Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
  • A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• 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 regulation or perturbation of the structure, expression, and/or activity of enzymes, thereby facilitating the detection and treatment or destruction of these cells. More specifically, these agents target and perturb the activity or regulation thereof of the altered mitochondrial energy metabolism observed in such locales as the modified pyruvate dehydrogenase (PDH) complex associated with most cancers.
• PDH modified pyruvate dehydrogenase
• Mitochondria are the primary control centers for energy production and cellular life- and-death processes in eukaryotes. While various mechanisms of communication with the rest of the cell exist, reversible phosphorylation is an important means of regulating mitochondrial functions. (Pagliarini D.J. and Dixon J.E. (2006). Mitochondrial modulation: reversible phosphorylation takes center stage? TRENDS in Biochem. Sci. 31 :26-34, passim, herein incorporated by reference) The steadily increasing number of reported mitochondrial kinases, phosphatases, and phosphoproteins suggests that phosphorylation is likely to emerge as a common theme in the regulation of mitochondrial processes. Pathological or genetic changes associated in mitochondrial enzyme structure, function and activity regulation contribute to, and may be important targets for, the treatment of disease.
• mitochondria In line with their role as both a point of convergence and regulator of diverse cellular functions, mitochondria have crucial roles in apoptosis, production of reactive oxygen species
• ROS ROS
• numerous metabolic processes including the production of more than 90% of cellular ATP.
• ROS ROS
• mitochondria must respond rapidly by tuning their ATP output.
• signal molecules to and from mitochondria include ions, gases, metabolites, hormones, transcription factors and proteins. Consequently, the recognition of mitochondria as centers for receiving, integrating, and transmitting cellular signals is an important advance in the design and testing of pharmaceuticals.
• a cornerstone of signalling in mitochondrial regulation is reversible phosphorylation.
• AA amino add (S. serine; T, threonine; Y, tyrosine); ANT, adenine nucleotide transporter; Axmito, mitochondrial annexin; ⁇ -OX, ⁇ -ox ⁇ Jatlon: 8 C KAD, branched chain keioacld dehydrogenase: CI-CV. respiratory chain complexes 1-5: CPT. carnitine palmitoyltransferase: CREB. cAMP-responave element (Crel-bindino prote i n: CYP, cytochrome P450. DBP. dodecamer binding protein: EF.
• elongation factor GST. glutathione S-tra ⁇ sterase; HSP. heat shock protein;
• IM Inner membrane: IMS. i m ⁇ rm ⁇ mbrane space: M. matrix; MnSOD, manganese super oxide di ⁇ rnutaie; mTERF, mitochondrial transcription termination factor; mtGAT. mitochondrial gt ⁇ cerol-3- phosphatase acet ⁇ ltransferase; mthsp. mitochondrial HSP; mtTBP. mitochondrial telomer ⁇ -binding protein; NDK. nucleoside diphosphate kinase: OM. outer membrane: OXPHOS.
• SSAT spermidine/spermine acetyltransferase: StAR, steroidogenic acute regulatory protein: TCA.
• tricarboxylic acid cycle TRAP-1, tumor-necrosis factor type 1 receptor-associated protein: VQAC. voltage-dependent anion channel.
• PKs protein kinases
• PTPs protein tyrosine phosphatases
• kinases and phosphatases have been implicated in mitochondrial functions in a surprising number of studies, and so far at least 25 kinases and eight phosphatases have been reported to localize to mitochondria, as seen in FIGURE 2.
• These kinases and phosphatases are clearly not restricted to one group or family; rather, they represent nearly every known mammalian kinase and phosphatase subgroup, reflecting the range of signalling pathways that are likely to influence the mitochondrion.
• These signalling molecules include kinases and phosphatases varying in substrate specificity, (e.g., tyrosine kinases, classic PTP subgroups, serine/threonine kinases, and dual-specific PTPs); in catalytic mechanisms (e.g., cysteine-based PTPs, aspartic acid- based PTPs, and metal-dependent phosphatases); and in evolutionary conservation (e.g., bacterially-related pyruvate dehydrogenase kinases (PDKs) and phosphatases (PDPs), branched chain ketoacid dehydrogenase kinase (BCKDK) and phosphatase (BCKDP), and many mammalian-specific enzymes).
