Application of chelerythrine and chelerythrine combined with sanguinarine in the preparation of cancer therapeutic drugs

Abstract

The present invention belongs to the field of medicine, and specifically relates to the use of chelerythrine and chelerythrine combined with sanguinarine in the preparation of cancer treatment drugs. In the drug, the mass ratio of chelerythrine, chelerythrine and sanguinarine is 1-5:1-5:1-5; the drug is used to promote cancer cell apoptosis, reduce cancer cell activity, inhibit cancer cell proliferation, and suppress cancer cell migration, and the cancer includes esophageal squamous cell carcinoma. The present invention first discovered and confirmed that chelerythrine has a strong anti-tumor effect, and chelerythrine, chelerythrine and sanguinarine have a synergistic effect. This result provides a new treatment option for patients with esophageal squamous cell carcinoma.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of chelerythrine and chelerythrine combined with sanguinarine in the preparation of cancer therapeutic drugs. Background Art

[0002] Esophageal cancer (EC) is one of the most common malignant tumors of the digestive tract, with 813,000 new cases of esophageal cancer worldwide in 2023. Esophageal cancer is divided into esophageal adenocarcinoma (EADC) and esophageal squamous cell carcinoma (ESCC). The first choice for the treatment of ESCC is surgery, while other treatments are also used. For some patients who cannot undergo surgery, radiotherapy and chemotherapy become the first choice for patients.

[0003] Chemotherapy drugs are drugs for treating tumors. Chemotherapy drugs can kill tumor cells. These drugs can act on different links of tumor cell growth and reproduction, inhibiting or killing tumor cells. Anti-tumor chemotherapy drugs have limited efficacy when used alone in clinical practice. Combination therapy can exert the synergistic effect of drugs and enhance the therapeutic effect, delay and reduce the development of drug resistance, and has important clinical significance.

[0004] Chelidonium majus is a species of the genus Chelidonium in the family Papaveraceae. It is from the book "Jiuhuang Bencao" and belongs to the whole herb category. It has the effects of clearing away heat and detoxification, anti-cancer and analgesic. At present, the anti-cancer effects of chelidonium and its active ingredients involve ovarian cancer, breast cancer, gastric cancer and other cancers, but there are no reports on its effect on esophageal squamous cell carcinoma. Summary of the invention

[0005] The inventors found in the process of studying tumor treatment that chelerythrine, a part of the active ingredients of chelidonium, has a strong anti-tumor effect, and its combination with chelidonine and sanguinarine can significantly enhance the effect of inhibiting esophageal squamous cell carcinoma.

[0006] In the early stage of the study, the inventors carried out the inhibitory effect of chelerythrine alone, and in combination with chelerythrine and sanguinarine, on the activity of two esophageal squamous cell carcinoma cells. The results showed that at the same concentration, the activity of esophageal squamous cell carcinoma cells was lower when the three compounds were used in combination than when chelerythrine was used alone, indicating that the three compounds had a synergistic effect (see Example 1), and the combination of the three compounds can significantly inhibit the invasion and migration of esophageal squamous cell carcinoma cells (see Example 2). In the in vivo experiment, in the KYSE450 and ECA109 cell xenograft tumor models, the combination of the three compounds can significantly inhibit the growth of the tumor without obvious drug toxicity (see Example 3).

[0007] Therefore, one of the objects of the present invention is to provide the use of chelerythrine in the preparation of cancer therapeutic drugs, wherein the cancer includes esophageal squamous cell carcinoma.

[0008] According to one aspect of the present invention, the cancer is meant to include tumors, neoplasia and any other malignant tissues or cells. The drug is used to promote apoptosis of cancer cells, reduce the activity of cancer cells, inhibit cancer cell proliferation, and suppress cancer cell migration.

[0009] Chelerythrine is an isoquinoline alkaloid with a CAS number of 6880-91-7. It is mainly found in plants such as chelerythrine and Macleaya cordata. It has multiple biological activities, but there are no reports on its anti-esophageal squamous cell carcinoma effect.

[0010] According to one aspect of the present invention, the application is the application of chelerythrine or chelerythrine combined with sanguinarine in the preparation of cancer therapeutic drugs.

