CN113440616A - Combination therapy for RAS or RAF mutant cancers - Google Patents

Combination therapy for RAS or RAF mutant cancers Download PDF

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CN113440616A
CN113440616A CN202110321666.7A CN202110321666A CN113440616A CN 113440616 A CN113440616 A CN 113440616A CN 202110321666 A CN202110321666 A CN 202110321666A CN 113440616 A CN113440616 A CN 113440616A
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田红旗
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KECHOW PHARMA Inc
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Abstract

The present invention relates to a combination therapy comprising administering to a mammal having cancer a therapeutically effective amount of a first component which is a MEK inhibitor and a second component which is a chemotherapeutic agent, a KRAS inhibitor, a SHP2 inhibitor, a RAF inhibitor, or a combination of said chemotherapeutic agent and/or a KRAS inhibitor and/or a RAF inhibitor.

Description

Combination therapy for RAS or RAF mutant cancers
Technical Field
The present invention relates to a method of combination therapy for a mammal having a cancer, the method comprising administering to a mammal having a cancer a therapeutically effective amount of a first component which is a MEK inhibitor and a second component which is a chemotherapeutic agent, a KRAS inhibitor, a RAF inhibitor, or a combination of the chemotherapeutic agent and/or a KRAS inhibitor and/or a RAF inhibitor, the cancer being a RAS or RAF mutant cancer.
Background
RAS family proteins are small molecule gtpases and the first oncogenes identified in human tumors and are widely involved in cell growth, differentiation and tumor development and progression. Mutations in the RAS protein cause many of the most aggressive tumors and the search for therapies for these mutant proteins has become an urgent problem.
The currently known RAS family shares three genes: KRAS, NRAS and HRAS. Mutations in the RAS enzyme are closely associated with tumorigenesis, and RAS mutation types vary among different types of tumors. In human tumors, KRAS mutations are most common, accounting for approximately 85%, NRAS and HRAS accounting for 12% and 3%, respectively. KRAS mutations account for an absolute majority in pancreatic, colorectal and lung cancers. NRAS mutations are most seen in melanoma and acute myeloid leukemia, and HRAS mutations are most seen in bladder cancer and head and neck cancer.
Non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers, and surgical treatment of lung cancer is mainly suitable for early and middle stages, but about 75% of patients are found to be in middle and advanced stages and have low 5-year survival rate. On the other hand, the tumor remission rate of chemotherapy for treating non-small cell lung cancer is 40-50%. However, chemotherapy generally fails to cure non-small cell lung cancer, and only prolongs the life of the patient, with side effects such as vomiting of malignancy, severe alopecia, congestion of the oral mucosa, decreased immunity, etc. It is worth noting that the targeted drug is an anticancer drug with strong pertinence and small side effect, and the combined treatment of the targeted drug and the traditional chemotherapeutic agent can reduce the dosage of the chemotherapeutic agent, reduce the side effect and bring better curative effect.
KRAS mutant lung carcinoma accounts for approximately 25% of NSCLC. Therefore, therapeutic approaches to KRAS mutations are urgently needed in NSCLC patients. The most common way of activating the KRAS gene is by point mutation, with 95% of KRAS mutations occurring predominantly at codon 12 (> 80%) and codon 13 of exon 2, and the common forms of mutation being KRAS-G12C (39%), KRAS-G12V (18-21%) and KRAS-G12D (17-18%) mutations. Mutations in other positions occurred predominantly at codon 61 (. about.0.1%).
At present, cancer treatment approaches aiming at KRAS mutation are mainly divided into two approaches, the first approach is to directly inhibit KRAS protein to inhibit tumor growth, and AMG510, MRTX849 and the like enter clinical experiments at present, but the drugs only aim at G12C mutation types and are ineffective to other mutations. The second is to inhibit tumor by inhibiting RAF-MEK-ERK of KRAS downstream pathway, such as AZD6244 and GSK1120212 of MEK.
AZD6244 was not effective in the third stage of the clinic, and it may be effective only at a certain mutation site, but ineffective at other mutations.
Currently, there is a lack of effective treatment for the common multiple KRAS mutated non-small cell lung cancers.
AMG-510 is a KRAS inhibitor derived from acrylamide, developed by Amgen, and is currently being clinically tested for solid tumors with KRAS G12C mutation. The structure of AMG-510 is as follows:
Figure RE-GDA0003205807320000021
however, clinical results indicate that drug resistance occurs in NSCLC patients administered AMG-510, and thus a combination drug capable of enhancing the therapeutic effect in combination with AMG-510 is required.
Thus, an effective therapy for cancer treatment with RAS gene mutants is desired; more specifically, drug combination therapies that are effective in treating the common multiple KRAS mutated non-small cell lung cancers and that are well-tolerated are desired.
RAF is a key downstream target of the RAS guanine-nucleotide binding/gtpase protein and mediates activation of the MAP kinase cascade consisting of RAF-MEK-ERK. The RAF gene encodes a highly conserved serine-threonine-specific protein kinase, which is recruited to the plasma membrane upon direct binding to the RAS, the initial event in RAF activation. As an inhibitor of the RAF pathway, one of the downstream kinases, a traditional BRAFV600 inhibitor has been approved for the treatment of BRAF mutated NSCLC and melanoma in combination with the MEK inhibitor trametinib. In addition, current combination treatment regimens for other BRAF mutation indications have not emerged, and thus there is a continuing need to try the treatment of other indications with a MEK inhibitor in combination with a BRAFV600 inhibitor.
Disclosure of Invention
Combination therapies effective for the treatment of cancer, such as NSCLC, including combination therapy of a MEK inhibitor with a conventional chemotherapeutic agent, such as Docetaxel (DOX), with a KRAS inhibitor, with a SHP2 inhibitor, with a pan RAF/BRAF mutation inhibitor are disclosed.
A combination therapy comprising administering to a mammal having cancer a therapeutically effective amount of a first component which is a MEK inhibitor and a second component which is a chemotherapeutic agent, a KRAS inhibitor, a pan RAF inhibitor, or a combination of the chemotherapeutic agent and/or KRAS inhibitor and/or pan RAF/BRAF inhibitor is disclosed.
