Pharmaceutical combination product for treating invasive B-cell lymphoma and treatment method thereof
By combining PRMT5 inhibitors and BTK inhibitors, synergistically act on invasive B-cell lymphoma, the problem of resistance to BTK inhibitors in the disease was solved, significantly inhibiting lymphoma cell growth and apoptosis, and improving the therapeutic effect.
Patent Information
- Application Number
- CN202411416976.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2024-12-27
AI Technical Summary
Problems with resistance to Bruton tyrosine protein kinase (BTK) inhibitors, resulting in limited efficacy, and protein arginine methyltransferase 5 (PRMT5) in drug-resistant lymphoma Overexpression affects prognosis.
A combination product is provided that includes a combination of PRMT5 inhibitors and BTK inhibitors that bind to BTK inhibitors to synergistically treat aggressive B-cell lymphomas by detecting PRMT5 expression and inhibiting its growth and proliferation.
Significantly inhibit the growth and apoptosis of invasive B-cell lymphoma cells, prolong patient survival, and improve therapeutic effects, especially in drug resistance.
Smart Images
Figure CN119185559A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a combination product for treating aggressive B-cell lymphoma and a treatment method thereof, and in particular to a combination product of a protein arginine methyltransferase 5 (PRMT5) inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor and a treatment method thereof, belonging to the field of medical technology. Background Art
[0002] Aggressive B-cell lymphoma is a highly malignant lymphoma with a poor prognosis, accounting for about 60% of non-Hodgkin's lymphoma. Although Bruton's tyrosine kinase (BTK) inhibitors have achieved good results as the main treatment for aggressive B-cell lymphoma, aggressive B-cell lymphoma in the relapsed and refractory stage is prone to multidrug resistance and has a very short overall survival, which has attracted public attention.
[0003] Bruton's tyrosine kinase (BTK) is a cytoplasmic kinase encoded by the BTK gene. It is widely expressed in all hematopoietic cells, especially B lymphocytes and terminally differentiated plasma cells. In the normal development of B lymphocytes, the correct expression of different protein regions of the BTK gene plays a key role in the function of B cells and multiple transduction pathways. Ibrutinib is the first BTK inhibitor approved by the FDA for the treatment of relapsed and refractory mantle cell lymphoma and other B cell lymphomas. However, although ibrutinib has promoted the treatment of aggressive B cell lymphoma, one-third of patients still develop primary resistance, and more patients will gradually develop acquired resistance, which limits the efficacy of BTK inhibitors in B cell lymphoma, especially aggressive B cell lymphoma, and has become a clinical problem that needs to be solved urgently.
[0004] Protein arginine methyltransferase 5 (PRMT5) is highly expressed in lymphoma tissues and can promote the growth and proliferation of lymphoma cells. Studies have shown that PRMT5 is overexpressed in lymphoma tissues that are relapsed and resistant to BTK inhibitors, and these patients have a poor prognosis. In addition, knocking out or inhibiting PRMT5 can significantly inhibit the growth of malignant lymphoma cells. Summary of the invention
[0005] The present invention aims to provide a combination product for treating aggressive B-cell lymphoma and a method for treating the same, particularly a combination product of a protein arginine methyltransferase 5 (PRMT5) inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor.
[0006] In a first aspect of the present invention, a combination product is provided comprising a PRMT5 inhibitor and a BTK inhibitor, wherein the PRMT5 inhibitor is protein arginine methyltransferase 5 and the BTK inhibitor is Bruton's tyrosine kinase.
[0007] In a preferred embodiment, the combination product is in the form of a pharmaceutical composition.
[0008] In another preferred embodiment, the PRMT5 inhibitor and the BTK inhibitor are each in the form of a separate preparation.
[0009] In another preferred embodiment, the PRMT5 inhibitor and the BTK inhibitor are administered simultaneously or sequentially.
[0010] In another aspect of the present invention, the combination product further comprises a pharmaceutically acceptable carrier, diluent or excipient.
[0011] In a preferred embodiment, the combination product is in the form of tablets, capsules, granules, syrups, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, aerosols, ointments, creams or injections.
[0012] In another aspect of the present invention, there is provided a method for treating aggressive B-cell lymphoma using the above combination product, comprising administering the combination product to a patient with aggressive B-cell lymphoma, thereby treating the aggressive B-cell lymphoma.
