Compositions and methods of use of gamma-ketoaldehyde scavengers for treating, preventing or improving nonalcoholic fatty liver disease (NAFLD), NASH, ALD or conditions related to the liver

Abstract

To provide methods for use in treating, preventing or improving diseases related to the liver in an animal, such as nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD) or nonalcoholic steatohepatitis (NASH).SOLUTION: The present invention provides a method for treating, preventing, or ameliorating liver disease in an animal with or at risk of liver disease, thereby inhibiting or treating the liver disease, comprising the step of administering to the animal an effective amount of a compound having a structure represented by a compound of the following formula, and stereoisomers and analogs thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. 62 / 383,895, filed September 6, 2016, and U.S. Patent Application No. 62 / 410,133, filed October 19, 2016, which are incorporated by reference in their entireties.

[0002] 2. Background of the Invention 1. Field The present invention relates to compositions comprising compounds that scavenge γ-ketoaldehyde (γ-KA), such as 2-hydroxybenzylamine (2-HOBA), and to methods of administering γ-KA scavengers for the treatment, prevention, or amelioration of liver-related diseases or conditions, such as non-alcoholic fatty liver disease (NAFLD) and / or alcoholic liver disease (ALD), and / or non-alcoholic steatohepatitis (NASH). [Background technology]

[0003] 2. background Alcohol-related chronic liver disease is a major cause of morbidity and mortality that continues to rise. After viral hepatitis, chronic excessive ethanol consumption is responsible for 25% of all deaths caused by liver cancer and cirrhosis. Chronic alcohol use frequently coexists with liver disease and cancer. Alcoholic liver disease (ALD) represents a significant health care burden, with costs estimated at $27 billion annually in the United States alone. Although abstinence is the best treatment for ALD, recidivism is the major risk, with recurrence rates ranging from 67% to 81% over the course of a year.

[0004] ALD encompasses various liver manifestations such as fatty liver (steatosis), hepatitis and cirrhosis / fibrosis, which may occur simultaneously in certain individuals. The spectrum of ALD ranges from simple steatosis to alcoholic steatohepatitis (ASH) and cirrhosis, and is exacerbated by obesity. There are many mechanisms by which alcohol induces liver injury, but inflammation is fundamental to ALD progression. Ethanol metabolism promotes antioxidant depletion and leads to the formation of harmful substances such as acetaldehyde, acetate, reactive oxygen species (ROS), and lipid peroxides that induce an inflammatory response. In addition, alcohol and its metabolites stimulate inflammation by promoting gastrointestinal leakage and stimulating immune cells (the so-called adaptive immune response) and / or activating innate immune pathways such as complement. Activation of innate immune components initiates alcohol-induced liver injury, but also triggers hepatoprotective, regenerative, and anti-inflammatory responses that reduce hepatocellular damage.

[0005] It is well known that lipid peroxidation and oxidative stress play a significant role in inflammation and pathogenesis of chronic liver diseases, especially ALD. Aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), form covalent protein adducts that disrupt normal protein function. Orders of magnitude more reactive than MDA and 4-HNE are the peroxidation products of arachidonic acid, termed acyclic γ-KA (also known as isolevulgandins or isoketals), which are major mediators of inflammation (Figure 1). γ-KA rapidly and covalently adducts to proteins and DNA, disrupting normal molecular function and forming protein-protein crosslinks (isoketals). γ-KA is produced by the F2-isoprostane (F2-isoP) pathway. γ-KA is involved in free radical-mediated arachidonic acid peroxidation in various pathophysiological conditions. It has been shown that ALD accumulates through the nonenzymatic formation of the atypical eicosanoids isoprostanes (isoP) and isofurans (isoF). Isofurans are similar to isoprostanes but contain a substituted tetrahydrofuran ring. It has been demonstrated that anti-γ-KA antibody titers are elevated in the serum of human subjects with ALD compared to subjects without ALD (Figure 2).

[0006] ALD is characterized by the progression of steatosis, inflammation, hepatocyte necrosis and apoptosis, with the concomitant development of fibrosis and cirrhosis. It is also well established that excessive alcohol intake causes oxidative injury to the liver. F2-isoPs have been shown to be the most accurate predictors of oxidative stress in vivo, and their levels are increased in alcoholic liver disease and chronic hepatitis. KA overproduction is involved in the pathogenesis of a number of chronic inflammatory diseases. More recently, it has been shown that ethanol feeding in mice induces hepatic γ-KA formation, which readily binds to proteins to form stable adducts. These γ-KA-protein adducts likely contribute to ethanol-induced liver injury by eliciting proinflammatory or adduct-specific immune responses.

[0007] There is great interest in identifying appropriate therapeutic interventions aimed at inhibiting inflammatory processes and interfering with immunogenic pathways associated with ALD. 2-Hydroxybenzylamine (2-HOBA), i.e., buckwheat fiber, was found to be a potent scavenger of γ-KA, capturing γ-KA 980 times faster than the rate of formation of γ-KA-lysyl-protein adducts. Importantly, this showed that this γ-KA scavenger does not inhibit cyclooxygenase enzymes. In a model of oxidant-mediated cell death (Figure 3), 2-HOBA almost completely prevented cell death induced by t-butyl hydroperoxide (tBHP). In addition, 2-HOBA was demonstrated to have a protective effect against oxidant-mediated cell death in HepG2 cells exposed to various concentrations of hydrogen peroxide (H2O2).

[0008] Despite the severe economic and health impact of ALD, little progress has been made in the management of patients with this severe clinical condition. Although abstinence is the cornerstone of treatment, there is considerable interest in identifying other therapeutic interventions and treatments for ALD. Current modalities for ALD treatment include corticosteroids and pentoxifylline. Corticosteroids improve short-term survival in severe forms of alcoholic hepatitis, but frequent use is contraindicated. Pentoxifylline is a competitive, nonselective phosphodiesterase inhibitor that improved short-term survival in severe acute alcoholic hepatitis and demonstrated an improved risk-benefit profile compared with prednisone, but did not confer additive benefit when used in combination with prednisone. Both of these treatments attenuate the inflammatory response but do not target the underlying inflammatory signal. Trapping toxic oxidized lipids with 2-HOBA is novel in that it reduces the formation of exacerbating protein adducts that perpetuate inflammation and drive liver damage. The present invention encompasses the use of 2-HOBA to prevent ALD and also attenuate the prevalence of alcoholic liver disease.

[0009] Epidemiological data indicate that nonalcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease in Western countries. NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis, and occurs frequently in the setting of obesity, dyslipidemia and insulin resistance. NASH can lead to cirrhosis and liver failure in 10–15% of patients. The mechanisms that distinguish steatosis from NASH are still poorly understood. It is becoming clear that inflammation and the resulting production of reactive oxygen species (ROS) and reactive lipid species (RLS) are key factors in the pathogenesis of NASH. In the liver, inflammatory and immune responses Alterations in the endothelial cell membrane exacerbate the production of ROS and proinflammatory cytokines, leading to the exacerbation of NASH.

