Use of gaboxadol for the treatment of alcoholism

Abstract

Disclosed is the use of gabexate for the treatment of alcoholism. More specifically, the present invention provides the use of gabexate (Gabexate) or a pharmaceutically acceptable salt thereof, or a metabolite thereof, in the manufacture of a pharmaceutical composition for the prevention or treatment of alcoholism and / or alcoholic liver disease. Also provided is the use of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof, in the manufacture of an acetaldehyde dehydrogenase 2 (ALDH2) agonist.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, to the new use of gabexate or its metabolite or its salt as an active ingredient in the preparation of a drug for preventing or treating alcoholism, alcoholic liver disease. BACKGROUND

[0002] Excessive alcohol intake at one time can inhibit the respiratory center of the brain, leading to respiratory arrest and even death. Long-term excessive drinking almost inevitably leads to cirrhosis of the liver, accompanied by symptoms such as abdominal distension and jaundice, which seriously affects the quality of life. Acute alcohol intoxication (AAI) is also known as acute ethanol poisoning, which 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 excitation to inhibition. The patient often shows abnormal behavior and consciousness, and in severe cases, organ function is damaged, leading to respiratory and circulatory failure, which can even endanger life. Alcohol poisoning has increasingly seriously threatened people's physical health. After drinking, ethanol is absorbed into the blood in the digestive tract, and it is absorbed faster on an empty stomach. The ethanol in the blood is detoxified by the liver, first converted to acetaldehyde by ethanol dehydrogenase, and then converted to acetic acid by acetaldehyde dehydrogenase. Acetic acid is further broken down into water and carbon dioxide. The whole process takes about 2-4 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. The main effect of this drug is to promote the awakening of alcohol poisoning patients and prevent patients from dying due to alcohol-induced respiratory depression, but its effect on accelerating ethanol metabolism and reducing alcohol-induced tissue damage is minimal. Because alcohol poisoning is dangerous, it is of great clinical significance to actively use 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, and can further develop into alcoholic hepatitis, liver fibrosis and cirrhosis. Some alcoholics or heavy drinkers will develop alcohol-related health problems, and ALD is the most common organ damage caused by alcohol. At present, the main treatment measures for alcoholic liver disease in clinical practice include alcohol abstinence, nutritional supplementation, and drug treatment for antioxidant and anti-inflammatory effects, but the effect is not good. So far, there is still a lack of effective treatment methods, and therefore, alcoholic liver disease urgently needs safe and effective treatment drugs.

[0004] The liver is the largest metabolic organ in the human body and is the main site of alcohol metabolism. About 90% of alcohol is absorbed through the gastrointestinal tract and enters the liver through the blood, where it is first oxidized to acetaldehyde by alcohol dehydrogenase (ADH), and then oxidized to acetic acid by acetaldehyde dehydrogenase 2 (ALDH2). Acetic acid is further metabolized and excreted from the body or involved in biosynthesis. ALDH2 is a rate-limiting enzyme for alcohol metabolism, and its substrate acetaldehyde is a strong electrophilic primary carcinogen. According to statistics, about 40% of East Asians (about 8% of the world's population) carry ALDH2 gene point mutations (ALDH2*2), which leads to aldehyde metabolism disorders and increases the risk of AAI and ALD. Studies have shown that ALDH2 agonists can promote the rate of ethanol / acetaldehyde metabolism and protect the liver from damage, and are expected to play a preventive and therapeutic role in alcoholic diseases. Acetaldehyde can affect the nervous and cardiovascular systems of the human body, causing symptoms such as headache, nausea, vomiting, and palpitations. Long-term drinking can cause liver damage and even cirrhosis and liver cancer. Therefore, reducing the concentration and time of alcohol and acetaldehyde in the body can significantly reduce the damage of alcohol to the body. SUMMARY

[0005] The purpose of the present application 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 pharmaceutical compositions for the prevention or treatment of alcoholism, alcoholic liver disease, acetaldehyde dehydrogenase 2 (ALDH2) agonists.

[0006] In a first aspect of the present application, the use of gabexate (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,

[0007]

[0008] In another preferred embodiment, the metabolite is selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof:

[0009]

[0010] In another preferred embodiment, the active ingredient in the pharmaceutical composition is gabexate or a pharmaceutically acceptable salt thereof.

[0011] In another preferred embodiment, the prevention or treatment of alcoholism is one or more selected from the group consisting of:

[0012] (1) accelerating the rate of ethanol and / or acetaldehyde metabolism;

[0013] (2) reducing the rate of intoxication;

[0014] (3) prolonging the incubation period of intoxication;

[0015] (4) shortening the duration of intoxication;

[0016] (5) improving the state and / or behavior after intoxication, such as shortening the time to recover grip strength, accelerating the recovery of limb coordination, etc.;

[0017] (6) reducing the mortality rate of alcoholism; and / or

[0018] (7) reducing alcohol-induced liver damage, such as hepatocyte infiltration, increase in inflammatory factors, etc.

[0019] In another preferred embodiment, the metabolite is 6-guanidino hexanoic acid, ethyl p-hydroxybenzoate, or a combination thereof.

[0020] In another preferred embodiment, the pharmaceutically acceptable salt of gaboxadol is selected from the group consisting of hydrochloride, sulfate, mesylate, maleate, phosphate, hydrobromide, malate, fumarate, p-toluene sulfonate, nitrate, citrate, preferably mesylate.

[0021] In another preferred embodiment, the alcoholism is acute alcoholism or chronic alcoholism.

[0022] 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.

[0023] In another preferred embodiment, preventing or treating alcoholic liver disease comprises one or more ways selected from the group consisting of:

[0024] (a) reducing alcohol-induced elevation of transaminases;

[0025] (b) reducing hepatocyte or liver tissue lipid accumulation;

[0026] (c) improving hepatocyte or liver antioxidant function;

[0027] (d) improving hepatocyte or liver anti-inflammatory function;

[0028] (e) reducing hepatocyte or liver active aldehyde accumulation; and / or

[0029] (f) delaying the progression of alcoholic liver disease.

[0030] In another preferred embodiment, preventing or treating alcoholic liver disease comprises one or more ways selected from the group consisting of:

[0031] (a) reducing alcohol-induced acute or chronic hepatocyte and liver damage;

[0032] (b) reducing alcohol-induced elevation of transaminases; and / or

[0033] (c) reducing alcohol-induced oxidative stress damage.

[0034] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0035] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, an injection preparation, or a topical preparation.

[0036] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of a tablet, a capsule, a powder, a granule, a pill, a lozenge, an effervescent tablet, a patch, an ointment, a lyophilized powder, or an injection solution.

[0037] In another preferred embodiment, the dosage form of the pharmaceutical composition is a lyophilized powder or an injection solution.

[0038] In another preferred embodiment, the lyophilized powder comprises one or more carriers selected from the group consisting of mannitol, lactose, hydrolyzed gelatin, sodium chloride, glucose, or a combination thereof.

[0039] In another preferred embodiment, the injection solution comprises one or more carriers selected from the group consisting of water for injection, mannitol, sodium chloride, glucose, or a combination thereof.

[0040] In a second aspect of the present application, there is provided a use of gaboxadol or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of an acetaldehyde dehydrogenase 2 (ALDH2) agonist.

[0041]

[0042] In another preferred embodiment, the metabolite is selected from the group consisting of a compound or a pharmaceutically acceptable salt thereof:

[0043]

[0044] In another preferred embodiment, the ALDH2 is ALDH2*1 and / or ALDH2*2.

[0045] In another preferred embodiment, the acetaldehyde dehydrogenase 2 (ALDH2) agonist is an ALDH2 covalent agonist, which can produce agonistic activity by covalently binding to ALDH2, such as gaboxadol.

[0046] In a third aspect of the present application, there is provided a method for preventing or treating alcoholism and / or alcoholic liver disease, comprising the step of administering one or more of gaboxadol or a metabolite thereof, or a pharmaceutically acceptable salt thereof, or administering the composition of the present application to a subject in need thereof, thereby preventing or treating alcoholism and / or alcoholic liver disease.

