WO2021077990A1 - Utilisation d'un composé à base de vitamine e - Google Patents

Utilisation d'un composé à base de vitamine e Download PDF

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WO2021077990A1
WO2021077990A1 PCT/CN2020/118187 CN2020118187W WO2021077990A1 WO 2021077990 A1 WO2021077990 A1 WO 2021077990A1 CN 2020118187 W CN2020118187 W CN 2020118187W WO 2021077990 A1 WO2021077990 A1 WO 2021077990A1
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vitamin
dtx
tumor
vns
tocopherol
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李青坡
姜新东
金召磊
邱伟根
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慧禹康成(杭州)医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the present invention relates to the use of vitamin E compounds in the preparation of drugs for lowering the level of oxidative stress caused by drugs, the use of drugs for improving immunity, and the preparation of drugs for alleviating bone marrow suppression or alleviating hemolysis. Use in medicine.
  • ROS Reactive oxygen species
  • Some chemotherapy drugs can also cause hemolytic toxicity, bone marrow suppression, anemia and allergic reactions.
  • vitamin E compounds can lower the level of oxidative stress caused by anti-tumor drugs and at the same time improve the anti-tumor effects of anti-tumor drugs. Moreover, vitamin E compounds and anti-tumor drugs have a synergistic effect in improving immunity, and vitamin E compounds have the effect of alleviating bone marrow suppression or hemolytic disorders.
  • one aspect of the present invention provides the use of vitamin E compounds in the preparation of drugs for down-regulating the level of oxidative stress caused by anti-tumor drugs.
  • Another aspect of the present invention provides the use of vitamin E compounds in combination with anti-tumor drugs in the preparation of drugs for improving immunity.
  • Another aspect of the present invention provides the use of vitamin E compounds in the preparation of medicines for alleviating bone marrow suppression caused by anti-tumor drugs.
  • Another aspect of the present invention provides the use of vitamin E compounds in the preparation of drugs for alleviating hemolytic disorders, especially for alleviating hemolytic disorders caused by anti-tumor drugs.
  • vitamin E compounds examples include d- ⁇ -tocopherol, dl- ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, d- ⁇ -tocopherol acetate, and dl- ⁇ -Tocopherol acetate, d- ⁇ -tocopherol succinate, dl- ⁇ -tocopherol succinate, polyethylene glycol vitamin E succinate (TPGS), etc.
  • antitumor drugs examples include docetaxel (DTX), paclitaxel, cabazitaxel, lalotaxel, vinorelbine (preferably vinorelbine bitartrate), hydroxycamptothecin, and the like.
  • the indications of the anti-tumor drugs include breast cancer, prostate cancer, lung cancer, gastric cancer, ovarian cancer, cervical cancer, head and neck cancer, etc., preferably breast cancer, lung cancer and the like.
  • Vitamin E compounds can be administered sequentially with anti-tumor drugs, or they can be administered at the same time. When simultaneously administered, it can be formulated into a compound pharmaceutical composition for administration.
  • the pharmaceutical dosage forms of vitamin E compounds can be emulsions, liposomes and micelles, preferably emulsions, and more preferably vitamin E nanoemulsions.
  • the preparation method of the vitamin E nanoemulsion includes:
  • Oil phase Mix egg yolk lecithin and absolute ethanol at 40-70°C and stir until clear, then add vitamin E, medium-chain triglycerides and oleic acid, stir until clear, after 0.22 ⁇ m Filter with a microporous membrane, and then evaporate the ethanol in a vacuum to obtain an oil phase;
  • Aqueous phase Add arginine and sucrose to the water for injection, stir until completely dissolved, and filter with a 0.22 ⁇ m microporous membrane to obtain the aqueous phase;
  • Colostrum first preheat the hot water phase and the oil phase to 50-70°C, then slowly add the water phase to the oil phase with a high-speed disperser under shearing and stirring at 5000 to 20000 rpm. Continuously shearing at 5000 ⁇ 20000rpm for 20 ⁇ 10 minutes, adjust the pH, and get colostrum;
  • Dispensing sterilization After filtering the final milk with a 0.45 ⁇ m microporous filter membrane, distributing it into a vial, filling it with nitrogen, sealing the lid, autoclaving at 121°C for 15 minutes, and then quickly Cool down to obtain vitamin E nanoemulsion.
