KR20110025168A - Pharmaceutical composition - Google Patents

Abstract

Control or disruption of the structure, expression and / or activity of at least one enzyme and / or enzyme complex or subunits thereof, such as through altered mitochondrial energy metabolism of pyruvate dehydrogenase (PDH) complexes in warm-blooded animals, including humans Pharmaceutically acceptable modulators and methods of use thereof affect the phosphorylation state of the complex, including an effective amount of at least one lipoic acid derivative and at least one pharmaceutically acceptable carrier. By increasing PDH kinase activity and / or decreasing PDH phosphatase activity, the modulator prevents the production of detoxified anaerobic glycolytic toxic metabolites through inhibition of the activity of the El subunit of the PDH complex, Increases mitochondrial oxidative phosphorylation activity. The mitochondrial membrane polarization is lost because cells characterized by hyperproliferation such as tumor cells cannot produce acetyl-CoA and NADH due to other actions by the modulator that inhibit the action of the E2 subunit of the PDH complex. It promotes cell death.

Description

Pharmaceutical composition {PHARMACEUTICAL COMPOSITION}

FIELD OF THE INVENTION The present invention relates to therapeutic and diagnostic compositions, more particularly showing selective uptake into cells characterized by hyperproliferation, including cancer cells, and regulating the modulation or perturbation of the structure, expression and / or activity of enzymes Thus, the present invention relates to a pharmaceutical composition and a method of using the same, which facilitates the detection and treatment or destruction of these cells. More specifically, these agents target and disrupt the activity or regulation of modified mitochondrial energy metabolism observed in locales such as the modified pyruvate dehydrogenase (PDH) complex associated with most cancers.

Mitochondria are the major regulatory centers for energy production and cellular life-and-kill processes in eukaryotic cells. While a variety of communication mechanisms and the remaining cells are present, the reversible phosphorylation is an important means of regulating mitochondrial function (Pagliarini DJ and Dixon JE (2006) Mitochondrial regulation:..? Reversible phosphorylation takes center stage TRENDS in Biochem . Sci . 31: 26-34, passim , incorporated herein by reference) The steady increase in the number of reported mitochondrial kinases, phosphatase and phosphoproteins suggests that phosphorylation may emerge as a common topic in the regulation of mitochondrial processes. Suggest. Pathological or genetic changes associated with the regulation of mitochondrial enzyme structure, function and activity contribute to the treatment of the disease and may be important targets for the treatment of the disease.

In combination with mitochondria's role as a convergence point and regulator of various cellular functions, mitochondria play an important role in apoptosis, generation of reactive oxygen species (ROS), and numerous metabolic processes, including production of more than 90% of cellular ATP. Do this. In addition, as cells grow and divide, new mitochondria must be made, which itself requires thorough coordination of nuclear and mitochondrial DNA transcription and translation. Finally, as cell energy demands change, mitochondria must respond to this by modulating their ATP production. Thus, mitochondria require complex communication systems with cellular functions. As is apparent from FIG. 1, signal molecules entering and exiting the mitochondria include ions, gases, metabolites, hormones, transcription factors and proteins. Thus, recognition of mitochondria as a center for the reception, integration and transmission of cellular signals is an important advance in pharmaceutical design and testing.

The basis of signaling in mitochondrial regulation is reversible phosphorylation. However, while the first demonstration of protein kinase events was reported in 1954, and it was discovered almost 40 years ago that key mitochondrial functions could be regulated by reversible phosphorylation, reporting mitochondrial phosphorylation events It is rare. This is true even in the 1980s and 1990s, with the recognition of numerous signal transduction phosphorylation cascades that cross the plasma membrane, and then through the cell substrate to the nucleus. This lack of knowledge is partly due to the fact that most of the mitochondrial protein base is behind two lipid bilayers, which prevents the mitochondrial proteins from reaching the cell substrate signaling cascades. In either case, mitochondria are not widely accepted to modulate signaling by reversible phosphorylation in a manner critical to disease management. Nevertheless, as shown in Table 1, by 2006, more than 60 proteins in all mitochondrial compartments (eg, substrates, inner membranes, interstitial spaces, and outer membranes including the plasma-faced outer surfaces) were found in various categories. It has been identified as phosphoproteins involved in mitochondrial function. Increasing data also demonstrate the importance of reversible phosphorylation of mitochondrial targets and the use of compositions targeting them to improve cancer treatment.

number protein location P site source function references One PDC E1α M Ser Various Acetyl-CoA Formation [11] 2 PDC E1β M Tyr Human sperm Acetyl-CoA Formation [8] 3 PDC E3 M Tyr Human sperm Regulation of PDC [8] 4 PDK isomorphism 2 ^b M Ser / Thr mouse TCA [27] 5 Aconitase M ? Cattle / Potato TCA [22] 6 NAD-isocitrate dehydrogenase M ? Cattle / Potato TCA [22] 7 NAD-malic acid dehydrogenase M ? potato TCA [22] 8 NAD-Malic Enzyme M ? potato TCA [22] 9 Succinyl-CoA-ligase α subunit M ? Mice / Potatoes TCA [22,47] 10 Succinyl-CoA-ligase β subunit M ? Mice / Potatoes TCA [22,47] 11 Formic acid dehydrogenase M Ser / Thr potato TCA [22,23] 12 Aconitase M Tyr Guinea pig synaptosome TCA [8] 13 BCKAD M Ser Various AA Ambassador [31] 14 BCDAD kinase ^b M Ser mouse AA Ambassador [54] 15 HSP22 M Ser corn Chaperone [39] 16 HSP 90 M ? potato Chaperon [22] 17 Chaperonin 60 M ? potato Chaperon [22] 18 mthSD75 M Tyr Mouse liver cancer cells Chaperon [29] 19 TRAP-1 ^c M Tyr Human sperm Chaperon [8] 20 CYP2E1 ^c M / IM? Ser COS Detoxification [43] 21 CYP2B1 ^c M / IM? Ser COS Detoxification [18] 22 GSTA 4-4 M Ser / Thr COS Detoxification [44] 23 DBP M ? leaven mRNA turnover [38] 24 MnSOD M ? potato Oxidative stress defense [22] 25 EF-Tu M Thr? Rabbit heart Protein synthesis [32] 26 Creatine Kinase M ? small Synthesis of phosphate creatine [45] 27 MTERF ^b M ? mouse Warrior Decision [42] 28 Abf2p M Ser / Thr? leaven mtDNA maintenance [26] 29 MtTBP ^b M ? leaven mtDNA maintenance [50] 30 NDK M / IMS Ser / His pea Nucleoside triphosphate balance [49] 31 StAR IMS Ser COS-1 Steroid hormone synthesis [19] 32 Axmito ^c M? Tyr mouse Regulation of PLA? [53] 33 SSAT ^b M? Ser? mouse Acetylation of spermidine [21] 34 Sab OM Ser Mouse Cardiac Myocytes SH3 region = binding protein [28] 35 CPT-1 OM Ser mouse β-OX [35] 36 MtGAT ^c OM Ser / Thr? mouse Glycerolipid Biosynthesis [41] 37 BAD ^c OM Ser FLS.12 cells Apoptosis [30] 38 BCL-2 OM Ser Jurkat Apoptosis [17,52] 39 Bcl-X _L OM Thr U-937 cells Apoptosis [36] 40 CREB M / IM Ser? mouse mtDNA Warrior? [24] 41 VDAC OM Tyr Guinea pig synaptosome transport [8,45] 42 Cl: ESSS IM Ser small OXPHOS [25] 43 Cl: 10 kDa IM Ser small OXPHOS [25] 44 Cl 42 kDa (2 sites) IM Ser & Thr small OXPHOS [45,55] 45 CIII Center I IM Tyr Human sperm OXPHOS [8] 46 CIII Center II IM ? small OXPHOS [45] 47 CIV I IM Tyr small OXPHOS [37] 48 CIV II IM Tyr Osteoclast OXPHOS [40] 49 CIV III ^b IM Ser / Thr? small OXPHOS [34] 50 CIV IV IM ? mouse OXPHOS [46] 51 CIV Vb ^b IM Ser / Thr? small OXPHOS [34] 52 CV α IM ? Cattle / Potato OXPHOS [22] 53 CV β IM Thr Human sk mousse OXPHOS [33] 54 CV a IM ? potato OXPHOS [48] 55 CV b IM ? potato OXPHOS [48] 56 ScIRP IM ? mouse OXPHOS [20 57 SDH-Fp IM ? Cattle / Potato OXPHOS [22] 58 bc1 complex, β-MPP subunit IM ? potato OXPHOS [22] 59 CIII Center I IM Tyr Human sperm OXPHOS [8] 60 NAD (P) hydrogen transferase IM ? small H ⁺ pump [45] 61 ANT IM ? small transport [45] 62 Phosphate carrier protein IM ? small transport [45] 63 Aldose reductase ^c ? S / T? Various cell lines Osmotic control [51]

