CA2717511A1 - Modulation of enzymatic structure, activity, and/or expression level - Google Patents

Abstract

A pharmaceutically-acceptable modulator of the structure, activity, or expression level of an enzyme complex, such as the pyruvate dehydrogenase (PD) complex, in the mitochondria of warm-blooded ani-mals, including humans, and methods of use thereof, comprises an effective amount of at least one lipoic acid derivative and at least one pharma-ceutically-acceptable carrier thereof. By preventing the detoxification of toxic metabolites of anaerobic glycolysis through inhibition of the activity of the PD complex E1.alpha. subunit, the modulator increases PD kinase activity and concomitantly decreases PD phosphatase activity, obliging increased oxidative phosphorylation activity in the mitochondrion. As a cell charac-terized by hyperprolif eration, such as a tumor cell, cannot also generate acetyl-CoA and NADH because of the action of the modulator in inhibiting the action of the E2 subunit of the PD complex, the mitochondrial mem-brane polarization is lost, facilitating cell death.

Description

Modulation of Enzymatic Structure, Activity, and/or Expression Level Field of the Invention This invention relates to therapeutic and diagnostic compositions, and more particularly to pharmaceutical compositions, and methods of use thereof, which demonstrate selective uptake into cells characterized by hyperproliferation, including cancer cells, and which modulate the structure, activity, and/or expression levels of enzymes, thereby facilitating the destruction of these cells, and more specifically targeting the modified mitochondrial pyruvate dehydrogenase (PDH) complex associated with most cancers.

Background of the Invention The vast majority of fast-growth tumor cells exhibits profound genetic, biochemical, and histological differences with respect to nontransformed cells, including a markedly-modified 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 02 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 02 concentrations, such as the partial hypoxia observed in poorly-oxygenated solid tumors.
In particular, since the concentration of 02 is lower than 20 pM 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.

As depicted in FIGURE 1, for years, the tricarboxylic acid (TCA) cycle was regarded as only 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 upregulated 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.

The role of lipoic acid in the PDH complex has been well studied. The PDH complex has three central subunits, El, E2, and E3 (pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoamide dehydrogenase, respectively). These complexes have 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 hemithioacetal by the reaction of pyruvate and thiamine pyrophosphate, this anion attacks the S1 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 Si 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.

The activity of the PDH complex in mitochondria is highly regulated by a variety of allosteric effectors and by covalent modification. PDH activity is regulated by its state of phosphorylation, being most active in the dephosphorylated state. PDH phosphorylation is catalyzed by PDH kinase (PDK).
PDK activity is enhanced by an increase in the level of ATP, NADH, and acetyl-CoA. Negative effectors of PDK are ADP, NAD+, CoA-SH, and pyruvate, the levels of which increase when ATP
levels fall. While the regulation of PDH phosphatase (PDP), the enzyme which activates PDH through dephosphorylation, is not completely understood, it is known that Mg+2 and Ca +2 activate PDP.

Two products of the complex, NADH and acetyl-CoA, are negative allosteric effectors of PDH-a, the dephosphorylated active form of PDH. These effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the PDH complex. In addition, NADH and acetyl-CoA are powerful positive effectors of PDK, the enzyme that inactivates PDH by converting it to the phosphorylated PDH-b form. Since NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP

levels also up-regulate PDK activity, reinforcing down-regulation of PDH activity in energy-rich cells. However, since pyruvate is a potent negative effector of PDK, when pyruvate levels rise, PDH-a will be favored even with high levels of NADH and acetyl-CoA.

Concentrations of pyruvate which maintain PDH-a are sufficiently high so that, in ATP-rich cells, the allosterically down-regulated, high K. form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA.
With large amounts of pyruvate in cells having high ATP and NADH levels, pyruvate carbon will be directed to the two main storage forms of carbon (glycogen via gluconeogenesis and fat production via fatty acid synthesis) where acetyl-CoA is the principal carbon donor. Although the regulation of PDP-b is not well understood, it is quite likely regulated to maximize pyruvate oxidation under diminished ATP concentrations and to minimize PDH activity under high ATP concentrations.

Tumor cells cannot indefinitely build up pyruvate and associated aldehydes and radicals, such as acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radicals, 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 (3-hydroxyethylthiamine pyrophosphate. This active acetaldehyde is in turn condensed with a second acetaldehyde, ultimately deacidifying or reducing the original pyruvate, by using either the amino acid glutamine or lipoic acid, 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 buildup due to the tumor cell's reliance of glycolysis as a source of ATP

production, however, there is little reference in the prior art to the effects of blocking the production of acetoin on tumor cell viability.

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. In this transition, there is enhanced signalling by hypoxia-inducing factor (HIF) in cancer cells, not surprising given HIF's significant role in the metabolism of glucose, as shown in FIGURE 2. Mutations that directly or indirectly instigate HIF signalling in fact appear to be a common mechanism in the development of cancer. HIF induces the overexpression of PDK1, which then acts to lower PDH
complex activity. Phosphorylation by PDK1 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, PDH complex activation may lead to the enhanced production of reactive oxygen species, which may in turn lead to apoptosis. However, alterations in PDK1 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, PDK1 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 PDK1 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 6,331,559 and 6,951,887 to Bingham et al., as well as US Provisional Application No. 60/912,598 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 patents 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 and/or expression level, and/or regulating the activity, of the PDH complex.

As it has been demonstrated that the structure and/or activity of the PDH complex is a critical determinant of tumor activity, then, it would be beneficial to provide for a pharmaceutically-acceptable modulator of the structure and/or activity, or even the expression level, of the PDH complex, and methods of use thereof.

Objects of the Invention and Industrial Applicability Consequently, it is an object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of a disease, condition, or syndrome characterized by cellular hyperproliferation, such as cancer, which exhibits selective activity in tumor cells.

It is a further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome which causes minimal side effects upon administration.

It is a still further object of the present invention to provide a pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome 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 pharmaceutical composition to be used in the treatment or diagnosis of such an aforementioned disease, condition, or syndrome which modulates the structure, activity, and/or expression level of the PDH complex in tumor cell mitochondria.

Summary of the Invention In accordance with the aforementioned aims, the present invention broadly provides a pharmaceutical composition useful for treating, diagnosing, or preventing a disease, condition, or syndrome characterized by an alteration of the structure UUMUZ

and/or activity of at least one enzyme complex having dehydrogenase activity, such as the PDH complex, including those characterized by cellular hyperproliferation, such as cancer, or symptoms thereof, in warm-blooded animals, including humans, wherein the pharmaceutical composition comprises an effective amount of at least one lipoic acid derivative, including those as described in US Patents 6,331,559 and 6,951,887 and US Provisional Application No.
60/912,598, all herein incorporated by reference, and at least one pharmaceutically-acceptable carrier thereof.

By inhibiting mitochondrial energy metabolism, the modulators 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. The modulators of the present invention may also inhibit mitochondrial energy metabolism by the activation of PDKs and/or inhibition of PDPs or by inhibiting the conversion of pyruvate to the less-toxic molecule acetoin through inhibition of the activity of the El subunit of the PDH complex. The inhibition of acetoin synthesis will distort other processes, including redox balance and may also cause the production of toxic by-products, including acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radical, these by-products themselves consequently causing irreversible damage to the mitochondrion of the diseased cell.

