Use of a composition containing amino acids for preparing a medicament for preventing and treating liver disease

Abstract

A composition for use in preventing and / or treating liver disease in a mammal, the composition comprising an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and the carboxylic acids citric acid, succinic acid, malic acid.

Description

Technical Field

[0001] The present disclosure generally relates to compositions comprising amino acids. More specifically, the present disclosure relates to compositions comprising amino acids for preventing and / or treating liver diseases. Background Art

[0002] Liver disease belongs to the most frequently occurring diseases caused by various unfavorable environmental conditions such as parasites and viruses, drugs, toxic substances, alcoholism and smoking. It is usually a chronic disease with worsening clinical features. Chronic liver disease is characterized by: liver parenchyma is gradually destroyed over time, with inflammatory response and fat accumulation, with activation of fibrotic damage and cell transformation, resulting in cancer in many cases. Therefore, liver disease can include fatty degeneration, fibrosis, cirrhosis and hepatocellular carcinoma as the progressive clinical disorder of the liver. Cirrhosis is the result of acute and chronic liver disease, and is characterized by: liver tissue is replaced by fibrotic scar tissue and regenerative nodules, resulting in progressive loss of liver function. Fibrosis and nodular regeneration result in the loss of the normal microscopic lobular structure of the liver. Fibrosis represents scar tissue growth caused by, for example, infection, inflammation, injury and even healing. Over time, fibrotic scar tissue slowly replaces normal functional liver tissue, resulting in a decrease in the amount of blood flowing to the liver, so that the liver cannot fully handle nutrients, hormones, drugs and poisons present in the blood flow. The more common causes of cirrhosis include alcoholism, hepatitis C virus infection, ingestion of toxins, and there are many other possible causes. As mentioned below, the most epidemiologically relevant disorder that progresses to cirrhosis and possibly cancer is increased body fat and associated liver fat accumulation. This fat accumulation can be attributed to two main clinical conditions: alcohol abuse and obesity.

[0003] Excessive and chronic alcohol consumption can lead to alcoholic liver disease (ALD), a major global health problem. The pathological features of ALD develop over a long period of time and include hepatic steatosis, steatohepatitis, cirrhosis and even cancer.

[0004] The pathogenesis of alcohol is multifactorial. Early events such as mitochondrial damage, reactive oxygen species (ROS) generation and fat accumulation have been shown to be direct consequences of ethanol metabolism and are common features between ALD and non-alcoholic liver disease or NAFLD (see below) (Mantena et al. 2008).

[0005] Cellular defense mechanisms against the deleterious effects of alcohol are not well understood. Autophagy, an important degradative cellular pathway for digesting cellular proteins and organelles for energy or eliminating damaged cellular structures, is thought to play a role in ALD, but understanding of these mechanisms remains fragmentary (Lin et al. 2015). Autophagy has been shown to play a key role in both hepatocytes and non-parenchymal cells (i.e., macrophages and hepatic stem cells), affecting insulin sensitivity, lipid accumulation, hepatocyte damage, and innate immune responses.

[0006] Recent studies in mice and cell models have shown that acute ethanol ingestion activates autophagy in the liver (Ding et al. 2010; Ni et al. 2013; Lin et al. 2013).

[0007] In contrast, chronic ethanol intoxication has been shown to inhibit hepatic autophagy (Thomes et al. 2015; Cho et al. 2014). Inhibition of autophagy has been shown to exacerbate ethanol-driven steatosis and liver damage in mice (Ding et al. 2010; Ni et al. 2013; Lin et al. 2013).

[0008] Conversely, pharmacological promotion of autophagy was shown to alleviate ethanol-driven hepatic steatosis and liver damage (Lin et al. 2013). As a result, autophagy is viewed as a protective mechanism against the cytotoxic effects of ethanol and has emerged as a target for the development of therapeutic agents for ALD.

[0009] As mentioned previously, the second leading cause of liver disease is obesity, which is widespread in developed countries and increasing epidemiologically in developing countries as well, and causes the so-called non-alcoholic fatty liver disease or NAFLD. Progression toward steatohepatitis, cirrhosis, and liver cancer is increasingly occurring worldwide. The biochemical and molecular mechanisms involved in this condition overlap with those commonly described in ALD.

