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Phosphorothioate modified nucleic acid aptamer medicine conjugate as well as a preparation method and application thereof

A technology of full phosphorothioate and nucleic acid aptamer, which can be applied in the field of medicine and can solve problems such as reduced efficacy

Active Publication Date: 2020-08-14
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The naked natural nucleic acid is easily recognized and cleaved by various nucleases in the body in the blood circulation, so it may have been degraded before reaching the target lesion site, resulting in a greatly reduced efficacy

Method used

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  • Phosphorothioate modified nucleic acid aptamer medicine conjugate as well as a preparation method and application thereof
  • Phosphorothioate modified nucleic acid aptamer medicine conjugate as well as a preparation method and application thereof
  • Phosphorothioate modified nucleic acid aptamer medicine conjugate as well as a preparation method and application thereof

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preparation example Construction

[0044] The second aspect of the present invention provides a method for preparing the all-phosphorothioate-modified nucleic acid aptamer-drug conjugate provided in the first aspect of the present invention, comprising: linking the drug molecule with a nucleic acid fragment substituted by a phosphorus-sulfur bond to provide The method for providing the phosphorothioate-modified nucleic acid aptamer-drug conjugate with phosphorus-sulfur bond-substituted nucleic acid fragments can specifically refer to the literature methods given above.

[0045] In the preparation method provided by the present invention, the preparation method may specifically include: 1) modifying the drug molecule with a linker molecule; 2) reacting the drug molecule modified with the linker molecule with a nucleic acid fragment substituted with a phosphorus-sulfur bond to provide the All phosphorothioate-modified nucleic acid aptamer-drug conjugates.

[0046] In the preparation method provided by the present...

Embodiment 1

[0056] A. Preparation of 4-nitrophenyl-4-(2-dimercaptopyridyl) ethyl carbonate:

[0057]

[0058] Add 88mg (0.4mmol) 2,2'-pyridine dimercapto, 31.3mg (0.4mmol) 2-mercaptoethanol, and 10mL dichloromethane into a 50mL three-neck round bottom flask. Stirring at room temperature for 2h, the reaction process was monitored by TLC. Dichloromethane was removed by rotary evaporation under reduced pressure, and purified by silica gel column reaction to obtain 20 mg of 2-(2-dimercaptopyridyl)ethanol as a yellow-white oily liquid with a yield of 53%.

[0059] Add 18.7mg (0.1mmol) 2-(2 dimercaptopyridyl) ethanol to 50mL three-neck round bottom flask, add 10mL anhydrous dichloromethane, add p-nitrophenyl chloroformate 40mg (0.2mmol), N , N'-diisopropylethylamine 25.9mg (0.2mmol), 4-dimethylaminopyridine 1.22mg (catalytic amount), stirred overnight at room temperature, and the reaction process was monitored by TLC. After the reaction is complete, add 0.01M hydrochloric acid solution to ...

Embodiment 2

[0067] The targeted uptake ability of the thionucleic acid aptamer-mitomycin C conjugate by tumor cells:

[0068] Further investigate the ability of the thionucleic acid aptamer drug conjugate S-ApDC (prepared in Example 1) to selectively enter tumor cells, and at the same time add the control sequence drug conjugate CpDC (5'-ATTGCACTTACTATATTGCACTTACTATATTGCACTTACTAT-3', SEQ ID NO .2, not thioxated, the preparation method refers to Example 1, only the nucleic acid sequence is different). PL45 cells or MCF-7 cells were digested and inoculated into 12-well plates at a density of 100,000 / dish. After culturing in a 37°C incubator for 24 hours, they were washed three times with DPBS, and then 200 μL of 250 nM Cy5-labeled single-chain (XQ- 2d or S-XQ-2d, unthio aptamer and thio aptamer respectively, neither drug-conjugated, SEQ ID NO.1) and S-ApDC / CpDC, incubated at 37°C for 2h and washed with DPBS After 3 times, it was digested with trypsin for 1-2min, and detected by flow cytome...

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Abstract

The invention relates to the field of medicines, in particular to a phosphorothioate modified nucleic acid aptamer medicine conjugate as well as a preparation method and an application thereof. The phosphorothioate modified nucleic acid aptamer drug conjugate comprises a drug molecule group and a phosphorus-sulfur bond substituted nucleic acid aptamer fragment, wherein the drug molecule group is selected from a mitomycin C group, and the drug molecule group is connected with the phosphorus-sulfur bond substituted nucleic acid aptamer fragment through a linking group. The phosphorothioate modified aptamer drug conjugate provided by the invention not only has all the advantages of an aptamer, but also greatly improves the enzyme digestion resistance of the aptamer-mitomycin C conjugate and prolongs the blood circulation half-life period by modifying phosphorothioate of a phosphate skeleton of an aptamer fragment; and in addition, the stability is improved, meanwhile, the original targeting property and specificity are reserved, and the high industrialization prospect is achieved.

Description

technical field [0001] The present invention relates to the field of medicines, in particular to a nucleic acid aptamer drug conjugate modified by phosphorothioate and its preparation method and application. Background technique [0002] Mitomycin is an anti-tumor antibiotic isolated from the culture medium of Streptomyces capitus, which is effective against a variety of solid tumors. It can form cross-links with the double helix structure of DNA, destroy the structure and function of DNA, and inhibit proliferation. DNA replication in the proliferating phase can kill cells in the proliferating phase, and can also act on cells in the quiescent phase, thereby inhibiting tumor cells. It is clinically applicable to digestive tract cancers, such as gastric cancer, intestinal cancer, liver cancer, and pancreatic cancer. [0003] Nucleic acid aptamer (Aptamer) is an oligonucleotide fragment obtained from a nucleic acid molecule library by using in vitro screening technology-systema...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/54A61K31/407A61P35/00C07H21/02C07H1/00
CPCA61K47/549A61K31/407A61P35/00C07H21/02C07H1/00
Inventor 谭蔚泓王雪强杨秋霞符婷
Owner HUNAN UNIV
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