[0013] In another preferred embodiment, the method for treating aggressive B-cell lymphoma specifically comprises the following steps: A). First, Western blotting was used to detect the expression of PRMT5 in different aggressive B-cell lymphoma cell lines; B). Then the cell viability luminescence method was used to detect the growth and proliferation of different aggressive B-cell lymphoma cell lines by inhibiting PRMT5; C). Flow cytometry was then used to examine the effect of PRMT5 inhibition on inducing apoptosis in aggressive B-cell lymphoma cells. D). Finally, the cell viability luminescence method was used to detect the growth and proliferation of different aggressive B-cell lymphoma cell lines after the combined use of PRMT5 inhibitors and BTK inhibitors.
[0014] The present invention provides a method for treating aggressive B-cell lymphoma by providing a combination product of a PRMT5 inhibitor and a BTK inhibitor, the method comprising administering the combination product of the present invention to an aggressive B-cell lymphoma patient, thereby treating the aggressive B-cell lymphoma. The present invention also provides the combination product for treating aggressive B-cell lymphoma. Specifically, the combination product of the present invention may have a synergistic effect when used to treat an aggressive B-cell lymphoma patient. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The expression of protein arginine methyltransferase 5 in different malignant lymphoma cell lines.
[0016] Figure 2 To inhibit protein arginine methyltransferase 5 in the growth and proliferation changes of aggressive B-cell lymphoma.
[0017] Figure 3 To investigate the changes in apoptosis by inhibiting protein arginine methyltransferase 5 in aggressive B-cell lymphoma.
[0018] Figure 4 Figure 2 shows changes in growth and proliferation of aggressive B-cell lymphoma cells treated as single agents and in combination. DETAILED DESCRIPTION
[0019] After extensive and in-depth research, the present inventors provide a combination product, wherein the combination product comprises a PRMT5 inhibitor and a BTK inhibitor, wherein the PRMT5 inhibitor is protein arginine methyltransferase 5, and the BTK inhibitor is Bruton's tyrosine kinase, wherein the combination product is in the form of a pharmaceutical composition.
[0020] In another embodiment, the PRMT5 inhibitor and the BTK inhibitor are each in the form of separate formulations.
[0021] In another embodiment, the PRMT5 inhibitor and the BTK inhibitor are administered simultaneously or sequentially.
[0022] Wherein, the combination product further comprises a pharmaceutically acceptable carrier, diluent or excipient.
[0023] Wherein, the combination product is in the form of tablets, capsules, granules, syrups, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, aerosols, ointments, creams or injections.
[0024] The method of using the above combination product to treat aggressive B-cell lymphoma comprises administering the combination product to a patient with aggressive B-cell lymphoma, thereby treating the aggressive B-cell lymphoma.
[0025] Treatment for aggressive B-cell lymphoma includes the following steps: A). Western blotting was used to detect the expression of PRMT5 in different aggressive B-cell lymphoma cell lines; The method includes: using rabbit anti-PRMT5 antibody to perform Western blotting to detect the expression level of PRMT5 protein, and using GAPDH as an internal reference and rabbit anti-GAPDH as a primary antibody for detection.
[0026] Experimental results: as attached Figure 1 As shown, at the protein level, PRMT5 is highly expressed in aggressive B-cell lymphoma cell lines.
[0027] B). The cell viability luminescence method was used to detect the growth and proliferation of different aggressive B-cell lymphoma cell lines by inhibiting PRMT5. The specific steps included: taking normal aggressive B-cell lymphoma cell lines Granta519, JVM-2, Z138, Maver-1, JeKo-1 and Rec-1 in the logarithmic growth phase and 2 x 10 5 Cells were inoculated at a density of 100 μL / well in a 96-well plate, and each cell line was equipped with 2 replica wells to detect changes in cell growth and proliferation in aggressive B-cell lymphoma cell lines after the addition of PRMT5 inhibitors GSK3326595 or LLY-283. The highest concentration was 20 μM, and 7 concentrations were diluted in 2-fold ratios in sequence, and cultured for 72 hours. After 72 hours of culture, 100 μL of CellTiter-Glo reagent was added to every 100 μL of cell suspension, and the mixture was gently shaken on an orbital shaker for 2 minutes, incubated at room temperature for 10 minutes, and the luminescent signal was detected.
[0028] Experimental results: as attached Figure 2 As shown, the horizontal axis represents the drug concentration; the vertical axis represents the cell viability of lymphoma cells, Figure 2 a represents the addition of PRMT5 inhibitor GSK3326595 to aggressive B-cell lymphoma cell lines, Figure 2 b indicates that LLY-283 was added to the aggressive B-cell lymphoma cell line, and both were able to inhibit the growth and proliferation of lymphoma cells. Figure 2 a shows that in the aggressive B-cell lymphoma cell lines Granta519, JVM2, Z138, Maver-1, JeKo-1 and Rec-1, the addition of PRMT5 inhibitor GSK3326595 significantly inhibited the growth and proliferation of lymphoma cells; Figure 2 b: In the aggressive B-cell lymphoma cell lines Granta519, JVM2, Z138, Maver-1, JeKo-1 and Rec-1, the addition of PRMT5 inhibitor LLY-283 significantly inhibited the growth and proliferation of lymphoma cells.
