An antitumor pharmaceutical composition and use thereof

Abstract

The application provides an antitumor pharmaceutical composition and application thereof, and belongs to the field of pharmacy. The antitumor pharmaceutical composition is composed of 5-fluorouracil and paclitaxel; the weight ratio of the 5-fluorouracil and the paclitaxel is 2:(2.3-10). Researches show that the 5-FU and the PTX with the specific weight ratio have a synergistic effect on inhibiting the proliferation of breast cancer cells in a certain concentration range. The 5-FU and the PTX with the specific weight ratio can be used for preparing a drug for treating breast cancer, and have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to an antitumor drug composition and its uses. Background Technology

[0002] Paclitaxel (PTX) is a complex secondary metabolite found in plants of the genus Taxus. Its molecular formula is C2. 47 H 51 NO 14 The weight-average molecular weight is 853.92. PTX is a widely used broad-spectrum antitumor drug in clinical practice. It inhibits the depolymerization of microtubules, maintaining the stability of the depolymerized state, thereby inhibiting the mitosis of tumor cells and ultimately achieving the goal of inhibiting tumor cell proliferation. However, PTX has extremely poor water solubility. Traditional formulations use polyoxyethylene castor oil (Cremophor EL) and anhydrous ethanol as solubilizing solvents, but Cremophor EL can cause a series of allergic reactions, requiring desensitization treatment before administration. In addition, due to the lack of target specificity, PTX has adverse reactions such as bone marrow suppression, allergic reactions, gastrointestinal reactions, and neurotoxicity during clinical use. Therefore, the clinical use of PTX is severely limited.

[0003] 5-Fluorouracil (5-FU) is obtained by replacing the hydrogen atoms in uracil with fluorine atoms. Its molecular formula is C4H3N2O2F, and its weight-average molecular weight is 130.8. 5-FU is a hydrophilic drug, slightly soluble in water (17 mg / mL), and soluble in dilute acids or alkalis. 5-FU is also a widely used antitumor drug in clinical practice, exhibiting antitumor activity against various cancers, including breast cancer, colorectal cancer, gastric cancer, head and neck cancer, and cervical cancer. Its main antitumor mechanism is that the metabolite fluorodeoxyuridine monophosphate (FdUMP) inhibits thymidylate synthase, thereby preventing DNA synthesis and inhibiting tumor growth. Oral absorption of 5-FU is incomplete and unpredictable; therefore, it is often administered by injection in clinical practice. However, 5-FU has a short half-life and is rapidly metabolized in the body, requiring frequent dosing, which is inconvenient. In addition, 5-FU has a series of side effects, mainly including gastrointestinal reactions and bone marrow suppression, as well as hair loss, nail changes, dermatitis, and increased skin pigmentation. Therefore, the clinical use of 5-FU is severely limited.

[0004] PTX and 5-FU are often used in combination for cancer treatment in clinical practice. Literature reports that the combination of PTX and 5-FU can treat advanced breast cancer, metastatic breast cancer, and advanced gastric cancer, with definite short-term efficacy, few adverse reactions, and significant improvement in quality of life, demonstrating high clinical value. However, the effect of combining PTX and 5-FU is not certain; their effects may be additive, synergistic, or antagonistic. These effects are often closely related to the dosage ratio between the drugs and the tumor cell type. Therefore, finding the optimal dosage ratio for PTX and 5-FU to exert a synergistic effect is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide an antitumor pharmaceutical composition and its use.

[0006] The present invention provides an antitumor drug composition comprising 5-fluorouracil and paclitaxel; wherein the weight ratio of 5-fluorouracil to paclitaxel is 2:(2.3-10).

[0007] Furthermore, the weight ratio of 5-fluorouracil to paclitaxel is (2:5) to (5:6);

[0008] Preferably, the weight ratio of 5-fluorouracil to paclitaxel is (2:5) to (13:20).

[0009] Furthermore, the tumor is a malignant tumor; preferably, the malignant tumor is breast cancer.

[0010] The present invention also provides a method for preparing the aforementioned antitumor drug composition, wherein the preparation method comprises the following steps: mixing 5-fluorouracil and paclitaxel raw materials according to the weight ratio, or preparing a solution by mixing;

[0011] Preferably, after the prepared solutions are mixed, the total concentration of 5-fluorouracil and paclitaxel in the mixed solution is not less than 0.0825 μg / mL;

[0012] More preferably, after the prepared solutions are mixed, the total concentration of 5-fluorouracil and paclitaxel in the mixed solution is 0.0825 μg / mL to 1.32 μg / mL;

[0013] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the mixed solution is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the mixed solution is 0.28 μg / mL to 1.32 μg / mL;

[0014] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the mixed solution is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the mixed solution is 0.33 μg / mL to 1.32 μg / mL.

[0015] The present invention also provides an antitumor drug formulation, which is a formulation prepared by using the aforementioned antitumor drug composition as the active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients;

[0016] Preferably, the total concentration of 5-fluorouracil and paclitaxel in the formulation is not less than 0.0825 μg / mL;

[0017] More preferably, the total concentration of 5-fluorouracil and paclitaxel in the formulation is 0.0825 μg / mL to 1.32 μg / mL;

[0018] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the formulation is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the formulation is 0.28 μg / mL to 1.32 μg / mL;

[0019] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the formulation is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the formulation is 0.33 μg / mL to 1.32 μg / mL.

