Application of cinnabaric acid in anti-tumor drug and anti-tumor drug

Abstract

The invention discloses application of cinnabine in antitumor drugs and a drug, the molecular formula of the cinnabine is C14H10N2O5, and tumors comprise at least one of colorectal cancer, pancreatic cancer, lung cancer, liver cancer, gastric cancer, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, breast cancer, myeloma, hemangioma and central nervous system tumor. The anti-tumor mechanism of the cinnabarine is to inhibit a KRAS G12D mutation subtype. The cinnabaric acid can effectively inhibit proliferation of various tumor cells, and is expected to be applied to tumor treatment as an anti-tumor small-molecular targeted drug.

Description

Technical Field

[0001] The present invention relates to an anti-tumor drug, in particular to the application of cinnamic acid in the anti-tumor drug and the drug. Background Art

[0002] Tumor diseases, with their diverse biological characteristics and complex molecular mechanisms, constitute one of the most serious health challenges worldwide and have become a huge burden on the world's public health system.

[0003] Due to the complex factors of rapid development, difficulty in detection and incomplete surgical cure of malignant solid tumors, they spread and metastasize. At this time, chemotherapy will be used as the main means of treatment. Chemotherapy drugs are cytotoxic drugs. While killing tumor cells, they will also kill normal cells. The toxic side effects limit the use of many chemotherapy drugs. Therefore, finding a treatment method with strong effectiveness and good safety is of great significance to prolonging the survival of cancer patients and improving their quality of life.

[0004] Kirsten rat sarcoma viral oncogene (KRAS) is one of the most frequently mutated oncogenes in human malignant tumors and an important cancer driver gene for various high-mortality tumors. Studies have shown that approximately 30% of human malignancies are associated with KRAS gene mutations, with the mutation rate of KRAS in pancreatic cancer reaching 97% and in colorectal cancer reaching 52%.

[0005] KRAS gene mutations include many different site mutations, the most common of which are G12D (35%), G12V (29%) and G12C (21%) mutations. Although different tumor cells contain KRAS mutations, their downstream signal transduction pathways and the mutual compensation and feedback regulation between various pathways show great differences. In addition, the KRAS protein itself is difficult to target. Therefore, KRAS mutant tumors have always been an insurmountable chasm.

[0006] With the continuous progress of research in recent years, scientists have found the heteromeric sites where KRAS G12C mutants bind to covalent inhibitors. Currently, two KRAS G12C inhibitors are on the market and have achieved surprising results in clinical studies. However, targeted therapy for KRAS G12D is still in the early stages of research, and there are currently no drugs on the market. Therefore, the clinical need to develop KRAS G12D inhibitors is imminent. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an application of rubellonic acid in anti-tumor drugs and a drug.

[0008] In order to solve the above technical problems, the present invention provides an application of cinnamic acid in anti-tumor drugs, wherein the molecular formula of cinnamic acid is C 14 H 10 N2O5, the structural formula is:

[0009]

[0010] Preferably, the tumor includes at least one of colorectal cancer, pancreatic cancer, lung cancer, liver cancer, gastric cancer, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, breast cancer, myeloma, hemangioma, and central nervous system tumors.

[0011] Preferably, the tumor is colorectal cancer or pancreatic cancer.

[0012] Preferably, the anti-tumor mechanism of ruthenic acid is to inhibit KRAS gene mutation.

[0013] Preferably, the anti-tumor mechanism of ruthenic acid is to inhibit the KRAS G12D mutant subtype.

[0014] The cinnamic acid anti-tumor drug of the present invention comprises cinnamic acid, wherein the molecular formula of the cinnamic acid is C 14 H 10 N2O5, the structural formula is:

[0015]

[0016] Preferably, a pharmaceutically acceptable carrier is also included, and the pharmaceutically acceptable carrier is one or more of an adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye, colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier.

[0017] Preferably, the drug is in the form of one or more of tablets, injections, granules, capsules, pills, sustained-release preparations, patches, and ointments.

[0018] Preferably, the drug is in the form of an enteric-coated preparation or an injection.

[0019] Preferably, the drug administration subject is a mammal, and the daily administration amount of cinnamic acid is 1 mg-1000 mg.

