Modified release amino acid formulations for oral administration

Abstract

Methods and formulations of modified-release amino acids having improved pharmacokinetics, metabolism, and utilization are provided for treating or managing diseases defined by impaired amino acid metabolism.

Description

[0001] This application is a divisional application of the international application with application number PCT / IB2016 / 055773 filed on September 27, 2016 and invention name “Modified Release Amino Acid Formulations for Oral Administration”. The international application entered the Chinese national phase on May 28, 2018, and its application number is 201680069481.9. Field of the Invention

[0002] The present invention relates to formulations and dosage forms for oral administration of amino acids with improved dosage accuracy, improved palatability and improved pharmacokinetics for optimal therapeutic effect. Background Art

[0003] Amino acids are essential building blocks for protein synthesis in the human body and are primarily derived from dietary protein sources. The human body uses 19 different L-configuration amino acids to synthesize proteins. Some of these amino acids are called "essential amino acids" because the body cannot synthesize them independently. Other amino acids are not "essential" because the body can synthesize them.

[0004] Digestion of dietary protein into amino acids begins in the stomach with the combined activity of pepsinogen, pepsin and hydrochloric acid to generate oligopeptides. After transit from the stomach through the pylorus, digestion continues in the duodenum where the combined activity of bile secretion, bicarbonate and pancreatic enzymes can generate 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 an active transport process that is stereospecific for the amino acids. Secretion of amino acids into the intestine is a concomitant mechanism. Di- and tripeptides can also be absorbed from the small intestine, but they are also finally hydrolyzed to amino acids in the intestinal epithelial cells. These processes are largely pH dependent. When food is ingested, the stomach secretes hydrochloric acid and the pH drops to about 2. As a result of alkaline bile secretion, the pH gradually increases from 4 to about 7 after passing the pyloric valve. The buffer system operates to prevent pH values ​​greater than about 7.5-7.8.

[0005] The time required to digest dietary protein 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 can vary depending on food intake, protein intake, disease and concomitant drug treatment, with food intake being the most common determining factor. Amidon et al. 1995 reported a gastric emptying time of about 3.5 hours after a high-fat meal, a gastric emptying time of about 1.5 hours after a normal meal, and a gastric emptying time of about 10 to 20 minutes after 250 mL of water. Keohane et al. 1985 reported that the time required for hydrolysis of amino acids in the small intestine averaged about 3 hours, and this time varied for individual proteins and amino acids. The hydrolysis begins to occur immediately after gastric emptying, after transit through the pyloric valve, and lasts for several hours in the first segment of the small intestine (i.e., the jejunum).

[0006] Amino acids are often given as a supplement to the diet to promote protein synthesis, especially where the goal is to increase muscle mass. Specially formulated combinations of amino acids are also given to support the nutritional health of individuals with special dietary intake restrictions and needs. For example, in phenylketonuria, the body is unable to metabolize phenylalanine to tyrosine due to a lack of an essential enzyme. Instead, phenylalanine breaks down into several toxic byproducts. Montgomery et al. 1988; Waisbren et al. 2007.

[0007] Phenylalanine is an essential amino acid that is normally metabolized to tyrosine by the enzyme phenylalanine hydroxylase. In phenylketonuria this enzyme is missing or does not function properly, and phenylalanine is decarboxylated to a variety of different compounds, three of which are toxic: phenylpyruvate, phenyllactate, and phenylacetate. Due to this metabolic inhibition, dietary intake of phenylalanine must be reduced while the diet is used to provide the amino acid tyrosine (as it is indispensable). Ney DM et al. 2014; Dioguardi, 2011; Waisbren et al. 2007.

[0008] An effective management strategy for phenylketonuria involves specially formulated amino acid preparations that omit phenylalanine but supplement with tyrosine. Since phenylalanine cannot be removed from most dietary protein sources, these amino acid supplements often replace dietary protein that has been completely eliminated 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 SS et al 1991.

[0009] Examples of amino acid preparations sold for the management of phenylketonuria include those marketed as Antifen TM 、Nutricia XP2 TM (Maxamaid) and Milupa PKU2TM Nutricia XP2 TM and Milupa PKU2 TM Vitamins and minerals are also included. Table A below gives the formulations of these products, based on approximately 100 g of amino acids in each formulation, omitting the vitamin and mineral content.

[0010] Table A

[0011]

[0012]

[0013] 1 http: / / www.dmfmetabolic.it / wp-content / uploads / 2013 / 10 / AntifenInteg-rale.pdf

[0014] 2 http: / / www.nutricia.it / prodotti / xp2-maxamaid /

[0015] 3 http: / / www.nutricia.it / prodotti / pku-2-secunda /

[0016] Other conditions for which specially formulated amino acid preparations are manufactured and administered using the same strategy to provide all the essential and non-essential amino acids but omit the amino acids that the body cannot metabolize, include tyrosinemia, hyperleucinosis, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia, and glutaric acidemia (Type II, IIA, or IIB).

[0017] Free amino acids may present a number of disadvantages over naturally occurring proteins, particularly when given in higher doses, due to the rapid absorption of the amino acids, imposing a higher dietary acid load.

[0018] In addition, some commercial amino acid preparations have an unpleasant taste and smell, which makes them extremely difficult for many people, especially infants and children, to take regularly. In addition, these preparations are released immediately when taken, resulting in a very different absorption pattern than when protein is taken.

[0019] It is an object of the present invention to limit the catabolism of amino acids caused when free amino acid uptake exceeds the body's metabolic capacity.

[0020] Another object of the present invention is to improve the taste of amino acid preparations when taken orally.

[0021] Another object of the present invention is to provide a unique amino acid dosage form that can be consumed directly without the need for rehydration.

[0022] It is yet another object of the present invention to provide amino acid formulations with modified release properties that more closely mimic the absorption pattern of proteins upon oral ingestion. Summary of the invention

[0023] Modified release amino acid formulations have been developed that overcome the aforementioned disadvantages. The formulations are more palatable and, most importantly, mimic the release profile of amino acids from naturally derived food sources. In particular, the formulations use a release delay mechanism that releases modified amino acids from the formulation at a rate that mimics the release rate of amino acids from naturally occurring proteins.

[0024] In the limited trials conducted to date, several individual amino acids and combinations of amino acids, when modified to achieve specific in vitro release criteria, have been demonstrated to provide statistically superior improvements in plasma amino acid concentrations over comparable free amino acid preparations. Thus, in a first major embodiment, the present invention provides an orally administrable amino acid formulation comprising one or more modified release amino acids, wherein said formulation comprising 2 g of a modified release amino acid is administered at <711> USP39NF 34, in a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1N hydrochloric acid (pH 1.2) with a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0025] In animal in vivo bioavailability studies, it has been unexpectedly discovered that isoleucine, leucine, tyrosine and valine, when modified to release as described herein, consistently have peak concentrations in the blood that are significantly less than the peak concentrations produced by comparable free amino acid preparations. Thus, in a second major embodiment, the present 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 is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. USP 39NF 34.

[0026] Another major embodiment relates to a particular group of amino acids - branched chain amino acids - and the unexpected discovery that the branched chain amino acids (in groups), when modified to release as described herein, produce C 2+ than comparable free amino acid preparations. max and C 终点 Significantly lower C max and significantly higher C 终点 Thus, in another main embodiment, the present invention provides an orally administrable amino acid formulation comprising one or more modified-release branched-chain amino acids, wherein the formulation comprising 2 g of modified-release branched-chain amino acids is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. USP 39NF34.

[0027] Yet another major embodiment relates to another particular group of amino acids - the essential amino acids - which, when modified to release as described herein, unexpectedly produce significantly lower C max and significantly higher C 终点 Thus, in yet another main embodiment, the present invention provides an orally administrable amino acid formulation comprising one or more modified-release essential amino acids, wherein the formulation comprising 2 g of modified-release essential amino acids is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. USP 39NF 34.

[0028] Yet another major embodiment relates to another group of amino acids - the large neutral amino acids (excluding phenylalanine, tyrosine and methionine) - which, when modified to release as described herein, unexpectedly produce significantly lower C max and significantly higher C 终点Thus, in yet another major embodiment, the present invention provides an orally administrable amino acid formulation comprising one or more modified-release large neutral amino acids, wherein the formulation comprising 2 g of modified-release large neutral amino acids is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. USP 39NF 34.

[0029] In a seventh major embodiment, the present invention provides an orally administrable amino acid formulation comprising a combination of modified release non-essential amino acids, wherein said formulation comprising 2 g of modified release amino acids is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. USP 39NF34.

[0030] The formulation is preferably in the form of edible granules that can be mixed with other foods such as yogurt, or in the form of large chewable tablets that cannot be swallowed whole but must be eaten in several bites like a biscuit. The amount of amino acid in the unit dose formulation will depend on the metabolic requirements of the individual being treated, generally depending on the individual's age, sex, weight and physical activity, and whether the individual suffers from a metabolic disorder that limits his or her intake of one or more amino acids, as described in more detail herein.

[0031] The formulations may contain one, all or any combination of essential and non-essential amino acids and may be specifically designed for any application of amino acid supplementation. The formulations are particularly well suited for the treatment of patients with amino acid processing disorders (e.g., phenylketonuria, hypertyrosinemia, hyperleucinemia, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia, and glutaric acidemia).

[0032] The modified release can be accomplished by various ingredients herein referred to as release delay agents, which are means for preferentially releasing amino acids in the jejunum, etc. These agents can cause modified release of amino acids from the formulation when ingested or when tested in a dissolution medium. Alternatively, they can prolong or delay the release of amino acids so that some amino acids are not released until later in the digestive process (e.g., after the formulation reaches the higher pH environment of the small intestine). In appropriately designed formulations, these agents can even release a small group of amino acids in the formulation at a different release rate than other amino acids. When tested at a fixed pH, the formulation is preferably released in its modified release mode.

[0033] Other aspects of the invention relate to the use of the formulations to treat or manage diseases that require 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 following conditions in a patient in need thereof, including phenylketonuria, hypertyrosinemia, hyperleucinosis, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia and glutaric acidemia, comprising orally administering to the patient an amino acid formulation of the invention.

[0034] Another embodiment provides a method for making a formulation of the present invention, and in this embodiment, the present invention provides a method for making a formulation of the present invention, comprising: (a) providing a first mixture comprising a single or more amino acids; (b) contacting the mixture with a wetting agent or solvent (e.g., alcohol and / or organic solvent and / or water) and a binder to form a wet mixture; (c) optionally passing the wet mixture through a screen to form uniform wet granules; (d) drying the uniform wet granules to form dry granules; (e) optionally passing the dry granules through a screen to form uniform dry granules, and (f) optionally coating the dry granules with one or more coating agents to provide modified release for the dry granules. Suitable coating agents include, for example, cellulosic polymers, fatty acids, waxes, and other coating agents capable of providing modified release for the dry granules. Suitable coating techniques include, for example, fluidized bed coating and melt granulation. The dry granules can be incorporated into any conventional dosage form, but are preferably used as dosage forms that can be easily mixed with other ingredients (such as flavors and inactive excipients or additives, or active ingredients such as vitamins, minerals, and carbohydrates) without additional modification. Alternatively, the dry granules may be compressed into chewable tablets.

[0035] Additional advantages of the present invention are set forth in part in the description that follows and are apparent to some extent from the description or may be learned through practice of the present invention. The advantages of the present invention will be realized and obtained by the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the subsequent detailed description of the present invention are merely exemplary and explanatory and are not restrictive, as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The 19 amino acids that the human body uses to synthesize proteins are listed and organized according to their structure and properties (i.e., aliphatic, aromatic, acidic, basic, sulfurized, and pK a ) to classify them.

[0037] Figure 2 Essential amino acids, ie those amino acids that are not produced by biochemical synthesis in the body and must be taken up through the diet, are listed as reported by Montgomery et al. 1988.

[0038] Figure 3 is a graphical illustration 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.

[0039] Figure 4 is a graphical illustration of the release profile of tryptophan from the formulation of Example 8 over time using a dissolution medium at a pH of 1.2 and a paddle speed of 50 rpm.

[0040] Figure 5 is a graphical illustration of the release profile over time of tryptophan from the formulation of Example 9 when subjected to the experimental conditions described in Example 8 but using a basket method instead of a paddle.