AMENDED SHEET - IPEA/US

A R M GlUU 1(,I(P 10 The pharmaceutical composition of the present invention may modulate the effects of PDK1, PDK2, PDK3, PDK4, and the mutants or isoforms of each thereof. The pharmaceutical compound may also modulate the effects of PDP1, PDP2 and the isoforms of each thereof.

The pharmaceutical composition 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 compound derived from a molecule fundamentally associated with the TCA cycle, and by extension glycolysis, the pharmaceutical composition of the present invention demonstrates selective uptake into tumor cells. Furthermore, such selective tumor cell uptake minimizes the side effects the administration of this pharmaceutical composition would have on healthy non-transformed cells and tissue.

In a first embodiment of the present invention, the.
modulator has a general formula (I):

~~(CH 2)X-R3 ( I ) wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl CõH2n+1, alkene CõH2n, alkenyl CnH2n-1, alkyne CnH2n-2r alkynyl CnH2n-3, alkyl sulfide AMENDED SHEET - IPEA/US

CH3 (CH2) n-S-, disulfide alkyl CH3CHt S-S-, thiocarbamic ester (CH2) õC=NH-, and semithioacetal CH3CH(OH)-S-, wherein n is 1-10 and t is 0-9, aromatic, acyl defined as R4C(O)-, heteroaryl, imidoyl defined as R5C(=NH)-, organometallic aryl, alkyl-organometallic aryl, semiacetal R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations thereof;

wherein R1 and R2 as defined above can be unsubstituted or substituted;

wherein R3 is selected from a group consisting of amino acids, carbohydrates, nucleic acids, lipids, and multimers thereof;

wherein R4 is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which can be substituted or unsubstituted;

wherein R5 is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, and =alkylheteroaryl, any of which can be substituted or unsubstituted;

wherein R6 is CC13, CF3, or COOH;
and wherein x is 0-16;

or salts thereof.

In a second embodiment of the present invention, the modulator is defined by a second general formula (II):
AMENDED SHEET - IPEAIUS

(in 1002851L

S

(CH2)x-R3 ( I I ) wherein M is a covalent bond, -[C(R1)(R2))1-, or a metal chelate or other metal complex where the metal is not palladium, wherein R1 and R2 are independently selected from the group consisting of hydrogen, acyl R4C (O) -, alkyl CnH2õ+1, alkenyl defined as CmH2n,_i, alkynyl defined as CmH2m-3, aryl, heteroaryl, alkyl sulfide CH3(CH2)n-S-, imidoyl defined as R4C(=NH)-, hemiacetal defined as R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations thereof, wherein R1 and R2 can be unsubstituted or substituted, wherein R3 is selected from a group consisting of CC13, CF3, COOH, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations of amino acids, carbohydrates, and lipids thereof, wherein R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, and heterocyclyl, any of which can be substituted or unsubstituted, and wherein x is 0-16, z is 0-5, n is 0-10 and m is 2-10, metabolites thereof, or salts thereof.

AMENDED SHEET - IPEA/US

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

In a further aspect of the present invention, there is provided a method of diagnosing, treating, or preventing a disease, condition, syndrome, or symptoms thereof, characterized by an alteration of the structure or function of at least one enzyme complex, such as the PDH complex, including those characterized by cellular hyperproliferation, such as cancer, in warm-blooded animals, including humans, wherein the method comprises administering to such an animal an effective amount of the pharmaceutical composition disclosed herein.

In a still further aspect of the present invention, there is provided a method of diagnosing and predicting benefit in a patient presenting symptoms of a disease, condition, or syndrome characterized by an alteration of the structure and/or activity of at least one enzyme complex, such as the PDH complex, including those characterized by cellular hyperproliferation, such as cancer, comprising obtaining a sample of cells from the patient, administering an effective amount of the pharmaceutical composition of the present.

invention to the cells in vitro, and obtaining the results therefrom.

AMENDED SHEET - IPEA/US

Brief Description of the Figures FIGURE 1 illustrates the structures of substrates and products in the glycolytic production of pyruvate, also showing ATP and NADH generation and associated enzymes.

FIGURE 2 shows the regulation of glucose metabolism by HIF-1.

FIGURE 3A illustrates the difference in energy metabolism between normal tissue and cancer tissue in vivo.

FIGURE 3B depicts the differences between the biogenic forms of lipoic acid in the PDH complex and the lipoic acid derivatives forming part of the pharmaceutical composition of the present invention.

FIGURE 3C presents the regulation of the PDH complex by lipoyl residue effects on PDK.

FIGURE 4 shows the effects of the pharmaceutical composition of the present invention on xenograft tumor growth.

FIGURE 5 shows the effect of treatment with the pharmaceutical composition of the present invention on three tumor cell types and a non-transformed cell.

FIGURE 6A shows ATP levels in lung cancer cells after treatment with the pharmaceutical composition of the present invention at or above the lethal threshold.

AMENDED SHEET - IPEA/US

FIGURE 6B compares the pharmaceutical composition of the present invention's inhibition of ATP synthesis in pyruvate-containing media versus glucose-containing media.

FIGURE 6C compares the pharmaceutical composition of the present invention's inhibition of ATP synthesis in breast cancer cells to that in normal breast cells.

FIGURE 6D compares the pharmaceutical composition of the present invention's inhibition of ATP synthesis with that of lipoic acid and an inactive form of the present invention in lung cancer cells.

FIGURE 7 illustrates the pharmaceutical composition of the present invention's effects on tumor cell mitochondrial levels of the PDH complex and alpha-ketoglutarate (aKDH) dehydrogenase enzymatic activities.

FIGURE 8A shows Western analyses of two-dimensional gels of extracts from lung cancer cells treated or mock-treated with the pharmaceutical composition of the present invention.

FIGURE 8B shows enlargements of paired two-dimensional gel samples treated and mock-treated with the pharmaceutical composition of the present invention.

FIGURE 9A depicts the regulatory role of PDKs as modulated by endogenous lipoate covalently bound to the PDH
complex E2 subunit.

FIGURE 9B depicts a possible mechanism for differential inactivation of tumor cell PDH complex by the pharmaceutical composition of the present invention.

AMENDED SHEET - IPEA/US

P 1(J~9~~~S'53 MP 10 FIGURE 10 presents the effects of the pharmaceutical composition of the present invention on mitochondrial membrane potential in H460 lung cancer cells.

FIGURE 11 shows Western blot analysis results wherein cell death pathways in diverse tumor cell types by the pharmaceutical composition of the present invention.

Detailed Description of the Invention The present invention is generally directed to pharmaceutical compositions for treating, diagnosing, or preventing a disease, condition, or syndrome characterized by an alteration of the structure and/or activity of at least one enzyme complex having dehydrogenase activity, such'as the PDH
complex, including those characterized by cellular hyperproliferation, such as cancer, or symptoms thereof, in warm-blooded animals. Such animals include those of the mammalian class, such as humans, horses, cattle, domestic animals including dogs and cats, and the like, subject to disease and other pathological conditions and syndromes characterized by cellular hyperproliferation, including cancer. The pharmaceutical composition of the present invention comprises an effective amount of at least one lipoic acid derivative, including those described in US Patents 6,331,559 and 6,951,887 and US Provisional Application No.

60/912,598, also known as a thioctan, and a pharmaceutically-acceptable carrier or excipient therefor. As a molecule which AMENDED SHEET - IPEA/US

02853 1 in AFEUMINNOW I O.A.%R

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 modulators of the present invention are particularly well-suited for the selective delivery into and effective concentration within the mitochondria of cells and tissues characterized by hyperproliferation, such as tumorous ones, thereby sparing normal cells and tissue from the effects of the composition.