• substrate specificity e.g., tyrosine kinases, classic PTP subgroups, serine/threonine kinases, and dual-specific PTPs
• catalytic mechanisms e.g.,
• PINKl PTEN-induced kinase
• PTPMTl the dual-specific PTP targeted to the mitochondrion PTPMTl
• Tim50 the aspartic-acid-based phosphatase/ ATPase Tim50.
• substrates for many of these proteins are currently unknown, it is clear from biological and genetic data that they possess crucial functions in mitochondria. For example, although its submitochondrial localization remains to be determined, PINKl is targeted to mitochondria by an N-terminal signal sequence.
• PTPMTl was recently identified as the first PTP that is localized primarily inside the mitochondrion and, like PINKl, is targeted to the mitochondrion by an N-terminal signal peptide and is found tightly associated with the matrix face of the inner mitochondrial membrane. PTPMTl is highly expressed in pancreatic ⁇ cells, whose mitochondria have the important function of coupling glucose metabolism to the secretion of insulin.
• Tim50 a key component of the TIM (translocase of the inner membrane) complex, has sequence homology to the CTD family of aspartic-acid-based phosphatases/ ATPases. Tim50, like other members of this family, might function as an ATPase, but it has been also shown to possess phosphatase activity against the phosphotyrosine analog /r ⁇ r ⁇ -nitrophenyl phosphate in vitro. Given the above, it can be seen that not only are kinases and phosphatases recruited to mitochondria from elsewhere in the cell, but the mitochondrion itself seems to possess a contingent of resident signalling molecules.
• FIGURE 3B The most well-established example of reversible phosphorylation acting as a regulatory mechanism in healthy cell mitochondria is that of the PDH complex in the matrix, a simplified cartoon of which is illustrated in FIGURE 3B.
• This complex catalyzes the conversion of glycolysis-derived pyruvate to acetyl-coenzyme A (CoA), the main precursor to the tricarboxylic acid (TCA) cycle.
• CoA acetyl-coenzyme A
• TCA tricarboxylic acid
• Phosphorylation and dephosphorylation of the PDH complex are carried out by PDKs and PDPs, respectively. At least four PDK isoforms and two PDP isoforms are known, all of which are associated with the E2 subunit of the PDH complex. The phosphorylation events occur on three separate serine residues of the El subunit, each leading to significant inactivation of this complex. Notably, there is now at least one report of a PDK itself being phosphorylated.
• phosphorylation within this organelle is not limited to serine and threonine residues.
• An example of tyrosine phosphorylation that affects mitochondrial energetics is seen in the regulation of cytochrome c oxidase (COX) in the inner membrane, depicted in FIGURE 3C.
• COX cytochrome c oxidase
• FIGURE 3C An example of tyrosine phosphorylation that affects mitochondrial energetics is seen in the regulation of cytochrome c oxidase (COX) in the inner membrane, depicted in FIGURE 3C.
• COX as the terminal enzyme in the respiratory chain, coordinately reduces oxygen to water while pumping protons across the inner membrane.
• COX is allosterically regulated by ATP and ADP, as well as the thyroid hormone T 2 and possibly Ca +2 ions.
• COX becomes phosphorylated in a cAMP-dependent manner both in vitro and in HepG2 cells in vivo.
• COX comprises thirteen subunits and has been crystallized as a dimer.
• the phosphorylation site has been identified as Tyr 304 of subunit 1, which is located at the dimer interface on the intermembrane.
• the phosphorylation event markedly inhibits COX activity, perhaps by disrupting dimer formation.
• tyrosine phosphorylation of COX In a second example of tyrosine phosphorylation of COX, a portion of the nonreceptor tyrosine kinase c-Src, similar to the Lyn tyrosine kinase localizes inside mitochondria and leads to tyrosine phosphorylation of COX on an unidentified site of subunit II in osteoclasts. The result of the phosphorylation event is opposite to that seen for subunit I, leading to enhanced COX activity.
• kinases and phosphatases are themselves regulated.
• the extent of some kinase activities within mitochondria might simply be dictated by the number of enzymes that are imported into the organelle.
• different regulatory means must be in place. Although these processes remain to be determined, it is likely that second messengers will have a key role.
• the activities of the PDK and PDP isoforms are known to be controlled by ions and small molecules such as Mg 2+ , Ca 2+ , K + and ADP.
