Application of suramine and derivative and pharmaceutical composition thereof

Abstract

The invention relates to an application of suramine in preparation of a medicine for treating malignant tumors, wherein the malignant tumors comprise melanoma. In addition, the invention relates to application of suramine derivatives in preparation of medicines for treating malignant tumors, and the malignant tumors comprise melanoma. Furthermore, the present invention relates to the use of a pharmaceutical composition for the preparation of a medicament for the treatment of malignant tumors including melanoma, the pharmaceutical composition comprising suramine or a suramine derivative. According to the present invention, the malignant tumor, especially melanoma, can be treated by using the small molecule compound suramine drug, such that the treatment response rate of the melanoma patient can be improved, the adverse reaction and the drug resistance can be reduced, and the long-term effective treatment can be provided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to the use of suramin, suramin derivatives, and pharmaceutical compositions comprising suramin or suramin derivatives in the preparation of a medicament for treating malignant tumors including melanoma. Background Art

[0002] In recent years, the role of epigenetic regulation in the field of cancer has been increasingly emphasized. In particular, the emerging research direction of RNA epigenetic modification has opened up a new path for cancer treatment. It has been found that the m 6 6A modification enzyme PCIF1 plays a key role in regulating Th1 cell differentiation, and the lack of PCIF1 in T cells can significantly inhibit the development of various tumors. This effect depends on the promotion of the anti-tumor activity of NK cells by Th1 cells. At the mechanistic level, PCIF1 inhibits the differentiation of Th1 cells by reducing the synthesis of STAT1 protein that depends on its catalytic activity. At the same time, other studies have also confirmed that PCIF1 can directly regulate the proliferation, migration, and invasion of tumor cells. Given the high invasiveness, poor prognosis, and increasing incidence of malignant tumors (such as melanoma), traditional treatment methods such as surgery, radiotherapy, and chemotherapy have poor effects on advanced patients, and there is also a lack of drugs that are significant and effective for treating, for example, melanoma in the market. Therefore, the development of effective therapeutic drugs has great clinical value for improving the survival rate of patients and the treatment effect.

[0003] Currently, for drugs for treating malignant tumors, especially melanoma, there are usually the following several types:

[0004] Immune checkpoint inhibitors, which include anti-PD-1 monoclonal antibodies (such as Pembrolizumab and Toripalimab) and anti-CTLA-4 inhibitors (such as Ipilimumab). These drugs significantly prolong the survival period of patients by enhancing the anti-tumor immune response of T cells. For example, the 3-year survival plateau of Ipilimumab is 21%, and in a randomized controlled trial, Pembrolizumab was evaluated in patients with completely resected stage III. The 1-year recurrence-free survival rate was 75.4%. However, the response rate of immune checkpoint inhibitors is limited, only some patients respond to immune checkpoint inhibitors, and the response time of some is not ideal; immune checkpoint inhibitors also have immune-related adverse reactions, such as they may cause side effects, sometimes may be fatal; immune checkpoint inhibitors are also prone to drug resistance, and some patients discontinue treatment due to immune-related adverse reactions, and there are drug resistance problems.

[0005] BRAF / MEK inhibitors, such as encorafenib combined with binimetinib. The median progression-free survival of patients receiving this combination therapy is 14.9 months, while that of patients receiving vemurafenib alone is only 7.3 months. This indicates that the combination therapy significantly improves the survival time of melanoma patients. However, although approximately 20% of patients achieve a progression-free survival (PFS) of 4-5 years, the long-term safety and efficacy of BRAF / MEK inhibitors are not yet clear; and among patients receiving BRAF / MEK inhibitor treatment, a certain proportion develop new primary cancers.

[0006] Tebentafusp: This is a T cell receptor bispecific antibody targeting GP100 and CD3, used for the treatment of HLA-A*02:01 positive adult patients with unresectable or metastatic uveal melanoma. In a phase III clinical trial with a 3-year follow-up, the median overall survival of Tebentafusp is 21.6 months, and the 3-year survival rate is 27%. However, currently, Tebentafusp is in the clinical phase III, and its long-term effects and safety remain to be further verified.

[0007] Therefore, there is an urgent need to find a more effective drug and apply it to the treatment of melanoma to overcome the deficiencies of existing drugs in the treatment of melanoma. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the present invention aims to develop the application of a more effective drug in the treatment of melanoma, which can improve the treatment response rate of melanoma patients, reduce adverse reactions and drug resistance, and provide a more long-term and effective treatment. For example, to improve the response rate: modify the m 6 Am-modified synthase PCIF1 as a new drug target to improve the response rate of patients to treatment; overcome drug resistance: explore new molecular mechanisms, including targeting PCIF1 can not only enhance the anti-tumor immune response function mediated by the body's T cells, but also directly inhibit the proliferation of melanoma cells by targeted drugs, achieving two-pronged approach to avoid drug resistance of patients to existing treatments. Based on this, the present invention proposes the application of suramin in the preparation of drugs for the treatment of malignant tumors including melanoma. By utilizing the dual action mechanism of suramin, the present invention provides a new, effective, and less side-effect drug application for the treatment of melanoma.

