CN110038006A - Application of epigallocatechin gallate combined with tyrosine kinase inhibitor in the preparation of cancer therapeutic drugs - Google Patents

Abstract

The invention relates to application of epigallocatechin gallate (EGCG) and a tyrosine kinase inhibitor in preparation of a cancer treatment drug, belongs to the technical field of biological medicines, and provides a medication method for combining epigallocatechin gallate (EGCG) serving as a lead compound and a tyrosine kinase inhibitor. The sensitivity of the inhibitor is improved, and the application range of the inhibitor is widened. The invention provides an effective treatment strategy for EGFR wild type cancer patients.

Description

Epigallocatechin gallate combined with tyrosine kinase inhibitor in the preparation of cancer application in the treatment of

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to an anticancer composition and its application.

Background technique

Tyrosine Kinase Receptor (TKR) Epidermal Growth Factor Receptor (EGFR) is widely expressed on various cell membranes except suspension cells. Excessive activation of EGFR protein is closely related to the occurrence, development, malignancy and prognosis of tumors. It can cause tumor cell proliferation, promote tumor tissue angiogenesis and tumor cell metastasis, and EGFR is one of the main targets for targeted therapy of human tumors. Tyrosine kinase inhibitors (TKIs) are a major area of targeted therapy drug development. Gefitinib, developed for wild-type EGFR tumors, is the first-generation TKI-type EGFR inhibitor, but it has been clinically found that Gefiitnib has no significant effect on patients with highly activated EGFR after the market. Studies have found that Gefitinb has an affinity for EGFR with L858R mutation that is 5-6 times that of wild-type EGFR, and its effective rate for patients with this mutation is as high as 80%, making it a special drug for these tumor patients. Patients with EGFRL858R only account for 6% of all tumor patients, and more than 60% of the total EGFR wild-type patients have no clinically effective TKI inhibitors available. Therefore, it is of great significance to develop new therapeutic strategies against wild-type EGFR.

SUMMARY OF THE INVENTION

In order to overcome the problem in the background art, that is, so far, there is currently no effective inhibitor for tumor patients with wild-type EGFR. The invention provides a treatment method of combining epigallocatechin gallate (EGCG) and a compound designed and synthesized with EGCG as a leading compound in part or in whole and one or more tyrosine kinase inhibitors. The combination of the two improves the sensitivity of wild-type EGFR to the original tyrosine kinase inhibitor, broadens the scope of application of the original inhibitor, and provides an effective treatment strategy for cancer patients with EGFR wild-type.

The present invention is achieved through the following technical solutions:

Application of epigallocatechin gallate (EGCG) combined with tyrosine kinase inhibitor in the preparation of cancer therapeutic drugs. The epigallocatechin gallate: including EGCG and compounds designed and synthesized with EGCG as the leading compound in part or in whole, and the tyrosine kinase inhibitor includes a combination of one or more tyrosine kinase inhibitors.

Preferably, the cancer is a tumor expressing wild-type EGFR.

Specifically, the application of epigallocatechin gallate (EGCG) in combination with gefitinib in the preparation of EGFR wild-type tumor treatment drugs is shown.

A pharmaceutical composition for treating cancer, comprising a combination of an effective amount of epigallocatechin gallate (EGCG) and one or more tyrosine kinase inhibitors, or comprising part or all of the design with EGCG as a lead compound Combinations of synthetic compounds and one or more tyrosine kinase inhibitors.

Preferably, the pharmaceutical composition is used in the preparation and treatment of EGFR wild-type lung cancer drugs.

Particularly preferably, the pharmaceutical composition for treating cancer includes epigallocatechin gallate (EGCG) and gefitinib. When preparing a drug against EGFR wild-type tumors, the dosage of the compound can be adjusted according to the route of administration, the age, body weight of the patient, the type and severity of the disease to be treated, etc. The dosage of gefitinib is 0.1-5 mg/kg body weight, and the dosage of EGCG 0.1-8mg/kg body weight.

Beneficial effects of the present invention:

The invention discloses a combination of EGCG and EGCG as a leading compound designed and synthesized in part or in whole with a tyrosine kinase inhibitor, especially the combination of EGCG and Gefitinib to prepare a pharmaceutical composition, which can be used for the treatment of EGFR wild-type tumor patients, and can be used in vitro. It was seen that the growth of tumor cells was significantly inhibited, and the increase in tumor volume of EGFR wild-type tumor cell xenografts was inhibited in vivo, and the combination of the two reduced the treatment risks and toxic side effects caused by anticancer drugs at higher doses.

