TCA cycle intermediates and methods of use thereof

文档序号：538215 发布日期：2021-06-01 浏览：47次中文

阅读说明：本技术 Tca循环中间体和其使用方法 (TCA cycle intermediates and methods of use thereof ) 是由 A·D·莱文 D·A·格罗斯 J·帕特尔于 2019-10-10 设计创作，主要内容包括：本发明总体上涉及TCA循环中间体的非口服制剂和使用这种制剂治疗代谢障碍的方法。在某些实施例中,本发明提供了被配制成用于非口服施用的含有琥珀酸盐的治疗组合物,和使用这种组合物治疗代谢障碍的方法。本发明还提供了联合疗法,其包括被配制成用于非口服施用的含有琥珀酸盐的组合物和含有柠檬酸盐的组合物(任选地被配制成用于口服施用),和使用这种联合疗法治疗代谢障碍的方法。(The present invention relates generally to non-oral formulations of TCA cycle intermediates and methods of using such formulations to treat metabolic disorders. In certain embodiments, the present invention provides succinate containing therapeutic compositions formulated for non-oral administration, and methods of using such compositions to treat metabolic disorders. The invention also provides combination therapies comprising a succinate containing composition and a citrate containing composition formulated for non-oral administration (optionally formulated for oral administration), and methods of treating metabolic disorders using such combination therapies.)

1. A combination therapy comprising:

a first TCA cycle intermediate or a prodrug, analog, derivative or salt thereof; and

a second TCA cycle intermediate, or a prodrug, analog, derivative or salt thereof, different from the first TCA cycle intermediate.

2. The combination therapy of claim 1, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are formulated for oral administration.

3. The combination therapy of claim 2, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a succinate prodrug, analog, or derivative, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a citrate salt or citrate salt prodrug, analog, derivative, or salt.

4. The combination therapy of claim 1, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are formulated for non-oral administration.

5. The combination therapy of claim 4, wherein the first TCA cycle intermediate or prodrug, analog, derivative or salt thereof is succinate or a succinate prodrug, analog, derivative or salt, and wherein the second TCA cycle intermediate or prodrug, analog, derivative or salt thereof is citrate or a citrate prodrug, analog, derivative or salt.

6. The combination therapy of claim 1, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is formulated for oral administration, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is formulated for non-oral administration.

7. The combination therapy of claim 6, wherein the first TCA cycle intermediate or prodrug, analog, derivative or salt thereof is a succinate prodrug, analog or derivative, and wherein the second TCA cycle intermediate or prodrug, analog, derivative or salt thereof is a citrate salt or citrate salt prodrug, analog, derivative or salt.

8. The combination therapy of claim 6, wherein the first TCA cycle intermediate or prodrug, analog, derivative or salt thereof is citrate or a prodrug, analog, derivative or salt of citrate, and wherein the second TCA cycle intermediate or prodrug, analog, derivative or salt thereof is succinate or a prodrug, analog, derivative or salt of succinate.

9. The combination therapy of claim 1, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided in a single formulation.

10. The combination therapy of claim 1, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided in separate formulations.

11. A method of treating a metabolic disorder in a subject, the method comprising providing to a subject having a metabolic disorder:

a first TCA cycle intermediate or a prodrug, analog, derivative or salt thereof; and

a second TCA cycle intermediate, or a prodrug, analog, derivative or salt thereof, different from the first TCA cycle intermediate.

12. The method of claim 11, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided orally.

13. The method of claim 12, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a succinate prodrug, analog, or derivative, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a citrate salt or citrate salt prodrug, analog, derivative, or salt thereof.

14. The method of claim 11, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided non-orally.

15. The method of claim 14, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is succinate or a succinate prodrug, analog, derivative, or salt, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is citrate or a citrate prodrug, analog, derivative, or salt.

16. The method of claim 11, wherein the first TCA cycle intermediate or prodrug, analog, derivative or salt thereof is provided orally and the second TCA cycle intermediate or prodrug, analog, derivative or salt thereof is provided non-orally.

17. The method of claim 16, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a succinate prodrug, analog, or derivative, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a citrate salt or citrate salt prodrug, analog, derivative, or salt thereof.

18. The method of claim 16, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is citrate or a prodrug, analog, derivative, or salt of citrate, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is succinate or a prodrug, analog, derivative, or salt of succinate.

19. The method of claim 11, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided in a single formulation.

20. The method of claim 11, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided in separate formulations.

21. A composition, comprising:

a first TCA cycle intermediate or a prodrug, analog, derivative or salt thereof; and

a second TCA cycle intermediate, or a prodrug, analog, derivative or salt thereof, different from the first TCA cycle intermediate.

22. The composition of claim 21, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided in separate formulations.

23. The composition of claim 22, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided as an oral formulation.

24. The composition of claim 23, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a succinate prodrug, analog, or derivative, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a citrate salt or citrate salt prodrug, analog, derivative, or salt.

25. The composition of claim 22, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided as a non-oral formulation.

26. The composition of claim 25, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is succinate or a succinate prodrug, analog, derivative, or salt, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is citrate or a citrate prodrug, analog, derivative, or salt.

27. The composition of claim 22, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is provided as an oral formulation, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is provided as a non-oral formulation.

28. The composition of claim 27, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a succinate prodrug, analog, or derivative, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a citrate salt or citrate salt prodrug, analog, derivative, or salt.

29. The composition of claim 27, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is citrate or a prodrug, analog, derivative, or salt of citrate, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is succinate or a prodrug, analog, derivative, or salt of succinate.

30. The composition of claim 21, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof and the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof are provided in a single formulation.

31. The composition of claim 30, wherein the formulation is an oral formulation.

32. The composition of claim 31, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a succinate prodrug, analog, or derivative, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is a citrate salt or citrate salt prodrug, analog, derivative, or salt.

33. The composition of claim 30, wherein the formulation is a non-oral formulation.

34. The composition of claim 33, wherein the first TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is succinate or a succinate prodrug, analog, derivative, or salt, and wherein the second TCA cycle intermediate or prodrug, analog, derivative, or salt thereof is citrate or a citrate prodrug, analog, derivative, or salt.

35. A composition comprising one or more TCA cycle intermediates or prodrugs, analogs or derivatives thereof formulated for non-oral administration.

36. The composition of claim 35, wherein the one or more TCA cycle intermediates comprise a succinate salt or a prodrug, analog or derivative thereof.

37. The composition of claim 36, wherein the composition is formulated for subcutaneous or intravenous administration.

38. The composition of claim 36, wherein the composition further comprises a buffering agent in an amount to buffer the pH of the composition to about 3.0 to about 8.0.

39. The composition of claim 36, wherein the composition comprises the succinate salt or prodrug, analog or derivative thereof at a concentration of from about 1mg/kg subject weight to about 5g/kg subject weight.

40. A method of treating a disorder associated with altered TCA cycle metabolism in a subject, the method comprising providing to a subject having a disorder associated with altered TCA cycle metabolism a composition comprising one or more TCA cycle intermediates or prodrugs, analogs or derivatives thereof, the composition formulated for non-oral administration.

41. The method of claim 40, wherein the one or more TCA cycle intermediates comprise a succinate salt or a prodrug, analog or derivative thereof.

42. The method of claim 41, wherein the composition is formulated for subcutaneous or intravenous administration.

43. The method of claim 41, wherein the composition further comprises a buffering agent in an amount to buffer the pH of the composition to about 3.0 to about 8.0.

44. The method of claim 41, wherein the succinate salt or prodrug, analog or derivative thereof is provided at about 1mg/kg subject weight to about 5g/kg subject weight.

45. The method of claim 41, wherein the disorder is selected from the group consisting of: disorders associated with POLG mutations, energy disorders (energetic disorders), glutaremia type 1 or 2, long chain fatty acid oxidation disorders, methylmalonemia (MMA), mitochondrial-related diseases, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy with broken red fibers (MERRF), mitochondrial myopathy, mitochondrial respiratory chain defects, muscular dystrophy (e.g., duchenne muscular dystrophy and becker muscular dystrophy), neurological disorders, pain or fatigue diseases, Propionemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, and succinyl CoA lyase deficiency.

46. A combination therapy for treating a disorder associated with altered TCA cycle metabolism in a subject, the combination therapy comprising:

a non-oral formulation comprising a succinate salt or a prodrug, analogue or derivative thereof; and

a formulation comprising citrate, citric acid or a prodrug, analog or derivative thereof.

47. The combination therapy of claim 46, wherein the non-oral formulation is formulated for subcutaneous or intravenous administration.

48. The combination therapy of claim 46, wherein said non-oral formulation comprises a buffering agent in an amount to buffer the pH of said non-oral formulation to from about 3.0 to about 8.0.

49. The combination therapy of claim 46, wherein the non-oral formulation comprises from about 1mg/kg subject weight to about 5g/kg subject weight of succinate salt or prodrug, analog or derivative thereof.

50. The combination therapy of claim 46, wherein the formulation comprising citrate, citric acid, or a prodrug, analog, or derivative thereof is an oral formulation.

51. The combination therapy of claim 50, wherein the formulation comprising citrate, citric acid, or a prodrug, analog, or derivative thereof comprises a buffering agent in an amount that buffers the pH of the formulation to from about 3.0 to about 8.0.

52. The combination therapy of claim 51, wherein said buffer comprises one selected from the group consisting of: an amino acid or a derivative thereof and a metal ion.

53. The combination therapy of claim 52, wherein said amino acid is lysine, ornithine or a derivative thereof.

54. The combination therapy of claim 53, wherein the disorder is selected from the group consisting of: disorder associated with POLG mutations, energy disorder, glutaremia type 1 or 2, long chain fatty acid oxidation disorder, methylmalonemia (MMA), mitochondrial-related disease, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy with broken red fibers (MERRF), mitochondrial myopathy, mitochondrial respiratory chain defects, muscular dystrophy (e.g., duchenne muscular dystrophy and becker muscular dystrophy), neurological disorder, pain or fatigue disease, Propionemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, and succinyl CoA lyase deficiency.

55. A method of treating a disorder associated with altered TCA cycle metabolism in a subject, the method comprising providing to a subject having a disorder associated with altered TCA cycle metabolism a combination therapy comprising:

a non-oral formulation comprising a succinate salt or a prodrug, analogue or derivative thereof; and

a formulation comprising citrate, citric acid or a prodrug, analog or derivative thereof.

56. The method of claim 55, wherein the non-oral formulation is provided by subcutaneous or intravenous administration.

57. The method of claim 55, wherein the non-oral formulation comprises a buffering agent in an amount to buffer the pH of the non-oral formulation to about 3.0 to about 8.0.

58. The method of claim 55, wherein the succinate salt or prodrug, analog or derivative thereof is provided at about 1mg/kg subject weight to about 5g/kg subject weight.

59. The method of claim 55, wherein the formulation comprising citrate, citric acid or a prodrug, analog or derivative thereof is an oral formulation.

60. The method of claim 59, wherein the formulation comprising citrate, citric acid, or a prodrug, analog, or derivative thereof comprises a buffer in an amount to buffer the pH of the formulation to about 3.0 to about 8.0.

61. The method of claim 60, wherein the buffer comprises one selected from the group consisting of: an amino acid or a derivative thereof and a metal ion.

62. The method of claim 61, wherein the amino acid is lysine, ornithine or a derivative thereof.

63. The method of claim 59, wherein the formulation comprising citrate, citric acid, or a prodrug, analog, or derivative thereof, is provided in multiple doses per day.

64. The method of claim 55, wherein the non-oral formulation and the formulation comprising citrate are provided sequentially.

65. The method of claim 64, wherein the non-oral formulation is provided first and the formulation comprising citrate is provided second.

66. The method of claim 65, wherein the non-oral formulation is provided in multiple doses over a period of time, and wherein the formulation comprising citrate is provided in multiple doses over a second period of time.

67. The method of claim 55, wherein the disorder is selected from the group consisting of: disorder associated with POLG mutations, energy disorder, glutaremia type 1 or 2, long chain fatty acid oxidation disorder, methylmalonemia (MMA), mitochondrial-related disease, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy with broken red fibers (MERRF), mitochondrial myopathy, mitochondrial respiratory chain defects, muscular dystrophy (e.g., duchenne muscular dystrophy and becker muscular dystrophy), neurological disorder, pain or fatigue disease, Propionemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, and succinyl CoA lyase deficiency.

68. A composition, comprising:

infant formula or human breast milk;

a non-salt form of citric acid or a prodrug, analog or derivative thereof; and

a buffer in an amount to buffer the pH of the composition to about 3.0 to about 8.0.

69. The composition of claim 68, wherein buffering agent comprises one selected from the group consisting of: an amino acid or a derivative thereof and a metal ion.

70. The composition of claim 69, wherein the amino acid is lysine, ornithine or a derivative thereof.

71. A method of treating a disorder associated with altered TCA cycle metabolism in a subject, the method comprising providing to a subject having a disorder associated with altered TCA cycle metabolism an oral formulation comprising a non-salt form of citric acid or a prodrug, analog or derivative thereof and a buffer in an amount to buffer the pH of the composition to from about 3.0 to about 8.0.

72. The method of claim 71, wherein the buffer comprises one selected from the group consisting of: an amino acid or a derivative thereof and a metal ion.

