Use of oxaliplatin in the preparation of medicaments for the treatment of cancer
By using oxcarbazine alone or in combination with 5-fluorouracil, the problems of high toxicity and low efficacy of existing colorectal cancer treatments have been solved, achieving better anti-tumor effects and fewer side effects, thus providing a new treatment option for colorectal cancer.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XUZHOU NORMAL UNIVERSITY
- Filing Date
- 2023-02-09
- Publication Date
- 2026-06-02
AI Technical Summary
Existing drugs for treating colorectal cancer, such as 5-fluorouracil, are highly toxic and have low efficacy at low doses. Targeted therapies and immunomodulators are expensive and have short durations of effectiveness, and chemotherapy drugs can lead to drug resistance. Therefore, it is necessary to find new and effective treatment methods.
Oxcarmine, alone or in combination with 5-fluorouracil, is used to prepare antitumor drugs at concentrations of 20-200 μMol for the treatment of colorectal cancer, breast cancer, and gastric cancer.
Oxcarpine showed significant inhibition of colon cancer cell growth in vitro and in vivo, with less toxicity than 5-fluorouracil. Combination therapy was more effective than monotherapy, exhibiting better anti-tumor effects and fewer side effects in nude mice.
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Figure CN117224512B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of oxcarbazone in the preparation of antitumor drugs. Technical Background
[0002] Colorectal cancer, especially CRC, is a highly malignant tumor of the gastrointestinal tract, characterized by its aggressive infiltration and tendency to metastasize. According to statistics from the World Health Organization, colorectal cancer consistently ranks among the top five most common cancers. In my country, the incidence rate of colorectal cancer is far higher than the global average. Current treatment methods for colorectal cancer include surgical resection, radiotherapy, chemotherapy, and immunosuppressive therapy. Chemotherapy remains a primary treatment, but its efficacy is unsatisfactory, with no significant increase in the overall five-year survival rate. Furthermore, chemotherapy drugs (such as 5-fluorouracil) are highly toxic, low-dose treatments are less effective, and targeted therapies and immunomodulators are expensive, have short durations of effectiveness, and often lead to strong drug resistance. Therefore, finding new and effective treatment methods is crucial for improving patient survival rates and reducing morbidity.
[0003] Natural products have historically been a major source of chemotherapy drugs, including anticancer drugs. However, with the rise of targeted therapy in the late 20th century, research into the anticancer effects of natural products essentially ceased. Because targeted therapies are prone to drug resistance, have short durations of effectiveness, and generally lack satisfactory overall efficacy, natural products returned to the center of anticancer chemotherapy drug development at the beginning of this century. From 2000 to 2010, all of the dozen or so cancer chemotherapy drugs approved by the FDA were natural products or their derivatives. Currently, natural products used to treat colorectal cancer still suffer from uncertain efficacy, low response rates, short overall survival, high drug toxicity, and high prices. Therefore, there is a need to develop a drug application strategy that uses natural extract monomers to inhibit colorectal cancer.
[0004] Bidens pilosa, belonging to the Asteraceae family, is widely distributed in China and has therapeutic effects on various diseases, such as inflammation, hypertension, and diabetes. Currently, both Bidens pilosa and Coreopsis grandiflora have been found to have anti-tumor activity. However, the natural products extracted from Bidens pilosa and Coreopsis grandiflora include flavonoids, phenylpropanoids, sesquiterpenes, and sterols, with numerous monomers, often dozens or even hundreds. It remains unclear which monomer has a definitive effect in inhibiting colorectal cancer tumors and can be used to replace or partially replace chemotherapy drugs.
[0005] Okanin is a flavonoid compound extracted from natural Chinese herbal medicines such as Bidens bipinnata Linn. and Coreopsis tinctoria Nutt.'s. Studies have reported that oxanin has anti-inflammatory and anti-thrombotic functions, but there are currently no reports on its anti-tumor activity. Summary of the Invention
[0006] To address the above problems, this invention proposes an application scheme for the use of the traditional Chinese medicine extract monomer oxaline in the preparation of drugs that inhibit tumors.
[0007] This invention discloses the use of oxcarbazin in the preparation of antitumor drugs, wherein oxcarbazin is used alone or in combination with 5-fluorouracil in the antitumor drugs.
[0008] Furthermore, the tumors are colorectal cancer, breast cancer, and stomach cancer.
[0009] Furthermore, when oxcarbazin is used to prepare anti-colorectal cancer drugs, the concentration is 20-200 μMol.
