Pharmaceutical composition for treating liver cancer and application thereof

Abstract

This application provides a pharmaceutical composition and its application for treating liver cancer, relating to the field of biomedical technology. The pharmaceutical composition includes active ingredients, namely the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009. By combining the proteasome inhibitor Bortezomib with the Rev-Erb agonist SR9009, synergistic anti-liver cancer activity is observed both in vitro and in vivo, enhancing the mechanism of protein toxicity-induced apoptosis in liver cancer cells. The combined effect of Bortezomib and Rev-Erb agonist SR9009 is significantly superior to either Bortezomib or Rev-Erb agonist alone, overcoming the poor efficacy of Bortezomib monotherapy in liver cancer, and demonstrating significant synergistic effects and clinical application value.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a pharmaceutical composition and its application for treating liver cancer. Background Technology

[0002] Liver cancer (primarily hepatocellular carcinoma, HCC) is one of the most common malignant tumors worldwide, characterized by high incidence, high mortality, and poor prognosis. Current clinical treatments for liver cancer include surgical resection, liver transplantation, interventional therapy, and drug therapy. However, due to the difficulty in early diagnosis, most patients are diagnosed at an advanced stage, losing the opportunity for radical surgical treatment. Therefore, systemic drug therapy has become a crucial approach.

[0003] Bortezomib, a proteasome inhibitor, is a well-established anti-tumor drug that primarily works by inhibiting the ubiquitin-proteasome pathway, leading to the accumulation of misfolded proteins in tumor cells and subsequently inducing endoplasmic reticulum stress and apoptosis. While this drug has shown significant efficacy in hematologic malignancies such as multiple myeloma, its widespread application in solid tumors like liver cancer is limited by inherent drug resistance, significant dose-limiting toxicities (such as peripheral neuropathy), and poor efficacy as a single agent. Therefore, developing a drug that can effectively inhibit the proliferation of liver cancer cells is a pressing issue in the field of liver cancer treatment. Summary of the Invention

[0004] The purpose of this application is to provide a pharmaceutical composition and application for treating liver cancer, aiming to solve the problems of poor efficacy and strong drug resistance of Bortezomib monotherapy in the prior art for treating liver cancer.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a pharmaceutical composition for treating liver cancer, the pharmaceutical composition comprising active ingredients, the active ingredients including the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009.

[0006] In one or more embodiments of this application, the chemical structural formula of the proteasome inhibitor Bortezomib is as follows: The chemical structural formula of Rev-Erb agonist SR9009 is as follows: .

[0007] In one or more embodiments of this application, the molar ratio of the proteasome inhibitor Bortezomib to the Rev-Erb agonist SR9009 is 1:2000 to 1:50.

[0008] In one or more embodiments of this application, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

[0009] In one or more embodiments of this application, the excipients include one or more of excipients, diluents, disintegrants, lubricants, adhesives, or stabilizers.

[0010] In one or more embodiments of this application, the dosage form of the pharmaceutical composition is an injection, a lyophilized powder for injection, or an oral preparation.

[0011] Secondly, this application provides the use of a proteasome inhibitor in combination with a Rev-Erb agonist in the preparation of a medicament for treating liver cancer.

[0012] In one or more embodiments of this application, the proteasome inhibitor is Bortezomib, and the Rev-Erb agonist is SR9009. The chemical structural formula of Bortezomib is [insert chemical formula here]. The chemical structural formula of SR9009 is .

[0013] In one or more embodiments of this application, the molar ratio of the proteasome inhibitor Bortezomib to the Rev-Erb agonist SR9009 is 1:2000 to 1:50.

[0014] In one or more embodiments of this application, the proteasome inhibitor combined with the Rev-Erb agonist includes administering the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009 simultaneously, separately, or sequentially.