[0009] To solve the above technical problems or achieve the above objectives, the present invention adopts the following technical solutions:

[0010] According to one aspect of the present invention, there is provided the application of suramin in the preparation of drugs for the treatment of malignant tumors, which malignant tumors include melanoma.

[0011] In one embodiment of the present invention, suramin inhibits the proliferation and migration ability of tumor cells and promotes CD4 + T cells to differentiate into Th1 cells.

[0012] In one embodiment of the present invention, suramin enhances the ability of tumor-infiltrating T cells to secrete IFN-γ and / or enhances the ability of tumor-infiltrating T cells to secrete IL-2.

[0013] In one embodiment of the present invention, suramin is administered by intraperitoneal injection.

[0014] In one embodiment of the present invention, suramin is administered once every two days.

[0015] In one embodiment of the present invention, the dosage of suramin per administration is 20 - 40 mg / kg.

[0016] In one embodiment of the present invention, the dosage of suramin per administration is 30 mg / kg.

[0017] In one embodiment of the present invention, the administration of suramin is used in combination with chemotherapy, radiotherapy, targeted therapy, and / or immunotherapy.

[0018] According to another aspect of the present invention, there is provided the use of a suramin derivative in the preparation of a drug for treating malignant tumors, which malignant tumors include melanoma.

[0019] According to still another aspect of the present invention, there is provided the use of a pharmaceutical composition in the preparation of a drug for treating malignant tumors, which malignant tumors include melanoma, and the pharmaceutical composition comprises an effective therapeutic amount of suramin or a suramin derivative.

[0020] The technical solution provided by the present invention has the following advantages compared with the prior art:

[0021] (1) In the present invention, when treating malignant tumors, especially melanoma, suramin can not only directly inhibit the proliferation of melanoma tumor cells, but also regulate the tumor immune microenvironment by promoting CD4 + T cells to differentiate into Th1 cells, thereby enhancing the anti-tumor effect of immune cells. This dual mechanism makes suramin more effective in inhibiting tumor growth.

[0022] (2) In the present invention, when treating malignant tumors, especially melanoma, suramin can improve the immune response in the tumor microenvironment and enhance the body's immune surveillance and clearance ability against tumors by promoting the differentiation of Th1 cells. This is of great significance for preventing tumor recurrence and metastasis.

[0023] (3) The dual mechanism of action of suramin in the present invention means that it may also be effective against a variety of other malignant tumors, not limited to specific tumor types, which provides new possibilities for the treatment of a wide range of cancers.

[0024] (4) In the present invention, when suramin is used to treat malignant tumors, especially melanoma, the action of suramin is not limited to a single pathway, and it is more difficult for tumor cells to develop resistance to suramin, which helps to prolong the duration of the therapeutic effect.

[0025] (5) In the present invention, when suramin is used to treat malignant tumors, especially melanoma, it can be used in combination with existing treatment methods (such as chemotherapy, radiotherapy, targeted therapy, and / or immunotherapy), thereby being able to improve the effectiveness of these treatments, especially in enhancing the immune response.

[0026] (6) In the present invention, when suramin is used to treat malignant tumors, especially melanoma, suramin can regulate the immune response. Compared with some traditional chemotherapy drugs, it has lower systemic side effects and can improve the quality of life of patients.

[0027] (7) In the present invention, when treating malignant tumors, especially melanoma, as a small molecule compound, suramin has relatively low production and application costs, which can make the treatment more economical and increase the chances of patients obtaining effective treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings herein are incorporated into and constitute a part of this specification, showing embodiments consistent with the present invention and, together with the specification, are used to explain the principles of the present invention.

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1Shows the comparison result graph of melanoma tumor cell proliferation in the control group (Ctrl, without suramin administration) and the experimental group (Suramin, with a suramin concentration of 200 μM) in an embodiment of the present invention (ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001). Among them, Figure A shows the statistical graph of tumor cell count (Cell number) after incubating B16-F10 tumor cells for 24 hours and 48 hours after plating B16-F10 tumor cells in the control group Ctrl and the experimental group Suramin; Figure B shows the result graph of detecting the proliferation (percentage of Ki67 marker) of B16-F10 tumor cells after incubating for 24 hours by flow analysis technology in the control group Ctrl and the experimental group Suramin; Figure C shows the result graph of detecting the apoptosis status (percentage of 7-AAD + Annexin V + marker) of B16-F10 tumor cells after incubating for 24 hours by flow analysis technology in the control group Ctrl and the experimental group Suramin; Figure D shows the result graph of detecting the effect on the migration of B16-F10 tumor cells after incubating for 24 hours by scratch assay in the control group Ctrl and the experimental group Suramin.

