Use of annocatin and / or dimethylbiguanide in the preparation of antitumor formulations

Abstract

The application relates to application of annocatin and / or dimethylbiguanide in preparation of antitumor preparations and belongs to the technical field of medicines. The application provides application of annocatin and / or dimethylbiguanide in preparation of antitumor medicines, wherein the concentration of annocatin is <=20 micromol / L, and the concentration of dimethylbiguanide is <=20 mmol / L. The annocatin and dimethylbiguanide are combined in the application, can play a synergistic effect, have stronger antitumor activity, can effectively reduce the toxic side effect of annocatin on normal cells, and have the characteristics of safety and high efficiency.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of anechoic acid and / or dimethyl biguanide in the preparation of antitumor formulations. Background Technology

[0002] Cancer is a serious disease that threatens human health. Its occurrence and progression involve multiple cell signaling pathways and various molecular mechanisms, making it difficult for single-drug therapy to comprehensively block the proliferation and spread of cancer cells. Cancer cells are highly susceptible to drug resistance during treatment, and existing anti-tumor drugs have significant toxic side effects on normal cells and tissues. Traditional Chinese medicine (TCM) components can regulate multiple cell signaling pathways at the molecular level, exhibiting selective toxicity against cancer cells while relatively protecting normal cells, thus demonstrating anti-tumor potential in scientific research and clinical trials. However, single TCM herbs or TCM components often have relatively few targets or cell signaling pathways, resulting in poor anti-tumor activity.

[0003] Annonacin is widely found in the branches and leaves of plants in the Litchiaceae family and possesses certain antitumor activity. However, it exhibits various toxic side effects on normal cells and tissues, including cytotoxicity, neurotoxicity, hepatotoxicity, cardiotoxicity, and interference with the reproductive system. Metformin is a type 2 diabetes treatment drug, and in recent years it has been found to exert antitumor effects through multiple pathways, including cellular metabolic regulation, AMPK pathway activation, antioxidant stress, inhibition of cancer stem cells, and anti-angiogenesis. However, to date, there is no research or application of combining annonacin with metformin in the preparation of antitumor drugs and the reduction of drug toxicity. Summary of the Invention

[0004] The purpose of this invention is to provide the application of anechoic acid and / or dimethyl biguanide in the preparation of antitumor formulations, so as to solve the problems of weak antitumor activity and strong toxic side effects on normal cells and tissues of single drugs in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the use of anechoic acid and / or dimethyl biguanide in the preparation of antitumor drugs.

[0007] Preferably, the tumor includes one or more of ovarian cancer, osteosarcoma, prostate cancer, and colorectal cancer.

[0008] Preferably, the concentration of anechoic acid in the preparation of antitumor drugs is ≤20 μmol / L;

[0009] The concentration of dimethyl biguanide in the preparation of antitumor drugs is ≤20 mmol / L.

[0010] Preferably, in the preparation of antitumor drugs by combining anechoic acid and dimethyl biguanide, the concentration of anechoic acid is ≤20 μmol / L and the concentration of dimethyl biguanide is ≤20 mmol / L.

[0011] The present invention also provides an antitumor formulation comprising anechoic acid and / or dimethyl biguanide.

[0012] Preferably, the concentration of anechoic acid is ≤20 μmol / L; and the concentration of dimethyl biguanide is ≤20 mmol / L.

[0013] The present invention has the following technical effects and advantages:

[0014] This invention combines anesthetic and dimethyl biguanide to achieve a synergistic effect, resulting in stronger antitumor activity and effectively reducing the toxic side effects of anesthetic on normal cells. It is safe and highly effective. Attached Figure Description

[0015] Figure 1 The figures show the cell-inhibitory activities of anesthetic and / or bimethyl guanidine. HCT116 represents the inhibitory effect of anesthetic and / or bimethyl guanidine on the human colorectal cancer cell line HCT116; MG63 represents the inhibitory effect of anesthetic and / or bimethyl guanidine on the human osteosarcoma cell line MG63; PC3 represents the inhibitory effect of anesthetic and / or bimethyl guanidine on the human prostate cancer cell line PC3; SKOV3 represents the inhibitory effect of anesthetic and / or bimethyl guanidine on the human ovarian cancer cell line SKOV3; MIHA represents the inhibitory effect of anesthetic and / or bimethyl guanidine on the human normal hepatocyte cell line MIHA; ANN represents the anesthetic alone group; MET represents the bimethyl guanidine alone group; and ANN+MET represents the anesthetic combined with bimethyl guanidine group.

