WO2014011895A2

WO2014011895A2 - High solubility acid salts, intravenous dosage forms, nutrition supplementation and methods of use thereof

Info

Publication number: WO2014011895A2
Application number: PCT/US2013/050100
Authority: WO
Inventors: Banavara L. Mylari; Frank C. SCIAVOLINO
Original assignee: Thetis Pharmaceuticals Llc
Priority date: 2012-07-11
Filing date: 2013-07-11
Publication date: 2014-01-16
Also published as: WO2014011895A3

Abstract

Disclosed herein are highly soluble and bioavailable ω-3 fatty acid salts. These fatty acid salts have high bioavailability when administered orally. They are also used for relatively high concentration intravenous dosage forms that can be used for the treatment of atrial defibrillation. These fatty acid salts can also be used to supplement food, drink and nutraceuticals with ω-3 polyunsaturated fatty acids.

Description

HIGH SOLUBILITY FATTY ACID SALTS, INTRAVENOUS DOSAGE FORMS, NUTRITION SUPPLEMENTATION AND METHODS OF USE THEREOF

RELATED APPLICATION

This application claims priority from U.S. Provisional Patent Application No.

61/670,262, filed on July 11, 2012, incorporated by reference herein in its entirety.

BACKGROUND

The beneficial effects of polyunsaturated fatty acids of the ω-3 series on multiple risk factors for cardiovascular illnesses are well known; for example the patents IT 1235879, U.S. Pat. No. 5,502,077, U.S. Pat. No. 5,656,667 and U.S. Pat. No. 5,698,594 refer respectively to hypertriglyceridemia, defects of the cholesterol level and hypertension. However, each of the cited documents deal with the treatment of risk factors, not with real and proclaimed illnesses.

ω-3 polyunsaturated and other fatty acid salts have poor solubility in aqueous solution. This prevents intravenous administration of ω-3 fatty acids to the tissue in which they have much of their therapeutic effect, i.e. the circulatory system. Further, this poor solubility increases the difficulty of combining ω-3 fatty acids with food, drink and nutraceuticals.

It is well known in the art that highly water soluble medicinal preparations, when administered orally, result in efficient absorption of such preparations from the

gastrointestinal tract into systemic circulation. Another hallmark of such preparations is the rapid rate at which they are absorbed into the systemic circulation resulting in a high concentration of the active agent in the blood. Also, water soluble preparations are especially suitable for parenteral administration, for example, intravenous administration.

Because of their polyunsaturated nature ω-3 polyunsaturated fatty acids are susceptible to lipid oxidation. This oxidation leads to formation of undesirable fishy and rancid off-flavors that prevent such products from being palatable. Increasing the solubility of ω-3 polyunsaturated fatty acids avoids the need for emulsions when trying to add these fatty acids to food. The oil-water interface of emulsions greatly influences oxidation of ω-3 polyunsaturated fatty acids. Its removal would prevent much oxidation. Further, solubility of ω-3 polyunsaturated fatty acids in the aqueous phase would allow the ω-3 polyunsaturated fatty acids greater access to aqueous soluble anti-oxidants that would prevent oxidation of the fatty acids. Thus, there exists a need in the art for higher solubility fatty acid salts, particularly ω- 3 fatty acid salts.

SUMMARY

Provided herein is a method of increasing the concentration of a fatty acid in aqueous solution comprising combining the anion fatty acid with a cation that increases the solubility of the fatty acid in aqueous solution. In other embodiments, the fatty acid is eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). In other embodiments, the cation is metformin, piperazine, meglumine or lysine.

In certain embodiments of this method, the maximum concentration of the fatty acid is greater than 15 or μg/mL in the plasma of the mammalian subject. In other embodiments, the maximum concentration of the fatty acid is greater than 10 μg/mL and less than 100 μg/mL; greater than 10 μg/mL and less than 50 μg/mL; greater than 10 μg/mL and less than 40 μg/mL; greater than 15 μg/mL and less than 40 μg/mL; or greater than 15 μg/mL and less than 35 μg/mL in the plasma of the mammalian subject.

The disclosure provides a composition comprising a salt of a fatty acid comprising a fatty acid and a cation, wherein the salt of the fatty acid has a solubility in aqueous solution greater than 50 and less than 1000 mg/mL; greater than 100 and less than 1000 mg/mL; greater than 50 and less than 500 mg/mL or greater than 100 and less than 500 mg/mL. In other embodiments, the solubility of the fatty acid salt in aqueous solution is 50 to 100 times the solubility of the ethyl ester of the fatty acid. In certain embodiments, when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is greater than 10 μg/mL or 15 μg/mL in the plasma of the mammalian subject. In certain embodiments, when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is greater 10 μg/mL and less than 100 μg/mL; greater than 10 μg/mL and less than 50 μg/mL; greater than 10 μg/mL and less than 40 μg/mL; greater than 15 μg/mL and less than 40 μg/mL; or greater than 15 μg/mL and less than 35 μg/mL in the plasma of the mammalian subject. In other embodiments, when the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is at least two times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form. In other embodiments, the maximum

concentration of the fatty acid is at least 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form.

In other embodiments, the fatty acid is eicosapentaenoic acid (EPA) or

docosahexaenoic acid (DHA). In other embodiments, the cation is metformin, piperazine, meglumine or lysine. In certain embodiments, the maximum concentration of the fatty acid is two times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form. In other embodiments, the maximum concentration of the fatty acid is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form.

In other embodiments, when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the area under the curve from time zero to 24 hours of the fatty acid is greater than 90, 100 or 120 μg*h/mL in the plasma of the mammalian subject. In certain embodiments, the area under the curve from time zero to 24 hours of the fatty acid is greater than 100 or 120 μg*h/mL in the plasma of the mammalian subject. In other embodiments, the area under the curve from time zero to 24 hours of the fatty acid is greater than 90 μg*h/mL and less than 160 μg*h/mL; greater than 100 μg*h/mL and less than 150 μg*h/mL; or greater than 110 μg*h/mL and less than 150 μg*h/mL in the plasma of the mammalian subject.

In other embodiments, the fatty acid is eicosapentaenoic acid (EPA) or

docosahexaenoic acid (DHA). In other embodiments, the cation is metformin, piperazine, meglumine or lysine.

The disclosure also a method of treating atrial fibrillation, comprising intravenously administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising piperazine, meglumine or lysine salts of EPA or DHA.

In one embodiment, the effective amount is between 5-100 mg/kg of the subject's body weight per day.

In other embodiments, the subject is cardiopathic or is affected by coronary ischemia, cardiac insufficiency, cardiac decompensation or diabetic pathology concomitant with cardiopathy.

The disclosure also provides a method of reducing the probability of an occurrence of a major cardiovascular event in a subject who is affected by atrial fibrillation, comprising intravenously administering to a subject in need thereof an effective amount of a

pharmaceutical composition comprising piperazine, meglumine or lysine salts of EPA or DHA. In one embodiment of this method, the effective amount is between 5-100 mg/kg of the subject's body weight per day.

In other embodiments of this method, the subject is cardiopathic or is affected by coronary ischemia, cardiac insufficiency, cardiac decompensation or diabetic pathology concomitant with cardiopathy. In another embodiment, the subject who is affected by atrial fibrillation has not undergone a previous infarct episode.

The disclosure also provides an intravenous dosage form comprising a salt of a fatty acid comprising a fatty acid and a cation, wherein the salt of the fatty acid concentration of greater than 50 and less than 1000 mg/mL; greater than 100 and less than 1000 mg/mL; greater than 50 and less than 500 mg/mL or greater than 100 and less than 500 mg/mL. In other embodiments, the solubility of the fatty acid salt is 50 to 100 times the solubility of the ethyl ester of the fatty acid. In certain embodiments, when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is greater than 10 μg/mL in the plasma of the mammalian subject. In certain embodiments, the maximum concentration of the fatty acid is greater than 15 or μg/mL in the plasma of the mammalian subject. In other embodiments, the maximum concentration of the fatty acid is greater than 10 μg/mL and less than 100 μg/mL; greater than 10 μg/mL and less than 50 μg/mL; greater than 10 μg/mL and less than 40 μg/mL; greater than 15 μg/mL and less than 40 μg/mL; or greater than 15 μg/mL and less than 35 μg/mL in the plasma of the mammalian subject.

