Medical application of berberine analogue

Abstract

The invention relates to an application of a compound or a pharmaceutically acceptable salt thereof in preparation of drugs for preventing and treating tumors: # imgabs0, wherein the tumors are selected from colorectal cancer, lung cancer, liver cancer, breast cancer, melanoma or gastric cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of a berberine analogue. Background Art

[0002] Malignant tumors are caused by malignant cell proliferation and are invasive and metastatic. The main classifications include carcinoma, sarcoma and carcinosarcoma. The cause of cancer is usually due to the long-term combined effects of chemical, physical, viral and other factors on the body, which trigger a series of abnormal genetic changes. The incidence of cancer is higher in high-risk groups such as those with a family history of cancer and cancer-related diseases, and people over 40 years old need to be more vigilant. Summary of the invention

[0003] The purpose of the present invention is to provide a new use of berberine analogs.

[0004] Specifically, the first aspect of the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating tumors:

[0005]

[0006] The tumor is selected from colorectal cancer, lung cancer, liver cancer, breast cancer, melanoma or gastric cancer.

[0007] In a preferred embodiment, the above compound is used as the sole active ingredient in the preparation of drugs for preventing and treating tumors.

[0008] The second aspect of the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating colorectal cancer metastasis:

[0009]

[0010] In a preferred embodiment, the above compound is used as the sole active ingredient in the preparation of a drug for preventing and treating colorectal cancer metastasis.

[0011] The details of various aspects of the present invention will be described in detail in the following sections. The features, objectives and advantages of the present invention will become more apparent through the following description and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1 B68 can inhibit the migration ability of LOVO and CT26 cells (a) After LOVO and CT26 cells were treated with different concentrations of B68 and B1, the migration ability of cells was detected; (b) The figure shows the statistical analysis results of (a), **P<0.01, ***P<0.001, ****P<0.0001.

[0013] Figure 2 (a) Modeling flow chart; (b) Representative images of the colon and rectum after AOM / DSS treatment; (c) Colorectal length of mice in the Control+B68, AOM / DSS, AOM / DSS+B68, and AOM / DSS+B1 groups; (d) Changes in body weight in the Control+B68, AOM / DSS, AOM / DSS+B68, and AOM / DSS+B1 groups during the experiment; (e) The number and diameter of tumors in the Control+B68, AOM / DSS, AOM / DSS+B68, and AOM / DSS+B1 groups; (f) Representative images of H&E staining of mouse colon tumors. DETAILED DESCRIPTION

[0014] The present invention provides the use of the compound of the following formula (I) (berberine analog B68) in the preparation of drugs for preventing and treating tumors:

[0015]

[0016] 10,11-dimethoxy-6,7-dihydro-[1,3]dioxolo[4”,5”:4',5']benzo[1',2':3,4]azepino[1,2-b]isoquinolin-9(5H)-one 10,11-dimethoxy-6,7-dihydro-[1,3]dioxolo[4”,5”:4',5']benzo[1',2':3,4]azepino[1,2-b]isoquinolin-9(5H)-one, molecular formula: C 21 H 19 NO5, molecular weight: 365.4g / mol.

[0017] The present invention also includes all pharmaceutically acceptable salts corresponding to the above compounds. These salts can be formed by a positively charged part (amino group) in the compound and a negatively charged acid (e.g., hydrochloric acid) with opposite charge; including but not limited to salts generated with the following acids: hydrochloric acid, sulfuric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfurous acid, nitric acid, carbonic acid, boric acid, selenious acid, phosphomolybdic acid, phosphorous acid, citric acid, maleic acid, D-malic acid, L-malic acid, DL-malic acid, D-lactic acid, L-lactic acid, DL-lactic acid, oxalic acid, sulfonic acid, benzenesulfonic acid, substituted benzenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, phthalic acid, tartaric acid, malonic acid, succinic acid, fumaric acid, citric acid, benzoic acid or substituted benzoic acid.

[0018] The compounds of the present invention can be purchased through commercial channels, and their purity meets the pharmaceutical standards.

[0019] The compounds of the present invention can be used alone or in the form of a pharmaceutical composition. The pharmaceutical composition comprises the compounds of the present invention as active ingredients and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition of the present invention contains 0.1 to 99.9% by weight of the compounds of the present invention as active ingredients. The "pharmaceutically acceptable carrier" will not destroy the pharmaceutical activity of the compounds of the present invention, and its effective amount, i.e., the amount that can play the role of a pharmaceutical carrier, is non-toxic to the human body.

