WO2020220607A1 - Use of chitooligosaccharides for protecting against and treating drug-induced liver injury - Google Patents

Abstract

Disclosed is a use of chitooligosaccharides (COST) having a molecular weight of ≤1000 in the preparation of a composition for protecting against and treating drug-induced liver injury, particularly a composition for protecting against and treating liver injury induced by acetaminophen. Also disclosed is a method for protecting against and treating drug-induced liver injury, comprising administering an effective amount of COST to a subject having a liver injury. Experiments confirmed that COST improved abnormal biochemical indices of liver function induced by acetaminophen, significantly reduced an abnormal increase in mouse serum ALT and AST induced by acetaminophen, ameliorated liver tissue injury induced by acetaminophen, increased an amount of glutathione (GSH) as a detoxicant in a liver tissue having acetaminophen-induced liver injury, increased an anti-oxidation ability of oxidatively damaged liver induced by acetaminophen, and decreased an amount of malondialdehyde (MDA) in oxidatively damaged liver induced by acetaminophen. The present invention can enable treatment of a clinical drug-induced liver injury, offering a better option for patients.

Description

Use of chitosan oligosaccharides for protection and treatment of drug-induced liver injury Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the medical use of chitooligosaccharides with a molecular weight of ≤1000 in protecting and treating drug-induced liver injury.

Background technique

Drug-induced liver injury (DILI) refers to the direct toxicity of the drug itself or its metabolites to the liver after the human body is exposed to conventional or high-dose drugs, or the human body is allergic to the drug or its metabolites. Liver damage caused by metabolic idiosyncratic reactions. Acetaminophen (APAP) is a widely used analgesic in clinical practice. At therapeutic doses (<4g/d), APAP can cause a transient increase in serum aminotransferase, especially in malnutrition and liver dysfunction In people who abuse alcohol, or take certain CYP450-inducing drugs, when overdose (more than 4g/d in adults, more than 50-75mg/kg·d in minors), severe liver damage and even acute liver failure may occur (Acute liver failure, ALF). It is estimated that about 2,000 people in European and American countries experience ALF each year, and nearly 50% of them are drug-induced liver injury caused by APAP. 61% of cases seem to be caused by the use of the median dose (34g/3d). Researchers found that the annual incidence of drug-induced liver injury in China is as high as 23.80 per 100,000 people. Among them, acute drug-induced liver injury accounted for 87%, and 13% of patients had chronic drug-induced liver injury. Among the drugs that cause liver damage, the number one drug category in China is various health products and traditional Chinese medicines, accounting for 26.81%, followed by anti-tuberculosis drugs, accounting for 21.99%. Although liver damage caused by APAP only It accounts for 3.8% of all DILI, but accounts for 50.8% of all DILI caused by analgesics and anti-inflammatory drugs. The domestic application of pain relief and anti-inflammation still mainly faces liver damage caused by acetaminophen, and the situation is severe.

Most APAP in the blood is combined with glucuronic acid and sulfate through UDP-glucuronyl transferase (UGT) and sulfotransferase (SULT) in the liver to form conjugated metabolites and a small amount Metabolites that have been hydroxylated and deacetylated are excreted in the urine. Under normal circumstances, about 5-9% of APAP undergoes liver metabolism through different pathways, namely cytochrome P450 enzymes (CYP450), mainly CYP2E1 and to a lesser extent CYP1A2, CYP2A6 and CYP3A4 metabolism, forming a highly active toxic intermediate The metabolite N-acetyl-p-benzoquinone imine (NAPQI). The sulfhydryl group of glutathione (GSH) then converts NAPQI into harmless metabolites excreted in the urine. However, when the phase II metabolic enzymes are saturated after APAP is excessive, the excess NAPQI depletes GSH, resulting in the covalent binding of NAPQI to sulfhydryl groups in cellular proteins, especially mitochondrial proteins. This leads to mitochondrial oxidative stress and dysfunction, which ultimately leads to liver cell necrosis. NAPQI also interferes with the complex complexes I and II of the mitochondrial electron transport chain (ETC), causing electrons to leak from ETC and combine with oxygen, thereby forming a large number of superoxide radicals (hydrogen peroxide H2O2 and peroxynitrate ONOO-) and other activities Oxygen (ROS) and reactive nitrogen (RNS) cause oxidative stress and protein nitration, which in turn leads to cytotoxicity.

