WO2020186683A1

WO2020186683A1 - Application of quercetin in preparation of drug for prevention and treatment of drug-induced liver injury

Info

Publication number: WO2020186683A1
Application number: PCT/CN2019/098902
Authority: WO
Inventors: 陈阿丽
Original assignee: 广东药科大学
Priority date: 2019-03-18
Filing date: 2019-08-01
Publication date: 2020-09-24
Also published as: CN109864987A

Abstract

The present invention relates to a medical use of quercetin in prevention and treatment of drug-induced acute liver injury, and in particular, to application of the quercetin in preparation of a drug for prevention and treatment of liver injury caused by acetaminophen. Animal experiments prove that the quercetin controls abnormal rise in biochemical indicators related to acetaminophen-induced liver functions and changes in pathological structures, and increases the content of in-vivo antidote GSH in acetaminophen-induced liver tissue injury; the quercetin is an oral preparation or injection preparation, the administration dosage may be changed according to the age, the weight, the intake amount of acetaminophen, the arrangement of a treatment course and the like of a patient, a recommended administration dosage for a person is 40 mg/kg/d, and an oral administration mode is recommended; the quercetin can effectively resist liver injury caused by acetaminophen, and can be used for preparing a drug for treating liver injury caused by acetaminophen.

Description

Application of quercetin in preparing medicine for protecting and treating drug-induced liver injury

Technical field

The invention belongs to the technical field of biomedicine, and relates to the medical use of quercetin in the protection and treatment of drug-induced acute liver injury, and in particular to the application of quercetin in the preparation of drugs for the protection and treatment of liver injury caused by acetaminophen.

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), also known as paracetamol and N-acetaminophen, is a widely used clinical analgesic. At a therapeutic dose of <4g/d, APAP can cause a transient increase in serum aminotransferase, especially in people with malnutrition, liver dysfunction, alcoholism, or taking certain CYP450-inducing drugs. When overdose, it is adult Severe liver damage and even acute liver failure (ALF) may occur when it is greater than 4g/d and minors greater than 50-75mg/kg·d. It is estimated that about 2,000 people in Europe and the United States experience ALF every year, of which nearly 50% are drug-induced liver injury caused by APAP, and 61% of cases seem to be caused by the use of a median dose of 34g/3d.

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, namely hydrogen peroxide H2O2 and peroxynitrate ONOO- etc. Oxygen (ROS) causes 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. Generally, excessive use of paracetamol and NAC within 8-12 hours have a good effect. The main adverse reactions are skin rash, nausea, vomiting, and fever. Therefore, its therapeutic effect is not ideal. Another main reason is that the mechanism of acetaminophen causing liver damage 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.

Quercetin (quercetin, 3,3′,4′,5,7-pentahydroxyflavone) and its derivatives are the most widely distributed flavonoids in the plant kingdom. They are widely found in the flowers, leaves and fruits of plants. The most important bioflavonoid in the human diet has many functions such as dilating blood vessels, lowering blood pressure, preventing coronary heart disease, preventing myocardial ischemia/reperfusion injury, and anti-thrombosis. In addition, quercetin also has a powerful antioxidant The biological activity of the toxic free radicals can quench the toxic free radicals and form resonance stable phenoxy free radicals, which can protect and treat liver oxidative damage. As a natural medicine that is non-toxic and non-triadic to the human body, quercetin has good antioxidant activity in vivo and in vitro and is expected to be used in clinical treatment of a series of diseases related to oxidation. In view of this, the inventor of the present application will provide the use of quercetin in the preparation of a drug for the protection and treatment of acetaminophen-induced drug-induced acute liver injury.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide new medical uses of quercetin in the protection and treatment of drug-induced acute liver injury, and specifically relates to the preparation of quercetin in the protection and treatment of liver injury caused by acetaminophen Application in medicine.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

The application of quercetin in the preparation of drugs for protecting and treating drug-induced liver injury, and the structural formula of quercetin is:

The present invention adopts BABL/C mouse intraperitoneal injection of paracetamol to induce acute liver injury model, and its pathogenesis is similar to that of the clinic. The experimental results show that quercetin can significantly weaken paracetamol-induced liver function related biochemical indicators Gu C The abnormal increase of aminotransferase (ALT) and aspartate aminotransferase (AST) (as shown in Figure 1), and the pathological results were significantly improved (as shown in Figure 2); Quercetin can significantly improve the liver tissue lesions induced by acetaminophen ( As shown in Figure 3), and dose-dependent; at the same time, quercetin also significantly increased the liver antioxidant glutathione (GSH) content (as shown in Figure 4). In the application of quercetin in the preparation of acetaminophen-induced liver injury drugs, its protective and therapeutic mechanism is that quercetin can effectively resist the oxidative liver injury of acetaminophen, and reduce AST and ALT Activity, improve the liver's antioxidant capacity, protect liver cells from oxidative stress damage and fight against the powerful oxidation of acetaminophen intermediate metabolite N-acetyl-p-benzoquinone imine (NAPQI), mainly through oxidative stress Affect the liver toxicity of acetaminophen to exert its protective and therapeutic effects on liver injury.

Preferably, the quercetin is an oral preparation or an injection preparation.

Preferably, the dosage of quercetin can be administered according to the patient's age, weight, intake of acetaminophen, and treatment schedule. The recommended dosage for humans is 40 mg/kg/d.

Preferably, the quercetin is administered orally or by injection, and oral administration is recommended.

The beneficial effects of the present invention:

(1) The present invention provides a new use of quercetin in the preparation of drugs for protecting medicinal acute liver injury, especially the use of quercetin in preparing drugs for protecting paracetamol-induced medicinal liver injury; Quercetin It can effectively resist the liver damage caused by acetaminophen, and can be used to prepare therapeutic drugs for liver damage caused by acetaminophen, which provides patients with better choices;

(2) Quercetin is derived from natural products, has simple preparation steps, low cost, low pollution, and is conducive to large-scale production. As a natural medicine that is non-toxic and has no triple effect on the human body, quercetin has a good body Internal and external antioxidant activity, it is expected to develop drugs for clinical treatment of liver injury caused by drugs;

(3) The oral administration of the present invention can exert significant curative effect. On the one hand, safety is improved, and on the other hand, the pain caused by injection administration is reduced.

Description of the drawings

Figure 1 shows the abnormal biochemical indicators of liver function in BABL/C mice induced by acetaminophen. BABL/C mice induced acute liver injury after intraperitoneal injection of 300 mg/kg acetaminophen. The serum AST showed a significant increase trend after acetaminophen was given for 2 hours (*p<0.05), while ALT showed a better and significant difference at 4 hours (**p<0.01), and the subsequent AST and ALT continued to increase.

Figure 2 shows that quercetin protects the abnormalities of acetaminophen-induced liver function-related biochemical indicators in BABL/C mice. After the intraperitoneal injection of 300 mg/kg acetaminophen for 2 hours, N-acetylcysteine was given (NAC, 300mg/kg), quercetin (100mg/kg, 200mg/kg, 400mg/kg) after intragastric administration, continue feeding until 24 hours later, compared with the model group, the AST and ALT of each treatment group are obvious Decrease, NAC, ***p<0.001; Que (100mg/kg), ALT*p<0.05, AST***p<0.001; Que (200mg/kg, 400mg/kg), ***p<0.001.

Figure 3 shows that the quercetin treatment group attenuated acetaminophen-induced liver damage, in which H&E staining is shown.

Figure 4 shows that quercetin treatment significantly improved the content of glutathione GSH in the liver.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention, not to limit the scope of the present invention.

Acetaminophen in the following examples was purchased from Dalian Meilun Biological Technology Co., Ltd, with a purity greater than 98.5%, dissolved in sterile PBS, and dissolved in a 40 degree water bath; Quercetin and acetylcysteine were purchased from Sigma -Aldrich; The experimental animals were purchased from SPF male C57BL/6 mice weighing 18-22g from the Experimental Animal Center of Southern Medical University. The feeding conditions of the mice are temperature 23±2°C, humidity 55±5% and 12h sunshine, and they are given diet and water that meet rodent feeding standards.

