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Autophagy inhibitor and arginase medicine compound and application thereof

An autophagy inhibitor and drug complex technology, applied in the field of medicine, can solve the problems that have not been seen before, and achieve the effects of prolonging the half-life, enhancing the killing effect and reducing the immunogenicity

Inactive Publication Date: 2015-02-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] So far, there have been no reports on the induction of tumor cell autophagy by human recombinant arginase and the combination of autophagy inhibitors and human recombinant arginase in the treatment of tumors

Method used

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  • Autophagy inhibitor and arginase medicine compound and application thereof
  • Autophagy inhibitor and arginase medicine compound and application thereof
  • Autophagy inhibitor and arginase medicine compound and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of human recombinant arginase.

[0035] The coding sequence encoding human arginase was obtained by PCR method, the gene coding sequence is 969bp in length, with restriction sites XhoI and EcoRI; the PCR product and the empty plasmid vector were double-digested with XhoI and EcoRI endonucleases respectively , connect the target gene to the carrier, transform the ampicillin resistance plate, pick several positive transformants, take one of them to amplify and extract the plasmid, and carry out double digestion identification with XhoI and EcoRI endonucleases; extract the recombinant plasmid, and convert the recombinant vector Digest with BglII to linearize, transform Pichia pastoris by electric shock; pick positive transformants; screen through RDB, MM and MD plates to obtain clones with the phenotype his+muts, and further induce expression at the shaker level, The expression of arginase on the shaker level was detected by SDS-PAGE protein electrop...

Embodiment 2

[0037] Embodiment 2: the prescription of autophagy inhibitor drug

[0038] (1) Preparation of chloroquine: Dissolve an appropriate amount of chloroquine in pure water to form a 10mmol / L storage solution, filter and sterilize with a 0.1 μm filter and store at 4°C, dilute 500-1000 times with PRMI-1640 medium for in vitro experiments Used to inhibit autophagy;

[0039] (2) Preparation of ammonium chloride: Dissolve an appropriate amount of ammonium chloride in water to prepare a 0.4 mol / L storage solution, filter and sterilize with a 0.1 μm filter, and store at 4°C. In vitro experiments were diluted 50-80 times to inhibit cell autophagy;

[0040] (3) Preparation of hydroxychloroquine: Dissolve an appropriate amount of hydroxychloroquine in pure water to form a 10mmol / L stock solution, filter and sterilize with a 0.1 μm filter, store at 4°C, and dilute 500-1000 times in the in vitro laboratory for inhibition autophagy;

[0041] (4) Preparation of 3-MA: Take an appropriate amoun...

Embodiment 3

[0045] Example 3: Human recombinant arginase can induce autophagy in melanoma A375 and breast cancer MDA-MB-231 and MCF-7

[0046] After A375 cells were treated with 3.2IU / ml arginase for 24h, they were paraffin-embedded, sectioned and stained, and the submicroscopic structure of the cells was observed under a transmission electron microscope. The results were as follows: figure 1 As shown in A, there are a large number of typical double-membrane autophagosomes in the cells of the administration group, but not found in the control group; the eGFP-LC3 plasmid was transferred into melanoma A375, and then 3.2IU / ml of arginine was used to Acidase was treated for 24 hours, and then the green fluorescent spots were observed under the laser confocal microscope, the results were as follows: figure 1 B shows that there are a large number of fluorescent spots in the cells of the administration group, while the control group has fewer;

[0047] The collected melanoma A375 cells were w...

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Abstract

The invention relates to the field of biological pharmacy, and provides a new medicine compound for treating tumors, and particularly relates to an autophagy inhibitor and arginase medicine compound and application thereof. The medicine compound is used for treating the tumors. According to the medicine compound, the antineoplastic activity of arginase can be obviously strengthened in vitro; the immunogenicity of a person for recombing the arginase can be reduced; and the half-life period of the arginase in vivo can be prolonged. The arginase modified by poly-sialic acid is provided; and the arginase modified by the poly-sialic acid is characterized in that the active activated poly-sialic acid is reacted with the arginase under the condition that sodium cyanoborohydride exists to form conjugates. By means of the autophagy inhibitor and arginase medicine compound, the killing effect of the arginase on the tumors can be strengthened, the curative effect of the arginase can be strengthened, and the curative effects of the arginase on treating the melanin tumor and the breast cancer can be strengthened.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a new drug compound for treating tumors; in particular, it relates to a drug compound of an autophagy inhibitor and arginase and its application; the drug compound is used for treating tumors, especially melanoma and liver cancer. Background technique [0002] Arginase is a key enzyme involved in the urea cycle in the mammalian liver, where it converts arginine into ornithine and urea. The prior art discloses that arginine is an essential amino acid for maintaining the growth of some tumor cells, and when the level of arginine in tumor cells is lower than a certain value, the cells will die. Arginase can specifically and efficiently degrade arginine in vitro and in vivo. Arginine plays an important role in the normal physiological functions of the human body. It is a semi-essential amino acid. Normal body cells can convert other amino acids into arginine through the urea cycle, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61P35/00A61K38/50
Inventor 鞠佃文王子玉赵舒薇李玉彬曾贤范佳君王绍飞李力
Owner FUDAN UNIV
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