Use of PRMT5 inhibitors in the preparation of drugs for treating drug-resistant solid tumors

Abstract

The present invention discloses the use of PRMT5 inhibitors in the preparation of drugs for treating drug-resistant solid tumors, and belongs to the field of medicine; the present invention constructs a triple-negative breast cancer cell line resistant to chemical drugs, and uses PRMT5 inhibitors to overcome the resistance to chemical drug treatment of triple-negative breast cancer, which greatly reverses the dilemma of tumor patients having no drugs available after the occurrence of chemical drug treatment resistance, delays the tumor progression rate, and improves the survival time of tumor patients, which has important clinical application prospects and needs. The present invention uses PRMT5 inhibitors to treat triple-negative breast cancer cell lines resistant to chemical drugs, which can effectively inhibit the proliferation and growth of tumors, and has broad application prospects in treating chemical drug-resistant cells.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to use of a PRMT5 inhibitor in preparing a drug for treating drug-resistant solid tumors. Background Art

[0002] Chemotherapy is the abbreviation of chemical drug therapy, which achieves the purpose of treatment by using chemotherapeutic drugs to kill cancer cells. Chemotherapy is currently one of the most effective means of treating cancer, and is known as one of the three major cancer treatments along with surgery and radiotherapy. Paclitaxel, one of the most widely used drugs, is a natural anticancer drug with a molecular formula of C 47 H 51 NO 14 , which has been widely used in the clinical treatment of various solid tumors, such as breast cancer, ovarian cancer, lung cancer and some head and neck cancers. Paclitaxel is a diterpene alkaloid compound with anticancer activity. Its main mechanism of action is to cause microtubules and microtubule dimers that make up microtubules to lose their dynamic balance, inhibit the mitosis of cancer cells and trigger cell apoptosis. Chemotherapy regimens containing taxanes are the preferred regimens recommended for the treatment of triple-negative breast cancer in major breast cancer diagnosis and treatment guidelines at home and abroad, and are widely used in clinical practice. Triple-negative breast cancer lacks effective therapeutic targets due to the lack of ER, PR, and HER2 expression. Chemical drugs, especially taxanes, play an important role.

[0003] When chemotherapy becomes resistant and the disease progresses, according to the recommendations of domestic and international guidelines, the subsequent options are gosatuzumab or other types of chemotherapy drugs. The former is a new type of antibody-drug conjugate, which has not yet been officially used in clinical practice in China. And from the relevant ASCENT clinical trial data, when triple-negative breast cancer progresses after the use of paclitaxel drugs, the median PFS time of the gosatuzumab treatment group is 5.6 months, which is higher than the 1.7 months of other chemotherapy regimens, but the efficacy is still far from meeting the treatment needs of patients. Therefore, after triple-negative breast cancer becomes resistant to chemotherapy, drugs with new mechanisms and new targets are urgently needed to overcome resistance and thus improve patient survival.

[0004] Therefore, how to overcome the resistance to chemotherapy in triple-negative breast cancer is a hot topic for clinicians, researchers and pharmaceutical companies. At present, there is no method in my country to overcome the resistance to chemotherapy in triple-negative breast cancer using protein arginine methyltransferase (PRMT) inhibitors. Summary of the invention

[0005] The purpose of the present invention is to use PRMT5 inhibitors to treat triple-negative breast cancer cell lines that are resistant to chemical drugs, which can effectively inhibit the proliferation and growth of tumors and has broad application prospects in treating chemical drug-resistant cells.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0007] Specifically, the present invention relates to the use of PRMT5 inhibitors in the preparation of drugs for preventing or treating drug resistance of solid tumors. The present invention constructs a triple-negative breast cancer cell line resistant to chemical drugs, and treats it with a PRMT5 inhibitor, which can effectively inhibit the growth and proliferation of the resistant cell line and delay the growth of the tumor, that is, a certain dose of PRMT5 inhibitor has a good therapeutic effect on the chemical drug-resistant cell line.

[0008] Specifically, the present invention relates to the use of PRMT5 inhibitors in the preparation of drugs for preventing or treating drug resistance of solid tumors, wherein the PRMT5 inhibitors include PRMT degraders.

[0009] In some preferred embodiments of the present invention, the PRMT5 inhibitors include PRMT5 inhibitors obtained by CRISPR-Cas9 knockout of PRMT, lentiviral transfection knockdown of PRMT (shPRMT), and small interfering RNA knockdown of PRMT (siPRMT).

