WO2025256111A1 - Utilisation de lactate d'éthyle pour traiter une lésion hépatique et une maladie hépatique associée à l'alcool - Google Patents

Utilisation de lactate d'éthyle pour traiter une lésion hépatique et une maladie hépatique associée à l'alcool

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Publication number
WO2025256111A1
WO2025256111A1 PCT/CN2024/143404 CN2024143404W WO2025256111A1 WO 2025256111 A1 WO2025256111 A1 WO 2025256111A1 CN 2024143404 W CN2024143404 W CN 2024143404W WO 2025256111 A1 WO2025256111 A1 WO 2025256111A1
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Prior art keywords
alcohol
ethyl lactate
liver
liver disease
alcoholic
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PCT/CN2024/143404
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English (en)
Chinese (zh)
Inventor
李于
张翠英
肖冬光
蒋洋
崔奥媛
赵久香
林良才
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Tianjin University of Science and Technology
Shanghai Institute of Nutrition and Health of CAS
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Tianjin University of Science and Technology
Shanghai Institute of Nutrition and Health of CAS
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Publication of WO2025256111A1 publication Critical patent/WO2025256111A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/04—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • This invention relates to the field of biomedicine, specifically to the application of ethyl lactate in liver injury and alcohol-related liver disease.
  • alcohol-related liver diseases More than 90% of long-term chronic drinkers will develop alcohol-related fatty liver disease, which, with long-term chronic drinking or occasional binge drinking, can gradually progress to alcohol-related steatohepatitis, fibrosis, and cirrhosis. Meanwhile, alcohol-associated liver disease (ALD) is a leading cause of morbidity and mortality from chronic liver disease. To date, there are no drugs approved by the U.S. Food and Drug Administration for the treatment of ALD.
  • the purpose of this invention is to provide a medicament or pharmaceutical composition for improving alcoholic liver damage or treating ALD.
  • ethyl lactate or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical remedy or pharmaceutical composition, said pharmaceutical remedy or pharmaceutical composition for one or more uses selected from the group consisting of:
  • the alcohol-related liver disease includes chronic alcohol-related liver disease, acute alcohol-related liver disease, or a combination of chronic-onset and acute alcohol-related liver disease.
  • the alcohol-related liver disease is selected from the group consisting of: liver injury, hepatic steatosis, alcoholic steatohepatitis, hepatic oxidative stress, alcoholic fatty liver, or combinations thereof.
  • the FGF21 agonist increases the activity of FGF21 in hepatocytes.
  • the SIRT1 agonist increases the activity of SIRT1 in hepatocytes.
  • the drug or pharmaceutical composition is also used for purposes selected from the group consisting of:
  • the alcohol-related liver disease inflammatory factors are selected from the group consisting of IL-1 ⁇ , IL-6, MCP1, ICAM1, CD11b, or combinations thereof.
  • the lipid synthesis-related factors are selected from the group consisting of SREBP-1c, ACC1, FAS, SCD1, DGAT1, ASCL4, or combinations thereof.
  • the drug or drug composition is applied to a subject selected from the group consisting of mammals or rodents.
  • the object has one or more features selected from the group consisting of:
  • the high expression means that the relative expression level ( Z1 ) of the mRNA of the alcohol-related liver disease inflammatory factor or lipid synthesis-related factor is ⁇ 1.2, preferably ⁇ 1.5 , and more preferably ⁇ 2.0 , compared with the baseline value ( Z0 ).
  • the fatty acid synthase content is high when the ratio (C1 / C0 ) of the fatty acid synthase content ( C1 ) to the reference value ( C0 ) is ⁇ 1.2, preferably ⁇ 1.5, and more preferably ⁇ 2.0.
  • the benchmark value refers to the relative mRNA expression level of inflammatory factors or lipid synthesis-related factors of alcohol-related liver disease in healthy individuals, or the content of fatty acid synthase.
  • the drug or drug composition may be used alone or in combination in the prevention and treatment of liver injury and alcohol-related liver disease.
  • the combined use includes: combined use with other drugs for the prevention and treatment of liver injury and alcohol-related liver disease.
  • the pharmaceutical composition further contains additional medications for treating liver injury and alcohol-related liver disease.
  • the additional medication for treating liver injury and alcohol-related liver disease is selected from the group consisting of: phosphatidylcholine, glycyrrhizic acid, glutathione, N-acetylcysteine, thioproline, silymarin, bicyclol, ursodeoxycholic acid, S-adenosylmethionine, cholestyramine, coenzyme A, coenzyme Q10, water-soluble vitamins (such as vitamin C, B complex vitamins), inosine, ornithine aspartate, alprostadil, or combinations thereof.
