Combination of MEK inhibitor with PLCbeta / PKC signaling pathway inhibitor and its use in treating BRAF(V600E) mutant tumors

Abstract

The application discloses a combination of a MEK inhibitor and a PLCbeta / PKC signal path inhibitor and application of the combination in treating BRAF(V600E) mutant tumors, and belongs to the field of biological medicines. The PLCbeta / PKC signal path inhibitor is combined with the MEK inhibitor, drug resistance can be reversed, stronger anti-BRAF(V600E) mutant tumor activity is exhibited, the growth of tumors can be more effectively inhibited, and the defects of poor treatment effect or large toxic side effects of single drug treatment are overcome. The application has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the combination therapy of MEK inhibitors and PLCβ / PKC signaling pathway inhibitors and their use in the treatment of BRAF(V600E) mutant tumors. Specifically, it involves the combination therapy of the MEK inhibitor trametinib with the PLCβ inhibitor U73122 or the PKC inhibitor sotrastaurin for the treatment of tumors or related diseases carrying BRAF V600E mutations. This invention is particularly applicable to therapeutic situations that overcome acquired resistance to MEK inhibitors. Background Technology

[0002] The BRAF gene is a key member of the Ras-Raf-MEK-MAPK / ERK signaling pathway, and its encoded BRAF protein kinase plays an important role in physiological processes such as cell growth, differentiation, and survival. In various tumor types, the mutation at amino acid position 600 of BRAF (valine (V) to glutamate (E) – BRAF V600E) is the most common acquired activating mutation, which leads to a significant increase in BRAF kinase activity. This, in turn, continuously activates the downstream MEK / ERK signaling pathway, driving tumor cell proliferation, metastasis, and anti-apoptosis, and is considered one of the important molecular pathogenesis mechanisms of various malignant tumors.

[0003] Clinical and molecular epidemiological studies have shown that the BRAF V600E mutation is widespread in various human tumors, particularly melanoma, papillary thyroid carcinoma, colorectal cancer, and non-small cell lung cancer, with an incidence rate as high as 40-60% in melanoma and 40-70% in papillary thyroid carcinoma. In addition, this mutation is also found in rare tumors and histioproliferative diseases such as gliomas (e.g., pilocytic astrocytoma), Langerhans histiocytosis (LCH), and Erdheim-Chester disease. Some literature also reports its presence in low-grade serous ovarian carcinoma, biliary tract cancer, and hepatocellular carcinoma. Studies have found that tumors carrying this mutation often exhibit greater biological invasiveness and poorer clinical prognosis.

[0004] Currently, treatment for BRAF V600E mutations primarily relies on small molecule kinase inhibitors, including BRAF inhibitors (such as vemurafenib and dabrafenib) and MEK inhibitors (such as trametinib). These drugs, used alone or in combination, have been approved for the treatment of various solid tumors, showing preliminary efficacy, particularly in advanced melanoma and non-small cell lung cancer. However, clinical research and practice indicate that patients often develop acquired resistance within a short period after receiving targeted therapy with BRAF and MEK inhibitors, either as monotherapy or in combination. Furthermore, some patients exhibit innate resistance from the initial stages of treatment, resulting in poor efficacy. The mechanisms of resistance are complex and diverse, mainly including imbalances in the negative feedback regulation of the MAPK pathway, compensatory activation of other signaling pathways induced by sustained inhibition (such as PI3K / AKT and PLC / PKC), and adaptive changes in the tumor microenvironment in response to treatment. Further exploration of other combination therapy strategies to improve efficacy, delay resistance, and reduce side effects is crucial for improving patient prognosis. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] To overcome the aforementioned treatment bottlenecks, the inventors propose an innovative combination therapy strategy: combining the MEK inhibitor trametinib with the PLC inhibitor U73122 or the PKC inhibitor sotrastaurin. This dual-targeting of the MEK-ERK and PLC / PKC signaling axes simultaneously inhibits tumor cell survival and proliferation signals at multiple key regulatory nodes. This combination is expected to effectively block compensatory activation of tumor signaling pathways, enhance tumor cell apoptosis, and delay or overcome acquired and primary drug resistance, making it particularly suitable for BRAF V600E-positive tumors that are insensitive to or resistant to conventional targeted therapies. Currently, no published literature or patents have systematically studied this combination strategy; therefore, this invention is significantly innovative in both its treatment approach and mechanism, possessing high clinical application potential and industrialization value.

[0007] Solution for solving the problem

[0008] Therefore, the present invention provides the following treatment options:

[0009] [1]. PLCβ / PKC signaling pathway inhibitors for any of the following uses (i) to (ii):

[0010] (i) Use of a combination of a PLCβ / PKC signaling pathway inhibitor and a MEK inhibitor in the preparation of a medicament or pharmaceutical composition for the prevention and / or treatment of tumors;

[0011] (ii) Use of PLCβ / PKC signaling pathway inhibitors in the preparation of reagents for enhancing the preventive and / or therapeutic effects of MEK inhibitors on tumors;

[0012] The tumor is a BRAF(V600E) mutant tumor.

[0013] [2]. According to the use described in [1], wherein the MEK inhibitor includes trametinib.

[0014] [3]. According to the use described in [1], wherein the PLCβ / PKC signaling pathway inhibitor includes U73122 and sotrastuxin.

[0015] [4]. The use according to any one of [1]-[3], wherein the tumor is a drug-resistant BRAF (V600E) mutant tumor.

[0016] [5]. The use according to any one of [1]-[3], wherein the tumor is a BRAF(V600E) mutant tumor resistant to MEK inhibitors.

[0017] [6]. According to any one of [1]-[3], wherein the BRAF(V600E) mutant tumor includes papillary thyroid carcinoma, leukemia, melanoma, undifferentiated thyroid carcinoma, serous ovarian cancer, breast cancer, colorectal cancer, glioma or non-small cell lung cancer.

[0018] [7]. According to the use described in [6], the BRAF(V600E) mutant tumors include melanoma.

[0019] [8]. The use according to any one of [1]-[3], wherein the improvement of the preventive and / or therapeutic effect of MEK inhibitors on tumors includes reversing the subject’s resistance to MEK inhibitors or increasing the subject’s sensitivity to MEK inhibitors.

[0020] [9]. According to any one of [1]-[3], wherein the molar ratio of the MEK inhibitor to the PLCβ / PKC signaling pathway inhibitor is (1~10). 4 ):(1~10 12 ).

[0021]

[10] . According to the use described in [9], wherein the molar ratio of the MEK inhibitor and the PLCβ / PKC signaling pathway inhibitor is (1~10). 3 ):(1~10 11 ).

[0022]

[11] . The use according to any one of [1]-[3], wherein the drug or pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0023]

[12] . The use according to any one of [1]-[3], wherein the administration of the drug or drug composition includes oral, transdermal, intramuscular, subcutaneous and intravenous injection.

[0024]

[13] . A pharmaceutical composition comprising a MEK inhibitor and a PLCβ / PKC signaling pathway inhibitor.

[0025]

[14] . The pharmaceutical composition according to

[13] , wherein the MEK inhibitor comprises trametinib.

[0026]

[15] . The pharmaceutical composition according to

[13] or

[14] , wherein the PLCβ / PKC inhibitor comprises U73122 and sotrastuxin.

[0027]

[16] . The pharmaceutical composition according to

[15] , wherein the molar ratio of the MEK inhibitor and the PLCβ / PKC signaling pathway inhibitor is (1~10). 3 ):(1~10 11 ).

[0028]

[17] . A pharmaceutical composition according to any one of

[13] -

[16] , wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0029]

[18] . A pharmaceutical composition according to any one of

[13] -

[17] , wherein the pharmaceutical composition is administered orally, transdermally, intramuscularly, subcutaneously, or intravenously.

[0030] The effects of the invention

[0031] This invention combines a MEK inhibitor with a PLCβ / PKC signaling pathway inhibitor and unexpectedly found that the combined use of the two can reverse the body's resistance to the MEK inhibitor. The two have a synergistic effect, which can effectively inhibit the growth of cancer cells in the body, significantly reduce tumor burden and invasiveness, and no obvious toxic side effects were observed. Attached Figure Description

[0032] Figure 1 To construct a lin-45(V627E) mutant nematode model to simulate acquired resistance to the MEK inhibitor (trametinib).

[0033] Figure 1Sequence comparison of A: Nematode LIN-45 (Q07292) and human BRAF (P15056).

[0034] Figure 1 B: Schematic diagram of experimental procedure: Wild-type and lin-45(V627E) mutant nematodes were treated with DMSO or different concentrations of trametinib, respectively. Then, the brood size of their F1 offspring was quantitatively analyzed, and RNA sequencing and transcriptome analysis were carried out.

