WO2006099779A1 - Peptide cyclique comportant une rgd et son liposome a ciblage actif - Google Patents

Peptide cyclique comportant une rgd et son liposome a ciblage actif Download PDF

Info

Publication number
WO2006099779A1
WO2006099779A1 PCT/CN2005/001258 CN2005001258W WO2006099779A1 WO 2006099779 A1 WO2006099779 A1 WO 2006099779A1 CN 2005001258 W CN2005001258 W CN 2005001258W WO 2006099779 A1 WO2006099779 A1 WO 2006099779A1
Authority
WO
WIPO (PCT)
Prior art keywords
liposome
cyclic peptide
rgd
ssl
amino acid
Prior art date
Application number
PCT/CN2005/001258
Other languages
English (en)
French (fr)
Inventor
Jiyao Wang
Shilin Du
Weiyue Lu
Original Assignee
Zhongshan Hospital, Fudan University
Fudan University
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.)
Filing date
Publication date
Application filed by Zhongshan Hospital, Fudan University, Fudan University filed Critical Zhongshan Hospital, Fudan University
Priority to US11/990,577 priority Critical patent/US20100098748A1/en
Publication of WO2006099779A1 publication Critical patent/WO2006099779A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • the present invention belongs to the field of pharmaceutical and clinical pharmacy, and relates to a polypeptide sequence and a cyclized form thereof, and an integrin receptor-mediated, integrin receptor-mediated ligand, Active targeting of liposomes and medical uses against hepatic stellate cells. Specifically, it relates to a cyclic peptide containing a sequence of refinery aspartate (RGD) and an active targeting liposome thereof, a preparation method and use for treating liver fibrosis.
  • RGD refinery aspartate
  • Hepatic fibrosis is the common pathological basis of all chronic liver diseases, and it is the early and necessary stage of cirrhosis. According to statistics, 25 % ⁇ 40% of them eventually develop into cirrhosis. Its pathological changes can be caused by different causes, such as viruses, ethanol, parasites and other chronic liver damage, activate hepatic stellate cells (HSC), promote collagen-based extracellular matrix (ECM) synthesis Increased, decreased P deflation or insufficient compensation, so that abnormal deposition of extracellular matrix in the liver causes liver fibrosis.
  • HSC hepatic stellate cells
  • ECM extracellular extracellular matrix
  • HSC proliferation and activation are the cytological basis for the development of hepatic fibrosis and are a common central link in the formation of various causes of liver fibrosis (Frieman S. L. Semin Liver Dis. 1990, 10 (1): 20-29). Therefore, targeted therapy for HSC has the potential to reverse liver fibrosis. Since HSC is located in the sinusoidal space of the liver and accounts for a small proportion (about 5%) of the entire liver cell population, it is difficult to design a specific treatment targeted to HSC.
  • the targeted preparation can utilize a carrier to selectively accumulate the drug at the site of action to exert a drug effect, thereby achieving the purpose of high efficiency and reducing toxic side effects, especially cytotoxic drugs.
  • Successful targeted preparations should have three factors: localized accumulation, controlled release, and non-toxic and biodegradable.
  • the targeting of liposomes is divided into passive targeting and active targeting depending on whether the surface has active groups. Passively targeting liposomes do not carry reactive groups, and the liposomes are selectively enriched in certain organs or lesions by utilizing the physiological characteristics and differences of various organs of the human body.
  • the active targeting is to enable liposomes to target specific cells by introducing active-mediated groups (such as ligands, monoclonal antibodies, etc.) on the surface of the liposome, depending on their affinity to the cells.
  • Active targeting is more specific than stimuli targeting, and can target the focus and organ of liposomes to cell-level targets. In theory, it can achieve controlled release in vivo, which is the best pharmacy research direction to improve drug efficacy and reduce toxicity.
  • SSL is superior to normal liposomes (CL) in that it extends the residence time in the circulation and reduces MPS uptake.
  • CL normal liposomes
  • Integrin is a family of cell membrane glycoprotein receptors that mediate cell adhesion to extracellular matrix (ECM).
  • ECM extracellular matrix
  • the ligand that binds to integrin is ECM. Integrin has two subunits, ⁇ and ⁇ , and different combinations use different ECMs as ligands to achieve different functions.
  • the aspartic acid (RGD) tripeptide sequence is a common binding site for integrin recognition.
  • the HSC surface can express integrin receptors, the resting HSCs only express ⁇ ⁇ , and the remaining subunits express little or no expression; while the activated HSCs can express multiple integrin receptors.
  • the object of the present invention is to provide a cyclic peptide containing a spermato aspartate (RGD) sequence and an active targeting liposome thereof, and the present invention connects a cyclic peptide with a liposome, and can further encapsulate a drug such as Interferon can achieve active targeted therapy for liver fibrosis against integrin receptors on the surface of hepatic stellate cells.
  • a further object of the invention is to provide a process for the preparation of said cyclic peptides and their actively targeted liposomes.
  • the RGD cyclic peptide of the present invention is an oligopeptide having eight amino acid residues, and the amino acid sequence is *cysteine-glyphos-sweet-glycoside-color-purine-lysine* (C*GRGDSPK*) , where * is the looping position.
  • the RGD sequence is a binding site for the integrin receptor on the surface of hepatic stellate cells.
  • RGD cyclic peptide is cyclized with a lysine residue by an amide bond (-C0-NH-) through a cysteine residue, and a cysteine has a free thiol group at one end.
  • the RGD cyclic peptide amino acid sequence is either X*GRGDSPZ*, * represents a ring position, X represents a cysteine residue, contains a free sulfhydryl group, and Z represents any one which can form a ring with a cysteine residue.
  • Amino The amino acid sequence of the RGD cyclic peptide is either X*YRGDYZ*, wherein * represents a ring position, X represents a cysteine residue, which contains a free sulfhydryl group, and Y represents alanine, arginine, and day.
  • Asparagine aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline
  • Z represents any one capable of interacting with a cysteine residue.
  • the free thiol group on the RGD cyclic peptide can be linked to a maleimide-polyethylene glycol lipid derivative (MAL-PEG-DOPE) in the liposome membrane material, and the RGD cyclic peptide is attached to the surface of the liposome.
  • MAL-PEG-DOPE maleimide-polyethylene glycol lipid derivative
  • the reaction equation is as follows.
  • the MAL-PEG-DOPE is a biodegradable material that is mainly excreted by the kidneys.
  • the artificially synthesized cyclic peptide containing the RGD sequence of the present invention has the following advantages: 1.
  • the RGD-containing cyclic aspartic acid (RGD) sequence contained in the RGD cyclic peptide is specifically bound to the HSC surface integrin receptor. The site, which meets the requirements for exogenous ligands, is specific for binding to HSC, time- and concentration-dependent, saturation, and competitive inhibition.
  • the RGD cyclic peptide is formed by an amide bond (-C0-NH-), which has a stable conformation and is not easily degraded.
  • the residue of cysteine in the RGD cyclic peptide contains an active thiol group (-SH) for easy modification.
  • RGD cyclic peptide is a synthetic functional peptide with a small molecular weight and is not susceptible to an immune response.
  • the liposome of the present invention is prepared by a rotary evaporation-thin film hydration-extrusion method.
  • the liposomes include normal liposome (CL), long-circulating liposome (SSL), RGD cyclic peptide-modified CL (RGD-CL), RGD cyclic peptide-modified SSL (RGD-SSL), and separate wrapping Interferon CL (CL-IFN), SSL (SSL-IFN), RGD cyclic peptide modified CL (RGD-CL-IFN:), RGD cyclic peptide modified SSL (RGD-SSL_IFN).
  • the membrane material consists of lecithin (EPC), cholesterol (Chol), maleic acid-polyethylene glycol lipid derivative (ML-PEG 334.- DOPE) in a molar ratio of 2:1:0. . 02.
  • the membrane material of the SSL is composed of EP (:, Chol, monomethoxypolyethylene glycol lipid derivative (mPEG 2 - DPO), MAL-PEG 334 - DOPE, and the molar ratio is 2 : 1 : 0. 1 : 0. 02. PEG accounted for 3. 2 mol%.
