A tetravalent platinum immunogenic death inducing agent and its preparation method and application

Abstract

The present invention discloses a tetravalent platinum immunogenic death inducing agent having a general formula I structure, and a preparation method and application thereof. The compound of the present invention has an AKR inhibitory effect, can induce the immunogenic death of tumors, and exhibits excellent activity against platinum drug-resistant tumor cells. The tetravalent platinum complex of the present invention, through its unique structural advantage, combines targeted AKR inhibition and immune response induction, and can be used to prepare a new chemotherapeutic immunodrug with multiple anti-tumor mechanisms to solve the problems of tumor resistance and lack of small molecule chemoimmunotherapeutic agents.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a tetravalent platinum immunogenic death inducer and a preparation method and application thereof; specifically, the tetravalent platinum immunogenic death inducer of the present invention has an aldehyde-keto reductase inhibitory effect. Background Art

[0002] Cancer is a chronic disease that poses a serious threat to human health. Platinum drugs are one of the most commonly used chemotherapy drugs in clinical practice due to their broad anti-tumor spectrum and significant anti-cancer effects. Since cisplatin was approved by the US FDA for the treatment of reproductive system tumors, platinum drugs have been developed to the third generation, with significant improvements in chemical stability, solubility, and anti-tumor effects. Despite this, platinum drugs still face two major challenges that need to be addressed, namely serious adverse reactions and drug resistance. This forces researchers to continue to develop new platinum drugs with milder adverse reactions and stronger targeting to make up for the shortcomings of traditional platinum drugs.

[0003] Aldo-keto reductase (AKR) is a superfamily of oxidoreductases with NADPH as a coenzyme. As the rate-limiting enzyme of the polyol metabolic pathway, it participates in a variety of biological processes in the body and has an intervening effect on the material metabolism of tumor cells in cancer. Studies have found that AKR is widely and highly expressed in human tumors, and the high expression of different subtypes has certain tumor specificity, which makes it a highly concerned anti-tumor target. In addition, AKR plays an important mediating role in tumor resistance to a variety of chemotherapeutic drugs, including platinum drugs, anthracyclines (danorubicin and doxorubicin), methotrexate, cyclophosphamide, gefitinib and other commonly used clinical anti-tumor drugs. Overexpression of AKR is an important manifestation of tumor resistance, and its loss of expression is a good prognostic indicator of cisplatin chemosensitivity. Among the various AKR subtypes, the AKR1C subtype is the most important factor in inducing platinum drug resistance, and tumor platinum drug resistance can be improved by inhibiting the expression and activity of AKR1C. Therefore, designing new AKR inhibitors is an effective strategy to overcome tumor platinum drug resistance.

[0004] Immunogenic cell death (ICD) is a type of regulated cell death and one of the important mechanisms of tumor immunotherapy. Under the stimulation of immunogenic death inducers, a variety of ICD damage-related molecular patterns such as calreticulin (CRT), high mobility group protein 1 (HMGB1), and ATP are secreted or transported to the extracellular space, promoting the maturation and activation of dendritic cells (DCs) and presenting antigens to cytotoxic T cells. Cytotoxic CD8 +T cells are recruited to the tumor microenvironment and phagocytize tumor cells, leading to tumor immune killing. Immunogenic death inducers cause tumor cells to shift from a "cold" immune state to a "hot" immune state, regulating the inhibitory tumor immune microenvironment, which is an effective anti-tumor immunotherapy strategy. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a tetravalent platinum immunogenic death inducer and its preparation method and application. The compound of the present invention is a tetravalent platinum complex, which has an AKR inhibitory effect, can induce the immunogenic death of tumors, and exhibits excellent activity against platinum-resistant tumor cells. The tetravalent platinum complex of the present invention, through its unique structural advantages, combines targeted AKR inhibition and immune response induction, and can be used to prepare a new type of chemotherapeutic immunodrug with multiple anti-tumor mechanisms to solve the problems of tumor resistance and lack of small molecule chemoimmunotherapeutic agents.

[0006] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0007] The present invention provides a tetravalent platinum immunogenic death inducing agent having a general formula I:

[0008]

[0009] in,

[0010] The structure is any of the following:

[0011] The tetravalent platinum immunogenic death inducing agent of the present invention is preferably the following compound:

[0012]

[0013] The above-mentioned compounds of general formula I of the present invention may also exist in the form of their salts, which are converted in vivo into compounds of general formula I. For example, within the scope of the present invention, the compounds of the present invention are converted into pharmaceutically acceptable salt forms according to processes known in the art, and they are used in the form of salts.

