CN112704677A

CN112704677A - Use of vitamin E compounds

Info

Publication number: CN112704677A
Application number: CN201911038679.2A
Authority: CN
Inventors: 李青坡; 姜新东; 金召磊; 邱伟根
Original assignee: Huiyu Kangcheng Hangzhou Pharmaceutical Technology Co ltd
Current assignee: Huiyu Kangcheng Hangzhou Pharmaceutical Technology Co ltd
Priority date: 2019-10-24
Filing date: 2019-10-24
Publication date: 2021-04-27
Also published as: WO2021077990A1

Abstract

The invention relates to an application of a vitamin E compound in preparing a medicine for reducing the oxidative stress level caused by an anti-tumor medicine, an application of the vitamin E compound in preparing a medicine for improving immunity by combining with the anti-tumor medicine, and an application of the vitamin E compound in preparing a medicine for relieving the bone marrow suppression effect caused by the anti-tumor medicine or relieving the hemolytic disease caused by the anti-tumor medicine.

Description

Use of vitamin E compounds

Technical Field

The invention relates to application of vitamin E compounds in preparing medicaments for reducing the level of oxidative stress caused by medicaments, application in preparing medicaments for improving immunity and application in preparing medicaments for relieving bone marrow suppression or relieving hemolysis.

Background

Many chemotherapeutic agents have been demonstrated to have clinically strong antitumor activity. However, the traditional Chinese medicine composition often shows obvious toxic and side effects in the treatment process, and the clinical application of the traditional Chinese medicine composition is severely restricted.

Reactive Oxygen Species (ROS) are highly reactive ions and radicals, including singlet oxygen, superoxide radicals, hydroxyl radicals, and peroxides, which play a mandatory role in many physiological processes. At lower concentrations, ROS can act as important second messengers that regulate cell signaling, adhesion, migration, and homeostasis. While high levels of reactive oxygen species can oxidize unsaturated fatty acids in lipids and amino acids in proteins, resulting in irreversible damage to vital organelles and DNA, ultimately leading to apoptosis and necrosis. Notably, due to the differences in redox states between cancer cells and normal cells, the basal levels of ROS-mediated signaling in malignant cells are elevated, making them more susceptible to the deleterious effects of exogenous ROS than normal cells. Many chemotherapeutic drugs generate active oxygen and free radicals, which are accumulated in cancer tissues and attack cancer cells to exert anticancer effects. However, normal tissue is also often damaged by elevated levels of oxidative stress.

Some chemotherapy drugs also cause hemolytic toxicity, bone marrow suppression, anemia, and anaphylaxis.

Disclosure of Invention

The inventor finds that the vitamin E compound can reduce the oxidative stress level caused by the antitumor drug and can improve the antitumor effect of the antitumor drug. Moreover, the vitamin E compound and the anti-tumor drug have synergistic effect in the aspect of improving immunity, and the vitamin E compound has the effect of relieving bone marrow suppression or hemolytic diseases.

In view of this, an aspect of the present invention provides the use of a vitamin E compound in the manufacture of a medicament for down-regulating the level of oxidative stress caused by an anti-tumor drug.

On the other hand, the invention provides the application of the vitamin E compound and an anti-tumor medicament in the preparation of the medicament for improving the immunity.

In another aspect of the invention, there is provided the use of a vitamin E compound in the manufacture of a medicament for alleviating myelosuppression caused by an anti-tumor drug.

In another aspect of the invention there is provided the use of a vitamin E compound in the manufacture of a medicament for alleviating a haemolytic condition, in particular caused by an anti-tumour medicament.

Examples of the vitamin E compound include d- α -tocopherol, dl- α -tocopherol, β -tocopherol, γ -tocopherol, δ -tocopherol, d- α -tocopherol acetate, dl- α -tocopherol acetate, d- α -tocopherol succinate, dl- α -tocopherol succinate, and polyethylene glycol vitamin E succinate (TPGS).

Examples of the antitumor agent include Docetaxel (DTX), paclitaxel, cabazitaxel, larotaxel, vinorelbine (preferably vinorelbine bitartrate), hydroxycamptothecin, and the like.

The indications of the antitumor drug comprise breast cancer, prostate cancer, lung cancer, gastric cancer, ovarian cancer, cervical cancer, head and neck cancer and the like, and preferably breast cancer, lung cancer and the like.

The vitamin E compound can be administered with the antineoplastic agent sequentially or simultaneously. When the medicine is taken simultaneously, the medicine can be prepared into a compound medicine composition for administration.

The pharmaceutical dosage form of the vitamin E compound may be an emulsion, a liposome and a micelle, preferably an emulsion, more preferably a vitamin E nanoemulsion.