In one embodiment, the MEK inhibitor is a compound of formula (I) or a pharmaceutically salt thereof,
Figure RE-GDA0003205807320000022
wherein,
x ═ O, S or C ═ O;
y ═ N or CH;
r1, R2, R4 and R5 are each independently selected from hydrogen, halogen or C1-6 alkyl;
R3selected from hydrogen, halogen, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy, halo C1-6Alkylthio radical, C1-6Alkyl and halo C1-6An alkyl group;
R6is-C (O) NR8R7or-NHSO2R7；
R7And R8Each independently selected from hydrogen, C substituted by hydroxy1-6Alkyl radical, C3-6Cycloalkyl radical, C3-6Cycloalkyl radical C1-6Alkyl and C substituted by hydroxy3-6Cycloalkyl radical C1-6An alkyl group.
In one embodiment, the MEK inhibitor is compound 1, compound 2, compound 3, or compound 4.
Figure RE-GDA0003205807320000031
The MEK inhibitors described above are disclosed in the patents granted by the chinese patent invention: ZL 201210189086.8, ZL 201210189087.2, and ZL 201210190520.4.
In one embodiment, the MEK inhibitor is administered to the mammal in an effective amount of from 0.1 to 1000mg/kg body weight; preferably, an effective amount of 0.5 to 10mg/kg body weight is administered to the mammal. The frequency of administration is twice a day or twice a week, depending on the particular condition of the mammal. Preferably, the administration is twice weekly.
In one embodiment, the chemotherapeutic agent is selected from cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, and pemetrexed.
In one embodiment, the KRAS inhibitor is AMG510, MRTX849, BAY-293, ARS-1620, BI1701963, or the like.
In one embodiment, the SHP2 inhibitor is JAB-3068, TNO155, BBP-398(IACS-15509), JAB-3312, SHP099, or the like.
In one embodiment, the RAF inhibitor is PLX4032, LGX818, GSK2118436, PLX8394, BI 882370, TAK580, LY3009120, Vemurafenib, and the like, wherein LY3009120 and Vemurafenib have the formula
Figure RE-GDA0003205807320000032
In one embodiment, the chemotherapeutic agent is administered at a dose of 0.1-30mg/kg body weight, such as 0.1-10mg/kg body weight,
in a preferred embodiment, the chemotherapeutic agent is docetaxel. In a preferred embodiment, the dose of docetaxel is 5-120mg/kg body weight, preferably 7.5-110mg/kg body weight. In the case where the mammal is a human, the dose of docetaxel is intravenously administered every 21 days over 1 hour at doses of 60 and 100mg/m2The body surface or (1.6-2.6 mg/kg body weight); or administered intravenously at a weekly dose of 20 and 40mg/m over 1 hour2(0.5-1.0 mg/kg body weight),administration was carried out for up to 6 months.
Although the dosage regimen of the MEK inhibitor and the chemotherapeutic agent is set out above, the dosage and frequency of administration are generally adjusted according to the general physical condition of the subject and the severity of the side effects caused, in particular, the severity of the side effects causing hematopoiesis, the nervous system and the renal system. The MEK inhibitors and chemotherapeutic agents disclosed herein may be administered in a variety of known ways, such as orally, topically, rectally, parenterally, by inhalation spray or via an implanted reservoir, although the most suitable route in any given case will depend on the particular host and the nature and severity of the condition for which the active ingredient is administered. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
In one embodiment, the MEK inhibitor and the chemotherapeutic agent are administered simultaneously, sequentially or intermittently. In one embodiment, the MEK inhibitor is administered concurrently with the chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is administered prior to the MEK inhibitor; or the chemotherapeutic agent is administered after administration of the MEK inhibitor; the interval between administrations depends on the patient and the half-life of the drug. In one embodiment, the MEK inhibitor is administered orally; the chemotherapeutic agent is administered intravenously.
In one embodiment, the mammal is a human, rat, mouse, or the like. In a preferred embodiment, the mammal is a human.
In one embodiment, the cancer is a RAS mutant cancer, such as a KRAS mutant cancer, NRAS mutant cancer, HRAS mutant cancer, or BRAF mutant cancer. In one embodiment, the RAS mutant cancer is pancreatic cancer, colorectal cancer, lung cancer, melanoma, acute myeloid leukemia, bladder cancer, or head and neck cancer, among others. In a preferred embodiment, the cancer is lung cancer. In a preferred embodiment, the cancer is non-small cell lung cancer.
In one embodiment, the KRAS comprises a mutation at one or more positions selected from codons 12, 13, 59, and 61. In one embodiment, the KRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, S17, P34, a59 and Q61. In one embodiment, the KRAS mutant form has one or more amino acid substitutions selected from: G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, a59E, a59G, a59T, Q61K, Q61L, Q61R, and Q61H. In one embodiment, the KRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, a59, Q61, K117 and a 146. In one embodiment, the KRAS mutant form has one or more amino acid substitutions selected from: G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, a59E, a59G, a59T, Q61K, Q61L, Q61R, Q61H, K117N, K117R, K117E, a146P, a146T, and a 146V. In one embodiment, the BRAF mutation is a BRAF V600E mutation.
In one embodiment, the NRAS comprises a mutation at one or more positions selected from codons 12, 13, 59, 61 and 146. In some embodiments, the NRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, a59, Q61, K117, and a 146. In some embodiments, the NRAS mutant form has one or more amino acid substitutions selected from the group consisting of: G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, a59D, a59T, Q61K, Q61L, Q61R, Q61H, K117N, K117R, K117E, a146P, a146T, and a 146V.
In a further preferred embodiment, the cancer is non-small cell lung cancer, which has a KRAS G12 mutation or a Q61 mutation. Still further, the mutation is a KRAS G12 mutation selected from G12C, G12S, or G12V; the mutation is KRAS Q61K mutation.
In one embodiment, the ratio of MEK inhibitor to KRAS inhibitor is 1:1 to 10:1, preferably 3:1 (molar in vitro concentration ratio) or 1:1 to 1:20, preferably 1:6 to 1:8 (mass in vivo concentration ratio).
In one embodiment, the ratio of MEK inhibitor and ubiquitin inhibitor is from 1:1 to 1:50, preferably from 1:40 (molar in vitro ratio) or from 1:1 to 1:50, preferably 1:30 (mass in vivo ratio).
The inventors of the present invention, by the combination therapy of MEK inhibitor (e.g. compound 1) of the present invention and chemotherapeutic agent Docetaxel (DOX), all had good effects, e.g. additive or synergistic effects, on NSCLC with four KRAS mutations (Q61K, G12C, G12S and G12V), especially on non-small cell lung cancer with KRAS G12V and Q61K mutations; the MEK inhibitors of the present invention and KRAS inhibitors such as AMG510 have a synergistic effect on non-small cell lung cancer with KRAS G12C mutation; the MEK inhibitors of the present invention have a synergistic effect with SHP2 inhibitors such as SHP099 on KRAS G12C mutant non-small cell lung cancer; and the MEK inhibitor of the present invention and a pan RAF inhibitor such as LY3009120 have synergistic effects on non-small cell lung cancer with the KRAS Q61K mutation. And the MEK inhibitors of the present invention and RAF inhibitors such as Vemurafenib have a synergistic effect on melanoma with RAF mutations.
