A pharmaceutical composition for preventing or treating copper overload-induced liver injury and use thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a medicine composition for preventing or treating copper overload induced liver injury and application thereof, and an active ingredient of the medicine composition is composed of VDAC oligomerization inhibitor VBIT-4 or a pharmaceutically acceptable salt thereof and NLRP3 inflammasome inhibitor MCC950 or a pharmaceutically acceptable salt thereof. The present application is based on a new mechanism that copper overload induces mitochondrial DNA release by promoting VDAC oligomerization, and then activates NLRP3 inflammasome and triggers hepatocyte pyroptosis, and proposes a combined drug strategy: VBIT-4 inhibits upstream VDAC oligomerization to reduce mitochondrial DNA release, and MCC950 inhibits downstream NLRP3 activation. The two drugs produce a synergistic effect, reduce the single drug dose and side effects. The present application provides a safe and effective new prevention and treatment scheme for copper overload induced liver injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a pharmaceutical composition for the prevention or treatment of copper overload-induced liver injury and its application. Background Technology

[0002] Copper (Cu) is an essential trace element for the body, playing a crucial role in basic biological processes as a key component of many enzymes. It is widely used as a growth-promoting trace element additive in livestock production. However, the widespread use of copper in agriculture and industry has led to serious environmental pollution problems, posing a serious threat to animal and human health through the spread and accumulation of pollutants in the food chain. The liver, as the body's main metabolic and detoxification organ, is a major target organ for environmental pollutants, including heavy metals. Studies have shown that copper exposure can induce various forms of cell death in hepatocytes, including copper proptosis, ferroptosis, and apoptosis.

[0003] Currently, clinical treatments for copper overload-related liver injury mainly include copper chelators (such as D-penicillamine, trientine, and ammonium tetrathiomolybdate), zinc salts, and hepatoprotective supportive therapy. However, existing treatments have significant limitations: copper chelators generally suffer from poor selectivity and severe adverse reactions; for example, long-term use of D-penicillamine can lead to serious side effects such as bone marrow suppression, nephrotoxicity, and autoimmune reactions; trientine is expensive and also carries risks of neurotoxicity; zinc salts have a slow onset of action and are only suitable for maintenance therapy; and treatment options for acute liver failure such as acetaminophen-induced liver injury remain very limited. Therefore, there is an urgent clinical need to develop new, safe, and effective strategies for the prevention and treatment of copper overload-induced liver injury.

[0004] Recent studies have found that copper overload-induced liver injury is closely related to the activation of the NLRP3 inflammasome. As a key sensor and effector of innate immunity, the NLRP3 inflammasome senses damage signals and recruits active stem cells (ASCs) through interactions with pyrin-containing domains, thereby recruiting and activating pro-caspase-1 to form an activated inflammasome complex. Activated caspase-1 not only processes pro-IL-1β and pro-IL-18 into their mature forms but also cleaves GSDMD, releasing its N-terminal domain. This domain forms pores in the cell membrane, mediating the release of inflammatory factors and pyroptosis. Studies have shown that the activation of the NLRP3 inflammasome is closely related to mitochondrial function. Mitochondrial stress can trigger the oligomerization of voltage-dependent anion channels (VDACs), leading to the formation of mitochondrial outer membrane pores and facilitating the escape of mitochondrial contents, including mitochondrial DNA (mtDNA), into the cytoplasm. Kim et al. (Science, 2019) reported that VDAC oligomers can form mitochondrial pores to release mtDNA fragments and promote the development of lupus-like diseases. Baik et al. (Science Immunology, 2023) further confirmed that the dissociation of hexokinase from mitochondria can promote the oligomerization of VDAC, thereby promoting the assembly and activation of the NLRP3 inflammasome.

[0005] While research on NLRP3 inflammasome inhibitors has made some progress, single-target intervention strategies face numerous challenges. MCC950 is one of the most representative known small-molecule NLRP3 inhibitors, showing positive effects in more than 50 animal inflammation models and having entered a phase II clinical trial for rheumatoid arthritis. However, this clinical trial was terminated early due to hepatotoxicity, and its efficacy was lower than expected, limiting its clinical application. VBIT-4, as a VDAC oligomerization inhibitor, has shown effects in preventing mitochondrial dysfunction and apoptosis in lupus and type 2 diabetes mouse models, but its efficacy as a standalone treatment is limited. Currently, there are no reports of combining VDAC oligomerization inhibitors with NLRP3 inflammasome inhibitors for the prevention or treatment of copper overload-induced liver injury.