^a Abbreviation: AA, amino acids (S, serine; T, threonine; Y, tyrosine); ANT, adenine nucleotide transporter; Axmito, mitochondrial annexin; β-OX, β-oxidation; BCKAD, branched chain keto acid dehydrogenase; CI-CV, respiratory chain complexes 1-5; CPT, carnitine palmitoyl transferase; CREB, cAMP-responsive element (Cre) -binding protein; CYP, cytochrome P450; DBP, dodecamer-binding protein; EF, prolongation factor; GST, glutathione S-transferase; HSP, heat shock protein; IM, intima; IMS, intima space; M, substrate; MnSOD, manganese peroxygen mutant enzyme; mTERF, mitochondrial transcription termination factor; mtGAT, mitochondrial glycerol-3-phosphatase acetyltransferase; mthsp, mitochondrial HSP; mtTBP, mitochondrial telomere-binding protein; NDK, nucleoside diphosphate kinase; OM, envelope; OXPHOS: oxidative phosphorylation; P site: phosphorylation site; PDC E1 / 3, pyruvate dehydrogenase complex E1 / 3 subunit; PDK, pyruvate dehydrogenase kinase; PLA, phospholipase A; ScIRP, subunit c-immunoreactive peptides; SSAT, spermidine / spermine acetyltransferase; StAR, steroid producing regulatory protein; TCA, tricarboxylic acid cycle; TRAP-1, tumor-necrosis factor type 1 receptor-associated protein; VDAC, voltage dependent anion channel.

^b phosphorylation is only observed in vitro on recombinant proteins.

cproteins are translocated to mitochondria (ie, proteins are not resident mitochondrial proteins).

In the human genome, protein kinases (PKs) and protein tyrosine phosphatases (PTPs), the maximum kinase and phosphatase families each have more than 500 elements and more than 100 elements. . Combined with a smaller family of kinases and phosphatases, these signal molecules comprise nearly 3 percent of all proteins encoded in the human genome. Similar to the aforementioned phosphoproteins, kinases and phosphatases are involved in mitochondrial function in a surprising number of studies, and so far at least 25 kinases and 8 phosphatases are shown in FIG. 2. It has been reported to be ubiquitous in mitochondria as shown in FIG. These kinases and phosphatases are clearly not limited to one group or family; Rather, they represent almost all known mammalian kinases and phosphatase subgroups, reflecting a range of signaling pathways that are likely to affect mitochondria. Such signaling molecules include other kinases and phosphatases in substrate specificity (eg, tyrosine kinases, classic PTP subgroups, serine / threonine kinases and bi-specific PTPs); Other kinases and phosphatases in the enzyme mechanism (eg, cysteine-based PTPs, aspartic acid-based PTPs and metal-dependent phosphatases); And other kinases and phosphatases (e.g., bacterial related pyruvate dehydrogenase kinases (PDKs) and phosphatases (PDPs), branched chain keto acid dehydrogenase kinases (BCKDK) and in evolutionary conservation and Phosphatase (BCKDP) and numerous mammal-specific enzymes).

Many of these signaling molecules have different non-mitochondrial roles in cells and are known to exist outside the mitochondria. In most proteins, little is known about the impetus or mechanism for their mitochondrial translocation. However, it is clear that kinases and phosphatases, like the phosphoproteins listed above, are present in all compartments of the mitochondria, as evident in FIG. 3, and their activity depends on various mitochondrial functions.

However, some signaling molecules appear to be predominantly mitochondria. In addition to PDKs and PDPs, this group includes PTEN-induced kinase PINK1, bi-specific PTPs targeted to mitochondrial PTMT1, and aspartic acid phosphatase / ATP hydrolase Tim50. Substrates for many of these proteins are currently unknown, but it is clear from biological and genetic data that they have important functions in mitochondria. For example, submitochondrial localization is determined, but PINK1 is targeted to the mitochondria by the N-terminal signal sequence. This kinase, which shares high sequence homology with the Ca + 2 / chalmodulin-regulated kinase family, appears to be involved in pro-survival activities first. Similarly, PTPMT1 has recently been identified as the first PTP ubiquitous mainly in the mitochondria, and is targeted to mitochondria by the N-terminal signal peptide, such as PINK1, and is known to be closely associated with the substrate surface of the mitochondrial lining. PTPMT1 is highly expressed in pancreatic β cells in which mitochondria have an important function of linking glucose metabolism to the secretion of insulin. Finally, Tim50, an important component of the TIM (intracellular translocse) complex, has sequence homology with the CTD family of aspartic acid phosphatase / ATP hydrolase. Like other elements in this family, Tim50 can function as an ATP hydrolase, but it has also been shown to have phosphatase activity against phosphotyrosine analog para-nitrophenyl phosphate in vitro. As given above, it is understood that kinases and phosphatases are not only supplemented with mitochondria from elsewhere in the cell, but also that the mitochondria themselves appear to have a contingent of resident signaling molecules.

The effect of phosphorylation on most known mitochondrial phosphoproteins is not clear, but some phosphorylation events are partly characterized, with responsive kinases and phosphatases being sometimes not identified. As with the phosphoproteins and signaling molecules discussed above, these examples are not limited to one field of mitochondria.

Phosphorylation on the mitochondrial outer membrane has an important role in the regulation of apoptosis. A particularly well defined event is the phosphorylation of BAD, a BCL-2 family of apoptotic precursors. After treatment with first-surviving cytokine interleukin-3, PKA was shown to translocate to the outer membrane. Once immobilized with A-kinase anchoring protein on the outer membrane, PKA phosphorylates BAD on Ser 112, contributing to inactivation and disassociation of BAD from mitochondria, the process shown in FIG. 3A. Phosphorylation of BAD by p70S6 kinase on Ser 136 and phosphorylation of BAD by unidentified kinase on Ser 155 are also related to BAD inactivation.

The best-proven example of reversible phosphorylation that acts as a regulatory mechanism in healthy cell mitochondria is the phosphorylation of the PDH complex in the substrate, the simplified illustration shown in Figure 3B. This complex catalyzes the conversion of glycolysis-derived pyruvate to acetyl-coenzyme A (CoA), a major precursor to the tricarboxylic acid (TCA) cycle. As the link between these two major energy-generating pathways, the PDH complex must be properly regulated to maintain cellular glucose homeostasis.

Since it has been identified as the first mitochondrial phosphoprotein, its regulation by PDH complex and reversible phosphorylation has been thoroughly studied. Phosphorylation and dephosphorylation of the PDH complex is performed 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. Phosphorylation events occur on three individual serine residues of the E1 subunit, each of which leads to significant inactivation of this complex. It is noteworthy that there is at least one report on the PDK itself that is currently phosphorylated. It is known that this phosphorylation carried out by PKC inactivates PDK, potentially demonstrating different levels of PDH complex regulation by reversible phosphorylation. Thus, PDH complexes are a prime example of mitochondria that use phosphorylation to add regulatory levels to other conserved processes.

As indicated by the mitochondrial tyrosine kinases and phosphatases listed above, phosphorylation in these organelles is not limited to serine and threonine residues. An example of tyrosine phosphorylation that affects mitochondrial energetics is observed in the regulation of cytochrome c oxidase (COX) in the inner membrane shown in FIG. 3C. In the respiratory chain, COX as the terminating enzyme reduces the oxygen coordination to water, pumping protons across the inner membrane. Similar to the PDH complex, COX is allosterically regulated by thyroid hormone T ₂ and possibly Ca ⁺² ions as well as ATP and ADP. In addition to this type of regulation, it has been shown that COX is phosphorylated in a c-AMP-dependent manner both in vitro and intracellularly of HepG2 cells. COX contains 13 subunits and crystallizes as dimers. The phosphorylation site is identified by Tyr 304 of subunit 1 located at the dimer interface on the intermembrane. Phosphorylation events significantly inhibit COX activity, perhaps by cleavage of dimer formation.