[0029] The data processing includes: using GraphPadPrism8.0 software to record data for statistical analysis and drawing; The CellTiter-Glo cell viability assay kit was purchased from Promega.
[0030] C). Flow cytometry was used to detect the effect of PRMT5 inhibition on inducing apoptosis in aggressive B-cell lymphoma cells. Specific methods: Normal aggressive B-cell lymphoma cell lines Z138, Granta519, JVM-2 and Maver-1 in the logarithmic growth phase were taken and cultured at 5 x 10 5 Cells were seeded at a density of 100 μL / well in a 24-well plate, and 1 μM PRMT5 inhibitor GSK3326595 was added respectively, and culture was continued for 5 days. After 5 days of culture, the cell suspension was collected, centrifuged and washed twice with pre-cooled PBS. After centrifugation, the cells were resuspended with 100 μL of 1x Annexin-V, and then 2 μL of PI was added to each 100 μL of cell suspension and gently mixed. Incubate at room temperature in the dark for 15 minutes. Detect on a flow cytometer within 30 minutes.
[0031] Data processing: FlowJo was used to analyze and plot the flow cytometry data of cell apoptosis; GraphPad Prism 8.0 software was used to record the data for statistical analysis and drawing.
[0032] Experimental results: as attached Figure 3 As shown, the horizontal axis represents the untreated group and the group treated with PRMT5 inhibitor GSK3326595; the vertical axis represents the apoptosis of lymphoma cells. Figure 3 a indicates that in the aggressive B-cell lymphoma cell line Z138, the apoptosis of lymphoma cells was significantly increased after treatment with GSK3326595 compared with the untreated group; Figure 3 b indicates that in the aggressive B-cell lymphoma cell line Granta519, the apoptosis of lymphoma cells was significantly increased after treatment with GSK3326595 compared with the untreated group; Figure 3 c indicates that in the aggressive B-cell lymphoma cell line JVM2, the apoptosis of lymphoma cells was significantly increased after treatment with GSK3326595 compared with the untreated group; Figure 3 d shows that in the aggressive B-cell lymphoma cell line Maver-1, the apoptosis of lymphoma cells was significantly increased after treatment with GSK3326595 compared with the untreated group.
[0033] Experimental results: After inhibiting PRMT5 in the normal invasive B-cell lymphoma cell lines Z138, Granta519, JVM-2 and Maver-1 in the growth period, the cell apoptosis rate was significantly increased compared with normal lymphoma cells, and the difference was statistically significant.
[0034] Annexin V was purchased from BD Biosciences; Propidium iodide (PI) was purchased from Invitrogen.
[0035] D). The cell viability luminescence method was used to detect the growth and proliferation of different aggressive B-cell lymphoma cell lines treated with the combined use of PRMT5 inhibitors and BTK inhibitors. The specific method included taking normal aggressive B-cell lymphoma cell lines Granta519, JVM-2, Z138 and JeKo-1 in the logarithmic growth phase and quantifying them at 2 x 10 5 Cells were seeded at a density of 100 μl / well in a 96-well plate, and each cell line was equipped with 2 replica wells to detect changes in cell growth and proliferation in aggressive B-cell lymphoma cell lines after the addition of single PRMT5 inhibitor GSK3326595 (GSK), single BTK inhibitor Pirtobrutinib (PBN), and combined PRMT5 inhibitor GSK3326595 and BTK inhibitor Pirtobrutinib, and cultured for 72 hours. After 72 hours of culture, 100 μl CellTiter-Glo reagent was added to each 100 μl of cell suspension, gently shaken on an orbital shaker for 2 minutes, incubated at room temperature for 10 minutes, and the luminescent signal was detected.