[0020] The present invention also provides a combination antitumor drug containing 5-fluorouracil and paclitaxel of the same or different strengths, administered simultaneously or separately, and a pharmaceutically acceptable carrier; wherein the weight ratio of 5-fluorouracil to paclitaxel is 2:(2.3-10);

[0021] Preferably, the weight ratio of 5-fluorouracil to paclitaxel is (2:5) to (5:6);

[0022] More preferably, the weight ratio of 5-fluorouracil to paclitaxel is (2:5) to (13:20).

[0023] Furthermore, in the drug, the total concentration of 5-fluorouracil and paclitaxel is not less than 0.0825 μg / mL;

[0024] Preferably, the total concentration of 5-fluorouracil and paclitaxel in the drug is 0.0825 μg / mL to 1.32 μg / mL;

[0025] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the drug is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the drug is 0.28 μg / mL to 1.32 μg / mL;

[0026] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the drug is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the drug is 0.33 μg / mL to 1.32 μg / mL.

[0027] Furthermore, the tumor is a malignant tumor; preferably, the malignant tumor is breast cancer.

[0028] The present invention also provides the use of 5-fluorouracil and paclitaxel in the preparation of antitumor drugs; wherein the weight ratio of 5-fluorouracil and paclitaxel is 2:(2.3-10);

[0029] Preferably, the weight ratio of 5-fluorouracil to paclitaxel is (2:5) to (5:6);

[0030] More preferably, the weight ratio of 5-fluorouracil to paclitaxel is (2:5) to (13:20).

[0031] Furthermore, in the drug, the total concentration of 5-fluorouracil and paclitaxel is not less than 0.0825 μg / mL;

[0032] Preferably, the total concentration of 5-fluorouracil and paclitaxel in the drug is 0.0825 μg / mL to 1.32 μg / mL;

[0033] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the drug is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the drug is 0.28 μg / mL to 1.32 μg / mL;

[0034] More preferably, when the weight ratio of 5-fluorouracil to paclitaxel in the drug is 13:20, the total concentration of 5-fluorouracil and paclitaxel in the drug is 0.33 μg / mL to 1.32 μg / mL.

[0035] Furthermore, the tumor is a malignant tumor; preferably, the malignant tumor is breast cancer.

[0036] In existing technologies, breast cancer treatment can involve local drug delivery, such as preparing the drug into an injectable formulation for local administration. This method of delivery allows the drug to act directly on the tumor tissue, avoiding dilution of the drug concentration after passing through the bloodstream or digestive tract, ensuring the drug concentration at the tumor tissue and exerting better efficacy. The drug compositions, formulations, and combined drugs of the present invention can also be formulated into local delivery formulations that act directly on the tumor tissue, ensuring that the drug concentration at the tumor tissue is not diluted.

[0037] This invention demonstrates that the combined use of 5-FU and PTX at a specific weight ratio exhibits a synergistic effect in inhibiting the proliferation of breast cancer cells within a certain concentration range. The specific weight ratio of 5-FU and PTX described in this invention can be used to prepare drugs for treating breast cancer, showing promising application prospects.

[0038] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0039] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0040] Figure 1 The effect of different concentration ratios of 5-FU and PTX on the proliferation of MCF-7 cells: a represents the effect of different concentration ratios of 5-FU and PTX on the activity of MCF-7 cells, where *p<0.05, **p<0.01, ***p<0.001 represent statistical comparisons between each test drug group and the negative control group; # p<0.05, ## p<0.01, ### p<0.001 indicates a statistical comparison between 5-FU solution or PTX solution and a mixture of 5-FU and PTX at the same concentration; the statistical comparison was performed using two-way ANOVA; b is the dose-equivalence curve after combined administration of 5-FU and PTX at different concentration ratios.

[0041] Figure 2 The effect of combined administration of 5-FU and PTX at different concentrations at a fixed ratio on the proliferation of MCF-7 cells: a) Effect of combined administration of 5-FU and PTX at different concentrations on the activity of MCF-7 cells, where *p<0.05, **p<0.01, ***p<0.001 represent statistical comparisons between each tested drug group and the negative control group; # p<0.05, ## p<0.01, ### p<0.001 indicates a statistical comparison between 5-FU solution or PTX solution and a mixture of 5-FU and PTX at the same concentration; the statistical comparison was performed using a two-way ANOVA; b is the dose-equivalence curve of 5-FU and PTX at different concentrations with a fixed concentration ratio.

[0042] Figure 3The figure shows the induction of apoptosis in MCF-7 cells by 5-FU, PTX, and their combination. In the figure, Q1-UL represents necrotic cells, Q1-UR represents cells in late apoptosis and secondary necrosis, Q1-LL represents live cells, and Q1-LR represents early apoptosis.