In a second example of tyrosine phosphorylation of COX, a portion of non-receptor tyrosine kinase c-Src similar to Lyn tyrosine kinase is ubiquitous inside the mitochondria and leads to tyrosine phosphorylation of COX on the unidentified site of subunit II in osteoclasts. The result of the phosphorylation event is the opposite of that observed in subunit I, which improves COX activity.

An important aspect of mitochondrial signaling is how kinases and phosphatases themselves are regulated. While residing somewhere in the cell, such as Abl, Akt, GSK3I3 and PKCδ, many of the kinases targeted to mitochondria appear to be only in their active state. Thus, the degree of some kinase activity in the mitochondria may simply be affected by the number of enzymes entering the organelles.

However, for resident signaling molecules, other regulatory means must be in place. While these processes remain undetermined, the second messengers are likely to play an important role. Activity of PDK and the same type PDP has been known to be regulated by the ions and Mg ^{2 +,} Ca ^{2 +,} K ⁺ and a small molecule, such as ADP. Characterization of mitochondrial nitric oxide synthases and the discovery of soluble adenylate cyclases in mitochondria provide another opportunity for second messengers to contribute to the regulation of mitochondrial signaling molecules. Finally, ROS, proven as a means of regulating signaling molecules somewhere in the cell, will almost certainly be involved in the regulation of kinases and phosphatases in the mitochondria, where most reactive oxygen species are produced. The relative expression levels of kinases and phosphatase isoforms will play an important role in pathology and are linked to other signal transduction events associated with the disease. In addition, changes in gene and expression levels can be correlated with such changes.

Even after some thorough proteomic investigations, only two-thirds of mammalian mitochondrial proteome are known. Most of the other 1/3 are likely to consist of low abundance proteins such as signaling proteins that are below the detection level of this mass spectrometry. Also evident in this study is the high variability in protein content between mitochondria from other tissues. For example, it was found that only about 50% of the proteins in their proteomic efforts were conserved in the four tissues examined (ie, brain, heart, liver and skeletal muscle). Other mitochondrial signaling pathways are likely to not only change from tissue to tissue in the same way, but also contribute to the observed mitochondrial diversity.

Nevertheless, there is plenty of evidence for the conclusion that reversible phosphorylation is involved in the regulation of mitochondrial processes. With more than 60 reported phosphoproteins, 30 kinases and phosphatases, and a variety of accessory signaling proteins, mitochondria are certainly an underestimated site for signaling by reversible phosphorylation, and in fact such regulation is associated with cancer. It may be useful for the treatment of such hyperproliferative diseases.

Most of the fast-growing tumor cells show profound genetic, biochemical and histological differences for the unmodified cells. Many of these are associated with altered energy metabolism compared to the developing tissue. The most notorious and well-known energy metabolic modification in tumor cells is their ability to increase in the presence of high O ₂ concentrations, a phenomenon also known as the Warburg effect.

Warburg first proposed that the impetus for enhanced glycolysis in tumor cells was an energy deficiency caused by irreversible impairment of mitochondria's ability to release glucose, similar to anaerobic muscle, to lactate through glycolysis and subsequently secreted. This increase in glycolytic flux in tumor cells has been suggested to be a metabolic strategy to ensure survival and growth in low O ₂ concentrations such as partial hypoxia observed in solid tumors with poor oxygenation. In particular, since the concentration of O ₂ is lower than 20 μM in many human hypoxia tumors, oxidative phosphorylation is limited in this regard. Thus, glycolysis seems to be a major energy pathway in solid tumors (eg, slow growing melanoma and breast adenocarcinoma).

A proportional relationship between cell proliferation rate and ATP feed rate was established for fast-growing tumor cells. Some authors suggest that activity correlates with tumor malignancy, so that the rate of glycolysis is higher in highly de-differentiated and rapidly growing tumors than in slower growing tumors or normal cells. In fact, high levels of lactate have been proposed as predictors of malignancy. It is possible that these events are associated with other signal transduction events and genetic changes, examples of which include the generation and release of hypoxia inducing and angiogenic factors.

 As shown in FIG. 4, for many years the tricarboxylic acid (TCA) cycle was considered to be uniquely biologically meaningful only for its role in the production of ATP as an energy source for the organism. However, recent studies show that TCA cycle activity affects signal transduction pathway functions, including cell growth and apoptosis determination, and that appropriate glycolysis and TCA cycle enzymes can be upregulated or downregulated. In addition, there is a direct correlation between tumor progression and the activity of the glycolysis enzyme hexokinase and phosphofructokinase (PFK), which are significantly increased in fast-growing tumor cells. Thus, it is assumed that tumor cells showing deficiency in their oxidative capacity are more malignant than tumor cells with active oxidative phosphorylation. Even under hypoxia or aerobic conditions, the dependence of cancer tissues on glycolysis is associated with an increase in malignancy.

The role of lipoic acid in the PDH complex of healthy cells is well studied. The PDH complex has three central subunits, E1, E2 and E3 (pyruvic acid dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoamide dehydrogenase, respectively). These complexes have a central E2 center, with other subunits surrounding the center to form the complex. In the gap between these two subunits, the lipoyl region carries intermediates between active sites. The refoil region itself is attached to the E2 center by a flexible linker. In the formation of hemithioacetal by reaction of pyruvate and thiamine pyrophosphate, this anion attacks S1 of the oxidized lipoate species attached to the lysine residue. Thus, lipoate S2 is substituted as a sulfide or sulfhydryl moiety, releasing thiazole due to the subsequent decay of the tetrahedral hemithioacetal, releasing TPP and generating thioacetic acid salt on S1 of the lipoate. At this point, the liporate-thioester functionality is translocated to the E2 active site, where the acyl transition reactions transfer acetyl from the "swinging arm" of the thioate to the thiol of coenzyme A. This produces acetyl-CoA, which is released from the enzyme complex and then enters the TCA cycle. Thereafter, the dihydrolipoates still bound to the lysine residues of the complex migrate to the E3 active site, where they undergo flavin-mediated oxidation and return to their lipoate dormant state, producing FADH ₂ (and ultimately NADH). And regenerate lipoates back into competitive acyl receptors. If this lipoate species is blocked, there will be no electron flow to FADH ₂ or generation of acetyl-CoA, resulting in toxic accumulation of pyruvate in the cell. In cancer cells, the production of acetoin has been suggested as a requirement for cell detoxification and survival.

As mentioned earlier, the activity of PDH complexes in mitochondria is highly regulated by various allosteric effectors and covalent modifications. PDH activity is regulated by the phosphorylation state that is most active in the dephosphorylated state. PDH phosphorylation is catalyzed by PDK. PDK activity is enhanced by increasing levels of ATP, NADH and acetyl-CoA. Negative factors of PDK are ADP, NAD ⁺ , CoA-SH and pyruvate, their levels increase as ATP levels drop. The regulation of PDP, an enzyme that activates PDH through dephosphorylation, is not fully understood, while Mg ⁺² and Ca ⁺² are known to activate PDP.

Two products of the complex, NADH and acetyl-CoA, are negative allosteric factors of PDH-a, the dephosphorylated active form of PDH. These factors reduce the enzyme's affinity for pyruvate, thus limiting carbon flow through the PDH complex. NADH and acetyl-CoA are also potent positive factors of PDK, an enzyme that converts PDH into phosphorylated PDH-b form to inactivate PDH. Since NADH and acetyl-CoA accumulate at high cellular energy charges, it is not surprising that high ATP levels also upregulate PDK activity, enhancing down-regulation of PDH activity in energy-rich cells. However, since pyruvate is a potent negative factor of PDK, when pyruvate levels rise, PDH-a will be favored even at high levels of NADH and acetyl-CoA.

The concentration of pyruvate maintaining PDH-a is high enough that the allosterically down-regulated high K _m form of PDH in ATP-rich cells can nevertheless convert pyruvate to acetyl-CoA. Due to the large amount of pyruvate in cells with high ATP and NADH levels, pyruvate carbon has two major carbon storage forms in which acetyl-CoA is the main carbon source (glycogen and fatty acid synthesis through gluconeogenesis). Fat production). Although the regulation of PDP-b is not well understood, it will be well controlled to maximize pyruvate oxidation under reduced ATP concentrations and to minimize PDH activity under high ATP concentrations.

