TCA CYCLE INTERMEDIATES AND METHODS OF USE THEREOF

Cross-Reference to Related Applications

This application claims the benefit of, and priority to, U.S. Provisional Application No. 62/744,251, filed October 11, 2018; U.S. Provisional Application No. 62/771,289, filed

November 26, 2018; U.S. Provisional Application No. 62/799,064, filed January 31, 2019; and U.S. Provisional Application No. 62/900,921, filed September 16, 2019, the contents of each of which are incorporated herein by reference.

Field of the Invention

The invention generally relates to TCA cycle intermediates and methods of using such compositions for treating metabolic disorders.

Background

Inherited metabolic disorders, such as propionic acidemia (PA) and methylmalonic acidemia (MMA), cause a variety of life-threatening symptoms, including seizures, strokes, and damage to the brain, heart, and liver. PA and MMA result from enzymatic deficiencies that impair the body's ability to convert certain fats and amino acids into succinate, an intermediate in the tricarboxylic acid (TCA) cycle. Consequently, individuals with PA or MMA must maintain a low-protein diet for their entire lives, and in some patients, even strict adherence to the diet fails to prevent neurological damage and other symptoms.

Efforts to treat afflicted individuals by providing additional succinate have failed.

Unadulterated succinate is poorly bioavailable, so providing succinate in quantities sufficient to remedy metabolic disorders is difficult. Thus, despite our understanding of the molecular basis of metabolic disorders like PA and MMA, these genetic disorders remain highly debilitating and often fatal.

Summary

The invention overcomes the challenge of delivering succinate at doses therapeutically effective to treat metabolic disorders such as PA and MMA by providing combination therapies that include multiple intermediates of the TCA cycle. By providing two or more TCA cycle intermediates to a subject, compositions and methods of the invention rely on the body's natural metabolism pathways to restore levels of succinate and allow delivery of a larger bolus of succinate than is possible with a single agent. In addition, because the compositions can provide various TCA cycle intermediates, they are useful in treating a broad spectrum of metabolic diseases, disorders, and conditions. For example, combinations of succinate and citrate are useful for treating PA and MMA, but other combinations may be used to treat different conditions.

The use of multiple TCA cycle intermediates allows flexibility in the formulation and administration of the compositions of the invention. Each TCA cycle intermediate may independently be provided to the subject by the optimal route of administration for the particular form of the intermediate. Thus, succinate-containing combinations may include non-oral delivery of succinate salts or oral delivery of succinate derivatives that have improved bioavailability. Moreover, in succinate-citrate combinations, both intermediates may be given orally, both may be given non-orally, or one may be given orally and the other may be given non-orally.

The invention also provides succinate-containing compositions formulated for non-oral administration. The compositions, which include formulations for subcutaneous or intravenous administration, have superior bioavailability to oral formulations. The non-oral formulations of succinate may be used in the combination therapies of the invention.

Compositions containing succinate or other TCA cycle intermediates can be readily administered to infants, including newborns. Many metabolic disorders such as PA and MMA are due to genetic defects, so the consequences of errant metabolism become manifest at birth when the child no longer has access to a nutritional supply from the mother. Therefore, early intervention is necessary to rectify metabolic imbalances before they lead to permanent symptoms. Because the invention provides succinate-containing compositions formulated for non-oral administration, such compositions do not require active suckling from the baby to deliver therapeutically effective doses of succinate. Consequently, treatment of infants can begin immediately to help prevent development of disease symptoms.

In an aspect, the invention provides compositions containing one or more TCA cycle intermediates or prodrugs, analogs, or derivatives thereof formulated for non-oral administration. The TCA cycle intermediate may be citrate, isocitrate, alpha-ketoglutarate, succinyl-coenzyme A, succinate, fumarate, malate, or oxaloacetate. Preferably, the TCA cycle intermediate is succinate.

In another aspect, the invention provides methods of treating a condition associated with altered TCA cycle metabolism in a subject. The methods include providing to a subject having a condition associated with altered TCA cycle metabolism a composition comprising one or more TCA cycle intermediates or prodrugs, analogs, or derivatives thereof formulated for non-oral administration. Preferably, the TCA cycle intermediate is succinate.

The composition may be formulated for administration by any non-oral route. For example, the composition may be formulated for injection or infusion. The injection or infusion may be subcutaneous, intravenous, intraarterial, or intramuscular. The composition may be formulated for non-oral enteral administration, such as rectal administration.

The composition may include a buffering agent that maintains a neutral or near-neutral pH of the composition. For example, the buffering agent may be present in amount sufficient to buffer the pH of the composition to from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 10.0, from about 5.0 to about 9.0, from about 5.0 to about 8.0 from about 5.0 to about 7.0, from about 6.0 to about 10.0, from about 6.0 to about 9.0, from about 6.0 to about 8.0 from about 7.0 to about 10.0, from about 7.0 to about 9.0, or from about 8.0 to about 10.0.

The TCA cycle intermediate may be provided at any therapeutically effective dose. For example, the TCA cycle intermediate may be provided at from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.2 mg/kg subject weight to about 5 g/kg subject weight, from about 0.5 mg/kg subject weight to about 5 g/kg subject weight, from about 1 mg/kg subject weight to about 5 g/kg subject weight, from about 2 mg/kg subject weight to about 5 g/kg subject weight, from about 5 mg/kg subject weight to about 5 g/kg subject weight, from about 0.1 mg/kg subject weight to about 2 g/kg subject weight, from about 0.2 mg/kg subject weight to about 2 g/kg subject weight, from about 0.5 mg/kg subject weight to about 2 g/kg subject weight, from about 1 mg/kg subject weight to about 2 g/kg subject weight, from about 2 mg/kg subject weight to about 2 g/kg subject weight, from about 5 mg/kg subject weight to about 2 g/kg subject weight, from about 0.1 mg/kg subject weight to about 1 g/kg subject weight, from about 0.2 mg/kg subject weight to about 1 g/kg subject weight, from about 0.5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 1 g/kg subject weight, from about 2 mg/kg subject weight to about 1 g/kg subject weight, from about 5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 500 mg/kg subject weight, from about 2 mg/kg subject weight to about 200 mg/kg subject weight, or from about 5 mg/kg subject weight to about 100 mg/kg subject weight.

The condition may be any disease, disorder, or condition associated with altered TCA cycle metabolism. For example, the condition may be a disorder related to POLG mutation, an energetic disorder, glutaric acidemia type 1 or type 2, a long chain fatty acid oxidation disorder, methylmalonic acidemia (MMA), a mitochondrial associated disease, a mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy and ragged-red fibers (MERRF), mitochondrial myopathy, a mitochondrial respiratory chain deficiency, muscular dystrophy (e.g., Duchenne’s muscular dystrophy and Becker’s muscular dystrophy), a neurologic disorder, a pain or fatigue disease, propionic acidemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, or succinyl CoA lyase deficiency. Preferably, the condition is MMA or PA.

In another aspect, the invention provides combination therapies for treating a condition associated with altered TCA cycle metabolism in a subject. The combination therapies include a non-oral formulation containing succinate or a prodrug, analog, or derivative thereof and a formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid.

In another aspect, the invention provides methods of treating a condition associated with altered TCA cycle metabolism in a subject. The methods include providing to a subject having a condition associated with altered TCA cycle metabolism a combination therapy including a non oral formulation containing succinate or a prodrug, analog, or derivative thereof and a formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid.

The non-oral formulation containing succinate or a prodrug, analog, or derivative thereof may be formulated for administration by any non-oral means, as described above.

The non-oral formulation containing succinate or a prodrug, analog, or derivative thereof may contain a buffering agent, as described above. The non-oral formulation containing succinate or a prodrug, analog, or derivative thereof may be provided at any therapeutically effective dose. For example, succinate or a prodrug, analog, or derivative thereof may be provided at from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.2 mg/kg subject weight to about 2 g/kg subject weight, from about 0.5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 500 mg/kg subject weight, from about 2 mg/kg subject weight to about 200 mg/kg subject weight, or from about 5 mg/kg subject weight to about 100 mg/kg subject weight.

The formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be formulated for administration by any suitable means. For example, the formulation may be formulated for oral, enteral, parenteral, subcutaneous, intravenous, intraarterial, or intramuscular administration. Preferably, the formulation is formulated for oral administration.

The formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may contain a buffering agent. For example, the buffering agent may be present in amount sufficient to buffer the pH of the composition to from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 10.0, from about 5.0 to about 9.0, from about 5.0 to about 8.0 from about 5.0 to about 7.0, from about 6.0 to about 10.0, from about 6.0 to about 9.0, from about 6.0 to about 8.0 from about 7.0 to about 10.0, from about 7.0 to about 9.0, or from about 8.0 to about 10.0. The buffering agent may be an amino acid or a metal ion. The amino acid may be a natural or non-natural amino acid. The amino acid may be lysine, ornithine, or a derivative thereof.

The formulation containing citrate or a prodrug, analog, or derivative thereof may be provided at any therapeutically effective dose. For example, citrate or a prodrug, analog, or derivative thereof may be provided at from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.1 mg/kg subject weight to about 5 g/kg subject weight,

from about 0.2 mg/kg subject weight to about 2 g/kg subject weight, from about 0.5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 500 mg/kg subject weight, from about 2 mg/kg subject weight to about 200 mg/kg subject weight, or from about 5 mg/kg subject weight to about 100 mg/kg subject weight.

The non-oral formulation containing succinate or a prodrug, analog, or derivative thereof, the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid, or both may be provided in multiple doses per day. For example, one or more of the formulations may be provided in 2, 3, 4, 5, 6, or more doses per day.

The non-oral formulation containing succinate or a prodrug, analog, or derivative thereof and the formulation containing citrate, citric acid, or a prodrug, analog, or derivative thereof may be provided simultaneously, sequentially in either order, or in an alternating manner. Sequential administration or alternating administration may include providing the non-oral formulation containing succinate or a prodrug, analog, or derivative thereof exclusively for a period of time and providing the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid exclusively for a period of time. Sequential administration may include an period of overlap in which the subject is provided both the non-oral formulation containing succinate or a prodrug, analog, or derivative thereof and the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid. The periods of exclusivity and periods of overlap may independently be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, 18 months, or 24 months.

The condition may be any disease, disorder, or condition associated with altered TCA cycle metabolism, as described above.

In another aspect, the invention provides compositions that include a nutritional source for infants, such as baby formula or human breast milk; a non-salt form of citric acid or a prodrug, analog, or derivative thereof; and a buffering agent in an amount to buffer a pH of the composition from about 3.0 to about 8.0.

The buffering agent may be present in amount sufficient to buffer the pH of the composition to from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 10.0, from about 5.0 to about 9.0, from about 5.0 to about 8.0 from about 5.0 to about 7.0, from about 6.0 to about 10.0, from about 6.0 to about 9.0, from about 6.0 to about 8.0 from about 7.0 to about 10.0, from about 7.0 to about 9.0, or from about 8.0 to about 10.0. The buffering agent may be an amino acid or a metal ion. The amino acid may be a natural or non-natural amino acid. The amino acid may be lysine, ornithine, or a derivative thereof.

In another aspect, the invention provides methods of treating a condition associated with altered TCA cycle metabolism in a subject. The methods include providing to a subject having a condition associated with altered TCA cycle metabolism an oral formulation containing citrate and a buffering agent in an amount to buffer a pH of the composition from about 3.0 to about 8.0.

The buffering agent may be present in amount sufficient to buffer the pH of the composition to from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 10.0, from about 5.0 to about 9.0, from about 5.0 to about 8.0 from about 5.0 to about 7.0, from about 6.0 to about 10.0, from about 6.0 to about 9.0, from about 6.0 to about 8.0 from about 7.0 to about 10.0, from about 7.0 to about 9.0, or from about 8.0 to about 10.0. The buffering agent may be an amino acid or a metal ion. The amino acid may be a natural or non-natural amino acid. The amino acid may be lysine, ornithine, or a derivative thereof.

The condition may be any disease, disorder, or condition associated with altered TCA cycle metabolism, as described above. The subject may be human. The human may be a child. For example, the child may be less than 18 years in age, less than 12 years in age, less than 10 years in age, less than 8 years in age, less than 6 years in age, less than 5 years in age, less than 4 years in age, less than 3 years in age, less than 2 years in age, or less than 1 year in age.

In another aspect, the invention provides methods of treating or preventing Leber's hereditary optic neuropathy in a subject. The methods include providing a subject having or at risk of developing Leber's hereditary optic neuropathy a non-oral formulation containing succinate or a prodrug, analog, or derivative thereof.

The non-oral formulation may be provided intraocularly. The non-oral formulation may be provided systemically. For example, the non-oral formulation may be provided intravenously or subcutaneously. The non-oral formulation may include a buffering agent that maintains a neutral or near neutral pH of the composition. For example, the buffering agent may be present in amount sufficient to buffer the pH of the composition to from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 10.0, from about 5.0 to about 9.0, from about 5.0 to about 8.0 from about 5.0 to about 7.0, from about 6.0 to about 10.0, from about 6.0 to about 9.0, from about 6.0 to about 8.0 from about 7.0 to about 10.0, from about 7.0 to about 9.0, or from about 8.0 to about 10.0.

The methods of treating or preventing Leber's hereditary optic neuropathy in a subject may include providing a formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid. The non-oral formulation and the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be a single formulation. Alternatively, the non-oral formulation and the formulation containing citrate, citric acid, or a prodrug, analog, or derivative thereof may be separate formulations.

The formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be provided by any route of administration. For example, the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be provided orally, intraocularly, subcutaneously, or intravenously.

The formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may contain a buffering agent. For example, the buffering agent may be present in amount sufficient to buffer the pH of the composition to from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 10.0, from about 5.0 to about 9.0, from about 5.0 to about 8.0 from about 5.0 to about 7.0, from about 6.0 to about 10.0, from about 6.0 to about 9.0, from about 6.0 to about 8.0 from about 7.0 to about 10.0, from about 7.0 to about 9.0, or from about 8.0 to about 10.0. The buffering agent may be an amino acid or a metal ion. The amino acid may be a natural or non-natural amino acid. The amino acid may be lysine, ornithine, or a derivative thereof. The non-oral formulation containing succinate or a prodrug, analog, or derivative thereof, the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid, or both may be provided in multiple doses per day. For example, one or more of the formulations may be provided in 2, 3, 4, 5, 6, or more doses per day.

In another aspect, the invention provides formulations that contain citric acid, or a prodrug, analog, derivative thereof; one or more citrate salts, or a prodrug, analog, or derivative thereof; and an amino acid buffering agent.

The citrate salts may include mono sodium citrate or monopotassium citrate.

The amino acid buffering agent may be lysine or ornithine.

The formulation may contain a sugar. The sugar may be sucrose, fructose, galactose, maltose, or lactose.

