Use of new-generation EGFR inhibitor single drug or combination thereof in preparing drugs for tumor diseases

Abstract

The present invention provides the use of a new-generation EGFR inhibitor single drug or a combination thereof in preparing drugs for tumor diseases.

Description

Application of new generation EGFR inhibitors, alone or in combination, in the preparation of drugs for tumor diseases Technical Field

[0001] The present application belongs to the field of medicine and relates to the use of a new generation of EGFR inhibitors, alone or in combination, in the preparation of drugs for tumor diseases, and specifically to the use of N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or its pharmaceutically acceptable salts, alone or in combination with EGFR inhibitors, in the preparation of cancer drugs. Background Art

[0002] Primary bronchogenic carcinoma (PBLC), commonly known as lung cancer, is the malignant tumor with the highest incidence and mortality in China. In 2022, lung cancer ranked first among all new malignant tumor cases in China, accounting for 18.06%. Lung cancer also accounted for 23.9% of all deaths from malignant tumors in China, also ranking first. Early-stage lung cancer often has no obvious symptoms, and most patients are already in the advanced stage by the time they present symptoms and seek medical attention. The overall five-year survival rate for patients with advanced lung cancer is approximately 20%.

[0003] Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer. Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutation in NSCLC. The prevalence of EGFR mutations in the Chinese NSCLC population is 28.2%, and in lung adenocarcinoma, the prevalence is as high as 50.2%. First- and second-generation EGFR tyrosine kinase inhibitors (TKIs) are used as first-line treatment for patients with advanced NSCLC harboring classic EGFR mutations (EGFR exon 19 deletions or exon 21 L858R point mutations). They can improve median progression-free survival (PFS) to 9.2-14.7 months, significantly superior to chemotherapy. However, disease progression is inevitable, with secondary EGFR T790M mutations being the most common.

[0004] Ametinib is the first Chinese-origin third-generation EGFR-TKI that can effectively inhibit the EGFR T790M mutation. It has been approved for the treatment of patients with advanced NSCLC who have progressed on previous EGFR-TKI treatment and are T790M-positive. It is also the world's first third-generation EGFR-TKI with a second-line PFS of more than 1 year. At the same time, ametinib first-line treatment significantly prolongs the median PFS compared to the first-generation EGFR-TKI gefitinib, and has been approved as a first-line standard treatment for patients with advanced NSCLC with classic EGFR mutations.

[0005] Although third-generation EGFR-TKIs have brought more survival benefits to patients with EGFR-mutated NSCLC, the problem of drug resistance is still unavoidable. Similar to the resistance mechanisms of first- and second-generation EGFR-TKIs, the currently reported resistance mechanisms of third-generation EGFR-TKIs mainly include EGFR secondary mutations, bypass activation, and histological transformation. Among EGFR secondary mutation resistance, the most common resistance mechanism is C797X mutation. Therefore, the use of new EGFR inhibitors in combination with ametinib to simultaneously cover EGFR classic mutations and resistance mutations may significantly improve efficacy and maximize the survival benefits of patients receiving ametinib treatment.

[0006] Summary of the Invention

[0007] The present invention provides the use of an EGFR inhibitor alone or in combination in the preparation of a medicament for treating tumor diseases, wherein the EGFR inhibitor is N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof, and the use of the EGFR inhibitor alone or in combination with other EGFR inhibitors in the preparation of a medicament for treating cancer.

[0008] The present invention also includes a method for treating cancer, comprising administering to a subject an effective dose of N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof, or N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof further in combination with other EGFR inhibitors.

[0009] The present invention further provides a pharmaceutical composition, characterized in that N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof, alone or further in combination with an EGFR inhibitor, and one or more pharmaceutically acceptable carriers, excipients or diluents.

[0010] In certain embodiments of the present invention, the pharmaceutically acceptable salt is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2,5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, lauryl phosphate, maltodextrin, maltodextrin, pyroglutamic acid, tartaric acid, t-butyl phthalate ... Alkyl sulfate, dibenzoyltartaric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactosonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1,5-naphthalene disulfonic acid, naphthalene-2-sulfonic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, n-butyric acid, p-hydroxybenzoic acid, or L-malic acid;

[0011] Phosphoric acid, hydrochloric acid, sulfuric acid, hydrobromic acid, citric acid, oxalic acid, maleic acid, salicylic acid or p-hydroxybenzoic acid is preferred.

