CN116036287A

CN116036287A - Application of Gefitinib combined with EGCG and/or EGF in preparation of medicines for treating EGFR wild type tumors

Info

Publication number: CN116036287A
Application number: CN202211609072.7A
Authority: CN
Inventors: 盛军; 王宣军; 黄艳苹; 字成庭; 向泽敏; 黄业伟; 赵云丽; 爨向丹; 徐欢欢; 罗瑞
Original assignee: Yunnan Daye Dihong Biotechnology Co ltd; Yunnan Agricultural University
Current assignee: Yunnan Daye Dihong Biotechnology Co ltd; Yunnan Agricultural University
Priority date: 2019-05-07
Filing date: 2019-05-07
Publication date: 2023-05-02
Also published as: CN110038006A

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of Gefitinib combined EGCG and/or EGF in preparation of medicines for treating EGFR wild-type tumors. The invention provides a drug administration method for combining a tyrosine kinase inhibitor Gefitinib and epigallocatechin gallate EGCG and/or EGF, which can obviously inhibit the growth of a human lung cancer cell strain NCI-H1666 by combined use, and reduces the treatment risk and toxic and side effects caused by an anticancer drug at a large dose. Improves the sensitivity of the inhibitor and widens the application range of the inhibitor. The invention provides an effective treatment strategy for EGFR wild type cancer patients.

Description

Application of Gefitinib combined with EGCG and/or EGF in preparation of medicines for treating EGFR wild type tumors

The application is a divisional application of application date 2019, 05-07, application number 201910374362.X and application of the invention name epigallocatechin gallate combined tyrosine kinase inhibitor in preparing cancer therapeutic drugs.

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to application of Gefitinib combined with EGCG and/or EGF in preparation of medicines for treating EGFR wild type tumors.

Background

Tyrosine Kinase Receptor (TKR) Epidermal Growth Factor Receptor (EGFR) is widely expressed on various cell membranes except suspension cells, and the excessive activation of EGFR protein has very close relation with the occurrence, development, malignancy degree and prognosis of tumors, can cause proliferation of tumor cells, promote angiogenesis of tumor tissues and metastasis of tumor cells, and EGFR is one of main targets for targeted treatment of human tumors. Tyrosine Kinase Inhibitors (TKIs) are a major area of targeted therapeutic drug development. Gefitinib (Gefitinib) developed against wild-type EGFR tumors is a first generation TKI-type EGFR inhibitor, but after marketing it was found clinically that Gefitinib had no significant effect on patients with high activation of EGFR. The research shows that the affinity of Gefitinb to EGFR with L858R mutation is 5-6 times that of wild EGFR, and the effective rate of Gefitinb to patients carrying the mutation is up to more than 80%, thus the Gefitinb is a specific drug for patients with tumors. Patients carrying EGFR 858R only account for 6% of all tumor patients, while more than 60% of EGFR wild type patients have no clinically effective TKI-type inhibitors available. Therefore, it is of great importance to develop new therapeutic strategies for wild-type EGFR.

Disclosure of Invention

In order to overcome the problems in the background art, i.e. to date, there are no effective inhibitors for tumor patients against wild-type EGFR. The invention provides a treatment method for combining epigallocatechin gallate (EGCG) and a compound which is partially or completely designed and synthesized by taking EGCG as a lead compound with one or more tyrosine kinase inhibitors, and the two are combined, so that the sensitivity of wild EGFR to the original tyrosine kinase inhibitors is improved, the application range of the original inhibitors is widened, and an effective treatment strategy is provided for EGFR wild cancer patients.

The invention is realized by the following technical scheme:

the application of epigallocatechin gallate (EGCG) combined with tyrosine kinase inhibitor in preparing medicine for treating cancer is provided. The epigallocatechin gallate: comprises EGCG and a compound which takes EGCG as a lead compound and is partially or totally designed and synthesized, wherein the tyrosine kinase inhibitor comprises one or a combination of a plurality of tyrosine kinase inhibitors.

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

Particularly preferred is the application of epigallocatechin gallate (EGCG) combined with Gefitinib (Gefitinib) in preparing EGFR wild type tumor therapeutic drugs.

A pharmaceutical composition for treating cancer comprising an effective amount of epigallocatechin gallate (EGCG) in combination with one or more tyrosine kinase inhibitors, or comprising a combination of a compound designed to be synthesized partially or fully with EGCG as a lead compound and one or more tyrosine kinase inhibitors.

