BR112018005943B1 - ORAL ADMINISTRATION AMINO ACIDS FORMULATIONS WITH MODIFIED RELEASE - Google Patents

Abstract

ORAL ADMINISTRATION AMINO ACIDS FORMULATIONS WITH MODIFIED RELEASE. Modified release amino acid formulations and methods for the treatment or management of diseases defined by impaired amino acid metabolism, with improved pharmacokinetics, metabolism and utilization are provided.

Description

FIELD OF THE INVENTION

[001] The present invention relates to orally administered formulations and amino acid dosage forms with improved dosing accuracy, improved palatability and improved pharmacokinetics for optimal therapeutic effect.

FUNDAMENTALS OF THE INVENTION

[002] Amino acids are necessary building blocks in the synthesis of proteins by the human body and are mainly derived from dietary sources of protein. The human body uses nineteen separate L-configuration amino acids to synthesize proteins. Some of these amino acids are referred to as “essential amino acids” because the body cannot synthesize them independently. Other amino acids are not “essential” because the body can synthesize them.

[003] The digestion of dietary proteins into amino acids begins in the stomach thanks to the combined activity of pepsinogen, pepsin and hydrochloric acid leading to oligopeptides. Digestion continues in the duodenum after transit from the stomach through the pylorus, where the combined activity of bile secretion, bicarbonates, and pancreatic enzymes is able to produce individual amino acids. Montgomery R. et al., 1988; Baracos, 2004. The released amino acids are then absorbed from the small intestine into the portal vein according to active stereospecific transport processes specific for amino acids. Intestinal secretion of amino acids is a concomitant mechanism. Dipeptides and tripeptides can also be absorbed from the small intestine, but they too are eventually hydrolyzed to amino acids in the enterocyte. These processes are largely dependent on pH. When food is ingested, the stomach secretes hydrochloric acid and the pH decreases to about 2. After the pyloric valve, the pH increases progressively from 4 to about 7 as a consequence of alkaline bile secretion. A buffer system operates to avoid pH values greater than approximately 7.5-7.8.

[004] The time required to digest dietary proteins is the sum of the time spent in the gastric emptying phase and the time required for hydrolysis in the small intestine. The time required for both processes may vary depending on food intake, protein intake, illness and concomitant drug treatments, with food intake being the most common determining factor. Amidon et al., 1995 report a gastric emptying time of approximately 3.5 hours after a high fat meal, approximately 1.5 hours after a medium meal and approximately 10 to 20 minutes after 250 ml of water. Keohane et al., 1985 report that the time required to hydrolyze amino acids in the small intestine, on average, is approximately three hours and that this time varies for individual proteins and amino acids. This hydrolysis begins to occur immediately after gastric emptying, after transit through the pyloric valve, and lasts for several hours in the first tract of the small intestine, i.e. the jejunum.

[005] Amino acids are commonly administered as dietary supplements to facilitate protein synthesis, particularly where increases in muscle mass are intended. Specially formulated combinations of amino acids are also administered to support the nutritional health of individuals with special dietary intake limitations and requirements. In phenylketonuria, for example, the body is unable to metabolize phenylalanine into tyrosine due to a lack of necessary enzymes. Phenylalanine instead breaks down to various toxic by-products. Montgomery et al., 1988; Waisbren et al., 2007.

[006] Phenylalanine is an essential amino acid that is normally metabolized to tyrosine by the enzyme phenylalanine hydroxylase. In phenylketonuria, this enzyme is absent or does not function properly, and phenylalanine is decarboxylated into several compounds, three of which are toxic: phenylpyruvate, phenyl lactate, and phenyl acetate. Due to this metabolic inhibition, the dietary intake of phenylalanine should be reduced, providing the amino acid tyrosine with the diet, as it is indispensable. Ney D.M. et al., 2014; Dioguardi, 2011; Waisbren et al., 2007.

[007] An effective management strategy for phenylketonuria consists of specially formulated amino acid formulations that omit phenylalanine while supplementing tyrosine. Since phenylalanine cannot be removed from most dietary sources of protein, these amino acid supplements often take the place of dietary proteins that are eliminated completely from the diet. Vliet Van D. et al., 2014. However, these patients can also be treated with a carefully controlled protein diet that contains very little phenylalanine. Gropper S.S. et al., 1991.

1 http://www.dmfmetabolic.it/wp-content/uploads/2013/10/AntifenIntegrale.pdf 2 http://www.nutricia.it/prodotti/xp2-maxamaid/ 3 http://www.nutricia.it/prodotti/pku-2-secunda/[008] Examples of amino acid formulations sold for the management of phenylketonuria include formulations sold as AntifenTM, 5 Nutricia XP2TM (Maxamaid), and Milupa PKU2TM (Secunda). Nutricia XP2TM and Milupa PKU2TM also contain vitamins and minerals. Table A below presents the formulations of these products based on approximately 100g of amino acids in each formulation, omitting the vitamin and mineral content. TABLE A

1 http://www.dmfmetabolic.it/wp-content/uploads/2013/10/AntifenIntegrale.pdf 2 http://www.nutricia.it/prodotti/xp2-maxamaid/ 3 http://www.nutricia. it/prodotti/pku-2-second/

[009] Other conditions for which specially formulated amino acid preparations are manufactured and administered, using the same strategy to supply all essential and non-essential amino acids, but omitting amino acids that the body cannot metabolize, include tyrosinemia, leucinosis, methylmalonic acidemia , homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia and glutaric acidemia (types II, IIA or IIB).

[0010] Free amino acids potentially have numerous disadvantages over naturally occurring proteins due to the rapid absorption of the amino acids, imposing a greater dietary acid load, particularly when higher doses are administered.

[0011] In addition, the taste and odor of some commercial amino acid formulations are not good, which makes it extremely difficult for many people to ingest regularly, especially infants and children. Furthermore, these formulations are released immediately when ingested, leading to a very different absorption pattern than when proteins are ingested.

[0012] One of the objectives of the present invention is to limit the amino acid catabolism caused when free amino acids are ingested and dominate the body's metabolic capacity.

[0013] Another object of the present invention is to improve the taste of amino acid formulations when ingested orally.

[0014] Yet another object of the present invention is to provide unique dosage forms of amino acids that can be directly ingested without the need for reconstitution.

[0015] Yet another object of the present invention is to provide amino acid formulations with modified release properties, which better mimic the absorption pattern of proteins when ingested orally.

SUMMARY OF THE INVENTION

[0016] Modified-release amino acid formulations have been developed that overcome the foregoing drawbacks. The formulations are more palatable and, most importantly, mimic the amino acid release profile from naturally derived food sources. In particular, the formulations employ a release delay mechanism that modifies the release of amino acids from the formulation to mimic the rate of release of amino acids from naturally occurring proteins.

[0017] In the limited trials performed to date, various individual amino acids and combinations of amino acids, when modified to meet specific in vitro release criteria, have demonstrated statistically superior improvements in plasma amino acid concentrations over comparable free amino acid formulations. Thus, in a first main embodiment, the invention provides an orally administered amino acid formulation comprising one or more modified release amino acids, wherein the formulation comprising 2 g of the modified release amino acids does not release more than x% of the modified release amino acids in 30 minutes of dissolution tests performed on a paddle apparatus <711> USP 39 NF 34, at 37 °C, in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0018] In an in vivo animal bioavailability study, it was unexpectedly found that isoleucine, leucine, tyrosine and valine, when modified for release as described herein, consistently have a peak blood concentration that is significantly lower to the maximum concentration produced by a comparable free amino acid formulation. Thus, in a second main embodiment, the invention provides an orally administered amino acid formulation comprising a modified-release amino acid selected from arginine, isoleucine, leucine, tyrosine and valine, wherein the formulation comprising 2 g of the modified-release amino acid no longer releases than x% of modified release amino acids in 30 minutes dissolution test performed on a paddle apparatus <711> USP 39 NF 34 at 37 °C in 450 or 500 mL 0.1 N hydrochloric acid (pH 1 ,2), blade speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0019] Another major embodiment relates to a particular group of amino acids - the branched-chain amino acids - and the unexpected discovery that branched-chain amino acids, in aggregate, when modified for release as described herein, produce a significantly higher Cmax. greater and greater Clast greater than the Cmax and Clast produced by a comparable free amino acid formulation. Thus, in another major embodiment, the invention provides an orally administered amino acid formulation comprising one or more modified-release branched-chain amino acids, wherein the formulation comprising 2 g of the modified-release branched-chain amino acids does not release more than x% of the modified-release amino acids in 30 minutes of dissolution tests performed on a paddle apparatus <711> USP 39 NF 34, at 37°C, in 450 or 500 mL, 0.1N hydrochloric acid (pH 1.2), speed blades 50 rpm, where x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0020] Yet another major embodiment relates to another particular group of amino acids - essential amino acids - which, when modified for release as described herein, unexpectedly produce a significantly lower Cmax and significantly higher Clast when compared to a comparable free amino acid formulation . Thus, in yet another major embodiment, the invention provides an orally administered amino acid formulation comprising one or more modified-release essential amino acids, wherein the formulation comprising 2 g of the modified-release essential amino acids does not release more than x% of the modified-release amino acids. modified in 30 minutes of dissolution tests performed on a <711> USP 39 NF 34 paddle apparatus at 37 °C in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50%, or even 40%.

[0021] Yet another major embodiment refers to another group of amino acids - large neutral amino acids (excluding Phenylalanine, Tyrosine and Methionine) - which, when modified for release as described herein, unexpectedly produce a significantly lower Cmax and significantly higher Clast when compared to a comparable free amino acid formulation. Thus, in yet another major embodiment, the invention provides an orally administered amino acid formulation comprising one or more large neutral modified-release amino acids, wherein the formulation comprising 2 g of the large neutral modified-release amino acids does not release more than x% of the amino acids. 30-minute modified-release dissolution tests performed on a <711> USP 39 NF 34 paddle apparatus at 37 °C in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), speed of blades 50 rpm, where x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0022] In a seventh major embodiment, the invention provides an orally administered amino acid formulation comprising a combination of the non-essential modified-release amino acids, wherein the formulation comprising 2 g of the modified-release amino acids does not release more than x% of the modified-release amino acids. 30-minute modified release dissolution tests performed on a <711> USP 39 NF 34 paddle apparatus at 37 °C in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50%, or even 40%.

[0023] The formulations are preferably in the form of edible granules that can be mixed with other foods such as yogurt or large chewable tablets that cannot be swallowed whole but must be eaten like a biscuit in several bites. The amount of amino acids in the unit dose formulations will depend on the metabolic demands of the individual treated generally depending on the individual's age, sex, weight and physical activities, and whether the individual suffers from a metabolic disorder that limits their intake of one or more amino acids, as described in more detail in this document.

[0024] The formulation may comprise any, all or any combination of essential and non-essential amino acids, and may be specifically designed for any application where amino acid supplementation is desired. The formulations are particularly well suited for the treatment of people with amino acid processing disorders such as phenylketonuria, tyrosinemia, leucinosis, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia and glutamic acidemia.

[0025] Modified release can be performed by ingredients mentioned herein variously as release delaying agents, means to preferentially release the amino acids in the jejunum, etc. These agents are capable of causing a modified release of amino acids from the formulation when ingested or when tested in dissolution media. Alternatively, they are able to prolong or delay the release of amino acids so that some of the amino acids are not released until later in the digestive process, such as after the formulation has reached the higher pH environment of the small intestine. These agents are even capable, in a properly designed formulation, of releasing a subset of amino acids in the formulation at a different release rate than other amino acids. Formulations are preferably released based on their modified release pattern when tested with a fixed pH value.

[0026] Other aspects of the present invention involve the use of the formulations to treat or manage a disease that requires close management of amino acid intake, usually due to impaired amino acid metabolism. Thus, in another embodiment, the invention provides a method of treating a metabolic disorder selected from the group consisting of phenylketonuria, tyrosinemia, leucinosis, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia and glutamic acidemia, in a patient in need thereof, comprising orally administering to the patient an amino acid formulation of the present invention.

[0027] Yet another embodiment provides methods for making the formulations of the present invention, and in this embodiment, the invention provides a method of preparing a formulation of the present invention comprising: (a) providing a first mixture comprising a single or a plurality of of amino acids; (b) contacting said mixture with a wetting agent or solvent (for example, an alcohol and/or an organic solvent and/or water) and a binder to form a wet mixture; (c) optionally passing said wet mixture through a screen to form a uniform wet granulate; (d) drying said uniform wet granulate to form a dry granulate; (e) optionally passing said dry granulate through a sieve to form a uniform dry granulate; and (f) optionally coating the dried granulate with one or more coating agents to provide modified release for said dry granulate. Suitable coating agents include, for example, cellulose polymers, fatty acids, waxes and other coating agents capable of providing modified release for said dried granulate. Suitable coating techniques include, for example, fluid bed coating and melt granulation. The dried granules can be incorporated into any conventional dosage form, but are preferably used as the dosage form, without further modification, where they can be easily mixed with other ingredients such as flavorings and excipients or inactive additives, or active ingredients such as vitamins, minerals and carbohydrates. Alternatively, the dried granules can be compressed into a chewable tablet.

