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CN115252603A - Application of indsulam in preparation of medicine for treating bladder cancer - Google Patents

Application of indsulam in preparation of medicine for treating bladder cancer Download PDF

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CN115252603A
CN115252603A CN202210805345.9A CN202210805345A CN115252603A CN 115252603 A CN115252603 A CN 115252603A CN 202210805345 A CN202210805345 A CN 202210805345A CN 115252603 A CN115252603 A CN 115252603A
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bladder cancer
cells
tumor
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indol
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黄卫人
宋斐
于沥博
吴果
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Shenzhen Second Peoples Hospital
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    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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Abstract

The invention fully proves that the indesulam can inhibit the cell proliferation of the bladder cancer from in vivo and in vitro levels, most importantly can cause the mass production of neoantigens of tumor cells, further activates anti-tumor immune response, finally shows ultrahigh bladder cancer clearing efficiency, and has unique advantages and good application prospects in the clinical treatment of the bladder cancer.

Description

Application of indsulam in preparation of medicine for treating bladder cancer
Technical Field
The invention relates to the field of medicine, in particular to application of indolium in preparation of a medicine for treating bladder cancer.
Background
Bladder cancer is one of the most common urological malignancies, and is particularly high in the elderly (> 55 years) male population, with over 57 million diagnosed bladder cancers worldwide per year and about 21 million deaths. Bladder cancer can be classified into non-muscle invasive bladder cancer (non-muscle invasive binder cancer) and muscle invasive bladder cancer (muscle invasive binder cancer) according to the degree of tumor cell infiltration. First-line treatment of non-muscle invasive bladder cancer is usually achieved by transurethral cystectomy (TURBT), followed by the infusion of BCG (Bacillus Calmette-Guerin, BCG) or chemotherapy to prevent recurrence. Muscle invasive bladder cancer adopts partial cystectomy or radical cystectomy according to the tumor development degree; chemotherapy can be applied before cystectomy or chemotherapy, i.e. the tumor volume is reduced by a neoadjuvant chemotherapy (neoadjuvant chemotherapy) mode, so as to achieve better treatment effect; or used after surgery (adjuvant chemotherapy) to prevent tumor recurrence. For the case of metastatic bladder cancer, the metastasis is not suitable for excision, the clinical treatment mainly comprises chemotherapy and radiotherapy, the five-year survival rate of local metastatic bladder cancer is about 38 percent after treatment, and the five-year survival rate of systemic metastatic bladder cancer is only 6 percent. Therefore, chemotherapy is crucial to the treatment of both early and late stage bladder cancer, and the search for new highly effective chemotherapeutic regimens is imperative.
The chemotherapy drugs commonly used for clinical treatment of bladder cancer include cisplatin (cisclin), mitomycin C (mitomycin C), methotrexate (methotrexate), gemcitabine (gemcitabine), doxorubicin (doxorubicin), vinblastine (vinblastine), carboplatin (carboplatin), etc. The single chemotherapeutic medicament has poor effect and poor persistence in the clinical treatment of the bladder cancer, and is easy to generate medicament resistance. The combined chemotherapy schemes of MVAC (methotrexate + vinblastine + adriamycin + cisplatin), GC (gemcitabine + cisplatin), CMV (cisplatin + methotrexate + vinblastine), CISCA (cisplatin + carboplatin) and the like can reduce the recurrence rate of bladder cancer and prolong the life of a patient, but the single drug administration cannot achieve the treatment effect, the treatment period is long, and the problems of drug resistance and the like are also faced.
Compared with chemotherapy, immunotherapy has higher targeting property and weaker side effect, and is widely applied to clinical treatment of bladder cancer. In addition to traditional BCG perfusion, immune Checkpoint Inhibitors (ICIs) can enhance the tumor clearance of immune cells by blocking inhibitory signals between tumor cells and immune cells. PD-1 antibodies such as Nivolumab (Nivolumab) and pembrolizumab (pembrolizumab), CTLA-4 antibodies such as yipriumamab (Ipilimumab), and PD-L1 antibodies such as atelizumab (Atezolizumab) and Nivolumab (Nivolumab) have been approved for sale. The single ICI therapy has poor overall treatment effect, the population response rate in bladder cancer patients is only 20-30%, and the ICI combined chemotherapy can effectively prolong the survival time of the patients. Therefore, highly effective therapies that can simultaneously target tumor killing and immune system activation are the main research direction for bladder cancer treatment at present.
