A pharmaceutical composition comprising chloφromazine or a pharmaceutically acceptable salt thereof and its use in the preparation of a medicament for treating liver cancer

Abstract

The application discloses a pharmaceutical composition containing chlorpromazine or a pharmaceutically acceptable salt thereof and application thereof in preparation of a medicine for treating liver cancer. 50 The application proves the effect of combined administration of chlorpromazine and sorafenib through in-vivo and in-vitro experiment verification, proves that the IC 30%-35% is reduced, and the sensitivity of the liver cancer cell to sorafenib is enhanced; and in a Hepa1-6 subcutaneous tumor animal model, the tumor size of the combined administration of chlorpromazine and sorafenib is reduced by 45%-50% relative to the sorafenib administration alone, the tumor weight is reduced by 50%-55%, the liver cell carcinoma progression is significantly delayed, and the treatment effect of the liver cell carcinoma is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a pharmaceutical composition comprising chlorpromazine or a pharmaceutically acceptable salt thereof and its use in the preparation of a drug for treating liver cancer. Background Technology

[0002] Sorafenib resistance is a major challenge that shortens the survival time of patients with hepatocellular carcinoma and increases treatment costs.

[0003] Sorafenib (Common name: Sorafenib; Chemical name: 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide; Molecular formula: C 21 H 16ClF3N4O3 (molecular weight: 464.82 g / mol), originally developed by Bayer AG, Germany, and marketed under the brand name Nexavar®, was approved by the FDA in 2005. It is a first-line treatment for systemic hepatocellular carcinoma, ushering in a new era of targeted therapy for liver cancer. Sorafenib, as a multi-target kinase inhibitor, can effectively block tumor angiogenesis by inhibiting the activity of receptors closely related to angiogenesis, such as vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGF), and block the RAF / MEK / ERK-mediated tumor cell proliferation signaling pathway, thereby exerting an anti-tumor effect (Sorafenib resistance and therapeutic strategies in hepatocellular carcinoma. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2025, 1880(3):189310). Sorafenib, as a first-line drug for the treatment of advanced liver cancer, has also been shown to be an inducer of ferroptosis in recent years. Sorafenib induces ferroptosis in liver cancer cells through multiple pathways, the most important of which is the blocking of the cysteine / glutamate reverse transporter System Xc-. Sorafenib impairs cysteine ​​uptake and GSH synthesis by inhibiting the transcription of SLC7A11, a key subunit of System Xc-. As an important intracellular antioxidant, the decrease in GSH levels prevents cells from effectively clearing lipid peroxides, ultimately leading to ferroptosis (Understanding sorafenib-induced ferroptosis and resistance mechanisms: Implications for cancer therapy. European Journal of Pharmacology, 2023, 955:175913).

[0004] Currently, there are two main solutions for sorafenib resistance in clinical practice. One is to use second-line targeted therapies such as regorafenib and apatinib. However, these second-line therapies have more side effects and higher treatment costs compared to sorafenib. The other is to use immune checkpoint inhibitors such as atezolizumab and bevacizumab in combination with anti-angiogenic drugs. This combination therapy can prolong the survival of hepatocellular carcinoma patients who do not respond to sorafenib treatment when the patient's condition is good and the tumor burden is under control. However, the treatment cost of immune checkpoint inhibitors is extremely high. For example, the unified medical insurance price of atezolizumab (trade name: Tecentriq) in the Chinese market is 32,800 yuan / vial (specification: 1200mg / 20ml), and this price has remained stable since it was included in medical insurance in 2023. If calculated according to the conventional treatment course (once every 3 weeks, 4 times for one course), the cost of a single course is approximately 131,200 yuan. Even with 50% reimbursement from medical insurance, patients would still need to pay more than 60,000 yuan out of pocket. Therefore, finding alternative drugs with lower treatment costs, mature clinical applications, and good efficacy is of great significance.

[0005] The paper “TDP-43 upregulates lipid metabolism modulator ABHD2 to suppress apoptosis in hepatocellular carcinoma” discloses that TDP-43 can inhibit apoptosis of liver cancer cells by upregulating the expression of ABHD2. However, no drugs targeting TDP-43 have been developed, and no drugs that inhibit ABHD2 have been found. Compared with existing drugs such as chlorpromazine, the research and development costs are too high, and the success rate is difficult to guarantee.

