Application of amygdalin in preparation of medicine for preventing and / or treating non-small cell lung cancer

Abstract

The invention discloses an application of amygdalin in preparation of a medicine for preventing and / or treating non-small cell lung cancer, and belongs to the technical field of biological medicines. It is found for the first time that the natural compound amygdalin can effectively inhibit growth of EGFR mutant lung cancer by promoting lysosome degradation of EGFR protein and inhibiting downstream signal channels of the EGFR protein; in addition, the C797S mutation-mediated tyrosine kinase inhibitor has a remarkable treatment potential on the drug resistance of the C797S mutation-mediated tyrosine kinase inhibitor. In-vivo research further proves that the combination of amygdalin and osimertinib can generate a synergistic effect and enhance the anti-tumor curative effect. It is worth noting that the amygdalin has no obvious cytotoxicity under the effective action concentration and has good safety. Results show that the amygdalin is expected to become a novel EGFR targeted degradation agent, and a new treatment strategy is provided for non-small cell lung cancer patients with tyrosine kinase inhibitor drug resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of amygdalin in the preparation of medicaments for the prevention and / or treatment of non-small cell lung cancer. Background Technology

[0002] Lung cancer is a malignant tumor with high incidence and mortality rates worldwide. Approximately 80-85% of these cases are non-small cell lung cancer (NSCLC). In recent years, epidermal growth factor receptor tyrosine kinase inhibitors (Epidermal growth factor receptor tyrosine kinase inhibitors) have been used to address this issue. EGFR-TKIs (ethylene glycol tyrosine kinase inhibitors) are the first-line drugs for patients with advanced EGFR-mutant non-small cell lung cancer. However, a large number of patients develop acquired resistance due to the T790M mutation within 10 to 14 months after receiving EGFR-TKI treatment. Fortunately, with the development of third-generation EGFR-TKIs (especially osimertinib), the problem of reduced EGFR-TKI sensitivity caused by the T790M resistance mutation has been solved.

[0003] However, patients treated with osimertinib usually develop resistance after about 10 months of treatment. The most common cause is the C797S mutation in exon 20, which accounts for 10%-26% of second-line osimertinib resistance cases and 7% of first-line osimertinib resistance cases.

[0004] Fourth-generation EGFR inhibitors specifically target the catalytically active Lys745 site, selectively altering the spatial conformation of mutant EGFR and preventing its binding to ligands. However, most of these drugs are still in the early stages of clinical trials. Therefore, there are currently no recommended standard treatment regimens for these drug-resistant patients. Summary of the Invention

[0005] Amygdalin is a naturally occurring cyanogenic glycoside widely found in various plants, particularly in the seeds of apricots, bitter almonds, and peaches, and it readily forms hydrates. Its toxicity originates from the benzaldehyde and hydrogen cyanide produced during oral administration; intravenous administration is significantly less toxic than oral administration. The human tolerance dose for oral amygdalin is 0.6-1 g / day, while the tolerance dose for intravenous administration can reach 10 g / day.

[0006] For centuries, amygdalin has been used in Asia, Europe, and other regions to treat a variety of diseases, including cough, asthma, nausea and vomiting, leprosy, and vitiligo. Recent studies have found that amygdalin can exert significant anti-tumor effects against cervical cancer, kidney cancer, and liver cancer cells by influencing cell cycle progression, inducing apoptosis, regulating immune function, and regulating gut microbiota.

[0007] This invention is the first to discover that the natural compound amygdalin can effectively inhibit the growth of EGFR-mutant lung cancer by promoting the lysosomal degradation of EGFR protein and inhibiting its downstream signaling pathways, and has significant therapeutic potential against C797S mutation-mediated resistance to tyrosine kinase inhibitors.

[0008] In view of this, the purpose of the present invention is to provide an application of amygdalin in reversing EGFR-TKI resistance.

[0009] In a first aspect, the present invention provides the use of amygdalin or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a medicament for the prevention and / or treatment of non-small cell lung cancer.

[0010] Furthermore, the use of amygdalin or a pharmaceutically acceptable salt or hydrate thereof in combination with EGFR-TKI in the preparation of medicaments for the prevention and / or treatment of non-small cell lung cancer.