Preferably, the pharmaceutical composition is applied to preparation and treatment of EGFR wild type lung cancer drugs.

Particularly preferred pharmaceutical compositions for treating cancer include epigallocatechin gallate (EGCG) and Gefitinib (Gefitinib). When preparing a medicament against EGFR wild type tumor, the dosage of the compound can be adjusted according to the administration route, age, weight of the patient, type and severity of the disease to be treated, etc., the dose of gefitinib is 0.1-5mg/kg body weight, and the dose of EGCG is 0.1-8mg/kg body weight.

The invention has the beneficial effects that:

the invention discloses EGCG and a compound which is partially or totally designed and synthesized by taking EGCG as a lead compound, and a tyrosine kinase inhibitor are combined, in particular to a pharmaceutical composition prepared by combining EGCG and Gefitinib, which can be used for treating EGFR wild type tumor patients, obviously inhibit the growth of tumor cells in vitro, inhibit the increase of tumor volume of EGFR wild type tumor cell xenograft in vivo, and reduce the treatment risk and toxic and side effects caused by anticancer drugs under a large dosage.

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

Drawings

FIG. 1 is the effect of EGCG in combination with Gefitinib on EGFR wild type and mutant cell proliferation;

FIG. 2 is the effect of EGCG in combination with Gefitinib on EGFR signaling pathway related proteins in EGFR wild type and mutant cells;

FIG. 3 is the effect of EGCG in combination with Gefitinib on body weight of A431 tumor-bearing mice;

FIG. 4 is a visual graph of the effect of EGCG in combination with Gefitinib on tumor growth in A431 nude mice with transplanted tumors;

FIG. 5 is the effect of EGCG in combination with Gefitinib on tumor volume of A431 nude mice with transplanted tumors (transplanted tumor growth curve);

FIG. 6 is the effect of EGCG in combination with Gefitinib on tumor weight of A431 nude mice with transplanted tumors;

FIG. 7 is the effect of EGCG in combination with Gefitinib on the body weight of NCI-H1975 tumor bearing mice;

FIG. 8 is a visual graph showing the effect of EGCG in combination with Gefitinib on tumor growth in NCI-H1975-grafted nude mice;

FIG. 9 is the effect of EGCG in combination with Gefitinib on tumor volume of NCI-H1975 nude mice with engraftment tumors (engraftment tumor growth curve);

FIG. 10 is the effect of EGCG in combination with Gefitinib on tumor weight in NCI-H1975-grafted tumor nude mice.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding by the skilled person.

The invention selects an EGFR wild type and high-expression cell line and an EGFR double-mutation cell line as research objects, mainly aims at the inhibition effect of EGFR wild type tumor cell proliferation and the inhibition effect of xenogeneic inhibition on the growth of nude mice tumor by combining EGFR generation inhibitors gefitinib and EGCG, and illustrates the action mechanism of the effect.

Example 1

Effect of EGCG in combination with Gefitinib on EGFR wild type and mutant cell proliferation effects in vitro

1. Experimental materials

Cell lines: human epidermoid squamous cell carcinoma A431, human lung carcinoma cell line NCI-H1666, NCI-H1975.

2. Detection principle: MTT method for detecting cell activity

Viability assay of MTT-detected cells: MTT concentration was 5mg/ml. Thus, 0.5 g of MTT was weighed, dissolved in 100ml of Phosphate Buffer (PBS) or phenol red-free medium, filtered through a 0.22 μm filter to remove bacteria in the solution, and after preparation, the solution was sub-packaged and stored at 4℃in the absence of light. The container is preferably wrapped in aluminum foil.

The test steps are as follows:

a: the method comprises collecting cells in log phase to adjust cell suspension concentration to 3×10 ⁴ Well, 200 μl was added per well (edge wells were filled with sterile PBS). At the same time, zeroing holes (dimethyl sulfoxide) and control holes (cells, drug dissolution medium (acid culture medium) with the same concentration, MTT, dimethyl sulfoxide) are arranged at 5% CO ² Culturing in an incubator at 37 ℃.

B: after the cells are attached, the cell monolayer can be seen to be fully paved at the bottom of the hole (96-hole flat bottom plate), the cells are washed by PBS, the PBS is discarded, the dosing can be started, 4-6 compound holes are arranged according to 200 mu l of each hole, otherwise, the real situation is difficult to react. After the treatment is completed, the same edge wells are filled with acidic medium. Preventing sample volatilization.

