A combined pharmaceutical composition for preventing or treating follicular lymphoma and use thereof

Abstract

The present application relates to a kind of combination drug composition for preventing or treating follicular lymphoma and its application, the combination drug composition includes VEGFR2 inhibitor and DGK alpha inhibitor.The present application research finds that VEGFR2 inhibitor combined with DGK alpha inhibitor can not only reduce the dosage of VEGFR2 inhibitor or DGK alpha inhibitor, improve the safety of drug, but also have more significant inhibitory effect on follicular lymphoma than single use VEGFR2 inhibitor or DGK alpha inhibitor, reach the effect of synergistic promotion.The present application first proves that it can significantly inhibit follicular lymphoma cell proliferation and induce apoptosis by follicular lymphoma cell strain;It can inhibit the expression of MAPK pathway related protein of follicular lymphoma cell.The present application provides effective drug combination strategy for the improvement or treatment of follicular lymphoma, and has very significant meaning.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a new method for the prevention or treatment of follicular lymphoma, specifically to a combination drug composition for the prevention or treatment of follicular lymphoma and its application. Background Technology

[0002] Follicular lymphoma (FL) is the most common indolent lymphoma, accounting for approximately 20% of all non-Hodgkin lymphoma (NHL) cases. The most common presentation of follicular lymphoma is painless lymphadenopathy, typically involving multiple sites of lymphoid tissue, and sometimes palpable supratrochlear lymphadenopathy.

[0003] Using current first-line regimens, most FL patients show an initial response to treatment. However, despite improvements from first-line therapy, standard treatment for FL is not curative, and approximately 20% of patients still experience treatment-resistant or early relapse. Moreover, this early relapse is often due to chemotherapy resistance, leading to a significantly shortened survival. Therefore, there is an urgent need to develop new treatment strategies to improve the survival rate of this poorly prognostic FL subgroup.

[0004] VEGFR is a receptor that binds to vascular endothelial growth factor (VEGF) and plays a crucial role in regulating angiogenesis. During tumor growth, tumor cells produce large amounts of VEGF, stimulating angiogenesis in surrounding tissues, providing nutrients and oxygen to the tumor, and promoting tumor growth and metastasis. VEGFR inhibitors are a class of drugs that target VEGFR, blocking the binding of VEGF to its receptor VEGFR, thereby inhibiting angiogenesis and tumor cell growth. They are used to treat some malignant tumors, such as lung cancer, kidney cancer, and colorectal cancer.

[0005] Diacylglycerol kinase (DGKα) plays a crucial role in regulating cell proliferation, apoptosis, and migration. In cancer cells, it catalyzes the conversion of diacylglycerol to phosphatidic acid, reducing apoptosis and promoting cell proliferation. Besides cancer cells, DGKα is abundant in T cells and induces a T cell unresponsive state, a major mechanism by which advanced cancers evade immune responses. Therefore, DGKα inhibition offers a novel therapeutic strategy for refractory cancers, and specific DGKα inhibitors hold promise as a dual-effect anti-cancer treatment, enhancing T cell function while inhibiting cancer cell proliferation. However, the applicability of DGKα inhibitors to follicular lymphoma treatment, particularly in combination with VEGFR2 inhibitors, and their mechanism of action on follicular lymphoma-related cell lines, remain unclear. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new method for the prevention or treatment of follicular lymphoma, specifically a combination drug composition for the prevention or treatment of follicular lymphoma and its application.

[0007] To achieve this objective, the present invention employs the following technical solution:

[0008] In a first aspect, the present invention provides a combination pharmaceutical composition for the prevention or treatment of follicular lymphoma, the combination pharmaceutical composition comprising a VEGFR2 inhibitor and a DGKα inhibitor.

[0009] This invention creatively combines VEGFR2 inhibitors and DGKα inhibitors as drugs for the prevention or treatment of follicular lymphoma. The study found that combining VEGFR2 inhibitors with DGKα inhibitors not only reduces the dosage of either inhibitor and improves safety, but also significantly inhibits follicular lymphoma more effectively than using either inhibitor alone, achieving a synergistic effect. This invention first demonstrates, using follicular lymphoma cell lines, that it can significantly inhibit follicular lymphoma cell proliferation and induce apoptosis; then, Western blot experiments demonstrate that it can inhibit the expression of MAPK pathway-related proteins in follicular lymphoma cells. This invention provides an effective drug combination strategy for the improvement or treatment of follicular lymphoma, which is of great significance.

