CN116889568A - A kind of anti-tumor combination drug and its use - Google Patents

Abstract

The invention provides a combined medicament, which comprises a MEK inhibitor, a compound shown in a formula (I) and pharmaceutically acceptable salt, wherein the combination of the MEK inhibitor, the compound shown in the formula (I) and the pharmaceutically acceptable salt shows better synergistic effect in an in-vitro cell proliferation inhibition test and an in-vivo tumor model.

Description

Antitumor combination medicine and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a combined medicine of a compound shown in a formula (I) and a MEK inhibitor and application thereof in preparing an anti-tumor medicine.

Background

Mitogen-activated protein kinase (MAPK) pathway is an important signal transduction pathway in cells, and RAS is converted from an inactive conformation to an active conformation by stimulation of upstream cytokines and other molecules, so that intracellular RAF is recruited to cell membranes and is dimerized and phosphorylated, and the activated RAF is sequentially phosphorylated to activate MEK and ERK, and finally regulate proliferation, differentiation, apoptosis and metastasis of cells. This pathway transmits signals from the outside of the cell into the nucleus through a specific cascade of RAS/RAF/MEK/ERK, ultimately leading to activation of specific genes. This pathway over-activation is closely related to the occurrence of a variety of tumors.

RAF is a very important serine/threonine protein kinase in the MAPK signaling pathway, located downstream of the RAS, and can be activated by the RAS. The RAF family comprises three subtypes ARAF, BRAF, CRAF (also known as RAF 1), with a high degree of homology and similar domains. RAF kinase is used as a key signal protein at the downstream of RAS, and has important research significance in the treatment of RAS mutant gene mutant tumor. RAS mutant tumors account for about 25% of human tumors on average, the highest of all genetic mutations, and 100 tens of thousands of human KRAS mutations die from tumors annually worldwide. By inhibiting RAF kinase, MAPK signal transduction is regulated, so that the proliferation of RAS mutant tumor cells is inhibited. Thus, RAF kinase has become an important target for clinical treatment of tumors.

The full name of KRAS gene Kirsten ratsarcoma viral oncogene homolog, turned to Chinese, is "Kirsten rat sarcoma virus oncogene homolog". The protein encoded by the KARS gene is a small gtpase (smallGTPase) belonging to the RAS superfamily of genes related to human tumors, three of which are HRAS, KRAS and NRAS, located on chromosomes 11, 12 and 1, respectively. The KRAS gene has a great influence on human cancers, and KRAS mutations are present in about 30% of cancer patients, including 90% of pancreatic cancers, 50% of colon cancers and 25% of lung cancers. In non-small cell lung cancer, KRAS gene mutation accounts for 20-30%, and is frequently present in lung adenocarcinoma, and is rare in lung squamous carcinoma. Of the KRAS mutation types, G12C mutations are most common, accounting for about 44% of all KRAS mutations, with the most common cancer species being non-small cell lung cancer. As early as several decades, KRAS was identified as an important therapeutic target for cancer, however, since KRAS proteins have no pockets on their surface suitable for binding small molecule inhibitors, i.e. such proteins lack distinct targets to allow small molecule drugs to bind to and impair their function. For many years, therefore, as regards the drug flexibility of KRAS gene targeted therapy, only one covalently modified targeted drug, sotoraib (code AMG 510), directed against the KRAS G12C mutation has been marketed by the FDA in 2021 at 5 months 29 for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) patients carrying the KRAS G12C mutation, which have previously received at least one systemic treatment.

Patent WO2021110141A1 discloses a small molecule pan-RAF kinase inhibitor, the structure of which is shown in formula (I), and the chemical name is N- (3- (2- ((1 r,5 s) -3-oxabicyclo [3.1.0] hex-1-yl) -6- (2-hydroxyethoxy) pyridin-4-yl) -4-methylphenyl) -2- (trifluoromethyl) isonicotinamide. The small molecule inhibitor has good RAF kinase inhibition activity and various cell antiproliferative activity, and meanwhile, the molecule has good pharmacokinetic properties, and is expected to be developed into clinical medicines.