[0035] Figure 1 C: Comparison of penetrance of the protruding vulva (Pvl) phenotype between wild-type, lin-45(V627E) mutant, and lin-45(V627E) mutant treated with 1 μM trametinib.

[0036] Figure 1 D: The dose-dependent effect of trametinib on the broodability of wild-type and lin-45(V627E) F1 generation nematodes. Statistical analysis was performed using the Mann-Whitney U test and Dunn's multiple comparison test. n > 35, *** p < 0.001, **** p < 0.0001, ns indicates no statistically significant difference.

[0037] Figure 2 Transcriptome analysis revealed that egl-30 / Gαq was upregulated in trametinib-resistant lin-45(V627E) mutant nematodes.

[0038] Figure 2 A: Principal component analysis (PCA) was performed on RNA-seq data from lin-45(V627E) mutant nematodes treated with 10 μM and 100 μM trametinib to assess changes in whole transcriptome expression patterns.

[0039] Figure 2 B: Volcano plot showing gene expression differences in lin-45(V627E) F1 generation under 100 μM trametinib treatment compared to the 10 μM treatment group. Red indicates significantly upregulated genes, and blue indicates significantly downregulated genes. Screening criteria were: |fold change| > 2 and corrected P-value (Padj) < 0.05. Each group contained 3 biological replicates (n=3).

[0040] Figure 2C: Dot plot showing the gene ontology (GO) enrichment analysis results of upregulated genes in lin-45(V627E) F1 generation after 100 μM trametinib treatment.

[0041] Figure 2 A comparison of the RNA expression profiles of the D:egl-30 gene in lin-45(V627E) F1 generations treated with 10 μM and 100 μM trametinib showed that the gene was upregulated in trametinib-resistant nematodes under high-dose treatment.

[0042] Figure 3 This study demonstrates the crucial role of the Gαq-PLCβ-PKC signaling pathway in trametinib resistance, as confirmed by genetic screening and pharmacological experiments.

[0043] Figure 3 Comparison of penetrance of the protruding vulva (Pvl) phenotype in wild-type, lin-45(V627E) mutant, and lin-45(V627E); egl-30(R25C) double mutant nematodes.

[0044] Figure 3 B: The dose-dependent effect of trametinib on the number of broodstock of the F1 generation of lin-45(V627E); egl-30(R25C) double mutant nematodes. Statistical analysis was performed using Dunn's multiple comparison test. n > 35, p < 0.0001, ns indicates no statistically significant difference.

[0045] Figure 3 C: Comparison of brood size between wild-type and lin-45(V627E) F1 generation nematodes under DMSO, 50 μM U73122, 50 μM sotrastaurin (Sotra), 100 μM trametinib, and their combined treatments. Statistical significance was also assessed using Dunn's multiple comparison test. n > 35, p < 0.0001.

[0046] Figure 4 Inhibiting the Gαq-PLCβ-PKC signaling pathway can enhance the anti-proliferative effect of trametinib on A375 melanoma cells.

[0047] Figure 4A: Left panel: Cell survival curves of A375 cells after 72 hours of combined treatment with different concentrations of U73122 and trametinib (n = 3 independent experiments, data are expressed as mean ± standard deviation); Right panel: The synergistic effect of the combined drug was assessed by the Combination Index (CI) calculated by the Chou-Talalay method: CI < 0.3 indicates strong synergistic effect (red); 0.3 ≤ CI ≤ 0.9 indicates synergistic effect; 0.90 < CI < 1.1 indicates additive effect; CI ≥ 1.1 indicates antagonistic effect (blue).

[0048] Figure 4 B: Left panel: Cell survival curves of A375 cells after 72 hours of combined treatment with different concentrations of sotrastaurin and trametinib (n = 3 independent experiments, data are expressed as mean ± standard deviation); Right panel: Synergistic effect of drug combination was also assessed by combination index (CI).

[0049] Figure 4 C:A375 cells were treated with DMSO, 10 μM U73122, or 5 μM sotrastaurin in combination with different concentrations of trametinib for 24 hours. Western blot analysis was then used to analyze the protein expression levels of phosphorylated ERK (pERK), total ERK, and α-Tubulin. The figure shows representative results from three independent experiments.

[0050] Figure 4 D: Comparison of pERK levels in A375 cells under treatment with 1 nM trametinib alone, 1 nM trametinib + 10 μM U73122, and 1 nM trametinib + 5 μM sotrastaurin. pERK signal intensity was normalized to the 1 nM trametinib alone group, and results are expressed as a percentage.

[0051] Figure 5 Inhibitors of the PLCβ / PKC signaling pathway can reverse acquired trametinib resistance in A375 melanoma cells.

[0052] Figure 5A: Parental A375 cells and trametinib-resistant cell line A375-TR were treated with different concentrations of trametinib and then subjected to a clonogenic assay to assess differences in cell growth capacity and drug responsiveness.

[0053] Figure 5 B: Cell viability curves of A375 and A375-TR cells after treatment with different concentrations of trametinib for 72 hours (n = 3 independent experiments, data are expressed as mean ± standard deviation).

[0054] Figure 5 C: Half-maximal inhibitory concentration (IC50) of trametinib in A375 and A375-TR cells 50 The comparison showed a significant reduction in drug sensitivity in the drug-resistant cell lines.

[0055] Figure 5 D: Left panel: Cell survival curves of A375-TR cells after 72 hours of combined treatment with different concentrations of U73122 and trametinib (n = 3); Right panel: Drug synergistic effect assessed by Chou-Talalay Combination Index (CI): CI < 0.3 indicates strong synergistic effect (red); 0.3 ≤ CI ≤ 0.9 indicates synergistic effect; 0.90 < CI < 1.1 indicates additive effect; CI ≥ 1.1 indicates antagonistic effect (blue).

[0056] Figure 5 E: Left panel: Cell survival curves of A375-TR cells after 72 hours of combined treatment with different concentrations of sotrastaurin and trametinib (n = 3); Right panel: The synergistic effect of the combined treatment was also assessed based on CI values.

[0057] Figure 6 To investigate the synergistic inhibition of melanoma growth in a mouse A375 xenograft model by combining trametinib with an inhibitor of the PLCβ / PKC signaling pathway.

[0058] A375 melanoma cells were subcutaneously injected into Balb / c-nude mice purchased from Beijing Huafukang. Tumors were cultured until the average tumor volume reached 200-250 mm. 3Mice were randomly divided into two groups for drug treatment: the first group received solvent control, trametinib, U73122, or U73122 + trametinib combination treatment (n = 6 in each group); the second group received solvent control, trametinib, sotrastaurin, or sotrastaurin + trametinib combination treatment (n = 6 in each group).

[0059] Figure 6 A- Figure 6 B: Growth curves of tumor volume changes over time in mice of each treatment group (mean ± SEM).

[0060] Figure 6 C- Figure 6 D: Tumor weight data of mice after sacrifice at specified time points: Day 8 (solvent control, trametinib, U73122, U73122+trametinib group), Day 10 (solvent control, sotrastaurin group), Day 18 (trametinib, sotrastaurin + trametinib group).

[0061] Figure 6 E- Figure 6 F: Image of the tumor removed when the mouse was sacrificed.

[0062] Figure 6 G- Figure 6 H: Curves of mouse body weight change over time (mean ± SEM) for each group of mice, used to assess overall tolerability during treatment. Detailed Implementation

[0063] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0064] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0065] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0066] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0067] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0068] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0069] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0070] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0071] In addition, unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0072] As used in this article, "melanoma" refers to a condition characterized by the growth of tumors originating from the melanocyte system in the skin and other organs. Most melanocytes are found in the skin, but they can also be found in the meninges, digestive tract, lymph nodes, and eyes. When melanoma occurs in the skin, it is called cutaneous melanoma. Melanoma can also occur in the eye, and is called ocular or intraocular melanoma. Melanoma rarely occurs in the meninges, digestive tract, lymph nodes, or other areas where melanocytes are found.

[0073] The terms “mutant melanoma” or “malignant melanoma” are used interchangeably to refer to melanocytic tumors containing defective (also known as “mutations”) melanoma cells. Malignant melanomas typically develop from or near nevi and consist of a large number of cells with a strong tendency to metastasize. 40–60% of melanomas carry an activating mutation in the gene encoding the serine-threonine protein kinase BRAF (BRAF). Of the BRAF mutations observed in melanoma, over 90% are at codon 600, and of these mutations, over 90% are single nucleotide mutations resulting in the substitution of valine for glutamic acid (BRAF V600E). The second most common mutation is BRAF V600K, which substitutes valine for lysine, representing 5–6% of melanomas, followed by BRAF V600R and BRAF V600D. (Ascierto PA, Kirkwood JM, Grob JJ, Simeone E, Grimaldi AM,Maio M, Palmieri G, Testori A, Marincola FM, Mozzillo N. The role of BRAFV600 mutation in melanoma. J Transl Med. 2012 Jul 9;10:85).

