An anti-tumor drug preparation, its preparation method and application

Abstract

This application belongs to the technical field of biopharmaceutical preparations, and provides an anti-tumor drug preparation, its preparation method and application. This anti-tumor drug preparation can inhibit the activity of tumor cells by enhancing the therapeutic effect of broad-spectrum anti-tumor drugs, and can effectively reduce the size of solid tumors. This anti-tumor drug preparation is a silica ore anti-tumor drug preparation, and this anti-tumor drug preparation includes: a silicate mineral containing one or more silicate ions and at least one broad-spectrum anti-tumor drug; wherein, the concentration range of silicon is 0.25 to 120 ppm.

Description

Technical Field

[0001] This application belongs to the technical field of biopharmaceutical preparations, and particularly relates to an anti-tumor drug preparation, its preparation method and application. Background Art

[0002] Tumor has always been a major disease endangering human health, which can cause damage to the structure and function of normal human tissues and organs, and in severe cases, can lead to the death of patients. Chemotherapy is one of the main ways to treat tumors currently.

[0003] Currently, the drugs mainly used for chemotherapy clinically include bortezomib, doxorubicin, cisplatin, etc. Usually, due to the large amount of drugs used in the chemotherapy process, a large number of chemotherapy drugs will cause side effects such as gastrointestinal discomfort, cell damage, and bone marrow suppression to patients to a certain extent. However, reducing the drug dosage will result in incomplete tumor clearance or even drug resistance of tumors, and these drugs are expensive and have a long treatment cycle, which will cause great pressure on patients both mentally and materially.

[0004] Therefore, there is an urgent need to provide an anti-tumor drug preparation that can not only reduce the dosage of chemotherapy drugs but also enhance the therapeutic effect of chemotherapy drugs on tumors. Summary of the Invention

[0005] In view of this, the embodiments of this application provide an anti-tumor drug preparation, its preparation method and application. This anti-tumor drug preparation can inhibit the activity of tumor cells by enhancing the therapeutic effect of broad-spectrum anti-tumor drugs, and can effectively reduce the size of solid tumors.

[0006] The first aspect of the embodiments of this application provides an anti-tumor drug preparation, which is a silica ore anti-tumor drug preparation. This anti-tumor drug preparation includes: a silicate mineral containing one or more silicate ions and at least one broad-spectrum anti-tumor drug; wherein, the concentration range of silicon is 0.25 - 120 ppm.

[0007] Combined with the first aspect, in the first possible implementation manner of the first aspect, the broad-spectrum anti-tumor drugs include: one or more of bortezomib, cisplatin, doxorubicin, PD-L1, paclitaxel, vinorelbine, oxaliplatin, docetaxel, and gemcitabine.

[0008] Combined with the first aspect, in the second possible implementation manner of the first aspect, the silicate minerals include one or more of calcium silicate, sodium silicate, melilite, zinc melilite, cuprosklodowskite, zinc silicate, copper silicate, bioactive glass, and copper- or zinc-doped mesoporous silica.

[0009] In combination with the first aspect, in the third possible implementation manner of the first aspect, the anti-tumor drug preparation includes: a silicate mineral with a silicon concentration of 30 ppm, doxorubicin, cisplatin, and bortezomib; wherein, the concentration of doxorubicin is 0-20 μM, the concentration of cisplatin is 0-10 μM, and the concentration of bortezomib is 0-500 nM.

[0010] In combination with the first aspect, in the fourth possible implementation manner of the first aspect, the anti-tumor drug preparation includes: a combination anti-tumor drug preparation of silicate ion / bortezomib, a composite anti-tumor drug preparation of calcium silicate / doxorubicin, and a composite anti-tumor drug preparation of doxorubicin / melilite / zirconolite / cuspidine powder.

[0011] The second aspect of the embodiments of the present application provides a preparation method of an anti-tumor drug preparation, including: preparing a silicate ion preparation with a silicon concentration range of 0.5-120 ppm from a silicate mineral; configuring a drug preparation of a broad-spectrum anti-tumor drug, and the drug preparation of the broad-spectrum anti-tumor drug includes a drug preparation with a preset concentration corresponding to at least one broad-spectrum anti-tumor drug; mixing the silicate ion preparation and the drug preparation of the broad-spectrum anti-tumor drug according to a preset ratio to obtain an anti-tumor drug preparation.

[0012] In combination with the second aspect, in the first possible implementation manner of the second aspect, the silicate ion preparation is prepared by sterile water, PBS, physiological saline or cell culture medium.

[0013] In combination with the second aspect, in the second possible implementation manner of the second aspect, the method further includes: dissolving one or more of the silicate minerals corresponding to the silicate ion preparation and a broad-spectrum anti-tumor drug in a polymer solution to prepare a product in the form of a microneedle, hydrogel or electrospinning; wherein, the polymer solution includes at least one of a sodium alginate solution, a hyaluronic acid solution or a gellan gum solution.

