Application of oxanin combined with epirubicin in the prevention and / or treatment of colon cancer

Abstract

The present invention discloses the use of oxanin combined with epirubicin in the prevention and / or treatment of colon cancer, and relates to the field of biomedicine technology. The technical points are: the pharmaceutical composition of the present invention comprises oxanin and epirubicin, preferably in a molar ratio of 15:1 or 7.5:1, and is intended to reduce the dosage of epirubicin through synergistic effects, reduce toxic side effects, and enhance therapeutic effects. The present invention also includes a method for detecting the effect of the pharmaceutical composition on the vitality and apoptosis of colon cancer cells, and an animal model for evaluating the effect on tumor growth in colon cancer-bearing mice. The pharmaceutical composition of the present invention can significantly inhibit the proliferation of colon cancer cells, enhance the pro-apoptotic effect of epirubicin, and show good anti-tumor effects and low organ toxicity in animal models. The present invention provides a safe and effective new treatment strategy for patients with colon cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of oxanin combined with epirubicin in preventing and / or treating colon cancer. Background Art

[0002] Colon cancer is one of the most common gastrointestinal malignancies worldwide. Although the specific causes of colon cancer remain unclear, it is known that over 80% of patients have no family history of the disease. However, lifestyle factors such as diet, obesity, smoking, and alcohol consumption may increase the risk of developing the disease. Currently, surgery-assisted chemoradiotherapy is the mainstay of treatment for colon cancer. While this treatment can prolong patient survival, the toxic side effects of chemotherapy drugs on normal tissues and the development of drug resistance in patients limit their effectiveness.

[0003] Epirubicin (Epi) is an antibiotic anti-tumor drug that interferes with the transcription process by intercalating between DNA nucleobase pairs, preventing the formation of mRNA, and thus inhibiting the synthesis of DNA and RNA. As a commonly used drug in chemotherapy, the use of epirubicin is limited by dose-related toxicity, and patients may develop drug resistance to it. Therefore, the use of oxanin combined with epirubicin in the prevention and / or treatment of colon cancer mentioned in the present invention is intended to reduce the dosage of epirubicin through synergistic effects, while reducing its toxic side effects, providing a new strategy for the treatment of colon cancer. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide the use of oxanin combined with epirubicin in the prevention and / or treatment of colon cancer. The combination of oxanin and epirubicin for colon cancer has obvious synergistic effect.

[0005] In order to achieve the above-mentioned purpose, the technical solution of the present invention is as follows: the use of oxanin combined with epirubicin in the prevention and / or treatment of colon cancer, the pharmaceutical composition comprises oxanin and epirubicin, wherein the molar ratio of oxanin to epirubicin is 15:1.

[0006] Furthermore, the molar ratio of oxanin to epirubicin in the pharmaceutical composition can also be 7.5:1.

[0007] Furthermore, the pharmaceutical composition is used to prepare a drug for inhibiting the activity of colon cancer cells.

[0008] Furthermore, the pharmaceutical composition is used to prepare a drug for inhibiting tumor growth in colon cancer-bearing mice.

[0009] Furthermore, the pharmaceutical composition includes but is not limited to injections, pills, tablets, granules, and capsules.

[0010] Another object of the present invention is to provide a method for detecting the effect of oxanin combined with epirubicin on the viability of colon cancer cells, the method comprising using the MTT method to detect the effect of different concentrations of oxanin and epirubicin on the viability of HCT116 cells, and calculating the cell viability.

[0011] Another object of the present invention is to provide a method for detecting the effect of oxanin combined with epirubicin on apoptosis of colon cancer cells, the method comprising using the Annexin V / PI method to detect the effect of oxanin and epirubicin on apoptosis of HCT116 cells.

[0012] Furthermore, the method evaluates the enhancing effect of oxanin on the pro-apoptotic effect of epirubicin by comparing the effects of epirubicin or oxanin alone with the combined use of the two drugs on the apoptosis of colon cancer cells.

[0013] Another object of the present invention is to provide an animal model for evaluating the effects of oxanin combined with epirubicin on tumor growth in colon cancer-bearing mice. The model is established by establishing a colon cancer CT26 transplanted tumor mouse model and administering oxanin and epirubicin in combination to evaluate the inhibitory effect on tumor growth and the toxic reaction to important organs.

