Application of ginsenoside Rc and derivatives thereof in treatment of early lung metastasis of Lewis lung cancer

Abstract

The invention discloses application of ginsenoside Rc and a derivative or salt thereof in preparation of a medicine for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combining other tumors. The ginsenoside Rc shows a better effect of preventing and treating early lung metastasis of Lewis lung cancer and higher safety than cis-platinum in a tumor-bearing mouse model, and no adverse reaction is found.

Description

Technical Field

[0001] The present invention discloses the use of ginsenoside Rc and its derivatives or salts in preparing medicine for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors. Background Art

[0002] Lung cancer is one of the most lethal types of cancer, characterized by rapid metastasis and frequent resistance to current chemotherapy and radiotherapy. Metastasis clearly marks a late and often incurable stage of tumor progression. Lewis lung carcinoma metastasis involves complex and redundant pathways between cancer cells and the microenvironment, mediating invasion at the primary site, survival and arrest in the bloodstream, and progressive growth at distant sites. A series of discrete biological processes mobilize Lewis cancer cells from ectopic tumors to lung tissue. This sequence can be summarized as cancer cells invading tissues surrounding the primary tumor, gaining access to the lymphatic system or bloodstream, surviving and eventually arresting in the circulation, and then extravasating into tissues and establishing at new sites, such as lung, liver, brain, and bone metastases. A drawback of experimental metastasis models is that they only simulate a portion of the metastatic process—the post-invasive phase. In humans, these final stages of the metastatic process are only observed when distant lesions are large enough for imaging. In spontaneous experimental models, tumor cells are injected into a site, establishing a primary tumor and developing metastatic lesions. However, it is better to inject cells into an ectopic location, that is, the tissue of origin. Although this is slower to complete, this experiment allows the measurement of the entire metastatic process.

[0003] Despite advances in surgical techniques, chemotherapy, and radiotherapy, the 5-year survival rate for lung cancer patients is less than 20%. This high mortality rate may be attributed to early metastasis. Metastatic spread is a key cause of death and treatment failure. Therefore, to reduce mortality, the development of new therapies for patients with metastatic lung cancer is necessary. Preventing recurrence and metastasis is a crucial step in the management of lung cancer patients. Lung cancer research has recently focused on the tumor microenvironment, recognizing that alterations in the tumor microenvironment are predictors of tumor development and metastasis. Currently, chemotherapeutic agents and targeted agents are commonly used for maintenance therapy. Pemetrexed and erlotinib are widely used as maintenance therapies for advanced non-small cell lung cancer; however, their adverse effects and high cost burden reduce patient compliance. Cisplatin and carboplatin are the two most commonly used platinum-based drugs in SCLC chemotherapy. In clinical trials, cisplatin is frequently selected for its potent antitumor activity, but its adverse effects include nephrotoxicity and nausea and vomiting. Therefore, to avoid nephrotoxicity, urine output should be monitored and high-dose infusions should be managed during cisplatin-based chemotherapy.

[0004] The tumor microenvironment plays a crucial role in tumor growth, progression, and metastasis. It is increasingly recognized that the tumor inflammatory microenvironment is closely associated with tumor metastasis. Tumor cell lines known to metastasize in vivo are manipulated to alter the expression or mutational status of a single gene, and cyclooxygenase-2 (Cox-2), a rate-limiting enzyme in prostaglandin biosynthesis, plays a crucial role in the tumor inflammatory microenvironment. Cox-2 is overexpressed in all cancer metastatic processes and is involved in angiogenesis, epithelial-mesenchymal transition (EMT) initiation, and extracellular matrix (ECM) destruction. Understanding these pathways and their dynamic interactions will help identify promising molecular targets for cancer therapy and key obstacles to their clinical development. The discovery of new, relatively nontoxic agents that can prevent this deadly disease is an important challenge with significant impact on clinical practice.

[0005] The prior art does not disclose the use of ginsenoside Rc and its derivatives claimed in the present invention for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors. Summary of the Invention

[0006] Based on this, the present invention provides a use of ginsenoside Rc and its derivatives or salts in the preparation of a medicament for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors.

[0007] According to another aspect of the present invention, there is provided a use of a pharmaceutical composition comprising ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors.

[0008] Furthermore, the ginsenoside Rc and its derivatives or salts thereof are used to prevent early lung metastasis of Lewis lung cancer through one or more of the following: inhibiting the migration ability of Lewis lung cancer cells, inhibiting the growth of Lewis lung cancer tumor volume and reducing the mass of Lewis lung cancer tumors.

[0009] Further, the other tumor is not hepatocellular carcinoma and / or melanoma.

[0010] According to another aspect of the present invention, there is provided a use of ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating tumors, including early lung metastasis of Lewis lung cancer, and excluding hepatocellular carcinoma and / or melanoma.

[0011] Furthermore, the pharmaceutical composition further comprises one or more other drugs for treating lung cancer and / or early lung metastasis of lung cancer.

[0012] Furthermore, the other drugs include chemotherapy drugs, targeted drugs and immunotherapy drugs.

