CN115154463A

CN115154463A - Combined pharmaceutical composition for inhibiting triple-negative breast cancer and application thereof

Info

Publication number: CN115154463A
Application number: CN202210701365.1A
Authority: CN
Inventors: 吴睿; 陈思晴; 张佳丽; 占扎君
Original assignee: Zhejiang University of Technology ZJUT; Hangzhou Institute of Advanced Studies of UCAS
Current assignee: Zhejiang University of Technology ZJUT; Hangzhou Institute of Advanced Studies of UCAS
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2022-10-11

Abstract

The invention discloses a combined medicine composition for inhibiting triple negative breast cancer and application thereof. In vivo and in vitro test data of the embodiment of the invention show that the combination of dihydroartemisinin and a natural product aurovertin B shows better drug effect than that of dihydroartemisinin and a natural product aurovertin B which are used for inhibiting three-negative breast cancer metastasis alone; particularly, in the constructed 4T1 mouse orthotopic lung metastasis model, the combined drug group of dihydroartemisinin and a natural product aurovertin B can more obviously inhibit the lung metastasis of tumor cells compared with a single drug group. Therefore, the invention provides a novel combined drug combination mode, can be used as an effective treatment strategy for intervening TNBC tumor metastasis, and has better social value.

Description

Combined pharmaceutical composition for inhibiting triple-negative breast cancer and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a combined medicine composition for inhibiting triple negative breast cancer and application thereof.

Background

Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer characterized by a deficiency in Estrogen Receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 (HER 2) accounting for approximately 15% -20% of all breast cancers. The breast cancer is high in malignancy degree, strong in invasiveness, easy to relapse and transfer and low in survival rate, so that the breast cancer is an important reason for high total death of breast cancer. Because of its negative expression of ER, PR and HER2, it is not sensitive to conventional endocrine therapy and molecular targeted therapy. Therefore, chemotherapy remains the standard therapeutic approach for TNBC. Therefore, the research and development of a lead compound with an anti-metastatic effect on TNBC or the discovery of a new drug combination mode have important significance for the treatment of TNBC.

The Dihydroartemisinin (DHA) is a first generation derivative of Artemisinin (ART), and has proved that the Dihydroartemisinin has significant anti-tumor activity in various malignant tumors and has no toxicity to normal cells under proper dosage. The anti-tumor effect of dihydroartemisinin mainly comprises the following aspects: 1. by up-regulating Bax expression, resulting in caspase-9 activation and down-regulating Bcl-2, bcl-xL, cyclin E, CDK2 and CDK4 expression, leading to esophageal cancer cell cycle arrest (Du X.; li Y. J.; wu C. L.; et al. Initiation of apoptosis, cell cycle array and autophagy of esophageal cancer cells by dihydroarminginin [ J ]. Biomed Pharmacother, 2013, 67 (5): 417-24.); 2. inducing apoptosis of gastric cancer cell BGC823 through the c-Jun NH 2-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p 38 MAPK) signaling pathways (Zhang S.; shi L.; ma H.; et al. Dihydroartemisin antigens apoptosis in human organic cancer cell line BGC-823 through activation of JNK1/2 and p38 MAPK signaling pathways J. J Recept Signal transduction Res., 2017, 37 (2): 174-80.); 3. inhibition of Cancer cell metastasis by down-regulating expression of The Snail and PI3K/AKT signaling pathways (Li N.; zhang S.; luo Q.; et al, the Effect of Dihydroartemisinin on The Malignancy and Epithelial-Mesenchyl transfer of scientific Cancer Cells [ J ]. Curr Pharm Biotechnol., 2019, 20 (9): 719-726.); 4. promotion of apoptosis and inhibition of angiogenesis in pancreatic cancer cells by induction of microRNA-mRNA regulatory networks (Li Y. L.; wang Y. W.; kong R.; et al. Dihydroartemisinin supressants across cellular vitamin a microRNA-mRNA regulatory network [ J. Oncotarget, 2016, 7 (38): 62460-73.); 5. promote the AIM2/caspase-1 inflammasome, induce nuclear and mitochondrial DNA damage, and finally promote autophagy of HepG2215 cells (Shi X.; wang L.; ren L.; et al. Dihydroartemisinine, an antisense drug, indeces present in melanoma 2 inflamome activation and autophagy in human hepatocyte cells Hecina 2215 cells J.,. Phyter Res., 2019, 33 (5): 3-25).