[0010] In a preferred embodiment of the present invention, solid tumors include benign solid tumors and malignant solid tumors; benign solid tumors include hamartomas, leiomyomas, hemangiomas, lymphangiomas, adenomas or adenomatous polyps; malignant solid tumors include Hodgkin's lymphoma, non-Hodgkin's lymphoma, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, as well as head and neck malignancies, urinary system malignancies, endometrial cancer, cervical cancer, osteosarcoma, chondrosarcoma, Ewing's sarcoma, thyroid cancer, hepatoblastoma, and Wilms' tumor.

[0011] In a preferred embodiment of the present invention, the solid tumor is triple-negative breast cancer.

[0012] In a preferred embodiment of the present invention, the prevention or treatment of solid tumor drug resistance is the drug resistance of breast cancer to paclitaxel.

[0013] In some preferred embodiments of the present invention, the PRMT5 inhibitor is selected from at least one of LLY-283, GSK-591, GSK3326595, EPZ015938, JNJ-64619178, MRTX719, EPZ05666, SH3765, PF-06939999, and GSK3326595.

[0014] In some preferred embodiments of the present invention, the dosage of the PRMT5 inhibitor is selected from: 5-150 mg in vivo and 0.5-15 μM in vitro.

[0015] In some preferred embodiments of the present invention, the dosage of the PRMT5 inhibitor is selected from: 10-100 mg in vivo; specifically preferably 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg; in vitro 1-10 μM; specifically preferably 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM.

[0016] The present invention also discloses a drug for preventing or treating triple-negative breast cancer cell lines resistant to chemical drugs, comprising: at least one of PRMT5 inhibitor LLY-283 and PRMT5 inhibitor GSK-591. The present invention uses PRMT5 inhibitor LLY-283 to treat triple-negative breast cancer cell lines resistant to chemical drugs, which can significantly inhibit the growth and proliferation of resistant cell lines to slow down the growth rate of tumors.

[0017] The present invention uses the PRMT5 inhibitor GSK-591 to treat triple-negative breast cancer cell lines that are resistant to chemical drugs. Compared with the G9a inhibitor UNC0642, it has better sensitivity to resistant cell lines.

[0018] The present invention utilizes PRMT5 inhibitors to effectively inhibit the growth and proliferation of triple-negative breast cancer cell lines that are resistant to chemotherapeutic drugs, which reverses the dilemma of tumor patients having no drugs available after developing resistance to chemotherapeutic drugs, slows down the progression of tumors, and increases the survival time of tumor patients, which has important clinical application prospects and needs. Therefore, the present invention is a method of using PRMT5 inhibitors to treat triple-negative breast cancer cell lines that are resistant to chemotherapeutic drugs, which can effectively inhibit the proliferation and growth of tumors, and has broad application prospects in treating chemotherapeutic drug-resistant cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of LLY-283 on the growth and proliferation of the test cell MDA-MB-43;

[0020] Figure 2 The effect of LLY-283 on the growth and proliferation of the test cell HS-578T;

[0021] Figure 3 The effect of LLY-283 on the growth and proliferation of the test cell MDA-MB-231;

[0022] Figure 4 The effect of GSK-591 on the growth and proliferation of the test cell MDA-MB-436;

[0023] Figure 5The effect of GSK-591 on the growth and proliferation of the test cell HS-578T;

[0024] Figure 6 The effect of GSK-591 on the growth and proliferation of the test cell MDA-MB-231;

[0025] Figure 7 The effects of GSK591 inhibitors and UNC0642 inhibitors on the growth of drug-resistant cell lines;

[0026] Figure 8 The effects of LLY-283 inhibitors, GSK591 inhibitors, and the combination of LLY-283 and GSK-591 on the growth of drug-resistant cell lines;

[0027] Fig. 9 The effects of LLY-283 inhibitor, 23-hydroxybetulinic acid, and LLY-283 combined with 23-hydroxybetulinic acid on the growth of drug-resistant cell lines;

[0028] Fig.10 It is the volume of tumor proliferation in mice after injection of normal saline and LLY-283;

[0029] Fig.11 The figure shows the changes in tumor volume over time in mice after injection of normal saline and LLY-283. DETAILED DESCRIPTION

[0030] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, each embodiment of the present invention will be described in detail below in conjunction with the embodiments. However, it will be appreciated by those skilled in the art that in each embodiment of the present invention, many technical details are proposed in order to make the reader better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can also be realized. The experimental method in the following embodiments, if not otherwise specified, is a conventional method. The test materials used in the following embodiments, if not otherwise specified, are purchased from conventional biochemical reagent stores. The quantitative tests in the following embodiments are all set with 5 repeated experiments, and the results are averaged.