[0010] Since altered amino acid metabolism is a hallmark of liver disease associated with both alcohol consumption and obesity, characterized in particular by decreased levels of circulating branched-chain amino acids (BCAAs) (Charlton, 2006), there is growing interest in developing new therapeutic approaches based on amino acid supplementation as treatments for liver disease. Summary of the invention

[0011] The object of the present specification is to provide a novel amino acid-based composition effective in the prevention and treatment of liver diseases.

[0012] According to the present description, the above objects are achieved thanks to the subject matter particularly mentioned in the appended claims, which are to be understood as constituting an integral part of the present disclosure.

[0013] One embodiment of the present specification provides a composition for treating liver disease in a mammal, the composition comprising an active agent comprising amino acids leucine, isoleucine, valine, threonine, lysine and carboxylic acids citric acid, succinic acid, malic acid.

[0014] In one or more embodiments, the active agent of the composition further comprises one or more amino acids selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, cysteine, and tyrosine.

[0015] In a preferred embodiment, the liver disease may be selected from alcoholic liver disease (ADL), non-alcoholic liver disease (NAFDL) lipodystrophy, hepatitis, cirrhosis, hepatocellular carcinoma (HCC).

[0016] Another embodiment of the present disclosure provides a method of treating liver disease in a mammal, the method comprising: selecting a composition comprising an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and the carboxylic acids citric acid, succinic acid and malic acid; administering the composition to treat the liver disease in the mammal. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0018] - Figure 1 Shown are gene expressions shown as mRNA levels in HepG2 cells treated with different amino acid-based compositions and ethanol (EtOH) for 9 days (*P value < 0.05 and **P < 0.01 relative to CTRL cells).

[0019] - Figure 2 Shown are p62 protein levels in HepG2 cells treated with different amino acid-based compositions and EtOH for 9 days (*P value < 0.05 and **P < 0.01 relative to CTRL cells; #P value < 0.05 relative to EtOH-treated cells). DETAILED DESCRIPTION

[0020] In the following description, numerous specific details are given to provide a thorough understanding of some embodiments. These embodiments may be implemented without one or more of the specific details, or with other methods, components, materials, etc. In other cases, well-known structures, materials, or operations are not shown or described in detail to avoid confusing some aspects of the embodiments.

[0021] Reference throughout this specification to "one embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Therefore, the phrase "in one embodiment" appearing in various places throughout this specification does not necessarily all refer to the same embodiment. In addition, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner. The headings provided herein are for convenience only and do not explain the scope or meaning of the embodiments.

[0022] Alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are major global health problems characterized by the development of pathological features over a long period of time, including hepatic steatosis, steatohepatitis, cirrhosis, and eventually cancer.

[0023] Early events in the pathogenesis of alcohol such as mitochondrial damage, reactive oxygen species (ROS) generation, and fat accumulation are common features between ALD and NAFLD.

[0024] Additionally, altered amino acid metabolism has been shown to be a distinguishing feature of liver disease, characterized by low circulating branched-chain amino acid (BCAA) levels, and BCAA supplementation has been shown to be associated with a reduced frequency of complications of cirrhosis when prescribed as maintenance therapy (Charlton 2006).

[0025] The inventors of the present application have found that high effectiveness against liver diseases such as ALD and NAFLD can be achieved by adding specific carboxylic acids to a composition comprising a combination of leucine, isoleucine, valine, threonine, and lysine.

[0026] The compositions disclosed herein have been shown to restore altered amino acid metabolism induced by ethanol, and thus prevent autophagy, the compositions comprising the amino acids leucine, isoleucine, valine, threonine, lysine in combination with three carboxylic acids as active agents, the three carboxylic acids being substrates for the tricarboxylic acid cycle, including specific amounts of citric acid, succinic acid, and malic acid. The tricarboxylic acid cycle (TCA cycle) (also known as the Krebs cycle and the citric acid cycle) carried out in the mitochondrial matrix is ​​the second stage of cellular respiration, a three-stage process by which living cells break down organic fuel molecules in the presence of oxygen to harvest the energy required for their growth and division.

