CN116271057A

CN116271057A - Combined pharmaceutical composition for preventing or treating follicular lymphoma and application thereof

Info

Publication number: CN116271057A
Application number: CN202310350628.3A
Authority: CN
Inventors: 徐兵; 檀金水; 查洁; 刘雅婷; 鲁先平; 潘德思; 付鑫
Original assignee: Shenzhen Chipscreen Biosciences Co Ltd; First Affiliated Hospital of Xiamen University
Current assignee: Shenzhen Chipscreen Biosciences Co Ltd; First Affiliated Hospital of Xiamen University
Priority date: 2023-04-04
Filing date: 2023-04-04
Publication date: 2023-06-23

Abstract

The invention relates to a combined pharmaceutical composition for preventing or treating follicular lymphoma and application thereof, wherein the combined pharmaceutical composition comprises an HDAC inhibitor and a PI3K inhibitor. The research of the invention discovers that the combination of the HDAC inhibitor and the PI3K inhibitor not only can reduce the dosage of the HDAC inhibitor or the PI3K inhibitor and improve the medication safety, but also has the effect of obviously inhibiting follicular lymphoma compared with the single HDAC inhibitor or the single PI3K inhibitor, and has the synergistic effect. The invention provides an effective drug combination strategy for improving or treating follicular lymphoma, and has very remarkable significance.

Description

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

Technical Field

The invention belongs to the technical field of biological medicines, relates to a novel prevention or treatment mode of follicular lymphoma, and in particular relates to a combined pharmaceutical composition for preventing or treating follicular lymphoma and application thereof.

Background

Follicular Lymphoma (FL) is the most common indolent lymphoma, accounting for about 20% of all non-Hodgkin's lymphoma (NHL) cases (DOI: 10.3322/CAAC.21357, DOI: 10.3322/CAAC.21654). Follicular lymphoma is most commonly manifested as painless lymphadenectasis, typically manifested as multiple site lymphoid tissue invasion, sometimes accessible to supratrochlear lymphadenectasis.

Using the current first line regimen, most FL patients had a preliminary response to treatment, with 40% to 80% showing a complete response (DOI: 10.1182/blood-2003-12-4434, DOI: 10.1002/ajh.25696). However, despite the improvement in first line therapy, conventional treatment of FL is incurable, and about 20% of patients still experience refractory or early recurrence, which occurs in the first 2 years after chemo-immunotherapy diagnosis and treatment (DOI: 10.1002/ajh.24492, DOI: 10.1200/JCO.2014.59.7534). Moreover, this early recurrence is often chemical resistant, resulting in a significant reduction in survival. Therefore, there is an urgent need to propose new therapeutic strategies to improve the survival of this poor prognosis FL subgroup.

The Sidamine is a novel molecular entity drug which is originally discovered by Shenzhen micro-core biological company and has global patent protection, is an original chemical novel drug which is marketed in batches for the first time in China, is also an oral inhibitor of global first subtype selective Histone Deacetylase (HDAC), and belongs to an epigenetic regulator drug with novel mechanism. (DOI: 10.1186/s13148-017-0377-8, DOI:10.1038/s 41419-020-02972-2.).

PI3K refers to phosphatidylinositol 3-kinase, an intracellular lipid phosphokinase, mainly composed of p85 regulatory subunit, p55 regulatory subunit and p110 catalytic subunit, and can prevent the development of tumor diseases. The PI3K inhibitor is used as a molecular targeting drug, achieves an antiproliferative effect on tumor cells, and can also cause cancer cell death by combined action on the tumor cells and an immune system. It is not clear whether the combination of cibutamine with PI3K inhibitors can be used in follicular lymphoma therapy, or the mechanism of action on follicular lymphoma-associated cell lines.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a novel prevention or treatment mode of follicular lymphoma, in particular to a combined drug composition for preventing or treating follicular lymphoma and application thereof.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the invention provides a combination pharmaceutical composition for the prevention or treatment of Follicular Lymphoma (FL), comprising an HDAC inhibitor and a PI3K inhibitor.

