CN100346830C - Pharmaceutical composition for treating B cell lymphoma - Google Patents

Abstract

The invention relates to a pharmaceutical composition for treating B-cell lymphoma. The medicine is composed of rituximab (Rituximab, Rituxan, MabThera) and SAHA (suberoylanilide hydroxamic acid, suberoylanilide hydroxamic acid). The medicine has obvious synergistic effect , Compared with single use, it can significantly inhibit the growth and proliferation of B lymphoma cells, synergistically induce apoptosis, synergistically inhibit NF-κB activity and down-regulate BCL-XL expression. The combination of the two drugs can greatly reduce the cost of treatment while improving the curative effect.

Description

A kind of pharmaceutical composition for treating B-cell lymphoma

technical field

The invention relates to a pharmaceutical composition for treating cancer, in particular to a pharmaceutical composition for treating B-cell lymphoma.

Background technique

Lymphoma is a kind of malignant tumor originating from lymphoid tissue. In recent years, the worldwide incidence rate has almost doubled, seriously endangering human health. B-cell lymphoma is the most common type of lymphoma, accounting for about 50-60% of lymphomas. Except for early-stage, localized, and low-grade B-cell lymphoma, which can be treated with radiotherapy alone, the current main treatment method is still combined chemotherapy. Combined chemotherapy can make about 50-60% of patients achieve disease remission, and 20-30% of patients achieve long-term survival, but most patients are ineffective or relapse rapidly after treatment, the disease progresses rapidly, and the prognosis is poor. More importantly, the side effects of chemotherapy are large, and patients, especially the elderly or those with other diseases, cannot tolerate it.

CD20 is widely expressed on the surface of B-cell lymphoma cells and is an ideal target for immunotherapy. In recent years, human-mouse chimeric anti-CD20 monoclonal antibody rituximab (Rituximab, Rituxan, MabThera) has been successfully used in clinics, through antibody-mediated immunotherapy against cell-specific surface markers, with low toxicity and low bone marrow The characteristics of inhibition and high specificity open up a new way for the treatment of B cell lymphoma. However, the treatment cost of patients is expensive, and some patients develop drug resistance in the early stage of treatment or when the disease relapses. Therefore, it is of great significance to study the feasibility of combined application of rituximab and other drugs to improve efficacy and reduce costs.

Histone deacetylase (HDAC) inhibitors can block the inhibition of gene expression caused by HDAC recruitment dysfunction by inhibiting HDAC, and have significant anti-tumor effects. Among them, SAHA (suberoylanilide hydroxamic acid, suberoylanilide hydroxamic acid, suberoylanilide hydroxamic acid) has entered phase II clinical trials, and has a certain curative effect in the treatment of patients with relapsed B-cell lymphoma. It is a highly potential targeted therapy drug.

Contents of the invention

The object of the present invention is to provide a pharmaceutical composition for treating B-cell lymphoma.

The object of the present invention is achieved through the following technical methods: the present invention is a pharmaceutical composition for treating B-cell lymphoma, which consists of rituximab and SAHA. The dosage of rituximab is 1-100 μg/ml, and the dosage of SAHA is 1-10 μM. The dosage of rituximab can be 1-50 μg/ml, and the dosage of SAHA can be 1-5 μM. The dosage of rituximab is preferably 20 μg/ml, and the dosage of SAHA is preferably 2.5 μM.

The present invention uses the following two human cell lines: the rituximab-sensitive follicular lymphoma SU-DHL-4 cell line, and the rituximab-resistant Burkitt's lymphoma Daudi cell line. The two human cell lines were treated with different concentrations of rituximab (0, 1, 20, 50, 100 μg/ml) and SAHA (0, 1, 2.5, 5, 10 μM), and the MTT experiment and flow analysis were carried out. Cytometry for annexin-V and mitochondrial transmembrane potential experiments, Western blot analysis experiments and nucleoprotein gel electrophoresis transfer detection experiments.

A pharmaceutical composition for treating B-cell lymphoma disclosed in the present invention has the advantages that: by using the pharmaceutical composition of the present invention, the synergistic effect of the two drugs can be synergistically inhibited the growth and proliferation of B-lymphoma cells, and synergistically inhibit the growth and proliferation of B-cell lymphoma cells. Induces apoptosis of B lymphoma cells, synergistically inhibits NF-κB activity and downregulates BCL-XL expression. The combination of the two drugs can greatly reduce the cost while improving the curative effect.