[0015] Based on the above technical solutions, the pharmaceutical composition and application for treating liver cancer of this application have at least the following beneficial technical effects: The active ingredients of the pharmaceutical composition for treating liver cancer in this application include the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009. By combining the proteasome inhibitor Bortezomib with the Rev-Erb agonist SR9009, a synergistic anti-liver cancer activity was observed both in vitro and in vivo. The combined effect of Bortezomib and Rev-Erb agonist SR9009 was significantly superior to either Bortezomib or SR9009 alone, enhancing the mechanism of protein toxicity-induced apoptosis in liver cancer cells and overcoming the poor efficacy of Bortezomib monotherapy in liver cancer. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the structures of the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009. Figure 1 A is a schematic diagram of the chemical structure of Bortezomib. Figure 1 Figure B is a schematic diagram of the chemical structure of SR9009.

[0018] Figure 2 This is a graph showing the in vitro experimental results of the synergistic anti-hepatocellular carcinoma study of Bortezomib (abbreviated as Bort) and SR9009. Figure 2 A shows the proliferation results of CCK8 cells in different experimental drug groups. Figure 2 B is a graph showing the synergistic effect of Bortezomib combined with SR9009 in three types of liver cancer cells in different experimental drug groups.

[0019] Figure 3 This is a diagram showing the in vivo experimental results of the synergistic anti-liver cancer study of Bortezomib (abbreviated as Bort) and SR9009. Figure 3 A is a schematic diagram of tumor tissue growth. Figure 3 B is a diagram illustrating the weight of the tumor tissue.

[0020] Figure 4 These are Western blot images of proteins from different drug groups.

[0021] Figure 5 The images show the expression levels of ubiquitinated protein (UB) and the internal reference protein β-actin in the 1% NP-40 insoluble fraction from different drug groups, as well as images of ubiquitinated misfolded protein aggregation detected by immunofluorescence staining. Figure 5 A is a graph showing the expression levels of ubiquitinated protein (UB) and internal reference protein β-actin in the 1% NP-40 insoluble fraction in different drug groups; Figure 5 B is an image of ubiquitinated misfolded protein aggregation in different drug groups.

[0022] Figure 6 This is a graph showing the experimental results of protein toxicity and apoptosis induction in liver cancer cells in different drug groups. Figure 6 A is a flow cytometry scatter plot of different drug groups. Figure 6 B is a statistical analysis graph of liver cancer cell apoptosis in different drug groups. Figure 6C is a statistical graph of liver cancer cell viability in different drug groups. Detailed Implementation

[0023] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0024] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0025] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0026] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0027] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0028] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0029] Bortezomib, a proteasome inhibitor, is a well-established anti-tumor drug that primarily works by inhibiting the ubiquitin-proteasome pathway, leading to the accumulation of misfolded proteins in tumor cells and subsequently inducing endoplasmic reticulum stress and apoptosis. While it has shown significant efficacy in hematologic malignancies such as multiple myeloma, its widespread application in solid tumors like liver cancer is limited by inherent drug resistance, significant dose-limiting toxicities (such as peripheral neuropathy), and poor efficacy as a monotherapy.

[0030] SR9009 is a Rev-Erb agonist initially investigated for its role in regulating circadian rhythms and metabolic diseases. However, its synergistic effects with proteasome inhibitors, particularly its combined use in inducing apoptosis in liver cancer cells through enhanced protein toxicity, are currently undocumented. Therefore, identifying combination therapy strategies that can sensitize the anti-liver cancer effects of Bortezomib while reducing its toxic side effects is of significant clinical value.

[0031] Based on this, this application provides a pharmaceutical composition for treating liver cancer, the pharmaceutical composition comprising active ingredients, the active ingredients being the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009. The chemical formula of the proteasome inhibitor Bortezomib is as follows: The chemical formula of Rev-Erb agonist SR9009 is [chemical formula missing]. ,like Figure 1 As shown.

[0032] The technical solution of this application will be described in detail below with reference to specific embodiments.

[0033] Example 1 The proteasome inhibitor Bortezomib, in combination with the Rev-Erb agonist SR9009, synergistically inhibits the growth of hepatocellular carcinoma cells in vitro.