[0028] Additional advantages of the invention are set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and achieved by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

DESCRIPTION OF FIGURES

[0029] Figure 1 tabulates the 19 amino acids used by the human body to synthesize proteins and classifies them according to structure and properties, ie, aliphatic, aromatic, acidic, basic, sulfuric and pKa.

[0030] Figure 2 tabulates essential amino acids, that is, amino acids that are not produced in the body through biochemical synthesis, but must be ingested with the diet, as reported by Montgomery et al., 1988.

[0031] Figure 3 is a graphical representation of the release profile over time of uncoated tryptophan made according to the method of Example 7, using two different paddle speeds, compared to tryptophan formulated according to the present invention.

[0032] Figure 4 is a graphical depiction of the tryptophan release profile over time from the formulation of Example 8 using a dissolution medium at pH 1.2 and a paddle speed of 50 rpm.

[0033] Figure 5 is a graphical description of the tryptophan release profile over time from the formulation of Example 9 when subjected to the experimental conditions described in Example 8, but using the basket method instead of the paddle.

[0034] Figure 6 is a graphical description of the weight release profiles of a formulation containing the mixture of amino acids over time from the formulation of Example 10, when subjected to the experimental conditions described in Example 10.

[0035] Figure 7 is a graphical depiction of the weight dissolution profile of the free amino acid formulation described in Example 11, Table 10b, tested according to the methods described in Example 11.

[0036] Figure 8 plots the dissolution profile for the 18 amino acids separated from the mixture of APR AA (bkT037/71)

[0037] Figure 9 is a graphical representation of the weight dissolution profile of four separate formulations of modified-release amino acids coated with ethylcellulose, made and tested according to the methods and formulation reported in Example 11 and Table 10d and 10e.

[0038] Figure 10 represents the dissolution profile for the 18 amino acids separated from APR Lot 2 (bkT037/72-2), Table 10f, when tested according to the methods described in Example 11.

[0039] Figure 11 plots the dissolution profile for the 18 amino acids separated from APR Lot 4 (bkT037/72-4), Table 10g, when tested according to the methods reported in Example 11.

[0040] Figure 12 plots the aggregated amino acid dissolution profile of four separate ethylcellulose/glyceryl coated modified-release amino acid formulations, manufactured and tested as described in Example 12, having the compositions described in Table 11a.

[0041] Figure 13 plots the dissolution profile of 18 separate amino acids from APR Lot 5 (bkT037/73-5) (Table 11c), when tested according to the methods reported in Example 12.

[0042] Figure 14 plots the dissolution profile of 18 separate amino acids from APR Lot 7 (bkT037/72-7) (Table 11d), when tested according to the methods reported in Example 12.

[0043] Figure 15 depicts the aggregate weight amino acid dissolution profiles of four separate test formulations of the current inventions (APR/bkT037/72-2 Lot 2, APR/bkT037/72-4 Lot 4, APR/bkT037/72-4 Lot 5 APR/bkT037/73-5, APR/bkT037/72-7 Lot 7) and the free amino acid mixture bkT037/71, manufactured and tested as described in Examples 11 and 12.

[0044] Figure 16 reports mean pig plasma concentrations of 14 amino acids over time, on an aggregated basis, from two separate modified-release amino acid formulations of the present invention, a free amino acid formulation and casein, as described in Example 14.

[0045] Figure 17 reports mean pig plasma concentrations of the essential amino acids over time, on an aggregated basis, from two separate modified-release amino acid formulations of the present invention, a free amino acid formulation and casein, as described in Example 14.

[0046] Figure 18 reports mean pig plasma concentrations of branched-chain amino acids over time, on an aggregated basis, from two separate modified-release amino acid formulations of the present invention, a free amino acid formulation and casein , as described in Example 14.

[0047] Figure 19 reports mean pig plasma concentrations of large neutral chain amino acids over time, on an aggregated basis, from two separate modified-release amino acid formulations of the present invention, a free and a free amino acid formulation. casein, as described in Example 14.

DETAILED DESCRIPTION Definitions

[0048] When the singular forms “um”, “uma” and “a/o” or similar terms are used in this document, they will be understood to include plural referents, unless the context clearly dictates otherwise. Thus, for example, reference to "a hydrocarbon" includes mixtures of two or more hydrocarbons and the like. The word “or” or similar terms as used in this document means any member of a specific list and also includes any combination of members of that list.

[0049] When used in this document, the term "about" or "ca" will compensate for the variability allowed in the pharmaceutical industry and inherent in pharmaceutical products, such as differences in product strength and bioavailability due to manufacturing variations and product degradation induced in time. The term allows for any variation that, in pharmaceutical practice, allows the evaluated product to be considered pharmaceutically equivalent or bioequivalent, or both, if the context requires, to the recited strength of a claimed product. It will be understood that all numerical values expressed in this document may be preceded by the term "about".

[0050] The term “independently” is used to mean that particular elements modified by a set of potential variables may or may not be defined by the same variable. Thus, for example, if a composition comprises z% of element A and z% of element B, and z may independently be 10% or 20%, the composition may comprise 10% of element A and 10% of element B or 10% of element B. A and 20% of element B, etc.

[0051] The term "amino acid" refers to any naturally occurring amino acid capable of participating in the synthesis of peptides and proteins. For ease of elaboration, the amino acid will often be written without its stereo configuration, although it is understood that the amino acid must be present as its naturally occurring stereoisomer.

[0052] In the formulations of the present invention, the amino acids may be present as the free base, as the hydrochloride salt or as another suitable salt. The term essential amino acid refers to any amino acid that the body is unable to produce that must be derived from an external source. Essential amino acids that can be included in the formulations of the present invention include L-Isoleucine, L-Leucine, L-Lysine, L-Threonine, L-Tryptophan, L-Valine, L-Methionine, L-Phenylalanine, L-Histidine and L -arginine or any of its pharmaceutically acceptable salts (these last two are considered essential only for breastfeeding women, although, for simplicity, they are mentioned as “essential amino acids in this document”).

[0053] The term non-essential amino acid refers to any amino acid other than an essential amino acid and therefore includes, for example, L-alanine, L-aspartic acid, L-cystine, L-glutamine and/or L-acid -glutamic, Glycine, L-Proline, L-Serine, Taurine, L-Tyrosine and L-Carnitine or any of their pharmaceutically acceptable salts. Any of these amino acids may be present, as the free base, the hydrochloride salt or any other suitable salt.

[0054] The term "branched-chain amino acids" refers to valine, isoleucine and leucine, or any of their pharmaceutically acceptable salts.

[0055] The term "large neutral amino acids" in one embodiment refers to tryptophan, threonine, valine, isoleucine, leucine, and histidine, or any of their pharmaceutically acceptable salts. In another embodiment, the term also includes phenylalanine, methionine and tyrosine.

[0056] The term "formulation" refers to a finished or semi-finished combination of food or pharmaceutical or medical food ingredients, including active ingredients and inactive excipients or additives. The term refers to in-process formulations, finished formulations and formulations packaged as a final unit dose.

[0057] Throughout the description and claims of this descriptive report, the word "comprehend" and variations of the word, such as "comprising" and "comprises", means "including, but not limited to", and is not intended to exclude , for example, other additives, components, integers or steps.

[0058] The terms “treating” and “treatment”, when used in this document, refer to the medical treatment of a patient with the intention of curing, improving, stabilizing or preventing a disease, pathology or disorder. This term includes active treatment, i.e., treatment directed specifically towards the amelioration of a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed towards removing the cause of the associated disease, pathological condition or disorder. In addition, this term includes palliative care, that is, treatment aimed at relieving symptoms rather than curing the disease, pathological condition or disorder; preventive treatment, i.e., treatment directed to partially or totally minimize or inhibit the development of the associated disease, pathological condition or disorder; and supportive care, that is, treatment employed to complement other specific therapy aimed at ameliorating the associated disease, pathological condition or disorder. The term also includes the dietary management of a patient who, because of a specific disease or medical condition, has limited or impaired ability to ingest, digest, adsorb or metabolize food or nutrients or who has other medically determined special nutrient requirements, the dietary management that cannot be achieved by normal diet modification alone.

[0059] The term "modified release" refers to any pharmaceutical formulation in which the release rate is intentionally altered to obtain a desired therapeutic or pharmacokinetic response. The term thus includes sustained-release formulations, in which drug release is prolonged over time, or a rate of release that is independent of the pH of the surrounding environment. The term also includes delayed-release formulations, in which the release of the formulation's active ingredient (or a portion thereof) is delayed to occur after initial ingestion. A delayed-release formulation is typically designed so that release predominantly occurs once the formulation reaches the small intestine.

[0060] As used herein, "therapeutically effective amount" refers to an amount sufficient to elicit the desired biological response. The therapeutically effective amount or dose will depend on the age, sex and weight of the patient, and the patient's current medical condition. One skilled in the art will be able to determine appropriate dosages depending on these and other factors in addition to the present disclosure. When used to refer to nutrients such as amino acids, minerals, vitamins, etc., the term refers to the amount generally recognized as necessary to support the metabolic needs of the human body.

[0061] "Pharmaceutically acceptable" means what is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and not biologically or otherwise undesirable and includes what is acceptable for veterinary use as well as for medical food use, human food and pharmaceutical. "Pharmaceutically acceptable salts" means salts which are pharmaceutically or medically or dietaryly acceptable, as defined above, and which possess the desired pharmacological or nutritional activity.

[0062] When intervals are expressed in this document by specifying alternate upper and lower bounds of the interval, it will be understood that the endpoints may be combined in any manner that is mathematically feasible. So, for example, a range from 50 or 80 to 100 or 70 could alternatively be expressed as a series of ranges from 50 to 100, 50 to 70, and 80 to 100. Lower limits are related using phase and/or, it will be understood that upper limits may be unbounded by lower links or combined with lower limits and vice versa. Thus, for example, a range greater than 40% and/or less than 80% includes ranges greater than 40%, less than 80%, and greater than 40% but less than 80%.

[0063] When a specific test methodology is provided by the United States Pharmacopoeia or other industry-accepted compendium herein, it will be understood that the test method may be modified in accordance with usual and customary practices without altering the basic physical principles or the final results based on the conventional skill of the person skilled in the art, and the examples provided herein, as, for example, may be necessary to test different dosage forms.

[0064] When percentages are given in this document, it will be understood that the percentages are percentages by weight and that the proportions are based on weight, unless otherwise indicated.

[0065] Discussion

[0066] The invention is defined in terms of main embodiments and submodalities, and it will be understood that main embodiments can be combined to define other main embodiments, that submodalities can be combined to define additional submodalities, and that submodalities and combinations of submodalities can be combined with all major embodiments to define other embodiments of the present invention. The ability to combine modalities and submodalities is limited only by what is mathematically or physically impossible.

[0067] In a first major embodiment, the invention provides an orally administered amino acid formulation comprising one or more modified-release amino acids, wherein the formulation comprising 2 g of the modified-release amino acids does not release more than x% of the modified-release amino acids in 30 minutes of dissolution tests performed on a paddle apparatus <711> USP 39 NF 34, at 37 °C, in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm , where x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0068] In a second main embodiment, the invention provides an orally administered amino acid formulation comprising a modified release amino acid selected from arginine, isoleucine, leucine, tyrosine and valine, wherein the formulation comprising 2 g of the modified release amino acids does not release more than x% of modified-release amino acids in 30-minute dissolution test performed on a paddle apparatus <711> USP 39 NF 34 at 37 °C in 450 or 500 mL 0.1 N hydrochloric acid (pH 1,2), blade speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0069] In a third major embodiment, the invention provides an orally administered amino acid formulation comprising modified-release branched-chain amino acids, wherein the formulation comprising 2 g of the modified-release amino acids does not release more than x% of the modified-release amino acids in 30 minutes of dissolution tests performed on a paddle apparatus <711> USP 39 NF 34, at 37 °C, in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm , where x% is 90%, 80%, 70%, 60%, 50% or even 40%. In a preferred embodiment, the formulation includes modified release valine, isoleucine and leucine.

[0070] In a fourth major embodiment, the invention provides an orally administered amino acid formulation comprising a combination of the modified-release essential amino acids, wherein the formulation comprising 2 g of the modified-release amino acids does not release more than x% of the modified-release amino acids modified in 30 minutes of dissolution tests performed on a <711> USP 39 NF 34 paddle apparatus at 37 °C in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50%, or even 40%. In a preferred embodiment, modified release amino acids include Arginine, Histidine, Isoleucine, Leucine, Tronin, Lysine and Tryptophan.

[0071] In a fifth major embodiment, the invention provides an orally administered amino acid formulation comprising large neutral modified-release amino acids, wherein the formulation comprising 2 g of the large neutral modified-release amino acids does not release more than x% of the modified-release amino acids modified in 30 minutes of dissolution tests performed on a <711> USP 39 NF 34 paddle apparatus at 37 °C in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50%, or even 40%. In a preferred embodiment, the formulation includes modified release tryptophan, threonine, valine, isoleucine, leucine and histidine.