Tumor cells are unable to infinitely accumulate pyruvate and related aldehydes and radicals, such as acetaldehyde, peroxide, hydrogen peroxide and hydroxyl radicals, which are cytotoxic at high concentrations, and through this mechanism the cellular pH is reduced. Because it lowers dramatically. Thus, active acetaldehyde decarboxylation by the E1 component of the PDH complex via β-hydroxyethylthiamine pyrophosphate bound to a significant portion of mitochondrial pyruvate for AS-30D and Ehrlich liver cancer. It is described. This active acetaldehyde is in turn condensed with a second acetaldehyde, ultimately deoxidizing or reducing the original pyruvate using either amino acid glutamine or lipoic acid (eg, maintaining pH homeostasis in the cell). Competitively inhibits acetoin (3-hydroxybutanone), a compound that is less toxic to cells than its pyruvate precursor. However, despite the importance of acetoin in the pathway of tumor cell detoxification as a result of pyruvate accumulation due to glycolysis dependence of tumor cells as a source of ATP production, There is little reference in the prior art.

Recent studies suggest that forcing tumor cells with stronger aerobic metabolism inhibits tumor growth. Therefore, metastasis to Warburg metabolism requires blocking of the PDH complex. In this metastasis, there is enhanced signaling by hypoxia-inducing factor (HIF) in tumor cells, which is not surprising to see the significant role of HIF in glucose metabolism as shown in FIG. 5. Mutations that directly or indirectly promote HIF signaling appear to be in fact a common mechanism in cancer development. HIF induces overexpression of PDK1, which in turn acts to lower PDH complex activity. Phosphorylation by PDK1 can be particularly effective in maintaining inactive PDH complexes, since this isoform uniquely phosphorylates three serine residues in the alpha subunit of E1, the first subunit of the PDH complex. Reactivation of El requires the removal of all three phosphate groups. In addition, PDH complex activation can lead to an improvement in ROS production, which in turn can lead to apoptosis. However, the change in PDK1 observed in cancer can be attributed not only to changes in its concentration, but also to its activity and possibly changes in its amino acid sequence between one tumor type or between one patient and another patient. In addition, PDK1 can form other complexes with various tumor associated molecules that depend on the tumor type. Thus, inhibiting PDK may be a potential target for apoptosis in tumors. However, to date, known PDK1 inhibitors have been demonstrated to cause maximal inhibition of only 60% of this isoenzyme.

While conventional chemotherapy targets proliferation of proliferating cells, all clinically acceptable chemotherapy uses high drug doses that induce profound damage to normal proliferating host cells. Therefore, more selective targeting is needed for the treatment of cancer. Another problem associated with chemotherapy is that in many tumor types, there is either inherent or acquired resistance to antitumor agents. Overall, conventional chemotherapy provides little long term therapeutic benefit to most malignant tumors and is often associated with side effects that reduce the length or quality of life. Thus, there is a need for a radically new approach that can provide long-term management of tumors while allowing for good quality of life.

Clearly, drug efficacy, delivery and side effects are issues that need to be addressed in the development of new chemotherapy. In solid tumors, delivery to the hypoxia region can be difficult if the drug does not readily penetrate other cell layers. In order to remove this uncertainty, the design of anticancer agents with metabolic inhibition constants at least in the submicromolar range seems to be appropriate. Cancer cells can be claimed to be genetic and phenotypicly heterogeneous from cell line to cell line. However, all tumor cell lines rely on glycolysis and oxidative phosphorylation for ATP supply. Focusing on the Warburg effect enables drug design based on physiological and biochemical energy differences between tumors and normal cells, which selectively affects tumor metabolism and growth without functionally affecting healthy host tissues and organs. Facilitate the design of delivery and treatment strategies.

U.S. Provisional Application Nos. 60 / 912,598 to Bingham et al., As well as U.S. Pat.Nos. 6,331,559 and 6,951,887, both incorporated herein by reference, are novel types that selectively target and kill both tumor cells and other types of diseased cells. Disclosed are therapeutic agents for lipoic acid derivatives. In addition, these patents disclose pharmaceutical compositions comprising the effective amount of the lipoic acid derivatives together with a pharmaceutically acceptable carrier and methods of use thereof. However, while these patents describe the structure and general use of these lipoic acid derivatives, no patent indicates that these derivatives are useful for modulating the structure and / or expression levels of the PDH complex and / or for regulating the activity of the PDH complex. none.

Since the structure and / or activity of the PDH complex has proven to be an important determinant of tumor activity, it would be beneficial to provide a pharmaceutically acceptable modulator of the structure and / or activity or expression level of the PDH complex and its use. .

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for use in the treatment or diagnosis of a disease, condition or syndrome that is characterized by cell hyperproliferation such as cancer and which exhibits selective activity in tumor cells.

It is another object of the present invention to provide a pharmaceutical composition which is used for the treatment or diagnosis of the above mentioned diseases, conditions or syndromes and which causes at least side effects upon administration.

It is another object of the present invention to provide a pharmaceutical composition which is used for the treatment or diagnosis of the above mentioned diseases, conditions or syndromes, which can be easily prepared at the lowest possible cost and stored for the longest possible time.

Provided herein are pharmaceutical compositions which are used in the treatment or diagnosis of the above mentioned diseases, conditions or syndromes, and which regulate mitochondrial energy metabolism via levels of structure, activity and / or expression of the PDH complex in tumor cell mitochondria. Another purpose.

In order to achieve the above-mentioned objects, the present invention relates to at least one enzyme, such as a PDH complex, comprising a disease, condition or syndrome or symptoms thereof characterized by a cell hyperproliferation such as cancer in warm-blooded animals, including humans and / or Or a pharmaceutical composition useful for the treatment, diagnosis or prevention of a disease, condition or syndrome, characterized by alterations in the modulation, expression and / or activity of enzyme complexes or subunits thereof or disturbances. Including an effective amount of at least one lipoic acid derivative, including the lipoic acid derivatives described in US Pat. Nos. 6,331,559 and 6,951,887 and US Provisional Application No. 60 / 912,598, which are incorporated by reference, and at least one pharmaceutically acceptable carrier thereof. A wide variety of pharmaceutical compositions are provided.

By inhibiting mitochondrial energy metabolism, lipoic acid derivatives of the present invention cause both a loss of mitochondrial membrane potential and other mitochondrial consequences in the diseased cell, causing an irreversible initiation of cell death. In addition, the lipoic acid derivatives of the present invention metabolize mitochondrial energy by inhibiting the conversion of pyruvate to acetoin, a less toxic molecule by activation of PDKs and / or inhibition of PDPs or through inhibition of activity of the E1 subunit of the PDH complex. Can be suppressed. Inhibition of acetoin synthesis will distort other processes, including redox balance, and also lead to the generation of toxic byproducts including acetaldehyde, peroxide, hydrogen peroxide and hydroxyl radicals, which in turn result in diseased cells May cause irreversible damage to the mitochondria.

The pharmaceutical composition of the present invention can modulate the effects of PDK1, PDK2, PDK3, PDK4 and their respective mutations or isoforms. In addition, the pharmaceutical composition can control the effects of PDP1, PDP2 and their respective isoforms.

In addition, the pharmaceutical composition of the present invention can regulate the expression level of phosphoylase, kinase and dehydrogenase enzyme components found in the PDH complex. Such regulation may occur at the transcriptional, translational or post-translational stage, including epigenetic silencing of the appropriate genes.

As compounds derived from molecules that are fundamentally associated with the TCA cycle and by prolonged glycolysis, the pharmaceutical compositions of the present invention demonstrate selective uptake into tumor cells. In addition, such selective tumor cell intake minimizes the side effects that can be had on healthy non-modified cells and tissues due to administration of the present pharmaceutical compositions.

In one embodiment of the invention, the lipoic acid derivatives are as general formula (I):

Wherein R ₁ and R ₂ are hydrogen, alkyl C _n H _{2n +1} , alkene C _n H _2n , alkenyl C _n H _{2n -1} , alkane C _n H _2n-2 , alkynyl C _n H _{2n -3} , Alkyl sulfide CH ₃ (CH ₂ ) _n -S-, disulfide alkyl CH ₃ CH _t -SS-. Thiocarbamic ester (CH ₂ ) _n C = NH- and semithioacetal CH ₃ CH (OH) -S-, where n is 1 to 10 and t is 0 to 9; Aliphatic; Acyl defined as R ₃ C (O)-; Heteroaryl; Imidoyl, defined as R ₄ C (═NH)-; Organometallic aryl; Alkyl-organometallic aryl; And semiacetal R ₅ CH (OH) -S-;

R ₁ and R _{2 as} defined above may be unsubstituted or substituted;

R ₃ is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which may be substituted or unsubstituted;

R ₄ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl and alkylheteroaryl, any of which may be substituted or unsubstituted;

R ₅ is CCl ₃ , CF ₃ or COOH;

And x is of formula (I) wherein 0-16;

Or salts thereof.

