Pharmaceutical composition and application thereof

Abstract

The invention relates to a pharmaceutical composition, and belongs to the technical field of biological medicines. The pharmaceutical composition comprises a proteasome inhibitor and a WEE family kinase inhibitor. A proteasome inhibitor and a WEE family kinase inhibitor are combined for use, the proteasome inhibitor can promote tumor cells to generate M-phase retardation and multipolar spindle bodies and finally pyroptosis in the M phase, and G2 retardation induced by the proteasome inhibitor can be effectively removed by using the WEE family kinase inhibitor, so that the tumor cells can be effectively prevented from being damaged. Therefore, the tumor cells are prevented from entering the M phase to escape pyroptosis, and the two act together to synergistically promote the pyroptosis of the tumor cells. Through combination of the pharmaceutical composition, primary drug resistance of solid tumors to proteasome inhibitors such as BTZ can be overcome, and the pharmaceutical composition has a good inhibition effect on tumors (especially solid tumors) and has very important clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a pharmaceutical composition and use thereof. Background Art

[0002] Proteasome is an ideal molecular target for tumor treatment. As early as 2003, its inhibitor Bortezomib (BTZ) was approved by the US FDA for the treatment of mantle cell lymphoma. It has now become a commonly used chemotherapy drug for hematological tumors in clinical practice, with the drug name Velcade.

[0003] BTZ has shown good efficacy in the treatment of multiple myeloma as a single drug. Multiple clinical trials have confirmed that the overall response rate of multiple myeloma patients to BTZ is close to 40%, and the median time without disease progression is more than 6 months. When BTZ is used in combination with other drugs, the efficacy is even stronger. Studies have shown that when BTZ is used in combination with Melphalan and Prednisone, the complete response rate of multiple myeloma patients reaches 30%, the partial response rate reaches 71%, and the median time without disease progression reaches 24 months.

[0004] However, compared with the success of BTZ in the treatment of hematological tumors, BTZ is almost ineffective in the treatment of solid tumors alone. Several clinical trials have confirmed that the overall response rate of BTZ monotherapy in patients with various solid tumors such as neuroendocrine tumors, renal cancer, liver cancer and lung cancer is 0% or close to 0%, and the median time without disease progression is less than 2 months. The combination therapy of BTZ with drugs such as Gemcitabine and Docetaxel is also very weak. The complete response rate in patients with solid tumors such as pancreatic cancer, non-small cell lung cancer, colon cancer and breast cancer is 0%, the partial response rate is less than 10%, and the median time without disease progression is only about 2 months.

[0005] Therefore, there is an urgent need to find reasonable and effective new drug combination regimens with solid theoretical basis to overcome the primary resistance of solid tumors to proteasome inhibitors such as BTZ, promote the application of proteasome inhibitors in the treatment of solid tumors, and use old drugs in new ways, which is of very important clinical significance. Summary of the invention

[0006] Based on this, it is necessary to provide a pharmaceutical composition that can work synergistically and has a better inhibitory effect on tumors (especially solid tumors) in order to address the problem of primary drug resistance when proteasome inhibitors are currently used alone to treat solid tumors, as well as the problem that their random combination with some traditional chemotherapy drugs has very weak efficacy.

[0007] The invention discloses a pharmaceutical composition, which comprises a proteasome inhibitor and a WEE family kinase inhibitor.

[0008] The present invention has found through experiments that proteasome inhibitors (such as BTZ, CFZ or specific siRNA, etc.) can induce G2 and mitosis (M) phase arrest in tumor cells, which can lead to M phase arrest and multipolar spindles in tumor cells, and finally pyroptosis in the M phase, but at the same time, it will cause a large proportion of cells to be arrested in the G2 phase. The G2 phase arrest may protect tumor cells from entering the M phase to a certain extent, allowing them to escape the fate of pyroptosis.

[0009] Based on this research, the present invention explored how to relieve the G2 phase arrest induced by BTZ to allow more tumor cells to enter the M phase, and found that WEE family kinase inhibitors can effectively relieve the G2 phase arrest induced by proteasome inhibitors, thereby synergistically promoting tumor cell pyroptosis.

[0010] In one embodiment, the WEE family kinase includes at least one of WEE1, WEE1B and PKMYT1.

