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

Abstract

The application discloses application of ginsenoside Rc and derivatives or salts thereof in preparation of a medicine for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combination with other tumors. The ginsenoside Rc of the application shows better prevention and treatment effects on early lung metastasis of Lewis lung cancer and higher safety than cisplatin in a tumor-bearing mouse model, and no adverse reactions are found.

Description

TECHNICAL FIELD

[0001] The application discloses application of ginsenoside Rc and derivatives or salts thereof in preparation of a medicine for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combining other tumors. BACKGROUND

[0002] Lung cancer is one of the most deadly cancer types, characterized by rapid metastasis and often developing resistance to current chemotherapy regimens and radiotherapy. Metastasis marks the late stage of tumor progression, which is often incurable, and Lewis lung cancer metastasis involves not only complex and redundant pathways of cancer cells and microenvironment, but also mediates tumor invasion at the primary site, survival and arrest in the blood, and gradual growth at distant sites. A series of discrete biological processes move Lewis cancer cells from ectopic tumors to lung tissue. This series of processes can be summarized as cancer cell invasion into the tissue surrounding the primary tumor, entry into the lymphatic system or blood circulation, survival and eventual arrest in circulation, and then extravasation into tissue and growth at new sites, such as lung metastasis, liver metastasis, brain metastasis and bone metastasis, etc. One drawback of experimental metastasis models is that they only mimic a part of the metastasis process, i.e. the post-invasion stage. In humans, only the final stage of the metastasis process is observed when the distant lesions are large enough to be imaged. In spontaneous experimental models, tumor cells are injected into a certain site, forming a primary tumor and developing metastatic foci. However, it is best to inject cells into ectopic locations, i.e. the tissue of origin, although it is slower to complete, this experiment can measure the entire metastasis process.

[0003] Despite advances in surgical techniques, chemotherapy and radiotherapy, the 5-year survival rate of lung cancer patients is less than 20%. High mortality rate can be attributed to early metastasis. Metastasis dissemination is a key reason for death and treatment failure. Therefore, in order to reduce mortality, it is necessary to develop new therapies for treating metastatic lung cancer patients. Preventing recurrence and metastasis is an important step in managing lung cancer patients. Lung cancer research has recently focused on tumor microenvironment and recognized that changes in tumor microenvironment are a predictor of tumorigenesis and metastasis. Currently, chemotherapy drugs and targeted drugs are commonly used for maintenance therapy. Pemetrexed and Erlotinib are widely used as maintenance therapy for advanced non-small cell lung cancer; however, their adverse effects and high economic burden reduce patient compliance. Cisplatin and carboplatin are the two most commonly used platinum drugs in SCLC chemotherapy. In clinical trials, cisplatin is often chosen for its strong anti-tumor activity, but its adverse reactions include nephrotoxicity, nausea and vomiting. Therefore, in order to avoid nephrotoxicity, urine output should be monitored and large dose infusion should be controlled in cisplatin-based chemotherapy.

[0004] The tumor microenvironment plays a crucial role in tumor growth, progression and metastasis. It is increasingly recognized that tumor inflammatory microenvironment is closely related to tumor metastasis. It is known that tumor cell lines capable of metastasis in vivo are manipulated to change the expression or mutation status of a single gene, cyclooxygenase-2 (Cox-2) is a rate-limiting enzyme of prostaglandin biosynthesis, which plays a crucial role in the tumor inflammatory microenvironment. Cox-2 is overexpressed in all cancer metastasis 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, as well as key obstacles to their clinical development. It is an important challenge to discover new relatively non-toxic agents that can prevent this deadly disease, which has a major impact on clinical practice.

[0005] The application of ginsenoside Rc and its derivatives for preventing and / or treating early lung metastasis of Lewis lung cancer and / or combined with other tumors is not found in the prior art. SUMMARY

[0006] Based on this, the application provides the use of 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.

[0007] According to another aspect of the application, there is provided the 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] Further, the effect of ginsenoside Rc and its derivatives or salts thereof for preventing early lung metastasis of Lewis lung cancer is by one or more of: 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.

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

[0010] According to another aspect of the application, there is provided the use of ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating a tumor, which includes early lung metastasis of Lewis lung cancer, and which does not include hepatocellular carcinoma and / or melanoma.

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

[0012] Further, the other drug includes a chemotherapeutic drug, a targeted drug and an immunotherapeutic drug.

