Application of a ruberythrin in an antitumor drug and the drug

Abstract

The application discloses application of coccinellin in antitumor drugs and the drugs, wherein the coccinellin has a molecular formula of C 14 H 10 N2O5, and 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 tumor. The antitumor mechanism of the coccinellin is to inhibit KRAS G12D mutant subtype. The coccinellin can effectively inhibit proliferation of various tumor cells, and is expected to be applied to tumor treatment as an antitumor small molecule targeted drug.

Description

Technical Field

[0001] This invention relates to an antitumor drug, and particularly to the application of cinnamicin in antitumor drugs and the drug itself. Background Technology

[0002] Cancer, with its variable biological characteristics and complex molecular mechanisms, constitutes one of the most serious health challenges worldwide and has become a huge burden on the world's public health system.

[0003] Malignant solid tumors are prone to spread and metastasis due to their rapid development, difficulty in detection, and incomplete surgical removal. Chemotherapy becomes the primary treatment in these cases. However, chemotherapy drugs are cytotoxic, killing both tumor and healthy cells, and their toxic side effects limit their use. Therefore, finding effective and safe treatments is crucial for prolonging the survival of cancer patients and improving their quality of life.

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

[0005] KRAS gene mutations include many different site mutations, the most common being G12D (35%), G12V (29%), and G12C (21%) mutations. Although different tumor cells contain KRAS mutations, the downstream signal transduction pathways and the compensatory and feedback regulation between these pathways show great differences. In addition, the KRAS protein itself is difficult to target. Therefore, KRAS-mutant tumors have always been a formidable obstacle.

[0006] With recent research advancements, scientists have identified isomeric sites where KRAS G12C mutants bind to covalent inhibitors. Two KRAS G12C inhibitors are currently on the market, demonstrating promising results in clinical trials. However, targeted therapies for KRAS G12D are still in early research stages, and no drugs are currently available. Therefore, the clinical need to develop KRAS G12D inhibitors is urgent. Summary of the Invention

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

[0008] To address the aforementioned technical problems, this invention provides an application of cinnabarin in antitumor drugs, wherein the molecular formula of cinnabarin is C2. 14 H 10 N2O5, structural formula: .

[0009] Preferably, the tumor includes at least one of colorectal cancer, pancreatic cancer, lung cancer, liver cancer, stomach cancer, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, breast cancer, myeloma, hemangioma, and central nervous system tumors.

[0010] Preferably, the tumor is colorectal cancer or pancreatic cancer.

[0011] Preferably, the antitumor mechanism of the cinnamicin is the inhibition of KRAS gene mutation.

[0012] Preferably, the antitumor mechanism of the cinnamicin is the inhibition of the KRAS G12D mutant subtype.

[0013] The present invention relates to a cinnabarin antitumor drug, comprising cinnabarin, wherein the molecular formula of cinnabarin is C2. 14 H 10 N2O5, structural formula: .

[0014] Preferably, it also includes a pharmaceutically acceptable carrier, which is one or more of the following: adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye, colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier.

[0015] Preferably, the drug is formulated in one or more of the following forms: tablets, injections, granules, capsules, pills, sustained-release formulations, patches, and ointments.

[0016] Preferably, the drug is formulated as an enteric-coated preparation or an injectable preparation.

[0017] Preferably, the drug is administered to mammals, and the daily dosage of the cinnamicin is 1 mg to 1000 mg.

[0018] The beneficial effects of this invention are: This invention provides an application of cinnamicin in anti-tumor drugs. The cinnamicin of this invention can effectively inhibit the proliferation of various tumor cells and can effectively inhibit malignant solid tumors such as colorectal cancer and pancreatic cancer caused by KRAS G12D mutation. It is expected to be used as an anti-tumor small molecule targeted drug for the treatment of tumors. Attached Figure Description

[0019] Figure 1 shows the results of the inhibition of tumor cells by cinnamicin; Figure 2 The results show the inhibition rates of cinnamicin on different tumor cell lines; Figure 3 The antitumor effect of cinnamicin on xenografts in KRAS G12D mice; Detailed Implementation

[0020] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0021] In the specific example, the molecular formula of cinnamicin is C2. 14 H 10 N2O5, structural formula: .

[0022] This invention uses six cell lines to determine the antitumor activity of cinnamicin. These 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).

[0023] (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 10 cm diameter culture dish and the dishes were placed in a 37°C 5% CO2 cell culture incubator.

[0024] (2) Cell passage: When the cell density reaches 80%, discard the old culture medium, wash the cells twice with 10 mL of PBS each time. Add 1 mL of trypsin to digest the cells, observe under a microscope, and when the cells become round, discard the trypsin, add a certain amount of fresh culture medium, and pipette 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, add the culture medium to 10 mL. Incubate at 37℃ in a 5% CO2 cell culture incubator.