[0072] In a sixth main embodiment, the invention provides a method of treating a metabolic disorder selected from the group consisting of phenylketonuria, tyrosinemia, leucinosis, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia and glutamic acidemia, in a patient in need thereof, comprising orally administering to the patient the formulation of the present invention defined by any of the main modalities or submodalities.

[0073] In a seventh major embodiment, the invention provides an orally administered amino acid formulation comprising a combination of the non-essential modified-release amino acids, wherein the formulation comprising 2 g of the modified-release amino acids does not release more than x% of the amino acids from 30-minute modified release dissolution tests performed on a <711> USP 39 NF 34 paddle apparatus at 37 °C in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, where x% is 90%, 80%, 70%, 60%, 50%, or even 40%.

[0074] In various submodalities, the formulation may comprise any, all, or any combination of essential and non-essential amino acids, and may be specifically designed for any application where amino acid supplementation is desired. The formulations are particularly well suited for the treatment of people with amino acid processing disorders such as phenylketonuria, tyrosinemia, leucinosis, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia and glutamic acidemia. Other more specific submodalities are described more specifically in Tables B-D. However, it will be understood that a certain amount of flexibility must be built into this formulation to account for, for example, the amino acid requirements of infants and children, which could cause some of the amino acids to fall outside the recited proportions.

[0075] Table B presents parts by relative weight of nineteen amino acids for general use as modified-release amino acids in formulations intended for amino acid supplementation, as well as phenylketonuria and tyrosinemia. Column A presents preferred ranges of suitable ratios of amino acids, while column B presents a more preferred range of suitable ratios. Table B only describes the relative proportions of amino acids in the formulation. Thus, other ingredients can be included. Aggregate amino acid release rates from any of the formulations described in Table B are preferably as reported in the first major embodiment. Table B

[0076] Other embodiments are defined when the first main embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered 5 by the general formulations of Table B (defined by Columns A or B) in the proportions recited in Columns A or B.

[0077] Other embodiments are defined when the first main embodiment includes as modified-release amino acids the essential amino acids, the branched-chain amino acids or the large neutral amino acids covered by the general formulations of Table B (defined by Columns A or B) in the proportions recited in columns A or B.

[0078] Other embodiments are defined when the first main embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the phenylketonuria formulations of Table B (defined by Columns A or B) in the proportions recited in Columns A or B.

[0079] Other embodiments are defined when the first major embodiment includes as modified-release amino acids the essential amino acids, branched-chain amino acids, or large neutral amino acids covered by the phenylketonuria formulations of Table B (defined by Columns A or B) in proportions recited in columns A or B.

[0080] Other modalities are defined when the first main 10 modality includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the tyrosinemia formulations of Table B ( defined by Columns A or B) in the proportions recited in Columns A or B.

[0081] Other embodiments are defined when the first major embodiment 15 includes as modified-release amino acids the essential amino acids, the branched-chain amino acids, or the large neutral amino acids covered by the tyrosinemia formulations of Table B (defined by Columns A or B) in proportions recited in columns A or B.

[0082] Table C presents relative weight parts of nineteen 20 amino acids for use as modified-release amino acids in leucinosis, methylmalonic acidemia, propionic acidemia, and glutaric acidemia. Column A presents preferred ranges of appropriate amino acid ratios for each of these metabolic disorders, while column B presents a more preferred range of appropriate ratios. Table C describes only the relative proportions of amino acids in the formulation. Thus, other ingredients can be included. Aggregate amino acid release rates from any of the formulations described in Table C are preferably as reported in the first major embodiment. Table C

[0083] Other embodiments are defined when the first major embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the 5 leucinosis formulations of Table C ( defined by Columns A or B) in the proportions recited in Columns A or B.

[0084] Other embodiments are defined when the first main embodiment includes as modified-release amino acids the essential amino acids, the branched-chain amino acids or the large neutral amino acids covered by the leucinosis formulations of Table C (defined by Columns A or B) in the proportions recited in columns A or B.

[0085] Other embodiments are defined when the first major embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the methylmalonic acidemia formulations of Table C ( defined by Columns A or B) in the proportions recited in Columns A or B.

[0086] Other embodiments are defined when the first major embodiment includes as modified-release amino acids the essential amino acids, branched-chain amino acids, or large neutral amino acids covered by the methylmalonic acidemia formulations of Table C (defined by Columns A or B) in proportions recited in columns A or B.

[0087] Other embodiments are defined when the first major embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the propionic acidemia formulations of Table C ( defined by Columns A or B) in the proportions recited in Columns A or B.

[0088] Other embodiments are defined when the first major embodiment includes as modified-release amino acids the essential amino acids, branched-chain amino acids, or large neutral amino acids covered by the propionic acidemia formulations of Table C (defined by Columns A or B) in proportions recited in columns A or B.

[0089] Other embodiments are defined when the first major embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the glutaric acidemia formulations of Table C ( defined by Columns A or B) in the proportions recited in Columns A or B.

[0090] Other embodiments are defined when the first major embodiment includes as modified-release amino acids the essential amino acids, branched-chain amino acids, or large neutral amino acids covered by the glutaric acidemia formulations of Table C (defined by Columns A or B) in proportions recited in columns A or B.

[0091] Table D presents parts by relative weight of nineteen amino acids for use as modified-release amino acids in isovaleric acidemia, homocystinuria, and hyperglycinemia. Column A presents preferred ranges of appropriate amino acid ratios for each of these metabolic disorders, while column B presents more preferred ranges of appropriate ratios. Table C describes the relative proportions of amino acids in the formulation. Thus, other ingredients may be included. Aggregate amino acid release rates from any of the formulations described in Table D are preferably as reported in the first major embodiment. Table D

[0092] Other embodiments are defined when the first major embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the isovaleric acidemia formulations of Table D ( defined by Columns A or B) in the proportions recited in Columns A or B.

[0093] Other embodiments are defined when the first major embodiment includes as modified-release amino acids the essential amino acids, the branched-chain amino acids, or the large neutral amino acids covered by the isovaleric acidemia formulations of Table D (defined by Columns A or B) in proportions recited in columns A or B.

[0094] Other embodiments are defined when the first major embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the hemocystinuria formulations of Table D (defined by Columns A or B) in the proportions recited in Columns A or B.

[0095] Other embodiments are defined when the first main embodiment includes as modified-release amino acids the essential amino acids, the branched-chain amino acids or the large neutral amino acids covered by the homocystinuria formulations of Table C (defined by Columns A or B) in the proportions recited in columns A or B.

[0096] Other embodiments are defined when the first major embodiment includes as modified-release amino acids three or more, five or more, 10 or more, or 15 or more, or even all amino acids covered by the hyperglycinemia formulations of Table D (defined by Columns A or B) in the proportions recited in Columns A or B.

[0097] Other embodiments are defined when the first main embodiment includes as modified-release amino acids the essential amino acids, the branched-chain amino acids or the large neutral amino acids covered by the hyperglycinemia formulations of Table D (defined by Columns A or B) in the proportions recited in columns A or B.

[0098] The additional submodalities are defined when the third main modality is limited by the relative proportions of branched-chain amino acids recited in table E1-E3.

[0099] The additional submodalities are defined when the fourth main modality is limited by the relative portions of essential amino acids (Arginine, Histidine, Isoleucine, Leucine, Lysine, Threonine, Tryptophan and Valine) recited in tables F1-F3. Table F1

[00100] Additional submodalities are defined when the fifth main modality is limited by the relative proportions of large neutral amino acids recited in table G1-G3. Table G1

[00101] In another submodality for the treatment of phenylketonuria, defined without regard to the proportions of amino acids, the formulation of the present invention comprises three or more of, five or more of, or all of, arginine, histidine, isoleucine, leucine, lysine, methionine , threonine, tryptophan and valine, or one or more pharmaceutically acceptable salts thereof, as modified-release essential amino acids.

[00102] In another submodality for the treatment of phenylketonuria, again defined without regard to the proportions of amino acids, the formulations of the present invention comprise three or more of, five or more of, or all of, arginine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan and valine, or one or more pharmaceutically acceptable salts thereof, as modified-release essential amino acids; and three or more of, five or more of, alanine, aspartic acid, carnitine, cystine, glutamine and/or glutamic acid, glycine, proline, serine, taurine and tyrosine, or one or more pharmaceutically acceptable salts thereof, as non-amino acids modified-release essentials.

[00103] In a certain submodality, which is suitable for the treatment of any of the metabolic disorders described herein, defined without regard to the proportions of ingredients, the formulation comprises arginine and histidine, or one or more pharmaceutically acceptable salts thereof, as modified-release essential amino acids; and alanine, aspartic acid, carnitine, cystine, glutamine and/or glutamic acid, proline, serine and taurine, or one or more acceptable salts thereof, as non-essential modified-release amino acids.

[00104] In a particularly preferred embodiment, the formulation is provided as a single unit dose form, comprising from 1 to 40 grams of amino acids, wherein the formulation is therapeutically effective to meet the dietary amino acid requirements of a human patient when administered three to four times a day. That is, the formulation is adequate to meet the patient's dietary needs for the amino acids that are actually present in the formulation.

[00105] In another main embodiment, the invention provides a method for preparing the formulation defined by any of the main embodiments or submodalities of the present invention, comprising: (a) providing a first mixture comprising a single or a plurality of amino acids; (b) contacting the mixture with a wetting agent and a binder to form a wet mixture; (c) optionally passing the wet mixture through a sieve to form a uniform wet granulate; (d) drying the wet granulate uniformly to form a dry granulate; (e) optionally passing the dried granulation through a sieve to form a uniform dry granulate; and (f) optionally coating the uniform dried granulate with a modified release composition.

[00106] The method is particularly suitable for mixing a large number of amino acids and can be further adapted by preparing more than one granulate with different mixtures of amino acids and combining the different granulates to form the final formulation. Thus, in a preferred embodiment of the manufacturing method, the first mixture may comprise only one type of amino acid, or up to 6 amino acids, further comprising repeating steps (a)-(d) and optionally (e) and (f) with a second different mixture of amino acids comprising only one amino acid, or up to 6 amino acids to form a second uniform dry pellet and combining the uniform dry pellets to form the formulation. In one embodiment, the granules are compressed to form a chewable tablet. In another embodiment, after forming the granulated mixture, it is further processed by, for example, adding flavorings or nutrients.

[00107] The formulations can also be defined based on the percentage of amino acids in the formulation. Thus, for example, the invention further provides formulations comprising greater than 20% by weight, 70% by weight or 90% by weight of amino acids, wherein (a) said amino acids comprise from about 10% by weight to about 80% % by weight, from about 20% by weight to about 60% by weight, from about 30 to about 50%, or from about 35 to about 45% essential amino acids; and (b) said essential amino acids comprise any or all combinations of L-Isoleucine, L-Leucine, L-Lysine, L-Threonine, L-Tryptophan, L-Valine, L-Methionine, L-Histidine and L-Arginine ( the last two are considered essential only for nursing mothers).

[00108] Alternatively, the modified release formulation can be defined based on the ratio of essential to non-essential amino acids in the formulation. Thus, in various embodiments, the invention provides an orally administered modified-release amino acid formulation comprising greater than 20% by weight, 50%, 70% by weight, or 90% by weight of amino acids, wherein (a) said amino acids comprise essential and non-essential amino acids in a weight ratio of about 10:90, 20:80. 30:70, 40:60, 50:50, 60:40, 70:30 80:20 to about 90:10, 80:20, 70:30, 60:40, 50:50. 40:60, 30:70 or 20:80; and (b) said essential amino acids comprise any or all combination of L-Isoleucine, L-Leucine, L-Lysine, L-Threonine, L-Tryptophan, L-Valine, L-Methionine, L-Histidine and L-Arginine ( the last two are considered essential only for nursing mothers).

[00109] The formulation preferably includes one or more excipients or additives capable of controlling the release of all amino acids in the formulation or a subset of amino acids. Such excipients or additives are referred to herein generally as "delayed release excipients or additives" and are capable of providing an extended release profile, a delayed release profile, a combination of immediate release and extended release profiles, or delayed release. In one embodiment, the excipients or additives are delayed release excipients or additives and are mentioned as "means for preferentially releasing said amino acids at a pH between 4 and 7" or "means for preferentially releasing said amino acids in the jejunum when administered via the oral.” In this case, the excipient is typically present in an amount effective to cause preferential release of said amino acids from said formulation at a pH between 4 and 7.

[00110] The term “preferential release” does not exclude the possibility that some of the excipients release amino acids in a lower pH environment, or even that the release rate in a lower pH environment is the same as that which occurs in an environment of lower pH. higher pH. The important point is that the excipient reduces the total amount of amino acids that would otherwise be released in the low pH environment of the stomach versus an immediate release dosage form. Thus, for example, the excipient may produce a slower release profile over time as the formulation passes through the GI tract than would otherwise occur with an immediate release formulation, or as modeled in an in vitro experiment. adequate. Alternatively, the excipient can delay release until the formulation reaches the higher pH environment of the jejunum.