[0014] The third aspect of the embodiments of the present application provides an application of an anti-tumor drug preparation, and the application includes the application of the anti-tumor drug preparation described in the first aspect in drug-resistant tumors.

[0015] In combination with the third aspect, in the first possible implementation manner of the third aspect, the drug-resistant tumors include one or more of tumors caused by acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchial lung cancer (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer, etc.

[0016] The beneficial effects of the embodiments of the present application compared with the prior art are:

[0017] The embodiments of the present application provide an anti-tumor drug preparation, its preparation method and application. The anti-tumor drug preparation is a silicon ore anti-tumor drug preparation, and the anti-tumor drug preparation includes: a silicate mineral containing one or more silicate ions and at least one broad-spectrum anti-tumor drug; wherein, the concentration range of silicon is 0.25-120 ppm. The anti-tumor drug preparation can inhibit the activity of tumor cells by enhancing the therapeutic effect of the broad-spectrum anti-tumor drug, and can effectively reduce the size of solid tumors. Moreover, this anti-tumor drug preparation can reduce the dosage of the drug while enhancing the therapeutic effect of the chemotherapeutic drug, and to a certain extent, relieve the burden on patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is a schematic diagram of the experimental results of silicate ions enhancing the apoptosis of human multiple myeloma cell lines induced by bortezomib provided by the embodiments of the present application;

[0020] Figure 2 It is a schematic diagram of the experimental results of silicate ions combined with bortezomib inducing apoptosis of newly diagnosed multiple myeloma cells provided by the embodiments of the present application;

[0021] Figure 3 It is a schematic diagram of the experimental results of silicate ions combined with bortezomib inducing apoptosis of refractory or relapsed multiple myeloma cells provided by the embodiments of the present application;

[0022] Figure 4 It is a schematic diagram of the experimental results of silicate ions enhancing the G2 / M cell cycle arrest of bortezomib on human multiple myeloma cell lines provided by the embodiments of the present application;

[0023] Figure 5 It is a schematic diagram of the experimental results of silicate ions enhancing the inhibition of the NF-κB pathway by bortezomib provided by the embodiments of the present application;

[0024] Figure 6 It is a schematic diagram of the effects of silicate ions combined with doxorubicin and cisplatin on the activities of osteosarcoma cells, breast cancer cells and melanoma cells provided by the embodiments of the present application;

[0025] Figure 7 It is a schematic diagram of the effects of different types of silicon ore combined with doxorubicin on melanoma cells provided by the embodiments of the present application;

[0026] Figure 8 It is a schematic diagram of the action process of silicate anions combined with bortezomib in multiple myeloma in mice provided by an embodiment of the present application. Detailed implementation manners

[0027] In the following description, specific details such as specific system structures and technologies are presented for the purpose of illustration rather than limitation, so as to thoroughly understand the embodiments of the present application. However, those skilled in the art should clearly understand that the present application can also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to avoid unnecessary details from interfering with the description of the present application.

[0028] The technical solutions provided by the present application are explained and described in detail below in conjunction with specific embodiments.

[0029] An embodiment of the present application provides an anti-tumor drug preparation, which includes: a silicate mineral containing silicate anions and at least one broad-spectrum anti-tumor drug; wherein, the concentration range of silicon in the silicate mineral containing silicate anions is 0.5-120 ppm.

[0030] In the anti-tumor drug preparation provided in this embodiment, the broad-spectrum anti-tumor drugs include: one or more of bortezomib, cisplatin, doxorubicin, paclitaxel, vinorelbine, oxaliplatin, docetaxel, and gemcitabine. Specifically, it can be selected according to actual needs when preparing the anti-tumor drug preparation, and no specific limitation is made in this embodiment.

[0031] In some embodiments, the silicate mineral includes one or more of calcium silicate, sodium silicate, melilite, zinc melilite, cuspidine, zinc silicate, copper silicate, bioactive glass, and copper- or zinc-doped mesoporous silica.

[0032] As an embodiment of the anti-tumor drug preparation provided by the present application, the anti-tumor drug preparation includes: a silicate mineral with a silicon concentration of 30 ppm, doxorubicin, cisplatin, and bortezomib. Among them, the concentration of doxorubicin is 0.5-20 μM, preferably 10 μM; the concentration of cisplatin is 0.5-10 μM, preferably 5 μM; the concentration of bortezomib is 0.5-500 nM, preferably 50 nM.

