Use of matrine F in the preparation of drugs used in combination with gefitinib

Abstract

This invention relates to the field of pharmaceutical applications, specifically disclosing the use of matrine F in the preparation of a drug for use in combination with gefitinib. The drug is used to treat non-small cell lung cancer, and the combination involves the synergistic effect of matrine F and gefitinib to inhibit tumor growth or reverse gefitinib resistance. This invention utilizes the combination of matrine F monomer and gefitinib, achieving precise dosage, a clear mechanism, and a significant synergistic effect, providing a novel and more targeted treatment strategy for addressing gefitinib resistance in non-small cell lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical applications and relates to the use of matrine F in the preparation of drugs used in combination with gefitinib. Background Technology

[0002] Kushenol F, also known as Sophora flavanol F, Sophora flavonoid, or Sophora flavonoid, is a natural flavonoid monomer compound with a dihydroflavonoid skeleton. It is found in the roots of Sophora flavescens and Sophora narrow-leaved plants or other plants of the same genus. Its structural feature is the combination of a benzopyran ring and an isopentenyl side chain, which gives it unique pharmacological activity.

[0003] Currently, the monomer mainly has two structures, including isoprene structures located at the 6- and 8-position C atoms of the flavonoid core, respectively. Molecular formula: C 25 H 28 O6, molecular weight: 424.49 g / mol, LogP (lipophilicity) 5.30-5.74. Its main pharmacological effects include antitumor activity, inhibiting MAPK signaling pathways (such as the ERK / JNK pathway)-induced apoptosis in breast cancer cells (MDA-MB-231) and leukemia cells (HL-60); regulating the expression of inflammatory factors, inhibiting pathways such as NF-κB, and scavenging free radicals to reduce oxidative damage.

[0004] Matrine F is currently used primarily for psoriasis and atopic dermatitis. In an imiquimod-induced psoriasis mouse model, matrine F significantly improved skin lesions, reduced the expression of pro-inflammatory factors such as IL-17A, IL-22, and IL-23 in the skin, and increased IL-10. Its efficacy is superior to the positive control drug calcipotriol, and it can inhibit the proliferation of HaCaT keratinocytes and the release of inflammatory factors.

[0005] Furthermore, in vitro studies have shown that matrine F induces mitochondrial-mediated apoptosis by activating caspase-3 and caspase-9, downregulating the anti-apoptotic protein Bcl-2 / Bcl-xL, and upregulating the pro-apoptotic protein Bax. In addition, it can inhibit the activity of MMP-2 / MMP-9 enzymes associated with tumor cell migration and invasion.

[0006] When gefitinib is used to treat non-small cell lung cancer (NSCLC), drug resistance symptoms are easily developed. However, when used in combination with adjuvant drugs, it can effectively inhibit tumor growth and reduce adverse reactions. The efficacy may be due to the synergistic or antagonistic effects of multiple pathways. It is necessary to combine the above-mentioned research on the function of matrine F in inhibiting inflammation and regulating metabolism, and propose a combination drug that can synergistically reduce drug resistance in non-small cell lung cancer and enhance efficacy with gefitinib, and reveal the mechanism behind its action. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a novel use of matrine F combined with gefitinib in the preparation of drugs for treating non-small cell lung cancer (NSCLC). This combination therapy, through the synergistic effect of the two drugs, significantly inhibits NSCLC cell proliferation and tumor growth, and effectively reverses acquired and natural resistance to gefitinib. Compared to complex compound preparations with significant batch-to-batch variations, the combination of matrine F monomer and gefitinib achieves precise dosage, a clear mechanism, and a significant synergistic effect, providing a novel and more targeted treatment strategy for addressing gefitinib resistance in NSCLC.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: the use of matrine F in the preparation of a drug for use in combination with gefitinib, the drug being used to treat non-small cell lung cancer, wherein the combination is a synergistic effect of matrine F and gefitinib to inhibit tumor growth or reverse gefitinib resistance.

[0009] Preferably, the non-small cell lung cancer is EGFR-mutant non-small cell lung cancer.

[0010] Preferably, the non-small cell lung cancer is non-small cell lung cancer that has developed resistance to gefitinib.

