A composition containing amino acids for use in the prevention and / or treatment of intestinal diseases

Abstract

A composition for preventing and / or treating intestinal diseases, which has an active agent, and the active agent includes amino acids such as leucine, isoleucine, valine, threonine, lysine, and citric acid, succinic acid, malic acid or salts thereof.

Description

Technical Field

[0001] This specification generally relates to compositions containing amino acids for use in the prevention and / or treatment of intestinal diseases, particularly diseases associated with inflammation and changes in permeability.

Background Art

[0002] The intestinal mucosal barrier (IMB) is extremely important for nutrient absorption and the health of both humans and animals. Recent publications from clinical and experimental studies have shown that nutrient-bacteria-host interactions are important for intestinal homeostasis. Dysfunction of nutrient-bacteria-host interactions has been reported to be associated with the development of metabolic disorders and intestinal diseases, such as irritable bowel syndrome and inflammatory intestinal diseases (IBD), two major types of IBD, ulcerative colitis (UC), and Crohn's disease (CD). On the other hand, systemic disorders including obesity and type 2 diabetes, and certain diseases such as kidney disorders, have been associated with IMB dysfunction and loss.

[0003] IBD causes chronic intestinal inflammation, and as a result, recurrent and remitting symptoms including abdominal pain, diarrhea, anemia, and weight loss (Solberg et al., Scand. J. Gastroenterol. 44, 431-440, 2009; Solberg et al., Clin. Gastroenterol. Hepatol. 5, 1430-1438, 2007). The clinical course of IBD varies widely both between individuals and over the lifetime of an individual. Periods of clinically inactive disease can be interrupted by acute flares, and medication, hospitalization, and sometimes intestinal surgery may be required. The pathogenesis of IBD is caused by an abnormal, long-term T cell-mediated immune response to the commensal gut microbiota that occurs in genetically susceptible individuals. Known IBD risk genes are associated with various immune functions, including innate immune functions such as physical barriers and autophagy. Current models suggest that multiple factors are involved in the pathogenesis of IBD, including histological and cellular level changes leading to macroscopic erosion and ulcers, including multi-layer mucosal injury. Disruption of the intestinal barrier can occur following depletion of the intestinal tight junction (TJ) structure, a protein structure located at the apical portion of the intestinal epithelial cells that connects both epithelial and endothelial cells. Disruption of the intestinal barrier (wounding) can cause the translocation of microorganisms and other antigens into the intestinal wall, which can result in uncontrolled immune activation, a major feature of IBD (Neurath, M.F. & Travis, S.P.G. Gut 61, 1619-1635, 2012). The structural basis for healed mucosa is an intact barrier that limits bacterial translocation and associated immune activation. Thus, a functional definition of mucosal healing can be described as mucosa with restored barrier function. Almost all currently available therapies for IBD act by suppressing inflammation, often by blocking specific inflammatory molecules. However, given the burden of infectious and neoplastic diseases associated with chronic immunosuppressive therapy, the goal of achieving mucosal healing without immunosuppression is attractive.

[0004] In addition to the physical barrier composed of epithelial cells, the production of proteins, TJs, and intracellular mechanisms to cope with invading pathogens is very important for maintaining homeostasis and must be regenerated during the process of mucosal healing. Notably, the intestinal epithelium is not static but very dynamic, renewed every 5 - 7 days, and can continuously protect immune cells from the abundant luminal bacterial flora. Mucin is a glycoprotein produced and secreted by goblet cells and is considered a major component of the mucus layer that separates commensal bacteria from the epithelium. Microbial control is an important part of the functional barrier. Paneth cells are located at the bottom of the intestinal crypts of the stem cell niche and are the main source of antimicrobial peptides (AMPs).

[0005] Over the past 20 years, complete mucosal healing has been confirmed as an important treatment goal for IBD. For example, in the Inflammatory Bowel South-Eastern Norway (IBSEN) study, mucosal healing at the outpatient clinic in the first year after diagnosis was associated with a reduced risk of future colectomy in patients with ulcerative colitis and a reduction in inflammation after 5 years and a decrease in future steroid treatment in patients with Crohn's disease (Froslie et al., Gastroenterology 133, 412 - 422, 2007).

[0006] Although treatment strategies that directly promote mucosal healing are still lacking, promoting mucosal healing without immunosuppression is an attractive goal. For this purpose, dozens of pathways that promote mucosal healing have been identified. However, most of them may lead to excessive proliferation of intestinal epithelium and, in turn, tumor growth.

Summary of the Invention

[0007] This specification aims to provide an amino acid-based composition that is particularly effective in preventing and / or treating intestinal diseases in subjects associated with changes in intestinal inflammation and permeability by promoting mucosal healing without exerting immunosuppressive effects and / or uncontrolled intestinal cell proliferation.

[0008] According to this specification, the above object is achieved by the subject matter specifically recited in the following claims, which are to be regarded as an integral part of this disclosure.

[0009] One embodiment of this specification provides a composition for preventing and / or treating intestinal diseases in a subject, the composition having an active agent, and the aforementioned active agent includes amino acids leucine, isoleucine, valine, threonine, lysine, and carboxylic acids citric acid, succinic acid, malic acid or acceptable salts thereof.

[0010] The intestinal disease may be selected from the group consisting of irritable intestinal disease (IBS) and inflammatory intestinal disease (IBD), and IBD may here be selected from between Crohn's disease and ulcerative colitis.

[0011] In one or more embodiments, the active agent of the composition may further include one or more amino acids selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, cysteine, tyrosine.

[0012] This disclosure also provides a method for preventing and / or treating intestinal diseases in a subject. The method can prevent and / or treat an intestinal disease selected from the group consisting of irritable intestinal disease (IBS) and inflammatory intestinal disease (IBD), where IBD may preferably be selected from between Crohn's disease and ulcerative colitis. The method comprises the steps of selecting a composition having an active agent, the aforementioned active agent including amino acids leucine, isoleucine, valine, threonine, lysine, and carboxylic acids citric acid, succinic acid, and malic acid, and administering the composition to the subject. The active agent may further include one or more amino acids selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, cysteine, tyrosine as disclosed in this specification.

Brief Description of the Drawings

[0013] Hereinafter, the present invention will be described for illustrative purposes only with reference to the accompanying drawings.

