Pharmaceutical applications of foxm1 inhibitors in combination with bub1 inhibitors

Abstract

This invention discloses a combined application of FOXM1 inhibitor and BUB1 inhibitor, which has significant anti-tumor effects, synergistic effects and low toxicity. It is particularly suitable for the treatment of liver cancer, triple-negative breast cancer and melanoma, and solves the problems of poor efficacy, narrow indications, easy drug resistance and easy recurrence of monotherapy, providing a new strategy for tumor treatment.

Description

Technical Field

[0001] This invention relates to the pharmaceutical application of a FOXM1 inhibitor combined with a BUB1 inhibitor, and more particularly to the pharmaceutical application of a FOXM1 inhibitor combined with a BUB1 inhibitor with synergistic effects. Background Technology

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

[0003] BUB1 is a mitotic serine / threonine kinase with multiple functions, including 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 related to tumorigenesis. Overexpression of BUB1 drives spontaneous tumorigenesis in transgenic mice and accelerates Myc-induced lymphoma progression. Furthermore, overexpression of BUB1 can induce tumorigenesis by activating Aurora-B, leading to missegregation of chromosomes. Studies have found that BUB1 is upregulated in basal-like breast cancer and is essential for maintaining cancer stem cell renewal in breast cancer cell lines. In conclusion, overexpression of BUB1 is closely associated with the occurrence and malignant progression of various human cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, lymphoma, and ovarian cancer.

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

[0005] Purpose of the invention: The present invention aims to provide a combined application of FOXM1 inhibitor and BUB1 inhibitor that has a synergistic effect in targeted tumor therapy.

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

[0007] Preferably, the cancer is an intermediate-to-late stage solid tumor or hematologic malignancy that highly expresses FOXM1 and BUB1.

[0008] Further 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 express FOXM1 and BUB1.

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

[0010] This invention reveals that BUB1 is a direct downstream target of the transcription factor FOXM1. The FOXM1 / BUB1 signaling axis promotes malignant tumor progression through multiple mechanisms, including regulating DNA damage repair, cell cycle progression, tumor cell stemming, and invasion and metastasis. Therefore, targeting the FOXM1 / BUB1 signaling axis holds promise for effective treatment of more malignant tumors. Synergistically inhibiting the expression, function, and activity of both FOXM1 and BUB1 will suppress the proliferation of malignant tumor cells, representing a novel strategy for targeted therapy of refractory cancers such as liver cancer, breast cancer, and melanoma that highly express FOXM1 and BUB1.

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

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

[0013] FDI-6 is a specific inhibitor of FOXM1, which exerts its anti-tumor effects by inhibiting the transcriptional function of FOXM1 and regulating tumor cell cycle progression and DNA damage repair pathways.

[0014]

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

[0016] Further preferred, the BUB1 inhibitor is BAY-1816032.

[0017] BAY-1816032 is a specific inhibitor of BUB1, which exerts its anti-tumor effect 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] Further preferably, the single dose of the FOXM1 inhibitor is 0.1–40 mg / kg, and the single dose of the BUB1 inhibitor is 0.1–40 mg / kg.

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

[0023] Further preferred, the single-use dose of FDI-6 is 1-10 mg / kg, and the single-use dose of BAY-1816032 is 1-10 mg / kg.

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

[0025] Further preferred, when using a simultaneous administration method, two drugs with single active ingredients can be administered simultaneously, or a compound drug preparation containing two active ingredients can be administered.

[0026] Further preferred, when using a sequential dosing method, there are no special requirements for the order of administration; more preferably, the FOXM1 inhibitor is administered first, followed by the BUB1 inhibitor.

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

[0028] Other FOXM1 inhibitors or their pharmaceutically acceptable salts, when used in combination with BUB1 inhibitors or their pharmaceutically acceptable salts, have the same therapeutic effect.

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

[0030] The combination therapy designed in this invention exhibits significant anti-tumor effects, with synergistic effects and low toxicity, making it particularly suitable for the treatment of liver cancer, triple-negative breast cancer, and melanoma. It overcomes the problems of poor efficacy, narrow indications, easy development of drug resistance, and high relapse rates associated with monotherapy, providing a new strategy for cancer treatment. Attached Figure Description

[0031] Figure 1This is a schematic diagram illustrating 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 < 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 apoptosis experiment in Example 1;

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

[0035] Figure 5 This is a statistical chart showing the in vivo tumor volume and weight of BAY-1816032 and / or FDI-6 in Example 2;

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

[0037] Figure 7 This is a schematic diagram illustrating the in vivo antitumor effects of BUB1 shRNA and / or FDI-6 in Example 3;

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

[0039] The technical solution of the present invention will be further described below with reference to the embodiments.

[0040] Materials and reagents used in the examples: Human hepatocellular carcinoma cells HUH7 and HepG2, and human malignant melanoma cells A-375 were cultured in DMEM medium and 10% fetal bovine serum (Bio-Channel) at 5% CO2 and 37°C; human triple-negative breast cancer cells MDA-MB-231 were cultured in L-15 medium and 10% fetal bovine serum at 5% CO2 and 37°C; BAY-1816032 and FDI-6 (MedChemExpress) were dissolved in DMSO. Apoptosis detection kit (KeyGEN) and crystal violet staining solution (KeyGEN) were also used.

