Composition comprising amino acids for preventing and / or treating bowel disease

Abstract

A composition for preventing and / or treating bowel disease, the composition comprising an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine, citric acid, succinic acid, malic acid.

Description

Technical Field

[0001] The present disclosure generally relates to compositions comprising amino acids for use in preventing and / or treating intestinal diseases, particularly intestinal diseases associated with inflammation and altered permeability. Background Art

[0002] Intestinal mucosal barrier (IMB) is crucial for the absorption of nutrition and the health of both humans and animals. Recent publications from clinical and experimental studies have shown the importance of nutrient-bacteria-host interactions for intestinal homeostasis. It is reported that the dysfunction of nutrient-bacteria-host interactions is related to the occurrence of metabolic disorders and enteropathy, and the enteropathy is for example, irritable bowel syndrome and inflammatory bowel disease (inflammatory intestinal disease, IBD), ulcerative colitis (ulcerative colitis, UC) and Crohn's disease (Crohn's disease, CD) are two main types of IBD. On the other hand, systemic disorders (including obesity and type 2 diabetes) and specific diseases (such as nephropathy) are relevant to IMB dysfunction and loss.

[0003] IBD causes chronic intestinal inflammation that leads to recurrence and remission of 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 is highly variable in two situations: between individuals and during the life of a given individual. Clinically inactive disease periods can be disrupted by acute attacks, resulting in the need for medication, hospitalization, and sometimes intestinal surgery. The pathogenesis of IBD is driven by abnormal and prolonged T cell-mediated immune responses against commensal intestinal microbiota that occur in genetically susceptible individuals. Known IBD risk genes are associated with a variety of immune functions, including innate immune functions, such as physical barriers and autophagy. Current models involve multiple factors in the pathogenesis of IBD, including multi-layer mucosal damage, such as histological and cellular changes that ultimately lead to macroscopic erosions and ulcers. The intestinal barrier can be destroyed after the intestinal tight junction (TJ) structure is exhausted. The intestinal tight junction (TJ) structure is a protein structure located at the top part of the intestinal epithelial cells connecting both epithelial cells and endothelial cells. The destruction (trauma) of the intestinal barrier can cause microorganisms and other antigens to shift (translocation) into the intestinal wall, thereby causing uncontrolled immune activation, which is a key feature of IBD (Neurath, MF & Travis, SP Gut 61, 1619-1635, 2012). The structural basis of the healed mucosa is a complete barrier that limits bacterial translocation and subsequent immune activation. Therefore, the functional definition of mucosal healing can be described as a mucosa with a reconstructed barrier function. Almost all currently available treatments for IBD are by suppressing inflammation, usually blocking specific inflammatory molecules to play a role. However, considering the burden of infectious and neoplastic diseases associated with long-term immunosuppressive therapy, the goal of achieving mucosal healing in the absence of immunosuppression is attractive.

[0004] In addition to the physical barrier constituted by epithelial cells, the production of proteins, TJs, and intracellular machinery to respond to invading pathogens are essential for maintaining homeostasis and must be regenerated during the process of mucosal healing; notably, the intestinal epithelium is not static but is highly dynamic and is renewed every 5 to 7 days, allowing for continuous protection of immune cells from the rich luminal microbiota. Mucins are glycoproteins produced and secreted by goblet cells and are considered the major components 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 in the stem cell niche and are the major source of antimicrobial peptides (AMPs).

[0005] Over the past 20 years, complete mucosal healing has been established as an important treatment goal for IBD. For example, in the Inflammatory Bowel South-Eastern Norway (IBSEN) study, mucosal healing at the 1-year follow-up visit after diagnosis was associated with a reduced risk of future colectomy in patients with ulcerative colitis and reduced inflammation and future steroid therapy in patients with Crohn's disease after 5 years (Froslie et al., Gastroenterology 133, 412–422, 2007).

[0006] Although it is an attractive goal to favor mucosal healing in the absence of immunosuppression, therapeutic strategies to directly promote mucosal healing are still lacking. To this end, dozens of pathways that promote mucosal healing have been identified; however, most of them can lead to hyperproliferation of the intestinal epithelium and, therefore, tumor growth. Summary of the invention

[0007] The object of the present description is to provide amino acid-based compositions that are particularly effective in preventing and / or treating intestinal diseases, particularly those associated with intestinal inflammation and altered permeability, in a subject by promoting mucosal healing without producing immunosuppressive effects and / or uncontrolled intestinal cell proliferation.

[0008] According to the present description, the above objects are achieved thanks to the subject matter particularly mentioned in the appended claims, which are understood to constitute an essential part of the present disclosure.

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

[0010] The bowel disease may be selected from irritable bowel disease (IBS) and inflammatory bowel disease (IBD), wherein the IBD may be selected from Crohn's disease and ulcerative colitis.

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

[0012] The present disclosure also provides a method for preventing and / or treating intestinal diseases in a subject. The method can prevent and / or treat intestinal diseases selected from irritable bowel disease (IBS) and inflammatory bowel disease (IBD), wherein IBD may preferably be selected from Crohn's disease and ulcerative colitis. The method includes selecting a 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 and malic acid; and administering the composition to the subject. The active agent may also include one or more amino acids selected from the following: histidine, phenylalanine, methionine, tryptophan, cysteine, tyrosine, as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0014] - Figure 1 The effect of E8 on the health status of Caco2 cells at different differentiation time points is shown. (A) Representative images of Caco2 cells differentiated for 14 or 21 days and treated with E7 (0.1% to 1.0%) for 24 hours (scale bar 20×); (B) Viability of Caco2 cells differentiated for 21 days and treated with E8 (0.05% to 1.0%) for 24 hours measured by MTT assay; (C to K) mRNA levels of intestinal permeability (C to G) and mitochondrial biogenesis (J, K) genes in Caco2 cells differentiated for 14 or 21 days and treated with E7 (0.1% to 1.0%) for 24 or 48 hours. The cycle number at which transcripts can be detected is compared to the cycle number of GAPDH and is expressed as relative expression relative to the control (CTRL) (shown as 1.0). Data are expressed as mean ± SEM (n = 3 to 4 samples / group). One-way ANOVA (B), two-way ANOVA (C to K), *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRL;

[0015] - Figure 2The effect of E8 treatment on cell viability in an in vitro intestinal inflammation model is shown. (A) In vitro intestinal inflammation model and E8 treatment; (B) Representative images of Caco2 cells differentiated for 14 days and treated with E7 (0.1% to 1.0%) for 24, 48 and 72 hours in the presence or absence of inflammatory stimuli [i.e., IL-1β (25ng / ml), TNF-α (50ng / ml) and LPS (10μg / ml)] (scale bar 20×); (C, D) Viability measured by MTT (C) and TOX6 (D) assays. Data are expressed as mean ± SEM (n = 3 to 4 samples / group). Two-way ANOVA, ***p<0.001vs CTRL, #p<0.05 and ###p<0.001vs inflammation;

[0016] - Figure 3 The effect of E8 treatment on intestinal permeability in an in vitro intestinal inflammation model is shown. (A to F) mRNA levels of intestinal permeability markers in Caco2 cells differentiated for 14 days and treated with E8 (0.1% to 1.0%) for 24, 48 hours and 72 hours. The cycle number when the transcript can be detected is compared with the cycle number of GAPDH and expressed as relative expression relative to the control (CTRL) (shown as 1.0). (G to H) Western blot analysis of zonulin 1 (ZO-1) and occludin levels in Caco2 cells differentiated for 14 days and treated with E7 (0.1% to 1.0%) for 48 hours. Data are expressed as mean ± SEM (n = 3 to 4 samples / group). Two-way ANOVA (A to F), one-way ANOVA (G to H), *p<0.05, **p<0.01, ***p<0.001 vs CTRL; #p<0.05 and ###p<0.001 vs inflammation.