While some longitudinal combinations of MAPK inhibitors are disclosed in the prior art as beneficial, it is not always possible to predict whether all permutations will have clinical benefit. The invention aims to provide a RAF and MEK combination drug which can potentially benefit clinically and can target KRAS gene therapy.

Disclosure of Invention

The invention aims to provide a combination medicament of a RAF inhibitor and a MEK inhibitor and application thereof in preparing antitumor medicaments.

In particular, the method comprises the steps of,

the invention provides a combination medicament, which comprises a MEK inhibitor, a compound shown in a formula (I) and pharmaceutically acceptable salts:

in some embodiments of the invention, the MEK inhibitor in the combination is selected from cobicitinib.

In some embodiments of the invention, the MEK inhibitor in the combination is selected from the group consisting of trametinib.

The invention also provides application of the combined medicine in preparing medicines for treating or preventing tumors.

In some embodiments of the invention, the tumor in the above uses is selected from malignant tumors that are mutated in MAPK signaling pathways.

In some embodiments of the invention, the tumor in the above uses is selected from the group consisting of RAS mutated lung cancer, RAS mutated pancreatic cancer, RAS mutated colorectal cancer.

In some embodiments of the invention, the tumor in the above uses is selected from the group consisting of RAF mutated lung cancer, RAF mutated pancreatic cancer, RAF mutated colorectal cancer.

In some embodiments of the invention, the RAS mutation described in the foregoing uses is selected from the group consisting of a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12V mutation, a KRAS G12S mutation, a KRAS G13D mutation, a KRAS Q61K mutation.

In some embodiments of the invention, the RAF mutation described in the foregoing uses is selected from the BRAF V600E mutation.

In some embodiments of the invention, the compound of formula (I) and the MEK inhibitor for the above uses may be administered simultaneously, sequentially or intermittently.

In some embodiments of the invention, the amount of the compound of formula (I) in the above uses is 100 mg/day, 200 mg/day, 400 mg/day or 800 mg/day.

In some embodiments of the invention, the MEK inhibitor is used in an amount of 2 mg/day for the above-described uses.

In some embodiments of the invention, the amount of the compound of formula (I) to MEK inhibitor used in the above-described use is 50:1, 100:1, 200:1 or 400:1.

The invention also provides a method of treating a tumor comprising administering to the subject a therapeutically effective amount of a MEK inhibitor and a compound of formula (I):

in some embodiments of the invention, the MEK inhibitor described in the above methods of treatment is selected from the group consisting of trametinib.

In some aspects of the invention, the MEK inhibitor in the above-described methods of treatment is selected from cobicitinib.

In some embodiments of the invention, the neoplasm in the above-described method of treatment is selected from malignant neoplasms in which MAPK signaling pathway mutations are present.

In some embodiments of the invention, the tumor in the above-described methods of treatment is selected from the group consisting of RAS mutated lung cancer, RAS mutated pancreatic cancer, RAS mutated colorectal cancer.

In some embodiments of the invention, the tumor in the above-described methods of treatment is selected from the group consisting of RAF mutated lung cancer, RAF mutated pancreatic cancer, RAF mutated colorectal cancer.

In some embodiments of the invention, the RAS mutation described in the above methods of treatment is selected from the group consisting of a KRAS G12C mutation, a KRAS G12D mutation, a KRAS G12V mutation, a KRAS G12S mutation, a KRAS G13D mutation, a KRAS Q61K mutation.

In some embodiments of the invention, the RAF mutation described in the above methods of treatment is selected from the BRAF V600E mutation.

In some embodiments of the invention, the compound of formula (I) and the MEK inhibitor in the above methods of treatment may be administered simultaneously, sequentially or intermittently.

In some embodiments of the invention, the amount of the compound of formula (I) used in the above-described methods of treatment is 100 mg/day, 200 mg/day, 400 mg/day or 800 mg/day.

In some embodiments of the invention, the MEK inhibitor is used in the above-described methods of treatment in an amount of 2 mg/day.

In some embodiments of the invention, the dosage ratio of the compound of formula (I) to MEK inhibitor in the above-described methods of treatment is 50:1, 100:1, 200:1 or 400:1.