Drawings
Figure 1 shows the tumor growth curve of compound 1 in combination with docetaxel in a Calu-6 lung cancer subcutaneous transplantation model.
Figure 2 shows the tumor growth curve of compound 1 in combination with docetaxel in the NCI-H358 lung cancer subcutaneous transplant model.
Figure 3 shows the tumor growth curve of compound 1 in combination with docetaxel in an a549 lung cancer subcutaneous transplantation model.
Figure 4 shows the tumor growth curve of compound 1 in combination with docetaxel in the NCI-H441 lung cancer subcutaneous transplantation model.
Figure 5 shows inhibition of Calu-6 tumor, weight change in experimental animals in each group, tumor weight at the end of experiment in each group, and tumor volume/tumor weight rate of change analysis using compound 1 in combination with LY 3009120.
Figure 6 shows an in vitro anti-proliferative dose response curve of compound 1 and Vemurafenib to tumor cells.
Figures 7A and 7B show the in vivo antiproliferative effect of compound 1 in combination with Vemurafenib on melanoma cells a 375.
Figure 8 shows the in vivo efficacy of compound 1 and AMG510 in combination on NCI-H358 lung cancer subcutaneous graft model.
Figure 9 shows the in vivo efficacy of compound 1 in combination with SHP099 on the NCI-H358 lung cancer subcutaneous transplant model.
Detailed Description
The invention is further illustrated by, but is not limited to, the following examples which illustrate the invention.
1. Pharmacodynamic evaluation experiment
The following example investigates the pharmacodynamic evaluation of compound 1 (obtained from a Binjiang drug, the same below) in combination with docetaxel in a different lung cancer model compared to compound 1 alone therapy.
1.1 design of the experiment
Figure RE-GDA0003205807320000051
Figure RE-GDA0003205807320000061
1.2 Experimental animal information
a) Calu-6/A549: BALB/c nude mice, females, week old at tumor cell inoculation time of 6-8 weeks, feeding environment: SPF grade.
b) NCI-H358: NOD/SCID mice, females, tumor cells were inoculated at week age 6-8 weeks and fed in SPF-grade.
c) NCI-H441 is NU/NU mouse, female, the week age of tumor cell inoculation is 6-8 weeks, and the breeding environment is SPF grade.
1.3 environmental conditions of laboratory animal raising Chambers
The experimental animals are all raised in independent ventilation boxes with constant temperature and humidity, the temperature of a raising room is 20-22 ℃, the humidity is 59-78%, the air exchange is carried out for 10-20 times/hour, and the light and shade alternation time is 12h/12h day and night; the complete granulated feed for the cobalt 60 radiation sterilized rat is continuously supplied, and can be freely taken in unlimited amount, tap water (used after high-pressure steam sterilization) is drunk, and the drinking water bottle continuously supplies water and can be freely taken in. The mouse raising box is a polysulfone mouse box, is used after autoclaving and has the specification of 325mm multiplied by 210mm multiplied by 180 mm; the padding is autoclaved corncobs, at most 6 animals are raised in each box, and the cage card is marked with an experiment number, experiment starting time, experimenters, animal sources, groups, animal numbers and the like; the experimental animals were marked with ear tags.
1.4 animal modeling and grouping
The following cells (density 5X 10) were subcutaneously inoculated on the right side of the mouse on Day 0 (Day 0)6) Cells were resuspended in 0.1mL of suspension (for Calu-6, the suspension is PBS; for NCI-H358, A549 and NCI-H441, all were 1:1 PBS: matrigel), establishing a corresponding subcutaneous lung cancer transplantation model. Several days after cell inoculation, tumors are grown to an average volume of the desired size (e.g., 201mm for NCI-H358 cells)3Left and right; for other cell lines, growth was to 154mm3Left and right) were randomly divided into 6 groups of 8 per group according to tumor volume, and administration was started.
1.5 Experimental observations
After tumor inoculation, routine monitoring includes the effects of tumor growth and treatment on the normal behavior of the animal, including activity, food intake and water intake, weight gain or loss, eyes, hair follicles and other abnormalities in the experimental animal.
After tumor inoculation, the body weight and tumor size of the mice were measured three times per week. Tumor size calculation formula:
tumor volume (mm)3) 0.5 × (tumor major diameter × tumor minor diameter)2)。
1.6 cellular information
TABLE 1 cell types and mutation status and culture conditions
Cell lines Mutational status Culture medium
NCI-H358 KRAS G12C RPMI1640+10％FBS
A549 KRAS G12S RPMI1640+10％FBS
Calu-6 KRAS Q61K MEM+10％FBS
NCI-H441 KRAS G12V RPMI1640+10％FBS
The cells were all cultured in an incubator at 37 degrees, 5% CO2, and 95% humidity. Cells in the exponential growth phase were collected and resuspended in PBS to appropriate concentration for subcutaneous inoculation of the right side of the mice.
1.7 formulation of test drugs
Compound 1 was dissolved in an ultrapure aqueous solution containing 0.5% HPMC and 0.2% Tween-80 and new solutions were prepared weekly. Docetaxel is used in clinical formulations in the required dosage.
1.8 judging the parameters or criteria
According to relative tumor inhibition ratio (TGI)RTV) And evaluating the curative effect, and evaluating the safety according to the weight change and death condition of the animals. The specific judgment parameters are as follows:
relative tumor inhibition ratio TGIRTV(％)：TGIRTV＝1-TRTV/CRTV(％)。
TRTV/CRTV% is the relative tumor proliferation rate, i.e., the percentage value of the relative tumor volume in the treated and control groups at a certain time point.
TRTVAnd CRTVRelative Tumor Volumes (TV) at a particular time point for the treated group and the PBS control group, respectively.
The calculation formula is as follows: t isRTV: treatment group mean RTV; cRTV: control group mean RTV; RTV ═ Vt/V0， V0Is the tumor volume of the animal in the group, VtIs the tumor volume of the animal after treatment.
