A pharmaceutical composition for treating tumor drug resistance and application thereof

Abstract

The application provides a kind of pharmaceutical composition and its application.The pharmaceutical composition includes aspirin, BRAF / MEK inhibitor.The pharmaceutical composition overcomes the effect of BRAF V600E / K mutant cancer drug resistance and can reduce the occurrence of fever AE, has a significant impact on the treatment of patients with advanced cancer, and is expected to obtain the effect of more than the sum of its parts.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical composition technology, and more particularly to a pharmaceutical composition for treating drug-resistant tumors and its application. Background Technology

[0002] Lung cancer is the leading cause of cancer incidence and mortality in China, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all lung cancer cases. V-Rafmurine sarcoma viral oncogene homolog B1 (BRAF) is a key molecule in the mitogen-activated protein kinase (MAPK) pathway. In NSCLC, the BRAF mutation rate ranges from 1.5% to 3.5%; BRAF V600 accounts for approximately 50% of all BRAF mutations, with the V600E mutation being the most common.

[0003] Patients with BRAF V600-mutant NSCLC have a poor prognosis and short overall survival (OS). Chemotherapy is a common treatment strategy for NSCLC patients, but the efficacy of chemotherapy and immunotherapy in BRAF V600-mutant NSCLC patients is not ideal. In addition, targeted therapies have been explored in patients with BRAF V600E-mutant advanced NSCLC. Dabrafenib, a potent and selective BRAF mutant kinase inhibitor, achieved an overall objective response rate (ORR) of 33% in the international multicenter, open-label phase II trial BRF113928. The median progression-free survival (PFS) assessed by investigators and IRC was 5.5 months, and the median OS was 15.4 and 12.7 months, respectively. In conclusion, for BRAF V600E-mutant advanced NSCLC, the efficacy of chemotherapy, immunotherapy, and BRAF inhibitor monotherapy is limited, and more effective treatment options need to be explored.

[0004] Recent studies have found that dual-target inhibition of BRAF and MEK can further enhance the inhibition of the MAPK signaling pathway, increase apoptosis, and delay the onset of drug resistance. In the BRF113928 trial, previously treated patients receiving combined treatment with the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib achieved an overall objective response rate (ORR) of up to 63.2%, a disease control rate (DCR) of 78.9%, and a median progression-free survival (PFS) of 9.7 months. Furthermore, whether used as first-line or later-line therapy, dabrafenib combined with trametinib demonstrates good efficacy in treating BRAF V600-mutant NSCLC patients, outperforming BRAF monotherapy. Currently, the latest NCCN and CSCO guidelines both recommend dabrafenib combined with trametinib as the preferred first-line treatment for BRAF V600E-mutant advanced NSCLC patients. Unfortunately, more than half of the patients treated with dabrafenib in combination with trametinib inevitably develop drug-related fever and must be treated with drugs such as nonsteroidal anti-inflammatory drugs to control it.

[0005] Meanwhile, the problem of targeted drug resistance is equally significant, severely limiting its clinical efficacy and becoming a bottleneck restricting the widespread clinical application of dabrafenib combined with trametinib. Therefore, solving the resistance problem of dabrafenib combined with trametinib is an urgent challenge. Studies have shown that the resistance mechanism of BRAF / MEK inhibitors is mainly mediated by the PI3K-AKT-mTOR and RAS-RAF-MEK pathways, such as alterations in cell cycle-related genes, activation of the PI3K-AKT pathway, and NRAS / KRAS mutations. Because the resistance mechanism after dabrafenib combined with trametinib is complex, and the opportunity for subsequent targeted therapies is low, there are no clear recommendations for subsequent treatment guidelines. Systemic therapies such as immunotherapy and chemotherapy are used, but the effects are not satisfactory and require further improvement.

[0006] In summary, among the currently known resistance mechanisms of dabrafenib combined with trametinib, no single resistance mechanism has a prominent incidence rate, and there is no effective clinical strategy to overcome resistance to dabrafenib combined with trametinib. Therefore, further exploration of the resistance mechanisms of dabrafenib combined with trametinib and its reversal strategies has significant scientific and clinical value. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a pharmaceutical composition for treating drug-resistant tumors and its application. It solves the problem of drug resistance in BRAF V600-mutant cancers existing in the prior art.

[0008] In a first aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of aspirin and a BRAF / MEK inhibitor.

[0009] In one embodiment of the present invention, the BRAF / MEK inhibitor includes at least one of dabrafenib, vemurafenib, encorafenib, trametinib, cobimetinib, and binimetinib.

[0010] In one embodiment of the present invention, the BRAF / MEK inhibitor is dabrafenib. In another embodiment of the present invention, the BRAF / MEK inhibitor is trametinib. In yet another embodiment of the present invention, the BRAF / MEK inhibitor is a combination of dabrafenib and trametinib.

[0011] In one embodiment of the present invention, the pharmaceutical composition includes aspirin and dabrafenib.

[0012] In one embodiment of the present invention, the pharmaceutical composition includes aspirin, dabrafenib, and trametinib.

[0013] In one embodiment of the present invention, the aspirin and BRAF / MEK inhibitor have a synergistic effect.

[0014] In one embodiment of the present invention, the aspirin and the BRAF / MEK inhibitor are administered sequentially or simultaneously. In one embodiment of the present invention, the aspirin is administered before the BRAF / MEK inhibitor is administered. In another embodiment of the present invention, the aspirin is administered after the BRAF / MEK inhibitor is administered.

[0015] In one embodiment of the present invention, aspirin and BRAF / MEK inhibitors are in their respective separate unit dosage forms. In some embodiments, aspirin and BRAF / MEK inhibitors are in the same unit dosage form.

