WO2010019225A1

WO2010019225A1 - Pharmaceutical composition

Info

Publication number: WO2010019225A1
Application number: PCT/US2009/004604
Authority: WO
Inventors: Robert Shorr; Robert Rodriguez; Paul Bingham; Zuzana Zachar; Patrick P. Zaretski
Original assignee: Robert Shorr; Robert Rodriguez; Paul Bingham; Zuzana Zachar; Zaretski Patrick P
Priority date: 2008-08-15
Filing date: 2009-08-12
Publication date: 2010-02-18

Abstract

Pharmaceutically-acceptable agents modulate the structure, function, activity, cellular location or compartmentalization, and/or expression levels of fatty acid-containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids in diseased cells, thereby facilitating the destruction of these cells. More specifically, these agents target and perturb the activity, or regulation thereof, of the altered mitochondrial energy metabolism observed in the modified pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (α- KDH), and/or branched-chain α-ketoacid dehydrogenase (BCAKDH) complexes associated with most cancers by modification of the oxidation-reduction state therein, such as but not limited to affecting concentrations of metabolic by-products of PDH, α-KDH, and/or BCAKDH complex activity, including reactive oxygen and nitrogen species.

Description

Pharmaceutical Composition

Field of the Invention

Management of metabolic by-products, such as reactive oxygen and nitrogen species (RONS), is key to regulating growth, differentiation, and/or homeostasis of normal cells and tissue. Other metabolic by-products may be toxic and need to be detoxified and/or removed from cells and tissue. Thus, many metabolic by-product levels are tightly regulated by a number of enzymatic pathways, as well as genetic and epigenetic transcription, translation, and post-translation events. In certain disease states, the ability to maintain homeostatic concentrations of metabolic by-products such as but not limited to RONS is altered, and cells that inappropriately overproduce or underproduce such by-products activate pathologically- associated signal transduction cascades and/or gene expression, such as those that trigger inflammation, hyperproliferation including tumor formation, or apoptosis or necrosis. Thus, in some disease states, such as in diabetes or cancer, aliphatic fatty acid-containing, -binding, or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids may be absent or otherwise altered in structure, function, activity, cellular location or compartmentalization, and/or expression levels. Compounds that effectively modulate signal transduction-linked or toxic metabolic by-product levels to achieve a beneficial therapeutic outcome are hence highly desirable. This invention relates to pharmaceutically-acceptable agents that alter metabolic by-products and their linkage to signal transduction, genetic or epigenetic expression, or detoxification events in diseased cells. These agents modulate the structure, function, activity, cellular location or compartmentalization, and/or expression levels of aliphatic fatty acid-containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, or nucleic acids in diseased cells, thereby remediating the pathology of these cells, such as via destruction of these cells. In a preferred embodiment, the aliphatic fatty acid moiety has a chain length of three to twenty carbons, such as but not limited to lipoate or analogs, congeners, or derivatives thereof. Examples of such proteins include without limitation acyl transferase proteins such as the modified pyruvate dehydrogenase (PDH) α- ketoglutarate dehydrogenase (α-KDH), and branched-chain α-ketoacid dehydrogenase (BCAKDH) complexes observed in most cancers and receptor- linked kinases such as the insulin receptor tyrosine kinase observed in cancer and diabetes. Such aliphatic fatty acid- containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, or nucleic acids associated with cancer are often localized in cancer cell mitochondria, which are dramatically altered from those in normal cells. This alteration is associated with modification of the oxidation-reduction ("redox") state therein, such as but not limited to affecting concentrations of metabolic by-products of PDH, α-KDH, and/or BCAKDH complex activity, including RONS, which ultimately lead to tumor cell apoptosis or necrosis.

Background of the Invention

Free radicals contain one or more unpaired electrons; since all molecules seek to have an equal number of protons and electrons, the unpaired electron spins of these radicals make the radicals highly reactive. Driven by changes in free energy associated with electron or hydrogen transfers, redox biochemistry is hence fundamental to life both because the ATP supply required by higher organisms for their energy needs depends heavily on such chemistry and because electron-transfer processes such as those involving RONS also play a key messenger role in biological systems. Crucial to such activities are reactive oxygen species, including oxygen radicals (e.g., O₂ ^'* and OH^') and nonradical O₂ derivatives (e.g.,

H₂O₂), and reactive nitrogen species (e.g., NO^*, ONOO^", and NO₂Cl), collectively known as

RONS.

RONS are produced continuously by the mitochondria of most cells (O₂ ^"", H₂O₂, NO^", ONOO^", and OH^"); cytochrome P450 (O₂ ^'" and H₂O₂); macrophages (O₂ ^", H₂O₂, and NO^'); and peroxisomes (H₂O₂). During mitochondrial oxidative metabolism, about 5% of oxygen is converted primarily into O₂ ^'" with the remainder reduced to water. Given such high reactivity, and especially as RONS have the potential to damage lipids in cell membranes, DNA, and proteins, it is not surprising that there is a cellular antioxidant defense system to manage RONS and their effects. This defense system includes mitochondrial, peroxisomal, and cytoplasmic antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione (GSH) peroxidase, glutaredoxin, peroxiredoxin and thioredoxin); nonclassic antioxidant enzymes (e.g., hemoxygenase-1); Phase Il detoxifying enzymes (e.g., GSH reductase, NQOl, and GSH transferase); and nonenzymatic antioxidants (e.g., lipoic acid, vitamins E and C, GSH, and catechins).

Mitochondria have numerous metabolic roles within the cell and are a crucial point of convergence for many cellular activities and processes. In line with their role as both a point of convergence and regulator of diverse cellular functions in eukaryotes, mitochondria have crucial roles in numerous metabolic processes, including the production of both over 90% of cellular ATP and RONS, and cell death. Hence, mitochondria require a complex system of communication with cellular functions. Pathological or genetic changes associated in mitochondrial enzyme structure, function, activity, and regulation contribute to disease, and thus may be important targets for the treatment of disease.

For example, the mitochondrial PDH complex plays a central role in the maintenance of glucose homoeostasis in mammals. Carbon flux through the PDH complex is meticulously regulated by elaborate mechanisms involving, among other things, short-term post- translational reversible phosphorylation and long-term transcriptional controls. Short-term post-translational reversible phosphorylation involves multiple phosphorylation sites and tissue-specific distribution of dedicated kinases and phosphatases. Indeed, such regulation is not only dependent on the interactions among the catalytic and regulatory components of the PDH complex but is also sensitive to the intramitochondrial redox state and metabolite levels as indicators of the energy status. (See Rigas B and Sun Y (2008). Induction of oxidative stress as a mechanism of action of chemopreventive agents against cancer. Brit. J. Cancer 98:1 157-1 \60, passim, herein incorporated by reference. See also Patel MS and Korotchkina LG (2006). Regulation of the pyruvate dehydrogenase complex. Biochem. Soc. Tram. 34:217-222, passim, herein incorporated by reference.) This may be especially true for cancer and other diseased cells, where enzyme function and regulation may differ from normal conditions. Changes in redox potential may also contribute to differential transcriptional controls of the regulatory components of the PDH complex between normal and disease states.