[0041] Figure 6 is a graphical illustration of the gravimetric release profile over time for a formulation comprising the amino acid mixture from the formulation of Example 10 when subjected to the experimental conditions described in Example 10.

[0042] Figure 7 is a graphical illustration of the gravimetric dissolution profiles of the free amino acid formulations described in Example 11, Table 10b, tested according to the method described in Example 11.

[0043] Figure 8 The solubility profiles of 18 different amino acids from the APR AA mixture (bkT037 / 71) were plotted.

[0044] Fig. 9is a graphical illustration of the gravimetric dissolution profiles of four different ethylcellulose coated modified release amino acid formulations made and tested according to the methods and formulations reported in Example 11 and Tables 10d and 10e.

[0045] Fig.10 The dissolution profiles of 18 different amino acids from APR batch 2 (bkT037 / 72-2), Table 10f, when tested according to the method reported in Example 11 are plotted.

[0046] Fig.11 The dissolution profiles of 18 different amino acids from APR batch 4 (bkT037 / 72-4), Table 10g, when tested according to the method reported in Example 11 are plotted.

[0047] Fig.12 The gravimetric dissolution profiles of a panel of amino acids from four different ethylcellulose / glyceryl dibehenate coated modified release amino acid formulations having the compositions reported in Table 11a, manufactured and tested as described in Example 12, are plotted.

[0048] Fig.13 The dissolution profiles of 18 different amino acids from APR batch 5 (bkT037 / 73-5) (Table 11c) when tested according to the method reported in Example 12 are plotted.

[0049] Fig.14 The dissolution profiles of 18 different amino acids from APR batch 7 (bkT037 / 72-7) (Table 11d) when tested according to the method reported in Example 12 are plotted.

[0050] Fig.15 Illustrated are the gravimetric dissolution profiles of the amino acid collection of four different experimental formulations of the invention (APR Batch 2 / bkT037 / 72-2, APR Batch 4 / bkT037 / 72-4, APR Batch 5 / bkT037 / 73-5, APR Batch 7 / bkT037 / 72-7) and the free amino acid mixture bkT037 / 71 manufactured and tested as described in Examples 11 and 12.

[0051] Fig.16 The mean plasma concentrations (as a sum) of the 14 amino acids in pigs over time from two different modified release amino acid preparations of the invention, one free amino acid preparation and casein as described in Example 14 are reported.

[0052] Fig.17The mean plasma concentrations (on a sum basis) of essential amino acids in pigs over time from two different modified release amino acid preparations of the invention, one free amino acid preparation and casein as described in Example 14 are reported.

[0053] Fig.18 The mean plasma concentrations (on a sum basis) of branched chain amino acids in pigs over time from two different modified release amino acid formulations of the invention, one free amino acid formulation and casein as described in Example 14 are reported.

[0054] Fig.19 The mean plasma concentrations (on a sum basis) of large neutral chain amino acids in pigs over time from two different modified release amino acid preparations of the invention, one free amino acid preparation and casein as described in Example 14 are reported. Detailed description

[0055] definition

[0056] When the singular forms "a," "an," and "the" or similar terms are used herein, they are to be understood to include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a hydrocarbon" includes a mixture of two or more such hydrocarbons, and the like. The word "or" or similar terms used herein means any one member of a particular list and also includes any combination of members of that list.

[0057] As used herein, the terms "about" or "ca." will compensate for variability allowed in the pharmaceutical industry and inherent in pharmaceutical products, such as differences in product strength and bioavailability due to manufacturing variations and time-induced product degradation. The terms allow for any variation that will allow the product being evaluated to be considered pharmaceutically equivalent or bioequivalent to the recited strength of the claimed product, or both if the context requires. It should be understood that all numerical values ​​expressed in this document may be preceded by the term "about."

[0058] The term "independently" is used to mean that a particular element modified by a set of possible 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%, then the composition may comprise 10% of element A and 10% of element B, or 10% of element A and 20% of element B, etc.

[0059] The term "amino acid" refers to any naturally occurring amino acid that can participate in the synthesis of polypeptides and proteins. For ease of drawing, the amino acids are usually not written with their stereo configuration, although it should be understood that the amino acids should exist as their naturally occurring stereoisomers.

[0060] In the formulations of the present invention, the amino acids may be present as free bases, hydrochlorides or another suitable salt. The term essential amino acid refers to any amino acid that the body cannot make on its own and must be derived from an external source. Essential amino acids that may 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 pharmaceutically acceptable salt thereof (the last two being considered essential only for infants, although - for simplicity - they are referred to as "essential amino acids herein").

[0061] The term "non-essential amino acid" refers to any amino acid other than the essential amino acids, and therefore includes, for example, L-alanine, L-aspartic acid, L-cystine, L-glutamine and / or L-glutamic acid, glycine, L-proline, L-serine, taurine, L-tyrosine and L-carnitine or any pharmaceutically acceptable salt thereof. Any of the amino acids may be present as free alkali, hydrochloride or any other suitable salt.

[0062] The term "branched chain amino acids" refers to valine, isoleucine and leucine, or any pharmaceutically acceptable salt thereof.

[0063] In one embodiment, the term "large neutral amino acids" refers to tryptophan, threonine, valine, isoleucine, leucine and histidine, or any pharmaceutically acceptable salt thereof. In another embodiment, the term also includes phenylalanine, methionine and tyrosine.

[0064] The term "formulation" refers to a finished or semi-finished combination of a drug or medical food or food ingredient, including both active ingredients and inactive excipients or additives. The term refers to the formulation in process, the finished formulation, and the formulation packaged as a final unit dose.

[0065] Throughout the description and claims of this application, the word "comprise" and variations of the word, such as "comprising" and "comprises" mean "including but not limited to" and are not intended to exclude, for example, other additives, components, integers or steps.

[0066] The terms "treating" and "treatment", when used herein, refer to the medical management of a patient with the intent to cure, alleviate, stabilize or prevent a disease, pathological condition or disorder. The terms include active treatment, i.e., treatment specifically directed to the improvement of a disease, pathological condition or disorder, and also include etiological treatment, i.e., treatment directed to the removal of the cause of the relevant disease, pathological condition or disorder. In addition, the terms include palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition or disorder; preventive treatment, i.e., treatment directed to minimizing or partially or completely inhibiting the development of a relevant disease, pathological condition or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to the improvement of a relevant disease, pathological condition or disorder. The terms also include dietary management of patients who have limited or impaired ability to ingest, digest, absorb or metabolize food or nutrients due to a specific disease or medical condition, or who have other special medically determined nutrient requirements that cannot be achieved by modification of a normal diet alone.

[0067] The term "modified release" refers to any pharmaceutical formulation in which the rate of release is intentionally altered to achieve a desired therapeutic response or pharmacokinetic response. Thus, the term includes extended release formulations in which the release of the drug is prolonged over time, or the rate of release is independent of the pH of the surrounding environment. The term also includes delayed release formulations in which the release of the active ingredient from the formulation (or portion thereof) is delayed until after initial ingestion. Delayed release formulations are typically designed so that release occurs primarily once the formulation reaches the small intestine.

[0068] As used herein, "therapeutically effective amount" refers to an amount sufficient to elicit a desired biological response. The therapeutically effective amount or dosage will depend on the patient's age, sex, and weight as well as the patient's current medical condition. One skilled in the art will be able to determine an appropriate dosage based 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 an amount generally recognized as necessary to support the metabolic needs of the human body.

[0069] "Pharmaceutically acceptable" means that it is useful in preparing pharmaceutical compositions, is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes pharmaceutical compositions that are acceptable for veterinary use as well as human medical foods, foods and pharmaceutical uses. "Pharmaceutically acceptable salts" means salts that are acceptable for pharmaceutical or medical foods or foods, as defined above, and which possess the desired pharmacological or nutritional activity.

[0070] When range is expressed in this article by indicating the alternative upper and lower limits of range, it should be understood that the endpoints can be combined in any mathematically feasible manner. Therefore, for example, the range of 50 or 80 to 100 or 70 can also be expressed as a series of ranges of 50 to 100, 50 to 70 and 80 to 100. When a series of upper and lower limits are used in words and / or associated, it should be understood that the upper limit may not be limited by the lower limit or may be combined with the lower limit, and vice versa. Therefore, for example, a range greater than 40% and / or a range less than 80% includes a range greater than 40%, less than 80% and a range greater than 40% but less than 80%.

[0071] When a specific test method from the U.S. Pharmacopoeia or other industry recognized compendium is given herein, it is understood that the test method may be modified in accordance with usual and customary practice without changing the underlying physical principle or the end result based on conventional techniques of those skilled in the art, and as the examples provided herein, for example, it may be necessary to test different dosage forms.

[0072] When percentages are given herein, it is to be understood that the percentages are percentages by weight and the ratios are by weight unless otherwise indicated.

[0073] discuss

[0074] The present invention is defined in terms of main embodiments and sub-embodiments, and it is understood that the main embodiments can be combined to define other main embodiments, the sub-embodiments can be combined to define supplementary sub-embodiments, and the combinations of sub-embodiments and sub-embodiments can be combined with all main embodiments to define other embodiments of the present invention. The ability to combine embodiments and sub-embodiments is limited only by what is mathematically or physically impossible.

[0075] In a first major embodiment, the invention provides an orally administrable amino acid formulation comprising one or more modified release amino acids, wherein said formulation comprising 2 g of a modified release amino acid is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. USP 39NF 34.

[0076] In a second major embodiment, the invention provides an orally administrable amino acid formulation comprising a modified release amino acid selected from the group consisting of arginine, isoleucine, leucine, tyrosine and valine, wherein said formulation comprising 2 g of a modified release amino acid is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. USP 39NF 34.

[0077] In a third major embodiment, the invention provides an orally administrable amino acid formulation comprising modified-release branched-chain amino acids, wherein said formulation comprising 2 g of modified-release amino acids is administered at <711> In a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified release amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. In a preferred embodiment, the formulation includes modified release of valine, isoleucine and leucine.

[0078] In a fourth major embodiment, the invention provides an orally administrable amino acid formulation comprising a combination of modified release essential amino acids, wherein said formulation comprising 2 g of modified release amino acids is administered at <711> USP 39NF 34, in a dissolution test in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2), at a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%. In a preferred embodiment, the modified released amino acids include arginine, histidine, isoleucine, leucine, threonine, lysine and tryptophan.

[0079] In a fifth major embodiment, the present invention provides an orally administrable amino acid formulation comprising a modified release large neutral amino acid, wherein said formulation comprising 2 g of a modified release large neutral amino acid is administered at <711> USP39NF 34, in a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1N hydrochloric acid (pH 1.2) at a paddle speed of 50 rpm, no more than x% of the modified release amino acid is released within 30 minutes, wherein 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.

[0080] In a sixth major embodiment, the present invention provides a method for treating a metabolic disorder selected from phenylketonuria, hypertyrosinemia, hyperleucinosis, methylmalonic acidemia, homocystinuria, hyperglycinemia, isovaleric acidemia, propionic acidemia and glutamateemia in a patient in need thereof, the method comprising orally administering to the patient a formulation of the present invention as defined by any of the major embodiments or sub-embodiments.

[0081] In a seventh major embodiment, the invention provides an orally administrable amino acid formulation comprising a combination of modified release non-essential amino acids, wherein said formulation comprising 2 g of modified release amino acids is administered at <711> USP 39NF34, in a dissolution test conducted in a paddle apparatus at 37°C in 450 or 500 mL of 0.1 N hydrochloric acid (pH 1.2) with a paddle speed of 50 rpm, no more than x% of the modified released amino acid is released within 30 minutes, wherein x% is 90%, 80%, 70%, 60%, 50% or even 40%.

[0082] In various sub-embodiments, the preparation can include one, all or any combination of essential and non-essential amino acids, and can be specifically designed for any application in which amino acid supplementation is desired. The preparation is particularly well suited for the treatment of patients with amino acid processing disorders (such as phenylketonuria, hypertyrosinemia, hyperleucinemia, methylmalonic acidemia, homocystinuria, hyperglycineemia, isovaleric acidemia, propionic acidemia and glutaric acidemia). Other more special sub-embodiments are more clearly recorded in Table BD. However, it should be understood that some flexibility should be included in these preparations, for example, to be responsible for the amino acid requirements of infants and children, which may cause some amino acids to fall outside the ratios listed.

