缩和纯化
在100～35℃反应1～5小时，用氮气吹干溶剂，得到切肽后的粗品。
可以将VVYP的粗品溶解在稀酸中，搅拌，过滤，滤液经过纳滤机纯化。
还可以将粗品经过C18柱分离纯化，例如将VVYP的粗品溶解在5％稀醋酸中，搅拌5小时，过滤，滤液经过C18柱分离纯化。
粗品如果溶解在不同的稀酸中，如硫酸、盐酸、三氟乙酸、氢溴酸、甲烷基磺酸、萘磺、醋酸、富马酸、马来酸、酒石酸、2，5-二羟基苯甲酸、甲磺酸、乙磺酸、苯磺酸和对甲苯磺酸等中，可以得到不同的VVYP盐的纯品。
VVYP的纯品可以以盐的形式存在，包括硫酸盐、盐酸盐、氢溴酸盐、三氟乙酸盐、甲烷基磺酸盐、萘磺酸、乙酸盐、富马酸盐、马来酸盐、酒石酸盐、2，5-二羟基苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐和对甲苯磺酸盐等。
此外，本发明还建立了四氯化碳、对乙酰氨基酚和酒精性的急性肝损伤模型，观察Val-Val-Tyr-Pro肽对四氯化碳、对乙酰氨基酚和酒精性肝损伤的保护作用及其作用机理，药理实验结果表明，在上述三种肝损伤模型中，Val-Val-Tyr-Pro肽具有非常好的保肝护肝，降低转氨酶尤其是降低AST水平，减轻肝脏组织病理性改变和修复肝损伤的作用。本发明的VVYP可以用于制备抗肝癌活性药物。
多肽类药物VVYP(即，Val-Val-Tyr-Pro所示的肽)可以按照本发明的前述方法制备，也可以按照已知的制备方法制备得到，还可以商业购买得到。该化合物具有很好的水溶性。
本发明的一个方面涉及化合物VVYP对于化学性物质引起的肝损伤(化学性肝损伤)肝损伤的作用。
在0.1％CCl4引起的小鼠肝损伤模型中，分别测定血清中丙氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)，总胆红素(TBIL)，总胆固醇(CHO1)和甘油三酯(TG)的水平，并处死动物，剖腹取肝，制备肝匀浆。检测肝匀浆中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)水平，同时对肝 组织进行病理学检查。
实验结果表明Val-Val-Tyr-Pro肽能显著降低ALT、AST和总胆红素(TBIL)的水平，降低ALT、AST和TBIL效果均明显优于阳性药双环醇，而双环醇对AST和TBIL水平降低效果不明显。
此外，CCl4模型组中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力显著降低，与正常对照组动物相比分别降低82.5％和23％，Val-Val-Tyr-Pro肽保护组(8mg/kg，16mg/kg，32mg/kg)能明显升高SOD和GSH-Px活力，SOD活性分别恢复至对照组的99％，50.89％和44.54％，GSH-Px活性分别恢复至对照组96.8％，94.9％和111.2％因此，Val-Val-Tyr-Pro肽的保护作用机制与减轻提高抗氧化酶活性，从而抑制氧化应激有关。
肝组织病理切片进一步显示，Val-Val-Tyr-Pro肽(32mg/kg，16mg/kg，8mg/kg)剂量依赖性减轻肝脏组织病理性改变，病理积分明显降低，分别降低至正常的34％，49％和45％。
本发明的另一个方面涉及本发明化合物VVYP对于酒精性肝损伤的作用。
众所周知，我国近年来由酒精所导致的急性肝损伤呈逐年上升趋势，酒精已经成为继病毒性肝炎之后的第二大导致肝损伤的病因。
本发明人在酒精引起的肝损伤小鼠模型中，通过用56％的红星二锅头灌胃小鼠造成急性酒精性肝损伤模型，通过检测血清肝功能生化指标(AST、ALT、TBIL、CHO1及TG)的变化评价Val-Val-Tyr-Pro肽对酒精性肝损伤的保护作用。
Val-Val-Tyr-Pro肽能显著降低动物给予酒精组小鼠血清中AST、ALT及TG的升高，显示了良好的作用，其中Val-Val-Tyr-Pro肽降低TG效果与双环醇相当，Val-Val-Tyr-Pro肽降低ALT和AST水平优于双环醇，而双环醇降低酒精性肝损伤小鼠AST水平无显著性差异。
本发明的又一个方面涉及本发明化合物VVYP对于病毒性和/或药物性肝损伤的作用。
在对乙酰氨基酚诱发小鼠急性肝损伤模型中，以双环醇(25mg/kg)作为阳性对照，用不同剂量(6.25mg/kg、12.5mg/kg、25mg/kg)的Val-Val-Tyr-Pro肽给药3次，每天2次，第二天下午用对乙酰氨基酚(140mg/kg)腹腔注射诱发小鼠急性肝损伤，16h后测定血清肝功能和肝匀 浆线粒体中谷草转氨酶(AST)，判定Val-Val-Tyr-Pro肽对对乙酰氨基酚诱发肝损伤的保护作用。
Val-Val-Tyr-Pro肽各剂量灌胃均能保护对乙酰氨基酚引起的小鼠肝损伤，使升高的血清转氨酶(ALT和AST)水平和总胆红素(TBIL)显著降低，并且Val-Val-Tyr-Pro肽能防止肝线粒体中AST水平的降低。
含有本发明的化合物(Val-Val-Tyr-Pro肽)的药物可以单独使用，或以药物组合物形式使用，这种药物组合物是如下制备的：按照本身已知的方法作为药物制剂的制备方法，将所述化合物和药理学可接受的载体混合，例如，得到片剂(包括糖包衣片剂，混悬剂，气雾剂，膜剂，膜包衣片剂，舌下片剂，口中可分解的片剂，口含片等等)，丸剂，粉剂，颗粒剂，胶囊剂(包括软胶囊，微囊)，糖浆剂，液剂，乳剂，小丸剂，鼻制剂，肺制剂(吸入剂)，注射剂(例如，皮下注射，静脉注射，肌内注射，腹膜内注射)，静脉滴注，透皮吸收制剂，软膏剂，洗剂，粘附剂，栓剂(例如，直肠栓剂，阴道栓剂)，滴眼剂，控制释放制剂(例如，瞬时释放制剂，缓释制剂，缓释微囊)，等等。
含有本发明的化合物的药物可以安全地口服或胃肠外给药(例如，静脉内，肌内，皮下，器官内，鼻内，皮内，滴注，脑内，直肠，阴道，腹膜内，肿瘤体内，肿瘤近端给药，直接给药到病变，等等)。