Hence, the PDH complex regulates mammalian carbohydrate and amino acid oxidation via reversible phosphorylation involving protein-protein interactions among PDH complex components and via sensing changes in the concentrations of intramitochondrial metabolites which affects and is affected by its redox state (e.g., pyruvate, acetyl-CoA, and NADH). Furthermore, the PDH complex exhibits tissue-specific regulation through the evolutionary development of multiple phosphorylation sites and the tissue-specific distribution of multiple isoenzymes of dedicated kinases such as pyruvate dehydrogenase kinase (PDK) and phosphatases such as pyruvate dehydrogenase phosphatase (PDP). Hormonal regulation at the transcriptional level of these regulatory enzymes adds long-term PDH complex regulation under both normal and disease states. The PDH complex is therefore unique in its exhibition of multiple mechanisms of mammalian glucose homeostasis regulation.

Overall, in typical solid tumors and hematological malignancies, the vast majority of fast-growth tumor cells exhibits profound genetic, biochemical, and histological differences with respect to nontransformed cells. Many of these are associated with altered energy metabolism in comparison to the tissue of origin. The most notorious and well-known energy metabolism alteration in tumor cells is an increased glycolytic capacity even in the presence of a high O₂ concentration, a phenomenon known as the Warburg effect. Warburg originally proposed that the driving force of the enhanced glycolysis in tumor cells was the energy deficiency caused by an irreversible damage of the mitochondrial function in which, similarly to anaerobic muscle, glucose is converted through glycolysis to lactate, which is later secreted. It has been proposed that this increase in the glycolytic flux in tumor cells is a metabolic strategy to ensure survival and growth in environments with low O₂ concentrations, such as the partial hypoxia observed in poorly-oxygenated solid tumors. In particular, since the O₂ concentration is lower than 20 μM in many human hypoxic tumors, oxidative phosphorylation is limited therein. Consequently, glycolysis seems to be the main energy pathway in solid tumors (e.g., slow-growing melanomas and mammary adenocarcinoma).

A proportional relationship between the rate of cellular proliferation and the rate of ATP supply has been established for fast-growth tumor cells. Some authors have proposed that the glycolytic activity correlates with the degree of tumor malignancy, so that the glycolytic rate is greater in highly de-differentiated and fast-growing tumors than in slower- growing tumors or normal cells. In fact, a high level of lactate has been proposed as a predictor of malignancy. That these events are linked to additional signal transduction events and genetic changes is likely, and examples include hypoxia-inducible factor (HIF)- lα and the production and release of angiogenic factors.

For years, the tricarboxylic acid (TCA) cycle was regarded as being biologically significant only for its role in the production of ATP as an energy source for the organism. However, recent studies have shown that TCA cycle activity also affects signal transduction pathway functions, including cell growth and apoptosis decisions, and that the pertinent glycolytic and TCA cycle enzymes are able to be up- or down-regulated. There is also a direct correlation between tumor progression and the activities of the glycolytic enzymes hexokinase and phosphofructokinase (PFK) 1 , which are greatly increased in fast-growth tumor cells. Accordingly, it has been postulated that tumor cells which exhibit deficiencies in their oxidative capacity are more malignant than those that have an active oxidative phosphorylation. No matter whether under hypoxic or aerobic conditions, then, cancer tissue's reliance on glycolysis is associated with increased malignancy.

Lipoic acid (l,2-dithione-3-pentanoic acid) is a sulfur-containing antioxidant with metal-chelating and anti-glycation capabilities. Unlike many antioxidants which are active only in either the lipid or the aqueous phase, lipoic acid is active in both lipid and aqueous phases. The anti-glycation capacity of lipoic acid combined with its capacity for hydrophobic binding enables lipoic acid to prevent glycosylation of albumin in the bloodstream. Lipoic acid is the oxidized part of a redox pair, capable of being reduced to dihydrolipoic acid (DHLA). Lipoic acid is readily absorbed from the diet and is rapidly converted to DHLA by NADH or NADPH in most tissues. Thus, the highest tissue concentrations of free lipoic acid likely to be achieved through oral supplementation are at least ten times lower than those of other intracellular antioxidants, such as vitamin C and GSH. Unlike GSH, however, for which only the reduced form is an antioxidant, both lipoic acid and DHLA are antioxidants. Lipoic acid is active against OH^*, HClO, and O₂, but not against O₂ ^" or H₂O₂. DHLA is active against OH^" and HClO, but not against H₂O₂ or O₂.

In general, DHLA has superior antioxidant activity to lipoic acid; by donating two hydrogens, DHLA can neutralize free radicals without itself becoming a free radical. Both lipoic acid and DHLA can directly scavenge physiologically-relevant RONS in vitro, but it is unclear whether lipoic acid acts directly to scavenge RONS in vivo. Moreover, free lipoic acid is rapidly eliminated from cells, so any increases in direct radical scavenging activity are unlikely to be sustained.

Lipoic acid exists as two enantiomers, R- and S-enantiomer. Naturally-occurring lipoic acid is the R-form, but synthetic lipoic acid (known as alpha lipoic acid) is a racemic mixture of R-form and S-form. Although the R-enantiomer is more biologically active than the S-enantiomer, administration of alpha lipoic acid actually results in greater formation of DHLA due to a synergistic effect which each enantiomer exerts on the reduction of the other. Both lipoic acid and DHLA can chelate heavy metals that could generate free radicals, having been found both to inhibit copper- and iron-mediated oxidative damage in vitro and to inhibit excess iron and copper accumulation in vivo. However, the R-form is more effective for chelation than alpha-lipoic acid.

In mitochondria, lipoic acid can compensate for the low mitochondrial concentrations of GSH, which plays a role in the detoxification and elimination of potential carcinogens and toxins, generally through the induction of cystine/cysteine uptake, which thereby increases GSH synthesis, and by increasing the expression of gamma-glutamylcysteine ligase, the rate- limiting enzyme in GSH synthesis. Studies in animals have found that GSH synthesis and tissue GSH levels are significantly lower in aged animals than in younger animals, leading to decreased ability of aged animals to respond to oxidative stress or toxin exposure. It has consequently been demonstrated in old rats supplemented with R-lipoic acid versus control that mitochondrial membrane potentials and oxygen consumption were significantly restored while at the same time malondialdehyde, a product of lipid peroxidation, was reduced to one- fifth of the control level. Lipoic acid has also been shown to restore GSH synthesis to more youthful levels in aging rat liver. Thus, it has been demonstrated that supplementation with both lipoid acid and acetyl-1-carnitine is an effective way of improving mitochondrial metabolic function without increasing oxidative stress. (See Best B. Lipoic acid. http://www.benbest.com/nutrceut/lipoic.html, accessed on May 6, 2008, passim, herein incorporated by reference. See also Higdon J. Lipoic acid. http://lpi.oregonstate.edu/infocenter/othernuts/la/, accessed on May 7, 2008, passim, herein incorporated by reference.) Given the important role of lipoic acid in the regulation of RONS metabolism, then, it may be inferred that modulation of fatty acid-containing, -binding, and or -interacting enzymes, proteins, metals, ions, lipids, or nucleic acids which are associated with lipoate would have a similar effect on RONS metabolism.

