Drug application of FOXM1 inhibitor combined with BUB1 inhibitor

Abstract

The invention discloses combined application of an FOXM1 inhibitor and a BUB1 inhibitor, which is remarkable in anti-tumor effect, has a synergistic effect and lower toxicity, is particularly suitable for treating liver cancer, triple-negative breast cancer and melanoma, and solves the problems of poor curative effect, narrow indication, easiness in drug resistance, easiness in relapse and the like of single medication. And a new strategy is provided for tumor treatment.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical application of a FOXM1 inhibitor combined with a BUB1 inhibitor, and in particular to a pharmaceutical application of a FOXM1 inhibitor combined with a BUB1 inhibitor having a synergistic effect. Background Art

[0002] FOXM1 is a transcription factor of the forkhead box family, which has an evolutionarily conserved "winged helix" DNA binding domain in the family. Members of the FOX transcription factor superfamily regulate the transcription of genetic information from DNA into mRNA by binding to specific DNA sequences. FOXM1 has been shown to be involved in regulating a variety of biological functions, including DNA damage repair, cell cycle progression, angiogenesis, invasion and metastasis, and cell apoptosis. FOXM1 has been shown to be overexpressed in a variety of tumors, including liver cancer, breast cancer, melanoma, pancreatic cancer, colon cancer, and gastric adenocarcinoma, and is involved in regulating the malignant proliferation, invasion and metastasis, and resistance to chemotherapeutic drugs of tumors.

[0003] BUB1 is a mitotic serine / threonine kinase with multiple functions in chromosome segregation, KT-MT interaction, and SAC function. Studies have shown that BUB1 phosphorylates Cdc20 and histone H2A, leading to active transcription in human cells. Dysregulation of BUB1 has been shown to be closely associated with tumorigenesis. Overexpression of BUB1 can drive spontaneous tumorigenesis in transgenic mice and accelerate Myc-induced lymphoma progression. In addition, overexpression of BUB1 can induce tumor formation by causing chromosome missegregation through activation of Aurora-B. Studies have shown that BUB1 is upregulated in basal-like breast cancer and is a factor required for maintaining cancer stem cell renewal in breast cancer cell lines. In summary, overexpression of BUB1 is closely associated with the occurrence and malignant progression of a variety of human cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, as well as lymphoma and ovarian cancer.

[0004] Although the above two types of inhibitors are potentially effective drugs for treating tumors, they are still limited by insufficient efficacy, narrow indications, easy development of drug resistance and easy recurrence when used in treating tumors. Summary of the invention

[0005] Purpose of the invention: The present invention aims to provide a combined use of a FOXM1 inhibitor and a BUB1 inhibitor having a synergistic effect in targeted tumor treatment.

[0006] Technical solution: The FOXM1 inhibitor of the present invention is combined with the BUB1 inhibitor for use in the preparation of drugs for treating cancer.

[0007] Preferably, the cancer is a mid- to late-stage solid tumor or hematological tumor that highly expresses FOXM1 and BUB1.

[0008] More preferably, the cancer is liver cancer, breast cancer, melanoma, pancreatic cancer, lung adenocarcinoma, gastric cancer, renal cell carcinoma, ovarian cancer, cervical cancer, prostate cancer or colon cancer that highly expresses FOXM1 and BUB1.

[0009] Preferably, the BUB1 inhibitor is a sensitizing drug for FOXM1 inhibitor, and inhibiting the expression of BUB1 can increase the sensitivity of tumors to FOXM1 inhibitors.

[0010] The present invention found that BUB1 is a direct downstream target of the transcription factor FOXM1. The FOXM1 / BUB1 signal axis promotes the malignant progression of tumors by regulating DNA damage repair, cell cycle progression, tumor cell stemness, invasion and metastasis. Therefore, targeting the FOXM1 / BUB1 signal axis is expected to achieve effective treatment of more malignant tumors. Joint inhibition of the expression, function and activity of FOXM1 and BUB1 will synergistically inhibit the proliferation of malignant tumor cells, becoming a new strategy for targeted treatment of refractory cancers such as liver cancer, breast cancer, melanoma, etc. that highly express FOXM1 and BUB1.

[0011] Preferably, the FOXM1 inhibitor is selected from FDI-6, FOXN1-IN-1, RCM-1, STL001, STL427944, thiostrepton or a pharmaceutically acceptable salt thereof.

