Application of Cistancheside F in the preparation of anti-tumor drugs

Abstract

The present invention provides the use of Cistancheside F or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug. In vitro experiments show that Cistancheside F has significant inhibitory activity against a variety of tumor strains, including human lung cancer cells A549, human gastric adenocarcinoma cells AGS-CAS9, human liver cancer cells SK-HEP-1, human bladder cancer cells RT112, human cervical cancer cells Hela, human prostate cancer cells PC-3, and human neuroblastoma cells SH-SY5Y. Animal experiments show that Cistancheside F has a good in vivo anti-tumor effect and has a significant therapeutic effect on a variety of tumors, such as bladder cancer and liver cancer. Cistancheside F has strong anti-tumor activity and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to an anti-tumor compound and a use thereof, and particularly relates to the medical use of cistancheside F in the preparation of anti-tumor drugs. Background Art

[0002] Malignant tumors are one of the major diseases that seriously threaten human health and life.

[0003] Bladder cancer is one of the most common malignant tumors of the urinary system. The most common clinical manifestations of bladder cancer are painless hematuria, followed by frequent urination, urgency, pain, dysuria, low back pain, etc. Its diagnosis mainly relies on cystoscopy, renal function assessment, and upper urinary tract imaging (including urinary system color Doppler ultrasound, CT, MRI, angiography, etc.). Currently, modern medicine divides it into non-muscle invasive bladder cancer and muscle invasive bladder cancer. 70%-80% of patients with newly diagnosed bladder cancer present with non-muscle invasive disease, and their treatment mainly includes transurethral bladder tumor resection and postoperative intravesical chemotherapy; the treatment of muscle invasive bladder cancer mainly includes radical cystectomy and related neoadjuvant chemotherapy.

[0004] While China has achieved some success in preventing and treating liver cancer through the promotion of hepatitis B vaccines in recent years, statistics show that the incidence of liver cancer in my country remains only lower than that of lung cancer, colorectal cancer, and gastric cancer, and its mortality rate is second only to lung cancer. Therefore, the treatment of liver cancer remains crucial. Currently, various treatment options are available, depending on the stage of liver cancer development, including surgery, liver transplantation, radiotherapy, chemotherapy, and traditional Chinese medicine.

[0005] Although the emergence of new drugs and treatments over the past decade has improved survival rates for cancer patients, cancer remains the second leading cause of death worldwide. Currently, chemotherapy is the mainstay of cancer treatment. However, chemotherapy drugs have low specificity and, while killing tumor cells, can also affect the biological functions of normal cells, resulting in "off-target" toxicity. Furthermore, chemotherapy drugs are prone to developing drug resistance, significantly reducing the effectiveness of cancer treatment.

[0006] Cistanoside F (CF) is a phenylethanoid glycoside compound existing in nature. It is currently mainly derived from traditional Chinese medicines with kidney-tonifying effects, such as Cistanche deserticola, Rehmannia glutinosa, Scrophularia ningpoensis, and Orobancha.

[0007] Ma Mengling (Ma Mengling, Tang Zeling, Zhu Weiliang, et al. Cistancheside F inhibits adipogenic differentiation of C2C12 cells via the AMPK / ACC pathway [J]. Journal of Traditional Chinese Medicine, 2023, 34(12): 2822-2825) found that Cistancheside F may inhibit adipogenic differentiation of C2C12 cells by regulating the AMPK / ACC pathway. Chinese patent CN116966193A discloses the medical use of Cistancheside F, specifically the use of Cistancheside F in the preparation of a drug for preventing and treating skeletal muscle atrophy.

[0008] At present, there is no report on the use of Cistancheside F in the preparation of anti-tumor drugs. Summary of the Invention

[0009] The present invention aims to provide the use of Cistancheside F or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug. The Cistancheside F or a pharmaceutically acceptable salt thereof of the present invention has a broad anti-tumor spectrum, high anti-tumor activity and high safety.

