Application of gabexate in treatment of alcoholism

Abstract

The invention discloses application of gabexate to treatment of alcoholism. More specifically, the present invention provides the use of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of a pharmaceutical composition for the prevention or treatment of alcoholism and / or alcoholic liver disease. The invention also provides application of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite of gabexate in preparation of an acetaldehyde dehydrogenase 2 (ALDH2) agonist. The invention also discloses application of gabexate or pharmaceutically acceptable salts thereof or metabolites thereof in preparation of health-care food with an auxiliary protection effect on alcoholic liver injury.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to a new use of gabexate or its metabolites or salts as active ingredients in the preparation of medicines for preventing or treating alcoholism and alcoholic liver disease. Background Art

[0002] Alcohol consumption is a global public health problem, causing about 3 million deaths each year. Excessive drinking has brought great harm to people. Drinking too much alcohol at one time can easily inhibit the respiratory center of the brain, causing respiratory arrest and death. Long-term excessive drinking will almost inevitably lead to cirrhosis, abdominal distension, jaundice and other symptoms. Acute alcohol intoxication (AAI), also known as acute ethanol poisoning, is a common disease in the emergency department, accounting for the majority of acute poisoning patients. The cause of the disease is that after a person drinks a large amount of alcohol in a short period of time, excessive ethanol will accumulate in the brain, and the central nervous system will gradually change from excitement to inhibition, often showing abnormal behavior and consciousness. In severe cases, organ function is damaged, leading to respiratory and circulatory failure, which can endanger life. Alcohol poisoning has become an increasingly serious threat to people's health. After drinking, ethanol is absorbed into the blood in the digestive tract, and drinking on an empty stomach is absorbed faster. The ethanol in the blood is detoxified by the liver. It is first converted into acetaldehyde under the action of alcohol dehydrogenase, and then converted into acetic acid under the action of acetaldehyde dehydrogenase. Acetic acid is further decomposed into water and carbon dioxide. The whole process takes about 2 to 4 hours. When the activity of acetaldehyde dehydrogenase in the body is low, the unmetabolized acetaldehyde has a strong toxic effect on cells. Clinically, the recognized drug for alcohol poisoning is mainly naloxone, which mainly functions to awaken patients with alcohol poisoning and prevent them from dying due to respiratory depression caused by alcohol, but it has little effect on accelerating ethanol metabolism and reducing alcoholic tissue damage. Because alcohol poisoning is a serious condition, it is of great clinical significance to actively adopt safe and effective treatment drugs.

[0003] Alcoholic liver disease (ALD) is a liver disease caused by long-term heavy drinking. In the early stage, it usually manifests as fatty liver, which can then develop into alcoholic hepatitis, liver fibrosis and cirrhosis. Some alcoholics or those who drink excessively will have alcohol-related health problems, among which ALD is the most common organ damage caused by alcohol. ALD has become one of the most important chronic liver diseases in my country. At present, the clinical treatment measures for alcoholic liver disease mainly include quitting drinking, nutritional supplements, antioxidant, anti-inflammatory and other drug treatments, but the effect is not good. So far, there is still a lack of effective treatment methods. Therefore, alcoholic liver disease is in urgent need of safe and effective therapeutic drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of gabexate or its metabolites or salts thereof, in particular, the use of gabexate or its metabolites or salts thereof as active ingredients in the preparation of a pharmaceutical composition for preventing or treating alcoholism, alcoholic liver disease, an acetaldehyde dehydrogenase 2 (ALDH2) agonist or a health food having an auxiliary protective effect on alcoholic liver damage.

[0005] In a first aspect, the present invention provides a use of Gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of a pharmaceutical composition for preventing or treating alcoholism and / or alcoholic liver disease.

[0006]

[0007] Wherein, the metabolite is selected from the following group of compounds or pharmaceutically acceptable salts thereof:

[0008]

[0009] In another preferred embodiment, the active ingredient in the pharmaceutical composition is gabexate or a pharmaceutically acceptable salt thereof.

[0010] In another preferred embodiment, the prevention or treatment of alcoholism is one or more selected from the following groups:

[0011] (1) Accelerate the metabolic rate of ethanol and / or acetaldehyde;

[0012] (2) Reduce the rate of drunkenness;

[0013] (3) Prolonging the incubation period of intoxication;

[0014] (4) shorten the duration of intoxication;

[0015] (5) Improve the state and behavior after being drunk;

[0016] (6) reduce mortality from alcoholism; and / or

[0017] (7) Reduce alcohol-induced liver damage.

[0018] In another preferred embodiment, the metabolite is 6-guanidinohexanoic acid, ethyl p-hydroxybenzoate, or a combination thereof.

[0019] In another preferred embodiment, the pharmaceutically acceptable salt of gabexate is selected from the group consisting of hydrochloride, sulfate, methanesulfonate, maleate, phosphate, hydrobromide, malate, fumarate, p-toluenesulfonate, nitrate, citrate, preferably methanesulfonate.

[0020] In another preferred embodiment, the alcohol poisoning is acute alcohol poisoning or chronic alcohol poisoning.

[0021] In another preferred embodiment, the alcoholic liver disease is selected from the group consisting of alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis, or a combination thereof.

[0022] In another preferred embodiment, the prevention or treatment of alcoholic liver disease comprises one or more methods selected from the following group:

[0023] (a) Alleviate acute or chronic hepatocellular and liver damage caused by alcohol;

[0024] (b) reduce alcohol-induced elevations in transaminases; and / or

[0025] (c) Reduce alcohol-induced oxidative stress damage.

[0026] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0027] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, an injection preparation or a topical administration preparation.

[0028] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: tablets, capsules, powders, granules, pills, lozenges, effervescent tablets, patches, ointments, lyophilized powder injections or injections.

[0029] In another preferred embodiment, the pharmaceutical composition is in the form of a lyophilized powder injection or an injection solution.

[0030] In another preferred embodiment, the lyophilized powder injection comprises one or more carriers selected from the following group: mannitol, lactose, hydrolyzed gelatin, sodium chloride, glucose, or a combination thereof.

[0031] In another preferred embodiment, the injection solution comprises one or more carriers selected from the following group: water for injection, mannitol, sodium chloride, glucose, or a combination thereof.

[0032] In a second aspect, the present invention provides use of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of an acetaldehyde dehydrogenase 2 (ALDH2) agonist.

[0033]

[0034] Wherein, the metabolite is selected from the following group of compounds or pharmaceutically acceptable salts thereof:

[0035]

[0036] In another preferred embodiment, the ALDH2 is ALDH2*1 and / or ALDH2*2.

[0037] The third aspect of the present invention provides the use of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of a health food having an auxiliary protective effect on alcoholic liver damage.

[0038]

[0039] Wherein, the metabolite is selected from the following group of compounds or pharmaceutically acceptable salts thereof:

[0040]

[0041] In a fourth aspect, the present invention provides a method for preventing or treating alcoholism and / or alcoholic liver disease, comprising the steps of administering gabexate or its metabolites, or one or more of their pharmaceutically acceptable salts, or administering the composition of the present invention to a subject in need thereof, thereby preventing or treating alcoholism and / or alcoholic liver disease.

