CN115364111A - Application of glycerophospholipid compound in treating tumor - Google Patents

Abstract

The invention relates to an application of glycerophospholipid compounds in treating tumors. Specifically, the invention relates to application of a glycerophospholipid compound containing a structure shown in a formula (I) in preparing a medicament for treating tumors, particularly malignant tumors. The compound can induce tumor cells to die, so that the compound has an excellent anti-tumor effect.

Description

Application of glycerophospholipids compound in treating tumor

Technical Field

The invention relates to the field of tumor treatment, in particular to application of a glycerophospholipid compound in preparing a medicament for treating tumors, particularly malignant tumors.

Background

Malignant tumors are one of the major diseases that threaten human health and affect life span. The top ten cancers with global incidence are in turn: breast cancer, lung cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer and bladder cancer. The first ten deaths had cancers that were in order: lung cancer, colorectal cancer, liver cancer, gastric cancer, breast cancer, esophageal cancer, pancreatic cancer, prostatic cancer, cervical cancer and leukemia. Therefore, the research and development of more and better anti-tumor drugs have great market value and important social significance.

At present, the malignant tumor still lacks an effective treatment means, and the 5-year survival rate of the whole tumor in China is only 40 percent ^[3] . Many treatments have been aimed at killing malignant tumor cells, but the effect is not ideal. The traditional radiotherapy and chemotherapy have poor specificity, and can kill malignant tumor cells and seriously damage other tissue cells such as immune cells, and weaken the anti-tumor immunity of the cells. Apoptosis is a specific mode of cell death discovered earlier, and in general, apoptosis is a mechanism by which the body removes infected or damaged cells and maintains the body's normal function, but malignant cells have a specific mechanism by which apoptosis can be evaded ^[4] . In addition, malignant tumors also have multiple immune escape capabilities for immune cell attack ^[5] . Therefore, the lack of specific and effective methods for killing malignant tumor cells has been one of the important reasons for hindering the therapeutic effect of malignant tumors.

In recent years, it has been found that cells have a variety of death modes, and iron death (ferroptosis) has attracted much attention. Iron death is iron ion dependent and cell death is caused by membrane lipid peroxidation ^[6] . More and more researches find that the iron death can be used as a new target for inhibiting tumors, and a plurality of genes related to the iron death mechanism are found ^[7] Targeting these genes to induce tumor cell death is a new strategy for developing therapeutic drugs for malignant tumors. Most of the existing iron death activators such as Erastin, RSL3 and the like are small molecular compounds ^[8] The function of inhibiting the cell antioxidant mechanism is suitable for some specific types of tumor cells, and the application range, side effects and the like of the tumor cells need to be further researched and evaluated. Therefore, there is a need to develop more various iron-like death activators with different action targets and different application ranges.

In recent years, the role of lipid metabolism disorder in the development of malignant tumor has been increasingly emphasized. Mammalian cells have a wide variety of lipids and multiple functions, and glycerophospholipids are the most abundant lipids, the main components of biological membranes, and important signal pathway molecules. Abnormal metabolism of glycerophospholipids associated with various tumors ^[9] . The research of the inventor finds that a class of glycerophospholipids can induce malignant tumor cells to generate iron death, and has no effect on various normal cells. The mechanism of inducing iron death of malignant tumor cells by glycerophospholipids is different from that of common iron death activators for inhibiting cell anti-oxidation mechanisms, and the glycerophospholipids are novel iron death activators, so that the development of novel medicaments for treating malignant tumors is expected on the basis.

Disclosure of Invention

The invention aims to provide an application of a glycerophospholipid compound in preparing a medicament for treating tumors, particularly malignant tumors.

Therefore, the invention provides the application of the compound containing the structure shown in the formula (I) or the medicinal salt thereof or the medicinal composition containing the compound in preparing the medicament for treating the tumor,

in a specific embodiment, the use according to the invention, wherein the compound comprising a structure according to formula (I) is according to formula (II) below:

wherein:

-OR is selected from-OH, choline, L-serine;

M ⁺ selected from Na ⁺ 、K ⁺ ；

Wherein when-OR is choline group, M ⁺ Is absent.

In another specific embodiment, the use according to the invention, wherein the compound comprising a structure according to formula (I) is selected from the following compounds:

18:2PC(DLPC)

18:2PA

18:2PS

in another specific embodiment, the use according to the present invention, wherein said tumor is a malignant solid tumor, preferably osteosarcoma, colon cancer, rectal cancer, melanoma, prostate cancer, cervical cancer.