[0014]

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Mode for Carrying Out the Invention

[0015] The following description sets forth numerous specific details in order to provide a thorough understanding of the embodiments. The embodiments may be practiced without one or more of the specific details, or in combination with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0016] References to "one embodiment" or "an embodiment" throughout this specification mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0017] This specification aims to provide an amino acid-based composition that is particularly effective for the prevention and / or treatment of intestinal diseases in a subject. The intestinal disease may be selected from the group consisting of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), where IBD may preferably be selected from between Crohn's disease and ulcerative colitis.

[0018] The composition for use in the prevention and / or treatment of intestinal diseases in a subject has an active agent, and the aforementioned active agent includes the amino acids leucine, isoleucine, valine, threonine, lysine, and citric acid, succinic acid, malic acid.

[0019] In one or more embodiments, the subject may be a subject suffering from chronic kidney disease (CKD), and the intestinal disease may preferably be selected from between irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), where IBD may be selected from between Crohn's disease and ulcerative colitis.

[0020] The inventors of the present application have surprisingly found that the compositions disclosed herein are particularly effective in improving intestinal inflammation and permeability, representing two mutually regulating factors where inflammation due to the presence of cytokines disrupts the TJs, the dysregulated TJs then enable intestinal translocation, and both local and systemic inflammation are activated.

[0021] Specifically, in subjects suffering from chronic kidney disease (CKD), the fecal levels of calprotectin, a marker of intestinal inflammation, and zonulin, a marker of IMB dysfunction, were measured, where IMB dysfunction is a risk factor for the progression of chronic CKD. In particular, in CKD, the combination of enteritis and intestinal permeability can cause disruption of the intestinal barrier, and luminal bacteria and toxic bacterial by-products (enterotoxins) migrate into the systemic circulation (endotoxemia). Endotoxemia is the cause of the onset of systemic inflammation, acceleration of CKD progression, uremic syndrome, cardiovascular disease, and increased risk of death, and has been recorded in both animals and humans with advanced CKD. In CKD, intestinal dysbiosis, i.e., changes in the intestinal microbiota, is a major factor in enteritis. For example, the duodenum and jejunum, where bacteria do not colonize under physiological conditions, are colonized by aerobic and anaerobic bacteria in CKD, causing chronic inflammation of the gastrointestinal tract such as esophagitis, gastritis, duodenitis, enteritis, and colitis. The increased intestinal permeability in patients with CKD follows the disruption and depletion of intestinal TJ structures caused by uremia, which leads to the loss of barrier homeostasis and increased intestinal permeability.

[0022] The measured increases in fecal calprotectin and zonulin in subjects with CKD were selected for the clinical studies provided in the following section because the two markers have previously been used for the diagnosis of intestinal inflammation (Ayling RM Adv Clin Chem 2018 87:161 - 190) and permeability (Fasano A Clin Gastroenterol Hepatol 2012 10:1096 - 1100), respectively.

[0023] The inventors of the present application provide herein evidence suggesting that baseline fecal calprotectin and zonulin are significantly higher in CKD patients compared to healthy subjects who have not ingested them, and that nephropathy promotes intestinal inflammation and IMB dysfunction.

[0024] After ingesting the compositions disclosed herein for 6 months, the levels of calprotectin and zonulin decreased significantly in fecal samples of CKD patients compared to their pre-treatment levels, thus providing evidence of the effectiveness of the compositions in the treatment of intestinal diseases related to inflammation and permeability.

[0025] The composition administered to CKD patients has an active agent containing citric acid, succinic acid, and malic acid in combination with leucine, isoleucine, valine, threonine, and lysine.

[0026] The weight ratio of the total amount of citric acid, succinic acid, and malic acid to the total amount of the amino acids leucine, isoleucine, valine, threonine, and lysine can be included in the range of 0.05 to 0.3, preferably 0.1 to 0.25.

[0027] In one or more embodiments, the composition can consist of leucine, isoleucine, valine, threonine, lysine, citric acid, succinic acid, and malic acid, and optionally vitamin B1 and / or vitamin B6.

[0028] In one or more embodiments, the active agent can further include one or more amino acids selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, cysteine, and tyrosine.

[0029] In one or more embodiments, the composition may have an active agent consisting of leucine, isoleucine, valine, threonine, lysine, histidine, phenylalanine, methionine, tryptophan, cysteine, tyrosine, and citric acid, succinic acid and malic acid, and the aforementioned amino acids are the only amino acids contained in the composition. In one or more embodiments, the composition does not contain any additional amino acids.

[0030] In one or more embodiments, the composition may consist of leucine, isoleucine, valine, threonine, lysine, histidine, phenylalanine, methionine, tryptophan, cysteine, citric acid, succinic acid and malic acid, and optionally vitamin B1 and / or vitamin B6.

[0031] The composition may contain the amino acids isoleucine, leucine and valine in an amount of 35% to 65% by weight, preferably 42% to 58% by weight, based on the weight of the composition.

[0032] The composition may contain the amino acids isoleucine, leucine, valine, lysine and threonine in an amount of 50% to 95% by weight, preferably 65% to 90% by weight, based on the weight of the composition. The composition may contain citric acid, malic acid, succinic acid in an amount of 5% to 50% by weight, preferably 8% to 35% by weight, based on the weight of the composition.

[0033] The weight ratio of leucine to citric acid may be included in the range of 1 to 5, preferably 2.50 to 3.90.

[0034] In one or more embodiments, 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 total weight or total molar amount of malic acid plus succinic acid. In a further embodiment, the weight ratio of citric acid to the sum of malic acid and succinic acid is included in the range of 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 included in the range of 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.

[0035] The preferred weight ratio of isoleucine:leucine is included in the range of 0.2 to 0.7, preferably in the range of 0.30 to 0.60, and / or the preferred weight ratio of valine:leucine is included in the range of 0.2 to 0.70, preferably in the range of 0.30 to 0.65.

[0036] The weight ratio of threonine:leucine can be included in the range of 0.10 to 0.90, preferably in the range of 0.20 to 0.70, and / or the weight ratio of lysine:leucine is included in the range of 0.20 to 1.00, preferably in the range of 0.40 to 0.90.

[0037] In a preferred embodiment, the ratio of the total weight of citric acid, malic acid, and succinic acid to the total weight of methionine, phenylalanine, histidine, and tryptophan is higher than 1.35.