The formulation may be a powder that is soluble in an aqueous medium. The formulation may be an aqueous solution.

The formulation, or the soluble components within the formulation, may contain citric acid, monosodium citrate, monopotassium citrate, lysine, and sucrose. The formulation may contain, by mass, from about 40% to about 60% citric acid; from about 1% to about 10% monosodium citrate; from about 0.1% to about 5% monopotassium citrate; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 49.2 % citric acid, about 2% monosodium citrate, about 0.3% monopotassium citrate, about 37.5% lysine, and about 11% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 44.2 % citric acid, about 8.3% monosodium citrate, about 2.3% monopotassium citrate, about 33.8% lysine, and about 11.5% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, from about 50% to about 60% citrate, citric acid, or a combination thereof; from about 0.2% to about 1% sodium; from about 0.02% to about 0.5% potassium; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose.

The formulation may be suitable for oral administration.

The formulation may be given to a subject to treat or prevent a condition associated with altered TCA cycle metabolism, such as any of those described above. In another aspect, the invention provides a formulation including citric acid, or a prodrug, analog, derivative thereof; at least one citrate salt, or a prodrug, analog, or derivative thereof; and an amino acid. The formulation may include citric acid and at least one citrate salt, for example, the at least one citrate salt may be selected from the group consisting of monosodium citrate and monopotassium citrate. In other embodiments, the formulation may be citric acid and at least two citrate salts, for example, the at least two citrate salts may be monosodium citrate and monopotassium citrate.

The formulation may include any natural or non-natural amino acid. In certain embodiments, the amino acid is a cationic amino acid, such as lysine or arginine. The formulation may include other components, such as a sugar, for example sucrose.

The formulation may be provided by any route of administration, and a preferable route is oral. An exemplary oral formulation is a powder that is soluble in an aqueous medium. In a particular embodiment, the formulation includes citric acid, monosodium citrate, monopotassium citrate, sucrose, and one or more of lysine and arginine.

Other aspects of the invention provide methods of treating a condition associated with altered TCA cycle metabolism in a subject. Such methods may involve providing to a subject having a condition associated with altered TCA cycle metabolism a formulation comprising (i) citric acid, or a prodrug, analog, derivative thereof; (ii) at least one citrate salt, or a prodrug, analog, or derivative thereof; and (iii) an amino acid, wherein a therapeutic effect is achieved in the subject by activity of a combination of two or more of components (i), (ii), and (iii) in the formulation. In certain embodiments, the amino acid is incorporated into the TCA cycle of the subject and contributes to the therapeutic effect that is achieved in the subject.

Exemplary conditions include a disorder related to POLG mutation, an energetic disorder, glutaric acidemia type 1 or type 2, a long chain fatty acid oxidation disorder, methylmalonic acidemia (MMA), a mitochondrial associated disease, a mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy and ragged-red fibers (MERRF), mitochondrial myopathy, a mitochondrial respiratory chain deficiency, muscular dystrophy (e.g., Duchenne’s muscular dystrophy and Becker’s muscular dystrophy), a neurologic disorder, a pain or fatigue disease, propionic acidemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, or succinyl CoA lyase deficiency. The formulation may include citric acid and at least one citrate salt, for example, the at least one citrate salt may be selected from the group consisting of monosodium citrate and monopotassium citrate. In other embodiments, the formulation may be citric acid and at least two citrate salts, for example, the at least two citrate salts may be monosodium citrate and monopotassium citrate.

The formulation may include any natural or non-natural amino acid. In certain embodiments, the amino acid is a cationic amino acid, such as lysine or arginine. The formulation may include other components, such as a sugar, for example sucrose.

The formulation may be provided by any route of administration, and a preferable route is oral. An exemplary oral formulation is a powder that is soluble in an aqueous medium. In a particular embodiment, the formulation includes citric acid, monosodium citrate, monopotassium citrate, sucrose, and one or more of lysine and arginine. The formulation may be provided in one or multiple doses per day. For example, one or more of the formulations may be provided in 1,

2, 3, 4, 5, 6, or more doses per day.

The formulation containing citrate or a prodrug, analog, or derivative thereof may be provided at any therapeutically effective dose. For example, citrate or a prodrug, analog, or derivative thereof may be provided at from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.1 mg/kg subject weight to about 5 g/kg subject weight,

from about 0.2 mg/kg subject weight to about 2 g/kg subject weight, from about 0.5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 500 mg/kg subject weight, from about 2 mg/kg subject weight to about 200 mg/kg subject weight, or from about 5 mg/kg subject weight to about 100 mg/kg subject weight.

The formulation, or the soluble components within the formulation, may contain citric acid, monosodium citrate, monopotassium citrate, lysine, and sucrose. The formulation may contain, by mass, from about 40% to about 60% citric acid; from about 1% to about 10% monosodium citrate; from about 0.1% to about 5% monopotassium citrate; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 49.2 % citric acid, about 2% monosodium citrate, about 0.3% monopotassium citrate, about 37.5% lysine, and about 11% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 44.2 % citric acid, about 8.3% monosodium citrate, about 2.3% monopotassium citrate, about 33.8% lysine, and about 11.5% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, from about 50% to about 60% citrate, citric acid, or a combination thereof; from about 0.2% to about 1% sodium; from about 0.02% to about 0.5% potassium; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose.

Brief Description of the Drawings

FIG. 1 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after subcutaneous administration of 13 C ₄ -succinate at 10 mg/kg.

FIG. 2 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after subcutaneous administration of 13 C ₄ -succinate at 10 mg/kg.

FIG. 3 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after subcutaneous administration of 13 C ₄ -succinate at 50 mg/kg.

FIG. 4 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after subcutaneous administration of 13 C ₄ -succinate at 50 mg/kg.

FIG. 5 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after subcutaneous administration of 13 C ₄ -succinate at 100 mg/kg.

FIG. 6 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after subcutaneous administration of C ₄ -succinate at 100 mg/kg.

FIG. 7 is graph showing the effects of citrate and succinate on basal respiration in cells from a patient with propionic acidemia (PA).

FIG. 8 is a graph of plasma concentration of C ₄ -succinate at various time points in individual rats after intravenous administration of 13 C ₄ -succinate at 10 mg/kg.

FIG. 9 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after intravenous administration of 13 C ₄ -succinate at 10 mg/kg.

FIG. 10 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after oral administration of 13 C ₄ -succinate at 50 mg/kg.

FIG. 11 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after oral administration of 13 C ₄ -succinate at 50 mg/kg.

FIG. 12 is a graph of plasma concentration of 13 C ₆ -citrate at various time points in individual rats after intravenous administration of 13 C ₆ -citrate at 10 mg/kg. FIG. 13 is a graph of average plasma concentration of 13 C ₆ -citrate at various time points in rats after intravenous administration of 13 C ₆ -citrate at 10 mg/kg.

FIG. 14 is a graph of plasma concentration of 13 C ₆ -Citrate at various time points in individual rats after oral administration of 13 C ₆ -Citrate at 50 mg/kg.

FIG. 15 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after oral administration of 13 C ₆ -Citrate at 50 mg/kg.

Detailed Description

The invention provides compositions and methods that allow efficient delivery of intermediates of the TCA cycle for treatment of metabolic disorders. In particular, the compositions and methods of the invention enable delivery of succinate by subcutaneous or intravenous administration. Synthesis of succinate is defective in propionic acidemia and methylmalonic acidemia, and the errant metabolism in patients with these disorders lead to a variety of potentially fatal effects, such as neurological damage, cardiomyopathy, and infections. The non-oral formulations of succinate permit delivery of the compound with much higher bioavailability that can be achieved with prior oral succinate formulations. Consequently, the non-oral formulations provide succinate at sufficient levels to prevent serious, long-term effects of defective TCA cycle metabolism.

The invention also provides combination therapies that include non-oral formulations of succinate and oral formulations of citrate, another intermediate of the TCA cycle. In contrast to succinate, citrate can administered orally at therapeutically effective doses. The combination therapies thus provide delivery of different TCA cycle intermediates by different means. The non-oral formulation of succinate and oral formulation of citrate may be provided sequentially or simultaneously. Sequential combination therapies allow patients, particularly infants, to transition from non-oral therapies to oral administration when they become old enough to ingest citrate in therapeutically effective quantities. Such therapies allow early intervention in newborns while permitting long-term care that can be administered without a health-care professional. Simultaneous combination therapies are useful when clinical issues, such as vomiting, skin rashes, etc., limit the amount of intermediate that can be delivered by either mode of administration. TCA Cycle and Associated Disorders

The TCA cycle is illustrated below:

i¾iCsl#^ j A Abnormal TCA cycle metabolism is associated with a variety of conditions. In hereditary metabolic disorders of the TCA cycle, such as 2-oxoglutaric aciduria, fumarase deficiency, and succinyl-CoA synthetase deficiency, genetic mutations affect enzymes of the TCA cycle or enzymes that catalyze related reactions. Consequently, individual reactions of the TCA cycle are impaired, leading to the depletion of intermediates required for the cycle to proceed. Such diseases typically present early with severe symptoms, such as mental retardation, microcephaly, deafness, and hypotonia and are often fatal in early childhood.

Other metabolic disorders affect pathways in which other metabolites, such as fatty acids and amino acids, are converted into intermediates of the TCA cycle. For example, propionyl- CoA is generated by oxidation of odd-chain fatty acids and breakdown of the amino acids isoleucine, valine, threonine, and methionine and is then converted to succinyl-CoA. The conversion of propionyl-CoA to succinyl-CoA involves the following sequence of reactions:

CoA

Propionic acidemia (PA) results from a deficiency in propionyl-CoA carboxylase. In patients with PA, excess propionyl-CoA is converted to propionic acid, which accumulates in the bloodstream. In an analogous manner, methylmalonic acidemia (MMA) results from a deficiency in methylmalonyl CoA mutase. In patients with MMA, excess methylmalonyl-CoA is converted to methylmalonic acid, which accumulates in the bloodstream. In both PA and MMA, the high levels of acid in the blood lead to a variety of potentially fatal effects, such as organ damage, stroke, and seizure.

Treatment of PA and MMA focuses on dietary management to avoid amino acids that trigger acid accumulation. Consequently, patients with PA or MMA must strictly adhere to a low-protein diet to minimize their intake of triggering amino acids. The restricted intake of amino acids in patients with PA or MMA often leads to a shortage of L-carnitine, a quaternary ammonium compound involved in transport of fatty acids across the mitochondrial membrane. Consequently, patients with PA or MMA typically receive supplemental L-camitine. The low- protein diet of infants with PA or MMA also puts them at risk for bacterial infections, so they may be given antibiotics prophylactically.

PA and MMA are autosomal recessive genetic disorders. Homozygous mutations in either of PCCA or PCCB, the genes that encode propionyl-CoA carboxylase, cause PA. MMA can result from homozygous mutations in MUT, which encodes methylmalonyl-CoA mutase. Methylmalonyl-CoA mutase requires vitamin B _l2 as a cofactor, and mutations in genes involved in vitamin B _I2 metabolism, such as LMBRD1, MCEE, MMAA, MMAB, MMACHC, and MMADHC, can also cause MMA. A dietary deficiency of vitamin B _I2 can also lead to MMA.

Abnormal TCA cycle metabolism is also observed in other diseases that do not have direct genetic links to this metabolic pathway. For example, altered TCA metabolism is observed in neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis, Alzheimer's disease, Parkinson's disease, or Huntington's disease, and in a wide variety of cancers. Although the symptoms this diverse set of diseases vary, in many cases decreased activity of specific TCA enzymes or decreased mitochondrial ATP production has been observed, and it is believed that boosting levels of TCA cycle intermediates would mitigate the symptoms and improve prognoses.

The invention provides methods for treating any disease, disorder, or condition associated with altered TCA cycle metabolism or that can be ameliorated by providing an intermediate of the TCA cycle. The condition may be an inherited disorder, such as PA, MMA, 2-oxoglutaric aciduria, fumarase deficiency, or succinyl-CoA synthetase deficiency. The condition may be a neurodegenerative disorder, such as Amyotrophic Lateral Sclerosis, Alzheimer's disease, Parkinson's disease, or Huntington's disease. The condition may be a cancer, such as pancreatic cancer, kidney cancer, cervical cancer, prostate cancer, muscle cancer, gastric cancer, colon cancer, glioblastoma, glioma, paraganglioma, leukemia, liver cancer, breast cancer, carcinoma, and neuroblastoma.

Additional metabolic diseases, disorders, or conditions that can be treated with compositions or methods of the invention include acute angina, acute kidney injury, acute starvation, adrenoleukodystrophy (ALD), adrenomyeloneuropathy (AMN), an age-associated disease, Alpers disease (progressive infantile poliodystrophy), Alzheimer's disease, amyotrophic lateral sclerosis (ALS), atrial fibrillation, autism and autism spectrum disorders (ASD), Barth syndrome (lethal infantile cardiomyopathy), beta-oxidation defects, bioenergetic metabolism deficiency, bipolar disorder, carnitine-acyl-camitine deficiency, carnitine deficiency, carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, a cerebral vascular accident, chronic progressive external ophthalmoplegia syndrome) (CPEO), co enzyme Q10 deficiency, complex I deficiency (NADH dehydrogenase (NADH-CoQ reductase) deficiency), complex II deficiency (succinate dehydrogenase deficiency), complex III deficiency (ubiquinone-cytochrome c oxidoreductase deficiency, complex IV deficiency/C OX deficiency, complex V deficiency (ATP synthase deficiency), coronary occlusion, COX deficiency, creatine deficiency syndromes (e.g., cerebral creatine deficiency syndromes (CCDS), guanidinoaceteate methyltransferase deficiency (GAMT deficiency), L-arginine: glycine amidinotransferase deficiency (AGAT deficiency), and SLC6A8-related creatine transporter deficiency (SLC6A8 deficiency)), diabetes, disorders related to POLG mutations, endotoxemia, an energetic disorder, epilepsy, Friedreich's ataxia (FRDA or FA), glutaric aciduria type II, Huntington's disease, hypoxia, ischemia, Keams-Sayre syndrome (KSS), lactic acidosis, long-chain acyl-CoA dehydrogenase deficiency) (FCAD, VFCAD, VFCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (FCHAD), Feigh disease or syndrome (subacute necrotizing encephalomyelopathy), Feber's hereditary optic neuropathy (FHON), Fuft disease, macular degeneration, male infertility, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial associated disease, mitochondrial cytopathy, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MEFAS), mitochondrial encephalopathy (including encephalomyopathy, encephalomyelopathy), mitochondrial DNA depletion, mitochondrial myopathy, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial respiratory chain deficiency, multiple organ dysfunction syndrome, mood disorders, motor neuron diseases, muscular dystrophy (e.g., Duchenne’s muscular dystrophy and Becker’s muscular dystrophy), myocardial infarction, myoclonic epilepsy and ragged-red fibers (MERRF),

myoneurogastointestinal disorder and encephalopathy (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), neurodegenerative disorders associated with Parkinson's, Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, refractory epilepsy, renal tubular acidosis, respiratory chain deficiencies, schizophrenia, sepsis, short-chain acyl-CoA dehydrogenase deficiency (SCAD), short-chain hydroxyacyl-CoA dehydrogenase deficiency (SCHAD, SCHADD), stroke, succinyl CoA lyase deficiency, systemic inflammatory response syndrome, and very long-chain acyl-CoA dehydrogenase deficiency (VFCAD). The disease, disorder, or condition may be secondary to, or associated with another disease, disorder, or condition. For example, the disease disorder or condition may be associated with cardiac disease, a gastrointestinal disorder, ischemia reperfusion injury, intoxication, liver disease, loss of motor control, muscle weakness and pain, mutations in the mitochondrial genome, a neurologic disease, disorder or condition, pain or fatigue disease, seizures, sensineural hearing loss, swallowing difficulties, tinnitus, or visual/hearing problems.