  • phosphatidylcholine glycyrrhizic acid
  • glutathione N-acetylcysteine
  • thioproline silymarin
  • bicyclol ursodeoxycholic acid
  • S-adenosylmethionine S-adenosylmethionine
  • cholestyramine coenzyme A
  • the pharmaceutical composition is a solid or liquid formulation.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of oral preparations, injectable preparations, enteric sustained-release preparations, and lyophilized preparations.
  • the carrier of the injectable formulation is selected from the group consisting of physiological saline, glucose, stabilizers, preservatives, suspending agents, emulsifiers, or combinations thereof.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of tablets, capsules, granules, powders, ointments, powders, injections, and aqueous solutions.
  • the dosage form of the pharmaceutical composition is an oral preparation, preferably a tablet, capsule, or granule.
  • the route of administration of the pharmaceutical composition is selected from the group consisting of subcutaneous, intravenous, and rectal administration.
  • the pharmaceutical composition comprises: (i) ethyl lactate or a pharmaceutically acceptable salt thereof as an active ingredient; and (ii) a pharmaceutically acceptable carrier.
  • the drug or drug composition improves liver damage and alcohol-related liver disease by inhibiting hepatic lipid synthesis.
  • the drug or drug composition improves liver damage and alcohol-related liver disease by inhibiting fatty acid synthase.
  • the present invention provides an alcoholic beverage product for preventing and treating liver damage and alcohol-related liver disease and/or improving hangovers, the alcoholic beverage product comprising ethyl lactate.
  • the content of ethyl lactate in the alcoholic beverage is 0.01–15 g/L, more preferably 0.1–10 g/L, and even more preferably 0.2–5 g/L.
  • the present invention provides a method for promoting SIRT1 expression in hepatocytes, comprising the steps of:
  • the method is in vitro.
  • the method is non-diagnostic and non-therapeutic.
  • the concentration of ethyl lactate is 0.01–15 g/L, more preferably 0.1–10 g/L, and even more preferably 0.2–5 g/L.
  • the liver cells are derived from mammals.
  • the hepatocytes are derived from human or non-human mammals.
  • the non-human mammals include rodents (such as rats and mice) and primates (such as monkeys).
  • the present invention provides a method for promoting FGF21 expression in hepatocytes, comprising the steps of:
  • the method is in vitro.
  • the method is non-diagnostic and non-therapeutic.
  • the concentration of ethyl lactate is 0.01–15 g/L, more preferably 0.1–10 g/L, and even more preferably 0.2–5 g/L.
  • the liver cells are derived from mammals.
  • the hepatocytes are derived from human or non-human mammals.
  • the non-human mammals include rodents (such as rats and mice) and primates (such as monkeys).
  • the present invention provides a method for inhibiting lipid synthesis in hepatocytes, comprising the steps of:
  • Ethyl lactate or its pharmaceutically acceptable salts are brought into contact with hepatocytes, thereby inhibiting lipid synthesis in hepatocytes.
  • ethyl lactate or a pharmaceutically acceptable salt thereof inhibits lipid synthesis by inhibiting fatty acid synthase.
  • the method is in vitro.
  • the method is non-diagnostic and non-therapeutic.
  • the concentration of ethyl lactate is 0.01–15 g/L, more preferably 0.1–10 g/L, and even more preferably 0.2–5 g/L.
  • the hepatocytes are primary hepatocytes.
  • the liver cells are derived from mammals.
  • the hepatocytes are derived from human or non-human mammals.
  • the non-human mammals include rodents (such as rats and mice) and primates (such as monkeys).
  • a sixth aspect of the present invention provides a method for preventing and treating liver damage and/or alcohol-related liver disease, comprising the steps of:
  • the alcohol-related liver disease includes chronic alcohol-related liver disease, acute alcohol-related liver disease, or a combination of chronic-onset and acute alcohol-related liver disease.
  • the alcohol-related liver disease is selected from the group consisting of: liver injury, hepatic steatosis, alcoholic steatohepatitis, hepatic oxidative stress, alcoholic fatty liver, or combinations thereof.
  • Figure 1 shows the dose-dependent improvement of liver injury and alcohol-related liver disease by ethyl lactate.
  • A Representative images of mouse liver H&E and Oil Red O staining.
  • B Mouse plasma ALT and AST activity levels.
  • C Mouse plasma triglyceride and total cholesterol levels.
  • D Representative images of mouse liver 4-HNE and MDA immunohistochemical staining.
  • E Representative images of mouse liver MPO and F4/80 immunohistochemical staining.
  • F Expression levels of inflammation-related genes in mouse liver. Data are expressed as mean ⁇ SEM. * indicates p ⁇ 0.05 compared to the EtOH+PBS group.
