Novel compositions containing branched chain amino acids

Abstract

This invention relates to compositions comprising branched-chain amino acids (BCAA) and the dipeptide L-alanyl-L-alanine, and their use in improving performance and recovery during physical activity and in preventing and / or treating muscle degeneration associated with pathological conditions or age-related conditions.

Description

TECHNICAL FIELD

[0001] The present invention relates to a combination of branched-chain amino acids (BCAAs) and the dipeptide L-alanyl-L-alanine, related pharmaceutical compositions, and the use of said compositions for improving performance and recovery during physical activity, and their use as a medicament for preventing and / or treating muscle wasting associated with pathological conditions or age-related conditions. BACKGROUND

[0002] Skeletal muscle is a plastic organ maintained by multiple pathways that regulate cell and protein turnover. Skeletal muscle proteins are constantly and simultaneously synthesized and degraded. The amount of protein in muscle and skeletal muscle mass in mature individuals is maintained depending on the net muscle protein balance (NBAL), which is the difference between muscle protein synthesis (MPS) and breakdown (MPB). Growth factors, hormones, cytokines, nutrients, and mechanical loads can activate cellular signaling pathways that favor protein synthesis or degradation, leading to an increase or decrease in skeletal muscle mass, respectively.

[0003] Muscle is not only important for movement and posture maintenance, but also provides the largest protein reservoir in the body and serves as a source of amino acids that can be used by various organs, including the heart, liver, and brain, for energy production and gluconeogenesis during catabolic processes.

[0004] Many situations are associated with a negative net muscle protein balance. In certain pathological conditions, an increased activation of the proteolytic system can occur, leading to the removal of contractile proteins and organelles from muscle, resulting in muscle atrophy. Muscle wasting occurs systemically in the elderly (a condition known as age-related sarcopenia), as a physiological response to fasting, malnutrition, or immobility (e.g., post-surgery), and in pathological conditions such as neuromuscular degenerative disorders (e.g., muscular dystrophy or atrophy), chronic obstructive pulmonary disorders, cancer-related cachexia, diabetes, renal failure, heart failure, Cushing syndrome, sepsis, burns, uremia, cirrhosis, and AIDS.

[0005] Excessive loss of muscle mass is associated with poor prognosis of several diseases.

[0006] Sarcopenia can also be observed as a result of sustained high-intensity exercise, such as running a marathon, which stimulates muscle catabolism to provide amino acids as substrates for energy production.

[0007] Athletes widely use sports supplements to ensure adequate carbohydrate and protein availability, thereby avoiding muscle catabolism.

[0008] Essential amino acids are biomolecules that the human body cannot synthesize and therefore must be obtained from the diet. Branched-chain amino acids (BCAAs) leucine, valine, and isoleucine account for 35% of the essential amino acids in muscles and are the essential amino acids consumed in the largest quantities during physical activity.

[0009] It has also been shown that serum concentrations of essential amino acids may affect the occurrence and severity of fatigue during endurance competitions.

[0010] Furthermore, theoretically, amino acids can enhance performance in a variety of ways, such as altering fuel utilization during exercise and preventing mental fatigue, as well as Newsholme EA et al, J Nutr. 2006, 136; 274S-6S; CPMSharp et al, Journal of Strength and Conditioning Research 2010, 24(4) / 1125–1130).

[0011] Many studies have shown that BCAAs serve as substrates for protein synthesis and energy production, and perform several metabolic and signal transduction functions, particularly by activating the mTOR signaling pathway.

[0012] These effects are achieved by BCAAs themselves, especially leucine, and their metabolites. In fact, leucine stimulates protein synthesis via the mTOR signaling pathway, as well as the phosphorylation of translation initiation factors and ribosomal proteins, while its inhibitory effect on proteolysis is mainly mediated by HMG (β-hydroxy-β-methylbutyric acid) and branched-chain keto acids.

[0013] Interestingly, unlike most amino acids, the initial steps of BCAA catabolism occur in skeletal muscle rather than the liver because branched-chain aminotransferase (BCAT) (the first enzyme responsible for the BCAA catabolism pathway) has higher activity in this tissue. This provides a unique advantage for BCAA-based nutritional formulations in enhancing muscle and brain function compared to others, as circulating BCAAs rapidly increase after protein intake and become readily available for use by extrahepatic tissues.

[0014] In light of the above, athletes now widely use BCAA supplements to limit the development of central fatigue and support muscle health and anabolism.

[0015] L-alanine is a gluconeogenic amino acid and one of the products of BCAA catabolism.

[0016] Dope Perform (Dompé Farmaceutici S.p.A.) comprises both BCCAs and L-alanine and is used to improve athletic performance and reduce fatigue.

[0017] There is still a need to develop improved, more effective compositions to be used as supplements for athletes. Moreover, there is a strong felt need to develop compositions effective in preventing and / or treating muscle wasting, for example, associated with age, fasting, malnutrition, immobility or pathological conditions. SUMMARY

[0018] The present inventors have surprisingly found that the oral administration of a combination containing BCCAs and the dipeptide L-alanyl-L-alanine (Ala-Ala) is particularly effective in improving muscle function, structure and metabolism in the case of intense training and in those cases where muscle wasting occurs.