Diseases, disorders, and conditions associated with altered TCA cycle metabolism often affect organs, tissues, and systems that have high energy demands, such as the brain, cochlea, endocrine system, heart, kidney, liver, respiratory system, retina, and skeletal muscles. Thus, the compositions and methods of the invention may be used to treat diseases, disorders, or conditions that affect one or more of these organs, tissues, or systems.

Another clinically important metabolic pathway is the mitochondrial electron transport chain. The electron transport chain uses a complex series of redox reactions to create a proton gradient across the mitochondrial inner membrane, and the chemiosmotic potential from the proton gradient is used to drive adenosine triphosphate (ATP) synthesis. The electron transport chain involves four enzymatic complexes in the mitochondrial inner membrane: NADH dehydrogenase, also called respiratory complex I; succinate dehydrogenase, also called respiratory complex II; coenzyme Q:cytochrome c reductase, also called respiratory complex III; and cytochrome c oxidase, also called respiratory complex IV. Electrons enter the transport chain in either of two ways. First, NADH dehydrogenase may transfer electrons from NADH to ubiquinone, the first intermediate electron carrier in the chain. Alternatively, electrons from succinate may be transferred to ubiquinone by succinate dehydrogenase. In the next step of the electron transport chain, electrons are transferred from ubiquinone to cytochrome c, the second intermediate electron carrier, by coenzyme Qxytochrome c reductase. In the final step, cytochrome c oxidase transfers electrons from cytochrome c to molecular oxygen to form water, the net product of electron transport. Succinate dehydrogenase is the only enzyme that participates in both the TCA cycle and the electron transport chain.

Leber's hereditary optic neuropathy (LHON) is a retinal degenerative condition caused by defects in the electron transport chain. LHON results from mutations in mitochondrial genes that encode components of NADH dehydrogenase, such as MT-ND1, MT-ND4, MT-ND4L, and MT-ND6. Because mutations that cause LHON are encoded by genes in the mitochondrial genome, which is transmitted to the embryo from the egg but not from the sperm, LHON can only be inherited maternally.

An insight of the invention is that succinate is useful for treatment of LHON. Due to reduced NADH dehydrogenase activity, electron transport and ATP synthesis are decreased in patients with LHON. In particular, formation of ubiquinone, the first intermediate electron carrier in the chain, is diminished. However, electron transport activity can be restored by providing supplemental succinate, which can donate electrons to form ubiquinone via succinate dehydrogenase. Thus, providing additional succinate to serve as electron donor for the electron transport chain in patients with LHON compensates for the insufficiency of electron transfer from NADH.

Compositions Containing TCA Cycle Intermediates

The invention provides compositions that contain one or more TCA cycle intermediates or prodrugs, analogs, or derivatives thereof formulated for non-oral administration. For example and without limitation, the TCA cycle intermediate or prodrug, analog, or derivative thereof may be citrate, cis-aconitate, D-isocitrate, a-ketoglutarate, succinate, fumarate, malate, oxaloacetate, acetone, acetoacetate, b-hydroxybutyrate, b-ketopentanoate, or b-hydroxypentanoate.

Preferably, the TCA cycle intermediate is succinate. The TCA cycle intermediate or prodrug, analog, or derivative thereof may be provided as a free acid, salt, or a non-salt form.

The compositions containing one or more TCA cycle intermediates or prodrugs, analogs, or derivatives thereof may be formulated for administration by any non-oral route. For example and without limitations, the compositions may be formulated for subcutaneous, intravenous, intraarterial, intramuscular, intradermal, or rectal administration. Preferably, the compositions are formulated for intravenous or subcutaneous administration.

A prodrug is a medication or compound that, after administration, is metabolized (i.e., converted within the body) into a pharmacologically active drug. The prodrug itself may be pharmacologically inactive. Prodrugs may be used to improve how a medicine is absorbed, distributed, metabolized, and excreted. The prodrug may improve the bioavailability of the active drug when the active drug is poorly absorbed from the gastrointestinal tract. The prodrug may improve how selectively the drug interacts with cells or processes that are not its intended target, thereby reducing unintended and undesirable side effects. The prodrug may be converted into a biologically active form (bioactivated) inside cells (a Type I prodrug) or outside cells (a Type II prodrug). The prodrug may bioactivated in the gastrointestinal tract, in systemic circulation, in metabolic tissue other than the target tissue, or in the target tissue.

TCA cycle intermediates or prodrugs, analogs, or derivatives thereof may be provided as pharmaceutically acceptable salts, such as nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,

glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a pharmaceutically acceptable salt is an alkali salt. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, a pharmaceutically acceptable salt is an alkaline earth metal salt. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

Prodrugs of succinate are known in the art and described in, for example, International Publication Nos. WO 1997/047584, WO 2014/053857, WO 2015/155230, WO 2015/155231, WO/2015/155238, WO 2017/060400, WO 2017/060418, and WO 2017/060422; European Patent Publication Nos. EP 2903609, EP 3129016, EP 3129364, EP 3129058, and EP 3391941; and U.S. Patent Publication Nos. US 2017/0100359, US 2017/0105960, and US 2017/0105961, the contents of each of which are incorporated herein by reference. Representative compounds from these documents are provided below.

Compounds of formulas (I) and (IA):

or a pharmaceutically acceptable salt thereof, wherein the dotted bond between A and B denotes an optional bond so as to form a ring closed structure,

and wherein

when the formula is Formula (I), Z is selected from -CH ₂ - (e.g., derived from malonic acid), -CH2-CH2-CH2 (e.g., derived from glutaric acid), -CH=CH2- (e.g., derived from fumaric acid), -CH2-CH(OH)- (e.g., derived from malic acid), -CH(OH)-CH2- (e.g., derived from malic acid), CH2C(OH)(COOH)-CH2- (e.g., derived from citric acid), -C(0)-CH ₂ -CH ₂ - (e.g., derived from alphaketoglutaric acid), -CH2-CH2-C(0)- (e.g., derived from alpha-ketoglutaric acid), - CH ₂ -C(COC(OH)-C(COOH)-CHOH)=CH- (e.g., derived from aconitic acid), -CH=C(COOH)- C¾- (e.g., derived from aconitic acid), -CH(OH)-CH(COOH)-CH2- (e.g., derived from isocitric acid), -CH ₂ -CH(C00H)-CH(0H)- (e.g., derived from isocitric acid), -CH ₂ -CH(COOH)-C(=0)- (e.g., derived from oxalosuccinic acid), -C(=0)-CH(COOH)-CH ₂ - (e.g., derived from

oxalosuccinic acid), -C(=0)-CH ₂ - (e.g., derived from oxaloacetatic acid), -CH ₂ -C(=0)- (e.g., derived from oxaloacetic acid); or

when the formula is Formula (IA), Z is selected from -CH(OH)-CH2(OH) and n is 0 e.g., derived from glyceric acid); or Z is absent or -CH2- and n is 1 and B is an alkyl group (e.g., derived from pyruvic acid or acetoacetic acid, respectively);

A and B are independently different or the same and are selected from -OR, -OR’, - NHR”, -SR’”

or -OH; wherein R is

or in those cases, where the compound is according to Formula (IA), then B is C ₁ -C ₄ - alkyl, branched or straight, preferably R is Me;

R’ is selected from the formula (II), (V) or (IX) below:

O R-s

_ RA XJ

3 (0 )

and both A and B are not -OH,

R’, R” and R’” are independently different or identical and are selected from formula

(VII- VIII) below:

Ri and R ₃ are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH ₂ Xalkyl, CH ₂ CH2CH20C(=0)CH2CH2C0X ₆ R8 or

X is selected from O, NH, NR ₆ , S,

R ₂ is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(0)CH ₃ , C(0)CH ₂ C(0)CH ₃ , C(0)CH ₂ CH(0H)CH ₃ ,

p is an integer and is 1 or 2,

R ₆ is selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII)

X ₅ is selected from -H, -COOH, -C(=0)XR ₆ ,C0NRiR ₃ or one of the formulas

R9 is selected from H, Me, Et or 0 ₂ CCH ₂ CH ₂ C0XR ₈ ,

Rio is selected from Oacyl, NHalkyl, NHacyl, or 0 ₂ CCH ₂ CH ₂ C0 X ₆ R ₈ ,

X ₆ is O or NRg, and R ₈ is selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t- butyl, acetyl, acyl, propionyl, benzoyl, succinyl, or formula (II), or formula (VIII),

R ₁₁ and R ₁₂ are independently the same or different and are selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, succinyl, acyl, - CH ₂ Xalkyl, -CH ₂ Xacyl, where X is selected from O, NR ₆ or S, R , R _H and R15 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, -COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH ₂ Xalkyl

R _c and R are independently CH ₂ Xalkyl, CH ₂ Xacyl, where X = O, NR ₆ or S, and alkyl is e.g. H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, and acyl is e.g. formyl, acetyl, propionyl, isopropionyl, byturyl, tert-butyryl, pentanoyl, benzoyl, succinyl, or the like,

R _f , R _g and R _h are independently selected from Xacyl, -CH ₂ Xalkyl, -CH ₂ X-acyl and R9, alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert- butyl, n-pentyl, neopentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, nonyl ordecyl, and acyl is selected from formyl, acetyl, propionyl, butyryl pentanoyl, benzoyl, succinyl and the like,

R _2O and R _2I are independently different or identical and are selected from H, lower alkyl, i.e. C1-C4 alkyl or R ₂ o and R _2I together may form a C4-C ₇ cycloalkyl or an aromatic group, both of which may optionally be substituted with halogen, hydroxyl or a lower alkyl, or

R _2O and R _2I may be

CH ₂ X-acyl, F, CH ₂ COOH, CH ₂ C0 ₂ alkyl, and

when there is a cyclic bond present between A and B the compound is

and acyls and alkyls may be optionally substituted.

Compounds of formulas (I) and (IA),or a pharmaceutically acceptable salt thereof, wherein the dotted bond between A and B denotes an optional bond so as to form a ring closed structure, and

wherein A is -ORi, and Ri is H or a pharmaceutically acceptable salt, or an optionally substituted alkyl group or a group of formula (II) and B in formula (I) is -OR ₂ and R ₂ is independently a group according to formula (II) where formula (II) is

wherein R ₃ and R ₄ are independently H, optionally substituted C ₁ -C ₃ alkyl, or are linked together to form a ring and where R ₅ is linked to Ri to form a ring or R ₅ is selected from OCOR _a , OCOOR _b , 0C0NR _c R _d ,S0 ₂ R _e , OPO(OR _f )(OR _g ) or CONR _c R _d where R _a is optionally substituted alkyl or optionally substituted cycloalkyl, R _b is optionally substituted alkyl, R _c and R _d are independently H, optionally substituted alkyl or are linked together to form a ring which may contain one or more further heteroatoms, R _e is optionally substituted alkyl, R _f and R _g are independently, H, methyl, ethyl or are linked together to form a ring;

wherein B in Formula (IA) is C1-C4 alkyl, branched or straight;

and wherein

when the formula is Formula (I), Z is selected from -CH ₂ - (e.g., derived from malonic acid), -CH ₂ -CH ₂ -CH ₂ (e.g., derived from glutaric acid), -CH=CH ₂ - (e.g., derived from fumaric acid), -CH ₂ -CH(OH)- (e.g., derived from malic acid), -CH(OH)-CH ₂ - (e.g., derived from malic acid), CH ₂ C(OH)(COOH)-CH ₂ - (e.g., derived from citric acid -C(O)- CH ₂ -CH ₂ - (e.g., derived from alpha-ketoglutaric acid), -CH ₂ -CH ₂ -C(0)- (e.g., derived from alpha-ketoglutaric acid), - CH ₂ -C(COC(OH)-C(COOH)-CHOH)=CH- (e.g., derived from aconitic acid), -CH=C(COOH)- CH ₂ - (e.g., derived from aconitic acid), -CH(OH)- CH(COOH)-CH ₂ - (e.g., derived from isocitric acid), -CH ₂ -CH(COOH)-CH(OH)- (e.g., derived from isocitric acid), -CH ₂ -CH(C00H)-C(=0)- (e.g., derived from oxalosuccinic acid), -C(=0)-CH(C00H)-CH ₂ - (e.g., derived from

oxalosuccinic acid), -C(=0)-CH ₂ - (e.g., derived from oxaloacetatic acid), -CH ₂ -C(=0)- (e.g., derived from oxaloacetic acid); or

when the formula is Formula (IA), Z is selected from -CH(OH)-CH ₂ (OH) and n is 0 e.g., derived from glyceric acid); or Z is absent or -CH ₂ - and n is 1 and B is an alkyl group (e.g., derived from pyruvic acid or acetoacetic acid, respectively). Compounds of formulas (I) and (IA),or a pharmaceutically acceptable salt thereof, wherein the dotted bond between A and B denotes an optional bond so as to form a ring closed structure,

and wherein

when the formula is Formula (I), Z is selected from -CH ₂ - (e.g., derived from malonic acid), -CH ₂ -CH ₂ -CH ₂ (e.g., derived from glutaric acid), -CH=CH ₂ - (e.g., derived from fumaric acid), -CH ₂ -CH(OH)- (e.g., derived from malic acid), -CH(OH)-CH ₂ - (e.g., derived from malic acid), CH ₂ C(OH)(COOH)-CH ₂ - (e.g., derived from citric acid), -C(0)-CH ₂ -CH ₂ - (e.g., derived from alphaketoglutaric acid), -CH ₂ -CH ₂ -C(0)- (e.g., derived from alpha-ketoglutaric acid), - CH ₂ -C(COC(OH)-C(COOH)-CHOH)=CH- (e.g., derived from aconitic acid), -CH=C(COOH)- CH ₂ - (e.g., derived from aconitic acid), -CH(OH)-CH(COOH)-CH ₂ - (e.g., derived from isocitric acid), -CH ₂ -CH(COOH)-CH(OH)- (e.g., derived from isocitric acid), -CH ₂ -CH(COOH)-C(=0)- (e.g., derived from oxalosuccinic acid), -C(=0)-CH(COOH)-CH ₂ - (e.g., derived from

oxalosuccinic acid), -C(=0)-CH ₂ - (e.g., derived from oxaloacetatic acid), -CH ₂ -C(=0)- (e.g., derived from oxaloacetic acid); or

when the formula is Formula (IA), Z is selected from -CH(OH)-CH ₂ (OH) and n is 0 e.g., derived from glyceric acid); or Z is absent or -CH ₂ - and n is 1 and B is an alkyl group (e.g., derived from pyruvic acid or acetoacetic acid, respectively);