[0016] In one embodiment of the present invention, the aspirin:BRAF / MEK inhibitor ratio, by mass, is (50-1000):(1-200). In another embodiment, the aspirin:BRAF inhibitor:MEK inhibitor ratio, by mass, is (50-1000):(5-200):(1-5). In yet another embodiment, the aspirin:BRAF inhibitor:MEK inhibitor ratio, by mass, is (80-900):(5-150):(1-3).

[0017] In one embodiment of the present invention, the effective dose of aspirin is 80-900 mg. In some embodiments, the aspirin is administered in doses of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 50 mg. Dosage of 0, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900 mg, or any range or value thereof.

[0018] In one embodiment of the present invention, the effective dose of the BRAF / MEK inhibitor is 1-200 mg. In some embodiments, the BRAF / MEK inhibitor is administered in the form of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 Dosage of 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 160, 165, 170, 175, 180, 190, 195, 200 mg, or any range or value thereof, which can be derived from these dosages.

[0019] Understandably, the appropriate effective dose can be determined based on the type of disease, the severity and progression of the disease, the individual's clinical condition, the individual's clinical history, and the response to treatment.

[0020] In one embodiment of the invention, the BRAF / MEK inhibitor and aspirin are administered at least once, two, or three times daily for several days. In some embodiments, the BRAF / MEK inhibitor and aspirin are administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times weekly for several weeks. In some embodiments, the BRAF / MEK inhibitor and aspirin are administered once daily for several days. In some embodiments, the BRAF / MEK inhibitor and aspirin are administered twice daily for several days.

[0021] In one embodiment of the present invention, the composition further includes other chemotherapeutic agents, immunotherapeutic agents, or combinations thereof.

[0022] In one embodiment of the invention, the composition is typically administered via any conventional route. This includes, but is not limited to, oral or intravenous administration. Alternatively, it can be administered orally, intradermally, subcutaneously, intramuscularly, intraperitoneally, or intranasally. Administration also includes in vitro or ex vivo administration. The composition and other therapeutic agents can be administered via the same route of administration or via different routes of administration.

[0023] In one embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient or carrier.

[0024] In one embodiment of the present invention, the pharmaceutical composition has a better effect on inhibiting tumor cell activity or proliferation compared with the administration of BRAF / MEK inhibitors alone.

[0025] In one embodiment of the present invention, the pharmaceutical composition exhibits better tumor cell killing activity compared to the administration of BRAF / MEK inhibitors alone.

[0026] In one embodiment of the present invention, the pharmaceutical composition has a better effect on promoting tumor cell apoptosis compared with the administration of BRAF / MEK inhibitors alone.

[0027] In a preferred embodiment of the present invention, the tumor cells are BRAF V600E / K mutant tumor cells.

[0028] In one embodiment of the present invention, the pharmaceutical composition has a better inhibitory effect on the activation of the PI3K / AKT and MAPK pathways compared with the administration of BRAF / MEK inhibitors alone.

[0029] In one embodiment of the present invention, compared with the administration of BRAF / MEK inhibitors alone, the pharmaceutical composition has a better effect on increasing the expression of apoptosis-related protein Bim and downregulating or inhibiting the expression of AXL, BXL-XL, and Caspase 9.

[0030] In one embodiment of the present invention, the pharmaceutical composition has a better effect of downregulating or inhibiting the expression of P-ERK and GPX4 compared with the administration of BRAF / MEK inhibitors alone.

[0031] In one embodiment of the present invention, the pharmaceutical composition can better improve the progression-free survival rate and overall survival rate of patients compared with the administration of BRAF / MEK inhibitors alone.

[0032] In one embodiment of the present invention, the pharmaceutical composition can better improve the median progression-free survival and median overall survival of patients compared with the administration of BRAF / MEK inhibitors alone.

[0033] In a second aspect, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a cancer treatment drug.

[0034] In one embodiment of the present invention, the cancer is a BRAF V600 mutant cancer. In another embodiment of the present invention, the BRAF V600 mutant cancer is a BRAF V600E / K mutant cancer.

[0035] In one embodiment of the present invention, the BRAF V600E / K mutant cancers include BRAF V600E / K mutant melanoma, BRAF V600E / K mutant non-small cell lung cancer (NSCLC), BRAF V600E / K mutant thyroid cancer, BRAF V600E / K mutant colorectal cancer, and other BRAF mutation-related cancers.

[0036] In one embodiment of the present invention, the pharmaceutical composition can reduce tumor volume and prolong patient survival.

[0037] In one embodiment of the present invention, the pharmaceutical composition can inhibit the activation of the PI3K / AKT and MAPK pathways.

[0038] In one embodiment of the present invention, the pharmaceutical composition can increase the expression of the apoptosis-related protein Bim and downregulate or inhibit the expression of AXL, BXL-XL, and Caspase 9.

[0039] In one embodiment of the present invention, the pharmaceutical composition can modulate or inhibit the expression of P-ERK and GPX4.

[0040] In a third aspect, the present invention provides the use of the above-described composition or aspirin in the preparation of formulations for treating BRAF / MEK inhibitor-resistant diseases.

[0041] In one embodiment of the present invention, the BRAF / MEK inhibitor resistance disease includes BRAF V600 mutant cancer resistance.

[0042] In one embodiment of the present invention, the BRAF V600 mutant cancer is a BRAF V600E / K mutant cancer. In another embodiment of the present invention, the BRAF V600E / K mutant cancer includes BRAF V600E / K mutant melanoma, BRAF V600E / K mutant non-small cell lung cancer (NSCLC), and other BRAF mutation-related cancers.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The pharmaceutical composition of the present invention has the effect of overcoming the drug resistance of BRAF V600E / K mutant cancers and can reduce the occurrence of febrile adverse events (AEs), which has a significant impact on the treatment of patients with advanced cancer and is expected to achieve twice the result with half the effort.

