Pharmaceutical composition for treating drug-induced liver injury and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating drug-induced liver injury and application of the pharmaceutical composition, the pharmaceutical composition comprises glucaric acid 1, 4-lactone and N-acetylcysteine, and the mass ratio of the glucaric acid 1, 4-lactone to the N-acetylcysteine is 1: (1-5). The pharmaceutical composition disclosed by the invention can be used for preparing a medicine for treating or preventing the drug-induced liver injury, and the drug-induced liver injury is liver injury caused by excessive acetaminophen or long-term administration. The pharmaceutical composition provides comprehensive liver protection through multi-target cooperation, and has a significant improvement effect on liver injury caused by acetaminophen.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a composite pharmaceutical composition for treating drug-induced liver injury and applications thereof. Background Art

[0002] Drug-induced liver injury (DILI) is a major cause of liver disease and acute liver failure (ALF) worldwide. According to statistics, DILI causes over 2 million deaths worldwide each year, accounting for 4% of all deaths. One in 25 deaths is related to liver disease. Acetaminophen (APAP) overdose is the leading cause of acute liver injury and failure in Europe and the United States, accounting for 46% of all ALF cases.

[0003] Clinically, the gold standard for the treatment of APAP poisoning is N-acetylcysteine (NAC), which neutralizes NAPQI (N-acetyl-p-benzoquinoneimine) by supplementing GSH precursors and directly scavenging free radicals. However, the efficacy of NAC is highly dependent on the time window of administration, and its effect is significantly reduced in patients with severe symptoms or delayed treatment. In addition, NAC monotherapy has limitations:

[0004] (1) Time limit: NAC cannot reverse the oxidative damage that has occurred after more than 8 hours of human poisoning (1.5-2 hours in animal experiments);

[0005] (2) Dosage bottleneck: High doses of NAC (e.g., 400 mg / kg) may induce allergic reactions or aggravate liver damage;

[0006] (3) Single mechanism: neutralization of NAPQI only by supplementing GSH.

[0007] In addition to NAC, clinically approved hepatoprotective drugs (such as glycyrrhizic acid preparations, silymarin, and polyene phosphatidylcholine) primarily exert their effects through anti-inflammatory, antioxidant, or hepatocyte membrane stabilization, but their efficacy still lags behind that of NAC. For example, silymarin enhances antioxidant capacity by activating the Nrf2 pathway but is unable to inhibit the CYP2E1-mediated APAP toxicity metabolism. Existing drugs often focus on a single pathological link (such as oxidative stress or inflammation) and are unable to block the multi-step cascade of APAP poisoning.

[0008] In recent years, natural products have become a research hotspot for DILI treatment due to their multi-target effects. Traditional Chinese medicine (TCM) compound formulas have demonstrated unique advantages in the treatment of liver diseases. For example, the Xiere Zhuyu formula disclosed in CN 105687383 B alleviates liver failure by regulating the HMGB1-TLR4-NF-κB pathway, and the TCM composition described in CN 106110274 B improves chronic hepatitis by soothing the liver and unblocking the meridians. However, these treatments primarily target viral hepatitis or liver failure, lack specific designs targeting APAP hepatotoxicity, and have a single mechanism of action, making it difficult to address the multi-step pathological process of DILI. Summary of the Invention

[0009] The present application provides a compound pharmaceutical composition for treating drug-induced liver injury and its application, wherein the pharmaceutical composition has a significant improvement effect on liver injury caused by acetaminophen (APAP).

[0010] The pharmaceutical composition comprises glucaric acid 1,4-lactone and N-acetylcysteine, wherein the mass ratio of glucaric acid 1,4-lactone to N-acetylcysteine is 1:1-5.