  • the dosage form of the anti-tumor drug is injection, which can be selected from emulsion, liposome and micelle.
  • an anti-tumor drug is loaded into a vitamin E compound drug. More preferably, the anti-tumor drug preparation is a vitamin E nanoemulsion loaded with an anti-tumor drug.
  • Oil phase at 40-70°C, mix and stir the anti-tumor drugs, egg yolk lecithin and absolute ethanol until clear, then add vitamin E, medium chain triglycerides and oleic acid, and stir until clear , Filtered through a 0.22 ⁇ m microporous membrane, and then evaporate the ethanol in a vacuum to obtain the oil phase;
  • Aqueous phase Add arginine and sucrose to the water for injection, stir until completely dissolved, and filter with a 0.22 ⁇ m microporous membrane to obtain the aqueous phase;
  • Colostrum first preheat the hot water phase and the oil phase to 50-70°C, then slowly add the water phase to the oil phase with a high-speed disperser under shearing and stirring at 5000 to 20000 rpm. Continuously shearing at 5000 ⁇ 20000rpm for 20 ⁇ 10 minutes, adjust the pH, and get colostrum;
  • Dispensing sterilization After filtering the final milk with a 0.45 ⁇ m microporous filter membrane, distributing it into a vial, filling it with nitrogen, sealing the lid, autoclaving at 121°C for 15 minutes, and then quickly Cool down to obtain vitamin E nanoemulsion loaded with anti-tumor drugs.
  • the particle size of the anti-tumor drug-loaded vitamin E nanoemulsion is 50-500 nm, preferably 80-350 nm, more preferably 100-200 nm.
  • the administration dose of the vitamin E compound is 1-40 mg vitamin E/kg body weight.
  • the ratio of the vitamin E compound to the anti-tumor drug is 1:5 to 1:20,
  • the route of administration of the vitamin E compound is injection.
  • vitamin E compounds can inhibit the increase in ROS levels in normal cells stimulated by anti-tumor drugs, and at the same time improve the anti-tumor effect.
  • vitamin E compounds and anti-tumor drugs have a synergistic effect in improving immunity.
  • vitamin E compounds significantly reduce the anticancer drug bone marrow cell growth inhibitory effect.
  • vitamin E compounds can significantly reduce hemolytic disorders.
  • vitamin E compounds can reduce the acute toxicity and neurotoxicity of antitumor drugs.
  • FIG. 1 Fluorescence micrographs of tumor cells 4T1 and normal cells LO2 after co-incubation with the preparations obtained in Example 1;
  • B tumor cells 4T1 and normal after co-incubation with the preparations obtained in Example 1 Fluorescence intensity map corresponding to ROS in cell LO2;
  • C The selectivity of different concentrations of docetaxel vitamin E nanoformulations on normal cells and tumor cells;
  • D The selection of different concentrations of Taxotere on normal cells and tumor cells Sex.
  • Figure 2 shows the anti-tumor activity in vivo, (A) tumor growth curve of mice inoculated with 4T1; (B) tumor growth curve of mice inoculated with MDA-MB-231; (C) tumor growth curve of mice inoculated with A549; (D) inoculated with 4T1 Representative tumor photos of mice after treatment; (E) Representative tumor photos of mice inoculated with A549 after treatment; (F) Fluorescence quantitative analysis of tumor tissue.
  • Figure 3. (A) Representative pictures of H&E staining of different tissues; (B) the body weight change curve of mice during the whole experiment.
