COMBINATION THERAPIES THAT INCLUDE AN AGENT THAT PROMOTES GLUCOSE OXIDATION AND AN INHIBITOR OF PYRUVATE DEHYDROGENASE KINASE

FIELD OF THE INVENTION

The invention relates to methods and compositions for treating conditions, such as diabetes, cancer, and cardiovascular conditions.

BACKGROUND

Millions of people suffer from conditions associated with defective mitochondrial metabolism. For example, abnormalities in mitochondrial metabolism lead to decreased cardiac efficiency in many forms of heart disease, are a causative factor in maternally inherited diabetes, and contribute to metastasis of certain types of cancer. Mitochondria produce the majority of high-energy molecules within a cell, and defects in mitochondrial metabolism result in reduced energy production. In some clinical manifestations, such as heart failure, ischemic heart disease, and diabetic cardiomyopathies, the reduction is attributable to the reliance of affected tissues on fatty acids rather glucose as a source of energy.

SUMMARY

Aspects of the invention recognize that glucose oxidation requires less oxygen than does fatty acid oxidation, so tissues that use the latter are more susceptible to damage when blood supply is diminished. Therefore, therapies that promote glucose oxidation are needed to treat or prevent a wide variety of diseases and conditions. Accordingly, an agent that shifts cellular metabolism from fatty acid oxidation to glucose oxidation, such as trimetazidine or an analog, derivative, or prodrug thereof, is beneficial for treating mitochondrial disorders.

The invention additionally recognizes that glucose catabolism includes sequential anaerobic (i.e., oxygen-independent) and aerobic (oxygen-requiring) processes. Consequently, the invention further recognizes that a single agent may not result in complete breakdown of glucose given the different aspects of glucose catabolism. It has been discovered that a combination therapy in which both anaerobic (i.e., oxygen-independent) and aerobic (oxygen-requiring) processes of glucose catabolism are addressed provides a highly efficient treatment for mitochondrial disorders. In that manner, the invention provides combination therapies, methods, and compositions, that direct cellular metabolism by providing a first agent that triggers cells to break down glucose rather than fatty acids and a second agent that drives the aerobic steps of glucose catabolism to achieve complete oxidation of glucose. Thus, the invention provides therapies that optimize energy production in a variety of pathological conditions.

In certain aspects, the invention provides combination therapies that include a compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and a pyruvate dehydrogenase kinase (PDK) inhibitor. Together, these agents address both the anaerobic and aerobic processes of glucose catabolism, thereby providing a new approach for treating mitochondrial disorders.

The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be trimetazidine, etomoxir, perhexiline, a PPAR agonist, a malonyl CoA decarboxylase inhibitor, dichloroacetate, or an analog, derivative, or prodrug of any of the aforementioned agents.

The compound that shifts cellular metabolism from fatty acid oxidation may be represented by formula (IV):

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in which:

R¹, R², and R³are independently selected from the group consisting of H and a (C₁-C₄)alkyl group;

R⁴and R⁵together are ═O, —O(CH₂)_mO—, or —(CH₂)_m—, wherein m=2-4, or R⁴is H and R⁵is OR¹⁴, SR¹⁴, or (CH₂CH₂O)_nH, wherein R¹⁴is H or a (C₁-C₄)alkyl group and n=1-15; and

R⁶is a single or multi-ring structure optionally substituted at one or more ring positions by a heteroatom, wherein each ring position optionally comprises one or more substituents.

One or more ring position of R⁶may be or include a substituent that includes a compound that promotes mitochondrial respiration, such as succinate, fumarate, malate, oxaloacetate, citrate, isocitrate, α-ketoglutarate, pyruvate, acetone, acetoacetic acid, β-hydroxybutpic acid, β-ketopentanoate, or β-hydroxypentanoate. The substituent may be or include a linker, such as (CH₂CH₂O)_x, in which x=1-15. The substituent may be or include a NAD⁺precursor molecule, such as nicotinic acid, nicotinamide, and nicotinamide riboside.

The substituent on a ring position of R⁶may be

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in which y=1-3.

The substituent on a ring position of R⁶may be

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in which y=1-3.

R⁶may be

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The compound of formula (IV) may have a structure represented by one of formulas (IX) and (X):

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The compound that shifts cellular metabolism from fatty acid oxidation may be represented by formula (V):

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in which R¹, R², and R³are independently H or a (C₁-C₄)alkyl group; R⁴and R⁸together are ═O, —O(CH₂)_mO—, or —(CH₂)_m—, in which m=2-4, or R⁴is H and R⁸is H, OR¹⁴, SR¹⁴, or (CH₂CH₂O)_nH, in which R¹⁴is H or a (C₁-C₄)alkyl group and n=1-15; R⁹, R¹⁰, R¹², and R¹³are independently H or (CH₂CH₂O)_zH, in which z=1-6; and R¹¹comprises a compound that promotes mitochondrial respiration.

The compound that promotes mitochondrial respiration may be an intermediate of the citric acid cycle or a molecule that can be metabolized to enter the citric acid cycle. For example, the compound may be succinate, fumarate, malate, oxaloacetate, citrate, isocitrate, α-ketoglutarate, pyruvate, acetone, acetoacetic acid, β-hydroxybutpic acid, β-ketopentanoate, or β-hydroxypentanoate.

R¹¹may include a linker, such as polyethylene glycol. For example, R¹¹may include (CH₂CH₂O)_x, in which x=1-15.

R¹¹may be

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in which y=1-3.

R¹¹may include a NAD⁺precursor molecule. For example, R¹¹may include nicotinic acid, nicotinamide, or nicotinamide riboside.

R¹¹may be

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in which y=1-3.

The compound that shifts cellular metabolism from fatty acid oxidation may be represented by formula (VI):

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in which:

at least one of positions A, B, C, D, E, and F is substituted with —(CH₂CH₂O)_nH and n=1-15.

The compound may have a substitution at position F. For example, the compound may be represented by formula (IX), as shown above.

The compound that shifts cellular metabolism from fatty acid oxidation may be represented by formula (VII):

$\begin{matrix} A - C, & (VIII) \end{matrix}$

in which A is a molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation, and C is a NAD⁺precursor molecule. A and C may be covalently linked.

The molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be trimetazidine, etomoxir, perhexiline, a PPAR agonist, a malonyl CoA decarboxylase inhibitor, or dichloroacetate.

The molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be PEGylated with an ethylene glycol moiety. The molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may have multiple ethylene glycol moieties, such as one, two three, four, five, or more ethylene glycol moieties. The ethylene glycol moiety may be represented by (CH₂CH₂O)_x, in which x=1-15. The ethylene glycol moiety may form a covalent linkage between the molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and the NAD⁺precursor molecule. The ethylene glycol moiety may be separate from a covalent linkage between the molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and the NAD⁺precursor molecule. The molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be a PEGylated form of trimetazidine.

The NAD⁺precursor molecule may be nicotinic acid, nicotinamide, or nicotinamide riboside.

The compound of formula (VII) may include nicotinic acid that is covalently linked to a PEGylated form of trimetazidine. The nicotinic acid may be covalently linked via the PEGylated moiety, i.e., via an ethylene glycol linkage. The nicotinic acid may be covalently linked via the trimetazidine moiety.

The compound of formula (VII) may have a structure represented by formula (X), as shown above.

The compound that shifts cellular metabolism from fatty acid oxidation may be represented by formula (VIII):

A-L-C (VIII),

in which A is a molecule that molecule that shifts metabolism from fatty acid oxidation to glucose oxidation, L is a linker, and C is a NAD⁺precursor molecule. A may be covalently linked to L, and L may be covalently linked to C.

The molecule that molecule that shifts metabolism from fatty acid oxidation to glucose oxidation, the linker, and the NAD⁺precursor molecule may be as described above in relation to compounds of other formulas.

The compound of formula (VIII) may have a structure represented by formula (X), as shown above.

The compound that shifts cellular metabolism from fatty acid oxidation may be represented by formula (I):

A-L-B (I),

in which A is a molecule that molecule that shifts metabolism from fatty acid oxidation to glucose oxidation, L is a linker, and B is a compound that promotes mitochondrial respiration.

The molecule that shifts metabolism from fatty acid oxidation to glucose oxidation may be trimetazidine, etomoxir, perhexiline, a PPAR agonist, a malonyl CoA decarboxylase inhibitor, or dichloroacetate.

The linker may be any suitable linker that can be cleaved in vivo. The linker may be an alkoxy group. The linker may be polyethylene glycol of any length. Preferably, the linker is represented by (CH₂CH₂O)_x, in which x=1-15.

The compound may include a NAD⁺precursor molecule covalently linked to another component of the compound. The NAD⁺precursor molecule may be nicotinic acid, nicotinamide, or nicotinamide riboside. The NAD⁺precursor molecule may be attached to the molecule that molecule that shifts metabolism, the compound that promotes mitochondrial respiration, or the linker. The NAD⁺precursor molecule may be attached to another component via an additional linker. Preferably, the NAD⁺precursor molecule is attached to the compound that promotes mitochondrial respiration via a 1,3-propanediol linkage.

The compound of formula (I) may be represented by formula (II):

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in which y=1-3.

The compound of formula (I) may be represented by formula (III):

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in which y=1-3.

Any of the compounds described above may include one or more atoms that are enriched for an isotope. For example, the compounds may have one or more hydrogen atoms replaced with deuterium or tritium. The isotopically enriched atom or atoms may be located at any position within the compound.

The PDK inhibitor may be (R)-3.3.3-trifluoro-2-hydroxy-2-methyl propionamide, 2-chloroproprionate, 4,5-diarylisoxazole, anilide tertiary carbinol, aromatic DCA derivative, betulinic acid, CPI-613, dichloroacetate (DCA), a DCA-loaded tertiary amine, a DCA-oxaliplatin derivative, a dihydrolipoamide mimetic, a furan carboxylic acid, a hemoglobin-DCA conjugate (e.g., fusion molecule of 1 Hgb:12 DCAs), honokiol DCA, an inositol ester (e.g., inositol hexa(N-methylnicotinate-dichloroacetate), an inositol ionic complex (e.g., tetra(dichloroacetyl) gluconate), M77976, mitaplatin, mito-DCA (e.g., fusion molecule of 1 triphenylphosphonium cation: 3 DCA), N-(2-aminoethyl)-2(3-chloro-4-((4-isopropylbenzyl)oxy)phenyl)acetamide, phenylbutyrate, pyruvate, a pyruvate analog containing a phosphinate or phosphonate group, radicicol, R-lipoic acid, a tetrahydroisoquinoline, a thiophene carboxylic acid, or VER-246608.

In another aspect, the invention provides methods of treating a condition in a subject by providing a compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and a pyruvate dehydrogenase kinase (PDK) inhibitor.

The condition may be aneurysm, angina, atherosclerosis, brain ischemia, cancer, cardiac failure, cardiomyopathy, cardiovascular disease, cataracts, cerebral apoplexy, cerebral ischemia, cerebral vascular disease, congenital heart disease, coronary artery disease, coronary heart disease, diabetes, diabetic cardiomyopathy, diabetic complications, dyslipidemia, heart attack, heart failure, high blood pressure (hypertension), hyperglycemia, hyperlactacidemia, insulin resistance syndrome, ischemic heart disease, metabolic syndrome, mitochondrial disease, mitochondrial encephalomyopathy, myocardial ischemia, nephropathy, neuropathy, obesity, pericardial disease, peripheral arterial disease, pulmonary hypertension, retinopathy, rheumatic heart disease, stroke, transient ischemic attacks, valvular heart disease, or ventricular hypertrophy.

The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and the PDK inhibitor may be provided in any suitable manner. They may be provided in a single composition. Alternatively, they may be provided in separate compositions. The agents may be provided simultaneously or sequentially. The agents may be provided at different intervals, with different frequency, or in different quantities.

The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may include any of the elements and have any of the structural features described above in relation to combination therapies of the invention.

The PDK inhibitor may include any of the elements described above in relation to combination therapies of the invention.

In another aspect, the invention provides pharmaceutical compositions that include compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and a pyruvate dehydrogenase kinase (PDK) inhibitor.

The PDK inhibitor may include any of the elements described above in relation to combination therapies of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table summarizing the effects of various compounds on mitochondrial function.

FIG. 2 is a table summarizing the effects of nicotinamide on various mitochondrial functional parameters.

FIG. 3 is a series of graphs showing the effects of nicotinamide on oxygen consumption rate and reserve capacity.

FIG. 4 is a series of graphs showing the effects of nicotinamide on extracellular acidification rate.