[0017] - Figure 4 The effect of E8 treatment on barrier integrity in an in vitro intestinal inflammation model is shown. (A) CaCo2 cells differentiated for 14 days cultured on 24-well inserts were pretreated with E7 (0.1% to 1.0%) for 24 hours and subsequently exposed to inflammatory stimuli for an additional 24 hours. IL-1b (25 ng / ml) and TNF-α (50 ng / ml) were applied to the basolateral side of the cells; LPS (5 μg / ml) was applied on both sides. (B) TEER was measured at the beginning and end of the experiment. Values ​​are expressed as a percentage relative to CTRL and represent mean ± SEM, n = 3 to 4. One-way ANOVA, *p<0.05, **p<0.01 vs CTRL;

[0018] - Figure 5The effect of E8 treatment on mitochondrial biogenesis in an in vitro intestinal inflammation model is shown. (A, B) Relative expression of mRNA of mitochondrial biogenesis markers in Caco2 cells differentiated for 14 days and treated with E7 (0.1% to 1.0%) for 24, 48 hours and 72 hours. The cycle number when the transcript can be detected is compared with the cycle number of GAPDH and expressed as relative expression relative to the control (CTRL) (shown as 1.0). (C) Western blot analysis of PGC1α in Caco2 cells differentiated for 14 days and treated with E7 (0.1% to 1.0%) for 48 hours. Data are expressed as mean ± SEM (n = 3 to 4 samples / group). Two-way ANOVA, *p<0.05, ***p<0.001vs CTRL; #p<0.05vs inflammation;

[0019] - Figure 6 The effect of E8 treatment on IL8 secretion in an in vitro intestinal inflammation model is shown. Caco-2 cells differentiated for 14 days were pretreated with E7 for 1 hour and subsequently exposed to inflammatory stimuli or not, as previously reported. IL8 secretion in the culture medium was evaluated by ELISA kit. Values ​​are expressed in pg / ml and represent mean ± SEM (n = 3 samples / group). Two-way ANOVA, ***p<0.001vs CTRL; #p<0.05 and ##p<0.01vs inflammation;

[0020] - Figure 7 A comparison of the efficacy and safety of E8 and EAAm compositions in an in vitro intestinal inflammation model is shown. (A) Caco2 cells differentiated for 14 days were pretreated with E7 (1.0%) and EAAm (1.0%) (i.e., a composition without TCA cycle intermediates) for 1 hour and subsequently exposed to inflammatory stimuli or not for 24 or 48 hours; (B) Viability of Caco2 cells differentiated for 14 days and treated with E7 or EAAm (1.0%) for 24 and 48 hours as measured by MTT assay; (C to J) Relative mRNA expression of intestinal permeability (C to H) and mitochondrial biogenesis (G to H) genes. The cycle number when transcripts can be detected is compared with the cycle number of GAPDH and expressed as relative expression relative to the control (CTRL) (shown as 1.0). Data are expressed as mean ± SEM (n = 3 to 4 samples / group). Two-way ANOVA, *p<0.05, ***p<0.001vs CTRL; #p<0.05vs inflammation, §p<0.05vs EAAm-treated cells;

[0021] - Figure 8The effect of 5aa-based compositions in an in vitro intestinal inflammation model is shown. (A) Caco2 cells differentiated for 14 days were pretreated with 5AA, 5AA-acid and E7 (all 1.0%) for 1 hour and exposed to inflammatory stimuli or not for 24 hours. (A to G) Relative expression of mRNA of intestinal permeability genes. The number of cycles when transcripts can be detected is compared with the number of cycles of GAPDH and expressed as relative expression relative to control (CTRL) (shown as 1.0). Data are expressed as mean ± SEM (n = 3 to 4 samples / group). Two-way ANOVA, *p<0.05vs CTRL; #p<0.05, ##p<0.01, ###p<0.001, as specifically reported in each figure. DETAILED DESCRIPTION

[0022] In the following description, numerous specific details are given to provide a comprehensive understanding of some embodiments. Some embodiments may be implemented without one or more 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 obscuring some aspects of some embodiments.

[0023] References throughout this specification to "one embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the phrase "in one embodiment" appearing in multiple locations throughout this specification does not necessarily all refer 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 explain the scope or meaning of the embodiments.

[0024] The purpose of the present description is to provide an amino acid-based composition that is particularly effective in preventing and / or treating intestinal diseases in a subject. The intestinal disease may be selected from irritable bowel disease (IBS) and inflammatory bowel disease (IBD), wherein the IBD may preferably be selected from Crohn's disease and ulcerative colitis.

[0025] The composition for preventing and / or treating intestinal diseases in a subject comprises an active agent comprising the amino acids leucine, isoleucine, valine, threonine, lysine and citric acid, succinic acid, malic acid.

[0026] In one or more embodiments, the subject may be a subject affected by chronic kidney disease (CKD), and the bowel disease may preferably be selected from irritable bowel disease (IBS) and inflammatory bowel disease (IBD), wherein the IBD may be selected from Crohn's disease and ulcerative colitis.

[0027] The inventors of the present application have unexpectedly discovered that the compositions disclosed herein are particularly effective in improving intestinal inflammation and permeability, which represent two mutually restrictive factors, because inflammation destroys TJs in the presence of cytokines, and dysregulated TJs in turn allow intestinal translocation and activation of both local and systemic inflammation.

[0028] Specifically, the feces (faecal) level of intestinal inflammation marker calprotectin and IMB dysfunction marker zonulin was measured in the object affected by chronic kidney disease (CKD), and IMB dysfunction is a risk factor for chronic CKD progress. Especially, in CKD, the combination of intestinal inflammation and intestinal permeability can cause intestinal barrier destruction, causing luminal bacteria and toxic bacterial byproducts (enterotoxins) to shift to systemic circulation (endotoxinemia). Endotoxemia is the reason that systemic inflammation occurs, CKD progresses accelerated, uremic syndrome, cardiovascular disease, and mortality risk improve, as recorded in both animals and people suffering from late CKD. In CKD, intestinal dysbiosis (i.e., the change of intestinal microbial population) is the key factor of intestinal inflammation. For example, the duodenum and jejunum that are not colonized by bacteria under physiological conditions are colonized by aerobic and anaerobic bacteria in CKD, which is the reason for chronic inflammation of the gastrointestinal tract (such as esophagitis, gastritis, duodenitis, enteritis, colitis). Intestinal permeability is increased in patients with CKD following the disruption and depletion of intestinal TJ architecture caused by elevated azotemia, and disruption and depletion of TJs leads to loss of barrier homeostasis and increased intestinal permeability.