[0074] In some exemplary embodiments of the present invention, the BRAF V600E sequence is as shown in SEQ ID NO:1:

[0075] MAALSGGGGGGAEPGQALFNGDMEPEAGAGAGAAASSAADPAIPEEVWNIKQMIKLTQEHIEALLDKFGGEHNPPSIYLEAYEEYTSKLDALQQREQQLLESLGNGTDFSVSSSASMDTVTSSSSSSLSVLPSSLSVFQNPTDVARSNPKSPQKPIVRVFLPNKQRTVVPARCGVTVRDSLKKALMMRGLI PECCAVYRIQDGEKKPIGWDTDISWLTGEELHVEVLENVPLTTHNFVRKTFFTLAFCDFCRKLLFQGFRCQTCGYKFHQRCSTEVPLMCVNYDQLDLLFVSKFFEHHPIPQEEASLAETALTSGSSPSAPASDSIGPQILTSPSPSKSIPIPQPFRPADEDHRNQFGQRDRSSSAPNVHINTIEPVNIDDLI RDQGFRGDGGSTTGLSATPPASLPGSLTNVKALQKSPGPQRERKSSSSSEDRNRMKTLGRRDSSDDWEIPDGQITVGQRIGSGSFGTVYKGKWHGDVAVKMLNVTAPTPQQLQAFKNEVGVLRKTRHVNILLFMGYSTKPQLAIVTQWCEGSSLYHHLHIIETKFEMIKLIDIARQTAQGMDYLHAKSIIH RDLKSNNIFLHEDLTVKIGDFGLATEKSRWSGSHQFEQLSGSILWMAPEVIRMQDKNPYSFQSDVYAFGIVLYELMTGQLPYSNINNRDQIIFMVGRGYLSPDLSKVRSNCPKAMKRLMAECLKKKRDERPLFPQILASIELLARSLPKIHRSASEPSLNRAGFQTEDFSLYACASPKTPIQAGGYGAFPVH

[0076] Unless otherwise stated, the term "melanoma" may also include recurrent or resistant melanoma. The terms "recurrent" or "resistant" refer to the repeated outbreaks or progression of melanoma, regardless of whether the disease was cured prior to the outbreaks or progression.

[0077] As used herein, the terms "synergy," "synergistic effect," or "synergistic action" refer to the therapeutic effect of a combination of compounds (PLCβ / PKC signaling pathway inhibitors and MEK inhibitors) that is greater than the additive effect of the compounds used in the drug combination. Advantageously, when combined, this synergistic effect between the active ingredients (therapeutic active compounds) allows for the use of smaller doses of one or both active ingredients to provide greater efficacy at the same dosage, and / or to prevent or delay the accumulation of multidrug resistance. The synergistic effect can be achieved by co-formulating the drug combinations or compositions of the present invention, by simultaneously administering the compounds in a unit dosage form, or by administering them as separate formulations simultaneously or sequentially.

[0078] <Detailed Description of the Invention>

[0079] In a first aspect of the invention, a PLCβ / PKC signaling pathway inhibitor is provided for any of the following uses (i) to (ii):

[0080] (i) Use of a combination of a PLCβ / PKC signaling pathway inhibitor and a MEK inhibitor in the preparation of a medicament or pharmaceutical composition for the prevention and / or treatment of tumors;

[0081] (ii) Use of PLCβ / PKC signaling pathway inhibitors in the preparation of reagents for enhancing the preventive and / or therapeutic effects of MEK inhibitors on tumors.

[0082] In some implementations, the improvement of the preventive and / or therapeutic effects of MEK inhibitors on tumors includes reversing the subject's resistance to MEK inhibitors or increasing the subject's sensitivity to MEK inhibitors.

[0083] (Tumor)

[0084] In some implementations, the tumor is a BRAF(V600E) mutant tumor.

[0085] In some alternative embodiments, the tumor is a drug-resistant BRAF (V600E) mutant tumor, including acquired drug-resistant BRAF (V600E) mutant tumors and inherently drug-resistant BRAF (V600E) mutant tumors.

[0086] In some specific embodiments, the resistant BRAF (V600E) mutant tumors are resistant to BRAF downstream MEK-targeting drugs. In some exemplary embodiments of the present invention, the BRAF downstream MEK-targeting drug is trametinib. In some embodiments of the present invention, the type of tumor described in the present invention is not limited, and can be any tumor with BRAF (V600E) mutations, including but not limited to: papillary thyroid carcinoma, leukemia, melanoma, undifferentiated thyroid carcinoma, serous ovarian cancer, prostate cancer, breast cancer, colorectal cancer, glioma, hairy cell leukemia, or non-small cell lung cancer, etc.

[0087] In some exemplary embodiments, the tumor is a melanoma.

[0088] In this invention, "reversing tumor drug resistance" refers to making drug-resistant tumor cell lines sensitive to tumor suppressor drugs and undergoing apoptosis. For example, in this invention, the PLCβ / PKC signaling pathway inhibitor acts on tumor cells, making the tumor cells more sensitive to MEK inhibitors. "Reversing tumor drug resistance" and "suppressing tumors" are two different concepts.

[0089] (Inhibitor)

[0090] As used herein, the term "MEK inhibitor" refers to an agent that interacts with mitogen-activated protein kinase (MEK) and inhibits its enzymatic activity. Inhibition of MEK enzyme activity, in turn, reduces MEK's ability to phosphorylate substrate peptides or proteins. MEK1 and MEK2 are protein kinases involved in the RAS-RAF-MEK-ERK signaling cascade. This cascade is involved in regulating a variety of processes, including apoptosis, cell cycle progression, cell migration, differentiation, metabolism, and proliferation. Therefore, the term "MEK inhibitor" includes, within its scope, compounds capable of inhibiting MEK.

[0091] In some specific embodiments of the present invention, the MEK inhibitor includes trametinib.

[0092] In some embodiments, the trametinib has the following CAS number: 871700-17-3, CBNumber: CB32514557, and molecular formula: C 26 H 23 FIN5O4, chemical formula as shown below:

[0093] .

[0094] In some optional embodiments, the trametinib can be obtained by any means known in the art, such as by purchasing it through commercial channels, for example, from MACKLIN, catalog number C16249920.

[0095] In this invention, the PLCβ / PKC signaling pathway inhibitor refers to a compound capable of specifically blocking the activity of the phospholipase Cβ (PLCβ) and / or protein kinase C (PKC) signaling pathways. PLCβ belongs to the phospholipase C family and, upon activation by a G protein-coupled receptor (GPCR), hydrolyzes PIP2 (phosphatidylinositol diphosphate) on the cell membrane, generating IP3 (inositol triphosphate) and DAG (diacylglycerol). PKC, activated by DAG and calcium ions, is a serine / threonine kinase that regulates cell proliferation, apoptosis, and inflammation by phosphorylating downstream target proteins (such as transcription factors and ion channels). The PLCβ / PKC signaling pathway includes: PLCβ activation → DAG / IP3 generation → PKC activation → downstream effects (such as NF-κB pathway activation and gene expression regulation). Specifically, the PLCβ inhibitor indirectly inhibits PKC activation by blocking the PLCβ catalytic domain, inhibiting PIP2 hydrolysis, and reducing DAG / IP3 generation.

[0096] In some exemplary embodiments, the PLCβ inhibitor includes U73122.

[0097] In some embodiments, the CAS number of U73122 is 112648-68-7, CBNumber is CB6237598, and the molecular formula is C. 29 H 40 N2O3, chemical formula as shown below:

[0098] .

[0099] In some exemplary embodiments, the PKC inhibitor includes sotrastaurin.

[0100] In some embodiments, the CAS number of the sotrastolin is 425637-18-9, CBNumber is CB92510056, and the molecular formula is C. 25 H 22 N6O2, chemical formula as shown below:

[0101] .

[0102] In some optional embodiments, the U73122 and sotrastolin can be obtained by any means known in the art, such as by purchasing through commercial channels, for example, U73122 purchased from MCE, catalog number HY-13419, and sotrastolin purchased from MCE, catalog number HY-10343.

