A combination drug for preventing and / or treating neuroendocrine prostate cancer, use thereof, and pharmaceutical composition

Abstract

The present application belongs to the field of chemical medicine, and particularly relates to a combined drug for preventing and / or treating neuroendocrine prostate cancer, use thereof and a pharmaceutical composition. The combined drug of the present application is fluphenazine and ilesidomosine-copper chloride; wherein fluphenazine can be used for treating neuroendocrine prostate cancer (NEPC) by increasing the oxygen consumption of NEPC cells and reducing the glycolytic capacity of NEPC cells; and ilesidomosine-copper chloride has no obvious killing effect on prostate cancer cell lines with NEPC phenotype, but when fluphenazine is combined with ilesidomosine-copper chloride, the combination has a significant therapeutic effect on prostate cancer with NEPC phenotype, and shows synergistic effect. The present application opens up a new direction for treating neuroendocrine prostate cancer, and has a broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical medicine, and particularly relates to a combination drug for preventing and / or treating neuroendocrine prostate cancer, use thereof and a pharmaceutical composition. BACKGROUND

[0002] Neuroendocrine prostate cancer (NEPC) is a special pathological subtype of prostate cancer, which has unique biological behavior and clinical characteristics. The main manifestation is that the tumor cells exhibit neuroendocrine differentiation characteristics. This type of tumor has poor differentiation, strong invasiveness, easy metastasis, and strong drug resistance, and resists conventional endocrine therapy. The current conventional treatment regimen for NEPC is: (1) chemotherapy, such as cisplatin + etoposide, which is a first-line chemotherapy regimen for NEPC, can inhibit tumor proliferation and improve survival rate; other drugs such as fluorouracil and pemetrexed are often used in combination to enhance efficacy; (2) targeted therapy and immunotherapy, targeting PIK3CA / AKT1 / PTEN gene mutations, using targeted drugs (such as PARP inhibitors) or immune checkpoint inhibitors may improve prognosis; (3) endocrine therapy: NEPC is not sensitive to traditional androgen deprivation therapy (ADT), but new endocrine drugs (such as abiraterone and enzalutamide) may delay progression in some patients. However, even with the above treatments, the 5-year survival rate of NEPC patients is still less than 10%, which is much lower than that of ordinary prostate cancer patients (more than 90%).

[0003] Most NEPC is induced by prostate adenocarcinoma after endocrine therapy. In the process of transformation of prostate adenocarcinoma into NEPC, the energy metabolism mode is changed from "oxidative phosphorylation dependence" to "anaerobic glycolysis dependence", which makes the tumor have stronger survival ability. It has been reported that ilesidomine can cause cells to undergo copper death, and its ability to kill cells depends on the degree of dependence of cells on oxidative phosphorylation; specifically, the more a cell depends on aerobic respiration (oxidative phosphorylation), the more susceptible it is to ilesidomine-induced copper death. This effect may apply to all cells in the biological world, not just tumor cells, but there is currently no report on NEPC being affected by ilesidomine-induced copper death. At the same time, the copper death promoter ilesidomine-copper chloride has stronger killing power on cells dependent on oxidative phosphorylation, but its effect on NEPC is still unknown.

[0004] Fluphenazine is a typical first-generation antipsychotic drug that mainly improves the symptoms of hallucinations and delusions in patients with mental illness by regulating dopamine levels in the brain. However, there is no report on the use of fluphenazine to treat NEPC.

[0005] Although the current drugs for treating neuroendocrine prostate cancer have achieved certain effects, they have large side effects and limited efficacy. Therefore, it is urgent to develop a new treatment strategy with good efficacy and less side effects to provide new options for prostate cancer patients with NEPC phenotype. SUMMARY

[0006] In view of the problems of the prior art, the present application provides a combination drug for preventing and / or treating neuroendocrine prostate cancer, use thereof and a pharmaceutical composition.

[0007] The present application provides a combination drug for preventing and / or treating neuroendocrine prostate cancer, wherein the combination drug is fluphenazine and a drug containing ilesidomine; the drug containing ilesidomine is ilesidomine or an ilesidomine complex.

[0008] Preferably, the molar concentration ratio of fluphenazine to the drug containing ilesidomine is 1:0.5-1.5.

[0009] Preferably, the molar concentration ratio of fluphenazine to the drug containing ilesidomine is 1:1.

[0010] Preferably, the ilesidomine complex is an ilesidomine-copper complex.

[0011] Preferably, the ilesidomine-copper complex is at least one selected from ilesidomine-copper chloride, ilesidomine-copper sulfate and ilesidomine-copper nitrate.

