Application of combination of tripterine and chloroquine in preparation of antitumor drugs

Abstract

The invention discloses an application of combination of tripterine and chloroquine in preparation of an antitumor drug. The antitumor drug comprises tripterine and chloroquine. The combined application of the tripterine and the chloroquine has stronger anti-tumor activity than a single component, and the combined pharmaceutical composition can significantly enhance the ICD effect, prolong the lifetime of tumor-bearing mice, and provide a new method and thought for cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to the application of triptolide and chloroquine in the preparation of antitumor drugs. Background Technology

[0002] Currently, the main treatments for cancer include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, with targeted therapy becoming a research hotspot in recent years. Targeted therapy can identify and attack molecules or signaling pathways specific to cancer cells, with less impact on normal cells, thus improving treatment efficacy while reducing side effects. Cell membrane biomimetic nanoparticles offer a solution for efficient drug delivery to tumor sites, but developing technologies that target tumor cell mitochondria remains a major challenge. Reactive oxygen species (ROS) play a crucial role in the occurrence and development of cancer; persistently elevated ROS levels can inhibit tumor cell proliferation, invasion, and metastasis. However, these can be effectively counteracted by the autophagy pathway, a self-protective mechanism that mitigates oxidative damage and acts as a survival-promoting pathway in cancer cells. Therefore, it is necessary to develop combined strategies that inhibit autophagy and further expand ROS targeting to mitochondria to restore and enhance immunogenic cell death (ICD)-mediated anti-tumor therapy.

[0003] Tripterygium wilfordii root bark is a pentacyclic triterpenoid compound derived from the root bark of the traditional Chinese medicine Tripterygium wilfordii. Tripterygium wilfordii can inhibit the development of prostate cancer, liver cancer, breast cancer, and gastric cancer by inducing apoptosis, autophagy, angiogenesis, and tumor metastasis. It also shows great potential in tumor immunotherapy, enhancing anti-tumor immune responses by inducing endoplasmic reticulum stress and ROS production in tumor cells, leading to ICD. Chloroquine is a lysosomal target agent that inhibits autophagy by suppressing lysosomal acidification. It can be used as a sensitizer for radiotherapy and chemotherapy to improve anti-cancer efficacy, inhibit cell growth and differentiation, and induce apoptosis. However, detailed studies on the combined use of these two drugs in tumor treatment and application are currently scarce. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide an application of triptolide and chloroquine in the preparation of antitumor drugs. Specifically, the combination of triptolide and chloroquine can inhibit the autophagy level of 4T1 cells, increase ROS, induce ICD, and the combined treatment of triple-negative breast cancer has significant efficacy.

[0005] Specifically, this can be achieved through the following technical solutions:

[0006] Application of triptolide and chloroquine in the preparation of antitumor drugs.

[0007] Furthermore, the triptolide is a triptolide monomer, and the chloroquine is a chloroquine monomer.

[0008] Furthermore, the molar ratio of triptolide to chloroquine is 1:0.1-10, preferably 1:1.

[0009] Furthermore, the mitochondrial membrane of 4T1 cells was extracted using sucrose density gradient centrifugation.

[0010] Furthermore, the drug is any pharmaceutically acceptable dosage form, including liposomes, nanoemulsions, nanoparticles, microcapsules, microspheres, micropellets, solid dispersions, preferably nanoparticles.

[0011] Furthermore, the average particle size of the self-assembled nanoparticles of triptolide and chloroquine is preferably 50-400 nm, more preferably 100-200 nm.

[0012] The self-assembled nanoparticles of triptolide and chloroquine further include a chelating agent, a co-solvent, and water. The chelating agent is preferably at least one selected from polyethylene and pyrrolidone, ethylenediamine, and ethylenediaminetetraacetic acid, more preferably polyethylene and pyrrolidone. The co-solvent is preferably at least one selected from n-butanol, ethanol, polyethylene glycol 400, glycerol, and 1,2-propanediol, more preferably ethanol.