[0019] It is therefore an object of the present application a composition comprising branched chain amino acids (BCCAs) and the peptide L-alanyl-L-alanine.

[0020] It is a further object of the present application the above-mentioned composition for use as a medicament for preventing and / or treating muscle wasting associated with pathological conditions, age-related conditions, malnutrition, immobility or fasting.

[0021] It is a further object of the present application the use of the above-mentioned composition for physical exercise to improve muscle performance and / or recovery and / or reduce muscle fatigue.

[0022] It is a further object of the present application a pharmaceutical composition comprising as active ingredient the above-mentioned combination of branched chain amino acids (BCCAs) and L-alanyl-L-alanine, together with at least one pharmaceutically or nutraceutically acceptable vehicle, excipient and / or adjuvant.

[0023] DEFINITIONS

[0024] As used herein, the term "branched chain amino acids (BCAAs)" means the amino acids L-isoleucine, L-leucine and L-valine.

[0025] As used herein, the term "L-alanyl-L-alanine" or "Ala-Ala" is used indifferently to indicate a dipeptide consisting of two L-alanine units connected by an amide linkage. While the term "Ala" refers to the single amino acid L-alanine. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1Gastrocnemius muscle homogenates of mice treated with vehicle, BCAA (BCAA), BCAA plus L-alanine (BCAA+2Ala) or BCAA plus L-alanyl-L-alanine (BCAA+Ala-Ala) as described in Example 1 are shown. (ANOVA at T3: ***BCAA+Ala-Ala vs BCAA+2Ala (p<0.005), vs BCAA (p<0.005) and vs. vehicle (p<0.005); §§ ANOVA at T4: **BCAA+Ala-Ala vs BCAA+2Ala (p<0.01), vs BCAA (p<0.01) and vs vehicle (p<0.005); §§ ANOVA at T4: **BCAA+Ala-Ala vs BCAA+2Ala (p<0.01), vs BCAA (p<0.01) and vs vehicle (p<0.005);

[0027] Figure 2 Gastrocnemius muscle homogenates of mice treated with vehicle, BCAA (BCAA), BCAA plus L-alanine (BCAA+2Ala) or BCAA plus L-alanyl-L-alanine (BCAA+Ala-Ala) as described in Example 1 are shown. (ANOVA at T3: ***BCAA+Ala-Ala vs BCAA+2Ala (p<0.005), vs BCAA (p<0.005) and vs. vehicle (p<0.005);

[0028] Figure 3 Gastrocnemius muscle homogenates of mice treated with vehicle, BCAA (BCAA), BCAA plus L-alanine (BCAA+2Ala) or BCAA plus L-alanyl-L-alanine (BCAA+Ala-Ala) as described in Example 1 are shown. (ANOVA at T3: ***BCAA+Ala-Ala vs BCAA+2Ala (p<0.005), vs BCAA (p<0.005) and vs. vehicle (p<0.005);

[0029] Figure 4 Gastrocnemius muscle homogenates of mice treated with vehicle, BCAA (BCAA), BCAA plus L-alanine (BCAA+2Ala) or BCAA plus L-alanyl-L-alanine (BCAA+Ala-Ala) as described in Example 1 are shown. (ANOVA at T3: ***BCAA+Ala-Ala vs BCAA+2Ala (p<0.005), vs BCAA (p<0.005) and vs. vehicle (p<0.005);

[0030] Figure 5 The area under the curve of 0 to 24 hours (AUCo-24h, pg / ml*min) of valine (panel A), leucine (panel B) and isoleucine (panel C) measured after administration of BCAAs only (BCAA), BCAAs plus L-alanine (BCAA+2Ala) or BCAAs plus L-alanyl-L-alanine (BCAA+Ala-Ala) to mice as described in Example 2 is shown.

[0031] Figure 6 The mean values ± SD of the mean residence time (MRT) of valine (panel A), leucine (panel B) and isoleucine (panel C) measured after administration of BCAAs only (BCAA), BCAAs plus L-alanine (BCAA+2Ala) or BCAAs plus L-alanyl-L-alanine (BCAA+Ala-Ala) to mice as described in Example 2 is shown.

[0032] Figure 7 The glycemic curve after administration of a glucose bolus to vehicle-, BCAAs plus L-alanine (BCAA+2Ala) or BCAAs plus L-alanyl-L-alanine (BCAA+Ala-Ala) pre-treated mice as described in Example 3 is shown (BCAA+Ala-Ala vs vehicle *** p<0.005; BCAA+2Ala vs vehicle §§§ p<0.005).

[0033] Figure 8 The incremental area under the curve (iAUC) measured within 120 minutes after administration of a glucose bolus to vehicle-, BCAAs plus L-alanine (BCAA+2Ala) or BCAAs plus L-alanyl-L-alanine (BCAA+Ala-Ala) pre-treated mice as described in Example 3 is shown.