FIG. 5 is a table summarizing the effects of a combination of trimetazidine and nicotinamide on various mitochondrial functional parameters.

FIG. 6 is a series of graphs showing the effects of a combination of trimetazidine and nicotinamide on oxygen consumption rate and reserve capacity.

FIG. 7 is a series of graphs showing the effects of a combination of trimetazidine and nicotinamide on extracellular acidification rate.

FIG. 8 is a table summarizing the effects of succinate on various mitochondrial functional parameters.

FIG. 9 is a series of graphs showing the effects of succinate on oxygen consumption rate and reserve capacity.

FIG. 10 is a series of graphs showing the effects of succinate on extracellular acidification rate.

FIG. 11 is a table summarizing the effects of compound CV-8816 on various mitochondrial functional parameters.

FIG. 12 is a series of graphs showing the effects of compound CV-8816 on oxygen consumption rate and reserve capacity.

FIG. 13 is a series of graphs showing the effects of compound CV-8816 on extracellular acidification rate.

FIG. 14 is a table summarizing the effects of compound CV-8814 on various mitochondrial functional parameters.

FIG. 15 is a series of graphs showing the effects of compound CV-8814 on oxygen consumption rate and reserve capacity.

FIG. 16 is a series of graphs showing the effects of compound CV-8814 on extracellular acidification rate.

FIG. 17 is a table summarizing the effects of trimetazidine on various mitochondrial functional parameters.

FIG. 18 is a series of graphs showing the effects of trimetazidine on oxygen consumption rate and reserve capacity.

FIG. 19 is a series of graphs showing the effects of trimetazidine on extracellular acidification rate.

FIG. 20 is a table summarizing the effects of compound CV-8815 on various mitochondrial functional parameters.

FIG. 21 is a series of graphs showing the effects of compound CV-8815 on oxygen consumption rate and reserve capacity.

FIG. 22 is a series of graphs showing the effects of compound CV-8815 on extracellular acidification rate.

FIG. 23 is a table summarizing the effects of a combination of succinate, nicotinamide, and trimetazidine on various mitochondrial functional parameters.

FIG. 24 is a series of graphs showing the effects of a combination of succinate, nicotinamide, and trimetazidine on oxygen consumption rate and reserve capacity.

FIG. 25 is a series of graphs showing the effects of a combination of succinate, nicotinamide, and trimetazidine on extracellular acidification rate.

FIG. 26 is a table summarizing the effects of a combination of trimetazidine analog 2 and nicotinamide on various mitochondrial functional parameters.

FIG. 27 is a series of graphs showing the effects of a combination of trimetazidine analog 2 and nicotinamide on oxygen consumption rate and reserve capacity.

FIG. 28 is a series of graphs showing the effects a combination of trimetazidine analog 2 and nicotinamide on extracellular acidification rate.

FIG. 29 is a table summarizing the effects of a combination of trimetazidine analog 1 and nicotinamide on various mitochondrial functional parameters.

FIG. 30 is a series of graphs showing the effects of a combination of trimetazidine analog 1 and nicotinamide on oxygen consumption rate and reserve capacity.

FIG. 31 is a series of graphs showing the effects of a combination of trimetazidine analog 1 and nicotinamide on extracellular acidification rate.

FIG. 32 is a table summarizing the effects of a combination of trimetazidine analog 3 and nicotinamide on various mitochondrial functional parameters.

FIG. 33 is a series of graphs showing the effects of a combination of trimetazidine analog 3 and nicotinamide on oxygen consumption rate and reserve capacity.

FIG. 34 is a series of graphs showing the effects of a combination of trimetazidine analog 3 and nicotinamide on extracellular acidification rate.

FIG. 35 is a table summarizing the effects of a combination of succinate and nicotinamide on various mitochondrial functional parameters.

FIG. 36 is a series of graphs showing the effects of a combination of succinate and nicotinamide on oxygen consumption rate and reserve capacity.

FIG. 37 is a series of graphs showing the effects of a combination of succinate and nicotinamide on extracellular acidification rate.

FIG. 38 is a schematic of the ischemia-reperfusion (IR) method used to analyze the effects of selected compositions on coronary flow.

FIG. 39 is a graph of coronary flow of after IR.

FIG. 40 is graph of left ventricular developed pressure (LVDP) after IR.

FIG. 41 shows images of TTC-stained heart slices after IR.

FIG. 42 is graph of infarct size after IR.

FIG. 43 is a schematic of the method used to analyze the effects of selected compositions on cardiac function.

FIG. 44 shows hearts from mice six weeks after transverse aortic constriction.

FIG. 45 is of graph of heart weight relative to body weight six weeks after transverse aortic constriction.

FIG. 46 is graph of heart weight six weeks after transverse aortic constriction.

FIG. 47 shows graphs of fractional shortening (FS) and ejection fraction (EF) at indicated time points after transverse aortic constriction.

FIG. 48 is a graph of left ventricular end-systolic diameter at indicated time points after transverse aortic constriction.

FIG. 49 is a graph of intraventricular septal dimension at indicated time points after transverse aortic constriction.

FIG. 50 is a graph of left ventricular mass at indicated time points after transverse aortic constriction.

FIG. 51 is a graph of isovolumic relaxation time at indicated time points after transverse aortic constriction.

FIG. 52 is a graph of the ratio peak velocity flow in early diastole vs. late diastole at indicated time points after transverse aortic constriction.

FIG. 53 is a graph of left ventricular developed pressure at six weeks after transverse aortic constriction.

FIG. 54 is a graph of the rate of left ventricle pressure rise at six weeks after transverse aortic constriction.

FIG. 55 is a graph showing levels of CV-8814 and trimetazidine after intravenous administration of CV-8834.

FIG. 56 is a graph showing levels of CV-8814 and trimetazidine after oral administration of CV-8834.

FIG. 57 is a graph showing levels of CV-8814 and trimetazidine after oral administration of CV-8834.

FIG. 58 is a graph showing levels of CV-8814 and trimetazidine after oral administration of CV-8834.

FIG. 59 is a graph showing levels of CV-8814 and trimetazidine after oral administration of CV-8834.

FIG. 60 is a graph showing levels of trimetazidine after oral administration of CV-8972 or intravenous administration of trimetazidine.

FIG. 61 is a graph showing levels of CV-8814 after oral administration of CV-8972 or intravenous administration of CV-8814.

FIG. 62 is a graph showing levels of CV-8814 after intravenous administration of CV-8834 or oral administration of CV-8834.