[0012] More preferably, the FOXM1 inhibitor is FDI-6.

[0013] FDI-6 is a specific inhibitor of FOXM1, which exerts its anti-tumor effect by inhibiting the transcriptional function of FOXM1 to regulate the tumor cell cycle progression, DNA damage repair pathway, etc.

[0014]

[0015] Preferably, the BUB1 inhibitor is selected from BAY-1816032, BAY-524, BAY-320, 2OH-BNPP1 or a pharmaceutically acceptable salt thereof.

[0016] More preferably, the BUB1 inhibitor is BAY-1816032.

[0017] BAY-1816032 is a specific inhibitor of BUB1, which exerts anti-tumor effects by inhibiting the activity of BUB1.

[0018]

[0019] Preferably, the molar ratio of the FOXM1 inhibitor to the BUB1 inhibitor is 1:(0.5-2).

[0020] Further preferably, the molar ratio of the FOXM1 inhibitor to the BUB1 inhibitor is selected from 1:0.5, 1:1, 1:1.2 or 1:2.

[0021] More preferably, the single dose of the FOXM1 inhibitor is 0.1 to 40 mg / kg, and the single dose of the BUB1 inhibitor is 0.1 to 40 mg / kg.

[0022] More preferably, the single dose of FDI-6 is 0.5-20 mg / kg, and the single dose of BAY-1816032 is 0.5-20 mg / kg.

[0023] Still more preferably, the single dose of FDI-6 is 1 to 10 mg / kg, and the single dose of BAY-1816032 is 1 to 10 mg / kg.

[0024] Preferably, the FOXM1 inhibitor and the BUB1 inhibitor are administered simultaneously or sequentially.

[0025] More preferably, when the simultaneous administration is adopted, two drugs with single active ingredients can be administered simultaneously, or a compound pharmaceutical preparation containing two active ingredients can be administered.

[0026] More preferably, when sequential administration is adopted, there is no special requirement for the order of administration; more preferably, the FOXM1 inhibitor is administered first and the BUB1 inhibitor is administered later.

[0027] The drug of the present invention can be made into various preparations, such as ordinary tablets, capsules, sustained-release tablets, sustained-release capsules, dispersible tablets, chewable tablets, orally disintegrating tablets, dropping pills, liposomes, solid dispersions, liquid preparations, etc.

[0028] Other FOXM1 inhibitors or pharmaceutically acceptable salts thereof can be used in combination with BUB1 inhibitors or pharmaceutically acceptable salts thereof to achieve the same therapeutic effect.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] The combined drug designed by the present invention has significant anti-tumor effects, synergistic effects and low toxicity, and is particularly suitable for the treatment of liver cancer, triple-negative breast cancer and melanoma. It solves the problems of poor efficacy, narrow indications, easy drug resistance and easy recurrence in single drug use, and provides a new strategy for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Schematic diagram of the synergistic inhibition of malignant proliferation of liver cancer, melanoma and triple-negative breast cancer by FDI-6 and BAY-1816032 in vitro in Example 1; wherein the drug synergy index CI is less than 1.0;

[0032] Figure 2 This is a schematic diagram of the clone formation experiment in Example 1;

[0033] Figure 3 This is a schematic diagram of the cell apoptosis experiment in Example 1;

[0034] Figure 4 This is a schematic diagram of the in vivo anti-tumor effect of BAY-1816032 and / or FDI-6 in Example 2;

[0035] Figure 5 The statistical graphs of tumor volume and weight in vivo of BAY-1816032 and / or FDI-6 in Example 2;

[0036] Figure 6 This is a schematic diagram of the in vivo toxicity evaluation of BAY-1816032 and / or FDI-6 in Example 2;

[0037] Figure 7 Schematic diagram of the in vivo anti-tumor effect of BUB1 shRNA and / or FDI-6 in Example 3;

[0038] Figure 8 This is a statistical graph of the tumor volume and weight in vivo in Example 3 after BUB1 shRNA and / or FDI-6. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with embodiments.

[0040] Materials and reagents in the examples: Human hepatoma cell lines HUH7, HepG2, and A-375 were cultured in DMEM medium and 10% fetal bovine serum (Bio-Channel) at 5% CO. 2 , 37°C; human triple-negative breast cancer cells MDA-MB-231 cells were cultured in L-15 medium and 10% fetal bovine serum at 5% CO 2 , cultured at 37°C; BAY-1816032 and FDI-6 (MedChemExpress) dissolved in DMSO. Cell apoptosis detection kit (KeyGEN), crystal violet staining solution (KeyGEN).