[0013] Furthermore, the chemotherapy drugs include one or more of the following: cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, pemetrexed and vinorelbine.

[0014] Furthermore, the targeted drug includes one or more of the following: EGFR mutation inhibitors, ALK inhibitors, KRASG12C inhibitors, ROS1 inhibitors, MET inhibitors, RET inhibitors and HER2 inhibitors.

[0015] Furthermore, the EGFR mutation inhibitors include gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib and vometinib.

[0016] Furthermore, the ALK inhibitors include crizotinib, alectinib, ceritinib, brigatinib and lorlatinib.

[0017] Furthermore, the KRAS G12C inhibitors include sotorasib and adagrasib.

[0018] Furthermore, the ROS1 inhibitors include entrectinib, ceritinib and lorlatinib.

[0019] Furthermore, the MET inhibitors include capmatinib and tepotinib.

[0020] Furthermore, the RET inhibitors include selpercatinib and pralatinib.

[0021] Furthermore, the HER2 inhibitor is trastuzumab (Enhertu).

[0022] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0023] Furthermore, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0024] Furthermore, the dosage form of the pharmaceutical composition is powder, tablet, pill, capsule, film, lozenge, granule, injection or oral solution.

[0025] Beneficial effects of the present invention:

[0026] The ginsenoside Rc of the present invention showed better effect in preventing and treating early lung metastasis of Lewis lung cancer and higher safety than cisplatin in a tumor-bearing mouse model, and no adverse reactions were found. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0028] Figure 1 Schematic diagram of the effect of saponins on the migration ability of Lewis lung cancer (LLC-luc) cells. (A) A total of 3×10 4 LLC-luc cells were evenly plated in Transwell chambers and treated with various drugs (ginsenoside Rc, ginsenoside Rg3, ginsenoside Rg5, ginsenoside Ra1, and notoginsenoside Ft1). After 36 hours of culture, the cells were stained with crystal violet and photographed to observe the effects of the drugs on cell migration. Three replicates were set up for each experiment. (B) Statistical analysis of the number of cells affected by the drugs in the different groups. Data are expressed as mean ± SEM. NS indicates P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0029] Figure 2 Schematic diagram of the effect of 10 mg / kg dose of ginsenoside Rc on the growth of subcutaneous tumors of Lewis lung cancer (LLC-luc) cells in mice. (A) A total of 5×10 5 LLC-luc cells were injected subcutaneously into the right side of the back of C57BL / 6J mice. The mice were randomly divided into a control group, a 10 mg / kg Rc group (n=5 per group), and a 5-Fu group. LLC-luc tumor growth was measured. (B) Tumor inhibition rates of tumor-bearing mice were calculated. (C) Body weight changes of mice in each dose group were measured and analyzed. (D) Images of LLC-luc tumor size in mice in each dose group were presented. (E) LLC-luc tumor weights were measured and analyzed. Data are expressed as mean ± SEM. NS indicates P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0030] Figure 3 Schematic diagram of the effect of ginsenoside Rc on early lung metastasis of Lewis lung cancer (LLC-luc) in mice. (A) When the cells were in the logarithmic growth phase, 1×10 6Luciferase-labeled Lewis lung cancer cells were subcutaneously injected into the left scapula of the back of mice to establish a Luciferase-labeled Lewis lung cancer tumor-bearing mouse model with lung metastasis. Mice were randomly divided into a control group, a cisplatin-treated group, and a 10 mg / kg Rc-treated group (n=3 in each group). Intravital fluorescence imaging was used to monitor Lewis lung metastasis. (B) Lung tissues were collected and photographed at the end of the experiment in the Rc-treated and vehicle-treated mice. (C) Fluorescence analysis of drug treatment in mice with Lewis lung cancer lung metastasis. (D) Images of lung tissue sections fixed with 4% paraformaldehyde and stained with HE. (E) Statistical analysis of lung tumor area in the Rc-treated and vehicle-treated mice. Data are expressed as mean ± SEM. NS indicates P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0031] Figure 4 Schematic diagram of the effect of 10 mg / kg dose of ginsenoside Rc on the growth of Heap1-6 liver cancer subcutaneous tumors. (A) A total of 1×10 6 Heap1-6 hepatocellular carcinoma cells were injected subcutaneously into the right side of the back of C57BL / 6J mice. The mice were randomly divided into a control group, a sorafenib-treated group, and a Rc 10 mg / kg-treated group (n=3 per group). Heap1-6 hepatocellular carcinoma tumor growth was measured. (B) Tumor inhibition rates of tumor-bearing mice were calculated. (C) Body weight changes of mice in each dose-treated group were measured and analyzed. (D) Images show the size of Heap1-6 hepatocellular carcinoma tumors in each treatment group. (E) Heap1-6 hepatocellular carcinoma tumor weights were measured and analyzed. Data are expressed as mean ± SEM. NS indicates P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0032] Figure 5 Schematic diagram of the effect of 10 mg / kg dose of ginsenoside Rc on the subcutaneous tumor growth of B16F10 melanoma cells. (A) A total of 2×10 4B16F10 melanoma cells were injected subcutaneously into the right side of the back of C57BL / 6J mice. The mice were randomly divided into a control group and a Rc 10 mg / kg treatment group (n=3 per group). B16F10 melanoma tumor growth was measured. (B) Tumor inhibition rate of tumor-bearing mice was calculated. (C) Body weight changes of mice in each treatment group were measured and analyzed. (D) Images of B16F10 melanoma tumor sizes in mice in each treatment group were presented. (E) B16F10 melanoma tumor weights were measured and analyzed in each treatment group. Data are expressed as mean ± SEM. NS indicates P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0034] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those skilled in the art in the field of the present invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0035] The present invention is intended to encompass all alternatives, modifications, and equivalents that may come within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0036] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] In the present invention, the term "comprising" is synonymous with "including." As used herein, the terms "comprises," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0038] As described in the background technology section, resistance to chemotherapy, targeted therapy, or immunotherapy is one of the key factors leading to low patient survival rates, and there is an urgent need to develop new drugs to improve the survival rate of patients with early-stage lung metastasis of Lewis lung cancer. At the same time, the prior art does not contain any reports on the use of the pharmaceutical composition of ginsenoside Rc and its derivatives or salts claimed in the present invention in the preparation of a medicament for preventing and / or treating early-stage lung metastasis of Lewis lung cancer and / or combined with other tumors. To address the above problems, the present invention provides a use of ginsenoside Rc and its derivatives or salts in the preparation of a medicament for preventing and / or treating early-stage lung metastasis of Lewis lung cancer and / or combined with other tumors.