[0020] The beneficial effects of the present invention are as follows: the present invention provides an application of cinnamic acid in anti-tumor drugs, the cinnamic acid of the present invention can effectively inhibit the proliferation of various tumor cells, can effectively inhibit malignant solid tumors such as colorectal cancer and pancreatic cancer caused by KRAS G12D mutation, and is expected to be used as an anti-tumor small molecule targeted drug in the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result diagram of the inhibition of tumor cells by cinnabaric acid;

[0022] Figure 2 The results are the inhibition rate of cinnamic acid on different tumor cell lines;

[0023] Figure 3 The anti-tumor effect of rubellonic acid on KRAS G12D mouse transplant tumors; DETAILED DESCRIPTION

[0024] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0025] In the specific example, the molecular formula of cinnamic acid is C 14 H 10 N2O5, the structural formula is:

[0026]

[0027] The present invention uses 6 cell lines to measure the anti-tumor activity of rutinic acid, and the six cell lines are A549 (human non-small cell lung cancer cells), Hela (human cervical cancer cells), Hep G2 (human liver cancer cells), HGC27 (human gastric cancer cells (undifferentiated)), RPM8826 (human multiple myeloma cell line), and HCT116 (KRAS G12D mutation) (human colon cancer cells).

[0028] (1) Cell culture: Tumor cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). 10 mL of complete culture medium was added to each culture dish with a diameter of 10 cm and the dishes were placed in a 37° C. 5% CO 2 cell culture incubator for culture.

[0029] (2) Cell passaging: When the cell density reaches 80%, discard the old culture medium and wash the cells twice with PBS, 10 mL each time. Add 1 mL of trypsin to digest the cells and observe under a microscope. When the cells become round, discard the trypsin and add a certain amount of fresh culture medium to beat the cells into single cells. Then divide the cell suspension into two equal parts and add them to two new 10 cm cell culture dishes. Finally, fill the culture medium to 10 mL. Place in a 37°C 5% CO2 cell culture incubator for culture.

[0030] (3) Cell cryopreservation: Select cells with good growth status, discard the old culture medium, and wash the cells twice with PBS, 10 mL each time. Add 1 mL of trypsin to digest the cells and observe under a microscope. When the cells become round, discard the trypsin, add a certain amount of fresh culture medium, blow off the cells completely, and then add the cell suspension to a 15 mL centrifuge tube. Centrifuge at room temperature at 1000 rpm for 3 minutes in a horizontal centrifuge. Resuspend the cells in the cell freezing solution prepared in advance, dispense them into cryopreservation tubes at 1 mL per tube, place the cryopreservation tubes in a gradient cooling cryopreservation box, and place them in a -80°C refrigerator. After 24 hours, transfer the cryopreservation tubes to a liquid nitrogen tank. All cell experiments need to be completed in a clean bench to ensure strict aseptic operation. The clean bench needs to be sterilized by ultraviolet for more than 30 minutes before use.

[0031] (4) Determination of cell number: Inoculate the cell suspension (100 μL / well) in a 96-well plate. Pre-incubate the plate in a humidified incubator (at 37°C, 5% CO2). Add 10 μL of CCK8 solution to each well of the plate. Be careful not to introduce bubbles into the wells as they will interfere with the OD reading. Incubate the plate in the incubator for 1-4 hours. Measure the absorbance at 450 nm using a microplate reader.

[0032] (5) Cell proliferation and cytotoxicity assay: Seed cells were cultured at 10 3 -10 4 Cells were cultured at a density of 100 μL of culture medium in a 96-well plate at 37°C in a CO2 incubator for 24 hours. 0 μM, 4 μM, 8 μM, 16 μM, 24 μM, and 32 μM of cinnamic acid were added to the 96-well plate, with 6 replicates for each concentration. The 96-well cell culture plate was incubated in the incubator for 24 hours. 10 μL of CCK8 solution was added to each well of the plate using a repeating pipette. Be careful not to introduce bubbles into the wells as they will interfere with the OD readings. The culture plates were incubated in the incubator for 1-4 hours.

[0033] Before reading the plate, it is important to mix gently on an orbital shaker for 1 minute to ensure an even distribution of color.