[0031] It should be noted that the PRMT5 inhibitor of the present invention is selected from LLY-283, GSK-591, GSK3326595, EPZ015938, JNJ-64619178, MRTX719, EPZ05666, SH3765, PF-06939999, and Formula I is at least one of the PRMT5 inhibitors Pemranmetostat (GSK3326595) that are currently undergoing clinical trials I for evaluating the safety and efficacy of treating solid tumors and non-Hodgkin's lymphoma (NCT02783300).

[0032] .

[0033] It should be noted that the solid tumors of the present invention include benign solid tumors and malignant solid tumors; benign solid tumors include but are not limited to: hamartoma, leiomyoma, hemangioma, lymphangioma, and various adenomas and adenomatous polyps, such as lipoma, uterine fibroids, breast fibroma, etc.; malignant solid tumors include but are not limited to: Hodgkin's lymphoma, non-Hodgkin's lymphoma, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, as well as head and neck malignant tumors, urinary system malignant tumors, endometrial cancer, cervical cancer, osteosarcoma, chondrosarcoma, Ewing's sarcoma, thyroid cancer, hepatoblastoma, and Wilms' tumor.

[0034] It should be noted that the administration route described in the embodiments of the present invention is selected from oral administration, parenteral administration, and transdermal administration, and the parenteral administration includes but is not limited to intravenous injection, subcutaneous injection, and intramuscular injection.

[0035] It should be noted that the use of PRMT5 inhibitors in the preparation of drugs for preventing or treating solid tumor resistance, wherein the administration frequency of PRMT5 inhibitors can be once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks or once a month.

[0036] The following is an exemplary description of one or more embodiments through the pictures in the corresponding drawings, and these exemplary descriptions do not constitute a limitation on the embodiments.

[0037] The following embodiments of the present invention take the experiments on triple-negative breast cancer chemotherapy-resistant cells and animal models as examples.

[0038] Example 1: In vitro pharmacodynamic study of PRMT5 inhibitor LLY-283 in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0039] 1. Drug under investigation

[0040] Drug name: LLY-283;

[0041] LLY-283 preparation method: DMSO was used to dissolve the PRMT5 inhibitor LLY-283, with a final concentration of 10uM; the concentration gradients were set to 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM, 0.15625μM, and 0.078125μM to be tested, for a total of 8 concentration gradients;

[0042] 2. Construction of chemotherapeutic drug-resistant cell lines

[0043] Breast cancer cells used in the test: MDA-MB-436, HS-578T, and MDA-MB-231 were purchased from the ATCC cell bank;

[0044] The culture medium used was high-glucose DMEM medium (10% fetal bovine serum);

[0045] Drugs used: Paclitaxel;

[0046] Cell culture: Test cells MDA-MB-436, HS-578T, and MDA-MB-231 were placed in culture medium and cultured at 37°C and 5% CO2 to obtain parental cells in a normal growth state and adherent state; paclitaxel was dissolved in DMSO to prepare paclitaxel solution, and paclitaxel was added to the culture medium, starting from a concentration of 5nM. After 24 hours of drug treatment, the culture medium was replaced, and the floating dead cells were washed with PBS. Cell culture medium containing 5nM paclitaxel was continued to be added until the cells grew normally and could be stably propagated; when the cells were stable at this concentration, the next concentration (10nM) was entered for gradient induction until the final maintenance concentration was 20nM, at which the cells could stably propagate and proliferate. Compared with the parent strain, the half-inhibitory concentration IC 50 If the expression rate exceeds 3-5 times, it can be determined that the drug-resistant triple-negative breast cancer cell line has been successfully constructed.