[0027] Compositions comprising the above active agents, as well as compositions comprising the above active agents with additional specific amino acids (listed in Table 1 below), are significantly more effective than similar amino acid compositions without such specific carboxylic acids.

[0028] In one or more embodiments, in the composition disclosed herein, the weight ratio between the total amount of citric acid, succinic acid and malic acid and the total amount of amino acids leucine, isoleucine, valine, threonine, lysine is 0.05 to 0.3, preferably 0.1 to 0.25.

[0029] In one or more embodiments, the active agent may further comprise one or more amino acids selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, cysteine, and tyrosine.

[0030] In one or more embodiments, the carboxylic acids included in the composition consist of citric acid, succinic acid, and malic acid.

[0031] In another embodiment, the active agent of the composition disclosed herein may further include aspartic acid and / or ornithine L-alpha ketoglutarate (OKG).

[0032] According to one embodiment, the composition comprises an active agent consisting of leucine, isoleucine, valine, threonine, lysine, histidine, phenylalanine, methionine, tryptophan, cysteine ​​and optionally tyrosine, and citric acid, succinic acid, and malic acid, the amino acids being the only amino acids contained in the composition. Citric acid, succinic acid and malic acid can be the only carboxylic acids contained in the composition.

[0033] In another embodiment, the composition may comprise the amino acids isoleucine, leucine and valine in an amount ranging from 35% to 65% by weight, preferably from 42% to 56% by weight, relative to the weight of the active agent.

[0034] In one or more embodiments, the weight ratio between leucine and citric acid is 5 to 1, preferably 2.50 to 3.50.

[0035] In another embodiment, the weight or molar amount of citric acid is greater than the weight or molar amount of each of malic acid and succinic acid. Preferably, the weight or molar amount of citric acid is greater than the weight or molar amount of malic acid plus succinic acid. In another embodiment, the weight ratio between citric acid and the sum of malic acid and succinic acid is 1.0 to 4.0, preferably 1.5 to 2.5. In a preferred embodiment, the weight ratio of citric acid: malic acid: succinic acid is 10: 1: 1 to 2: 1.5: 1.5, preferably 7: 1: 1 to 1.5: 1: 1, more preferably 5: 1: 1 to 3: 1: 1. In a preferred embodiment, the weight ratio of citric acid: malic acid: succinic acid is 4: 1: 1.

[0036] According to some embodiments of the present disclosure, the preferred isoleucine:leucine molar ratio is 0.2 to 0.7, preferably 0.30 to 0.60, and / or the preferred valine:leucine weight ratio is 0.2 to 0.70, preferably 0.30 to 0.65.

[0037] In another embodiment, the threonine:leucine molar ratio is from 0.10 to 0.90, preferably from 0.20 to 0.70, and / or the lysine:leucine weight ratio is from 0.20 to 1.00, preferably from 0.40 to 0.90.

[0038] In a preferred embodiment, the ratio between the total molar amount of citric acid, malic acid, succinic acid and the total molar amount of methionine, phenylalanine, histidine and tryptophan is higher than 1.35.

[0039] In one or more embodiments, the weight ratio between the sum of citric acid, malic acid, succinic acid and the sum of branched-chain amino acids leucine, isoleucine, valine is 0.1 to 0.4, preferably 0.15 to 0.35.

[0040] In another embodiment, the total weight of the branched-chain amino acids leucine, isoleucine, valine plus threonine and lysine is greater than the total weight of three carboxylic acids, such as citric acid, malic acid and succinic acid. Preferably, the weight of a single carboxylic acid (citric acid, succinic acid or malic acid) is less than the weight of each of the single amino acids leucine, isoleucine, valine, threonine and lysine.

[0041] In another embodiment, the total molar amount of lysine and threonine is higher than the total molar amount of the three carboxylic acids citric acid, succinic acid, malic acid. Preferably, the ratio between the total molar amount of the three carboxylic acids citric acid, succinic acid, malic acid and the total molar amount of lysine and threonine is 0.1 to 0.7, preferably 0.15 to 0.55.

[0042] In one or more embodiments, the composition disclosed herein further comprises a vitamin, preferably selected from vitamin B, such as vitamin B1 and / or vitamin B6.

[0043] In another embodiment of the present disclosure, the composition may comprise carbohydrates, additives and / or flavoring substances.