[0038] In one or more embodiments, the weight ratio of the sum of citric acid, malic acid, and succinic acid to the sum of the branched-chain amino acids leucine, isoleucine, and valine is included in the range of 0.1 to 0.4, preferably 0.15 to 0.35.

[0039] In a further embodiment, the total weight of threonine and lysine added to the branched-chain amino acids leucine, isoleucine, and valine is greater than the total weight of the three acids citric acid, malic acid, and succinic acid. Preferably, the weight of a single acid (citric acid, succinic acid, or malic acid) is less than the weight of each of the single amino acids of leucine, isoleucine, valine, threonine, and lysine.

[0040] In a further embodiment, the total weight of lysine and threonine is greater than the total weight of the three acids citric acid, succinic acid, and malic acid. Preferably, the ratio of the total weight of the three acids citric acid, succinic acid, and malic acid to the total weight of lysine and threonine is included in the range of 0.1 to 0.7, preferably 0.15 to 0.55.

[0041] In one or more embodiments, the amino acids are present in the composition in free form. The advantages associated with the use of the compositions described herein are that, through the use of the free-form amino acids contained in the active agent, and through known production processes that are widely used in the field of preparing compositions based on free amino acids, such compositions can be produced at a relatively very low cost with respect to synthetic proteins and growth factors. However, the field of application of the present invention can also be extended to amino acids obtained by genetic engineering or any other artificial method.

[0042] In one or more embodiments, the compositions disclosed herein may further comprise vitamins selected preferably from the group of B vitamins, such as vitamin B1 and / or vitamin B6. The vitamins may be contained in an amount greater than 0.001% in the composition, and preferably may be contained in the range of 0.001 to 0.5% by weight of the composition.

[0043] In addition, the composition may further comprise carbohydrates and / or flavoring substances.

[0044] In one or more embodiments, the composition may be a pharmaceutical composition further comprising a pharmaceutically acceptable vehicle. The composition may also contain pharmaceutically acceptable excipients such as, for example, vitamins, carbohydrates, natural and artificial sweeteners and / or flavoring substances. In a preferred embodiment, the pharmaceutically acceptable excipient may be selected from maltodextrin, fructose, fish oil, sucralose, sucrose esters, vitamin D3.

[0045] Furthermore, arginine, which is an amino acid, is preferably avoided, especially when preparing the composition according to the present disclosure, specifically the active agent. In addition, further amino acids that may be specifically excluded by the composition disclosed herein are serine, proline, alanine. Such amino acids may be counterproductive or even harmful at certain concentrations or stoichiometric ratios within the composition.

[0046] The amino acids disclosed herein can be replaced with their respective pharmaceutically acceptable derivatives, i.e., salts. The carboxylic acids of the active agent can be replaced with their respective pharmaceutically acceptable derivatives, i.e., salts.

[0047] For oral use, the composition described herein can be in the form of tablets, capsules, granules, gels, jelly powders, powders.

[0048] The present disclosure also provides a method for preventing and / or treating intestinal diseases in a subject. The intestinal disease can be selected from the group consisting of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), where IBD can preferably be selected from between Crohn's disease and ulcerative colitis. The method comprises the steps of selecting a composition having an active agent, the aforementioned active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine, and the carboxylic acids citric acid, succinic acid, and malic acid, and administering the composition to the subject. The active agent may further comprise one or more amino acids selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, cysteine, tyrosine as disclosed herein.

[0049] Further specifications regarding the amounts and ratios of the various amino acids provided by the composition are included in the appended claims, which form an essential part of the technical teachings provided herein with respect to the present invention. Examples

[0050] Examples of the compositions of the present disclosure are shown in Table 1 below. Table 1 [Table 1] The compositions in Table 1 above can be first prepared by sieving all components through a 0.8 mesh. To obtain a premix, each component (in an amount less than 10% by weight of the total amount) is taken in a polyethylene bag together with a portion of L-lysine HCl to obtain about 10% of the total weight of the composition. The bag is then shaken by hand for 5 minutes. The premix is then placed in a mixer (Planetaria) together with the remaining components and mixed for a period of 15 minutes at 120 rpm to obtain a uniform final composition.

[0051] Method - Part 1 Patients This study was approved by the Ethics Committee of the local health authority (regulation 74 / 2018) after obtaining written informed consent from all study subjects.

[0052] Twenty-nine elderly (age > 65 years) patients with stage 3b - 4 CKD (estimated glomerular filtration rate (eGFR) of 44 - 29 ml / min / 1.73 m 2Patients diagnosed as were preselected. The reason for selecting elderly subjects is that aging is associated with TJ loss, abnormal microbial balance, and intestinal barrier dysfunction, so there is a high possibility of changes in intestinal barrier integrity. Among these patients, patients with concurrent acute and chronic inflammatory diseases (n = 3), diabetic patients (n = 5), cancer patients (n = 0), patients with autoimmune diseases (n = 1), patients receiving steroid / immunosuppressive therapy (n = 0), patients with chronic obstructive pulmonary disease (n = 7), patients with heart failure (n = 2), and patients with liver diseases (n = 1) were excluded. Furthermore, since tobacco smoking is associated with changes in intestinal tight junctions, patients who smoke were excluded. Therefore, 9 out of the first 29 patients were investigated. The patients were on a diet therapy with a protein intake of 0.6 - 0.7 g / kg (50% from animal sources), a calorie intake > 30 kcal / kg / day, and a phosphate intake < 1000 mg / day. In addition to eGFR, renal function was evaluated by the Modification of Diet in Renal Disease (MDRD) (Menon V Am J Kidney dis 2099 53:208 - 217) in the study group of renal diseases normalized by body surface area.

[0053] These patients were required to ingest the composition disclosed herein and shown as "Composition E7" in Table 1 for 6 months. Measurements taken 12 months after the investigation were used as the baseline values of the study. 11 healthy controls were also enrolled in the study as a control group (CTRL).