[0010] Preferably, the VEGFR2 inhibitor is selected from any one or a combination of at least two of Chiauranib (CS2164) or its pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0011] Preferably, the DGKα inhibitor is selected from ritanserin or any one or a combination of at least two of its pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0012] Preferably, the combined pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0013] Preferably, the combined pharmaceutical composition of the present invention can be administered alone or in combination with excipients to form an appropriate dosage form for administration. The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0014] Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0015] Preferably, the combined pharmaceutical composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

[0016] The combined drug composition can be a single compound preparation or a combination of two separate preparations; when it is a combination of two separate preparations, it can be administered simultaneously, alternately, or sequentially.

[0017] Preferably, the preparation is any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, etc.

[0018] In a second aspect, the present invention provides the use of the combined pharmaceutical composition according to the first aspect in the preparation of a medicament for the prevention, improvement or treatment of non-Hodgkin's lymphoma.

[0019] Preferably, the non-Hodgkin lymphoma includes indolent non-Hodgkin lymphoma.

[0020] Preferably, the indolent non-Hodgkin lymphoma includes follicular lymphoma.

[0021] Thirdly, the present invention provides the use of the pharmaceutical composition according to the first aspect in the preparation of a proliferation inhibitor of follicular lymphoma cells.

[0022] The present invention also provides the use of the combination pharmaceutical composition according to the first aspect in the preparation of a proliferation inhibitor of follicular lymphoma cells for a non-therapeutic purpose.

[0023] According to the research results of this invention, the combined pharmaceutical composition has a significant inhibitory effect on the proliferation of follicular lymphoma cells. Therefore, this result indicates that the combined pharmaceutical composition can be used as an in vitro experimental reagent in scientific research, such as studying the growth and metabolic mechanisms or behavior of follicular lymphoma cells, and screening drugs for the treatment of follicular lymphoma. The proliferation inhibitor claimed in this invention is not intended to eliminate the cause or lesion; that is, it is an application in the preparation of a proliferation inhibitor for follicular lymphoma cells for a non-therapeutic purpose.

[0024] Fourthly, the present invention provides the use of the pharmaceutical composition according to the first aspect in the preparation of an apoptosis promoter for follicular lymphoma cells.

[0025] The present invention also provides the use of the combined pharmaceutical composition according to the first aspect in the preparation of an apoptosis promoter for follicular lymphoma cells for a non-therapeutic purpose.

[0026] According to the research results of this invention, the combined pharmaceutical composition significantly promotes apoptosis in follicular lymphoma cells. Therefore, this result indicates that the combined pharmaceutical composition can be used as an in vitro experimental reagent in scientific research, such as for studying the apoptosis and metabolic mechanisms or behavior of follicular lymphoma cells, and screening drugs for the treatment of follicular lymphoma. The apoptosis promoter claimed in this invention is not intended to eliminate the cause or lesion; that is, it is an application in the preparation of an apoptosis promoter for follicular lymphoma cells for a non-therapeutic purpose.

[0027] Preferably, the follicular lymphoma cells include any one or a combination of at least two of DOHH2 cells, SU-DHL-4 cells, or Karpas422 cells.

[0028] Fifthly, the present invention provides the use of the combination pharmaceutical composition according to the first aspect in the preparation of MAPK pathway inhibitors.

[0029] The present invention also provides the use of the combination pharmaceutical composition according to the first aspect in the preparation of MAPK pathway inhibitors for non-therapeutic purposes.