indisulam(C14H12ClN3O4S2Also known as E7070) is an arylsulfonamide drug which can inhibit the expression of CDK2 and Cyclin E, block the cell cycle to the G1 phase, and thus inhibit cell proliferation. At present, indisulam is applied to clinical tests of cancer species such as colorectal cancer, leukemia, pancreatic cancer, lung cancer, melanoma and the like, but is not reported in clinical treatment of bladder cancer.
Disclosure of Invention
According to a first aspect, there is provided the use of N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide, or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, for the manufacture of a medicament for the treatment of bladder cancer.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to inhibit bladder cancer cell proliferation.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to induce apoptosis in bladder cancer cells.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used for targeted degradation of RBM39 proteins.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to induce tumor neoantigens and thereby activate immune responses.
According to a second aspect, in one embodiment, there is provided a medicament for the treatment of bladder cancer, comprising N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, and a pharmaceutically acceptable carrier.
According to the application of the N- (3-chloro-1H-indol-7-yl) -1, 4-benzene disulfonamide in the preparation of the drugs for treating bladder cancer, the invention fully proves that the indolium can not only inhibit the cell proliferation of the bladder cancer, but also can cause a large amount of neoantigens of tumor cells to be generated most importantly, so as to activate the anti-tumor immune response, and finally shows the ultrahigh bladder cancer elimination efficiency, thereby having unique advantages and good application prospects in the clinical treatment of the bladder cancer.
Drawings
FIGS. 1A and 1B are graphs showing the results of measuring the proliferation level of MB49 under the treatment of gradient concentration indolium by flow cytometry in example 1;
FIG. 2 is a graph showing the results of measuring the apoptosis level of MB49 under the gradient concentration ndisulam treatment in example 1 by flow cytometry;
FIG. 3 is a WB assay result of targeted degradation of RBM39 protein by indigulam in example 1;
FIG. 4 is a graph showing the results of the indolium inhibition of tumor formation of MB49 in immunodeficient mice in example 1;
FIG. 5 is a graph of the results of the indolium significantly inhibiting the tumorigenesis of MB49 in fully immunized mice in example 1;
FIG. 6 is the IHC staining results and statistical analysis chart of the tumor tissues of the indisulam treated group and the control group in example 1;
FIG. 7 is a graph showing the results of ELIspot verification of the immuno-response of indesulam activation in example 1.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art would readily recognize that some of the features may be omitted in different instances or may be replaced by other materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the description of the methods may be transposed or transposed in order, as will be apparent to a person skilled in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.
The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning.
As used herein, "indisulam" refers to N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide, also known as E7070, CAS number 165668-41-7, molecular formula C14H12ClN3O4S2。
The term "salt" is to be understood as any form of the active compound used by the present invention, wherein said compound may be in ionic form or charged or coupled to a counter ion (cation or anion) or in solution. The definition may also include quaternary ammonium salts and complexes of the active molecule with other molecules and ions, particularly complexes through ionic interactions. This definition includes in particular physiologically acceptable salts, which term is to be understood as equivalent to "pharmacologically acceptable salts".
The term "pharmaceutically acceptable salt" generally refers to any salt (generally speaking) that is physiologically tolerable when used in an appropriate manner for treatment, particularly when applied or used in humans and/or mammalsMeaning that it is non-toxic, particularly as a result of the counter ion). These physiologically acceptable salts may be formed with cations or bases and in the context of the present invention, especially when administered in humans and/or mammals, they are to be understood as being salts formed by at least one compound provided according to the invention, usually an acid (deprotonated), such as an anion, and at least one physiologically tolerated cation, preferably an inorganic cation. In the context of the present invention, salts with alkali metals and alkaline earth metals, and ammonium cations (NH) may be included in particular4+) The salt to be formed may specifically include, but is not limited to, salts with (mono) or (di) sodium, (mono) or (di) potassium, magnesium or calcium. These physiologically acceptable salts may also be formed with anions or acids, and in the context of the present invention, in particular when administered in humans and/or mammals, they are to be understood as being salts formed from at least one compound provided according to the invention, usually protonated (e.g. on nitrogen), such as a cation and at least one physiologically tolerable anion. In the context of the present invention, salts formed with physiologically tolerable acids, i.e. salts of the particular active compounds with physiologically tolerable organic or inorganic acids, may in particular be included, but are not limited to, salts with hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid or citric acid.