FIG. 63 is a graph showing levels of CV-8814 after intravenous administration of CV-8814 or oral administration of CV-8814.

FIG. 64 is a graph showing the HPLC elution profile of a batch of CV-8972.

FIG. 65 is a graph showing analysis of molecular species present in a batch of CV-8972.

FIG. 66 is a pair of graphs showing HPLC elution profiles of molecular species present in a batch of CV-8972.

FIG. 67 is a pair of graphs showing HPLC elution profiles of molecular species present in a batch of CV-8972.

FIG. 68 is a graph showing X-ray powder diffraction analysis of a batch of CV-8972.

FIG. 69 is a graph showing X-ray powder diffraction analysis of batches of CV-8972.

FIG. 70 is a graph showing differential scanning calorimetry and thermal gravimetric analysis of a batch of CV-8972.

FIG. 71 is a graph showing dynamic vapor sorption (DVS) of a batch of CV-8972.

FIG. 72 is a graph showing differential scanning calorimetry and thermal gravimetric analysis of a batch of CV-8972.

FIG. 73 is a graph showing dynamic vapor sorption (DVS) of a batch of CV-8972.

FIG. 74 is a graph showing X-ray powder diffraction analysis of samples of CV-8972.

FIG. 75 is a graph showing differential scanning calorimetry and thermal gravimetric analysis of a batch of CV-8972.

FIG. 76 is a graph showing X-ray powder diffraction analysis of samples of CV-8972.

FIG. 77 is a graph showing X-ray powder diffraction analysis of samples of CV-8972.

FIG. 78 is a graph showing differential scanning calorimetry and thermal gravimetric analysis of samples containing form A of CV-8972.

FIG. 79 is a graph showing differential scanning calorimetry and thermal gravimetric analysis of a sample containing form A of CV-8972.

DETAILED DESCRIPTION

Regulation of cellular metabolism for the production of energy is critical to the progression and management of a variety of diseases, disorders, and conditions. Glucose oxidation and fatty acid oxidation are energy-producing metabolic pathways that compete with each other for substrates. In glucose oxidation, glucose is broken down to pyruvate via glycolysis in the cytosol of the cell. Pyruvate then enters the mitochondria, where it is converted to acetyl coenzyme A (acetyl-CoA) by the pyruvate dehydrogenase complex (PDC). In beta-oxidation of fatty acids, which occurs in the mitochondria, two-carbon units from long-chain fatty acids are sequentially converted to acetyl-CoA.

The remaining steps in energy production from oxidation of glucose or fatty acids are common to the two pathways. Acetyl-CoA is oxidized to carbon dioxide (CO₂) via the citric acid cycle (also called the tricarboxylic cycle, TCA cycle, and Krebs cycle), which results in the conversion of nicotinamide adenine dinucleotide (NAD⁺) to its reduced form, NADH. NADH, in turn, drives the mitochondrial electron transport chain. The electron transport chain comprises a series of four mitochondrial membrane-bound complexes that transfer electrons via redox reactions and pump protons across the membrane to create a proton gradient. The redox reactions of the electron transport chain require molecular oxygen (O₂). Finally, the proton gradient enables another membrane-bound enzymatic complex to form high-energy ATP molecules, the primary intracellular molecule that drives energy-requiring reactions.

Because the PDC links glycolysis to the citric acid cycle, regulation of its activity plays a key gate-keeping function. When PDC activity is low, conversion of pyruvate to acetyl CoA is blocked, and pyruvate is instead converted to lactate in a reaction that also regenerates (NAD⁺) from NADH. Thus, down-regulation of PDC activity uncouples glycolysis from the citric acid cycle and allows energy to be derived from glucose in oxygen-independent manner. Phosphorylation of the E1 pyruvate dehydrogenase subunit of PDC by pyruvate dehydrogenase kinase (PDK, also called PDHK and PDC kinase) deactivates the complex, and dephosphorylation of the same subunit by pyruvate dehydrogenase phosphatase (PDP) activates the complex.

Catabolism of fatty acids and glucose differ in ways that make each energy source advantageous under certain physiological conditions. Compared to glucose, fatty acids provide twice as much ATP per mass unit and thus are more efficient than glucose as a source of stored energy. However, ATP can be produced faster from glucose than from fatty acids. In addition, fatty acid oxidation requires more oxygen than does glucose oxidation. Glucose oxidation includes both glycolysis, which yields ATP in a series of oxygen-independent reactions, and post-glycolytic reactions, i.e., pyruvate decarboxylation and the citric acid cycle, which require oxygen and generate the majority of ATP produced by glucose oxidation. Consequently, in physiological conditions in which energy is needed rapidly and/or oxygen is scarce, such as in muscles during intensive exercise or in ischemic tissue, catabolism of glucose is preferred.

Cellular regulation of metabolic pathways for energy production is critical in a variety of diseases and conditions. For example, in cardiovascular conditions that lead to reduced blood flow to tissues, oxygen levels are insufficient to support fatty acid oxidation. Therefore, providing agents that promote glucose oxidation can provide therapeutic benefits. For example, in cases of angina, restoration of energy production in cardiac tissue can reduce the risk of myocardial infarction. In some cases of diabetes and obesity, overexpression of PDK4, which encodes an isoform of PDK, inhibits activity of the PDC and impairs glucose oxidation. Genes encoding other PDK isoforms, such as PDK1 and PDK3, are overexpressed in certain cancers. Without wishing to be bound by any particular theory, is thought that inhibition of PDC allows tumor cells to rely exclusively on glycolysis for energy production and avoid apoptotic signals that would otherwise be generated by cells in hypoxic conditions. Survival under hypoxic conditions is a key adaptation that permits metastatic invasion of tumor cells into other tissues.

The invention provides combination therapies that correct metabolic defects, such as those described above, by promoting glucose oxidation. The combination therapies include an agent that shifts cellular metabolism from fatty acid oxidation to glucose oxidation, such as trimetazidine or an analog, derivative, or prodrug thereof, and an inhibitor of pyruvate dehydrogenase kinase. The first agent promotes the use of glucose as an energy source, and the second agent shunts the pyruvate produced from glycolysis into the citric acid cycle. By driving complete oxidation of glucose, the therapies ensure that the energy yield from glucose catabolism is maximized and that mitochondria produce apoptotic signals under appropriate conditions. The combination therapies may also include a NAD⁺precursor molecule. The invention includes compositions containing the therapeutic combinations of the invention and methods of treating conditions using such combinations.