[0010] In order to achieve the above-mentioned purpose of the invention, the following technical solutions are provided:

[0011] The structural formula of Cistancheside F is as follows:

[0012]

[0013] The present invention provides use of cistancheside F or a pharmaceutically acceptable salt thereof in preparing a drug for preventing and / or treating tumors.

[0014] In vitro experiments showed that the IC of Cistancheside F of the present invention was 50 The mean range is: 0.73-4.55μg / ml, among which the IC values ​​for human bladder cancer cell RT112, human liver cancer cell SK-HEP-1, and human lung cancer cell A549 are 50 The value is the lowest.

[0015] In vivo test results show that the Cistancheside F of the present invention has a high tumor inhibition rate on RT112 human bladder cancer and SK-HEP-1 human liver cancer. Cistancheside F has a good in vivo anti-tumor effect.

[0016] Based on this, the present invention requests protection for the use of Cistancheside F in the preparation of anti-tumor drugs.

[0017] Preferably, the tumor is lung cancer, gastric adenocarcinoma, liver cancer, bladder cancer, cervical cancer, prostate cancer, or neuroblastoma.

[0018] More preferably, the tumor is bladder cancer or liver cancer.

[0019] The administration routes of the Cistancheside F or its pharmaceutically acceptable salt described in the present invention include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0020] Preferably, the administration route is oral administration.

[0021] For oral administration, the pharmaceutical composition can be prepared by mixing Cistancheside F with pharmaceutically acceptable carriers well known in the art. These carriers enable the compound of the present invention to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc. for oral administration to patients. For example, a pharmaceutical composition for oral administration can be obtained in the following manner: the active ingredient is combined with one or more solid carriers, the resulting mixture is granulated if necessary, and a small amount of excipients is added if necessary to process it into a mixture or granules to form a tablet or tablet core. The tablet core can be combined with an optionally enteric-compatible coating material to form a coated formulation that is more conducive to absorption by an organism (e.g., a human).

[0022] A pharmaceutical composition, characterized in that it comprises cistancheside F or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0023] The beneficial effects of the present invention are:

[0024] The present invention discloses the anti-tumor activity of Cistancheside F. In vitro experiments show that Cistancheside F has significant inhibitory activity on various tumor lines, including human lung cancer cells A549, human gastric adenocarcinoma cells AGS-CAS9, human liver cancer cells SK-HEP-1, human bladder cancer cells RT112, human cervical cancer cells Hela, human prostate cancer cells PC-3, and human neuroblastoma cells SH-SY5Y, and inhibits tumor cell proliferation. 50 Low value.

[0025] Animal studies have shown that Cistancheside F has a strong anti-tumor effect in vivo, with significant therapeutic effects on various tumors, including bladder cancer and liver cancer. Cistancheside F has strong anti-tumor activity and has broad application prospects.

[0026] Definition of terms

[0027] In the present invention, unless otherwise specified, the terms used have the following meanings:

[0028] The term "pharmaceutically acceptable" means that salts, solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

[0029] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared with relatively nontoxic, pharmaceutically acceptable acids. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent. Pharmaceutically acceptable acids include inorganic acids, including but not limited to hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, phosphorous acid, sulfuric acid, bisulfate, and the like. The pharmaceutically acceptable acid includes organic acids, including but not limited to acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compound of the present invention contains a relatively basic functional group, it can be converted into an acid addition salt.

[0030] The term "treatment" as used herein means any treatment of a disease in a mammal, including: (1) preventing the disease, i.e., causing clinical symptoms of the disease not to develop; (2) inhibiting the disease, i.e., preventing the development of clinical symptoms; and (3) alleviating the disease, i.e., causing the clinical symptoms to subside.

[0031] As used herein, "prevention" refers to a reduction in the risk of acquiring or developing a disease or disorder.