Technical effects

The combination of the compound of formula (I) and a MEK inhibitor, especially the combination with trametinib, has better synergistic effect on cell proliferation inhibition experiments and in-vivo tumor models.

Definition and description

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

The term "combination" means that the two agents are contained in separate units of formulation of different specifications and can be administered simultaneously, sequentially or at intervals.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

The term "therapeutically effective amount" refers to a sufficient amount of a compound of the present invention or a pharmaceutically acceptable salt to treat a disorder at a reasonable effect/risk ratio applicable to any medical treatment and/or prophylaxis. It will be appreciated, however, that the total daily amount of pharmaceutically acceptable salts and compositions of the compounds of formula I of the present invention must be determined by the physician within the scope of sound medical judgment. For any particular patient, the particular therapeutically effective dose level will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the specific composition employed; age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular compound employed; duration of treatment; a medicament for use in combination with or simultaneously with the particular compound employed; and similar factors well known in the medical arts. For example, it is common in the art to start doses of the compound at levels below that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

As used herein, the term "administering" means physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Routes of administration of anti-VEGF antibodies include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In certain embodiments, the anti-VEGF antibody is administered by a non-parenteral route, in certain embodiments, orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, e.g., intranasal, vaginal, rectal, sublingual or topical. Administration may also be performed, for example, one, multiple times, and/or over one or more extended periods of time.

The term "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In certain embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably in certain contexts herein.

In the term "KRAS G12C", KRAS refers to a mutant gene, G12C refers to a mutation site and the type of mutation by amino acid. The specific meaning of the other mutations is the same as that of "KRAS G12C".

Drawings

FIG. 1 is a graph showing the synergistic effect of a compound of formula (I) below in combination with trametinib on the A549 cell line.

FIG. 2 is a graph showing the synergistic effect of a Calu-6 cell line of a compound of formula (I) in combination with trametinib.

FIG. 3 is a graph showing the synergistic effect of HCT116 cell line of a compound of formula (I) in combination with trametinib.

FIG. 4 is a graph showing the synergistic effect of NCIH2122 cell line of the following formula (I) in combination with trametinib.

FIG. 5 is a graph showing the synergistic effect of the combination of a compound of formula (I) with trametinib on the Panc1005 cell line.

FIG. 6 is a graph showing the synergistic effect of HT-29 cell lines of a compound of formula (I) in combination with trametinib.

FIG. 7 is a graph showing tumor volume change in experimental study of CO-04-0307PDX model.

Detailed Description

The present invention is described in detail below by way of examples, which are provided for illustration only and do not limit any scope of the present invention. Also, the invention is not limited to any particular preferred embodiment described herein. It should be understood by those skilled in the art that equivalent substitutions and corresponding modifications of the technical features of the present invention are included in the scope of the present invention.

Example 1: cell proliferation inhibition assay for the combination of a compound of formula (I)

1. Purpose of experiment

The combination of the compound of formula (I) with trametinib was studied by the CTG (Cell Titer-Glo Luminescent viability assay) assay for aberrant activation of MAPK pathway tumor Cell lines (Calu-6 (KRAS) ^Q61K )，HCT116(KRAS ^G13D )，PANC1005(KRAS ^G12D )，HT-29(BRAF ^V600E )，A549(KRAS ^G12S )，NCIH2112(KRAS ^G12C ) To evaluate the effect of the combination thereof.

2. Experimental materials

2.1 cell information

TABLE 1 cell information

2.2 sample information

Trametinib was purchased from seleck, cat# S2673, lot S267310, and stored at-20 ° after 30mM stock solution in DMSO.

The compounds of formula (I) are prepared by themselves with reference to WO2021110141A 1.

2.3 other reagents and consumables

TABLE 2 other reagent, consumable information

3. Test method

3.1 Experimental design

The inhibition of Calu-6, HCT116, PANC1005, SW1417, A549, and NCIH2112 cell proliferation by compounds (I) and Qu Meiti was analyzed and two replicates were set with compound ((I) concentration ranging from 4.6 to 30000nM and trametinib concentration ranging from 0.14 to 500nM.