  • Figure 4 shows the effect of synergistic immunotherapy in vivo;
  • A Representative flow cytometry showing different groups of T cells in splenic lymphocytes;
  • B Using image processing to calculate the fluorescence of CD8+CTL and IFN- ⁇ in distant tumors Intensity;
  • C Calculate the percentage of T cells in the spleen based on (A).
  • ELISA was used to detect the levels of IL-4, IL-2 and IFN- ⁇ in the spleen tissue (D) and tumor tissue (E) of each group of mice separated at the end of different treatments.
  • Figure 5 shows the detoxification effect of DTX-VNS.
  • A Bone marrow cells from mice were incubated with VNS, DTX-VNS, Taxotere and Taxotega at a dose of 0.1 ⁇ g/mL, 1 ⁇ g/mL or 10 ⁇ g/mL in methylcellulose-based medium VE 14 days. The mock-treated cells are used as a control; the scale bar is 100 ⁇ m.
  • B Count the colonies of CFU-GMs colony forming units with an inverted microscope, which can produce granulocytes and macrophages.
  • C Hemolysis photos of VNS, DTX-VNS, Taxotere plus VE, DTX-NS and Taxotere mixed at a moderate concentration (5 ⁇ g/mL) for 3 hours. The darkening of the color indicates severe hemolysis.
  • D The percentage of hemolysis observed after incubating different experiments with RBC to 3 hours. The data is expressed as the mean ⁇ standard error of three experiments. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005.
  • Figure 6 shows the detoxification effect of DTX-VNS.
  • A Bone marrow cells from mice and blank VNS, DTX-VNS, Taxotere and Taxotere plus VE were cultured in methylcellulose-based culture at a dose of 0.1 ⁇ g/mL, 1 ⁇ g/mL or 10 ⁇ g/mL Base culture for 14 days. Counting was performed using an inverted microscope, (B) producing burst forming units of red blood cells and (C) producing colony forming units of granulocytes, red blood cells, macrophages and megakaryocytes. The data is expressed as the mean ⁇ standard error of three experiments. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005.
  • Figure 7 shows the toxicity evaluation.
  • A Throughout the experiment, different treatments have different effects on the body weight of rats.
  • B The relative organ weight of rats in different treatments for 22 days.
  • Figure 8 shows the tolerated dose study.
  • A Survival rate of mice after injection of Taxotere and DTX-VNS.
  • Figure 9 shows DiO- and DiI-loaded DTX-VNS diluted with 100 times water respectively (Integrated VNS: complete VNS; Cracked VNS:: cracked VNS)
  • Figure 10 shows the selective release of DTX-VNS in vitro.
  • the DiO-, DiI-loaded DTX-VNS, 4T1 tumor cells (A) and NIH 3T3 normal cells (B) were incubated at 37°C for a predetermined time, and the fluorescence images were checked by a confocal fluorescence microscope. Using image processing, calculate the fluorescence intensity of lysed VNS and integrated VNS in 4T1 tumor cells (C) or NIH 3T3 normal cells (D) based on (A) or (B).
  • the scale bar is 20 ⁇ m.
  • Figure 11 shows the selective release of DTX-VNS in tumor cells.
  • the DiO-, DiI-loaded DTX-VNS and A549 (A) and MDA-MB-231 (B) tumor cells were incubated at 37° C. for a predetermined time, and the fluorescence image was checked by a confocal fluorescence microscope. Using image processing, the fluorescence intensity of the lysed VNS and the integrated VNS in the A549 (C) or MDA-MB-231 tumor cells (D) is calculated based on (A) or (B).
  • the scale bar is 20 ⁇ m.
  • Figure 12 shows the selective release of DTX-VNS in normal cells.
  • the scale bar is 20 ⁇ m.
  • Figure 13 shows the selective release of DTX-VNS in vitro.
  • A Spectra of NR-, DiD-loaded DTX-VNS diluted with 100X water and 100X alcohol, respectively. Fluorescence imaging of NR-, DiD-loaded DTX-VNS diluted with 100X alcohol (left, representing lysed VNS) and 100X water (right, representing integrated VNS). The excitation wavelength is 590nm, and the emission wavelength is 600-630nm (B) or 670-700nm (C).
