Autophagy inhibitor and afatinib medicine composition and application of composition to preparation of tumor synergist

An autophagy inhibitor, afatinib technology, applied in the field of medicine, can solve the problems such as no reports of autophagy inhibitor and afatinib for lung adenocarcinoma, and achieve the effect of enhancing killing and protecting tumor cells

Inactive Publication Date: 2019-03-05
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] According to literature search, so far, there is no relevant report on the combination of autophagy inhibitors and afatinib and its use in the treatment of lung adenocarcinoma.

Method used

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  • Autophagy inhibitor and afatinib medicine composition and application of composition to preparation of tumor synergist
  • Autophagy inhibitor and afatinib medicine composition and application of composition to preparation of tumor synergist
  • Autophagy inhibitor and afatinib medicine composition and application of composition to preparation of tumor synergist

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Experiment of afatinib inducing tumor cell apoptosis

[0027] After lung adenocarcinoma H1975 and H1650 cells have grown to the logarithmic stage, they are 5*10 5 The concentration of cells / mL was transferred to the cell culture plate for culture, and 0μM, 2.5μM, 5μM, 10μM, 20μM afatinib were given, and the culture was continuously for 48 hours. 10μL of MTT reaction solution was added and incubated at 37℃ for 4 hours in the dark; The intracellular succinate dehydrogenase fully reacted to produce formazan, and 150 μL DMSO was added to dissolve formazan. The OD value of each group was measured at 570nm, and the OD of the control group was 100% for cell viability percentage conversion, and the graphpad was used for statistics. Analysis, the results are as figure 1 , Shown in 2;

[0028] Collect H1975 and H1650 cells treated with 0μM, 2.5μM, 5μM, 10μM, 20μM afatinib for 24h and 10μM afatinib for 0h, 3h, 6h, 12h, 24h, and lyse with RIPA lysis solution to extract total c...

Embodiment 2

[0029] Example 2 Afatinib induces tumor cell autophagy experiment

[0030] H1975 and H1650 cells and control cells treated with 10 μM afatinib for 48 hours were stained with Cyto-ID autophagy-related protein LC3-II protein staining reagent and observed under a laser confocal microscope with an excitation wavelength of 560 nm. The results are as follows Figure 4 It shows that there are more green fluorescent spots in the cytoplasm of H1975 and H1650 cells treated with 10μM afatinib for 48 hours, and the number of green fluorescent spots depends on the expression level of autophagosome-related protein Lc3-II, so it is different from Compared with the control group, afatinib can significantly up-regulate the expression of LC3-II in H1975 and H1650 cells, that is, induce autophagy in two tumor cells;

[0031] Collect H1975 and H1650 cells treated with 0μM, 2.5μM, 5μM, 10μM, 20μM afatinib for 24h and 10μM afatinib for 0h, 3h, 6h, 12h, 24h, and lyse with RIPA lysis solution to extract to...

Embodiment 3

[0032] Example 3 Experiments on inhibiting autophagy to increase tumor cell apoptosis induced by afatinib

[0033] Based on the fact that chloroquine itself is weakly alkaline and has lysosome properties, it will destroy the acidic environment of the lysosome, inhibit the activity of monoacylglycerol lipase, phospholipase A2 and other enzymes, and make the long-lived protein in the autophagosome package Other substances cannot be degraded by lysosomes to cause autophagic vesicles to accumulate in the cell; and, 3-MA is an inhibitor of class III phosphatidylinositol 3-kinase, which inhibits the formation of the Beclinl-PI3KC3 complex by inhibiting the formation of LC3- The conversion of I to Lc3II causes the ratio of Lc3-II / Lc3-I to decrease, and the decrease in the ratio means that the formation of autophagosomes is inhibited;

[0034] In this experiment, 2.5μM afatinib was used to interfere with H1975 and H1650 cells, and 3-MA or CQ was added 2h before administration to inhibit au...

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Abstract

The invention belongs to the technical field of medicines, and relates to an autophagy inhibitor and afatinib medicine composition and an application of the composition to preparation of a tumor synergist. According to a combination medicine, afatinib remarkably inhibits proliferation of tumor cells and induces apoptosis and autophagy of tumor cells, autophagy protects the tumor cells when the tumor cells are killed and wounded by the afatinib, so that autophagy inhabitation is obviously enhanced, and the killing and wounding effects of the afatinib on the tumor cells are improved. According to the composition, treatment dosage of the afatinib, toxic and side effects generated by medicines and medicine resistance can be reduced, and treatment cost is reduced. The composition is used for intervention treatment of chronic myelogenous leukemia, liver cancer, breast cancer, non-small cell lung cancer and tumors caused by EGFR (epidermal growth factor receptor) and HER2 (human epidermal growth factor receptor 2) mutation in a united or sequential mode.

Description

Technical field [0001] The invention belongs to the technical field of medicine, and relates to a new pharmaceutical composition for treating tumors; in particular, it relates to an autophagy inhibitor and afatinib pharmaceutical composition and its use in the preparation of tumor synergistic preparations, in particular to the combined medicine It is used in the preparation of synergistic drugs for treating non-small cell lung cancer, breast cancer, liver cancer and chronic myeloid leukemia containing EGFR mutations through combined administration or sequential administration. Background technique [0002] The prior art discloses that Afatinib is a second-generation TKI drug and a polycyclic drug. It can bind to EGFR and Her2 proteins strongly and irreversibly, thereby blocking its corresponding signal pathways and inhibiting The growth of cells has obvious curative effect on tumor cells containing EGFR mutations. [0003] According to data, lung cancer is the most common tumor di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/517A61K31/4706A61K31/52A61P35/00A61P35/02
Inventor 冯美卿胡祥祥施思于晓晨王欢
Owner FUDAN UNIV
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