[0033] The anti-tumor drug preparation provided in this embodiment is applied to drug-resistant tumors, which include tumors caused by acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, squamous cell carcinoma of the head and neck, testicular cancer, gastric cancer, liver cancer, etc., one or more of them.

[0034] The following is a detailed explanation of the preparation method of the anti-tumor drug preparation provided in the embodiments of the present application.

[0035] The preparation method of the anti-tumor drug preparation provided in the embodiments of the present application includes the following steps S101 to S105.

[0036] S101. Prepare a silicate ion preparation with the concentration range of silicon being 0.5 - 120 ppm from a silicate mineral.

[0037] S102. Configure a drug preparation of a broad-spectrum anti-tumor drug, and the drug preparation of the broad-spectrum anti-tumor drug includes drug preparations with preset concentrations corresponding to at least one broad-spectrum anti-tumor drug.

[0038] S103. Mix the silicate ion preparation and the drug preparation of the broad-spectrum anti-tumor drug according to a preset ratio to obtain an anti-tumor drug preparation.

[0039] S104. Dissolve one or more of the silicate minerals corresponding to the silicate ion preparation and the broad-spectrum anti-tumor drug in a polymer solution to prepare products in the form of microneedles, hydrogels or electrospun fibers;

[0040] Among them, the polymer solution includes at least one of sodium alginate solution, hyaluronic acid solution or gellan gum.

[0041] The following further explains the preparation method of the above anti-tumor drug preparation with specific examples.

[0042] As an example of the preparation method of the anti-tumor drug preparation provided by this application, a silicate ion / bortezomib (BOR) combined anti-tumor drug preparation is prepared. Specifically, the preparation method of the silicate ion preparation is as follows: Dissolve calcium silicate powder in deionized water at a solid / liquid ratio of 1 mg / mL, and add it to RPMI-1640 culture medium according to a certain ratio to prepare an ion preparation with a concentration of 0.25-120 ppm (μg / mL); the preparation method of the BOR preparation is as follows: Dissolve BOR in phosphate buffered saline (PBS) at a solid / liquid ratio of 1 mM, and add it to RPMI-1640 culture medium according to a certain ratio to prepare a drug preparation with a concentration of 0-500 nM; the preparation method of the silicate ion / bortezomib (BOR) combined anti-tumor drug preparation is as follows: Mix the above silicate ion preparation and BOR drug preparation according to a preset ratio, where the concentration of the silicon preparation is 0.25-120 ppm (μg / mL), and the concentration of the BOR drug preparation is 0-100 nM.

[0043] As an example of the preparation method of the anti-tumor drug preparation provided by this application, a calcium silicate / broad-spectrum anti-tumor drug composite preparation is prepared. Specifically, the preparation method of the broad-spectrum anti-tumor drug preparation is as follows: Dissolve any one of doxorubicin and cisplatin in PBS at a solid / liquid ratio of 1 mM, and add it to the tumor culture medium according to a certain ratio to prepare a broad-spectrum anti-tumor drug preparation with a concentration of 0-20 μM; the preparation method of the calcium silicate preparation is as follows: Dissolve calcium silicate powder in deionized water at a solid / liquid ratio of 1 mg / mL, and add it to the special cell culture medium for drug-resistant tumor cells according to a certain ratio to prepare an ion preparation with a concentration of 0.25-120 ppm (μg / mL); the preparation method of the calcium silicate-enhanced broad-spectrum anti-tumor drug preparation is as follows: Mix the above several preparations according to a preset ratio, where the concentration of the broad-spectrum anti-tumor drug is 0-20 μM, and the concentration of calcium silicate is 0.25-120 ppm (μg / mL).

[0044] In addition, in some embodiments, the present embodiment also provides a method for preparing a broad-spectrum anti-tumor drug / various silicate ore composite drug preparation. Specifically, the preparation method of the broad-spectrum anti-tumor drug preparation is as follows: Dissolve doxorubicin in PBS at a solid / liquid ratio of 1 mM, and add it to the tumor culture medium in a certain proportion to prepare a broad-spectrum anti-tumor drug preparation with a concentration of 0-20 μM; the preparation method of the various silicate ore preparations is as follows: According to ISO / EN 10993-12, prepare a series of diluted ion extracts of melilite / willemite / roselite powders. First, add melilite / willemite / roselite powders to the serum-free tumor cell culture medium, incubate at 37 °C for 24 hours at a solid / liquid ratio of 200 mg / mL, centrifuge the mixture at 4000 rpm for 10 minutes, and then sterilize and collect the supernatant through a filter membrane (Millipore, 0.22 μm) to obtain a stock solution. Then, dilute the stock solution with the tumor cell culture medium to prepare a suitable ion extract; the preparation method of the various silicate ore enhanced broad-spectrum anti-tumor drug preparation is as follows: Mix the above several preparations in a preset proportion, where the concentration of doxorubicin is 0-20 μM and the concentration of silicate ore is 0.25-120 ppm (μg / mL).

