RU2777605C2 - Use of amino acid additive for improved muscle protein synthesis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns an amino acid composition for stimulation of muscle protein synthesis. A method for stimulation of muscle protein synthesis includes the administration to a subject of a composition containing from 1 g to 4 g of following concentrations of amino acids in terms of % wt./wt.: from 1 to 2% of histidine, from 9 to 11% of isoleucine, from 35 to 38% of leucine, from 14 to 17% of lysine, from 2 to 4% of methionine, from 5 to 7% of phenylalanine, from 8 to 9% of threonine, from 9 to 11% of valine, from 0.005 to 0.8% of tryptophane, and from 8 to 11% of arginine. Moreover, the stimulation of muscle protein synthesis is increased by about 50% compared to a subject, who is not administered with the specified amino acid composition. The proposed composition stimulates muscle protein synthesis by more than 50% in doses from 1 g to 4 g with exercises or without exercises.

EFFECT: invention allows for an increase in muscle protein synthesis in a subject, when using the claimed composition in claimed doses.

12 cl, 2 tbl, 1 ex

Description

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application 62/580861, filed Nov. 02, 2017, the disclosure of which is incorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

[0002] This invention was made with government support AR052293 awarded by the National Institutes of Health. The government has certain rights to this invention.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0003] This disclosure relates generally to the use of an anabolic amino acid composition to stimulate muscle protein synthesis. In particular, compositions and methods for their use are disclosed for preventing and/or treating loss of any one of muscle mass, muscle strength, muscle function, and physical function, or any combination thereof, in a mammal, especially an adult mammal.

BACKGROUND OF THE INVENTION

[0004] Large doses of dietary protein are needed to stimulate muscle protein synthesis. The anabolic effects of nutrition are primarily due to the transfer and incorporation of amino acids from dietary protein sources into skeletal muscle proteins. The purpose of this is to compensate for muscle protein that is lost during fasting (post-absorption) periods due to, for example, amino acid oxidation and/or carbon donation for hepatic gluconeogenesis. However, as we age, the body's ability to absorb protein becomes less efficient. Loss of muscle mass begins at age 30 at a rate of 3-8% per decade and accelerates from age 60. This loss reaches 35-40% in the elderly over 70 years of age, and therefore sarcopenia is especially noticeable in the elderly. Therefore, age-related progressive loss of muscle mass increases the risk of injury and disability.

[0005] Thus, there is a need for a nutritional supplement to accelerate the rate of muscle protein synthesis to a greater extent than would be expected from dietary protein intake.

SUMMARY OF THE INVENTION

[0006] Among the various aspects of this disclosure are methods and compositions for stimulating muscle protein synthesis. In general, the present invention provides a nutritional composition comprising effective amounts of amino acids that stimulate muscle protein synthesis in a subject compared to a subject not administered the composition.

[0007] One aspect of this disclosure is directed to providing a composition containing histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, tryptophan, and arginine. In one aspect, the present invention provides a composition comprising the following concentrations of amino acids on a % w/w basis: about 1 to 2% histidine, about 9 to 11% isoleucine, about 35 to 38% leucine, about 14 to 17% lysine, about 2 to 4% methionine, about 5 to 7% phenylalanine, about 8 to 9% threonine, about 9 to 11% valine, about 0.005 to 0.8% tryptophan, and about 8 to 11% arginine.

[0008] In another aspect, muscle protein synthesis is improved by approximately 50% at a minimum dose of 3.6 grams. In another aspect, the composition contains the following concentrations of amino acids in terms of % w/w: 1.5% histidine, 9.7% isoleucine, 36.4% leucine, 15.2% lysine, 3% methionine, 6, 1% phenylalanine, 8.5% threonine, 10% valine, 0.6% tryptophan and 9% arginine.

[0009] Another aspect of this invention relates to a method of administering a nutritional composition containing amino acids, at a dose of 12 g once a day, at a dose of 3.6 g three times a day, at a dose of 3.6 g once a day, in at a dose of 3.6 g twice a day, at a dose of 6.7 g once a day, at a dose of 6.7 g twice a day or at a dose of 6.7 g three times a day.

[0010] In an additional aspect, a composition is provided comprising the following amino acids; histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, tryptophan, and arginine, which stimulates muscle protein synthesis by over 50% at less than 12 grams with or without exercise.

[0011] In yet another aspect, this disclosure provides a composition and method of using the same to stimulate muscle protein synthesis using a dosage of less than 12 grams. In some embodiments, the composition contains the following concentrations of amino acids on a % w/w basis: about 1 to 2% histidine, about 9 to 11% isoleucine, about 35 to 38% leucine, about 14 to 17% lysine , about 2 to 4% methionine, about 5 to 7% phenylalanine, about 8 to 9% threonine, about 9 to 11% valine, about 0.005 to 0.8% tryptophan, and about 8 to 11% arginine .

[0012] While many embodiments of the invention have been disclosed, still other embodiments of the invention will be apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. Accordingly, the detailed description is to be considered illustrative and not restrictive.

DETAILED DESCRIPTION OF THE INVENTION

[0013] This disclosure generally provides methods and compositions comprising a specific amino acid composition capable of stimulating the rate of muscle protein synthesis. Applicants have found that the compositions described herein surprisingly stimulate muscle protein synthesis in a very effective manner. In particular, the compositions disclosed herein stimulate the production of more muscle protein than the total weight of the components taken. According to the present invention, a formulation containing free essential amino acids (EAAs) and arginine in certain amounts has been found to be well suited for stimulating muscle protein synthesis. In particular, nutritional compositions containing a mixture of essential amino acids and arginine, including significant amounts of leucine, have been found to be effective in stimulating muscle protein synthesis. Advantageously, the composition has been found to be effective in adults, where stimulation of muscle protein synthesis is less effective. Muscle protein synthesis is improved compared to subjects who do not receive said amino acid composition and occurs with or without exercise.

[0014] Disclosed are the components to be used in the preparation of the disclosed compositions, as well as the compositions themselves to be used in the methods disclosed herein. Data and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, while specific reference to each individual and collective combination and permutation of these compounds may not be disclosed. explicitly, each is specifically discussed and described in this document. For example, if a particular compound is disclosed and discussed, and a number of modifications that can be made to a number of compound molecules are discussed, each combination and permutation in the compound is specifically discussed, as well as modifications that are possible unless specifically indicated to the contrary. Thus, if the molecular class A, B, and C, as well as the molecular class D, E, and F, and an example of a combined molecule, A-D, are disclosed, then, even if each is not specified individually, each is considered individually and collectively, meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are considered open. Also disclosed similarly, any subset or combination thereof. So, for example, subgroups A-E, B-F, and C-E would be considered open. This concept applies to all aspects of this application, including, but not limited to, steps in the methods of making and using the disclosed compositions. Thus, if there are many additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular embodiment, or combination of embodiments, of the disclosed methods.

[0015] Various aspects of the present invention are described in more detail in the following sections.

(I) COMPOSITIONS

[0016] Provided herein are anabolic amino acid compositions useful for stimulating muscle protein synthesis. In some embodiments, the composition may contain the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, and tryptophan. In preferred embodiments, the composition may contain the amino acid arginine in addition to the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, and tryptophan.

[0017] Typically, amino acids can be L-amino acids, D-amino acids, or mixtures thereof. In preferred embodiments, the amino acids are L-amino acids. Those skilled in the art will appreciate that the amino acids of the composition may be free amino acids or amino acid salts. The amino acid composition of this invention may also be in the form of an intact protein or peptide, provided that the protein or peptide contains the amino acids of this invention in the correct concentration relative to each other. In preferred embodiments, the amino acids of the invention are free amino acids or amino acid salts. Individual amino acids, if not obtained commercially, can be obtained by methods well known in the art, including chemical synthesis or the use of recombinant microorganisms.

[0018] The amino acids can be the canonical amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, tryptophan, and arginine, or non-canonical amino acid derivatives (eg, an amino acid precursor). Non-limiting examples of non-canonical amino acids that can be used in this invention include L-lysine acetate, L-tyrosine derivatives, ornithine, keto acid analogs, hydrochloride salt (L-cysteine HCL.H ₂ O), and various amino acid N-acetyl derivatives. In preferred embodiments of the invention, the amino acids used in the compositions and solutions of this invention may be in free form or in salt form.

[0019] In one aspect, the amino acid composition of this invention includes one or more essential amino acids. An essential amino acid (EAA) is an amino acid that cannot be synthesized de novo by the subject, and therefore must be included in their diet. Amino acids considered essential for humans are phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine. In some embodiments, the EEA combination includes phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine. In preferred embodiments of the invention, the amino acid composition also contains at least one conditionally essential amino acid.