The pharmaceutical composition of the present invention may modulate the effects of PDK1, PDK2, PDK3, PDK4, and the isoforms of each thereof. The pharmaceutical composition may also modulate the effects of PDP1, PDP2, and the isoforms and/or mutants of each thereof. Such modulation may occur.

through either promotion or inhibition of kinase or phosphatase activity.

By inhibiting mitochondrial energy metabolism, the modulators 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. The modulators of the present invention may also inhibit mitochondrial energy metabolism by the activation of PDKs and/or inhibition of PDPs or by inhibiting the conversion of pyruvate to the less-toxic molecule acetoin through inhibition of the activity of the El subunit of the PDH complex. The inhibition of acetoin AMENDED SHEET - IPEA/US

PCT/US IIQS/UQ 5 .D'f Z1JR10 synthesis will distort other processes, including redox balance and may also cause the production of toxic by-products, including acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radical, these by-products themselves 5 consequently causing irreversible damage to the mitochondrion of the diseased cell.

In a first embodiment of the present invention, the modulator has a general formula (I):

I I
S

(CH2)x-R3 (I) 10 wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl CnH2n+1, alkene C,H2n, alkenyl CnH2n-1r alkyne CnH2n_2, alkynyl CnH2n-3, alkyl sulfide CH3(CH2)n-S-, disulfide alkyl CH3CHt S-S-, thiocarbamic ester (CH2)nC=NH-, and semithioacetal CH3CH(OH)-S-, wherein n is 1-10 15 and t is 0-9, aromatic, acyl defined as R4C(O)-, heteroaryl, imidoyl defined as R5C(=NH)-, organometallic aryl, alkyl-organometallic aryl, semiacetal R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations thereof;

20 wherein R1 and R2 as defined above can be unsubstituted or substituted;

wherein R3 is selected from a group consisting of amino acids, carbohydrates, nucleic acids, lipids, and multimers thereof;

AMENDED SHEET - IPEA/US

wherein R4 is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which can be substituted or unsubstituted;

wherein R5 is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, any of. which can be substituted or unsubstituted;

wherein R6 is CC13, CF3, or COOH;
and wherein x is 0-16;

or salts thereof.

In a second embodiment of the present invention, the modulator is defined by a second general formula (II):

(CH2)x R3 (II) wherein M is a covalent bond, -(C(R1)(R2)]=-, or a metal chelate or other metal complex where the metal is not palladium, wherein R1 and R2 are independently selected from the group consisting of hydrogen, acyl R4C (O) -, alkyl CnH211, alkenyl defined as CmH2m_i, alkynyl defined as CõH2i_3, aryl, heteroaryl, alkyl sulfide CH3(CH2)n-S-1 imidoyl defined as R4C(=NH)-, hemiacetal defined as R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations thereof, AMENDED SHEET - IPEA/US

wherein R1 and R2 can be unsubstituted or substituted, wherein R3 is selected from a group consisting of CC131 CF3r COOH, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations of amino acids, carbohydrates, and lipids thereof, wherein R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, and heterocyclyl, any of which can be substituted or unsubstituted, and wherein x is 0-16, z is 0-5, n is 0-10 and m is 2-10, metabolites thereof, or salts thereof.

It has been observed that, in both the first-and second general formulae, the (R)-isomer of each particular lipoic acid derivative possesses greater physiological activity than does the (S)-isomer. Hence, while both isomers are contemplated for use in the present invention, in particularly preferred embodiments, it is specifically contemplated that the (R)-isomer be preferentially used, or that the (R)-isomer be present in a mixture with the (S)-isomer.

The pharmaceutical composition of the present invention may also modulate the expression levels of the phosphatase, 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.

AMENDED SHEET - IPEA/US

The compositions of the present invention may further include a pharmaceutically-acceptable carrier or excipients.
Examples of pharmaceutically-acceptable carriers are well known in the art and include those conventionally used in pharmaceutical compositions, such as, but not limited to, antioxidants, buffers, chelating agents, flavorants, colorants, preservatives, absorption promoters to enhance bioavailability, antimicrobial agents, and combinations thereof. The amount of such additives depends on the properties desired, which can readily be determined by one skilled in the art.

The pharmaceutical compositions of the present invention may routinely contain salts, buffering agents, preservatives, and compatible carriers, optionally in combination with other therapeutic ingredients. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically-acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically- and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically-acceptable salts can be prepared as alkaline AMENDED SHEET - IPEA/US

_qqnnRin metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

The present invention additionally provides methods for treating or diagnosing a patient with therapeutic or diagnostic agents by delivering an effective amount of at least one therapeutic or diagnostic agent to cells for implementing the prevention, diagnosis, or treatment of a disease, condition, or syndrome characterized by alteration of the structure and/or activity of at least one enzyme complex, including those characterized by cellular hyperproliferation, or symptoms thereof. Modulating the PDH complex as an improved treatment of cancer is especially contemplated, including treatment of primary tumors by the control of tumoral cell proliferation, angiogenesis, metastatic growth, apoptosis, and treatment of the development of micrometastasis after or concurrent with surgical removal; and radiological or other chemotherapeutic treatment of a primary tumor. The pharmaceutical composition of the present invention is useful in such cancer types as primary or metastatic melanoma, lymphoma, sarcoma, lung cancer, liver cancer, Hodgkin's and non-Hodgkin's lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer.

For therapeutic and diagnostic applications, the pharmaceutical composition can be administered directly to a AMENDED SHEET - IPEA/US

P IN OUS/DR0 515 b'I % 10 patient when combined with a pharmaceutically-acceptable carrier. This method may be practiced by administering the therapeutic or diagnostic agent alone or in combination with an effective amount of another therapeutic or diagnostic 5 agent, which may be, but is not limited to, a glycolytic inhibitor, a microtubule-interacting agent, a cytostatic agent, a folic acid inhibitor, an alkylating agent, a topoisomerase inhibitor, a tyrosine kinase inhibitor, podophyllotoxin or derivatives thereof, an antitumor 10 antibiotic, a chemotherapeutic agent, an apoptosis-inducing agent, an anti-angiogenic agent, nitrogen mustards, nucleic acid intercalating agents, and combinations thereof. Such therapeutic agents may further include other metabolic inhibition reagents. Many such therapeutic agents are known 15 in the art. The combination treatment 'method provides for simultaneous, sequential, or separate use in treating such conditions as needed to amplify or ensure patient response to the treatment method.

The methods of the present invention may be practiced 20 using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Although formulations specifically suited for parenteral administration are preferred, the compositions of the present invention can 25 also be formulated for inhalational, oral, topical, transdermal, nasal, ocular, pulmonary, rectal, transmucosal, AMENDED SHEET - IPEA/US

F (: Tu92&8M %Iq ? 1 o intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, intrapleural, intrauterine, intratumoral, or infusion methodologies or administration, in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. If such.a formulation is desired, other additives well-known in the art may be included to impart the desired consistency and other properties to the formulation.

Those skilled in the art will recognize that the particular mode of administering the therapeutic or diagnostic agent depends on the particular agent selected; whether the administration is for treatment, diagnosis, or prevention of a disease, condition, syndrome, or symptoms thereof; the severity of the medical disorder being treated or diagnosed;

and the dosage. required for therapeutic efficacy. For example, a preferred mode of administering an anticancer agent for treatment of leukemia would involve intravenous administration, whereas preferred methods for treating skin cancer could involve topical or intradermal administration.