[0016] Figure 2 The figure shows the inhibition of cell colony formation by anechoic acid and / or dimethyl biguanide. In the figure, HCT116 represents the inhibition of cell colony formation by anechoic acid and / or dimethyl biguanide on human colorectal cancer cell line HCT116, and MIHA represents the inhibition of cell colony formation by anechoic acid and / or dimethyl biguanide on human normal liver cell line MIHA.

[0017] Figure 3 Figure A shows the inhibition of tumor cell metastasis by anechoic acid and / or dimethyl biguanide. Figure B shows the inhibition of cell metastasis by anechoic acid and / or dimethyl biguanide on human colorectal cancer cell line HCT116.

[0018] Figure 4 The figures show the apoptosis-inducing activity of anechoic acid and / or dimethyl biguanide. In the figure, HCT116 represents the apoptosis-inducing effect of anechoic acid and / or dimethyl biguanide on the human colorectal cancer cell line HCT116, and MIHA represents the apoptosis-inducing effect of anechoic acid and / or dimethyl biguanide on the human normal hepatocyte cell line MIHA.

[0019] Figure 5 The figure shows the inhibition of cytochrome P450 enzyme activity by anechoic acid and / or bimethyl guanidine. CYP2B6 represents the inhibition of cytochrome P450 enzyme CYP2B6 by anechoic acid and / or bimethyl guanidine, CYP3A4 represents the inhibition of cytochrome P450 enzyme CYP3A4 by anechoic acid and / or bimethyl guanidine, and CYP2C9 represents the inhibition of cytochrome P450 enzyme CYP2C9 by anechoic acid and / or bimethyl guanidine. Detailed Implementation

[0020] This invention provides the use of anechoic acid and / or dimethyl biguanide in the preparation of antitumor drugs.

[0021] In this invention, the tumor includes one or more of ovarian cancer, osteosarcoma, prostate cancer, and colorectal cancer.

[0022] In this invention, the concentration of anechoic acid in the preparation of antitumor drugs is ≤20 μmol / L, preferably ≤10 μmol / L, and more preferably 10 μmol / L;

[0023] The concentration of dimethyl biguanide in the preparation of antitumor drugs is ≤20 mmol / L, preferably ≤10 mmol / L, and more preferably 10 mmol / L.

[0024] In this invention, in the preparation of an antitumor drug by combining anechoic acid and bimethyl biguanide, the concentration of anechoic acid is ≤20 μmol / L, preferably ≤10 μmol / L, and more preferably 10 μmol / L; the concentration of bimethyl biguanide is ≤20 mmol / L, preferably ≤10 mmol / L, and more preferably 10 mmol / L.

[0025] The present invention also provides an antitumor formulation comprising anechoic acid and / or dimethyl biguanide.

[0026] In this invention, the concentration of anechoic acid is ≤20 μmol / L, preferably ≤10 μmol / L, and more preferably 10 μmol / L;

[0027] The concentration of dimethyl biguanide is ≤20 mmol / L, preferably ≤10 mmol / L, and more preferably 10 mmol / L.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] The biological materials used in this invention, including the human colorectal cancer cell line HCT116, the human osteosarcoma cell line MG63, the human prostate cancer cell line PC3, the human ovarian cancer cell line SKOV3, and the human normal hepatocyte cell line MIHA, were all purchased from the China Center for Type Culture Collection (CCTCC).

[0030] In the test reagents of this invention: DMEM high glucose medium, fetal bovine serum (FBS), penicillin / streptomycin, and trypsin were purchased from Thermo Fisher Scientific, Inc., USA; phosphate-buffered saline (PBS) was purchased from HyClone, Inc., USA.

[0031] Complete culture medium preparation: Mix 500 mL of DEME high glucose medium, 50 mL of LBS, and 1 mL of penicillin / streptomycin.

[0032] Example 1: Assay of antitumor cell viability and toxicity of anechoic acid and / or dimethyl biguanide

[0033] (1) Inoculation: HCT116, MG63, PC3, SKOV3 and MIHA cell lines in the logarithmic growth phase were inoculated into 96-well plates. The inoculation volume was 160 μL / well and the inoculation density was 5000 cells / well. The cells were divided into an anthocyanin monotherapy group (ANN), a bimethyl guanidine monotherapy group (MET), an anthocyanin combined with bimethyl guanidine group (ANN+MET), a blank group and a negative control group.