The role of lipoic acid in the PDH complex of healthy cells has been well studied. The PDH complex has a central E2 (dihydrolipoyl transacetylase) subunit core surrounded by the El (pyruvate dehydrogenase) and E3 (dihydrolipoyl dehydrogenase) subunits to form the complex. In the gap between the El and E3 subunits, the lipoyl domain ferries intermediates between the active sites. The lipoyl domain itself is attached by a flexible linker to the E2 core. Upon formation of a hemithioacetal by the reaction of pyruvate and thiamine pyrophosphate, this anion attacks the SI of an oxidized lipoate species that is attached to a lysine residue. Consequently, the lipoate S2 is displaced as a sulfide or sulfhydryl moiety, and subsequent collapse of the tetrahedral hemithioacetal ejects thiazole, releasing the TPP cofactor and generating a thioacetate on the Sl of the lipoate. At this point, the lipoate- thioester functionality is translocated into the E2 active site, where a transacylation reaction transfers the acetyl from the "swinging arm" of lipoate to the thiol of coenzyme A. This produces acetyl-CoA, which is released from the enzyme complex and subsequently enters the TCA cycle. The dihydrolipoate, still bound to a lysine residue of the complex, then migrates to the E3 active site, where it undergoes a flavin-mediated oxidation back to its lipoate resting state, producing FADH₂ (and ultimately NADH) and regenerating the lipoate back into a competent acyl acceptor. Should this lipoate species be interrupted, then, there would be no flow of electrons to FADH₂ or generation of acetyl-CoA, and, as a consequence, a toxic buildup of pyruvate within the cell. In cancer cells, the production of acetoin has been suggested to be a requirement for cellular detoxification and survival.

The role of lipoic acid in the α-KDH complex has also been well studied. The α- KDH complex is an enzyme complex in the mitochondrial matrix composed of three central subunits, Elk, E2k, and E3 (α-ketoglutarate dehydrogenase, dihydrolipoyl succinyltransferase, and dihydrolipoyl dehydrogenase, respectively). Elk and E2k are unique to the α-KDH complex, whereas E3 is also a component of the PDH and BCAKDH complexes. Cofactors used with the α-KDH complex are thiamin pyrophosphate (TPP), which is bound to El and decarboxylates α ketoglutarate, yielding a hydroxyethyl-TTP carbanion, with lipoic acid as the prosthetic group; lipoic acid, which is covalently linked to a lysine residue on E2 in a lipoamide linkage and which accepts the hydroxyethyl carbanion from TPP as an succinyl group; coenzyme A (CoA), which is a substrate for E2 and accepts the succinyl group from the lipoamide; flavin adenine dinucleotide (FAD), which is bound to E3 and which is reduced by lipoamide, and nicotinamide adenine dinucleotide (NAD⁺), which is a substrate for E3 and is reduced by FADH₂. The α-KDH complex has a central E2 core, with the other subunits surrounding this core to form the complex. In the gap between these two subunits, the lipoyl domain ferries intermediates between the active sites. The lipoyl domain itself is attached by a flexible linker to the E2 core. Upon formation of a hemithiosuccinal by the reaction of pyruvate and thiamine pyrophosphate, this anion attacks the Sl of an oxidized lipoate species that is attached to a lysine residue. Consequently, the lipoate S2 is displaced as a sulfide or sulfhydryl moiety, and subsequent collapse of the tetrahedral hemithiosuccinal ejects thiazole, releasing the TPP cofactor and generating a thiosuccinate on the Sl of the lipoate. At this point, the lipoate-thioester functionality is translocated into the E2 active site, where a transacylation reaction transfers the succinyl from the "swinging arm" of lipoate to the thiol of coenzyme A. This produces succinyl-CoA, which is released from the enzyme complex and subsequently enters the TCA cycle. The dihydrolipoate, still bound to a lysine residue of the complex, then migrates to the E3 active site, where it undergoes a flavin-mediated oxidation back to its lipoate resting state, producing FADH₂ (and ultimately NADH) and regenerating the lipoate back into a competent acyl acceptor. Should this lipoate-mediated function be blocked, then, there would be no flow of electrons to FADH₂ or generation of acetyl-CoA, and, as a consequence, a toxic buildup of pyruvate within the cell. On the whole, the chemical aspect of the co-enzymatic action of lipoic acid is to mediate the transfer of electrons and activate acyl groups, resulting from the decarboxylation and oxidation of α-KDH within the complex. In this process, lipoic acid is itself transiently reduced to dihydrolipoic acid; this reduced form is the acceptor of the activated succinyl groups. Its dual role of electron and acyl-group acceptor enables lipoic acid to act as a shuttle and couple the two processes. The overall metabolic reaction in the TCA cycle which the α-KDH complex is responsible for is: α -ketoglutarate + NAD⁺ + CoA ^« succinyl CoA + CO₂ + NADH Allosterically, the α-KDH complex is inhibited by ATP and high succinyl CoA and activated by ADP and elevated NAD⁺/NADH and CoA/succinyl CoA ratios.

Additionally, as illustrated in FIGURE I ₅ the E3 subunit of the PDH and α-KDH complexes themselves are each capable of RONS generation upon stimulation by an appropriate agent or under the appropriate cellular conditions. (See Starkov A, Fiskum G, Chinopoulos C, Lorenzo B, I Browne S, Patel M, and Beal M (2004). Mitochondrial α- ketoglutarate dehydrogenase complex generates reactive oxygen species. J. Neurosci. 24:7779-7788, passim, herein incorporated by reference.) Given that all three enzymes share the same E3 subunit, while this proposition is unproven, it is likely that, under the appropriate conditions and stimuli, that BCAKDH generates RONS as well. Similarly, the signal transduction pathway-associated protein kinase C (PKC), and specifically its beta-II isoform (PKCpH), has been demonstrated to increase RONS production under hyperglycemic conditions, and, in fact, that PKCβll is itself activated by RONS through a positive-feedback system. {See Inoguchi T, Sonta T, Tsubouchi H, Etoh T, Kakimoto M, Sonoda N, Sato N, Sekiguchi N, Kobayashi K₅ Sumimoto H, Utsumi H, and Nawata H (2003). Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD(P)H oxidase. J. Am. Soc. Nephrol. 14:S227-S232, passim, herein incorporated by reference. See also Kim H, Sik Bang S, Seok Choi J, Han H, and Kim I (2005). Involvement of PKC and ROS in the cytotoxic mechanism of anti-leukemic decursin and its derivatives and their structure-activity relationship in human K562 erythroleukemia and U937 myeloleukemia cells. Cancer Lett. 223: 191-201, passim, herein incorporated by reference. See also Li PF, Maasch C, Haller H, Dietz R, and Von Harsdorf R (1999). Requirement for protein kinase C in reactive oxygen species-induced apoptosis of vascular smooth muscle cells. Circulation 100:967-973, passim, herein incorporated by reference.) Accordingly, tumor cells cannot build up pyruvate, associated aldehydes, and RONS indefinitely, as these molecules are cytotoxic at high levels through such mechanisms as drastically lowering cellular pH. It has thus been described, for AS-30D and Ehrlich hepatomas, that a significant fraction of mitochondrial pyruvate is decarboxylated to an active acetaldehyde by the El component of the PDH complex via bound β- hydroxyethylthiamine pyrophosphate. This active acetaldehyde is in turn condensed with a second acetaldehyde, ultimately deacidifying or reducing the original pyruvate, by using either lipoic acid or the amino acid glutamine, to generate acetoin (3-hydroxybutanone), a compound which both competitively inhibits PDH and which is less toxic to the cell than its pyruvate precursor {e.g., by maintaining pH homeostasis within the cell). Despite the importance of acetoin in the pathway of tumor cell detoxification as a result of pyruvate and RONS buildup due to the tumor cell's reliance of glycolysis as a source of ATP production, however, there was little reference in the prior art to the effects of blocking the production of acetoin and consequent RONS buildup on tumor cell viability.