[0042] In another preferred embodiment, the subject is a subject with elevated aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT). For example, compared with the normal control value C0, the aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) level C1 in the subject's blood or serum is higher than C0, preferably at least 10%, at least 20%, at least 30%, at least 50%, at least 80%, at least 90% or even at least 100% higher.

[0043] In another preferred embodiment, the subject carries an ALDH2*2 mutation.

[0044] In another preferred embodiment, the subject is a human or a mammal, such as a rat, a mouse or a monkey.

[0045] In another preferred embodiment, the subject suffers from pancreatitis.

[0046] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The test results of gabexate promoting ethanol metabolism in hepatocytes

[0048] Figure 2 Results of the experiment on different salt forms of gabexate promoting ethanol metabolism in hepatocytes

[0049] Figure 3 Gabexate administration before alcohol improves ethanol metabolism in mice

[0050] Figure 4Gabexate administration after drinking promotes ethanol metabolism in mice

[0051] Figure 5 Gabexate reduces acetaldehyde levels in mice 1 hour after drinking

[0052] Figure 6 Gabexate reduces ethanol content in rats 2 hours after drinking

[0053] Figure 7 Gabexate metabolites and their combination reduce ethanol content at 3 and 6 hours after drinking in mice

[0054] Figure 8 The results of the test on improving the alcohol tolerance time and drunkenness duration of mice by administering gabexate before drinking

[0055] Fig. 9 Gabexate administration before alcohol improves the results of mouse grasping test

[0056] Fig.10 The results of the test on the effect of gabexate administration after drinking on improving the alcohol tolerance time and drunkenness duration of mice

[0057] Fig.11 The results of the test on the improvement of the grasping force and hanging endurance of mice by gabexate administration after drinking

[0058] Fig.12 Gabexate improves the climbing time and climbing score test results of drunken behavior in mice

[0059] Fig.13 The results of the rotarod time test of drunkenness behavior in mice were improved by gabexate administration before drinking

[0060] Fig.14 Results of the cell survival test of gabexate in the alcohol-induced hepatocyte injury model

[0061] Fig.15 Results of cell survival test of different salt forms of gabexate in alcohol-induced hepatocyte injury model

[0062] Fig.16 Results of a trial on reducing mortality from acute alcohol poisoning with gabexate

[0063] Fig.17 The test results of gabexate's ability to protect the liver of mice with acute alcoholic liver injury

[0064] Fig.18 The test results of gabexate's ability to protect the liver of mice with acute alcoholic liver injury

[0065] Fig.19 The test results of gabexate on liver function indexes (AST, ALT) in mice with acute alcoholic liver injury

[0066] Fig. 20 The results of the test on the antioxidant index superoxide dismutase activity of gabexate in mice with acute alcoholic liver injury

[0067] Fig.21 The test results of gabexate on malondialdehyde content in the liver of mice with acute alcoholic liver injury

[0068] Fig. 22 The test results of gabexate on the reduced glutathione content in the liver of mice with acute alcoholic liver injury

[0069] Fig.23 The activity curve of gabexate stimulating mutant ALDH2*2 and wild-type ALDH2*1

[0070] Fig.24 The activity curve of gabexate inhibiting serine protease

[0071] Fig.25 Results of the test on the activity of key enzymes (alcohol dehydrogenase and acetaldehyde dehydrogenase) of ethanol metabolism in mice with acute alcoholic liver injury

[0072] Fig.26 Results of the test of Gabexate on inflammatory indicators (interleukin-1β and TNF-α) in mice with acute alcoholic liver injury DETAILED DESCRIPTION

[0073] After extensive and in-depth research, through a large number of screening and testing, the inventors have provided for the first time the use of gabexate or its metabolites or salts as active ingredients for preparing a pharmaceutical composition for preventing or treating alcoholism and alcoholic liver disease. The inventors unexpectedly found that gabexate or its metabolites have acetaldehyde dehydrogenase agonist activity, can accelerate the metabolic rate of ethanol and / or acetaldehyde, and significantly reduce various drunkenness-related symptoms caused by drinking, thereby preventing or treating alcoholism and alcoholic liver disease. On this basis, the present invention was completed.

[0074] the term

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0076] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0077] Active ingredients

[0078] The active ingredient of the present invention is gabexate or its metabolite.

[0079] The structural formula of gabexate is shown below, and its CAS number is 39492-01-8.

[0080]

[0081] In the present invention, the gabexate metabolite is selected from the group consisting of:

[0082]

[0083] The gabexate and its metabolites of the present invention are intended to include their pharmaceutically acceptable salts.

[0084] The preferred salt of gabexate or 6-guanidinohexanoic acid is a salt thereof formed with an organic acid or an inorganic acid. Typically, the pharmaceutically acceptable salt of gabexate or 6-guanidinohexanoic acid independently includes (but is not limited to): hydrochloride, sulfate, methanesulfonate, maleate, phosphate, hydrobromide, malate, fumarate, p-toluenesulfonate, nitrate, citrate, preferably methanesulfonate.

[0085] The preferred salt of ethyl hydroxybenzoate or p-hydroxybenzoic acid is a salt thereof formed with an organic base or an inorganic base. Typically, the pharmaceutically acceptable salts of ethyl p-hydroxybenzoate or p-hydroxybenzoic acid independently include (but are not limited to): ammonium salts, alkali metal salts such as sodium, lithium, potassium salts, alkaline earth metal salts such as calcium, magnesium salts and salts formed with organic bases (such as organic amines).

[0086] The organic substance of the present invention may be gabexate or a pharmaceutically acceptable salt thereof, or a combination of one or more of the metabolites or pharmaceutically acceptable salts thereof.

[0087] Alcohol metabolism

[0088] When the human body drinks alcohol, the alcohol is absorbed into the blood and then metabolized mainly by the liver. The main metabolic process is that the alcohol dehydrogenase (ADH) in the liver metabolizes the alcohol into acetaldehyde, and then the acetaldehyde is metabolized into acetic acid by acetaldehyde dehydrogenase (ALDH). Acetic acid is eventually converted into carbon dioxide and water, and then excreted from the body through breathing and urine.

[0089] Among them, acetaldehyde is the main toxic metabolite, which can cause harm to the human body. Acetaldehyde can affect the human nervous system and cardiovascular system, causing symptoms such as headaches, nausea, vomiting, and palpitations. Long-term drinking can cause liver damage and even cause cirrhosis and liver cancer.

[0090] Therefore, reducing the concentration and time of alcohol and acetaldehyde in the body can significantly reduce the damage caused by alcohol to the body.

[0091] Aldehyde dehydrogenase

[0092] The enzyme responsible for the conversion of acetaldehyde in the human body is acetaldehyde dehydrogenase (ALDH) in the liver. It has two isoenzymes, which are distributed in the cytosol (ALDH1) and mitochondria (ALDH2). There is a significant difference in the catalytic rate between the two. The KM of ALDH2 for acetaldehyde is lower than that of ALDH1, about 1 / 10 of the latter, and it is the isoenzyme mainly responsible for the conversion of acetaldehyde.