[0011] Preferably, the matrine F reverses gefitinib resistance by inhibiting the expression or function of matrix interaction molecule 1 (STIM1).

[0012] Preferably, the inhibition of STIM1 by matrine F leads to a decrease in intracellular calcium ion concentration, downregulation of nuclear factor κB (NF-κB) signaling pathway activity, and / or downregulation of tumor necrosis factor (TNF) signaling pathway activity.

[0013] Preferably, the matrine F and gefitinib are prepared into a single pharmaceutical composition for simultaneous administration.

[0014] Preferably, the matrine F and gefitinib are prepared as separate pharmaceutical units for sequential administration.

[0015] Preferably, the dosage ratio of matrine F to gefitinib is 3:2.

[0016] The basic principle of the protocol is that matrine F directly targets the STIM1 protein, inhibiting its function, which leads to a decrease in intracellular calcium ion concentration. This, in turn, inhibits the activity of the CaMKII, IKKβ, and NF-κB signaling axes, disrupts the TNF-α autocrine loop, and weakens the Ras-ERK signaling pathway. Ultimately, these factors collectively lead to the downregulation of EGFR expression and the inhibition of its activation, thereby restoring the sensitivity of drug-resistant tumor cells to gefitinib.

[0017] The beneficial effects of this invention are:

[0018] 1. This invention is the first to discover that the combination of matrine F and gefitinib has a significant synergistic effect, which can greatly reduce the half-maximal inhibitory concentration (IC50) of the two drugs when used alone. It exhibits strong antitumor activity against both gefitinib-sensitive and gefitinib-resistant strains, especially reducing the IC50 of gefitinib against the gefitinib strain PC9-GR. 50 It reduced the drug resistance by 24 times, effectively reversing the common clinical problem of gefitinib resistance.

[0019] 2. This invention breaks through the bottleneck of unclear mechanism of action of compound preparations. Through advanced technologies such as DARTS and Lip-MS, the direct molecular target of matrine F is accurately identified as STIM1. The molecular mechanism by which it downregulates EGFR expression and activation through the "STIM1-calcium signaling-NF-κB / TNF / ERK" pathway axis is fully elucidated, providing a solid theoretical basis for combination therapy and realizing the leap from "extensive compound preparations" to "precise targeting".

[0020] 3. Compared with traditional Chinese medicine compound preparations with complex ingredients and large fluctuations in the content of main components between batches, this invention uses high-purity matrine F monomer in combination with gefitinib, which increases the dosage from a rough milliliter level to a precise milligram level, greatly ensuring the consistency and reliability of the drug's therapeutic effect and laying the foundation for the standardized production and clinical translation of the drug.

[0021] 4. Matrine F has an IC50 effect on normal lung cells, such as BEAS-2B and HPMEC. 50 Much higher than the IC50 for tumor cells 50 It showed good selective cytotoxicity, indicating that the combination strategy has a wide therapeutic window and a lower risk of potential adverse reactions.

[0022] 5. This invention provides two feasible pharmaceutical formulations. Preparing them as a single pharmaceutical composition, such as compound tablets or capsules, simplifies the dosing regimen, improves patient compliance, and ensures that the two active ingredients are absorbed and utilized in the optimal ratio. Preparing them as independent pharmaceutical units, such as individual tablets or separately packaged injections, provides greater flexibility in clinical medication, allowing physicians to adjust the dosage and timing of administration of the two drugs according to the patient's specific condition, tolerance, and other factors, achieving individualized precision treatment.

[0023] 6. Developing the two drugs as independent pharmaceutical units for combination therapy can be based on existing safety data of gefitinib, focusing primarily on the additional efficacy and safety of the combination therapy, which may accelerate the clinical trial process and regulatory approval speed. Attached Figure Description

[0024] Figure 1 This is the chemical structural formula of matrine F.

[0025] Figure 2 Here is the chemical structural formula of gefitinib.

[0026] Figure 3 This is a schematic diagram illustrating the mechanism by which matrine F reverses gefitinib resistance.

[0027] Figure 4 This describes the inhibitory effect of matrine F on the proliferation of PC9 cells.

[0028] Figure 5 This study investigated the inhibitory effect of matrine F on the proliferation of PC9-GR cells.