[0030] According to the research results of this invention, the combined pharmaceutical composition significantly inhibits the expression of MAPK pathway-related genes and proteins. Therefore, this result indicates that the combined pharmaceutical composition can be used as an in vitro experimental reagent in the field of scientific research. The MAPK pathway inhibitor claimed in this invention is not intended to eliminate the cause or lesion; that is, it is an application in the preparation of MAPK pathway inhibitors for a non-therapeutic purpose.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention creatively combines VEGFR2 inhibitors and DGKα inhibitors as drugs for the prevention or treatment of follicular lymphoma. The study found that combining VEGFR2 inhibitors with DGKα inhibitors not only reduces the dosage of either inhibitor and improves safety, but also significantly inhibits follicular lymphoma more effectively than using either inhibitor alone, achieving a synergistic effect. This invention first demonstrates, using follicular lymphoma cell lines, that it can significantly inhibit follicular lymphoma cell proliferation and induce apoptosis; then, Western blot experiments demonstrate that it can inhibit the expression of MAPK pathway-related proteins in follicular lymphoma cells. This invention provides an effective drug combination strategy for the improvement or treatment of follicular lymphoma, which is of great significance. Attached Figure Description

[0033] Figure 1AThis is a graph showing the cell proliferation inhibition rate of DOHH2 cells after 24 hours of treatment with Cioroni combined with Litanserin;

[0034] Figure 1B This is a graph showing the cell proliferation inhibition rate of SU-DHL-4 cells after 24 hours of treatment with Cioronide combined with Litanserin.

[0035] Figure 1C This is a graph showing the cell proliferation inhibition rate of Karpas422 cells after 24 hours of treatment with a combination of cioronide and litanserin.

[0036] Figure 2A This is a flow cytometry result of DOHH2 cells treated with cioronide and litanserin for 48 hours to detect apoptosis levels.

[0037] Figure 2B This is a graph showing the statistical results of apoptosis rate in DOHH2 cells;

[0038] Figure 3A This is a flow cytometry result of SU-DHL-4 cells treated with Cioronide and Litanserin for 48 hours to detect apoptosis levels.

[0039] Figure 3B This is a graph showing the statistical results of apoptosis rate in SU-DHL-4 cells;

[0040] Figure 4A This is a flow cytometry result of apoptosis level in Karpas422 cells treated with Cioronide and Litanserin for 48 hours.

[0041] Figure 4B This is a graph showing the statistical results of apoptosis rate in Karpas422 cells;

[0042] Figure 5A This is a flow cytometry result of EDU staining of DOHH2 cells after 24 hours of treatment with cioronide and litanserin.

[0043] Figure 5B This is a statistical chart of EDU staining results of DOHH2 cells after 24 hours of treatment with cioroni and litanserin.

[0044] Figure 6A This is a flow cytometry result of EDU staining of SU-DHL-4 cells after 24 hours of treatment with cioroni and litanserin.

[0045] Figure 6B This is a statistical chart of EDU staining results of SU-DHL-4 cells after 24 hours of treatment with Cioronide and Litanserin.

[0046] Figure 7A This is a flow cytometry result of EDU staining of Karpas422 cells after 24 hours of treatment with cioroni and litanserin.

[0047] Figure 7B This is a statistical chart of EDU staining results of Karpas422 cells after 24 hours of treatment with Cioronide and Litanserin.

[0048] Figure 8 This is a Western blot result of the combined use of cioroni and litanserin to detect the expression of MAPK pathway anti-apoptosis-related proteins in FL cell lines.

[0049] Figure 9A This is a comparison of body weight in a DOHH2-CDX mouse model after treatment with a combination of cioroni and litanserin.

[0050] Figure 9B This is a graph showing the subcutaneous tumor volume of a CDX mouse model after treatment with a combination of cioroni and litanserin.

[0051] Figure 9C This is a graph showing the subcutaneous tumor weight of a CDX mouse model after treatment with a combination of cioroni and litanserin.

[0052] Figure 9D This is an anatomical view of the tumor in a CDX mouse model after treatment with cioroni. Detailed Implementation

[0053] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0054] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0055] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0056] The drug Chiauranib (CS2164) used in the following examples was provided by Shenzhen Chipscreen Biosciences Co., Ltd.; the drug Ritanserin was provided by Taoshu Biotechnology Co., Ltd.

[0057] FL cell lines (including DOHH2 cells, SU-DHL-4 cells, and Karpas422 cells) were provided by the Institute of Hematology, School of Medicine, Xiamen University.

[0058] Example 1

[0059] The CCK8 assay was used to verify the inhibitory effect of the combined drug combination on the proliferation of FL cell lines.