[0093] About 40% of the population in China carries a point mutation in the ALDH2 gene, where the normal allele is ALDH2*1 and the mutant allele is ALDH2*2. ALDH2*2 carriers have low ALDH2 enzyme activity, and this defect leads to the accumulation of acetaldehyde in the body after drinking, manifested as characteristic flushing, headache, nausea, dizziness and palpitations after drinking, and increases the risk of alcoholic liver disease.

[0094] In particular, the active ingredient of the present invention has an ALDH2 agonist effect, which can promote the conversion of acetaldehyde into acetic acid and avoid acetaldehyde accumulation. In particular, the active ingredient of the present invention has excellent agonist activity on both ALDH2*1 and ALDH2*2, and is suitable for normal people and ALDH2*2 carriers.

[0095] Alcoholism

[0096] Alcohol poisoning refers to a toxic disease caused by excessive drinking or excessive intake of ethanol, which first excites and then inhibits the central nervous system. Patients mainly show abnormal behavior and consciousness. In severe cases, it can cause multi-system damage (the most serious damage to the nervous system and liver), and even endanger life. According to the speed of onset, it can be divided into acute alcohol poisoning and chronic alcohol poisoning.

[0097] Acute alcohol poisoning refers to a state of central nervous system dysfunction caused by consuming a large amount of alcohol or alcoholic beverages in a short period of time. Patients often show abnormal behavior and consciousness. In severe cases, organ function is damaged, leading to respiratory and circulatory failure, which is life-threatening. It is also called acute ethanol poisoning.

[0098] Chronic alcoholism refers to the damage to various organs caused by long-term heavy drinking. The clinical manifestations are mainly central and peripheral nervous system disorders, which may be accompanied by cardiovascular and digestive system symptoms. In severe cases, it can lead to coma or even death. It is also called alcohol dependence or alcohol addiction.

[0099] The present invention is the first to discover that the active ingredient of the present invention has the effect of preventing or treating alcohol poisoning. When a person or animal ingests alcohol, the alcohol content in the subject's body is lower, the concentration is lower, and / or the existence time is shorter than that without using the active ingredient or composition of the present invention, thereby reducing the impact or damage of alcohol on the body and mind. The therapeutic effect of the present invention may specifically include one or more of the following effects: (1) accelerating the metabolic rate of ethanol and / or acetaldehyde; (2) reducing the drunkenness rate; (3) prolonging the incubation period of drunkenness; (4) shortening the duration of drunkenness; (5) improving the state and behavior after drunkenness; (6) reducing the mortality rate of alcohol poisoning; and / or (7) reducing alcohol-induced liver damage.

[0100] In addition, the therapeutic effects of the present invention include preventing alcohol poisoning, accelerating ethanol metabolism, allowing ethanol to quickly decompose into acetaldehyde, and finally decomposing into carbon dioxide and water, thereby reducing or delaying the inhibition of alcohol on the central nervous system, reducing the possibility of the subject reaching alcohol poisoning, alleviating alcohol-induced behavioral disorders, or prolonging the incubation period of drunkenness. In addition, the therapeutic effects of the present invention also include "sobering up", allowing the subject to wake up from the state of alcohol poisoning, restore consciousness or restore self-control.

[0101] Alcoholic liver disease

[0102] As used herein, alcoholic liver disease refers to liver disease caused by long-term heavy drinking. The liver is the main organ for the decomposition of alcohol. Drinking alcohol may lead to liver damage, and even alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis and alcoholic liver failure. The higher the level of alcohol in the body and the longer the drinking time, the more severe the liver damage tends to be. Experiments have shown that the active ingredient or composition of the present invention can alleviate alcohol damage to liver cells, improve alcohol's reduction in liver function, improve liver cell antioxidant and anti-inflammatory functions, and / or delay the progression of alcoholic liver disease.

[0103] The words "prevention", "treatment", "improvement", "acceleration", "lowering", "prolongation", "shortening", "reduction", "mitigation" and the like described in the present invention include delaying and stopping the progression of the disease or symptoms, or eliminating the disease, and do not require 100% inhibition, elimination and reversal. In some embodiments, the composition of the present invention can alleviate, shorten or reverse one or more symptoms or indicators of alcoholism or alcoholic liver disease, such as alleviating, shortening and / or reversing by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the levels observed in the absence of the active ingredient or composition of the present invention.

[0104] Some representative indicators for treating alcohol poisoning (but not limited to): righting reflex loss rate, righting reflex loss time and recovery time, pole climbing time, pole climbing score, rotating rod time, serum ethanol concentration, liver ADH and ALDH activity of drunk mice.

[0105] Some representative indicators for the treatment of alcoholic liver disease (but not limited to): mortality rate of acute alcohol poisoning, liver-to-body ratio, ALT content, AST content, SOD activity, MDA content, GSH content, TNF-α, IL-1β content.

[0106] Composition

[0107] The present invention provides a pharmaceutical composition, comprising one or more active ingredients of the present invention and a pharmaceutically acceptable carrier. In particular, the pharmaceutical composition is used for preventing or treating alcoholism and / or alcoholic liver disease.

[0108] The present invention also provides an aldehyde dehydrogenase 2 (ALDH2) agonist, which may include one or more active ingredients of the present invention and a pharmaceutically acceptable carrier. The ALDH2 is ALDH2*1 and / or ALDH2*2.

[0109] The present invention also provides a health food, which may include one or more active ingredients of the present invention and a pharmaceutically or health food acceptable carrier. In particular, the function of the health food is to have an auxiliary protective effect on alcoholic liver damage.

[0110] In the composition, the active ingredient of the present invention can be used alone as the only active ingredient, or can be used in combination with other active ingredients.

[0111] Generally, the composition of the present invention contains an effective amount of the active ingredient. The term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0112] As used herein, the term "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0113] The composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier, which can be in solid or liquid form. The carrier includes (but is not limited to): water, NaCl, physiological saline solution, lactated Ringer's solution, conventional sucrose, conventional glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates, such as lactose, straight-chain starch or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine and pigments. Such preparations can be sterilized and, if necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants and / or aromatic substances that do not react harmfully with the compounds provided herein or interfere with the activity of the compounds provided herein. Those of ordinary skill in the art will recognize that other carriers and excipients are suitable for the disclosed compounds.

[0114] The compositions of the present invention can be administered to the subject by any suitable route, including orally, subcutaneously, intravenously, intramuscularly, transdermally, by inhalation spray, topically, nasally, buccally, with the active compound being administered to the subject. Preferably, the active compound is administered orally, subcutaneously, intravenously, intramuscularly, transdermally.

[0115] Compositions of the invention suitable for administration will typically be discrete units in solid form, such as tablets, capsules, powders, granules, pills, patches, gels, ointments, suppositories, or in liquid form, such as oral liquids, injectable or infusible solutions or suspensions, or lyophilized powder injections.