[0006] Chlorpromazine (Common English name: Chlorpromazine; Chemical name: 3-(2-chloro-10H-phenthiazin-10-yl)-N,N-dimethylpropyl-1-amine; Molecular formula: C2 17 H 19ClN2S (molecular weight: 318.86 g / mol) belongs to the first generation of phenothiazines. Chlorpromazine was first approved for marketing by the U.S. Food and Drug Administration (FDA) in 1957 with chlorpromazine hydrochloride as the active ingredient. The original developer was Rhône-Poulenc in France, and the patent was subsequently licensed to the SmithKline & French Corporation (SKF) in the United States, and marketed under the brand name Thorazine® (History of the Discovery and Clinical Introduction of Chlorpromazine. Annals of Clinical Psychiatry. 2005;17 (3),113-135). Subsequently, many companies have produced generic versions of it, and it has been used clinically for over 70 years.

[0007] The main mechanism of action of chlorpromazine is to block neurotransmitter receptors such as D2 receptor, α1-adrenergic receptor, and 5-HT2A receptor to exert sedative and antipsychotic effects. Its molecular structure consists of a tricyclic phenothiazine core + propylamine side chain + terminal tertiary amine + chlorine atom at position 2. The phenothiazine core provides a hydrophobic plane, which binds to dopamine D2 receptor, histamine H1 receptor, and α1-adrenergic receptor through π-π stacking and van der Waals forces, forming the basic framework for drug-receptor recognition. Its rigid structure also maintains the specific spatial orientation of the drug into the pocket. The sulfur atom on the ring enhances the lipophilicity of the molecule, making it easy to cross the blood-brain barrier. The propylamine side chain keeps the terminal nitrogen atom 5-7 Å away from the phenothiazine plane, allowing the drug to bind to two key sites of the D2 receptor simultaneously. The terminal tertiary amine binds to the aspartic acid residue of the D2 receptor at physiological pH, which is the key driving force for drug action (ACSChemical Neuroscience. 2019;10(1),79-88).

[0008] As a clinically mature drug, chlorpromazine has a well-defined traditional application area and definite efficacy. Its core indications are schizophrenia and manic-depressive disorder, especially effective for "positive symptoms" such as hallucinations, delusions, thought disorders, and behavioral disturbances, but less effective for negative symptoms such as emotional blunting and social withdrawal (the past, present, and future of psychosis management. Br Med Bull. 2025 Sep 22;156 (1):ldaf016). With the advent of atypical antipsychotics with better efficacy and fewer side effects, chlorpromazine is no longer a first-line treatment for antipsychotics. However, based on its central nervous system depressant mechanism, chlorpromazine has been developed for many other therapeutic uses. Based on its medullary depressant effect, chlorpromazine is used to treat intractable hiccups and is currently the only FDA-approved treatment for this condition (Pharmacologic Interventions for Intractable and Persistent Hiccups: A Systematic Review. JEmerg Med. 2017 Oct; 53 (4): 540-549). Due to its muscle relaxant effect, chlorpromazine has also been used as adjunctive therapy for tetanus to relieve tetanic spasms and paroxysmal convulsions (Stephen D. Silberstein, hael J. Marmura & Hsiangkuo Yuan. Essential Neuropharmacology. Published online by Cambridge University Press: 06 October 2020. 25-Chlorpromazine). However, there are no reports of its use as adjunctive therapy for hepatocellular carcinoma. Summary of the Invention

[0009] Purpose of the invention: The first objective of this invention is to provide a pharmaceutical composition comprising chlorpromazine or a pharmaceutically acceptable salt thereof.

[0010] A second object of the present invention is to provide the use of a pharmaceutical composition comprising chlorpromazine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of liver cancer.

[0011] Technical solution: In order to achieve the above objectives, the present invention provides a pharmaceutical composition comprising chlorpromazine or a pharmaceutically acceptable salt thereof, the pharmaceutical composition further comprising sorafenib or a pharmaceutically acceptable salt thereof.

[0012] The chlorpromazine or its pharmaceutically acceptable salts include chlorpromazine or chlorpromazine hydrochloride.

[0013] The mass ratio of chlorpromazine or its pharmaceutically acceptable salt to sorafenib is 1:3 to 1:6.

