CN110840868A

CN110840868A - Application of bromhexine in preparation of anti-cancer drugs

Info

Publication number: CN110840868A
Application number: CN201910457821.0A
Authority: CN
Inventors: 赵承光; 杨乐和; 石灵西; 陈丝雨; 林梦莎; 朱宇; 李继法; 朱建东; 黄晓颖; 王良兴
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2019-05-29
Filing date: 2019-05-29
Publication date: 2020-02-28
Anticipated expiration: 2039-05-29
Also published as: CN110840868B

Abstract

The invention discloses an application of bromhexine in preparing an anti-cancer drug, and test results show that the bromhexine can obviously inhibit the proliferation of lung cancer cells, and when the bromhexine is combined with cisplatin, erlotinib, gefitinib or afatinib, a strong synergistic effect is shown, so that the bromhexine can be used as a drug combination to achieve a better anti-cancer effect.

Description

Application of bromhexine in preparation of anti-cancer drugs

Technical Field

The invention belongs to the field of medicine preparation, and particularly relates to application of bromhexine in preparation of an anti-cancer medicine.

Background

Lung cancer is one of the most common malignant tumors, the morbidity and mortality are high, and the morbidity is still rising due to the aggravation of atmospheric pollution, smoking and other factors. The lung is also the most frequently occurring metastatic site of other malignancies. The first treatment means for lung cancer and lung metastatic cancer is surgical resection, however, patients with surgical resection chances are less than 30%, and other treatment means commonly used comprise targeted drug therapy, chemotherapy, radiotherapy and the like, and have the defects of easy generation of drug resistance, large side effect and the like although the treatment means has a certain curative effect. The factors of complicated pathogenesis, poor treatment effect, high recurrence and metastasis rate, large treatment side effect, poor accuracy and the like of the lung cancer are main reasons for poor overall treatment effect. Therefore, it is of great research significance to elucidate lung cancer pathogenesis and to study new therapeutic strategies.

Bromhexine is a phlegm-resolving medicament widely applied clinically at present. Bromhexine can crack polysaccharide cellulose in the sputum, reduce the viscosity of the sputum, and also can cause the respiratory tract to secrete mucus type small-molecule mucin, so that the sputum becomes thin. The main indications are patients who are difficult to cough mucus caused by chronic bronchitis, asthma and the like.

Disclosure of Invention

The invention provides an application of bromhexine in preparing anticancer drugs, and experimental results show that the bromhexine has a certain anticancer effect and can be used as a new anticancer drug.

Preferably, the anticancer drug is used for treating lung cancer.

Test results show that the bromhexine can obviously inhibit the proliferation of the lung cancer cells PC-9, A549 and H460, and preferably, the anticancer medicament is used for inhibiting the proliferation of the lung cancer cells PC-9, A549 and H460.

The test result shows that the anticancer drug can also be used for inhibiting the colony formation of NSCLC cells.

Preferably, the anticancer drug comprises an active ingredient;

the active ingredients consist of bromhexine and other anti-lung cancer drugs.

Cell tests show that the bromhexine can be combined with certain specific medicines to obtain better synergistic effect, and preferably, the other anti-lung cancer medicines are one or more of cisplatin, erlotinib, gefitinib and afatinib.

Preferably, the active ingredients consist of bromhexine and erlotinib;

the anticancer drug is used for inhibiting the proliferation of lung cancer cells H460 and PC-9.

As a further preference, the molar ratio of bromhexine to erlotinib is 1: 0.1-2, and at the moment, the combination of the two has a strong synergistic effect, so that the anticancer effect is obviously improved.

Preferably, the active ingredients consist of bromhexine and cisplatin;

the anticancer drug is used for inhibiting the proliferation of lung cancer cells H460.

As a further preference, the molar ratio of bromhexine to cisplatin is 1: 0.1-2, and at the moment, the combination of the two has a strong synergistic effect, so that the anticancer effect is obviously improved.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention develops the new anticancer application of the phlegm-reducing drug bromhexine, in particular to the application on lung cancer;

(2) the bromhexine and the existing anti-lung cancer drugs are combined, so that a strong synergistic effect can be generated between the bromhexine and the existing anti-lung cancer drugs, the anti-cancer effect is improved, and the dosage of the drugs is reduced.

