CN116589502A - Tetravalent platinum type immunogenic death inducer, preparation method and application thereof - Google Patents
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- CN116589502A CN116589502A CN202310614109.3A CN202310614109A CN116589502A CN 116589502 A CN116589502 A CN 116589502A CN 202310614109 A CN202310614109 A CN 202310614109A CN 116589502 A CN116589502 A CN 116589502A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System compounds of the platinum group
- C07F15/0086—Platinum compounds
- C07F15/0093—Platinum compounds without a metal-carbon linkage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/28—Compounds containing heavy metals
- A61K31/282—Platinum compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a tetravalent platinum type immunogenic death inducer with a general formula I structure, and a preparation method and application thereof. The compound has AKR inhibition effect, can induce the immunogenic death of tumors, and shows excellent activity on platinum drug resistant tumor cells. The tetravalent platinum complex of the invention integrates the targeted AKR inhibition effect and the induced immune response effect through the unique structural advantages thereof, and can be used for preparing novel compounds with multiple antitumor mechanismsChemotherapeutic immune medicine to solve the problems of tumor resistance and lack of small molecule chemotherapeutic agent.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a tetravalent platinum immunogenic death inducer, a preparation method and application thereof; in particular, the tetravalent platinum type immunogenic death inducer of the present invention has an aldehyde ketone reductase inhibitory effect.
Background
Cancer is a type of chronic disease that is severely dangerous to human health. The platinum medicine has wide anti-tumor spectrum and obvious anticancer effect, and is one of the most commonly used chemotherapeutics in clinic. Since cisplatin was approved by the FDA in the united states for the treatment of tumors in the reproductive system, platinum-based drugs have been developed to the third generation, and have been significantly improved in terms of chemical stability, solubility, and antitumor effects. Nonetheless, platinum drugs still face two challenges to be resolved, namely serious adverse reactions and drug resistance. This forces researchers to continue to develop new platinum drugs with lighter adverse reactions and stronger targeting to remedy the shortcomings of the traditional platinum drugs.
Aldehyde Ketone Reductase (AKR) is a super family of oxidoreductases with NADPH as a coenzyme, which acts as a rate-limiting enzyme for polyol metabolic pathways, and is involved in various biological processes in vivo, and has an intervention in the metabolism of substances in tumor cells in cancer. The research shows that AKR is widely and highly expressed in human tumors, and the high expression of different subtypes has certain tumor specificity, so that AKR becomes a target of anti-tumor which is of great concern. In addition, AKR plays an important mediating role in tumor drug resistance to various chemotherapeutics, including platinum drugs, anthracyclines (danobicin and doxorubicin), methotrexate, cyclophosphamide, gefitinib and other clinically used antitumor drugs. Overexpression of AKR is an important manifestation of tumor-induced drug resistance, while its lack of expression is a good prognostic indicator of cisplatin chemosensitivity. Among the individual subtypes of AKR, the subtype AKR1C is the most important factor for initiating platinum drug resistance, and can improve the platinum drug resistance of tumors by inhibiting the expression and activity of AKR 1C. Therefore, the design of novel AKR inhibitors is an effective strategy for overcoming tumor platinum drug resistance.
Immunogenic Cell Death (ICD) is a regulator of cell death and is one of the important mechanisms of action in tumor immunotherapy. Upon stimulation with an immunogenic death inducer, a variety of ICD damage-associated molecular patterns such as Calreticulin (CRT), high mobility group protein 1 (HM)GB 1), ATP is secreted or transported outside the cell, promoting Dendritic Cell (DC) maturation and activation and antigen presentation to cytotoxic T cells. Cytotoxic CD8 + T cells are recruited to the tumor microenvironment, phagocytizing tumor cells, resulting in tumor immune killing. The immunogenic death inducer changes the tumor cells from a cold immune state to a hot immune state, and regulates the inhibitory tumor immune microenvironment, thus being an effective anti-tumor immunotherapy strategy.
Disclosure of Invention
The invention aims to: the invention aims to provide a tetravalent platinum immunogenic death inducer, a preparation method and application thereof. The compound is tetravalent platinum complex, has AKR inhibition effect, can induce immunogenic death of tumor, and shows excellent activity on platinum drug-resistant tumor cells. The tetravalent platinum complex integrates the targeted AKR inhibition effect and the induced immune response effect through the unique structural advantages, can be used for preparing novel chemotherapeutic immune medicines with multiple anti-tumor mechanisms, and solves the problems of tumor resistance and lack of small molecule chemical immune therapeutic agents.
The technical scheme is as follows: the aim of the invention is achieved by the following technical scheme:
the invention provides a tetravalent platinum type immunogenic death inducer with a general formula I:
wherein ,
the structure of (2) is any one of the following: />
The tetravalent platinum type immunogenic death inducer disclosed by the invention is preferably selected from the following compounds:
the compounds of the general formula I according to the invention mentioned above may also be present in the form of their salts, which are converted in vivo into compounds of the general formula I. For example, within the scope of the present invention, the compounds of the present invention are converted to pharmaceutically acceptable salt forms and used in salt form according to procedures known in the art.