Optionally, the vitamin E nanoemulsion preparation method comprises:

(1) oil phase: mixing egg yolk lecithin and absolute ethyl alcohol at 40-70 ℃, stirring until the mixture is clear, adding vitamin E, medium-chain triglyceride and oleic acid, stirring until the mixture is clear, filtering through a 0.22 mu m microporous filter membrane, and then volatilizing the ethyl alcohol in vacuum to obtain an oil phase;

(2) water phase: adding arginine and sucrose into water for injection, stirring to dissolve completely, and filtering with 0.22 μm microporous membrane to obtain water phase;

(3) colostrum: firstly, preheating a water phase and an oil phase to 50-70 ℃, then slowly adding the water phase into the oil phase by using a high-speed dispersion machine under the shearing and stirring of 5000-20000 rpm, continuously shearing at 5000-20000 rpm for 20 +/-10 minutes after the water phase is completely added, and adjusting the pH value to obtain primary emulsion;

(4) final milk: transferring the primary emulsion into a high-pressure homogenizer, and homogenizing for 3-8 times at 25-45 ℃ under the condition of 500-1500 bar;

(5) subpackaging and sterilizing: filtering the final emulsion with 0.45 μm microporous membrane, subpackaging in penicillin bottles, charging nitrogen gas, sealing with gland, sterilizing with high pressure steam at 121 deg.C for 15 min, and rapidly cooling to obtain vitamin E nanoemulsion.

The antitumor drug is in the form of injection, and can be selected from emulsion, liposome and micelle.

Preferably a preparation formed by loading an anti-tumor medicament in a vitamin E compound medicament. More preferably, the anti-tumor drug preparation is vitamin E nanoemulsion loaded with anti-tumor drugs.

The preparation method comprises the following steps:

(1) oil phase: mixing the antitumor drug, egg yolk lecithin and absolute ethyl alcohol at 40-70 deg.C, stirring to clarify, adding vitamin E, medium chain triglyceride and oleic acid, stirring to clarify, filtering with 0.22 μm microporous membrane, and vacuum volatilizing ethanol to obtain oil phase;

(5) subpackaging and sterilizing: filtering the final emulsion with 0.45 μm microporous membrane, subpackaging in penicillin bottles, charging nitrogen gas, sealing with gland, sterilizing with high pressure steam at 121 deg.C for 15 min, and rapidly cooling to obtain vitamin E nanoemulsion loaded with antitumor drug.

Preferably, the particle size of the vitamin E nano-emulsion loaded with the anti-tumor drug is 50-500nm, preferably 80-350nm, and more preferably 100-200 nm.

Preferably, the vitamin E compound is administered in a dose of 1-40mg vitamin E per kg body weight.

Preferably, the ratio of the vitamin E compound to the antitumor drug is as follows: 1:5 to 1:20 of a surfactant,

preferably, the route of administration of the vitamin E compound is by injection.

Has the advantages that:

on the one hand, vitamin E compounds can inhibit the increase of ROS level in normal cells stimulated by antitumor drugs, and simultaneously improve antitumor effect.

On the other hand, the vitamin E compound and the antitumor drug have synergistic effect on improving the immunity.

On the other hand, the vitamin E compound obviously reduces the growth inhibition effect of the bone marrow cells of the antitumor drug.

On the other hand, vitamin E compounds can significantly reduce hemolytic disorders.

On the other hand, the vitamin E compound can reduce the acute toxicity and the neurotoxicity of the antitumor drugs.

Drawings

FIG. 1, (A) fluorescence micrographs corresponding to ROS in tumor cells 4T1 and normal cells LO2 after co-incubation with each of the formulations obtained in example 1; (B) fluorescence intensity profiles corresponding to intracellular ROS in tumor cells 4T1 and normal cells LO2 after co-incubation with each of the formulations obtained in example 1; (C) the docetaxel vitamin E nano-preparations with different concentrations have selectivity on normal cells and tumor cells; (D) selectivity of taxotere against normal and tumor cells at different concentrations.

FIG. 2 shows in vivo anti-tumor activity, (A) tumor growth curves of 4T1 vaccinated mice; (B) inoculating MDA-MB-231 mice with tumor growth curves; (C) inoculating a549 mouse tumor growth curve; (D) photographs of representative tumors after termination of treatment with 4T1 vaccinated mice; (E) photographs of representative tumors after the completion of treatment of a549 vaccinated mice; (F) tumor tissue fluorescence quantitative analysis. FIG. 3, (A) H & E staining representative pictures of different tissues; (B) body weight profile of mice throughout the experiment.

FIG. 4 is a graph of in vivo synergistic immunotherapy effect; (A) representative flow cytograms showing different sets of T cells in splenic lymphocytes; (B) calculating fluorescence intensities of CD8+ CTL and IFN- γ in distant tumors using image processing; (C) the percentage of T cells in the spleen was calculated based on (a). The spleen tissue (D) and tumor tissue (E) of each group of mice isolated at the end of the different treatments were tested for IL-4, IL-2 and IFN- γ levels using ELISA. P <0.05, p <0.01, p < 0.005. The error represents the standard deviation from the mean (n-5).

