Preparation method of compound medicine for treating small cell lung cancer

Abstract

The invention relates to a preparation method of a compound medicine for treating small cell lung cancer, and relates to the technical field of medicine preparation, the method comprises the following steps: S1, mixing allicin, phospholipid and cholesterol by adopting a film dispersion method; s2, carrying out a step-by-step temperature control program on the mixture prepared in the step S1 to obtain an arsenic-vanadium sample; s3, soaking the arsenic-vanadium sample obtained in the step S2 in an organic solvent for liquid-phase stripping; s4, centrifuging the mixture obtained in the step S3 to remove uncrushed large arsenic-vanadium particles, and collecting supernate to obtain an arsenic-vanadium solution; and S5, removing the organic solvent from the arsenic-vanadium solution obtained in the step S4 to obtain the arsenic-vanadium nanodot. The invention has the advantages that the problems of high toxicity, poor targeting property, low allicin stability, low bioavailability and lack of an effective synergistic delivery system of the existing arsenic are solved, and a new pharmaceutical composition and a technical path are provided for individualized treatment of small cell lung cancer, especially under the background of p53 mutation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine preparation, more particularly, to a preparation method of a composite medicine for treating small cell lung cancer. BACKGROUND

[0002] Small cell lung cancer (SCLC) is a highly invasive neuroendocrine tumor, accounting for 15% of all lung cancers, characterized by rapid proliferation, early metastasis and poor prognosis, with a 5-year survival rate of only 7.2%. 60%-70% of SCLC patients have blood metastasis when diagnosed, losing the opportunity for surgical treatment and only being able to receive systemic treatment. Despite decades of active research, the treatment options for SCLC are still limited. The current domestic clinical treatment is still mainly based on platinum combined with etoposide, a DNA-damaging chemotherapy drug, but it is only sensitive in the early stage and rapidly relapses and develops resistance within a few months. Therefore, there is currently a lack of effective targeted treatment for SCLC, especially for patients carrying mutant p53, whose treatment is more challenging.

[0003] Lung cancer is caused by deficiency of vital energy, imbalance of yin and yang, invasion of evil toxin into the lung, stagnation of evil in the lung, leading to dysfunction of the lung, failure of dispersion and descent, stagnation of lung qi, loss of distribution of body fluid, accumulation of body fluid into phlegm, stagnation of phlegm and qi, obstruction of collaterals by blood stasis and toxin, and finally formation of lung mass after long-term accumulation of blood stasis and toxin. DNA damage response (DDR) is an important mechanism for cell self-repair and protection of DNA damage. Modern medicine believes that accumulation of excessive damaged DNA due to defects in DNA damage and repair is an important factor in the formation of malignant tumors. Both traditional Chinese medicine theory and modern medical research emphasize that accumulation of harmful substances in the body leads to cancer, suggesting that defects in the DNA damage response mechanism during tumor development may have common characteristics with toxic stasis in traditional Chinese medicine. Therefore, the combination of the two can provide a new idea for the treatment of SCLC.

[0004] As a tumor suppressor gene, p53 can recognize different types of damage, activate complex signaling networks, block cell cycle to promote DNA repair, or remove cells that fail to repair DNA damage through apoptosis, senescence and other pathways to prevent tumor formation; according to statistics, the mutation rate of p53 gene in SCLC is as high as 90%; after mutation of p53 gene, its anti-tumor activity is lost, and it can hinder DDR through G1 / S checkpoint defects, up-regulate trans-damage DNA synthesis, and induce DNA damage sensitivity to promote tumor development; therefore, targeting the abnormal DDR caused by p53 mutation in SCLC is a direct and effective treatment strategy to reverse the structure of mutant p53 protein and restore its tumor suppressor activity; but this strategy is limited by the smooth surface of mutant p53 protein, the lack of broad-spectrum allosteric regulation sites, and the difficulty of compound binding, making it difficult to develop drugs targeting mutant p53 protein to restore its tumor suppressor activity; as a new emerging tumor treatment strategy, ferroptosis has attracted much attention in recent years; unlike existing cell death forms such as apoptosis, autophagy, necrosis and pyrolysis, ferroptosis is a new iron and reactive oxygen-dependent regulatory cell death form caused by abnormal accumulation of iron-dependent lipid peroxide (LPO); the accumulation of LPO is a dynamic process affected by both LPO production and elimination; in tumor cells, ferrous ions (Fe 2+ ) can react with high levels of hydrogen peroxide (H2O2) to produce hydroxyl radicals, which then oxidize unsaturated fatty acids (PUFA) to LPO, which damages cell membrane structure and induces ferroptosis; in addition, tumor cells have high levels of glutathione (GSH), and glutathione peroxidase 4 (GPX4) with GSH as a cofactor can eliminate LPO to inhibit ferroptosis; based on this, nanomaterials with the ability to consume GSH have been shown to induce ferroptosis; compared with traditional chemotherapy and radiotherapy, this ferroptosis-inducing treatment has lower toxicity and higher efficacy, making targeting ferroptosis a promising tumor treatment strategy.