[0032] The "pharmaceutical composition" of the present invention refers to a preparation of one or more compounds of the present invention or their salts and a carrier generally accepted in the art for delivering biologically active compounds to an organism (e.g., a human). The purpose of a pharmaceutical composition is to facilitate administration and delivery to an organism.

[0033] The term "pharmaceutically acceptable carrier" refers to a substance that is co-administered with an active ingredient and facilitates the administration of the active ingredient, including but not limited to any glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the State Food and Drug Administration for use in humans or animals (e.g., livestock). Examples include but are not limited to calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1The inhibitory activity of the Cistancheside F of the present invention on tumor cells (IC 50 value, μg / ml). DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to fully understand the present invention, the present invention is further described below through specific examples. However, those skilled in the art should be aware that the examples of the present invention do not limit the present invention in any way.

[0036] Example 1 In vitro antitumor test of the compounds of the present invention

[0037] In vitro experiments are primarily used to screen candidate compounds and gain a preliminary understanding of the tumor types and potency of the test compound, providing a reference for subsequent in vivo experiments. This experiment, based on the "Guiding Principles for Nonclinical Studies of Cytotoxic Antitumor Drugs," selected human tumor cells to evaluate the in vitro antitumor effects of Cistancheside F. This provided a preliminary understanding of the potency of Cistancheside F's antitumor effects, providing a reference for subsequent in vivo experiments.

[0038] 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt

[0039] The anti-tumor activity of the compounds of the present invention was tested by colorimetry (CCK-8 method).

[0040] Instruments: clean bench (SW-CJ-1FD, AIRTECH, Sujing Antai), constant temperature CO2 incubator (3111, Thermo, USA), inverted biological microscope (IX71, OLYMPUS, Japan), enzyme-linked immunosorbent assay (Model 680, BIO-RAD, USA), plate shaker (Kylin-bell lab Instruments), high pressure sterilizer (YXO.SG41.280, Shanghai Huaxian), centrifuge (SIGMA).

[0041] Reagents: DMEM (GIBCO), fetal bovine serum (GIBCO), trypsin (SIGMA), DMSO (SIGMA).

[0042] 1.1 Experimental Materials

[0043] Cell lines: human lung cancer cells A549, human gastric adenocarcinoma cells AGS-CAS9, human liver cancer cells SK-HEP-1, human bladder cancer cells RT112, human cervical cancer cells Hela, human prostate cancer cells PC-3, and human neuroblastoma cells SH-SY5Y.

[0044] 1.2 Experimental methods

[0045] Take cells in logarithmic growth phase and adjust the cell concentration to 4.0×10 4 Cells were seeded in a 96-well plate at a density of approximately 4,000 cells / ml, with 100 μl per well. After 24 hours of incubation in an incubator, the complete medium was replaced with 100 μl of complete medium containing various concentrations of drug. For the control group, 100 μl of complete medium without drug was added to each well. Three replicates were set for both the control and drug groups. A blank control (medium alone without cells) was also established as a zeroing control. After 48 hours of incubation in a 37°C, 5% CO2 incubator, 10 μl of CCK-8 solution was added to each well. After an additional 1.5 hours of incubation, the OD value was measured at 450 nm using a microplate reader.

[0046] Cell inhibition rate (%) = [(OD value of the drug administration group + blank group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)] × 100%.

[0047] IC was calculated according to Koch's modified formula 50 Value (μg / ml).

[0048] 1.3 Results Figure 1 .

[0049] The IC50 values ​​of Cistanoside F (CF) of the present invention on 7 human tumor cells are in the range of 0.73-4.55 μg / ml, among which the IC50 values ​​on human bladder cancer cell RT112, human liver cancer cell SK-HEP-1 and human lung cancer cell A549 are the highest. 50 The value is the lowest.

