Combined pharmaceutical composition for improving or treating follicular lymphoma and application thereof

Abstract

The invention relates to a combined pharmaceutical composition for improving or treating follicular lymphoma, which is characterized in that the active components of the combined pharmaceutical composition consist of a first active component and a second active component, the first active component is selected from any one or a combination of at least two of simvastatin, pharmaceutically acceptable salt, isomer, solvate and metabolite of simvastatin; the second active component is selected from any one or a combination of at least two of dovirisad, pharmaceutically acceptable salt, isomer, solvate and metabolite of dovirisad. The research finds that the simvastatin and the dovirisail not only can reduce the dosage of the simvastatin or the dovirisail and improve the medication safety, but also have the effect of improving or treating follicular lymphoma more remarkably than that of single simvastatin or dovirisail, and play a synergistic promotion effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a new combined drug intervention strategy for follicular lymphoma, and specifically to a combined drug composition for improving or treating follicular lymphoma and its application. Background Art

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

[0003] Using current first-line regimens, most FL patients have an initial response to treatment and even show a complete response. However, despite improvements in first-line treatment, conventional treatment of FL is not curative, and some patients still experience refractory or early relapse. Moreover, this early relapse is often resistant to chemotherapy, resulting in a significantly shortened survival. Therefore, there is an urgent need to propose new treatment strategies to improve the survival of this poor prognosis FL subgroup.

[0004] Simvastatin is a widely used statin, mainly used to treat hypercholesterolemia and related cardiovascular diseases. It reduces cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and is a commonly used drug for the treatment of hypercholesterolemia and atherosclerosis.

[0005] Duvilisse is an innovative oral dual inhibitor of PI3K-δ and PI3K-γ introduced into China by Shijiazhuang Pharmaceutical Group. Its inhibition of PI3K-δ leads to apoptosis of malignant tumor cells, while its inhibition of PI3K-γ reduces the differentiation and metastasis of supporting cells in the tumor microenvironment.

[0006] Whether simvastatin combined with duvelithib can be used to treat follicular lymphoma and its mechanism of action on follicular lymphoma-related cell lines is still unclear. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide a new combined drug intervention strategy for follicular lymphoma, specifically to provide a combined drug composition for improving or treating follicular lymphoma and its application.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a combined pharmaceutical composition for improving or treating follicular lymphoma, wherein the active component of the combined pharmaceutical composition consists of a first active component and a second active component;

[0010] The first active ingredient is selected from simvastatin, any one of its pharmaceutically acceptable salts, isomers, solvates, metabolites, or a combination of at least two thereof;

[0011] The second active ingredient is selected from any one of duvilisse, its pharmaceutically acceptable salts, isomers, solvates, metabolites, or a combination of at least two thereof.

[0012] The combined pharmaceutical composition involved in the present invention creatively combines simvastatin and duvilisse (or pharmaceutically acceptable salts, isomers, solvates, metabolites of the aforementioned compounds) as a drug for improving or treating follicular lymphoma. The present invention has found that simvastatin and duvilisse can not only reduce the dosage of simvastatin or duvilisse, improve the safety of medication, but also have a more significant effect of improving or treating follicular lymphoma than single simvastatin or duvilisse, and play a synergistic effect. The present invention first proves that it can significantly inhibit the proliferation of follicular lymphoma cells and induce apoptosis through follicular lymphoma cell lines; it proves that it can inhibit the expression of Pan-Kla in follicular lymphoma cells through cell WB experiments; and finally proves that the combined pharmaceutical composition can inhibit the tumor formation process of mice and improve the survival rate of mice through the CDX mouse model. The present invention provides an effective drug combination strategy for the improvement or treatment of follicular lymphoma, which is of great significance.

[0013] Preferably, the combined pharmaceutical composition further contains a pharmaceutically acceptable excipient.

[0014] Preferably, the combination pharmaceutical composition of the present invention can be administered alone or in combination with excipients to make an appropriate dosage form for administration. The pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0015] Preferably, the combination pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0016] Preferably, the combined pharmaceutical composition is a combination of two separate preparations, and the two separate preparations are administered simultaneously or sequentially.