[0083] Table B shows the relative weight portions of nineteen amino acids commonly used as modified release amino acids in formulations intended for amino acid supplementation and phenylketonuria and hypertyrosinemia. Column A shows the preferred ranges of suitable ratios of amino acids, while column B shows the more preferred ranges of suitable ratios. Table B only records the relative ratios of amino acids in the formulations. Therefore, other ingredients may be included. The total release rate of amino acids from any formulation recorded in Table B is preferably as listed in the first main embodiment.

[0084] Table B

[0085]

[0086]

[0087] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the general formulation of Table B (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0088] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the general formulation of Table B (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0089] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the phenylketonuria preparation of Table B (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0090] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the phenylketonuria preparation of Table B (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0091] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the hypertyrosinemia preparation of Table B (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0092] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the hypertyrosinemia preparation of Table B (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0093] Table C shows the relative weight portions of the nineteen amino acids used as modified release amino acids in hyperleucinosis, methylmalonic acidemia, propionic acidemia, and glutaric acidemia. Column A shows the preferred ranges of suitable ratios of amino acids for each metabolic disorder, while column B shows the more preferred ranges of suitable ratios. Table C only records the relative ratios of the amino acids in the formulation. Therefore, other ingredients may be included. The overall release rate of the amino acids from any formulation recorded in Table C is preferably as listed in the first main embodiment.

[0094] Table C

[0095]

[0096]

[0097] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the hyperleucinosis preparation of Table C (defined by column A or B) in the ratios recited in column A or B, further embodiments are defined.

[0098] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the hyperleucinosis preparations of Table C (defined by columns A or B) in the proportions listed in columns A or B, further embodiments are defined.

[0099] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the methylmalonic acidemia preparation of Table C (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0100] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the methylmalonic acidemia preparation of Table C (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0101] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the propionic acidemia preparation of Table C (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0102] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the propionic acidemia preparation of Table C (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0103] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the glutaric acidemia preparation of Table C (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0104] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the glutaric acidemia preparation of Table C (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0105] Table D shows the relative weight portions of the nineteen amino acids used as modified release amino acids in isovaleric acidemia, homocystinuria and hyperglycinemia. Column A shows the preferred ranges of suitable ratios of amino acids for each metabolic disorder, while column B shows the more preferred ranges of suitable ratios. Table D only records the relative ratios of the amino acids in the formulation. Therefore, other ingredients may be included. The total release rate of the amino acids from any formulation recorded in Table D is preferably as listed in the first main embodiment.

[0106] Table D

[0107]

[0108]

[0109] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the isovaleric acidemia preparation of Table D (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0110] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the isovaleric acidemia preparation of Table D (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0111] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the homocystinuria preparation of Table D (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0112] When the first main embodiment comprises as modified release amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the homocystinuria preparation of Table D (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0113] When the first main embodiment comprises as modified released amino acids three or more, five or more, 10 or more, or 15 or more or even all of the amino acids comprised by the hyperglycinemia preparation of Table D (defined by column A or B) in the ratios listed in column A or B, further embodiments are defined.

[0114] When the first main embodiment comprises as modified released amino acids the essential amino acids, branched chain amino acids or large neutral amino acids comprised by the hyperglycinemia formulation of Table D (defined by column A or B) in the proportions listed in column A or B, further embodiments are defined.

[0115] When the third major embodiment is limited by the relative proportions of the branched chain amino acids listed in Tables E1-E3, additional sub-embodiments are defined.

[0116] Table E1

[0117]

[0118] Table E2

[0119]

[0120] Table E3

[0121]

[0122] When the fourth main embodiment is limited by the relative proportions of the essential amino acids listed in Tables F1-F3 (arginine, histidine, isoleucine, leucine, lysine, threonine, tryptophan and valine), supplementary sub-embodiments are defined.

[0123] Table F1

[0124]

[0125] Table F2

[0126]

[0127] Table F3

[0128]

[0129] While the fifth major embodiment is limited by the relative proportions of the large neutral amino acids listed in Tables G1-G3, additional sub-embodiments are defined.

[0130] Table G1

[0131]

[0132] Table G2

[0133]

[0134]

[0135] Table G3

[0136]

[0137] In another sub-embodiment for the treatment of phenylketonuria, regardless of the definition of the amino acid ratio, the formulation of the present invention comprises three or more, five or more, or all of the following amino acids: arginine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan and valine or one or more pharmaceutically acceptable salts thereof as modified release essential amino acids.

[0138] In another sub-embodiment for the treatment of phenylketonuria, again without regard to the definition of the amino acid ratio, the formulation of the present invention comprises three or more, five or more, or all of the following amino acids: 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 comprises three or more, five or more, or all of the following amino acids: 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 modified-release non-essential amino acids.

[0139] In a particular sub-embodiment, which is suitable for the treatment of any metabolic disorder described herein, regardless of the definition of the ingredient ratio, the formulation comprises arginine and histidine or one or more pharmaceutically acceptable salts thereof as modified-release essential amino acids; and comprises alanine, aspartic acid, carnitine, cystine, glutamine and / or glutamic acid, proline, serine and taurine or one or more pharmaceutically acceptable salts thereof as modified-release non-essential amino acids.

[0140] In a particularly preferred embodiment, the formulation is provided as a unit dosage form of a single dose, comprising 1 to 40 grams of amino acids, wherein the formulation is therapeutically effective to meet the amino acid dietary needs of a human patient when administered 3 to 4 times per day. That is, the formulation is sufficient to meet the patient's dietary needs for the amino acids actually present in the formulation.

[0141] In another major embodiment, the present invention provides a method for manufacturing a formulation defined in any major embodiment or sub-embodiment of the present invention, comprising: (a) providing a first mixture comprising a single or multiple 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 screen to form uniform wet granules; (d) drying the uniform wet granules to form dry granules; (e) optionally passing the dry granules through a screen to form uniform dry granules, and (f) optionally coating the uniform dry granules with a modified release composition.

[0142] The method is particularly well suited for mixing large quantities of amino acids and can be applied additionally by preparing more than one granule with different amino acid mixtures, and combining the different granules to make a 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, and further comprises repeating steps (a)-(d) and optionally (e) and (f) using a second different amino acid mixture comprising only one amino acid, or up to 6 amino acids, to form a second uniform dry granule, and combining the uniform dry granules to make a formulation. In one embodiment, the granules are compressed to form a chewable tablet. In another embodiment, after the granule mixture is formed, it is further processed, for example by adding flavors or nutrients.

[0143] The formulation can also be defined based on the percentage of amino acids in the formulation. Thus, for example, the present invention also provides a formulation comprising greater than 20 wt%, 70 wt% or 90 wt% of amino acids, wherein (a) the amino acids comprise from about 10 wt% to about 80 wt%, from about 20 wt% to about 60 wt%, from about 30 wt% to about 50 wt% or from about 35 wt% to about 45 wt% of essential amino acids; and (b) the essential amino acids comprise all of the following amino acids or any combination thereof: L-isoleucine, L-leucine, L-lysine, L-threonine, L-tryptophan, L-valine, L-methionine, L-histidine and L-arginine (the last two being considered essential only for infants).

[0144] Alternatively, the modified release formulation can be defined based on the ratio of essential amino acids to non-essential amino acids in the formulation. Thus, in various embodiments, the invention provides a modified release, orally administrable amino acid formulation comprising greater than 20 wt%, 50 wt%, 70 wt% or 90 wt% amino acids, wherein (a) the 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) the essential amino acids comprise all or any combination of the following amino acids: L-isoleucine, L-leucine, L-lysine, L-threonine, L-tryptophan, L-valine, L-methionine, L-histidine and L-arginine (the last two of which are considered essential only for infants).

[0145] The formulation preferably includes one or more excipients or additives that can control the release of all amino acids or a subset of amino acids in the formulation. These excipients or additives are generally referred to as "release-delayed excipients or additives" in this article, and can provide a combination of extended release profiles, delayed release profiles, immediate release and extended release or delayed release profiles. In one embodiment, the excipient or additive is a delayed release excipient or additive and is referred to as "a tool that preferentially releases the amino acids between pH 4 and 7" or "a tool that preferentially releases the amino acids in the jejunum when administered orally". In this case, the excipient is usually present in an amount that effectively causes the amino acids to preferentially release from the formulation between pH 4 and 7.

[0146] The term "preferential release" does not exclude the possibility that some of the excipients will release amino acids in a lower pH environment, or even that the release rate in a lower pH environment will be equal to the release rate that occurs in a higher pH environment. The 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 compared to an immediate release dosage form. Thus, for example, when the formulation passes through the GI tract, the excipient may produce a slower release profile over time than would otherwise occur using an immediate release formulation, or as simulated in a suitable in vitro experiment. Alternatively, the excipient may delay the release until the formulation reaches the higher pH environment of the jejunum.

[0147] Modified release excipients or additives / coatings 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, hydroxypropyl methylcellulose, carrageenan, alginic acid and its salts, hydroxyethylcellulose, hydroxypropyl cellulose, gum karaya, gum arabic, gum tragacanth, 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, sodium carboxymethylcellulose, mono- and diglycerides of fatty acids, fatty acids and their esters or derivatives including glyceryl behenate and glyceryl dipalmitostearate, starch and its derivatives such as corn acetate.

[0148] preparation

[0149] These compositions of the present invention can be prepared by a method well known in the pharmaceutical and food supplement fields, and can be administered by a variety of routes depending on whether local or systemic treatment is required. The most preferred route of administration is oral. In addition to amino acids, the composition will typically contain one or more acceptable carriers (excipients or additives). In the manufacture of the composition of the present invention, the active ingredient is typically mixed with an excipient, diluted with an excipient, or enclosed in a carrier in the form of, for example, a capsule, sachet, paper or other container. When the excipient acts as a diluent, it can be a solid, semisolid or liquid material that acts as a vehicle, carrier or medium for the active ingredient. Therefore, the composition can be in the following forms: tablets, pills (including soft and hard gelatin capsules), powders, granules, microspheres, lozenges, sachets (i.e., encapsulated powders or granules or microspheres) sachets, elixirs, suspensions, emulsions, solutions and syrups, containing, for example, up to 90% by weight of amino acids.

[0150] Using this technology, a variety of different amino acid products can be designed in which all or only a selected portion of the amino acids have a modified release profile. For example, a formulation can be designed in which:

[0151] All or only a portion of the amino acids in the formulation are formulated for modified release. That is, greater than 20%, 40%, 60% or 80%, and / or less than 80%, 60%, 40% or 20% of the amino acids are formulated for modified release.

[0152] All or only a portion of the essential amino acids in the formulation are formulated for modified release. That is, greater 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.

[0153] No essential amino acids are formulated for modified release.

[0154] All or only a portion of the non-essential amino acids in the formulation are formulated for modified release. That is, greater than 20%, 40%, 60% or 80%, and / or less than 80%, 60%, 40% or 20% of the non-essential amino acids are formulated for modified release.

[0155] No non-essential amino acids are formulated for modified release.

[0156] Any combination of the above strategies.

[0157] For example, using the formulation strategies described in this application, an amino acid dosage form can be formulated so that only essential amino acids are formulated for modified release, and non-essential amino acids are not. Alternatively, an amino acid dosage form can be formulated so that only non-essential amino acids are formulated for modified release, and essential amino acids are not. Alternatively, depending on the results of release testing for a particular formulation, it may be best to retain some amino acids, such as tyrosine, as immediate release amino acids.

[0158] Depending on the desired pharmacokinetic profile, various strategies are available to control the release of amino acids from the formulation. These strategies will typically involve pH-independent excipients or additives that release the amino acids independently of their location in the gastrointestinal tract or pH-dependent excipients or additives to delay the release of the amino acids until the formulation reaches the higher pH environment of the small intestine.

[0159] pH-independent 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, hydroxypropyl methylcellulose, carrageenan, alginic acid and its salts, hydroxyethylcellulose, hydroxypropyl cellulose, xanthan gum, karaya gum, gum arabic, tragacanth gum, locust bean gum, guar gum, sodium carboxymethylcellulose, methylcellulose, beeswax, carnauba wax, cetyl alcohol, hydrogenated vegetable oils, stearyl alcohol, fatty acids and their esters or derivatives including, for example, glyceryl behenate and stearyl dipalmitin, xanthan gum, starch and starch derivatives such as corn acetate.

