Application of polymyxin E sodium methanesulfonate in the preparation of drugs for treating triple-negative breast cancer

Abstract

The invention discloses the application of polymyxin E sodium methanesulfonate in the preparation of a drug for treating triple-negative breast cancer, and belongs to the field of biomedicine technology. The invention uses polymyxin E sodium methanesulfonate as the only active ingredient, verifies that polymyxin E sodium methanesulfonate inhibits the viability and proliferation of triple-negative breast cancer cells, blocks the cell cycle at the G2 / M phase, promotes cell apoptosis, and can inhibit the migration and invasion of triple-negative breast cancer cells, and can become an effective drug for treating triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of polymyxin E sodium methanesulfonate in preparing a drug for treating triple-negative breast cancer. Background Art

[0002] Breast cancer is a disease in which mammary epithelial cells proliferate uncontrollably under the action of multiple carcinogens. Breast cancer is divided into different subtypes according to the different receptor expressions in breast tissue. Triple Negative Breast Cancer (TNBC) is a common subtype of breast cancer, which refers to negative expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2), accounting for about 15-20% of all newly diagnosed breast cancers. Compared with other subtypes, it is highly invasive and metastatic, with a worse prognosis and higher grade, and is more common in young women. Due to the lack of receptor expression, TNBC is insensitive to targeted therapy. The treatment of early diagnosed TNBC includes neoadjuvant chemotherapy and surgery. For patients with recurrent / refractory TNBC, there is currently no standard chemotherapy regimen and drug resistance is serious. The treatment response is usually short-lived, followed by rapid recurrence and metastasis. There is a lack of effective treatment options in clinical practice.

[0003] Polymyxin E is a polypeptide antibiotic produced by Paenibacillus polymyxa. Its basic structure is a heptapeptide ring connected by a tripeptide chain, and the fatty acid at the tail is connected to the end of the tripeptide through an amide bond. Polymyxin E is mostly used clinically in the form of colistimethate sodium (CS) as a prodrug. Colistimethate sodium is a polypeptide antibiotic composed of multiple different components. It is hydrolyzed into polymyxin E in the body and has good clinical efficacy in treating infections caused by multidrug-resistant Gram-negative bacteria, especially severe infections caused by Pseudomonas aeruginosa and Acinetobacter baumannii.

[0004] CS is mainly used clinically to control various Gram-negative bacilli, especially various infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, such as sepsis, peritonitis, respiratory system infections, bile duct infections, urinary tract infections, burn wound infections, corneal infections, etc. CS is usually used in critical situations as the last line of defense for antimicrobial drugs. Its antibacterial mechanism of action includes cell membrane lysis, capsule-capsule interaction, and killing bacteria by inducing oxygen stress. The most common side effects of CS are nephrotoxicity and neurotoxicity, which to some extent limit the widespread application of CS.

[0005] Therefore, finding effective therapeutic drugs for TNBC patients has become a hot topic and difficulty in clinical research, and developing drug repositioning for CS has good academic value and clinical significance. Summary of the invention

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides the use of polymyxin E sodium methanesulfonate in the preparation of a drug for treating triple-negative breast cancer, provides a new method for the clinical treatment of triple-negative breast cancer, and develops new uses for polymyxin E sodium methanesulfonate.

[0007] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides the use of polymyxin E sodium methanesulfonate in the preparation of a drug for treating triple-negative breast cancer.

[0009] In a second aspect, the present invention provides the use of polymyxin E sodium methanesulfonate in the preparation of a drug for inhibiting the proliferation of triple-negative breast cancer cells.

[0010] In a third aspect, the present invention provides the use of polymyxin E sodium methanesulfonate in the preparation of a drug for promoting apoptosis of triple-negative breast cancer cells.

[0011] Optionally, the drug is a medicament prepared with polymyxin E sodium methanesulfonate as the only active ingredient and pharmaceutically acceptable excipients.

[0012] Optionally, the effective dosage concentration of polymyxin E sodium methanesulfonate in the medicament is 2.5-10 mg / kg / d.

[0013] In the present invention, polymyxin E sodium methanesulfonate has good effects of inhibiting the viability and proliferation of MDA-MB-231 cells, blocking the cell cycle at the G2 / M phase, promoting cell apoptosis, and inhibiting cell migration and invasion at 5-20 μM (preferably 10 μM). It is calculated that an adult (60 kg) who orally takes 2.5-10 mg / kg body weight (preferably 5 mg / kg body weight) of polymyxin E sodium methanesulfonate per day has a significant therapeutic effect on triple-negative breast cancer.