[0005] The rapid development of nanotechnology provides more possibilities for the application of ferroptosis in tumor treatment; as a carrier, nanomaterials are more likely to selectively accumulate in target tissues due to their unique size and surface effects, thereby improving treatment efficacy; however, current clinically used nanomedicines still have some difficulties in terms of complexity, high cost and uncontrollable toxicity and side effects of the preparation process, and there is an urgent need to develop a new type of high-efficiency and low-toxicity nanomedicine to address these problems.

[0006] Arsenic-containing compounds have been used as medicines to treat diseases for more than 2000 years in ancient and modern times at home and abroad, especially arsenic trioxide (As2O3) with effective component arsenic trioxide (ATO) is most famous for its great success in treating acute promyelocytic leukemia (APL); however, traditional inorganic / organic arsenic drugs including arsenic trioxide are often limited by tumor drug resistance, resulting in poor treatment effect; in addition, the non-specificity of ATO easily causes toxic side effects on normal tissues, seriously affecting the quality of life of patients; studies have shown that the toxicity and efficacy of ATO are mainly related to the valence state of arsenic element, and the toxicity and efficacy of trivalent arsenic (As 3+ ) are significantly higher than those of pentavalent arsenic (As 5+ ).

[0007] The foregoing statements are intended to provide general background information and do not necessarily constitute the prior art. SUMMARY

[0008] The purpose of the present application is to provide a preparation method of a complex drug for treating small cell lung cancer, which solves the problems of high toxicity, poor targeting, low stability of allicin, low bioavailability and lack of effective synergistic delivery system of existing arsenic trioxide drugs, and provides a new drug combination and technical path for individualized treatment of small cell lung cancer, especially in the presence of p53 mutation.

[0009] The present application provides a preparation method of a complex drug for treating small cell lung cancer, which comprises the following steps:

[0010] S1: mixing allicin, phospholipid and cholesterol by using a thin film dispersion method;

[0011] S2: obtaining an arsenic vanadium sample by using a step-by-step temperature control program for the mixture prepared in step S1;

[0012] S3: soaking the arsenic vanadium sample obtained in step S2 in an organic solvent for liquid phase stripping;

[0013] S4: removing the unbroken arsenic vanadium large particles by centrifugation, collecting the supernatant, and obtaining an arsenic vanadium solution;

[0014] S5: removing the organic solvent from the arsenic vanadium solution obtained in step S4 to obtain arsenic vanadium nanodots.

[0015] Further, the step S1 comprises weighing (2, 3-dioxypropyl) trimethylammonium chloride, (2, 3-dioxypropyl) trimethylammonium chloride, cholesterol, DSPE-PEG2000, allicin and octreotide acetate in a 100mL round-bottom flask containing 10mL dichloromethane and 5mL methanol for mixing.

[0016] Further, the step S2 comprises ultrasonicating the mixture prepared in step S1 until completely dissolved.

[0017] Further, the step S3 comprises rotary evaporating the completely dissolved mixture obtained in step S2 at 40℃ under reduced pressure, removing the organic solvent, and drying the mixture to obtain an arsenic-vanadium sample.

[0018] Further, the step S4 comprises mixing the arsenic-vanadium sample obtained in step S3 with hydrogen sodium arsenate heptahydrate in room temperature water, and then taking out the mixture into an EP tube and ultrasonicating for 3 minutes.

