Application of Heterocyclic Compounds in Protecting Drug-Induced Liver Injury

Abstract

The present invention relates to the fields of biology and medicine, and discloses the use of heterocyclic compounds in protecting against drug-induced liver injury. The inventors of the present invention have found that treatment with heterocyclic compounds can protect against drug-induced acute liver injury, including increasing the survival of mice, reducing the release of transaminases in serum, and inhibiting liver necrosis; the heterocyclic compounds alone or in combination with antidotes can significantly treat drug-induced acute liver injury or liver failure. Therefore, the heterocyclic compounds can be used as potential drugs for preventing and / or treating drug-induced liver injury.

Description

Technical Field

[0001] The present invention relates to the fields of biology and medicine, and particularly to the application of heterocyclic compounds in protecting against drug-induced liver injury. Background Art

[0002] Drug-induced liver injury (DILI) refers to liver injury caused by the use of chemical drugs, biological products, traditional Chinese patent medicines and other drugs in clinical treatment of diseases. In recent years, with the continuous development of society and the improvement of people's living standards, the types of liver diseases in China have gradually changed from viral hepatitis to drug-induced liver injury. At present, the clinical treatment methods for drug-induced liver injury or acute liver failure are extremely limited. The only clinically approved antidote is N-acetylcysteine (NAC), but the efficacy and use of NAC have obvious limitations. Therefore, it is of great clinical significance to discover new drugs that can effectively protect and treat drug-induced liver injury. Thiolutin (THL for short in this article) is a sulfur-based microbial antibiotic produced by Streptomyces, which has broad-spectrum antibacterial activity, but its research in drug-induced liver injury has not been reported. Summary of the Invention

[0003] In order to overcome the above problems existing in the prior art, the present invention provides the application of heterocyclic compounds in protecting against drug-induced liver injury.

[0004] In a first aspect, the present invention provides the application of at least one of the heterocyclic compounds represented by Formula I in the preparation of a drug for preventing and / or treating drug-induced liver injury.

[0005]

[0006] In a second aspect, the present invention provides a pharmaceutical composition, which contains at least one of the heterocyclic compounds and an additional antidote.

[0007] In a third aspect, the present invention provides the application of the pharmaceutical composition in the preparation of a drug for preventing and / or treating drug-induced liver injury.

[0008] In a fourth aspect, the present invention provides the application of at least one of the heterocyclic compounds or the pharmaceutical composition in the preparation of a drug for increasing the survival rate of patients with drug-induced liver injury, reducing the release of transaminase in serum or inhibiting liver necrosis.

[0009] The heterocyclic compounds shown in Formula I (such as thiogallin or holomycin) can protect against drug-induced acute liver injury (such as acetaminophen (APAP) and thioacetamide (TAA)) in a dose-dependent manner, including increasing patient survival rate (especially the survival rate under lethal-dose drug usage), reducing the release of transaminases in serum, and inhibiting liver necrosis; the heterocyclic compounds alone or in combination with an antidote (such as N-acetylcysteine, NAC) can significantly treat drug-induced acute liver injury or liver failure. Therefore, the heterocyclic compounds can be used as potential drugs for preventing and / or treating drug-induced liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It shows that in Example 1 of the present invention, thiogallin improves APAP-induced drug-induced liver injury in a dose-dependent manner;

[0011] Figure 2 It shows that in Example 2 of the present invention, thiogallin significantly improves acute liver failure induced by lethal-dose APAP;

[0012] Figure 3 It shows that in Example 3 of the present invention, thiogallin can rescue APAP-induced drug-induced liver injury;

[0013] Figure 4 It shows that in Example 4 of the present invention, treatment with thiogallin significantly increases the survival rate of mice treated with lethal-dose APAP;

[0014] Figure 5 It shows that in Example 5 of the present invention, thiogallin can improve the release of transaminases in a TAA-induced drug-induced liver injury model;

[0015] Figure 6 It shows that in Example 6 of the present invention, holomycin can improve APAP-induced drug-induced liver injury. DETAILED DESCRIPTION OF THE INVENTION

[0016] The endpoints and any values disclosed herein are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0017] The present invention provides the use of at least one of the heterocyclic compounds shown in Formula I in the preparation of a drug for preventing and / or treating drug-induced liver injury,

[0018]

[0019] Among them, R1 is H or an alkyl group with 1 to 6 carbon atoms, and R2 is an alkyl group with 1 to 6 carbon atoms. The heterocyclic compound can alleviate the symptoms of drug-induced liver injury in a dose-dependent manner.