  • Figure 2 shows the direct effect of ethyl lactate on hepatocytes to inhibit alcohol-induced lipid synthesis.
  • A Representative images of FAS and GLUL immunofluorescence staining of mouse liver sections; the locations of the central and portal veins are marked.
  • B Expression levels of lipid synthesis genes in mouse liver. * indicates p ⁇ 0.05 compared to the EtOH+PBS group.
  • C Representative images of BODIPY staining in primary mouse liver cells.
  • Figure 3 shows that ethyl lactate increases the expression of SIRT1 and FGF21 in hepatocytes.
  • A FGF21 expression level in mouse liver and plasma FGF21 content.
  • B SIRT1 expression level in mouse liver. * indicates p ⁇ 0.05 compared to the EtOH+PBS group.
  • D Correlation analysis of SIRT1 expression level in mouse liver with FGF21 expression level, plasma FGF21 content, and plasma triglyceride content.
  • E FGF21 gene expression.
  • SIRT1 gene expression * indicates p ⁇ 0.05 compared to the control group, # indicates p ⁇ 0.05 compared to 100 mM EtOH.
  • G SIRT1 protein level.
  • Figure 4 shows that under starvation conditions, ethyl lactate induces FGF21 in the liver to inhibit lipid degeneration.
  • Figure 5 shows that ethyl lactate improves acute alcohol gavage-induced hangovers in mice.
  • Figure 6 shows that lactate does not affect alcohol-induced hepatic steatosis.
  • ethyl lactate can effectively improve liver damage and/or alcohol-related liver disease.
  • Animal experiments showed that ethyl lactate dose-dependently improved alcohol-induced hepatic steatosis, damage, oxidative stress, and inflammatory responses.
  • ethyl lactate regulates hepatic metabolism and improves alcoholic steatohepatitis by directly inhibiting alcohol-induced lipid synthesis through direct interaction with hepatocytes and by increasing SIRT1 activity and promoting the expression and secretion of hepatic FGF21. Based on these findings, this invention was completed.
  • the term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
  • any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).
  • prevention and treatment and “treatment and/or prevention” are used interchangeably and are generally used to describe a comprehensive strategy for dealing with a disease or health problem, including both preventative and therapeutic measures.
  • active ingredient of the present invention and “ethyl lactate of the present invention” are used interchangeably to refer to an active ingredient capable of preventing and treating liver damage and/or alcohol-related liver disease.
  • Ethyl lactate ( C5H10O3 ) is a natural ester flavor compound, mainly synthesized from lactic acid and ethanol under the catalysis of microbial esterification enzymes. It is typically found in higher concentrations (0.5-3 g/L) in solid-state fermented baijiu ( Chinese white liquor). In baijiu, it enhances its richness, body, flavor profile, and prolongs the aftertaste. Due to its flavor characteristics, as well as its non-toxicity, good solubility, low volatility, and biodegradability, ethyl lactate is also added to alcoholic beverages, chewing gum, baked goods, and frozen drinks. However, there are currently no reports on whether ethyl lactate possesses in vivo biological activity or regulates biological processes.
  • Fibroblast growth factor 21 is a stress-induced hormone that plays a crucial role in regulating energy homeostasis and glucose-lipid homeostasis via a heterodimeric receptor complex composed of FGF receptor 1 (FGFR1) and ⁇ -klotho.
  • FGF21 FGF receptor 1
  • Numerous long-acting FGF21 analogs and agonistic monoclonal antibodies targeting the FGFR1- ⁇ -klotho receptor complex have been developed and entered clinical trials. In these trials, substantial improvements have been observed in serum markers of dyslipidemia, hepatic steatosis, and liver fibrosis in patients with non-alcoholic steatohepatitis (NASH). Therefore, drugs targeting hepatic FGF21 hold significant potential for application in metabolic-related diseases.
  • NASH non-alcoholic steatohepatitis
  • SIRT1 Silent Information Regulator 1
  • SIRT1 is a widely expressed protein that plays a complex role in the pathology, progression, and treatment of various diseases.
  • SIRT1 is an NAD + -dependent deacetylase that regulates gene expression through histone deacetylation.
  • SIRT1 has been reported to play a regulatory role in various diseases, such as aging, obesity, and fatty liver. Under fasting conditions, SIRT1 also mediates the expression and secretion of hepatic FGF21.
  • Fatty acid biosynthesis affects a variety of cellular functions, and its dysfunction is associated with diseases such as cancer, obesity, and non-alcoholic fatty liver disease.
  • Cellular fatty acid biosynthesis is carried out by fatty acid synthases (FAS). These enzymes catalyze the synthesis of long-chain fatty acids from acetyl-CoA and malonyl-CoA.