[0031] Figure 2 Shows the comparison result graph of promoting Th1 polarization in vitro in the control group (without suramin administration, 0 μM) and the experimental group (with suramin concentrations of 15 μM and 30 μM) in an embodiment of the present invention (ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001). Among them, Figure A shows that in the control group (without suramin, 0 μM) and the experimental group (with suramin concentrations of 15 μM and 30 μM), CD4 + T cells were plated in a 96-well U-bottom plate and cultured under Th1 polarization conditions for 96 hours, and then the ability of CD4 + T cells (stained with CD4-BV421 fluorescent dye) to secrete IFN-γ (stained with IFN-γ-APC fluorescent dye) was detected by flow cytometry; Figure B shows the statistical graph of IFN-γ (IFN-γ + T cells (CD4 + T cell) secreted IFN-γ (IFN-γ + ) in the control group (without suramin, 0 μM) and the experimental group (with suramin concentrations of 15 μM and 30 μM).

[0032] Figure 3Shows the comparative result graphs of the anti-tumor effects in the B16-F10 melanoma model of mice in the control group (Ctrl, without suramin administration) and the experimental group (Suramin, intraperitoneally injected with suramin once every two days, with a dosage of 30 mg / kg each time) in an embodiment of the present invention (ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001), where Figure A shows that after subcutaneously injecting 4×10 5 B16-F10 cells into 8-week-old, specific pathogen-free C57 / BL6 male mice in the control group Ctrl and the experimental group Suramin to establish a melanoma model, the growth of the mice tumors was recorded and monitored, and a representative tumor result graph on the 18th day (Day) is shown; Figure B shows the result graph of the influence on the tumor volume (Tumor Volume (mm 3 )) of tumor-bearing mice in the control group Ctrl and the experimental group Suramin; Figure C shows the result graph of the influence on the tumor weight (Tumor Weight (g)) of tumor-bearing mice in the control group Ctrl and the experimental group Suramin.

[0033] Figure 4 Shows the comparative result graphs of the anti-tumor effects in the mouse PCIF1 - / - B16-F10 melanoma model in the control group (Ctrl, without suramin administration) and the experimental group (Suramin, intraperitoneally injected with suramin once every two days, with a dosage of 30 mg / kg each time) in an embodiment of the present invention (ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001), where Figure A shows that after subcutaneously injecting 4×10 5 PCIF1 - / - B16-F10 cells into 8-week-old, specific pathogen-free C57 / BL6 male mice in the control group Ctrl and the experimental group Suramin to establish a melanoma model, the growth of the mice tumors was recorded and monitored, and a representative tumor result graph on the 18th day (Day) is shown; Figure B shows the result graph of the influence on the tumor volume (Tumor Volume (mm 3 )) of tumor-bearing mice in the control group Ctrl and the experimental group Suramin; Figure C shows the result graph of the influence on the tumor weight (Tumor Weight (g)) of tumor-bearing mice in the control group Ctrl and the experimental group Suramin.

[0034] Figure 5 Shows the control group (Ctrl, without suramin administration) and the experimental group (Suramin, injected with suramin once every two days, with a dosage of 30 mg / kg each time) in PCIF1 in an embodiment of the present invention- / - Comparison results of the effects on the functions of tumor-infiltrating T cells in the B16-F10 melanoma model (ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001). Figure A shows the subcutaneous injection of 4×10 5 PCIF1 - / - B16-F10 cells into 8-week-old, specific pathogen-free C57 / BL6 male mice to construct a melanoma model, and then detect the functions of tumor-infiltrating T cells on the 18th day. Representative flow cytometry shows the results of tumor-infiltrating CD4 + T cells (stained with CD4-BV421 fluorescent dye) secreting IFN-γ (stained with IFN-γ-APC fluorescent dye); Figure B shows the IFN-γ (IFN-γ + in tumor-infiltrating CD4 + CD4 + T cell (%)) secretion in the control group Ctrl and the experimental group Suramin; Figure C shows the results of representative flow cytometry of tumor-infiltrating CD4 + T cells (stained with CD4-BV421 fluorescent dye) secreting IL-2 (stained with IL-2-FITC fluorescent dye) in the control group Ctrl and the experimental group Suramin; Figure D shows the IL-2 (IL-2 + in tumor-infiltrating CD4 + CD4 + T cell (%)) secretion in the control group Ctrl and the experimental group Suramin. Detailed implementation manners

[0035] In order to more clearly understand the above objects, features, and advantages of the present invention, the embodiments of the present invention will be further described below. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0036] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only a part of the embodiments of the present invention, rather than all the embodiments.