Description of drawings

Figure 1A shows the effect of rituximab alone in inhibiting the growth of B lymphoma cells.

Figure 1B shows the effect of SAHA alone on inhibiting the growth of B lymphoma cells.

Figure 1C shows the effect of combined use of rituximab and SAHA to inhibit the growth of B lymphoma cells.

Fig. 2A shows the effects of the flow cytometry detection of the apoptosis control group of the follicular lymphoma SU-DHL-4 cell line.

Fig. 2B shows the effect of rituximab alone inducing the apoptosis of follicular lymphoma SU-DHL-4 cell line.

Fig. 2C shows the effect of SAHA alone in inducing the apoptosis of follicular lymphoma SU-DHL-4 cell line.

Fig. 2D shows the effect of combined administration of rituximab and SAHA to induce apoptosis of follicular lymphoma SU-DHL-4 cell line.

Fig. 2E shows the synergy index effect diagram of the combined use of rituximab and SAHA to induce apoptosis of follicular lymphoma SU-DHL-4 cell line.

Fig. 2F shows the effects of detecting Burkitt's lymphoma Daudi cell line apoptosis control group by flow cytometry.

Figure 2G shows the effect of rituximab alone inducing the apoptosis of Burkitt's lymphoma Daudi cell line.

Figure 2H shows the effect of SAHA alone inducing the apoptosis of Burkitt's lymphoma Daudi cell line.

Figure 2I shows the effect of the combination of rituximab and SAHA inducing the apoptosis of Burkitt's lymphoma Daudi cell line.

Figure 2J shows the synergy index effect diagram of the combination of rituximab and SAHA inducing the apoptosis of Burkitt's lymphoma Daudi cell line.

Fig. 3 shows the results of mitochondrial transmembrane potential after single and combined administration of rituximab or SAHA for 24 hours.

Fig. 4A shows the effect of single and combined administration of rituximab or SAHA on Caspase-3, PARP and BCL-XL of follicular lymphoma cell line SU-DHL-4.

Fig. 4B is a graph showing the effect of single and combined administration of rituximab or SAHA on Caspase-3, PARP and BCL-XL of Burkitt's lymphoma Daudi cell line.

Figure 5 shows the results of single and combined use of rituximab or SAHA on B lymphoma cell NF-κB activity

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1

Preparation of a pharmaceutical composition for treating B-cell lymphoma

The invention relates to a pharmaceutical composition for treating B-cell lymphoma, which is prepared by mixing rituximab and SAHA according to a certain concentration ratio. The dosage of rituximab was 20 μg/ml, and the dosage of SAHA was 2.5 μM.

Example 2

Preparation of a pharmaceutical composition for treating B-cell lymphoma

The invention relates to a pharmaceutical composition for treating B-cell lymphoma, which is prepared by mixing rituximab and SAHA according to a certain concentration ratio. The dosage of rituximab was 1 μg/ml, and the dosage of SAHA was 10 μM.

Example 3

Preparation of a pharmaceutical composition for treating B-cell lymphoma

The invention relates to a pharmaceutical composition for treating B-cell lymphoma, which is prepared by mixing rituximab and SAHA according to a certain concentration ratio. The dosage of rituximab was 100 μg/ml, and the dosage of SAHA was 1 μM.

Example 4

Efficacy evaluation of a pharmaceutical composition for treating B-cell lymphoma

(1) Reagents:

Rituximab was purchased from Shanghai Roche Company and stored at -4°C. SAHA was donated by Shanghai MSD, dissolved in DMSO, the stock solution concentration was 100mM, and stored at -20°C. MTT, propidium iodide (PI), rhodamine 123 (Rh123), and dithiothreitol were purchased from Sigma. Anti-PARP, caspase-3, BCL-XL, NF-κB (P65), peroxidase-conjugated goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody were purchased from Santa Cruz Company. Anti-β-actin was purchased from Abcam.