[0034] Figure 9 The force-frequency curve (N*mm / kg) produced by the torque of the flexor digitorum muscle of control mice and mice subjected to hindlimb-unloading and treated with vehicle, BCAAs or BCAAs plus L-alanyl-L-alanine (BCAA+Ala-Ala) as described in Example 4 is shown. DETAILED DESCRIPTION

[0035] As will be shown in the experimental part, the inventors have surprisingly found that a formulation comprising a combination of BCAAs with the dipeptide L-alanyl-L-alanine is particularly effective in improving muscle function, structure and metabolism. Surprisingly, this effect can be observed both under high intensity training and under pathological conditions of muscle deterioration. The data obtained also show that the dipeptide L-alanyl-L-alanine significantly improves the absorption of BCAAs and induces their preferential distribution in muscle when they are administered orally.

[0036] Therefore, a first object of the present application is a composition comprising the branched chain amino acids (BCAAs) L-leucine, L-valine and L-isoleucine in combination with the peptide L-alanyl-L-alanine.

[0037] Preferably, in the above composition, the weight ratio L-leucine : L-valine : L-isoleucine is comprised between 2:1 :1 and 8:1 :1.

[0038] According to a preferred embodiment of the present application, L-leucine, L-valine and L-isoleucine are present in the composition in a weight ratio of 2:1 :1, respectively.

[0039] According to another preferred embodiment of the present application, L-leucine, L-valine and L-isoleucine are present in the composition in a weight ratio of 4:1 :1.

[0040] According to another preferred embodiment of the present application, L-leucine, L-valine and L-isoleucine are present in the composition in a weight ratio of 8:1 :1.

[0041] Preferably, in the composition of the present application, the weight ratio between BCAAs and the peptide L-alanyl-L-alanine is comprised between 5:1 and 1 :1, preferably between 3:1 and 1.5:1, more preferably between 2:1 and 1.6:1.

[0042] According to a particularly preferred embodiment, in the composition of the present application, the weight ratio L-leucine : L-valine : L-isoleucine : L-alanyl-L-alanine is comprised between 2:1 :1 :1 and 2:1 :1 :3.6, more preferably between 2:1 :1 :2.5 or 2:1 :1 :2.

[0043] Preferably, the composition of the present application does not contain additional amino acids or peptides other than BCAAs and L-alanyl-L-alanine.

[0044] Preferably, the composition of the present application is suitable for oral administration.

[0045] According to an embodiment, the composition according to the first object of the present application is a pharmaceutical composition.

[0046] The pharmaceutical composition preferably comprises the BCAAs and the dipeptide L-alanyl-L-alanine in admixture with at least one pharmaceutically acceptable carrier, excipient or adjuvant. The composition can comprise any pharmaceutically acceptable carrier, excipient or adjuvant known in the art. For example, the pharmaceutical composition according to the present application can contain usual ingredients such as fillers, binders, lubricants, flavoring agents, stabilizers, pH adjusting agents, disintegrants, preservatives.

[0047] Preferably, the pharmaceutical composition of the present application is suitable for oral administration.

[0048] The pharmaceutical composition for oral administration of the present application can be a solid or a liquid formulation. Preferred formulations of the present application contemplate a pharmaceutical form selected from the group consisting of granules, tablets, capsules, effervescent tablets, oral suspensions, emulsions, powders, solutions or syrups. The most preferred pharmaceutical form according to the present application is a granule for oral suspension or dissolution in a liquid, preferably water.

[0049] Preferably, the composition of the present application, preferably as a pharmaceutical composition, is formulated to contain per unit dose the following amounts of BCAAs and dipeptide: 0.6 to 1 g, preferably 0.8 g of L-leucine; 0.3 to 0.5 g, preferably 0.4 g of L-isoleucine; 0.3 to 0.5 g, preferably 0.4 g of L-valine; and 0.5 g to 1.5 g, preferably 0.8 g to 1.5 g, more preferably 0.8 g of L-alanyl-L-alanine.

[0050] A second object of the present application is the composition disclosed above for use as a medicament.

[0051] As indicated in the experimental section, the composition according to the present application significantly improves neuromuscular function of the flexor hallucis muscle when administered in an animal model of muscle wasting.

[0052] Therefore, in a preferred embodiment, the composition described above is used for preventing, ameliorating and / or treating muscle wasting associated with:

[0053] i. a pathological condition, preferably selected from the group consisting of neuromuscular degenerative disorders, such as muscular dystrophy or muscle atrophy; chronic obstructive pulmonary disorders; cancer-related cachexia; diabetes; renal failure; heart failure; Cushing syndrome; sepsis; burns; uremia; cirrhosis; and AIDS.

[0054] ii. an age-related condition, preferably sarcopenia; or

[0055] iii. malnutrition, immobility or fasting;

[0056] In all the above-mentioned applications, the composition according to the present application is preferably administered twice a day.

[0057] As indicated in the experimental part, the composition of the present application is particularly suitable for being administered before, during or after physical exercise to improve muscle performance and / or recovery and / or reduce muscle fatigue.

[0058] Another object of the present application is the use of the above composition to improve muscle performance and / or recovery and / or reduce muscle fatigue before, during or after physical exercise.

[0059] In the above use, the composition is administered as a single dose of 0.6 to 1 g, preferably 0.8 g of L-leucine; 0.3 to 0.5 g, preferably 0.4 g of L-isoleucine; 0.3 to 0.5 g, preferably 0.4 g of L-valine; and 0.5 g to 1.5 g, preferably 0.8 g to 1.5 g, more preferably 0.8 g of L-alanyl-L-alanine, as required.