[0011] The structural formula of the glucaric acid 1,4-lactone is as follows:

[0012]

[0013] The detoxification effect of glucaric acid 1,4-lactone (1,4-GL) in APAP poisoning has not been explored, and there is no research on its combination with N-acetylcysteine (NAC). This application combines 1,4-GL with NAC, wherein NAC neutralizes the toxic product NAPQI, and 1,4-GL acts on the target cytochrome P4502E1 (CYP2E1) and c-Jun amino-terminal kinase (JNK), respectively. On the one hand, by inhibiting the activity of CYP2E1 enzyme, the survival of the toxic product NAPQI is reduced, and the intervention mechanism of NAC neutralizing the toxic product synergizes with each other, breaking through the time limit of NAC through the dual mechanism of "inhibiting toxic metabolism + neutralizing toxic products". On the other hand, 1,4-GL also acts on the target JNK, exerting a liver protective effect by regulating the JNK pathway and its downstream effector molecules, and further strengthening the detoxification and protective effects on liver damage on the basis of exerting the detoxification effect through "inhibiting toxic metabolism + neutralizing toxic products".

[0014] The combination of 1,4-GL and NAC in this application breaks through the time limitation of NAC through the dual mechanism of "inhibiting toxic metabolism + neutralizing toxic products", combines 1,4-GL to regulate the toxic metabolism of the signaling pathway (JNK), and synergizes multiple targets to provide all-round liver protection.

[0015] Optionally, the mass ratio of glucaric acid 1,4-lactone to N-acetylcysteine is 1:1-2.

[0016] Further preferably, the mass ratio of glucaric acid 1,4-lactone to N-acetylcysteine is 1:1.

[0017] The present application also provides a use of the pharmaceutical composition in the preparation of a drug for treating or preventing drug-induced liver injury, wherein the drug-induced liver injury is liver injury caused by an overdose or long-term use of acetaminophen.

[0018] Optionally, the drug for treating or preventing drug-induced liver injury comprises an effective therapeutic amount of the pharmaceutical composition and a pharmaceutically acceptable carrier.

[0019] A pharmaceutically acceptable carrier may be added to a therapeutically effective amount of the pharmaceutical composition. The carrier may be a diluent, a binder, a disintegrant, a lubricant, a flavoring agent, an aromatic agent, and the like.

[0020] Optionally, the dosage form of the drug for treating or preventing drug-induced liver injury is an injection or an oral preparation.

[0021] Optionally, the drug can be prepared into an injection, wherein the concentration of glucaric acid 1,4-lactone in the injection is 20-50 μM, and the concentration of N-acetylcysteine is 50-100 μM.

[0022] Optionally, the drug can be prepared into oral liquid, tablets, granules, capsules, powders, pellets or micropills, but is not limited to oral preparations. The drug in the dosage form can be prepared using current pharmaceutical preparation methods.

[0023] The present application also provides a drug for treating or preventing drug-induced liver injury, comprising an effective therapeutic amount of the pharmaceutical composition and a pharmaceutically acceptable carrier.

[0024] The present application also provides a use of the pharmaceutical composition in the preparation of a cytochrome P4502E1 (CYP2E1) inhibitor.

[0025] Compared with the prior art, this application has the following beneficial effects:

[0026] (1) The first combination therapy: 1,4-GL was used in combination with NAC for the first time, breaking through the time limit of NAC through the dual mechanism of "inhibiting toxic metabolism + neutralizing toxic products". Animal experiments showed that 1,4-GL combined with NAC still had significant therapeutic effects 6 hours after poisoning, significantly breaking through the time limit of 1.5 to 2 hours in NAC animal experiments.

[0027] (2) Multi-target synergy: NAC neutralizes toxic products and combines 1,4-GL metabolic inhibition (CYP2E1) and signal pathway regulation (JNK) to provide comprehensive liver protection. Serum indicators show that after the detoxification of 1,4-GL combined with NAC, the serum ALT, AST, TBIL and MDA levels were significantly reduced, and the GSH and SOD levels were significantly increased.

[0028] (3) Clinical translation potential: The significant therapeutic effect was verified in animal models, providing theoretical support for the development of new antidotes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the survival results of mice poisoned with 700 mg / kg APAP in different treatment groups.