[0036] As attached Figure 4 As shown in the figure, the horizontal axis represents the untreated group, the single application of PRMT5 inhibitor GSK3326595 (GSK), the single application of BTK inhibitor Pirtobrutinib (PBN), and the combination of GSK and PBN treatment (Combo); the vertical axis represents the viability of lymphoma cells. Among them, Figure 4 a shows that in the aggressive B-cell lymphoma cell line Z138, compared with the untreated group, the lymphoma cell viability decreased after single application of GSK or single application of PBN treatment; the decrease in lymphoma cell viability after combined GSK and PBN treatment was more obvious than that in the single drug group. Figure 4 b: In the aggressive B-cell lymphoma cell line Granta519, compared with the untreated group, the lymphoma cell viability decreased after single application of GSK or single application of PBN treatment; the decrease in lymphoma cell viability after combined treatment with GSK and PBN was more obvious than that in the single-drug group. Figure 4 c: In the aggressive B-cell lymphoma cell line JVM2, compared with the untreated group, the lymphoma cell viability decreased after single application of GSK or single application of PBN treatment; the decrease in lymphoma cell viability after combined GSK and PBN treatment was more obvious than that in the single drug group. Figure 4d: In the aggressive B-cell lymphoma cell line JeKo-1, compared with the untreated group, the lymphoma cell viability decreased after single application of GSK or single application of PBN treatment; the decrease in lymphoma cell viability after combined GSK and PBN treatment was more obvious than that in the single drug group.
[0037] Experimental results: The combined use of PRMT5 inhibitor GSK3326595 (GSK) and BTK inhibitor Pirtobrutinib (PBN) in aggressive B-cell lymphoma cell lines can synergistically inhibit the growth and proliferation of lymphoma cells.
[0038] Data processing: GraphPad Prism 8.0 software was used to record data for statistical analysis and drawing. CellTiter-Glo cell viability detection kit was purchased from Promega.
[0039] The method combines a PRMT5 inhibitor and a BTK inhibitor and applies the experimental method to prove that knocking out or inhibiting PRMT5 can significantly inhibit the growth of malignant lymphoma cells. The method also uses the following experimental methods and experimental results: detecting the expression of PRMT5 in different aggressive B-cell lymphoma cell lines by using a Western blotting method, detecting the growth and proliferation of PRMT5 inhibition in different aggressive B-cell lymphoma cell lines by using a cell viability luminescence method, detecting the effect of inducing apoptosis of aggressive B-cell lymphoma cells after inhibiting PRMT5 by using a flow cytometry method, and detecting the growth and proliferation of the combined use of a PRMT5 inhibitor and a BTK inhibitor in different aggressive B-cell lymphoma cell lines by using a cell viability luminescence method. It is concluded that the combined use of a PRMT5 inhibitor GSK3326595 and a BTK inhibitor Pirtobrutinib in aggressive B-cell lymphoma cell lines can synergistically inhibit the growth and proliferation of lymphoma cells, so that the combination product of the present invention can be used to treat aggressive B-cell lymphoma and has a synergistic effect when used to treat aggressive B-cell lymphoma patients.
Claims
1. A combination product for the treatment of aggressive B-cell lymphoma, characterized in that: The combination product comprises a PRMT5 inhibitor and a BTK inhibitor, wherein the PRMT5 inhibitor is protein arginine methyltransferase 5 and the BTK inhibitor is Bruton's tyrosine kinase.
2. The combination product according to claim 1, characterized in that: The combination product is in the form of a pharmaceutical composition.
3. The combination product according to claim 1, characterized in that: The PRMT5 inhibitor and the BTK inhibitor are each in the form of separate preparations.
4. The combination product according to claim 1, characterized in that: The PRMT5 inhibitor and the BTK inhibitor are administered simultaneously or sequentially.
5. The combination product according to claim 1, characterized in that: The combination product further comprises a pharmaceutically acceptable carrier, diluent or excipient.
6. The combination product according to any one of claims 1 to 5, characterized in that: The combination product is in the form of tablets, capsules, granules, syrups, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, aerosols, ointments, creams or injections.
7. A method for treating aggressive B-cell lymphoma using the combination product according to any one of claims 1 to 6, characterized in that: Includes using the combination product in patients with aggressive B-cell lymphoma to treat aggressive B-cell lymphoma.
8. The method for treating aggressive B-cell lymphoma according to claim 7, characterized in that: The specific steps include: A). Western blotting was used to detect the expression of PRMT5 in different aggressive B-cell lymphoma cell lines; B). Cell viability luminescence assay was used to detect the growth and proliferation of different aggressive B-cell lymphoma cell lines by inhibiting PRMT5; C). Flow cytometry was used to detect the effect of PRMT5 inhibition on inducing apoptosis in aggressive B-cell lymphoma cells; D). Cell viability luminescence assay was used to detect the growth and proliferation of different aggressive B-cell lymphoma cell lines treated with a combination of PRMT5 inhibitors and BTK inhibitors.