[0043] Figure 4 The images show fluorescence images of apoptosis observed using Hoechst 33258 staining, magnified 20×. Small arrows indicate nuclear shrinkage, mitosis, and apoptotic bodies: a is the negative control group; b is 5-FU solution at a concentration of 0.52 μg / mL used alone; c is PXT solution at a concentration of 0.8 μg / mL used alone; d is a mixture of 5-FU and PXT solutions of equal volumes (0.52 μg / mL and 0.8 μg / mL); e is 5-FU solution at a concentration of 1.04 μg / mL used alone; f is PXT solution at a concentration of 1.60 μg / mL used alone; g is a mixture of 5-FU and PTX solutions of equal volumes (1.04 μg / mL and 1.60 μg / mL). Detailed Implementation

[0044] 1. Experimental instruments and equipment

[0045] The raw materials and equipment used in the specific embodiments of this invention are all known products, obtained by purchasing commercially available products. The main instruments and equipment are shown in Table 1, and the main raw materials and reagents used are shown in Table 2.

[0046] Table 1. Instruments and Equipment

[0047]

[0048]

[0049] Table 2 Raw Materials and Reagents

[0050]

[0051] 2. Cell lines used in the experiment

[0052] The cell line used in the specific embodiments of the present invention is the human breast cancer cell line (MCF-7).

[0053] 3. Experimental Methods

[0054] In the specific embodiments of the present invention, the solution preparation and cell experiments involved all adopt conventional cell experiment methods (unless otherwise specified). Some solution preparation methods and cell experiment methods are as follows.

[0055] (1) Preparation of complete culture medium

[0056] Prepare complete culture medium in 100 mL units: Place 10 mL of FBS solution in a sterile graduated glass bottle, add 1 mL of penicillin-streptomycin solution (10000 U / mL, 10 mg / mL), and finally add 89 mL of DMEM medium. Mix well to obtain a final concentration of 10% FBS and 1% penicillin-streptomycin solution (DMEM medium). Store the complete culture medium at 4°C, prepare fresh, and do not store for more than one week.

[0057] (2) Preparation of cell cryopreservation solution

[0058] Remove fetal bovine serum (FBS) from the -20°C freezer. After thawing, mix well and set aside. Mix 700 μL of FBS with 300 μL of culture medium at a ratio of 7:3 to obtain 1 mL of cryopreservation solution. For long-term storage, keep in a -20°C freezer.

[0059] (3) Preparation of phosphate-buffered saline (PBS)

[0060] Take one packet of phosphate buffer powder, dissolve it in an appropriate amount of ultrapure water, adjust the pH to 7.2-7.6, dispense it into 100mL glass bottles, autoclave at 121℃, and store in a sealed container at 4℃.

[0061] (4) Cultivation conditions

[0062] Cells were cultured in a constant temperature cell culture incubator containing 5% CO2 at 37°C.

[0063] (5) Passage of tumor cells

[0064] Observe cell growth under an inverted microscope. Take a flask of well-grown cells, ensuring the adherent cells cover 85%–95% of the bottom area of ​​the flask. Preheat the flask with 0.25% trypsin at 37°C. Discard the old culture medium, wash the cells 2–3 times with PBS buffer, and then digest them with the preheated trypsin. Immediately after digestion, add an appropriate amount of culture medium, gently pipette the adherent cells, and transfer the cell suspension to a 15 mL sterile centrifuge tube. Centrifuge at 800 rpm for 5 min. After centrifugation, discard the supernatant, resuspend the cells in fresh culture medium, and passage them at a ratio of 1:2 or 1:3.

[0065] (6) Cryopreservation of tumor cells

[0066] Take a flask of cells in good growth condition, with adherent cells covering more than 90% of the bottom area of ​​the culture flask. Perform PBS washing, digestion, and centrifugation according to the cell passage procedure described in (5) above. Resuspend the centrifuged cells in 1 mL of cryopreservation buffer, transfer them to sterile cryovials, seal them, and label them. Pre-freeze the cells at -20℃ and -80℃ for 12 hours sequentially using the temperature gradient method, and then transfer them to a liquid nitrogen tank for long-term storage.

[0067] (7) Tumor cell resuscitation

[0068] Set the temperature of the constant-temperature water bath to 37.5℃. Once the temperature stabilizes, quickly remove the cells from the liquid nitrogen container, place them in PE gloves, and continuously agitate them in the 37℃ water bath for about 1-2 minutes until they just thaw. Immediately transfer the cell suspension to preheated culture medium and centrifuge at 800 rpm for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in freshly prepared complete culture medium. After mixing well, transfer the resuspended cells to a culture flask for further culture. Newly revived cells need to adhere for approximately 4 hours to observe their adhesion and growth status, confirming successful revival.

[0069] Example 1: Synergistic anti-breast cancer cell proliferation effect of different concentration ratios of 5-fluorouracil and paclitaxel

[0070] 1. Experimental Methods

[0071] (1) Cell inoculation

[0072] MCF-7 cells in the logarithmic growth phase were collected and washed, digested, and centrifuged according to the methods described in "Passaging of Tumor Cells." The resulting cells were resuspended in DMEM medium. A small amount of the cell suspension was diluted 10-fold with PBS solution and counted using flow cytometry. Based on the counting results, the cell suspension was then diluted to 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / mL into each well of a 96-well plate. 200 μL of PBS solution was added around the cells to prevent evaporation of the culture medium. The seeded plates were labeled and incubated overnight in a 37°C, 5% CO2 incubator.