[0014] The present invention also provides a method for preparing the tetravalent platinum immunogenic death inducing agent, comprising the following steps:

[0015] (1) reacting a divalent platinum complex with hydrogen peroxide H2O2 to obtain a tetravalent platinum complex;

[0016] Wherein, the divalent platinum complex is selected from any one of the following compounds:

[0017]

[0018] (2) Flufenamic acid (FFA) The carboxyl group of the product was activated with N-hydroxysuccinimide NHS and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI to obtain flufenamic acid activated ester (FFA-NHS)

[0019] (3) adding the tetravalent platinum complex prepared in step (1) and the flufenamic acid activated ester prepared in step (2) to the DMF solution, heating and stirring until the reaction is complete;

[0020] (4) Centrifuge, concentrate the supernatant, add methanol to the concentrate and mix well, then add it to diethyl ether to precipitate a solid, centrifuge, discard the supernatant, retain the precipitate, repeatedly wash the precipitate with methanol and diethyl ether, and dry it to obtain the target product.

[0021] Specifically, a preferred preparation method of the present invention comprises the following steps:

[0022] (1) reacting a divalent platinum complex with hydrogen peroxide H2O2 to obtain a tetravalent platinum complex;

[0023] (2) Flufenamic acid (FFA) The carboxyl groups were activated with N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI);

[0024] (3) After the reaction is completed, the mixture is repeatedly extracted with water, and the organic phase is retained. The solvent is evaporated to obtain a yellow powder, which is the activated ester of flufenamic acid (FFA-NHS).

[0025] (4) adding the tetravalent platinum complex prepared in step (1) and the flufenamic acid activated ester prepared in step (2) to the DMF solution, stirring to dissolve, and heating in an oil bath to 65° C.;

[0026] (5) After the reaction is completed, the precipitate is removed by centrifugation, and the concentrate is concentrated under reduced pressure. A small amount of methanol is added to the concentrate and mixed evenly, and then the concentrate is added to a large amount of ether to precipitate a solid. The mixed solution is centrifuged and the supernatant is discarded, and the precipitate is retained. The precipitate is repeatedly washed with methanol and ether, and vacuum dried to obtain the target product.

[0027] The present invention also provides a pharmaceutical composition, comprising the tetravalent platinum immunogenic death inducing agent and a pharmaceutically acceptable carrier or excipient.

[0028] The pharmaceutical composition of the present invention can be administered in various known ways, such as orally, parenterally, by inhalation spray or via an implanted reservoir. The pharmaceutical composition of the present invention can be administered alone or in combination with other drugs. Oral compositions can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, suspensions, dispersions and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, adhesives, colorants, fillers, emulsifiers, etc.

[0029] Sterile injectable compositions can be formulated using suitable dispersing agents or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.

[0030] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected depends on a variety of factors, including the activity of the specific compound of the present invention or its salt used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0031] The present invention also provides the use of the tetravalent platinum immunogenic death inducer in the preparation of anti-tumor drugs.

[0032] The tumor is lung cancer, liver cancer, gastric cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, prostate cancer, head and neck cancer or glioma.

[0033] The invention also provides a chemical immune anticancer agent, the effective component of which is Pt-FFA.

[0034] Beneficial effects:

[0035] The invention provides a tetravalent platinum immunogenic death inducing agent with AKR inhibitory effect, which is obtained by connecting flufenamic acid (FFA) to a cisplatin derivative to obtain a tetravalent platinum complex Pt-FFA. The preparation method is simple and easy, and the synthetic raw materials are easily available.

[0036] The tetravalent platinum complex prepared by the present invention maintains low activity in plasma and normal tissues, and is reduced to a divalent platinum complex and flufenamic acid in the reducing environment of tumor cells and exerts its efficacy. Studies have found that Pt-FFA can not only exert tumor killing activity through chemotherapy effects such as DNA damage, cell cycle arrest, and induction of cell apoptosis, but also induce the immunogenic death of tumors, promote the increase of cytotoxic T cells in the tumor immune microenvironment, and achieve tumor immunosuppression.

[0037] It is worth noting that Pt-FFA has a dual inhibitory effect on the expression and enzyme activity of AKR1C1, a chemotherapy resistance-related target in cisplatin-resistant lung adenocarcinoma strains, thereby overcoming the platinum resistance of tumors.

[0038] Therefore, the tetravalent platinum immunogenic death inducer provided by the present invention can be used to prepare anti-tumor drugs, and has great application prospects in overcoming tumor platinum drug resistance and developing chemotherapeutic immunological anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0040] Figure 1 is the Pt-FFA in Example 1 of the present invention 1 H-NMR spectrum (DMSO-d6, 400 MHz);

[0041] Figure 2 is the Pt-FFA in Example 1 of the present invention 13 C-NMR spectrum (DMSO-d6, 400 MHz);

[0042] Figure 3 is the Pt-FFA in Example 1 of the present invention 195 Pt-NMR spectrum (DMSO-d6, 86 MHz);

[0043] Figure 4 HR-ESI-MS (CH3OH) spectrum of Pt-FFA in Example 1 of the present invention;