[0030] Figure 2 These are the results of serum biochemical indicators in mice poisoned with 700 mg / kg APAP in different treatment groups.

[0031] Figure 3 These are the results of serum biochemical indicators in mice poisoned by 300 mg / kg APAP in different treatment groups.

[0032] Figure 4 These are the HE staining results of liver tissue in mice poisoned with 300 mg / kg APAP in different treatment groups.

[0033] Figure 5 The results of serum biochemical indicators of mice in different treatment groups 1 and 6 hours after taking APAP.

[0034] Figure 6 These are the results of ROS levels in fresh livers of mice poisoned with 300 mg / kg APAP after intervention with different treatment groups.

[0035] Figure 7 The results show the effects of different treatment groups on the expression levels of mouse liver proteins.

[0036] Figure 8 The results show the effects of different treatment groups on the mRNA expression levels in mouse liver. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0039] Example 1: Preparation of pharmaceutical composition

[0040] Both 1,4-GL and NAC are commercially available products. 1,4-GL was purchased from Toronto Research Chemical Company, Canada, with a purity greater than 98%. NAC was purchased from Beijing Bailingwei Technology Co., Ltd., with a purity greater than 99%.

[0041] The structural formula of 1,4-GL is:

[0042]

[0043] A certain amount of 1,4-GL and NAC were accurately weighed and dissolved in an appropriate amount of physiological saline at different mass ratios to prepare an oral solution, which was then filtered and sterilized to prepare a solution. In the following examples, the drug composition used for oral gavage treatment of the intervention group was prepared at a mass ratio of 1:1.

[0044] At the same time, 1,4-GL solution and NAC solution were prepared separately and used for oral gavage treatment of the intervention group in the following examples.

[0045] Alternatively, accurately weigh a certain amount of 1,4-GL and NAC according to the ratio and dissolve them in an appropriate amount of normal saline to prepare an injection, which is then filtered and sterilized to prepare a lyophilized powder injection.

[0046] Example 2: Verification of therapeutic efficacy in lethal animal model:

[0047] Trial groups:

[0048] A total of 86 C57BL / 6J mice were randomly divided into 6 groups: 6 normal control groups (Control group), 16 700 mg / kg APAP-induced hepatotoxicity mouse model groups (Model group), 16 200 mg / kg NAC intervention groups (NAC group) during 700 mg / kg APAP-induced hepatotoxicity, 16 200 mg / kg 1,4-GL intervention groups (1,4-GL group) during 700 mg / kg APAP-induced hepatotoxicity, 16 100 mg / kg 1,4-GL combined with 100 mg / kg NAC intervention groups (1,4-GL+NAC group) during 700 mg / kg APAP-induced hepatotoxicity, and 16 400 mg / kg NAC intervention groups (DNAC group) during 700 mg / kg APAP-induced hepatotoxicity.

[0049] Test process:

[0050] Normal control group (Control group): normal diet.

[0051] Each intervention treatment group, under normal diet, was gavaged once every 24 hours with the corresponding intervention group drug and the corresponding concentration requirements (i.e., each gavage was performed according to the concentration requirements as described above, for example, the 100 mg / kg 1,4-GL combined with 100 mg / kg NAC intervention group was gavaged with 100 mg / kg 1,4-GL combined with 100 mg / kg NAC each time, and the same was true for other treatment groups) for 5 days. On the 6th day, APAP was gavaged once at 700 mg / kg. 3 hours after APAP induction administration, the corresponding intervention group drug was gavaged again. 24 hours after APAP induction administration, blood and liver samples of surviving mice were collected.

[0052] The Model group was fed a normal diet for the first 5 days and then orally administered with 700 mg / kg APAP on the 6th day. Blood and liver samples were collected from surviving mice 24 hours after APAP induction.