[0029] Figure 6 This demonstrates the inhibitory effect of gefitinib on PC9 cell proliferation.

[0030] Figure 7 This study investigated the inhibitory effect of gefitinib on the proliferation of PC9-GR cells.

[0031] Figure 8 This study investigated the synergistic inhibitory effect of matrine F combined with gefitinib on the proliferation of PC9-GR cells.

[0032] Figure 9 Photographs showing the tumor size of CDX models in each group of mice.

[0033] Figure 10 This is a bar chart showing tumor growth in an in vivo CDX model.

[0034] Figure 11 Principal component analysis plot of global gene expression profile for tumor samples.

[0035] Figure 12 To verify the direct binding results of matrine F and STIM1 in the DARTS experiment.

[0036] Figure 13 Volcano plot of differentially expressed genes in the combination therapy group / control group.

[0037] Figure 14 Cluster heatmap showing significant differential expression after treatment with a combination of matrine F and gefitinib.

[0038] Figure 15 This is a graph showing the enrichment of the KEGG pathway. Detailed Implementation

[0039] The specific implementation method is described below with reference to the accompanying drawings.

[0040] Example 1

[0041] The basics are as follows: Figures 1 to 4The image shows the use of matrine F in the preparation of a drug for use in combination with gefitinib for the treatment of non-small cell lung cancer, wherein the combination is a synergistic effect of matrine F and gefitinib to inhibit tumor growth or reverse gefitinib resistance.

[0042] The non-small cell lung cancer is EGFR-mutant non-small cell lung cancer, and the non-small cell lung cancer is non-small cell lung cancer that has developed resistance to gefitinib.

[0043] Matrine F reverses gefitinib resistance by inhibiting the expression or function of matrix interaction molecule 1 (STIM1). Inhibition of STIM1 by matrine F leads to decreased intracellular calcium ion concentration, downregulation of nuclear factor κB signaling pathway activity, and / or downregulation of tumor necrosis factor signaling pathway activity.

[0044] This study investigated the structure of isoprene at the C-8 position of the flavonoid core. In non-small cell lung cancer (NSCLC) cell and animal models, matrine F monomer, at low doses, inhibited the proliferation and growth of NSCLC and also showed inhibitory effects on EGFR-TKI-resistant NSCLC. When combined with gefitinib, it demonstrated better efficacy against gefitinib-resistant NSCLC than gefitinib monotherapy; the two may have synergistic anti-tumor effects.

[0045] The molecular formula of matrine F is C0. 25 H 28 O5, molecular weight 424.49 g / mol, pale yellow to colorless crystalline powder, dissolves in DMSO. Gefitinib, molecular formula C 22 H 24 ClFN4O3, molecular weight 446.9 g / mol, is a white to off-white crystalline powder that can be dissolved in DMSO.

[0046] The specific experimental procedure is as follows:

[0047] I. Cell Experiments

[0048] Experimental methods

[0049] 1. Cell lines and culture conditions

[0050] The following five human non-small cell lung cancer (NSCLC) cell lines were selected for this study:

[0051] A549: EGFR wild type, carrying the KRAS G12S activation mutation.

[0052] H1650: Carries an EGFR exon 19 deletion mutation (ex19del) and a PTEN gene deletion.

[0053] PC9: Carries the EGFR ex19del mutation and is highly sensitive to gefitinib.

[0054] H1975: Carries double mutations in EGFR L858R and T790M, and is naturally resistant to gefitinib.

[0055] HCC827: Carries the EGFR ex19del mutation.

[0056] All cell lines were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and routinely cultured in a constant temperature and humidity incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0057] 2. Compounds and Treatment Schemes

[0058] Kushenol F was dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution, which was then aliquoted and stored at -20°C. Before use, it was diluted with complete culture medium to the required working concentration, ensuring that the final concentration of DMSO in each experimental group was consistent and below 0.1% (v / v) to eliminate the influence of the solvent on cell viability.

[0059] After cells were seeded in culture plates and allowed to adhere overnight, they were replaced with fresh complete culture medium containing different concentrations of matrine F (5 μM, 10 μM, 20 μM) or an equal volume of DMSO (as a solvent control). The treatment time was determined based on subsequent assay parameters (such as 48-72 hours for MTT assay of cell viability).

