CN105963302A - Low-dosage medicine composition containing EGFR (epidermal growth factor receptor) tyrosine kinase and application thereof in preparation of anti-tumor transfer medicines - Google Patents

Abstract

The invention relates to a low-dosage medicine composition containing EGFR (epidermal growth factor receptor) tyrosine kinase and an application thereof in preparation of anti-tumor transfer medicines. The low-dosage medicine composition with anti-tumor transfer function is characterized by comprising ursolic acid and gefitinib according to the weight ratio of 10:(1-5). The low-dosage medicine composition has the advantage that after the gefitinib with targeting function is jointly used together with the anti-tumor natural product of ursolic acid with high efficiency and low toxicity, the good anti-tumor transfer function on cancer cells is realized, and a safe and reliable novel candidate medicine is obtained.

Description

A low-dose pharmaceutical composition containing EGFR tyrosine kinase and its preparation and prevention Application in Tumor Metastasis Drugs

technical field

The invention belongs to the field of anti-tumor metastasis drugs, in particular, the invention relates to a low-dose pharmaceutical composition for anti-tumor metastasis.

Background technique

Ursolic acid is 3β-hydroxy-ursolic-12-en-28-acid (3β-hydroxy-urs-12-en-28-oic acid, referred to as UA), also known as ursolic acid, which belongs to a-amyresinol (a-amyrin)-type pentacyclic triterpenoids, with a relative molecular weight of 456.68, a molecular formula of C ₃₀ H ₄₈ O ₃ , and a structure as shown in Formula I, are widely distributed natural active compounds in nature, mainly in the form of free or glycosides It exists in the form of loquat leaf, bearberry, hawthorn, hedyotis diffusa and other natural plants. It is also one of the main active ingredients of many traditional Chinese medicines and has a wide range of pharmacological effects, such as anti-cancer, liver protection, Anti-inflammation, anti-virus, anti-oxidation, etc. Among them, the anti-cancer activity is the most significant. It not only has resistance to various carcinogens, but also has inhibitory effects on various tumor cells both in vivo and in vitro. Because of its small side effects and low toxicity, it shows great potential for clinical application. In recent years, anti-tumor research on UA has been deepened at home and abroad, and it has been found that it has unique advantages and potential application prospects in tumor prevention, treatment, and prevention of late recurrence and metastasis. Patent N201510097801.9 discloses a pharmaceutical composition containing ursolic acid and cyclophosphamide, which can not only enhance the anti-tumor efficacy of a single component, but also reduce its toxicity to normal tissues, thereby improving tumor The effect of treatment has great application prospects in the field of tumor treatment. Patent N03150714.X discloses that ursolic acid is effective on human liver cancer cells, human breast cancer cells, human lymphoma cells, human lymphoblastic leukemia and human acute lymphoblastic leukemia, human acute promyelocytic leukemia and human chronic myelogenous leukemia. Has a cytotoxic effect.

Gefitinib (chemical name: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinepropoxy)quinazolin-4-amine), English name : Gifitinid, whose structure is shown in formula II, is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, suitable for patients with sensitive mutations in the epidermal growth factor receptor tyrosine kinase (EGFR TK) gene Third-line, second-line or even first-line treatment for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC). Gefitinib is a poorly soluble drug, and its solubility in aqueous solution is pH-dependent, that is, the lower the pH, the greater the solubility in aqueous solution, and it is almost insoluble in water with a pH value of about 7; Patients with mutations have a certain therapeutic effect, but patients generally relapse or metastasize within 1-2 years, or develop new mutations that are resistant to gefitinib, so even if gefitinib (casein NSK inhibitors) treatment still does not improve five-year overall survival in patients with non-small cell lung cancer. Patent N201210566665.X discloses a tablet containing gefitinib, which can release active ingredients continuously and steadily, and achieve good therapeutic effect on locally advanced lung cancer or metastatic non-small cell lung cancer who have previously received chemotherapy.

Taking different cancer cell lines as the research objects, through the combination of ursolic acid and gefitinib, the anticancer activity of different cancer cell lines was tested in vitro. The results showed that the combined use of ursolic acid and gefitinib was Under the condition of low dose, it has a significant inhibitory effect on the proliferation, migration and invasion of cancer cells, especially non-small cell lung cancer, and inhibits the proliferation of cancer cells in a concentration- and time-dependent manner.

Contents of the invention

The purpose of the present invention is to provide a low-dose pharmaceutical composition for anti-tumor metastasis. By combining gefitinib with targeting effect and high-efficiency and low-toxicity anti-tumor natural product ursolic acid, the combined drug is investigated. The anti-metastatic effect on cancer cells is expected to be a relatively safe and reliable new drug candidate.