[0019] Further, the step S5 comprises centrifuging the mixed solution obtained in step S4 at 8000 rpm for 10 minutes to remove empty vesicles and phospholipid fragments, thereby obtaining arsenic-vanadium nanodots.

[0020] Further, the molar ratio of arsenic and vanadium in the arsenic-vanadium nanodots is 1:1 / 2-2.

[0021] Further, the temperature control procedure in step S2 comprises increasing the temperature to 650℃ within 8 hours, maintaining the temperature for 5 hours; then cooling to 550℃ within 8 hours, maintaining the temperature for 6 hours; then slowly cooling to 500℃ within 2 hours, maintaining the temperature for 8 hours; and finally cooling to room temperature within 20 hours, thereby obtaining an arsenic-vanadium sample.

[0022] Further, the molar ratio of phospholipid and cholesterol in step S1 is 1-5:1.

[0023] Further, the mass ratio of allicin and lipid in step S1 is 1:4 or 1:8 or 1:12 or 1:16 or 1:20.

[0024] The preparation method of the complex drug for treating small cell lung cancer of the present application encapsulates the arsenic trioxide prodrug (hydrogen sodium arsenate heptahydrate) and allicin in liposomes, targets small cell lung cancer tissues, fully plays the synergistic effect of the two in the antitumor mechanism, especially in the synergistic anticancer effect in regulating the mutant p53 signal pathway and the DNA damage response (DDR) pathway, thereby improving the drug efficacy, reducing the toxic side effects, and improving the clinical treatment effect. The present application can solve the problems of the existing arsenic trioxide drug, such as high toxicity, poor targeting, low stability of allicin, low bioavailability, and lack of effective synergistic delivery system, and provides a new drug combination and technical path for individualized treatment of small cell lung cancer, especially in the presence of p53 mutation background. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The flowchart of the preparation method of the complex drug for treating small cell lung cancer of the present application is provided.

[0026] Figure 2 The schematic diagram of the preparation encapsulation rate comparison results of different molar ratio of phospholipid and cholesterol provided for the embodiment 1 of the present application.

[0027] Figure 3 The schematic diagram of the preparation encapsulation rate comparison results of different molar ratio of pentavalent arsenic and lipid provided for the embodiment 2 of the present application.

[0028] Figure 4 The schematic diagram of the preparation encapsulation rate comparison results of different molar ratio of allicin and lipid provided for the embodiment 3 of the present application.

[0029] Figure 5 The schematic diagram of the preparation encapsulation rate comparison results under different temperature provided for the embodiment 4 of the present application.

[0030] Figure 6 The transmission electron microscope diagram of the preparation method of the complex drug for treating small cell lung cancer.

[0031] Figure 7 The stability diagram of the preparation method of the complex drug for treating small cell lung cancer within 7 days.

[0032] Figure 8 The comet experiment diagram of different preparation groups treating different cells.

[0033] Figure 9 The γ-H2AX fluorescence diagram of different preparation groups treating different cells. DETAILED DESCRIPTION

[0034] The specific embodiments of the present application will be further described in detail below in combination with the drawings and examples. The following examples are used to illustrate the present application, but not to limit the scope of the present application.

[0035] The terms "first", "second", "third", "fourth" and the like in the specification and claims of the present application are used to distinguish similar objects, and do not necessarily have to describe a specific order or sequence.

[0036] Embodiment 1

[0037] Figure 1 The schematic diagram of the flow of the preparation method of the complex drug for treating small cell lung cancer provided for the embodiment 1 of the present application. Please refer to Figure 1 The preparation method of the complex drug for treating small cell lung cancer provided by the embodiment of the present application comprises the following steps:

[0038] S1: adopting the thin film dispersion method, mixing allicin, phospholipid and cholesterol;

[0039] Specifically, the step S1 comprises weighing (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOPC), (2, 3-dioleyloxypropyl) trimethylammonium chloride, cholesterol, DSPE-PEG2000, allicin and octreotide acetate into a 100 mL round-bottom flask containing 10 mL dichloromethane and 5 mL methanol for mixing; wherein the molar ratio of phospholipid to cholesterol is 1-5:1.