In another specific embodiment, the use according to the present invention, wherein said tumor is a hematological malignancy, preferably a leukemia.

In another particular embodiment, the use according to the invention, characterized in that said compound treats said tumor by inducing the death of tumor cells, preferably by inducing the death of tumor cells by iron.

In another particular embodiment, the use according to the invention is characterized in that said compound treats said solid malignant tumors by alleviating pathological symptoms and signs, preferably by slowing the growth rate of the tumor, and/or by reducing the volume of the tumor, and/or by enhancing the efficacy of other treatments, and/or by reducing the rate of tumor recurrence after other treatments, and/or by prolonging the time of tumor recurrence after other treatments.

In another particular embodiment, the use according to the invention is characterized in that said compound treats said hematological malignancy by alleviating pathological symptoms and signs, preferably by reducing the number of tumor cells, and/or enhancing the efficacy of other treatments, and/or reducing the proportion of tumor recurrence after other treatments, and/or prolonging the time for tumor recurrence after other treatments.

In another specific embodiment, the use according to the present invention, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound comprising a structure represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier or excipient.

In another specific embodiment, the use according to the present invention, wherein the compound comprising the structure represented by formula (I) is used in combination with another therapeutic method or agents, preferably radiotherapy, chemotherapy, immunotherapy, targeted therapy, preferably another agent for the treatment of tumors.

The compound containing the structure shown in the formula (I) is characterized in that: the fatty acyl side chain in the phospholipid is a double-chain octadecadienoic acid, and the phosphate group of the fatty acyl side chain binding part can be linked to different polar heads to form different glycerophospholipids, preferably 1, 2-dilinoleoyl-sn-glycerol-3-phosphatidylcholine (1, 2-dilinoleoyl-sn-glycero-3-phosphatidylcholine) (18.

The choline group of the invention is

A group; the L-serine is

A group.

The "pharmaceutically acceptable salts" of the present invention refer to pharmaceutically non-toxic acid addition salts and base addition salts. The acid addition salt is formed by the compound and suitable inorganic acid or organic acid, and comprises hydrochloride, phosphate, hydrogen phosphate, sulfate, hydrogen sulfate, sulfite, acetate, oxalate, and the like,Malonate, valerate, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, methanesulfonate, malate, tartrate, benzoate, pamoate, salicylate, vanillic acid, mandelate, succinate, gluconate, lactobionate, laurylsulfonate, and the like. The base addition salt is formed by the compound and a suitable inorganic base or organic base, and comprises salts formed by alkali metal, amine or quaternary ammonium compounds, such as sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt, amine salt, tetramethyl quaternary ammonium salt, tetraethyl quaternary ammonium salt, choline salt, especially sodium salt and choline salt; amine salts, including with ammonia (NH) ₃ ) Salts with primary, secondary or tertiary amines, such as methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, ethanolamine, serine, lysine and arginine salts, in particular serine salts.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of: sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a pleasant to the eye and palatable pharmaceutical preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be inert excipients, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as microcrystalline cellulose, croscarmellose sodium, corn starch or alginic acid; binding agents, for example starch, gelatin, polyvinylpyrrolidone or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or they may be coated by known techniques which mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, water soluble taste masking substances such as hydroxypropylmethyl cellulose or hydroxypropyl cellulose, or time extending substances such as ethyl cellulose, cellulose acetate butyrate may be used.

Oral formulations may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with a water soluble carrier, for example polyethylene glycol, or an oil vehicle, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone and acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain fatty alcohols, for example heptadecaethyleneoxycetanol (heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyethylene oxide sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene oxide sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl paraben, one or more colouring agents, one or more flavouring agents and one or more sweetening agents, for example sucrose, saccharin or aspartame.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable preparation. These compositions can be preserved by the addition of antioxidants such as butylated hydroxyanisole or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent or one or more preservatives. Suitable dispersing or wetting agents and suspending agents are described above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifiers may be naturally occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyethylene oxide sorbitol monooleate. The emulsions may also contain sweetening agents, flavouring agents, preservatives and antioxidants. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a colorant and an antioxidant.