However, within the last few years, cellular redox changes initially thought of solely as deleterious have emerged as a pivotal and proximal event in cancer. Consequently, altering the redox status of the cancer cell by natural and pharmaceutical agents represents a valid strategy for cancer treatment or prevention.

As it was assumed for years that all RONS are bad for the cell, the consequent response was to attempt to suppress RONS, hoping to prevent or even reverse RONS-related biological damage. However, it is now recognized that there exists a network of redox-based regulatory mechanisms that are often quite relevant to carcinogenesis. Additionally, while at low concentrations RONS protect the cell and at higher concentrations can damage many biological molecules (e.g., DNA, proteins, and lipids), it is now recognized that, at such higher concentrations, RONS may also help prevent cancer by initiating the death of the transformed cell.

Redox signalling is a reversible phase of physiological regulatory reactions occurring over shorter time periods which primarily concern the main cellular redox systems (e.g., GSH, ascorbate, vitamin E, lipoic acid, NADPH, or NADH). The oxidative reactions, leading to post-translational protein modification, are returned to the resting state by reductive pathways. Such modifications include glutathiolation, S-nitrosylation, methionine sulfoxidation, and oxidations with disulphide formation. In contrast, oxidative stress denotes a longer-lasting, often irreversible oxidative shift that characterizes a pathophysiological state. It has been defined as an imbalance between oxidants and antioxidants in favor of the former, resulting in increased cellular levels of RONS, and has been implicated in the pathogenesis of several diseases, including cancer. There is consequently significant evidence for a role of RONS in cancer, including genotoxicity; promotion of transformed cell growth; angiogenesis; and regulation of apoptosis.

For example, a "persistent oxidative stress in cancer" has been hypothesized. (Toyokuni S., Okamoto K., Yodoi J., Hiai H. (1995). Persistent oxidative stress in cancer. FEBS Lett 358: 1 -3, herein incorporated by reference). Such oxidative stress was considered to contribute to oncogene activation, genomic instability, chemotherapy resistance, and even invasion and metastasis. Nuclear factor-κB (NF-κB), MAPK cascades, and GSH and related antioxidant pathways were hypothesized to be the mediators. Chronic inflammation, widely connected to carcinogenesis, is also a source of RONS, and a linkage of inflammation-generated RONS to cancer has also been postulated. HIF- lα is linked to cancer through its regulation by RONS; in particular, RONS signalling may account for the high levels of HIF-I α in normoxic areas of tumors. HlF- l α promotes survival in low oxygen conditions, like those encountered in cancer, by upregulating an array of hypoxia-induced genes, including the vascular endothelial factor, which promotes angiogenesis. Finally, RONS have been associated with the induction of apoptotic and necrotic cell death, the specific outcome depending, among others, on the cellular levels of RONS. However, as it has been noted that treatment of tumor cells with NO-donating aspirin, n-3 polyunsaturated fatty acids, and other conventional non-steroidal anti-inflammatory drugs generates redox changes, through increased RONS production, which lead either to reversible redox signalling or irreversible oxidative stress, these changes in turn culminating in tumor cell death through apoptosis or necrosis (Rigas and Sun, supra), RONS-generating therapeutic agents may be useful in the treatment of cancer.

Also, it has been demonstrated in a murine tumor cell line that mutations in mitochondrial DNA which are associated with overproduction of reactive oxygen species (and therefore likely RONS) can contribute to tumor progression by enhancing the metastatic potential of tumor cells, as murine tumor cell lines which were poorly metastatic acquired increased metastatic potential upon the transfer of mitochondrial DNA bearing mutations of the NADH dehydrogenase subunit 6 gene from a murine tumor cell line which was highly metastatic. (See Ishikawa K. et al (2008). ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 320:661-664, herein incorporated by reference.) Hence, it may be seen that there is also a genetic component to the production and/or metabolism of RONS which may be exploited in the treatment of cancer.

Recent studies suggest that forcing cancer cells into more aerobic metabolism suppresses tumor growth. The transition to Warburg metabolism therefore requires shutting down the PDH complex. Phosphorylation by PDKl can be particularly effective for maintaining an inactive PDH complex since this isoform uniquely phosphorylates three serine residues in the alpha subunit of El , the first subunit of the PDH complex. Reactivation of El requires the removal of all three phosphate groups. Furthermore, RONS production by E3 propagates a chain of events leading in due course to PDH complex inactivation, which eventually leads to cell death. However, alterations in PDKl observed in cancer may not only be due to changes in its concentration but also to changes in its activity and possibly in its amino acid sequence, even between one tumor type or one patient to another. Additionally, PDKl may form different complexes with various molecules associated with tumors depending upon tumor type. Thus, inhibition of PDK may be a potential target in generating apoptosis in tumors. However, to date, known PDKl inhibitors have been demonstrated to cause maximally only 60% inhibition of this isozyme.

While traditional chemotherapy targets dividing, proliferating cells, all clinically- accepted chemotherapeutic treatments use large drug doses that also induce profound damage to normal, proliferative host cells. Therefore, more selective targeting is required for the treatment of cancer. Another problem associated with chemotherapy is that, in many tumor types, there is either inherent or acquired resistance to antineoplastic drugs. Overall, traditional chemotherapy currently offers little long-term benefit for most malignant tumors and is often associated with adverse side-effects that diminish the length or quality of life. Hence, radical new approaches are required that can provide long-term management of tumors while permitting a decent quality of life.

Certainly, drug efficacy, delivery, and side-effects are problems that need to be solved in developing new chemotherapies. In solid tumors, delivery to a hypoxic region may be difficult when the drug does not permeate through the different cellular layers easily. To eliminate these uncertainties, it seems relevant to design anticancer agents having metabolic inhibition constants in, at least, the submicromolar range. It may be argued that cancer cells are genetic and phenotypically heterogeneous from line to line. However, all tumor cell lines depend on glycolysis and oxidative phosphorylation for ATP supply. Concentrating on the Warburg effect allows for designing drugs based on the physico- and biochemical energetic differences between tumor and normal cells to facilitate the design of delivery and therapeutic strategies that selectively affect solely tumor metabolism and growth, without affecting healthy host tissue and organ functionality.