[0060] 3. Induction and culture of PC9 gefitinib-resistant cell line (PC9-GR)

[0061] Acquired resistance to the drug was induced in PC9 cells using a "pulse-recovery" method with increasing concentration gradients.

[0062] Initial IC 50 Confirmation: The half-maximal inhibitory concentration (IC50) of gefitinib against parental PC9 cells was determined through previous experiments. 50 The value is determined to be 10 nM.

[0063] Induction process: using IC 50 Starting with a concentration of 10 nM, cells were induced and passaged. The treatment procedure for each generation of cells is as follows:

[0064] (1) Pulsed exposure: When the cells grow to about 60-70% confluence, replace with complete culture medium containing 1 μM gefitinib and treat continuously for 24 hours.

[0065] (2) Recovery culture: Discard the drug-containing culture medium, gently wash the cells twice with pre-warmed PBS, and then replace it with drug-free complete culture medium and continue culturing for 48 hours.

[0066] (3) Increasing concentration and continuous induction: The above "pulse-recovery" process is repeated. As the cells gradually adapt, the concentration of gefitinib during pulse exposure can be gradually increased (e.g., from 1 μM to 2 μM, 4 μM...) according to the cell survival status, until it can stably proliferate at a high concentration of gefitinib (e.g., 2 μM). The drug is withdrawn 7 days before the experiment to eliminate drug residue effects.

[0067] 4. Experimental Results

[0068] Combination Figures 4 to 8 As shown in Table 1, matrine F has an IC50 effect on normal lung cell lines BEAS-2B and HPMEC. 50 The values ​​were 106.67 μM and 70.19 μM, respectively, indicating relatively low toxicity to normal cells. In contrast, matrine F showed stronger inhibitory activity against the proliferation of all tested NSCLC cell lines, with an IC50 value of 106.67 μM and 70.19 μM. 50 The values ​​ranged from 14.07 μM (H1975) to 22.68 μM (H1650). Notably, the antitumor activity of matrine F did not appear to be clearly associated with EGFR mutation types (including wild-type, ex19del, and L858R / T790M), suggesting that its mechanism of action may not depend on the EGFR pathway.

[0069] Table 1. Half-maximal inhibitory concentrations (IC50) of matrine F on different cell lines 50 )

[0070]

[0071] A PC9 cell line (PC9-GR) resistant to gefitinib was successfully induced using a pulse-recovery concentration gradient escalation method. The parental PC9 cells were highly sensitive to gefitinib, with an IC50 concentration of [missing value]. 50 The concentration was 182 nM; however, the induced PC9-GR cells exhibited extremely strong drug resistance, with an IC50 of 182 nM. 50 The drug resistance index reached 11.03 μM, exceeding 60 times, confirming the successful construction of the PC9-GR cell model, which can be used for subsequent combination drug research.

[0072] To investigate the combined effect of matrine F and gefitinib, combination therapy experiments were conducted on both the susceptible PC9 cell line and the resistant PC9-GR cell line. The combination of low-concentration gefitinib (10 nM) and matrine F significantly enhanced the latter's cytotoxic effect on PC9 cells. The single-agent IC50 of matrine F was... 50The concentration was 16.65 μM, while the combination therapy reduced its IC50 to 16.65 μM. 50 At a concentration lowered to 4.1 μM, sensitivity increased by 4.06 times. Combination therapy strategies demonstrated a stronger effect in reversing drug resistance. Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown.

[0073] A fixed concentration of matrine F of 5 μM showed low inhibitory rates against PC9-GR cells on its own, but when combined with gefitinib, it increased the IC50 of gefitinib against PC9-GR cells. 50 The concentration dropped dramatically from 11.03 μM to 458 nM, reducing drug resistance by 24 times and effectively reversing its drug resistance phenotype.

[0074] A fixed concentration of gefitinib (1 μM) combined with matrine F also resulted in a lower IC50 value for matrine F against PC9-GR. 50 The sensitivity was increased fourfold by reducing the M value from 23.29 μM to 6.5 μM.