[0010] Preferably, when oxcarbazin is used to prepare anti-colorectal cancer drugs, the concentration is 50-200 μMol.
[0011] Furthermore, when oxcarbazine and 5-fluorouracil are used in combination to prepare drugs for colorectal cancer, the concentrations of oxcarbazine and 5-fluorouracil are the same, both ranging from 20 to 200 μmol.
[0012] The beneficial effects of this invention are as follows:
[0013] 1. This invention discovers that oxcarbinine can inhibit the growth of colon cancer cells both in vivo and in vitro, and nude mice exhibit good tolerance to this natural compound. Existing technologies have not reported significant effects of oxcarbinine, a monomer extracted from traditional Chinese medicine, in anti-tumor drugs. Therefore, this natural product has the potential to be developed into a novel anti-cancer drug, such as for the treatment of colon cancer.
[0014] 2. This invention, through comparison of the anti-colonic cancer effects of oxcarnitine with other analogues of Coreopsis grandiflora and Bidens pilosa extracts, found that at drug concentrations of 20-200 μMol, oxcarnitine had a better anti-colonic cancer effect than other analogues of Coreopsis grandiflora and Bidens pilosa extracts, such as maricin, quercetin, and flavomargin.
[0015] 3. Existing drugs used to treat colorectal cancer include 5-fluorouracil. This invention, by comparing the antitumor effects of oxcarbazine and 5-fluorouracil on colorectal cancer, found that oxcarbazine has a better antitumor effect and fewer side effects in nude mice compared to 5-fluorouracil. This indicates that compared to the currently used conventional colorectal cancer drug 5-fluorouracil, oxcarbazine has better anticancer effects and lower toxicity, and can be used as an alternative to 5-fluorouracil in treating colorectal cancer. Based on the analysis of the mechanisms and properties of cancer, oxcarbazine can also be used to treat breast cancer and gastric cancer.
[0016] 4. This invention has found that the combined use of oxcarbazine and 5-fluorouracil is more effective than the sum of the effects of the two drugs at the same concentration, providing a new research and development approach for anti-colorectal cancer drugs. Attached Figure Description
[0017] Figure 1 This is a diagram of the oxcarpine molecular structure.
[0018] Figure 2 Results of HPLC purification of oxcarpine;
[0019] Figure 3 The results are from the nuclear magnetic resonance (NMR) test of oxcarmine.
[0020] Figure 4 The results are from oxcanin mass spectrometry (MS) detection.
[0021] Figure 5 This is a schematic diagram illustrating the effect of oxcarbamate concentration on the viability of HCT116 colon cancer cells in vitro.
[0022] Figure 6 This is a schematic diagram illustrating the effect of oxcarbamate concentrations within the range of 200 μMol on the viability of NCM460 cells in vitro.
[0023] Figure 7 This is a schematic diagram illustrating the effect of oxcarbazone analogues (masarin, quercetin, and flavomarin) at concentrations within 200 μmol on the viability of HCT116 colon cancer cells in vitro.
[0024] Figure 8 A schematic diagram illustrating the ability of oxcarbamate to induce apoptosis in HCT116 cells;
[0025] Figure 9 The fitted curves of oxcarbazone and 5-fluorouracil drug concentrations versus cell activity;
[0026] Figure 10 A schematic diagram of the combined use index of oxcarbazine and 5-fluorouracil;
[0027] Figure 11 This is a schematic diagram illustrating the effects of oxcarbazone and 5-fluorouracil on tumor size in mice with colorectal cancer during animal experiments.
[0028] Figure 12 This is a schematic diagram illustrating the effect of oxcarbazine and 5-fluorouracil on mouse body weight in animal experiments. Detailed Implementation
[0029] The present invention will be further described below with reference to the embodiments.
[0030] Unless otherwise specified, all raw materials and reagents used in the following examples were obtained commercially.
[0031] Example 1: In vitro verification of whether oxcarbazone has an inhibitory effect on colon cancer cell activity.
[0032] 1. Experimental Samples:
[0033] (1) Okanin
[0034] Oxycarbin was purchased from Sichuan Dester Biotechnology Co., Ltd., CAS No.: 487-76-4, molecular formula: C15H12O6, molecular weight: 288.25. The structural formula is as follows: Figure 1 As shown, the purification method was HPLC, and the purity was 99.83%. Figure 2 The compound was identified using NPM and Mass; the results are shown below. Figure 3 and Figure 4 .