The invention also provides a preparation method of the tetravalent platinum immunogenic death inducer, which comprises the following steps:
(1) Bivalent platinum complex and hydrogen peroxide H 2 O 2 The tetravalent platinum complex is prepared by reaction;
wherein the divalent platinum complex is selected from any one of the following compounds:
(2) FFA of flufenamic acidActivated with N-hydroxysuccinimide NHS and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI to give the activated ester of flufenamic acid (FFA-NHS)
(3) Adding the tetravalent platinum complex prepared in the step (1) and the flufenamic acid activated ester prepared in the step (2) into a DMF solution, heating and stirring until the reaction is finished;
(4) Centrifuging, concentrating the supernatant, adding methanol and concentrated solution, mixing, adding into diethyl ether to precipitate solid, centrifuging, removing supernatant, retaining precipitate, repeatedly washing precipitate with methanol and diethyl ether, and drying to obtain target product.
Specifically, the preparation method of the invention comprises the following steps:
(1) Bivalent platinum complex and hydrogen peroxide H 2 O 2 The tetravalent platinum complex is prepared by reaction;
(2) Flufenamic Acid (FFA)Is activated with N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI);
(3) Repeatedly extracting with water after the reaction, retaining organic phase, and steaming solvent to obtain yellow powder (FFA-NHS)
(4) Adding the tetravalent platinum complex prepared in the step (1) and the flufenamic acid activated ester prepared in the step (2) into DMF solution, stirring and dissolving, and heating to 65 ℃ in an oil bath;
(5) After the reaction is finished, centrifuging to remove precipitate, concentrating under reduced pressure, adding a small amount of methanol and concentrated solution, uniformly mixing, adding into a large amount of diethyl ether to precipitate solid, centrifuging the mixed solution, discarding supernatant, retaining precipitate, repeatedly washing the precipitate with methanol and diethyl ether, and vacuum drying to obtain the target product.
The invention also provides a pharmaceutical composition, which comprises the tetravalent platinum immunogenic death inducer and a pharmaceutically acceptable carrier or auxiliary material.
The pharmaceutical compositions of the present invention may be administered in a variety of known ways, such as orally, parenterally, by inhalation spray, or via an implanted reservoir. The pharmaceutical composition of the invention can be administered alone or in combination with other drugs. The oral composition may be any orally acceptable dosage form including, but not limited to, tablets, capsules, emulsions, suspensions, dispersions, and solutions. Common pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, and the like.
Sterile injectable compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.
The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for the particular patient, composition and mode of administration, and that is non-toxic to the patient. The dosage level selected will depend on a variety of factors including the activity of the particular compound of the invention or salt thereof employed, the route of administration, the time of administration, the rate of excretion of the particular composition employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, general health and past medical history of the patient being treated, and like factors well known in the medical arts.
The invention also provides application of the tetravalent platinum immunogenic death inducer in preparation of antitumor drugs.
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.
The invention also provides a chemical immune anticancer agent, the active ingredient of which is Pt-FFA.
The beneficial effects are that:
the tetravalent platinum type immunogenic death inducer with AKR inhibition effect provided by the invention is obtained by connecting flufenamic acid (FFA) to cis-platinum derivatives. The preparation method is simple and feasible, and the synthetic raw materials are easy to obtain.
The tetravalent platinum complex prepared by the invention keeps low activity in plasma and normal tissues, and is reduced into a divalent platinum complex and flufenamic acid in a tumor cell reducing environment to exert drug effects. The research shows that Pt-FFA can exert tumor killing activity through the chemotherapy effects such as DNA damage, cell cycle retardation, induction of apoptosis and the like; but also can induce the immunogenic death of the tumor, promote the increase of cytotoxic T cells in the tumor immune microenvironment and realize the immunosuppression of the tumor.
It is worth noting that Pt-FFA has double inhibition effects on the expression and enzyme activity of a chemotherapy drug resistance related target AKR1C1 of a lung adenocarcinoma cisplatin-resistant strain, so that the platinum drug resistance of tumors can be overcome.