[0013] Further, the chemotherapeutic drug comprises one or more of cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, pemetrexed and vinorelbine.

[0014] Further, the targeted drug comprises one or more of EGFR mutation inhibitor, ALK inhibitor, KRAS G12C inhibitor, ROS1 inhibitor, MET inhibitor, RET inhibitor and HER2 inhibitor.

[0015] Further, the EGFR mutation inhibitor comprises gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, amatinib and futmerinib.

[0016] Further, the ALK inhibitor comprises crizotinib, alectinib, ceritinib, brigatinib and lorlatinib.

[0017] Further, the KRAS G12C inhibitor comprises sotorasib and adagrasib.

[0018] Further, the ROS1 inhibitor comprises entrectinib, ceritinib and lorlatinib.

[0019] Further, the MET inhibitor comprises capmatinib and tepotinib.

[0020] Further, the RET inhibitor comprises selpercatinib and pralsetinib.

[0021] Further, the HER2 inhibitor is Enhertu.

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

[0023] Further, the excipient is selected from one or more of diluent, wetting agent, binding agent, disintegrating agent, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersing agent, plasticizing agent, light-shielding agent and antioxidant.

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

[0025] Advantages of the present application:

[0026] The ginsenoside Rc of the present application shows better prevention and treatment effect on early lung metastasis of Lewis lung cancer than cisplatin in a tumor-bearing mouse model, and has higher safety, and no adverse reactions are found. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art according to these drawings without exceeding the scope of the present application.

[0028] Figure 1 The results of the effect of ginsenosides on the migration ability of Lewis lung cancer (LLC-luc) cells are shown in the schematic diagram. (A) A total of 3×10 4 LLC-luc cells in the logarithmic growth phase were evenly spread in the Transwell chamber, and drugs (ginsenoside Rc, ginsenoside Rg3, ginsenoside Rg5, ginsenoside Ra1 and notoginsenoside Ft1) were added, respectively, and cultured for 36 h. Cell crystal violet staining was performed, and the drug effects on cell migration were observed by taking pictures. Three parallel replicates were set up for each group of controls. (B) The number of cells affected by different groups of drugs was counted. The data are expressed as mean ± SEM, NS is P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0029] Figure 2 The results of the effect of 10 mg / kg dose of ginsenoside Rc on the growth of subcutaneous tumors of mouse Lewis lung cancer (LLC-luc) cells are shown in the schematic diagram. (A) A total of 5×10 5 LLC-luc cells were injected subcutaneously into the right dorsal side of C57BL / 6J mice. The mice were randomly divided into a control group, a Rc 10 mg / kg drug administration group (5 mice in each group) and a chemotherapy drug 5-Fu administration group, and the LLC-luc tumor growth was measured; (B) the tumor growth inhibition rate of the tumor-bearing mice was counted; (C) the weight change rate of the mice in each different dose administration group was measured and counted; (D) the LLC-luc tumor size pictures of the mice in each administration group were shown; (E) the LLC-luc tumor weight of the administration group was measured and counted. The data are expressed as mean ± SEM, NS is P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0030] Figure 3 The results of the effect of ginsenoside Rc on the early lung metastasis of mouse Lewis lung cancer (LLC-luc) are shown in the schematic diagram. (A) When the cells were in the logarithmic growth phase, 1×10 6Luciferase+ labeled Lewis lung cancer cells were injected subcutaneously into the left dorsal scapular region of mice to establish a Luciferase+ labeled Lewis lung cancer lung metastasis model. The mice were randomly divided into a control group, a cisplatin administration group, and a Rc 10 mg / kg administration group (3 mice per group), and the Lewis lung metastasis was monitored by live imaging fluorescence; (B) The lung tissues of the Rc administration group and the solvent group were photographed at the end of the experiment; (C) The fluorescence was counted to determine the treatment of the Lewis lung cancer lung metastasis model mice by the drugs; (D) The HE-stained section of the lung tissue of the administration group mice after 4% paraformaldehyde fixation was shown; (E) The lung tumor area of the Rc administration group and the solvent group mice was counted. The data are expressed as mean ± SEM, NS P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0031] Figure 4 The results of the effect of ginsenoside Rc at a dose of 10 mg / kg on the growth of Heap1-6 hepatoma subcutaneous tumors are shown in the schematic diagram. (A) A total of 1×10 6 Heap1-6 hepatoma cells were injected subcutaneously into the right dorsal side of C57BL / 6J mice, and the mice were randomly divided into a control group, a sorafenib administration group, and a Rc 10 mg / kg administration group (3 mice per group). The Heap1-6 hepatoma tumor growth was measured; (B) The tumor growth inhibition rate of the tumor-bearing mice was counted; (C) The weight change rate of the mice in each different dose administration group was measured and counted; (D) The tumor size of the Heap1-6 hepatoma cells of the mice in each administration group was shown; (E) The tumor weight of the Heap1-6 hepatoma cells of the administration group was measured and counted. The data are expressed as mean ± SEM, NS P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 5 The results of the effect of ginsenoside Rc at a dose of 10 mg / kg on the growth of B16F10 melanoma subcutaneous tumors are shown in the schematic diagram. (A) A total of 2×10 4B16F10 melanoma cells were injected into the back right side of C57BL / 6J mice subcutaneously, and the mice were randomly divided into a control group, a Rc 10 mg / kg administration group (3 in each group), and the B16F10 melanoma tumor growth was measured; (B) the tumor growth inhibition rate of the drug on the tumor-bearing mice was statistically analyzed; (C) the weight change rate of the mice in each different dose administration group was measured and statistically analyzed; (D) the B16F10 melanoma cell tumor size pictures of the mice in each administration group were displayed; (E) the B16F10 melanoma cell tumor weight of the administration group was measured and statistically analyzed. The data is expressed as mean ± SEM, NS is P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0034] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application or the field to which the term applies. Although any methods, conditions, materials, or agents similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods, conditions, materials, or agents are described herein.