[0040] S2: The mixture prepared in step S1 is subjected to a step-by-step temperature control program to obtain an arsenic-vanadium sample;

[0041] Specifically, the mixture prepared in step S1 is ultrasonically dissolved; wherein the step-by-step temperature control program comprises heating to 650°C within 8 hours, and maintaining for 5 hours; then cooling to 550°C within 8 hours, and maintaining at this temperature for 6 hours; then slowly cooling to 500°C within 2 hours, and maintaining for 8 hours; and finally cooling to room temperature within 20 hours to obtain an arsenic-vanadium sample.

[0042] S3: The arsenic-vanadium sample obtained in step S2 is immersed in an organic solvent for liquid phase stripping;

[0043] Specifically, the completely dissolved mixture obtained in step S2 is rotary evaporated at 40°C under reduced pressure to remove the organic solvent, and is sufficiently dried to obtain an arsenic-vanadium sample (AcP@LP).

[0044] S4: The mixture obtained in step S3 is centrifuged to remove unbroken arsenic-vanadium large particles, and the supernatant is collected to obtain an arsenic-vanadium solution;

[0045] Specifically, the step S4 comprises mixing the arsenic-vanadium sample obtained in step S3 with disodium hydrogen arsenate heptahydrate at room temperature for hydration, and then taking out to an EP tube for ultrasonic treatment for 3 minutes (ultrasonic power: 50%, ultrasonic for 3 seconds and stop for 7 seconds).

[0046] S5: The arsenic-vanadium solution obtained in step S4 is removed of the organic solvent to obtain arsenic-vanadium nanodots.

[0047] Specifically, the mixed solution obtained in step S4 is centrifuged at 8000 rpm for 10 min to remove empty vesicles and phospholipid fragments, and the arsenic-vanadium nanodots (AsAcP@LP) are obtained.

[0048] Figure 2 The preparation of different molar ratios of phospholipid to cholesterol for Example 1 of the present application is shown in the schematic diagram of the comparison results of the encapsulation efficiency. Figure 2 As shown, when the ratio of phospholipid to cholesterol is 3:1, the liposome constructed has the best particle size uniformity (PDI <0.3) and drug encapsulation efficiency, and therefore the ratio of phospholipid is preferably 3:1.

[0049] Further, the molar ratio of arsenic and vanadium in the arsenic-vanadium nanodots is 1:1 / 2-2.

[0050] Example 2

[0051] The preparation method of this example includes the following steps:

[0052] S1: Weigh (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOPC), (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), cholesterol, DSPE-PEG2000, allicin, octreotide acetate into a 100 mL round-bottom flask containing 10 mL dichloromethane and 5 mL methanol for mixing;

[0053] S2: The mixture prepared in step S1 is ultrasonicated until completely dissolved;

[0054] S3: The completely dissolved mixture obtained in step S2 is rotary evaporated at 40°C under reduced pressure to remove the organic solvent and dry it thoroughly to obtain the AcP@LP sample.

[0055] S4: The AcP@LP sample obtained in step S3 is mixed with the white arsenic prodrug (disodium hydrogen arsenate heptahydrate) at room temperature and then taken out to an EP tube and ultrasonicated for 3 min (ultrasonic power: 50%, ultrasonic for 3 s and stop for 7 s), and the molar ratio of pentavalent arsenic to lipid is 1:1-5;

[0056] S5: The mixed solution obtained in step S4 is centrifuged at 8000 rpm for 10 min to remove empty vesicles and phospholipid fragments, and AsAcP@LP is obtained.

[0057] Figure 3 The preparation of different molar ratios of pentavalent arsenic to lipid for Example 2 of the present application is shown in the schematic diagram. The results are shown in Figure 3 When the molar ratio of pentavalent arsenic to lipid is 1:4, the liposome constructed has the best drug encapsulation rate, so the molar ratio of pentavalent arsenic to lipid is determined to be 1:4.

[0058] Example 3

[0059] The preparation method of this example includes the following steps:

[0060] S1: Weigh (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOPC), (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), cholesterol, DSPE-PEG2000, allicin, octreotide acetate into a 100 mL round-bottom flask containing 10 mL dichloromethane and 5 mL methanol for mixing, and the mass ratio of allicin to lipid is 1:4 or 1:8 or 1:12 or 1:16 or 1:20;

[0061] S2: The mixture prepared in step S1 is ultrasonicated until completely dissolved;

[0062] S3: The completely dissolved mixture obtained in step S2 was evaporated under reduced pressure at 40°C to remove the organic solvent and dried to obtain an AcP@LP sample.