[0034] (1) The liver cancer cells and drugs involved.

[0035] Human liver cancer cells: PLC / PRF5, Huh7 and HepG2 cells, all purchased from the Cell Bank of the Chinese Academy of Sciences.

[0036] Drugs: Proteasome inhibitor Bortezomib and Rev-Erb agonist SR9009. Bortezomib was purchased from MCE (catalog number HY-10227), and SR9009 was purchased from MCE (catalog number HY-16989).

[0037] (2) Experimental methods.

[0038] The three different types of liver cancer cells were cultured in complete cell culture medium (DMEM medium + 10% fetal bovine serum) until the cell confluence was approximately 80%. After digestion, the cells were seeded into 96-well plates at a rate of 3000 cells per well. After culturing for another 16 hours, the cells were treated with different drug concentrations as shown in the following groups for 48 hours. Then, the culture medium was discarded, and 100 μL of DMEM (containing 10% CCK8 assay reagent) was added to each well. After culturing in a cell incubator for 1 hour, the OD value at a wavelength of 450 nm was measured using a multi-mode microplate reader.

[0039] The drug treatment groups are as follows: Group 1: Liver cancer cells were treated with Bortezomib (abbreviated as Bort) at working concentrations of 0 nM, 12.5 nM, 25 nM, 50 nM and 100 nM, respectively.

[0040] Group 2: Liver cancer cells were treated with Bortezomib (abbreviated as Bort) at working concentrations of 0 nM, 12.5 nM, 25 nM, 50 nM and 100 nM, respectively, in combination with 10 μM of SR9009.

[0041] Group 3: Liver cancer cells were treated with Bortezomib (abbreviated as Bort) at working concentrations of 0 nM, 12.5 nM, 25 nM, 50 nM and 100 nM, respectively, in combination with 20 μM of SR9009.

[0042] After the experiment, the CCK8 assay value of 0 nM Bortezomib in the first group was taken as 100% cell viability, and the percentage of cell viability after all treatments was calculated. The experiment was repeated three times, and finally, cell viability curves for different drug treatments were plotted based on the experimental data. Figure 2 As shown in Figure A. Following the standard operating procedure of the Synergy Finder website (https: / / synergyfinder.org / ), Synergy Finder is a standalone web application for interactive analysis and visualization of multi-dose combination response data for multiple drugs (≥2 types). Using Bliss as the analytical reference model, cell viability under different drug combinations was input, and the Bliss synergy scores of Bortezomib (abbreviated as Bort) and SR9009 were calculated. A value greater than 10 was considered a synergistic relationship, and a value greater than 0 and less than / equal to 10 was considered an additive relationship. The results are shown below. Figure 2 As shown in B.

[0043] (3) Experimental results.

[0044] Experimental results are as follows Figure 2 As shown in A, Figure 2Figure A shows the proliferation results of CCK8 cells in different experimental drug groups. Figure 2 As shown in A, the combination of Bortezomib with 10 μM or 20 μM SR9009 can significantly reduce the IC50 value of Bortezomib (abbreviated as Bort) against liver cancer cells, indicating that Bortezomib combined with SR9009 has a strong inhibitory effect on liver cancer cells. Liver cancer cells are highly sensitive to the drug combination of Bortezomib and SR9009, and the inhibitory effect on liver cancer cells is stronger with the increase of SR9009 concentration.

[0045] Experimental results are as follows Figure 2 As shown in B, Figure 2 B shows the synergistic effect of Bortezomib combined with SR9009 in three types of liver cancer cells in different experimental drug groups. Figure 2 As shown in B, the Bliss synergistic scores of Bortezomib combined with SR9009 were all greater than 10, indicating that Bortezomib combined with SR9009 can significantly synergistically inhibit the growth of HCC cells in vitro.

[0046] Example 2 The proteasome inhibitor Bortezomib, in combination with the Rev-Erb agonist SR9009, synergistically inhibits the growth of liver cancer cells in vivo.

