A small molecule coupling compound of traditional Chinese medicine and its application in drug-resistant tumors

Abstract

The present invention relates to the field of biomedicine technology, and discloses a Chinese medicine small molecule coupling compound and its application in drug-resistant tumors. The present invention utilizes a Val-Cit linker that responds to cathepsin and a linker linker that responds to PH to couple Toosendanin and luteolin, thereby obtaining a Chinese medicine small molecule coupling compound with a novel structure; the Chinese medicine small molecule coupling compound obtained by the present invention has good anti-tumor effect and tumor targeting, can increase the concentration of drugs in tumor tissues, and reduce the toxic and side effects of drugs on normal tissues, and the coupled Toosendanin and luteolin can synergistically inhibit the proliferation of tumor cells, induce cell apoptosis, and have the effect of reversing the drug resistance of non-small cell lung cancer to osimertinib.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a Chinese medicine small molecule coupling compound and application thereof in drug-resistant tumors. Background Art

[0002] Lung cancer is a malignant tumor with high morbidity and mortality rates worldwide, which seriously threatens human life and health. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 80% of all lung cancers. With the continuous deepening of molecular biology and oncology research, targeted therapy has become one of the important means of treating lung cancer. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI), such as osimertinib, have achieved significant therapeutic effects in the treatment of lung cancer. However, EGFR-TKI drugs will inevitably develop drug resistance, which has become a key bottleneck restricting its clinical application and long-term survival of patients.

[0003] The mechanisms of EGFR-TKI resistance are complex and diverse, including secondary mutations of the EGFR gene, bypass activation, reactivation of downstream signaling pathways, and changes in the tumor microenvironment. Among them, the T790M mutation of the EGFR gene is a common cause of resistance to the first and second generation EGFR-TKIs, and after the initial significant treatment effect of the third generation EGFR-TKI osimertinib, a variety of resistance mechanisms including the C797S mutation gradually emerged. The emergence of these resistance problems has put the treatment of lung cancer patients in a difficult situation, and new treatment strategies are urgently needed to overcome resistance, prolong patients' survival time and improve their quality of life.

[0004] In the search for strategies to overcome EGFR-TKI resistance, combination therapy is a common approach. However, traditional combination therapy regimens often have problems such as drug interactions, toxicity superposition, and poor treatment compliance. Therefore, the development of new drug combinations or innovative drug forms has become a research hotspot.

[0005] Traditional Chinese medicine has a long history of application and rich clinical experience in China, and many of its small molecule compounds have potential anti-tumor activity. Toosendanin and luteolin are two small molecules of traditional Chinese medicine with good biological activity. Toosendanin is a tetracyclic triterpenoid compound extracted from Toosendan bark, which has multiple biological activities such as anti-inflammatory, anti-tumor, and immune regulation. Studies have shown that Toosendanin can inhibit the proliferation of tumor cells, induce cell apoptosis, and has a certain effect of reversing tumor multidrug resistance. Luteolin is widely present in many plants and is a flavonoid compound with multiple pharmacological effects such as antioxidant, anti-inflammatory, and anti-tumor. Luteolin can inhibit the growth, invasion and metastasis of tumor cells by regulating multiple signaling pathways, and it also has a certain cardioprotective effect.

[0006] In view of the potential anti-tumor activity of toosendan and luteolin and their complementarity in mechanism of action, if toosendan and luteolin are linked by chemical bonds to construct a Chinese medicine small molecule coupling compound, it is expected to provide new ideas and methods for overcoming the drug resistance of EGFR-TKI. Summary of the invention

[0007] The purpose of the present invention is to provide a Chinese medicine small molecule coupled compound and its application in drug-resistant tumors. The Chinese medicine small molecule coupled compound has good anti-tumor effect and tumor targeting, and has the effect of reversing the drug resistance of non-small cell lung cancer to osimertinib.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] One of the technical solutions of the present invention:

[0010] A Chinese medicine small molecule coupling compound is obtained by coupling toosendanin and luteolin through a Val-Cit linker that responds to cathepsin and a linker linker that responds to pH.

[0011] Furthermore, the structural formula of the Chinese medicine small molecule coupling compound is:

[0012]

[0013] The second technical solution of the present invention:

[0014] Application of the above-mentioned Chinese medicine small molecule coupling compound in the preparation of drugs for preventing and / or treating tumors.