[0045] In the above manner, anti-tumor drug preparations such as silicate ion / bortezomib (BOR) combination anti-tumor drug preparation, calcium silicate / broad-spectrum anti-tumor drug composite preparation, and broad-spectrum anti-tumor drug / various silicate ore composite drug preparation can be prepared. The anti-tumor drug preparation prepared by the preparation method in the embodiment of the present application can inhibit the activity of tumor cells by enhancing the therapeutic effect of the broad-spectrum anti-tumor drug.

[0046] The following will take specific experimental examples to explain in detail the enhancement of the therapeutic effect of the broad-spectrum anti-tumor drug by the anti-tumor drug preparation prepared by the preparation method of the anti-tumor drug preparation provided in the embodiment of the present application.

[0047] First is the effect of silicate ions on enhancing bortezomib-induced apoptosis in human multiple myeloma cell lines (HMCLs). Specifically, in one embodiment of the present application, an anti-tumor drug preparation of silicate ions / bortezomib combination was used to evaluate apoptosis and cell cycle of HMCLs. First, HMCLs were treated with the anti-tumor drug preparation of silicate ions / bortezomib combination and cultured for 24 hours, stained with Annexin V-FITC / PI (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions, and then analyzed by flow cytometry (BD Biosciences) to detect apoptosis. Apoptosis data was evaluated using Flow Jo software (v10, Tree Star, Ashland, United States). HMCLs were treated with the solution configured above and cultured for 24 hours, stained with PI (Multi-Sciences, Lianke Bio, China) according to the manufacturer's instructions, and then analyzed by flow cytometry (BD Biosciences) to detect the cell cycle. Mod-Fit LT software (v3.1, Verity software House, Inc., Topsham, ME, United States) was used to evaluate cell cycle data.

[0048] Next is the effect of silicate ions on enhancing bortezomib in newly diagnosed multiple myeloma (NDMM) and relapsed / refractory multiple myeloma (RRMM). In one embodiment of the present application, an anti-tumor drug preparation of silicate ions / bortezomib combination was used to evaluate human peripheral blood mononuclear cells (PBMCs), primary NDMM cells isolated from NDMM patients, and primary RRMM cells isolated from RRMM patients.

[0049] Next is the effect of calcium silicate enhanced doxorubicin / cisplatin preparation on the activity of drug-resistant tumor cells. In one embodiment of the present application, three drug-resistant tumor cells are selected in this experiment, MNNG (osteosarcoma cells), 4T1 (breast cancer cells), B16F10 (melanoma cells). The drugs selected are doxorubicin hydrochloride and cisplatin (typical broad-spectrum anti-tumor drugs in clinical practice). The silicate ion solution is prepared with calcium silicate, and the concentrations are selected as 0, CS / 1 / 8, CS / 1 / 4, CS / 1 / 2. For the well-cultured tumor cells, aspirate the culture medium, drop in 1 - 2 ml of trypsin, then place in the incubator for 1 min, add an equal amount of culture medium to neutralize, centrifuge (1000 r, 3 min), then aspirate the supernatant, add 5 ml of culture medium, pipette, place about 1000 cells in each well plate, place in the incubator for one day. Wait until the tumor cells are completely adherent, then aspirate the culture medium, add 100 μl of the drug solution containing silicate ions (the solution filtered through a bacterial membrane), place in the incubator for 2 d. After taking out, aspirate the waste liquid, then add 100 μl of CCK8 solution, and then place in the incubator for 2 h. The results are obtained by testing with an enzyme-linked immunosorbent assay (ELISA) reader.

[0050] Furthermore, it is the efficacy of Si / BOR combination therapy on multiple myeloma (ARP-1) in mice. In one embodiment of the present application, 6-week-old NOD-SCID (non-obese diabetic-congenitally mutant immunodeficient) mice are subcutaneously injected with ARP-1 (5×106) cells on both sides to establish an MM (multiple myeloma) xenograft model. After 7 days, when the tumors form, the mice are randomly divided into four groups (n = 6 in each group), including a vehicle, BOR, Si, and Si / BOR combination groups. BOR (1 mg / kg) is intraperitoneally injected once every three days for 2 weeks, and Si (200 μl) is subcutaneously injected around the tumor once a day for 2 weeks. The tumor size is monitored with calipers every two days. Tumor tissues are taken from tumor-bearing NOD / SCID mice, and the tumor masses are fixed in 4% paraformaldehyde and then embedded in paraffin before sectioning. Finally, immunohistochemical staining is performed to analyze cleaved caspase-3.