[0116] The composition is preferably prepared under sterile conditions. The choice of carrier should match the mode of administration, which is well known to those skilled in the art.

[0117] The effective amount of the active ingredient of the present invention may vary with the mode of administration and the amount of alcohol consumed by the subject, the degree of intoxication, the severity of intoxication behavior, etc. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease or symptom to be treated or improved by the subject, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, the active ingredient of the present invention can be administered at a dose of about 0.1 mg / kg-1 g / kg or 1 mg / kg-100 mg / kg of animal body weight per day. For example, several divided doses may be administered per day, or the dose may be reduced proportionally, depending on the urgency of the subject's condition.

[0118] The present invention also provides a method for preventing or treating alcoholism and / or alcoholic liver disease, comprising the steps of administering the active ingredient of the present invention or the composition of the present invention to a subject in need thereof, thereby preventing or treating alcoholism and / or alcoholic liver disease.

[0119] The active ingredient or composition of the present invention can be administered before, during or after drinking, especially from 6 hours before drinking to the beginning of drinking, such as 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 0.5 hours, 10 minutes, 5 minutes, 1 minute before drinking, any time during drinking, or within 6 hours after drinking, such as 1 minute, 5 minutes, 10 minutes, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours or 5 hours after drinking.

[0120] In another preferred embodiment, the subject is a human or non-human mammal, such as a rat, a mouse, or a monkey.

[0121] The main advantages of the present invention include:

[0122] A) The present invention first discovered the use of gabexate and its metabolites in preparing a pharmaceutical composition for preventing or treating alcoholism and / or alcoholic liver disease.

[0123] B) The present invention first discovered the use of gabexate and its metabolites in the preparation of aldehyde dehydrogenase 2 (ALDH2) agonists.

[0124] C) The present invention is the first to discover the use of gabexate and its metabolites in the preparation of health food with auxiliary protective effects on alcoholic liver damage.

[0125] D) The active ingredient gabexate of the present invention is a marketed drug with a large amount of clinical application experience, is safe and reliable, and is more conducive to the development of new products of the present invention.

[0126] The present invention will be further described below in conjunction with specific implementation. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0127] If not otherwise specified, the gabexate used in the examples is gabexate mesylate.

[0128] Example 1 Experiment on the promotion of ethanol metabolism in hepatocytes by gabexate

[0129] 1.1 The alcohol metabolism rate at the cellular level reflects the direct effect of drugs on the alcohol-promoting effect of cells. The effect of gabexate on the ethanol metabolism rate at the cellular level was investigated. The experimental plan was as follows: the experimental groups were divided into alcohol group, positive drug control group (Alda-1, 100 μM) and gabexate group.

[0130] HepG2 cells were cultured normally for 12 hours, and the positive drug and gabexate stock solution were diluted with culture medium to the concentration to be tested (final concentration of 10 and 100 μmol / L), and the corresponding test solution was added to each well. After incubation with cells for 6 hours, alcohol modeling was performed, and the culture medium was incubated with a predetermined concentration of ethanol solution for 16 hours. After the modeling was completed, the supernatant was taken, and the ethanol concentration of the culture medium was detected by the Megazyme-Alcohol kit. The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0131] Experimental results: Figure 1 As shown, compared with the alcohol group, gabexate has the ability to promote ethanol metabolism in hepatocytes, and the ability of gabexate to promote ethanol metabolism is concentration-dependent, * indicates P < 0.05, ** or ## indicates P < 0.01.

[0132] 1.2 To investigate the effects of different salt forms of gabexate on the ethanol metabolic rate at the cellular level, the experimental scheme was as follows: the experimental groups were divided into an alcohol group, free gabexate, and groups of different salt forms of gabexate (mesylate, hydrochloride, sulfate, acetate, maleate, etc.).

[0133] HepG2 cells were cultured normally for 12 hours, and the positive drug and different salt-type gabexate stock solutions were diluted with culture medium to the concentration to be tested (final concentration of 10 and 100 μmol / L), and the corresponding test solution was added to each well. After incubation with the cells for 6 hours, alcohol modeling was performed, and the culture medium was incubated with a predetermined concentration of ethanol solution for 16 hours. After the modeling was completed, the supernatant was taken, and the ethanol concentration of the culture medium was detected by the Megazyme-Alcohol kit. The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0134] Experimental results: Figure 2 As shown, compared with the alcohol group, other salts of gabexate also have the ability to promote hepatocyte ethanol metabolism, among which acetate and hydrochloride gabexate have the same ability to promote ethanol metabolism as methanesulfonate, * indicates P < 0.05, ** or ## indicates P < 0.01, *** indicates P < 0.001.

[0135] Example 2 Gabexate promotes ethanol metabolism in mice

[0136] 2.1 To investigate the effect of gabexate administration before alcohol on ethanol metabolism in mice under high-dose alcohol, the experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into an alcohol group (alcohol dose 7 g / kg), an alcohol + metadoxine (500 mg / kg) group, an alcohol + low-dose gabexate (10 mg / kg) group, and an alcohol + high-dose gabexate (30 mg / kg) group according to their body weight.

[0137] All groups of mice were fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, alcohol + metadoxine (500 mg / kg) group and alcohol + low-dose gabexate (10 mg / kg) group and alcohol + high-dose gabexate (30 mg / kg) group were administered by oral gavage. The alcohol group was gavaged with the same volume of solvent. After that, all groups were gavaged with alcohol. Blood was collected from the eye sockets after drinking. Blood was collected from mice 1, 3, 6, and 12 hours after drinking. Serum was centrifuged and collected after standing at room temperature. The serum ethanol concentration was detected by Megazyme-Alcohol kit. The data were summarized, the curve was fitted with GraphPad, and the significant difference (t-test) was calculated.

[0138] Experimental results: Figure 3 As shown in the figure, the blood ethanol concentration of mice increased first and then decreased after drinking, and reached a peak between 2 and 3 hours. Compared with the alcohol group, two doses of gabexate reduced serum alcohol concentration at all time points and restored serum ethanol concentration to normal levels more quickly, indicating that gabexate administered before drinking can effectively promote ethanol metabolism in drunk mice.

[0139] 2.2 To investigate the effect of gabexate administration after alcohol on ethanol metabolism in mice under high-dose alcohol, the experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into an alcohol group (alcohol dose 7 g / kg), an alcohol + metadoxine (60 mg / kg) group, an alcohol + low-dose gabexate (1 mg / kg) group, an alcohol + medium-dose gabexate (3 mg / kg) group, and an alcohol + high-dose gabexate (10 mg / kg) group according to their body weight.

[0140] All groups of mice were fasted for 12 hours before the experiment, and all groups were gavaged with alcohol. 30 minutes after the alcohol was given, the alcohol + metadoxine (60 mg / kg) group, alcohol + low-dose gabexate (1 mg / kg) group, alcohol + medium-dose gabexate (3 mg / kg) group and alcohol + high-dose gabexate (10 mg / kg) group were administered by intraperitoneal injection, and the alcohol group was gavaged with the same volume of solvent. Blood was collected from the eye sockets after drinking, and the blood of mice was collected 0.5, 1, 2, 4, 6, 9, 12, 18 and 24 hours after drinking. After standing at room temperature, the serum was centrifuged and the serum ethanol concentration was detected by Megazyme-Alcohol kit. The data were summarized, the curve was fitted with GraphPad, and the significant difference (t-test) was calculated.