[0054] After enrollment, the patients received the following measures: 1. Biological temperature variables including 24-hour urinary protein content. The protein intake was estimated by calculating the Urea Nitrogen Appearance (UNA) (Maroni BJ Kidney Int 1985 27:58 - 65; Masud T Kidney Int 2002 62:1750 - 56). 2. Body composition, including total body water (TBW), extracellular water (ECW), intracellular water (ICW) (all in liters), and percentages relative to body weight, resistance (RZ, in ohms), conductance (XC, in ohms), and phase angle (in degrees), which is considered an index of skeletal muscle mass (Sunario JJ, Parent Enter Nutr 2021 45:1089-1099), were measured by bioelectrical impedance analysis (BIA) using the same operator with the same device (renal EFG 50Hz,; EFG diagnostic Ltd, Belfast, Northern Ireland). 3. Determination of AA in plasma Free amino acid concentrations in plasma of the ingested patients and control patients were measured by cation exchange chromatography and detected by spectrophotometer (570 nm and 440 nm) after post-column reaction with ninhydrin reagent using a Biochrom 30+ amino acid analyzer (Biochrom Ltd, ERRECI s.r.l, Pieve Emanuele, Italy) (n = 8-9 samples per group). Plasma samples were mixed 1:1 with a 5% sulfosalicylic acid (SSA)-based protein precipitation reagent containing L-norleucine (Sigma Aldrich, Milan, Italy) as an internal standard (C f = 250 mmol / L) and incubated at 4°C for 30 minutes. After centrifugation (10,000 xg, 5 minutes at 4°C), the supernatant was collected and filtered through a filter column (0.22 mm) (Millipore, Milan, Italy) to completely remove proteins (12,000 xg, 10 minutes at 4°C). Calibration standards (C of each amino acid fA solution of 250 mmol / L (Biochrom Ltd, Cambridge Research Park, UK) was treated with an L-norleucine SSA 5% - based buffer as a sample to equalize the pH during loading. Standard solutions and samples maintained at low temperature during the experiment were injected into the analyzer (60 ml / sample). Amino acid separation was achieved by a cation exchange chromatography process. Amino acid concentration was detected by a spectrophotometer after a post-column reaction with ninhydrin under controlled reaction conditions at 135 °C. 4. Determination of calprotectin and zonulin levels in feces The determination of calprotectin in fecal samples was performed by immunoenzymatic method and measured using a Chrous TRIO instrument (DIESSE Diagnostica Senese S.p.A., Italy) according to the manufacturer's instructions. A calprotectin value of less than 50 μg / g per fecal sample was considered normal.

[0055] Intestinal permeability was evaluated as the concentration of zonulin in feces (ng / ml) using a commercially available ELISA kit (Zonulin Stool ELISA, DRG Instruments GmbH, Germany). The normal amount of zonulin in the feces of healthy subjects is considered to be less than 60 ng / ml.

[0056] Eleven elderly and healthy non-smoker subjects with the same age, gender, and body mass index (BMI, Kg / m 2 ) were used as a control (CTRL). The CTRL received the same treatment as CKD, but only at the time of recruitment.

[0057] After completing the above measures, the composition was only prescribed to the patients. Specifically, the patients were instructed to take 2 packets per day (1 packet in the morning and 1 packet in the afternoon diluted with 150 - 200 ml of water) for 6 months. The composition of each packet is shown in Table 2. Table 2

Table 2

[0058] The increase in plasma glutamine and alanine levels follows an increase in the muscle utilization of branched-chain amino acids (BCAAs; included in the composition). Furthermore, to confirm BCAA-induced glutamine and alanine formation, the increases in plasma levels of two ratios, glutamine / (BCAA + aspartic acid + asparagine + glutamic acid) and alanine / (BCAA + aspartic acid + asparagine + glutamic acid), were added. In fact, aspartic acid, asparagine, and glutamic acid also contribute to the formation of glutamine and alanine. 6. Estimation of the effect of the composition on urine production Blood urea nitrogen (BUN) was evaluated with a Biochrom 30+ amino acid analyzer. This measurement can be particularly important for differentiating whether an increase in BUN, when GFR progresses, is due to an increase in nitrogen intake due to composition intake and / or due to deterioration of renal insufficiency. An increase or decrease in the BUN / total plasma amino acids (AA) ratio in fasting patients indicated no excessive nitrogen intake.

[0059] 7. Statistical methods The central tendency and dispersion of continuous variables were reported as mean ± standard deviation. Discrete variables were reported as number (N) and percentage frequency. Since some variables violated the normality assumption (Shapiro–Wilk test), nonparametric statistics were chosen for hypothesis testing. Within-group and between-group comparisons of continuous variables were performed using the Wilcoxon signed-rank test and the Mann–Whitney U test, respectively. Categorical variables were compared using the chi-square test or Fisher's exact test as needed. All tests were two-sided tests. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using the SAS / STAT statistical package, release 9.4 (SAS Institute Inc., Cary, NC, USA).

[0060] Results - Part 1 Patient baseline characteristics Table 3 refers to the baseline characteristics of the control group (CTR) and patients with chronic kidney disease (CKD). The reported p-values are by the Mann–Whitney U test or *Fisher's exact test. CKD patients and CTR patients were similar in age and weight (as BMI), but differed in body composition. Indeed, patients showed an increase in total body weight (TBW; p < 0.05) and extracellular body weight (EBW; p < 0.01), and a decrease in phase angle, which is considered an indicator of reduced skeletal muscle mass (p < 0.01).

[0061] Compared with the control group, CKD showed mild anemia, with lower levels of iron (p < 0.01) and ferritin (p < 0.05) in serum, and their mean values remained within the normal range of our institute's values (hyperparathyroidism).

[0062] From a renal perspective, patients had moderate to severe renal insufficiency (MDRD 30.83 ± 7.88 ml / min / 1.73m 2 ) and compensated metabolic acidosis (decrease in plasma bicarbonate level compared with controls, p < 0.01).

[0063] Blood urea nitrogen (BUN) was higher than that of the control group (p<0.001), while the protein intake from UNA was lower (p<0.05), but the absolute value was higher than the prescribed value (0.7~0.6 g / kg / day). Table 3

Table 3

Table 4

[0064] Table 5

Table 5

Table 6

Table 7

[0065] This study shows that elderly patients with CKD have reduced intestinal integrity due to increased intestinal permeability caused by inflammation and TJ depletion. These results further show that patients are compliant with the intake of the composition associated with improved intestinal inflammation and permeability.

[0066] The mitochondrial intermediates (malic acid, succinic acid, citric acid) of the composition may have promoted a more balanced inflammatory / adaptive immune response against adaptive immunity and acted synergistically with the amino acids of the composition.