[0015] Furthermore, the tumor is non-small cell lung cancer.

[0016] Furthermore, the non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0017] The third technical solution of the present invention:

[0018] A drug for preventing and / or treating tumors, wherein the active ingredient of the drug for preventing and / or treating tumors comprises the above-mentioned traditional Chinese medicine small molecule coupled compound or a pharmaceutically acceptable salt thereof.

[0019] The present invention provides a drug for preventing and / or treating tumors, comprising an active ingredient within a safe and effective amount, namely, the Chinese medicine small molecule coupled compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient;

[0020] Among them, the professional term "safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects;

[0021] The professional term "pharmaceutically acceptable excipients" refers to: one or more compatible solid / liquid fillers or gel substances, suitable for human use, and must be of sufficient purity and sufficiently low toxicity;

[0022] The professional term "compatibility" means that the solid / liquid filler or gel material can be mixed with the active ingredient Chinese medicine small molecule coupling compound or its pharmaceutically acceptable salt of the present invention without significantly reducing the efficacy of the active ingredient Chinese medicine small molecule coupling compound or its pharmaceutically acceptable salt;

[0023] Pharmaceutically acceptable excipients include: cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil), polyols (such as propylene glycol, glycerol, mannitol, sorbitol), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives and pyrogen-free water, etc.;

[0024] When administering a drug for preventing and / or treating tumors provided by the present invention, a safe and effective amount of the active ingredient, a small molecule Chinese medicine coupling compound or a pharmaceutically acceptable salt thereof, is administered to a mammal (human) in need of treatment, wherein the administration dose is a pharmaceutically effective administration dose, and the specific administration dose should take into account factors such as the administration route and the patient's health status, and is a conventional technical means of those skilled in the art;

[0025] The administration method of the drug for preventing and / or treating tumors provided by the present invention is not particularly limited, and representative administration methods include but are not limited to: oral, intratumoral, rectal, parenteral (intravenous, intramuscular / subcutaneous);

[0026] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules;

[0027] The solid dosage form for oral administration is prepared by mixing the active ingredient with at least one conventional inert excipient or carrier, such as sodium citrate, dicalcium phosphate, or with the following ingredients:

[0028] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0029] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic;

[0030] (c) humectants, for example, glycerin;

[0031] (d) disintegrating agents, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0032] (e) a buffering solvent, for example, paraffin;

[0033] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0034] (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate;

[0035] (h) adsorbents, for example, kaolin;

[0036] (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof;

[0037] The solid dosage form for oral administration can also be prepared by coating and shell materials, such as enteric coating, polymeric material, wax material or other materials known in the art. The solid dosage form for oral administration prepared by coating and shell materials can release the active ingredient, the small molecule coupled compound of traditional Chinese medicine or its pharmaceutically acceptable salt in the digestive tract slowly, thereby improving the efficacy;

[0038] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures; in addition to the active ingredient, the Chinese medicine small molecule coupling compound or its pharmaceutically acceptable salt, the liquid dosage form for oral administration may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances; in addition to these inert diluents, the liquid dosage form may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices;

[0039] Preparations for parenteral injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions, suitable aqueous and non-aqueous carriers, diluents, solvents or excipients (including water, ethanol, polyols) and suitable mixtures;

[0040] The Chinese medicine small molecule coupled compound of the present invention can be administered alone, or can be used in combination with other known drugs for treating or improving similar symptoms.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention utilizes a Val-Cit linker that responds to cathepsin and a linker linker that responds to pH to couple toosendan and luteolin, thereby obtaining a novel structural Chinese medicine small molecule coupling compound.

[0043] The Chinese medicine small molecule coupled compound obtained by the present invention has good anti-tumor effect and tumor targeting, can increase the concentration of the drug in tumor tissue, and reduce the toxic side effects of the drug on normal tissues. Moreover, the coupled toosendanin and luteolin can synergistically inhibit the proliferation of tumor cells, induce cell apoptosis, and have the effect of reversing the drug resistance of non-small cell lung cancer to osimertinib.