[00111] Release-modifying excipients or additives/glazing agents are well known to those skilled in the art and include, for example, ethylcellulose, cellulose acetate, vinyl acetate/vinyl chloride copolymers, acrylate/methacrylate copolymers, polyethylene oxide, hydroxypropylmethylcellulose, carrageenan, alginic acid and its salts, hydroxyethylcellulose, hydroxypropylcellulose, karaya gum, acacia gum, tragacanth gum, locust bean gum, guar gum, xanthan gum, sodium carboxymethylcellulose, methylcellulose, beeswax , carnauba wax, cetyl alcohol, hydrogenated vegetable oils, stearyl alcohol, acrylic acid copolymers, carrageenan, pectin, a sodium carboxymethylcellulose, mono- and diglycerides of fatty acids, fatty acids and their esters or derivatives, including glyceryl behenates and dipalmitostearate of glyceryl, starches and their derivatives, such as corn acetate. Formulations

[00112] These compositions of the present invention can be prepared in a manner well known in the pharmaceutical and food supplement arts and can be administered by a variety of routes, depending on whether local or systemic treatment is desired. The most preferred route of administration is oral. The compositions usually contain, in addition to the amino acids, one or more acceptable carriers (excipients or additives). In preparing the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient, or contained within such a carrier in the form, for example, of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills (including soft and hard gelatine capsules), powders, granules, microspheres, lozenges, sachets (i.e. powders or granules or packaged microspheres), sachets, elixirs, suspensions, emulsions , solutions and syrups, containing, for example, up to 90% by weight of amino acids.

[00113] By using this technology, it is possible to design various amino acid products, in which all or only a selected portion of amino acids have the modified release profile. For example, it is possible to design a formulation where:

[00114] All or just a portion of the amino acids in the formulation are formulated for modified release. That is, more than 20%, 40%, 60% or 80% and/or less than 80%, 60%, 40% or 20% of the amino acids are formulated for modified release.

[00115] All or just a portion of the essential amino acids in the formulation are formulated for modified release. That is, more than 20%, 40%, 60% or 80% and/or less than 80%, 60%, 40% or 20% of the essential amino acids are formulated for modified release.

[00116] None of the essential amino acids are formulated for modified release.

[00117] All or just a portion of the non-essential amino acids in the formulation are formulated for modified release. That is, more than 20%, 40%, 60% or 80% and/or less than 80%, 60%, 40% or 20% of non-essential amino acids are formulated for modified release.

[00118] None of the non-essential amino acids are formulated for modified release.

[00119] Any combination of the above strategies.

[00120] For example, using the formulation strategies described in the present application, it is possible to formulate amino acid dosage forms such that only essential amino acids are formulated for modified release and non-essential amino acids are not. Alternatively, it is possible to formulate amino acid dosage forms such that only non-essential amino acids are formulated for modified release and essential amino acids are not. Alternatively, depending on the release test results for a particular formulation, it may be better to keep some of the amino acids, such as tyrosine, as immediate release amino acids.

[00121] Several strategies are available to control the release of amino acids from the formulation, depending on the desired pharmacokinetic profile. These strategies will typically involve non-pH dependent excipients or additives so that the formulation releases amino acids regardless of their location in the GI tract or pH dependent excipients or additives to delay the release of amino acids until the formulation reaches the higher pH environment of the small intestine.

[00122] Non-pH dependent sustained release agents/coatings that may be included in the composition include, but are not limited to, ethylcellulose, cellulose acetate, vinyl acetate/vinyl chloride copolymers, acrylate/methacrylate copolymers, polyethylene oxide, hydroxypropylmethylcellulose, carrageenan, alginic acid and its salts, hydroxyethylcellulose, hydroxypropylcellulose, xanthan gum, karaya gum, acacia gum, tragacanth gum, locust bean gum, guar gum, sodium carboxymethylcellulose, methylcellulose, wax beeswax, carnauba wax, cetyl alcohol, hydrogenated vegetable oils, stearyl alcohol, fatty acids and their esters or derivatives including, for example, glyceryl behenate and glyceryl dipalmitostearate, xanthan gum, starches and starch derivatives such as corn acetate .

[00123] In general, at least one non-pH dependent modified release agent is present in the composition in an amount of from about 1% by weight or 5% by weight to about 50% by weight or 25% by weight, depending on preferably from about 1% by weight to about 30% by weight. It should be understood, however, that the scope of the present invention is not limited to any specific non-pH dependent sustained release agents.

[00124] The pH-dependent agents that increase the release rate of amino acids from the formulation at a pH greater than 5.5 include, but are not limited to, polymers that expand at a pH in excess of 5.5, enteric agents for coating the amino acid and agents that increase the solubility of the amino acids at a pH greater than 5.5, maintaining an acidic microenvironment in the formulation, for example an organic acid. Suitable organic acids include, for example, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid, citric acid, benzoic acid and carbonic acid. Any combination of these strategies can be employed to promote amino acid release at pH greater than 5.5. At least one pH-dependent agent is typically present in the composition in an amount of from about 0.5% by weight to about 40% by weight, preferably from about 1% by weight to about 20% by weight. %.

[00125] Polymers that expand at a pH greater than 5.5 include, but are not limited to, acrylic acid copolymers, methacrylic acid copolymers (including, for example, those sold under the brand name Eudragit®), sodium alginate , carrageenan, alginic acid, pectin and sodium carboxymethylcellulose.

[00126] Enteric agents include, but are not limited to, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, methacrylic acid copolymers, cellulose acetate trimellitate, hydroxypropylmethylcellulose acetate, succinate, shellac and zein.

[00127] Agents that increase the solubility of amino acids at a pH greater than 5.5 include, but are not limited to, organic acids. These organic acids maintain an acidic microenvironment in the tablet and include, but are not limited to, citric acid, fumaric acid, tartaric acid, adipic acid, glucono delta-lactone, and malic acid.

[00128] The compositions of the present invention are typically prepared first by wet granulating, coating and, if a biscuit is desirable, subsequently compressed into a biscuit dosage form after mixing with other suitable excipients or additives, such as lubricants, anti-adherents and agents disintegrants. Flavoring agents can be added during the granulation steps so that they are part of the granules or "intragranular", or subsequently mixed with the granulates, prior to compression of the granules and, optionally, prior to being compressed into a tablet/biscuit.

[00129] In the wet granulation method, at least one amino acid and other ingredients are granulated with a granulation fluid (for example, isopropyl alcohol, ethyl alcohol, trichloromethane or water) in a planetary mixer, high shear mixer or bed granulator fluidized. The binding agents can be contained in the granulation fluid or they can be in the dry mix. The wet granules are dried in an oven or fluidized bed dryer and then sieved through a suitable screen to obtain free flowing granules which can then be coated with functional excipients or additives capable of providing a modified or extended or delayed release profile.

[00130] Bulking agents can be included in the formulation of granules or cookies and include, but are not limited to, microcrystalline cellulose, mannitol, xylitol, dicalcium phosphate, calcium sulfate, starches, lactose, sucrose, dextrose, sorbitol, fructose and cellulose powder. When present, the bulking agent can be present in the composition in an amount from about 5% by weight to about 90% by weight, preferably from about 10% by weight to about 50% by weight. %.

[00131] Disintegrants that may be included in the formulations include, but are not limited to, microcrystalline cellulose, starches, crospovidone, sodium starch glycollate and croscarmellose sodium. When present, the disintegrating agent can be present in the composition in an amount from about 0.5% by weight to about 30% by weight, preferably from about 1% by weight to about 15% by weight. %.

[00132] Non-stick and glidants that can be used in the composition include, but are not limited to, talc, corn starch, silicon dioxide, sodium lauryl sulfate and metallic stearates. When present, the non-stick or glidant can be present in the composition in an amount from about 0.2% by weight to about 15% by weight, preferably from about 0.5% by weight to about 5% by weight. . %.

[00133] Lubricants that can be used in the composition include, but are not limited to, magnesium stearate, calcium stearate, sodium stearate, stearic acid, sodium stearyl fumarate, hydrogenated cottonseed oil, talc and waxes, including but not limited to beeswax, carnauba wax, cetyl alcohol, glyceryl stearate, glyceryl palmitate, glyceryl behenate, hydrogenated vegetable oils and stearyl alcohol. When present, the lubricant can be present in an amount from about 0.2% by weight to about 20% by weight, preferably from about 0.5% by weight to about 5% by weight. %.

[00134] Binding agents ("binders") that may be used include, but are not limited to, polyvinylpyrrolidone, starch, methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sucrose solution, dextrose solution, guar gum, xanthan gum, acacia, tragacanth, locust bean gum and sodium alginate or any other salt of alginic acid. When present, the binding agent can be present in the composition in an amount from about 0.2% by weight to about 10% by weight, preferably from about 0.5% by weight to about 5% by weight. . %.

[00135] Glazing agents that may be included in the composition include, but are not limited to, ethylcellulose, cellulose acetate, vinyl acetate/vinyl chloride copolymers, acrylate/methacrylate copolymers, polyethylene oxide, hydroxypropylmethylcellulose, carrageenan , alginic acid and its salts, hydroxyethylcellulose, hydroxypropylcellulose, karaya gum, acacia gum, tragacanth gum, locust bean gum, guar gum, sodium carboxymethylcellulose, methylcellulose, beeswax, carnauba wax, cetyl alcohol, vegetable oils hydrogenated compounds, stearyl alcohol, fatty acids and their esters or derivatives, including glyceryl behenates and glyceryl dipalmitostearate, xanthan gum, starches and starch derivatives, such as corn acetate.

[00136] Flavor Masking Materials a. Amino acids are inherently bitter tasting and, in one embodiment of the present invention, these bitter amino acids are microencapsulated with a taste masking material. Materials useful for masking the taste of pharmaceutical formulations include materials capable of microencapsulating the amino acid, thereby protecting the senses from its bitter taste. The taste masking materials of the present invention provide for superior pharmaceutical formulations, for example, creating a more palatable pharmaceutical formulation compared to pharmaceutical formulations and/or creating a dosage form that requires less of traditional taste or flavor masking agents.

[00137] Taste masking materials include, for example, hydroxypropyl ethers of cellulose (HPC); low-substituted hydroxypropyl ethers (L-HPC); hydroxypropylmethyl ethers of cellulose (HPMC); methylcellulose polymers; ethylcelluloses (EC) and mixtures thereof; Polyvinyl Alcohol (PVA); hydroxyethylcelluloses; carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC); copolymers of polyvinyl alcohol and polyethylene glycol; monoglycerides, triglycerides, polyethylene glycols, modified food starch, acrylic polymers and blends of acrylic polymers with cellulose ethers; cellulose acetate phthalate; sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins and mixtures of these materials.

[00138] In addition to microencapsulating the amino acids with a taste masking material or a material that improves the shelf life of the formulation as described herein, the pharmaceutical formulations of the present invention may also comprise one or more flavoring agents. "Flavoring agents" or "sweeteners" useful in the pharmaceutical formulations of the present invention include, for example, acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream, berry, blackcurrant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus juices, citrus cream, cotton candy, cocoa, cola, fresh cherry, fresh citrus, cyclamate, dextrose, eucalyptus, eugenol, fructose, fruit juice, ginger, glycyrrhinoate, glycyrrhiza (licorice) syrup, grapefruit, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glycyrrhizinate, maltol, mannitol, maple, marshmallow, menthol, cream of mint , mixed berry, neohesperidin DC, neotame, orange, pear, peach, mint, peppermint cream, raspberry, root, rum, saccharin, safrole, sorbitol, mint, cream mint, strawberry, strawberry cream, stevia, sucralose , sucrose, saccharin sodium, saccharin, aspartame, neotame, acesulfame potassium, mannitol, talin, xylitol, sucralose, sorbitol, Swiss cream, tagatose, mandarin orange, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry, winter seed , xylitol or any combination of these flavoring ingredients, e.g. anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, cream of orange, vanilla-mint and their mixtures. In other embodiments, sodium chloride is incorporated into the pharmaceutical formulation.

[00139] Based on the amino acids and excipients or additives, one skilled in the art could determine the best combination of flavors to provide the ideal tasting product for consumer demand and compliance. See, for example, Roy et al., Modifying Bitterness: Mechanism, Ingredients, and Applications (1997).

Microencapsulation Methods

[00140] The amino acid can be microencapsulated by methods known to one skilled in the art. Such known methods include, for example, spray drying processes, disk spinning processes, hot melt processes, spray cooling methods, fluidized bed, electrostatic deposition, centrifugal extrusion, rotational suspension separation, liquid gas polymerization or solid-gas interface, pressure extrusion or solvent extraction bath spraying. In addition, various chemical techniques, for example, complex coacervation, solvent evaporation, polymer-polymer incompatibility, interfacial polymerization in liquid medium, polymerization in situ, drying in liquid and desolvation in liquid medium can also be used. In addition, other methods such as dry granulation (i.e. roller compaction and aggregative fluidization), extrusion/spheronization or nanoparticle coating may also be used.

[00141] When microencapsulated, amino acids can be present in particle sizes ranging from 1 micron to 1000 microns, 5 microns to 200 microns, or 10 microns to 100 microns.