[0011] In one embodiment, the proteasome inhibitor is selected from at least one of small molecule compounds, CRISPR-Cas9, PROTAC, and siRNA; the WEE family kinase inhibitor is selected from at least one of small molecule compounds, CRISPR-Cas9, PROTAC, and siRNA. It is understandable that small molecule compounds refer to conventional small molecule drugs / compounds, such as bortezomib, carfilzomib, etc.; CRISPR-Cas9 system refers to gene editing technology, which can change protein activity or content by gene editing, silencing target, etc.; PROTAC (proteolysis-targeting chimeras) is a reagent system that uses the ubiquitin-proteasome system (UPS) to degrade target proteins; siRNA refers to small interfering RNA that can reduce the abundance of important subunits of the proteasome and inhibit its activity, which can be designed in a conventional manner.

[0012] In one embodiment, the proteasome inhibitor is selected from: Bortezomib, Carfilzomib, Ixazomib, Oprozomib (a proteasome inhibitor PR-047), Delanzomib (CEP-18770) or at least one of siRNAs of other proteasome subunits.

[0013] In one embodiment, the WEE family kinase inhibitor is selected from at least one of MK-1775, Zn-C3, WEE1-IN-5 and PD0166285.

[0014] In one embodiment, the proteasome inhibitor is bortezomib; the WEE family kinase inhibitor is PD0166285.

[0015] In one embodiment, the mass dosage ratio of the proteasome inhibitor to the WEE family kinase inhibitor is: 1:0.01-100, further 1:1-100, and further 1:10±5.

[0016] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0017] The present invention also discloses the use of a proteasome inhibitor and a WEE family kinase inhibitor in combination in the preparation of a drug for inhibiting tumors.

[0018] It can be understood that the combination of the proteasome inhibitor and the WEE family kinase inhibitor includes but is not limited to preparing them as a composition.

[0019] The invention also discloses the use of the above-mentioned pharmaceutical composition in preparing a drug for inhibiting tumors.

[0020] In some embodiments, the tumor is a solid tumor, or the tumor includes: osteosarcoma, neuroendocrine tumor, renal cancer, breast cancer, gastric cancer, cervical cancer, ovarian cancer, melanoma, head and neck cancer, esophageal cancer, colon cancer, liver cancer or lung cancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] A pharmaceutical composition of the present invention uses a proteasome inhibitor and a WEE family kinase inhibitor in combination, wherein the proteasome inhibitor can promote M phase arrest and multipolar spindles in tumor cells, and ultimately pyroptosis in the M phase, and the WEE family kinase inhibitor can effectively relieve the G2 arrest induced by the proteasome inhibitor, thereby preventing tumor cells from entering the M phase and escaping pyroptosis. The two work together to synergistically promote tumor cell pyroptosis.

[0023] The combined use of the drug composition can overcome the primary drug resistance of solid tumors to proteasome inhibitors such as BTZ, has a good inhibitory effect on tumors (especially solid tumors), and has very important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a representative diagram of the cell cycle phase distribution after BTZ treatment;

[0025] Figure 2 This is a statistical diagram of the proportion of cell cycle phases after BTZ treatment;

[0026] Figure 3 This is a statistical diagram of the proportion of cell cycle phases after CFZ treatment;

[0027] Figure 4 The effect of siRNA knocking down the mRNA level of proteasome ATPase subunits;

[0028] Figure 5 This is a statistical diagram of the cell cycle phase ratio after knocking down the proteasome ATPase subunit;

[0029] Figure 6 The effect of siPSMC5-2 knocking down PSMC5 mRNA level (A) and the statistical diagram of cell cycle phase ratio;

[0030] Figure 7 This is the fate map of M-phase tumor cells after BTZ treatment and the M-phase time statistics;

[0031] Figure 8 This is the fate map of M-phase tumor cells and the M-phase time statistics after knocking down PSMC5;

[0032] Fig. 9 This is a statistical diagram of the fate of M-phase cells after BTZ treatment;

[0033] Fig.10 This is a statistical diagram of the fate of M-phase cells after knockdown of PSMC5;

[0034] Fig.11 This is the result of BTZ inducing multipolar spindles in M ​​phase cells;

[0035] Fig.12 This is the result of knocking down PSMC5 to induce multipolar spindles in M ​​phase cells;

[0036] Fig.13 Western blot images of BTZ-induced upregulation of CHK1, WEE1, PKMYT1, and inhibitory CDK1;

[0037] Fig.14 Schematic diagram of drug treatments to evaluate the ability of different drugs to relieve BTZ-induced G2 arrest;