[0053] Experimental results:

[0054] Results showed that, except for the control group, mice in the Model, NAC, 1,4-GL, 1,4-GL + NAC, and DNAC groups experienced significant deterioration in mental state, activity, and appetite, as well as extreme lethargy, after administration of a lethal dose of 700 mg / kg APAP. Deaths first occurred in the Model group 12 hours after APAP administration, followed by deaths in all other groups from 13 to 14 hours. From 12 to 22 hours after APAP administration, mouse mortality was recorded hourly, and survival curves were plotted for each APAP group. By the 22nd hour, a significant number of mice had died, with survival rates of only 25% in the Model group, 50% in the NAC group, 37.5% in the 1,4-GL group, 69% in the 1,4-GL + NAC group, and 43.75% in the DNAC group. The 1,4-GL + NAC group had the highest survival rate, while the Model group suffered the most severe mortality. The survival curves of the mice in each APAP group were drawn for statistical analysis, and it was found that only the survival of the mice in the 1,4-GL+NAC group was significantly improved compared with the Model group (p < 0.05, Figure 1 ), with a survival rate of 69%, which was significantly higher than that of the NAC group (50%) and 1,4-GL (37.5%) with the same dosage, and the DNAC group with a high dosage (43.75%), indicating that there was a significant synergistic effect between 1,4-GL and NAC.

[0055] The serum biochemical indicators of mice in each group that took a lethal dose of 700 mg / kg APAP were compared with those in the control group (measured using the ELASA kit according to the instructions). It was found that the serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBIL (total bilirubin) and MDA (serum malondialdehyde) levels in the Model group were significantly increased compared with the Control group (p < 0.05, Figure 2 AC, F), the contents of GSH (glutathione) and SOD (superoxide dismutase) decreased significantly (p < 0.05, Figure 2-1 Among the intervention groups, only the 1,4-GL+NAC group had significantly lower serum ALT and TBIL levels than the Model group (p<0.05, Figure 2 In addition, although the serum AST level in the NAC group was significantly lower than that in the Model group (p < 0.05, Figure 2 B), the serum GSH and SOD levels in the NAC and DNAC groups were significantly restored compared with those in the Model group, and the serum MDA level was also significantly reduced (p < 0.05, Figure 2 DF), but the improvement of these indicators in the 1,4-GL+NAC group was more significant than that in the NAC and DNAC groups (p<0.05, Figure 2 (B, DF).

[0056] In the liver injury induced by lethal dose of APAP in mice, the 1,4-GL+NAC group showed the most significant improvement in serum biochemical indicators. Compared with the Model group, serum ALT decreased by 49.04%, AST decreased by 74.71%, TBIL decreased by 22.19%, GSH increased by 494.93%, SOD increased by 120.19%, and MDA decreased by 39.42%, all of which were significantly improved (p < 0.05, Figure 2 Medium AF).

[0057] In addition, the improvement effect of the 1,4-GL+NAC group was more significant than that of the NAC and 1,4-GL monotherapy groups. Among them, serum AST and MDA were significantly lower than those of the NAC, 1,4-GL and DNAC groups (AST: 250.78±130.62 vs 561.32±187.55, 850.27±254.74, 696.43±237.13; MDA: 15.17±1.15 vs 20.23±3.68, 20.91±4.3, 20.11±1.09), GSH and SOD were significantly increased (GSH: 24.63±5.82 vs 15.48±4.96, 14.3±5.97, 14.04±3.04; SOD: 307.62±31.58 vs 218.77±28.7, 201.01±33.63, 218.52±29.92)(p<0.05, see Figure 2 (B, DF).

[0058] Example 3: Non-lethal animal model efficacy verification:

[0059] Experimental groups and drug administration: 51 C57BL / 6J mice were randomly divided into 6 groups: 6 normal control groups (Control group), 9 300 mg / kg APAP-induced hepatotoxicity mouse model groups (Model group), 9 200 mg / kg NAC intervention groups (NAC group) during 300 mg / kg APAP-induced hepatotoxicity, 9 200 mg / kg 1,4-GL intervention groups (1,4-GL group) during 300 mg / kg APAP-induced hepatotoxicity, 9 100 mg / kg 1,4-GL combined with 100 mg / kg NAC intervention groups (1,4-GL+NAC group) during 300 mg / kg APAP-induced hepatotoxicity, and 9 400 mg / kg NAC intervention groups (DNAC group) during 300 mg / kg APAP-induced hepatotoxicity.