[0141] Experimental results: Figure 4As shown in the figure, the blood ethanol concentration of mice after drinking alcohol showed a trend of first increasing and then decreasing, and reached a peak between 1-2 hours. Compared with the alcohol group, the medium dose (3 mg / kg) and high dose (10 mg / kg) of gabexate reduced the serum alcohol concentration at all time points and restored the serum ethanol concentration to normal levels more quickly. The high dose of gabexate (10 mg / kg) restored the ethanol concentration to normal levels at 4 hours, indicating that gabexate administered after drinking can effectively promote ethanol metabolism in drunk mice. Compared with the positive control metadoxine, gabexate has a low dose, a fast onset, and a significantly greater reduction in ethanol.

[0142] Example 3 Gabexate promotes acetaldehyde metabolism in mice

[0143] To investigate the effect of gabexate on acetaldehyde metabolism in mice under high-dose alcohol, the experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into an alcohol group (alcohol dose 7 g / kg), an alcohol + metadoxine (500 mg / kg) group, an alcohol + low-dose gabexate (1 mg / kg) group, and an alcohol + high-dose gabexate (3 mg / kg) group according to their body weight.

[0144] All groups of mice were fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, alcohol + metadoxine (500 mg / kg) group and alcohol + low-dose gabexate (1 mg / kg) group and alcohol + high-dose gabexate (3 mg / kg) group were administered by oral gavage. The alcohol group was gavaged with the same volume of solvent. After that, all groups were gavaged with alcohol. Blood was collected from the eye sockets after drinking. Blood was collected from mice 1 hour after drinking. After standing at room temperature, serum was centrifuged and the serum acetaldehyde concentration was detected by HPLC derivatization. The data were summarized, the curve was fitted with GraphPad, and the significant difference (t-test) was calculated.

[0145] Experimental results: Figure 5 As shown, the serum acetaldehyde concentration did not decrease in the metadoxine (500 mg / kg) group compared with the alcohol group.

[0146] Unexpectedly, at very low doses (1 mg / kg and 3 mg / kg), gabexate significantly reduced serum acetaldehyde concentrations at the test time points, indicating that gabexate can effectively promote acetaldehyde metabolism in drunken mice, thereby significantly reducing the harm caused by acetaldehyde.

[0147] Example 4 Gabexate promotes rat ethanol metabolism experiment

[0148] To investigate the effect of gabexate on ethanol metabolism in rats under high-dose alcohol, the experimental scheme was as follows: 8-week-old SD male rats were divided equally according to their body weight into an alcohol group (alcohol dose 7 g / kg), an alcohol + metadoxine (30 mg / kg) group, an alcohol + low-dose gabexate (5 mg / kg) group, and an alcohol + high-dose gabexate (15 mg / kg) group.

[0149] All groups of mice were fasted for 12 hours before the experiment. 30 minutes before alcohol administration, the alcohol + metadoxine (30 mg / kg) group, alcohol + low-dose gabexate (5 mg / kg) group and alcohol + high-dose gabexate (15 mg / kg) group were administered by intraperitoneal injection. The alcohol group was gavaged with the same volume of solvent. After that, all groups were gavaged with alcohol. Blood was collected by cutting the tail to test the ethanol concentration in the rats 2 hours after alcohol administration. The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0150] Experimental results: Figure 6 As shown, in the ethanol metabolism experiment in rats with acute alcohol intoxication, both doses of gabexate reduced the serum alcohol concentration at the test time point compared with the alcohol group, * indicates P < 0.05.

[0151] Example 5 Experiment on the promotion of ethanol metabolism in mice by gabexate metabolites

[0152] To investigate the effects of gabexate metabolites on ethanol metabolism in mice under high-dose alcohol, the experimental scheme was as follows: 8-week-old ICR male mice were divided equally according to body weight into an alcohol group (alcohol dose 7 g / kg), an alcohol + metadoxine (60 mg / kg) group, an alcohol + gabexate (10 mg / kg) group, an alcohol + guanidinohexanoic acid (5.02 mg / kg) group, an alcohol + ethyl paraben (3.98 mg / kg) group, and an alcohol + combination (guanidinohexanoic acid, 5.02 mg / kg; ethyl paraben, 3.98 mg / kg) group.

[0153] All groups of mice were fasted for 12 hours before the experiment. 30 minutes before alcohol administration, the alcohol + metadoxine (60 mg / kg) group, alcohol + gabexate (10 mg / kg) group, alcohol + guanidocanoic acid (5.02 mg / kg) group, alcohol + ethyl paraben (3.98 mg / kg) group and alcohol + combination (guanidocanoic acid, 5.02 mg / kg; ethyl paraben, 3.98 mg / kg) group were administered by intraperitoneal injection. The alcohol group was gavaged with the same volume of solvent. After that, all groups were gavaged with alcohol. Blood ethanol concentration in mice was tested 3h and 6h after alcohol administration by removing eyeballs. The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0154] Experimental results: Figure 7 As shown, compared with the alcohol group, gabexate metabolites and their combination had a significant effect of reducing blood alcohol, * indicates P < 0.05, ** indicates P < 0.01.

[0155] Example 6 Gabexate improves the behavior of mice drunkenness

[0156] 6.1 To investigate whether gabexate administration before alcohol has a wake-promoting effect on drunken mice under higher doses of alcohol, a behavioral righting reflex experiment was conducted. The experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into a control group, an alcohol group (alcohol dose 8 g / kg), an alcohol + metadoxine (60 mg / kg) group, an alcohol + low-dose gabexate (10 mg / kg) group, and an alcohol + high-dose gabexate (30 mg / kg) group according to their body weight.

[0157] Each group of mice fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, the alcohol + metadoxine (60 mg / kg) group, alcohol + low-dose gabexate (10 mg / kg) group, and alcohol + high-dose gabexate (30 mg / kg) group were administered by intraperitoneal injection. The control group and the alcohol group were gavaged with the same volume of solvent. After that, except for the control group, which was gavaged with the same volume of solvent, the rest of the groups were gavaged with alcohol. After drinking, the righting reflex of mice disappeared and the recovery time, as well as the gripping force of mice detected by the gripping force detector, were counted, and the drunkenness rate, drunkenness latency, and sobering time were calculated. The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0158] Experimental results: Figure 8 , 9 As shown, compared with the alcohol group (drunkenness rate 100%), the alcohol + low-dose gabexate (10 mg / kg) group reduced the drunkenness rate of mice (75%), and significantly prolonged the incubation period of drunkenness, * indicates P < 0.05, and shortened the duration of drunkenness, ** indicates P < 0.01. Nine hours after drinking, the gripping force of the alcohol group was significantly reduced compared with the control group, ### indicates P < 0.001, indicating that the physical strength of the drunk mice was still not restored nine hours after drinking; compared with the alcohol group, gabexate can significantly increase the gripping force of drunk mice, * indicates P < 0.05, indicating that gabexate has a good alcohol sobering effect and can restore the physical strength of mice faster.