1.9 statistical analysis
Tumor volume and animal body weight results are expressed as Mean (Mean) ± SEM (Mean standard error). Statistical analysis of relative tumor volumes between groups the grouped relative tumor volume data was selected. The independent sample T test method was used to compare the relative tumor volumes between the two groups for significant differences. All data were analyzed using SPSS 18.0. p <0.05 is significantly different; p <0.01 is a very significant difference.
2. Examples of the embodiments
Pharmacodynamic evaluations were performed according to the above pharmacodynamic evaluation experiments with compound 1 administered alone, docetaxel administered alone, compound 1+ docetaxel administered in combination, compound 1+ KRAS inhibitor AMG510 administered in combination, compound 1+ SHP2 inhibitor SHP099 administered in combination, and compound 1+ RAF inhibitor such as LY3009120 administered in combination to the four cell lines or the lung cancer subcutaneous transplantation model described above.
2.1 example A- -antitumor Activity of Compound 1 alone or in combination in a Calu-6 Lung cancer subcutaneous transplantation model
At day 45 after tumor cell inoculation (day 28 after administration), the tumor volume of the control group was 3529.0mm as shown in Table 23The tumor volumes of the G2 group, the G3 group, the G4 group, the G5 group and the G6 group were 1069.7mm respectively3、 1350.2mm3、1360.7mm3、420.8mm3And 493.3mm3Relative tumor inhibition ratio TGIRTV(%) 73%, 66%, 65%, 89%, 87%, respectively. Analysis by independent sample T test method, relative controlGroup tumor volumes were statistically very significantly different (p)<0.01), which indicates that each administration group has significant anti-tumor activity. And the p values of the single administration group and the corresponding combined administration group (G2 vs G5, G4 vs G5, G3 vs G6, G4 vs G6) are all compared<0.001, indicating that the combination has synergistic effect. (the tumor volumes of the treatment groups and the control group are shown in FIG. 1)
TABLE 2 pharmacodynamic analysis in Calu-6 Lung cancer subcutaneous transplantation model at day 45 (28 days of drug administration) after cell inoculation
Figure RE-GDA0003205807320000071
Figure RE-GDA0003205807320000081
Note a comparison with control
2.2 example B- -antitumor Effect of Compound 1 alone or in combination in the NCI-H358 Lung cancer subcutaneous transplantation model
At day 33 after tumor cell inoculation (day 26 after grouping), the tumor volumes of the control group were 1876.5mm3, G2, G3, G4, G5 and G6, respectively, 167.4mm3、496.4mm3、229.4mm3、64.9 mm3And 77.3mm3The relative tumor inhibition rates TGIRTV (%) were 91%, 74%, 89%, 97%, and 96%, respectively, and the tumor volumes were statistically significantly different from those of the control group (p values were all < 0.001).
The tumor growth of each treatment group and control group is shown in FIG. 2, and the pharmacodynamic analysis is shown in Table 3.
TABLE 3 pharmacodynamic analysis of tumor volume at day 26 post experimental grouping in NCI-H358 Lung cancer subcutaneous transplantation model
Figure RE-GDA0003205807320000082
Note that:
a, comparison with control group
b,G2 vs G5,p＝0.017；G4 vs G5,p＝0.006；G3 vs G6,p＜0.001；G4 vs G6,p＝0.008.
The combination showed better tumor growth inhibition than the single drug group. Compared with the corresponding combined administration groups (G2 vs G5, G4 vs G5, G3 vs G6 and G4 vs G6), the single administration group has significant difference, which indicates that the combined administration has synergistic effect.
2.3 example C- -antitumor Activity of Compound 1 alone or in combination in a BALB/C nude mouse A549 Lung cancer subcutaneous transplantation model
A549: as shown in Table 4, on day 30 after the grouping, the tumor volumes of the control group were 1364.5mm3, and those of the G2 group, G4 group, G5 group and G6 group were 541.2mm, respectively3、584.6mm3、246.4mm3And 284.2mm3The relative tumor inhibition ratios TGIRTV (%) were 60%, 61%, 85%, and 81%, respectively, and the tumor volumes were statistically significantly different from those of the control group (p)<0.05) showed better tumor growth inhibition with the combination compared to the single administration. The tumor volume of the G3 group was 1012.3mm3The relative tumor inhibition ratios TGIRTV (%) were 26%, respectively, and there was no statistically significant difference in tumor volume (p) with respect to the control group>0.05). Compared with the corresponding combined administration groups (G2 vs G5, G4 vs G5, G3 vs G6 and G4 vs G6), the single administration group has significant differences (P values are all less than 0.05), which indicates that the combined administration has synergistic effect.
TABLE 4 pharmacodynamic analysis of tumor volume at day 30 after experimental grouping in A549 Lung cancer subcutaneous transplantation model
Figure RE-GDA0003205807320000091
Note a: compared with a control group
2.4 example D- -antitumor Effect of Compound 1 alone or in combination in the subcutaneous transplantation model of NCI-H441 Lung cancer in NU/NU mice
As shown in Table 5, the tumor volume of the control group was 827.3mm at day 20 after the grouping3The tumor volumes of the G2 group, the G3 group, the G4 group, the G5 group and the G6 group were 294.6mm respectively3、336.8mm3、276.1mm3、132.9mm3And 103.6 mm3The relative tumor inhibition rates TGIRTV (%) were 66%, 61%, 69%, 85%, 88%, respectively, and the tumor volumes were statistically significantly different from those of the control group (p value < 0.001).
By 20 days after administration, G3 (compound 1BIW) was significantly different from the corresponding combination group G6 in relative tumor volume (P <0.05), and G2 (compound 1BID) was also significantly different from G5 at the end of the experiment, indicating that the effect of the combination therapy was superior to that of the compound 1 monotherapy. Compared with the group G4 in which DOX is singly administered, the combined treatment group G6 (compound 1BIW, DOX QD) has obvious difference in relative tumor volume at the experimental end point, which indicates that the combined treatment effect is also superior to that of DOX single-drug treatment. However, there was no significant difference between G4(DOX QW) and G5 (compound 1BID, DOX QD) (P >0.05), suggesting that the mode of administration of compound 1 (BID or BIW) in the combination treatment regimen directly affected the therapeutic effect.
TABLE 5 pharmacodynamic analysis of tumor volumes at day 20 after experimental grouping in the NCI-H441 Lung cancer subcutaneous transplantation model
Figure RE-GDA0003205807320000092
Note a: compared with a control group
b,G2 vs G5,p＝0.001；G4 vs G5,p＝0.084；G3 vs G6,p<0.001；G4 vs G6,p＝0.042
From the above results, it can be seen that compound 1 administered in combination with docetaxel (dox) showed additive or synergistic effects in four subcutaneous lung cancer transplantation models (or four KRAS mutant non-small cell lung cancers) compared to compound 1 administered alone.