Figure 5 shows the detoxification of DTX-VNS. (A) Bone marrow cells from mice were incubated in methylcellulose-based medium at a dose of 0.1. mu.g/mL, 1. mu.g/mL or 10. mu.g/mL for 14 days VNS, DTX-VNS, taxotere and taxotere plus VE. Mock-treated cells were used as controls; the scale bar is 100 μm. (B) Colonies of CFU-GMs colony forming units that produce granulocytes and macrophages were counted using an inverted microscope. (C) Hemolyzed photographs of VNS, DTX-VNS, taxotere plus VE, DTX-NS and taxotere mixed for 3 hours at medium concentration (5. mu.g/mL) all darkened in color indicating severe hemolysis. (D) Percent hemolysis observed after incubation of the different experiments with RBC up to 3 hours. Data are presented as mean ± standard error of three experiments. P <0.05, p <0.01, p < 0.005.

Figure 6 shows the detoxification of DTX-VNS. (A) Bone marrow cells from mice were cultured with VNS, DTX-VNS, taxotere and taxotere plus VE at a dose of 0.1. mu.g/mL, 1. mu.g/mL or 10. mu.g/mL in methylcellulose-based medium for 14 days. Counting using an inverted microscope, (B) producing a burst forming unit of erythrocytes and (C) producing a colony forming unit of granulocytes, erythrocytes, macrophages and megakaryocytes. Data are presented as mean ± standard error of three experiments. P <0.05, p <0.01, p < 0.005. (D) Hemolyzed photographs of different concentrations (high: 10 mg/mL; low: 1mg/mL) mixed with blank VNS, DTX-VNS, taxotere plus VE, DTX-NS and taxotere for 3 hours all darkened in color indicating severe hemolysis.

FIG. 7 is a toxicity evaluation. (A) The effect of different treatments on the body weight of the rats varied throughout the experiment. (B) Rats were treated differently for 22 days relative to the weight of the organs.

Figure 8 is a tolerated dose study. (A) Survival of mice injected with taxotere and DTX-VNS. (B) Individual body weights (n-3) of mice were obtained throughout the experiment.

FIG. 9 is a DiO-, DiI-loaded DTX-VNS (Integrated VNS: complete VNS; Cracked VNS: disrupted VNS) diluted with 100-fold water, respectively

Figure 10 is an in vitro selective release of DTX-VNS. DiO-, DiI-loaded DTX-VNS was incubated with 4T1 tumor cells (A) and NIH3T3 normal cells (B) at 37 ℃ for a predetermined time and fluorescence images were examined by confocal fluorescence microscopy. Using image processing, fluorescence intensities of the lytic VNS and the integrated VNS in 4T1 tumor cells (C) or NIH3T3 normal cells (D) were calculated based on (a) or (B). The scale bar is 20 μm.

FIG. 11 is a graph of the selective release of DTX-VNS in tumor cells. DiO-, DiI-loaded DTX-VNS was incubated with A549(A) and MDA-MB-231(B) tumor cells for a predetermined time at 37 ℃ and fluorescence images were examined by confocal fluorescence microscopy. Using image processing, fluorescence intensities of lysed VNS and integrated VNS in a549(C) or MDA-MB-231 tumor cells (D) were calculated based on (a) or (B). The scale bar is 20 μm.

FIG. 12 is a graph of the selective release of DTX-VNS in normal cells. DiO-, DiI-loaded DTX-VNS was incubated with LO2(A) and HEK293 normal cells (B) at 37 ℃ for a predetermined time and fluorescence images were examined by confocal fluorescence microscopy. Using image processing, the fluorescence intensity of the integrated VNS in lysed VNS and LO2(C) or HEK293 normal cells (D) was calculated based on (a) or (B). The scale bar is 20 μm.

FIG. 13 shows in vitro selective release of DTX-VNS. (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 cleaved 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) Optical photographs of various tissues (heart), liver (liver), lung (lung), kidney (kinney), bone (bone), fat (fat), stomach (stomach), intestines (intestines), spleen (spleen), brain (brain), ovary (ovary) and tumor (tumor)).

Figure 14 is selective release of DTX-VNS in vivo and ex vivo. In vivo and ex vivo images of various tissues (heart, liver, lung, kidney, bone, fat, stomach, intestine, spleen, brain, ovary, tumor) of mice bearing a549 tumors were obtained at predetermined times after intravenous injection, and either DTX-vns (a) or DTX-ns (b) was injected. The% ID/g (percentage of injected dose per gram of tissue) was analyzed to show the accumulation of ruptured vns (ns) and intact vns (ns) in various tissues 48 hours after intravenous injection, either DTX-vns (c) or DTX-ns (d) was injected. (E) DTX concentration (n ═ 5) in various tissues (heart, liver, spleen, lung, kidney, stomach, brain and MDA-MB-231 tumors) of mice 24 hours after intravenous injection of DTX-VNS and Taxotere.

Figure 15 shows the in vivo and ex vivo selective release of DTX-VNS. In vivo and ex vivo images of various tissues from 4T 1-bearing tumor mice (A) or MDA-MB-231-bearing tumor mice (B) were obtained at predetermined times after intravenous administration of DTX-VNS. The% ID/g (percentage of injected dose per gram of tissue) was analyzed to show the accumulation of lysed VNS and intact VNS (24 hours post-iv) in various tissues of 4T1 tumor bearing mice (C) or MDA-MB-231 tumor bearing mice (D).