The other compound Aurovertin B is obtained by extracting and separating from fermentation liquor of the fungus aureobasidium denticola (the preparation method is shown in Wang F., luo D.Q., liu J.K. Aurovertin E, a new polyethylene pyrone from the basic ethylene Albatellus confluens [ J ]. J.Antibiot., 2005, 58: 412-415.). Aurovertin compounds, as an ATP synthase inhibitor, have a strong inhibitory effect on the proliferation of cancer cells and are considered as potential cancer therapeutics. aurovertin B can be used as a useful new medicine, and shows potential antitumor activity on breast cancer. Inhibits cell proliferation by cell cycle arrest at G0/G1 stage, has better apoptosis promoting effect on Breast cancer MCF-7 cells, and has smaller influence on normal cell line MCF-10A (Huang T. C.; chang H. Y.; hsu C. H.; et al. Targeting Therapy for Breast Carcinoma by ATP Synthase Inhibitor auroverretin B [ J ]. J Proteome Res, 2008, 7 (4): 1433-1444.). In vitro anti-triple negative breast cancer metastasis studies have shown that aurovertin B can significantly inhibit the migration, adhesion and invasion of TNBC cells. And the in vivo mouse breast pad in situ inoculation transfer model proves that Aurovertin B has better in vivo anti-tumor transfer activity (Yanrubia, aurovertin B and analogues thereof have anti-triple negative breast cancer transfer activity evaluation and DUSP 1-mediated anti-transfer mechanism research [ D ]. Zhejiang: zhejiang industrial university college of medicine, 2021.). The results show that aurovertin B may be a better candidate compound for treating TNBC, and can be used for treating cancer and used as a candidate for combined medication.

Based on the limitation of a three-negative breast cancer treatment scheme and the positive effects of dihydroartemisinin and aurovertin B on the aspect of anti-tumor treatment, the research discovers for the first time that dihydroartemisinin and aurovertin B can enhance the anti-TNBC tumor metastasis activities of dihydroartemisinin and aurovertin B in vitro and in vivo. The invention provides a novel drug combination mode and a drug combination application method thereof, and the drug combination mode has obvious effect of resisting three-negative breast cancer metastasis.

Disclosure of Invention

The invention aims to provide a combined medicine composition for inhibiting triple-negative breast cancer and application thereof, and the combined medicine of dihydroartemisinin and a natural product aurovertin B is applied to inhibiting triple-negative breast cancer metastasis, and can be used as new application of the combined medicine for inhibiting triple-negative breast cancer metastasis.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a pharmaceutical composition for inhibiting triple negative breast cancer comprises dihydroartemisinin and aurovertin B.

Furthermore, the dosage form of the combination comprises any one of pharmaceutically acceptable dosage forms.

Further, the combined medicine is a combination of two separate preparations, namely a dihydroartemisinin preparation and an aurovertin B preparation.

Further, the two separate formulations are administered simultaneously or sequentially.

The combined medicine composition provided by the invention has good application in preparing a triple-negative breast cancer resistant medicine.

The pharmaceutical composition for combination in the application improves macrophage M1 polarization in triple negative breast cancer tissues.

In the application, the dihydroartemisinin and aurovertin B are used in combination to inhibit the adhesion and invasion of triple-negative breast cancer cells and inhibit the in-vivo lung metastasis of triple-negative breast cancer.

The combined medication mode of the dihydroartemisinin and the natural product aurovertin B has more obvious effect of resisting the three-negative breast cancer metastasis than the single medication.

The beneficial effects of the invention are as follows: the dihydroartemisinin and the natural product aurovertin B can obviously inhibit the adhesion and invasion of the triple negative breast cancer cell 4T 1; in the constructed 4T1 mouse orthotopic lung metastasis model, the combined drug combination of dihydroartemisinin and a natural product aurovertin B can more obviously inhibit tumor cell metastasis compared with the single drug combination of dihydroartemisinin and natural product aurovertin B. Therefore, the invention provides a novel combined medication combination mode, can be used as an effective treatment strategy for intervening TNBC tumor metastasis, and has better social value and significance. Provides a theoretical basis for subsequent research, and opens up a new direction for the development of treatment and research of TNBC in the future.

Drawings

FIG. 1 is a graph showing the effect of dihydroartemisinin in combination with aurovertin B on 4T1 adhesion of triple negative breast cancer cells.

FIG. 2 shows the effect of dihydroartemisinin in combination with aurovertin B on the invasion of triple negative breast cancer cells 4T 1.

FIG. 3 shows the effect of dihydroartemisinin in combination with aurovertin B on the in situ vaccination of 4T1 breast cancer tumor-bearing mice with model on in vivo anti-tumor metastasis.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention is further illustrated by the following examples, but the scope of the invention is not limited by any of the specific examples, but is defined by the claims.