[0020] Preferably, R1 is H or an alkyl group with 1 to 4 carbon atoms, more preferably H or methyl.

[0021] Preferably, R2 is an alkyl group with 1 to 4 carbon atoms, more preferably methyl.

[0022] In a particularly preferred embodiment of the present invention, the heterocyclic compound is thiolutin (THL, the structure is shown in Formula I-1) and / or holomycin (HL, the structure is shown in Formula I-2).

[0023]

[0024] The present invention also provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition contains at least one of the heterocyclic compounds shown in Formula I and an additional antidote.

[0025] In the present invention, the weight ratio of the heterocyclic compound to the additional antidote is preferably 1:250 - 350, such as 1:250, 1:270, 1:290, 1:310, 1:330, 1:350 or any value between the above values.

[0026] In the present invention, the additional antidote can be an active substance commonly used in the art that has the effect of alleviating drug-induced liver injury. Preferably, the additional antidote is N-acetylcysteine.

[0027] The present invention also provides the use of the pharmaceutical composition as described above in the preparation of a drug for preventing and / or treating drug-induced liver injury.

[0028] In the present invention, the drug-induced liver injury (or liver failure) is a common drug-induced liver injury, such as drug-induced liver injury induced by at least one of antibiotics (such as quinolone antibiotics, penicillin antibiotics, cephalosporin antibiotics), anti-inflammatory, analgesic and antipyretic drugs or antiplatelet aggregation drugs (such as aspirin), lipid-lowering drugs (such as lipid-lowering statin drugs) and antitumor drugs. Preferably, it is drug-induced liver injury induced by acetaminophen and / or thioacetamide. Prevention and / or treatment include increasing the survival rate of patients, reducing the release of transaminase in the serum and inhibiting liver necrosis.

[0029] Acetaminophen (APAP) overdose is one of the most common causes of drug-induced liver injury worldwide. APAP is a widely used antipyretic and analgesic drug that is converted into N-acetyl-para-benzo-quinone imine (NAPQI) in the liver under the catalysis of cytochrome P450 (CYP450). Glutathione (GSH) in the liver normally combines with mercaptoacetic acid and cysteine to promote the excretion of electrophilic NAPQI. When glutathione is deficient, oxidative stress or mitochondrial dysfunction occurs, the toxic NAPQI cannot be normally metabolized and binds to intracellular proteins, leading to hepatocyte dysfunction and necrosis, and ultimately resulting in liver failure and death of the organism.

[0030] The present invention also relates to the use of at least one of the heterocyclic compounds represented by formula I or the pharmaceutical composition in the preparation of a medicament for increasing the survival rate of patients with drug-induced liver injury, reducing the release of transaminases in serum or inhibiting liver necrosis.

[0031] The present invention also relates to a method for preventing and / or treating drug-induced liver injury, which is characterized in that the method comprises: administering the heterocyclic compound or the pharmaceutical composition to a patient with drug-induced liver injury.

[0032] In the present invention, there is no particular limitation on the administration mode of the heterocyclic compound. For example, the administration route may include at least one of intravenous injection, intramuscular injection and subcutaneous injection. Those skilled in the art can formulate the heterocyclic compound into a specific dosage form, such as an injection, according to the administration mode.

[0033] In the present invention, the patients to be prevented and / or treated may include mammals, such as mice or humans.