A is selected from -SR, -OR and NHR, and R is

when Z is CH ₂ and R is Me, Et, propyl, butyl, pentyl, hexyl, heptyl, octyl or succinyl, then X5, R15, R14 and R13 cannot all be H;

when Z is -CH ₂ -CH ₂ -CH ₂ , -CH=CH-, -CH ₂ -CH(OH)-, -CH(OH)-CH ₂ -, - CH ₂ C(OH)(COOH)-CH ₂ -, -C(0)-CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -C(0)-, -CH ₂ -C(COOH)=CH-, - CH=C(COOH)-CH ₂ -, -CH(OH)-CH(COOH)-CH2-, -CH2-CH(COOH)-CH(OH)-, -CH2- CH(C00H)-C(=0)-, -C(=0)-CH(C00H)-CH2-, -C(=0)-CH2-, or -CH2-C(=0)- and Ri is Me, octyl or succinyl, then X5, R 5, R14 and R13 cannot all be H;

Ri cannot contain the motif -CH2CH2N-acyl; Ri cannot be glutamate.; when in formula (IA), Z is-CH(OH)-CH2(OH) and n is 0, or Z is absent and n is 1 and B is an alkyl group (e.g., derived from pyruvic acid) then Ri cannot be Me when X5 is -COOH, or -C(=0)XR ₆ ;

B is selected from -0-R\ -NHR”, -SR’” or -OH; and R’ is selected from the formula (II) to (IX) below:

or in those cases, where the compound is according to Formula (IA), then B is C1-C4- alkyl, branched or straight, preferably R is Me;

R’, R” and R’” are independently different or identical and is selected from formula (VII- VIII) below:

Ri and R ₃ are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, -CH ₂ Xalkyl, - CH ₂ X-acyl, F, -CH ₂ COOH, -CH ₂ C0 ₂ alkyl,

X is selected from O, NH, NR ₆ , S,

R ₂ is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, -C(0)CH ₃ , - C(0)CH ₂ C(0)CH ₃ , -C(0)CH ₂ CH(0H)CH ₃ ,

p is an integer and is 1 or 2

R ₆ is selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII)

X ₅ is selected from -H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, -COOH, - C(=0)XR ₆ , CONR _I R ₃ or is formula

X7 is selected from Ri, -NR _I R ₃ ,

R9 is selected from H, Me, Et or 0 ₂ CCH ₂ CH ₂ C0XR ₈

Rio is selected from -Oacyl, -NHalkyl,- NHacyl, or 0 ₂ CCH ₂ CH ₂ C0X ₆ R ₈

X ₆ is selected from O, NR ₈ , NR ₆ R ₈ , wherein R ₆ and R ₈ are independently different or identical and are is selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII),

R ₁₁ and R _I2 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, propionyl, benzoyl, -CH ₂ Xalkyl, -CH ₂ Xacyl, where X is O, NR ₆ or S,

R _c and R _d are independently different or identical and are selected from CH ₂ Xalkyl, CH ₂ Xacyl, where X = O, NR ₆ or S,

R _I3 , R ₁₄ and R ₁₅ are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, -COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH ₂ Xalkyl; Substituents on R _l3 and R _I4 or R _l3 and R ₁₅ may bridge to form a cyclic system to form cycloalkyl, heterocycloalkyl, lactone or lactams.

R _f , R _g and R _h are independently different or identical and are selected from Xacyl, - CH ₂ Xalkyl, -CH ₂ X-acyl and Rg,

alkyl is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,

acyl is selected from formyl, acetyl, propionyl, isopropionyl, buturyl, tert-butyryl, pentanoyl, benzoyl, succinyl.

acyl and/or alkyl may be optionally substituted, and

when the dotted bond between A and B is present, the compound according to formula (I) is

om -COOH, -C(=0)XR ₆ ,

A compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein the dotted bond between A and B denotes an optional bond so as to form a ring closed structure, and wherein

Z is selected from -CH ₂ -CH ₂ - or >CH(CH ₃ ),

A is selected from -SR, -OR and NHR, and R is

B is selected from -0-R\ -NHR”, -SR’” or -OH; and R’ is selected from the formula (II) to (IX) below:

R’, R” and R’” are independently different or identical and is selected from formula (IV- VIII) below:

Ri and R ₃ are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH ₂ Xalkyl, CH ₂ X-acyl, F, CH ₂ COOH, CH ₂ C0 ₂ alkyl,

X is selected from O, NH, NR ₆ , S,

R ₂ is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(0)CH ₃ ,

C(0)CH ₂ C(0)CH ₃ , C(0)CH ₂ CH(0H)CH ₃ ,

p is an integer and is 1 or 2

R ₆ is selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII)

X ₅ is selected from -H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, -COOH, - C(=0)XR ₆ , CONR _I R ₃ or is formula

X7 is selected from Ri, -NR _I R ₃ ,

R9 is selected from H, Me, Et or 0 ₂ CCH ₂ CH ₂ C0XR ₈

Rio is selected from Oacyl, NHalkyl, NHacyl, or 0 ₂ CCH ₂ CH ₂ C0X ₆ R ₈

X ₆ is selected from O, NR ₈ , NR ₆ R ₈ , wherein R ₆ and R ₈ are independently different or identical and are is selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII),

R ₁₁ and R _I2 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, propionyl, benzoyl, -CH ₂ Xalkyl, - CH ₂ Xacyl, where X is O, NR ₆ or S,

R _c and R _d are independently different or identical and are selected from CH ₂ Xalkyl, CH ₂ Xacyl, where X = O, NR ₆ or S,

R _I3 , R ₁₄ and R ₁₅ are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, -COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH ₂ Xalkyl; substituents on R _l3 and RI ₄ or R _l3 and R ₁₅ may bridge to form a cyclic system to form cycloalkyl, heterocycloalkyl, lactone or lactams.

R _f , R _g and R _h are independently different or identical and are selected from Xacyl, - CH ₂ Xalkyl, -CH ₂ X-acyl and Rg,

alkyl is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl,

acyl is selected from formyl, acetyl, propionyl, isopropionyl, buturyl, tert-butyryl, pentanoyl, benzoyl, succinyl.

acyl and/or alkyl may be optionally substituted, and

when the dotted bond between A and B is present, the compound according to formula (I) is

A compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein the dotted bond between A and B denotes an optional bond so as to form a ring closed structure, and wherein

Z is selected from -CH ₂ -CH ₂ - or >CH(CH ₃ ),

A and B are independently different or the same and are selected from -OR, -OR’, -

NHR”, -SR’” or -OH; wherein R is

R’ is selected from the formula (II), (V) or (IX) below:

and both A and B are not -OH,

R’, R” and R’” are independently different or identical and are selected from formula (VII- VIII) below:

Ri and R ₃ are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH ₂ Xalkyl, CH ₂ CH2CH20C(=0)CH2CH2C0X ₆ R8 or

X is selected from O, NH, NR ₆ , S,

R ₂ is selected from Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, C(0)CH ₃ ,

C(0)CH ₂ C(0)CH ₃ , C(0)CH ₂ CH(0H)CH ₃ ,

p is an integer and is 1 or 2,

R ₆ is selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, or formula (II), or formula (VIII)

X ₅ is selected from -H, -COOH, -C(=0)XR ₆ , CONR _I R ₃ or one of the formulas

R9 is selected from H, Me, Et or 0 ₂ CCH ₂ CH ₂ C0XR ₈ ,

R10 is selected from Oacyl, NHalkyl, NHacyl, or 0 ₂ CCH ₂ CH ₂ C0 X ₆ R ₈ ,

X ₆ is O or NR ₈ , and R ₈ is selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, succinyl, or formula (II), or formula (VIII),

R ₁₁ and R _I2 are independently the same or different and are selected from H, alkyl, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, acetyl, acyl, propionyl, benzoyl, succinyl, acyl, - CH ₂ Xalkyl, -CH ₂ Xacyl, where X is selected from O, NR ₆ or S,

Ris, RM and RI5 are independently different or identical and are selected from H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, -COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH ₂ Xalkyl

R _c and R are independently CH ₂ Xalkyl, CH ₂ Xacyl, where X = O, NR ₆ or S, and alkyl is e.g. H, Me, Et, propyl, i-propyl, butyl, iso-butyl, t-butyl, and acyl is e.g. formyl, acetyl, propionyl, isopropionyl, byturyl, tert-butyryl, pentanoyl, benzoyl, succinyl, or the like, R _f , R„. and R _h are independently selected from Xacyl, -CH ₂ Xalkyl, -CH ₂ X-acyl and R9, alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n pentyl, neopentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, nonyl or decyl, and acyl is selected from formyl, acetyl, propionyl, butyryl pentanoyl, benzoyl, succinyl and the like,

R _2O and R _2I are independently different or identical and are selected from H, lower alkyl, i.e. C1-C4 alkyl or R ₂₀ and R _2I together may form a C ₄ -C ₇ cycloalkyl or an aromatic group, both of which may optionally be substituted with halogen, hydroxyl or a lower alkyl, or

R _2O and R _2I may be

CH ₂ X-acyl, F, CH ₂ COOH, CH ₂ C0 ₂ alkyl, and

when there is a cyclic bond present between A and B the compound is

and acyls and alkyls may be optionally substituted.

A compound of formula (A),

and wherein R ₃ is selected from H, or optionally substituted Ci_C ₃ alkyl such as e.g., methyl, ethyl, propyl or iso-propyl and wherein R5 is -0C(=0)R _a , wherein R _a is methyl or formula (C)

wherein R-\ is H or a pharmaceutically acceptable salt, or an optionally substituted alkyl group or a group of formula (II) and R ₂ is independently a group according to formula (II) where formula (II) is

wherein R ₃ and R ₄ are independently H, optionally substituted C ₁ -C ₃ alkyl, or are linked together to form a ring and where R ₅ is linked to Ri to form a ring or R ₅ is selected from OCOR _a , OCOOR _b , OCONR _c R _d ,S0 ₂ R _e , OPO(OR _f )(OR _g ) or CONR _c R _d where R _a is optionally substituted alkyl or optionally substituted cycloalkyl, R _b is optionally substituted alkyl, R _c and R _d are independently H, optionally substituted alkyl or are linked together to form a ring which may contain one or more further heteroatoms, R _e is optionally substituted alkyl, R _f and R _g are independently, H, methyl, ethyl or are linked together to form a ring and wherein the compound is not succinic acid bis (2,2-dimethylpropionyloxymethyl) ester; succinic acid

dibutyryloxymethyl ester; or succinic acid bis-(l-butyryloxy-ethyl) ester.

The invention also provides therapeutic compositions that contain citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid as one component of combination therapies that also include a composition containing a TCA cycle intermediate or prodrug, analog, or derivative thereof. The compositions that contain citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be formulated for oral administration, or they may be formulated for non-oral administration.

The compositions of the invention may contain a buffering agent. Any suitable buffering agent may be used. The buffering agent may be an amino acid or a derivative thereof. For example, the amino acid may be alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic Acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. Preferably, the amino acid is lysine or ornithine. The buffering agent may be or may contain a metal ion. For example, the metal ion may be Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , or Cu ²⁺ .

The buffering agent may maintain the pH of the composition at neutral or close to neutral. For example and without limitation, the buffering agent may maintain the pH of the composition at from about 3.0 to about 10.0, from about 3.0 to about 9.0, from about 3.0 to about 8.0 from about 3.0 to about 7.0, from about 3.0 to about 6.0, from about 4.0 to about 10.0, from about 4.0 to about 9.0, from about 4.0 to about 8.0 from about 4.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 10.0, from about 5.0 to about 9.0, from about 5.0 to about 8.0 from about 5.0 to about 7.0, from about 6.0 to about 10.0, from about 6.0 to about 9.0, from about 6.0 to about 8.0 from about 7.0 to about 10.0, from about 7.0 to about 9.0, or from about 8.0 to about 10.0.

In another aspect, the invention provides a formulation including citric acid, or a prodrug, analog, derivative thereof; at least one citrate salt, or a prodrug, analog, or derivative thereof; and an amino acid. The formulation may include citric acid and at least one citrate salt, for example, the at least one citrate salt may be selected from the group consisting of monosodium citrate and monopotassium citrate. In other embodiments, the formulation may be citric acid and at least two citrate salts, for example, the at least two citrate salts may be monosodium citrate and monopotassium citrate.

The formulation may include any natural or non-natural amino acid. In certain embodiments, the amino acid is a cationic amino acid, such as lysine or arginine. The formulation may include other components, such as a sugar, for example sucrose.

The formulation may be provided by any route of administration, and a preferable route is oral. An exemplary oral formulation is a powder that is soluble in an aqueous medium. In a particular embodiment, the formulation includes citric acid, monosodium citrate, monopotassium citrate, sucrose, and one or more of lysine and arginine.

Other aspects of the invention provide methods of treating a condition associated with altered TCA cycle metabolism in a subject. Such methods may involve providing to a subject having a condition associated with altered TCA cycle metabolism a formulation comprising (i) citric acid, or a prodrug, analog, derivative thereof; (ii) at least one citrate salt, or a prodrug, analog, or derivative thereof; and (iii) an amino acid, wherein a therapeutic effect is achieved in the subject by activity of a combination of two or more of components (i), (ii), and (iii) in the formulation. In certain embodiments, the amino acid is incorporated into the TCA cycle of the subject and contributes to the therapeutic effect that is achieved in the subject.

Exemplary conditions include a disorder related to POLG mutation, an energetic disorder, glutaric acidemia type 1 or type 2, a long chain fatty acid oxidation disorder, methylmalonic acidemia (MMA), a mitochondrial associated disease, a mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy and ragged-red fibers (MERRF), mitochondrial myopathy, a mitochondrial respiratory chain deficiency, muscular dystrophy (e.g., Duchenne’s muscular dystrophy and Becker’s muscular dystrophy), a neurologic disorder, a pain or fatigue disease, propionic acidemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, or succinyl CoA lyase deficiency.

