Application of lurasidone in targeting male tumor

Abstract

The invention relates to the field of targeted therapy and tumor medicine, in particular to application of lurasidone in targeting male tumors. Specifically, the invention discloses application of lurasidone shown in a formula I or pharmaceutically acceptable salt thereof, and the lurasidone or pharmaceutically acceptable salt thereof can promote senescence neutrophil ferroptosis and is used for preparing a medicine or a medicine composition for treating male tumors. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of targeted therapy and tumor medicine, and in particular to a therapeutic strategy and application of lurasidone targeting male tumors. Background Art

[0002] Tumors are neoplasms formed by the proliferation of local tissue cells under the action of various tumorigenic factors. Tumors are divided into benign tumors and malignant tumors. The former are not harmful to human health, while the latter will produce toxic substances, damage normal organs, and threaten human health. Affected by factors such as the aging of the population, the incidence and mortality of cancer have continued to rise in recent years. According to a 2019 World Health Organization report, cancer has become the top two causes of death for people under 70 years old in most countries, seriously hindering the extension of human life.

[0003] According to clinical data, the occurrence and development of most tumors are related to gender. Among them, the incidence and mortality of most tumors in male patients are higher than those in female patients. Therefore, gender-differentiated treatment plans for tumors may be able to provide precise treatment for male and female tumor patients.

[0004] Tumor immunotherapy has shown good therapeutic effects on a variety of solid tumors. However, in actual treatment, the treatment effect on males is not as good as that on females.

[0005] Therefore, there is a need to develop effective therapeutic drugs and approaches for male tumor patients. Summary of the invention

[0006] The purpose of the present invention is to provide an active ingredient for treating male tumors and a new use thereof in inhibiting male tumors.

[0007] Specifically, the present invention provides the use of lurasidone represented by formula I and a composition thereof in inhibiting male tumors.

[0008] In the first aspect of the present invention, there is provided a use (or pharmaceutical use) of lurasidone or a pharmaceutically acceptable salt thereof as shown in Formula I,

[0009]

[0010] Furthermore, lurasidone represented by formula I or a pharmaceutically acceptable salt thereof is used to prepare (a) an agent for promoting ferroptosis of senescent neutrophils; and / or (b) a drug or a pharmaceutical composition for treating male tumors.

[0011] In another preferred embodiment, the male tumor includes but is not limited to bladder cancer, melanoma, or a combination thereof.

[0012] In another preferred embodiment, the male tumor includes a tumor that is insensitive to neutrophil ferroptosis.

[0013] In another preferred embodiment, the lurasidone or a pharmaceutically acceptable salt thereof targets Lcn2 to promote ferroptosis of aging neutrophils.

[0014] In another preferred embodiment, the lurasidone or a pharmaceutically acceptable salt thereof activates T cells in the tumor microenvironment.

[0015] In another preferred embodiment, the pharmaceutical composition further comprises: a PD-1 inhibitor.

[0016] In another preferred embodiment, the medicine includes: oral preparations and parenteral preparations.

[0017] In another preferred embodiment, the preparation includes: powder, granules, capsules, injections, tinctures, oral liquids, tablets, lozenges, or pills.

[0018] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0019] (a) Lurasidone or a pharmaceutically acceptable salt thereof represented by formula I:

[0020]

[0021] (b) PD-1 inhibitors

[0022] and (c) a pharmaceutically acceptable carrier.

[0023] In another preferred embodiment, the PD-1 inhibitor includes: PD-1 antibody and PD-L1 antibody.

[0024] In the third aspect of the present invention, the use of the pharmaceutical composition according to the second aspect of the present invention is provided, which is used to prepare (a) promoting ferroptosis of senescent neutrophils by targeting Lcn2; and / or (b) activating CD8 in the tumor microenvironment + T cells; and / or (c) inhibiting the proliferation of male tumor cells; and / or (d) drugs for treating male tumors.