[0012] Preferably, the fluphenazine can kill neuroendocrine prostate cancer cells.

[0013] The present application also provides use of the above-mentioned combination drug in the preparation of a drug for preventing and / or treating prostate cancer.

[0014] Preferably, the prostate cancer is neuroendocrine prostate cancer.

[0015] Preferably, the drug can kill neuroendocrine prostate cancer cells.

[0016] The present application also provides a pharmaceutical composition for preventing and / or treating neuroendocrine prostate cancer, which is prepared by taking fluphenazine and a drug containing ilesidomine as active ingredients and adding pharmaceutically acceptable adjuvants; the drug containing ilesidomine is ilesidomine or an ilesidomine complex; and the molar concentration ratio of fluphenazine to the drug containing ilesidomine is 1:0.5-1.5.

[0017] Preferably, the molar concentration ratio of fluphenazine to the drug containing ilesidomine is 1:1.

[0018] Preferably, the elamidex complex is an elamidex-copper complex.

[0019] The combination drug of the present application is fluphenazine and elamidex-copper; wherein fluphenazine can be used for treating neuroendocrine prostate cancer (NEPC) by increasing the oxygen consumption of NEPC cells and reducing their glycolytic capacity; and elamidex-copper has no obvious killing effect on prostate cancer cell lines with NEPC phenotype, but the combination of fluphenazine and elamidex-copper can be used for treating prostate cancer with NEPC phenotype, and the two show a synergistic effect. The present application opens up a new direction for the treatment of neuroendocrine prostate cancer and has broad application prospects.

[0020] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present application.

[0021] The above content of the present application will be further described in detail through the specific embodiments below. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Enzalutamide induces NEPC model. (A) Observe the morphological changes of C4-2B cells after enzalutamide induction; (B) Western blot analysis of protein expression levels of androgen receptor (AR) and neuron-specific enolase (NSE).

[0023] Figure 2 The effect of different concentrations of fluphenazine on the cell viability of prostate cancer cells with NEPC phenotype. p <0.05; ** indicates p <0.01; *** indicates p <0.001.

[0024] Figure 3 The effect of fluphenazine on the energy metabolism pattern of prostate cancer cells with NEPC phenotype. (A) Basal oxygen consumption and maximum oxygen consumption value; (B) Basal glycolysis value and glycolysis reserve value.

[0025] Figure 4 The effect of different concentrations of elamidex-copper on the cell viability of NEPC phenotype prostate cancer cells.

[0026] Figure 5 The effect of low concentration of Flu+ES-Cu solution on the cell viability of NEPC phenotype prostate cancer cells.

[0027] Figure 6 Therapeutic effect of Flu+ES-Cu combination in vivo on NEPC phenotype prostate cancer. DETAILED DESCRIPTION

[0028] In the following examples and experimental examples, reagents and materials not specifically mentioned are commercially available.

[0029] Example 1 Combination drug for preventing and / or treating neuroendocrine prostate cancer

[0030] The present example provides a combination drug for preventing and / or treating neuroendocrine prostate cancer, which is a combination drug comprising 5 mg / kg of fluphenazine (Flu, purchased from MCE, item number: HY-119980) and 5 mg / kg of ilesrumab-copper chloride, and the combination drug has a synergistic effect.

[0031] The technical solutions of the present application are further described below through experiments.

[0032] Experimental Example 1 Fluphenazine has strong killing effect on prostate cancer cells with NEPC phenotype

[0033] I. Experimental method

[0034] 1. Establish a cell model of prostate cancer cells induced by enzalutamide (ENZA) to NEPC transformation

[0035] (1) Human prostate cancer bone metastasis subline cells (C4-2B cells) were inoculated in a 6 cm dish and cultured in 1640 medium. When the cells covered 60% of the bottom surface of the culture dish, the endocrine therapy drug enzalutamide was added to a final concentration of 5 μM.

[0036] (2) Continue to culture C4-2B cells, replace the medium every 3 days during the period, if the cells cover more than 90% of the bottom surface of the culture dish, discard the culture medium, and gently blow the adherent cells with PBS (phosphate buffered saline) to make them fall off. Collect the cell suspension, centrifuge at 100g for 3 minutes, discard the supernatant. Resuspend the cells with 1 ml of PBS, take 0.4 ml and mix with 3.6 ml of 1640 medium containing ENZA (1 μM). After 2 days, discard the culture medium and replace it with 1640 medium containing ENZA (5 μM) for continuous culture.

[0037] (3) After about two months of culture, observe the morphological changes of C4-2B cells.