[00142] Spray drying is often more readily available to scale up to a commercial scale. In various embodiments, the material used in the dry spray encapsulation process is emulsified or dispersed in the core material in a concentrated form, for example, 10-60% solids. In some embodiments of the present invention, solid loading is between about 10-20%, or between about 10-40%, or between about 40-60%. The microencapsulation material is, in one embodiment, emulsified into droplets of about 1 to 3 µm. In other embodiments, the microencapsulation material is emulsified into droplets of about 1 to 200 µm, or to droplets of about 1 to 100 µm. Once a dispersion of amino acid and encapsulating material is obtained, the emulsion is fed as droplets into the heated chamber of the spray dryer. In some embodiments, the droplets are sprayed into the chamber or centrifuged on a rotating disk. The microspheres are then dried in the heated chamber and dropped to the bottom of the spray drying chamber where they are collected.

[00143] Coacervation involves the microencapsulation of materials such as active pharmaceutical ingredients and involves a three-part process of particle or droplet formation, coascervate wall formation, and capsule isolation. This method can produce very small particle size microcapsules (10-70 microns).

[00144] Extrusion/spheronization is another method involving the wet mass of active pharmaceutical ingredients, followed by extruding the wet mass through a perforated plate to produce short cylindrical rods. These rods are subsequently placed in a rapidly rotating spheronizer to shape the cylindrical rods into uniform spheres. The spheres are subsequently dried using a fluidized bed dryer and then coated with a functional coating using a fluidized bed equipped with a Wurster insert and spray nozzle. This method produces smooth, even spheres that are ideal for receiving a functional coating. Drug loadings of up to 80% are possible (depending on drug characteristics).

Dry Coating Methods

[00145] In addition to microencapsulation, the stability or release time of the amino acids used in the present invention can be increased by alternative methods such as dry coating and nanoparticle coating. Dry coating involves the formation of coated amino acid granules which are then mixed with other components. Dry granulation is achieved by forming dense compacts which are subsequently milled to a desired particle size and then blended with other components of the pharmaceutical composition. Dry granulation and nanoparticle coating can provide greater stability and taste masking characteristics to the active pharmaceutical by diluting and isolating such components in a granular matrix of compatible ingredients that can increase the shelf life of amino acid products as well as mask the Bitter taste if sweeteners or flavorings are used in the coating material.

[00146] The typical technique for dry granulation is the use of aggregative fluidization or roller compaction. During the slugging process, the dry powders are compressed using a conventional tableting machine, or more generally, a large heavy duty rotary press. The resulting compacts or “ingots” are then milled to a desired particle size. Roller compaction is a gentler alternative method; the powder mixture being squeezed between two rollers to form a compressed sheet. The leaf is normally weak and brittle and immediately breaks into flakes. These flakes need gentler handling to break them down into granules, and this can usually be achieved by screening alone. Parikh, D.M., Handbook of Pharmaceutical Granulation Technology, (Marcel Dekker ed. 1997).

Additional Ingredients

[00147] Other ingredients may also be incorporated into the formulations of the present invention, including vitamins, minerals, fats, fatty acids such as DHA, EPA and ARA (arachidonic acid), carbohydrates and other compounds, preferably in therapeutically effective amounts. Suitable minerals include, for example, calcium, chloride, chromium, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sulphur, sodium and zinc. Examples of suitable vitamins include Vitamin A (Retinol, Retinyl Palmitate Acetate, Beta-Carotene), Thiamine (Vitamin B1), Riboflavin (Vitamin B2), Niacin (Vitamin B3, Nicotinic Acid), Panthenoic Acid (Vitamin B5), Vitamin B6 (pyridoxine), Vitamin B12 (cobalamin) Biotin, Vitamin C (ascorbic acid), Vitamin D (calciferol), Vitamin E (alpha-tocopherol), Folic Acid (folate) and Vitamin K (phylloquinone, menaquinone).

[00148] In one embodiment, the formulation comprises one or more, five or more, 10 or more, or all of the following nutrients in addition to the amino acids present in the formulation, in therapeutically effective amounts: choline, inositol, vitamin A, vitamin D, vitamin E , vitamin K, vitamin C, thiamine, riboflavin, niacin, vitamin B6, folate, vitamin B12, biotin, pantothenic acid, potassium, calcium, magnesium, iron, zinc, copper, manganese, selenium, chromium, molybdenum, iodine, sodium, phosphorus, chloride, docosahexaenoic acid, arachidonic acid and lutein.

[00149] Of course, it will be understood that any of the foregoing nutrients may be present in a form well known to those skilled in the art which provide similar nutritional value, such as salts and chelates and esters and other derivatives of the foregoing nutrients. Table H describes an example of various nutrients, effective doses of nutrients, and nutrient derivatives capable of incorporating the formulations of the present invention. The formulation can include any one or combination of these nutrients. Table H

final formulation

[00150] In a particularly preferred embodiment, the formulation comprises granules of amino acids coated with one or more release-modifying excipients, also mentioned in this document as "coating means to delay the rate of amino acid release" or "coating means to achieve the indicated release rate”. Granulates can be prepared by wet or dry granulation techniques, as discussed above, but are preferably made by wet granulation. They are also preferably confined to a specific size range, such as 0.1-3 mm, 0.5-2.0 mm, 0.5-1.0 mm, 0.5-2.0 mm or 1 .0-2.0mm. Each amino acid can be contained in its own granules, but modified-release amino acids are preferably mixed within the granules.

[00151] The modified release properties are preferably obtained with a suitable release modifier coating or coatings applied to the granulate, in an amount of 1% by weight to 30% by weight, or from 5% by weight to 25% by weight based on the weight of amino acids. Suitable release-delaying excipients for coating are described elsewhere in this document, but a preferred composition comprises a coating from 1% by weight to 15% by weight, from 2% by weight to 10% by weight, or from 5% by weight to 7.5% by weight ethylcellulose based on the weight of amino acids. Another preferred composition comprises a first coating of ethylcellulose (as described above) and a second coating of 5% to 15% or about 10% by weight of glyceryl dibehenate based on the weight of amino acids.

EXAMPLES

[00152] The following examples are presented in order to provide those skilled in the art with a complete description and description of how the methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope than the inventors consider their invention. Efforts have been made to ensure accuracy with respect to numbers (eg amounts, temperature, etc.), but some errors and deviations must be considered.

[00153] Example 1 - Exemplary Amino Acid Mixtures for Phenylketonuria 5

[00154] Table 1 lists four separate representative amino acid mixtures for the dietary management of phenylketonuria, on a percentage basis. Table 1

Example 2 - Exemplary Powder Formulations and Manufacturing Process

1 Produto 1 sem Sabor; Produto 2 sem Sabor; Produto 3 Sabor de Caramelo; Produto 4 Sabor de Baunilha; Produto 5 Sabor de Banana[00155] The following table describes the formulations of five separate products containing 18 amino acids (excluding methionine) using 5 Plasdone K26/32 as a binding agent and a taste masking agent (Products 2, 3, 4 and 5) . The reference formulation (Product 1) is without Plasdone K26/32 and without flavors. Table 2a. Composition of Products 1, 2, 3, 4 and 5

1 Product 1 without Flavor; Product 2 Unflavored; Product 3 Caramel flavor; Product 4 Vanilla flavor; Product 5 Banana Flavor

[00156] Each of the formulations was manufactured according to the following general method. Step 1 Manufacturing Equipment: Scales of Various Types High Sharing Mixer: Diosna Laboratory P1/6 Mixer Sieves Static Oven Mixing Machine Amino Acid Mix Product 1

[00157] Precisely weigh the amino acids and mix for 20 minutes with the mixing machine.

Unflavored granulated preparation Product 2

[00158] Precisely weigh the amino acids and mix for 20 minutes with the mixing machine. Transfer the mixture to the Diosna and blend for 1 minute at a blade speed of 250 rpm. Wet the mixture with an alcoholic solution of Plasdone K26/32 (10% in ethanol solution - 10 g to 100 g of Ethanol). After one minute and 30 seconds, add the solution and granulate and mix for three minutes at a blade speed of 250 rpm and a pulsator speed of 500 rpm. Unload the granules and pass through a sieve with a width of 0.8 mm. Dry in a static oven for about four hours at 40°C. Unload and pass again through a sieve with a width of 0.8 mm.

Preparation of flavored granules Products 3, 4 and 5

[00159] Precisely weigh the amino acids and mix for 20 minutes with the mixing machine. Transfer the mixture to the Diosna, and mix for 1 minute and 30 seconds with a blade speed of 250 rpm. Moisten the mixture with an alcoholic solution of Plasdone K26/32 (10% content in ethanol solution - 10 g for 100 g AA) and taste. The amount of flavors added to the alcoholic solution of Plasdone K26/32 is as follows:

[00160] -1.13 g of 58% Caramel in Ethanol (for Product 3)

[00161] -1.13 g of 60% Vanilla content in Ethanol (for Product 4)

[00162] -1.46 g of 14% Banana content in Ethanol (for Product 5)

[00163] After one minute and 30 seconds, mix the solution and granulate and mix for three minutes at a blade speed of 250 rpm and a pulsator speed of 500 rpm. Unload the granules and pass through a sieve with a width of 0.8 mm. Dry in a static oven for about four hours at 40°C. Unload and pass the mixture through a sieve with a width of 0.8 mm.

Step 2

[00164] Preparation of methionine using a Plasdone K26/32 as binding agent and flavor masking agent plus a very small amount of Caramel flavor using a manufacturing method similar to the method used for Products 3, 4 and 5 reported in step 1. Table 2b. Composition Granulated L-Methionine Product 6

Step 3

2,115 g de L-Metionina granular no Produto 7 correspondendo a 2,050 g de Metionina[00165] Preparation of the final formulation by mixing the powder/granules from Step 1 plus the granules from Step 2 in the amount reported below. Table 2c. Composition of the final formulation of Products 7, 8, 9, 10 and 11

2.115 g of granular L-Methionine in Product 7 corresponding to 2.050 g of Methionine

[00166] 2 Product 7 No Flavor (only a small amount of caramel derived from Methionine powder), Product 8 No Flavor (only a small amount of caramel derived from Methionine powder), Product 9 Caramel Flavor, Product 10 Caramel and Vanilla, Product 11 Caramel and Banana Flavor

[00167] All the products mentioned above were manufactured by mixing 10 the powder or granules of Products 2, 3, 4 and 5 with the granules of Product 6.

Example 3 - Taste Test

Legenda: - Odor/Sabor: 0 = ruim 5 = bom - Persistência (mau gosto na boca): 0 = baixa persistência 5 = longa persistência - Dispersibilidade: 0 = ruim 5 = bom[00168] In the final formulations reported in Example 2 (Products 8, 9, 10 and 11), the following test was performed on 6 healthy adults. Reported results are average values: Table 3. Taste Test Results

Legend: - Odor/Taste: 0 = bad 5 = good - Persistence (bad taste in the mouth): 0 = low persistence 5 = long persistence - Dispersibility: 0 = bad 5 = good

[00169] As the above data shows, the formulations of the present invention are better than the marketed reference product and better than the formulation without Plasdone K26/32 in terms of dispersibility and taste.

Example 4 - Exemplary Cookie Formulations and Manufacturing Process

1 2,115 g de L-Metionina granulada correspondendo a 2.050 g de Metionina 2 Produto 1 Sabor de Caramelo, Produto 2 Sabor de Baunilha, Produto 3 Sabor de Banana[00170] Various biscuit formulations were prepared starting with Granulated Products similar to those reported in Example 2 (slightly altering the amounts of flavors). Table 4a

1 2.115 g of granulated L-Methionine corresponding to 2.050 g of Methionine 2 Product 1 Caramel Flavor, Product 2 Vanilla Flavor, Product 3 Banana Flavor

1 Produto 4 Sabor de Caramelo, Produto 5 Sabor de Baunilha, Produto 6 Sabor de banana 2 2,115 g de L-Metionina granulado correspondendo a 2,050 g de Metionina[00171] To the above-mentioned products sucrose, silicon dioxide and glyceryl dibehenate were added through a simple mixing process. The final formulations were as follows: Table 4b

1 Product 4 Caramel Flavor, Product 5 Vanilla Flavor, Product 6 Banana Flavor 2 2.115 g granulated L-Methionine corresponding to 2.050 g Methionine

1 2.115 g de L-Metionina granulado correspondem a 2,050 g de Metionina Exemplo 5 - Teste de Sabor da Formulação de Biscoito[00172] The tablets were prepared using a 5 monopunch tablet machine with an average weight between 5.5 and 6.2 g. To correct some puncture adhesion problems, a new formulation was prepared from Product 1: Table 4-C

1 2,115 g of granulated L-Methionine corresponds to 2,050 g of Methionine Example 5 - Biscuit Formulation Taste Test

Legenda: -Odor-Sabor-Aspecto: 0 = mau 5 = bom -Persistência (mau gosto na boca): 0 = baixa persistência 5 = persistência longa -Dispersibilidade: 0 = ruim 5 = bom[00173] Products 4, 5 and 6 reported in Example 4 were tested for odor and taste on 6 healthy adults. Reported results are mean values. Table 5

Legend: -Odor-Taste-Appearance: 0 = bad 5 = good -Persistence (bad taste in the mouth): 0 = low persistence 5 = long persistence -Dispersibility: 0 = bad 5 = good

Example 6 - Preparation of Modified Release Formulations Mixture Preparation

[00174] Weigh and sieve each individual amino acid, diluent (if used) and HPMC and mix for 20 minutes in a suitable mixing machine.