  • D Various tissues (heart, liver, lung, kidney, bone, fat, stomach, intestines, spleen) ), optical photographs of brain, ovary and tumor.
  • Figure 14 shows the selective release of DTX-VNS in vivo and in vitro.
  • Figure 15 shows the selective release of DTX-VNS in vivo and in vitro.
  • Accumulation in tissue (D) 24 hours after intravenous injection).
  • Figure 16 is a graph showing the fluorescence intensity corresponding to ROS in tumor cells 4T1 and normal cells LO2 after co-incubation with the preparations obtained in Example 2, Example 3 and Example 4.
  • VNS (Blank-VNS): VE nano formulation
  • DTX-VNS Vitamin E nano-formulation loaded with DTX
  • Taxotere Taxotere
  • Taxotere plus VE Taxotere plus VE
  • Docetaxel (DTX) was purchased from Fujian South Pharmaceutical Co., Ltd. (Fujian, China).
  • MCT Medium chain triglycerides
  • S100 soybean lecithin
  • Vitamin E (VE, ⁇ -tocopherol acetate) was purchased from BASF (China) Co. Ltd. Shanghai
  • Corn oil and soybean oil were purchased from Xinxing (Tieling) Pharmaceutical Co., Ltd. (Liaoning, China)
  • DMEM medium RPMI 1640 medium, trypsin, fetal bovine serum (FBS) and penicillin/streptomycin (100U/mL) were all from Jinuo Biotechnology Co., Ltd. (Zhejiang, China).
  • the live dead cell kit [L-3224] was purchased from Life Technologies (Carlsbad, CA).
  • Chemicals and solvents are of analytical grade and used as is.
  • 4T1 mouse breast cancer
  • A549 human lung cancer
  • MDA-MB-231 human breast cancer
  • LO2 human liver cells
  • NIH-3T3 mouse embryonic fibroblasts
  • HEK 293 cell lines were purchased from Cell Biology, Institute of Biochemistry (Shanghai, China)).
  • Cells were cultured in RPMI 1640 medium or DMEM at 37°C, 5% CO 2 and supplemented with 10% fetal bovine serum (Life Technologies, Inc., Carlsbad, CA) and 100U/mL penicillin and 100U/mL chain Mycin.
  • Test method 4T1 tumor cells and LO2 normal cells were incubated with Blank-VNS, DTX-VNS, Taxotere or Taxotere plus VE at a DTX concentration of 10 ⁇ g/mL for 48 hours. The cells were then treated with DCFH-DA (10mM) for 20 minutes to detect intracellular ROS. Then, check the fluorescence of the sample with a fluorescence microscope.
  • the MTT assay was used to study the killing effects of DTX-VNS and Taxotere on various tumor cells (A549, 4T1, MDA-MB-231) and normal cells (LO2, NIH 3T3, HEK 293). It was found that Taxotere has similar lethality to normal cells and tumor cells. Under the same incubation conditions, DTX-VNS killed tumor cells significantly more, and more normal cells could survive. For example, DTX-VNS caused nearly 70% of tumor cells to be killed, while almost 60% of normal cells survived at an incubation concentration of 50 ⁇ gDTX/mL (Figure 1C). After treatment with the same concentration of Taxotere, 60-70% of cancer cells and normal cells died ( Figure 1D). These results indicate that DTX-VNS exhibits selective killing power in normal cells and tumor cells.
  • Test method The anti-tumor effect was evaluated on 4T1, MDA-MB-231 and A549 tumor models.
  • 4T1 or MDA-MB-231 cells (1 ⁇ 10 6 ) were injected into the upper breast pad of each female BALB/c or nude mouse to obtain 4T1 and MDA-MB-231 tumor models.
  • A549 cells (1 ⁇ 10 6 ) were injected subcutaneously into the right flank of each BALB/c mouse to establish an A549 tumor model. When the tumor diameter reached 50-100 mm 3 , the mice were randomly divided into 4 groups (5 in each group).