The term "prodrug" is used in its broadest sense and includes those derivatives that can be converted in vivo to the compounds of the invention. Methods for preparing prodrugs of the named functional compounds will be known to those skilled in the art, and may be found, for example, in connection with the disclosure of Krogsgaard-Larsen et al, "Textbook of Drug design and Discovery" Taylor & Francis (2002, 4 months).
The term "solvate" refers generally to any form of substance obtained by non-covalent bonding of an active compound according to the invention to another molecule, usually a polar solvent, and may include in particular, but not limited to, hydrates and alcoholates, such as methanolate. In some embodiments, the solvent includes, but is not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and ethanolamine.
As used herein, "EdU" refers to 5-ethynyl-2 'deoxyuridine, the English name 5-ethenyl-2' -deoxyuridine.
According to a first aspect, there is provided the use of N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof for the manufacture of a medicament for the treatment of bladder cancer.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to inhibit bladder cancer cell proliferation.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to induce apoptosis in bladder cancer cells.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used for targeted degradation of RBM39 proteins.
In one embodiment, the N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to induce tumor neoantigens, thereby activating an immune response.
According to a second aspect, in one embodiment, there is provided a medicament for the treatment of bladder cancer, comprising N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, and a pharmaceutically acceptable carrier. In the present invention, the drug may include one or more pharmaceutically acceptable carriers, which generally refer to carriers for administration of the therapeutic agent, which do not themselves induce the production of antibodies harmful to the individual receiving the drug, and which are not unduly toxic upon administration. Such carriers are well known to those skilled in the art, and relevant information regarding pharmaceutically acceptable carriers is disclosed, for example, in Remington's Pharmaceutical Sciences (Mack pub. Co., n.j.1991). In particular, the carrier may be a combination including, but not limited to, one or more of saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and the like.
In the medicine provided by the invention, the compound or the pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof can be a single effective component, and can also be combined with other active components to form a combined preparation. Other active ingredients may be other various drugs that may be used to treat bladder cancer. The amount of active ingredient in the medicament is generally a safe and effective amount which should be adjusted by the person skilled in the art. For example, the administration amount of the above-mentioned compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, and the active ingredient of a medicament as an active ingredient generally depends on the body weight of the patient, the type of application, the condition and severity of the disease. For example, the above-mentioned compound or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof as an active ingredient may be administered in an amount of usually 0.1 to 1000mg/kg/day, 0.1 to 0.5mg/kg/day, 0.5 to 1mg/kg/day, 1 to 2mg/kg/day, 2 to 3mg/kg/day, 3 to 4mg/kg/day, 4 to 5mg/kg/day, 5 to 6mg/kg/day, 6 to 8mg/kg/day, 8 to 10mg/kg/day, 10 to 20mg/kg/day, 20 to 30mg/kg/day, 30 to 40mg/kg/day, 40 to 60mg/kg/day, 60 to 80mg/kg/day, 80 to 100mg/kg/day, 100 to 150mg/kg/day, 150 to 200mg/kg/day, 200 to 300mg/kg/day, or 400 to 300mg/kg/day, more preferably, 400 to 600mg/kg/day, or 800 mg/kg/day.
In some embodiments, the compounds provided herein may be adapted for any form of administration, either orally or parenterally, for example, by pulmonary, nasal, rectal and/or intravenous injection, and more particularly intradermal, subcutaneous, intramuscular, intraarticular, intraperitoneal, pulmonary, buccal, sublingual, nasal, transdermal, vaginal, oral or parenteral administration. One skilled in the art can select an appropriate formulation according to the mode of administration, for example, a formulation suitable for oral administration may include, but is not limited to, pills, tablets, chewables, capsules, granules, drops or syrups, and the like, and for example, a formulation suitable for parenteral administration may include, but is not limited to, solutions, suspensions, reconstitutable dry preparations or sprays, suppositories, and the like.