[0067] These findings highlight the beneficial effect of this composition on IMB integrity in patients with damaged or defective intestinal barriers (which may be due to gastrointestinal diseases or other disorders including dysbiosis, systemic inflammation, CKD), or critically ill subjects in the intensive care unit.

[0068] In the following section, experimental results are provided to analyze the comparison of the effects of different compositions. Specifically, an analysis was performed to show the synergistic effect achieved by the composition when the three patented acids (succinic acid, citric acid, malic acid) were administered in combination with the patented amino acids.

[0069] Method - Part 2 Cell culture and treatment Human colon adenocarcinoma Caco2 cells were purchased from the American Type Culture Collection (ATCC-HTB-37, LGC, Milan, Italy). Caco2 cells were cultured in a standard medium: EMEM (ATCC30-2003, LGC) supplemented with 10% fetal bovine serum (FBS, ATCC30-2020, LGC), penicillin (100 U / ml), and streptomycin (100 μg / ml) (Euroclone, Milan, Italy) under a controlled atmosphere of 5% CO2 and 37 °C.

[0070] In each experiment, confluent Caco2 cells (passages 10 - 20) were grown in standard medium for 14 days to obtain fully differentiated cells (i.e., cells that spontaneously formed tight junctions between cells and had a brush border) (Wang et al. 2011). The differentiated Caco2 cells were pretreated for 1 hour with the following four compositions: 1) "E8", an essential amino acid composition enriched with tricarboxylic acid (TCA) cycle intermediates (i.e., citric acid, succinic acid, and malic acid, ratio 4:1:1) according to an embodiment of the present application, 0.1 and 1.0% (p / v); 2) "EAAm", an essential amino acid composition without TCA cycle intermediates, 1.0% (p / v); 3) "5aa", a composition containing only the five claimed amino acids (i.e., leucine, lysine, isoleucine, valine, and threonine), 1.0% (p / v); 4) "5aa acid", the composition "5aa" enriched with TCA intermediates such as E8 (i.e., citric acid, succinic acid, and malic acid, ratio 4:1:1) according to an embodiment of the present application, 1.0% (see Table 8 for the composition). After the pretreatment, all conditions were tested in the presence or absence of the inflammatory mediators IL-1β (25 ng / ml), TNF-α (50 ng / ml), LPS (10 μg / ml) and cultured for 24, 48 or 72 hours in EMEM supplemented with 1% heat-inactivated FBS, penicillin (100 U / ml), and streptomycin (100 μg / ml) (Van De Valle J et al. 2010).

[0071] Table 8

Table 8

[0072] Gene expression analysis Total RNA was isolated from Caco2 cells using the RNeasy Mini Kit (Qiagen, Milan, Italy) and treated with DNase according to the manufacturer's protocol (Bio-Rad Laboratories, Milan, Italy). cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories) and amplified by real-time quantitative PCR using iTaq Universal SYBR Green SuperMix (Bio-Rad Laboratories) on a CFX Connect Real-Time PCR System (Bio-Rad Laboratories). Primers were designed using Primer3 (version 0.4.0) software (Table 9). The number of cycles at which various transcripts were detectable (threshold cycle: CT) was compared to the housekeeping CT and called ΔCT. Gene relative levels were expressed as 2 -ΔΔCT and ΔΔCT corresponded to the difference between the ΔCT of any treatment group and the ΔCT of the control group (Ruocco et al. 2020).

[0073] Table 9

Table 9

[0074] Immunoblot analysis Protein extracts were obtained from differentiated Caco2 cells as per the manufacturer's instructions using mammalian protein extraction reagent (M-PER, Pierce, Thermo Fisher Scientific, Merck, Milano, Italy) in the presence of a protease and phosphatase inhibitor cocktail (Sigma-Aldrich, Merck, Milano, Italy). Protein content was determined by bicinchoninic acid protein assay (BCA, Euroclone, Milano, Italy). Appropriate amounts of protein were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions for immunoblotting. The separated proteins were then semi-dry transferred onto nitrocellulose membranes (Bio-Rad Laboratories), and the proteins of interest were revealed using specific antibodies: anti-zonulin 1 and anti-occludin (both Invitrogen, Thermo Fisher Scientific) and anti-PGC-1a (Abcam, Prodotti Gianni, Milano, Italy) each at a 1:1,000 dilution. Anti-vinculin (1:3,000; Sigma-Aldrich, Milano, Italy) was used as a loading control. Immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulins (Corsetti et al. 2014). The amount of each protein was measured using SuperSignal Substrate (Euroclone), analyzed with Chemidoc XRS+ and quantified with ImageLab software (both Bio-Rad Laboratories).

[0075] Cell viability assay The viability of differentiated Caco2 cells was determined using the standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MTT is a yellow dye that is reduced by multiple redox enzymes including mitochondrial succinate dehydrogenase to formazan, a blue product. Differentiated Caco2 cells seeded in 96-well plates (100 μl / well) were treated with each composition for 24 and 48 hours in the presence or absence of inflammatory mediators. After the treatment, MTT (5 mg / ml in PBS, 20 μl) was added and incubated for an additional 4 hours. The purple formazan crystals were dissolved in 5% SDS / 0.1 M HCl (100 μL / well) overnight at 37 °C, and the absorbance was recorded at two wavelengths of 570 nm / 655 nm using a microplate reader (Ragni et al. 2022). Alternatively, the cell viability was determined using a sulforhodamine B-based in vitro toxicity assay kit (TOX6, Sigma Aldrich) according to the manufacturer's instructions.

[0076] IL-8 secretion in cell culture supernatant To evaluate IL8 secretion by differentiated Caco2 cells treated with the E8 composition for 24, 48, and 72 hours in the presence or absence of an inflammatory regulator, the extracellular medium was collected and centrifuged at 2,000 g for 10 minutes. The release of IL-8 was evaluated using a sandwich ELISA method (ab214030 - Human IL-8 ELISA Kit, Abcam) and quantified in pg / ml units using the standard provided with the kit.