The combined pharmaceutical composition creatively combines the HDAC inhibitor and the PI3K inhibitor to be used as medicines for preventing or treating follicular lymphoma, and researches show that the combination of the HDAC inhibitor and the PI3K inhibitor not only can reduce the dosage of the HDAC inhibitor or the PI3K inhibitor and improve the medication safety, but also has the effect of obviously inhibiting the follicular lymphoma compared with the single HDAC inhibitor or the PI3K inhibitor and has the synergistic effect. The invention firstly proves that the strain can obviously inhibit the proliferation of follicular lymphoma cells and induce the follicular lymphoma cells to undergo apoptosis; mitochondrial membrane potential detection proves that the preparation can induce mitochondrial membrane potential increase to cause follicular lymphoma cells to undergo apoptosis; cell WB experiments prove that the expression of c-MYC and PCNA of follicular lymphoma cells can be inhibited; finally, the CDX mouse model proves that the combined drug composition can inhibit the tumorigenic process of mice and improve the survival rate of the mice. The invention provides an effective drug combination strategy for improving or treating follicular lymphoma, and has very remarkable significance.

Preferably, the HDAC inhibitor is selected from Entinostat (Entinostat), vorinostat (Vorinostat), panobinostat (Panobinostat), mo Xisi he (Mocetinostat), belinostat (Belinostat), prazistat (Pracinostat), romidepsin (Romidepsin), cidamide (Chidamide, CS 055), or any one or a combination of at least two of the foregoing compounds in pharmaceutically acceptable salts, isomers, solvates, metabolites.

Preferably, the PI3K inhibitor has inhibitory activity of PI3K subunit, and is selected from any one or a combination of at least two of dulcis (Duvelisib), 3-Methyladenine (3-Methyladenine), bupirinib (Buparlisib), copanib (copanib), alpha-Linolenic acid (alpha-linolic acid), regrettin (rigsertib), lin Puli plug (linperlisib), or pharmaceutically acceptable salts, isomers, solvates and metabolites of the above compounds.

Preferably, the pharmaceutical combination further comprises pharmaceutically acceptable excipients.

Preferably, the pharmaceutical composition for combined use of the present invention may be administered alone or in combination with an auxiliary material to prepare a suitable dosage form, and the pharmaceutically acceptable auxiliary material includes any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an emulsifying agent, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

Preferably, the combined 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 formulations, which are administered simultaneously or sequentially.

The combined medicine composition can be in the form of a single compound preparation or a combination of two independent preparations; when two separate formulations are combined, they may be administered simultaneously, or they may be administered in cross-over or sequentially.

Preferably, the preparation is any pharmaceutically acceptable dosage form, such as tablet, powder, suspension, granule, capsule, solution, enema, emulsion, etc.

In a second aspect, the invention provides the use of a combination pharmaceutical composition according to the first aspect for the manufacture of a medicament for the prevention, amelioration or treatment of non-hodgkin's lymphoma.

Preferably, the non-hodgkin's lymphoma comprises indolent non-hodgkin's lymphoma.

Preferably, the indolent non-hodgkin's lymphoma comprises follicular lymphoma.

In a third aspect, the present invention provides the use of a combination pharmaceutical composition according to the first aspect for the preparation of a proliferation inhibitor of follicular lymphoma cells or an apoptosis promoter of follicular lymphoma cells.

In a fourth aspect, the invention also provides the use of a combination pharmaceutical composition according to the first aspect for the preparation of a proliferation inhibitor of follicular lymphoma cells or an apoptosis promoter of follicular lymphoma cells at a non-therapeutic destination.

According to the research result of the invention, the combined medicine composition has the effects of obviously inhibiting the proliferation of follicular lymphoma cells and promoting the apoptosis of follicular lymphoma cells, so the result shows that the combined medicine composition can be used as an in vitro experimental reagent in the scientific research field, for example, the research on the growth, the apoptosis and the metabolic mechanism or the behavior of follicular lymphoma cells, and the screening of medicines for treating follicular lymphoma and the like.