[0037] The present invention provides the application of suramin in the preparation of a drug for treating malignant tumors, and the malignant tumors include melanoma.

[0038] In the above application, suramin inhibits the proliferation and migration abilities of tumor cells, and promotes CD4 +T cells differentiate into Th1 cells. Preferably, suramin promotes CD4 + T cells to secrete IFN-γ.

[0039] In the above application, suramin enhances the anti-tumor ability of tumor-infiltrating T cells and inhibits the growth of tumor cells. Preferably, suramin enhances the ability of tumor-infiltrating T cells to secrete IFN-γ and / or enhances the ability of tumor-infiltrating T cells to secrete IL-2.

[0040] In the above application, suramin is administered by intraperitoneal injection.

[0041] In the above application, suramin is administered once every two days.

[0042] In the above application, the dosage of suramin per administration is 20 - 40 mg / kg. Preferably, the optimal dosage of suramin per administration is 30 mg / kg.

[0043] In the above application, the administration of suramin can be used in combination with chemotherapy, radiotherapy, targeted therapy, and / or immunotherapy.

[0044] In addition, the present invention provides the application of suramin derivatives in the preparation of drugs for treating malignant tumors, which include melanoma.

[0045] Furthermore, the present invention provides the application of a pharmaceutical composition in the preparation of drugs for treating malignant tumors, which include melanoma, and the pharmaceutical composition includes an effective therapeutic amount of suramin or suramin derivatives.

[0046] The above technical solutions of the present invention will be described and elaborated in detail below in conjunction with specific embodiments.

[0047] In all the following examples, the cells, compounds, dosing doses, and detection methods used are carried out according to the following instructions.

[0048] I. Material Preparation

[0049] 1. Cell line: The mouse melanoma tumor cell line B16-F10 was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0050] 2. Experimental animals: 8-week-old male C57 / BL6 mice were purchased from Shanghai Lingchang Experimental Animal Co., Ltd. and used to construct the B16-F10 mouse tumor model.

[0051] 3. Reagents: CD4 + T cell isolation kit( CD4 +The T cell sorting kit was purchased from STEMCELL Technologies; high-glucose RPMI-1640 and DMEM media were both purchased from Hyclone; fetal bovine serum, penicillin-streptomycin solution, trypsin, etc. were all purchased from GIBCO; murine IL-2 was purchased from Peprotech; murine IL-12 was purchased from GenScript; phorbol myristate acetate (PMA), Ionomycin were purchased from Sigma; the reagent GolgiPlug was purchased from BD Biosciences.

[0052] 4. Instruments: CO 2 cell incubator; microscope; cell culture laminar flow hood; cell counter; microplate reader; flow cytometer; cell culture dish; ophthalmic scissors; syringe and 96-well U-bottom plate, etc.

[0053] 5. Antibodies: anti-CD3, anti-CD28, anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-TCR-β, anti-IL-4, anti-IFN-γ and anti-IL-2 antibodies were all purchased from Univ-bio.

[0054] II. Drug Preparation

[0055] Animal experiment: Suramin was dissolved in sterilized ddH 2 O, and the stock solution concentration was 7.5 mg / mL.

[0056] III. In vitro Th1 Differentiation and Flow Cytometry Detection

[0057] 1. One day in advance, plate the required 96-well U-bottom plate with 200 μL of anti-CD3 mAb (2 μg / mL), and incubate overnight at 4°C.

[0058] 2. Prepare 50 mL of the medium required for Th1 differentiation: high-glucose RPMI-1640 + 10% FBS + 0.384 μL of 2-mercaptoethanol + 1% penicillin-streptomycin solution + anti-CD28 mAb (2 μg / mL). The added cytokines were IL-12 (10 ng / mL), anti-IL-4 mAb (10 μg / mL), and IL-2 (2 ng / mL).

[0059] 3. Isolate the spleens of C57 / BL6 mice and use CD4 + T cell isolation kit to enrich and purify the CD4 + T cells are plated according to requirements, with 2×10 5 T cells per well. Add the Th1 differentiation medium prepared in step 2 and culture and differentiate in a 37°C, 5% CO 2 cell incubator for 96 hours.