In other embodiments, the fatty acid is eicosapentaenoic acid (EPA) or

docosahexaenoic acid (DHA). In other embodiments, the cation is metformin, piperazine, meglumine or lysine. In other embodiments, the maximum concentration of the fatty acid is two times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form. In other embodiments, the maximum concentration of the fatty acid is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form.

The disclosure also provides an intravenous dosage form comprising a salt of a fatty acid comprising a fatty acid and a cation, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the area under the curve from time zero to 24 hours of the fatty acid is greater than 90 μg*h/mL in the plasma of the mammalian subject. In certain embodiments, the area under the curve from time zero to 24 hours of the fatty acid is greater than 100 or 120 μg*h/mL in the plasma of the mammalian subject. In other embodiments, the area under the curve from time zero to 24 hours of the fatty acid is greater than 90 μg*h/mL and less than 160 μg*h/mL; greater than 100 μg*h/mL and less than 150 μg*h/mL; or greater than 110 μg*h/mL and less than 150 μg*h/mL in the plasma of the mammalian subject.

In other embodiments, the fatty acid is eicosapentaenoic acid (EPA) or

The disclosure also provides a nutraceutical, food product or drink product comprising a salt of a fatty acid wherein the fatty acid is eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). In certain embodiments, the cation of the salt is metformin, piperazine, meglumine or lysine.

The disclosure also provides a method of making a fatty acid supplemented nutraceutical, food product or drink product comprising mixing an aqueous solution of a fatty acid comprising a fatty acid and a cation wherein the fatty acid is eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). In other embodiments, the cation is metformin, piperazine, meglumine or lysine. In other embodiments, the cation is metformin, piperazine, meglumine or lysine.

In certain embodiments of the nutraceutical, food product or drink product, the fatty acid the concentration of the fatty acid in the aqueous solution is between greater than 50 and less than 1000 mg/mL; greater than 100 and less than 1000 mg/mL; greater than 50 and less than 500 mg/mL or greater than 100 and less than 500 mg/mL. In other embodiments, the solubility of the fatty acid salt is 50 to 100 times the solubility of the ethyl ester of the fatty acid.

In any of the above embodiments, the fatty acid piperazine salt of EPA or DHA can comprise a compound of the structural Formula I, II, III, IV, V, VI, VII or VIII:

Formula I

Formula I I

Formula IV

Formula VIII

wherein X^" is an anion of a pharmaceutically acceptable acid compound, or a mixture

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a line graph showing mean plasma metformin concentrations ^g/mL) rats administered a single oral dose of metformin EPA or metformin HCl plotted against time. 3 male and 3 female rats were used for each time point. Figure 2 is a line graph showing mean plasma metformin concentrations ^g/mL) in male and female rats administered a single oral dose of metformin EPA, metformin HC1, EPA FFA or EPA ethyl ester plotted against time.

Figure 3 is a line graph showing the same data as Figure 3 where the plasma metformin levels are shown in log 10 scale.

Figure 4 is a line graph showing mean plasma EPA concentrations ^g/mL) in rats administered a single oral dose of metformin EPA, EPA FFA or EPA ethyl ester plotted against time. 3 male and 3 female rats were used for each time point.

Figure 5 is a line graph showing mean plasma metformin concentrations ^g/mL) in male and female rats administered a single oral dose of metformin EPA, metformin HC1, EPA FFA or EPA ethyl ester plotted against time.

Figure 6 is a line graph showing the same data as Figure 5 where the plasma EPA levels are shown in log 10 scale. DETAILED DESCRIPTION

The disclosure provides salts of fatty acids with high solubility and bioavailability. High solubility of the fatty acids allows for them to be better absorbed when administered orally to subjects and allows for intravenous administration of these fatty acids at higher dosages. Further, the high solubility and bioavailability of these fatty acid salts allows them to be easily used as additives to food and nutraceuticals.

In certain embodiments, the fatty acids are ω-3 polyunsaturated fatty acids, ω-3 polyunsaturated fatty acids include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Cations that are used to form fatty acid salts include metformin, piperazine, meglumine and lysine. Examples of fatty acid salts made with these cations are provided below.

Metformin eicosapentaenoate is represented below:

wherein any of the nitrogen atoms of metformin can be protonated . This is also referred to, herein, as TP-101. Metformin docosahexaenoate is represented below:

wherein any of the nitrogen atoms of metformin can be protonated.

Provided herein are piperazine salts of EPA and DHA, as well as meglumine salts of EPA and DHA. In one aspect, provided herein is a compound of Formula I:

Formula I

In another aspect, provided herein is a compound of Formula II:

Formula II

The compound of Formula I and II are, respectively, the mono-salt of EPA with piperazine and the mono-salt of DHA with piperazine.

In ano Formula IV:

Formula IV

The compounds of Formula III and IV are, respectively, the di-salt of EPA with piperazine and the di-salt of DHA with piperazine. In still ano V and VI:

Formula V

Formula VI

wherein X^" is a pharmaceutically acceptable counter anion. The pharmaceutically acceptable counter anion can derived from acid compounds listed in Table 1, pp 406 - 407, Handbook of Pharmaceutical Salts, P. Heinrich Stahl Camille G. Wermuth (Eds.), incorporated by reference herein. In an embodiment, the pharmaceutically acceptable counter anion is selected from mineral acids, such as hydrochloric acid, hydrobromic acid, and phosphoric acid. In another embodiment, the pharmaceutically acceptable counter anion is selected from carboxylic acids, poly-carboxylic acids, and poly-hydroxy carboxylic acids, such as acetic acid, propionic acid, succinic acid, maleic acid, malic acid, tartaric acid, lactic acid, citric acid, and benzoic acid. In another embodiment, the pharmaceutically acceptable counter anion is selected from sulfonic acids and hydroxyl-sulfonic acids, including, but not limited to, methanesulfonic acid, isethionic acid, ethanesulfonic acid, 2 -hydro xy- ethanesulfonic acid, and benzenesulfonic acid. In another embodiment, the pharmaceutically acceptable counter anion is selected from amino acids, including, but not limited to, glycine, alanine, lysine, arginine, aspartic acid, or glutamic acid. In another embodiment, X^" is an omega-3 polyunsaturated acid, such as eicosapentaenoic acid or docosahexaenoic acid.

In an embodiment, provided herein are the following compounds: the hydrochloride salt of Formula V, the hydrobromide salt of Formula V, the phosphate salt of Formula V, and the sulfate salt of Formula V.

In another embodiment, provided herein are the following compounds: the hydrochloride salt of Formula VI, the hydrobromide salt of Formula VI, the phosphate salt of Formula VI, and the sulfate salt of Formula VI.

The present invention also relates to compounds of the Formula V and Formula VI wherein X^" is a pharmaceutically acceptable counter anion derived from naturally occurring amino acids. Examples of the amino acids include, but are not limited to, glycine, alanine, lysine, and glutamic acid.

In another aspect, provided herein are compounds of the Formula VII and formula VIII. These

Formula VIII

The compounds of Formula I, II, III, IV, V, VI, VII, or VIII also include isomers and enantiomers wherever it is applicable.

Also provided herein are solvates (e.g., hydrates) of the compounds of Formula I, II,

III, IV, V, VI, VII, or VIII. As used herein, the term "solvate" refers to any form of the compounds of the invention that are bound by a non-covalent bond to another molecule (such as a polar solvent). Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. When the solvent is water, the solvate formed is a hydrate. Example hydrates include hemihydrates, mono hydrates, dihydrates, etc.