在本发明的制剂中，本发明化合物的含量根据制剂形式而不同，但相对于每个制剂的总重量，作为本发明化合物的含量通常约为0.01至100％重量，优选0.1至50％重量，更优选0.5至20％重量。
尽管剂量根据给药途径、症状、患者年龄等等而不同，但剂量是，例如，每1千克体重每天口服给药患有肝损伤的成年患者大约0.005-50mg，优选大约0.05-10mg，更优选大约0.2-4mg的本发明化合物，可以将其分为1至3份给药。
当本发明的药物组合物是缓释制剂时，其剂量根据本发明化合物的种类和含量、剂型、药物释放的持续时间、给药目标动物(例如，哺乳动物，例如人类，大鼠，小鼠，狗，兔子等等)和给药目的而不同。对于肠胃外给药，例如，一周释放大约0.1-大约100mg的本发明化合物。
上述药理学可接受的载体的例子包括赋形剂(例如，淀粉，乳糖，蔗糖，碳酸钙，磷酸钙等等)，粘合剂(例如，淀粉，阿拉伯胶，羧甲纤维素，羟丙基纤维素，结晶纤维素，海藻酸，凝胶，聚乙烯吡咯烷酮等等)，润滑剂(例 如，硬脂酸镁，硬脂酸钙，滑石粉等等)，崩解剂(例如，羧甲纤维素钙，滑石粉等等)，稀释剂(例如，注射用水，盐水等等)，添加剂(例如，稳定剂，防腐剂，着色剂，调味剂，溶解助剂，乳化剂，缓冲剂，等渗剂等等)，等等。
本发明的预防或治疗药物还可以与其它药物一起使用。在本发明化合物和其他药物的组合形式中，对本发明化合物和其他药物的给药时间没有限制，本发明化合物或其药物组合物和其他药物或其药物组合物可以同时给药患者，或可以在不同的时间给药患者。可以按照临床上使用的给药数量来确定其他药物的剂量，并且可以根据给药患者、给药途径、疾病、组合药等等恰当地选择。
对其他药物的给药模式没有特别限制，只要本发明的化合物和其他药物在给药过程中组合即可。这种给药模式的例子包括下列方法：
(1)同时配制本发明的化合物或其药物组合物和其他药物，得到单一给药制剂。(2)单独配制本发明的化合物或其药物组合物和其他药物或其药物组合物，得到两种制剂，通过相同给药途径同时给药。(3)单独配制本发明的化合物或其药物组合物和其他药物或其药物组合物，得到两种制剂，通过相同给药途径、在不同时间给药。(4)单独配制本发明的化合物或其药物组合物和其他药物或其药物组合物，得到两种制剂，通过不同给药途径同时给药。(5)单独配制本发明的化合物或其药物组合物和其他药物或其药物组合物，得到两种制剂，通过不同给药途径、在不同时间给药(例如，以本发明的化合物或其药物组合物和其他药物或其药物组合物的顺序给药，或反顺序给药)。
在本发明的组合药物中，可以恰当地按照给药患者、给药途径、疾病等等来确定本发明化合物和其他药物的混合比例。
例如，尽管本发明化合物在本发明组合药物中的含量根据制剂形式而变化，但相对于整个制剂，通常为大约0.01至大约100wt％，优选大约0.1至大约50wt％，更优选大约0.5至大约20wt％。
其他药物在本发明组合药物中的含量根据制剂形式而变化，但相对于整个制剂，通常为大约0.01至大约100wt％，优选大约0.1至大约50wt％，更优选大约0.5至大约20wt％。
添加剂(例如载体等等)在本发明组合药物中的含量根据制剂形式而变化，但相对于整个制剂，通常为大约1至大约99.99wt％，优选大约10至大约90wt％。
实施例
下面参照实施例来详细说明本发明，但本发明并不限定于此，可以在本发明范围内进行变化。
实施例中所采用的原料列表如下：
Figure PCTCN2017090552-appb-000001
在以下各实施例中：
所述的接肽试剂是DMF；
所述的脱帽试剂是PIP∶DMF＝1∶4；体积比。
实施例1：VVYP的合成
1)制备Fmoc-L-Pro-2-CTC树脂：
取100g 2-CTC树脂(1.1mmol/g树脂，140mmol)用800mL干燥的二氯甲烷浸泡，使树脂充分溶胀，抽除二氯甲烷。
取70.8g Fmoc-L-Pro-OH(FW：337.4，210mmol)和180mL N，N-二异丙基乙氨(DIEA，1190mmol)，用800毫升无水DMF溶解，加入反应器中，25℃反应2小时，然后加入200mL甲醇和DIEA的混合溶剂(9∶1，体积比)继续反应1h，封闭未反应的位点，抽干，分别用DMF、甲醇和二氯甲烷各洗涤三遍，真空干燥，获得Fmoc-L-Pro-2-CTC resin树脂。
采用Fmoc-OSu试剂作为对照，测定氨基酸的上载率是0.98mmol/g树脂。收率是89％。
2)制备Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂：
用800mL脱帽试剂加入到Fmoc-L-Pro-2-CTC resin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取183.8g Fmoc-L-Tyr(tBu)-OH(FW：459.5，400mmol)、151.7g HBTU(FW：379.24，400mmol)、59.5g HOBT(FW：135.13，440mmol)和121mLN，N-二异丙基乙氨(DIEA，800mmol)混合物，25℃反应60min，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF和二氯甲烷洗涤，获得Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂。
3)制备Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