Preparation of unflavored granules

[00175] Transfer the mixture to the Diosna mixer, mixing for 1 minute with the blade speed set to 250 rpm. Moisten the mixture with an alcoholic solution of EC (15% content). After one minute and 30 seconds, add the solution and granulate for three minutes at a blade speed of 250 rpm and a pulsator speed of 500 rpm. Unload the granules and pass through a sieve with a width of 0.63 mm. Dry in a static oven for about four hours at 45°C. Unload and pass through a sieve with a width of 0.8 mm.

[00176] It is also possible to process 4-5 amino acids into granules at a time, and mix all the granulated amino acids together to produce the final product. Some examples of potential formulations are reported in Example 1, Table 1.

Example 7 - Modified Release Formulation

1 correspondente a 3 g de CE[00177] The following formula was prepared to determine the amount of hydroxypropyl methylcellulose (HPMC) and ethylcellulose (EC) required to modify the release of tryptophan in a Dissolution Test. Table 6. Product Composition

1 corresponding to 3 g of EC

Mixture Preparation

[00178] Weigh and sift the single amino acids, mannitol and HPMC and mix for 20 minutes in a suitable container with the mixing machine. Preparation of unflavored granules

[00179] Transfer the mixture to the Diosna mixer, mixing for 1 minute with the blade speed at 250 rpm. Wet the mixture with an alcoholic solution of EC (ethylcellulose) (15% strength). After one minute and 30 seconds add the solution and granulate for three minutes with the blade speed at 250 rpm and the pulsator speed at 500 rpm. Unload the granules and pass through a sieve with a width of 0.63 mm. Dry in a static oven for about four hours at 45°C. Unload and pass through a sieve with a width of 0.8 mm.

[00180] The formulation was evaluated under the dissolution conditions described below to determine the rate of tryptophan release from the formulation (pH 1.2 for 2 hours, increased to pH 6.8 after 2 hours) under basket conditions at 50 and 100 rpm versus a simple blend of uncoated AA. The Dissolution Test results are reported in Figure 3. The analysis was performed by HPLC.

Dissolution Test Conditions

[00181] Device:^i^iPaddles

[00182] Temperature: 37°C ± 5°C

[00183] Medium: 0.1 N Hydrochloric Acid

[00184] pH adjustment: 0.2 M solution of trisodium phosphate dodecahydrate. Medium volume: 450 ml of 0.1 N HCl. After 120 minutes, adjust the dissolution medium to pH 6.8 ± 0.05 (according to European Pharmacopoeia 2.9.3 Delayed Release Solid Dosage Forms Method A ).

[00185] Speed: 50 rpm or 100 rpm

[00186] Sampling time: 5 and 15 minutes for uncoated formulation

[00187] 5, 15, 30, 60, 120, 125, 135, 150, 180 minutes for coated formulation (after 120 minutes, the pH of the medium was changed to pH 6.8)

[00188] Vessel concentration: about 90 μg/ml (a fraction of the delayed formulation powder remains as surfactant during the dissolution test)

Example 8 - Modified Release Formulation without Mannitol

[00189] The following formulation was prepared according to the procedures set out in Example 7 and subjected to dissolution tests under the conditions described below. The tryptophan release rate from the formulation is shown in Figure 4. The analysis was performed by HPLC. Table 7. Product Composition

[00190] Dissolution Test Conditions

[00191] Device:^i^iPaddles

[00192] Temperature: 37°C ± 5°C

[00193] Medium: 0.1 N Hydrochloric Acid 54/86

[00194] Average volume: 450ml

[00195] Speed: 50 rpm

[00196] Sampling times: 5', 15', 30', 60', 120', 180'

[00197] Vessel concentration: about 90μg/ml (a fraction of the delayed formulation powder remains as surfactant during the dissolution test)

Example 9 - Modified Release Formulation without Mannitol or HPMC (Methocel 4KM)

1correspondendo a 67 g de uma solução de etanol de Etilcelulose a 15%[00198] The following formulation was prepared according to the procedures set out in Example 7 and subjected to dissolution tests under the conditions described below. The tryptophan release rate from the formulation is shown in Figure 5. The analysis was performed by HPLC. Table 8. Product Composition

1 corresponding to 67 g of a 15% Ethylcellulose ethanol solution

Mixture Preparation

[00199] Weigh and sift the individual amino acids and mix for 20 minutes in a suitable container with the mixing machine. Preparation of unflavored granules

[00200] Transfer the mixture to the Diosna mixer, mixing for 1 minute with the blade speed at 250 rpm. Wet the mixture with an alcoholic solution of EC (ethylcellulose) (15% strength). After one minute and 30 seconds, add the solution and granulate for three minutes with the blade speed at 500 rpm and the pulsator speed at 1500 rpm. Unload the granules and pass through a sieve with a width of 0.8 mm. Dry in a static oven for about 5 hours and 30 minutes at 45°C. Unload and pass through a sieve with a width of 1.4 mm.

[00201] The formulation was evaluated under the dissolution conditions described below to determine the tryptophan release rate of the formulation (pH 1.2) under basket conditions at 50 rpm. Dissolution test results are reported in Figure 5.

[00202] Dissolution Test Conditions

[00203] Device: Basket

[00204] Temperature: 37°C ± 5°C

[00205] Medium: 0.1 N Hydrochloric Acid

[00206] Average volume: 450ml

[00207] Speed: 50 rpm

[00208] Sampling Times: 5', 15', 30', 60', 120', 180', 240', 300' and 360'

[00209] Vessel concentration: about 90 μg/ml (a fraction of the delayed formulation powder remains as surfactant during the dissolution test)

Example 10 - Modified Release Formulation in 19 amino acids coated with ethylcellulose and glyceryl dibehenate

1 correspondendo a 4,84 g de base de L-lisina 2 correspondendo a 2,49 g de base de L-Histidina 3 correspondendo a 98,21 g se a L-lisina e a L-histidina forem consideradas como base[00210] The following formulation was prepared according to the procedures described below. Table 9. Product Composition

1 corresponding to 4.84 g of L-lysine base 2 corresponding to 2.49 g of L-Histidine base 3 corresponding to 98.21 g if L-lysine and L-histidine are considered as base

[00211] Manufacturing equipment used to prepare the Product

[00212] Equipment used for production:

[00213] Scales of various types

[00214] Configuration ProCepT Mipro 900ml

[00215] Sieves

[00216] Static Oven

[00217] ProCepT fluidized bed bottom-up coating (melting nozzle) (or any other suitable apparatus for granular melt coating)

Mixture of Amino Acids

[00218] Mix all the amino acids in a suitable device

Preparation of Granules

[00219] Transfer the amino acid mixture to the ProCepT Mipro, add the granulation solution consisting of a 2% sodium alginate water solution and granulate. After granulation, sieve the granulate; use the fraction with 0.5 mm <PSD <1 mm for the next step.

Ethyl Cellulose Coating (Polymer Coating)

[00220] Transfer the filtered granulated fraction into the fluidized bed apparatus and place it with a 15% (w/w) methanol solution of ethylcellulose. The total amount of added polymer should equal approximately 5.00% of the weight of the granulate.

Glyceryl Dibehenate Coating (Hot Melt Coating)

[00221] Transfer the ethylcellulose coated granulate into the fluidized bed apparatus and coat it with melted glyceryl dibehenate. The total amount of added polymer should equal approximately 15.00% of the granulated weight.

[00222] Product dissolution test

[00223] Dissolution medium: pH 1.2 ± 0.1 medium (0.1 N hydrochloric acid)

[00224] Apparatus: Paddle Apparatus (Apparatus 2, USP <711> modified); 50 rpm

[00225] Time: 30 - 60 - 120 -180 minutes

[00226] Temperature: 37 ± 0.5°C

[00227] Average Volume: 500 mL

[00228] Sample: 2.0 g of amino acid mixture

[00229] Ponderal dissolution test: 30-60-120-180 minutes a. The results of the Weight Dissolution Test take into account that ethylcellulose and glyceryl dibehenate are not soluble in the HCl solution. At 30-60-120-180 minutes, the dissolution medium was filtered through a vacuum filter paper. The filtered powder and filter were dried for ± 4 hours in a vacuum oven at 50°C until constant weight was achieved and the samples were weighed. For the difference, the amount of amino acids released was calculated and reported in Figure 6. Example 11 - Manufacturing and Dissolution Test of Free Amino Acid Formulation and Modified Release Formulations

[00230] Table 10a reports the qualitative and quantitative composition of a representative AA amino acid formulation mixture (bkT037 / 71). Table 10a

[00231] Manufacturing Method (general)

[00232] Manufacturing equipment used to prepare the Product

[00233] Scales of various types

[00234] Sieves

[00235] Static Oven

[00236] Mixing Machine

[00237] Sift and mix all the amino acids in a suitable device

[00238] The amino acids are mechanically passed through a 350μm stainless steel sieve and mixed with a mixer. for 30 minutes in

[00239] Product dissolution test

[00240] Dissolution medium: pH 1.2 ± 0.1 medium (0.1 N hydrochloric acid)

[00241] Apparatus: Paddle Apparatus (Apparatus 2, modified USP <711>) 50 rpm

[00242] Time: 30 – 60 minutes

[00243] Temperature: 37 ± 0.5°C

[00244] Average Volume: 500 mL

[00245] Sample: 2.0 g of amino acid mixture

[00246] Time: 30 - 60 minutes Temperature: 37 ± 0.5°C Medium Volume: 500 mL Sample: 2.0 g of amino acid mixture Dissolution Test Weight: 30-60-120-180 minutes

[00247] Each point of time has its own dissolution vessel. At 30-60-120180 minutes, the dissolution medium was filtered through a vacuum filter paper. The filtered powder and filter were dried for ± 4 hours in a vacuum oven at 50°C until constant weight was achieved and the samples were weighed. For the difference, the released amount of amino acids was calculated.

Results

[00248] The results of the weight dissolution test are reported in Table 10b and figure 7. Table 10b

[00249] Analytical conditions: • HPLC: Agilent 1200 series • Detector: Agilent 1200 series fluorimeter • Chromatographic column: YMC-Triart C18 12nm S (250 x 4.6 mm - 5 μm)

[00250] The amino acids are analyzed after extraction (in different ways) and derivatization with FMOC (9-Fluorenyl methyl chloroformate). Note: Carnitine is analyzed by LC/MS.

[00251] Dissolution test results for Mixture AA (bkT037/71) are reported in Table 10c and Figure 8. Table 10c

[00252] Table 10d informs the quantitative and qualitative formulations of four different batches of amino acid particles coated with ethylcellulose. Table 10d

[00253] Manufacturing Method (general)

[00254] Manufacturing equipment used to prepare the Product

[00255] Scales of various types

[00256] Configuration ProCepT Mipro 900ml

[00257] Sieves

[00258] Static Oven

[00259] ProCepT fluidized bed bottom-up coating (melt nozzle) (or any other apparatus suitable for granular melt coating)

Mixture of Amino Acids

[00260] Mix all the amino acids in a suitable device.

Preparation of Granules

[00261] Transfer the amino acid mixture to the ProCepT Mipro, add the granulation solution composed of a 2% sodium alginate water solution and granulate. After granulation, sieve the granulate; use the fraction with 0.5 mm < PSD < 1 mm and 0.5 mm < PSD < 1.5 mm for the next step.

Ethyl Cellulose Coating (Polymer Coating)

[00262] Transfer the sieved fraction of granulate to the fluidized bed apparatus and coat it with a 15% (w/w) methanol solution of ethylcellulose. The total amount of added polymer should equal approximately 5.00% and 7.50% of the granulated weight.

[00263] Product Dissolution Test

Dissolution Conditions:

[00264] Dissolution medium: pH 1.2 ± 0.1 medium (0.1 N hydrochloric acid)

[00265] Apparatus: Paddle Apparatus (Apparatus 2. USP <711> modified); 50 rpm

[00266] Time: 30 - 60 - 120 -180 - 240 minutes

[00267] Temperature: 37 ± 0.5°C

[00268] Average Volume: 500 mL

[00269] Sample: 2.0 g of amino acid mixture

[00270] Weight Dissolution Test: Sampling Time: 30-60- 120-180 minutes

[00271] Each point of time has its own dissolution vessel.

[00272] At 30-60-120-180 minutes, the dissolution medium was filtered through a paper filter under vacuum. The filtered powder and filter were dried for ± 4 hours in a vacuum oven at 50°C until constant weight was achieved and the samples were weighed. For the difference, the released amount of amino acids was calculated. Results a. The results of the weight dissolution test are reported in Table 10e and Figure 9. Table 10e

[00273] Single amino acid release analyzes were performed only on APR Batch 2 and APR Batch 4 prototypes.

Analytical conditions:

[00274] HPLC: Agilent 1200 series

[00275] Detector: Agilent 1200 Series Fluorimeter

[00276] Chromatographic Column: YMC-Triart C18 12nm S (250 x 4.6 mm - 5 μm)

[00277] The amino acids are analyzed after extraction (in different ways) and derivatization with FMOC (9-Fluorenyl methyl chloroformate). Note: Carnitine is analyzed by LC/MS.