[0011] Furthermore, the non-small cell lung cancer is non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutation.

[0012] Furthermore, the non-small cell lung cancer is non-small cell lung cancer that has developed resistance to EGFR-TKIs. That is, the non-small cell lung cancer is EGFR-TKI-resistant non-small cell lung cancer.

[0013] Furthermore, the non-small cell lung cancer is EGFR C797S mutation-mediated drug-resistant non-small cell lung cancer (NSCLC).

[0014] Further, the EGFR-TKI includes at least one of osimertinib, ametinib, vometinib, befotinib, afatinib, dacomitinib, gefitinib, erlotinib, and icotinib; preferably osimertinib.

[0015] In a second aspect, the present invention provides the use of amygdalin, particularly a composition comprising amygdalin, more preferably a composition comprising amygdalin and osimertinib, in the preparation of a reversal medicament for reversing acquired resistance to osimertinib in non-small cell lung cancer.

[0016] In in vivo and in vitro studies of non-small cell lung cancer, this invention found that: 1. Amygdalin can effectively inhibit the expression level of EGFR protein at doses without significant cytotoxicity; 2. The combination of osimertinib and amygdalin has a significant anti-tumor effect; 3. This combination regimen has a significant inhibitory effect on the C797S mutant strain.

[0017] In a third aspect, the present invention provides a pharmaceutical composition for treating non-small cell lung cancer, the active ingredients of which include amygdalin or a pharmaceutically acceptable salt thereof, hydrate thereof, and osimertinib.

[0018] Furthermore, the pharmaceutical composition also includes pharmaceutical excipients.

[0019] Furthermore, the pharmaceutical excipients include at least one of fillers, binders, disintegrants, lubricants, flavoring agents, or preservatives.

[0020] Furthermore, the dosage form of the drug includes oral dosage forms and injectable dosage forms.

[0021] Compared with the prior art, the present invention has the following advantages: This invention is the first to discover that the natural compound amygdalin can effectively inhibit the growth of EGFR-mutant lung cancer by promoting lysosomal degradation of EGFR protein and inhibiting its downstream signaling pathways, and it also shows significant therapeutic potential against C797S mutation-mediated tyrosine kinase inhibitor resistance. In vivo studies further confirmed that amygdalin, when used in combination with osimertinib, produces a synergistic effect, enhancing anti-tumor efficacy. Notably, amygdalin exhibits no significant cytotoxicity at effective concentrations and demonstrates good safety. These results suggest that amygdalin holds promise as a novel EGFR-targeting degradation agent, providing a new treatment strategy for patients with non-small cell lung cancer resistant to tyrosine kinase inhibitors. Attached Figure Description

[0022] Figure 1 The figures show the experimental results of amygdalin showing no significant cytotoxicity and downregulating EGFR protein expression. Among them, a is a schematic diagram of the high-throughput screening process for traditional Chinese medicine monomers; b is a scatter plot of the initial screening results; c is the chemical structure of amygdalin; d is the EGFR immunofluorescence image of NCI-H1993 cells; e is the Western blot result of EGFR protein expression; and fi is the cytotoxicity detection graph of NCI-H1993 and NCI-H1975 cells.

[0023] Figure 2 This is a graph showing the experimental results of the effect of amygdalin on EGFR expression in EGFR mutant / wild-type lung cancer cells. In the graph, ac is the Western blot result of EGFR / p-EGFR in mutant cells; df is the flow cytometry quantification of EGFR on the surface of mutant cells; gi is the Western blot result of total EGFR in normal / wild-type cells; and jl is the flow cytometry quantification of EGFR on the surface of normal / wild-type cells.

[0024] Figure 3This is an experimental result diagram showing how amygdalin weakens the activity of downstream signaling pathways by inhibiting EGFR. Among them, ab and de are staining diagrams of cell colony formation assay; c and f are CCK-8 assay diagrams of mutant cell viability; gh is staining diagram of wild-type / EGF-stimulated wild-type cell colony formation assay; i is CCK-8 assay diagram of corresponding cell proliferation; and jl is Western blot result diagram of EGFR and downstream signaling proteins in cells.