[0014] Compared with the prior art, the beneficial effects of this solution are as follows: the present invention combines oxanin with epirubicin, and the present invention achieves a synergistic effect of the two, significantly inhibits the proliferation of colon cancer cells HCT116, and reduces the dosage of epirubicin, thereby reducing the dose-related toxic and side effects. Secondly, the experimental results show that this combination regimen can significantly enhance the apoptosis-promoting effect of epirubicin on colon cancer cells, and the apoptosis rate of the combination group is significantly increased compared with the use of either drug alone. In addition, in the colon cancer-bearing mouse model, the tumor inhibition effect of oxanin combined with epirubicin is significantly better than that of either drug alone, with a tumor inhibition rate of 75.18%, showing strong in vivo anti-tumor activity. At the same time, the scheme can also improve the organ index of mice and reduce the toxic reaction of epirubicin to important organs. In summary, this scheme not only improves the therapeutic effect, but also effectively reduces the toxic and side effects of chemotherapy drugs, providing a safer and more effective treatment option for patients with colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the effects of okanamin and epirubicin on HCT116 cell viability in Example 1 of the present invention;

[0016] Figure 2 Schematic diagram showing the effect of okanamin combined with epirubicin on HCT116 cell viability in Example 1 of the present invention;

[0017] Figure 3This is a schematic diagram showing the calculation of the combined index of oxanin and epirubicin on HCT116 cells in Example 1 of the present invention;

[0018] Figure 4 This is a graph showing the experimental results of detecting the effect of oxanin combined with epirubicin on apoptosis of HCT116 cells using the Annexin V / PI method in Example 1 of the present invention;

[0019] Figure 5 This is a graph showing the apoptosis-promoting effect of okanamin combined with epirubicin on colon cancer cells HCT116 in Example 1 of the present invention;

[0020] Figure 6 This is a schematic diagram of the results of an in vivo pharmacodynamic study of okananin combined with epirubicin on a colon cancer CT26 xenograft mouse model in Example 2 of the present invention;

[0021] Figure 7 Schematic diagram of the effect of okanamin combined with epirubicin on tumor growth in tumor-bearing mice in Example 2 of the present invention;

[0022] Figure 8 This is a schematic diagram of the effects of okanamin combined with epirubicin on organ indexes of tumor-bearing mice in Example 2 of the present invention. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0024] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0025] The present invention provides the use of okanin combined with epirubicin (Epi) in the preparation of a drug for preventing and / or treating colon cancer. The okanin combined with epirubicin of the present invention significantly inhibits the activity of colon cancer cells, and the okanin combined with epirubicin significantly inhibits the growth of tumors in colon cancer-bearing mice. The sources of the okanin and epirubicin of the present invention are not particularly limited and can be commercially available products well known to those skilled in the art, or can be obtained by preparation methods disclosed in the prior art.

[0026] In the present invention, the molar ratio of oxanin to epirubicin is 15: 1 and 7.5: 1, preferably 15: 1. The combined index of oxanin and epirubicin in the present invention on colon cancer cells is less than 1, showing significant synergistic effect.

[0027] In the present invention, the drug comprises oxanin and epirubicin, and pharmaceutical excipients compatible with oxanin and epirubicin, to prepare a dosage form that is convenient for administration, such as, but not limited to, aqueous solution injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder aerosols, sustained-release agents, and controlled-release agents. The pharmaceutical excipients described in the present invention can be those conventionally used in various preparations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; or they can be selected for compatibility with the substance, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants.