73. The method of claim 72, wherein the amino acid is lysine, ornithine or a derivative thereof.

74. The method of claim 71, wherein the subject is a human less than five years of age.

75. The method of claim 71, wherein the composition further comprises infant formula or human breast milk.

76. A method of treating or preventing Leber's hereditary optic neuropathy in a subject, the method comprising providing a subject having or at risk of having Leber's hereditary optic neuropathy with a non-oral formulation comprising succinate salt or a prodrug, analogue or derivative thereof.

77. The method of claim 76, wherein the non-oral formulation is provided intraocularly.

78. The method of claim 76, wherein the non-oral formulation is provided systemically.

79. The method of claim 78, wherein the non-oral formulation is provided intravenously or subcutaneously.

80. The method of claim 76, wherein the non-oral formulation comprises a buffering agent in an amount to buffer the pH of the non-oral formulation to about 3.0 to about 8.0.

81. The method of claim 76, further comprising providing a formulation comprising citrate, citric acid, or a prodrug, analog, or derivative thereof.

82. The method of claim 81, wherein the formulation comprising citrate, citric acid or a prodrug, analog or derivative thereof is provided in the eye.

83. The method of claim 81, wherein the formulation comprising citrate, citric acid or a prodrug, analog or derivative thereof is provided orally, subcutaneously or intravenously.

84. The method of claim 81. Wherein the non-oral formulation and the formulation comprising citrate, citric acid or a prodrug, analog or derivative thereof are provided as a single formulation.

85. The method of claim 81, wherein the formulation comprising citrate, citric acid, or a prodrug, analog, or derivative thereof comprises a buffer in an amount to buffer the pH of the formulation to about 3.0 to about 8.0.

86. The method of claim 85, wherein said buffer comprises one selected from the group consisting of: an amino acid or a derivative thereof and a metal ion.

87. The method of claim 86, wherein the amino acid is lysine, ornithine or a derivative thereof.

88. The method of claim 81, wherein at least one of the non-oral formulation and the formulation comprising citrate, citric acid, or a prodrug, analog, or derivative thereof, is provided in multiple doses per day.

89. A formulation, comprising:

citric acid or a prodrug, analog, derivative thereof;

at least one citrate salt or a prodrug, analog or derivative thereof; and

an amino acid buffer.

90. The formulation of claim 89, wherein the formulation comprises citric acid and at least one citrate salt.

91. The formulation according to claim 90, wherein said at least one citrate salt is selected from one of the group consisting of: monosodium citrate and monopotassium citrate.

92. The formulation of claim 89, wherein the amino acid buffer is lysine.

93. The formulation of claim 89, wherein the formulation comprises citric acid and at least two citrates.

94. The formulation of claim 93, wherein the at least two citrates comprise monosodium citrate and monopotassium citrate.

95. The formulation of claim 89, wherein the formulation comprises a sugar.

96. The formulation of claim 95, wherein the sugar is sucrose.

97. The formulation of claim 89, wherein the formulation is a powder that is soluble in an aqueous medium.

98. The formulation of claim 97, wherein the formulation comprises citric acid, monosodium citrate, monopotassium citrate, lysine, and sucrose.

99. The formulation of claim 98, wherein the formulation comprises by mass:

about 40% to about 60% citric acid;

about 1% to about 10% monosodium citrate;

about 0.1% to about 5% monopotassium citrate;

about 30% to about 40% lysine; and

from about 10% to about 15% sucrose.

100. The formulation of claim 99, wherein the formulation comprises by mass:

about 49.2% citric acid;

about 2% monosodium citrate;

about 0.3% monopotassium citrate;

about 37.5% lysine; and

about 11% sucrose.

101. The formulation of claim 99, wherein the formulation comprises 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.

102. A formulation, comprising:

citric acid or a prodrug, analog, derivative thereof;

at least one citrate salt or a prodrug, analog or derivative thereof; and

an amino acid.

103. The formulation of claim 102, wherein the formulation comprises citric acid and at least one citrate salt.

104. The formulation of claim 103, wherein the at least one citrate salt is selected from the group consisting of: monosodium citrate and monopotassium citrate.

105. The formulation of claim 102, wherein the amino acid is lysine or arginine.

106. The formulation of claim 102, wherein the formulation comprises citric acid and at least two citrates.

107. The formulation of claim 106, wherein the at least two citrates comprise monosodium citrate and monopotassium citrate.

108. The formulation of claim 102, wherein the formulation further comprises a sugar.

109. The formulation of claim 108, wherein the sugar is sucrose.

110. The formulation of claim 102, wherein the formulation is a powder that is soluble in an aqueous medium.

111. The formulation of claim 102, wherein the formulation comprises citric acid, monosodium citrate, monopotassium citrate, sucrose, and one or more of lysine and arginine.

112. A method of treating a disorder associated with altered TCA cycle metabolism in a subject, the method comprising providing to a subject having a disorder associated with altered TCA cycle metabolism a formulation comprising (i) citric acid or a prodrug, analogue, 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 the activity of a combination of two or more of components (i), (ii) and (iii) in the formulation.

113. The method of claim 112, wherein the amino acid is incorporated into the TCA cycle of the subject and contributes to the therapeutic effect achieved in the subject.

114. The method of claim 112, wherein the disorder is selected from the group consisting of: disorder associated with POLG mutations, energy disorder, glutaremia type 1 or 2, long chain fatty acid oxidation disorder, methylmalonemia (MMA), mitochondrial-related disease, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy with broken red fibers (MERRF), mitochondrial myopathy, mitochondrial respiratory chain defects, muscular dystrophy (e.g., duchenne muscular dystrophy and becker muscular dystrophy), neurological disorder, pain or fatigue disease, Propionemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, and succinyl CoA lyase deficiency.

115. The method of claim 112, wherein the formulation comprises citric acid and at least one citrate salt.

116. The method of claim 115, wherein the at least one citrate salt is selected from the group consisting of: monosodium citrate and monopotassium citrate.

117. The method of claim 112, wherein the amino acid is lysine or arginine.

118. The method of claim 112, wherein the formulation comprises citric acid and at least two citrates.

119. The method of claim 118, wherein the at least two citrates comprise monosodium citrate and monopotassium citrate.

120. The method of claim 112, wherein the formulation further comprises a sugar.

121. The method of claim 120, wherein the sugar is sucrose.

122. The method of claim 112, wherein the formulation is provided orally.

123. The method of claim 122, wherein the formulation is a powder that is soluble in an aqueous medium.

124. The method of claim 112, wherein the formulation comprises citric acid, monosodium citrate, monopotassium citrate, sucrose, and one or more of lysine and arginine.

Technical Field

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

Background

Inherited metabolic disorders such as Propionemia (PA) and methylmalonic acidemia (MMA) can lead to a variety of life-threatening symptoms including seizures, stroke, and damage to the brain, heart, and liver. PA and MMA are caused by enzyme deficiencies that impair the body's ability to convert certain fats and amino acids to succinate, an intermediate of the tricarboxylic acid (TCA) cycle. Thus, patients with PA or MMA must maintain a low protein diet for life, and in some patients, even strict adherence to diet does not prevent neurological damage and other symptoms.

Efforts to treat affected individuals by providing additional succinate have failed. Undoped succinate has low bioavailability and thus it is difficult to provide a sufficient amount of succinate to treat metabolic disorders. Thus, although we have an understanding of the molecular basis of metabolic disorders such as PA and MMA, these genetic disorders are still highly disabling and often fatal.

Disclosure of Invention

The present invention overcomes the challenge of delivering succinate in therapeutically effective doses to treat metabolic disorders such as PA and MMA by providing a combination therapy comprising a plurality of TCA cycle intermediates. By providing two or more TCA cycle intermediates to a subject, the compositions and methods of the invention rely on the body's natural metabolic pathways to restore succinate levels and allow delivery of larger doses of succinate than would be possible with a single agent. In addition, because the compositions can provide a variety of TCA cycle intermediates, they are useful in the treatment of a broad spectrum of metabolic diseases, disorders, and conditions. For example, a combination of succinate and citrate may be used to treat 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. For particular forms of intermediates, each TCA cycle intermediate can be independently provided to a subject by an optimal route of administration. Thus, succinate containing combinations may include non-oral delivery of succinate or oral delivery of succinate derivatives with improved bioavailability. Furthermore, in the succinate-citrate combination, both intermediates may be administered orally, both may be administered non-orally, or one may be administered orally and the other may be administered non-orally.

The invention also provides succinate containing compositions formulated for non-oral administration. The compositions (including formulations for subcutaneous or intravenous administration) have superior bioavailability to oral formulations. Non-oral formulations of succinate salts may be used in the combination therapy of the present 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 caused by genetic defects, and therefore, when a child is no longer able to obtain a nutritional supply from the mother, the consequences of incorrect metabolism manifest themselves at birth. Therefore, early intervention is necessary before metabolic imbalance leads to permanent symptoms. Because the present invention provides succinate containing compositions formulated for non-oral administration, such compositions do not require active sucking by the infant to deliver a therapeutically effective dose of succinate. Thus, treatment of the infant can be started immediately to help prevent the development of disease symptoms.

In one aspect, the invention provides compositions comprising 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, α -ketoglutarate, succinyl-CoA, succinate, fumarate, malate or oxaloacetate. Preferably, the TCA cycle intermediate is succinate.

In another aspect, the invention provides methods of treating a disorder associated with altered TCA cycle metabolism in a subject. The method comprises providing a composition formulated for non-oral administration containing one or more TCA cycle intermediates or prodrugs, analogs or derivatives thereof to a subject having a disorder associated with altered TCA cycle metabolism. 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 comprise a buffer which maintains the neutral or near neutral pH of the composition. For example, the buffering agent may be present in the following amounts: sufficient to buffer the pH of the composition to about 3.0 to about 10.0, about 3.0 to about 9.0, about 3.0 to about 8.0, about 3.0 to about 7.0, about 3.0 to about 6.0, about 4.0 to about 10.0, about 4.0 to about 9.0, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 5.0 to about 10.0, about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 10.0, about 6.0 to about 9.0, about 6.0 to about 8.0, about 7.0 to about 10.0, about 7.0 to about 9.0, or about 8.0 to about 10.0.

The TCA cycle intermediates can be provided in any therapeutically effective dose. For example, TCA cycle intermediates can be provided in the following amounts: about 0.1mg/kg subject weight to about 5g/kg subject weight, about 0.2mg/kg subject weight to about 5g/kg subject weight, about 0.5mg/kg subject weight to about 5g/kg subject weight, about 1mg/kg subject weight to about 5g/kg subject weight, about 2mg/kg subject weight to about 5g/kg subject weight, about 5mg/kg subject weight to about 5g/kg subject weight, about 0.1mg/kg subject weight to about 2g/kg subject weight, about 0.2mg/kg subject weight to about 2g/kg subject weight, about 0.5mg/kg subject weight to about 2g/kg subject weight, about 1mg/kg subject weight to about 2g/kg subject weight, From about 2mg/kg subject weight to about 2g/kg subject weight, from about 5mg/kg subject weight to about 2g/kg subject weight, from about 0.1mg/kg subject weight to about 1g/kg subject weight, from about 0.2mg/kg subject weight to about 1g/kg subject weight, from about 0.5mg/kg subject weight to about 1g/kg subject weight, from about 1mg/kg subject weight to about 1g/kg subject weight, from about 2mg/kg subject weight to about 1g/kg subject weight, from about 5mg/kg subject weight to about 1g/kg subject weight, from about 1mg/kg subject weight to about 500mg/kg subject weight, from about 2mg/kg subject weight to about 200mg/kg subject weight, or from about 5mg/kg subject weight to about 100mg/kg subject weight.

The condition can be any disease, disorder or condition associated with altered TCA cycle metabolism. For example, the disorder can be a disorder associated with POLG mutations, energy disorders (energetic disorders), glutaremia type 1 or 2, long chain fatty acid oxidation disorders, methylmalonate (MMA), mitochondrial-related diseases, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy with broken red fibers (MERRF), mitochondrial myopathy, mitochondrial respiratory chain defects, muscular dystrophy (e.g., duchenne's muscular dystrophy and becker's muscular dystrophy), neurological disorders, pain or fatigue diseases, Propionemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, or succinyl CoA lyase deficiency. Preferably, the disorder is MMA or PA.

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

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

Non-oral formulations containing the succinate salt or prodrug, analog or derivative thereof may be formulated for administration by any non-oral means, as described above.

Non-oral formulations containing a succinate salt or a prodrug, analog or derivative thereof may contain a buffering agent, as described above.

Non-oral formulations containing the succinate salt or prodrug, analog or derivative thereof may be provided in any therapeutically effective dose. For example, the succinate salt or prodrug, analog or derivative thereof can be provided in the following amounts: from about 0.1mg/kg subject weight to about 5g/kg subject weight, from about 0.2mg/kg subject weight to about 2g/kg subject weight, from about 0.5mg/kg subject weight to about 1g/kg subject weight, from about 1mg/kg subject weight to about 500mg/kg subject weight, from about 2mg/kg subject weight to about 200mg/kg subject weight, or from about 5mg/kg subject weight to about 100mg/kg subject weight.

