Tumor targeting polypeptide-medicine coupling derivative, and preparation method and application thereof

A tumor-targeting and targeting peptide technology, which can be used in anti-tumor drugs, drug combinations, pharmaceutical formulations, etc., to solve problems such as limitations, reduced efficacy, and weak penetration

Inactive Publication Date: 2018-04-24
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many antineoplastic drugs currently in clinical use, but traditional chemotherapeutic drugs, such as doxorubicin, maytansine, auristatin, and paclitaxel, generally have strong cytotoxicity. While potently killing tumor cells, it will also...

Method used

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  • Tumor targeting polypeptide-medicine coupling derivative, and preparation method and application thereof
  • Tumor targeting polypeptide-medicine coupling derivative, and preparation method and application thereof
  • Tumor targeting polypeptide-medicine coupling derivative, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Preparation method of tumor targeting, long-acting bifunctional polypeptides ABD-RGDK and ABD-RPARPAR

[0077] (1) Construct the amino acid sequence:

[0078] ABD-RGDK: CGSLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGSGGGSGGGSSRGDK

[0079] ABD-RPARPAR: CGSLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGSGGGSGGGSSRPARPAR

[0080] (2) Nucleotide sequence:

[0081] ABD-RGDK: GGTTGCGGTTCTCTGGCTGAAGCTAAAGTCCTGGCAAATCGTGAGCTGGACAAATATGGTGTGTCCGATTTCTACAAACGCCTGATCAACAAAGCGAAGACCGTTGAAGGTGTAGAAGCACTGAAACTGCACATTCTGGCCGCGCTGCCGGGTGGCGGTTCCGGTGGCGCAGCGGCGGCGGTAGCTCTCGTGGCGACAAA

[0082]ABD-RPARPAR: GGTTGCGGTTCTCTGGCTGAAGCTAAAGTCCTGGCAAATCGCGAGCTGGACAAGTATGGCGTGTCCGATTTCTACAAACGTCTGATCAACAAAGCGAAAACCGTAGAAGGTGTTGAAGCGCTGAAACTGCACATTCTGGCCGCGCTGCCTGGCGGTGGCTCCGTCGGCGTAGCGGTGGCGGTTCTAGCCGTCACCGGCACG;

[0083] The electrophoresis of the fusion peptide is shown in figure 1 (a)-(b);

[0084] (3) The plasmid type is selected as pET-30a, and the host bacteria is E....

Embodiment 2

[0087] Example 2: Preparation method of tumor targeting, long-acting bifunctional polypeptide-doxorubicin derivative

[0088] (1): Synthesis of ABD-RGDK-DOXO and ABD-RPARPAR-DOXO:

[0089] Synthesis of ABD-RGDK-DOXO: Weigh 60 mg of lyophilized powder of ABD-RGDK peptide, dissolve in 6 mL of 100 mM Na at pH 8.5-9.5 2 CO 3 -NaHCO 3 Add 320μL 50mM TCEP mother solution to the solution, reduce at room temperature for 20min, then weigh 18.57mg of DOXO-EMCH solid, fully dissolve in 4mL DMF, then add dropwise to the peptide solution at a speed of 30μL / min, stir gently, and Protect from light for 2-4h. Finally, centrifuge at 10,000 rpm at 4°C for 5 min to remove unreacted precipitates to obtain the crude product of ABD-RGDK-DOXO.

[0090] Synthesis of ABD-RPARPAR-DOXO: Weigh 60mg of lyophilized powder of ABD-RGDK peptide, dissolve in 6mL of 100Mm Na with pH8.5-9.5 2 CO 3 -NaHCO 3 Add 280μL 50mM TCEP mother solution to the solution, reduce it at room temperature for 20min, then w...

Embodiment 3

[0102] Example 3: Uptake of drugs by tumor cells

[0103] A549 cells in good growth state were selected, digested with trypsin, and plated in a 96-well plate with a plate density of 4×10 4 cells / hole. Cells were incubated at 37°C, 5% CO 2 Incubate in the incubator for 24 hours to allow the cells to adhere to the wall, then remove the medium and replace it with 100 μL drug-containing medium, the drugs are DOX, ABD-RGDK-DOXO, ABD-RPARPAR-DOXO, ABD-RGDK-DOXO+RGDK peptide , ABD-RPARPAR-DOXO+RPARPAR peptide, the administration concentration is 20uMDOXeqv and 500μM peptide, and three replicate wells are set up for each group of drugs. After the drug was applied for 1, 2, and 8 hours, the drug was removed, the wells were washed three times with PBS, and then 100 μL PBS was added to measure the absorption value of each well with a fluorescent microplate reader. The excitation wavelength was 480 nm, and the emission wavelength was 580 nm. Plot the cellular uptake fluorescence intens...

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Abstract

The invention belongs to the technical field of biological pharmacy, and relates to a tumor targeting polypeptide-medicine coupling derivative, and a preparation method and application thereof. The tumor targeting polypeptide-medicine coupling derivative comprises targeting polypeptides, long effect polypeptides and medicine molecules, wherein the targeting polypeptides are connected onto the endC of the long effect polypeptides through flexible connecting peptides; fusion peptides are used as carriers of the medicine molecules; the medicine carriers and the long effect polypeptides in the fusion peptides are connected through chemical bonds; and the long effect polypeptides are polypeptide or protein structural domains having the affine mutual effects with human serum albumin. The tumortargeting polypeptide-medicine coupling derivative can be combined with the human serum albumin; the remaining half-life period in the blood circulation system can be obviously prolonged; the long-term effectiveness in the body is realized; the targeting and seepage into the tumor tissues or cells can be realized; then, free effect molecules are released in tumor tissue micro acid environments orin cells in through an acid hydrolysis mechanism; and a better anti-tumor efficiency is achieved.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a tumor-targeting polypeptide-drug coupling derivative, a preparation method and application thereof. Background technique [0002] Malignant tumor is a common disease that seriously threatens human life, and its treatment has always been a worldwide problem. There are many antineoplastic drugs currently in clinical use, but traditional chemotherapeutic drugs, such as doxorubicin, maytansine, auristatin, and paclitaxel, generally have strong cytotoxicity. While effectively killing tumor cells, it will also cause damage to normal tissues and cells of the human body. In addition, such drugs have defects such as weak penetration in cells and rapid metabolism in the body, which greatly reduce their curative effect and greatly limit their clinical application. Therefore, researchers have been working to find strategies to increase drug efficacy and safety. With the develop...

Claims

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Application Information

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IPC IPC(8): A61K47/64A61K31/704A61P35/00
CPCA61K31/704
Inventor 刘永东张纯苏志国
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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