Preferably, the follicular lymphoma cells comprise DOHH2 cells and/or SU-DHL-4 cells.

In a fifth aspect, the present invention provides the use of a combination pharmaceutical composition according to the first aspect for the preparation of an inhibitor of follicular lymphoma cell HDAC family expression.

In a sixth aspect, the invention provides the use of a combination pharmaceutical composition according to the first aspect for the preparation of an inhibitor of follicular lymphoma cell HDAC family expression for a non-therapeutic destination.

According to the research result of the invention, the combined pharmaceutical composition has the effect of remarkably inhibiting the expression of the follicular lymphoma cell HDAC family, so the result shows that the combined pharmaceutical composition can be used as an in vitro experimental reagent in the scientific research field, for example, the physiological metabolism mechanism related to the lymphoma cell HDAC family is researched.

In a seventh aspect, the invention provides the use of a combination pharmaceutical composition according to the first aspect for the preparation of an inhibitor of follicular lymphoma cell PCNA/c-MYC expression.

In an eighth aspect, the invention provides the use of a combination pharmaceutical composition according to the first aspect for the preparation of an inhibitor of PCNA/c-MYC expression of follicular lymphoma cells at a non-therapeutic destination.

According to the research result of the invention, the combined pharmaceutical composition has the effect of obviously inhibiting the expression of PCNA/c-MYC of follicular lymphoma cells, so the result shows that the combined pharmaceutical composition can be used as an in vitro experimental reagent in the scientific research field, for example, the physiological metabolism mechanism related to PCNA/c-MYC protein of lymphoma cells is researched.

Compared with the prior art, the invention has the following beneficial effects:

Drawings

FIG. 1A is a graph showing the results of 24h inhibition of cell proliferation of DOHH2 cells treated with cilobroma in combination with dulcitol;

FIG. 1B is a graph showing the results of 24h inhibition of cell proliferation of SU-DHL-4 cells treated with cilnidin in combination with dulcitol;

FIG. 2A is a graph showing the results of 24h apoptosis horizontal flow assay of DOHH2 cells treated with cilobroma in combination with dulcitol;

FIG. 2B is a graph of apoptosis rate statistics of DOHH2 cells;

FIG. 3A is a graph showing the results of 24h apoptosis horizontal flow assay of SU-DHL-4 cells treated with cilnidin in combination with dulcitol;

FIG. 3B is a graph showing statistics of apoptosis rate of SU-DHL-4 cells;

FIG. 4A is a graph showing the results of mitochondrial membrane potential measurements of cells from DOHH2 cells treated with cilobroma in combination with dulcitol for 24 hours;

FIG. 4B is a statistical plot of mitochondrial membrane potential measurements of DOHH2 cells;

FIG. 5A is a graph showing the results of mitochondrial membrane potential measurements of cells from 24h after treatment of SU-DHL-4 cells with cilnidin in combination with dulcitol;

FIG. 5B is a statistical chart of mitochondrial membrane potential measurements of SU-DHL-4 cells;

FIG. 6 is a graph of Western blot results of protein expression levels of histone deacetylase/apoptotic pathway/proliferation markers following 24h treatment of DOHH2/SU-DHL-4 cells with cilidamide in combination with dulcitol;

FIG. 7A is a graph of body weight comparison results after treatment of a DOHH2-CDX mouse model with cilobroma in combination with dulcitol;

FIG. 7B is a graph showing the results of subcutaneous tumor volumes in mice after treatment of CDX mouse models with cilazamine in combination with dulcitol;

FIG. 7C is a graph of the subcutaneous tumor weight results of mice treated with cilazamine in combination with doviriser;

FIG. 7D is a graph of the size results of mice treated with the CDX mouse model in combination with the cilazamine and the dulcitol;

FIG. 8A is a graph showing the results of inhibition of cell proliferation after 24h of DOHH2 cells treated with cilobroma in combination with Lin Puli plug;

FIG. 8B is a graph showing the results of 24h apoptosis horizontal flow assay of DOHH2 cells treated with cetamazine in combination with Lin Puli plug;