[0051] The above experimental process will be explained below in the form of a specific implementation process.

[0052] Example 1: Effect of silicate ions (Si) on enhancing bortezomib (BOR)-induced apoptosis in human multiple myeloma cell lines (HMCLs).

[0053] The preparation method of the calcium silicate preparation is as follows: Dissolve calcium silicate powder in deionized water at a solid / liquid ratio of 1 mg / mL, and add it to RPMI-1640 culture medium according to a certain ratio to prepare an ion preparation with a concentration of 0.25 - 120 ppm (μg / mL).

[0054] The preparation method of the BOR preparation is as follows: Dissolve BOR in PBS at a solid / liquid ratio of 1 mM, and add it to RPMI-1640 culture medium in an appropriate ratio to prepare a drug preparation with a concentration of 0-500 nM.

[0055] The preparation method of the Si-enhanced BOR preparation is as follows: Mix the above several preparations in a specific ratio, where the concentration of the calcium silicate preparation is 0.25-120 ppm (μg / mL), and the concentration of the BOR preparation is 0-100 nM

[0056] Based on the above-prepared solution, HMCLs were treated and cultured for 24 hours, then stained with Annexin V-FITC / PI (Dojindo, Kumamoto, Japan), and analyzed by flow cytometry (BD Biosciences) to detect apoptosis. Apoptosis data was evaluated using Flow Jo software (v10, Tree Star, Ashland, United States).

[0057] Based on the above-prepared solution, HMCLs were treated and cultured for 24 hours, then stained with PI staining (Multi-Sciences, Lianke Bio, China), and analyzed by flow cytometry (BD Biosciences) to detect the cell cycle. Mod-Fit LT software (v3.1, Verity software House, Inc., Topsham, ME, United States) was used to evaluate the cell cycle data.

[0058] First, determine whether Si has cytotoxicity to MM cells according to the data shown: HMCLs (ARP-1, CAG, NCI-H929, JJN3, LP1, MM1.S, U266, U266 / BOR (BOR-resistant cells), and RPMI-8226) were treated with different concentrations of CS for 24 hours, and then apoptosis was analyzed by flow cytometry. The results showed that different concentrations of Si did not increase the apoptosis of MM cells. Then, judge the synergistic effect of the Si / BOR combination on different MM cells: HMCLs including ARP-1, U266, and U266 / BOR were incubated with Si, BOR, and the Si / BOR combination for 24 hours, and apoptosis was analyzed by flow cytometry. From the cell survival rate in the Q4 quadrant, it can be obtained that, as Figure 1As shown in Part A, untreated MM cells had a higher survival rate (ARP-1: 90.3%; U266: 90.0%; U266 / BOR: 82.5%), while Si alone had no effect on cell survival rate (ARP-2: 88.2%; U264: 92.4%; U266 / BOR: 81.1%). When two non-resistant MM cells were treated with low-dose BOR (ARP-1: 2 nM; U266: 5 nM), the cell survival rate decreased slightly (ARP1: 84.0%; U26: 81.2%), while the Si / BOR combination significantly reduced the cell survival rate (ARP-1: 259.2%; U266: 52.2%). For the drug-resistant MM cell line (U266 / BOR), it was observed that treating U266 / BOR with a high dose of 50 nM BOR resulted in a cell survival rate of 56.1%, while the Si / BOR combination with the same BOR dose (50 nM) significantly reduced the cell survival rate to 36.5%. In addition, the half-maximal inhibitory concentration (IC50) of BOR was calculated based on the results of flow cytometry. It can be seen that the IC50 of BOR in U266 / BOR (IC50: 65.58 nM) was much higher than that in BOR-sensitive cell lines (ARP-1 and U266; IC50: 5.31 nM and 7.87 nM). Additionally, the research data showed that the Si / BOR combination treatment significantly reduced the IC50 of BOR, especially for the BOR-resistant cell lines (ARP-1, IC50: 3.01 nM; U266, IC50: 4.59 nM; U266 / BOR, IC50: 18.44 nM), as Figure 1 shown in Part B. To confirm the above results, western blotting was used to evaluate the expression of apoptosis-related proteins in this example. As Figure 1 shown in Part C, combination treatment significantly increased the expression of cleaved caspase-3 and cleaved PARP1. In summary, this experiment showed that Si, as a sensitizer, could increase the cytotoxic effect of BOR on HMCLs (including BOR-resistant cell lines).

[0059] Example 2: Silicate ions (Si) enhance bortezomib (BOR) treatment for newly diagnosed multiple myeloma (NDMM) and refractory or relapsed multiple myeloma (RRMM).

[0060] Myeloma sample acquisition: Bone marrow samples were obtained from MM patients, and peripheral blood was obtained from healthy donors.