[0044] The dipeptide linker Val-Cit in the Chinese medicine small molecule coupling compound obtained by the present invention is a specific recognition sequence of cathepsin B, and cathepsin B is mainly highly expressed in the tumor phase microenvironment; in addition, in normal human tissue, the pH value is about 7.4, while the microenvironment in which tumor cells are located is slightly acidic, with a pH value as low as about 6.5, and the pH values ​​of the endosomes / lysosomes of tumor cells are also different at different stages: the pH value of the endosomes and lysosomes in early tumor cells is about 6.0, while the pH value is about 5.0 in the late stage; utilizing the characteristics of high cathepsin B concentration and low pH value in the tumor microenvironment, the Chinese medicine small molecule coupling compound with dual response to the cathepsin B and esterase tumor microenvironment designed by the present invention can achieve precise drug release at the tumor site, has tumor responsiveness, reduces the systemic toxicity of the drug, can greatly reduce the toxic and side effects of the drug, and improves its safety of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0046] Figure 1 is the structural formula of the Chinese medicine small molecule coupling compound LT-19 of the present invention;

[0047] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of the Chinese medicine small molecule coupling compound LT-19 of the present invention;

[0048] Figure 3 It is the carbon nuclear magnetic resonance spectrum of the Chinese medicine small molecule coupling compound LT-19 of the present invention;

[0049] Figure 4 IC of LT-19 to PC9 / OR for CCK8 experiments 50 Test result graph;

[0050] Figure 5 IC of osimertinib on PC9 / OR for CCK8 experiments 50 Test result graph;

[0051] Figure 6 The synergy index calculation results of LT-19 and osimertinib were performed using Synergy Finder;

[0052] Figure 7 The results of Transwell migration assay of LT-19, where A is the visual field observation result of Blank group, B is the visual field observation result of 5μM osimertinib group, C is the visual field observation result of 50nM LT-19 group, D is the visual field observation result of 5μM osimertinib + 50nm LT-19 group, and E is the comparison of the number of cells in the visual field;

[0053] Figure 8 The results of flow cytometry apoptosis test of LT-19, where A is the Blank group, B is the 5μM osimertinib group, C is the 50nM LT-19 group, D is the 5μM osimertinib + 50nm LT-19 group, and E is the comparison of cell apoptosis percentage;

[0054] Fig. 9 The results of the Edu cell proliferation test of LT-19, where A is the fluorescence detection result and B is the comparison of the percentage of Edu cells;

[0055] Fig.10 Comet assay results of LT-19, where A is the Blank group, B is the 5μM osimertinib group, C is the 50nM LT-19 group, and D is the 5μM osimertinib + 50nm LT-19 group. DETAILED DESCRIPTION

[0056] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0057] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0058] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0059] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0060] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0061] In the following examples, all reagents, materials and raw materials were obtained from commercial sources.

[0062] Example

[0063] A Chinese medicine small molecule coupling compound LT-19, the structural formula of which is as follows:

[0064]

[0065] It is obtained by coupling toosendanin and luteolin through a Val-Cit linker that responds to cathepsin and a linker that responds to pH.

[0066] The Chinese medicine small molecule coupling compound obtained in the example was scanned by nuclear magnetic resonance hydrogen spectrum, and the nuclear magnetic resonance hydrogen spectrum was as follows: Figure 2 As shown, the hydrogen spectrum data is shown below;

[0067] 1HNMR: δ7.64(5H),7.62(1H),7.37(1H),7.20(1H),6.95(4H),6.81(1H),6.74(1H),6.45(2H),6.2 3(1H),6.08(1H),5.82(1H),5.53(5H),5.37(5H),4.87(1H),4.30(1H),4.21(2H),4.20(2H),4.19( 1H),4.00(2H),3.56(2H),3.47(2H),3.33(1H),3.15(2H),2.79(2H),2.62(1H),2.42(1H),2.06(6H ),1.96(1H),1.90(4H),1.72(2H),1.63(1H),1.57(2H),1.26(3H),1.13(6H),0.87(3H),0.85(3H).