The pharmaceutical compositions of the present invention may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated to form a microemulsion by adding to a mixture of water and glycerol. The injection solution or microemulsion may be injected into the bloodstream of a patient by local bulk injection. Alternatively, it may be desirable to administer the solutions and microemulsions in a manner that maintains a constant circulating concentration of the compounds of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any blend of fixed oils including synthetic mono-or diglycerides can be used for this purpose. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

It is well known to those skilled in the art that the dosage of a drug administered depends on a variety of factors, including, but not limited to: the activity of the particular compound employed, the age of the patient, the weight of the patient, the health of the patient, the patient's integument, the patient's diet, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like. In addition, the optimal treatment regimen, such as mode of treatment, daily amount of the compound of formula (la) or type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

The compound or the pharmaceutically acceptable salt thereof containing the structure shown in the formula (I) can be used as an active ingredient, mixed with a pharmaceutically acceptable carrier or excipient to prepare a composition, and prepared into a clinically acceptable dosage form.

The compounds of the present invention may be used in combination with other active ingredients as long as they do not produce other adverse effects, such as allergic reactions and the like. The compounds of the present invention may be used as the sole active ingredient or in combination with other drugs. Combination therapy is achieved by administering the individual therapeutic components simultaneously, separately or sequentially.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be used alone or in combination with another treatment or therapeutic agents conventionally used clinically, such as radiation therapy, chemotherapy, immunotherapy, targeted therapy, and the like, including but not limited to the following antineoplastic agents and treatments:

1) Alkylating agents such as cisplatin, oxaliplatin, chlorambucil, cyclophosphamide, mechlorethamine, melphalan, temozolomide, busulfan, nitrosoureas;

2) Antineoplastic antibiotics such as doxorubicin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin C, actinomycin, mithramycin; antimitotic drugs such as vincristine, vinblastine, vindesine, vinorelbine, paclitaxel, taxotere, polo kinase inhibitors;

3) Antimetabolic and antifolic agents such as fluoropyrimidine, methotrexate, cytarabine, azacitidine, decitabine, tikitasamide, hydroxyurea, IDH1/IDH2 mutant inhibitors;

4) Topoisomerase inhibitors such as epipodophyllotoxin, camptothecin, irinotecan;

5) Cytostatic agents such as antiestrogens/antiandrogens, e.g., tamoxifen, fulvestrant, toremifene, reynolds xifen, dronoxifene, idoxifene, bicalutamide, flutamide, nilutamide, cyproterone acetate;

LHRH antagonists or LHRH agonists such as goserelin, leuprorelin, and buserelin, progestogens such as megestrol acetate;

aromatase inhibitors such as anastrozole, letrozole, vorozole, exemestane, 5 a-reductase inhibitors such as finasteride;

6) Antibodies against invasive agents such as inhibitors of the c-Src kinase family, metalloproteinase inhibitors, inhibitors of urokinase plasminogen activator receptor function, or heparanase;

7) Inhibitors of cell growth function include tyrosine kinase inhibitors and inhibitors of serine/threonine kinases such as Ras/Raf signaling inhibitors, cell signaling inhibitors of MEK and/or AKT kinases, c-Kit inhibitors, c-Met inhibitors, PDGFR inhibitors, ABL kinase inhibitors, PI3 kinase inhibitors, CSF-1R kinase inhibitors, EGFR family kinase inhibitors, FGFR family kinase inhibitors, IGF receptor kinase inhibitors, aurora kinase inhibitors, cyclin-dependent kinase inhibitors such as CDK2 and/or CDK4, CDK6 inhibitors, nuclear transport protein CRM1 inhibitors, wnt/beta-catenin inhibitors;

8) Inhibitors of anti-apoptotic proteins such as BCL2 inhibitors (venetocalax) and MCL1 inhibitors;

9) PARP inhibitors such as Olaparib and Rucaparib, etc.;

10 Anti-angiogenesis inhibitors such as VEGFR inhibitors;

11 Epigenetic inhibitors such as Histone Deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors;

12 Tumor immunotherapy includes any in vitro or in vivo method of increasing the immunogenicity of patient tumor cells, such as transfection with the cytokines IL-2, IL-4, or GM-CSF; methods of reducing the anergic effects of T cells such as anti-PD-1/PD-L monoclonal antibody; methods using transfected immune cells such as cytokine-transfected dendritic cells; methods of using cytokine-transfected tumor cell lines; a functional method of reducing immunosuppressive cells such as regulatory T cells, myeloid-derived suppressor cells, or dendritic cells expressing indoleamine 2, 3-deoxyenzyme; and tumor-associated antigenic proteins or peptides;

13 Chimeric antigen receptor T cell immunotherapy (CAR T);

14 Tumor gene therapy such as CRISPR-Cas 9, rnai and gene transduction.

The term "alleviating pathological symptoms and signs" as used herein refers to the reduction of tumor mass or growth rate, pain, ulcer area, bleeding, anemia, obstruction, tumor infiltration and metastasis.