[0060] 4. After culturing for 96 hours, centrifuge the cells, discard the supernatant, and resuspend them with a complete medium containing PMA (50 ng / mL), Innomycin (1 μg / mL), and GolgiPlug (1 μL / mL). Take out after stimulating and culturing in a 37°C, 5% CO 2 cell incubator for 4 hours, stain with surface antibodies, fix, perform cytokine staining, and resuspend with PBS into a flow tube for loading onto the machine.

[0061] IV. Homologous tumor cell line xenograft model

[0062] 1. Select the B16-F10 tumor cell line in the logarithmic growth phase, separate and remove the medium by centrifugation. Wash the cells twice with pre-cooled phosphate buffer (PBS) to remove the residual serum in the cell medium, resuspend the cells with PBS and count, and adjust the cell density to 4×10 5 cells per 250 μL injection solution.

[0063] 2. Inject the cell suspension subcutaneously into the left hind limb of each mouse, ensuring that each mouse receives 4×10 5 cells, and randomly assign the mice to different experimental groups.

[0064] 3. Starting from the second day after tumor inoculation, inject suramin into the mice at a dose of 30 mg / kg. Observe the tumor formation in the mice, and use a vernier caliper to measure the longest diameter a and the shortest diameter b of the tumor every three days. According to the formula: tumor volume = a×b 2 / 2 to calculate the tumor volume.

[0065] 4. Record the tumor growth data, plot the tumor growth curve and the mouse body weight change curve, and when the tumor volume of the control group mice reaches the maximum volume specified by ethics, euthanize the mice, take out the tumor tissue and weigh it, record the data and plot the tumor weight graph.

[0066] V. Flow cytometry of tumor-infiltrating T cells

[0067] 1. At the end of the experiment, dissect the mice and take out the tumor tissue.

[0068] 2. Gently grind the tumor tissue into a paste in a 70-μm nylon mesh, and add 1640 medium containing 2% fetal bovine serum (FBS) during the process for rinsing to obtain a cell filtrate.

[0069] 3. Collect the cell suspension using a 15-mL centrifuge tube, centrifuge at a relative centrifugal force of 600 g (g is the acceleration due to gravity), remove the supernatant, resuspend the cells with 3 mL of 42% Percoll, and lay 70% Percoll at the bottom of 42% Percoll.

[0070] 4. Centrifuge at a relative centrifugal force of 1260 g for 10 minutes, collect the cells in the middle layer (about 1.5 mL), resuspend them to 10 mL with PBS, and centrifuge again at a relative centrifugal force of 600 g for 10 minutes.

[0071] 5. Resuspend the cell pellet with RPMI 1640 medium containing 10% FBS, take it out after stimulating with PMA, Ionomycin, and GolgiPlug for 4 hours, perform surface marker staining, perform cytokine staining after fixation, and finally resuspend it with PBS for analysis using a flow cytometer.

[0072] Experimental Example 1

[0073] Study on the inhibition of the proliferation of B16-F10 tumor cell line by suramin

[0074] In this example, by comparing with the control group, the inhibition of suramin on the B16-F10 tumor cell line in the experimental group was studied, and the molecular mechanism of suramin on B16-F10 tumor cells was explored.

[0075] Experimental steps:

[0076] Seed 2×10 5 B16-F10 cells in a 96-well U-bottom plate for culture, and set the control group Ctrl and the experimental group Suramin. The concentration of suramin in the experimental group Suramin was preferably set at 200 μM. After culturing for 24 hours and 48 hours, count the number of cells in the control group Ctrl and the experimental group Suramin respectively, and use flow cytometry analysis technology to detect the proliferation ability and apoptosis status of tumor cells in the control group Ctrl and the experimental group Suramin after culturing for 24 hours. At the same time, use the scratch assay to observe the migration ability of tumor cells in the control group Ctrl and the experimental group Suramin after culturing for 24 hours.

[0077] Results:

[0078] As Figure 1As shown in Figure A in the middle, it shows a statistical chart of the tumor cell count after incubating B16-F10 tumor cells for 24 hours and 48 hours after plating B16-F10 tumor cells in the control group Ctrl and the experimental group Suramin. It can be seen from this statistical chart that after incubating B16-F10 tumor cells for 24 hours and 48 hours, the tumor cell count in the experimental group Suramin is significantly lower than that in the control group Ctrl. Therefore, 200 μM suramin in the experimental group Suramin significantly inhibits the increase in the number of B16-F10 cells.

[0079] As Figure 1 As shown in Figure B in the middle, it shows a result chart of detecting the proliferation (percentage of Ki67 marker) of B16-F10 tumor cells after incubating for 24 hours by flow analysis technology in the control group Ctrl and the experimental group Suramin. It can be seen from this result chart that suramin in the experimental group Suramin significantly inhibits the proliferation ability of B16-F10 cells.

