Anti-tumor pharmaceutical composition and application thereof

Abstract

The invention discloses an anti-tumor pharmaceutical composition. The active ingredients in the pharmaceutical composition are tizanidine and docetaxel. The two effective components of the pharmaceutical composition generate a synergistic effect, so that the drug sensitivity can be enhanced, and in addition, the docetaxel dosage and the toxic and side effects can be reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of breast cancer treatment, and particularly relates to application of a combined regimen of a synergistic drug combination for inhibiting growth of triple-negative breast cancer cells. BACKGROUND

[0002] Basal-like breast cancer (BLBC) is a subtype of breast cancer with strong invasiveness, high histological grade, small age of onset and the worst prognosis among all breast cancer subtypes. The BLBC subtype is not sensitive to endocrine therapy and trastuzumab treatment due to the negative expression of ER, PR and HER2 (i.e., triple-negative breast cancer), and chemotherapy is the main drug treatment means for the BLBC subtype. Although chemotherapy is initially effective in the early stage, the BLBC patients are prone to early local recurrence and distant metastasis due to the emergence of drug resistance or resistance to chemotherapy, and the 5-year survival rate of metastatic patients is less than 30%. With the development of gene sequencing technology, targeted gene variation and immunotherapy are two directions of BLBC treatment research, such as PARP inhibitors, HSP90 inhibitors, PI3K / AKT / mTOR signal pathway inhibitors and immune checkpoint inhibitors. According to the current research results, a perfect treatment scheme has not been obtained, and therefore, a new targeted treatment method is urgently needed for triple-negative breast cancer. How to improve the cure rate of BCBL patients and how to improve the prognosis of patients so as to reduce the risk of recurrence and metastasis are the keys to improving the survival rate of breast cancer patients and are also the hotspots and difficulties in the current research on breast cancer. SUMMARY

[0003] In order to overcome the shortcomings of the prior art, the application provides a combined drug composition, which comprises tiazemidine and docetaxel. The application also provides use of tiazemidine and docetaxel in preparation of a combined drug for treating breast cancer.

[0004] In the drug composition, the molar concentration ratio of tiazemidine to docetaxel is 250000:1-500000:1.

[0005] In the drug composition, the molar concentration ratio of tiazemidine to docetaxel is 375000:1.

[0006] The drug composition contains effective amounts of tiazemidine and docetaxel as active ingredients, and is prepared by adding pharmaceutically acceptable adjuvants.

[0007] The excipients are starch, dextrin, magnesium stearate, microcrystalline cellulose, polysaccharide chitosan, hydroxypropyl methylcellulose HPMC, hydroxypropyl cellulose HPC, hydroxyethyl cellulose HEC, methylcellulose MC, ethylcellulose EC, sodium carboxymethyl starch CMC-Na, carbopol, polycarboxyvinyl, or a mixture of one or more thereof.

[0008] The medicament is an oral preparation; further, the oral preparation is a tablet, a capsule, a granule, an oral liquid, or a pill. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Effects of different concentrations of tazemetostat on the survival rates of human breast cancer cell lines and human normal breast epithelial cells, Figure 2 Effects of tazemetostat on the migration (A) and colony formation (B) of Hs578T cells, Figure 3 Effects of different concentrations of tazemetostat on the tumorigenic ability of human triple-negative breast cancer cell subcutaneous xenografts in nude mice, Figure 4 Drug combination index CI graph and inhibition rate of tazemetostat and docetaxel combination on triple-negative breast cancer Hs578T (A, B) and SUM159. DETAILED DESCRIPTION

[0010] Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0011] Example 1: Preparation of stock solution and working solution of the drug Preparation of the stock solution of tazemetostat: A certain amount (10 mg) of tazemetostat powder was accurately weighed into an EP tube, and a certain amount (197 μL) of DMSO was added to prepare a 200 mM stock solution (TZN200) after ultrasonic dissolution, which was divided into 20 uL / tube and stored in a-80℃ refrigerator; Preparation of the stock solution of docetaxel: A certain amount (10 mg) of docetaxel powder was accurately weighed into an EP tube, and a certain amount (123.78 μL) of DMSO was added to prepare a 100 mM stock solution (DXT100) after vortexing and dissolving, which was divided into 10 uL / tube and stored in a-80℃ refrigerator; Preparation of the working solution: freshly prepared, a certain volume of the stock solution was diluted with DMSO to different concentration gradients of the working solution, and then diluted with complete culture medium to the required concentration by 500 or 1000 times before adding to the cells.