FIG. 16 is a graph of fluorescence intensity of ROS in tumor cells 4T1 and normal cells LO2 after co-incubation with each of the formulations obtained in example 2, example 3, and example 4.

Detailed Description

The invention is further illustrated by the following examples and figures. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Description of terms:

DTX: docetaxel

VE: vitamin E

VNS (Blank-VNS): VE nano preparation

DTX-VNS: DTX-loaded vitamin E nano preparation

Taxotere: teddi

Taxotere plus VE: tesofidi plus VE

DTX-NS: DTX nano preparation

Experimental materials:

docetaxel (DTX) was purchased from south drug industry ltd, fujian, china.

Medium Chain Triglycerides (MCT), soy lecithin (S100) was purchased from Lipoid co. (Ludwigshafen, Germany).

Vitamin E (VE, alpha-tocopherol acetate) was purchased from basf (china) co

Corn oil and soybean oil were purchased from emerging (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 Biotech, Inc. (Zhejiang, China).

3- [4, 5-Dimethylthiazol-2-yl ] -2, 5-diphenyltetrazolium bromide (MTT reagent) and Hochest33324 were obtained from Sigma-Aldrich Inc. (St Louis, MO, USA).

Live and dead cell kit [ L-3224] was purchased from Life Technologies (Carlsbad, Calif.).

3,3' -octacosylcarbonylphthalocyanine perchlorate (DiO), 1,1' -octacosanyl-3, 3,3',3' -tetramethylindocyanine perchlorate (DiI), 1,1' -octacosanyl-3, 3,3',3' -tetramethyltricosenyl propidium iodide (DiD) and Nile Red (NR) were purchased from Invitrogen Co. (Carlsbad, Calif., USA).

Chemicals and solvents were of analytical grade and used as received.

Cell culture:

4T1 (mouse breast cancer), A549 (human lung cancer), MDA-MB-231 (human breast cancer), LO2 (human liver cell), NIH-3T3 (mouse embryonic fibroblast) and HEK293 cell lines were purchased from institute of Biochemical research, cell biology (Shanghai, China). Cells were incubated at 37 ℃ with 5% CO₂In RPMI 1640 medium or DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc., Carlsbad, Calif.) and 100U/mL penicillin and 100U/mL streptomycin.

Animals: a nude mouse; SD rat

Example 1

Prescription 1(VE nano preparation)

Prescription 2 (docetaxel-loaded VE nano preparation)

Prescription 3: (DTX Nanometers DTX-NS)

Test example 1

The test method comprises the following steps: 4T1 tumor cells and LO2 normal cells were incubated with Blank-VNS, DTX-VNS, Taxotere or Taxoteplus VE at a DTX concentration of 10. mu.g/mL for 48 hours. Cells were then treated with DCFH-DA (10mM) for 20 minutes to detect intracellular ROS. Then, the fluorescence of the sample was examined with a fluorescence microscope.

The experimental results are as follows: incubation of DTX-VNS, Blank-VNS and Taxotere plus VE in 4T1 tumor cells all showed weak green fluorescence, indicating the ability of DTX-VNS, Blank-VNS and Taxotere plus VE to significantly down-regulate ROS levels in cancer cells. In normal cells, significant green fluorescence was observed upon incubation of taxotere, resulting in a significant increase of ROS in normal cells, indicating that cells may upregulate oxidative stress, whereas cells have weak green fluorescence intensity before and after DTX-VNS treatment, indicating that low intracellular ROS levels were maintained (fig. 1A, fig. 1B).

Test example 2

The MTT assay was used to study the killing effect of DTX-VNS and taxotere on various tumor cells (A549, 4T1, MDA-MB-231) and normal cells (LO2, NIH3T3, HEK 293). Taxotere was found to have similar lethality to normal cells and tumor cells. Under the same incubation conditions, DTX-VNS killed tumor cells significantly more, while more normal cells could survive. For example, DTX-VNS resulted in approximately 70% of tumor cells being killed, while almost 60% of normal cells survived at 50 μ g DTX/mL incubation concentration (fig. 1C). After treatment with the same concentration of taxotere, 60-70% of cancer cells and normal cells died (fig. 1D). These results indicate that DTX-VNS exhibits selective lethality in normal and tumor cells.

Test example 3

The test method comprises the following steps: the anti-tumor effect was evaluated on 4T1, MDA-MB-231 and A549 tumor models. 4T1 or MDA-MB-231 cells (1X 10)⁶) 4T1 and MDA-MB-231 tumor models were obtained by injection into the upper breast pad of each female BALB/c or nude mouse, respectively. A549 cells (1X 10)⁶) A549 tumor model was established by subcutaneous injection into the right flank of each BALB/c mouse. When the tumor diameter reaches 50-100mm³At the time, the mice were randomly divided into 4 groups (5 mice per group). Groups 1-4 mice were injected intravenously with saline, VNS, DTX-VNS (10mg DTX/kg per injection) and Taxotere (Taxotere, 10mg DTX/kg per injection) for 6 times (twice weekly), respectively. Tumor volume and body weight of mice were monitored. After treatment, tumors and major organs were collected, weighed, and sectioned for H&E, Ki-67 or Tunel staining.