[0077] Intestinal barrier integrity Caco2 cells were seeded at 1x10 5Seeded at a cell / well density. Once the cells reached confluence, they were grown for 14 days to obtain differentiated Caco2 cells. The cell culture insert allows access to both the basolateral and apical sides of the cells, representing the basolateral and apical poles of the intestinal epithelium, respectively. The differentiated Caco2 cells were pretreated with the E8 composition (0.1 and 1.0%) on the apical side of the cells and after 24 hours, the cells were either exposed or not exposed to an inflammatory stimulus. IL-1β (25 ng / ml) and TNF-α (100 ng / ml) were applied to the basolateral side of the cells. In contrast, LPS was applied bilaterally (5 μg / ml / side), which on one hand represents a typical luminal microbiota and on the other hand represents an increase in the presence of bacterial components in the serum during inflammatory diseases (Van De Valle J et al. 2010). Trans-epithelial electrical resistance (TEER) was measured at the start of the experiment, after 24 hours of pretreatment, and at the end of the experiment using an Epithelial Volt / Ohm Meter EVOM3 (World Precision Instruments, MatTek In Vitro Life Science Laboratories, Slovak Republic) (Hiebl et al. 2020).

[0078] Statistical analysis The sample size of each experiment reflects the number of independent biological replicates as provided in the figure legends. Data are reported as mean ± SEM. Statistical analysis was performed using Prism 6.0 software (GraphPad Software, Inc.) by one-way or two-way analysis of variance tests. Values with p < 0.05 were considered statistically significant.

[0079] Results - Part 2 Composition E8 improved the intestinal permeability and mitochondrial biosynthesis of Caco2, an in vitro model of the intestinal barrier. The human epithelial cell line Caco2, isolated from human colorectal adenocarcinoma by Fogh & Trempe in 1974, has been the most widely used cell line as an intestinal model for absorption, transport, and bioavailability studies in the past few decades (Fogh & Trempe 1975; Haddad et al. 2023). Confluent Caco2 cells spontaneously initiate differentiation from day 14 to day 21 and form a polarized cell monolayer with a brush border having apical membranes and basolateral membranes, junctional complexes, and apical microvilli typical of human intestinal epithelial cells (Engle et al. 1998, Hidalgo et al. 1989).

[0080] To identify the appropriate differentiation time to study the effect of E8 on healthy cell states, Caco2 was treated on days 14 and 21 of differentiation. After exposure with increasing doses of E8 (0.05 - 1.0%), no cytotoxic effect of the composition on Caco2 cells differentiated for 21 days was observed (A, B in Figure 1). Notably, E8 (0.1% and 1.0%) increased Caco2 viability, probably due to the stimulation of mitochondrial function, as the MTT assay indirectly evaluates the succinate dehydrogenase activity of mitochondria (A, B in Figure 1).

[0081] Therefore, the effects of E8 (0.1 - 1.0%) on the relative mRNA expression of the most important proteins involved in intestinal permeability regulation were evaluated in Caco2 cells differentiated for 14 or 21 days: 1) zonula occludens-1 (ZO-1), a peripheral membrane adapter protein that functions as a bridge connecting integral membrane proteins to the actin cytoskeleton and other signaling proteins; 2) occludin and claudin, integral membrane proteins; 3) E-cadherin, a major component of adherens junctions involved in intestinal homeostasis and barrier function (Lee et al. 2018; Schneider et al. 2010). Data showed that treatment with E8 for 24 and 48 hours increased the relative expression of genes regulating intestinal permeability, mainly in Caco2 cells differentiated for 14 days (C - G in Figure 1).

[0082] Since E8 provides essential amino acids and TCA cycle intermediates involved in mitochondrial regulation, the mRNA expression of two very important mitochondrial genes was also evaluated: 1) Tfam, which is essential for transcription, replication, and packaging of mtDNA into nucleoids (Stiles et al. 2016); 2) Cyt c (i.e., cytochrome c), a central component of the mitochondrial electron transport system.

[0083] According to the results obtained from the MTT assay, treatment with the E8 composition stimulated the gene expression of Tfam and Cyt c, confirming the potential benefits of E8 on mitochondrial biogenesis under basal conditions (J, K in Figure 1).

[0084] Considering the preliminary results, the most valuable differentiation time point at which E8 could improve Caco2 integrity and mitochondrial biogenesis under basal conditions was considered to be the 14-day period.

[0085] E8 maintained intestinal barrier integrity in an in vitro model of enteritis. Caco2 cells have also been shown to be able to produce inflammatory markers (such as cytokines) in response to specific stimuli such as IL-1β, TNF-α, and LPS (Ponce de León-Rodríguez et al. 2019). Due to this property, differentiated Caco2 cells become a valuable in vitro model of enteritis (Rodríguez-Ramiro et al. 2013, Hollebeeck et al. 2012, Van De Walle et al. 2008). Differentiated Caco2 cells were pretreated with composition E8 (0.1% and 1.0%) for 1 hour, and then the cells were exposed to inflammatory stimuli [i.e., IL-1β (25 ng / ml), TNF-α (100 ng / ml), and LPS (10 μg / ml)] in the presence or absence of composition E8. The first response of Caco2 cells after inflammation is cytokine production that occurs within 24 hours, and permeability damage occurs only after continuous incubation with inflammatory mediators (e.g., decreased expression of ZO-1, occludin, and claudin), so the evaluation was extended to 24, 48, and 72 hours. (A, B of Figure 2).

[0086] The MTT assay showed an increase in cell viability after treatment with the E8 composition under all experimental conditions, but inflammation did not affect the health of the cells (C of Figure 2). The viability of CaCo2 cells was also evaluated by the TOX6 assay (i.e., a measure of total biomass) after inflammation and E8 treatment, and no significant cytotoxicity was observed (D of Figure 2). Next, the effectiveness of the E8 composition (0.1 - 1.0%) in maintaining intestinal permeability during inflammation at different times was evaluated (Figure 3).

[0087] As shown previously, under basal conditions, E8 treatment increased the mRNA relative expression of the major regulatory genes of intestinal permeability and maintained this for 72 hours (A - G in Figure 3). Furthermore, E8 treatment at a higher concentration (1.0%) stimulated the expression of all the genes analyzed even in the presence of inflammation (A - G in Figure 3). The etiology of inflammatory bowel disease is associated with impairment of intestinal permeability - regulating proteins (Schneider et al., 2010, Bruewer et al., 2006). Notably, under the experimental conditions here, 48 - hour inflammation induced a decrease in the gene expression of occludin, claudin 3, claudin 4, and E - cadherin, but this was efficiently prevented by E8 treatment (1.0%) (B and D - F in Figure 3). Furthermore, by treating with E8 at a higher concentration (1.0%), the damage induced by 48 - hour inflammation was prevented, particularly at the occludin protein level (G, H in Figure 3).