[0060] The test process was the same as in Example 1, except that the dosage of APAP was 300 mg / kg.

[0061] Experimental results:

[0062] The results showed that compared with the healthy mice in the Control group, the serum ALT, AST, TBIL, and MDA levels of the Model group mice that took a non-lethal dose of 300 mg / kg APAP were significantly increased, and GSH and SOD were consumed in large quantities and their synthesis was significantly insufficient (p < 0.05, see Figure 3The intervention groups of NAC, 1,4-GL, 1,4-GL+NAC and DNAC all corrected the changes in serum biochemical indicators of APAP-poisoned mice. The serum ALT, AST, TBIL and MDA were significantly lower than those of the Model group, while GSH and SOD were significantly increased compared with the Model group (p < 0.05, see Figure 3 Medium AF).

[0063] Among them, the improvement effect of 1,4-GL+NAC group was the most outstanding. Compared with the Model group, ALT decreased by 40.74%, AST decreased by 45.28%, TBIL decreased by 76.63%, GSH increased by 101.21%, SOD increased by 51.89%, and MDA decreased by 32.75%, all of which were significantly improved (p < 0.05, see Figure 3 Medium AF).

[0064] The improvement effect of the 1,4-GL+NAC group was more significant than that of the NAC and 1,4-GL monotherapy groups. The serum ALT was significantly lower than that of the NAC, 1,4-GL and DNAC groups (2370.37±282.75 vs 3175.42±271.30, 3350.09±590.66, 3099.90±259.31), and SOD was significantly increased (353.55±28.28 vs 309.90±18.22, 283.45±35.72, 285.77±12.13) (p < 0.05, see Figure 3 Compared with the NAC and 1,4-GL groups, serum TBIL was significantly lower (2.32±0.97 vs 4.90±0.88, 5.83±1.25), and GSH was significantly higher (33.24±1.67 vs 25.56±1.38, 24.52±1.46) (p<0.05, see Figure 3 The serum MDA was significantly lower than that of the 1,4-GL and DNAC groups (9.98±1.38 vs 12.47±0.97, 12.50±1.74) (p<0.05, see Figure 3 Of course, the serum AST level in the 1,4-GL+NAC group was also the lowest (see Figure 3 Middle B).

[0065] The liver lobule structure of mice in the control group was intact, the size and morphology of liver cells were normal, the hepatic cords were arranged regularly and orderly, no liver cell degeneration and necrosis and inflammatory cell infiltration were observed, and the liver sinusoid structure was intact (see Figure 4In the non-lethal dose of 300 mg / kg APAP, the liver tissue of the mice in the Model group showed large-scale hemorrhage and necrosis centered on the central vein in the hepatic lobules, unclear hepatic lobule structure, disordered hepatic cords, inflammatory cell infiltration, and obvious swelling and severe degeneration of most hepatocytes. Some cells were lysed, cell nuclei were fragmented, and the boundaries between cells were unclear. There was also congestion of the hepatic sinusoids (see Figure 4 In the intervention NAC ( Figure 4 C), 1,4-GL( Figure 4 D), 1,4-GL+NAC( Figure 4 Middle E) and DNAC group ( Figure 4 Middle F) Pathological damage in the liver of mice was significantly alleviated compared with that in the Model group, with a significantly reduced area of necrosis, preserved liver lobule structure, disordered hepatic cords, inflammatory cell infiltration, mild to moderate swelling and hydropic degeneration in most hepatocytes, lysis of some cells, unclear boundaries between cells, nuclear fragmentation, and hepatic sinusoidal congestion. The pathological improvement in the 1,4-GL+NAC group was the most obvious ( Figure 4 Middle E).