[0034] In the present invention, the dosage of the heterocyclic compound can be determined according to the condition of the patient. Taking mice as an example, the dosage of the heterocyclic compound can be 0.1 - 5 mg / kg body weight / day.

[0035] The present invention will be described in detail below by way of examples. In the following examples, the mice are female C57BL / 6J mice about 8 - 10 weeks old.

[0036] Example 1

[0037] This example is used to illustrate that thiolutin improves APAP-induced drug-induced liver injury in a dose-dependent manner.

[0038] First, the sublethal acute liver injury model induced by 300 mg / kg APAP was used to study the hepatoprotective effect of different concentrations of THL. Mice were intraperitoneally injected with 300 mg / kg APAP after fasting for 18 hours, and intraperitoneally injected with 0 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg THL 2 hours before APAP treatment. The control group (Ctrl) was only injected with solvent control. The number of mice in each group was 8. Liver and serum samples were collected 12 hours after APAP treatment. The results of pathological tissue section HE staining (Solebol hematoxylin and eosin (HE) staining kit, G1120) showed that APAP treatment caused severe liver tissue damage in mice, manifested as severe congestion, typical acute large-scale necrosis around the central vein, nuclear fragmentation or dissolution, accompanied by swelling and degeneration of hepatocytes and a large number of inflammatory cell infiltration. Pretreatment with 0.1mg / kg THL can effectively reduce liver tissue congestion, and pretreatment with 0.25mg / kg THL can significantly reduce liver tissue necrosis; the liver tissue damage of mice in the 0.5mg / kg THL pretreatment group was very mild, and there was no obvious congestion in the liver tissue. Although cell swelling still existed around the central vein, large pieces of necrosis basically disappeared; there was almost no abnormality in the liver tissue of mice in the 1mg / kg THL pretreatment group ( Figure 1 A). According to statistics, the area of ​​liver tissue necrosis in mice in the 0mg / kg group was as high as 41.7%, while the areas of liver tissue necrosis in mice in the 0.1, 0.25, and 0.5mg / kg THL groups were 36.95%, 23.3%, and 1.30%, respectively. There was no liver tissue necrosis in the 1mg / kg THL group ( Figure 1 B). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are important indicators commonly used to evaluate liver tissue damage. APAP can induce a significant increase in serum ALT and AST levels, while THL pretreatment can significantly reduce serum ALT and AST levels in a dose-dependent manner. The inhibition rate of 0.25 mg / kg THL on ALT and AST exceeded 50%, while the inhibition rate of 0.5 mg / kg THL exceeded 95%. The serum ALT and AST levels of mice in the 1 mg / kg THL group were basically close to those of normal mice ( Figure 1 C). Based on the above experimental results, THL pretreatment 2 hours in advance can reduce 300 mg / kg APAP-induced liver injury in a dose-dependent manner, and 1 mg / kg THL can almost completely block APAP-induced liver injury. Figure 1 In the table, “*” indicates p<0.05, “**” indicates p<0.01, “***” indicates p<0.001, and “****” indicates p<0.0001, the same below.

[0039] Example 2

[0040] This example is used to illustrate that thiolutin can significantly improve acute liver failure induced by a lethal dose of APAP.

[0041] The aforementioned experiment demonstrated that 1 mg / kg THL could completely block liver injury induced by a sub-lethal dose (300 mg / kg) of APAP. Subsequently, the effect of THL on acute liver failure induced by a lethal dose of APAP was explored. First, a liver failure model was established using the commonly used lethal dose of 500 mg / kg APAP. In the THL group, 1 mg / kg THL was intraperitoneally injected 2 hours before APAP treatment. The number of mice in each group was 10. The survival of the mice was observed and recorded within 96 hours after the injection of APAP. The results showed that the survival rate of the control group mice decreased to 18.18% within 27 hours and all died within 36 hours. However, within 96 hours, no mice in the THL group died, and the survival rate was 100% ( Figure 2 A). When the APAP dose was increased to 700 mg / kg, all the control group mice died within 18 hours. After 96 hours of observation, the survival rate of the THL group mice still reached 72%, 73% ( Figure 2 B). When the APAP dose was further increased to 900 mg / kg, all the control group mice died within 9 hours. The mortality rate of the THL group reached 36.36% at 9 hours, and the survival rate could still be maintained at 27.27% at the end of the 96-hour observation period ( Figure 2 C). The above results indicate that 1 mg / kg THL can significantly improve the resistance of mice to acute liver failure induced by a lethal dose of APAP. In the range of 500 - 900 mg / kg APAP, THL can significantly increase the survival rate of mice.