[0088] Therefore, the effect of E8 treatment on the barrier integrity of Caco2 cells was evaluated by measuring the transepithelial electrical resistance (TEER), which quantifies the movement of ions across the paracellular pathway (A in Figure 4).

[0089] Caco2 cells were pretreated with E8 (0.1 - 1.0%) for 24 hours and then an inflammatory stimulus was applied for 24 hours. As expected, the inflammatory stimulus impaired intestinal barrier integrity (-29.3% compared to CTRL), and only E8 treatment at 1.0% protected the cells partially from damage (-17% compared to CTRL, +12% compared to inflammation) (B in Figure 4).

[0090] E8 stimulated mitochondrial biogenesis in an in vitro model of intestinal inflammation In an in vitro model of enteritis, the effect of E8 treatment (0.1 - 1.0%) on mitochondrial biogenesis was analyzed. After 48 hours, inflammation decreased the gene expression of PGC1α and COXIV, and the PGC1α protein level in Caco2 cells (A - C in Figure 5).

[0091] E8 (1.0%) already had the activity to improve mitochondrial biogenesis under basal conditions. However, after 48 - 72 hours of treatment, even in the presence of inflammation, the expression of PGC1α gene and protein, as well as COX IV gene expression, increased (A - C in Figure 5).

[0092] These results suggest that E8 prevents mitochondrial damage induced by inflammation.

[0093] E8 attenuated the inflammatory response in vitro in intestinal inflammation IL8 is a chemokine produced by other cell types including macrophages and intestinal epithelial cells, and is secreted to attract and activate neutrophils during the acute phase of inflammation (Hoffmann et al. 2002). IL8 induces the chemotaxis of neutrophils and granulocytes, moves them towards the site of infection, and stimulates phagocytosis upon arrival.

[0094] To evaluate the potential anti - inflammatory effect of E8, IL8 release in an in vitro model of enteritis was analyzed. As expected, inflammatory mediators stimulated IL8 release from Caco2 intestinal epithelial cells after 24 - hour exposure, which was maintained for 72 hours (Figure 6). Treatment with E8 at the lowest concentration (0.1%) decreased IL8 secretion after 48 - hour treatment (-9% against inflammation, Figure 6). After continuous treatment, both doses (0.1 - 1.0%) of E8 significantly attenuated the inflammatory response, reducing IL8 release by -13% and -9% respectively compared to the inflammatory state (Figure 6).

[0095] TCA cycle intermediates enhanced the healthy benefits of the amino acid composition in an in vitro model of intestinal inflammation. The E8 composition containing the stoichiometric ratio of balanced essential amino acids and branched - chain amino acids, as well as TCA cycle intermediates (i.e., citric acid, succinic acid, and malic acid), was designed to suppress mitochondrial damage and improve intestinal barrier function in inflammatory bowel disease.

[0096] To demonstrate that TCA cycle intermediates cooperate with essential amino acids to enhance the benefits on intestinal permeability and intestinal mitochondrial biogenesis, in an in vitro model of enteritis, the effect of E8 (i.e., an essential amino acid composition containing TCA cycle intermediates) was compared with an amino acid composition (EAAm, Table 1) provided with essential amino acids and without TCA cycle intermediates (A of Figure 7).

[0097] Notably, in the absence of inflammation, EAAm treatment for 24 hours and 48 hours had an adverse effect on cell viability, whereas conversely, E8 treatment showed no cytotoxic effect (B of Figure 7). Furthermore, the results indicate that only E8 prevents the inflammation-induced damage of the intestinal barrier and stimulates the expression of genes of major regulatory factors of intestinal permeability and mitochondrial biogenesis more efficiently than EAAm (C-J of Figure 7).

[0098] The effect of a partial composition (i.e., leucine, lysine, isoleucine, valine, and threonine - "5aa" composition) composed of the most representative amino acids in the E8 composition was also studied in comparison with the same composition (the "5aa acid" composition; Table 1) added with TCA cycle intermediates at the same ratio provided by E8. Differentiated Caco2 cells were treated with compositions 5aa and 5aa acid, and E8 (each 1%) for 24 hours in the presence or absence of inflammatory stimuli (A of Figure 8).

[0099] The results provided herein indicate that under basal conditions, the 5aa acid composition could stimulate the mRNA expression of intestinal permeability genes more efficiently than the 5aa composition and even more efficiently than the E8 composition (B-G of Figure 8). During inflammation, the E8 composition could also suppress the damage induced by inflammatory stimuli (B-G of Figure 8).

[0100] Overall, data obtained from the comparison of an essential amino acid composition without TCA cycle intermediates (i.e., EAAm) and 5aa-based compositions (i.e., 5aa and 5aa acid) also demonstrated that TCA cycle intermediates serve as important boosters in the health of intestinal epithelium and can fully achieve beneficial effects during inflammation.

[0101] Overall, the results provided herein demonstrate that the compositions of this application can improve intestinal permeability and intestinal mitochondrial biogenesis under basal conditions in an in vitro model of the intestinal barrier. Notably, the compositions prevented intestinal permeability and inflammatory damage induced in the mitochondrial compartment in an in vitro model of enteritis. The compositions of this application exhibited an anti-inflammatory effect and decreased IL8 secretion by intestinal epithelial cells. TCA cycle intermediates enhanced the effect of essential amino acids on intestinal permeability and intestinal mitochondrial biogenesis. Overall, the data provided the effect of the compositions in the prevention and management of IMB integrity in patients with intestinal barrier damage or insufficiency.

[0102] References Bruewer, M., Samarin, S., Nusrat, A. Inflammatory bowel disease and the apical junctional complex. Ann. N Y Acad. Sci. 2006;1072, 242 - 252.

[0103] Corsetti G, D'Antona G, Ruocco C, et al. Dietary supplementation with essential amino acids boosts the beneficial effects of rosuvastatin on mouse kidney. Amino Acids. 2014;46:2189 - 2203.