Example 1 adhesion test to examine the Effect of Dihydroartemisinin in combination with aurovertin B on cell adhesion ability

Dihydroartemisinin mother liquor: dihydroartemisinin powder was prepared in DMSO to give a 10mM stock solution of dihydroartemisinin, which was administered at a concentration of 5. Mu.M in subsequent experiments.

aurovertin B stock: aurovertin powder was prepared in DMSO to give 13 mM aurovertin B stock solution, and the concentration of the subsequent experiment was 0.05. Mu.M.

Setting a control group (DMEM medium without any medicine), a dihydroartemisinin group (the dihydroartemisinin mother liquor is diluted to the final concentration of 5 mu M by the DMEM medium), an aurovertin B group (the aurovertin B mother liquor is diluted to the final concentration of 0.05 mu M by the DMEM medium), and the dihydroartemisinin is combined with the aurovertin B group (the dihydroartemisinin mother liquor and the aurovertin B mother liquor are diluted in the DMEM medium together to the final concentrations of 5 mu M and 0.05 mu M respectively), applying the macrophages for 24 hours, replacing serum-free fresh DMEM medium for 48 hours, and collecting the supernatant of the macrophages of each group, namely the conditioned medium for treating different medicines.

Fibronectin was diluted to a concentration of 10 μ g/mL with PBS phosphate buffer, 50 μ L was added to each well of a 96-well plate, coated for 3 h at 37 ℃, and washed 2 times with PBS after gelation. Murine triple negative breast cancer cell 4T1 (purchased from cell bank of Chinese academy of sciences) at 2X 10 ⁴ Concentration of wells per well plate, 0.2 mL of different drug treated conditioned media was added per well. Adding at 37 deg.C, 5% ₂ The cells were cultured in an incubator for 24 hours, observed under a microscope for the adherent state of the cells and photographed (100 μm), and the results are shown in FIG. 1. The experimental result shows that with the addition of the drug, the cell morphology changes and does not show a fusiform adherent state any more; the dihydroartemisinin and aurovertin B combination group has more mellow cell morphology and hardly adheres compared with the single drug group. The results show that the dihydroartemisinin combined with aurovertin B can inhibit 4T1 cell adhesion.

Example 2Transwell experiment to examine the influence of dihydroartemisinin in combination with aurovertin B on the invasion ability of cells

Setting a control group (DMEM culture medium without any medicine), a dihydroartemisinin group (the DMEM culture medium is used for diluting dihydroartemisinin mother liquor to the final concentration of 5 mu M), an aurovertin B group (the DMEM culture medium is used for diluting aurovertin B mother liquor to the final concentration of 0.05 mu M), and a dihydroartemisinin combined aurovertin B group (the dihydroartemisinin mother liquor and the aurovertin B mother liquor are diluted in the DMEM culture medium together to the final concentrations of 5 mu M and 0.05 mu M respectively), applying macrophages for 24 h, replacing fresh DMEM culture medium without fetal calf serum for 48 h, and collecting supernatants of macrophages of each group, namely the conditioned medium processed by different medicines.

Transwell invasion assay Transwell chambers were used as Transwell chambers and were placed in plates with the Transwell Chamber as the upper Chamber and the lower Chamber, the upper and lower layers of culture medium being separated by a 8 μm pore size polycarbonate filter. 25. Mu.g of Matrigel (BD Co.) was applied to the surface of the filter in each well, and the membrane was left to stand at 37 ℃ for 30 min for gel coating, followed by 0.2 mL of 4T1 cell suspension (1.5X 10) in PRMI1640 medium containing 10% fetal bovine serum in each Matrigel upper chamber ⁵ one/mL). After the cells adhere to the wall, the culture solution in the upper chamber is replaced by a conditioned medium treated by different medicines. 0.6 mL of PRMI1640 medium containing 10% fetal bovine serum was added to its lower chamber. At 37 ℃ 5% CO ₂ Incubate 24 h, observe cell invasion to lower chamber, use cotton swab from polycarbonate filter membrane upper layer to remove all non-mobile cells; the migrated cells were fixed by adding 600 μ L of 4% paraformaldehyde in the lower chamber and stained with 0.1% crystal violet after 20 min. After observation and photographing under a microscope (K15445, nikon, japan) (100 μm), the cells invaded into the lower chamber were quantified, extracted with 33% acetic acid (v/v), OD values were measured at 630 nm, and the invasion rate was calculated using the following formula.

Attack Rate = [1- (OD) _CM -OD _blank )/(OD _control -OD _blank )]×100％

OD _CM For different drug-treated media assay wells, OD _control As control wells, OD _blank Untreated raw blank wells.