[0050] Example 2 Inhibitory effect of Cistancheside F of the present invention on human bladder cancer cell RT112 tumor growth

[0051] In vivo animal testing reflects the combined efficacy and toxicity of a drug and is a crucial step in the drug screening process that cannot be replaced by in vitro assays and models. Currently, the most widely studied tumor model is the transplant tumor model, which is easy to manipulate, produces stable and uniform tumors in animals, has a high incidence rate, exhibits minimal variability in tumor formation time, and is susceptible to the introduction of interfering factors. In vivo testing typically utilizes animal tumor transplant models and human cancer xenograft models.

[0052] 1 Experimental animals and materials

[0053] 1.1 SPF BALB / c nude mice, male, 18-22 g, 48 mice.

[0054] 1.1 Human bladder cancer cell line RT112 was purchased from the Wuhan University Collection Center and subcultured and preserved by the College of Life Sciences, Inner Mongolia Agricultural University.

[0055] 2 Establishment of RT112 bladder cancer mouse model

[0056] 2.1 Take out the frozen RT112 cell line from liquid nitrogen and shake it in a 40℃ water bath to dissolve it quickly. Then centrifuge at 800rpm for 3min, spray the outer surface of the cryopreservation tube with 75% alcohol, and move the cryopreservation tube into the clean bench. Discard the supernatant and use complete culture medium (DMEM medium containing 10% FBS) to inoculate the cells in a 10cm cell culture dish for subculture. Before inoculation, use trypsin (0.25% trypsin + 0.02% EDTA) to digest the cells, blow them away, suspend them with serum-free DMEM, and adjust the cell concentration to 1.0×10 7 The cells / mL were inoculated subcutaneously in the right axilla of BALB / c nude mice (0.2 mL / mouse).

[0057] 2.2 Grouping and Dosing

[0058] There were 8 animals in each group.

[0059]

[0060]

[0061] 3. Detection indicators:

[0062] 3.1 General Observation: Observe the animal's behavior, reaction, diet, and death after administration, and record the body weight every other day.

[0063] 3.2 Tumor weight and tumor transplantation rate (%): After blood collection, mice were killed by cervical dislocation, and the tumors were removed and weighed on an analytical balance. The tumor weight was recorded, and the tumor inhibition rate (%) was calculated according to the following formula.

[0064] Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of drug-treated group) / tumor weight of model group × 100%.

[0065] 3.3 Effects on immune organs

[0066] The thymus and spleen were removed and weighed using an analytical balance. The organ weights were recorded and the organ index was calculated using the following formula.

[0067] Organ coefficient = organ weight (mg) / body weight (10g).

[0068] 4The experimental results were expressed as mean ± standard deviation, and the t test was used for comparison between groups.

[0069] 5 Results

[0070] 5.1 General Observations

[0071] As can be seen from the table below, compared with the model group, the body weight of mice in the Cistancheside F group, the echinacoside group, and the verbascoside group were significantly reduced, which was statistically significant.

[0072] Compared with the model group, the tumor weights of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly reduced, which was statistically significant; however, at the same dose, the tumor inhibition effects of the echinacoside group and verbascoside group were weaker than that of the Cistancheside F group.

[0073] Table 1 Effects of Cistancheside F of the present invention on body weight, tumor weight and tumor inhibition rate of RT112 bladder cancer-bearing mice

[0074]

[0075] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0076] 5.2 Effects of Cistancheside F on Organ Indexes in RT112 Bladder Cancer-Bearing Mice

[0077] Table 2 Effects of Cistancheside F of the present invention on thymus index and spleen index of RT112 bladder cancer-bearing mice

[0078]

[0079] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0080] Compared with the model group, the thymus index and spleen index of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly decreased, which was statistically significant; however, at the same dose, the thymus index and spleen index of mice in the echinacoside group and verbascoside group were higher than those in the Cistancheside F group.

[0081] The test results show that Cistanoside F (CF) of the present invention has a high tumor inhibition rate against RT112. Compared with echinacoside and verbascose, Cistanoside F shows better in vivo anti-tumor effect.

[0082] Example 3 Inhibitory effect of Cistancheside F of the present invention on human liver cancer cell SK-HEP-1 tumor growth

[0083] 1 Experimental animals and materials

[0084] 1.1 SPF BALB / c nude mice, male, 18-22 g, 48 mice.