  • mice in groups 1-4 were injected intravenously with saline, VNS, DTX-VNS (10mg DTX/kg per injection) and Taxotere (10mg DTX/kg per injection) for 6 times (twice a week) ). Monitor the tumor volume and body weight of the mice. After treatment, the tumors and major organs were collected, weighed, and sliced for H&E, Ki-67 or Tunel staining.
  • mice bearing 4T1 tumors were divided into 5 groups (5 mice in each group). Mice in groups 1-5 were injected intravenously with normal saline, Blank-VNS (100mg VE/kg), Taxotere (10mg DTX/kg), DTX-VNS (10mg DTX/kg and 100mg VE/kg) and Taxotere (10mg DTX/kg) ) plus VE (100mg/kg) three times. After one week of treatment, the mice were sacrificed and tumors and spleens were collected. Fresh tumor tissues were sectioned to analyze CD8+ T cells and IFN- ⁇ levels using immunofluorescence.
  • T cells (CD3+, CD4+ and CD8+) in the spleen were separated and analyzed by flow cytometry, and the levels of IL-4, IL-2 and IFN- ⁇ in the tumor and spleen tissues were checked with an ELISA kit.
  • mice bearing 4T1 tumors were sacrificed after various treatments, and tumor sections were used to analyze the level of IFN- ⁇ and the infiltration of CD8-positive cytotoxic T lymphocytes (CD8+CTL). It was found that VE alone (blank-VNS group) or DTX alone (taxotere group) only caused a slight increase in IFN- ⁇ levels and CD8+CTL infiltration in tumors in the saline group. After treatment with the combination of DTX and VE, the levels of IFN- ⁇ and CD8+CTL in the tumor were significantly improved, as shown in the DTX-VNS and Taxotere plus VE groups ( Figure 4D).
  • the amount of CD3, CD8 and CD4 positive T cells was measured in the spleen, and the DTX-VNS and Taxotere plus VE groups showed stronger proliferation than other groups ( Figure 4A, Figure 4C).
  • ELISA was used to further monitor the immune response of Th1 and Th2 in the tumor and spleen.
  • the Th1 immune response increases the secretion of IL-2 and IFN- ⁇ , while the Th2 response is significantly down-regulated, which can be confirmed by the significant reduction of IL-4 secretion in the DTX-VNS and Taxotere plus VE groups (Figure 4D, Figure 4E) .
  • the results show that compared with VE or DTX alone, VE can more effectively enhance the immune response in the tumor microenvironment under the action of DTX.
  • Two-week-old SD rats were sacrificed, and bone marrow cells were isolated from the tibia. Then treat the cells with Blank-VNS, DTX-VNS, Taxotere or Taxotere plus VE (5 ⁇ 10 3 cells/well in MethoCult medium), with DTX concentrations of 0.1, 1 and 10 ⁇ g/mL. The cells were incubated at 37°C and 5% CO 2 for about 14 days until colonies were formed, and the counts were observed under a microscope after staining with crystal violet.
  • CFU is counted under the microscope, including CFU-GMs, BFU-Es and CFU-GEMM.
  • Taxotere caused a significant decrease in the amount of CFU-GM, BFU-Es and CFU-GEMM at the incubation concentration of 0.1-10 ⁇ g/mL DTX.
  • the decrease in the number of CFU after DTX-VNS or Taxotere plus VE treatment was greatly reduced ( Figure 5A, 5B, Figure 6A, Figure 6B, Figure 6C).
  • mice For acute toxicity studies, SD rats (200-225g) were divided into 6 groups (7 rats/group). Rats in groups 1-6 were injected intravenously with normal saline, Blank-VNS, DTX-VNS (low dose, 8mg DTX/kg), DTX-VNS (high dose, 16mg DTX/kg), Taxotere (low dose, 8mg DTX/kg) ) And Taxotere (high dose, 16mg DTX/kg) four times (once a week). The rats were weighed and visually observed changes in mortality, behavior patterns, and physical signs of disease once a day during this period. At the end of the experiment, blood was collected for biochemical and hematological analysis. Then, the rats were sacrificed, and the organs were weighed to calculate the organ coefficient.
