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. Also provided is the use of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of an acetaldehyde dehydrogenase 2 (ALDH2) agonist.

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] Excessive alcohol intake at one time may inhibit the respiratory center of the brain, leading to respiratory arrest and death. Long-term excessive drinking will almost inevitably lead to liver cirrhosis, accompanied by symptoms such as abdominal distension and jaundice, which seriously affects the quality of life. Acute alcohol intoxication (AAI), also known as acute ethanol poisoning, is a common disease in the emergency department and accounts 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, unmetabolized acetaldehyde has a strong toxic effect on cells. Clinically, the recognized drug for alcohol poisoning is mainly naloxone, which mainly helps to wake up 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 alcohol-induced tissue damage. Since 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. At present, the clinical treatment measures for alcoholic liver disease mainly include quitting drinking, nutritional supplements, antioxidant and anti-inflammatory drug treatments, but the effect is not good. So far, there is still a lack of effective treatments. Therefore, alcoholic liver disease is in urgent need of safe and effective therapeutic drugs.

[0004] The liver is the largest metabolic organ in the human body and the main site of alcohol metabolism. About 90% of alcohol enters the liver through the blood after being absorbed by the gastrointestinal tract. It is first oxidized to acetaldehyde by alcohol dehydrogenase (ADH), and then oxidized to acetic acid by acetaldehyde dehydrogenase 2 (ALDH2). Acetic acid is excreted from the body or participates in biosynthesis through further metabolism. ALDH2 is the rate-limiting enzyme in alcohol metabolism, and its substrate acetaldehyde is a strongly electrophilic first-class 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 metabolic rate of ethanol / acetaldehyde, protect liver damage, and are expected to play a preventive and therapeutic role in alcoholic diseases. 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. Therefore, reducing the concentration and time of alcohol and acetaldehyde in the body can significantly reduce the damage caused by alcohol to the body. Summary of the invention

[0005] The present invention aims 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 preventing or treating alcoholism and alcoholic liver disease, and aldehyde dehydrogenase 2 (ALDH2) agonists.

[0006] 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.

[0007]

[0008] In another preferred embodiment, the metabolite is selected from 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 following groups:

[0012] (1) Accelerate the metabolic rate of ethanol and / or acetaldehyde;

[0013] (2) Reduce the rate of drunkenness;

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

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

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

[0017] (6) reduce mortality from alcoholism; and / or

[0018] (7) Reduce alcohol-induced liver damage, such as hepatocyte infiltration and increased inflammatory factors.

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

[0020] 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.

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

[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, the prevention or treatment of alcoholic liver disease comprises one or more methods selected from the following group:

[0024] (a) Reduce the increase in transaminase caused by alcohol;

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

[0026] (c) improving the antioxidant function of hepatocytes or liver;

[0027] (d) improving the anti-inflammatory function of hepatocytes or liver;

[0028] (e) reducing the accumulation of reactive aldehydes in hepatocytes or the liver; and / or

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

[0030] In another preferred embodiment, the prevention or treatment of alcoholic liver disease comprises one or more methods selected from the following group:

[0031] (a) Alleviate acute or chronic hepatocellular and liver damage caused by alcohol;

[0032] (b) reduce alcohol-induced elevations in transaminases; and / or

[0033] (c) Reduce 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 administration preparation.

[0036] 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.

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

[0038] 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.

[0039] 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.

[0040] In a second aspect of the present invention, there is provided a use of gabexate 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 following compounds or pharmaceutically acceptable salts 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, and gabexate and the like can covalently bind to ALDH2 to produce agonistic activity.

[0046] In a third 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.

[0047] 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.

[0048] In another preferred embodiment, the subject carries an ALDH2*2 mutation.