[0041] MTT assay for cell proliferation: Cells were seeded at a density of 8000–10000 cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. After treatment with the appropriate drug for 3 days, cells were incubated with MTT dye for 3–4 h. The culture medium was discarded, and the cells were thoroughly dissolved and mixed with DMSO before cell proliferation was assessed. Cell growth inhibition rate = (1 - absorbance value of experimental group / absorbance value of control group) × 100%.

[0042] Clonal formation assay: 500-800 cells / well were seeded in 12-well plates and cultured overnight to allow cell adhesion. After adding the appropriate drug, the cells were cultured for 14 days. The cells were fixed with 4% paraformaldehyde for 15 minutes, then stained with 0.1% crystal violet for 10 minutes. The cells were then observed and photographed using an inverted fluorescence microscope.

[0043] Apoptosis assay: Cells were seeded at a density of 30%–50% per dish in 35 mm culture dishes and cultured overnight to allow cell adhesion. Then, the appropriate drugs were added for 3 days. Cells were digested with trypsin and washed twice with PBS. After that, cells were stained with propidium iodide and annexin V-FITC. Finally, the apoptosis status was analyzed using an Agilent NovoCyteQuanton flow cytometer.

[0044] Statistical analysis methods: SPSS 19.0 software was used for statistical analysis; continuous data are expressed as mean ± standard deviation (x±s). Student's t-test was used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, and LSD test was used for pairwise comparisons between groups. *P < 0.05 # P < 0.05 indicates that the difference is statistically significant.

[0045] Example 1: Evaluation of the in vitro inhibitory effects of FOXM1 inhibitor FDI-6 combined with BUB1 inhibitor BAY-1816032 on the proliferation of human hepatocellular carcinoma cells HUH7, HepG2, A-375, and MDA-MB-231.

[0046] 1. Experimental Methods

[0047] (1) MTT assay for cell proliferation

[0048] Four types of tumor cells were treated with DMSO (control), FDI-6, BAY-1816032, and a combination of FDI-6 and BAY-1816032. In the combination treatment group, the molar ratio of FDI-6 to BAY-1816032 was 1:(0.5–2). Cell proliferation was detected by MTT assay after 3 days.

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

[0050] The effect of combination drugs on cell proliferation was examined using a colony formation assay. Four types of tumor cell lines were treated with BAY-1816032 in combination with FDI-6. Specifically, for HUH7 cells, the dosage of FDI-6 monotherapy was 1.0 μM, the dosage of BAY-1816032 monotherapy was 1.0 μM, the dosage of the combined FDI-6 treatment was 0.5 μM, and the dosage of the combined BAY-1816032 treatment was 0.5 μM. For HepG2 cells, the dosage of FDI-6 monotherapy was 4.0 μM, the dosage of BAY-1816032 monotherapy was 4.0 μM, the dosage of the combined FDI-6 treatment was 2.0 μM, and the dosage of the combined BAY-1816032 treatment was 2.0 μM. For A375 cells, the dosage of FDI-6 monotherapy was 5.0 μM, the dosage of BAY-1816032 monotherapy was 5.0 μM, the dosage of FDI-6 combined with BAY-1816032 was 2.5 μM, and the dosage of BAY-1816032 combined with FDI-6 was 2.5 μM. For MDA-MB-231 cells, the dosage of FDI-6 monotherapy was 3.0 μM, the dosage of BAY-1816032 monotherapy was 3.0 μM, the dosage of FDI-6 combined with BAY-1816032 was 1.5 μM, and the dosage of BAY-1816032 combined with FDI-6 was 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 using an inverted fluorescence microscope.

[0051] (3) Apoptosis assay

[0052] After treating HUH7 cells with BAY-181603240.5μM combined with FDI-60.5μM for 3 days, cell apoptosis was detected by flow cytometry.

[0053] 2. Experimental Results

[0054] (1) MTT assay for cell proliferation

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

[0056] like Figure 1 As shown, when the molar ratio of FDI-6 and BAY-1816032 is in the range of 1:(0.5 to 2), the CI value of all three types of tumor cells is less than 1, indicating that BAY-1816032 combined with FDI-6 can synergistically inhibit the malignant proliferation of tumor cells within the measured molar ratio range.

[0057] (2) Number of cell clones

[0058] When human hepatocellular carcinoma cells HUH7 and HepG2, human malignant melanoma cells A-375, and human breast cancer cells MDA-MB-231 were treated with FDI-6 and BAY-1816032 in a 1:1 molar ratio, the number of cell clones was significantly lower than in the groups treated with FDI-6 or BAY-1816032 alone. Figure 2 This further validated the synergistic inhibitory effect of FDI-6 combined with BAY-1816032 on tumor cell growth.

[0059] (3) Apoptosis

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

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

[0062] Example 2: FDI-6, in combination with BAY-1816032, inhibited the growth of HUH7 xenograft tumors of human liver cancer cells in vivo.