[0047] In another preferred embodiment, the subject is a subject having elevated levels of aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) in blood or serum, e.g. a subject having at least 10%, at least 20%, at least 30%, at least 50%, at least 80%, at least 90% or even at least 100% higher levels of aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) in blood or serum compared to a normal control value CO.

[0048] In another preferred embodiment, the subject is a subject carrying the ALDH2*2 mutation.

[0049] In another preferred embodiment, the subject is a human or a mammal, e.g. a rat, a mouse or a monkey.

[0050] In another preferred embodiment, the subject is suffering from pancreatitis.

[0051] It should be understood that, within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Test results for the promotion of ethanol metabolism in hepatocytes by gaboxadol

[0053] Figure 2 Test results for the promotion of ethanol metabolism in hepatocytes by different salt forms of gaboxadol

[0054] Figure 3 Promotion of ethanol metabolism kinetics in mice by gaboxadol administered before alcohol

[0055] Figure 4 Promotion of ethanol metabolism kinetics in mice by gaboxadol administered after alcohol

[0056] Figure 5 Reduction of acetaldehyde levels in mice 1 hour after alcohol by gaboxadol

[0057] Figure 6 Reduction of ethanol levels in rats 2 hours after alcohol by gaboxadol

[0058] Figure 7 Reduction of ethanol levels in mice 3 and 6 hours after alcohol by gaboxadol metabolites and combinations

[0059] Figure 8 Test results for the improvement of alcohol tolerance time and drunkenness duration in mice by gaboxadol administered before alcohol

[0060] Figure 9 Test results for the improvement of grip strength in mice by gaboxadol administered before alcohol

[0061] Figure 10Results of tests for gabexate ester to improve alcohol tolerance time and drunkenness duration in mice when administered after alcohol consumption

[0062] Figure 11 Results of tests for gabexate ester to improve drunkenness behavior grip strength and hanging endurance in mice when administered after alcohol consumption

[0063] Figure 12 Results of tests for gabexate ester to improve drunkenness behavior pole climbing time and pole climbing score in mice

[0064] Figure 13 Results of tests for gabexate ester to improve drunkenness behavior pole climbing time and pole climbing score in mice

[0065] Figure 14 Results of tests for gabexate ester to improve cell survival rate in an alcohol-induced hepatocyte damage model

[0066] Figure 15 Results of tests for gabexate ester to improve survival rate in acute alcoholism

[0067] Figure 16 Results of tests for gabexate ester to protect liver in acute alcoholic liver injury in mice

[0068] Figure 17 Results of tests for gabexate ester to protect liver in acute alcoholic liver injury in mice

[0069] Figure 18 Results of tests for gabexate ester to improve liver function indicators (glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase) in acute alcoholic liver injury in mice

[0070] Figure 19 Results of tests for gabexate ester to improve antioxidant indicators (superoxide dismutase activity) in acute alcoholic liver injury in mice

[0071] Figure 20 Results of tests for gabexate ester to improve malondialdehyde content in liver in acute alcoholic liver injury in mice

[0072] Figure 21 Results of tests for gabexate ester to improve reduced glutathione content in liver in acute alcoholic liver injury in mice

[0073] Figure 22 Results of tests for gabexate ester to improve liver function indicators (glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase) in chronic alcoholic liver injury in mice

[0074] Figure 23 Results of tests for gabexate ester to improve serum inflammatory indicators (interleukin-6 and TNF-α) in chronic alcoholic liver injury in mice

[0075] Figure 24 Results of tests for gabexate ester to improve serum lipid indicators (triglycerides and cholesterol) in chronic alcoholic liver injury in mice

[0076] Figure 25 Results of tests for gabexate on liver inflammation indicators (interleukin-6 and TNF-α) of chronic alcoholic liver injury mice

[0077] Figure 26 Results of tests for gabexate on liver lipid indicators (triglyceride and cholesterol) of chronic alcoholic liver injury mice

[0078] Figure 27 Results of pathological section and oil red O staining for gabexate on chronic alcoholic liver injury mice

[0079] Figure 28 Activity curve of gabexate on mutant ALDH2*2 and wild type ALDH2*1

[0080] Figure 29 Binding curve of gabexate with wild type ALDH2*1 and mutant ALDH2*2

[0081] Figure 30 Mass spectrum results of covalent binding of gabexate with wild type ALDH2*1 and mutant ALDH2*2

[0082] Figure 31 Activity curve of gabexate on inhibiting serine protease

[0083] Figure 32 Results of tests for gabexate on key enzymes (alcohol dehydrogenase and acetaldehyde dehydrogenase) of ethanol metabolism of acute alcoholic liver injury mice

[0084] Figure 33 Results of tests for gabexate on inflammation indicators (interleukin-1β and TNF-α) of acute alcoholic liver injury mice DETAILED DESCRIPTION

[0085] The present inventors, through extensive and in-depth research, by a large number of screening and testing, first provided the use of gabexate or its metabolites or salts thereof as an active ingredient for preparing a pharmaceutical composition for preventing or treating alcoholism and alcoholic liver disease. The present inventors unexpectedly found that gabexate or its metabolites have acetaldehyde dehydrogenase agonist activity, can accelerate the ethanol and / or acetaldehyde metabolism rate, and significantly reduce various drunk-related symptoms caused by drinking to prevent or treat alcoholism and alcoholic liver disease. On this basis, the present application was completed.

[0086] TERMS

[0087] Unless otherwise defined, 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 application belongs.

[0088] As used herein, the terms "containing" or "comprising" can be open, semi-closed and closed. In other words, the terms also include "consisting essentially of or "consisting of.

[0089] Active ingredient

[0090] The active ingredient of the present application is gaboxadol or a metabolite thereof.

[0091] Gaboxadol has the following structural formula, and its CAS number is 39492-01-8.

[0092]

[0093] In the present application, the gaboxadol metabolite is selected from the group consisting of:

[0094]

[0095] The gaboxadol or a metabolite thereof of the present application is intended to include their pharmaceutically acceptable salts.

[0096] The gaboxadol or 6-guanidino hexanoic acid preferably salt is the salt formed with an organic acid or an inorganic acid, typically, the pharmaceutically acceptable salt of gaboxadol or 6-guanidino hexanoic acid independently includes (but is not limited to): hydrochloride, sulfate, methanesulfonate, maleate, phosphate, hydrobromide, malate, fumarate, p-toluenesulfonate, nitrate, citrate, preferably methanesulfonate.

[0097] The ethyl parahydroxybenzoate or parahydroxybenzoic acid preferably salt is the salt formed with an organic base or an inorganic base, typically, the pharmaceutically acceptable salt of ethyl parahydroxybenzoate or parahydroxybenzoic acid independently includes (but is not limited to): ammonium salt, alkali metal salt such as sodium, lithium, potassium salt, alkaline earth metal salt such as calcium, magnesium salt and salt formed with an organic base (such as organic amine).

[0098] The organic substance of the present application can be gaboxadol or a pharmaceutically acceptable salt thereof, a combination of one or more of the metabolites or a pharmaceutically acceptable salt thereof.

[0099] Alcohol metabolism

[0100] When an individual ingests alcohol, alcohol (ethanol) is rapidly absorbed into the blood circulation and subsequently undergoes a metabolic process, mainly in the liver. In the liver, ethanol is first catalytically converted to acetaldehyde by alcohol dehydrogenase (ADH), which is the rate-limiting step of alcohol metabolism. Subsequently, acetaldehyde is further metabolized to acetic acid under the action of acetaldehyde dehydrogenase (ALDH). Acetic acid is a relatively harmless metabolite, which can be completely oxidized in the body and ultimately converted into carbon dioxide and water. These end products are excreted out of the body through the respiratory and urinary systems.

[0101] Among them, acetaldehyde is the main toxic metabolite, which can cause harm to the human body. Acetaldehyde can affect the nervous system and cardiovascular system of the human body, causing symptoms such as headache, nausea, vomiting, palpitations, etc. Long-term drinking can cause liver damage, and even cause cirrhosis and liver cancer.

[0102] Therefore, reducing the concentration and time of alcohol and acetaldehyde in the body can significantly reduce the damage of alcohol to the body.