As used herein, "effective amount" refers to the dosage or multiple dosages of the therapeutic or diagnostic agent at which the desired therapeutic or diagnostic effect is achieved. Generally, an effective amount of the therapeutic or diagnostic agent may vary with the activity of the specific agent employed; the metabolic stability and length of action of that agent; the species, age, body weight, general health, AMENDED SHEET - IPEA/US

FQRTU'W OM99%.11:480P o dietary status, sex and diet of the subject; the mode and time of administration; rate of excretion; drug combination, if any; and extent of presentation and/or severity of the particular condition being treated. The precise dosage can be determined by an artisan of ordinary skill in the art without undue experimentation, in one or several administrations per day, to yield the desired results, and the dosage may be adjusted by the individual practitioner to achieve a desired therapeutic effect or in the event of any complication.

Importantly, when used to treat cancer, the dosage amount of the therapeutic agent used should be sufficient to inhibit or kill tumor cells while leaving normal cells substantially unharmed.

The therapeutic or diagnostic agent included in the pharmaceutical compositions of the present invention can be prepared in any amount desired up to the maximum amount that can be administered safely to a patient. The amount of the diagnostic agent or therapeutic agent may range from less than 0.01 mg/mL to greater than 1000 mg/mL, preferably about 50 mg/mL.

Generally, the pharmaceutical composition of the present invention will be delivered in a manner sufficient to administer to the patient an amount effective to modulate the structure and/or activity of the PDH complex. The. dosage amount may thus range from about 0.3 mg/m2 to 2000 mg/m2, preferably about 60 mg/m2. The dosage amount may be AMENDED SHEET - IPEA/US

P IUSZUUMU, S-115.~1'~ 10 administered in a single dose or in the form of individual divided doses, such as from one to four or more times per day.
In the event that the response in a subject is insufficient at a certain dose, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent of patient tolerance. Multiple doses per day are contemplated to achieve appropriate systemic or targeted levels of the therapeutic or diagnostic agent.

In yet another embodiment of the present invention, the lipoic acid derivatives of the present invention may be used as diagnostic and predictive agents in vitro. As stated earlier, depending on the specific tumor cell or cell type in question, different lipoic acid derivatives may be more or less effective at inhibiting distinct tumor classes through the modulation of the PDH complex. Thus, for example, in cases where diagnosis or selection of an appropriate chemotherapeutic strategy may be difficult, testing of a culture of tumor cells in vitro with lipoic acid derivatives known to target specific tumor cell types provides an alternative approach for identifying tumor types and effective treatments.

Turning to the figures, FIGURE 3A illustrates one of the many likely differences in energy metabolism in normal tissues and tumor cells in vivo. Tumor cells often rely more heavily on cytoplasmic glycolysis than mitochondrial energy metabolism for ATP generation than do normal cells under corresponding AMENDED SHEET - IPEA/US

AFTNOM %ORIMPO

conditions. Changes in expression and regulation of the PDH
complex are apparently part of this tumor-specific adaptation.
The decreases in the levels of PDH catalytic components and/or increases in the levels of inhibitory PDKs producing these effects may render tumor cells much more vulnerable to agents attacking the PDH complex than are normal cells.

FIGURE 3B depicts the structures of lipoic acid as it catalyzes the normal reactions involved in synthesizing acetyl-CoA from pyruvate in the PDH complex. In vivo lipoic acid is joined through its carboxyl terminus in a non-peptide amide linkage to an epsilon amino group of a lysine in the E2 lipoyl domain active sites. Notice also. that the oxidation/reduction/acetylation state of PDH E2-bound lipoate is monitored by the kinases and phosphatases that control PDH

activity by controlling the phosphorylation inactivation of the Ela PDH subunit. This figure also depicts the structure of. the three representative lipoic acid derivatives which may be used in the present invention. While CPI-613 and CPI-045 have high anticancer potency, CPI-157 has little or no activity in cell culture and is useful as a control in several experiments.

FIGURE 3C presents the relationship between PDH complex components, including E2 with its bound lipoates, El, and the regulatory PDK. High levels of acetyl-lipoate or dihydrolipoate (not diagrammed) activate PDKs which, in turn, suppress further flux through the PDH complex by inactivating AMENDED SHEET - IPEA/US

Ela, the subunit catalyzing the first step in PDH complex catalysis. This process acts as a governor for carbon/energy flow through the PDH complex, and this regulatory process is apparently substantially altered to support the variant energy 5 metabolism of tumor cells, as seen in FIGURE 3A.

FIGURE 4 shows the effects of the pharmaceutical composition of the present invention on xenograft tumor growth. Cells were implanted subcutaneously on the dorsal flank as described in EXAMPLE 2. Mice were then injected with 10 the drug (or vehicle alone; "mock") intraperitoneally starting on days as indicated in the figure. The left panel shows a pancreatic tumor model injected three times weekly with the present invention at 1 mg/kg or the vehicle control. This experiment is representative of two'done with BxPC-3 cells and 15 of two done with AsPC-1 cells. The right three panels show an H460 lung tumor model injected with the concentrations indicated either once weekly (circles), three times weekly (inverted triangles), or five times weekly (triangles for vehicle treatment and squares for drug treatment). This 20 experiment is representative of four done with H460 cells.

FIGURE 5 shows the effect of treatment with the pharmaceutical composition of the present invention on three tumor cell types and a non-transformed cell type (MDCK) either at 200-300 pM ("Treated") or mock treatment ("Mock treated").

25 Cells were treated in appropriate tissue culture media containing 10% serum or 48 hours. Extensive cell death by AMENDED SHEET - IPEA/US

apoptosis or apoptosis-like pathways (see also FIGURE 11) in the three cancer cell lines is observed through the methodology described in EXAMPLE 2. In contrast, the non-transformed MDCK cells are apparently unaffected by drug treatment at this dose.

FIGURE 6A shows ATP levels in H460 lung cancer cells after treatment with the pharmaceutical composition of the present invention at or above the lethal threshold (200pM in 10% serum). Dashed lines represent treatment at the concentrations indicated. Solid lines of corresponding texture represent treatment for the time indicated, followed by removal of the drug and 60 minutes of recovery in the drug-free medium. Block arrows indicated intervals of ATP
recovery.

FIGURE 6B compares inhibition of ATP synthesis in media in which pyruvate (in the form of methyl-pyruvate) is the primary carbon source (dashed lines) and in which glucose is the primary carbon source (solid lines). Notice that the pharmaceutical composition of the present invention ultimately produces cell death at the same threshold concentrations in both media; however, early depletion of total cellular ATP
levels is high in pyruvate-containing media and absent in glucose-containing media. Also, the onset of cell death is more rapid in the 300pM than the 200pM drug concentration..

FIGURE 6C compares the pharmaceutical composition of the present invention's inhibition of ATP synthesis in SK-Br-3 AMENDED SHEET - IPEA/US

PGPCT
T/MBYA8ffi ?5VMW10 breast cancer cells and HMEC normal breast cells. In contrast to the experiments whose results are depicted in FIGURES 6A
and 6B, these experiments were done in serum-free medium (MEBM). As a result, the drug's lethal threshold is lower, approximately 50pM. Note that the small depression in ATP
levels in the 22-hour normal cell samples is not related to drug dose and reflects normal experimental variation.