[0160] Typically, the at least one pH-independent modified release agent is present in the composition in an amount of about 1 wt% or 5 wt% to about 50 wt% or 25 wt%, preferably about 1 wt% to about 30 wt%. However, it should be understood that the scope of the present invention is not limited to any specific pH-independent sustained release agent.

[0161] 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 swell at a pH greater than 5.5, enteric agents that coat amino acids, and agents that increase the solubility of amino acids by maintaining an acid microenvironment in the formulation at a pH greater than 5.5, such as organic acids. 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 used to promote the release of amino acids at a pH greater than 5.5. The at least one pH-dependent agent is typically present in the composition in an amount of about 0.5 wt % to about 40 wt %, preferably about 1 wt % to about 20 wt %.

[0162] Polymers that swell at a pH above 5.5 include, but are not limited to, acrylic acid copolymers, methacrylic acid copolymers (including, for example, those brand), sodium alginate, carrageenan, alginic acid, pectin, and sodium carboxymethylcellulose.

[0163] Enteric agents include, but are not limited to, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, methacrylic acid copolymers, cellulose acetate trimellitate, hydroxypropyl methylcellulose acetate, succinates, shellac, and zein.

[0164] Agents that increase the solubility of amino acids at a pH greater than 5.5 include, but are not limited to, organic acids. Such organic acids maintain an acid 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.

[0165] The compositions of the present invention are usually first prepared by wet granulation, coated and, if a cookie shape is desired, mixed with other suitable excipients or additives such as lubricants, anti-adherents and disintegrants, and then compressed into a cookie-type dosage form. Flavoring agents can be added during the granulation step, such that they are part of the granules or "intragranular" or subsequently mixed with the granules, before compression of the granules and optionally before compression into tablets / cookies.

[0166] In a wet granulation process, the at least one amino acid and other ingredients are granulated in a wrap-around mixer, high shear mixer or fluidized bed granulator using a granulation liquid (e.g., isopropanol, ethanol, chloroform or water). The binder may be included in the granulation liquid or may be in the dry mixture. 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 may then be coated with functional excipients or additives capable of imparting a modified or extended or delayed release profile.

[0167] Fillers can be included in granular or cake-type formulations and include, but are not limited to, microcrystalline cellulose, mannitol, xylitol, dicalcium phosphate, calcium sulfate, starch, lactose, sucrose, dextrose, sorbitol, fructose and cellulose powder. When present, the filler can be present in the composition in an amount of about 5wt% to about 90wt%, preferably about 10wt% to about 50wt%.

[0168] Disintegrants that may be included in the formulation include, but are not limited to, microcrystalline cellulose, starch, crospovidone, sodium starch glycolate, and croscarmellose sodium. When present, the disintegrant may be present in the composition in an amount of about 0.5 wt % to about 30 wt %, preferably about 1 wt % to about 15 wt %.

[0169] Anti-adherents and glidants that can be used in the composition include, but are not limited to, talc, corn starch, silicon dioxide, sodium lauryl sulfate, and metal stearates. When present, the anti-adherent or glidant can be present in the composition in an amount of about 0.2 wt % to about 15 wt %, preferably about 0.5 wt % to about 5 wt %.

[0170] 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 corn seed oil, talc and waxes, including, but not limited to, beeswax, carnauba wax, cetyl alcohol, glyceryl stearate, glyceryl palmitate, glyceryl behenate, hydrogenated vegetable oil and stearyl alcohol. When present, the lubricant can be present in an amount of about 0.2 wt % to about 20 wt %, preferably about 0.5 wt % to about 5 wt %.

[0171] Binders ("binders") that can be used include, but are not limited to, polyvinyl pyrrolidone, starch, methylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, sucrose solution, dextrose solution, guar gum, xanthan gum, gum arabic, tragacanth gum, locust bean gum, and sodium alginate or any other alginates. When present, the binder may be present in the composition in an amount of about 0.2 wt % to about 10 wt %, preferably about 0.5 wt % to about 5 wt %.

[0172] Coating 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, hydroxypropyl methylcellulose, carrageenan, alginic acid and its salts, hydroxyethylcellulose, hydroxypropyl cellulose, gum karaya, gum arabic, gum tragacanth, locust bean gum, guar gum, sodium carboxymethylcellulose, methylcellulose, beeswax, carnauba wax, cetyl alcohol, hydrogenated vegetable oils, stearyl alcohol, fatty acids and their esters or derivatives including glyceryl behenate and stearyl dipalmitin, xanthan gum, starch and starch derivatives including corn acetate.

[0173] Taste masking materials

[0174] Amino acids have an inherently bitter taste and in one embodiment of the invention, these bitter amino acids are microencapsulated using a taste masking material. Materials useful for masking the taste of pharmaceutical preparations include those that can microencapsulate amino acids, thereby protecting the senses from their bitter taste. The taste masking materials of the present invention provide superior pharmaceutical preparations by, for example, creating pharmaceutical preparations that are more palatable relative to pharmaceutical preparations and / or by creating dosage forms that require less traditional flavoring or taste masking agents.

[0175] Taste-masking materials include, for example, cellulose hydroxypropyl ether (HPC); low-substituted hydroxypropyl ether (L-HPC); cellulose hydroxypropyl methyl ether (HPMC); methylcellulose polymers; ethylcellulose (EC) and mixtures thereof; polyvinyl alcohol (PVA); hydroxyethylcellulose; carboxymethylcellulose and salts of carboxymethylcellulose (CMC); copolymers of polyvinyl alcohol and polyethylene glycol; monoglycerides, triglycerides, polyethylene glycol, modified food starch, acrylic acid polymers and mixtures of acrylic acid polymers with cellulose ethers; cellulose acetate phthalate; ceto-films such as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures of these materials.

[0176] In addition to the use of taste masking materials or materials that increase the shelf life of the formulations described herein to microencapsulate amino acids, the pharmaceutical formulations of the present invention may also include one or more flavoring agents. "Flavoring agents" or "sweeteners" useful in the pharmaceutical formulations of the present invention include, for example, gum arabic syrup, acesulfame potassium, alitose, anise, apple, aspartame, banana, Bavarian cream, berries, blackcurrant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus liqueur, citrus cream, marshmallows, cocoa, cola, cool cherry, cool citrus, tartrate, dextrose, eucalyptus, eugenol, fructose, fruit wine, ginger, glycyrrhizinate, glycyrrhizin (licorice) syrup, grapes, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glycyrrhizinate, maltol, mannitol, maple syrup, marshmallows, menthol, mint cream, mixed berries, neohesperidin dihydrochalcone ketone, neotame, orange, pear, peach, mint, mint cream, raspberry, magen root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, saccharin sodium, saccharin, aspartame, neotame, acesulfame potassium, mannitol, talinose, xylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine, African arrowroot sweet, tutu cream, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of the flavoring ingredients, for example, anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint, and mixtures thereof. In other embodiments, sodium chloride is incorporated into the pharmaceutical formulation.

[0177] Based on the amino acids and excipients or additives, one skilled in the art will be able to determine the best combination of flavors to provide the best flavor product for consumer demand and compliance. See, e.g., Roy et al. Modifying Bitterness: Mechanism, Ingredients, and Applications (1997).

[0178] Microencapsulation

[0179] Amino acids can be microencapsulated by methods known to those of ordinary skill in the art. Such known methods include, for example, spray drying, rotating disc method, hot melt method, spray cooling method, fluidized bed, electrostatic deposition method, centrifugal extrusion method, rotary suspension separation method, polymerization at liquid-gas or solid-gas interface, pressurized extrusion method or spray solvent extraction tank. In addition to these methods, several chemical techniques can also be used, for example, complex coacervation method, solvent evaporation method, polymer-polymer incompatibility, interfacial polymerization method in liquid medium, in-situ polymerization method, liquid drying method and desolvation method in liquid medium. In addition, other methods such as dry granulation method (i.e., roller extrusion and dry granulation method), extrusion / spheronization method or nanoparticle coating method can also be used.

[0180] When microencapsulated, the amino acids may be present in a particle size ranging from 1 μm to 1000 μm, from 5 μm to 200 μm, or from 10 μm to 100 μm.

[0181] Spray drying is generally more accessible to expansion to commercial scale. In various embodiments, the materials used in the spray-drying encapsulation process are emulsified or dispersed into the core material in a concentrated form, for example, 10-60% solids. In some embodiments of the present invention, the solid loading is between about 10-20% or between about 10-40% or between about 40-60%. In one embodiment, the microencapsulation material is emulsified until droplets of about 1 to 3 μm are obtained. In other embodiments, the microencapsulation material is emulsified until droplets of about 1 to 200 μm are obtained or until droplets of about 1 to 100 μm are obtained. Once a dispersion of amino acids and encapsulation materials is obtained, the emulsion is injected into the heating chamber of a spray dryer as droplets. In some embodiments, the droplets are sprayed into the heating chamber or centrifuged from a rotating disk. The microspheres are then dried in the heating chamber and fall to the bottom of the spray drying chamber and collected there.

[0182] Coacervation involves microencapsulation of materials such as active pharmaceutical ingredients and involves a three-part process of particle or droplet formation, coacervate wall formation and capsule isolation. This method can produce microcapsules of very small particle size (10-70 μm).

[0183] Extrusion / spheronization is another method that involves forming a wet mass from the active pharmaceutical ingredient, which is then extruded through a perforated disk to produce short cylindrical rods. These rods are then placed in a rapidly rotating spheronizer to shape the cylindrical rods into uniform spheres. The spheres are then dried using a fluid bed dryer and then coated with a functional coating using a fluid bed equipped with a Wurster core and nozzle. This method produces smooth, uniform spheres that are ideal for receiving functional coatings. Drug loadings up to 80% are possible (depending on the drug properties).

[0184] Dry coating method

[0185] In addition to microencapsulation, the stability or release time of the amino acid used in the present invention can be increased by alternative methods such as dry coating and nanoparticle coating. Dry coating involves the formation of particles coated with amino acids, which are then mixed with other components. Dry granulation is achieved by forming dense compressed blocks, then grinding into the desired particle size, and then mixing with the other components of the pharmaceutical composition. Dry granulation and nanoparticle coating can provide enhanced stability and taste masking properties for active drugs by diluting and separating such components that can improve the shelf life and bitterness masking of amino acid products (if sweeteners or spices are used in the coating material) in a granulated matrix of compatible ingredients.

[0186] Typical techniques for dry granulation are to use slugging or roller compression. During the dry granulation process, dry powders are compressed using a conventional tablet press or more commonly a large, heavy-duty rotary compressor. The resulting compressed mass or "slug" is then ground to the desired particle size. Roller compression is an alternative, gentler method; the powder mixture is squeezed between two rollers to form a compressed tablet. The tablets are usually fragile and brittle and break into fragments immediately. These fragments require more gentle treatment to break them into particles, and this can usually be achieved only by sieving. Parikh, DM, Handbook of Pharmaceutical Granulation Technology, (Marcel Dekker 1997 edition).

[0187] Additional Ingredients

[0188] Other ingredients can also be included in the preparation of the present invention, including vitamins, minerals, fats, fatty acids such as DHA, EPA and ARA (arachidonic acid), carbohydrates and other compounds, preferably with therapeutically effective amounts. Suitable minerals include, for example, calcium, chlorine, chromium, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sulfur, sodium and zinc. Suitable examples of vitamins include vitamin A (retinol, retinol acetate and palmitate, beta-carotene), thiamine (vitamin B1), riboflavin (vitamin B2), nicotinic acid (vitamin B3, nicotinic acid), pantothenic 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).

[0189] In one embodiment, in addition to the amino acids present in the formulation, the formulation comprises one or more, five or more, ten or more, or all of the following nutrients in therapeutically effective amounts: choline, inositol, vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, thiamine, riboflavin, niacin, vitamin B6, folic acid, 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.

[0190] Of course, it should be understood that any of the aforementioned nutrients can be present in a manner well known to those skilled in the art that provides similar nutritional value, such as salts and chelates and esters and other derivatives of the aforementioned nutrients. Table H records various nutrients, effective nutrient doses, and an example of nutrient derivatives that can be incorporated into the formulation of the present invention. The formulation may contain any one or a combination of these nutrients.