[0063] S4: The AcP@LP sample obtained in step S3 was mixed with a white arsenic prodrug (disodium hydrogen arsenate heptahydrate) and hydrated at room temperature, and then taken out and ultrasonicated in an EP tube for 3 min (ultrasonic power: 50%, ultrasonic for 3 s and stop for 7 s);

[0064] S5: The mixed solution obtained in step S4 was centrifuged at 8000 rpm for 10 min to remove empty vesicles and phospholipid fragments, and AsAcP@LP was obtained.

[0065] Figure 4 The results of the comparison of the encapsulation rates of different molar ratios of allicin and lipids provided for Example 3 of the present application are shown in the schematic diagram. Figure 4 As shown, the liposome constructed when the molar ratio of allicin to lipid is 1:8 has the best drug encapsulation rate, and the PDI is still less than 0.3, so it is determined that the molar ratio of allicin to lipid is 1:8.

[0066] Example 4

[0067] The preparation method of this example includes the following steps:

[0068] S1: (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOPC), (2, 3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), cholesterol, DSPE-PEG2000, allicin, octreotide acetate were weighed and placed in a 100 mL round-bottom flask containing 10 mL dichloromethane and 5 mL methanol for mixing;

[0069] S2: The mixture prepared in step S1 was ultrasonicated until completely dissolved;

[0070] S3: The completely dissolved mixture obtained in step S2 was evaporated under reduced pressure at different temperatures to remove the organic solvent and dried to obtain an AcP@LP sample. The temperature range is 20°C to 60°C;

[0071] S4: The AcP@LP sample obtained in step S3 was mixed with a white arsenic prodrug (disodium hydrogen arsenate heptahydrate) and hydrated at room temperature, and then taken out and ultrasonicated in an EP tube for 3 min (ultrasonic power: 50%, ultrasonic for 3 s and stop for 7 s);

[0072] S5: The mixed solution obtained in step S4 was centrifuged at 8000 rpm for 10 min to remove empty vesicles and phospholipid fragments, and AsAcP@LP was obtained.

[0073] Figure 5The schematic diagram of the comparison results of the encapsulation efficiency of the preparation prepared at different temperatures for the embodiment 4 of the present application is shown in the following table. Figure 5 As shown in the following table, when the reaction temperature is 40℃, the liposome has the optimal encapsulation efficiency and higher stability, and the PDI is controlled within the ideal range. Therefore, 40℃ is finally selected as the optimal reaction temperature for the preparation process of the liposome.

[0074] Embodiment 5

[0075] Figure 6 The transmission electron microscopy (TEM) image of the preparation method of the complex drug for treating small cell lung cancer, Figure 7 The stability diagram of the preparation method of the complex drug for treating small cell lung cancer within 7 days. Please refer to Figure 6 , Figure 7 It should be noted that, in this embodiment, the arsenic-vanadium nanodots (AsAcP@LP) prepared in embodiment 1 are observed by transmission electron microscopy for sample preparation process and stability detection, which specifically includes the following steps:

[0076] The complex liposome delivery system AsAcP@LP is dispersed in pure water, an appropriate amount of solution is diluted to an appropriate multiple, and a drop is dropped on a copper mesh, and then it is dried;

[0077] The morphology is observed by transmission electron microscopy (TEM).

[0078] The TEM result shows that it is spherical, regular in shape, clear in structure, and presents a typical double-layer vesicle structure, with a particle size mainly distributed between 100-200nm, indicating that its size is moderate, and the result is shown in the following table. Figure 6

[0079] Further, in order to investigate the storage stability, the liposome is placed at room temperature for 7 days of standing storage, and the changes of particle size, Zeta potential and PDI and other indicators are measured.