[0061] Myeloma cell culture: Bone marrow mononuclear cells (BMC) and peripheral blood mononuclear cells (PBMC) were isolated using lymphocyte separation medium (Corning, USA), and then cultured in RPMI-1640 medium containing 20% FBS under an atmosphere of 37°C and 5% CO2. Primary NDMM and RRMM cells were labeled with CD138 flow antibody.

[0062] To further verify the activity and safety of the Si / BOR combination, in this example, peripheral blood mononuclear cells (PBMC) from healthy individuals, primary NDMM cells isolated from NDMM patients, and primary RRMM cells isolated from RRMM patients were specifically selected. Treatment with a series of concentrations of BOR and / or 1 / 2 concentration of CS in vitro could not induce apoptosis of PBMC isolated from healthy donors. For primary NDMM cells, treatment with BOR alone led to a significant increase in apoptosis, characterized by a CD138+ immunophenotype, while treatment with a 1 / 2 concentration of Si solution alone could not induce apoptosis (P<0.05). Compared with treatment with BOR alone, the Si / BOR combination treatment led to a significant increase in apoptosis, as shown in Figure 2 Parts A and B. Then, in this example, it was further evaluated whether the Si / BOR combination could still induce apoptosis of primary RRMM cells isolated from RRMM patients. As shown in Figure 3 Parts A and B, in 5 RRMM patient samples, compared with BOR alone, the Si / BOR combination treatment led to a significant increase in apoptosis of CD38+ cells. These results indicate that the combination of Si and BOR not only effectively killed primary NDMM cells isolated from NDMM patients, but also significantly inhibited RRMM cells isolated from RRMM patients, see Figure 2 and Figure 3 shown. More importantly, Si / BOR did not kill normal hematopoietic stem cells, indicating its good biosafety.

[0063] Example 3 Si enhances the G2 / M cell cycle arrest of HMCLs induced by BOR.

[0064] HMCLs cells were treated with the Si / BOR combination drug for 24 hours.

[0065] The cells were stained with PI (Multi-Sciences, Lianke Bio, China).

[0066] Flow cytometry (BD Biosciences) was used for analysis to detect the cell cycle.

[0067] Mod-Fit LT software (v3.1, Verity Software House, Inc., Topsham, ME, United States) was used to evaluate cell cycle data.

[0068] G2 / M cell cycle arrest is one of the major cellular changes induced by BOR. To further investigate the mechanism of action of Si / BOR combination therapy, the cell cycle status was evaluated in this example. Figure 4 As shown, in the U266 / BOR cell line, after 24 hours of exposure to BOR alone, the percentage of cells in the G2 / M phase increased significantly, and the proportion in the G1 phase decreased accordingly, but the S phase was also significantly reduced. For ARP-1 and U266 cells, BOR treatment led to an increase in the G2 / M and G1 phases, but a decrease in the S phase. The Si / BOR combination treatment caused a more significant cell cycle arrest in the G2 / M phase in HMCLs than BOR alone, which may be related to the synergistic effect of inducing apoptosis in multiple myeloma. In contrast, Si treatment alone did not affect the distribution of the cell cycle.

[0069] Example 4 Si enhances the inhibition of NF-κB pathway by BOR.

[0070] The cells were washed with PBS and proteins were isolated using RIPA lysis buffer.

[0071] The protein content was determined by BCA kit (Shanghai Epizyme Biomedical Technology, China).

[0072] Total protein of 20 mg of sample was loaded on 4%-20% polyacrylamide gel and transferred to PVDF membrane (Merck Millipore, Darmstadt, Germany). After soaking in blocking buffer for 1 h at room temperature, the membrane was incubated with various primary antibodies at 4°C overnight and then incubated with secondary antibodies for 1 h.

[0073] Studies have shown that activation of nuclear factor κB (NF-κB) leads to poor prognosis in MM patients. BOR inhibits proteasome-mediated κ, where IκBa binds to phosphorylated nuclear factor-κ light chain enhancer in activated B cells, preventing its translocation to the nucleus. Therefore, the effect of Si / BOR combination on NF-κB signaling was further explored in this example. Figure 5As shown, the results in this example indicate that treatment with BOR alone in HMCLs significantly increased the expression of IκBa and phosphorylated IκB, and inhibited the activation of the NF-κB pathway, as manifested by reduced expression of phosphorylated P65, P105, and P50, while no regulatory effect on phosphorylated P65, P105, and P50 was observed. This result indicates that Si alone has a very weak inhibitory ability on the NF-κB pathway. It can be seen that the SI / BOR combination treatment led to a significantly higher inhibition of NF-κB-related protein expression than BOR treatment alone, indicating the synergistic effect of SI and BOR on HMCLs.