[0029] In subjects affected by CKD, measurements of elevated fecal calprotectin and zonulin have been selected for the clinical studies presented in the following sections because these two markers have been previously used to diagnose intestinal inflammation (Ayling RMAdv Clin Chem 2018 87:161-190) and permeability (Fasano AClin Gastroenterol Hepatol 2012 10:1096-1100), respectively.

[0030] The inventors of the present application provide evidence herein that baseline fecal calprotectin and zonulin are significantly higher in CKD patients compared to unsupplemented healthy subjects, suggesting that kidney disease promotes intestinal inflammation and IMB dysfunction.

[0031] In stool samples of CKD patients, calprotectin and zonulin levels were significantly reduced after 6 months of supplementation with the composition disclosed herein compared to the levels of calprotectin and zonulin before treatment, thus providing evidence of the effectiveness of the composition in treating intestinal diseases associated with inflammation and permeability.

[0032] The composition administered to the CKD patient comprises an active agent comprising citric acid, succinic acid, and malic acid in combination with leucine, isoleucine, valine, threonine, and lysine.

[0033] 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 may be 0.05 to 0.3, preferably 0.1 to 0.25.

[0034] In one or more embodiments, the composition may consist of leucine, isoleucine, valine, threonine, lysine, citric acid, succinic acid, and malic acid, and optionally vitamin B1 and / or vitamin B6.

[0035] 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, tyrosine.

[0036] In one or more embodiments, the composition may comprise an activating agent consisting of leucine, isoleucine, valine, threonine, lysine, histidine, phenylalanine, methionine, tryptophan, cysteine, tyrosine, and citric acid, succinic acid, and malic acid, the amino acids being the only amino acids included in the composition. In one or more embodiments, the composition does not comprise any additional amino acids.

[0037] 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.

[0038] 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 58% by weight relative to the weight of the composition.

[0039] The composition may comprise the amino acids isoleucine, leucine, valine, lysine and threonine in an amount of 50% to 95% by weight, preferably 65% ​​to 90% by weight, relative to the weight of the composition. The composition may comprise citric acid, malic acid, succinic acid in an amount of 5% to 50% by weight, preferably 8% to 35% by weight, relative to the weight of the composition.

[0040] The weight ratio of leucine to citric acid may be 1 to 5, preferably 2.50 to 3.90.

[0041] In one or more embodiments, the weight or molar amount of citric acid is higher than the weight or molar amount of each of malic acid and succinic acid. Preferably, the weight or molar amount of citric acid is higher than the total 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.

[0042] A preferred isoleucine:leucine weight ratio is from 0.2 to 0.7, preferably from 0.30 to 0.60, and / or a preferred valine:leucine weight ratio is from 0.2 to 0.70, preferably from 0.30 to 0.65.

[0043] The weight ratio of threonine:leucine may be from 0.10 to 0.90, preferably from 0.20 to 0.70, and / or the weight ratio of lysine:leucine may be from 0.20 to 1.00, preferably from 0.40 to 0.90.

[0044] In a preferred embodiment, the ratio between the total weight of citric acid, malic acid, succinic acid and the total weight of methionine, phenylalanine, histidine and tryptophan is higher than 1.35.

[0045] In one or more embodiments, the weight ratio of the sum of citric acid, malic acid, and succinic acid to the sum of branched-chain amino acids leucine, isoleucine, and valine is 0.1 to 0.4, preferably 0.15 to 0.35.

[0046] 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 the three acids citric acid, malic acid, 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 leucine, isoleucine, valine, threonine and lysine.

[0047] In another embodiment, the total weight of lysine and threonine is higher than the total weight of the three acids citric acid, succinic acid, and malic acid. Preferably, the ratio between the total weight of the three acids citric acid, succinic acid, and malic acid and the total weight of lysine and threonine is 0.1 to 0.7, preferably 0.15 to 0.55.

[0048] In one or more embodiments, the amino acid is present in the composition in free form. The advantage associated with the use of the composition described herein is the fact that the use of the amino acid in free form contained in the active agent allows such a composition to be produced by production methods known per se at a relatively low cost relative to synthetic proteins and growth factors, and is widely used in the field of preparing compositions based on free amino acids. 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.

[0049] In one or more embodiments, the compositions disclosed herein may further comprise vitamins, preferably selected from vitamin B (e.g., vitamin B 1 and / or vitamin B 6 The vitamins may be included in the composition in an amount higher than 0.001%, preferably 0.001% to 0.5% by weight of the composition.

[0050] Additionally, the composition may also contain carbohydrates and / or flavoring substances.

[0051] In one or more embodiments, the composition can be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The composition can also comprise a pharmaceutically acceptable excipient, such as, for example, vitamins, carbohydrates, natural and artificial sweeteners and / or flavoring substances. In a preferred embodiment, the pharmaceutically acceptable excipient can be selected from maltodextrin, fructose, fish oil, sucralose, sucrose esters, vitamin D3.

[0052] 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 the compositions disclosed herein may specifically exclude are serine, proline, alanine. Such amino acids may be counterproductive or even harmful in the composition at some concentrations or stoichiometric ratios.

[0053] The amino acids disclosed in this specification may be replaced by corresponding pharmaceutically acceptable derivatives (ie, salts). The carboxylic acids of the active agents may be replaced by corresponding pharmaceutically acceptable derivatives (ie, salts).

[0054] For oral use, the composition according to the present specification may be in the form of tablets, capsules, granules, gels, jellify powders, powders.

[0055] The present disclosure also provides a method for preventing and / or treating intestinal diseases in a subject. The intestinal disease may be selected from irritable bowel disease (IBS) and inflammatory bowel disease (IBD), wherein the IBD may preferably be selected from Crohn's disease and ulcerative colitis. The method comprises selecting a 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 and malic acid; and administering the composition to the subject. The active agent may also comprise one or more amino acids selected from the following: histidine, phenylalanine, methionine, tryptophan, cysteine, tyrosine, as disclosed herein.

[0056] Additional descriptions regarding the amounts of various amino acids provided by the composition and the ratios therebetween are contained in the appended claims, which form an essential part of the technical teaching related to the present invention provided herein.

[0057] Example

[0058] Some examples of compositions of the present disclosure are shown in Table 1 below.

[0059] Table 1

[0060]

[0061] The composition of Table 1 above can be prepared by first sieving all components with a 0.8 mesh. 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 about 10% of the 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 for a period of 15 minutes at 120 rpm to obtain a uniform final composition.