2.5 example E-synergistic Effect of the combination of Compound 1 and KRASi AMG510 on NSCLC H358 in vitro
The cell line NSCLC H358 was seeded at 3k/well density in 96-well plates with 5% CO2Culturing at 37 deg.C. The next day, different concentrations (1-10000nM) of Compound 1 and AMG510 (obtained from ambroxol) were added and incubated for 5 days under normal conditions. Adding MTT (5mg/ml), and using enzymeThe absorbance of each concentration well at 490nm was measured using a standard instrument using the formula viatility ═ 100% (OD)experimental-ODblank)/(ODMedia-ODblank) Conversion to cell viability. IC of two inhibitors was calculated separately by fitting dose-response curves to graphpad6 software50See table 6 for values. Cells were seeded at the same density and both inhibitors were diluted to their IC50Different fold (see table 7), added to the same cell culture well and two single inhibitor control sets were set up and incubated for 5 days.
Table 6: IC50 values on H358 for both inhibitors Compound 1 and AMG510
Inhibitors IC50 value
Compound
1 117.4nM
AMG510 38.2nM
Table 7: inhibitor concentrations for the combination experiments were formulated as IC50 from Table 6
Well No. Compound 1 AMG510
1 0 0
2 0.0625IC50 0.0625IC50
3 0.125IC50 0.125IC50
4 0.25IC50 0.25IC50
5 0.5IC50 0.5IC50
6 IC50 IC50
7 2IC50 2IC50
8 4IC50 4IC50
9 8IC50 8IC50
10 16IC50 16IC50
The cell viability was calculated by MTT assay (method as above) and the synergy Index (CI) was calculated for both compounds using CalcuSyn software, following the formula where CI <1 is synergistic, CI >1 is antagonistic and CI ═ 1 is additive. See table 8 for experimental results.
Table 8: synergistic coefficient of AMG510 and Compound 1
Figure RE-GDA0003205807320000101
Figure RE-GDA0003205807320000111
The results are shown in the table above, with CI values consistently less than 1 at each concentration, indicating that compound 1 and AMG510 in combination can produce a synergistic effect on H358 cell lines cultured in vitro.
In vivo experimental methods and results
H358 was inoculated at a density of 5e 6/mouse on the right dorsal side of nude mice. Over 10 days, groups were started with mean 281.9mm tumor volume per group3. The following table was followed for 21 days, and tumor size was measured once every two days according to the formula TGIRTV(％)： TGIRTV＝1-TRTV/CRTV(%) the administration group TGI was calculated.
TABLE 9 in vivo Experimental dosing regimen
Figure RE-GDA0003205807320000112
And finishing the experiment on the 22 nd day, weighing the whole tumor, and calculating the tumor weight inhibition rate of each group. The results of the experiment are shown in FIG. 8. The combined use of the compound 1 and the AMG510 has an inhibiting effect on the growth of H358 tumor, the tumor growth inhibition rate (TGI) of the combined group is greater than that of the single drug of the compound 1 and the single drug of the AMG510 (shown in a table in the figure), and meanwhile, the tumor weight Inhibition Rate (IR) of the combined group is also greater than that of the two single drugs. The combination group (compound 1+ AMG510) showed significant differences in tumor weight and tumor volume (P <0.05) compared to the drug alone, as shown by the t-test.
2.6 example F-synergistic Effect of Compound 1 and the pan-RAF inhibitor LY3009120 on NSCLC Calu-6 in vitro and in vivo combination
In vitro test method
The cell line NSCLC Calu-6 was seeded at 3k/well density in 96-well plates and cultured under normal conditions. The next day, different concentrations (1-10000nM) of Compound 1 and LY3009120 (from Bidak medicine) were added and incubated for 72h in an incubator containing 5% carbon dioxide at 37 ℃. MTT (5mg/ml) was added, and the absorbance of each concentration well at a wavelength of 570nm was measured by a microplate reader, using the formula viatility ═ 100% (OD)experimental-ODblank)/(ODMedia-ODblank) Conversion to cell viability. IC of two inhibitors was calculated separately by fitting dose-response curves to graphpad6 software50See table 9 for values. Cells were seeded at the same density and both inhibitors were diluted to their IC50Different fold (see table 10), added to the same cell culture well and two single inhibitor controls were set up and incubated for 3 days under normal conditions.
Table 10: IC50 values on Calu-6 for both inhibitors Compound 1 and LY3009120
Inhibitors IC50 value
Compound
1 21.38nM
LY3009120 812.44nM
Table 11: inhibitor concentrations for the combination experiments were formulated as IC50 from Table 9
Well No. Compound 1 LY3009120
1 0 0
2 0.0625IC50 0.0625IC50
3 0.125IC50 0.125IC50
4 0.25IC50 0.25IC50
5 0.5IC50 0.5IC50
6 IC50 IC50
7 2IC50 2IC50
8 4IC50 4IC50
9 8IC50 8IC50
10 16IC50 16IC50
The cell viability was calculated by MTT assay (method as above) and the synergy Index (CI) was calculated for both compounds using CalcuSyn software, following the formula where CI <1 is synergistic, CI >1 is antagonistic and CI ═ 1 is additive. The results are shown in Table 11.
Table 12: synergy coefficient of LY3009120 and Compound 1
Compound 1 LY3009120 Fa (inhibition rate) CI
(nM) (nM)
1.33623 50.775 0.184994 0.831
2.67245 101.55 0.309832 0.515
5.34491 203.1 0.468952 0.417
10.6898 406.2 0.661708 0.330
21.3796 812.4 0.756792 0.393
42.7592 1624.8 0.836352 0.454
85.5185 3249.6 0.889392 0.551
171.037 6499.2 0.913325 0.819
342.074 12998.4 0.952781 0.803
The results show that at each concentration, CI values consistently were less than 1, indicating that compound 1 in combination with LY3009120 was able to produce a synergistic effect on the in vitro cultured Calu-6 cell line.
In vivo experimental method
Calu-6 was inoculated at 5e 6/mouse into the right dorsal side of nude mice (B/C nude mice, purchased from Wintolite), and divided into groups over 7 days, with the average tumor volume of each group being 264mm3Administered for 21 days as follows, tumor size was measured twice a week according to the formula TGI ═ 1- (treatment-treatment 0)/(vehicle-vehicle 0)]100% of TGI in the administration group was calculated, the experiment was terminated on day 22, the whole tumor was weighed, and the tumor weight inhibition rate of each group was calculated.