[0038] Fig.15 For tumor cells Fig.14 Statistical graphs of cell cycle phase ratios after indicated treatments;

[0039] Fig.16 The result diagram of BTZ / PD0166285 combination inducing more tumor cells to have pyroptosis morphology;

[0040] Fig.17 The result graph shows that the combination of BTZ / PD0166285 more significantly induced the increase of Annexin V / PI double-positive cells;

[0041] Fig.18 The immunoblot image shows that the combination of BTZ / PD0166285 more significantly induces GSDME cleavage;

[0042] Fig.19 The colony formation staining results of tumor cells treated with BTZ and MK-1775 or PD0166285 are shown;

[0043] Fig. 20 This is the relative quantitative result of colony of tumor cells treated with BTZ and MK-1775 or PD0166285;

[0044] Fig.21 is the dose-effect curve of BTZ treatment of tumor cells in the presence of Vehicle or PD0166285;

[0045] Fig. 22 is the dose-effect curve of BTZ treating tumor cells in the presence of Vehicle or Rabusertib;

[0046] Fig.23 This is a picture of tumors from an experiment in which BTZ / PD0166285 combination therapy inhibited tumor formation in mice;

[0047] Fig.24 This is a graph showing the tumor weight results of an experiment in which BTZ / PD0166285 combined with other drugs inhibited tumor formation in mice;

[0048] Fig.25 This is a picture of a tumor from an experiment in which BTZ / PD0166285 combined with other drugs inhibited tumor growth in mice;

[0049] Fig.26 This is the tumor growth curve of the BTZ / PD0166285 combination inhibition experiment on tumor growth in mice;

[0050] Fig. 27 The tumor weight of the BTZ / PD0166285 combination in mice was used to inhibit tumor growth.

[0051] Fig.28 The immunoblot results of GSDME in tumor tissues of the BTZ / PD0166285 combination inhibition experiment in mice. DETAILED DESCRIPTION

[0052] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0054] The reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the following examples, unless otherwise specified, are all achievable by conventional methods.

[0055] In the examples and figures, * means P<0.05; ** means P<0.01; *** means P<0.001; **** means P<0.0001; ns means no significant difference (no significance).

[0056] Example 1

[0057] 1. Exploring the effects of inhibiting proteasome activity on mitosis

[0058] The inventors found in previous studies and research that targeting the mitosis process is an effective anti-tumor method. Proteasomes play an important role in the mitosis process. Therefore, in order to explore the effect of inhibiting proteasome activity on mitosis, the following experiment was designed.

[0059] 1. Experimental methods

[0060] 1) The human liver cancer cell line SNU449 and the human cervical cancer cell line HeLa (purchased from ATCC) were treated with proteasome inhibitors Bortezomib (BTZ, purchased from Selleck, Catalog No. S1013), Carfilzomib (CFZ, purchased from Selleck, Catalog No. S2835) or small interfering RNA (siRNA) of proteasome ATPase subunits.

[0061] Among them, each siRNA is as follows:

[0062] NC: UUCUCCGAACGUGUCACGU, negative control, not targeting any human gene;

[0063] siPSMC1:GAACGUAGAAUGAAAGUAA (SEQ ID NO. 1);

[0064] siPSMC2: GUGCUUCAUUCGAGUUAUU (SEQ ID NO. 2);

[0065] siPSMC3: CCAUGAAGGACAAGAUAAA (SEQ ID NO. 3);

[0066] siPSMC4:GGGAAGACCAUGUUGGCAA (SEQ ID NO.4);

[0067] siPSMC5-1:AGAAGAAAGUGUUGGUCAA (SEQ ID NO.5);

[0068] siPSMC5-2:GGAACGAACUAAAUGCUAA (SEQ ID NO. 6);

[0069] siPSMC6: AGAGUUAUUUCAGCGUGUA (SEQ ID NO. 7).

[0070] The above siPSMC1-6 indicates that its corresponding target is the ATPase subunit PSMC1-6 of the proteasome, and each siRNA has dTdT added to the 3' end to increase the resistance of siRNA to nucleases.

