AU2007362357A1

AU2007362357A1 - Organic salts of beta-alanine

Info

Publication number: AU2007362357A1
Application number: AU2007362357A
Authority: AU
Inventors: Joseph Macdougall; Michele Molino
Original assignee: Northern Innovations and Formulations Corp
Current assignee: Northern Innovations and Formulations Corp
Priority date: 2007-12-12
Filing date: 2007-12-12
Publication date: 2009-06-18
Also published as: EP2231588A4; WO2009073947A1; EP2231588A1

Description

WO 2009/073947 PCT/CA2007/002264 Organic salts of 3-Alanine Field of the Invention The present invention relates to a structure and method for producing stable salts of# alanine and organic acids. More specifically, formed salts of the present invention are particularly 5 well suited for oral administration thereby the formed salts may provide enhanced nutritional and/or therapeutical efficacy in relation to the individual components alone. Background of the Invention It is commonly known that increased muscle mass, strength and extended muscular performance occur in the most effective manner when exercise routines are done to complete 10 exhaustion. However, during extended periods of exercise, metabolites from the breakdown of adenosine triphosphate (ATP), mainly hydrogen ions (H), begin to accumulate leading to a decline in the pH levels of blood and muscle, which can be problematic or undesirable. The increase in acidity of the muscle, as a result of the accumulation of H' ions, is directly linked to muscle fatigue, which ultimately causes a decrease in the duration of intensive bouts of exercise (Cooke R, Pate E. 15 The effects of ADP and phosphate on the contraction of muscle fibers. Biophys J. 1985 Nov;48(5):789-98). This fatigue is a result of inhibition of enzymes, by decreased pH, which are vital for energy production and the force-producing capacity of muscles (Febbraio MA, Dancey J. Skeletal muscle energy metabolism during prolonged, fatiguing exercise. J Appl Physiol. 1999 Dec;87(6):2341-7). 20 Carnosine is one of the most effective buffers, or pH stabilizers, in human skeletal muscle, and as such is very efficient at mopping up excess H+ ions. When carnosine is ingested as food, it must be broken down to its constituent amino acids (f-alanine and histidine) in order cross cell membranes, after which it is then reassembled. Due to lack of absorption, in addition to the financial cost of the raw material, carnosine, administration of the constituent amino acids has been 25 explored. For example, administration of exogenous f-alanine has been shown to increase the 1 WO 2009/073947 PCT/CA2007/002264 levels of carnosine in skeletal muscle cells (Bate-Smith EC. The buffering of muscle in rigour: protein, phosphate, and carnosine. J Physiol. 1938;92:336-43). Additionally, other methods for increasing the duration of exercise have been explored. One such method is the administering of compounds that are essential for ATP synthesis and are 5 depleted during exhaustive exercise, such as malic acid. Malic acid is a naturally occurring compound found in a large number of fruits and vegetables, as well as all living cells; which plays a key role in the transportation of NADH from the cytosol to the mitochondria for energy production (ATP production). Malic acid is part of the initiation of the Krebs cycle and is one of the only metabolites that actually decrease in concentration during exercise. Thus, administration of 10 exogenous malic acid will result in increased ATP production as a result of attenuation of malic acid depletion. Supplementation with other deprotonated organic acids can be used for attenuation of metabolic acidosis. For example, citrate lacks all of its acidic protons, yielding three carboxylate functionalities that can readily take up free protons. Since all of acidic protons are removed, the 15 citrate has three sites which are capable of taking up free protons in serum and working muscle. Therefore, administration of deprotonated organic acids can inhibit the decrease in pH, which is a result of ATP hydrolysis, thereby leading to less fatigue resulting from the inhibition of enzymes that are vital for energy production and the force-producing capacity of muscles. Summary of the Invention 20 In the present invention, compounds and methods for their production are disclosed. Specifically, the compounds are salts comprising an organic acid and fl-alanine, and having a structure of Formula 1: Formula 1