[0060] Experimental part

[0061] All experiments were performed in accordance with the Italian Guidelines for Care and Use of Laboratory Animals (D.L. 116 / 92) and the European Directive (2010 / 63 / UE). The present study belongs to the research approved by the National Ethics Committee for the Research Animal Welfare of the Italian Ministry of Health, in terms of ethical issues and respect for the law. Most of the experimental procedures used in the present study were approved by the International Scientific Network for the Study of Animal Models of Neuromuscular Diseases (http: / / www.treat-nmd.eu / research / preclinical / SOPs / ).

[0062] Statistics

[0063] All experimental data are expressed as mean ± standard error of the mean (S.E.M.). Multiple statistical comparisons between groups were performed by one-way ANOVA, with Bonferroni post-hoc correction when the null hypothesis was rejected (p < 0.05) to better evaluate the variability within and between groups and avoid false positives. If necessary, single comparisons were performed between the two methods (modified formulation vs. vehicle or vs. standard formulation).

[0064] Example 1 - Efficacy of the formulation during training

[0065] a) Experimental groups and protocol

[0066] C57BL / 6J wild type (WT) male mice, 10 weeks old, purchased from Charles River (Calco, Italy), were used in the study. All mice were acclimated in the animal facility for one week before the beginning of the experimental protocol; then, based on their body weight and absolute and normalized forelimb grip strength values, they were assigned to each treatment group to create homogeneous cohorts at the beginning of the study.

[0067] The experiments, data collection and analysis were performed in blind by the experimental personnel.

[0068] The mice were divided into the following groups:

[0069] 1. Control group (n=5):

[0070] Mice that did not receive the physical exercise protocol and drank water vehicle;

[0071] 2. Vehicle group (n=5):

[0072] Mice that received the physical exercise protocol described below and drank the vehicle (filtered tap water).

[0073] 3. BCAA group (n=8):

[0074] Mice that received the physical exercise protocol described below and drank an aqueous solution containing a mixture of L-leucine, L-valine and L-isoleucine in a weight ratio of 2:1:1. Each mouse received a total amount of 328 mg / Kg of L-leucine, 164 mg / Kg of L-valine and 164 mg / Kg of L-isoleucine.

[0075] 4. BCAA and L-Ala group (n=8):

[0076] Mice that received the physical exercise protocol described below and drank an aqueous solution containing a mixture of L-leucine, L-valine, L-isoleucine and L-alanine in a weight ratio of 2:1:1:2. Each mouse received a total amount of 328 mg / Kg of L-leucine, 164 mg / Kg of L-valine, 164 mg / Kg of L-isoleucine and 328 mg / Kg of L-alanine.

[0077] 5. BCAA and L-ala-L-ala (n=8):

[0078] Mice received the following physical exercise regimen and drank water containing a mixture of L-leucine, L-valine, L-isoleucine and L-alanyl-L-alanine dipeptide in a weight ratio of 2:1 :1 :2. Each mouse received a total of 328 mg / Kg of L-leucine, 164 mg / Kg of L-valine, 164 mg / Kg of L-isoleucine and 328 mg / Kg of L-alanyl-L-alanine.

[0079] The aqueous solutions for groups 3 to 5 were prepared by dissolving the amino acids and dipeptide in filtered tap water to obtain the desired final concentration. This was obtained directly by taking into account the amount of water consumed by each mouse per week and its body weight.

[0080] Mice from groups 2 to 5 received the following physical exercise regimen.

[0081] Specifically, all the mice ran on an electrically powered horizontal treadmill (Columbus Instruments, USA) according to the following two-phase plan:

[0082] Phase 1 : This consisted of a 3-week pre-treatment exercise period (45 minutes, 5 days / week) to acclimatize the mice to the procedure and gradually reach the target workload. All the mice started running with a 5-minute warm-up at 5 m / min and then increased the speed by 1 m / min every minute until reaching the maximum speed, which was maintained until the end of the exercise bout. The maximum speed was increased once a week until reaching the target speed of 25 m / min.

[0083] Phase 2: This phase consisted of a 4-week exercise period at maximum workload (45 minutes, 5 days / week) starting with a 15-minute warm-up at 10 m / min and then increasing the speed by 1 m / min every minute until reaching the maximum speed of 25 m / min, which was maintained until the end of the exercise bout.

[0084] Treatment with water (group 2) or different solutions (groups 3 to 5) was carried out simultaneously with phase 2.

[0085] During the entire study period, all the mice were maintained on a controlled diet with a daily food intake of 4 to 5 g / mouse.

[0086] b) Analysis of parameters in mice

[0087] i. Methods

[0088] In vivo and ex vivo parameters were collected from the animals.

[0089] • In vivo parameters collection

[0090] The following data were monitored during the exercise regimen:

[0091] 1. Body weight change and water intake.

[0092] 2. Forelimb grip strength

[0093] Forelimb strength was measured by means of a grip strength meter (Columbus Instruments, USA) in each mouse at days 0 (TO), 7 (T1), 14 (T2), 21 (T3) and 28 (T4) of exercise regimen phase 2. The maximum force (absolute and normalized for body weight) exerted by each animal during 5 determinations against a gentle pull off the grip was used for statistical analysis.