[0159] 6.2 To investigate whether gabexate administration after alcohol administration has the effect of promoting awakening and accelerating physical recovery in drunk mice under higher doses of alcohol, a behavioral righting reflex experiment was conducted. The experimental plan was as follows: 8-week-old ICR male mice were evenly divided into a control group, an alcohol group (alcohol dose 8 g / kg), an alcohol + metadoxine (60 mg / kg) group, an alcohol + low-dose gabexate (1 mg / kg) group, an alcohol + medium-dose gabexate (3 mg / kg) group and an alcohol + high-dose gabexate (10 mg / kg) group according to their body weight.

[0160] All groups of mice were fasted for 12 hours before the experiment. Except for the control group, which was gavaged with the same volume of solvent, the rest of the groups were gavaged with alcohol. 30 minutes after the alcohol was given, the alcohol + metadoxine (60 mg / kg) group, alcohol + low-dose gabexate (1 mg / kg) group, alcohol + medium-dose gabexate (3 mg / kg) group and alcohol + high-dose gabexate (10 mg / kg) group were administered by intraperitoneal injection. The control group and the alcohol group were gavaged with the same volume of solvent. After drinking, the righting reflex of mice disappeared and the recovery time, as well as the grasping force of mice detected by the grasping force detector and the hanging endurance of mice tested by the hanging device, and the intoxication latency and sobering time were counted. The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0161] Experimental results: Fig.10 , 11 As shown, compared with the alcohol group, the alcohol + low-dose gabexate (1g / kg) group and the alcohol + high-dose gabexate (10g / kg) group shortened the duration of drunkenness, ** indicates P<0.01. Nine hours after drinking, the gripping force and hanging endurance of the alcohol group were significantly reduced compared with the control group, # indicates P<0.05, ### indicates P<0.001, indicating that the physical strength of the drunk mice was still not restored after nine hours of drinking; compared with the alcohol group, three doses of gabexate can significantly increase the gripping force of drunk mice, ** indicates P<0.01, ** indicates P<0.001, indicating that gabexate has a good alcohol sobering effect and can restore the physical strength of mice faster.

[0162] 6.3 To investigate whether gabexate has the effect of improving motor coordination in drunken mice, a behavioral pole climbing experiment was conducted. The experimental plan was as follows: 8-week-old ICR male mice were evenly divided into a control group, an alcohol group (alcohol dose 4.5 g / kg), an alcohol + metadoxine (500 mg / kg) group, an alcohol + low-dose gabexate (10 mg / kg) group, and an alcohol + high-dose gabexate (30 mg / kg) group according to their body weight.

[0163] Before the formal experiment, all groups of mice were trained to climb poles for three days. All groups of mice were fasted for 12 hours before the experiment. 30 minutes before alcohol administration, the alcohol + metadoxine (500 mg / kg) group and the alcohol + low-dose gabexate (10 mg / kg) group and the alcohol + high-dose gabexate (30 mg / kg) group were administered by oral gavage, and the control group and the alcohol group were gavaged with the same volume of solvent. After that, except for the control group, which was gavaged with the same volume of solvent, the rest of the groups were gavaged with alcohol. Ten minutes after drinking, the mouse climbing behavior test was performed, and the mouse climbing time was recorded and scored according to the mouse movement state (Table 1). The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0164] Table 1. Scoring criteria for the movement status of the pole climbing experiment

[0165]

[0166] Experimental results: Fig.12 As shown in the figure, compared with the control group, the alcohol group significantly reduced the climbing time of mice and increased the climbing score of mice, ### indicates P<0.001; compared with the alcohol group, both doses of gabexate significantly increased the climbing time of mice and reduced the climbing score of mice, * indicates P<0.05, ** indicates P<0.01. This shows that gabexate can effectively improve the motor ability of mice after drunkenness and play a role in sobering up.

[0167] 6.4 To investigate whether gabexate has the effect of improving motor coordination in drunken mice, a behavioral rotarod experiment was conducted. The experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into a control group, an alcohol group (alcohol dose 4.5 g / kg), an alcohol + Alda-1 (60 mg / kg) group, and an alcohol + gabexate (100 mg / kg) group according to their body weight.

[0168] Before the formal experiment, all groups of mice were trained in the rotarod for three days. All groups of mice fasted for 12 hours before the experiment. 30 minutes before alcohol administration, the alcohol + Alda-1 (60 mg / kg) and alcohol + Gabexate (100 mg / kg) groups were administered by oral gavage and intraperitoneal injection. The control group and the alcohol group were gavaged with the same volume of solvent. After that, except for the control group, which was gavaged with the same volume of solvent, the other groups were gavaged with alcohol. The mouse rotarod behavior test was performed 10 minutes after drinking, and the time the mouse spent on the rod was recorded. The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0169] Experimental results: Fig.13As shown in the figure, compared with the control group, the alcohol group significantly reduced the time of mice on the rod, ### indicates P<0.001; compared with the alcohol group, the alcohol + gabexate (100 mg / kg) group significantly increased the time of mice on the rod, ** indicates P<0.01. This shows that gabexate can effectively improve the motor ability of mice after being drunk and play a role in sobering up.

[0170] Example 6 Gabexate improves ethanol-induced hepatocyte injury experiment

[0171] 6.1 To investigate the effect of gabexate on ethanol-induced liver injury at the cellular level, the experimental scheme was as follows: the experimental groups were divided into an alcohol group, a positive drug control group (silybin, 100 μM and Alda-1, 10 μM) and a gabexate group.

[0172] HepG2 cells were cultured normally for 12 hours, and the positive drug and gabexate stock solution were diluted with culture medium to the concentration to be tested (the final concentration was 1-10 μmol / L), and the corresponding test solution was added to each well. After incubation with the cells for 6 hours, alcohol modeling was performed, and the culture medium contained a predetermined concentration of 350mM ethanol solution and incubated for 16 hours. After the modeling was completed, the supernatant was discarded, and the cell survival rate was detected by Cell Counting Kit-8 (CCK8). The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0173] Experimental results: Fig.14 As shown, compared with the alcohol group, gabexate had a protective effect on hepatocytes, * indicates P < 0.05, ** indicates P < 0.01.

[0174] 6.2 The effects of different salt forms of gabexate on ethanol-induced liver injury at the cellular level were investigated. The experimental scheme was as follows: the experimental groups were divided into alcohol group, positive drug control group (metformin, 100 μM), free gabexate and different salt forms of gabexate (hydrochloride, sulfate, methanesulfonate, maleate, etc.).