[0039] According to another aspect of the present invention, there is provided a use of a pharmaceutical composition comprising ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors.

[0040] In a preferred embodiment, the ginsenoside Rc and its derivatives or salts thereof are used to prevent early lung metastasis of Lewis lung cancer by one or more of the following: inhibiting the migration ability of Lewis lung cancer cells, inhibiting the growth of Lewis lung cancer tumor volume and reducing the mass of Lewis lung cancer tumor.

[0041] In a preferred embodiment, the other tumor is not hepatocellular carcinoma and / or melanoma.

[0042] According to another aspect of the present invention, there is provided a use of ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating tumors, including early lung metastasis of Lewis lung cancer, and excluding hepatocellular carcinoma and / or melanoma.

[0043] In a preferred embodiment, the pharmaceutical composition further comprises one or more other drugs for treating lung cancer and / or early lung metastasis of lung cancer.

[0044] In a preferred embodiment, the other drugs include chemotherapy drugs, targeted drugs and immunotherapy drugs.

[0045] In a preferred embodiment, the chemotherapy drug comprises one or more of the following: cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, pemetrexed and vinorelbine.

[0046] In a preferred embodiment, the targeted drug includes one or more of the following: EGFR mutation inhibitors, ALK inhibitors, KRAS G12C inhibitors, ROS1 inhibitors, MET inhibitors, RET inhibitors and HER2 inhibitors.

[0047] In a preferred embodiment, the EGFR mutation inhibitors include gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib and vometinib.

[0048] In a preferred embodiment, the ALK inhibitors include crizotinib, alectinib, ceritinib, brigatinib and lorlatinib.

[0049] In a preferred embodiment, the KRAS G12C inhibitors include sotorasib and adagrasib.

[0050] In a preferred embodiment, the ROS1 inhibitors include entrectinib, ceritinib and lorlatinib.

[0051] In a preferred embodiment, the MET inhibitors include capmatinib and tepotinib.

[0052] In a preferred embodiment, the RET inhibitors include selpercatinib and pralatinib.

[0053] In a preferred embodiment, the HER2 inhibitor is trastuzumab (Enhertu).

[0054] In a preferred embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0055] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, glidants, lubricants, dispersants, plasticizers, opacifiers and antioxidants.

[0056] In a preferred embodiment, the pharmaceutical composition is in the form of powder, tablet, pill, capsule, film, lozenge, granule, injection or oral solution.

[0057] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or conditions recommended by the manufacturer.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0059] The above-mentioned features of the present invention or the features described in the embodiments may be combined in any combination. All features disclosed in this patent specification may be used in any combination, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal, or similar purpose. Therefore, unless otherwise specified, the features disclosed are only general examples of equal or similar features.

[0060] The present invention is divided into two parts: lung cancer related pharmacodynamics experiments and liver cancer / melanoma pharmacodynamics experiments.

[0061] Objective: To explore the preventive and therapeutic effects of ginsenoside Rc on lung cancer based on animal models.

[0062] Methods: (1) LLC-luc lung cancer cell migration was studied by administering various ginsenosides at their respective effective doses. After a certain treatment time, the cells were fixed and stained with crystal violet to observe the number of migrating cells under a microscope, and the number of cells in different drug groups was counted. (2) In a subcutaneous LLC-luc lung cancer cell tumor-bearing mouse model, ginsenoside Rc was used to treat the mice, and the tumor volume and mouse body weight were monitored to observe the effect of the drug on the animal body weight; (3) In a C57BL / 6J mouse allograft Lewis lung cancer early lung metastasis model, ginsenoside Rc was used to treat the mice, and the mice were monitored by fluorescence live imaging of Lewis cell lung metastasis; (4) A Heap1-6-luc liver cancer subcutaneous tumor model was established in C57BL / 6j mice, and ginsenoside Rc was used to treat the mice. The tumor diameter was measured twice a week to monitor the tumor size; (5) A B16F10 subcutaneous melanoma model was established in C57BL / 6j mice, and ginsenoside Rc was used to treat the mice and the tumor growth was monitored.