[0074] Example 5: Preparation of calcium silicate-enhanced doxorubicin / cisplatin formulation and its effect on the activity of drug-resistant tumor cells.

[0075] The preparation method of the broad-spectrum anti-tumor drug formulation is as follows: Dissolve any one of doxorubicin and cisplatin in PBS at a solid / liquid ratio of 1 mM, and add it to the tumor culture medium in a certain proportion to prepare a broad-spectrum anti-tumor drug formulation with a concentration of 0 - 20 μM.

[0076] The preparation method of the calcium silicate formulation is as follows: Dissolve calcium silicate powder in deionized water at a solid / liquid ratio of 1 mg / mL, and add it to the special cell culture medium for drug-resistant tumor cells in a certain proportion to prepare an ionic formulation with a concentration of 0.25 - 120 ppm (μg / mL).

[0077] The preparation method of the calcium silicate-enhanced broad-spectrum anti-tumor drug formulation is as follows: Mix the above several formulations in a specific proportion, where the concentration of the broad-spectrum anti-tumor drug is 0 - 20 μM and the concentration of calcium silicate is 0.25 - 120 ppm (μg / mL).

[0078] Take the above-prepared solution, and add fetal bovine serum, growth supplements, penicillin, and streptomycin in proportion. Inoculate drug-resistant tumor cells into a 96-well culture plate with an initial density of 1×103 cells per well. After culturing for 24 hours, replace the cell culture medium with the prepared calcium silicate / anti-tumor drug formulation. After culturing for another 48 hours, evaluate the cell activity by measuring the absorbance of all samples at 450 nm using a microplate reader with CCK-8 (Cell Counting Kit-8).

[0079] See Figure 6As shown, according to the data of CCK8, when no drugs are added, the survival rate of cells is not affected by the Si concentration. Therefore, the Si concentration selected in this example is a safe concentration. When Si is not added, doxorubicin at 5 μM has an inhibitory effect on MNNG, 4T1, and B16F10 tumor cells, but only has an inhibition rate of 20% - 30%. As the Si concentration increases, Si binds to doxorubicin, and the survival rate of the three types of tumor cells continuously decreases. When the Si concentration is equal to CS / 1 / 2, the inhibition rate of the three types of tumor cells reaches 40% - 50%, almost doubling, indicating that Si can effectively enhance the therapeutic effect of doxorubicin. When doxorubicin is 10 μM, when the Si concentration is 0 and when the Si concentration is equal to CS / 1 / 2, the cell survival rate of the 4T1 cell group significantly decreases to 21%. When the cisplatin concentration is 5 μM, the inhibition rate of cisplatin on the three tumors reaches 50% - 60%. As the Si concentration increases, the inhibition rate of the tumors continuously increases. When the Si concentration is equal to CS / 1 / 2, the inhibition rate of the three tumors reaches 70% - 80%. When the cisplatin concentration is 10 μM, the tumor cells can be completely inhibited, and the survival rate of the three types of tumor cells is all below 20% (at this time, it is default that the tumor cells have been completely killed, so introducing Si does not further improve the therapeutic effect).

[0080] Example 6: Preparation of various silica minerals enhanced doxorubicin preparations and their effects on the activity of melanoma cells.

[0081] The preparation method of the broad-spectrum anti-tumor drug preparation is as follows: Dissolve doxorubicin in PBS at a solid / liquid ratio of 1 mM, and add it to the tumor culture medium in a certain proportion to prepare a broad-spectrum anti-tumor drug preparation with a concentration of 0 - 20 μM.

[0082] The preparation method of the silica mineral preparation is as follows: According to ISO / EN 10993-12, a series of diluted ion extracts of melilite / zinc melilite / willemite powders were prepared. First, add melilite / zinc melilite / willemite powders to serum-free tumor cell culture medium, incubate at 37 °C for 24 hours at a solid / liquid ratio of 200 mg / mL, centrifuge the mixture at 4000 rpm for 10 minutes, and then sterilize and collect the supernatant through a filter membrane (Millipore, 0.22 μm) to obtain a stock solution. Subsequently, dilute the stock solution with melanoma cell culture medium to prepare a suitable ion extract.

[0083] The preparation method of various silica minerals enhanced broad-spectrum anti-tumor drug preparations is as follows: Mix the above several preparations according to a preset ratio, where the concentration of doxorubicin is 0 - 20 μM and the concentration of silica minerals is 0.25 - 120 ppm (μg / mL).

[0084] Take the above-prepared solution and add fetal bovine serum, growth supplements, penicillin, and streptomycin in proportion. Inoculate melanoma cells into a 96-well culture plate at an initial density of 1×10³ cells per well. After culturing for 24 hours, replace the cell culture medium with the prepared calcium silicate / doxorubicin preparation. After culturing for another 48 hours, evaluate the activity of melanoma cells by measuring the absorbance of all samples at 450 nm using a microplate reader with a CCK-8 (Cell Counting Kit-8).