  • the RGD cyclic peptide was added to a membrane material for preparing CL or SSL according to a molar ratio of MAL-PEG-DOPE to RGD cyclic peptide of 10:1, and the thiol group of the RGD cyclic peptide cysteine was covalently linked to MAL-PEG-DOPE. , the RGD cyclic peptide can be attached to the surface of the liposome. The unbound RGD cyclic peptide is removed by gel column chromatography to obtain RGD-CL or RGD-SSL.
  • the interferon (IFN) solution to be encapsulated is further added to CL, SSL, RGD-CL or RGD-SSL, vortexed for 30 min in an ice bath, and unencapsulated IFN is removed by gel column chromatography. .
  • the encapsulation efficiency of the interferon was 35.6%, and the drug loading rate was 10 4 U IFN / mol phospholipid.
  • the particles are uniform by extrusion, and the particle size ranges from 50 to 200 nm, preferably 100 nm.
  • the active targeting liposome constructed by the present invention against the integrin receptor on the surface of hepatic stellate cells achieves a targeted therapeutic effect on experimental liver fibrosis by a receptor-mediated pathway. That is, by the specific interaction between the HSC surface receptor and the synthetic RGD cyclic peptide, the RGD cyclic peptide-labeled, interferon-loaded active targeting liposome is targeted to the fibrotic liver, and a good anti-fiber is obtained. The efficacy.
  • Rat HSCs were isolated by in vitro experiments and confirmed by fluorescent tracing that the actively targeted liposomes specifically bind to HSC.
  • Figure 1 shows the histological staining of liver in each group.
  • HE staining (X 100): liver fibrosis group (A); interferon-liposome group (C); RGD cyclic peptide-liposome-interferon group (E); Desmin staining (X 400): Hepatic fibrosis group (B); interferon-liposome group (D); RGD cyclic peptide-liposome-interferon group (F).
  • Figure 2 shows the liver function indicators of each group of rats.
  • 1 sham operation group
  • 2 liver fibrosis group
  • 3 interferon-liposome group
  • 4 RGD cyclic peptide-liposome-interferon group.
  • sham operation group ⁇ ⁇ ⁇ 0.05
  • BDL group * ⁇ ⁇ 0.05
  • BDL+IFN-SSL ⁇ ⁇ 0.05.
  • ALT propylaminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • TBIL total bilirubin
  • ⁇ -GT glutamic acid transferase
  • Figure 3 shows the serum fibrosis index of each group of rats.
  • 1 sham operation group
  • 2 liver fibrosis group
  • 3 interferon-liposome group
  • 4 RGD cyclic peptide-liposome-interferon group.
  • sham group ⁇ ⁇ ⁇ 0 05;. .
  • BDL group * ⁇ ⁇ 0 05;.
  • BDL + IFN-SSL A P ⁇ 0 05.
  • HA hyaluronic acid
  • PCIII type III procollagen
  • LN laminin
  • C IV type IV collagen
  • Figure 4 shows the content of hydroxyproline (HYP) in liver tissue of each group (mg/g liver tissue).
  • 1 sham operation group
  • 2 liver fibrosis group
  • 3 interferon-liposome group
  • 4 RGD cyclic peptide-liposome-interferon group.
  • Figure 5 shows the expression of type I collagen mRNA in liver tissue of rats in each group.
  • 1 sham operation group
  • 2 liver fibrosis group
  • 3 interferon-liposome group
  • 4 RGD cyclic peptide-liposome-interferon group.
  • A type I collagen mRNA electrophoresis band
  • B type I collagen mRNA / GAPDH mRNA relative gray value.
  • Figure 6 shows the expression of ⁇ -actin-SMA in liver tissue of rats in each group.
  • Liposomes were prepared by the following procedure using rotary evaporation-film hydration-extrusion.
  • EPC, Choi, MAL-PEG 34M - DOPE were accurately weighed in a ratio of 2:1:0. 02 (molar ratio), dissolved in chloroform, and evaporated to a transparent film in a water bath at 40 ° C, and the organic solvent was evaporated.
  • the lOOnm filter was repeatedly extruded 15 times with a Mini Extruder to obtain a uniform ordinary liposome (CL).
  • the RGD cyclic peptide was added to CL or SSL in PBS at a molar ratio of 10:1 to MAL-PEG-D0PE, shaken at room temperature (25 ° C) overnight, and unbound by gel column (CL-4B) chromatography. RGD cyclic peptide, you can get RGD-CL or RGD-SSL.
  • interferon (IFN_ a lb) solution to be encapsulated was separately added to CL, SSL, RGD-CL, RGD-SSL, vortexed for 30 min in an ice bath, and removed by gel column (CL-4B) chromatography. By encapsulating IFN, CL-IFN, SSL-IFN, RGD-CL-IFN or RGD-SSL-IFN can be obtained.
  • encapsulation efficiency The entrapment efficiency of interferon was determined by enzyme-linked immunosorbent assay (ELISA) to be 35.6%, and the drug loading rate was 10 4 U IFN / ⁇ pity. The activity of the liposome-coated interferon was determined by virus inhibition method.
  • ELISA enzyme-linked immunosorbent assay
  • the fluorescein isothiocyanate (FITC)-labeled RGD cyclic peptide was co-incubated with HSC according to the nature of receptor-ligand binding, ie, specificity, concentration and time-dependent, competitive inhibition.
  • the results show that the binding properties of RGD cyclic peptide to HSC are consistent with the basic characteristics of acceptor ligands.
  • HSCs were seeded on 6-well plates, adhered, and cultured overnight in 0.25% FBS-DMEM. Pre-blocking with 1% BSA-DMEM before the experiment. The relative fluorescence intensity of HSC binding was measured by flow cytometry.
  • RGD-SSL-CF RGD cyclic peptide-modified calcein-coated liposomes
  • SSL-CF calcein-coated liposomes
  • the HSC was inoculated on a 33 cm 2 cell culture dish and cultured overnight with 0.25% FBS-DMEM. Pre-blocking with 1% BSA-DMEM before the experiment.
  • RGD-SSL-CF and SSL-CF were added to incubate for 4 h. The cells were scraped off and dissolved in PBS (1% Triton X-100).
  • the binding of RGD-SSL-CF to HSC was shown by fluorescence microscopy.
  • Example 5 93 ⁇ 4 ⁇ ( 99m Tc) labeled liposome
  • Liposomes were prepared according to Example 2 by adding DTPA-DOPE and phospholipids to the liposome membrane material at a ratio of 1:10 (molar ratio).
  • RGD cyclic peptide-liposome labeling 99m Tc was carried out using a stannous chloride (SnCl 2 ) reduction method.
  • SPECT single photon emission computed tomography scanning imaging was performed at different time points by injecting 99m Tc-RGD-SSL and 99m Tc-SSL 0.5 ml (2mCi ) into the tail vein of normal rats and liver fibrosis rats.
  • the probe is kept at a distance of 2cm from the rat, and the positive phase of the rat is collected.
  • the ⁇ matrix is 512 X 512, 50 sec/frame, and the region of interest of each organ is drawn, and the total count of the organ/the organ of interest is determined. The size (counts/pixel), which is the count per pixel per organ.
  • Example 7 Treatment of Hepatic Fibrosis Rats by RGD Cyclic Peptide Modified Interferon Liposome
  • RGD-SSL-IFN RGD cyclic peptide-liposome-interferon
  • the results showed that the liver function index, serum liver fibrosis index, liver tissue hydroxyproline content and liver pathological changes in the treatment group were significantly improved compared with the interferon-liposome (SSL-IFN) group; liver type I collagen mRNA and The expression of ⁇ -agonin in hepatic stellate cells was significantly decreased (Figs. 1 to 6).
  • Rats were randomly divided into 4 groups, sham operation group, model (BDL) group, IFN-SSL treatment (BDL+IFN-SSL) group, interferon-liposome treatment (BDL+IFN). - RGD-SSL) Each group of 10 members. Except for the sham operation group, the other groups were double-ligated and the common bile duct was cut. In the sham operation group, the laparotomy was exposed, and the upper part of the common bile duct was about 1 cm, and then the abdomen was closed. From the day of ligation, the BDL+IFN-SSL group was injected once a week with 0.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • ADP alkaline phosphatase
  • y-GT Y-glutamine
  • liver tissue homogenate hydroxyproline (Hyp) by colorimetry, expressed as Hyp content per g of liver tissue.
  • RNA extraction from liver tissue was analyzed by gel system.
  • the relative content of collagen mRNA was compared with the ratio of X-type absorbance X area to GAPDH absorbance X area. Said.
  • Rat HSCs were isolated by two-step collagenase perfusion and density gradient centrifugation. Protein extraction, protein concentration determination, denaturing SDS polyacrylamide gel electrophoresis, electroporation on nylon membrane, 5% skim milk powder blocked, primary antibody (rabbit anti-mouse antibody) and horseradish-labeled secondary antibody (anti-rabbit) Antibody) Incubation, exposure development. The specific bands of Western blot were scanned by Graymad using Biomad's graphic analysis software. The intensity of the specific band signal was expressed by the integral absorbance AXmin 2 , and the expression level of the target protein was semi-quantitatively compared.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Dispersion Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Description