[0047] (1) The liver cancer cells, animals and drugs involved.

[0048] Mouse-derived liver cancer cells: Hepa1-6 cells, purchased from the Cell Bank of the Chinese Academy of Sciences.

[0049] Animals: Six-week-old female C57BL / 6 mice, purchased from Vital Rivers.

[0050] Drugs: proteasome inhibitor Bortezomib (purchased from MCE, catalog number HY-10227) and Rev-Erb agonist SR9009 (purchased from MCE, catalog number HY-16989).

[0051] (2) Experimental methods.

[0052] Six-week-old female C57BL / 6 mice were subcutaneously inoculated with Hepa1-6 cells at a rate of 1 million cells per mouse. One week later, all mice were randomly divided into four groups of seven mice each, and then administered the drug via intraperitoneal injection (twice a week) according to the following grouping: (1) Control group: control solvent DMSO.

[0053] (2) Bortezomib monotherapy group (abbreviated as Bort group): 0.5 mg / Kg.

[0054] (3) SR9009 monotherapy group: 60 mg / Kg.

[0055] (4) Bortezomib + SR9009 combined administration group: 0.5 mg / Kg Bortezomib + 60 mg / Kg SR9009.

[0056] After four consecutive administrations, the mice were euthanized, the tumor tissue was removed, and weighed.

[0057] (3) Experimental results.

[0058] Experimental results are as follows Figure 3 As shown in A, Figure 3 A is a schematic diagram of tumor tissue growth, from which... Figure 3 As shown in Figure A, compared with the control group, the Bortezomib monotherapy group, and the SR9009 monotherapy group, the tumor volume of the Bortezomib + SR9009 combination therapy group was significantly reduced. Figure 3 B is a schematic diagram of tumor weight. Compared with the control group, the Bortezomib monotherapy group, and the SR9009 monotherapy group, the tumor weight of the Bortezomib + SR9009 combination therapy group was significantly reduced.

[0059] Example 3 The proteasome inhibitor Bortezomib, combined with the Rev-Erb agonist SR9009, enhances the protein toxicity and induces apoptosis in liver cancer cells.

[0060] (1) The liver cancer cells, animals and drugs involved.

[0061] Human liver cancer cells (purchased from the Chinese Academy of Sciences Cell Bank): PLC / PRF5, HepG2, and Huh7 cells.

[0062] Drugs: proteasome inhibitor Bortezomib (purchased from MCE, catalog number HY-10227), Rev-Erb agonist SR9009 (purchased from MCE, catalog number HY-16989), and apoptosis inhibitor Z-VAD-FMK (purchased from MCE, catalog number HY-16658B).

[0063] (2) Experimental scheme.

[0064] Experimental scheme 1: The aforementioned liver cancer cells were treated in the following groups for 48 hours: (1) Control group: control solvent DMSO.

[0065] (2) Bortezomib monotherapy group (abbreviated as Bort group): working concentration 20 nM.

[0066] (3) SR9009 monotherapy group: working concentration 20 μM.

[0067] (4) Bortezomib (working concentration 20 nM) + SR9009 (working concentration 20 μM). After drug treatment, the following tests were performed: a. Western blot detection of ER stress molecular markers: After drug treatment, cells were discarded, washed once with PBS, and then lysed using 1x SDS loading buffer. The cell lysate was boiled at 100°C for 10 minutes, followed by SDS-PAGE gel separation and PVDF membrane transfer. The PVDF membrane after protein transfer was blocked with 5% skim milk TBS buffer and incubated with the ER stress molecular markers ATF4, CHOP, and the internal control protein β-actin primary antibody. After incubation overnight at 4°C, the PVDF membrane was washed three times, followed by incubation with HRP-labeled secondary antibody at room temperature for 1 hour. The PVDF membrane was washed three times with TBST solution and then exposed to ECL chemiluminescence solution for detection.