Compositions

The invention includes combination therapies that include a compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and an inhibitor of pyruvate dehydrogenase kinase. Compounds that shift cellular metabolism from fatty acid oxidation to glucose oxidation are described in, for example, International Patent Publication No. WO 2018/236745, the contents of which are incorporated herein by reference.

Compounds that Shift Cellular Metabolism from Fatty Acid Oxidation to Glucose Oxidation

The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be represented by formula (I):

$\begin{matrix} A - L - B, & (I) \end{matrix}$

in which A is a molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation, L is a linker, and B is a compound that promotes mitochondrial respiration.

Component A may be any suitable molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation. Such compounds can be classified based on their mechanism of action. See Fillmore, N., et al., Mitochondrial fatty acid oxidation alterations in heart failure, ischemic heart disease and diabetic cardiomyopathy, Brit. J. Pharmacol. 171:2080-2090 (2014), incorporated herein by reference.

One class of glucose-shifting compounds includes compounds that inhibit fatty acid oxidation directly. Compounds in this class include inhibitors of malonyl CoA decarboxylase (MCD), carnitine palmitoyl transferase 1 (CPT-1), or mitochondrial fatty acid oxidation. Mitochondrial fatty acid oxidation inhibitors include trimetazidine and other compounds described in International Patent Publication No. WO 2002/064,576, the contents of which is incorporated herein by reference. Trimetazidine binds to distinct sites on the inner and outer mitochondrial membranes and affects both ion permeability and metabolic function of mitochondria. Morin, D., et al., Evidence for the existence of [³H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore, Brit. J. Pharmacol. 123:1385-1394 (1998), incorporated herein by reference. MCD inhibitors include CBM-301106, CBM-300864, CBM-301940, 5-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-4,5-dihydroisoxazole-3-carboxamides, methyl 5-(N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)morpholine-4-carboxamido)pentanoate, and other compounds described in Chung, J. F., et al., Discovery of Potent and Orally Available Malonyl-CoA Decarboxylase Inhibitors as Cardioprotective Agents, J. Med. Chem. 49:4055-4058 (2006); Cheng J. F. et al., Synthesis and structure-activity relationship of small-molecule malonyl coenzyme A decarboxylase inhibitors, J. Med. Chem. 49:1517-1525 (2006); U.S. Patent Publication No. 2004/0082564; and International Patent Publication No. WO 2002/058,698, the contents of which are incorporated herein by reference. CPT-1 inhibitors include oxfenicine, perhexiline, etomoxir, and other compounds described in International Patent Publication Nos. WO 2015/018,660; WO 2008/109,991; WO 2009/015,485; and WO 2009/156479; and U.S. Patent Publication No. 2011/0212072, the contents of which are incorporated herein by reference.

Another class of glucose-shifting compounds includes compounds that stimulate glucose oxidation directly. Examples of such compounds are described in U.S. Patent Publication No. 2003/0191182; International Patent Publication No. WO 2006/117,686; U.S. Pat. No. 8,202,901, the content of each of which are incorporated herein by reference.

Another class of glucose-shifting compounds includes compounds that decrease the level of circulating fatty acids that supply the heart. Examples of such compounds include agonists of PPARα and PPARγ, including fibrate drugs, such as clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and fenofibrate, and thiazolidinediones, GW-9662, and other compounds described in U.S. Pat. No. 9,096,538, which is incorporated herein by reference.

Component L may be any suitable linker. Preferably, the linker can be cleaved in vivo to release components A and B. The linker may be an alkoxy group. The linker may be polyethylene glycol of any length. The linker may be represented by (CH₂CH₂O)_x, in which x=1-15 or (CH₂CH₂O)_x, in which x=1-3. Other suitable linkers include 1,3-propanediol, diazo linkers, phosphoramidite linkers, disulfide linkers, cleavable peptides, iminodiacetic acid linkers, thioether linkers, and other linkers described in Leriche, G., et al., Cleavable linkers in chemical biology, Bioorg. Med. Chem. 20:571-582 (2012); International Patent Publication No. WO 1995/000,165; and U.S. Pat. No. 8,461,117, the contents of which are incorporated herein by reference.

Component B may be any compound that promotes mitochondrial respiration. For example, component B may be an intermediate of the citric acid cycle or a molecule that can be metabolized to enter the citric acid cycle, such as succinate, fumarate, malate, oxaloacetate, citrate, isocitrate, α-ketoglutarate, pyruvate, acetone, acetoacetic acid, β-hydroxybutpic acid, β-ketopentanoate, or β-hydroxypentanoate. Intermediates of the citric acid cycle may become depleted if these molecules are used for biosynthetic purposes, resulting in inefficient generation of ATP from the citric acid cycle. However, due to the anaplerotic effect, providing one intermediate of the citric acid cycle leads to restoration of all intermediates as the cycle turns. Thus, intermediates of the citric acid cycle can promote mitochondrial respiration.

The compound may include a NAD⁺precursor molecule. NAD⁺is an important oxidizing agent that acts as a coenzyme in multiple reactions of the citric acid cycle. In these reactions, NAD⁺is reduced to NADH. Conversely, NADH is oxidized back to NAD⁺when it donates electrons to mitochondrial electron transport chain. In humans, NAD⁺can be synthesized de novo from tryptophan, but not in quantities sufficient to meet metabolic demands. Consequently, NAD⁺is also synthesized via a salvage pathway, which uses precursors that must be supplied from the diet. Among the precursors used by the salvage pathway for NAD⁺synthesis are nicotinic acid, nicotinamide, and nicotinamide riboside. By providing a NAD⁺precursor, such as nicotinic acid, nicotinamide, or nicotinamide riboside, the compound facilitates NAD⁺synthesis.

The inclusion of a NAD⁺precursor allows the compounds to stimulate energy production in cardiac mitochondria in multiple ways. First, component A shifts cellular metabolism from fatty acid oxidation to glucose oxidation, which is inherently more efficient. Next, component B ensures that the intermediates of the citric acid cycle are present at adequate levels and do not become depleted or limiting. As a result, glucose-derived acetyl CoA is efficiently oxidized. Finally, the NAD⁺precursor provides an essential coenzyme that cycles between oxidized and reduced forms to promote respiration. In the oxidized form, NAD⁺drives reactions of the citric acid cycle. In the reduced form, NADH promotes electron transport to create a proton gradient that enables ATP synthesis. Consequently, the chemical potential resulting from oxidation of acetyl CoA is efficiently converted to ATP that can be used for various cellular functions.