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

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

[0051] 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

[0052] Figure 1 The test results of gabexate promoting ethanol metabolism in hepatocytes

[0053] Figure 2 Results of the experiment on different salt forms of gabexate promoting ethanol metabolism in hepatocytes

[0054] Figure 3 Gabexate administration before alcohol improves ethanol metabolism in mice

[0055] Figure 4 Gabexate administration after alcohol consumption promotes ethanol metabolism in mice

[0056] Figure 5 Gabexate reduces acetaldehyde levels in mice 1 hour after drinking

[0057] Figure 6 Gabexate reduces ethanol content in rats 2 hours after drinking

[0058] Figure 7 Gabexate metabolites and their combination reduce ethanol content at 3 and 6 hours after drinking in mice

[0059] Figure 8 The results of the test on improving the alcohol tolerance time and drunkenness duration of mice by administering gabexate before drinking

[0060] Fig. 9 Gabexate administration before alcohol improves the results of mouse grasping test

[0061] Fig.10The results of the test on the effect of gabexate administration after drinking on improving the alcohol tolerance time and drunkenness duration of mice

[0062] Fig.11 The results of the test on the grip strength and hanging endurance of mice after administration of gabexate

[0063] Fig.12 Gabexate improves the climbing time and climbing score test results of drunken behavior in mice

[0064] Fig.13 Gabexate administration after drinking improves the results of the rotarod time test of drunkenness behavior in mice

[0065] Fig.14 Results of the cell survival test of gabexate in the alcohol-induced hepatocyte injury model

[0066] Fig.15 Results of an experiment to improve the survival rate of acute alcohol poisoning with gabexate

[0067] Fig.16 The test results of gabexate's ability to protect the liver of mice with acute alcoholic liver injury

[0068] Fig.17 Results of pathological section test of gabexate on acute alcoholic liver injury in mice

[0069] Fig.18 The test results of gabexate on liver function indexes (AST, ALT) in mice with acute alcoholic liver injury

[0070] Fig.19 The results of the test on the antioxidant index superoxide dismutase activity of gabexate in mice with acute alcoholic liver injury

[0071] Fig. 20 The test results of gabexate on malondialdehyde content in the liver of mice with acute alcoholic liver injury

[0072] Fig.21 The test results of gabexate on the reduced glutathione content in the liver of mice with acute alcoholic liver injury

[0073] Fig. 22 The test results of gabexate on liver function indexes (AST, ALT) in mice with chronic alcoholic liver injury

[0074] Fig.23 Results of the test of gabexate on serum inflammatory indicators (interleukin-6 and TNF-α) in mice with chronic alcoholic liver injury

[0075] Fig.24 Results of the test of gabexate on serum lipid indexes (triglycerides and cholesterol) in mice with chronic alcoholic liver injury

[0076] Fig.25 Results of the test of gabexate on liver inflammation indicators (interleukin-6 and TNF-α) in mice with chronic alcoholic liver injury

[0077] Fig.26 Results of the test of gabexate on liver lipid indexes (triglycerides and cholesterol) in mice with chronic alcoholic liver injury

[0078] Fig. 27 Pathological sections and Oil Red O staining results of gabexate on chronic alcoholic liver injury in mice

[0079] Fig.28 The activity curve of gabexate stimulating mutant ALDH2*2 and wild-type ALDH2*1

[0080] Fig.29 Binding curves of gabexate with wild-type ALDH2*1 and mutant ALDH2*2

[0081] Fig.30 Mass spectrometry results of covalent binding of gabexate to wild-type ALDH2*1 and mutant ALDH2*2

[0082] Fig.31 The activity curve of gabexate inhibiting serine protease

[0083] Fig.32 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

[0084] Fig.33 Results of the test of Gabexate on inflammatory indicators (interleukin-1β and TNF-α) in mice with acute alcoholic liver injury DETAILED DESCRIPTION

[0085] 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.

[0086] the term

[0087] 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.

[0088] 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".

[0089] Active ingredients

[0090] The active ingredient of the present invention is gabexate or its metabolite.

[0091] The structural formula of gabexate is shown below, and its CAS number is 39492-01-8.

[0092]

[0093] In the present invention, the gabexate metabolite is selected from the group consisting of:

[0094]

[0095] The gabexate or its metabolite of the present invention is intended to include their pharmaceutically acceptable salts.

[0096] 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.

[0097] The preferred salt of ethyl p-hydroxybenzoate or p-hydroxybenzoic acid is a salt 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).

[0098] 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.