[0062] Methods - Part 1

[0063] patient

[0064] The study was approved by the Ethics Committee of the Local Health Authorities (Reg 74 / 2018) after the entire study population provided their written informed consent.

[0065] Patients diagnosed with stage 3b-4 CKD (estimated glomerular filtration rate - eGFR - 44 to 29 ml / min / 1.73 m2) have been preselected. 2) were selected from twenty-nine elderly (age>65 years) patients. The reason for selecting elderly subjects is that since aging is associated with loss of TJs, microbial dysbiosis, and intestinal barrier dysfunction, there is a higher probability of finding alterations in the integrity of the intestinal barrier. Among these patients, patients with concomitant acute and chronic inflammatory diseases (n=3), diabetes (n=5), cancer (n=0), autoimmune diseases (n=1), treatment with steroids / immunosuppressants (n=0), chronic obstructive pulmonary disease (n=7), heart failure (n=2), and liver disease (n=1) were excluded. In addition, smoking patients were excluded because smoking is associated with alterations in intestinal tight junctions. Therefore, out of the initial 29 patients, 9 patients were studied. The patients were treated with a diet with a protein intake of 0.6 to 0.7 g / kg (50% animal origin), a calorie intake of >30 Kcal / kg / day, and a phosphate intake of <1000 mg / day. In addition to eGFR, renal function was assessed by the Modified Dietary Predictions in Renal Disease Study Group (MDRD) (Menon VAm J Kidney dis 2099 53:208-217) normalized to body surface area.

[0066] These patients were asked to undergo 6 months of supplementation with the composition disclosed herein and shown as "Composition E7" in Table 1. The measurements collected after the 12-month survey were used as baseline values ​​for the study. Eleven healthy controls were also recruited for the study as a control group (CTRL).

[0067] After recruitment, patients underwent the following measurements:

[0068] 1. Biological fluid variables, including 24-hour urine protein content. Urea nitrogen appearance (UNA) was calculated to estimate protein intake (Maroni BJ Kidney Int 1985 27:58-65; Masud T Kidney Int 2002 62:1750-56).

[0069] 2. Body composition. Total body water (TBW), extracellular water (ECW), intracellular water (ICW), all in litre (l) and percentage of body weight, resistance (RZ, in ohms), conductance (XC, in ohms), and phase angle (in degrees), which are considered to be indicators of skeletal muscle mass, were measured by bioimpedance analysis (BIA) by the same operator using the same instrument (renal EFG 50 Hz; EFG diagnostic Ltd, Belfast Northern Ireland) (Sunario J J Parent Enter Nutr 2021 45:1089-1099).

[0070] 3. Determination of plasma AA

[0071] Free amino acid concentrations in plasma from supplemented and control patients were measured by cation exchange chromatography on a Biochrom 30+ amino acid analyzer (Biochrom Ltd, ERRECI srl Pieve Emanuele, Italy) and detected spectrophotometrically (570 nm and 440 nm) after post-column reaction with ninhydrin reagent (n = 8 to 9 samples / group). Plasma samples were diluted in an aqueous solution containing L-norleucine (Sigma Aldrich, Milan, Italy) as an internal standard (C f =250mmol / L) in a 5-sulfosalicylic acid 5% (SSA) based deproteinization reagent 1:1 and incubated at 4°C for 30 minutes. After centrifugation (10,000×g for 5 minutes at 4°C), the supernatant was collected and filtered in a filter column (0.22 mm) (Millipore, Milan, Italy) to completely remove proteins (12,000×g for 10 minutes at 4°C). Calibration standards (C f =250mmol / L) (Biochrom Ltd, Cambridge Research Park, UK) solutions were treated with L-norleucine SSA 5% based buffer as samples to ensure equal pH at loading. Standard solutions and samples kept cool during the experiment were injected into the analyzer (60ml / sample). Amino acid separation was achieved by cation exchange chromatography. Amino acid concentrations were measured spectrophotometrically after post-column reaction with ninhydrin under control reaction conditions at 135°C.

[0072] 4. Determination of Fecal Calprotectin and Zonulin Levels

[0073] Calprotectin determination in stool samples was performed by an immunoenzymatic method and measured by a Chrous TRIO instrument (DIESSE Diagnostica Senese Sp: A., Italy) according to the manufacturer's instructions. Calprotectin values ​​< 50 μg / g for each stool sample were considered normal.

[0074] Intestinal permeability was assessed as fecal zonulin concentration (ng / ml) using a commercial ELISA kit (Zonulin Stool ELISA, DRG Instruments Gmbh, Germany).The normal amount of zonulin in the feces of healthy subjects is considered to be <60 ng / ml.

[0075] The age, gender, and body mass index (BMI, Kg / m 2 ) matched elderly healthy non-smoking subjects were used as controls (CTRL). CTRL underwent the same procedure as CKD, but only at the time of their recruitment.

[0076] After completing the above measurements, the patient was prescribed only this composition; specifically, the patient was instructed to consume 2 packets / day for 6 months (1 packet in the morning and 1 packet in the afternoon, diluted in 150 to 200 ml of water). The composition of each packet is shown in Table 2.

[0077] Table 2

[0078] Element mg / pack L-Leucine 1200.00 L-Isoleucine 600.00 L-Valine 600.00 L-Lysine 900.00 L-Threonine 700.00 L-Histidine 150.00 L-Phenylalanine 100.00 L-Methionine 50.00 L-Tryptophan 50.00 L-Cysteine 150.00 Citric Acid 409.00 Succinic acid 102.5 Malic Acid 102.5 Vitamin B6 0.85 Vitamin B1 0.70

[0079] After 6 months of composition supplementation, the above procedures were repeated in CKD patients.

[0080] 5. Evaluation of Patient Compliance with the Composition

[0081] Patients were considered compliant with the prescribed composition if they achieved significant increases in plasma glutamine and alanine relative to baseline values ​​after an overnight fast within 6 months from the start of supplementation.

[0082] As muscle utilization of branched chain amino acids (BCAA (Branched Chain Amino Acid); included in the composition) increases, plasma glutamine and alanine levels also increase. In addition, as a determination of BCAA-induced glutamine and alanine formation, two ratios of increased plasma levels were added: glutamine / (BCAA+aspartic acid+asparagine+glutamate) and alanine / (BCAA+aspartic acid+asparagine+glutamate). In fact, aspartic acid, asparagine, and glutamate also contribute to glutamine and alanine formation.

[0083] 6. Evaluation of the Effect of Compositions on Urea Production

[0084] Blood urea nitrogen (BUN) was evaluated with a Biochrom 30+ amino acid analyzer. This measurement can be particularly important in the case of GFR progression in order to distinguish whether a possible increase in BUN is due to increased nitrogen intake and / or worsening renal dysfunction caused by supplementation with the composition. The absence of an increase or decrease in the BUN / plasma total amino acid (AA) ratio in fasting patients indicates the absence of nitrogen intake overload.