[0063] Results: (1) Compared with other saponin small molecule compounds, ginsenoside Rc has the most significant inhibitory effect on the migration function of Lewis lung cancer cells (LLC-luc cells). (2) Compared with the solvent group, ginsenoside Rc has a significant inhibitory effect on the growth of LLC-luc lung cancer, and has no organ toxicity and no adverse reactions. (3) By administering ginsenoside Rc to the lung metastasis model of C57BL / 6J mice with homologous Lewis lung cancer (LLC-luc cells), it was found that ginsenoside Rc can significantly inhibit the lung metastasis of lung cancer, and compared with the first-line lung cancer chemotherapy drug cisplatin, it has higher safety and more significant efficacy. (3) By establishing the Heap1-6-luc liver cancer subcutaneous tumor model in C57BL / 6j mice, it was found that ginsenoside Rc has no therapeutic effect on hepatocellular carcinoma. (4) By establishing the B16F10 subcutaneous melanoma model in C57BL / 6j mice, it was found that ginsenoside Rc has no effect on the growth of melanoma.

[0064] Conclusion: Ginsenoside Rc showed better prevention and treatment effects on lung metastasis and higher safety than cisplatin in the Lewis lung cancer lung metastasis mouse model, and no adverse reactions were found.

[0065] Example 1 Exploring the Effect of Traditional Chinese Medicine Saponin Small Molecule Drugs on LLC-luc Cell Migration Ability

[0066] 1.1 Experimental Materials

[0067] Murine LLC-luc lung cancer cells (Lewis lung cancer cells LLC-luc is a highly malignant spontaneous undifferentiated epithelial carcinoma of the lung) were purchased from Zhongqiao Xinzhou and cultured in DMEM containing 10% fetal bovine serum, 100 U / L penicillin, and 100 mg / L streptomycin in an incubator at 37°C, 5% CO2, and saturated humidity. Cells in the logarithmic growth phase were used for experiments.

[0068] The experimental instruments of Example 1 are shown in Table 1.

[0069] Table 1 Experimental instruments of Example 1

[0070]

[0071] The experimental reagents of Example 1 are shown in Table 2.

[0072] Table 2 Experimental reagents of Example 1

[0073]

[0074]

[0075] 1.2 Experimental Methods

[0076] (1) Observe cell growth. Remove the culture medium from cells that are growing well and starve them for 24 hours with serum-free medium. Harvest the cells by trypsinization by adding 1 ml of 0.25% trypsin-EDTA and incubating at room temperature. Continuously examine cell adhesion under a microscope. After a few minutes, when cells detach, add 10 to 20 ml of complete growth medium containing 10% FBS to inactivate trypsin.

[0077] (2) Add 100 μL of 0.5% trypan blue to 100 μL of cell sample and then count the number of cells using a hemocytometer. To ensure the accuracy of the count, the optimal counting concentration is 2–5 × 10 5cells / ml. Centrifuge at 200 to 300g and 4°C for 5 minutes to precipitate the remaining cells, then wash the cell pellet twice with 10ml PBS. Adjust the cell concentration with serum-free culture medium according to the cell counting results, and add the cells to the chamber at a ratio of sample: cell suspension = 1:1. First add 400μL of dosing working solution (the final concentration of ginsenosides Rg3 and Rg5 is 30μM, the concentration of Panax notoginseng saponin Ft1 is 10μM, and the final concentration of ginsenosides Ra1 and Rc is 3000μM) and then add 400uL of cell suspension. At this time, the sample concentration is diluted 2 times, that is, the final concentration is 3×10 4 Then, take 500 μl of complete culture medium containing 10% FBS and add it to the lower chamber of the 24-well plate. Use tweezers to place the Transwell chamber into the 24-well plate.

[0078] (3) Incubate the 24-well plate at 37°C, 5% CO2, and 90% humidity for 24-48 hours. Remove the Transwell chamber, remove the culture medium, and gently wipe the matrix gel and cells inside the chamber with a cotton swab or cotton soaked in PBS. Add 600 μl of 4% paraformaldehyde fixative to a clean well of the 24-well plate and place the chamber in the fixative for 30 minutes. Discard the fixative and wash the inside and outside of the chamber once with PBS.

[0079] (4) Add 600 μL of crystal violet staining solution to a clean well of a 24-well plate and place the chamber in the staining solution for 10 minutes. Remove the chamber and wash the inside and outside of the chamber three times with PBS. After proper air drying, observe under a microscope for qualitative analysis; for quantitative analysis, take photos of 3-5 fields of view and use ImageJ to count and average the results.