[0080] As Figure 1 As shown in Figure C in the middle, it shows a result chart of detecting the apoptotic status (percentage of 7-AAD + Annexin V + marker) of B16-F10 tumor cells after incubating for 24 hours by flow analysis technology in the control group Ctrl and the experimental group Suramin. It can be seen from this result chart that there is little difference in the apoptotic status between the control group Ctrl and the experimental group Suramin, which is not significant. Therefore, suramin in the experimental group Suramin has no significant effect on tumor cell apoptosis.

[0081] As Figure 1 As shown in Figure D in the middle, it shows a result chart of detecting the effect on the proliferation of B16-F10 tumor cells after incubating for 24 hours by scratch assay in the control group Ctrl and the experimental group Suramin. It can be seen from this result chart that suramin in the experimental group Suramin significantly inhibits the migration ability of B16-F10 cells.

[0082] Conclusion:

[0083] 200 μM suramin significantly inhibits the proliferation and migration abilities of B16-F10 melanoma cells.

[0084] Experimental Example 2

[0085] Study the effect of suramin in promoting Th1 cell differentiation in vitro

[0086] In this example, by comparing with the control group, the effect of different concentrations of suramin in the experimental group on Th1 cell differentiation was studied.

[0087] Experimental steps:

[0088] Two times ten 5 cells CD4 + T cells were seeded in a 96-well U-bottom plate for culture, and a control group and an experimental group were set up. Suramin was not applied in the control group, 0 μM, and suramin was applied in the experimental group at concentrations of 15 μM and 30 μM. They were cultured under Th1 polarization conditions in vitro for 96 hours, and flow cytometry was used to detect the ability of CD4 + T cells to secrete IFN-γ as an evaluation of the Th1 polarization level.

[0089] Results:

[0090] As Figure 2 shown in Figure A in the middle, it shows the CD4 in the control group (without suramin, 0 μM) and the experimental group (applied with 15 μM and 30 μM suramin) CD4 + T cells were seeded in a 96-well U-bottom plate and cultured under Th1 polarization conditions for 96 hours, and then flow cytometry was used to detect the ability of CD4 + T cells (stained with CD4-BV421 fluorescent dye) to secrete IFN-γ (stained with IFN-γ-APC fluorescent dye) (representative flow cytometry diagram for IFN-γ secretion detection). From this result diagram, it can be seen that the CD4 CD4 + T cells treated with 30 μM suramin in the experimental group secreted more or stronger IFN-γ.

[0091] As Figure 2 shown in Figure B in the middle, it shows the statistical chart of IFN-γ secreted by CD4 + T cells (CD4 + T cell) in the control group (without suramin, 0 μM) and the experimental group (applied with 15 μM and 30 μM suramin) (statistical chart of IFN-γ + secretion level). From this statistical chart, it can be seen that the CD4 CD4 + T cells treated with 30 μM suramin in the experimental group secreted more IFN-γ, and 30 μM suramin significantly promoted the secretion of IFN-γ by CD4 + T cells.

[0092] Conclusion:

[0093] Treatment of CD4 + T cells with 30 μM suramin in vitro can promote the differentiation of Th1 cells.

[0094] Experimental Example 3

[0095] Study on the inhibitory effect of suramin on tumors in B16-F10 cell-bearing mice

[0096] Experimental procedures:

[0097] The groups were set as the control group Ctrl and the drug treatment experimental group Suramin. The experimental animals were 8-week-old, specific pathogen-free C57 / BL6 male mice. A tumor model was constructed by subcutaneously injecting 4×10 5 B16-F10 cells into the left abdominal subcutaneous area of the mice. After modeling, suramin was not administered in the control group Ctrl, and suramin was injected once every two days in the experimental group Suramin, with a dosage of 30 mg / kg each time. The growth of tumors in the mice of the two groups was recorded and monitored respectively, and samples were collected within the scope of ethical requirements.

[0098] Results:

[0099] As Figure 3 shown in Figure A, the growth of tumors in the 8-week-old, specific pathogen-free C57 / BL6 male mice in the control group Ctrl and the experimental group Suramin was recorded and monitored after subcutaneously injecting 4×10 5 B16-F10 cells to construct a melanoma model, and a representative tumor result graph on the 18th day was shown. It can be seen from this graph that the growth of tumors in the mice in the experimental group Suramin was small and inhibited.

[0100] As Figure 3 shown in Figure B, the result graph of the influence on the tumor volume of tumor-bearing mice in the control group Ctrl and the experimental group Suramin was shown. It can be seen from this graph that from the 14th day to the 18th day of the growth of tumor cells, compared with the control group, the tumor volume of the tumor-bearing mice in the experimental group Suramin was significantly smaller, indicating that suramin has a significant inhibitory effect on the tumor volume.