The formulation may include citric acid and at least one citrate salt, for example, the at least one citrate salt may be selected from the group consisting of monosodium citrate and monopotassium citrate. In other embodiments, the formulation may be citric acid and at least two citrate salts, for example, the at least two citrate salts may be monosodium citrate and monopotassium citrate.

The formulation may include any natural or non-natural amino acid. In certain embodiments, the amino acid is a cationic amino acid, such as lysine or arginine. The formulation may include other components, such as a sugar, for example sucrose.

The formulation may be provided by any route of administration, and a preferable route is oral. An exemplary oral formulation is a powder that is soluble in an aqueous medium. In a particular embodiment, the formulation includes citric acid, monosodium citrate, monopotassium citrate, sucrose, and one or more of lysine and arginine. The formulation may be provided in one or multiple doses per day. For example, one or more of the formulations may be provided in 1,

2, 3, 4, 5, 6, or more doses per day.

The formulation containing citrate or a prodrug, analog, or derivative thereof may be provided at any therapeutically effective dose. For example, citrate or a prodrug, analog, or derivative thereof may be provided at from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.1 mg/kg subject weight to about 5 g/kg subject weight,

from about 0.2 mg/kg subject weight to about 2 g/kg subject weight, from about 0.5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 500 mg/kg subject weight, from about 2 mg/kg subject weight to about 200 mg/kg subject weight, or from about 5 mg/kg subject weight to about 100 mg/kg subject weight.

The formulation, or the soluble components within the formulation, may contain citric acid, monosodium citrate, monopotassium citrate, lysine, and sucrose. The formulation may contain, by mass, from about 40% to about 60% citric acid; from about 1% to about 10% monosodium citrate; from about 0.1% to about 5% monopotassium citrate; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 49.2 % citric acid, about 2% monosodium citrate, about 0.3% monopotassium citrate, about 37.5% lysine, and about 11% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 44.2 % citric acid, about 8.3% monosodium citrate, about 2.3% monopotassium citrate, about 33.8% lysine, and about 11.5% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, from about 50% to about 60% citrate, citric acid, or a combination thereof; from about 0.2% to about 1% sodium; from about 0.02% to about 0.5% potassium; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose.

Formulations of the invention may contain aqueous suspensions of one or more TCA cycle intermediates or prodrugs, analogs, or derivatives thereof. The aqueous suspensions may contain the TCA cycle intermediate in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium

carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the TCA cycle intermediate in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the TCA cycle intermediate in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified, for example sweetening, flavoring and coloring agents, may also be present.

The compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soya bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Compositions of the invention may include other pharmaceutically acceptable carriers, such as sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin (glycerol), erythritol, xylitol. sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

Compositions containing TCA cycle intermediates may be in a form suitable for oral use. For example, oral formulations may include tablets, troches, lozenges, fast-melts, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations. Tablets contain citrate or citric acid in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration in the stomach and absorption lower down in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Patents 4,256,108, 4,166,452 and 4,265,874, to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsules in which the citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the compound is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

An alternative oral formulation, where control of gastrointestinal tract hydrolysis of the citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid is sought, can be achieved using a controlled-release formulation, where a compound of the invention is encapsulated in an enteric coating.

Syrups and elixirs for oral administration of citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and agents for flavoring and/or coloring. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in l,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The methods of the invention include compositions that are provided as intraocular formulations. Intraocular formulations include any formulation suitable for delivery of an agent to the eye. For example and without limitation, intraocular formulations include aqueous gels, contact lenses, dendrimers, emulsions, emulsions, eye drops, implants, in situ thermosensitive gels, liposomes, microneedles, nanomicelles, nanoparticles, nanosuspensions, ointments, and suspensions. Ocular formulations are known in the art and describe in, for example, Patel, A., et al., Ocular drug delivery systems: An overview, World J Pharmacol. 2013; 2(2): 47-64.

doi: l0.5497/wjp.v2.i2.47; U.S. Patent No. 9,636,347; U.S. Publication Nos. 2017/0044274 and 2009/0148527; and International Publication No. WO 2015/105458, the contents of each of which are incorporated herein by reference.

The invention also provides therapeutic compositions that contain the following components: a non-salt form of citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid; a nutritional source for infants, such as baby formula or human breast milk; and a buffering agent. The compositions are suitable for oral administration to human infants and provide both basic nutrition and a supplemental source of citrate or citric acid.

The nutritional source may contain one or more of a fat source, a carbohydrate source, and a source of protein or amino acids. The protein source may contain whey and/or casein. The protein source may contain lactose. The fat source may contain a vegetable oil. The nutritional source may contain vitamins and/or minerals.

The nutritional source may be engineered to fulfill the dietary needs of an infant with a metabolic disorder, such as PA or MMA. For example, the source of protein or amino acids may be substantially free of valine, isoleucine, threonine, and methionine. The fat source may be free of odd-chain fatty acids. The nutritional source may be supplemented with carnitine or antibiotics. The nutritional source may have a reduced content of protein and/or amino acids compared to a recommended value for a healthy infant, or it may be substantially free of protein and/or amino acids.

The buffering agent in the compositions containing a non-salt form of citrate or citric acid and a nutritional source may contain any buffering agent, such as those described above.

Preferably, the buffering agent is an amino acid, such as lysine or ornithine. The buffering agent may be or may contain a metal ion.

The compositions of the invention may contain one or more antibiotics. The invention also provides formulations that allow oral delivery of citrate in sufficient quantities to remedy diseases, disorders, and conditions associated with altered TCA cycle metabolism without providing an excessive amount of any mineral or metal ion. The

formulations contain amounts of citrate salts such that when the formulation is administered to a person, the cationic component of each salt provided to the person does not exceed the recommended daily amount of that component. In preferred embodiments, the formulation contains each citrate salt in an amount that does not exceed the recommended daily amount of the cation in that citrate salt. Alternatively or additionally, the formulation may comprise a formulation in which the amount of each citrate salt released into the subject's body does not exceed the recommended daily amount of the cation in that citrate salt.

Preferably, the formulation contains the following components: citric acid, or a prodrug, analog, or derivative thereof; one or more citrate salts, or prodrugs, analogs, derivatives thereof; and an amino acid (as an active agent, a buffering agent or both).

The formulation may be a powder that is soluble in an aqueous medium. For example, the formulation may be provided as a dry powder to which water can be added to provide an aqueous solution or suspension that can be consumed orally by a subject. Alternatively, the formulation may be provided as an aqueous solution. The formulation may be provided in a single-serving or a multiple-serving format.

The amino acid may be any amino acid, such as any of those described above.

Preferably, the amino acid is lysine, arginine, or ornithine.

Any suitable citrate salt or combination of citrate salts may be used in the formulation. The salt may contain sodium, lithium, potassium, calcium, magnesium, or manganese. The salt may contain citrate in its monobasic, dibasic, or tribasic state. Preferably, the salt is

monosodium citrate or monopotassium citrate.

Preferred formulations include citric acid and a combination of citrate salts, such as a combination monosodium citrate and monopotassium citrate. Such combinations allow delivery of ample citrate, in various ionization states, without providing an excess of any individual metal ion.

The recommended daily amount of a salt or mineral may be any amount determined to be suitable for intake for a person in one day. The recommended daily amount may be a minimum amount that should be taken in, a maximum amount that should not be exceeded, or a value or range of values between the two. The recommended daily amount may correspond to a standard known in the art, such as a standard promulgated by a private, governmental, medical, or health agency. For example and without limitation, the standard may be the acceptable daily intake (ADI), issued by the Food and Agriculture Organization and the World Health Organization; the daily intake guide (DIG), issued by the Australian Food and Grocery Council; the daily value (DV) and reference daily intake (RDI), issued by Health Canada; the daily value (DV) or reference daily intake (RDI), issued by the Food and Drug Administration (FDA) in the United States; the dietary reference intake (DRI), estimated average requirement (EAR), or

recommended dietary/daily allowance (RDA), issued by the Institute of Medicine (IOM) of the National Academies in the United States; the dietary reference value (DRV), lower reference nutrient intake (LRNI), or reference nutrient intake (RNI), issued by the United Kingdom

Department of Health; the dietary reference value (DRV), lower reference nutrient intake (LRNI), or reference nutrient intake (RNI), issued by the European Union's European Food Safety Authority; the guideline daily amount (GDA), issued by the Institute of Grocery

Distribution; or the adequate intake (AI), issued by the National Institutes of Health in the United States.

The recommended daily amount may account for conditions related to the individual, such as age, sex, weight, pregnancy status, lactation status, or menopausal status. For example, the recommended daily amounts of several minerals, such as calcium, iron, and potassium, are higher for women who are pregnant and/or lactating than for non-pregnant, non-lactating women. Other groups of patients whose mineral needs may vary include children, (i.e., pediatric patients), post-menopausal women, and the elderly.

For example and without limitation, the recommended daily amounts for specific minerals may be as follows: about 700 mg, about 800 mg, about 1000 mg, about 1200 mg, about 1300 mg, about 1500 mg, about 2000 mg, or about 2500 mg, of calcium; about 0.2 mg, about 0.22 mg, about 0.34 mg, about 0.44 mg, about 0.7 mg, about 0.89 mg, about 0.9 mg, about 1 mg, about 1.3 mg, about 2 mg, about 4 mg, about 6 mg, about 8 mg, or about 10 mg of copper; about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 13 mg, about 16 mg, about 18 mg, about 24 mg, about 27 mg, about 30 mg, about 36 mg, about 40 mg, or about 45 mg of iron; about 80 mg, about 150 mg, about 200 mg, about 250 mg, about 310 mg, about 320 mg, about 350 mg, about 360 mg, about 400 mg, or about 420 mg of magnesium; about 1.2 mg, about 1.5 mg, about 1.6 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 2.2 mg, about

2.3 mg, about 2.6 mg, about 3 mg, about 4 mg, about 6 mg, about 8 mg, about 10 mg, or about 11 mg of manganese; about 3000 mg, about 3500 mg, about 3800 mg, about 4000 mg, about 4500 mg, about 4700 mg, or about 5100 mg of potassium; about 1500 mg, about 2000 mg, about 2300 mg, or about 2400 mg, about 3000 mg, or about 3400 mg of sodium; and about 8 mg, about

9.4 mg, about 11 mg, about 12 mg, about 13 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, or about 40 mg of zinc. The formulation may contain one or more sugars to improve the flavor when the formulation is provided orally to a subject. For example and without limitation, the sugar may be sucrose, fructose, galactose, maltose, or lactose.

The formulation, or the soluble components within the formulation, may contain citric acid, monosodium citrate, monopotassium citrate, lysine, and sucrose. The formulation may contain, by mass, from about 40% to about 60% citric acid; from about 1% to about 10% monosodium citrate; from about 0.1% to about 5% monopotassium citrate; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 49.2 % citric acid, about 2% monosodium citrate, about 0.3% monopotassium citrate, about 37.5% lysine, and about 11% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, about 44.2 % citric acid, about 8.3% monosodium citrate, about 2.3% monopotassium citrate, about 33.8% lysine, and about 11.5% sucrose. The formulation, or the soluble components within the formulation, may contain, by mass, from about 50% to about 60% citrate, citric acid, or a combination thereof; from about 0.2% to about 1% sodium; from about 0.02% to about 0.5% potassium; from about 30% to about 40% lysine; and from about 10% to about 15% sucrose.

Methods of Treating Metabolic Disorders

The invention provides methods of treating metabolic disorders, diseases, or conditions by providing one or more TCA cycle intermediates or prodrugs, analogs, or derivatives thereof formulated for non-oral administration. The compositions may be administered by any non-oral route. For example and without limitation, the composition may be administered

subcutaneously, intravenously, intraarterially, intramuscularly, intradermally, or rectally.

Preferably, the composition is administered subcutaneously or intravenously. The composition containing a TCA cycle intermediate or prodrug, analog, or derivative thereof formulated for non-oral administration may be any of the one described above, such as citrate, cis-aconitate, D-isocitrate, a-ketoglutarate, succinate, fumarate, malate, oxaloacetate, acetone, acetoacetate, b-hydroxybutyrate, b-ketopentanoate, or b-hydroxypentanoate.

Preferably, the TCA cycle intermediate is succinate.

The metabolic disorder, disease, or condition may be any disease, disorder, or condition associated with altered TCA cycle metabolism or that can be ameliorated by providing an intermediate of the TCA cycle, such as any of those described above.

The TCA cycle intermediate or prodrug, analog, or derivative thereof may be provided at any therapeutically effective dose. For example and without limitation, the TCA cycle intermediate or prodrug, analog, or derivative thereof may be provided at from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.2 mg/kg subject weight to about 5 g/kg subject weight, from about 0.5 mg/kg subject weight to about 5 g/kg subject weight, from about 1 mg/kg subject weight to about 5 g/kg subject weight, from about 2 mg/kg subject weight to about 5 g/kg subject weight, from about 5 mg/kg subject weight to about 5 g/kg subject weight, from about 0.1 mg/kg subject weight to about 2 g/kg subject weight, from about 0.2 mg/kg subject weight to about 2 g/kg subject weight, from about 0.5 mg/kg subject weight to about 2 g/kg subject weight, from about 1 mg/kg subject weight to about 2 g/kg subject weight, from about 2 mg/kg subject weight to about 2 g/kg subject weight, from about 5 mg/kg subject weight to about 2 g/kg subject weight, from about 0.1 mg/kg subject weight to about 1 g/kg subject weight, from about 0.2 mg/kg subject weight to about 1 g/kg subject weight, from about 0.5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 1 g/kg subject weight, from about 2 mg/kg subject weight to about 1 g/kg subject weight, from about 5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 500 mg/kg subject weight, from about 2 mg/kg subject weight to about 200 mg/kg subject weight, or from about 5 mg/kg subject weight to about 100 mg/kg subject weight.

The composition may be provided according to any suitable schedule. For example and without limitation, the composition may be provided as single dose every 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or more. Each formulation independently may be provided once, twice, three times, four times, or more per day. The composition may be provided over a period of time. For example and without limitation, the composition may be provided for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, 18 months, or 24 months. One or more boundaries of the period may be defined by the age of the patient. For example and without limitation, the period of providing the composition may start or end when the patient is born, 1 week old, 2 weeks old, 3 weeks old, 4 weeks old, 6 weeks old, 8 weeks old, 10 weeks old, 12 weeks old, 3 months old, 3 months old, 4 months old, 5 months old, 6 months old, 8 months old, 10 months old, 12 months old, 18 months old, or 24 months old.