[0103] Acetaldehyde dehydrogenase

[0104] The main enzyme responsible for the conversion of acetaldehyde in the human body is acetaldehyde dehydrogenase in the liver, which has two isozymes, distributed in cytosol (ALDH1) and mitochondria (ALDH2). There is a significant difference in catalytic rate between the two, ALDH2 has a K m lower than ALDH1, about 1 / 10 of the latter, which is the isozyme mainly responsible for the conversion of acetaldehyde.

[0105] In China, about 40% of the population carries a single nucleotide polymorphism of the ALDH2 gene, among which the normal allele is ALDH2*1, and the mutant allele is ALDH2*2. Individuals carrying the ALDH2*2 mutant gene have significantly reduced ALDH2 enzyme activity. This genetic defect can slow down the metabolism of acetaldehyde in the body after drinking, causing acetaldehyde to accumulate in the body. This accumulation often manifests as a unique facial flushing, headache, nausea, dizziness, and palpitations after drinking, and may increase the risk of individuals developing alcoholic liver disease.

[0106] In particular, the active ingredient of the present application has an ALDH2 agonist effect, which can promote the conversion of acetaldehyde to acetic acid and avoid the accumulation of acetaldehyde. In particular, the active ingredient of the present application has excellent agonist activity for ALDH2*1 and ALDH2*2, and can be suitable for normal population and ALDH2*2 carriers.

[0107] Alcoholism

[0108] Alcoholism refers to excessive drinking or excessive consumption of ethanol, leading to toxic diseases of central nervous system excitation followed by inhibition. Patients mainly show abnormal behavior and consciousness, and severe cases can cause multiple system damage (most severely damaging the nervous system and liver), and even endanger life. According to the size of the amount of alcohol consumed at one time and the length of time of drinking, it can be divided into acute alcoholism and chronic alcoholism.

[0109] Acute alcoholism refers to the state of central nervous system dysfunction that occurs after a short period of time after ingesting a large amount of alcohol or alcohol-containing beverages. Patients often show abnormal behavior and consciousness, and severe cases can damage organ function, leading to respiratory and circulatory failure, and even endanger life, also known as acute alcohol poisoning.

[0110] 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 can be accompanied by cardiovascular and digestive system symptoms. Alcohol can also interfere with the activity of liver enzymes, increasing the burden on the liver and easily causing fatty liver and the formation of alcoholic liver cirrhosis. Also known as alcohol dependence or alcohol addiction.

[0111] The present application first discovered that the active ingredients of the present application have the effect of preventing or treating alcoholism. When a human or animal ingests alcohol, the active ingredients or compositions of the present application make the alcohol content in the body of the subject less, the concentration lower and / or the presence time shorter compared to not using the active ingredients or compositions of the present application, thereby making the impact or damage of alcohol on the body and mind less. The therapeutic effects of the present application can specifically include one or more of the following effects: (1) accelerating the metabolism rate of ethanol and / or acetaldehyde; (2) reducing the rate of intoxication; (3) prolonging the latency of intoxication; (4) shortening the duration of intoxication; (5) improving the state or behavior after intoxication; (6) reducing the mortality rate of alcoholism; and / or (7) and reducing liver damage caused by alcohol.

[0112] In addition, the therapeutic effects of the present application include preventing alcoholism, accelerating ethanol metabolism, rapidly decomposing ethanol into acetaldehyde, and ultimately decomposing into carbon dioxide and water, thereby reducing or delaying the inhibition of the central nervous system by alcohol, reducing the likelihood of the subject reaching alcoholism, reducing the action disorder caused by alcohol, or prolonging the latency of intoxication. In addition, the therapeutic effects of the present application also include "sobering up", making the subject sober from the state of alcoholism, regaining consciousness or regaining self-control.

[0113] Alcoholic liver disease

[0114] As used herein, alcoholic liver disease is a liver disease caused by long-term heavy drinking. The liver is the main organ for the metabolic decomposition of alcohol, and drinking alcohol can cause damage to liver function, and even alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis, or alcoholic liver failure, etc. The higher the level of alcohol in the body, the longer the time of drinking, and the more severe the liver damage. Experiments have shown that the active ingredients or compositions of the present application can alleviate the damage of alcohol to liver cells, improve the decline of liver function caused by alcohol, improve the antioxidant and anti-inflammatory functions of liver cells, and / or delay the progression of alcoholic liver disease.

[0115] The terms "prevent," "treat," "ameliorate," "accelerate," "reduce," "prolong," "shorten," "decrease," or "alleviate" as used herein include delaying and stopping the progression of a disease or symptoms, or eliminating the disease, and does not require 100% inhibition, elimination, and reversal. In some embodiments, the compositions described herein are capable of alleviating, shortening, or reversing one or more symptoms, or indicators, of alcoholism or alcoholic liver disease, e.g., by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, as compared to levels observed in the absence of the active ingredients or compositions of the present application.

[0116] Some representative indicators of treating alcoholism, but not limited to, are the rate of loss of righting reflex, the time of loss of righting reflex and recovery time, pole climbing time, pole climbing score, rotarod time on bar, serum ethanol concentration, liver ADH and ALDH activity.

[0117] Some representative indicators of treating alcoholic liver disease, but not limited to, are the mortality rate of acute alcoholism, liver-body ratio, ALT content, AST content, SOD activity, MDA content, GSH content, TNF-α, IL-1β content.

[0118] Compositions

[0119] The present application provides a pharmaceutical composition comprising one or more active ingredients of the present application; and a pharmaceutically acceptable carrier. In particular, the pharmaceutical composition is used for preventing or treating alcoholism and / or alcoholic liver disease.

[0120] The present application also provides an acetaldehyde dehydrogenase 2 (ALDH2) agonist, which can comprise one or more active ingredients of the present application; and a pharmaceutically acceptable carrier. The ALDH2 is ALDH2*1 and / or ALDH2*2.

[0121] In the compositions described herein, the active ingredients of the present application can be used alone or in combination with other active ingredients.

[0122] Generally, the compositions of the present application contain an effective amount of the active ingredients. The term "effective amount" or "effective dose" refers to an amount adequate to function or activate and acceptable to a human and / or animal.

[0123] As used herein, the term "pharmaceutically acceptable" refers to those substances that are appropriate for use with humans and / or animals without undue adverse side effects such as toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of therapeutic agents including various excipients and diluents.

[0124] The compositions of the present application contain a safe and effective amount of the active ingredients of the present application and a pharmaceutically acceptable carrier, and can exist in a variety of solid or liquid forms. The carrier includes, but is not limited to, water, NaCl, normal saline solution, lactated Ringer's solution, conventional sucrose, conventional dextrose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorants, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acids esters, hyrmethylcellulose, polyvinylpyrrolidone, and pigments. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings and / or aromatics, and the like which do not deleteriously react with the compounds provided herein or interfere with their activity. Those of ordinary skill in the art will recognize other carriers and excipients suitable for use with the disclosed compounds.

[0125] The compositions of the present application can be administered to a subject by any suitable route, including orally, subcutaneously, intravenously, intramuscularly, transdermally, by inhalation spray, topically, nasally, buccally. Preferably, administration is oral, oral, subcutaneous, intravenous, intramuscular, transdermal.

[0126] The compositions of the present application suitable for administration typically will be discrete units such as tablets, capsules, powders, granules, pellets, patches, gels, ointments, suppositories, or in a liquid form such as oral fluids, injectable or infusible solutions or suspensions, or freeze dried powders.

[0127] The compositions are preferably manufactured under aseptic conditions. The choice of carrier will depend on the desired means of administration and is well within the skill of those in the art.

[0128] The effective amount of the active ingredients of the present application can vary depending on the mode of administration and the subject's alcohol consumption, the severity of the drunken behavior, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art (e.g., through clinical trials) based on various factors. The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease or symptoms to be treated or ameliorated in the subject, the patient's body mass, the patient's immune status, the route of administration, etc. Generally, the active ingredients of the present application can be administered at a dose of about 0.1 mg / kg to 1 g / kg or 1 mg / kg to 100 mg / kg of animal body weight per day. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the subject's situation.