[0080] 1.3 Experimental data processing method

[0081] The results were expressed as (X±S). GraphdhPad statistical software was used to compare the parameters between the two groups. The t-test method was used for statistical analysis. The data were presented as mean±SEM. NS indicates P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0082] 1.4 Experimental Results

[0083] The results are as follows Figure 1 As shown, after crystal violet staining, the cells were observed and photographed under a 40× microscope. Statistical analysis showed that compared with the experimental control group, ginsenosides Rc and Rg5 both had a significant inhibitory effect on LLC-luc cell migration, with ginsenoside Rc having the most significant inhibitory effect on LLC-luc cell migration. However, ginsenosides Rg3, Ra1, and notoginsenoside Ft1 did not show a significant inhibitory effect on cell migration. This result suggests that ginsenosides may have an inhibitory effect on LLC lung cancer metastasis.

[0084] Example 2 Pharmacodynamic Evaluation of Ginsenoside Rc on LLC-luc Lung Cancer Cells in C57BL / 6J Mouse Subcutaneous Lung Tumor Model

[0085] 2.1 Experimental Materials

[0086] Animals: 30 male SPF C57BL / 6J mice, 5-6 weeks old, weighing 20 ± 2 g, were purchased from Weitonglihua (certificate number: SCXK (Beijing) 2007-0001). All mice were provided with free access to food. All animal experiments were conducted in strict accordance with the relevant regulations on animal use at the Animal Center of Shanghai University of Traditional Chinese Medicine and approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine.

[0087] Cell line: Murine colon cancer cells LLC-luc were purchased from Zhongqiao Xinzhou (Lewis lung cancer cells LLC-luc is a spontaneous undifferentiated pulmonary epithelial carcinoma with a high degree of malignancy). They were subcultured in DMEM containing 10% fetal bovine serum, 100 U / L penicillin, and 100 mg / L streptomycin in an incubator at 37°C, 5% CO2, and saturated humidity. Cells in the logarithmic growth phase were used for experiments.

[0088] The experimental instruments of Example 2 are shown in Table 3.

[0089] Table 3 Experimental instruments of Example 2

[0090]

[0091]

[0092] The experimental reagents of Example 2 are shown in Table 4.

[0093] Table 4 Experimental reagents of Example 2

[0094]

[0095] The drug formulation of Example 2 is shown in Table 5.

[0096] Table 5 Drug configuration of Example 2

[0097]

[0098]

[0099] Note: Administer the drug intraperitoneally at 10 μl / g, and adjust the dosage according to the experimental changes later.

[0100] 2.2 Experimental Methods

[0101] 2.2.1 Experimental system

[0102] Animals: 30 C57BL / 6J male mice, SPF grade, 5-6 weeks old, weighing 20±2 g, purchased from Weitonglihua Company.

[0103] Cell line: Murine LLC-luc lung cancer cells were purchased from Zhongqiao Xinzhou and cultured in DMEM containing 10% fetal bovine serum, 100 U / L penicillin, and 100 mg / L streptomycin in an incubator at 37°C, 5% CO2, and saturated humidity. Cells in the logarithmic growth phase were used for experiments.

[0104] 2.2.2 Model establishment

[0105] Construction of tumor-bearing mouse model: LLC-luc lung cancer cells were collected in the logarithmic growth phase and the cell suspension concentration was adjusted to 5×10 6 Cells / ml (cell suspension: matrix gel is 9:1), and 0.10 ml / mouse was injected subcutaneously under sterile conditions into the right back of C57BL / 6J mice (5×105 cells per mouse). The number of mice inoculated with LLC-luc was 30, and the SPF grade was routinely raised for 5-10 days. The experimental animals were selected with a tumor volume of 50-100 mm 3 The specific experimental plan is shown in Table 6.

[0106] Table 6 Experimental groups and drug administration methods

[0107]

[0108]

[0109] Note: Dosing began immediately after grouping, using a 1mL syringe with a precision of 0.02mL. QD means once a day. After 20 consecutive doses, the tumor volume of a single mouse exceeded 2000mm. 3 The mice were euthanized, and the mean tumor volume of each group of mice reached 2000 mm 3 The experiment was terminated when .

[0110] 2.2.3 Experimental evaluation indicators and testing

[0111] 1) Tumor diameter: Tumor diameter was the primary indicator of detection, and measurements were taken every 3-4 days. The relative tumor growth rate was calculated based on the measurement results, and the anti-tumor effect of the test substance was dynamically observed. Tumor volume (TV) = 1 / 2 × a × b2 (where a and b are the longest and shortest diameters, respectively); relative tumor volume (RTV) = Vt / V0 (where V0 is the tumor volume measured on day 0 of group dosing, and Vt is the tumor volume at each measurement). The anti-tumor effect was evaluated using the relative tumor growth rate (TGI) (%) = (1-(TRTV / CRTV)) × 100% (where TRTV is the RTV of the treatment group and CRTV is the RTV of the control group).