[0047] 3. Experimental Procedure

[0048] The IncuCyte live cell analysis system or CCK8 detection experiment verifies that the chemotherapy-resistant cells are more sensitive to PRMT5 inhibitors than the parental triple-negative breast cancer cells; the specific steps are as follows:

[0049] Appropriate amount of cells were seeded into 384-well or 96-well plates; after cells adhered to the plate, LLY-283 inhibitor was added and treated for 5 days; Incucyte live cell analysis system: cells were placed in an incubator, and cell proliferation was analyzed using a live cell analysis system (Sartorius, Incucyte S3) with a 10X objective lens according to the instrument instructions; MTT method: cell viability assay reagent CCK8 (Biosharp, BS350B) was added to each well to reach a final concentration of 10%. After gently shaking the plate and incubating in the dark at 37°C for 1 hour, the values ​​were measured in a multimode microplate reader (TECAN, Spark).

[0050] Example 2: In vitro pharmacodynamic study of PRMT5 inhibitor GSK-591 in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0051] 1. Drug under investigation

[0052] Drug name: GSK-591;

[0053] GSK-591 preparation method: DMSO was used to dissolve the PRMT5 inhibitor GSK-591, with a final concentration of 10 μM; the concentration gradients were set to 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, and 0.078125 μM to be tested, for a total of 8 concentration gradients;

[0054] 2. Construction of chemotherapeutic drug-resistant cell lines

[0055] Breast cancer cells used in the test: MDA-MB-436, HS-578T, MDA-MB-231;

[0056] The culture medium used was high-glucose DMEM medium (10% fetal bovine serum);

[0057] Cell culture: Test cells MDA-MB-436, HS-578T, and MDA-MB-231 were placed in culture medium and cultured at 37°C and 5% CO2 to obtain parental cells in a normal growth state and adherent state; paclitaxel was dissolved in DMSO to prepare paclitaxel solution, and paclitaxel was added to the culture medium, starting from a concentration of 5nM. After 24 hours of drug treatment, the culture medium was replaced, and the floating dead cells were washed with PBS. Cell culture medium containing 5nM paclitaxel was continued to be added until the cells grew normally and could be stably propagated; when the cells were stable at this concentration, the next concentration (10nM) was entered for gradient induction until the final maintenance concentration was 20nM, at which the cells could stably propagate and proliferate. Compared with the parent strain, the half-inhibitory concentration IC 50If the expression rate exceeds 3-5 times, it can be determined that the drug-resistant triple-negative breast cancer cell line has been successfully constructed.

[0058] 3. Experimental Procedure

[0059] The IncuCyte live cell analysis system or CCK8 detection experiment verifies that the chemotherapy-resistant cells are more sensitive to PRMT5 inhibitors than the parental triple-negative breast cancer cells; the specific steps are as follows:

[0060] Appropriate amount of cells were seeded into 384-well or 96-well plates; after cells adhered to the plate, GSK-591 inhibitor was added and treated for 5 days; Incucyte live cell analysis system: cells were placed in an incubator, and cell proliferation was analyzed using a live cell analysis system (Sartorius, Incucyte S3) with a 10X objective lens according to the instrument instructions; MTT method: cell viability assay reagent CCK8 (Biosharp, BS350B) was added to each well to reach a final concentration of 10%. After gently shaking the plate and incubating at 37°C in the dark for 1 hour, the values ​​were measured in a multimode microplate reader (TECAN, Spark).

[0061] Example 3: Study on the in vivo efficacy of PRMT5 inhibitors in chemotherapy treatment of multidrug-resistant triple-negative breast cancer PDX models

[0062] 1. Drug under investigation

[0063] Drug name: PRMT5 inhibitor LLY-283;

[0064] Preparation of drug solution: PRMT5 inhibitor (LLY-283) was dissolved in DMSO and prepared immediately before use; drug carrier preparation: 0.4g HEC (1%) + 0.1ml Tween80 (0.25%) + 0.02g Antifoam (0.05%) + PBS. The final dosage of PRMT5 inhibitor was 60mg / kg.

[0065] 2. Experimental Animals

[0066] Nude mice (Nudemouse), 6-8 weeks old, female, breeding environment: SPF grade;

[0067] Breeding environment: control the temperature at 20~26℃; control the relative humidity at 40%~70%; automatic lighting, alternating light and dark every 12 hours.