含精一甘一天冬氨酸序列环肽及其主动靶向脂质体 技术领域
本发明属制药和临床药学领域， 涉及一种含有精一甘一天冬氨酸（RGD)序 列的整合素受体配体的多肽序列及其环合形式， 以及构建整合素受体介导的、针 对肝星状细胞的主动靶向脂质体和医药用途。 具体涉及含有精一甘一天冬氨酸 (RGD)序列的环肽及其主动靶向脂质体， 制备方法和治疗肝纤维化的用途。 背景技术
肝纤维化是一切慢性肝病共同病理学基础， 是形成肝硬化的早期和必经阶 段， 据统计， 其中 25 %〜40%最终发展为肝硬化。 其病理学变化可由不同病因 所致， 如病毒、 乙醇、 寄生虫等引起慢性肝损害后， 激活肝星状细胞 （hepatic stellate cell, HSC), 促进以胶原为主的细胞外基质（ECM)合成增多、 P牟解减 少或代偿不足， 从而使细胞外基质在肝脏内异常沉积引起肝纤维化。 研究表明， HSC的增生和激活是肝纤维化发生的细胞学基础， 是各种病因肝纤维化形成的共 同中心环节 (Frieman S. L. Semin Liver Dis. 1990, 10 (1) : 20 - 29)。 因此针对 HSC的靶向治疗有可能逆转肝纤维化。 由于 HSC位于肝脏窦周隙内， 在整个肝脏 细胞群中所占比例少 （约 5 % ), 设计靶向到 HSC的特异性治疗存在困难。
靶向制剂可利用载体将药物选择性地积集于作用部位而发挥药效， 以达到 高效而降低毒副作用的目的，尤其是细胞毒性药物。成功的靶向制剂应具备定位 蓄积、控制释药以及无毒、可生物降解三个要素。脂质体的靶向依其表面是否带 有活性基团分为被动靶向、主动靶向。被动靶向脂质体不带有活性基团， 是利用 人体各器官的生理特性和差异而使脂质体选择性地富集于某些器官或病灶。而主 动靶向是通过在脂质体表面引入活性介导基团 （如配体、 单克隆抗体等）， 依靠 其与细胞之间的亲和力，使得脂质体能够靶向到特异性的细胞。相对于祯动靶向， 主动靶向的选择特异性强， 能将脂质体的病灶和器官靶向提高到细胞水平的靶 向。 理论上可以实现体内控释， 是提高药效、 降低毒性的最佳药剂学研究方向。
1990年 Klibanov等（Klibanov AL, et al. FEBS Lett, 1990, 268 (2) : 235； Bume G, Biochim Biophys Acta, 1990, 1029 (1) :91 )， 研制出一种能在循环中 存在更长时间的脂质体， 其膜中含有棕榈酰葡萄糖苷酸或聚乙二醇（PEG) 的类 脂衍生物 （如 PEG-DSPE), 这类脂质体常称之为空间稳定脂质体 （sterically stabilized liposome, SSL) 或长循环脂质体 ( long circulating liposome, LCL)。由于它的表面含有的亲水基团髙度水合，能有效地阻止血液中许多不同组 分 (Lasic DD， Martin FJ, Gabizon A, et al. Biochim Biophys Acta, 1991， 1070 (1) : 187)， 特别是调理素与之结合， 从而抑制单核吞噬细胞系统 （MPS)对 其的吞噬作用。由于 SSL能延缓释药并提高对特定靶组织的选择性，使其在许多 方面更适于应用。 研究表明 (Lasic DD， et al. Biochim Biophys Acta, 1991, 1070 (1) : 187), SSL优于普通脂质体（CL)之处在于延长了在循环中的滞留时间、 可减少被 MPS摄取的速度与程度、 能增加靶部位如肿瘤组织及感染部位的吸收、 在体内具有穿透生物屏障的能力以及具有在动物及人体内的非剂量依赖性，即具 有线性药动学特性。
整合素（integrin)是一类细胞膜表面糖蛋白受体家族， 主要介导细胞与细 胞外基质 （ECM) 的粘附， 与整合素结合的配体是 ECM。 整合素有 α、 β两个亚 单位， 不同组合以不同的 ECM为配基， 实现不同的功能。 精一甘一天门冬氨酸 (RGD)三肽序列为整合素识别的共同结合位点。 HSC表面可以表达整合素受体， 静止态的 HSC仅表达 α ι， 其余亚单位表达较少或不表达； 而激活态的 HSC可以 表达多种整合素受体。
目前尚无关于针对 HSC、 整合素受体介导的主动靶向脂质体治疗肝纤维化的 报导。 发明内容
本发明的目的是提供一种含有精一甘一天冬氨酸（RGD)序列的环肽及其主 动靶向脂质体，本发明将环肽与脂质体进行连接，并可进一步包裹药物如干扰素， 能针对肝星状细胞表面上整合素受体，实现主动靶向治疗肝纤维化。本发明的进 一步目的是提供所述环肽及其主动靶向脂质体的制备方法。
本发明所述的 RGD环肽为含有八个氨基酸残基的寡肽， 氨基酸序列为为 *半 胱-甘 -精-甘-天冬 -色-脯-赖氨酸 * (C*GRGDSPK*)， 其中 *为成环位置。其中 RGD 序列是肝星状细胞表面整合素受体的结合位点。
上述 RGD环肽通过半胱氨酸残基与赖氨酸残基以酰胺键（-C0-NH-)成环， 使半胱氨酸一端含有游离巯基。
所述的 RGD环肽氨基酸序列或为 X*GRGDSPZ*， *表示成环位置， X代表半胱 氨酸残基， 含有一个游离的巯基， Z代表任意一个能与半胱氨酸残基成环的氨基 所述的 RGD环肽氨基酸序列或为 X*YRGDYZ*，其中， *表示成环位置， X代表 半胱氨酸残基， 其含有一个游离的巯基， Y代表丙氨酸、 精氨酸、 天冬酰胺、 天 冬氨酸、 半胱氨酸、 谷氨酰胺、 谷氨酸、 甘氨酸、 组氨酸、 异亮氨酸、 亮氨酸、 赖氨酸、 蛋氨酸、 苯丙氨酸、 脯氨酸、 丝氨酸、 苏氨酸、 色氨酸、 酪氨酸、 缬氨 酸中至少一个氨基酸或者任意长度的氨基酸序列， 不影响与靶向受体结合， Z代 表任意一个能与半胱氨酸残基成环的氨基酸。
RGD环肽上的游离巯基可以与脂质体膜材料中的马来酰胺-聚乙二醇类脂衍 生物（MAL-PEG- DOPE)相连， 将 RGD环肽连接于脂质体的表面上， 反应方程式如 下述。 所述的 MAL-PEG- DOPE属于生物可降解材料， 主要通过肾脏排泄。
Figure imgf000005_0001