[0035] The present application is intended to encompass all alternatives, modifications, and equivalents that can be included within the scope of the present application as defined by the claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The application is not limited to the described embodiments.

[0036] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

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

[0038] As described in the background section, chemotherapy, targeted therapy or immunotherapy resistance is one of the key factors leading to low patient survival rate, and there is an urgent need to develop new drugs to improve the survival rate of patients with Lewis lung cancer early lung metastasis, and there is no relevant report on the use of the ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating Lewis lung cancer early lung metastasis and / or combined with other tumors. In order to solve the above problems, the present application provides the use of ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating Lewis lung cancer early lung metastasis and / or combined with other tumors.

[0039] According to another aspect of the present application, there is provided the 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 Lewis lung cancer early lung metastasis and / or combined with other tumors.

[0040] In a preferred embodiment, the effect of the ginsenoside Rc and its derivatives or salts thereof in preventing Lewis lung cancer early lung metastasis is achieved by one or more of: inhibiting Lewis lung cancer cell migration ability, inhibiting Lewis lung cancer tumor volume growth, and reducing Lewis lung cancer tumor mass.

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

[0042] According to another aspect of the present application, there is provided the use of ginsenoside Rc and its derivatives or salts thereof in the preparation of a medicament for preventing and / or treating Lewis lung cancer early lung metastasis and / or combined with other tumors.

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

[0044] In a preferred embodiment, the other drug comprises a chemotherapy drug, a targeted drug and an immunotherapy drug.

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

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

[0047] In a preferred embodiment, the EGFR mutant inhibitor comprises gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, amivantamab, and futmerib.

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

[0049] In a preferred embodiment, the KRAS G12C inhibitor comprises sotorasib and adagrasib.

[0050] In a preferred embodiment, the ROS1 inhibitor comprises entrectinib, ceritinib, and lorlatinib.

[0051] In a preferred embodiment, the MET inhibitor comprises capmatinib and tepotinib.

[0052] In a preferred embodiment, the RET inhibitor comprises selpercatinib and pralsetinib.

[0053] In a preferred embodiment, the HER2 inhibitor is 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: diluent, wetting agent, binding agent, disintegrating agent, inclusion agent, flavoring agent, sustained release agent, glidant, lubricant, dispersing agent, plasticizer, light shielding agent, and antioxidant.

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

[0057] The present application is further described in conjunction with the following specific examples. It should be understood that these examples are only intended to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, unless otherwise specified, are generally performed according to conventional conditions or the conditions recommended by the manufacturer.

[0058] 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 application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are intended to be illustrative only and are not intended to be limiting.

