A medicament, liposome, preparation method and use for treating tumors

Abstract

The application belongs to the technical field of medicine, and particularly relates to a medicine for treating tumors, a liposome, a preparation method and use; comprising cantharidin and staurosporine, and the weight ratio of the cantharidin and the staurosporine is 350-2500:1; the application effectively improves the effect of treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a drug for treating tumors, liposomes, preparation methods, and uses. Background Technology

[0002] Cancer treatment drugs are generally divided into three types: conventional chemotherapy drugs, targeted drugs, and immunotherapy drugs.

[0003] 1. Conventional chemotherapy drugs:

[0004] Their mechanisms of action mainly include blocking DNA replication, disrupting DNA structure, affecting RNA transcription, inhibiting protein synthesis, preventing cell division, and blocking tumor angiogenesis. Examples include taxanes such as docetaxel and paclitaxel; platinum-based drugs such as oxaliplatin and cisplatin; fluorouracil-based drugs such as capecitabine and tegafur; and anthracycline-based drugs such as doxorubicin and pirarubicin.

[0005] 2. Targeted therapies:

[0006] These drugs primarily exert their inhibitory or killing effects on tumor cells by specifically binding to oncogenic sites within tumor cells and to targets associated with tumor cell proliferation and angiogenesis, while having minimal impact on normal cells and tissues. Examples include gefitinib, sorafenib, and tyrosine phosphatase inhibitors.

[0007] 3. Immunotherapy drugs:

[0008] Their mechanism of action is to help patients enhance their individual immune system and immune cells, thereby enabling them to automatically identify cancer cells in the body and eliminate them under the action of the immune system. Examples include pembrolizumab, nivolumab, and toripalimab.

[0009] Cantharidin is a compound derived from insects in nature and possesses a variety of biological activities. In clinical studies, cantharidin is mainly used to treat cancer, showing efficacy against malignant tumors such as liver cancer, lung cancer, stomach cancer, and colorectal cancer.

[0010] The efficacy of cantharidin needs further improvement. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a drug, liposome, preparation method and use for treating tumors, so as to effectively improve the effect of tumor treatment.

[0012] This invention provides a drug for treating tumors, comprising cantharidin and asteroidin, wherein the weight ratio of cantharidin to asteroidin is 390-2350:1, preferably 390-2350:1, preferably 400-1200:1, and preferably 465-1176:1.

[0013] Preferably, the weight ratio of cantharidin to asteroidin is 465-588:1.

[0014] More preferably, the weight ratio of cantharidin to asteroidin is 588:1.

[0015] Preferably, the drug for treating tumors consists of cantharidin and asteroidin.

[0016] This invention provides a liposome comprising the aforementioned drug for treating tumors, preferably further comprising folic acid.

[0017] Preferably, the mass concentration of cantharidin in the liposomes is 0.05-0.5 μg / mL, more preferably 0.05-0.2 μg / mL, and even more preferably 0.05-0.1 μg / mL.

[0018] Preferably, the liposomes further include lecithin, cholesterol, phospholipid-polyethylene glycol, and phospholipid-polyethylene glycol-folic acid.

[0019] This invention provides a method for preparing liposomes, comprising the following steps: mixing lecithin, cholesterol, phospholipid-polyethylene glycol, phospholipid-polyethylene glycol-folic acid, cantharidin, and astrocytocin and dissolving them in a solvent; removing the solvent under reduced pressure; adding a buffer solution; stirring; and filtering to obtain the liposomes.

[0020] This invention provides a use of the liposomes for preparing drugs to treat tumors, specifically liver cancer, lung cancer, gastric cancer, colorectal cancer, or stomach cancer. Preferably, the tumor is liver cancer.

[0021] The beneficial effect of this invention is that both cantharidin and asteroidin have anti-tumor effects, and the higher their concentration, the better the effect. Based on cantharidin, this invention selects asteroidin for combination. The inventors have discovered that the combination of cantharidin and asteroidin, especially at a cantharidin concentration of 0.2 μg / mL and an asteroidin concentration of 0.34 ng / mL, can significantly enhance the anti-tumor effect.

[0022] Cantharidin has certain toxic side effects. This application combines astrosarcine and cantharidin, and it was found that, under the same dosage of cantharidin, the inhibitory effect on tumor cells can be significantly improved, that is, under the same efficacy, the toxic side effects on normal cells can be significantly reduced. Attached Figure Description

[0023] Figure 1 The inhibitory effects of different liposomes on the proliferation of H22 cells in embodiments of the present invention are shown.

[0024] Figure 2The present invention presents experimental results of different liposomes inducing apoptosis in liver cancer cells at different stages.