[0041] MTT assay for cell proliferation: cells were seeded in a 96-well plate at a density of 8,000 to 10,000 cells / well and incubated in 5% CO 2After culturing at 37°C for 24 hours, the corresponding drugs were treated for 3 days, and thiazolyl blue dye (MTT) was added for incubation for 3-4 hours. The culture medium was discarded, DMSO was added to fully dissolve and mix, and cell proliferation was detected. Cell growth inhibition rate = (1-absorbance value of experimental group / absorbance value of control group) × 100%.

[0042] Cloning experiment: 500-800 cells were seeded in a 12-well plate, cultured overnight to allow the cells to adhere to the wall, and then cultured for 14 days after adding the corresponding drugs; fixed with 4% paraformaldehyde for 15 minutes, then stained with 0.1% crystal violet stain for 10 minutes, and observed and photographed with an inverted fluorescence microscope.

[0043] Cell apoptosis experiment: The cells were seeded in 35 mm culture dishes at a density of 30% to 50% per dish, cultured overnight to allow the cells to adhere to the wall, and then treated with corresponding drugs for 3 days; the cells were digested with trypsin and washed twice with PBS; then the cells were stained with propidium iodide and annexinV-FITC. After completion, the cell apoptosis was analyzed using Agilent NovoCyteQuanteon flow cytometer.

[0044] Statistical analysis method: SPSS19.0 software was used for statistical analysis; measurement data were expressed as x±s, Student's t test was used for comparison between two groups, one-way analysis of variance was used for comparison between multiple groups, and LSD test was used for comparison between two groups. *P<0.05, # P<0.05 indicates that the difference is statistically significant.

[0045] Example 1: Evaluation of the in vitro inhibition of proliferation of human hepatoma cell lines HUH7, HepG2, A-375, and MDA-MB-231 by FOXM1 inhibitor FDI-6 combined with BUB1 inhibitor BAY-1816032

[0046] 1. Experimental methods

[0047] (1) MTT assay for cell proliferation

[0048] Four tumor cells were treated with DMSO (control), FDI-6, BAY-1816032, and FDI-6 and BAY-1816032 in combination, wherein the molar ratio of FDI-6 to BAY-1816032 in the combined treatment group was 1:(0.5-2). After 3 days, cell proliferation was detected by MTT.

[0049] (2) Clone formation assay to detect cell number

[0050] The effect of the combined drug on cell proliferation was detected by cloning assay, and four tumor cells were treated with BAY-1816032 and FDI-6. The single-drug FDI-6 dose for HUH7 cells was 1.0 μM, the single-drug BAY-1816032 dose was 1.0 μM, the combined FDI-6 dose was 0.5 μM, and the combined BAY-1816032 dose was 0.5 μM. The single-drug FDI-6 dose for HepG2 cells was 4.0 μM, the single-drug BAY-1816032 dose was 4.0 μM, the combined FDI-6 dose was 2.0 μM, and the combined BAY-1816032 dose was 2.0 μM. A375 cells were treated with FDI-6 at a single dose of 5.0 μM, BAY-1816032 at a single dose of 5.0 μM, FDI-6 at a combined dose of 2.5 μM, and BAY-1816032 at a combined dose of 2.5 μM. MDA-MB-231 cells were treated with FDI-6 at a single dose of 3.0 μM, BAY-1816032 at a single dose of 3.0 μM, FDI-6 at a combined dose of 1.5 μM, and BAY-1816032 at a combined dose of 1.5 μM. After 14 days, the cells were fixed with 4% paraformaldehyde for 15 min, stained with crystal violet for 10 min, and observed and photographed with an inverted fluorescence microscope.

[0051] (3) Cell apoptosis assay

[0052] HUH7 cells were treated with BAY-18160324 0.5 μM and FDI-6 0.5 μM for 3 days, and cell apoptosis was detected by flow cytometry.

[0053] 2. Experimental results

[0054] (1) MTT assay for cell proliferation

[0055] Results The synergistic index (CI) was calculated using the Chou-Talalay formula, where CI < 1.0 indicated a synergistic effect, CI = 1.0 indicated an additive effect, and CI > 1.0 indicated an antagonistic effect.