[0013] The method for preparing the self-assembled nanoparticles of triptolide and chloroquine includes the following steps:

[0014] (1) Mix the chelating agent, solubilizer and tripterygium tincture evenly at a stirring speed of 500 r / min;

[0015] (2) Chloroquine aqueous solution was added dropwise at a stirring speed of 500 r / min to obtain triptolide and chloroquine self-assembled nanoparticles.

[0016] Furthermore, nanoparticles were co-extruded to coat the mitochondrial membrane of 4T1 cells.

[0017] Furthermore, the drug also includes any one or more combinations of pharmaceutically acceptable pharmaceutical excipients.

[0018] Furthermore, the pharmaceutical excipients include calcium chloride and polyvinylpyrrolidone.

[0019] Furthermore, the drug is administered via any one of the following methods: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration, with intravenous injection being preferred.

[0020] Furthermore, the tumor includes any one of melanoma, gastric cancer, lung cancer, pancreatic cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, skin cancer, prostate cancer, thyroid cancer, leukemia, lymphoma, esophageal cancer, oral cancer, intestinal cancer, nasal cancer, and head and neck cancer, with breast cancer being preferred.

[0021] The combination of triptolide and chloroquine provided by this invention has a synergistic effect on enhancing tumor immunity. The nanoparticles provided by this invention effectively increase the solubility of triptolide and chloroquine on the one hand, and on the other hand, by inhibiting compensatory mitophagy, they relieve their protective effect, effectively increase ROS, synergistically trigger ICD, and enhance the immune killing effect on tumor-bearing mice. Attached Figure Description

[0022] Figure 1 This is a transmission electron microscope image of the self-assembled nanoparticles of triptolide and chloroquine of this invention.

[0023] Figure 2 This is a particle size distribution diagram of the self-assembled nanoparticles of triptolide and chloroquine of the present invention;

[0024] Figure 3 The results show the stability of the self-assembled nanoparticles of triptolide and chloroquine in this invention.

[0025] Figure 4 To investigate the in vitro release rate of the self-assembled nanoparticles of triptolide and chloroquine of this invention;

[0026] Figure 5 This diagram illustrates the therapeutic effect of self-assembled triptolide and chloroquine nanoparticles on an animal model of breast cancer in situ tumors. A represents a schematic diagram of the nanoparticle administration regimen; B represents the change in tumor luminescence intensity in tumor-bearing mice; and C represents the BLI (Bioluminescence Intensity) of different drug treatment groups at different time points. rel Ratio curve; D is the change in tumor volume in tumor-bearing mice; E is the tumor removed from tumor-bearing mice on day 20 after different drug treatments; F is the tumor inhibition rate after different drug treatments; G is the tumor weight removed from tumor-bearing mice on day 20 after drug treatment; H is the survival time of tumor-bearing mice.

[0027] Figure 6 To detect changes in ICD markers in tumor tissues of tumor-bearing mice after different drug treatments using flow cytometry; where A represents changes in dendritic cells (DCs) in tumor tissues after different drug treatments; and B represents changes in CD8+. + Changes in T cells; C and F represent CD8+. + Changes in T cell functional markers GZMB and IFN-γ; D represents CD4+. +Changes in T cells; E represents changes in macrophages; G represents changes in Treg cells; H represents changes in myeloid-derived suppressor cells (MDSCs) in tumor tissue;

[0028] Figure 7 The image shows the H&E staining results (scale bar is 100μm) of the major organs and tumor tissues of tumor-bearing mice, as well as the blood biochemical indicators.

[0029] Figure 8 The expression level of autophagy in tumor tissue was determined by PCR experiment (Note: Compared with the control group, *P<0.05, **P<0.01, ***P<0.001; #P<0.05, ##P<0.01, ###P<0.001; Compared with the triptolide and chloroquine self-assembled nanoparticle group, P<0.05, &&P<0.01, &&&P<0.001. The t-test was used to assess the significance of the difference, and the results are expressed as mean ± SD). Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments in order to better understand the technical solution.

[0031] Example 1: Preparation of self-assembled nanoparticles of triptolide and chloroquine

[0032] Formula 1: The molar ratio of triptolide and chloroquine self-assembled nanoparticles is 1:1.

[0033]

[0034] Formula 2: The molar ratio of triptolide and chloroquine self-assembled nanoparticles is 1:10.