[0025] Figure 4 This is a graph showing the experimental results of the combined use of amygdalin and osimertinib to enhance the inhibitory effect on EGFR-mutant lung cancer cells. Among them, ac is the cell proliferation CCK-8 detection graph; df is the Western blot result of EGFR and downstream signaling proteins in cells; g is the xenograft growth curve; h is the actual image of the xenograft; i is the xenograft weight statistics graph; and jk is the Western blot result of EGFR and downstream signaling proteins in tumor tissue.

[0026] Figure 5 This is an experimental result diagram showing the effect of amygdalin promoting EGFR degradation via the lysosomal pathway. Among them, ab is the q-PCR detection diagram of EGFR mRNA in cells; cd is the Co-IP detection diagram of EGFR ubiquitination; ef is the Western blot result of EGFR protein half-life; g is the immunofluorescence colocalization diagram of EGFR and LAMP1; hi is the Western blot result of EGFR in lysosomal components; jk is the Western blot result of EGFR after inhibitor pretreatment.

[0027] Figure 6 This is an experimental result diagram of amygdalin reversing EGFR C797S mutation-mediated osimertinib resistance. Among them, ab is the Western blot result of EGFR / p-EGFR in C797S mutant cells; cd is the CCK-8 assay result of mutant cell proliferation; e, g, i, k, m, o are the Western blot results of EGFR / p-EGFR in cells from 6 patients; f, h, j, l, n, p are the CCK-8 assay results of cell proliferation from the corresponding patients. Detailed Implementation

[0028] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] Unless otherwise specified, all materials and reagents used in this invention are available from commercially available products in the field.

[0031] 1. The experimental method of this invention includes: 1.1 Cell Culture: NCI-H1975, HCC827, PC9, NCI-H1993, NCI-H460, and BEAS-2B cell lines were purchased from the American Type Culture Collection (ATCC). Cells were cultured in a 37°C, 5% CO2 incubator using RPMI-1640 or Duchenne Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS), and mycoplasma contamination was periodically checked.

[0032] 1.2 Cell transfection, lentiviral infection, and construction of stable cell lines: EGFR-GFP was overexpressed in NCI-H1993 cells via lentivirus-mediated transformation, as follows: The pSin-EF2-EGFR-GFP plasmid was co-transfected with the packaging plasmids psPAX2 (gag, pol) and pMD2.G into HEK293T cells using either calcium phosphate co-precipitation or polyethyleneimine (PEI, Polysciences) transfection protocol.

[0033] 72 hours after transfection, the cell culture supernatant was collected, filtered through a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore), and then used to infect NCI-H1993 cells in the presence of 10 μg / mL polybrene (Sigma).

[0034] After infection, stable monoclonal cell lines were selected using 0.5–2 μg / mL puromycin, and then expanded for subsequent drug susceptibility experiments.

[0035] 1.3. Protein immunoblotting and co-precipitation: Protein immunoblotting and co-immunoprecipitation experiments were performed according to previous methods. The specific procedures were as follows: Cell lysis: After cell collection, cells were lysed using RIPA lysis buffer (50 mmol / L Tris-HCl, pH 7.4; 150 mmol / L NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS). A protease inhibitor mixture (Selleck, B14001) and benzyl sulfonyl fluoride (PMSF, Servicebio, G2008) were added to the lysis buffer. After incubation on ice for 30 min, cells were centrifuged at 4 °C and 12000 × g for 10 min, and the supernatant was collected.

[0036] Immunoblot analysis: The supernatant was mixed with 5×SDS loading buffer, separated by 10% SDS-PAGE, and then subjected to subsequent immunoblotting detection.

[0037] Immunoprecipitation analysis: Cell lysate was incubated overnight with specific antibody and Protein A / G magnetic beads (Pierce, 20333 / 20399), followed by washing three times with PBS containing 0.5% Triton X-100, and the immunoprecipitation complex was analyzed by Western blot.