[0010] Furthermore, patients with NAFLD and NASH have higher mortality and morbidity compared to the general population. NAFLD exhibits high cardiovascular mortality, while NASH exhibits higher liver-related mortality. Recent observations have concluded that NASH is currently the third most common indication for liver transplantation in the United States and is expected to become the most common indication for liver transplantation within the next decade. NASH is an inflammatory form of NAFLD, characterized by excess hepatic fat, inflammation, and hepatocyte ballooning, with or without coexisting fibrosis. NASH is most worrisome for progression to end-stage liver disease, or cirrhosis. The mechanisms and conditions that support NASH are not histologically clear, but there are similarities to alcoholic steatohepatitis. It is well known that lipid peroxidation and oxidative stress play an important role in the pathogenesis of chronic liver diseases, including NASH. Reactive oxygen species (ROS) promote the formation of lipid peroxides and trigger the generation of bifunctional electrophiles (BFEs), which are major mediators of inflammation. Among these BFEs, 4-hydroxynonenal (4-HNE), acrolein, malondialdehyde (MDA), methylglyoxal (MGO) and levuglandin (LG) have been shown to mediate oxidative damage by covalently modifying lipids, proteins and DNA. BFEs are highly reactive compounds that form covalent adducts with proteins and DNA, disrupting normal molecular function and forming cross-links with proteins, phosphoethanolamine and DNA. Summary of the Invention [Problem to be solved by the invention]

[0011] Nonalcoholic steatohepatitis (NASH) is inflammation and damage of the liver caused by the accumulation of fat in the liver. NASH is similar to ALD, but occurs in people who consume little alcohol. Two to five percent of Americans suffer from NASH, most often middle-aged, overweight or obese people. The present invention encompasses the use of 2-HOBA to prevent and / or treat NASH. [Means for solving the problem]

[0012] The present invention encompasses the use of 2-HOBA to scavenge toxic oxidized lipids (ketoaldehydes), effectively regulating the inflammatory program and reversing liver damage associated with inflammation.

[0013] The present invention encompasses the use of 2-HOBA to target γ-KA to prevent lipid peroxidation and the resulting γ-KA-specific immune response in alcoholic liver disease. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram depicting how γ-KA reacts with lysine or other primary amines to form a reversible Schiff base adduct. [Diagram 2] FIG. 2 is a graph depicting serum γ-KA antibody titers in hospitalized ICU patients with and without alcoholic liver disease. [Diagram 3] FIG. 3 is a graph depicting the protective effect of 2-HOBA against oxidant-mediated cell death in mouse hepatocytes upon increasing t-butyl hydroperoxide. [Figure 4] 4A and 4B depict ROS in HepaRG at increasing ethanol doses and the protective effect of 2-HOBA at increasing ethanol concentrations. [Diagram 5] 5A and 5B depict ALT and AST levels in the medium of HepaRG cells pretreated with different levels of 2-HOBA. [Figure 6] FIG. 6 shows the effect of 2-HOBA pretreatment on ethanol-mediated γ-KA formation. [Figure 7] FIG. 7 shows the identification of histones-H3 and -H4 modified by γ-KA in mouse lung. [Figure 8]FIG. 8 is a graph depicting liver and kidney isoprostanes (IsoP) and isofurans (IsoF). [Figure 9] FIG. 9 depicts evidence for the formation of isoLG in human NAFLD liver. [Figure 10] FIG. 10 depicts an increase in MPO positive cells in NASH. [Figure 11-1] 11A-11E depict the development of NAFLD in STAM mice. [Figure 11-2] Figures 11F-11I depict the development of NAFLD in STAM mice. [Figure 12-1] Figures 12A-T describe the effects of 2-HOBA on STAM mice. [Figure 12-2] Figures 12A-T describe the effects of 2-HOBA on STAM mice. [Figure 12-3] Figures 12A-T describe the effects of 2-HOBA on STAM mice. [Figure 13] 13A-F show insulin signaling and hepatic inflammasomes in STAM™ mice. [Figure 14-1] 14A-G depict the development of NAFLD in DIAMOND™ mice. [Figure 14-2] 14A-G depict the development of NAFLD in DIAMOND™ mice. [Figure 15-1] FIG. 15 summarizes the results of the study in DIAMOND™ mice. [Figure 15-2] FIG. 15 summarizes the results of the study in DIAMOND™ mice. [Figure 16-1] FIG. 16 summarizes the results of the study in DIAMOND™ mice. [Figure 16-2] FIG. 16 summarizes the results of the study in DIAMOND™ mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention encompasses a novel nutritional therapy that is expected to reduce liver injury by preventing the formation of γ-KA-protein adducts and their differential effects on innate and adaptive immune response.This nutritional therapy can be used to treat, prevent or improve liver-related conditions or diseases, such as but not limited to NAFLD, ALD and NASH.Nutritional therapy can be used to improve overall liver health and maintain healthy liver function.

[0016] The present invention includes a means to specifically prevent the formation of γ-KA adducts in the liver using a class of bifunctional electrophile (BFE) "scavenger" molecules. The buckwheat natural product 2-hydroxybenzylamine (2-HOBA), a series of phenolic amines including pyridoxamine and its water-soluble derivative, constitutes a preferred embodiment. 2-HOBA specifically reacts with γ-KA 980 times faster than with lysine, preventing the formation of adducts with proteins and lipids in vitro and in vivo.

[0017] The compositions and methods of the present invention are directed to animals, including humans and non-human animals. The animals may be healthy or may suffer from a disease or condition.

[0018] The term administering or administration refers to providing a composition to a mammal, administering ... Ingesting, administering or administering the active ingredient(s) to the subject, including administering or administering to the subject, and combinations thereof.

[0019] The present invention encompasses compositions and methods for using 2-HOBA, which is also referred to as salicylamine, SAM, 2-hydroxybenzylamine, and pentylpyridoxamine (PPM).

[0020] An embodiment of the present invention is a compound of the formula: [ka] (In the formula, R is N or C; R2 is independently H, hydroxy, halogen, nitro, CF3, C1-6 alkyl, C1-6 alkoxy, C3- 10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R2, R3 and R4 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R5; 3~8 may form a membered ring; R3 is H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 5-membered ring, cyclized together with one or more R2 or R5 to an optionally substituted C, O, S or N-containing 3~8 may form a membered ring; R4 is H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R5; 3~8 may form a membered ring; R5 is a bond, H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R4; 3~8(May form a membered ring) and stereoisomers and analogs thereof, and further includes their use as medicaments in methods for treating, preventing, or ameliorating a liver condition or disease, such as NAFLD, ALD, and NASH, in a subject having or at risk of a liver condition or disease, such as NAFLD, ALD, and NASH, thereby inhibiting or treating such a liver condition or disease.