[0071] The specific experimental methods are as follows:

[0072] The BTZ and CFZ drug powders were dissolved in DMSO to a 5 mM mother solution. Among them, BTZ was used to treat SNU449 or HeLa cells with gradient concentrations of 0nM, 10nM, 15nM and 20nM, and CFZ was used to treat SNU449 cells with gradient concentrations of 0nM, 10nM, 15nM, 20nM and 50nM. All siRNAs (NC, siPSMC1, siPSMC2, siPSMC3, siPSMC4, siPSMC5-1 or -2, siPSMC6) were used at a concentration of 20nM. The transfection method is as follows. All embodiments used Invitrogen's Lipofectamine RNAiMAX transfection reagent for siRNA transfection. Taking 24-well cell culture plates as an example, other specification well plates can increase or decrease the liquid volume in proportion to the size, and prepare 100 μL opti-MEM per well. TM(reduced serum medium), add a certain amount of siRNA to make the final concentration of 20nM, shake thoroughly to mix, then add 1μL Lipofectamine RNAi MAX, gently flick to mix, let stand at room temperature for 15 minutes, then add the transfection complex to a 24-well plate, and finally add 500μL of cell suspension of appropriate concentration (SNU449 or HeLa, confluence 30-40%), shake horizontally and place in a cell culture incubator.

[0073] 2) Cells with a confluence of 30-40% were treated with BTZ (SNU449 or HeLa) and CFZ (SNU449) drugs (such as Figure 1-3 indicated concentrations) for 30 h or siRNA (20 nM, Figure 4-6 ) 60 hours after transfection, cells were stained with propidium iodide (PI) and histone 3 serine 10 phosphorylation (pH3-S10, indicating mitosis) antibodies and detected by flow cytometry. Cells in the G0 / G1 phase, S phase, G2 phase, and M phase were distinguished based on the signals of PI and pH3-S10. The specific method is as follows:

[0074] After the cells were treated, 0.25% trypsin (0.02% EDTA) was used to digest and collect the cells, centrifuged at 300g for 5 minutes, the supernatant was removed, and the cells were washed once with 4°C pre-cooled 1×PBS, centrifuged under the same conditions, the supernatant was removed, 300μL 1×PBS was used to resuspend the cells, and 700μL-20°C pre-cooled 1×PBS was added to fix the cells, adding dropwise while shaking, and placed in a -20°C refrigerator for pre-cooling overnight. Next, the fixed cell suspension was pipetted into a 1.5mL EP tube, centrifuged at 600g for 5 minutes, the supernatant was removed, the cells were flicked, 250μL 0.25% Triton X-100in 1×PBS was added, the membrane was broken at room temperature for 10 minutes, 1mL pre-cooled 1×PBS was added, centrifuged at 600g for 5 minutes, the supernatant was removed, 120μL 1% BSA in 1×PBS / T (0.1% Tween-20) was added to each tube, and the tube was rotated and blocked in an incubator at room temperature for 30 minutes. Add 0.25 μL Phospho-Histone 3 (S10)-AF647 antibody to each tube, incubate at room temperature for 1 hour, add 1 mL of pre-cooled 1× PBS, wash with rotation for 5 minutes, centrifuge at 600 g for 5 minutes, flick the cells, add appropriate amount of 1× PBS to resuspend, add 1 μL of propidium iodide (PI; 2 mg / mL), filter through a 400-mesh filter into the sample tube, and detect on the machine (blue and red light channels).

[0075] 2. Experimental results

[0076] The results are as follows Figure 1-6 As shown, Figure 1 The figure is a representative figure of the flow cytometry results of SNU449 or HeLa cells treated with BTZ. The numbers in the boxes indicate the percentage of cells in the gate, and the numbers above the boxes indicate the drug concentration (nM). Figure 2 for Figure 1 The statistical graph of the percentage of cells in each phase of three independent repeated experiments shows that BTZ treatment can significantly increase the proportion of cells in the G2 phase and M phase, and as the drug concentration increases, the effect of increasing the number of G2 phase cells is stronger, while the increase of M phase cells is weakened.

[0077] Figure 3 This is a statistical graph of the percentage of cells in each phase after SNU449 cells were treated with CFZ. The dosage used is shown in the legend. It can be seen from the figure that CFZ treatment can also significantly increase the proportion of cells in the G2 phase and the M phase.

[0078] Figure 4 Figure 2. Effect of knocking down the mRNA level of the six proteasome ATPase subunits after transfection of SNU449 or HeLa cells with siRNA. Figure 5 This is a statistical graph of the percentage of cells in each phase of the cell cycle after knocking down the corresponding ATPase subunit. It can be seen from the figure that inhibiting proteasome activity by knocking down PSMC5 can also significantly increase the proportion of cells in the G2 phase and M phase.