[0038] 2. Western blot (WB) analysis

[0039] RIPA lysis buffer was used to extract cell proteins for Western blot analysis.

[0040] 3. CCK8 method

[0041] (1) The above method was used to induce C4-2B cells to produce NEPC phenotype. C4-2B cells that had not been treated with ENZA and C4-2B cells with NEPC phenotype were seeded into 96-well plates, with 2000 cells per well. Each cell type was seeded in 30 wells in total and divided into 6 groups with 5 wells in each group.

[0042] (2) Prepare a culture medium containing fluphenazine (Flu): Weigh 5 mg of fluphenazine, add 1142.8 μl of DMSO, and sonicate to obtain a stock solution with a concentration of 10 mM. Take 1.6 μl of the stock solution and add it to 1998.4 μl of 1640 complete medium. Mix thoroughly to obtain a Flu solution with a concentration of 8 μM. Take 1 ml of the 8 μM Flu solution and add it to another 1 ml of 1640 complete medium to obtain a Flu solution with a concentration of 4 μM. Take 1 ml of the 4 μM Flu solution and add it to another 1 ml of 1640 complete medium to obtain a Flu solution with a concentration of 2 μM. Take 1 ml of the 2 μM Flu solution and add it to another 1 ml of 1640 complete medium to obtain a Flu solution with a concentration of 1 μM. Take 1 ml of the 1 μM Flu solution and add it to another 1 ml of 1640 complete medium to obtain a Flu solution with a concentration of 0.5 μM. Add 0.8 μl of DMSO to 1 ml of 1640 complete medium to serve as the control culture medium.

[0043] (3) After the cells adhere to the wall, discard the culture medium and add 100 μl of the above-mentioned Flu solution of different concentrations and the control group culture medium to each well. Each group has 5 replicate wells.

[0044] (4) After 48 hours of culture, 10 μl of CCK-8 reagent was added to each well. After incubation for 1 hour, the absorbance at 450 nm was measured using a microplate reader.

[0045] 4. Detection of oxygen consumption and lactic acid release

[0046] (1) The original C4-2B cells and C4-2B cells with NEPC phenotype (C4-2B ENZA ) were seeded in Seahorse XF24 cell culture plates (Agilent, Cat. No. 102342-100), with 2000 cells per well, for a total of 10 wells. Two cell culture plates were plated for this experiment: one for measuring oxygen consumption and the other for measuring lactate release.

[0047] (2) After the cells adhered to the wall, discard the culture medium and mix the C4-2B cells and C4-2B ENZACells were divided into two groups, each group with 5 well replicates, and were treated with control medium (0.8 μL DMSO in 1 mL 1640 complete medium) or 1 μM Flu, respectively, to obtain C4-2B control, C4-2B-Flu, C4-2B ENZA control and C4-2B ENZA -Flu groups.

[0048] (3) After 48 hours of continuous culture, Seahorse XFe24 analyzer was used to detect the relative oxygen consumption rate of mitochondria in each group according to the method provided by Agilent Seahorse XF Cell Mito Stress Test Kit (Cat No: 103015-100); and the relative lactic acid release amount of each group was detected according to the method provided by Seahorse XF Glycolysis Stress Test Kit (Cat No: 103020-100).

[0049] II. Experimental results

[0050] The results are shown in Figure 1 A, C4-2B cells grew branches similar to synapses, and after ENZA induction, the C4-2B cells had irregular nuclei and grew tentacles, similar to the synaptic structure of nerve cells, and showed obvious neuroendocrine differentiation characteristics; the WB results are shown in Figure 1 B, after ENZA induction, the expression of androgen receptor (AR) gradually decreased, and the expression of neuron-specific enolase (NSE) gradually increased. The above results show that the NEPC cell model induced by ENZA is successfully constructed.

[0051] The results are shown in Figure 2 , in the original C4-2B cells without ENZA treatment, the cytotoxicity of fluphenazine (Flu) reached 4 μM, and the cytotoxicity was statistically significant, and when the Flu concentration reached 8 μM, the cell activity decreased by about 30%; in C4-2B (C4-2B ENZA ) with NEPC phenotype, the cytotoxicity of Flu reached 0.5 μM, and the cytotoxicity was statistically significant, and when the Flu concentration reached 8 μM, the cell activity decreased by about 60%.