The invention not only provides an effective treatment strategy for EGFR wild-type tumor patients, but also expands the applicable scope of the original tyrosine kinase inhibitor.

Description of drawings

Figure 1 is the effect of EGCG combined with Gefitinib on the proliferation of EGFR wild-type and mutant cells;

Figure 2 is the effect of EGCG combined with Gefitinib on related proteins in EGFR signaling pathway in EGFR wild-type and mutant cells;

Figure 3 is the effect of EGCG combined with Gefitinib on the body weight of A431 tumor-bearing mice;

Figure 4 is a visual diagram of the effect of EGCG in combination with Gefitinib on tumor growth in A431 xenografted nude mice;

Figure 5 is the effect of EGCG combined with Gefitinib on the tumor volume of A431 xenografted nude mice (transplanted tumor growth curve);

Figure 6 is the effect of EGCG combined with Gefitinib on the tumor weight of A431 xenografted nude mice;

Figure 7 is the effect of EGCG combined with Gefitinib on the body weight of NCI-H1975 tumor-bearing mice;

Figure 8 is a visual diagram of the effect of EGCG combined with Gefitinib on tumor growth of NCI-H1975 xenografted nude mice;

Figure 9 is the effect of EGCG combined with Gefitinib on the tumor volume of NCI-H1975 xenografted nude mice (transplanted tumor growth curve);

Figure 10 is the effect of EGCG combined with Gefitinib on tumor weight of NCI-H1975 xenografted nude mice.

Detailed ways

In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate the understanding of the skilled person.

The present invention selects EGFR wild-type and high-expressing cell lines and EGFR double-mutated cell lines as research objects, and mainly aims at the inhibition of EGFR wild-type tumor cell proliferation and the inhibition of heterogeneity by the combined use of gefitinib and EGCG, a first-generation inhibitor of EGFR. Inhibitory effect of tumor growth in nude mice, and elucidated the mechanism of this effect.

Example 1 The effect of EGCG combined with Gefitinib on the proliferation of EGFR wild-type and mutant cells in vitro

1. Experimental materials

Cell lines: human epidermal squamous cell carcinoma A431, human lung cancer cell lines NCI-H1666, NCI-H1975.

2. Detection principle: MTT method to detect cell viability

MTT assay for cell viability experiment: MTT concentration is 5mg/ml. Therefore, you can weigh 0.5 g of MTT, dissolve it in 100 ml of phosphate buffered saline (PBS) or phenol red-free medium, and filter it with a 0.22 μm filter to remove the bacteria in the solution. Store at ℃ protected from light. The container is best wrapped in aluminum foil.

Test steps: A: We use the adherent cell operation method. First, collect log-phase cells to adjust the cell suspension concentration, so that the density of the cells to be tested is adjusted to 3×10 ⁴ /well, and 200 μl is added to each well. PBS filled). At the same time, set the zero-adjustment well (dimethyl sulfoxide), the control well (cells, the same concentration of drug dissolution medium (acidic medium), MTT, dimethyl sulfoxide) in a 5% CO ₂ , 37 ℃ incubator nourish.

B: After the cells adhere to the wall, you can see that the cell monolayer covers the bottom of the well (96-well flat bottom plate), first wash the cells with PBS, discard the PBS, and then start dosing, according to 200 μl per well, set 4-6 replicates holes, otherwise it is difficult to reflect the real situation. After the treatment was completed, the same marginal wells were filled with acidic medium. Prevent sample volatilization.

C: 5% CO ₂ , incubate at 37°C for 24 hours, and observe the cell state under an inverted microscope.

D: Add 20 μl MTT solution (5 mg/ml, ie 0.5% MTT) to each well, and continue to culture for 4 h. If the drug can react with MTT, the culture medium can be discarded after centrifugation, carefully washed with PBS, and then the culture medium containing MTT can be added.

E: Terminate the culture, carefully aspirate the medium in the well.

F: Add 150 μl of dimethyl sulfoxide to each well, and place it on a shaker for 10 min at low speed to fully dissolve the crystals. The absorbance value of each well was measured at OD490nm of enzyme-linked immunosorbent assay instrument. The absorbance value (OD value) was detected at 630 nm as the reference.

G: The calculation formula of cell viability is: viability = OD average value of experimental group - OD average value of blank group/1) × 100%, and the obtained data can be obtained in a column chart in excel.