[0129] The present application also provides a method for preventing or treating alcoholism and / or alcoholic liver disease, comprising the step of administering the active ingredient of the present application, or administering the composition of the present application to a subject in need thereof, thereby preventing or treating alcoholism and / or alcoholic liver disease.

[0130] The active ingredient or the composition of the present application can be administered before, during or after drinking. In particular, it can be administered at any time during the 6 hours before drinking, such as 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 0.5 hour, 10 minutes, 5 minutes, 1 minute before drinking, at any time during drinking, or within 6 hours after drinking, such as 1 minute, 5 minutes, 10 minutes, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours or 5 hours after drinking.

[0131] In another preferred embodiment, the subject is a human or a non-human mammal, such as a rat, a mouse or a monkey.

[0132] The main advantages of the present application include:

[0133] A) The present application first discovers the use of gabexate and its metabolites in the preparation of a pharmaceutical composition for preventing or treating alcoholism and / or alcoholic liver disease.

[0134] B) The present application first discovers the use of gabexate and its metabolites in the preparation of an acetaldehyde dehydrogenase 2 (ALDH2) agonist.

[0135] C) The active ingredient of the present application, gabexate, is a marketed drug with a large amount of clinical application experience, safe and reliable, and more conducive to the development of new products of the present application.

[0136] The present application will be further described in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, which are usually carried out according to the conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0137] Unless otherwise specified, the gabexate used in the examples is gabexate mesilate.

[0138] Example 1 Gabexate promotes ethanol metabolism in hepatocytes

[0139] 1.1 The alcohol metabolism rate at the cellular level reflects the direct effect of drugs on cells to promote alcohol, and the effect of gabexate on the alcohol metabolism rate at the cellular level is investigated, and the experimental scheme is as follows: the experimental groups are divided into alcohol group, positive drug control group (Alda-1, 100 μM) and gabexate group.

[0140] HepG2 cells were cultured normally for 12 h, and the positive drug and gaboxadol stock solution were diluted with culture medium to the target concentration (final concentration of 10 and 100 μmol / L, respectively), and then incubated with cells for 6 h. After that, alcohol induction modeling was performed, and the culture medium containing a predetermined concentration of ethanol was added to the cells, and the incubation was continued for 3 h. After the modeling was completed, the supernatant of the culture medium was collected, and the concentration of ethanol therein was detected using a Megazyme-Alcohol kit. The data were summarized, and a column chart was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0141] The experimental results are shown in Table 1. Figure 1 As shown in Table 1, compared with the alcohol group, gaboxadol has the ability to promote liver cell ethanol metabolism, and the gaboxadol-promoted ethanol metabolism is concentration-dependent, * represents P < 0.05, and ** represents P < 0.01.

[0142] 1.2 Investigation of the effect of different salt types of gaboxadol on the ethanol metabolism rate at the cell level, and the experimental scheme is as follows: the experimental groups are divided into an alcohol group, a free gaboxadol group, and different salt type gaboxadol groups (mesylate, hydrochloride, sulfate, acetate, maleate, etc.).

[0143] HepG2 cells were cultured normally for 12 h, and the positive drug and different salt type gaboxadol stock solution were diluted with culture medium to the target concentration (final concentration of 10 and 100 μmol / L, respectively), and then incubated with cells for 6 h. After that, alcohol induction modeling was performed, and the culture medium containing a predetermined concentration of ethanol was added to the cells, and the incubation was continued for 3 h. After the modeling was completed, the supernatant of the culture medium was collected, and the concentration of ethanol therein was detected using a Megazyme-Alcohol kit. The data were summarized, and a column chart was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0144] The experimental results are shown in Table 2. Figure 2 As shown in Table 2, compared with the alcohol group, other salt type gaboxadol also has the ability to promote liver cell ethanol metabolism, and the acetate and hydrochloride gaboxadol-promoted ethanol metabolism is comparable to that of mesylate, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[0145] Example 2 Gaboxadol promotes ethanol metabolism in mice

[0146] 2.1 Investigation of the effect of gaboxadol pre-drinking on ethanol metabolism in mice under high-dose alcohol, and the experimental scheme is as follows: 8-week-old ICR male mice were divided into an alcohol group (alcohol dose of 7 g / kg), an alcohol + metadoxine group (500 mg / kg), an alcohol + low-dose gaboxadol group (10 mg / kg), and an alcohol + high-dose gaboxadol group (30 mg / kg) according to the body weight.

[0147] All mice were fasted for 12 h before the experiment. The alcohol + metadoxine group (500 mg / kg) was administered orally 30 min before alcohol administration, while the alcohol + low-dose gaboxadol group (10 mg / kg) and the alcohol + high-dose gaboxadol group (30 mg / kg) were administered intraperitoneally. The alcohol group was administered the same volume of solvent orally. All groups were administered alcohol orally. Blood samples were collected by orbital bleeding at 1, 3, 6, and 12 h after alcohol administration. The blood was left to stand at room temperature and then centrifuged to obtain the serum. The ethanol concentration in the serum was determined using the Megazyme-Alcohol kit. The data were pooled, fitted with a curve using GraphPad, and the significant differences were calculated (t-test).

[0148] Results: As shown in Figure 1, the blood ethanol concentration in mice increased after alcohol administration and then decreased, reaching a peak between 2 and 3 h. Compared with the alcohol group, both doses of gaboxadol reduced the serum ethanol concentration at all time points and returned the serum ethanol concentration to normal more quickly, indicating that pre-alcohol administration of gaboxadol can effectively promote ethanol metabolism in intoxicated mice. Figure 3 2.2 Investigation of the effect of post-alcohol administration of gaboxadol on ethanol metabolism in mice at a high alcohol dose, according to the following experimental protocol: 8-week-old ICR male mice were divided into the alcohol group (alcohol dose 7 g / kg), the alcohol + metadoxine group (60 mg / kg), the alcohol + low-dose gaboxadol group (1 mg / kg), the alcohol + medium-dose gaboxadol group (3 mg / kg), and the alcohol + high-dose gaboxadol group (10 mg / kg) according to body weight.

[0149] All mice were fasted for 12 h before the experiment. Then, all groups were administered alcohol orally. Thirty minutes after alcohol administration, the following groups were administered intraperitoneally: the alcohol + metadoxine group (60 mg / kg), the alcohol + low-dose gaboxadol group (1 mg / kg), the alcohol + medium-dose gaboxadol group (3 mg / kg), and the alcohol + high-dose gaboxadol group (10 mg / kg). As a control, the alcohol group was administered the same volume of solvent orally. Blood samples were collected by orbital bleeding at 0.5, 1, 2, 4, 6, 9, 12, 18, and 24 h after alcohol administration. The blood was left to stand at room temperature and then centrifuged to isolate and obtain the serum samples. The ethanol concentration in the serum was determined using the Megazyme-Alcohol kit. The data were pooled, fitted with a curve using GraphPad, and the significant differences were calculated (t-test). Results: As shown in Figure 2, the blood ethanol concentration in mice increased after alcohol administration and then decreased, reaching a peak between 2 and 3 h. Compared with the alcohol group, both doses of gaboxadol reduced the serum ethanol concentration at all time points and returned the serum ethanol concentration to normal more quickly, indicating that post-alcohol administration of gaboxadol can effectively promote ethanol metabolism in intoxicated mice.

[0150] Figure 4 ​As shown in Figure 2, the blood ethanol concentration in mice showed a trend of first increasing and then decreasing after drinking, and reached a peak between 1-2 hours. Compared with the alcohol group, gaboxadol medium dose (3 mg / kg) and high dose (10 mg / kg) reduced the serum alcohol concentration at all time points, and recovered the serum ethanol concentration to normal level more quickly. The ethanol concentration of high dose gaboxadol (10 mg / kg) returned to normal level at 4 hours, which indicated that gaboxadol given after drinking can effectively promote the ethanol metabolism of drunk mice. Compared with the positive control metadoxine, gaboxadol has lower dose, faster onset and significantly greater reduction in ethanol.