[0006] Therefore, developing a drug composition based on a novel mechanism of action that targets key pathological processes of copper overload-induced liver injury is of significant clinical importance and application value. Summary of the Invention

[0007] In order to overcome the above-mentioned defects of the prior art, the present invention provides a pharmaceutical composition for preventing or treating copper overload-induced liver injury and its application, so as to solve the problems existing in the background art.

[0008] The present invention provides the following technical solution: To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition for preventing or treating copper overload-induced liver injury, wherein the active ingredient of the pharmaceutical composition comprises: (a) The first active ingredient: VDAC oligomerization inhibitor VBIT-4, or a pharmaceutically acceptable salt thereof; (b) Second active ingredient: NLRP3 inflammasome inhibitor MCC950, or a pharmaceutically acceptable salt thereof.

[0009] The chemical name of VBIT-4 is (E)-3-(4-(tert-butyl)phenyl)-N-(2-(4-(tert-butyl)phenyl)-2-oxoethyl)acrylamide, CAS number 2086257-77-2. It is a voltage-dependent anion channel (VDAC1) oligomerization inhibitor with a Kd value of 17 μM. VBIT-4 reduces the formation of mitochondrial outer membrane pores by inhibiting the oligomerization of VDAC on the mitochondrial outer membrane, thereby reducing the release of mitochondrial contents (including mtDNA) into the cytoplasm.

[0010] The chemical name of MCC950 is N-((1,2,3,5,6,7-hexahydro-s-indarsen-4-yl)carbamoyl)-4-(2-hydroxypropane-2-yl)furan-2-sulfonamide, CAS number 210826-40-7. It is a selective NLRP3 inflammasome inhibitor that targets the NACHT domain (Walker B motif) of NLRP3. MCC950 inhibits pyroptosis by directly inhibiting the assembly and activation of the NLRP3 inflammasome, blocking the activation of downstream caspase-1 and the cleavage of GSDMD.

[0011] This invention is the first to propose the combined use of the two active ingredients mentioned above for the prevention or treatment of copper overload-induced liver injury. Research results show that copper overload can promote VDAC oligomerization, leading to the release of mtDNA into the cytoplasm. The released mtDNA, as a damage-associated molecular pattern (DAMP), is recognized and activated by the NLRP3 inflammasome, thereby triggering hepatocyte pyroptosis. The combined application of VBIT-4 and MCC950 intervenes simultaneously upstream and downstream of the key pathological signaling cascade pathway of "VDAC oligomerization—mtDNA release—NLRP3 activation," producing a significant synergistic effect.

[0012] In some embodiments of the present invention, the mass ratio of VBIT-4 or a pharmaceutically acceptable salt thereof to MCC950 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.1 to 10):1.

[0013] In some preferred embodiments of the present invention, the mass ratio of VBIT-4 or a pharmaceutically acceptable salt thereof to MCC950 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.5 to 5):1.

[0014] In some more preferred embodiments of the present invention, the mass ratio of VBIT-4 or a pharmaceutically acceptable salt thereof to MCC950 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (1-3):1.

[0015] In a second aspect, the present invention provides the use of the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of copper overload-induced liver injury.

[0016] The liver injury induced by copper overload includes, but is not limited to: liver injury caused by excessive dietary copper intake, liver injury caused by industrial copper exposure, liver injury caused by environmental copper pollution exposure, liver injury related to copper metabolism disorders (such as Wilson's disease), and liver injury related to drug-induced copper overload.

[0017] The liver injury mentioned includes pathological changes such as hepatocyte pyroptosis, hepatocyte necrosis, hepatocyte apoptosis, liver inflammation, and liver fibrosis.

[0018] Thirdly, the present invention provides the use of the pharmaceutical composition in the preparation of the following pharmaceuticals: (1) Drugs for preventing or treating hepatocyte pyroptosis; (2) Drugs that inhibit VDAC oligomerization; (3) Drugs that inhibit the release of mitochondrial DNA (mtDNA); (4) Drugs that inhibit the activation of the NLRP3 inflammasome; (5) Medications for the prevention or treatment of copper overload-related inflammatory diseases.