Therefore, the tetravalent platinum immunogenic death inducer provided by the invention can be used for preparing anti-tumor drugs, and has a large application prospect in overcoming the drug resistance of tumor platinum drugs and developing chemotherapy immune anti-cancer drugs.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
FIG. 1 is a sample of the Pt-FFA in example 1 of the present invention 1 H-NMR spectrum (DMSO-d) 6 ，400MHz)；
FIG. 2 shows the Pt-FFA of example 1 of the present invention 13 C-NMR spectrum (DMSO-d) 6 ，400MHz)；
FIG. 3 shows the Pt-FFA of example 1 of the present invention 195 Pt-NMR spectrum (DMSO-d) 6 ，86MHz)；
FIG. 4 shows the HR-ESI-MS (CH) of Pt-FFA in example 1 of the present invention 3 OH) spectra;
FIG. 5 shows the effect of Pt-FFA on the expression of the DNA damage marker gamma-H2 AX in cisplatin-resistant lung cancer cells A549/DDP; effect on gamma-H2 AX protein expression profile (fig. 5A); gamma-H2 AX protein expression WB band gray scale analysis statistical graph (FIG. 5B);
FIG. 6 is a graph showing the effect of Pt-FFA on cycle arrest of cisplatin-resistant lung cancer cells A549/DDP;
FIG. 7 is a graph showing the effect of Pt-FFA on apoptosis of cisplatin-resistant lung cancer cells A549/DDP;
FIG. 8 shows the inhibitory effect of Pt-FFA on ARK1C1 protein expression and AKR1C1 enzyme activity in cisplatin-resistant lung cancer cells A549/DDP; an inhibition effect on ARK1C1 protein expression graph (fig. 8A); ARK1C1 protein expression WB band gray scale analysis statistical graph (FIG. 8B); inhibitory effect on AKR1C1 enzyme activity (fig. 8C);
FIG. 9 shows the effect of Pt-FFA on CRT expression on the cell surface of cisplatin-resistant lung cancer cells A549/DDP (FIG. 9A), ATP in culture (FIG. 9B), and HMGB1 release assay (FIG. 9C);
FIG. 10 shows the effect of Pt-FFA "cancer vaccine" on inhibition of C57BL/6J mice LLC lung cancer transplanted tumor growth;
FIG. 10A is a photograph of a subcutaneous graft taken after a drug administration treatment; FIG. 10B is a graph showing the number change of tumor-free mice; FIG. 10C shows the change in tumor volume;
FIG. 11 shows the effect of tail vein injection of Pt-FFA on inhibiting tumor growth in LLC 57BL/6J mice with LLC lung cancer transplantation (FIGS. 11A-11C) and weight change in mice (FIG. 11D);
FIG. 12 shows T helper cells (CD 4) in Pt-FFA versus C57BL/6J mice LLC lung cancer transplanted tumor tissue + T cells) (fig. 12A), cytotoxic T cells (CD 8) + T cells) (fig. 12B), mature dendritic cells (CD 86) + CD80 + DC cells) (fig. 12C); and changes in the pro-inflammatory factors IFN-gamma, IL-6, TNF-alpha in serum (FIG. 12D);
FIG. 13 is a schematic diagram showing the mechanism of action of the Pt-FFA of the present invention.
Detailed Description
The technical scheme of the present invention is described in detail below through specific examples, but the scope of the present invention is not limited to the examples.
The starting materials, reagents, etc. used in the examples of the present invention are all commercially available. The present invention can be prepared in salt form using salt forming methods commonly used in the art, for example: at room temperature, dissolving the compound into ethanol hydrochloride to react to generate hydrochloride; or adding benzenesulfonic acid into the mixture to react to obtain benzenesulfonate.
The experimental methods of the present invention, in which specific conditions are not specified, are generally performed according to conventional conditions or according to conditions suggested by the manufacturer of the raw materials or goods.
EXAMPLE 1 Synthesis of tetravalent platinum Complex Pt-FFA
(1) 1g of cis-platinum (Cisplatin) is placed in a flask, 40mL of water is added and stirred until the cis-platinum (Cisplatin) is dissolved, then 50mL of 30% hydrogen peroxide is slowly added dropwise, the mixture is heated to 75 ℃ and stirred for 5 hours in a dark place, then the mixture is filtered when the mixture is hot, the mixture is placed in a refrigerator at 4 ℃ for two days, and the mixture is dried in vacuum to obtain tetravalent Oxoplatin light yellow crystals, wherein the yield is 87%.
(2) 1g of flufenamic acid (FFA) was placed in a flask, and 10ml of methylene chloride was added to dissolve the same; 491.08mg of N-hydroxysuccinimide (NHS) and 817.98mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) are respectively weighed and dissolved in dichloromethane, slowly added into FFA solution in a dropwise manner, stirred at room temperature for 24 hours, repeatedly extracted with water, the organic phase is reserved, and the solvent is rotationally distilled to obtain yellow powder, namely the flufenamic acid activated ester (FFA-NHS), and the yield is 55%.
(3) 100mg Oxoplatin,132.4mg flufenamic acid activated ester (FFA-NHS) was dissolved in 10ml DMF and stirred in an oil bath at 65℃for 24h.