Table 13: in vivo experimental dosing regimen
Figure RE-GDA0003205807320000131
As shown in fig. 5, the combined use of compound 1 and LY3009120 has an inhibitory effect on the growth of Calu-6 tumor, and the tumor growth inhibition ratio (TGI) of the combined group is greater than that of the compound 1 single drug and that of LY3009120 single drug (shown in the table), and the tumor weight Inhibition Ratio (IR) of the combined group is also greater than that of both single drugs. According to the formula of the combination evaluation, q is TGIAB/(TGIA + TGIB-TGIA TGIB) (A is compound 1, B is LY3009120, q is 1, the two drugs act together, q >1 is synergistic effect, q <1 is antagonistic effect), and q is 1.07. The combination group (compound 1+ LY3009120) was significantly different (P <0.05) compared to the two groups when administered alone, regardless of tumor volume or tumor weight, as determined by t-test. Thus, compound 1 in combination with LY3009120 produced a good synergistic effect on the xenograft model of Calu-6(KRAS Q61K) NSCLC (xenograft).
2.7 example G-Compound 1 alone or in combination with tumor cells Colo-205, HT-29 and A375 in vitro antitumor Activity
The following cells were cultured in complete medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin. The relative humidity of the culture environment is 95%, and CO is2Sterile incubator at 5% concentration.
TABLE 14 cell information
Cell line name Culture medium formula Cellular mutation sites
Colo-205 RPMI-1640(Hyclone)+10％FBS(BI) BRAFV600E
HT-29 McCoy’s 5a(Gibco)+10％FBS(BI) BRAFV600E
A375 DMEM(Hyclone)+10％FBS(BI) BRAFV600E
Inoculating the cells into a cell culture bottle, adding a proper amount of complete culture medium, placing the cell culture bottle in an aseptic incubator for culture, and carrying out subculture when the confluency of the cells reaches more than 80%.
Weighing a proper amount of compound 1 or Vemurafenib into a 1.5ml centrifuge tube, adding a corresponding volume of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 20mM, subpackaging, and keeping away from light and storing in a refrigerator at-20 ℃ for later use.
Administration alone
The proliferation of the cells cultured in vitro was examined by the MTT/MTS method. Adherent cells in the logarithmic growth phase were digested with trypsin or suspension cells were collected by centrifugation, counted, and 90. mu.l of cell suspension was inoculated into a 96-well plate at a cell inoculation density of 1000-3000/well, and 10. mu.l of compound at a final concentration 10-fold diluted in medium was added 24 hours later (the concentration settings are shown in Table 1.). Wells to which the same volume of 5% DMSO was added served as controls, with a final DMSO concentration of 0.5%. After 3 days of drug treatment, MTT/MTS was examined for cell viability. The specific method comprises the following steps: after 20. mu.l of MTT was added to each well and the resulting mixture was placed in an incubator and incubated for 4 hours, the supernatant was discarded and 150. mu.l of DMSO was added to dissolve crystalline formazan, and 490nM absorbance was detected by an enzyme-labeling instrument, or 490nM absorbance was detected directly by an enzyme-labeling instrument after incubation for 4 hours in an incubator. The GraphPad Prism 6 software makes dose-response curves and calculates IC 50. The experiment was repeated three times and the average value of IC50 was calculated.
TABLE 15 Compound 1 and Vemurafenib Final concentrations of Compounds for in vitro anti-cell proliferation assays
Figure RE-GDA0003205807320000141
C represents the concentration of the compound
Experiments the in vitro antiproliferative activity of compound 1 and Vemurafenib on BRAF mutated tumor cells was tested using the tetrazolium salt (MTT/MTS) method. The results show that compound 1 has strong proliferation inhibition effect on 2 BRAF mutant colon cancer cell lines. The dose-response curves of the compounds are shown in FIG. 6, and the corresponding IC50 is shown in tables 14-16.
TABLE 16 IC50 values for Compound 1 and Vemurafenib on Colo-205 cells
Drugs 1st(nM) 2nd(nM) 3rd(nM) Mean±SD(nM)
Compound 1 4.9 4.8 4.7 4.8±0.1
Vemurafenib 2279.5 1869.5 2196.9 2115.3±216.8
TABLE 17 IC50 values for HT-29 cells for Compound 1 and Vemurafenib
Drugs 1st(nM) 2nd(nM) 3rd(nM) Mean±SD(nM)
Compound 1 2.8 2.7 3.5 3±0.4
Vemurafenib 2316.6 1645.9 2008.7 1990.4±335.7
TABLE 18 IC50 values for Compound 1 and Vemurafenib on A375 cells
A375 1st(nM) 2nd(nM) 3rd(nM) Mean±SD(nM)
Compound 1 3.0 2.9 2.7 2.9±0.2
Vemurafenib 716.4 1068.5 563.7 782.9±258.9
Combined administration
Drug concentrations for the combination experiments were set according to the mean IC50 on both cells for compound 1 and Vemurafenib obtained in the first part of the experiments. Specific dosages are shown in table 17.
TABLE 19 concentration settings for Combined drug experiments with Compound 1 and Vemurafenib
Figure RE-GDA0003205807320000142
Figure RE-GDA0003205807320000151
Inoculating the cells into a cell culture bottle, adding a proper amount of complete culture medium, placing the cell culture bottle in an aseptic incubator for culture, and carrying out subculture when the confluency of the cells reaches more than 80%.
The proliferation of cells was detected by MTS/MTT method. Adherent cells in the logarithmic growth phase were trypsinized or suspension cells were collected by centrifugation, counted, 80. mu.l of cell suspension was inoculated into a 96-well plate at a cell inoculation density of 1000-. Wells with the same volume of 2.5% DMSO added were used as controls, with a final DMSO concentration of 0.5%, and single drug controls with only one compound were set up with three duplicate wells per concentration. After 3 days of drug treatment, MTT/MTS was examined for cell viability. The specific method comprises the following steps: after 20. mu.l of MTT was added to each well and the resulting mixture was placed in an incubator and incubated for 4 hours, the supernatant was discarded and 150. mu.l of DMSO was added to dissolve crystalline formazan, and 490nM absorbance was detected by an enzyme-labeling instrument, or 490nM absorbance was detected directly by an enzyme-labeling instrument after incubation for 4 hours in an incubator. The Combination Index (CI) was calculated with drug synergy evaluation software Calcusyn. The experiment was repeated 2-3 times.