[0103] In some embodiments, the molar ratio of the MEK inhibitor and the PLCβ / PKC signaling pathway inhibitor is (1~10). 4 ):(1~10 12 ), preferably (1~10 3 ):(1~10 11 For example, 1:1, 1:10 0.7 1:10, 1:10 1.7 1:10 2 1:10 2.7 1:10 3 1:10 3.7 1:10 4 1:10 4.7 1:10 5 1:10 5.7 1:10 6 1:10 6.7 1:10 7 1:10 7.7 1:10 8 1:10 8.7 1:10 9 1:10 9.7 1:10 10 1:10 10.7 1:10 11 1:10 11.7 1:10 12 2:1, 2:10 0.7 2:10, 2:10 1.7 2:10 2 2:10 2.7 2:10 3 2:10 3.7 2:10 4 2:10 4.7 2:10 5 2:10 5.7 2:10 6 2:10 6.7 2:10 7 2:10 7.7 2:10 8 2:10 8.7 2:10 9 2:109.7 、2:10 10 、2:10 11 、2:10 12 、3:1、3:10 0.7 、3:10、3:10 1.7 、3:10 2 、3:10 2.7 、3:10 3 、3:10 3.7 、3:10 4 、3:10 4.7 、3:10 5 、3:10 5.7 、3:10 6 、3:10 6.7 、3:10 7 、3:10 7.7 、3:10 8 、3:10 8.7 、3:10 9 、3:10 9.7 、3:10 10 、3:10 11 、3:10 12 、4:1、4:10 0.7 、4:10、4:10 1.7 、4:10 2 、4:10 2.7 、4:10 3 、4:10 3.7 、4:10 4 、4:10 4.7 、4:10 5 、4:10 5.7 、4:10 6 、4:10 6.7 、4:10 7 、4:10 7.7 、4:10 8 、4:10 8.7 、4:10 9 、4:10 9.7 、4:10 10 、4:10 11 、4:10 12 、5:1、5:10 0.7 、5:10、5:10 1.7 、5:10 2 、5:10 2.7 、5:10 3 、5:10 3.7 、5:10 4 、5:10 5.7 、5:104.7 、5:10 5 、5:10 6 、5:10 6.7 、5:10 7 、5:10 7.7 、5:10 8 、5:10 8.7 、5:10 9 、5:10 9.7 、5:10 10 、5:10 11 、5:10 12 、6:1、6:10 0.7 、6:10、6:10 1.7 、6:10 2 、6:10 2.7 、6:10 3 、6:10 3.7 、6:10 4 、6:10 4.7 、6:10 5 、6:10 5.7 、6:10 6 、6:10 6.7 、6:10 7 、6:10 7.7 、6:10 8 、6:10 8.7 、6:10 9 、6:10 9.7 、6:10 10 、6:10 11 、6:10 12 、7:1、7:10 0.7 、7:10、7:10 1.7 、7:10 2 、7:10 2.7 、7:10 3 、7:10 3.7 、7:10 4 、7:10 4.7 、7:10 5 、7:10 5.7 、7:10 6 、7:10 6.7 、7:10 7 、7:10 7.7 、7:10 8 、7:10 8.7 、7:10 9 、7:10 9.7 、7:10 10 、7:10 11 、7:10 12、8:1、8:10 0.7 、8:10、8:10 1.7 、8:10 2 、8:10 2.7 、8:10 3 、8:10 3.7 、8:10 4 、8:10 4.7 、8:10 5 、8:10 5.7 、8:10 6 、8:10 6.7 、8:10 7 、8:10 7.7 、8:10 8 、8:10 8.7 、8:10 9 、8:10 9.7 、8:10 10 、8:10 11 、8:10 12 、9:1、9:10 0.7 、9:10、9:10 1.7 、9:10 2 、9:10 2.7 、9:10 3 、9:10 3.7 、9:10 4 、9:10 4.7 、9:10 5 、9:10 5.7 、9:10 6 、9:10 6.7 、9:10 7 、9:10 7.7 、9:10 8 、9:10 8.7 、9:10 9 、9:10 9.7 、9:10 10 、9:10 11 、9:10 12 、10:1、10:10 0.7 、10:10、10:10 1.7 、10:10 2 、10:10 2.7 、10:10 3 、10:10 3.7 、10:10 4 、10:10 4.7 、10:10 5 、10:10 5.7 、10:10 6 、10:106.7 、10:10 7 、10:10 7.7 、10:10 8 、10:10 8.7 、10:10 9 、10:10 9.7 、10:10 10 、10:10 11 、10:10 12 、100:1、100:10、100:10 2 、100:10 3 、100:10 4 、100:10 5 、100:10 6 、100:10 7 、100:10 8 、100:10 9 、100:10 10 、100:10 11 、100:10 12 、10 3 :1、10 3 :10、10 3 :10 2 、10 3 :10 3 、10 3 :10 4 、10 3 :10 5 、10 3 :10 6 、10 3 :10 7 、10 3 :10 8 、10 3 :10 9 、10 3 :10 10 、10 3 :10 11 、10 3 :10 12 、10 4 :1、10 4 :10、10 4 :10 2 、10 4 :10 3 、10 4 :10 4 、10 4 :10 5 、10 4 :10 610 4 :10 7 10 4 :10 8 10 4 :10 9 10 4 :10 10 10 4 :10 11 10 4 :10 12 .

[0104] In some embodiments, the MEK inhibitor is present at a dose not less than 1 fM, not less than 10 fM, not less than 20 fM, not less than 30 fM, not less than 40 fM, not less than 50 fM, not less than 60 fM, not less than 70 fM, not less than 80 fM, not less than 90 fM, not less than 1 nM, not less than 2 nM, not less than 3 nM, not less than 4 nM, not less than 5 nM, not less than 6 nM, not less than 7 nM, not less than 8 nM, not less than 7 nM, not less than 10 nM, not less than 20 nM, not less than 30 nM, not less than 40 nM, not less than 50 nM, not less than 60 nM, not less than 70 nM, not less than 80 nM, 90 nM, or 100 nM. nM, not less than 1μM, not less than 2μM, not less than 3μM, not less than 4μM, not less than 5μM, not less than 6μM, not less than 7μM, not less than 8μM, not less than 9μM, not less than 10μM, not less than 20μM, not less than 30μM, not less than 40μM, not less than 50μM, not less than 60μM, not less than 70μM, not less than 80μM, not less than 90μM, not less than 100μM.

[0105] (Pharmaceutical composition)

[0106] In some optional embodiments of the invention, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers; further, the pharmaceutically acceptable carriers include one or more combinations of solvents, solubilizers, cosolvents, emulsifiers, flavoring agents, odorants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants, and preservatives.

[0107] The combined drug of this invention is not limited in dosage form, as long as it can effectively deliver the active ingredient into the body, including: injections, tablets, film-coated tablets, enteric-coated tablets, capsules, lozenges, granules, powders, ointments, pills, suspensions, pills, powders, sprays, drops, suppositories, creams, patches, etc.; preferred dosage forms include: liquid injections, powder injections, tablets for injection, etc.; injection methods include, but are not limited to, the original drug form, drug-loaded nanoparticles, etc.; injection sites include, but are not limited to, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and spinal injection, etc.

[0108] The combined drug of the present invention can also be formulated with acceptable excipients to create a pharmaceutical preparation for the prevention and / or treatment of tumors. The pharmaceutical preparation can be administered to patients via various routes of administration, including but not limited to oral, transdermal, intramuscular, subcutaneous, and intravenous injection.

[0109] The compositions described herein may be in forms suitable for oral administration, such as solid dosage forms like tablets, capsules, lozenges, or granules; liquid dosage forms like emulsions, solutions, or suspensions; for parenteral administration (including intravenous, subcutaneous, intramuscular, intravascular, or infusion), such as as sterile solutions, suspensions, or emulsions; or for topical administration, such as as ointments, creams, gels, or lotions.

[0110] Compositions for oral administration may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, capsules, emulsions, syrups, or elixirs. Orally administered compositions may contain one or more optional agents, such as sweeteners (fructose, aspartame, or saccharin); flavoring agents (e.g., peppermint, wintergreen oil, or cherry); coloring agents; and preservatives, to provide a pharmaceutically palatable formulation. Selective permeation membranes surrounding permeation-driven compounds are also suitable for the oral administration of compounds (CDK inhibitors, and / or anticancer agents selected from BRAF inhibitors and / or MEK inhibitors) contained in pharmaceutical compositions according to the invention. Compositions suitable for oral administration may include standard mediators such as mannitol, lactose, starch, corn starch, magnesium stearate, talc, sodium saccharin, cellulose, magnesium carbonate, etc. Such mediators are preferably pharmaceutical grade.

[0111] For ointments and creams, the active ingredients (CDK inhibitors and / or anticancer agents selected from BRAF inhibitors and / or MEK inhibitors) are formulated in an oil-in-water or water-in-oil matrix.

[0112] For intramuscular, intraperitoneal, subcutaneous, and intravenous administration, a sterile solution of the active ingredient (CDK inhibitor and / or an anticancer agent selected from BRAF inhibitors and / or MEK inhibitors) is typically used, and the pH of the solution should be appropriately adjusted and buffered.