[0025] In a fourth aspect of the present invention, there is provided an in vitro non-therapeutic and non-diagnostic method for inhibiting the proliferation of male tumor cells, comprising the steps of:

[0026] The lurasidone represented by formula I or its pharmaceutically acceptable salt is contacted with male tumor cells to inhibit the proliferation of male tumor cells.

[0027] In another preferred embodiment, lurasidone or a pharmaceutically acceptable salt thereof,

[0028] Targeting Lcn2 promotes ferroptosis of senescent neutrophils and activates CD8 in the tumor microenvironment + T cells, thereby inhibiting the proliferation of male tumor cells.

[0029]

[0030] In another preferred embodiment, the subject to be treated is a male tumor patient.

[0031] In another preferred embodiment, the concentration of Formula I or its pharmaceutically acceptable salt in the method is 0.5-20 μM, preferably 1-10 μM.

[0032] In another preferred embodiment, the method is an in vitro method.

[0033] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0034] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 showed that differences in neutrophil infiltration affected the growth differences between male and female tumors.

[0036] Figure 2 It was shown that tumor tissues of male individuals were infiltrated with more senescent neutrophils.

[0037] Figure 3 It shows that senescent neutrophils have a stronger immunosuppressive effect.

[0038] Figure 4 It was shown that removal of senescent neutrophils could significantly inhibit tumor growth in male mice.

[0039] Figure 5 We show that low-level ferroptosis leads to the accumulation of neutrophils in male senescent cells.

[0040] Figure 6 Lurasidone was shown to specifically induce ferroptosis in senescent neutrophils.

[0041] Figure 7 Lurasidone was shown to promote neutrophil ferroptosis by targeting Lcn2.

[0042] Figure 8 Lurasidone was shown to significantly slow the growth of MB49 tumors in male mice.

[0043] Fig. 9The combination of lurasidone and PD-1 antibody was shown to further inhibit tumor growth.

[0044] Fig.10 LCN2 is shown + Senescent neutrophils can serve as a prognostic marker in male cancer patients. DETAILED DESCRIPTION

[0045] After extensive and in-depth research, the inventors unexpectedly discovered for the first time a new strategy for targeted inhibition of male tumor growth. The present invention found that lurasidone can effectively inhibit the growth of malignant tumors, especially inhibit the growth of male malignant tumors. The PD-1 antibody and lurasidone combination therapy of the present invention can produce a synergistic effect and have an excellent inhibitory effect on male tumors. For single PD-1 antibody or lurasidone therapy, the tumor is at the same growth level. After the PD-1 antibody and lurasidone are used in combination, tumor growth is significantly inhibited. The present invention was completed on this basis.

[0046] Specifically, animal experiments have shown that in the MB49 mouse model, low levels of ferroptosis lead to the accumulation of male senescent neutrophils, affecting tumor growth. After administering lurasidone, ferroptosis of senescent neutrophils is induced by targeting Lcn2, thereby clearing senescent neutrophils. Removal of senescent neutrophils can significantly inhibit tumor growth in male mice. Lurasidone has the effect of alleviating tumor growth. In addition, the combination of lurasidone and PD-1 antibodies can further inhibit tumor growth and has a synergistic effect.

[0047] the term

[0048] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0049] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., is understood to include the described elements or components but does not exclude other elements or components.

[0050] As used herein, the term "male tumor" refers to male tumor-related diseases including but not limited to bladder cancer, leukemia, lymphoma, gastric cancer, esophageal cancer, Wilms' tumor, liver cancer, prostate cancer, and rectal cancer.

[0051] Iron Death

[0052] Ferroptosis is a new type of iron-dependent programmed cell death. Its main mechanism is that under the action of divalent iron or esteroxygenase, it catalyzes unsaturated fatty acids highly expressed on the cell membrane, causing lipid peroxidation, thereby inducing cell death. In previous studies, ferroptosis has been associated with tumor suppression. Studies have shown that tumor cell proliferation can be promoted by inhibiting ferroptosis. Conversely, ferroptosis inducers are effective in inhibiting cancer, including but not limited to triple-negative breast cancer, diffuse large B-cell lymphoma, and renal and ovarian clear cell carcinoma.