[0025] Figure 3 The results show the experimental effects of different liposomes inducing apoptosis in liver cancer cells according to embodiments of the present invention. Detailed Implementation

[0026] Example 1

[0027] Preparation method of FA / CTD-STS / Lip

[0028] Liposomes were prepared using a thin-film dispersion method. 65 mg of soybean lecithin, 5 mg of cholesterol, 12 mg of DSPE-PEG2000 (phospholipid-polyethylene glycol 2000), 2 mg of CTD (cantharidin), 5 mg of DSPE-PEG2000-FA (phospholipid-polyethylene glycol 2000-folic acid), and 340 μL of STS (astrosporin) (10 μg / mL) were placed in a 250 mL round-bottom flask and dissolved in 10 mL of organic solvent (chloroform:methanol (volume ratio 3:1)). The organic solvent was removed by rotary evaporation under reduced pressure in a 55 °C water bath. Add 10 mL of PBS buffer and hydrate at 55 °C for 1 h with stirring. Sonicate with the probe for 10 min (total power 100 W, sonication for 2 s, interval for 2 s). Filter through a 0.22 μm microporous membrane to obtain FA / CTD-STS / Lip with a volume of 10 mL. The mass concentration of CTD in FA / CTD-STS / Lip was determined to be 0.1 mg / mL by high performance liquid chromatography.

[0029] Using the method of Example 1, 2 mg of CTD (cantharidin) was removed from the raw material to prepare FA / STS / Lip. The concentration of FA / STS / Lip is equivalent to the mass concentration of STS corresponding to a CTD mass concentration of 0.1 mg / mL in FA / CTD-STS / Lip.

[0030] Using the method of Example 1, 340 μL of raw material STS (astrosporin) (10 μg / mL) was removed to prepare FA / CTD / Lip. The mass concentration of CTD in FA / CTD-STS / Lip was determined to be 0.1 mg / mL by high performance liquid chromatography.

[0031] Using the method of Example 1, 5 mg of DSPE-PEG2000-FA (phospholipid-polyethylene glycol 2000-folic acid) was removed from the raw material to prepare CTD-STS / Lip. The mass concentration of CTD in CTD-STS / Lip was determined to be 0.1 mg / mL by high performance liquid chromatography.

[0032] Example 2

[0033] Inhibitory effects of CTD and STS on the proliferation of H22 cells

[0034] The inhibitory effects of CTD and STS on the proliferation of H22 cells were determined using the CCK-8 (Cell Counting Kit-8) method.

[0035] H22 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 H22 cells / well were cultured for 15 h in 100 μL of 10% FBS (fetal bovine serum) and 1% penicillin-dextrose antibody-containing RPMI 1640 medium. The supernatant was not discarded, and different concentrations of 0.2, 0.4, 1, 2, 4, 8, and 10 μg / mL CCT D were directly added. Since the 100 μL of complete medium added at the time of inoculation was not discarded, the final concentrations were 0.1, 0.2, 0.5, 1, 2, 4, and 5 μg / mL. Each group was divided into 6 replicates. After incubation for 48 h, 20 μL of CCK8 solution was added to each well, and after another 1 h of incubation, the absorbance was measured at a wavelength of 450 nm using a microplate reader to calculate the H22 cell viability. The results are shown in Table 1.

[0036] H22 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-dextrose antibody (PDDA) for 15 h. The supernatant was not discarded, and different concentrations of 0.2, 1, 2, 50, 100, 200, and 1000 ng / mL LTS were directly added. Since the initial 100 μL of complete medium was not discarded, the final concentrations were 0.1, 0.5, 1, 25, 50, 100, and 500 ng / mL. Six replicates were used per group. Cells were incubated for 48 h, and 20 μL of CCK8 solution was added to each well. After another 1 h of incubation, the absorbance was measured at 450 nm using a microplate reader, and the viability of H22 cells was calculated. The results are shown in Table 2.

[0037] Table 1. Inhibitory effect of CTD on H22 cell proliferation

[0038]

[0039] Table 2. Inhibitory effect of STS on H22 cell proliferation

[0040]

[0041] Exploring the Collaboration Ratio of CTD and STS

[0042] Based on the IC50 of CTD and STS, and using the Combination Index (CI) to prove that the two drugs have a synergistic effect, CI<1 indicates that the two drugs have a synergistic effect, CI=1 indicates that the two drugs have an additive effect, and CI>1 indicates that the two drugs have an antagonistic effect.