FIGURE 6D compares inhibition of ATP synthesis in H460 lung cancer cells by the pharmaceutical composition of the present invention (left graph), lipoic acid (center graph), and an inactive form of the present invention (right graph).
As in FIGURE 6C, these experiments are done in serum-free medium so that the drug's lethal threshold is approximately 5OpM.

FIGURE 7 illustrates the pharmaceutical composition of the present invention's effects (at 400pM in DMEM with 10%
serum) on tumor cell mitochondrial levels of PDH (PDC) and (aKDH) enzymatic activities. Notice that PDH is strongly inhibited whereas aKDH is not. Enzyme activity levels are measured in extracts of purified mitochondria using resazurin reduction in response to added carbon source, as described in EXAMPLE 2. The background line corresponds to resazurin reduction in the absence of added carbon source.

Next, in FIGURE 8A, Western analyses of two-dimensional gels of extracts from H460 lung cancer cells treated (+) or mock treated (-) with the pharmaceutical composition of the AMENDED SHEET - IPEA/US

present invention (at 400pM for 120 minutes in RPMI medium with 10% serum) were performed. The Western transfers were probed with a cocktail of monoclonal antibodies against Ela and E2 subunits of the PDH complex. The Western transfers are aligned at E2. Notice the substantially higher levels of hyper-phosphorylated and the reduced levels of hypo-phosphorylated forms of El in the drug-treated sample. The left vertical white line illustrates one of the criteria for aligning the gels, the mobility of the E2 subunit. The right vertical white line passes through the less phosphorylated Ela form, the presumptively-enzymatically-active component.

FIGURE 8B shows enlargements of paired two-dimensional gel samples treated and mock-treated with the pharmaceutical composition of the present invention. Element A is an enlargement of a portion of FIGURE 8A. Element B is SK-Br-3 breast cancer cells mock-treated (-) and treated (+) for 180 minutes with 8OpM of the composition in MEBM serum-free breast epithelial cell medium. Element C is SK-Br-3 breast cancer cells mock-treated (-) and treated (+) for 240 minutes with 80pM of the composition in MEBM serum-free breast epithelial cell medium. Element D is HMEC normal breast epithelial cells mock-treated (-) and treated (+) for 240 minutes with 80pM of the composition in MEBM serum-free breast epithelial cell medium. The vertical white. line- passes through the less...

phosphorylated Ela form, the presumptively-enzymatically-active component.

AMENDED SHEET - IPEA/US

FIGURES 9A and 9B depict a working hypothesis for the strong, selective anticancer effects of the pharmaceutical composition of the present invention in vivo. FIGURE 9A, for example, shows the regulatory role of PDKs as modulated by endogenous lipoate covalently bound to the PDC E2 subunit.
PDKs normally inactivate the PDC in response to high levels of reduced and/or acetylated lipoate, a process that is apparently substantially modified in tumor cells.

Concomitantly, FIGURE 9B shows the large quantitative difference in the ratio of PDK to its substrate PDC-El in the PDC, believed to distinguish normal and tumor cells in vivo.
In normal cells the low level of PDK is thought to "walk"
hand-over-hand (through its two dimeric subunits) around the PDH complex, gradually phosphorylating El. This phosphorylation is in steady state equilibrium with PDP
dephosphorylation (not diagrammed). On the working hypothesis diagrammed here, thioctans stimulate PDKs through the same sites that normally bind acetyl-lipoate and/or dihydrolipoate, thereby artificially stimulating one or more PDK isoform to inacative Ela. In cancer cells, the much higher levels of PDK might make this stimulation by thioctans much more effective in shutting down PDC enzymatic activity and mitochondrial energy metabolism.

The following examples are provided to facilitate understanding of the pharmaceutical compositions of the present invention.

AMENDED SHEET - IPEA/US

CHEMICAL SYNTHESIS OF THIOCTANS

Lipoic acid derivatives (i.e., thioctans) CPI-613 and CPI-157 were synthesized by using a modified procedure described in US 6,331,559 B1 and US 6,951,887 B2 with 6, 8-bismercaptooctanoic acid as the starting material. Thioctan CPI-045 was synthesized as described in US 6,331,559 B1.

Structure analyses for the three thioctans are below.
Multiple independent syntheses of CPI-045 and CPI-613 were indistinguishable in their anti-cancer properties. Purity. of CPI-613 used in the xenograft (FIGURE 4) and ATP measurements (FIGURE 6) was in excess of 99%. All other preparations were greater than 98% pure.

CPI-613: 6,8-Bis-benzylsulfanyloctanoic acid: White crystalline solid, m.p. 65-66 C (lit.' 67.5-69 )--; 1H-NMR (250 MHz, CDC13): S 7.15-7.4 (m, 10H), 3.66 (s, 2H), 3.64 (s, 2H), 2.52-2.62 (m, 1H), 2.50 (t, j = 7.6 Hz, 2H), 2.29 (t, J = 7.6 Hz, 2H), 1.2-1.8 (m, 8H); 13C-NMR (62.9 MHz, CDC13): S 179.6, 138.6, 138.5, 128.9, 128.8, 128.5, 128.4, 126.9, 44.1, 36.4, 35.1, 34.4, 33.8, 28.7, 26.0, 24.4.

CPI-157: 6,8-Bis-ethylsulfanyloctanoic acid: Colorless oil; TLC (EtAc:Hexanes:HAc, 200:200:1 v/v): Rf = 0.60; 'H-NMR
(300 MHz, CDC13) S 2.64-2.76 (m, 1H), 2.65 (t, J = 7.5 Hz, 2H), 2.52 (q, J = 7.5 Hz, 2H), 2.49 (q, J = 7.2 Hz, 2H), 2.36 AMENDED SHEET - IPEA/US

(t, J = 7.4 Hz, 2H), 1.40-1.85 (m, 8H), 1.25 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.5 Hz, 3H); 13C-NMR (75 MHz, CDC13): S
180.0, 44.3, 34.6, 33.9, 28.9, 26.2, 25.9, 24.5, 24.2, 14.9, 14.7; IR (film): 2963, 1708, 1449, 1423, 1283, 1263 cm-1.

CPI-045: 6,8-Bis-benzoylsulfanyloctanoic acid: Colorless, viscous oil; TLC (Hexanes:EtAc:HAc, 100:50:1 v/v): Rf = 0.30;
1H-NMR (250 MHz, CDC13): 6 7.9-8.1 (m, 4H), 7.38-7.60 (m, 6H), 3.8-4.0 (m, 1H), 3.0-3.3 (m, 2H), 2.34 (t, J = 7.1 Hz, 2H), 1.4-2.2 (m, 8H); 13C-NMR (62.9 MHz, CDC13): 6 191.7, 191.5, 179.7, 137.0, 136.9, 133.3, 128.5, 127.3, 127.1, 43.6, 35.0, 34.6, 33.8, 26.4, 26.2, 24.3; IR (film): 2973, 1710, 1704, 1667, 1665, 1662, 1448, 1207, 1175, 911, 773, 757, 733, 688, 648 cm -1 METHODS USED TO DETERMINE THIOCTAN ANTI-CANCER EFFECTS
Cells: Human tumor cell lines were obtained from ATCC and propagated according to ATCC recommendations. Human Mammary Epithelial Cells (HMEC), Small Airway Epithelial Cells (SAEC), and Normal Human Epidermal Keratinocytes (NHEK) primary cells were obtained from LONZA Walkersville, Inc (Walkersville, MD).
Each cell line was maintained and propagated in appropriate media developed by and bought from the supplier according to the supplier's instructions. Experiments reported here used normal cells at passage three to six.