[0066] Example 4: Comparative study on the efficacy of 1,4-GL, NAC or their combination on mice intoxicated with APAP for 1 and 6 hours

[0067] Eighty-seven C57BL / 6J mice were randomly divided into 10 groups: 6 normal control groups (Control group), 9 300 mg / kg APAP-induced hepatotoxicity mouse model groups (Model group), 9 300 mg / kg APAP-induced hepatotoxicity 1 hour later 200 mg / kg NAC intervention groups (NAC 1h group), 9 300 mg / kg APAP-induced hepatotoxicity 6 hours later 200 mg / kg NAC intervention groups (NAC 6h group), 9 300 mg / kg APAP-induced hepatotoxicity 1 hour later 200 mg / kg 1,4-GL intervention groups (1,4-GL 1h group), 9 300 mg / kg APAP-induced hepatotoxicity 6 hours later 200 mg / kg 1,4-GL intervention groups (1,4-GL 6h group), 300 mg / kg APAP-induced hepatotoxicity 1 hour later 100 mg / kg 1,4-GL combined with 100 mg / kg There were 9 rats in the NAC intervention group (1,4-GL+NAC 1h group), 9 rats in the 100 mg / kg 1,4-GL combined with 100 mg / kg NAC intervention group (1,4-GL+NAC 6h group) after 300 mg / kg APAP-induced hepatotoxicity for 6 h, 9 rats in the 400 mg / kg NAC intervention group (DNAC 1h group) after 300 mg / kg APAP-induced hepatotoxicity for 1 h, and 9 rats in the 400 mg / kg NAC intervention group (DNAC 6h group) after 300 mg / kg APAP-induced hepatotoxicity for 6 h.

[0068] Normal control group (Control group): normal diet.

[0069] Each intervention group was gavaged 1 hour after APAP induction: under normal diet, the corresponding intervention group drug was gavaged once every 24 hours at the corresponding concentration requirements for 5 days. On the 6th day, APAP was gavaged once at 300 mg / kg. One hour after APAP induction, the intervention group drug of the corresponding concentration was gavaged again. The drugs gavaged each time in each intervention group were: NAC 1h group: 0.2mL 20mg / mL NAC solution; 1,4-GL 1h group: 0.2mL 20mg / mL 1,4-GL solution; 1,4-GL+NAC 1h group: 0.1mL 20mg / mL1,4-GL+0.1mL 20mg / mL NAC solution; DNAC 1h group: 0.2mL 40mg / mL NAC solution.

[0070] Each intervention group was gavaged 6 hours after APAP induction: under normal diet, the corresponding intervention group drug was gavaged once every 24 hours at the corresponding concentration requirement for 5 days. On the 6th day, APAP was gavaged once at 300 mg / kg. 6 hours after APAP induction, the intervention group drug was gavaged again with the corresponding concentration requirement. The drugs for each intervention group were gavaged as follows: NAC 6h group: 0.2mL 20mg / mL NAC solution; 1,4-GL 6h group: 0.2mL 20mg / mL 1,4-GL solution; 1,4-GL+NAC 6h group: 0.1mL 20mg / mL1,4-GL+0.1mL 20mg / mL NAC solution; DNAC 6h group: 0.2mL 40mg / mL NAC solution.

[0071] The Model group was fed a normal diet for the first 5 days and then orally administered with 300 mg / kg APAP on the 6th day.

[0072] 24 hours after APAP induction administration, blood and liver samples were collected from mice.