US Patents 6,090,842, 6,235,772, and 6,387,945 all to Packer et al., all herein incorporated by reference, teach lipoic acid analogues of various specific structures, and/or methods of production thereof, which may be used to treat conditions involving reactive oxygen species or redox mechanisms, including cancer. However, not only is it contemplated that the various ailments associated with these species or mechanisms are associated with aging, it is the ultimate aim of these analogs to reduce the concentration of reactive oxygen species within a diseased cell, and not to increase mitochondrial RONS concentrations to lead to tumor cell death. US Patents 6,331,559 and 6,951,887 to Bingham et al., as well as US Patent Application No. 12/105,096 by Bingham et al., all herein incorporated by reference, disclose a novel class of lipoic acid derivative therapeutic agents which selectively target and kill both tumor cells and certain other types of diseased cells. These teachings further disclose pharmaceutical compositions, and methods of use thereof, comprising an effective amount of such lipoic acid derivatives along with a pharmaceutically acceptable carrier. However, while these patents describe the structures of and general use for these lipoic acid derivatives, there is no indication in either patent that these derivatives are useful in modulating the structure, function, activity, cellular location or compartmentalization, and/or expression level of aliphatic fatty acid-containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids via increasing cellular RONS concentrations, leading to tumor cell death.

As it has been demonstrated that the structure, function, activity, cellular location or compartmentalization, and/or expression level of aliphatic fatty acid-containing, -binding, and/or interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids is a critical determinant of tumor activity, then, and as such structure, function, activity, cellular location or compartmentalization, and/or expression level may be influenced by the mitochondrial redox state, it would be beneficial to provide for a modulator of the structure, function, activity, cellular location or compartmentalization, and/or the expression level, of aliphatic fatty acid-containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids which generates increased mitochondrial RONS levels and thus culminates in apoptotic or necrotic tumor cell death. Objects of the Invention and Industrial Applicability

Consequently, it is an object of the present invention to provide a pharmaceutically- acceptable agent to be used in diseased cells which causes minimal side effects upon administration. It is a further object of the present invention to provide a pharmaceutically-acceptable agent to be used in diseased cells which is easily manufactured at the least possible cost and is capable of being stored for the longest possible period.

It is a still further object of the present invention to provide a pharmaceutically- acceptable agent to be used in diseased cells which modulates mitochondrial energy metabolism, especially via the structure, function, activity, cellular location or compartmentalization, and/or expression level of aliphatic fatty acid-containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids and other dehydrogenases, kinases, and phosphatases in tumor cell mitochondria.

It is a still further object of the present invention to provide a physiologically-active agent to be used in hyperproliferative cells which modulates the structure, function, activity, cellular location or compartmentalization, and/or expression level of aliphatic fatty acid- containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids in diseased cell mitochondria through the altered production of metabolic byproducts, such as but not limited to RONS, in the mitochondrion, which in turn culminates in apoptotic or necrotic tumor cell death.

Summary of the Invention

To achieve the aforementioned aims, the present invention broadly discloses pharmaceutically-acceptable agents which affect metabolic by-product production, such as but not limited to RONS, through modulation of the structure, function, activity, cellular location or compartmentalization, and/or expression levels of aliphatic fatty-acid-containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids in diseased cells. In a preferred embodiment of the present invention, the aliphatic fatty acid has a chain length of three to twenty carbons, and in a more preferred embodiment, the fatty acid is lipoate or analogs, congeners, and derivatives thereof. The proteins may be carrier proteins, channel proteins, receptor proteins, or enzymes. In one preferred embodiment, the receptor protein is the insulin receptor tyrosine kinase. In other preferred embodiments, the proteins may be a dehydrogenase, such as dihydrolipoyl dehydrogenase or the PDH, α-KDH, or BCAKDH complexes or the mutants of each thereof; a kinase, such as PKC and its PKCβll isoform and the mutants of each thereof, or PDK and its isoforms PDKl, PDK2, PDK3, PDK4, and the mutants of each thereof; or a phosphatase, such as PDP and its isoforms PDPl and PDP2 and the mutants of each thereof.

The agents of the present application work through an initial generation of metabolic by-products such as RONS across tumor cell types. Through the initial generation of RONS by stimulation of the E3 dihydrolipoyl dehydrogenase subunit of the PDH, α-KDH, and/or BCAKDH complexes, these RONS themselves stimulate further RONS production by PKC, forming a positive-feedback loop. By concomitantly also inhibiting mitochondrial energy metabolism through the activation of PDKs and/or inhibition of PDPs which thereby inhibit the activity of the El subunit of the PDH complex, thereby further increasing RONS production within the mitochondrion, the agents of the present invention cause both the loss of mitochondrial membrane potential and other mitochondrial consequences in the diseased cell, resulting in the irreversible initiation of cell death through either apoptosis or necrosis.

The agents of the present invention may also modulate the expression level of the phosphatase, kinase, and dehydrogenase enzyme constituents found in the PDH, α-KDH, and/or BCAKDH complexes. This modulation may occur at the transcriptional, translational, or post-translational stage, including epigenetic silencing of the appropriate genes.

As a compound which exerts influence on molecules fundamentally associated with the TCA cycle, and by extension glycolysis, the agents of the present invention may demonstrate selective uptake into diseased cells. Furthermore, such selective diseased cell uptake should minimize the side effects the administration of this agent would have on healthy non-transformed cells and tissue.

In one embodiment of the present invention, the agents have the general formula:

wherein x is 0- 16, mixed saturated or unsaturated;

Ria is CH₂, O, S, N, H, phenyl, or a metal; R_2a is CH₂, O, S, N, H, phenyl, or a metal; R₃ is -OH, -COH, -NH₂OH, -CCl₃, -CF₃, or -COOH;

R₄ is H, -OH, -COH, -NH₂OH, -CCI₃, -CF₃, -COOH, amino acids, carbohydrates, nucleic acids, lipids, and multimers thereof; Rib and R_2b can be independently:

(1) an acyl group R₅C(O)-, where R₅ is an alkyl, aryl, or organometallic aryl group, linked through a thioester linkage, including but not limited to acetyl and butaryl, with a specific example being bis-acetyl lipoate; (2) an aromatic group linked through a thioester linkage, including but not limited to benzoyl or a benzoyl derivative, with a specific example being bis-benzoyl lipoate;

(3) an alkyl group C_nH_2n+I, where n is 1-10, linked through a thioether linkage with such alkyl groups substituted with other moieties such as, for example, -OH, -Cl or -NH₂, including but not limited to methyl, ethyl, butyl, decanyl, and 6,8-bis carbomoyl methylipoate;

(4) an alkenyl group C_nH_2n-_I, where n is 2-10, linked through a thioether linkage, including but not limited to propylene, 2,3 dimethyl-2-butene, and heptene; (5) an alkynyl group C_nH_2n ^, where n is 2- 10, linked through a thioether linkage, including but not limited to acetylene, propyne, and octyne;