  • the total DTX dose is 32 or 64 mg/kg.
  • the main blood routine (Table 1) and blood biochemical (Table 2) values did not show significant differences between the DTX-VNS, Blank-VNS and saline groups at high and low therapeutic doses.
  • some pathological indicators such as MONO and CK values in Taxotere group were significantly abnormal compared with normal saline group.
  • No significant weight loss was found in the DTX-VNS group ( Figure 7A) and the relative organ weight changes of the animals ( Figure 7B).
  • mice in the DTX-VNS group survived. However, in the Taxotere group, the death of the mice occurred in the second and third weeks. In the second week of administration (80 mg DTX/kg in total), the hind limbs of the Taxotere group mice showed slight stiffness, which became more severe with continued administration. In the DTX-VNS group, slight stiffness (120 mg DTX/kg in total) occurred only after the third week of administration.
  • DTX-VNS H DTX-VNS high-dose group
  • Taxotere N Taxotere low-dose group
  • Taxotere H Taxotere high-dose group
  • DTX-VNS H DTX-VNS high-dose group
  • Taxotere N Taxotere normal dose group
  • Taxotere H Taxotere high-dose group
  • mice carrying various tumors 4T1, MDA-MB-231 and A549).
  • DTX-VNS was co-labeled with NR and DiD.
  • the mice were anesthetized and FRET images were observed using a Maestro imaging system (CRI, Inc., Woburn, MA) (590nm excitation, 600-630nm emission for NR and 670-700nm emission for DiD).
  • the mice were then sacrificed and major organs including tumors were collected and further imaged.
  • Use Image J to calculate the fluorescence intensity of the disintegrated and integrated DTX-VNS in various organs 24 hours after injection.
  • the mice carrying A549 were injected with NR-, DiD-loaded DTX-NS, and fluorescent images were obtained.
  • the toxicity of DTX-VNS to tumors and normal cells was determined by MTT assay. Expose A549, 4T1, MDA-MB-231, LO2, NIH 3T3 or HEK 293 cells to DTX-VNS and Taxotere for 48 hours. The data is expressed as the percentage of viable cells and is reported as the average of 5 measurements.
  • DTX-VNS with 480nm excitation FRET effect was obtained by co-encapsulating DiO and DiI into the nanosystem ( Figure 9).
  • the release behavior of DiO or DiI from DTX-VNS was monitored by analyzing the fluorescence intensity at 575nm and 505nm. By observing the red fluorescence, DTX-VNS observed red fluorescence after 1 hour incubation, indicating that it can quickly enter 4T1 tumor cells; after 4 hours of incubation, green fluorescence was observed, and DiO or DiI began to be released from DTX-VNS ( Figure 10A).
  • the uptake of DTX-VNS by normal cells was significantly lower than that by tumor cells. After incubation, normal NIH3T3 cells were observed. Red fluorescence was observed after 2 hours, but green fluorescence was not observed until 12 hours later ( Figure 10B).
  • the semi-quantitative results of fluorescence intensity can also show that the drug encapsulated in the liver is significantly higher than the released drug signal, while the drug released signal in the tumor is stronger than the encapsulated drug.
  • Figure 14C, Figure 15C, Figure 15D the results all show that the selective drug release behavior of DTX-VNS, the drug release in tumors is significantly faster than normal tissues.
  • DTX-NS NR and DiD loaded nanosystems
  • Figure 14E shows the use of HPLC-MS to determine the concentration of DTX in major organs (including tumors) 24 hours after the injection of DTX-VNS and Taxotere. Compared with Taxotere, DTX-VNS resulted in a significant increase in DTX retention in organs, especially in tumors (DTX concentration more than 50 times).