FIG. 9A is a graph showing statistics of 24h apoptosis rate of DOHH2 cells treated with cetabamine in combination with Lin Puli plug;

FIG. 9B is a graph showing statistics of 24h apoptosis rate of SU-DHL-4 cells treated with ciladalimine in combination with Lin Puli plug;

FIG. 10A is a graph of body weight comparison results after treatment of a DOHH2-CDX mouse model with cilobroma in combination with Lin Puli plug;

FIG. 10B is a graph showing the results of subcutaneous tumor volumes in mice treated with cetamazine in combination with Lin Puli plug in CDX mouse models;

FIG. 10C is a graph of the subcutaneous tumor weight results of mice treated with cetamazine in combination with Lin Puli plug in CDX mouse models;

FIG. 10D is a graph of the size results of mice treated with Sidaminode in combination with Lin Puli plug in CDX mouse models;

wherein, the CS055 in the drawing refers to the Sidamide (Chidamide).

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited. The experimental methods in each example, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions recommended by the manufacturer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control.

The drug ciladamine (Chidamide) referred to in the following examples is provided by Shenzhen micro-core organism company.

The drug divrillic (Duvelisib) referred to in the examples below is offered by the company europathic pharmaceutical industry limited.

The drug Lin Puli plug (linperlisib) according to the following examples is provided by Jiangsu, uygur medicine Co.

AML cell lines (including DOHH2 cells, SU-DHL-4 cells) were supplied by the university of Xiamen medical institute of blood.

Example 1

Inhibition of FL cell line proliferation by combination pharmaceutical compositions

The operation method comprises the following steps: the number is 2 multiplied by 10 ⁴ Inoculating FL cell lines (including DOHH2 and SU-DHL-4 cell lines) in logarithmic growth phase into a 96-well plate, and setting a single drug group of the Sidamide, a single drug group of the Duvillous, a combined group of the Sidamide and the Duvillous, and a control group;

the single drug concentration of the Sidamide in the cells of the experimental group is 125nM, 250nM, 500nM and 1000nM, the single drug concentration of the Duvalicarb in the cells of the experimental group is 50nM, 100nM, 200nM and 400nM, the combined group concentration of the Sidamide and the Duvalicarb is 125nM+50nM, 250nM+100nM, 500nM+200nM, 1000nM+400nM (the former is the Sidamide and the latter is the Duvalicarb), wherein the cells of the control group are treated with the same volume of DMSO; after mixing the corresponding volumes of drug or DMSO with the cells in the 96-well cell culture plates with gentle shaking, the level of cell proliferation was detected with CCK8 kit (MCE, shanghai) after culturing for 24h in a cell incubator (Thermo).

The results of the cell proliferation level of each group are shown in FIG. 1A (DOHH 2) and FIG. 1B (SU-DHL-4);

as can be seen from the results shown in fig. 1A and 1B, the combination of the cidobutamine and the duloxetine (250nm+100nm) can significantly reduce the use amount of the two drugs on the basis of increasing the inhibition level, i.e., can simultaneously ensure low drug toxic and side effects and excellent therapeutic effects against follicular lymphoma, compared with the high concentration (500 nM) of the cidobutamine single drug or the high concentration (200 nM) of the duloxetine single drug.

Example 2

Apoptosis inducing effect of pharmaceutical composition in combination on FL cell line

The operation method comprises the following steps: the number is 2 multiplied by 10 ⁵ Inoculating FL cell strain (DOHH 2 cell strain) in logarithmic growth phase into 24-well plate, and setting single drug group of Sidamide, single drug group of Duvalicarb, combined group of Sidamide and Duvalicarb, and control group;

the single drug concentration of the cilazamine in the cells of the experimental group is 250nM and 500nM respectively, the single drug concentration of the dulcitol in the cells of the experimental group is 100nM and 200nM respectively, and the combined group concentration of the cilazamine and the dulcitol is 250nM+100nM and 500nM+200nM respectively (the former is the cilazamine and the latter is the dulcitol), wherein the cells of the control group are treated with the same volume of DMSO; after a corresponding volume of the drug or DMSO is mixed with the cells in the 24-well cell culture plate by slight shaking, the cells are collected by centrifugation at 300g for 5min at 4 ℃ after being cultured for 24 hours in a cell culture box, washed once by PBS, and then the apoptosis level is detected and the apoptosis rate is counted by an Annexin V/PI (thermo filter, USA) flow staining method.