Drawings

FIG. 1 is a graph of the inhibition of lung cancer cell proliferation by bromhexine in the MTT assay of example 1;

FIG. 2 is a graph of inhibition of colony formation of NSCLC cells by bromhexine in example 2;

FIG. 3 is the synergistic anti-NSCLC activity of the combination of bromhexine and erlotinib in example 3;

FIG. 4 is a graph of the synergistic anti-NSCLC activity of the combination of bromhexine and cisplatin in example 4.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments.

Example 1

Three human non-small cell lung cancer cell strains PC-9, A549 and H460 are selected, and the influence of bromhexine on the proliferation of the lung cancer cells is observed through an MTT (methyl thiazolyl tetrazolium) experiment. Cells are paved into a 96-well plate according to 3000 cells per well, bromhexine (solvent is DMSO) with different drug concentrations is added after the cells adhere to the wall, and 20ul of MTT solution (5mg/ml) per well is added after the drug acts for 48 hours. After further incubation for 4 hours, the culture was terminated and the culture supernatant from the wells was carefully aspirated. Crystals were dissolved by adding 150ul DMSO per well. The light absorption value of each well is measured by an enzyme-linked immunosorbent instrument at 490nm wavelength, and the influence of bromhexine on the proliferation of the lung cancer cells is observed, and the result is shown in figure 1. The results show that: after 48 hours of action, the proliferation of NSCLC cell strains can be inhibited in a dose-dependent manner, wherein the IC50 of PC-9 is the lowest and the drugs are the most sensitive.

Example 2 bromhexine inhibits NSCLC cell colony formation.

Tumor cells can be propagated indefinitely to form cell colonies, and the influence of the compound on cell proliferation can be known through colony formation experiments. Cells are evenly paved in a 6-well plate according to 1000 cells per well, after the cells are attached to the wall, bromhexine (25 mu M,50 mu M,100 mu M and DMSO as a solvent) with different concentrations is added to act on NSCLC cells, the drugs are removed after the NSCLC cells act for 24 hours, fresh culture medium is added for continuous culture, after the cells form macroscopic colonies, 4% paraformaldehyde is fixed and then crystal violet is used for staining, the size and the number of the colonies among the wells are compared, and the result is shown in figure 2. The results show that bromhexine can inhibit the colony formation of the two NSCLC cells in a dose-dependent manner.

Example 3 bromhexine-sensitizing EGFR inhibitors exert anti-NSCLC effects

EGFR-TKIs represented by erlotinib, gefitinib and afatinib are first-line therapeutic drugs for EGFR mutant non-small cell lung cancer. However, patients who are initially sensitive to EGFR-TKIs almost end up developing drug resistance, so that the clinical efficacy and application of the drugs are limited. Through MTT experiments, the bromhexine can be used for sensitizing erlotinib to play an anti-tumor role. We used bromhexine and different concentration gradients of erlotinib (0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M) to act together on lung cancer cells H460 and PC-9, and the results are shown in FIG. 3, where (A) bromhexine and erlotinib, alone or in combination, inhibited H460 cell survival. (B) Bromhexine and erlotinib, alone or in combination, inhibit PC-9 cell survival. Bromhexine (5. mu.M in concentration), E: erlotinib (at concentrations of 0.5. mu.M 1. mu.M 2.5. mu.M 5. mu.M and 10. mu.M, respectively).

For H460 cells, 5 μ M bromhexine in combination with 0.5 μ M erlotinib, drug combination index 0.04527; 5 μ M bromhexine in combination with 1 μ M erlotinib, drug combination index 0.04092; 5 μ M bromhexine in combination with 2.5 μ M erlotinib, drug combination index 0.04212; 5 μ M bromhexine in combination with 5 μ M erlotinib, drug combination index 0.07563; 5 μ M bromhexine in combination with 10 μ M erlotinib had a drug combination index of 0.04235.

For PC-9 cells, 5 μ M bromhexine in combination with 0.5 μ M erlotinib, the drug combination index is 30718; 5 μ M bromhexine in combination with 1 μ M erlotinib, drug combination index 0.29851; 5 μ M bromhexine in combination with 2.5 μ M erlotinib, drug combination index 0.33573; 5 μ M bromhexine in combination with 5 μ M erlotinib, drug combination index 0.41023; 5 μ M bromhexine in combination with 10 μ M erlotinib had a drug combination index of 0.42154.