C：5％CO ₂ The cells were incubated at 37℃for 24 hours and observed under an inverted microscope.

D: mu.l MTT solution (5 mg/ml, i.e.0.5% MTT) was added to each well and the incubation was continued for 4h. If the drug reacts with MTT, the culture broth may be centrifuged and discarded, and after careful washing with PBS, the MTT-containing culture broth may be added.

E: the culture was terminated and the in-well culture solution was carefully aspirated.

F: 150 μl of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at low speed for 10min to dissolve the crystals sufficiently. Absorbance was measured for each well at OD490nm in an enzyme-linked immunosorbent assay. Absorbance values (OD values) were detected at 630nm as reference.

G: the cell viability calculation formula is: activity = experimental OD mean-blank OD mean/1) X100%, and the resulting data is plotted in excel.

3. Experimental results

As shown in fig. 1, gefitinib alone treatment group had no effect on survival of a431 cells and significant inhibitory effect on NCI-H1666 cells, but consistent with our expected results, gefitinib combined with EGF and EGCG treatment groups, respectively, did not have significant inhibitory effect on cell line growth of two wild-type EGFR. However, gefitinib can significantly inhibit the growth of EGFR wild-type cells when EGCG and EGF are present at the same time, but has no effect on double mutant NCI-H1975 cells. Preliminary demonstration of the selective inhibitory effect of this combination of cells on cell growth showed very pronounced inhibitory effect on cells A431 and NCI-H1666 of wild-type EGFR, but not on double mutated NCI-H1975 cells.

Example 2

Effect of Gefitinib on EGFR Signaling pathway-related protein phosphorylation in wild-type and mutant cells in the Co-presence of EGCG and EGF

1. Experimental materials

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

2. Detection principle: westernBlot detection of changes in related proteins in cells

3. The experimental method comprises the following steps:

a: and (3) treating cells, wherein after the cells are attached, starving treatment is carried out, and after overnight treatment, EGCG and EGF are treated. And (3) performing experiments according to specific experimental groups, directly adding an acidic medium into a blank control group, adding 10mL of the acidic medium into the rest experimental groups, so that the final concentration of EGCG is 20 mug/mL, diluting EGF mother liquor to 20ng/mL, adding the EGF mother liquor into the acidic medium, and placing the EGF mother liquor into a cell incubator after the dosing is completed, and setting corresponding treatment time according to the experimental groups.

b: extracting protein.

c: protein quantification: the protein assay principle uses BCA protein quantification.

d: the SDS-PAGE principle separates proteins of different molecular weights by electric field strength, depending on the amount of charge carried by the different proteins. The isolated protein is then transferred to a PVDF membrane to adsorb the protein in a non-covalent manner without disrupting the biological activity of the protein polypeptide. Then, the protein transferred onto the membrane is used as an antigen, the corresponding antibody is combined with the antigen overnight or at room temperature for two hours, and then the target protein expression can be detected through substrate color development after being combined with the HRP-labeled secondary antibody at room temperature for 1 hour.

4. Experimental results

The results are shown in FIG. 2: gefitinib is able to significantly inhibit phosphorylation of ERK, an EGFR family and its downstream proteins, in wild-type EGFRA431 cells in the presence of both EGCG and EGF. However, this result was not seen in double mutated EGFRRNCI-H1975 cells. The experimental results are consistent with the cell viability experiments.

Example 3

Inhibition of EGFR wild type AA431 cell carcinoma xenograft tumor model by EGCG and Gefitinib in combination

1. Experimental materials

A431 cells and culture: human epidermoid squamous cell carcinoma A431 was cultured in complete medium containing 10% FBS in DMEM high-sugar, and the cells were diluted to 2X 10 per mL of culture broth ⁶ Individual cells. Inoculated into a petri dish. Culturing in a cell culture box. Taking cells in logarithmic growth phase, preparing single cell suspension by digestion with 0.5% pancreatin solution, centrifuging at 15000rpm for 3min, discarding supernatant, and adjusting cell density to 5×10 with PBS ⁶ /mL。

Nude mice and their feeding: male BALB/Cnu/nu clean grade mice, supplied by Kwangsi laboratory animals Inc., of Changzhou, jiangsu, for 6-8 weeks. The nude mice are raised in a sterile and ventilated closed animal house, the temperature is 25 ℃, the light is timed in the daytime, the dark environment is adopted at night, and food and drinking water are free. The cage of nude mice and water are sterilized at high temperature and high pressure. And (3) regularly replacing the clean cage, padding, drinking water and the like for the nude mice, and keeping the growth environment of the nude mice clean.