[0073] (2) Preparation of test solution

[0074] Weigh an appropriate amount of 5-fluorouracil (5-FU) raw material, dissolve it in DMEM medium to prepare a stock solution of 100 μg / mL, and then dilute the stock solution with DMEM medium to prepare 5-FU solutions of different concentrations: 0.08 μg / mL, 0.15 μg / mL, 0.26 μg / mL, 0.4 μg / mL, 0.5 μg / mL and 0.7 μg / mL.

[0075] Weigh an appropriate amount of paclitaxel (PTX) raw material, dissolve it thoroughly in DMSO, and prepare a solution of 1×10 5 The mother liquor was prepared at a concentration of μg / mL and then diluted with DMEM medium to prepare PTX solutions of different concentrations: 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL and 0.8 μg / mL.

[0076] Then, 5-FU solutions of different concentrations were mixed with PTX solutions to obtain 5-FU and PTX mixed solutions with different concentration ratios:

[0077] Equal volumes of 5-FU solution with a concentration of 0.08 μg / mL and PTX solution with a concentration of 0.2 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.04 μg / mL, the concentration of PTX was 0.1 μg / mL, and the concentration ratio of 5-FU to PTX was 2:5.

[0078] Equal volumes of 5-FU solution with a concentration of 0.15 μg / mL and PTX solution with a concentration of 0.3 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.075 μg / mL, the concentration of PTX was 0.15 μg / mL, and the concentration ratio of 5-FU to PTX was 1:2.

[0079] Equal volumes of 5-FU solution with a concentration of 0.26 μg / mL and PTX solution with a concentration of 0.4 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.13 μg / mL, the concentration of PTX was 0.2 μg / mL, and the concentration ratio of 5-FU to PTX was 13:20.

[0080] Equal volumes of 5-FU solution with a concentration of 0.4 μg / mL and PTX solution with a concentration of 0.5 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.2 μg / mL, the concentration of PTX was 0.25 μg / mL, and the concentration ratio of 5-FU to PTX was 4:5.

[0081] Equal volumes of 5-FU solution with a concentration of 0.5 μg / mL and PTX solution with a concentration of 0.6 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.25 μg / mL, the concentration of PTX was 0.3 μg / mL, and the concentration ratio of 5-FU to PTX was 5:6.

[0082] Equal volumes of 0.7 μg / mL 5-FU solution and 0.8 μg / mL PTX solution were mixed. The concentration of 5-FU in the mixed solution was 0.35 μg / mL, the concentration of PTX was 0.4 μg / mL, and the concentration ratio of 5-FU to PTX was 7:8.

[0083] (3) Experimental group

[0084] Negative control group: cells + DMEM medium;

[0085] Test drug groups: cells + mixed solutions of 5-FU and PTX at different concentration ratios, cells + 5-FU solutions at different concentrations, and cells + PTX solutions at different concentrations.

[0086] Control group: No cells were added, only DMEM medium was added.

[0087] (4) Administration

[0088] After the cells had fully adhered to the plate, the original culture medium was aspirated. According to the experimental groups: 100 μL of culture medium was added to the negative control group; 100 μL of a mixture of 5-FU and PTX at different concentrations, or 100 μL of 5-FU at different concentrations, or 100 μL of PTX at different concentrations were added to the test drug group; 100 μL of culture medium was added to the blank group. After administration, the cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0089] (5) Measurement of OD value and calculation of cell viability

[0090] 24 hours after drug administration, discard the original culture medium in the well plate, add 100 μL of fresh DMEM medium, and then add 10 μL of CCK-8 reagent to each well. Incubate at 37°C and 5% CO2 for 4 hours. Remove the plate, shake to mix for 30 seconds, and finally place the 96-well plate on a microplate reader to measure the absorbance (OD value) at 25°C and 450 nm. Calculate the cell viability using the following formula: Cell viability (%) = (OD value / (OD value)) 供试药液组 -OD 空白组 ) / (OD 阴性对照组 -OD 空白组 )×100%. OD 供试药液组 The OD value measured for the test drug solution group is OD 空白组 The OD values ​​measured for the blank group are OD. 阴性对照组 The OD values ​​were measured for the negative control group.

[0091] (6) Analysis of synergistic effects

[0092] Based on a pharmacological interaction model of two drugs, the interaction between 5-FU and PTX was assessed using the synergy index-equivalence line equation (see reference: Chou T C. Drug combination studies and their synergy quantification using the Chou-Talalay method.[J]. Cancer Research, 2010, 70(2):440-446.). A combination index (CI) of 1 indicates an additive effect between the two drugs; CI < 1 indicates a synergistic effect; and CI > 1 indicates an antagonistic effect. 1.0 (ComboSyn, Paramus, NJ, USA.) Calculate dose-equivalence curves and synergism indices. See the literature (Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. [J]. Pharmacological Reviews, 2006, 58(3): 621-681.).

[0093] 2. Experimental Results

[0094] The effect of combined administration of 5-FU and PTX at different concentration ratios on the proliferation of breast cancer MCF-7 cells, as follows: Figure 1 As shown. Figure 1 a represents the effect of different concentration ratios of 5-FU and PTX combined on the viability of MCF-7 cells. Figure 1 As shown in Figure a, the combined use of 5-FU and PTX at different concentration ratios to inhibit tumor cell proliferation in MCF-7 breast cancer cells was superior to the effects of 5-FU or PTX alone. However, there was no significant increasing trend in effect with increasing dosage.