[0017] The combination pharmaceutical composition may be in the form of a single compound preparation or a combination of two separate preparations; when it is a combination of two separate preparations, the administration method may be simultaneous administration, cross administration or sequential administration.

[0018] Preferably, the preparation is any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, etc.

[0019] In a second aspect, the present invention provides use of the combined pharmaceutical composition according to the first aspect in the preparation of a drug for improving or treating follicular lymphoma.

[0020] In a third aspect, the present invention provides use of the combined pharmaceutical composition according to the first aspect in the preparation of a follicular lymphoma cell proliferation inhibitor.

[0021] In a fourth aspect, the present invention provides use of the combined pharmaceutical composition according to the first aspect in the preparation of a follicular lymphoma cell proliferation inhibitor for non-therapeutic purposes.

[0022] Preferably, the follicular lymphoma cells include DOHH2 cells and / or SU-DHL-4 cells.

[0023] In a fifth aspect, the present invention provides use of the combined pharmaceutical composition according to the first aspect in the preparation of an agent for promoting apoptosis of follicular lymphoma cells.

[0024] In a sixth aspect, the present invention provides use of the combined pharmaceutical composition according to the first aspect in the preparation of an agent for promoting apoptosis of follicular lymphoma cells for non-therapeutic purposes.

[0025] Preferably, the follicular lymphoma cells include DOHH2 cells and / or SU-DHL-4 cells.

[0026] According to the research results of the present invention, the combined pharmaceutical composition has the effect of significantly inhibiting the proliferation of follicular lymphoma cells and promoting the apoptosis of follicular lymphoma cells. Therefore, the results indicate that the combined pharmaceutical composition can be used as an in vitro experimental reagent in the field of scientific research, such as studying the growth, apoptosis and other metabolic mechanisms or behaviors of follicular lymphoma cells, screening drugs for treating follicular lymphoma, etc.

[0027] In a seventh aspect, the present invention provides use of the combined pharmaceutical composition according to the first aspect in the preparation of an inhibitor of Pan-Kla expression in follicular lymphoma cells.

[0028] Preferably, the follicular lymphoma cells include DOHH2 cells and / or SU-DHL-4 cells.

[0029] According to the research results of the present invention, the combined pharmaceutical composition has the effect of significantly inhibiting the expression of Pan-Kla in follicular lymphoma cells. Therefore, the results indicate that the combined pharmaceutical composition can be used as an in vitro experimental reagent in the field of scientific research, such as studying the physiological metabolic mechanism related to Pan-Kla protein in lymphoma cells.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The combined pharmaceutical composition involved in the present invention creatively combines simvastatin and duvilisse (or pharmaceutically acceptable salts, isomers, solvates, metabolites of the aforementioned compounds) as a drug for improving or treating follicular lymphoma. The present invention has found that simvastatin and duvilisse can not only reduce the dosage of simvastatin or duvilisse, improve the safety of medication, but also have a more significant effect of improving or treating follicular lymphoma than single simvastatin or duvilisse, and play a synergistic effect. The present invention first proves that it can significantly inhibit the proliferation of follicular lymphoma cells and induce apoptosis through follicular lymphoma cell lines; it proves that it can inhibit the expression of Pan-Kla in follicular lymphoma cells through cell WB experiments; and finally proves that the combined pharmaceutical composition can inhibit the tumor formation process of mice and improve the survival rate of mice through the CDX mouse model. The present invention provides an effective drug combination strategy for the improvement or treatment of follicular lymphoma, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A This is the statistical result of the cell proliferation inhibition rate of DOHH2 cells after 24 hours of treatment with simvastatin combined with duvelithide;

[0033] Figure 1B This is a statistical result graph of the cell proliferation inhibition rate of SU-DHL-4 cells after 24 hours of treatment with simvastatin combined with duvelithide;

[0034] Figure 2A This is the result of flow cytometry detection of cell apoptosis in DOHH2 cells treated with simvastatin combined with duvelith for 24 hours;