[0012] In certain embodiments of the present invention, the molar ratio of N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof to other EGFR inhibitors is 1:10-10:1, preferably 1:1-10:1, more preferably 2:1-6:1, for example 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1.

[0013] In certain embodiments of the present invention, the EGFR inhibitor is a small molecule inhibitor or a large molecule monoclonal antibody; preferably a small molecule EGFR-TKI inhibitor; more preferably a third-generation EGFR-TKI inhibitor; further preferably ametinib, osimertinib, vumetinib, befortinib or omotinib; most preferably ametinib.

[0014] In certain embodiments of the present invention, the third-generation EGFR-TKI inhibitor may be in a free state, a crystalline state, or a salt or its crystalline state, wherein the salt is selected from hydrochloride, phosphate, phosphate, sulfate, sulfate, sulfite, acetate, oxalate, malonate, valerate, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, methanesulfonate, isethionate, maleate, malate, tartrate, benzoate, pamoate, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate or laurylsulfonate; preferably methanesulfonate.

[0015] In certain embodiments of the present invention, the cancer is selected from breast cancer, ovarian cancer, prostate cancer, melanoma, brain tumor, esophageal cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, intestinal cancer, kidney cancer, skin cancer, glioblastoma, neuroblastoma, sarcoma, liposarcoma, osteochondroma, osteoma, osteosarcoma, seminoma, testicular tumor, uterine cancer, head and neck cancer, multiple myeloma, malignant lymphoma, polycythemia vera, leukemia, thyroid tumor, ureteral tumor, bladder tumor, gallbladder cancer, bile duct cancer or choriocarcinoma; preferably non-small cell lung cancer.

[0016] In certain embodiments of the present invention, the non-small cell lung cancer is selected from non-small cell lung cancer with EGFR mutation, preferably non-small cell lung cancer with one or more gene mutations of L858R mutation, exon 19 deletion mutation, T790M mutation, and C797X mutation.

[0017] In certain embodiments of the present invention, the non-small cell lung cancer is selected from non-small cell lung cancer resistant to the third-generation EGFR-TKI; preferably non-small cell lung cancer resistant to osimertinib or ametinib.

[0018] In certain embodiments of the present invention, the single administration dose range of N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof is selected from 0.1-1000 mg / kg; preferably 0.5-200 mg / kg; more preferably 1-150 mg / kg; most preferably 10-150 mg / kg; preferred examples: 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80mg / kg, 90mg / kg, 100mg / kg, 110mg / kg, 120mg / kg, 130mg / kg, 140mg / kg, 150mg / kg; or, a single dose range is selected from 1-1000mg; preferably 10-1000mg; more preferably 50-500mg; preferred examples: 10mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg.

[0019] In certain embodiments of the present invention, the administration frequency of N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof is once a day, twice a day or three times a day.

[0020] In certain embodiments of the present invention, the N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof is administered once a day at a dose selected from 1-150 mg / kg; or, administered once a day at a dose selected from 10-1000 mg.

[0021] In certain embodiments of the present invention, the single administration dose range of the EGFR inhibitor is selected from 0.1-1000 mg / kg; preferably 0.5-200 mg / kg; more preferably 1-100 mg / kg; further preferably 1-20 mg / kg; most preferably 1-10 mg / kg; or, the single administration dose range is selected from 1-1000 mg; preferably 10-200 mg; more preferably 55 mg or 110 mg.

[0022] In certain embodiments of the present invention, the EGFR inhibitor is administered once a day, twice a day, or three times a day.

[0023] In certain embodiments of the present invention, the EGFR inhibitor is administered once a day at a dose selected from 1-10 mg / kg; or, is administered once a day at a dose selected from 55 mg or 110 mg.

[0024] In certain embodiments of the present invention, the N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof and the EGFR inhibitor can be used simultaneously, concurrently, independently or sequentially.

[0025] In certain embodiments of the present invention, X in C797X is any amino acid, preferably C797S.