Formulations containing citrate, citric acid or a citrate or prodrug, analog or derivative of 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.

Formulations containing citrate, citric acid or a citrate or prodrug, analog or derivative of citric acid may contain a buffering agent. For example, the buffering agent may be present in the following amounts: sufficient to buffer the pH of the composition to about 3.0 to about 10.0, about 3.0 to about 9.0, about 3.0 to about 8.0, about 3.0 to about 7.0, about 3.0 to about 6.0, about 4.0 to about 10.0, about 4.0 to about 9.0, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 5.0 to about 10.0, about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 10.0, about 6.0 to about 9.0, about 6.0 to about 8.0, about 7.0 to about 10.0, about 7.0 to about 9.0, or about 8.0 to about 10.0. The buffer may be an amino acid or a metal ion. The amino acid may be a natural amino acid or an unnatural 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 in any therapeutically effective dose. For example, citrate or a prodrug, analog or derivative thereof can be provided in the following amounts: from about 0.1mg/kg subject weight to about 5g/kg subject weight, from about 0.2mg/kg subject weight to about 2g/kg subject weight, from about 0.5mg/kg subject weight to about 1g/kg subject weight, from about 1mg/kg subject weight to about 500mg/kg subject weight, from about 2mg/kg subject weight to about 200mg/kg subject weight, or from about 5mg/kg subject weight to about 100mg/kg subject weight.

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

The non-oral formulation comprising a succinate salt or prodrug, analog or derivative thereof and the formulation comprising a citrate salt, citric acid or prodrug, analog or derivative thereof may be provided simultaneously, sequentially in either order, or in an alternating manner. Sequential or alternating administration may include providing exclusively for a period of time a non-oral formulation containing succinate or a prodrug, analog or derivative thereof and providing exclusively for a period of time a formulation containing citrate, citric acid or a prodrug, analog or derivative of citrate. Sequential administration may include overlapping periods in which a subject is provided with a non-oral formulation containing a succinate salt or prodrug, analog or derivative thereof and a formulation containing a citrate salt, citric acid or prodrug, analog or derivative of citric acid. The exclusive period and the overlapping period may be independently 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 can be any disease, disorder or condition associated with altered TCA cycle metabolism, as described above.

In another aspect, the present invention provides compositions comprising a source of nutrition for an infant, such as infant 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 the pH of the composition from about 3.0 to about 8.0.

The buffer may be present in the following amounts: sufficient to buffer the pH of the composition to about 3.0 to about 10.0, about 3.0 to about 9.0, about 3.0 to about 8.0, about 3.0 to about 7.0, about 3.0 to about 6.0, about 4.0 to about 10.0, about 4.0 to about 9.0, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 5.0 to about 10.0, about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 10.0, about 6.0 to about 9.0, about 6.0 to about 8.0, about 7.0 to about 10.0, about 7.0 to about 9.0, or about 8.0 to about 10.0. The buffer may be an amino acid or a metal ion. The amino acid may be a natural amino acid or an unnatural amino acid. The amino acid may be lysine, ornithine or a derivative thereof.

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

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

In another aspect, the invention provides a method of treating or preventing Leber's hereditary optic neuropathy in a subject. The method comprises providing a subject at risk of or having Leber's hereditary optic neuropathy with a non-oral formulation comprising a succinate salt or a prodrug, analog or derivative thereof.

Non-oral formulations may be provided intraocularly. Non-oral formulations may be provided systemically. For example, the non-oral formulation may be provided intravenously or subcutaneously.

The non-oral formulation may include a buffer to maintain a neutral or near neutral pH of the composition. For example, the buffering agent may be present in the following amounts: sufficient to buffer the pH of the composition to about 3.0 to about 10.0, about 3.0 to about 9.0, about 3.0 to about 8.0, about 3.0 to about 7.0, about 3.0 to about 6.0, about 4.0 to about 10.0, about 4.0 to about 9.0, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 5.0 to about 10.0, about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 10.0, about 6.0 to about 9.0, about 6.0 to about 8.0, about 7.0 to about 10.0, about 7.0 to about 9.0, or about 8.0 to about 10.0.

A method 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 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.

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

In another aspect, the present invention provides a formulation comprising citric acid or a prodrug, analog, derivative thereof; one or more citrate salts or a prodrug, analog or derivative thereof; and an amino acid buffer.

The citrate salt may include monosodium citrate or monopotassium citrate.

The amino acid buffer 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 soluble components of the formulation may contain citric acid, monosodium citrate, monopotassium citrate, lysine and sucrose. The formulation may contain about 40% to about 60% by mass of citric acid; about 1% to about 10% monosodium citrate; about 0.1% to about 5% monopotassium citrate; about 30% to about 40% lysine; and about 10% to about 15% sucrose. The formulation or soluble components of 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 soluble components in 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 soluble components in the formulation may contain from about 50% to about 60% by mass of citrate, citric acid, or a combination thereof; from about 0.2% to about 1% sodium; about 0.02% to about 0.5% potassium; about 30% to about 40% lysine; and about 10% to about 15% sucrose.

The formulation may be suitable for oral administration.

The formulation can be administered to a subject to treat or prevent a disorder associated with altered TCA cycle metabolism, such as any of the disorders described above.

In another aspect, the present invention provides a formulation comprising 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 comprise 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 agent may be citric acid and at least two citrates, for example, the at least two citrates may be monosodium citrate and monopotassium citrate.

The formulation may comprise any natural or unnatural amino acid. In certain embodiments, the amino acid is a cationic amino acid, such as lysine or arginine. The formulation may comprise other components such as sugars, for example sucrose.

Other aspects of the invention provide methods of treating a disorder associated with altered TCA cycle metabolism in a subject. Such methods may involve providing 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 the 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 achieved in the subject.

Exemplary conditions include disorders associated with POLG mutations, energy disorders, glutaremia type 1 or 2, long chain fatty acid oxidation disorders, methylmalonate (MMA), mitochondrial-related diseases, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), myoclonic epilepsy with broken red fibers (MERRF), mitochondrial myopathy, mitochondrial respiratory chain defects, muscular dystrophy (e.g., duchenne muscular dystrophy and becker muscular dystrophy), neurological disorders, pain or fatigue diseases, Propionaemia (PA), pyruvate carboxylase deficiency, refractory epilepsy, or succinyl CoA lyase deficiency.

The formulation may comprise 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 agent may be citric acid and at least two citrates, for example, the at least two citrates may be monosodium citrate and monopotassium citrate.

The formulations may be provided by any route of administration, and the preferred route is oral. Exemplary oral formulations are powders that are soluble in aqueous media. In a particular embodiment, the formulation comprises citric acid, monosodium citrate, monopotassium citrate, sucrose, and one or more of lysine and arginine. The formulation may be provided once or more times per day. For example, one or more formulations may be provided in 1,2, 3, 4, 5, 6 or more doses per day.

Drawings

FIG. 1 shows subcutaneous administration of 10mg/kg¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 2 is a 10mg/kg subcutaneous administration¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

FIG. 3 is a schematic representation of subcutaneous administration of 50mg/kg¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 4 is a drawing showing subcutaneous administration of 50mg/kg¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

FIG. 5 is a subcutaneous administration of 100mg/kg¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 6 is a subcutaneous administration of 100mg/kg¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

Fig. 7 is a graph showing the effect of citrate and succinate on basal respiration in cells of a patient suffering from Propionemia (PA).

FIG. 8 is a graph of intravenous administration of 10mg/kg¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 9 is a graph of intravenous administration of 10mg/kg¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

FIG. 10 shows the administration of 50mg/kg orally¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 11 shows 50mg/kg of the drug administered orally¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

FIG. 12 is a graph of intravenous administration of 10mg/kg¹³C₆Citrate followed by different time points in individual rats¹³C₆-graph of plasma concentration of citrate.

FIG. 13 is a graph of intravenous administration of 10mg/kg¹³C₆Citrate followed by different time points in rats¹³C₆-graph of mean plasma concentrations of citrate.

FIG. 14 is a graph of oral administration of 50mg/kg¹³C₆Citrate followed by different time points in individual rats¹³C₆-graph of plasma concentration of citrate.

FIG. 15 shows 50mg/kg of the drug administered orally¹³C₆Citrate followed by different time points in rats¹³C₆-graph of mean plasma concentrations of citrate.

Detailed Description

The present invention provides compositions and methods that allow for the efficient delivery of TCA cycle intermediates for the treatment of metabolic disorders. In particular, the compositions and methods of the present invention are capable of delivering succinate by subcutaneous administration or intravenous administration. Succinate synthesis is defective in propionemia and methylmalonic acidemia, and incorrect metabolism in patients with these disorders can lead to various potentially fatal effects, such as nerve damage, cardiomyopathy, and infection. Non-oral formulations of succinate salts allow delivery of compounds with a bioavailability that is much higher than can be achieved with existing oral succinate salt formulations. Thus, non-oral formulations provide succinate at sufficient levels to prevent the serious long-term effects of defective TCA cycle metabolism.

The invention also provides combination therapies comprising a non-oral formulation of succinate and an oral formulation of citrate, another intermediate of the TCA cycle. In contrast to succinate, citrate can be administered orally at therapeutically effective doses. Thus, combination therapy provides for the delivery of different TCA cycle intermediates in different ways. The non-oral formulation of the succinate salt and the oral formulation of the citrate salt may be provided sequentially or simultaneously. Sequential combination therapy allows a patient (especially an infant) to transition from non-oral therapy to oral administration when the patient's age reaches a level sufficient to ingest a therapeutically effective amount of citrate. Such therapies allow early intervention in the newborn, while allowing long-term care that can be administered without healthcare professionals. Simultaneous combination therapy is useful when clinical problems such as vomiting, rash, etc. limit the amount of intermediate that can be delivered by either mode of administration.

TCA cycle and related disorders

The TCA cycle is as follows:

abnormal TCA cycle metabolism is associated with a variety of conditions. In inherited metabolic disorders of the TCA cycle, such as 2-oxoglutarate uraemia, fumarase deficiency and succinyl-CoA synthetase deficiency, genetic mutations affect the enzymes of the TCA cycle or the enzymes catalyzing the relevant reactions. Thus, the individual reactions of the TCA cycle suffer, leading to the depletion of intermediates required for the cycle to proceed. Such diseases usually occur at an early stage, with severe symptoms such as mental retardation, microcephaly, deafness and hypotonia, and are often fatal in infancy.

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

propionemia (PA) is caused by a deficiency of propionyl CoA carboxylase. In patients with PA, excess propionyl-CoA is converted to propionic acid, which accumulates in the bloodstream. In a similar manner, methylmalonemia (MMA) is caused by a deficiency in methylmalonyl CoA mutase. In patients with MMA, excess methylmalonyl CoA is converted to methylmalonate, which accumulates in the bloodstream. In PA and MMA, high levels of acid in the blood lead to various potentially lethal effects such as organ damage, stroke and epilepsy.

Treatment of PA and MMA is focused on dietary management to avoid amino acids that trigger acid accumulation. Therefore, patients with PA or MMA must strictly adhere to a low protein diet to minimize their trigger amino acid intake. The limited intake of amino acids in patients with PA or MMA often leads to a shortage of l-carnitine, a quaternary ammonium compound involved in the transport of fatty acids across the mitochondrial membrane. Thus, patients with PA or MMA typically receive supplemental l-carnitine. Infants with PA or MMA also are at risk for bacterial infection on their low protein diet, so they can be given prophylactic antibiotics.

PA and MMA are autosomal recessive genetic disorders. Homozygous mutation of the gene PCCA or PCCB encoding propionyl-CoA carboxylase results inAnd (6) PA. MMA can be produced by homozygous mutations in MUT encoding methylmalonyl-CoA mutase. methylmalonyl-CoA mutase requires vitamin B₁₂As a cofactor and is involved in vitamin B₁₂Mutations in the genes of metabolism (such as LMBRD1, MCEE, MMAA, MMAB, MMACHC and MMADHC) can also lead to MMA. Vitamin B₁₂MMA can also be caused by dietary deficiency of (a).

Abnormal TCA cycle metabolism is also observed in other diseases that are not directly genetically linked 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 variety of cancers. Although the symptoms vary from one disease to another, in many cases, reduced activity of specific TCA enzymes or reduced mitochondrial ATP production has been observed, and it is thought that increasing levels of TCA cycle intermediates will alleviate symptoms and improve prognosis.