本发明所述的人工合成的含有 RGD序列环肽具有以下的优点： 1. RGD环肽 中含有的精一甘一天冬氨酸（RGD)序列的是与 HSC表面整合素受体特异性的结 合位点， 符合作为外源性配基的要求， 即与 HSC的结合具有特异性、时间和浓度 的依赖性、 饱和性以及竞争性抑制作用。 2. RGD环肽中由酰胺键（- C0-NH-) 成 环，空间构象稳定，不易降解。 3. RGD环肽中半胱氨酸的残基含有活性巯基 (-SH), 便于进行修饰。 4. RGD环肽属于人工合成的功能肽， 分子量小， 不易引起免疫 反应。
本发明所述的脂质体采用旋转蒸发一薄膜水化一挤压法制备。所述的脂质体 包括普通脂质体（CL)、长循环脂质体（SSL)、 RGD环肽修饰的 CL (RGD-CL)、 RGD 环肽修饰的 SSL (RGD- SSL)以及分别包裹干扰素的 CL (CL-IFN)、 SSL (SSL- IFN)、 RGD环肽修饰的 CL (RGD- CL- IFN：)、 RGD环肽修饰的 SSL (RGD- SSL_IFN)。
所述的 的膜材料由卵磷脂（EPC)、 胆固醇（Chol)、 马来酰胺-聚乙二醇 类脂衍生物（ML- PEG334。- DOPE)组成， 其摩尔比为 2 : 1 : 0. 02。
所述的 SSL 的膜材料由 EP (：、 Chol、 单甲氧基聚乙二醇类脂衍生物 (mPEG2。。。- D0PE)、 MAL- PEG334。- DOPE组成， 其摩尔比为 2 : 1 : 0. 1 : 0. 02。 PEG 占 3. 2mol%。
按照 MAL-PEG-DOPE与 RGD环肽摩尔比为 10： 1将 RGD环肽加入到制备 CL或 SSL的膜材料中， 通过 RGD环肽半胱氨酸的巯基与 MAL- PEG- DOPE共价连接， 即 可将 RGD环肽连接到脂质体表面。 通过凝胶柱层析法去除未结合的 RGD环肽， 即可得到 RGD- CL或 RGD- SSL。
本发明进一步将待包封的干扰素（IFN)溶液分别加入到 CL、 SSL、 RGD-CL 或 RGD-SSL中， 冰浴下旋涡振荡 30min， 通过凝胶柱层析法除去未包入的 IFN。 干扰素的包封率为 35. 6%, 载药率为 104U IFN / mol磷脂。
CL、 SSL, RGD-CL, RGD-SSL, CL- IFN、 SSL-IFN, RGD- CL- IFN和 RGD- SSL-IFN 通过挤压法粒子均匀一致， 粒径范围为 50〜200nm， 优选 100nm。
本发明构建的针对肝星状细胞表面上整合素受体的主动靶向脂质体，通过受 体介导途径实现了对实验性肝纤维化的靶向治疗作用。即借助 HSC表面受体与人 工合成的 RGD环肽的特异性相互作用，将 RGD环肽标记的、载有干扰素的主动靶 向脂质体靶向到纤维化肝脏， 取得了良好的抗纤维化疗效。
经体外实验， 分离大鼠 HSC， 荧光示踪法证实， 所述的主动靶向脂质体能够 特异性地与 HSC结合。
体内实验， SPET显像显示 RGD-SSL主要分布于肝脏， 持续达 24小时， 主要 经肾脏和胆道排泄。通过胆总管结扎制备大鼠肝纤维化模型，采用尾静脉注射给 药的方法，观察 RGD- SSL-IFN对肝纤维化大鼠的治疗作用。结果显示治疗组肝功 能指标、 血清肝纤维化指标、 肝组织羟脯氨酸含量和肝脏病理学改变均较 SSL-IFN组有明显改善； 肝脏 I型胶原 mRNA和肝星状细胞的 α -激动蛋白表达量 明显下降， 显示了主动靶向脂质体对大鼠肝纤维化具有良好的治疗作用。 附图说明
图 1 是各组大鼠肝脏组织学染色，
其中， HE染色 （X 100): 肝纤维化组（A); 干扰素一脂质体组 （C); RGD 环肽一脂质体一干扰素组 （E); Desmin染色 （X 400): 肝纤维化组 （B); 干扰 素一脂质体组（D); RGD环肽一脂质体一干扰素组（F)。
图 2是各组大鼠肝功能指标，
其中， 1 : 假手术组； 2: 肝纤维化组； 3: 干扰素一脂质体组； 4: RGD环 肽一脂质体一干扰素组。 与假手术组相比， ΔΡ〈0. 05; 与 BDL组相比， * Ρ<0. 05; 与 BDL+IFN- SSL相比， ΑΡ<0· 05。 (ALT: 丙氨基氨基转齊酶； AST: 天冬氨酸氨基 转移酶； ALP: 碱性磷酸酶； TBIL: 总胆红素； γ - GT: Υ谷氨酰转移酶）。
图 3是各组大鼠血清肝纤维化指标，
其中， 1 : 假手术组； 2: 肝纤维化组； 3: 干扰素一脂质体组； 4: RGD环 肽一脂质体一干扰素组。 与假手术组相比， ΔΡ<0. 05； 与 BDL组相比， * Ρ<0. 05； 与 BDL+IFN-SSL相比， AP<0. 05。 (HA: 透明质酸； PCIII ： III型前胶原; LN: 层粘蛋白； CIV: IV型胶原）。
图 4是各组大鼠肝组织羟脯氨酸（HYP)含量（mg/g肝组织），
其中， 1 : 假手术组； 2: 肝纤维化组； 3: 干扰素一脂质体组； 4: RGD环 肽一脂质体一干扰素组。 与假手术组相比， ΔΡ<0. 05； 与 BDL组相比， * P〈0. 05; 与 BDL+IFN- SSL相比， AP〈0. 05。
图 5是各组大鼠肝组织 I型胶原 mRNA表达，
其中， 1 : 假手术组； 2: 肝纤维化组； 3: 干扰素一脂质体组； 4: RGD环 肽一脂质体一干扰素组。 A: I型胶原 mRNA 电泳条带； B: I型胶原 mRNA / GAPDH mRNA相对灰度值。 与假手术组相比， ΔΡ<0. 05； 与 BDL组相比， * Ρ<0. 05； 与 BDL+IFN-SSL相比， AP<0. 05。
图 6是各组大鼠肝组织 α肌动蛋白 - SMA ) 的表达，
其中， 1 : 空白对照； 2: 假手术组； 3: 肝纤维化组； 4: 干扰素一脂质体组； 5： RGD环肽一脂质体一干扰素组。 具体实施方式 实施例 1 制备 RGD多肽