Wherein, CI which is more than or equal to 0.9 and less than or equal to 1.1 is an overlapping effect, CI which is more than or equal to 0.8 and less than or equal to 0.9 is a low-degree synergistic effect, CI which is more than or equal to 0.6 and less than or equal to 0.8 is a medium synergistic effect, CI which is more than or equal to 0.4 and less than or equal to 0.6 is a high synergistic effect, and CI which is more than or equal to 0.2 and less than or equal to 0.4 is a strong synergistic effect, therefore, the combined drug with a specific proportion can obviously improve the anti-tumor effect of the two drugs, simultaneously reduce the concentration of. The bromhexine combined with erlotinib was found to significantly inhibit the proliferation of NSCLC cell lines after 48h compared to single drugs.

Example 4 bromhexine-sensitizing cisplatin exerts an anti-NSCLC effect

Cisplatin is a first-line drug for the treatment of a variety of solid tumors. Platinum-containing dual-drug chemotherapy is one of the treatment options for patients with advanced non-small cell lung cancer without driver mutations. In addition, cisplatin-based combined chemotherapy is also a main treatment scheme for advanced ovarian cancer, osteosarcoma and neuroblastoma, and is effective on multiple squamous cell carcinoma and transitional cell carcinoma such as head and neck, cervical, esophageal and urinary tumors when combined with ADM, CTX and the like. Through MTT experiments, the bromhexine can sensitize cisplatin to play an anti-tumor role. We used bromhexine and cisplatin (0.5. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M) at different concentration gradients to act together on H460, and the results are shown in FIG. 4, where (A) bromhexine and cisplatin alone or in combination inhibited H460 cell survival. Bromhexine (at a concentration of 5. mu.M), Cis: cisplatin (at concentrations of 0.5. mu.M 1. mu.M 2.5. mu.M 5. mu.M and 10. mu.M, respectively).

For H460 cells, 5 μ M bromhexine in combination with 0.5 μ M cisplatin, at a combination index of 0.11780; 5 μ M bromhexine in combination with 1 μ M cisplatin, at a combination index of 0.06475; 5 μ M bromhexine in combination with 2.5 μ M cisplatin, at a combination index of 0.15718; 5 μ M bromhexine in combination with 5 μ M cisplatin at a combination index of 0.08367; 5 μ M bromhexine in combination with 10 μ M cisplatin, the combination index was 0.05789.

5 μ M bromhexine in combination with 0.5 μ M cisplatin for PC-9 cells at a combination index of 0.26602; 5 μ M bromhexine in combination with 1 μ M cisplatin, at a combination index of 0.23788; 5 μ M bromhexine in combination with 2.5 μ M cisplatin, at a combination index of 0.17607; 5 μ M bromhexine in combination with 5 μ M cisplatin at a combination index of 0.28761; 5 μ M bromhexine in combination with 10 μ M cisplatin, the combination index was 0.26874.

Bromhexine combined with cisplatin was found to significantly inhibit the proliferation of H460 after 48H compared to single drug.

Claims

1. An application of bromhexine in preparing anticancer medicine.

2. Use of bromhexine in the manufacture of an anticancer drug according to claim 1, wherein the anticancer drug is for the treatment of lung cancer.

3. The use of bromhexine in the manufacture of an anticancer drug according to claim 1, wherein the anticancer drug is used to inhibit the proliferation of lung cancer cells PC-9, a549 and H460.

4. The use of bromhexine in the preparation of an anticancer agent according to claim 1, wherein said anticancer agent is used to inhibit the colony formation of NSCLC cells.

5. The use of bromhexine according to any one of claims 1 to 4 in the preparation of an anticancer drug, wherein the anticancer drug comprises an active ingredient;

the active ingredients consist of bromhexine and other anti-lung cancer drugs.

6. The use of bromhexine in the preparation of an anticancer agent according to claim 5, wherein the other anti-lung cancer drugs are one or more of cisplatin, erlotinib, gefitinib and afatinib.

7. The use of bromhexine in the manufacture of a medicament for the treatment of cancer according to claim 5, wherein the active ingredients consist of bromhexine and erlotinib;

8. The use of bromhexine in the manufacture of a medicament for the treatment of cancer according to claim 5, wherein the active ingredient consists of bromhexine and cisplatin;