The present invention provides methods of treating any disease, disorder or condition associated with altered metabolism of the TCA cycle or which can be ameliorated by providing intermediates of the TCA cycle. The disorder may be a genetic disorder such as PA, MMA, 2-oxoglutarate uropathy, fumarase deficiency or succinyl CoA synthetase deficiency. The disorder can be a neurodegenerative disorder, such as amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease, or huntington's disease. The condition may be cancer, such as pancreatic cancer, renal 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 may be treated with the compositions or methods of the invention include acute angina, acute kidney injury, acute hunger, Adrenoleukodystrophy (ALD), Adrenomyeloneuropathy (AMN), age-related diseases, Alpers disease (progressive infantile polio dystrophy), alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), atrial fibrillation, autism, and Autism Spectrum Disorders (ASD), Barth syndrome (fatal infantile cardiomyopathy), beta-oxidation deficiency, bioenergetic metabolic deficiency, bipolar disorder, carnitine-acyl carnitine deficiency, carnitine palmitoyl transferase i (cpt i) deficiency, carnitine palmitoyl transferase ii (cpt ii) deficiency, cerebrovascular accident, chronic progressive external eye paralysis syndrome (CPEO), coenzyme Q10 deficiency, deficiency in acute angina, acute renal injury, acute hunger, Adrenal Leukodystrophy (ALD), adrenomyeloneuropathy (amniosis), age-related diseases, Alpers disease (progressive infantile poliomyelitis dystrophy), alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), amygdalasia, ALS), atrial fibrillation, auti, Deficiency of Complex I (deficiency of NADH dehydrogenase (NADH-CoQ reductase)), deficiency of Complex II (deficiency of succinate dehydrogenase), deficiency of Complex III (deficiency of ubiquinone-cytochrome c oxidoreductase, deficiency of Complex IV/COX, deficiency of Complex V (deficiency of ATP synthase), occlusion of coronary arteries, deficiency of COX, deficiency of creatine syndrome (e.g., Cerebral Creatine Deficiency Syndrome (CCDS), deficiency of guanidinoacetic methyltransferase (GAMT deficiency), deficiency of L-arginine: glycinamidine transferase (AGAT deficiency), and deficiency of the creatine transporter associated with SLC6A8 (SLC6A 8)), diabetes, disorders associated with POLG mutations, endotoxemia, energy disorders, epilepsy, Friedrich's ataxia (FRDA or FA), glutaruria type II, Huntington's disease, hypoxia, ischemia, Carnss-Seer syndrome (KSS), Lactic acidosis, long-chain acyl-CoA dehydrogenase deficiency (LCAD, VLCAD, VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), Leigh disease or syndrome (subacute necrotizing encephalomyelopathy), Leber Hereditary Optic Neuropathy (LHON), Luft disease, macular degeneration, male infertility, medium-chain acyl-CoA dehydrogenase deficiency (MCAD), mitochondrial-related diseases, mitochondrial cytopathies, mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS), mitochondrial encephalopathies (including encephalomyopathy, encephalomyelitis), mitochondrial DNA depletion, mitochondrial myopathy, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial respiratory chain defects, multi-organ dysfunction syndrome, mood disorders, motor neuron diseases, muscular dystrophy (e.g., duchenne muscular dystrophy and becker muscular dystrophy), myocardial infarction, myoclonic epilepsy with broken red fibers (MERRF), myocardial infarction, stroke, myoneurotropic gastrointestinal disorders (myoneuregular disorder) and encephalopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), neurodegenerative disorders associated with Parkinson's disease, Pearson syndrome, pyruvate carboxylase deficiency, pyruvate dehydrogenase deficiency, refractory epilepsy, renal tubular acidosis, respiratory chain deficiency, schizophrenia, sepsis, short-chain acyl CoA dehydrogenase deficiency (SCAD), short-chain hydroxy acyl CoA dehydrogenase deficiency (SCHAD, SCHADD), stroke, succinyl CoA lyase deficiency, systemic inflammatory response syndrome and very long-chain acyl CoA dehydrogenase deficiency (VLCAD).

A 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 heart disease, gastrointestinal disorders, ischemia reperfusion injury, poisoning, liver disease, loss of motor control, muscle weakness and pain, mutations in the mitochondrial genome, neurological diseases, disorders or conditions, pain or fatigue diseases, seizures, sensorineural hearing loss, dysphagia, tinnitus, or vision/hearing problems.

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

Another clinically important metabolic pathway is the mitochondrial electron transport chain. The electron transport chain utilizes a complex series of redox reactions to generate a proton gradient on the inner mitochondrial membrane, and the chemical motif potential from the proton gradient is used to drive the synthesis of Adenosine Triphosphate (ATP). The electron transport chain involves four enzyme complexes in the inner mitochondrial membrane, NADH dehydrogenase, also known as respiratory complex I; succinate dehydrogenase, also known as respiratory complex II; coenzyme Q cytochrome c reductase, also known as respiratory complex III; and cytochrome c oxidase, also known as respiratory complex IV. Electrons enter the transfer chain in either of two ways. First, NADH dehydrogenase can transfer electrons from NADH to ubiquitin (the first intermediate electron carrier in the chain). Alternatively, the electron from succinate can be transferred to ubiquinone by succinate dehydrogenase. In the next step of the electron transport chain, electrons are transferred from ubiquinone to the second intermediate electron carrier cytochrome c via coenzyme Q, cytochrome c reductase. In the last step, cytochrome c oxidase transfers electrons from cytochrome c to molecular oxygen to form water (the net product of electron transfer). Succinate dehydrogenase is the only enzyme involved in the TCA cycle and the electron transport chain.

Leber Hereditary Optic Neuropathy (LHON) is a retinal degenerative disorder caused by defects in the electron transport chain. LHON is caused by mutations in mitochondrial genes encoding components of NADH dehydrogenase such as MT-ND1, MT-ND4, MT-ND4L and MT-ND 6. Because the mutations that lead to LHON are encoded by genes in the mitochondrial genome (which is transmitted to the embryo from the ovum, rather than the sperm), LHON can only be inherited through maternity.

The insight of the present invention is that succinate salts can be used for the treatment of LHON. Due to reduced NADH dehydrogenase activity, electron transport and ATP synthesis are reduced in patients with LHON. In particular, the formation of the first intermediate electron carrier ubiquinone in the chain is reduced. However, electron transport activity can be restored by providing supplemental succinate, which can donate electrons by succinate dehydrogenase to form ubiquinone. Thus, providing additional succinate as an electron donor for the electron transfer chain in patients with LHON compensates for the deficiency in electron transfer by NADH.

Compositions containing TCA cycle intermediates

The present invention provides compositions comprising one or more TCA cycle intermediates or prodrugs, analogs or derivatives thereof formulated for non-oral administration. For example, but not limited to, a TCA cycle intermediate or prodrug, analog or derivative thereof can be citrate, aconitate, D-isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, oxaloacetate, acetone, acetoacetate, beta-hydroxybutyrate, beta-ketovalerate or beta-hydroxyvalerate. Preferably, the TCA cycle intermediate is succinate. The TCA cycle intermediate or prodrug, analog or derivative thereof may be provided in free acid, salt or non-salt form.

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, but not limited to, the composition may be formulated for subcutaneous, intravenous, intra-arterial, intramuscular, intradermal, or rectal administration. Preferably, the composition is formulated for intravenous administration or subcutaneous administration.

A prodrug is a drug or compound that is metabolized (i.e., converted in vivo) to a pharmacologically active drug upon administration. The prodrug itself may not have pharmacological activity. Prodrugs can be used to improve the manner in which drugs are absorbed, distributed, metabolized, and excreted. Prodrugs may improve the bioavailability of an active drug when the active drug is poorly absorbed through the gastrointestinal tract. Prodrugs can improve the way in which a drug interacts selectively with cells or processes that are not their intended target, thereby reducing unintended and undesirable side effects. The prodrug can be converted to the biologically active form (bioactivated) either intracellularly (type I prodrug) or extracellularly (type II prodrug). The prodrug may be bioactivated in the gastrointestinal tract, in the systemic circulation, in metabolic tissues other than the target tissue, or in the target tissue.

The TCA cycle intermediate or prodrug, analog or derivative thereof may be provided as a pharmaceutically acceptable salt, such as a non-toxic acid addition salt, which is a salt of an amino group formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with an organic acid 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, adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, dodecylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoate, pectinates, persulfates, 3-phenylpropionates, salts of oleic acid, oxalates, palmitates, pamoate, pectinates, persulfates, 3-phenylpropionates, salts of acetic acid, Phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, pentanoates, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, the pharmaceutically acceptable salt is an alkali metal salt. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is an alkaline earth metal salt. In some embodiments, pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions, such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl groups having 1 to 6 carbon atoms, sulfonate, and arylsulfonate.

Prodrugs of succinates are known in the art and are described, for example, in 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 formulae (I) and (IA):

or a pharmaceutically acceptable salt thereof, wherein the point bond between a and B represents an optional bond to form a closed ring structure, and wherein

When formula is formula (I), Z is selected from-CH₂- (e.g. derived from malonic acid), -CH₂-CH₂-CH₂(e.g., derived from glutaric acid),. alpha.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 (O) -CH₂-CH₂- (e.g. derived from alpha-ketoglutaric acid), -CH₂-CH₂-C (O) - (e.g. derived from alpha-ketoglutaric acid), -CH₂-C (coc (oh) -C (cooh) -CHOH) ═ CH- (e.g., derived from aconitic acid), -CH ═ C (cooh) -CH₂- (e.g. derived from aconitic acid), -CH (OH) -CH (COOH) -CH₂- (e.g. derived from isocitric acid), -CH₂-CH (COOH) -CH (OH) - (e.g. derived from isocitric acid), -CH₂-CH (cooh) -C (═ O) - (e.g. derived from oxalyl succinic acid), -C (═ O) -CH (cooh) -CH₂- (e.g. derived from oxalyl succinic acid), -C (═ O) -CH₂- (e.g. derived from oxaloacetic acid), -CH₂-C (═ O) - (e.g., derived from oxaloacetate); or

When 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-CH₂And 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, which may be branched or straight chain₁-C₄-alkyl, preferably R is Me;

r' is selected from the following formulae (II), (V) or (IX):

and neither A nor B is-OH,

r ', R "and R'" are independently different or the same and are selected from the following formulae (VII-VIII):

R₁and R₃Independently different or the same and selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, O-acyl, O-alkyl, N-acyl, N-alkyl, X acyl, CH₂X alkyl, CH₂CH₂CH₂OC(＝O)CH₂CH₂COX₆R₈Or

X is selected from O, NH, NR₆、S，

R₂Selected from Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, C (O) CH₃、C(O)CH₂C(O)CH₃、C(O)CH₂CH(OH)CH₃，

p is an integer and is 1 or 2,

R₆selected from H, alkyl, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl or formula (II) or formula (VIII),

X₅selected from-H, -COOH, -C (═ O) XR₆、CONR₁R₃Or one of the following formulae

R₉Selected from H, Me, Et or O₂CCH₂CH₂COXR₈，

R₁₀Selected from the group consisting of Oacyl, NH alkyl, NH acyl or O₂CCH₂CH₂COX₆R₈，

X₆Is O or NR₈And R is₈Selected from H, alkyl, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl, succinyl or formula (II) or formula (VIII),

R₁₁and R₁₂Independently the same or different, and is selected from H, alkyl, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl, succinyl, acyl, -CH₂X alkyl, -CH₂X acyl, wherein X is selected from O, NR₆Or the number of the S-beams is,

R₁₃、R₁₄and R₁₅Independently different or the same and selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, -COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, X acyl, CH₂The alkyl group of X is a group selected from,

R_cand R_dIndependently is CH₂X alkyl, CH₂X acyl, wherein X is O, NR₆Or S, and alkyl is, for example, H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, and acyl is, for example, formyl, acetyl, propionyl, isopropionyl, butyryl, tert-butyryl, valeryl, benzoyl, succinyl, and the like,

R_f、R_gand R_hIndependently selected from X acyl, -CH₂X alkyl, -CH₂The acyl group X and the group 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, valeryl, benzoyl, succinyl and the like,

R₂₀and R₂₁Independently different or the same, and is selected from H, lower alkyl, i.e. C₁-C₄Alkyl, or R₂₀And R₂₁Together may form C₄-C₇Cycloalkyl or aromatic radicals, both of which may optionally be substituted by halogen, hydroxy or lower alkylIs substituted, or

R₂₀And R₂₁Can be

CH₂X-acyl radical F, CH₂COOH、CH₂CO₂Alkyl radical, and

when a ring bond is present between A and B, the compound is

And the acyl and alkyl groups may be optionally substituted.

