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

Abstract

The present application relates to a kind of combined drug composition for improving or treating follicular lymphoma and its application, the active component of the combined drug composition is composed of first active component and second active component;The first active component is selected from any one or combination of at least two of ritanserin or its pharmaceutically acceptable salt, isomer, solvate, metabolite;The second active component is selected from any one or combination of at least two of donepezil or its pharmaceutically acceptable salt, isomer, solvate, metabolite.The combined drug composition has excellent improvement or treatment effect of follicular lymphoma, two active components not only can reduce the dosing of ritanserin or donepezil when inhibiting follicular lymphoma, improve drug safety, but also have more significant inhibiting effect of follicular lymphoma than using single active component, reach the effect of synergistic promotion.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a new method for improving or treating follicular lymphoma, specifically to a combination pharmaceutical composition for improving or treating 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] Ritanserine was initially discovered as a serotonin receptor antagonist and used to treat schizophrenia and depression, improve sleep quality, and alleviate alcohol addiction. Subsequent studies revealed that ritanserine is a diacylglycerol kinase-α (DGKα) inhibitor, effectively combating the growth and progression of cancer cells both in vitro and in vivo. It is a promising anti-tumor drug that can also alleviate anxiety and improve sleep to some extent. Duverixetine, introduced to China by CSPC Pharmaceutical Group, is an innovative oral dual inhibitor of PI3K-δ and PI3K-γ. Its inhibition of PI3K-δ leads to apoptosis in malignant tumor cells, while its inhibition of PI3K-γ reduces the differentiation and metastasis of supporting cells in the tumor microenvironment.

[0005] Whether the combination of ritanserin and duveritase can be used to treat follicular lymphoma, and its mechanism of action on follicular lymphoma-related cell lines, remains unclear. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a new method for improving or treating follicular lymphoma, specifically a combination pharmaceutical composition for improving or treating follicular lymphoma and its application.

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

[0008] In a first aspect, the present invention provides a combination pharmaceutical composition for improving or treating follicular lymphoma, wherein the active component of the combination pharmaceutical composition is composed of a first active component and a second active component;

[0009] The first active ingredient is selected from any one or a combination of at least two of ritanserin or its pharmaceutically acceptable salts, isomers, solvates, and metabolites;

[0010] The second active ingredient is selected from any one or a combination of at least two of duvirise or its pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0011] This invention develops a novel drug combination therapy, which combines ritanserin (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites) and duvelice (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites). This combined drug composition exhibits excellent improvement or therapeutic effects in follicular lymphoma. The invention's research found that the two active components, when inhibiting follicular lymphoma, not only reduce the dosage of ritanserin (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites) or duvelice (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites), improving drug safety, but also have a more significant inhibitory effect on follicular lymphoma than using a single active component, achieving a synergistic effect.

[0012] This invention first demonstrates, using follicular lymphoma cell lines (DOHH2 and SU-DHL-4 cells), that compared to ritanserin and duveritol monotherapy, the combined drug composition significantly inhibits follicular lymphoma cell proliferation and induces apoptosis. Western blot experiments show that the combined drug composition inhibits follicular lymphoma cell proliferation and promotes apoptosis by affecting targets related to the PI3K / AKT / mTOR pathway and the classical MAPK pathway. Furthermore, by constructing a DOHH2 tumor-bearing mouse model, it is demonstrated that the combined drug composition reduces tumor burden in DOHH2 tumor-bearing mice. This invention provides an effective drug combination strategy for the improvement or treatment of follicular lymphoma and is of significant importance.

[0013] In this invention, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0014] Preferably, the combined pharmaceutical composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

[0015] 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.

[0016] In this invention, the formulation is any pharmaceutically acceptable dosage form.

[0017] Preferably, the combined pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0018] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0019] 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 improving or treating follicular lymphoma.

[0020] This invention develops a novel approach to improve or treat follicular lymphoma by combining ritanserin (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites) and duvelice (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites). This combined drug composition exhibits excellent efficacy in improving or treating follicular lymphoma. The invention's research found that the two active components, when inhibiting follicular lymphoma, not only reduce the dosage of ritanserin (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites) or duvelice (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites), improving medication safety, but also have a more significant inhibitory effect on follicular lymphoma than using a single active component, achieving a synergistic effect.

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

[0022] 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, apoptosis, and metabolic mechanisms or behaviors of follicular lymphoma cells, and screening therapeutic drugs. 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 follicular lymphoma cell proliferation inhibitor for a non-therapeutic purpose.

[0023] Preferably, the follicular lymphoma cells include the DOHH2 cell line or the SU-DHL-4 cell line.