• 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.
• 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.
• 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 hypoxic inducing factor and the production and release of angiogenic factors.
• 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.
• glycolytic enzymes hexokinase and phosphofructokinase (PFK) 1, which are greatly increased in fast-growth tumor cells.
• tumor cells which exhibit deficiencies in their oxidative capacity are more malignant than those that have an active oxidative phosphorylation.
• cancer tissue's reliance on glycolysis is associated with increased malignancy.
• the role of lipoic acid in the PDH complex of healthy cells 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.
• 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.
• 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 FADH 2 (and ultimately NADH) and regenerating the lipoate back into a competent acyl acceptor.
• Negative effectors of PDK are ADP, NAD + , CoA- SH, and pyruvate, the levels of which increase when ATP levels fall. While the regulation of PDP, the enzyme which activates PDH through dephosphorylation, is not completely understood, it is known that Mg +2 and Ca +2 activate PDP.
• NADH and acetyl-CoA 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.
• 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 m form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA.
• pyruvate carbon 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.
• PDP-b 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 ⁇ -hydroxyethylthiamine pyrophosphate.
• aldehydes and radicals such as acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radicals
• 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).
• HIF induces the overexpression of PDKl, which then acts to lower PDH complex activity.
• 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.
• PDH complex activation may lead to the enhanced ROS production, which may in turn lead to apoptosis.
• 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.
• 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.
• 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.
• 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 regulation or perturbation of the structure, expression, and/or activity of at least one enzyme and/or enzyme complex, or subunit thereof, 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.
• the lipoic acid derivatives of the present invention By inhibiting mitochondrial energy metabolism, the lipoic acid derivatives 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 lipoic acid derivatives 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.
• 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 byproducts themselves consequently causing irreversible damage to the mitochondrion of the diseased cell.
• toxic by-products including acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radical
• the pharmaceutical composition of the present invention may modulate the effects of PDKl, PDK2, PDK3, PDK4, and the mutants or isoforms of each thereof.
• the pharmaceutical compound may also modulate the effects of PDPl, 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.
• 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.
• the lipoic acid derivatives have the general formula (I):
• M is a covalent bond, -[C(Ri )(R 2 )] Z -, or a metal chelate or other metal complex where the metal is not palladium;
• the lipoic acid derivatives have a third general formula (III):
• the lipoic acid derivatives are defined by a fourth general formula (IV):
• M is a covalent bond, -[C(Ri )(R 2 )] Z -, or a metal chelate or other metal complex where the metal is not palladium;
• 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.
• 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.
• a method of diagnosing, treating, or preventing a disease, condition, syndrome, or symptoms thereof which includes an alteration of the regulation or perturbation of the structure, expression, and/or function of at least one enzyme and/or enzyme complex, or subunit thereof, 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.
• a method of diagnosing and predicting benefit in a patient presenting symptoms of a disease, condition, or syndrome, or symptoms thereof, which includes an alteration of the regulation or perturbation of the structure, expression, and/or activity of at least one enzyme and/or enzyme complex, or subunit thereof, 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.
• FIGURE 1 depicts general signal transduction molecules targeted both into and out from mitochondria and the effects thereof.
• FIGURE 2 shows a list of mitochondrial kinases and phosphatases and the locations within the mitochondrion thereof.
• FIGURE 3A illustrates the phosphorylation of BAD by PKA in the outer mitochondrial membrane, and the effects of reversible phosphorylation thereupon.
• FIGURE 3 B presents the conversion of pyruvate into acetyl-CoA in the mitochondrial matrix by the action of the PDH complex, and the effects of reversible phosphorylation thereupon.
• FIGURE 3 C shows the action of COX in reducing oxygen to water and pumping protons across the inner mitochondrial membrane, and the effects of reversible phosphorylation thereupon.
• FIGURE 4 illustrates the structures of substrates and products in the glycolytic production of pyruvate, also showing ATP and NADH generation and associated enzymes.
• FIGURE 5 shows the regulation of glucose metabolism by HIF-I.
• FIGURE 6A illustrates the difference in energy metabolism between normal tissue and cancer tissue in vivo.
• FIGURE 6B 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 6C presents the regulation of the PDH complex by lipoyl residue effects on PDK.