The term "slowing the growth rate of a tumor" or "reducing the volume of a tumor" as used herein refers to primarily slowing the growth rate of a solid tumor, which usually has a fast growth rate and a significant increase in volume in the short term, or reducing the volume of a tumor. Or a reduction in the number of abnormal cells in a hematologic tumor.

The invention relates to a method for enhancing the curative effect of other treatments, which mainly refers to enhancing the curative effect of other methods for treating tumors, such as surgical operation treatment, chemotherapy, radiotherapy, immunotherapy and the like.

The invention relates to a method for reducing the recurrence rate of tumor and/or prolonging the recurrence time of tumor after other treatments, which mainly means that after the tumor is reduced or disappeared by adopting the treatment means such as surgical operation treatment, chemotherapy, radiotherapy, immunotherapy and the like, and after a period of time, the number ratio of the patients with tumor and corresponding symptom sign reappearance (tumor recurrence) is reduced; or the time interval from the reduction or disappearance of the tumor to the reappearance of the tumor and the corresponding symptom signs (tumor recurrence) is prolonged after the tumor is reduced or disappeared by the treatment procedures of surgical operation treatment, chemotherapy, radiotherapy, immunotherapy and the like.

The invention proves that the compound containing the glycerophospholipid structure can effectively induce the death of tumor cell iron so as to kill tumor cells through specific experiments.

Drawings

FIG. 1 is a graph of the number of cell clones formed 6 days after the action of 0.1mM 18 PC (DLPC) and solvent BSA Negative Control (NC) on mouse colon cancer MC38 cell line; where a is a photograph of clones from solvent BSA treated MC38 cells, B is a photograph of clones from 0.1mm 18 pc (DLPC) treated MC38 cells, C is a histogram of the number of clones from 0.1mm 18 pc (DLPC) and solvent BSA Negative Control (NC), and the values of the two sets of histograms were subjected to a statistical t-test, where x indicates a p value <0.001, indicating that there was a very significant statistical difference between the two sets of data.

Figure 2 is a graph of the rate of cell survival after 24 hours of exposure of 18.

FIG. 3 is a photograph showing the cell morphology observed under an optical microscope after a reaction of 18.

FIG. 4 is a graph of staining of cellular PI 24 hours after exposure of 18.

Figure 5 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 6 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 7 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 8 is a graph of the rate of cell survival after 24 hours of exposure of 18 pc (DLPC) and solvent BSA Negative Control (NC) to the human colon cancer SW480 cell line, and the values of several groups of histograms were subjected to a statistical t-test, where a p value <0.05 indicates a statistical difference in the group compared to the NC group, a p value <0.001 indicates a very significant statistical difference in the group compared to the NC group.

Figure 9 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 10 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 11 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 12 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 13 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 14 is a graph of the rate of cell survival after 24 hours of exposure of 18.

Figure 15 is a graph of the rate of cell survival after 24 hours of exposure of 18 ps and solvent BSA Negative Control (NC) to mouse colon cancer MC38 cell line, and statistical t-tests were performed on the values of several groups of histograms, where a value for p represents a significant statistical difference from the group of data vs. the NC group of data, and a value for p represents a value <0.001, representing a very significant statistical difference from the group of data vs.

FIG. 16 is a graph showing the cell survival rate of 18.

Figure 17 solvent BSA Negative Controls (NC), 18 pc (DLPC), 18.

Figure 18 is a graph of the cell survival rate after 24 hours of solvent BSA Negative Control (NC), 18.

FIG. 19 is a flow cytometric image of BODIPY 581/591C11 staining after 4 hours of solvent BSA Negative Control (NC), 18.

FIG. 20 is an SEM image of the cell morphology of 18.

Detailed Description

The invention is further described below by way of specific examples, but it should be understood that these examples are only for illustrating the invention and do not limit the scope of the invention in any way.

Experimental Material

The structures of 18:

18:2PC(DLPC)

18:2PA

18:2PS

18.