[0026] The term "effective dose" refers to the amount of a drug that is effective in treating a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent and preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent and preferably prevent) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with the condition to a certain extent. Depending on the extent to which the drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, in vivo efficacy can be measured by assessing duration of survival, progression-free survival (PFS) duration, response rate (RR), duration of response and / or quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Combined index of inhibition of PC-9 cell proliferation by drug A combined with drug B

[0028] Figure 2 Combined index of inhibition of HCC827 cell proliferation by drug A combined with drug B

[0029] Figure 3 Combined index of inhibition of NCI-H1975 cell proliferation by drug A combined with drug B

[0030] Figure 4 Efficacy of drug A combined with drug B on subcutaneous xenograft tumor model of human non-small cell lung cancer LD1-0025-200717

[0031] Figure 5 Efficacy of drug A combined with drug B on the Ba / F3 EGFR-L858R cell line in the NOD SCID mouse subcutaneous homograft tumor model

[0032] Figure 6 Efficacy of drug A combined with drug B in the subcutaneous xenograft tumor model of human non-small cell lung cancer LD1-0025-390629 DETAILED DESCRIPTION

[0033] The present application will be explained in more detail below with reference to the embodiments. The embodiments of the present application are only used to illustrate the technical solutions of the present application and are not intended to limit the essence and scope of the present application.

[0034] Biological evaluation methods

[0035] Example 1. Evaluation of the inhibitory effect of drug A and drug B combined on the proliferation of human lung cancer cells with EGFR mutations.

[0036] 1. Experimental Materials

[0037] 1.1 Cell lines and experimental reagents

[0038] Human non-small cell lung cancer cells PC-9 were purchased from Guangzhou Geneo, and HCC-827 and NCI-H1975 were purchased from Nanjing Kebai.

[0039] Drug A: Prepared using the method described in PCT / CN2023 / 082178, the drug was dissolved in DMSO;

[0040] Drug B: prepared using the method disclosed in WO2016054987, and dissolved in DMSO;

[0041] Luminescent Cell Viability Assay was purchased from Promega, catalog number G7573;

[0042] 1640 culture medium was purchased from Gibco, catalog number 11995073;

[0043] FBS was purchased from Gibco, catalog number 10091148;

[0044] Pancreatin was purchased from Gibco, catalog number 25200056;

[0045] PBS was purchased from Gibco, catalog number 10010023.

[0046] 1.2 Instruments:

[0047] Microplate reader (BioTek Synergy H1); pipette (Eppendorf & Rainin).

[0048] 2. Experimental methods and processing results

[0049] 2.1 Evaluation of the combined effects of drug A and drug B on the growth inhibition of EGFR mutant human lung cancer cells

[0050] HCC827, PC-9 and NCI-H1975 cells in good growth condition were seeded into 96-well plates. After overnight adherence and growth, different concentrations of the test substances were administered, and the two test substances were cross-acted at different concentrations.

[0051] The starting concentration of test substance A was set at 300.00 nM, with a 1:3 dilution, for a total of 6 concentration gradients; the starting concentration of test substance B was set at 50.00 nM, with a 1:3 dilution, for a total of 6 concentration gradients. The specific drug action schedule is shown in Table 1. After 6 days of combined application of the test substances to each cell type, the CTG method was used to detect the in vitro proliferation inhibitory effect of the test substances on human lung cancer cells.

[0052] Table 1 Dosage regimen of drug A and drug B

[0053] According to the signal value measured by the microplate reader, the cell growth inhibition rate was calculated according to the following formula:

[0054] Growth inhibition rate (%) = [1-(measured value - minimum mean - background value) / (maximum mean - background value - minimum mean)] × 100% (measured value: compound well reading; minimum value: ZPE well reading (well reading without cell culture medium); maximum value: HPE well reading (DMSO-treated cell reading); background value: cell background measurement value before compound addition).

[0055] The results of the cell growth inhibition rate of the combined action of Drug A and Drug B are shown in Tables 2 to 4. Drug A and Drug B can inhibit the growth of EGFR mutant lung cancer cells in a concentration-dependent manner within different concentration ranges. The combination of the two drugs can enhance the cell growth inhibition, and the drug effects have an additive or synergistic effect.