[0053] Neutrophils

[0054] Neutrophils are the first line of defense for the host against invading pathogens. They have inherent phagocytic ability and can absorb nanoparticles and phagocytize dead red blood cells. In previous studies, neutrophils are important immune cells in the tumor microenvironment. They have strong heterogeneity and have an important impact on the growth and progression of solid tumors. They have a dual role in promoting and inhibiting tumors in tumor diseases. This shows that neutrophils have become a potential target for tumor treatment.

[0055] In the study of this application, animal experiments have demonstrated that accumulated senescent neutrophils in male mice can significantly promote tumor growth, and that removal of senescent neutrophils can significantly slow down tumor growth in male mice.

[0056] Active compounds and active ingredients of the invention

[0057] In the present invention, an active ingredient that can effectively inhibit male tumors is provided, which is selected from the following group: lurasidone, or a pharmaceutically acceptable salt thereof, or a combination thereof, or a crystal thereof, or a solvate thereof.

[0058] The structural formula of lurasidone is as follows:

[0059]

[0060] The experiment showed that the active ingredient of the present invention promoted ferroptosis of senescent neutrophils by targeting Lcn2 and activated CD8 + T cells, thereby inhibiting the proliferation of male tumors, and then treating and / or alleviating male tumor-related diseases.

[0061] As used herein, "active compound of the present invention" and "active compound for inhibiting male tumors of the present invention" are used interchangeably and refer to compounds having the ability to inhibit male tumors, including lurasidone, or a pharmaceutically acceptable salt thereof, or a combination thereof, or a crystal thereof, or a solvate thereof.

[0062] It should be understood that the active ingredients of the present invention include the active compounds of the present invention that target Lcn2 to promote ferroptosis of senescent neutrophils, or pharmaceutically acceptable salts, enantiomers, diastereomers or racemates thereof, or prodrugs thereof. It should be understood that the active ingredients of the present invention also include crystalline forms, amorphous compounds, and deuterated compounds of the active compounds of the present invention.

[0063] The "pharmaceutically acceptable salt" is a conventional non-toxic salt formed by the reaction of the active compound of the present invention with an inorganic acid or an organic acid. For example, conventional non-toxic salts can be prepared by the reaction of the active compound of the present invention with an inorganic acid or an organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid, etc.; or the active compound of the present invention forms an ester with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid, and then forms a sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt with an inorganic base; or the active compound of the present invention forms an ester with lysine, arginine, or ornithine, and then forms a corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or a corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

[0064] In addition, the active ingredients of the present invention are particularly suitable for use in combination with other antiviral drugs. Representative other antiviral drugs include (but are not limited to): reverse transcriptase inhibitors, protease inhibitors, coreceptor antagonists, retroviral integrase inhibitors, viral adsorption inhibitors, specific viral transcription inhibitors, antibodies, or combinations thereof.

[0065] Pharmaceutical compositions and applications

[0066] The present invention also provides the use of the active ingredient of the present invention, or its or its pharmaceutically acceptable salt, or its prodrug, or its extract, or a mixture of one or more of its medicinal materials as an effective ingredient in the preparation of a drug for treating male tumor-related diseases.

[0067] The pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99wt%, and the preferred ratio is that the active compound of the present invention as the active ingredient accounts for 0.1wt% to 90wt% or 1wt% to 50wt% of the total weight, and the rest is a pharmaceutically acceptable carrier, diluent or solution or saline solution.

[0068] When necessary, the drug of the present invention may also include a pharmaceutically acceptable carrier, which includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0069] The compounds and pharmaceutical compositions provided by the present invention may be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and may be present in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or instillation.

[0070] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the preparation formula generally contains 0.05-400 mg of the active compound of the present invention, preferably, the unit dosage of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.