(6) alkyl, alkenyl, and alkynyl groups which can be either open chains or alicyclics, with the alicyclic groups having additions or substitutions of any of the carbons to form heterocyclics, including but not limited to cyclopropane, cyclopentene, and 6,8 methyl- succinimido lipoate;

(7) alkyl, alkenyl, and alkynyl groups which can have additions on any of their carbons, including but not limited to hydroxyls and amines;

(8) an aromatic or aryl group linked through a thioether linkage which can be a benzene or a benzene derivative, including but not limited to toluene and aniline; (9) alkyl sulfide groups CH₃(CH₂VS-, where n can be but is not limited to 0-9, linked through a disulfide linkage;

(10) imidoyl groups CHR₆Q=NH)-, where n can be but is not limited to 1-10, linked through a thioamide linkage; and

(1 1) semiacetal groups R₇CH(OH)-S-, where R₅ is limited to compounds with strongly electron withdrawing substituents, including but not limited to trichloroacetaldehyde and pyruvic acid; or salts, prodrugs, or solvates thereof.

Ri_b and R_2b as defined above can be unsubstituted or substituted and may also comprise thioesters that can be oxidized to produce sulfoxides or sulfones, for example, C- S(O)-R and C-S(O)₂-R, respectively. Ri and R₂ may further comprise disulfides that can be oxidized to thiosulfinic or thiosulfonic acids, for example C-S(O)-S-R and C-S(O)₂-S-R, respectively.

Re is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which can be substituted or unsubstituted. Similarly, R₇ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, any of which can be substituted or unsubstituted.

Furthermore, as any or all of these general structures may be metabolized within the cell or mitochondrion, it is expressly intended that metabolites of the above-referenced structure is within the scope of the present invention.

In each of the general formulae, the (R)-isomer of each particular agent possesses greater physiological activity than does the (S)-isomer. Consequently, the agent should be present either solely in its (R)-isomer form or in a mixture of the (R)- and (S)-isomers.

Brief Description of the Figures

The following drawings are illustrative of embodiments of the invention and are not intended to limit the scope of the application as encompassed by the entire specification and claims.

FIGURE 1 illustrates the general principal of RONS generation by stimulation of the E3 subunit of the PDH and/or α-KDH complexes.

FIGURE 2 shows the positive-feedback loop created by RONS generation of both the PDH and/or α-KDH complexes and PKC in tumor cells when stimulated by the agents of the present invention as compared to that in normal cells under identical stimulation.

FIGURE 3 depicts the general results of RONS generation by the agents of the present invention upon both the PDH complex and the energy metabolism of cancer cells. FIGURE 4 illustrates the relative effects of the agents of the present invention on the PDH complex versus those on the α-KDH complex over time.

FIGURE 5 demonstrates dose/response results of FACS assays of dichlorofluorescein (DCF) oxidation in MDA cancer cells using the agents of the present invention. FIGURE 6 shows the mean of FACS distribution as a function of time of exposure to the agents of the present invention.

FIGURE 7 shows the accumulation of the agents of the present invention in treated HepG2 cells versus mock-treated cells.

FIGURE 8 illustrates the effect of PKC inhibitors on the cytotoxic effects of the agents of the present invention on H460 NSCLC tumor cells.

FIGURE 9 depicts the effects of GSH depletion by DL-Buthionine-[S,R]-sulfoximine (BSO) at various concentrations of the agents of the present invention.

FIGURE 10 depicts the effect on H₂O₂ production by the PDH complex of increasing concentration of the agents of the present invention. FIGURE 1 1 illustrates the effect on H₂O₂ production by the PDH complex of the agents of the present invention versus that of other compounds.

FIGURE 12 depicts the effect on H₂O₂ production by the α-KDH complex of increasing concentration of the agents of the present invention.

FIGURE 13 illustrates the effect on H₂O₂ production by the α-KDH complex of the agents of the present invention versus that of other compounds.

FIGURE 14 shows an idealized graph showing the effects of antioxidant compounds versus that of RONS-enhancing compounds coupled with that of the agents of the present invention on tumor cell survivability.

FIGURE 15 demonstrates that mitochondrial Iysates from tumor cells are fifty times more sensitive to RONS than those from normal cells. Detailed Description of the Invention

The present invention is generally directed to pharmaceutically-acceptable agents that affect metabolic by-product production, such as but not limited to RONS production, through modulation of the structure, function, activity, cellular location or compartmentalization, and/or expression levels of aliphatic fatty-acid-containing, -binding, and/or -interacting proteins in diseased cells. In a preferred embodiment of the present invention, the fatty acid has a chain length of three to twenty carbons, and in a more preferred embodiment, the fatty acid is lipoate, or analogs, congeners, and derivatives thereof. The proteins may be carrier proteins, channel proteins, receptor proteins, or enzymes. In one preferred embodiment, the receptor protein is the insulin receptor tyrosine kinase binding site for lipoate. In other preferred embodiments, the proteins may be a dehydrogenase, such as dihydrolipoyl dehydrogenase or the PDH or α-KDH complexes; a kinase, such as PKC and its PKCβll isoform and the mutants thereof, or PDK and its isoforms PDKl, PDK2, PDK3, PDK4, and the mutants of each thereof; or a phosphatase, such as PDP and its isoforms PDPl and PDP2 and the mutants of each thereof.

The agents of the present application work through an initial generation of RONS across tumor cell types. Through the initial generation of RONS by stimulation of the E3 dihydrolipoyl dehydrogenase subunit of the PDH and/or α-KDH complexes, these RONS themselves stimulate further RONS production by PKC, forming a positive-feedback loop, as seen in FIGURE 2. By concomitantly also inhibiting mitochondrial energy metabolism through the activation of PDKs and/or inhibition of PDPs which thereby inhibit the activity of the El subunit of the PDH complex, thereby increasing RONS production within the mitochondrion, as shown in FIGURE 3, the agents of the present invention cause both the loss of mitochondrial membrane potential and other mitochondrial consequences in the diseased cell, resulting in the irreversible initiation of cell death through either apoptosis or necrosis.

The agents of the present invention may also modulate the expression level of the phosphorylase, kinase, and dehydrogenase enzyme constituents found in the PDH complex. This modulation may occur at the transcriptional, translational, or post-translational stage, including epigenetic silencing of the appropriate genes.