[0044] Figure 5 The effect of Pt-FFA on the expression of DNA damage marker γ-H2AX in cisplatin-resistant lung cancer cells A549 / DDP; the effect diagram of the effect on γ-H2AX protein expression ( Figure 5 A); γ-H2AX protein expression WB band grayscale analysis statistics ( Figure 5 B);

[0045] Figure 6The effect of Pt-FFA on the cell cycle arrest of cisplatin-resistant lung cancer cells A549 / DDP;

[0046] Figure 7 The effect of Pt-FFA on apoptosis of cisplatin-resistant lung cancer cells A549 / DDP;

[0047] Figure 8 The inhibitory effect of Pt-FFA on ARK1C1 protein expression and AKR1C1 enzyme activity in cisplatin-resistant lung cancer cells A549 / DDP; the inhibitory effect on ARK1C1 protein expression ( Figure 8 A); ARK1C1 protein expression WB band grayscale analysis statistics ( Figure 8 B); Inhibitory effect on AKR1C1 enzyme activity ( Figure 8 C);

[0048] Fig. 9 Effect of Pt-FFA on CRT expression on the cell surface of cisplatin-resistant lung cancer cells A549 / DDP ( Fig. 9 A) ATP in culture medium ( Fig. 9 B) and HMGB1 release detection ( Fig. 9 C);

[0049] Fig.10 The inhibitory effect of Pt-FFA "cancer vaccine" on the growth of LLC lung cancer transplanted tumors in C57BL / 6J mice;

[0050] Fig.10 A is a picture of the subcutaneous transplanted tumor removed after drug treatment; Fig.10 B shows the change in the number of tumor-free mice; Fig.10 C is the change of tumor volume;

[0051] Fig.11 The inhibitory effect of Pt-FFA injected into the tail vein on the growth of LLC lung cancer transplanted tumors in C57BL / 6J mice ( Fig.11 A- Fig.11 C) and mouse body weight changes ( Fig.11 D)

[0052] Fig.12 Pt-FFA was used to treat helper T cells (CD4 + T cells)( Fig.12 A), cytotoxic T cells (CD8 + T cells)( Fig.12 B), mature dendritic cells (CD86 + CD80 + DC cells) Fig.12C); and the changes in serum pro-inflammatory factors IFN-γ, IL-6, and TNF-α ( Fig.12 D)

[0053] Fig.13 Schematic diagram of the mechanism of action of Pt-FFA of the present invention. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0055] The starting materials, reaction reagents, etc. used in the specific embodiments of the present invention are all commercially available. The present invention can be prepared in the form of salt by using a salt-forming method commonly used in the art, for example, dissolving the compound in hydrochloric acid ethanol at room temperature to react to generate a hydrochloride; or adding benzenesulfonic acid to react to generate a benzenesulfonate.

[0056] The experimental methods in the examples of the present invention where specific conditions are not specified are generally carried out under conventional conditions or under conditions recommended by raw material or product manufacturers.

[0057] Example 1 Synthesis of tetravalent platinum complex Pt-FFA

[0058] (1) Place 1 g of cisplatin in a flask, add 40 ml of water and stir until dissolved, then slowly dropwise add 50 ml of 30% hydrogen peroxide, heat to 75°C and stir for 5 h in the dark, filter while hot, place in a refrigerator at 4°C for two days, and vacuum dry to obtain pale yellow crystals of tetravalent oxoplatin with a yield of 87%.

[0059]

[0060] (2) 1 g of flufenamic acid (FFA) was placed in a flask, and 10 ml of dichloromethane was added to dissolve it; 491.08 mg of N-hydroxysuccinimide (NHS) and 817.98 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were weighed and dissolved in dichloromethane, and then slowly added dropwise to the FFA solution. After stirring at room temperature for 24 hours, the mixture was repeatedly extracted with water, the organic phase was retained, and the solvent was evaporated to obtain a yellow powder, which was flufenamic acid activated ester (FFA-NHS), with a yield of 55%.

[0061]

[0062] (3) Take 100 mg of Oxoplatin and 132.4 mg of flufenamic acid activated ester (FFA-NHS) and dissolve them in 10 ml of DMF. Stir in an oil bath at 65°C for 24 h.

[0063] (4) After the reaction is completed, the precipitate is removed by centrifugation. After the reaction solution is concentrated under reduced pressure, about 3 mL of methanol is added and mixed evenly. The mixture is slowly dripped into 30 mL of ether to precipitate yellow flocs. The mixture is washed twice with methanol and twice with ether to obtain a pure solid, which is a tetravalent platinum complex Pt-FFA. The yield is 58%.

[0064] Tetravalent platinum complex Pt-FFA 1 H-NMR spectrum (DMSO-d6, 400MHz) Figure 1 , 13 C-NMR spectrum (DMSO-d6, 400MHz) Figure 2 , 195 Pt-NMR spectrum (DMSO-d6, 400MHz) Figure 3 , high resolution mass spectrometry (CH3OH) Figure 4 .