In some embodiments, the compounds of Formula I, II, III, IV, V, VI, VII, or VIII are crystalline, which is useful not only for drug delivery purposes, but is also useful in the preparation of pharmaceutical formulations, and will improve general handling,

manipulation, and storage of the drug compound. In one embodiment, the crystalline form of the compound of Formula I, II, III, IV, V, VI, VII, or VIII is in a particular polymorph form.

The ability of a substance to exist in more than one crystal form is defined as polymorphism; the different crystal forms of a particular substance are referred to as "polymorphs." In general, polymorphism is affected by the ability of a molecule of a substance to change its conformation or to form different intermolecular or intra-molecular interactions, particularly hydrogen bonds, which is reflected in different atom arrangements in the crystal lattices of different polymorphs. In contrast, the overall external form of a substance is known as "morphology," which refers to the external shape of the crystal and the planes present, without reference to the internal structure. Crystals can display different morphology based on different conditions, such as, for example, growth rate, stirring, and the presence of impurities.

gastrointestinal tract into systemic circulation. Another hallmark of such preparations is the rate at which they are absorbed into systemic circulation resulting in high concentration of the active agent or agents in the blood. Moreover, for delivery of xenobiotics via the intravenous route, they must be presented as a clear solution. PUFAs and esters of PUFAs are practically insoluble in water. In fact, they form soap-like emulsions when mixed with water. Therefore, the potential to derive optimum therapeutic benefits of PUFAs should be markedly facilitated by delivery of water soluble PUFAs. The compounds of the present invention are markedly more water soluble to achieve high oral absorption and to enable the preparation of intravenous dosage forms.

Fatty acid salts according to the disclosure are able to be diluted in aqueous solution at high concentrations. In certain embodiments, the fatty acid salts described herein have a solubility in aqueous solution greater than 50 and less than 1000 mg/mL; greater than 100 and less than 1000 mg/mL; greater than 50 and less than 500 mg/mL; greater than 100 and less than 500 mg/mL in solution. In other embodiments, fatty acid salts described herein have a solubility in aqueous solution of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg/mL. The fatty acid salts have solubility in aqueous solution that is 50-100 times greater than the solubility of the ethyl ester of the fatty acid. In other embodiments, the solubility of fatty acid salts is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times greater than the solubility of the ethyl ester of the fatty acid.

Moreover, the fatty acid salts described herein have high bioavailability when administered to human subjects. For example, the maximum plasma concentration (Cmax), dose normalized maximum plasma concentration (Cmax/dose), area under the curve from time zero to 24 hours (AUC(0-24)), dose normalized area under the curve (AUC/dose), or average plasma concentration are higher for a given dose of fatty acid salt described herein, than for fatty acid salts described in the prior art.

For example, when metformin EPA is administered to a mammalian subject at 52 mg/kg it produces a Cmax of between 10 μg/mL and 50 μg/mL of EPA. In other embodiments, when metformin EPA is administered to a mammalian subject at 52 mg/kg it produces a Cmax of between 10 μg/mL and 40 μg/mL; 10 μg/mL and 30 μg/mL; 10 μg/mL and 20 μg/mL·^■, 10 μg/mL and 15 μg/mL; 15 μg/mL and 50 μg/mL; 15 μg/mL and 40 μg/mL·^■, 15 μ /ηιΕ and 30 μg/mL; 15 μ /ηιΕ and 20 μg/ L·, 20 μ /ηιΕ and 50 μg/mL^■, 20 μ /ηιΕ and 40 μg/ L·, 20 μ /ηιΕ and 30 μg/ L·, 30 μ /ηιΕ and 50 μg/mL^■, 30 μ /ηιΕ and 40 μg/ L·, and 40 μ /ηιΕ and 50 μg/mh of EPA in plasma of the mammalian subject. In certain embodiments, the Cmax is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 μg/mL of EPA in plasma of the mammalian subject. In other embodiments, the Cmax of EPA is 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times the Cmax of EPA from the same dose of the ethyl ester of the fatty acid. In certain embodiments, the Cmax is between 2 and 10, 3 and 8 or 4 and 6 times the Cmax of EPA from the same dose of the ethyl ester of the fatty acid. In other embodiments, the Cmax is about 5 times the Cmax of EPA from the same dose of the ethyl ester of the fatty acid.

In other embodiments, when metformin EPA is administered to a mammalian subject at 104 mg/kg it produces a Cmax of between 10 μg/mL and 50 μg/mL of EPA in plasma of the mammalian subject. In other embodiments, when metformin EPA is administered to a mammalian subject at 104 mg/kg it produces a Cmax of between 10 μg/mL and 40 μg/mL; 10 μg/mL and 30 μg/mL; 10 μg/mL and 20 μg/mL; 10 μg/mL and 15 μg/mL; 15 μg/mL and 50 μg/mL; 15 μg/mL and 40 μg/mL; 15 μg/mL and 30 μg/mL; 15 μg/mL and 20 μg/mL; 20 μg/mL and 50 μg/mL; 20 μg/mL and 40 μg/mL; 20 μg/mL and 30 μg/mL; 30 μg/mL and 50 μg/mL; 30 μg/mL and 40 μg/mL; and 40 μg/mL and 50 μg/mL of EPA in plasma of the mammalian subject. In certain embodiments, the Cmax is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 μg/mL of EPA in plasma of the mammalian subject. In other embodiments, the Cmax of EPA is 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times the Cmax of EPA from the same dose of the ethyl ester of the fatty acid. In certain embodiments, the Cmax is between 2 and 10, 3 and 8 or 4 and 6 times the Cmax of EPA from the same dose of the ethyl ester of the fatty acid. In other embodiments, the Cmax is about 5 times the Cmax of EPA from the same dose of the ethyl ester of the fatty acid.

In other embodiments, when metformin EPA is administered to a mammalian subject at 52 mg/kg it produces a AUC(0-24) of between 90 μg*h/mL and 200 μg*h/mL of EPA in plasma of the mammalian subject. In other embodiments, when metformin EPA is administered to a mammalian subject at 52 mg/kg it produces a AUC(0-24) of between 90 μg*h/mL and 180 μg*h/mL; 90 μg*h/mL and 160 μg*h/mL; 90 μg*h/mL and 140 μg*h/mL; 90 μg*h mL and 120 μg*h mL; 90 μg*h mL and 100 μg*h mL; 110 μg*h mL and 200 μg*h/mL; 110 μg*h/mL and 180 μg*h/mL; 110 μg*h/mL and 160 μg*h/mL; 110 μg*h/mL and 140 μg*h mL; 110 μg*h/mL and 120 μg*h/mL; 130 μg*h mL and 200 μg*h/mL; 130 μg*hJmL· and 180 μ *ίι/ηιΕ; 130 μ *ίι/ηιΕ and 160 μ *ίι/ηιΕ; 130 μg*hJmL· and 140 μg*h/mL·, 150 μ *ίι/ηιΕ and 200 μg*h/mL^■, 150 μ *ίι/ηιΕ and 180 μg*h/mL·, 150 μ *ίι/ηιΕ and 160 μg*h/mL^■, 170 μ *ίι/ηιΕ and 200 μg*h/mL·, 170 μ *ίι/ηιΕ and 180 μ *ίι/ηιΕ; and 190 μ *ίι/ηιΕ and 200 μ *ίι/ηιΕ of EPA in plasma of the mammalian subject. In certain embodiments, the AUC(0-24) is at least 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 μ *1ι/ιηΕ of EPA in plasma of the mammalian subject. In other embodiments, the AUC(0-24) of EPA is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times the AUC(0-24) of EPA from the same dose of the ethyl ester of the fatty acid. In certain embodiments, the AUC(0-24) is between 1.5 and 8, 2 and 6 or 3 and 4 times the AUC(0-24) of EPA from the same dose of the ethyl ester of the fatty acid. In other embodiments, the AUC(0-24) is about 2 times the AUC(0-24) of EPA from the same dose of the ethyl ester of the fatty acid.