[0035] (2) Cells
[0036] HCT116 human colorectal cancer cells and NCM460 human immortalized small intestinal epithelial cells were both purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were cultured in DMEM medium containing 10% PBS, 100 U / m penicillin, and 100 mg / L streptomycin in a constant temperature closed incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.
[0037] (3) Reagents and test kits
[0038] 5-Fluorouracil was purchased from Merck & Co Inc., CAS number 51-21-8. The MTT assay kit and the TUNEL cell apoptosis detection kit were both purchased from Beyotime Biotechnology Co., Ltd., product numbers C0009S and C1086, respectively.
[0039] 2. Operation process:
[0040] HCT116 and NCM460 cells in logarithmic growth phase were harvested and their cell density adjusted to 8 x 10⁻⁶ cells / year. 3Cells were seeded in 96-well plates and, after adhesion, were treated with oxcarbazin or its analogues masalacin, quercetin, and noroside at final concentrations of 0, 20, 50, 100, and 200 μM in cell culture medium. After 24 and 48 hours of treatment, cell morphology changes were observed under an inverted microscope. The MTT assay was used to detect the effect of the experimental samples on tumor cell viability.
[0041] The experimental results showed that, 24 hours after drug administration, compared with the control group, culture medium solutions containing oxcarbazin at final concentrations of 20, 50, 100, and 200 μM significantly inhibited the activity of HCT116 cells (P < 0.01). Figure 5 However, it did not significantly inhibit immortalized human small intestinal epithelial cells NCM460; on the contrary, cell activity increased with increasing drug concentration. Figure 6 Furthermore, compared to its analogues mariglobin, quercetin, and flavonoidin, oxcarbazin exhibited better inhibitory activity against colorectal cancer cells. Figure 7 The effect of oxcarbazin on the HCT116 colon cancer cell line was dose-dependent. This result suggests that oxcarbazin in this invention has an inhibitory effect on colon cancer cell activity, but does not exhibit significant toxicity to immortalized normal colon cells at concentrations below 200 μM.
[0042] Example 2: Oxcarnitine induces apoptosis in colon cancer cells
[0043] To detect the level of apoptosis induced by oxcarbamate in HCT116 colorectal cancer cells.
[0044] Operation process:
[0045] (1) Cell sample preparation: Place cell crawling slides in 24-well plates, collect HCT116 colon cancer cells in the logarithmic growth phase, and adjust the cell density to 1×10⁻⁶. 5 / well, inoculated into 24-well culture plates, incubated for 24 h, supernatant removed, DMEM medium solution containing 100 μM oxcarbazine added, and cultured for 12 and 24 h.
[0046] (2) Cell fixation: Cells on the slide were fixed with 4% paraformaldehyde for 30 min and then washed twice with PBS.
[0047] (3) Cell permeability: Resuspend cells in 0.3% Triton X-100 PBS, incubate at room temperature for 5 min, and wash twice with PBS.
[0048] (4) Cell staining: Add 50 μL of Tuol staining solution to the cell slide, incubate at 37°C for 60 min, and wash with PBS 3 times for 5 min each time after staining.
[0049] (5) Mounting and microscopic examination: Mount the slide with an anti-quenching agent and then observe it under a fluorescence microscope. The excitation wavelength range is 450-500nm and the emission wavelength range is 515-565nm (green).
[0050] result:
[0051] Experimental results are as follows Figure 8 A and Figure 8 As shown in Figure B, the apoptosis level of HCT116 colon cancer cells was significantly increased after treatment with 100 μM oxcarbazine for 12 and 24 hours. Fluorescence value statistics showed that the apoptosis level of HCT116 colon cancer cells increased by 4 to 8 times after oxcarbazine treatment for 12 and 24 hours.
[0052] Example 3: Verification of the synergistic effect of oxcarbazine and 5-fluorouracil
[0053] Operation process:
[0054] Logarithmic growth phase colon cancer cells HCT116 and normal colon cells NCM460 were selected, and the cell density was adjusted to 8 x 10⁻⁶. 3 Cells were seeded in 96-well plates and incubated for 24 hours. The supernatant was discarded. For the single-drug groups, oxcarbazine or 5-fluorouracil at final concentrations of 0, 20, 50, 100, and 200 μM were added to the cell culture medium. For the combined-drug groups, equal concentrations of oxcarbazine and 5-fluorouracil were added simultaneously (oxcarbazine + 5-fluorouracil: 0+0, 20+20, 50+50, 100+100, 200+200 μM). After 48 hours, cell morphology changes were observed under an inverted microscope. The MTT assay was used to detect the effect of the experimental samples on tumor cell viability, and the combined-drug index of oxcarbazine and 5-fluorouracil was analyzed using Compusyn.