According to a third aspect, in an embodiment, there is provided a method of treatment comprising: administering to the subject a therapeutically effective amount of N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof.
In the present invention, a "subject" generally includes human, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., which would benefit from treatment with the formulation, kit or combined formulation.
In the present invention, a "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, is capable of achieving the effect of treating the diseases as listed above.
Example 1
Tumor killing in combination with immune system activation is currently the main direction of research in the treatment of bladder cancer. The indosulam is already used as an anti-tumor drug to be applied to clinical tests, shows better tumor killing capability, and can improve the tumor removal efficiency of an immune system by inducing a tumor neoantigen. At present, clinical trials of indisulam mainly focus on various tumors such as colorectal cancer, leukemia, pancreatic cancer, lung cancer, melanoma and the like, but the treatment effect in bladder cancer is not yet verified. In addition, the indisulam has a plurality of action targets, on one hand, indisulam can inhibit the proliferation of tumor cells by blocking the cell cycle; on the other hand, indisulam can promote tumor cells to generate new antigens and activate anti-tumor immune response by interfering the alternative splicing of pre-mRNA in the tumor cells. Therefore, the mechanism by which indosulam exerts an antitumor effect remains to be further confirmed. The embodiment mainly discloses the treatment effect and the anti-tumor mechanism of the Indianula applied to the bladder cancer, and aims to provide a new solution for the clinical treatment of the bladder cancer.
Based on the above purposes, this example first examined the proliferation and apoptosis levels of mouse bladder cancer cell line MB-49 after indisula in vitro treatment to verify its effect on the proliferation and apoptosis of bladder cancer cells. In addition, MB49 cell lysate after the indisula m in vitro treatment and mouse spleen cells are subjected to in vitro co-culture, and activation condition of immune cells is detected through ELIspot, so as to verify that indisula m promotes generation of tumor neoantigens; meanwhile, MB49 cells were inoculated subcutaneously in C57BL/6 mice and BALB/C Nude mice, respectively, to compare the tumorigenic volumes in fully immunized and immunodeficient mice, thereby further confirming at the in vivo level that indisulam is involved in immune activation.
The specific method of this embodiment is as follows:
MB49 cell proliferation assay
The compound indesulam used in this example was purchased from Selleck under the accession number S9742. The starting solvent for this compound was DMSO, diluted to the desired concentration with DMEM medium before use.
MB49 was treated in vitro at a gradient concentration (0,0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M) for 96h, and cells from different treatment groups were treated at 7.5X 105Inoculating the mixture into a 24-well plate, and culturing overnight in an incubator at 37 ℃; then, the medium was replaced with a medium containing EdU (the ratio of the volume of EdU to the volume of the medium before addition of EdU was 1: 2000), and the mixture was incubated at 37 ℃ for 1 hour in an incubator; then the medium was aspirated off, washed once with PBS, the cells were digested with pancreatin (without EDTA), centrifuged at 4 ℃ at 1500rpm for 5min and the supernatant discarded; fixing the cells with 4% paraformaldehyde at room temperature for 30min, and removing the fixing solution; adding 2mg/mL glycine, incubating at room temperature for 5min, and removing the glycine solution; after being washed by PBS, 1 XApollo staining reaction liquid is added into cells, the cells are incubated for 30min at room temperature in a dark place, and the staining reaction liquid is discarded; washing the cells with a permeabilizing agent (PBS containing 0.5% TritonX-100) for 2 times, and discarding the permeabilizing agent; finally, the proliferation level of MB49 under the treatment of gradient concentration indisula was detected by a flow cytometer. As shown in FIGS. 1A and 1B, the cell proliferation rate was decreased sequentially as the concentration of indsulam added to the medium was increased, demonstrating that indsulam can inhibit the proliferation of the bladder cancer cell line MB 49.