[0094] 3. Determination of salivary IgA levels by enzyme-linked immunosorbent assay (ELISA)

[0095] Determination of salivary IgA levels in vivo.

[0096] Saliva samples were collected from the oral cavity of each mouse by using a micropipette about 5 minutes after intraperitoneal injection of pilocarpine (1 mg / kg, Sigma-Aldrich, USA) to stimulate salivation. Then, saliva was immediately placed in a microfuge tube containing PMSF (Sigma-Aldrich, USA) to inhibit proteases and kept on ice. Samples were clarified by centrifugation at 16,000 x g for 10 minutes at 4°C; supernatants were stored at -80°C until analysis. Salivary IgA levels were quantified using the Mouse IgA Ready-SET-Go! ELISA kit (eBioscience, Vienna, Austria) according to the manufacturer protocol.

[0097] 4. Determination of plasma levels of lactate dehydrogenase (LDH) by spectrophotometry.

[0098] Blood was obtained from cardiac puncture of the left ventricle with heparinized insulin syringe and collected in heparinized tubes. Samples were processed within 30 minutes from collection. Platelet-poor plasma was obtained after two consecutive centrifugation steps (20 minutes, 4000 rpm, 4°C; 10 minutes, 12000 rpm, 4°C). Lactate dehydrogenase (LDH) activity was determined by a commercially available kit (CKNAC LR and LDH LR, SGM Italy). The instrument was set at 37°C, wavelength 340 nm.

[0099] • Ex vivo parameters collection

[0100] At the end of week 4 of phase 2, animals were anesthetized and sacrificed with a combination of ketamine (100 mg / kg) and xylazine (16 mg / kg) intraperitoneally. Gastrocnemius and tibialis muscles were dissected from each mouse and frozen until analysis.

[0101] These samples were stored at -80°C for the planned endpoint evaluation. The following analyses were performed: 1. In gastrocnemius muscle Determination of protein content by Bradford assay.

[0102] The buffer used in the assay was composed of the following ingredients dissolved in 100 mL of water: 1 mL of Triton X-100, 2 mL of Tris HCL pH8, 2.74 mL of NaCl (5M), 10 mL of glycerol, 1 mL of EDTA 0.5M pH8. 100 mM PMSF (protease inhibitor) in EtOH was added before use. Gastrocnemius muscles were homogenized with a Bertin homogenizer using CK14 tubes (Precellys Lysing Kit) in buffer at dilution 1 / 5 w / v (g / mL). The samples were then centrifuged after homogenization and the lysate was analyzed for total protein quantification. The determination of the protein content was performed by the Bradford test according to the following protocol: 1) 250 μL of Brilliant Blue was added to the plate; 2) the samples were diluted 1:10 for the test, while those outside the calibration range were thus diluted 1:20 in saline and retested. 3) 5 μL of saline solution (blank), 5 μL of sample were added to the plate. The calibration as well as the samples were performed in triplicate; 4) the samples were shaken for 30 seconds; 5) the plate was incubated at room temperature for 10 minutes; 6) the plate was read at 595 nm.

[0103] ii. Results

[0104] 1. Health and body weight

[0105] All mice received the exercise / treatment regimen without showing any signs of stress (lack of appetite, abnormal weight loss, hair loss, stereotypic or aggressive behavior, etc.) or macroscopic changes in important functions. No significant changes in the body weight of the animals (whether treated or not) were observed throughout the experimental window.

[0106] 2. Forelimb grip strength

[0107] The values of the maximum forelimb force normalized for body weight measured at each weekly time point are shown as Figure 1

[0108] At T0, the values of the different experimental groups substantially overlap.

[0109] ​From T1 to T4, all mice treated with BCAA, BCAA and L-alanine, and BCAA and L-alanyl-L-alanine showed higher force values relative to vehicle-treated mice.

[0110] The addition of L-alanine or L-alanyl-L-alanine to BCAA resulted in a more powerful forelimb.

[0111] At T3 and T4, mice treated with BCAA and L-alanyl-L-alanine were significantly more powerful than mice treated with BCAA and L-alanine Figure 1 ).

[0112] These data show that the supplementation of L-alanyl-L-alanine improves muscle function and force after treatment: this indicates that the formulation is effective in providing protection against muscle weakness in physiological and pathological conditions.

[0113] 3. Determination of protein content in muscle

[0114] Gastrocnemius homogenates analyzed with the Bradford assay showed a significant increase in total protein content in all treatment groups compared to vehicle (125 ± 13 mg / g of tissue). In particular, the BCAA and L-alanyl-L-alanine group (192 ± 8 mg / g of tissue) showed a higher protein content compared to BCAA and L-alanine (162 ± 18 mg / g of tissue) and BCAA groups (142 ± 10 mg / g of tissue).

[0115] Data are reported as mean ± S.E.M. in Figure 2 .

[0116] These data show that the supplementation of L-alanyl-L-alanine improves muscle protein content: this can lead to the preservation of muscle weight, which can be beneficial for the functional recovery of the affected skeletal muscle.