A low-dose pharmaceutical composition with anti-tumor metastasis and its application in the preparation of anti-tumor metastasis drugs, characterized in that the pharmaceutical composition comprises ursolic acid and gefitinib in a substance ratio of 10:1-5 Ni, the combination of the two can play a synergistic anti-cancer effect.

Description of drawings

Figure 1. The inhibition results of ursolic acid and gefitinib used alone or in combination for 24 h on the proliferation of A549 cells;

Figure 2. The inhibition results of ursolic acid and gefitinib used alone or in combination for 24 h on the proliferation of H1975 cells;

Figure 3. The inhibition results of ursolic acid and gefitinib alone and in combination for 24 h on the proliferation of H1650 cells;

Figure 4. The inhibition results of ursolic acid and gefitinib alone and in combination for 24 h on the invasion ability of A549 cells;

Figure 5. The inhibitory rate of ursolic acid and gefitinib used alone or in combination for 24 h on the invasion of A549 cells;

Figure 6. The inhibitory rate of ursolic acid and gefitinib used alone or in combination for 24 h on the invasion of H1975 cells;

Figure 7. Inhibitory results of ursolic acid and gefitinib alone and in combination with the migration ability of A549 cells for 24 h;

Figure 8. The inhibitory rate of ursolic acid and gefitinib alone and in combination for 24 h on the migration of A549 cells;

Figure 9. The inhibitory rate of ursolic acid and gefitinib alone and in combination for 24 h on the migration of H1975 cells;

Figure 10. Effects of ursolic acid and gefitinib alone and in combination on the protein expression levels of RGD, ICAM-1, EGFR, and VCAM-1 in H1975 cells;

Figure 11. Effects of ursolic acid and gefitinib alone and in combination on the protein expression of RGD, ICAM-1, EGFR, and VCAM-1 in H1975 cells.

detailed description

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

Example 1

Inhibitory effects of ursolic acid and gefitinib alone and in combination for 24 hours on the proliferation of different lung cancer cell lines: The standard MTT colorimetric method was used to measure the effect of different combination ratios of ursolic acid and gefitinib on different lung cancer cell lines Proliferation inhibitory activity, the results are shown in Figures 1-3.

As shown in Figure 1-3, when ursolic acid (10 μM) and gefitinib (1-5 μM) were administered in combination in a low concentration range for 24 hours, it had no significant inhibitory effect on cancer cell proliferation and had no significant effect on normal cells. Therefore, we chose to combine ursolic acid with a safe and effective low-dose concentration (no obvious cell killing effect) and gefitinib in the follow-up, and continue to study its ability to inhibit the invasion and metastasis of lung cancer cells, in order to explore Its potential application in the field of anti-tumor metastasis.

Example 2

Effects of ursolic acid and gefitinib alone and in combination on the invasion ability of A549 cell line

A549 cells were first added with serum-free phenol red-free medium and starved overnight, and the cells collected by digestion and centrifugation were suspended in blank medium containing different drug concentrations. Take a 12-well plate, add 500 μL of cell suspension containing different drug concentrations (about 5×10 ⁵ /well) to the upper chamber of the small chamber, add 500 μl of medium containing 10% calf serum to the lower chamber, and the drug effect is 24 h, take out the small chamber, fix with 4% paraformaldehyde for 20 min, wipe off the upper layer of non-migrated cells with a cotton swab, wash 3 times with PBS, stain with 0.1% crystal violet for 40 min, wash 3 times with PBS, and clean the bottom membrane of the small chamber Take it off, seal it up and save it, take pictures under an inverted microscope at 5 random fields of view, and count the number of migrated cells. The results are shown in Figures 4 and 5.

The experimental results are shown in Figures 4 and 5. The detection of the invasion ability of A549 cell line after ursolic acid and gefitinib were used alone or in combination for 24 h showed that the blank control group, UA=10 μM and gefitinib (Under the condition of 5 μM) in the group used alone, the number of cells passing through the microporous membrane was significantly higher than that in the combined treatment group of ursolic acid and gefitinib, and the invasion ability of the cells was weakened, and the invasion ability was significantly inhibited.

Example 3

The inhibitory rate of ursolic acid and gefitinib alone and in combination to the invasion ability of H1975 cell line, the steps are the same as in Example 2, and the results are shown in Figure 6.

The experimental results are shown in Figure 6. The inhibition rate of ursolic acid and gefitinib alone and in combination for 24 h on the invasion ability of H1975 cell line showed that the blank control group, UA (under 10 μM condition) and Gefitinib About 70-80% of H1975 cells invaded through the chamber in the nontinib (5 μM condition) alone group, and only about 35% of the H1975 invaded through the chamber when the two were administered together , the combination of the two significantly reduced the invasion ability of H1975 cells.