MB49 apoptosis assay
MB49 was treated in vitro with a gradient concentration (0,0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M) of indesulam, 96h later the cells were digested with pancreatin (without EDTA), centrifuged at 4 ℃ and 1500rpm for 5min and the supernatant discarded; washing cells with PBS pre-cooled at 4 deg.C, centrifuging at 1500rpm at 4 deg.C for 5min, and discarding PBS; resuspend cells in 100. Mu.L of precooled 1 × Annexin V Binding Buffer, then add 5. Mu.L FITC-Annexin V and 5. Mu.L PI and mix well, incubate for 15min at room temperature in the dark; the cells were then resuspended in 400. Mu.L of a pre-cooled 1 × Annexin V Binding Buffer and the level of apoptosis of MB49 was measured by flow cytometry under gradient concentration indesulam treatment. As shown in FIG. 2, the apoptosis ratio of the cells was increased in turn as the concentration of indosulam added to the medium was increased, demonstrating that indosulam can induce apoptosis of MB 49.
3. Indesulam drug effect persistence verification
Experiment A: MB49 is treated by gradient concentration (0,0.1 MuM, 1 MuM, 10 MuM, 100 MuM) of indulam, cells are collected after 96h, and the degradation level of RBM39 protein after treatment by different concentrations of indulam is detected by Western Blot (WB) after lysis by RIPA.
Experiment B: MB49 was treated with 1. Mu.M of indesulam, drugs were removed after 96 hours and sufficiently eluted with PBS, and then cells were collected on days 0, 1, 2, 3, 4, 5, and 6 after elution, respectively, and the level of lysis of RBM39 protein (RNA-binding motif 39) was measured by WB after lysis with RIPA to examine the persistence of indesulam-targeted degradation of RBM 39.
FIG. 3 is a WB detection result chart of indesulm targeted degradation of RBM39 protein, the left graph is a result chart of experiment A, and the right graph is a result chart of experiment B. From the left panel, it can be seen that the degradation of RBM39 protein is more pronounced with increasing indisulam concentration. As can be seen from the right panel, RBM39 protein degradation in bladder cancer cells was still effectively maintained for at least 6 days after indisulam was eluted.
The results in fig. 3 demonstrate that indisulam can also mediate ubiquitination degradation of the splicing factor RBM39 protein in bladder cancer cells, and participate in the regulation of alternative splicing of pre-mRNA. RNA splicing abnormality caused by indiscriminate can induce the generation of tumor neoantigens, and has potential activation effect on an immune system.
BALB/c Nude mice subcutaneous neoplasia
BALB/c Nude mice at 6-8 weeks were divided into two groups (treatment group)&Control group), 10 cells per group, and MB49 cells of drug control group (DMSO) or drug-treated group (1 μ M indesulam) were inoculated subcutaneously, two sites were inoculated per mouse, and 5 × 10 cells were inoculated per site5A cell; the length (L, mm) and width (W, mm) of the subcutaneous tumor were measured with a vernier caliper at 7, 10, 13, 16, 19 days after the inoculation, respectively, in accordance with 1/2 (L.times.W)2) Tumor volume was calculated.
Fig. 4 is a graph showing the results of inhibition of tumor formation of MB49 in immunodeficient mice by indesulam, and from the left, it can be seen that the subcutaneous tumor formation volume of MB49 in nude mice after indesulam treatment is smaller than that of the control group (DMSO) at different time points, i.e., indesulam can limit the in vivo tumor formation of MB49 by inhibiting cell proliferation. As can be seen from the right panel, the subcutaneous tumor volume was significantly less in the indisulam treated group than in the control group at day 19.
C57BL/6 mice subcutaneous neoplasia
The 6-8 week wild type C57BL/6 mice were divided into two groups (treatment group)&Control group), 10 cells per group, and two sites per mouse were inoculated with 5 × 10 cells per site, with control (DMSO) or treated (1 μ M insulam) MB49 cells subcutaneously5A cell; the length (L, mm) and width (W, mm) of the subcutaneous tumor were measured continuously with a vernier caliper at 1/2 (L.times.W) on days 7, 10, 13, 16, and 19 after the inoculation, respectively2) Tumor volume was calculated.