[0044] In a preferred embodiment, the composition is specifically for the prevention and / or treatment of a liver disease selected from the group consisting of fatty liver disease, lipodystrophy, hepatitis, cirrhosis, hepatocellular carcinoma (HCC).

[0045] In one or more embodiments, the liver disease is fatty liver disease. Fatty liver disease can be caused by alcohol consumption (alcoholic fatty liver disease, ALD).

[0046] In one or more embodiments, the fatty liver disease is non-alcoholic fatty liver disease (NFLD).

[0047] In one or more embodiments, fatty liver disease can be caused by drugs or toxins, such as, for example, amiodarone, methotrexate, diltiazem, highly active antiretroviral therapy, glucocorticoids, tamoxifen, mushroom poisoning.

[0048] Furthermore, in particular when preparing compositions according to the present disclosure and in particular active agents, the amino acid arginine is preferably avoided. In addition, other amino acids that are preferably avoided in the compositions disclosed herein may be serine, proline, alanine. Such amino acids may be counterproductive or even harmful in the composition at certain concentrations or stoichiometric ratios.

[0049] The amino acids disclosed in this application may be replaced by the corresponding pharmaceutically acceptable derivatives (ie, salts).

[0050] As will become apparent hereinafter, administration of compositions according to the present disclosure is particularly effective in preventing and / or treating liver disease.

[0051] In a preferred embodiment, the disclosed compositions can be used to treat alcoholic liver disease (ALD).

[0052] According to another embodiment, the amino acid composition may comprise a pharmaceutically acceptable excipient, such as, for example, protein, vitamins, carbohydrates, natural and artificial sweeteners and / or flavoring substances. In a preferred embodiment, the pharmaceutically acceptable excipient may be selected from whey protein, maltodextrin, fructose, calcium caseinate, fish oil, sucralose, sucrose esters, vitamin D3, B vitamins.

[0053] For oral use, the composition according to the present description may be in the form of tablets, capsules, granules, gels, gellable powders or powders.

[0054] Further descriptions on the amounts of various amino acids provided by the composition and the ratios therebetween are contained in the appended claims, which form an integral part of the technical teaching provided herein with respect to the present invention.

[0055] Example

[0056] Table 1 shows two different amino acid based compositions tested in vitro on hepatocytes (HepG2 cells) and in vivo on ethanol consuming rats, as disclosed below.

[0057] The composition hereinafter referred to as "BCAAem" comprises an active agent containing the amino acids leucine, lysine, isoleucine, valine, threonine, cysteine, histidine, phenylalanine, methionine, tyrosine, tryptophan.

[0058] A composition called "alpha 5m (α5m)" contains an active agent comprising the same amino acids plus citric acid, succinic acid and malic acid.

[0059] Table 1

[0060]

[0061] The composition of Table 1 above can be prepared by first screening all components with a 0.8 sieve. To obtain a premix, each ingredient (in an amount <10% by weight of the total amount) is placed in a polyethylene bag together with a portion of L-Lysine HCl to obtain 10% weight of the total composition. The bag is then manually shaken for 5 minutes. The premix is ​​then loaded in a mixer (Planetaria) with the remaining ingredients and mixed at 120 rpm for a period of 15 minutes to obtain a uniform final composition.

[0062] method

[0063] Animals and handling

[0064] The experimental protocol was approved and performed according to the European Communities Council Directive of 24 November 1986 (86 / 609 / EEC) and the Italian Ministry of Heath and complied with the National Animal Protection Guidelines.

[0065] Male Wistar rats (3 months old) from Charles River (Calco, Como, Italy) were used for the experimental analyses disclosed below.