[0021] Another embodiment of the present invention is a compound of the formula: [ka] (In the formula, R is N or C; R2 is independently H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R2, R3 and R4 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R5; 3~8 may form a membered ring; R3 is H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 5-membered ring, cyclized together with one or more R2 or R5 to an optionally substituted C, O, S or N-containing 3~8 may form a membered ring; R4 is H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R5; 3~8 may form a membered ring; R5 is a bond, H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R4; 3~8 (May form a membered ring) and stereoisomers and analogs thereof, and further includes uses thereof in methods for the treatment, prevention or amelioration of liver conditions or diseases, such as NAFLD, ALD and NASH, in subjects having or at risk for such liver conditions.

[0022] In certain embodiments, the compound can be selected from the compounds disclosed herein. In a preferred embodiment, the compound can be a salicylamine.

[0023] Another embodiment of the present invention is a method for treating, preventing, or ameliorating a liver condition or disease, such as NAFLD, ALD, and NASH, in a subject having or at risk for a liver condition or disease, such as NAFLD, ALD, and NASH, thereby inhibiting or treating such liver condition, comprising administering to the subject at least one compound, in a dosage and amount effective to treat the functional abnormality in a mammal, in conjunction with a drug having a known side effect of treating, preventing, or ameliorating a liver condition or disease, such as NAFLD, ALD, and NASH, having the following formula: [ka] (In the formula, R is N or C; R2 is independently H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R2, R3 and R4 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R5; 3~8 may form a membered ring; R3 is H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 5-membered ring, cyclized together with one or more R2 or R5 to an optionally substituted C, O, S or N-containing 3~8 may form a membered ring; R4 is H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10 Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R5; 3~8 may form a membered ring; R5 is a bond, H, hydroxy, halogen, nitro, CF3, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~10Cycloalkyl, C containing C, O, S or N optionally substituted with one or more of R4, R2 and R3 3~8 cyclic ring, optionally substituted C, O, S or N-containing ring, which is cyclized with one or more of R2, R3, or R4; 3~8 (May form a membered ring) and stereoisomers and analogs thereof.

[0024] Examples of compounds that can be used with the methods disclosed herein include, but are not limited to, compounds having the formula: [ka] (In the formula, R is N or C; R2 is independently H, substituted or unsubstituted alkyl; R3 is H, halogen, alkoxy, hydroxyl, nitro; R4 is H, substituted or unsubstituted alkyl, carboxyl. and pharma- ceutically acceptable salts thereof.

[0025] In a preferred embodiment, the compound is a salicylamine (2-hydroxybenzylamine or 2-HOBA).

[0026] The compound may be selected from the following or a pharma- ceutically acceptable salt thereof: [ka]

[0027] The compound may also be selected from the following, or a pharma- ceutically acceptable salt thereof: [ka]

[0028] The compound or analog may also be selected from the following, or a pharma- ceutically acceptable salt thereof: [ka]

[0029] The compound may also be selected from the following, or a pharma- ceutically acceptable salt thereof: [ka]

[0030] The compound may also be selected from the following, or a pharma- ceutically acceptable salt thereof: [ka]

[0031] The compounds of the present invention can be administered by any method known to those skilled in the art, including, but not limited to, oral, transdermal, inhalation, nasal, topical, intravaginal, ocular, intraaural, intracerebral, rectal, and parenteral administration, such as by injection, including intravenous, intraarterial, intramuscular, and subcutaneous. The compounds may be administered therapeutically to treat an existing disease or condition, or prophylactically to prevent a disease or condition.

[0032] While any suitable pharmaceutical vehicle containing the present composition may be utilized within the scope of the present invention, preferably the present composition is combined with a suitable pharmaceutical carrier such as dextrose or sucrose.

[0033] Methods for calculating how often the composition is administered are well known in the art, and any suitable administration frequency (e.g., one 6 g dose / day or two 3 g doses / day) can be used within the scope of the present invention for any suitable period of time (e.g., a single dose can be administered over 5 minutes or 1 hour, or alternatively, multiple doses can be administered over an extended period of time). The compositions of the present invention may be administered over an extended period of time, such as weeks, months or years. The compositions are divided into separate servings each containing one dose per day, such that a daily serving is produced that contains the total amount of the composition administered in a day or 24 hour period. The dosage may be administered in a single serving, or in individual servings containing more than one dose (multiple individual servings).

[0034] Any suitable dose of the composition of the present invention can be used within the scope of the present invention. Methods for calculating appropriate doses are well known in the art.

[0035] "Treatment" or "treating" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. The term encompasses active treatment, i.e., treatment aimed specifically at ameliorating the disease, pathological condition, or disorder, and also encompasses causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, pathological condition, or disorder. In addition, the term encompasses palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at preventing or reducing the severity of an associated disease, pathological condition, or disorder; "treatment" includes treatment aimed at minimizing or partially or completely suppressing the occurrence of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder. EXAMPLES

[0036] Example 1 Fully differentiated day 21 HepaRG hepatocytes (Bio-Predict International) were incubated with the readily fluorescent MRP2 substrate CDFD. We present clear epithelial cells surrounding a colony of hepatocytes containing numerous bile canaliculi that metabolize A. HepaRG cells are a unique and well-established human hepatocyte culture system with high fidelity to the absorption, distribution, metabolism, and excretion of A in the human liver. Consistent with the known mechanism of action of 2-HOBA, pretreatment of HepaRG cells with 2-HOBA (0, 250, 500 and 1000 µM) for 24 h, followed by increasing concentrations of ethanol (0-200 mM; 24 h), had no effect on reactive oxygen species formation (Figure 4A). However, 2-HOBA almost completely prevented ethanol-induced cell death (Figure 4B). Similarly, pretreatment of HepaRG cells with 2-HOBA (0, 250, 500 and 1000 μM) for 24 h followed by increasing concentrations of ethanol (0-200 mM; 24 h) nearly normalized ALT (Figure 5A) and AST (Figure 5B) levels in culture medium harvested from HepaRG hepatocytes. Currently, there is little knowledge regarding the identity of proteins susceptible to γ-KA modification. We investigated the identity of proteins that endogenously form adducts using HepaRG hepatocyte cultures that were previously exposed to ethanol with or without 2-HOBA pretreatment (0, 250 or 500 μM, 24 h). Cells were harvested and total proteins were analyzed by SDS-PAGE and then probed with D11SCFv, a well-characterized single-chain antibody that recognizes peptides and proteins modified by γ-KA isomers (Figure 6). Consistent with the theory that there is a "pool" of proteins susceptible to γ-KA modification, all treatments showed similar levels of "background" anti-D11 cross-reactivity. However, there was a robust increase in cross-reactivity of a single band (arrow) at approximately 18 kD in hepatocytes treated with ethanol but not protected with 2-HOBA. In contrast, the intensity of this band was restored by 2-HOBA pretreatment (250 or 500 μM 2-HOBA) to levels observed in untreated controls. Interestingly, this band has the same molecular weight as that recently identified as an adduct with γ-KA and anti-D11 antibodies in a mouse model of pulmonary fibrosis (Figure 7).These data imply that ETOH promotes the formation of proteins that form adducts with γ-KA and that this formation can be blocked by 2-HOBA.