[0045] The pharmaceutical composition of the present invention overcomes BRAF V600E / K mutant cancer drug resistance by inhibiting the activation of the PI3K / AKT and MAPK pathways.

[0046] The pharmaceutical composition of the present invention has the advantages of being safe, economical, and readily available, and has clinical safety. Attached Figure Description

[0047] Figure 1 This is a technical roadmap for Example 3;

[0048] Figure 2 A549 BARF V600E Visual representation of the effect;

[0049] Figure 3 This is a graph showing the activity of the triple drug combination against tumor cells.

[0050] Figure 4 The graph shows the cell survival rate results of the triple drug combination against tumor cells.

[0051] Figure 5 For the dual drug combination against A549 BARF V600E The results of the activity were shown in the graph.

[0052] Figure 6 This is a graph showing the effect of a triple drug combination on tumor cell proliferation.

[0053] Figure 7 This is a graph showing the effect of the dual-drug combination on tumor cell proliferation.

[0054] Figure 8 This is a graph showing the immunofluorescence results of the dual-drug combination on tumor cells;

[0055] Figure 9 This is a graph showing the results of the dual-drug combination's killing effect on tumor cells;

[0056] Figure 10 This is a graph showing the survival rate of tumor cells against the dual-drug combination.

[0057] Figure 11 This image shows the TUNEL staining results of the dual-drug combination on tumor cells.

[0058] Figure 12 This is a graph showing the results of changes in tumor volume in mice;

[0059] Figure 13 This is a graph showing the survival curve results for mice;

[0060] Figure 14 This is a figure showing the results of Western blot detection of apoptosis-related proteins;

[0061] Figure 15 This is a map of the viral vector. Detailed Implementation

[0062] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that 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.

[0063] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0064] Those skilled in the art will understand that dosage units of μg / kg or mg / kg are converted and expressed in equivalent concentration units of μg / ml or mM. Concentration conversions are well known and allow those skilled in the art to make reasonable comparisons and conclusions regarding the dosages, potency, and results described herein.

[0065] The terms “pharmaceutical-grade” or “pharmaceutical-acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other adverse reactions when administered to animals or humans.

[0066] The term "pharmaceutical-grade carrier" includes any solvent, dispersion medium, coating agent, preservative, isotonic agent, and absorption delay agent. Solvents or dispersion media contain, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Coating agents include, for example, lecithin. Isotonic agents include, for example, sugars or sodium chloride. Delayed absorption agents include, for example, aluminum monostearate and gelatin.

[0067] The term “therapeutic effective amount” as used herein is synonymous with “effective amount,” “therapeutic effective dose,” and / or “effective dose,” and refers to the amount of a drug (or combination of drugs) sufficient to produce the desired result or exert the desired effect on the particular condition being treated.

[0068] Pharmaceutical compositions can be prepared, packaged, and / or sold as unit dosage forms and / or as multiple unit dosage forms. A “unit dosage form” is a composition containing a predetermined amount of a pharmaceutical component. The amount of the pharmaceutical component is generally equal to the dose of the pharmaceutical ingredient to be administered to an individual and / or a fraction of that dose, such as one-half, one-third, or one-quarter of such a dose. Pharmaceutical compositions can be in the form of unit dosage forms. In some embodiments, a unit dosage form contains a first active pharmaceutical component (e.g., aspirin) and a second active pharmaceutical component (e.g., dabrafenib) such that there is physical contact between the first and second active pharmaceutical components. In some embodiments, there is substantially no physical contact between the first and second active pharmaceutical components. In some embodiments, the first and second active pharmaceutical components do not physically contact each other. In some embodiments, a pharmaceutical composition contains multiple unit dosage forms containing aspirin and dabrafenib in separate unit dosage forms. When a pharmaceutical composition contains multiple unit dosage forms (where aspirin and dabrafenib are in separate unit dosage forms), the unit dosage forms of aspirin and dabrafenib can be administered sequentially or simultaneously.

[0069] "BRAF / MEK inhibitors" refer to a combination of drugs that target BRAF kinase and its downstream effector molecule MEK in the MAPK signaling pathway. By inhibiting the kinase activity of BRAF (such as the V600E mutant) and blocking MEK phosphorylation of ERK, they synergistically downregulate the abnormally activated RAS-RAF-MEK-ERK signaling cascade, thereby inducing tumor cell cycle arrest, apoptosis, and inhibiting proliferation. This combination strategy can overcome the compensatory pathway activation (such as CRAF or RTK signaling) caused by BRAF inhibitor monotherapy and reduce adverse reactions such as skin toxicity. Clinically, it is mainly used for malignant tumors such as advanced melanoma and non-small cell lung cancer that are positive for BRAF V600 mutations. However, its long-term efficacy is limited by acquired resistance mechanisms (such as NRAS mutations and BRAF splicing variants).

[0070] The term "dabrafenib" refers to the β-Raf inhibitor represented by the structure of formula (I):

[0071] Or its pharmaceutically acceptable salt.

[0072] Dabrafenib is preferably administered in the methanesulfonate form of N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazolyl-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide methanesulfonate. Dabrafenib, together with its pharmaceutically acceptable salts, is disclosed and claimed in PCT Patent Publication No. WO2009 / 137391 as a BRAF activity inhibitor, particularly in the treatment of cancer. Dabrafenib is a potent and selective RAF kinase inhibitor of human wild-type BRAF and CRAF enzymes, as well as mutant forms BRAFV600E, BRAFV600K, and BRAFV600D.

[0073] The term "trametinib" refers to a MEK inhibitor represented by the structure of formula (II):

[0074] Or its pharmaceutically acceptable salt or its solvate.