用800mL脱帽试剂加入到Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取135.8g Fmoc-L-Val-OH(FW：339.4，400mmol)、151.7g HBTU(FW：379.24，400mmol)、59.5g HOBT(FW：135.13，440mmol)和121mLN，N-二异丙基乙氨(DIEA，800mmol)混合物，25℃反应60min，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF和二氯甲烷洗涤，获得Fmoc-L-Val-L-Tyr(tBu)-L-Pro-2-CTC树脂。
4)制备Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
用800mL脱帽试剂加入到Fmoc-L-Val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取135.8g Fmoc-L-Val-OH(FW：339.4，400mmol)、151.7g HBTU(FW：379.24，400mmol)、59.5g HOBT(FW：135.13，440mmol)和121mLN，N-二异丙基乙氨(DIEA，800mmol)混合物，25℃反应60min，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF和二氯甲烷洗涤，获得Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂。
5)制备L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂
用800mL脱帽试剂加入到Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTCresin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干，得到L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC resin树脂。
6)切肽
将L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC resin加入1000mL的切肽试剂(TFA∶TSI∶H2O等于95∶2.5∶2.5，体积比)，25℃反应4h，抽滤，收集滤液，氮气吹干，得到切肽后的VVYP粗品。
7)VVYP分离纯化
将VVYP粗品溶解在5％的醋酸仲，搅拌5h，用纳滤机纯化，氮气吹干浓缩液，冻干，得到VVYP纯品(以VVYP乙酸盐的形式存在，VVYP·CH3COOH，见图1)，纯度是99.3％(见图2)。得到VVYP纯品48.3克，收率是82％
实施例2：AVLP的合成
1)制备Fmoc-L-Pro-2-CTC resin：
取100g 2-CTC树脂(1.4mmol/g树脂，140mmol)用800mL干燥的DMF浸泡，使树脂充分溶胀，抽除二氯甲烷。
取70.8g Fmoc-L-Pro-OH(FW：337.4，210mmol)和131mLN-甲基吗啉(NMM，1190mmol)，用800毫升无水DMF溶解，加入反应器中，10℃反应4小时，然后加入200mL甲醇继续反应1h，封闭未反应的位点，抽干，用DMF洗涤六遍，真空干燥，获得Fmoc-L-Pro-2-CTC resin。
采用Fmoc-OSu试剂作为对照，测定氨基酸的上载率是1.26mmol/g。
2)制备Fmoc-L-Leu-L-Pro-2-CTC resin：
用800mL脱帽试剂加入到Fmoc-L-Pro-2-CTC resin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取2178.1g Fmoc-L-Leu-OH(FW：353.4，504mmol)、191.3g HBTU(FW：379.24，504mmol)、74.3g HOBT(FW：135.13，550mmol)和111mLN-甲基吗啉(NMM，1010mmol)混合物，10℃反应4小时，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF洗涤，获得Fmoc-L-Leu-L-Pro-2-CTCresin。
3)制备Fmoc-L-Val-L-Leu-L-Pro-2-CTC resin：
用800mL脱帽试剂加入到Fmoc-L-Leu-L-Pro-2-CTC resin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取183.2g Fmoc-L-Val-OH(FW：339.4，504mmol)、191.3g HBTU(FW：379.24，504mmol)、74.3g HOBT(FW：135.13，550mmol)和111mLN-甲基吗啉(NMM，1010mmol)混合物，10℃反应4小时，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF洗涤，获得Fmoc-L-Val-L-Leu-L-Pro-2-CTC resin。
4)制备Fmoc-L-ala-L-Val-L-Leu-L-Pro-2-CTC resin：
用800mL脱帽试剂加入到Fmoc-L-Val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取156.9g Fmoc-L-ala-OH(FW：311.33，504mmol)、191.3g HBTU(FW：379.24，504mmol)、74.3g HOBT(FW：135.13，550mmol)和111mLN-甲基吗啉(NMM，1010mmol)混合物，10℃反应4小时，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF和二氯甲烷洗涤，获得Fmoc-L-ala-L-Val-L-Leu-L-Pro-2-CTC resin。