[0065] 1 H-NMR(500MHz,DMSO-d6)δ(ppm):9.83(s,1H),7.98(s,1H),7.90-7.89(d,1H),7.54-7. 53(d,2H),7.39-7.38(d,2H),7.27-7.25(d,1H),6.89-6.86(t,1H),6.23-5.97(m,6H).

[0066] 13 C-NMR (600MHz, DMSO-d6, ppm): 174.28, 161.79, 144.46, 132.22, 130.75, 121.77, 120.23, 119.19, 115.65, 35.80, 28.59.

[0067] 195 Pt-NMR (86MHz, DMSO-d6, ppm): 1032.11.

[0068] ESI-MS(positive,m / z):596.0209,597.0234,598.0223,599.0212,600.0217.

[0069] Example 2 Cytotoxicity test of compound Pt-FFA

[0070] The compound Pt-FFA prepared in Example 1 of the present invention was subjected to a tumor cell proliferation inhibition test using the CCK-8 method.

[0071] Tumor cell lines A549, A549 / DDP, SK-HEP-1, HepG-2, SGC7901, Panc-1 and normal cell line L02 were selected, and the cell lines were purchased from the Cell Bank of the Committee for the Collection of Typical Cultures of the Chinese Academy of Sciences. The culture conditions are as follows:

[0072] A549: 10% FBS RPMI-1640 complete medium;

[0073] A549 / DDP: 10% FBS RPMI-1640 complete medium;

[0074] SK-HEP-1: 10% FBS MEM complete medium + sodium pyruvate;

[0075] HepG2: 10% FBS DMEM complete medium;

[0076] SGC7901: 10% FBS DMEM complete medium;

[0077] Panc-1: 10% FBS RPMI-1640 complete medium;

[0078] L02: 10% FBS RPMI-1640 complete medium.

[0079] The fetal bovine serum and culture medium were purchased from Gibco.

[0080] Pt-FFA and cisplatin (CDDP) were used as the drugs to be tested. After the drugs were exposed to the cells for 48 hours, the survival of the cells was observed and the half inhibitory concentration of the test cells was calculated. The specific steps are as follows:

[0081] (1) Collect the above-mentioned logarithmic phase cells, resuspend them in the above-mentioned complete medium, and adjust the concentration of the cell suspension to 5×10 4 / mL, inoculated in a 96-well plate, 200 μL per well, set up 3 replicates;

[0082] (2) The above experimental cells were placed in a cell culture incubator with a CO2 concentration of 5% and cultured at 37°C for 24 h until the cells adhered to the wall;

[0083] (3) Preparation of drug solution: Pt-FFA and cisplatin were prepared as stock solutions with DMSO, diluted with serum-free medium (Gibco) at a certain gradient multiple, added to the above-mentioned 96-well plate filled with cells, with 3 replicates for each concentration, and incubated for 48 h;

[0084] (4) CCK-8 reagent (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was diluted 10 times with serum-free medium, and 100 μL of medium containing 10 μL of CCK-8 reagent was added to each well and incubated for 1 h;

[0085] (5) Detect the absorbance of each well at 450 nm using an enzyme reader;

[0086] (6) Calculate the cell survival rate according to the following formula:

[0087]

[0088] Wherein, Abs(sample) is the absorbance value of cells in the sample group; Abs(blank) is the absorbance value of the liquid in the blank group; Abs(control) is the absorbance value of cells in the control group that have not been treated with drugs;

[0089] (7) Draw a cell survival rate-concentration curve and calculate the half inhibitory concentration (IC) of the compound. 50 ), the results are shown in Table 1.

[0090] Table 1 IC of Pt-FFA and cisplatin on different cells 50 Value (μM, 48h)

[0091]

[0092] The results in Table 1 show that Pt-FFA showed high toxicity to various tumor cell lines tested, and the effect was significantly better than cisplatin. For the cisplatin-resistant cell line A549 / DDP, Pt-FFA still showed high toxicity, about 70 times that of cisplatin. This shows that Pt-FFA may have the potential to improve platinum resistance in non-small cell lung cancer. In addition, Pt-FFA has a certain selectivity and is less toxic to normal cells.

[0093] Example 3 Study on the Damage of Tumor Cell DNA by Compound Pt-FFA

[0094] The protein expression level of γ-H2AX in cells was tested by Western blot technology, and the DNA damage caused by Pt-FFA to A549 / DDP tumor cells was detected.

[0095] The specific implementation is as follows:

[0096] (1) Collect logarithmic phase cells and inoculate them on 6-well plates, with about 500,000 cells per well. Incubate overnight at 37°C and 5% CO2. Aspirate and discard the old culture medium, dilute CDDP to 2 μM and PFA to three different concentrations of 0.5, 1, and 2 μM with serum-free culture medium, add 1 mL to each well, and continue incubating in the incubator for 24 h.