[0095] Figure 1 b shows the dose-equivalence curves after combined use of 5-FU and PTX at different concentration ratios, and Table 3 shows the synergistic index (CI) of combined use of 5-FU and PTX at different concentration ratios. Figure 1As shown in b, when 5-FU and PTX are used in combination, five points on the dose-equivalence curve are below 1, i.e., CI < 1. CI < 1 indicates a synergistic effect between the two drugs, suggesting that the five combined drug concentration ratios have a synergistic effect on inhibiting MCF-7 cell proliferation. These five ratios are 5-FU to PTX concentrations of 2:5, 1:2, 13:20, 4:5, and 5:6. Table 3 also provides the results. Figure 1 b yielded the same results. The synergistic effect was optimal when the concentration ratio of 5-FU to PTX was 13:20.

[0096] Table 3. Synergistic index of 5-FU and PTX at different concentration ratios

[0097]

[0098] Example 2: Synergistic anti-breast cancer cell proliferation effect of 5-fluorouracil and paclitaxel at the same concentration ratio

[0099] 1. Experimental Methods

[0100] (1) Cell inoculation

[0101] Same as “(1) Cell seeding” in Example 1.

[0102] (2) Preparation of test solution

[0103] Weigh an appropriate amount of 5-fluorouracil (5-FU) raw material, dissolve it in DMEM medium to prepare a stock solution of 100 μg / mL, and then dilute the stock solution with DMEM medium to prepare 5-FU solutions of different concentrations: 0.0325 μg / mL, 0.065 μg / mL, 0.13 μg / mL, 0.26 μg / mL, 0.52 μg / mL and 1.04 μg / mL.

[0104] Weigh an appropriate amount of paclitaxel (PTX) raw material, dissolve it thoroughly in DMSO, and prepare a solution of 1×10 5 The mother liquor was prepared at a concentration of μg / mL and then diluted with DMEM medium to prepare PTX solutions of different concentrations: 0.05 μg / mL, 0.10 μg / mL, 0.20 μg / mL, 0.40 μg / mL, 0.80 μg / mL, and 1.60 μg / mL.

[0105] With a fixed concentration ratio of 13:20 for the combined use of 5-FU and PTX, 5-FU and PTX solutions were mixed separately to obtain 5-FU and PTX mixtures of different concentrations:

[0106] Equal volumes of 5-FU solution with a concentration of 0.0325 μg / mL and PTX solution with a concentration of 0.05 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.01625 μg / mL, the concentration of PTX was 0.025 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 0.04125 μg / mL.

[0107] Equal volumes of 5-FU solution with a concentration of 0.065 μg / mL and PTX solution with a concentration of 0.10 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.0325 μg / mL, the concentration of PTX was 0.05 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 0.0825 μg / mL.

[0108] Equal volumes of 5-FU solution with a concentration of 0.13 μg / mL and PTX solution with a concentration of 0.20 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.065 μg / mL, the concentration of PTX was 0.10 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 0.165 μg / mL.

[0109] Equal volumes of 5-FU solution with a concentration of 0.26 μg / mL and PTX solution with a concentration of 0.40 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.13 μg / mL, the concentration of PTX was 0.20 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 0.33 μg / mL.

[0110] Equal volumes of 5-FU solution with a concentration of 0.52 μg / mL and PTX solution with a concentration of 0.80 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.26 μg / mL, the concentration of PTX was 0.40 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 0.66 μg / mL.

[0111] Equal volumes of 5-FU solution with a concentration of 1.04 μg / mL and PTX solution with a concentration of 1.60 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.52 μg / mL, the concentration of PTX was 0.80 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 1.32 μg / mL.

[0112] (3) Experimental group

[0113] Negative control group: cells + DMEM medium;

[0114] Test drug groups: cells + mixed solutions of 5-FU and PTX at different concentrations, cells + 5-FU solutions at different concentrations, and cells + PTX solutions at different concentrations.

[0115] Control group: No cells were added, only DMEM medium was added.

[0116] (4) Administration

[0117] After the cells had fully adhered to the plate, the original culture medium was aspirated. According to the experimental groups: 100 μL of culture medium was added to the negative control group; 100 μL of a mixture of 5-FU and PTX at different concentrations, or 100 μL of 5-FU at different concentrations, or 100 μL of PTX at different concentrations were added to the test drug group; 100 μL of culture medium was added to the blank group. After administration, the cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0118] (5) Measurement of OD value and calculation of cell viability

[0119] Same as in Example 1, “(5) Measurement of OD value and calculation of cell viability”.

[0120] (6) Analysis of synergistic effects

[0121] Same as “(6) Analysis of synergistic effect” in Example 1.

[0122] 2. Experimental Results

[0123] With a fixed concentration ratio of 13:20 for the combined use of 5-FU and PTX, the effects of different concentrations of the combined 5-FU and PTX on the proliferation of breast cancer MCF-7 cells were investigated. Figure 2 As shown. Figure 2 a represents the effect of combined administration of 5-FU and PTX at different concentrations on the activity of MCF-7 cells. Figure 2 As shown in Figure a, when the concentration ratio of 5-FU to PTX is fixed, but the total concentration of the 5-FU and PTX mixture varies, the combined use of 5-FU and PTX significantly inhibits MCF-7 cell proliferation more effectively than either 5-FU or PTX alone. Furthermore, the inhibitory effect on MCF-7 cell proliferation increases with increasing total concentration of 5-FU and PTX in the mixture, eventually stabilizing. The inhibitory effect on MCF-7 cell proliferation significantly increases when the total concentrations of 5-FU and PTX in the mixture reach 0.33 μg / mL, 0.66 μg / mL, and 1.32 μg / mL.