The experimental results are as follows: DTX-VNS was tested for anti-tumor activity in 4T1, MDA-MB-231 and A549 tumor models. At the same DTX dose, DTX-VNS showed a stronger ability to inhibit growth of three tumors than taxotere (fig. 2A, B, C). At the end of the experiment, tumors were collected for each group, and the average tumor weight in the DTX-VNS group was the smallest among the groups (fig. 2D, fig. 2E). Among them, 4T1 tumors in the DTX-VNS group showed more severe cancer cell apoptosis and significantly reduced proliferative activity (FIG. 2A, FIG. 2D). A quantitative analysis of Ki-67 or Tunel staining of group 4T1 or A549 tumor tissues is shown in FIG. 2F.

In addition, the killing of normal cells by DTX-VNS was tested in 4T1, MDA-MB-231 and A549 tumor models. The tissue section results showed that normal tissues of the DTX-VNS group did not show significant damage or inflammation compared to the Saline group (fig. 3A), and the body weight of the mice did not decrease significantly throughout the experiment (fig. 3B).

Test example 4

The test method comprises the following steps:

mice bearing the 4T1 tumor were divided into 5 groups (5 mice per group). Groups 1-5 mice were injected intravenously with physiological 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. One week after treatment, mice were sacrificed and tumors and spleens were collected. Fresh tumor tissue was sectioned to analyze CD8+ T cell and IFN- γ levels using immunofluorescence. T cells (CD3+, CD4+ and CD8+) in the spleen were isolated and analyzed using flow cytometry, and factors such as IL-4, IL-2 and IFN- γ levels in tumor and spleen tissues were examined using ELISA kits.

And (3) test results:

mice bearing 4T1 tumors were sacrificed after various treatments and tumors sectioned for analysis of IFN- γ levels and infiltration of CD8 positive cytotoxic T lymphocytes (CD8+ CTLs). VE alone (blank-VNS group) or DTX alone (taxotere group) was found to cause only slight increases in IFN- γ levels and CD8+ CTL infiltration in tumors in the saline group. Both IFN-. gamma.and CD8+ CTL levels were significantly improved in tumors after treatment with the combination of DTX and VE, as shown in DTX-VNS and Taxotere plus VE groups (FIG. 4D). The amount of CD3, CD8 and CD4 positive T cells was measured in the spleen and showed stronger proliferation in the DTX-VNS and Taxotere plus VE groups than the other groups (fig. 4A, fig. 4C). Immune responses of Th1 and Th2 were further monitored in tumors and spleen using ELISA after treatment. The Th1 immune response was significantly down-regulated by increasing IL-2 and IFN- γ secretion, while the Th2 response was demonstrated by a significant reduction in IL-4 secretion in the DTX-VNS and Taxotere plus VE groups (FIG. 4D, FIG. 4E). The results indicate that VE can enhance immune responses occurring in the tumor microenvironment more effectively under the action of DTX than VE or DTX alone.

Test example 5

Two-week-old SD rats were sacrificed and bone marrow cells were isolated from the tibia. Cells (5X 103 cells/well in MethoCult medium) were then treated with Blank-VNS, DTX-VNS, Taxotere or Taxotere plus VE at DTX concentrations of 0.1,1 and 10. mu.g/mL. Cells were incubated at 37 ℃ and 5% CO 2 for about 14 days until colonies were formed and observed under a microscope after crystal violet staining.

And (3) test results:

CFU counts were taken under the microscope and included CFU-GMs, BFU-Es and CFU-GEMM. Tesoffit caused a significant reduction in the amount of CFU-GM, BFU-Es and CFU-GEMM at incubation concentrations of 0.1-10. mu.g/mLDTX. However, the decrease in the number of CFUs after DTX-VNS or Taxotere plus VE treatment was greatly reduced over the same concentration range (fig. 5A, 5B, fig. 6A, 6B, 6C). These results indicate that, unlike taxotere alone, DTX-VNS is effective in alleviating the myelosuppressive effects caused by DTX.

Test example 6

Fresh RBCs with DTX concentrations ranging from 1-10 μ g/mL were used to assess the hemolytic effects of DTX-VNS.

Fresh blood was collected from a healthy rabbit heart. The blood was diluted with physiological saline (1: 10, v/v) and centrifuged at 1300rmp for 15 minutes to separate erythrocytes. Cells in saline were incubated with various DTX concentrations of Blank-VN, DTX-VNS, Taxotere, Taxotere plus VE, DTX loaded nanosystems (DTX-NS). Cells treated with pure water and physiological saline alone served as positive and negative controls, respectively. After 3 hours incubation at 37 ℃, the samples were centrifuged at 3000rpm for 5 minutes, the supernatant carefully collected and spectrally analyzed with an ultraviolet spectrophotometer (UNIC-7200, china) at 545 nm. Hemolysis parameters were calculated according to the formula (ODt-ODn)/(ODp-ODn). times.100%, where ODt, ODn and ODp represent the absorbance of the test, negative and positive groups, respectively.