(4) After the reaction is finished, the precipitate is removed by centrifugation, the reaction solution is concentrated under reduced pressure, and then is added with about 3mL of methanol to be uniformly mixed, and is slowly dripped into 30mL of diethyl ether to precipitate yellow floccules, and the yellow floccules are respectively washed twice with methanol and diethyl ether to obtain pure solids, namely tetravalent platinum complex Pt-FFA, and the yield is 58%.
Tetravalent platinum complex Pt-FFA 1 H-NMR spectrum (DMSO-d) 6 400 MHz) is shown in FIG. 1, 13 C-NMR spectrum (DMSO-d) 6 400 MHz) is shown in FIG. 2, 195 Pt-NMR spectrum (DMSO-d) 6 400 MHz) is shown in FIG. 3, high resolution mass spectrometry (CH 3 OH) is shown in FIG. 4.
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)。
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。
195 Pt-NMR(86MHz,DMSO-d6,ppm):1032.11。
ESI-MS(positive,m/z):596.0209,597.0234,598.0223,599.0212,600.0217。
Example 2 test of cytotoxic Activity of Compound Pt-FFA
The compound Pt-FFA prepared in the embodiment 1 of the invention is subjected to a tumor cell proliferation inhibition test, and the test method adopts a CCK-8 method.
Tumor cell lines A549, A549/DDP, SK-HEP-1, hepG-2, SGC7901, panc-1 and normal cell line L02 are selected respectively, and the cell lines are all purchased from a cell bank of the national academy of sciences' typical culture preservation committee. The culture conditions were as follows:
a549:10% FBS RPMI-1640 complete medium;
a549/DDP:10% FBS RPMI-1640 complete medium;
SK-HEP-1:10% FBS MEM complete medium+sodium pyruvate;
HepG2:10% FBS DMEM complete medium;
SGC7901:10% FBS DMEM complete medium;
panc-1:10% FBS RPMI-1640 complete medium;
l02:10% FBS RPMI-1640 complete medium.
The above fetal bovine serum and culture medium were purchased from Gibco corporation.
Taking Pt-FFA and Cisplatin (CDDP) as medicaments to be detected, allowing a substance to be detected to act on cells for 48 hours, observing the survival condition of the cells, and calculating the half inhibition concentration of the cells to be tested, wherein the specific operation steps are as follows:
(1) Collecting the logarithmic phase cells, re-suspending with the complete culture medium, and adjusting the concentration of the cell suspension to 5×10 4 Inoculating the cells into 96-well plates with 200 mu L of each well, and arranging 3 compound wells;
(2) Placing the above experimental cells in CO 2 In a cell incubator with a concentration of 5%, 37 DEG CCulturing for 24h until the cells adhere to the wall;
(3) Preparing a medicine solution: preparing mother solution of Pt-FFA and cisplatin with DMSO, diluting with serum-free culture medium (Gibco) according to a certain gradient multiple, adding into the above cell-paved 96-well plate, arranging 3 compound wells for each concentration, and incubating for 48h;
(4) CCK-8 reagent (available from Nanjinouzan Biotechnology Co., ltd.) was diluted 10-fold with serum-free medium, 100. Mu.L of medium containing 10. Mu.L of CCK-8 reagent was added to each well, and incubation was continued for 1 hour;
(5) Detecting the absorbance value of each hole at 450nm of the enzyme label instrument;
(6) Cell viability was calculated according to the following formula:
wherein Abs (sample) is the absorbance value of the cells of the sample group; abs (blank) is the absorbance value of the liquid in the blank; abs (control) is the absorbance value of the control cells not treated with the drug;
(7) Cell viability-concentration curves were made and the semi-Inhibitory Concentration (IC) of the compound was calculated 50 ) The results are shown in Table 1.
TABLE 1Pt-FFA, cisplatin versus different cells IC 50 Value (mu M,48 h)
The results in Table 1 show that Pt-FFA shows higher toxicity to various tumor cell lines tested, and the effect is significantly better than that of cisplatin. For cisplatin resistant cell line A549/DDP, pt-FFA still shows high toxicity, about 70 times that of cisplatin. This suggests that Pt-FFA may have the potential to improve platinum drug resistance for non-small cell lung cancer. And Pt-FFA has certain selectivity and has smaller toxicity to normal cells.
EXAMPLE 3 study of Compound Pt-FFA on DNA damage of tumor cells
Protein expression level of gamma-H2 AX in cells is tested by Western blot technology, and DNA damage caused by Pt-FFA to A549/DDP tumor cells is detected.