[0038] Antibody incubation and signal detection: Primary antibody was used to incubate total cell lysate and immunoprecipitate, followed by the addition of appropriate secondary antibody and chemiluminescent substrate for detection. Images were acquired and analyzed using the ChemiDoc MP imaging system (Bio-Rad) and Image Lab Software 6.0 (Bio-Rad).

[0039] 1.3. Cellular immunofluorescence: After appropriate treatment, NCI-H1993, NCI-H1975, and HCC827 cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. After washing with phosphate buffer, the cells were blocked with 3% bovine serum albumin (BSA) at 25°C for 1 hour. The cells were then incubated overnight at 4°C with primary antibody. The next day, after washing, the cells were incubated at room temperature with secondary antibody for 1 hour. Before staining, the cell nuclei were stained with an antifluorescence quencher containing 4',6-diamidinyl-2-phenylindole (DAPI).

[0040] 1.4 Flow cytometry: Cell surface protein detection: Single-cell suspensions of cultured cells were prepared and stained with fluorescently labeled primary antibodies at 4°C for 30 minutes. Intracellular protein detection: A commercially available fixation / permeabilization kit was used according to the instructions. After fixation and permeabilization, fluorescently labeled primary antibodies were added for incubation. After washing with staining buffer, secondary antibody staining was performed if necessary. Detection was performed using a CytoFLEXLX flow cytometer equipped with 405nm, 488nm, and 638nm lasers. Fluorescence signals of fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) were detected using 525 / 40nm, 585 / 42nm, and 660 / 20nm filters, respectively. At least 10,000-100,000 cell events were collected per sample, and data acquisition and analysis were performed using CytExpert 2.4 software.

[0041] 1.5 Cloning experiment: The colony formation assay was used to evaluate the effect of amygdalin on the long-term proliferative capacity of cells. The procedure was as follows: 1000 cells per well were seeded into 6-well plates, medium containing a specified concentration of amygdalin was added, and the cells were cultured for 14 days to form colonies; the colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet; imaging and colony counting were performed using a cell imaging system.

[0042] 1.6 Cell viability assay (CCK-8): Cell viability was assessed using the CCK-8 assay: Cells were seeded at a density of 3000 cells per well in a 96-well plate. After appropriate treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C with 5% carbon dioxide for 2 hours. The absorbance at 450 nm was measured using an ELISA reader.

[0043] 1.7 Protein half-life experiment: Cells were treated with 40 μg / mL cyclohexylimide to inhibit protein synthesis, and cells were collected at 0, 8, 16, and 24 hours. The stability of EGFR protein was detected by Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the gray value of the EGFR band relative to GAPDH was detected at each time point.

[0044] 1.8 Real-time quantitative polymerase chain reaction: After treating non-small cell lung cancer cells with different concentrations of amygdalin for 24 hours, total RNA was extracted using the chloroform-guanidine isothiocyanate-phenol method (Trizol). The RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using a commercially available reverse transcription kit. Using GAPDH as an internal reference gene, the mRNA expression level of EGFR was detected by real-time quantitative polymerase chain reaction, and gene expression analysis was performed using the 2-ΔΔCT method.

[0045] 1.9 Lysosome isolation: Lysosomes were isolated from cultured cells using the Minute™ Lysosome Isolation Kit according to the manufacturer's instructions. Brief procedure: Cells were collected, washed with cold phosphate buffer, and resuspended in buffer A; incubated on ice for 10 minutes, followed by vigorous vortexing and centrifugation at 16000×g for 30 seconds before filtering; the filtrate was centrifuged multiple times to remove cell nuclei, mitochondria, and cell debris; the supernatant was added to buffer B and incubated on ice to precipitate lysosomes; the lysosomes were collected by centrifugation at 11000×g for 10 minutes, and the precipitate was washed and resuspended in a detergent-containing buffer for later use; the purity of the isolated lysosomes was verified by Western blotting using antibodies against lysosomal markers (such as LAMP1 and LAMP2).