In other embodiments, when metformin EPA is administered to a mammalian subject at 104 mg/kg it produces a AUC(0-24) of between 150 μg*h/mL and 300 μg*h/mL of EPA in plasma of the mammalian subject. In other embodiments, when metformin EPA is administered to a mammalian subject at 104 mg/kg it produces a AUC(0-24) of between 150 μg*h/mL and 300 μg*h/mL; 150 μg*h/mL and 280 μg*h/mL; 150 μg*h/mL and 260 μg*h/mL; 150 μg*h/mL and 240 μg*h/mL; 150 μg*h/mL and 220 μg*h/mL; 150 μg*h/mL and 200 μg*h mL; 150 μg*h/mL and 180 μg*h/mL; 150 μg*h mL and 160 μg*h/mL; 170 μg*h mL and 300 μg*h/mL; 170 μg*h mL and 280 μg*h/mL; 170 μg*h/mL and 260 μg*h/mL; 170 μg*h/mL and 240 μg*h/mL; 170 μg*h/mL and 220 μg*h/mL; 170 μg*h/mL and 200 μg*h mL; 170 μg*h/mL and 180 μg*h/mL; 190 μg*h mL and 300 μg*h/mL; 190 μg*h/mL and 280 μg*h/mL; 190 μg*h/mL and 260 μg*h/mL; 190 μg*h/mL and 240 μg*h/mL; 190 μg*h/mL and 220 μg*h/mL; 190 μg*h/mL and 200 μg*h/mL; 210 μg*h/mL and 300 μg*h mL; 210 μg*h/mL and 280 μg*h/mL; 210 μg*h mL and 260 μg*h/mL; 210 μg*h/mL and 240 μg*h/mL; 210 μg*h/mL and 220 μg*h/mL; 230 μg*h/mL and 300 μg*h/mL; 230 μg*h/mL and 280 μg*h/mL; 230 μg*h/mL and 260 μg*h/mL; 230 μg*h/mL and 240 μg*h mL; 250 μg*h/mL and 300 μg*h/mL; 250 μg*h mL and 280 μg*h/mL; 250 μg*h/mL and 260 μg*h/mL; 270 μg*h/mL and 300 μg*h/mL; 270 μg*h/mL and 280 μg*h/mL and 290 μg*h/mL and 300 μg*h/mL EPA in plasma of the mammalian subject. In certain embodiments, the AUC(0-24) is at least 150, 100, 110, 120, 130, 140, 150, 160, 170, 180, 1150, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 μg*h/mL of EPA in plasma of the mammalian subject. In other embodiments, the AUC(0-24) of EPA is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times the AUC(0-24) of EPA from the same dose of the ethyl ester of the fatty acid. In certain embodiments, the AUC(0- 24) is between 1.5 and 8, 2 and 6 or 3 and 4 times the AUC(0-24) of EPA from the same dose of the ethyl ester of the fatty acid. In other embodiments, the AUC(0-24) is about 2 times the AUC(0-24) of EPA from the same dose of the ethyl ester of the fatty acid.

In certain embodiments, the bioavailability of the above dosages are the result of oral administration. In other embodiments, the bioavailability of the above dosages are the result of intravenous administration.

This disclosure provides methods for the use of polyunsaturated fatty acids of the ω-3 series for the preparation of a drug useful in the primary prevention of a major cardiovascular event in subjects who have not undergone previous infarct episodes, wherein the fatty acids comprise eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) and/or at least one pharmaceutically acceptable derivative thereof, in quantities greater than or equal to 25 wt % on the total fatty acid weight. In certain embodiments, the polyunsaturated fatty acids of the ω-3 series are administered intravenously.

Intravenous Administration

The disclosure also provides intravenous dosage forms of fatty acid salts as well as methods of using these intravenous dosage forms for the treatment of disease in mammalian subjects. The dosage forms can include ω-3 polyunsaturated fatty acids, ω-3 polyunsaturated fatty acids include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Cations that are used to form fatty acid salts include metformin, piperazine, meglumine and lysine. Examples of fatty acid salts made with these cations are provided above.

These intravenous dosage forms can contain fatty acids at a concentration greater than 50 and less than 1000 mg/mL; greater than 100 and less than 1000 mg/mL; greater than 50 and less than 500 mg/mL; greater than 100 and less than 500 mg/mL in solution. In other embodiments, the intravenous dosage forms can contain concentrations of fatty acids of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg/mL. The fatty acid salts have solubility in aqueous solution that is 50-100 times greater than the solubility of the ethyl ester of the fatty acid. In other embodiments, the solubility of fatty acid salts is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times greater than the solubility of the ethyl ester of the fatty acid. In certain embodiments, the fatty acids are ω-3 polyunsaturated fatty acids. In other embodiments, the fatty acids are EPA or DHA. The total daily dosages of EPA and DHA can range from 5 to 1000 mg/kg. In other embodiments, the total daily dosage can range between 5, 10, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 and 950 mg/kg and 10, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 mg/kg. These intravenous dosage forms can be administered 1-10 times daily.

In certain embodiments, these intravenous dosage forms are used for the treatment, prevention or alleviation of symptoms associated with atrial fibrillation.

In the present description, the expression "polyunsaturated fatty acids of the ω-3 series" means those long-chain polyunsaturated fatty acids, generally C16-C24, containing fish oils, in particular those having a C20-C22 chain, which are predominant in purification processes. Examples of these polyunsaturated fatty acids of the ω-3 series include EPA and DHA.

As used herein, the term "mammalian subject" refers to rodents, livestock, primates or pets. Rodents include rats, mice or rabbits. Livestock include sheep, goats, llamas, camels, cattle, or buffalo. Primates include monkeys, apes or humans. Pets include dogs or cats.

As used herein the term "major cardiovascular event" refers to particular those events which involve reversible or irreversible cardiovascular damage, such as infarct of the myocardium and of individual coronary branches, death from cardiac causes, sudden death, etc., besides to infarct, broadly speaking, ictus etc., and those conditions prodromal to such major events, such as myocardial fibrillation, atrial and/or ventricular fibrillation, etc. Said major cardiovascular events are usually induced by various cardio circulatory and

cardiorespiratory pathologies such as coronary ischemic illness not displayed by previous infarct episodes, and by serious hypoxic/anoxic states caused by a sudden lack of oxygen (for example during anesthesia, surgery, etc.), possibly in the presence of conditions which contemplate an increase in the oxygen requirement, (accentuated physical stress, drug abuse, acute hypertensive crises, etc.) and analogous acute and chronic pathologies due to cardiac defects of electrical and/or mechanical type.

As used herein, "atrial fibrillation" is a form of cardiac arrhythmia. Symptoms associated with atrial fibrillation include rapid heart rate, irregular heart rate, palpitations, exercise intolerance, angina, shortness of breath, edema, stroke, transient ischemic attack, fainting and congestive heart failure. Atrial fibrillation can be associated with central sleep apnea, left atrial enlargement, mitral stenosis, hypertension, coronary artery disease, mitral regurgitation, hypertrophic cardiomyopathy, pericarditis, congenital heart disease, previous heart surgery, lung diseases, excessive alcohol consumption, hyperthyroidism, carbon monoxide poisoning, Friedrich's ataxia or rheumatoid arthritis. The subjects affected by pathologies of the cardiocirculatory and cardiorespiratory system, hence not simply at prospective risk due to hypertriglyceridemia, hypertension or other, are representative of subjects definable at various levels as cardiopaths, by being affected, for example, by coronary ischemia detectable by coronarography, scintigraphy of the myocardium, electrocardiogram (ECG) under stress, etc., against which interventions of revascularization (angioplasty) or other possible pharmacological or invasive treatments have been proposed, and of subjects affected by electrical hyperexcitability of the myocardium cells, disorder of the diffusion of electrical excitement or of electrical conduction

(arrhythmia, fibrillation, etc.) or by other defects of mechanical type (cardiac insufficiency, decompensation), possibly aggravated by concomitant pathologies such as diabetes.