[0075] Trametinib is preferably administered as a solvate in the form of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide dimethyl sulfoxide. The compound of formula (I) is N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide. Trametinib and its pharmaceutically acceptable salts, or solvates thereof, disclosed in WO 2005 / 121142, can be used as MEK activity inhibitors, particularly in the treatment of cancer. Preferably, trametinib is in the form of a dimethyl sulfoxide solvate. Preferably, trametinib is in the form of a sodium salt. More preferably, trametinib is in the form of a solvate selected from: hydrates, acetic acid, ethanol, nitromethane, chlorobenzene, 1-pentanol, isopropanol, ethylene glycol, and 3-methyl-1-n-butanol. These solvates and salt forms can be prepared by those skilled in the art according to WO 2005 / 121142.

[0076] Example 1: Cell Experiment

[0077] 1. Lentiviral packaging

[0078] The BRAF V600E cDNA sequence (amino acid sequence as shown in SEQ ID NO.1, nucleic acid sequence as shown in SEQ ID NO.2) was synthesized from the whole genome and cloned into the lentiviral backbone plasmid GV492 (element sequence: Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin), cloning site: BamHI / AgeI. Figure 15 As shown, the expression vector GV492-Ubi-BRAF V600E-Puro (containing puromycin resistance) was obtained. The primer sequences used were: BRAF-P1: AGGTCGACTCTAGAGGATCCCGCCACCATGGCGGCGCTGAGCGGTGGCGGTG (SEQ ID NO:4); BRAF-P2: TCCTTGTAGTCCATACCGTGGACAGGAAACGCACCATATCCCC (SEQ ID NO:5). The sequencing results are shown in SEQ ID NO:3. Using Lipofectamine 3000, 10 μg of the expression vector carrying the target gene, 7.5 μg of the packaging plasmid psPAX2 (gag / pol), and 2.5 μg of pMD2.G (VSV-G envelope) were co-transfected into 293T cells. After 6 h, the culture medium was replaced with fresh medium (containing 1% FBS). Viral supernatants were collected 48 h and 72 h after transfection, filtered through a 0.45 μm filter membrane, and stored at -80 °C or concentrated before use.

[0079] 2. Lentiviral infection of target cells

[0080] (1) Cell culture: Baf3 cells were cultured in RPMI-1640 medium containing 10% FBS and 1 ng / mL IL-3. PC-9 / HCC827 / A549 cells were cultured in DMEM containing 10% FBS. The cell density was adjusted to 2 × 10⁶ cells / mL 24 h before infection. 5 cells / mL.

[0081] (2) Viral infection:

[0082] Lentiviral virus carrying the BRAF V600E target gene was added to the above cells, and the culture medium was replaced with fresh medium 24 hours after infection. 72 hours after infection, puromycin-containing medium was added, and the medium was changed every 2 days for continuous selection for 7-10 days. After all control group cells died, the surviving cell population was retained to obtain stable BRAF V600E mutant Baf3, PC-9, HCC827, and A549 cell lines (hereinafter referred to as Baf3). BARF V600E PC-9 BARF V600E HCC827 BARF V600E A549 BARF V600E The successful construction of a cell line overexpressing the BRAF V600E mutation was verified. Taking A549 cells as an example, the results are as follows... Figure 2 As shown. The results indicate that A549 BARF V600E The cells successfully expressed the BARF V600E mutant protein. Using the same method, similar results were obtained for the other three cell types.

[0083] 3. Effects of the aspirin + trametinib + dabrafenib drug combination on the activity of drug-resistant cells.

[0084] Baf3, which is in the logarithmic growth phase BARF V600E PC-9 BARF V600E HCC827 BARF V600E Cells were seeded in 96-well plates. After 24 hours, the cells had fully adhered and resumed normal growth. Culture medium containing different concentrations (0.01, 0.1, 1, 10, 100 μM / L) of aspirin + trametinib + dabrafenib (molar ratio 10:0.1:1) was added, and the cells were incubated for another 48 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another hour. The positive control group was incubated for 48 hours with an equal volume of trametinib + dabrafenib. Negative control: cells + culture medium only (no drugs). Blank control: culture medium only (no cells + no drugs). OD values ​​were read at 450 nm using a microplate reader, and the cell growth inhibition rate was calculated using the following formula: Cell viability (%) = [(OD experimental group - OD blank) / (OD negative control - OD blank)] × 100. Results are as follows. Figure 3 As shown. The IC50 value was calculated using software such as GraphPad Prism to fit the curve. The results are as follows. Figure 4 As shown.

[0085] The results showed that in BARF V600E transfected cell lines, the combination of aspirin, trametinib, and dabrafenib significantly exhibited better cell-killing activity than the combination of trametinib and dabrafenib alone. The IC50 of the aspirin-trametinib-dabrafenib combination was significantly lower than that of the trametinib-dabrafenib combination alone, indicating that the addition of ASP further enhanced the efficacy. This suggests that the aspirin-trametinib-dabrafenib combination can sensitize Baf3 cells. BARF V600E PC-9 BARF V600E HCC827 BARF V600E Cellular sensitivity to trametinib and dabrafenib. Trametinib and dabrafenib exhibit synergistic effects with aspirin.

[0086] 4. Effects of the aspirin + dabrafenib drug combination on the activity of drug-resistant cells.

[0087] Similar to step 3, the difference is that the experimental group received aspirin plus dabrafenib (molar ratio 10:1), while the positive control group received equal amounts of aspirin alone and dabrafenib alone. The cells used were A549. BARF V600E .

[0088] Experimental results are as follows Figure 5As shown in the figure, the results indicate that the aspirin + dabrafenib combination has significantly better cell-killing ability than either the aspirin alone or the dabrafenib alone group. A synergistic effect exists between aspirin and dabrafenib.

[0089] 5. Effects of the aspirin + trametinib + dabrafenib drug combination on the proliferation of drug-resistant cells.