[0037] Taken together, these findings demonstrate that γ-KA is a major mediator of liver injury caused by lipid peroxidation and that the use of 2-HOBA protects against cell death induced by such oxidants or by exposure to ethanol. Targeting γ-KA can be used as a method to prevent lipid peroxidation and the resulting γ-KA-specific immune responses in alcoholic liver disease.

[0038] Example 2 C57 / BL6J mice were co-administered with 2-HOBA and ethanol to establish the efficacy of 2-HOBA in attenuating ethanol-mediated liver dysfunction (increased AST and ALT), reducing γ-KA formation, favorably altering immune responses, and stimulating beneficial intracellular signaling pathways. Results show that pretreatment with 2-HOBA (1.0 mg / ml) significantly reduces liver injury (elevated γ-KA and liver function enzymes) activated upon exposure to ethanol.

[0039] Prior to the 14-day ethanol treatment, 2-HOBA (0.5 g / L, Lieber-DeC The efficacy of 2-HOBA in attenuating liver injury was examined by administering ethanol (5% v / v) to mice fed a liquid diet of arli (LDLD). The NIAAA model (10 days of ethanol in LDLD + indulgence) was used to administer ethanol (5% v / v). Food intake and body weight were not significantly affected by the addition of 0.5 g / L 2-HOBA to the LDLD (data not shown). With ethanol, the number of leukocytes and lymphocytes was reduced, whereas the number of eosinophils and basophils was increased, compared to the maltodextrin-fed diet (Table 1). This was consistent with other reports of alcoholic liver injury. In these studies, such a response was not observed in the presence of 2-HOBA. 2-HOBA at 0.5 g / L did not attenuate the increase in serum AST or ALT (Table 2). [ka] [ka]

[0040] An additional cohort of mice was pretreated with 2-HOBA (1.0 g / L) for 14 days prior to ethanol administration using the NIAAA model. Food intake in ethanol-treated mice was lower and, as a result, they weighed approximately 2.5 g less than maltodextrin-fed controls at the end of the study.

[0041] [ka]

[0042] Liver triglyceride content tended to be lower in ETOH-stimulated mice pretreated with 2-HOBA (1.0 g / L). Liver injury, as indicated by changes in serum ALT, tended to be lower in 2-HOBA pretreated (164.4 ± 20.8 U / L) compared to control + ETOH mice (253.0 ± 62.9 U / L), but these did not reach statistical significance. AST was lower in mice pretreated with 2-HOBA + ETOH (676.6 ± 86.7 U / L) compared to control (906.1 ± 186.1 U / L), but these did not reach statistical significance. These changes were accompanied by significant reductions in hepatic isoprostanes (IsoP) and isofurans (IsoF), as shown in Figure 8, and also resulted in a significant reduction in renal isoprostanes levels (1.3±0.1 ng / g vs. 2.0±0.3 ng / g in control+ETOH; P<0.05).

[0043] Pretreatment with 1.0 g / L 2-HOBA for 14 days in the NIAAA model reduced liver injury and mitigated the significant increase in hepatic isoprostanes and isofurans content observed with ETOH exposure, as well as reducing renal isoprostanes formation.

[0044] Example 3 - NASH F2-isoprostanes (F2-isoP) are prostaglandin-like compounds formed in vivo via a nonenzymatic mechanism involving free radical-initiated peroxidation of arachidonic acid and have been shown to be the most accurate predictor of oxidative stress in vivo. Elevated plasma or urinary levels of F2-isoP have been reported in patients with alcoholic liver disease and in animals with NAFLD / NASH, and increased hepatic F2-isoP has also been reported in humans and animals with NASH. F2-isoP values ​​are also increased in chronic hepatitis. Preliminary data indicate that livers from mice and human liver tissues with proven NAFLD / NASH show a dramatic increase in isoLG protein adduct levels compared to controls (Figure 9). These observations were accompanied by increased staining of myeloperoxidase-positive neutrophils in samples characteristic of biopsy-proven NASH (Figure 10), suggesting enhanced inflammation. A number of agents appear to be associated with enhanced inflammatory processes.

[0045] It was demonstrated that increased plasma lipopolysaccharide (LPS) and free fatty acids (FFA) in the plasma of NASH patients are associated with increased hepatic TLR4-Myd88-independent signaling. It was also demonstrated that palmitate and LPS induce NF-kB activity in cultured HepaRG cells that can block chemical or short interfering RNA-mediated TLR4 inhibition. Liang et al. recently demonstrated that alternative metabolic ligands (cholesterol and carbohydrates) may also initiate steatosis, hepatocyte hypertrophy, and enhanced mixed (neutrophil and mononuclear cell) inflammation via inflammasome-mediated mechanisms. It proved that there is.

[0046] Inflammasomes are multimeric protein complexes of the innate immune system that activate caspase-1 upon binding of PAMPs (pathogen-associated molecular patterns) or DAMPs (damage-associated molecular patterns), either directly or via the adaptor molecule ASC. Caspase-1 in turn activates downstream signaling pathways, and the resulting changes in biological function are highly diverse depending on the cell type and the initiating ligand. Inflammasomes are activated by members of the (NOD)-like receptor (NLR) family (NLRP1-3, NLRP6-7, NLRP12, NLRC4), NAIP, and Aim2. Activation of the NLRP3 inflammasome triggers the cleavage of inactive pro-IL-1β, pro-IL-18, and pro-IL-33 into their active forms by caspase-1. Known NLRP3-inflammasome activators include fatty acids, cholesterol, and protein aggregates, among others. Molecular triggers of the NASH inflammasome that have already been described include DNA, saturated fatty acids and LPS, NLRP-1. This suggests that isoLGs are also additional potent inflammasome activators through mechanisms that have yet to be fully detailed. Indeed, defective NLRP1 and NLRP3 signaling / activation by isoLG ligands may underlie altered hepatic immune metabolism, host-gut microbiome adaptation, and defective NLRP-inflammasome sensing that are hallmarks of the transition from NAFL to NASH.

[0047] 2-Hydroxybenzylamine (2-HOBA) has a 980-fold higher reactivity with γKA than lysine and, importantly, does not inhibit the cyclooxygenase enzyme. In a model of oxidant-mediated cell death, 2-HOBA almost completely prevented t-butyl hydroperoxide (tBHP)-induced hepatic cell death. This is a surprising finding considering the fact that the pathogenesis of oxidative injury is extremely complex and multifaceted. In addition, 2-HOBA was demonstrated to have a protective effect against oxidant-mediated cell death in HepG2 cells exposed to various concentrations of H2O2. Taken together, these findings suggest that γKA may be a major mediator of liver injury caused by lipid peroxidation and, furthermore, that the use of 2-HOBA may provide protection from such oxidant-induced cell death in the liver. Clearly, the efficacy of 2-HOBA depends on its accumulation in target tissues, which was recently supported by pharmacokinetic studies in an in vivo mouse model. Administration of 1, 3, and 10 g / L 2-HOBA for 7 days was associated with a dose-dependent increase in plasma and liver 2-HOBA levels, with liver levels being approximately 10-fold higher than in plasma.