[0099] Alcohol metabolism

[0100] When an individual ingests alcohol, alcohol (ethanol) is rapidly absorbed into the bloodstream and then undergoes a metabolic process primarily in the liver. In the liver, ethanol is first converted to acetaldehyde by the enzyme alcohol dehydrogenase (ADH), which is the rate-limiting step in alcohol metabolism. Acetaldehyde is then further metabolized to acetic acid by the enzyme acetaldehyde dehydrogenase (ALDH). Acetic acid is a relatively harmless metabolite that can be completely oxidized in the body and ultimately converted to carbon dioxide and water. These end products are excreted from 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 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.

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

[0103] Aldehyde dehydrogenase

[0104] The enzyme responsible for the conversion of acetaldehyde in the human body is acetaldehyde dehydrogenase in the liver. It has two isoenzymes, one in the cytosol (ALDH1) and the other in the mitochondria (ALDH2). The two have obvious differences in catalytic rate. The K of ALDH2 to acetaldehyde is m It is lower than ALDH1, about 1 / 10 of the latter, and is the isoenzyme mainly responsible for the conversion of acetaldehyde.

[0105] In China, approximately 40% of the population carries a single nucleotide polymorphism of the ALDH2 gene, where 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 slows down the metabolism of acetaldehyde in the body after drinking, causing acetaldehyde to accumulate in the body. This accumulation phenomenon often manifests as characteristic facial flushing, headache, nausea, dizziness, and palpitations after drinking, and may increase an individual's risk of alcoholic liver disease.

[0106] 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.

[0107] Alcoholism

[0108] 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 amount of alcohol consumed at one time and the length of time of drinking, it can be divided into acute alcohol poisoning and chronic alcohol poisoning.

[0109] 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.

[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 may be accompanied by cardiovascular and digestive system symptoms. Alcohol can also interfere with the activity of liver enzymes, increase the burden on the liver, and easily cause the formation of fatty liver and alcoholic cirrhosis. It is also called alcohol dependence or alcohol addiction.

[0111] 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 or behavior after drunkenness; (6) reducing the mortality rate of alcohol poisoning; and / or (7) reducing liver damage caused by alcohol.

[0112] 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.

[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 decomposition of alcohol. Drinking alcohol may cause liver damage, and may even cause alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, alcoholic cirrhosis or 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.

[0115] The words "prevention", "treatment", "improvement", "acceleration", "lowering", "prolongation", "shortening", "reduction" or "mitigation" 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.

[0116] 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.

[0117] 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.

[0118] Composition

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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, at 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.

[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 invention include:

[0133] 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.

[0134] B) The present invention first discovered the use of gabexate and its metabolites in the preparation of aldehyde dehydrogenase 2 (ALDH2) agonists.

[0135] C) 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.

[0136] 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.

[0137] If not otherwise specified, the gabexate used in the examples is gabexate mesylate.

[0138] Example 1 Experiment on the promotion of ethanol metabolism in hepatocytes by gabexate

[0139] 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.

[0140] HepG2 cells were cultured normally for 12 hours, and the positive drug and gabexate mother solution were diluted to the target concentration with culture medium (final concentrations were 10 and 100 μmol / L, respectively). After incubation with cells for 6 hours, alcohol-induced modeling was performed, and culture medium containing a predetermined concentration of ethanol was added to the cells and incubated for another 3 hours. After modeling was completed, the culture supernatant was collected and the concentration of ethanol therein was detected using the Megazyme-Alcohol kit. The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0141] 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, ** indicates P < 0.01.

[0142] 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.).

[0143] HepG2 cells were cultured normally for 12 hours, and the positive drug and the gabexate stock solution of different salt forms were diluted to the target concentration with culture medium (the final concentrations were 10 and 100 μmol / L, respectively). After incubation with the cells for 6 hours, alcohol-induced modeling was performed, and the culture medium containing the predetermined concentration of ethanol was added to the cells and continued to be incubated for 3 hours. After the modeling was completed, the culture supernatant was collected, and the concentration of ethanol therein was detected using the Megazyme-Alcohol kit. The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0144] 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, ** indicates P < 0.01, *** indicates P < 0.001.

[0145] Example 2 Gabexate promotes ethanol metabolism in mice

[0146] 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 group (500 mg / kg), an alcohol + low-dose gabexate group (10 mg / kg) and an alcohol + high-dose gabexate group (30 mg / kg) according to their body weight.