利用文献报道的方法（Schnolzer M， Alewood P, Jones A, Alewood D, Kent SB. Int J Pept Protein Res. 1992, 40 (3-4) : 180-93)， 在蛋白合成仪上固相合 成 半 胱 - 甘 - 精 - 甘 - 天 冬 - 色 - 脯 - 赖 硫 脂 PAM (CysGlyArgGlyAspSerProLys- SCH2C0- Leu- PAM)树脂。 与文献不同的是， Boc— 氨基酸（2. 2ramol )在含缩合剂 （HBTU 2. 0 mmol )与 N， N-二异丙基乙胺（DIEA 20%, v/v) 的 N， N-二甲基甲酰胺 （DMF)溶液中活化 3rain， 然后依次加入到树 脂 （0. 25mmol ) 中反应 10min。 N— Boc保护基团用三氟乙酸（TFA) 去除； 合成 的整个过程中， DMF与二氯甲烷 （DCM)用来清洗树脂。 所用氨基酸的侧链保护 基为： Arg (Tosyl ), Asp (OcHxl ), Cys (4MeBzl)， Lys (2C1Z)， Ser (Bzl)。 树脂合成完成后， 将其在 0°C于含 5%对甲酚 （p-cresol ) 的无水氟化氢中搅拌 lh， 冷乙醚沉淀得粗品， 高压制备液相纯化。 将纯化的样品溶于含 6M盐酸呱啶 ( GuHCl ) 的 0. 25M磷酸盐缓冲液 （pH 7. 5 )。 然后加入 2% ( v/v ) 苯硫酚 (thiophen0l)。 将溶液纯化、 冷冻干燥得 RGD环肽。 将制得的 RGD环肽与异硫 氰荧光素（FITC)在 pH9. 0的磷酸盐缓冲液中反应，纯化、冷冻干燥得 FITC - RGD 环肽。 经 HPLC鉴定纯度 >95%。 实施例 2制备脂质体
采用旋转蒸发一薄膜水化一挤压法， 通过如下步骤制备脂质体。
1. 按 2 : 1 : 0. 1 : 0. 02 (摩尔比）精密称取蛋磷脂（EPC)、 胆固醇（Chol )、 单甲氧基聚乙二醇-（mPEG2。。。_D0PE)、 马来酰亚胺-聚乙二醇 -二棕榈酰磷脂酰乙 醇胺 （MAL-PEG345。-D0PE)， 溶于氯仿， 40 °C水浴旋转蒸发成透明薄膜， 蒸干有机 溶剂。 加入磷酸盐缓冲液 (PBS, pH7. 4, 22 °C ) 充分水化。 用 Mini Extruder 反复挤压 15次过 lOOnm滤膜，得到均一的长循环脂质体（SSL)。 PEG占 3. 2mol%。
按 2 : 1 : 0. 02 (摩尔比）精密称取 EPC、 Choi 、 MAL- PEG34M- DOPE,溶于氯仿， 40°C水浴旋转蒸发成透明薄膜，蒸干有机溶剂。加入磯酸盐缓冲液（PBS， pH7. 4, 22 °C ) 充分水化。 用 Mini Extruder反复挤压 15次过 lOOnm滤膜， 得到均一的 普通脂质体 （CL)。
将 RGD环肽按照与 MAL-PEG-D0PE摩尔比 10： 1加入 CL或 SSL的 PBS溶液中， 室温（25°C )振荡过夜， 通过凝胶柱 （CL-4B)层析法去除未结合的 RGD环肽， 即可得到 RGD- CL或 RGD-SSL。
将待包封的干扰素（IFN_ a lb)溶液分别加入到 CL、 SSL、 RGD-CL、 RGD- SSL 中， 冰浴下旋涡振荡 30min， 通过凝胶柱（CL-4B)层析法除去未包入的 IFN， 即 可制得 CL- IFN、 SSL- IFN、 RGD- CL- IFN或 RGD- SSL- IFN。
在制备 CL、 SSL, RGD- CL或 RGD-SSL的过程中， 以钙黄绿素（calcein, CF) 溶液（50mmol/L)代替 PBS作为水化液， 其余步骤同前， 即可制得 CF脂质体。 以荧光分光光度计测得 CF的包封率为 10% ( λ ex=492nm， λ em=512nm)0
2. 形态和粒径研究： 取少量 SSL和 RGD-SSL稀释后， 用 1 %磷钨酸负染， 透射电子显微镜进行观察。可见脂质体经 RGD环肽修饰以及包裹干扰素后形态未 改变， 且分布均匀，呈典型的指紋结构。用激光粒度散射仪测定脂质体平均粒径 为 98.1±23. lnm。
包封率的测定： 釆用酶联免疫吸附法（ELISA)测定干扰素包封率为 35.6%， 载药率为 104U IFN /μπιοΐ憐脂。 采用病毒抑制法测定脂质体包裹干扰素后活性 不变。
3. 其他特性： 选择测定 5批 RGD-SSL- IFN进行检测。 ρΗ计测定的 ρΗ值为 7.21±0.08。 采用醋酸纤维素膜电泳法测定 RGD- SSL- IFN带负电荷 （测定条件： 上下电极液 0.01mol/L， pH 7.4磷酸盐缓冲液， 电压 200V)。 按照细管法原理用 乌氏粘度计测定相对粘度， 为（1.0323X10_3±3.7460X10—5) Pa. 用比重瓶 法测定相对密度为 1.0025 ±1.37X10一 4。 实施例 3 采用荧光素和放射性同位素示踪法考察人工合成的含有 RGD序列 环肽作为整合素受体配基可行性
根据受体一配体结合的特性， 即特异性、浓度和时间依赖性、竞争性抑制作 用， 将异硫氰酸荧光素（FITC)标记的 RGD环肽与 HSC共同孵育。 结果显示 RGD 环肽与 HSC结合性质符合作为受体配基的基本特征。 采用 ¾标记 RGD环肽进行 放射性配基 Scatchard分析， 平衡解离常数 （Kd)和每个细胞上的结合位点数 (Bmax) 分别为 7.05X 10—9 mmol/L和 6.79X 105。
步骤如下-
1. 分离大鼠 HSC
2. 荧光示踪法考察激活态 HSC与环肽的结合
将 HSC接种于 6孔板上， 贴壁后， 以 0.25%FBS-DMEM培养过夜。 实验前用 1%BSA-DMEM预封闭。 以流式细胞仪检测 HSC结合的相对荧光强度。
2.1 浓度一效应关系： 每孔加入不同浓度的环肽， 分别于 4°C和 37°C培养 。
2.2 时间一效应关系： 每孔加入环肽 200nmol/L， 分别于 4°C和 37°C分别孵 育 〜
2.3竞争抑制试验：每孔先加入不同浓度未标记环肽，然后于反应体系中加 入 200 nmol/L FITC标记的环肽， 再结合 4h。
3. 放射性配基结合分析法测定 RGD环肽与 HSC结合的平衡解离常数（Kd) 和每个细胞上的结合位点数（Bmax)