[0124] Figure 2 b represents the dose-equivalence curves of 5-FU and PTX at different concentrations when used in combination, with a fixed concentration ratio. Table 4 shows the synergistic index (CI) of 5-FU and PTX at different concentrations. Figure 2As shown in Table b, when 5-FU and PTX were used in combination, the dose-equivalence curves revealed that five out of the six combined drug concentrations (0.0825 μg / mL, 0.165 μg / mL, 0.33 μg / mL, 0.66 μg / mL, and 1.32 μg / mL) exhibited a synergistic effect in inhibiting MCF-7 cell proliferation. As shown in Table 4, no synergistic effect was observed at a total concentration of 0.04125 μg / mL. The synergistic effect was optimal at a total concentration of 0.33 μg / mL.

[0125] Table 4. Synergistic index of 5-FU and PTX at different concentrations

[0126]

[0127] Example 3: Flow cytometry detection of apoptosis induced by the synergistic effect of 5-fluorouracil and paclitaxel in breast cancer cells at a fixed ratio.

[0128] 1. Experimental Methods

[0129] (1) Cell inoculation

[0130] MCF-7 cells in the logarithmic growth phase were collected and washed, digested, and centrifuged according to the methods described in "Passaging of Tumor Cells." The resulting cells were resuspended in DMEM medium. A small amount of the cell suspension was diluted 20-fold with PBS solution and counted using flow cytometry. Based on the counting results, the cell suspension was then diluted to 2 × 10⁻⁶ cells / mL. 5 Cells / mL were evenly seeded into 12-well plates at a rate of 1000 μL per well. The seeded culture plates were then labeled and incubated overnight in a 37°C, 5% CO2 incubator.

[0131] (2) Preparation of test solution

[0132] Weigh an appropriate amount of 5-fluorouracil (5-FU) raw material, dissolve it in DMEM medium to prepare a stock solution of 100 μg / mL, and then use DMEM medium to dilute the stock solution into 5-FU solutions of different concentrations, namely 0.52 μg / mL and 1.04 μg / mL.

[0133] Weigh an appropriate amount of paclitaxel (PTX) raw material, dissolve it thoroughly in DMSO, and prepare a solution of 1×10 5 The mother liquor was prepared at a concentration of μg / mL and then diluted with DMEM medium to prepare PTX solutions of different concentrations: 0.80 μg / mL and 1.60 μg / mL.

[0134] With a fixed concentration ratio of 13:20 for the combined use of 5-FU and PTX, 5-FU and PTX solutions were mixed separately to obtain 5-FU and PTX mixtures of different concentrations:

[0135] Equal volumes of 5-FU solution with a concentration of 0.52 μg / mL and PTX solution with a concentration of 0.80 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.26 μg / mL, the concentration of PTX was 0.40 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 0.66 μg / mL.

[0136] Equal volumes of 5-FU solution with a concentration of 1.04 μg / mL and PTX solution with a concentration of 1.60 μg / mL were mixed. The concentration of 5-FU in the mixed solution was 0.52 μg / mL, the concentration of PTX was 0.80 μg / mL, and the total concentration of 5-FU and PTX in the mixed solution was 1.32 μg / mL.

[0137] (3) Experimental group

[0138] Negative control group: cells + DMEM medium;

[0139] Test drug groups: cells + mixed solutions of 5-FU and PTX at different concentrations, cells + 5-FU solutions at different concentrations, and cells + PTX solutions at different concentrations.

[0140] (4) Administration

[0141] After the cells had fully adhered to the plate, the original culture medium was aspirated. According to the experimental groups: 1000 μL of culture medium was added to the negative control group; 1000 μL of a mixture of 5-FU and PTX at different concentrations, or 1000 μL of 5-FU at different concentrations, or 1000 μL of PTX at different concentrations were added to each group. After administration, the cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0142] (5) Cell staining and detection analysis

[0143] First, aspirate the original culture medium from the well plate. Add 500 μL of EDTA-free trypsin to each well for digestion. Then, centrifuge at 2000 rpm for 5 min, wash twice with PBS, collect the cells, resuspend the cells in 500 μL of Binding Buffer, and then add 5 μL of Annexin V-FITC and 5 μL of Propidiun Iodide. Mix well and incubate at room temperature in the dark for 15 min. Detect the cells using flow cytometry within 1 h, using channels FL1 and FL2 or FL3, with an excitation wavelength Ex = 488 nm and an emission wavelength Em = 530 nm.