[0056] Table 2 Inhibitory effect of drug A and drug B combined on HCC827 cell growth

[0057] Table 3 Inhibitory effect of drug A and drug B on PC-9 cell growth

[0058] Table 4 Inhibitory effect of drug A and drug B on the growth of NCI-H1975 cells

[0059] 2.2 Evaluation of the combined inhibitory effect of drug A and drug B on the growth of EGFR-mutant human lung cancer cells

[0060] Based on the experimental results of Experiment 2.1, the cell growth inhibition rate was converted into cell survival rate, and the combination index of the two drugs was analyzed using Combenefit software.

[0061] Figures 1, 2, and 3 show that the combination index of the two drugs is between -10 and 10 at most drug combination concentrations, indicating that the combination of drug A and drug B has an additive effect; at a small number of concentrations, the combination index is greater than 10, indicating that the combination of drug A and drug B has a synergistic effect.

[0062] 3. Experimental conclusions:

[0063] The combination of drug A and drug B can enhance the growth inhibitory effect on HCC827, PC-9 and NCI-H1975 cells compared with single drug.

[0064] Example 2. In vivo pharmacodynamics of the combination of drug A and drug B against human non-small cell lung cancer LD1-0025-200717 in a NU / NU mouse subcutaneous xenograft tumor model

[0065] 1. Experimental Materials

[0066] 1.1 Human Non-Small Cell Lung Cancer LD1-0025-200717 PDX Model Information

[0067] LD1-0025-200717, human non-small cell lung cancer tumor tissue, passaged to FP4+6, was used in this efficacy study and was provided by Shanghai Lidi Biotechnology Co., Ltd.

[0068] Table 5. Model information

[0069] 1.2 Experimental animals

[0070] NU / NU mice, female, weighing 18–21 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SCXK (Beijing) 2021-0006).

[0071] 1.3 Test drugs

[0072] 1) Drug A: Prepared using the method described in PCT / CN2023 / 082178, using 0.5% HPMC for drug preparation.

[0073] 2) Drug B: Prepared by the method disclosed in WO2016054987, and the drug is formulated with pH 4.18 acetate buffer solution.

[0074] 2. Experimental method

[0075] The tumor mass of the LD1-0025-200717 human-derived non-small cell lung cancer xenograft in vivo was cut into tumor tissues with a size of about 3 mm × 3 mm × 3 mm (about 45 - 60 mg), and inoculated subcutaneously on the right back of NU / NU mice. The mice were observed after inoculation and the growth of the tumor was monitored. On the 28th day after inoculation, the average tumor volume of the tumor-bearing mice was 179.69 mm 3 At this time, grouping and drug administration were carried out, and the day of grouping and drug administration was defined as day 0. The specific grouping information is shown in Table 6.

[0076] Table 6 Grouping and dosing regimen of animals in the in vivo efficacy experiment

[0077] The experimental index was to investigate the effect of the drug on tumor growth. The specific index was the relative tumor proliferation rate T / C (%) or the tumor growth inhibition rate TGI (%).

[0078] Tumor volume measurement: Measured twice a week using a vernier caliper. The formula for calculating the tumor volume is V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively.

[0079] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). According to the results of tumor measurement, the relative tumor volume (RTV) was calculated. The formula is RTV = Vt / V0, where V0 is the average tumor volume measured at the time of grouping and drug administration (i.e., d0), Vt is the average tumor volume at a certain measurement, and TRTV and CRTV take data on the same day.

[0080] Tumor growth inhibition rate TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100 (Ti > T0) or TGI (%) = [1 - (Ti - T0) / T0] × 100 (Ti < T0), where Ti is the average tumor volume after the start of drug administration in the compound group, T0 is the average tumor volume at the first drug administration in the compound group, V0 is the average tumor volume at the first drug administration in the vehicle control group, and Vi is the average tumor volume after the start of drug administration in the vehicle control group.

[0081] The body weight of all tumor-bearing mice was measured daily. The experiment was completed, the experimental endpoint was reached, or the average tumor volume reached 2000mm 3 The animals were killed by CO2 anesthesia, and then the tumors were removed by dissection and photographed.