[0080] The results show that, during the entire observation period, the fluctuation of each parameter is small, and there is no significant change, indicating that the liposome has good physical stability, and is suitable for subsequent pharmacodynamic study, and the result is shown in the following table. Figure 7

[0081] Embodiment 6

[0082] Figure 8 The comet experiment diagram of different cell treatments by different preparation groups. Please refer to Figure 8 It should be noted that, in this embodiment, the synergistic anticancer effect of AsAcP@LP prepared in embodiment 1 in regulating mutant p53 signaling pathway and DNA damage response (DDR) pathway is detected, and the cell DNA damage degree is evaluated, which specifically includes the following steps:

[0083] ​​Construction of lentivirus-transfected H1299-P53 non-small cell lung cancer cells: adherent cells were seeded in 24-well plates at a density of 1 x 10 5 cells / well, ensuring that the cell density reached about 2 x 10 5 cells / well at the time of transfection; on the day of transfection, the original culture medium was discarded, 2 mL of fresh complete culture medium containing 6 μg / mL Polybrene was added, followed by the addition of the virus suspension, and incubation at 37°C, 5% CO2 for 24 hours; then the virus-containing culture medium was discarded, washed twice with PBS and replaced with fresh complete culture medium for continuous culture for 48 hours. 72 hours after transfection, 2 μg / mL of puromycin was added to the final concentration for resistance screening until all negative control cells died.

[0084] The degree of DNA damage in cells was evaluated using a comet electrophoresis kit (Beyotime Biotechnology, China);

[0085] The cells to be tested were collected, mixed with low-melting-point agarose, and then coated on a pretreated glass slide;

[0086] After lysis treatment, DNA unwinding was performed under alkaline conditions, and electrophoresis was performed at 25V for 30 min.

[0087] The glass slide was then neutralized, stained with DNA fluorescent dye provided in the kit, and observed under a fluorescence microscope.

[0088] The comet tail length and tail moment parameters were analyzed by CASP software to quantitatively evaluate the degree of DNA damage.

[0089] The results showed that in H1299 cells, the Tail DNA% of the Control group and the AsAcP@LP group were both below 10%, belonging to mild damage or no damage; while in H1299-P53 cells, the Tail DNA% of the AsAcP@LP group was >40%, indicating high damage. The results are shown in Figure 8 .

[0090] Example 7

[0091] Figure 9 The γ-H2AX fluorescence images of different cells treated with different formulations. Please refer to Figure 9 , it should be noted that this example is to detect the synergistic anticancer effect of AsAcP@LP prepared in Example 1 in regulating the mutant p53 signaling pathway and the DNA damage response (DDR) pathway, and to evaluate the degree of DNA damage in cells, which includes the following steps:

[0092] Cellular DNA damage was assessed using a DNA damage detection kit (Beyotime Biotechnology, China).

[0093] After fixing and permeabilizing the cells, fluorescently labeled γ-H2AX antibody was added for incubation.

[0094] After washing, DAPI staining was used for nuclear staining.

[0095] Fluorescent images were collected using a confocal microscope, and the degree of DNA double-strand breakage was evaluated by the number of γ-H2AX fluorescent spots.

[0096] As shown in the immunofluorescence results, γ-H2AX was expressed very little in H1299 cells, and there was no obvious fluorescent signal focus in the H1299-P53 cell control group. In the AsAcP@LP group, the fluorescence intensity and particle accumulation gradually increased. These results showed that the combination of white arsenic and allicin induced more extensive DNA double-strand breakage damage in small cell lung cancer cells containing mutant P53; the results are shown in Figure 9 .

[0097] Example 8

[0098] This example evaluates the cytotoxicity of the different preparation groups described above in different cells (human umbilical vein endothelial cells (HUVEC) and mouse breast cancer cells (4T1)), and includes the following steps:

[0099] Logarithmic growth phase 4T1 cells were taken and seeded at 1×105 cells per well in a 96-well plate, and cultured at 37°C for 12 hours.

[0100] The culture medium was discarded, and blank culture medium and different concentrations of different group drugs (arsenicene group, vanadocene group, arsenic-vanadium group) were added to treat the cells for 24 hours.

[0101] The drug-containing culture solution was discarded, and thiazolyl blue (MTT) was added for incubation in the dark for 4 hours. After discarding the MTT solution, dimethyl sulfoxide (DMSO) solution was added, and the absorbance was measured at 490 nm to calculate the cell survival rate.