The invention provides combination therapies for treating metabolic disorders, diseases, or conditions. The combination therapies include providing a non-oral formulation containing succinate or a prodrug, analog, or derivative thereof and another formulation containing citrate, citric acid, or a prodrug, analog, or derivative thereof. The non-oral formulation may be administered by any non-oral route. For example and without limitation, the non-oral formulation may be administered subcutaneously, intravenously, intraarterially, intramuscularly, intradermally, or rectally. Preferably, the non-oral formulation is administered subcutaneously or intravenously. The formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be administered by any route, such as orally, enterally, parenterally, subcutaneously, intravenously, intraarterially, intramuscularly, intradermally, or rectally. Preferably, the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid is provided orally.

The combination therapies of the invention may be used to treat any disease, disorder, or condition associated with altered TCA cycle metabolism or that can be ameliorated by providing an intermediate of the TCA cycle, such as any of those described above.

In the combination therapies of the invention, each of succinate or a prodrug, analog, or derivative thereof and citrate or a prodrug, analog, or derivative of citrate or citric acid is provided at therapeutically effective dose. For example, each independently may be provided at from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.1 mg/kg subject weight to about 5 g/kg subject weight, from about 0.2 mg/kg subject weight to about 2 g/kg subject weight, from about 0.5 mg/kg subject weight to about 1 g/kg subject weight, from about 1 mg/kg subject weight to about 500 mg/kg subject weight, from about 2 mg/kg subject weight to about 200 mg/kg subject weight, or from about 5 mg/kg subject weight to about 100 mg/kg subject weight.

In the combination therapies of the invention, the non-oral formulation containing succinate or a prodrug, analog, or derivative thereof and the other formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be provided in any temporal relationship. For example, the two formulations may be provided simultaneously, sequentially in either order, or in alternating sequence. In a preferred method, the non-oral formulation containing succinate or a prodrug, analog, or derivative thereof is provided first, and the formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid is provided subsequently.

In the combination therapies of the invention, each formulation independently may be provided according to any suitable schedule, as described above.

In the combination therapies of the invention, each formulation independently may be provided over a period of time, as described above.

In the combination therapies of the invention, the non-oral formulation containing succinate or a prodrug, analog, or derivative thereof and the other formulation containing citrate, citric acid, or a prodrug, analog, or derivative of citrate or citric acid may be provided over distinct periods, coextensive periods, or overlapping periods. Thus, the combination therapies may include transitions from a period in which one formulation is provided to a period in which the other formulation is provided. The transitions may be discrete, or they may contain periods in which both formulations are provided. The transitions may be gradual and may include a decrease in dosage of the first formulation and an increase in dosage of the second formulation.

Examples

Example 1: Subcutaneous Administration of Succinate

Summary

The bioavailability of 13 C ₄ -succinate was determined following subcutaneous (SC) administration in male Sprague-Dawley rats. C ₄ -succinate was dosed subcutaneously at 10 mg/kg, 50 mg/kg, and 100 mg/kg. Blood samples were collected up to 8 hours post-dose, and plasma concentrations of the test article were determined by LC-MS/MS. Pharmacokinetic parameters were determined using Phoenix WinNonlin (v8.0) software. Following SC dosing of 13 C ₄ -succinate at 10 mg/kg, maximum plasma concentrations (average of 3480 ± 1512 ng/mL) were observed at 15 minutes post dosing. The average half-life following subcutaneous dosing could not be determined; however, the half-life for one rat was 0.453 hours. The average exposure based on the dose-normalized AUCi _ast was 160 ± 55.4 hr*kg*ng/mL/mg. Based on the IV data from a previous study, the average bioavailability of 1 ³ C ₄ -succinate at 10 mg/kg was 74.4 ± 25.7%.

Following SC dosing of 13 C ₄ -succinate at 50 mg/kg, maximum plasma concentrations (average of 17333 ± 2214 ng/mL) were observed at 15 minutes post dosing. The average half-life following subcutaneous dosing could not be determined because the correlation coefficient (r ) was less than 0.85. The average exposure based on the dose-normalized AUCi _ast was 210 ± 41.0 hr*kg*ng/mL/mg. Based on the IV data from a previous study, the average bioavailability of 1 ³ C ₄ -succinate at 50 mg/kg was 97.6 ± 19.0%.

Following SC dosing of C ₄ -succinate at 100 mg/kg, maximum plasma concentrations (average of 31000 ± 7451 ng/mL) were observed at 15 minutes post dosing. The average half-life following subcutaneous dosing was 1.07 hours. The average exposure based on the dose- normalized AUCi _ast was 216 ± 52.9 hr*kg*ng/mL/mg. Based on the IV data from a previous study, the average bioavailability of C ₄ -succinate at 100 mg/kg was 100 ± 24.6%.

Following subcutaneous dosing of 13 C ₄ -succinate at 10, 50, and 100 mg/kg, there was a nonlinear increase in exposure with increasing dose. The exposure of the higher doses was dose proportional, but greater than dose proportional to the low dose exposure.

Observations and Adverse Reactions

No adverse reactions were observed following subcutaneous dosing of C ₄ -succinate in male Sprague-Dawley rats.

Dosing Solution Analysis

The dosing solutions were analyzed by LC-MS/MS. The measured dosing solution concentrations are shown in Table 1. The dosing solutions were diluted into rat plasma and analyzed in triplicate. All concentrations are expressed as mg/mL of the free base. The nominal dosing level was used in all calculations.

Table 1. Measured Dosing Solution Concentrations (mg/mL)

Quantitative Plasma Sample Analysis

Plasma samples were extracted and analyzed using the methods described below in the section on Sample Extraction. Individual and average plasma concentrations are shown in Table 2 through Table 4. All data are expressed as ng/mL of the free base. Samples that were below the limit of quantification were not used in the calculation of averages. Concentrations versus time data are plotted in Figure 1 through Figure 6.

Data Analysis

Pharmacokinetic parameters were calculated from the time course of the plasma concentration and are presented in Table 2 through Table 4. Pharmacokinetic parameters were determined with Phoenix WinNonlin (v8.0) software using a non-compartmental model. The maximum plasma concentration (C _max ) and the time to reach maximum plasma concentration (t _max ) after SC dosing were observed from the data. The area under the time-concentration curve (AUC) was calculated using the linear trapezoidal rule with calculation to the last quantifiable data point, and with extrapolation to infinity if applicable. Plasma half-life (ti _/2 ) was calculated from 0.693/slope of the terminal elimination phase. Mean residence time, MRT, was calculated by dividing the area under the moment curve (AUMC) by the AUC. Bioavailability was determined by dividing the individual dose-normalized PO AUCi _ast values by the average dose- normalized IV AUCi _ast value from study 18CARNP11. Any samples below the limit of quantitation (1.00 ng/mF) were treated as zero for pharmacokinetic data analysis. Table 2. Individual and Average Plasma Concentrations (ng/mL) for 13 C ₄ -Succinate After Subcutaneous Administration at 10 mg/kg in Male Sprague-Dawley Rats

C _max : maximum plasma concentration; t _max : time of maximum plasma concentration; ti _/2 : half-life, data points used for half-life determination are in bold; MRTi _ast : mean residence time, calculated to the last observable time point; AUCi _ast : area under the curve, calculated to the last observable time point; AUC _¥ : area under the curve, extrapolated to infinity; ND: not determined; BLOQ: below the limit of quantitation (1 ng/mL).

' Dose-normalized by dividing the parameter by the nominal dose in mg/kg.

Bioavailability determined by dividing the individual oral dose-normalized AUCi _ast values by the average IV dose normalized AUCi _ast value 215 hr*kg*ng/mL/mg from previous study.

3 Not determined because the line defining the terminal elimination phase had an r 2 <0.85.

FIG. 1 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after subcutaneous administration of 13 C ₄ -succinate at 10 mg/kg.

FIG. 2 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after subcutaneous administration of 13 C ₄ -succinate at 10 mg/kg. Table 3. Individual and Average Plasma Concentrations (ng/mL) for 13 C ₄ -Succinate After Subcutaneous Administration at 50 mg/kg in Male Sprague-Dawley Rats

C _max : maximum plasma concentration; t _max : time of maximum plasma concentration; ti _/2 : half-life, data points used for half-life determination are in bold; MRTi _ast : mean residence time, calculated to the last observable time point; AUCi _ast : area under the curve, calculated to the last observable time point; AUC _¥ : area under the curve, extrapolated to infinity; ND: not determined; BLOQ: below the limit of quantitation (1 ng/mL).

' Dose-normalized by dividing the parameter by the nominal dose in mg/kg.

2Bioavailability determined by dividing the individual oral dose-normalized AUCi _ast values by the average IV dose normalized AUCi _ast value 215 hr*kg*ng/mL/mg from previous study.

3 Not determined because the line defining the terminal elimination phase had an r 2 <0.85.

FIG. 3 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after subcutaneous administration of 13 C ₄ -succinate at 50 mg/kg.

FIG. 4 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after subcutaneous administration of 13 C ₄ -succinate at 50 mg/kg. Table 4. Individual and Average Plasma Concentrations (ng/mL) for 13 C ₄ -Succinate After Subcutaneous Administration at 100 mg/kg in Male Sprague-Dawley Rats

C _max : maximum plasma concentration; t _max : time of maximum plasma concentration; ti _/2 : half-life, data points used for half-life determination are in bold; MRTi _ast : mean residence time, calculated to the last observable time point; AUCi _ast : area under the curve, calculated to the last observable time point; AUC _¥ : area under the curve, extrapolated to infinity; ND: not determined; BLOQ: below the limit of quantitation (1 ng/mL).

'Dose-normalized by dividing the parameter by the nominal dose in mg/kg.

2Bioavailability determined by dividing the individual oral dose-normalized AUCi _ast values by the average IV dose normalized AUCi _ast value 215 hr*kg*ng/mL/mg from previous study.

3 Not determined because the line defining the terminal elimination phase had an r 2 <0.85.

FIG. 5 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after subcutaneous administration of 13 C ₄ -succinate at 100 mg/kg.

FIG. 6 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after subcutaneous administration of 13 C ₄ -succinate at 100 mg/kg. Dosing Solution Preparation and Animal Data Sheets

Materials List _ Animal Dosing & Specimen Collection and Processing _

Study#: 18CARNP16 Date: 8/21/2018

Group#: 1 Animal: Male Sprague Dawley

_ _ Rats _

Test Article: 13C ₄ -Succmate Dosing Vehicle: Physiologic Saline

_ buffered to pH 6.5-7

Dose: _ 10 mg/kg _ Specimen Collected: _ Blood _

Dosing sol’n Cone: 2 mg/mL Anticoagulant/Additive Sodium heparin

Dosing Volume: 5 mL/kg Route of Blood Jugular Vein Cannulae

Collection:

Dosing Route: SC Approx. Vol. Collected: ~ 0.3 mL

Food was withheld from the animals over night before dosing, until four hours post-dose, at which time food was returned.

* EE, 1616, Is, 8/21/18

Balance Used: AS-1273

Comments/Observations:

All animals show normal activities during experiment CS, EE, 8/21/18, CS 8/21/18

Animal Dosing & Specimen Collection and Processing

Study#: 18CARNP16 Date: 8/21/2018

Group#: 2 Animal: Male Sprague Dawley

_ Rats _

Test Article: 13C ₄ -Succinate Dosing Vehicle: Physiologic Saline

buffered to pH 6.5-7

Dose: Specimen Collected: _ Blood _

Dosing sol’n Cone: 10 mg/mL Anticoagulant/Additive: Sodium heparin Dosing Volume: 5 mL/kg Route of Blood Jugular Vein Cannulae

Collection:

Dosing Route: SC Approx. Vol. Collected: ~ 0.3 mL

Food was withheld from the animals over night before dosing, until four hours post-dose, at which time food was returned.

Balance Used: AS-1273

Comments/Observations:

All animals show normal activities during study - CS 8/21/18

Animal Dosing & Specimen Collection and Processing

Study#: 18CARNP16 Date: 8/21/2018

Group#: 3 Animal: Male Sprague Dawley

_ Rats _

Test Article: 13C ₄ -Succinate Dosing Vehicle: Physiologic Saline

buffered to pH 6.5-7

Dose: 100 mg/kg Specimen Collected: _ Blood _

Dosing sol’n Cone: 20 mg/mL Anticoagulant/Additive: Sodium heparin Dosing Volume: 5 mL/kg Route of Blood Jugular Vein Cannulae

Collection:

Dosing Route: SC Approx. Vol. Collected: ~ 0.3 mL

Food was withheld from the animals over night before dosing, until four hours post-dose, at which time food was returned.

Balance Used: AS- 1273

Comments/Observations:

All animals show normal activities during study - CS 8/21/18

Analytical Stock Solution Preparation

Analytical stock solutions (1.00 mg/mL of the free drug) were prepared in DMSO. Standard Preparation

Standards were prepared in Sprague-Dawley rat plasma containing sodium heparin as the anticoagulant. Working solutions were prepared in 50:50 acetonitrile:water and then added to the rat plasma to make calibration standards to final concentrations of 2000, 1000, 500, 100, 50.0, 10.0, 5.00, and 1.00 ng/mL. Standards were treated identically to the study samples

Sample Extraction

Plasma samples were manually extracted using acetonitrile in a 96-well plate as outlined in Table 5. Table 5. Preparation of Plasma Samples

HPLC Conditions

Instrument; Waters Aequity OPLC ^®

Column; Waters HSS Ύ3, 50x2.1 mm id, 1 ,8 pm

Aqueous Reservoir (A): 0.1% formic acid in water

Organic Reservoir (B); 0.1% formic acid in acetonitrile

Gradient Program;

Flo Rate: 800 mΐ ./min

formic acid Weak Autosampler Wash: 4 nil ammonium formate

Mass Spectrometer Conditions

instrument PE Seiex ARM000

Interface; Electrospray (‘ urix> ion Spray”)

Mode: Multiple reactions monitoring (MRM)

Gases: CUR 30, CAD i 0, GS 1 50, GS2 50

IS; to» Spray Voltage; DP; Dechjstenng Potential; EP: Entrance Potential; CE: Collision Energy; CXP: Collision Cell Exit Potential; * AO setings are in volts Example 2: Effects of Citrate and Succinate on Basal Respiration

FIG. 7 is graph showing the effects of citrate and succinate on basal respiration in cells from a patient with propionic acidemia (PA). Fibroblasts from a normal patient (sample 826) and fibroblasts from a patient with PA were cultured for 72 hours in culture medium alone, culture medium supplemented with 0.2 mM citrate, or culture medium supplemented with 4 mM succinate, as indicated. Oxygen consumption rate (OCR) was measured with an XFe96 Extracellular Flux Analyzer, Seahorse Bioscience. Data shown are mean values ± SD and are normalized to protein amount mean value. ****p<0.000l,**P<0.0l compared between the groups shown are calculated in GraphPad Prism 7 using unpaired t test for samples from a single biological replicate and 8 technical replicates.