[0071] The compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, and can be administered by oral, nasal, skin, lung or gastrointestinal tract routes. Injection is the most preferred. The most preferred daily dose is 0.01-1 mg / kg body weight, taken at one time, or 0.01-0.5 mg / kg body weight taken in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Usually, a small dose is started and the dose is gradually increased until the most suitable dose is found.

[0072] The drugs or inhibitors of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or wrapped with other substances to introduce into the body. From the standpoint of easy administration, the preferred pharmaceutical composition is a liquid composition, especially an injection.

[0073] The main advantages of the present invention include:

[0074] 1) The experiments of the present invention show that lurasidone can induce ferroptosis of senescent neutrophils by targeting Lcn2, thereby inhibiting the proliferation of tumor cells and further inhibiting the development of cancer.

[0075] 2) The present invention provides a new therapy for treating tumors by combining lurasidone with PD-1 antibody therapy, which has a significant synergistic effect.

[0076] 3) The present invention provides a new approach for the treatment of tumors.

[0077] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0078] Example 1: Differences in neutrophil infiltration affect differences in tumor growth between males and females

[0079] 1.1 Methods

[0080] The bladder cancer cell line MB49 in the logarithmic growth phase was taken at 1×10 6 The subcutaneous transplant tumor model was established by inoculating male and female C57 background mice with 100 cells. The growth of the transplanted tumor was observed and recorded every 2 days, and the death time of the mice was also observed (if the volume of the transplanted tumor in the mouse was greater than 2000mm 3 The patients were also considered dead and were killed according to regulations. At 14 days after subcutaneous inoculation, the tumor tissue was digested into a single cell suspension with type IV collagenase. The tumor tissues of male and female bladder cancer patients were collected and digested into a single cell suspension, and then incubated with the corresponding antibodies. Flow cytometry was used to detect neutrophils and activated CD8 + The proportion of T cells. Male and female C57 mice were injected with 100ug anti-Ly6G neutralizing antibody (Bioxcell, Cat#BP0075) through the tail vein to remove neutrophils. The injection was performed every three days, and the bladder cancer cell line MB49 in the logarithmic growth phase was subcutaneously inoculated. The growth of the transplanted tumor was observed and recorded every 2 days, and the activated CD8 + The proportion of T cells.

[0081] 1.2 Results

[0082] MB49 tumors inoculated subcutaneously in male mice grew faster than in female mice, and male mice had a higher mortality rate after MB49 tumor inoculation ( Figure 1 (middle AB).

[0083] Flow cytometry results showed that more neutrophils were infiltrated in MB49 tumors of male mice, and more neutrophils were infiltrated in tumor tissues of male bladder cancer patients than in female bladder cancer patients. + T cell activation is weaker, and tumor-infiltrating CD8 + T cells are less activated ( Figure 1 Medium CD).

[0084] In addition, neutrophil depletion significantly reduced MB49 tumor growth in male mice and enhanced tumor-infiltrating CD8 +T cell activation, MB49 tumor growth and CD8 + T cell activation was barely affected ( Figure 1 Medium EF).

[0085] The above results indicate that the difference in tumor-infiltrating neutrophils between male and female mice affects the difference in tumor growth.

[0086] Example 2 Male tumor tissues are infiltrated with more senescent neutrophils

[0087] 2.1 Methods

[0088] Flow cytometry analysis of CD45 infiltrating cells in male and female MB49 tumor tissues + Single-cell sequencing analysis was performed on immune cells, and the cells were grouped using the T-SNE dimensionality reduction algorithm. The neutrophil subpopulation cells were further grouped using the T-SNE dimensionality reduction algorithm, and the differences between male and female neutrophil subpopulations were statistically analyzed. The neutrophils were then pseudo-time-series analyzed using the Monocle algorithm. The expression of Cxcr4 and CD62L surface proteins infiltrating neutrophils in male and female MB49 tumors was detected by flow cytometry to reflect the aging state of neutrophils. The β-galactosidase activity of infiltrating neutrophils in MB49 tumor tissues of male and female tumor mice and tumor tissues of male and female bladder cancer patients was detected by FDG flow cytometry to judge the degree of neutrophil aging.

