Pharmaceutical composition containing Pt-coated PCN-Cu and PD-L1 antibodies and application of pharmaceutical composition in tumor treatment

Abstract

The invention discloses a pharmaceutical composition containing Pt-coated PCN-Cu and PD-L1 antibodies and an application of the pharmaceutical composition in tumor treatment. Pancreatic cancer is a malignant tumor which seriously threatens health of Chinese people at present, the death rate is high, prognosis is poor, and the immunotherapy effect is not ideal. It is found that Pt-coated PCN-Cu not only can induce tumor cells to generate copper death, but also can promote high expression of tumor cells PD-L1. Compared with independent use of the PD-L1 antibody, the combined application of the Pt-coated PCN-Cu and the PD-L1 antibody can significantly enhance the immunotherapy effect, and is expected to be applied to clinical treatment of pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition containing Pt@PCN-Cu and PD-L1 antibody, and also relates to the application of the pharmaceutical composition in tumor treatment. The present invention belongs to the technical field of tumor immunotherapy drugs. Background Art

[0002] The information disclosed in the background technology part of the present invention is intended to increase the understanding of the present invention, and should not be regarded as admitting or suggesting in any form that the information constitutes the prior art known to ordinary technicians in the field.

[0003] Pancreatic cancer has an insidious onset and progresses rapidly. It is a highly malignant digestive system tumor. In my country, with the aging of the population, changes in eating habits and increased life pressure, the incidence of pancreatic cancer continues to rise, and the five-year survival rate of patients is less than 10%. Although the overall survival rate of many cancer patients has significantly improved, it is regrettable that the survival rate of pancreatic cancer patients remains at a low level, becoming a major public health issue threatening people's health. Traditional pancreatic cancer treatments include surgical resection, chemotherapy and radiotherapy. Due to the limitations of traditional treatments, immunotherapy has become a new treatment strategy that has attracted much attention. Immunotherapy can better identify and attack tumor cells by activating the immune system. Since 2016, anti-programmed cell death protein ligand 1 antibodies (αPD-L1) have been approved by the US Food and Drug Administration for tumor treatment. Based on this, the development of pancreatic cancer immunotherapy has always been the direction of scientific research. However, immunotherapy still faces a series of problems and challenges such as immune tolerance. On the one hand, pancreatic cancer has rich stroma, low immunogenicity, low mutation load, and a "cold" immune microenvironment, making it difficult for classic immunotherapy strategies to play an effective anti-cancer role. On the other hand, PD-L1 expression in pancreatic cancer patients shows spatial heterogeneity within the tumor, and there are significant differences in the PD-L1 positivity rate. Therefore, new regimens based on PD-L1 antibody therapy and combined drug treatment for pancreatic cancer have attracted the attention of researchers.

[0004] Recent studies have shown that copper (Cu) ion levels in tumors can affect the expression of PD-L1. Copper is an extremely important intracellular trace element that plays a key role in maintaining redox homeostasis. On the one hand, copper ions are cofactors for proteases with key cellular functions, including mitochondrial respiration and biosynthesis. On the other hand, copper ion imbalance may lead to cytotoxicity. The proliferation and metastasis of tumor cells are more dependent on copper ions than normal tissue cells, and the serum copper ion levels of tumor patients are significantly increased. Recently, a new type of cell death that depends on copper ions, namely copper death, has been identified. Unlike other forms of cell death, copper death cannot be inhibited by known cell death inhibitors. Therefore, regulating copper ion levels in tumor cells seems to be an increasingly promising tumor treatment method.

[0005] Hexokinase (HK) is the rate-limiting enzyme of aerobic glycolysis, which can convert glucose into glucose-6-phosphate. There are four isoforms in the HK family: HK1, HK2, HK3, and HK4. Among them, HK2 binds to the voltage-dependent anion channel protein of the mitochondrial outer membrane, enabling HK2 to use ATP produced by mitochondria for glycolysis. HK2 not only has the function of a rate-limiting enzyme in glycolysis, but also has a non-metabolic enzyme function. Recent studies have shown that HK2 acts as a protein kinase to promote PD-L1 expression. Our previous studies found that copper ion accumulation in pancreatic cancer cells can participate in the anti-tumor process through the dual action of inducing copper death and increasing the high expression of PD-L1 in an HK2-dependent manner. Therefore, the combined use of inducing copper death of tumor cells and immunotherapeutics may be a more effective tumor treatment. Summary of the invention

[0006] The present invention aims to provide a combined immunotherapy method and a pharmaceutical composition suitable for tumors and their use.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] A pharmaceutical composition of the present invention comprises at least: (a) a copper-death-promoting anti-tumor nanomaterial drug Pt@PCN-Cu; and (b) a monoclonal antibody against immune checkpoints.