5)制备L-ala-L-Val-L-Leu-L-Pro-2-CTC resin
用800mL脱帽试剂加入到Fmoc-L-ala-L-Val-L-Leu-L-Pro-2-CTC resin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干，得到L-ala-L-Val-L-Leu-L-Pro-2-CTC resin。
6)切肽
将L-ala-L-Val-L-Leu-L-Pro-2-CTC resin加入1000mL的切肽试剂(TFA∶H2O等于90∶5，体积比)，25℃反应4h，抽滤，收集滤液，用氮气吹干溶剂，得到切肽后的AVLP粗品。
7)AVLP纯化
将AVLP粗品溶解在5％的醋酸中，搅拌5h，过滤，用300Da纳滤膜纯化AVLP粗品，收率AVLP的精制品溶液，用氮气吹干溶剂，冷冻干燥，得到AVLP纯品(以AVLP乙酸盐的形式存在，AVLP·CH3COOH，见图3)，纯度是98.5％，AVLP的收率是82.1％(见图4)。
实施例3：IGFP的合成
1)制备Fmoc-L-Pro-2-CTC resin：
取100g 2-CTC树脂(1.4mmol/g树脂，140mmol)用800mL干燥的DMF浸泡，使树脂充分溶胀，抽除二氯甲烷。
取70.8g Fmoc-L-Pro-OH(FW：337.4，210mmol)和131mLN-甲基吗啉(NMM，1190mmol)，用800毫升无水DMF溶解，加入反应器中，10℃反应4小时，然后加入200mL甲醇继续反应1h，封闭未反应的位点，抽干，用DMF洗涤六遍，真空干燥，获得Fmoc-L-Pro-2-CTC resin。
采用Fmoc-OSu试剂作为对照，测定氨基酸的上载率是1.21mmol/g树脂。
2)制备Fmoc-L-phe-L-Pro-2-CTC resin：
用800mL脱帽试剂加入到Fmoc-L-Pro-2-CTC resin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取187.6g Fmoc-L-phe-OH(FW：387.5，484mmol)、183.6g HBTU(FW：379.24，484mmol)、71.9g HOBT(FW：135.13，532.4mmol)和106mLN-甲基吗啉(NMM，968mmol)混合物，10℃反应4小时，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF洗涤，获得Fmoc-L-phe-L-Pro-2-CTC resin。
3)制备Fmoc-Gly-L-phe-L-Pro-2-CTC resin：
用800mL脱帽试剂加入到Fmoc-L-phe-L-Pro-2-CTC resin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；取143.9g Fmoc-Gly-OH(FW：297.3，484mmol)、183.6g HBTU(FW：379.24，484mmol)、71.9g HOBT(FW：135.13，532.4mmol)和106mLN-甲基吗啉(NMM，968mmol)混合物，10℃反应4小时，取少许树脂做茚三酮检测，呈阴性。抽干，然后用DMF洗涤，获得Fmoc-Gly-L-phe-L-Pro-2-CTC resin。
4)制备Fmoc-L-Ile-Gly-L-phe-L-Pro-2-CTC resin：
用800mL脱帽试剂加入到Fmoc-Gly-L-phe-L-Pro-2-CTC resin树脂中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干；
取171.0g Fmoc-L-Ile-OH(FW：353.4，484mmol)、183.6g HBTU(FW：379.24，484mmol)、71.9g HOBT(FW：135.13，532.4mmol)和106mLN-甲基吗啉(NMM，968mmol)混合物，10℃反应4小时，取少许树脂 做茚三酮检测，呈阴性。抽干，然后用DMF和二氯甲烷洗涤，获得Fmoc-L-Ile-Gly-L-phe-L-Pro-2-CTC resin。
5)制备L-Ile-Gly-L-phe-L-Pro-2-CTC resin
用800mL脱帽试剂加入到Fmoc-L-Ile-Gly-L-phe-L-Pro-2-CTC resin中，25℃反应10min，重复操作2次。用无水的DMF洗涤，抽干，得到L-Ile-Gly-L-phe-L-Pro-2-CTC resin。
6)切肽
将L-Ile-Gly-L-phe-L-Pro-2-CTC resin加入1000mL的切肽试剂(TFA∶H2O等于90∶5，体积比)，25℃反应4h，抽滤，收集滤液，用氮气吹干溶剂，得到切肽后的IGFP粗品。
7)IGFP纯化
将IGFP粗品溶解在5％的醋酸中，搅拌5h，过滤，用300Da纳滤膜纯化IGFP粗品，收率IGFP的精制品溶液，用氮气吹干溶剂，冷冻干燥，得到IGFP纯品(以IGFP乙酸盐的形式存在，IGFP·CH3COOH，见图5)，纯度是99.8％(见图6)，IGFP的收率是82.6％。
实施例4Val-Val-Tyr-Pro肽对四氯化碳所致小鼠急性肝损伤的保护作用
一、材料与方法
(1)材料与主要试剂
受试动物：清洁级KM小鼠，雄性，120只，18-22g
受试药物：
Val-Val-TVr-Pro肽：99％纯度，由江西本草天工科技有限责任公司提供