3.2 Experimental procedure

(1) Preparation of working solution concentration of compound

The compound to be tested is prepared and added into a designated hole of T8 cassette according to a software prompt during drug addition.

(2) Cell seeding and drug treatment

The day before compound treatment, cells were digested, counted by MUSE cell counter, diluted to the indicated concentration with the corresponding medium according to the counting result, and added to the corresponding 384-well microwell plate as required using multichannel liquid separation system, and cultured overnight in an incubator at 37 ℃.

The overnight cultured cells were removed, and the compounds were added in accordance with the microplate layout using D300E, and incubated in an incubator for 5 days. After the incubation, the plate was taken out, equilibrated to room temperature for 30 minutes, and the Cell prepared in advance was taken outThe reagents were equilibrated to room temperature for 30 minutes and mixed well, and 20. Mu.LCell +.>The reagent is vibrated for 10 minutes under the condition of 500 revolutions per minute of a microplate constant temperature vibrator, and the above operation is carried out under the condition of light shielding. The enzyme label instrument is started and preheated 15 minutes in advance, and the luminescence value is detected at 590 nm.

3.3 data analysis

Drug synergy assessment was performed using combineflit software.

4. Experimental results

Table 3. ^a Proliferation inhibition rate of combination of compound of formula (I) and trametinib at clinical Crough concentration

The clinical Ctrough concentration is 20nM for trametinib and 400nM for the compound of formula (I);

the synergistic effect of the compounds of formula (I) with trametinib in different cell lines is shown in FIGS. 1-6.

Conclusion of the experiment

As can be seen from table 3 and fig. 1-6, the compound of formula (I) has a better synergy with trimetinib on both KRAS mutant and BRAF mutant cell lines (the darker the blue region the stronger the synergy, greater than 20 being the synergy and greater than 30 being the strong synergy).

Example 2: clinical C on in vitro organoid model _trough Proliferation inhibition ratio of trimetinib in combination with Compound of formula (I) at (absorption steady-state trough concentration)

1. Purpose of experiment

The proliferation inhibition effect of the compound of formula (I) in combination with trametinib on organoids derived from in vitro cultures of tumor tissues of different patients was studied by CTG (Cell Titer-Glo Luminescent viability assay) assay to evaluate the effect of both combinations.

2. Experimental materials

2.1 organoid information

TABLE 4 organoid information

2.2 sample information

Trametinib was purchased from seleck, cat# S2673, lot S267310, prepared in DMSO as 30mM stock solution and stored at-20 ℃.

The compounds of formula (I) are prepared by themselves with reference to WO2021110141A 1.

2.3 other reagents and consumables

TABLE 5 other reagent, consumable information

3. Test method

3.1 Experimental design

The experiments were divided into a drug-alone experimental group, a drug-combination experimental group, a negative control group (DMSO, dimethyl sulfoxide) and 2 positive control groups (BEZ 235 and Staurosporine). Negative and positive controls were used to calculate the signal and noise of the experiment and evaluate the quality of the experimental data.

3.2 Experimental procedure

(1) Tumor organoid resuscitating culture

Frozen tumor organoids were removed from liquid nitrogen, rapidly dissolved at 37 ℃, added with 5mL of 10% fbs-containing Advance DMEM medium, centrifuged at 300g for 5 min, the pellet was collected, resuspended in GAS medium, added with pre-chilled diluted matrigel, thoroughly mixed, added to 24-well cell culture dishes, incubated at 37 ℃ for 30 min, added GAS medium, continued culture for 1 week, and medium was supplemented every 3 days until passage.

(2) Organoid inoculation

After the tumor organoids can be passaged, the matrix glue is dissolved at 4 ℃ for standby. Cultured tumor organoids were collected with a papanicolaou tube and hydrolyzed with Tryple to form single cell suspensions. After cell counting, the cell concentration was adjusted to 1.6X10 with GAS medium ⁵ cells/mL, 2mL of cell suspension was taken and kept on ice for further use. Preparing matrigel mixed solution on ice for standby, mixing cell suspension and matrigel, adding 50 mu L cell suspension into 96-well plate, incubating at 37 ℃ for 30 min,after adding 40. Mu.L of GAS medium and culturing in a cell culture incubator at 37℃for 2 days, the initiation of tumor organoids formation and growth were observed.