Example 4

Effects of ursolic acid and gefitinib alone and in combination on the migration ability of A549 cell line

Cells in the logarithmic growth phase (about 3×10 ⁶ /well) were inoculated on the plate, and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. Draw three parallel lines vertically at 1/4, 1/2, and 3/4 of the orifice plate, wash the cells 3 times with PBS, take a wide band of scratches under a fluorescent microscope, and record the coordinate axes of the pictures taken. Add medium containing different drug concentrations, place in an incubator at 37°C and 5% CO ₂ for 24, 48, and 72 h. Take pictures again at the same site to measure the migration distance, and compare the migration distances of the two photographs in each group. The results are shown in Figures 7 and 8.

The experimental results are shown in Figures 7 and 8. After ursolic acid and gefitinib were used alone or in combination for 24 h, the cell migration inhibition rate of A549 cell line was detected. There was no significant effect on the migration distance between groups. After 24 hours of administration, cells in the blank control group migrated significantly, and the scratch broadband narrowed; when the UA concentration was 10 μM and gefitinib was 5 μM, The lower cells also obviously migrated; but when the two were used in combination, in the case of gefitinib at 5 μM, the scratch broadband was significantly larger than that of gefitinib alone. Therefore, the combination of the two drugs can synergistically inhibit the migration ability of cells, and the scratch width is significantly different from that of the blank control group and has statistical significance (P<0.01).

Example 5

The inhibitory rate of ursolic acid and gefitinib alone and in combination on the migration ability of H1975 cell line, the steps are the same as in Example 4, and the results are shown in Figure 9.

The experimental results are shown in Figure 9. After ursolic acid and gefitinib were used alone or in combination for 24 h, the cell migration inhibition rate of H1975 cell line was detected. The results showed that: at 0 h, the administration intervention group was compared with the blank control group. The migration distance was not significantly affected. After 24 hours of administration, the scratch broadband of the blank control group narrowed, and the cells obviously migrated; when the UA concentration was 10 μM and the gefitinib was 5 μM, the cells Migration also occurred obviously, but the migration distance was not as obvious as that of the blank control group; but when the two were used in combination, in the case of gefitinib at 5 μM, the scratch broadband was significantly larger than that of gefitinib and gefitinib alone. Big ursolic acid. Therefore, the combination of the two drugs can synergistically inhibit the migration ability of cells, and the scratch width is significantly different from that of the blank control group and has statistical significance (P<0.01).

Example 6

Effects of ursolic acid and gefitinib alone and in combination on the protein expression levels of RGD, ICAM-1, EGFR, and VCAM-1 in H1975 cells

The cells were seeded into a 6-well plate. After the cells reached more than 80% and different drugs were added to intervene the cells for 24 h, the culture medium was discarded, the cells were rinsed twice with pre-cooled PBS, and freshly prepared PBS was added to each well of the 6-well plate. Cell lysate RIPA (radioimmunopre-cipitation assay) 200 μL [add 10 μL protease inhibitor, 10 μL phosphatase inhibitor and 5 μL phenylmethanesulfonylfluoride (PMSF) to 1 mL RIPA], place on ice for 30 min , shake once every 5 min, centrifuge at 12000×g 4°C for 15 min, take the supernatant, and prepare the total protein; use the bicincho-ninic acid (BCA) protein concentration assay kit to determine the protein concentration, and use β- The actin level was loaded as an equal amount of protein, and 20 μg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, and then the separated protein was transferred to polyvinylidene (polyvinylidene, PVDF) membrane, blocked with TBST containing 5% skimmed milk powder at room temperature for 1 h, added primary antibody to react overnight at 4 °C, washed the membrane with TBST at room temperature for 3 times (10 min/time) the next day, and then added HRP-labeled secondary Antibody (diluted 1:5000) was incubated at room temperature for 1 h, and the membrane was washed three times with TBST at room temperature (10 min/time), and finally developed by electrochemiluminescence. The image analysis software Image J was used to analyze the gray value of the strips, and the results are shown in Figures 10 and 11.

The experimental results are shown in Figures 10 and 11. The blank control group did not affect the expression of RGD, ICAM-1, EGFR, and VCAM-1 protein levels; UA concentration of 0 μM and gefitinib at 5 μM had no effect on RGD, ICAM-1, EGFR, and VCAM-1 protein expression levels have little effect; but when the two are administered together, they can play a synergistic effect and can significantly inhibit RGD, ICAM-1, EGFR, VCAM-1 Protein expression level, by down-regulating the levels of RGD, ICAM-1, EGFR and VCAM-1, thereby inhibiting the proliferation of cancer cells and inhibiting cell metastasis.

Claims (2)

1. a pharmaceutical composition with anti metastasis, it is characterised in that comprise the amount of material than the ursolic acid for 10:1-5 And gefitinib.

2. pharmaceutical composition application in preparing medicine for anti transfer of tumor as claimed in claim 1.