N-acetylcysteine (NAC) is the only detoxification drug approved by the FDA in 2011 for the treatment of intrinsic DILI caused by acetaminophen. However, acetylcysteine has a short half-life and is only effective for early treatment. The main adverse reactions are skin rash, nausea, vomiting, and fever. The therapeutic effect is not ideal. Another main reason is that acetaminophen causes liver damage. The mechanism is very complicated. In view of this, it is very urgent and necessary to research and develop drug-induced liver injury drugs with good therapeutic effects.

Chitin (chitin), the most abundant biomass after cellulose, is commonly found in the shells of crustaceans, insect epidermis, and fungal cell walls. Treating chitin with an alkaline solution converts it into chitosan: chitin in its fully or partially deacetylated form. Chitosan can be defined as a natural non-toxic biopolymer, linear polysaccharides are composed of β-1,4-GlcNAc and β-1,4-GlcN. Chitosan contains rather unstable glycosidic bonds, which allows it to be cleaved by hydrolyzing agents to produce chitosan oligomers with a variable degree of polymerization (DP). Chitosan with a DP of less than 20 and an average MW of less than 3.9kDa is called oligochitosan (COS). COS has a wide range of biological activities and has broad application prospects in many fields such as medicine, cosmetics, food, and agriculture, as well as applications In the report of liver protection drugs, for example, CN201610639820.4 discloses the use of chitosan oligosaccharides in the preparation of drugs for sobering up and protecting the liver. The drugs are commonly used oral dosage forms, including tablets, capsules, granules, oral liquids, and suspensions. liquid. New uses are added for chito-oligosaccharides, which effectively solves the problem of sobering and protecting the liver of drunken persons, and avoids alcohol damage to the human body as much as possible. But there are problems such as inaccurate curative effect.

Summary of the invention

The technical problem to be solved by the present invention is to provide a method for protecting and treating drug-induced liver injury, so as to provide patients with drug-induced liver injury, especially patients with DILI caused by acetaminophen.

In order to solve the above technical problems, the present invention discloses a specific molecular weight chitosan oligosaccharide (molecular weight≤1000), referred to as the use of COST, for preparing a composition for protecting and treating drug-induced liver injury, especially for preparing protection and treatment The composition for drug-induced acute liver injury, especially for preparing a composition for protecting and treating liver injury caused by acetaminophen. The composition is used for systemic administration or parenteral administration, preferably intravenous administration.

COST is derived from natural products, with simple preparation steps, low cost and low pollution, which is conducive to large-scale production. The researchers of the present invention found that COST, which is non-toxic and non-triadic to the human body, has good antioxidant activity in vivo and in vitro, and can effectively resist the liver damage caused by acetaminophen. Tests have confirmed that COST can regulate acetaminophen. Induced abnormal liver function biochemical indicators, significantly reduced the abnormal elevation of ALT and AST in the serum of mice induced by acetaminophen; improved liver tissue damage induced by acetaminophen; improved liver injury induced by acetaminophen The content of antidote GSH in the tissues; increase the antioxidant capacity of acetaminophen-induced liver oxidative damage; reduce the content of MDA in acetaminophen-induced liver oxidative damage. It is expected to be developed and applied to the clinical treatment of liver injury caused by drugs, providing patients with better choices. Its mechanism of protection and treatment may be that chitooligosaccharides can effectively resist the oxidative liver damage of acetaminophen, and reduce the activity of AST and ALT, improve the antioxidant capacity of the liver, and protect liver cells from oxidative stress damage. Against the powerful oxidation of N-acetyl-p-benzoquinone imine (NAPQI), an intermediate metabolite of acetaminophen, through anti-oxidative stress to affect the hepatotoxicity of acetaminophen, thereby exerting its protection and treatment of liver injury effect.