[0085] See Figure 7 As shown in [reference], according to the CCK8 data, it can be found that when doxorubicin is not added, the cell viability is not affected by any concentration of silicate minerals. Therefore, the silicate mineral concentration selected in this example is a safe concentration. When no silicate minerals are added, doxorubicin at 5 μM and 10 μM has an inhibitory effect on melanoma cells, but only with an inhibition rate of 20% - 30%. As the concentration of different types of silicate minerals increases, the silicate minerals release Si and metal ions that bind to doxorubicin, continuously reducing the viability of melanoma cells. When the Si concentration is equal to CS / 1 / 2 and the doxorubicin concentration is equal to 10 μM, the tumor cell inhibition rate of the melilite group is 75%, see Figure 8 as shown in part A of [reference]; when the Si concentration is equal to CS / 1 / 8 and the doxorubicin concentration is equal to 10 μM, the tumor cell inhibition rate of the linarite group is equal to 85%, see Figure 8 as shown in part C of [reference]. Therefore, the inhibitory effect of copper ions in linarite on tumor cells is better than that of magnesium ions in melilite. When the concentrations of melilite and johannsenite are equal to CS / 1 / 2 and the concentration of linarite is equal to CS / 1 / 8, the tumor cell inhibition rates of the three groups of different silicate minerals all reach 70% - 80%. It can be found that the combination of different types of silicate minerals and doxorubicin also enhances the drug activity.

[0086] Example 7: Efficacy of Si / BOR combination therapy on multiple myeloma (ARP-1) in mice.

[0087] Establish a multiple myeloma xenograft model: Subcutaneously inject ARP-1 (5×10⁶) cells into the left and right sides of 6-week-old NOD-SCID mice to establish a multiple myeloma xenograft model. After 7 days, when the tumors form, randomly divide the mice into four groups (n = 6 per group), including a vehicle control group, an Si group, a BOR group, and a group combined with Si and BOR. Inject BOR (1 mg / kg) intraperitoneally every three days for 2 consecutive weeks, and inject Si (200 μL) subcutaneously around the tumor once a day for 2 weeks. Monitor the tumor size with a caliper every two days.

[0088] Tumor tissue section analysis: Tumor tissues were taken from tumor-bearing NOD / SCID mice. The tumor masses were fixed in 4% paraformaldehyde and then embedded in paraffin before sectioning. Finally, immunohistochemical staining was performed to analyze cleaved caspase3.

[0089] As Figure 8 shown, compared with the vector control group, Si group, and BOR group, combination therapy led to a more significant reduction in tumor burden, manifested as a decrease in tumor volume and tumor weight, as shown in parts A, B, and C of Figure 8 . In addition, no significant toxicity was observed in the mice receiving combination therapy, as the body weights of the mice did not decrease significantly during the treatment, as shown in part D of Figure 8 . Next, immunohistochemistry was used in this example to verify the expression of cleaved caspase-3. The results showed that the combination therapy of calcium silicate and BOR increased the expression of cleaved caspase-3, as shown in part E of Figure 8 . In summary, the experiments in this example showed that the Si / BOR combination therapy was very effective in vivo.

[0090] In summary, the embodiments of the present application provide an anti-tumor drug preparation, its preparation method, and application. The anti-tumor drug preparation is a silica-based anti-tumor drug preparation, and the anti-tumor drug preparation includes: a silicate mineral containing one or more silicate ions and at least one broad-spectrum anti-tumor drug; wherein, the concentration range of silicon is 0.25-120 ppm. The anti-tumor drug preparation can inhibit the activity of tumor cells by enhancing the therapeutic effect of the broad-spectrum anti-tumor drug, and can effectively reduce the size of solid tumors. Moreover, the anti-tumor drug preparation can reduce the amount of drug used while enhancing the therapeutic effect of the chemotherapy drug, and to a certain extent, reduce the burden on patients.

[0091] It should be noted that relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that an article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to this process, method, article or device. Without further limitation, an element defined by the statement "including a..." does not exclude the existence of another identical element in the process, method, article or device including the said element.

[0092] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included within the protection scope of the present application.

Claims

1. Use of an anti-tumor drug preparation in the preparation of a drug for treating multiple myeloma, characterized in that, The anti-tumor drug preparation contains calcium silicate and bortezomib, wherein the concentration range of silicon is 0.25 to 120 ppm, and the concentration range of bortezomib is 0.5 to 500 nM. The calcium silicate enhances the apoptosis of human multiple myeloma cell line cells induced by bortezomib.

Images