[0056] like Figure 1 As shown, when the combined molar ratio of FDI-6 and BAY-1816032 was in the range of 1:(0.5-2), the CI values ​​in the three tumor cells were all less than 1, indicating that BAY-1816032 combined with FDI-6 could synergistically inhibit the malignant proliferation of tumor cells within the determined molar ratio range.

[0057] (2) Number of cell clones

[0058] After FDI-6 and BAY-1816032 were co-treated with a molar ratio of 1:1, the number of cell clones was significantly less than that of the groups treated with FDI-6 or BAY-1816032 alone ( Figure 2 ). This further verified the synergistic inhibitory effect of FDI-6 combined with BAY-1816032 on tumor cell growth.

[0059] (3) Cell apoptosis

[0060] The analysis of cell apoptosis data showed that compared with the single-drug treatment group, the number of apoptotic cells in the FDI-6 and BAY-1816032 (molar ratio 1:1) combined treatment group increased significantly, indicating that the combination of FDI-6 and BAY-1816032 can synergistically promote HUH7 cell apoptosis ( Figure 3 ).

[0061] The above experimental results show that the combination of FDI-6 and BAY-1816032 has a synergistic anti-tumor effect.

[0062] Example 2: FDI-6 inhibits the growth of human hepatoma cell HUH7 xenograft tumors in vivo by sequential combination with BAY-1816032

[0063] 1. Experimental methods

[0064] (1) Test drug

[0065] FOXM1 inhibitor: FDI-6, BUB1 inhibitor: BAY-1816032.

[0066] Preparation method: FDI-6 is prepared with 10% DMSO, 10% Tween, 40% PEG400 and 40% saline; BAY-1816032 is prepared with 10% DMSO, 10% Tween, 30% PEG400 and 50% saline.

[0067] (2) Experimental animals

[0068] BALB / cA-nude mice, 7-8 weeks old, female; breeding environment: SPF grade.

[0069] (3) Experimental procedures

[0070] Human liver cancer HUH7 cell line was inoculated into the armpit of nude mice to construct a human liver cancer cell HUH7 transplanted tumor model. When the tumor grew to 50-70 mm 3The animals were randomly divided into HUH7 model group, FDI-6 administration group, BAY-1816032 administration group and FDI-6 / BAY-1816032 sequential administration group. The FDI-6 administration group was intraperitoneally injected with FDI-6 (20 mg / kg) once a day for 21 days. The BAY-1816032 administration group was intraperitoneally injected with BAY-1816032 (20 mg / kg) once a day for 21 days. The sequential administration group was intraperitoneally injected with FDI-6 (20 mg / kg) once a day for 10 days, and then intraperitoneally injected with BAY-1816032 (20 mg / kg) once a day for 11 days (the molar ratio of FDI-6 to BAY-1816032 was 1:1.2). The volume of the transplanted tumor was measured every two days, the weight of the mice was weighed, the data was recorded, and the nude mice were killed on the 21st day. The tumor tissue blocks were surgically removed and weighed, and the tumor inhibition rate (%) was calculated based on the weight and volume of the tumor tissue blocks.

[0071] 2. Experimental results

[0072] Compared with the model group, the tumor growth of the FDI-6 and BAY-1816032 groups was inhibited to a certain extent. Compared with the single drug treatment group and the model group, the FDI-6 / BAY-1816032 sequential administration group had a significant reduction in tumor weight and volume (the order of sequential administration is not limited to the order of first giving FDI-6 and then giving BAY-1816032) ( Figure 4-5 ).

[0073] The inhibitory effect of the FDI-6 / BAY-1816032 sequential administration group on human liver cancer cell HUH7 xenograft tumors was stronger than that of the BAY-1816032 single-use group or the FDI-6 single-use group, and the difference was significant, indicating that the sequential combination of BAY-1816032 and FDI-6 can exert a better anti-tumor effect in vivo.

[0074] The preliminary toxicity evaluation results showed that compared with the model group, the body weight of mice in the single-dose group and the sequential-dose group did not change significantly, and the heart, liver, spleen, lung and kidney of the mice were also significantly damaged ( Figure 6 ). FDI-6 and BAY-1816032 showed low toxicity whether administered alone or in combination in a sequential manner.