And (3) test results:

in the low DTX concentration range 1-5. mu.g/mL, similar to Blank-VNS, DTX-VNS did not cause significant hemolysis. Even at the highest DTX concentration of 10. mu.g/mL, only 7.53% of hemolysis occurred. However, at the same concentration, DTX-NS and taxotere alone showed significantly increased levels of hemolysis (39.69 ± 1.14% and 42.89 ± 2.05%, respectively) (fig. 5C, fig. 5D, fig. 6D). These data indicate that DTX-VNS has significantly reduced hemolysis compared to Taxotere.

Test example 7

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 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 per week). Rats were weighed and observed visually once a day during this period for mortality, behavioral patterns, and changes in the apparent signs of disease. At the end of the experiment, blood was collected for biochemical and hematological analysis. Then, the rats were sacrificed and the respective organs were weighed to calculate the organ coefficients.

The experimental results are as follows:

the total DTX dose was 32 or 64 mg/kg. The values of the major blood routine (Table 1) and blood biochemistry (Table 2) showed no significant differences between the DTX-VNS, Blank-VNS and saline groups at the high and low therapeutic doses. However, some of the pathological indicators of the Taxotere group, such as the MONO and CK values, were significantly abnormal compared to the saline group. No significant weight loss (fig. 7A) and no relative organ weight change (fig. 7B) of the animals was found in the DTX-VNS group. These results strongly suggest that DTX-VNS has low systemic toxicity, even at injected doses up to 64 mg/kg.

After four weeks of treatment (160 mg DTX/kg total), all mice in the DTX-VNS group survived. However, in the Taxotere group, death of mice occurred in the second and third weeks. In the second week of dosing (total 80mg DTX/kg), the hind limbs of the Taxotere group mice showed slight stiffness, which became more severe with continued dosing. In the DTX-VNS group, only slight stiffness occurred after the third week of dosing (120 mg DTX/kg total). These results indicate that the neurotoxicity of DTX-VNS is significantly reduced compared to Taxotere. Within one week after cessation of treatment, stiffness was significantly reduced and the mice returned to normal body weight (figure 8). The dose tolerance of the animals to DTX-VNS was significantly improved compared to that of taxotere.

TABLE 1 changes in hematological indices

Remarking: DTX-VNS N: DTX-VNS Low dose group

DTX-VNS H: DTX-VNS high dose group

Taxotere N: taxotere low dose group

Taxotere H: taxotere high dose group

TABLE 2 blood biochemical index changes

Remarking: DTX-VNS N: DTX-VNS Low dose group

DTX-VNS H: DTX-VNS high dose group

Taxotere N: taxotere normal dose group

Taxotere H: taxotere high dose group

Test example 8

Drug release was studied. DiO and DiI (1: 5, w/w) were co-encapsulated into DTX-VNS. Cancer cells (4T1, MDA-MB-231 and A549) and normal cells (LO2, HEK293 and NIH-3T3) were incubated with DiO and DiI-labeled DTX-VNS for different periods of time. FRET images were obtained using fluorescence microscopy (480nm excitation, DiO emission at 490-520nm, DiI emission at 560-650 nm). All images were acquired and processed using Fluo-View software (Olympus LX83-FV 3000). The fluorescence intensity of disintegrated and encapsulated DTX-VNS in cells was calculated using Image J software.

Drug release was further studied in mice bearing various tumors (4T1, MDA-MB-231 and A549) after intravenous injection of DTX-VNS, co-labeled with NR and DiD. At predetermined time points, mice were anesthetized and FRET images were observed using Maestro imaging system (CRI, inc., Woburn, MA) (590nm excitation, 600-. Mice were then sacrificed and major organs including tumors were collected and further imaged. The fluorescence intensity of DTX-VNS disintegrated and integrated in various organs 24 hours after injection was calculated using Image J. Mice carrying A549 were injected with NR-, DiD-loaded DTX-NS and fluorescence images were obtained.

The toxicity of DTX-VNS to tumor and normal cells was determined by MTT assay according to the instructions. A549, 4T1, MDA-MB-231, LO2, NIH3T3 or HEK293 cells were exposed to DTX-VNS and Taxotere for 48 hours. Data are expressed as a percentage of viable cells and reported as the average of 5 measurements.

And (3) test results:

DTX-VNS with FRET effect of 480nm excitation was obtained by co-encapsulation of DiO and DiI into nanosystems (fig. 9). DiO or DiI release behavior from DTX-VNS was monitored by analyzing fluorescence intensity at 575nm and 505 nm. By observing red fluorescence, DTX-VNS observed red fluorescence after 1 hour of incubation, indicating rapid entry into 4T1 tumor cells; after 4 hours of incubation, the appearance of green fluorescence was observed and DiO or DiI began to be released from DTX-VNS (fig. 10A). The uptake of DTX-VNS by normal cells was significantly lower than that by tumor cells. After incubation, NIH3T3 normal cells were observed, red fluorescence was observed after 2 hours, but green fluorescence was not observed until after 12 hours (fig. 10B).