[0089] 2.2 Results

[0090] Neutrophils infiltrating MB49 tumors can be divided into four subgroups. Statistical analysis found that male MB49 tumors were infiltrated with more N3 neutrophil subgroups than female MB49 tumors. Pseudo-sequential analysis showed that N3 neutrophils were at the end of differentiation, so N3 may be a group of senescent neutrophils ( Figure 2 (in AC).

[0091] Flow cytometry results showed that more senescent neutrophils (CD62L low CXCR4 high ), and the FDG test results showed that the β-galactosidase activity of neutrophils infiltrating MB49 tumors in male mice was stronger, indicating that the neutrophils were more aged ( Figure 2 (in DF).

[0092] The above results indicate that more senescent neutrophils are infiltrated in the tumor tissues of male individuals.

[0093] Example 3: Aged neutrophils have stronger immunosuppressive effects

[0094] 3.1 Methods

[0095] The algorithm analyzes the score values ​​of the immunosuppressive ability of four different neutrophil subsets infiltrating MB49 tumor tissues in mice, and then selects genes with high expression in N3 to form a signature gene set. The TCGA database is used to analyze the correlation between the signature gene set with high expression in the N3 subset and patient survival, and the training set data is verified using the validation database. cre iDTR mice (used to remove the original neutrophils in the recipient mice) were subcutaneously inoculated with bladder cancer cell line MB49 in the logarithmic growth phase. The growth of transplanted tumors was observed and recorded every 2 days, and the activated CD8 + T cells and CD4 + The ratio of T cells is used to study the effects of senescent neutrophils versus young neutrophils on tumor growth and T cell activation.

[0096] 3.2 Results

[0097] The analysis found that N3 subpopulation neutrophils have the strongest immunosuppressive ability and are significantly negatively correlated with the survival of bladder cancer patients ( Figure 3 (middle AB).

[0098] The results of adoptive transfer experiments showed that aged neutrophils have a stronger ability to inhibit T cell activation than young neutrophils, and have a stronger effect on promoting the growth of MB49 subcutaneous tumors ( Figure 3 Medium CD).

[0099] The above results demonstrate that aged neutrophils have stronger immunosuppressive effects than young neutrophils.

[0100] Example 4 Removal of senescent neutrophils can significantly inhibit tumor growth in male mice

[0101] 4.1 Methods

[0102] By analyzing single-cell data in the tumor microenvironment, tumor cells and various immune cells (including CD4 + T cells, CD8 +The expression of Csf3r, S100a9, Retnlg and Lcn2 genes in T cells, dendritic cells, macrophages and N0, N1, N2, N3 neutrophil subsets. Retnlg-DTR-GFP transgenic mice were customized by Saiye Biotechnology Co., Ltd. (the exogenous fragment "P2A-DTR-GFP" was inserted after the last exon of the Retnlg gene through CRISPR Cas9 technology) for MB49 subcutaneous tumor experiments. Flow cytometry sorting of GFP + and GFP - Tumor-infiltrating neutrophils were detected, and the expression of Retnlg was detected to verify whether the mice were successfully constructed. Diphtheria toxin was injected intraperitoneally into male and female Retnlg-DTR-GFP mice and littermate control mice, and then bladder cancer cell line MB49 in the logarithmic growth phase was subcutaneously inoculated. The growth of transplanted tumors was observed and recorded every 2 days; the concentration of Lcn2 protein in serum was detected by ELISA; FDG + and FDG - neutrophils were detected to detect the expression of Lcn2; flow cytometry was used to detect the expression of Lcn2 protein in male and female tumor-infiltrating neutrophils, and ELISA was used to detect the concentration of Lcn2 protein in the serum of male and female tumor-bearing mice, as well as the concentration of LCN2 in the serum of male and female bladder cancer patients.