[0085] 7. Statistical methods

[0086] Central tendency and dispersion of continuous variables are reported as mean ± standard deviation. Discrete variables are reported as number (number, N) and frequency percentage. Several variables violate the normality assumption (Shapiro-Wilk test), so nonparametric statistics are preferred for hypothesis testing. The intra-group and inter-group comparisons of continuous variables are carried out by Wilcoxon signed rank test and Mann-Whitney U test (Mann-Whitney U-test) respectively. Categorical variables are compared (if applicable) by chi-square test (Chi-square test) or Fisher's exact test. All tests are two-tailed. P value <0.05 is considered to be statistically significant. All statistical analyses are carried out using SAS / STAT statistical software package version 9.4 (SAS Institute Inc., Cary, NC, USA).

[0087] Results - Part 1

[0088] Baseline characteristics of patients

[0089] Table 3 relates to the baseline characteristics of controls (CTR) and patients with chronic kidney disease (CKD). The reported p values ​​are from the Mann-Whitney U test or *Fisher's exact test. CKD patients and CTRL had similar age, weight (such as BMI), but had different body composition. In fact, patients showed an increase in total body weight (Total Body Weight, TBW; p < 0.05) and extracellular body weight (Extracellular Body Weight, EBW; p < 0.01), and a decrease in the phase angle (p < 0.01), considered as an indicator of reduced skeletal muscle mass.

[0090] When compared with controls, CKD showed mild anemia, lower serum iron levels (p < 0.01) and ferritin levels (p < 0.05), whose means were still within the normal range of values ​​for hyperparathyroidism in our laboratory.

[0091] From a renal perspective, the patient presented with moderate to severe renal failure (MDRD 30.83±7.88 ml / min / 1.73 m 2 ) and compensatory metabolic acidosis (decreased plasma bicarbonate levels, p < 0.01 compared with controls).

[0092] Blood urea nitrogen (BUN) was higher than that in controls (p < 0.001), whereas protein intake was lower (p < 0.05) according to UNA, but the absolute values ​​were higher than the prescribed values ​​(0.7 to 0.6 g / kg / d).

[0093] Table 3

[0094] CTR CKD p Gender Male N (%) 4(36％) 4(50％) 0.66* Age (years) 72.27±3.74 74.56±6.90 0.32 Weight(Kg) 64.50±12.80 62.76±9.05 0.61 Body surface (UM) 1.72±0.20 1.66±0.15 0.40 fatt_corr_sup_corp 1.01±0.10 1.05±0.09 0.40 Rz(Ohm) 536.3±40.0 515.5±113.2 0.99 Xc(Ohm) 52.15±12.42 42.25±17.38 0.17 Phase angle (degrees) 5.16±0.60 4.22±0.67 0.008 Total water (%) 35.65±16.60 55.93±9.20 0.012 Extracellular water (%) 19.25±8.48 55.75±4.39 <0.0001 ICW(%) 44.65±4.10 40.69±10.06 0.59 Fat mass (Kg) 21.91±2.40 19.97±9.96 0.48 Lean body mass (Kg) 27.55±2.85 44.00±10.03 0.00011 Cell mass (Kg) 24.86±2.79 19.84±6.52 0.065 Muscle mass (Kg) 27.55±2.85 24.21±6.07 0.32 BMI(Kgm-2) 24.86±2.79 25.31±2.31 0.50 BasicMet 1385±68 1299±145 0.44 MDRD 88.20±16.59 30.83±7.88 <0.0001 Hb 14.16±1.20 11.85±1.12 0.0006 Albuminemia (g / dL) 4.21±0.23 4.13±0.18 0.43 Sideremia (ug / dL) 106.09±26.69 72.38±32.76 0.005 Ferritin (ng / mL) 175.60±93.28 78.97±60.65 0.016 BUN (mg / dL) 11.47±3.52 43.86±16.09 <0.0001

[0095] Table 4 shows baseline measurements associated with intestinal mucosal inflammation (calprotectin) and permeability (zonulin) in controls (CTR) and patients with chronic kidney disease (CKD). The reported p values ​​are from the Mann-Whitney U test. Patients exhibited intestinal mucosal inflammation (higher fecal calprotectin, p = 0.005 vs control) and higher permeability (higher fecal zonulin concentration, p < 0.001 vs control).

[0096] Table 4

[0097] CTR CKD p Calprotectin 30.25±27.62 95.60±53.26 0.005 Zonulin 54.96±32.73 219.38±171.50 0.001

[0098] Changes in baseline variables after 6 months of E7 supplementation (T6 to T12)

[0099] As shown in Table 5, after 6 months of supplementation with the composition, the resulting fecal levels of both calprotectin and zonulin were significantly reduced (where p-values ​​are from Wilcoxon signed-rank test), indicating reduced intestinal inflammation and decreased intestinal permeability.

[0100] Table 5

[0101] variable Baseline After 6 months of AA p Calprotectin 95.60±53.26 37.92±22.04 0.008 Zonulin 219.4±171.5 70.5±35.9 0.05

[0102] Patients with CKD had good compliance with the prescribed combination supplementation, as shown by the increased glutamine / (BCAA+aspartate+asparagine+glutamate) ratio (p=0.008) and alanine / (BCAA+aspartate+asparagine+glutamate) ratio (p=0.016) (Table 6).

[0103] Table 6

[0104] variable Baseline After 6 months of AA p-value Gln / (BCAA+Asp+Asn+Glu) 0.43±0.23 0.91±0.29 0.008 Ala / (BCAA+Asp+Asn+Glu) 0.61±0.08 0.88±0.25 0.008

[0105] Table 7 below shows the Bun values, blood urea nitrogen / total amino acids (AA) ratios, and blood urea nitrogen / non-essential amino acids (non-essential AA) ratios of CKD patients at baseline and after 6 months of supplementation with the composition. Given that BUN / total AA is similar to baseline values, the composition does not cause an increase in urea formation.

[0106] Table 7

[0107] variable Baseline After 6 months of AA p-value Bun 43.86±16.09 50.33±9.69 0.20 Bun / (Total AA) 0.020±0.012 0.018±0.006 0.84 Bun / (AA optional) 0.030±0.018 0.025±0.009 0.55

[0108] Therefore, the results presented herein compare i) the differences in measured variables between CKD patients and the CTRL group before supplementation, and ii) the changes in variables in CKD patients at baseline and after 6 months of supplementation with the composition.

[0109] This study demonstrates that elderly patients with CKD have reduced intestinal integrity due to increased intestinal permeability caused by inflammation and TJ depletion; in addition, the results indicate that patients have good compliance with the composition supplementation, which is associated with improved intestinal inflammation and permeability.

[0110] The mitochondrial intermediates (malate, succinate, citrate) in the composition act synergistically with the amino acids in the composition to promote a more balanced inflammatory / adaptive immune response with respect to adaptive immunity.