[0035] Figure 2B This is the statistical result of apoptosis rate of DOHH2 cells;

[0036] Figure 3A This is the result of flow cytometry detection of cell apoptosis in SU-DHL-4 cells treated with simvastatin combined with duvelithide for 24 hours;

[0037] Figure 3B This is the statistical result diagram of apoptosis rate of SU-DHL-4 cells;

[0038] Figure 4 This is the Western blot result of the protein expression level of pan-histone lactylation in DOHH2 cells treated with simvastatin combined with duvelithide for 24 hours;

[0039] Figure 5AThis is a comparison of the tumor weights of mice in each group after simvastatin combined with duvelithide treatment of CDX mouse model;

[0040] Figure 5B This is a comparison of the tumor volumes of mice in each group after simvastatin combined with duvelithide treatment of CDX mouse model;

[0041] Figure 5C This is the anatomical appearance of the tumors in each group of mice in the CDX mouse model after simvastatin combined with duvelithide treatment;

[0042] Figure 5D This is the HE staining result of tumor tissue of each group of mice after the CDX mouse model was treated with simvastatin combined with duvelith. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0044] The drug simvastatin involved in the following examples was provided by Jiangsu Nanjing Taoshu Biological Company; the drug duvelisib involved was provided by Shijiazhuang Pharmaceutical Group Ouyi Pharmaceutical Co., Ltd.; and the follicular lymphoma cell lines (including DOHH2 cells and SU-DHL-4 cells) were provided by the Institute of Hematology, Xiamen University School of Medicine.

[0045] Example 1

[0046] Inhibitory effect of combined drug combination on proliferation of FL cell line

[0047] The operation method is: take the quantity as 2×10 4 FL cell lines (including DOHH2 and SU-DHL-4 cell lines) in the logarithmic growth phase were seeded in 96-well plates, and simvastatin alone group (Simvastatin), duvelisib alone group (Duvelisib), simvastatin and duvelisib combination group (Combo), and control group were set up;

[0048] The single-drug concentrations of simvastatin in the experimental group of cells were 0.6μM, 0.7μM, 0.8μM, 0.9μM, 1μM, 2μM, and 3μM, respectively; the single-drug concentrations of duvilice in the experimental group of cells were 0.06μM, 0.07μM, 0.08μM, 0.09μM, 0.1μM, 0.2μM, and 0.3μM, respectively; the concentrations of simvastatin and duvilice in the combined group were 0.6μM+0.06μM, 0.7μM+0.07μM, and 0.8μM+0.0 8μM, 0.9μM+0.09μM, 1μM+0.1μM, 2μM+0.2μM, 3μM+0.3μM (the former is simvastatin and the latter is duvilisep), wherein the control group cells were treated with the same volume of DMSO; the corresponding volume of drug or DMSO was gently shaken and mixed with the cells in the above 96-well cell culture plate, and then cultured in a cell culture incubator (Thermo) for 24 hours, and the cell proliferation level was detected using a CCK8 kit (MCE, Shanghai).

[0049] The results of cell proliferation levels in each group are shown in Figure 1A (DOHH2) and Figure 1B (SU-DHL-4)

[0050] pass Figure 1A and Figure 1B The results show that compared with higher concentrations of simvastatin alone or higher concentrations of duviliser alone, the combination of simvastatin and duviliser can significantly reduce the dosage of both drugs while improving the inhibition level, that is, it can simultaneously ensure low drug toxicity and side effects and excellent anti-follicular lymphoma therapeutic effects.

[0051] Example 2

[0052] Apoptosis-inducing effect of combined drug combination on FL cell line (DOHH2 cell line)

[0053] The operation method is: take the quantity as 2×10 5 FL cell lines (DOHH2 cell lines) in the logarithmic growth phase were seeded in 24-well plates, and simvastatin alone group (Simvastatin), duvelisib alone group (Duvelisib), simvastatin and duvelisib combination group (Combo), and control group were set up;