[0020] The pharmaceutically acceptable carriers include, but are not limited to, phospholipids, aluminum stearate, aluminum oxide, ion exchange materials, self-emulsifying drug delivery systems, Tween or other surfactants, serum proteins, buffer substances such as phosphates, aminoacetic acid, sorbic acid, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, magnesium silicate, partial glyceride mixtures of saturated fatty acids, and the like.

[0021] Other commonly used pharmaceutical excipients include binders (such as microcrystalline cellulose), fillers (such as starch, glucose, anhydrous lactose and lactose beads), disintegrants (such as cross-linked PVP, cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricants (such as magnesium stearate), as well as absorption enhancers, adsorption carriers, flavoring agents, sweeteners, excipients, diluents, wetting agents, etc.

[0022] The compounds of the present invention and their pharmaceutical compositions can be prepared according to conventional methods in the art and can be administered by enteral, parenteral or topical routes. Oral preparations include capsules, tablets, oral liquids, granules, pills, powders, pills, ointments, etc.; parenteral preparations include injections, etc.; topical preparations include creams, patches, ointments, sprays, etc. Oral preparations are preferred.

[0023] The compounds of the present invention and their pharmaceutical compositions can be administered orally, sublingually, intramuscularly or subcutaneously, intravenously, urethrally, vaginally, and the like.

[0024] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually performed under normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0025] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0026] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0027] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Human colorectal cancer cells RKO, HCT116 and mouse colorectal cancer cells MC38 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured in MEM (MeilunBio), 1640 (MeilunBio), McCoy's 5A (MeilunBio) and DMEM (MeilunBio) culture media containing 10% fetal bovine serum (Biological Industries, Cromwell, CT, USA), 100 mg / mL streptomycin (Meilunbio, China) and 100 U / mL penicillin (Meilunbio, China) at 37°C and 5% CO2. Berberine analog B68 was synthesized by our research group, and berberine (B1) was purchased from Yuanye Biotechnology Co., Ltd. Other reagents: CCK-8 kit (Meilunbio, China) was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0028] Male C57BL / 6J mice, 6-8 weeks old, weighing 18-20 g, ethics number: PZSHUTCM2302060015, were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. (Shanghai, China) and were kept in the animal center of Shanghai University of Traditional Chinese Medicine in a specific pathogen free (SPF) environment for animal experiments, with a humidity of 45%-60%, a constant temperature of (23±2)℃, and 12 h of light per day. The food and daily drinking water of the experimental animals were sterilized by conventional high pressure sterilization.

[0029] Example 1 Effect of berberine derivative B68 on the activity of tumor cells

[0030] 1.1 Experimental methods

[0031] The CCK-8 experiment was used to verify the effect of B68 on tumor cell proliferation in vitro. The specific steps are as follows:

[0032] Different types of tumor cells in the logarithmic growth phase (colorectal cancer cells RKO and HCT116, lung cancer cells H1975, liver cancer cells Hepg2, breast cancer cells 4T1 and MCF-7, melanoma cells B16F10, gastric cancer cells MGC803) were inoculated in 96-well plates at a density of 5×10 per well. 3 cells, and then placed in an incubator at 37°C and 5% CO2 for incubation; after the cells adhered to the wall and expanded, the drug was prepared to the concentration required for the experiment. After 24 hours of administration, the drug-containing culture medium was discarded and replaced with 100 μL / well of CCK-8 solution, and incubated at 37°C for 1-4 hours. The operation should be kept away from light throughout the process. Then, an ELISA reader (BioTek, USA) was used to measure the absorbance value (OD value) at a wavelength of 450nm to calculate the cell survival rate. Cell viability (%) = [OD (drug) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0033] 1.2 Experimental Results

[0034] Table 1 Inhibitory activity of B68 on tumor cells (IC50, μM)

[0035]

[0036] As shown in Table 1, B68 has strong anti-tumor activity against colorectal cancer cells (RKO and HCT116), lung cancer (H1975), liver cancer (HepG2), gastric cancer cells (MGC-803), breast cancer (4T1 and MCF-7) and melanoma (B16-F10), which are superior to the anti-tumor activity of the natural product berberine (B1), indicating that B68 has a certain broad-spectrum anti-tumor activity.