Results (Unique Amino Acids)

[00278] Dissolution test results for APR Lot 2 (bkT037/72-2) are reported in Table 10f and Figure 10. Table 10f

[00279] Dissolution test results for APR Lot 4 (bkT037/72-4) are reported in Table 10g and Figure 11. Table 10g

Example 12 - Fabrication and Dissolution Testing of Modified Release Formulations Using Ethyl Cellulose and Glyceryl Dibehenate as Coating Agents

[00280] Table 11a informs the qualitative and quantitative composition of the separate formulation of 4 amino acids coated with ethylcellulose and glyceryl dibehenate as release rate modifying agents. Table 11a

[00281] Manufacturing Method (general) Manufacturing equipment used to prepare the Product

[00282] Scales of various types

[00283] Configuration ProCepT Mipro 900ml

[00284] Sieves

[00285] Static Oven

[00286] ProCepT fluidized bed bottom-up coating (melt nozzle) (or any other suitable apparatus for granular melt coating)

Mixture of Amino Acids

[00287] Mix all the amino acids in a suitable device

Preparation of Granules

[00288] Transfer the amino acid mixture to the ProCepT Mipro, add the granulation solution consisting of a 2% sodium alginate water solution and granulate. After granulation, sieve the granulate; use the fraction with 0.5 mm < PSD < 1 mm or 0.5 mm < PSD < 1.6 mm for the next step.

Ethyl Cellulose Coating (Polymer Coating)

[00289] Transfer the filtered granulated fraction into the fluidized bed apparatus and place it with a 15% (w/w) methanol solution of ethylcellulose. The total amount of added polymer should equal approximately 5.00% and 7.50% of the granulated weight.

Glyceryl Dibehenate Coating (Hot Melt Coating)

[00290] Transfer the ethylcellulose coated granulate into the fluidized bed apparatus and coat it with melted glyceryl dibehenate. The total amount of added polymer should equal approximately 10.00% of the granulated weight.

Analytical Conditions:

[00291] Product dissolution test

[00292] Dissolution medium: pH 1.2 ± 0.1 medium (0.1 N hydrochloric acid)

[00293] Apparatus: Paddle Apparatus (Apparatus 2. USP <711> modified); 50 rpm

[00294] Time: 30 - 60 - 120-180 - 240 minutes

[00295] Temperature: 37 ± 0.5°C

[00296] Average Volume: 500 mL

[00297] Sample: 2.0 g of amino acid mixture

[00298] Weight Dissolution Test: Sampling Time: 30-60 120-180 minutes i. At 30-60-120-180 minutes, the dissolution medium was filtered through a vacuum filter paper. The filtered powder and filter were dried for ± 4 hours in a vacuum oven at 50°C until constant weight was achieved and the samples were weighed. For the difference, the released amount of amino acids was calculated.

[00299] Results

[00300] The results of the weight dissolution test are reported in Table 11b and Figure 12. Table 11b

[00301] The analyzes were performed on APR Lot 5 and APR Lot 7 prototypes

Analytical conditions:

[00302] HPLC: Agilent 1200 series

[00303] Detector: Agilent 1200 Series Fluorimeter

[00304] Chromatographic Column: YMC-Triart C18 12nm S (250 x 4.6 mm - 5 μm)

[00305] The amino acids are analyzed after derivatization with FMOC (9-Fluorenyl methyl chloroformate)

[00306] Note: Carnitine is analyzed by LC/MS.

Results (Unique Amino Acids)

[00307] Single amino acid dissolution test results for APR Lot 5 (bkT037/73-5) are reported in Table 11c and Figure 13. Table 11c

[00308] Results of single amino acid dissolution assays for APR Lot 7 (bkT037/72-7) are reported in Table 11d and Figure 14. Table 11d

[00309] Table 11e and Figure 15 report the total amino acids released (Weighted Dissolution Test) at various time points from each of the four test formulations compared to an immediate-release amino acid formulation. Table 11e

Tabela 11g

Tabela 11h

Tabela 11i

Tabela 11j[00310] Tables 11f-11j report the amount of individual amino acids released from four different test formulations at different time points. Table 11f

Table 11g

Table 11am

Table 11i

Table 11j

Example 13 - Final Composition of Amino Acids

[00311] The quantitative phenylketonuria composition of the amino acid mixture was optimized according to the recommendations of nutritionists; the single amino acid percentages are reported in Table 12a. Table 12a

Example 14 - In Vivo Bioavailability in Pigs

[00312] An in vivo bioavailability study was performed in pigs to model the anticipated pharmacokinetics and metabolism of the formulations of the present invention, compared to a comparable mixture of free amino acids, with similar proportions and total amounts of amino acids, and 78/ 86 commercially available formulation of milk proteins (casein). The following formulations were tested:

[00313] APR-04 (bkT037/72-4)

[00314] APR-07 (bkT037/73-7)

[00315] Free amino acid (bkT037/71)

[00316] Casein

In Vivo Study Design:

[00317] Subjects: 8 pigs

[00318] Design: 4 crossover formulations

[00319] Route and method of administration: oral, by fattening. A dose of the product was administered to each animal mixed with a small amount of water (300 ml) to ensure rapid supposition (< 5 minutes). The entire amount of product was administered using syringes. The animals were fasted for 13.5±0.5 hours before treatment and access to water was suspended one hour before and one hour after treatment.

[00320] Dose, frequency and duration of administration: The amount of product administered was based on body weight and equaled 0.8 g amino acid/kg body weight for each test product. The products were administered by a single administration in the morning of each treatment day.

[00321] Washing: 48-72 hours

[00322] Blood sample: 0.75 h before treatment (T), 0.5 h before T, 0.25 h before T, 0.25 h after T, 0.5 h after T, 0, 75h after T, 1h after T, 1.25h after T, 1.5h after T, 2h after T, 2.5h after T, 3h after T, 4h after T, 5h after T

[00323] Analysis: Plasma concentrations of 14 amino acids (alanine, arginine, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, threonine, tryptophan, tyrosine, valine) were measured and analyzed. Methionine and aspartic acid were excluded from the analysis due to stability issues. The following parameters were evaluated: Area Under the Concentration/Time Curve (AUC0-last), Peak Concentration (Cmax), Time to Peak Concentration (Tmax) and Clast. The behavior of the two APR formulations was compared to the Free Amino Acids and Casein formulation.

Results:

[00324] The plasma concentration of an amino acid (AA) is the result of its rates of appearance (Ra) and disappearance (Rd) from the plasma. Factors that control Ra include protein intake and tissue release. Factors that control Rd include tissue absorption and body loss through urine, sweat, etc. Hormones also help to regulate plasma AA concentrations, particularly insulin and glucagon, which induce hypoaminoacidemia (but for quite different reasons) and cortisol, which induces hyperaminoacidemia. Furthermore, in pathological states, catechol amines, thyroid hormones and cytokines can modulate plasma levels of AA. The peripheral availability of Aas after protein ingestion is controlled by the liver, with activation of ureagenesis in the feed and repression of hyperprotein during a low-protein diet (Cynober, 2002). All these factors can influence the plasma amino acid concentration reported in this document. 1) Mean Concentrations of Aggregate Plasma Amino Acids

[00325] Mean plasma concentrations of the 14 amino acids over time, in aggregate form, are reported in Figure 16 and Table 14a. Baseline values were not subtracted before performing the calculations. Table 14a

[00326] The following observations can be made:

[00327] The APR formulations show a lower plasma peak of aggregated amino acids (Cmax) when compared to the free amino acid formulation.

[00328] If we consider the single amino acid curve, the Repeated Measures ANOVA applied to Ln-transformed data shows a significant difference (p <0.05) in Cmax from APR-04 versus Free Amino Acids for Arginine, Isoleucine, Leucine , Tyrosine and Valine.

[00329] If we consider the single amino acid curve, the Repeated Measures ANOVA applied to Ln-transformed data shows a significant difference (p <0.05) in Cmax from APR-07 versus Free Amino Acids for Isoleucine, Leucine, Tyrosine and Valina.

[00330] The difference in Cmax is even more evident, if we consider the BC data, in which the baseline amino acid values are subtracted, as shown below in Table 14b, although the difference is not statistically significant for the mean of the whole aggregate of amino acids: Table 14b

[00331] The highest initial peak plasma amino acid concentration 10 (Cmax) observed for the Free Amino Acid Formulation rapidly decreases, as evidenced by the parameter "Clast. “Clast” is the plasma amino acid concentration in the last sampling (5h)

[00332] If we compare the curve of single amino acids, in the group treated with APR-04, the Clast is higher (but not statistically significant, except for Valine) versus the Free Amino Acids Group for Alanine, Arginine, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Proline, Serine, Threonine, Tryptophan and Valine (13 amino acids out of 14)

[00333] If we compare the single singles curve, in the group treated with APR-07, the Clast is higher (but not statistically significant, except for Valine, 20 Isoleucine and Leucine) in relation to the Free Amino Acids Group for Arginine, Histidine, Isoleucine, Leucine, Lysine, Proline, Tryptophan and Valine (8 amino acids out of 14)

[00334] The trend in Cmax and Clast observed in the plasma concentration curve for aggregated amino acids becomes statistically significant if the subgroup of Essential Amino Acids and the subgroup of BCAAs (Branched Chain Amino Acids) are considered (see points 2 and 3).

2) Mean Concentrations of Essential Amino Acids Analyzed (Arginine, Histidine, Isoleucine, Leucine, Threonine, Lysine, Tryptophan and Valine).

[00335] Mean plasma concentrations of the essential amino acids tested, in aggregate form, are reported in Figure 17 and Table 14c. The aggregated amino acids for analysis were Arginine, Histidine, Isoleucine, Leucine, Threonine, Lysine, Tryptophan and Valine. This subgroup was chosen for analysis because these amino acids cannot be synthesized endogenously. As a result, their plasma concentrations from jugular vein sampling may be more representative of intestinal adsorption of the tested formulations than non-essential amino acids whose presence in the blood can be generated independently by the body. Table 14c

[00336] Statistical analysis for the date reported in Table 14c (for AUC, Cmax, and Clast: One-way repeated measures ANOVA with post hoc analysis with Bonferroni adjustment; for Tmax: Friedman test followed by multiple sign test for pairwise comparison) is reported in Table 14d. Table 14d

3) Average concentration of BCAAs (Branched chain amino acids: valine, isoleucine and leucine).

[00337] Mean plasma concentrations of the branched-chain amino acids tested, in aggregate form, are reported in Figure 18 and Table 14e. The amino acids pooled together for analysis were valine, isoleucine, and leucine. This subgroup of essential amino acids was chosen for analysis because these essential amino acids are unique among amino acids in that their first catabolic step cannot occur in the liver. As a consequence, they largely escape first-pass splanchnic metabolism (Brosnan et al.). Table 14e

[00338] Statistical analysis for the date reported in Table 14e (for AUC, Cmax, and Clast: One-way repeated measures ANOVA with post hoc analysis with 15 Bonferroni adjustment; for Tmax: Friedman test followed by multiples test signals for pairwise comparison) is reported in Table 14f. Table 14f

4) Mean total concentration of LNAAs (Large Neutral Amino Acids: phenylalanine, tyrosine, tryptophan, threonine, methionine, valine, isoleucine, leucine, histidine).

[00339] Plasma concentrations of the large neutral amino acids tested, in aggregate form, are reported in Figure 19 and Table 14g. The amino acids pooled together for analysis were tryptophan, threonine, valine, isoleucine, leucine, histidine. Tyrosine was excluded because of the anomalous behavior it exhibited in dissolution and in vivo tests, and methionine because it was not stable in the samples. Phenylalanine was excluded from the analysis because it was not present in the tested formulations. This subgroup of essential amino acids was chosen for analysis because LNAA shares a common transport system with phenylalanine for entering the brain. High plasma concentrations of LNAAs may reduce absorption into the brain (Van Spronsen et al.) and thus may have clinical advantages. In particular, if the subgroup analysis shows an increase in Clast for the APR formulations versus the free amino acid formulations, it can be speculated that the patient may have some kind of “prolonged protection/tolerance” to phenylalanine. Table 14g

[00340] Statistical analysis for the date reported in Table 14g (for AUC, Cmax, and Clast: one-way repeated measures ANOVA with post hoc analysis with Bonferroni adjustment; for Tmax: Friedman test followed by multiple sign test for pairwise comparison) is reported in Table 14h. Table 14h

[00341] Throughout this application various publications are referenced. The disclosures of these publications are incorporated herein by reference in order to more fully describe the prior art to which this invention belongs. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will become apparent to those skilled in the art from a consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

REFERENCES CITED

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[00343] BARACOS V.E. Animal models of amino acids metabolism: a focus on the intestine. J. Nutria. 134:1656S-1659S (2004)

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[00345] CYNOBER LA. Plasma amino acid levels with a note on membrane transport: characteristics. regulation. and metabolic significance. Nutrition. 2002 Sep;18(9):761-6.