[0068] 3. Experimental Procedure

[0069] The PDX model of triple-negative breast cancer with multidrug resistance and chemotherapy was donated by the Department of Breast Surgery, Affiliated Cancer Hospital of University of Chinese Academy of Sciences: A tumor tissue sample was taken from a patient with triple-negative breast cancer with multidrug resistance and chemotherapy and induced in nude mice. The nude mice that had grown to a certain volume were euthanized, and the tumor tissue was removed and divided into tumor masses of uniform size, which were inoculated subcutaneously in the right ribs of 20 nude mice through a trocar. When the average tumor volume of the mice could be measured, they were randomly divided into 2 groups, with 10 mice in each group. Six experiments were conducted in parallel, and normal saline (NC) and LLY-283 were orally administered according to the protocol after grouping. The tumor volume was measured once every two days, the body weight was weighed, and the data was recorded. The calculation formula for tumor volume (V) is as follows:

[0070] V=0.52×a×b2

[0071] Where: a, b represent length and width respectively.

[0072] Table 1 Experimental groups and dosing regimen

[0073] Group Grouping Dosage (mg / kg) Dosage regimen Dosage Group 1 Control group (normal saline) -- QDx14 po Group 2 LLY-283 monotherapy group 60 QDx14 po

[0074] Note: QDx14: once a day, 14 times; po: oral administration.

[0075] Example 4: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0076] The present invention uses a compound of PRMT5 inhibitor LLY-283 and GSK-591, wherein the weight ratio of LLY-283 to GSK-591 is 1:1-2, and the two are compounded and used in a specific ratio, which further improves the therapeutic effect of PRMT5 inhibitor on chemical drug-resistant triple-negative breast cancer resistant cell lines.

[0077] 1. Drug under investigation

[0078] Drug name: LLY-283 and GSK-591 complex; the weight ratio of LLY-283 to GSK-591 is 1:1, denoted as S1;

[0079] Preparation method of LLY-283 and GSK-591 complex: DMSO dissolves the PRMT5 inhibitor complex respectively, and the final concentration is 10 μM; set to 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.078125 μM to be tested, a total of 8 concentration gradients;

[0080] 2. Construction of chemotherapeutic drug-resistant cell lines

[0081] Breast cancer cells used in the test: MDA-MB-436;

[0082] The culture medium used was high-glucose DMEM medium (10% fetal bovine serum);

[0083] Cell culture: The test cell MDA-MB-436 was placed in a culture medium and cultured at 37°C and 5% CO2 to obtain parent cells in a normal growth state and an adherent state; paclitaxel was dissolved in DMSO to prepare a paclitaxel solution, and paclitaxel was added to the culture medium, starting from a concentration of 5nM. After 24 hours of drug treatment, the culture medium was replaced, and the floating dead cells were washed with PBS. Cell culture medium containing 5nM paclitaxel was continued to be added until the cells grew normally and could be stably propagated; when the cells were stable at this concentration, the next concentration (10nM) was entered for gradient induction until the final maintenance concentration was 20nM, at which the cells could stably propagate and proliferate. Compared with the parent strain, the half-inhibitory concentration IC 50 If the expression rate exceeds 3-5 times, it can be determined that the drug-resistant triple-negative breast cancer cell line has been successfully constructed.

[0084] 3. Experimental Procedure

[0085] IncuCyte live cell analysis system or CCK8 detection experiment verifies that compared with the parental triple-negative breast cancer strain, the chemotherapy-resistant strain cells are more sensitive to the combination of LLY-283 and GSK-591; the specific steps are as follows:

[0086] An appropriate amount of cells was seeded into a 384-well plate or a 96-well plate; after the cells adhered to the plate, the LLY-283 and GSK-591 complex inhibitor was added and treated for 5 days; Incucyte live cell analysis system: the cells were placed in an incubator and cell proliferation was analyzed using a live cell analysis system (Sartorius, Incucyte S3) with a 10X objective lens according to the instrument instructions; MTT method: the cell viability assay reagent CCK8 (Biosharp, BS350B) was added to each well to reach a final concentration of 10%. After gently shaking the plate and incubating in the dark at 37°C for 1 hour, the values ​​were measured in a multimode microplate reader (TECAN, Spark).

[0087] Example 5: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0088] 1. Drug under investigation

[0089] Drug name: LLY-283 and GSK-591 complex; the weight ratio of LLY-283 to GSK-591 is 1:2, denoted as S2;

[0090] The other steps are the same as those in Example 4.