[0079] Figure 6 After SNU449 or HeLa cells were transfected with the second siRNA targeting PSMC5 (siPSMC5-2, with a different sequence from siPSMC5-1), the effect of knocking down the PSMC5 mRNA level (A) and its effect on the cell cycle phase distribution (B). As can be seen from the figure, transfection with siPSMC5-2 can also significantly increase the ratio of G2 phase to M phase cells, further confirming that inhibiting proteasome activity by knocking down PSMC5 can significantly increase the ratio of G2 phase to M phase cells.

[0080] Based on this, the results of cell cycle analysis showed that the three ways of inhibiting proteasome activity, namely BTZ, CFZ or siRNA, could induce G2 or M phase arrest in tumor cells, and siPSMC5-1 had the best blocking effect on G2 and M phases.

[0081] 2. Observing the fate of M-phase tumor cells after inhibition of proteasome activity

[0082] On the basis of the above studies, in order to further observe whether inhibition of proteasome activity can lead to the death of tumor cells accumulated in the M phase, we constructed an SNU449 cell line that stably expresses Histone H2B-EGFP (green, indicating the cell nucleus) and mCherry-α-Tubulin (red, indicating microtubules). After the cells were treated with BTZ or siPSMC5, they were placed in a live cell real-time imager for photography.

[0083] 1. Methods

[0084] 1.1 Construction of SNU449 cell line stably expressing Histone H2B-EGFP and mCherry-α-Tubulin.

[0085] First, according to the routine operation, we used the fusion PCR method to clone the fusion fragment of H2B-EGFP and mCherry-α-Tubulin, and then connected it to the pCDH vector using the BamHI / XbaI restriction site to complete the construction of H2B-EGFP and mCherry-α-Tubulin expression vectors, named pCDH-H2B-EGFP and pCDH-mCherry-α-Tubulin, respectively.

[0086] Then, we performed lentiviral packaging experiments on these two plasmids and inoculated 3×10 5 / well HEK293T cells (purchased from ATCC), and the next day 200ng PMD2G, 600ng psPA×2 lentiviral packaging plasmid and 1200ng target plasmid (pCDH-H2B-EGFP or pCDH-mCherry-α-Tubulin) were co-transfected into the cells by calcium phosphate transfection method.

[0087] The process of calcium phosphate transfection is briefly described as follows: add 105μL of sterile water to a 1.5mL EP tube, then add 15μL of 2M CaCl2 solution to the bottom of the water, mix the three plasmids on the tube cap and add the water layer, then slowly add 125μL of 2×HBS solution above the water layer, finally insert the tip of the gun into the bottom of the tube, slowly blow out bubbles, bring the bottom CaCl2 to the upper layer and mix with the plasmid and HBS buffer, let stand at room temperature for 15 minutes, replace 2mL of fresh culture medium in the plate wells, add the transfection complex dropwise into the plate wells, replace fresh culture medium 12 hours later, aspirate the culture medium 48 hours after replacing the culture medium, centrifuge at 1000g for 10 minutes, aspirate an appropriate amount of supernatant for cell infection or divide into portions and store in a -80 degree refrigerator. Inoculate SNU449 cells with a confluence of 30% in a 24-well plate, dilute the virus solution collected above by one half with fresh culture medium, add polybrene with a final concentration of 10ug / mL, mix well and add to the well plate, replace fresh culture medium for 24 hours after infection and continue to culture, add puromycin with a final concentration of 2ug / mL to the culture medium when the cells are full of passage for selection, and replace normal culture medium for 24 hours and continue to culture. First infect the virus expressing H2B-EGFP, select with puromycin, and then infect the virus expressing mCherry-α-Tubulin. After infection, use MoFlo Astrios Eqs flow sorter for cell sorting, obtain SNU449 cells that can simultaneously express H2B-EGFP and mCherry-α-Tubulin (double positive), and expand the culture to complete the construction of the cell line.

[0088] 1.2 Observation of the fate of tumor cells accumulated in the M phase after inhibition of proteasome activity

[0089] Control solvent (Vehicle) or BTZ (30nM) was added to SNU449 cells stably expressing H2B-EGFP (green) and mCherry-α-Tubulin (red), or NC (20nM) or siPSMC5-1 / 2 (two siRNAs mixed: 10nM siPSMC5-1+10nM siPSMC5-2) were transfected, and then the cells were placed in a living cell imager for real-time photography every 20 minutes.