[0075] Example 3: FDI-6 combined with BUB1 shRNA inhibits the growth of human hepatocellular carcinoma cell HUH7 xenografts in vivo

[0076] 1. Experimental methods

[0077] (1) Test drug

[0078] FOXM1 inhibitor: FDI-6. BUB1 shRNA lentiviral vector (pGV112-shBUB1) and NC shRNA lentiviral vector (pGV112-shNC) were constructed by Shanghai GeneCare Gene Medical Technology Co., Ltd.

[0079] Preparation method: FDI-6 is prepared with 10% DMSO, 10% Tween, 40% PEG400 and 40% saline.

[0080] (2) Experimental animals

[0081] BALB / cA-nude mice, 7-8 weeks old, female; breeding environment: SPF grade.

[0082] (3) Experimental procedures

[0083] The BUB1 shRNA lentiviral stable cell line HUH7 BUB1 shRNA and the NC shRNA lentiviral stable cell line HUH7 NC shRNA were constructed. The HUH7 BUB1 shRNA and HUH7 NC shRNA cells in the logarithmic growth phase were inoculated into the armpits of nude mice to construct the corresponding cell xenograft tumor models. When the HUH7 NC shRNA xenograft tumors grew to 60-80 mm 3 The animals were randomly divided into NC shRNA group and NC shRNA + FDI-6 administration group, and the HUH7 BUB1 shRNA xenograft tumor model was randomly divided into BUB1 shRNA group and BUB1 shRNA + FDI-6 administration group. The FDI-6 administration group was intraperitoneally injected with FDI-6 (20 mg / kg) once a day for 21 days. The volume of the transplanted tumor was measured every two days, the mice were weighed, the data were recorded, and the nude mice were killed on the 21st day. The tumor tissue blocks were surgically removed and weighed, and the tumor inhibition rate (%) was calculated based on the weight and volume of the tumor tissue blocks.

[0084] 2. Experimental results

[0085] The results showed that compared with the NC shRNA group, the tumor growth of the NC shRNA+FDI-6 group and the BUB1 shRNA group was inhibited to a certain extent. Compared with the BUB1 shRNA group, the tumor growth of the BUB1 shRNA+FDI-6 group was significantly inhibited ( Figure 7-8 ).

[0086] Compared with the NC shRNA group, inhibition of BUB1 expression significantly inhibited the growth of human liver cancer xenografts. Compared with the NC shRNA+FDI-6 administration group, inhibition of BUB1 expression significantly increased the sensitivity of xenografts to FDI-6, indicating that targeting BUB1 and FOXM1 can exert a better anti-tumor effect in vivo.

[0087] In summary, the targeted combination drug (combined administration or sequential administration) designed by the present invention can significantly inhibit the growth of tumor cells in vivo and in vitro, and has good clinical application prospects.

Claims

1. Use of a FOXM1 inhibitor combined with a BUB1 inhibitor in the preparation of a drug for treating cancer.

2. The use according to claim 1, characterized in that: The cancer is a mid- to late-stage solid tumor or hematological tumor that highly expresses FOXM1 and BUB1.

3. The use according to claim 2, characterized in that: The cancer is liver cancer, breast cancer, melanoma, pancreatic cancer, lung adenocarcinoma, gastric cancer, renal cell carcinoma, ovarian cancer, cervical cancer, prostate cancer or colon cancer with high expression of FOXM1 and BUB1.

4. The use according to claim 1, characterized in that: The BUB1 inhibitor is a sensitizing drug for the FOXM1 inhibitor.

5. The use according to claim 1, characterized in that: The FOXM1 inhibitor is selected from FDI-6, FOXN1-IN-1, RCM-1, STL001, STL427944, thiostrepton or a pharmaceutically acceptable salt thereof.

6. The use according to claim 5, characterized in that: The FOXM1 inhibitor is FDI-6.

7. The use according to claim 1, characterized in that: The BUB1 inhibitor is selected from BAY-1816032, BAY-524, BAY-320, 2OH-BNPP1 or a pharmaceutically acceptable salt thereof.

8. The use according to claim 7, characterized in that: The BUB1 inhibitor is BAY-1816032.

9. The use according to claim 1, characterized in that: The molar ratio of the FOXM1 inhibitor to the BUB1 inhibitor is 1:(0.5-2).

10. The use according to claim 1, characterized in that: The FOXM1 inhibitor and the BUB1 inhibitor are administered simultaneously or sequentially.

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