[0059] The features mentioned in the present invention, or the features mentioned in the embodiments, can be combined arbitrarily. All the features disclosed in the present patent specification can be used with any combination, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent 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 experiments were conducted by administering various ginsenosides at their respective effective doses. After a certain treatment time, the number of cell migrations was observed under a microscope after fixing the cells and staining with crystal violet, and the number of cells in different drug groups was counted. (2) In LLC-luc lung cancer cell subcutaneous tumor-bearing mouse models, ginsenoside Rc was used for treatment, and the tumor volume and mouse body weight were monitored to observe the effect of the drug on animal body weight. (3) In C57BL / 6J mouse allograft Lewis lung cancer early lung metastasis models, ginsenoside Rc was used for treatment, and fluorescence in vivo imaging was performed on mice to monitor Lewis cell lung metastasis. (4) C57BL / 6j mouse Heap1-6-luc liver cancer subcutaneous tumor models were established, and ginsenoside Rc was used for treatment, and the tumor size was monitored by measuring the tumor diameter twice a week. (5) C57BL / 6j mouse B16F10 subcutaneous melanoma models were established, and ginsenoside Rc was used for treatment and tumor growth was monitored.

[0063] Results: (1) Compared with other saponin small molecule compounds, ginsenoside Rc had 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 had a significant inhibitory effect on the growth of LLC-luc lung cancer, and had no organ toxicity or adverse reactions. (3) Through drug treatment of the C57BL / 6J mouse homologous Lewis lung cancer (LLC-luc cell) transplanted lung cancer lung metastasis model, it was found that ginsenoside Rc could significantly inhibit lung metastasis of lung cancer, and compared with the first-line lung cancer chemotherapy drug cisplatin, it had higher safety and more significant efficacy. (3) By establishing a C57BL / 6j mouse Heap1-6-luc liver cancer subcutaneous tumor model, it was found that ginsenoside Rc had no therapeutic effect on hepatocellular carcinoma. (4) By establishing a C57BL / 6j mouse B16F10 subcutaneous melanoma model, it was found that ginsenoside Rc had 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 One explores the effect of Chinese saponin small molecule drugs on the migration ability of LLC-luc cells

[0066] 1.1, Experimental materials

[0067] LLC-luc lung cancer cells (Lewis lung cancer cells LLC-luc are a strain of highly malignant spontaneous undifferentiated epithelioid carcinoma) were purchased from Zhijiao Xin Zhou and cultured in a 37°C, 5% CO2 and saturated humidity incubator with DMEM culture medium containing 10% fetal bovine serum, 100 U / L penicillin and 100 mg / L streptomycin. The logarithmic growth phase cells were used for experiments.

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

[0069] Table 1 Experimental instruments of Example One

[0070]

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

[0072] Table 2 Experimental reagents of Example One

[0073]

[0074]

[0075] 1.2, Experimental method

[0076] (1) Observe the cell growth state, remove the culture medium from the cells in good growth state, and add serum-free medium to starve for 24 h. Harvest the cells by adding 1 ml 0.25% trypsin-EDTA and incubating at room temperature, and check the cell adhesion under a microscope. After a few minutes, when the cells fall off, add 10 to 20 ml complete growth medium containing 10% FBS to inactivate the trypsin.

[0077] (2) Add 100 μL 0.5% trypan blue to 100 μL cell sample, then count the number of cells using a hemocytometer. To ensure the accuracy of the count, the optimal count concentration is 2-5 x 10 5cells / ml. Centrifuge at 200-300g, 4°C for 5 min to pellet the remaining cells, then wash the cell pellet twice with 10 ml PBS. Adjust the cell concentration with serum-free medium according to the cell counting result, and add the cells into the chamber at a sample: cell suspension ratio of 1:1. First add 400 μL of the drug-containing working solution (the final concentrations of ginsenosides Rg3 and Rg5 are 30 μM, the concentration of notoginsenoside Ftl is 10 μM, and the final concentrations of ginsenosides Ra1 and Rc are 3000 μM), and then add 400 uL of the cell suspension. At this time, the sample concentration is diluted 2-fold, i.e., the final concentration is 3 x 10 4 / 800 μL. Take 500 μl of complete medium containing 10% FBS and add it to the lower chamber of the 24-well plate, and use tweezers to place the Transwell chamber into the 24-well plate.

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

[0079] (4) Add 600 uL of crystal violet staining solution to the clean wells of the 24-well plate, and place the chamber in the staining solution for 10 minutes. Take out the chamber, and wash the chamber inside and outside 3 times with PBS. After appropriate air-drying, observe and qualitatively study under a microscope; take 3-5 fields of view and take photographs, and then use ImageJ to count and take the average value for quantitative study.