Example 3: Pharmacokinetics, Excretion, and Tissue Distribution of Orally-administered Citrate or Intravenously-administered Succinate

Summary

The plasma- time course, routes and rates of excretion, and tissue distribution of radioactivity following a single oral dose of ¹⁴ C-labeled citrate or single oral dose of ¹⁴ C-labeled succinate were analyzed in rats.

Dose Formulation and Preparation

¹⁴ C-succinate and ¹⁴ C-citrate were dissolved in either deionized water for oral administration or 0.9% sodium chloride for intravenous injection. Formulations were prepared on the day prior to dosing.

Test System

CD ^® rats were obtained from Charles River Faboratories. Animals were 9-11 weeks in age at beginning of study.

Study Design

Treatment groups are shown in Table 6.

Table 6. Treatment Groups

Animals given oral ¹⁴ C-citrate were kept in a fasting state for 8 hours prior to dosing and for 4 hours after dosing. ¹⁴ C-citrate was given by oral gavage. Animals given intravenous ¹⁴ C- succinate were not kept in a fasting state. ¹⁴ C-succinate was administered by tail vein injection. Sample Collection and Analysis

Following oral dosing, the tube and dose site were wiped with gauze, which was collected for subsequent radioanalysis. Following IV dosing, the needle and dose site were wiped with gauze, which was collected for subsequent radioanalysis. The dose site wipe total radioactivity was subtracted from the total dose administered, to determine dose loss, according to SOP ADME.

Blood and plasma were collected by cardiac puncture for terminal collection at 0.5, 1, 2, 4, 8, and 24 hours post-dose. Blood was stored on an ice block or wet ice until centrifugation to obtain plasma. Triplicate aliquots were weighed, mixed with scintillation cocktail, and counted by LSC.

Urine and feces were collected at 0-24 hours post-dose and stored on wet ice. Expired C0 ₂ was collected at 0-8 hours and 8-24 hours post-dose by drawing air through traps containing 2 N NaOH according to MPI Research SOP. Samples were stored at ambient temperature and weighed. Aliquots were submitted for radioanalysis.

At 24 hours post-dose, cages were rinsed with a 1% trisodium phosphate (TSP) solution and wiped with gauze pads. The final cage rinse samples and cage wipe samples were collected into separate appropriate containers, and the weight of each cage rinse and cage wipe were recorded.

Samples were analyzed for radioactivity by liquid scintillation counting (LSC) for at least 5 minutes or 100,000 cumulative counts. Samples were analyzed in triplicate.

At 0.5, 1, 2, 4, 8, and 24 hours post-dose, after terminal blood collections, euthanized animals were frozen in a hexane/dry ice bath and embedded in a 5% carboxymethylcellulose matrix according to MPI Research SOP IMG-23.

Embedded carcasses were sectioned according to MPI Research SOP EQP-145 on a Leica CM3600 Cyromacrotome set to maintain -10 to -30°C. Sections of approximately 30 pm thickness were taken in the sagittal plane. Appropriate sections selected at various levels of interest in the block were collected to encompass the following tissues, organs, and biological fluids, where possible: adrenal gland, bladder and contents (urine), blood (cardiac), bone, bone marrow, brain, epididymis, eye, fat (brown), fat (white), heart, kidney, large intestine/cecum and contents, liver, lung, muscle, pancreas, prostate/uterus, salivary glands, skin, small intestine and contents, spleen, stomach and contents, testes/ovaries, thymus, thyroid, and mesenteric lymph nodes. Sections were mounted, exposed to phosphor imaging screens, and scanned at 50 pm using the Storm 860 image acquisition system according to MPI Research SOP EQP-146.

Quantification, relative to the calibration standards, was performed by image

densitometry using MCID™ image analysis software according to MPI Research SOP IMG-24. A standard curve was constructed from the integrated response (MDC/mm ) and the nominal concentrations of the [ ¹⁴ C]calibration standards. The concentrations of radioactivity were expressed as pCi/g and converted to pg or ng equivalents of [ ¹⁴ C]Test Article per gram sample (ng-eq/g or pg-eq/g) using the specific activity of administered [ ¹⁴ C]Test Article dose formulation. A lower limit of quantification (LLOQ) was applied to the data. The LLOQ was determined using the radioactive concentration of the lowest calibration standard used to generate a calibration curve divided by the specific activity of the dose formulation (pCi/mg). Artifacts, such as those produced by dislodged contents of the alimentary canal, were excluded from analysis during image analysis.

Samples were stored as indicated in Table 7.

Table 7. Sample Storage Conditions

Results

The percentages of radioactivity recovered in various samples from animals sacrificed 24 hours after treatment with either a single dose of oral ¹⁴ C-citrate or a single dose of intravenous 1 ⁴ C-succinate are shown in Table 8. Table 8. Percentage Recovery of Radioactive Dose

The plasma concentrations of radioactive material in rats treated with either a single dose of oral ¹⁴ C-citrate or a single dose of intravenous ¹⁴ C-succinate are shown in Table 9.

Table 9. Plasma Concentrations of Radioactive Material

The concentrations of radioactivity in blood and tissues in rats treated with a single dose of intravenous ¹⁴ C-succinate are shown in Table 10. Table 10. Radioactivity in Tissues of Rats Treated with IV ¹⁴ C-Succinate

ng Equivalents test article/g

Animal Number (Timepoint)

1001 1002 1003 1004 1005 1006

Tissue (0.5 Hour) (1 Hour) (2 Hours) (4 Hours) (8 Hours) (24 Hours)

Adrenal gland 4060 1441 1188 1192 840 581

Blood 891 1398 912 BLQ BLQ BLQ

Bone 3128 3200 1989 2330 2042 1322

Bone marrow 1314 1750 1398 1419 1019 769

Cecum BLQ BLQ BLQ BLQ BLQ BLQ

Cecum contents 540 882 757 750 806 BLQ

Cerebellum 709 847 529 503 BLQ BLQ

Cerebrum 596 853 477 543 348 BLQ

Epididymis 699 785 442 BLQ BLQ BLQ

Eye 569 584 371 413 BLQ BLQ

Fat (brown) 996 BLQ BLQ BLQ BLQ BLQ

Fat (white) BLQ BLQ BLQ 348 BLQ BLQ

Harderian gland BLQ 1983 1478 BLQ BLQ BLQ

Kidney 3154 3060 2704 2075 2031 1120

Lacrimal gland (Ex-orbital) BLQ 1557 1655 1993 1388 BLQ

Lacrimal gland (Intra-orbital) BLQ 2323 1355 BLQ BLQ BLQ

Large intestinal contents BLQ 519 NR 810 593 BLQ

Large intestine BLQ 1787 1716 1361 BLQ BLQ

Liver 2431 3607 2005 1561 1190 715

Lung 920 1185 683 677 491 371

Lymph node 938 1376 NR BLQ BLQ BLQ

Medulla 606 714 501 BLQ 323 BLQ

Muscle 563 732 773 638 401 341

Myocardium 1032 1908 1058 771 496 395

Nasal turbinates 941 1302 978 1055 NR 525 Olfactory lobe 708 747 466 725 BLQ BLQ

Pancreas 1676 3492 2193 2404 641 385

Preputial gland 1660 1347 1073 ND ND ND

Prostate gland 675 1220 1045 995 NR 497

S alivary gland 1518 2826 2286 2458 1533 596

Seminal vesicle 326 469 591 513 BLQ BLQ

Skin 863 857 531 717 445 464

Small intestinal contents 715 711 847 774 400 BLQ

Small intestine BLQ BLQ BLQ BLQ BLQ BLQ

Spinal cord 544 715 BLQ BLQ 394 BLQ

Spleen 788 1058 810 796 736 556

Stomach BLQ 1798 ND 935 BLQ BLQ

Stomach contents BLQ BLQ BLQ BLQ BLQ BLQ

Testes 475 580 410 583 BLQ BLQ

Thymus 801 1212 838 NR 654 596

Thyroid gland ND 4809 3675 NR 644 589

Urine 3531 5000 3016 1974 BLQ BLQ

BLQ: Below limit of quantitation (<320 ng equivalents test article/g).

ND: Not detectable (sample shape not discernible from background or surrounding tissue). NR: Not represented (tissue not represented on section). The concentrations of radioactivity in blood and tissues in rats treated with a single dose of oral ¹⁴ C-citrate are shown in Table 11.

Table 11. Radioactivity in Tissues of Rats Treated with Ora ¹⁴ C-Citrate

ng Equivalents test article/g

Animal Number (Timepoint)

2001 2002 2003 2004 2005 2006

Tissue _ (0.5 Hour) (1 Hour) (2 Hours) (4 Hours) (8 Hours) (24 Hours)

Adrenal gland NR 20538 17413 13375 15645 10316

Blood 15935 12808 11183 8800 BLQ BLQ

Bone 49396 58936 43368 42499 47959 17943

Bone marrow 9150 18940 21841 20023 21000 11005 Cecum BLQ ND ND 21290 ND BLQ

Cecum contents 3593 7501 9083 37320 8242 4081

Cerebellum 5447 5944 6952 5953 4300 3183

Cerebrum 4464 5907 6789 5659 4073 3231

Epididymis 4469 6854 6691 7917 6583 4188

Esophageal contents 3395667 NR 455859 NR BLQ BLQ

Eye BLQ 3086 4533 6017 BLQ BLQ

Fat (brown) BLQ BLQ BLQ BLQ BLQ BLQ

Fat (white) BLQ BLQ BLQ BLQ BLQ BLQ Harderian gland 7011 NR 17809 22082 30362 11021 Kidney 31411 35017 25508 21332 17379 8211 Lacrimal gland (Ex-orbital) 9751 13243 16362 21167 17875 7542

Lacrimal gland (Intra-orbital) NR 27199 23343 20093 22551 NR

Large intestinal contents 3638 4169 5302 11545 12377 5971

Large intestine BLQ 16238 15856 28662 11353 BLQ

Liver 35567 34866 31094 22356 20156 10050

Lung 11418 11240 10832 8280 6727 4911

Lymph node BLQ BLQ BLQ BLQ BLQ BLQ

Medulla 4578 6118 7787 6270 4556 3479

Muscle 3783 5745 4737 4311 3965 3490

Myocardium 7466 8932 9941 7845 5336 4390

Nasal turbinates 12703 12918 14959 24265 9529 NR

Olfactory lobe NR 5917 8491 8727 NR 4121

Pancreas 22855 49518 48038 52562 13125 5990

Preputial gland ND ND ND ND ND ND

Prostate gland 4912 8819 8011 12828 7804 7448

Salivary gland 12638 24116 28408 35370 33304 6456

Seminal vesicle 3710 4087 5590 NR 8840 NR

Skin 7114 10352 9019 8095 7843 6049

Small intestinal contents 765641 155596 79444 61651 25276 4687

Small intestine ND 11950 ND ND ND 21231

Spinal cord 3633 4913 6114 6416 5079 3837

Spleen 8138 11559 12833 11528 14371 7745

Stomach 120191 57312 20761 14630 ND 6169

Stomach contents 3239644 1081479 856445 88064 BLQ BLQ

Testes 3146 5217 5382 5009 4224 3751

Thymus 5369 9433 10568 10357 10658 8768

Thyroid gland 6773 22240 9678 5807 NR BLQ

Urine 54074 145954 NR 119053 NR 3684

BLQ: Below limit of quantitation (<320 ng equivalents test article/g).

ND: Not detectable (sample shape not discernible from background or surrounding tissue).

NR: Not represented (tissue not represented on section). Example 4: Oral Bioavailability of Succinate

Summary

The oral bioavailability of 13 C ₄ -succinate was evaluated in male Sprague-Dawley rats.

13 C ₄ -succinate was dosed by intravenous (IV) and oral (PO) routes of administration at 10 mg/kg and 50 mg/kg, respectively. Blood samples were collected up to 8 hours post-dose, and plasma concentrations of the test articles were determined by LC-MS/MS. Pharmacokinetic parameters were determined using Phoenix WinNonlin (v8.0) software.

Following IV dosing of C ₄ -succinate at 10 mg/kg, the average half-life was 2.46 ± 2.04 hours. Its average clearance rate was 4.61 ± 0.341 L/hr/kg. The average volume of distribution was 0.982 ± 0.584 L/kg. Following PO dosing of C ₄ -succinate at 50 mg/kg, maximum plasma concentrations (average of 211 ± 186 ng/mL) were observed at 15 minutes post dosing. The average half-life following oral dosing could not be determined because the correlation coefficient (r ) was less than 0.85, or due to a lack of quantifiable data points trailing the C _max . The average exposure based on the dose-normalized AUCi _ast was 1.84 ± 1.58 hr*kg*ng/mL/mg.

The average oral bioavailability of 13 C ₄ -succinate after dosing at 50 mg/kg was 0.854 ± 0.735%.

Observations and Adverse Reactions

No adverse reactions were observed following intravenous and oral dosing of C ₄ - succinate in male Sprague-Dawley rats.

Dosing Solution Analysis

The dosing solutions were analyzed by LC-MS/MS. The measured dosing solution

concentrations are shown in Table 12. The dosing solutions were diluted into rat plasma and analyzed in triplicate. All concentrations are expressed as mg/mL of the free base. The nominal dosing level was used in all calculations.

Table 12. Measured Dosing Solution Concentrations (mg/mL)

*5% dextrose solution

Quantitative Plasma Sample Analysis

Plasma samples were extracted and analyzed using the methods described in the section on Sample Extraction. Individual and average plasma concentrations are shown in Table 13 and Table 14. All data are expressed as ng/mL of the free base. Samples that were below the limit of quantification were not used in the calculation of averages. Concentrations versus time data are plotted in FIGS. 8-11.

Data Analysis

Pharmacokinetic parameters were calculated from the time course of the plasma concentration and are presented in Table 13 and Table 14. Pharmacokinetic parameters were determined with Phoenix WinNonlin (v8.0) software using a non-compartmental model. The maximum plasma concentrations (Co) after IV dosing were estimated by extrapolation of the first two time points back to t=0. The maximum plasma concentration (C _max ) and the time to reach maximum plasma concentration (T _max ) after PO dosing were observed from the data. The area under the time concentration curve (AUC) was calculated using the linear trapezoidal rule with calculation to the last quantifiable data point, and with extrapolation to infinity if applicable. Plasma half-life (ti _/2 ) was calculated from 0.693/slope of the terminal elimination phase. Mean residence time, MRT, was calculated by dividing the area under the moment curve (AUMC) by the AUC. Clearance (CL) was calculated from dose/ AUC. Steady-state volume of distribution (Vss) was calculated from CL*MRT (mean residence time). Bioavailability was determined by dividing the individual dose-normalized PO AUCi _ast values by the average dose-normalized IV AUCi _ast value. Any samples below the limit of quantitation (1 ng/mL) were treated as zero for pharmacokinetic data analysis.