[0082] Experimental data were analyzed and graphed using GraphPad Prism 9.4.0. Tumor volume data for each group at different time points were statistically analyzed using Dunnett's multiple comparisons test in a two-way ANOVA to assess intergroup differences. Differences in tumor volume between the two groups were analyzed using the t-test, with a P < 0.05 defining the difference as statistically significant.

[0083] 3. Experimental Results

[0084] The growth inhibitory effects of drug A combined with drug B on subcutaneous xenograft tumors of human non-small cell lung cancer LD1-0025-200717 are shown in Table 7 and Figure 4.

[0085] Table 7. Evaluation of the anti-tumor efficacy of the test drugs in the LD1-0025-200717PDX NU / NU mouse tumor model Note: Data are expressed as Mean ± SEM. Statistical analysis was performed using one-way ANOVA compared with the vehicle group. Additionally, statistical analysis using t-tests was performed for the comparison of the drug A 50 mg / kg combined with drug B 10 mg / kg group and the drug A 50 mg / kg group, with p values ​​< 0.001.

[0086] At the end of the experiment, the average tumor volume of tumor-bearing mice in the Vehicle group was 949.86 mm 3 The average tumor volume of the drug A group and the drug A combined with drug B group was 62.07 mm 3 and 0mm 3 The tumor inhibition rates were 165.38% and 200%, respectively, both significantly different from the vehicle group (p<0.001). Comparison of tumor volume between the treatment groups on day 28 post-dose revealed that the combination of Drug A and Drug B significantly outperformed Drug A (p<0.001). Throughout the experiment, tumor-bearing mice tolerated both Drug A alone and in combination with Drug B well, with no significant weight loss.

[0087] 4. Experimental Conclusion

[0088] In the subcutaneous xenograft tumor model of human non-small cell lung cancer LD1-0025-200717, drug A monotherapy had significant therapeutic effects, with a tumor inhibition rate of 165.38%; compared with drug A monotherapy, drug A combined with drug B showed stronger anti-tumor effects, with a tumor inhibition rate of 200%, achieving a synergistic effect without showing obvious toxic side effects.

[0089] Example 3. In vivo pharmacodynamics of the combination of drug A and drug B on the Ba / F3 EGFR-L858R cell line in a NOD SCID mouse subcutaneous homograft tumor model

[0090] 1. Experimental Materials

[0091] 1.1 Cells

[0092] Cell line: Ba / F3 EGFR-L858R

[0093] Source: Kangyuan Broad Biotechnology (Beijing) Co., Ltd.

[0094] 1.2 Experimental animals

[0095] NOD SCID mice, female, weighing 17–24 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0096] 1.3 Test drugs

[0097] 1) Drug A: Prepared using the method described in PCT / CN2023 / 082178, using 0.5% HPMC for drug preparation.

[0098] 2) Drug B: prepared using the method disclosed in WO2016054987, using pH 4.18 acetic acid buffer for drug preparation.

[0099] 2. Experimental Methods

[0100] 2.1 Cell culture

[0101] Ba / F3 EGFR-L858R cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1 μg / mL Puromycin in a 37°C, 5% CO2 incubator. Ba / F3 EGFR-L858R cells in the logarithmic growth phase were collected and counted. Cells were resuspended in PBS to 2×10 7 cells / mL and then inoculated.

[0102] 2.2 Cell seeding and grouping

[0103] 0.1 mL of Ba / F3 EGFR-L858R cell suspension (containing 2×106 The cells were subcutaneously inoculated on the right side of the back of NOD SCID mice. The tumor growth was observed and the animals were randomly divided into groups according to the tumor volume and weight. The average tumor volume at the time of grouping was 152 mm 3 The day of grouping was defined as day 0, i.e., PG-D0. Specific grouping information is shown in Table 8.

[0104] Table 8 Animal groups and dosing regimens for in vivo efficacy experiments Note: After 2 weeks of administration, the drug was discontinued and the animals in all groups were observed until the average tumor volume exceeded 2000 mm. 3 .

[0105] The experimental indicator is to examine whether tumor growth is inhibited, delayed or cured. The tumor diameter is measured with a vernier caliper twice a week. The tumor volume is calculated as: V = 0.5 × D × d 2 , V represents the tumor volume, D and d represent the length and width of the tumor, respectively.