[0075] Combination Figure 8 As shown, the Combination Index (CI) was calculated to quantitatively evaluate the nature of the combined effect. Under the two combined schemes mentioned above:

[0076] Fixed with 5 μM matrine F + 458 nM gefitinib (IC50) 50 points): CI=(458 / 11030)+(5 / 23.29)=0.256

[0077] Fixed with 1 μM gefitinib + 6.5 μM matrine F (IC50) 50 points): CI=(1000 / 11030)+(6.5 / 23.29)=0.37

[0078] Both CI values ​​were much less than 1, clearly indicating a strong synergistic effect between matrine F and gefitinib in inhibiting PC9-GR cell proliferation. The regimen with a fixed matrine F concentration (CI = 0.256) showed a more significant synergistic effect.

[0079] Example 2

[0080] The difference from the above embodiments is that, as shown in the appendix Figure 5 , Figure 6 As shown: Matrine F and gefitinib are prepared into a single pharmaceutical composition, or into separate pharmaceutical units for simultaneous or sequential administration.

[0081] In this embodiment, a simultaneous administration strategy is adopted, and the dosage ratio of matrine F to gefitinib is 3:2.

[0082] Animal experiments were conducted based on the above experimental results:

[0083] II. In vivo mouse CDX model experiment

[0084] 1. Establishment of xenograft tumor (CDX) model

[0085] PC9-GR cells in the logarithmic growth phase were digested with trypsin, counted, and resuspended in pre-chilled serum-free medium. The cell suspension was thoroughly mixed with Matrigel at a 1:1 volume ratio on ice, and kept at a low temperature to prevent gel solidification. The final cell density was adjusted to 5 × 10⁶ cells per mouse. 6 A mixture of cells. Using a sterile 1mL syringe, 200 μL of the cell-Matrix gel mixture was subcutaneously injected into the right dorsal side of each mouse. The mice's condition and tumor formation were observed daily.

[0086] 2. Grouping and Dosing

[0087] Grouping: When the tumor volume grew to approximately 100 mm³, the tumor-bearing mice were randomly assigned (n=6 / group) to the following four experimental groups:

[0088] Control group: Administered an equal volume of drug solvent (e.g., physiological saline containing 5% DMSO + 5% Cremophor EL).

[0089] Gefitinib group: 20 mg / kg / day, orally by gavage.

[0090] Sophoflavescenol F group: 30 mg / kg / day, orally by gavage.

[0091] Combination group: Gefitinib and Matrine F were administered simultaneously at a dose of 20 mg / kg / d, in the same manner as the monotherapy group.

[0092] Dosing regimen: Mice in each group were administered the drug continuously for 17 days. Mouse body weight and tumor volume were recorded daily.

[0093] 3. Tumor volume and body weight monitoring

[0094] Tumor measurements: Measure the longest diameter (a) and shortest diameter (b) of the tumor every 2-3 days using calipers. Tumor volume (TV) is calculated using the formula: TV(mm³) = 0.5×a×b².

[0095] Weight monitoring: Mice were weighed daily to assess potential systemic toxicity from the drug.

[0096] Experimental endpoint: Seventeen days after drug administration, all mice were humanely euthanized using the carbon dioxide inhalation method. Tumor tissue was dissected, weighed, and photographed. Some tumor tissue was frozen at -80°C or fixed in 4% paraformaldehyde for subsequent analysis.

[0097] 4. Transcriptome sequencing (RNA-Seq) analysis

[0098] Sample preparation: PC9-GR tumor tissues (n=3 / group) from the four groups of mice in the above experiment (control group, gefitinib group, matrine F group and combined drug administration group) were rapidly ground into powder in liquid nitrogen.

[0099] RNA extraction: Total RNA was extracted from tumor tissues in each group using the TRIzol method. RNA concentration was detected by NanoDrop, and RNA integrity (RIN value) was assessed using an Agilent 2100 Bioanalyzer to ensure that the sample quality met the requirements for library preparation.

[0100] Library construction and sequencing: Qualified samples underwent mRNA enrichment, fragmentation, and cDNA synthesis to construct PE150 sequencing libraries. High-throughput sequencing was performed on the Illumina NovaSeq 6000 platform.

[0101] Bioinformatics analysis: Raw data underwent quality control, alignment (reference genome GRCh38), and gene expression quantification. The screening criteria for differentially expressed genes (DEGs) were set as |log2(Fold Change)| > 1 and adjusted p-value < 0.05. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on DEGs to reveal significant signaling pathway changes between groups.