[0012] Example 2: Drug cell culture and drug addition Cells and reagents Human triple-negative breast cancer cell lines Hs578T and SUM159 were donated by Professor Dong Chenfang of Zhejiang University School of Medicine; Tizanidine (TZN, Selleck) and Docetaxel (DXT, Selleck) formulations were purchased from Shanghai Lanmu Chemical Co., Ltd.; DMEM high-glucose medium, phosphate-buffered saline (PBS) and 0.5% trypsin were purchased from Jiangsu Kaiji Biotechnology Co., Ltd.; high-quality fetal bovine serum was purchased from Hangzhou Nuoyang Biotechnology Co., Ltd.; and the enhanced CCK-8 assay kit was purchased from Jiangsu Kaiji Biotechnology Co., Ltd.

[0013] Cell culture The human triple-negative breast cancer cells Hs578T and SUM159, donated by Professor Dong Chenfang of Zhejiang University School of Medicine, were cultured in DMEM medium containing 10% FBS, 5% CO2, and at 37°C in a cell culture incubator.

[0014] After cell digestion and counting, cells were seeded at a specific density in 96-well plates: Hs578T / SUM159 (1500 cells / well), 100 μL of cell suspension per well, and incubated overnight. After 24 hours, the pre-prepared working solution was prepared and diluted 1000-fold with 1 mL of complete culture medium. The old culture medium in each well was discarded, and then 100 μL of freshly prepared drug-containing medium was added, along with 0.1% DMSO. Repeat the process in 4 wells. The final drug concentrations of tizanidine and docetaxel in the two cell lines are shown in Table 1. Table 1. Concentrations of tizanidine and docetaxel in Hs578T and SUM159 cell culture media

[0015]

[0016] Cell viability CCK8 detection and calculation After treating cells with the drug for 72 h, the old culture medium was discarded and replaced with freshly prepared DMEM medium containing 1X CCK8 solution (10X), 100 μL per well. After incubation at 37℃ in the dark for 1–2 h, the absorbance at 490 nm was read using a microplate reader. A Blank control group in DMEM medium was also included. Cell viability (%) was calculated based on the percentage of absorbance in the drug-treated group and DMSO, and plotted on the ordinate with drug concentration on the x-axis using Prism 5.0 to obtain a dose-response curve. The drug concentrations at IC50 and IC20 were then calculated.

[0017] Among four triple-negative breast cancer cell lines (Hs578T, MDA231, BT474, and SUM159), compared with normal human breast epithelial cells MCF10A, in vitro CCK-8 assays showed that only Hs578T cells were sensitive to tizanidine after 72 h of treatment. Additionally, the relatively insensitive SUM159 cells were selected for further comparison. Figure 1 As shown in the figure, AC is a fitted curve after CCK8 detection, with the vertical axis representing cell viability (%) and the horizontal axis representing drug concentration (μM); D is a bar graph of the median lethal concentration (IC50) of tizanidine for each cell line.

[0018] Example 3: Effects of tizanidine on the proliferation of triple-negative breast cancer cell lines and subcutaneous breast cancer xenografts Six- to seven-week-old female Balb / C nude mice were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Each mouse was subcutaneously inoculated with 5 x 10^6 Hs578T cells. Tumors were induced when the average volume reached 50-100 mm. 3 At that time, the mice were divided into three groups based on the size of the tumor and treated accordingly. Tumor volume and body weight were monitored and recorded every two days. Tumor volume was calculated using the formula: L × W² / 2, where L is the tumor length and W is the tumor width. Relative tumor volume was calculated as the tumor volume per centimeter (ci).

[0019] The inhibition rate corresponding to each concentration was calculated based on cell viability and plotted on the ordinate with drug concentration on the abscissa using Prism 5.0 to obtain the dose-response curve and its parameters. Then, Compusyn software was used to calculate the combination index (CI) and dosing reduction index (DRI) for each concentration combination of tizanidine and docetaxel. Median-drug effect analysis was performed on the combination of these two drugs. CI > 1.2 indicates an antagonistic effect, CI < 0.8 indicates a synergistic effect, and CI between 0.8 and 1.2 is considered an additive effect. DRI is defined as the multiple of drug dose reduction achieved by the combination of drugs compared to single-drug administration to achieve the same level of inhibitory effect. A DRI > 1 is of positive significance for clinical use.

[0020] 1) Effects of tizanidine on the proliferation of triple-negative breast cancer cell lines Based on the results of Example 2, the Hs578T and SUM159 cell lines were selected as the subjects for further research. This invention utilizes cell migration and monoclonal experiments (results are shown in...). Figure 2As shown in the figure, tizanidine has a certain degree of inhibitory effect on the proliferation, colony formation and migration of triple-negative breast cancer cells.