[0046] 1.10 Animal experiments: Nude mice aged 6 weeks and weighing approximately 20 grams were purchased from Shanghai Southern Model Biotechnology Co., Ltd. and housed in a specific pathogen-free (SPF) environment. All experimental mice were housed under the same conditions, including temperature, humidity, and light cycle, to ensure uniform experimental conditions. Standardized feed was used to ensure consistent dietary composition and avoid interference from dietary factors on experimental results. All experimental procedures were strictly in accordance with the regulations of the Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University.

[0047] Constructing xenograft tumor models to evaluate the in vivo effects of drugs: 2×10 6 NCI-H1975 cells were subcutaneously inoculated into mice. After tumor cell inoculation, mice were randomly divided into groups. When the tumor volume reached 50 cubic millimeters, drug treatment was initiated. Mice in the amygdalin group and osimertinib group were injected intraperitoneally with the corresponding drug every two days (osimertinib dose was 10 mg / kg), while the control group was injected intraperitoneally with an equal volume of solvent. The treatment was continued for 21 days.

[0048] The body weight and tumor volume of the mice were measured with calipers every 3 days, and the daily survival rate was recorded. The tumor volume was calculated using the formula: Tumor volume (cubic millimeters) = π / 6 × length × width². After 21 days of treatment, all mice were sacrificed, the tumors were removed and weighed.

[0049] 1.11 Quantitative and Statistical Analysis: Statistical analysis and plotting were performed using GraphPad Prism 10 software. One-way / two-way ANOVA combined with Dukär's multiple comparison test, unpaired t-test, and chi-square test were used to assess the statistical significance of differences between groups.

[0050] 2. Results 2.1 Amygdalin has no significant cytotoxicity and can downregulate EGFR protein expression. As cancer treatment strategies shift from traditional highly toxic radiotherapy and chemotherapy to safer molecularly targeted therapies, this invention aims to screen compounds with low cytotoxicity and the ability to effectively regulate EGFR expression from a library of traditional Chinese medicine monomers. To screen for traditional Chinese medicine monomers with EGFR-regulating potential, this invention uses the non-small cell lung cancer cell line NCI-H1993 as an experimental model.

[0051] Since exogenous overexpression plasmids are usually driven by strong promoters (such as cytomegalovirus promoters), their expression is difficult to regulate with drugs. Therefore, we used a multi-concentration lentivirus gradient infection method to establish a cell model with stable overexpression of EGFR-green fluorescent protein (GFP) and screened out monoclonal cell lines with EGFR messenger ribonucleic acid (mRNA) expression levels increased by 1.5 to 2 times for subsequent drug screening.

[0052] This invention performed high-throughput phenotypic screening on 1103 monomeric compounds from traditional Chinese medicines. The screening conditions were a treatment with a concentration of 20 μmol for 24 hours. First, well sites with cytotoxicity were excluded. Then, the expression level of EGFR-GFP was assessed by fluorescence intensity. The overall screening process is described below. Figure 1 a. The results showed that the fluorescence intensity of the amygdalin-treated group was significantly reduced ( Figure 1 (b) suggests that it has the potential to downregulate EGFR expression; the chemical structure of amygdalin is shown in [see image]. Figure 1 c.

[0053] To further investigate its mechanism of action, we treated NCI-H1993 cells with 20 μmol of amygdalin and performed immunofluorescence analysis. The results showed that EGFR expression was downregulated and membrane localization disappeared. At the same time, no significant changes in nuclear morphology or typical apoptotic features were observed, indicating that at this concentration, amygdalin only reduced EGFR expression and did not induce apoptosis. Figure 1 d). Western blotting further confirmed that as the concentration of amygdalin increased, the levels of both endogenous and exogenous EGFR proteins decreased. Figure 1 e).

[0054] Furthermore, we evaluated the cytotoxicity of this compound and found that treatment of NCI-H1993 and NCI-H1975 cells with different concentrations of amygdalin for 24 hours, even at a concentration of 120 μmol, did not induce significant cytotoxicity. Figure 1 The f-1i indicates that it has good safety within the effective concentration range for downregulating EGFR.

[0055] In summary, amygdalin is a non-cytotoxic traditional Chinese medicine monomer that can effectively downregulate EGFR expression and has the potential to become a targeted therapy drug for lung cancer.