[0066] Animals were housed individually in clean polypropylene cages and divided into six groups:

[0067] 1) Pair-fed group (pair-fed CTRL, n = 6) was fed with a control liquid diet in which ethanol (EtOH) was replaced by isocaloric maltodextrin;

[0068] 2) EtOH group (EtOH, n = 7) fed ad libitum with a Lieber-DeCarli liquid diet containing EtOH [the amount of EtOH was gradually increased to reach 36% of caloric intake after 1 week, corresponding to a final concentration of 6.2% (vol / vol)];

[0069] 3) BCAAem group (BCAAem, n = 6), which was fed with a control liquid diet in which EtOH was replaced by isocaloric maltodextran and supplemented with a branched-chain amino acid composition providing 1.5 g / kg / day BCAAem (“BCAAem” in Table 1 );

[0070] 4) α5 group (α5m, n = 6), which was fed with a control liquid diet in which EtOH was replaced by isocaloric maltodextran and supplemented with an amino acid composition providing 1.5 g / kg / day (“α5m” in Table 1 );

[0071] 5) an EtOH plus BCAAem group (EtOH+BCAAem, n=7), which was fed ad libitum with a Lieber-DeCarli liquid diet containing a combination of EtOH and BCAAem; and

[0072] 6) EtOH plus α5m group (EtOH+α5m, n=7), which was fed ad libitum with a Lieber-DeCarli liquid diet containing a combination of EtOH and α5m.

[0073] Sample preparation

[0074] The liver (n = 4 animals / group) was weighed, homogenized in cold methanol: water (v / v, 1:1), and extracted according to Want et al. (Want et al. 2013). The vacuum-dried samples were suspended in 120 μl / 50 mg of methanol: 1 mM TDFHA = 1:1 and centrifuged at 16,000g for 10 minutes at 4°C. 2 μl of the supernatant was directly loaded onto the UPLC mass spectrometer and analyzed as reported below. Four technical replicates were performed for each sample using three different methods.

[0075] Chromatography and Quantification of Amino Acids in Liver

[0076] Standard amino acids were purchased from Sigma (Milan, Italy). Each amino acid stock solution was prepared at 1 mg / ml in water, diluted to a final concentration of 3 pmol / μl, and injected directly into a TripleTOF 5600+ mass spectrometer (AB Sciex, Milan, Italy) at 10 μl / min via a syringe. Therefore, the declustering potential (DP) and collision energy (CE) were optimized for each amino acid.

[0077] Next, three amino acid mixtures were prepared based on DP and CE values: MIX 1, containing threonine, asparagine, tyrosine and serine, and analyzed with DP: 30 V, CE: 15 V; MIX 2, containing glycine, alanine, leucine, isoleucine, valine, proline, histidine, methionine, aspartic acid, glutamine and phenylalanine, and analyzed with DP: 40 V, CE: 15 V; and MIX 3, containing glutamate, lysine, arginine and tryptophan, and analyzed with DP: 80 V, CE: 18 V.

[0078] Source parameters were: Gas 1: 33 psi, Gas 2: 58 psi, Curtain Gas: 25 psi, Temperature: 500°C, and ISVF (Ion Spray Voltage Floating): 5500V.

[0079] To obtain the calibration curve, different amounts (10, 33, 50, 100, 200, 400 pm ol) of the three mixtures were injected in quadruplicate into the mass spectrometer after UPLC separation using UPLC 1290 (Agilent Technologies Italia, Cernusco sul Naviglio, Milan, Italy). The chromatographic column was from Waters, Acquity HSS T3 C18 2.1 x 100 mm, 1.7 μm, while the mobile phase was A: 1 mM TDFHA (tridecafluoroheptanoic acid) in water; B: 1 mM TDFHA in acetonitrile. A gradient of B from 12.5% ​​to 26.5% in 4 minutes and then from 26.5% to 92% in 3.5 minutes was used to separate all amino acids, with a flow rate of 0.35 ml / min and a column temperature of 65°C, as described (Le et al., 2014).

[0080] The autosampler was set to 4°C. Calibration curves were drawn using chromatographic peak areas and weighted regression (all compounds were 1 / x except asparagine, tyrosine, valine and glutamic acid were fitted to 1 / x2) by MultiQuant software version 2.1 (SCIEX). The quantitative values ​​(pmol) of each amino acid in rat liver samples were obtained by correlating the chromatographic peak areas with the chromatographic peak areas of calibration standards derived from external runs and normalizing for tissue (mg).

[0081] Cell culture and treatment

[0082] Human HCC HepG2 cells were purchased from the American Type Culture Collection (HB-8065; ATCC, Manassas, VA). Cells were routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 μg / mL) at 37°C in an atmosphere with 5% CO2. Each 75 cm 2 Flasks (Corning Inc., Corning, NY) were seeded with 2 million HepG2 cells.