The method for protecting and treating drug-induced liver injury disclosed in the present invention includes: administering an effective amount of COST to a subject with liver injury, the effective amount being 8-13 mg/kg/day, preferably 11 mg/kg/day.

Description of the drawings

Figure 1 shows the changes of ALT and AST in serum of mice after intraperitoneal injection of acetaminophen (APAP);

Figure 2 is a comparison of the biochemical indicators of liver function of mice in each group after 24 hours of administration;

Figure 3 is a H&E staining pathological structure diagram of the liver tissue of each group of mice after 24 hours of administration;

Figure 4 shows the comparison of GSH content in the liver of mice in each group 24 hours after administration;

Figure 5 is a comparison of the total antioxidant capacity (T-AOC) of the liver tissue of each group of mice after 24 hours of administration;

Figure 6 is a comparison of MDA content in liver tissues of mice in each group after 24 hours of administration;

Figure 7 is a comparison of GSH-Px content in liver tissues of mice in each group after 24 hours of administration;

Figure 8 shows the comparison of SOD content in liver tissues of mice in each group after 24 hours of administration;

Figure 9 is a comparison of CAT content in liver tissues of mice in each group 24h after administration;

Figure 10 shows the changes of ALT and AST in the supernatant of liver L02 cells in each group after 24 hours of administration.

Detailed ways

The above-mentioned content of the present invention will be further described in detail below through specific embodiments. However, it should not be understood that the scope of the above-mentioned subject of the present invention is limited to the following embodiments. Without departing from the above technical idea of the present invention, various substitutions or changes made based on common technical knowledge and conventional means in the field should all be included in the scope of the present invention.

To illustrate the present invention, we use the BABL/C mouse intraperitoneal injection of acetaminophen-induced acute liver injury model and the in vitro APAP-induced human liver cell L02 injury model to further develop the method disclosed in the present invention. Description.

1. Exploration of the pathological process of acetaminophen-induced liver injury in mice

25 male BALB/C mice 6-8 weeks, adaptive feeding for 3 days, randomly divided into acetaminophen (APAP) 0h group, 2h group, 4h group, 6h group, 8h group, 5 mice in each group , Fasting for 12 hours before the experiment, intraperitoneal injection of acetaminophen 300mg/kg, the corresponding group of mice were sacrificed every 2h from time 0, blood was collected, left at room temperature for 1 hour, and centrifuged at 4500 rpm for 15 minutes. The supernatant, the serum, was tested for ALT and AST. The results showed that ALT and AST in the serum of mice induced by acetaminophen increased significantly from 2-4 hours after administration. Among them, serum AST was significantly increased after acetaminophen was administered for 2 hours. The trend of increasing (*p<0.05), ALT was significantly different at 4h (**p<0.01), and the subsequent AST and ALT continued to increase. It can be seen that the onset of acute liver injury induced by acetaminophen is 2-4 hours after administration. The acute liver injury induced by paracetamol after model administration starts from 2-4 hours. The specific results are shown in Table 1 and Figure 1:

Table 1

Mean±SEM	APAP-0h group	APAP-2h group	APAP-4h group	APAP-6h group
ALT(U/L)	15.7±2.3	59.2±7.7	236.9±62.1	565.7±72.7
AST(U/L)	25.6±1.5	98.9±19.1	123.3±30.1	231.3±21.3