NH

3 O A 2 n 2 WO 2009/073947 PCT/CA2007/002264 wherein: A represents a deprotonated organic acid selected from the group consisting of: malate, citrate and fumarate; and where A- is malate or fumarate, n = 2, and 5 where A- is citrate; n = 3. Detailed Description of the Invention In the following description, for the purposes of explanations, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one of ordinary skill in the art that the present invention may be practiced without these 10 specific details. The present invention is directed towards the structures and synthesis of salts of 0-alanine and organic acids. The present invention provides for the production of stable salts, which may afford a combination of f-alanine and an organic acid, free of physiologically unsafe additives to an 15 individual upon administration to said individual. Furthermore, the present invention is particularly well suited for use in tablets, capsules, powders, granules, powdered beverage mixes and other forms known in the art of dietary supplements. 3-alanine combined with an organic acid forms a non-hygroscopic crystalline powder, which is stable in storage and can be processed without special precautions. Due to the non 20 hygroscopic nature of the 3-alanine salt it would be understood by one of skill in the art, that the salt is easy to process and is particularly suitable for processing with rapidly running machines, since it does not tend to stick together or become lumpy. As used herein, 'o-alanine' refers to the chemical beta-alanine, also known as 3 aminopropionic acid. Additionally, as used herein, 'f-alanine' also includes derivatives of3 25 alanine such as esters, amides, and salts, as well as other derivatives, including derivatives having pharmacoproperties upon metabolism to an active form. 3 WO 2009/073947 PCT/CA2007/002264 As used herein, 'malic acid' refers to the chemical 1-Hydroxy-1,2-ethanedicarboxylic acid, (CAS Registry No. 6915-15-17), also known as, hydroxybutanedioic acid, hydroxysuccinic acid, malate, or 2-hydroxybutanedioate. Additionally, as used herein, 'malic acid' also includes derivatives of malate such as esters, amides, and salts, as well as other derivatives, including 5 derivatives having pharmacoproperties upon metabolism to an active form. As used herein, 'citric acid' refers to the chemical 2-hydroxy-1,2,3-propane-tricarboxylic acid, (CAS Registry No. 77-92-9), also known as, 0-hydroxytricarboxylic acid. Additionally, as used herein, 'citric acid' also includes derivatives of citrate such as esters, amides, and salts, as well as other derivatives, including derivatives having pharmacoproperties upon metabolism to an active 10 form. As used herein, 'fumaric acid' refers to the chemical (E)-2-butenedioic acid, (CAS Registry No. 1 10-17-8), also known as, trans-1,2-ethylenedicarboxylic acid, allomaleic acid, and boletic acid. Additionally, as used herein, 'fumaric acid' also includes derivatives of fumarate such as esters, amides, and salts, as well as other derivatives, including derivatives having 15 pharmacoproperties upon metabolism to an active form. As used herein, the term 'organic acid' refers to organic compounds which contain carboxylic acids (-C(O)OH). Typical examples of organic acids include, but are not limited to; malic acid, fumaric acid, citric acid, orotic acid, lactic acid, pyruvic acid, and tartaric acid. As used herein, the term 'pharmaceutically acceptable excipients' refers to substances added 20 to produce quality tablets, chewable tablets, capsules, granulates or powders, but which do not provide nutritive value. A non-exhaustive list of examples of excipients includes monoglycerides, magnesium stearate, modified food starch, gelatin, microcrystalline cellulose, glycerin, stearic acid, silica, yellow beeswax, lecithin, hydroxypropylcellulose, croscarmellose sodium, and crosprovidone. 25 According to the present invention, the compounds disclosed herein comprise molecules of 1-alanine combined with an organic acid to form a salt having a structure according to Formula 1. 4 WO 2009/073947 PCT/CA2007/002264 The aforementioned compound being prepared according to the reaction as set forth for the purposes of the description in Scheme 1: Scheme 1 1) Heat