2. Experimental method

a: establishing cells of the nude mice transplanted tumor model are revived and passaged, and after the cell state is regulated for one week, the growth environment of the cells tends to be stable. After the cells were digested and counted, the number density of viable cells was adjusted to 5X 10 per 200. Mu.L of suspension ⁶ Individual cells were kept ready for use. Then 1500g was centrifuged for 3min. After the medium was aspirated, the medium was gently and gently swirled with physiological saline to prepare a cell suspension for use. In a sterile super clean bench, nude mice were grouped and ear-labeled according to the principle of uniform average body weight, and 200. Mu.L of cell suspension (about 5X 10) was injected subcutaneously into the back of each nude mouse ⁶ Individual cells). Every other day, the major (Dmax) and minor (Dmin) diameters of the tumors were measured with a vernier caliper, and the volume V of the tumor was calculated (v=dmax x Dmin 2/2).

b: animals were grouped and dosed nude mice grouped according to the above were dosed (1) blank group: injecting physiological saline into the abdominal cavity every six days every week, and lavaging the stomach with the physiological saline containing 1% -Tween 80; (2) gefitinib group: lavage (twice a week) at a dose of 50 mg/kg; (3) EGCG group: the dosage is 20mg/kg or 40mg/kg once daily (i.p. injection) every six days of week; (4) The combination administration group was tested in accordance with the administration mode of 20mg/kg or 40mg/kg by intraperitoneal injection, respectively, at a dose of 50mg/kg of Gefitinib 2 times per week every six days (once daily). Tumor volumes were determined three times a week prior to dosing. And the weight of nude mice was weighed, and the frequency was once per week, and experimental records were made. Nude mice were sacrificed after dosing was completed, tumor masses were removed and photographed. Solid tumors were preserved according to the follow-up schedule.

3. Experimental results

As shown in fig. 3, no significant weight loss was found between the groups compared to the normal control animals. However, EGCG alone treatment group promoted weight gain in nude mice after 3 weeks of administration, and showed significance compared to control group. It shows that the medicines in each group have no obvious toxic or side effect on animals. After the completion of the administration, the tumor of the nude mice was removed. As shown in fig. 4, the effect of combined use on tumor growth is shown. Tumor growth curves were plotted by measuring tumor tumors transplanted from nude mice every other day. As shown in fig. 5, gefitinib combined group significantly inhibited tumor volume growth from the 29 th measurement of tumor volume compared to Gefitinib alone treated group; as the results of the previous experiments are consistent, the selectivity of Gefitinib in vitro is better than that of the Gefitinib in vivo. The experiment also shows that Gefitinib has better inhibition phenomenon on the growth of the tumor of the nude mice, and can show very obvious inhibition effect after being combined with EGCG, thereby basically completely inhibiting the growth of tumor cells. The tumor was weighed out, and as a result, as shown in fig. 6, the inhibition rate of tumor was 52% in the Gefitinib alone group and the control group, and the inhibition rate of solid tumor was 69% and 81% in the Gefitinib alone group and the control group. There was no statistical difference, but the combination group inhibited tumor growth to some extent compared to alone. No animals died during the whole experiment.

Example 4

Inhibition of EGFR double mutant NCI-H1975 cell carcinoma xenograft tumor model by combination of EGCG and Gefitinib

1. Experimental materials

NCI-H1975 cells and cultures: human non-small cell lung carcinoma NCI-H1975 was cultured in complete medium of DMEM1640 containing 10% FBS, and the cells were diluted to 2X 10 ⁶ /mL. Inoculated into a petri dish. Culturing in a cell culture box. Taking cells in logarithmic growth phase, preparing single cell suspension by digestion with 0.5% pancreatin solution, centrifuging at 15000rpm for 3min, discarding supernatant, and adjusting cell density to 5×10 with PBS ⁶ /mL。