[0028] Example 1: In vitro experiments on colon cancer cells HCT116 using oxanin and epirubicin

[0029] 1. MTT assay to detect the effects of oxanin and epirubicin on HCT116 cell viability

[0030] HCT116 cells in logarithmic phase were collected and 8×10 3 The cells were plated into 96-well plates and cultured for 24 hours at 37°C and 5% CO2. The experiment was divided into a blank group and a drug-treated group. After the cells adhered to the wall, PBS solution was added to the blank group, and different concentrations of oxanide (12.5, 25, 50, and 100 μM) and doxorubicin (0.5, 1, 2, 4, 8, and 16 μM) were added to the drug-treated group, with 3 parallel wells in each group. The cell morphology was observed by continuing to culture for 48 hours, adding 10 μL of MTT solution to each well, and culturing for 4 hours at 37°C and 5% CO2. The supernatant was discarded, and 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. After shaking on a microporous oscillator for 10 minutes, the absorbance of each well was measured at a wavelength of 490 nm using an enzyme reader, and the cell viability was calculated according to the following formula. The results are as follows Figure 1 shown.

[0031] Cell viability = OD value of the drug-treated group / OD value of the blank group × 100%.

[0032] The results are as follows Figure 1 As shown in the results, oxanin and epirubicin can inhibit the proliferation of HCT116 cells, and with the increase of the administration concentration of oxanin and epirubicin, the effect of inhibiting cell proliferation gradually increased, which was significantly different from the blank group.

[0033] 2. MTT assay to detect the effect of oxadiazine combined with epirubicin on HCT116 cell viability and calculate the combined index

[0034] MTT assay was used to determine the cell viability of HCT116 cells treated with oxanin (10, 20, and 30 μM) and epirubicin (1, 2, and 4 μM) for 48 h. Figure 2 shown.

[0035] CompuSyn software was used to calculate the combined index CI value after drug combination. When CI < 1, it indicates that the two drugs have a synergistic effect; when CI = 1, it indicates that the two drugs have an additive effect; when CI > 1, it indicates that the two drugs have an antagonistic effect. Figure 3 shown.

[0036] like Figure 3 As shown in the figure, when the concentration ratio of oxanin to epirubicin was 7.5:1 and 15:1, the combined effect of the two drugs on inhibiting HCT116 cell proliferation was more significant than that of single drugs, with the corresponding CI values ​​less than 1, indicating a synergistic effect. The combined use of oxanin and epirubicin can reduce the dosage of epirubicin and the cumulative dose of epirubicin to produce the same inhibitory effect on colon cancer cells, thus reducing the probability of toxic side effects caused by the dosage of epirubicin. Based on the above, a concentration ratio of oxanin to epirubicin = 15:1 was selected for subsequent experiments.

[0037] 3. Detection of cell apoptosis by Annexin V / PI method

[0038] HCT116 cells in logarithmic phase were collected and plated at 100 × 10 4The cells were plated into 6-well plates and cultured for 24 hours at 37°C and 5% CO2. The experiment was divided into blank group, epirubicin group, okanin group and combination drug group. PBS solution was added to the blank group, and 2 mL of solution with a final concentration of 2 μM Epi, 30 μM Okanin and a combination of the two drugs (2 μM Epi + 30 μM Okanin) was added to the drug group in sequence. After culturing for 24 hours, the cells to be tested were digested with EDTA-free trypsin and collected into EP tubes, and centrifuged at 1000 rpm for 3 minutes. Carefully discard the supernatant, wash the cell pellet with 1 mL of pre-cooled PBS buffer, and discard the supernatant. One of the blank groups was divided into two parts. One part was treated in boiling water for 1 minute, then mixed with the untreated part, centrifuged and discarded the supernatant, and pre-cooled 1×Binding Buffer was added to make up to 1.5 mL. The solution was divided into 3 tubes, one of which was a blank control tube and the other two were single-stained tubes. 5 μL Annexin V-FITC or 10 μL PI were added to the single-stained tubes and incubated at room temperature in the dark for 5 min. The drug-treated group was resuspended in 500 μL 1× Binding Buffer and then added with 5 μL Annexin V-FITC and 10 μL PI. The cells were incubated at room temperature in the dark for 5 min and detected and analyzed by flow cytometry. Each experimental group was repeated three times. The experimental results are shown in Figure 2. Figure 4 shown.

