Application of pristimerin in preparation of medicine for treating colorectal cancer

Abstract

The invention relates to an application of a natural product pristimerin in preparation of a medicine for treating colorectal cancer. The natural product disclosed by the invention has a remarkable inhibiting effect on intractable colorectal cancer, so that the colorectal adenocarcinoma can be relieved or treated.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically relates to the use of natural product pristimerin in the preparation of a drug for treating colorectal cancer. Background Art

[0002] Colorectal cancer (CRC), as a common malignant tumor of the digestive system clinically, most patients show symptoms such as hematochezia and abdominal pain as the disease progresses. The prognosis is poor and the mortality rate is high, which is the second leading cause of cancer-related death globally (9.4%). Colorectal adenocarcinoma refers to adenocarcinoma between the dentate line and the transitional part of the sigmoid colon and rectum, accounting for 75% - 85% of colorectal cancer.

[0003] Colorectal cancer is a series of highly heterogeneous diseases. There are significant differences in the efficacy and survival period of colorectal cancer with the same pathological type and clinical stage. These characteristics increase the treatment difficulty of colorectal cancer. In the past, the treatment of colorectal adenocarcinoma mainly relied on chemotherapy, and the available options included oxaliplatin + fluorouracil (FOLFOX), irinotecan + fluorouracil (FOLFIRI), oxaliplatin + capecitabine (XELOX or Cape OX), oxaliplatin + irinotecan + fluorouracil (FOLFOXIRI), etc. In recent years, a large number of molecular targeted drugs such as anti-vascular endothelial growth factor and its receptor, anti-epidermal growth factor receptor, inhibitor of murine sarcoma virus oncogene homolog B1, and anti-human epidermal growth factor receptor-2 have been applied clinically, further prolonging the overall survival period of patients and improving the quality of life. However, during the use of chemotherapy drugs such as oxaliplatin and irinotecan and molecular targeted drugs such as bevacizumab and cetuximab, patients may experience adverse reactions such as peripheral sensory neuropathy, early-onset and late-onset diarrhea, gastrointestinal perforation, and skin toxicity, which will also reduce physical function and life happiness and affect the quality of life of patients.

[0004] Therefore, there is an urgent need for new drugs for treating colorectal cancer. Summary of the Invention

[0005] The object of the invention is to provide a class of drugs for safely and effectively treating colorectal adenocarcinoma and their applications.

[0006] In the first aspect of the present invention, there is provided the use of pristimerin or a pharmaceutically acceptable salt thereof for the preparation of a preparation or composition, wherein the preparation or composition is used for the following uses: (a) inhibiting the activity of colorectal cancer Caco-2 cells; and / or (b) treating colorectal cancer.

[0007] In another preferred example, the colorectal cancer is CMS2 type colorectal cancer.

[0008] In another preferred embodiment, the colorectal cancer is refractory colorectal cancer.

[0009] In another preferred embodiment, the colorectal cancer is resistant to paclitaxel and / or 5-fluorouracil.

[0010] In another preferred embodiment, the composition is a pharmaceutical composition.

[0011] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: oral preparations, injections, patches, and transdermal agents.

[0012] In another preferred embodiment, the oral preparations are selected from the group consisting of: capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, and tinctures.

[0013] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the group consisting of: oral, intravenous, intramuscular, parenteral, transdermal, intraperitoneal, or a combination thereof.

[0014] In another preferred embodiment, the preparation is a preparation for laboratory use.

[0015] In a second aspect of the present invention, a method for inhibiting the proliferation of colorectal cancer cells in vitro is provided. The method includes:

[0016] (i) Culturing colorectal cancer cells in the presence of celastrol, thereby inhibiting the proliferation of colorectal cancer cells,

[0017] wherein the colorectal cancer cells are Caco-2 cells.

[0018] In another preferred embodiment, the concentration of celastrol is 0.1 - 1000 μM; preferably 0.5 - 500 μM; more preferably 1 - 500 μM, and even more preferably 1 - 100 μM.

[0019] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0020] In a third aspect of the present invention, an active ingredient combination is provided. The combination includes:

[0021] (a1) A first active ingredient, which includes celastrol or a pharmaceutically acceptable salt thereof;

[0022] (a2) A second active ingredient, which is a drug different from the first active ingredient and used for inhibiting colorectal cancer; the drug is selected from: platinum inhibitors, camptothecin derivatives, BRAF inhibitors, VEGF inhibitors, EGFR inhibitors, or a combination thereof.

[0023] In a fourth aspect of the present invention, a pharmaceutical composition is provided. The pharmaceutical composition includes:

[0024] (1) The combination of active ingredients as described in the third aspect of the present invention; and

[0025] (2) A pharmaceutically acceptable carrier.

[0026] In another preferred example, the second active ingredient includes: cisplatin, oxaliplatin, irinotecan, capecitabine, bevacizumab, cetuximab, or a combination thereof.