[0019] Fourthly, the present invention provides a complete pharmaceutical preparation for preventing or treating copper overload-induced liver injury, comprising: (a) A first pharmaceutical preparation containing a therapeutically effective amount of VBIT-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; (b) A second pharmaceutical preparation containing a therapeutically effective amount of MCC950 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; The first drug formulation and the second drug formulation are administered simultaneously, separately, or sequentially.

[0020] Fifthly, the present invention provides a pharmaceutical formulation comprising the pharmaceutical composition described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0021] Pharmaceutically acceptable carriers include, but are not limited to: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, flavoring agents, sweeteners, preservatives, antioxidants, etc., which can be conventionally selected according to the route of administration and dosage form.

[0022] The pharmaceutical preparation can be any clinically acceptable dosage form, including but not limited to: oral preparations (such as tablets, capsules, granules, powders, oral liquids, suspensions, syrups, etc.), injectable preparations (such as injection solutions, sterile powders for injection, lyophilized powders for injection, etc.), topical preparations (such as creams, ointments, gels, patches, etc.), inhaled preparations, suppositories, drops, etc.

[0023] The technical effects and advantages of this invention are as follows: This invention is the first to combine the VDAC oligomerization inhibitor VBIT-4 with the NLRP3 inflammasome inhibitor MCC950 for the prevention and treatment of copper overload-induced liver injury, pioneering a novel treatment strategy for copper overload-induced liver injury based on the "mtDNA release-NLRP3 activation" pathway.

[0024] The pharmaceutical composition of this invention exhibits a unique "dual blocking" effect in its mechanism of action: VBIT-4 acts on the upstream stage, inhibiting VDAC oligomerization and mtDNA release, thereby reducing the activation signaling of the NLRP3 inflammasome at its source; MCC950 acts on the downstream stage, directly inhibiting the activation of the NLRP3 inflammasome and blocking pyroptosis. The combined use of the two active ingredients produces a significant synergistic effect, which is superior to the effect of using either drug alone.

[0025] The synergistic effect of the pharmaceutical composition of the present invention allows for a reduction in the effective dose of each individual drug when used in combination, thereby reducing the potential toxic side effects of high-dose single-drug use (such as the risk of hepatotoxicity at high doses of MCC950 and the inhibitory effect of VBIT-4 on mitochondrial respiration at high concentrations), and significantly improving drug safety.

[0026] This invention provides a precise intervention method for the key pathological signaling cascade pathway of "mitochondrial damage-inflammasome activation-pyroptosis" in copper overload-induced liver injury. Compared with traditional copper chelation therapy strategies, this invention more directly targets the downstream pathological consequences caused by copper overload and can complement copper chelation therapy, providing a new option for the comprehensive treatment of copper overload-related liver injury. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments are only for explaining the invention and do not constitute a limitation on the scope of protection of this invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art without departing from the spirit and principles of this invention should be included within the scope of protection of this invention.

[0028] Example 1: Establishment and Mechanism Verification of a Copper Overload-Induced Porcine Liver Injury Model This embodiment verifies the damaging effect of copper overload on porcine liver through in vivo experiments and elucidates the objective existence of the signaling pathway "VDAC oligomerization-mtDNA release-NLRP3 inflammasome activation-hepatocyte pyroptosis", providing a theoretical basis for the application of the pharmaceutical composition of the present invention.

[0029] 1.1 Laboratory Animals and Grouping Treatment Twenty weaned piglets of similar weight and good health were randomly divided into two groups: a control group and a copper treatment group, with 10 piglets in each group. The control group was fed a basal diet (copper content meeting NRC standards), while the copper treatment group had copper sulfate added to their basal diet. The diet was designed to contain 250 mg / kg of copper (high-copper diet). The trial lasted 60 days. After the trial, blood, liver tissue, and stool samples were collected for testing.

[0030] 1.2 Experimental Methods (1) Detection of liver copper content and biochemical indicators Liver tissue treated with concentrated nitric acid ( After digestion at 120℃ until completely dissolved, the concentration of copper ions in liver, serum, and feces was determined using an Agilent 7500cx inductively coupled plasma mass spectrometer (ICP-MS), with results expressed as wet weight. The activities of alkaline phosphatase (ALP), gamma-glutamyl transferase (γ-GT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in serum were determined using a Mindray BS-380 fully automated biochemical analyzer.