[0090] Take Baf3 cells in the logarithmic growth phase BARF V600E PC-9 BARF V600E HCC827 BARF V600E Resuspend the cells in complete culture medium and add 2 mL of cell suspension (500 cells / well) to each well of a 6-well plate. Control and experimental groups were set up. The experimental group received a combination of aspirin, trametinib, and dabrafenib (molar amounts of 150 μm + 1.5 μm + 15 μm, respectively). The positive control group received an equal amount of trametinib and dabrafenib combination. The negative control group received only cells and culture medium (no drugs). Cells were cultured at 37°C and 5% CO2 for 10 days. The culture medium was discarded, and the cells were gently washed once with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes and washed once with PBS. Staining was performed with 0.5% crystal violet for 20 minutes, followed by slow rinsing with running water until the background was clear. Colony formation was observed. Results are as follows: Figure 6 As shown.

[0091] The results showed that the number of cells in the experimental group was significantly less than that in the control group, and aspirin effectively enhanced the effects of trametinib and dabrafenib on Baf3. BARF V600E PC-9 BARF V600E HCC827 BARF V600E Trametinib and dabrafenib have a synergistic effect with aspirin in killing cells and effectively inhibiting cell proliferation.

[0092] 6. Effects of the aspirin + dabrafenib drug combination on the proliferation of drug-resistant cells.

[0093] (1) Cell cloning experiment: Similar to step 5, except that the experimental group was aspirin + dabrafenib (molar amounts of 150μm + 15μm respectively), the positive control group was an equal amount of aspirin alone and an equal amount of dabrafenib alone, and the cells used were A549. BARF V600E The result is as follows Figure 7 As shown.

[0094] The results showed that the number of cells in the experimental group was significantly less than that in the control group, and aspirin effectively enhanced the effect of dabrafenib on A549. BARF V600E Its cytotoxic ability effectively inhibits cell proliferation.

[0095] (2) Cell immunofluorescence assay: The effects of different treatments (such as single or combined drug treatment) on tumor cell proliferation were detected using Ki67 (proliferation marker) and DAPI (nuclear dye). Specific steps included:

[0096] ① Cell culture: A549 BARF V600E Cells at 5 × 10 4 Seeds were planted at a density of / wells into 24-well plates and cultured for 24 hours until adherence.

[0097] ② Drug treatment: Change the culture medium to contain different drugs and treat for 48 hours. Set up a control group and an experimental group. The experimental group was aspirin + dabrafenib (molar amounts of 150μm + 15μm, respectively), and the control group was aspirin alone and dabrafenib alone.

[0098] ③ Immunofluorescence staining: Fix with 4% paraformaldehyde, block with 5% BSA for 1 hour, add Ki67 primary antibody (1:200) and incubate at 4°C overnight, then add Alexa Fluor 488 secondary antibody (1:500) and incubate in the dark for 1 hour.

[0099] ④ DAPI counterstaining: Stain with DAPI (1 μg / mL) for 5 minutes, then wash 3 times with PBS. Confocal microscopy observation: Five fields of view were randomly selected from each well, and images of the Ki67 (green) and DAPI (blue) channels were captured respectively. Results are as follows. Figure 8 As shown in the fluorescence image: the control group cells have dense nuclei and are strongly Ki67 positive (green), while the combined group cells are sparse and have weak Ki67 signal.

[0100] The results showed that the combined treatment (dabrafenib and aspirin) significantly reduced the proportion of Ki67 positive cells, and its effect was significantly better than that of the aspirin monotherapy group and the dabrafenib monotherapy group, indicating that there is a synergistic effect between dabrafenib and aspirin.

[0101] 7. Effects of the aspirin + dabrafenib drug combination on the killing and apoptosis of drug-resistant cells.

[0102] (1) Cell viability detection (MTT assay)

[0103] Cell seeding: Seed cells in 96-well plates at approximately 5 × 10⁶ cells per well. 3 One cell was cultured for 24 hours until it adhered to the cell wall.

[0104] Drug treatment: Different drug treatments were added (NC control, dabrafenib monotherapy, aspirin monotherapy, and combination therapy) for 24-48 hours.

[0105] Staining: Add MTT (5 mg / mL) (10 μL / well) and incubate at 37°C for 2-4 hours.

[0106] Termination of reaction: Dissolve formazan in DMSO and shake to mix.

[0107] OD value determination: The absorbance was measured at 570 nm using a micro spectrophotometer.

[0108] Data analysis: Calculate cell viability (OD treatment group / OD control group × 100%) and perform statistical analysis (such as t-test).

[0109] The results are as follows Figure 9 , 10 As shown, the combined treatment group had the lowest cell survival rate, which was significantly better than the single treatment group, indicating that the combined treatment may significantly enhance cytotoxicity or induce apoptosis.

[0110] (2) TUNEL staining to detect cell apoptosis

[0111] Cell Seeding and Treatment: Seed cells on coverslips or culture dishes and culture for 24 hours. Then, treat with the drug (NC control, dabrafenib monotherapy, aspirin monotherapy, or combined treatment) for 24-48 hours. Fixation: Fix cells with 4% PFA for 30 minutes and wash three times with PBS at room temperature. Permeabilization: Treat cells with 0.1% Triton X-100 for 5-10 minutes and wash three times with PBS. TUNEL Staining: Add TUNEL reaction mixture (containing TdT enzyme and dUTP label) according to the kit instructions, incubate at 37°C in the dark for 1 hour, and wash three times with PBS. DAPI Staining: Stain nuclei with DAPI (1 μg / mL) for 5 minutes and wash three times with PBS. Observation and Imaging: Observe using a fluorescence microscope (TUNEL: red, DAPI: blue) and capture merged images. Results are as follows: Figure 11 As shown.