[0191] Table H

[0192]

[0193]

[0194] Final formulation

[0195] In a particularly preferred embodiment, the formulation comprises amino acid particles coated with one or modified release excipients (also referred to herein as "coating means for delaying the amino acid release rate", or "coating means for achieving the recited release rate"). The particles can be made by wet or dry granulation techniques, as discussed above, but they are preferably made by wet granulation. The particles 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 1.0-2.0 mm. Each amino acid can be contained within its own particle, but the modified release amino acids are preferably mixed within the particles.

[0196] The modified release properties are preferably achieved using a suitable modified release coating or coating applied to the granules in an amount of 1 wt% to 30 wt% or 5 wt% to 25 wt% based on the weight of the amino acids. Suitable release delaying excipients for the coating are described elsewhere in this document, but one preferred composition comprises a coating of 1 wt% to 15 wt%, 2 wt% to 10 wt% or 5 wt% to 7.5 wt% ethylcellulose based on the weight of the amino acids. Another preferred composition comprises a first coating of ethylcellulose (as described above) and a second coating of 5 wt% to 15 wt% or about 10 wt% glyceryl dibehenate based on the weight of the amino acids. Example

[0197] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the methods claimed herein are made and evaluated, and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numerical values ​​(e.g., amounts, temperature, etc.), but some errors and deviations should still be accounted for.

[0198] Example 1 - Exemplary Amino Acid Mixture for Phenylketonuria

[0199] Table 1 lists four different representative amino acid mixtures used in the dietary management of phenylketonuria, in percentages.

[0200] Table 1

[0201]

[0202]

[0203] 1. Weight based on free base

[0204] Example 2 - Exemplary Powder Formulation and Manufacturing Process

[0205] The table below documents the formulation of five different products containing 18 amino acids (excluding methionine) using polyvinylpyrrolidone (Plasdone) K26 / 32 as a binder and taste masking agent (products 2, 3, 4 and 5). The reference formulation (product 1) does not contain polyvinylpyrrolidone K26 / 32 and does not contain fragrance.

[0206] Table 2a. Composition of products 1, 2, 3, 4 and 5

[0207]

[0208]

[0209] 1 Product 1 is unflavored; Product 2 is unflavored; Product 3 is caramel flavored; Product 4 is vanilla flavored; Product 5 is banana flavored

[0210] Each of the formulations was prepared according to the following general method.

[0211] Step 1

[0212] Manufacturing equipment:

[0213] - Various types of scales

[0214] - High shear mixer: Diosna Laboratory mixer P1 / 6

[0215] -Screen

[0216] -Static oven

[0217] -Mixing machines

[0218] Amino Acid Mixture Product 1

[0219] The amino acids were accurately weighed and mixed using a mixing machine for 20 minutes.

[0220] Preparation of fragrance-free granular products 2

[0221] Accurately weigh the amino acid and use a mixing machine to mix for 20 minutes. Transfer the mixture to Diosna and mix for 1 minute at a blade speed of 250rpm. Use an alcohol solution of polyvinylpyrrolidone K26 / 32 (10% in ethanol solution - 10g to 100g ethanol) to moisten the mixture. After 1 minute and 30 seconds, add the solution and particles and mix for 3 minutes at a blade speed of 250rpm and a shear paddle speed of 500rpm. Discharge the particles and pass through a sieve with a width of 0.8mm. Dry in a static oven at 40°C for about 4 hours. Discharge and pass through a sieve with a width of 0.8mm again.

[0222] Preparation of Flavored Granular Products 3, 4 and 5

[0223] The amino acids were accurately weighed and mixed using a mixing machine for 20 minutes. The mixture was transferred to a Diosna and mixed at a blade speed of 250 rpm for 1 minute and 30 seconds. The mixture was moistened with an alcohol solution of polyvinylpyrrolidone K26 / 32 (10% content in ethanol solution - 10 g to 100 g AA) and perfume. The amount of perfume added to the alcohol solution of polyvinylpyrrolidone K26 / 32 was as follows:

[0224] - 1.13 g of 58% caramel in ethanol (product 3)

[0225] - 1.13 g of 60% vanilla in ethanol (product 4)

[0226] - 1.46 g of 14% banana in ethanol (product 5)

[0227] After 1 minute and 30 seconds, the solution and particles were mixed for 3 minutes at a blade speed of 250 rpm and a shear paddle speed of 500 rpm. The particles were discharged and passed through a screen with a width of 0.8 mm. Drying was carried out in a static oven at 40° C. for about 4 hours. The mixture was discharged and passed through a screen with a width of 0.8 mm.

[0228] Step 2

[0229] Preparation of Methionine Using a manufacturing process similar to that reported in Step 1 for products 3, 4 and 5, using polyvinylpyrrolidone K26 / 32 as a binder and taste masking agent, plus a very small amount of caramel flavor.

[0230] Table 2b. Composition of Product 6 Granular L-Methionine

[0231] Product 6 Components g <![CDATA[L-Methionine 1 > 250.0 <![CDATA[Polyvinylpyrrolidone K26 / 32 2 > 30 <![CDATA[Caramel flavor 3 > 3,6 sum 289

[0232] 1 100g L-methionine is equivalent to 103.2g L-methionine granules

[0233] 2 Polyvinylpyrrolidone K26 / 32 20% in ethanol solution: 30 g is equivalent to 6 g of polyvinylpyrrolidone K26 / 32

[0234] 3 Caramel in 58% ethanol solution: 3.6 g equivalent to 2.088 g pure caramel

[0235] Step 3

[0236] Preparation of the final formulation The powders / granules from step 1 plus the granules from step 2 were mixed in the amounts reported below.

[0237] Table 2c. Composition of final formulation products 7, 8, 9, 10 and 11

[0238]

[0239]

[0240] 1 2.115 g of granulated L-methionine in product 7 is equivalent to 2.050 g of methionine

[0241] 2 Product 7 has no flavor (only a small amount of caramel derived from methionine powder), Product 8 has no flavor (only a small amount of caramel derived from methionine powder), Product 9 has caramel flavor, Product 10 has caramel and vanilla flavor, Product 11 has caramel and banana flavor

[0242] All the above mentioned products were prepared by mixing the powders or granules of products 2, 3, 4 and 5 and the granules of product 6.

[0243] Example 3 - Taste Test

[0244] The tests described below were performed on 6 healthy adults on the final formulations reported in Example 2 (products 8, 9, 10 and 11). The reported results are mean values.

[0245] Table 3. Results of taste test

[0246]

[0247] illustrate:

[0248] - Smell / Taste: 0 = bad 5 = good

[0249] - Persistence (bad taste in mouth): 0 = low persistence 5 = long persistence

[0250] -Dispersibility: 0 = poor 5 = good

[0251] As shown in the above data, the formulation of the present invention is superior to the commercial reference product, and is superior to the formulation without polyvinylpyrrolidone K26 / 32 in terms of dispersibility and taste.

[0252] Example 4 - Exemplary Cookie Formulation and Manufacturing Process

[0253] Starting from a granular product similar to those reported in Example 2 (slightly varying the amount of flavor), several cake-type formulations were prepared.

[0254] Table 4a

[0255]

[0256] 1 2.115g granulated L-methionine is equivalent to 2.050g methionine

[0257] 2 Product 1 Caramel Flavor, Product 2 Vanilla Flavor, Product 3 Banana Flavor

[0258] Sucrose, silicon dioxide and glyceryl dibehenate were added to the above mentioned products through a simple mixing process. The final formulation was as follows:

[0259] Table 4b

[0260]

[0261] 1 Product 4 Caramel Flavor, Product 5 Vanilla Flavor, Product 6 Banana Flavor

[0262] 2 2.115g granulated L-methionine is equivalent to 2.050g methionine

[0263] Tablets with an average weight between 5.5 and 6.2 g were prepared using a single-hole tablet press. To correct some sticking problems during tableting, a new formulation starting with Product 1 was prepared:

[0264] Table 4c

[0265] Product 7 Components g L-Alanine 5.240 L-Arginine 6.140 L-Aspartic Acid 9.180 L-Cystine 2.030 L-Glutamine 12.490 Glycine 4.480 L-Histidine HCl 3.040 L-Isoleucine 5.110 L-Leucine 9.180 L-Lysine HCL 7.390 L-Proline 7.210 L-Serine 5.900 Taurine 0.188 L-Threonine 4.540 L-Tryptophan 1.680 L-Tyrosine 7.510 L-Valine 6.360 L-Carnitine 0.116 <![CDATA[L-Methionine 1 > 2.115 sum 99.899 Polyvinylpyrrolidone 1.050 Caramel Spice 0.986 Comprizuker 50.000 Silicon dioxide 2.500 Glyceryl dibehenate 2.500

[0266] 1 2.115g granulated L-methionine is equivalent to 2.050g methionine

[0267] Example 5 - Taste Test of Cookie Formulations

[0268] Products 4, 5 and 6 reported in Example 4 were tested for smell and taste on 6 healthy adults. The reported results are mean values.

[0269] Table 5

[0270]

[0271] illustrate:

[0272] - Smell - Taste - Review: 0 = bad 5 = good

[0273] - Persistence (bad taste in mouth): 0 = low persistence 5 = long persistence

[0274] -Dispersibility: 0 = poor 5 = good

[0275] Example 6 Preparation of modified release dosage forms

[0276] Mixing preparation

[0277] The individual amino acids, diluent (if used) and HPMC were weighed and sieved and mixed in a suitable mixer for 20 minutes.

[0278] Fragrance-free granule preparation

[0279] Transfer the mixture to a Diosna mixer and mix for 1 minute using a blade speed set at 250 rpm. Wet the mixture with an alcohol solution of EC (15% content). After 1 minute 30 seconds, add the solution and particles for 3 minutes at a blade speed of 250 rpm and a shear paddle speed of 500 rpm. Discharge the particles and pass through a screen with a width of 0.63 mm. Dry in a static oven at 45° C. for about 4 hours. Discharge and pass through a screen with a width of 0.8 mm.

[0280] It is also possible to process 4-5 amino acids into granules at a time and mix all the amino acid granules together to prepare the final product. Some examples of potential formulations are reported in Table 1 of Example 1.

[0281] Example 7 - Modified Release Formulations

[0282] The following formulations were prepared to determine the amounts of hydroxypropylmethylcellulose (HPMC) and ethylcellulose (EC) required to modify the release of tryptophan in a dissolution assay.

[0283] Table 6. Composition of the products

[0284]

[0285]

[0286] 1 Equivalent to 3g EC

[0287] Mixing preparation

[0288] The individual amino acids, mannitol and HPMC were weighed and sieved and mixed in a suitable container using a mixing machine for 20 minutes.

[0289] Fragrance-free granule preparation

[0290] Transfer the mixture to a Diosna mixer and mix for 1 minute using a blade speed of 250 rpm. Wet the mixture with an alcohol solution of EC (ethyl cellulose) (15% content). After 1 minute and 30 seconds, add the solution and particles for 3 minutes at a blade speed of 250 rpm and a shear paddle speed of 500 rpm. Discharge the particles and pass through a screen with a width of 0.63 mm. Dry in a static oven at 45° C. for about 4 hours. Discharge and pass through a screen with a width of 0.8 mm.

[0291] The formulations were evaluated under the dissolution conditions reported below to determine the release rate of tryptophan from the formulations (pH 1.2 for 2 hours, increased to pH 6.8 after 2 hours) relative to a simple mixture of uncoated AA, in basket conditions at 50 and 100 rpm. The results of the dissolution tests are reported in Figure 3 The analysis was performed by HPLC.

[0292] Dissolution test conditions

[0293] Device: stirring paddle

[0294] Temperature: 37℃±5℃

[0295] Medium: 0.1N hydrochloric acid

[0296] pH adjustment: 0.2 M solution of trisodium phosphate dodecahydrate. Medium volume: 450 mL 0.1 N HCl. After 120 minutes, adjust the dissolution medium to pH 6.8 ± 0.05 (according to Ph. Eur. 2.9.3 Delayed Release Solid Dosage Forms Method A).