[0102] 5. Drug Affinity Target Stabilization Technology (DARTS) and LiP-MS Target Fishing

[0103] Preparation of cell lysates

[0104] Culture PC9-GR cells, collect cells in the logarithmic growth phase, wash with pre-chilled PBS, add lysis buffer (such as NP-40 buffer containing protease inhibitors), and lyse on ice. Centrifuge, collect the supernatant, and quantify the total protein concentration.

[0105] 6. DARTS Experiment

[0106] Drug incubation: Total protein was gently incubated with different concentrations of matrine F (0 μM, 20 μM, 200 μM) at 4°C for 1 hour to allow the drug to bind to potential targets.

[0107] Enzymatic hydrolysis: Divide the above reaction system into two tubes. Add Pronase (final concentration 0.5 μg / μL) to one tube and add an equal volume of solvent as a control to the other tube. Perform a limited enzymatic hydrolysis reaction at room temperature (the time needs to be optimized in the preliminary experiment, such as 30 minutes).

[0108] Termination and denaturation: Add SDS loading buffer and boil to terminate the enzymatic digestion reaction.

[0109] Western blotting (WB) analysis: The degree of degradation of specific candidate target proteins was detected using SDS-PAGE and WB techniques. Compared with the control group, the protein bands protected by matrine F were brighter in the enzyme-added group.

[0110] 7. Limited Protease Digestion Mass Spectrometry (LiP-MS)

[0111] Drug incubation and enzymatic hydrolysis: After incubating the protein extract with 20 μM matrine F or solvent control, Pronase was added for brief enzymatic hydrolysis.

[0112] Protease inactivation and peptide preparation: After terminating the reaction, complete enzymatic hydrolysis was performed using conventional trypsin to generate peptides.

[0113] Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: LC-MS / MS analysis of peptides.

[0114] Data analysis: By comparing the differences in peptide abundance between the matrine F-treated group and the control group, peptides whose proteolytic patterns changed after being bound by matrine F were identified, thereby directly identifying the direct target proteins of matrine F in cells.

[0115] 8. Experimental Results

[0116] (1) Matrine F combined with gefitinib significantly inhibited the growth of PC9-GR drug-resistant tumors in vivo.

[0117] To evaluate the in vivo efficacy of matrine F alone and in combination with gefitinib against gefitinib-resistant tumors, we constructed a PC9-GR cell xenograft (CDX) model in mice subcutaneously. Figure 9 and Figure 10As shown, compared with the control group, gefitinib monotherapy (20 mg / kg / d) only slightly inhibited tumor growth, which is consistent with the drug resistance characteristics of PC9-GR cells. Matrine F monotherapy (30 mg / kg / d) showed moderate antitumor effects, indicating that it has certain antitumor activity. However, the combination therapy group showed the most significant tumor growth inhibition, with tumor volume growth almost stopping throughout the entire treatment cycle (17 days), and the final tumor weight was significantly lower than that of other groups. There was no significant decrease in body weight in any group of mice during treatment, indicating good tolerability of the treatment regimen. This in vivo experiment confirms that matrine F can effectively enhance the efficacy of gefitinib against drug-resistant tumors, and the two have a clear synergistic effect.

[0118] (2) Transcriptome sequencing revealed that combined drug therapy synergistically regulates multiple survival-promoting signaling pathways.

[0119] To explore the mechanism of synergistic effects at the molecular level, we performed transcriptome sequencing (RNA-Seq) on four groups of in vivo tumor tissues. Principal component analysis (PCA) was then combined with... Figure 11 The results showed that the gene expression profiles of the combination therapy group were significantly different from those of the control group and the two single-drug groups, indicating that the combination therapy triggered a unique global transcriptional reprogramming.

[0120] Combination Figure 13 , Figure 14 and Figure 15 As shown, gene set enrichment analysis (GSEA) further revealed that, compared with monotherapy, combination therapy synergistically downregulated multiple signaling pathways closely related to tumor proliferation, survival, and drug resistance. Among these, the downregulation of the TNF signaling pathway, NF-κB signaling pathway, and MAPK signaling pathway was most significant. This suggests that the synergistic effect of combination therapy is not achieved through a single pathway, but rather by simultaneously targeting multiple redundant pro-survival signaling networks, thereby more thoroughly dismantling the tumor's drug resistance defense system.