[0048] One model that has been used in studying the pathogenesis of NASH, cirrhosis and HCC is the STAM model (Stelic Inc., Tokyo, Japan). The STAM model provides histological and With strong fidelity to human NASH in both function and physiology (high fasting glucose, liver biochemistry, intrahepatic lipids, dyslipidemia; FIG. 11), it advances features that have been difficult to achieve in genetic knockout or dietary models of NAFLD. We demonstrated that 2-HOBA can reduce the severity of NAFLD in STAM mice (FIG. 12).

[0049] In the STAM™ model, NASH is induced in C57BL / 6 mice by a single subcutaneous injection of 200 μg of streptozotocin (STX) solution on postnatal day 2 and by starting high-fat diet feeding at 4 weeks of age. At 3 weeks of age, 12 mice that received STZ injections were divided into two groups: 1) 2-HOBA (n=6), and 2) vehicle control (n=6). Mice in the 2-HOBA group were given 2-HOBA in drinking water (1 g in 1 L of water), while the vehicle control group was given regular water without 2-HOBA. At 4 weeks of age, all mice were given free access to high-fat food. All mice were given 2-HOBA in drinking water (1 g in 1 L of water) and 2-HOBA in drinking water (1 L of water). All mice were given 2-HOBA in drinking water ( ... All mice were maintained on a high-fat diet and received 2-HOBA supplemented water or regular water based on their group assignment throughout the study protocol. Body weight and food / water intake were monitored weekly. Animals were sacrificed at 9 weeks of age (6 weeks 2-HOBA or vehicle treatment) and tissues and serum were collected for analysis.

[0050] Liver sections were stained with hematoxylin and eosin (to score steatosis, hepatocyte ballooning, and inflammation), Sirius red (to assess fibrosis), and for F4 / 80+ macrophages. Scoring was performed in a blinded fashion for steatosis, ballooning, inflammation, and necrosis using the following criteria: Steatosis (0-4): 0=<5%; 1=5-25%; 2=25-50%; 3=50-75%; 4=75-100%. Ballooning (0-3): 0=none; 1=mild (foci involving less than three hepatocytes); 2=moderate (foci involving more than three hepatocytes or multifocal); 3=moderate (multifocal with more than three foci of three or more hepatocytes). Inflammation (0-4): 0=none; 1=minimal (0 to 1 lesion per 20 areas); 2=mild (2 lesions); 3=moderate (3 lesions); 4=severe (4 or more lesions). Serum levels of glucose, insulin, alanine transaminase, aspartate transaminase, triglycerides, cholesterol, and F2-isoprostanes were measured. Serum and tissue levels of the following inflammatory markers: IL-1α, IL-6, IL-1β, IL-10, IL-17, MCP-1, and TNFα were measured using a multiplex assay (Luminex, Millipore). Hepatic 2-HOB was measured using a 2-HOB kit (Millipore, Billerica, MA). A, F2-isoprostane, and isofuran levels were determined by LC / MS / MS methods. Liver mRNA expression was assessed by RT-qPCR for the following genes: Gck, Pck1, Pdk4, Irs1, Irs2, Pgc1a, Cpt1a, Gyk, Srepb1c, Acc1, Fxr, Cox1, Cox2, Nox4, catalase, Gpx1, Gp41phx, and p22phox. Liver protein content and phosphorylation status were determined for Akt, GSK3β, mTOR, ERK, JNK, FOXO1, and NALP. Endpoints were compared between 2-HOBA-treated and vehicle-treated groups using independent samples two-tailed t-tests. Significance was set at α=0.05.

[0051] Survival, clinical signs and behavior were monitored daily. Mean body weight and food intake were similar between groups, while liver weight and liver to body weight ratio were significantly reduced (Figure 12). Serum glucose levels were similar between groups, while insulin, ALT and lipids all showed a trend towards reduction. Importantly, serum isoprostanes (a marker of BFE and isoLG adduct formation) and histological NASH severity were significantly reduced with 2-HOBA treatment.

[0052] To better understand the criteria for liver function and phenotype improvement described in Figure 13, livers from STAM mice were subjected to immunoblot analysis. 2-HOBA treatment dramatically increased the phosphorylation of AKT and GSK3β without significantly altering signaling through mTOR, ERK, or FOXO1. In addition, the data showed that the expression of pyruvate dehydrogenase kinase 4 (pdk4), a key protein in the regulation of mitochondrial fuel metabolism, was significantly decreased, suggesting that the reduction in liver weight, improvement in NASH severity, and reduction in serum isoprostanes by 2-HOBA treatment may be mediated by a γKA-mediated mechanism that affects mitochondrial function. Furthermore, the data show a decrease in NALP1 by 2HOBA treatment. Recently, the role of NRLP3-inflammasome in regulating mitochondrial function has been described.

[0053] These data in the STAM model suggest that isoLG-protein adducts mediate hepatic injury. These results imply that NLRP1-inflammasome activation is the cause of harm and that 2-HOBA strongly reduces NASH severity and restores markers of hepatic insulin sensitivity.Importantly, the data show that oral administration of 2-HOBA blocks NLRP1-inflammasome activation in this model, suggesting a role for 2-HOBA in blocking isoLG-mediated inflammasome NASH responses.

[0054] Example 4 DIAMOND (Dietary-Induced Animal Model of Nonalcoholic Fatty Liver Disease) is a patented syngeneic mouse strain that sequentially develops nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, fibrosis, and hepatocellular carcinoma in response to a high-fat, high-sugar diet. Disease progression in DIAMOND mice closely resembles that of humans, including histopathological features. The DIAMOND mouse model is unique in that it is the only mouse model of NAFLD / NASH that develops NASH solely as a result of a Western diet (high fat, sugar water) and not due to gene knockouts or toxins that induce liver pathology (Figure 14).

[0055] Twelve 8-week-old male DIAMOND mice were placed on a high-fat diet (Harlan-ENVIGOTD.88317) and water containing glucose (18.9% w / v) and fructose (23.1% w / v) with free access to the diet. All mice continued on this diet throughout the study protocol. At 12 weeks of age, mice were divided into two groups: 1) 2-HOBA (n=6), and 2) vehicle control (n=6). Animals in the 2-HOBA group received 2-HOBA in drinking water (1 g per liter of water containing glucose and fructose). The vehicle control group received water (containing glucose and fructose) without 2-HOBA. Body weight and food intake were measured weekly. At approximately 23 weeks of age, all animals underwent a glucose tolerance test (GTT) and MRI imaging to assess liver fat. For GTT, animals were fasted for 12 hours and then glucose (100 mg / mL glucose in sterile water at 2 g / kg body weight) was administered by oral gavage. Blood was sampled at 0, 15, 30, 45, 60, 90, and 120 minutes after glucose administration and the area under the curve was calculated. Animals were sacrificed at 24 weeks of age (12 weeks of 2-HOBA or vehicle treatment). Tissues and serum were collected for analysis.