[0035]

[0036] Formula 3: The molar ratio of triptolide and chloroquine self-assembled nanoparticles is 1:0.1.

[0037]

[0038] Weigh out the prescribed amounts of triptolide, calcium chloride, and polyoxyethylene pyrrolidone for each of the above formulations and place them in a 5ml vial. Stir at 500 rpm for 24 hours at room temperature. Slowly add an aqueous solution of chloroquine to form self-assembled nanoparticles. Continue stirring at 500 rpm for 12 hours. Add the same volume of pure water and stir for 6 hours. Remove impurities at 500 rpm. Collect the supernatant at 10,000 rpm and sonicate for 2 minutes. Finally, co-extract the mitochondrial membrane and supernatant using a liposome extruder.

[0039] The particle size analysis results of self-assembled nanoparticles of triptolide and chloroquine with different ratios were similar. The average particle size of nanoparticles with a triptolide to chloroquine ratio of 1:10 was (193.8±0.31) nm, the average particle size of nanoparticles with a triptolide to chloroquine ratio of 1:1 was (165.8±0.44) nm, and the average particle size of nanoparticles with a triptolide to chloroquine ratio of 1:0.01 was (224.7±0.80) nm. The preferred ratio of triptolide to chloroquine was 1:1 for subsequent experiments.

[0040] Example 2: Characterization of self-assembled nanoparticles of triptolide and chloroquine

[0041] The prepared sample was diluted to a certain extent, and its average particle size and Zeta potential were measured using a laser particle size analyzer. After the sample was dropped onto a copper grid, it was negatively stained with 2% (w / v) phosphotungstic acid, and the morphology and particle size of the nanoemulsion were observed by transmission electron microscopy (TEM).

[0042] Test results as follows Figure 1 and Figure 2 As shown: Electron microscopy images stained with 2% phosphotungstic acid reveal the self-assembled nanoparticles of triptolide and chloroquine. Figure 1 The surface is intact, the particle size distribution is uniform, and the particle size is around 160 nm. The self-assembled nanoparticles of triptolide and chloroquine (measured by a Malvern laser particle size analyzer) have intact surfaces, uniform particle size distribution, and are all around 160 nm in size. Figure 2 The particle size distribution shows that the nanoparticles have a uniform particle size distribution, with an average particle size of approximately (165.8±0.44) nm, a polydispersity index (PDI) of 0.115±0.02, and a zeta potential of (-39±1.35) mV.

[0043] Example 3: Stability Study of Self-Assembled Nanoparticles of Tripterygium wilfordii and Chloroquine

[0044] The effect of heating-cooling cycles on the stability of self-assembled nanoparticles of triptolide and chloroquine was investigated. After the heat-cooling cycle test, there was no significant difference in particle size and PDI between the nanoparticles and the untreated ones. Figure 3 Tripterygium wilfordii and chloroquine self-assembled nanoparticles exhibit good stability at 4℃ and 25℃.

[0045] Example 4: In vitro release rate study of self-assembled nanoparticles containing triptolide and chloroquine

[0046] The in vitro release rates of self-assembled nanoparticles of triptolide and chloroquine were investigated. Three parallel aliquots of 1 mL each of triptolide and chloroquine nanoparticles were prepared and placed in pre-treated dialysis bags, with both ends tied tightly with cotton thread. The bags were then placed in 30 mL of phosphate-buffered saline (PBS) at pH 5.0 and pH 7.4, respectively, ensuring the bags were completely submerged. The mixture was incubated at 37°C with constant shaking (75 rpm). At the end of 0.1, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, and 24 hours, 2 mL of dialysis solution was aspirated from the centrifuge tube, and the remaining solution was replenished with an equal volume of release medium at the same pH. After filtration through a 0.45 μm microporous membrane, the triptolide content was determined by UV spectrophotometry, and a cumulative release curve was plotted. Figure 4 The results showed that triptolide and chloroquine self-assembled nanoparticles encapsulating the 4T1 mitochondrial membrane were released more quickly under acidic conditions.