[0052] The results are shown in Figure 3 A, Flu had no significant effect on the oxygen consumption of C4-2B cells, but could effectively increase the basal oxygen consumption and maximum oxygen consumption of C4-2B ENZA ; in addition, Flu increased the basal glycolysis of C4-2B cells, inhibited the basal glycolysis of C4-2B ENZA , and reduced the glycolysis reserve capacity of C4-2B Figure 3B). In general, Flu has no significant effect on the energy metabolism of C4-2B cells except for improving the basal glycolysis of tumors. ENZA For tumors, Flu can increase their aerobic respiration and reduce their glycolysis ability. It is currently recognized that glycolysis ability is positively correlated with the malignancy of the tumor.

[0053] The above results indicate that fluphenazine has a stronger killing effect on prostate cancer cells with NEPC phenotype. By increasing the oxygen consumption of NEPC cells and reducing their glycolysis capacity, it can effectively prevent NEPC from acquiring the characteristics of "anaerobic glycolysis dependence" and reduce their tolerance to hypoxia, and can be used to treat NEPC.

[0054] Experimental Example 2: Elisemol-copper chloride has no significant killing effect on NEPC phenotype prostate cancer cells

[0055] 1. Experimental Methods

[0056] 1. CCK8 method

[0057] (1) The original C4-2B cells and C4-2B cells with NEPC phenotype (C4-2B ENZA ) were seeded in 96-well plates, with 30 wells of each cell type and 2000 cells per well.

[0058] (2) Preparation of ES-Cu solution: Weigh 10 mg of elixirmol (ES, brand: MCE, catalog number: HY-12040), add 1248.4 μl of DMSO, and sonicate to obtain a 20 mM ES stock solution. Separately, weigh 10 mg of copper chloride dihydrate (CuCl2·2 H2O, brand: Sigma, catalog number: C3279), add 2932.9 μl of PBS, and dissolve thoroughly to obtain a 20 mM Cu stock solution.

[0059] (3) Take 3.2 μL of ES stock solution and Cu stock solution respectively, add them to 1993.6 μL of 1640 complete medium, mix thoroughly to obtain a 32 μM ES-Cu solution; take 1 mL of 32 μM ES-Cu solution and add it to another 1 mL of 1640 complete medium to obtain a 16 μM ES-Cu solution; take 1 mL of 16 μM ES-Cu solution and add it to another 1 mL of 1640 complete medium to obtain an 8 μM ES-Cu solution; take 1 mL of 8 μM ES-Cu solution and add it to another 1 mL of 1640 complete medium to obtain a 4 μM ES-Cu solution; take 1 mL of 4 μM ES-Cu solution and add it to another 1 mL of 1640 complete medium to obtain a 2 μM ES-Cu solution. Add 1.6 μL of DMSO to 1 mL of 1640 complete medium as the control medium.

[0060] (4) After the cells in the 96-well plate adhere to the wall, discard the culture medium and add 100 μL of the control group culture medium or ES-Cu solution of different concentrations, with 5 replicates for each concentration.

[0061] (5) Continue culturing for 48 hours, add 10 μL of CCK-8 reagent to each well, continue culturing for 1 hour, and use a microplate reader to detect cell activity.

[0062] 2. Experimental Results

[0063] The results are as follows Figure 4 As shown in the figure, ES-Cu began to inhibit C4-2B cells at a concentration of 8 μM; while it inhibited C4-2B cells with NEPC phenotype. ENZA For cells, the ES-Cu concentration needs to reach 32μM to show slight killing effect. The results showed that the use of ES-Cu alone had no significant killing effect on NEPC phenotype prostate cancer cells.

[0064] Experimental Example 3 Therapeutic Effect of Flu Combined with ES-Cu on Prostate Cancer with NEPC Phenotype

[0065] 1. Experimental Methods

[0066] 1. In vitro experiment: CCK-8 assay to detect cell viability

[0067] (1) The method mentioned in Experimental Example 1 was used to construct C4-2B cells with NEPC phenotype (C4-2B ENZA ), and seeded them into 96-well plates, with a total of 20 wells and 2000 cells per well.

[0068] (2) Prepare Flu solution and ES-Cu solution with concentrations of 1 μM and 2 μM respectively; mix the 2 μM Flu solution and ES-Cu solution at a volume ratio of 1:1 to obtain a 1 μM Flu + ES-Cu solution; add 0.1 μL DMSO to 1 mL 1640 complete culture medium as the control culture medium.

[0069] (3) After the cells adhered to the wall, the culture medium was discarded and 1 μM Flu solution, ES-Cu solution, Flu+ES-Cu solution and control group culture medium were added. Each group had 5 replicate wells and the cell activity was detected by CCK method.