双环醇片：国药准字H20040467，北京协和药厂
水飞蓟宾：国药准字H20040299，天津天士力圣特制药有限公司。
四氯化碳购于天津市福晨化学试剂厂
谷丙转氨酶(ALT)试剂盒、谷草转氨酶(AST)、总胆红素(TBIL)、总胆固醇(CHO1)和甘油三脂(TG)试剂盒购于日本和光纯药工业株式会社
超氧化物歧化酶(SOD)试剂盒购于南京建成生物工程研究所
谷胱甘肽过氧化物酶(GSH-Px)测定试剂盒购于南京建成生物工程研究所
(2)主要仪器
RM2016型石蜡切片机(德国Leica公司)
LEICA DM 1000光学显微镜
上海恒平FB224电子天平
德国Sigma1-16K高速离心机
日立7100全自动生化分析仪
(3)方法
实验动物分组：将昆明小鼠随机分为空白对照组，模型组，水飞蓟宾组(150mg/kg)，双环醇组(25mg/kg)Val-Val-Tyr-Pro肽高剂量组，Val-Val-Tyr-Pro肽中剂量组，Val-Val-Tyr-Pro肽低剂量组，每组12只动物。
实验动物给药：给药组各组小鼠，按随机顺序每天上午给予相应药物1次，给药方式为灌胃给药，给药容量2ml/100g，模型组和空白组给予等量的0.5％羧甲基纤维素钠。连续给药5天。
实验动物造模：给药第4天下午4点开始，模型组和给药组腹腔注射0.1％四氯化碳(10ml/kg)，造模第二天上午8点继续给药1次，给药3h眼眶静脉采血和取肝脏。
肝功能测定：采集到的血样(约600μl)在室温放置2h后，以3500rpm离心10min分离出血清，多功能生化仪测定血清中ALT、AST和ALP的活性。
肝脏切片检测：肝组织病理学检查取小鼠肝大叶大致相同部位的一小块肝组织，用10％甲醛溶液固定后，石蜡包埋，切片(片厚5μm)，苏木素-伊红(HE)染色，然后在光镜下观察肝脏组织病理学改变。根据肝脏病变程度以半定量方法积分，并按各种病变重要性乘以不同加权数。加权数：肝细胞坏死×3，肝细胞水样变×1，淤血、出血×1，炎性细胞浸润×1。计算每只动物肝病变的总积分，然后进行统计学处理。
GSH-Px、SOD测定：按试剂盒说明书进行。
(4)统计学方法
采用SPSS 16.0进行统计学分析，剂量资料多组间比较采用单因素方差分析(One-wayANOVN)，各组均数的多重比较采用最小显著插值法(LSD)，P＜0.05为差异有统计学意义。
二、结果
(1)Val-Val-Tyr-Pro肽对0.1％CCl4所致小鼠急性肝损伤中血清ALT，AST，TBIL，CHO1和TG的影响
模型组小鼠血清中ALT和AST的活性较正常对照组明显升高，且具有统计学差异，说明本次实验模型造模成功。Val-Val-Tyr-Pro肽高、中、低剂量均能降低肝损伤小鼠血清中ALT，AST的活性，与模型组比较均具有统计学差异(*P＜0.05，**P＜0.01)，高、中、低剂量降转氨酶效果优于阳性药水飞蓟宾和双环醇组，25mg/kg双环醇不具有降低AST效果。
此外，Val-Val-Tyr-Pro肽高剂量能降低四氯化碳损伤小鼠TBIL的增加，双环醇能降低正常和模型组小鼠TG的含量，而模型组和给药组对CHO1含量无影响(表1)。
以上结果表明，在0.1％四氯化碳造模下，昆明小鼠口服Val-Val-Tyr-Pro肽高剂量、中剂量和低剂量都具有保肝降酶效果，对抗四氯化碳诱导肝损伤小鼠的作用优于阳性药双环醇和水飞蓟宾。
表1 Val-Val-Tyr-Pro肽对0.1％CCl4所致小鼠急性肝损伤中血清ALT，AST，TBIL，CHO 1和TG的影响
注：与正常对照组比较，#P＜0.05，##P＜0.01；与模型组比较*P＜0.05，**P＜0.01.
Figure PCTCN2017090552-appb-000002
(2)Val-Val-Tyr-Pro肽对0.1％CCl4致急性肝损伤小鼠肝脏病理改变的影响
病理切片结果表明，正常对照组小鼠的肝组织结构正常，肝细胞没有变性、坏死等病理性变化(图1)，模型组小鼠肝脏切片结果显示：肝索状排列紊乱或细胞索变窄或溶解，出现以中央静脉为中心的局灶性肝细胞变性坏死及伴炎性细胞浸润，细胞核固缩等病理性改变(病理积分为1.00士0.00)。阳性药和Val-Val-Tyr-Pro肽各剂量组小鼠的肝细胞坏死，变性以及炎性细胞浸润等病变得到了很大程度的减轻。病理积分明显降低(0.34士0.25；0.49士0.22；0.45士0.32)，与模型组比较有显著差异(表2)，以上结果进一步表明Val-Val-Tyr-Pro肽具有较好的保护肝细胞作用，减轻上述病理改变。
表2.Val-Val-Tyr-Pro肽对0.1％CCl4致急性肝损伤小鼠肝脏病理积分的影响
注：与正常对照组比较，#P＜0.05；与模型组比较*P＜0.05.
Figure PCTCN2017090552-appb-000003
(3)Val-Val-Tyr-Pro肽对0.1％CCl4致急性肝损伤小鼠肝组织的SOD，GSH-Px水平的影响
机体中还存在多种抗氧化物酶，包括SOD，GSH-Px，过氧化氢酶(CAT)，谷胱甘肽还原酶(GR)等，可分别催化不同的氧化还原反应，使机体维持相对稳定的氧化还原状态。本研究表明，四氯化碳造模组SOD和GSH-Px活性均发生显著变化，与正常对照组动物相比分别降低82.5％和23％。Val-Val-Tyr-Pro肽给药(8，16和32mg/kg)可显著抑制SOD和GSH-Px活性的降低，使SOD活性分别恢复至对照组的99％，50.89％和44.54％，GSH-Px活性分别恢复至对照组96.8％，94.9％和111.2％(表3).
表3.Val-Val-Tyr-Pro肽对0.1％CCl4致急性肝损伤小鼠肝组织的SOD，GSH-Px水平的影响
注：与正常对照组比较，#P＜0.05，##P＜0.01；与模型组比较*P＜0.05，**P＜0.01.