[0097] (2) Protein extraction: cells were collected and total protein was extracted using protease inhibitors (Thermo Fisher Scientific, USA), phosphatase inhibitors (Thermo Fisher Scientific, USA) and RIPA lysis buffer (Shanghai Biotech Biotechnology Co., Ltd.), lysed on ice for 30 min, centrifuged at high speed (12000 rpm) for 15 min, and the supernatant was taken to determine the protein concentration using the BCA method;

[0098] (3) Sample preparation: The protein concentration of each sample was diluted to 1 μg / μL by adding water and 5× Loading buffer (Biyuntian Biotechnology Co., Ltd., Shanghai, China) and incubated in a metal bath at 100°C for 15 min to obtain the protein sample;

[0099] (4) Perform electrophoresis in electrophoresis buffer (mainly composed of Tris, glycine and SDS), add 10 μL of each protein sample to each well, and perform electrophoresis under the conditions of 80 V for 30 min in the concentration phase and 120 V for 60 min in the separation phase. After the electrophoresis, transfer to the membrane under the conditions of 0.32 A for 60 min.

[0100] (5) After blocking with blocking solution (5% BSA dilution) at room temperature for 1 h, the PVDF strips were incubated with the corresponding primary antibody γ-H2AX (Wuhan Abotek Biotechnology Co., Ltd.) at 4°C overnight and washed three times with 1× TBST, each time for 7 min;

[0101] (6) HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA) was used for incubation at room temperature for 1 h, washed three times with 1× TBST, and exposed to chemiluminescence. The results are shown in Figure 5 .

[0102] Depend on Figure 5 The AB results show that compared with the control group without drug treatment, Pt-FFA can cause a significant upregulation of γ-H2AX in A549 / DDP cells, and the degree of induction is significantly higher than that of CDDP, indicating that Pt-FFA causes significant damage to DNA and the DNA damage signaling pathway is being activated.

[0103] Example 4 Study on the effect of compound Pt-FFA on tumor cell cycle arrest

[0104] Cell cycle arrest was tested by flow cytometry.

[0105] The specific implementation is as follows:

[0106] (1) A549 / DDP cells were cultured at 3×10 6The cells were seeded at a density of 100 cells / well on a 6-well plate. After the cells attached to the wall, the culture medium was discarded and CDDP was diluted to 2 μM and PFA was diluted to three different concentrations of 0.5, 1, and 2 μM using serum-free culture medium (Gibco). 1 mL was added to each well and cultured for 24 h at 37°C and 5% CO2.

[0107] (2) After the incubation, the drug-containing medium was discarded, the cells were washed twice with PBS, digested with trypsin (Gibco), centrifuged at 1000 rpm for 3 min, resuspended with PBS, and washed twice with centrifugation. Then, 1 mL of pre-cooled 70% ethanol was added to each tube, and the cells were gently pipetted to make single cells and evenly suspended, and fixed at 4°C overnight (12 h).

[0108] (3) The fixed cells were centrifuged and washed twice with pre-cooled PBS. PI reagent and RNase A were mixed in a ratio of 9:1 in advance. 500 μL of the mixed working solution was added to each sample, and the mixture was incubated at 37°C in the dark for 30 min. The incubated samples were loaded on the flow cytometer (the PI reagent, RNase A and working solution used were all from the cell cycle detection kit purchased from Jiangsu Keygene Biotech Co., Ltd.). The results are shown in Figure 6 .

[0109] Depend on Figure 6 It can be seen that compared with the control group without drug treatment, 0.5, 1, and 2 μM Pt-FFA acted on A549 / DDP, which produced a blocking effect on the cell cycle, reduced G0 / G1 phase cells, and increased S phase cells, that is, it mainly affected the period of cell DNA replication, and its blocking degree was concentration-dependent. Among them, compared with the same concentration of cisplatin (CDDP), 2 μM Pt-FFA blocked the S phase of the cell cycle more significantly.

[0110] Example 5 Study on the apoptosis of tumor cells induced by compound Pt-FFA

[0111] The apoptosis of tumor cells after drug administration was tested by flow cytometry using Annexin V-FITC / PI double staining method.

[0112] The specific implementation is as follows:

[0113] (1) A549 / DDP cells were cultured at 3×10 6 The cells were inoculated at a density of 100 cells / well on a 6-well plate. After the cells adhered to the wall, the culture medium was discarded and CDDP was diluted to 2 μM and Pt-FFA was diluted to three different concentrations of 0.5, 1, and 2 μM using serum-free culture medium (Gibco). 1 mL was added to each well and cultured for 48 h at 37°C and 5% CO2.

[0114] (2) After incubation, discard the drug-containing medium, wash twice with PBS, digest the cells with EDTA-free trypsin (Gibco), collect the cells after digestion, centrifuge at 1000 rpm and 4°C for 5 min, and discard the supernatant. Wash the cells twice with pre-cooled PBS, centrifuge at 1000 rpm and 4°C for 5 min each time, and discard the supernatant.