3. Experimental results

The results are shown in Figure 1. Gefitinib alone treatment group had no effect on the survival rate of A431 cells, but had a significant inhibitory effect on NCI-H1666 cells. However, consistent with our expected results, Gefitinib combined with EGF and EGCG treatment group had no effect on the two There was no significant inhibitory effect on the growth of wild-type EGFR cell lines. However, when Gefitinib coexists with EGCG and EGF, it can significantly inhibit the growth of EGFR wild-type cells, but has no effect on double mutant NCI-H1975 cells. It is preliminarily demonstrated that this combined treatment of cells has a selective inhibitory effect on cell growth. It has a very obvious inhibitory effect on wild-type EGFR cells A431 and NCI-H1666, but has no effect on double mutant NCI-H1975 cells.

Example 2 The effect of Gefitinib on phosphorylation of EGFR signaling pathway-related proteins in wild-type and mutant cells in the presence of EGCG and EGF

1. Experimental materials

Cell lines: human epidermal squamous cell carcinoma A431, human lung cancer cell line NCI-H1975.

2. Detection principle: Western Blot detects changes in related proteins in cells

3. Experimental methods: a: Treat the cells. After the cells have adhered to the wall, starvation treatment is performed. After overnight treatment, EGCG and EGF treatment are performed. Experiments were carried out according to specific experimental groups. The blank control group was directly added to the acidic medium, and the remaining experimental groups were added to 10 mL of acidic medium to make the final concentration of EGCG at 20 μg/mL. The EGF stock solution was diluted to 20 ng/mL and added to the acidic medium. After finishing, put it in a cell incubator, and set the corresponding treatment time according to the experimental group.

b: Extract protein

c: Protein quantification: The principle of protein determination adopts the BCA protein quantification method

d: According to the principle of SDS-PAGE, proteins with different molecular weights are separated by the electric field strength according to the different charges. Then the separated protein is transferred to PVDF membrane, and the protein is adsorbed in the form of non-covalent bond without destroying the biological activity of the protein polypeptide. Then, the protein transferred to the membrane is used as the antigen, and the corresponding antibody can be combined with the antigen overnight or at room temperature for two hours, and then combined with the HRP-labeled secondary antibody for 1 hour at room temperature, and the target protein can be detected by the substrate color development. expression.

4. Experimental results

The results are shown in Figure 2: In wild-type EGFRA431 cells, in the presence of both EGCG and EGF, Gefitinib can significantly inhibit the phosphorylation of EGFR family and its downstream protein ERK. But this result was not seen in double mutant EGFR NCI-H1975 cells. The experimental results were consistent with the cell viability experiments.

Example 3 Inhibitory effect of EGCG combined with Gefitinib on tumor in EGFR wild-type AA431 cell carcinoma xenograft model

1. Experimental Materials

A431 cells and culture: Human epidermal squamous cell carcinoma A431 was cultured in DMEM high glucose complete medium containing 10% FBS, and the cells were diluted to 2ⅹ106 cells per mL of culture medium. inoculated into petri dishes. Incubate in a cell incubator. The cells in the logarithmic growth phase were taken, digested with 0.5% trypsin solution to prepare a single cell suspension, centrifuged at 15000 rpm for 3 min, the supernatant was discarded, and the cell density was adjusted to 5ⅹ10 ⁶ /mL with PBS.

Nude mice and their rearing: male BALB/C nu/nu clean-grade mice, 6-8 weeks old, provided by Changzhou Cavens Laboratory Animal Co., Ltd., Jiangsu Province. Nude mice were housed in a sterile and ventilated closed animal room at a temperature of 25°C, with regular light during the day and a dark environment at night, with free food and water. The cage and water of nude mice were sterilized by high temperature and high pressure. Change nude mice to clean cages, bedding and drinking water regularly to keep the growing environment of nude mice clean.

2. Experimental method

a: Establishment of nude mouse transplanted tumor model. Cells were recovered and passaged. After adjusting the cell state for one week, the growth environment of the cells tended to be stable. After the cells were digested and counted, the viable cell density was adjusted to contain 5ⅹ10 ⁶ cells per 200µL suspension for use. Then centrifuge at 1500g for 3min. After the medium was aspirated, it was gently blown with normal saline to prepare a cell suspension for later use. In a sterile ultra-clean bench, nude mice were grouped according to the principle of the same average body weight and ear marks were cut, and 200 µL of cell suspension (about 5ⅹ10 ⁶ cells) was subcutaneously injected into the back of each nude mouse. Every other day, the long diameter (Dmax) and short diameter (Dmin) of the tumor were measured with a vernier caliper, and the tumor volume V was calculated (V= Dmax ⅹ Dmin2/2).