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

[0025] According to the research results of this invention, the combined pharmaceutical composition has a significant effect in inducing 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 studying the growth, apoptosis, and metabolic mechanisms or behaviors of follicular lymphoma cells, and screening therapeutic drugs. 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 apoptosis promoters for follicular lymphoma cells for a non-therapeutic purpose.

[0026] Preferably, the follicular lymphoma cells include the DOHH2 cell line or the SU-DHL-4 cell line.

[0027] Fifthly, the present invention provides the use of the pharmaceutical composition according to the first aspect in the preparation of an inhibitor of the MAPK / ERK signaling pathway in follicular lymphoma cells.

[0028] According to the research results of this invention, the combined drug composition has a significant inhibitory effect on the MAPK / ERK signaling pathway in follicular lymphoma cells. Therefore, this result indicates that the combined drug composition can be used as an in vitro experimental reagent in the field of scientific research, such as studying the growth, apoptosis and metabolic mechanisms or behavior of follicular lymphoma cells, and screening drugs for the treatment of follicular lymphoma.

[0029] In a sixth aspect, the present invention provides the use of the combined pharmaceutical composition according to the first aspect in the preparation of an inhibitor of the PI3K / AKT / mTOR signaling pathway in follicular lymphoma cells.

[0030] According to the research results of this invention, the combined drug composition has a significant inhibitory effect on the PI3K / AKT / mTOR signaling pathway in follicular lymphoma cells. Therefore, this result indicates that the combined drug composition can be used as an in vitro experimental reagent in scientific research, such as to study the growth, apoptosis and metabolic mechanisms or behavior of follicular lymphoma cells, and to screen drugs for the treatment of follicular lymphoma.

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

[0032] This invention develops a novel drug combination therapy, which combines ritanserin (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites) and duvelice (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites). This combined drug composition exhibits excellent improvement or therapeutic effects in follicular lymphoma. The invention's research found that the two active components, when inhibiting follicular lymphoma, not only reduce the dosage of ritanserin (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites) or duvelice (or its pharmaceutically acceptable salts, isomers, solvates, and metabolites), improving drug safety, but also have a more significant inhibitory effect on follicular lymphoma than using a single active component, achieving a synergistic effect. Attached Figure Description

[0033] Figure 1A This is a graph showing the statistical results of the cell proliferation inhibition rate of DOHH2 cells after 24 hours of drug treatment in Example 1;

[0034] Figure 1B This is a graph showing the statistical results of the cell proliferation inhibition rate of SU-DHL-4 cells after 24 hours of drug treatment in Example 1;

[0035] Figure 2A This is a graph showing the statistical results of the apoptosis rate of DOHH2 cells treated with the drug for 24 hours in Example 2;

[0036] Figure 2B This is a graph showing the statistical results of the apoptosis rate of SU-DHL-4 cells treated with the drug for 24 hours in Example 2;

[0037] Figure 3A This is a Western blot result of the expression levels of drug targets (PI3Kδ, PI3Kγ, DGAα) and apoptosis-related proteins in DOHH2 or SU-DHL-4 cells after 24 hours of drug treatment in Example 3.

[0038] Figure 3B This is a Western blot result showing the expression levels of PI3K / AKT / mTOR pathway-related proteins in DOHH2 or SU-DHL-4 cells after 24 hours of drug treatment in Example 3.

[0039] Figure 3C This is a Western blot result showing the expression levels of MAPK classical pathway-related proteins in DOHH2 or SU-DHL-4 cells after 24 hours of drug treatment in Example 3.

[0040] Figure 4A This is a graph showing the changes in body weight of a tumor-bearing mouse model after drug treatment in Example 4;

[0041] Figure 4B This is a graph showing the changes in tumor volume after drug treatment in a tumor-bearing mouse model in Example 4;

[0042] Figure 4C This is a graph showing the changes in tumor weight after drug treatment in a tumor-bearing mouse model in Example 4;

[0043] Figure 4D This is an anatomical diagram of the tumor in the tumor-bearing mouse model after drug treatment in Example 4. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The drug ritanserin used in the following examples is product HY-10791 provided by MedChemExpress (MCE), and the drug duverisol is product T1988 provided by Taoshu Biotechnology Co., Ltd.

[0048] Follicular lymphoma cell lines (DOHH2 and SU-DHL-4 cells) were provided by the Institute of Hematology, School of Medicine, Xiamen University.

[0049] CB17-SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and bred by the Laboratory Animal Center of Xiamen University.