[0021] 2) The effect of tizanidine on subcutaneous xenografts of breast cancer in nude mice After establishing the nude mouse subcutaneous tumor model, wait until the subcutaneous tumor volume reaches 50-100 mm. 3 Subsequently, mice were divided into three groups based on tumor size: a control group (PBS), a low-dose tizanidine group (10 mg / kg), and a high-dose tizanidine group (30 mg / kg). After 7 days of continuous gavage, observation continued until day 21. Tumor growth was observed and recorded every other day, and the tumor volume growth curve is shown below. Figure 3 As shown in Figure A, the results indicate that the tumor growth curves in the low-dose group almost overlapped with those in the control group, suggesting that low-dose tizanidine had no inhibitory effect on mouse tumors. Although tumors in the high-dose group also continued to grow, the growth rate was relatively slow, especially after the 7-day administration period, when the inhibitory trend on tumor growth became increasingly apparent. At the end of the observation period, mice were sacrificed, and subcutaneous tumor tissue was harvested and weighed. The tumor weight results (…) Figure 3 B) showed that the tumors in the high-dose group were relatively mild (p=0.0456), indicating that tizanidine only had a weak inhibitory effect on tumor growth at high doses.

[0022] Example 4: Inhibitory effects of tizanidine and docetaxel on the proliferation of Hs578T and SUM159 cells. The dose ratio of the two drugs was set, and based on the CCK8 experimental results, dose-response curves of tizanidine and docetaxel, used alone and in combination, were plotted on Hs578T and SUM159 cells. Figure 4 As shown.

[0023] The relevant results are shown in Table 2. The dose required for the drug to inhibit cell activity by 50% at IC50 is given. The R-value is the correlation coefficient for curve fitting.

[0024] Table 2. Median lethal doses of tizanidine and docetaxel in Hs578T and SUM159 cells.

[0025]

[0026] To evaluate the efficacy of the two-drug combination, Compusyn software was used to calculate the corresponding CI and DRI values. For example... Figure 4 As shown, in both cell lines, when the cell activity inhibition rate was above 50%, the CI value of the combined group was less than 0.8, indicating that the combination of tizanidine and docetaxel has a relatively strong synergistic effect, and the CI value decreased as the inhibition rate increased, indicating that the synergistic effect was enhanced.

[0027] Based on the DRI (dose reduction index) values ​​of the tizanidine and docetaxel combination group in Table 3, it was found that when the cell activity inhibition rate of the combination group reached 50%, docetaxel could reduce the dosage of tizanidine by 6.3 and 4.1 times, respectively, for Hs578T and SUM159 cells. CI (Cycle Index) is the combination drug index; CI < 0.8 indicates a synergistic effect, CI > 1.2 indicates an antagonistic effect, and 0.8 ≤ CI ≤ 1.2 indicates an additive effect. DRI is defined as the factor by which the drug dosage is reduced when the combination drug is used compared to the single drug at the same level of cell activity inhibition. Furthermore, tizanidine not only increased the inhibition rate of docetaxel on triple-negative breast cancer cells but also reduced the dosage by approximately 2.5 times.

[0028] Table 3. CI and DRI values ​​of tizanidine combined with docetaxel

[0029]

[0030] This invention confirms that the synergistic effect of TZN as an adjuvant in combination with DXT not only enables DXT to exert stronger tumor-killing activity with a smaller dose, but also reduces the adverse reactions of DXT to patients.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0032] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art. The fact that the steps of the methods are listed in a specific order in this invention does not constitute any limitation on the order of the method steps.

Claims

1. A pharmaceutical composition, characterized by, The composition contains: tiazemine and docetaxel.

2. The pharmaceutical composition of claim 1, wherein, The molar concentration ratio of tiazemine and docetaxel is 250000:1-500000:

1.

3. The pharmaceutical composition of claim 2, wherein, The molar concentration ratio of tiazemine and docetaxel is 375000:

1.

4. The composition according to any one of claims 1 to 3, characterized in that, The pharmaceutical composition further comprises pharmaceutically acceptable adjuvants, and the adjuvants are a mixture of one or more of starch, dextrin, magnesium stearate, microcrystalline cellulose, polysaccharide chitosan, hydroxypropyl methyl cellulose HPMC, hydroxypropyl cellulose HPC, hydroxyethyl cellulose HEC, methyl cellulose MC, ethyl cellulose EC, sodium carboxymethyl starch CMC-Na, carbopol, polycarboxyvinyl, etc.

5. Use of the composition of any one of claims 1-4 in the preparation of a combined medicament for the treatment of breast cancer.

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