A compound of formulae (I) and (IV) or a pharmaceutically acceptable salt thereof, wherein the point bond between A and B represents an optional bond to form a closed ring structure, and

wherein A is-OR₁And R is₁Is H OR a pharmaceutically acceptable salt thereof, OR an optionally substituted alkyl group OR a group of formula (II), and B in formula (I) is-OR₂And R is₂Independently is a radical according to formula (II), wherein formula (II) is

Wherein R is₃And R₄Independently is H, optionally substituted C₁-C₃Alkyl, or are linked together to form a ring, and wherein R₅Is connected to R₁To form a ring, or R₅Selected from OCOR_a、OCOOR_b、OCONR_cR_d、SO₂R_e、OPO(OR_f)(OR_g) Or CONR_cR_dWherein R is_aIs optionally substituted alkyl or optionally substituted cycloalkyl, R_bIs optionally substituted alkyl, R_cAnd R_dIndependently is H, optionally substituted alkyl or is linked together to formRings which may contain one or more further hetero atoms, R_eIs optionally substituted alkyl, R_fAnd R_gIndependently H, methyl, ethyl or are linked together to form a ring;

wherein B in formula (IA) is a branched or straight chain C₁-C₄An alkyl group;

and wherein

When formula is formula (I), Z is selected from-CH₂- (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 (O) CH₂-CH₂- (e.g. derived from alpha-ketoglutaric acid), -CH₂-CH₂-C (O) - (e.g. derived from alpha ketoglutaric acid), -CH₂-C (coc (oh) -C (cooh) -CHOH) ═ CH- (e.g., derived from aconitic acid), -CH ═ C (cooh) -CH₂- (e.g. derived from aconitic acid), -CH (OH) -CH (COOH) -CH₂- (e.g. derived from isocitric acid), -CH₂-CH (COOH) -CH (OH) - (e.g. derived from isocitric acid), -CH₂-CH (cooh) -C (═ O) - (e.g. derived from oxalyl succinic acid), -C (═ O) -CH (cooh) -CH₂- (e.g. derived from oxalyl succinic acid), -C (═ O) -CH₂- (e.g. derived from oxaloacetic acid), -CH₂-C (═ O) - (e.g., derived from oxaloacetate); or

A compound of formulae (I) and (IA) or a pharmaceutically acceptable salt thereof, wherein the point bond between A and B represents an optional bond to form a closed ring structure,

and wherein

When formula is formula (I), Z is selected from-CH₂- (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 (O) -CH₂-CH₂- (e.g. derived from alpha-ketoglutaric acid), -CH₂-CH₂-C (O) - (e.g. derived from alpha-ketoglutaric acid), -CH₂-C (coc (oh) -C (cooh) -CHOH) ═ CH- (e.g., derived from aconitic acid), -CH ═ C (cooh) -CH₂- (e.g. derived from aconitic acid), -CH (OH) -CH (COOH) -CH₂- (e.g. derived from isocitric acid), -CH₂-CH (COOH) -CH (OH) - (e.g. derived from isocitric acid), -CH₂-CH (cooh) -C (═ O) - (e.g. derived from oxalyl succinic acid), -C (═ O) -CH (cooh) -CH₂- (e.g. derived from oxalyl succinic acid), -C (═ O) -CH₂- (e.g. derived from oxaloacetic acid), -CH₂-C (═ O) - (e.g., derived from oxaloacetate); or

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

When Z is CH₂And R is Me, Et, propyl, butyl, pentyl, hexyl, heptyl, octyl or succinyl, then X₅、R₁₅、R₁₄And R₁₃Cannot all be H;

when Z is-CH₂-CH₂-CH₂、-CH＝CH-、-CH₂-CH(OH)-、-CH(OH)-CH₂-、-CH₂C(OH)(COOH)-CH₂-、-C(O)-CH₂-CH₂-、-CH₂-CH₂-C(O)-、-CH₂-C(COOH)＝CH-、-CH＝C(COOH)-CH₂-, -CH (oh) -CH (cooh) -CH2-, -CH2-CH (cooh) -CH (oh) -, -CH2-CH (cooh) -C (═ O) -, -C (═ O) -CH (cooh) -CH2-, -C (═ O) -CH 2-or-CH 2-C (═ O) -, and R₁When Me, octyl or succinyl, X₅、R₁₅、R₁₄And R₁₃Cannot all be H;

R₁does not contain the motif-CH 2N-acyl; r₁Must not 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 when X is₅is-COOH or-C (═ O) XR₆When R is₁Cannot be Me;

b is selected from-O-R ', -NHR ", -SR'" or-OH; and R' is selected from the following formulae (II) to (IX):

or in those cases where the compound is according to formula (IA), then B is C, which may be branched or straight chain₁-C₄-alkyl, preferably R is Me;

r ', R "and R'" are independently different or the same and are selected from the following formulae (VII-VIII):

x is selected from O, NH, NR₆、S，

R₂Selected from Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, -C (O) CH₃、-C(O)CH₂C(O)CH₃、-C(O)CH₂CH(OH)CH₃，

p is an integer and is 1 or 2,

R₆selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl or formula (II) or formula (VIII),

X₅selected from-H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, -COOH, -C (═ O) XR₆、CONR₁R₃Or is of the formula

X₇Is selected from R₁、-NR₁R₃，

R₉Selected from H, Me, Et or O₂CCH₂CH₂COXR₈，

R₁₀Selected from-Oacyl, -NH alkyl, -NH acyl or O₂CCH₂CH₂COX₆R₈，

X₆Selected from O, NR₈、NR₆R₈Wherein R is₆And R₈Independently different or the same and selected from H, alkyl, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl or formula (II) or formula (VIII),

R₁₁and R₁₂Independently different or the same, and is selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, propionyl, benzoyl, -CH₂X alkyl, -CH₂X acyl, wherein X is O, NR₆Or the number of the S-beams is,

R_cand R_dIndependently different or the same, and is selected from CH₂X alkyl, CH₂X acyl, wherein X is O, NR₆Or the number of the S-beams is,

R₁₃、R₁₄and R₁₅Independently different or the same, and is selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, -COOH, O-acylRadical, O-alkyl, N-acyl, N-alkyl, X acyl, CH₂X is alkyl;

R₁₃and R₁₄Or R₁₃And R₁₅The substituents on (a) may be bridged to form a cyclic system, forming a cycloalkyl, heterocycloalkyl, lactone or lactam.

R_f、R_gAnd R_hIndependently different or the same, and is selected from X acyl, CH₂X alkyl, -CH₂X acyl and R_g，

The alkyl is selected from Me, Et, propyl, isopropyl, butyl, isobutyl and tert-butyl,

the acyl is selected from formyl, acetyl, propionyl, isopropionyl, butyryl, tert-butyryl, valeryl, benzoyl and succinyl.

The acyl and/or alkyl groups may be optionally substituted, and

when a point bond is present between A and B, the compounds according to formula (I) are

Wherein X₄Selected from-COOH, -C (═ O) XR₆，

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

and wherein

Z is selected from-CH₂-CH₂-or>CH(CH₃)，

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

B is selected from-O-R ', -NHR ", -SR'" or-OH; and R' is selected from the following formulae (II) to (IX):

r ', R "and R'" are independently different or the same and are selected from the following formulae (IV-VIII):

x is selected from O, NH, NR₆、S，

R₂Selected from Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, C (O) CH₃、C(O)CH₂C(O)CH₃、C(O)CH₂CH(OH)CH₃，

p is an integer and is 1 or 2,

R₆selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl or formula (II) or formula (VIII),

X₅selected from-H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, -COOH, -C (═ O) XR₆、CONR₁R₃Or a compound of the formula,

X₇is selected from R₁、-NR₁R₃，

R₉Selected from H, Me, Et or O₂CCH₂CH₂COXR₈，

R₁₀Selected from the group consisting of Oacyl, NH alkyl, NH acyl or O₂CCH₂CH₂COX₆R₈，

R₁₁and R₁₂Independently different or the same, and is selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, propionyl, benzoyl, -CH₂X alkyl, CH₂X acyl, wherein X is O, NR₆Or the number of the S-beams is,

R_cand R_dIndependently different or the same, and is selected from CH₂X alkyl, CH₂X acyl, wherein X is O, NR₆Or the number of the S-beams is,

R₁₃and R₁₄Or R₁₃And R₁₅The substituents on (a) may be bridged to form a cyclic system, forming a cycloalkyl, heterocycloalkyl, lactone or lactam.

R_f、R_gAnd R_hIndependently different or the same, and is selected from X acyl, -CH₂X alkyl, -CH₂X is an acyl group and Rg,

the alkyl is selected from Me, Et, propyl, isopropyl, butyl, isobutyl and tert-butyl,

the acyl is selected from formyl, acetyl, propionyl, isopropionyl, butyryl, tert-butyryl, valeryl, benzoyl and succinyl.

The acyl and/or alkyl groups may be optionally substituted, and

when a point bond is present between A and B, the compounds according to formula (I) are

Wherein X₄Selected from-COOH, -C (═ O) XR₆，

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

and wherein

Z is selected from-CH₂-CH₂-or>CH(CH₃)，

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 following formulae (II), (V) or (IX):

and neither A nor B is-OH,

r ', R "and R'" are independently different or the same and are selected from the following formulae (VII-VIII):

X is selected from O, NH, NR₆、S，

R₂Selected from Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, C (O) CH₃、C(O)CH₂C(O)CH₃、C(O)CH₂CH(OH)CH₃，

p is an integer and is 1 or 2,

R₆selected from H, alkyl, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl or formula (II) or formula (VIII),

X₅selected from-H, -COOH, -C (═ O) XR₆、CONR₁R₃Or one of the following formulae

R9 is selected from H, Me, Et or O₂CCH₂CH₂COXR₈，

R10 is selected from the group consisting of Oacyl, NH alkyl, NH acyl, or O₂CCH₂CH₂COX₆R₈，

X₆Is O or NR₈And R is₈Selected from H, alkyl, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, acetyl, acyl, propionyl, benzoyl, succinyl or formula (II) or formula (VIII),

ris, RM and RI5 are independently different or the same and are selected from H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, -COOH, O-acyl, O-alkyl, N-acyl, N-alkyl, Xacyl, CH₂X alkyl, R_cAnd R_dIndependently is CH₂X alkyl, CH₂X acyl, wherein X is O, NR₆Or S, and alkyl is, for example, H, Me, Et, propyl, isopropyl, butyl, isobutyl, tert-butyl, and acyl is, for example, formyl, acetyl, propionyl, isopropionyl, butyryl, tert-butyryl, valeryl, benzoyl, succinyl, and the like,

R_f、R_gand R_hIndependently selected from X acyl, -CH₂X alkyl, -CH₂The acyl group X and the group R9,

R₂₀and R₂₁Independently different or the same, and is selected from H, lower alkyl, i.e. C₁-C₄Alkyl, or R₂₀And R₂₁Together may form C₄-C₇Cycloalkyl or aromatic, both of which may be optionally substituted by halogen, hydroxy or lower alkyl, or

R₂₀And R₂₁Can be

CH₂X-acyl radical F, CH₂COOH、CH₂CO₂Alkyl radical, and

when a ring bond is present between A and B, the compound is

And the acyl and alkyl groups may be optionally substituted.

A compound of the formula (A),

wherein R is neutralized₂Identical or different and selected from the formula (B)

And wherein R₃Selected from H or optionally substituted C_1-C₃Alkyl, such as, for example, methyl, ethyl, propyl or isopropyl, and wherein R₅is-OC (═ O) R_aWherein R is_aIs methyl or formula (C)

A compound of formula (XX),

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

Wherein R is₃And R₄Independently is H, optionally substituted C₁-C₃Alkyl, or are linked together to form a ring, and wherein R₅Is connected to R₁To form a ring, or R₅Selected from OCOR_a、OCOOR_b、OCONR_cR_d、SO₂R_e、OPO(OR_f)(OR_g) Or CONR_cR_dWherein R is_aIs optionally substituted alkyl or optionally substituted cycloalkyl, R_bIs optionally substituted alkyl, R_cAnd R_dIndependently is H, optionally substituted alkyl or is linked together to form a ring which may contain one or more additional heteroatoms, R_eIs optionally substituted alkyl, R_fAnd R_gIndependently H, methyl, ethyl or are linked together to form a ring, and wherein the compound is not bis (2, 2-dimethylpropionyloxymethyl) succinate; dibutyryloxymethyl succinate; or bis- (l-butyryloxy-ethyl) succinate.

The invention also provides therapeutic compositions comprising citrate, citric acid or a prodrug, analog or derivative of citrate or citric acid as a component of a combination therapy which also includes compositions comprising a TCA cycle intermediate or prodrug, analog or derivative thereof. Compositions containing 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 present invention may contain a buffering agent. Any suitable buffer may be used. The buffer may be an amino acid or a derivative thereof. For example, the amino acid can 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 buffer may be or may contain metal ions. For example, the metal ion may be Na⁺、K⁺、Ca²⁺、Mg²⁺Or Cu² ⁺。

The buffering agent may maintain the pH of the composition at or near neutral. For example, but not limited to, the buffering agent may maintain the pH of the composition at about 3.0 to about 10.0, about 3.0 to about 9.0, about 3.0 to about 8.0, about 3.0 to about 7.0, about 3.0 to about 6.0, about 4.0 to about 10.0, about 4.0 to about 9.0, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 5.0 to about 10.0, about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 10.0, about 6.0 to about 8.0, about 7.0 to about 10.0, about 7.0 to about 9.0, or about 8.0 to about 10.0.

The formulations of the present invention may contain an aqueous suspension of one or more TCA cycle intermediates or prodrugs, analogs, or derivatives thereof. Aqueous suspensions may contain the TCA cycle intermediates 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 naturally occurring phosphatides, 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 (heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as condensation products of polyethylene oxide 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 (e.g. arachis oil, olive oil, sesame oil or coconut oil) or 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 antioxidant such as ascorbic acid.

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

The compositions of the present 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 thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

The compositions of the present invention may include other pharmaceutically acceptable carriers such as sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gum 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, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol (glycerin), erythritol, xylitol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible materials used in pharmaceutical formulations.

Compositions containing TCA cycle intermediates can be in a form suitable for oral use. For example, oral formulations may include tablets, troches, lozenges, fast-melt agents (fast-melt), aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Compositions for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring 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, such as 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 down 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. Pat. nos. 4,256,108, 4,166,452 and 4,265,874 to form osmotic therapeutic tablets for controlled release.