[0011] Chelidonine is a 20 H 19 NO5 is an organic compound with a molecular weight of 353.369 and a CAS number of 476-32-4. It is an alkaloid found in plants of the genus celandine. Sanguinarine is an alkaloid with a molecular formula of C 20 H 14 NO4, molecular weight is 332.33, CAS number is 2447-54-3.

[0012] According to one aspect of the present invention, in the drug, the mass ratio of chelerythrine, chelidonine and sanguinarine is 1-5:1-5:1-5.

[0013] According to one aspect of the present invention, the chelerythrine, chelidonine and sanguinarine are administered simultaneously or continuously in any order. When administered at the same time, each component is used in the same or independent formulation form. When used at different times, each component can use the same or different dosage forms.

[0014] According to one aspect of the present invention, the terms "combination" or "combination use" or "co-administration" or "combination drug" used throughout the specification refer to the use of therapeutic agents included in the same or separate pharmaceutical preparations at the same time or different times.

[0015] According to one aspect of the present invention, the drug further comprises pharmaceutically acceptable excipients. The excipients include conventional diluents (such as water for injection, microcrystalline cellulose, etc.), fillers (such as mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, vaseline, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), binders (such as carbomer, gum arabic, starch, cellulose, gelatin, polyvinyl pyrrolidone, polyacrylamide, etc.), disintegrants (such as sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, etc.), lubricants (such as talc, hard At least one of magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, micro-powdered silica gel, etc.), a wetting agent (such as at least one of propylene glycol, glycerol, ethanol, etc.), a stabilizer (such as at least one of disodium ethylenediaminetetraacetic acid, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, niacinamide, vitamin C, etc.), an osmotic pressure regulator (such as at least one of sodium chloride, glucose, etc.), a pH regulator (such as at least one of triethanolamine, sodium hydroxide, sodium citrate, etc.), a preservative (such as at least one of chlorobutanol, parabens, ethylparaben, benzalkonium bromide, etc.).

[0016] Another object of the present invention is to provide a cancer treatment drug, comprising chelidonine, sanguinarine, chelerythrine, and conventional pharmacological excipients.

[0017] According to one aspect of the present invention, the cancer includes esophageal squamous cell carcinoma.

[0018] According to one aspect of the present invention, the dosage form of the drug is a conventional dosage form in the field of pharmacy, wherein different types of drugs are packaged separately or together, and different types of drugs are in the same dosage form or in different dosage forms.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides a new use of chelerythrine and chelerythrine combined with sanguinarine in preparing cancer therapeutic drugs. The research results of the present invention show that the combined use of chelerythrine, chelerythrine and sanguinarine has a significant inhibitory effect on the proliferation of esophageal cancer cells, and the effect is better than that of a single drug; in ESCC cell-bearing mice, the combined use of chelerythrine, chelerythrine and sanguinarine can significantly inhibit the growth of tumors and prolong the survival time of mice; the results provide a new treatment option for ESCC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The inhibition of ESCC cell viability by treatment with different concentrations of compounds, A is after treatment with chelerythrine; B is after treatment with chelerythrine, C is after treatment with sanguinarine, D is the cell viability of KYSE450 cells treated with the mixture, and E is the cell viability of ECA109 cells treated with the mixture.

[0022] Figure 2 Crystal violet staining images of ESCC cells treated with different compounds.

[0023] Figure 3 Figure 2 shows the inhibition of ESCC cell migration and invasion by different compounds. A shows the effect of different compounds on the migration ability of KYSE450 cells. B shows the effect of different compounds on the invasion ability of KYSE450 cells. C shows the effect of different compounds on the migration ability of ECA109 cells. D shows the effect of different compounds on the invasion ability of ECA109 cells.

[0024] Figure 4 These are pictures of ESCC tumors after treatment with different compounds in the in vivo mouse tumor-bearing experiment.