[0090] 2. Results

[0091] The results are as follows Figure 7-8 As shown. Among them, Figure 7 and 8Representative diagrams of the fate of M phase cells after nuclear envelope breakdown (NEBD) by BTZ and siPSMC5 (Scale bar = 5 μm). It can be seen from the figure that BTZ or siPSMC5 treatment leads to M phase arrest, the spindle changes from normal bipolar to abnormal multipolar, and finally the cells show dynamic characteristics of pyroptosis.

[0092] Fig. 9 and 10 They are Figure 7 and 8 From the temporal fate statistics of cells in the M phase, it can be seen that most cells die in the M phase.

[0093] Fig.11 and 12 After the cells were treated with BTZ (15 nM) or siPSMC5 (20 nM), immunofluorescence staining was performed using antibodies against Pericentrin (PCNT, red, indicating centrosomes) and α-tubulin (TUBA, green, indicating microtubules) and DAPI (blue, indicating chromosomes) dye (Scale bar = 2.5 μm). The above results further confirmed that both treatments can induce multipolar spindles in M ​​phase cells.

[0094] The above results show that inhibiting proteasome activity will lead to M phase arrest and multipolar spindle in tumor cells, and eventually pyroptosis in the M phase.

[0095] 3. Explore how to relieve BTZ-induced G2 arrest

[0096] The above studies show that although inhibiting proteasome activity can induce abnormal M phase and pyroptosis in tumor cells, it will also cause a large proportion of cells to be blocked in the G2 phase. The G2 phase block may protect tumor cells from entering the M phase to a certain extent, allowing them to escape the fate of pyroptosis.

[0097] Therefore, we next explored how to relieve BTZ-induced G2 phase arrest to allow more tumor cells to enter the M phase and undergo pyroptosis.

[0098] 1. Changes in the levels of CHK family kinases (CHK1 and CHK2), WEE family kinases (WEE1 and PKMYT1), and inhibitory CDK1

[0099] We detected the protein levels of the key kinases of the G2 / M transition checkpoint, CHK family kinases (CHK1 and CHK2) and WEE family kinases (WEE1 and PKMYT1), as well as their downstream effector molecule inhibitory CDK1 (phosphorylated at tyrosine 15, abbreviated as pCDK1-Y15), as follows:

[0100] After SNU449 cells were treated with BTZ (20 nM) for 18 hours, proteins were collected for immunoblotting.

[0101] The results are as follows Fig.13 As shown, except CHK2, CHK1, WEE1, PKMYT1 and pCDK1-Y15 were all up-regulated by BTZ.

[0102] 2. Effects of inhibition of CHK1 and WEE family kinases on BTZ-induced G2 arrest

[0103] Next, we investigated whether inhibition of CHK1 and WEE family kinases could relieve BTZ-induced G2 arrest. We used the CHK1 inhibitor Rabusertib and the WEE family inhibitors MK-1775 (which mainly inhibits WEE1 activity) and PD0166285 (which can simultaneously inhibit WEE1 and PKMYT1 activity).

[0104] like Fig.14 As shown in the treatment schematic diagram, after the cells were treated with BTZ (20 nM) for 18 hours, the above three drugs or control solvents were added at a concentration of 0.5 μM. After further culture for 2 hours, the cells were collected for flow cytometry analysis to detect cell cycle distribution.

[0105] The results are as follows Fig.15 As shown, groups ①-⑥ are as follows: Group ① was treated with the control solvent Vehicle for 18 hours; Group ② was treated with 20 nM BTZ for 18 hours; Group ③ was treated with 20 nM BTZ for 18 hours, followed by the addition of the control solvent Vehicle for a further 2 hours; Group ④ was treated with 20 nM BTZ for 18 hours, followed by the addition of the WEE1 inhibitor 0.5 μM MK-1775 for a further 2 hours; Group ⑤ was treated with 20 nM BTZ for 18 hours, followed by the addition of the WEE1 and PKMYT1 co-inhibitor 0.5 μM PD0166285 for a further 2 hours; Group ⑥ was treated with 20 nM BTZ for 18 hours, followed by the addition of the CHK1 inhibitor 0.5 μM Rabusertib for a further 2 hours. Compared with the control solvent-treated group, WEE family kinase inhibitors MK-1775 and PD0166285 were able to significantly reduce the proportion of cells in the G2 phase and increase the proportion of cells in the M phase, suggesting that they can effectively relieve BTZ-induced G2 phase arrest, while the CHK1 inhibitor Rabusertib had no such effect.