[0068] The small molecule coupling compound of traditional Chinese medicine obtained in the example was scanned by nuclear magnetic resonance carbon spectrum. The nuclear magnetic resonance carbon spectrum is as follows: Figure 3 As shown, the carbon spectrum data are as follows;

[0069] 13CNMR: δ210.70(1C),182.75(1C),173.90(1C),171.90(1C),171.00(1C),170.74(1C),170.25(1C) ,169.40(1C),162.53(1C),162.13(1C),161.10(1C),156.90(1C),155.30(1C),154.80(1C),146.10 (1C),145.60(1C),142.70(1C),140.20(1C),138.09(1C),137.65(1C),133.00(1C),131.02(1C),12 9.88(4C),122.50(1C),122.00(1C),119.90(1C),117.90(4C),115.90(1C),114.00(1C),110.60(1C) ),107.95(1C),105.40(1C),104.38(1C),100.40(1C),98.80(1C),78.60(1C),76.80(1C),75.70(2C) ),72.40(1C),62.50(1C),62.10(1C),61.00(1C),58.80(1C),52.10(1C),51.50(1C),50.00(1C),45 .91(1C),44.00(1C),42.50(1C),41.02(1C),40.80(2C),40.40(1C),34.50(1C),32.70(1C),30.64( 1C),27.30(1C),26.70(2C),26.23(1C),25.35(1C),21.00(2C),19.00(1C),18.80(1C),13.02(1C).

[0070] Depend on Figure 2 and Figure 3 It can be seen that the present invention successfully coupled to obtain a Toosendanin-Luteolin Chinese Medicine small molecule coupling compound, and the Toosendanin-Luteolin Chinese Medicine small molecule coupling compound is recorded as LT-19.

[0071] Effect verification

[0072] 1. Synergistic experiment of LT-19 and osimertinib

[0073] The CCK8 experiment was used to verify whether LT-19 has synergistic resistance with the third-generation EGFR-TKI drug osimertinib. The experimental steps are as follows:

[0074] 1.1 Detection of IC of LT-19 and osimertinib against PC9 / OR 50

[0075] (1) PC9 / OR cells in the logarithmic growth phase were inoculated into a 96-well plate (100 μL / well), and the number of cells per well was set at 5×10 3 , complete medium, cultured in an incubator at 37°C, 5% CO2 for 12 h;

[0076] (2) preparing 100 mM LT-19 stock solution and 100 mM osimertinib stock solution into drug dilution solutions with concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390 μM, 0.195 μM, 0.0977 μM, 0.0488 μM and 0.0244 μM, respectively, and then adding the drug dilution solutions to the 96-well plate after culturing for 12 h in step (1), and culturing in an incubator at 37° C. and 5% CO 2 for 24 h;

[0077] (3) After culturing for 24 h in step (2), the 96-well plate was washed twice with PBS, and then 100 μL of premixed CCK8 solution (complete culture medium: CCK8 working solution = 9:1) was added to each well, and the culture was continued in an incubator at 37°C and 5% CO2 for 2 h;

[0078] (4) After continuing to culture for 2 hours in step (3), the absorbance D(λ) of each well was measured at a wavelength of 450 nm using an ELISA reader, and the D(λ) values ​​of the experimental group, control group, and zero-adjustment group were analyzed. The groups are shown below, and the cell survival rate was calculated. The cell survival rate reflects the cytotoxicity of the drug;

[0079] Experimental group: containing PC9 / OR cells, CCK-8, and the drug to be tested;

[0080] Control group: contains PC9 / OR cells, CCK-8, but no drug to be tested;

[0081] Zero adjustment group: containing CCK-8, but not PC9 / OR cells or the drug to be tested;

[0082]

[0083] IC of LT-19 to PC9 / OR in CCK8 experiment 50 The test results are shown in the figure Figure 4 As shown, the IC of osimertinib against PC9 / OR in CCK8 experiment 50 The test results are shown in the figure Figure 5 As shown;

[0084] Depend on Figure 4 and Figure 5 It can be seen that LT-19 has an IC of PC9 / OR 50The IC of osimertinib for PC9 / OR is 82.46nM. 50 It was 3.952 μM, which indicated that both LT-19 and osimertinib had a certain inhibitory effect on PC9 / OR;

[0085] 1.2 Based on the IC of LT-19 and osimertinib for PC9 / OR 50 Conduct synergistic experiments between LT-19 and osimertinib, and calculate the synergistic coefficient using Synergy Finder software

[0086] (1) PC9 / OR cells in the logarithmic growth phase were inoculated into a 96-well plate (100 μL / well), and the number of cells per well was set at 5×10 3 , complete medium, cultured in an incubator at 37°C, 5% CO2 for 12 h;