[0115] (3) First add 100μl 1×Binding Buffer to each sample, then add 5μl Annexin V-FITC and 5μl PI Staining Solution, blow gently to mix, and incubate at room temperature in the dark for 10 min; finally add 400μl 1×Binding Buffer and mix gently. After staining, the samples were detected by flow cytometry within 1 hour (Annexin V-FITC, PI Staining Solution and 1×Binding Buffer used were all from the cell apoptosis detection kit, purchased from Nanjing Novozymes Biotech Co., Ltd.). The results are as follows Figure 7 shown.

[0116] Depend on Figure 7 The results showed that compared with the control group without drug treatment, Pt-FFA induced apoptosis at 0.5 μM, and 1 and 2 μM could significantly induce apoptosis of A549 / DDP cells in a concentration-dependent manner. However, 2 μM CDDP had a weaker ability to induce apoptosis of A549 / DDP cells.

[0117] Example 6 Test of the inhibitory ability of compound Pt-FFA on ARK1C1 protein expression and enzyme activity in A549 / DDP cells

[0118] The changes in the expression level of ARK1C1 protein in cells were tested by Western blot technology, and the implementation method was the same as in Example 3. Figure 8 AB. Figure 8 AB shows that Pt-FFA down-regulated the expression level of AKR1C1 protein in A549 / DDP cells in a concentration-dependent manner, indicating that Pt-FFA can effectively reduce the expression of AKR1C1 in cells, providing a basis for its ability to improve cisplatin resistance.

[0119] The effect of Pt-FFA on the catalytic ability of AKR1C1 protein in the presence of coenzyme was detected by ultraviolet method. The specific implementation method is as follows:

[0120] (1) Prepare enzyme activity reaction buffer according to Table 2, 200 μL for each sample system, add 2 μg purified protein (Abnova) and inhibitor FFA as well as CDDP and Pt-FFA, mix well, and incubate at 37°C for 30 min;

[0121] (2) Add 0.2 mM NADP + (Shanghai Bioengineering), quickly measure the absorbance at 340nm with a microplate reader at a frequency of 1 time / min, that is, measure the relative content of NADPH generated, continue measuring for 60 minutes, and draw a relative enzyme activity curve. The results are as follows Figure 8 As shown in C.

[0122] Table 2 Enzyme activity reaction buffer

[0123]

[0124] Note: α-Tetrahydronaphthol was purchased from Adamas Reagent Co., Ltd.

[0125] Depend on Figure 8 C The results show that compared with the control group without drug treatment, 2μM CDDP has no effect on the relative content of NADPH, that is, it does not affect the enzyme activity of AKR1C1; while different concentrations of Pt-FFA and 2μM inhibitor FFA can significantly affect the amount of NADPH generated, that is, it has a significant inhibitory effect on the enzyme activity of AKR1C1, and the effect of Pt-FFA on the enzyme activity of AKR1C1 is concentration-dependent. The inhibition of Pt-FFA on the enzyme activity of AKR1C1 can affect its biological function and further affect the sensitivity of tumor cells to cisplatin.

[0126] Example 7 Detection of Molecular Markers of Immunogenic Death of Tumor Cells Induced by Pt-FFA

[0127] The early molecular mechanism of immunogenic death after the cells were treated with Pt-FFA prepared in Example 1 of the present invention was tested by immunofluorescence and ELISA. The cell line was A549 / DDP. After the cells were treated with Pt-FFA for 24 hours, the calreticulin (CRT) on the cell surface, the ATP secreted outside the cells, and the high mobility group protein B1 (HMGB1) content released outside the cells at a later stage were detected. The specific operation steps are as follows:

[0128] (1) A549 / DDP cells were inoculated on a slide in a 24-well plate and cultured at 37°C for 24 h. Drug-containing serum-free medium was added and cultured for another 24 h.

[0129] (2) After incubation, the cells were washed twice with cold PBS, fixed with 4% paraformaldehyde for 15 min, blocked with 5% goat serum albumin (Gibco) diluted with 3% BSA for 1 h, and CRT antibody (Abcam, USA) (1:50, diluted with 5% BSA) was added and incubated overnight at 4°C;

[0130] (3) The cells were washed three times with cold PBS, incubated with FITC fluorescent secondary antibody (Cell Signaling Technology) (1:500, TBST dilution) in the dark for 1 h, washed with PBS and stained with DAPI for 5 min. After washing with PBS, the cell fluorescence was photographed by laser scanning confocal microscopy. The results are shown in Fig. 9 A.

[0131] (4) A549 / DDP cells were seeded in 6-well plates at a number of 3×10 6 After culturing for 24 hours, the culture medium was discarded and 1 mL of serum-free culture medium containing drugs was added again. The culture was continued for 24 hours. The supernatant was carefully collected and the ATP concentration in the supernatant was detected using an enhanced ATP detection kit (Shanghai Biotech Biotechnology Co., Ltd., S0027) according to the instructions provided by the manufacturer. The results are shown in Fig. 9 B.