[0014] The dosage form of the pharmaceutical composition includes tablets, capsules, granules, oral solutions, emulsions, or suspensions. The injection method includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, or intracavitary injection.

[0015] The present invention also provides the use of a pharmaceutical composition comprising chlorpromazine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of liver cancer.

[0016] The pharmaceutical composition includes sorafenib and chlorpromazine or a pharmaceutically acceptable salt thereof.

[0017] The mass ratio of chlorpromazine or its pharmaceutically acceptable salt to sorafenib is 1:3 to 1:6.

[0018] The liver cancer mentioned includes hepatocellular carcinoma.

[0019] The pharmaceutical composition is a composition that utilizes chlorpromazine to inhibit the expression of ABHD2, thereby increasing the sensitivity of liver cancer cells to ferroptosis and alleviating the resistance of liver cancer cells to sorafenib.

[0020] The liver cancer cells mentioned include one or more of Hepa1-6 cells, Huh7 cells, or HCCLM3 cells.

[0021] Furthermore, the effects of the application are as follows: in a subcutaneous tumor animal model, the combination of chlorpromazine and sorafenib reduced tumor size by more than 45% and tumor weight by more than 50% compared to the sorafenib-only group.

[0022] Furthermore, the steps for establishing the subcutaneous tumor animal model are as follows:

[0023] (1) Hepatocellular carcinoma cells were injected subcutaneously into mice. When the orally inoculated tumors grew to a size that was easy to handle, the tumor tissue was separated and cut into 1 mm pieces. 3 The tissue blocks of that size were transferred subcutaneously to a new batch of mice;

[0024] (2) When the tumor grows to a volume of 100 mm³, mice are divided into groups for treatment;

[0025] (3) During the treatment, the growth of the tumor was closely monitored and recorded. After the treatment, the mice were sacrificed, and the tumors were weighed, H&E stained and Ki67 stained.

[0026] Furthermore, the liver cancer cells described in step (1) include, but are not limited to, Hepa1-6 cells.

[0027] Furthermore, the mice described in step (1) include, but are not limited to, C57BL / 6J mice.

[0028] Furthermore, the grouped dosing treatment described in step (2) includes sorafenib treatment alone and sorafenib combined with chlorpromazine treatment.

[0029] Furthermore, in the in vivo mouse experiments, chlorpromazine was prepared at 10 mg / kg and administered intraperitoneally immediately before use; sorafenib was prepared at 30 mg / kg and administered by gavage immediately before use.

[0030] Beneficial Effects: The combination of chlorpromazine and sorafenib provided by this invention has a significant inhibitory effect on the growth of hepatocellular carcinoma. After combined administration of chlorpromazine and sorafenib, the sorafenib IC50 of Hepatocellular carcinoma cells Hepa1-6 was significantly reduced. 50 It reduced tumor size by 30%-35% and enhanced the sensitivity of liver cancer cells to sorafenib. In the Hepa1-6 subcutaneous tumor animal model, the combination of chlorpromazine and sorafenib reduced tumor size by 45%-50% and tumor quality by 50%-55% compared to the sorafenib-only group, significantly delaying the progression of hepatocellular carcinoma and improving the treatment effect. Attached Figure Description

[0031] Figure 1 In the figure, A represents the verification diagram of different liver cancer cell lines overexpressing ABHD2. Figure 1 B in the figure represents the validation diagram of ABHD2 knockdown / knockout in different liver cancer cell lines;

[0032] Figure 2 The graph shows the effect of lipohydrolase ABHD2 on the antitumor effect of sorafenib. Figure 2 In the figure, A represents the cell survival rate of ABHD2-expressing hepatocellular carcinoma cell lines after treatment with different concentrations of sorafenib. Figure 2 In the figure, B represents the IC50 of ABHD2-expressing hepatocellular carcinoma cell lines treated with different concentrations of sorafenib. 50 Curve graph Figure 2 In the graph, C represents the growth curve of a sorafenib-treated ABHD2-expressing hepatocellular carcinoma cell line. Figure 2 In the figure, D represents the cell survival rate of ABHD2 hepatocellular carcinoma cell lines treated with different concentrations of sorafenib. Figure 2 E in the figure represents the IC50 of different concentrations of sorafenib in the treatment of ABHD2 knockdown / knockout hepatocellular carcinoma cell lines. 50 curve, Figure 2 F in the figure represents the growth curve of the ABHD2 hepatocellular carcinoma cell line treated with sorafenib;

[0033] Figure 3Figure showing the expression level of ABHD2 protein in Hepa1-6 cells treated with chlorpromazine.