[0020] The amino acids arginine, cysteine, glycine, glutamine, proline, serine, and tyrosine are considered conditionally essential, that is, they are not normally required in the diet, but must be supplied exogenously to specific populations that do not synthesize them in sufficient quantities. For example, under normal conditions, the body produces enough arginine to meet metabolic needs. Therefore, arginine supplementation is not necessary to stimulate muscle protein synthesis for many subjects. However, in certain clinical settings, including the elderly with heart failure, endogenous arginine production is not sufficient to meet all needs. It is advantageous for such a population that the combinations of this invention contain arginine or the amino acid precursor arginine. The term "amino acid precursor" refers to the metabolic precursor of an amino acid. For example, serine is the metabolic precursor of cysteine and glycine; and 3-phosphoglycerate is the metabolic precursor of serine and hence cysteine and glycine. As a second example, citrulline is the metabolic precursor of arginine. The metabolic pathways that synthesize amino acids are well known in the art and the skilled artisan may refer to them to identify metabolic precursors of other amino acids. In some embodiments, the combination of this invention includes phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, histidine, and at least one conditionally essential amino acid. For example, the combination may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conditionally essential amino acids. In other embodiments, the combination of the invention includes phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, histidine, and at least one conditionally essential amino acid precursor. For example, the combination may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conditionally essential amino acid precursors. In still other embodiments, the combination of the invention includes phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, histidine, and at least one non-essential amino acid and at least one conditionally essential amino acid precursor.

[0021] In some embodiments of the invention, the composition may contain the following concentrations of histidine in terms of % wt./mass.: from about 1% to about 2%, from about 1.1% to about 2%, from about 1.2% to about 2%, about 1.3% to about 2%, about 1.4% to about 2%, about 1.5% to about 2%, about 1.6% to about 2%, about 1.7% to about 2%, about 1.8% to about 2%, about 1.9% to about 2%, about 1% to about 1.9%, about 1% to about 1.8%, about 1% to about 1, 7%, about 1% to about 1.6%, about 1% to about 1.5%, about 1% to about 1.4%, about 1% to about 1.3%, about 1% to about 1, 2% or about 1% to about 1.1% histidine.

[0022] In some embodiments of the invention, the composition may contain the following concentrations of isoleucine in terms of % wt./mass.: from about 9% to about 11%, from about 9.1% to about 11%, from about 9.2% to about 11%, from about 9.3% to about 11%, from about 9.4% to about 11%, from about 9.4% to about 11%, from about 9.4% to about 11%, from about 9.5% to about 11%, about 9.6% to about 11%, about 9.7% to about 11%, about 9.8% to about 11%, about 9.9% to about 11%, about 10% to about 11%, about 10.1% to about 11%, about 10.2% to about 11%, about 10.3% to about 11%, about 10.4% to about 11% , about 10.5% to about 11%, about 10.6% to about 11%, about 10.7% to about 11%, about 10.8% to about 11%, about 10.9% to about 11% , from about 9% to about 10.9%, from about 9% to about 10.8%, from about 9% to about 10.7%, from about 9% to about 10.6%, from about 9% to about 10.5%, about 9% to about 10.4%, about 9% to about 10.3%, about 9% to about 10.2%, about 9% to about 1 0.1%, about 9% to about 10%, about 9% to about 9.9%, about 9% to about 9.8%, about 9% to about 9.7%, from about 9 % to about 9.6%, about 9% to about 9.5%, about 9% to about 9.4%, about 9% to about 9.3%, about 9% to about 9.2 % or from about 9% to about 9.1% isoleucine.

[0023] In some embodiments of the invention, the composition may contain the following concentrations of leucine in terms of % wt./mass.: from about 35% to about 38%, from about 35.2% to about 38%, from about 35.4% to about 38%, from about 35.6% to about 38%, from about 35.8% to about 38%, from about 36% to about 38%, from about 36.2% to about 38%, from about 36 .4% to about 38%, about 36.6% to about 38%, about 36.8% to about 38%, about 37% to about 38%, about 37.2% to about 38%, about 37.4% to about 38%, about 37.6% to about 38%, about 37.8% to about 38%, about 35% to about 37.8%, about 35% to about 37 .6%, about 35% to about 37.4%, about 35% to about 37.2%, about 35% to about 37%, about 35% to about 36.8%, from about 35% to about 36.6%, from about 35% to about 36.4%, from about 35% to about 36.2%, from about 35% to about 36%, from about 35% to about 35.8%, from about 35% to about 35.6%, about 35% to about 35.4%, or about 35% to about 35.2% leucine.

[0024] In some embodiments of the invention, the composition may contain the following concentrations of lysine in terms of % wt./mass.: from about 14% to about 17%, from about 14.2% to about 17%, from about 14.4% to about 17%, about 14.6% to about 17%, about 14.8% to about 17%, about 15% to about 17%, about 15.2% to about 17%, about 15.4% to about 17% , about 15.6% to about 17%, about 15.8% to about 17%, about 16% to about 17%, about 16.2% to about 17%, about 16.4% to about 17%, from about 16.6% to about 17%, about 16.8% to about 17%, about 14% to about 16.8%, about 14% to about 16.6%, about 14% to about 16.4%, from about 14% to about 16.2%, about 14% to about 16%, about 14% to about 15.8%, about 14% to about 15.6%, about 14% to about 15.4%, or about 14 % to about 15.2% lysine.

[0025] In some embodiments of the invention, the composition may contain the following concentrations of methionine in terms of % wt./mass.: from about 2% to about 4%, about 2.1% to about 4%, about 2.2% to about 4%, about 2.3% to about 4%, about 2.4% to about 4%, about 2.5% to about 4%, about 2.6% to about 4%, about 2.7% to about 4%, about 2.8% to about 4%, about 2.9% to about 4%, about 3% to about 4%, about 3.1% to about 4%, about 3.2% to about 4%, about 3.3% to about 4%, about 3.4% to about 4%, about 3.5% to about 4%, about 3.6% to about 4%, from about 3.7% to about 4%, about 3.8% to about 4%, about 3.9% to about 4%, about 2% to about 3.9%, about 2% to about 3.8% , about 2% to about 3.7%, about 2% to about 3.6%, about 2% to about 3.5%, about 2% to about 3.4%, about 2% to about 3.3% , about 2% to about 3.2%, about 2% to about 3.1%, about 2% to about 3%, about 2% to about 2.9%, about 2% to about 2.8%, about 2% to about 2.7%, about 2% to about 2.6%, about 2% to about about 2.5%, about 2% to about 2.4%, about 2% to about 2.3%, about 2% to about 2.2%, or about 2% to about 2.1% methionine.

[0026] In some embodiments of the invention, the composition may contain the following concentrations of phenylalanine in terms of % wt./mass.: from about 5% to about 7%, from about 5.1% to about 7%, from about 5.2% to about 7%, from about 5.3% to about 7%, from about 5.4% to about 7%, from about 5.5% to about 7%, from about 5.6% to about 7%, from about 5.7% to about 7%, about 5.8% to about 7%, about 5.9% to about 7%, about 6% to about 7%, about 6.1% to about 7 %, from about 6.2% to about 7%, from about 6.3% to about 7%, from about 6.4% to about 7%, from about 6.5% to about 7%, from about 6, 6% to about 7%, about 6.7% to about 7%, about 6.8% to about 7%, about 6.9% to about 7%, about 5% to about 6.9% , from about 5% to about 6.8%, from about 5% to about 6.7%, from about 5% to about 6.6%, from about 5% to about 6.5%, from about 5% to about 6.4%, about 5% to about 6.3%, about 5% to about 6.2%, about 5% to about 6.1%, about 5% to about 6%, from about 5% to about 5.9%, from about 5% to about 5 .8%, about 5% to about 5.7%, about 5% to about 5.6%, about 5% to about 5.5%, about 5% to about 5.4%, from about 5% to about 5.3%, about 5% to about 5.2%, or about 5% to about 5.1% phenylalanine.