[0056] Liver sections were stained with hematoxylin and eosin (to score steatosis, hepatocyte ballooning, and inflammation) and Sirius red (to assess fibrosis). Scoring was performed in a blinded fashion for steatosis, ballooning, inflammation, and necrosis using the following criteria: Steatosis (0-4): 0=<5%; 1=5-25%; 2=25-50%; 3=50-75%; 4=75-100%. Ballooning (0-3): 0=none; 1=mild (foci involving less than three hepatocytes); 2=moderate (foci involving more than three hepatocytes or multifocal); 3=moderate (multifocal with more than three foci of three or more hepatocytes). Inflammation (0-4): 0=none; 1=minimal (0 to 1 lesion per 20 areas); 2=mild (2 lesions); 3=moderate (3 lesions); 4=severe (4 or more lesions). Serum levels of glucose, alanine transaminase, and aspartate transaminase were measured. Liver mRNA expression was assessed by RT-qPCR for the following genes: Tnfa, Nlrp1a, Il1b, Il18, Timp1, Col1a1, ProCard, Nlrp3, Casp1, ProIl1b, Tgfb1, Bambi, Pdk4, and Gapdh. Independent samples two-tailed t-tests were used to compare endpoints between 2-HOBA-treated and vehicle-treated groups. Significance was set at α=0.05.

[0057] Figures 15-16 summarize the results of the DIAMOND mouse study. In the case of 2-HOBA supplementation, a trend towards reduced liver weight and associated significant reductions in the liver enzymes ALT and AST are demonstrated. These findings support the efficacy of 2-HOBA in preventing the onset or attenuating the severity of NASH.