[0087] (2) LT-19 IC 50 The IC of osimertinib was 82.46 nM. On this basis, seven concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 1.25 nM and 0 nM were selected for synergistic experiments. 50 The concentration of LT-19 was 3.952 μM. On this basis, seven concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 12.5 μM, 6.25 μM and 0 μM were selected for synergistic experiments. Different concentrations of LT-19 were mixed with osimertinib, and then the mixed drugs were added to the 96-well plate after culturing for 12 hours in step (1), and cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0088] (3) After culturing for 24 h in step (2), the 96-well plate was washed twice with PBS, and then 100 μL of premixed CCK8 solution (complete culture medium: CCK8 working solution = 9:1) was added to each well, and the culture was continued in an incubator at 37°C and 5% CO2 for 2 h;

[0089] (4) After continuing to culture for 2 hours in step (3), the absorbance D(λ) of each well was measured at a wavelength of 450 nm using an ELISA reader, and the D(λ) values ​​of the experimental group, control group, and zero-adjustment group were analyzed. The groups are shown below, and the cell survival rate was calculated. The cell survival rate reflects the cytotoxicity of the drug;

[0090] Experimental group: containing PC9 / OR cells, CCK-8, and the drug to be tested;

[0091] Control group: contains PC9 / OR cells, CCK-8, but no drug to be tested;

[0092] Zero adjustment group: containing CCK-8, but not PC9 / OR cells or the drug to be tested;

[0093]

[0094] The survival rate calculation results were input into the Synergy Finder website to calculate the synergy index. The synergy index calculation results of LT-19 and osimertinib using Synergy Finder are as follows: Figure 6 As shown;

[0095] Depend on Figure 6 It can be seen that the synergistic index (HSA) of LT-19 and osimertinib is 11.95, which indicates that there is a certain synergistic effect between LT-19 and osimertinib (HSA values ​​below 0 are antagonistic, 0-10 are additive, and above 10 are synergistic); in addition, when the concentration of LT-19 is 50nM and the concentration of osimertinib is 5μM, LT-19 and osimertinib have the best synergistic effect;

[0096] 2. Transwell migration assay of PC9 / OR cells

[0097] 2.1 PC9 / OR cells in logarithmic growth phase were cultured at 2×10 5 / mL inoculation density in 6-well plates, complete medium, cultured in an incubator at 37°C, 5% CO2 until the cell density reached 90%, then the complete medium in the 6-well plates was replaced with medium without FPS, and starved for 24h to eliminate the effect of serum on the experiment;

[0098] 2.2 The PC9 / OR cells starved in step 2.1 were digested with trypsin, and then resuspended in medium without FPS. The cells were counted with a cell counter and the cell concentration was adjusted to 5×10 5 / mL, and a cell suspension was obtained;

[0099] 2.3 Add 600 μL of culture medium containing 10% serum to the 24-well plate of the Transwell nested 24-well plate, take 100 μL of the cell suspension obtained in step 2.2, and add it to the Transwell chamber of the Transwell nested 24-well plate, and divide the Transwell nested 24-well plate after adding the cell suspension into four groups, namely Blank group, 5 μM osimertinib group, 50 nM LT-19 group and 5 μM osimertinib + 50 nm LT-19 group, wherein the Blank group did not add any drug to the Transwell chamber, the 5 μM osimertinib group added 5 μM osimertinib to the Transwell chamber, the 50 nM LT-19 group added 50 nM LT-19 to the Transwell chamber, and the 5 μM osimertinib + 50 nm LT-19 group added 5 μM osimertinib and 50 nm LT-19 to the Transwell chamber, and then place the Transwell nested 24-well plate at 37°C and 5% Incubate in a CO2 incubator for 24 h.

[0100] 2.4 Step 2.3 After 24 hours of routine culture, the Transwell chamber was removed, the culture medium was discarded, washed twice with PBS, fixed with methanol for 15 minutes, air-dried appropriately, stained with 0.1% crystal violet for 20 minutes, the non-migrated cells in the upper layer were wiped off, washed three times with PBS, and the cells were observed in five random fields under a 4x microscope and counted;

[0101] The results of the Transwell migration assay of LT-19 are as follows Figure 7 As shown, A is the visual field observation result of the Blank group, B is the visual field observation result of the 5μM osimertinib group, C is the visual field observation result of the 50nM LT-19 group, D is the visual field observation result of the 5μM osimertinib + 50nm LT-19 group, and E is the comparison of the number of cells in the visual field;

[0102] Depend on Figure 7 It can be seen that LT-19 can reverse the osimertinib resistance in PC9 / OR cells.