[0037] Example 2 Detection of the ability of B68 to inhibit the migration of LOVO and CT26 cells by scratch assay

[0038] 2.1 Experimental methods

[0039] 6 × 10 5 cells, and the cell fusion rate reached 100% overnight; the next day, use a 20μL gun tip to scratch vertically. After the scratching is completed, wash the cells 3 times with sterile PBS to wash away the cells that do not adhere to the wall, that is, the cells on the streak, and then replace with fresh serum-free medium. Dilute the B68 mother solution (20mM) with culture medium to 0, 2, 5, 10μM, B1-100μM, add 1mL to each well, and then put the cells in a 37℃ 5% CO2 incubator. Take out the cells after 18h, observe and measure the width of the scratch under a microscope, take pictures, and use Image J software to statistically analyze the distance between cells.

[0040] 2.2 Experimental Results

[0041] As shown in Figure 1, both B68 and B1 can significantly inhibit the migration of colorectal cancer cells, but the ability of B1 to inhibit the migration of colorectal cancer cells is far less than that of B68, and B68 inhibits the migration of colorectal cancer cells (LOVO and CT26) in a concentration-dependent manner.

[0042] Example 3 Construction of AOM / DSS model to comprehensively evaluate the effects of berberine derivative B68 on the occurrence and development of colorectal cancer 3.1 Experimental methods

[0043] Azoxymethane (AOM) and dextran sulfate sodium salt (DSS) are two drugs that are often used to establish a classic colitis carcinogenic mouse model, which can more realistically reflect the occurrence and development of intestinal cancer. For the AOM / DSS animal model experiment, we used 6-8 week-old C57BL / 6J mice (male). First, the mice were intraperitoneally injected with 12.5 mg / kg of AOM (Sigma, St. Louis, MO) for one week for induction. After drinking normal water for one week, the mice were allowed to drink drinking water containing 2.5% DSS (MP Biomedicals, USA) for one week to induce colitis. Then the mice were replaced with normal drinking water for two weeks. Repeat the above steps: 3 consecutive cycles (i.e., DSS drinking water-normal drinking water) to fully simulate the carcinogenesis process of human ulcerative colitis. In the third cycle, DSS was adjusted from 2.5% to 2%. On the 65th day, the mice were killed and multiple indicators including intestinal appearance, length, tumor incidence, mouse weight and immunohistochemistry were tested.

[0044] 3.2 Experimental Results

[0045] To further explore the therapeutic effect of B68 on colorectal cancer in mice, this experiment used the AOM / DSS model to simulate the pathogenesis of human inflammatory colorectal cancer and divided them into four groups: Control+B68, AOM / DSS, AOM / DSS+B68, and AOM / DSS+B1. The administration method and frequency were as follows Figure 2a The results showed that the tumor burden in the colon tissue of mice in the AOM / DSS group was higher, and B68 treatment had a significant inhibitory effect on AOM / DSS-induced tumors. B1 was able to inhibit the development of colorectal cancer, although the therapeutic effect was between the AOM / DSS group and the B68 treatment group, but the therapeutic effect was significantly inferior to that of B68 ( Figure 2bCompared with the AOM / DSS group, B68 treatment reduced the percentage of tumors (diameter>4mm) and tumors (diameter2-4mm) by 85.2% and 66.7%, respectively, while B1 treatment reduced the percentage of tumors (diameter>4mm) and tumors (diameter2-4mm) by 44.4% and 39.4%, respectively. This indicates that the therapeutic effect of B68 is significantly better than that of B1 ( Figure 2e ). The mice in the AOM / DSS+B68 group showed longer colons and heavier body weight than those in the AOM / DSS group ( Figure 2c and 2d ), which suggests that mice in the AOM / DSS group are more likely to develop tumors, because DSS-induced shortening of the colon length in mice is an indicator of the severity of inflammation, which is also a potential cause of colon cancer. In addition, H&E results showed that the effect of B68 treatment was significantly better than that of B1 treatment ( Figure 2f ). Taken together, these results further suggest that B68 inhibits the occurrence and progression of colorectal cancer by promoting tumor cell senescence.

[0046] The various aspects of the present invention have been described above. However, it should be understood that, without departing from the spirit of the present invention, equivalent changes and modifications may be made by those skilled in the art, and the changes and modifications also fall within the scope of the claims of the present application.

Claims

1. Use of the compound of formula (I) or its pharmaceutically acceptable salt in the preparation of drugs for preventing and treating tumors: The tumor is selected from colorectal cancer, lung cancer, liver cancer, breast cancer, melanoma or gastric cancer.

2. Use of the compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound is used as the sole active ingredient for preparing medicine for preventing and treating tumors.

3. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating colorectal cancer metastasis:

4. Use of the compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound is used as the sole active ingredient for preparing medicine for preventing and treating colorectal cancer metastasis.

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