Cell autophagy inhibitor, preparation method thereof and application

An autophagy inhibitor and inhibitor technology, applied in the field of medicine, to achieve the effect of mature extraction process, wide sources, and broad clinical application prospects

Active Publication Date: 2017-12-22
CHINA PHARM UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no Rubiaceae-type cyclic peptide to inhibit KRAS mutation, especially KRAS-dependent tumor cell protective autophagy to promote its apoptosis, and the preparation method and application of nanomicelle injection as an anti-tumor drug

Method used

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  • Cell autophagy inhibitor, preparation method thereof and application
  • Cell autophagy inhibitor, preparation method thereof and application
  • Cell autophagy inhibitor, preparation method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] RA-V(1) inhibits KRAS-dependent protective autophagy in tumor cells:

[0030] Cell viability was measured by MTT assay. The KRAS-independent A549 and H460 and KRAS-dependent H441 and H358 cell lines cultured overnight in 10% FBS medium were digested with trypsin to form a cell suspension, and seeded on a 96-well plate at an appropriate concentration, 100 μl / well , at CO 2 Cultivate in the incubator for 24 hours until the cells are completely attached to the wall, add RA-V(1) at a final concentration of 0, 50, 100, 200 nM, and after 24 hours of action, add 20 μl of MTT solution (5 mg / ml prepared in PBS, pH= 7.4), continue to incubate for 4 hours, terminate the culture, and carefully aspirate and discard the culture supernatant in the well. Add 150 μl DMSO to each well and shake for 10 minutes to fully melt the crystals. Select a wavelength of 490nm, measure the light absorption value of each well on an enzyme-linked immunosorbent assay instrument, record the results, ...

Embodiment 2

[0035] RA-V(1) significantly induces apoptosis of KRAS-dependent tumor cells:

[0036] The KRAS-independent A549 and H460 and KRAS-dependent H441 and H358 cell lines cultured overnight in 10% FBS medium were digested with trypsin to form a cell suspension, which was seeded on a 6-well plate at an appropriate concentration, and added after 24 hours RA-V(1), treated for 24 hours, digested with trypsin, centrifuged at room temperature 2000rpm for 5-10min, collected cells; resuspended cells once in pre-cooled 1×PBS (4°C), centrifuged at 2000rpm for 5-10min, washed cells; added Suspend cells in 300 μL of 1×Binding Buffer; add 5 μL of Annexin V-FITC to mix well, protect from light, and incubate at room temperature for 15 minutes; add 5 μL of PI for staining 5 minutes before going to the machine, and detect cells by flow cytometry.

[0037] The KRAS-independent A549 and H460, KRAS-dependent H441 and H358 cells cultured overnight in 10% FBS medium were planted in a 24-well plate at an...

Embodiment 3

[0040] Preparation method of mPEG200-PDLLA2000 micelles:

[0041] (1) Dissolution: Prepare drug-loaded nanomicelles according to the weight ratio of mPEG2000-PDLLA2000 and RA-V(1) 30-10:1, accurately weigh mPEG2000-PDLLA2000 and RA-V(1) according to Table 1, and mix them Dissolve in a pear-shaped bottle containing 100mL of dichloromethane, shake continuously until the drug and materials are dissolved, then add 25mL of methanol, shake continuously until the drug and materials are completely dissolved, and obtain a clear solution after about 5 minutes.

[0042] (2) Evaporation of solvent: place the pear-shaped bottle on a rotary evaporator, vacuum rotary evaporation, the rotating speed is 100 rpm, the control temperature is 60 ° C, and the organic solvent is removed. After it is completely removed, the temperature is reduced to 40 °C, continue vacuum rotary evaporation for 3 hours to remove the residual organic solvent to obtain a transparent gel-like mixed drug film of RA-V (1)...

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Abstract

The invention discloses a cell autophagy inhibitor taking rubiaceae-type cyclopeptides as effective components. The cell autophagy inhibitor is particularly used for inhibiting KRAS mutation, particularly KRAS dependent tumor cell protective autophagy, and promoting tumor cell apoptosis. The inhibitor is preferably a nano-micelle injection and can be applied to preparation of medicines for treating and preventing KRAS related cancers such as colon cancers, rectal cancers, lung cancers and pancreatic cancers. The cell autophagy inhibitor can effectively inhibit tumor cell protective autophagy, the rubiaceae-type cyclopeptides serving as the effective components are wide in source, the extraction process is mature, dosage forms and pharmacy modes are diversified, and the inhibitor can be applied to treatment and prevention of the KRAS related cancers and has a wide clinical application prospect.

Description

technical field [0001] The invention belongs to medical technology, and specifically relates to a cell autophagy inhibitor which uses Rubiaceae type cyclic peptide as an active ingredient, and a preparation method and application thereof. technical background [0002] The KRAS gene is a signal transduction protein in the "downstream region" of the intracellular signal transduction pathway, which has an important influence on the growth, survival and differentiation of cells. Under normal physiological conditions, signaling pathways such as EGFR are activated after cells are stimulated by the outside world. Wild-type KRAS is temporarily activated after being phosphorylated by active tyrosine kinases such as EGFR. The activated KRAS can activate signaling proteins downstream of the signaling pathway, and then KRAS Rapid inactivation, KRAS activation / inactivation effects are controlled. The mutant KRAS protein leads to abnormal protein function, and it is still activated in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/12A61K9/19A61K9/107A61K47/34A61P35/00
CPCA61K9/0019A61K9/1075A61K9/19A61K38/12A61K47/34A61K31/7052A61K38/00A61P1/00A61P1/18A61P11/00C07K7/64
Inventor 谭宁华陈俐娟宋立华杨建洪汪哲
Owner CHINA PHARM UNIV
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