釆用氚标记 cRGD (3H-cRGD 将 0.5ml 细胞悬液分别与不同浓度 3H-cRGD 0. 1ml 置于反应管内， 补足体积至 lml， 4°C孵育 3h， 冰 Hank， s液中止反应， 离心弃上清， 沉淀与甲酸水浴， 测量沉淀放射性计数。 各水平样品均设 3复管， 设有总结合管 （Total binding, TB ) 和非特异性结合管（Non- specif ic binding: NSB)。 按 Scatchard模型求出 HSC与 ¾- cRGD结合的解离常数 Kd和每个细胞表 面最大结合位点（Bmax)。 实施例 4采用荧光素示踪法表征表面修饰有 RGD环肽脂质体与 HSC结合的 特性
按照实施例 2制备 RGD环肽修饰的包裹钙黄绿素的脂质体（RGD- SSL-CF)和 包裹钙黄绿素的脂质体 （SSL-CF)。 将 RGD-SSL-CF和 SSL- CF分别与 HSC共同孵 育 4h。结果显示 HSC摄取 RGD-SSL- CF为 SSL-CF 的 5. 4倍； RGD-SSL- CF能够特 异性的与 HSC结合。
步骤如下：
1. 分离大鼠的 HSC与培养
2.荧光示踪法考察激活态 HSC 与 RGD 环肽修饰的包裹钙黄绿素的 (RGD-SSL-CF) 的结合
将 HSC接种于 33cm2细胞培养皿上， 以 0. 25%FBS-DMEM培养过夜。 实验前用 1%BSA-DMEM预封闭。 分别加入 RGD- SSL- CF和 SSL- CF共同孵育 4h。 刮除细胞， 溶解于 PBS ( l %Triton X- 100)。 荧光分光光度计法检测细胞结合的荧光强度 (Ex=492nm, Em=512nm)。 以荧光显微镜显示 RGD-SSL- CF与 HSC结合情况。 实施例 5 9¾锝（99mTc)标记脂质体
将 DTPA— DOPE与磷脂按照 1 : 10 (摩尔比）加入脂质体膜材料中， 按照实施 2制备脂质体。 采用氯化亚锡 （SnCl2)还原法进行 RGD环肽一脂质体标记 99mTc。
可通过如下步骤： 将 SnCl2溶解于 0. 15mol/L的盐酸（HC1 )溶液中， 制备成 10ug/ul的 SnCl2- HC1溶液。 按照 0. 5ml脂质体加入 SnCl2 100ug, 再加入 99raTc04 一洗脱液 2mCi， 混勾， 室温放置 15分钟即可备用。 实施例 6 99mTc标记 RGD环肽修饰的脂质体（""Tc-RGD- SSL)在正常和肝纤 维化大鼠体内分布及显像研究
采用尾静脉注射法，考察 99mTc- RGD-SSL在正常大鼠和肝纤维化大鼠体内分布 行为及 SPET显像研究。结果显示 RGD- SSL主要在肝脏浓集， 持续达 24小时， 经 肾脏和胆道排泄。
通过如下步骤：
通过正常大鼠和肝纤维化大鼠尾静脉分别注射 99mTc-RGD- SSL和 99mTc-SSL 0. 5ml (2mCi ), 于不同时间点进行 SPECT ( single photon emission computed tomography)扫描显像， 仪器为 Phi lips— IRIX三探头 SPECT。 探头与大鼠保持 2cm的距离， 采集大鼠的正位相， 釆集矩阵为 512 X 512， 50秒 /帧， 绘制出各脏 器感兴趣区， 测定某脏器总计数 /脏器感兴趣区大小（counts/pixel ), 即每个脏 器每象素的计数。 实施例 7 RGD环肽修饰的干扰素脂质体对肝纤维化大鼠的治疗作用 采用尾静脉注射给药法， 观察 RGD环肽一脂质体一干扰素 （RGD-SSL-IFN) 对肝纤维化大鼠的治疗作用。 结果显示治疗组肝功能指标、 血清肝纤维化指标、 肝组织羟脯氨酸含量和肝脏病理学改变均较干扰素一脂质体 （SSL-IFN) 组有明 显改善；肝脏 I型胶原 mRNA和肝星状细胞的 α -激动蛋白表达量明显下降（图 1〜 6)。
通如下过步骤：
1. 动物模型的制备和实验分组 大鼠随机分成 4组，假手术组、模型（BDL) 组、 IFN- SSL治疗（BDL+IFN-SSL)组、 干扰素 -脂质体治疗（BDL+IFN- RGD- SSL) 组各 10只。 除假手术组外， 其余各组均行双重结扎并剪断胆总管。 假手术组， 剖腹后暴露、 游离胆总管上段约 lcm， 随即关腹。 于结扎之日起 BDL+IFN-SSL组 每周尾静脉注射 1次 0. 2ml (相当于 5 X 104U IFN)的 IFN- SSL,； BDL+IFN- RGD-SSL 组每周尾静脉注射 1次 0. 2ml IFN- RGD- SSL (相当于 5 X 10 U IFN)的 IFN-RGD-SSL; BDL组和假手术组分别给予等量的生理盐水处理。各组大鼠连续实验 4周后， 最 后 1次给药 24 h后处死动物， 留取血清和肝组织。
2. 观察指标及检测的内容和方法
2. 1观察大鼠一般状态以及黄疸情况。
2. 2 HE和 a -SMA免疫组织化学染色观察各组大鼠肝组织病理改变。
2. 3釆用自动生化分析仪测定血清谷丙转氨酶（ALT)、 谷草转氨酶（AST)、 总胆红素 （TBIL)、 碱性磷酸酶（AKP)、 y -GT ( Y -谷氨酰胺）。
2. 4放免法测定血清透明质酸（ΗΑ)、 III型前胶原（PCIII )、层粘蛋白（LN：)、 IV型胶原 (CIV)含量。
2. 5采用比色法测定肝组织匀浆羟脯氨酸（Hyp)， 以每 g肝组织 Hyp含量表 示。
2. 6 RT-PCR法检测肝组织 I型胶原 mRNA表达。
肝组织总 RNA的提取、 cDNA 的合成、共扩增定量 PCR及扩增产物定量分析， 扩增产物以凝胶系统分析，胶原 mRNA相对含量用 I型胶原吸光度 X面积与 GAPDH 吸光度 X面积的比值表示。
2. 7 Western blot检测 HSC a -SMA表达。
采用两步胶原酶灌注、 密度梯度离心法分离大鼠 HSC。 提取蛋白， 测定蛋白 浓度， 进行变性 SDS聚丙烯酰胺凝胶电泳， 电转在尼龙膜上， 5%脱脂奶粉封闭， 用一抗（兔抗鼠抗体）和辣根酶标记的二抗（驴抗兔抗体）孵育， 曝光显影。采 用 Biomad公司图形分析软件对 Western blot的特异性条带进行灰度扫描， 以积 分吸光度 AXmin2表示特异性条带信号的强弱， 从而对目的蛋白表达水平进行半 定量比较。