[0027] In some embodiments of the invention, the composition may contain the following concentrations of threonine in terms of % wt./mass.: from about 8% to about 9%, from about 8.1% to about 9%, from about 8.2% to about 9%, about 8.3% to about 9%, about 8.4% to about 9%, about 8.5% to about 9%, about 8.6% to about 9%, about 8.7% to about 9%, about 8.8% to about 9%, about 8.9% to about 9%, about 8% to about 8.9%, about 8% to about 8.8%, about 8% to about 8, 7%, about 8% to about 8.6%, about 8% to about 8.5%, about 8% to about 8.4%, about 8% to about 8.3%, about 8% to about 8, 2% or about 8% to about 8.1% threonine

[0028] In some embodiments of the invention, the composition may contain the following concentrations of valine in terms of % wt./mass.: from about 9% to about 11%, from about 9.1% to about 11%, from about 9.2% to about 11%, from about 9.3% to about 11%, from about 9.4% to about 11%, from about 9.4% to about 11%, from about 9.4% to about 11%, from about 9.5% to about 11%, about 9.6% to about 11%, about 9.7% to about 11%, about 9.8% to about 11%, about 9.9% to about 11%, about 10% to about 11%, about 10.1% to about 11%, about 10.2% to about 11%, about 10.3% to about 11%, about 10.4% to about 11% , about 10.5% to about 11%, about 10.6% to about 11%, about 10.7% to about 11%, about 10.8% to about 11%, about 10.9% to about 11% , from about 9% to about 10.9%, from about 9% to about 10.8%, from about 9% to about 10.7%, from about 9% to about 10.6%, from about 9% to about 10.5%, about 9% to about 10.4%, about 9% to about 10.3%, about 9% to about 10.2%, about 9% to about 10.1% , about 9% to about 10%, about 9% to about 9.9%, about 9% to about 9.8%, about 9% to about 9.7%, about 9% to about 9 .6%, about 9% to about 9.5%, about 9% to about 9.4%, about 9% to about 9.3%, about 9% to about 9.2%, or about 9% to about 9.1% valine.

[0029] In some embodiments of the invention, the composition may contain the following concentrations of tryptophan in terms of % wt./mass.: from about 0.005% to about 0.8%, from about 0.006% to about 0.8%, from about 0.007% to about 0.8%, from about 0.008% to about 0.8%, from about 0.009% to about 0.8%, from about 0.01% to about 0.8%, from about 0.02% to about 0.8%, about 0.03% to about 0.8%, about 0.04% to about 0.8%, about 0.05% to about 0.8%, from about 0.06% to about 0.8%, from about 0.07% to about 0.8%, from about 0.08% to about 0.8%, from about 0.09% to about 0.8%, from about 0, 1% to about 0.8%, about 0.2% to about 0.8%, about 0.3% to about 0.8%, about 0.4% to about 0.8%, from about 0.5% to about 0.8%, about 0.6% to about 0.8%, about 0.7% to about 0.8%, about 0.005% to about 0.7%, from about 0.005% to about 0.6%, about 0.005% to about 0.5%, about 0.005% to about 0.4%, about 0.005% to about 0.3%, about 0.005% to about 0, 2%, from about 0.005% to about 0.1%, from about 0 005% to about 0.09%, about 0.005% to about 0.08%, about 0.005% to about 0.07%, about 0.005% to about 0.06%, about 0.005% to about 0 .05%, about 0.005% to about 0.04%, about 0.005% to about 0.03%, about 0.005% to about 0.02%, about 0.005% to about 0.01%, from about 0.005% to about 0.009%, about 0.005% to about 0.008%, about 0.005% to about 0.007%, or about 0.005% to about 0.006% tryptophan

[0030] In some embodiments, the composition may contain the following concentrations of arginine on a % w/w basis: about 8% to about 11%, about 8.1% to about 11%, about 8.2% to about 11%, about 8.3% to about 11%, about 8.4% to about 11%, about 8.5% to about 11%, about 8.6% to about 11%, about 8.7% to about 11%, about 8.8% to about 11%, about 8.9% to about 11%, about 9% to about 11%, about 9.1% to about 11%, about 9.2% to about 11%, about 9.3% to about 11%, about 9.4% to about 11%, about 9.4% to about 11%, about 9.4% to about 11%, from about 9.5% to about 11%, about 9.6% to about 11%, about 9.7% to about 11%, about 9.8% to about 11%, about 9.9% to about 11%, about 10% to about 11%, about 10.1% to about 11%, about 10.2% to about 11%, about 10.3% to about 11%, about 10.4% to about 11%, about 10.5% to about 11%, about 10.6% to about 11%, about 10.7% to about 11%, about 10.8% to about 11%, from about about 10.9% to about 11%, about 8% to about 10.8%, about 8% to about 10.7%, about 8% to about 10.6%, about 8% to about 10.5%, from about 8% to about 10.4%, about 8% to about 10.3%, about 8% to about 10.2%, about 8% to about 10.1%, about 8% to about 10%, from about 8% to about 9.9%, about 8% to about 9.8%, about 8% to about 9.7%, about 8% to about 9.6%, about 8% to about 9.5%, from about 8% to about 9.4%, about 8% to about 9.3%, about 8% to about 9.2%, about 8% to about 9.1%, about 8% to about 9%, from about 8% to about 8.9%, from about 8% to about 8.8%, from about 8% to about 8.7%, from about 8% to about 8.6%, from about 8% to about 8.5%, from about 8% up to about 8.4%, about 8% to about 8.3%, about 8% to about 8.2%, or about 8% to about 8.1% arginine

[0031] In some embodiments, the composition may contain the following concentrations of amino acids on a % w/w basis: about 1% to about 2% histidine, about 9% to about 11% isoleucine, about 35% to about 38% leucine, about 14% to about 17% lysine, about 2% to about 4% methionine, about 5% to about 7% phenylalanine, about 8% to about 9% threonine, about 9% to about 11 % valine, about 0.005% to about 0.8% tryptophan, and about 8% to about 11% arginine. In exemplary embodiments of the invention, the composition may contain, consist essentially of, or consist of 1.5% histidine, 9.7% isoleucine, 36.4% leucine, 15.2% lysine, 3% methionine, 6.1% phenylalanine, 8 .5% threonine, 10% valine, 0.6% tryptophan and 9% arginine.

[0032] In another aspect, the combinations of this invention may additionally contain one or more additional nutrients. The term "nutrient", as used herein, refers to probiotics, vitamins, carbohydrates, fiber, fatty acids, sulfates, minerals, antioxidants, and other food ingredients used in a subject's metabolism that are derived from the subject's environment. Suitable vitamins may include, but are not limited to: vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12, lipoic acid, vitamin A, biotin, vitamin K, vitamin C, vitamin D, and vitamin E Suitable minerals may include, but are not limited to, compounds containing: iron, copper, magnesium, manganese, molybdenum, nickel, and zinc. Suitable enzyme cofactors may include, but are not limited to: adenosine triphosphate (ATP), S-adenosylmethionine (SAM), coenzyme B, coenzyme M, coenzyme Q, glutathione, heme, methanofuran, and nucleotide sugars. Suitable lipids may include, but are not limited to, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides. Non-limiting examples of fatty acids include myristolic acid, palmitoleic acid, sapienoic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid. acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotinic acid, hexadecatrienic acid, alpha-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentacosahexanoic acid, clupano docosaenoic acid acid; and tetracosapentaenoic acid; and tetracosahexaenoic acid. Additional non-limiting examples of nutrients may include thiamine, riboflavin, niacin, folate, pantothenic acid, calcium, phosphorus, magnesium, manganese, iron, zinc, copper, selenium, sodium, potassium, betacarotene, retinol, alphatocopherol, betatocopherol, gammatocopherol, deltatocopherol, alphatocotrienol, betatocotrienol, gammatocotrienol, deltatocotrienol, apo-8-carotenal, trans-lycopene, cis-lycopene, trans-beta-carotene and cis-beta-carotene, caffeine. In some embodiments of the invention, the combinations of this invention additionally contain one nutrient. In other embodiments, the combinations of this invention further comprise at least one nutrient. For example, a combination of the invention may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nutrients. In still other embodiments, the combinations of the invention further comprise two or more nutrients. In preferred embodiments, the combinations of this invention further comprise at least one nutrient selected from the group consisting of omega-3 fatty acid and biotin. In other preferred embodiments of the invention, the combinations of this invention additionally contain two nutrients selected from the group consisting of omega-3 fatty acid and biotin. The total contribution of one or more nutrients to the total mass of the combination is significantly less than the contribution of multiple amino acids. Typically, the content of one or more nutrients is no more than about 10% by weight, preferably no more than about 5% by weight, more preferably no more than about 3% by weight. combinations. In preferred embodiments, one or more of the nutrients is biotin. In other preferred embodiments of the invention, one or more nutrients are one or more omega-3 fatty acids. In other preferred embodiments, the one or more nutrients are biotin and one or more omega-3 fatty acids.