[0043] H22 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 H22 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-dextrose antibody for 15 h. The supernatant was not discarded; a physical mixture containing 1.4 μg / mL CTD and 1.2 ng / mL STS was directly added to each well, with 6 replicates per group. Cells were incubated for 48 h, followed by incubation with 20 μL of CCK8 solution per well for another 1 h. The absorbance was measured at 450 nm using a microplate reader to calculate the H22 cell viability, and CI was calculated using Compu Syn software. The results are shown in Table 3. The results indicate that the mass concentrations of CTD and STS at 0.7 μg / mL:0.6 ng / mL have a good synergistic effect.

[0044] Table 3. Calculation of CI Values ​​for CTD and STS

[0045] CTD (μg / mL): STS (ng / mL) Cell viability CI 0.5:0.85 0.12167539 0.22246 0.7:0.6 16.7813658 0.34115 0.7:0.5 49.514734 0.66213 0.7:0.3 37.956591 0.52864 0.7:0.45 40.374096 0.56087

[0046] Example 3

[0047] H22 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells per well were incubated at 37°C in a 5% CO2 incubator for 15 h. Different concentrations of FA / STS / Lip, FA / CTD / Lip, CTD-STS / Lip, and FA / CTD-STS / Lip (with a fixed CTD concentration of 0.2 μg / mL and STS concentrations of 0.34, 0.43, 0.51, and 0.6 ng / mL) were added to each well in 6 replicates. After incubation for 48 h, 20 μL of LCK-8 solution was added to each well for another 1 h. Finally, the absorbance was measured at 450 nm using a microplate reader, and the cell count was calculated, as shown in Table 4.

[0048] (1) The preparation method of liposomes with a concentration ratio of 0.2 μg / mL LCT D: 0.17 ng / mL LCT D is as follows:

[0049] Liposomes were prepared using a thin-film dispersion method. 65 mg of soybean lecithin, 5 mg of cholesterol, 12 mg of DSPE-PEG2000 (phospholipid-polyethylene glycol 2000), 2 mg of CTD (cantharidin), 5 mg of DSPE-PEG2000-FA (phospholipid-polyethylene glycol 2000-folic acid), and 170 μL of STS (astrosporin) (10 μg / mL) were accurately weighed and placed in a 250 mL round-bottom flask, and dissolved in 10 mL of organic solvent (chloroform:methanol (volume ratio 3:1)). The organic solvent was removed by rotary evaporation under reduced pressure in a 55 °C water bath. Add 10 mL of PBS buffer and hydrate at 55 °C for 1 h with stirring. Sonicate with the probe for 10 min (total power 100 W, sonication for 2 s, interval for 2 s). Filter through a 0.22 μm microporous membrane to obtain FA / CTD-STS / Lip. Dilute according to the method in Table 5 to obtain liposomes with a concentration ratio of 0.2 μg / mCTD:0.17 ng / mLSTS.

[0050] (2) The preparation method of liposomes with a concentration ratio of 0.2 μg / mL LCT D: 0.34 ng / mL LCT D is as follows:

[0051] Liposomes were prepared using a thin-film dispersion method. 65 mg of soybean lecithin, 5 mg of cholesterol, 12 mg of DSPE-PEG2000 (phospholipid-polyethylene glycol 2000), 2 mg of CTD (cantharidin), 5 mg of DSPE-PEG2000-FA (phospholipid-polyethylene glycol 2000-folic acid), and 340 μL of STS (astrosporin) (10 μg / mL) were accurately weighed and placed in a 250 mL round-bottom flask, and dissolved in 10 mL of organic solvent (chloroform:methanol (volume ratio 3:1)). The organic solvent was removed by rotary evaporation under reduced pressure in a 55 °C water bath. Add 10 mL of PBS buffer and hydrate at 55 °C for 1 h with stirring. Sonicate with the probe for 10 min (total power 100 W, sonication for 2 s, interval for 2 s). Filter through a 0.22 μm microporous membrane to obtain FA / CTD-STS / Lip. Dilute according to the method in Table 5 to obtain liposomes with a concentration ratio of 0.2 μg / mCTD:0.17 ng / mLSTS.