[0080] 1.3, Experimental data processing method

[0081] The results are expressed as (X ± S), and the parameters between the two groups are compared using the GrapdhPad statistical software and the t-test method for statistical analysis. The data are shown as mean ± SEM, NS is P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0082] 1.4, Experimental results

[0083] The results are shown in Table 1. Figure 1 After crystal violet staining, the cells were observed and photographed under a 40x microscope, and it was found that, compared with the experimental control group, ginsenosides Rc and Rg5 had a good inhibitory effect on LLC-luc cell migration. Ginsenoside Rc had the most obvious inhibitory effect on LLC-luc cell migration, while ginsenosides Rg3, Ra1 and notoginsenoside Ftl did not have a more obvious 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 Panax notoginseng saponin Rc on LLC-luc lung cancer cells in C57BL / 6J mice

[0085] 2.1 Experimental materials

[0086] Animals: 30 C57BL / 6J mice, male, SPF level, 5-6 weeks old, weighing 20±2g, purchased from Vanteli Huaxin Company (License No.: SCXK (Jing) 2007-0001). All mice were free to eat. All animal experiments were strictly in accordance with the relevant regulations of the Animal Center of Shanghai University of Traditional Chinese Medicine, and were approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine.

[0087] Cell line: LLC-luc, a mouse colon cancer cell line, was purchased from Zhijia Xinzhou (Lewis lung cancer cell LLC-luc is a high-grade spontaneous intra-pulmonary undifferentiated epithelioid carcinoma). DMEM medium containing 10% fetal bovine serum, 100 U / L penicillin and 100 mg / L streptomycin was used for subculture at 37℃, 5% CO2 and saturated humidity in a incubator. The logarithmic growth phase cells 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 preparation of Example 2 is shown in Table 5.

[0096] Table 5 Drug preparation of Example 2

[0097]

[0098]

[0099] Note: Intraperitoneal administration according to 10 μl / g, and adjustment according to experimental dosage changes in later period.

[0100] 2.2 Experimental methods

[0101] 2.2.1 Experimental system

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

[0103] Cell line: LLC-luc lung cancer cells of mouse origin were purchased from Zhongqinewzhou, and were subcultured in DMEM medium 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. The cells in the logarithmic growth phase were used for experiments.

[0104] 2.2.2 Establishment of model

[0105] Tumor-bearing mouse model construction: collect LLC-luc lung cancer cells in the logarithmic growth phase, adjust the cell suspension concentration to 5×10 6 6J mice under sterile conditions, 0.10 ml per mouse (5×105 cells per mouse), 30 mice were inoculated with LLC-luc, and the mice were routinely fed for 5-10 days. The experimental animals with tumor volume of 50-100 mm 3 were randomly divided. The specific experimental scheme is shown in Table 6.

[0106] Table 6 Experimental grouping and administration method

[0107]

[0108]

[0109] Note: After grouping, drug administration was started immediately. A 0.02 mL precision 1 mL syringe was used for administration. QD means once a day. Continuous administration for 20 times, the tumor volume of a single mouse exceeds 2000 mm 3 The mouse was euthanized, and the mean tumor volume of each group of mice reached 2000 mm 3 when the experiment was terminated.

[0110] 2.2.3, experimental evaluation index and detection

[0111] 1) Tumor diameter: The tumor diameter was used as the main detection index, and was measured once every 3-4 days. The relative tumor proliferation rate was calculated according to the measurement results, and the anti-tumor effect of the test substance was dynamically observed. Tumor volume (TV), TV = 1 / 2 x a x b2 (where a and b are the longest diameter and the shortest diameter, respectively); relative tumor volume (RTV), RTV = Vt / V0 (where V0 is the tumor volume measured at d0 when the test was divided into groups, and Vt is the tumor volume at each measurement). The evaluation index of the anti-tumor effect was the relative tumor proliferation rate TGI (%), TGI (%) = (1-(TRTV / CRTV)) x 100% (where TRTV is the RTV of the treatment group, and CRTV is the RTV of the model control group).

[0112] 2) Tumor weight: After the experiment, the animals were sacrificed, the tumor mass was dissected and peeled off, the tumor weight was measured, and then the tumor was photographed. If the average tumor weight of the model control group is less than 1 g, or 20% of the tumor weight is less than 400 mg, it indicates that the tumor growth is poor, and the experiment is invalid. The tumor growth inhibition rate was calculated according to the weight results, and the tumor growth inhibition rate = [(average tumor weight of the model control group- average tumor weight of the treatment group) / average tumor weight of the model control group] x 100%.