As a molecule which is not only a derivative of one which is found normally within mitochondria but also one which is instrumental to the increased glycolytic activity of tumor cells as seen in the Warburg effect, the agents of the present invention are particularly well- suited for the selective delivery into and effective concentration within the mitochondria of tumor cells and tissues, thereby sparing normal cells and tissue from the effects of the agents.

wherein x is 0-16, mixed saturated or unsaturated; R,_a is CH₂, O, S, N, H, phenyl, or a metal;

R_2a is CH₂, O, S, N, H, phenyl, or a metal;

R₃ is -OH, -COH, -NH₂OH, -CCI₃, -CF₃, or -COOH;

R₄ is H, -OH, -COH, -NH₂OH, -CCl₃, -CF₃, -COOH, amino acids, carbohydrates, nucleic acids, lipids, and multimers thereof; Rib and R₂b can be independently:

(1) an acyl group RsC(O)-, where Rs is an alkyl, aryl, or organometallic aryl group, linked through a thioester linkage, including but not limited to acetyl and butaryl, with a specific example being bis-acetyl lipoate; (2) an aromatic group linked through a thioester linkage, including but not limited to benzoyl or a benzoyl derivative, with a specific example being bis-benzoyl lipoate;

(3) an alkyl group C_nH_2n+!, where n is 1-10, linked through a thioether linkage with such alkyl groups substituted with other moieties such as, for example, -OH, -Cl or -NH₂, including but not limited to methyl, ethyl, butyl, decanyl, and 6,8-bis carbomoyl methylipoate;

(4) an alkenyl group C_nH_2n-I, where n is 2-10, linked through a thioether linkage, including but not limited to propylene, 2,3 dimethyl-2-butene, and heptene;

(5) an alkynyl group C_nH_2n_₂, where n is 2-10, linked through a thioether linkage, including but not limited to acetylene, propyne, and octyne;

(6) alkyl, alkenyl, and alkynyl groups which can be either open chains or alicyclics, with the alicyclic groups having additions or substitutions of any of the carbons to form heterocyclics, including but not limited to cyclopropane, cyclopentene, and 6,8 methyl- succinimido lipoate; (7) alkyl, alkenyl, and alkynyl groups which can have additions on any of their carbons, including but not limited to hydroxyls and amines;

(8) an aromatic or aryl group linked through a thioether linkage which can be a benzene or a benzene derivative, including but not limited to toluene and aniline;

(9) alkyl sulfide groups CH₃(CH₂VS-, where n can be but is not limited to 0-9, linked through a disulfide linkage;

(10) imidoyl groups CHR6C(=NH)-, where n can be but is not limited to 1-10, linked through a thioamide linkage; and

Rib and R₂b as defined above can be unsubstituted or substituted and may also comprise thioesters that can be oxidized to produce sulfoxides or sulfones, for example, C- S(O)-R and C-S(O)₂-R, respectively. R| and R₂ may further comprise disulfides that can be oxidized to thiosulfinic or thiosulfonic acids, for example C-S(O)-S-R and C-S(O)₂-S-R, respectively.

Furthermore, as any or all of these general structures may be metabolized within the cell or mitochondrion, it is expressly intended that metabolites of the above-referenced structures are within the scope of the present invention. In each of the general formulae, the (R)-isomer of each particular lipoic acid derivative possesses greater physiological activity than does the (S)-isomer. Consequently, the lipoic acid derivative should be present either solely in its (R)-isomer form or in a mixture of the (R)- and (S)-isomers.

Turning to the figures, that the agents of the present invention have a greater effect over time on the PDH complex alone and not on the α-KDH complex is seen given the data from PDH and αKDH complex enzyme assays revealed in FIGURE 4. Tumor cells were grown to 80% confluence in 15cm plates and treated with the agents of the present invention as indicated. Mitochondria were isolated according to the method of Moreadith and Fiskum.

Mitochondria were lysed in 0.4% lauryl maltoside. 50μl of mitochondrial lysate was added to 96 well plates. 50μl of reaction mix (5OmM Tris, pH 7.5, 2mM β-NAD⁺, 225μM TPP, 2mM pyruvate or α-ketoglutarate, 150μM coenzyme A, 2.6mM cysteine, ImM MgCl₂ ) was added to mitochondrial lysates and the mixture was incubated for 45 minutes at 37⁰C. At this time, 15μM resazurin and 0.5U/ml diaphorase were added to the mixture and incubated for an additional five minutes. NADH production was monitored by measuring fluorescence using an excitation wavelength of 530nm and an emission wavelength of 590nm in a microplate reader (Fluorostar). All measurements were performed in duplicate and showed high consistency. It is evident upon inspection of the results that merely activating the αKDH complex is insufficient to demonstrate and explain the effects of the agents of the present invention. Thus, there must be an additional and/or synergistic phenomenon occurring within the mitochondrion of a tumor cell upon administration of these agents.

FIGURE 5 illustrates how DCF oxidation measures RONS generation in MDA cancer cells in response to treatment with the agents of the present invention at given concentrations. These experiments were done in DMEM medium with 10% serum. The control contained the same amount of solvent as the 400μM experimental sample. Experiments were performed according to manufacturer's directions (Molecular Probes; Invitrogen). It is evident from the graph that RONS generation in the tumor cells increased as the concentration of the agents increased. Thus, FIGURE 6 similarly shows that, as the concentration of the agent increases over time, the greater the degree of DCF oxidation in tumor cells. Indeed, FIGURE 7 plainly shows the accumulation of the agents of the present invention in HepG2 human hepatocellular liver carcinoma cells 35 minutes after both treatment with the agent at 300μM and 5μM DCF (CM-H2DCFDA) versus the lack of accumulation seen in mock-treated cells.

FIGURE 8 illustrates the effect of PKC inhibitors on the cytotoxic effects of the agents of the present invention on H460 NSCLC tumor cells. H460 NSCLC cells were seeded on a 96-welI microtiter plate. The cells were treated with either the agents of the present invention at concentrations of OμM, 25μM, or 50μM. Non-control cells were also co- treated with one of three separate PKC inhibitors: 2μM Go6976 (12-(2-cyanoethyl)- 6,7, 12, 13-tetrahydro- 13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole), 4μM

Go6983 (2-[l-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(lH-indol-3-yl)maleimide), or 4μM bisindolylmaleimide. After 24 hours of treatment, cell survival was assayed using CellTiter-Glo (Promega) cell viability assay (based on ATP steady state concentrations). In each case, tumor cell survival was increased upon treatment with a PKC inhibitor versus that seen with control. Consequently, PKC must be a significant contributor to the cytotoxic effects of the agents of the present invention. Turning to FIGURE 9, an experiment was performed where H460 cells were seeded in a 96-well plate and treated with both the agents of the present invention at concentrations of OμM, 20μM, 30μM, 40μM, 50μM, and 60μM and with BSO, an inhibitor of glutathione synthesis which leads to increased cellular susceptibility to RONS, at concentrations of OμM, 25μM, 50μM, lOOμM, and 200μM. After 24 hours of treatment, cell survival was assayed using CellTiter-Glo (Promega) cell viability assay (based on ATP steady state concentrations). As seen in FIGURE 9, at all concentrations of BSO, increasing the concentration of the agents of the present invention leads to decreased levels of glutathione, which leads to decreased scavenging of RONS in the mitochondrion and therefore potentiates the cytotoxicity of the tumor cell to the agents of the present invention, thus establishing that increased RONS levels contributes to the agents' cytotoxicity.