• FIGURE 7 shows the effects of the pharmaceutical composition of the present invention on xenograft tumor growth.
• FIGURE 8 shows the effect of treatment with the pharmaceutical composition of the present invention on three tumor cell types and a non-transformed cell.
• FIGURE 9 A shows ATP levels in lung cancer cells after treatment with the pharmaceutical composition of the present invention at or above the lethal threshold.
• FIGURE 9B compares the pharmaceutical composition of the present invention's inhibition of ATP synthesis in pyruvate-containing media versus glucose-containing media.
• FIGURE 9C compares the pharmaceutical composition of the present invention's inhibition of ATP synthesis in breast cancer cells to that in normal breast cells.
• FIGURE 9D 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 10 illustrates the pharmaceutical composition of the present invention's effects on tumor cell mitochondrial levels of the PDH complex and alpha-ketoglutarate ( ⁇ KDH) dehydrogenase enzymatic activities.
• FIGURE HA 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 1 IB shows enlargements of paired two-dimensional gel samples treated and mock-treated with the pharmaceutical composition of the present invention.
• FIGURE 12A depicts the regulatory role of PDKs as modulated by endogenous lipoate covalently bound to the PDH complex E2 subunit.
• FIGURE 12B depicts a possible mechanism for differential inactivation of tumor cell PDH complex by the pharmaceutical composition of the present invention.
• FIGURE 13 presents the effects of the pharmaceutical composition of the present invention on mitochondrial membrane potential in H460 lung cancer cells.
• FIGURE 14 shows Western blot analysis results wherein cell death pathways in diverse tumor cell types by the pharmaceutical composition of the present invention.
• the present invention is generally directed to pharmaceutical compositions for treating, diagnosing, or preventing a disease, condition, or syndrome, or symptoms thereof, which includes an alteration of the regulation or perturbation of the structure, expression, and/or activity of at least one enzyme and/or enzyme complex, or subunit thereof, such as the
• PDH complex including those characterized by cellular hyperproliferation, such as cancer, or symptoms thereof, in warm-blooded animals.
• 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.
• the lipoic acid derivatives 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 PDKl, PDK2, PDK3, PDK4, and the isoforms of each thereof via reversible phosphorylation.
• the pharmaceutical composition may also modulate the effects of PDPl, PDP2, and the isoforms and/or mutants of each thereof also by reversible phosphorylation. Such modulation may occur through either promotion or inhibition of kinase or phosphatase activity.
• the lipoic acid derivatives of the present invention By inhibiting mitochondrial energy metabolism, the lipoic acid derivatives 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 lipoic acid derivatives 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.
• 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.
• toxic by-products including acetaldehyde, superoxide, hydrogen peroxide, and hydroxyl radical
• the lipoic acid derivatives are defined by a first general formula (I):
• Ri and R 2 can be independently:
• an acyl group R 3 C(O)- where R 3 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;
• 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;
• alkyl group C n H 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 -NH 2 , including but not limited to methyl, ethyl, butyl, decanyl, and 6,8-bis carbomoyl methylipoate;
• alkynyl group C n H 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;
• 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;
• Ri and R 2 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 R 2 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.
• R 3 is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which can be substituted or unsubstituted.
• R 4 is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, any of which can be substituted or unsubstituted.
• R 5 is -CCl 3 , -CF 3 , or -COOH.
• M is a covalent bond, -[C(Ri)(R 2 )] z -, or a metal chelate or other metal complex where the metal is not palladium; wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, acyl R 4 C(O)-, alkyl C n H 2n+!
• alkenyl defined as C m H 201-I
• alkynyl defined as C m H 2m-3
• aryl, heteroaryl alkyl sulfide CH 3 (CH 2 ) n -S-
• Ri and R 2 as defined above can be unsubstituted or substituted
• R 3 is selected from a group consisting of amino acids, carbohydrates, nucleic acids, lipids, and multimers thereof
• R 4 and R 5 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
• R 5 is selected from the group consisting
• the (R)- isomer of each particular lipoic acid derivative possesses greater physiological activity than does the (S)-isomer.
• 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.
• compositions of the present invention may further include a pharmaceutically- acceptable carrier or excipients.
• 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.
• the salts 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.