[0025] (3) Cell cryopreservation: Select cells in good growth condition, discard the old culture medium, wash the cells twice with 10 mL of PBS each time. Add 1 mL of trypsin to digest the cells, observe under a microscope, and when the cells become round, discard the trypsin, add a certain amount of fresh culture medium, blow the cells completely off, and then add the cell suspension to a 15 mL centrifuge tube. Centrifuge horizontally at room temperature at 1000 rpm for 3 min. Resuspend the cells using the pre-prepared cell cryopreservation solution, aliquot 1 mL into cryovials, place the cryovials in a gradient cooling cryopreservation box, and store them in a -80℃ freezer. After 24 h, transfer the cryovials to a liquid nitrogen tank. All cell experiments must be performed in a laminar flow hood to ensure strict aseptic operation. The laminar flow hood must be sterilized with ultraviolet light for at least 30 min before use.

[0026] (4) Cell count determination: Seed the cell suspension (100 μL / well) into 96-well plates. Pre-incubate the plates in a humidified incubator (37°C, 5% CO2). Add 10 μL of CCK8 solution to each well of the plate. Avoid introducing air bubbles into the wells, as they will interfere with OD readings. Incubate the plates in the incubator for 1–4 hours. Measure the absorbance at 450 nm using a microplate reader.

[0027] (5) Cell proliferation and cytotoxicity assay: Seed cells were subjected to 10 3 -10 4 Cells were cultured at a density of 100 μL of medium in 96-well plates. Cells were incubated at 37°C for 24 hours in a CO2 incubator. 0 μM, 4 μM, 8 μM, 16 μM, 24 μM, and 32 μM cinnamicin were added to the 96-well plates, with 6 replicates for each concentration. The 96-well cell culture plates were incubated for 24 hours. Using a repeat pipette, 10 μL of CCK8 solution was added to each well of the plate. Care was taken not to introduce air bubbles into the wells, as they would interfere with OD readings. The culture plates were incubated for 1–4 hours.

[0028] Before reading the orifice plate, it is important to gently mix it on a track vibrator for 1 minute to ensure even color distribution.

[0029] The absorbance at 450 nm was measured using an ELISA reader.

[0030] Test results are as follows Figure 2 As shown, the final product of this invention, cinnamicin, exhibited good inhibitory effects on cell proliferation in all six tumor cell lines, making it a broad-spectrum antitumor drug. The IC50 values ​​of cinnamicin against different tumor cell lines are shown in Table 1.

[0031] Table 1 <![CDATA[IC 50 (uM)]]> 5 8.304 3.908 12.16 9.762 13.83 like Figure 1 As shown, the cells in the control group (Control) are morphologically intact, grow vigorously, and are densely packed. After drug administration (Drug), a significant decrease in the number of cells was observed under the microscope, and upon magnification, cell shrinkage, blistering, and necrosis could be observed.

[0032] Trial of cinnamicin on mouse xenografts Test drug: Cinnamomum cassia Male M-NSG mice aged 4-6 weeks were used. After purchasing and raising them for a 7-day quarantine period, vaccination was initiated.

[0033] Culture KRAS G12D mutant colon cancer cells at 5*10 6 Mice were inoculated at a concentration of 200 μL / cell. The inoculation site was usually subcutaneous. Subcutaneously inoculated cells typically formed tumors within 1-2 weeks. After tumor formation, close monitoring of the animal's condition, weight, and tumor volume was necessary. Tumor volume was generally measured using the formula V=ab. 2 / 2 Calculations are performed, where a and b are the two vertical diameters of the tumor. When the tumor volume is greater than 100 mm... 3 Treatment began at the specified time. The administration methods were as follows: the control group received 0.1 mL of physiological saline / day, the positive control group received 7.5 mg / kg / day of 5-fluorouracil (5-Fu), the medium-dose group received 5 mg / kg / day of cinnamicin, and the high-dose group received 7.5 mg / kg / day of cinnamicin. Tumor volume and mouse weight were measured every 4 days during treatment. After 21 days of continuous treatment, the mice were sacrificed and their tissues were collected.

[0034] Depend on Figure 3 As can be seen, cinnamicin can significantly reduce the volume of transplanted tumors, exhibiting a significant anti-tumor effect. Furthermore, it has little impact on the body weight of mice, demonstrating good safety.

[0035] In pharmaceutical manufacturing, cinnamicin can be incorporated into a pharmaceutically acceptable carrier, which includes one or more of the following: adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes, colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers. The drug can be formulated as one or more of the following: enteric-coated formulations, tablets, injections, granules, capsules, pills, sustained-release formulations, patches, and ointments.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of a cinnamicin in the preparation of antitumor drugs, characterized in that: The molecular formula of the cinnamicin is C 14 H 10 N2O5, structural formula: ， The antitumor mechanism of the cinnamicin is the inhibition of KRAS gene mutation; The tumor mentioned is one or more of the following: cervical cancer, liver cancer, stomach cancer, and multiple myeloma.

2. The application of cinnamicin according to claim 1 in the preparation of antitumor drugs, characterized in that: The antitumor mechanism of the cinnamicin is to inhibit the KRAS G12D mutant subtype.

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