[0055] result:
[0056] like Figure 9-10 As shown, Figure 9 The horizontal axis represents concentration, and the vertical axis represents inhibition rate. The three curves represent the inhibition rates of oxcarbazine, 5-fluorouracil, and the combination therapy on tumor cell activity as concentration changes. A larger value on the vertical axis indicates a stronger inhibition rate. Figure 9 The results showed that both oxcarbazine and 5-fluorouracil had significant inhibitory effects on colorectal cancer cells in the concentration range of 0-200 μM. Moreover, compared with 5-fluorouracil, oxcarbazine and the combination drug showed more significant inhibitory effects under the same concentration conditions.
[0057] Figure 10This is a diagram of the combined drug use index. The horizontal axis represents the cell activity inhibition rate, and the vertical axis represents the combined drug use index. A combined drug use index less than 1 indicates that the two drugs have a combined effect, meaning that the combined use of the two drugs is better than the sum of the effects of the two drugs at the same concentration. Figure 10 The combined drug results showed that oxcarbazine and 5-fluorouracil exhibited a certain combined inhibitory effect on colorectal cancer cells.
[0058] Example 4: Animal experiments verifying the inhibitory effect of oxcarbazin on the growth of colon cancer cells.
[0059] Operation process:
[0060] HCT116 cells in logarithmic growth phase were harvested and their density adjusted to 1×10⁻⁶. 7 A tumor model was constructed by injecting 100 μL of the solution into mice. After the model was constructed, the control group, the oxcarnitine treatment group, and the 5-fluorouracil treatment group were injected daily with equal volumes of physiological saline, 30 mg / kg / d oxcarnitine solution, and 20 mg / kg / d 5-fluorouracil solution, respectively. The size of the tumor and the weight of the mice were measured every three days. After 16 days, the mice were sacrificed, and tumor tissue blocks were dissected and their size was measured.
[0061] result:
[0062] like Figure 11 A and Figure 11 B shows that the tumors in the oxcarbazine-treated mice grew slowly over the 16-day experiment, while the tumor cells in the control group grew rapidly. This indicates that oxcarbazine 30 mg / kg / d significantly inhibited the growth of colorectal cancer tumor cells in mice after 16 days of treatment. The 5-fluorouracil-treated mice also showed slower tumor cell growth compared to the control group; however, compared to the oxcarbazine-treated group, the colorectal cancer cells in the 5-fluorouracil-treated group still exhibited a faster growth rate. This suggests that oxcarbazine showed a better inhibitory effect on colorectal cancer cell growth than 5-fluorouracil.
[0063] Example 5: Toxicity of Oxcarpine in Mice
[0064] The mouse intestinal cancer model is an in situ tumor formed by subcutaneous injection of intestinal cancer cells, which has little impact on the growth of mice. Therefore, without treatment, the mice in the intestinal cancer model can grow normally during the experiment. If drug treatment significantly inhibits the growth of mice and causes weight loss, it indicates that the drug is highly toxic to mice, and vice versa.
[0065] Operation process:
[0066] HCT116 cells in logarithmic growth phase were harvested and their density adjusted to 1×10⁻⁶. 7A tumor model was constructed by injecting 100 μL of the solution into mice. After the model was constructed, the control group, the oxcarnitine treatment group, and the 5-fluorouracil treatment group were injected daily with equal volumes of physiological saline, 30 mg / kg / d oxcarnitine solution, and 20 mg / kg / d 5-fluorouracil solution, respectively. The weight of the mice was measured every three days. The mice were sacrificed after 16 days and their weight was measured.
[0067] result:
[0068] like Figure 12 As shown, during the 16-day experiment, the body weight of mice injected with oxcarbazin did not show a significant difference compared to the control group (colon cancer model mice, untreated), indicating that oxcarbazin at an injection dose of 30 mg / kg / day had no significant toxic effect on mice. However, 5-fluorouracil showed a significant decrease in body weight compared to both the control and oxcarbazin-treated groups, indicating that 5-fluorouracil has strong toxic side effects on mice. This suggests that oxcarbazin has the potential to be developed as a therapeutic drug for colon cancer, or as an adjuvant drug for colon cancer treatment.
[0069] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
[0070] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. The application of oxcarbazine in combination with 5-fluorouracil in the preparation of antitumor drugs, characterized in that, The tumor is colorectal cancer; the concentrations of oxcarbazine and 5-fluorouracil are the same, ranging from 20 to 200 μmol.