[0009] Among them, preferably, the copper-killing anti-tumor nanomaterial drug Pt@PCN-Cu is constructed with PCN-Cu as a metal organic framework compound by incorporating Pt nanoparticles stabilized by polyvinyl pyrrolidone into PCN-Cu, wherein the PCN-Cu is obtained by dissolving tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in N,N-dimethylformamide solution, and heating, stirring, cooling and centrifuging.

[0010] Preferably, the method for constructing the copper-death-promoting anti-tumor nanomaterial drug comprises the following steps:

[0011] (1) dissolving tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in an N,N-dimethylformamide solution, heating, stirring, cooling and centrifuging to obtain PCN-Cu nanoparticles;

[0012] (2) dissolving polyvinyl pyrrolidone in methanol, adding chloroplatinic acid thereto, refluxing the mixture with air, removing methanol, precipitating with acetone, and centrifuging to obtain Pt nanoparticles. The obtained Pt nanoparticles are further purified with chloroform and hexane, and redispersed in N,N-dimethylformamide solution;

[0013] (3) The PCN-Cu nanoparticles prepared in step (1) are mixed with the Pt nanoparticles prepared in step (2), heated, stirred, cooled, and centrifuged, and washed multiple times with N,N-dimethylformamide solution to obtain a copper-promoting nanomaterial drug Pt@PCN-Cu.

[0014] Among them, preferably, the mass ratio of tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid described in step (1) is 1.1:3:29, the heating temperature is 90°C, the stirring speed is 300rpm, the time is 5h, the centrifugal speed is 12000rpm, and the centrifugal time is 20min.

[0015] Preferably, the amount of polyvinyl pyrrolidone used in step (2) is 266 mg, the amount of methanol used is 90 mL, the amount of 50 mM chloroplatinic acid used is 10 mL, the air reflux time is 3 h, the precipitation time is 3 h, the centrifugal speed is 6000 rpm, and the centrifugal time is 5 min.

[0016] Preferably, the heating temperature in step (3) is 90° C., the stirring speed is 300 rpm, the time is 5 h, the centrifugal speed is 12000 rpm, and the centrifugal time is 20 min.

[0017] Among them, preferably, the monoclonal antibodies of the immune checkpoints include but are not limited to one or a combination of PD-1 antibodies and PD-L1 antibodies.

[0018] Among them, preferably, the monoclonal antibody of the immune checkpoint is PD-L1 antibody.

[0019] Furthermore, the present invention also proposes the use of the pharmaceutical composition in the preparation of drugs for treating tumors, wherein the tumors include but are not limited to one of pancreatic cancer, gastric cancer and colorectal cancer.

[0020] Among them, preferably, in the pharmaceutical composition, the use of Pt@PCN-Cu and PD-L1 antibody includes simultaneous use, separate use or sequential use.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention provides a combined immunotherapy method and a pharmaceutical composition suitable for tumors, wherein the pharmaceutical composition comprises a copper-death-promoting anti-tumor nanomaterial drug Pt@PCN-Cu and a monoclonal antibody for immune checkpoints. Through research, the present invention found that Pt@PCN-Cu can not only induce copper death in tumor cells, but also promote high expression of PD-L1 in an HK2-dependent manner, activate immune pathways and synergistically enhance the effect of tumor immunotherapy. Compared with the use of PD-L1 antibodies alone, the combined use of Pt@PCN-Cu and PD-L1 antibodies can significantly enhance the immunotherapy effect, and is expected to be applied to the clinical treatment of tumors, especially pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is the synthesis process of Pt@PCN-Cu in Example 1;

[0025] Figure 2 is a transmission electron microscope image of Pt@PCN-Cu in Example 1;

[0026] Figure 3 is the X-ray diffraction pattern of Pt@PCN-Cu in Example 1;

[0027] Figure 4 is the X-ray photoelectron spectrum of Pt@PCN-Cu in Example 1;

[0028] Figure 5 The uptake of Pt@PCN-Cu by PANC-1 cells in the in vitro experiment in Example 2;