(2) Cell culture, cell survival and cell morphology:

Two human cell lines were used in this study: the follicular lymphoma SU-DHL-4 cell line, which is sensitive to rituximab, and the Burkitt's lymphoma Daudi cell line, which is rituximab-resistant. Lymphoma cells were cultured in RPMI-1640 medium (Gibco/BRL) under the conditions of 5% CO ₂ -95% air, saturated humidity and 37°C, and 10% fetal bovine serum was added. In each experiment, cells were seeded at a density of 2 x 10 ⁵ /ml. Cell viability was detected by trypan blue exclusion assay. Cell morphology was observed by Wright's staining.

(3) MTT experiment:

In 96-well plates, cells were treated with different concentrations of rituximab or SAHA. After 72 hours, add 0.1mg MTT to each well, and after incubating at 37°C for 4 hours, measure the absorbance value of the specimen at 570nm in a spectrophotometer.

(4) Detection of annexin-V and mitochondrial transmembrane potential by flow cytometry:

Apoptosis was analyzed with ApoAlert Annexin V-FITC Apoptosis kit (BD). In the detection of mitochondrial transmembrane potential, cells were washed with PBS, incubated with 10 μg/ml Rh123 for 30 minutes at 37°C, and then stained with 50 μg/ml PI. Fluorescence intensity was measured by flow cytometry.

(5) Western blot analysis:

5×10 ⁶ cells were lysed with 200 ml Laemmli lysis buffer (0.5M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS and 5% β-mercaptoethanol). Protein lysates (20 μg) were used for 10% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked in 5% skim milk in TBS/0.05% Tween 20, and then incubated with primary antibody for 2 hours at room temperature and with horseradish peroxidase-conjugated secondary antibody for 1 hour. Immune complexes were detected with a horseradish peroxidase chemiluminescence detection kit.

(6) Nucleoprotein separation:

1×10 ⁷ cells were washed with lysis buffer (10mM HEPES, 10mM KCl, 1.5mM MgCl ₂ , 0.5mM DTT, pH 7.9) without NP-40, and then resuspended in extraction buffer (20mM HEPES, pH 7.9, 420mM NaCl, 0.5mM DTT, 0.2mM EDTA and 25% glycerol), and incubated on ice for 20 minutes. After centrifugation at 12000×g for 10 minutes, the supernatant is nucleoprotein.

(7) Gel electrophoresis transfer detection experiment:

Daudi nuclear protein extracts were quantified using Coomassie brilliant blue reagent (Pierce). Nucleoprotein (10 μg) was incubated with 32P-labeled double-chain NF-κB (5'-AGTTGAGGGGACTTTCCCAGGC-3') consensus sequence, then separated by 4% non-denaturing PAGE gel, and the labeled band was detected by autoradiography.

(8) Statistical analysis:

The experimental results are expressed as the mean and standard deviation of three independent experiments, and the differences are compared by t test. A P value less than 0.05 was considered statistically significant. All statistics use SAS8.2 software.

(9) Results:

CD20-positive B lymphoma cell lines SU-DHL-4 and Daudi cells were used for culture, and the effects of rituximab and SAHA alone and in combination on cell growth and proliferation were observed by analyzing the growth curves. B lymphoma cells were treated with different concentrations of rituximab (0, 1, 20, 50, 100 μg/ml) and SAHA (0, 1, 2.5, 5, 10 μM). The inhibitory effects of different doses of rituximab or SAHA alone and in combination on B lymphoma cells are shown in Figure 1A, Figure 1B, and Figure 1C. Rituximab (20 μg/ml) combined with SAHA (2.5 μM) for 48 hours can significantly inhibit SU - Proliferation of DHL-4 and Daudi cells.