[0112] 2) Tumor Weight: After the experiment, animals were sacrificed, tumors were dissected and weighed, and then photographed. If the average tumor weight in the model control group was less than 1 g, or if 20% of the tumors weighed less than 400 mg, it indicated poor tumor growth and the experiment was discarded. Tumor growth inhibition rate was calculated based on the weight results: Tumor growth inhibition rate = [(average tumor weight in the model control group - average tumor weight in the treatment group) / average tumor weight in the model control group] × 100%.

[0113] 3) Body weight: The animals were weighed every 3-4 days to observe the effects of the test drugs on their body weight.

[0114] 2.2.4. Collect samples

[0115] At the end of the experiment, tumor tissues of 5 mice were collected from each of the solvent group, the 5-Fu and ginsenoside Rc treatment groups with the best effects. The tumor size was about the size of soybeans, and then made into single-cell suspensions for CYTOF experiments. In addition, tumors of the remaining 3 experimental animals in each group were collected and stored in a -80℃ refrigerator for RNA-seq experiments. Spleens of 3 mice in each group were collected and placed on ice for flow cytometry detection of mCD4+, mCD8+ and mIFN-γ experiments.

[0116] 2.2.5 Experimental data processing method

[0117] For cell-based experiments, each experimental group provided at least three replicates, and for in vivo animal experiments, at least five independent data sets were provided. All experimental results are presented as mean ± SEM. Statistical analysis was performed using Graphpad Prism 9 (GraphPad, San Diego, California). Unpaired t-tests were used to test the significance of each experimental data. A P value of 0.05 or above was considered nonsignificant; a P value of 0.05 or below was considered significant.

[0118] 2.2.6 Experimental Results

[0119] The results are as follows Figure 2 As shown in the experiment, it was found that the same dose of chemotherapy drug 5-Fu was less effective in inhibiting the growth of Lewis lung tumors than ginsenoside Rc monomer ( Figure 2 D: Three subcutaneous tumors disappeared in the Rc monomer-treated group, demonstrating superior efficacy to 5-Fu. Tumor removal at the end of the experiment revealed that tumor weights in the Rc-treated group were comparable to those in the 5-Fu-treated group at the same dose. No toxic side effects were observed in the Rc-treated mice.

[0120] 2.2.7 Summary and Discussion

[0121] Ginsenoside Rc at a dose of 10 mg / kg significantly inhibited the growth of LLC-luc cell subcutaneous tumors.

[0122] Example Pharmacodynamic evaluation of ginsenoside Rc in a Lewis lung cancer early metastasis model in C57BL / 6J mice

[0123] 3.1 Experimental Materials

[0124] The experimental apparatus is the same as that in Example 1.

[0125] 3.2 Experimental Methods

[0126] 3.2.1 Experimental system

[0127] The experimental system is the same as that of Example 1.

[0128] 3.2.2 Model establishment

[0129] 1) Tumor-bearing Mouse Model Establishment: Depending on the LLC-Luc cell line used, the number of cells to be injected, and the number of mice, several tissue culture flasks may be required to culture sufficient cells to establish an orthotopic lung tumor model. When the LLC-Luc cell culture reaches approximately 80% confluence, harvest the cells by trypsinization by adding 4 ml of 0.25% trypsin-EDTA and incubating at room temperature. Continuously inspect cell adhesion under a microscope. After several minutes, when cells have detached, add 10 to 20 ml of complete growth medium containing 10% FBS to inactivate the trypsin.

[0130] 2) Add 100 μL of 0.5% trypan blue to 100 μL of cell sample and count the cells using a hemocytometer. To ensure the accuracy of the count, the optimal counting concentration is 2–5×10 5 Cells / ml. Single-cell suspensions with >90% viability should be used for in situ injection. Centrifuge at 200 to 300 g at 4°C for 5 minutes to pellet remaining cells, then wash the cell pellet twice with 10 ml of PBS.

[0131] The cell pellet was resuspended in ice-cold 10% growth factor-reduced Matrigel (1× in PBS) to give the number of cells injected per 25 μL per mouse (25 μL injected per animal).

[0132] Cell seeding should be performed within 1 to 2 hours of preparing the cell suspension.

[0133] 3) Once the mouse is fully anesthetized, place it in lateral recumbency on a heating pad covered with a sterile towel. Apply a generous amount of 10% iodine solution to the left chest, above the lower rib line, and below the lower edge of the shoulder blade. Shave the left chest area before treating the incision site with iodine and 70% isopropyl alcohol. Maintain aseptic technique from this point until the end of the procedure.

[0134] 4) Gently pipette the tumor cells up and down to ensure a single cell suspension, then draw 25 μL of the tumor cell suspension into a 3 / 10-cc insulin syringe equipped with a 30-G hypodermic needle. 6 Luciferase-labeled Lewis lung cancer cells were subcutaneously injected into the left scapula of mice to establish a Luciferase-labeled Lewis lung cancer cell tumor-bearing mouse model.

[0135] 5) After surgery, inject buprenorphine (0.1 mg / kg) (optional if available) and place the mouse in left lateral recumbency under a heating lamp. Monitor the animal until it fully recovers from anesthesia.

[0136] 6) Weigh and examine mice daily for signs of infection, bleeding, weight loss, lethargy, and changes in food and / or water intake.