Formulations for oral use may also be presented as hard gelatin capsules wherein 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 in the form of soft gelatin capsules wherein the compound is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

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

Syrups and elixirs for oral administration of citrate, citric acid or a citrate or prodrug, analogue or derivative of 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 an agent for flavoring and/or coloring. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. Such suspensions 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 a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 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 diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The methods of the present invention include compositions provided as intraocular formulations. Intraocular formulations include any formulation suitable for delivery of a medicament to the eye. For example, but not limited to, intraocular formulations include aqueous gels, contact lenses, dendrimers, emulsions, eye drops, implants, in situ thermosensitive gels, liposomes, microneedles, nanomicelles, nanoparticles, nanosuspensions, ointments, and suspensions. Ophthalmic formulations are known in the art and are described, for example, in Patel, a. et al, ophthalmic drug delivery systems: overview, World journal of pharmacology (World J Pharmacol.) 2013; 47-64 of Doi:10.5497/wjp.v2.i 2.47; 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 present invention also provides a therapeutic composition comprising: a non-salt form of citrate, citric acid or a citrate salt or a prodrug, analog or derivative of citric acid; infant nutritional sources, such as infant formula or human breast milk; and a buffering agent. The composition is suitable for oral administration to human infants and provides basic nutrition and a supplemental source of citrate or citric acid.

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

The nutritional source may be designed to meet the dietary needs of infants suffering from metabolic disorders such as PA or MMA. For example, the source of protein or amino acids can be substantially free of valine, isoleucine, threonine, and methionine. The fat source may be free of odd-chain fatty acids. The nutrient source may be supplemented with carnitine or antibiotics. The nutrient source may have a reduced protein and/or amino acid content compared to recommended values for healthy infants, or it may be substantially free of protein and/or amino acids.

The buffer in the composition comprising citrate or a non-salt form of citric acid and a nutrient source may comprise any buffer, such as those described above. Preferably, the buffer is an amino acid, such as lysine or ornithine. The buffer may be or may contain metal ions.

The compositions of the present invention may contain one or more antibiotics.

The present invention also provides formulations that allow for oral delivery of citrate in sufficient amounts to treat diseases, disorders, and conditions associated with altered TCA cycle metabolism without providing an excess of any minerals or metal ions. The formulation contains citrate in an amount such that when the formulation is administered to a human, the cationic component of each salt provided to the human does not exceed the recommended daily amount of that component. In a preferred embodiment, the formulation contains an amount of each citrate salt that does not exceed the recommended daily dosage of the cation in the citrate salt. Alternatively or additionally, the formulation may comprise a formulation wherein the amount of each citrate salt released into the subject does not exceed the recommended daily amount of cation in the 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 amino acids (as active agents, buffers, or both).

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

The amino acid may be any amino acid, such as any of the amino acids described above. Preferably, the amino acid is lysine, arginine or ornithine.

Any suitable citrate or combination of citrates may be used in the formulation. The salt may contain sodium, lithium, potassium, calcium, magnesium or manganese. The salt may contain a citrate salt in its mono-, di-or tri-basic state. Preferably, the salt is monosodium citrate or monopotassium citrate.

Preferred formulations comprise a combination of citric acid and a salt of citric acid, such as a combination of monosodium citrate and monopotassium citrate. Such combinations allow for the delivery of large amounts of citrate in various ionization states without providing an excess of any individual metal ion.

The recommended daily amount of salt or mineral may be any amount determined to be suitable for one person to ingest a day. The recommended daily amount may be the minimum amount that should be ingested, the maximum amount that should not be exceeded, or a value or range of values between the two. The recommended daily amount may correspond to standards known in the art, such as standards promulgated by private, governmental, medical, or health authorities. For example, but not limited to, the standard may be an Acceptable Daily Intake (ADI) issued by Food and Agriculture organizations and World Health organizations (Food and agricultural Organization and the World Health Organization); a Daily Intake Guide (DIG) issued by the Australian Food and Grocery Council (Australian Food and Grocery Council); daily Value (DV) and Reference Daily Intake (RDI) issued by the Health Canada (Health Canada); daily numerical values (DV) or Reference Daily Intake (RDI) issued by the U.S. Food and Drug Administration (FDA); dietary Reference Intake (DRI), estimated average demand (EAR), or recommended diet/daily allowance (RDA) issued by the Institute of Medicine (IOM) of the National academy of sciences in the United States); a Dietary Reference Value (DRV), a Lower Reference Nutrient Intake (LRNI) or a Reference Nutrient Intake (RNI) issued by the british ministry of health; a Dietary Reference Value (DRV), a Lower Reference Nutrient Intake (LRNI) or a Reference Nutrient Intake (RNI) issued by the european union european food safety agency; a daily guide amount (GDA) issued by the food and Grocery Distribution Institute (Institute of Grocery Distribution); or Appropriate Intake (AI) as issued by the national institutes of health.

The recommended daily amount may take into account conditions associated with the individual, such as age, sex, weight, pregnancy status, lactation status, or menopausal status. For example, for pregnant and/or lactating women, the recommended daily amount of several minerals such as calcium, iron and potassium is higher than for non-pregnant non-lactating women. Other patient populations in which the mineral demand may vary include children (i.e., pediatric patients), postmenopausal women, and the elderly.

For example, but not limiting of, the recommended daily amount of a particular mineral may be as follows: calcium in an amount of about 700mg, about 800mg, about 1000mg, about 1200mg, about 1300mg, about 1500mg, about 2000mg, or about 2500 mg; about 0.2mg, about 0.22mg, about 0.34mg, about 0.44mg, about 0.7mg, about 0.89mg, about 0.9mg, about 1mg, about 1.3mg, about 2mg, about 4mg, about 6mg, about 8mg, or about 10mg of copper; about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 11mg, about 13mg, about 16mg, about 18mg, about 24mg, about 27mg, about 30mg, about 36mg, about 40mg, or about 45mg of iron; about 80mg, about 150mg, about 200mg, about 250mg, about 310mg, about 320mg, about 350mg, about 360mg, about 400mg, or about 420mg of magnesium; about 1.2mg, about 1.5mg, about 1.6mg, about 1.8mg, about 1.9mg, about 2mg, about 2.2mg, about 2.3mg, about 2.6mg, about 3mg, about 4mg, about 6mg, about 8mg, about 10mg, or about 11mg of manganese; about 3000mg, about 3500mg, about 3800mg, about 4000mg, about 4500mg, about 4700mg, or about 5100mg of potassium; about 1500mg, about 2000mg, about 2300mg, or about 2400mg, about 3000mg, or about 3400mg of sodium; and about 8mg, about 9.4mg, about 11mg, about 12mg, about 13mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, or about 40mg of zinc. The formulation may contain one or more sugars to improve flavor when the formulation is provided orally to a subject. For example, but not limited to, the sugar may be sucrose, fructose, galactose, maltose, or lactose.

Methods of treating metabolic disorders

The present 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 composition may be administered by any non-oral route. For example, but not limited to, the composition may be administered subcutaneously, intravenously, intraarterially, intramuscularly, intradermally, or rectally. Preferably, the composition is administered subcutaneously or intravenously.

A composition formulated for non-oral administration containing a TCA cycle intermediate or a prodrug, analog or derivative thereof may be any of the above, such as citrate, aconitate, D-isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, oxaloacetate, acetone, acetoacetate, beta-hydroxybutyrate, beta-ketovalerate or beta-hydroxyvalerate. 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 which may be ameliorated by providing intermediates of the TCA cycle, such as any of those described above.

The TCA cycle intermediate or prodrug, analog or derivative thereof may be provided in any therapeutically effective dose. For example, but not limited to, TCA cycle intermediates or prodrugs, analogs or derivatives thereof can be provided in the following amounts: about 0.1mg/kg subject weight to about 5g/kg subject weight, about 0.2mg/kg subject weight to about 5g/kg subject weight, about 0.5mg/kg subject weight to about 5g/kg subject weight, about 1mg/kg subject weight to about 5g/kg subject weight, about 2mg/kg subject weight to about 5g/kg subject weight, about 5mg/kg subject weight to about 5g/kg subject weight, about 0.1mg/kg subject weight to about 2g/kg subject weight, about 0.2mg/kg subject weight to about 2g/kg subject weight, about 0.5mg/kg subject weight to about 2g/kg subject weight, about 1mg/kg subject weight to about 2g/kg subject weight, From about 2mg/kg subject weight to about 2g/kg subject weight, from about 5mg/kg subject weight to about 2g/kg subject weight, from about 0.1mg/kg subject weight to about 1g/kg subject weight, from about 0.2mg/kg subject weight to about 1g/kg subject weight, from about 0.5mg/kg subject weight to about 1g/kg subject weight, from about 1mg/kg subject weight to about 1g/kg subject weight, from about 2mg/kg subject weight to about 1g/kg subject weight, from about 5mg/kg subject weight to about 1g/kg subject weight, from about 1mg/kg subject weight to about 500mg/kg subject weight, from about 2mg/kg subject weight to about 200mg/kg subject weight, or from about 5mg/kg subject weight to about 100mg/kg subject weight.

The composition may be provided according to any suitable schedule. For example, but not limited to, the composition may be provided in a 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 may be provided independently once, twice, three times, four times or more per day.

The composition may be provided over a period of time. For example, but not limited to, the composition can 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. The limits of one or more time periods may be defined by the age of the patient. For example, but not limited to, the period of time for providing the composition can begin or end at the time of birth, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, 18 months, or 24 months of the patient.

The present invention provides combination therapies for treating metabolic disorders, diseases or conditions. The combination therapy includes providing a non-oral formulation comprising a succinate salt or prodrug, analog or derivative thereof and another formulation comprising a citrate salt, citric acid or prodrug, analog or derivative thereof. The non-oral formulation may be administered by any non-oral route. For example, but not limited to, non-oral formulations can 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 citrate or a prodrug, analog or derivative of citric acid may be administered by any route, such as oral, enteral, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intradermal or rectal administration. Preferably, the formulation containing citrate, citric acid or a citrate or prodrug, analog or derivative of 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 which may be ameliorated by providing TCA cycle intermediates, such as any of those described above.

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

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

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

As noted above, in the combination therapy of the present invention, each formulation may be provided independently over a period of time.

In the combination therapies of the present invention, the non-oral formulation comprising a succinate salt or prodrug, analog or derivative thereof and the other formulation comprising a citrate salt, citric acid or prodrug, analog or derivative of citric acid may be provided at different periods, at the same delay or at overlapping periods. Thus, the combination therapy may include a transition from the period of providing one formulation to the period of providing another formulation. The transitions may be discrete, or they may contain periods where both formulations are provided. The transition may be gradual, and may include a decrease in the dosage of the first formulation and an increase in the dosage of the second formulation.

Examples of the invention

Example 1: subcutaneous administration of succinate salt

Summary of the invention

Subcutaneous (SC) administration in male Sprague-Dawley rats¹³C₄After succinate, its bioavailability was determined.¹³C₄Succinate is administered subcutaneously at 10mg/kg, 50mg/kg and 100 mg/kg. Blood samples were taken within 8 hours after dosing and plasma concentrations of the test products were determined by LC-MS/MS. Pharmacokinetic parameters were determined using Phoenix WinNonlin (v8.0) software.

At SC 10mg/kg¹³C₄After succinate, the maximum plasma concentration (mean 3480 ± 1512ng/mL) was observed 15 min after dosing. The mean half-life after subcutaneous administration cannot be determined; however, the half-life of one rat was 0.453 hours. AUC based on dose normalization_lastThe mean exposure of (d) was 160 ± 55.4h kg ng/mL/mg. Based on the IV data from the previous study,¹³C₄-the average bioavailability of succinate at 10mg/kg is 74.4 ± 25.7%.

At SC 50mg/kg¹³C₄After succinic acid, the maximum plasma concentration was observed 15 min after administration (mean 17333 ± 2214 ng/mL). The mean half-life after subcutaneous administration cannot be determined because of the correlation coefficient (r)²) Less than 0.85. AUC based on dose normalization_lastAverage exposure of 210. + -. 41.0h kg ng/mL/mg. Based on the IV data from the previous study,¹³C₄the mean bioavailability of succinate at 50mg/kg was 97.6 ± 19.0%.

At SC 100mg/kg¹³C₄Maximum plasma concentrations (mean 31000 ± 7451ng/mL) were observed 15 min after administration, after succinate. The mean half-life after subcutaneous administration was 1.07 hours. AUC based on dose normalization_lastThe mean exposure of (d) was 216 ± 52.9h kg ng/mL/mg. Based on the IV data from the previous study,¹³C₄-the average bioavailability of succinate at 100mg/kg is 100 ± 24.6%.

Administered subcutaneously at 10mg/kg, 50mg/kg and 100mg/kg¹³C₄After succinic acid, the exposure increased non-linearly with increasing dose. The exposure of the higher dose is proportional to the dose, but greater than the dose proportional to the low dose exposure.

Observations and adverse reactions

Subcutaneous administration in male Sprague-Dawley rats¹³C₄After succinate, no adverse effects were observed.