[0147] All groups of mice were fasted for 12 h before the experiment. Thirty minutes before alcohol administration, the alcohol + metadoxine group (500 mg / kg) was administered by oral gavage, while the alcohol + low-dose gabexate group (10 mg / kg) and the alcohol + high-dose gabexate group (30 mg / kg) were administered by intraperitoneal injection. The alcohol group was gavaged with an equal volume of solvent as a control. All groups were given alcohol by gavage. After drinking, blood samples of mice were collected by orbital blood sampling at 1, 3, 6 and 12 hours after drinking. The collected blood was allowed to stand at room temperature and then centrifuged to obtain serum. The ethanol concentration in serum was detected using the Megazyme-Alcohol kit. The data were summarized, the curves were fitted using GraphPad, and the significant differences (t-test) were calculated.

[0148] 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.

[0149] 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 group (60 mg / kg), an alcohol + low-dose gabexate group (1 mg / kg), an alcohol + medium-dose gabexate group (3 mg / kg) and an alcohol + high-dose gabexate group (10 mg / kg) according to their body weight.

[0150] All groups of mice were fasted for 12 hours before the experiment. Subsequently, all groups were given alcohol by gavage. 30 minutes after the administration of alcohol, the following groups were given drugs by intraperitoneal injection: alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gabexate group (1 mg / kg), alcohol + medium-dose gabexate group (3 mg / kg), and alcohol + high-dose gabexate group (10 mg / kg). As a control, the alcohol group was gavaged with an equal volume of solvent. Blood samples of mice were collected at 0.5, 1, 2, 4, 6, 9, 12, 18 and 24 hours after drinking alcohol by orbital blood sampling. The collected blood was allowed to stand at room temperature and then centrifuged to separate and obtain serum samples. The ethanol concentration in serum was detected using the Megazyme-Alcohol kit. The data were summarized, the curves were fitted using GraphPad, and the significant differences (t-test) were calculated. 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.

[0151] Example 3 Gabexate promotes acetaldehyde metabolism in mice

[0152] 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 group (500 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.

[0153] All groups of mice were fasted for 12 h before the experiment. Thirty minutes before the administration of alcohol, the alcohol + metadoxine group (500 mg / kg) was administered by oral gavage, while the alcohol + low-dose gabexate group (1 mg / kg) and the alcohol + high-dose gabexate group (3 mg / kg) were administered by intraperitoneal injection. The alcohol group was gavaged with an equal volume of solvent as a control. Thereafter, all groups were given alcohol by gavage. Blood samples of mice were collected 1 hour after drinking by orbital blood sampling. The collected blood was allowed to stand at room temperature and then centrifuged to obtain serum. The concentration of acetaldehyde in serum was detected using HPLC derivatization. The data were summarized, bar graphs were drawn using GraphPad, and significant differences (t-test) were calculated.

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

[0155] 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.

[0156] Example 4 Gabexate promotes rat ethanol metabolism experiment

[0157] 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 group (30 mg / kg), an alcohol + low-dose gabexate group (5 mg / kg) and an alcohol + high-dose gabexate group (15 mg / kg).

[0158] All rats were fasted for 12 h before the experiment. Thirty minutes before alcohol administration, the following groups were treated by intraperitoneal injection: alcohol + metadoxine group (30 mg / kg), alcohol + low-dose gabexate group (5 mg / kg), and alcohol + high-dose gabexate group (15 mg / kg). The alcohol group was gavaged with an equal volume of vehicle as a control. Thereafter, all groups were given alcohol by gavage. Blood samples of rats 2 h after drinking were collected by tail cutting. The collected blood was centrifuged after standing at room temperature to separate and obtain serum samples. The ethanol concentration in serum was detected using the Megazyme-Alcohol kit. The data were summarized, and a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0159] 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.

[0160] Example 5 Experiment on the promotion of ethanol metabolism in mice by gabexate metabolites

[0161] 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 evenly divided according to body weight into an alcohol group (alcohol dose 7 g / kg), an alcohol + metadoxine group (60 mg / kg), an alcohol + gabexate group (10 mg / kg), an alcohol + guanidinohexanoic acid group (5.02 mg / kg), an alcohol + ethyl paraben group (3.98 mg / kg), and an alcohol + combination group (guanidinohexanoic acid, 5.02 mg / kg; ethyl paraben, 3.98 mg / kg).