[0113] 3) Body weight: The body weight of the animals was measured every 3-4 days to observe the effect of the test drug on the body weight of the animals.

[0114] 2.2.4, Collecting samples

[0115] At the end of the experiment, 5 mice from each of the solvent group, 5-Fu group and ginsenoside Rc group were selected, and the tumor tissues were taken out when the tumor size was about the size of a soybean. Then, single-cell suspensions were prepared for CYTOF experiments. In addition, 3 remaining experimental animals from each group were taken out, and the tumor tissues were stored in a-80°C refrigerator for RNA-seq experiments. The spleens of 3 mice from each group were taken out and placed on ice for flow cytometry detection of mCD4+, mCD8+ and mIFN-γ experiments.

[0116] 2.2.5, Experimental data processing method

[0117] In the cell experiment, at least 3 replicates were provided for each experimental group, and at least 5 independent data were provided for the in vivo animal experiment data. Each experimental result was expressed as the mean SEM. Statistical analysis was performed using Graphpad Prism 9 (GraphPad, San Diego, California). In order to test the significance of the experimental data, a non-paired T test was used. A P value of 0.05 or above was not significant; a P value of 0.05 or less was considered to be significantly different.

[0118] 2.2.6, Experimental results

[0119] Results are shown in Figure 2 Figure 6. It was found that the same dose of 5-Fu had less effect on the growth of Lewis lung tumor than ginsenoside Rc monomer (Rc) in the experiment. The three subcutaneous tumors in the Rc treatment group disappeared, and the effect was better than that of 5-Fu. At the end of the experiment, it was found that the tumor weight in the Rc treatment group was similar to that in the 5-Fu treatment group at the same dose. No toxic side effects were found in the mice in the Rc treatment group. Figure 2 D: Rc monomer treatment group three subcutaneous tumors disappeared, the effect is better than 5-Fu). At the end of the experiment, it was found that the tumor weight in the Rc treatment group was similar to that in the 5-Fu treatment group at the same dose. No toxic side effects were found in the mice in the Rc treatment group.

[0120] 2.2.7, Summary and discussion

[0121] 10mg / kg dose of ginsenoside Rc had a significant inhibitory effect on the growth of LLC-luc cell subcutaneous tumors.

[0122] Example Three Pharmacodynamic evaluation of ginsenoside Rc drug in C57BL / 6J mouse background Lewis lung cancer early lung metastasis model

[0123] 3.1, Experimental materials

[0124] The experimental instruments are the same as in Example One.

[0125] 3.2, Experimental methods

[0126] 3.2.1 Experimental system

[0127] The experimental system is the same as in Example One.

[0128] 3.2.2 Model establishment

[0129] 1) Construction of tumor-bearing mouse model: According to the LLC-luc cell line used, the number of cells to be injected, and the number of mice, several tissue culture bottles may be needed to culture a sufficient number of cells to establish a lung orthotopic tumor model. When the LLC-Luc cells are cultured to about 80% confluence, the cells are harvested by trypsinization by adding 4 ml of 0.25% trypsin-EDTA and incubating at room temperature. The cell adhesion is checked under a microscope. After a few minutes, when the cells are detached, 10 to 20 ml of complete growth medium containing 10% FBS is added to inactivate the trypsin.

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

[0131] Resuspend the cell pellet in ice-cold 10% growth factor reduced Matrigel (1x PBS solution) to achieve the number of cells per mouse per 25 μL injected (25 μL injected per animal).

[0132] Cell inoculation should be performed within 1 to 2 hours after the cell suspension is prepared.

[0133] 3) After the mouse is sufficiently anesthetized, place it on its side on a heated 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 inferior border of the scapula. Shave the left chest area before treating the incision site with iodine and 70% isopropyl alcohol. From this point until the end of the procedure, maintain a sterile field.

[0134] 4) Ensure a single cell suspension by gently pipetting the tumor cells up and down, then draw 25 μL of the tumor cell suspension into a 3 / 10-cc insulin syringe fitted with a 30-G hypodermic needle. Inject 1*10 6 Luciferase-labeled Lewis lung cancer cells were injected subcutaneously into the left dorsal scapular region of the mouse to establish a mouse model of Luciferase-labeled Lewis lung cancer cell tumor.