[0112] The results showed that the combined treatment group had the most TUNEL-positive cells, which was significantly better than the single treatment group, indicating that the combined treatment of dabrafenib and aspirin significantly enhanced the apoptosis-inducing ability and may overcome drug resistance or enhance the anti-tumor effect through a synergistic mechanism.

[0113] The combined treatment drug composition exerts its antitumor effect by inhibiting cell survival (reducing OD value) and inducing apoptosis (increasing TUNEL-positive cells). This indicates that combining it with aspirin can partially overcome drug resistance or reduce side effects (such as fever).

[0114] Example 2 Animal Experiment

[0115] Six- to eight-week-old immunodeficient mice (BALB / c nude), five mice per group. Treatment group: Aspirin + trametinib + dabrafenib combination (doses of trametinib 150 mg / kg + 1 mg / kg + 40 mg / kg, respectively). Control group: Equal doses of trametinib + dabrafenib combination. Digestion of logarithmic growth phase PC-9 cells. BARF V600E Resuspended in PBS to 5×10⁻⁶ 6 cells / mL. 100 μL of cell suspension was injected subcutaneously into the right hind limb groin area, recorded as Day 0. Tumor volume reached 50-100 mm. 3 At approximately 10 days post-inoculation, the tumor was administered orally via gavage once daily for 2-3 weeks. Tumor volume was measured at the end of the experiment. Results are as follows: Figure 12 As shown. At the end of the experiment, the volume of the control group reached 120 mm. 3 The treatment group only had 30-40mm 3 Survival curves: The survival rate of the treatment group was significantly higher than that of the control group during the 30-day observation period. The survival rate of the treatment group decreased slowly, remaining at approximately 20% by day 30, while the survival rate of the control group decreased rapidly, approaching 0% after day 20. This shows that the treatment group had a much better survival ability than the control group. Survival curves were plotted using the Kaplan-Meier method, and the Log-rank test was used to compare the differences between the groups. Results are as follows... Figure 13 As shown.

[0116] The results showed that in the nude mouse xenograft model constructed using PC-9BARF V600E cells, aspirin combined with trametinib and dabrafil significantly reduced the volume of xenografts in nude mice and significantly prolonged their survival.

[0117] Example 3 Clinical Study

[0118] 1. Study population

[0119] Primarily targeting patients with primary stage IIIB-IV BRAF V600E-mutated advanced non-small cell lung cancer, the proposed treatment involves trametinib and dabrafenib for advanced lung cancer patients.

[0120] 2. Research Plan

[0121] Screening Period: After researchers have preliminarily identified subjects who basically meet the requirements of this trial and obtained their informed consent, the screening period begins. During the screening period, inclusion and exclusion criteria are assessed based on the subjects' medical history, physical examination, and laboratory tests.

[0122] Treatment period: Subjects will be treated with aspirin in combination with trametinib and dabrafenib. Subjects will receive drug treatment for 36 months, and treatment may be discontinued early unless the subject experiences tumor progression (worsening) or intolerable toxicity.

[0123] The treatment regimen consists of dabrafenib 150 mg twice daily and trametinib 2 mg once daily. If treatment toxicity occurs in a subject during treatment, the investigator must promptly discontinue or adjust the dosage of dabrafenib or trametinib. In cases of severe drug toxicity, the subject must discontinue the medication. The treatment regimen also consists of aspirin 100 mg / tablet, once daily (QD). If treatment toxicity occurs in a subject during treatment, the investigator must promptly discontinue or adjust the dosage of aspirin. In cases of severe drug toxicity, the subject must discontinue the medication.

[0124] Observation period: The study observation period was 30 months, with 36 patients enrolled. Successfully screened and randomly assigned subjects participated in 19 telephone visits (V3–V21) at the research center. These visits assessed whether patients experienced drug side effects, whether medication was discontinued or reduced, and whether disease progression occurred. A summary of each visit was required during the treatment period. Adverse events and concomitant medications were recorded promptly.

[0125] Survival follow-up: Subjects who terminate the trial early (except for those who die, are excluded, or are lost to follow-up) shall be followed up once a month from the date of randomization until 36 months or death.

[0126] 3. Key In / Out Criteria:

[0127] (1) Inclusion criteria:

[0128] Patients with inoperable stage IIIB-IV non-small cell lung cancer;

[0129] It has the BRAF V600E mutation;

[0130] The proposed treatment regimen is trametinib and dabrafenib, with an estimated survival period of more than 3 months.

[0131] ECOGPS 0 / 1;

[0132] The diameter of the primary lesion is at least 1 cm;

[0133] Patients who have previously taken or are currently taking aspirin are permitted to receive treatment.

[0134] (2) Exclusion criteria:

[0135] Currently receiving other anticoagulation therapy;

[0136] Previous systemic anti-NSCLC treatment;

[0137] Other positive driver mutations include EGFR, ALK, ROS1, MET14 exon skipping, RET, etc.

[0138] There are contraindications to the use of dabrafenib or trametinib and aspirin;

[0139] Patients who refuse follow-up visits;

[0140] (3) Criteria for early termination of the test:

[0141] The subject requested it voluntarily;

[0142] Subjects lost to follow-up;

[0143] The subjects experienced intolerable toxic reactions;

[0144] Researchers may request the termination of a trial for subjects (e.g., due to safety concerns or because they believe that early termination is in the best interests of the patients);

[0145] The subject died;

[0146] Subjects seriously violated the study protocol;

[0147] Patients incorrectly included or excluded (e.g., patients do not meet the inclusion / exclusion criteria);

[0148] 4. Data Analysis

[0149] 4.1 Therapeutic Dataset

[0150] (1) Full Analysis Set (FAS)

[0151] Based on the principle of intention-to-treat (ITT), all participating subjects were included in the full analysis set.