The specific implementation mode is as follows:
(1) Collecting log phase cells, inoculating into 6-well plate, and collecting about 50 ten thousand cells per well, and collecting 5% CO at 37deg.C 2 Incubate overnight under conditions. Old culture medium is sucked and removed, CDDP is diluted to 2 mu M by serum-free culture medium, PFA is diluted to three different concentrations of 0.5, 1 and 2 mu M, 1mL is added to each hole, and incubation is continued in an incubator for 24 hours;
(2) Protein extraction: collecting cells, extracting whole protein by protease inhibitor (Thermo company of America), phosphatase inhibitor (Thermo company of America) and RIPA lysate (bio-technology limited of Shanghai Biyun) for 30min, performing ice lysis, centrifuging at high speed (12000 rpm) for 15min, collecting supernatant, and measuring protein concentration by BCA method;
(3) Sample preparation: diluting the protein concentration of each sample to 1 mug/mu L by adding water and 5×loading buffer (Shanghai Biyun biotechnology Co., ltd., china), and incubating for 15min at 100deg.C in a metal bath to obtain protein samples;
(4) Electrophoresis is carried out in electrophoresis buffer solution (the main components are Tris, glycine and SDS), 10 mu L of each protein sample is added into each hole, the electrophoresis condition is that the voltage is 80V in a concentration stage, 30min, 120V in a separation stage and 60min, and after the electrophoresis is finished, membrane transfer is carried out, wherein the membrane transfer condition is 0.32A and 60min;
(5) After blocking with blocking solution (5% BSA dilution) at room temperature for 1H, the PVDF strips were incubated with the corresponding primary antibody gamma-H2 AX (WUF Botaike Biotechnology Co., ltd.) overnight at 4℃and washed three times with 1 XTBE for 7min each;
(6) HRP-labeled goat anti-rabbit secondary antibody (Abcam, usa) was selected and incubated for 1h at room temperature, washed three times with 1×tbst, and exposed to light by a chemiluminescent apparatus, the results of which are shown in fig. 5.
As can be seen from the results of fig. 5A-B: compared with the Control group without drug treatment, pt-FFA can cause significant up-regulation of gamma-H2 AX in A549/DDP cells, and the degree of induction of the up-regulation is significantly higher than CDDP, indicating that Pt-FFA causes significant damage to DNA and that DNA damage signaling pathway is being activated.
EXAMPLE 4 investigation of tumor cell cycle arrest by the Compound Pt-FFA
Cell cycle arrest was tested by flow cytometry.
The specific implementation mode is as follows:
(1) A549/DDP cells were cultured at 3X 10 6 Density of each well was seeded on 6-well plates, after cell attachment, the medium was discarded, CDDP was diluted to 2. Mu.M with serum-free medium (Gibco), PFA was diluted to three different concentrations of 0.5, 1, 2. Mu.M, 1mL of each well was added at 37℃and 5% CO 2 Culturing was continued for 24 hours.
(2) After the incubation, the drug-containing medium was discarded, washed twice with PBS, digested with pancreatin (Gibco), centrifuged at 1000rpm for 3min, resuspended in PBS for 2 times, and then 1mL of pre-chilled 70% ethanol was added to each tube, and the cells were gently beaten with a pipette to form single cells and suspended evenly and fixed overnight at 4 ℃ (12 h).
(3) The fixed cells were centrifuged, washed 2 times with pre-chilled PBS, PI reagent and RNase A were mixed in advance at a ratio of 9:1, 500. Mu.L of the mixed working solution was added to each sample, incubated at 37℃for 30min in the dark, and the incubated samples were loaded on a flow cytometer (PI reagent, RNase A and working solution used were all from cell cycle detection kit, available from Jiangsu Kaiki Biotechnology Co., ltd.). The results are shown in FIG. 6.
As is clear from FIG. 6, when 0.5, 1, 2. Mu.M of Pt-FFA acts on A549/DDP, it has a blocking effect on the cell cycle, and the G0/G1 phase cells decrease, and the S phase cells increase, that is, the period of cell DNA replication is mainly affected, and the degree of blocking has a concentration dependence, compared with the Control group without the drug treatment. Among them, the 2. Mu.M Pt-FFA is more pronounced in blocking the S phase of the cell cycle than Cisplatin (CDDP) at the same concentration.
EXAMPLE 5 study of Compound Pt-FFA to induce apoptosis of tumor cells
Tumor cells were tested for apoptosis following administration by flow cytometry Annexin V-FITC/PI double staining.
The specific implementation mode is as follows:
(1) A549/DDP cells were cultured at 3X 10 6 Individual/holesAfter cell attachment, the medium was discarded, CDDP was diluted to 2. Mu.M with serum-free medium (Gibco) and Pt-FFA was diluted to three different concentrations of 0.5, 1, 2. Mu.M with 1mL of 5% CO per well at 37 ℃ 2 Culturing was continued for 48 hours.
(2) After the incubation, the drug-containing medium was discarded, washed twice with PBS, cells were digested with pancreatin (Gibco) without EDTA, and after termination of the digestion, the cells were collected and centrifuged at 1000rpm at 4℃for 5min, and the supernatant was discarded. The cells were washed twice with pre-chilled PBS, centrifuged at 1000rpm at 4℃for 5min each, and the supernatant was discarded.