(II) COMPOSITIONS

[0033] In each of the above embodiments, the amino acids and nutrients (if present) can be formulated for use in animals or humans. In some embodiments, each amino acid and nutrient (if present) is formulated separately. In other embodiments of the invention, two or more amino acids and nutrients (if present) are formulated together. In yet other embodiments of the invention, all amino acids and nutrients that make up the combination according to this invention are formulated together. The one or more compositions can then be formulated into one or more dosage forms that can be administered together, sequentially, or over a period of time (e.g., 1 minute, 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours or more). The introduction can be performed using standard effective methods, including oral, parenteral (eg, intravenous, intraperitoneal, subcutaneous, intramuscular), buccal, sublingual or suppository administration. The term orally, as used herein, refers to any form of oral administration, including adding the composition to an animal feed or other food product. The composition of pharmaceutical compositions is discussed, for example, in Hoover, John E., Remington Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, New York (1980).

[0034] Methods for preparing compositions for use in animals or humans are well known in the art. For example, typically the composition may be formulated as a liquid composition, a solid composition, or a semi-solid composition. Liquid compositions include, but are not limited to, aqueous suspensions, solutions, emulsions, elixirs or syrups. The liquid composition typically includes a carrier solvent selected from a polar solvent, a non-polar solvent, or a combination thereof. The choice of solvent will be influenced by the properties of the components of the composition. For example, if the components are water soluble, a polar solvent may be used. Alternatively, if the components of the composition are lipid soluble, a non-polar solvent may be used. Suitable polar and non-polar solvents are known in the art. Semi-solid compositions include douches, suppositories, creams, and topical preparations. Dry compositions include, but are not limited to, reconstituted powders, chewable tablets, instant tablets, effervescent tablets, multilayer tablets, bilayer tablets, capsules, soft gelatin capsules, hard gelatin capsules, capsules, lozenges, chewable lozenges, pellets, powders, granules, particles, microparticles and dispersible granules. Compositions may include a combination of this invention together with an excipient. Non-limiting examples of excipients include binders, diluents (fillers), disintegrants, effervescent disintegrants, preservatives (antioxidants), flavor modifiers, lubricants and glidants, dispersants, colorants, pH modifiers, chelating agents, antimicrobial agents, release control polymers, and combinations of any of these agents.

[0035] Non-limiting examples of binders suitable for formulations of various embodiments of the invention include starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyl oxoazolidone, polyvinyl alcohols, C12-C18 fatty acid alcohols, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof. The polypeptide can be any composition of proteins in the range from about 100 to about 300,000 daltons. In one embodiment, the binder may be incorporated into the granulation mixture in solid form, including, but not limited to, a crystal, particle, powder, or any other finely divided solid form known in the art. In another embodiment of the invention, the binder may be dissolved or suspended in a solvent and sprayed onto the mixture in a granulation machine as a binding liquid during granulation.

[0036] Non-limiting examples of diluents (also referred to as "fillers" or "thinner") include carbohydrates, inorganic compounds, and biocompatible polymers such as polyvinylpyrrolidone (PVP). Other non-limiting examples of diluents include dibasic calcium sulfate, tribasic calcium sulfate, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, saccharides such as sucrose, dextrose, lactose, microcrystalline cellulose, fructose, xylitol and sorbitol, polyhydric alcohols; starches; ready-made diluents of direct compression; and mixtures of any of the above.

[0037] Disintegrants may be effervescent or non-effervescent. Non-limiting examples of non-effervescent disintegrants include starches such as corn starch, potato starch, its pregelatinized and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar agar, guar gum, locust bean gum wood, karaya gum, pectin and gum tragacanth. Suitable effervescent disintegrants include, but are not limited to, sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0038] Non-limiting examples of preservatives include, but are not limited to, ascorbic acid and its salts, ascorbyl palmitate, ascorbyl stearate, anoxomer, N-acetylcysteine, benzyl isothiocyanate, m-aminobenzoic acid, o-aminobenzoic acid, p-aminobenzoic acid (PABA), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid, canthaxanthin, alpha-carotene, beta-carotene, beta-apocaratic acid, carnosol, carvacrol, catechins, cetyl gallate, chlorogenic acid, citric acid and its salts, clove extract, extract coffee beans, p-coumaric acid, 3,4-dihydroxybenzoic acid, N,N'-diphenyl-p-phenylenediamine (DPPD), dilauryl thiodipropionate, distearyl thiodipropionate, 2,6-di-tert-butylphenol, dodecyl gallate, edetic acid, ellagic acid , erythorbic acid, sodium erythorbate, esculetin, esculin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, ethyl gallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA), eucalyptus extract, evg enol, ferulic acid, flavonoids (eg, catechin, epicatechin, epicatechin gallate, epigallocatechin (EGC), epigallocatechin gallate (EGCG), epigallocatechin-3-gallate polyphenol), flavones (eg, apigenin, chrysin, luteolin), flavonols (eg, datiscetin myricetin, kaemferol (daemfero)), flavanones, frexetin, fumaric acid, gallic acid, gentian extract, gluconic acid, glycine, gum arabic, hesperetin, alpha-hydroxybenzylphosphinic acid, hydroxycinnamic acid, hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid, hydroxytyrosol , hydroxyurea, rice bran extract, lactic acid and its salts, lecithin, lecithin citrate; R-alpha-lipoic acid, lutein, lycopene, malic acid, maltol, 5-methoxytryptamine, methyl gallate, monoglyceride citrate; monoisopropyl citrate; morine, beta-naphthoflavone, nordihydroguaiaretic acid (NDGA), octyl gallate, oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine, phosphoric acid, phosphates, phytic acid, phytylubichromel, allspice extract, propyl gallate, polyphosphates, quercetin, trans-resveratrol, rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin, sinapinic acid, succinic acid, stearyl citrate, syringic acid, tartaric acid, thymol, tocopherols (i.e. alpha, beta, gamma and delta tocopherol), tocotrienols (i.e. e. alpha, beta, gamma and delta tocotrienols), tyrosol, vanillic acid, 2,6-di-tert-butyl-4-hydroxymethylphenol (i.e. Ionox 100), 2,4-(tris- 3',5'-bi-tert-butyl-4'-hydroxybenzyl)mesitylene (i.e. Ionox 330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxybutyrophenone, tryptamine, tyramine, uric acid, vitamin K and derivatives, vitamin Q10, wheat germ oil, zeaxanthin or their combinations. In an exemplary embodiment, the preservatives are an antioxidant such as α-tocopherol or ascorbate and antimicrobial agents such as parabens, chlorobutanol or phenol.

[0039] Suitable flavor modifiers include flavors, taste-masking agents, sweeteners, and the like. Flavors include, but are not limited to, synthetic fragrance oils and aromatic fragrances and/or natural oils, extracts of plants, leaves, flowers, fruits, and combinations thereof. Other non-limiting examples of flavoring agents include cinnamon oils, wintergreen oil, peppermint oils, clover oil, hay oil, anise oil, eucalyptus, vanilla citrus oils such as lemon oil, orange oil, grape and grapefruit oils, fruit essences including apple, peach , pear, strawberry, raspberry, cherry, plum, pineapple and apricot essences.

[0040] Taste-masking agents include, but are not limited to, hydroxypropyl cellulose ethers (HPCs) such as Klucel®, Nisswo HPC, and PrimaFlo HP22; low-substituted hydroxypropyl ethers (L-HPC); hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC, Pharmacoat®, Metolose SR, Opadry YS, PrimaFlo, MP3295A, Benecel MP824 and Benecel MP843; methylcellulose polymers such as Methocel® and Metolose®; ethyl cellulose (EC) and mixtures thereof such as E461, Ethocel®, Aqualon®-EC, Surelease; polyvinyl alcohol (PVA) such as Opadry AMB; hydroxyethylcelluloses such as Natrosol®; carboxymethylcelluloses and carboxymethylcellulose (CMC) salts such as Aualon®-CMC; copolymers of polyvinyl alcohol and polyethylene glycol such as Kollicoat IR®; monoglycerides (Myverol), triglycerides (KLX), polyethylene glycols, modified food starch, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit® EPO, Eudragit® RD100 and Eudragit® E100; cellulose acetate phthalate; sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins and mixtures of these materials. In other embodiments, the invention contemplates additional taste-masking agents described in US Pat.

[0041] Non-limiting examples of sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (unless used as a carrier); saccharin and its various salts such as the sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia rebaudiana (stevioside); sucrose chloro derivatives such as sucralose; sugar alcohols such as sorbitol, mannitol, silitol, hydrogenated starch hydrolysates and the synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, in particular the potassium salt (acesulfame-K) and their sodium and calcium salts.