[0117] 4. Salivary IgA levels

[0118] IgA is a marker of the level of inflammation of the upper respiratory tract. IgA levels (ng / μg) normalized to total protein values in saliva samples collected before each mouse was sacrificed are shown in Figure 3 . Saliva IgA levels of mice treated with BCAA, BCAA and L-alanine, and BCAA and L-alanyl-L-alanine were significantly lower compared to the saliva IgA levels of vehicle-treated mice. IgA levels of mice treated with BCAA and L-alanyl-L-alanine were significantly lower compared to any other group.

[0119] These data indicate that supplementation with BCAAs and L-alanyl-L-alanine has improved efficacy in protecting muscle from the depletion in pathological conditions and preventing the occurrence of overtraining compared to BCAAs and L-alanine.

[0120] 5. Plasma levels of lactate dehydrogenase (LDH)

[0121] Figure 4 Plasma concentrations of LDH enzyme evaluated in mouse plasma samples are shown in Table 2. All three treatments showed a clear and significant trend of reduced LDH plasma levels relative to vehicle. Mice treated with BCAAs and L-alanyl-L-alanine had significantly lower LDH levels than any other group.

[0122] These data indicate that supplementation with BCAAs and L-alanyl-L-alanine has improved efficacy in protecting the structural integrity and metabolism of skeletal muscle during long-term exercise compared to BCAAs and L-alanine.

[0123] Example 2 - Pharmacokinetic study

[0124] The aim of this study was to compare the exposure in plasma of L-leucine-13C6,15N, L-isoleucine-13C6,15N, L-valine-13C5,15N after administration alone or in combination with L-alanine or L-alanyl-L-alanine in mice. Plasma samples were analyzed by UPLC-MSMS after clean-up and derivatization using EZfaast TM Amino acid analysis kit (Phenomenex) and analyzed by UPLC-MSMS.

[0125] i. Methods

[0126] Animals and maintenance

[0127] The study used CD1 mice with a body weight of 25 to 30 grams at the time of treatment, approximately age of about 4 weeks according to the growth curve. These animals were initially provided by Harlan, Italy. Once the animals were received from the supplier, the animals were subjected to a health check and acceptance. The animals were housed in cages appropriate for the species, five animals per group. The animals were acclimated to the local housing conditions for about 5 days. The animals were normally maintained in the following environment, except for short periods specified in the experimental procedures. The animals were housed in separate, dedicated rooms, with air controlled to provide at least 15 air changes / hour. The environmental control was set to maintain the temperature in the range of 22°C and the relative humidity in the range of 50% to 60%, with a cycle of approximately 12 hours of light and 12 hours of darkness automatically controlled. Food (Mucedola standard GLP diet) and water were available ad libitum throughout the study. All animals were weighed on the day of each treatment and randomly assigned to the groups before the experiment, which were uniquely identified on the back with a colored spray. Clinical signs were monitored periodically throughout the study to assess any reaction to the treatment. The experiment was carried out in compliance with Italian law (D.L.vo 26 / 2014). Blood samples (50 to 60 μί) were collected in heparinized eppendorf tubes (Heparin Vister 5000 U.I / mL), mixed gently and immediately placed on ice; the eppendorf tubes were then centrifuged (3500 x g, 15 minutes at 4°C) and the resulting plasma was collected and transferred into uniquely labeled eppendorf tubes and frozen at -80°C until analysis. At the end of the study, the animals were sacrificed by exsanguination under deep isoflurane anesthesia.

[0128] Groups and doses

[0129] The fasted animals were divided into 4 groups of 6 mice each and the different groups were treated as follows. The administration volume was 15 mL / kg, while the vehicle used for the study was a 1.5% w / w aqueous solution of citric acid.

[0130] 1) BCAA group:

[0131] L-leucine-13C6,15N 328 mg / kg; L-isoleucine-13C6,15N 164 mg / kg; L-valine-13C5,15N 164 mg / kg.

[0132] 2) BCAA + Ala group:

[0133] L-leucine-13C6,15N 328 mg / kg; L-isoleucine-13C6,15N 164 mg / kg; L-valine-13C5,15N 164 mg / kg and L-alanine 328 mg / kg.

[0134] 3) BCAA + Ala-Ala group

[0135] L-leucine-13C6,15N 328 mg / kg; L-isoleucine-13C6,15N 164 mg / kg; L-valine-13C5,15N 164 mg / kg plus L-alanine 328 mg / kg.

[0136] Bleeding schedule and feeding protocol

[0137] Animals were fasted overnight prior to administration and food was reintroduced in the cages 3 hours after administration. Serial sampling was performed at 15 min, 30 min, 1 h, 3 h, 8 h, 24 h, collecting about 50-60 μΐ^ of blood at each time point.

[0138] ii. Results

[0139] AUC ± SD for each single labeled amino acid was calculated over the time interval 0-24 h and reported in Figure 5 A, B and C. The mean value ± SD of the mean residence time (MRT) of each labeled amino acid was reported in Figure 6 A, B and C.

[0140] Plasma concentrations of labeled amino acids were significantly increased with the administration of alanine and L-alanyl-L-alanine, the latter showing the highest concentrations Figure 5 A, B, C). The mean residence time values were significantly increased compared to the BCAA group, with the mice treated with BCAA plus L-alanyl-L-alanine showing the highest values. No differences were found among the three groups in the other calculated pharmacokinetic parameters (Cmax, Tmax and T1 / 2).