The use of polyunsaturated fatty acids of the ω-3 series according to the invention is particularly indicated if the occurrence of a major event is predicted, such as an infarct, in particular of the myocardium, death from a cardiological cause, or sudden death, and where such an occurrence takes place in cardiopathic subjects affected, for example, by coronary ischemia, arrhythmia, atrial and/or ventricular fibrillation, electrical hyperexcitability of the myocardium cells, disorder of the diffusion of electrical excitement or of electrical conduction of the myocardium, or cardiac disorders of mechanical type, for example cardiac insufficiency or cardiac decompensation, possibly affected by diabetic pathology concomitant with the cardiopathy.

Preferably, the content of EPA and/or DHA and/or of the at least one derivative thereof is between 50% and 100%, in particular between 75% and 95%, and more preferably about 85% by weight on the total fatty acid weight. The preferred EPA and/or DHA derivatives are selected from the corresponding C1 -C3 alkyl esters and/or from their salts with metformin, piperazine, meglumine and lysine.

According to another aspect, the invention relates to a method for the primary prevention of a major cardiovascular event in subjects who have not undergone previous infarct episodes, comprising the administration of an effective dose of a drug comprising polyunsaturated fatty acids of the ω-3 series as hereinbefore described. In particular, the method of the invention is indicated whenever the occurrence of a major cardiac event is predicted such as an infarct, in particular of the myocardium, death from a cardiological cause or sudden death.

Nutraceutical, Food and Drink Additive The disclosure also provides compositions including food, drink, and nutraceutical products that are supplemented with ω-3 polyunsaturated fatty acid salt forms described herein. These fatty acids can be supplied in aqueous solution through formation of the fatty acid salts described herein. These fatty acids can also be used to supplement food, drink, and nutraceutical products in solid form. The formation of these fatty acid salts allows for higher concentrations of fatty acids to be formed in aqueous solution. Forming higher concentration aqueous solutions of ω-3 polyunsaturated fatty acids can be used to decrease their oxidation. When not in aqueous solution, ω-3 polyunsaturated fatty acids are often supplied as emulsions. Emulsions allow for enhanced oxidation of ω-3 polyunsaturated fatty acids along the oil-water interface. Further, emulsions can prevent antioxidants that are soluble in aqueous solution from preventing oxidation of ω-3 polyunsaturated fatty acids in the organic phase. Oxidation of ω-3 polyunsaturated fatty acids can make supplemented food, drink, and nutraceutical unpalatable.

The disclosure provides food, drink and nutraceuticals supplemented with aqueous solutions of fatty acid salts, described above.

Foods/Drinks

The fatty acid salts described herein can be incorporated into food or drink. There is no specific limitation on the foods/drinks to which the fatty acid salts described herein can be incorporated. Examples of such foods/drinks include processed foods based on meat, poultry meat, fish/shellfish and the like; soup; seasonings including sweetener and the like; rice seasonings; instant foods; frozen foods; snacks; various types of functional foods such as supplements, nutritional drinks and the like; canned foods; dairy products; confectionery such as chewing gum, candy, gummy candy, chocolate, baked sweets and the like; ice cream; soft drinks such as tea, coffee, cocoa, fruit juice, sports drink, carbonated drink, vegetable drink and the like; liquors; soya milk; lactic acid bacteria beverages; and chlorophyll juice.

The amount of the fatty acid salts described herein added to the food or drink varies in accordance with the type of food or drink and the amount that one wishes to supplement a diet with one or more fatty acids, particularly ω-3 fatty acids, and the method of applying the composition described herein, the method of use and the like. For example, the composition described herein is generally used in an amount of about 0.000001 to 20% by weight, and more preferably in an amount of about 0.00001 to 10% by weight, with respect to the food/drink. Nutraceutical Products

The fatty acid salts described herein can be incorporated into nutraceutical products to be used as a fatty acid supplement, particularly ω-3 fatty acids. There is no specific limitation on the nutraceutical products into which the fatty acid salts can be incorporated. Examples of the nutraceutical products include liquid, granular, and powdery substances. The amount of the fatty acid salt described herein varies in accordance with the amount of fatty acid that is desired in the nutraceutical, the type of nutraceutical product and the method of applying nutraceutical. For example, the fatty acid salt described herein is generally used in an amount of about 0.000001 to 20% by weight, and more preferably in an amount of about 0.00001 to 10% by weight, with respect to the pharmaceutical product. Nutraceuticals further include mouse washes, various types of dentifrices, chewing gum, candy, tablets, capsules, mouth sprays, and films.

The following examples illustrate the invention but without limiting it. EXAMPLES

List of Abbreviations

% Percent

°C Degrees Celsius

AUC(0-24) Area under the curve from time zero to 24 hours

AUC/Dose Dose normalized area under the curve

BLQ Below the limit of quantitation

Cmax Maximum observed concentration

Cmax/Dose Dose normalized maximum observed concentration

Cone Concentration

g Gram

h or hr Hour

kg Kilogram

M Male

mg Milligram

mg/kg Milligram per kilogram

mg/mL Milligram per milliliter

min Minute

mL Milliliter

mL/kg Milliliter per kilogram No. Number

PO Oral

rpm Revolutions per minute

SOP Standard Operating Procedure

Tl/2 Apparent plasma terminal phase half-life

Tmax Time when the maximum concentration was observed

Vol Volume

Example 1. Determination of plasma pharmacokinetic data of test substances in male and female rats following a single dose oral administration.

Methods

Five groups of 3 male and 3 female fasted Sprague-Dawley rats were administered a single oral gavage dose of one of five test article formulations in deionized water as detailed in Table 1 below:

Serial blood samples (approximately 0.3 mL) were obtained via a jugular vein catheter from the animals in each group at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours post-dose. Derived plasma samples were stored frozen at approximately -70°C until shipment to BASi, Inc. Results

All dosings were uneventful and all animals received their respective full dose. An oral dose administration of either TP-101 (52 or 104 mg/kg), metformin HC1 (20 mg/kg), EPA FFA (40 mg/kg) and EPA ethyl ester (40 mg/kg), each formulated in deionized water, was well tolerated by male and female Sprague Dawley rats. Animals appeared normal at all blood sampling timepoints through 24 hours post-dose following each administration. All blood samples were collected at their targeted times and derived plasma samples were stored frozen until shipment to BASi, Inc for analysis. The plasma sample assay results and pharmacokinetic analyses were provided to Calvert by the Sponsor.

TP- 101 is an ionic salt that dissociates into EPA free fatty acid and metformin. Plasma EPA levels in rats administered a 52 mg/kg oral dose of TP-101 (Group 1) were markedly higher than EPA levels following either a 40 mg/kg oral dose of EPA FFA (Group 4) or a 40 mg/kg oral dose of EPA ethyl ester (Group 5). Mean Cmax values were 23.6 ± 14.10, 13.6 ± 9.16 and 4.65 ± 1.320 μg/mL for Groups 1, 4 and 5, respectively. Mean AUC (0-24) values were 133 ± 39.1 , 89.9 ± 24.00 and 47.8 ± 12.00 μg*h mL, respectively. A doubling of the TP-101 oral dose to 104 mg/kg (Group 2) increased the mean AUC (0-24) value to 210 ± 40.9 μg*hr/mL, an approximate 60% increase versus the 52 mg/kg dose. (See Table 2).

Plasma metformin levels for the 52 mg/kg dose of TP-101 were similar to the levels following the 20 mg/kg oral dose of metformin HCl. The 104 mg/kg dose of TP-101 resulted in mean AUC(0-24) metformin levels approximately two-fold higher than the 52 mg/kg dose.