[0054] The single-drug concentrations of simvastatin in the experimental group cells were 2 μM, 3 μM, 4 μM, and 5 μM, respectively; the single-drug concentrations of duvilisse in the experimental group cells were 0.03 μM, 0.04 μM, 0.05 μM, and 0.06 μM, respectively; the concentrations of simvastatin and duvilisse in the combined group were 2 μM+0.03 μM, 3 μM+0.04 μM, 4 μM+0.05 μM, and 5 μM+0.06 μM, respectively (the former was simvastatin, and the latter was duvilisse). The cells in the control group were treated with the same volume of DMSO; the corresponding volume of drug or DMSO was gently shaken and mixed with the cells in the above-mentioned 24-well cell culture plate, and then cultured in a cell culture incubator for 24 hours, and then centrifuged at 4°C, 300g for 5 minutes to collect the cells, washed once with PBS, and then the cell apoptosis level was detected by Annexin V / PI (Thermofisher, USA) flow staining and the cell apoptosis rate was calculated.

[0055] Figure 2A and Figure 2B They are flow cytometry results of apoptosis level and statistical results of apoptosis rate of DOHH2 cells after 24 hours of treatment with simvastatin combined with duvelithide.

[0056] From the results shown in the above figure, it can be seen that compared with higher concentrations of simvastatin alone or higher concentrations of duviliser alone, the combination of simvastatin and duviliser can significantly reduce the dosage of both drugs while improving the level of cell apoptosis promotion, that is, it can simultaneously ensure low drug toxicity and side effects and excellent anti-follicular lymphoma therapeutic effects.

[0057] Example 3

[0058] Apoptosis-inducing effect of combined drug composition on FL cell line (SU-DHL-4 cell line)

[0059] The operation method is: take the quantity as 2×10 5 FL cell lines (SU-DHL-4 cell lines) in the logarithmic growth phase were seeded in 24-well plates, and simvastatin alone group (Simvastatin), duvelisib alone group (Duvelisib), simvastatin and duvelisib combination group (Combo), and control group were set up;

[0060] The single-drug concentrations of simvastatin in the experimental group cells were 2 μM, 3 μM, 4 μM, and 5 μM, respectively; the single-drug concentrations of duvilisse in the experimental group cells were 0.03 μM, 0.04 μM, 0.05 μM, and 0.06 μM, respectively; the concentrations of simvastatin and duvilisse in the combined group were 2 μM+0.03 μM, 3 μM+0.04 μM, 4 μM+0.05 μM, and 5 μM+0.06 μM, respectively (the former was simvastatin, and the latter was duvilisse). The cells in the control group were treated with the same volume of DMSO; the corresponding volume of drug or DMSO was gently shaken and mixed with the cells in the above-mentioned 24-well cell culture plate, and then cultured in a cell culture incubator for 24 hours, and then centrifuged at 4°C, 300g for 5 minutes to collect the cells, washed once with PBS, and then the cell apoptosis level was detected by Annexin V / PI (Thermofisher, USA) flow staining and the cell apoptosis rate was calculated.

[0061] Figure 3A and Figure 3B They are flow cytometry results of apoptosis level and statistical results of apoptosis rate in SU-DHL-4 cells treated with simvastatin combined with duvelithide for 24 hours.

[0062] From the results shown in the above figure, it can be seen that compared with higher concentrations of simvastatin alone or higher concentrations of duviliser alone, the combination of simvastatin and duviliser can significantly reduce the dosage of both drugs while improving the level of cell apoptosis promotion, that is, it can simultaneously ensure low drug toxicity and side effects and excellent anti-follicular lymphoma therapeutic effects.

[0063] Example 4

[0064] Inhibitory effect of combined drug composition on PAN-KLA expression in FL cell line

[0065] The operation method is: take the quantity as 1×10 6 FL cell lines (including DOHH2) in the logarithmic growth phase were seeded in 12-well plates, and simvastatin alone group (Simvastatin), duvelisib alone group (Duvelisib), simvastatin and duvelisib combination group (Combo), and control group were set up;

[0066] The single-drug concentration of simvastatin in the experimental group cells was 2 μM, the single-drug concentration of duvilisse in the experimental group cells was 0.03 μM, and the concentrations of simvastatin and duvilisse combined group were 2 μM+0.03 μM (the former was simvastatin, and the latter was duvilisse), and the cells in the control group were treated with the same volume of DMSO; the corresponding volume of drug or DMSO was gently shaken and mixed with the cells in the above-mentioned 12-well cell culture plate, and then cultured in a cell culture incubator for 24 hours, and then centrifuged at 4°C and 300g for 5 minutes to collect the cells, washed once with PBS, and lysed on ice for 1 hour with 200 μL RIPA lysis buffer (Thermo, USA), and the total protein was extracted for Western blot to detect the protein level expression of histone lactylation.