[0056] 2.2. Regulatory effect of amygdalin on EGFR expression in EGFR mutant and wild-type lung cancer cells To further investigate the regulatory effect of amygdalin on EGFR, we expanded the scope of our validation, selecting various lung cancer cell lines and normal bronchial epithelial cells for experiments. Experimental models included EGFR mutant cell lines (NCI-H1975, HCC827, PC9) and EGFR wild-type cell lines (NCI-H292, NCI-H460), with the normal bronchial epithelial cell line BEAS-2B serving as a control.

[0057] After treating cells with amygdalin at concentrations of 10, 20, and 40 μmol for 24 hours, the total EGFR protein and membrane-localized EGFR levels in all tested cell lines were significantly reduced. Figure 2 a-2l), and the level of phosphorylated EGFR (p-EGFR) in EGFR mutant cells was significantly inhibited ( Figure 2 The above results indicate that amygdalin can effectively downregulate EGFR expression in various lung cancer cells, demonstrating the broad applicability of its regulatory effect.

[0058] 2.3 Amygdalin weakens downstream signaling pathway activity by inhibiting EGFR. Based on the non-cytotoxic nature of amygdalin, we further explored its antitumor efficacy. Using clonogenic assays and the Cell Counting Kit-8 (CCK-8) proliferation assay, we examined the effects of amygdalin at gradient concentrations of 10, 20, and 40 μmol in EGFR mutant lung cancer cell lines (NCI-H1975, HCC827) and the EGFR wild-type cell line NCI-H460.

[0059] The results showed that amygdalin has a significant anti-tumor effect. Figure 3 a-3b and 3d-3e can effectively inhibit the proliferation of EGFR-mutant lung cancer cells. Figure 3 c, 3f). Western blotting experiments further confirmed that amygdalin effectively inhibited EGFR phosphorylation in NCI-H1975 and HCC827 cells, as well as the activation of downstream signaling pathways STAT3, AKT, and mitogen-activated protein kinase (MAPK). Figure 3 j、3k).

[0060] In the EGFR wild-type cell line NCI-H460, amygdalin alone did not produce a significant anti-tumor effect; however, after epidermal growth factor (EGF) induced EGFR activation, amygdalin effectively inhibited cell proliferation. Figure 3 g-3i) and reduces EGFR phosphorylation levels and activation of downstream STAT3, AKT, and MAPK pathways. Figure 3The above results indicate that amygdalin exerts its antitumor effect in an EGFR-dependent manner by regulating EGFR activity and its downstream signaling pathways.

[0061] 2.4. The combined use of amygdalin and osimertinib enhances the inhibitory effect on EGFR-mutant lung cancer cells. EGFR-TKIs are the core targeted therapies for EGFR-mutant lung cancer, but many patients develop acquired resistance after treatment, leading to treatment failure. Based on previous research results, we hypothesized that amygdalin may have a synergistic effect with the third-generation EGFR-TKI osimertinib, enhancing anti-tumor efficacy, and verified this hypothesis through a series of in vivo and in vitro experiments.

[0062] In in vitro experiments, CCK-8 proliferation assays showed that, compared with monotherapy, amygdalin combined with osimertinib significantly enhanced the inhibitory effect on the proliferation of EGFR mutant cell lines (NCI-H1975, HCC827, PC9). Figure 4 a-4c). Further Western blotting experiments revealed that the combination therapy had a stronger inhibitory effect on EGFR and its downstream STAT3 / AKT / MAPK signaling pathway. Figure 4 d-4f).

[0063] To evaluate in vivo efficacy, we established an NCI-H1975 cell xenograft tumor model and randomly divided mice into a solvent control group, an amygdalin monotherapy group, an osimertinib monotherapy group, and a combination therapy group. Results showed that amygdalin monotherapy moderately inhibited tumor growth, while the combination therapy group exhibited the most significant inhibitory effect on tumor progression. Figure 4 g-4i).