[0144] 2. Experimental Results

[0145] To demonstrate that the combined use of 5-FU and PTX induces apoptosis in MCF-7 cells, Annexin-FITC / PI double staining was used to stain and detect cells treated with either drug alone or in combination. The results are as follows: Figure 3 As shown. Figure 3 The results showed that when 5-FU and PTX were used in combination to treat MCF-7 cells, the apoptosis induction rates were 27.6% (5-FU concentration 0.26 μg / mL, PTX concentration 0.40 μg / mL, and total concentration of 5-FU and PTX 0.66 μg / mL) and 27.1% (5-FU concentration 0.52 μg / mL and PTX concentration 0.80 μg / mL), respectively. The total concentration of 5-FU and PTX in the mixed drug solution was 1.32 μg / mL. When 5-FU was used alone, the apoptosis induction rates were 10.4% (5-FU concentration 0.52 μg / mL) and 12.5% ​​(5-FU concentration 12.5 μg / mL), respectively. When PTX was used alone, the apoptosis induction rates were 15.8% (PTX concentration 0.8 μg / mL) and 18.2% (PTX concentration 1.60 μg / mL), respectively. The experimental results indicate that the combined use of 5-FU and PTX at specific dosage ratios significantly increased the apoptosis induction rate of MCF-7 cells compared to the apoptosis induction rate of either drug alone, demonstrating a potential apoptosis induction and synergistic effect.

[0146] Example 4: Effect of 5-fluorouracil and paclitaxel on nuclear morphology in the synergistic induction of apoptosis in breast cancer cells at a fixed ratio.

[0147] 1. Experimental Methods

[0148] (1) Cell inoculation

[0149] Take a clean coverslip (soaked in 75% ethanol), wash it twice with PBS solution, then wash it twice with culture medium, and then place it in a six-well plate for later use.

[0150] MCF-7 cells in the logarithmic growth phase were collected and washed, digested, and centrifuged according to the methods described in "Passaging of Tumor Cells." The resulting cells were resuspended in DMEM medium. A small amount of the cell suspension was diluted 20-fold with PBS solution and counted using flow cytometry. Based on the counting results, the cell suspension was then diluted to 5 × 10⁻⁶ cells / mL. 5Cells / mL were evenly seeded onto coverslips in each well of a 6-well plate at a rate of 2000 μL per well. The seeded culture plates were then labeled and incubated overnight in a 37°C, 5% CO2 incubator until the cells were approximately 60%–80% confluent.

[0151] (2) Preparation of test solution

[0152] Same as "(2) Preparation of test solution" in Example 3.

[0153] (3) Experimental group

[0154] Same as “(3) Experimental group” in Example 3.

[0155] (4) Administration

[0156] After the cells had fully adhered to the plate, the original culture medium was aspirated. According to the experimental groups: 2000 μL of culture medium was added to the negative control group; 2000 μL of a mixture of 5-FU and PTX at different concentrations, or 2000 μL of 5-FU at different concentrations, or 2000 μL of PTX at different concentrations were added to each group. After administration, the cells were incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0157] (5) Cell staining and detection analysis

[0158] First, aspirate the existing culture medium from the well plate and wash twice with PBS solution. Then, add 500 μL of fixative and fix the cells for about 15 min. After that, aspirate the fixative and wash twice with PBS solution for 3 min each time, shaking constantly to ensure complete washing. Next, add 500 μL of Hoechst 33258 staining solution and stain for at least 15 min. Aspirate the staining solution and wash twice more with PBS solution for 3 min each time. Place one drop (about 10 μL) of anti-fluorescence quenching solution on a glass slide, then cover it with a coverslip containing the cells, allowing the cells to contact the mounting solution, avoiding air bubbles as much as possible. Observe the blue cell nuclei using an inverted fluorescence microscope with an excitation wavelength of 345 nm and an emission wavelength of 487 nm.

[0159] 2. Experimental Results

[0160] To visually observe the nuclear morphology of tumor cell apoptosis, Hoechst staining was used to stain the nuclei of cells treated with 5-FU and PTX alone and in combination to observe the apoptotic effect. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that: the negative control group ( Figure 4 a) The cell nuclei are regularly round or nearly round, with a uniform blue color distribution, and the nuclei are of uniform size; the test drug solution group ( Figure 4b is 0.52 μg / mL 5-FU, Figure 4 c is 0.8 μg / mL PXT. Figure 4 d is 0.52μg / mL 5-FU+0.8μg / mL PXT, Figure 4 e is 1.04 μg / mL 5-FU, Figure 4 f is 1.6 μg / mL PXT, Figure 4 Cell nuclei treated with a solution of 1.04 μg / mL 5-FU + 1.6 μg / mL PTX showed dense, deep blue staining, with some nuclei exhibiting shrinkage and division, indicating clear apoptotic characteristics. Particularly when 5-FU and PTX were used in combination, the number of cell nuclei showing apoptotic characteristics was significantly higher than when 5-FU or PTX were used alone, and the number of apoptotic cells increased with increasing concentration of the 5-FU and PTX mixture. This experiment clearly demonstrates that the combined action of 5-FU and PTX has a significant apoptosis-inducing effect on MCF-7 cells.

[0161] Examples 1 to 4 above show that:

[0162] (1) The combined use of 5-FU and PTX was confirmed by the CCK-8 assay to have a synergistic inhibitory effect on human breast cancer MCF-7 cells. The synergistic index obtained by Compusyn software analysis further confirmed the synergistic effect of the combined use of 5-FU and PTX. The synergistic effect was related to both the concentration ratio of the two drugs and the total dose.