Figure PCTCN2017090552-appb-000004
结果表明，0.1％CCl4肝损伤模型组小鼠血清中ALT和AST水平较正常对照组明显升高(##P＜0.01)，Val-Val-Tyr-Pro肽高、中、低剂量均能降低肝损伤小鼠血清中ALT和AST的活性，与模型组比较均具有统计学差异(*P＜0.05，**P＜0.01)，高、中、低剂量降酶效果优于阳性药水飞蓟宾和双环醇组。此外，Val-Val-Tyr-Pro肽高剂量能防止CCl4引起的小鼠血清总胆红素含量的升高。0.1％CCl4肝损伤模型组小鼠的肝组织出现严重的损伤，肝大叶结构有明显的破坏，大量炎性细胞浸润其中，大部分肝细胞出现浑浊肿胀等病理性改变(病理积分为1.00士0.00)，并且血清中AST、ALT水平也显著升高。Val-Val-Tyr-Pro肽各剂量组小鼠肝细胞受损程度明显减少，其中，Val-Val-Tyr-Pro肽高、中剂量血清中ALT和AST的水平显著低于CCl4模型组，且降酶效果具有剂量依赖性，表明Val-Val-Tyr-Pro肽具有保护肝细胞膜，明显对抗0.1％CCl4诱导的肝损伤的作用。SOD是体内重要的抗氧化酶，已知真核细胞中，根据SOD活性中心所含金属离子不同，分为铜锌超氧化物岐化酶(Cu，Zn-SOD)存在胞外和胞液中；锰超氧化物岐化酶(Mn-SOD)存在于线粒体中，氧自由基(O2-)生成过多时可诱导Mn-SOD的合成。当体内代谢过程中产生自由基时，SOD可催化氧自由基(O2-)反应产生过氧化氢，过氧化氢与GSH在GSH-Px催化下生成水，从而清除自由基。
本研究发现模型组SOD和GSH-Px活力显著降低，Val-Val-Tyr-Pro肽保护组能明显升高SOD和GSH-Px活力，因此，Val-Val-Tyr-Pro肽的保护作用机制 与减轻提高抗氧化酶活性，从而抑制氧化应激有关。
实施例5：Val-Val-Tyr-Pro肽对小鼠急性酒精性肝损伤的保护作用
一、材料与方法
(1)材料
酒精度56％，购自北京红星酒业有限公司
其他同实施例1
(2)主要仪器
同实施例1
(3)方法
实验动物分组：将70只昆明小鼠随机分为空白对照组，模型组，阳性药水飞蓟宾组(75mg/kg)，阳性药双环醇组(25mg/kg)，Val-Val-Tyr-Pro肽高剂量组(25mg/kg)，Val-Val-Tyr-Pro肽中剂量组(12.5mg/kg)，Val-Val-Tyr-Pro肽低剂量组(6.25mg/kg)，每组10只动物。
实验动物给药：给药组各组小鼠，按随机顺序首日上午、下午和次日上午给予相应药物1次，给药方式为灌胃给药，给药容量2ml/100g。
实验动物造模：末次给药8h，除正常组外，每组灌胃给予红星二锅头20ml/kg造模，正常组给予等体积的蒸馏水，禁食不禁水16h后眼眶静脉采血。
血清肝功能测定：采集到的血样(约600μl)在室温放置2h后，以3 000rpm离心10min分离出血清，多功能生化仪测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)、胆固醇(CHO1)和甘油三酯(TG)的水平。
二、结果
Val-Val-Tyr-Pro肽对急性酒精性肝损伤小鼠血清ALT/AST/TBIL/CHO 1/TG的影响：与正常组比较，模型组小鼠血清中ALT、AST和TG的水平显著上升，Val-Val-Tyr-Pro肽高、中、低剂量组预先灌胃3次能使二锅头所引起的血清ALT和TG水平的升高明显降低，且高剂量Val-Val-Tyr-Pro肽能降低血清AST水平，双环醇25mg/kg和水飞蓟宾75mg/kg可明显降低ALT水平，但降AST水平的作用不明显，结果见表4。
表4.Val-Val-Tyr-Pro肽对急性酒精性肝损伤小鼠血清ALT/AST/TBIL/CHO1/TG的影响
注：与正常对照组比较，##P＜0.01；与模型组比较*P＜0.05，**P＜0.01.