[0141] Example 3 - Oral glucose tolerance test in mice

[0142] An oral glucose tolerance test (OGTT) was evaluated in mice pre-treated with 15 mL / kg vehicle (1.5% w / w aqueous citric acid solution) or with the compositions of BCAA plus L-alanine or BCAA plus L-alanyl-L-alanine at the doses described in Example 1 and dissolved in 1.5% w / w aqueous citric acid solution. The administration volume of each solution was 15 mL / kg. Mice were fasted overnight, weighed and each of the above treatments was administered orally as described above.

[0143] Forty-five minutes after oral administration of the pre-treatment, mice were orally gavaged with 20% glucose (2 mg / g body weight) and blood glucose was measured at time -45', 0', 15', 30', 60', 90' and 120' by tail vein bleeds using a portable glucometer (Johnson & Johnson).

[0144] Figure 7 The blood glucose curves obtained in mice treated with each pre-treatment are shown. After glucose gavage administration, mice treated with BCAAs plus L-alanyl-L-alanine showed significantly lower blood glucose levels at 15, 30 and 60 minutes, while BCAAs plus L-alanine showed a significant reduction in blood glucose levels only at 15 minutes.

[0145] The data obtained indicate that L-alanyl-L-alanine has a greater capacity to improve glucose tolerance in mice than L-alanine (i.e. its efficacy at more time points).

[0146] The incremental area under the curve of the blood glucose excursion from t=0 to t=120 minutes (iAUCo-120) was then calculated and is shown in Figure 8 .

[0147] As can be seen in the figure, BCAAs plus L-alanyl-L-alanine administration affected glucose tolerance in a significant manner compared to mice treated with vehicle and BCAAs plus L-alanine.

[0148] These data indicate that oral administration of BCAAs plus L-alanyl-L-alanine induces an improvement in glucose tolerance in healthy C57BL / 6 mice. This is consistent with the data obtained in the pharmacokinetic study, which indicates that L-alanyl-L-alanine improves the exposure of BCAAs compared to L-alanine. Therefore, this increase in plasma BCAAs leads to an increase in insulin secretion after glucose gavage, resulting in a decrease in plasma glucose content. In summary, it is shown that the dipeptide L-alanyl-L-alanine has a greater effect on reducing plasma glucose than L-alanine.

[0149] Example 4 - Preclinical evaluation of efficacy in improving muscle function in a mouse model of muscle atrophy (HU mice).

[0150] i. Methods

[0151] Experiments were performed in accordance with the Italian Guidelines for the use of laboratory animal (Legislative Decree 2014 n. 26), which are in compliance with the European Union Directive for the protection of experimental animals (2011 / 63 / EU) and were approved by the Italian Ministero della Salute (DM n. 133 / 2000-B).

[0152] Adult male C57BL / 6J mice (12-14 weeks old) were purchased from Charles River (Calco, Italy). Mice were subdivided into 4 groups of 8 animals each, 1 control and 3 hindlimb-unloaded (HU) groups. In detail:

[0153] Control group :

[0154] Mice were housed individually under control conditions for 4 weeks.

[0155] Hindlimb unloading (HU) group:

[0156] A muscle unloading protocol was used, similar to the one previously used in HU rats (Pierno S, et al. J Physiol 2007; 584: 983-95). Specifically, animals were individually suspended in a dedicated cage for 2 weeks. A thin cord was connected at one end to the tail by a rubber band and at the other end to the top of the cage. The length of the cord was adjusted to allow the animals to move freely with the forelimbs, while the body was inclined 30° to 40° from the horizontal plane. All mice had free access to water and received 8 g of standard rodent chow daily. The food remaining at the end of the day was weighed to calculate daily food consumption.

[0157] The different HU groups were treated as follows:

[0158] Vehicle: Mice were housed individually for 4 weeks and subjected to hindlimb unloading for the last 2 weeks. During this period, they received the vehicle (water).

[0159] BCAA: Mice were housed individually for 4 weeks, received BCAA (L-leucine 328 mg / kg; L-isoleucine 164 mg / kg; L-valine 164 mg / kg) once a day for 4 weeks and were subjected to hindlimb unloading for the last 2 weeks.

[0160] BCAA + Ala-Ala: Mice were housed individually for 4 weeks, receiving once daily for 4 weeks a mixture of BCAAs plus L-alanyl-L-alanine (L-leucine 328 mg / kg; L-isoleucine 164 mg / kg; L-valine 164 mg / kg plus L-alanyl-L-alanine 328 mg / kg) and hindlimb unloading during the last 2 weeks.

[0161] Each formulation was prepared by dissolving the powder in filtered tap water to obtain the final concentration. This was obtained by direct preparation, taking into account the amount of water consumed by each mouse per week and its body weight. The duration of the treatment was 4 weeks.