[0085] 1.1 Human hepatocellular carcinoma cells SK-HEP-1 were purchased from the Wuhan University Collection Center and subcultured and preserved by the College of Life Sciences, Inner Mongolia Agricultural University.

[0086] 2 Establishment of SK-HEP-1 liver cancer tumor-bearing mouse model

[0087] 2.1 SK-HEP-1 cells cultured in RPMI-1640 medium (containing 10% FBS) were digested with trypsin (0.25% trypsin + 0.02% EDTA), and then dispersed with serum-free RPMI-1640 medium and the cell concentration was adjusted to 7.5×10 6 The cells / mL were inoculated subcutaneously in the right axilla of BALB / c nude mice (0.2 mL / mouse).

[0088] 2.2 Grouping and Dosing

[0089] There were 8 animals in each group.

[0090]

[0091]

[0092] 3. Detection indicators:

[0093] 3.1 General Observation: Observe the animal's behavior, reaction, diet, and death after administration, and record the body weight every other day.

[0094] 3.2 Tumor weight and tumor transplantation rate (%): After blood collection, mice were killed by cervical dislocation, and the tumors were removed and weighed on an analytical balance. The tumor weight was recorded, and the tumor inhibition rate (%) was calculated according to the following formula.

[0095] Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of drug-treated group) / tumor weight of model group × 100%.

[0096] 3.3 Effects on immune organs

[0097] The thymus and spleen were removed and weighed using an analytical balance. The organ weights were recorded and the organ index was calculated using the following formula.

[0098] Organ coefficient = organ weight (mg) / body weight (10g).

[0099] 4The experimental results were expressed as mean ± standard deviation, and the t test was used for comparison between groups.

[0100] 5 Results

[0101] 5.1 General Observations

[0102] As can be seen from the table below, compared with the model group, the body weight of mice in the Cistancheside F group, the echinacoside group, and the verbascoside group were significantly reduced, which was statistically significant.

[0103] Compared with the model group, the tumor weights of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly reduced, which was statistically significant; however, at the same dose, the tumor inhibition effects of the echinacoside group and verbascoside group were weaker than that of the Cistancheside F group.

[0104] Table 3 Effects of Cistancheside F of the present invention on body weight, tumor weight and tumor inhibition rate of SK-HEP-1 liver cancer-bearing mice

[0105]

[0106] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0107] 5.2 Effects of Cistancheside F on Organ Indexes in SK-HEP-1 Liver Cancer-Bearing Mice

[0108] Table 4 Effects of Cistancheside F of the present invention on thymus index and spleen index of SK-HEP-1 liver cancer-bearing mice

[0109]

[0110] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0111] Compared with the model group, the thymus index and spleen index of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly decreased, which was statistically significant; however, at the same dose, the thymus index and spleen index of mice in the echinacoside group and verbascoside group were higher than those in the Cistancheside F group.

[0112] The test results show that the Cistancheside F of the present invention has a high tumor inhibition rate on SK-HEP-1. Compared with echinacoside and verbascoside, Cistancheside F shows better in vivo anti-tumor effect.

[0113] The above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description, and any such variations or modifications are still within the scope of protection of the present invention.

Claims

1. Use of cistancheside F or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating tumors, characterized in that: The tumor is lung cancer, gastric adenocarcinoma, liver cancer, bladder cancer, cervical cancer, prostate cancer or neuroblastoma.

2. The use according to claim 1, characterized in that The tumor is bladder cancer.

3. The use according to claim 1, characterized in that The tumor is liver cancer.

4. The use according to claim 1, characterized in that The administration routes of the cistancheside F or its pharmaceutically acceptable salt are oral, transmucosal, enteral, or intramuscular, subcutaneous, or intravenous administration.

5. The use according to claim 4, characterized in that The administration route is oral administration.

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