[0052] (3) The preparation method of liposomes with a concentration ratio of 0.2 μg / mL LCT D: 0.43 ng / mL LCT D is as follows:

[0053] Liposomes were prepared using a thin-film dispersion method. 65 mg of soybean lecithin, 5 mg of cholesterol, 12 mg of DSPE-PEG2000 (phospholipid-polyethylene glycol 2000), 2 mg of CTD (cantharidin), 5 mg of DSPE-PEG2000-FA (phospholipid-polyethylene glycol 2000-folic acid), and 430 μL of STS (astrosporin) (10 μg / mL) were accurately weighed and placed in a 250 mL round-bottom flask, and dissolved in 10 mL of organic solvent (chloroform:methanol (volume ratio 3:1)). The organic solvent was removed by rotary evaporation under reduced pressure in a 55 °C water bath. Add 10 mL of PBS buffer and hydrate at 55 °C for 1 h with stirring. Sonicate with the probe for 10 min (total power 100 W, sonication for 2 s, interval for 2 s). Filter through a 0.22 μm microporous membrane to obtain FA / CTD-STS / Lip. Dilute according to the method in Table 5 to obtain liposomes with a concentration ratio of 0.2 μg / mCTD:0.17 ng / mLSTS.

[0054] (4) The preparation method of liposomes with a concentration ratio of 0.2 μg / mL LCT D: 0.51 ng / mL LCT D is as follows:

[0055] Liposomes were prepared using a thin-film dispersion method. 65 mg of soybean lecithin, 5 mg of cholesterol, 12 mg of DSPE-PEG2000 (phospholipid-polyethylene glycol 2000), 2 mg of CTD (cantharidin), 5 mg of DSPE-PEG2000-FA (phospholipid-polyethylene glycol 2000-folic acid), and 510 μL of STS (astrosporin) (10 μg / mL) were accurately weighed and placed in a 250 mL round-bottom flask, and dissolved in 10 mL of organic solvent (chloroform:methanol (volume ratio 3:1)). The organic solvent was removed by rotary evaporation under reduced pressure in a 55 °C water bath. Add 10 mL of PBS buffer and hydrate at 55 °C for 1 h with stirring. Sonicate with the probe for 10 min (total power 100 W, sonication for 2 s, interval for 2 s). Filter through a 0.22 μm microporous membrane to obtain FA / CTD-STS / Lip. Dilute according to the method in Table 5 to obtain liposomes with a concentration ratio of 0.2 μg / mCTD:0.17 ng / mLSTS.

[0056] (5) The preparation method of liposomes with a concentration ratio of 0.2 μg / mL LCT D: 0.60 ng / mL LCT D is as follows:

[0057] Liposomes were prepared using a thin-film dispersion method. 65 mg of soybean lecithin, 5 mg of cholesterol, 12 mg of DSPE-PEG2000 (phospholipid-polyethylene glycol 2000), 2 mg of CTD (cantharidin), 5 mg of DSPE-PEG2000-FA (phospholipid-polyethylene glycol 2000-folic acid), and 600 μL of STS (astrosporin) (10 μg / mL) were accurately weighed and placed in a 250 mL round-bottom flask, and dissolved in 10 mL of organic solvent (chloroform:methanol (volume ratio 3:1)). The organic solvent was removed by rotary evaporation under reduced pressure in a 55 °C water bath. Add 10 mL of PBS buffer and hydrate at 55 °C for 1 h with stirring. Sonicate with the probe for 10 min (total power 100 W, sonication for 2 s, interval for 2 s). Filter through a 0.22 μm microporous membrane to obtain FA / CTD-STS / Lip. Dilute according to the method in Table 5 to obtain liposomes with a concentration ratio of 0.2 μg / mCTD:0.17 ng / mLSTS.

[0058] Table 4. Survival rate data of H22 cells for liposomes with different concentration ratios.

[0059]

[0060]

[0061] A series of concentration ratios were tested using FA / CTD-STS / Lip as the main component. The table shows that the optimal inhibition rate can be achieved by using 0.2 μg / mL CTD and 0.34 ng / mL STS in combination, indicating that this ratio can reduce cell viability.

[0062] Example 4

[0063] The inhibitory effects of FA / STS / Lip, FA / CTD / Lip, CTD-STS / Lip, and FA / CTD-STS / Lip on the proliferation of H22 cells were determined using the CCK-8 assay.

[0064] H22 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3Cells were cultured in RPMI 1640 medium (100 μL of 10% FBS (fetal bovine serum) and 1% penicillin-dextrose antibody per well) for 15 h. The supernatant was not discarded, and different concentrations of FA / STS / Lip, FA / CTD / Lip, CTD-STS / Lip, and FA / CTD-STS / Lip were directly added (0.1, 0.2, 0.4, 0.6, 0.8, 1 μg / mL). Since the 100 μL of complete medium added at the time of inoculation was not discarded, the final concentrations were 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 μg / mL, respectively. Six replicates were made per group, and the cells were incubated for 48 h. 20 μL of CCK8 solution was added to each well, and the cells were incubated for another 1 h. The absorbance was measured at 450 nm using a microplate reader, and the viability of H22 cells was calculated. Results are shown in Table 6. Figure 1 As shown.