Claims

权利要求书
1、 一种含有精一甘一天冬氨酸序列的环肽， 其特征是： 含有精一甘一天冬氨酸 序列， 由酰胺键（- C0-NH- ) 成环， 其半胱氨酸一端含有活性巯基。
2、 按权利要求 1所述的含有精一甘一天冬氨酸序列的环肽， 其特征是所述的环 肽的氨基酸序列为 X*YRGDYZ*， 其中， *表示成环位置， X代表半胱氨酸残基其含 有一个游离的巯基， Y代表至少一个氨基酸或者足够长度的氨基酸序列并且不影 响与靶向受体结合， Z代表任意一个能与半胱氨酸残基成环的氨基酸。
3、 按权利要求 1或 2所述的含有精一甘一天冬氨酸序列的环肽， 其特征是所述 的环肽的氨基酸序列为 X*GRGDSPZ*， 其中， *表示成环位置， X代表半胱氨酸残 基， 其含有一个游离的巯基， Z代表至少一个氨基酸或足够长的氨基酸序列。
4、 按权利要求 1或 2或 3所述的含有精一甘一天冬氨酸序列的环肽， 其特征是 所说的环肽的氨基酸序列为 X*GRGDSPK*， 其中， *表示成环位置， X代表半胱氨 酸残基， 其含有一个游离的巯基。
5、 按权利要求 1或 2或 3或 4所述的含有精一甘一天冬氨酸序列的环肽在制备 与肝星状细胞表面整合素受体结合的外源性配基中的用途。
6、 一种主动靶向脂质体， 其特征是由权利要求 1的 RGD环肽与脂质体组成， 通 过下述方法制备，
将 RGD环肽的半胱氨酸残基上的游离巯基与脂质体膜材料中的马来酰亚胺 通过共价键连接， 反应方程式为：
Figure imgf000013_0001
7、 按权利要求 6所述主动靶向脂质体， 其中所述的脂质体的膜材料由卵磷脂、 胆固醇、 单甲氧基聚乙二醇类脂衍生物和马来酰胺-聚乙二醇类脂衍生物组成。
8、 按权利要求 7所述主动靶向脂质体， 其中所述的脂质体的膜材料卵磷脂、 胆 固醇、 单甲氧基聚乙二醇类脂衍生物和马来酰胺 -聚乙二醇类脂衍生物的摩尔比 为 2 : 1 : 0. 1 : 0. 02。 ' '
9、 按权利要求 7所述主动靶向脂质体， 其中所述的脂质体的膜材料中聚乙二醇 占 3. 2mol%o
10、按权利要求 6所述主动靶向脂质体，其特征是所述主动靶向脂质体包裹干扰 素制成载药主动靶向脂质体。
11、按权利要求 6所述主动靶向脂质体，其中所述的 RGD环肽上的半胱氨酸残基 所含的游离巯基， 是与脂质体的结合位点。
12、 按权利要求 6所述主动靶向脂质体， 其中所述的脂质体粒径范围为 50〜 200nm。
13、 按权利要求 6所述主动靶向脂质体， 其中所述的脂质体粒径为 100nm。
14、 按权利要求 10所述的主动靶向脂质体在制备治疗肝纤维化药物中的用途。
15、 按权利要求 10所述的主动靶向脂质体在制备静脉注射治疗肝纤维化药物中 的用途。
PCT/CN2005/001258 2005-03-25 2005-08-15 Peptide cyclique comportant une rgd et son liposome a ciblage actif WO2006099779A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/990,577 US20100098748A1 (en) 2005-03-25 2005-08-15 Arg-Gly-Asp (RGD) Sequence Containing Cyclic Peptide and Its Active Targeting Liposomes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200510024656.8 2005-03-25
CNB2005100246568A CN100436477C (zh) 2005-03-25 2005-03-25 含精-甘-天冬氨酸序列环肽及其主动靶向脂质体