[0151] Example 3 Gaboxadol promotes acetaldehyde metabolism in mice

[0152] The effect of gaboxadol on acetaldehyde metabolism in mice under high dose alcohol was investigated, and the experimental scheme was as follows: 8-week-old ICR male mice were divided into alcohol group (alcohol dose 7 g / kg), alcohol + metadoxine group (500 mg / kg), alcohol + low dose gaboxadol group (1 mg / kg) and alcohol + high dose gaboxadol group (3 mg / kg) according to body weight.

[0153] Each group of mice was fasted for 12 hours before the experiment. 30 minutes before alcohol administration, the alcohol + metadoxine group (500 mg / kg) was administered by oral gavage, while the alcohol + low dose gaboxadol group (1 mg / kg) and the alcohol + high dose gaboxadol group (3 mg / kg) were administered by intraperitoneal injection, and the alcohol group was administered with the same volume of solvent as a control. Thereafter, all groups were administered alcohol by gavage. Blood samples were collected from mice 1 hour after drinking by orbital blood collection method. The collected blood was allowed to stand at room temperature, and then centrifuged to obtain serum. The concentration of acetaldehyde in serum was detected by HPLC derivatization method. The data were summarized, and the column chart was drawn by GraphPad, and the significant difference (t-test) was calculated.

[0154] Experimental results: As shown in Figure 3, compared with the alcohol group, the serum acetaldehyde concentration in the metadoxine group (500 mg / kg) was not reduced. Figure 5

[0155] Unexpectedly, at very low doses (1 mg / kg and 3 mg / kg), gaboxadol significantly reduced the serum acetaldehyde concentration at the test time points, which indicated that gaboxadol can effectively promote the acetaldehyde metabolism of drunk mice, thereby greatly reducing the harm caused by acetaldehyde.

[0156] Example 4 Gaboxadol promotes ethanol metabolism in rats

[0157] ​To investigate the effect of gaboxadol on ethanol metabolism in rats under high dose of alcohol, the experimental protocol was as follows: 8-week-old SD male rats were divided into alcohol group (alcohol dose 7 g / kg), alcohol + metadoxine group (30 mg / kg), alcohol + low dose gaboxadol group (5 mg / kg) and alcohol + high dose gaboxadol group (15 mg / kg) according to body weight.

[0158] Each group of rats was fasted for 12 h before the experiment. 30 minutes before alcohol administration, the following groups were administered by intraperitoneal injection: alcohol + metadoxine group (30 mg / kg), alcohol + low dose gaboxadol group (5 mg / kg) and alcohol + high dose gaboxadol group (15 mg / kg), and the alcohol group was given the same volume of solvent by gavage as a control. Thereafter, all groups were given alcohol by gavage. Blood samples were collected from rats 2 h after drinking by cutting the tail, and the collected blood was centrifuged after standing at room temperature to separate and obtain serum samples. The Megazyme-Alcohol kit was used to detect the ethanol concentration in the serum. The data were summarized, and the bar chart was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0159] Experimental results: as shown in Figure 6 , in the ethanol metabolism experiment of acute alcohol poisoning rats, compared with the alcohol group, gaboxadol at two doses reduced the serum alcohol concentration at the test time point, * indicates P < 0.05.

[0160] Example 5 Gaboxadol metabolites promote ethanol metabolism experiment in mice

[0161] To investigate the effect of gaboxadol metabolites on ethanol metabolism in mice under high dose of alcohol, the experimental protocol was as follows: 8-week-old ICR male mice were divided into alcohol group (alcohol dose 7 g / kg), alcohol + metadoxine group (60 mg / kg), alcohol + gaboxadol group (10 mg / kg), alcohol + guanidino hexanoic acid group (5.02 mg / kg), alcohol + p-hydroxybenzoic acid ethyl ester group (3.98 mg / kg) and alcohol + combination group (guanidino hexanoic acid, 5.02 mg / kg; p-hydroxybenzoic acid ethyl ester, 3.98 mg / kg) according to body weight.

[0162] Each group of mice was fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, the following groups were administered by intraperitoneal injection: alcohol + metadoxine (60 mg / kg), alcohol + gaboxadol (10 mg / kg), alcohol + glyceryl guacetin (5.02 mg / kg), alcohol + p-hydroxybenzoic acid ethyl ester (3.98 mg / kg), and alcohol + combination (glyceryl guacetin, 5.02 mg / kg; p-hydroxybenzoic acid ethyl ester, 3.98 mg / kg), and the alcohol group was administered the same volume of vehicle by gavage. Subsequently, all groups were administered alcohol by gavage. Blood samples were collected from the mice 3 and 6 hours after drinking by enucleation. The blood was allowed to stand at room temperature and then centrifuged to separate and obtain serum samples. The ethanol concentration in the serum was detected using the Megazyme-Alcohol kit. The data were summarized, and bar graphs were drawn using GraphPad, and significant differences were calculated (t-test).

[0163] Experimental results: As shown in Figure 7 , the metabolites of gaboxadol and their combinations had a significant effect on reducing blood ethanol compared with the alcohol group, * indicates P < 0.05, and ** indicates P < 0.01.

[0164] Example 6 Gaboxadol improves the behavior of drunk mice

[0165] 6.1 To investigate whether gaboxadol administered before drinking has a wake-up effect on drunk mice, a behavior righting reflex experiment was performed, and the experimental scheme is as follows: 8-week-old ICR male mice were divided into the control group, alcohol group (alcohol dose 8 g / kg), alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gaboxadol group (10 mg / kg), and alcohol + high-dose gaboxadol group (30 mg / kg) according to body weight.

[0166] Each group of mice was fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, the following groups were administered by intraperitoneal injection: alcohol + metadoxine (60 mg / kg), alcohol + low-dose gaboxadol group (10 mg / kg), and alcohol + high-dose gaboxadol group (30 mg / kg), and the control group and alcohol group were administered the same volume of vehicle by gavage. Subsequently, all groups were administered alcohol by gavage, except that the control group continued to be administered the same volume of vehicle by gavage. The time for the disappearance and recovery of the righting reflex of the mice after drinking was recorded, and the grip strength of the mice was detected by a grip strength detector 9 hours after drinking, and the drunk rate, drunk latency, and sober-up time were counted. The data were summarized, and bar graphs were drawn using GraphPad, and significant differences were calculated (t-test).

[0167] Experimental results: As shown in Figure 8 , 9As shown, compared with the alcohol group (drunk rate 100%), the alcohol + low-dose gaboxadol group (10 mg / kg) reduced the drunk rate of mice (75%) and significantly prolonged the drunk latency, *P < 0.05, and shortened the drunk duration, **P < 0.01. Nine hours after drinking, the alcohol group had a significant decrease in grip strength compared with the control group, ###P < 0.001, indicating that the drunk mice did not recover their strength at nine hours after drinking; compared with the alcohol group, gaboxadol significantly increased the grip strength of drunk mice, *P < 0.05, indicating that gaboxadol had a good effect of dispelling alcohol and could make the mice recover their strength faster.

[0168] 6.2 To investigate whether gaboxadol given after drinking has the effect of promoting wakefulness and accelerating the recovery of strength in drunk mice at a higher alcohol dose, a behavioral righting reflex experiment was performed, and 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 group (60 mg / kg), an alcohol + low-dose gaboxadol group (1 mg / kg), an alcohol + medium-dose gaboxadol group (3 mg / kg), and an alcohol + high-dose gaboxadol group (10 mg / kg) according to the body weight.

[0169] The mice in each group were fasted for 12 hours before the experiment, and except for the control group which was given the same volume of solvent by gavage, the other groups were given alcohol by gavage. Thirty minutes after alcohol administration, the following groups were given drug treatment by intraperitoneal injection: the alcohol + metadoxine group (60 mg / kg), the alcohol + low-dose gaboxadol group (1 mg / kg), the alcohol + medium-dose gaboxadol group (3 mg / kg), and the alcohol + high-dose gaboxadol group (10 mg / kg). As a control, the control group and the alcohol group were given the same volume of solvent by gavage. The time of disappearance and recovery of righting reflex in mice after drinking was recorded, and the grip strength of mice at 9 hours after drinking was detected by a grip strength detector and the suspension endurance of mice at 9 hours after drinking was tested by a suspension device, and the drunk latency and sober-up time were counted. The data were summarized, column charts were drawn by GraphPad, and significant differences (t-test) were calculated. The experimental results are as follows: Figure 10 、 11 As shown, compared with the alcohol group, the alcohol + low-dose gaboxadol group (1 g / kg) and the alcohol + high-dose gaboxadol group (10 g / kg) shortened the drunk duration, **P < 0.01. Nine hours after drinking, the alcohol group had a significant decrease in grip strength and suspension endurance compared with the control group, #P < 0.05, ###P < 0.001, indicating that the drunk mice did not recover their strength at nine hours after drinking; compared with the alcohol group, gaboxadol at three doses significantly increased the grip strength of drunk mice, **P < 0.01, ***P < 0.001, indicating that gaboxadol had a good effect of dispelling alcohol and could make the mice recover their strength faster.