[0132] (5) A549 / DDP cells were cultured at 3×10 6 The cells were inoculated at a density of 100 cells / well on a 6-well plate. After the cells adhered to the wall, the culture medium was discarded and 1 mL of serum-free culture medium containing drugs was added again. The culture was continued for 36 h, and the supernatant was collected. According to the instructions provided by the manufacturer, the HMGB1 in the supernatant was detected using an ELISA detection kit (Shenzhen Xinbosheng Biotechnology Co., Ltd., NOV-NB-S11133). The results are shown in Fig. 9 C.

[0133] Fig. 9 The results showed that compared with the control group without drug treatment, after Pt-FFA acted on A549 / DDP cells, there was a more obvious CRT exposure, and CRT was spotted and unevenly distributed on the surface of the cells ( Fig. 9 A); released ATP ( Fig. 9 B) and HMGB1( Fig. 9 C) significantly increased. Through the verification of these markers, it was further confirmed that Pt-FFA has a good potential to induce ICD.

[0134] Example 8: "Cancer Vaccine" Test of Compound Pt-FFA

[0135] Currently, the only gold standard for identifying whether it is an ICD inducer is the vaccination experiment. In order to detect the in vivo immune tumor suppression effect of Pt-FFA, a "cancer vaccine" test was performed in a mouse model. The specific implementation method is as follows:

[0136] (1) “Cancer vaccine” injection: LLC cells (Wuhan Pronocell Life Science Co., Ltd.) that had been given cisplatin, CDDP, or Pt-FFA for 24 h were digested with 0.25% trypsin (Gibco) and the cell concentration was adjusted to 1×10 7 / mL, and 0.1mL of cell suspension was injected into C57BL / 6J mice (SPF grade, female, weighing 20-22g, 6 weeks old, purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) subcutaneously below the left armpit.

[0137] (2) One week later, LLC cells in the logarithmic growth phase were digested with 0.25% trypsin and the cell concentration was adjusted to 1×10 7 / mL, and 0.1mL of cell suspension was injected subcutaneously under the right armpit of the mouse.

[0138] (3) Observe the growth of transplanted tumors in mice. When the tumor in the blank control group grows to 5 mm x 5 mm, the transplanted tumor model is successfully established. Measure the weight of mice and the length and width of tumors every day. Calculate the tumor volume according to volume = length * width * width / 2. Fig.10 .

[0139] Fig.10 The results showed that the blank control group and CDDP group failed to inhibit tumor growth, while the tumor cell "vaccine" treated with Pt-FFA completely inhibited tumor growth ( Fig.10 A, C), effectively prolonged the survival of tumor-implanted mice ( Fig.10 B) Thus, Pt-FFA was confirmed to be an effective ICD inducer in vivo.

[0140] Example 9 In vivo tumor inhibition experiment of compound Pt-FFA

[0141] (1) Subcutaneous tumor transplantation: LLC cells in the logarithmic growth phase were digested with 0.25% trypsin and the cell concentration was adjusted to 1×10 7 / mL, take 0.1mL of cell suspension and inject it subcutaneously under the left armpit of C57BL / 6J mice. Observe the growth of tumor. When the tumor grows to 5mmx5mm, the transplanted tumor model is successfully established. Measure the length and width of the tumor every day, and calculate the tumor volume according to volume = length*width*width / 2.

[0142] (2) When the tumor volume reaches 100 mm 3Afterwards, the above 18 mice were randomly divided into 3 groups (Control group, CDDP group and Pt-FFA group). The two drug-treated groups were injected with 5 mg / kg cisplatin and 10 mg / kg Pt-FFA through the tail vein, respectively, and the control group was given an equal volume of normal saline, once every two days, for a total of 4 injections. After the end of the drug administration, the tumor was removed, weighed and photographed, and the volume of the subcutaneous tumor during the drug administration period was recorded and observed.

[0143] (3) Data processing: Data were expressed as mean ± standard deviation (Mean ± SD) and analyzed using Graphpad Prime 7.0 software; unpaired two-sided t-test was used for comparison between the two groups. Fig.11 .

[0144] Depend on Fig.11 It can be seen that there was no significant change in the body weight of mice at the end of Pt-FFA treatment ( Fig.11 D), indicating that the compound Pt-FFA has lower systemic toxicity; however, the tumor volume ( Fig.11 A, B) and weight ( Fig.11 C) is smaller than that of the cisplatin-treated group, indicating that tumor growth has been effectively inhibited and the effect is better than that of cisplatin.

[0145] Example 10 In vivo immune response monitoring of compound Pt-FFA

[0146] Flow cytometry was used to characterize the cytotoxic T cells (CD8 + T cells), helper T cells (CD4 + T cells), mature DC cells (CD80 + CD86 + The levels of pro-inflammatory cytokines (IFN-γ, IL-6, TNF-α) in mouse serum were detected by ELISA technology.