[0014] Optionally, the auxiliary material includes at least one of an excipient, a flavoring agent, a disintegrant, a preservative, a lubricant, a wetting agent, a binder, a solvent, a thickener or a solubilizing agent.

[0015] Optionally, the dosage form of the medicament includes tablets, capsules, granules, oral liquid preparations or injections.

[0016] The beneficial effects of the present invention are as follows: the present invention provides the use of polymyxin E sodium methanesulfonate in the preparation of a drug for treating triple-negative breast cancer. The present invention proves through cell experiments that polymyxin E sodium methanesulfonate can inhibit the viability and proliferation of MDA-MB-231 cells, block the cell cycle at the G2 / M phase, promote cell apoptosis, and inhibit the migration and invasion of MDA-MB-231 cells. The present invention may provide a drug with good effects for the treatment of triple-negative breast cancer, and develop a new use of polymyxin E sodium methanesulfonate.

[0017] Polymyxin E sodium methanesulfonate is used as the only active ingredient and is combined with pharmaceutically acceptable excipients to prepare a drug having the effect of treating triple-negative breast cancer. When the dosage of polymyxin E sodium methanesulfonate is 2.5-120 mg / kg / d, it has a significant effect of treating triple-negative breast cancer.

[0018] The drug prepared with polymyxin E sodium methanesulfonate as the only active ingredient can be prepared into various dosage forms, among which injection is more conducive to absorption and may have better effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 CS inhibits the viability and proliferation of MDA-MB-231 cells; A is the concentration of CS that inhibits the viability of MDA-MB-231 cells screened by CCK8 method (n=6); B is the effect of CS at different concentrations (10, 15, 20 μM) on the proliferation of MDA-MB-231 cells at different time points (12, 24, 36, 48, 60, 72 h) detected by CCK8 method (n=6). CS, polymyxin E sodium methanesulfonate; data are expressed as mean ± SD; compared with the Ctrl group, *P<0.05, **P<0.01.

[0020] Figure 2CS blocks the cell cycle of MDA-MB-231 cells; A is the effect of different concentrations (10, 15, 20 μM) of CS on the cell cycle of MDA-MB-231 cells detected by flow cytometry (n=3); B is the proportion of each phase (G0 / G1, G2 / M, S phase) of the cell cycle of MDA-MB-231 cells under the action of different concentrations (10, 15, 20 μM) of CS. CS, polymyxin E sodium methanesulfonate; data are expressed as mean ± SD; compared with the Ctrl group, *P < 0.05, **P < 0.01.

[0021] Figure 3 CS induced apoptosis of MDA-MB-231 cells; A is the TUNEL experiment to detect the effect of different concentrations (10, 15, 20 μM) of CS on apoptosis of MDA-MB-231 cells (n=3); TUNEL is green fluorescence, DAPI is blue fluorescence (400×); B is the percentage of TUNEL / DAPI cells under the action of different concentrations (10, 15, 20 μM) of CS. CS, polymyxin E sodium methanesulfonate; data are expressed as mean ± SD; compared with the Ctrl group, *P < 0.05, **P < 0.01.

[0022] Figure 4 CS inhibits the migration of MDA-MB-231 cells; A is the effect of different concentrations (5, 10, 15 μM) of CS on the migration of MDA-MB-231 cells detected by cell scratch assay (n=6); the spacing of red lines is the distance of cell migration (100×); B is the percentage of relative migration rate of MDA-MB-231 cells under the action of different concentrations (10, 15, 20 μM) of CS. CS, polymyxin E sodium methanesulfonate; data are expressed as mean ± SD; compared with the Ctrl group, *P<0.05, **P<0.01.

[0023] Figure 5 CS inhibits the invasion of MDA-MB-231 cells; A is the effect of different concentrations (5, 10, 15 μM) of CS on the invasion of MDA-MB-231 cells by Transwell assay (n=3) (100×); B is the relative invasion rate of MDA-MB-231 cells under the action of different concentrations (5, 10, 15 μM) of CS. CS, polymyxin E sodium methanesulfonate; data are expressed as mean ± SD; compared with the Ctrl group, *P < 0.05, **P < 0.01. DETAILED DESCRIPTION

[0024] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0025] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0026] The MDA-MB-231 cells used in the embodiments of the present invention are TNBC cells, which can be purchased through conventional channels in the market.

[0027] Example 1 CS inhibits MDA-MB-231 cell viability and proliferation

[0028] Cell viability and proliferation assay: MDA-MB-231 cells were treated with CS at concentrations of 0, 2.5, 5, 10, 15, 20, 25, 30, 40, and 60 μM for 12, 24, 36, 48, 60, and 72 h, and the CCK-8 kit was used to detect the cell viability and proliferation of MDA-MB-231 cells.