Figure PCTCN2017090552-appb-000005
本实验采用二锅头灌胃的方法诱导小鼠急性酒精性肝损伤模型，肝脏ALT、AST和TG水平显著上升，说明本实验成功制备了小鼠急性酒精性肝损伤模型，且小鼠酒精灌胃后肝脏出现明显的脂质蓄积，血清的ALT和AST是反映肝细胞受损的重要指标，本研究表明Val-Val-Tyr-Pro肽组能显著降低血清ALT、AST水平，且可显著抑制肝脏甘油三酯蓄积，显示Val-Val-Tyr-Pro肽对小鼠的急性酒精性肝损伤有保护作用。
实施例6 Val-Val-Tyr-Pro肽对对乙酰氨基酚所致小鼠急性肝损伤的保护作用
一、材料与方法
(1)材料
酒精度56％，购自北京红星酒业有限公司
其他同实施例1
(2)主要仪器：同实施例1
(3)方法
实验动物分组：将72只昆明小鼠随机分为空白对照组，模型组，双环醇低剂量组(25mg/kg)，双环醇高剂量组(50mg/kg)，Val-Val-Tyr-Pro肽高剂量组(32mg/kg)，Val-Val-Tyr-Pro肽中剂量组(16mg/kg)，Val-Val-Tyr-Pro肽低剂量组(8mg/kg)，每组12只动物。
实验动物给药：给药组各组小鼠，按随机顺序首日上午、下午和次日上午给予相应药物1次，给药方式为灌胃给药，给药容量2ml/100g。
实验动物造模：末次给药8h后腹腔注射对乙酰氨基酚120mg/kg，注射16h后眼眶静脉采血和取肝脏。
血清和肝线粒体转氨酶测定：采集到的血样(约600μl)在室温放置2h后，以3 000rpm离心10min分离出血清，多功能生化仪测定血清中ALT、AST、TBIL、CHO1和TG的水平。
二、结果
(1)Val-Val-Tyr-Pro肽对小鼠腹腔注射AP 16h后血清ALT/AST/TBIL/TG的影响
小鼠于腹腔注射AP16h后，血清转氨酶水平明显升高，Val-Val-Tyr-Pro肽高、中、低剂量组预先灌胃3次能使AP所引起的ALT、AST和TBIL水平的升高明显降低，双环醇25mg/kg可明显降低ALT，但降AST的作用不明显，结果见表5。
表5.Val-Val-Tyr-Pro肽对小鼠腹腔注射AP 16h后血清ALT/AST/TBIL/TG的影响
注：与正常对照组比较，##P＜0.01，##P＜0.01；与模型组比较*P＜0.05，**P＜0.01.
Figure PCTCN2017090552-appb-000006
(2)Val-Val-Tyr-Pro肽对对AP引起小鼠肝线粒体AST降低的保护作用
小鼠腹腔注射AP16h后线粒体中AST水平均显著下降，而预先给予双环醇25mg/kg×3次能防止肝脏线粒体中AST的降低，当给予不同剂量Val-Val-Tyr-Pro肽后，与模型组相比，肝线粒体中AST水平均显著升高且升 高水平高于阳性药双环醇组，给药组各组间没有显著差异(P＞0.05)，结果见表6.
表6.Val-Val-Tyr-Pro肽对小鼠腹腔注射AP后不同时间肝线粒体中AST水平的影响
本实验中，小鼠腹腔注射对乙酰氨基酚(AP)16h后ALT和AST水平均显著增加，而预先给予Val-Val-Tyr-Pro肽高、中、低剂量灌胃3次后，血清中ALT、AST和TBIL水平均降低，几乎保持在正常水平，AP毒性机制主要是AP转化为半醌自由基(NAPQI)造成的，由于线粒体氧化呼吸功能可被多种醌类化合物所抑制，因此AP代谢产生的半醌自由基能影响肝脏线粒体的功能，造成线粒体的损伤。Val-Val-Tyr-Pro肽对AP引起的肝损伤有明显的保护作用，表现在血清AST转氨酶的升高明显减轻，另外，本实验中观察到，在AP损伤肝线粒体晚期(16h)，Val-Val-Tyr-Pro肽可明显防止AP引起的肝线粒体中AST的降低，阻止了线粒体内AST的外泄。
综上所述，Val-Val-Tyr-Pro肽既能明显减轻AP引起的整个肝细胞的损伤，又能通过对线粒体膜的保护作用而防止线粒体结构和功能的损伤从而阻止线粒体内AST等酶的释放。
工业实用性
本发明可以实现提供一种工艺简单、毒性低、产率高、纯度高的肽类化合物的制备方法，进一步地，利用本发明的制备方法，还可以提供新的肽化合物，例如VVYP；AVLP；IGFP；LLIIVP和STAP以及它们的盐。此外，本发明人还发现，多肽类化合物VVYP具有优异的保肝护肝的作用，降低转氨酶尤其是降低AST水平，减轻肝脏组织病理性改变和修复肝损伤 的作用，对于肝损伤，例如化学性、病毒性、药物性、或酒精性(例如急、慢性)肝损伤、以及急性酒精性中毒的预防和治疗具有优异的效果。
。
本发明如上所述进行了描述。当然本发明在其范围中包含各种方式的变化，这些变化并不偏离本发明的范围。此外，对于对本领域技术人员而言明显是本发明的变形的改变，都包括在权利要求的范围内。

Claims (19)

  1. 肽或其盐的制备方法，该方法包括：
    步骤A：以树脂作为起始原料，在碱性试剂的存在下，将具有α-氨基保护基团的构成肽的C端的氨基酸连接至树脂，
    步骤B：用脱帽试剂将所述保护基团脱去，
    步骤C：在碱性试剂和偶联试剂存在下，按照肽的从C端到N端的顺序依次连接组成肽的各个氨基酸进行反应，从而形成肽连接树脂，其中，每个氨基酸的连接都是先连接具有α-氨基保护基团的氨基酸进行接肽，然后用脱帽试剂将所述保护基团脱去进行脱帽，
    步骤D：进行切肽，得到肽或其盐的粗品，
    步骤E：纯化粗品，得到所述的肽或其盐。
  2. 权利要求1的制备方法，其中，步骤A中的所述树脂为2-氯三苯甲基氯树脂，碱性试剂选自：N，N-二异丙基乙氨(DIEA)和N-甲基吗啉(NMM)；步骤C中的碱性试剂选自：N，N-二异丙基乙氨和N-甲基吗啉；步骤C中偶联试剂选自O-苯并三氮唑-N，N，N′，N′-四甲基脲四氟硼酸(TBTU)，O-苯并三氮唑-四甲基脲六氟磷酸盐(HBTU)、1-羟基苯并三唑(HOBT)、六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop)。
  3. 权利要求1-2中任一项的制备方法，其中，所述各步骤中接肽反应的温度是10～35℃，反应时间是0.5～8小时，脱帽反应的温度是20～35℃，反应时间是5～60分钟。