[0103] 4.2 Results

[0104] Single-cell data analysis results showed that Retnlg and Lcn2 genes were specifically highly expressed in the N3 neutrophil subset ( Figure 4 (A).

[0105] QPCR detection of Retnlg-DTR-GFP transgenic mice revealed that GFP + Compared with GFP - The expression of Retnlg in neutrophils of Retnlg-DTR-GFP mice was much higher. And after intraperitoneal injection of diphtheria toxin to remove senescent neutrophils in Retnlg-DTR-GFP mice, it was found that the MB49 tumors in male mice were significantly reduced ( Figure 4 BC), indicating that the removal of senescent neutrophils can specifically inhibit the growth of MB49 tumors in male mice.

[0106] In addition, QPCR analysis revealed that FDG + The expression of Lcn2 is stronger in aged neutrophils ( Figure 4 (middle D).

[0107] Flow cytometry analysis showed that Lcn2 expression was higher in tumor-infiltrating neutrophils of male mice ( Figure 4 EF), and ELISA results also showed that the serum of male tumor-bearing mice contained higher levels of Lcn2 protein ( Figure 4 In G), male bladder cancer patients also had more LCN2 protein in their serum than female bladder cancer patients ( Figure 4 Middle H).

[0108] More interestingly, Lcn2 protein was significantly decreased in the serum of male Retnlg-DTR-GFP mice treated with DT ( Figure 4 Middle I).

[0109] The above results suggest that Lcn2 and Retnlg are both markers of senescent neutrophils.

[0110] Example 5 Low-level ferroptosis leads to accumulation of male senescent neutrophils

[0111] 5.1 Methods

[0112] Subcutaneous tumors of male and female tumor-bearing mice were removed, embedded in OTC and made into frozen sections. Immunofluorescence staining was used to detect the level of neutrophils in male and female MB49 tumor tissues. TUNEL staining was used to detect dead cells. + The number of dead neutrophils was statistically analyzed, and the expression of ferroptosis-related genes in the infiltrating neutrophils in male and female MB49 tumors in the single cell data was analyzed. The C11-BODIPY probe (Thermo Fisher Scientific, Cat#D3861) was used to detect the level of lipid reactive oxygen species in the infiltrating neutrophils in the male and female MB49 tumors and the tumor tissues of male and female bladder cancer patients.

[0113] 5.2 Results

[0114] Immunofluorescence experiments showed that there were fewer dead neutrophils in the tumor tissues of male MB49 than in female MB49 ( Figure 5 (middle AB).

[0115] Single-cell data analysis revealed that the expression of ferroptosis-related genes in infiltrating neutrophils in male mouse MB49 tumors was lower ( Figure 5 Middle C).

[0116] The results of C11-BODIPY probe detection showed that the lipid reactive oxygen levels of neutrophils infiltrating in the MB49 tumors of male mice and the tumor tissues of male bladder cancer patients were lower, indicating that ferroptosis was weaker ( Figure 5 middle DE).

[0117] Example 6 Lurasidone specifically induces ferroptosis in senescent neutrophils

[0118] 6.1 Methods

[0119] Neutrophils from the bone marrow of normal mice were plated in 24-well plates and treated with 5uM lurasidone and control reagent DMSO for 24 hours. The survival of neutrophils was detected by flow cytometry and the activity of β-galactosidase was detected by FDG. - and FDG + Neutrophils were treated with 5uM lurasidone and control reagent DMSO for 24h, and the lipid reactive oxygen levels of neutrophils were detected by C11-BODIPY probe. MB49 tumor cell line was cultured in 24-well plates and treated with 5uM lurasidone and control reagent DMSO for 24h. The survival of MB49 cells and the level of lipid reactive oxygen were detected by flow cytometry. CD4 + T cells and CD8 + T cells were plated in a U-shaped 96-well plate pre-coated with CD3 / CD28 antibodies and treated with 5uM lurasidone and control reagent DMSO for 24h. CD4 + T cells and CD8 + Lipid reactive oxygen species levels in T cells.