[0135] 5) Inject buprenorphine (0.1 mg / kg) postoperatively (optional, if available), and place the mouse on its left side under a heat lamp. Monitor the animal until it has fully recovered from the anesthesia.

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

[0137] Previous studies have shown that no lung cancer cells were found in the lungs 14 days after the experiment, and this time point was set as the pre-metastatic niche of the mouse lung. Lewis lung cancer cells were found in the lungs of mice 21 days after the experiment. The mice were given daily medication when the LLC-luc cells were transferred to the lungs to form the pre-metastatic niche, and the effect of the drug on the early metastasis of Lewis lung cancer was studied.

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

[0139] Table 7 Experimental grouping and drug administration method two

[0140]

[0141]

[0142] Note: A 1 mL syringe with a precision of 0.02 mL is used for administration. QD means once a day.

[0143] 3.3, Evaluation index and detection of the experiment

[0144] 1) Body weight: The body weight of the animals was weighed three times a week, and the body weight of the animals was recorded to observe the effect of the test drug on the body weight of the animals.

[0145] 2) Lewis lung cancer early metastasis model: C57BL / 6J mice were monitored weekly after surgery for the presence of LLC-luc cell metastasis to the lungs using fluorescence live imaging, and the mice were anesthetized using intraperitoneal injection of 4.16 mg / mL of Xylazine 50 combined with 0.416 mg / mL of Xylazine hydrochloride, with a dose of 10 mL / kg. Fluorescence live imaging was performed 10 minutes after intraperitoneal injection of 150 mg / mL of luciferin potassium salt substrate, with a dose of 10 mL / kg.

[0146] 3.4, Experimental data processing method

[0147] The results data are represented by (X ± S), and the parameters between the two groups are compared using the GrapdhPad statistical software and the t-test method for statistical analysis. The data are represented by the mean ± SEM, NS is P>0.05, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0148] 3.2.5, Experimental results

[0149] Metastasis is the most important biological behavior of malignant tumors and is also the main factor leading to treatment failure in tumor patients, but the molecular mechanism of early metastasis of lung cancer is still unclear. The concept of pre-metastatic niche provides a theoretical basis for the molecular mechanism of early metastasis. The present application establishes a Lewis lung cancer early metastasis model for Rc administration treatment research, and the results are shown in Figure 3 A, ginsenoside Rc has a significant inhibitory effect on Lewis lung cancer metastasis. The results show that compared with the control group, the in vivo fluorescence imaging of the drug administration group shows that the metastatic foci are smaller, and under the same drug dose, the toxicity of the cisplatin group is large, leading to a sharp decline in the body weight of the mice, and strong lethality, while the Rc administration group has no any toxic side effects. At the end of the experiment, the lung tissue of the mice was taken and it was found that the lung tumor size of the drug administration group was significantly different from that of the control group. As shown in Figure 3 A, ginsenoside Rc significantly inhibits LLC-luc lung cancer early metastasis.

[0150] 3.6, Summary and discussion

[0151] 10 mg / kg dose of ginsenoside Rc has a significant inhibitory effect on LLC-luc lung cancer cell lung metastasis.

[0152] Pharmacodynamic Evaluation of Rc Drug on the Homologous Transplantation Tumor Model of Murine Heap1-6 Hepatocarcinoma Cells in C57BL / 6J Mice

[0153] 4.1 Experimental Materials

[0154] The experimental instruments are the same as those in Example 1. The murine hepatocarcinoma cell line Heap1-6 (preserved in this laboratory). 9 C57BL / 6J mice, female, SPF grade, 5-6 weeks old, weighing 20±2 g. Purchased from SpfBaiFu Co., Ltd. (certificate number: SCXK(Beijing)2019-0010).

[0155] 4.2 Experimental Methods

[0156] The experimental system is the same as that in Example 1. The number of subcutaneous inoculated murine Heap1-6 cells is 1×10 6 cells. The dosage of sorafenib is 30 mg / kg.

[0157] 4.3 Experimental Evaluation Indexes and Detection

[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 Figure 4 shown. It was found in the experiment that the chemotherapeutic drug sorafenib with the same dosage had a significant inhibitory effect on the growth of hepatocellular carcinoma tumors, but the ginsenoside Rc monomer, as Figure 4 shown in D, instead promoted the growth of subcutaneous tumors of hepatocellular carcinoma. At the end point of the experiment, it was found that there was a huge difference in tumor weight between the ginsenoside Rc administration group and the solvent group and the chemotherapeutic drug sorafenib group at the same dosage. 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 hepatocarcinoma cells.