• pharmaceutically-acceptable salts can be prepared as alkaline 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, or symptoms thereof, which includes an alteration of the regulation or perturbation of the structure, expression, and/or activity of the at least one enzyme and/or enzyme complex, or subunit thereof, including those characterized by cellular hyperproliferation.
• Modulating the PDH complex as an improved treatment of cancer is especially contemplated, including treatment of primary tumors by the control of tumor 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.
• 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.
• the pharmaceutical composition can be administered directly to a 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 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 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 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 using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
• compositions of the present invention can also be formulated for inhalational, oral, topical, transdermal, nasal, ocular, pulmonary, rectal, transmucosal, 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.
• 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.
• 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.
• an 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.
• 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, 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 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.
• 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/m 2 to 2000 mg/m 2 , preferably about 60 mg/m 2 .
• the dosage amount may be 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.
• the lipoic acid derivatives of the present invention may be used as diagnostic and predictive agents in vitro.
• different lipoic acid derivatives may be more or less effective at inhibiting distinct tumor classes through the modulation of the PDH complex.
• 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.
• FIGURE 6A 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 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 6B depicts the structures of lipoic acid as it catalyzes the normal reactions involved in synthesizing acetyl-CoA from pyruvate in the PDH complex.
• 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.
• 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 El ⁇ 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 6C 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 El ⁇ , 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 metabolism of tumor cells, as seen in FIGURE 3 A.
• FIGURE 7 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 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 of two done with AsPC-I 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 experiment is representative of four done with H460 cells.
• FIGURE 8 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
• FIGURE 9A shows ATP levels in H460 lung cancer cells after treatment with the pharmaceutical composition of the present invention at or above the lethal threshold (200 ⁇ M 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 9B 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 300 ⁇ M than the 200 ⁇ M drug concentration.
• FIGURE 9C compares the pharmaceutical composition of the present invention's inhibition of ATP synthesis in SK-Br-3 breast cancer cells and HMEC normal breast cells.
• these experiments were done in serum-free medium (MEBM).
• MEBM serum-free medium
• the drug's lethal threshold is lower, approximately 50 ⁇ M.
• 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 9D 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 9C, these experiments are done in serum-free medium so that the drug's lethal threshold is approximately 50 ⁇ M.
• FIGURE 10 illustrates the pharmaceutical composition of the present invention's effects (at 400 ⁇ M in DMEM with 10% serum) on tumor cell mitochondrial levels of PDH (PDC) and ( ⁇ KDH) enzymatic activities. Notice that PDH is strongly inhibited whereas ⁇ KDH 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.
• FIGURE HA Western analyses of two-dimensional gels of extracts from H460 lung cancer cells treated (+) or mock treated (-) with the pharmaceutical composition of the present invention (at 400 ⁇ M for 120 minutes in RPMI medium with 10% serum) were performed.
• the Western transfers were probed with a cocktail of monoclonal antibodies against El ⁇ 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 El ⁇ form, the presumptively- enzymatically-active component.
• FIGURE 1 IB 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 80 ⁇ M 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 80 ⁇ M 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 80 ⁇ M of the composition in MEBM serum-free breast epithelial cell medium.
• the vertical white line passes through the less phosphorylated El ⁇ form, the presumptively-enzymatically-active component.
• FIGURES 12A and 12B depict a working hypothesis for the strong, selective anticancer effects of the pharmaceutical composition of the present invention in vivo.
• FIGURE 12 A 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.
• FIGURE 12B 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.
• 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).
• 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 El ⁇ .
• 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.
• Lipoic acid derivatives (i.e., thioctans) CPI-613 and CPI- 157 were synthesized by using a modified procedure described in US 6,331,559 Bl 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 Bl .
• Human tumor cell lines were obtained from ATCC and propagated according to ATCC recommendations.
• Human Mammary Epithelial Cells HMEC
• Small Airway Epithelial Cells SAEC
• 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.
• mice were implanted with human BxPC-3 or AsPC-I 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.
• IP intraperitoneally
• Drug or vehicle was injected at ca. 2ml per 25gm of body weight. Drug concentration was 1.25mg/ml (ca. 3.ImM) 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.
• FIG. 9 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) 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.
• drug solvent triethanolamine in water at 2.8mM in serum-containing media and 0.7mM in serum-free media
• 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 (- 1 ⁇ 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 10) The integrity of cells remaining at 72 hours, if any, was tested by trypan blue exclusion.