[0073] Experimental results:

[0074] In the time-limited experiment, the serum ALT and AST levels of the Model group mice taking 300 mg / kg APAP were significantly higher than those of the healthy mice in the Control group (p < 0.05, see Figure 5 In the NAC, 1,4-GL, 1,4-GL+NAC and DNAC groups, which were intervened 1 hour after taking APAP, the serum ALT and AST levels of mice were significantly decreased (p < 0.05, see Figure 5The serum ALT and AST levels in the 1,4-GL+NAC group were decreased by 76.05% and 70.90% compared with those in the Model group, respectively, and were significantly lower than those in the NAC and 1,4-GL groups (ALT: 792.62±180.95 vs 1322.3±513.05, 2328.72±453.44; AST: 224.49±55.55 vs 352.79±63.48, 506.76±97.06).

[0075] In the comparison of serum ALT and AST levels in mice treated 6 hours after taking APAP, it was found that only the serum ALT and AST levels in the 1,4-GL and 1,4-GL+NAC groups were significantly lower than those in the Model group (p < 0.05, see Figure 5 Among them, the serum ALT and AST levels in the 1,4-GL+NAC group decreased by 27.58% and 25.15% respectively compared with the Model group, and were significantly lower than those in the NAC and DNAC groups (ALT: 2397.09±446.63 vs 2998.5±453.6, 3393.27±455.16; AST: 577.47±61.11 vs 678.66±99.56, 740.76±61.65) (p < 0.05, see Figure 5 (middle AB).

[0076] The above experimental results show that the combined use of 1,4-GL+NAC was more effective than either drug alone when administered 1 hour and 6 hours after APAP induction. More importantly, the combined use of 1,4-GL+NAC also showed significant efficacy when administered 6 hours after APAP induction, while NAC had little effect at this time.

[0077] Example 5: Mechanism Studies

[0078] Three mice were randomly selected from each group in Example 2, and the ROS (reactive oxygen species) levels in the fresh livers of the mice in each group were measured and compared. The fluorescence intensity of the DHE staining in the livers of healthy mice in the control group was the weakest. The fluorescence intensity of the livers of mice in each group treated with APAP was enhanced, with the fluorescence intensity of the model group being the strongest. The fluorescence intensity of the 1,4-GL+NAC group was the weakest among the APAP-treated groups and was closest to the fluorescence intensity of the control group (see Figure 6 The ROS content in the liver of the Model group mice was significantly higher than that in the Control group (p < 0.05, see Figure 6The ROS levels in the livers of mice in the intervention groups of NAC, 1,4-GL, 1,4-GL+NAC and DNAC were down-regulated by 18.96%, 13.60%, 44.34% and 22.25% respectively compared with those in the Model group, all with significant therapeutic effects (p < 0.05, see Figure 6 Among them, the improvement effect of 1,4-GL+NAC group was more significant than that of NAC and 1,4-GL alone group (p<0.05, see Figure 6 Middle B) has the strongest ability to reduce ROS levels in the liver of APAP-injured mice, with a ROS downregulation ratio of up to 44.34%, which is significantly better than other treatment groups.

[0079] Compared with the Control group, the expression levels of liver CYP2E1 (cytochrome P4502E1), JNK (c-Jun amino-terminal kinase), P-JNK, FOS and JUN proteins in the Model group were significantly increased (P < 0.05, see Figure 7 Compared with the Model group, the expression levels of CYP2E1, JNK and P-JNK proteins in the liver of mice in the 1,4-GL group were significantly downregulated by 40.12%, 20.01% and 34.06%, respectively (P < 0.05, see Figure 7 In the 1,4-GL+NAC group, CYP2E1 protein was downregulated by 40.27%, JNK by 26.98%, P-JNK by 36.53%, FOS by 19.27%, and JUN by 26.08%, all of which were significantly downregulated compared with the protein expression levels in the Model group (P < 0.05, see Figure 7 Among them, the expression levels of CYP2E1 protein in the 1,4-GL and 1,4-GL+NAC groups were similar, with no significant difference. However, the expression levels of JNK and P-JNK proteins in the 1,4-GL+NAC group were more significantly downregulated than those in the 1,4-GL group (P < 0.05, see Figure 7 The expression levels of CYP1A2 protein in the liver of mice in each APAP group showed an upward trend compared with the control group, but there was no significant difference (see Figure 7 Middle A).