Example 1. Abnormalities of biochemical indicators of liver function in mice induced by acetaminophen

Male BALB/C mice 6-8 weeks, adaptively fed for 3 days, divided into acetaminophen 0h group, acetaminophen 2h group, acetaminophen 4h group, and acetaminophen 6h group , Paracetamol group for 8 hours, fasting for 12 hours before the experiment, intraperitoneal injection of paracetamol 300mg/kg, from time 0, the mice of the corresponding group were killed every 2 hours, blood was taken, and left at room temperature for 1 hour. Centrifuge at 4500 rpm for 15 minutes, take the supernatant, the serum, and perform ALT and AST tests. The results show that the ALT and AST in the serum of mice induced by acetaminophen increase significantly from 2-4 hours after administration. Acute liver injury induced by acetaminophen after the drug starts from 2-4 hours (as shown in Figure 1).

Example 2. Quercetin regulates the abnormalities of biochemical indicators of liver function in mice induced by acetaminophen

BABL/C mice were fasted for 12 hours before the experiment and were divided into normal control group, model group (APAP, 300mg/kg), quercetin administration group (100mg/kg, 200mg/kg and 400mg/kg) and positive drugs In the treatment group (NAC, 300mg/kg), quercetin and positive drug NAC were given intraperitoneal injection of acetaminophen 300mg/kg 2-4 hours before intraperitoneal administration, and sacrificed 24 hours later. Serum and liver tissue were collected , ALT and AST test results show that quercetin can significantly reduce the abnormal rise of ALT and AST in mice serum induced by acetaminophen; compared with the positive drug NAC, 200mg/kg of quercetin is comparable to it Therapeutic effect. The 400mg/kg quercetin treatment group showed significantly lower AST and ALT compared with positive NAC (##p<0.01), showing a better effect than the positive drug (as shown in Figure 2).

Example 3. Quercetin improves liver tissue damage induced by acetaminophen

The liver tissues of mice were fixed overnight in 4% paraformaldehyde, sliced with paraffin embedded and then stained with H&E. The pathological structure was observed and photographed with a microscope. The experimental results showed that quercetin can significantly improve acetaminophen The induced liver tissue lesions are dose-dependent, and the therapeutic effect of 400 mg/kg quercetin is even better than that of the positive drug (as shown in Figure 3).

Example 4. Quercetin increases the content of antidote GSH in liver tissue induced by acetaminophen

Weigh an appropriate 50 mg of liver tissue of the mouse, add physiological saline according to 1:9, homogenize, centrifuge at 10,000 rpm to take the supernatant, and determine the liver tissue GSH. The results show that it is compared with the model group quercetin Supplement can significantly increase the content of GSH in the liver, thereby increasing the antioxidant capacity and achieving the detoxification effect (as shown in Figure 4).

The above-mentioned embodiments only exemplarily illustrate the principles and effects of the present invention, and are not used to limit the present invention. Anyone familiar with this technology can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims

Application of quercetin in preparing medicines for protecting and treating drug-induced liver injury.
The use of quercetin in the preparation of drugs for protecting and treating drug-induced liver injury according to claim 1, wherein the drug-induced acute liver injury is drug-induced liver injury induced by acetaminophen.
The application of quercetin in the preparation of a medicine for protecting and treating drug-induced liver injury according to claim 2, wherein the quercetin improves acetaminophen-induced liver function-related biochemical indicators ALT and AST The abnormal elevation of the disease and the changes of the pathological structure.
The application of quercetin in the preparation of drugs for protecting and treating drug-induced liver injury according to claim 2, wherein the quercetin increases the antidote GSH in liver tissue induced by acetaminophen. content.
The use of the quercetin according to any one of claims 1 to 4 in the preparation of a medicament for protecting and treating drug-induced liver injury, wherein the quercetin is an oral preparation or an injection preparation.
The application of the quercetin according to any one of claims 1 to 4 in the preparation of a medicament for the protection and treatment of drug-induced liver injury, wherein the dosage of the quercetin can be based on the age, weight, and intake of the patient Include changes in the amount of acetaminophen and the schedule of treatment for administration, and the recommended dosage for humans is 40 mg/kg/d.
The use of the quercetin according to any one of claims 1 to 4 in the preparation of a medicament for protecting and treating drug-induced liver injury, wherein the quercetin is administered orally or by injection, and oral administration is recommended the way.