[0162] All groups of mice were fasted for 12 h before the experiment. Thirty minutes before alcohol administration, the following groups were treated by intraperitoneal injection: alcohol + metadoxine group (60 mg / kg), alcohol + gabexate group (10 mg / kg), alcohol + guanidocanoic acid group (5.02 mg / kg), alcohol + ethyl paraben group (3.98 mg / kg), and alcohol + combination group (guanidocanoic acid, 5.02 mg / kg; ethyl paraben, 3.98 mg / kg). The alcohol group was gavaged with an equal volume of solvent as a control. Thereafter, all groups were given alcohol by gavage. Blood samples of mice were collected 3 h and 6 h after drinking by removing the eyeballs. The collected blood was centrifuged after standing at room temperature to separate and obtain serum samples. The ethanol concentration in serum was detected using the Megazyme-Alcohol kit. The data were summarized, and a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated.

[0163] 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.

[0164] Example 6 Gabexate improves the behavior of mice drunkenness

[0165] 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 group (60 mg / kg), an alcohol + low-dose gabexate group (10 mg / kg), and an alcohol + high-dose gabexate group (30 mg / kg) according to their body weight.

[0166] All groups of mice were fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, the following groups were treated by intraperitoneal injection: alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gabexate group (10 mg / kg) and alcohol + high-dose gabexate group (30 mg / kg). The control group and alcohol group were gavaged with an equal volume of solvent as a control. Subsequently, except for the control group, which continued to be gavaged with the same volume of solvent, the remaining groups were gavaged with alcohol. The disappearance of the righting reflex and the recovery time of the mice after drinking were recorded, and the gripping force of the mice 9 hours after drinking was tested by a gripping force detector, and the drunkenness rate, drunkenness 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.

[0167] Experimental results: Figure 8 , 9As shown, compared with the alcohol group (drunkenness rate 100%), the alcohol + low-dose gabexate group (10 mg / kg) 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.

[0168] 6.2 To investigate whether gabexate administration after alcohol administration has the effect of promoting awakening and accelerating physical recovery in drunken 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 group (60 mg / kg), an alcohol + low-dose gabexate group (1 mg / kg), an alcohol + medium-dose gabexate group (3 mg / kg) and an alcohol + high-dose gabexate group (10 mg / kg) according to their body weight.

[0169] 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 vehicle, the other groups were gavaged with alcohol. 30 minutes after the administration of alcohol, the following groups were treated with drugs by intraperitoneal injection: alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gabexate group (1 mg / kg), alcohol + medium-dose gabexate group (3 mg / kg) and alcohol + high-dose gabexate group (10 mg / kg). As a control, the control group and the alcohol group were gavaged with the same volume of vehicle. The disappearance of the righting reflex and the recovery time of the mice after drinking were recorded, and the grasping force of the mice 9 hours after drinking was tested by a grasping force detector and the hanging endurance of the mice 9 hours after drinking was tested by a hanging device, and the intoxication latency and sobering time were calculated. The data were summarized, a bar graph was drawn using GraphPad, and the significant difference (t-test) was calculated. Experimental results: Fig.10 , 11 As shown, compared with the alcohol group, the alcohol + low-dose gabexate group (1g / kg) and the alcohol + high-dose gabexate group (10g / kg) 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.

[0170] 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 group (500 mg / kg), an alcohol + low-dose gabexate group (10 mg / kg) and an alcohol + high-dose gabexate group (30 mg / kg) according to their body weight.

[0171] 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 the administration of alcohol, the alcohol + metadoxine group (500 mg / kg) was administered by oral gavage, while the alcohol + low-dose gabexate group (10 mg / kg) and the alcohol + high-dose gabexate group (30 mg / kg) were administered by intraperitoneal injection. The control group and the alcohol group were gavaged with an equal volume of solvent as a control. Subsequently, except for the control group, which continued to be gavaged with an equal volume of solvent, the remaining 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's movement status (Table 1). The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0172] Table 1. Scoring criteria for movement status in pole climbing experiment

[0173]

[0174] 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.

[0175] 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 group (60 mg / kg), and an alcohol + gabexate group (100 mg / kg) according to their body weight.