[0039] like Figure 5 As shown in the results, compared with the blank group, the apoptosis rates of the epirubicin and oxadiazine groups increased, but compared with the epirubicin and oxadiazine groups, the apoptosis rate of the combination group was significantly increased, and the difference was statistically significant (P < 0.05). This shows that compared with the use of epirubicin or oxadiazine alone, the combination of the two has a better apoptosis-promoting effect on colon cancer cells HCT116, indicating that oxadiazine can enhance the apoptosis-promoting effect of epirubicin on colon cancer cells HCT116.

[0040] Example 2: In vivo pharmacodynamics of okananin combined with epirubicin in a mouse model of colon cancer CT26 xenografts

[0041] 1. Establishment of a CT26 Colon Cancer Xenograft Mouse Model

[0042] Forty 6-week-old male BALB / C mice were fed with normal drinking water and feed and adapted to the animal house for 1 week. The experiment was divided into a normal group, a model group, an epirubicin group, an oxadiazine group, and a combination group, with 8 mice in each group. CT26 cells were cultured, washed with sterile PBS, digested with trypsin, and then digested with complete medium. The cells were collected and centrifuged at 1000 rpm / min for 5 minutes. The supernatant was discarded and washed twice with sterile PBS. The cells were resuspended in sterile PBS and the cell concentration was adjusted to 5×10 7Except for the normal group, each mouse was subcutaneously injected with 100 μL of CT26 cell suspension at the right armpit.

[0043] 2. Medication

[0044] Tumor grows to 80-100 mm 3 BALB / C mice were randomly divided into four groups. The model group received an equal volume of normal saline intraperitoneally every day, the epirubicin group received an intraperitoneal injection of 2.5 mg / kg epirubicin solution, the oxadiazine group received an intraperitoneal injection of 40 mg / kg oxadiazine solution, and the combination group received an intraperitoneal injection of 2.5 mg / kg epirubicin plus 40 mg / kg oxadiazine.

[0045] Weigh the mice before administration every day and record the details. After 10 days of administration, weigh the mice the day after the last administration, sacrifice the mice, take and weigh them, take and weigh the heart, liver, spleen, lung, kidney and tumor tissues and weigh them, and explore the effect of okanamin combined with epirubicin on tumor growth in tumor-bearing mice and the toxic reaction to important organs. Calculate the tumor inhibition rate and organ index. Specific results are as follows Figure 6 shown.

[0046] Tumor inhibition rate (%) = (average tumor weight of mice in the model group - average tumor weight of mice in the drug administration group) / average tumor weight of mice in the model group × 100%

[0047] Organ index (%) = mouse organ mass (mg) / mouse body mass (g) × 100% Figure 7-8 As shown, the tumor size and mass of the epirubicin group, oxadiazine group, and combination group were all smaller than those of the model group, indicating that epirubicin, oxadiazine, and the combination of the two drugs can inhibit the growth of colon cancer tumors in vivo. The tumor inhibition rates of epirubicin and oxadiazine alone were 54.70% and 37.65%, respectively. After the two drugs were used in combination, the tumor inhibition rate reached 75.18%, indicating that the combination of the two drugs is more effective in preventing colon cancer than the use of epirubicin or oxadiazine alone. In addition, compared with the epirubicin group, the combined administration of oxadiazine and epirubicin improved the organ index of mice, indicating that oxadiazine can reduce the adverse reactions of epirubicin.

[0048] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A pharmaceutical composition for preventing and / or treating colon cancer, characterized by: The pharmaceutical composition consists of oxanin and epirubicin, wherein the concentration ratio of oxanin to epirubicin is 15:

1.

2. A pharmaceutical composition for preventing and / or treating colon cancer, characterized by: The pharmaceutical composition consists of oxanin and epirubicin, and the concentration ratio of oxanin to epirubicin in the pharmaceutical composition is 7.5:

1.

3. The pharmaceutical composition according to any one of claims 1 or 2, characterized in that: The pharmaceutical composition is used for preparing a drug for inhibiting the activity of colon cancer cells.

4. The pharmaceutical composition according to any one of claims 1 or 2, characterized in that: The pharmaceutical composition is used for preparing a medicine for inhibiting tumor growth in colon cancer-bearing mice.

5. The pharmaceutical composition according to any one of claims 1 or 2, characterized in that: The pharmaceutical compositions include but are not limited to injections, pills, tablets, granules, and capsules.

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