[0103] 3. Flow cytometry apoptosis assay

[0104] 3.1 PC9 / OR cells in logarithmic growth phase were cultured at 2×10 5 / mL inoculation density in 12-well plates with complete medium and cultured in an incubator at 37°C and 5% CO2 for 24 h;

[0105] 3.2 After culturing for 24 h in step 3.1, the 12-well plate was divided into four groups, namely, Blank group, 5 μM osimertinib group, 50 nM LT-19 group and 5 μM osimertinib + 50 nm LT-19 group, wherein the Blank group was not added with any drug, the 5 μM osimertinib group was added with 5 μM osimertinib, the 50 nM LT-19 group was added with 50 nM LT-19, and the 5 μM osimertinib + 50 nm LT-19 group was added with 5 μM osimertinib and 50 nm LT-19, and then cultured in an incubator at 37 ° C, 5% CO2 for 24 h;

[0106] 3.3 After culturing for 24 h in step 3.2, the PC9 / OR cells were digested with trypsin and washed with PBS to ensure that the trypsin was completely washed away. The cells were counted using a cell counter and the cell concentration was adjusted to 1×10 5 / mL, and a cell suspension was obtained;

[0107] 3.4 Add the cell suspension obtained in step 3 to a 1.5 mL centrifuge tube, centrifuge at 1000 g, remove the supernatant, then add 5 μL Annexin V-FITC dye solution and 10 μL PI dye solution, incubate at room temperature in the dark for 10 to 20 minutes, then perform flow cytometric apoptosis detection on the incubated cells using a Beckman flow cytometer, and perform data analysis using Flowjo software;

[0108] The results of flow cytometry apoptosis assay of LT-19 are as follows Figure 8 As shown, A is the Blank group, B is the 5 μM osimertinib group, C is the 50 nM LT-19 group, D is the 5 μM osimertinib + 50 nm LT-19 group, and E is the comparison of cell apoptosis percentage;

[0109] Depend on Figure 8 It can be seen that when 50nM LT-19 was used in combination, the resistant cells regained their sensitivity to osimertinib.

[0110] 4. Edu cell proliferation assay

[0111] 4.1 PC9 / OR cells in the logarithmic growth phase were seeded in a 96-well plate (100 μL / well), and the number of cells per well was set to 5×10 3 , complete medium, cultured in an incubator at 37°C, 5% CO2 for 12 h;

[0112] 4.2 After culturing for 24 h in step 4.1, the 96-well plate was divided into four groups, namely, Blank group, 5 μM osimertinib group, 50 nM LT-19 group and 5 μM osimertinib + 50 nm LT-19 group, wherein the Blank group was not added with any drug, the 5 μM osimertinib group was added with 5 μM osimertinib, the 50 nM LT-19 group was added with 50 nM LT-19, and the 5 μM osimertinib + 50 nm LT-19 group was added with 5 μM osimertinib and 50 nm LT-19, and then cultured in an incubator at 37 ° C, 5% CO2 for 24 h;

[0113] 4.3 Use complete medium to dilute 10 mM EdU to 2×EdU working solution, then preheat 2×EdU working solution and add an equal volume of complete medium containing PC9 / OR cells cultured for 24 hours in step 4.2, and incubate for 2 hours;

[0114] 4.4 After incubation for 2 hours in step 4.3, remove the culture medium, wash twice with PBS, then add 50mL 4% paraformaldehyde to each well and fix for 15 minutes. After fixation, discard the paraformaldehyde and wash three times with PBS containing 3% BSA, each wash for 3 to 5 minutes;

[0115] 4.5 Mix 43 μL Click Reaction Buffer, 2 μL CuSO4, 0.1 μL Azide488 and 5 μL Click Additive Solution to prepare a reaction system, then add the prepared reaction system to a 96-well plate, incubate at room temperature in the dark for 30 minutes, and then wash three times with PBS containing 3% BSA, each washing for 3 to 5 minutes;

[0116] 4.6 After washing, add 30 μL of ready-to-use DAPI stain to each well, incubate for 15 min in the dark, and wash three times with PBS containing 3% BSA, each wash for 3 to 5 min;

[0117] 4.7 After washing, add 50 μL of 50% glycerol to each well to prevent fluorescence quenching;

[0118] 4.8 Using a high-content imaging system, fluorescence detection was performed using the 405 nm channel and the 488 nm channel respectively;

[0119] The results of the LT-19 Edu cell proliferation assay are as follows Fig. 9 As shown, A is the fluorescence detection result, and B is the comparison of Edu cell percentage;

[0120] Depend on Fig. 9 It can be seen that when 50nM LT-19 was used in combination, the resistant cells regained their sensitivity to osimertinib.