[0176] Before the formal experiment, all groups of mice were trained in the rotarod for three days. All groups of mice were fasted for 12 hours before the experiment. 30 minutes before the administration of alcohol, the following groups were treated by intraperitoneal injection: alcohol + Alda-1 group (60 mg / kg) and alcohol + Gabexate group (100 mg / kg). The control group and alcohol group were gavaged with an equal volume of solvent as a control. Subsequently, except for the control group, which continued to be gavaged with the same volume of solvent, the remaining groups were gavaged with alcohol. The mouse rotarod behavioral 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.

[0177] Experimental results: Fig.13 As 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 group (100 mg / kg) 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.

[0178] Example 7 Gabexate improves ethanol-induced hepatocyte injury experiment

[0179] 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.

[0180] HepG2 cells were cultured normally for 12 hours, and the positive drug and gabexate mother solution were diluted to the target concentration (final concentration ranged from 1 to 10 μmol / L) with culture medium. After incubation with cells for 6 hours, alcohol-induced modeling was performed, and culture medium containing a predetermined concentration of ethanol was added to the cells, and 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, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0181] 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.

[0182] Example 8 Gabexate improves survival rate in acute alcohol poisoning

[0183] 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 group (15 mg / kg), an alcohol + three doses of metadoxine group (20 mg / kg) and an alcohol + three doses of gabexate group (10 mg / kg) according to their body weight.

[0184] Each group of mice fasted for 12 hours before the experiment. The dosing time points for the two-dose groups were before and 30 minutes after alcohol administration. For the three-dose group, in addition to dosing before and 30 minutes after alcohol administration, an additional dose was given 1 hour after alcohol administration. The control group and the alcohol group were gavaged with the same volume of solvent at the corresponding time points as a control. At the same time, except for the control group that continued to be gavaged with the same volume of solvent, the other groups were gavaged with alcohol. Observe and record the death of mice. Summarize the data and draw a bar graph using GraphPad.

[0185] Experimental results: Fig.15 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.

[0186] Example 9 Experiment on reducing liver damage in mice with acute alcohol poisoning with gabexate

[0187] 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 group (60 mg / kg), an alcohol + low-dose gabexate group (3 mg / kg) and an alcohol + high-dose gabexate group (10 mg / kg) according to their body weight.

[0188] All groups of mice were fasted for 12 hours before the experiment. Thirty minutes before alcohol administration, the following groups were treated by intraperitoneal injection: alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gabexate group (3 mg / kg), and alcohol + high-dose gabexate group (10 mg / kg). The control group and alcohol group were gavaged with an equal volume of solvent as a control. Subsequently, except for the control group, which continued to be gavaged with the same volume of solvent, the remaining groups were gavaged with alcohol. Nine hours after alcohol gavage, the mice were killed and dissected to assess the health of the liver. The specific steps included photographing the appearance of the mouse liver, weighing the liver, and calculating the liver-body ratio. H&E staining was also performed to observe histological changes. By removing the eyeballs and taking blood, the changes in the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum and the changes in the level of superoxide dismutase (SOD) in the plasma were tested 9 hours after alcohol administration. The contents of reduced glutathione (GSH) and malondialdehyde (MDA) in liver tissue samples 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).

[0189] Experimental results: Fig.16 , 17 As shown in Figures 18, 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.

[0190] like Fig.19 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.

[0191] like Fig. 20As 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.

[0192] like Fig.21 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.

[0193] Example 10 Experiment on reducing chronic alcoholic liver disease in mice with gabexate