In A549 tumor cells, by observing red fluorescence, DTX-VNS observed after 1 hour of incubation, it was shown that it can rapidly enter A549 tumor cells; after 4 hours of incubation, the appearance of green fluorescence was observed and DiO or DiI began to be released from DTX-VNS (fig. 11A).

In MD-MBA-231 tumor cells, by observing red fluorescence, DTX-VNS can observe red fluorescence after 1 hour of incubation, which indicates that MD-MBA-231 tumor cells can be rapidly accessed; after 4 hours of incubation, the appearance of green fluorescence was observed and DiO or DiI began to be released from DTX-VNS (fig. 11B).

The uptake of DTX-VNS by normal cells of LO2 was significantly lower than that by tumor cells. LO2 normal cells were observed after incubation, red fluorescence was observed after 4 hours, but green fluorescence was not observed until after 12 hours (fig. 12A).

The uptake of DTX-VNS by HEK293 normal cells was significantly lower than that by tumor cells. HEK293 normal cells were observed after incubation, red fluorescence was observed after 2 hours, but green fluorescence was not observed until after 12 hours (fig. 12B).

It was further demonstrated by semiquantitative results of fluorescence intensity that the signal (green fluorescence) in released DiO or DiI induced tumor cells such as 4T1, a549 and MB-MDA-231 will be progressively stronger than the signal (red fluorescence) generated by the encapsulated fluorescers (fig. 10C, 11D), whereas the green fluorescence intensity in normal cells such as NIH3T3, LO2 and HEK293 is always significantly lower than the intensity of red fluorescence (fig. 10D, 12C, 12D). These data indicate selective cellular internalization and drug release behavior of DTX-VNS.

Drug release behavior was further studied in tumor-bearing mice after intravenous injection with DTX-VNS co-encapsulated with NR and DiD. The FRET effect was generated under excitation at 590nm and the emission peaks were 618-and 667-nm, representing the signal for the released (Cracked VNS) and encapsulated (Integrated VNS), respectively (FIG. 13). The strong fluorescence signal at the wavelength of 600-630nm indicates drug release, and the strong fluorescence signal at the wavelength of 670-700nm indicates encapsulated drug signal. One hour after injection, many DTX-VNS were located in the liver. Its accumulation at the site of the a549 tumor gradually increased over time and reached a maximum 48 hours after injection. No strong fluorescence signal at 600-630nm was observed in the liver within 1-6 hours, which means that no NR or DiD was released from the nanosystems. However, a signal was clearly observed at the tumor site at 8 hours post-injection and gradually increased from 8 hours to 48 hours, indicating a clear drug release behavior in the tumor tissue (fig. 14A, fig. 14C). The nanosystems showed consistent drug release behavior in mice bearing 4T1 or MDA-MB-231 tumors as in mice bearing a549 tumors, with a clear signal of drug release observed at the tumor site at 8 hours, with a gradual increase in signal over time (fig. 15). However, no strong fluorescence signal for drug release was observed in normal tissues, and the results of semiquantitative fluorescence intensity also show that, for example, the signal for drug release in liver is significantly higher for encapsulated drug than for released drug, while the signal for drug release in tumor is stronger than for encapsulated drug (FIG. 14C, FIG. 15D), all of which indicate the selective drug release behavior of DTX-VNS, with drug release significantly faster in tumor than in normal tissues.

NR and DiD loaded nanosystems (DTX-NS) were prepared and injected into a549 tumor-bearing mice, and a significant reduction in the difference in drug release rates between tumor and normal tissues was found (fig. 14B, fig. 14D).

The in vivo DTX profile following DTX-VNS or Taxotere administration was further investigated. Figure 14E shows the concentration of DTX in major organs (including tumors) 24 hours after injection of DTX-VNS and Taxotere using HPLC-MS. DTX-VNS resulted in a significant increase in DTX retention in organs, especially in tumors (DTX concentrations over 50-fold), compared to Texorid.

Example 2

Prescription

The preparation method comprises the following steps:

(1) oil phase: mixing paclitaxel, egg yolk lecithin, vitamin E, medium chain triglyceride, oleic acid and anhydrous ethanol at 20-40 deg.C, stirring to clarify, and vacuum evaporating ethanol to obtain oil phase;

(2) water phase: adding sucrose into water for injection, stirring to dissolve completely, and filtering with 0.22 μm microporous membrane to obtain water phase;

(3) colostrum: firstly, preheating a water phase and an oil phase to 50-70 ℃, then slowly adding the water phase into the oil phase by using a high-speed dispersion machine under the shearing and stirring of 5000-20000 rpm, and continuously shearing at 15000rpm for 20 minutes after the water phase and the oil phase are completely added to obtain primary emulsion;

(4) final milk: cooling the primary emulsion to room temperature, transferring the primary emulsion to a high-pressure homogenizer, and homogenizing for 4 times at 25-45 ℃ under the condition of 1000 bar;

(5) and (3) filtering and subpackaging: filtering the final emulsion with 0.22 μm microporous membrane, subpackaging the filtered final emulsion in penicillin bottles, and introducing nitrogen;

the measurement results of the obtained nano-formulation are shown in Table 4.