(3) Compound formulation and dilution

After organoids were cultured in gel phase + aqueous phase (medium) for 2 days, compound gradient dilutions were prepared and added to the culture system.

Stock drug dissolved in DMSO was dissolved sufficiently at room temperature, if insoluble, and was observed after heating with ultrasound for 5 minutes until dissolved.

(4) Adding the medicine to be tested

After the beginning of tumor organoids formation and growth, 5 μl of the day prepared 20x stock solution was added sequentially to the microplate layout. Incubate at 37℃under 5% carbon dioxide for 5 days.

(5) Chemiluminescent assay

Cell ATP levels were measured using chemiluminescence, and cell viability was assessed. Specific operations according to the instruction, after culturing, adding 50 mu L CTG solution, mixing, transferring the cleavage mixture into an ELISA plate, and collecting chemiluminescence data in an ELISA apparatus after 5-10 minutes. Data were analyzed using Excel software, and IC was calculated using GraphPad Prism 7 software, fitting a pharmacodynamic dose curve from chemiluminescent data ₅₀ 。

Control and quality control

The test uses Z' factor as a quality control index. Z' factor is defined by 4 parameters: mean (μ) and standard deviation (σ) of positive (p) and negative (n) controls.

The calculation formula is as follows:

Z’factor＝1-(3*(σp+σn)/|(μp-μn)|)

the negative control group was an untreated group with the addition of solvent (DMSO); positive controls were run with BEZ235 added at 2.5uM or Staurosporine added at 1 uM.

Functional detection of general cellular levels Z' factor requires >0.3; the quality control passing value Z' factor of the test is set to be >0.5.

(6) Data analysis

Drug effect dose curve and IC ₅₀ The calculation method comprises the following steps: calculating the corresponding drug concentration when the survival rate is 50% by using GraphPad Prism 7 software, namely the IC of the drug on the organoids ₅₀ Values. Heat maps of the combined dual drug effect were fitted with synergy finder.r.

4. Experimental results

TABLE 6 proliferation inhibition of the combination of the compound of formula (I) with trametinib at clinical Ctrough concentration (20 nM for trametinib, 400nM for the compound of formula (I))

Conclusion of the experiment

From Table 6, it can be seen that the compound of formula (I) has a better proliferation inhibiting effect on part of the KRAS G12V mutant pancreatic cancer organoids when administered in combination with trametinib in the Kras mutant organoids.

Example 3: CO-04-0307 (intestinal cancer, KRAS G12C mutation) PDX model experiment study 1. Purpose of experiment

The in vivo efficacy of compound (I) in combination with trimetinib in a human colon cancer CO-04-0307 (KRASG 12C) subcutaneous xenograft tumor model was evaluated.

2. Experimental materials

(1) Experimental animal

Species: a mouse

Strain: BALB/c nude mice

Arrival week: 6-8 weeks of age

Gender: female

Weight of the goods: 18-22 g

The suppliers: beijing Vitolihua laboratory animal technology Co.Ltd

Animal pass number: 20210715Abzz0619000330

(2) Information on Compounds

TABLE 7 information on Compounds

Name of the name	Lot number	Purity (%)	Preservation conditions	Source
					Compound (I)	210323S	97.6	-20℃	Medicine Mingkang De
Trametinib	P1721180	99	-20℃	GSK

(3) Tumor tissue information

Establishment of the model for human colon cancer CO-04-0307 was originally derived from a clinical sample from surgical excision, and was defined as the P0 generation after implantation into nude mice. The implantation of the tumor tissue of the P0 generation into the next generation is called the P1 generation. And so on, is continuously implanted in nude mice. Wherein the tumor of FP3 is recovered by P2 generation resuscitating. The next generation generated by the FP3 generation was defined as FP4, and similarly, the tumor tissue of the FP7 generation would be used for this pharmacodynamic test.

3. Experimental methods and procedures

(1) Tumor tissue preparation

Tumor-bearing mice of the FP7 CO-04-0307 model are euthanized, the tumor is taken out and put into prepared PBS (containing 1% diabody) for cleaning and necrosis elimination,cutting the tumor mass into about 30mm in culture medium (containing 1% diabody) ³ For inoculation.