[0147] The specific implementation plan is as follows:

[0148] (1) After the C57BL / 6J mice in Example 9 were killed, the tumors and lymph nodes were collected to prepare a single cell suspension. The tumor was minced and digested with an enzyme mixture (type I collagenase, hyaluronidase, DNase I, Sigma) at 37°C for 1 h. During the digestion process, the mixture was inverted and mixed every 15 min. After the digestion was completed, the mixture was gently ground and filtered through a 200-mesh sieve to obtain a single cell suspension.

[0149] (2) The lymph nodes were placed in 1× PBS, gently crushed with a syringe plunger, and filtered through a 200-mesh sieve to obtain a single-cell suspension. Red blood cells in the single-cell suspension were removed using red blood cell lysis buffer (Sigma), and washed twice with 1× PBS.

[0150] (3) The collected cells were blocked with anti-mouse CD16 / 32 (BioLegend, USA), and lymph node cells were stained (incubation staining) with PE / Cyanine7 anti-mouse CD11c (CD11c-PE / Cyanine7), FITC anti-mouse IA / IE 6 (IA / IE 6-FITC), PE anti-mouse CD80 (CD80-PE), and APC anti-mouse CD86 (CD86-APC) according to the supplier's (BioLegend, USA) protocol. Tumor cells were incubated and stained with APC anti-mouse CD3 (CD3-APC), PE anti-mouse CD8a (CD8a-PE), and FITC anti-mouse CD4 (CD4-FITC). Finally, the stained cells were analyzed by flow cytometry. The results are shown in Fig.12 A- Fig.12 C.

[0151] (4) The eyeballs of the mice were removed and blood was collected. The blood was placed at room temperature for 1 h, allowed to coagulate and stratify, and then centrifuged at 3500 rpm for 15 min. The upper serum was carefully collected and the levels of cytokines IL-6, IFN-γ, and TNF-α in the serum were detected using an ELISA kit (Shenzhen Xinbosheng Biotechnology Co., Ltd.). The results are as follows: Fig.12 As shown in D.

[0152] Fig.12 The results showed that at the end of treatment, helper T cells (CD4 + T cells, Fig.12 A), cytotoxic T cells (CD8 + T cells, Fig.12 B), mature DC cells (CD80 + CD86 + cell, Fig.12 C) all showed significant increases, indicating that Pt-FFA promoted the maturation of dendritic cells and activated the killing ability of T cells against tumor cells, further confirming the activity of Pt-FFA in inducing tumor immunogenic death. In serum samples, proinflammatory cytokines (IFN-γ, IL-6, TNF-α, Fig.12 D) significantly increased, further demonstrating that Pt-FFA can activate a strong immune response.

[0153] In summary, if Fig.13As shown, Pt-FFA has multiple anti-tumor activities. Pt-FFA can improve tumor platinum drug resistance by inhibiting aldehyde-keto reductase, induce cell apoptosis to inhibit tumors, and can also induce tumor immunogenic death, and has synergistic anti-tumor properties of chemotherapy-immunotherapy. Therefore, the tetravalent platinum immunogenic death inducer provided by the present invention can be used to prepare anti-tumor drugs, and has great application prospects in overcoming tumor platinum drug resistance and developing chemotherapy-immune anticancer drugs.

[0154] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A tetravalent platinum immunogenic death inducing agent, characterized in that: It has the following structural formula:

2. A method for preparing the tetravalent platinum immunogenic death inducing agent according to claim 1, characterized in that: The following steps are involved: (1) reacting a divalent platinum complex with hydrogen peroxide H2O2 to obtain a tetravalent platinum complex; Wherein, the divalent platinum complex is: (2) Flufenamic acid (FFA) The carboxyl group of the product was activated with N-hydroxysuccinimide NHS and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI to obtain flufenamic acid activated ester (FFA-NHS) (3) adding the tetravalent platinum complex prepared in step (1) and the flufenamic acid activated ester prepared in step (2) to the DMF solution, heating and stirring until the reaction is complete; (4) Centrifuge, concentrate the supernatant, add methanol to the concentrate and mix well, then add it to diethyl ether to precipitate a solid, centrifuge, discard the supernatant, retain the precipitate, repeatedly wash the precipitate with methanol and diethyl ether, and dry it to obtain the target product.

3. A pharmaceutical composition, characterized in that It comprises the tetravalent platinum immunogenic death inducing agent according to claim 1 and a pharmaceutically acceptable carrier or excipient.

4. Use of the tetravalent platinum immunogenic death inducing agent according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor is lung cancer, liver cancer, stomach cancer, or pancreatic cancer.

5. A chemoimmunoassay anticancer agent, characterized in that: The active ingredient is the Pt-FFA described in claim 1.

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