[0165] Pharmacodynamic Evaluation of Rc Drug on the Homologous Transplantation Tumor Model of Murine Melanoma Cells B16-F10 in C57BL / 6J Mice

[0166] 5.1 Experimental Materials

[0167] Experimental instruments are the same as in Example One, and the mouse melanoma cell line B16F10 (preserved in the laboratory) is used. C57BL / 6J mice, 10 in number, female, SPF level, 5-6 weeks old, and weighing 20±2g. Purchased from Sibeifu Company (qualified certificate number SCXK (Jing) 2019-0010).

[0168] 5.2, Experimental method

[0169] The experimental system is the same as in Example One. C57BL / 6j mice are subcutaneously inoculated with 2×10 4 6 mice per group, and the number of mouse-derived melanoma B16F10 cells is 2×10

[0170] 5.3, Experimental evaluation index and detection

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

[0172] 5.4, Experimental data processing method

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

[0174] 5.5, Experimental results

[0175] The results are shown in Figure 5 The experiment found that ginsenoside Rc had no therapeutic effect on subcutaneous melanoma B16F10 cells at the same dose of 10mg / kg. The tumor weight at the end of the experiment showed that ginsenoside Rc monomers such as Figure 4 D had no difference in tumor size compared with the solvent group. The experiment proved that Rc had no therapeutic effect on B16F10 melanoma cells.

[0176] 5.6, Summary and discussion

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

[0178] Results: (1) Compared with other ginsenosides, 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, and had no organ toxicity and no adverse reactions. (3) Through the administration of C57BL / 6J mice with homologous LLC-luc lung cancer cell lung metastasis model, 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 had higher safety (no lethality at the same dose) and more significant efficacy. (4) Through the establishment of C57BL / 6j mouse Heap1-6-luc hepatocellular carcinoma subcutaneous tumor model, it was found that ginsenoside Rc had no therapeutic effect on hepatocellular carcinoma. (5) Through the establishment of C57BL / 6j mouse B16F10 subcutaneous melanoma model, it was found that ginsenoside Rc had no effect on the growth of melanoma.

[0179] The above describes the embodiments of the present application in detail, and the principles and implementation modes of the present application are described by applying specific examples. The above description of the embodiments is only used to help understand the method of the present application and its core idea. Meanwhile, the changes or deformations made by the skilled in the art according to the principles of the present application, based on the specific implementation modes and application scope of the present application, all belong to the scope of protection of the present application. In summary, the content of the specification should not be understood as a limitation of the present application.

Claims

1. Application of ginsenoside Rc in the preparation of drugs for the prevention and / or treatment of early lung metastases in Lewis lung cancer.

2. The use of a pharmaceutical composition comprising ginsenoside Rc in the preparation of a medicament for the prevention and / or treatment of early lung metastases in Lewis lung cancer.

3. The application according to claim 1 or 2, characterized in that, The ginsenoside Rc is 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 quality of Lewis lung cancer tumors.

4. The application 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 metastases of lung cancer.

5. The application according to claim 4, characterized in that, The other drugs include chemotherapy drugs, targeted drugs, and immunotherapy drugs.

6. The application 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 application 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.

8. The application according to claim 7, characterized in that, The EGFR mutation inhibitors include gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, and vometinib.

9. The application according to claim 7, characterized in that, The ALK inhibitors include crizotinib, alectinib, ceritinib, brigatinib, and lolatinib.

10. The application according to claim 7, characterized in that, The KRAS G12C inhibitors include sotorasib and adagrasib.

11. The application according to claim 7, characterized in that, The ROS1 inhibitors include entrectinib, ceritinib, and lorlatinib.

12. The application according to claim 7, characterized in that, The MET inhibitors include carmatinib and terpoxtinib.

13. The application according to claim 7, characterized in that, The RET inhibitors include selpercatinib and pralatinib.

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

15. The application according to claim 14, characterized in that, The excipients are selected from one or more of the following: diluents, wetting agents, adhesives, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, opacifiers, and antioxidants.

16. The application according to claim 2, characterized in that, The dosage form of the pharmaceutical composition is powder, tablet, drop, capsule, film, lozenge, granule, injection or oral liquid.

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