[0034] (2) Drug administration: 12 h after inoculation of each cell line, 40 μL of complete culture medium containing 0.3125 μmol / L, 0.625 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, and 20 μmol / L anthocyanin were added to the ANN group wells, respectively; 40 μL of complete culture medium containing 0.3125 mmol / L, 0.625 mmol / L, 1.25 mmol / L, 2.5 mmol / L, 5 mmol / L, 10 mmol / L, and 20 mmol / L biguanide were added to the MET group wells, respectively; and 40 μL of complete culture medium containing 0.3125 μmol / L anthocyanin was added to the ANN+MET group wells, respectively. 40 μL of complete culture medium was prepared for the following conditions: lycheein + 0.3125 mmol / L bimethyl guanidine, 0.625 μmol / L anthocyanin + 0.625 mmol / L bimethyl guanidine, 1.25 μmol / L anthocyanin + 1.25 mmol / L bimethyl guanidine, 2.5 μmol / L anthocyanin + 2.5 mmol / L bimethyl guanidine, 5 μmol / L anthocyanin + 5 mmol / L bimethyl guanidine, 10 μmol / L anthocyanin + 10 mmol / L bimethyl guanidine, and 20 μmol / L anthocyanin + 20 mmol / L bimethyl guanidine. 40 μL of complete culture medium was added to the inoculation wells of the blank group and the negative control group, respectively. Each 96-well plate was incubated at 37°C with 5% CO2 for 44 h.

[0035] (3) Measurement: 20 μL of 5 mg / mL LMTT solution was added to each well of the ANN group, MET group, ANN+MET group, and negative control group. After incubation for 4 h, the supernatant was discarded, and 100 μL of DMSO was added to each well. The mixture was shaken for 15 s to fully dissolve the formazan. The OD of each well was then measured using a microplate reader. 492nm The values ​​were calculated, and the cell viability of each cell line was determined. The results are as follows: Figure 1 As shown.

[0036] The results showed that, compared with the ANN and MET groups, the ANN+MET group was more effective in reducing the cell viability of HCT116, MG63, PC3, and SKOV3 cell lines, and also effectively reduced the cytotoxicity of anechoic acid to MIHA cell line. In the ANN+MET group, the cell viability of each tumor cell line was lowest in the wells treated with 10 μmol / L anechoic acid + 10 mmol / L bimethyl guanidine. This indicates that the combination of anechoic acid and bimethyl guanidine has stronger antitumor activity and can effectively reduce the cytotoxicity of anechoic acid to normal cells.

[0037] Example 2: Effects of anechoic acid and / or dimethyl biguanide on cell colony formation

[0038] (1) Inoculation: HCT116 and MIHA cell lines were inoculated into 6-well plates containing 2 mL of complete culture medium, with an inoculation density of 5 × 10⁶ cells / well. 3 Each cell / well was divided into four groups: an anthocyanin monotherapy group, a bimethyl guanidine monotherapy group, an anthocyanin + bimethyl guanidine combination group, and a control group.

[0039] (2) Drug administration: The cells in each 6-well plate were cultured in an incubator at 37°C and 5% CO2 for 6 hours until they adhered to the wall. The complete culture medium was then discarded. 2 mL of complete culture medium containing 10 μmol / L anthocyanin was added to the wells of the anthocyanin-only group. 2 mL of complete culture medium containing 10 mmol / L bimethyl guanidine was added to the wells of the bimethyl guanidine-only group. 2 mL of complete culture medium containing 10 μmol / L anthocyanin and 10 mmol / L bimethyl guanidine was added to the wells of the anthocyanin + bimethyl guanidine combination group. 2 mL of complete culture medium containing 0.1% DMSO was added to the wells of the control group. The cells were cultured and the corresponding complete culture medium was changed every 2 days.

[0040] (3) Observation: After 10 days of continuous culture, the formation of HCT116 and MIHA cell colony clones in each inoculation well was observed. The results are as follows: Figure 2 As shown.

[0041] The results showed that 10 μmol / L anthocyanin effectively inhibited the formation of HCT116 and MIHA cell colonies; 10 mmol / L bimethyl guanidine had a slight inhibitory effect on the formation of HCT116 cell colonies but no significant effect on the formation of MIHA cell colonies; the combined use of 10 μmol / L anthocyanin and 10 mmol / L bimethyl guanidine completely inhibited the formation of HCT116 cell colonies and significantly reduced the inhibitory effect of anthocyanin on the formation of MIHA cell colonies. This indicates that the combination of anthocyanin and bimethyl guanidine has excellent anti-tumor effects while significantly reducing the toxic side effects of anthocyanin on normal cells.