The apoptosis of B lymphoma cells was detected by flow cytometry (double staining method of Annexin V and PI) after single and combined drugs. After SU-DHL-4 was treated with rituximab 20 μg/mL, SAHA 2.5 μM and rituximab 20 μg/mL and SAHA 2.5 μM for 48 hours, Annexin V positive cells were: control group 3.4% (see Figure 2A) 17.3% in the rituximab group (see Figure 2B); 26.3% in the SAHA group (see Figure 2C); 56.7% in the combined use of rituximab and SAHA (see Figure 2D). In Daudi cells: control group 3.0% (see Figure 2F); rituximab group 4.2% (see Figure 2G); SAHA group 24.4% (see Figure 2H); rituximab combined with SAHA group 61.7% (see Figure 2I ). At the same time, we used the isobologram established by Berenbaum to analyze whether the combined application of rituximab and SAHA was synergistic, additive or antagonistic. The positive rate of Annexin V when rituximab and SAHA were used alone or in combination was 30%±5%, and the combined index (CI) was calculated: CI=d1/D1+d2/D2. D1 and D2 represent the drug doses required to produce 30%±5% effect when drugs 1 and 2 are used alone, and d1 and d2 represent the drug doses when drugs 1 and 2 are used in combination to produce the same effect. CI>1, the graph is convex, the combination of the two is antagonistic, CI=1, the graph is a straight line, the combination of the two is additive, CI<1, the graph is concave, and the combination of the two is synergistic effect. Rituximab and SAHA acted on SU-DHL-4 and Daudi cells, and the graphs were both concave (see Figure 2E, Figure 2J), proving that they act synergistically.

The mitochondrial transmembrane potential (Δψm) and the integrity of the cell membrane can be obtained by double staining with the lipophilic anionic dye Rh123 and PI to detect a single cell. The former is due to the mitochondrial uptake is proportional to the mitochondrial Δψm, and the decrease in the uptake indicates a decrease in Δψm; PI cannot pass through the intact In the early stage of cell apoptosis, the cell membrane remained intact, and PI could not enter the cell. On the contrary, in the early stage of necrotic cell membrane destruction, PI staining increased. Please refer to Figure 3, Δψm decreased after rituximab (20 μg/ml) combined with SAHA (2.5 μM) for 24 hours.

Changes in protein expression in SU-DHL-4 cells and Daudi cells when rituximab and SAHA were used alone or in combination are shown in Figure 4A and Figure 4B. The activation of Caspase-3 by rituximab alone is not obvious, and the expression of Caspase-3 can be induced by SAHA alone. The corresponding substrate PARP is cut into 89KD fragments, which cannot perform normal functions and lead to apoptosis. Rituximab and This effect is more obvious when SAHA is used in combination. Therefore, rituximab-induced apoptosis of B lymphoma cell lines does not require the participation of activated Caspases, while SAHA-induced apoptosis of B lymphoma cell lines proceeds through activated Caspases, which can also partly explain the mechanism of the synergistic effect of the two. BCL-XL is an important apoptosis inhibitor of the BCL-2 family, which can stabilize the mitochondrial membrane permeability, reduce the release of apoptotic factors, and inhibit apoptosis. Rituximab and SAHA can down-regulate the expression of BCL-XL protein, and the down-regulation of BCL-XL in the combined group is more obvious, thereby promoting the apoptosis of lymphoma cells (Figure 4A and Figure 4B).

NF-κB is a signal transduction family, which mostly acts in the form of heterodimers and regulates the expression of many genes related to cell malignant transformation, immune and inflammatory responses in the nucleus. BCL-XL is the target gene of NF-κB, NF-κB activation can up-regulate the expression of BCL-XL. Rituximab (20 μg/ml) combined with SAHA (2.5 μM) applied for 48 hours could inhibit the expression of NF-κB (Fig. 5A). In Daudi cells, nuclear protein gel electrophoresis transfer detection further confirmed that the combination of the two can lead to the reduction of NF-κB transcriptional activity (Fig. 5B).

In summary, the combination of anti-CD20 monoclonal antibody rituximab and histone deacetylase inhibitors can inhibit the activity of NF-κB, down-regulate the expression of BCL-XL, and synergistically inhibit the apoptosis of lymphoma cells. Another effective treatment method.

Claims (3)

1, a kind of pharmaceutical composition for the treatment of B cell lymphoma is characterized in that this medicine is made up of rituximab and SAHA, and wherein, the rituximab consumption is 1-100 μ g/ml, and the SAHA consumption is 1-10 μ M.

2, pharmaceutical composition as claimed in claim 1 is characterized in that the rituximab consumption is 1-50 μ g/ml, and the SAHA consumption is 1-5 μ M.

3, pharmaceutical composition as claimed in claim 1 is characterized in that the rituximab consumption is 20 μ g/ml, and the SAHA consumption is 2.5 μ M.

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