[0034] Figure 4 The image shows the effect of chlorpromazine combined with sorafenib on liver cancer cells. Figure 4 In the graph, A represents the survival rate of liver cancer cells. Figure 4 B in the text represents sorafenib IC 50 Line graph;

[0035] Figure 5 A schematic diagram illustrating the combined administration of chlorpromazine and sorafenib for the treatment of hepatocellular carcinoma.

[0036] Figure 6 The image shows the effect of combined administration of chlorpromazine and sorafenib on subcutaneous tumors. Figure 6 In the image, A represents a gross image of the transplanted tumor. Figure 6 B in the image represents the tumor weight measurement. Figure 6 In the graph, C represents the tumor growth monitoring status.

[0037] Figure 7 In the image, A represents the results of H&E staining and Ki67 staining of the tumor. Figure 7 B in the graph represents the quantitative result of Ki67 staining. Detailed Implementation

[0038] The technical solution provided by the present invention will be described in detail below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions are all carried out according to conventional steps.

[0039] Example 1: Chlorpromazine combined with sorafenib inhibits liver cancer cells.

[0040] 1. The effect of lipid hydrolase ABHD2 on the therapeutic effect of sorafenib

[0041] The sorafenib used in this embodiment is catalog number S125098, purchased from Aladdin, and prepared at a concentration of 10 mM using dimethyl sulfoxide (DMSO). The mouse hepatocellular carcinoma cells Hepa1-6 were purchased from ATCC, and the human hepatocellular carcinoma cell lines Huh7 and HCCLM3 were purchased from the Tumor Cell Bank of the Chinese Academy of Medical Sciences. They were cultured in DMEM high-glucose medium (containing double antibiotics) with 10% FBS.

[0042] ABHD2 was overexpressed in Hepa1-6 and Huh7 liver cancer cells, respectively. The overexpression plasmid pLVX-ABHD2 (1 μg, pLVX vector sequence shown in SEQ ID NO. 1, pLVX-ABHD2 sequence shown in SEQ ID NO. 2) was mixed with ExFectTransfection Reagent (2 μl, purchased from Vazyme, catalog number: T101-01) and 50 μl of DMEM high-glucose medium (Jiangsu Kaiji Biotechnology Co., Ltd.). After incubation at room temperature for 15 min, the mixture was added to 450 μl of DMEM high-glucose medium in Hepa1-6 cells or Huh7 cells at a cell density of 75%, respectively. After culturing for 8-12 h, the medium was replaced with DMEM high-glucose complete medium. The overexpression effect is shown in the figure. Figure 1 As shown in Figure A, compared with the empty vector plasmid group, the ABHD2 expression level in cells overexpressing ABHD2 was significantly increased, indicating that ABHD2 was successfully overexpressed.

[0043] The next step was to knock down / knock out ABHD2 in Hepa1-6 and HCCLM3 liver cancer cells, respectively. The knockdown plasmid pSUPER-retor-puor-vertor-Sh-ABHD2-1 (sequence shown in SEQ ID NO. 17) was constructed on the vector pSUPER-retor-puor-vertor (empty vector sequence shown in SEQ ID NO. 16) using primers mABHD2-shABHD2#1-F (sequence shown in SEQ ID NO. 3) and mABHD2-shABHD2#1-R (sequence shown in SEQ ID NO. 4); the knockdown plasmid pSUPER-retor-puor-vertor-Sh-ABHD2-2 (sequence shown in SEQ ID NO. 18) was constructed on the vector pSUPER-retor-puor-vertor using primers mABHD2-shABHD2#2-F (sequence shown in SEQ ID NO. 5) and mABHD2-shABHD2#2-R (sequence shown in SEQ ID NO. 6); and the mABHD2- scramble-F (sequence shown in SEQ ID NO. 7) was used to construct the knockdown plasmid pSUPER-retor-puor-vertor-Sh-ABHD2-2 on the vector pSUPER-retor-puor-vertor. A control plasmid (SEQ ID NO. 19) was constructed on the vector pSUPER-retor-puor-vertor using scramble-R (sequence shown in SEQ ID NO. 8) to knock down the plasmid. The knockout plasmid Lenti-CRISPR-V2-sg-ABHD2 (sequence shown in SEQ ID NO. 14) was constructed on the vector Lenti-CRISPR-V2 (sequence shown in SEQ ID NO. 13) using primers hABHD2-Len-sg-1 (sequence shown in SEQ ID NO. 9) and hABHD2-Len-sg-2 (sequence shown in SEQ ID NO. 10); the control plasmid Lenti-CRISPR-V2-ctr (sequence shown in SEQ ID NO. 15) was constructed using primers hABHD2-Len-ctr-1 (sequence shown in SEQ ID NO. 11) and hABHD2-Len-ctr-2 (sequence shown in SEQ ID NO. 12).