• Table 2 provides data regarding the action of thioctans against tumor cells in vitro.
• Table 2 provides data regarding the action of thioctans against tumor cells in vitro.
• "+” indicates that the cells underwent apoptosis or necrosis- like cell death at doses of approximately 200-3 OO ⁇ M (in the presence of 10% serum) and approximately 50 ⁇ M in serum-free medium.
• FIGURES 8 and 9 "--" indicates that these cells required approximately five-fold higher drug doses to induce cell death in the corresponding medium, "nt” indicates non-tested combination. All tumor lines were analyzed in the appropriate media with 10% serum, as were the MDCK normal cells in FIGURE 8.
• 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
• 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 9.
• Methyl pyruvate medium in FIGURE 9 consisted of RPMI without glucose (Invitrogen), supplemented with 10% dialyzed fetal bovine serum, 5mM HEPES (pH 7.4), and 1OmM methylpyruvate (Sigma-Aldrich), and the matched glucose medium was conventional RPMI (Invitrogen).
• PDH and ⁇ KDH 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 of Moreadith and Fiskum. 1 Mitochondria were lysed in 0.4% lauryl maltoside.
• 50 ⁇ l of mitochondrial lysate was added to 96-well plates.
• 50 ul of reaction mix (5OmM Tris, pH 7.5, 2mM ⁇ -NAD+, 225uMv TPP, 2mM pyruvate or ⁇ -ketoglutarate, 150 ⁇ M coenzyme A, 2.6mM cysteine, ImM MgCl 2 ) was added to mitochondrial lysates, and the mixture was incubated for 45 minutes at 37 0 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 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 ⁇ % of the measured value. As a result, error bars were omitted from FIGURE 10.
• 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 ⁇ l Lysis buffer A [455 ⁇ l Zoom 2D protein solubilizer 1 (Invitrogen), 2.5 ⁇ l IM Tris base, 5 ⁇ l IOOX 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, 2.5 ⁇ l of dimethylacrylamide (DMA, Sigma- Aldrich) was added, and lysates were incubated for an additional 10 minutes. 5 ⁇ l of 2M DTT were added to neutralize excess DMA. Lysates were centrifuged at 16,00Ox g for 15 minutes.
• DMA dimethylacrylamide
• 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 0.8 ⁇ l pH 3-10 ampholytes, 0.75 ⁇ l 2M DTT and brought up to 150 ⁇ l with Zoom 2D protein solubilizer 1. 150 ⁇ l 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, 15 min 2000V, 30min). Strips were treated for 15 minutes in IX loading buffer, followed by 15 minutes in IX loading buffer plus 16OmM iodoacetatic acid. Strips were electrophoresed on NuPAGE 4-12% Bis Tris ZOOM gels (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 600Ox g. Pellet was lysed with lysis buffer C (4M urea, 10% glycerol, 2% SDS, 0.003% BPB; 5% 2-mercaptoethanol).
• Mitochondrial Ca +2 detection Cells were seeded on 35 mm glass bottom plates (BD Biosciences) at 3x10 5 , 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 ⁇ M, Invitrogen) in phenol red-free media and incubated at 37°C for 10 minutes. Cells were washed once with PBS, and images were 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 13), indicating that these dyes were measuring a mitochondrial Ca +2 signal. 3"4
• Gerencser AA and Adam- Vizi V Selective, high-resolution fluorescence imaging of mitochondrial Ca 2+ 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 Ca 2+ uptake in apoptosis. Cell Calcium 40, 553-560 (2006).
• the substrate effects on thioctan inhibition of ATP synthesis 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 1 might be compromised at lethal threshold doses and above. Using the potential-sensitive dye TMRE we observe the expected effect.
• FIGURE 13 Mitochondrial membrane potential declines rapidly with initiation of drug treatment. The kinetics of membrane potential decline is very similar to the loss of ATP synthesis in the presence of mitochondrial substrates. (FIGURE 9) ATP depletion in mitochondria is known to provoke a homeostatic response that includes the uptake of Ca +2 released from cytoplasmic stores, including the endoplasmic reticulum.
• lipoic acid derivative analogs presented below have been manufactured and are herein disclosed.

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