[0106] 4. Verification of synergistic effect

[0107] From the above results, it can be seen that inhibiting proteasome activity can induce M phase arrest, multipolar spindle and pyroptosis in M ​​phase cells, but at the same time it will cause a large proportion of cells to be arrested in the G2 phase. The inventors considered that since WEE family kinase inhibitors can effectively relieve the G2 phase arrest induced by proteasome inhibitors, thereby promoting more tumor cells to enter the M phase, the inventors creatively combined proteasome inhibitors with WEE family kinase inhibitors to examine their ability to synergistically promote tumor cell pyroptosis.

[0108] 1. Methods

[0109] After 48 hours of treatment with BTZ (20nM) and PD0166285 (0.25μM) alone or in combination, SNU449 or HeLa cells were photographed in bright field to observe the proportion of cells with pyroptotic morphology (hot air balloon-like, flat and transparent), and the cells treated as above were stained with Annexin V / PI and detected by flow cytometry to count the proportion of Annexin V / PI double-positive cells. In addition, according to the literature, pyroptosis caused by chemotherapy drugs is often mediated by the N-terminal fragment (GSDME-N) produced after GSDME cleavage. We also collected protein samples of cells treated as above and analyzed the cleavage of GSDME by immunoblotting.

[0110] 2. Results

[0111] The results are as follows Figure 16-17 As shown, Fig.16 The upper part is a representative image of phase contrast microscope bright field photos of cells after being treated with BTZ and PD0166285 alone or in combination, and the lower part is a statistical graph of the proportion of cells with pyroptotic morphology in each group (the upper part is a representative image, and the lower part is a statistical graph). Fig.17 It is the proportion of Annexin V / PI double-positive cells after cells were treated with BTZ and PD0166285 alone or in combination. Fig.18 The results of GSDME immunoblot detection after cells were treated with BTZ and PD0166285 alone or in combination.

[0112] From the above results, it can be seen that compared with the control group, a few cells with pyroptosis appeared in the BTZ-treated group, very few pyroptotic cells were found in the PD0166285-treated group, and a large number of pyroptotic cells appeared in the combined group ( Fig.16 ). Consistent with this, the number of Annexin V / PI double-positive cells increased only slightly in the single-drug treatment group, but increased significantly in the combination group ( Fig.17 ), GSDME had only a small amount of shearing in the single-drug treatment group, but significant shearing occurred in the combination group ( Fig.18 ), which indicates that the combination of BTZ / PD0166285 can promote more cell pyroptosis.

[0113] 5. Cytotoxicity Assay

[0114] 1. Methods

[0115] 1) Colony formation assay of SNU449 and HeLa cells

[0116] On the first day, 1000 (SNU449) or 1500 (HeLa) cells were attached to a 24-well plate; on the second day, cells were treated with BTZ / PD0166285 alone or in combination, and the drugs were changed every three days for a total of 9 days. When the cells were collected, they were first rinsed twice with 1mL 1×PBS, then fixed with methanol at room temperature for 15 minutes, then 500μL 0.1% crystal violet stain was added, stained at room temperature for 10 minutes, rinsed three times with 1mL 1×PBS, dried, scanned and photographed, and after the staining and photography were completed, the crystal violet was redissolved in 200μL methanol, and its OD570 value was measured using an ELISA instrument to perform relative quantification of surviving cells.

[0117] 2) Cell viability assay

[0118] On the first day, cells were seeded at appropriate density in a 96-well plate, and treated with corresponding drugs on the second day. After 48 hours, cell viability was determined using an Alamar blue kit, and a dose-effect curve was drawn.

[0119] 2. Results

[0120] The results are as follows Figure 19-22 As shown, Fig.19 Photographs of crystal violet staining of SNU449 or HeLa cells treated with BTZ / MK-1775 or BTZ / PD0166285 alone or in combination. Fig. 20 Relative quantitative results of viable cells after SNU449 or HeLa cells were treated with BTZ / MK-1775 or BTZ / PD0166285 alone or in combination. Fig.21 and 22 The BTZ dose-effect curves were drawn based on the results of SNU449 cell viability detection after treatment with BTZ and PD0166285 or BTZ and Rabusertib.