The NAD⁺precursor molecule may be covalently attached to the compound in any suitable manner. For example, it may be linked to A, L, or B, and it may be attached directly or via another linker. Preferably, it is attached via a linker that can be cleaved in vivo. The NAD⁺precursor molecule may be attached via a 1,3-propanediol linkage.

The compound may be covalently attached to one or more molecules of polyethylene glycol (PEG), i.e., the compound may be PEGylated. In many instances, PEGylation of molecules reduces their immunogenicity, which prevents the molecules from being cleared from the body and allows them to remain in circulation longer. The compound may contain a PEG polymer of any size. For example, the PEG polymer may have from 1-500 (CH₂CH₂O) units. The PEG polymer may have any suitable geometry, such as a straight chain, branched chain, star configuration, or comb configuration. The compound may be PEGylated at any site. For example, the compound may be PEGylated on component A, component B, component L, or, if present, the NAD⁺precursor. The compound may be PEGylated at multiple sites. For a compound PEGylated at multiple sites, the various PEG polymers may be of the same or different size and of the same or different configuration.

The compound may be a PEGylated form of trimetazidine. For example, the compound may be represented by formula (VI):

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in which one or more of the carbon atoms at positions A, B, C, D, and E and/or the nitrogen atom at position F are substituted with —(CH₂CH₂O)_nH and n=1-15. The carbon atoms at positions A, B, C, D, and E may have two PEG substituents. In molecules that have multiple PEG chains, the different PEG chains may have the same or different length.

The compounds of formula (I) may be represented by formula (II):

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in which y=1-3.

The compounds of formula (I) may be represented by formula (III):

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in which y=1-3.

The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be represented by formula (IV):

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in which R¹, R², and R³are independently H or a (C₁-C₄)alkyl group; R⁴and R⁵together are ═O, —O(CH₂)_mO—, or —(CH₂)_m—, in which m=2-4, or R⁴is H and R⁵is OR¹⁴, SR¹⁴, or (CH₂CH₂O)_nH, in which R¹⁴is H or a (C₁-C₄)alkyl group and n=1-15; and R⁶is a single or multi-ring structure optionally substituted at one or more ring positions by a heteroatom, in which each ring position optionally comprises one or more substituents.

R⁶may be a single or multi-ring structure of any size. For example, the structure may contain 3-22 atoms, not including hydrogen atoms bonded to atoms in ring positions. The structure may include one or more alkyl, alkenyl, or aromatic rings. The structure may include one or more heteroatoms, i.e., atoms other than carbon. For example, the heteroatom may be oxygen, nitrogen, or sulfur, or phosphorus.

One or more ring position of R⁶may include a substituent that includes a compound that promotes mitochondrial respiration, as described above in relation to component B of formula (I). The substituent may include a linker, as described above in relation to component L of formula (I). The substituent may include a NAD⁺precursor molecule, as described above in relation to compounds of formula (I).

The substituent on a ring position of R⁶may be

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in which y=1-3.

The substituent on a ring position of R⁶may be

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in which y=1-3.

R⁶may be

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For some compounds that include trimetazidine prodrugs, analogs, derivatives, it is advantageous to have the trimetazidine moiety substituted with a single ethylene glycol moiety. Thus, compositions of the invention may include compounds of formulas (I) and (VIII) that contain linkers in which x=1, compounds of formulas (II) and (III) in which y=1, compounds of formula (V) in which z=1, compounds of formula (VI) in which n=1, and compounds of formula (VII) in which A is linked to C via a single ethylene glycol moiety. Without wishing to be bound by theory, the attachment of a single ethylene glycol moiety to the trimetazidine moiety may improve the bioavailability of trimetazidine.

The compound of formula (IV) may have a structure represented by formula (IX) or formula (X):

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The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be represented by formula (V):

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in which R¹, R², and R³are independently H or a (C₁-C₄)alkyl group; R⁴and R⁸together are ═O, —O(CH₂)_mO—, or —(CH₂)_m—, in which m=2-4, or R⁴is H and R⁸is H, OR¹⁴, SR¹⁴, or (CH₂CH₂O)_nH, in which R¹⁴is H or a (C₁-C₄)alkyl group and n=1-15; R⁹, R¹⁰, R¹², and R¹³are independently H or (CH₂CH₂O)_zH, in which z=1-15; and R¹¹comprises a compound that promotes mitochondrial respiration, as described above in relation to component B of formula (I). R¹¹may include a linker, as described above in relation to component L of formula (I). R¹¹may be

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in which y=1-3.

R¹¹may include a NAD⁺precursor molecule, as described above in relation to compounds of formula (I).

R¹¹may be

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in which y=1-3.

In some embodiments described above, the compound includes multiple active agents joined by linkers in a single molecule. It may be advantageous to deliver multiple active agents as components of a single molecule. Without wishing to be bound by a particular theory, there are several reasons why co-delivery of active agents in a single molecule may be advantageous. One possibility is that a single large molecule may have reduced side effects compared to the component agents. Free trimetazidine causes symptoms similar to those in Parkinson's disease in a fraction of patients. However, when trimetazidine is derivatized to include other components, such as succinate, the molecule is bulkier and may not be able to access sites where free trimetazidine can causes unintended effects. Trimetazidine derivatized as described above is also more hydrophilic and thus may be less likely to cross the blood-brain barrier to cause neurological effects. Another possibility is that modification of trimetazidine may alter its pharmacokinetic properties. Because the derivatized molecule is metabolized to produce the active agent, the active agent is released gradually. Consequently, levels of the active agent in the body may not reach peaks as high as when a comparable amount is administered in a single bolus. Another possibility is that less of each active agent, such as trimetazidine, is required because the compositions of the invention may include compounds that have multiple active agents. For example, trimetazidine shifts metabolism from fatty acid oxidation to glucose oxidation, and succinate improves mitochondrial respiration generally. Thus, a compound that provides both agents stimulates a larger increase in glucose-driven ATP production for a given amount of trimetazidine than does a compound that delivers trimetazidine alone.

The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be represented by formula (VII):

A-C (VII),

in which A is a molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation, and C is a NAD⁺precursor molecule. A and C may be covalently linked.