[0113] Furthermore, the action of compounds contained in a pharmaceutical composition (i.e., CDK inhibitors and / or anticancer agents selected from BRAF inhibitors and / or MEK inhibitors) can be delayed or prolonged through appropriate formulation. For example, slowly soluble pellets of the compound can be prepared and incorporated into tablets or capsules. This technique can be improved by preparing several pellets with different dissolution rates and filling capsules with a mixture of pellets. Tablets or capsules can be coated with a film that resists dissolution for a predictable period of time. Even parenteral formulations can be made into long-acting formulations by dissolving or suspending the compound in an oily or emulsified carrier that allows it to disperse slowly in serum.

[0114] In a second aspect of the invention, a pharmaceutical composition is provided comprising a MEK inhibitor and a PLCβ / PKC signaling pathway inhibitor.

[0115] In some implementations, the MEK inhibitor includes trametinib as described above.

[0116] In some implementations, the PLCβ / PKC signaling pathway inhibitors include U73122 and sotrastaurin, as described above.

[0117] In some embodiments, the molar ratio of the MEK inhibitor and the PLCβ / PKC signaling pathway inhibitor is (1~10). 4 ):(1~10 12 ), preferably (1~10 3 ):(1~10 11 For example, 1:1, 1:10 0.7 1:10, 1:10 1.7 1:10 2 1:10 2.7 1:10 3 1:10 3.7 1:10 4 1:10 4.7 1:10 5 1:10 5.7 1:10 6 1:10 6.7 1:10 7 1:10 7.7 1:10 8 1:10 8.7 1:10 9 1:10 9.7 1:10 10 1:10 10.7 1:10 11 1:10 11.7 1:1012 、2:1、2:10 0.7 、2:10、2:10 1.7 、2:10 2 、2:10 2.7 、2:10 3 、2:10 3.7 、2:10 4 、2:10 4.7 、2:10 5 、2:10 5.7 、2:10 6 、2:10 6.7 、2:10 7 、2:10 7.7 、2:10 8 、2:10 8.7 、2:10 9 、2:10 9.7 、2:10 10 、2:10 11 、2:10 12 、3:1、3:10 0.7 、3:10、3:10 1.7 、3:10 2 、3:10 2.7 、3:10 3 、3:10 3.7 、3:10 4 、3:10 4.7 、3:10 5 、3:10 5.7 、3:10 6 、3:10 6.7 、3:10 7 、3:10 7.7 、3:10 8 、3:10 8.7 、3:10 9 、3:10 9.7 、3:10 10 、3:10 11 、3:10 12 、4:1、4:10 0.7 、4:10、4:10 1.7 、4:10 2 、4:10 2.7 、4:10 3 、4:10 3.7 、4:10 4 、4:10 4.7 、4:10 5 、4:10 5.7 、4:10 6 、4:106.7 、4:10 7 、4:10 7.7 、4:10 8 、4:10 8.7 、4:10 9 、4:10 9.7 、4:10 10 、4:10 11 、4:10 12 、5:1、5:10 0.7 、5:10、5:10 1.7 、5:10 2 、5:10 2.7 、5:10 3 、5:10 3.7 、5:10 4 、5:10 5.7 、5:10 4.7 、5:10 5 、5:10 6 、5:10 6.7 、5:10 7 、5:10 7.7 、5:10 8 、5:10 8.7 、5:10 9 、5:10 9.7 、5:10 10 、5:10 11 、5:10 12 、6:1、6:10 0.7 、6:10、6:10 1.7 、6:10 2 、6:10 2.7 、6:10 3 、6:10 3.7 、6:10 4 、6:10 4.7 、6:10 5 、6:10 5.7 、6:10 6 、6:10 6.7 、6:10 7 、6:10 7.7 、6:10 8 、6:10 8.7 、6:10 9 、6:10 9.7 、6:10 10 、6:10 11 、6:10 12 、7:1、7:10 0.7 、7:10、7:10 1.7 、7:102 、7:10 2.7 、7:10 3 、7:10 3.7 、7:10 4 、7:10 4.7 、7:10 5 、7:10 5.7 、7:10 6 、7:10 6.7 、7:10 7 、7:10 7.7 、7:10 8 、7:10 8.7 、7:10 9 、7:10 9.7 、7:10 10 、7:10 11 、7:10 12 、8:1、8:10 0.7 、8:10、8:10 1.7 、8:10 2 、8:10 2.7 、8:10 3 、8:10 3.7 、8:10 4 、8:10 4.7 、8:10 5 、8:10 5.7 、8:10 6 、8:10 6.7 、8:10 7 、8:10 7.7 、8:10 8 、8:10 8.7 、8:10 9 、8:10 9.7 、8:10 10 、8:10 11 、8:10 12 、9:1、9:10 0.7 、9:10、9:10 1.7 、9:10 2 、9:10 2.7 、9:10 3 、9:10 3.7 、9:10 4 、9:10 4.7 、9:10 5 、9:10 5.7 、9:10 6 、9:10 6.7 、9:10 7 、9:10 7.7 、9:10 8、9:10 8.7 、9:10 9 、9:10 9.7 、9:10 10 、9:10 11 、9:10 12 、10:1、10:10 0.7 、10:10、10:10 1.7 、10:10 2 、10:10 2.7 、10:10 3 、10:10 3.7 、10:10 4 、10:10 4.7 、10:10 5 、10:10 5.7 、10:10 6 、10:10 6.7 、10:10 7 、10:10 7.7 、10:10 8 、10:10 8.7 、10:10 9 、10:10 9.7 、10:10 10 、10:10 11 、10:10 12 、100:1、100:10、100:10 2 、100:10 3 、100:10 4 、100:10 5 、100:10 6 、100:10 7 、100:10 8 、100:10 9 、100:10 10 、100:10 11 、100:10 12 、10 3 :1、10 3 :10、10 3 :10 2 、10 3 :10 3 、10 3 :10 4 、10 3 :10 5 、10 3 :10 6 、10 3 :10 7 、10 3 :108 10 3 :10 9 10 3 :10 10 10 3 :10 11 10 3 :10 12 10 4 1, 10 4 10, 10 4 :10 2 10 4 :10 3 10 4 :10 4 10 4 :10 5 10 4 :10 6 10 4 :10 7 10 4 :10 8 10 4 :10 9 10 4 :10 10 10 4 :10 11 10 4 :10 12 .

[0118] In some embodiments, the MEK inhibitor is present at a dose not less than 1 fM, not less than 10 fM, not less than 20 fM, not less than 30 fM, not less than 40 fM, not less than 50 fM, not less than 60 fM, not less than 70 fM, not less than 80 fM, not less than 90 fM, not less than 1 nM, not less than 2 nM, not less than 3 nM, not less than 4 nM, not less than 5 nM, not less than 6 nM, not less than 7 nM, not less than 8 nM, not less than 7 nM, not less than 10 nM, not less than 20 nM, not less than 30 nM, not less than 40 nM, not less than 50 nM, not less than 60 nM, not less than 70 nM, not less than 80 nM, 90 nM, or 100 nM. nM, not less than 1μM, not less than 2μM, not less than 3μM, not less than 4μM, not less than 5μM, not less than 6μM, not less than 7μM, not less than 8μM, not less than 9μM, not less than 10μM, not less than 20μM, not less than 30μM, not less than 40μM, not less than 50μM, not less than 60μM, not less than 70μM, not less than 80μM, not less than 90μM, not less than 100μM.

[0119] The pharmaceutical composition further includes one or more pharmaceutically acceptable carriers; further, the pharmaceutically acceptable carriers include one or more combinations of solvents, solubilizers, cosolvents, emulsifiers, flavoring agents, odor-correcting agents, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants, and preservatives.

[0120] The combined drug of this invention is not limited in dosage form, as long as it can effectively deliver the active ingredient into the body, including: injections, tablets, film-coated tablets, enteric-coated tablets, capsules, lozenges, granules, powders, ointments, pills, suspensions, pills, powders, sprays, drops, suppositories, creams, patches, etc.; preferred dosage forms include: liquid injections, powder injections, tablets for injection, etc.; injection methods include, but are not limited to, the original drug form, drug-loaded nanoparticles, etc.; injection sites include, but are not limited to, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and spinal injection, etc.

[0121] The combined drug of the present invention can also be formulated with acceptable excipients to create a pharmaceutical preparation for the prevention and / or treatment of tumors. The pharmaceutical preparation can be administered to patients via various routes of administration, including but not limited to oral, transdermal, intramuscular, subcutaneous, and intravenous injection.

[0122] The compositions described herein may be in forms suitable for oral administration, such as solid dosage forms like tablets, capsules, lozenges, or granules; liquid dosage forms like emulsions, solutions, or suspensions; for parenteral administration (including intravenous, subcutaneous, intramuscular, intravascular, or infusion), such as as sterile solutions, suspensions, or emulsions; or for topical administration, such as as ointments, creams, gels, or lotions.