Analysis of dosing solutions

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

TABLE 1 measured dosing solution concentration (mg/mL)

Quantitative plasma sample analysis

Plasma samples were extracted and analyzed using the methods described below in the sample extraction section. Individual and mean plasma concentrations are shown in tables 2 to 4. All data are expressed as ng/mL of free base. Samples below the limit of quantitation were not used for the calculation of the mean. The concentration versus time data is plotted in fig. 1-6.

Data analysis

Pharmacokinetic parameters were calculated from the time course of plasma concentrations and are presented in tables 2 to 4. Pharmacokinetic parameters were determined by Phoenix WinNonlin (v8.0) software using a non-compartmental model. Maximum plasma concentration (C) following SC administration was observed according to the data_max) And the time to reach maximum plasma concentration (t)_max). The area under the time-concentration curve (AUC) was calculated using the linear trapezoidal rule, where to the last quantifiable data point was calculated, and where, if applicable, extrapolated to infinity. Plasma half-life (t)_1/2) From the slope calculation of the 0.693/end elimination phase. The mean residence time MRT is calculated by dividing the area under the moment curve (AUMC) by AUC. Bioavailability PO AUC normalized by single dose from study 18CARNP11_lastValue divided by mean dose normalized IV AUC_lastA value is determined. For pharmacokinetic data analysis, any sample below the limit of quantitation (1.00ng/mL) was considered zero.

TABLE 2 subcutaneous administration of 10mg/kg in male Sprague-Dawley rats¹³C₄Post succinate individual and mean plasma concentrations (ng/mL)

C_max: maximum plasma concentration; t is t_max: time of maximum plasma concentration; t is t_1/2: half-life, data points for half-life determination, shown in bold; MRT_last: average residence time, calculated to the last observable time point; AUC_last: calculating the area under the curve 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).

¹Dose normalization by dividing the parameter by the nominal dose (in mg/kg).

²AUC by normalizing individual oral doses_lastValues divided by AUC from mean IV dose normalization from previous studies_lastValue 215h kg ng/mL/mg determined bioavailability.

³Is undetermined because the line defining the endpoint elimination phase has r²<0.85。

FIG. 1 shows subcutaneous administration of 10mg/kg¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 2 is a 10mg/kg subcutaneous administration¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

TABLE 3 subcutaneous administration of 50mg/kg in male Sprague-Dawley rats¹³C₄Post succinate individual and mean plasma concentrations (ng/mL)

¹By dividing the parameter by the nominal dose (in m)g/kg) was performed.

²AUC by normalizing individual oral doses_lastValues divided by AUC from mean IV dose normalization from previous studies_lastValue 215h kg ng/mL/mg determined bioavailability.

³Is undetermined because the line defining the endpoint elimination phase has r²<0.85。

FIG. 4 is a drawing showing subcutaneous administration of 50mg/kg¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

TABLE 4 subcutaneous administration of 100mg/kg in male Sprague-Dawley rats¹³C₄Post succinate individual and mean plasma concentrations (ng/mL)

¹Dose normalization by dividing the parameter by the nominal dose (in mg/kg).

²AUC by normalizing individual oral doses_lastValues divided by AUC from mean IV dose normalization from previous studies_lastValue 215h kg ng/mL/mg determined bioavailability.

³Is undetermined because the line defining the endpoint elimination phase has r²<0.85。

FIG. 5 is a subcutaneous administration of 100mg/kg¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 6 is a subcutaneous administration of 100mg/kg¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

Dosing solution preparation and animal data sheet

Bill of materials

Device inventory
		Name (R)	ASI/other ID numbering
Analytical balance	AS265
		10ml	AS1007

Animal administration and sample collection and handling

Food was withdrawn from the animals overnight before dosing until four hours after dosing.

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

The balance used was: AS-1273

Review/observation results:

all animals showed normal activity during the experiment-CS, EE, 8/21/18, CS 8/21/18

Animal administration and sample collection and handling

Food was withdrawn from the animals overnight before dosing until four hours after dosing.

The balance used was: AS-1273

Review/observation results:

all animals showed normal activity-CS 8/21/18 during the study period

Animal administration and sample collection and handling

Food was withdrawn from the animals overnight before dosing until four hours after dosing.

The balance used was: AS-1273

Review/observation results:

all animals showed normal activity-CS 8/21/18 during the study period

Analytical stock solution preparation

Assay stock solutions (1.00mg/mL free drug) were prepared in DMSO.

Standard article

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

Sample extraction

Plasma samples were extracted manually in 96-well plates using acetonitrile, as outlined in table 5.

TABLE 5 preparation of plasma samples

HPLC conditions

The instrument comprises the following steps: waters Acquity

Column: waters HSS T3, 50X 2.1mm id,1.8 μm

Aqueous reservoir (a): 0.1% aqueous formic acid solution

Organic layer (B): 0.1% formic acid in acetonitrile

Gradient planning:

time (minutes)	Gradient curve	％A	％B
				0.00	6	99.9	0.1
0.75	6	0.1	99.9
				0.80	6	99.9	0.1
1.00	6	99.9	0.1

Flow rate: 800. mu.L/min

Injection volume: 10 μ L

Operating time: 1.0 minute

Column temperature: 40 deg.C

Sample temperature: 8 deg.C

Cleaning a strong autosampler: 0.2% formic acid in 1:1: l (v: v: v) water methanol isopropanol

And (3) cleaning a weak autosampler: 4mM ammonium formate

Conditions of the Mass spectrometer

The instrument comprises the following steps: PE Sciex API4000

Interface: electrospray ("turbine ion spray")

Mode (2): multiple Reaction Monitoring (MRM)

Gas: CUR 30, CAD 10, GS 150, GS 250

Source temperature: 500 deg.C

Monitored voltage and ions:

IS: an ion spray voltage; DP: declustering potential (declustering potential); EP: an inlet voltage; CE: collision energy; CXP: a collision cell exit voltage; all settings are in volts

Example 2: effect of citrate and succinate on basal respiration

Fig. 7 is a graph showing the effect of citrate and succinate on basal respiration in cells of a patient suffering from Propionemia (PA). Fibroblasts from normal patients (sample 826) and fibroblasts from patients with PA were cultured in medium alone, medium supplemented with 0.2mM citrate, or medium supplemented with 4mM succinate for 72 hours, as indicated. Oxygen Consumption Rate (OCR) was measured using an XFe96 extracellular flux analyzer (Seahorse Bioscience). Data shown are mean ± SD and normalized to the mean protein amount. In GraphPad Prism 7, unpaired t-tests were used on samples from single biological and 8 technical replicates to calculate P <0.0001, P <0.01 compared between the groups shown.

Example 3: pharmacokinetics, excretion and tissue distribution of orally administered citrate or intravenously administered succinate

Summary of the invention

In rats a single oral administration was analyzed¹⁴C-labelled citrate or single oral administration¹⁴Plasma time course after C-labeled succinate, route and rate of excretion, and tissue distribution of radioactivity.

Dosage formulations and preparation

Will be provided with¹⁴C-succinate salt and¹⁴the C-citrate salt is dissolved in deionized water (for oral administration) or 0.9% sodium chloride (for intravenous injection). The formulations were prepared the day prior to administration.

Test system

Rats were from Charles River Laboratories. At the beginning of the study, animals were 9-11 weeks of age.

Design of research

Treatment groups are shown in table 6.

TABLE 6 treatment groups

Oral administration¹⁴Animals with C-citrate remained in a fasting state for 8 hours prior to dosing and for 4 hours after dosing.¹⁴The C-citrate is administered by oral feeding. Intravenous administration¹⁴Animals with C-succinate did not maintain a fasted state.¹⁴The C-succinate salt was administered by tail vein injection.

Sample collection and analysis

After oral administration, the tube and site of administration were swabbed with gauze, which was collected for subsequent radioactivity analysis. After IV administration, the needle and the site of administration were swabbed with gauze, which was collected for subsequent radioactivity analysis. The total radioactivity of the site wipe was subtracted from the total dose administered according to SOP ADME to determine the dose loss.

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

Urine and feces were collected 0-24 hours after dosing and stored on wet ice. Expired CO was collected by drawing air through a trap containing 2N NaOH at 0-8 hours and 8-24 hours post-dosing according to MPI Research SOP₂. The samples were stored at ambient temperature and weighed. Aliquots were submitted for radioactivity analysis.

At 24 hours post-dosing, the cages were rinsed with a 1% trisodium phosphate (TSP) solution and wiped with a gauze pad. The final cage wash solution samples and cage wipe samples were collected in separate suitable containers and the weight of each cage wash solution and cage wipe was recorded.

The samples were subjected to radioactivity analysis 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 collection, euthanized animals were frozen in a hexane/dry ice bath and embedded in 5% carboxymethyl cellulose matrix according to MPI Research SOP IMG-23.

Sections of embedded cadavers were sectioned on a Leica CM3600 cyromotome set to maintain-10 ℃ to-30 ℃ according to MPI Research SOP EQP-145. Slices of approximately 30 μm thickness were taken on the sagittal plane. Appropriate slices selected at different levels of interest in the block were acquired, where possible, to cover as much as possible the following tissues, organs and biological fluids: adrenal gland, bladder and contents (urine), blood (heart), bone marrow, brain, epididymis, eye, fat (brown), fat (white), heart, kidney, large/cecum and contents, liver, lung, muscle, pancreas, prostate/uterus, salivary glands, skin, small intestine and contents, spleen, stomach and contents, testis/ovary, thymus, thyroid, and mesenteric lymph nodes. Sections were mounted using a Storm 860 image acquisition system, exposed to a fluorescence imaging screen, and scanned at 50 μm according to MPI Research SOP EQP-146.

Use of MCID in accordance with MPI Research SOP IMG-24^TMImage analysis software quantitated by image densitometry against calibration standards. According to the integrated response (MDC/mm)²) And 2¹⁴C]Calibration standards nominal concentrations standard curves were constructed. The concentration of radioactivity is expressed in μ Ci/g, and the administered [ 2 ]¹⁴C]The specific activity of the dosage formulation of the test article is converted to [ 2 ] per gram of the sample¹⁴C]The articles were tested for μ g or ng equivalents (ng-eq/g or μ g-eq/g). A lower quantitative limit (LLOQ) was applied to the data. LLOQ was determined using the radioactive concentration of the lowest calibration standard used to generate the calibration curve divided by the specific activity of the dose formulation (μ Ci/mg). During image analysis, artifacts (such as those resulting from displacement of gut contents) are excluded from the analysis.

Samples were stored as shown in table 7.

TABLE 7 sample storage conditions

Results

In Table 8 is shown the results obtained from single dose oral administration¹⁴C-citrate or single dose intravenous¹⁴Percentage of radioactivity recovered in various samples from animals sacrificed 24 hours after C-succinate treatment.

TABLE 8 percent recovery of radioactive dose

In Table 9 is shown the oral administration in a single dose¹⁴C-citrate or single dose intravenous¹⁴Plasma concentration of the radioactive material in C-succinate treated rats.

TABLE 9 plasma concentration of radioactive substances

The veins are shown in Table 10 in a single dose¹⁴Radioactivity concentration in blood and tissues in C-succinate treated rats.

TABLE 10 in use IV¹⁴Radioactivity in tissues of C-succinate treated rats

BLQ: below the limit of quantitation (<320ng equivalent test article/g).

ND: undetectable (no shape of the sample can be discerned from the background or surrounding tissue).

NR: not shown (no tissue is shown on the section).

In Table 11 is shown the oral administration of a single dose¹⁴Radioactivity concentration in blood and tissues in C-citrate treated rats.

TABLE 11 oral administration¹⁴Radioactivity in tissues of C-citrate treated rats

BLQ: below the limit of quantitation (<320ng equivalent test article/g).

ND: undetectable (no shape of the sample can be discerned from the background or surrounding tissue).

NR: not shown (no tissue is shown on the section).

Example 4: oral bioavailability of succinate salts

Summary of the invention

Evaluation in male Sprague-Dawley rats¹³C₄-oral bioavailability of succinate.¹³C₄Succinate is administered by Intravenous (IV) and oral (PO) administration routes at doses of 10mg/kg and 50mg/kg, respectively. Blood samples were taken within 8 hours after dosing and plasma concentrations of the test products were determined by LC-MS/MS. Pharmacokinetic parameters were determined using Phoenix WinNonlin (v8.0) software.

At IV 10mg/kg¹³C₄After succinate, the mean half-life is 2.46. + -. 2.04 hours. The mean clearance was 4.61. + -. 0.341L/h/kg. The average distribution volume was 0.982. + -. 0.584L/kg. 50mg/kg in PO¹³C₄Maximum plasma concentrations (average 211 ± 186ng/mL) were observed 15 min after administration, after succinate. The mean half-life after oral administration cannot be determined because of the correlation coefficient (r)²) Less than 0.85, or due to lack of following C_maxCan be used to quantify the data points. AUC based on dose normalization_lastThe mean exposure of (a) was 1.84 ± 1.58h kg ng/mL/mg. After administration at 50mg/kg,¹³C₄the average oral bioavailability of succinate is 0.854 ± 0.735%.

Observations and adverse reactions

Intravenous and oral administration in male Sprague-Dawley ratsMedicine¹³C₄After succinate, no adverse effects were observed.

Analysis of dosing solutions

The dosing solution was analyzed by LC-MS/MS. The measured dosing solution concentrations are shown in table 12. Dosing solutions were diluted into rat plasma and analyzed in triplicate. All concentrations are expressed in mg/mL of free base. The nominal dosing level was used in all calculations.