[0106] The tumor inhibition efficacy of the compounds was evaluated by TGI (%) and T / C (%).

[0107] Calculation of TGI (%): If the tumor did not regress, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / (average tumor volume at the end of treatment for the vehicle control group - average tumor volume at the time of grouping for the vehicle control group)] × 100%. If the tumor regressed, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / average tumor volume at the time of grouping for that treatment group] × 100%.

[0108] Calculation of T / C (%): T / C (%) = average tumor volume of a treatment group at the end of drug administration / average tumor volume of the vehicle control group at the end of treatment × 100%.

[0109] All data are presented as mean ± SEM. Tumor volume data for each group at different time points were statistically analyzed using Dunnett's multiple comparisons test in two-way ANOVA to assess intergroup differences. Tumor volume and weight were analyzed between groups using Dunnett's multiple comparisons test in one-way ANOVA. Differences in tumor volume between the two groups were analyzed using the t-test. All data were analyzed using GraphPad Prism 9, and p < 0.05 was considered significant.

[0110] 3. Experimental Results

[0111] The inhibitory effects of drug A combined with drug B on the growth of subcutaneous homograft tumors of cell line Ba / F3 EGFR-L858R in NOD SCID mice are shown in Table 9 and Figure 5 .

[0112] Table 9. Evaluation of the anti-tumor efficacy of the test drugs on the cell line Ba / F3 EGFR-L858R NOD SCID mouse tumor model Note: Based on the tumor volume on day 28 after administration, the p values ​​of the drug A 50 mg / kg combined with drug B 10 mg / kg treatment group were compared with the drug A 50 mg / kg group and the drug B 10 mg / kg group, respectively, which were statistically significant.

[0113] On the 14th day after administration, the average tumor volume of tumor-bearing mice in the Vehicle group was 2359 mm 3 All animals in the drug A, drug B, and drug A combined with drug B groups experienced complete tumor regression, with a tumor inhibition rate of 200%, which was significantly different from the vehicle group (p<0.0001). Based on the tumor volume on the 28th day after administration (i.e., 14 days after drug withdrawal), the tumor inhibition effect of the drug A combined with drug B group was significantly better than that of the drug A group (p=0.0315) and the drug B group (p=0.0011). In the drug A group, drug B group, and drug A combined with drug B group, 6 / 7, 7 / 7, and 2 / 7 mice had tumor volumes greater than 300 mm, respectively. 3 The results indicate that the combination therapy significantly delayed tumor recurrence compared to the monotherapy group. Throughout the experiment, tumor-bearing mice treated with either drug A alone, drug B alone, or the combination of drug A and drug B showed no significant weight loss, demonstrating good tolerance to treatment with each drug at the experimental dose.

[0114] 4. Experimental Conclusion

[0115] In the cell line Ba / F3 EGFR-L858R homologous transplant tumor model, both drug A and drug B alone and their combination therapy significantly inhibited tumor growth. During the drug withdrawal observation period, the effect of drug A combined with drug B treatment group in delaying tumor recurrence was significantly better than that of drug A and drug B monotherapy groups, showing a stronger anti-tumor effect without showing obvious toxic side effects.

[0116] Example 4. In vivo pharmacodynamic study of the combination of drug A and drug B on human non-small cell lung cancer LD1-0025-390629 in a subcutaneous xenograft tumor model of NU / NU mice

[0117] 1. Experimental Materials

[0118] 1.1 Human non-small cell lung cancer LD1-0025-390629 model information

[0119] LD1-0025-390629, human non-small cell lung cancer tumor tissue, a third-generation EGFR inhibitor-resistant PDX model, passaged to the FP4+2 generation for this efficacy study, was provided by Shanghai Lidi Biotechnology Co., Ltd.

[0120] Table 10. Model information

[0121] 1.2 Experimental animals

[0122] NU / NU mice, female, weighing 18–21 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SCXK (Beijing) 2021-0006).

[0123] 1.3 Test drugs

[0124] 1) Drug A: Prepared using the method described in PCT / CN2023 / 082178, using 0.5% HPMC for drug preparation.