[0170] 6.3 To investigate whether gaboxadol can improve the motor coordination of intoxicated mice, a behavioral pole climbing test was performed. The experimental protocol was as follows: 8-week-old ICR male mice were divided into control group, alcohol group (alcohol dose 4.5 g / kg), alcohol + metadoxine group (500 mg / kg), alcohol + low-dose gaboxadol group (10 mg / kg), and alcohol + high-dose gaboxadol group (30 mg / kg) according to body weight.

[0171] Before the formal experiment, all groups of mice were trained to climb the pole for three days. The mice in each group were fasted for 12 h before the experiment. The alcohol + metadoxine group (500 mg / kg) was administered by oral gavage 30 minutes before alcohol administration, while the alcohol + low-dose gaboxadol group (10 mg / kg) and the alcohol + high-dose gaboxadol group (30 mg / kg) were administered by intraperitoneal injection. The control group and the alcohol group were given the same volume of solvent by gavage as a control. Subsequently, except for the control group which continued to be given the same volume of solvent, the rest of the groups were given alcohol by gavage. The mouse pole climbing behavior test was performed 10 minutes after alcohol consumption, and the mouse pole climbing time and score according to the mouse movement state were recorded (Table 1). The data were summarized and plotted as a bar graph using GraphPad, and the significant differences (t-test) were calculated.

[0172] Table 1. Pole climbing test movement state scoring criteria

[0173]

[0174] Experimental results: As shown in Figure 12 compared with the control group, the alcohol group significantly reduced the pole climbing time of the mice and increased the pole climbing score of the mice, ### indicates P < 0.001; compared with the alcohol group, the two dose groups of gaboxadol significantly increased the pole climbing time of the mice and reduced the pole climbing score of the mice, * indicates P < 0.05, and ** indicates P < 0.01. This indicates that gaboxadol can effectively improve the motor ability of intoxicated mice and has a sobering effect.

[0175] 6.4 To investigate whether gaboxadol can improve the motor coordination of intoxicated mice, a behavioral pole climbing test was performed. The experimental protocol was as follows: 8-week-old ICR male mice were divided into control group, alcohol group (alcohol dose 4.5 g / kg), alcohol + Alda-1 group (60 mg / kg), alcohol + gaboxadol group (100 mg / kg) according to body weight.

[0176] Before the formal experiment, all groups of mice were trained for three days on the rotarod. The mice in each group were fasted for 12 hours before the experiment. Thirty minutes before the administration of alcohol, the following groups were administered by intraperitoneal injection: alcohol + Alda-1 group (60 mg / kg) and alcohol + gaboxadol group (100 mg / kg), and the control group and alcohol group were administered the same volume of solvent by gavage as a control. Subsequently, in addition to the control group, which was continuously administered the same volume of solvent by gavage, the other groups were administered alcohol by gavage. Ten minutes after drinking, the mice were tested for rotarod behavior, and the time on the rod was recorded. The data were summarized, and a column chart was drawn using GraphPad, and the significant differences (t-test) were calculated.

[0177] The experimental results are shown in Table 1. Figure 13 As shown in Table 1, compared with the control group, the alcohol group significantly reduced the time on the rod of the mice, and ### indicates P < 0.001; compared with the alcohol group, the alcohol + gaboxadol group (100 mg / kg) significantly increased the time on the rod of the mice, and ** indicates P < 0.01. This indicates that gaboxadol can effectively improve the motor ability of mice after intoxication and has a sobering effect.

[0178] Example 7: Gaboxadol improves ethanol-induced liver cell damage experiment

[0179] To investigate the effect of gaboxadol on ethanol-induced liver damage at the cellular level, the experimental scheme is as follows: the experimental groups are divided into an alcohol group, a positive drug control group (silybin, 100 μM and Alda-1, 10 μM), and a gaboxadol group.

[0180] HepG2 cells were normally cultured for 12 hours, and the positive drug and gaboxadol stock solutions were diluted with culture medium to the target concentration (final concentration of 1-10 μmol / L). After 6 hours of co-incubation with the cells, alcohol-induced modeling was performed, and the culture medium containing the predetermined concentration of ethanol was added to the cells, and the incubation was continued for 16 hours. After the modeling was completed, the supernatant was discarded, and the cell survival rate was detected using the Cell Counting Kit-8 (CCK8) method. The data were summarized, and a column chart was drawn using GraphPad, and the significant differences (t-test) were calculated.

[0181] The experimental results are shown in Table 2. Figure 14 As shown in Table 2, compared with the alcohol group, gaboxadol has a protective effect on liver cells, and * indicates P < 0.05, and ** indicates P < 0.01.

[0182] Example 8: Gaboxadol improves the survival rate of acute alcoholism

[0183] To investigate the effect of gabexate on mortality in mice poisoned by extremely high doses of alcohol, the experimental protocol was as follows: Eight-week-old male ICR mice were randomly divided into a control group, an alcohol group (alcohol dose 10 g / kg), an alcohol group plus gabexate administered twice (15 mg / kg), an alcohol group plus metadoxine administered three times (20 mg / kg), and an alcohol group plus gabexate administered three times (10 mg / kg).

[0184] Mice in each group were fasted for 12 hours before the experiment. The two-drug groups received the drug before and 30 minutes after alcohol administration. For the three-drug groups, in addition to administration before and 30 minutes after alcohol administration, an additional drug was administered 1 hour after alcohol administration. The control and alcohol groups received the same volume of solvent via gavage at the corresponding time points as controls. While the control group continued to receive the same volume of solvent via gavage, all other groups received alcohol via gavage. Mouse mortality was observed and recorded. Data were summarized and a bar chart was created using GraphPad.

[0185] Experimental results: such as Figure 15 As shown, compared with the alcohol group, the survival rate of gabexate after two administrations was 33.33%, and the survival rate after three administrations was 66.67%, indicating that gabexate significantly improved the survival rate of mice with acute alcohol poisoning.

[0186] Example 9: Gabexate's Experiment on Reducing Liver Injury in Mice with Acute Alcohol Poisoning

[0187] To investigate the liver-protective effect of gabexate under high-dose alcohol, the experimental protocol was as follows: Eight-week-old male ICR mice were randomly divided into a control group, an alcohol group (7 g / kg alcohol), an alcohol + metadoxine group (60 mg / kg alcohol), an alcohol + low-dose gabexate group (3 mg / kg alcohol), and an alcohol + high-dose gabexate group (10 mg / kg alcohol).

[0188] Each group of mice was fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, the following groups were administered by intraperitoneal injection: alcohol + metadoxine (60 mg / kg), alcohol + low-dose gaboxadol (3 mg / kg), and alcohol + high-dose gaboxadol (10 mg / kg). The control group and the alcohol group were administered the same volume of vehicle by gavage. Subsequently, all groups were administered alcohol by gavage, except for the control group, which was administered the same volume of vehicle by gavage. Nine hours after alcohol gavage, the mice were sacrificed and dissected to assess liver health. The steps included taking photographs of the mouse liver, weighing the liver, and calculating the liver-body ratio. H&E staining was performed to observe histological changes. Blood was collected by enucleation to test the changes in serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the changes in plasma levels of superoxide dismutase (SOD) nine hours after alcohol administration. The contents of reduced glutathione (GSH) and malondialdehyde (MDA) in liver tissue samples were determined according to the kit instructions. The data were summarized, and column charts were drawn using GraphPad, and significant differences were calculated (t-test).