2. COST regulates the abnormal biochemical indicators of liver function in mice induced by acetaminophen

60 BABL/C mice, fasted for 12 hours before the experiment, were randomly divided into normal control group (CON), model group (APAP300mg/kg), COST high, medium and low doses (L-25mg/kg, M-50mg/kg and H-100mg/kg) administration group, acetylcysteine (NAC) 300mg/kg positive control group, intraperitoneal injection of acetaminophen 300mg/kg 2 hours before the administration of COST and positive NAC, 24 hours after administration They were sacrificed, and serum and liver tissue were collected. The ALT and AST test results showed that compared with the model group, the AST and ALT of each treatment group were significantly lower (*p<0.05, **p<0.01, ***p<0.001, vs APAP), and presents a certain concentration dependence. As the concentration of COSM is higher, the therapeutic effect is more obvious. There is basically no significant difference (#p<0.05, ##p<0.01, ###p<0.001vs NAC). That is, COST can significantly reduce the abnormal increase of ALT and AST in the serum of mice induced by acetaminophen. The specific results are shown in Table 2 and Figure 2:

Table 2

3.COST improves liver tissue damage induced by acetaminophen

The liver tissues of each group of mice were fixed overnight in 4% paraformaldehyde, embedded in paraffin and sectioned and stained with H&E. The pathological structure was observed with a microscope and photographed. The experimental results showed that the COST treatment group weakened the acetaminophen According to the induced liver injury (H&E staining), and presents a certain dose-dependent, the therapeutic effect of 100mg/kg COST is even better than that of positive drugs. COST can significantly improve the liver tissue lesions induced by acetaminophen. The specific results are shown in Figure 3.

4. COST increases the content of the antidote GSH in the liver tissue of acetaminophen-induced liver injury

Weigh 50 mg of liver tissue of each group of mice, add physiological saline according to 1:9, homogenize, centrifuge at 10,000 rpm to take the supernatant, and measure the liver tissue glutathione (GSH). The results show Compared with the APAP model group, the COST group significantly increased the content of GSH, and showed a certain concentration dependence, and the COST high-dose group (COST-H) had the same effect as NAC. (*p<0.05, **p<0.01, ***p<0.001vs APAP; #p<0.05, ##p<0.01, ###p<0.001vs NAC). Compared with the model group, COST can significantly increase the content of liver GSH, thereby achieving the detoxification effect. The specific results are shown in Table 3 and Figure 4:

table 3

5. COST improves the antioxidant capacity of acetaminophen-induced liver oxidative damage

Weigh 50 mg of liver tissue of each group of mice, add the extract according to 1:9, homogenize, centrifuge at 10,000 rpm to take the supernatant, and determine the total antioxidant capacity (T-AOC) of liver tissue. The results showed that the total antioxidant capacity (T-AOC) of COST medium dose (COST-M) and high dose (COST-H) was significantly enhanced compared with APAP model group, and showed a certain concentration dependence. And the effect of T-AOC and NAC of COST high-dose group (COST-H) was similar. (*p<0.05, **p<0.01, ***p<0.001vs APAP; #p<0.05, ##p<0.01, ###p<0.001vs NAC). Compared with the model group, COST-H and COST-M can significantly increase the content of liver GSH, thereby achieving the effect of improving liver injury. The specific results are shown in Table 4 and Figure 5:

Table 4

6.COST reduces the content of MDA in acetaminophen-induced liver oxidative damage

Weigh 50 mg of liver tissue of each group of mice, add the extract according to 1:9, homogenize, centrifuge at 10,000 rpm to take the supernatant, and measure the content of MDA in liver tissue. The result shows that COST The treatment significantly reduced MDA, the product of liver fatty acid and ROS, and improved liver oxidative damage. Compared with the APAP model group, the MDA of the COST high, medium and low dose group was significantly reduced, and showed a certain concentration dependence. COSM reduced APAP-induced liver oxidative damage (*P<0.05, **p<0.01,***p<0.001vs APAP;), the high school administration group has the same effect as the positive drug NAC. The specific results are shown in Table 5 and Figure 6:

table 5

7. COST increases GSH-Px content in acetaminophen-induced liver oxidative damage

Weigh 50 mg of liver tissue of each group of mice, add the extract according to 1:9, homogenize, centrifuge at 10,000 rpm to take the supernatant, and perform liver tissue glutathione peroxidase (GSH-Px) ) Content determination, the results showed that COST treatment significantly increased the content of liver GSH-Px and improved liver oxidative damage. Compared with the APAP model group, the GSH-Px of the COST high, medium and low dose group was significantly increased, and showed a certain concentration-dependent , COST reduces APAP-induced oxidative damage to the liver (*p<0.05, **p<0.01, ***p<0.001 vs APAP;) The specific results are shown in Table 6 and Figure 7:

Table 6

8.COST increases the SOD content in acetaminophen-induced liver oxidative damage

Weigh 50 mg of liver tissue of each group of mice, add the extract according to 1:9, homogenize, centrifuge at 10,000 rpm to take the supernatant, and determine the content of superoxide dismutase (SOD) in liver tissue. The results showed that COST treatment significantly increased liver SOD content and improved liver oxidative damage. Compared with the APAP model group, the SOD of the COST high, medium and low dose group was significantly increased, and COST reduced APAP-induced liver oxidative damage (*p<0.05, * *p<0.01,***p<0.001vs APAP;), the effect of the high, medium and low dose group is equivalent to that of the positive drug NAC. The specific results are shown in Table 7 and Figure 8:

Table 7

9.COSM increases the SOD content in acetaminophen-induced liver oxidative damage

Weigh 50 mg of liver tissue of each group of mice, add the extract according to 1:9, homogenize, centrifuge at 10,000 rpm to take the supernatant, and determine the content of catalase (CAT) in liver tissue. It was shown that COST treatment significantly increased liver CAT content and improved liver oxidative damage. Compared with APAP model group, COST high-dose group had a significant increase in CAT. COST high-dose reduced APAP-induced liver oxidative damage (*p<0.05, ** p<0.01,***p<0.001vs APAP;), the effect of the high dose group is equivalent to that of the positive drug NAC. The specific results are shown in Table 8 and Figure 9:

Table 8

10. COST regulates the protective effect of paracetamol-induced injury of human liver cells L02 in vitro and improves the abnormality of biochemical indicators

Culture human normal hepatocytes LO2 in vitro. Cells are spread on a 96-well plate at 15000-20000 cells/well and cultured for 8-12h; according to the preliminary toxicity experiment, they are grouped into COST high, medium and low (1.0mg/mL, 0.5 mg/mL, 0.25mg/mL), the normal group and the model group have 6 replicate holes in each group. After adding COS, each group is cultured for 12 hours and then 8mM APAP is added to continue the culture for 12 hours; observe the pathological state and treatment effect under the microscope, then collect The culture broth was centrifuged to take the supernatant, the kit was used to measure the release of AST and ALT, and the remaining cells were added with CCK-8 reagent to measure the cell viability. The results showed that COST can significantly inhibit APAP-induced liver cell damage or death. Compared with the model group, high and medium doses of COST significantly increased the survival rate of liver cells and improved APAP-induced AST and ALT liver injury indicators (* p<0.05, **p<0.01, ***p<0.001, vs APAP), the specific results are shown in Table 9, Table 10 and Figure 10:

Table 9

Table 10

Claims (8)

1. A use of chitosan oligosaccharide (COST) with a molecular weight of ≤1000 is used to prepare a composition for protecting and treating drug-induced liver injury.
2. The use according to claim 1, characterized in that it is used to prepare a composition for protecting and treating drug-induced acute liver injury.
3. The use according to claim 1, characterized in that it is used to prepare a composition for protecting and treating liver damage caused by acetaminophen.
4. The use according to claim 1 or 2, wherein the composition is used for systemic administration or parenteral administration.
5. The use according to claim 4, wherein the composition is used for intravenous administration.
6. A method for protecting and treating drug-induced liver injury includes: giving an effective amount of COST to a subject with liver injury.
7. 8. The method of claim 6, wherein the effective amount is 8-13 mg/kg/day.
8. 8. The method according to claim 7, wherein the effective amount is 11 mg/kg/day.

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