[0058] References Albano E, Vidali M. Immune mechanisms in alcoholic liver disease. Genes Nutr 2010; 5(2):141-147.PM:19809845 Mottaran E, Stewart SF, Rolla R et al. Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease. Free Radic Biol Med 2002; 32(1):38-45.PM:11755315 Stewart SF, Vidali M, Day CP, Albano E, Jones DE. Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease. Hepatology 2004; 39(1):197-203.PM:14752838 Lee GS, Yan JS, Ng RK, Kakar S, Maher JJ. Polyunsaturated fat in the methionine-choline-deficient diet influences hepatic inflammation but not hepatocellular injury. J Lipid Res 2007; 48(8):1885-1896.PM:17526933 1. Williams R. The pervading influence of alcoholic liver disease in hepatology. Alcohol Alcohol 2008; 43(4):393-397.PM:18385413 2. Murray CJ, Richards MA, Newton JN et al. UK health performance: findings of the Global Burden of Disease Study 2010. Lancet 2013; 381(9871):997-1020.PM:23668584 3. Basra S, Anand BS. Definition, epidemiology and magnitude of alcoholic hepatitis. World J Hepatol 2011; 3(5):108-113.PM:21731902 4. Mackie J, Groves K, Hoyle A et al. Orthotopic liver transplantation for alcoholic liver disease: a retrospective analysis of survival, recidivism, and risk factors predisposing to recidivism. Liver Transpl 2001; 7(5):418-427.PM:11349262 5. Miguet M, Monnet E, Vanlemmens C et al. Predictive factors of alcohol relapse after orthotopic liver transplantation for alcoholic liver disease. Gastroenterol Clin Biol 2004; 28(10 Pt 1):845-851.PM:15523219 6. Miller WR, Walters ST, Bennett ME. How effective is alcoholism treatment in the United States? J Stud Alcohol 2001; 62(2):211-220.PM:11327187 7. O'Shea RS, Dasarathy S, McCullough AJ. Alcoholic liver disease. Hepatology 2010; 51(1):307-328.PM:20034030 8. Wang HJ, Gao B, Zakhari S, Nagy LE. Inflammation in alcoholic liver disease. Annu Rev Nutr 2012; 32:343-368.PM:22524187 9. Roychowdhury S, McMullen MR, Pritchard MT et al. An early complement-dependent and TLR-4-independent phase in the pathogenesis of ethanol-induced liver injury in mice. Hepatology 2009; 49(4):1326-1334.PM:19133650 10. Cohen JI, Roychowdhury S, McMullen MR, Stavitsky AB, Nagy LE. Complement and alcoholic liver disease: role of C1q in the pathogenesis of ethanol-induced liver injury in mice. Gastroenterology 2010; 139(2):664-74, 674.PM:20416309 11. Pritchard MT, McMullen MR, Medof ME, Stavitsky A, Nagy LE. Role of complement in ethanol-induced liver injury. Adv Exp Med Biol 2008; 632:175-186.PM:19025122 12. Gao B, Bataller R. Alcoholic liver disease: pathogenesis and new therapeutic targets. Gastroenterology 2011; 141(5):1572-1585.PM:21920463 13. Lieber CS. Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis. Alcohol 2004; 34(1):9-19.PM:15670660 14. Rouach H, Fataccioli V, Gentil M, French SW, Morimoto M, Nordmann R. Effec t of chronic ethanol feeding on lipid peroxidation and protein oxidation in relation to liver pathology. Hepatology 1997; 25(2):351-355.PM:9021946 15. Seki S, Kitada T, Yamada T, Sakaguchi H, Nakatani K, Wakasa K. In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases. J Hepatol 2002; 37(1):56-62.PM:12076862 16. Benedetti A, Comporti M, Esterbauer H. Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids. Biochim Biophys Acta 1980; 620(2):281-296.PM:6254573 17. Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990; 9(6):515-540.PM:2079232 18. Morrow JD, Awad JA, Boss HJ, Blair IA, Roberts LJ. Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids. Proc Natl Acad Sci U S A 1992; 89(22):10721-10725.PM:1438268 19. Roychowdhury S, McMullen MR, Pritchard MT, Li W, Salomon RG, Nagy LE. Formation of gamma-ketoaldehyde-protein adducts during ethanol-induced liver injury in mice. Free Radic Biol Med 2009; 47(11):1526-1538.PM:19616618 20. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci U S A 1990; 87(23):9383-9387.PM:2123555 21. Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc 2013; 8(3):627-637.PM:23449255 22. Fessel JP, Hulette C, Powell S, Roberts LJ, Zhang J. Isofurans, but not F2-isoprostanes, are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease. J Neurochem 2003; 85(3):645-650.PM:12694390 23. Binder CJ. Naturally occurring IgM antibodies to oxidation-specific epitopes. Adv Exp Med Biol 2012; 750:2-13.PM:22903662 24. Chou MY, Hartvigsen K, Hansen LF et al. Oxidation-specific epitopes are important targets of innate immunity. J Intern Med 2008; 263(5):479-488.PM:18410591 25. Lieber CS. Role of oxidative stress and antioxidant therapy in alcoholic and nonalcoholic liver diseases. Adv Pharmacol 1997; 38:601-628.PM:8895826 26. Wu D, Cederbaum AI. Alcohol, oxidative stress, and free radical damage. Alcohol Res Health 2003; 27(4):277-284.PM:15540798 27. Milne GL, Sanchez SC, Musiek ES, Morrow JD. Quantification of F2-isoprostanes as a biomarker of oxidative stress. Nat Protoc 2007; 2(1):221-226.PM:17401357 28. Ivester P, Roberts LJ, Young T et al. Ethanol self-administration and alterations in the livers of the cynomolgus monkey, Macaca fascicularis. Alcohol Clin Exp Res 2007; 31(1):144-155.PM:17207113 29. Raszeja-Wyszomirska J, Safranow K, Milkiewicz M, Milkiewicz P, Szynkowska A, Stachowska E. Lipidic last breath of life in patients with alcoholic liver disease. Prostaglandins Other Lipid Mediat 2012; 99(1-2):51-56.PM:22706383 30. Konishi M, Iwasa M, Araki J et al. Increased lipid peroxidation in patients with non-alcoholic fatty liver disease and chronic hepatitis C as measured by the plasma level of 8-isoprostane. J Gastroenterol Hepatol 2006; 21(12):1821-1825.PM:17074020 31. Davies SS, Bodine C, Matafonova E et al. Treatment with a gamma-ketoaldehy de scavenger prevents working memory deficits in hApoE4 mice. J Alzheimers Dis 2011; 27(1):49-59.PM:21709376 32. Salomon RG. Isolevuglandins, oxidatively truncated phospholipids, and atherosclerosis. Ann N Y Acad Sci 2005; 1043:327-342.PM:16037255 33. Salomon RG, Subbanagounder G, O'Neil J et al. Levuglandin E2-protein adducts in human plasma and vasculature. Chem Res Toxicol 1997; 10(5):536-545.PM:9168251 34. Smathers RL, Galligan JJ, Shearn CT et al. Susceptibility of L-FABP- / - mice to oxidative stress in early-stage alcoholic liver. J Lipid Res 2013; 54(5):1335-1345.PM:23359610 35. Stewart SF, Vidali M, Day CP, Albano E, Jones DE. Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease. Hepatology 2004; 39(1):197-203.PM:14752838 36. Mottaran E, Stewart SF, Rolla R et al. Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease. Free Radic Biol Med 2002; 32(1):38-45.PM:11755315 37. Chedid A, Mendenhall CL, Moritz TE et al. Cell-mediated hepatic injury in alcoholic liver disease. Veterans Affairs Cooperative Study Group 275. Gastroenterology 1993; 105(1):254-266.PM:8514042 38. Lin F, Taylor NJ, Su H et al. Alcohol dehydrogenase-specific T-cell responses are associated with alcohol consumption in patients with alcohol-related cirrhosis. Hepatology 2013; 58(1):314-324.PM:23424168 39. Rao R. Endotoxemia and gut barrier dysfunction in alcoholic liver disease. Hepatology 2009; 50(2):638-644.PM:19575462 40. Hritz I, Mandrekar P, Velayudham A et al. The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88. Hepatology 2008; 48(4):1224-1231.PM:18792393 41. Soares JB, Pimentel-Nunes P, Roncon-Albuquerque R, Leite-Moreira A. The role of lipopolysaccharide / toll-like receptor 4 signaling in chronic liver diseases. Hepatol Int 2010; 4(4):659-672.PM:21286336 42. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990; 249(4975):1431-1433.PM:1698311 43. Markiewski MM, Lambris JD. The role of complement in inflammatory diseases from behind the scenes into the spotlight. Am J Pathol 2007; 171(3):715-727.PM:17640961 44. Li W, Laird JM, Lu L et al. Isolevuglandins covalently modify phosphatidylethanolamines in vivo: detection and quantitative analysis of hydroxylactam adducts. Free Radic Biol Med 2009; 47(11):1539-1552.PM:19751823 45. Bykov I, Jauhiainen M, Olkkonen VM et al. Hepatic gene expression and lipid parameters in complement C3(- / -) mice that do not develop ethanol-induced steatosis. J Hepatol 2007; 46(5):907-914.PM:17321001 46. Bykov I, Junnikkala S, Pekna M, Lindros KO, Meri S. Complement C3 contributes to ethanol-induced liver steatosis in mice. Ann Med 2006; 38(4):280-286.PM:16754259 47. Kalant D, Cain SA, Maslowska M, Sniderman AD, Cianflone K, Monk PN. The chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77 / acylation-stimulating protein. J Biol Chem 2003; 278(13):11123-11129.PM:12540846 48. Schieferdecker HL, Schlaf G, Koleva M, Gotze O, Jungermann K. Induction of functional anaphylatoxin C5a receptors on hepatocytes by in vivo treatment of r ats with IL-6. J Immunol 2000; 164(10):5453-5458.PM:10799912 49. Mack C, Jungermann K, Gotze O, Schieferdecker HL. Anaphylatoxin C5a actions in rat liver: synergistic enhancement by C5a of lipopolysaccharide-dependent alpha(2)-macroglobulin gene expression in hepatocytes via IL-6 release from Kupffer cells. J Immunol 2001; 167(7):3972-3979.PM:11564816 50. Zagol-Ikapitte I, Amarnath V, Jadhav S, Oates JA, Boutaud O. Determination of 3-methoxysalicylamine levels in mouse plasma and tissue by liquid chromatography-tandem mass spectrometry: application to in vivo pharmacokinetics studies. J Chromatogr B Analyt Technol Biomed Life Sci 2011; 879(15-16):1098-1104.PM:21489890 51. Davies SS, Brantley EJ, Voziyan PA et al. Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H2O2-mediated cytotoxicity. Biochemistry 2006; 45(51):15756-15767.PM:17176098 52. Carithers RL, Jr., Herlong HF, Diehl AM et al. Methylprednisolone therapy in patients with severe alcoholic hepatitis. A randomized multicenter trial. Ann Intern Med 1989; 110(9):685-690.PM:2648927 53. Lucey MR, Mathurin P, Morgan TR. Alcoholic hepatitis. N Engl J Med 2009; 360(26):2758-2769.PM:19553649 54. Mathurin P, Mendenhall CL, Carithers RL, Jr. et al. Corticosteroids improve short-term survival in patients with severe alcoholic hepatitis (AH): individual data analysis of the last three randomized placebo controlled double blind trials of corticosteroids in severe AH. J Hepatol 2002; 36(4):480-487.PM:11943418 55. Naveau S, Chollet-Martin S, Dharancy S et al. A double-blind randomized controlled trial of infliximab associated with prednisolone in acute alcoholic hepatitis. Hepatology 2004; 39(5):1390-1397.PM:15122768 56. Mathurin P, Abdelnour M, Ramond MJ et al. Early change in bilirubin levels is an important prognostic factor in severe alcoholic hepatitis treated with prednisolone. Hepatology 2003; 38(6):1363-1369.PM:14647046 57. Akriviadis E, Botla R, Briggs W, Han S, Reynolds T, Shakil O. Pentoxifylline improves short-term survival in severe acute alcoholic hepatitis: a double-blind, placebo-controlled trial. Gastroenterology 2000; 119(6):1637-1648.PM:11113085 58. De BK, Gangopadhyay S, Dutta D, Baksi SD, Pani A, Ghosh P. Pentoxifylline versus prednisolone for severe alcoholic hepatitis: a randomized controlled trial. World J Gastroenterol 2009; 15(13):1613-1619.PM:19340904 59. Mathurin P, Louvet A, Duhamel A et al. Prednisolone with vs without pentoxifylline and survival of patients with severe alcoholic hepatitis: a randomized clinical trial. JAMA 2013; 310(10):1033-1041.PM:24026598 60. Albano E, Vidali M. Immune mechanisms in alcoholic liver disease. Genes Nutr 2010; 5(2):141-147.PM:19809845 61. Stewart SF, Vidali M, Day CP, Albano E, Jones DE. Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease. Hepatology 2004; 39(1):197-203.PM:14752838 62. Stewart SF, Vidali M, Day CP, Albano E, Jones DE. Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease. Hepatology 2004; 39(1):197-203.PM:14752838 63. Cresci GA, Bush K, Nagy LE. Tributyrin supplementation protects mice from acute ethanol-induced gut injury. Alcohol Clin Exp Res 2014; 38(6):1489-1501.PM:24890666 64. Lieber CS. The discovery of the microsomal ethanol oxidizing system and it s physiologic and pathologic role. Drug Metab Rev 2004; 36(3-4):511-529.PM:15554233 65. Perrot N, Nalpas B, Yang CS, Beaune PH. Modulation of cytochrome P450 isozymes in human liver, by ethanol and drug intake. Eur J Clin Invest 1989; 19(6):549-555.PM:2515975 66. Lieberman MA, Marks AD. Ethanol Metabolism. Basic Medical Biochemistry: A Clinical Approach. Wolters Kluwer; 2012. 67. Russell TD, Schaack J, Orlicky DJ et al. Adipophilin regulates maturation of cytoplasmic lipid droplets and alveolae in differentiating mammary glands. J Cell Sci 2011; 124(Pt 19):3247-3253.PM:21878492 68. Zagol-Ikapitte IA, Matafonova E, Amarnath V et al. Determination of the Pharmacokinetics and Oral Bioavailability of Salicylamine, a Potent gamma-Ketoaldehyde Scavenger, by LC / MS / MS. Pharmaceutics 2010; 2(1):18-29.PM:21822464 69. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J 2008; 22(3):659-661.PM:17942826 70. Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. Center for Drug Evaluation and Research. Rockville, MD, USA: U.S. Food and Drug Administration; 2005.