[0111] These findings highlight the beneficial effects of the composition on IMB integrity in critically ill subjects in the intensive care unit or patients suffering from intestinal barrier damage or insufficiency, which may result from gastrointestinal disease or other conditions, including dysbiosis, systemic inflammation, CKD.

[0112] In the following sections, experimental results are provided to analyze the comparison of the effects of different compositions; specifically, an analysis has been performed to show the synergistic effect achieved by the composition when the three claimed acids (succinic acid, citric acid, malic acid) are administered in combination with the claimed amino acids.

[0113] Methods - Part 2

[0114] Cell culture and treatment

[0115] Human colon adenocarcinoma Caco2 cells were purchased from the American Type Culture Collection (ATCC-HTB-37, LGC Milan, Italy). Caco2 cells were routinely cultured in standard culture medium: EMEM (ATCC30-2003, LGC) supplemented with 10% fetal bovine serum (FBS (fetal bovine serum), ATCC 30-2020, LGC), penicillin (100 U / ml) and streptomycin (100 μg / ml) (Euroclone, Milan, Italy) at 37°C in a controlled atmosphere with 5% CO2.

[0116] For each experiment, confluent Caco2 cells (from passages 10 to 20) were grown in standard culture medium for 14 days to obtain fully differentiated cells (ie, spontaneously forming tight junctions between cells and possessing a brush border) (Wang et al. 2011). Differentiated Caco2 cells were pretreated for 1 hour with the following four compositions: 1) "E8", an essential amino acid composition enriched in tricarboxylic acid (TCA) cycle intermediates (i.e., citric acid, succinic acid, and malic acid in a ratio of 4:1:1) according to some embodiments 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 five claimed amino acids (i.e., leucine, lysine, isoleucine, valine, and threonine), 1.0% (p / v); 4) "5aa-acid", a composition "5aa" enriched in TCA intermediates as in E8 (i.e., citric acid, succinic acid, and malic acid in a ratio of 4:1:1) according to some embodiments of the present application, 1.0% (for compositions, see Table 8). At the end of pretreatment, all conditions were tested in the presence or absence of inflammatory mediators IL-1β (25 ng / ml), TNF-α (50 ng / ml) and LPS (10 μg / ml) and cultured in EMEM supplemented with 1% heat-inactivated FBS, penicillin (100 U / ml) and streptomycin (100 μg / ml) for 24, 48 or 72 h (Van DeValle J et al. 2010).

[0117] Table 8

[0118] Amino Acids E8 EAAm 5aa 5aa-acid L-Leucine 22.45 30.01 22.45 22.45 L-Lysine (hydrochloride) 21.13 19.58 21.13 21.13 L-Isoleucine 11.23 15.00 11.23 11.23 L-Valine 11.23 15.00 11.23 11.23 L-Threonine 13.1 8.40 13.1 13.10 L-Cysteine 2.81 3.60 - - L-Histidine 2.81 3.60 - - L-Phenylalanine 1.87 2.40 - - L-Methionine 0.94 1.20 - - L-Tyrosine - 0.72 - - L-Tryptophan 0.94 0.48 - - Vitamin B1 (Thiamine Hydrochloride) 0.02 - - - Vitamin B6 (Pyridoxine Hydrochloride) 0.02 - - - TCA intermediates 20 Citric Acid 7.65 - - 7.65 Malic Acid 1.92 - - 1.92 Succinic acid 1.92 - - 1.92

[0119] All values ​​are reported as percentage (g / 100 g).

[0120] Gene expression analysis

[0121] 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 (BioRad Laboratories) on a CFX connected real-time PCR system (Bio-Rad Laboratories). Primers were designed using Primer3 (version 0.4.0) software (Table 9). The number of cycles (threshold cycle, CT) at which multiple transcripts can be detected was compared with the housekeeper CT, referred to as ΔCT. The relative level of the gene is expressed as 2 -ΔΔCT , where ΔΔCT corresponds to the difference between the ΔCT of any treatment group and the ΔCT of the control group (Ruocco et al. 2020).

[0122] Table 9

[0123]

[0124] T a , annealing temperature; COX-IV, cytochrome c oxidase (complex IV); Cyt c, cytochrome c; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGC1α, peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α; Tfam, mitochondrial transcription factor A.

[0125] Immunoblot analysis

[0126] Protein extracts were obtained from differentiated Caco2 cells using Mammalian Protein Extraction Reagent (M-PE (Mammalian Protein Extraction Reagent) R, Pierce, Thermo Fisher Scientific, Merck, Milan, Italy) according to the manufacturer's instructions in the presence of a protease and phosphatase inhibitor cocktail (Sigma-Aldrich, Merck, Milan, Italy). Protein content was determined using the bicinchoninic acid protein assay (BCA, Euroclone, Milan, 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 to a nitrocellulose membrane (Bio-Rad Laboratories) and the target proteins were visualized with specific antibodies: anti-zonulin 1 and anti-occludens (all from Invitrogen, Thermo Fisher Scientific) and anti-PGC-1a (Abcam, Prodotti Gianni, Milan, Italy), each diluted 1:1,000. Anti-vinculin (1:3000; Sigma-Aldrich, Milan, 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 by ImageLab software (both from Bio-Rad Laboratories).

[0127] Cell viability assay

[0128] The standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolyl MTT is a yellow dye that is reduced by multiple oxidoreductases, including mitochondrial succinate dehydrogenase, to a blue product, formazan. 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. At the end of the treatment, MTT (5 mg / ml in PBS, 20 μl) was added and incubated for a further 4 hours. Purple formazan was added. Crystals were dissolved in 5% SDS / 0.1 M HCl (100 μL / well) overnight at 37°C and absorbance was recorded on a microplate reader at dual wavelengths of 570 nm / 655 nm (Ragni et al. 2022). Alternatively, cell viability was determined using a sulforhodamine B-based in vitro toxicology assay kit (TOX6, Sigma Aldrich) according to the manufacturer's instructions.

[0129] IL-8 secretion in cell culture supernatants

[0130] To evaluate IL8 secretion from differentiated Caco2 cells treated with E8 compositions for 24, 48, and 72 hours in the presence or absence of inflammatory mediators, the extracellular medium was collected and centrifuged at 2,000 g for 10 minutes. IL-8 release was evaluated using a sandwich ELISA method (ab214030 - Human IL-8 ELISA Kit, Abcam) and quantified using the standards provided in the kit in pg / ml.