[0029] In order to verify the inhibitory effect of CS on MDA-MB-231 cells, we first cultured MDA-MB-231 cells and analyzed their cell viability using the CCK-8 method. The results showed that when the CS concentration was ≥10 μM, the drug could significantly inhibit cell viability, and when the CS concentration was ≤5 μM, the drug had no significant effect on cell viability ( Figure 1 A). Based on the cell viability experiment, the inhibitory concentrations of CS on MDA-MB-231 cells were selected as 10, 15, and 20 μM. The CCK-8 method was used to detect the effect of CS on the proliferation of MDA-MB-231 cells at different time points of 12, 24, 36, 48, 60, and 72 h. The results showed that with the increase of the action time, the OD 450nm The values ​​gradually increased, but at each time point, the higher the drug concentration, the higher the OD 450nm The lower the value ( Figure 1 B).

[0030] Example 2 CS blocks the cell cycle and induces apoptosis in MDA-MB-231 cells

[0031] Cell cycle and apoptosis experiments: MDA-MB-231 cells were treated with CS at concentrations of 0, 10, 15, and 20 μM for 24 h (0 μM CS was the Ctrl group), and the cell cycle (PI staining) kit and TUNEL kit were used to detect cell cycle and apoptosis.

[0032] Based on the above cell viability experiment, an effective drug concentration was screened. In this experiment, 10, 15, and 20 μM CS were selected to treat MDA-MB-231 cells for 24 h. Flow cytometry results showed that 10 μM CS could significantly reduce the G2 / M phase and increase the proportion of S phase cells, confirming that CS significantly blocked the MDA-MB-231 cell cycle at the G2 / M phase ( Figure 2 A- Figure 2 B).

[0033] The results of TUNEL experiments showed that as the drug concentration increased, the number of apoptotic cells (green fluorescence) gradually increased, and the proportion of apoptotic cells gradually increased. When the CS concentration was 10 μM, the drug could significantly induce cell apoptosis, with an apoptotic ratio of 14.1%. When the CS concentration was 20 μM, the apoptotic ratio was 20.7% ( Figure 3 A- Figure 3 B).

[0034] Example 3 CS inhibits the migration and invasion of MDA-MB-231 cells

[0035] Cell migration and invasion assay: MDA-MB-231 cells were treated with CS at concentrations of 0, 5, 10, and 15 μM for 24 h (0 μM CS was the Ctrl group), and cell scratch assay and Transwell chamber were used to detect cell migration and invasion.

[0036] In this experiment, CS at concentrations of 0, 5, 10, and 15 μM was used to treat MDA-MB-231 cells for 24 h. The scratch results showed that CS could significantly inhibit the migration of MDA-MB-231 cells at a concentration (5 μM) that had no significant effect on cell viability. Compared with the Ctrl group, the relative migration rate was significantly reduced (85.6%) ( Figure 4 A- Figure 4 B); At the same time, the Transwell results showed that compared with the Ctrl group, CS concentration of 5 μM could significantly inhibit the invasion of MDA-MB-231 cells (63.33%) ( Figure 5 A- Figure 5 B). In addition, with the increase of drug concentration (10, 15 μM), the inhibition rate of CS on MDA-MB-231 cell migration and invasion increased significantly.

[0037] In summary, CS can inhibit the viability and proliferation of MDA-MB-231 cells, block the cell cycle at the G2 / M phase, induce cell apoptosis, and significantly inhibit the migration and invasion of MDA-MB-231 cells. CS may become a candidate drug for the future clinical treatment of TNBC.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of polymyxin E sodium methanesulfonate in the preparation of a drug for treating triple-negative breast cancer, characterized in that: The drug is a medicament prepared with polymyxin E sodium methanesulfonate as the only active ingredient and pharmaceutically acceptable excipients; The effective dosage concentration of polymyxin E sodium methanesulfonate in the medicament is 2.5-10 mg / kg / d.

2. Application of polymyxin E sodium methanesulfonate in the preparation of drugs for inhibiting the proliferation of triple-negative breast cancer cells.

3. Application of polymyxin E sodium methanesulfonate in the preparation of drugs that promote apoptosis of triple-negative breast cancer cells.

4. The use according to claim 1, characterized in that: The auxiliary materials include at least one of excipients, flavoring agents, disintegrants, preservatives, lubricants, wetting agents, adhesives, solvents, thickeners or solubilizers.

5. The use according to claim 4, characterized in that: The dosage form of the medicament includes tablets, capsules, granules, oral liquid preparations or injections.

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