  • FAS activity is regulated by various factors, including nutritional status, hormone levels, and gene expression. Abnormalities in FAS activity levels are closely related to the occurrence and development of many diseases.
  • Intoxication refers to symptoms such as ataxia and coma that occur shortly after drinking alcohol. It is positively correlated with blood ethanol concentration and decreases as ethanol is metabolized and cleared.
  • a hangover is a prolonged physical and psychological discomfort that occurs when ethanol is completely metabolized and eliminated from the body, such as fatigue, difficulty concentrating, and nausea.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a safe and effective amount of the ethyl lactate of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient.
  • the ethyl lactate of the present invention or a pharmaceutically acceptable salt thereof may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
  • compositions may also contain pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent. This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerin, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.
  • therapeutic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions prior to injection, using liquid carriers.
  • compositions of the present invention can be administered via conventional routes, including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or local administration.
  • routes including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or local administration.
  • the subjects of prevention or treatment may be animals; particularly humans.
  • compositions of the present invention are used for actual treatment, various dosage forms of the pharmaceutical compositions may be used depending on the application.
  • Preferred is an intravenous formulation.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally by adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in the conventional manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in the conventional manner depending on the dosage form.
  • the preparation of eye drops can be carried out as follows: the ethyl lactate of the present invention or its pharmaceutically acceptable salt is dissolved together with the base substance in sterile water (in which a surfactant is dissolved), the osmotic pressure and pH are adjusted to physiological state, and suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and thickeners may be added as appropriate, and then the solution is completely dissolved.
  • compositions of the present invention can also be administered in a sustained-release form.
  • ethyl lactate or a pharmaceutically acceptable salt thereof of the present invention can be incorporated into a pill or microcapsule carried by a sustained-release polymer, and then the pill or microcapsule can be surgically implanted into the tissue to be treated.
  • sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc., and preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the dosage of the ethyl lactate of the present invention or its pharmaceutically acceptable salt as the active ingredient can be reasonably determined according to the weight, age, sex, and symptom severity of each patient to be treated.
  • Ethyl lactate has a dose-dependent effect on improving liver injury and/or alcohol-related liver disease, and has great potential for drug development.
  • Ethyl lactate as a food-derived small molecule with metabolic regulation function, can be applied to the food field to regulate the body's metabolic homeostasis.
  • Ethyl lactate can be used as an effective FGF21 inducer.
  • mice were induced using a slow-on-acute alcohol feeding method. Specifically, two male or female mice aged 12-14 weeks were per cage. They were first acclimatized to a liquid alcohol diet for 5 days, with the alcohol volume content gradually increased from 0% to 4%, followed by 10 days of a 5% volume alcohol diet. On day 16 at 9:00 AM, the mice were administered 5 g/kg alcohol via gavage. Nine hours after gavage, the mice were sacrificed, and blood and liver samples were rapidly collected. Control mice were fed isocaloric maltodextrin instead of ethanol. During the alcohol feeding and gavage, ethyl lactate was added to the alcohol (1 g, 3 g, 10 g/L of ethyl lactate in 52 vol% alcohol), while control mice received PBS.
  • mice liver 20-50 mg was placed in a 1.5 ml centrifuge tube containing 1 ml of Trizol and two steel balls. The tube was then homogenized in a pre-chilled homogenizer at 60 Hz for 120 seconds to ensure complete tissue lysis. The lysed tissue was centrifuged at 12000 rpm for 15 minutes at 4°C. 800 ⁇ l of the supernatant was transferred to a new 1.5 ml centrifuge tube. 200 ml of chloroform was added, and the tube was vigorously inverted for 15 seconds. The tube was then incubated at room temperature for 3 minutes and centrifuged at 9000 rpm for 15 minutes at 4°C.
  • RNA Discard the isopropanol in the centrifuge tube, add 1 ml of 70% ethanol (prepared with DEPC water), and centrifuge at 8000 rpm for 10 min at 4°C to wash the RNA. Discard the ethanol in the centrifuge tube, and let it stand at room temperature for a while to allow the ethanol to evaporate as much as possible. After the ethanol has evaporated, add 100-200 ⁇ l of DEPC water to dissolve the RNA. Analyze the concentration of the fully dissolved RNA.
  • RNA concentrations from the samples were diluted to 12 ⁇ l of 3000 ng total volume with DEPC water.
  • the previously extracted RNA was then reverse transcribed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit.
  • mice Normally fed C57BL/6 mice were anesthetized with isoflurane, and a 24G indwelling intravenous catheter was inserted into their portal vein.
  • the mouse livers were perfused with 50 ml of PBS (calcium and magnesium-free) containing 0.5 mM EDTA.