  4. 权利要求1-3中任一项的制备方法，其中，所述各步骤中切肽试剂是三氟乙酸、三异丙基硅烷和水的混合物，三氟乙酸的比例是90～95％，三异丙基硅烷的比例是0～5％，水的比例是0～5％，在20～35℃切肽反应1～5小时。
  5. 权利要求1-4中任一项的制备方法，其中，所述步骤E中的纯化是利用纳滤机进行的。
  6. 权利要求1-5中任一项的制备方法，其中，所述步骤D中利用氮气吹干的方法得到了浓缩的粗品。
  7. 权利要求1-6中任一项的制备方法，其中，
    在步骤A和步骤C中，具有α-氨基保护基团的氨基酸的投料量为，以摩尔数计，是所连接的树脂的1.5～6倍。
    在步骤A中，碱性试剂的投料量为(以摩尔数计)是所连接的树脂的2～10倍；在步骤C中，在连接每个氨基酸的接肽反应中，HBTU的投料量与具有α-氨基保护基团的氨基酸的量相等；HOBT的投料量为，以摩尔数计，是具有α-氨基保护基团的氨基酸的1.1～5倍；碱性试剂的投料量为，以摩尔数计，是具有保护基团的氨基酸的2～10倍，
    在步骤A-E中，氨基酸的α-氨基保护基团均为Fmoc；接肽试剂选自二氯甲烷，N，N-二甲基甲酰胺(DMF)，N，N-二甲基乙酰胺，四氢呋喃和乙酸乙酯中的一种或更多种；脱帽试剂为哌啶(PIP)∶DMF等于1∶2～10(体积比)；脱帽试剂的加入量和待脱帽的树脂的重量比例是5～25ml脱帽试剂/g树脂。
  8. 权利要求1-7中任一项的制备方法，其中，所述肽选自：
    VVYP；AVLP；IGFP；LLIIVP和STAP，
    其中
    Y代表：L-酪氨酸；
    V代表：L-缬氨酸；
    P代表：L-脯氨酸；
    A代表：L-丙氨酸；
    L代表：L-亮氨酸；
    I代表：L-异亮氨酸；
    G代表：L-甘氨酸；
    F代表：L-苯丙氨酸；
    S代表：L-丝氨酸；
    T代表：L-苏氨酸；
    所述肽的盐选自：硫酸盐、盐酸盐、氢溴酸盐、三氟乙酸盐、甲烷基磺酸盐、萘磺酸、乙酸盐、富马酸盐、马来酸盐、酒石酸盐、2，5-二羟基苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐和对甲苯磺酸盐。
  9. 权利要求1-8中任一项的制备方法，其中，所述肽为VVYP，该方法包括：
    步骤(1)：制备Fmoc-L-Pro-2-CTC树脂：
    取2-CTC树脂，用干燥的二氯甲烷浸泡，然后加入用接肽溶剂溶解的Fmoc-L-Pro-OH和N，N-二异丙基乙氨的混合物进行反应后，加入醇继续反应，抽干，洗涤，干燥，获得Fmoc-L-Pro-2-CTC树脂；
    步骤(2)：制备Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂：
    在步骤(1)获得的Fmoc-L-Pro-2-CTC树脂中，加入脱帽试剂，反应，抽干，洗涤；加入用接肽溶剂溶解的Fmoc-L-Tyr(tBu)-OH、HBTU、HOBT和N，N-二异丙基乙氨的混合物进行反应，抽干，洗涤，干燥，获得Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂；
    步骤(3)：制备Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
    在步骤(2)中获得的Fmoc-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂进行反应，抽干，洗涤；加入接肽溶剂溶解的Fmoc-L-val-OH、HBTU、HOBT和N，N-二异丙基乙氨混合物进行反应，抽干，洗涤，干燥，获得Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂；
    步骤(4)：制备Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂：
    在步骤(3)获得的Fmoc-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂进行反应，抽干，洗涤；加入接肽溶剂溶解的Fmoc-L-val-OH、HBTU、HOBT和N，N-二异丙基乙氨混合物进行反应，抽干，洗涤，干燥，获得Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂；
    步骤(5)：制备L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂
    在步骤(4)获得中的Fmoc-L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂中，加入脱帽试剂进行反应，获得L-val-L-val-L-Tyr(tBu)-L-Pro-2-CTC树脂。
  10. 一种肽或其盐，其选自下列：VVYP；AVLP；IGFP；LLIIVP和STAP以及它们的盐。
  11. 序列Val-Val-Tyr-Pro所示的肽在制备预防或治疗肝损伤的药物中的用途。
  12. 权利要求11所述的用途，其中，肝损伤是病毒性肝损伤。
  13. 权利要求11所述的用途，其中，肝损伤是药物性肝损伤。
  14. 权利要求11所述的用途，其中，肝损伤是酒精性肝损伤。
  15. 权利要求11所述的用途，其中，肝损伤是化学性肝损伤。
  16. 序列Val-Val-Tyr-Pro所示的肽在制备预防或治疗急性酒精中毒的药物中的用途。
  17. 序列Val-Val-Tyr-Pro所示的肽在制备降低转氨酶的药物中的用途。
  18. 权利要求17的用途，其中，转氨酶是谷草转氨酶。
  19. 用于预防或治疗肝损伤的药物，其包含序列Val-Val-Tyr-Pro所示的肽。
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