(3) Each sample was added with 100. Mu.l of 1 Xbinding Buffer, followed by 5. Mu.l of Annexin V-FITC and 5. Mu. lPI Staining Solution, gently swirled, and incubated at room temperature for 10min in the absence of light; finally, 400. Mu.l of 1 Xbinding Buffer was added and gently mixed. The stained samples were examined by flow cytometry over 1h (Annexin V-FITC, PI Staining Solution and 1 Xbinding Buffer all from apoptosis detection kits, available from Nanjinouzan Biotechnology Co., ltd.). The results are shown in FIG. 7.
As can be seen from the results of FIG. 7, pt-FFA induced apoptosis at 0.5. Mu.M, 1, 2. Mu.M significantly induced apoptosis of A549/DDP cells, and showed concentration dependence, compared to the Control group without drug treatment. Whereas 2. Mu.M CDDP was less capable of inducing apoptosis in A549/DDP.
EXAMPLE 6 test of the inhibitory Capacity of Compound Pt-FFA to ARK1C1 protein expression and enzymatic Activity in A549/DDP cells
The procedure of example 3 was repeated to test the change in the intracellular ARK1C1 protein expression level by Westren blot technique. The results are shown in FIGS. 8A-B. From FIGS. 8A-B, it is known that concentration dependence downregulation occurs on the expression level of AKR1C1 protein in A549/DDP cells by Pt-FFA, which indicates that Pt-FFA can effectively reduce the expression of AKR1C1 in cells, and provides a basis for improving cisplatin resistance.
The influence of Pt-FFA on the catalytic ability of AKR1C1 protein in the presence of coenzyme is detected by an ultraviolet method, and the specific implementation method is as follows:
(1) Preparing enzyme activity reaction buffer according to Table 2, wherein each sample system is 200 mu L, adding 2 mu g purified protein (Abnova company) and inhibitor FFA and CDDP, pt-FFA, mixing, and incubating at 37deg.C for 30min;
(2) Adding 0.2mM NADP + The absorbance (of Shanghai Biotechnology) was measured rapidly at 340nm with a microplate reader at a frequency of 1 time/min, i.e.the relative amount of NADPH produced was measured, and the relative enzyme activity was plotted by continuous measurement for 60 min. The results are shown in FIG. 8C.
TABLE 2 enzyme reaction buffers
Note that: alpha-tetrahydronaphthol was purchased from Aldammars reagent Co., ltd
As can be seen from the results in FIG. 8C, 2. Mu.M CDDP has no effect on the relative amount of NADPH, i.e., does not affect the enzymatic activity of AKR1C1, compared to the Control group without drug treatment; the Pt-FFA and the inhibitor FFA with different concentrations and 2 mu M can obviously influence the generation amount of NADPH, namely, the Pt-FFA has obvious inhibition effect on the enzyme activity of AKR1C1, and the influence of the Pt-FFA on the enzyme activity of AKR1C1 has concentration dependence. Inhibition of AKR1C1 enzymatic activity by Pt-FFA can affect its biological function and further affect the sensitivity of tumor cells to cisplatin.
EXAMPLE 7 detection of Pt-FFA induced tumor cell immunogenic death molecular markers
For the early molecular mechanism of immunogenic death after the Pt-FFA treatment cells prepared in the embodiment 1 of the invention, the test method adopts immunofluorescence and ELISA methods. After Pt-FFA is acted on cells for 24 hours, the content of Calreticulin (CRT), extracellular secreted ATP and high mobility group protein B1 (HMGB 1) released to the outside in a later period are detected, and the specific operation steps are as follows:
(1) Inoculating A549/DDP cells on a climbing sheet of a 24-pore plate for culture, culturing at 37 ℃ for 24 hours, and adding a medicine-containing serum-free culture medium for continuous culture for 24 hours;
(2) After the incubation was completed, 2 times with cold PBS, 4% paraformaldehyde fixed for 15 minutes, 5% goat serum albumin (Gibco) diluted with 3% bsa, blocked for 1 hour, CRT antibodies (Abcam, usa) (1:50, 5% bsa dilution) were added and incubated overnight at 4 ℃;
(3) Cells were incubated with FITC fluorescent secondary antibody (Cell Signaling Technology) (1:500, TBST dilution) three times with cold PBS for 1h in the absence of light, washed with PBS, stained with DAPI for 5min, washed with PBS, and fluorescence of cells was captured by laser scanning confocal microscopy, as shown in FIG. 9A.
(4) a549/DDP cells were seeded in 6-well plates in 3×10 numbers 6 After 24 hours incubation, the medium was discarded, 1mL of serum-free medium containing the drug was added again, incubation was continued for 24 hours, the supernatant was carefully collected, and the ATP concentration in the supernatant was measured using an enhanced ATP assay kit (shanghai bi yun biotechnology ltd, S0027) according to the instructions provided by the manufacturer. The result is shown in FIG. 9B.