Fig. 5 is a graph of the results of the indesulam significantly inhibiting the tumorigenesis of MB49 in fully immunized mice, and as can be seen from the left panel of fig. 5, the tumor volume of the indisulam-treated group was significantly smaller than that of the DMSO control group at the time point, the longer the time, the more significant the difference. As can be seen from the right panel of fig. 5, at day 19, the tumor volume of the indisulam-treated group was significantly smaller than that of the DMSO control group. Notably, the inhibitory effect of MB49 after treatment with indosulam on neoplasia in C57BL/6 mice was more pronounced than in BALB/C Nude mice, i.e. the anti-tumor response of indosulam involved activation of the immune system. From the IHC staining results shown in the left panel of FIG. 6, it can be seen that the tumor tissue of the indisula-treated group was more infiltrated with CD8 than the tumor tissue of the control group+T cells and DCs, further demonstrating that indesulam-treated MB49 cells produce neoantigens and thus are more potentCan recruit more immune cells into the tumor microenvironment. The right panel of fig. 6 is the statistical analysis result of IHC.
6.ELIspot
C57BL/6 mouse bone marrow cells were isolated and cultured with 1640 medium containing 10ng/mL mouse recombiant IL-4, 20ng/mL mouse recombiant GM-CSF and 10% FBS for 7 days in a 37 ℃ incubator with medium replacement every 2 days to induce dendritic cell Differentiation (DCs). Respectively collecting 1 × 107MB49 cells of a drug control group (DMSO), a drug treatment group (1 mu M indoulam) and a positive control group (over-expression chicken ovalbumin OVA) are repeatedly frozen and thawed for 5 times to obtain cell lysate. Incubating the differentiated DCs with MB49 lysate for 24h to allow sufficient uptake of MB49 antigen; washing the DCs with PBS to remove residual lysate, then co-culturing with mouse spleen cells (1); removing cells in a 96-well plate, washing with PBS 5 times (200. Mu.L/well), adding 1. Mu.g/mL R4-6A2-biotin, and incubating at room temperature for 2h; washing with PBS 5 times (200. Mu.L/well), adding 100. Mu.L/well 1 × Streptavidin-ALP, and incubating at room temperature for 1h; washing with PBS for 5 times (200 muL/hole), adding 100 muL/hole substrate BCIP/NBT plus for color reaction, and stopping color development after 5-10 min; the 96-well plate was washed with running water, dried, and photographed by reading the plate.
Fig. 7 is a graph of ELIspot verifying that indesulam activates immune response, and it can be seen that the enzyme-linked spots induced by the MB49 cell lysate of the indesulam-treated group are significantly larger than those induced by the control group (DMSO), which proves that indesulam can increase the expression of the neoantigen on the surface of MB49 cells, thereby activating immune response.
In one embodiment, the invention provides a double-effect treatment application of indisulam in bladder cancer, and relates to tumor killing and immune activation functions of a small-molecule chemotherapeutic drug indisulam (E7070) in bladder cancer. The invention fully proves that the indolium can inhibit the cell proliferation of the bladder cancer from in vivo and in vitro levels, most importantly, can cause the generation of a large amount of neoantigens of tumor cells, further activate the anti-tumor immune response, finally show ultrahigh bladder cancer removal efficiency, and have unique advantages and good application prospects in the clinical treatment of the bladder cancer.
The present invention has been described in terms of specific examples, which are provided to aid in understanding the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

Claims (6)

  1. Use of n- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof for the preparation of a medicament for the treatment of bladder cancer.
  2. 2. The use according to claim 1, wherein N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to inhibit bladder cancer cell proliferation.
  3. 3. The use according to claim 1, wherein N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used for inducing apoptosis in bladder cancer cells.
  4. 4. The use according to claim 1, wherein N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used for targeted degradation of RBM39 protein.
  5. 5. The use according to claim 1, wherein N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide is used to induce tumor neoantigens and thereby activate immune responses.
  6. 6. A medicament for the treatment of bladder cancer comprising N- (3-chloro-1H-indol-7-yl) -1, 4-benzenedisulfonamide or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, and a pharmaceutically acceptable carrier.
CN202210805345.9A 2022-07-08 2022-07-08 Application of indsulam in preparation of medicine for treating bladder cancer Pending CN115252603A (en)

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