[0120] 6.2 Results

[0121] Lurasidone treatment significantly promoted neutrophil cell death and reduced neutrophil senescence ( Figure 6 Further experiments found that lurasidone specifically promoted FDG + Ferroptosis of senescent neutrophils ( Figure 6 E), and had no effect on MB49 tumor cell lines and CD4 + T cells and CD8 + T cell ferroptosis Figure 6 (in FH).

[0122] The above results indicate that lurasidone can specifically induce ferroptosis of senescent neutrophils, thereby slowing down the senescent state of neutrophils.

[0123] Example 7 Lurasidone promotes neutrophil ferroptosis by targeting Lcn2

[0124] 7.1 Methods

[0125] The binding of lurasidone and LCN2 protein was detected by microthermophoresis (MST). Neutrophils from the bone marrow of wild-type and Lcn2-KO mice (obtained from the Army Medical University, in Lcn2-KO, the Lcn2 gene is homozygous inactivated) were plated in 24-well plates and cultured. 5uM lurasidone and control reagent DMSO were given for 24h, and the Fe in neutrophils was detected by FerroOrange reagent (DOJINDO, Cat#F374). 2+ The content of Lcn2-KO was detected by C11-BODIPY probe, and the level of lipid reactive oxygen species in neutrophils was detected. The bone marrow of wild-type male mice was intravenously injected into male mice irradiated with a lethal dose, and the bone marrow of wild-type male mice, Lcn2-KO male mice and wild-type female mice was intravenously injected into female mice irradiated with a lethal dose. After 6-8 weeks, the bladder cancer cell line MB49 in the logarithmic growth phase was subcutaneously inoculated, and the growth of the transplanted tumor was observed and recorded every 2 days.

[0126] 7.2 Results

[0127] Since Lcn2 is a Fe 2+ Lcn2 is a chelating protein that is involved in ferroptosis. Lcn2 is a protein specifically expressed by aging neutrophils in the tumor microenvironment. The inventors speculate that lurasidone is likely to promote neutrophil ferroptosis by acting on Lcn2.

[0128] The results of MST experiments showed that there was a strong binding interaction between lurasidone and LCN2 protein (K d =0.37±0.08mM)( Figure 7 (A).

[0129] Flow cytometry analysis revealed that Fe in neutrophils after Lcn2 knockout 2+ The content of ferroptosis increased, and lurasidone was no longer able to promote ferroptosis of neutrophils ( Figure 7 Chinese BC).

[0130] The results of bone marrow chimera experiments showed that Lcn2 can indeed promote tumor growth ( Figure 7 (middle D).

[0131] The above results indicate that lurasidone acts on Lcn2 to promote neutrophil ferroptosis.

[0132] Example 8 Lurasidone significantly slows down the growth of MB49 tumors in male mice

[0133] 8.1 Methods

[0134] Male and female C57 mice were intraperitoneally injected with 1 mg / kg of lurasidone, followed by subcutaneous inoculation of bladder cancer cell line MB49 in the logarithmic growth phase. The growth of transplanted tumors was observed and recorded every 2 days. Subcutaneous tumor tissues of mice were digested into single cell suspensions, and the proportion of neutrophils infiltrating tumor tissues, neutrophil senescence status, and CD8 + T cell activation level.

[0135] 8.2 Results

[0136] Lurasidone significantly reduced the growth of MB49 tumors in male mice, but had little effect on the growth of MB49 tumors in female mice ( Figure 8 (A).

[0137] At the same time, flow cytometry analysis showed that lurasidone could significantly reduce the proportion of infiltrating neutrophils in MB49 tumors in male mice and reduce their senescence state, while enhancing CD8 + Expression of IFNγ in T cells ( Figure 8 (in BD).