(5) A549/DDP cells were cultured at 3X 10 6 The density of the cells/well was inoculated on a 6-well plate, after the cells were attached, the medium was discarded, 1mL of serum-free medium containing the drug was added again, the culture was continued for 36 hours, the supernatant was collected, and HMGB1 in the supernatant was detected using ELISA detection kit (Shenzhen Xinbo Biotechnology Co., ltd., NOV-NB-S11133) according to the instructions provided by the manufacturer. The result is shown in FIG. 9C.
The results in FIG. 9 show that there was a significant CRT exposure after Pt-FFA was applied to A549/DDP cells, and that the CRTs were spotted and unevenly distributed on the surface of the cells, compared to the Control group without drug treatment (FIG. 9A); the released ATP (fig. 9B) and HMGB1 (fig. 9C) increased significantly. By validation of these markers, it was further demonstrated that Pt-FFA has good ICD induction potential.
EXAMPLE 8 "cancer vaccine" test of Compound Pt-FFA
The only gold standard currently identifying whether it is an ICD inducer is vaccination experiments. To examine the in vivo immunosuppressive tumor-suppressing effect of Pt-FFA, it was tested for "cancer vaccine" in a mouse model. The specific implementation mode is as follows:
(1) "cancer vaccine" injection: digestion with 0.25% pancreatin (Gibco)LLC cells (Wohplaxel Life technologies Co., ltd.) were administered with cisplatin CDDP and Pt-FFA for 24h, respectively, and the cell concentration was adjusted to 1X 10 7 0.1mL of the cell suspension was injected into C57BL/6J mice (SPF grade, female, weight 20-22g,6 weeks old, animals purchased from Hangzhou child source laboratory animal technologies Co., ltd.) subcutaneously under the left armpit.
(2) After one week, LLC cells in the logarithmic growth phase were digested with 0.25% pancreatin to adjust the cell concentration to 1X 10 7 0.1mL of the cell suspension was injected subcutaneously under the right underarm of the mice.
(3) And (5) observing the growth condition of the transplanted tumor of the mice, and successfully molding the transplanted tumor of the mice when the tumor of the blank control group grows to 5mmx5 mm. Mice body weight, tumor length, width were measured daily and tumor volume was calculated as volume = length x width/2. The results are shown in FIG. 10.
The results in fig. 10 show that the blank and CDDP groups failed to inhibit tumor growth, whereas Pt-FFA treated tumor cell "vaccine" completely inhibited tumor growth (fig. 10A, C), effectively prolonging survival of the tumor-bearing mice (fig. 10B). Pt-FFA was thus demonstrated to be a potent ICD inducer in vivo.
EXAMPLE 9 in vivo tumor suppression experiment with Compound Pt-FFA
(1) Subcutaneous tumor transplantation: LLC cells in logarithmic growth phase were digested with 0.25% pancreatin, and cell concentration was adjusted to 1X 10 7 0.1mL of the cell suspension was injected subcutaneously under the left underarm of C57BL/6J mice. And observing the growth condition of the tumor, and successfully transplanting the tumor to form a mold when the tumor grows to 5mmx5 mm. Tumor length, width were measured daily and tumor volume was calculated from volume = length, width/2.
(2) When the tumor volume reaches 100mm 3 After that, the above 18 mice were randomly divided into 3 groups (Control group, CDDP group and Pt-FFA group), and 5mg/kg cisplatin, 10mg/kg Pt-FFA were administered by tail vein injection respectively, and an equal volume of physiological saline was administered to the Control group, 1 time every two days, 4 times total injection, and after the end of the administration, tumors were taken and weighed and photographed, and the volume of subcutaneous tumor during the administration was recorded and observed.
(3) And (3) data processing: data are expressed as Mean ± standard deviation (Mean ± SD), analyzed using Graphpad Prime 7.0 software; comparison between the two groups used a unpaired two-sided t-test. The results are shown in FIG. 11.
As can be seen from fig. 11, there was no significant change in mouse body weight at the end of Pt-FFA treatment (fig. 11D), indicating that compound Pt-FFA has lower systemic toxicity; however, the tumor volume (fig. 11, A, B) and weight (fig. 11, C) were smaller than those of cisplatin-administered groups, indicating that tumor growth was effectively inhibited and that the effect was superior to cisplatin.
EXAMPLE 10 in vivo monitoring of immune response of Compound Pt-FFA
Cytotoxic T cells (CD 8) in the tumor microenvironment of mice after administration by flow cytometry + T cells), helper T cells (CD 4 + T cells), mature DC cells (CD 80 + CD86 + Cells) are detected; the levels of proinflammatory cytokines (IFN-. Gamma., IL-6, TNF-. Alpha.) in the serum of mice were detected by ELISA technique.