[0152] (2) Modified intention-to-treat population (mITT)

[0153] Based on the intention-to-treat (ITT) principle, this includes all subjects who have received at least one treatment and have had one efficacy assessment.

[0154] (3) Conforms to the protocol set (PPS)

[0155] Based on the protocol compliance principle, all participants who adhered fully to the study design (i.e., without significant protocol violations) will be included in the compliance set. The precise definition of "significant protocol violation" will be finalized during data review and generally may include the following: significant deviation from the trial procedure (e.g., measurement of the primary efficacy endpoint outside the window), serious violation of inclusion / exclusion criteria, randomization error, use of investigational contraindicated drugs in combination therapy, poor adherence (adherence should be ≥80% and ≤120%), significant out-of-window follow-up, or significant missing follow-up items. Efficacy analyses will be conducted using the compliance set separately.

[0156] 4.2 Security Analysis Dataset

[0157] Subjects who participated in and received at least one treatment were included in the safety analysis set. Data from all safety analyses were applied to this dataset.

[0158] 4.3 Effectiveness Assessment / Therapeutic Effect Analysis:

[0159] Univariate analysis of the relationship between lung cancer and treatment regimen: DFS and OS will be compared using the log-rank test to determine whether there is a statistically significant difference (P value less than or equal to 0.05) between the experimental and control groups throughout the survival curves; Multivariate analysis: PFS and OS will be compared using the Cox proportional hazards model to estimate the relative hazard ratio and explore other factors affecting efficacy.

[0160] 5. Experimental Results: In the aforementioned large-sample, multicenter clinical study, compared with osimertinib alone, the combination therapy of osimertinib and aspirin significantly prolonged progression-free survival (PFS) and overall survival (OS), effectively delaying disease progression and reducing the risk of death. This demonstrates that aspirin can enhance the efficacy of osimertinib. More importantly, aspirin has a good safety profile with a low incidence of adverse events, laying a solid foundation for its effective combination therapy with dalatinib and trametinib.

[0161] Example 4 Mechanism Study

[0162] A549 cells in logarithmic growth phase were taken. BARF V600ECells were resuspended in complete culture medium, and 2 mL of cell suspension (500 cells / well) was added to each well of a 6-well plate. The plates were then treated with culture medium containing different drugs for 48 h. Control and experimental groups were set up: the experimental group received an aspirin + dabrafenib combination, and the control group received equal amounts of dabrafenib alone and aspirin alone. Western blot analysis was then performed. Total protein was extracted with RIPA lysis buffer, separated by SDS-PAGE, and transferred to a PVDF membrane. Primary antibodies were added: Cleaved Caspase-3 (1:1000), Bcl-2 (1:1000), p-ERK (1:1000), and β-Actin (1:5000). Secondary antibody: HRP-labeled anti-rabbit / mouse IgG (1:5000). Imaging results are shown below. Figure 14 As shown.

[0163] Western blotting results showed that aspirin combined with dabrafenib increased the expression of the apoptosis-related protein Bim and downregulated the expression of AXL, BXL-XL, and Caspase 9, confirming that the combination of aspirin and dabrafenib increased the induction of apoptosis. Simultaneously, it downregulated the expression of P-ERK in the MAPK pathway and inhibited the ferroptosis molecule GPX4 (glutathione peroxidase 4), suggesting that this drug combination may inhibit tumor development by sensitizing ferroptosis.

[0164] Possible mechanisms by which aspirin overcomes resistance to dabrafenib combined with trametinib: Aspirin may overcome resistance to dabrafenib combined with trametinib by inhibiting the activation of the PI3K / AKT and MAPK pathways. Combining aspirin with dabrafenib may enhance the sustained efficacy of BRAF+MEKi by improving T-cell therapy, thereby modulating the immune microenvironment, enhancing the efficacy of T-cell therapy, and improving anti-cancer immunity. Aspirin may overcome resistance in BRAF V600 ENSCLC through the co-targeting of COX2 and BRAF+EGFR.

[0165] The possible mechanism by which aspirin combined with dabrafenib and trametinib reduces febrile adverse events (AEs): The most common AE of dabrafenib combined with trametinib is fever. Previous studies have found that the mechanism of fever may be related to the upregulation of pro-inflammatory cytokines IL-1B and IL-6. IL-1B and IL-6 can cross the blood-brain barrier and begin the synthesis of PGE2 in the hypothalamus, thereby altering the body's temperature set point. Therefore, pro-inflammatory cytokine-mediated fever is one possible explanation. As a COX inhibitor, aspirin can inhibit the production of PGE2, thus exerting an antipyretic effect.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0167] SEQ ID NO:1

[0168] MAALSGGGGGGAEPGQALFNGDMEPEAGAGAGAAASSAADPAIPEEVWNIKQMIKLTQEHIEALLDKF

[0169] GGEHNPPSIYLEAYEEYTSKLDALQQREQQLLESLGNGTDFSVSSSASMDTVTSSSSSSLSVLPSSLSVFQN

[0170] PTDVARSNPKSPQKPIVRVFLPNKQRTVVPARCGVTVRDSLKKALMMRGLIPECCAVYRIQDGEKKPIG

[0171] WDTDISWLTGEELHVEVLENVPLTTHNFVRKTFFTLAFCDFCRKLLFQGFRCQTCGYKFHQRCSTEVPL

[0172] MCVNYDQLDLLFVSKFFEHHPIPQEEASLAETALTSGSSPSAPASDSIGPQILTSPSPSKSIPIPQPFRPADED

[0173] HRNQFGQRDRSSSAPNVHINTIEPVNIDDLIRDQGFRGDGGSTTGLSATPPASLPGSLTNVKALQKSPGPQ

[0174] RERKSSSSSEDRNRMKTLGRRDSSDDWEIPDGQITVGQRIGSGSFGTVYKGKWHGDVAVKMLNVTAPTP

[0175] QQLQAFKNEVGVLRKTRHVNILLFMGYSTKPQLAIVTQWCEGSSLYHHLHIIETKFEMIKLIDIARQTAQG

[0176] MDYLHAKSIIHRDLKSNNIFLHEDLTVKIGDFGLATEKSRWSGSHQFEQLSGSILWMAPEVIRMQDKNPY

[0177] SFQSDVYAFGIVLYELMTGQLPYSNINNRDQIIFMVGRGYLSPDLSKVRSNCPKAMKRLMAECLKKKRD

[0178] ERPLFPQILASIELLARSLPKIHRSASEPSLNRAGFQTEDFSLYACASPKTPIQAGGYGAFPVH

[0179] SEQ ID NO:2

[0180]