Claims

1. 1. A method for inhibiting or treating liver disease by treating, preventing or ameliorating liver disease in an animal having or at risk of liver disease, comprising administering to a patient a compound of the formula: 【Chemistry 1】 (In the formula: R is N or C; R ２ are independently H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ２ , R ３ and R ４ C containing C, O, S or N optionally substituted with ３～８ 1-membered ring, one or more R ２ , R ３ , or R ５ and optionally substituted C, O, S or N-containing ３～８ may form a membered ring; R ３ is H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ４ , R ２ and R ３ C containing C, O, S or N optionally substituted with ３～８ 1-membered ring, one or more R ２ Or R ５ and optionally substituted C, O, S or N-containing ３～８ may form a membered ring; R ４ is H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ４ , R ２ and R ３ C containing C, O, S or N optionally substituted with ３～８ 1-membered ring, one or more R ２ , R ３ , or R ５ and optionally substituted C, O, S or N-containing ３～８ may form a membered ring; R ５ is a bond, H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ４ , R ２ and R ３ C containing C, O, S or N optionally substituted with ３～８ 1-membered ring, one or more R ２ , R ３ , or R ４ and optionally substituted C, O, S or N-containing ３～８ (May form a membered ring) and stereoisomers and analogs thereof.

2. 2. The method of claim 1, wherein the liver disease further comprises non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD) or non-alcoholic steatohepatitis (NASH).

3. The compound has the formula: 【Chemistry 2】 (In the formula: R is N or C; R ２ is independently H, substituted or unsubstituted alkyl; R ３ is H, halogen, alkoxy, hydroxyl, nitro; R ４ is H, substituted or unsubstituted alkyl, carboxyl. and pharma- ceutically acceptable salts thereof.

4. 2. The method of claim 1, wherein the compound is salicylamine (2-hydroxybenzylamine or 2-HOBA).

5. The compound has the formula: 【Chemistry 3】 or a pharma- ceutically acceptable salt thereof.

6. The compound has the formula: 【Chemistry 4】 or a pharma- ceutically acceptable salt thereof.

7. The compound has the formula: 【Chemistry 5】 or a pharma- ceutically acceptable salt thereof.

8. The compound has the formula: 【Chemistry 6】 or a pharma- ceutically acceptable salt thereof.

9. The compound has the formula: 【Chemistry 7】 or a pharma- ceutically acceptable salt thereof.

10. A pharma- ceutical acceptable carrier and a compound having the formula: 【Chemistry 8】 (In the formula: R is N or C; R ２ are independently H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ２ , R ３ and R ４ C containing C, O, S or N optionally substituted with ３～８ member ring, and one or more R ２ , R ３ , or R ５ and optionally substituted C, O, S or N-containing ３～８ may form a membered ring; R ３ is H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ４ , R ２ and R ３ C containing C, O, S or N optionally substituted with ３～８ 1-membered ring, one or more R ２ Or R ５ and optionally substituted C, O, S or N-containing ３～８ may form a membered ring; R ４ is H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ４ , R ２ and R ３ C containing C, O, S or N optionally substituted with ３～８ 1-membered ring, one or more R ２ , R ３ , or R ５ and optionally substituted C, O, S or N-containing ３～８ may form a membered ring; R ５ is a bond, H, hydroxy, halogen, nitro, CF ３ , C １～６ Alkyl, C １～６ Alkoxy, C ３～１０ cycloalkyl, one or more R ４ , R ２ and R ３ C containing C, O, S or N optionally substituted with ３～８ 1-membered ring, one or more R ２ , R ３ , or R ４ and optionally substituted C, O, S or N-containing ３～８ (May form a membered ring) and nutritional compositions comprising the compound of formula (I), as well as stereoisomers and analogs thereof, and their use as pharmaceutical agents in methods for inhibiting or treating liver disease by treating, preventing or ameliorating liver disease in animals having or at risk of liver disease.

Images