[0031] (2) Histopathological and ultrastructural observation Liver tissue was fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histopathological changes were observed under a light microscope. Another 1... Fresh liver tissue was prefixed with 2.5% glutaraldehyde and postfixed with 1% osmium tetroxide, embedded in epoxy resin, and then ultrathinly sectioned for observation of hepatocyte ultrastructure and mitochondrial morphology under a transmission electron microscope.

[0032] (3) Quantitative analysis of cytoplasmic mitochondrial DNA (mtDNA) release Liver tissue was homogenized in ice-cold mitochondrial separation buffer, centrifuged at 950×g to remove cell nuclei and debris, and the supernatant was collected by centrifugation at 17,000×g to collect cytoplasmic fraction. Total cytoplasmic DNA was extracted using a DNA separation kit, and the copy numbers of the mtDNA-specific sequences D-loop and mtCOX1 were detected by real-time quantitative PCR (qRT-PCR), with β-actin as an internal control.

[0033] (4) VDAC oligomerization level detection (BN-PAGE) Liver tissue was collected and homogenized in ice-cold homogenizing buffer (250 mM sucrose, 20 mM imidazole / HCl, pH 7.0). The mixture was centrifuged at 20,000 × g for 10 min, and the precipitate was resuspended. Digitalis saponins (20% final concentration) were added and the mixture was lysed on ice for 10 min. The supernatant was collected after centrifugation and subjected to blue-green gel electrophoresis (BN-PAGE). After transfer to a membrane, oligomeric bands were detected using anti-VDAC1 antibody.

[0034] (5) Detection of pyroptosis-related gene and protein expression Total RNA was extracted from liver tissue using the Trizol method. After reverse transcription, the mRNA expression of NLRP3, GSDMD, Caspase-1, IL-18, and IL-1β was detected by qRT-PCR using the SYBR Green method. Total protein was extracted using RIPA lysis buffer. The protein expression levels of NLRP3, cleaved Caspase-1 (C-Caspase1), N-terminal fragment of GSDMD (N-GSDMD), IL-18, and IL-1β were detected by Western blotting, with GAPDH used as an internal control.

[0035] 1.3 Experimental Results (1) Results of copper accumulation and liver injury indicators ICP-MS results showed that the copper ion content in the liver, serum, and feces of the copper-treated group was significantly higher than that of the control group (P<0.05). Biochemical analysis showed that, compared with the control group, the activities of ALP, γ-GT, AST, and ALT in the serum of the copper-treated group were significantly increased (P<0.05), as detailed in Table 1. This indicates that copper overload led to significant hepatocellular damage and abnormal liver function.

[0036] Table 1. Effects of copper exposure on biochemical indicators of pig liver function (Mean ± SD) (2) Histopathological and ultrastructural results Gross observation showed that the livers of the control group were uniformly reddish-brown, while the livers of the copper-treated group were lighter in color, appearing pale yellow. H&E staining and microscopic examination revealed that the liver tissue structure of the control group was clear and intact; the hepatocytes of the copper-treated group showed diffuse vacuolar degeneration, dilated and congested hepatic sinusoids and central veins, significantly widened interchordal spaces, and abundant infiltration of lymphocytes and neutrophils in the portal areas and lobules. Transmission electron microscopy showed that the mitochondria in the copper-treated group were swollen, with broken cristae, and numerous mitophagosomes formed by double-membrane structures encapsulating the damaged mitochondria.

[0037] (3) mtDNA release and VDAC oligomerization results qRT-PCR results showed that the copy number of mtDNA (D-loop and mtCOX1) in the cytoplasmic fraction of liver tissue in the copper-treated group increased by approximately 3.5 times compared with the control group (P<0.01). BN-PAGE results showed that VDAC in the control group mainly existed in monomeric form, while the copper-treated group showed obvious VDAC oligomer bands at molecular weights of approximately 120-240 kDa, indicating that copper overload significantly promoted VDAC oligomerization.