[0297] Speed: 50rpm or 100rpm

[0298] Sampling time: 5 and 15 minutes for uncoated preparations 5, 15, 30, 60, 120, 125, 135, 150, 180 minutes for coated preparations (after 120 minutes, the pH of the medium becomes pH 6.8)

[0299] Container concentration: approximately 90 μg / mL (a small portion of the powder from the delayed formulation remains as a surfactant during the dissolution test)

[0300] Example 8 - Modified Release Formulation Without Mannitol

[0301] The following formulations were prepared according to the procedure set forth in Example 7 and subjected to dissolution testing under the conditions reported below. The release rate of tryptophan from the formulations was Figure 4 The analysis was performed by HPLC.

[0302] Table 7. Composition of the products

[0303]

[0304] Dissolution test conditions

[0305] Device: stirring paddle

[0306] Temperature: 37℃±5℃

[0307] Medium: 0.1N hydrochloric acid

[0308] Medium volume: 450mL

[0309] Speed: 50rpm

[0310] Sampling time: 5, 15, 30, 60, 120, 180 minutes

[0311] Container concentration: approximately 90 μg / mL (a small portion of the powder from the delayed formulation remains as a surfactant during the dissolution test)

[0312] Example 9 - Modified Release Formulation Without Mannitol or HPMC (Methocel 4KM)

[0313] The following formulations were prepared according to the procedure set forth in Example 7 and subjected to dissolution testing under the conditions reported below. The release rate of tryptophan from the formulations was Figure 5 The analysis was performed by HPLC.

[0314] Table 8. Composition of the products

[0315]

[0316]

[0317] 1 Equivalent to 67g of 15% ethylcellulose ethanol solution

[0318] Mixing preparation

[0319] The individual amino acids were weighed and sieved and mixed in a suitable container using a mixing machine for 20 minutes.

[0320] Fragrance-free granule preparation

[0321] Transfer the mixture to a Diosna mixer and mix for 1 minute using a blade speed of 250 rpm. Wet the mixture with an alcohol solution of EC (ethyl cellulose) (15% content). After 1 minute and 30 seconds, add the solution and particles for 3 minutes at a blade speed of 500 rpm and a shear paddle speed of 1500 rpm. Discharge the particles and pass through a screen with a width of 0.8 mm. Dry in a static oven at 45° C. for about 5 hours and 30 minutes. Discharge and pass through a screen with a width of 1.4 mm.

[0322] The formulations were evaluated under the dissolution conditions reported below to determine the release rate of tryptophan from the formulations (pH 1.2) in basket conditions at 50 rpm. The results of the dissolution tests are given in Figure 5 Reported in China.

[0323] Dissolution test conditions

[0324] Device: Basket

[0325] Temperature: 37℃±5℃

[0326] Medium: 0.1N hydrochloric acid

[0327] Medium volume: 450mL

[0328] Speed: 50rpm

[0329] Sampling time: 5, 15, 30, 60, 120, 180, 240, 300 and 360 minutes

[0330] Container concentration: approximately 90 μg / mL (a small portion of the powder from the delayed formulation remains as a surfactant during the dissolution test)

[0331] Example 10 - Modified release formulation of 19 amino acids coated with ethylcellulose and dibehenyl glycerol

[0332] The following formulations were prepared according to the procedures described below.

[0333] Table 9. Composition of the products

[0334] Components Final mixture (g) Final mixture (%) L-Isoleucine 3.20 4.18 L-Lysine HCl 4.63 <![CDATA[6.05 1 ]]> L-Threonine 3.31 4.33 L-Tryptophan 1.05 1.37 L-Leucine 5.75 7.52 L-Valine 4.00 5.23 L-Methionine 1.28 1.67 L-Cystine 1.27 1.66 L-Glutamine 7.82 10.22 L-Histidine HCl 2.35 <![CDATA[3.07 2 ]]> L-Alanine 3.76 4.91 L-Arginine 3.84 5.02 L-Serine 3.69 4.82 Glycine 2.81 3.67 Proline 4.52 5.91 L-Aspartic Acid 5.76 7.53 Taurine 0.16 0.21 L-Tyrosine 4.70 6.14 L-Carnitine 0.10 0.13 Glyceryl dibehenate 9.12 11.92 Sodium alginate 0.02 0.03 Ethyl cellulose 3.37 4.40 Total 76.51 <![CDATA[100.00 3 ]]>

[0335] 1 Equivalent to 4.84g L-Lysine Base

[0336] 2 Equivalent to 2.49g L-histidine base

[0337] 3 Equivalent to 98.21 g if L-lysine and L-histidine are considered as bases

[0338] Manufacturing equipment for the preparation of the product

[0339] Equipment used for production:

[0340] - Various types of scales

[0341] -ProCepT Mipro 900mL setting

[0342] -Screen

[0343] -Static oven

[0344] -ProCepT fluidized bed coating from bottom to top (melt nozzle) (or any other device suitable for hot melt coating of particles)

[0345] Amino Acid Mix

[0346] Mix all amino acids in a suitable apparatus.

[0347] Particle Preparation

[0348] Transfer the amino acid mixture to ProCepT Mipro and add a granulation solution consisting of a 2% aqueous sodium alginate solution and particles. After granulation, sieve the particles; use the fraction with 0.5 mm < PSD < 1 mm for the next step.

[0349] Coat with ethylcellulose (polymer coating)

[0350] Transfer the sieved fraction of the particles to a fluidized bed apparatus and spray-coat with a methanol solution of 15% (w / w) ethylcellulose. The total amount of polymer added should be approximately equal to 5.00% of the weight of the particles.

[0351] Coat with glyceryl dibehenate (hot melt coating)

[0352] Transfer the particles coated with ethylcellulose to a fluidized bed apparatus and spray-coat with molten glyceryl dibehenate. The total amount of polymer added should be approximately equal to 15.00% of the weight of the particles.

[0353] Dissolution test of the product

[0354] Dissolution medium: Medium pH 1.2 ± 0.1 (0.1 N hydrochloric acid)

[0355] Apparatus: Stirring paddle apparatus (Apparatus 2, modified USP <711>); 50 rpm

[0356] Time: 30 - 60 - 120 - 180 minutes

[0357] Temperature: 37 ± 0.5 °C

[0358] Volume of medium: 500 mL

[0359] Sample: 2.0 g amino acid mixture

[0360] Weight dissolution test: 30 - 60 - 120 - 180 minutes

[0361] The results of the weight dissolution test take into account that ethylcellulose and glyceryl dibehenate are insoluble in the HCl solution. At 30 - 60 - 120 - 180 minutes, the dissolution medium is filtered through a paper filter under vacuum. The filtered powder and filter paper are dried in a vacuum oven at 50 °C ± 4 hours until a constant weight is reached, and the sample is weighed. For the difference, the amount of amino acid released is calculated and reported in Figure 6

[0362] Example 11 – Manufacture and dissolution test of free amino acid preparation and modified release preparation ​

[0363] Table 10a reports the qualitative and quantitative composition of a representative amino acid formulation AA mixture (bkT037 / 71).

[0364] Table 10a

[0365] Amino Acids %(Contains HCl) Amino Acids % (as base) L-Alanine 5.87 L-Alanine 5.87 L-Arginine 6.01 L-Arginine 6.01 L-Cystine 1.99 L-Cystine 1.99 L-Glutamine 12.22 L-Glutamine 12.22 Glycine 4.38 Glycine 4.38 L-Histidine HCl 3.67 L-Histidine 2.98 L-Aspartic Acid 9.00 L-Aspartic Acid 9.00 L-Proline 7.06 L-Proline 7.06 L-Serine 5.77 L-Serine 5.77 Taurine 0.24 Taurine 0.24 L-Tyrosine 7.35 L-Tyrosine 7.35 L-Carnitine 0.16 L-Carnitine 0.16 L-Isoleucine 5.00 L-Isoleucine 5.00 L-Lysine HCl 7.23 L-Lysine 5.78 L-Threonine 5.17 L-Threonine 5.17 L-Tryptophan 1.64 L-Tryptophan 1.64 L-Leucine 8.98 L-Leucine 8.98 L-Valine 6.25 L-Valine 6.25 L-Methionine 2.00 L-Methionine 2.00 Total 100 Total 97.86

[0366] Manufacturing method (general)

[0367] Manufacturing equipment for the preparation of the product

[0368] - Various types of scales

[0369] -Screen

[0370] -Static oven

[0371] -Mixing machines

[0372] All amino acids were sieved and mixed in a suitable apparatus.

[0373] The amino acids were mechanically passed through a 350 μm stainless steel screen and mixed in a mixer for 30 minutes.

[0374] Dissolution test of the product

[0375] Dissolution medium: pH 1.2±0.1 (0.1N hydrochloric acid)

[0376] Device: stirring paddle device (device 2, USP <711> Modified) 50rpm

[0377] Time: 30-60 minutes

[0378] Temperature: 37±0.5℃

[0379] Medium volume: 500mL

[0380] Sample: 2.0g amino acid mixture

[0381] Gravimetric dissolution test: 30-60-120-180 minutes

[0382] Each time point has its own dissolution container. At 30-60-120-180 minutes, the dissolution medium is filtered through a paper filter under vacuum. The filtered powder and filter paper are dried in a vacuum oven at 50°C for ±4 hours until a constant weight is reached, and the sample is weighed. For the difference, the release of the amino acid is calculated.

[0383] result

[0384] The results of the gravimetric dissolution tests are given in Tables 10b and Figure 7 Reported in China.

[0385] Table 10b

[0386] Time (min) % Released AA 0 0 30 99.96 60 100.00 120 99.95 180 99.99

[0387] Analysis conditions:

[0388] HPLC: Agilent Series 1200

[0389] Detector: Fluorometer Agilent Series 1200

[0390] Column: YMC-Triart C18 12nm S (250x 4.6mm–5μm)

[0391] Amino acids were analyzed after extraction (using different methods) and derivatization with FMOC (9-fluorenylmethyl chloroformate). Note: Carnitine was analyzed by LC / MS.

[0392] The results of the dissolution test of the AA mixture (bkT037 / 71) are shown in Tables 10c and Figure 8 Reported in China.

[0393] Table 10c

[0394]

[0395] Table 10d reports the quantitative and qualitative formulations of four different batches of ethylcellulose-coated amino acid particles.

[0396] Table 10d

[0397]

[0398]

[0399] Manufacturing method (general)

[0400] Manufacturing equipment for product preparation

[0401] - Various types of scales

[0402] -ProCepT Mipro 900mL setting

[0403] -Screen

[0404] -Static oven

[0405] - ProCepT fluidized bed coating from bottom to top (melt nozzle) (or any other device suitable for hot melt coating of particles)

[0406] Amino Acid Mix

[0407] Mix all amino acids in a suitable apparatus.

[0408] Particle preparation

[0409] Transfer the amino acid mixture to ProCepT Mipro, and add a granulation solution consisting of a 2% aqueous sodium alginate solution and particles. After granulation, sieve the particles; use the fractions with 0.5 mm < PSD < 1 mm and 0.5 mm < PSD < 1.5 mm for the next step.

[0410] Coat with ethyl cellulose (polymer coating)

[0411] Transfer the sieved fraction of the particles to a fluidized bed apparatus, and spray and coat with an ethanol solution of 15% (w / w) ethyl cellulose. The total amount of polymer added should be approximately equal to 5.00% and 7.50% of the particle weight.

[0412] Dissolution test of the product

[0413] Dissolution conditions:

[0414] Dissolution medium: Medium pH 1.2 ± 0.1 (0.1 N hydrochloric acid)

[0415] Apparatus: Stirring paddle apparatus (Apparatus 2, modified USP <711>); 50 rpm

[0416] Time: 30 - 60 - 120 - 180 - 240 minutes

[0417] Temperature: 37 ± 0.5 °C

[0418] Medium volume: 500 mL

[0419] Sample: 2.0 g amino acid mixture

[0420] Weight dissolution test: Sampling time: 30 - 60 - 120 - 180 minutes. Each time point has its own dissolution container.

[0421] At 30 - 60 - 120 - 180 minutes, the dissolution medium is filtered through a paper filter under vacuum. The filtered powder and filter paper are dried in a vacuum oven at 50 °C ± 4 hours until a constant weight is reached, and the sample is weighed. For the difference, calculate the release amount of amino acids.

[0422] result

[0423] The results of the weight dissolution test are reported in Table 10e and Fig. 9 are reported in.

[0424] Table 10e

[0425]

[0426]

[0427] Analysis of single amino acid release has only been performed on prototype APR Batch 2 and APR Batch 4.