[0029] Figure 6 The results of the in vitro experiment in Example 3 showing that Pt@PCN-Cu inhibited the growth of PANC-1 cells;

[0030] Figure 7 This is the WB result of Pt@PCN-Cu on copper death-related markers of PANC-1 cells in Example 4;

[0031] Among them, ①PBS, ②ES, ③CuCl2+ES, ④Pt@PCN-Cu;

[0032] Figure 8 This is the WB result of the in vitro experiment in Example 5, in which Pt@PCN-Cu enhances PD-L1 expression through HK2;

[0033] Among them, (a) the first group; (b) the second group; (c) the third group;

[0034] Fig. 9 It is the experimental flow chart of Example 6;

[0035] Fig.10 This is a diagram showing the effect of Pt@PCN-Cu combined with PD-L1 antibody in the in vivo experiment in Example 6 on the subcutaneous tumor formation in C57 mice;

[0036] Fig.11 This is a schematic diagram of the changes in subcutaneous tumor volume of each group of mice with treatment time in Example 6. DETAILED DESCRIPTION

[0037] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0038] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0039] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0040] Tetracarboxyphenylporphyrin copper (TCPP-Cu), zirconium oxychloride octahydrate (ZrOCl2·8(H2O)), benzoic acid, N,N-dimethylformamide solution, polyvinyl pyrrolidone, methanol, chloroplatinic acid, acetone, chloroform, and hexane were purchased from China National Pharmaceutical Group Co., Ltd.

[0041] PD-L1 antibody was purchased from BioXcell, USA.

[0042] C57 / BL6 mice were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0044] Example 1 Preparation and characterization of Pt@PCN-Cu

[0045] according to Figure 1 The method shown in the figure is used to prepare Pt@PCN-Cu. The specific steps are as follows:

[0046] (1) In N,N-dimethylformamide solution (20 mL), tetracarboxyphenylporphyrin copper (22 mg), zirconium oxychloride octahydrate (60 mg) and benzoic acid (580 mg) were dissolved and magnetically stirred at 90°C for 5 h at a speed of 300 rpm. After the solution was cooled to room temperature, PCN-Cu was collected by centrifugation (12000 rpm, 20 min) and washed with N,N-dimethylformamide solution several times. The obtained PCN-Cu was then redispersed in N,N-dimethylformamide solution for storage;

[0047] (2) Polyvinyl pyrrolidone (266 mg) was dissolved in methanol (90 mL), and then chloroplatinic acid (10 mL, 50 mM) was added to the solution. The mixture was refluxed in air for 3 h. The methanol was then removed using a rotary evaporator, and the Pt nanoparticles were precipitated by acetone for 3 h and collected by centrifugation (6000 rpm, 5 min). The obtained Pt nanoparticles were further purified with chloroform and hexane and then redispersed in N,N-dimethylformamide solution;

[0048] (3) The PCN-Cu nanoparticles prepared in step (1) were mixed with the Pt nanoparticles prepared in step (2), and the mixture was stirred (300 rpm) at 90°C for 5 h. Pt@PCN-Cu was then obtained by centrifugation and washing with N,N-dimethylformamide solution for multiple times. The obtained Pt@PCN-Cu was redispersed in N,N-dimethylformamide solution and stored for later use.

[0049] The formation of Pt@PCN-Cu was confirmed by transmission electron microscopy. Figure 2 As shown, Pt@PCN-Cu exhibits a uniform morphology with an average size of 200 nm. Figure 3 is the X-ray diffraction pattern of Pt@PCN-Cu. In addition, X-ray photoelectron spectroscopy analysis was performed to explore the valence state and composition of Pt@PCN-Cu nanoparticles. Figure 4 As shown, X-ray photoelectron spectroscopy shows that Pt@PCN-Cu is composed of Cu, C, N, Zr and Pt elements.

[0050] Example 2 In vitro experiments demonstrate the uptake of Pt@PCN-Cu by PANC-1 cells

[0051] PANC-1 cells (human pancreatic cancer cells) were added to the cell culture dish and incubated overnight in a 37°C cell culture incubator. PANC-1 cells were co-cultured with Pt@PCN-Cu (20 nM) for 1 h, 4 h, and 8 h, respectively. Next, the cell culture medium was removed and washed three times with PBS, and the PANC-1 cell nuclei were stained with DAPI. Subsequently, images were collected by confocal microscopy. The results are shown in Figure 2. Figure 5 As shown, with the increase of co-culture time of PANC-1 cells and Pt@PCN-Cu, the content of Pt@PCN-Cu in PANC-1 cells gradually increased.