Publications (1)

Publication Number Publication Date
WO2006099779A1 true WO2006099779A1 (fr) 2006-09-28

Family

ID=35305160

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2005/001258 WO2006099779A1 (fr) 2005-03-25 2005-08-15 Peptide cyclique comportant une rgd et son liposome a ciblage actif

Country Status (3)

Country Link
US (1) US20100098748A1 (zh)
CN (1) CN100436477C (zh)
WO (1) WO2006099779A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115028683A (zh) * 2022-06-13 2022-09-09 陕西慧康生物科技有限责任公司 五肽化合物及其在制备治疗肝纤维化疾病药物中的应用
CN115028683B (zh) * 2022-06-13 2024-05-14 陕西慧康生物科技有限责任公司 五肽化合物及其在制备治疗肝纤维化疾病药物中的应用

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101327190B (zh) * 2008-07-29 2011-04-27 北京大学 一种供注射用的抗肿瘤长循环靶向脂质体
US20130202652A1 (en) * 2010-07-30 2013-08-08 Alnylam Pharmaceuticals, Inc. Methods and compositions for delivery of active agents
CN101991867B (zh) * 2010-10-22 2013-01-23 复旦大学附属中山医院 用于早期肝纤维化诊断的多模式靶向探针及其制备方法
CN102008736B (zh) * 2010-12-10 2012-10-31 复旦大学附属中山医院 一种靶向环肽修饰的脂质体微泡及其制备方法
US9775803B2 (en) 2011-10-19 2017-10-03 Samsung Electronics Co., Ltd. Liposome comprising elastin-like polypeptide and tumor cell targeting material and use thereof
JP2016522213A (ja) * 2013-06-03 2016-07-28 ユニヴァーシティ オブ サザン カリフォルニアＵｎｉｖｅｒｓｉｔｙ ｏｆ Ｓｏｕｔｈｅｒｎ Ｃａｌｉｆｏｒｎｉａ 目標の架橋多重膜リポソーム
WO2016207281A1 (en) * 2015-06-26 2016-12-29 Spiber Technologies Ab Cyclic rgd cell-binding motif and uses thereof
CN106310220A (zh) * 2016-08-17 2017-01-11 浙江中医药大学 一种包封鳖甲肽的rgd‑ssl脂质体及其制备方法
CN112480245B (zh) * 2020-12-19 2023-08-18 上海佰君生物科技有限公司 一种疏水性环状肽配基纯化人免疫球蛋白g的应用
CN113332442A (zh) * 2021-06-10 2021-09-03 愈美明德(成都)生物医药科技有限公司 一种靶向递送分子、粒子及其制备方法、应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5770565A (en) * 1994-04-13 1998-06-23 La Jolla Cancer Research Center Peptides for reducing or inhibiting bone resorption
US20050186264A1 (en) * 2000-10-12 2005-08-25 Kiani Mohammad F. Targeting drug/gene carriers to irradiated tissue
MXPA03009760A (es) * 2001-04-25 2005-10-05 Eidgenoessische Technische Hoc Matrices de suministro de farmacos para mejorar la curacion de heridas.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YANG Y. ET AL.: "Progress in the Study on the Synthesis of RGD-Containing Cyclopeptide and Its Analogues", CHINESE JOURNAL OF ORGANIC CHEMISTRY, vol. 22, no. 4, 2002, pages 239 - 247 *
ZHANSHAN L.I. ET AL.: "Study on the Tumor Targeting of RGD-PEG-liposomes", CHINA PHARMACY, vol. 14, no. 7, 2003, pages 393 - 394 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115028683A (zh) * 2022-06-13 2022-09-09 陕西慧康生物科技有限责任公司 五肽化合物及其在制备治疗肝纤维化疾病药物中的应用
CN115028683B (zh) * 2022-06-13 2024-05-14 陕西慧康生物科技有限责任公司 五肽化合物及其在制备治疗肝纤维化疾病药物中的应用

Also Published As

Publication number Publication date
CN1687118A (zh) 2005-10-26
CN100436477C (zh) 2008-11-26
US20100098748A1 (en) 2010-04-22

Similar Documents

Publication Publication Date Title
WO2006099779A1 (fr) Peptide cyclique comportant une rgd et son liposome a ciblage actif
JP6789823B2 (ja) 両末端ｐｅｇ化インテグリン結合性ペプチドおよびその使用方法
US6248864B1 (en) Compounds and methods and modulating tissue permeability
ES2525649T3 (es) Derivados peptídicos y su uso como vectores de moléculas en forma de conjugados
Wang et al. Self-assembling peptide-based nanodrug delivery systems
US20150218252A1 (en) Self-assembling peptides, peptide nanostructures and uses thereof
AU773028B2 (en) Compounds and methods for modulating claudin-mediated functions
Sekido et al. Novel drug delivery system to bone using acidic oligopeptide: pharmacokinetic characteristics and pharmacological potential
US8828928B2 (en) Amphiphilic peptides and peptide particles
TW201031424A (en) Breast cancer-targeting peptides and use thereof
WO2021167107A1 (ja) ヒトトランスフェリンレセプター結合ペプチド
Ruan et al. Stapled RAP12 peptide ligand of LRP1 for micelles-based multifunctional glioma-targeted drug delivery
US9566347B2 (en) Peptides and methods using same
CN109563135B (zh) α(V)β(6)整合素结合肽及其使用方法
RU2013146690A (ru) Циклические пептиды с антинеопластической и антиангионенной активностью
US20030170752A1 (en) Alpha-fetoprotein peptides and uses thereof
US20080138284A1 (en) Novel Peptides That Promote Lipid Efflux
WO2021205001A9 (en) Peptides and uses thereof
US20080199398A1 (en) Novel Peptides That Promote Lipid Efflux
US20080227686A1 (en) Novel Peptides that Promote Lipid Efflux
US20080206142A1 (en) Novel Peptides That Promote Lipid Efflux
CN114073774A (zh) 药物和野生型穿膜肽衍生物的组合物
US10167326B2 (en) Alpha-fetoprotein “ring and tail” peptides
WO2021260068A1 (en) Vasopressin-2 receptor antagonist peptides and uses thereof
NZ622045B2 (en) Peptide nanoparticles and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

NENP Non-entry into the national phase

Ref country code: RU

WWW Wipo information: withdrawn in national office

Country of ref document: RU

WWE Wipo information: entry into national phase

Ref document number: 11990577

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 05774473

Country of ref document: EP

Kind code of ref document: A1

WWW Wipo information: withdrawn in national office

Ref document number: 5774473

Country of ref document: EP