[0068] b. Expression levels of ubiquitinated protein (UB) and internal control protein β-actin in the 1% NP-40 insoluble fraction: After drug treatment, cells were discarded from the culture medium, washed once with PBS, and then lysed on ice for 30 minutes with 1% NP40 cell lysis buffer (150 mM NaCl, 1 mM EDTA, 1% NP-40, 50 mM Tris-HCl, pH 8.0, and protease inhibitor). Cells were then centrifuged at 14,000 × g at 4°C for 15 minutes, the supernatant was discarded, and the precipitate (1% NP-40 insoluble fraction) was dissolved in 1 x SDS loading buffer. The precipitate solution was boiled at 100°C for 10 minutes, followed by SDS-PAGE gel separation and PVDF membrane transfer. The PVDF membrane after protein transfer was blocked with 5% skim milk TBS buffer and incubated with ubiquitinated protein and internal control protein β-actin primary antibodies. After incubation at 4°C overnight, the PVDF membrane was washed three times, followed by incubation with HRP-labeled secondary antibody at room temperature for 1 hour. The PVDF membrane was then washed three times with TBST solution and exposed to ECL luminescent solution for detection.

[0069] c. Immunofluorescence staining for the aggregation of ubiquitinated misfolded proteins: After drug treatment, cells were discarded from the culture medium, washed once with PBS, and fixed with 4% paraformaldehyde for 15 minutes. Then, cells were permeabilized with 0.2% Triton X-100 PBS for 15 minutes, followed by blocking with 3% BSA PBS for 1 hour. After blocking, cells were blocked overnight at 4°C with UB-K48 antibody dilution buffer, incubated at room temperature for 1 hour with fluorescent secondary antibody dilution buffer, washed three times with PBST, and stained with DAPI solution for 2 minutes. Finally, cells were washed three times with PBS, and the staining was observed and images were acquired using a fluorescence microscope.

[0070] d. Annexin-PI double staining combined with flow cytometry for apoptosis detection: Cells treated with the drug (including floating cells) were collected and washed once with PBS. The cells were then resuspended in 1× binding buffer, and the density was adjusted to 500,000 cells / mL. 100 μL of cell suspension was taken, and 5 μL of Annexin V-FITC and 5 μL of PI were added sequentially. The mixture was gently mixed and incubated at room temperature in the dark for 15 minutes. After incubation, 400 μL of 1× binding buffer was added, and the cells were then analyzed by flow cytometry. Annexin V-positive cells were considered apoptotic cells, and the percentage of apoptotic cells under different treatment conditions was calculated.

[0071] Experimental scheme 2: The aforementioned liver cancer cells were treated in the following groups for 48 hours: (1) Control group: control solvent DMSO.

[0072] (2) Bortezomib monotherapy group (abbreviated as Bort group): working concentration 20 nM.

[0073] (3) SR9009 monotherapy group: working concentration 20 μM.

[0074] (4) Bortezomib + SR9009 combined administration group: Bortezomib (working concentration 20 nM) + SR9009 (working concentration 20 μM).

[0075] (5) Bortezomib + SR9009 combined administration group + apoptosis inhibitor group: Bortezomib (working concentration 20 nM) + SR9009 (working concentration 20 μM) + Z-VAD-FMK (working concentration 5 μM).

[0076] (6) Bortezomib + SR9009 combined administration group + apoptosis inhibitor group: Bortezomib (working concentration 20 nM) + SR9009 (working concentration 20 μM) + Z-VAD-FMK (working concentration 10 μM).

[0077] After drug treatment, the culture medium was discarded, and 100 μL of DMEM (containing 10% CCK8 detection reagent) was added to each well. After culturing in a cell incubator for 1 hour, the OD value at a wavelength of 450 nm was detected by a multi-functional microplate reader. The control group was taken as 100% cell viability, and the cell viability percentage of each group was calculated.

[0078] (3) Experimental results.