[0121] 5. Comet Experiment

[0122] 5.1 PC9 / OR cells in logarithmic growth phase were cultured at 2×10 5 / mL inoculation density in 12-well plates with complete medium and cultured in an incubator at 37°C and 5% CO2 for 24h;

[0123] 5.2 After culturing for 24 h in step 3.1, the 12-well plate was divided into four groups, namely, Blank group, 5 μM osimertinib group, 50 nM LT-19 group and 5 μM osimertinib + 50 nm LT-19 group, wherein the Blank group was not added with any drug, the 5 μM osimertinib group was added with 5 μM osimertinib, the 50 nM LT-19 group was added with 50 nM LT-19, and the 5 μM osimertinib + 50 nm LT-19 group was added with 5 μM osimertinib and 50 nm LT-19, and then cultured in an incubator at 37 ° C, 5% CO2 for 24 h;

[0124] 5.3 Remove the culture medium, wash with pre-cooled PBS, collect the cells by centrifugation, resuspend them in PBS, count them with a cell counter, and adjust the cell concentration to 1×10 5 / mL, and a cell suspension was obtained;

[0125] 5.4 Production

[0126] First layer of agarose: Add a small amount of normal melting point agarose (1%) on a frosted glass slide, cover it with a coverslip to spread it evenly, and solidify it at 4°C for 5-10 minutes;

[0127] The second layer of cell agarose mixture: 20 μL of the cell suspension obtained in step 5.3 was mixed with 80 μL of low melting point agarose (1%) to obtain a cell agarose mixture. Then, the cover glass of the first layer was removed, and the cell agarose mixture was quickly added dropwise. The cover glass was covered to spread evenly and solidified at 4°C for 5-10 minutes.

[0128] 5.5 Remove the coverslip, immerse the slide in pre-cooled lysis buffer, and lyse at 4°C for 2 hours;

[0129] 6.6 Take out the slide, rinse it with distilled water, and then immerse the slide in alkaline electrophoresis buffer (300mM NaOH, 1mM Na2EDTA, pH>13), and unwind at 4℃ for 20-40min;

[0130] 6.7 Adjust the electrophoresis tank so that the liquid level is 0.25 cm above the glass slide, control the voltage to 25 V, the current to 300 mA, and run the electrophoresis for 40 min;

[0131] 6.8 After electrophoresis, remove the slides and neutralize them with neutralizing solution (0.4 M Tris-HCl, pH 7.5) three times, 5 min each time, and then rinse with distilled water;

[0132] 6.9 Add 20 μL P I dye to the slide, cover with a coverslip, incubate in the dark for 20 min, observe under a fluorescence microscope, and randomly select multiple fields of view to take pictures;

[0133] The comet assay results of LT-19 are as follows Fig.10 As shown, A is the Blank group, B is the 5 μM osimertinib group, C is the 50 nM LT-19 group, and D is the 5 μM osimertinib + 50 nm LT-19 group;

[0134] Depend on Fig.10 It can be seen that when used in combination with 50nM LT-19, osimertinib restored its ability to damage the DNA of drug-resistant cells.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A Chinese medicine small molecule coupling compound, characterized in that: The Chinese medicine small molecule coupling compound is obtained by coupling toosendanin and luteolin through a Val-Cit linker that responds to cathepsin and a linker linker that responds to pH; The structural formula of the Chinese medicine small molecule coupling compound is: 。 2. Use of the Chinese medicine small molecule coupled compound as claimed in claim 1 in the preparation of a drug for restoring the sensitivity of non-small cell lung cancer to osimertinib.

3. The use according to claim 2, characterized in that: The non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

4. A drug for restoring the sensitivity of non-small cell lung cancer to osimertinib, characterized in that: The active ingredient of the drug for restoring the sensitivity of non-small cell lung cancer to osimertinib includes the traditional Chinese medicine small molecule coupled compound or a pharmaceutically acceptable salt thereof as claimed in claim 1.

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