The compound of formula (VII) may include nicotinic acid that is covalently linked to a PEGylated form of trimetazidine. The nicotinic acid may be covalently linked via a PEGylated moiety, i.e., via an ethylene glycol linkage. The nicotinic acid may be covalently linked via the trimetazidine moiety.

The compound that shifts cellular metabolism from fatty acid oxidation to glucose oxidation may be represented by formula (VIII):

A-L-C (VIII),

in which A is a molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation, L is a linker, and C is a NAD⁺precursor molecule. A may be covalently linked to L, and L may be covalently linked to C.

The molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation, the linker, and the NAD⁺precursor molecule may be as described above in relation to compounds of other formulas.

The compounds may be provided as co-crystals with other compounds. Co-crystals are crystalline materials composed of two or more different molecules in the same crystal lattice. The different molecules may be neutral and interact non-ionically within the lattice. Co-crystals may include one or more of the compounds described above and one or more other molecules that stimulate mitochondrial respiration or serve as NAD⁺precursors. For example, a co-crystal may include any of the following combinations: (1) a molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and (2) a NAD⁺precursor molecule; (1) a compound that promotes mitochondrial respiration and (2) a NAD⁺precursor molecule; (1) a molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and (2) a compound that promotes mitochondrial respiration; (1) a molecule comprising a molecule that shifts cellular metabolism from fatty acid oxidation to glucose oxidation covalently linked to a compound that promotes mitochondrial respiration and (2) a NAD⁺precursor molecule. In specific embodiments, a co-crystal may include (1) a compound of formula (I), (III), (IV), or (V) and (2) nicotinic acid, nicotinamide, or nicotinamide riboside.

The compounds may include one or more atoms that are enriched for an isotope. For example, the compounds may have one or more hydrogen atoms replaced with deuterium or tritium. Isotopic substitution or enrichment may occur at carbon, sulfur, or phosphorus, or other atoms. The compounds may be isotopically substituted or enriched for a given atom at one or more positions within the compound, or the compounds may be isotopically substituted or enriched at all instances of a given atom within the compound.

Inhibitors of PDK

The PDK inhibitor may be any agent that inhibits one or more isoforms of pyruvate dehydrogenase kinase. The PDK inhibitor may be any suitable class of molecule. For example and without limitation, the PDK inhibitor may be a small molecule, protein, peptide, polypeptide, nucleic acid (e.g., RNA, siRNA, shRNA, miRNA, mRNA, DNA, nucleic acid with one or more modified nucleotides, etc.), or combination thereof. For example and without limitation, the PDK inhibitor may be (R)-3.3.3-trifluoro-2-hydroxy-2-methyl propionamide, 2-chloroproprionate, 4,5-diarylisoxazole, anilide tertiary carbinol, aromatic DCA derivative, betulinic acid, CPI-613, dichloroacetate (DCA), a DCA-loaded tertiary amine, a DCA-oxaliplatin derivative, a dihydrolipoamide mimetic, a furan carboxylic acid, a hemoglobin-DCA conjugate (e.g., fusion molecule of 1 Hgb:12 DCAs), honokiol DCA, an inositol ester (e.g., inositol hexa(N-methylnicotinate-dichloroacetate), an inositol ionic complex (e.g., tetra(dichloroacetyl) gluconate), M77976, mitaplatin, mito-DCA (e.g., fusion molecule of 1 triphenylphosphonium cation: 3 DCA), N-(2-aminoethyl)-2(3-chloro-4-(4-isopropylbenzyl)oxy)phenyl)acetamide, phenylbutyrate, pyruvate, a pyruvate analog containing a phosphinate or phosphonate group, radicicol, R-lipoic acid, a tetrahydroisoquinoline, a thiophene carboxylic acid, or VER-246608. PDK inhibitors are known in the art and described in, for example, U.S. Patent Publication No. 2017/0001958; U.S. Pat. No. 8,871,934; International Patent Publication Nos. WO 2015/040,424 and WO 2017/167,676; and Peter W. Stacpoole, Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer, JNCI: Journal of the National Cancer Institute, Volume 109, Issue 11, 1 Nov. 2017, djx071, doi: 10.1093/jnci/djx071, the contents of each of which are incorporated herein by reference.

Formulations

The invention provides pharmaceutical compositions containing one or more of the compounds described above. A pharmaceutical composition containing the compounds may be in a form suitable for oral use, for example, as tablets, troches, lozenges, fast-melts, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the compounds in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration in the stomach and absorption lower down in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated to form osmotic therapeutic tablets for control release by the techniques described in U.S. Pat. Nos. 4,256,108, 4,166,452 and 4,265,874, the contents of which are incorporated herein by reference. Preparation and administration of compounds is discussed in U.S. Pat. No. 6,214,841 and U.S. Pub. 2003/0232877, the contents of which are incorporated by reference herein in their entirety.

Formulations for oral use may also be presented as hard gelatin capsules in which the compounds are mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the compounds are mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

An alternative oral formulation, where control of gastrointestinal tract hydrolysis of the compound is sought, can be achieved using a controlled-release formulation, where a compound is encapsulated in an enteric coating.

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

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

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

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

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and agents for flavoring and/or coloring. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 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 di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compositions of the invention are useful for improving cardiac efficiency. A variety of definitions of cardiac efficiency exist in the medical literature. See, e.g.. Schipke, J. D. Cardiac efficiency, Basic Res. Cardiol. 89:207-40 (1994); and Gibbs, C. L. and Barclay, C. J. Cardiac efficiency, Cardiovasc. Res. 30:627-634 (1995), incorporated herein by reference. One definition of cardiac mechanical efficiency is the ratio of external cardiac power to cardiac energy expenditure by the left ventricle. See Lopaschuk G. D., et al., Myocardial Fatty Acid Metabolism in Health and Disease, Phys. Rev. 90:207-258 (2010), incorporated herein by reference. Another definition is the ratio between stroke work and oxygen consumption, which ranges from 20-25% in the normal human heart. Visser, F., Measuring cardiac efficiency: is it useful? Hear Metab. 39:3-4 (2008), incorporated herein by reference. Another definition is the ratio of the stroke volume to mean arterial blood pressure. Any suitable definition of cardiac efficiency may be used to measure the effects of compositions of the invention

The compositions of the invention may contain an agent that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and an inhibitor of PDK in a single formulation. Alternatively, the compositions of the invention may contain an agent that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and an inhibitor of PDK in separate formulations. The compositions may contain a NAD⁺precursor molecule in a formulation that contains an agent that shifts cellular metabolism from fatty acid oxidation to glucose oxidation and/or an inhibitor of PDK, or the NAD⁺precursor molecule may be provided in a separate formulation.