[0044] In Hepa1-6, knockdown plasmids pSUPER-retor-puor-vertor-Sh-ABHD2-1 and pSUPER-retor-puor-vertor-Sh-ABHD2-2 were used (the control plasmid was transformed into the control plasmid (sequence shown in SEQ ID NO.19)). In HCCLM3, knockout plasmid Lenti-CRISPR-V2-sg-ABHD2 was used (the control plasmid Lenti-CRISPR-V2-ctr was transformed into the control plasmid (sequence shown in SEQ ID NO.15)).

[0045] The knockdown / knockout plasmid was mixed with ExFect Transfection Reagent (2 μl, purchased from Vazyme, catalog number: T101-01) and 50 μl of DMEM high glucose medium (Jiangsu Kaiji Biotechnology Co., Ltd.), and incubated at room temperature for 15 min. Then, it was added to 450 μl of DMEM high glucose medium culture system of Hepa1-6 cells or HCCLM3 cells at a cell density of 75%. After culturing for 8-12 h, the medium was replaced with DMEM high glucose complete medium.

[0046] Knockdown / knockout efficiency, such as Figure 1 As shown in B, compared with the control group (NC), the expression level of ABHD2 in cells transfected with knockdown / knockout plasmids was significantly reduced, indicating that ABHD2 knockdown / knockout was successful.

[0047] Sorafenib was prepared at concentrations of 2 μM, 4 μM, 6 μM, and 8 μM and administered to ABHD2-overexpressing hepatocellular carcinoma cells (n=3) and a control group transfected with empty vector plasmids, respectively. Cell viability was determined by the MTT assay 72 h later.

[0048] Experimental results are as follows Figure 2 As shown in Figure A, with increasing sorafenib concentration, the viability of Huh7 and Hepa1-6 cells significantly decreased. However, compared to the empty vector group, the viability of liver cancer cells overexpressing ABHD2 significantly rebounded, reversing the cell damage induced by sorafenib. Meanwhile, inhibition curves were plotted with the drug concentration (lg) as the x-axis and cell survival rate as the y-axis, as shown in Figure A. Figure 2 As shown in B, the calculated drug concentration, IC50, corresponds to a 50% inhibition of cell growth. 50 The results showed that overexpression of ABHD2 in liver cancer cells increased the IC50 value of sorafenib. 50 value.

[0049] Both the ABHD2-overexpressing hepatocellular carcinoma cell group and the control group transfected with empty vector plasmid were treated with 5 μM sorafenib for 4 days. Growth curves were plotted with growth time on the x-axis and cell viability on the y-axis. Figure 2 As shown in C, the results show that overexpression of ABHD2 reduces the inhibitory effect of sorafenib on tumor cell growth and proliferation, proving that overexpression of ABHD2 can reduce the sensitivity of liver cancer cells to sorafenib.

[0050] Next, sorafenib concentration gradients were prepared and administered to the ABHD2 knockdown / knockout hepatocellular carcinoma group and the blank control group, respectively. Cell viability and IC50 were measured using the MTT assay after 72 h. 50 The result is as follows Figure 2As shown in D, with increasing sorafenib concentration, the viability of HCCLM3 and Hepa1-6 cells significantly decreased, and compared with the control (NC) group, the decrease in cell viability was more significant after knockdown / knockout of ABHD2. Figure 2 As shown in E, the IC50 of sorafenib in hepatocellular carcinoma cells with knockdown / knockout of ABHD2 is... 50 Significantly reduced.