[0052] Example 3 In vitro experiments verify that Pt@PCN-Cu inhibits the growth of PANC-1 cells

[0053] PANC-1 cells were seeded in a cell culture dish and co-cultured with PBS, Elesclomol (ES, 50 nM), CuCl2+ES (5 μM+50 nM), and Pt@PCN-Cu (20 nM) for 24 h. Next, the cell culture medium was removed and the PANC-1 cells were co-stained for 30 min using the CalceinAM / PI double staining kit. Green fluorescence (live cells) and red fluorescence (dead cells) were recorded in different fluorescence channels using a confocal laser scanning microscope. The results are shown in Figure 2. Figure 6 As shown in the figure, the green fluorescence of PANC-1 cells in the Pt@PCN-Cu group decreased relatively, and the red fluorescence increased relatively, indicating that Pt@PCN-Cu can promote tumor cell death and inhibit tumor cell growth.

[0054] Example 4 In vitro experiment Pt@PCN-Cu reduces the expression of DLAT, FDX1 and LIAS in PANC-1 cells

[0055] PANC-1 cells were divided into four groups for Western Blot experiments: ①PBS, ②ES, ③CuCl2+ES, ④Pt@PCN-Cu. The specific operation was as follows: PANC-1 cells were 1×10 6Cells were seeded in 6-well plates at a density of 100 cells / well and cultured overnight. PANC-1 cells were then treated with PBS, Elesclomol (ES, 50nM), CuCl2+ES (5μM+50nM) and Pt@PCN-Cu (20nM), respectively. After incubation at 37°C for 24h, the cell lysate was added to the culture dish to extract cell protein, and the protein concentration was determined using a BCA protein assay kit. 20μg of protein sample was added to each lane and separated by electrophoresis on a 12.5% ​​polyacrylamide gel and transferred to a PVDF membrane using a gel electrophoresis apparatus. After blocking with 5% skim milk for 1h, the PVDF membrane was incubated with DLAT, LIAS, FDX1 and β-actin antibodies at 4°C overnight. Subsequently, the PVDF membrane was washed three times and incubated with HRP-conjugated antibodies for 1h at room temperature. 200μL of ECL chemiluminescent reagent was added to the PVDF membrane, and Western Blot images were obtained using a gel imaging system. The results are shown in Figure 2. Figure 7 As shown in the figure, after the cells were treated with Pt@PCN-Cu, the expression of DLAT, FDX1 and LIAS proteins in PANC-1 cells was reduced, indicating that Pt@PCN-Cu may induce copper death of PANC-1 cells.

[0056] Example 5 In vitro experiment Pt@PCN-Cu enhances PD-L1 expression via HK2

[0057] PANC-1 cells in the logarithmic growth phase were evenly inoculated into 6-well plates, and after 24 hours of adherence culture, different groups of drugs were added for treatment, and finally Western Blot detection was performed. The specific groups are shown in Table 1:

[0058] Table 1 Experimental grouping

[0059]

[0060] Note: “+” indicates that the substance is added, and “-” indicates that the substance is not added.

[0061] The results are as follows Figure 8 As shown in (a), compared with the control group, the Pt@PCN-Cu group can increase the expression of PD-L1 protein; Figure 8 As shown in (b), the shHK2 group can reduce the expression of PD-L1 protein, and there is no significant change in the expression of PD-L1 protein after the addition of G-6-P; Figure 8 As shown in (c), the addition of Pt@PCN-Cu and shHK2 decreased the expression of PD-L1 protein. These results indicate that Pt@PCN-Cu can increase the expression of PD-L1 in an HK2-dependent manner.

[0062] Example 6 In vivo experiment Pt@PCN-Cu combined with PD-L1 antibody to treat subcutaneous tumor formation in C57 mice

[0063] In vivo experimental process Fig. 9 As shown (showing the time point of drug tail vein injection). The KPC pancreatic cancer tumor model was used as a model tumor model, and KPC tumor cells (seeding density of 5×10 6 mice, KPC tumor cells from ATCC, No. CRL-2539), conventional feeding, when the tumor volume reached about 100mm 3 At the same time, the mice were randomly divided into 4 groups and given PBS, PD-L1 antibody (10 mg / kg), Pt@PCN-Cu (5 mg / kg) and Pt@PCN-Cu + PD-L1 antibody (Pt@PCN-Cu: ​​5 mg / kg; PD-L1 antibody: 10 mg / kg) through the tail vein. The changes in the subcutaneous tumor volume of the mice were observed and counted on days 3, 6, 9, 12 and 15 after administration. The mice were sampled on the 15th day after administration, and the tumors of the mice were dissected for analysis.