In a second embodiment of the invention, the lipoic acid derivatives are represented as second general formula (II):

Wherein M is a covalent bond-[C (R ₁ ) (R ₂ )] _z -or a metal chelate or other metal complex, wherein the metal is not palladium;

R ₁ and R ₂ is hydrogen, acyl R ₃ C (O) -, alkyl _{C n H 2n +1, C m} H 2m - 1 alkenyl, alkynyl defined as C _m H _2m-3 defined by, aryl , Heteroaryl, alkyl sulfide CH ₃ (CH ₂ ) _n -S-, imidoyl as defined by R ₃ C (= NH)-and hemiacetal as defined by R ₄ CH (OH) -S- Is selected;

R ₁ and R _{2 as} defined above may be unsubstituted or substituted;

R ₃ and R ₄ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclyl, any of which may be substituted or unsubstituted and ;

R ₅ is selected from the group consisting of CCl ₃ , CF ₃ or COOH;

And x is 0-16, z is 0-5, n is 0-10 and m is 2-10;

Or salts thereof.

In a third embodiment of the invention, the riboic acid derivatives are as the third general formula (III):

Wherein R ₁ and R ₂ are hydrogen, alkyl C _n H _{2n +1} , alkene C _n H _2n , alkenyl C _n H _{2n -1} , alkane C _n H _2n-2 , alkynyl C _n H _{2n -3} , Alkyl sulfide CH ₃ (CH ₂ ) _n -S-, disulfide alkyl CH ₃ CH _t -SS-. Thiocarbamic ester (CH ₂ ) _n C═NH— and semithioacetal CH ₃ CH (OH) —S—, where n is 1 to 10 and t is 0 to 9, aliphatic, R ₄ C (O) — Imyl, heteroaryl, imidoyl defined as R ₅ C (= NH)-, organometallic aryl, alkyl-organometallic aryl, semiacetal R ₆ CH (OH) -S-, amino acids, carbohydrates, Independently selected from the group consisting of nucleic acids, lipids and multimers and combinations thereof;

R ₁ and R _{2 as} defined above may be unsubstituted or substituted;

R ₃ is selected from the group consisting of amino acids, carbohydrates, nucleic acids, lipids and multimers thereof;

R ₄ is independently selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which may be substituted or unsubstituted;

R ₅ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl and alkylheteroaryl, any of which may be substituted or unsubstituted;

R ₆ is CCl ₃ , CF ₃ or COOH;

And x is from 3 to 16 in general formula (III);

Or salts thereof.

In a fourth embodiment of the invention, the lipoic acid derivatives are as the fourth general formula (IV):

Wherein M is a covalent bond-[C (R ₁ ) (R ₂ )] _z -or a metal chelate or other metal complex, wherein the metal is not palladium;

R ₁ and R ₂ is hydrogen, acyl R ₄ C (O) -, alkyl _{C n H 2n +1, C m} H 2m - 1 alkenyl, alkynyl defined as C _m H _2m-3 defined by, aryl , Heteroaryl, alkyl sulfide CH ₃ (CH ₂ ) _n -S-, imidoyl as defined by R ₄ C (= NH)-, hemiacetal as defined by R ₆ CH (OH) -S-, amino acids, carbohydrates Are independently selected from the group consisting of nucleotides, nucleic acids, lipids, and multimers and combinations thereof;

R ₁ and R _{2 as} defined above may be unsubstituted or substituted;

R ₃ is independently selected from the 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 may be substituted or unsubstituted and ;

R ₅ is selected from the group consisting of CCl ₃ , CF ₃ or COOH;

And x is 0 to 16, z is 0 to 5, n is 0 to 10 and m is 2 to 10;

Or salts thereof.

In addition, since any such general structure can be metabolized in cells or mitochondria, it is expressly intended that the metabolites of the above-referenced structures are within the scope of the present invention.

In each of the above formulas, the (R) -isomer of each particular lipoic acid derivative has higher physiological activity than the (S) -isomer. Thus, the lipoic acid derivative must be present alone, either in (R) -isomer form or in a mixture of (R)-and (S) -isomers.

In another aspect of the invention, at least one enzyme and / or enzyme complex, or a PDH complex, comprising a disease, condition or syndrome or symptoms thereof characterized by a cell hyperproliferation such as cancer in a warm blooded animal, including humans A method of treating, diagnosing, or preventing a disease, condition, symptom muscle, or symptom thereof, including a change in the regulation or disturbance of the structure, expression, and / or function of a subunit, the method comprising the steps of: Provided is a method comprising administering an effective amount.

In another aspect of the invention, at least one enzyme and / or enzyme complex or subunit thereof, such as a PDH complex, comprising symptoms of a disease, condition or syndrome that is characterized by cell hyperproliferation such as cancer A method of diagnosing and predicting a therapeutic benefit in a patient suffering from diseases, conditions, symptoms of symptoms or symptoms thereof, including a change in structure, expression and / or activity or a disturbance, wherein a sample of cells is obtained from the patient And administering an effective amount of the composition of the invention to said cells in vitro and obtaining a result therefrom.

The following drawings illustrate embodiments of the invention and are not intended to limit the scope of the present application to what is included in the entire specification and claims.
1 depicts general signal transduction agents targeted to and from mitochondria and their effects.
2 shows a list of mitochondrial kinases and phosphatases and their positions within the mitochondria.
3A shows the effect of phosphorylation of BAD by PKA and irreversible phosphorylation on it in the mitochondrial envelope.
3B shows the effect of the conversion of pyruvate to acetyl-CoA and its irreversible phosphorylation in mitochondrial substrate by action of PDH complex.
FIG. 3C shows the effect of COX and irreversible phosphorylation on it in reducing oxygen to water and pumping protons across the mitochondrial lining.
4 shows the structure of the product in the corresponding production of substrate and pyruvate, and also shows ATP and NADH generation and associated enzymes.
5 shows the regulation of glucose metabolism by HIF-1.
6A shows the difference in energy metabolism between normal tissue and cancer tissue in vivo.
6B shows the differences between the bioproducing forms of lipoic acid in the PDH complex and the lipoic acid derivatives forming part of the pharmaceutical composition of the present invention.
6C shows the modulation of PDH complex by lipoyl residue effect on PDK.
Figure 7 shows the effect of the pharmaceutical composition of the present invention on xenograft tumor growth.
8 shows the effect of treatment with the pharmaceutical composition of the present invention on three tumor cell types and non-modified cells.
9A depicts ATP levels in lung tumor cells after treatment with the pharmaceutical composition of the present invention above the lethal threshold.
9B compares the inhibition of ATP synthesis of the pharmaceutical compositions of the present invention in pyruvate-containing medium versus glucose-containing medium.
9C compares the inhibition of ATP synthesis of the pharmaceutical composition of the present invention in breast cancer cells with the inhibition of ATP synthesis of the pharmaceutical composition of the present invention in normal breast cells.
9D compares the inhibition of ATP synthesis of the pharmaceutical composition of the present invention in lung cancer cells with the inhibition of lipoic acid and the inactive form of the present invention.
10 shows the effects of the pharmaceutical composition of the present invention on tumor cell mitochondrial levels of PDH complex and alpha-ketoglutarate (αKDH) dehydrogenase enzyme activity.
FIG. 11A shows Western analysis of two-dimensional gels of extracts extracted from treated or mock-treated lung cancer cells with the pharmaceutical composition of the present invention. FIG.
FIG. 11B shows magnified paired two-dimensional gel specimens treated and false-treated with the pharmaceutical composition of the present invention. FIG.
12A shows the regulatory role of PDKs regulated by endogenous lipoate covalently bound to the PDH complex E2 subunit.
12B depicts possible mechanisms for the differential inactivation of tumor cell PDH complexes by the pharmaceutical compositions of the present invention.
Figure 13 shows the effect of the pharmaceutical composition of the present invention on mitochondrial membrane potential in H460 lung cancer cells.
FIG. 14 shows Western blot analysis results, which are cell death pathways by the pharmaceutical composition of the present invention in various tumor cell types.