[0051] HCCLM3 and Hepa1-6 cells in the ABHD2 knockdown / knockout group and the control group were treated with sorafenib (HCCLM3, 7 μM; Hepa1-6, 3 μM) for 4 days. Growth curves were plotted with growth time on the x-axis and cell viability on the y-axis. The results are as follows: Figure 2 As shown in F, knocking down / knockout of ABHD2 enhances the inhibitory effect of sorafenib on tumor cell growth and proliferation, demonstrating that knocking down / knockout of ABHD2 increases the sensitivity of liver cancer cells to sorafenib and aggravates sorafenib-induced cell death.

[0052] Figure 2 The AF data were analyzed using the t-test and expressed as mean ± standard error (SEM) (n=3); differences were considered statistically significant when p < 0.05 (*) and p < 0.01 (**).

[0053] 2. Chlorpromazine inhibits ABHD2 expression.

[0054] The chlorpromazine used in this embodiment has the approval number H31021061 and was purchased from Shanghai Hefeng Pharmaceutical Co., Ltd.

[0055] Hepa1-6 cells were treated with chlorpromazine at three different concentrations (10, 20, and 30 μM). After 24 hours, the cells were harvested, and protein was extracted for Western blot analysis to determine the expression level of ABHD2 protein. The experimental results are as follows: Figure 3 As shown, chlorpromazine administration significantly reduced the expression of ABHD2 in liver cancer cells.

[0056] 3. Chlorpromazine increases the sensitivity of sorafenib to inhibit liver cancer cells by inhibiting ABHD2 expression.

[0057] Hepa1-6 cells overexpressing ABHD2 and those in the empty vector control group were treated with a combination of chlorpromazine (CPZ) and sorafenib. Chlorpromazine was prepared at a concentration of 5 μM, and sorafenib was prepared at gradient concentrations of 2 μM, 4 μM, 6 μM, and 8 μM. The negative control was DMSO alone. Cell viability was determined by the MTT assay after 72 h, and the IC50 was calculated. 50 The result is as follows Figure 4 As shown.

[0058] The results showed that, compared with the group treated with sorafenib alone, whether ABHD2 was overexpressed or the empty plasmid was transfected, the cell viability of the combined administration was significantly decreased, and the ICsub 50 subof sorafenib dropped significantly. Among them, when the empty plasmid was transfected, the ICsub 50 subof sorafenib was 3.537 when administered alone, and dropped to 2.430 when combined with chlorpromazine, a decrease of 31.3%, indicating that chlorpromazine had a sensitizing effect on sorafenib. In the case of combined administration, the decrease in cell viability in the group overexpressing ABHD2 was smaller than that in the control group transfected with the empty plasmid, and the ICsub 50 subof sorafenib increased, indicating that the sensitizing effect of chlorpromazine was inhibited, proving that chlorpromazine affected the drug sensitivity of sorafenib through the lipid hydrolase target ABHD2 and assisted in exerting the anti-hepatocellular carcinoma effect.

[0059] Example 2 Chlorpromazine combined with sorafenib inhibits mouse Hepa1-6 subcutaneous tumors

[0060] [[ID=((15))]]The experimental animals used in this example were SPF-grade C57BL / 6J mice, male, 18 - 20 g, 6 - 8 weeks old, purchased from the Comparative Medicine Center of Yangzhou University, with the production license number SCXK (Jiangsu) 2022 - 0009. The laboratory room temperature was 20 - 22 °C, the relative humidity was 40% - 60%, ventilated by an exhaust fan, with natural light for 12 h / day, cage-raised, 6 mice per cage, and the cage was cleaned once every three days. The mice were randomly divided into 3 groups, with 4 mice in each group.

[0061] In the in vivo experiment of mice, chlorpromazine was prepared into 10 mg / kg at the time of use, freshly prepared before use, and administered by intraperitoneal injection; sorafenib was prepared into 30 mg / kg at the time of use, freshly prepared before use, and administered by gavage. <o000179>As Figure 5 shown in the experimental schematic diagram, Hepa1-6 cells were subcutaneously injected into C57BL / 6J mice. When the orthotopically inoculated tumor grew to a size convenient for operation, the tumor tissue was isolated and cut into tissue blocks of about 1 mm³, and then transferred subcutaneously to a new batch of C57BL / 6J mice. The gross image of the transplanted tumor is as shown in Figure 6 A in it. When the tumor grew to a volume of about 100 mm³, the mice were grouped for treatment, and were respectively given single sorafenib treatment (30 mg / kg, once every 2 days, for 3 weeks), or combined treatment with chlorpromazine (10 mg / kg, once every 2 days, for 3 weeks). The blank control of this experiment was an equal volume of normal saline, and the positive drug control was single administration of sorafenib.