[0064] Molecular analysis of tumor tissue confirmed that intraperitoneal injection of amygdalin downregulated EGFR expression and inhibited downstream signaling pathways. Figure 4 j), the combined treatment group showed a more thorough inhibition of these downstream signaling pathways (j), Figure 4 k).

[0065] In summary, the combination of amygdalin and osimertinib can synergistically inhibit the EGFR signaling pathway and enhance anti-tumor activity, providing a theoretical basis for further exploring the application of this combination strategy in EGFR-mutant lung cancer.

[0066] 2.5 Amygdalin promotes EGFR degradation via the lysosomal pathway. To elucidate the molecular mechanism by which amygdalin regulates EGFR and enhances therapeutic efficacy, we treated EGFR mutant cell lines (NCI-H1975 and HCC827) with amygdalin at concentrations of 10, 20, and 40 μmol for 24 hours. The results showed that amygdalin had no effect on EGFR mRNA expression levels. Figure 5(a, 5b) suggests that it functions through post-transcriptional regulation.

[0067] To investigate whether amygdalin affects the stability of EGFR protein, we performed a cyclohexylimide (CHX) tracking experiment in cells of the control group and the amygdalin-treated group, collecting cells at 0, 8, 16, and 24 hours for analysis. The results showed that amygdalin significantly shortened the half-life of EGFR protein. Figure 5 e, 5f), suggesting that it promotes the degradation of EGFR protein.

[0068] EGFR renewal is mainly mediated by the ubiquitin-proteasome system and the endosome-lysosome pathway. Therefore, we detected the ubiquitination level of EGFR using an immunoprecipitation assay and found that amygdalin treatment did not increase the ubiquitination modification of EGFR. Figure 5 c, 5d), which excludes the proteasome-dependent degradation pathway.

[0069] Immunofluorescence colocalization assays further revealed that EGFR translocates from the cell membrane to the cytoplasm over time, and the degree of colocalization with the lysosomal marker lysosome-associated membrane protein 1 (LAMP1) gradually increases. Figure 5 g). Subsequent lysosomal isolation and purification experiments directly confirmed that amygdalin can induce the enrichment of EGFR in lysosomes ( Figure 5 h、5i).

[0070] To verify the functional role of lysosomes in this process, we inhibited lysosomal acidification with bafloxacin A1 and chloroquine, respectively. Both treatments effectively reversed amygdalin-mediated EGFR downregulation. Figure 5 (j, 5k), while the proteasome inhibitor MG132 had no rescue effect.

[0071] In summary, amygdalin promotes EGFR degradation through the lysosomal pathway, and this process does not affect transcriptional regulation and ubiquitin-proteasome activity, providing a mechanistic basis for its role in enhancing the efficacy of EGFR-targeted therapy.

[0072] 2.6 Amygdalin Reverses Osimertinib Resistance Mediated by EGFR C797S Mutation Some patients develop acquired resistance to osimertinib, a third-generation tyrosine kinase inhibitor, with EGFR C797S mutation being the primary resistance mechanism. Currently, effective treatment options for these patients remain very limited. We hypothesize that amygdalin may inhibit this resistance mutation and are conducting relevant validation experiments.

[0073] We constructed stable EGFR C797S mutant overexpression cell lines in NCI-H1993 and NCI-H1975 cell backgrounds, and treated the cells with amygdalin (20, 40 μmol) or osimertinib (5, 10 μmol) for 24 hours. The results showed that osimertinib failed to inhibit the proliferation and EGFR phosphorylation of C797S mutant cells, while amygdalin treatment significantly inhibited cell proliferation and substantially reduced the levels of total EGFR and phosphorylated EGFR. Figure 6 (a-6d), suggesting its potential to overcome drug resistance mediated by C797S mutation.

[0074] To further validate the clinical relevance of the findings, we isolated and cultured primary cells from tumor tissues of six patients carrying secondary C797S mutations. Under the same experimental conditions, amygdalin exhibited significant antitumor activity, even when osimertinib failed to inhibit cell proliferation, it still reduced the expression of EGFR and phosphorylated EGFR. Figure 6 e-6o).

[0075] In summary, amygdalin can effectively inhibit the growth of EGFR C797S mutant tumors and is expected to become a potential treatment strategy for patients resistant to third-generation tyrosine kinase inhibitors.