b: Animal grouping and administration according to the above-mentioned grouping of nude mice. (1) Blank control group: intraperitoneal injection of normal saline every six days a day, and intragastric administration of normal saline containing 1%-Tween 80; (2) Gefitinib Nitrogen group: gavage at a dose of 50 mg/kg (twice a week); (3) EGCG group: once a day (intraperitoneal injection) for six days a week at a dose of 20 mg/kg or 40 mg/kg; (4) combined administration The groups were administered six days a week (once a day) with a dose of 20 mg/kg or 40 mg/kg by intraperitoneal injection, and Gefitinib was administered at a dose of 50 mg/kg twice a week. Tumor volume was measured before administration, three times a week. And the body weight of nude mice was weighed once a week, and the experimental records were made. Nude mice were sacrificed after administration was completed, and tumor masses were removed and photographed. Solid tumors were preserved according to subsequent experimental arrangements.

3. Experimental results

As shown in Figure 3, it was found that there was no significant weight loss between the groups compared to the normal control animals. However, EGCG alone treatment group can promote the increase of body weight of nude mice after 3 weeks of administration, which is significant compared with the control group. It shows that each group of drugs has no obvious toxic and side effects on animals. After the administration was completed, the tumors of the nude mice were removed. As shown in Figure 4, the effect of the combination on tumor growth is shown. The tumor growth curve was drawn by measuring the xenograft tumors of nude mice every other day. As shown in Figure 5, compared with the Gefitinib-treated group, the Gefitinib combination group significantly inhibited the growth of tumor volume from the 29th measurement of tumor volume. Consistent with previous experimental results, the selectivity of Gefitinib in vitro was higher than that in vivo Experiments are very selective. This experiment also shows that Gefitinib has a good inhibitory effect on tumor growth in nude mice. When combined with EGCG, it can show a very obvious inhibitory effect, basically completely inhibiting the growth of tumor cells. The removed tumor was weighed, and the results are shown in Figure 6. Compared with the blank control group, the tumor inhibition rate was 52% in the Gefitinib treatment group alone. Compared with the Gefitinib combination group and the Gefitinib treatment group alone, the inhibition rates of solid tumors were respectively 69% and 81%. There was no statistical difference, but the combination group inhibited tumor growth to a certain extent compared to the monotherapy group. No animals died during the entire experiment.

Example 4 Inhibitory effect of EGCG combined with Gefitinib on tumor in EGFR double mutant NCI-H1975 cell carcinoma xenograft model

1. Experimental Materials

NCI-H1975 cells and culture: Human non-small cell lung cancer NCI-H1975 was cultured in DMEM1640 complete medium containing 10% FBS, and the cells were diluted to 2ⅹ10 ⁶ /mL. inoculated into petri dishes. Incubate in a cell incubator. The cells in the logarithmic growth phase were taken, digested with 0.5% trypsin solution to prepare a single cell suspension, centrifuged at 15000 rpm for 3 min, the supernatant was discarded, and the cell density was adjusted to 5ⅹ10 ⁶ /mL with PBS.

Nude mice and their rearing: male BALB/C nu/nu clean-grade mice, 6-8 weeks old, provided by Changzhou Cavens Laboratory Animal Co., Ltd., Jiangsu Province. Nude mice were housed in a sterile and ventilated closed animal room at a temperature of 25°C, with regular light during the day and a dark environment at night, with free food and water. The cage and water of nude mice were sterilized by high temperature and high pressure. Change nude mice to clean cages, bedding and drinking water regularly to keep the growing environment of nude mice clean.

2. Experimental method

a: Establishment of nude mouse transplanted tumor model. Cells were recovered and passaged. After adjusting the cell state for one week, the growth environment of the cells tended to be stable. After the cells were digested and counted, the density of viable cells was adjusted to contain 3ⅹ10 ⁶ cells per 200 µL suspension for use. Then centrifuge at 1500g for 3min. After aspirating the medium, gently blow with normal saline to make a cell suspension. In a sterile ultra-clean bench, group nude mice according to the principle of the same average body weight and cut ear marks. Inject 200 µL of cell suspension (approximately 3 x 10 ⁶ cells) subcutaneously on the back. Every other day, the long diameter (Dmax) and short diameter (Dmin) of the tumor were measured with a vernier caliper, and the tumor volume V was calculated (V= Dmax ⅹ Dmin2/2).