[0083] Six hours after inoculation, 75mM (0.34%) EtOH and 1% BCAAem or α5m were added alone or in combination. Untreated cell plates were inoculated as controls. Every 24 hours, the culture medium in the control flask and the treatment flask was replaced with fresh culture medium with or without EtOH and BCAAem or α5m, respectively. Four days after inoculation, the cells were trypsinized and inoculated into new flasks with 2 million living cells per flask, and the culture medium was changed every day, as previously described (Pochareddy et al. 2012). Five days after the separation process (9 days in total, with or without EtOH, BCAAem or α5m, or EtOH plus BCAAem or α5m), the harvested cells were reported as follows for different determinations.

[0084] Quantitative RT-PCR analysis

[0085] Quantitative RT-PCR reactions were performed as described (Tedesco et al. 2008) and run on the iCycler iQ Real-Time PCR Detection System (Bio-Rad) using iQ SybrGreen I SuperMix (Bio-Rad; Segrate, Italy).

[0086] In short, use RNA was isolated from tissues using the QIAQUICK tissue mini kit (Qiagen, Milan, Italy). cDNA was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad Laboratories, Segrate, Italy).

[0087] Primers were designed using Beacon Designer 2.6 software from Premier Biosoft International (sequences are reported in Table 2 below). The cycle number at which multiple transcripts can be detected (threshold cycle, CT) was compared to the cycle number for TBP, referred to as ΔCT. Relative gene levels were expressed as 2-(ΔΔCT), where ΔΔCT equals ΔCT of rats treated with EtOH- or BCAAem- or CAA mixtures (or treated HepG2 cells) minus ΔCT of control rats (or untreated HepG2 cells).

[0088] Table 2

[0089]

[0090] T a Annealing temperature (°C); Accession number PGC-1α: NM_013261; Accession number Tfam: NM_009360.4; Accession number NRF1: NM_005011; Accession number Cytc: JF919224.1; Accession number TBP: NG_051572 were used to normalize gene expression.

[0091] Western blot analysis

[0092] Protein extracts were obtained from livers using T-PER mammalian protein extraction reagent (Pierce, ThermoScientific, Rockford, USA) in the presence of a protease and phosphatase inhibitor cocktail (Sigma Aldrich, Milan, Italy) as described by the manufacturer. Protein content was measured by bicinchoninic acid protein assay (BCA, Pierce, Euroclone, Milan, Italy), and 50 μg of protein was run on SDS-PAGE under reducing conditions. The separated proteins were then electrophoretically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Segrate, Italy). The target proteins were visualized with specific antibodies: anti-p62 and anti-α-actin (all from Cell Signaling, Euroclone, Milan, Italy), each diluted 1:1000. Immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulins at room temperature for 1 hour. The amount of protein was measured using SuperSignal substrate (Pierce, Euroclone, Milan, Italy) and quantified by densitometry using the IMAGEJ software image analyzer.

[0093] Statistical analysis

[0094] For all gene expression data, values ​​between control cells and treated cells were compared using a two-sided paired sample t-test. P values ​​< 0.05 were considered statistically significant.

[0095] result

[0096] Combination α5m is more effective than BCAAem combination in restoring hepatic mitochondrial biogenesis impaired by EtOH consumption and functions

[0097] The ability of the BCAAem and α5m combination to improve mitochondrial biogenesis and function impaired by EtOH exposure was evaluated.To investigate the molecular mechanisms involved in the effects of the BCAAem and α5m combination, an in vitro model of hepatic EtOH toxicity was used.

[0098] To this end, hepatic HepG2 cells were exposed to 75 mM EtOH for 9 days in the presence or absence of BCAAemα5m compositions. Proliferator-activated receptor gamma coactivator 1α (PGC-1α), nuclear respiratory factor-1 (NRF-1), mitochondrial DNA transcription factor A (Tfam), and cyt c mRNA levels were unchanged or slightly lower in HepG2 cells exposed to 75 mM EtOH for 9 days compared to untreated control cells ( Figure 1 ).