TABLE 4

*: PDI: polydispersity index

Example 3

Prescription

The preparation method comprises the following steps:

(1) oil phase: mixing tartaric acid, egg yolk lecithin, vitamin E, medium chain triglyceride, oleic acid and anhydrous ethanol at 20-40 deg.C, stirring to clarify, and vacuum evaporating ethanol to obtain oil phase;

(3) colostrum: firstly, preheating a water phase and an oil phase to 50-70 ℃, then slowly adding the water phase into the oil phase by using a high-speed dispersion machine under the shearing and stirring of 5000-20000 rpm, and continuously shearing at 15000rpm for 30 minutes after the water phase and the oil phase are completely added to obtain primary emulsion;

(4) final milk: cooling the primary emulsion to room temperature, transferring the primary emulsion to a high-pressure homogenizer, and homogenizing for 4 times at 25-45 ℃ under 1200 bar;

the measurement results of the obtained nano-formulation are shown in Table 5.

TABLE 5

*: PDI: polydispersity index

Example 4

Prescription

The preparation method comprises the following steps:

(1) oil phase: mixing docetaxel, yolk lecithin, vitamin E, medium chain triglyceride, oleic acid and absolute ethanol at 20-40 deg.C, stirring to clarify, and vacuum evaporating ethanol to obtain oil phase;

(4) final milk: cooling the primary emulsion to room temperature, transferring the primary emulsion to a high-pressure homogenizer, and homogenizing for 6 times at 25-45 ℃ under the condition of 1000 bar;

the measurement results of the obtained nano-formulation are shown in table 6.

TABLE 6

*: PDI: polydispersity index

Test example 9

The test method comprises the following steps: the 4T1 tumor cells and LO2 normal cells are incubated with the vitamin E nano preparation loaded with paclitaxel, the vitamin E nano preparation loaded with vinorelbine bitartrate and the vitamin E nano preparation loaded with hydroxycamptothecine, and the concentrations of the paclitaxel, the vinorelbine bitartrate and the hydroxycamptothecine are all 10 mu g/mL and last for 48 hours. Cells were then treated with DCFH-DA (10mM) for 20 minutes to detect intracellular ROS. Then, the fluorescence of the sample was examined with a fluorescence microscope.

The experimental results are as follows: after the cells are treated by the vitamin E nano preparation loaded with paclitaxel, the vitamin E nano preparation loaded with vinorelbine bitartrate and the vitamin E nano preparation loaded with hydroxycamptothecin, the low intracellular ROS level is maintained (figure 16).

Claims

1. Use of a vitamin E compound in the manufacture of a medicament for down-regulating the level of oxidative stress caused by an anti-neoplastic drug.

2. The vitamin E compound and the antineoplastic medicine are used together to prepare the medicine for improving the immunity.

3. Use of vitamin E compounds in the preparation of a medicament for alleviating the myelosuppressive effects caused by antineoplastic agents.

4. Use of a vitamin E compound in the manufacture of a medicament for alleviating a hemolytic disorder, in particular a hemolytic disorder caused by an anti-tumor drug.

5. Use according to any one of claims 1 to 4, wherein the vitamin E compound is selected from: one or more of d-alpha-tocopherol, dl-alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, d-alpha-tocopherol acetate, dl-alpha-tocopherol acetate, d-alpha-tocopherol succinate, dl-alpha-tocopherol succinate, or polyethylene glycol vitamin E succinate (TPGS).

6. Use according to any one of claims 1 to 5, wherein the antineoplastic drug is selected from: docetaxel (DTX), paclitaxel, cabazitaxel, larotaxel, vinorelbine (preferably vinorelbine bitartrate) or hydroxycamptothecin.

7. Use according to any one of claims 1 to 6, wherein the indications for the antineoplastic agent include breast cancer, prostate cancer, lung cancer, gastric cancer, ovarian cancer, cervical cancer, head and neck cancer, preferably breast cancer or lung cancer.

8. Use according to any of claims 1 to 7, wherein the pharmaceutical dosage form of vitamin E compounds is an emulsion, a liposome or a micelle, preferably an emulsion, more preferably a vitamin E nanoemulsion.

9. Use according to any one of claims 1 to 8, wherein the antineoplastic medicament is administered in the form of an injection, optionally an emulsion, a liposome and a micelle;

optionally, the anti-tumor drug preparation is a preparation formed by loading an anti-tumor drug in a vitamin E compound drug; optionally, the anti-tumor drug preparation is vitamin E nanoemulsion loaded with an anti-tumor drug;

10. Use according to any one of claims 1 to 9,

preferably, the vitamin E compound is administered in a dose of 1-40mg vitamin E per kg body weight;

preferably, the ratio of the vitamin E compound to the antitumor drug is as follows: 1: 5-1: 20;