[0065] H22 cells are suspension cells, so when processing them, there is no need to discard the supernatant; liposomes of different mass concentrations can be added directly.

[0066] The stock solution of FA / STS / Lip, FA / CTD / Lip, CTD-STS / Lip and FA / CTD-STS / Lip is 0.1 mg / mL, and the specific preparation method is shown in Example 1.

[0067] The preparation method of each liposome at 8 μg / mL is as follows: take 160 μL of 0.1 mg / mL FA / STS / Lip, FA / CTD / Lip, CTD-STS / Lip and FA / CTD-STS / Lip stock solution and add 1840 μL of 10% FBS complete culture medium. The specific configuration of each concentration of liposome is shown in Table 5.

[0068] Table 5. Liposome samples prepared using cantharidin concentration as the standard.

[0069]

[0070] Table 6. Inhibitory effects of different liposomes on H22 cell proliferation

[0071]

[0072] As shown in Table 6 and Figure 1As shown, after 48 hours of treatment with different drug formulations at different concentrations, all drug formulations exhibited concentration-dependent cytotoxicity against H22 cells. Compared with other single-load CTD or STS, the combined strategy based on CTD and STS promoted the anti-proliferative effect. The survival rate of hepatocellular carcinoma cells in the FA / CTD-STS / Lip group was significantly lower than that in the FA / STS / Lip and FA / CTD / Lip groups, exhibiting stronger cytotoxicity, indicating that CTD and STS synergistically can significantly inhibit the proliferation of hepatocellular carcinoma cells. The cytotoxicity of the FA / CTD-STS / Lip group was significantly stronger than that of the CTD-STS / Lip group, indicating that folic acid-mediated CTD-STS nanoliposome co-delivery significantly reduced cell survival.

[0073] Example 5

[0074] To detect apoptosis, 2 × 10⁶ H22 cells were used. 6 Cells were seeded per well in 6-well plates and cultured for 15 h. Cells were then treated with CTD-STS, CTD-STS / Lip, FA / CTD / Lip, FA / CTD / Lip, and FA / CTD-STS / Lip (final CTD concentration 0.3 μg / mL) for 48 h. Cells were then collected by centrifugation into 10 mL centrifuge tubes and washed twice with PBS to remove free drugs. 100 μL of diluted 1×Annexin V Binding Buffer was added to each centrifuge tube, and the mixture was pipetted thoroughly. 2.5 μL of Annexin V-FITC and 2.5 μL of PI were added. The mixture was gently vortexed and incubated at room temperature in the dark for 20 min. 400 μL of diluted 1×Annexin V Binding Buffer was then added, and the sample was mixed. The apoptosis rate of H22 cells was detected by flow cytometry.

[0075] Results and Analysis

[0076] Flow cytometry was used to quantitatively analyze the apoptosis induced by FA / STS / Lip, FA / CTD / Lip, CTD-STS / Lip, and FA / CTD-STS / Lip in H22 cells. Figure 2-3As shown, the apoptosis rates induced by FA / STS / Lip, FA / CTD / Lip, CTD-STS / Lip, and FA / CTD-STS / Lip were 7.36%, 23.48%, 18.99%, and 34.80%, respectively. Compared with other control groups, the FA / CTD-STS / Lip group increased the apoptosis level of H22 cells, indicating that the combined application of CTD and STS can effectively induce apoptosis in liver cancer cells. The apoptosis rate in the FA / CTD-STS / Lip group was significantly higher than that in the CTD-STS / Lip group, indicating that folic acid modification can enhance the apoptosis induced by CTD-STS / Lip in H22 cells.

[0077] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0078] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A drug for treating tumors, characterized in that, The active ingredients consist of cantharidin and asteroidin, with a weight ratio of 588:1, and the tumor is liver cancer.

2. A liposome, characterized in that, Including the tumor treatment drug as described in claim 1.

3. The liposomes as described in claim 2, characterized in that, It also includes lecithin, cholesterol, phospholipid-polyethylene glycol, and phospholipid-polyethylene glycol-folic acid.

4. A method for preparing liposomes as described in claim 3, characterized in that, Lecithin, cholesterol, phospholipid-polyethylene glycol, phospholipid-polyethylene glycol-folic acid, cantharidin, and astrocytocin were mixed and dissolved in a solvent. The solvent was removed under reduced pressure, buffer solution was added, the mixture was stirred, and then filtered to obtain the liposomes.

5. Use of the liposomes as described in claim 2 or 3, characterized in that, The liposomes are used to prepare drugs for treating liver cancer.

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