[0102] The results show that the arsenicene preparation group has greater toxicity, and the cell viability of the arsenic-vanadium preparation group in normal cells is more than 85%, and the toxicity is smaller. In tumor cells, the cell survival rate of the cells treated by the arsenic-vanadium preparation group is less than 30%, and the arsenic-vanadium preparation group has good killing power on tumor cells.

[0103] Example 9

[0104] This example evaluates the production of reactive oxygen species in cells by the different preparation groups described above, and includes the following steps:

[0105] Take the logarithmic growth phase of 4T1 cells, 5x105 cells per well were inoculated in laser confocal dish, 37℃ incubation for 12 hours.

[0106] Discard the culture medium, add blank medium and 20 μg / mL of different groups of drugs (arsenic group, vanadium group, arsenic vanadium group) to treat cells for 24 hours.

[0107] Discard the culture medium containing drugs, add active oxygen probe DCFH-DA (10 mM) and incubate in the dark for 20 minutes, and finally observe the green fluorescence intensity under 488 nm wavelength by laser confocal microscope (CLSM).

[0108] The results show that the arsenic vanadium preparation group produces a large amount of active oxygen, indicating that the cells enter the oxidative stress state.

[0109] Example 10

[0110] This example analyzes the GPX4 protein blotting experiment of different preparation groups in the above-mentioned cells, which specifically includes the following steps:

[0111] Take the logarithmic growth phase of 4T1 cells 5x105 cells per well were inoculated in 6 well plates, set Control group (blank control group, add blank culture medium), As group (arsenic group), V group (vanadium group), AsV group (arsenic vanadium group), each group was given 20 μg / mL, and cultured for 12 hours after drug administration.

[0112] Further, the cells were lysed with RIPA lysis buffer, and the protein content of the extracted protein was quantified by BCA protein determination kit.

[0113] Further, the lysate was loaded into a 15% sodium dodecyl sulfate polyacrylamide gel, which was further transferred to a PVDF membrane.

[0114] Further, the membrane was blocked with protein-free rapid blocking buffer at room temperature for 1 hour, and then incubated with GPX4 and β-actin primary antibody at 4℃ overnight.

[0115] Further, wash 3 times with TBST buffer solution at pH 7.4, and then incubate with secondary antibody at room temperature for 2 hours.

[0116] Finally, develop with chemiluminescence instrument, and observe the GPX4 protein level.

[0117] The results show that the arsenic vanadium preparation group significantly reduces the content of intracellular GPX4, indicating that the GPX4 enzyme in vivo is inactivated, further inducing iron death.

[0118] Example 11

[0119] The embodiment detects the lipid peroxide (MDA) in cells of different preparation groups in the above-mentioned method, and specifically comprises the following steps:

[0120] Further, 4T1 cells in the logarithmic growth phase are inoculated in a 6-well plate at 1*106 per well, and cultured at 37 DEG C for 12 hours.

[0121] Further, the culture medium is discarded, and the cells are treated with blank culture medium and 20ug / mL of different groups of drugs (arsenicene group, vanadocene group, arsenic-vanadium group) for 12 hours.

[0122] Further, the cells are collected and washed with PBS buffer solution with pH 7.4 for 3 times.

[0123] Further, the intracellular MDA content is detected by using a lipid peroxide detection kit.

[0124] The results show that the intracellular MDA content of the arsenic-vanadium preparation group is significantly increased, which is consistent with the expected effect of ferroptosis.

[0125] Embodiment 12

[0126] The embodiment detects the pharmacodynamic effect of different preparation groups of mice in the above-mentioned method, and specifically comprises the following steps:

[0127] Further, 4T1 cells in the logarithmic growth phase are inoculated in a 6-well plate at 1*106 per well, and cultured at 37 DEG C for 12 hours.

[0128] Further, after the modeling is successful, the mice are given drugs every two days, and each mouse is given 4mg / kg of drugs.

[0129] Further, the tumor volume of the mice is measured before each drug administration, and the measurement results are recorded.

[0130] The results show that the tumor growth rate of the arsenic-vanadium preparation group is obviously lower than that of other preparation groups, indicating that the arsenic-vanadium preparation has good pharmacodynamic effect and can play an anti-tumor role.