(2) Tumor tissue inoculation and grouping

Each animal was inoculated at a volume of about 30mm at the right back position ³ CO-04-0307FP7 tumor mass, the average tumor volume reaches 159mm ³ At this time, the administration was started using a random packet. The experimental groupings and dosing schedules are shown in the following table.

TABLE 8 grouping of experimental animals and dosing regimen

Note that:

number of mice per group

2. Dosing volume parameters: based on the weight of the mice, 10. Mu.L/g. If the weight is reduced by more than 15%, stopping administration until the weight is recovered to be within 10% and then administration is performed.

(3) Preparation of test article

TABLE 9 preparation of test substances

Note that: the drug needs to be gently mixed well before administration to animals. The compound formulation cycle is daily.

(4) Daily observations of laboratory animals

The development of this protocol and any modifications passed the evaluation approval of the laboratory animal administration and use committee (IACUC) of the new drug development company, inc. The use and welfare of experimental animals was performed in compliance with the international committee for laboratory animal assessment and approval (AAALAC) specifications. Animals were monitored daily for health and mortality, and routine examinations included observation of tumor growth and the effects of drug treatment on daily performance of the animals, such as behavioral activity, intake of water (visual inspection only), weight changes (twice a week), physical signs of appearance, or other abnormalities. The number of animal deaths and side effects in the groups were recorded based on the number of animals in each group.

(5) Tumor measurement and experimental index

Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b ² A and b represent the major and minor diameters of the tumor, respectively.

The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) =trtv/crtv×100% (TRTV: treatment group RTV mean; CRTV: negative control group RTV mean). The relative tumor volume (relative tumor volume, RTV) was calculated from the results of the tumor measurements, with the calculation formula rtv=vt/V0, where V0 is the tumor volume measured at the time of group administration (i.e., D0), vt is the tumor volume at the time of a certain measurement, and TRTV and CRTV take the same day data.

TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group)/(mean tumor volume at the end of treatment of solvent control group-mean tumor volume at the beginning of treatment of solvent control group) ]x100%.

(6) Statistical analysis

Statistical analysis was performed using SPSS software based on RTV data at the end of the test. The comparison between groups was analyzed by one-way ANOVA, with variance (significant differences in F values), and examined by using the gas-Howell method. p <0.05 was considered a significant difference.

4. Experimental results

TABLE 10 tumor inhibition rate

Note that: * p <0.05 p <0.01

5. Conclusion of the experiment

As can be seen from Table 10 and FIG. 7, the compounds of formula (I) are mixed withCombination of trametinib for human colon cancer CO-04-0307 (KRAS) ^G12C ) Tumor proliferation of subcutaneous xenograft tumor model has combined synergistic effect.

Claims (9)

1. A combination drug, characterized in that it comprises a MEK inhibitor, a compound of formula (I), and a pharmaceutically acceptable salt: 2. The combination drug as described in claim 1, wherein the MEK inhibitor is selected from trametinib. 3. Use of the combination drug as described in any one of claims 1-2 in the preparation of a drug for treating or preventing tumors. 4. The use as described in claim 3, wherein the tumor is selected from malignant tumors with mutations in the MAPK signaling pathway. 5. The use as described in claim 4, wherein the tumor is selected from RAS-mutated lung cancer, RAS-mutated pancreatic cancer, and RAS-mutated colorectal cancer. 6. The use as described in claim 4, wherein the tumor is selected from RAF-mutated lung cancer, RAF-mutated pancreatic cancer, and RAF-mutated colorectal cancer. 7. The use as described in claim 5, wherein the RAS mutation is selected from KRAS G12C mutation, KRAS G12D mutation, KRAS G12V mutation, KRAS G12S mutation, KRAS G13D mutation, and KRAS Q61K mutation. 8. The use as described in claim 6, wherein the RAF mutation is selected from the BRAF V600E mutation. 9. The use as described in any one of claims 3-8, characterized in that the compound of formula (I) and the MEK inhibitor can be administered simultaneously, sequentially, or at intervals.