TABLE 12 measured dosing solution concentration (mg/mL)

5% glucose solution

Quantitative plasma sample analysis

Plasma samples were extracted and analyzed using the methods described below in the sample extraction section. Individual and mean plasma concentrations are shown in tables 13 and 14. All data are expressed as ng/mL of free base. Samples below the limit of quantitation were not used for the calculation of the mean. The concentration versus time data are plotted in fig. 8-11.

Data analysis

Pharmacokinetic parameters were calculated from the time course of plasma concentrations and are presented in tables 13 and 14. Pharmacokinetic parameters were determined by Phoenix WinNonlin (v8.0) software using a non-compartmental model. Maximum plasma concentration (C) after IV administration₀) The estimation is done by extrapolating the first two time points back to t-0. Data were used to observe maximum plasma concentration (C) following PO administration_max) And the time to maximum plasma concentration (T)_max). The area under the time concentration curve (AUC) was calculated using the linear trapezoidal rule, where to the last quantifiable data point was calculated, and where, if applicable, extrapolated to infinity. Plasma half-life (t)_1/2) From the slope calculation of the 0.693/end elimination phase. The mean residence time MRT is calculated by dividing the area under the moment curve (AUMC) by AUC. Clearance (CL) was calculated as dose/AUC. Volume of steady state distribution (V)_ss) Calculated from CL MRT (mean residence time).Bioavailability PO AUC by normalizing individual doses_lastValue divided by mean dose normalized IV AUC_lastA value is determined. For pharmacokinetic data analysis, any sample below the limit of quantitation (1ng/mL) was considered zero.

TABLE 13 intravenous administration of 10mg/kg in male Sprague-Dawley rats¹³C₄Post-succinate individual and mean plasma concentrations (ng/mL) and pharmacokinetic parameters

C₀: maximum plasma concentration extrapolated to t ═ 0; t is t_max: time of maximum plasma concentration; t is t_1/2: half-life, data points for half-life determination, shown in bold; MRTlast: average residence time, calculated to the last observable time point; CL: the clearance rate; v_ss: a steady state distribution volume; AUC_last: area under the curve, calculating 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).

¹Extrapolate to t-0.

²Dose normalization by dividing the parameter by the nominal dose (in mg/kg).

FIG. 8 is a graph of intravenous administration of 10mg/kg¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 9 is a graph of intravenous administration of 10mg/kg¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

TABLE 14 oral administration of 50mg/kg in male Sprague-Dawley rats¹³C₄Post succinate individual and mean plasma concentrations (ng/mL)

¹Dose normalization by dividing the parameter by the nominal dose (in mg/kg).

²By single oral AUC_lastValue divided by mean IV AUC_lastValue-determined bioavailability.

³Is undetermined because the line defining the endpoint elimination phase has r²<0.85。

⁴Not determined due to lack of follow C_maxCan be used to quantify the data points.

FIG. 10 shows the administration of 50mg/kg orally¹³C₄Succinate followed by different time points in individual rats¹³C₄-graph of plasma concentration of succinate.

FIG. 11 shows 50mg/kg of the drug administered orally¹³C₄After succinate at different time points in rats¹³C₄-graph of mean plasma concentration of succinate.

Dosing solution preparation and animal data sheet

Animal administration and sample collection and handling

Food was withdrawn from the animals overnight before dosing until four hours after dosing.

The balance used was: AS-1273

Review/observation results:

all animals showed normal behaviour during the experiment-CS 6/15/18

Animal administration and sample collection and handling

Food was withdrawn from the animals overnight before dosing until four hours after dosing.

The balance used was: AS-1273

Review/observation results:

all animals showed normal behaviour during the experiment-CS 6/15/18

Analytical stock solution preparation

Assay stock solutions (1.00mg/mL free drug) were prepared in DMSO.

Standard article

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

Sample extraction

Plasma samples were extracted manually in 96-well plates using acetonitrile, as outlined in table 15.

TABLE 15 preparation of plasma samples

HPLC conditions

The instrument comprises the following steps: waters Acquity

Column: waters HSS T3, 50X 2.1mm id,1.8 μm

Aqueous reservoir (a): 0.1% aqueous formic acid solution

Organic layer (B): 0.1% formic acid in acetonitrile

Gradient planning:

time (minutes)	Gradient curve	％A	％B
				0.00	6	99.9	0.1
0.75	6	0.1	99.9
				0.80	6	99.9	0.1
1.00	6	99.9	0.1

Flow rate: 800. mu.L/min

Injection volume: 10 μ L

Operating time: 1.0 minute

Column temperature: 40 deg.C

Sample temperature: 8 deg.C

Cleaning a strong autosampler: 0.2% formic acid in 1:1:1(v: v: v) water methanol isopropanol

And (3) cleaning a weak autosampler: 4mM ammonium formate

Conditions of the Mass spectrometer

The instrument comprises the following steps: PE Sciex API4000

Interface: electrospray ("turbine ion spray")

Mode (2): multiple Reaction Monitoring (MRM)

Gas: CUR 30, CAD 10, GS 150, GS 250

Source temperature: 500 deg.C

Monitored voltage and ions:

analyte	Polarity	Precursor ion	Product ion	IS	DP	EP	CE	CXP
									13C 4-succinate salt	Negative pole	121.1	76	-4500	-36	-10	-18	-10
Warfarin (internal standard)	Negative pole	307.1	250.0	-4500	-80	-10	-30	-17

IS: an ion spray voltage; DP: de-clustering voltage; EP: an inlet voltage; CE: collision energy; CXP: collision cell outlet potential; all settings are in volts

Example 5: oral bioavailability of citrate salts

Summary of the invention

Evaluation in male Sprague-Dawley rats¹³C₆-oral bioavailability of citrate.¹³C₆Citrate is administered by Intravenous (IV) and oral (PO) administration routes at 10mg/kg and 50mg/kg, respectively. Blood samples were taken within 8 hours after dosing and plasma concentrations of the test products were determined by LC-MS/MS. Pharmacokinetic parameters were determined using Phoenix WinNonlin (v8.0) software.

At IV 10mg/kg¹³C₆After citrate, the mean half-life is 2.34. + -. 0.207 h. The average clearance rate is 2.96 plus or minus 0.345L/h/kg. The average distribution volume was 1.75. + -. 0.223L/kg. 50mg/kg in PO¹³C₆After citrate, the maximum plasma concentration (mean 1580 ± 191ng/mL) was observed 15 min after administration. The mean half-life after oral administration cannot be determined; however, the half-life of one rat was 0.865 hours. The mean exposure of AUClast, normalized to dose, was 33.2 ± 10.9h kg ng/mL/mg. After administration at 50mg/kg,¹³C₆the mean oral bioavailability of citrate is 9.86 ± 3.24%.

Observations and adverse reactions

Oral administration in male Sprague-Dawley rats¹³C₆After citrate, no adverse effects were observed.

Analysis of dosing solutions

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

Table 16: measured dosing solution concentration (mg/mL)

5% glucose solution

Quantitative plasma sample analysis

Plasma samples were extracted and analyzed using the methods described below in the sample extraction section. Individual and mean plasma concentrations are shown in tables 17 and 18. All data are expressed as ng/mL of free base. Samples below the limit of quantitation were not used for the calculation of the mean. The concentration versus time data are plotted in fig. 12-15.

Data analysis

Pharmacokinetic parameters were calculated from the time course of plasma concentrations and are presented in tables 17 and 18. Pharmacokinetic parameters were determined by Phoenix WinNonlin (v8.0) software using a non-compartmental model. Maximum plasma concentration (C) after IV administration₀) The estimation is done by extrapolating the first two time points back to t-0. Data were used to observe maximum plasma concentration (C) following PO administration_max) And the time to maximum plasma concentration (T)_max). The area under the time concentration curve (AUC) was calculated using the linear trapezoidal rule, where to the last quantifiable data point was calculated, and where, if applicable, extrapolated to infinity. Plasma half-life (t)_1/2) From the slope calculation of the 0.693/end elimination phase. The mean residence time MRT is calculated by dividing the area under the moment curve (AUMC) by AUC. Clearance (CL) was calculated as dose/AUC. Volume of steady state distribution (V)_ss) Calculated from CL MRT (mean residence time). Bioavailability PO AUC by normalizing individual doses_lastValue divided by mean dose normalized IV AUC_lastA value is determined. For pharmacokinetic data analysis, any sample below the limit of quantitation (1ng/mL) was considered zero.

TABLE 17 in malesIntravenous administration of 10mg/kg in Sprague-Dawley rats¹³C₆Individual and mean plasma concentration after citrate (ng/mL) and pharmacokinetic parameters

C₀: maximum plasma concentration extrapolated to t ═ 0; t is t_max: time of maximum plasma concentration; t is t_1/2: half-life, data points for half-life determination, shown in bold; MRTlast: average residence time, calculated to the last observable time point; CL: the clearance rate; vss: a steady state distribution volume; AUC_last: area under the curve, calculating 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).

¹Extrapolate to t-0.

²Dose normalization by dividing the parameter by the nominal dose (in mg/kg).

FIG. 12 is a graph of intravenous administration of 10mg/kg¹³C₆Citrate followed by different time points in individual rats¹³C₆-graph of plasma concentration of citrate.

FIG. 13 is a graph of intravenous administration of 10mg/kg¹³C₆Citrate followed by different time points in rats¹³C₆-graph of mean plasma concentrations of citrate.

TABLE 18 oral administration of 50mg/kg in male Sprague-Dawley rats¹³C₆Post-citrate individual and mean plasma concentrations (ng/mL)

¹Dose normalization by dividing the parameter by the nominal dose (in mg/kg).

²By single oral AUC_lastValue divided by mean IV AUC_lastValue-determined bioavailability.

³Is undetermined because the line defining the endpoint elimination phase has r²<0.85。

FIG. 14 is a graph of oral administration of 50mg/kg¹³C₆Citrate followed by different time points in individual rats¹³C₆-graph of plasma concentration of citrate.

FIG. 15 shows 50mg/kg of the drug administered orally¹³C₆Citrate followed by different time points in rats¹³C₆-graph of mean plasma concentrations of citrate.

Dosing solution preparation and animal data sheet

Animal administration and sample collection and handling

Food was withdrawn from the animals overnight before dosing until four hours after dosing.

The balance used was: AS-1273

Review/observation results:

all animals showed normal behaviour during the experiment-CS 6/6/18

Animal administration and sample collection and handling

Food was withdrawn from the animals overnight before dosing until four hours after dosing.

The balance used was: AS-1273

Review/observation results:

all animals showed normal behaviour during the experiment-CS 6/6/18

Analytical stock solution preparation

Assay stock solutions (1.00mg/mL free drug) were prepared in DMSO.

Standard article

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

Sample extraction

Plasma samples were extracted manually in 96-well plates using acetonitrile, as outlined in table 19.

TABLE 19 preparation of plasma samples

HPLC conditions

The instrument comprises the following steps: waters Acquity

Column: acquityHSS T3,50×2.1mm id,1.8μm

Aqueous reservoir (a): 0.1% aqueous formic acid solution

Organic layer (B): 0.1% formic acid in acetonitrile

Gradient planning:

time (minutes)	Gradient curve	％A	％B
				0.00	6	99.0	0.1
0.75	6	0.1	99.9
				0.80	6	99.9	0.1
1.00	6	99.9	0.1

Flow rate: 800. mu.L/min

Injection volume: 10 μ L

Operating time: 1.0 minute

Column temperature: 40 deg.C

Sample temperature: 8 deg.C

Cleaning a strong autosampler: 0.2% formic acid in 1:1:1(v: v: v) water methanol isopropanol

And (3) cleaning a weak autosampler: 4mM ammonium formate

Conditions of the Mass spectrometer

The instrument comprises the following steps: PE Sciex API4000

Interface: electrospray ("turbine ion spray")

Mode (2): multiple Reaction Monitoring (MRM)

Gas: CUR 30, CAD 10, GS 150, GS 250

Source temperature: 500 deg.C

Monitored voltage and ions:

IS: an ion spray voltage; DP: de-clustering voltage; EP: an inlet voltage; CE: collision energy; CXP: collision cell outlet potential; all settings are in volts

Example 6: solubility of citric acid in lysine buffered solutions

The effect of lysine buffer on the solubility of citric acid at different pH values was analyzed. The results are shown in table 20.

TABLE 20 solubility of citric acid in lysine buffered solutions

Example 7: formulations containing citrate

Formulations containing citrate according to embodiments of the present invention are provided in table 21.

TABLE 21 formulations containing citrate

Is incorporated by reference

Throughout this disclosure, other documents, such as patents, patent applications, patent publications, periodicals, books, treatises, web content, have been cited and referenced. All such documents are hereby incorporated by reference herein in their entirety for all purposes.

Equivalents of

Various modifications of the invention, as well as many further embodiments thereof, in addition to those shown and described herein will become apparent to those skilled in the art from the entirety of this document, including reference to the scientific and patent literature cited herein. The subject matter herein contains important information, paradigms, and guidance that may be useful in practicing the invention in its various embodiments and its equivalents.

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