[0047] Example 5: Antitumor effect of self-assembled nanoparticles of triptolide and chloroquine

[0048] Mouse 4T1 breast cancer cells in logarithmic growth phase were digested with 0.25% trypsin, centrifuged at 900 rpm for 3 minutes, and the cell concentration was adjusted to an appropriate level. The cells were then placed on ice for later use. After mixing the cells, 7 × 10⁶ cells were drawn using a syringe. 5 A tumor was implanted in the first pair of mammary fat pads in the left axilla of a mouse. Figure 5 A).

[0049] Mice were randomly divided into five groups: Control group, triptolide group (Cel), triptolide calcium chloride group (Cel-Ca), triptolide calcium chloride chloroquine group (Cel-Ca / CQ), and triptolide and chloroquine self-assembled nanoparticle coating group (Cel-Ca / CQ@OMM), with 5-6 mice in each group. The drugs were administered daily via tail vein injection, and changes in tumor luminescence intensity were detected using in vivo fluorescence technology in mice. Figure 5 B) Calculate the BLI of different drug treatment groups at different time points. rel Ratio curve ( Figure 5 C). Measure the tumor diameter every two days and calculate the tumor volume. Figure 5 D), tumor-bearing mice were sacrificed on day 20 after drug administration, and the dissected tumors were obtained. Figure 5 E), calculate the tumor inhibition rate after different drug treatments ( Figure 5 F), recording the tumor weight after tumor removal from tumor-bearing mice (F). Figure 5 G) and the survival time of tumor-bearing mice ( Figure 5 H).

[0050] Experimental results showed that, compared with Control and Cel, the self-assembled nanoparticles of triptolide and chloroquine significantly reduced breast cancer growth, and the combined application of the 4T1 mitochondrial membrane matrix exhibited stronger antitumor activity than the single component. In summary, the self-assembled nanoparticles of triptolide and chloroquine can also significantly reduce tumor growth in cancer cells such as melanoma, gastric cancer, lung cancer, pancreatic cancer, liver cancer, kidney cancer, cervical cancer, uterine cancer, skin cancer, prostate cancer, thyroid cancer, leukemia, lymphoma, esophageal cancer, oral cancer, colorectal cancer, nasal cancer, and head and neck cancer.

[0051] Example 6: ICD index analysis in tumor tissue

[0052] Tumor immunogenic cell death (ICD), a process that releases tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), can significantly promote dendritic cell maturation and antigen presentation, thereby activating T-cell anti-tumor immunity.

[0053] Approximately 100 mg of tumor tissue was collected and placed in a centrifuge tube containing PBS. The tumor was cut into small pieces, digestive enzyme solution was added, and single cells were separated by grinding. After centrifugation, the cells were collected, and red blood cell lysis buffer was added to lyse the red blood cells. The cells were washed twice with PBS, centrifuged, and resuspended to obtain a single-cell suspension. CD4 count was analyzed by flow cytometry. + T cells, CD8 + T cell, Treg, DCs, MDSCs, M1 / M2 ratio status, such as Figure 6 As shown in the figure. A represents the changes in dendritic cells (DCs) in tumor tissue after treatment with different drugs; B represents the changes in CD8+ cells. + Changes in T cells; C and F represent CD8+. + Changes in T cell functional markers GZMB and IFN-γ; D represents CD4+. + Changes in T cells; E represents changes in macrophages; G represents changes in Treg cells; H represents changes in myeloid-derived suppressor cells (MDSCs) in tumor tissue.

[0054] Experimental results showed that, compared with the control group, the combined application of triptolide and chloroquine self-assembled nanoparticles significantly increased CD4 levels. + T cells, CD8 + T cells, DCs, and the M1 / M2 ratio were all significantly upregulated, while Tregs and MDSCs were significantly downregulated, indicating that the combined application of triptolide and chloroquine self-assembled nanoparticles can significantly induce ICD and enhance the efficacy of tumor immunotherapy.