[0121] From the results of colony formation, it can be seen that the combination of proteasome inhibitor BTZ and WEE family kinase inhibitor MK-1775 or PD0166285 can synergistically kill tumor cells ( Figure 19-20 ). The results of Alamar blue cell viability assay showed that compared with the control group, PD0166285 significantly reduced the 50% maximum inhibitory concentration (IC50) of BTZ on tumor cells ( Fig.21), while the CHK1 inhibitor Rabusertib had no such effect ( Fig. 22 ), suggesting that WEE family kinase inhibitors can significantly enhance the sensitivity of tumor cells to BTZ, while CHK1 inhibitors have no such sensitizing effect, which is consistent with their ability to relieve BTZ-induced G2 arrest.

[0122] Example 2

[0123] BP-Combo (BTZ / PD0166285 combination) inhibits tumor formation in mice.

[0124] First, 5 million HeLa cells were inoculated subcutaneously into the hind limbs of nude mice. One day later, vehicle, 0.65 mg / kg BTZ, 0.256 mg / kg PD0166285, or BP-Combo (same dose as single drug) were intraperitoneally injected, followed by administration every three days for a total of three times. Tumor tissues were collected 30 days after subcutaneous inoculation.

[0125] The results are as follows Figure 23-24 As shown, Fig.23 The upper part shows the schematic diagram of drug treatment, and the lower part shows the tumor photo; Fig.24 The results show that compared with the control and single-drug treatment groups, the BP-Combo group did not form obvious tumors as a whole, and the tumor weight was the lightest, indicating that BP-Combo can more significantly inhibit tumor formation.

[0126] Example 3

[0127] BP-Combo (BTZ / PD0166285 combination) inhibits tumor growth in mice.

[0128] First, 5 million HeLa cells were inoculated subcutaneously into the hind limbs of nude mice. After 38 days, when the tumor grew to about 50 mm3, vehicles, 1 mg / kg BTZ, 0.256 mg / kg PD0166285, or BP-Combo (same dose as single drug) were intraperitoneally injected, and then administered once every three days for a total of five times. Tumor tissues were collected 53 days after subcutaneous inoculation.

[0129] The results are as follows Figure 25-28 As shown, Fig.25 The upper part shows the schematic diagram of drug treatment, and the lower part shows the tumor photo; Fig.26 are the tumor growth curves of each group; Fig. 27 is a statistical graph of tumor weight; Fig.28This is an immunoblot image to detect the shearing of GSDME in tumor tissues of each group. The results showed that compared with the control and single-drug treatment groups, the tumors in the BP-Combo group grew slower and weighed less, suggesting that BP-Combo can more significantly inhibit tumor growth in mice ( Figure 25-27 ). In addition, by extracting and analyzing the protein of tumor tissue, it was found that ( Fig.28 ), BP-Combo can more significantly induce the cleavage of GSDME in vivo, suggesting that BP-Combo may inhibit the development of tumors in mice by inducing tumor pyroptosis.

[0130] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0131] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A pharmaceutical composition, characterized in that These include proteasome inhibitors and WEE family kinase inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that The WEE family kinases include at least one of WEE1, WEE1B and PKMYT1.

3. The pharmaceutical composition according to claim 1, characterized in that The proteasome inhibitor is selected from at least one of small molecule compounds, CRISPR-Cas9, PROTAC, and siRNA; the WEE family kinase inhibitor is selected from at least one of small molecule compounds, CRISPR-Cas9, PROTAC, and siRNA.

4. The pharmaceutical composition according to claim 3, characterized in that The proteasome inhibitor is selected from: at least one of Bortezomib, Carfilzomib, Ixazomib, Oprozomib, Delanzomib or other proteasome subunit siRNA.

5. The pharmaceutical composition according to claim 1 or 3, wherein the WEE family kinase inhibitor is selected from at least one of MK-1775, Zn-C3, WEE1-IN-5 and PD0166285.

6. The pharmaceutical composition according to claim 5, characterized in that The proteasome inhibitor is bortezomib; the WEE family kinase inhibitor is PD0166285.

7. The pharmaceutical composition according to claim 1, characterized in that The mass dosage ratio of the proteasome inhibitor to the WEE family kinase inhibitor is 1:10±5.

8. Use of a proteasome inhibitor and a WEE family kinase inhibitor in combination for preparing a drug for inhibiting tumors.

9. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for inhibiting tumors.

10. The use according to claim 8 or 9, characterized in that The tumor is a solid tumor, or the tumor includes: osteosarcoma, neuroendocrine tumor, renal cancer, breast cancer, gastric cancer, cervical cancer, ovarian cancer, melanoma, head and neck cancer, esophageal cancer, colon cancer, liver cancer or lung cancer.

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