AMENDED SHEET - IPEA/US

Tumor growth inhibition studies: CD1-Nu/Nu female mice were implanted with human BxPC-3 or AsPC-1 pancreatic tumor cells or H460 NSCLC by subcutaneous (SC) injection.
Approximately 8-12 days later the mice were injected intraperitoneally (IP) at doses and schedule as indicated in the figure legend. Drug or vehicle was injected at ca. 2ml per 25gm of body weight. Drug concentration was 1.25mg/ml (ca. 3.1mM) or less. The vehicle/solvent consisted of triethanolamine in water at 25mM or less. The vehicle injected in mock treated animals was always identical to the solvent in which the highest drug dose for that experiment was injected. Mice were monitored daily for physical condition and mortality. Body weight and tumor volume were assessed daily before treatment, and approximately three times weekly during and after treatment. Mice were kept on a 12 hour light/dark cycle, were fed ad libitum and were housed at Stony Brook University Animal Facility in accordance with institutional guidelines.

Cell death assays: For most assessments of cell viability CellTiter-Glo assay (Promega) was used at times sufficiently long not to be confounded by early thioctan inhibition of ATP
synthesis. (FIGURE 6) In a typical experiment, cells were plated in black, clear bottom, 96-well plates at 5,000 cells per well. 18-25 hours later, medium was replaced with fresh medium containing drug solvent (triethanolamine in water at 2.8mM in serum-containing media and 0.7mM in serum-free media) AMENDED SHEET - PEA/US

FF'tTN9ffAM%-1A1MP' o or thioctan CPI-613 in the same solvent. The assay was performed at 24 or 48 hours after drug addition, depending on drug dose, according the manufacturer's directions.

In some cases, cells were plated in 48-well plates at 10,000 cells per well, and medium was replaced 18-25 hours later with fresh medium containing drug solvent (~-+l% EtOH
final concentration) or different concentrations of thioctan CPI-045 in the same solvent. Cells remained in solvent or drug-containing medium for the remainder of the experiment.

Plates were inspected at 24, 48 and 72 hours post drug addition, and cell numbers were estimated as a confluence percentage. Under these conditions, thioctan-induced cell death is highly apoptotic at near-threshold doses, and cell number estimates are very reliable indicators of death.

(FIGURE 7) The integrity of cells remaining at 72 hours, if any, was tested by trypan blue exclusion.

Table 1 provides data regarding the action of thioctans against tumor cells in vitro. Listed are the human tumor and primary human cells we have investigated for sensitivity to CPI-613 and/or CPI-045 killing. "+" indicates that the cells underwent apoptosis or necrosis-like cell death at doses of approximately 200-300 M (in the presence of 10% serum) and approximately 50 pM in serum-free medium. (FIGURES 5 and 6) "--" indicates that these cells required approximately five-fold higher drug doses to induce cell death in the corresponding medium. "nt" indicates non-tested combination.
AMENDED SHEET - IPEA/US

All tumor lines were analyzed in the appropriate media with 10% serum, as were the MDCK normal cells in FIGURE 5. In addition, HMEC, SAEC, NHKC primary human cells, and SK-BR-3, A549 and H460 tumor lines were also analyzed in the appropriate serum-free media. Primary cells were contact inhibited and transformed cells were at comparable densities.
ATP assay: Cells were plated in black, clear bottom, 96-well plates at 5,000 cells per well. 18-25 hours later, medium was replaced with fresh medium containing drug solvent (triethanolamine) or thioctan (CPI-613 or CPI-157) or lipoic acid for time interval and at drug concentration as indicated.
Cell viability and integrity was assessed by recovery after drug withdrawal by trypan blue exclusion. ATP was measured using CellTiter-Glo luminescence assay (Promega) according to manufacturer's directions. All measurements were performed in duplicate and showed high consistency. The standard error of the mean ranged from 0.1-2% of the measured value. As a result, error bars were omitted from FIGURE 6. Methyl pyruvate medium in FIGURE 6 consisted of RPMI without glucose (Invitrogen), supplemented with 10% dialyzed fetal bovine serum, 5mM HEPES (pH 7.4), and 10mM methylpyruvate (Sigma-Aldrich), and the matched glucose medium was conventional RPMI
(Invitrogen).

PDH and aKDH enzyme assays: Tumor cells were grown to 80%
confluence in 15 cm plates and treated with CPI-613 as indicated. Mitochondria were isolated according to the method AMENDED SHEET - IPEA/US

r~~r3~~~8n :b9r~~1o of Moreadith and Fiskum.' Mitochondria were lysed in 0.4%
lauryl maltoside. 50pl of mitochondrial lysate was added to 96-well plates. 50 ul of reaction mix (50mM Tris, pH 7.5, 2mM
[3-NAD+, 225uMv TPP, 2mM pyruvate or a-ketoglutarate, 150 M

5 coenzyme A, 2.6mM cysteine, 1mM MgC12) was added to mitochondrial lysates, and the mixture was incubated for 45 minutes at 37 C. At this time, 15 M resazurin and 0.5U/ml diaphorase were added to the mixture and incubated for an additional five minutes. NADH production was monitored by 10 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. The standard error of the mean ranged from 0.3-4% of the measured value. As a result, error 15 bars were omitted from FIGURE 7.

Ela phosphorylation:

Cell lysates for 2-D gels: Cells were grown to 95%
confluence in 60 mm dishes and treated with drug or solvent as indicated. Cells were lysed in situ with 450 pl Lysis buffer 20 A [455pl Zoom 2D protein solubilizer 1 (Invitrogen), 2.5 l 1M
Tris base, 5p1 10OX protease inhibitor cocktail (Complete min, EDTA-free, Roche); 5 l 2M DTT). Cell lysate was transferred to 1.5ml microfuge tubes and sonicated on ice for 15 passes at 50% power. After 10 minute incubation at room temperature, 25 2.5pl of dime thy1acrylamide (DMA, Sigma-Aldrich) was added, and lysates were incubated for an additional 10 minutes. 5pl AMENDED SHEET - IPEA/US

?&3mal: ~ o of 2M DTT were added to neutralize excess DMA. Lysates were centrifuged at 16,000x g for 15 minutes.

2-D gels: We used Zoom Benchtop proteomics system (Invitrogen) according to the manufacturer's direction.
Briefly, 30-50 l of lysate were mixed with 0.8p1 pH 3-10 ampholytes, 0.75pl 2M DTT and brought up to 150 p1 with Zoom 2D protein solubilizer 1. 150pl of sample was loaded into IPG
runner, and pH 3-10NL IPG strips were added. Strips were soaked overnight at room temperature. A step protocol was used for isolectric focusing (250V, 20min.; 450V, 15min; 750V, min 2000V, 30min). Strips were treated for 15 minutes in 1X loading buffer, followed by 15 minutes in 1X loading buffer plus 160mM iodoacetatic acid. Strips were electrophoresed on NuPAGE 4-12% Bis Tris ZOOM gels (Invitrogen).