The specific embodiment is as follows:
(1) After C57BL/6J mice in example 9 were sacrificed, tumors and lymph nodes were collected to prepare single cell suspensions. Tumors were minced and digested with enzyme mixtures (collagenase type I, hyaluronidase, dnase I, sigma) for 1h at 37 ℃. The mixture was gently ground and filtered through a 200 mesh screen after digestion was completed by mixing once every 15min to obtain a single cell suspension.
(2) Lymph nodes were placed in 1 XPBS, gently crushed with a syringe plunger, and filtered through a 200 mesh screen to obtain a single cell suspension. The red blood cells in the single cell suspension were removed with red blood cell lysate (sigma) and washed twice with 1 x PBS.
(3) The collected cells were blocked with anti-mouse CD16/32 (BioLegend Co., U.S.A.), and lymph node cells were stained (incubation stained) with PE/Cyanine7 anti-mouse CD11c (CD 11c-PE/Cyanine 7), FITC anti-mouse I-A/I-E6 (I-A/I-E6-FITC), PE anti-mouse CD80 (CD 80-PE), APC anti-mouse CD86 (CD 86-APC), and tumor cells were incubated with APC anti-mouse CD3 (CD 3-APC), PE anti-mouse CD8a (CD 8 a-PE) and FITC anti-mouse CD4 (CD 4-FITC) according to the protocol of the supplier (BioLegend Co., U.S.A.). Finally, stained cells were analyzed by flow cytometry. The results are shown in FIGS. 12A-12C.
(4) The mice were subjected to eyeball removal and blood collection, the obtained blood was left at room temperature for 1h, and after the blood was coagulated and layered, the blood was centrifuged at 3500rmp for 15min, the supernatant serum was carefully collected and the levels of cytokines IL-6, IFN- γ and TNF- α in the serum were detected by ELISA kit (Shenzhen Biotechnology Co., ltd.), and the results were shown in FIG. 12D.
The results in FIG. 12 show that at the end of treatment, helper T cells (CD 4 + T cells, fig. 12A), cytotoxic T cells (CD 8 + T cells, fig. 12B), mature DC cells (CD 80 + CD86 + Cells, fig. 12C), all had significant increases, demonstrating that Pt-FFA promoted maturation of dendritic cells, activated the killing ability of T cells to tumor cells, further confirming the activity of Pt-FFA to induce tumor immunogenic death. The significant increase in pro-inflammatory cytokines (IFN-gamma, IL-6, TNF-alpha, FIG. 12D) in serum samples further demonstrated that Pt-FFA can activate a powerful immune response.
In summary, as shown in FIG. 13, pt-FFA has multiple antitumor activities. Pt-FFA can inhibit aldehyde-ketone reductase and further improve the drug resistance of a tumor platinum drug, induce apoptosis to inhibit the tumor, and also can induce immunogenic death of the tumor, so that the Pt-FFA has the anti-tumor property of chemotherapy-immunotherapy synergy. Therefore, the tetravalent platinum immunogenic death inducer provided by the invention can be used for preparing anti-tumor drugs, and has a large application prospect in overcoming the drug resistance of tumor platinum drugs and developing chemotherapy immune anti-cancer drugs.
As described above, although the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limiting the invention itself. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. A tetravalent platinum class immunogenic death inducer having the general formula I:
wherein ,
the structure of (2) is any one of the following: />
2. The tetravalent platinum type immunogenic death inducer according to claim 1, characterized by being selected from the group consisting of:
3. a method of preparing a tetravalent platinum type immunogenic death inducer according to any one of claims 1 to 2, comprising the steps of:
(1) Bivalent platinum complex and hydrogen peroxide H 2 O 2 The tetravalent platinum complex is prepared by reaction;
wherein the divalent platinum complex is selected from any one of the following compounds:
(2) FFA of flufenamic acidActivated with N-hydroxysuccinimide NHS and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI to give the activated ester of flufenamic acid (FFA-NHS)
(3) Adding the tetravalent platinum complex prepared in the step (1) and the flufenamic acid activated ester prepared in the step (2) into a DMF solution, heating and stirring until the reaction is finished;
(4) Centrifuging, concentrating the supernatant, adding methanol and concentrated solution, mixing, adding into diethyl ether to precipitate solid, centrifuging, removing supernatant, retaining precipitate, repeatedly washing precipitate with methanol and diethyl ether, and drying to obtain target product.
4. A pharmaceutical composition comprising a tetravalent platinum type immunogenic death inducer according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier or adjuvant.
5. Use of the tetravalent platinum class immunogenic death inducer according to any one of claims 1 to 2 in the preparation of an antitumor drug.
6. The use according to claim 5, wherein the tumor is lung cancer, liver cancer, stomach cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, prostate cancer, head and neck cancer or glioblastoma.
7. A chemical immune anticancer agent is characterized in that the effective component is Pt-FFA.
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