[0131] Intestinal barrier integrity

[0132] Caco2 cells were seeded on polycarbonate membrane transwell inserts (Brand, Sigma Aldrich) with a pore size of 0.4 μm at a density of 1×105 cells / well. When the cells reached confluence, they were grown for 14 days to obtain differentiated Caco2 cells. The cell culture inserts allow access to both the basolateral and apical sides of the cells, which represent the circulatory pole and the luminal pole of the intestinal epithelium, respectively. Differentiated Caco2 cells were pretreated with E8 compositions (0.1% and 1.0%) on the apical side of the cells, and after 24 hours, the cells were exposed to inflammatory stimuli or not. IL-1β (25ng / ml) and TNF-α (100ng / ml) were applied to the basolateral side of the cells; in contrast, LPS was applied on both sides (5μg / ml / side), representing typical luminal microbial communities on the one hand, and on the other hand, representing the increased presence of bacterial components in serum during inflammatory diseases (Van De Valle J et al. 2010). Trans-epithelial electrical resistance (TEER) was measured at the beginning of the experiment, after 24 h pretreatment, and at the end of the experiment using an epithelial Volt / OhmMeter EVOM3 (World Precision Instruments, MatTek In Vitro Life Science Laboratories, Slovak Republic) (Hiebl et al. 2020).

[0133] Statistical analysis

[0134] The sample size for each experiment reflects the number of independent biological replicates, as provided in the legend. Data are reported as mean ± SEM. Statistical analysis was performed using Prism 6.0 software (GraphPad Software, Inc.) by one-way or two-way ANOVA tests. Values ​​of p < 0.05 were considered statistically significant.

[0135] Results - Part 2

[0136] Composition E8 improves intestinal permeability and mitochondrial biogenesis in the Caco2 in vitro intestinal barrier model

[0137] The human epithelial cell line Caco2, isolated from human colorectal adenocarcinoma by Fogh & Trempe in 1974, is the cell line most widely used as an intestinal model for absorption, transport and bioavailability studies in recent decades (Fogh & Trempe 1975; Haddad et al. 2023). Confluent Caco2 cells spontaneously begin to differentiate from day 14 to 21 and form a polarized cell monolayer with apical and basolateral membranes, junctional complexes, and a brush border with microvilli on the apical side, which is typical of human intestinal epithelial cells (Engle et al. 1998, Hidalgo et al. 1989).

[0138] To determine the correct differentiation time to study the effect of E8 on the healthy cell state, Caco2 was treated on days 14 and 21 of differentiation. After exposure to increasing doses of E8 (0.05% to 1.0%), no cytotoxic effects of the composition were observed on Caco2 cells differentiated for 21 days ( Figure 1 A, B). Notably, E8 (0.1% and 1.0%) increased Caco2 viability, which may be due to the stimulation of mitochondrial function, since the MTT assay indirectly evaluates the activity of mitochondrial succinate dehydrogenase ( Figure 1 A, B).

[0139] Therefore, the effect of E8 (0.1% to 1.0%) on the relative expression of mRNA of the most important proteins involved in the regulation of intestinal permeability in Caco2 cells differentiated for 14 or 21 days has been evaluated: 1) zonula occludens-1 (ZO-1), which is a peripheral membrane adaptor protein that acts as a bridge connecting integral membrane proteins to the actin cytoskeleton and other signaling proteins; 2) occludens and claudins, which are integral transmembrane proteins; 3) E-cadherin, which is a major component of adherens junctions and is involved in intestinal homeostasis and barrier function (Lee et al. 2018; Schneider et al. 2010). The data show that E8 treatment for 24 and 48 hours increases the relative expression of genes regulating intestinal permeability, mainly in Caco2 cells differentiated for 14 days ( Figure 1 C to G).

[0140] Since E8 provides essential amino acids and TCA cycle intermediates involved in mitochondrial regulation, the mRNA expression of two key mitochondrial genes was also evaluated: 1) Tfam, which is essential for the transcription, replication, and packaging of mtDNA into the nucleoid (Stiles et al. 2016); and 2) Cyt c (i.e., cytochrome c), which is a central component of the electron transport chain in mitochondria.

[0141] According to the results obtained in the MTT assay, treatment with the E8 composition stimulated the gene expression of Tfam and Cytoc, which confirms the potential benefit of E8 on mitochondrial biogenesis under basal conditions ( Figure 1 J, K).

[0142] Based on preliminary results, the 14-day period is considered to be the most valuable differentiation time point, at which E8 can improve Caco2 integrity and mitochondrial biogenesis in already basal conditions. E8 maintains intestinal inflammation in an in vitro model Barrier integrity

[0143] It has also been shown that Caco2 cells are able to produce inflammatory markers (e.g., cytokines) in response to specific stimuli (e.g., IL-1β, TNF-α, and LPS) (Ponce de León-Rodríguez et al. 2019). This feature makes differentiated Caco2 cells a valuable in vitro model of intestinal inflammation (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. Since the first response of Caco2 cells after inflammation is cytokine production during 24 h, and permeability impairment (e.g., decreased expression of ZO-1, occludin, and claudin) occurs only after continued incubation with inflammatory mediators, the evaluation was extended to 24, 48, and 72 h ( Figure 2 A, B).

[0144] MTT assays showed that under all experimental conditions, cell viability was enhanced after E8 composition treatment, while inflammation did not affect cell health ( Figure 2 C). CaCo2 cell viability was also assessed using the TOX6 assay (i.e., measurement of total biomass) following inflammation and E8 treatment, and no significant cytotoxicity was observed ( Figure 2 D). The efficacy of E8 compositions (0.1% to 1.0%) at different time points in maintaining intestinal permeability during inflammation was then evaluated. Figure 3 ).

[0145] As shown above, under basal conditions, E8 treatment increased the relative mRNA expression of key regulatory genes of intestinal permeability, which was maintained for 72 h ( Figure 3A to G). In addition, treatment with a higher concentration (1.0%) of E8 stimulated the expression of all genes analyzed, even in the presence of inflammation ( Figure 3 A to G). The pathogenesis of inflammatory bowel disease is associated with impaired proteins involved in intestinal permeability regulation (Schneider et al., 2010, Bruewer et al., 2006). It is noteworthy that under the current experimental conditions, the decrease in gene expression of occludin, claudin 3, claudin 4, and E-cadherin induced by 48 h of inflammation was effectively prevented by E8 treatment (1.0%) ( Figure 3 B and D to F). In addition, treatment with a higher concentration (1.0%) of E8 prevented the damage induced by 48 h of inflammation, especially at the level of occludin ( Figure 3 G, H).

[0146] 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 ( Figure 4 A).

[0147] Caco2 cells were pretreated with E8 (0.1% to 1.0%) for 24 hours, and then inflammatory stimulation was applied for 24 hours. As expected, inflammatory stimulation impaired intestinal barrier integrity (-29.3% vs CTRL), and only E8 treatment 1.0% partially protected cells from damage (-17% vs CTRL, and +12% vs inflammation) ( Figure 4 B).

[0148] E8 stimulates mitochondrial biogenesis in an in vitro model of intestinal inflammation

[0149] In an in vitro intestinal inflammation model, the effect of E8 treatment (0.1% to 1.0%) on mitochondrial biogenesis was analyzed. After 48 h, inflammation impaired the gene expression of PGC1α and COXIV in Caco2 cells and the PGC1α protein level ( Figure 5 A to C).