[0076] This invention is the first to discover that the natural compound amygdalin can effectively inhibit the growth of EGFR-mutant lung cancer by promoting lysosomal degradation of EGFR protein and inhibiting its downstream signaling pathways, and has significant therapeutic potential against C797S mutation-mediated resistance to tyrosine kinase inhibitors.

[0077] The core discovery of this invention is the elucidation of a novel antitumor mechanism of amygdalin. Traditional EGFR-TKIs (such as gefitinib and osimertinib) inhibit EGFR phosphorylation and downstream oncogenic signaling pathways by competitively binding to the ATP-binding pocket of the EGFR kinase domain (mimicking the adenine portion of ATP); while amygdalin reduces the protein stability of EGFR by promoting lysosomal-mediated EGFR degradation.

[0078] Multiple experimental evidences support this mechanism: immunofluorescence experiments show that amygdalin promotes the translocation of EGFR from the cell membrane to the cytoplasm and increases its co-localization with lysosomes; lysosomal dissociation experiments directly confirm the increased transport of EGFR to lysosomes; furthermore, inhibition of lysosomal acidification can completely reverse amygdalin-mediated EGFR degradation. This unique mode of action, independent of kinase activity inhibition, provides a new strategy for overcoming drug resistance problems associated with kinase domain mutations.

[0079] EGFR mutations are a key target for targeted therapy in non-small cell lung cancer, but acquired resistance—especially resistance to third-generation TKIs induced by C797S mutations—remains a significant challenge in clinical treatment. Therefore, the role of amygdalin in effectively degrading EGFR has important clinical significance.

[0080] This invention demonstrates that amygdalin significantly inhibits the proliferation of osimertinib-resistant C797S mutant cells in both in vitro cell lines and patient-derived primary culture models, providing a new treatment option for patients who have failed third-generation TKIs. Furthermore, the combination of amygdalin and osimertinib exhibits synergistic antitumor activity, suggesting that this traditional Chinese medicine monomer has the potential to become an important component of combination therapy, potentially delaying or even overcoming the development of drug resistance.

[0081] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0082] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of amygdalin or its pharmaceutically acceptable salts or hydrates in the preparation of drugs for the prevention and / or treatment of non-small cell lung cancer.

2. The use of amygdalin or a pharmaceutically acceptable salt or hydrate thereof in combination with EGFR-TKI in the preparation of medicaments for the prevention and / or treatment of non-small cell lung cancer.

3. The application according to claim 1 or 2, characterized in that, The non-small cell lung cancer mentioned refers to non-small cell lung cancer with epidermal growth factor receptor mutation.

4. The application according to claim 1 or 2, characterized in that, The non-small cell lung cancer mentioned refers to non-small cell lung cancer that has developed resistance to EGFR-TKI.

5. The application according to claim 1 or 2, characterized in that, The non-small cell lung cancer mentioned is drug-resistant non-small cell lung cancer mediated by EGFR C797S mutation.

6. The application according to claim 2, characterized in that, The EGFR-TKI includes at least one of osimertinib, ametinib, vometinib, befotinib, afatinib, dacomitinib, gefitinib, erlotinib, and icotinib; preferably osimertinib.

7. The use of a composition containing amygdalin in the preparation of a reversal medicament for reversing acquired resistance to osimertinib in non-small cell lung cancer, preferably, the composition containing amygdalin further comprising an EGFR-TKI, more preferably, the composition containing amygdalin being a composition comprising amygdalin and osimertinib.

8. A pharmaceutical composition for treating non-small cell lung cancer, characterized in that, Its active ingredients include amygdalin or its pharmaceutically acceptable salts, hydrates, and osimertinib.

9. The pharmaceutical composition according to claim 8, characterized in that, The drug also includes pharmaceutical excipients, which include at least one of fillers, binders, disintegrants, lubricants, flavoring agents, or preservatives.

10. The pharmaceutical composition according to claim 8, characterized in that, The dosage forms of the drug include oral dosage forms and injectable dosage forms.

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