[0101] As Figure 3 shown in Figure C, the result graph of the influence on the tumor weight of tumor-bearing mice in the control group Ctrl and the experimental group Suramin was shown. It can be seen from this graph that the tumor weight of the tumor-bearing mice in the experimental group Suramin was significantly smaller, indicating that suramin has a significant inhibitory effect on the tumor weight.

[0102] Thus, it can be seen that the growth of tumors in mice was significantly inhibited after suramin administration.

[0103] Conclusion:

[0104] By injecting suramin subcutaneously into the abdomen once every two days, with a dosage of 30 mg / kg each time, the occurrence and development of melanoma were significantly inhibited.

[0105] Example 4

[0106] Experimental steps for studying the inhibition of tumor cell growth by suramin through enhancing the anti-tumor ability of tumor-infiltrating T cells:

[0107] Construct PCIF1 through gene editing technology - / - B16-F10 tumor cell line. Set the groups as the control group Ctrl and the drug treatment experimental group Suramin. Select 8-week-old, specific pathogen-free C57 / BL6 male mice as experimental animals, and construct a tumor model by subcutaneously injecting 4×10 5 PCIF1 - / - B16-F10 cells into the left abdominal subcutaneous area of the mice. After tumor modeling, suramin was not administered in the control group Ctrl, and suramin was injected once every two days in the experimental group Suramin, with a dosage of 30 mg / kg each time. Record and monitor the growth of the mice tumors respectively, and collect samples within the scope of ethical requirements. After data recording, isolate and purify the infiltrating T cells from the tumor tissues, and use flow cytometry analysis technology to detect the secretion of related cytokines in the T cells.

[0108] Results:

[0109] As Figure 4 shown in Figure A in the middle, it shows the results of recording and monitoring the growth of the subcutaneous tumors of 8-week-old, specific pathogen-free C57 / BL6 male mice in the control group Ctrl and the experimental group Suramin after injecting 4×10 5 PCIF1 - / - B16-F10 cells to construct a melanoma model, and show a representative tumor result graph on the 18th day. It can be seen from this figure that the growth of the tumors in the mice in the experimental group Suramin is small and inhibited.

[0110] As Figure 4 shown in Figure B in the middle, it shows the result graph of the influence on the tumor volume of tumor-bearing mice in the control group Ctrl and the experimental group Suramin. It can be seen from this figure that from the 14th day to the 18th day of tumor cell growth, compared with the control group, the tumor volume of the tumor-bearing mice in the experimental group Suramin is significantly smaller, indicating that suramin has a significant inhibitory effect on the tumor volume.

[0111] As Figure 4 shown in Figure C in the middle, it shows the result graph of the influence on the tumor weight of tumor-bearing mice in the control group Ctrl and the experimental group Suramin. It can be seen from this figure that the tumor weight of the tumor-bearing mice in the experimental group Suramin is significantly smaller, indicating that suramin has a significant inhibitory effect on the tumor weight.

[0112] Therefore, it can be seen from Figure 4 that after suramin administration, the mouse PCIF1 - / -The tumor growth of B16-F10 was significantly inhibited.

[0113] As Figure 5 shown in Figure A in the middle, it shows the function of tumor-infiltrating T cells detected on the 18th day after subcutaneous injection of 4×10 5 PCIF1 - / - cells into 8-week-old, specific pathogen-free C57 / BL6 male mice in the control group Ctrl and the experimental group Suramin to construct a melanoma model, and the results of the secretion of IFN-γ (stained with IFN-γ-APC fluorescent dye) by tumor-infiltrating CD4 + T cells (stained with CD4-BV421 fluorescent dye) were shown by representative flow cytometry (representative flow cytometry for IFN-γ secretion detection). It can be seen from this result graph that the PCIF1 - / - in the experimental group Suramin secreted more IFN-γ in tumor-infiltrating T cells of B16-F10 tumor-bearing mice.

[0114] As Figure 5 shown in Figure B in the middle, it shows the statistical graph of the secretion of IFN-γ (IFN-γ + CD4 + T cell (%)) in tumor-infiltrating CD4 + T cells in the control group Ctrl and the experimental group Suramin (statistical graph of IFN-γ secretion level). It can also be seen from this statistical graph that the PCIF1 - / - in the experimental group Suramin secreted more IFN-γ in tumor-infiltrating T cells of B16-F10 tumor-bearing mice.

[0115] Therefore, it can be seen from Figure 5 Figure A and Figure B in it that suramin in the experimental group Suramin significantly enhanced the ability of PCIF1 - / - to secrete IFN-γ in tumor-infiltrating T cells of B16-F10 tumor-bearing mice.