[0137] Previous research on the project showed that no lung cancer cells were found in the lungs 14 days after the experiment, and this time point was defined as the pre-metastatic microenvironment of the mouse lungs. Lewis lung cancer cells were detected in the mouse lungs 21 days after the experiment. The mice were given daily dosing starting when LLC-luc cells metastasized to the lungs and formed a pre-metastatic microenvironment to study the effect of the drug on the early metastasis of Lewis lung cancer.

[0138] The specific experimental plan is shown in Table 7

[0139] Table 7 Experimental groups and drug administration methods 2

[0140]

[0141]

[0142] Note: Use a 1mL syringe with a precision of 0.02mL for administration. QD means once daily.

[0143] 3.3 Experimental evaluation indicators and testing

[0144] 1) Body weight: Weigh and record the animals' body weight three times a week to observe the effects of the test drugs on the animals' body weight.

[0145] 2) Lewis Lung Cancer Early Metastasis Model: Lung metastasis of LLC-luc cells was monitored weekly postoperatively in C57BL / 6J mice using fluorescence in vivo imaging. Prior to imaging, mice were anesthetized with an intraperitoneal injection of 4.16 mg / mL of Zotai 50 combined with 0.416 mg / mL of xylazine hydrochloride (10 mL / kg). Fluorescence in vivo imaging was performed 10 minutes after intraperitoneal injection of 150 mg / mL of luciferin potassium substrate (10 mL / kg).

[0146] 3.4 Experimental data processing method

[0147] The results were expressed as (X±S). GraphdhPad statistical software was used to compare the parameters between the two groups. The t-test method was used for statistical analysis. The data were presented as mean±SEM. NS indicates P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0148] 3.2.5 Experimental Results

[0149] Metastasis is the most important biological behavior of malignant tumors and the main factor leading to treatment failure in tumor patients. However, the molecular mechanism of early metastasis of lung cancer is still unclear. The concept of pre-metastatic niche provides a theoretical research basis for explaining the molecular mechanism of early metastasis. The present invention establishes a Lewis lung cancer early metastasis model for Rc drug administration treatment research, and the results are as follows Figure 3 As shown in A, ginsenoside Rc has a significant inhibitory effect on the metastasis of Lewis lung cancer. The results showed that compared with the control group, the in vivo fluorescence imaging of the mice in the drug-treated group showed that the metastatic foci in the drug-treated group were smaller, and at the same dosage, the cisplatin group was more toxic, causing a sharp drop in the weight of the mice and strong lethality, while the Rc-treated group had no toxic side effects. At the end of the experiment, the lung tissue of the mice was taken and it was found that the size of the lung tumors in the drug-treated group was significantly different from that in the control group. Figure 3 As shown in A, ginsenoside Rc significantly inhibited the early metastasis of LLC-luc lung cancer.

[0150] 3.6 Summary and Discussion

[0151] Ginsenoside Rc at a dose of 10 mg / kg had a significant inhibitory effect on the lung metastasis of LLC-luc lung cancer cells.

[0152] Example 4 Pharmacodynamic Evaluation of Rc Drug on Murine Heap1-6 Hepatocellular Carcinoma Cells in a Syngeneic Transplanted Tumor Model in C57BL / 6J Mice

[0153] 4.1 Experimental Materials

[0154] The experimental equipment was the same as in Example 1. The mouse hepatocellular carcinoma cell line Heap1-6 (maintained in our laboratory) was cultured. Nine female C57BL / 6J mice, SPF grade, 5-6 weeks old, weighing 20 ± 2 g, were purchased from Spafor (certificate number SCXK (Beijing) 2019-0010).

[0155] 4.2 Experimental Methods

[0156] The experimental system was the same as that in Example 1. The number of mouse Heap1-6 cells inoculated subcutaneously was 1×10 6 The dose of sorafenib was 30 mg / kg.

[0157] 4.3 Experimental evaluation indicators and testing

[0158] The experimental evaluation criteria are the same as those in Example 1.

[0159] 4.4 Experimental data processing method

[0160] The experimental data processing method is the same as that in Example 1.

[0161] 4.5 Experimental Results

[0162] The results are as follows Figure 4 As shown in the experiment, it was found that the same dose of chemotherapy drug sorafenib had a significant inhibitory effect on the growth of hepatocellular carcinoma tumors, but ginsenoside Rc monomers such as Figure 4 As shown in D, it actually promoted the growth of subcutaneous hepatocellular carcinoma tumors. Tumor removal at the end of the experiment revealed significant differences in tumor weight between the ginsenoside Rc group and the vehicle group and the chemotherapy drug sorafenib group at the same dose. Ginsenoside Rc had no therapeutic effect on heap1-6 hepatocellular carcinoma.

[0163] 4.6 Summary and Discussion

[0164] Ginsenoside Rc at a dose of 10 mg / kg had no inhibitory effect on the growth of subcutaneous tumors of Heap1-6 liver cancer cells.