Table 13. Individual and Average Plasma Concentrations (ng/mL) and Pharmacokinetic

Parameters for 13 C ₄ -succinate After Intravenous Administration at 10 mg/kg in Male Sprague- Dawley Rats

Co: maximum plasma concentration extrapolated to t=0; t _max : time of maximum plasma concentration; ti _/2 : half-life, data points used for half-life determination are in bold; MRTlast: mean residence time, calculated to the last observable time point; CL: clearance; Vss: steady state volume of distribution; AUCi _ast : area under the curve, calculated to the last observable time point; AUC¥: area under the curve, extrapolated to infinity; ND: not determined; BLOQ: below the limit of quantitation (1 ng/mL).

Extrapolated to t=0.

² Dose-normalized by dividing the parameter by the nominal dose in mg/kg.

FIG. 8 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after intravenous administration of 13 C ₄ -succinate at 10 mg/kg.

FIG. 9 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after intravenous administration of 13 C - succinate at 10 mg/kg. Table 14. Individual and Average Plasma Concentrations (ng/mL) for 13 C ₄ - succinate After Oral Administration at 50 mg/kg in Male Sprague-Dawley Rats

C _max : maximum plasma concentration; t _max : time of maximum plasma concentration; ti _/2 : half-life, data points used for half-life determination are in bold; MRTlast: mean residence time, calculated to the last observable time point; AUCi _ast : area under the curve, calculated to the last observable time point; AUC¥: area under the curve, extrapolated to infinity; ND: not determined; BLOQ: below the limit of quantitation (1 ng/mL).

2 'Dose-normalized by dividing the parameter by the nominal dose in mg/kg.

Bioavailability determined by dividing the individual oral AUCi _ast values by the average IV AUCi _ast value.

3 Not determined because the line defining the terminal elimination phase had an r 2 <0.85. 4Not determined due to a lack of quantifiable data points trailing the C _max .

FIG. 10 is a graph of plasma concentration of 13 C ₄ -succinate at various time points in individual rats after oral administration of 13 C ₄ -succinate at 50 mg/kg.

FIG. 11 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after oral administration of 13 C ₄ -succinate at 50 mg/kg. Dosing Solution Preparation and Animal Data Sheets

Animal Dosing & Specimen Collection and Processing

Study#: 18CARNP11 _ Date: 6/15/2018

Group#: 1 Animal: Male Sprague Dawley

Rats

Test Article: 13C ₄ -Succinate Dosing Vehicle: 5% dextrose solution Dose: 10 mg/kg Specimen Collected: Blood

Dosing sol’n Cone: 2 mg/mL Antico

Dosing Volume: 5 mL/kg Route of Blood Jugular Vein Cannulae

_ Collection:

Dosing Route: IV Approx. Vol. Collected: ~ 0.3 mL

Food was withheld from the animals over night before dosing, until four hours post-dose, at which time food was returned.

Balance Used: AS- 1273

Comments/Observations:

All animals show normal behavior during experiment - CS 6/15/18

Animal Dosing & Specimen Collection and Processing

Study#: 18CARNP11 _ Date: 6/15/2018

Group#: 2 Animal: Male Sprague Dawley

_ Rats _

Test Article: 13C ₄ -Succinate Dosing Vehicle: _ water _

Dose: 50 mg/kg Specimen Collected: _ Blood _

Dosing sol’n Cone: 10 mg/mL Anticoagulant/Additive: Sodium heparin Dosing Volume: 5 mL/kg Route of Blood Jugular Vein Cannulae

_ Collection:

Dosing Route: PO Approx. Vol. Collected: ~ 0.3 mL

Food was withheld from the animals over night before dosing, until four hours post-dose, at which time food was returned.

Balance Used: AS- 1273

Comments/Observations:

All animals show normal behavior during experiment - CS 6/15/18

Analytical Stock Solution Preparation

Analytical stock solutions (1.00 mg/mL of the free drug) were prepared in DMSO.

Standard Preparation

Standards were prepared in Sprague-Dawley rat plasma containing sodium heparin as the anticoagulant. Working solutions were prepared in 50:50 acetonitrile:water and then added to the rat plasma to make calibration standards to final concentrations of 2000, 1000, 500, 100, 50.0, 10.0, 5.00, 2.50, and 1.00 ng/mL. Standards were treated identically to the study samples.

Sample Extraction

Plasma samples were manually extracted using acetonitrile in a 96-well plate as outlined in Table 15.

Table 15. Preparation of Plasma Samples

HPLC Conditions

Instrument: Waters Acquity IIPLC*

Column: Waters H$S T3, 50 x 2, 1 mm id, I pm

Aqueous Reservoir (A): 0. 1 % formic acid in water

Organic Reservoir ΐ S : QJ% formic acid in acetonitrile

Grads ent Program :

Flow Rale: 800 mILhίh

formic add Weak Atrtesaniplef Wash: 4 fiiM ammoniisB formate

Mass Spectrometer Conditions

Mass Spectrometer Conditions

Instrument: PE Sciex ARΪ4000

Interface: Bleetrospray (^Turbo Ion Spray-"}

Mode: Multiple reactions monitoring (MRM)

Cases: CUR 30, CAD 10, GSi 50, GS2 50

ouret Temperature: 5O0°C

oltages and Ions A o tmed :

IS- k S ay Voltage; DP: Dccl tcfiog Potential; EP: Eatr&nce Potential; CE; Collision Energy; CXP: Collision Ceii E it .Potecaia) : "A H setings are its vohs

Example 5: Oral Bioavailability of Citrate

Summary

The oral bioavailability of 13 C ₆ -citrate was evaluated in male Sprague-Dawley rats. 13 C ₆ - citrate was dosed by intravenous (IV) and oral (PO) routes of administration at 10 mg/kg and 50 mg/kg, respectively. Blood samples were collected up to 8 hours post-dose, and plasma concentrations of the test articles were determined by LC-MS/MS. Pharmacokinetic parameters were determined using Phoenix WinNonlin (v8.0) software.

Following IV dosing of C ₆ -citrate at 10 mg/kg, the average half-life was 2.34 ± 0.207 hours. Its average clearance rate was 2.96 ± 0.345 L/hr/kg. The average volume of distribution was 1.75 ± 0.223 L/kg. Following PO dosing of C ₆ -citrate at 50 mg/kg, maximum plasma concentrations (average of 1580 ± 191 ng/mL) were observed at 15 minutes post dosing. The average half-life following oral dosing could not be determined; however, the half-life for one rat was 0.865 hours. The average exposure based on the dose-normalized AUCi _ast was 33.2 ± 10.9 hr*kg*ng/mL/mg. The average oral bioavailability of C ₆ -citrate after dosing at 50 mg/kg was 9.86 + 3.24%.

Observations and Adverse Reactions

No adverse reactions were observed following intravenous and oral dosing of C ₆ -citrate in male Sprague-Dawley rats.

Dosing Solution Analysis

The dosing solutions were analyzed by LC-MS/MS. The measured dosing solution concentrations are shown in Table 16. The dosing solutions were diluted into rat plasma and analyzed in triplicate. All concentrations are expressed as mg/mL of the free base. The nominal dosing level was used in all calculations.

Table 16: Measured Dosing Solution Concentrations (mg/mL)

*5% dextrose solution

Quantitative Plasma Sample Analysis

Plasma samples were extracted and analyzed using the methods described in the section on Sample Extraction. Individual and average plasma concentrations are shown in Table 17 and Table 18. All data are expressed as ng/mL of the free base. Samples that were below the limit of quantification were not used in the calculation of averages. Concentrations versus time data are plotted in FIGS. 12-15.

Data Analysis

Pharmacokinetic parameters were calculated from the time course of the plasma concentration and are presented in Table 17 and Table 18. Pharmacokinetic parameters were determined with Phoenix WinNonlin (v8.0) software using a non-compartmental model. The maximum plasma concentrations (Co) after IV dosing were estimated by extrapolation of the first two time points back to t=0. The maximum plasma concentration (C _max ) and the time to reach maximum plasma concentration (T _max ) after PO dosing were observed from the data. The area under the time-concentration curve (AUC) was calculated using the linear trapezoidal rule with calculation to the last quantifiable data point, and with extrapolation to infinity if applicable. Plasma half-life (ti _/2 ) was calculated from 0.693/slope of the terminal elimination phase. Mean residence time, MRT, was calculated by dividing the area under the moment curve (AUMC) by the AUC. Clearance (CL) was calculated from dose/ AUC. Steady-state volume of distribution (Vss) was calculated from CL*MRT (mean residence time). Bioavailability was determined by dividing the individual dose-normalized PO AUCi _ast values by the average dose-normalized IV AUCi _ast value. Any samples below the limit of quantitation (1 ng/mL) were treated as zero for pharmacokinetic data analysis.

Table 17. Individual and Average Plasma Concentrations (ng/mL) and Pharmacokinetic

Parameters for 13 C ₆ -citrate After Intravenous Administration at 10 mg/kg in Male Sprague- Dawley Rats

C ₀ : maximum plasma concentration extrapolated to t=0; t _max : time of maximum plasma concentration; ti _/2 : half-life, data points used for half-life determination are in bold; MRTlast: mean residence time, calculated to the last observable time point; CL: clearance; Vss: steady state volume of distribution; AUCi _ast : area under the curve, calculated to the last observable time point; AUC¥: area under the curve, extrapolated to infinity; ND: not determined; BLOQ: below the limit of quantitation (1 ng/mL).

1 Extrapolated to t=0.

2Dose-normalized by dividing the parameter by the nominal dose in mg/kg.

FIG. 12 is a graph of plasma concentration of 13 C ₆ -citrate at various time points in individual rats after intravenous administration of 13 C ₆ -citrate at 10 mg/kg.

FIG. 13 is a graph of average plasma concentration of 13 C ₆ -citrate at various time points in rats after intravenous administration of 13 C ₆ -citrate at 10 mg/kg. Table 18. Individual and Average Plasma Concentrations (ng/mL) for 13 C ₆ -citrate After Oral Administration at 50 mg/kg in Male Sprague-Dawley Rats

C _max : maximum plasma concentration; t _max : time of maximum plasma concentration; ti _/2 : half-life, data points used for half-life determination are in bold; MRTlast: mean residence time, calculated to the last observable time point; AUCi _ast : area under the curve, calculated to the last observable time point; AUC¥: area under the curve, extrapolated to infinity; ND: not determined; BLOQ: below the limit of quantitation (1 ng/mL).

'Dose-normalized by dividing the parameter by the nominal dose in mg/kg.

² Bioavailability determined by dividing the individual oral AUCi _ast values by the average IV AUCi _ast value.

3 Not determined because the line defining the terminal elimination phase had an r 2 <0.85. FIG. 14 is a graph of plasma concentration of 13 C ₆ -Citrate at various time points in individual rats after oral administration of 13 C ₆ -Citrate at 50 mg/kg.

FIG. 15 is a graph of average plasma concentration of 13 C ₄ -succinate at various time points in rats after oral administration of 13 C ₆ -Citrate at 50 mg/kg. Dosing Solution Preparation and Animal Data Sheets

Animal Dosing & Specimen Collection and Processing

Study#: 18CARNP9 Date: 6/6/2018

Group#: 1 Animal: Male Sprague Dawley

Rats

Test Article: 13 C ₆ - Citrate Dosing Vehicle: 5% dextrose solution

Dose: Specimen Collected: _ Blood _

Dosing sol’n Cone: 2 mg/mL Anticoagulant/Additive: Sodium heparin Dosing Volume: 5 mL/kg Route of Blood Collection: Jugular Vein Cannulae

IV Approx. Vol. Collected: ~ 0.3 mL

Food was withheld from the animals over night before dosing, until four hours post-dose, at which time food was returned.

Balance Used: AS- 1273

Comments/Observations:

All animals show normal behavior during experiment CS 6/6/18

Animal Dosing & Specimen Collection and Processing

Study#: 18CARNP9 Date: 6/6/2018

Group#: 2 Animal: Male Sprague Dawley

_ Rats _

Test Article: 13 C ₆ - Citrate Dosing Vehicle: _ Water _

Dose: 50 mg/kg Specimen Collected: _ Blood _

Dosing sol’n Cone: 10 mg/mL Anticoagulant/Additive: Sodium heparin Dosing Volume: 5 mL/kg Route of Blood Collection: Jugular Vein Cannulae Dosing Route: PO Approx. Vol. Collected: ~ 0.3 mL

Food was withheld from the animals over night before dosing, until four hours post-dose, at which time food was returned.

Balance Used: AS- 1273

Comments/Observations:

All animals show normal behavior during experiment CS 6/6/18

Analytical Stock Solution Preparation

Analytical stock solutions (1.00 mg/mL of the free drug) were prepared in DMSO.

Standard Preparation

Standards were prepared in Sprague-Dawley rat plasma containing sodium heparin as the anticoagulant. Working solutions were prepared in 50:50 acetonitrile:water and then added to the rat plasma to make calibration standards to final concentrations of 2000, 1000, 500, 100, 50.0, 10.0, 5.00, 2.50, and 1.00 ng/mL. Standards were treated identically to the study samples.

Sample Extraction

Plasma samples were manually extracted using acetonitrile in a 96-well plate as outlined in Table 19.

Table 19. Preparation of Plasma Samples

HPLC Conditions

Instrument Waters Ac uiiv UPLC ^K

Column: Acquity UPLC* H$$ T3, 50 x 2,1 mm id 1.8 sun

Aqueous Reservoir (A): 0. 1% formic acid m water

Organic Reservoir (R): 0. 1 % formic acid in acetonitrile

Gradient Program;

Flow Rate: 800 uL/min

Injection Volume: i 0 gt

Run Time: I 0 min

Column Temperature: 4I)°C

Sum p i e Temperature: 8°C

Strong Autesaffipler Wash: 1 : 1: t.(v:v:v) waienmeihi ol isoptopanol with 0,2% formic acid Weak Aytosampler Wash: 4 aiM m onium formate

Mass Spectrometer Conditions

histrumcnt; PE Sciex API4CXM

interface: Electrospray (“Turbo Ion Spray”)

Mode: Multiple reactions monitoring (MRM)

Gases: CUR 30, CAD 10, GS1 50, GS2 50

Source Temperature; 500°C

Voltages and Ions Monitored*

IS; ton Spray Voltage; DP: Deeiasiering Poteni ; EP; Entrance Potential; CE: Collision Ene*¾y; CXP: Collision Celt Exit Potential; *Afl settings ar® in volts

Example 6: Solubility of Citric Acid in Lysine-buffered Solutions

The effect of lysine buffers on the solubility of citric acid at various pH values was analyzed. Results are shown in Table 20. Table 20. Solubility of Citric Acid in Lysine -buffered Solutions

Example 7: Citrate-containing Formulations

Citrate-containing containing formulations according to embodiments of the invention are provided in Table 21.

Table 21. Citrate-containing Formulations

Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes Equivalents

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.