[0150] E8 (1.0%), which already had the activity of improving mitochondrial biogenesis under basal conditions, increased PGC1α gene and protein expression as well as COX IV gene expression after 48 to 72 hours of treatment, even in the presence of inflammation ( Figure 5 A to C).

[0151] These results suggest that E8 prevents mitochondrial damage induced by inflammation.

[0152] E8 attenuates inflammatory responses in an in vitro intestinal inflammation model

[0153] IL8 is a chemokine produced by macrophages and other cell types (including intestinal epithelial cells), which secrete IL8 during the acute phase of inflammation to attract and activate neutrophils (Hoffmann et al. 2002). IL8 induces chemotaxis of neutrophils and granulocytes, causing them to migrate to the site of infection and, once they arrive, stimulates phagocytosis.

[0154] To evaluate the potential anti-inflammatory effects of E8, IL8 release was analyzed in an in vitro intestinal inflammation model. As expected, inflammatory mediators stimulated IL8 release from Caco2 enterocytes after 24 h of exposure, which was maintained for 72 h ( Figure 6 The lowest concentration (0.1%) of E8 treatment reduced IL8 secretion after 48 h of treatment (-9% vs inflammation, Figure 6 After continuous treatment, both doses (0.1% to 1.0%) of E8 significantly attenuated the inflammatory response, with IL8 release reduced by -13% and -9%, respectively, compared with inflammatory conditions ( Figure 6 ).

[0155] TCA cycle intermediates enhance the health benefits of amino acid composition in an in vitro model of intestinal inflammation

[0156] It is believed that E8 compositions comprising balanced essential and branched-chain amino acid stoichiometric ratios and TCA cycle intermediates (ie, citrate, succinate, and malate) can counteract mitochondrial damage and improve intestinal barrier function in inflammatory bowel disease.

[0157] To demonstrate that TCA cycle intermediates act synergistically with essential amino acids and enhance the benefits on intestinal permeability and intestinal mitochondrial biogenesis, the present inventors compared the effects of E8 (i.e., an essential amino acid composition with TCA cycle intermediates) with an amino acid composition that provides essential amino acids but does not contain TCA cycle intermediates (EAAm, Table 1) in an in vitro intestinal inflammation model ( Figure 7 A).

[0158] Notably, in the absence of inflammation, EAAm treatment for 24 and 48 h negatively affected cell viability; in contrast, E8 treatment did not show any cytotoxic effects ( Figure 7 B). In addition, the results showed that only E8 prevented inflammation-induced damage to the intestinal barrier and stimulated the expression of key regulatory genes for intestinal permeability and mitochondrial biogenesis more effectively than EAAm ( Figure 7 C to J).

[0159] The effects of a partial composition consisting of the most representative amino acids in the E8 composition (i.e., leucine, lysine, isoleucine, valine, and threonine—the "5aa" composition) were also studied in comparison with the same composition added to the TCA cycle intermediates at the same ratios provided in E8 (the "5aa-acid" composition; Table 1). Differentiated Caco2 cells were treated with the compositions 5aa and 5aa-acids and E8 (each at 1%) for 24 h in the presence or absence of inflammatory stimuli. Figure 8 A).

[0160] The results presented herein demonstrate that under basal conditions, the 5aa-acid composition was able to stimulate mRNA expression of intestinal permeability genes more effectively than the 5aa composition, and even more effectively than the E8 composition ( Figure 8 B to G). During inflammation, the E8 composition is also able to counteract the damage induced by inflammatory stimuli ( Figure 8 B to G).

[0161] In summary, the data obtained from the comparison of essential amino acid compositions without TCA cycle intermediates (i.e., EAAm) and 5aa-based compositions (i.e., 5aa and 5aa-acids) suggest that TCA cycle intermediates represent important promoters of intestinal epithelial health while allowing for full beneficial effects during inflammation.

[0162] In summary, the results provided herein indicate that the composition of the present application is able to improve intestinal permeability and intestinal mitochondrial biogenesis under basal conditions in an in vitro intestinal barrier model. It is noteworthy that the composition prevents inflammatory damage to intestinal permeability and mitochondrial compartments in an in vitro intestinal inflammation model. The composition of the present application shows an anti-inflammatory effect and reduces IL8 secretion of intestinal epithelial cells. TCA cycle intermediates enhance the effects of essential amino acids on intestinal permeability and intestinal mitochondrial biogenesis. In general, the data provide the role of the composition in preventing and managing IMB integrity in patients with intestinal barrier damage or dysfunction.

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Claims

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

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

3. The composition for use according to claim 1 or claim 2, wherein the weight ratio between the sum of citric acid, malic acid, and succinic acid and the sum of branched-chain amino acids leucine, isoleucine, valine plus lysine and threonine is 0.05 to 0.3, preferably 0.1 to 0.

25.

4. A composition for use according to any one of the preceding claims, wherein the weight ratio between the total amount of citric acid, malic acid, succinic acid and the total amount of branched-chain amino acids leucine, isoleucine, valine is 0.1 to 0.4, preferably 0.15 to 0.

35.

5. Composition for use according to any one of the preceding claims, wherein the weight ratio between citric acid and the sum of malic acid and succinic acid is from 1.0 to 4.0, preferably from 1.5 to 2.

5.

6. A composition for use according to any one of the preceding claims, wherein the weight ratio of citric acid:malic acid:succinic acid is from 10:1:1 to 2:1.5:1.5, preferably from 7:1:1 to 1.5:1:1, more preferably from 5:1:1 to 3:1:

1.

7. The composition for use according to any one of the preceding claims, wherein the active agent further comprises at least one amino acid selected from the group consisting of histidine, phenylalanine, methionine, tryptophan, tyrosine, cysteine.

8. A composition for use according to any one of the preceding claims, wherein the active agent further comprises histidine, phenylalanine, methionine, tryptophan, cysteine.

9. Composition for use according to any one of the preceding claims, wherein the ratio between the total weight of citric acid, malic acid, succinic acid and the total weight of methionine, phenylalanine, histidine and tryptophan is higher than 1.

35.

10. Composition for use according to any of the preceding claims, wherein the ratio between the total weight of the three acids citric acid, succinic acid, malic acid and the total weight of lysine and threonine is between 0.10 and 0.70, preferably between 0.15 and 0.

55.

11. Composition for use according to any of the preceding claims, wherein the amino acids isoleucine, leucine and valine are present in an amount ranging from 35% to 65% by weight, preferably from 42% to 58% by weight relative to the weight of the composition.

12. A composition for use according to any one of the preceding claims, wherein the weight or molar amount of citric acid is greater than the total weight or molar amount of malic acid and succinic acid.

13. Composition for use according to any one of the preceding claims, wherein the weight ratio between leucine and citric acid is from 1 to 5, preferably from 2.50 to 3.

90.

14. A composition for use according to any one of the preceding claims, wherein the composition does not contain arginine.

15. A composition for use according to any one of the preceding claims, wherein the composition is free of serine, proline and alanine.

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