[0138] The above results indicate that lurasidone can significantly slow down the growth of MB49 tumors in male mice.

[0139] Example 9 Combination of lurasidone and PD-1 antibody to further inhibit tumor growth

[0140] 9.1 Methods

[0141] Male C57 mice were injected intraperitoneally with 1 mg / kg of lurasidone, intravenously with 100 mg of PD-1 monoclonal antibody (Bioxcell, clone number: J43, Cat#BP0033), or injected with lurasidone and PD-1 monoclonal antibody at the same time, and then subcutaneously inoculated with bladder cancer cell line MB49 in the logarithmic growth phase. The growth of transplanted tumors was observed and recorded every 2 days, and the tumor growth of female mice was used as a control.

[0142] 9.2 Results

[0143] Both lurasidone and PD-1 monoclonal antibody can effectively inhibit tumor growth in male mice, and the combination of the two can further slow down tumor growth in male mice, even slower than that in female mice ( Fig. 9 ). Therefore, the combination of lurasidone and PD-1 has a very significant effect on inhibiting the growth of male tumors.

[0144] Example 10 LCN2 + Senescent neutrophils may serve as a prognostic marker for male cancer patients

[0145] 10.1 Methods

[0146] Paraffin sections of male and female bladder cancer patients with follow-up information were collected, and the expression of MPO (a marker of human neutrophils) and LCN2 protein was detected by immunofluorescence staining after antigen retrieval. + The proportion of neutrophils and LCN2 + The high and low levels of neutrophils divided male and female bladder cancer patients into two groups, and the survival status of the two groups was statistically analyzed.

[0147] 10.2 Results

[0148] Immunofluorescence experiments showed that male bladder cancer patients had more LCN2 infiltrates in their tumors + Neutrophils ( Fig.10 (A).

[0149] LCN2 in male patients + The higher the proportion of neutrophils, the worse the prognosis of patients, and LCN2 in female patients + There was little correlation between neutrophils and survival ( Fig.10 (in BE).

[0150] Therefore, LCN2 + Senescent neutrophils can serve as a prognostic marker in male cancer patients.

[0151] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of lurasidone or a pharmaceutically acceptable salt thereof represented by formula I, It is characterized in that For preparation: (a) an agent that promotes ferroptosis of senescent neutrophils; and / or (b) A drug or a pharmaceutical composition for treating male tumors.

2. The use according to claim 1, characterized in that The male tumors include tumors that are insensitive to neutrophil ferroptosis.

3. The use according to claim 1, characterized in that The lurasidone or a pharmaceutically acceptable salt thereof targets Lcn2 to promote ferroptosis of aging neutrophils.

4. The use according to claim 1, characterized in that The lurasidone or a pharmaceutically acceptable salt thereof activates T cells in the tumor microenvironment.

5. The use according to claim 1, characterized in that The medicine includes oral preparations and parenteral preparations.

6. The use according to claim 1, characterized in that The pharmaceutical composition further comprises: a PD-1 inhibitor.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) Lurasidone or a pharmaceutically acceptable salt thereof represented by formula I: (ii) PD-1 inhibitors; and (iii) a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that The PD-1 inhibitors include: PD-1 antibodies and PD-L1 antibodies.

9. Use of the pharmaceutical composition according to claim 7, characterized in that: Used to prepare drugs for inhibiting the proliferation of male tumors.

10. An in vitro non-therapeutic and non-diagnostic method for inhibiting the proliferation of male tumor cells, characterized in that: Includes steps: Contacting lurasidone represented by formula I or a pharmaceutically acceptable salt thereof with male tumor cells, thereby inhibiting the proliferation of male tumor cells; In another preferred embodiment, lurasidone or a pharmaceutically acceptable salt thereof targets Lcn2 to promote ferroptosis of senescent neutrophils and activate CD8 + T cells, thereby inhibiting the proliferation of male tumor cells.

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