[0042] Example 3: Effects of anechoic acid and / or dimethyl biguanide on cell migration

[0043] (1) Inoculation: HCT116 cell line was inoculated into 6-well plates at a density of 2 × 10⁶ cells / well. 5 Each cell / well was divided into four groups: an anthocyanin monotherapy group, a bimethyl guanidine monotherapy group, an anthocyanin + bimethyl guanidine combination group, and a control group.

[0044] (2) Drug administration: After the cells adhered to the walls of each 6-well plate in an incubator at 37℃ and CO2 concentration of 5%, 2 mL of complete culture medium containing 10 μmol / L anthocyanin was added to the wells of the anthocyanin-only group, 2 mL of complete culture medium containing 10 mmol / L bimethyl guanidine was added to the wells of the bimethyl guanidine-only group, 2 mL of complete culture medium containing 10 μmol / L anthocyanin and 10 mmol / L bimethyl guanidine was added to the wells of the anthocyanin + bimethyl guanidine combination group, and 2 mL of complete culture medium containing 0.1% DMSO was added to the wells of the control group. After incubation for 48 h, 0.5 mL of trypsin was added to each well for 2 min for cell counting.

[0045] (3) Cell migration: Take 1×10 5 One cell was mixed with 100 μL of FBS-free complete medium and seeded into a Transwell chamber. 600 μL of complete medium containing 10% FBS was added to the bottom of the Transwell chamber, and the cells were cultured for 72 h at 37 °C and 5% CO2. Cells that had not migrated to the inner layer of the Transwell chamber were wiped clean with a cotton swab. 0.5 mL of 4% paraformaldehyde was added to each well and fixed at room temperature for 15 min. After washing along the side wall of the Transwell chamber with PBS and drying, 500 μL of crystal violet was added to each well and stained in the dark for 5 min. Excess crystal violet was then washed along the side wall of the Transwell chamber with ddH2O and dried. 500 μL of 33% acetic acid was added to each well to fully dissolve the crystal violet.

[0046] (4) Detection: Take 100 μL of glacial acetic acid in which crystal violet is fully dissolved, and measure the OD on an ELISA reader. 560nm Value. Result as follows Figure 3 As shown.

[0047] The results showed that 10 μmol / L anthocyanin effectively inhibited the metastasis of HCT116 cells, while 10 mmol / L bimethyl guanidine had a slight inhibitory effect on HCT116 cell metastasis. The combined use of 10 μmol / L anthocyanin and 10 mmol / L bimethyl guanidine significantly inhibited HCT116 cell metastasis, and the inhibitory effect was much stronger than simply adding the concentrations of anthocyanin and bimethyl guanidine. This indicates that the combination of anthocyanin and bimethyl guanidine has an excellent effect on inhibiting tumor cell metastasis.

[0048] Example 4: Effects of anechoic acid and / or dimethyl biguanide on apoptosis

[0049] (1) Seeding: HCT116 and MIHA cell lines were seeded into 6-well plates at a density of 2 × 10⁶ cells per well. 5 Each cell / well was divided into four groups: an anthocyanin monotherapy group, a bimethyl guanidine monotherapy group, an anthocyanin + bimethyl guanidine combination group, and a control group.

[0050] (2) Drug administration: After the cells adhered to the cell walls, each 6-well plate was cultured in an incubator at 37℃ and CO2 concentration of 5% for 6 hours. Then, 2 mL of complete culture medium containing 10 μmol / L anthocyanin was added to the wells of the anthocyanin-only group, 2 mL of complete culture medium containing 10 mmol / L bimethyl guanidine was added to the wells of the bimethyl guanidine-only group, 2 mL of complete culture medium containing 10 μmol / L anthocyanin and 10 mmol / L bimethyl guanidine was added to the wells of the anthocyanin + bimethyl guanidine combination group, and 2 mL of complete culture medium containing 0.1% DMSO was added to the wells of the control group. The cells were cultured for another 48 hours.

[0051] (3) Assay: 0.5 mL of EDTA-free trypsin was added to each well for 2 min of digestion. All cells from each well were collected, centrifuged at 8000 rpm for 6 min, washed twice with 1×PBS, and the supernatant was discarded to obtain the cells from each well; refer to Alexa. Instructions for using the 488annexin V / Dead Cell Apoptosis Kit: Perform cell culture on each inoculation well. Fluor 488 annexin V and PI double staining was performed, and the results were analyzed and processed using flow cytometry and FlowJo software. The results are as follows: Figure 4 As shown.