[0123] Compositions for oral administration may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, capsules, emulsions, syrups, or elixirs. Orally administered compositions may contain one or more optional agents, such as sweeteners (fructose, aspartame, or saccharin); flavoring agents (e.g., peppermint, wintergreen oil, or cherry); coloring agents; and preservatives, to provide a pharmaceutically palatable formulation. Selective permeation membranes surrounding permeation-driven compounds are also suitable for the oral administration of compounds (CDK inhibitors, and / or anticancer agents selected from BRAF inhibitors and / or MEK inhibitors) contained in pharmaceutical compositions according to the invention. Compositions suitable for oral administration may include standard mediators such as mannitol, lactose, starch, corn starch, magnesium stearate, talc, sodium saccharin, cellulose, magnesium carbonate, etc. Such mediators are preferably pharmaceutical grade.

[0124] For ointments and creams, the active ingredients (CDK inhibitors and / or anticancer agents selected from BRAF inhibitors and / or MEK inhibitors) are formulated in an oil-in-water or water-in-oil matrix.

[0125] For intramuscular, intraperitoneal, subcutaneous, and intravenous administration, a sterile solution of the active ingredient (CDK inhibitor and / or an anticancer agent selected from BRAF inhibitors and / or MEK inhibitors) is typically used, and the pH of the solution should be appropriately adjusted and buffered.

[0126] Furthermore, the action of compounds contained in a pharmaceutical composition (i.e., CDK inhibitors and / or anticancer agents selected from BRAF inhibitors and / or MEK inhibitors) can be delayed or prolonged through appropriate formulation. For example, slowly soluble pellets of the compound can be prepared and incorporated into tablets or capsules. This technique can be improved by preparing several pellets with different dissolution rates and filling capsules with a mixture of pellets. Tablets or capsules can be coated with a film that resists dissolution for a predictable period of time. Even parenteral formulations can be made into long-acting formulations by dissolving or suspending the compound in an oily or emulsified carrier that allows it to disperse slowly in serum.

[0127] Example

[0128] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0129] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. The following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0130] 1. Materials and Methods:

[0131] Cultivation and Drug Treatment Methods of Caenorhabditis elegans Strains

[0132] The *Caenorhabditis elegans* used in this invention were conventionally reared at 20 °C on nematode growth medium (NGM) plates inoculated with *Escherichia coli* OP50. The NGM medium was prepared as follows: 3.0 g NaCl, 17.0 g Bacto-agar, and 2.5 g Bacto-peptone were weighed and diluted to 1 L with deionized water. The medium was then autoclaved at 121 °C for 30 min, cooled to approximately 60 °C, and then 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, and 25 mL of 1 M KPO4 (pH = 6, with 108.3 g KH2PO4 and 35.6 g K2HPO4 added, and diluted to 1 L with deionized water) were added sequentially. The mixture was thoroughly mixed and dispensed into sterile plastic culture dishes. The medium was allowed to solidify before use.

[0133] During the drug treatment process, after cooling the above NGM medium to about 60 °C, add trametinib (C16249920, MACKLIN), U73122 (HY-13419, MCE), sotrastaurin (HY-10343, MCE) or an equal volume of DMSO solvent control (196055, MP Biomedicals) as required for the experiment, mix thoroughly, and then pour the drug treatment medium plates.

[0134] The hermaphroditic Caenorhabditis elegans (P0 generation) on day 1 of adulthood were transferred to the above-mentioned drug-treated culture medium plates for drug exposure experiments. The hermaphroditic adults were also used for subsequent in vivo imaging experiments.

[0135] The wild-type Caenorhabditis elegans strain Bristol N2 used in this invention was derived from the Caenorhabditis Genetics Center (CGC). The lin-45 (V627E) mutant (with the sequence shown in NCBI accession number: Q07292, where position V is mutated to E at position 627) was constructed by Fujian Shangyuan Biotechnology Co., Ltd. using CRISPR / Cas9 gene editing technology according to the requirements of this invention.

[0136] 2. Microscopic imaging

[0137] Adult hermaphroditic *C. elegans* samples were selected and anesthetized with 1 μM levamisole in M9 buffer. The anesthetized nematodes were fixed on 3% agarose gels and immediately subjected to microscopic imaging at room temperature. Bright-field imaging was performed using a Zeiss Axio Observer system.

[0138] 3. Forward genetic screening of Caenorhabditis elegans

[0139] The inventors used forward genetic screening to isolate the genetic repressor of the lin-45 (V627E) mutant. Lin-45 (V627E) mutant (P0) nematodes were synchronized to late L4 stage, collected in 4 mL M9 buffer, and mutagenized at room temperature for 4 hours with 50 mM ethyl methanesulfonate (EMS, Sigma M0880). They were then washed three times with M9, transferred to NGM plates, and cultured under standard conditions. After 20 hours, the adults were bleached. F1 generation eggs were distributed onto approximately 800 9 cm NGM plates, with 50-100 eggs per plate. F2 generation adults were screened from each plate. Mutants exhibiting normal vulva were cultured separately, and their progeny were further observed. The inventors identified egl-30 (R25C) located on chromosome I through whole-genome sequencing.

[0140] 4. Determination of the reproductive capacity of Caenorhabditis elegans

[0141] Synchronized L4-stage nematodes were individually transferred to NGM plates inoculated with E. coli OP50. They were transferred to fresh NGM plates every 24 hours until oviposition ceased. The number of offspring in the L3 / L4 stage was counted. The experiment was independently replicated at least three times, and total reproduction was statistically analyzed using GraphPad Prism 10.

[0142] 5. RNA sequencing of Caenorhabditis elegans

[0143] Synchronized nematodes were cultured on NGM plates inoculated with OP50. After 24 hours of treatment with the drug alone or in combination, adult nematodes on day 1 were collected and lysed using TRIzol reagent (Invitrogen). Total RNA was extracted according to the reagent instructions, and SUPERase·In™ RNase inhibitors (Invitrogen) were used at each step to prevent degradation. RNA concentration was determined using the Qubit RNA High Sensitivity Detection Kit (Invitrogen), and RNA quality was assessed using an Agilent 2100 bioanalyzer. Samples with an RNA integrity index (RIN) > 6.0 were used for library construction. 50-500 ng of total RNA was used to prepare libraries using the KAPA RNAHyperPrep Kit (KAPA Biosystems). The libraries were quality controlled and quantified using Agilent 2100 and Qubit, and sequenced on the Illumina HiSeq platform to generate 150 bp paired-end sequencing data.

[0144] 6. Cell Culture

[0145] The human melanoma cell line A375 was purchased from ATCC (ATCC number: CRL-1619). TM The cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Yeasen) and 1% penicillin-streptomycin (Yeasen) at 37 ℃ and 5% CO2. A375-TR (trametinib-resistant strain) was established by gradually increasing the concentration of trametinib over a long period of time, and single clones were screened by limiting dilution.

[0146] 7. Cell viability assay

[0147] Cell viability was determined using the Cell-ATP Viability Assay Kit (HY-K0302; MedChemExpress). Cells were incubated in complete culture medium with the drug, alone or in combination, for 72 hours, with at least three independent replicates. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 10 software (GraphPad Software, San Diego, CA, USA).

[0148] 8. Cloning experiment

[0149] Cells were seeded at a low density (100 cells / well) in six-well plates and cultured for 10 days under drug treatment. Cells were then fixed with methanol and stained with 1× crystal violet. Colony count and average area were analyzed using ImageJ.

[0150] 9. Protein Blotting Analysis Methods

[0151] In this embodiment of the invention, cells were washed three times with cold PBS solution to remove culture medium and extracellular residues, and then lysed with RIPA lysis buffer (catalog number C0121, Beyotime) containing a total protease inhibitor and a phosphatase inhibitor (phosSTOP, Roche). The resulting lysate was centrifuged at 14,000 × g for 15 min at 4 °C to remove insoluble components, and the supernatant was collected and stored at -80 °C until subsequent analysis.

[0152] The obtained protein samples were added to 5× SDS loading buffer (catalog number 20315ES20, Yeasen) and denatured by heating at 95 °C for 5 min. The denatured protein samples were then separated by electrophoresis on a FastPAGE precast gel (catalog number TSP024-15, TSINGKE) and subsequently transferred to a PVDF membrane (catalog number B36011, ESAYBIO). The membrane was blocked with 5% bovine serum albumin (BSA) solution and incubated overnight with primary antibody at 4 °C.

[0153] After incubation, the samples were washed three times with 10× TBST buffer containing 5‰ Tween-20 (catalog number T1081, Solarbio), and then incubated with secondary antibody for 1 h on a shaker. Protein band development was obtained by chemiluminescence detection using the SuperSignal West Dura (Thermo Fisher Scientific, catalog number 34076).