[0428] Analysis conditions :

[0429] HPLC: Agilent Series 1200

[0430] Detector: Fluorometer Agilent Series 1200

[0431] Column: YMC-Triart C18 12nm S (250x 4.6mm–5μm)

[0432] Amino acids were analyzed after extraction (using different methods) and derivatization with FMOC (9-fluorenylmethyl chloroformate). Note: Carnitine was analyzed by LC / MS.

[0433] Results (single amino acid)

[0434] The dissolution test results of APR batch 2 (bkT037 / 72-2) are shown in Tables 10f and Fig.10 Reported in China.

[0435] Table 10f

[0436]

[0437]

[0438] The results of the dissolution test of APR batch 4 (bkT037 / 72-4) are shown in Tables 10g and Fig.11 Reported in China.

[0439] Table 10g

[0440]

[0441] Example 12 - Manufacture and dissolution testing of modified release formulations using ethylcellulose and glyceryl dibehenate as coating agents

[0442] Table 11a reports the qualitative and quantitative compositions of four different amino acid formulations coated with ethylcellulose and glyceryl dibehenate as release rate modifiers.

[0443] Table 11a

[0444]

[0445]

[0446] Manufacturing method (general)

[0447] Manufacturing equipment for product preparation

[0448] - Various types of balances

[0449] - ProCepT Mipro 900mL setting

[0450] - Sieve

[0451] - Static oven

[0452] - ProCepT fluidized bed coating from bottom to top (melt nozzle) (or any other device suitable for hot melt coating of particles)

[0453] Amino acid mixture

[0454] Mix all amino acids in a suitable device.

[0455] Particle preparation

[0456] Transfer the amino acid mixture to ProCepT Mipro and add a granulation solution consisting of an aqueous solution of 2% sodium alginate and particles. After granulation, sieve the particles; use the fraction with 0.5mm < PSD < 1mm or 0.5mm < PSD < 1.6mm for the next step.

[0457] Coating with ethylcellulose (polymer coating)

[0458] Transfer the sieved fraction of the particles to a fluidized bed device and spray-coat with a methanol solution of 15% (w / w) ethylcellulose. The total amount of polymer added should be approximately equal to 5.00% and 7.50% of the particle weight.

[0459] Coating with glyceryl dibehenate (hot melt coating)

[0460] Transfer the particles coated with ethylcellulose to a fluidized bed device and spray-coat with molten glyceryl dibehenate. The total amount of polymer added should be approximately equal to 10.00% of the particle weight.

[0461] Analysis conditions:

[0462] Dissolution test of the product

[0463] Dissolution medium: medium pH 1.2 ± 0.1 (0.1N hydrochloric acid)

[0464] Device: paddle device (device 2, USP <711> modified); 50 rpm

[0465] Time: 30-60-120-180-240 minutes

[0466] Temperature: 37±0.5℃

[0467] Medium volume: 500mL

[0468] Sample: 2.0g amino acid mixture

[0469] Gravimetric dissolution test: Sampling time: 30-60-120-180 minutes At 30-60-120-180 minutes, the dissolution medium is filtered through a paper filter under vacuum. The filtered powder and filter paper are dried in a vacuum oven at 50°C for ±4 hours until a constant weight is reached, and the sample is weighed. For the difference, the release amount of the amino acid is calculated.

[0470] result

[0471] The results of the gravimetric dissolution tests are given in Tables 11b and Fig.12 Reported in China.

[0472] Table 11b

[0473]

[0474]

[0475] The analysis has been performed on prototype APR Batch 5 and APR Batch 7.

[0476] Analysis conditions:

[0477] HPLC: Agilent Series 1200

[0478] Detector: Fluorometer Agilent Series 1200

[0479] Column: YMC-Triart C18 12nm S (250x 4.6mm–5μm)

[0480] Amino acids were analyzed after derivatization with FMOC (9-fluorenylmethyl chloroformate). Note: Carnitine was analyzed using LC / MS.

[0481] Results (single amino acid)

[0482] The dissolution test results of the single amino acids of APR batch 5 (bkT037 / 73-5) are shown in Tables 11c and Fig.13 Reported in China.

[0483] Table 11c

[0484]

[0485] The dissolution test results of the single amino acids of APR batch 7 (bkT037 / 72-7) are shown in Tables 11d and Fig.14 Reported in China.

[0486] Table 11d

[0487]

[0488] Table 11e and Fig.15 The amino acid pool released (gravimetric dissolution assay) from each of the four test formulations at different time points is reported compared to the immediate release amino acid formulation.

[0489] Table 11e

[0490]

[0491]

[0492] Tables 11f-11j report the amounts of individual amino acids released from the four different test formulations at different time points.

[0493] Table 11f

[0494]

[0495]

[0496] Table 11g

[0497]

[0498]

[0499] Table 11h

[0500]

[0501] Table 11i

[0502]

[0503]

[0504] Table 11j

[0505]

[0506]

[0507] Example 13 - Final Amino Acid Composition

[0508] The quantitative composition of the amino acid mixture for phenylketonuria has been optimized according to nutritionists' recommendations; the percentages of the individual amino acids are reported in Table 12a.

[0509] Table 12a

[0510]

[0511]

[0512] Example 14 - In vivo bioavailability in pigs

[0513] In vivo bioavailability studies were conducted in pigs to model the pharmacokinetics and metabolism of the intended formulations of the invention compared to comparable commercially available formulations of free amino acids and milk protein (casein) having similar amino acid ratios and total amounts of amino acids. The following formulations were tested:

[0514] ·APR-04(bkT037 / 72-4)

[0515] ·APR-07(bkT037 / 73-7)

[0516] Free amino acids (bkT037 / 71)

[0517] Casein

[0518] In vivo study design:

[0519] Subjects: 8 pigs

[0520] Design: Crossover of 4 preparations

[0521] · Administration route and method: Oral, by gavage. The product dose given to each animal was mixed with a small amount of water (300 mL) to ensure rapid (≤ 5 minutes) absorption. The total amount of the product was administered using a syringe. Animals were fasted for 13.5 ± 0.5 hours before treatment, and water intake was suspended one hour before and one hour after treatment.

[0522] • Dosage, frequency and duration of administration: The amount of product administered is based on body weight and is equal to 0.8 g amino acid / kg body weight for each test product. The products are administered by a single administration in the morning of each treatment day.

[0523] Wash out: 48-72 hours

[0524] Blood sampling: 0.75h before treatment (T), 0.5h before T, 0.25h before T, 0.25h after T, 0.5h 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

[0525] 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 aspartate were excluded from the analysis due to stability issues. The following parameters were evaluated: area under the concentration / time curve (AUC0-endpoint), peak concentration (C max ) and the time to reach peak concentration (T max ) and C 终点 The behavior of the two APR preparations was compared with that of free amino acids and casein preparations.

[0526] result:

[0527] The plasma concentration of amino acids (AA) is the result of their rate of appearance (Ra) in plasma and their rate of disappearance (Rd) from plasma. Factors controlling Ra include protein uptake and tissue release. Factors controlling Rd include tissue absorption and body loss through urine, sweat, etc. Hormones also help regulate plasma AA concentrations, particularly insulin and glucagon, both of which induce hypoaminoacidemia (but for completely different reasons), and cortisol, which induces hyperaminoacidemia. In addition, in pathological conditions, catecholamines, thyroid hormones, and cytokines can regulate plasma AA levels. The peripheral availability of amino acids (AAs) after protein ingestion is controlled by the liver, which activates urea production during high-protein meals and inhibits urea production during low-protein meals (Cynober 2002). All of these factors can affect the plasma amino acid concentrations reported in this article.

[0528] 1) Average total plasma concentration of amino acids

[0529] The mean plasma concentrations of 14 amino acids over time, as a pool, were Fig.16 and are reported in Table 13a. No baseline values ​​were subtracted before performing the calculations.

[0530] Table 13a

[0531] 14AA (original data) APR-04 APR-07 Free amino acids Casein AUC 0-endpoint 1184.1 1141.8 1200.7 1125.6 <![CDATA[C max ]]> 339.0 341.9 419.2 324.8 <![CDATA[C 终点 ]]> 190.8 168.7 163.5 172.9

[0532] The following observations can be made:

[0533] The APR formulation showed lower plasma peaks (C max ).

[0534] If we consider the curves for individual amino acids, repeated measures ANOVA was applied to the Ln-transformed data in the C from APR-04 with respect to the free amino acids arginine, isoleucine, leucine, tyrosine and valine. max The above showed significant difference (p<0.05).

[0535] If we consider the curves for individual amino acids, relative to the free amino acids isoleucine, leucine, tyrosine and valine, repeated measures ANOVA applied to the Ln-transformed data was performed on the C from APR-07. max There were significant differences (p<0.05).

[0536] If we consider BC data, in C max The differences were even more pronounced in the BC data when the baseline values ​​of the amino acids were subtracted, as shown below in Table 13b, although the differences were not statistically significant for the means of the entire set of amino acids.

[0537] Table 13b

[0538] 14AA (BC data) APR-04 APR-07 Free amino acids Casein AUC 0-endpoint 332.3 302.3 382.9 258.9 <![CDATA[C max ]]> 168.6 172.5 251.5 147.3

[0539] The higher initial peak of amino acid plasma concentration (C max ) decreases rapidly, such as "C 终点 "The parameters prove the same. "C 终点 ” is the plasma concentration of amino acids at the last sampling (5 h)

[0540] If we compare the curves of single amino acids in the group treated with APR-04, the C 终点 Higher (but not statistically significant, except for arginine)

[0541] If we compare the curves of single amino acids in the group treated with APR-07, the C 终点 Higher (but not statistically significant, except for arginine)

[0542] If the essential amino acid subgroups and the BCAAs (branched chain amino acids) subgroups are considered (see points 2 and 3), the C observed in the plasma concentration curves of the combined amino acids is max and C终点 The trend becomes statistically significant.

[0543] 2) Mean values ​​of the concentrations of the essential amino acids analyzed (arginine, histidine, isoleucine, leucine, threonine, lysine, tryptophan, and valine)

[0544] The mean plasma concentrations of the essential amino acids tested, as a pool, were Fig.17 and are reported in Table 13c. The amino acids grouped together for analysis were arginine, histidine, isoleucine, leucine, threonine, lysine, tryptophan, and valine. The subgroup was selected for analysis because the amino acids cannot be synthesized endogenously. As a result, their concentrations in plasma from samples taken from the jugular vein may be more representative of the intestinal absorption of the test formulation than non-essential amino acids, whose presence in the blood may be independently produced by the organism.

[0545] Table 13c

[0546] RequiredAA(raw data) APR-04 APR-07 Free amino acids Casein AUC 0-endpoint 1020.5 1029.0 1098.5 969.9 <![CDATA[C max ]]> 277.0 292.9 409.0 299.1 <![CDATA[T max ]]> 1.50 1.19 0.88 1.16 <![CDATA[C 终点 ]]> 170.7 166.2 138.2 153.0

[0547] Statistical analysis of the data reported in Table 13c (AUC, C max and C 终点 : One-way repeated measures ANOVA with Bonferroni correction using post hoc analysis; T max : Friedman test followed by multiple signal test for pairwise comparisons) are reported in Table 13d.

[0548] Table 13d

[0549] Raw data AUC <![CDATA[C max ]]> <![CDATA[T max ]]> <![CDATA[C 终点 ]]> Free AA relative to APR-04 NS <0.01 NS 0.029 Free AA relative to APR-07 NS <0.01 NS 0.017

[0550] 3) Average BCAAs concentration (branched-chain amino acids: valine, isoleucine, and leucine)

[0551] The mean plasma concentration of the tested branched-chain amino acids, as a pool, was Fig.18 and are reported in Table 13e. The amino acids grouped together for analysis were valine, isoleucine, and leucine. This subset 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. Therefore, they largely bypass first-pass splanchnic metabolism (Brosnan et al.).

[0552] Table 13e

[0553] BCAA (raw data) APR-04 APR-07 Free amino acids Casein AUC 0-endpoint 1365.8 1367.3 1556.1 1365.3 <![CDATA[C max ]]> 350.9 335.8 628.3 401.1 <![CDATA[T max ]]> 2.41 1.72 0.63 1.16 <![CDATA[C 终点 ]]> 253.7 256.2 197.0 228.7

[0554] Statistical analysis of the data reported in Table 13e (AUC, C max and C 终点: One-way repeated measures ANOVA with Bonferroni correction using post hoc analysis; T max : Friedman test followed by multiple signal test for pairwise comparisons) are reported in Table 13f.