Fig. 2A and fig. 2B are graphs of apoptosis horizontal flow detection results and apoptosis rate statistics after 24h treatment of DOHH2 cells with cidamine in combination with dulcitol, respectively.

The results shown in the above graph show that, compared with the single drug of the cilobutyramine with high concentration (500 nM) or the single drug of the duloxetine with high concentration (200 nM), the combination of the cilobutyramine and the duloxetine (250 nM+100 nM) can obviously reduce the use amount of the two drugs on the basis of improving the level of promoting apoptosis, namely, can simultaneously ensure low toxic and side effects of the drugs and excellent therapeutic effects of resisting follicular lymphoma.

Example 3

The operation method comprises the following steps: the number is 2 multiplied by 10 ⁵ Inoculating FL cell lines (SU-DHL-4 cell lines) in logarithmic growth phase into 24-well plates, and setting a single drug group of the Sidamide, a single drug group of the Devillous, a combined group of the Sidamide and the Devillous, and a control group;

the single drug concentration of the ciladalimmine in the cells of the experimental group is 2000nM and 4000nM respectively, the single drug concentration of the dulcitol in the cells of the experimental group is 800nM and 1600nM respectively, and the combined group concentration of the ciladalimmine and the dulcitol is 2000nM+800nM and 4000nM+1600nM respectively (the former is the ciladalimmine and the latter is the dulcitol), wherein the cells of the control group are treated with the same volume of DMSO; after a corresponding volume of the drug or DMSO is mixed with the cells in the 24-well cell culture plate by slight shaking, the cells are collected by centrifugation at 300g for 5min at 4 ℃ after being cultured for 24 hours in a cell culture box, washed once by PBS, and then the apoptosis level is detected and the apoptosis rate is counted by an Annexin V/PI (thermo filter, USA) flow staining method.

Fig. 3A and 3B are graphs of apoptosis horizontal flow detection results and apoptosis rate statistics after 24h treatment of SU-DHL-4 cells with cidamine in combination with dulcitol, respectively.

The results shown in the above figures show that the combination of the cidamine and the dulcitol can obviously reduce the use amount of the two medicines on the basis of improving the level of promoting apoptosis, namely, the low toxic and side effects of the medicines and the excellent curative effect of resisting follicular lymphoma can be ensured simultaneously.

Example 4

Mitochondrial membrane potential effect of combination pharmaceutical compositions on FL cell lines

the single drug concentration of the cilazamine in the cells of the experimental group is 250nM and 500nM respectively, the single drug concentration of the dulcitol in the cells of the experimental group is 100nM and 200nM respectively, and the combined group concentration of the cilazamine and the dulcitol is 250nM+100nM and 500nM+200nM respectively (the former is the cilazamine and the latter is the dulcitol), wherein the cells of the control group are treated with the same volume of DMSO; after the corresponding volume of the drug or DMSO is mixed with the cells in the 24-hole cell culture plate by slight shaking, the cells are collected by centrifugation at 300g for 5min at 4 ℃ after being cultured for 24 hours in a cell culture box, washed once by PBS, and then the mitochondrial membrane potential in the cells is detected by JC-1 flow staining method and the positive rate is counted.

FIGS. 4A and 4B are graphs of intracellular mitochondrial membrane potential results and graphs of statistical results, respectively, of 24h after treatment of DOHH2 cells with cilobutylamine in combination with dulcitol.