Cells used for the experiments:

example 1:18

(1) Cell clone formation assay

The experimental procedure was as follows:

appropriate 18. MC38 cells in the logarithmic growth phase were taken, digested with Trypsin 0.25% (purchased from GE Healthcare) for 5 to 15 minutes, centrifuged at 1000rpm for 5 minutes to collect the cells, resuspended in a single cell suspension with the medium, and a 1/1000 volume of 0.1m 18 pc (DLPC) stock solution of the single cell suspension was added to make the final concentration of 18. 2000 cells were seeded into one well of a 6-well plate, 2ml of medium per well. 37 ℃ and 5% CO ₂ Culturing in a cell culture box, growing the cells for 6 days, and replacing a fresh culture medium during the culture period to ensure that the cells obtain sufficient nutrition. After 6 days, the medium was decanted and 1ml of 3.7% formaldehyde was added to fix the cells. After the formaldehyde was decanted, the column was washed twice with 1 XPBS for 5 minutes each. Pour out 1 XPBS, stain with 1ml of crystal violet (ex Seville) for 7 minutes, blot out crystal violet and wash twice with 1 XPBS. After taking a picture with a general camera, the number of clones was counted with Image J software.

As a result:

FIG. 1 is a graph of the number of cell clones formed 6 days after 0.1mM 18 PC (DLPC) and solvent 0.5% BSA Negative Control (NC) were allowed to act on the mouse colon cancer MC38 cell line. Cell clone formation experiments examined the viability of individual cells, and it can be seen from a, B and C of fig. 1 that the number of 18.

(2) CCK-8 experiment

The experimental procedure was as follows:

taking MC38 cells in logarithmic phase, digesting the adherent growing cells with trypsinase 0.25% (purchased from GE Healthcare company) for 5-15 minutes, centrifuging at 1000rpm for 5 minutes to collect the cells, centrifuging the suspended growing cells at 1000rpm for 5 minutes to collect the cells, resuspending the cells with a cell culture medium, and adjusting the cell density to be proper. The cell suspension was seeded into 96-well plates at 2000 cells/well with 100uL of medium per well. When the cells had grown to a density of 60% to 70%, the medium was carefully discarded, the cells were washed once with 1 XPBS, and fresh medium containing 0.1mM, 0.2mM or 0.5mM 18 ₂ After 24 hours of incubation in the Cell culture chamber, the medium was carefully discarded, the cells were washed once with 1 XPBS, fresh medium without the acting agent was added at 100 uL/well, 10uL of CCK8 solution of Cell Counting Kit-8 (available from Dojindo Co.) was added to each well, 37 ℃ C., 5% CO ₂ The cells were incubated in an incubator for 45 minutes, and absorbance at 450nm was measured using a multifunctional microplate reader FlexStation3 from Molecular Devices.

As a result:

FIG. 2 is a graph of cell survival rates of 18. The CCK-8 experiment examined the surviving cell ratio, and from this figure, the 18.

(3) Observation of cell morphology

The experimental procedure was as follows:

18. Fresh medium containing 0.1mM, 0.2mM or 0.5mM 18 PC (DLPC) or 0.5% BSA, 100 uL/well, 37 ℃,5% ₂ After 24 hours incubation in the cell culture chamber, the cell morphology was observed under an OLYMPUS light microscope (model TH 4-200) and photographed.

As a result:

FIG. 3 is a photograph of the morphology of cells observed under an optical microscope after 18. From this figure, it is clear that the NC group cells, the 18.

(4) PI staining

The experimental procedure was as follows:

18. Culturing the cells on a 96-well cell culture dish, and when the density of the cells reaches 60% to 70%, treating the cells instead with fresh medium containing 0.5% BSA or 0.1mM, 0.2mM or 0.5mM 18 2PC (DLPC). After 24 hours, the medium was discarded and the cells were washed once with 1 × PBS. 100uL of fresh medium was added. PI dye (purchased from Biolegend) at a concentration of 0.5mg/mL was added to the medium at a dilution of 1 ₂ The cells were incubated in a cell culture chamber for 15 to 30 minutes in the absence of light and observed under a green excitation light by an OLYMPUS optical microscope (model TH 4-200).

As a result:

FIG. 4 is a graph of staining of cellular PI 24 hours after exposure of 18. PI staining detects the cell rate of cell membrane damage, and positive PI staining indicates that the cell membrane is damaged and is characterized by dead cells. As can be seen from fig. 4, the number of PI-staining positive cells in the NC group was small, the ratio of PI-staining positive cells in the 18.

Further, by the same experimental method as described above, except that 18.

The survival cell rate was determined by the CCK-8 assay as described above, and the results were as follows:

figure 5 is a graph of the cell survival rate of 18. As can be seen from the graph, the 18.

FIG. 6 is a graph of the cell survival rate of 18. As can be seen from the figure, the 18.

FIG. 7 is a graph of cell survival rates of 18. From this figure, the 0.5mm 18 pc (DLPC) group exhibited a significantly lower percentage of viable cells than the NC group.

FIG. 8 is a graph of cell survival rates of 18. As can be seen from the graph, the 18.

