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Cyclic peptide containing arginine, glycine, asparagicacid-sequence and active target liposome

一种天冬氨酸、序列的技术，应用在主动靶向脂质体,治疗肝纤维化领域，达到良好抗纤维化疗效、分子量小、便于修饰的效果

Inactive Publication Date: 2008-11-26
ZHONGSHAN HOSPITAL FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no report on HSC, integrin receptor-mediated active targeting liposomes for the treatment of liver fibrosis

Method used

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  • Cyclic peptide containing arginine, glycine, asparagicacid-sequence and active target liposome
  • Cyclic peptide containing arginine, glycine, asparagicacid-sequence and active target liposome
  • Cyclic peptide containing arginine, glycine, asparagicacid-sequence and active target liposome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 prepares RGD polypeptide

[0037] Using the method reported in the literature (Schnolzer M, Alewood P, Jones A, Alewood D, KentSB. Int J Pept Protein Res.1992, 40 (3-4): 180-93), solid-phase synthesis of cysteine ​​on a protein synthesizer -Glyn-ArgGlyAspSerProLys-Sch 2 CO-Leu-PAM) resin. Different from the literature, Boc-amino acid (2.2mmol) in N,N-dimethylformaldehyde containing condensing agent (HBTU 2.0mmol) and N,N-diisopropylethylamine (DIEA20%, v / v) Activated in amide (DMF) solution for 3 minutes, and then sequentially added to resin (0.25 mmol) to react for 10 minutes. The N-Boc protecting group was removed with trifluoroacetic acid (TFA); DMF and dichloromethane (DCM) were used to wash the resin throughout the synthesis. The side chain protecting groups of the amino acids used are: Arg (Tosyl), Asp (OcHxl), Cys (4MeBzl), Lys (2ClZ), Ser (Bzl). After the resin was synthesized, it was stirred at 0°C for 1 h in anhydrous hydrogen fluoride contain...

Embodiment 2

[0038] Embodiment 2 prepares liposome

[0039] Using rotary evaporation-film hydration-extrusion method, the liposomes were prepared through the following steps.

[0040] 1. Precisely weigh lecithin (EPC), cholesterol (Chol), monomethoxypolyethylene glycol-(mPEG) according to 2:1:0.1:0.02 (molar ratio) 2000 -DOPE), maleimide-polyethylene glycol-dipalmitoylphosphatidylethanolamine (MAL-PEG 3450 -DOPE), dissolved in chloroform, 40 ℃ water bath rotary evaporation to form a transparent film, evaporated to dry organic solvent. Phosphate buffered saline (PBS, pH 7.4, 22° C.) was added to fully hydrate. Use the Mini Extruder to repeatedly squeeze 15 times through the 100nm filter membrane to obtain uniform long-circulating liposomes (SSL). PEG accounts for 3.2 mol%.

[0041] Accurately weigh EPC, Chol, MAL-PEG according to 2:1:0.02 (molar ratio) 3450 -DOPE, dissolved in chloroform, evaporated in a water bath at 40°C to form a transparent film, and evaporated the organic solvent ...

Embodiment 3

[0048] Example 3 Using fluorescein and radioisotope tracer method to investigate the feasibility of artificially synthesized cyclic peptide containing RGD sequence as integrin receptor ligand

[0049] According to the properties of receptor-ligand binding, namely specificity, concentration and time dependence, competitive inhibition, fluorescein isothiocyanate (FITC)-labeled RGD cyclic peptide was co-incubated with HSCs. The results showed that the binding properties of RGD cyclic peptide to HSC conformed to the basic characteristics of receptor ligand. use 3 H-labeled RGD cyclic peptide was analyzed by radioligand Scatchard, and the equilibrium dissociation constant (Kd) and the number of binding sites (Bmax) on each cell were 7.05×10 -9 mmol / L and 6.79×10 5 .

[0050] Proceed as follows:

[0051] 1. Isolation of Rat HSCs

[0052] 2. Fluorescence tracer method to investigate the binding of activated HSC and cyclic peptide

[0053] HSCs were seeded on 6-well plates, and ...

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Abstract

The present invention belongs to the field of pharmaceutical technology and clinical pharmacy, relates to a cyclopeptide containing arginine-glycine-aspartic acid (RGD) sequence and its active liposome targeting, its preparation and application. The described cyclopeptide structure meets the requirements as ligand, and is cyclized by amido bond, its spatial conformation is stable, and is not easily degraded, its one end contains active sulphydryl group, and is convenient for modifying artificially-synthetic function peptide, its molecular weight is small, so that it is not easy to result in immune reaction, and can be used as ligand, and can be combined with hepatic stellate cell (HSC) surface integrator acceptor to construct active liposome targeting and implement target therapy for experimental hepatic fibroid cell. Said invention can obtain good therapeutic effect for curing hepatitis fibrosis.

Description

technical field [0001] The invention belongs to the field of pharmacy and clinical pharmacy, and relates to a polypeptide sequence of an integrin receptor ligand containing arginine-glycine-aspartic acid (RGD) sequence and its cyclic form, and the construction of integrin receptor-mediated , Actively targeting liposomes against hepatic stellate cells and medicinal use. It specifically relates to a cyclic peptide containing arginine-glycyl-aspartic acid (RGD) sequence and its active targeting liposome, a preparation method and an application for treating liver fibrosis. Background technique [0002] Liver fibrosis is the common pathological basis of all chronic liver diseases, and it is the early and necessary stage of liver cirrhosis. According to statistics, 25% to 40% of them eventually develop into liver cirrhosis. Its pathological changes can be caused by different etiologies. For example, after chronic liver damage caused by viruses, ethanol, and parasites, hepatic ste...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/64A61K47/42A61K47/48A61K9/127A61P1/16
CPCA61K9/0019A61K47/48238A61K9/127A61K9/1271A61K47/48815C07K7/64A61K47/62A61K47/6911A61P1/16
Inventor 王吉耀杜施霖陆伟跃
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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