[0060] The operation method is as follows: Take a quantity of 2 × 10 4 Logarithmic growth phase FL cell line (DOHH2 cell line) was seeded in 96-well plates and set up cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group;

[0061] The concentrations of cioronide monotherapy in the experimental groups were 2 μM, 4 μM, 6 μM, and 8 μM, respectively. The concentrations of ritanserin monotherapy in the experimental groups were 10 μM, 12 μM, 14 μM, and 16 μM, respectively. The concentrations of cioronide and ritanserin combination therapy were 2 μM+10 μM, 4 μM+12 μM, 6 μM+14 μM, and 8 μM+16 μM, respectively (the former being cioronide and the latter ritanserin). The control group cells were treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the 96-well cell culture plates, the cells were cultured in a cell culture incubator (Thermo) for 24 h and the cell proliferation level was detected using a CCK8 assay kit (MCE, Shanghai).

[0062] The cell proliferation levels of each group are as follows: Figure 1A As shown;

[0063] pass Figure 1A The results show that, compared with high-concentration (8 μM) cioronide monotherapy or high-concentration (16 μM) ritanserin monotherapy, the combination of cioronide and ritanserin (4 μM + 12 μM) can reduce the dosage of both drugs while improving the inhibition level, thus ensuring both low drug toxicity and excellent antifollicular lymphoma treatment efficacy.

[0064] Example 2

[0065] The CCK8 assay was used to verify the inhibitory effect of the combined drug combination on the proliferation of FL cell lines.

[0066] The operation method is as follows: Take a quantity of 2 × 10 4 Logarithmic growth phase FL cell line (SU-DHL-4 cell line) was seeded in 96-well plates and set up cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group;

[0067] The concentrations of cioronide monotherapy in the experimental groups were 5 μM, 10 μM, 15 μM, and 20 μM, respectively. The concentrations of ritanserin monotherapy in the experimental groups were 10 μM, 15 μM, 20 μM, and 25 μM, respectively. The concentrations of cioronide and ritanserin combination therapy were 5 μM+10 μM, 10 μM+15 μM, 15 μM+20 μM, and 20 μM+25 μM, respectively (the former being cioronide and the latter ritanserin). The control group cells were treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the 96-well cell culture plates, the cells were cultured in a cell culture incubator (Thermo) for 24 h and the cell proliferation level was detected using a CCK8 assay kit (MCE, Shanghai).

[0068] The cell proliferation levels of each group are as follows: Figure 1B As shown;

[0069] pass Figure 1B The results show that, compared with high-concentration (20 μM) cioronide monotherapy or high-concentration (25 μM) ritanserin monotherapy, the combination of cioronide and ritanserin (15 μM + 20 μM) can reduce the dosage of both drugs while improving the inhibition level, thus ensuring both low drug toxicity and excellent antifollicular lymphoma treatment efficacy.

[0070] Example 3

[0071] The CCK8 assay was used to verify the inhibitory effect of the combined drug combination on the proliferation of FL cell lines.

[0072] The operation method is as follows: Take a quantity of 2 × 10 4 Logarithmic growth phase FL cell line (Karpas422 cell line) was seeded in 96-well plates and set up cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group;

[0073] The concentrations of cioronide monotherapy in the experimental groups were 4 μM, 12 μM, 16 μM, and 20 μM, respectively. The concentrations of ritanserin monotherapy in the experimental groups were 10 μM, 16 μM, 20 μM, and 24 μM, respectively. The concentrations of cioronide and ritanserin combination therapy were 4 μM+10 μM, 12 μM+16 μM, 16 μM+20 μM, and 20 μM+24 μM, respectively (the former being cioronide and the latter ritanserin). The control group cells were treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the 96-well cell culture plates, the cells were cultured in a cell culture incubator (Thermo) for 24 h and the cell proliferation level was detected using a CCK8 assay kit (MCE, Shanghai).

[0074] The cell proliferation levels of each group are as follows: Figure 1C As shown;

[0075] pass Figure 1C The results show that, compared with high-concentration (20 μM) cioronide monotherapy or high-concentration (24 μM) ritanserin monotherapy, the combination of cioronide and ritanserin (16 μM + 20 μM) can reduce the dosage of both drugs while improving the inhibition level, thus ensuring both low drug toxicity and excellent therapeutic effect against follicular lymphoma.