Test Article

1. Test Article 1

Identification: TP-101

Lot/Batch No.: 8471-9 A

Physical Description: Light brown solid

Storage Conditions: Room temperature (15 - 30 °C), stored under nitrogen

2. Test Article 2

Identification: Metformin HCl

Lot/Batch No.: J06X013

Physical Description: White powder

Storage Conditions: Room temperature (15 - 30 °C)

3. Test Article 3

Identification: EPA Free Fatty Acid (FFA)

Lot/Batch No.: Ml 10044

Physical Description: Liquid

Storage Conditions: Room temperature (15 - 30 °C)

4. Test Article 4

Identification: EPA Ethyl Ester

Lot/Batch No.: Ml 10024

Physical Description: Liquid

Storage Conditions: Room temperature (15 - 30 °C) Vehicle

Identification: Deionized water

Lot/Batch No.: Lot 30 Jan 2012; expiration date: 12 Feb 2012 and Lot 1

Feb 2012; expiration date: 15 Feb 2012

Physical Description: Clear, colorless liquid

Storage Conditions: Room temperature

Dose Preparations

Group 1 : TP-101 - 5.2 mg/mL: On 31 Jan 2012, 130.00 mg of TP-101 was transferred into a glass beaker, 21 mL of deionized water was added and the preparation was stirred/mixed for 32 minutes. The pH of the preparation was measured at 10.1 and then adjusted to 8.2 by the addition of 0.1 mL of 0.1N HC1. The preparation was brought to a final volume of 25 mL with the addition of 3.9 mL of deionized water. The final formulation appeared as a homogeneous, cloudy, off- white liquid and was transferred to a prelabeled amber glass bottle and stored at room temperature.

Group 2: TP-101 - 10.4 mg/mL: On 31 Jan 2012, 260.01 mg of TP-101 was transferred into a glass beaker, 21 mL of deionized water was added and the preparation was stirred/mixed for 26 minutes. The pH of the preparation was measured at 9.9 and then adjusted to 8.3 by the addition of 0.2 mL of 0.1N HC1. The preparation was brought to a final volume of 25 mL with the addition of 3.8 mL of deionized water. The final formulation appeared as a homogeneous, cloudy, off- white liquid and was transferred to a prelabeled amber glass bottle and stored at room temperature.

Group 3: Metformin HC1 - 2 mg/mL: On 1 Feb 2012, 50.08 mg of Metformin HC1 was transferred into a glass beaker, 21 mL of deionized water was added and the preparation was stirred/mixed for 25 minutes. The pH of the preparation was measured at -5.5. The preparation was brought to a final volume of 25 mL with the addition of 4 mL of deionized water. The final formulation appeared as a clear, colorless liquid and was transferred to a prelabeled amber glass bottle and stored at room temperature.

Group 4: EPA FFA - 4 mg/mL: On 1 Feb 2012, 100.80 mg of EPA FFA was transferred into a glass beaker, 21 mL of deionized water was added and the preparation was stirred/mixed for 15 minutes. The pH of the preparation was measured at -4.5. The preparation was brought to a final volume of 25 mL with the addition of 4 mL of deionized water. The final formulation appeared as a homogeneous, cloudy liquid and was transferred to a prelabeled amber glass bottle and stored at room temperature.

Group 5: EPA Ethyl Ester - 4 mg/ml: On 1 Feb 2012, 100.11 mg of EPA ethyl ester was transferred into a glass beaker, 21 ml of deionized water was added and the preparation was stirred/mixed for 10 minutes. The pH of the preparation was measured at -4.5. The preparation was brought to a final volume of 25 ml with the addition of 4 ml of deionized water. The final formulation appeared as a

homogeneous, cloudy liquid and was transferred to a prelabeled amber glass bottle and stored at room temperature.

With one exception, all dosing preparations were utilized within one hour or less of completion of preparation. The dose to Rat 672 (Group 1 ) was approximately 65 minutes following the completion of preparation. System (Animals and Animal Care)

escription

Species: Rat

Stock: Sprague Dawley Crl:CD(SD)

Total Number: 30; each animal was surgically implanted by the vendor with a catheter in a jugular vein using polyurethane tubing (0.025 x 0.040) and a dextrose/heparin preparation as the lumen lock solution.

Gender: 15 males and 15 females

Age Range: Males: 9 to 10 weeks; Females: 13 to 14 weeks. Records of dates of birth for animals used in this study will be retained in the Calvert archives.

Body Weight Range: 241 to 300 g at time of dosing

Animal Source: Charles River Laboratories, Inc. (Kingston, NY)

Experimental History: Purpose-bred and experimentally naive at the outset of the study.

Identification: Eartag and cage card ationale for Choice of Species and Number of Animals

The rat is a standard rodent species used in preclinical pharmacokinetic studies of new chemical entities and different test article formulations.¹ Thirty animals were considered to be the minimum number necessary to properly perform this parallel oral pharmacokinetic discovery-type study involving five distinct test article formulations. Three animals per sex per group permitted the generation of descriptive statistical analysis.

usbandry

Housing: Animals were individually housed in plastic totes upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals." The room in which the animals were kept was documented in the study records. No other species were kept in the same room.

Lighting: 12 hours light/12 hours dark except when the room

lights were turned on during the dark cycle to accommodate required study procedures.

Room Temperature: 19 to 22°C

Relative Humidity: 34 to 66%

Food: All animals had access to Harlan Teklad Rodent Diet

(certified) or equivalent ad libitum. Animals were deprived of food overnight prior to dosing and fed approximately 4 hours post-dosing. The lot number(s) and specifications of each lot used are archived at Calvert. No contaminants are known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which are maintained in the Calvert archives.

Water: Water was available ad libitum to each animal. The water is routinely analyzed for contaminants as per Calvert SOPs. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis are maintained in the Calvert archives.

Acclimation: Study animals were acclimated to their laboratory

environment for a minimum of 5 days prior to their dosing.

4. Prestudy Health Screen and Selection Criteria

All animals received for this study were assessed as to their general health by a member of the veterinary staff or other authorized personnel. During the acclimation period, each animal was observed at least once daily for any abnormalities or for the development of infectious disease. Only animals that were determined to be suitable for use were assigned to this study.

5. Assignment to Study Groups

Animals were manually assigned to the study and study groups based on body weight and catheter patency. Animals were assigned a permanent identification number and then ear tagged.

6. Humane Care of Animals

Treatment of animals was in accordance with the study protocol and also in accordance with Calvert SOPs which adhere to the regulations outlined in the USDA Animal Welfare Act (9 CFR Parts 1 , 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, NRC, 2011 , The National Academies Press). The Calvert Institutional Animal Care and Use Committee (IACUC) approved the study protocol prior to finalization to insure compliance with acceptable standard animal welfare and humane care.

Test Article Administration

1. Treatments and Dosing Details

3M/3F I EPA ethyl ester PO 40 4.0

2. Dosing

Routes: Oral administration

Frequency: Single dose

Procedures A single oral gavage administration was performed using a ball-tipped, stainless steel 16 gauge gavage needle attached to a 3 ml plastic syringe.

Individual dosing syringes were weighed loaded and unloaded.

3. Justification for Route, Dose Levels and Dosing Schedule

The oral route was selected by the Sponsor as this is the potential route of administration in humans. The dose levels were selected by the Sponsor based on previously published metformin and EPA toxicity data.

D. In-Life Observations and Measurements

1. Clinical Observations

Frequency: Animals were observed at least once daily and at all times of blood sampling.

2. Body Weight

Frequency: Body weight was determined on the day of test article administration just prior to dosing.

E. Sample Collection

1. Dosing Syringes

The actual dose administered to the animal was the difference between the loaded and unloaded dosing syringe weights.

2. Blood Sampling

Serial blood samples were obtained from each animal by a jugular vein catheter and then transferred into pre-labeled tubes containing K₂EDTA anticoagulant. Just before each blood sampling timepoint, blood was withdrawn from the jugular vein catheter (a volume slightly larger than the catheter tubing void volume) and discarded. A blood sample (approximately 0.3 mL) was then obtained from the catheter using a syringe with blunted needle and the sample was immediately transferred into a blood collection tube containing anticoagulant. Following each 0.3 mL blood sample collection, the catheter was flushed with an anticoagulant solution in sterile saline (a volume slightly larger than the catheter tubing void volume).

Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours post-dose. Collected blood samples were gently inverted several times and stored on chill packs protected from light until centrifugation.

3. Plasma Harvesting

Blood samples were centrifuged at -1500 g for 15 minutes at +4 °C within 30 minutes of collection. Derived plasma was split into two aliquots; one aliquot contained 50 μΐ_^ and the other contained the remainder of the plasma sample.

Plasma aliquots tubes were labeled with the Calvert study number, treatment group number, species, animal number, dose level/route, date of collection, time of collection, and sample type. Samples were placed immediately on dry ice until stored frozen at approximately -70 °C.

F. Terminal Procedures

All animals were euthanized by C0₂ asphyxiation following their final blood sampling. No gross necropsy was performed.

Results

G. Animal Dosing, Clinical Observations and Sample Collection

Individual and mean animal body weights and dosing details are presented in Table 4, below. Clinical observations and blood sampling/processing times are summarized in Table 5.

Table 4 - Individual and Mean Animal Body Weights and Dosing Details

%RSD 4.3 3.2 1.1 3.2 1.1

Calculated as the difference between full and empty syringe weights

bcalculated based upon the respective nominal formulation cone, and the dose amount (g) administered

Table 4 (continued) - Individual and Mean Animal Body Weights and Dosing Details

Calculated as the difference between full and empty syringe weights

Calculated as the difference between full and empty syringe weights

Table 5

Group 1

Grou 2

Group 3

Grou 4

All dosings were uneventful and all animals received their respective full dose. An oral dose administration of either TP-101 (52 or 104 mg/kg), metformin HCl (20 mg/kg), EPA FFA (40 mg/kg) and EPA ethyl ester (40 mg/kg), each formulated in deionized water, was well tolerated by male and female Sprague Dawley rats. TP-101 is an ionic salt that dissociates into EPA free fatty acid and metformin. Animals appeared normal at all blood sampling timepoints through 24 hours post-dose following each administration. All blood samples were collected at their targeted times and derived plasma samples were stored frozen until shipment to BASi, Inc. for analysis.

Metformin Plasma Pharmacokinetic Results

Individual plasma metformin concentrations are presented in Table 6. Mean plasma metformin concentrations are presented in Table 7. Individual plasma pharmacokinetic parameters are presented in Table 8. Mean plasma pharmacokinetic parameters are presented in Table 8.

Table 6 - Individual Plasma Metformin Concentrations

Table 7 - Median Plasma Metformin and EPA Concentrations

Table 8 - Individual Pharmacokinetic Parameters of Metformin

Reig essiofi used Sse isst ihfee aFsalyiinasiy measu?3 $e s

[aj = R*2 « 0.S52

a*gf€sss» fails fssr aii EPA FFA. and EPA Et y! Ether fr aSsi

Table 9 - Mean Pharmacokinetic Parameters of Metformin in Rat Plasma Following a Single Oral Dose of TP-101 or Metformin HC1

SD Not calculated for n<3

NC - not calculated

Regression used the last three analytically measurable concentrations (4, 8, and 24 hours)

Mean plasma metformin concentrations plotted as a function of time are presented in Figures 1-3. Plasma metformin concentration-time profiles following a single 52 mg/kg oral dose of TP-101 to rats in Group 1 (52 mg/kg is an equivalent dose of 20 mg/kg metformin HC1), a 104 mg/kg dose of TP-101 to rats in Group 2, or a 20 mg/kg oral dose of metformin HC1 to rats in Group 3 demonstrated a bi-phasic decline through 24 hours post-dose following a Tmax at -0.5 to 0.7 hours. The mean terminal phase half-life (Tl/2) calculated using the 4 hour, 8 hour and 24 hour timepoints was slightly longer for the TP-101 dose groups (-5.1 to 6.3 hours) versus the metformin HC1 dose group (-3.4 hours).

Group 1 and Group 3 mean Cmax (Group 1 - 1.87 + 0.511 μg/mL; Group 3 - 2.32 + 0.333 μg/mL) and AUC(0-24) values (Group 1 - 5.01 + 0.397 μg*hr/mL; Group 3 - 5.18 + 0.484 μg*h/mL) were similar. Plasma metformin concentrations following a 104 mg/kg oral dose of TP-101 (Group 2) were approximately two-fold higher than were observed for Groups 1 and 3 with mean Cmax and AUC (0-24) values of 4.26 + 1.200 μg/mL and 11.6 + 2.34 μg*h/mL, respectively. These data suggest a dose-proportional response for plasma metformin levels with increasing TP-101 dose. No gender-related differences were observed for the metformin plasma pharmacokinetic parameters (exposure and elimination).

EPA Plasma Pharmacokinetic Results

Individual plasma EPA concentrations are presented in Table 10. Mean plasma EPA concentrations are presented in Table 7, above. Individual plasma pharmacokinetic parameters are presented in Table 11. Mean plasma pharmacokinetic parameters are presented in Table

11. summarized in Table 12.

Table 10 - Individual Plasma EPA Concentrations

Table 11 - Individual Pharmacokinetic Parameters of EPA

Regression usssf t & Sast t rse aRasy¾iaa% {ReasuisfeSe «xwarsSatiaBS i4. S, ans 24 hssjrsS

[aj = R*2 ί 9-S5S

[bj = Pesstwe terminal steps

Regression faSts for *l? TP- C1 104 mg.¾g, kteSfewRis HCi, EPA FFA

asset EPA &m≠ Efeert»a»ii ara^ate for either |aj «rfb]

Table 12 - Mean Pharmacokinetic Parameters of EPA in Rat Plasma Following a Single Oral Dose of TP-101, EPA FFA or

EPA Ethyl Ester

SD Not calculated for n<3

NC - not calculated

[a] = R^A2 < 0.850

Regression failed for all TP-101 104 mg/kg, Metformin HCl, EPA FFA and EPA Ethyl Ester treated

animals for reasons of either [a] or a positive terminal slope

Mean plasma EPA concentrations plotted as a function of time are presented in Figured 4-6. A 52 mg/kg dose of TP-101 (Group 1), a 40 mg/kg dose of EPA FFA (Group 4), and a 40 mg/kg dose of EPA ethyl ester (Group 5) contained equivalent doses of EPA. Endogenous levels of EPA were a component of all reported plasma concentrations, and presumably accounted for the plasma EPA levels reported for Group 3 (20 mg/kg dose of metformin HC1).

Plasma EPA levels in the rats administered a 52 mg/kg oral dose of TP-101 were higher than concurrent levels following either a 40 mg/kg oral dose of EPA FFA or a 40 mg/kg oral dose of EPA ethyl ester. Mean Cmax values were 23.6 + 14.10, 13.6 + 9.16 and 4.65 + 1.320 μg/mL for Groups 1, 4 and 5, respectively. Mean AUC(0-24) values were 133 + 39.1, 89.9 + 24.00 and 47.8 + 12.00 μg*h/mL, respectively. Mean AUC(0-24) values following a 104 mg/kg oral dose of TP-101 (Group 2) were -60% higher than the 52 mg/kg dose. The mean Cmax for the 104 mg/kg dose was also higher, 27.1 + 10.80 μg/mL. Plasma EPA levels declined gradually, from an observed Tmax in the majority of rats at 1 to 2 hours post-dose, through 24 hours post-dose. Calculation of a terminal phase half-life of EPA in each dose group was not calculated due to either an R² value <0.850 or a positive terminal slope. No appreciable gender-related differences were observed for the EPA plasma pharmacokinetic parameters (exposure and elimination).

Conclusions

52 mg/kg and 104 mg/kg oral doses of TP-101 were well tolerated by Sprague Dawley rats.