Cell survival under dual drug combination was tested using the MTS/MTT cell proliferation assay, CI values were calculated using a drug synergy model established with TING-cha CHOU by comparing compound 1 and Vemurafenib single drug tests performed simultaneously, and whether synergy was present was judged by CI values (CI >1 antagonism; CI ═ 1 additivity; CI <1 synergy).
On Colo-205, compound 1 and Vemurafenib have synergistic effects in the concentration range from 1/8-fold IC50 to 4-fold IC50 and have strong synergistic effects in the vicinity of IC50 by comprehensive judgment of the results of three experiments. See table 18.
TABLE 20 summary of CI values for colo-205
Figure RE-GDA0003205807320000152
Fa value represents the inhibition rate of the drug on cells
On HT-29, compound 1 and Vemurafenib showed synergistic effects in the concentration range from 1/16-fold IC50 to 4-fold IC50, and strong synergistic effects around 1/2-fold IC50, as judged by a combination of the results of three experiments. See table 19.
TABLE 21 summary of CI values for HT-29
Figure RE-GDA0003205807320000161
Fa value represents the inhibition rate of the drug on cells
At a375, compound 1 and Vemurafenib showed synergistic effects in the concentration range from 1/4-fold IC50 to 4-fold IC50, and around IC50, strong synergistic effects, as judged by a combination of the results of three experiments. See table 20.
TABLE 22 summary of CI values for A375
Figure RE-GDA0003205807320000162
Fa value represents the inhibition rate of the drug on cells
2.8 example H-in vivo antitumor Effect of Compound 1 alone or in combination in tumor cells A375
A375 was inoculated at a density of 5e 6/mouse on the right dorsal side of nude mice. Over 7 days, groups were started with mean 188mm tumor volume per group3. The following table was followed for 9 days, and tumor size was measured twice a week according to the formula TGI ═ 1- (treatment-treatment 0)/(vehicle-vehicle 0)]100% TGI calculated for the dosing group.
TABLE 23 dosing regimens
Figure RE-GDA0003205807320000171
And finishing the experiment on the 10 th day, weighing the whole tumor, and calculating the tumor weight inhibition rate of each group. The results of the experiment are shown in FIGS. 7A and 7B. The combination of compound 1 and Vemurafenib has an inhibitory effect on the growth of A375 tumors, and the Tumor Growth Inhibition (TGI) of the combined group is greater than that of the single drug of compound 1 and that of Vemurafenib (shown in the table in the figure), and meanwhile, the tumor weight Inhibition (IR) of the combined group is greater than that of the two single drug groups. According to the formula of the combined drug evaluation, q is TGIAB/(TGIA + TGIB-TGIA TGIB) (A is compound 1, B is Vemurafenib, q is 1, the two drugs are added, q is 1, the two drugs are synergistic, q is 1, the two drugs are antagonistic), and the q value is 1.51. The combined group (compound 1+ Vemurafenib) showed significant differences in tumor weight and tumor volume (P <0.05) compared to Vemurafenib administered alone by t-test.
The results show that the compound 1 and Vemurafenib have synergistic effect on BRAF V600E mutant tumor cells (two colon cancer cells, namely a melanoma cell), and the results show that the compound 1 and Vemurafenib have obvious inhibition effect on cell proliferation as single drugs.
2.9 example I-Compound 1 alone or in combination with the anti-tumor Activity of SHP2 inhibitor SHP099
The following cells were cultured in complete medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin. The relative humidity of the culture environment is 95%, and CO is2Sterile incubator at 5% concentration.
TABLE 24 cell information
Cell line name Culture medium formula Cellular mutation sites
H358 RPMI-1640(Gibco)+10％FBS(Yeason) KRASG12C
Inoculating the cells into a cell culture bottle, adding a proper amount of complete culture medium, placing the cell culture bottle in an aseptic incubator for culture, and carrying out subculture when the confluency of the cells reaches more than 80%.
Weighing a proper amount of compound 1 or SHP099 into a 1.5ml centrifuge tube, adding a corresponding volume of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 20mM, subpackaging, and storing in a refrigerator at-20 ℃ in a dark place for later use.
Administration alone
The proliferation of the cells cultured in vitro was examined by the MTT method. Adherent cells in the logarithmic growth phase were trypsinized or suspension cells were collected by centrifugation, counted, and 90. mu.l of cell suspension was seeded at a cell seeding density of 3000 cells/well in 96-well plates, and 10. mu.l of compound was added at a final concentration of 10-fold diluted in medium 24 hours later (concentration settings are shown in Table 1.). Wells to which the same volume of 5% DMSO was added served as controls, with a final DMSO concentration of 0.5%. After 5 days of drug treatment, MTT measures cell viability. The specific method comprises the following steps: after 10. mu.l of MTT was added to each well and the resulting mixture was placed in an incubator for further 4 hours, the supernatant was discarded, 150. mu.l of DMSO was added thereto to dissolve formazan crystals, and the absorbance of the formazan crystals was detected at 490nM by a microplate reader. The GraphPad Prism 6 software makes dose-response curves and calculates IC 50. The experiment was repeated three times and the average value of IC50 was calculated.
TABLE 25 Compound 1 and SHP099 Final concentration of Compound in vitro anti-cell proliferation assay
Figure RE-GDA0003205807320000181
C represents the concentration of the compound
The in vitro antiproliferative activity of compound 1 and SHP099 on KRAS mutated tumor cells was tested using the tetrazolium salt (MTT) method. The results show that compound 1 has strong proliferation-inhibiting effect on KRAS mutated lung cancer cell lines. The dose-response curves for these compounds are shown in FIG. 9, along with the corresponding IC50 in Table 26.
TABLE 26 IC50 values for H358 cells for Compound 1 and SHP099
Drugs IC50(nM)
Compound 1 7.24
SHP099 5808.51
Combined administration
The drug concentrations in the combination experiments were set according to the IC50 values on both cells for compound 1 and SHP099 obtained in the first part of the experiments. Specific dosages are shown in table 17.
TABLE 27 concentration settings for combination experiments with Compound 1 and SHP099
Figure RE-GDA0003205807320000182
Inoculating the cells into a cell culture bottle, adding a proper amount of complete culture medium, placing the cell culture bottle in an aseptic incubator for culture, and carrying out subculture when the confluency of the cells reaches more than 80%.