[0116] As Figure 5 shown in Figure C in the middle, it shows the result graph of the secretion of IL-2 (stained with IL-2-FITC fluorescent dye) by tumor-infiltrating CD4 + T cells (stained with CD4-BV421 fluorescent dye) in the control group Ctrl and the experimental group Suramin (representative flow cytometry for IL-2 secretion detection). It can be seen from this result graph that the PCIF1 - / - in the experimental group Suramin secreted more IL-2 in tumor-infiltrating T cells of B16-F10 tumor-bearing mice.

[0117] As Figure 5 shown in Figure D in the middle, it shows tumor-infiltrating CD4+ IL-2 in T cells (IL-2 + CD4 + Statistical chart of the secretion of T cell (%) (Statistical chart of IL-2 secretion level). It can also be seen from this statistical chart that in the experimental group Suramin, PCIF1 - / - The tumor-infiltrating T cells of B16-F10 tumor-bearing mice secrete more IL-2.

[0118] Therefore, from Figure 5 Figures C and D, it can be seen that in the experimental group Suramin, suramin significantly enhanced PCIF1 - / - The ability of tumor-infiltrating T cells of B16-F10 tumor-bearing mice to secrete IL-2.

[0119] Conclusion:

[0120] Suramin can inhibit the growth of tumor cells by enhancing the function of tumor-infiltrating T cells.

[0121] Therefore, it can be seen from the above Examples 1-4 that suramin can be applied in drugs for treating melanoma. Suramin can inhibit the proliferation and migration ability of melanoma cells, and promote CD4 + T cells to differentiate into Th1 cells, and suramin can enhance the anti-melanoma ability of tumor-infiltrating T cells to inhibit the growth of melanoma cells. Among them, suramin is preferably intraperitoneally injected once every two days, and the dosage of suramin each time is 30 mg / kg.

[0122] In addition, in the alternative embodiments, the suramin used in the above embodiments can be replaced by suramin derivatives and pharmaceutical compositions including suramin or suramin derivatives. In addition, the administration method, administration time, administration dose, etc. of suramin can also be adjusted according to actual needs.

[0123] As shown in Table 1 below, the chemical names, molecular weights and chemical formulas of suramin and three suramin derivatives are shown. Suramin derivative 1, suramin derivative 2 and suramin derivative 3 in Table 1 were experimented according to the above Examples 1-4, and the results were the same as those of the above Examples 1-4 using suramin, and no repeated description will be made here. For the specific experimental results, refer to the descriptions and explanations in the above Examples 1-4 and Figures 1 - 5 the descriptions.

[0124] Table 1 - Suramin and Suramin Derivatives

[0125]

[0126] In summary, in the present invention, the small molecule compound suramin or its derivatives and pharmaceutical compositions can be used to treat malignant tumors, especially melanoma, which can improve the treatment response rate of melanoma patients, reduce adverse reactions and drug resistance, and provide more long-term and effective treatment.

[0127] It should be noted that in this text, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to the said process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the said element.

[0128] The above are only embodiments of the present invention, which enable those skilled in the art to understand and implement the present invention. Various modifications to the said embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but rather to the broadest scope consistent with the principles and features disclosed herein.

Claims

1. Use of suramin in preparing a drug for treating malignant tumors, characterized in that: The malignant tumor includes melanoma.

2. The use according to claim 1, characterized in that: Suramin inhibits the proliferation and migration of tumor cells and promotes CD4 + T cells differentiate into Th1 cells.

3. The use according to claim 1, characterized in that: Suramin enhances the ability of tumor-infiltrating T cells to secrete IFN-γ and / or enhances the ability of tumor-infiltrating T cells to secrete IL-2.

4. The use according to claim 1, characterized in that: Suramin was administered by intraperitoneal injection.

5. The use according to claim 1, characterized in that: Suramin was administered every two days.

6. The use according to claim 5, characterized in that: The dosage of suramin per administration is 20-40 mg / kg.

7. The use according to claim 6, characterized in that: The dosage of suramin was 30 mg / kg each time.

8. The use according to claim 1, characterized in that: Administration of suramin is used in conjunction with chemotherapy, radiation therapy, targeted therapy, and / or immunotherapy.

9. Use of suramin derivatives in the preparation of drugs for treating malignant tumors, characterized in that: The malignant tumor includes melanoma.

10. Use of a pharmaceutical composition in preparing a drug for treating malignant tumors, characterized in that: The malignant tumor includes melanoma, and the pharmaceutical composition includes an effective therapeutic amount of suramin or a suramin derivative.

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