[0181] SEQ ID NO:3

[0182]

Claims

1. A pharmaceutical composition, characterized in that: This includes therapeutically effective doses of aspirin and BRAF / MEK inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that: The BRAF / MEK inhibitors include at least one of dabrafenib, vemurafenib, encorafenib, trametinib, cobimetinib, and binimetinib; Preferably, the BRAF / MEK inhibitor is dabrafenib; preferably, the BRAF / MEK inhibitor is trametinib; preferably, the BRAF / MEK inhibitor is both dabrafenib and trametinib.

3. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition includes aspirin and dabrafenib; Preferably, the pharmaceutical composition includes aspirin, dabrafenib, and trametinib.

4. The pharmaceutical composition according to claim 1, characterized in that: The aspirin and BRAF / MEK inhibitors have a synergistic effect. Preferably, the aspirin and BRAF / MEK inhibitor are administered sequentially or simultaneously; Preferably, aspirin and BRAF / MEK inhibitors are in their respective separate unit formulations; or aspirin and BRAF / MEK inhibitors are in the same unit formulation.

5. The pharmaceutical composition according to claim 1, characterized in that: The aspirin:BRAF / MEK inhibitor ratio, by mass, is (50-1000):(1-200). In one embodiment of the present invention, the aspirin:BRAF inhibitor:MEK inhibitor ratio, by mass, is (50-1000):(5-200):(1-5). In another embodiment of the present invention, the aspirin:BRAF inhibitor:MEK inhibitor ratio, by mass, is (80-900):(5-150):(1-3).

6. The pharmaceutical composition according to claim 1, characterized in that: The composition also includes other chemotherapeutic agents, immunotherapeutic agents, or combinations thereof; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable excipient or carrier.

7. The pharmaceutical composition according to claim 1, characterized in that: Compared with the administration of BRAF / MEK inhibitors alone, the pharmaceutical composition has a better effect on inhibiting tumor cell activity or proliferation; And / or, the pharmaceutical composition exhibits better tumor cell killing activity compared to the administration of BRAF / MEK inhibitors alone; And / or, the pharmaceutical composition has a better effect on promoting tumor cell apoptosis compared with the administration of BRAF / MEK inhibitors alone; preferably, the tumor cells are BRAF V600E / K mutant tumor cells; And / or, compared with the administration of BRAF / MEK inhibitors alone, the pharmaceutical composition has a better inhibitory effect on PI3K / AKT and MAPK pathway activation; And / or, compared with the administration of BRAF / MEK inhibitors alone, the pharmaceutical composition has a better effect in increasing the expression of apoptosis-related protein Bim and downregulating or inhibiting the expression of AXL, BXL-XL, and Caspase 9; And / or, compared with the administration of BRAF / MEK inhibitors alone, the pharmaceutical composition has a better effect in downregulating or inhibiting the expression of P-ERK and GPX4; And / or, compared with the administration of BRAF / MEK inhibitors alone, the pharmaceutical composition can better improve the progression-free survival rate and overall survival rate of patients; And / or, compared with the administration of BRAF / MEK inhibitors alone, the pharmaceutical composition can better improve the median progression-free survival and median overall survival of patients.

8. The use of the pharmaceutical composition according to any one of claims 1-7 in the preparation of a medicament for treating cancer. Preferably, the cancer is a BRAF V600 mutant cancer; more preferably, the BRAF V600 mutant cancer is a BRAF V600E / K mutant cancer; even more preferably, the BRAF V600E / K mutant cancer includes BRAF V600E / K mutant melanoma, BRAF V600E / K mutant non-small cell lung cancer (NSCLC), BRAF V600E / K mutant thyroid cancer, BRAF V600E / K mutant colorectal cancer, and other BRAF mutation-related cancers. Preferably, the pharmaceutical composition can reduce tumor volume and prolong patient survival; Preferably, the pharmaceutical composition can inhibit the activation of the PI3K / AKT and MAPK pathways; Preferably, the pharmaceutical composition can increase the expression of the apoptosis-related protein Bim and downregulate or inhibit the expression of AXL, BXL-XL, and Caspase 9; Preferably, the pharmaceutical composition can modulate or inhibit the expression of P-ERK and GPX4; Preferably, the pharmaceutical composition can better improve the progression-free survival rate and overall survival rate of patients; Preferably, the pharmaceutical composition can better improve patients' median progression-free survival and median overall survival.

9. The use of any one of the compositions of claims 1-7 or aspirin in the preparation of formulations for treating BRAF / MEK inhibitor-resistant diseases.

10. The application as described in claim 9, characterized in that: The BRAF / MEK inhibitor resistance diseases include BRAFV600 mutant cancer resistance; Preferably, the BRAF V600 mutant cancer is a BRAF V600E / K mutant cancer. Preferably, the BRAF V600E / K mutant cancer includes BRAF V600E / K mutant melanoma, BRAF V600E / K mutant non-small cell lung cancer (NSCLC), and other BRAF mutation-related cancers.

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