15 Westerns: Proteins were blotted onto PVDF 4.5pm membranes. PDH Ela and E2 were detected using mAbs (Invitrogen).

Caspase-3 and PARP cleavage: Cleaved caspase-3 was detected on Western blots according to Roy and Nicholson.2 Briefly, after drug or solvent treatment cells were scraped and the medium/cell/apoptotic bodies mixture was centrifuged at 6000x g. Pellet was lysed with lysis buffer C (4M urea, 10% glycerol, 2% SDS, 0.003% BPB; 5% 2-mercaptoethanol). 30 pg of total cell lysate protein per well were loaded on 12%

Bis-Tris gels. Proteins blotted onto PVDF 4.5pm membranes.
Pro- and active-Caspase-3 were detected with anti-caspase-3 AMENDED SHEET - IPEA/US

mAb (mouse monocolonal [31A1067]; abcam). PARP cleavage was detected using monoclonal anti-poly (ADP-ribose) polymerase antibody, clone C-2-10 (Sigma-Aldrich).

Mitochondrial Ca *2 detection: Cells were seeded on 35 mm glass bottom plates (BD Biosciences) at 3x105, grown overnight and treated with drug or solvent as indicated. Cells were then loaded with calcium dye Fluo-4, X-Rhod-1 or Rhod-2 (4 pM, Invitrogen) in phenol red-free media and incubated at 37 C for minutes. Cells were washed once with PBS, and images were 10 captured using an Axiovert 200M, (Zeiss) deconvolution microscope at a fixed exposure time, using FITC filter.
Quantification of fluorescence was performed using software provided by the manufacturer. X-Rhod-1 and Rhod-2 gave similar results (FIGURE 10), indicating that these dyes were measuring a mitochondrial Ca+2 signal.3_4 AMENDED SHEET - IPEA/US

Table 1: Effect of thioctans against tumor and primary cells in vitro brain, glioblastoma U-87 MG + +
brain, glioblastoma LN-229 + nt breast, adenocarcinoma SK-Br-3 + +
breast, adenocarcinoma MCF7 nt +
bone, osteosarcoma Saos-2 nt +
cervical, adenocarcinoma HeLa + +
colorectal, adenocarcinoma SW480 nt +
hepatocellular, carcinoma Hep G2 + +
kidney, carcinoma A-498 + nt O lung, carcinoma A459 + +
E
lung, carcinoma H460 + +
muscle, rhabdomyosarcoma RD nt +
ovarian, carcinoma SKOV-3 + nt pancreatic, adenocarcinoma AsPC-1 + nt pancreatic, adenocarcinoma BxPC-3 + nt prostate, carcinoma LnCaP nt +
uterine, sarcoma Mes_SA + +
uterine, sarcoma, MDR Mes-SA/dx5 + +

uJ mammary epithelial cells HMEC - -c~
small airway epithelial cells SAEC nt keratinocytes NHKC nt AMENDED SHEET - IPEA/US

References:

1. Moreadith RW and Fiskum G. Isolation of mitochondria from ascites tumour-cells permeabilized with digitonin. Analytical Biochemistry 137, 360-367 (1984).

2. Roy S and Nicholson DW. Criteria for identifying authentic caspase substrates during apoptosis. Apoptosis 322,110-125 (2000).

3. Gerencser AA and Adam-Vizi V. Selective, high-resolution fluorescence imaging of mitochondrial Caz+
concentration. Cell Calcium 30, 311-321 (2001).

4. Gyorgy H, Gyorgy C, Das S, Garcia-Perez C, Saotome M, Roy SS, and Yi MQ. Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial CaZ+ uptake in apoptosis. Cell Calcium 40, 553-560 (2006).

THIOCTANS PERTURB MITOCHONDRIAL MEMBRANE POTENTIAL AND Ca+z UPTAKE
The substrate effects on thioctan inhibition of ATP

synthesis (FIGURE 6) indicate that the drug is interfering with the TCA cycle in the mitochondrial matrix. If this is the case, we anticipate that mitochondrial membrane potential' might be compromised at lethal threshold doses and above.
Using the potential-sensitive dye TMRE we observe the expected effect. (FIGURE 10) Mitochondrial membrane potential declines rapidly with initiation of drug treatment. The AMENDED SHEET - IPEA/US

PG lU5ZUUUMU O0.55I O.,D~1' Z0 P 10 kinetics of membrane potential decline is very similar to the loss of ATP synthesis in the presence of mitochondrial substrates. (FIGURE 6) ATP depletion in mitochondria is known to provoke a 5 homeostatic response that includes the uptake of Ca +2 released from cytoplasmic stores, including the endoplasmic reticulum.2 Moreover, import of this Ca +2 into the mitochondrial matrix is thought to require the mitochondrial membrane potential.
Thus, we anticipate that thioctan treatment at or above the 10 lethal threshold may produce a sustained cytoplasmic release of Ca +2 with transient mitochondrial uptake of the ion in view of progressively compromised membrane potential. Using X-Rhod-l and Rhod-2 to measure mitochondrial Ca +2 and Fluo-4 to measure cytoplasmic Ca+2, we observe these expected effects.
15 (FIGURE 10) By around two hours at CPI-doses at and slightly above the lethal threshold (compare FIGURES 6 and 10) - as mitochondrial membrane potential declines - this initial mitochondrial Ca+2 transient decays. It is followed at 4-6 20 hours by a second large mitochondrial Ca+2 peak presumably associated with initiation of the calcium-dependent cell death pathways.3 References:
1. Garrett R and Grisham CM. Biochemistry. Thomson 25 Brooks/Cole, Southbank, Vic., Australia; Belmont, CA. (2007).

2. Graier WF, Frieden M, and Malli R. Mitochondria and AMENDED SHEET - IPEA/US

?FTM3 Q81UU W'.3 UT Z 10 Ca 2+ signaling: old guests, new functions. Pflugers Archiv-European Journal of Physiology 455, 375-396 (2007).

3. Gyorgy H, Gyorgy C, Das S. Garcia-Perez C, Saotome M. Roy SS, and Yi MQ. Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40, 553-560 (2006).

THIOCTANS INDUCE DIVERSE CELL DEATH PROGRAMS

Reduction in mitochondrial energy metabolism is known to correlate with the decision to enter a cell death pathway in some circumstances, though detailed mechanisms remain incompletely understood.'-3 At thioctan doses above threshold but within -2-fold of this minimal killing dose, all tested cancer cell types undergo cell death that morphologically resembles apoptosis predominantly. (FIGURE 5) Apoptotic death was confirmed under these conditions by conventional Annexin immunostaning and TUNEL DNA end-labelling assays (results not shown).

At higher drug doses (more than --2-fold over threshold), active thioctans induce cell death (as assessed by replating viability assays and trypan blue or propidium exclusion) without the morphological correlates of apoptosis, suggesting a necrosis-like pathway (results not shown).

These data confirm that the thioctan CPI-613 inhibition mitochondrial energy metabolism correlates precisely with AMENDED SHEET - IPEA/US

induction of cell death.

The observation that diverse tumor cells, known or presumed to contain inactivating mutations for distinct and diverse cell death pathways4 are all killed at very similar thioctan doses (FIGURE 5 and Table I) is striking. This observation suggests that these drugs induce a master signal that is capable of engaging multiple, potentially redundant distal cell death execution pathways.5 Consistent with this possibility, we find that the Z-VAD-FMK generic caspase inhibitor subtly alters the morphology of cell death in thioctan treated cells, but has no discernible effect on the lethal threshold dose of the drug.