[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% physiological saline; BAY-1816032 is prepared with 10% DMSO, 10% Tween, 30% PEG400 and 50% physiological saline.

[0067] (2) Laboratory animals

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

[0069] (3) Experimental steps

[0070] Human hepatocellular carcinoma HUH7 cell line was inoculated into the axillae of nude mice to construct a HUH7 xenograft model of human hepatocellular carcinoma cells. The tumors were allowed to grow to 50–70 mm. 3Animals were randomly divided into four groups: a HUH7 model group, an FDI-6 administration group, a BAY-1816032 administration group, and a sequential FDI-6 / BAY-1816032 administration group. The FDI-6 administration group received FDI-6 (20 mg / kg) intraperitoneally once daily for 21 days. The BAY-1816032 administration group received BAY-1816032 (20 mg / kg) intraperitoneally once daily for 21 days. The sequential administration group received FDI-6 (20 mg / kg) intraperitoneally once daily for 10 days, followed by BAY-1816032 (20 mg / kg) intraperitoneally once daily for 11 days (the molar ratio of FDI-6 to BAY-1816032 was 1:1.2). Tumor volume and mouse weight were measured every two days, and data were recorded. Nude mice were sacrificed on day 21. The tumor tissue block was surgically removed and weighed. The tumor inhibition rate (%) was calculated based on the weight and volume of the tumor tissue block.

[0071] 2. Experimental Results

[0072] Compared to the model group, tumor growth was inhibited to some extent in the FDI-6 and BAY-1816032 monotherapy groups. Compared to the monotherapy groups and the model group, the FDI-6 / BAY-1816032 sequential dosing group showed a significant reduction in both tumor weight and volume (the sequential dosing order is not limited to administering FDI-6 first and then BAY-1816032). Figures 4-5 ).

[0073] The sequential administration of FDI-6 / BAY-1816032 showed stronger inhibitory effects on HUH7 xenografts of human liver cancer cells than either the BAY-1816032 monotherapy group or the FDI-6 monotherapy group alone, with significant differences. This indicates that the sequential combination of BAY-1816032 and FDI-6 can exert a better anti-tumor effect in vivo.

[0074] Preliminary toxicity evaluation results showed that, compared with the model group, there was no significant change in body weight in the single-drug group and the sequential-drug group, but the heart, liver, spleen, lungs and kidneys of the mice were significantly damaged. Figure 6 Both FDI-6 and BAY-1816032 exhibited low toxicity, whether administered alone or in a sequential combination.

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

[0076] 1. Experimental Methods

[0077] (1) Test drug

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

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

[0080] (2) Laboratory animals

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

[0082] (3) Experimental steps

[0083] A stable cell line HUH7 BUB1 shRNA lentivirus and a stable cell line HUH7 NC shRNA lentivirus were constructed. HUH7 BUB1 shRNA and HUH7 NC shRNA cells in logarithmic growth phase were inoculated into the axillae of nude mice to construct xenograft tumor models of the corresponding cells. The HUH7 NC shRNA xenograft tumors were developed when they grew to 60–80 mm. 3 Animals were randomly divided into an NC shRNA group and an NC shRNA + FDI-6 administration group. Similarly, HUH7 BUB1 shRNA xenograft tumor models were randomly divided into a BUB1 shRNA group and a BUB1 shRNA + FDI-6 administration group. The FDI-6 administration group received FDI-6 (20 mg / kg) intraperitoneally once daily for 21 days. Tumor volume and mouse weight were measured every two days, and data were recorded. Nude mice were sacrificed on day 21. 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, tumor growth was inhibited to some extent in both the NC shRNA+FDI-6 group and the BUB1 shRNA group. Compared with the BUB1 shRNA group, tumor growth was significantly inhibited in the BUB1 shRNA+FDI-6 group. Figures 7-8 ).

[0086] Compared with the NC shRNA group, inhibiting BUB1 expression significantly suppressed the growth of human hepatocellular carcinoma xenografts. Compared with the NC shRNA + FDI-6 administration group, inhibiting 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 therapy (combined or sequential administration) designed in this invention can significantly inhibit the growth of tumor cells in vitro and in vivo, and has good prospects for clinical application.

Claims

1. The use of a FOXM1 inhibitor in combination with a BUB1 inhibitor in the preparation of a drug for treating cancer, wherein the cancer is liver cancer, triple-negative breast cancer, or malignant melanoma that highly expresses FOXM1 and BUB1, wherein the FOXM1 inhibitor is selected from FDI-6 or a pharmaceutically acceptable salt thereof, and the BUB1 inhibitor is selected from BAY-1816032 or a pharmaceutically acceptable salt thereof.

2. The application according to claim 1, characterized in that, The BUB1 inhibitor is a sensitizing agent for FOXM1 inhibitors.

3. The application according to claim 1, characterized in that, The FOXM1 inhibitor mentioned is FDI-6.

4. The application according to claim 1, characterized in that, The BUB1 inhibitor mentioned is BAY-1816032.

5. The application according to claim 1, characterized in that, The molar ratio of the FOXM1 inhibitor to the BUB1 inhibitor is 1:(0.5~2).

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

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