[0034] The absorbance at 450 nm was measured using a microplate reader.

[0035] The test results are as follows Figure 2 As shown, the final cinnamic acid of the present invention exhibits a good effect of inhibiting cell proliferation in 6 tumor cell lines and is a broad-spectrum anti-tumor drug. The IC50 values ​​of cinnamic acid on different tumor cell lines are shown in Table 1.

[0036] Table 1

[0037]

[0038] like Figure 1 As shown, the cells in the control group (Control) are intact in morphology, vigorous in growth, and tightly arranged. After drug administration (Drug), it is clearly seen that the number of cells under the microscope is reduced, and after magnification, it can be observed that the cells are wrinkled, blistering, and necrotic.

[0039] Experimental study on rutinic acid in mouse transplanted tumors

[0040] Experimental drug: Veruminous acid

[0041] Male M-NSG mice aged 4 to 6 weeks were used and inoculation was started after a 7-day quarantine period after purchase.

[0042] KRAS G12D mutant colon cancer cells were cultured at 5*10 6 The mice are inoculated with a concentration of 10 cells / 200 μL / mouse. The inoculation site is usually subcutaneous. The subcutaneously inoculated cells usually form tumors within 1-2 weeks. After the tumor is formed, the animal's condition, weight and tumor volume need to be closely observed. The tumor volume is generally measured using the formula V = ab 2 / 2

[0043] Calculate, where a and b are the two perpendicular diameters of the tumor. 3 The administration was started at 14:00 pm. The administration method was as follows: the control group was given 0.1 mL of normal saline per day, the positive control group was given 5-fluorouracil (5-Fu) 7.5 mg / kg / day, the medium-dose group was given 5 mg / kg / day of cinnabaric acid, and the high-dose group was given 7.5 mg / kg / day of cinnabaric acid. During the treatment, the tumor volume and the weight of the mice were measured every 4 days. After 21 consecutive days of treatment, the mice were killed and the samples were collected.

[0044] Depend on Figure 3 It can be seen that cinnamic acid can significantly reduce the volume of transplanted tumors and has a significant anti-tumor effect. It also has little effect on the weight of mice and has good safety.

[0045] When preparing the medicine, cinnamic acid can be added to a pharmaceutically acceptable carrier, which includes one or more of an adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye, colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier. The drug preparation form is one or more of an enteric-coated preparation, tablet, injection, granule, capsule, pill, sustained-release preparation, patch, and ointment.

[0046] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application of cinnamic acid in anti-tumor drugs, characterized in that: The molecular formula of the cinnamic acid is C 14 H 10 N2O5, the structural formula is:

2. The use of cinnamic acid in antitumor drugs according to claim 1, characterized in that: The tumor includes at least one of colorectal cancer, pancreatic cancer, lung cancer, liver cancer, gastric cancer, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, breast cancer, myeloma, hemangioma, and central nervous system tumors.

3. The use of cinnamic acid in antitumor drugs according to claim 2, characterized in that: The tumor is colorectal cancer or pancreatic cancer.

4. The use of cinnamic acid in antitumor drugs according to claim 1, characterized in that: The anti-tumor mechanism of rubellonic acid is to inhibit KRAS gene mutation.

5. The use of cinnamic acid in antitumor drugs according to claim 1, characterized in that: The anti-tumor mechanism of rubellonic acid is to inhibit the KRAS G12D mutant subtype.

6. A cinnamic acid anti-tumor drug, characterized in that: Including cinnabar acid, the molecular formula of cinnabar acid is C 14 H 10 N2O5, the structural formula is:

7. The anti-tumor drug according to claim 6, characterized in that: Also included are pharmaceutically acceptable carriers, which include one or more of adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes, colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.

8. The anti-tumor drug according to claim 6, characterized in that: The drug is in the form of one or more of tablets, injections, granules, capsules, pills, sustained-release preparations, patches, and ointments.

9. The anti-tumor drug according to claim 6, characterized in that: The drug is in the form of an enteric-coated preparation or an injection.

10. The anti-tumor drug according to claim 6, characterized in that: The drug administration object is a mammal, and the daily administration amount of the cinnamic acid is 1 mg-1000 mg.

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