[0042] Lubricants can be used to lubricate the ingredients that form the composition of this invention. As a glidant, the lubricant facilitates the removal of solid dosage forms during the manufacturing process. Non-limiting examples of lubricants and glidants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, stereotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. The composition will typically contain from about 0.01% to about 10% of the mass. lubricant. In some embodiments of the invention, the composition will contain from about 0.1% to about 5% of the mass. lubricant. In an additional embodiment of the invention, the composition will contain from about 0.5% to about 2% of the mass. lubricant.

[0043] Dispersants may include, but are not limited to, starch, alginic acid, polyvinyl pyrrolidones, guar gum, kaolin, bentonite, refined wood pulp, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high hydrophilic surfactants. lipophilic balance (HLB).

[0044] Depending on the embodiment of the invention, it may be desirable to include a coloring agent. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C). These colorants or dyes, together with their respective lacquer dyes and certain natural and derivative dyes, may be suitable for use in various embodiments of the invention.

[0045] Non-limiting examples of pH modifiers include citric acid, acetic acid, tartaric acid, malic acid, fumaric acid, lactic acid, phosphoric acid, sorbic acid, benzoic acid, sodium carbonate, and sodium hydrogen carbonate.

[0046] A chelating agent may be included as an excipient to immobilize oxidizing groups, including but not limited to metal ions, to inhibit oxidative degradation of morphinan by these oxidizing groups. Non-limiting examples of chelating agents include lysine, methionine, glycine, gluconate, polysaccharides, glutamate, aspartate, and disodium ethylenediaminetetraacetate (Na ² EDTA).

[0047] An antimicrobial agent may be included as an excipient to minimize degradation of the compound of this invention by microbial agents, including but not limited to bacteria and fungi. Non-limiting examples of antimicrobials include parabens, chlorobutanol, phenol, calcium propionate, sodium nitrate, sodium nitrite, Na ² EDTA, and sulfites, including but not limited to sulfur dioxide, sodium bisulfite, and potassium hydrosulfite.

[0048] Release-controlling polymers may be included in various embodiments of solid dosage formulations comprising the compounds of this disclosure. In one embodiment of the invention, release-controlling polymers can be used as a coating for tablets. In other embodiments, including but not limited to bilayer tablets, the release controlling polymer can be mixed with granules and other excipients to form a tablet in a known manner, including but not limited to compression into a tablet mold. Suitable release control polymers include, but are not limited to, hydrophilic polymers and hydrophobic polymers.

[0049] Suitable hydrophilic release control polymers include, but are not limited to, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose ethers, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, nitrocellulose, cross-linked starch, agar, casein, chitin, collagen, gelatin, maltose, mannitol, maltodextrin, pectin, pullulan, sorbitol, xylitol, polysaccharides, ammonium alginate, sodium alginate, calcium alginate, potassium alginate, sodium alginate, propylene glycol alginate, carmellose sodium, carmellose calcium, carrageenan, fucoidan, furcellaran, gum arabic , carrageenan gum, Prosopis panicle gum (ghaftigum), guar gum, karaya gum, locust bean gum, bark gum, tragacanth gum, scleroglucan gum, xanthan gum, hypnoia, laminaran, acrylic polymers, acrylate polymers of maledrydin polymers, carboxyvinyl polymers and styrene, with copolymers of maleic anhydride and ethylene, propylene copolymers of maleic anhydride or copolymers of maleic anhydride and isobutylene), cross-linked polyvinyl alcohol and poly-N-vinyl-2-pyrrolidone, polyglucan diesters, polyacrylamides, polyacrylic acid, polyamides, polyethylene glycols, polyethylene oxides, poly(hydroxyalkyl methacrylate ), polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride, polystyrenes, polyvinylpyrrolidone, anionic and cationic hydrogels, and combinations thereof.

[0050] The amino acid compositions described herein may also include compositions that can be formulated as a powder that can be added to foods, as baked goods (eg, cookies and cakes), and as a concentrate. The concentrate can be added to water or other ingested liquid to create a nutritional drink. A nutritional supplement is usually contained in a disposable or reusable container such as a package, box, carton, wrapper, bottle or jar. If the dietary supplement is in the form of a concentrate that can be added to and mixed with a drink, the bottle can be used to package the concentrate. The nutritional supplement may also include water.

(a) DOSE OF AMINO ACID

[0051] As one of skill in the art will appreciate, the dosage of the amino acid composition of this invention can and will vary depending on the body weight, sex, age and/or health of the subject, the intensity of the subject's exercise, method of administration. Non-limiting examples of species may include a human, a domestic animal, a laboratory animal, a zoo animal, or a farm animal. Routine experimentation can easily establish the required dosage. Typical doses of amino acids for oral administration may be about 7 g per dose. In some embodiments, an amino acid dose of about 3, 4, 5, 10, or 12 grams of the amino acid composition may be administered. In other embodiments, an amino acid dose of about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 grams of the amino acid composition may be administered. In an exemplary embodiment of the invention, the dose of amino acid may be about 7 grams per dose.

[0052] If necessary, the introduction of several doses of the amino acid composition per day can also be used to provide the desired stimulation of muscle protein synthesis. For example, one, two, three, four or more doses of the amino acid composition may be administered per day. In a preferred embodiment of the invention, one dose of the amino acid composition may be administered per day. In another preferred embodiment of the invention, two doses of the amino acid composition may be administered per day. In yet another preferred embodiment of the invention, three doses of the amino acid composition may be administered per day.

[0053] The time and duration of administration of the composition of this invention can and will vary. For example, when the composition is administered to stimulate muscle protein synthesis with or without exercise, the composition may be administered without exercise, before exercise, during exercise, or after exercise. Alternatively, when the composition is administered to improve muscle protein synthesis in a subject prone to muscle problems, such as an older subject, the composition may be administered on a regular basis.

[0054] In an exemplary embodiment of the invention, when the subject is a human, three doses of 3 g per dose of the amino acid composition may be administered per day. In another exemplary embodiment of the invention, two doses of 7 g per dose of the amino acid composition may be administered per day. In yet another exemplary embodiment of the invention, a unit dose of 7 g per dose of the amino acid composition may be administered per day.

(b) Introduction

[0055] The composition of this invention can be administered by intravenous, intramuscular, subcutaneous injection or parenteral route. In some embodiments, the composition may be formulated for administration by injection (eg, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these compositions are preferably combined with pharmaceutically acceptable carrier vehicles such as saline, Ringer's solution, dextrose solution, and the like.

[0056] The amino acid composition of this invention may contain a pharmaceutical carrier (or excipient). Such a carrier may be any solvent or solid encapsulation material which is non-toxic. The carrier may impart shape or texture or act as a diluent. Suitable pharmaceutical carriers may include liquid carriers such as saline and other non-toxic salts at or near physiological concentrations, and solid carriers not for human use such as talc or sucrose, or animal feed. Carriers may also include stabilizing agents, wetting and emulsifying agents, salts of varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Carriers and excipients, as well as formulations for parenteral and non-parenteral drug delivery, are set forth in Remington Pharmaceutical Sciences 19th ed. Mack Publishing (1995).

[0057] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal) the composition may be an aqueous solution or an oil-based solution. Aqueous solutions may contain a sterile diluent such as water, saline, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate; and/or a tonicity agent such as sodium chloride, dextrose or a polyalcohol such as mannitol or sorbitol. The pH of the aqueous solution can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may additionally contain sesame, peanut, olive oil or mineral oil.

[0058] In a preferred embodiment of the invention, the amino acid composition of this invention can be administered orally. Non-limiting examples of oral formulations that can be used to administer the amino acid composition of this invention can be a nutritional formulation, a health food, a health food drink in the form of a whole meal, part of a meal, as a powder supplement for dissolution, in the form of a pharmaceutical formulation such as tablets, pills, sachets or capsules, or by tube feeding, for example by nasogastric, nasoduodenal, esophagostomy, gastrostomy or jejunotomy, or as peripheral or total parenteral nutrition. In an exemplary embodiment of the invention, the compositions of this invention may be administered orally as a dietary supplement.