[0091] Example 6: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0092] 1. Drug under investigation

[0093] Drug name: LLY-283 and GSK-591 complex; the weight ratio of LLY-283 to GSK-591 is 1:0.5, denoted as S3;

[0094] The other steps are the same as those in Example 4.

[0095] Embodiment 7:

[0096] Drug name: LLY-283 and GSK-591 complex; the weight ratio of LLY-283 to GSK-591 is 1:2.5, denoted as S4;

[0097] The other steps are the same as those in Example 4.

[0098] Example 8: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0099] The preferred technical scheme of the present invention also includes: using the PRMT5 inhibitor LLY-283 in combination with 23-hydroxybetulinic acid to treat triple-negative breast cancer resistant cell lines resistant to chemical drug treatment, wherein the weight ratio of LLY-283 to 23-hydroxybetulinic acid is 1:0.5-1.5; using LLY-283 in combination with 23-hydroxybetulinic acid to treat triple-negative breast cancer resistant cell lines resistant to chemical drug treatment, further improving its therapeutic effect.

[0100] 1. Drug under investigation

[0101] Drug name: LLY-283 combined with 23-hydroxybetulinic acid complex; wherein the weight ratio of LLY-283 to 23-hydroxybetulinic acid is 1:0.5, denoted as Y1;

[0102] Preparation method of LLY-283 combined with 23-hydroxybetulinic acid complex: DMSO dissolves LLY-283 combined with 23-hydroxybetulinic acid complex respectively, with a final concentration of 10 μM; set to 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.078125 μM to be tested, a total of 8 concentration gradients;

[0103] 2. Construction of chemotherapeutic drug-resistant cell lines

[0104] Breast cancer cells used in the test: MDA-MB-231;

[0105] The culture medium used was high-glucose DMEM medium (10% fetal bovine serum);

[0106] Cell culture: The test cell MDA-MB-436 was placed in a culture medium and cultured at 37°C and 5% CO2 to obtain parent cells in a normal growth state and an adherent state; doxorubicin was dissolved in DMSO to prepare a doxorubicin solution, and doxorubicin was added to the culture medium, starting from a concentration of 5nM. After 24 hours of drug treatment, the culture medium was replaced, and the floating dead cells were washed with PBS. Cell culture medium containing 5nM doxorubicin was continued to be added until the cells grew normally and could be stably propagated; when the cells were stable at this concentration, the next concentration (10nM) was entered for gradient induction until the final maintenance concentration was 20nM, at which the cells could stably propagate and proliferate. Compared with the parent strain, the half-inhibitory concentration IC 50 If the expression rate exceeds 3-5 times, it can be determined that the drug-resistant triple-negative breast cancer cell line has been successfully constructed.

[0107] 3. Experimental Procedure

[0108] IncuCyte live cell analysis system or CCK8 detection experiment verifies that compared with the parental strain of triple-negative breast cancer, the chemotherapy-resistant strain cells are more sensitive to the LLY-283 combined with 23-hydroxybetulinic acid complex; the specific steps are as follows:

[0109] An appropriate amount of cells was seeded into a 384-well plate or a 96-well plate; after the cells adhered to the plate, the LLY-283 and GSK-591 complex inhibitor was added and treated for 5 days; Incucyte live cell analysis system: the cells were placed in an incubator and cell proliferation was analyzed using a live cell analysis system (Sartorius, Incucyte S3) with a 10X objective lens according to the instrument instructions; MTT method: the cell viability assay reagent CCK8 (Biosharp, BS350B) was added to each well to reach a final concentration of 10%. After gently shaking the plate and incubating in the dark at 37°C for 1 hour, the values ​​were measured in a multimode microplate reader (TECAN, Spark).

[0110] Example 9: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0111] 1. Drug under investigation

[0112] Drug name: LLY-283 combined with 23-hydroxybetulinic acid complex; wherein the weight ratio of LLY-283 to 23-hydroxybetulinic acid is 1:1, denoted as Y2;

[0113] The other steps are the same as those in Example 8.

[0114] Example 10: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0115] 1. Drug under investigation

[0116] Drug name: LLY-283 combined with 23-hydroxybetulinic acid complex; the weight ratio of LLY-283 to 23-hydroxybetulinic acid is 1:1.5, denoted as Y3;

[0117] The other steps are the same as those in Example 8.