2. Experimental method

a: establishing cells of the nude mice transplanted tumor model are revived and passaged, and after the cell state is regulated for one week, the growth environment of the cells tends to be stable. After the cells were digested and counted, the number density of viable cells was adjusted to 3X 10 per 200. Mu.L of suspension ⁶ Individual cells were kept ready for use. Then 1500g was centrifuged for 3min. After the medium has been sucked away, the medium is removed,gently blowing with physiological saline to obtain cell suspension, grouping nude mice according to the principle of uniform weight average, cutting ear marks, and subcutaneously injecting 200 μl cell suspension (about 3×10) into back of each nude mouse ⁶ Individual cells). Every other day, the major (Dmax) and minor (Dmin) diameters of the tumors were measured with a vernier caliper, and the volume V of the tumor was calculated (v=dmax x Dmin 2/2).

b: animals were grouped and dosed following day after tumor cell inoculation, dosing was performed according to the grouped nude mice (1) placebo: injecting physiological saline into the abdominal cavity every six days every week, and lavaging the stomach with the physiological saline containing 1% -Tween 80; because Gefitinib is dissolved in 1% Tween-80 to prepare suspension; (2) Gefitinib is infused twice a week and is administered at 50mg/kg or 100mg/kg Gefitinib; (3) The EGCG group is administrated once daily according to six days per week, and the dosage of intraperitoneal injection is EGCG40mg/kg; (4) The combination administration group was administered at a dose of 40mg/kg of EGCG once daily and 50mg/kg or 100mg/kg of Gefitinib twice weekly for six days per week. Tumor volume measurement frequency was measured every other day, and nude mice body weight was weighed every two days and recorded. After the completion of the administration, the nude mice were sacrificed, and the tumors were removed and photographed. Solid tumors were preserved according to the follow-up schedule. 6-8 week old nude mice are selected in the modeling experiment and are divided into 6 groups, and 5-6 nude mice are fed with sterile feed.

3. Experimental results

The following day after tumor cell injection, the body weight of nude mice was examined every 3 days during the administration period, and the results are shown in fig. 7: the body weight of NCI-H1975 tumor-bearing mice is not affected differently by each group of medicines, and the nude mice which are not brought by the medicines die in the whole process, so that the medicines have no toxic or side effect on the nude mice. Inhibition of tumor growth in NCI-H1975 tumor-bearing mice by each group of drugs. As shown in FIG. 8, it can be seen that tumor growth was not inhibited in NCI-H1975 tumor-bearing nude mice after the administration of Gefitinib and EGCG in combination, and there was no difference in tumor growth between groups. The results show that the combined use has no effect on EGFR double mutant cell NCI-H1975 transplantation tumor, and further proves that the combined use only has an inhibitory effect on EGFR wild type tumor. Tumor growth curves were plotted as measured every other day for tumor volumes of transplanted tumor nude mice, and the results are shown in fig. 9, where combined use had no effect on tumor volume of NCI-H1975 cells; the excised tumors were weighed and the results are shown in fig. 10, with the combined use having no effect on the tumor weight of the transplanted tumors of EGFR double mutant 1975 cells.

According to the invention, an EGFR wild type and high-expression epidermoid squamous cell line and an EGFR double-mutation lung cancer cell line are selected as controls, and a series of cell experiments (MTT, western-blotting) prove that after gefitinib and EGCG and derivatives thereof are used in combination, the EGFR wild type tumor cells are obviously inhibited, and the EGFR double-mutation non-small cell lung cancer is not effective. Indicating that this combination is selective.

In vivo experiments further prove that the gefitinib and EGCG combined use can obviously inhibit the growth of EGFR wild type tumor cell transplantation tumor, has no obvious influence on the body weight, and has no obvious toxic or side effect.

In conclusion, the combined use of the epigallocatechin gallate (EGCG) and the EGFR inhibitor gefitinib can obviously inhibit the growth of EGFR wild type tumor cells, and can not cause harm to organisms, and the combination of the EGCG and the EGFR inhibitor can reduce the treatment risk and toxic and side effects caused by anticancer drugs at a large dosage.

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

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. Use of a tyrosine kinase inhibitor Gefitinib in combination with one or more of the following a) to b) for the preparation of a medicament for the treatment of EGFR wild type tumors:

a) Epigallocatechin gallate;

b)EGF；

the EGFR wild type tumor is human lung cancer cell strain NCI-H1666.

2. A pharmaceutical composition for treating human lung cancer cell strain NCI-H1666, characterized in that: comprises a first component and a second component;

the component I comprises a tyrosine kinase inhibitor Gefitinib;

the second component comprises epigallocatechin gallate and/or EGF.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is in the form of an injection.