  • Oil phase Mix paclitaxel, egg yolk lecithin, vitamin E, medium-chain triglycerides, oleic acid and absolute ethanol at 20-40°C and stir until it is clear, and then evaporate the ethanol in a vacuum. Oil phase
  • Aqueous phase add sucrose to the water for injection, stir until it is completely dissolved, and filter with a 0.22 ⁇ m microporous membrane to obtain the aqueous phase;
  • Colostrum first preheat the hot water phase and the oil phase to 50-70°C, then slowly add the water phase to the oil phase with a high-speed disperser under shearing and stirring at 5000 to 20000 rpm. Continuously shear at 15000rpm for 20 minutes to obtain colostrum;
  • Filtration and packaging filter the final milk with a 0.22 ⁇ m microporous filter membrane, and then aliquot the filtered final milk into a vial and fill it with nitrogen;
  • Oil phase Mix bitartrate, egg yolk lecithin, vitamin E, medium-chain triglycerides, oleic acid and absolute ethanol at 20-40°C and stir until it is clear, then evaporate the ethanol in a vacuum. Get the oil phase
  • Aqueous phase add sucrose to the water for injection, stir until it is completely dissolved, and filter with a 0.22 ⁇ m microporous membrane to obtain the aqueous phase;
  • Colostrum first preheat the hot water phase and the oil phase to 50-70°C, then slowly add the water phase to the oil phase with a high-speed disperser under shearing and stirring at 5000 to 20000 rpm. Continuously shear at 15000rpm for 30 minutes to obtain colostrum;
  • Filtration and packaging filter the final milk with a 0.22 ⁇ m microporous filter membrane, and then aliquot the filtered final milk into a vial and fill it with nitrogen;
  • Oil phase Mix docetaxel, egg yolk lecithin, vitamin E, medium-chain triglycerides, oleic acid and absolute ethanol at 20-40°C and stir until it is clear, then evaporate the ethanol in a vacuum , That is, the oil phase is obtained;
  • Aqueous phase add sucrose to the water for injection, stir until it is completely dissolved, and filter with a 0.22 ⁇ m microporous membrane to obtain the aqueous phase;
  • Colostrum first preheat the hot water phase and the oil phase to 50-70°C, then slowly add the water phase to the oil phase with a high-speed disperser under shearing and stirring at 5000 to 20000 rpm. Continuously shear at 15000rpm for 30 minutes to obtain colostrum;
  • Filtration and packaging filter the final milk with a 0.22 ⁇ m microporous filter membrane, and then aliquot the filtered final milk into a vial and fill it with nitrogen;
  • Test method 4T1 tumor cells and LO2 normal cells were incubated with paclitaxel-loaded vitamin E nano-preparation, vitamin E nano-preparation loaded with vinorelbine bitartrate, and vitamin E nano-preparation loaded with hydroxycamptothecin, paclitaxel and vinorelbite bitartrate The concentrations of Bin and hydroxycamptothecin were both 10 ⁇ g/mL for 48 hours. The cells were then treated with DCFH-DA (10mM) for 20 minutes to detect intracellular ROS. Then, check the fluorescence of the sample with a fluorescence microscope.

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Abstract

L'invention concerne l'utilisation d'un composé à base de vitamine E dans la préparation d'un médicament permettant de réguler à la baisse le niveau du stress oxydatif provoqué par un médicament antitumoral, l'utilisation d'un composé à base de vitamine E en association avec un médicament antitumoral dans la préparation d'un médicament permettant d'améliorer l'immunité, et l'utilisation d'un composé à base de vitamine E dans la préparation d'un médicament permettant d'atténuer l'atteinte de la moelle osseuse provoquée par un médicament antitumoral ou d'atténuer des troubles hémolytiques provoqués par un médicament antitumoral.
PCT/CN2020/118187 2019-10-24 2020-09-27 Utilisation d'un composé à base de vitamine e WO2021077990A1 (fr)

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