NH

2 0 2) Cool (RT to 4 C) NH 3 0 A + HA II OH Lower Alcohol [ OH. n 1 2 3 5 With reference to Scheme 1, in the first step of the reaction the fl-alanine (1) is dissolved in an excess of hot lower alcohol. The lower alcohol is considered to be hot, as would be known by one of ordinary skill in the art. Preferably the lower alcohol is considered to be hot when heated to a temperature about 5'C below the boiling point of the corresponding lower alcohol. In various embodiments of the present invention, the lower alcohol is selected from the 10 group consisting of methanol, ethanol, propanol, and isopropanol. These lower alcohols may be used singly or in admixture containing two or more alcohols. Concurrently, in the second step of the reaction the organic acid (2) is dissolved into an excess of hot lower alcohol. The lower alcohol is considered to be hot, as would be known by one of ordinary skill in the art. Preferably the lower alcohol is considered to be hot when heated to a 15 temperature about 5'C below the boiling point of the corresponding lower alcohol. Both solutions above are then mixed together and heated to about the boiling point of the corresponding lower alcohol. If there are solids still present after heating, the solution is filtered while hot to remove any unreacted starting materials. The solution is then allowed to cool to room temperature, covered and refrigerated or cooled until crystallization occurs, preferably for between 20 about 24 to about 48 hours. The resultant crystals are filtered under vacuum and washed with ice cold lower alcohol, yielding a crystalline powder, the f-alanine organic acid salt (3). In larger scale preparations of the present invention, diethyl ether can be added until the cloud point, as would be known to one of skill in the art, is reached after the mixture is cooled to 5 WO 2009/073947 PCT/CA2007/002264 room temperature, after which the solution is refrigerated or cooled to allow crystallization to complete. This will facilitate greater precipitation of the product thus yielding more of the / alanine organic acid salt (3), which would be desired in industrial settings. fl-alanine organic acid salts are used advantageously alone or with additional active 5 ingredients, such as, trace elements, vitamins, mineral substances, or other amino acids as well as, optionally, excipients usually used for the preparation of the respective forms of administration. The forms of administration include, particularly, all varieties of tablets, both those that are swallowed without being chewed, and tablets to be chewed or dissolved in the mouth of an individual, as well as those that are dissolved in a liquid before being ingested by an individual. 10 The tablet forms include uncoated tablets, one-layer or multilayer or encased forms or effervescent tablets. Further preferred forms of administration are capsules of hard and soft gelatin, the latter being particularly suitable to include a liquid core. Additionally, 0-alanine organic acid salts can be used advantageously for the preparation of solutions and suspensions and as a powder, either effervescent or granulated. 15 The examples given below explain the execution of the invention with respect to the production of #-alanine organic acid salts. Provided below is a basic method for producing 0 alanine organic acid salts. However, those of skill in the art will appreciate certain changes may be made in the process of "scaling-up" the reaction to manufacture larger batches of 0-alanine organic acid salts which may be required for commercial uses and supply requirements. Other methods of 20 synthesis may also be apparent to those of skill in the art. 6 WO 2009/073947 PCT/CA2007/002264 Examples Example 1 0 1) Heat F . NH2O O 2) Cool (RT to4 0 C) NH 0 O + HO EtOH OH OH OH O _ OH2 OH O2 1 2 3 178.18 g (2 mol) of #-alanine (1) is dissolved into 400mL of hot ethanol, solution 1. 5 Concurrently, 134.09 g (1 mol) of malic acid (2) is dissolved in 200mL of hot ethanol, solution 2. Solution 2 is added to solution 1 with stirring and the resultant solution is heated to the boiling point. If there are solids still present the solution is filtered at this temperature to remove unreacted starting materials. The solution is then allowed to cool to room temperature and then covered and refrigerated to allow crystallization to complete; about 24 hours. The resultant crystals are filtered 10 under vacuum and washed with ice cold ethanol, yielding a crystalline powder, the -alanine malate (3). Example 2 0 1) Heat N O + HOOH 2) Cooro(RT to 4 C) NH OH 0 L- OH HO -' Propanol K OH] [0 1 2 3 15 178.18 g (2 mol) of -alanine (1) is dissolved into 400mL of hot propanol, solution 1. Concurrently, 116.07 g (1 mol) of fumaric acid (2) is dissolved in 200mL of hot propanol, solution 2. Solution 2 is added to solution 1 with stirring and the resultant solution is heated to the boiling point. If there are solids still present the solution is filtered at this temperature to remove unreacted starting materials. The solution is then allowed to cool to room temperature and refrigerated to 20 allow crystallization to complete; about 24 hours. The resultant crystals are filtered under vacuum and washed with ice cold ethanol, yielding a crystalline powder, the -alanine fumarate (3). 7 WO 2009/073947 PCT/CA2007/002264 Example 3 0 - o HO OH1) HeatI NH2O HO OH Cool tto4 OC) H 0 OH 0 OH O Isopropanol OH O OH o HO LO.3 1 2 3 267.27 g (3 mol) of #-alanine (1) is dissolved into 600mL of hot isopropanol, solution 1, Concurrently, 192.12 g (1 mol) of citric acid (2) is dissolved in 300mL of hot isopropanol, solution 5 2. Solution 2 is added to solution 1 with stirring and the resultant solution is heated to the boiling point. If there are solids still present the solution is filtered at this temperature to remove unreacted starting materials. The solution is then allowed to cool to room temperature and refrigerated to allow crystallization to complete; about 24 hours. The resultant crystals are filtered under vacuum and washed with ice cold ethanol, yielding a crystalline powder, the f-alanine citrate (3). 8 WO 2009/073947 PCT/CA2007/002264 Extensions and Alternatives In the foregoing specification, the invention has been described with a specific embodiment thereof; however, it will be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. 9