  • the livers were then perfused with collagenase buffer (66.7 mM NaCl, 6.7 mM KCl, 6.3 mM CaCl2, 0.05% collagenase 2 , 0.226 mM BSA, 100 mM HEPES, pH 7.4) for 3-5 min to digest the livers.
  • collagenase buffer (66.7 mM NaCl, 6.7 mM KCl, 6.3 mM CaCl2, 0.05% collagenase 2 , 0.226 mM BSA, 100 mM HEPES, pH 7.4
  • livers were transferred to 10 cm culture dishes to release hepatocytes, followed by filtration through a 40 ⁇ m filter.
  • the hepatocytes were washed with PBS and centrifuged at 500 rpm for 5 min at 4 °C. After centrifugation, the cells were resuspended in low-glucose medium containing 10% FBS. Cells were seeded into 6-well plates according to experimental requirements and cultured in a 5% CO2 incubator at 37 °C.
  • Freshly isolated primary mouse liver cells were evenly seeded in 6-well plates with coverslips at the bottom and cultured overnight in low-glucose medium. Serum-free low-glucose medium containing 100 mM ethanol was then prepared for ethanol treatment, along with ethyl lactate treatment. The serum-free medium and ethyl lactate were replaced with fresh ethanol every 24 hours for a total of 48 hours.
  • a 5 ⁇ M BODIPY dye working solution was prepared by diluting the stock solution 2500 times to a concentration of 5 mM (1.3 mg BODIPY added to 1 mL DMSO), and aliquoted at -20°C. After 48 hours of cell treatment, the cells were washed three times with 3 mL PBS per well.
  • 3 mL of PBS was then applied to each well for staining, and the cells were incubated at 37°C for 15 min. From this point onward, the cells were kept away from light as much as possible. After staining, the cells were washed twice with 3 mL PBS. The cells were fixed with 3 mL of 4% paraformaldehyde solution at room temperature for 30 min. The fixative was removed, and the cells were washed three times with 3 mL PBS for 5 min each time. Incubate with 2 ml of 2 ⁇ g/ml Dipy solution for 10 min, then wash three times with PBS. Mount with glycerol gelatin, store at 4°C, and photograph.
  • the mouse plasma ALT and AST activity assay kit was used. According to the instructions, 7.5 ⁇ l of plasma and 150 ⁇ l of reagent one were added to a 96-well plate and incubated at 37°C for 10 min. Then, 50 ⁇ l of reagent two was added, and the absorbance was measured at 340 nm. A total of 15 cycles were performed.
  • ALT/AST(U/L) ⁇ OD 340 / ⁇ T s ⁇ 60 ⁇ 207.5/6.22/7.5/0.6 ⁇ 1000.
  • a mouse plasma TG and TC assay kit was used. Following the instructions, 2.5 ⁇ l of plasma or standard solution was added to each well of a 96-well plate, followed by 250 ⁇ l of working solution. The plate was incubated at 37°C for 10 min, and the wavelength of each well was measured using a microplate reader at 500 nm. Distilled water was used as a blank control. The concentrations in the samples were calculated using the standard concentrations.
  • the experimental data are expressed as mean ⁇ standard error.
  • the two-tailed unpaired t-test method was used in Excel to compare the differences between the two groups. A statistically significant difference was considered to be P less than or equal to 0.05.
  • Example 1 Ethyl lactate dose-dependently improves alcohol-related liver disease.
  • This study investigated the effect of ethyl lactate on ALD in mice using a slow-on-acute alcohol feeding model (NIAAA or Gao-Binge model).
  • NIAAA slow-on-acute alcohol feeding model
  • Gao-Binge model simulates the drinking pattern of long-term heavy drinking followed by recent binge drinking in humans and reproduces the pathological characteristics of human ALD.
  • the Gao-Binge model was used. During the slow-on-acute alcohol feeding process, alcohol was added to the mice at dose gradients of 1 g/L, 3 g/L, and 10 g/L (groups), and the dose-response effect of ethyl lactate in improving ALD was verified.
  • ethyl lactate tends to improve liver damage in ALD mice and has a certain dose-response effect.
  • ethyl lactate can significantly reduce hyperlipidemia in mice induced by long-term alcohol feeding, and the effect becomes more significant with increasing dose of ethyl lactate.
  • Oxidative stress and inflammation are considered two key characteristics of ALD (Alcoholic Dietary Disorder). Prolonged alcohol consumption leads to a compensatory increase in the hepatic microsomal alcohol metabolism pathway. While ethanol is metabolized via CYP2E1, a large amount of reactive oxygen species (ROS) are generated, causing oxidative stress in hepatocytes. These ROS directly lead to lipid peroxidation, increasing the levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) in hepatocytes. These ROS then form adducts with intracellular proteins, producing cytotoxicity and exacerbating oxidative stress. Therefore, immunohistochemical staining of liver sections for 4-HNE and MDA can be used to determine hepatic ROS production and oxidative stress levels.