[0194] The protective effect of gabexate on the liver of mice after long-term intake of LDC liquid feed containing alcohol was investigated. The experimental scheme was as follows: 8-week-old ICR male mice were evenly divided into a control group (taking liquid feed without alcohol with the same calories), 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] During the first five days of the experiment, the concentration of alcohol in the liquid feed was gradually increased until it reached a liquid feed containing 4% alcohol. Starting from the sixth day, mice in the alcohol + low-dose gabexate group (1 mg / kg) and the high-alcohol + dose gabexate group (3 mg / kg) were administered daily by intraperitoneal injection. At the same time, mice in the alcohol + metadoxine group (250 mg / kg) were administered by gavage. As a control, mice in the alcohol group and the control group were gavaged with an equal volume of solvent. After 37 days of continuous feeding, on the morning of the 38th day, the liquid feed containing alcohol was removed and the mice were gavaged with alcohol. After 9 hours, the mice were killed and dissected to assess their liver health. The specific steps 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 evaluated by Elisa method. The data were summarized, bar graphs were drawn using GraphPad, and significant differences (t-test) were calculated. Experimental results: As shown in Fig. 22 , 23As shown in Figures 24, 25, and 26, compared with the control group, the serum transaminase level, serum and liver inflammatory factors and lipid content in the alcohol group were significantly increased, ### indicates P < 0.001, indicating that acute alcohol poisoning has obvious damage to the liver, causing mice to have two characteristics of alcoholic liver disease, alcoholic fatty liver and alcoholic hepatitis; compared with the alcohol group, both doses of gabexate can significantly improve the serum transaminase level, serum and liver inflammatory factors and lipid increases, *** indicates P < 0.001, indicating that gabexate can protect the liver function of mice with chronic alcoholic liver damage.

[0196] like Fig. 27 As shown in the results, compared with the control group, the alcohol group showed fat vacuoles of varying sizes in the cytoplasm of hepatocytes in HE and Oil Red O staining, with obvious characteristics of fatty fatty degeneration of hepatocytes; compared with the alcohol group, both doses of gabexate significantly reduced the size of fat vacuoles in hepatocytes. This indicates that gabexate can protect the hepatocyte morphology of mice with chronic alcoholic liver damage and reduce liver damage caused by alcohol.

[0197] Example 11 ALDH2 enzyme activity test

[0198] The effect of gabexate on ALDH2 activity was investigated. The experimental method was as follows: gabexate was dissolved in 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 a 96-well plate to achieve different final concentrations from 0 to 400 μmol / L. The above samples were incubated at 37°C for 5 minutes. After the incubation, 40 mmol / L acetaldehyde solution was added to make the total sample volume reach 200 μL. The 96-well plate was quickly placed in an ELISA reader for time kinetic scanning and the absorbance readings at a wavelength of 340 nm were recorded.

[0199] 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.

[0200] Experimental results: Fig.28 As shown in the figure, gabexate has a certain stimulatory activity of ALDH2*2, with the maximum stimulatory multiple being about 2 and the half effective concentration (EC 50 ) value was 35.39μmol / L, and the maximum agonist factor 2 of positive drug Alda-1 was 6.37, and the half effective concentration (EC50 ) value was 1.532 μmol / L; Gabexate had significant ALDH2*1 stimulating activity, with the maximum stimulating multiple being >1.9 and the half effective concentration (EC 50 ) value was 34.06μmol / L, positive drug Alda-1, the maximum agonist multiple was 2, and the half effective concentration (EC 50 ) value is 0.2862μmol / L.

[0201] Example 12 Gabexate and ALDH2 Microthermophoresis Experiment

[0202] The binding ability of gabexate to ALDH2 was investigated. 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 a concentration of 100 nM using PBS-T buffer, and the protein was diluted to 800 nM. The protein and dye were mixed evenly in a 1:1 ratio and incubated at room temperature for 30 minutes, after which the supernatant was separated by centrifugation. The gabexate stock solution was diluted in a gradient manner, and 10 μL was added to PCR tubes (ranging from 0.5 to 1000 μmol / L). 10 μL of labeled protein was added to each PCR tube, mixed thoroughly, and continued to incubate at room temperature for 2 hours. The sample was aspirated using a capillary for detection, and the data was recorded and analyzed using a fluorescence scanner.

[0203] Experimental results: Fig.29 As shown in Figure 2, gabexate has a strong binding affinity with ALDH2. The binding constant (K d ) was 10.53 μmol / L, and the binding constant (K d ) is 8.55μmol / L.

[0204] Example 13 Gabexate covalently binds to ALDH2

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

[0206] Experimental results: Fig.30 As shown, by screening modifications with target mass changes, gabexate shared 12 potential binding sites with ALDH2*1 and ALDH2*2, respectively, among which the S338 site had the most spectral matches (PSM), and the response was enhanced with increasing compound concentration.