[0189] The experimental results are shown in Figures Figure 16 , 17 , 18. Compared with the control group, the liver of the alcohol group was significantly whitened and yellowed, but the liver volume did not change significantly, and the liver glycogen in the pathological section was significantly reduced, and the contents of ALT and AST were significantly increased, indicating that acute alcoholism had a significant damage to the liver; compared with the alcohol group, the liver color of the gaboxadol group recovered, and the liver-body ratio did not change significantly, and the liver glycogen in the pathological section increased significantly, and the contents of ALT and AST decreased significantly, indicating that gaboxadol could protect the liver function of acute alcoholism mice.

[0190] As shown in Figure Figure 19 , compared with the control group, the SOD inhibition rate of the alcohol group was significantly reduced, and the SOD activity was also significantly reduced, indicating that ethanol could cause the body's ability to scavenge oxygen free radicals to decrease, causing liver damage; compared with the alcohol group, the high-dose gaboxadol (10 mg / kg) group could significantly improve the SOD inhibition rate, and the SOD enzyme activity, and the body's ability to scavenge oxygen free radicals, reducing alcohol-induced liver damage.

[0191] As shown in Figure Figure 20As shown, compared with the control group, the MDA content in the alcohol group increased (P=0.0518), indicating that ethanol leads to an increase in malondialdehyde content in the body, which increases the degree of free radical attack. Compared with the alcohol group, the high-dose gabexate (10mg / kg) group reduced the MDA content (P=0.0689), which reduced the attack of free radicals on the body's cells.

[0192] like Figure 21 As shown, compared with the control group, the alcohol group had a lower GSH content (P=0.0569), indicating that ethanol leads to an increase in oxidative stress in the body. Compared with the alcohol group, the high-dose gabexate (10mg / kg) group reduced MDA content (P=0.0689), alleviating the body's oxidative stress and reducing alcohol-induced liver damage.

[0193] Example 10: Gabexate Reduction of Chronic Alcoholic Liver Disease in Mice

[0194] To investigate the protective effect of gabexate on the liver of mice after long-term intake of LDC liquid diet containing alcohol, the experimental protocol was as follows: Eight-week-old male ICR mice were randomly divided into a control group (receiving the same calorie non-alcoholic liquid diet), an alcohol group, an alcohol + metadoxine group (250 mg / kg), an alcohol + low-dose gabexate group (1 mg / kg), and an alcohol + high-dose gabexate group (3 mg / kg) according to their body weight.

[0195] For the first five days of the experiment, the concentration of alcohol in the liquid diet was gradually increased until it reached 4% alcohol. Starting on day six, mice in the alcohol + low-dose gabexate group (1 mg / kg) and the high-alcohol + high-dose gabexate group (3 mg / kg) received the medication daily via intraperitoneal injection. Simultaneously, mice in the alcohol + metadoxine group (250 mg / kg) received the medication via gavage. As controls, mice in the alcohol group and the control group received an equal volume of the solvent via gavage. After 37 days of continuous feeding, on the morning of day 38, the alcohol-containing liquid diet was removed, and the mice were administered alcohol via gavage. Nine hours later, the mice were sacrificed and dissected to assess their liver health. Specific procedures included photographing the appearance of the mouse liver and performing H&E staining and Oil Red O staining to observe histological changes. Serum transaminase levels, as well as lipid and inflammatory factor-related indicators in serum and liver, were assessed using ELISA. Data were summarized, bar charts were generated using GraphPad, and significance differences (t-test) were calculated. Experimental results: as shown. Figure 22 , 23, 24, 25, 26, compared with the control group, alcohol group serum transaminase level, serum and liver inflammation factor and lipid content increased significantly, ### indicates P < 0.001, shows that acute alcoholism has obvious damage to the liver, and the mice have two characteristics of alcoholic fatty liver and alcoholic hepatitis; Compared with the alcohol group, both doses of gabexate can significantly improve the serum transaminase level, the increase of serum and liver inflammatory factors and lipids, *** indicates P < 0.001, indicating that gabexate can protect the liver function of chronic alcohol-induced liver injury mice.

[0196] As shown in Figure 27 , compared with the control group, the alcohol group showed that the cytoplasm of hepatocytes contained fat vacuoles of different sizes on HE and oil red O staining, and had obvious hepatocyte fatty fatty degeneration characteristics; Compared with the alcohol group, both doses of gabexate significantly reduced the size of fat vacuoles in hepatocytes. It shows that gabexate can protect the hepatocyte morphology of chronic alcohol-induced liver injury mice and reduce alcohol-induced liver damage.

[0197] Example 11 ALDH2 enzyme activity test

[0198] To investigate the effect of gabexate on ALDH2 activity, the experimental method is as follows: dissolve gabexate in DMSO to prepare a 10 mmol / L stock solution. In a 96-well plate, add Tris buffer solution (50 mmol / L, pH = 8.0), 50 mmol / L NAD solution, ALDH2*2 protein or ALDH2*1 protein, and mix well. Dilute the gabexate stock solution in gradient, and add it to the 96-well plate to achieve different final concentrations of 0 to 400 μmol / L. Incubate the above sample at 37°C for 5 minutes, and then add 40 mmol / L acetaldehyde solution to make the total volume of the sample 200 μL. Quickly put the 96-well plate into the enzyme marker, carry out time kinetic scanning, and record the absorbance reading at 340 nm wavelength.

[0199] About 40% of the population in China carries ALDH2 gene point mutations, of which the normal allele is ALDH2*1 and the mutant allele is ALDH2*2. ALDH2*2 carriers have lower ALDH2 enzyme activity, and this defect leads to accumulation of acetaldehyde in the body after drinking, which is characterized by flushing, headache, nausea, dizziness and palpitations after drinking, and increases the risk of alcoholic liver disease.

[0200] The experimental results are shown in Figure 28 , gabexate has certain agonistic ALDH2*2 activity, the maximum agonistic multiple is about 2, the half effective concentration (EC 50 ) value is 35.39 μmol / L, the positive drug Alda-1, the maximum agonistic multiple 2 is 6.37, the half effective concentration (EC50 The concentration of gabexate was 1.532 μmol / L; gabexate exhibited significant ALDH2*1 agonist activity, with a maximum agonist factor >1.9 and a half-maximal effective concentration (EC50) of 1.532 μmol / L. 50 The value was 34.06 μmol / L, for the positive control drug Alda-1, with a maximum agonistic fold of 2 and a half-maximal effective concentration (EC50). 50 The value was 0.2862 μmol / L.

[0201] Example 12: Microthermophoretic Experiment with Gabexate and ALDH2

[0202] To investigate the binding affinity of gabexate to ALDH2, the experimental method was as follows: Gabexate was dissolved in PBS-T to prepare a 10 mmol / L stock solution. RED-tris-NTA dye was diluted to 100 nM using PBS-T buffer, while the protein was diluted to 800 nM. The protein and dye were mixed thoroughly at a 1:1 ratio and incubated at room temperature for 30 minutes, followed by centrifugation to separate the supernatant. The gabexate stock solution was serially diluted, and 10 μL was added to each PCR tube (ranging from 0.5 to 1000 μmol / L). 10 μL of labeled protein was added to each PCR tube, mixed thoroughly, and incubated at room temperature for another 2 hours. Samples were aspirated using capillary tubes for detection, and data were recorded and analyzed using a fluorescence scanner.

[0203] Experimental results: such as Figure 29 As shown, both gabexate and ALDH2 have strong binding affinity, and the binding constant (K) between gabexate and ALDH2*1 is... d The concentration of gabexate was 10.53 μmol / L, and the binding constant (K) between gabexate and ALDH2*2 was... d The concentration was 8.55 μmol / L.