Next, FIGURE 10 depicts the effect on H₂O₂ production by the PDH complex of increasing concentration of the agents of the present invention. Porcine heart PDH complex enzyme was purchased from Sigma. Production of H₂O₂ by the PDH complex upon administration of the agents of the present invention at concentrations of OμM, 50μM, 1 OOμM, 200μM, and 400μM was assayed at administration and 20-minute intervals up to 100 minutes post-administration using Amplex Red H?O₂/peroxidase assay kit (Invitrogen) by its ability to oxidize Amplex Red to rizafurin in the presence of peroxidase. As is evident from the data, H₂O₂ production increased as agent concentration increased.

Similarly, FIGURE 1 1 illustrates the effect on H₂O₂ production by the PDH complex of the agents of the present invention versus that of other compounds. Porcine heart PDH complex enzyme was purchased from Sigma. Production of H₂O₂ by the PDH complex upon administration of the agents of the present invention at concentrations of 200μM; administration of three less active compounds each at 200μM; and untreated and solvent-only control was assayed at administration and at 10-minute intervals up to 50 minutes post- administration using Amplex Red H?O₂/peroxidase assay kit (Invitrogen) by its ability to oxidize Amplex Red to rizafurin in the presence of peroxidase. As seen in the figure, H₂O₂ production by the agents of the present invention is nearly triple that of control.

H₂O₂ production by the α-KDH complex of increasing concentration of the agents of the present invention is illustrated in FIGURE 12. Porcine heart α-KDH complex enzyme was purchased from Sigma. Production of H₂O₂ by the α-KDH complex upon administration of the agents of the present invention at concentrations of lOOμM, 200μM, and 400μM with control was assayed at administration and 10-minute intervals up to 50 minutes post- administration using Amplex Red H₂O₂/peroxidase assay kit (Invitrogen) by its ability to oxidize Amplex Red to rizafurin in the presence of peroxidase. As is evident from the data, H₂O₂ production increased as agent concentration increased.

Finally, FIGURE 13 similarly illustrates the effect on H₂O₂ production by the α-KDH complex of the agents of the present invention versus that of other compounds. Porcine heart α-KDH complex enzyme was purchased from Sigma. Production of H₂O₂ by the α-KDH complex upon administration of the agents of the present invention at concentrations of 200μM; administration of three less active compounds each at 200μM; and solvent-only control was assayed at administration and at 10-minute intervals up to 50 minutes post- administration using Amplex Red H₂O₂/peroxidase assay kit (Invitrogen) by its ability to oxidize Amplex Red to rizafurin in the presence of peroxidase. As seen in the figure, H₂O₂ production by the agents of the present invention is nearly double that of control. The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. Furthermore, while exemplary embodiments have been expressed herein, others practiced in the art may be aware of other designs or uses of the present invention. Thus, while the present invention has been described in connection with exemplary embodiments thereof, it will be understood that many modifications in both design and use will be apparent to those of ordinary skill in the art, and this application is intended to cover any adaptations or variations thereof. It is therefore manifestly intended that this invention be limited only by the claims and the equivalents thereof.

Claims

The invention to be claimed is:

1. Pharmaceutically-acceptable agents which affect the production of metabolic by-products, such as but not limited to reactive oxygen and nitrogen species (RONS), by modulating the structure, function, activity, cellular location or compartmentalization, and/or expression level of aliphatic fatty acid-containing, -binding, and/or -interacting enzymes, proteins, metals, ions, lipids, and/or nucleic acids.

2. The agents of claim 1, wherein the fatty acid has a chain length of three to twenty carbons.

3. The agents of claim 2, wherein the fatty acid is lipoate or analogs, congeners, or derivatives thereof.

4. The agents of claim 1, wherein the protein is selected from the group consisting of carrier proteins, receptor proteins, channel proteins, and enzymes.

5. The agents of claim 4, wherein the protein is a dehydrogenase.

6. The agents of claim 5, wherein the protein is dihydrolipoyl dehydrogenase and isoforms and mutants thereof.

7. The agents of claim 5, wherein the protein is a kinase.

8. The agents of claim 7, wherein the kinase is protein kinase C (PKC) and isoforms and mutants thereof.

9. The agents of claim 8, wherein the PKC is PKCβ and isoforms and mutants thereof.

10. The agents of claim 9, wherein the PKCβ is PKCβll and isoforms and mutants thereof.

1 1. The agents of claim 7, wherein the kinase is pyruvate dehydrogenase kinase (PDK) 1 , PDK2, PDK3, PDK4, and isoforms and mutants of each thereof.

12. The agents of claim 4, wherein the protein is a phosphatase.

13. The agents of claim 12, wherein the phosphatase is pyruvate dehydrogenase phosphatase (PDP) 1 , PDP2, and isoforms and mutants of each thereof.

14. The agents of claim 4, wherein the protein is the insulin receptor tyrosine kinase and isoforms and mutants thereof.

15. The agents of claim 1, wherein the protein is mitochondrial.

16. The agents of claim 15, wherein the protein is transported into the mitochondrion.

17. The agents of claim 15, wherein the protein is native to the mitochondrion.

18. The agents of claim 15, wherein the protein is exported from the mitochondrion.

19. The agents of claim 1 , wherein the agent has the structure:

wherein x is 0-16, mixed saturated or unsaturated; Ria is CH₂, O, S, N, H, phenyl, or a metal; R_2a is CH₂, O, S, N, H, phenyl, or a metal;

R₃ is -OH, -COH, -NH₂OH, -CCl₃, -CF₃, or -COOH;

R₄ is H, -OH, -COH, -NH₂OH, -CCl₃, -CF₃, -COOH, alkyl, alkenyl, and alkynyl groups, amino acids, carbohydrates, nucleic acids, lipids, and multimers thereof;

Rib and R₂b can be independently: (1) an acyl group R₅C(O)-, where R₅ is an alkyl, aryl, or organometallic aryl group, linked through a thioester linkage, including but not limited to acetyl and butaryl, with a specific example being bis-acetyl lipoate; (2) an aromatic group linked through a thioester linkage, including but not limited to benzoyl or a benzoyl derivative, with a specific example being bis-benzoyl lipoate;

(5) an alkynyl group C_nH_2n-₂, where n is 2-10, linked through a thioether linkage, including but not limited to acetylene, propyne, and octyne;

(9) alkyl sulfide groups CHs(CHi)_n-S-, where n can be but is not limited to 0-9, linked through a disulfide linkage;

(10) imidoyl groups CHR₆C(=NH)-, where n can be but is not limited to 1-10, linked through a thioamide linkage; and

(1 1) semiacetal groups RvCH(OH)-S-, where R₅ is limited to compounds with strongly electron withdrawing substituents, including but not limited to trichloroacetaldehyde and pyruvic acid; Rib and R_2b can be unsubstituted or substituted and may further comprise:

(1) thioesters that can be oxidized to produce sulfoxides or sulfones, for example, C- S(O)-R and C-S(O)₂-R, respectively;

(2) disulfides that can be oxidized to thiosulfinic or thiosulfonic acids, for example C- S(O)-S-R and C-S(O)₂-S-R, respectively;

Rδ is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which can be substituted or unsubstituted;

R₇ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, any of which can be substituted or unsubstituted; or salts, prodrugs, or solvates thereof.

20. The agent of claim 1 , wherein the agent is used to treat diseased cells and tissue.