[0042] Example 3

[0043] This example is used to illustrate that thiolutin can treat drug-induced liver injury induced by APAP.

[0044] First, the therapeutic effect and therapeutic window of 1 mg / kg THL on 300 mg / kg APAP-induced liver injury were detected. Mice were divided into the APAP- group, the APAP group, the THL group, and the NAC group. Mice in the APAP group, the THL group, and the NAC group were intraperitoneally injected with 300 mg / kg APAP. Three dosing time points were set in the THL treatment group, and 1 mg / kg THL was intraperitoneally injected at 1, 2, and 4 hours after APAP treatment. Three dosing time points were also set in the NAC treatment group, and 400 mg / kg NAC was intraperitoneally injected at 1, 2, and 4 hours after APAP treatment. The APAP- group was the untreated control group, and the same volume of normal saline as that in the APAP group was intraperitoneally injected. The number of mice in each group was 8. Mouse liver and serum samples were collected 12 hours after APAP treatment. The results of HE staining and serum transaminase detection showed that obvious liver damage occurred in the mice in the APAP group, manifested as acute liver injury phenotypes such as an increased area of liver tissue necrosis and an elevated level of serum transaminase. However, when THL and NAC treatments were given at 1 or 2 hours after APAP treatment, no obvious necrotic areas appeared in the liver tissues of the mice, and the level of serum transaminase only increased slightly. It can be seen that both THL and NAC had significant therapeutic effects when administered within 2 hours after APAP treatment, and there was no obvious difference in the therapeutic effects between the two( Figure 3 A-C).

[0045] Example 4

[0046] This example is used to illustrate that the treatment with thiochrysin significantly improves the survival rate of mice treated with a lethal dose of APAP.

[0047] To further clarify the protective effect of THL on APAP-induced acute liver injury in mice, explore whether the survival rate of mice treated with a lethal dose of APAP can be improved by THL treatment, the range of APAP lethal doses at which THL can increase the survival rate of mice, and compare the therapeutic effects of THL and NAC, a therapeutic model of THL and NAC was established on the basis of the aforementioned lethal dose APAP mouse model. The modeling method was to divide all mice into five groups, namely Ctrl, THL 1, THL 2.5, NAC 300, and NAC 600 groups. After fasting for 18 hours, all groups were intraperitoneally injected with 500 mg / kg APAP. One hour after APAP treatment, the THL 1, THL 2.5, NAC 300, and NAC 600 groups were intraperitoneally injected with 1 mg / kg THL, 2.5 mg / kg THL, 300 mg / kg NAC, and 600 mg / kg NAC, respectively. The Ctrl group was intraperitoneally injected with the same volume of normal saline as the 1 mg / kg THL group. The number of mice in each group was 10, and the number of surviving mice within 96 hours after APAP injection was counted. Mouse models treated with 700 mg / kg APAP and 900 mg / kg APAP were established in the same way, and a THL 1+NAC 300 group treated with a combination of 1 mg / kg THL and 300 mg / kg NAC was added respectively. The experimental results are as Figure 4 shown. All the mice in the Ctrl group treated with 500 mg / kg, 700 mg / kg, and 900 mg / kg APAP died within 48, 24, and 12 hours respectively; when modeling with 500 mg / kg APAP, the survival rates of the THL 1, THL 2.5, NAC 300, and NAC 600 groups were significantly higher than that of the Ctrl group, reaching 100%, 72.7%, 90.9%, and 90.9% respectively ( Figure 4 A); when modeling with 700 mg / kg APAP, the survival rates of the THL 1, THL 2.5, NAC 300, and NAC 600 groups were still significantly higher than that of the Ctrl group, being 27.3%, 45.5%, 18.2%, and 45.5% respectively. Additionally, it is worth noting that the therapeutic effect of the THL 1+NAC 300 group was significant, and the survival rate was 100%, which was better than that of THL and NAC used alone ( Figure 4 B); when modeling with 900 mg / kg APAP, the improvement in the survival rate by THL and NAC alone was not significant, but it could prolong the survival time of mice. However, the combined treatment of THL 1+NAC 300 could still increase the survival rate to 63.64% ( Figure 4 C). Based on the above experimental results, both THL and NAC alone can improve the survival of mice treated with a lethal dose of 500 - 900 mg / kg APAP. The therapeutic effects of the two alone are comparable, but the combined use of THL and NAC can achieve excellent therapeutic effects on high-dose APAP.