  • ROS reactive oxygen species
  • Neutrophils and macrophages are the most prevalent infiltrating immune cells in the liver of patients with atrial fibrillation (ALD), responding to the inflammatory immune response in the liver.
  • Myeloperoxidase (MPO) is highly expressed in neutrophils and is a common marker protein for neutrophil detection.
  • F4/80 is a specific marker protein for macrophages.
  • a certain number of resident macrophages (Kupffer cells) exist within hepatocytes, and activated macrophages aggregate in clusters.
  • Inflammatory cells and pro-inflammatory factors are associated with genes such as pro-inflammatory cytokines IL-1 ⁇ (Interleukin-1 ⁇ ) and IL-6 (Interleukin-6), chemokines MCP1 (Monocyte Chemoattractant Protein-1, MCP1) (also known as CCL2, (Chemokine(CC-motif)ligand 2)), and adhesion molecules required for immune cell function such as ICAM1 (Intercellular Adhesion Molecule 1) and CD11b.
  • IL-1 ⁇ Interleukin-1 ⁇
  • IL-6 Interleukin-6
  • chemokines MCP1 Monocyte Chemoattractant Protein-1, MCP1
  • CCL2 Chemokine(CC-motif)ligand 2
  • adhesion molecules required for immune cell function such as ICAM1 (Intercellular Adhesion Molecule 1) and CD11b.
  • ethyl lactate dose-dependently downregulated the expression of inflammation-related genes in the liver of ALD mice.
  • ethyl lactate significantly improved liver inflammation.
  • Ethyl lactate inhibits lipid synthesis in hepatocytes, reducing lipotoxicity caused by excessive lipid accumulation and thus decreasing hepatocyte damage and death. This, in turn, reduces damage-associated molecular patterns (DAMPs) released from dead hepatocytes, which to some extent reduces the hepatic immune response and improves liver inflammation in ALD mice.
  • DAMPs damage-associated molecular patterns
  • ethyl lactate has a good effect on improving alcohol-related liver disease and has a significant dose-response effect.
  • ethyl lactate significantly improves ALD, and at doses of 3 g/L and 10 g/L, it also significantly improves ALD, demonstrating potential for treating liver injury and/or alcohol-related liver disease.
  • Example 2 Ethyl lactate directly inhibits alcohol-induced lipid synthesis in hepatocytes.
  • Simple lipid deposition is an early stage of ALD development. Excessive lipid accumulation leads to hepatic steatosis, eventually developing into alcoholic fatty liver (AFL). With continued alcohol consumption, hepatocyte death and inflammation occur in the liver, and AFL gradually progresses to alcoholic steatohepatitis (ASH). Therefore, excessive alcohol metabolism-induced lipid synthesis is a key early step in ALD development. Ethanol is ultimately metabolized in the liver into acetyl-CoA, which directly participates in de novo fatty acid synthesis. This process is regulated by the transcription factor sterol regulatory element-binding protein 1c (SREBP-1c).
  • SREBP-1c transcription factor sterol regulatory element-binding protein 1c
  • ACC1 acetyl-CoA carboxylase 1
  • FAS fatty acid synthase
  • SCD1 stearoyl-CoA desaturase 1
  • DGAT1 diglyceride acyltransferase 1
  • ASCL4 long-chain fatty acid-CoA ligase 4
  • FAS fatty acid saturates
  • ethyl lactate Treatment with ethyl lactate significantly reduced the level of fatty acids (FAS) in mouse livers, and also weakened its spatial distribution. This indicates that ethyl lactate can inhibit key genes and proteins involved in lipid synthesis at both the transcriptional and protein levels, thereby reducing de novo lipid synthesis and improving hepatic steatosis.
  • FOS fatty acids
  • ethyl lactate significantly downregulated the expression of the aforementioned lipid synthesis-related genes and exhibited a dose-response effect, indicating that ethyl lactate improves slow-on-acute induced ALD in mice by inhibiting alcohol metabolism-induced de novo fatty acid synthesis and reducing hepatic lipid deposition.
  • Example 3 Ethyl lactate increases the expression of SIRT1 and FGF21 in hepatocytes.
  • Fibroblast growth factor 21 is a hepatic secretory factor primarily secreted by the liver with metabolic regulatory functions, acting on various metabolic organs throughout the body, such as adipose tissue and the liver. Following alcohol consumption, FGF21 is significantly induced within a short period, regulating the metabolic stress induced by excessive alcohol intake. Therefore, the applicant hypothesizes whether FGF21 is a potential downstream target for ethyl lactate to improve ALD.
  • FGF21 transcription is regulated by SIRT1.