[0050] The experimental results were statistically analyzed using GraphPad Prism 8.0. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0051] Example 1

[0052] The inhibitory effect of the combined drug combination on the proliferation of follicular lymphoma cell lines:

[0053] The operation method is as follows: take the number of items in the logarithmic growth phase as 2 × 10.4 Follicular lymphoma cell lines (DOHH2 and SU-DHL-4) were seeded in 96-well plates. A blank control group, ritanserin monotherapy group, duveritol monotherapy group, and ritanserin and duveritol combination group were set up.

[0054] The concentrations of ritanserin monotherapy were ①0.625 μM, ②1.25 μM, ③2.5 μM, ④5 μM, and ⑤10 μM; the concentrations of duverithione monotherapy were ①50 nM, ②100 nM, ③200 nM, ④400 nM, and ⑤800 nM; the concentrations of ritanserin and duverithione combination therapy were ①0.625 μM + 50 nM, ②1.25 μM + 100 nM, ③2.5 μM + 200 nM, ④5 μM + 400 nM, and ⑤10 μM + 800 nM (the former being ritanserin and the latter being duverithione); the blank control group received the same volume of DMSO. DMSO or the drug was added to the cells in the 96-well cell culture plate and gently shaken to mix. The cells were placed in a cell culture incubator (Thermo) for 24 hours, and then incubated for another 3 hours with CCK8 reagent (MCE, Shanghai) at 37 degrees Celsius to detect cell proliferation levels.

[0055] Experimental results: Cell viability results obtained after drug application are as follows Figure 1A (DOHH2) Figure 1B (SU-DHL-4) is shown. (By...) Figure 1A , Figure 1B The results showed that, in both cell lines of follicular lymphoma, the combination of ritanserin and duveritase showed better inhibitory effects on cell proliferation at lower concentrations compared to the ritanserin monotherapy group and the duveritase monotherapy group, indicating the synergistic effect of the two drugs.

[0056] Simultaneously, the combination index (CI) of ritanserin and duvirise was calculated. The results showed that, with the combination of 0.625 μM + 50 nM: CI = 0.46849 (DOHH2), CI = 0.35124 (SU-DHL-4); with the combination of 1.25 μM + 100 nM: CI = 0.40134 (DOHH2), CI = 0.29270 (SU-DHL-4); with the combination of 2.5 μM + 200 nM: CI = 0.40134 (DOHH2), CI = 0.29270 (SU-DHL-4); In the combined use: CI = 0.41444 (DOHH2), CI = 0.24433 (SU-DHL-4); in the 5μM + 400nM combined use: CI = 0.52672 (DOHH2), CI = 0.51425 (SU-DHL-4); in the 10μM + 800nM combined use: CI = 0.45490 (DOHH2), CI = 0.53212 (SU-DHL-4).

[0057] Example 2

[0058] The combined drug combination induced apoptosis in follicular lymphoma cell lines:

[0059] The operation method is as follows: take the number of items in the logarithmic growth phase as 2 × 10. 5 Follicular lymphoma cell lines (DOHH2 and SU-DHL-4) were seeded in 24-well plates. A blank control group, ritanserin monotherapy group, duveritine monotherapy group, and ritanserin and duveritine combination group were set up.

[0060] In the DOHH2 cell line, the concentrations of ritanserin monotherapy were ①10 μM and ②20 μM; the concentrations of duverithione monotherapy were ①400 nM and ②800 nM; and the concentrations of ritanserin and duverithione combination therapy were ①10 μM + 400 nM and ②20 μM + 800 nM (the former being ritanserin and the latter being duverithione). In the SU-DHL-4 cell line, the concentrations of ritanserin monotherapy were ①15 μM and ②20 μM; the concentrations of duverithione monotherapy were ①1200 nM and ②1600 nM; and the concentrations of ritanserin and duverithione combination therapy were ①15 μM + 1200 nM and ②20 μM + 1600 nM (the former being ritanserin and the latter duverithione). The blank control group cells were all treated with the same volume of DMSO. DMSO or the drug was added to the cells in a 24-well cell culture plate and gently vortexed to mix. The plate was then incubated in a Thermo cell culture incubator for 24 hours. After 24 hours of culture, the cells were centrifuged at 300g for 5 minutes, stained with Annexin V / PI (Thermofisher, USA), and the apoptosis level was detected by flow cytometry. The apoptosis rate was then statistically analyzed.

[0061] Experimental results: Statistical results of cell apoptosis rate are as follows Figure 2A (DOHH2) Figure 2B As shown in (SU-DHL-4). The results show that in follicular lymphoma cells, compared with the ritanserin monotherapy group and the duverithione monotherapy group, the ritanserin and duverithione combination group can better promote cell apoptosis at a lower concentration, and the apoptosis effect gradually increases with increasing drug concentration.