The present invention generally relates to the structure, expression of at least one enzyme and / or enzyme complex or subunit thereof, such as a PDH complex, comprising a disease, condition or syndrome or symptoms thereof characterized by a cell hyperproliferation such as cancer in a warm blooded animal. And / or a pharmaceutical composition for treating, diagnosing, or preventing a disease, condition, syndrome, or symptom thereof, including altering activity or altering disturbances. Such animals include animals of mammalian species, such as humans, horses, cattle, livestock animals including dogs and cats, and others suffering from diseases characterized by cell hyperproliferation including cancer and other pathological conditions and syndromes. The pharmaceutical composition of the present invention comprises an effective amount of at least one lipoic acid derivative, also known as thioctan, comprising the derivatives described in US Pat. Nos. 6,331,559 and 6,951,887 and US Provisional Application No. 60 / 912,598, and pharmaceuticals therefor And acceptable carriers or excipients. As well as derivatives of molecules normally found in mitochondria, as well as molecules that help increase the corresponding activity of tumor cells observed in the Warburg effect, lipoic acid derivatives of the present invention are characterized by cells and tissues characterized by hyperproliferation such as tumor cells and tissues. It is particularly suitable for the selective delivery of to the mitochondria and the effective concentration in the mitochondria, thereby making normal cells and tissues ineffective in the composition.

The pharmaceutical composition of the present invention can modulate the effects of PDK1, PDK2, PDK3, PDK4 and their respective isoforms through reversible phosphorylation. In addition, the pharmaceutical composition may modulate the effects of PDP1, PDP2 and their respective isoforms and / or mutations by reversible phosphorylation. Such regulation may occur through the promotion or inhibition of kinase or phosphatase activity.

By inhibiting mitochondrial energy metabolism, lipoic acid derivatives of the invention cause both a loss of mitochondrial membrane potential in diseased cells and other mitochondrial consequences, resulting in an irreversible initiation of cell death. In addition, lipoic acid derivatives of the present invention metabolize mitochondrial energy by inhibiting the conversion of pyruvate to acetoin, a less toxic molecule by activation of PDKs and / or inhibition of PDPs or through inhibition of activity of the E1 subunit of the PDH complex. Can be suppressed. Inhibition of acetoin synthesis will distort other processes, including redox balance, and also lead to the generation of toxic byproducts including acetaldehyde, peroxide, hydrogen peroxide and hydroxyl radicals, which in turn result in diseased cells May cause irreversible damage to the mitochondria.

In a first embodiment of the invention, the lipoic acid derivatives are represented as first general formula (I):

Here, x is defined by a general formula of 0 to 16. R ₁ and R ₂ are independently:

(1) Acyl group R ₃ C (O)-, wherein R ₃ includes, but is not limited to, acetyl and butaryl, and specific examples are alkyl, aryl linked via thioester bonds which are bis-acetyl lipoates Or an acyl group which is an organometallic aryl group;

(2) benzoyl or benzoyl derivatives, including but not limited to, specific examples include aromatic groups linked through thioester bonds which are bis-benzoyl lipoates;

(3) an alkyl group C _n H _{2n +1} , wherein n is 1 to 10, and includes, but is not limited to, methyl, butyl, decanyl and 6,8-biscarbomoyl methyllipoate; Alkyl groups linked via thioether bonds having alkyl groups substituted with other moieties such as —OH, —Cl or —NH ₂ ;

(4) an alkenyl group C _n H _{2n -1} , wherein n is 2 to 10 and an alkenyl group linked through a thioether bond including, but not limited to, propylene, 2,3 dimethyl-2-butene and heptene;

(5) an alkynyl group C _n H _{2n -2} , wherein n is 2 to 10 and an alkynyl group linked through a thioether bond, including, but not limited to, acetylene, propyne and octin;

(6) alkyl, alkenyl and alkynyl groups, which may be either open chains or alicyclics, wherein the alicyclic groups are added or substituted with arbitrary carbons to form cyclopropane, cyclopentene and 6,8 methyl Alkyl, alkenyl and alkynyl groups that form heterocyclics, including but not limited to succinimides;

(7) alkyl, alkenyl and alkynyl groups that can be added on any of the carbons including, but not limited to, hydroxyls and amines;

(8) aromatic or aryl groups linked through thioether bonds, which may be benzene or benzene derivatives, including but not limited to toluene and aniline;

(9) alkyl sulfide groups CH ₃ (CH ₂ ) _n -S-, n may be 0 to 9, but is not limited thereto, and alkyl sulfide groups linked through disulfide bonds;

(10) imidoyl groups CHR ₄ C (═NH) —, where n may be from 1 to 10, but not limited to, imidoyl groups linked via a thioamide bond; And

(11) Semiacetal groups R ₅ CH (OH) -S-, wherein R ₅ includes trichloroacetaldehyde and pyruvic acid, but has compounds with strong electron withdrawing substituents, including but not limited to Semiacetal groups not limited to these;

Or salts thereof.

R ₁ and R _{2 as} defined above may be unsubstituted or substituted and may also be oxidized to produce, for example, sulfoxides or sulfones, respectively, CS (O) -R and CS (O) ₂ -R. Thioesters can be included.

R ₁ and R ₂ may include disulfides that are, for example, CS (O) SR and CS (O) ₂ —SR, which may be oxidized to thiosulfonic acid or thiosulfonic acid, respectively.

R ₃ is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which may be substituted or unsubstituted. Likewise, R ₄ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl and alkylheteroaryl, any of which may be substituted or unsubstituted.

R ₅ is -CCl ₃ , -CF ₃ or -COOH.

In a second embodiment of the invention, the lipoic acid derivatives are represented as second general formula (II):

Wherein M is a covalent bond-[C (R ₁ ) (R ₂ )] _z -or a metal chelate or other metal complex, wherein the metal is not palladium;

R ₁ and R ₂ is hydrogen, acyl R ₃ C (O) -, alkyl _{C n H 2n +1, C n} H 2n - 1 alkenyl, alkynyl defined as C _n H _2n-3 defined by, aryl , Heteroaryl, alkyl sulfide CH ₃ (CH ₂ ) _n -S-, imidoyl as defined by R ₃ C (= NH)-and hemiacetal as defined by R ₄ CH (OH) -S- Is selected;

R ₁ and R _{2 as} defined above may be unsubstituted or substituted;

R ₃ and R ₄ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclyl, any of which may be substituted or unsubstituted and ;

R ₅ is selected from the group consisting of CCl ₃ , CF ₃ or COOH;

And second general formula (II) wherein x is 0-16, z is 0-5, n is 0-10;

Or salts thereof.

In a third embodiment of the invention, the lipoic acid derivatives are as the third general formula (III):

Wherein R ₁ and R ₂ are hydrogen, alkyl C _n H _{2n +1} , alkene C _n H _2n , alkenyl C _n H _{2n -1} , alkane C _n H _2n-2 , alkynyl C _n H _{2n -3} , Alkyl sulfide CH ₃ (CH ₂ ) _n -S-, disulfide alkyl CH ₃ CH _t -SS-. Thiocarbamic ester (CH ₂ ) _n C═NH— and semithioacetal CH ₃ CH (OH) —S—, where n is 1 to 10 and t is 0 to 9, aliphatic, R ₄ C (O) — Imyl, heteroaryl, imidoyl defined as R ₅ C (= NH)-, organometallic aryl, alkyl-organometallic aryl, semiacetal R ₆ CH (OH) -S-, amino acids, carbohydrates, Independently selected from the group consisting of nucleic acids, lipids and multimers and combinations thereof;

R ₁ and R _{2 as} defined above may be unsubstituted or substituted;

R ₃ is selected from the group consisting of amino acids, carbohydrates, nucleic acids, lipids and multimers thereof;

R ₄ is independently selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which may be substituted or unsubstituted;

R ₅ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl and alkylheteroaryl, any of which may be substituted or unsubstituted;

R ₆ is CCl ₃ , CF ₃ or COOH;

And x is from 3 to 16 in general formula (III);

Or salts thereof.

In a fourth embodiment of the invention, the lipoic acid derivatives are as the fourth general formula (IV):

Wherein M is a covalent bond-[C (R ₁ ) (R ₂ )] _z -or a metal chelate or other metal complex, wherein the metal is not palladium;

R ₁ and R ₂ is hydrogen, acyl R ₄ C (O) -, alkyl _{C n H 2n +1, C m} H 2m - 1 alkenyl, alkynyl defined as C _m H _2m-3 defined by, aryl , Heteroaryl, alkyl sulfide CH ₃ (CH ₂ ) _n -S-, imidoyl as defined by R ₄ C (= NH)-, hemiacetal as defined by R ₆ CH (OH) -S-, amino acids, carbohydrates Are independently selected from the group consisting of nucleotides, nucleic acids, lipids, and multimers and combinations thereof;

R ₁ and R _{2 as} defined above may be unsubstituted or substituted;

R ₃ is independently selected from the 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 may be substituted or unsubstituted and ;

R ₅ is selected from the group consisting of CCl ₃ , CF ₃ or COOH;

And x is 0 to 16, z is 0 to 5, n is 0 to 10 and m is 2 to 10;

Or salts thereof.