  • Liver-specific knockout of SIRT1 reduces FGF21 expression and exacerbates starvation-induced physiological steatosis of the liver.
  • the cells were cultured in a medium containing 100 mM ethanol for 48 hours to simulate the ethanol exposure of hepatocytes in vitro, while simultaneously being treated with ethyl lactate (1 mM or 10 mM) for 48 hours.
  • ethanol exposure increased the expression of FGF21 in hepatocytes, and ethyl lactate treatment further increased the expression of FGF21, exhibiting a dose-response effect.
  • Ethyl lactate treatment can increase the gene expression and protein level of SIRT1, which are downregulated after alcohol exposure (F and G in Figure 3).
  • ethyl lactate increases the expression of SIRT1 in the liver and promotes the expression and secretion of FGF21.
  • FGF21 as a metabolic regulator, improves hepatic steatosis and reduces liver lipids.
  • Ethyl lactate treatment induces increased FGF21, which downregulates the transcription of genes related to hepatic lipid synthesis, thereby reducing hepatic lipid synthesis, decreasing hepatic lipid deposition, and thus improving liver damage and inflammation.
  • Example 4 Under starvation conditions, ethyl lactate induced hepatic FGF21 to inhibit lipid degeneration.
  • FGF21 an energy homeostasis regulator in response to metabolic stress
  • Prolonged fasting leads to lipolysis of adipose tissue, resulting in a large amount of free fatty acids in the plasma within a short period.
  • the liver absorbs and utilizes these fatty acids, leading to short-term physiological fat accumulation in the liver.
  • This study investigated whether ethyl lactate could induce FGF21 in mouse liver under physiological conditions using a 24-hour fasting mouse model.
  • Wild-type mice were fed a standard diet and allowed free access to water containing 3 g/L ethyl lactate for one month. During the final week, the mice received an additional daily intraperitoneal injection of 30 mg/kg ethyl lactate. Prior to sacrifice, the mice were subjected to a 24-hour fast with free access to water. Liver tissue was collected and stained with H&E and Oil Red O to determine the degree of hepatic steatosis.
  • liver weight index of fasted mice treated with ethyl lactate was significantly lower than that of the control ( Figure 4B). This indicates that most of the free fatty acids absorbed in the liver of mice treated with ethyl lactate were broken down and utilized, resulting in fewer lipid droplets in the liver and a lighter liver weight.
  • the expression and secretion of FGF21 in the liver of mice significantly increased after fasting, and ethyl lactate treatment further increased the expression and secretion of FGF21 in the liver. Furthermore, ethyl lactate also significantly increased the expression of FGF21 in the liver of mice under fed conditions, indicating that ethyl lactate can effectively induce FGF21 in the liver, thereby exerting a metabolic improvement effect.
  • fatty acid breakdown genes such as carnitine palmitoyl transferase 1 ⁇ (CPT1 ⁇ ) and medium-chain acyl-CoA dehydrogenase (MCAD), and lipid synthesis genes such as FAS and SCD1.
  • ethyl lactate After treatment with ethyl lactate, the fatty acid oxidation gene was significantly increased, which corresponds to the increased expression of FGF21. This further indicates that ethyl lactate inhibits starvation-induced steatosis in mouse liver by increasing the expression and secretion of FGF21 in the liver, thereby increasing downstream fatty acid oxidation, so that the free fatty acids absorbed by the liver are fully utilized, and thus reducing the accumulation of lipid droplets in hepatocytes.
  • ethyl lactate can inhibit steatosis in mouse liver under physiological conditions by increasing the expression and secretion of FGF21.
  • Example 5 Ethyl lactate improves acute alcohol gavage-induced hangover in mice.
  • mice were induced to become intoxicated by gavage with 2.5 g/kg body weight of ethanol. The degree of intoxication was assessed 1 hour later using open field and rotarod tests. Mice were then induced to experience hangovers by gavage with 5 g/kg body weight of ethanol. The hangover condition was assessed 6 hours later using open field and rotarod tests. Each mouse underwent only one behavioral test after gavage. The effects of ethyl lactate (10 g/L of ethyl lactate in 52 vol% ethanol) on intoxication and hangovers were investigated by simultaneously adding ethyl lactate to the alcohol.
  • ethyl lactate In a hangover state (5 g/kg ethanol administered by gavage, 6 h), ethyl lactate significantly increased the distance traveled in the open field and the time spent on the rotarod in mice, indicating that ethyl lactate can improve the hangover state in mice. Simultaneously, ethyl lactate requires a certain amount of time to induce FGF21 in vivo to counteract alcohol-induced hangover.
  • Example 6 Lactic acid does not affect alcohol-induced hepatic steatosis.