[0027] In another preferred example, the concentration ratio of the first active ingredient to the second active ingredient is from 1000:1 to 1:1000; preferably, from 100:1 to 1:100; more preferably, from 10:1 to 1:10.

[0028] In another preferred example, the mass ratio of the first active ingredient to the second active ingredient is from 1:500 to 500:1; preferably, from 100:1 to 1:100; more preferably, from 1:50 to 50:1.

[0029] The fifth aspect of the present invention provides the use of the combination of active ingredients as described in the third aspect of the present invention for preparing a preparation or composition, characterized in that the preparation or composition is used for the following uses: (a) inhibiting the activity of colorectal cancer Caco-2 cells; and / or (b) treating colorectal cancer.

[0030] The sixth aspect of the present invention provides a method for treating colorectal adenocarcinoma, comprising: administering to a subject in need a therapeutically effective amount of pristimerin or a pharmaceutically acceptable salt thereof.

[0031] In another preferred example, the subjects include rodents (such as mice, rats), and primate mammals (humans).

[0032] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Detailed Embodiments

[0033] Through extensive and in-depth research, by screening a large number of drug libraries, the inventors unexpectedly found that the natural product pristimerin has a significantly excellent therapeutic effect on refractory colorectal adenocarcinoma, and its inhibitory effect on the drug-absorption-poor CMS2-type colon cancer cell line CaCo2 is significantly better than that of paclitaxel and 5-fluorouracil. The present invention has been completed on this basis.

[0034] Pristimerin

[0035] Pristimerin (CAS: 1258-84-0) is a pentacyclic triterpenoid compound with strong pharmacological effects, having significant anti-tumor and anti-inflammatory activities, and can be isolated and extracted from plants such as Tripterygium wilfordii and Celastrus orbiculatus. The chemical formula is shown as follows:

[0036]

[0037] CMS2-type colorectal cancer

[0038] The Consensus Molecular Subtypes (CMS) classification is a molecular classification method for colorectal cancer (CRC). Colorectal cancer is divided into CMS1-MSI immune type, CMS2-classical type, CMS3-metabolic type, and CMS4-mesenchymal type. CMS2 colorectal cancer is one of the main subtypes in the molecular classification (Consensus Molecular Subtypes, CMS) of colorectal cancer, accounting for about 37% of early colorectal cancer. Its characteristics are the activation of the WNT / β-catenin and MYC signaling pathways, mostly originating from the left colon or rectum, and having a relatively good prognosis. However, it still faces many difficulties in clinical treatment, and related research also has important scientific and clinical significance. Compared with other classifications, the core characteristics of CMS2 are as follows: 1. Molecular characteristics: driven by chromosomal instability (CIN), activation of the WNT / β-catenin pathway: obvious epithelial cell differentiation, lacking immune or stromal characteristics. 2. Clinical behavior: high incidence in the left colon, well-differentiated, mainly local progression: may be effective for EGFR-targeted therapy (RAS / BRAF wild-type required). 3. Treatment strategy: prioritize surgery combined with chemotherapy (such as FOLFOX); targeted therapy options: cetuximab (for patients with RAS / BRAF wild-type).

[0039] Caco-2 cells are derived from human colorectal adenocarcinoma cells and are a polyclonal continuous cell line. Under specific in vitro culture conditions, Caco-2 develops, differentiates, and polarizes, thus forming a phenotype, morphology, and function similar to those of small intestinal epithelial cells. Caco-2 cells belong to the CMS2 classical type (activation of the WNT / β-catenin signal) and express intestinal cell differentiation markers (such as sucrase-isomaltase).

[0040] Active ingredient combination

[0041] The present invention also provides an active ingredient combination for preparing a drug for treating colorectal cancer, and the active ingredient contained therein is selected from pristimerin or a pharmaceutically acceptable salt thereof.

[0042] The present invention also provides a pharmaceutical composition, which comprises a combination of active ingredients within a safe and effective amount range, and a pharmaceutically acceptable carrier.

[0043] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that the components in the composition can be blended with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0044] There is no particular limitation on the administration mode of the active ingredient or pharmaceutical composition of the present invention. Representative administration modes include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0045] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0046] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers. For example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweetening agents, flavoring agents and fragrances.

[0047] In addition to the active ingredient, suspensions may contain suspending agents, such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide and agar or mixtures of these substances, etc.

[0048] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstituting into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyhydric alcohols and their suitable mixtures.

[0049] The main advantages of the present invention include:

[0050] (1) The active ingredient of the present invention has significant inhibitory activity against the drug-resistant colon cancer cell line Caco-2 cells.

[0051] (2) The drug of the present invention is simple and convenient to obtain, low in price, and suitable for large-scale production.

[0052] (3) Compared with synthetic drugs, natural drugs are more easily metabolized by the body, reducing the risk of toxic accumulation.