[0555] Table 13f

[0556] Raw data AUC <![CDATA[C max ]]> <![CDATA[T max ]]> <![CDATA[C 终点 ]]> Free AA relative to APR-04 NS <0.01 <0.01 0.045 Free AA relative to APR-07 NS <0.01 0.031 <0.01

[0557] 4) Average value of total LNAAs concentration (large neutral amino acids: phenylalanine, tyrosine, tryptophan, threonine, methionine, valine, isoleucine, leucine, histidine)

[0558] Plasma concentrations of the tested large neutral amino acids, as aggregates, were Fig.19 and reported in Table 13g. The amino acids grouped together for analysis were tryptophan, threonine, valine, isoleucine, leucine, histidine. Tyrosine was excluded because of its anomalous behavior in dissolution and in vivo testing, and methionine was excluded because of its instability in the samples. Phenylalanine was excluded from the analysis because it was not present in the test preparations. This subset of essential amino acids was chosen for analysis because LNAAs share a common transport system with phenylalanine to enter the brain. High plasma concentrations of LNAAs may reduce uptake into the brain (Van Spronsen et al.) and may therefore confer clinical advantages. In particular, if analysis of the subset of APR preparations shows increased C 终点 , it can be inferred that the patient may have some "prolonged protection / tolerance" to phenylalanine.

[0559] Table 13g

[0560] LNAA (raw data) APR-04 APR-07 Free amino acids Casein AUC 0-endpoint 925.0 921.8 1028.3 868.9 <![CDATA[C max ]]> 244.3 240.0 383.9 256.6 <![CDATA[T max ]]> 2.16 1.22 0.69 1.16 <![CDATA[C 终点 ]]> 166.9 161.8 132.5 143.0

[0561] Statistical analysis of the data reported in Table 13g (for AUC, C max and C 终点 : One-way repeated measures ANOVA with Bonferroni correction using post hoc analysis; T max : Friedman test followed by multiple signal test for pairwise comparisons) are reported in Table 13h.

[0562] Table 13h

[0563]

[0564] Throughout this application, various publications are cited. The disclosures of these publications are incorporated by reference to more fully describe the state of the art to which the present invention pertains. It will be apparent to those skilled in the art that various modifications and variations may be made in the present invention without departing from the scope or spirit of the present invention. Other embodiments of the present invention will be apparent to those skilled in the art from considerations of the specification and practice of the present invention disclosed herein. It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the present invention being indicated by the following claims.

[0565] References cited

[0566] AMIDON GL S H.SHAH VPCRISON JRA theoretical basis for a biopharmaceutical drug classification: the correlation of in vitro drugproduct dissolution and in vivo bioavailability.Pharm.Res.12:414-420(1995)

[0567] BARACOS VEAnimal models of amino acids metabolism:a focus on theintestine.J.Nutria.134:1656S-1659S(2004)

[0568] BROSNAN JT. BROSNAN ME. Branched-chain amino acids: enzyme and substrate regulation. J Nutr. 2006 Jan; 136 (1 Suppl): 207S-11S.

[0569] CYNOBER LA.Plasma amino acid levels with a note on membranetransport:characteristics.regulation.and metabolicsignificance.Nutrition.2002Sep;18(9):761-6.

[0570] DIOGUARDI F.S.Clinical use of amino acids as dietary supplement:prosand cons.J.Cochexia Sarcopenia Muscle.2:75-80(2011)

[0571] EMA 2010.Guideline on the investigation of bioequivalence CPMP / EWP / QWP / 1401 / 98 Rev.1.London.20 January 2010

[0572] GIOVANNINI M..RIVA E..SALVATICI E..CEFALO G..RADAELLI G.Randomizedcontrolled trial of a protein substitute with prolonged release on theprotein statatus of children with phenylketonuria.J.Am.Coll.Nutr.33.103-110(2014)

[0573] GROPPER S.S.ACOSTA P.B.Effect of simultaneous ingestion of L-aminoacids and whole protein on plasma amino acid and urea nitrogen concentrationsin humans.J.Parenteral Enteral Nutr.15:48-53(1991)

[0574] KEOHANE P.P.GRIMBLE G.K.BROWN B.SPILLER R.C.Influence of proteincomposition and hydrolysis method on intestinal absorption of protein inman.Gut.26:907-913(1985)

[0575] MONTGOMERY R.DRYER R.L.CONWAY T.W.SPECTOR A.A.Biochimica.Aspettimedico biologici.Translation by Berra B..Ragnotti G..Tettamanti G..Edi-Ermes.Milan.1988

[0576] Chapter 1:Nutrizione.pp.1-47

[0577] Chapter 2:Struttura delle proteine.pp.49-121

[0578] Chapter 10:Metabolismo degli amminoacidi.pp.584-639.

[0579] NEY D.M.BLANK R.D.HANSEN K.E.Advances in the nutritional andpharmacological management of phenylketonuria.Co-Clinical Nutrition.Com.17:61-68(2014)

[0580] NEY DM.Does the PKU diet contribute to impaired renal function?JInherit Metab Dis.2013 Sep;36(5):903-4.

[0581] PENA MJ.ROCHA JC AND BORGES N.Amino Acids.Glucose Metabolism andClinical Relevance for Phenylketonuria Management.Ann Nutr Disord&Ther–Volume2 Issue 3–2015

[0582] VAN SPRONSEN FJ.DE GROOT MJ.HOEKSMA M.REIJNGOUD DJ.VAN RIJN M.Largeneutral amino acids in thetreatment of PKU:from theory to practice.J InheritMetab Dis.2010Dec;33(6):671-6.

[0583] VLIET D.VAN.DERKS T.G.J.RIJN M.VAN.DE GROOT M.J.MCDonald A.etal.Single amino acid supplementation in aminoacidopatients:a systemicreview.Orphanet J.Rare Dis.9:1-14(2014)

[0584] WAISBREN S.E.NOEL K.FAHRDACH K.CELLA C.FRAME D.DORENBAUM A.LEVYH.Phenylalanine blood levels and clinical outcomes in phenylketonuria:asystemic literature review and meta-analysis.Mol.Genet.Metab.92:63-70(2007)

[0585] Branched Chain Amino Acids in Clinical Nutrition-Volume1.Rajendram.R;Preedy.V.R.;Patel.V.B.(Eds)2015.XXViii.270 p.91illus.incolor.Hardcover

[0586] WHANG K.Y.AND EASTER R.A.Asian-Aus.J.Anim.Sci.2000Vol.13.No 6:811-816

Claims

1. A modified release amino acid formulation comprising a plurality of modified release particles and a plurality of particles comprising tyrosine, The modified release particles comprise: a) a sodium alginate binder mixed with an amino acid component, the amino acid component comprising at least 15 amino acids selected from the group consisting of: 2.0 to 12.0 parts by weight of L-alanine, 3.0 to 10.5 parts by weight of L-arginine, 5.0 to 10.5 parts by weight of L-aspartic acid, 1.5 to 4.0 parts by weight of L-cystine, 7.0 to 25.0 parts by weight of L-glutamic acid + L-glutamine, 3.5 to 15.0 parts by weight of glycine, 2.0 to 6.5 parts by weight of L-histidine, 2.0 to 8.5 parts by weight of L-isoleucine, 8.0 to 15.0 parts by weight of L-leucine, 4.5 to 10.5 parts by weight of 1.0 to 3.0 parts by weight of L-lysine, 1.0 to 3.0 parts by weight of L-methionine, 4.0 to 7.5 parts by weight of L-phenylalanine, 3.5 to 15.0 parts by weight of L-proline, 2.0 to 8.5 parts by weight of L-serine, 4.0 to 7.5 parts by weight of L-threonine, 1.0 to 4.0 parts by weight of L-tryptophan, 2.5 to 10.0 parts by weight of L-valine, 0.0 to 0.2 parts by weight of L-carnitine, and 0.05 to 1 part by weight of taurine; b) an ethylcellulose coating coating the modified release particles; and in, The formulation comprises 2 g of modified release particles and the formulation is <711> USP 39NF 34, in a dissolution test conducted in a paddle apparatus at 37°C in 500 mL of 0.1 N hydrochloric acid at a paddle speed of 50 rpm, not more than 70% of the amino acids in the formulation are released within 30 minutes; And wherein L-tyrosine is present in the preparation in an amount of 2.5 parts by weight to 14.0 parts by weight.

2. The modified release amino acid formulation of claim 1, wherein the modified release particles comprise 1 wt% to 15 wt% ethylcellulose, based on the weight of the amino acids.

3. The modified release amino acid formulation of claim 2, wherein the formulation further comprises one or more ingredients selected from vitamins, minerals and carbohydrates.

4. The modified release amino acid formulation of claim 2, wherein the formulation further comprises one or more ingredients selected from the group consisting of choline, inositol, vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, thiamine, riboflavin, niacin, vitamin B6, folic acid, 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.

5. The modified release amino acid formulation of claim 2, wherein the formulation further comprises one or more fillers selected from the group consisting of lactose, sucrose, dextrose, sorbitol, fructose and cellulose powder.

6. The modified release amino acid formulation of claim 2, wherein the formulation further comprises one or more disintegrants selected from microcrystalline cellulose, starch, crospovidone, sodium starch glycolate and croscarmellose sodium.

7. The modified release amino acid formulation of claim 2, wherein the formulation further comprises at least one of a glidant and a lubricant.

8. The modified release amino acid formulation of claim 7, wherein the glidant or lubricant is selected from the group consisting of talc, corn starch, silicon dioxide, sodium lauryl sulfate, magnesium stearate, calcium stearate, sodium stearate, stearic acid, sodium stearyl fumarate, hydrogenated cottonseed oil, talc, wax, cetyl alcohol, glyceryl stearate, glyceryl palmitate, glyceryl behenate, hydrogenated vegetable oil, and stearyl alcohol.

9. The modified release amino acid formulation of claim 2, wherein the formulation further comprises a taste masking agent selected from the group consisting of cellulose hydroxypropyl ether (HPC); low-substituted hydroxypropyl ether (L-HPC); cellulose hydroxypropyl methyl ether (HPMC); methylcellulose polymers; ethylcellulose (EC); polyvinyl alcohol (PVA); hydroxyethylcellulose; carboxymethylcellulose and salts of carboxymethylcellulose (CMC); copolymers of polyvinyl alcohol and polyethylene glycol; monoglycerides, triglycerides, polyethylene glycol, modified food starch, acrylic acid polymers, and mixtures of acrylic acid polymers and cellulose ethers; cellulose acetate phthalate; mixtures of HPMC and stearic acid, cyclodextrins, and mixtures of any of the above.

10. The modified release amino acid formulation of claim 2, wherein the formulation further comprises a flavoring selected from the group consisting of acacia syrup, acesulfame potassium, alitose, anise, apple, aspartame, banana, Bavarian cream, berry, black currant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus liqueur, citrus cream, marshmallow, cocoa, cola, cool cherry, cool citrus, tactile essence, tactile essence, dextrose, eucalyptus, eugenol, fructose, fruit wine, ginger, licorice syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon Lemon cream, maltol, mannitol, maple syrup, marshmallow, menthol, mint cream, mixed berry, neohesperidin dihydrochalcone, neotame, orange, pear, peach, mint, mint cream, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, neotame, acesulfame potassium, mannitol, talinose, xylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine, african arrowroot sweetener, tutu cream, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, and combinations thereof.

11. The modified release amino acid formulation of claim 1 , wherein the amino acid component of the modified release particles necessarily comprises L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-proline, L-serine, L-threonine, L-tryptophan, L-valine, L-carnitine, and taurine.

12. The modified release amino acid formulation of claim 1, wherein the amino acid component of the modified release particles comprises L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-proline, L-serine, L-threonine, L-tryptophan, L-valine, L-carnitine, and taurine.

13. The modified release amino acid formulation of claim 1, wherein the amino acid component of the modified release particles comprises L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-glutamine, glycine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-proline, L-serine, L-threonine, L-tryptophan, L-valine, L-carnitine, and taurine.

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