[0165] Example 5 Pharmacodynamic evaluation of Rc drug on murine melanoma cell B16-F10 in a homologous transplant tumor model in C57BL / 6J mice

[0166] 5.1 Experimental Materials

[0167] The experimental equipment was the same as in Example 1. The mouse melanoma cell line B16F10 (maintained in our laboratory) was cultured. Ten female C57BL / 6J mice, SPF grade, 5-6 weeks old, weighing 20 ± 2 g, were purchased from Spafor (certificate number SCXK (Beijing) 2019-0010).

[0168] 5.2 Experimental Methods

[0169] The experimental system was the same as that in Example 1. C57BL / 6j mice were subcutaneously inoculated with 2×10 mouse melanoma B16F10 cells. 4 After the tumor was formed, the drug was intraperitoneally injected at a dose of 10 mg / kg every day.

[0170] 5.3 Experimental evaluation indicators and testing

[0171] The experimental evaluation criteria are the same as those in Example 1.

[0172] 5.4 Experimental Data Processing Method

[0173] The experimental data processing method is the same as that in Example 1.

[0174] 5.5 Experimental Results

[0175] The results are as follows Figure 5 As shown in the experiment, it was found that the same dose of 10mg / kg ginsenoside Rc had no therapeutic effect on melanoma B16F10 subcutaneous tumors. At the end of the experiment, the tumor was weighed and the ginsenoside Rc monomer was found to be Figure 4 D. There was no difference in tumor size compared with the solvent group. The experiment showed that Rc had no therapeutic effect on B16F10 melanocytoma.

[0176] 5.6 Summary and Discussion

[0177] Ginsenoside Rc at a dose of 10 mg / kg had no inhibitory effect on the growth of subcutaneous tumors of B16 F10 melanoma cells.

[0178] Results: (1) Compared with other ginsenoside administration experiments, ginsenoside Rc had the most significant inhibitory effect on LLC-luc cell migration. (2) Compared with the solvent group, ginsenoside Rc had a significant inhibitory effect on the subcutaneous growth of LLC-luc lung cancer, without organ toxicity or adverse reactions. (3) By administering ginsenoside Rc to the lung metastasis model of homologous LLC-luc lung cancer cells in C57BL / 6J mice, it was found that ginsenoside Rc could significantly inhibit the early lung metastasis of Lewis lung cancer, and compared with the first-line lung cancer chemotherapy drug cisplatin, it was safer (no lethality at the same dose) and more effective. (4) By establishing a Heap1-6-luc liver cancer subcutaneous tumor model in C57BL / 6j mice, it was found that ginsenoside Rc had no therapeutic effect on hepatocellular carcinoma. (5) By establishing a B16F10 subcutaneous melanoma model in C57BL / 6j mice, it was found that ginsenoside Rc had no effect on the growth of melanoma.

[0179] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method of the present invention and its core concept. At the same time, changes or modifications made by those skilled in the art based on the concept of the present invention, the specific implementation methods of the present invention, and the scope of application are all within the scope of protection of the present invention. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. Use of ginsenoside Rc and its derivatives or salts in the preparation of drugs for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors.

2. Use of a pharmaceutical composition comprising ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors.

3. The use according to claim 1 or 2, characterized in that The ginsenoside Rc and its derivatives or salts thereof are used to prevent early lung metastasis of Lewis lung cancer through one or more of the following: inhibiting the migration ability of Lewis lung cancer cells, inhibiting the growth of Lewis lung cancer tumor volume and reducing the mass of Lewis lung cancer tumor; Preferably, the other tumor is not hepatocellular carcinoma and / or melanoma.

4. Use of ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating tumors, characterized in that: The tumor includes early lung metastasis of Lewis lung cancer, and the tumor does not include hepatocellular carcinoma and / or melanoma.

5. The use according to claim 2, characterized in that The pharmaceutical composition further comprises one or more other drugs for treating lung cancer and / or early lung metastasis of lung cancer; Preferably, the other drugs include chemotherapy drugs, targeted drugs and immunotherapy drugs.

6. The use according to claim 5, characterized in that The chemotherapy drugs include one or more of the following: cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, pemetrexed and vinorelbine.

7. The use according to claim 5, characterized in that The targeted drugs include one or more of the following: EGFR mutation inhibitors, ALK inhibitors, KRAS G12C inhibitors, ROS1 inhibitors, MET inhibitors, RET inhibitors and HER2 inhibitors; Preferably, the EGFR mutation inhibitors include gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib and vometinib; Preferably, the ALK inhibitors include crizotinib, alectinib, ceritinib, brigatinib and lorlatinib; Preferably, the KRAS G12C inhibitors include sotorasib and adagrasib; Preferably, the ROS1 inhibitors include entrectinib, ceritinib and lorlatinib; Preferably, the MET inhibitors include capmatinib and tepotinib; Preferably, the RET inhibitors include selpercatinib and pralatinib; Preferably, the HER2 inhibitor is trastuzumab (Enhertu).

8. The use according to claim 2, characterized in that The pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that The auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant.

10. The use according to claim 2, characterized in that The dosage form of the pharmaceutical composition is powder, tablet, dripping pill, capsule, film, lozenge, granule, injection or oral solution.

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