[0059] Compositions for oral administration typically contain excipients in addition to the amino acid ingredients of the composition. Formulations for oral administration may be enclosed in gelatin capsules or compressed into tablets. Common excipients used in such formulations include pharmaceutically compatible excipients/diluents such as microcrystalline cellulose, hydroxypropyl methylcellulose, starch, lactose, sucrose, glucose, mannitol, sorbitol, dibasic calcium phosphate or calcium carbonate; binding agents such as alginic acid, carboxymethylcellulose, microcrystalline cellulose, gelatin, gum tragacanth or polyvinylpyrrolidone; disintegrants such as alginic acid, cellulose, starch or polyvinylpyrrolidone; lubricants such as calcium stearate, magnesium stearate, talc, silicon dioxide or sodium stearyl fumarate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavors such as peppermint, methyl salicylate, or citrus flavors; coloring agents; and preservatives such as antioxidants (eg vitamin A, vitamin C, vitamin E or retinyl palmitate), citric acid or sodium citrate. Oral formulations may also be administered as aqueous suspensions, elixirs or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents and, if desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.

[0060] The compositions of this invention may be nutritionally complete, that is, they may include vitamins, minerals, trace elements, as well as sources of nitrogen, carbohydrates and fats and/or fatty acids, so that they can be used as the sole source of nutrition, providing almost all food, the necessary daily amounts of vitamins, minerals, carbohydrates, fats and / or fatty acids, proteins and the like.

(II) METHODS

[0061] The invention also provides methods for increasing muscle protein synthesis, preventing and/or treating loss of any one of muscle mass, muscle strength, muscle function, and physical function, or any combination thereof. The method includes administering to the subject a composition containing a plurality of essential amino acids and optionally essential amino acids and/or nutrients. Suitable combinations and formulations for administration are described above in Section I and Section II , respectively.

[0062] The nutritional composition of this disclosure can advantageously be used in the manufacture of a medicament for the prevention or treatment of a disease or condition associated with muscle wasting in a mammal, especially in an adult mammal. Alternatively, the nutritional composition of the present invention may advantageously be used in the manufacture of a medicament for the prevention or treatment of a disease or condition selected from the group of sarcopenia, muscle wasting, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscular dystrophy, muscle catabolism. , muscle wasting, loss of muscle strength, loss of muscle mass, loss of muscle function, loss of physical performance, loss of physical performance, impaired mobility, weakness, surgery, disability, risk of falling, and risk of fall-related fractures. Suitable subjects may include a human, livestock, pet, laboratory animal, or zoological animal. In a preferred embodiment of the invention, the subject is a human. The magnitude of the response may depend, in part, on the given dose, the exact combination of amino acids, the physiological state and/or age of the subject, and/or the time of administration depending on exercise performance.

[0063] Preferably, said subject is an elderly person. In this regard, it is stated that in the context of this application, an elderly person is a person aged 50 years or more, in particular aged 55 years or more, in particular aged 60 years or more, in particular aged 65 years and over. . This rather broad definition takes into account the fact that the average age varies between different populations, on different continents, etc. In most of the developed countries of the world, the chronological age of 65 years is accepted as the definition of "old" or old person (related to the age at which one can start receiving pension benefits), but like many Western concepts, this does not apply very well to the situation in Africa. At the moment there is no United Nations (UN) standard numerical criterion, but the UN-agreed limit is 60+ years to refer to the elderly population in the Western world. More traditional African definitions of an elderly or "older" person correspond to a chronological age of 50 to 65 years, depending on the setting, region and country.

[0064] The nutritional composition of this disclosure can advantageously be used to prevent or treat muscle wasting, in particular muscle loss, during or after weight maintenance, during or after energy restriction, during or after bed rest, or during recovery. after physical injury. In a particularly preferred embodiment of the invention, the compositions of this invention are used in the treatment of a subject, for example an overweight or obese subject, said subject is following a weight loss program, an energy restriction program and/or an exercise program. Said subject may be a child, adolescent, adult, or elderly subject. In an embodiment of the invention, said subject is a child, adolescent, or adult.

[0065] With aging, muscle mass and function are gradually lost, so by the age of 60, many otherwise healthy people have reached the threshold at which function begins to be impaired. In one aspect, administration of a composition of this invention to any healthy subject may increase muscle protein synthesis, increase muscle strength, increase muscle function, or a combination thereof, although the magnitude of the response may vary depending on the age and/or physiological state of the subject.

[0066] Loss of body protein often accompanies disease, whether the disease is short-term or long-term. In another aspect, administering a combination of this invention to a subject who has lost a body protein as a result of disease may increase muscle protein synthesis, increase muscle strength, increase muscle function, or a combination of the two in the subject. In another aspect, administering a combination of this invention to a subject at risk for or experiencing muscle atrophy may increase muscle protein synthesis, increase muscle strength, increase muscle function, or a combination thereof. Non-limiting examples of subjects at risk for or experiencing muscle atrophy include people with a sedentary lifestyle, people with a sedentary job, health conditions that restrict movement, subjects who are bedridden, subjects who are outside the Earth's gravity, subjects with nerve injury, that connects to a muscle, and subjects who are at risk of disease or are diagnosed with a disease that affects the nerves that control muscles. Non-limiting examples of diseases, disorders or pathological conditions in which muscle wasting occurs include poliomyelitis, Guillain-Barré syndrome, chronic obstructive pulmonary disease, congestive heart failure, acute coronary syndrome, chronic heart failure, cardiac cachexia, cancer, sarcopenia, spinal cord injury , osteoarthritis, arthritis, stroke, malnutrition, muscular dystrophy (eg, Becker, congenital, Duchenne, distal, Emery-Dreyfus, humerofacial, lumbar, oculopharyngeal), spinal muscular atrophy (eg, ALS, motor neuron disease, childhood progressive muscular atrophy of the spine, intermediate muscular atrophy of the spine, juvenile muscular atrophy of the spine, muscular atrophy of the spine in adults), inflammatory myopathies (eg, dermatomyositis, polymyositis), peripheral nerve diseases (eg, Charcot-Marie dental disease, Dejerine-Sott disease, ataxia Friedr Eich), neuromuscular junction diseases (myasthenia gravis, Lambert-Eaton syndrome), metabolic muscle diseases (acid maltase deficiency, carnitine deficiency, carnitine palmityl transferase deficiency, branch point hydrolase deficiency, lactate dehydrogenase deficiency, mitochondrial deaminase deficiency, phosphorylase deficiency, Phosphofructokinase deficiency, phosphoglycerate kinase deficiency), other myopathies (central rod disease, hyperthyroid myopathy, myotonia congenita, myotubular myopathy, paramitonia congenita, periodic paralysis-hypokalemia-hyperkalemia) or any other disease or condition requiring immobilization or bed rest, especially chronic heart failure and heart failure cachexia.

[0067] The method of this invention typically includes administering the amino acid composition to a subject. Suitable subjects include animals such as mammals and humans. Non-limiting examples of suitable animals include pets such as cats, dogs, rodents and horses; research animals such as mice, rats and other rodents; farm animals such as cows, cattle, pigs, goats, sheep, horses, deer, chickens and other poultry; zoo animals; and primates such as chimpanzees, monkeys and gorillas. In a preferred embodiment of the invention, the subject is a human. In another preferred embodiment of the invention, the subject is a mammal.

[0068] The frequency of administration may be one, two, three times or more times a day, or one, two, three times or more times a week or a month, if necessary to achieve the desired effect. The amount administered to a subject can and will vary depending on the subject (eg, age, weight, health status) and the particular route of administration. Those skilled in the art will appreciate that dosages may also be determined using Goodman & Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493, and using a physician's handbook.

[0069] The person may be of any age. In some embodiments of the invention, the age of the human subject may be about 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 years or older. In some preferred embodiments of the invention, the age of the human subject is 40 years or older. In other preferred embodiments of the invention, the age of the human subject is 45 years or older. In the remaining preferred embodiments of the invention, the age of the human subject is 50 years or older. In still other preferred embodiments of the invention, the age of the human subject is 55 years or older. In other preferred embodiments of the invention, the age of the human subject is 60 years or older. In the remaining preferred embodiments of the invention, the age of the human subject is 65 years or older. In still other preferred embodiments of the invention, the age of the human subject is 70 years or older. In other preferred embodiments of the invention, the age of the human subject is 75 years or older. In still other preferred embodiments of the invention, the age of the human subject is 80 years or older. In the remaining preferred embodiments of the invention, the age of the human subject is 85 years or older. In still other preferred embodiments of the invention, the age of the human subject is 90 years or older.

[0070] In some embodiments, the subject may be an alternative embodiment for subjects who lack daily exercise.

[0071] In other embodiments of the invention, the subject may be an alternative to the embodiments of the invention for subjects who lack the ability to effectively stimulate muscle protein synthesis.