[0204] Example 13 Gabexate covalently binds to ALDH2

[0205] To investigate the covalent binding of gabexate with ALDH2, the experimental method is as follows: the protein sample is diluted to a concentration of 2 mg / mL, and DMSO or small molecules are added. The mixed solution is placed at room temperature in the dark and incubated for 2 hours. Then, the small molecules in the solution are removed by Zeba column separation technology. 10 μg of protein is taken out, diluted with PBS buffer, and then denatured with solid urea. Next, DTT is added and mixed at 37°C for 30 minutes. Then, IAA is added and mixed at room temperature for 30 minutes. Then, DTT is added and mixed at room temperature for 15 minutes. Finally, LysC and Trypsin are added, and a certain concentration of DDM solution is added, and incubated at 37°C for 16 hours. Desalination is performed using a C18 column, and after drying, the sample is resuspended in an aqueous solution containing 0.015% (v / v) DDM, and then subjected to LC-MS / MS analysis.

[0206] Experimental results: as shown in Figure 30 , by screening modifications with target mass changes, gabexate is covalently bound to 12 potential binding sites of ALDH2*1 and ALDH2*2, respectively, among which the spectrum matching (PSM) of S338 site is the most, and the response is enhanced with the increase of compound concentration.

[0207] Example 14 Serine protease activity test

[0208] To investigate the effect of gabexate on serine protease activity, the experimental method is as follows: gabexate is dissolved in DMSO to prepare a 10 mmol / L stock solution. N-phenyl-L-arginine ethyl ester (1 mmol / L) and hydrochloric acid (1 mmol / L) are pre-added to a 96-well quartz plate and mixed well. The gabexate stock solution is gradiently diluted and added to the 96-well quartz plate (with a final concentration of 0-25 μmol / L). Serine protease (such as trypsin or thrombin) is quickly added, and mixed again to make the total volume of the sample 200 μL. Then, the 96-well quartz plate is quickly placed in a microplate reader for time kinetic scanning, and the absorbance change at 253 nm wavelength is recorded.

[0209] Experimental results: as shown in Figure 31 , gabexate can inhibit the activity of serine protease, and the half inhibitory concentration (IC 50 ) value of gabexate for trypsin is 4.082 μmol / L, and the half inhibitory concentration (IC 50 ) value of gabexate for thrombin is 4.054 μmol / L.

[0210] This suggests that gabexate can inhibit serine proteases (such as trypsin and thrombin), and thus inhibit inflammation in the liver and other organs (such as the pancreas) caused by alcohol, especially alcoholic liver disease and / or concurrent acute pancreatitis and other conditions caused by alcohol poisoning.

[0211] Example 15 Gaboxadol reduces liver injury in acute alcohol intoxication mice

[0212] To investigate the mechanism of gaboxadol in promoting ethanol metabolism and protecting liver injury at animal level under high-dose alcohol condition, the experimental method is as follows: 8-week-old ICR male mice are divided into control group, alcohol group (alcohol dose 7 g / kg), alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gaboxadol group (3 mg / kg) and alcohol + high-dose gaboxadol group (10 mg / kg) according to body weight.

[0213] Each group of mice is fasted for 12 hours before the experiment. 30 minutes before alcohol administration, the following groups are administered by intraperitoneal injection: alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gaboxadol group (3 mg / kg) and alcohol + high-dose gaboxadol group (10 mg / kg), and the control group and alcohol group are given the same volume of solvent by gavage as a control. Subsequently, except for the control group which continues to be given the same volume of solvent by gavage, the rest of the groups are given alcohol by gavage. At 9 hours after alcohol, the mice are sacrificed and the liver tissue is removed. Then, according to the instructions of the kit, the activities of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase in the liver, as well as the contents of inflammatory factors (including IL-1β and TNF-α) are accurately determined. The data are summarized and column charts are drawn using GraphPad, and the significant differences (t-test) are calculated.

[0214] Experimental results: as shown in Figure 32 compared with the control group, the activity of acetaldehyde dehydrogenase 2 in the liver of the alcohol group decreased significantly, ### indicates P<0.001; compared with the alcohol group, both doses of gaboxadol can significantly increase the activity of ALDH2 in vivo, *** indicates P<0.001; the activity of alcohol dehydrogenase does not change significantly after drinking alcohol, and each group of gaboxadol also has no significant effect on the activity of alcohol dehydrogenase.

[0215] As shown in Figure 33 compared with the control group, the IL-1β and TNF-α of the alcohol group increased, and a single dose of alcohol can cause an increase in inflammatory factors, leading to alcoholic liver injury; compared with the alcohol group, the two doses of gaboxadol can reduce the increase of IL-1β and TNF-α, * indicates P<0.05, and reduce alcohol-induced liver injury.

[0216] Discussion

[0217] Gabexate is a non-peptide proteolytic enzyme inhibitor, usually marketed in the form of its mesylate salt. The preparation is a lyophilized injection with the characteristics of small molecular weight, small side effects, and no immunogenicity, which can inhibit the activity of trypsin, chymotrypsin, fibrinolysin, plasma kallikrein, pancreatic kallikrein, urokinase and thrombin, etc. It is mainly used in the treatment of acute pancreatitis (AP) and disseminated intravascular coagulation (DIC) in clinic. In recent years, it has been found that it can inhibit the inflammatory response after arterial thrombosis, and has an anti-coagulation effect when permeating. So far, there is no record and report about the preventive or therapeutic effect of gabexate on alcoholism and alcoholic liver disease in the prior art.

[0218] Surprisingly, the experimental results of the present application 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), reduce the symptoms of drunkenness, improve the behavior after drunkenness, and reduce the mortality rate of acute alcoholism, and can be used for the treatment of alcoholism; further, gabexate and its metabolites can improve ethanol-induced liver damage, improve the morphology and structure of liver tissue and cells in alcoholism, reduce the markers of liver damage, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), improve the antioxidant capacity of liver tissue and reduce the generation of free radicals, reduce the risk of alcoholic liver disease caused by alcoholism, and thus can be used for the prevention and treatment of alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis and other alcoholic liver diseases.

[0219] Based on this, it can be seen that gabexate and its metabolites as active ingredients can effectively prevent damage to the human body caused by alcohol, and can effectively treat alcoholism and reduce the health risks caused by drinking. In addition, gabexate as a marketed drug has a large amount of clinical application experience, is safe and reliable, and is conducive to the development of new products of the present application, and has very good application prospect.

[0220] The preferred embodiments disclosed above are only used to help explain the present application. The preferred embodiments do not describe all the details, nor limit the application to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of the present application. The embodiments are selected and described in detail in order to better explain the principles and practical applications of the present application, so that those skilled in the art can well understand and utilize the present application. The present application is limited only by the claims and their full scope and equivalents.

Claims

1. Use of gaboxadol or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for the prevention or treatment of alcoholism and / or alcoholic liver disease; wherein, The alcoholic liver disease is selected from the group consisting of alcoholic fatty liver, alcoholic hepatitis, or a combination thereof. The gaboxadol has the following structure: 。 2. Use according to claim 1, characterized in that, The active ingredient in the pharmaceutical composition is gaboxadol or a pharmaceutically acceptable salt thereof.

3. Use according to claim 1, characterized in that, The alcoholism is acute alcoholism or chronic alcoholism.

4. The use according to claim 1, characterized in that, Preventing or treating alcoholism is one or more selected from the group consisting of: (1) increasing the rate of ethanol and / or acetaldehyde metabolism; (2) reducing the rate of intoxication; (3) prolonging the latency of intoxication; (4) shortening the duration of intoxication; (5) improving the state and / or behavior after intoxication; (6) reducing the mortality rate of alcoholism; and / or (7) reducing alcohol-induced liver damage.

5. The use according to claim 1, characterized in that, The alcoholic liver disease is alcoholic fatty liver.

6. The use according to claim 1, characterized in that, Preventing or treating alcoholic liver disease includes one or more selected from the group consisting of: (a) reducing alcohol-induced transaminase elevation; (b) reducing lipid accumulation in hepatocytes or liver tissue; (c) improving the antioxidant function of hepatocytes or liver; (d) improving the anti-inflammatory function of hepatocytes or liver; (e) reducing active aldehyde accumulation in hepatocytes or liver; and / or (f) delaying the progression of alcoholic liver disease.

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

8. Use of gaboxadol or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for preventing or treating alcoholic liver damage, wherein the gaboxadol has the following structure: 。 9. Use according to claim 8, characterized in that, The alcoholic liver damage is acute alcoholic liver damage or chronic alcoholic liver damage.

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