[0099] However, administration of the BCAAem and α5m combination for 9 days increased PGC-1α and Tfam mRNA levels relative to untreated cells and EtOH-treated cells ( Figure 1 ).

[0100] Notably, the potency of the α5m composition in improving markers of hepatic mitochondrial biogenesis was statistically greater than that of BCAAem.

[0101] Autophagy analysis

[0102] Autophagic flux can be inferred by combining several markers, including p62 / SQSTM1 protein levels and LC3II / LC3I ratio, Beclin1 and Atg7, and 4EBP1 phosphorylation (Klionsky et al., 2016). p62 / SQSTM1 has been shown to be incorporated into autophagosomes and degraded in autolysosomes. Therefore, reduced p62 / SQSTM1 protein levels indicate increased autophagy (Klionsky et al. 2016).

[0103] As this application Figure 2 As shown in , p62 protein levels were lower in HepG2 cells exposed to 75 mM EtOH for 9 days than in untreated control cells.

[0104] However, after 9 days of administration of the BCAAem or α5m composition, p62 protein levels were higher relative to p62 levels in untreated and EtOH-treated cells.

[0105] Also in this case, the potency of the α5m composition was statistically higher than that of the BCAAem composition.

[0106] Liver amino acid quantification

[0107] As disclosed above, altered amino acid metabolism, specifically low circulating BCAA levels, has been shown to be a distinguishing feature of alcoholic liver disease (Charlton, 2006).

[0108] The data presented below refer to the effects of BCAAem or α5m compositions on amino acid metabolism in the liver of chronic EtOH-depleted rats.

[0109] For this purpose, free amino acid levels in liver tissue of rats consuming EtOH alone or in combination with the tested compositions were measured using chromatographic analysis.

[0110] Supplementation with the BCAAem and α5m combination had no effect on the levels of arginine, leucine, and tryptophan, as reported in Table 3. In contrast, EtOH consumption resulted in a decrease in the levels of these amino acids in the liver.

[0111] Interestingly, administration of the BCAAem and α5m combination prevented the decrease in arginine, leucine, and tryptophan in EtOH-depleted rats.

[0112] Furthermore, isoleucine, serine, tyrosine, and valine concentrations were lower in the liver of mice exposed to the EtOH-containing diet.

[0113] Although BCAAem supplementation could not prevent their decrease, α5m supplementation was able to effectively prevent the EtOH-induced decrease in isoleucine and valine.

[0114] The concentrations of the remaining amino acids had no statistical differences among the groups.

[0115] These results are consistent with the specific ability of the α5m composition compared to the BCAAem composition to re-normalize BCAA levels in liver samples of EtOH-depleted rats.

[0116] Table 3

[0117]

[0118] Values ​​are reported as mean ± SD (pmol / mg tissue), n = 4 animals / group; *P < 0.05 relative to the CTRL group;

[0119] # Compared with the EtOH group, P value < 0.05.

[0120] In summary, the in vitro and in vivo results indicate that dietary supplementation with a composition comprising active agents including a combination of leucine, isoleucine, valine, threonine, lysine, citric acid, succinic acid, and malic acid is significantly effective in preventing mitochondrial damage in hepatocytes exposed to EtOH.

[0121] This effect was also accompanied by a decrease in autophagy (ie, increased p62 protein levels) that was enhanced by EtOH exposure (ie, decreased p62 protein levels).

[0122] EtOH itself may enhance autophagy to compensate for EtOH toxicity and, in particular, mitochondrial damage, which is evident in alcohol-exposed hepatocytes and in the liver of alcohol-consuming animals.

[0123] In fact, autophagy is a cellular mechanism aimed at eliminating dysfunctional organelles, including mitochondria. When hepatocytes are exposed to alcohol and mitochondrial function is reduced, increased autophagy, as also disclosed in this application, can be used to eliminate dysfunctional mitochondria.

[0124] The amino acid composition disclosed herein can prevent mitochondrial damage so that cells do not need autophagy to maintain their vitality function, and the composition comprises a combination of leucine, isoleucine, valine, threonine, lysine and citric acid, succinic acid and malic acid. Therefore, autophagy is reduced (as shown by the increase in p62 protein levels shown in the application).