Fig. 9 is a graph of the cell survival ratio of 18. As can be seen from the graph, the 18.

FIG. 10 is a graph showing the cell survival rate of 18. As can be seen from the graph, the 18.

Figure 11 is a graph of the cell survival rate of 18. As can be seen from the graph, the 18.

Fig. 12 is a graph showing the cell survival rate of 18. As can be seen from the graph, the 18.

Fig. 13 is a graph showing the cell survival rate of 18. As can be seen from the graph, the 18.

Figure 14 is a graph of cell survival rates 24 hours after exposure of 18. As can be seen from the figure, the 18.

FIG. 15 is a graph of cell survival rates of 18. As can be seen from the figure, the 18.

Example 2: survival effect of 18

The experimental procedure was as follows:

the surgical instruments to be used are autoclaved in advance, and the aseptic operation of the whole process of material drawing is noticed. C57BL/6 mice (purchased from Jackson lab) were sacrificed by decapitation and disinfected with alcohol, the abdominal cavity of the mice was cut with surgical scissors, the spleen was separated, washed 2-3 times with 1 XPBS, and other tissues around the spleen were removed. Grinding on 200 mesh metal screen with inner core of syringeThe mouse spleen was ground, and after the grinding was completed, the cell suspension was collected by washing the grinder with RPMI-1640 medium (purchased from Hyclone) and centrifuged at 800rpm for 3 minutes to collect cells. To the collected cells, 3 volumes of erythrocyte lysate (from Solarbio) was added. The mixture was placed on ice for 15 minutes, during which time the red blood cells were lysed by vortexing the mixture gently twice. The leukocytes were pelleted by centrifugation at 450 Xg for 10 minutes, the supernatant was carefully aspirated, and the cells were washed 3 times with RPMI-1640 medium and harvested by centrifugation at 800rpm for 3 minutes each. Adding 10% fetal calf serum-containing RPMI-1640 culture medium into the cell precipitate, and resuspending the cells to obtain a cell density of 1 × 10 ⁷ Individual cells/mL, at 37 ℃,5% ₂ Cultured in a cell culture box.

The procedure for treating cells with 18.

As a result:

fig. 16 is a graph of cell survival ratios after exposure of 18. From this figure, the 18. It was shown that 18.

Example 3:18

(1) The iron death inhibitor blocks tumor cell death caused by 18

There are various types of cell death, for example: iron death, scorching, apoptosis, and the like. Fer-1 (Ferrostatin-1) and Lip-1 (Liproxstatin-1) are specific inhibitors of iron death, and Fer-1 can inhibit oxidative, iron-dependent cell death by blocking cystine transport and glutathione production. Lip-1 blocks lipid peroxidation. The results of the previous experiments show that 18.

The experimental procedure was as follows:

18 PC (DLPC) and 0.5% BSA, as compared to the above-described examplesThe same as described in example 1. MC38 cells in logarithmic growth phase are taken, digested for 5-15 minutes by trypsinase 0.25% (purchased from GE Healthcare), centrifuged for 5 minutes at 1000rpm to collect the cells, the cells are resuspended in cell culture medium, and the cell density is adjusted to be proper. The cell suspension was seeded into 96-well plates at 2000 cells/well with 100uL of medium per well. When the cells were grown to a density of 60% to 70%, the medium was carefully discarded, the cells were washed once with 1 × PBS, and a medium containing 0.5mM 18 PC (DLPC) or 0.5mM 18 PC +10uM Fer-1 or 0.5mM 18 ₂ After 24 hours incubation in the Cell incubator, the medium was carefully discarded, the cells were washed once with 1 XPBS, fresh medium without the acting agent was added at 100 uL/well, 10uL of CCK8 solution of Cell Counting Kit-8 (available from Dojindo) was added to each well, 37 ℃ C., 5% CO ₂ The cells were incubated in a cell incubator for 45 minutes, and the absorbance at 450nm was measured using a multifunctional microplate reader FlexStation3 from Molecular Devices.

As a result:

FIG. 17 is a graph showing the cell survival rate of solvent BSA Negative Controls (NC), 18. As can be seen from this figure, the 18. This result shows that the iron death inhibitors Fer-1 and Lip-1 can prevent 18.

(2) Iron chelators reduce tumor cell death by 18

There are various types of cell death, among which iron death is a cell necrosis mediated by iron-catalyzed hyperoxidation of polyunsaturated fatty acids. The iron chelator, deferoxamine mesylate (DFO), can form a complex with iron ions by means of chemical bonding, reduce the content of iron ions in cells, thereby preventing or alleviating the onset of iron death in cells. When cells were treated with 18.