The results shown in the above graph show that, compared with the single drug of the cilobutyramine with high concentration (500 nM) or the single drug of the duloxetine with high concentration (200 nM), the combination of the cilobutyramine and the duloxetine (250 nM+100 nM) can obviously reduce the use amount of the two drugs on the basis of increasing the mitochondrial membrane potential of cells, namely, can simultaneously ensure low drug toxic and side effects and excellent therapeutic effects on follicular lymphoma.

Example 5

the single drug concentration of the ciladalimmine in the cells of the experimental group is 2000nM and 4000nM respectively, the single drug concentration of the dulcitol in the cells of the experimental group is 800nM and 1600nM respectively, and the combined group concentration of the ciladalimmine and the dulcitol is 2000nM+800nM and 4000nM+1600nM respectively (the former is the ciladalimmine and the latter is the dulcitol), wherein the cells of the control group are treated with the same volume of DMSO; after the corresponding volume of the drug or DMSO is mixed with the cells in the 24-hole cell culture plate by slight shaking, the cells are collected by centrifugation at 300g for 5min at 4 ℃ after being cultured for 24 hours in a cell culture box, washed once by PBS, and then the mitochondrial membrane potential in the cells is detected by JC-1 flow staining method and the positive rate is counted.

FIGS. 5A and 5B are graphs of intracellular mitochondrial membrane potential results and graphs of statistical results, respectively, of Sidamine in combination with dulcitol treated SU-DHL-4 cells for 24 h.

The results shown in the above graph show that, compared with the single drug of the cilobutyramine with high concentration (4000 nM) or the single drug of the duloxetine with high concentration (1600 nM), the combination of the cilobutyramine and the duloxetine (2000 nM+800 nM) can obviously reduce the use amount of the two drugs on the basis of increasing the mitochondrial membrane potential of cells, namely, can simultaneously ensure low drug toxic and side effects and excellent therapeutic effects on follicular lymphoma.

Example 6

Inhibition of HDAC expression by combination pharmaceutical compositions

The operation method comprises the following steps: the number is 1 multiplied by 10 ⁶ Inoculating FL cell lines (including DOHH2 and SU-DHL-4 cell lines) in logarithmic growth phase into a 12-well plate, and setting a single drug group of the Sidamide, a single drug group of the Duvillous, a combined group of the Sidamide and the Duvillous, and a control group;

the single drug concentration of the cetrapamide of the cells of the experimental group is 500nM respectively, the single drug concentration of the dulcitol of the cells of the experimental group is 200nM respectively, the combined group concentration of the cetrapamide and the dulcitol is 500nM+200nM respectively (the former is the cetrapamide and the latter is the dulcitol), wherein the cells of the control group are treated with DMSO of the same volume; after a corresponding volume of the drug or DMSO was mixed with the cells in the 12-well cell culture plate by slight shaking, the cells were collected by centrifugation at 300g for 5min at 4℃after culturing for 24 hours in a cell culture box, washed once with PBS, lysed with 200. Mu.L of RIPA lysate (Thermo, USA) on ice for 1 hour, western blot was performed on the total protein extracted, and protein level expression of apoptosis-related protein, proliferation-related protein and histone deacetylase was detected.

FIG. 6 shows Western blot detection results demonstrating that the expression of DOHH2/SU-DHL-4 cell HDAC1/3, anti-apoptotic and proliferation-related proteins in FL cell lines is inhibited by cidamine and divalise.

Example 7

Effect of combination pharmaceutical compositions on FL tumorigenesis

The specific operation method is as follows:

(1) The method comprises the steps of setting a control group, a single-drug group of the Sidamide, a single-drug group of the DOWN-Williams, and a combined group of the Sidamide and the DOWN-Williams, wherein the Sidamide is prepared into a suspension by using 0.5% of sodium carboxymethyl cellulose, and the DOWN-Williams is prepared into a suspension by using 10% DMSO,45% PEG300,5% Tween80 and 40% Saline.

(2) Construction of CDX mouse model

Taking 1×10 ⁷ DOHH2 cells in logarithmic growth phase and injected subcutaneously into NOD-Prkdc ^-/- IL2rg ^-/- (NSG) mice were tumor-developed subcutaneously and CDX mouse models were established.