[0125] Furthermore, and most importantly, the disclosed compositions have been found to be highly effective in restoring free BCAA, arginine, and tryptophan concentrations in the liver of EtOH-depleted rats.

[0126] Notably, preliminary results indicate that the amino acid composition disclosed herein comprising a combination of leucine, isoleucine, valine, threonine, lysine and citric acid, succinic acid and malic acid can also reduce the diameter of lipid droplets in the hepatocytes of mice exposed to a high-fat diet (HFD, 60% of calories from fat) for 6 months, which is a widely used mouse model of NAFLD. In summary, these results strongly support the view that the amino acid composition disclosed herein and active in alcohol-dependent hepatotoxicity can be healthy in preventing the occurrence and exacerbation of NAFLD.

[0127] From the foregoing it is apparent that the compositions according to the present disclosure may be used to prevent and / or treat liver diseases which affect a large portion of the adult population worldwide and increase societal costs.

[0128] References

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Claims

1. A composition for preparing a medicament for preventing and / or treating liver disease in a mammal, the composition comprising an active agent, the active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and the carboxylic acids citric acid, succinic acid, malic acid, in, The composition avoids the amino acids arginine, serine, proline, alanine, The weight ratio of the total amount of citric acid, malic acid, and succinic acid to the total amount of leucine, isoleucine, valine, lysine, and threonine is 0.05 to 0.

3. Wherein, the liver disease is selected from alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD).

2. The use of the composition according to claim 1, wherein the weight ratio between the total amount of citric acid, malic acid, and succinic acid and the total amount of leucine, isoleucine, valine, lysine, and threonine is 0.1 to 0.

25.

3. The use of the composition according to claim 1 or 2, wherein the weight ratio between the total amount of citric acid, malic acid, succinic acid and the total amount of leucine, isoleucine, and valine is 0.15 to 0.

35.

4. Use of a composition according to claim 1 or 2, wherein the weight ratio between citric acid and the sum of malic acid and succinic acid is from 1.0 to 4.

0.

5. Use of a composition according to claim 4, wherein the weight ratio between citric acid and the sum of malic acid and succinic acid is between 1.5 and 2.

5.

6. Use of the composition according to claim 1 or 2, wherein the weight ratio of citric acid:malic acid:succinic acid is 10:1:1 to 2:1.5:1.

5.

7. Use of the composition according to claim 6, wherein the weight ratio of citric acid:malic acid:succinic acid is 7:1:1 to 1.5:1:

1.

8. Use of the composition according to claim 7, wherein the weight ratio of citric acid:malic acid:succinic acid is 5:1:1 to 3:1:

1.

9. The use of the composition according to claim 1 or 2, wherein the active agent further comprises at least one amino acid selected from histidine, phenylalanine, methionine, tryptophan, tyrosine, and cysteine.

10. Use of the composition according to claim 9, wherein the active agent further comprises histidine, phenylalanine, methionine, tryptophan, cysteine ​​and tyrosine.

11. Use of the composition according to claim 9, wherein the ratio between the total molar amount of citric acid, malic acid, succinic acid and the total molar amount of methionine, phenylalanine, histidine and tryptophan is higher than 1.

35.

12. Use of the composition according to claim 1 or 2, wherein the ratio between the total molar amount of the three carboxylic acids citric acid, succinic acid, malic acid and the total molar amount of lysine and threonine is 0.10 to 0.

70.

13. Use of the composition according to claim 12, wherein the ratio between the total molar amount of the three carboxylic acids citric acid, succinic acid, malic acid and the total molar amount of lysine and threonine is 0.15 to 0.

55.

14. Use of the composition according to claim 1 or 2, wherein the molar amount of citric acid is higher than the total molar amount of malic acid and succinic acid.

15. Use of the composition according to claim 1 or 2, wherein the weight ratio between leucine and citric acid is 5 to 1.

16. Use of the composition according to claim 15, wherein the weight ratio between leucine and citric acid is 2.50 to 3.

50.

17. Use of the composition according to claim 1 or 2, wherein the composition further comprises one or more vitamins.

18. Use of a composition according to claim 17, wherein the one or more vitamins are selected from vitamin B.

19. Use of the composition according to claim 18, wherein the one or more vitamins are vitamin B1 and / or vitamin B6.

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