b: Animal grouping and administration From the second day after inoculation with tumor cells, the administration was performed according to the grouped nude mice (1) Blank control group: intraperitoneal injection of normal saline every six days, gavage containing 1%-Tween 80 Normal saline; because Gefitinib was dissolved in 1% Tween-80 to prepare a suspension; (2) Gefitinib was administered by gavage twice a week, and 50 mg/kg or 100 mg/kg Gefitinib was administered; (3) EGCG group was administered according to each Six days, the frequency of administration was once a day, and the intraperitoneal dose was 40 mg/kg of EGCG; (4) The combined administration group was administered 40 mg/kg of EGCG once a day for six days a week and 50 mg/kg or 100 mg of Gefitinib twice a week. The doses of mg/kg were administered. The frequency of tumor volume measurement was every other day, and the body weight of nude mice was weighed every two days and recorded. After the administration was completed, the nude mice were sacrificed, and the tumors were removed and photographed. Solid tumors were preserved according to subsequent experimental arrangements. In the modeling experiment, nude mice aged 6-8 weeks were selected and divided into 6 groups, with 5-6 mice in each group, and were fed with sterile feed.

3 Experimental results

After the injection of tumor cells, the drug was administered on the second day. During the administration period, the body weight of nude mice was detected every 3 days. The results are shown in Figure 7: there is no difference in the effect of each group of drugs on the body weight of NCI-H1975 tumor-bearing mice. The whole process There is no drug-induced death in nude mice, indicating that the drug has no toxic and side effects on nude mice. The inhibitory effect of each group of drugs on tumor growth in NCI-H1975 tumor-bearing mice. As shown in Figure 8, it can be seen from the figure that after the combined administration of Gefitinib and EGCG, the tumor growth of NCI-H1975 tumor-bearing nude mice was not inhibited, and there was no difference in tumor growth among the groups. The results showed that this combination had no effect on the EGFR double mutant NCI-H1975 xenograft tumor, which further proved that this combination only had an inhibitory effect on EGFR wild-type tumors. The tumor volume of the transplanted nude mice was measured every other day, and the tumor growth curve was drawn. The results are shown in Figure 9. The combined use had no effect on the tumor volume of NCI-H1975 cells that inhibited the tumor; the peeled tumor was weighed. , the results are shown in Figure 10, the combined use had no effect on the tumor weight of the xenografted tumor of EGFR double mutant 1975 cells.

In the present invention, the epidermal squamous cell line with EGFR wild-type and high expression and the lung cancer cell line with EGFR double mutation are selected as controls. , showed a very obvious inhibitory effect on EGFR wild-type tumor cells, but had no effect on EGFR double-mutated non-small cell lung cancer. It shows that this combination drug is selective.

The in vivo experiment further proved that the combination of gefitinib and EGCG can significantly inhibit the growth of EGFR wild-type tumor cell xenografts, and has no obvious effect on the body weight, indicating that this combination has no obvious side effects.

In conclusion, the combined use of epigallocatechin gallate (EGCG) and the EGFR inhibitor gefitinib can significantly inhibit the growth of EGFR wild-type tumor cells without causing harm to the body. The present invention provides an effective treatment strategy for EGFR wild-type cancer patients, improves the sensitivity of the original inhibitor, and broadens the applicable scope of the original inhibitor.

When preparing a drug for EGFR wild-type lung cancer, the dosage of the compound can be adjusted according to the route of administration, the patient's age, body weight, the type and severity of the disease to be treated, etc. The dosage of gefitinib is 0.1-5 mg/kg body weight, the dosage of EGCG 0.1-8mg/kg body weight.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should Various changes may be made in details without departing from the scope of the invention as defined by the claims.

Claims (4)

1. the application of epigallocatechin gallate (EGCG) combined with tyrosine kinase inhibitor in the preparation of cancer treatment medicine, it is characterized in that: described epigallocatechin gallate comprises: EGCG and EGCG as Part or all of the lead compound is designed to be a synthetic compound, the tyrosine kinase inhibitor comprising a combination of one or more tyrosine kinase inhibitors. 2. A pharmaceutical composition for the treatment of cancer, characterized in that: comprising the combination of an effective amount of epigallocatechin gallate EGCG and one or more tyrosine kinase inhibitors according to claim 1, or comprising A combination of a partially or fully designed and synthesized compound with EGCG as the lead compound and one or more tyrosine kinase inhibitors. 3. The use according to claim 1, wherein the cancer is an EGFR wild-type tumor. 4. The application of the pharmaceutical composition according to claim 2 in the preparation and treatment of EGFR wild-type tumor drugs.