[0063] During the whole experiment, the growth status of the tumor was closely monitored, and the results are as Figure 6 ​​As shown in C; after the experiment, the mice were euthanized, the tumors were removed and weighed, as shown in Figure C. Figure 6 As shown in B in the diagram; Figure 6 The combined results of BC and BC studies indicate that the combination of chlorpromazine and sorafenib can inhibit the progression of hepatocellular carcinoma, manifested by a significant reduction in tumor weight and a significant delay in tumor growth. The mean tumor weight in the sorafenib monotherapy group was 0.413 g, while the mean weight in the combination therapy group decreased to 0.185 g, a reduction of 55%. The mean tumor volume in the sorafenib monotherapy group was 743.3 mm. 3 The average volume after combined administration was 388.8 mm. 3 This represents a decrease of 47.7%.

[0064] Figure 6 The BC data were analyzed using one-way ANOVA, and the results are expressed as mean ± standard error (mean ± SEM) (n = 4). Compared with the sorafenib monotherapy group, p>0.05 was considered not statistically significant (ns), p<0.05 was considered statistically significant (*), and p<0.01 was considered highly significant (**).

[0065] The extracted tumor was routinely embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (HE) and Ki67. The experimental results are as follows: Figure 7 The results showed that the combined use of drugs exacerbated the damage to tumor tissue. Figure 7 As shown in A, H&E staining results indicate that the number of vacuoles in the combination therapy was significantly increased compared to sorafenib alone; at the same time, the positive rate of Ki67 staining in tumor cells was significantly reduced in the combination therapy, indicating a significant decrease in the proliferative capacity of tumor cells.

[0066] The Ki67 staining results were quantitatively analyzed using ImageJ software. The results are as follows: Figure 7 As shown in B, the Ki67 positive cell rate decreased from 19.8% with sorafenib alone to 7.6% with combination therapy, a reduction of 61.6%, indicating that combination therapy weakened the proliferative capacity of tumor cells.

[0067] Tumor H&E staining and Ki67 staining results (scale bar = 100 μm) and analytical data are expressed as mean ± standard error (mean ± SEM) (n = 3). Compared with the sorafenib monotherapy group, p > 0.05 was considered not statistically significant (ns), p < 0.05 was considered statistically significant (*), and p < 0.01 was considered highly statistically significant (**).

Claims

1. A pharmaceutical composition comprising chlorpromazine or a pharmaceutically acceptable salt thereof, characterized in that, The pharmaceutical composition also includes sorafenib or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, characterized in that, The chlorpromazine or its pharmaceutically acceptable salts include chlorpromazine or chlorpromazine hydrochloride.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The mass ratio of chlorpromazine or its pharmaceutically acceptable salt to sorafenib is 1:3 to 1:

6.

4. The pharmaceutical composition according to claim 1 or 2, characterized in that, The dosage forms of the pharmaceutical composition include tablets, capsules, granules, solutions, emulsions, or suspensions.

5. The use of the pharmaceutical composition comprising chlorpromazine or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 4 in the preparation of a medicament for the prevention and / or treatment of liver cancer.

6. The application according to claim 5, characterized in that, The mass ratio of chlorpromazine to sorafenib in the composition is 1:3 to 1:

6.

7. The application according to claim 5, characterized in that, The liver cancer mentioned includes hepatocellular carcinoma.

8. The application according to claim 5, characterized in that, The pharmaceutical composition is a composition that utilizes chlorpromazine to inhibit the expression of ABHD2, thereby increasing the sensitivity of liver cancer cells to ferroptosis and alleviating the resistance of liver cancer cells to sorafenib.

9. The application according to claim 8, characterized in that, The liver cancer cells mentioned include one or more of Hepa1-6 cells, Huh7 cells, or HCCLM3 cells.

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