[0125] 2) Drug B: prepared using the method disclosed in WO2016054987, using pH 4.18 acetic acid buffer for drug preparation.

[0126] 2. Experimental Methods

[0127] LD1-0025-390629 human non-small cell lung cancer xenografts were cut into approximately 3 mm × 3 mm × 3 mm (approximately 45-60 mg) pieces of tumor tissue and inoculated subcutaneously on the right back of NU / NU mice. The mice were observed and tumor growth was monitored. On day 21 of inoculation, the average tumor volume of the tumor-bearing mice was 158.18 mm. 3 The day of group administration was defined as day 0. Specific group information is shown in Table 11.

[0128] Table 11 Animal groups and dosing regimens for in vivo efficacy experiments

[0129] The experimental indicators are to examine the effect of drugs on tumor growth, and the specific indicators are relative tumor proliferation rate T / C (%) or tumor inhibition rate TGI (%).

[0130] Tumor volume measurement: Use a vernier caliper to measure twice a week. The tumor volume is calculated as V = 0.5a × b 2 , a and b represent the long diameter and wide diameter of the tumor, respectively.

[0131] Relative tumor growth rate (T / C) (%): The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) was calculated based on the tumor measurement results using the formula RTV = V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., d0), V t is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0132] Tumor growth inhibition rate TGI (%) = [1-(T i -T0) / (V i -V0)]×100(T i >T0) or TGI (%) = [1-(T i -T0) / T0]×100(T i <T0),T i is the average tumor volume of the compound group after the start of administration, T0 is the average tumor volume of the compound group at the first administration, V0 is the average tumor volume of the vehicle control group at the first administration, and V i The mean tumor volume of the vehicle control group after the start of drug administration.

[0133] The body weight of all tumor-bearing mice was measured daily. The experiment was completed, the experimental endpoint was reached, or the average tumor volume reached 2000mm 3 The animals were killed by CO2 anesthesia, and then the tumors were removed by dissection and photographed.

[0134] Experimental data were analyzed and graphed using GraphPad Prism 9.4.0. Tumor volume data for each group at different time points were statistically analyzed using Dunnett's multiple comparisons test in a two-way ANOVA to assess intergroup differences. Differences in tumor volume between the two groups were analyzed using the t-test, with a p < 0.05 defining the difference as statistically significant.

[0135] 3. Experimental Results

[0136] The growth inhibitory effects of drug A combined with drug B on subcutaneous xenograft tumors of human non-small cell lung cancer LD1-0025-390629 are shown in Table 12 and Figure 6.

[0137] Table 12. Evaluation of the anti-tumor efficacy of the test drugs in the LD1-0025-390629PDX NU / NU mouse tumor model Note: Data are expressed as Mean ± SEM and compared with the Vehicle group. Statistical analysis was performed using one-way ANOVA. Additionally, the 50 mg / kg plus 10 mg / kg drug A group compared with the 30 mg / kg (BID) drug A group and the 10 mg / kg drug B group were statistically analyzed using t-tests. The p-values ​​were 0.0028 and 0.0112, respectively.

[0138] At the end of the experiment, the average tumor volume of tumor-bearing mice in the Vehicle group was 1671.0 mm 3 The average tumor volumes of drug A group, drug B group, and drug A combined with drug B group were 603.1, 981.4, and 207.9 mm, respectively. 3 Compared with the Vehicle group, the Drug A and Drug B single-drug groups showed a tendency to inhibit tumor growth, but did not reach statistical significance (p values ​​were 0.0502 and 0.1761, respectively). The Drug A combined with Drug B group could significantly inhibit tumor growth (p = 0.0022). Based on the comparison of tumor volume between the drug groups on the 28th day after administration, statistics showed that the tumor inhibition effect of the Drug A combined with Drug B group was significantly better than that of the Drug A group (p = 0.0028) and the Drug B group (p = 0.0112). During the entire experiment, the tumor-bearing mice did not experience significant weight loss symptoms in the Drug A single-drug group, the Drug B single-drug group, and the Drug A combined with Drug B group, and tolerated the treatment of each drug at this experimental dose well.