[0175] HepG2 cells were cultured normally for 12 hours, and the positive drug and different salt-type gabexate stock solutions were diluted with culture medium to the concentration to be tested (final concentration of 10 and 100 μmol / L), and the corresponding test solution was added to each well. After incubation with the cells for 6 hours, alcohol modeling was performed, and the culture medium contained 350mM ethanol solution and incubated for 16 hours. After the modeling was completed, the supernatant was discarded, and the cell survival rate was detected by Cell Counting Kit-8 (CCK8). The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0176] Experimental results: Fig.15As shown, compared with the alcohol group, other salt forms of gabexate also have the effect of protecting liver cells, * indicates P < 0.05.

[0177] Example 7 Gabexate reduces mortality from acute alcohol poisoning

[0178] The effect of gabexate on the mortality of mice poisoned by extremely high doses of alcohol was investigated. The experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into a control group, an alcohol group (alcohol dose 10 g / kg), an alcohol + two doses of gabexate (15 mg / kg) group, an alcohol + three doses of metadoxine (20 mg / kg) group and an alcohol + three doses of gabexate (10 mg / kg) group according to their body weight.

[0179] Each group of mice fasted for 12 hours before the experiment. The two-dose group was given the drug 30 minutes before and after drinking. The three-dose group was given the drug one hour after drinking, in addition to 30 minutes before and after drinking. The control group and the alcohol group were gavaged with the same volume of solvent. After that, except for the control group, which was gavaged with the same volume of solvent, the rest of the groups were gavaged with alcohol. The death of mice was recorded. The data were summarized and a bar graph was drawn using GraphPad.

[0180] Experimental results: Fig.16 As shown, compared with the alcohol group, the survival rate of gabexate was 33.33% after two administrations and 66.67% after three administrations, which indicated that gabexate significantly improved the survival rate of mice with acute alcohol intoxication.

[0181] Example 8 Experiment on reducing liver damage in mice with acute alcohol poisoning with gabexate

[0182] To investigate the protective effect of gabexate on the liver under high-dose alcohol, the experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into a control group, an alcohol group (alcohol dose 7 g / kg), an alcohol + metadoxine (60 mg / kg) group, an alcohol + low-dose gabexate (3 mg / kg) group, and an alcohol + high-dose gabexate (10 mg / kg) group according to their body weight.

[0183] All groups of mice were fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, alcohol + metadoxine (60 mg / kg), alcohol + low-dose gabexate (3 mg / kg) and alcohol + high-dose gabexate (10 mg / kg) were administered by intraperitoneal injection. The alcohol group was gavaged with the same volume of solvent. All groups were then gavaged with alcohol. Nine hours after alcohol gavage, the mice were killed and dissected, the appearance of the mouse liver was photographed, the liver weight was weighed, and the liver-to-body ratio was calculated. The liver was fixed, paraffin sectioned and H&E stained, and the changes in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and plasma superoxide dismutase (SOD) levels were tested by removing the eyeballs and taking blood 9 hours after alcohol administration. The liver tissue was taken and the contents of reduced glutathione (GSH) and malondialdehyde (MDA) were determined according to the method in the kit instructions. The data were summarized, a bar graph was drawn using GraphPad, and significant differences were calculated (t-test).

[0184] Experimental results: Fig.17 , 18 As shown in Figures 19, compared with the control group, the liver of the alcohol group turned significantly whiter and yellower, but there was no significant change in liver volume. In the pathological sections, liver glycogen was significantly reduced compared with the control group, and ALT and AST contents were significantly increased. ### indicates P<0.001, indicating that acute alcohol poisoning has obvious damage to the liver. Compared with the alcohol group, the liver color of the gabexate group was restored, and there was no significant change in the liver-to-body ratio. In the pathological sections, liver glycogen was significantly increased, and ALT and AST contents were significantly reduced. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001, indicating that gabexate can protect the liver function of mice with acute alcohol poisoning.

[0185] like Fig. 20 As shown, compared with the control group, the SOD inhibition rate in the alcohol group was significantly reduced, ** indicates P<0.01, and the SOD activity was also significantly reduced, ** indicates P<0.01, indicating that ethanol can reduce the body's ability to scavenge oxygen free radicals and cause liver damage; compared with the alcohol group, the high-dose gabexate (10 mg / kg) group can significantly increase the SOD inhibition rate, * indicates P<0.05, and increase the SOD enzyme activity, * indicates P<0.05, thereby improving the body's ability to scavenge oxygen free radicals and reducing alcohol-induced liver damage.

[0186] like Fig.21 As shown, compared with the control group, the MDA content in the alcohol group increased, P = 0.0518, indicating that ethanol can increase the content of malondialdehyde in the body and increase the degree of attack by free radicals; compared with the alcohol group, the high-dose gabexate (10 mg / kg) group can reduce the MDA content, P = 0.0689, reducing the attack of free radicals on the body's cells.

[0187] like Fig. 22 As shown, compared with the control group, the GSH content in the alcohol group was reduced, P = 0.0569, indicating that ethanol can increase the degree of oxidative stress in the body; compared with the alcohol group, the high-dose gabexate (10 mg / kg) group can reduce the MDA content, P = 0.0689, alleviate the body's oxidative stress, and reduce alcohol-induced liver damage.

[0188] Example 9 ALDH2 enzyme activity test

[0189] The effect of gabexate on ALDH2 activity was investigated. The experimental method was as follows: gabexate was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mmol / L stock solution. Tris buffer solution (50 mmol / L, pH = 8.0), 50 mmol / L NAD solution, ALDH2*2 protein or ALDH2*1 protein were pre-added to a 96-well plate and mixed. The gabexate stock solution was gradiently diluted and added to the 96-well plate (final concentration was 0-400 μmol / L). The above samples were incubated at 37°C for 5 min. After the incubation, 40 mmol / L acetaldehyde solution was added. The total sample volume was 200 μL. The 96-well plate was quickly placed in an ELISA reader for time kinetic scanning and the 340 nm reading was recorded.

[0190] About 40% of the population in China carries a point mutation in the ALDH2 gene, where the normal allele is ALDH2*1 and the mutant allele is ALDH2*2. ALDH2*2 carriers have low ALDH2 enzyme activity, and this defect leads to the accumulation of acetaldehyde in the body after drinking, manifested as characteristic flushing, headache, nausea, dizziness and palpitations after drinking, and increases the risk of alcoholic liver disease.

[0191] Experimental results: Fig.23 As shown, gabexate has a certain ALDH2*2 stimulating activity, with a maximum stimulating multiple of 2 and a half effective concentration (EC50) value of 35.39 μmol / L. For the positive drug Alda-1, the maximum stimulating multiple 2 is 6.37 and the half effective concentration (EC50) value is 1.532 μmol / L. Gabexate has a significant ALDH2*1 stimulating activity, with a maximum stimulating multiple of >1.9 and a half effective concentration (EC50) value of 34.06 μmol / L. For the positive drug Alda-1, the maximum stimulating multiple is 2 and the half effective concentration (EC50) value is 0.2862 μmol / L.