[0064] The results are as follows Fig.10 As shown in Figure 2, the tumors in the Pt@PCN-Cu+PD-L1 antibody group were significantly smaller than those in the other groups. During the entire treatment cycle, the tumor volumes of each group changed over time. Fig.11 As shown, the tumor volume of mice in the PBS group and the PD-L1 antibody group increased rapidly and significantly over time, the tumor volume of mice in the PD-L1 antibody group increased slowly over time, and the tumor volume of mice in the Pt@PCN-Cu+PD-L1 antibody group did not change significantly over time, indicating that the addition of Pt@PCN-Cu can effectively enhance the anti-tumor effect of PD-L1 antibody.

[0065] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The composition at least includes: (a) copper-death-promoting anti-tumor nanomaterial drug Pt@PCN-Cu; and (b) monoclonal antibodies for immune checkpoints.

2. The pharmaceutical composition according to claim 1, characterized in that The copper-death-promoting anti-tumor nanomaterial drug Pt@PCN-Cu is constructed by incorporating Pt nanoparticles stabilized by polyvinyl pyrrolidone into PCN-Cu using PCN-Cu as a metal organic framework compound, wherein the PCN-Cu is obtained by dissolving tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in an N,N-dimethylformamide solution, and then heating, stirring, cooling and centrifuging.

3. The pharmaceutical composition according to claim 2, characterized in that The method for constructing the copper-death-promoting anti-tumor nanomaterial drug comprises the following steps: (1) dissolving tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid in an N,N-dimethylformamide solution, heating, stirring, cooling and centrifuging to obtain PCN-Cu nanoparticles; (2) dissolving polyvinyl pyrrolidone in methanol, adding chloroplatinic acid thereto, refluxing the mixture with air, removing methanol, precipitating with acetone, and centrifuging to obtain Pt nanoparticles. The obtained Pt nanoparticles are further purified with chloroform and hexane, and redispersed in N,N-dimethylformamide solution; (3) The PCN-Cu nanoparticles prepared in step (1) are mixed with the Pt nanoparticles prepared in step (2), heated, stirred, cooled, and centrifuged, and washed multiple times with N,N-dimethylformamide solution to obtain a copper-promoting nanomaterial drug Pt@PCN-Cu.

4. The pharmaceutical composition according to claim 3, characterized in that The mass ratio of tetracarboxyphenylporphyrin copper, zirconium oxychloride octahydrate and benzoic acid described in step (1) is 1.1:3:29, the heating temperature is 90°C, the stirring speed is 300rpm, the time is 5h, the centrifugal speed is 12000rpm, and the centrifugal time is 20min.

5. The pharmaceutical composition according to claim 3, characterized in that The amount of polyvinyl pyrrolidone used in step (2) is 266 mg, the amount of methanol used is 90 mL, the amount of 50 mM chloroplatinic acid used is 10 mL, the air reflux time is 3 h, the precipitation time is 3 h, the centrifugal speed is 6000 rpm, and the centrifugal time is 5 min.

6. The pharmaceutical composition according to claim 3, characterized in that The heating temperature in step (3) is 90° C., the stirring speed is 300 rpm, the time is 5 h, the centrifugal speed is 12000 rpm, and the centrifugal time is 20 min.

7. The pharmaceutical composition according to claim 1, characterized in that The monoclonal antibodies for the immune checkpoints include, but are not limited to, one or a combination of PD-1 antibodies and PD-L1 antibodies.

8. The pharmaceutical composition according to claim 7, characterized in that The monoclonal antibody for the immune checkpoint is PD-L1 antibody.

9. Use of the pharmaceutical composition according to any one of claims 1 to 8 in the preparation of a drug for treating tumors, wherein the tumor includes but is not limited to one of pancreatic cancer, gastric cancer and colorectal cancer.

10. The use according to claim 9, characterized in that In the pharmaceutical composition, the use of Pt@PCN-Cu and PD-L1 antibody includes simultaneous use, separate use or sequential use.

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