[0076] Example 4

[0077] Apoptosis-inducing effect of combined drug combination on FL cell line

[0078] The operation method is as follows: Take a quantity of 2 × 10 5 Logarithmic growth phase FL cell line (DOHH2 cell line) was seeded in 24-well plates and set up cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group;

[0079] The concentrations of cioronide monotherapy in the experimental groups were 6 μM and 8 μM, the concentrations of ritanserin monotherapy in the experimental groups were 8 μM and 12 μM, and the concentrations of cioronide and ritanserin combination groups were 6 μM+8 μM and 8 μM+12 μM, respectively (the former being cioronide and the latter ritanserin). The control group cells were treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the 24-well cell culture plates, the cells were cultured in a cell culture incubator for 48 h. The cells were then collected by centrifugation at 300 g for 5 min at 4 °C, washed once with PBS, and the apoptosis level and apoptosis rate were detected by Annexin V / PI (Thermofisher, USA) flow cytometry.

[0080] Figure 2A , Figure 2B The figures show the flow cytometry results of apoptosis levels and the statistical results of apoptosis rate in DOHH2 cells after 48 hours of treatment with cioronide combined with litanserin.

[0081] The results shown in the figure above indicate that, compared to high-concentration (8 μM) Cioronide monotherapy or high-concentration (12 μM) Ritanserine monotherapy, the combination of Cioronide and Ritanserine (6 μM + 8 μM) can reduce the dosage of both drugs while increasing the level of apoptosis promotion, thus simultaneously ensuring low drug toxicity and excellent therapeutic effects against follicular lymphoma. The combination indices for Cioronide and Ritanserine were calculated to be CI = 0.636 (6 μM + 8 μM) and CI = 0.386 (8 μM + 12 μM), respectively.

[0082] Example 5

[0083] Apoptosis-inducing effect of combined drug combination on FL cell line

[0084] The operation method is as follows: Take a quantity of 2 × 10 5 Logarithmic growth phase FL cell line (SU-DHL-4 cell line) was seeded in 24-well plates and set up cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group;

[0085] The concentrations of cioronide monotherapy in the experimental group cells were 12 μM and 16 μM, respectively; the concentrations of ritanserin monotherapy in the experimental group cells were 16 μM and 20 μM, respectively; and the concentrations of cioronide and ritanserin combination group cells were 12 μM+16 μM and 16 μM+20 μM, respectively (the former being cioronide and the latter ritanserin). The control group cells were treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the above 24-well cell culture plates, the cells were cultured in a cell culture incubator for 48 h. The cells were then collected by centrifugation at 300 g for 5 min at 4 °C, washed once with PBS, and the apoptosis level and apoptosis rate were detected by Annexin V / PI (Thermofisher, USA) flow cytometry.

[0086] Figure 3A , Figure 3B The figures show the flow cytometry results of apoptosis levels and the statistical results of apoptosis rate in SU-DHL-4 cells after 48 hours of treatment with cioronide combined with litanserin.

[0087] The results shown in the figure above indicate that, compared to high-concentration (16 μM) Cioronide monotherapy or high-concentration (20 μM) Ritanserine monotherapy, the combination of Cioronide and Ritanserine (12 μM + 16 μM) can reduce the dosage of both drugs while increasing the level of apoptosis promotion, thus simultaneously ensuring low drug toxicity and excellent therapeutic effects against follicular lymphoma. The combination indices for Cioronide and Ritanserine were calculated to be CI = 0.994 (12 μM + 16 μM) and CI = 0.585 (16 μM + 20 μM), respectively.

[0088] Example 6

[0089] Apoptosis-inducing effect of combined drug combination on FL cell line

[0090] The operation method is as follows: Take a quantity of 2 × 10 5 Logarithmic growth phase FL cell line (Karpas422 cell line) was seeded in 24-well plates and set up cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group;

[0091] The concentrations of cioronide monotherapy in the experimental group cells were 12 μM and 16 μM, respectively; the concentrations of ritanserin monotherapy in the experimental group cells were 16 μM and 20 μM, respectively; and the concentrations of cioronide and ritanserin combination group cells were 12 μM+16 μM and 16 μM+20 μM, respectively (the former being cioronide and the latter ritanserin). The control group cells were treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the above 24-well cell culture plates, the cells were cultured in a cell culture incubator for 48 h. The cells were then collected by centrifugation at 300 g for 5 min at 4 °C, washed once with PBS, and the apoptosis level and apoptosis rate were detected by Annexin V / PI (Thermofisher, USA) flow cytometry.