[0070] 2. In vivo experiment: Detecting the fluorescence intensity of mouse tumors using a small animal in vivo imaging device

[0071] (1) C4-2B was injected subcutaneously into the thigh of 10 4-week-old male Balbc nude mice (weighing about 20 g / mouse). ENZACells, 100,000 tumor cells per mouse were inoculated.

[0072] (2) On the 3rd day after inoculation, the bilateral testes of all mice were removed by surgery to simulate clinical androgen deprivation therapy.

[0073] (3) Drug preparation: PBS was used as a control group for injecting drugs; 5 mg of fluphenazine was weighed, ultrasonically assisted to dissolve, fully dissolved in 100 μL of DMSO, and diluted with 4900 μL of PBS to obtain a Flu injection; 5 mg of iltostat was weighed, ultrasonically assisted to dissolve, fully dissolved in 100 μL of DMSO, and diluted with 4900 μL of PBS to obtain a 1 mg / mL ES solution, and 2.13 mg of CuCl2·2H2O was added, fully dissolved and mixed to obtain an ES-Cu injection; 5 mg of cisplatin (brand: MCE, item number: HY-17394) was weighed, fully dissolved in 5 mL of PBS to obtain a cisplatin injection; 5 mg of fluphenazine and iltostat was weighed, fully dissolved in 100 μL of DMSO, and diluted with 4900 μL of PBS, and 2.13 mg of CuCl2·2H2O was added, fully dissolved and mixed to obtain a Flu+ES-Cu injection.

[0074] (3) On the 7th day after inoculation, 100 μL of PBS, Flu injection, ES-Cu injection, cisplatin injection or Flu+ES-Cu injection was injected into the mice by intraperitoneal injection, a total of 5 groups, 2 mice per group. Injection was performed once every 48 hours, a total of 10 times.

[0075] (4) After the last injection of drugs, the mice were cultured for another 2 days, and the fluorescence intensity of the tumors of the mice was detected using a small animal live imaging instrument.

[0076] II. Experimental results

[0077] The results are shown in Figure 5 Compared with the control group, 1 μM of Flu can reduce the activity of C4-2B ENZA cells to about 75%; 1 μM of ES-Cu solution has no effect on the activity of C4-2B ENZA cells; and 1 μM: 1 μM of Flu+ES-Cu solution can reduce the activity of C4-2B ENZA cells to about 20%. The results show that the combined use of Flu and ES-Cu has a synergistic effect on the killing of C4-2B ENZA cells, which is much stronger than the killing effect of the two drugs alone.

[0078] The results are shown in Figure 6As shown, compared with the control group of mice, Flu and ES-Cu alone treatment, tumors with NEPC phenotype almost no shrinkage; the current common chemotherapy drug cisplatin weak effect on the treatment of such tumors; and Flu and ES-Cu combined use, NEPC tumor growth in vivo was significantly inhibited, better than the effect of the two drugs alone, with a synergistic effect. The above results show that Flu combined with ES-Cu on NEPC phenotype of prostate cancer has a significant therapeutic effect.

[0079] In summary, Fluphenazine by increasing the oxygen consumption of NEPC cells, reduce its glycolytic ability, thereby preventing NEPC obtain "anaerobic glycolysis dependent" characteristics, and reduce its tolerance to hypoxia, can be used for the treatment of NEPC; and ilesimod-copper chloride on prostate cancer cell line with NEPC phenotype has no obvious killing effect, but the combination of fluphenazine and ilesimod-copper chloride, the tumor volume was significantly reduced, and better than the effect of the two alone, on NEPC phenotype of prostate cancer has a significant therapeutic effect.

Claims

1. A combination medicament for preventing and / or treating neuroendocrine prostate cancer, characterized by comprising: The combination drug is fluphenazine and ilesrum-copper complex; the molar concentration ratio of fluphenazine to ilesrum-copper complex is 1:0.5-1.

5. ​ 2. The combination of claim 1, wherein: The ilesrum-copper complex is at least one selected from ilesrum-copper chloride, ilesrum-copper sulfate and ilesrum-copper nitrate.

3. Use of the combination drug of claim 1 or 2 in the preparation of a drug for preventing and / or treating prostate cancer.

4. Use according to claim 3, characterized in that, The prostate cancer is neuroendocrine prostate cancer.

5. A pharmaceutical composition for preventing and / or treating neuroendocrine prostate cancer, characterized by: It is prepared by taking fluphenazine and ilesrum-copper complex as active ingredients and adding pharmaceutically acceptable adjuvants; the molar concentration ratio of fluphenazine to ilesrum-copper complex is 1:0.5-1.5.

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