The experimental result is shown in figure 2, the amount of the cells invading the lower chamber is obviously reduced by the combined drug combination of dihydroartemisinin and aurovertin B. The quantitative analysis is carried out on the cells invading the lower chamber, and the result shows that the combination of dihydroartemisinin and aurovertin B can obviously reduce the invasion rate of 4T1 cells compared with a control group, a dihydroartemisinin group and an aurovertin B group. The dihydroartemisinin and aurovertin B can obviously inhibit the invasion of 4T1 cells.

Example 3 evaluation of the Effect of dihydroartemisinin in combination with aurovertin B on in vivo anti-tumor metastasis by the in situ inoculation model of tumor-bearing mice with T4T 1 mammary cancer

CMC-Na aqueous solution: 0.5 Adding the CMC-Na solid into 100 mL of deionized water, heating, stirring and dissolving for 3 h, and storing at normal temperature.

Dihydroartemisinin liquid medicine: the dihydroartemisinin powder is prepared into 70 mg/ml CMC-Na water solution and administered by intragastric administration once a day.

aurovertin B liquid medicine: aurovertin B powder is prepared into 10mg/ml CMC-Na water solution for intraperitoneal injection once every other day.

Bouin stationary liquid: mixing saturated picric acid, formaldehyde and glacial acetic acid at a ratio of 70: 25: 5 (v/v/v), stirring well, and storing in a cool and dry place.

(1) Adherent growth of 4T1 cells in RPMI1640 complete medium containing 10% fetal bovine serum at 37 ℃ with 5% CO ₂ And carrying out conventional culture in a saturated humidity incubator, and carrying out liquid change once passage for 2 to 3 days. Collecting and concentrating to 4 × 10 ⁶ Injecting 0.1 mL of cell suspension into breast pads of a second pair of breasts of Balb/c mice, observing for one week until the length diameter of a tumor body is 8-10mm, randomly dividing the mice into 4 groups, namely a control group (CMC-Na aqueous solution, and performing intraperitoneal injection once every other day (the application volume is 0.1 mL/10 g)); aurovertin B dose group (10 mg/kg, intraperitoneal injection once every other day (the application volume is 0.1 mL/10 g)); a dihydroartemisinin dosage group (70 mg/kg, and intragastric administration once a day (administration volume is 0.1 mL/10 g)); the combined dose group (10 mg/kg aurovertin B liquid medicine is injected into abdominal cavity once every other day (the application volume is 0.1 mL/10 g), and 70mg/kg dihydroartemisinin liquid medicine is administrated once a day by intragastric administration (the application volume is 0.1 mL/10 g)). Mice were observed for survival at each dose.

(2) The mice were sacrificed 6 weeks after cell injection, the lungs were removed, immersed in Bouin fixative for fixed staining, and the number of lung metastatic nodules was counted. The calculation method comprises the following steps:

the number of lung metastasis nodules = grade I × 1 + grade II × 2 + grade III × 3 + grade IV × 4 (which is classified into 4 grades according to nodule diameter: grade I < 0.5 mm, grade II < 0.5 mm < 1 mm, grade III < 2 mm, grade IV > 2 mm).

The experimental results are shown in FIG. 3, the number of tumor nodules in each group is counted to obtain 171.8 + -6.05 for the control group, 77.5 + -13.01 for the aurovertin B10 mg/kg group, 88.2 + -8.45 for the dihydroartemisinin 70mg/kg group, and 14.5 + -1.12 for the dihydroartemisinin combined with aurovertin B group. The number of lung metastasis foci of the combined drug group of dihydroartemisinin and aurovertin B is obviously reduced, and the significant difference exists compared with the single drug group. The result shows that the dihydroartemisinin and aurovertin B have obvious in-vivo anti-tumor metastasis effect on the triple negative breast cancer cells.

In conclusion, the dihydroartemisinin and aurovertin B have better in-vitro and in-vivo anti-tumor metastasis activity of the three-negative breast cancer cells.

Claims

1. A combined pharmaceutical composition for inhibiting triple negative breast cancer is characterized by comprising dihydroartemisinin and aurovertin B.

2. The use of a combination according to claim 1 for the preparation of a medicament against triple negative breast cancer.

3. Use of a combination according to claim 2 for the manufacture of a medicament for the treatment of triple negative breast cancer, wherein the combination increases macrophage M1 polarization in triple negative breast cancer tissue.

4. The use of the combination pharmaceutical composition according to claim 2 for the preparation of a medicament for treating triple negative breast cancer, wherein the combination pharmaceutical composition inhibits the adhesion and migration of triple negative breast cancer cells.

5. Use of a combination pharmaceutical composition as claimed in claim 2 in the manufacture of a medicament for the treatment of triple negative breast cancer wherein the combination pharmaceutical composition is a combination of two separate formulations, a dihydroartemisinin formulation and an aurovertin B formulation, for simultaneous or sequential administration.