[0121] (3) Target fishing identification STIM1 as the direct target of matrine F

[0122] To identify the direct molecular target of matrine F, we employed Drug Affinity Target Stability Technology (DARTS). The results combined... Figure 11 and Figure 12 The results showed that as the concentration of matrine F (0, 20, 200 μM) increased, the STIM1 protein's resistance to pronase degradation increased, indicating that matrine F can directly bind to the STIM1 protein and protect it from enzymatic degradation.

[0123] This finding was further validated by limited protease digestion-mass spectrometry (LiP-MS). In samples treated with matrine F (20 μM), multiple peptides of the STIM1 protein exhibited unique protease digestion protection patterns, confirming that STIM1 is a direct intracellular interacting protein of matrine F.

[0124] (4) Elucidation of the mechanism by which matrine F inhibits calcium signaling and reverses drug resistance by targeting STIM1.

[0125] Based on the above findings, we proposed and verified the molecular mechanism by which matrine F reverses EGFR-TKI resistance by targeting STIM1, combined with... Figure 3 As shown:

[0126] Matrine F targets STIM1 and inhibits calcium signaling: Matrine F binds to and inhibits the function of STIM1, resulting in weakened storage-operated calcium influx (SOCE) and a sustained decrease in intracellular calcium ion concentration ([Ca²⁺]i).

[0127] Downregulation of calcium signaling inhibits the NF-κB pathway, leading to decreased intracellular calcium ion concentration and consequently reduced activity of the calcium-dependent kinase CaMKII. Inactivated CaMKII cannot effectively phosphorylate and activate IKKβ, thus hindering the degradation of IκBα. The stability of IκBα imprisons the NF-κB p65 subunit in the cytoplasm, preventing it from entering the nucleus to initiate the transcription of downstream genes (including pro-survival and inflammatory factors). The downregulation of the NF-κB pathway in transcriptomic data perfectly aligns with this finding.

[0128] Downregulation of calcium signaling disrupts the TNF autocrine loop and affects EGFR. The low-calcium environment caused by STIM1 inhibition disrupts the positive feedback loop of TNF-α autocrine secretion, reducing the production of TNF-α ligands. This leads to reduced activation of the TNFR1 receptor and decreased expression of its downstream adaptor proteins TRAF2 / 5 and RIPK1, ultimately reducing the production and release of EGFR ligands such as EGF, thus weakening the abnormal activation of EGFR at its source.

[0129] Downregulation of calcium signaling inhibits the MAPK / ERK pathway, and calcium ions are an important regulator of Ras GTPase activity. Decreased intracellular calcium concentration directly inhibits Ras-GTP loading, thereby blocking the downstream Raf-MEK-ERK signaling cascade. ERK inactivation not only reduces EGFR feedback phosphorylation but also affects its protein stability.

Claims

1. The use of a combination of matrine F and gefitinib in the preparation of a medicament for treating non-small cell lung cancer with secondary resistance to gefitinib, said medicament for treating non-small cell lung cancer, characterized in that, The drug inhibits tumor growth or reverses gefitinib resistance through the synergistic effect of matrine F and gefitinib.

2. The use according to claim 1, characterized in that, The non-small cell lung cancer mentioned is EGFR-mutant non-small cell lung cancer.

3. The use according to claim 1, characterized in that, The matrine F reverses gefitinib resistance by inhibiting the expression or function of matrix interaction molecule 1.

4. The use according to claim 3, characterized in that, The inhibition of matrix interaction molecule 1 by matrine F leads to a decrease in intracellular calcium ion concentration, downregulation of nuclear factor κB signaling pathway activity, and / or downregulation of tumor necrosis factor signaling pathway activity.

5. The use according to claim 1, characterized in that, The matrine F and gefitinib were prepared into a single pharmaceutical composition for simultaneous administration.

6. The use according to claim 1, characterized in that, The matrine F and gefitinib are prepared as separate pharmaceutical units for sequential administration.

7. The use according to claim 1, characterized in that, The dosage ratio of matrine F to gefitinib is 3:2.

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