In addition, since any such general structure can be metabolized in cells or mitochondria, it is expressly intended that the metabolites of the above-referenced structures are within the scope of the present invention.

In both the first and second general formulas above, the (R) -isomer of each particular lipoic acid derivative was observed to have higher physiological activity than the (S) -isomer. Thus, while both isomers are contemplated for use in the present invention, particularly preferred embodiments, the (R) -isomer is used preferentially or the (R) -isomer is in a mixture with the (S) -isomer. It is specifically considered to exist.

In addition, the pharmaceutical composition of the present invention can regulate the expression level of human hydrolase, kinase and dehydrogenase enzyme components found in the PDH complex. Such regulation may occur at the transcriptional, translational or post-translational stages, including epigenetic silencing of appropriate genes.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers are known in the art and are not limited to antioxidants, buffers, chelating agents, flavoring agents, colorants, preservatives, absorption enhancers that enhance bioavailability, antimicrobial agents and combinations thereof. And conventionally used in pharmaceutical compositions such as acceptable carriers. The amount of such additives depends on the desired properties, which can be readily determined by one skilled in the art.

The pharmaceutical compositions of the present invention may routinely contain salts, buffers, preservatives and fusion carriers, which may optionally be used in combination with other therapeutic ingredients. When used in medicine, the salts must be pharmaceutically acceptable, but salts that are not pharmaceutically acceptable can be readily used to prepare the pharmaceutically acceptable salts and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, palmicylic Acids, p-toluene sulfonic acid, tartaric acid, citric acid, methane sulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzene sulfonic acid. In addition, pharmaceutically acceptable salts may be prepared as alkali metal or alkaline earth salts such as sodium, potassium or calcium salts of the carboxylic acid groups.

In addition, the present invention is directed to altering the modulation or disruption of the structure, expression and / or activity of at least one enzyme and / or enzyme complex or subunit thereof, including diseases, conditions or syndromes or symptoms thereof characterized by cell hyperproliferation. A method of treating or diagnosing a patient or using diagnostic agents by delivering an effective amount of at least one treatment or diagnostic agent to cells for the prevention, diagnosis or treatment of a disease, condition, syndrome or symptom comprising the same. to provide. Treatment of primary tumors that regulate the treatment of development of micrometastasis after or concurrent with tumor cell proliferation, angiogenesis, metastatic growth, apoptosis and surgical removal; And modulating the PDH complex as an improved cancer treatment, including radiation or other chemotherapy treatments of primary tumors. The pharmaceutical compositions of the present invention are primary or metastatic melanoma, lymphoma, sarcoma, lung cancer, liver cancer, Hodgkin's and non-Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer and breast cancer, prostate cancer, ovary It is useful for cancer types such as adenocarcinomas such as cancer and pancreatic cancer.

For therapeutic and diagnostic applications, the pharmaceutical composition may be administered directly to the patient when combined with a pharmaceutically acceptable carrier. This method can be used alone or as a glycolytic inhibitor, microtubule-interacting agent, cell growth inhibitor, folate inhibitor, alkylating agent, phase isomerase inhibitor, tyrosine kinase inhibitor, grapephytotoxin or derivatives thereof, antitumor antibiotics, chemical Therapeutic agents, apoptosis-inducing agents, anti-angiogenic agents, nitrogen mustard, nucleic acid intercalating agents, and combinations thereof, but may be carried out in combination with an effective amount of other therapeutic or diagnostic agents, including but not limited to Can be. Such therapeutic agents may further comprise other metabolic inhibitory reagents. Many such therapeutic agents are known in the art. Combination treatment methods provide simultaneous, sequential or separate use in the treatment of such conditions as necessary to amplify or assure patient response to the treatment method.

The methods of the present invention can be performed using any mode of administration that produces an effective level of active compounds without causing medically acceptable and clinically unacceptable side effects. Although formulations suitable specifically for parenteral administration are preferred, the compositions of the present invention may be inhaled, oral, topical, transdermal, nasal, ocular in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. , Lung, rectal, transmucosal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, pleural, intrauterine, intratumoral or infusion methods or for administration. If such a formulation is desired, other additives known in the art can be included to impart desired consistency and other properties to the formulation.

Those skilled in the art will appreciate that the particular mode of administration of the therapeutic or diagnostic agent may be selected from a particular agent; Whether the administration is for the treatment, diagnosis or prevention of a disease, condition, symptom muscle or symptoms thereof; The severity of the medical condition being treated or diagnosed; And one dose required for therapeutic efficacy. For example, a preferred mode of administration of antitumor agents for the treatment of leukemia involves intravenous administration, while a preferred method of treating skin cancer will include topical or intradermal administration.

As used herein, "effective amount" refers to one or multiple doses of a therapeutic or diagnostic agent for which a desired therapeutic or diagnostic effect is achieved. In general, an effective amount of the therapeutic or diagnostic agent will depend on the activity of the specific agent employed; Metabolic stability and length of action of the agent; The species, age, body weight, general health, dietary condition, sex and diet of the subject; Mode and time of administration; Excretion rate; Drug combination if necessary; And the extent of presentation and / or severity of the particular condition being treated. The exact dosage may be determined by one of ordinary skill in the art without performing excessive experiments with one or several daily administrations to produce the desired results, and the dosage may be determined to achieve the desired therapeutic effect or Any complications can be controlled by individual specialists. Importantly, when used to treat cancer, the dose of therapeutic agent used should be sufficient to inhibit or kill tumor cells without substantially harming normal cells.

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

In general, the pharmaceutical compositions of the present invention will be delivered to the patient in a manner sufficient to administer an amount effective to modulate the structure and / or activity of the PDH complex. Thus, the dosage may range from about 0.3 mg / m 2 to 2000 mg / m 2, preferably about 60 mg / m 2. The dosage may be administered in a single dose or in the form of separate divided doses, such as once to four or more times daily. If the response from the subject is insufficient when administered at a particular dose, more doses (or more effective doses by different, more localized delivery routes) may be employed up to the extent of patient resistance. Multiple doses per day are contemplated to reach an appropriate systemic or target level of the therapeutic or diagnostic agent.

In another embodiment of the present invention, the lipoic acid derivatives of the present invention can be used as in vitro diagnostic and anticipated agents. As mentioned above, depending on the specific tumor cell or cell type of interest, other lipoic acid derivatives may be more or less effective in the inhibition of different tumor classes through the regulation of the PDH complex. Thus, for example, where diagnosis or selection of appropriate chemotherapy strategies may be difficult, tumor types and tumors may be due to testing of cultures of tumor cells in vitro using lipoic acid derivatives known to target specific tumor cell types. Other approaches are provided to identify effective treatments.

Returning to the figure, FIG. 6A shows one of the many differences that can occur in energy metabolism in normal tissues and tumor cells in vitro. Tumor cells often rely more on cytoplasmic glycolysis for mitochondrial energy metabolism for ATP production than normal cells under these conditions. Changes in the expression and regulation of PDH complexes are clearly part of this tumor-specific adaptation. Increasing the level of PDH catalyst components and / or increasing the level of inhibitory PDKs that produce this effect may render tumor cells more vulnerable to agents that attack the PDH complex than normal cells.

6B shows the structure of lipoic acid catalyzing the normal reactions involved in synthesizing acetyl-CoA from pyruvate in the PDH complex. In vivo lipoic acid is linked via a carboxyl terminus at a non-peptide amide bond with the epsilon amino group of lysine at the E2 lipoyl region active sites. Note that the oxidation / reduction / acetylation status of PDH E2-binding lipoate is monitored by kinases and phosphatases that modulate PDH activity by regulating phosphorylation inactivation of the El alpha PDH subunit. This figure also shows the structure of three representative pipeoic acid derivatives that can be used in the present invention. Whereas CPI-613 and CPI-045 have high antitumor titers, CPI-157 has little or no activity in cell culture and is useful as a control in some experiments.