  • mice with lactic acid Since ethyl lactate may be hydrolyzed into lactic acid in vivo, we treated mice with lactic acid to investigate whether the improving effect of ethyl lactate depends on its hydrolysis product, lactic acid.
  • This invention discovers the effects and mechanisms of a food-derived, natural small-molecule compound, ethyl lactate, in improving liver injury and/or alcohol-related liver disease.
  • ethyl lactate was found to dose-dependently improve alcohol-induced hepatic steatosis, injury, oxidative stress, and inflammatory responses.
  • ethyl lactate treatment significantly reduced the expression levels of key proteins in hepatic lipid synthesis.
  • In vitro cell experiments revealed that ethyl lactate inhibits alcohol-induced lipid synthesis through direct interaction with hepatocytes.
  • mechanistic studies showed that ethyl lactate regulates hepatic metabolism and improves alcoholic steatohepatitis by increasing the transcriptional activity of SIRT1 and promoting the expression and secretion of FGF21 in the liver.
  • This invention marks the first discovery that ethyl lactate possesses in vivo biological activity, participating in the regulation of ALD development and exhibiting significant ameliorative and therapeutic effects on liver damage and/or alcohol-related liver disease in mice.
  • This provides new insights and methods for improving liver damage caused by excessive alcohol consumption or treating alcoholic steatohepatitis.
  • ethyl lactate's downstream target, hepatic FGF21, and its analogues have demonstrated good effects in clinical trials on weight loss, lowering blood lipids, reducing liver lipids, and improving fatty liver and fibrosis.
  • hepatic FGF21 As a highly effective in vivo inducer of hepatic FGF21, ethyl lactate holds promise for improving other metabolic-related diseases as well.

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Abstract

L'invention concerne l'utilisation de lactate d'éthyle dans la prévention et le traitement d'une lésion hépatique et/ou d'une maladie hépatique associée à l'alcool. Le lactate d'éthyle inhibe la synthèse de lipides induite par l'alcool, et régule les niveaux du métabolisme du foie par augmentation de l'activité SIRTI et FGF21, ce qui permet d'atténuer une maladie hépatique associée à l'alcool.
PCT/CN2024/143404 2024-06-12 2024-12-27 Utilisation de lactate d'éthyle pour traiter une lésion hépatique et une maladie hépatique associée à l'alcool Pending WO2025256111A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
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CN1093403A (zh) * 1994-04-10 1994-10-12 吉林市酿酒总厂 营养配制型白酒

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1093403A (zh) * 1994-04-10 1994-10-12 吉林市酿酒总厂 营养配制型白酒

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Title
"Chinese Master's Theses Full-text Database, Medical and Health Sciences", 1 June 2020, article WEI MENGJAO: "Liquor Exposure Induced Toxic Effects and Molecular Mechanism in Mice Liver and Kidney", XP093381324, DOI: 10.27284/d.cnki.gsxiu.2020.001711 *
HUANGFU JIE, LU JUN, LI CHANGWEN, WANG DELIANG, LUAN CHUNGUANG, JIANG XIN, SONG TAO, JIANG WEI, HAN XINLIN, FENG JING, LIU YANLI, : "Evaluating and forecasting the associated main flavor components in Baijiu (Chinese distilled spirits) with alcohol metabolism and hangover symptoms through mice acute withdrawal model", FOOD SCIENCE & NUTRITION, JOHN WILEY & SONS, INC, vol. 11, no. 1, 1 January 2023 (2023-01-01), pages 334 - 343, XP093381325, ISSN: 2048-7177, DOI: 10.1002/fsn3.3064 *
JIANG YANG, WEI SHUANG, SHEN SHIMING, LIU YUXIAO, SU WEITONG, DING DONG, ZHENG ZENGPENG, YU HAOKAI, ZHANG TINGTING, YANG QIULI, ZH: "Ethyl Lactate Ameliorates Hepatic Steatosis and Acute‐on‐Chronic Liver Injury in Alcohol‐Associated Liver Disease by Inducing Fibroblast Growth Factor 21", ADVANCED SCIENCE, JOHN WILEY & SONS, INC, GERMANY, vol. 12, no. 5, 1 February 2025 (2025-02-01), Germany, XP093381320, ISSN: 2198-3844, DOI: 10.1002/advs.202409516 *
MARCUS HOLLENBACH ET AL.: "Ethyl Pyruvate and Ethyl Lactate Down-Regulate the Production of Pro-Inflammatory Cytokines and Modulate Expression of Immune Receptors", BIOCHEMICAL PHARMACOLOGY, vol. 76, no. 5, 31 December 2008 (2008-12-31), XP023904404, ISSN: 0006-2952, DOI: 10.1016/j.bcp.2008.06.006 *

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