[0053] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0054] Examples

[0055] Instruments and materials:

[0056] Cell counting slides (model: JSP-GM, Guangzhou Boda Boju Technology Co., Ltd.); cell counter (model: JSY-FL-045N, Guangzhou Boda Boju Technology Co., Ltd.); cell culture incubator (Thermo Fisher Scientific, USA); inverted fluorescence microscope (model: TS100, Nikon Corporation); digital display constant temperature water bath (Xicheng Xinrui Instrument Factory, Jintan District); 96-well cell culture plate (Corning Life Sciences, USA); cell culture dish (Corning Life Sciences, USA); DMEM medium (Thermo Fisher Scientific, USA); cell proliferation-toxicity detection kit Cell Counting Kit-8 (Langjieke Technology Co., Ltd.); fetal bovine serum FBS (Merck, Germany)

[0057] Example 1

[0058] The IC 50 value of picrophylline against Caco-2 cells was determined by the CCK-8 method, and the research showed the inhibitory effect of picrophylline on Caco-2 cells.

[0059] 1.1 Test method

[0060] In this experiment, Cell Counting Kit-8 was used to determine the inhibitory activity of picrophylline against Caco-2 cells.

[0061] The CCK-8 kit (CellCountingKit-8) was used to measure the absorbance to calculate the cell viability, so as to reflect the drug activity.

[0062] Calculation formula

[0063] Cell viability = [(As - Ab) / (Ac - Ab)] × 100%

[0064] Inhibition rate = [(Ac - As) / (Ac - Ab)] × 100%

[0065] As: Absorbance of the experimental well (containing cells, culture medium, CCK-8 solution and drug solution);

[0066] Ac: Absorbance of the control well (containing cells, culture medium, CCK-8 solution, without drug);

[0067] Ab: Absorbance of the blank well (containing culture medium, CCK-8 solution, without cells and drug);

[0068] 1.1.1 Experimental procedure

[0069] The cells were cultured, passaged, counted and inoculated into 96-well plates (cell concentration per well 10 5 / 100 μl), administered with drugs, after 24 h of culture, 10% CCK-8 reagent (100 μl) was added, incubated in the dark for 1 h, the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader (450 nm), and the cell viability and its IC 50 value were calculated.

[0070] 1.1.2 Drug preparation

[0071] The concentration of the drug stock solution was 10 mM, and the experimental well concentration range (10, 5, 2.5, 1 μM) was prepared by serial dilution, and three replicates were set for each concentration.

[0072] 1.2 Experimental results

[0073] The IC 50 value of pristimerin on Caco-2 cells

[0074] Compound <![CDATA[IC 50 value]]> Pristimerin 2.24 μM Paclitaxel > 100 μM 5-Fluorouracil > 100 μM

[0075] The experimental results showed that Caco-2 cells were resistant to paclitaxel and 5-fluorouracil, while pristimerin had significant inhibitory activity on Caco-2 cells.

[0076] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of prismaticol or a pharmaceutically acceptable salt thereof for preparing a preparation or composition, wherein: The preparation or composition is used for a purpose selected from the group consisting of: (a) inhibiting the activity of colorectal cancer Caco-2 cells; and / or (b) treating colorectal cancer.

2. The use according to claim 1, characterized in that The colorectal cancer is CMS2 type colorectal cancer.

3. The use according to claim 1, characterized in that The colorectal cancer is refractory colorectal cancer.

4. The use according to claim 3, characterized in that The colorectal cancer is resistant to paclitaxel and / or 5-fluorouracil.

5. The use according to claim 1, characterized in that The composition is a pharmaceutical composition.

6. A method for inhibiting the proliferation of colorectal cancer cells in vitro, the method comprising: (i) culturing colorectal cancer cells in the presence of prismaticin, thereby inhibiting the proliferation of colorectal cancer cells, Wherein, the colorectal cancer cells are Caco-2 cells.

7. The method according to claim 6, characterized in that The concentration of prismatin is 0.1-1000 μM; preferably 0.5-500 μM; more preferably 1-500 μM, more preferably 1-100 μM.

8. An active ingredient combination, characterized in that The combination comprises: (a1) a first active ingredient, the first active ingredient comprising prismatin or a pharmaceutically acceptable salt thereof; (a2) a second active ingredient, which is a drug for inhibiting colorectal cancer that is different from the first active ingredient; the drug is selected from: platinum inhibitors, camptothecin derivatives, BRAF inhibitors, VEGF inhibitors, EGFR inhibitors, or a combination thereof.

9. A pharmaceutical composition, comprising: (1) The active ingredient combination according to claim 8; and (2) A pharmaceutically acceptable carrier.

10. Use of the active ingredient combination according to claim 8 for preparing a preparation or composition, characterized in that: The preparation or composition is used for a purpose selected from the group consisting of: (a) inhibiting the activity of colorectal cancer Caco-2 cells; and / or (b) treating colorectal cancer.

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