The proliferation of cells was detected by MTS/MTT method. Adherent cells in the logarithmic growth phase were digested with trypsin or suspension cells were collected by centrifugation, counted, 80. mu.l of cell suspension was inoculated into a 96-well plate at a cell inoculation density of 1000-. Wells with the same volume of 2.5% DMSO added were used as controls, with a final DMSO concentration of 0.5%, and single drug controls with only one compound were set up with three duplicate wells per concentration. After 3 days of drug treatment, MTT/MTS was examined for cell viability. The specific method comprises the following steps: after 10. mu.l of MTT was added to each well and the resulting mixture was placed in an incubator and incubated for 4 hours, the supernatant was discarded and 150. mu.l of DMSO was added to dissolve crystalline formazan, and 490nM absorbance was detected by an enzyme-labeling instrument, or after incubation for 4 hours in an incubator, 10. mu.l of MTS was added to each well, 490nM absorbance was detected by an enzyme-labeling instrument. The Combination Index (CI) was calculated with drug synergy evaluation software Calcusyn. The experiment was repeated 2-3 times.
Cell survival under dual drug combination was tested using the MTS/MTT cell proliferation assay, CI values were calculated using a drug synergy model established with TING-cha CHOU by comparing compound 1 and SHP099 single drug tests performed simultaneously, and whether synergy was present was judged by CI values (CI >1 antagonism; CI ═ 1 additivity; CI <1 synergy).
Compound 1 and SHP099 had synergistic effects at H358 in the concentration range from 1/8-fold IC50 to 4-fold IC50, and in the vicinity of IC50, had strong synergistic effects. See table 18.
TABLE 28 summary of CI values on H358 for combination of Compound 1 and SHP099
Figure RE-GDA0003205807320000191
Fa value represents the inhibition rate of the drug on cells
2.10 example J-Compound 1 alone or in combination with anti-tumor in vivo efficacy in tumor cells H358
H358 was inoculated at a density of 5e 6/mouse on the right dorsal side of nude mice. Over 10 days, groups were started with mean 281.9mm tumor volume per group3. The following table was followed for 21 days, and tumor size was measured once every two days according to the formula TGIRTV(％)： TGIRTV＝1-TRTV/CRTV(%) the administration group TGI was calculated.
TABLE 29 dosing regimen
Figure RE-GDA0003205807320000192
And finishing the experiment on the 22 nd day, weighing the whole tumor, and calculating the tumor weight inhibition rate of each group. The results of the experiment are shown in FIG. 9. The combination of the compound 1 and the SHP099 has an inhibiting effect on the growth of H358 tumor, the tumor growth inhibition rate (TGI) of the combination group is greater than that of the single drug of the compound 1 and that of the single drug of the SHP099 (shown in a table in the figure), and the tumor weight Inhibition Rate (IR) of the combination group is also greater than that of the two single drugs. The combination group (compound 1+ SHP099) showed significant differences in tumor weight and tumor volume (P <0.05) compared to the drug alone, as shown by the t-test.
The foregoing examples and description of certain embodiments should be taken as illustrative, not in a limiting sense, and the present invention is defined by the claims. It will be readily understood that various modifications and combinations of the above-described features may be utilized without departing from the invention as set forth in the claims. All such variations are intended to be included within the scope of the present invention. All references cited are incorporated by reference into this application in their entirety.

Claims (16)

1. A combination therapy comprising administering to a mammal having cancer a therapeutically effective amount of a first component which is a MEK inhibitor and a second component which is a chemotherapeutic agent, a KRAS inhibitor, a SHP2 inhibitor, a RAF inhibitor, or a combination of said chemotherapeutic agent and/or a KRAS inhibitor and/or a RAF inhibitor.
2. The combination therapy of claim 1, wherein the MEK inhibitor is a compound of formula (I) or a pharmaceutically salt thereof,
Figure RE-FDA0003205807310000011
wherein,
x ═ O, S or C ═ O;
y ═ N or CH;
R1、R2、R4and R5Each independently selected from hydrogen, halogen or C1-6 alkyl;
R3selected from hydrogen, halogen, C1-6Alkoxy radical, C1-6Alkylthio, halo C1-6Alkoxy, halo C1-6Alkylthio radical, C1-6Alkyl and halo C1-6An alkyl group;
R6is-C (O) NR8R7or-NHSO2R7；
R7And R8Each independently selected from hydrogen, C substituted by hydroxy1-6Alkyl radical, C3-6Cycloalkyl radical, C3-6Cycloalkyl radical C1-6Alkyl and C substituted by hydroxy3-6Cycloalkyl radical C1-6An alkyl group.
3. The combination therapy of claim 1, wherein the MEK inhibitor is compound 1, compound 2, compound 3, or compound 4.
Figure RE-FDA0003205807310000012
4. The combination therapy of claim 1, wherein the MEK inhibitor is administered to the mammal in an effective amount of from 0.1 to 1000mg/kg body weight twice a day or twice a week.
5. The combination therapy of claim 1, wherein the chemotherapeutic agent is selected from cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, and pemetrexed.
6. The combination therapy of claim 5, wherein the chemotherapeutic agent is docetaxel.
7. The combination therapy of claim 1, wherein the cancer is a RAS gene mutated cancer.
8. The combination therapy of claim 7, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, melanoma, acute myeloid leukemia, bladder cancer, or head and neck cancer.
9. The combination therapy of claim 8, wherein the cancer has a mutation selected from KRAS or NRAS or BRAF.
10. The combination therapy of claim 9, wherein the KRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, S17, P34, a59 and Q61.
11. The combination therapy of claim 9, wherein the KRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, a59, Q61, K117 and a 146.
12. The combination therapy of claim 9, wherein the BRAF mutation is a BRAF V600E mutation.
13. The combination therapy of claim 7, wherein the cancer is non-small cell lung cancer.
14. The combination therapy of claim 1, wherein the KRAS inhibitor is AMG-510, MRTX849 or BI 1701963.
15. The combination therapy of claim 1, wherein the RAF inhibitor is LY3009120, PLX4032, LGX818, GSK2118436, BI 882370, TAK580, or Vemurafenib.
16. The combination therapy of claim 1, wherein the SHP2 inhibitor is JAB-3068, TNO155, BBP-398(IACS-15509), JAB-3312, or SHP 099.
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WO2024165044A1 (en) * 2023-02-10 2024-08-15 上海科州药物研发有限公司 Polymorph as protein kinase mek inhibitor, and preparation method therefor and use thereof

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