[0192] Example 10 Serine protease activity test

[0193] The effect of gabexate on the activity of serine proteases was investigated. The experimental method was as follows: gabexate was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mmol / L stock solution. BAEE (1 mmol / L) and HCl (1 mmol / L) were added to a 96-well quartz plate in advance and mixed. The gabexate stock solution was diluted in a gradient manner and added to the 96-well quartz plate (final concentration was 0-25 μmol / L), serine proteases (trypsin, thrombin) were quickly added and mixed. The total sample volume was 200 μL, and the 96-well quartz plate was quickly placed in an ELISA reader for time kinetic scanning and recording of 253 nm readings.

[0194] Experimental results: Fig.24 As shown, gabexate can inhibit the activity of serine proteases. The half-maximal inhibitory concentration (IC50) value of gabexate for trypsin is 4.082 μmol / L, and the half-maximal inhibitory concentration (IC50) value of gabexate for thrombin is 4.054 μmol / L.

[0195] This suggests that gabexate can inhibit serine proteases (such as trypsin and thrombin), thereby inhibiting alcohol-induced inflammation in the liver and other organs (such as pancreas), especially alcoholic liver disease and / or concurrent acute pancreatitis caused by alcohol poisoning.

[0196] Example 11 Experiment on reducing liver damage in mice with acute alcohol poisoning with gabexate

[0197] The mechanism of gabexate in promoting ethanol metabolism and protecting liver damage at the animal level under high-dose alcohol was investigated. The experimental method was as follows: 8-week-old ICR male mice were evenly divided into a control group, an alcohol group (alcohol dose 7g / kg), an alcohol + metadoxine (60mg / kg) group, an alcohol + low-dose gabexate (3mg / kg) group, and an alcohol + high-dose gabexate (10mg / kg) group according to their body weight.

[0198] All groups of mice were fasted for 12 hours before the experiment. 30 minutes before alcohol administration, the alcohol + metadoxine (60 mg / kg) group, alcohol + low-dose gabexate (3 mg / kg) group and alcohol + high-dose gabexate (10 mg / kg) group were administered by intraperitoneal injection. The alcohol group was gavaged with the same volume of solvent. After that, all groups were gavaged with alcohol. The mice were killed 9 hours after drinking, and the liver tissue was taken. The activity of liver ALDH2 and ADH, and the content of inflammatory factors (IL-1β, TNF-α) in the liver were determined according to the method in the kit instructions. The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0199] Experimental results: Fig.25As shown, compared with the control group, the activity of acetaldehyde dehydrogenase 2 in the liver of mice in the alcohol group was significantly decreased, ### indicates P<0.001; compared with the alcohol group, both doses of gabexate could significantly increase the activity of ALDH2 in vivo, *** indicates P<0.001; there was no significant change in the activity of alcohol dehydrogenase after drinking, and the gabexate groups had no significant effect on the activity of alcohol dehydrogenase.

[0200] like Fig.26 As shown, compared with the control group, the IL-1β and TNF-α in the alcohol group were increased. A single drinking can cause an increase in inflammatory factors, leading to alcoholic liver damage. Compared with the alcohol group, the two-dose group of gabexate can reduce the increase of IL-1β and TNF-α, * indicates P<0.05, reducing alcohol-induced liver damage.

[0201] discuss

[0202] Gabexate is a non-peptide proteolytic enzyme inhibitor, first listed in Japan, and currently listed in many countries including China, usually listed in the form of its mesylate. The preparation is a lyophilized injection, has the characteristics of small molecular weight, small side effects, and no immunogenicity, can inhibit the activities of trypsin, chymotrypsin, fibrinase, plasma kallikrein, pancreatic kallikrein, urokinase and thrombin, and is mainly used clinically to treat acute pancreatitis (AP) and disseminated intravascular coagulation (DIC). In recent years, it has been found that it can inhibit the inflammatory response after arterial thrombosis, and has an anticoagulant effect during penetration. So far, there is no record or report in the prior art about the preventive or therapeutic effect of gabexate on alcoholism and alcoholic liver disease.

[0203] Surprisingly, the experimental results of the present invention prove that gabexate and its metabolites can promote the metabolism of ethanol and acetaldehyde in cells and animals (especially by increasing the activity of ALDH2 enzyme), alleviate the symptoms of drunkenness, improve the behavior after drunkenness, reduce the mortality rate of acute alcohol poisoning, and can be used to treat alcohol poisoning; further, gabexate and its metabolites can improve ethanol-induced liver damage, improve the morphology and structure of liver tissue and cells in alcohol poisoning, reduce markers of liver damage, including aspartate aminotransferase (AST), alanine aminotransferase ALT), improve the antioxidant capacity of liver tissue, reduce the generation of free radicals, and reduce the risk of alcoholic liver disease caused by alcohol poisoning, so that it can be used to prevent and treat alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis and other alcoholic liver diseases.

[0204] Based on this, it can be seen that gabexate and its metabolites as active ingredients can effectively prevent human body from being damaged by alcohol, and can effectively treat alcohol poisoning and reduce health risks caused by drinking. In addition, gabexate, as a marketed drug, has a large amount of clinical application experience, is safe and reliable, is conducive to the development of new products of the present invention, and has very good application prospects.

[0205] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Use of Gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of a pharmaceutical composition for preventing or treating alcoholism and / or alcoholic liver disease, in, The metabolite is selected from the following compounds or pharmaceutically acceptable salts thereof:

2. The use according to claim 1, characterized in that The active ingredient in the pharmaceutical composition is gabexate or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, characterized in that Prevention or treatment of alcoholism is one or more selected from the following group: (1) Accelerate the metabolic rate of ethanol and / or acetaldehyde; (2) Reduce the rate of drunkenness; (3) Prolonging the incubation period of intoxication; (4) shorten the duration of intoxication; (5) Improve the state and behavior after being drunk; (6) reduce mortality from alcoholism; and / or (7) Reduce alcohol-induced liver damage.

4. The use according to claim 1, characterized in that The metabolite is 6-guanidinohexanoic acid, ethyl p-hydroxybenzoate, or a combination thereof.

5. The use according to claim 1, characterized in that The pharmaceutically acceptable salt of gabexate is selected from the group consisting of hydrochloride, sulfate, methanesulfonate, maleate, phosphate, hydrobromide, malate, fumarate, p-toluenesulfonate, nitrate, citrate, preferably methanesulfonate.

6. The use according to claim 1, characterized in that The alcohol poisoning is acute alcohol poisoning or chronic alcohol poisoning.

7. The use according to claim 1, characterized in that The alcoholic liver disease is selected from the group consisting of alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis, or a combination thereof.

8. The use according to claim 1, characterized in that Prevention or treatment of alcoholic liver disease includes one or more methods selected from the following group: (a) Alleviate acute or chronic hepatocellular and liver damage caused by alcohol; (b) reduce alcohol-induced elevations in transaminases; and / or (c) Reduce alcohol-induced oxidative stress damage.

9. Use of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of an aldehyde dehydrogenase 2 (ALDH2) agonist, in, The metabolite is selected from the following compounds or pharmaceutically acceptable salts thereof:

10. Use of gabexate or its pharmaceutically acceptable salt, or its metabolite in the preparation of a health food having an auxiliary protective effect on alcoholic liver damage, in, The metabolite is selected from the following compounds or pharmaceutically acceptable salts thereof:

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