[0048] Example 5

[0049] This example is used to illustrate that thioguanone can also protect against drug-induced liver injury induced by TAA.

[0050] A drug-induced liver injury model was established using 350 mg / kg of thioacetamide (TAA). The THL group was intraperitoneally injected with 1 mg / kg of THL 2 hours before APAP treatment. The number of mice in each group was 8. Liver and serum samples were collected 12 hours after TAA treatment. The results showed that TAA could induce a significant increase in serum ALT and AST levels, while THL pretreatment could significantly reduce serum ALT and AST levels ( Figure 5 ). This indicates that thioguanone can also protect against drug-induced liver injury induced by TAA, suggesting the universality of thioguanone in the protection of drug-induced liver injury.

[0051] Example 6

[0052] This example is used to illustrate that the structure analogue holomycin with "thioguanone" as the core can also protect against drug-induced liver injury induced by APAP.

[0053] A drug-induced liver injury model was established using 300 mg / kg of APAP. The holomycin group was intraperitoneally injected with 1 mg / kg of holomycin (HL) 2 hours before APAP treatment. The number of mice in each group was 8. Liver and serum samples were collected 12 hours after APAP treatment. The results showed that APAP could induce a significant increase in serum ALT and AST levels, while HL pretreatment could significantly reduce serum ALT and AST levels and provide protection against liver failure induced by a lethal dose of APAP ( Figure 6 ). This indicates that holomycin can also protect against drug-induced liver injury induced by APAP, suggesting that analogues with "thioguanone" as the core also play a protective role in drug-induced liver injury.

[0054] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of technical features. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Use of at least one of the heterocyclic compounds of formula I in the preparation of a medicament for preventing and / or treating drug-induced liver injury induced by acetaminophen and / or thioacetamide, in, R1 is H or a C1-C6 alkyl group, and R2 is a C1-C6 alkyl group.

2. The use according to claim 1, wherein: R1 is H or a C1-C4 alkyl group.

3. The use according to claim 1 or 2, wherein: R2 is a C1-C4 alkyl group.

4. The use according to claim 1, wherein: R1 is H or methyl.

5. The use according to claim 1 or 4, wherein: R2 is methyl.

6. Use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating drug-induced liver injury induced by acetaminophen and / or thioacetamide, wherein: The pharmaceutical composition contains at least one of the heterocyclic compounds of Formula I and an additional antidote; Wherein, R1 is H or a C1-C6 alkyl group, and R2 is a C1-C6 alkyl group.

7. The use according to claim 6, wherein: R1 is H or a C1-C4 alkyl group.

8. The use according to claim 6 or 7, wherein: R2 is a C1-C4 alkyl group.

9. The use according to claim 6, wherein: R1 is H or methyl.

10. The use according to claim 6 or 9, wherein: R2 is methyl.

11. The use according to claim 6, wherein: The weight ratio of the heterocyclic compound to the additional antidote is 1:250-350.

12. The use according to claim 6 or 11, wherein: An alternative antidote is N-acetylcysteine.

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