[0052] The results showed that HCT116 and MIHA cells treated with anechoic acid alone underwent apoptosis, while no apoptosis was observed in HCT116 and MIHA cells treated with bimethyl guanidine alone. Compared with the anechoic acid and bimethyl guanidine alone groups, the combination of anechoic acid and bimethyl guanidine increased the apoptosis rate of HCT116 cells and decreased the apoptosis rate of MIHA cells. This indicates that the combination of anechoic acid and bimethyl guanidine has stronger antitumor activity and lower toxicity to normal cells.

[0053] Example 5: Effects of anechoic acid and / or dimethyl biguanide on cytochrome P450 enzyme activity

[0054] (1) Grouping: Bupropion, midazolam and tolbutamide were used as probe substrates for detecting the enzyme activities of cytochrome P450 enzymes CYP2B6, CYP3A4 and CYP2C9, respectively. Each probe substrate was divided into an acetaminophen monotherapy group, a dimethyl biguanide monotherapy group, an acetaminophen + dimethyl biguanide combination group and a control group.

[0055] (2) Drug administration: Anthocyanin was added to the probe substrate of the anthocyanin monotherapy group, dimethyl biguanide was added to the probe substrate of the dimethyl biguanide monotherapy group, anthocyanin and dimethyl biguanide were added to the probe substrate of the anthocyanin + dimethyl biguanide combination group, and DMSO was added to the probe substrate of the control group. Then, mouse liver microsomes and Tris-HCl buffer without NADPH were added to each probe substrate in sequence and pre-incubated at 37°C for 5 min. After adding NADPH and incubating at 37°C for 60 min, 400 μL of ice-cold acetonitrile was added immediately to terminate the reaction. Finally, 0.2 mL of diazepam was added to each probe substrate as an internal standard. After vortexing for 2 min, the supernatant was obtained by centrifugation at 13000 rpm for 10 min. The amount and concentration of each reagent added are shown in Table 1.

[0056] (3) Detection: Take 100 μL of each supernatant and add it to ZORBAX Eclipse Plus. In a C18 column (1.8 μm, 2.1 × 50 mm), LC / MS was performed using an Agilent 1100 high-performance liquid chromatograph (quaternary pump) with multiple reaction monitoring (MRM) in positive electrospray ionization (PSI) mode. Specifically, the mobile phase consisted of solvent A (0.1 wt% formic acid aqueous solution) and solvent B (acetonitrile), which increased the polarity of mobile phase A to improve separation efficiency. The elution temperature was 35 °C, the flow rate was 0.4 mL / min, and the elution program was as follows: 0–0.5 min, linear transition from 80% solvent A to 5% solvent A; 0.5–1.5 min, 5% solvent A; 1.5–1.6 min, linear transition from 5% solvent A to 80% solvent A; then a 1.3 min stop to equilibrate the C18 column, for a total elution time of 1.8 min. Instrument control and data acquisition were performed using MassHunter software (VB07.00). Results are as follows: Figure 5 As shown.

[0057] Table 1. Cytochrome P450 enzyme activity detection system

[0058]

[0059]

[0060] The results showed that the activities of cytochrome P450 enzymes CYP2B6, CYP3A4, and CYP2C9 were significantly reduced in the anthocyanin-only group, while the activities of each cytochrome P450 enzyme showed no significant change in the bimethyl guanidine-only group. However, the activities of each cytochrome P450 enzyme were significantly increased in the anthocyanin + bimethyl guanidine combination group compared to the anthocyanin-only group. This indicates that the combination of anthocyanin and bimethyl guanidine can effectively alleviate the inhibitory effect of anthocyanin on cytochrome P450 enzyme activity.

[0061] As can be seen from the above embodiments, the present invention provides the application of anechoic acid and / or dimethyl biguanide in the preparation of antitumor formulations. The combination of anechoic acid and dimethyl biguanide in this invention can exert a synergistic effect, exhibiting stronger antitumor activity, and can effectively reduce the toxic side effects of anechoic acid on normal cells, thus possessing the advantages of safety and high efficiency.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of anechoic acid combined with dimethyl biguanide in the preparation of antitumor drugs, characterized in that, The tumor is selected from one or more of ovarian cancer, osteosarcoma, prostate cancer, and colorectal cancer.

2. The application according to claim 1, characterized in that, The concentration of anechoic acid in the preparation of antitumor drugs is 0.3125~20 μmol / L; the concentration of dimethyl biguanide in the preparation of antitumor drugs is 0.3125~20 mmol / L.

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