[0118] Example 11: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0119] 1. Drug under investigation

[0120] Drug name: LLY-283 combined with 23-hydroxybetulinic acid complex; wherein the weight ratio of LLY-283 to 23-hydroxybetulinic acid is 1:0.25, denoted as Y4;

[0121] The other steps are the same as those in Example 8.

[0122] Example 12: In vitro pharmacodynamic study of PRMT5 inhibitors in overcoming chemotherapy-resistant triple-negative breast cancer cell lines

[0123] 1. Drug under investigation

[0124] Drug name: LLY-283 combined with 23-hydroxybetulinic acid complex; the weight ratio of LLY-283 to 23-hydroxybetulinic acid is 1:2, denoted as Y5;

[0125] The other steps are the same as those in Example 8.

[0126] Comparative Example 1:

[0127] 1. Drug under investigation

[0128] Drug name: G9a inhibitor UNC0642;

[0129] UNC0642 preparation method: G9a inhibitor UNC0642 was dissolved in DMSO, with a final concentration of 10 μM; the concentration gradients were set to 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, and 0.078125 μM to be tested, for a total of 8 concentration gradients;

[0130] 2. Construction of chemotherapeutic drug-resistant cell lines

[0131] Breast cancer cells used in the test: MDA-MB-436;

[0132] The culture medium used was high-glucose DMEM medium (10% fetal bovine serum);

[0133] Cell culture: The test cell MDA-MB-436 was placed in a culture medium and cultured at 37°C and 5% CO2 to obtain parent cells in a normal growth state and an adherent state; paclitaxel was dissolved in DMSO to prepare a paclitaxel solution, and paclitaxel was added to the culture medium, starting from a concentration of 5nM. After 24 hours of drug treatment, the culture medium was replaced, and the floating dead cells were washed with PBS. Cell culture medium containing 5nM paclitaxel was continued to be added until the cells grew normally and could be stably propagated; when the cells were stable at this concentration, the next concentration (10nM) was entered for gradient induction until the final maintenance concentration was 20nM, at which the cells could stably propagate and proliferate. Compared with the parent strain, the half-inhibitory concentration IC 50 If the expression rate exceeds 3-5 times, it can be determined that the drug-resistant triple-negative breast cancer cell line has been successfully constructed.

[0134] 3. Experimental Procedure

[0135] Appropriate amount of cells were seeded into 384-well or 96-well plates; after cells adhered to the plate, G9a inhibitor UNC0642 was added and treated for 5 days; Incucyte live cell analysis system: cells were placed in an incubator, and cell proliferation was analyzed using a live cell analysis system (Sartorius, Incucyte S3) with a 10X objective lens according to the instrument instructions; MTT method: cell viability assay reagent CCK8 (Biosharp, BS350B) was added to each well to reach a final concentration of 10%. After gently shaking the plate and incubating at 37°C in the dark for 1 hour, the values ​​were measured in a multimode microplate reader (TECAN, Spark).

[0136]

Performance test

[0137] 1. The therapeutic effect of PRMT5 inhibitors on drug-resistant triple-negative breast cancer cell lines

[0138] PAR represents the parental cell line; 20A, 20B, and 20C are successfully constructed drug-resistant cell lines and different clones, respectively.

[0139] (1) LLY-283 test results,

[0140] Figure 1 , Figure 2 , Figure 3The effects of LLY-283 on the growth and proliferation of the test cells MDA-MB-436, HS-578T, and MDA-MB-231 are respectively; Figure 1 It can be seen that with the increase of LLY-283 concentration, the growth and proliferation of the drug-resistant MDA-MB-436 cell line was significantly reduced compared with the parental cell line, and the proliferation rate was relatively slow, indicating that the PRMT5 inhibitor LLY-283 has a good therapeutic effect on the drug-resistant MDA-MB-436 cell line; similarly, Figure 2 , Figure 3 It can be seen that compared with the parental cell line, the RMT5 inhibitor LLY-283 has a good inhibitory effect on the growth and proliferation of the drug-resistant HS-578 cell line and the drug-resistant MDA-MB-231 cell line, that is, the RMT5 inhibitor LLY-283 has excellent sensitivity in treating the drug-resistant HS-578 cell line and the drug-resistant MDA-MB-231 cell line.