[0154] The primary and secondary antibodies used in this invention include, but are not limited to: α-Tubulin (DM1A) Mouse mAb (catalog number 3873T, CST), anti-mouse IgG HRP conjugated secondary antibody (catalog number 7076P2, CST), anti-rabbit IgG HRP conjugated secondary antibody (catalog number 7074P2, CST), p44 / 42 MAPK (Erk1 / 2) (137F5) Rabbit mAb (catalog number 4695T, CST), and phosphorylated p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (D13.14.4E) XP Rabbit mAb (catalog number 4370T, CST).

[0155] 10. Mouse xenotransplantation and in vivo drug treatment methods

[0156] In this embodiment of the invention, all animal experiments were conducted under conditions approved by the animal ethics committee. BALB / c nude mice were housed in a temperature-controlled environment at approximately 21°C, with a light / dark cycle of 12 hours of light / 12 hours of darkness, and were allowed free access to food and water.

[0157] Will contain 5×10 6A suspension of A375 BRAF (V600E) melanoma cells was prepared in PBS buffer and subcutaneously injected into the bilateral inguinal regions of 5-week-old immunodeficient BALB / c nude female mice, with at least 6 animals per group. Tumor formation was monitored twice weekly, and the major and minor axes of the tumor were measured using calipers. The tumor volume was determined according to the modified ellipsoid formula (tumor volume = 1 / 2 × length × width). 2 Calculate the tumor volume. When the tumor volume reaches approximately 0.5 cm... 3 Animals were randomly divided into different treatment groups, including no treatment group, MEK inhibitor trametinib monotherapy group, PLCβ inhibitor U73122 or PKC inhibitor sotrastaurin monotherapy group, and trametinib combined with U73122 / sotrastaurin group.

[0158] In drug preparation, sotrastaurin (catalog number HY-10343, MCE) or trametinib (catalog number C16249920, MACKLIN) powder was dissolved in a mixed solvent containing 5% DMSO (catalog number 196055, MP Biomedicals), 40% PEG300 (catalog number HY-Y0873, MCE), 5% Tween-80 (catalog number HY-Y1891, MCE), and 50% sterile physiological saline (volume percentage) to prepare working solutions of 80 mg / kg sotrastaurin and 0.2 mg / kg trametinib, respectively. The solutions were thoroughly mixed and used as administration solutions. The drugs were administered orally to each animal at a dose of 100 μL, three times a week. U73122 (product number HY-13419, MCE) was ground into powder and suspended in sterile saline to prepare a homogeneous suspension, which was then used to prepare a working solution of 50 mg / kg U73122. The resulting suspension was administered to each animal via intraperitoneal injection at a dose of 100 μL, three times a week. The control group animals were given the corresponding solvent or sterile saline, with the same route of administration and frequency as the corresponding treatment group.

[0159] Example 1: Establishing an in vivo model of acquired trametinib resistance using the Caenorhabditis elegans lin-45 (V627E) mutant model (functionally similar to human BRAF V600E).

[0160] Through preliminary research, the inventors discovered that the Raf family serine / threonine protein kinase lin-45 (V627E) mutant model in *Caenorhabditis elegans* is functionally similar to human BRAF V600E. Figure 1 The sequences of A, nematode LIN-45 (NCBI accession number: Q07292) and human BRAF (NCBI accession number: P15056) were compared, and the lin-45V627 corresponding to BRAF V600 was mutated to E).

[0161] To establish a nematode model that mimics BRAF(V600E)-driven tumor-acquired trametinib resistance, this study exposed adult day 1 wild-type (WT) and lin-45(V627E) mutant hermaphroditic nematodes (P0 generation) to trametinib, an FDA-approved MEK inhibitor, at concentrations ranging from 1 to 100 μM (Figure 1, B). All lin-45(V627E) mutants (n = 300) exhibited the protruding vulva (Pvl) phenotype (Figure 1, C). Trametinib treatment effectively suppressed the Pvl phenotype in 90% of the lin-45(V627E) P0 generation mutants (N = 300; Figure 1, C), indicating that trametinib treatment effectively inhibited the MAPK signaling pathway.

[0162] To assess the effect of trametinib on fertility, this study quantified the fertility of F1 offspring produced by hermaphrodites in the P0 generation. Compared with the DMSO control group, the number of chicks born in wild-type F1 offspring treated with 1 μM trametinib was significantly reduced (250 ± 49 vs. 23 ± 20, p < 0.0001; Figure 1, D). Wild-type F1 offspring treated with 10 μM or 100 μM trametinib were completely sterile (Figure 1, D), indicating that the MEK / ERK pathway was completely suppressed.

[0163] DMSO-treated lin-45(V627E) F1 progeny maintained normal brood numbers, comparable to DMSO-treated wild-type animals (241 ± 59 vs. 250 ± 49, p = 0.54; Figure 1, D). Notably, 1 μM trametinib had no significant effect on brood numbers in lin-45(V627E) F1 progeny compared to DMSO-treated mutants (236 ± 53 vs. 241 ± 59, p > 0.99; Figure 1, D), indicating that this concentration was insufficient to inhibit persistent MAPK activity in this mutant background. Conversely, 10 μM trametinib almost completely inhibited reproductive capacity (4 ± 7 vs. 241 ± 59, p < 0.0001; Figure 1, D), demonstrating that this concentration achieved effective pathway inhibition. Surprisingly, treatment with 100 μM trametinib partially restored the reproductive capacity of F1, reaching 63% of that in the DMSO control group (153 ± 61 vs. 241 ± 59, p = 0.0001; D in Figure 1), suggesting that the lin-45(V627E) mutant animals have acquired resistance to high-dose MEK inhibition.

[0164] This dose-dependent resistance phenotype may be achieved through MAPK pathway reactivation or activation of alternative signaling pathways. This phenomenon is similar to the mechanism by which BRAF-mutant melanoma cells acquire resistance after continuous exposure to trametinib in clinical practice. 1, 2 In summary, the lin-45 (V627E) nematode can serve as a genetically controllable in vivo model for elucidating the molecular mechanisms of acquired trametinib resistance.

[0165] Example 2: Transcriptome analysis and genetic screening revealed that the Gαq-PLCβ-PKC signaling axis is a key pathway mediating high-dose trametinib resistance.

[0166] To elucidate the molecular mechanism of acquired trametinib resistance in the lin-45(V627E) mutant, this example performed transcriptome analysis on trametinib-sensitive and resistant lin-45(V627E) mutants. Specifically, this example compared the F1 generation progeny of hermaphroditic lin-45(V627E) nematodes treated with 10 μM trametinib (sensitive) and 100 μM trametinib (resistant). Principal component analysis (PCA) of RNA-seq showed significant segregation between the two populations, indicating different gene expression patterns. Figure 2(A) Differential expression analysis identified 443 genes that were significantly upregulated in trametinib-resistant animals (fold change > 2 and corrected P-value (Padj) < 0.05; Figure 2, B). Gene ontology (GO) analysis of these genes revealed significant upregulation of genes related to the G protein signaling pathway, including egl-30 (fold change = 2.3, Padj = 0.008; Figure 2, B-D). This gene encodes the Gαq subunit of nematodes and is homologous to human GNAQ / GNA11 in the heterotrimeric G protein complex.

[0167] Supporting this finding, a positive genetic suppressor screening identified a recessive allele, egl-30(R25C), which suppressed the Pvl phenotype in the lin-45(V627E) mutant (Figure 3A). To assess the functional role of egl-30 in trametinib resistance, this example analyzed the brood number of chicks from the lin-45(V627E); egl-30(R25C) double mutant. Under DMSO treatment, the double mutant exhibited severe fertility defects (Figure 3B), producing significantly fewer offspring than the wild type (13 ± 7 vs. 250 ± 49) or the lin-45(V627E) single mutant (13 ± 7 vs. 241 ± 59, p < 0.0001). Similar to the lin-45(V627E) single mutant, the double mutant remained fertile at 1 μM trametinib but became infertile at 10 μM (Figure 3, B). Notably, while 100 μM trametinib partially restored fertility in the lin-45(V627E) single mutant (reaching 63% of the DMSO control), this rescue effect was severely impaired in the double mutant, with chick numbers decreasing to 12% of the DMSO control (2 ± 2 vs. 13 ± 7; Figure 3, B). These findings suggest that reduced EGL-30 / Gαq activity attenuates the resistance phenotype and supports the crucial role of the Gαq signaling pathway in mediating high-dose trametinib resistance.

[0168] To further investigate how EGL-30 / Gαq mediates drug resistance, this study investigates its classic downstream signaling pathway. Gαq typically activates phospholipase Cβ (PLCβ) to hydrolyze PIP2 into IP3 and DAG, thereby activating protein kinase C (PKC). PKC can phosphorylate downstream targets, including C-Raf, thereby activating the MAPK pathway. 3This invention assumes that the EGL-30 / Gαq-PLCβ-PKC axis enables the lin-45(V627E) mutant to maintain MAPK activity and proliferative capacity even under high concentrations of trametinib.