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[00347] EMA 2010. Guideline on the investigation of bioequivalence CPMP/EWP/QWP/1401/98 Rev. 1. London. 20 January 2010

[00348] GIOVANNINI M.. RIVA E.. SALVATICI E.. CEFALO G.. RADAELLI G. Randomized controlled trial of a protein substitute with prolonged release on the protein status of children with phenylketonuria. J. Am. Col. Nourish 33. 103-110 (2014)

[00349] GROPPER S.S. ACOSTA P.B. Effect of simultaneous ingestion of Lamino acids and whole protein on plasma amino acid and urea nitrogen concentrations in humans. J. Parenteral Enteral Nutr. 15:48-53 (1991)

[00350] KEOHANE P. P. GRIMBLE G. K. BROWN B. SPILLER R. C. Influence of protein composition and hydrolysis method on intestinal absorption of protein in man. Gut. 26:907-913 (1985)

[00351] MONTGOMERY R. DRYER R. L. CONWAY T. W. SPECTOR A. A. Biochimica. Aspetti medico biologici. Translation by Berra B.. Ragnotti G.. Tettamanti G.. Edi-Ermes. Milan. 1988

[00352] Chapter 1: Nutrizione. pp. 1-47

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[00356] NEY DM. Does the PKU diet contribute to impaired renal function? J Inherit Metab Dis. 2013 Sep;36(5):903-4.

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[00358] VAN SPRONSEN FJ. DE GROOT MJ. HOEKSMA M. REIJNGOUD DJ. VAN RIJN M. Large neutral amino acids in the treatment of PKU: from theory to practice. J Inherit Metab Dis. 2010 Dec;33(6):671-6.

[00359] VLIET D. VAN. DERKS T.G.J. RIJN M. VAN. DE GROOT M.J. MCDONALD A. et al. Single amino acid supplementation in aminoacidopatients: a systemic review. Orphanet J. Rare Dis. 9:1-14 (2014)

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Claims (21)

1. Orally administered amino acid formulation, comprising: a. one or more modified-release amino acids; B. hydroxypropylmethylcellulose or ethylcellulose or a combination thereof integrally mixed with said amino acids; or c. a coating on said modified release amino acids comprising ethylcellulose or a combination of ethylcellulose and glyceryl dibehenate; wherein the formulation comprises 2 g of the modified-release amino acids comprising a release profile that releases not more than 70% of the modified-release amino acids in 30 minutes of dissolution tests performed in a paddle apparatus <711> USP 39 NF 34 , at 37 °C, in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, and said modified release is defined by said release profile. 2. Formulation, according to claim 1, characterized in that it comprises five or more of the following amino acids as modified-release amino acids in the aforementioned parts by weight: Amino acid Parts by weight L-Alanine 2.0-12.0 L-Arginine 3, 0-10.5 L-Aspartic Acid 5.0-10.5 L-Cystine 1.5-4.0 L-Glutamic Acid + Glutamine 7.0-25.0 Glycine 3.5-15.0 L-Histidine 2.0-6.5 L-Isoleucine 2.0-8.5 L-Leucine 8.0-15.0 L-Lysine 4.5-10.5 L-Methionine 1.0-3.0 L-Phenylalanine 4.0-7.5 L-Proline 3.5-15.0 L-Serine 2.0-8.5 L-Threonine 4.0-7.5 L-Tryptophan L-Tyrosine L-Valine L-Carnitine Taurine 3. Formulation according to claim 2, characterized in that it comprises ten or more of said amino acids as modified-release amino acids in the indicated parts by weight. 4. Formulation according to claim 2, characterized in that it comprises fifteen or more of said amino acids as modified-release amino acids in the indicated parts by weight. 5. Formulation, according to claim 2, characterized in that it comprises the indicated essential amino acids, branched chain amino acids, and mentioned large neutral amino acids, such as modified release amino acids in the indicated parts by weight. 6. Formulation, according to claim 1, characterized in that it comprises five or more of the following amino acids as modified-release amino acids in the cited parts by weight: Amino acid Parts by weight L-Alanine 2.0-12.0 L-Arginine 3, 0-10.5 L-Aspartic Acid 5.0-10.5 L-Cystine 1.5-4.0 L-Glutamic Acid + Glutamine 7.0-25.0 Glycine 3.5-15.0 L-Histidine 2.0-6.5 L-Isoleucine 2.0-8.5 L-Leucine 8.0-15.0 L-Lysine 4.5-10.5 L-Methionine 1.0-3.0 L-Phenylalanine 4.0-7.5 L-Proline 3.5-15.0 L-Serine 2.0-8.5 L-Threonine 4.0-7.5 L-Tryptophan 1.0-4.0 L-Tyrosine 2.5-14.0 L-Valine 2.5-10.0 L-Carnitine 0.0-0.2 Taurine 0.05-1.0 comprising 0.0 parts by weight of L-phenylalanine. 7. Formulation, according to claim 1, characterized in that it comprises five or more of the following amino acids as modified-release amino acids in the aforementioned parts by weight: Amino acid Parts by weight L-Alanine 2.0-12.0 L-Arginine 3, 0-10.5 L-Aspartic Acid 5.0-10.5 L-Cystine 1.5-4.0 L-Glutamic Acid + Glutamine 7.0-25.0 Glycine 3.5-15.0 L-Histidine 2.0-6.5 L-Isoleucine 2.0-8.5 L-Leucine 8.0-15.0 L-Lysine 4.5-10.5 L-Methionine 1.0-3.0 L-Phenylalanine 4.0-7.5 L-Proline 3.5-15.0 L-Serine 2.0-8.5 L-Threonine 4.0-7.5 L-Tryptophan 1.0-4.0 L-Tyrosine 2.5-14.0 L-Valine 2.5-10.0 L-Carnitine 0.0-0.2 Taurine 0.05-1.0 comprising 0.0 part by weight of L-phenylalanine and 0.0 parts by weight of L-tyrosine. 8. Formulation according to claim 1, characterized in that the formulation is a powder or granules. 9. Formulation, according to claim 1, characterized in that it is in the form of a tablet, a pill, a soft or hard gelatine capsule, a powder, a granule, a microsphere, a pastille, a sachet of powders or granules or packaged microspheres, an elixir, a suspension, an emulsion, a chewable tablet or a syrup. 10. Formulation according to claim 1, characterized in that the formulation further comprises one or more additional ingredients selected from the group consisting of: a. vitamins, minerals and carbohydrates; or b. choline, inositol, vitamin A, vitamin D vitamin E, vitamin K, vitamin C, thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, biotin, pantothenic acid, potassium, calcium, magnesium, iron, zinc, copper, manganese , selenium, chromium, molybdenum, iodine, sodium, sulfur, phosphorus, docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid and lutein, and salts, chelates and esters thereof. 11. Formulation according to claim 1, characterized in that the formulation comprises 2 g of modified release amino acids comprising a release profile that releases no more than 60% of modified release amino acids in 30 minutes of dissolution tests performed in a paddle apparatus <711> USP 39 NF 34, at 37 °C, in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, and said modified release is defined by said release profile. 12. Orally administered amino acid formulation comprising one or more modified-release amino acids, characterized in that the formulation comprises 2 g of the modified-release amino acids comprising a release profile that releases no more than 70% of the modified-release amino acids within 30 minutes of dissolution tests carried out in a paddle apparatus <711> USP 39 NF 34, at 37 °C, in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, with the said modified release is defined by said release profile, wherein said modified release amino acids are granules coated with 5% to 25% by weight of one or more excipients release modifiers based on the weight of amino acids, each of the granules comprises a plurality of modified-release amino acids, wherein said formulation comprises: a. hydroxypropylmethylcellulose or ethylcellulose or a combination thereof integrally mixed with said amino acids; or b. a coating on said modified release amino acids comprising ethylcellulose or a combination of ethylcellulose and diglyceryl dibehenate. 13. Formulation according to claim 12, characterized in that it further comprises one or more release-modifying excipients selected from ethylcellulose and a combination of ethylcellulose and diglyceryl dibehenate. 14. Formulation according to claim 12, characterized in that it further comprises: a. a bulking agent selected from lactose, sucrose, dextrose, sorbitol, fructose and cellulose powder; B. a disintegrating agent selected from microcrystalline cellulose, starches, crospovidone, sodium starch glycollate and croscarmellose sodium; w. a glidant or lubricant selected from talc, corn starch, silicon dioxide, sodium lauryl sulfate, magnesium stearate, calcium stearate, sodium stearate, stearic acid, sodium stearyl fumarate, hydrogenated cottonseed oil, talc, waxes , cetyl alcohol, glyceryl stearate, glyceryl palmitate, glyceryl behenate, hydrogenated vegetable oils and stearyl alcohol; d. a taste masking agent selected from hydroxypropyl ethers of cellulose (HPC); low-substituted hydroxypropyl ethers (L-HPC); hydroxypropylmethyl ethers of cellulose (HPMC); methylcellulose polymers; ethylcelluloses (EC) and mixtures thereof; Polyvinyl Alcohol (PVA); hydroxyethylcelluloses; carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC); copolymers of polyvinyl alcohol and polyethylene glycol; monoglycerides, triglycerides, polyethylene glycols, modified food starch, acrylic polymers and blends of acrylic polymers with cellulose ethers; cellulose acetate phthalate; sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins and mixtures of these materials; It is. a flavoring agent selected from acacia syrup, acesulfame K, alitame, aniseed, apple, aspartame, banana, bavarian cream, berry, blackcurrant, butterscotch, calcium citrate, camphor, caramel, cherry, cream cherry, chocolate, Cinnamon, Bubble Gum, Citrus Fruits, Citrus Juices, Citrus Cream, Cotton Candy, Cocoa, Cola, Fresh Cherry, Fresh Citrus Fruits, Cyclamate, Dextrose, Eucalyptus, Eugenol, Fructose, Fruit Juice, Ginger, Glycyrrhinoate, Glycyrrhiza Syrup (Liquorice) , grapefruit, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glycyrrhizinate, maltol, mannitol, maple, marshmallow, menthol, cream mint, mixed berry, neohesperidin DC, neotame, orange, pear, peach, mint , cream mint, raspberry, root, rum, saccharin, safrole, sorbitol, mint, cream mint, strawberry, cream strawberry, stevia, sucralose, sucrose, saccharin sodium, saccharin, aspartame, neotame, acesulfame potassium, mannitol, talin, xylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry, winter seed, xylitol or a combination thereof; f. one or more release-modifying excipients selected from the group consisting of ethylcellulose, glyceryl dibehenate, cellulose acetate, vinyl acetate/vinyl chloride copolymers, acrylate/methacrylate copolymers, polyethylene oxide, hydroxypropylmethylcellulose, carrageenan, alginic acid and their salts, hydroxyethylcellulose, hydroxypropylcellulose, karaya gum, acacia gum, tragacanth gum, locust bean gum, guar gum, sodium carboxymethylcellulose, methylcellulose, beeswax, carnauba wax, cetyl alcohol, hydrogenated vegetable oils, stearyl alcohol, copolymers acrylic acid, sodium alginate, carrageenan, alginic acid, pectin, sodium carboxymethylcellulose, a starch derivative, or a combination thereof; or g. a binder selected from polyvinylpyrrolidone, starch, methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sucrose solution, dextrose solution, guar gum, xanthan gum, acacia, tragacanth, locust bean gum and sodium alginate or a salt of alginic acid. 15. Formulation according to claim 12, characterized in that it further comprises a binder selected from sodium alginate or an alginic acid salt. 16. Formulation according to claim 1, characterized in that the tyrosine in the formulation is not coated with a modified release coating. 17. Method of manufacturing the formulation as defined in claim 12, characterized in that it comprises: a. providing a first mixture comprising a plurality of amino acids; B. contacting the mixture with a wetting agent and a binder to form a wet mixture; w. optionally passing the wet mixture through a sieve to form a uniform wet granulate; d. drying the wet granulate uniformly to form a dry granulate; It is. optionally passing the dry granulation through a sieve to form a uniform dry granulate; and f. coating the uniformly dried granulate with a modified release composition. 18. Orally administered amino acid formulation comprising one or more modified-release amino acids, characterized in that the formulation comprises 2 g of the modified-release amino acids comprising a release profile that releases no more than 70% of the modified-release amino acids within 30 minutes of dissolution tests carried out in a paddle apparatus <711> USP 39 NF 34, at 37 °C, in 450 or 500 mL, 0.1 N hydrochloric acid (pH 1.2), paddle speed 50 rpm, with the said modified release is defined by said release profile, said modified release amino acids being granules coated with 5% to 25% by weight of one or more release modifying excipients based on the weight of the amino acids, and each of the granules comprise a plurality of modified-release amino acids, further comprising one or more release-modifying excipients selected from ethylcellulose and a combination of ethylcellulose and glyceryl dibehenate. 19. Formulation according to claim 18, characterized in that it further comprises a binder selected from sodium alginate or an alginic acid salt. 20. Formulation according to claim 18, characterized in that the tyrosine in the formulation is not coated with a modified release coating. Formulation according to claim 18, characterized in that it comprises granulates having a size between 0.1 to 1.5 mm.

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