Exposure to EPA was greater after an oral dose of TP-101 than after an equivalent oral dose of EPA, FFA or EPA ethyl ester. Plasma EPA levels following a 52 mg/kg oral dose of TP-101 were markedly higher than EPA levels following 40 mg/kg oral doses of EPA FFA or EPA ethyl ester. Plasma EPA AUC(0-24) following a 104 mg/kg oral dose of TP-101 was approximately 60% higher than measured for the 52 mg/kg dose.

Plasma metformin AUC(0-24) values following a 52 mg/kg oral dose of TP-101 and a 20 mg/kg oral dose of metformin HC1 were similar and increased proportionately following a 104 mg/kg oral dose of TP-101.

There were no appreciable gender related differences in EPA or metformin plasma pharmacokinetic parameters (exposure or elimination) following an oral dose of TP-101.

Claims

1. A method of increasing the concentration of a fatty acid in aqueous solution comprising combining the anion fatty acid with a cation that increases the solubility of the fatty acid in aqueous solution.

2. The method of claim 1, wherein the cation is metformin, piperazine, meglumine or lysine.

3. The method of claim 1, wherein the fatty acid is eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

4. The method of claim 1, wherein the maximum concentration of the fatty acid is greater than 15 μg/mL or 20 μg/mL in the plasma of a mammalian subject when administered to the mammalian subject at 52 mg/kg.

5. The method of claim 2, wherein the maximum concentration of the fatty acid is at least two times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form.

6. The method of claim 1, wherein the maximum concentration of the fatty acid is greater than 10 μg/mL and less than 100 μg/mL; greater than 10 μg/mL and less than 50 μg/mL; 10 μg/mL and less than 40 μg/mL; 15 μg/mL and less than 40 μg/mL; or 15 μg/mL and less than 35 μg/mL in the plasma of a mammalian subject when administered to the mammalian subject at 52 mg/kg.

7. A composition comprising a salt of a fatty acid comprising a fatty acid and a cation, wherein the salt of the fatty acid has a solubility in aqueous solution greater than 50 and less than 1000 mg/niL; greater than 100 and less than 1000 mg/niL; greater than 50 and less than 500 mg/niL or greater than 100 and less than 500 mg/niL.

8. The composition of claim 7, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is greater than 10 μg/mL; 15 μg/mL; or 20 μg/mL in the plasma of the mammalian subject.

9. The composition of claim 7, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is greater than 10 μg/mL and less than 100 μg/mL; 10 μg/mL and less than 50 μg/mL; 10 μg/mL and less than 40 μg/mL; 15 μg/mL and less than 40 μg/mL; 15 μg/mL and less than 35 μg/mL in the plasma of the mammalian subject.

10. The composition of claim 7, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is at least two times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form.

11. The composition of claim 7, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the area under the curve from time zero to 24 hours of the fatty acid is greater than 90 μg*h/mL; 100 μg*h/mL; or 120 μg*h/mL in the plasma of the mammalian subject.

12. The composition of claim 7, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the area under the curve from time zero to 24 hours of the fatty acid is greater than 90 μg*h/mL and less than 160 μg*h/mL; 100 μg*h/mL and less than 150 μg*h/mL; or 110 μg*h/mL and less than 150 μg*h/mL in the plasma of the mammalian subject.

13. The composition of claim 7, wherein the fatty acid is eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

14. The composition of claim 7, wherein the cation is metformin, piperazine, meglumine or lysine.

15. A method of treating atrial fibrillation, comprising intravenously administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a metformin, piperazine, meglumine or lysine salt form of an co-3 fatty acid salt.

16. The method of claim 15, wherein the effective amount is between 5-100 mg/kg of the subject's body weight per day.

17. The method of claim 15, wherein the subject is cardiopathic.

18. The method of claim 15, wherein the subject is affected by coronary ischemia, cardiac insufficiency, cardiac decompensation or diabetic pathology concomitant with cardiopathy.

19. A method of reducing the probability of an occurrence of a major cardiovascular event in a subject who is affected by atrial fibrillation, comprising intravenously administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising comprising a metformin, piperazine, meglumine or lysine salt form of an co-3 fatty acid salt.

20. The method of claim 19, wherein the effective amount is between 5-100 mg/kg of the subject's body weight per day.

21. The method of claim 19, wherein the subject is cardiopathic.

22. The method of claim 19, wherein the subject is affected by coronary ischemia, cardiac insufficiency, cardiac decompensation or diabetic pathology concomitant with cardiopathy.

23. The method of claim 19, wherein the subject who is affected by atrial fibrillation has not undergone a previous infarct episode.

24. A method of reducing the probability of atrial fibrillation in a cardiopathic subject, comprising intravenously administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a metformin, piperazine, meglumine or lysine salt form of an co-3 fatty acid salt.

25. The method of claim 24, wherein the effective amount is between 5-100 mg/kg of the subject's body weight per day.

26. The method of claim 24, wherein the subject is cardiopathic.

27. The method of claim 24, wherein the subject is affected by coronary ischemia, cardiac insufficiency, cardiac decompensation or diabetic pathology concomitant with cardiopathy.

28. The method of claim 24, wherein the subject who is affected by atrial fibrillation has not undergone a previous infarct episode.

29. An intravenous dosage form comprising a salt of a fatty acid comprising a fatty acid and a cation, wherein the fatty acid has a concentration greater than 50 and less than 1000 mg/mL; greater than 100 and less than 1000 mg/mL; greater than 50 and less than 500 mg/mL or greater than 100 and less than 500 mg/mL g/mL.

30. The intravenous dosage form of claim 29, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is greater than 10 μg/mL; 15 μg/mL; or 20 μg/mL in the plasma of the mammalian subject.

31. The intravenous dosage form of claim 29, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the maximum concentration of the fatty acid is greater than 10 μg/mL and less than 100 μg/mL; 10 μg/mL and less than 50 μg/mL; 10 μg/mL and less than 40 μg/mL; 15 μg/mL and less than 40 μg/mL; or 15 μg/mL and less than 35 μg/mL in the plasma of the mammalian subject.

32. The intravenous dosage form of claim 29, when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the area under the curve from time zero to 24 hours of the fatty acid is greater than 90 μg*h/mL; 100 μg*h/mL; or 120 μg*h/mL in the plasma of the mammalian subject.

33. The intravenous dosage form of claim 29, wherein when 52 mg/kg of the salt of the fatty acid is administered to a mammalian subject the area under the curve from time zero to 24 hours of the fatty acid is greater than 90 μg*h/mL and less than 160 μg*h/mL; 100 μg*h/mL and less than 150 μg*h/mL; or 110 μg*h/mL and less than 150 μg*h/mL; in the plasma of the mammalian subject.

34. The intravenous dosage form of claim 29, wherein the fatty acid is eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

35. The intravenous dosage form of claim 29, wherein the cation is metformin, piperazine, meglumine or lysine.

36. The intravenous dosage form of claim 35, wherein the maximum concentration of the fatty acid is at least two times greater in the plasma of a mammalian subject than when the same amount of fatty acid is administered to the mammalian subject in the ethyl ester form.

37. A nutraceutical, food product or drink product comprising a metformin, piperazine, meglumine or lysine salt form of an co-3 fatty acid salt.

38. A method of making a fatty acid supplemented nutraceutical, food product or drink product comprising mixing an aqueous solution of a fatty acid comprising a fatty acid and a cation with a nutraceutical, food product or drink product, wherein the solubility of the fatty acid in the aqueous solution is greater than 50 and less than 1000 mg/mL; greater than 100 and less than 1000 mg/mL in solution; greater than 50 and less than 500 mg/mL or greater than 100 and less than 500 mg/mL.

39. The method of claim 38, wherein the fatty acid is eicosapentaenoic acid (EPA) or

docosahexaenoic acid (DHA).

40. The method of claim 38, wherein the cation is metformin, piperazine, meglumine or lysine.

41. The compositions and methods of any of the preceding claims wherein the fatty acid salt a compound of the structural Formula I, II, III, IV, V, VI, VII or VIII:

Formula II

Formula IV

wherein X^" is an anion of a pharmaceutically acceptable acid compound, or a combination thereof.