[0055] Example 7: H&E staining analysis of major organs and tumor tissues and blood biochemical indicators in tumor-bearing mice

[0056] After drug administration, organs (heart, liver, spleen, lung, and kidney) and tumor tissues from tumor-bearing mice were collected, fixed in 4% paraformaldehyde, and immediately prepared into paraffin sections. The paraffin sections were dewaxed, stained with hematoxylin for 5 min, differentiated with hydrochloric acid and ethanol for 5 s, stained with eosin for 5 min, and then dehydrated using a gradient of ethanol and xylene. Finally, they were mounted with neutral resin. The sections were observed and photographed under a 200x microscope. Figure 7 A). In each drug treatment group, the cells of the major organs (heart, liver, spleen, lung, and kidney) were relatively intact, with no significant enlargement of the nuclei and no significant damage. In the PBS group, tumor cells were clustered and arranged in cords, with large, deeply stained nuclei and visible red-stained nucleoli. The tumor cells varied in size and shape, and there was basically no obvious apoptosis or necrosis. In the drug-treated group, the tumor cells showed focal necrosis, with darker nuclear staining, reduced density, and visible cavities, indicating that some tumor cells underwent apoptosis and necrosis.

[0057] After drug administration, blood was collected from the eyeballs of tumor-bearing mice. 1.5 mL of blood was collected in an EP tube, placed on ice for 30 min, and then centrifuged (3500 rpm, 10 min, 4℃). The supernatant was collected for detecting liver and kidney function indicators. The results are shown in the figure. Figure 7 B) The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (BUN) in tumor-bearing mice after administration did not show significant changes compared to the PBS group, indicating that none of the administration groups had significant liver and kidney toxicity.

[0058] Example 8: Detection of autophagy levels in tumor tissue

[0059] After drug administration, tumor tissue from tumor-bearing mice was collected, and 200 μL of Trizol reagent was added. The sample was ground and brought to a final volume of 800 μL, then vigorously vortexed to mix. The mixture was incubated on ice for 15–30 min, then centrifuged at 12,000 rpm at 4°C for 10 min. After centrifugation, the supernatant was carefully transferred to a new 1.5 mL centrifuge tube. 160 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s to emulsify thoroughly.

[0060] The results are as follows Figure 8 As shown, the expression of both ATG5 and ATG7 genes was reduced in tumor tissues of tumor-bearing mice, indicating that the self-assembled nanoparticles with added chloroquine could significantly inhibit autophagy. These experiments demonstrate that the combined application of triptolide and chloroquine has a good anti-tumor effect.

Claims

1. Application of triptolide and chloroquine in the preparation of antitumor drugs.

2. The application of triptolide and chloroquine in combination as described in claim 1 in the preparation of antitumor drugs, characterized in that, The triptolide is a triptolide monomer, and the chloroquine is a chloroquine monomer.

3. The application of triptolide and chloroquine in combination as described in claim 1 in the preparation of antitumor drugs, characterized in that, The molar ratio of triptolide to chloroquine is 1:0.1-10, preferably 1:

1.

4. The application of triptolide and chloroquine in combination as described in claim 1 in the preparation of antitumor drugs, characterized in that, The drug can be any pharmaceutically acceptable dosage form, including liposomes, nanoemulsions, nanoparticles, microcapsules, microspheres, micro pellets, and solid dispersions, preferably nanoparticles.

5. The application of triptolide and chloroquine in combination as described in claim 1 in the preparation of antitumor drugs, characterized in that, The drug also includes any one or more combinations of pharmaceutically acceptable pharmaceutical excipients.

6. The application of triptolide and chloroquine in combination as described in claim 5 in the preparation of antitumor drugs, characterized in that, The pharmaceutical excipients include calcium chloride and polyvinylpyrrolidone.

7. The application of triptolide and chloroquine in combination as described in claims 1-3 in the preparation of antitumor drugs, characterized in that, The drug can be administered via any one of the following methods: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration, with intravenous injection being preferred.

8. The application of triptolide and chloroquine in combination as described in claims 1-3 in the preparation of antitumor drugs, characterized in that, The tumor includes any one of melanoma, gastric cancer, lung cancer, pancreatic cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, uterine cancer, skin cancer, prostate cancer, thyroid cancer, leukemia, lymphoma, esophageal cancer, oral cancer, intestinal cancer, nasal cancer, and head and neck cancer, with breast cancer being preferred.

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