[0067] Figure 4 The results of Western blot test showed that compared with the single-drug group, the combination of simvastatin and duvilisib had a more significant effect in inhibiting the expression of PAN-KLA protein in FL cell lines.

[0068] Example 5

[0069] Effects of combined drug combinations on the tumorigenicity of FL

[0070] The specific operation method is as follows:

[0071] (1) A simvastatin monotherapy group (Simvastatin), a duvelisib monotherapy group (Duvelisib), a simvastatin and duvelisib combination group (Combo), and a control group were set up; simvastatin was prepared into a suspension using 0.5% sodium carboxymethylcellulose, and duvelisib was prepared into a suspension using a mixed solvent (10% DMSO, 45% PEG300, 5% Tween80, 40% Saline).

[0072] (2) Construction of CDX mouse model

[0073] Take 1×10 7 DOHH2 cells in logarithmic growth phase were injected subcutaneously into NOD-Prkdc - / - IL - / - The CDX mouse model was established by subcutaneous tumor formation in NSG mice.

[0074] (3) Seven days after injection, the drug was administered, with a dose of 5 mg / kg / day for simvastatin and 12.5 mg / kg / day for duvelith. The drug was administered for 14 consecutive days. The tumor progression of mice was detected by measuring the size of subcutaneous tumors. The day of drug administration was defined as day 0, and the experimental results and survival rate of mice were statistically analyzed. NSG mice were purchased from the Experimental Animal Center of Xiamen University and were raised by the Experimental Animal Center.

[0075] The statistics of tumor weight of each group of mice are shown in 5A, and the statistics of tumor volume are shown in Figure 5B The anatomical appearance of the tumor is shown in Figure 5C The HE staining results of mouse tumor tissue are shown in Figure 5D As shown, the above experimental results indicate that compared with the single-drug group, the simvastatin combined with duvelithide group is more effective in inhibiting the tumor formation process of the CDX model.

[0076] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0077] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

Claims

1. A combined pharmaceutical composition 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 simvastatin, any one of its pharmaceutically acceptable salts, isomers, solvates, metabolites, or a combination of at least two thereof; The second active ingredient is selected from any one of duvilisse, its pharmaceutically acceptable salts, isomers, solvates, metabolites, or a combination of at least two thereof.

2. The combined pharmaceutical composition according to claim 1, characterized in that: The combined pharmaceutical composition further contains 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 carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

4. The combined pharmaceutical composition according to claim 1, characterized in that: The combination pharmaceutical composition is a single compound preparation or a combination of two separate preparations; Preferably, the combination pharmaceutical composition is a combination of two separate preparations, and the two separate preparations are administered simultaneously or sequentially; Preferably, the preparation is in any pharmaceutically acceptable dosage form.

5. Use of the combined pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for improving or treating follicular lymphoma.

6. Use of the combined pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a follicular lymphoma cell proliferation inhibitor.

7. The use according to claim 6, characterized in that: The follicular lymphoma cells include DOHH2 cells and / or SU-DHL-4 cells.

8. Use of the combined pharmaceutical composition according to any one of claims 1 to 4 in the preparation of an agent for promoting apoptosis of follicular lymphoma cells.

9. The use according to claim 8, characterized in that: The follicular lymphoma cells include DOHH2 cells and / or SU-DHL-4 cells.

10. Use of the combined pharmaceutical composition according to any one of claims 1 to 3 in the preparation of an inhibitor of Pan-Kla expression in follicular lymphoma cells.

Images