[0079] Experimental results are as follows Figure 4 As shown, compared with the control group and the single-drug group, the expression of the molecular markers ATF4 and CHOP of ER stress was significantly upregulated in the Bortezomib and SR9009 combination therapy group. Figure 5 As shown in Figure A, compared with the control group and the single-drug group, the level of ubiquitinated protein in the 1% NP-40 insoluble fraction was significantly upregulated in the Bortezomib and SR9009 combination therapy group. Figure 5 As shown in Figure B, compared with the control group and the single-drug group, the aggregation of ubiquitinated misfolded protein (UB-K48 ubiquitinated protein indicated by green fluorescence) was significantly aggravated in the Bortezomib and SR9009 combination therapy group. Figure 6 As shown in Figures A and 6B, compared with the control group and the single-drug group, the proportion of Annexin V positive cells in the Bortezomib and SR9009 combination therapy group was significantly increased, indicating a significant increase in the number of apoptotic cells. Figure 6 As shown in Figure C, the apoptosis inhibitor Z-VAD-FMK successfully reversed the decrease in cell viability induced by the combined use of Bortezomib and SR9009, indicating that the combination of Bortezomib and SR9009 inhibits cell viability by intensifying apoptosis. These experimental results demonstrate that the combination of Bortezomib and SR9009 enhances the protein toxicity of liver cancer cells and induces apoptosis.

[0080] This study demonstrates that Bortezomib combined with SR9009 significantly upregulates unfolded protein response markers and aggregated ubiquitinated misfolded proteins in hepatocellular carcinoma (HCC) cells. It also confirms that apoptosis inhibitors can reverse the anti-HCC effect of the combination therapy. This indicates that the combination therapy induces endoplasmic reticulum stress-mediated apoptosis in HCC cells by enhancing protein toxicity stress, thereby achieving a synergistic anti-tumor effect. Cell viability was detected using the CCK-8 assay, and SynergyFinder analysis showed that the BLISS score of the combination of SR9009 and Bortezomib was greater than 10 in all three HCC cell types, suggesting a good synergistic effect between SR9009 and Bortezomib in anti-HCC. A mouse model of HCC bearing tumors was established, and Bortezomib was treated with the combination of SR9009 and SR9009. The results showed that the tumor weight in the combination therapy group was significantly smaller than that in the single-drug group and the control group, indicating that the combination therapy has good safety and significant tumor-suppressing effect in vivo.

[0081] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A pharmaceutical composition for treating liver cancer, characterized in that, The pharmaceutical composition includes active ingredients, which include the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009.

2. The pharmaceutical composition according to claim 1, characterized in that, The chemical structural formula of the proteasome inhibitor Bortezomib is as follows: The chemical structural formula of the Rev-Erb agonist SR9009 is as follows: .

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The molar ratio of the proteasome inhibitor Bortezomib to the Rev-Erb agonist SR9009 is 1:2000 to 1:

50.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition also includes one or more pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The excipients include one or more of the following: excipients, diluents, disintegrants, lubricants, adhesives, or stabilizers.

6. The pharmaceutical composition according to claim 1, characterized in that, The dosage form of the pharmaceutical composition is an injection, a lyophilized powder injection, or an oral preparation.

7. Application of proteasome inhibitors in combination with Rev-Erb agonists in the preparation of drugs for the treatment of liver cancer.

8. The application according to claim 7, characterized in that, The proteasome inhibitor is Bortezomib, the Rev-Erb agonist is SR9009, and the chemical structural formula of Bortezomib is as follows: The chemical structural formula of SR9009 is as follows: .

9. The application according to claim 8, characterized in that, The molar ratio of the proteasome inhibitor Bortezomib to the Rev-Erb agonist SR9009 is 1:2000 to 1:

50.

10. The application according to claim 8, characterized in that, The proteasome inhibitor combined with Rev-Erb agonist includes administering the proteasome inhibitor Bortezomib and the Rev-Erb agonist SR9009 simultaneously, separately, or sequentially.

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