[0072] In other embodiments, the subject may be recovering from a lack of exercise. During high-intensity muscle exercise, the rate of muscle work performed can exceed the blood supply's ability to deliver oxygen, creating conditions that cause regenerative stress in muscle cells and can lead to muscle damage, impaired performance, injury susceptibility, and prolonged recovery times. Non-limiting examples of exercise that can lead to muscle damage, impaired performance, injury proneness, and long recovery times may include flexibility exercises such as stretching, aerobic exercises such as cycling, swimming, walking, jump rope, rowing, running, hiking or playing tennis, anaerobic exercise such as strength training, functional training, eccentric training or sprinting, strength training, agility training or eccentric training.

(a) Improved muscle protein synthesis

[0073] The method of this invention improves muscle protein synthesis in a subject receiving an amino acid composition relative to a subject who has not received said amino acid composition.

[0074] An increase in muscle protein synthesis can be measured by any method known in the art. For example, rates of protein synthesis and breakdown in muscle and blood have been measured for many years using radioactively labeled amino acids or amino acids labeled with stable isotopes. See, for example, Yale J Biol Med 1997; 70(1): 65-76, incorporated herein by reference. Measurement of urinary excretion of 3-methylhistidine or arterial-venous difference in 3-methylhistidine is used as a measure of the unidirectional rate of muscle protein breakdown. Total skeletal body mass can be measured using dual energy X-ray (DEXA) absorptiometry or CT; see, for example, Am J Clin Nutr 2002; 76:378-83, and J Appl Physiol 1985 are incorporated herein by reference. Methods for measuring muscle strength or function are also well known in the art. For example, changes in muscle strength and function can be assessed using basic standardized tests as described in B J Nutr 2012; 108: S88-93 or in Lu et al. (2012) “Strength and Functional Measurement for Patients with Muscular Dystrophy” Muscular Dystrophy, Dr. Madhuri Hegde (Ed.), each is hereby incorporated by reference.

[0075] In some embodiments, the method of this invention can increase muscle protein synthesis by at least 2-fold. For example, muscle protein synthesis can be increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8 times, at least 9 times, at least 10 times or more after administration of the combination of this invention. In some embodiments of the invention, the method according to this invention can increase muscle strength by at least 2 times. For example, muscle strength can increase at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least at least 8 times, at least 9 times, at least 10 times or more after administration of the combination of this invention. In some embodiments of the invention, the method according to this invention can increase muscle function at least 2 times. For example, muscle function synthesis can be increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8 times, at least 9 times, at least 10 times or more after administration of the combination of this invention. In other embodiments, the method of the invention may increase muscle protein synthesis, muscle strength and muscle function, or combinations thereof. Methods for measuring muscle protein synthesis, muscle strength and muscle function are known in the art and are described in more detail in the examples.

(III) SETS

[0076] Kits are also provided. Such kits may include the composition described herein and, in some embodiments, instructions for administration. Such kits may facilitate the implementation of the methods described herein. When supplied as a kit, the various components of the composition may be packaged in separate containers and mixed immediately prior to use. Components include, but are not limited to, the amino acid composition as described herein. Such packaging of the components individually may, if desired, be provided in a package or dispenser device which may contain one or more unit dosage forms containing the composition. The package may, for example, contain metal or plastic foil, for example in the form of a blister pack. Such individual packaging of the components may also, in some cases, allow for long-term storage without loss of activity of the components.

[0077] The kits may also include reagents in separate containers, such as, for example, sterile water or saline, for addition to the lyophilized active ingredient packaged separately. For example, sealed glass ampoules may contain the lyophilized component and sterile water in a separate ampoule, sterile saline, or sterile solution, each packaged under an atmosphere of a neutral non-reactive gas such as nitrogen. The ampoules may be of any suitable material such as glass, organic polymers such as polycarbonate, polystyrene, ceramic, metal, or any other material commonly used to store reagents. Other examples of suitable containers include bottles, which may be made from ampoule-like substances, and shells, which may consist of an inner surface covered with a foil such as aluminum or alloy. Other containers include test tubes, vials, flasks, vials, syringes, and the like. The containers may have a sterile access port, such as a bottle with a stopper, which can be pierced with a hypodermic needle. Other containers may have two compartments that are separated by an easily removable membrane which, once removed, allows the components to mix. The removable membranes may be glass, plastic, rubber, and the like.

[0078] In certain embodiments, kits may be provided with instructions. The instructions may be printed on paper or other substrate and/or may be provided in a readable electronic medium such as a floppy disk, mini CD-ROM, CD-ROM, DVD-ROM, zip drive, video cassette, audio cassette, etc. Detailed instructions cannot be physically linked to a kit; instead, the user may be directed to an Internet website provided by the manufacturer or distributor of the kit.

DEFINITIONS

[0079] It should be understood that all terminology used herein is only intended to describe specific embodiments of the invention and is not intended to be limiting in any manner or scope. It should be understood that all terminology used in this specification is only intended to describe specific embodiments of the invention and is not intended to be limiting in any way or scope. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. In addition, all units, prefixes, and symbols may be denoted in the accepted SI form.

[0080] In the context of this invention, the following definitions apply, unless otherwise indicated. For the purposes of the present invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version and Handbook of Chemistry and Physics, 75th Ed. 1994. In addition, the general principles of organic chemistry are described in “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999 and “March Advanced Organic Chemistry,” 5th ed., Smith, M. B. and March, J., eds. . John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0081] The term "mmol", in the context of this document, is intended to mean millimoles. The term "equiv.", in the context of this document, is intended to mean equivalent. The term "ml", in the context of this document, is intended to mean milliliter. The term "g", in the context of this document, is intended to mean grams. The term "kg", in the context of this document, is intended to mean kilogram. The term "mcg", in the context of this document, is intended to refer to micrograms. The term "h", in the context of this document, is intended to mean the hour. The term "min", in the context of this document, is intended to mean minutes. The term "M", in the context of this document, is intended to denote molarity. The term "µl", in the context of this document, is intended to mean microliter. The term "µM", in the context of this document, is intended to mean micromolar concentration. The term "nM", in the context of this document, is intended to mean nanomolar concentration. The term "N", in the context of this document, is intended to mean normal concentration. The term a. m.u., in the context of this document, is intended to denote the atomic mass unit. The term "°C", in the context of this document, is intended to mean degrees Celsius. The term "mass/mass", in the context of this document, is intended to mean mass/mass. The term "v/v", as used herein, is intended to mean volume/volume. The term "MS", in the context of this document, is intended to refer to mass spectrometry. The term "HPLC", in the context of this document, is intended to mean high performance liquid chromatography. The term "room temp.", in the context of this document, is intended to refer to room temperature. The term "for example", in the context of this document, is intended to refer to an example. The term "NI", in the context of this document, is intended to mean that which has not been investigated.

[0082] The numerical ranges specified in the description include numbers defining the range, and include every integer within the defined range. Throughout this disclosure, various aspects of the invention are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range is to be considered as specifically disclosing all possible subranges, fractions, and individual numeric values within a given range. For example, a description of a range such as 1 to 6 should be considered a specifically disclosed subrange such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and . etc., as well as individual numbers in this range, such as 1, 2, 3, 4, 5, and 6, and decimals and fractions, such as 1.2, 3.8, 1½, and 4¾. This applies regardless of the range width. In the context of the present application, the term "at least" also includes the starting point of the open range. For example, the amount of "at least 95% of the mass." means any amount equal to 95% of the mass. or higher.

[0083] The terms "mass fraction", "% wt.", "percent by mass", "% by mass" and their variations in the context of this document refer to the concentration of a substance as the mass of this substance, divided by the total weight of the composition and multiplied by 100. It is understood that, as used herein, "percentage", "%" and the like are intended to mean a synonym for "mass fraction", "% mass." etc.

[0084] The term "about", in the context of this document, refers to a change in numerical value, which can occur, for example, due to typical measuring methods and equipment, in relation to any quantitative variable, including, but not limited to, mass and volume. In addition, given the processing procedures for solids and liquids used in the real world, there is some unintentional error and variation, which is likely due to differences in the manufacture, source or purity of the ingredients used to make the compositions or carry out the methods and the like.

[0085] The methods and compositions of this invention may include, consist essentially of, or consist of the components and ingredients of this invention, as well as other ingredients described herein. In the context of this document, the term "consisting essentially of" means that methods and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not significantly change the main and new characteristics of the claimed methods and compositions. .

[0086] Since various changes can be made to the above compositions and methods without departing from the scope of this invention, it is intended that all material contained in the above description and in the examples below should be interpreted as illustrative and not as limiting. .