(3) Starting the administration after about 7 days of injection, the dose of the sitagliptin was 5mg/kg/day, the dose of the dulcitol was 12.5mg/kg/day, and the administration was continued for 14 days, the progress of the tumor formation of the mice was examined by measuring the size of the subcutaneous tumor, and the starting diary for the administration was 0 days, and the results of the statistical experiment and the survival rate of the mice were counted. Wherein NSG mice are purchased from and bred by a laboratory animal center at Xiamen university.

The body weight change of each group of mice is shown in fig. 7A, the tumor volume change is shown in fig. 7B, the tumor weight change is shown in fig. 7C, and the anatomical appearance of the tumor is shown in fig. 7D. The combination of the cidamine and the wiglissade is shown to inhibit the tumorigenesis process of the CDX model.

Example 8

The operation method comprises the following steps: the number is 2 multiplied by 10 ⁴ Inoculating FL cell lines (including DOHH2 and SU-DHL-4 cell lines) in logarithmic growth phase into a 96-well plate, and setting a single drug group of the Sidamine, a single drug group of the Lin Puli plug, a combined group of the Sidamine and the Lin Puli plug and a control group;

the single drug concentration of the cetrimide in the cells of the experimental group is 125nM, 250nM, 500nM and 1000nM, the single drug concentration of the Lin Puli plug in the cells of the experimental group is 100nM, 200nM, 400nM and 800nM, the combined group concentration of the cetrimide and Lin Puli plug is 125nM+100nM, 250nM+200nM, 500nM+400nM, 1000nM+800nM (the former is the cetrimide and the latter is the Lin Puli plug), wherein the cells of the control group are treated with the same volume of DMSO; after mixing the corresponding volumes of drug or DMSO with the cells in the 96-well cell culture plates with gentle shaking, the level of cell proliferation was detected with CCK8 kit (MCE, shanghai) after culturing for 24h in a cell incubator (Thermo).

The results of the cell proliferation level of each group are shown in FIG. 8A (DOHH 2) and FIG. 8B (SU-DHL-4);

as can be seen from the results shown in fig. 8A and 8B, the use of the combination of the cetrimide and Lin Puli plug (250 nm+200 nM) can significantly reduce the use amount of the two drugs on the basis of increasing the inhibition level, i.e., can simultaneously ensure low drug toxic and side effects and excellent therapeutic effects against follicular lymphoma, compared to the high concentration (500 nM) of the cetrimide single drug or the high concentration (400 nM) of the Lin Puli plug single drug.

Example 9

The operation method comprises the following steps: the number is 2 multiplied by 10 ⁵ The FL cell strain in logarithmic growth phase (comprising DOHH2 and SU-DHL-4 cell strain) is inoculated in a 24-well plate, and a single drug group of the Sidamine, a single drug group of the Lin Puli plug, a combined group of the Sidamine and the Lin Puli plug and a control group are arranged;

the single drug concentration of the cetrimide in the cells of the experimental group is 250nM and 500nM respectively, the single drug concentration of the Lin Puli plug in the cells of the experimental group is 100nM and 200nM respectively, the combined group concentration of the cetrimide and Lin Puli plug is 250nM+100nM and 500nM+200nM respectively (the former is the cetrimide and the latter is the Lin Puli plug), wherein the cells of the control group are treated with the same volume of DMSO; after a corresponding volume of the drug or DMSO is mixed with the cells in the 24-well cell culture plate by slight shaking, the cells are collected by centrifugation at 300g for 5min at 4 ℃ after being cultured for 24 hours in a cell culture box, washed once by PBS, and then the apoptosis level is detected and the apoptosis rate is counted by an Annexin V/PI (thermo filter, USA) flow staining method.

Fig. 9A and 9B are graphs of apoptosis horizontal flow assay and apoptosis rate statistics, respectively, of cidamine in combination with Lin Puli plug treatment of DOHH2 cells for 24 h.