[0038] (4) Results of NLRP3 inflammasome and pyroptosis pathway activation qRT-PCR results showed that the mRNA expression levels of NLRP3, GSDMD, Caspase-1, IL-18, and IL-1β in liver tissue of the copper-treated group were upregulated by 2.8-fold, 3.1-fold, 2.5-fold, 2.3-fold, and 3.5-fold, respectively (P<0.05). Western blot results showed that the expression levels of NLRP3 protein, activated C-Caspase1 fragment, N-GSDMD fragment, and mature IL-1β protein were significantly increased in the copper-treated group (P<0.05), while the expression of IL-18 protein showed an increasing trend but did not reach statistical significance (P>0.05).

[0039] The above results indicate that copper overload can promote mitochondrial VDAC oligomerization, induce mtDNA release into the cytoplasm, thereby activating the NLRP3 inflammasome and ultimately triggering Caspase-1-dependent hepatocyte pyroptosis. Establishing this pathway provides a clear intervention target for the combination drug strategy of this invention.

[0040] Example 2: Verification of the protective effect of VBIT-4 against copper-induced hepatocyte injury This embodiment verifies the inhibitory effect of VDAC oligomerization inhibitor VBIT-4 on copper-induced mtDNA release and pyroptosis in porcine hepatocytes.

[0041] 2.1 Cell Culture and Treatment: The porcine liver cell line (purchased from NewGain Biotechnology Co., Ltd.) was cultured in high-glucose DMEM medium containing 10% fetal bovine serum and incubated at 37°C with 5% [unclear - likely referring to a specific temperature range]. Incubator. The CCK-8 assay was used for determination. The half-maximal inhibitory concentration (IC50) against hepatocytes was 360.8 μM. Subsequent experiments were conducted with the following groups: blank control group, ... Treatment group (360 μM), VBIT-4 monotherapy group (4 μM), VBIT-4+ Combined treatment group (4 μM VBIT-4 pretreated for 2 h followed by addition of 360 μM) Cells were collected and analyzed 24 hours after treatment.

[0042] 2.2 Experimental Results (1) Changes in cell viability CCK-8 test results showed that, compared with the control group, 360 μM Treatment reduced cell viability to 52.3% ± 6.1% of the control group (P<0.05). VBIT-4 monotherapy had no significant effect on cell viability (97.2% ± 4.5%). VBIT-4+ treatment... The cell viability in the combined treatment group significantly recovered to 78.5% ± 5.8% of the control group, compared to... The treatment group alone showed significant improvement (P<0.05).

[0043] (2) Changes in mtDNA release and VDAC oligomerization Cytoplasmic mtDNA quantification results showed that The copy numbers of cytoplasmic D-loop and mtCOX1 in the treatment group were 4.2 times and 3.8 times that of the control group, respectively (P<0.05); after combined use of VBIT-4, the copy numbers significantly decreased to 1.9 times and 1.7 times that of the control group, respectively (compared to...). Group comparison, P<0.05). BN-PAGE results showed that VBIT-4 effectively inhibited... Enhancement of induced VDAC oligomer bands.

[0044] (3) Changes in the expression of pyroptosis-related proteins Western blot results showed that, compared with Compared with the single treatment groups, the VBIT-4 combined treatment group showed a decrease of approximately 45% in NLRP3 protein expression, a decrease of approximately 52% in the N-GSDMD / GSDMD ratio, a decrease of approximately 48% in the C-Caspase1 / Caspase1 ratio, and a significant reduction in the mature expression of IL-1β and IL-18 (P<0.05). This indicates that inhibiting VDAC oligomerization can block downstream pyroptosis signaling at its source.

[0045] Example 3: Inhibition of copper-induced hepatocyte pyroptosis by MCC950 and verification of the necessity of NLRP3 This embodiment uses the NLRP3-specific inhibitor MCC950 and the activator Nigericin to verify the key role of NLRP3 in copper-induced hepatocyte pyroptosis.

[0046] 3.1 Cell treatment grouping Set up a blank control group. Group (360 μM), MCC950 group (10 μM), MCC950+ Group, Nigermycin group (3 μM), Nigermycin + Group. Cells were collected 24 hours after treatment for Western blot analysis.