EXAMPLES

[0087] The following examples are included to demonstrate various embodiments of this disclosure. Those skilled in the art will appreciate that the methods described in the following examples demonstrate techniques that the present inventors have found to work well in the practice of the present invention, and therefore may be considered the preferred means of practicing the present invention. However, in light of this description, those skilled in the art will appreciate that numerous changes can be made to the specific embodiments of the invention described while still obtaining similar or analogous results, without deviating from the general idea and scope of the present invention.

Example 1 Experimental data on the effect of a mixture of 3.6 g of EAA plus arginine.

[0088] The following study was performed to quantify responses to two doses of the following amino acid composition:

1-2% histidine

9-11% isoleucine

35-38% leucine

14-17% lysine

2-4% methionine

5-7% phenylalanine

8-9% threonine

9-11% valine

0.002-0.8% tryptophan

8-11% arginine

[0089] An acute stimulation of muscle protein synthesis as a result of taking 3.6 or 10.8 g of the amino acid composition described above was determined in the group of elderly people (see Table 1 ). The 3.6 g dose was chosen due to the commercial viability of this dose based on cost and taste. The 3.6g response was compared to a 10.8g dosage, as it was shown that the higher dosage not only stimulates muscle FSR protein, but also results in long-term benefits in terms of strength and physical function (13). Muscle protein synthesis was determined before and after oral administration of the amino acid formulation.

Table 1 Characteristics of the subject

The male Woman General Age (years) 69±3 68±3 69±2 Floor 6 6 12 Body weight (kg) 98.2±5.3 71.7 ± 6.3 83.7 ± 5.8 Height (cm) 183.8±4.1 164.6 ± 1.7 173.3 ± 3.6 BMI (kg/ ^m2 ) 29.0±1.2 26.3±1.9 27.6 ± 1.2

[0090] Subjects were studied after an overnight fast. The study procedure included a 5 hour fasting period and a 3 hour post-treatment EAA formula. Subjects were given a continuous infusion of trace amounts of ² H ₅ -phenylalanine throughout the experiment. Muscle biopsies were taken after 2 and 5 hours of indicator infusion in the basal period and three hours after administration of the amino acid formula. For each biopsy sample, muscle and protein-bound phenylalanine enrichment was determined and muscle protein fractional synthesis rate (FSR) was calculated at basal and up to 3 hours post formula. Muscle protein FSR reflects the proportion of total muscle mass that is produced per unit of time (an hour in this experiment). Because in such a short period of time as this experiment, there is no chance that the amount of muscle protein has changed from the beginning to the end of the study, FSR muscle protein is a direct reflection of the absolute rate of muscle protein synthesis. Values were calculated as post-injection value - provisional value. Thus, each subject served as its own control, as various studies have shown that fasting muscle protein synthesis does not change over the time period of this study (eg, 14). The post-oral value is the integrated response for all 3 hours after taking the amino acid preparation.

[0091] Muscle FSR increased by 0.0568%/hr after the 3.6 g dose. It was 57% greater than basal FSR. The corresponding value was an increase of 0.0692%/h, which is 76% more than the base value. Plasma EAA levels were also measured ( Table 2 ).

Table 2 Amino acid concentrations (mol/l/3 hours)*

Dose EAA Leucine Total BCAA** 190 ± 23.2 42.6 ± 7.1 96.5 ± 11.3 272.7 ± 34.6 82.9 ± 13.1 153.8 ± 22.8

* Increase from the corresponding base value, area under the curve

**BCAAs are branched chain amino acids, leucine, valine and isoleucine

[0092] Although protein was tripled at the higher dosage, the increase in muscle protein FSR at the higher dosage was only marginally greater than in response to the 3.6 g dose. Part of the explanation for the disproportionately greater response to the lower dosage is that the amino acid concentrations illustrated in Table 2 do not represent the magnitude of the difference in protein intake. This likely reflected the accelerated oxidation of EAA when offered at a higher dosage and therefore less efficient use of that dosage. Expressed differently, the signal at the muscle level to increase synthesis was disproportionately greater in response to the lower dose. This also indicates that the 3.6 g dosage was sufficient to activate the synthetic response in the muscles. Another plausible explanation for the disproportionate response to the 3.6 g dose is that the amount of leucine in the 3.6 g formulation was sufficient to cause maximum activation of initiation factors, and that the relatively small increase in FSR at the higher dose fully reflected the increased provision of precursors. for synthesis.

[0093] The newly synthesized protein contains all 20 amino acids, but the composition contains only 9 amino acids. The remaining amino acids are derived from the more efficient reuse of amino acids derived from protein breakdown. Thus, some of the amino acids, including alanine, which are normally released from muscle as the primary means of transporting nitrogen to the liver for incorporation into ammonia and urea for final excretion in the urine, are instead reused for protein synthesis. Consistent with this interpretation, an earlier study noted that alanine concentrations decreased after ingestion of the EAA blend (15), reflecting increased reuse for the protein synthesis process. Therefore, unlike supplementation with amino acid blends that include alanine and other NEAAs, there was no increase in urea production following EAA supplementation (15).

[0094] The results are unexpected due to the low dose of this optimized ratio of EAA and arginine to stimulate muscle protein synthesis. A typical protein containing amino acids would require the dosage to be well above 20 g for the effect to be much lower than 3.6 g of this composition.

Claims (12)

1. A method for stimulating muscle protein synthesis, comprising administering to a subject a composition containing from 1 g to 4 g of the following concentrations of amino acids in terms of % w/w: from 1 to 2% histidine, from 9 to 11% isoleucine, from 35 to 38% leucine, 14 to 17% lysine, 2 to 4% methionine, 5 to 7% phenylalanine, 8 to 9% threonine, 9 to 11% valine, 0.005 to 0.8% tryptophan and 8 up to 11% arginine, and the stimulation of muscle protein synthesis is increased by approximately 50% compared to a subject who is not administered the specified amino acid composition. 2. The method according to claim 1, characterized in that muscle protein synthesis is improved without exercise. 3. The method of claim 1, wherein the subject is a mammal. 4. The method of claim 1, wherein the subject is a human. 5. The method of claim 1, wherein the subject is a human 40 years of age or older. 6. The method according to claim 1, characterized in that the composition contains the following concentrations of amino acids in terms of % wt./mass.: 1.5% histidine, 9.7% isoleucine, 36.4% leucine, 15.2% lysine , 3% methionine, 6.1% phenylalanine, 8.5% threonine, 10% valine, 0.6% tryptophan and 9% arginine. 7. The method according to claim 1, characterized in that a dose of the composition of 3.6 g is given to a human subject three times a day. 8. The method according to claim 1, characterized in that the dose of the composition of 3.6 g is given to the human subject once a day. 9. The method according to claim 1, characterized in that the dose of the composition of 3.6 g is given to the human subject twice a day. 10. Composition containing the following amino acids in terms of % w/w: 1 to 2% histidine, 9 to 11% isoleucine, 35 to 38% leucine, 14 to 17% lysine, 2 to 4% methionine, 5 to 7% phenylalanine, 8 to 9% threonine, 9 to 11% valine, 0.005 to 0.8% tryptophan and 8 to 11% arginine, which stimulates muscle protein synthesis by more than 50% in doses of 1 g to 4 g with or without exercise. 11. A method of stimulating muscle protein synthesis using a dosage of less than 12 g, comprising administering to a subject a composition containing the following concentrations of amino acids in terms of % w/w: 1 to 2% histidine, 9 to 11% isoleucine, 35 to 38% leucine, 14 to 17% lysine, 2 to 4% methionine, 5 to 7% phenylalanine, 8 to 9% threonine, 9 to 11% valine, 0.005 to 0.8% tryptophan and 8 to 11% arginine, wherein the composition is administered using a dosage of 1 g to 4 g of amino acids, wherein the stimulation of muscle protein synthesis is increased relative to a subject who is not administered said amino acid composition with exercise. 12. A method for stimulating muscle protein synthesis using a dosage of less than 12 g, comprising administering to a subject a composition containing the following concentrations of amino acids in terms of % w/w: 1 to 2% histidine, 9 to 11% isoleucine, 35 to 38% leucine, 14 to 17% lysine, 2 to 4% methionine, 5 to 7% phenylalanine, 8 to 9% threonine, 9 to 11% valine, 0.005 to 0.8% tryptophan and 8 to 11% arginine, wherein the composition is administered using a dosage of 1 g to 4 g of amino acids, wherein stimulation of muscle protein synthesis is increased relative to a subject not administered said amino acid composition without exercise.