The experimental procedure was as follows:

18 PC (DLPC) and 0.5% BSA were prepared in the same manner as described in example 1 above. MC38 cells in logarithmic growth phase are taken, digested for 5-15 minutes by trypsinase 0.25% (purchased from GE Healthcare), centrifuged for 5 minutes at 1000rpm to collect the cells, the cells are resuspended in cell culture medium, and the cell density is adjusted to be proper. The cell suspension was seeded into 96-well plates at 2000 cells/well with 100uL of medium per well. When the cells had grown to a density of 60% to 70%, the medium was carefully discarded, the cells were washed once with 1 XPBS, and culture medium containing 0.2mM or 0.5mM 18 PC (DLPC) or 0.2mM/0.5mM 18 ₂ After 24 hours incubation in the Cell incubator, the medium was carefully discarded, the cells were washed once with 1 XPBS, fresh medium without the acting agent was added at 100 uL/well, 10uL of CCK8 solution of Cell Counting Kit-8 (available from Dojindo) was added to each well, 37 ℃ C., 5% CO ₂ The cells were incubated in an incubator for 45 minutes, and absorbance at 450nm was measured using a multifunctional microplate reader FlexStation3 from Molecular Devices.

As a result:

fig. 18 is a graph showing the cell survival rate of mouse colon cancer MC38 cell lines after 24 hours of the solvent BSA Negative Controls (NC), 18. As can be seen from the figure, the 18. This result shows that the iron chelator DFO completely prevented 0.2mm 18 pc (DLPC) from causing tumor cell death, and partially prevented 0.5mm 18.

(3) 18

One of the features of cellular iron death is the occurrence of lipid peroxidation. BODIPY 581/591C11 is a lipid peroxidation fluorescent probe, and can be used for detecting Reactive Oxygen Species (ROS) in cells and cell membranes. The polyunsaturated butadiene-m-diene oxidation of the dye can cause the fluorescence emission peak to shift from 590nm to 510nm, and the visible fluorescence peak shifts to the right when the dye is detected by a flow cytometer. After treatment of cells with 18.

The experimental procedure was as follows:

18 PC (DLPC) and 0.5% BSA were prepared in the same manner as described in example 1 above. MC38 cells in logarithmic growth phase are taken, digested for 5-15 minutes by trypsinase 0.25% (purchased from GE Healthcare), centrifuged for 5 minutes at 1000rpm to collect the cells, the cells are resuspended in cell culture medium, and the cell density is adjusted to be proper. The cell suspension was seeded into 96-well plates at 2000 cells/well with 100uL of medium per well. When the cells were grown to a density of 60% to 70%, the medium was carefully discarded, the cells were washed once with 1 XPBS, and medium containing 0.5mM 18 ₂ After 4 hours incubation in the Cell incubator, the medium was carefully discarded, the cells were washed once with 1 XPBS, fresh medium without working reagent was added at 100 uL/well, 10uL of CCK8 solution of Cell Counting Kit-8 (available from Dojindo) was added to each well, 37 ℃ C., 5% CO ₂ The cells were incubated in a cell incubator for 45 minutes, and the absorbance at 450nm was measured using a multifunctional microplate reader FlexStation3 from Molecular Devices. After staining with BODIPY 581/591C11 (from Invitrogen) for 30 minutes, flow cytometric detection was performed with an Accuri C6 flow sorter (from BD).

As a result:

FIG. 19 is a flow cytometric assay of lipid peroxidation 4 hours after the action of solvent BSA Negative Controls (NC), 18. From the figure, the fluorescence peak of the 18. It is demonstrated that 18.

(4) 18

Another common feature of iron death in cells is that the intracellular mitochondrial membrane is damaged, the mitochondria in cells that undergo iron death under electron microscopy become smaller, the mitochondrial cristae is blurred or missing, and the mitochondria become darker in color. The cells treated with 18.

The experimental procedure was as follows:

culturing mouse colon cancer MC38 cells in a 6-well plate at a cell density of 60-70%, replacing with fresh medium containing 0.5% BSA or 0.5mM 18 ₂ After incubation in the cell incubator for 12 hours, the original medium was discarded, and a mixed solution of a fixative (2.5% glutaraldehyde (purchased from Sigma) and a cell culture medium (1: mixed)) preheated at 37 ℃ was added and fixed at room temperature for 10 minutes. The mixed solution of the fixing solution and the culture medium was discarded. The fixative was added and fixed at room temperature for 1 hour. After staining with uranyl acetate for 25 minutes and then with lead nitrate for 5 minutes, photographs were observed with an electron transmission microscope (purchased from Hitachi, ltd.) of type H7650B.