[0092] Figure 4A , Figure 4B The figures show the flow cytometry results of apoptosis levels and the statistical results of apoptosis rate in Karpas422 cells after 48 hours of treatment with cioronide combined with litanserin.

[0093] The results shown in the figure above indicate that, compared to high-concentration (16 μM) Cioronide monotherapy or high-concentration (20 μM) Ritanserine monotherapy, the combination of Cioronide and Ritanserine (12 μM + 16 μM) can reduce the dosage of both drugs while increasing the level of apoptosis promotion, thus ensuring both low drug toxicity and excellent therapeutic effect against follicular lymphoma. The combination indices for Cioronide and Ritanserine were calculated as CI = 0.424 (12 μM + 16 μM) and CI = 0.508 (16 μM + 20 μM), respectively.

[0094] Example 7

[0095] EDU staining was used to verify the inhibitory effect of the combined drug combination on the proliferation of FL cell lines.

[0096] The operation method is as follows: Take a quantity of 2 × 10 6 Logarithmic growth phase FL cell lines (including DOHH2, SU-DHL-4 and Karpas422 cell lines) were seeded in 6-well plates, and cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group were set up;

[0097] In DOHH2 cells, the concentration of cioronide monotherapy in the experimental group was 6 μM, the concentration of ritanserin monotherapy was 15 μM, and the concentration of cioronide and ritanserin combination group was 6 μM + 15 μM (the former being cioronide and the latter being ritanserin).

[0098] In SU-DHL-4 cells, the concentration of cioronide monotherapy in the experimental group was 12 μM, the concentration of ritanserin monotherapy was 16 μM, and the concentration of cioronide and ritanserin combination group was 12 μM + 16 μM (the former being cioronide and the latter being ritanserin).

[0099] In Karpas422 cells, the concentration of cioronide monotherapy in the experimental group was 20 μM, the concentration of ritanserin monotherapy was 24 μM, and the concentration of cioronide and ritanserin combination group was 20 μM + 24 μM (the former being cioronide and the latter being ritanserin).

[0100] The control group was treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the above 6-well cell culture plates, the cells were cultured in an incubator for 24 h, incubated with 10 μM EdU for 2 h, centrifuged at 300 g for 5 min at 4 °C to collect the cells, washed once with PBS, and then the cell proliferation level was detected by EDU flow cytometry and the cell proliferation rate was calculated.

[0101] Figure 5A and Figure 5B The figures show the cell proliferation and statistical results of DOHH2 cells after 24 hours of treatment with cioronide and litanserin, respectively. Figure 6A and Figure 6B The figures show the cell proliferation and statistical results of SU-DHL-4 cells after 24 hours of treatment with Cioronide combined with Litanserin. Figure 7A and Figure 7B The figures show the cell proliferation and statistical results of Karpas422 cells after 24 hours of treatment with cioronide combined with litanserin.

[0102] As shown in the figure above, compared with cioronide and ritanserin monotherapy, the combination of cioronide and ritanserin can significantly inhibit cell proliferation, thus ensuring excellent antifollicular lymphoma treatment efficacy.

[0103] Example 8

[0104] Inhibitory effect of combined drug combination on MAPK pathway expression

[0105] The operation method is as follows: Take a quantity of 1×10 6 Logarithmic growth phase FL cell lines (DOHH2, SU-DHL-4 cell lines) were seeded in 12-well plates, and cioronide monotherapy group, ritanserin monotherapy group, cioronide and ritanserin combination group, and control group were set up;

[0106] In DOHH2 cells, the concentration of cioronide monotherapy in the experimental group was 6 μM, the concentration of ritanserin monotherapy was 15 μM, and the concentration of cioronide and ritanserin combination group was 6 μM + 15 μM (the former being cioronide and the latter being ritanserin).