[0169] To verify this hypothesis, this invention treated adult lin-45 (V627E) nematodes with the PLCβ inhibitor U73122 or the PKC inhibitor Sotrastaurin, alone or in combination with trametinib. Sotrastaurin is currently in Phase II clinical trials and exhibits minimal off-target activity even at elevated concentrations. 4 Compared to the DMSO control, treatment with 50 μM U73122 or 50 μM Sotrastaurin alone had minimal effect on fertility (U73122: 240 ± 32 vs. 241 ± 59, p = 0.33; Sotrastaurin: 222 ± 40 vs. 241 ± 59, p = 0.01; Figure 3, C). In contrast, combined use of 100 μM trametinib and either inhibitor resulted in complete infertility (Figure 3, C), mimicking the genetically repressive phenotype of egl-30. In summary, these findings establish the EGL-30 / Gαq-PLCβ-PKC signaling axis as a key mediator of adaptive MEK inhibitory resistance and highlight its potential therapeutic target for MAPK-driven malignancies.

[0170] Example 3: In human A375 melanoma cells, inhibition of the Gαq-PLCβ-PKC pathway was demonstrated to significantly enhance the antitumor effect of trametinib.

[0171] To assess whether the drug resistance mechanisms found in *C. elegans* are conserved in human cancer cells, this invention further investigated whether the PLCβ inhibitor U73122 and the PKC inhibitor Sotrastaurin could enhance the sensitivity of BRAF(V600E) mutant melanoma cells to the MEK inhibitor trametinib. In this embodiment, the human melanoma cell line A375 carrying the BRAF(V600E) mutation was used to analyze the effect of combined treatment with U73122 or Sotrastaurin and trametinib on A375 cell proliferation. Through multi-dose combination experiments, this embodiment observed significant synergistic inhibitory effects of trametinib combined with U73122 or Sotrastaurin at multiple concentration gradients (Figures 4A-4B). Notably, in most of these dose ratios, the calculated combination index (CI) was less than 0.3, indicating a strong synergistic effect of the two drugs used in combination.

[0172] To further verify whether the aforementioned synergistic effect is achieved by enhancing the inhibition of the MAPK / ERK signaling pathway, this embodiment examined the expression level of phosphorylated ERK (pERK), a key downstream signaling molecule. pERK is a marker of MAPK / ERK pathway activity, and its expression level typically reflects the activation state of this pathway. Western blot analysis showed that treatment with U73122 or Sotrastaurin alone had no significant effect on pERK levels, while treatment with trametinib alone significantly inhibited pERK expression (Figure 4C). Furthermore, compared with treatment with 1 nM trametinib alone, treatment with 5 μM U73122 or 10 μM Sotrastaurin further reduced pERK expression levels (Figure 4C-Figure 4D), suggesting that these two types of inhibitors synergistically enhance the antitumor effect of trametinib by strengthening the inhibition of the MAPK / ERK pathway.

[0173] Example 4: U73122 or sotrastaurin can reverse acquired trametinib resistance in melanoma.

[0174] To determine whether PLCβ / PKC activation is necessary for maintaining acquired trametinib resistance, this invention established a polyclonal trametinib-resistant A375 cell line (A375-TR) (Figure 5, A) by prolonged exposure to escalating concentrations of trametinib. As expected, the parental A375 cells were highly sensitive to trametinib (IC50-100%). 50= 0.5 nM), while the IC50 of A375-TR cells is 0.5 nM. 50 Significantly elevated (260 nM, p = 0.01) (Fig. 5B-5C). Treatment with U73122 or sotrastaurin alone effectively reduced the viability of A375-TR cells. Combined treatment with trametinib further enhanced the antiproliferative effects of both inhibitors, suggesting a synergistic effect (Fig. 5D-5E). These findings indicate that the sustained PLCβ / PKC signaling pathway is crucial for maintaining the drug-resistant phenotype, and that pharmacologically blocking this pathway restores the sensitivity of trametinib to drug-resistant melanoma cells.

[0175] Example 5: Trametinib combined with a PLCβ / PKC signaling pathway inhibitor can inhibit tumor growth in vivo.

[0176] To evaluate the in vivo efficacy of combined MEK and PLCβ / PKC inhibition, this invention established an A375 xenograft tumor model in nude mice. When the tumor volume reached approximately 200-250 mm... 3 Forty-eight tumor-burdened mice were randomly divided into two groups (n = 24 mice per group). The first group received either solvent control, trametinib, U73122, or U73122 combined with trametinib (n = 6 mice per treatment); the second group received either solvent control, trametinib, sotrastaurin, or sotrastaurin combined with trametinib (n = 6 mice per treatment). In the solvent control group, tumor growth was rapid; however, trametinib, U73122, or sotrastaurin alone significantly delayed tumor progression. Notably, the combination therapy (U73122 + trametinib and sotrastaurin + trametinib) was significantly superior to either single-agent therapy in tumor suppression (Figures 6A-6F), indicating a synergistic antitumor effect in vivo. Trametinib, sotrastaurin, or the combination of both had no significant effect on mouse body weight, suggesting good tolerability (Figure 6H). In contrast, mice treated with U73122 or in combination with trametinib showed a mild but measurable decrease in body weight, suggesting that there may be some systemic toxicity at this dose (G in Figure 6).

[0177] References:

[0178] 1. Hartman, M.L., M. Sztiller-Sikorska, A. Gajos-Michniewicz, and M.Czyz. 2020. Dissecting Mechanisms of Melanoma Resistance to BRAF and MEKInhibitors Revealed Genetic and Non-Genetic Patient- and Drug-SpecificAlterations and Remarkable Phenotypic Plasticity. Cells. 9.

[0179] 2. Tripathi, R., Z. Liu, A. Jain, A. Lyon, C. Meeks, D. Richards, J.Liu, D. He, C. Wang, M. Nespi, A. Rymar, P. Wang, M. Wilson, and R. Plattner.2020. Combating acquired resistance to MAPK inhibitors in melanoma bytargeting Abl1 / 2-mediated reactivation of MEK / ERK / MYC signaling. Nat Commun.11:5463.

[0180] 3. Kolch, W., G. Heidecker, G. Kochs, R. Hummel, H. Vahidi, H.Mischak, G. Finkenzeller, D. Marme, and U.R. Rapp. 1993. Protein kinase Calpha activates RAF-1 by direct phosphorylation. Nature. 364:249-252.

[0181] 4. Klaeger, S., S. Heinzlmeir, M. Wilhelm, H. Polzer, B. Vick, PAKoenig, M. Reinecke, B. Ruprecht, S. Petzoldt, C. Meng, J. Zecha, K. Reiter,H. Qiao, D. Helm, H. Koch, M. Schoof, G. Canevari, E. Casale, SR Depaolini,A. Feuchtinger, Z. Wu, T. Schmidt, L. Rueckert, W. Becker, J. Huenges, AKGarz, BO Gohlke, DP Zolg, G. Kayser, T. Vooder, R. Preissner, H. Hahne, N. Tonisson, K. Kramer, K. Gotze, F. Bassermann, J. Schlegl, HC Ehrlich, S.Aiche, A. Walch, PA Greif, S. Schneider, ER Felder, J. Ruland, G. Medard,I. Jeremias, K. Spiekermann, and B. Kuster. 2017. The target landscape of clinical kinase drugs. Science. 358.

[0182] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0183] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Use of PLCβ / PKC signaling pathway inhibitors in combination with MEK inhibitors in the preparation of medicaments or pharmaceutical compositions for the prevention and / or treatment of tumors; The tumor is a BRAF (V600E) mutant tumor; the BRAF (V600E) mutant tumor is melanoma; The MEK inhibitor is trametinib; the PLCβ / PKC signaling pathway inhibitor is U73122 or sotrastuxin.

2. The use according to claim 1, wherein, The drug or drug composition can reverse a subject's resistance to MEK inhibitors or increase a subject's sensitivity to MEK inhibitors.

3. The use according to claim 1, wherein, The molar ratio of the MEK inhibitor and the PLCβ / PKC signaling pathway inhibitor is (1~10). 4 ):(1~10 12 ).

4. The use according to claim 3, wherein, The molar ratio of the MEK inhibitor and the PLCβ / PKC signaling pathway inhibitor is (1~10). 3 ):(1~10 11 ).

5. The use according to claim 1, wherein, The drug or drug composition may also include one or more pharmaceutically acceptable carriers.

6. The use according to claim 1, wherein, The drug or drug composition may be administered orally, transdermally, intramuscularly, subcutaneously, or intravenously.

7. A pharmaceutical composition comprising a MEK inhibitor and a PLCβ / PKC signaling pathway inhibitor; wherein the MEK inhibitor is trametinib; and the PLCβ / PKC signaling pathway inhibitor is U73122 or sotrastuxin.

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