As a result:

FIG. 20 is an electron micrograph of the cell morphology of 18. As can be seen from the figure, in the 18. These morphological changes in mitochondria followed the mitochondrial morphological changes common to iron-dead cells, suggesting that the type of tumor cell death caused by 18.

Reference documents

[1]World Health Statistics 2020.World Health Organization.

[2]World Cancer Report 2020.International Agency for Research on Cancer,World Health Organization.

[3] The 2020 work report national cancer center.

[4] MicroRNA-519a-3p media applications resistance in breaker cells and the same expression from recognition by natural killer cells Christian Breunig, jens Pahl, moritz Kublbeck, et al Cell Death Dis.2017;8 (8): e2973.

[5] Activation of the VEGFC/VEGFR3 Pathway indexes Tumor Immune Escape in colorimetric Cancer. Carlotta TACCONI, federica Ungaro, carmen Correale, et al Cancer Res.2019;79 (16):4196-4210.

[6] Ferroptosis, a regulated cell death nexus linking metabolism, redox biology, and disease, brent R.Stockwell, jose Pehydro Friedmann Angeli, hulya Bair, et al cell 2017;171 (2):273-285.

[7] MDM2 and MDMX promoter by PPARalpha-modified. Venkatesh D, O' Brien NA, zandkarimi F, et al Genes Dev. 2020;34 (7-8):526-543.

[8]Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer.Ghoochani A,Hsu EC,Aslan M,Rice MA,Nguyen HM,Brooks JD, Corey E,Paulmurugan R,Stoyanova T.Cancer Res.2021 Mar 15；81(6):1583-1594.

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Claims (11)

1. The application of the compound containing the structure shown in the formula (I) or the medicinal salt thereof or the medicinal composition containing the compound in preparing the medicament for treating the tumor,

2. the use according to claim 1, wherein the compound having the structure of formula (I) is represented by the following formula (II):

wherein:

-OR is selected from-OH, choline, L-serine group;

M ⁺ selected from Na ⁺ 、K ⁺ ；

Wherein when-OR is a choline group, M ⁺ Is absent.

3. Use according to claim 1 or 2, wherein the compound comprising the structure of formula (I) is selected from the following compounds:

4. the use according to any one of claims 1 to 3, wherein the tumor is a solid tumor or a tumor of the hematological system, such as melanoma, skin cancer, liver cancer, kidney cancer, lung cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, gallbladder cancer, bile duct cancer, chorioepithelial cancer, pancreatic cancer, polycythemia vera, pediatric tumor, cervical cancer, ovarian cancer, breast cancer, bladder cancer, urothelial cancer, ureteral tumor, prostate cancer, seminoma, testicular tumor, leukemia, head and neck tumor, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, sarcoma, osteoma, osteosarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma.

5. Use according to any one of claims 1 to 4, wherein the tumour is a malignant solid tumour, preferably osteosarcoma, colon carcinoma, rectal carcinoma, melanoma, prostate carcinoma, cervical carcinoma.

6. Use according to any one of claims 1 to 4, wherein the tumour is a haematological malignancy, preferably a leukaemia.

7. Use according to any one of claims 1 to 6, characterized in that said compound treats said tumor by inducing tumor cell death, preferably by inducing tumor cell iron death.

8. Use according to claim 5, characterized in that said compound treats said solid malignant tumor by alleviating pathological symptoms and signs, preferably by slowing the growth rate of the tumor, and/or reducing the volume of the tumor, and/or enhancing the efficacy of other treatments, and/or reducing the proportion of tumor recurrence after other treatments, and/or prolonging the time of tumor recurrence after other treatments.

9. Use according to claim 6, characterized in that said compound treats said hematological malignancy by alleviating pathological symptoms and signs, preferably by reducing the number of tumor cells, and/or enhancing the efficacy of other treatments, and/or reducing the proportion of tumor recurrence after other treatments, and/or prolonging the time of tumor recurrence after other treatments.

10. The use according to any one of claims 1 to 9, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound comprising the structure of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier or excipient.

11. Use according to any one of claims 1 to 10, wherein the compound comprising the structure of formula (I) or a pharmaceutically acceptable salt thereof is used in combination with another therapeutic method or agents, preferably radiotherapy, chemotherapy, immunotherapy, targeted therapy, preferably another agent for the treatment of tumors.

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