[0107] In SU-DHL-4 cells, the concentration of cioronide monotherapy in the experimental group was 16 μM, the concentration of ritanserin monotherapy was 20 μM, and the concentration of cioronide and ritanserin combination group was 16 μM + 20 μM (the former being cioronide and the latter being ritanserin).

[0108] Control group cells were treated with the same volume of DMSO. After gently shaking and mixing the corresponding volume of drug or DMSO with the cells in the 12-well cell culture plate, the cells were cultured in a cell culture incubator for 24 h. Cells were then collected by centrifugation at 300g for 5 min at 4°C, washed once with PBS, and lysed with 200 μL RIPA lysis buffer (Thermo, USA) on ice for 1 h. Total protein was extracted and subjected to Western blot to detect the protein expression levels of drug targets, downstream related proteins, and apoptosis-related proteins.

[0109] Figure 8 The results of Western blot analysis showed that the combination of cioronide and ritanserin significantly inhibited the expression of anti-apoptotic proteins in the MAPK pathway in FL cell lines compared with either cioronide or ritanserin alone.

[0110] Example 9

[0111] Effects of combined drug combinations on the tumorigenesis of FL

[0112] The specific operating method is as follows:

[0113] (1) A control group, a cioronide monotherapy group, a ritanserin monotherapy group, and a cioronide and ritanserin combination group were set up. Cioronide was used as a suspension prepared with 0.1% sodium carboxymethyl cellulose solution, and ritanserin was used as a suspension prepared with a mixed solution (formulation of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% physiological saline).

[0114] (2) Constructing a CDX mouse model

[0115] Take 1×10 7 Logarithmic growth phase DOHH2 cells were injected subcutaneously into CB17-SCID mice to induce tumor formation, thus establishing a CDX mouse model.

[0116] (3) Seven days after injection, the drug was administered at a dose of 5 mg / kg / day for cisplatin and 2 mg / kg / day for 14 consecutive days. The tumor progression in mice was detected by measuring the size of the subcutaneous tumor. The experimental results and mouse survival rate were statistically analyzed with the start of the drug administration diary as day 0. CB17-SCID mice were purchased from the Experimental Animal Center of Xiamen University and were raised by the Experimental Animal Center.

[0117] The changes in body weight of mice in each group are as follows: Figure 9A As shown, the changes in tumor volume are as follows: Figure 9B As shown, the changes in tumor weight are as follows: Figure 9C As shown, the anatomical appearance of the tumor is as follows Figure 9D As shown, this indicates that cioroni combined with ritanserin inhibits the tumorigenesis process in the CDX model.

[0118] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0119] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0120] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A combination pharmaceutical composition for the prevention or treatment of follicular lymphoma, characterized in that, The combined drug composition is a combination of a VEGFR2 inhibitor and a DGKα inhibitor; The VEGFR2 inhibitor is cioronide or a pharmaceutically acceptable salt thereof; The DGKα inhibitor is ritanserin or a pharmaceutically acceptable salt thereof.

2. The combined pharmaceutical composition according to claim 1, characterized in that, The combined pharmaceutical composition also includes pharmaceutically acceptable excipients.

3. The combined pharmaceutical composition according to claim 2, characterized in that, The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

4. The combined pharmaceutical composition according to claim 1, characterized in that, The combined drug composition is a single compound preparation or a combination of two separate preparations.

5. The combined pharmaceutical composition according to claim 1, characterized in that, The combined drug composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

6. The combined pharmaceutical composition according to claim 4 or 5, characterized in that, The formulation can be any pharmaceutically acceptable dosage form.

7. Use of the combination pharmaceutical composition according to any one of claims 1-6 in the preparation of a medicament for the prevention, improvement or treatment of follicular lymphoma.

8. The use of the combination pharmaceutical composition according to any one of claims 1-6 in the preparation of a proliferation inhibitor of follicular lymphoma cells.

9. The use of the combined pharmaceutical composition according to any one of claims 1-6 in the preparation of an apoptosis promoter for follicular lymphoma cells.

10. The application according to claim 8 or 9, characterized in that, The follicular lymphoma cells include any one or a combination of at least two of DOHH2 cells, SU-DHL-4 cells, or Karpas422 cells.

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