[0207] Example 14 Serine protease activity test

[0208] The effect of gabexate on the activity of serine proteases was investigated. The experimental method was as follows: gabexate was dissolved in DMSO to prepare a 10 mmol / L stock solution. N-phenylacyl-L-arginine ethyl ester (1 mmol / L) and hydrochloric acid (1 mmol / L) were pre-added to a 96-well quartz plate and mixed thoroughly. The gabexate stock solution was diluted in a gradient manner and added to the 96-well quartz plate (final concentration varied from 0 to 25 μmol / L). Serine proteases (such as trypsin or thrombin) were quickly added and mixed again to a total sample volume of 200 μL. Subsequently, the 96-well quartz plate was quickly placed in a microplate reader for time-dynamic scanning and the absorbance changes at a wavelength of 253 nm were recorded.

[0209] Experimental results: Fig.31 As shown in Figure 2, gabexate can inhibit the activity of serine proteases, and the half inhibitory concentration (IC 50 ) value was 4.082 μmol / L, and the half inhibitory concentration (IC 50 ) value was 4.054μmol / L.

[0210] 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.

[0211] Example 15 Experiment on reducing liver damage in mice with acute alcohol poisoning with gabexate

[0212] The mechanism of gabexate in promoting ethanol metabolism and protecting liver damage at the animal level was investigated under high-dose alcohol conditions. The experimental method 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 group (60 mg / kg), an alcohol + low-dose gabexate group (3 mg / kg) and an alcohol + high-dose gabexate group (10 mg / kg) according to their body weight.

[0213] All groups of mice were fasted for 12 hours before the experiment. Thirty minutes before the administration of alcohol, the following groups were treated by intraperitoneal injection: alcohol + metadoxine group (60 mg / kg), alcohol + low-dose gabexate group (3 mg / kg) and alcohol + high-dose gabexate group (10 mg / kg). The control group and alcohol group were gavaged with an equal volume of solvent as a control. Subsequently, except for the control group, which continued to be gavaged with the same volume of solvent, the remaining groups were gavaged with alcohol. Nine hours after drinking, the mice were killed and the liver tissue was removed. Subsequently, according to the instructions of the kit, the activity of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase in the liver, as well as the content of inflammatory factors (including IL-1β and TNF-α) were accurately determined. The data were summarized, a bar graph was drawn using GraphPad, and significant differences (t-test) were calculated.

[0214] Experimental results: Fig.32 As 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.

[0215] like Fig.33 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.

[0216] discuss

[0217] Gabexate is a non-peptide proteolytic enzyme inhibitor, which is usually marketed in the form of its mesylate. The preparation is a lyophilized injection, which has the characteristics of small molecular weight, small side effects, and no immunogenicity. It can inhibit the activities of trypsin, chymotrypsin, fibrinase, plasma kallikrein, pancreatic kallikrein, urokinase and thrombin, and is mainly used to treat acute pancreatitis (AP) and disseminated intravascular coagulation (DIC) clinically. 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.

[0218] 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.

[0219] 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.

[0220] 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 its pharmaceutically acceptable salt, or its metabolite in the preparation of a pharmaceutical composition for preventing or treating alcoholism and / or alcoholic liver disease 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 The alcohol poisoning is acute alcohol poisoning or chronic alcohol poisoning.

4. 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 / or behavior after intoxication, such as shortening the time to recover grip and accelerating the recovery of limb coordination; (6) reduce mortality from alcoholism; and / or (7) Reduce alcohol-induced liver damage, such as hepatocyte infiltration and increased inflammatory factors.

5. 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.

6. 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) Reduce the increase in transaminase caused by alcohol; (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 the accumulation of reactive aldehydes in hepatocytes or the liver; and / or (f) Delay the progression of alcoholic liver disease.

7. The use according to claim 1, characterized in that The metabolite is selected from the following compounds or pharmaceutically acceptable salts thereof:

8. 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.

9. Use of gabexate or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the preparation of an acetaldehyde dehydrogenase 2 (ALDH2) agonist 10. The use according to claim 9, characterized in that The aldehyde dehydrogenase 2 (ALDH2) agonist is an ALDH2 covalent agonist, which can generate agonistic activity through covalent binding.

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