[0025] Figure 5 The changes in ESCC tumor volume after treatment with different compounds in the in vivo mouse tumor-bearing experiment. A is the tumor established by KYSE450 cells, and B is the tumor established by ECA109 cells. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0027] Example 1: CCK8 assay to detect the inhibitory effects of three compounds on different cancer cells (IC 50 )

[0028] Tumor cells (KYSE450 cells and ECA109 cells) in the logarithmic growth phase were counted and prepared into a concentration of 6×10 4100 μL of cell suspension of 100 μL / mL was added to a 96-well plate and cultured overnight in a 37°C, 5% CO2 cell culture incubator. After the cells adhered to the wall, chelidonine, chelerythrine, sanguinarine and a mixture of the three in a 1:1:1 mass ratio were added, with a concentration gradient of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 4 μM, 8 μM, and 10 μM. Three replicates were set for each group, and the drug solvent (DMSO) was used as the control group (Vehicle). After 48 hours of culture, 10 μL of CCK-8 working solution was added, and the cells were incubated in the cell culture incubator for another 3 hours. The OD value was detected at 450 nm and calculated.

[0029] The results are as follows Figure 1 As shown, chelerythrine, chelerythrine or sanguinarine alone can effectively reduce the viability of KYSE450 cells and ECA109 cells, and the combination of the three can produce a synergistic effect. The nanomolar dose treatment can reduce the viability of KYSE450 cells and ECA109 cells to less than 50%.

[0030] Example 2: Transwell assay to determine the effects of three compounds on ESCC cell invasion and migration

[0031] Add 60 μL of diluted Matrigel to the upper chamber of the Transwell and place it in a cell culture incubator for 1 hour until the Matrigel polymerizes into a gel (Matrigel is not added for migration experiments). Add 600 μL of chemokine to the lower chamber. Take cells in the logarithmic growth phase and inoculate them into the upper chamber. The number of cells in each upper chamber is 5×10 6 The cell suspension volume was 200 μL (chelidonine 2 μM, chelidonine 4 μM, sanguinarine 5 μM, and a 1:1:1 mixture of the three). After culturing for 24-48 h, the upper chamber medium was discarded, and the cells that did not pass through the membrane were wiped off with a cotton swab, fixed with methanol for 15 min, stained with crystal violet for 15 min, and the excess dye was washed off with deionized water. After drying naturally, the cells were photographed and the number of cells that passed through the membrane was counted.

[0032] The results are as follows Figure 2 and Figure 3 As shown, chelerythrine, chelerythrine or sanguinarine alone can effectively inhibit the invasion and migration of KYSE450 cells and ECA109 cells, and the combination of the three can produce a synergistic effect.

[0033] Example 3: In vivo mouse tumor experiment

[0034] 1-5×10 KYSE4509 cells and ECA109 cells in logarithmic growth phase were collected. 6 The tumor size was detected every 2 days and the volume of the tumor was 100 mm.3 The mice were given drugs by intraperitoneal injection (10 mg / kg, 100 μL, every 2 days) and divided into blank control group, chelerythrine group, chelerythrine group, sanguinarine group and mixture (the mass ratio of chelerythrine group, chelerythrine group and sanguinarine was 1:1:1). The treatment was terminated after 2 weeks (the tumor volume was detected every 2 days during this period), and the tumor tissues of mice in each group were collected for statistical analysis.

[0035] The results are as follows Figure 4 and Figure 5 As shown, compared with the blank control group, the tumor volume and weight of the chelidonium group, chelerythrine group, sanguinarine group and the mixture were significantly reduced, and the mixture had the best effect, indicating that the three compounds have a synergistic effect.

[0036] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0037] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0038] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The use of chelerythrine and chelerythrine combined with sanguinarine in the preparation of cancer treatment drugs, characterized in that: The cancer is esophageal squamous cell carcinoma. In the drug, the mass ratio of chelerythrine, chelidonine and sanguinarine is 1:1:

1.

2. The use of chelerythrine or chelerythrine combined with sanguinarine in the preparation of cancer therapeutic drugs according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

3. The use of chelerythrine or chelerythrine combined with sanguinarine in the preparation of cancer therapeutic drugs according to claim 2, characterized in that: The auxiliary materials include diluents, binders, disintegrants, lubricants, wetting agents, stabilizers, osmotic pressure regulators or preservatives.

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