[0141] (2) GSK-591 test results

[0142] Figure 4 , Figure 5 , Figure 6 The effects of GSK-591 on the growth and proliferation of test cells MDA-MB-436, HS-578T, and MDA-MB-231 are respectively; Figure 4 It can be seen that with the increase of GSK-591 concentration, the growth and proliferation of the drug-resistant MDA-MB-436 cell line was significantly reduced compared with the parental cell line, and the proliferation rate was relatively slow, indicating that the PRMT5 inhibitor LLY-283 has a good therapeutic effect on the drug-resistant MDA-MB-436 cell line; similarly, Figure 5 , Figure 6 It can be seen that compared with the parental cell line, the PRMT5 inhibitor LLY-283 has better sensitivity in treating the drug-resistant HS-578 cell line and the drug-resistant MDA-MB-231 cell line.

[0143] Figure 7 The effects of GSK591 inhibitors and UNC0642 inhibitors on the growth of drug-resistant cell lines; Figure 7 It can be seen that compared with GSK591, the G9a inhibitor UNC0642 (Comparative Example 1) has no killing effect on the drug-resistant MDA-MB-231 cells, which once again verifies that the PRMT5 inhibitors LLY-283 and GSK-591 have a good therapeutic effect on the drug-resistant triple-negative breast cancer cells constructed in the present invention.

[0144] (3) Test results of LLY-283 and GSK-591 complex

[0145] Figure 8The effects of LLY-283 inhibitors, GSK591 inhibitors, and the combination of LLY-283 and GSK-591 on the growth of drug-resistant cell lines; Figure 8 It can be seen that compared with the use of LLY-283 inhibitor and GSK591 alone, the use of the combination of LLY-283 and GSK-591 has a better therapeutic effect on the drug-resistant triple-negative breast cancer cells constructed in the present invention, and when the weight ratio of LLY-283 to GSK-591 in the combination of LLY-283 and GSK-591 is 1:1-2, it has a better therapeutic effect on drug-resistant triple-negative breast cancer cells.

[0146] (4) Test results of LLY-283 combined with 23-hydroxybetulinic acid complex

[0147] Fig. 9 The effects of LLY-283 inhibitor, 23-hydroxybetulinic acid, and LLY-283 combined with 23-hydroxybetulinic acid on the growth of drug-resistant cell lines; Fig. 9 It can be seen that compared with the use of LLY-283 inhibitor and 23-hydroxybetulinic acid alone, the use of LLY-283 combined with 23-hydroxybetulinic acid complex has a better therapeutic effect on the drug-resistant triple-negative breast cancer cells constructed in the present invention, and when the weight ratio of LLY-283 to 23-hydroxybetulinic acid in the LLY-283 combined with 23-hydroxybetulinic acid complex is 1:0.5-1.5, it has a better therapeutic effect on drug-resistant triple-negative breast cancer cells.

[0148] 2. In vivo efficacy in drug-resistant triple-negative breast cancer PDX models

[0149] Fig.10 is the volume of tumor proliferation in mice after injection of normal saline or LLY-283; Figure 8 It can be seen that the tumor volume of mice after injection of LLY-283 was significantly lower than that of the control group (NC); this indicates that the PRMT5 inhibitor LLY-283 can effectively inhibit tumor proliferation in the chemoresistant PDX model of triple-negative breast cancer.

[0150] Fig.11 The changes in tumor volume over time after injection of saline and LLY-283 in mice; Fig. 9 It can be seen that as time goes by, the tumor volume growth rate in mice injected with LLY-283 is significantly lower than that in mice injected with normal saline, further demonstrating that the PRMT5 inhibitor LLY-283 can effectively inhibit tumor proliferation in the chemoresistant PDX model of triple-negative breast cancer.

[0151] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0152] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a PRMT5 inhibitor in the preparation of a drug for preventing or treating drug resistance in solid tumors, wherein the PRMT5 inhibitor is selected from LLY-283 and GSK-591; the prevention or treatment of drug resistance in solid tumors is drug resistance of breast cancer to paclitaxel; and the solid tumor is triple-negative breast cancer.

2. The use according to claim 1, characterized in that: The dosage of the PRMT5 inhibitor is selected from: 5-150 mg in vivo and 0.5-15 μM in vitro.

3. The use according to claim 2, characterized in that: The dosage of the PRMT5 inhibitor is selected from: 10-100 mg in vivo and 1-10 μM in vitro.

Images