[0062] Example 3

[0063] The inhibitory effect of the combined drug combination on the expression of proteins related to the PI3K / AKT / mTOR pathway and the classical MAPK pathway:

[0064] The operation method is as follows: take the number of units in the logarithmic growth phase as 1×10. 6Follicular lymphoma cell lines (including DOHH2 and SU-DHL-4) were seeded in 12-well plates. A blank control group, a ritanserin monotherapy group, a duveritol monotherapy group, and a ritanserin and duveritol combination group were set up.

[0065] The drug concentrations of ritanserin monotherapy group were 5 μM; the drug concentrations of duveritase monotherapy group were 400 nM; the drug concentrations of combination groups were 5 μM + 400 nM (the former being ritanserin and the latter being duveritase); and the blank control group cells were given the same volume of DMSO.

[0066] DMSO or the drug was applied to cells in a 12-well cell culture plate, and the mixture was gently agitated. The plate was then incubated in a Thermo cell culture incubator for 24 hours. After 24 hours of culture, the cells were centrifuged at 300g for 5 minutes, washed once with PBS, centrifuged again at 300g for 5 minutes, the supernatant was discarded, and cell clumps were collected. 200 μL of RIPA lysis buffer was added, along with a proteasome inhibitor and a phosphatase inhibitor (Thermo, USA). The cells were lysed at 4°C for 1 hour, centrifuged at 12000g for 30 minutes, and the supernatant was collected as the extracted total intracellular protein. Western blot analysis was performed using the extracted protein to detect any alterations in the expression levels of proteins related to the PI3K / AKT / mTOR pathway and the classical MAPK pathway.

[0067] Experimental results: The results are as follows Figure 3A , Figure 3B , Figure 3C As shown in the results, ritanserin and duverithione monotherapy have little effect on the MAPK / ERK and PI3K / AKT / mTOR pathways, and even slightly activate the expression of some molecules. However, ritanserin combined with duverithione significantly downregulated the phosphorylation levels of Ras, RAF, MEK, ERK, AKT and m-TOR.

[0068] Example 4

[0069] Effects of combined drug combinations on the in vivo tumorigenesis process of follicular lymphoma cells:

[0070] The operation method is as follows:

[0071] (1) CB17-SCID mice (weighing 16.1-20.1g) were divided into four groups of five mice each: blank control group, ritanserin monotherapy group, duveritide monotherapy group, and ritanserin and duveritide combination group. Both ritanserin and duveritide were dissolved in a solution prepared with 10% DMSO, 45% PEG300, 5% Tween80, and 40% sodium chloride to prepare suspensions for gavage.

[0072] (2) Constructing a CDX mouse model

[0073] Take 1×10 during the logarithmic growth phase 7 A CDX mouse model was established by subcutaneously injecting DOHH2 cells into the back of CB17-SCID mice to induce tumor formation.

[0074] (3) Fourteen days after injection of DOHH2 cells, mice were administered medication by gavage at a dose of 2.5 mg / kg / day for ritanserin and 12.5 mg / kg / day for duveritase. The administration was continued for 14 days, and tumor progression in mice was detected by measuring the size of subcutaneous tumors. Day 0 was recorded in the medication initiation diary.

[0075] Experimental results: Changes in mouse body weight are as follows Figure 4A As shown; the changes in tumor volume in each group of tumor-bearing mice are as follows. Figure 4B As shown; the changes in tumor weight in each group of tumor-bearing mice are as follows: Figure 4C As shown; the tumor anatomy diagrams of each group of tumor-bearing mice after the experiment are shown below. Figure 4D As shown in the results above, ritanserin combined with dovilixe can better reduce the burden of follicular lymphoma and effectively inhibit the tumorigenesis process in follicular lymphoma-bearing mice compared to ritanserin or dovilixe alone.

[0076] 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.

[0077] 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.

[0078] 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. The use of a combination pharmaceutical composition in the preparation of a medicament for improving or treating follicular lymphoma, characterized in that, The active component of the combined pharmaceutical composition consists of a first active component and a second active component; The first active ingredient is selected from ritanserin or a pharmaceutically acceptable salt thereof; The second active ingredient is selected from duvelith or a pharmaceutically acceptable salt thereof.

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

3. The application according to claim 2, characterized in that, The combined drug composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

4. The application according to claim 2, characterized in that, The formulation can be any pharmaceutically acceptable dosage form.

5. The application according to claim 1, characterized in that, The combined pharmaceutical composition also contains pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients are selected from any one or a combination of at least two of the following: carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

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