[0131] Based on the above description, the advantages of the present application are as follows:

[0132] 1. The preparation method of the composite drug for treating small cell lung cancer of the present application, the arsenic trioxide prodrug (disodium hydrogen arsenate heptahydrate) is combined with allicin to be encapsulated in liposomes, targets small cell lung cancer tissue, fully plays the synergistic effect of both in the antitumor mechanism, especially in the synergistic anticancer effect in regulating mutant p53 signal pathway and DNA damage response (DDR) pathway, thereby improving the drug efficacy, reducing the toxic side effects, and improving the clinical treatment effect; the present application can solve the problems of the existing arsenic trioxide drug, such as high toxicity, poor targeting, low stability of allicin, low bioavailability, and lack of effective synergistic delivery system, and provide a new drug combination and technical path for individualized treatment of small cell lung cancer, especially in the presence of p53 mutation background.

[0133] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a complex drug for treating small cell lung cancer, characterized by, The method comprises the following steps: S1: mixing allicin, phospholipid and cholesterol by using a thin film dispersion method; S2: obtaining an arsenic-vanadium sample by using a step-by-step temperature control program on the mixture prepared in step S1; S3: soaking the arsenic-vanadium sample obtained in step S2 in an organic solvent for liquid phase exfoliation; S4: removing unbroken arsenic-vanadium large particles by centrifugation on the mixture obtained in step S3, collecting the supernatant, and obtaining an arsenic-vanadium solution; S5: removing the organic solvent from the arsenic-vanadium solution obtained in step S4 to obtain arsenic-vanadium nanodots.

2. The method of claim 1, wherein the method is for treating small cell lung cancer. The step S1 comprises weighing (2, 3-dioleyloxypropyl) trimethylammonium chloride, (2, 3-dioleyloxypropyl) trimethylammonium chloride, cholesterol, DSPE-PEG2000, allicin and octreotide acetate in a 100mL round-bottom flask containing 10mL dichloromethane and 5mL methanol for mixing.

3. The method of claim 1, wherein the method is for preparing a complex drug for treating small cell lung cancer. The step S2 comprises ultrasonicating the mixture prepared in step S1 until complete dissolution.

4. The method of claim 1, wherein the method is for preparing a complex drug for treating small cell lung cancer. The step S3 comprises rotary evaporation of the completely dissolved mixture obtained in step S2 at 40℃ under reduced pressure to remove the organic solvent, and fully drying to obtain an arsenic-vanadium sample.

5. The method of claim 1, wherein the method is for preparing a complex drug for treating small cell lung cancer. The step S4 comprises mixing the arsenic-vanadium sample obtained in step S3 with disodium hydrogen arsenate heptahydrate at room temperature, then taking it out to an EP tube and ultrasonicating for 3 minutes.

6. The method of claim 1, wherein the method is for preparing a complex drug for treating small cell lung cancer. The step S5 comprises centrifuging the mixed solution obtained in step S4 at 8000rpm for 10min to remove empty vesicles and phospholipid fragments, thereby obtaining arsenic-vanadium nanodots.

7. The method of claim 1, wherein the method is for preparing a complex drug for treating small cell lung cancer. The molar ratio of arsenic and vanadium in the arsenic-vanadium nanodots is 1:1 / 2-2.

8. The method of claim 1, wherein the method is for preparing a complex drug for treating small cell lung cancer. The step-by-step temperature control program in step S2 comprises heating to 650℃ within 8 hours, keeping the temperature for 5 hours; then cooling to 550℃ within 8 hours, keeping the temperature for 6 hours; then slowly cooling to 500℃ within 2 hours, keeping the temperature for 8 hours; and finally cooling to room temperature within 20 hours to obtain an arsenic-vanadium sample.

9. The method of claim 1, wherein the method is for preparing a complex drug for treating small cell lung cancer. The molar ratio of phospholipid to cholesterol in step S1 is 1-5:

1.

10. The method of claim 1, wherein the method is for preparing a complex pharmaceutical for treating small cell lung cancer. The mass ratio of allicin to lipid in step S1 is 1:4 or 1:8 or 1:12 or 1:16 or 1:20.

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