Methods of Treating Diseases, Disorders, and Conditions

The invention also provides methods of treating diseases, disorders, and conditions using the combination therapies described herein. The combination therapies are useful for treating any disease, disorder, or condition for which a shift in cellular metabolism from fatty acid oxidation to glucose oxidation would be advantageous.

The combination therapies are useful for treating or preventing diseases relating to glucose utilization, such as diabetes (e.g., type 1 diabetes, type 2 diabetes), insulin resistance syndrome, metabolic syndrome, hyperglycemia, and hyperlactacidemia. The combination therapies may also be used to treat complications of the aforementioned disorders, such as neuropathy, retinopathy, nephropathy, and cataracts.

The combination therapies may be used to treat or prevent diseases, disorders, or conditions associated with a limited supply of energy substrates to tissues, such as cardiac failure, cardiomyopathy, myocardial ischemia, dyslipidemia, atherosclerosis, and cerebral ischemia.

The combination therapies may be used to treat or prevent diseases, disorders, or conditions associated with mitochondrial dysfunction, such as mitochondrial disease and mitochondrial encephalomyopathy.

Other categories of diseases that can be treated or prevented with combination therapies of the invention include cardiovascular disease, cancer, and pulmonary hypertension.

For example and without limitation, the combination therapies may be used to treat or prevent aneurysm, angina, atherosclerosis, brain ischemia, cancer, cardiac failure, cardiomyopathy, cardiovascular disease, cataracts, cerebral apoplexy, cerebral ischemia, cerebral vascular disease, congenital heart disease, coronary artery disease, coronary heart disease, diabetes, diabetic cardiomyopathy, diabetic complications, dyslipidemia, heart attack, heart failure, high blood pressure (hypertension), hyperglycemia, hyperlactacidemia, insulin resistance syndrome, ischemic heart disease, metabolic syndrome, mitochondrial disease, mitochondrial encephalomyopathy, myocardial ischemia, nephropathy, neuropathy, obesity, pericardial disease, peripheral arterial disease, pulmonary hypertension, retinopathy, rheumatic heart disease, stroke, transient ischemic attacks, valvular heart disease, or ventricular hypertrophy.

The compositions may be provided by any suitable route of administration. For example and without limitation, the compositions may be administered buccally, by injection, dermally, enterally, intraarterially, intravenously, nasally, orally, parenterally, pulmonarily, rectally, subcutaneously, topically, transdermally, or with or on an implantable medical device (e.g., stent or drug-eluting stent or balloon equivalents).

EXAMPLES
Protocol

The effects of selected compounds on mitochondrial function were analyzed. HepG2 cells were dosed with test compound and in real time the extracellular oxygen levels and pH were measured using the XFe96 flux analyzer (Seahorse Biosciences). XFe Technology uses solid-state sensors to simultaneously measure both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine effects on oxidative phosphorylation (OXPHOS) and glycolysis simultaneously. The cells were then subjected to sequential exposure to various inhibitors of mitochondrial function to assess cellular metabolism.

Data Interpretation

A compound was identified as positive mitochondrial-active compound when it caused a change in oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) in the absence of cytotoxicity. Cytotoxicity was determined when both OXPHOS (OCR) and glycolysis (ECAR) were inhibited.

Definition of Mitochondrial Parameters

Oxygen consumption rate (OCR) is a measurement of oxygen content in extracellular media. Changes in OCR indicate effects on mitochondrial function and can be bi-directional. A decrease is due to an inhibition of mitochondrial respiration, while an increase may indicate an uncoupler, in which respiration is not linked to energy production.

$OCR = \frac{compound OCR - non mitochondrial OCR}{basal OCR - non mitochondrial OCR}$

Extracellular acidification rate (ECAR) is the measurement of extracellular proton concentration (pH). An increase in signal means an increase in rate in number of pH ions (thus decreasing pH value) and seen as an increase in glycolysis. ECAR is expressed as a fraction of basal control (rate prior to addition of compound).

$reserve capacity = \frac{FCCP OCR - non mitochondrial OCR}{basal OCR - non mitochondrial OCR}$

Reserve capacity is the measured ability of cells to respond to an increase in energy demand. A reduction indicates mitochondrial dysfunction. This measurement demonstrates how close to the bioenergetic limit the cell is.

$ECAR = \frac{compound ECAR}{basal ECAR}$

Mitochondrial Stress Test

A series of compounds were added sequentially to the cells to assess a bioenergetics profile, effects of test compounds on parameters such as proton leak, and reserve capacity. This can be used to assist in understanding potential mechanisms of mitochondrial toxicity. The following compounds were added in order: (1) oligomycin, (2) FCCP, and (3) rotenone and antimycin A.

Oligomycin is a known inhibitor of ATP synthase and prevents the formation of ATP. Oligomycin treatment provides a measurement of the amount of oxygen consumption related to ATP production and ATP turnover. The addition of oligomycin results in a decrease in OCR under normal conditions, and residual OCR is related to the natural proton leak.

FCCP is a protonophore and is a known uncoupler of oxygen consumption from ATP production. FCCP treatment allows the maximum achievable transfer of electrons and oxygen consumption rate and provides a measurement of reserve capacity.

Rotenone and antimycin A are known inhibitors of complex I and III of the electron transport chain, respectively. Treatment with these compounds inhibits electron transport completely, and any residual oxygen consumption is due to non-mitochondrial activity via oxygen requiring enzymes.

Definition of Mechanisms

An electron transport chain inhibitor is an inhibitor of mitochondrial respiration that causes an increase in glycolysis as an adaptive response (e.g. decrease OCR and increase in ECAR).

The inhibition of oxygen consumption may also be due to reduced substrate availability (e.g. glucose, fatty acids, glutamine, pyruvate), for example, via transporter inhibition. Compounds that reduce the availability of substrates are substrate inhibitors. A substrate inhibitor does not result in an increase in glycolysis (e.g. OCR decrease, no response in ECAR).

Compounds that inhibit the coupling of the oxidation process from ATP production are known as uncouplers. These result in an increase in mitochondrial respiration (OCR) but inhibition of ATP production.