[0047] 3.2 Experimental Results and Compared to the standalone processing group, MCC950+ In the combined treatment group, NLRP3 protein expression was significantly downregulated, with the N-GSDMD / GSDMD ratio and C-Caspase1 / Caspase1 ratio decreasing by approximately 65% ​​and 58%, respectively, and IL-1β and IL-18 secretion significantly reduced (P<0.05). Conversely, nigermycin + The above protein indicators in the combined treatment group were lower than those in the combined treatment group. The treatment alone resulted in an additional 30%-40% increase (P<0.05). These results strongly suggest that NLRP3 is a necessary signaling node for copper-induced hepatocyte pyroptosis, and that inhibiting NLRP3 is an effective strategy for treating copper overload liver injury.

[0048] Example 4: Validation of the synergistic effect of combined use of VBIT-4 and MCC950 This embodiment verifies whether the core inventive point of this invention—the combined application of VBIT-4 and MCC950—has a synergistic effect on copper-induced hepatocyte damage.

[0049] 4.1 Cell treatment grouping Setup: Control group, The drug-containing groups were: VBIT-4 monotherapy group (360 μM), VBIT-4 monotherapy group (4 μM), MCC950 monotherapy group (10 μM), and VBIT-4 + MCC950 combination therapy group (4 μM + 10 μM). All of the above drug-containing groups were treated with 360 μM. The process took 24 hours.

[0050] 4.2 Experimental Results (1) Quantitative Detection of Cytoplasmic mtDNA Release (Table 2) The results showed that the combined drug group had a significantly better inhibitory effect on mtDNA release than the individual drug groups.

[0051] Table 2. Effects of different treatments on the copy number of porcine hepatocyte cytoplasmic mtDNA (relative fold change, Mean ± SD) (2) Changes in the expression of pyroptosis-related proteins (Table 3) The combined drug group showed the highest inhibition rate of NLRP3 inflammasome and downstream pyroptosis executive protein, demonstrating a significant synergistic effect.

[0052] Table 3. Effects of different treatments on the expression of pyroptosis-related proteins (relative gray value / GAPDH, Mean ± SD) 4.3 Conclusion Analysis The experimental data above clearly demonstrate that the combined application of VBIT-4 and MCC950 produces a synergistic effect of "1+1>2". Mechanistically, VBIT-4 blocks upstream VDAC oligomerization to reduce mtDNA leakage, while MCC950 blocks downstream NLRP3 assembly to inhibit the inflammatory response. Both act simultaneously at two key nodes in the signaling pathway, which not only significantly improves the therapeutic effect but also reduces the potential risks associated with high-concentration single-drug use (such as the liver burden of MCC950 or excessive inhibition of mitochondrial respiration by VBIT-4).

[0053] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A pharmaceutical composition for preventing or treating copper overload-induced liver injury, characterized in that, The active ingredient of the pharmaceutical composition comprises: The primary active ingredient is VBIT-4, a VDAC oligomerization inhibitor, or a pharmaceutically acceptable salt thereof. Second active ingredient: NLRP3 inflammasome inhibitor MCC950, or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of the VBIT-4 or its pharmaceutically acceptable salt to the MCC950 or its pharmaceutically acceptable salt is (0.1–10):

1.

3. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio of the VBIT-4 or its pharmaceutically acceptable salt to the MCC950 or its pharmaceutically acceptable salt is (0.5–5):

1.

4. The pharmaceutical composition according to claim 3, characterized in that, The mass ratio of the VBIT-4 or its pharmaceutically acceptable salt to the MCC950 or its pharmaceutically acceptable salt is (1-3):

1.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The liver injury induced by copper overload includes liver injury caused by excessive dietary copper intake, liver injury caused by industrial copper exposure, liver injury caused by environmental copper pollution exposure, liver injury related to copper metabolism disorders, and liver injury related to drug-induced copper overload.

6. The pharmaceutical composition according to claim 5, characterized in that, The liver injury includes one or more of the following: hepatocyte pyroptosis, hepatocyte necrosis, hepatocyte apoptosis, liver inflammation, or liver fibrosis.

7. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is an oral preparation, an injectable preparation, a topical preparation, an inhaled preparation, a suppository, or a drop.

9. The pharmaceutical composition according to claim 8, characterized in that, The oral preparations are tablets, capsules, granules, powders, oral liquids, suspensions, or syrups; the injectable preparations are injection solutions, sterile powders for injection, or lyophilized powders for injection.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for the prevention or treatment of copper overload-induced liver injury.

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