[0162] Throughout the HU, the behavior, cleanliness, appearance of hair and eyes, food and water consumption of the HU animals were checked daily. Daily food intake was measured as the average of the amount of commercial food (in grams) consumed by the mice belonging to the CTRL and HU groups per day over 14 days. The mice were given a standard amount of food (10 g) in the morning every day and the remaining food was weighed in the morning of the following day. The daily intake was obtained by subtraction and the average daily intake over 14 days was calculated for each animal. The final result reported the average ± SEM of n animals in each experimental group. At the end of the treatment period, the mice were weighed and deeply anesthetized with an intraperitoneal injection of urethane (1.2 g / kg body weight) to allow the removal of the hindlimb skeletal muscles, i.e. the soleus (Sol), the extensor digitorum longus (EDL) and the gastrocnemius (Gas) muscles. These muscles were immediately used for electrophysiological and functional studies, or frozen in liquid nitrogen and stored at -80°C for further biochemical and gene expression analysis. After surgery, the animals were euthanized by an overdose of urethane. All efforts were made to minimize animal suffering.

[0163] In vivo torque protocol

[0164] In vivo maximal isometric torque of the plantar flexor muscle group (GC and soleus) was assessed at multiple time points over the long-term exercise or cage activity-based protocol. Mice were anesthetized by inhalation (about 4% isoflurane and 1.5% O2 l / min) and placed on a temperature-controlled table; anesthesia was maintained by a nose cone (about 2% isoflurane and 1.5% O2 l / min). The right hind limb was shaved and aseptically prepared and the foot was placed on a pedal connected to a servo motor (300C-LR model; Aurora Scientific, Aurora, ON, Canada). Transcutaneous electrical stimulation of the tibial nerve by needle electrodes (Chalgren Enterprises) connected to a stimulator (701B model; Aurora Scientific) was used to induce contractions to elicit plantar flexor muscle group contractions. Current was adjusted from 30 to 50 mA until maximal isometric torque was reached. A series of stimulations was then performed at increasing frequencies: 1, 10, 30, 50, 80 and 100 Hz with a pulse train of 200 ms. Data were analyzed using dynamic muscle analysis software (DMA v5.201; Aurora Scientific) to obtain torque, which was normalized to mouse body weight. Normalized values were used to construct torque-frequency curves.

[0165] ii. Results

[0166] Health and body weight

[0167] All mice received the exercise / treatment protocol without showing any signs of stress (lack of appetite, abnormal weight loss, hair loss, stereotypic or aggressive behavior, etc.) or macroscopic changes in important functions. No significant changes in body weight of the animals (either treated or not) were observed throughout the experimental window.

[0168] In vivo torque

[0169] In vivo neuromuscular function was assessed by measuring the torque generated by the hind limb plantar flexor muscles in anesthetized mice (independent of the animal’s will). As shown in Figure 9 The results of mice treated with BCAAs plus L-alanyl-L-alanine had the significantly highest torque-frequency curve among the treatment groups. In particular, the maximum torque value found at 100 Hz was found to almost overlap with the torque value of the sedentary mice group (398 ± 7 vs 379 ± 8 N*mm / kg; n = 8).

[0170] Torque measurements in anesthetized animals confirmed the muscle disuse (i.e., weakness) of the hind limb unloaded mice compared to the control group.

[0171] Moreover, the data clearly indicate that the treatment with BCAAs plus L-alanyl-L-alanine improves the neuromuscular function of the plantar flexor muscles (mainly GC and soleus) with respect to the treatment with vehicle and BCAAs, and the values of torque force are close to those observed in the control group.

Claims

1. A composition comprising the combination of branched chain amino acids (BCAAs) L-leucine, L-valine and L-isoleucine and the peptide L-alanyl-L-alanine, wherein the weight ratio L-leucine : L-valine : L-isoleucine : L-alanyl-L-alanine is from 2:1 :1 :2 to 2:1 :1 :3.

6.

2. The composition according to claim 1, wherein the weight ratio L-leucine : L-valine : L-isoleucine : L-alanyl-L-alanine is 2:1 :1 :2.5 or 2:1 :1 :

2.

3. The composition according to claim 1 or 2, which is a pharmaceutical composition.

4. The composition according to claim 1 or 2, which is for oral administration.

5. The composition according to claim 1 or 2, which contains the following amounts of BCAAs and dipeptide per unit dose: 0.6 to 1 g L-leucine; 0.3 to 0.5 g L-isoleucine; 0.3 to 0.5 g L-valine; and 0.5 to 1.5 g L-alanyl-L-alanine.

6. The composition according to claim 5, which contains 0.8 g L-leucine per unit dose.

7. The composition according to claim 5, which contains 0.4 g L-isoleucine per unit dose.

8. The composition according to claim 5, which contains 0.4 g L-valine per unit dose.

9. The composition according to claim 5, which contains 0.8 g L-alanyl-L-alanine per unit dose.

10. Use of a composition according to claims 1 to 9 for the manufacture of a medicament for preventing, ameliorating and / or treating muscle wasting associated with: i. a pathological condition selected from the group consisting of: muscular dystrophy; chronic obstructive pulmonary condition; cancer-related cachexia; heart failure and sepsis; ii. an age-related condition; or iii. malnutrition, immobility or fasting.

11. Use according to claim 10, wherein the muscle wasting is associated with the age-related condition sarcopenia.

12. Use of a composition according to claims 1 to 9 for improving muscle performance and / or recovery and / or reducing muscle fatigue before, after or during physical exercise.

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