[0139] 4. Experimental Conclusion

[0140] In the subcutaneous xenograft tumor model of human non-small cell lung cancer LD1-0025-390629, Drug A and Drug B alone tended to inhibit tumor growth, with tumor inhibition rates of 70.63% and 45.68%, respectively; Drug A combined with Drug B showed significantly improved anti-tumor effect compared with Drug A alone and Drug B alone, with a tumor inhibition rate of 96.72%, and no obvious toxic side effects were observed.

Claims

1. Use of N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or its pharmaceutically acceptable salt alone or in combination with EGFR inhibitors in the preparation of drugs for treating cancer.

2. The use according to claim 1, characterized in that: The EGFR inhibitor is a small molecule inhibitor or a large molecule monoclonal antibody; preferably a small molecule EGFR-TKI inhibitor; more preferably a third-generation EGFR-TKI inhibitor; further preferably ametinib, osimertinib, vometinib, befortinib or omotinib; most preferably ametinib.

3. The use according to claim 1, characterized in that: The cancer is selected from breast cancer, ovarian cancer, prostate cancer, melanoma, brain tumor, esophageal cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, intestinal cancer, kidney cancer, skin cancer, glioblastoma, neuroblastoma, sarcoma, liposarcoma, osteochondroma, osteoma, osteosarcoma, seminoma, testicular tumor, uterine cancer, head and neck cancer, multiple myeloma, malignant lymphoma, polycythemia vera, leukemia, thyroid tumor, ureteral tumor, bladder tumor, gallbladder cancer, bile duct cancer or choriocarcinoma; preferably non-small cell lung cancer.

4. The use according to claim 3, characterized in that: The non-small cell lung cancer is selected from non-small cell lung cancer with EGFR mutation; preferably non-small cell lung cancer with one or more gene mutations of L858R mutation, exon 19 deletion mutation, T790M mutation, and C797X mutation.

5. The use according to claim 3, characterized in that: The non-small cell lung cancer is selected from non-small cell lung cancer resistant to the third-generation EGFR-TKI; preferably non-small cell lung cancer resistant to osimertinib or ametinib.

6. The use according to any one of claims 1 to 5, characterized in that: The single administration dose range of the N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidine-3-yl)-N-methylmethanesulfonamide or its pharmaceutically acceptable salt is selected from 0.1-1000 mg / kg; preferably 0.5-200 mg / kg; more preferably 1-150 mg / kg; most preferably 10-150 mg / kg; or, the single administration dose range is selected from 1-2000 mg; preferably 10-1000 mg; more preferably 50 mg to 500 mg.

7. The use according to any one of claims 1 to 6, characterized in that: The administration frequency of the N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof is once a day, twice a day or three times a day.

8. The use according to any one of claims 1 to 5, characterized in that: The N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidine-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof is administered once a day at a dosage selected from 1-150 mg / kg; or, administered once a day at a dosage selected from 10-1000 mg.

9. The use according to any one of claims 1 to 5, characterized in that: The single dose range of the EGFR inhibitor is selected from 0.1-1000 mg / kg; preferably 0.5-200 mg / kg; more preferably 1-100 mg / kg; further preferably 1-20 mg / kg; most preferably 1-10 mg / kg; or, the single dose range is selected from 1-1000 mg; preferably 10-200 mg; more preferably 55 mg or 110 mg.

10. The use according to any one of claims 1 to 6, characterized in that: The EGFR inhibitor is administered once a day, twice a day, or three times a day.

11. The use according to any one of claims 1 to 5, characterized in that: The EGFR inhibitor is administered once a day at a dosage selected from 1-10 mg / kg; or, is administered once a day at a dosage selected from 55 mg or 110 mg.

12. A pharmaceutical composition comprising N-((2R,3S)-1-(3-((2-(3-chloro-1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-fluoro-5-isopropylisoquinolin-8-yl)-2-methylazetidin-3-yl)-N-methylmethanesulfonamide or a pharmaceutically acceptable salt thereof and one or more other EGFR inhibitors, and one or more pharmaceutically acceptable carriers, excipients or diluents, preferably, the other EGFR inhibitors are selected from ametinib, osimertinib, vometinib, befortinib or omotinib; most preferably ametinib.

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