To further test the possibility that thioctan-induced cell death can proceed through multiple terminal execution mechanisms we examined caspase-3 and PARP-1 cleavage -diagnostic of distinct cell death pathways.5 We find that both thioctans CPI-613 and CPI-045 induce highly variable levels of these two cleavage events in different cells. (FIGURE 11) Collectively, these results indicate that the thioctans are able to induce a strategic commitment to death that, depending on drug dose and cell type, is agnostic about the terminal, tactical execution of that decision.

References:
1. Watabe M and Nakaki T. ATP depletion does not account for apoptosis induced by inhibition of mitochondrial electron transport chain in human dopaminergic cells.
AMENDED SHEET - IPEA/US

FAG IO03WAfPM5' 5.I1:% 10 Neuropharmacology 52, 536-541 (2007).

2. Yuneva M, Zamboni N, Oefner P, Sachidanandam R, and Lazebnik Y. Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells. Journal of Cell Biology 178, 93-105 (2007).

3. Skulachev VP. Bioenergetic aspects of apoptosis, necrosis and mitoptosis. Apoptosis 11, 473-485 (2006).

4. Johnstone RW, Ruefli AA, and Lowe SW. Apoptosis: A
link between cancer genetics and chemotherapy. Cell 108, 153-164 (2002).

5. Cregan SP, Dawson VL, and Slack RS. Role of AIF in caspase-dependent and caspase-independent cell death.
Oncogene 23, 2785-2796 (2004).

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, AMENDED SHEET - IPEAIUS

P F913 t /UuZ 02853ts0i DA W 10 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.

AMENDED SHEET - IPEA/US

Claims

1. A modulator of the phosphorylation state of at least one enzyme complex having dehydrogenase activity in the mitochondria of diseased cells of warm-blooded animals, including humans.

3. The modulator of claim 1, wherein the enzyme complex is the pyruvate dehydrogenase (PDH) complex.

4. The modulator of claim 1, wherein the modulator promotes or inhibits kinase activity.

5. The modulator of claim 4, wherein the kinase is selected from a group comprising pyruvate dehydrogenase kinase (PDK) 1, PDK2, PDK3, PDK4, and isoforms of each thereof.

6. The modulator of claim 1, wherein the modulator promotes or inhibits phosphatase activity.

7. The modulator of claim 6, wherein the phosphatase is selected from a group comprising pyruvate dehydrogenase phosphatase (PDP) 1, PDP2, and isoforms of each thereof.

8. The modulator of claim 1, wherein the modulator promotes or inhibits dehydrogenase activity.

9. The modulator of claim 1, wherein the modulation is achieved by alteration of the phosphorylation state of the PDH
complex.

10. The modulator of claim 9, wherein the modulation occurs on the E1.alpha. subunit of the PDH complex.

11. The modulator of claim 10, wherein the modulation occurs by inactivation of PDP and the isoforms and mutations thereof.

12. The modulator of claim 10, wherein the modulation occurs by activation of PDK and the isoforms and mutations thereof.

13. The modulator of claim 9, wherein the modulator prevents the detoxification of toxic metabolites of anaerobic energy metabolism.

14. The modulator of claim 13, wherein the metabolites are selected from a group consisting of acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radical.

15. The modulator of claim 13, wherein the effect of modulation is observed by a decrease in acetoin production.

16. The modulator of claim 9, wherein the phosphorylation or dephosphorylation is irreversible.

17. The modulator of claim 16, wherein the effect of the phosphorylation or dephosphorylation results in cell death.

18. The modulator of claim 17, wherein the effect is apoptosis.

19. The modulator of claim 17, wherein the effect is necrosis.

20. The modulator of claim 1, wherein the modulator affects the expression level of PDK and the isoforms and mutations thereof.

21. The modulator of claim 1, wherein the modulator affects the expression level of PDP and the isoforms and mutations thereof.

22. The modulator of claim 20 or 21, wherein the expression level is altered at the level of transcription, translation, or post-translation.

23. The modulator of claim 22, wherein the alteration is epigenetic.

26. The modulator of claim 1, wherein the modulator has the formula (I):

wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl CnH2n+1, alkene C n H2n, alkenyl C n H2n-1, alkyne C n H2n-2, alkynyl C n H2n-3, alkyl sulfide CH3(CH2)n-S-, disulfide alkyl CH3CH t-S-S-, thiocarbamate (CH2)n-S-C(O)-NH-CH2, and semithioacetal CH3CH(OH)-S-, wherein n is 1-and t is 0-9, aromatic, acyl defined as R4C(O)-, heteroaryl, imidoyl defined as R5C(=NH)-, organometallic aryl, alkyl-organometallic aryl, semiacetal R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations of amino acids, carbohydrates, and lipids thereof;

wherein R1 and R2 can be unsubstituted or substituted, wherein R3 is selected from a group consisting of CCl3, CF3, COOH, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations of amino acids, carbohydrates, and lipids thereof, wherein R4, R5, and R6 is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which can be substituted or unsubstituted, and wherein x is 0-16, metabolites thereof, or salts thereof.

27. The modulator of claim 1, wherein the modulator has the formula:

wherein M is a covalent bond, -[C(R1)(R2)]z-, or a metal chelate or other metal complex where the metal is not palladium, wherein R1 and R2 are independently selected from the group consisting of hydrogen, acyl R4C(O)-, alkyl C n H2n+1, alkenyl defined as C m H2m-1, alkynyl defined as C m H2m-3, aryl, heteroaryl, alkyl sulfide CH3(CH2)n-S-, imidoyl defined as R4C(=NH)-, hemiacetal defined as R6CH(OH)-S-, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations thereof, wherein R1 and R2 can be unsubstituted or substituted, wherein R3 is selected from a group consisting of CCl3, CF3, COOH, amino acids, carbohydrates, nucleic acids, lipids, and multimers and combinations of amino acids, carbohydrates, and lipids thereof, wherein R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, and heterocyclyl, any of which can be substituted or unsubstituted, and wherein x is 0-16, z is 0-5, n is 0-10 and m is 2-10, metabolites thereof, or salts thereof;

28. The modulator of claim 1, wherein the modulator is present solely as the (R)-isomer thereof.

29. The modulator of claim 1, wherein the modulator is present as a mixture of the (R)-isomer and the (S)-isomer thereof.

30. The modulator of claim 1, wherein the modulator is useful in the treatment and diagnosis of a disease, condition, or syndrome characterized by an alteration of the structure and/or activity of the PDH complex.

31. The modulator of claim 30, wherein the disease, condition, or syndrome is further characterized by cellular hyperproliferation.

32. The modulator of claim 31, wherein the disease, condition, or syndrome is cancer.

33. A method of modulating the PDH complex in a patient presenting a disease, condition, or syndrome characterized by an alteration of the structure and/or activity of the PDH

complex, comprising administration of an effective amount of the modulator of claim 1.

34. The method of claim 33, wherein the disease, condition, or syndrome is further characterized by cellular hyperproliferation.

35. The method of claim 34, wherein the disease, condition, or syndrome is cancer.

36. A method of diagnosing and predicting benefit in a patient presenting symptoms of a disease, condition, or syndrome characterized by an alteration of the structure and/or activity of the PDH complex, comprising obtaining a sample of cells from the patient, administering an effective amount of the modulator of claim 1 to the cells in vitro, and obtaining the results therefrom.

37. The method of claim 36, wherein the disease, condition, or syndrome is further characterized by cellular hyperproliferation.

38. The method of claim 37, wherein the disease, condition, or syndrome is cancer.

39. The method of claim 33, wherein the modulator is combined with at least one pharmaceutically-acceptable carrier therefor.