[0080] In addition, in the comparison between the Model group and the group taking 200 mg / kg NAC intervention, it was found that the expression levels of CYP2E1 and CYP1A2 proteins in the liver of the two groups of mice were similar, and there was no significant difference between the groups (see Figure 7 Middle B).

[0081] Compared with the Control group, the expression level of CYP2E1 mRNA in the liver of the Model group mice was significantly increased (P < 0.05, see Figure 8 A), CYP1A2 mRNA also showed an upward trend (see Figure 8 The expression levels of CYP2E1 mRNA in the liver of mice in the 1,4-GL and 1,4-GL+NAC groups were down-regulated by 56.82% and 63.21%, respectively, compared with those in the Model group. The down-regulation rates of the two groups were similar but significantly down-regulated compared with those in the Model group (P < 0.05, see Figure 8 The expression level in the NAC group was close to that in the Model group, and there was no significant difference between the groups (see Figure 8 The expression levels of CYP1A2 mRNA in the liver of mice in each APAP group showed an upward trend compared with the control group, but there was no significant difference (see Figure 8 There was no significant difference between the NAC group and the Model group (see Figure 8 Middle D).

[0082] The expression levels of MAPK8, FOS and JUN mRNA in the liver of mice in the Model group were significantly increased compared with those in the healthy mice in the Control group (P < 0.05, see Figure 8 Compared with the model, the expression levels of MAPK8 mRNA in the liver of mice in the 1,4-GL group and the 1,4-GL+NAC group were significantly downregulated by 39.97% and 70.20%, FOS mRNA was downregulated by 52.55% and 95.03%, and JUN mRNA was downregulated by 35.97% and 74.57%, respectively (P < 0.05, see Figure 8 Most notably, the down-regulation of these mRNAs in the 1,4-GL+NAC group was more significant than that in the 1,4-GL group (P < 0.05, see Figure 8 (EG).

[0083] The above results show that, on the one hand, the combination therapy mainly reduces ROS generation through the dual detoxification mechanism of 1,4-GL inhibiting NAPQI production and NAC synthesizing GSH to neutralize NAPQI; on the other hand, 1,4-GL regulates the JNK pathway and its downstream effector molecules to exert a liver-protective effect, thereby breaking the time limit of existing antidotes and providing new ideas for the treatment of severe DILI patients in clinical practice.

[0084] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A pharmaceutical composition, characterized in that The invention comprises glucaric acid 1,4-lactone and N-acetylcysteine, wherein the mass ratio of glucaric acid 1,4-lactone to N-acetylcysteine is 1:1-5. 。 2. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the glucaric acid 1,4-lactone to N-acetylcysteine is 1:1-2.

3. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the glucaric acid 1,4-lactone to N-acetylcysteine is 1:

1.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a drug for treating or preventing drug-induced liver injury, wherein the drug-induced liver injury is liver injury caused by an overdose or long-term use of acetaminophen.

5. The use according to claim 4, characterized in that The drug for treating or preventing drug-induced liver injury comprises an effective therapeutic amount of the pharmaceutical composition and a pharmaceutically acceptable carrier.

6. The use according to claim 4, characterized in that The dosage form of the drug for treating or preventing drug-induced liver injury is an injection or an oral preparation.

7. The use according to claim 6, characterized in that The concentration of glucaric acid 1,4-lactone in the injection is 20-50 μM, and the concentration of N-acetylcysteine is 50-100 μM.

8. The use according to claim 4, characterized in that The pharmaceutical composition simultaneously neutralizes N-acetyl-p-benzoquinone imine, inhibits cytochrome P450 2E1, and regulates the c-Jun amino-terminal kinase pathway.

9. A drug for treating or preventing drug-induced liver injury, wherein the drug-induced liver injury is liver injury caused by acetaminophen overdose or long-term use, characterized in that: The invention comprises a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

10. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a cytochrome P450 2E1 inhibitor.

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