The results shown in the above figures show that, compared with the high-concentration single cetrimide or the high-concentration single Lin Puli plug, the combination of the cetrimide and the Lin Puli plug can obviously reduce the use amount of the two medicaments on the basis of improving the apoptosis promotion level, i.e. can simultaneously ensure low toxic and side effects of the medicaments and excellent therapeutic effects of resisting follicular lymphoma.

Example 10

Effect of combination pharmaceutical compositions on FL tumorigenesis

The specific operation method is as follows:

(1) The method comprises the steps of setting a control group, a single-drug group of the Sidamine, a single-drug group of the Lin Puli plug, and a combined group of the Sidamine and the Lin Puli plug, wherein the Sidamine is prepared into a suspension by using 0.5% of sodium carboxymethyl cellulose, and the Lin Puli plug is prepared into the suspension by using 10% DMSO,45% PEG300,5% Tween80 and 40% Saline.

(2) Construction of CDX mouse model

(3) Starting the administration after about 7 days of injection, the dose of the sitagliptin was 5mg/kg/day, the Lin Puli plug dose was 12.5mg/kg/day, the administration was continued for 14 days, the tumor development of the mice was examined by measuring the subcutaneous tumor size, and the starting diary for the administration was 0 days, and the experimental results and the survival rate of the mice were counted. Wherein NSG mice are purchased from and bred by a laboratory animal center at Xiamen university.

The body weight change of each group of mice is shown as 10A, the tumor volume change is shown as 10B, the tumor weight change is shown as 10C, and the anatomical appearance of the tumor is shown as 10D. It was shown that the CDX model was inhibited by the cilobroma in combination with Lin Puli plug.

The applicant states that the present invention is described by way of the above examples as a combination pharmaceutical composition for preventing or treating follicular lymphoma and its use, but the present invention is not limited to, i.e. it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. A combination pharmaceutical composition for preventing or treating follicular lymphoma, wherein the combination pharmaceutical composition comprises an HDAC inhibitor and a PI3K inhibitor.

2. The pharmaceutical combination according to claim 1, wherein the HDAC inhibitor is selected from the group consisting of entinostat, vorinostat, panobinostat, mo Xisi he, belinostat, pravastatin, romidepsin, cidamide, or any one or a combination of at least two of the foregoing compounds in pharmaceutically acceptable salts, isomers, solvates, metabolites.

3. The pharmaceutical combination according to claim 1, wherein the PI3K inhibitor has inhibitory activity of PI3K subunit and is selected from the group consisting of dulcitol, 3-methyladenine, bupanimate, copanite, α -linolenic acid, regoratine, lin Puli plug, and pharmaceutically acceptable salts, isomers, solvates, metabolites, and combinations of any one or at least two of the foregoing.

4. The combination pharmaceutical composition of any one of claims 1-3, further comprising a pharmaceutically acceptable adjuvant;

preferably, the pharmaceutically acceptable auxiliary materials comprise 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, bacteriostats or buffers;

preferably, the combined 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 formulations, both of which are administered simultaneously or sequentially;

preferably, the preparation is any one of pharmaceutically acceptable dosage forms.

5. Use of a combination pharmaceutical composition according to any one of claims 1-4 for the preparation of a medicament for the prevention, amelioration or treatment of non-hodgkin's lymphoma.

6. The use of claim 5, wherein the non-hodgkin's lymphoma comprises indolent non-hodgkin's lymphoma;

preferably, the indolent non-hodgkin's lymphoma comprises follicular lymphoma.

7. Use of a combination pharmaceutical composition according to any one of claims 1-4 for the preparation of a proliferation inhibitor of follicular lymphoma cells or an apoptosis promoter of follicular lymphoma cells.

8. The use according to claim 7, wherein the follicular lymphoma cells comprise DOHH2 cells and/or SU-DHL-4 cells.

9. Use of a combination pharmaceutical composition according to any one of claims 1-4 for the preparation of inhibitors of follicular lymphoma cell HDAC family expression.

10. Use of a combination pharmaceutical composition according to any one of claims 1-4 for the preparation of an inhibitor of follicular lymphoma cell PCNA/c-MYC expression.