[0104] Engle, M.J.; Goetz, G.S.; Alpers, D.H. Caco - 2 cells express a combination of colonocyte and enterocyte phenotypes. J. Cell. Physiol. 1998, 174, 362 - 369.

[0105] Fogh, J., and G. Trempe. 1975. New human tumor cell lines. In Human tumor cells in vitro. ed. J. Fogh, 115 - 159. New York: Springer。

[0106] Haddad, M. J.; Sztupecki, W.; Delayre - Orthez, C.; Rhazi, L.; Barbezier, N.; Depeint, F.; Anton, P. M。

[0107] Hidalgo I J, Raub T J, Borchardt R T. Characterization of the human colon carcinoma cell line (Caco - 2) as a model system for intestinal epithelial permeability. Gastroenterology. 1989;96:736 - 749。

[0108] Hiebl V, Schachner D, Ladurner A, Heiss E H, Stangl H, Dirsch V M. Caco - 2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations. Biol Proced Online. 2020;22:7。

[0109] Hoffmann E, Dittrich - Breiholz O, Holtmann H, Kracht M. Multiple control of interleukin - 8 gene expression. J Leukoc Biol. 2002;72:847 - 855。

[0110] Hollebeeck S, Winand J, Herent MF, et al. Anti-inflammatory effects of pomegranate (Punica granatum L.) husk ellagitannins in Caco-2 cells, an in vitro model of human intestine. Food Funct. 2012;3:875-885。

[0111] Lee B, Moon KM, Kim CY. Tight Junction in the Intestinal Epithelium: Its Association with Diseases and Regulation by Phytochemicals. J Immunol Res. 2018;2018:2645465。

[0112] Ponce de Leоn-Rodriguez MDC, Guyot JP, Laurent-Babot C. Intestinal in vitro cell culture models and their potential to study the effect of food components on intestinal inflammation [published correction appears in Crit Rev Food Sci Nutr. 2019;59(13):2166-2168]. Crit Rev Food Sci Nutr. 2019;59:3648-3666。

[0113] Ragni M, Ruocco C, Tedesco L, Carruba MO, Valerio A, Nisoli E. An amino acid-defined diet impairs tumour growth in mice by promoting endoplasmic reticulum stress and mTOR inhibition. Mol Metab. 2022;60:101478。

[0114] Rodriguez-Ramiro I, Ramos S, López-Oliva E, et al. Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-α-stimulated Caco-2 cells. Br J Nutr. 2013;110:206-215。

[0115] Ruocco C, Ragni M, Rossi F, et al. Manipulation of Dietary Amino Acids Prevents and Reverses Obesity in Mice Through Multiple Mechanisms That Modulate Energy Homeostasis. Diabetes. 2020;69:2324-2339。

[0116] Schneider MR, Dahlhoff M, Horst D, et al. A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation. PLoS One. 2010;5:e14325。

[0117] Stiles, A.R., Simon, M.T., Stover, A., Eftekharian, S., Khanlou, N., Wang, H.L., Magaki, S., Lee, H., Partynski, K., Dorrani, N., Chang, R., Martinez-Agosto, J.A., Abdenur, J.E. Mutations in TFAM, encoding mitochondrial transcription factor A, cause neonatal liver failure associated with mtDNA depletion. Molec. Genet. Metab. 2016, 119:91-99。

[0118] Van De Walle J, Romier B, Larondelle Y, Schneider YJ. Influence of deoxynivalenol on NF-kappaB activation and IL-8 secretion in human intestinal Caco-2 cells. Toxicol Lett. 2008;177:205-214。

[0119] Wang W, Liu Q, Wang C, Meng Q, Kaku T, Liu K. Effects of JBP485 on the expression and function of PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells. Peptides. 2011;32:946-955。

Claims

1. A composition for preventing and / or treating intestinal diseases, having an active agent, wherein the active agent includes amino acids such as leucine, isoleucine, valine, threonine, lysine, and citric acid, succinic acid, malic acid or salts thereof.

2. The composition according to claim 1, wherein the intestinal disease is selected from the group consisting of irritable bowel disease (IBS) and inflammatory bowel disease (IBD), and the IBD is preferably selected from between Crohn's disease and ulcerative colitis.

3. The composition according to claim 1 or claim 2, wherein the weight ratio of the total of citric acid, malic acid, succinic acid to the total of leucine, isoleucine, valine, which are branched-chain amino acids, plus lysine and threonine is included in the range of 0.05 to 0.3, preferably 0.1 to 0.

25.

4. The composition according to claim 1, wherein the weight ratio of the total amount of citric acid, malic acid, succinic acid to the total amount of leucine, isoleucine, valine, which are branched-chain amino acids, is included in the range of 0.1 to 0.4, preferably 0.15 to 0.

35.

5. The composition according to claim 1, wherein the weight ratio of citric acid to the total of malic acid and succinic acid is included in the range of 1.0 to 4.0, preferably 1.5 to 2.

5.

6. The composition according to claim 1, wherein the weight ratio of citric acid:malic acid:succinic acid is included in the range of 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.

7. The composition according to claim 1, wherein the active agent further includes at least one amino acid selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, tyrosine, and cysteine.

8. The composition according to claim 1, wherein the active agent further includes histidine, phenylalanine, methionine, tryptophan, and cysteine.

9. The composition according to claim 1, wherein the ratio of the total weight of citric acid, malic acid, succinic acid to the total weight of methionine, phenylalanine, histidine, and tryptophan is higher than 1.

35.

10. The composition according to claim 1, wherein the ratio of the total weight of the three acids of citric acid, succinic acid, malic acid to the total weight of lysine and threonine is included in the range of 0.10 to 0.70, preferably 0.15 to 0.

55.

11. The composition according to claim 1, wherein the amino acids isoleucine, leucine and valine are present in an amount of 35% to 65% by weight, preferably 42% to 58% by weight, based on the weight of the composition.

12. The composition according to claim 1, wherein the weight or molar amount of citric acid is greater than the total weight or total molar amount of both malic acid and succinic acid.

13. The composition according to claim 1, wherein the weight ratio of leucine to citric acid is included in the range of 1 to 5, preferably 2.50 to 3.

90.

14. The composition according to claim 1, wherein the composition does not contain arginine.

15. The composition according to claim 1, wherein the composition does not contain serine, proline, or alanine.

Images