Claims

1. A salt of -alanine and an organic acid, having the general formula: NH 3 O *AP OH wherein: A- represents a deprotonated organic acid selected from the group consisting of: malate, citrate and fumarate; and where A is malate or fumarate, n = 2, and where A is citrate; n = 3.

2. A composition comprising the salt of -alanine and an organic acid of claim 1, wherein the composition further comprises pharmaceutically acceptable excipients.

3. The composition of claim 2 wherein the pharmaceutically acceptable excipients are selected from the group consisting of monoglycerides, magnesium stearate, modified food starch, gelatin, microcrystalline cellulose, glycerin, stearic acid, silica, yellow beeswax, lecithin, hydroxypropylcellulose, croscarmellose sodium, and crosprovidone.

4. The salt of O-alanine and an organic acid of claim I wherein said salt is provided in a dosage form selected from the group consisting of tablets, chewable tablets, capsules, granulates or powders.

5. The composition of claim 2 wherein the composition is provided in a dosage form selected from the group consisting of tablets, chewable tablets, capsules, granulates or powders.

6. The salt of 0-alanine and an organic acid of claim 4 wherein said salt is administered to a mammal.

7. The composition of claim 5 wherein the composition is administered to a mammal.

8. A method for producing a f-alanine organic acid salt comprising at least the steps of: 10 WO 2009/073947 PCT/CA2007/002264 a) dissolving f-alanine in hot lower alcohol; b) dissolving an organic acid in hot lower alcohol; c) mixing the resultant solutions of a) and b); d) cooling the resultant mixture; and e) isolating the resulting O-alanine organic acid salt.

9. The method of claim 8 wherein the lower alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, and isopropanol.

10. The method of claim 8 wherein the -alanine and the organic acid are present in a 2:1 ratio.

11. The method of claim 10 wherein the organic acid is selected from the group consisting of malic acid and fumaric acid.

12. The method of claim 11 wherein the f-alanine and the organic acid are present in a 3:1 ratio.

13. The method of claim 12 wherein the organic acid is citric acid.

14. The method of claim 8 wherein the resultant mixture is cooled until crystallization occurs.

15. The method of claim 8 wherein the 0-alanine organic acid salt is isolated by vacuum filtration followed by washing of the filtrate with cold lower alcohol.

16. The -alanine organic acid salt of claim 8 having the molecular structure of: [N H 3 O SAG OH - - n wherein: A- represents a deprotonated organic acid selected from the group consisting of: malate, citrate and fumarate; and where A is malate or fumarate, n = 2, and where A- is citrate; n = 3. 11