[0163] (2) Annexin V-FITC / PI double staining assay showed that 5-FU and PTX both induced apoptosis in human breast cancer MCF-7 cells. The apoptosis induction rate increased significantly after the two drugs were used in combination, indicating a synergistic effect in inducing MCF-7 cell apoptosis. Hoechst's experimental results further confirmed this conclusion.

[0164] In summary, this invention demonstrates that the combined use of 5-FU and PTX at a specific weight ratio exhibits a synergistic effect in inhibiting the proliferation of breast cancer cells within a certain concentration range. The specific weight ratio of 5-FU and PTX presented in this invention can be used to prepare drugs for treating breast cancer, showing promising application prospects.

Claims

1. An antitumor drug composition, characterized in that: It is composed of 5-fluorouracil and paclitaxel; the concentration ratio of 5-fluorouracil to paclitaxel is 2:5, 1:2, 13:20, 4:5 or 5:6; the tumor is breast cancer; The antitumor drug composition is prepared according to the following steps: When the concentration ratio of 5-fluorouracil to paclitaxel is 2:5, equal volumes of 5-fluorouracil solution with a concentration of 0.08 μg / mL and paclitaxel solution with a concentration of 0.2 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 1:2, equal volumes of 5-fluorouracil solution with a concentration of 0.15 μg / mL and paclitaxel solution with a concentration of 0.3 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 13:20, equal volumes of 5-fluorouracil solution with a concentration of 0.26 μg / mL and paclitaxel solution with a concentration of 0.4 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.065 μg / mL and paclitaxel solution with a concentration of 0.10 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.13 μg / mL and paclitaxel solution with a concentration of 0.20 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.52 μg / mL and paclitaxel solution with a concentration of 0.80 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 1.04 μg / mL and paclitaxel solution with a concentration of 1.60 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 4:5, equal volumes of 5-fluorouracil solution with a concentration of 0.4 μg / mL and paclitaxel solution with a concentration of 0.5 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 5:6, equal volumes of 5-fluorouracil solution with a concentration of 0.5 μg / mL and paclitaxel solution with a concentration of 0.6 μg / mL are mixed.

2. An antitumor drug formulation, characterized in that: It is a formulation prepared by using the antitumor drug composition of claim 1 as the active ingredient, plus pharmaceutically acceptable excipients.

3. A combination antitumor drug, characterized in that: It contains 5-fluorouracil and paclitaxel of the same or different strengths, administered simultaneously or separately, and a pharmaceutically acceptable carrier; the concentration ratio of 5-fluorouracil to paclitaxel is 2:5, 1:2, 13:20, 4:5, or 5:6; the tumor is breast cancer; When the concentration ratio of 5-fluorouracil to paclitaxel is 2:5, equal volumes of 5-fluorouracil solution with a concentration of 0.08 μg / mL and paclitaxel solution with a concentration of 0.2 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 1:2, equal volumes of 5-fluorouracil solution with a concentration of 0.15 μg / mL and paclitaxel solution with a concentration of 0.3 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 13:20, equal volumes of 5-fluorouracil solution with a concentration of 0.26 μg / mL and paclitaxel solution with a concentration of 0.4 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.065 μg / mL and paclitaxel solution with a concentration of 0.10 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.13 μg / mL and paclitaxel solution with a concentration of 0.20 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.52 μg / mL and paclitaxel solution with a concentration of 0.80 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 1.04 μg / mL and paclitaxel solution with a concentration of 1.60 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 4:5, equal volumes of 5-fluorouracil solution with a concentration of 0.4 μg / mL and paclitaxel solution with a concentration of 0.5 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 5:6, equal volumes of 5-fluorouracil solution with a concentration of 0.5 μg / mL and paclitaxel solution with a concentration of 0.6 μg / mL are mixed. 4.5-Fluorouracil and paclitaxel in combination for the preparation of antitumor drugs; wherein the concentration ratio of 5-fluorouracil to paclitaxel is 2:5, 1:2, 13:20, 4:5 or 5:6; wherein the tumor is breast cancer; When the concentration ratio of 5-fluorouracil to paclitaxel is 2:5, equal volumes of 5-fluorouracil solution with a concentration of 0.08 μg / mL and paclitaxel solution with a concentration of 0.2 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 1:2, equal volumes of 5-fluorouracil solution with a concentration of 0.15 μg / mL and paclitaxel solution with a concentration of 0.3 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 13:20, equal volumes of 5-fluorouracil solution with a concentration of 0.26 μg / mL and paclitaxel solution with a concentration of 0.4 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.065 μg / mL and paclitaxel solution with a concentration of 0.10 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.13 μg / mL and paclitaxel solution with a concentration of 0.20 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 0.52 μg / mL and paclitaxel solution with a concentration of 0.80 μg / mL are mixed; or, equal volumes of 5-fluorouracil solution with a concentration of 1.04 μg / mL and paclitaxel solution with a concentration of 1.60 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 4:5, equal volumes of 5-fluorouracil solution with a concentration of 0.4 μg / mL and paclitaxel solution with a concentration of 0.5 μg / mL are mixed. When the concentration ratio of 5-fluorouracil to paclitaxel is 5:6, equal volumes of 5-fluorouracil solution with a concentration of 0.5 μg / mL and paclitaxel solution with a concentration of 0.6 μg / mL are mixed.

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