FIG. 6C shows the relationship between PDH complex components, including lipoate bound with E2, E1 and E2 with regulatory PDK. High levels of acetyl-lipoate or dihydrolipoate (not shown) activate PDKs, which in turn inactivates the subunit E1α, which catalyzes the first step in PDH complex catalysis, thereby reducing the flux through the PDH complex. Suppress more. The dl process acts as a governor for carbon / energy flow through the PDH complex, and this regulatory process is obviously substantially changed to support the different energy metabolism of the tumor cells shown in FIG. 3A.

7 shows the effect of the pharmaceutical composition of the present invention on xenograft tumor growth. Cells were implanted subcutaneously in the dorsal flank as described in Example 2. The mice were then injected with the drug (or vehicle alone; "mock") intraperitoneally, beginning on the date shown in the figure. The left panel shows a pancreatic tumor model injected three times weekly using the present invention or carrier control at a concentration of 1 mg / kg. This experiment shows two performed with BxPC-3 cells and two performed with AsPC-2 cells. The three panels on the right model H460 lung tumors injected at the concentrations indicated, either once weekly (circular), three times weekly (inverted triangle) or five times weekly (triangle for carrier therapy and square for drug therapy). To show. This experiment shows four things performed using H460 cells.

8 shows three cell types and non-modified cells treated with either treatment 200-300 μM (“treated”) or false treatment (“false treated”) due to treatment with the pharmaceutical composition of the present invention. The effect on the type (MDCK) is shown. Cells were treated for 48 hours in an appropriate tissue culture medium containing 10% serum. Extensive cell death by apoptosis or apoptosis-like pathways (see also FIG. 11) in three cancer cell lines is observed via the methodology described in Example 2. In contrast, non-modified MDCK cells are not clearly affected by drug treatment treated with this dose.

9A depicts ATP levels in H460 lung tumor cells after treatment with the pharmaceutical compositions of the present invention at or above the lethal threshold (200 μM in 10% serum). Dashed lines represent treatment at the indicated concentrations. The solid lines indicate that after treatment for the indicated time, the drug was removed and recovered for 60 seconds in the drug-free medium. Arrow blocks indicate the interval of ATP recovery.

9B compares the inhibition of ATP synthesis in medium with pyruvate (in the form of methyl-pyruvate) as the primary carbon source (dashed line) and medium with glucose as the primary carbon source (solid line). Note that the pharmaceutical composition of the present invention ultimately causes cell death at the same threshold concentrations in both media; However, early depletion of total cell ATP levels was high in pyruvate-containing medium and absent in glucose-containing medium. In addition, the onset of cell death is more rapid at the 300 μM than the 200 μM drug concentration.

9C compares the inhibition of ATP synthesis of the pharmaceutical composition of the present invention in SK-Br-3 breast cancer cells with the inhibition of ATP synthesis of the pharmaceutical composition of the present invention in HMEC normal breast cells. The results were compared in the experiments shown in Figures 6A and 6B, these experiments were performed in serum-free medium (MEBM). As a result, the drug's lethal threshold is lower, about 50 μM. Note that the small decrease in ATP levels in 22-hour normal cell samples is not related to drug dose and reflects normal experimental changes.

9D compares the inhibition of ATP synthesis in H460 lung cancer cells by the composition of the invention (left graph), lipoic acid (center graph) and the inactive form of the invention (right graph). As in FIG. 9C, these experiments were performed in serum-free medium so that the drug's lethal limit was about 50 μΜ.

10 shows the effect of the pharmaceutical composition of the present invention (400 μM concentration in DMEM supplemented with 10% serum) on tumor cell mitochondrial levels of PDH (PDC) and (αKDH) enzyme activities. Note that PDH is strongly inhibited, whereas αKDH is not. Enzyme activity levels are measured in extracts of mitochondria purified using resazurin reduction in response to the added carbon source, as described in Example 2. The background line corresponds to the resazurin reduction without the added carbon source.

Next, in FIG. 11A, of the extracts extracted from H460 lung cancer cells treated (+) or falsely treated (−) for 120 minutes at 400 μM concentration in RPMI medium supplemented with 10% serum Western analyzes of two-dimensional gels were performed. Western transfers were probed using a cocktail of monoclonal antibodies against the El and E2 subunits of the PDH complex. Western delivery is aligned at E2. Note that substantially higher levels of the over-phosphorylated form of E1 and reduced levels of low-phosphorylated El form are present in the drug-treated sample. The left vertical white line shows the mobility of the E2 subunit, one of the criteria for aligning the gels. The right vertical ringworm penetrates the less phosphorylated E1α forms that are believed to be enzymatically active ingredients.

FIG. 11B shows magnified false-treated paired two-dimensional gel specimens treated with the pharmaceutical composition of the present invention. FIG. Element A is an enlargement of the portion of FIG. 8A. Element B is SK-Br-3 breast cancer cells that were false-treated (−) and treated (+) for 180 minutes with 80 μM composition in MEBM serum free breast epithelial cell medium. Element C is SK-Br-3 breast cancer cells that were false-treated (−) and treated (+) (+) for 240 minutes with 80 μM composition in MEBM serum free breast epithelial cell medium. Element D is HMEC normal breast cells that were false-treated (−) and treated (+) for 240 minutes with 80 μM composition in MEBM serum free breast epithelial cell medium. Vertical ringworm penetrates the less phosphorylated E1α forms that are believed to be enzymatically active components.

12A and 12B show a working hypothesis for the potent and selective anticancer effects in vivo of the pharmaceutical compositions of the present invention. 12A shows the regulatory role of PDKs regulated, for example, by endogenous lipoate covalently bound to the PDC E2 subunit. PDKs inactivate PDC mainly in response to high levels of reduced and / or acetylated lipoates, a process that is clearly substantially modified in tumor cells.

Incidentally, FIG. 12B shows a large quantitative difference in the ratio of PDK to PDC-E1 that is believed to distinguish normal and tumor cells in vivo. In normal cells, low levels of PDK are thought to gradually phosphorylate E1 by "walking" hand-over-hand around the PDH complex (through its two dimeric subunits). This phosphorylation is in steady state equilibrium with PDP dephosphorylation (not shown). For the working hypothesis shown here, thioctans primarily stimulate PDKs through the same sites that bind acetyl-lipoates and / or dihydrolipoates, thereby artificially stimulating one or more PDK isoforms to elute E1α. Deactivate it. In tumor cells, even higher levels of PDK make this stimulation by thioctans much more effective at closing PDC enzyme activity and mitochondrial energy metabolism.

The following non-limiting examples are provided to facilitate understanding of the pharmaceutical composition of the present invention.

Example 1

Tioctane  Chemical synthesis

Lipoic acid derivatives (ie, thioctanes), CPI-613 and CPI-157, are modified procedures described in US Pat. No. 6,331,559 B1 and US Pat. No. 6,951,887 B2 as starting materials for 6,8-bismercaptooctanoic acid. It was synthesized using. Tioctane CPI-045 was synthesized as described in US Pat. No. 6,331,559 B1.

The structural analysis of the three thioctanes is as follows. Many independent synthesis of CPI-045 and CPI-613 were indistinguishable in anti-cancer properties. The purity of CPI-613 used for xenografts (FIG. 7) and ATP measurements (FIG. 9) exceeded 99%. All other preparations exceeded 98% purity.

CPI-613: 6,8-bis-benzylsulfanyloctanoic acid: white crystalline solid, melting point 65-66 ° C. (lit. 67.5-69 °); ¹ H-NMR (250 MHz, CDCl ₃ ): δ 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); ¹³ C-NMR (62.9 MHz, CDCl 3): δ 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; TLC (EtAc: hexanes: HAc, 200: 200: 1 v / v): R _f = 0.60; ¹ H-NMR (300 MHz, CDCl 3): δ 2.64-2.76 (m, 1H), 2.65 (t, J = 7.5 Hz, 2H), 2.52 (q, J = 7.5 Mz, 2H), 2.49 (q, J = 7.2 Hz, 2H), 2.36 (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 ); ¹³ C-NMR (75 MHz, CDC13): δ 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): R _f = 0.30; ¹ H-NMR (250 MHz, CDCl 3): δ 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); ¹³ C-NMR (62.9 MHz, CDC13): δ 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

Example 2

Tioctane  Methods used to determine anti-cancer effects

Cells: Human tumor cell lines were obtained from ATCC and propagated according to ATCC recommendations. Human breast epithelial cells (HMEC), small airway epithelial cells (SAEC) and normal human epidermal keratinocytes (NHEK) primary cells were obtained from LONZA Walkersville (Walkersville, MD). Each cell line was developed and maintained by the supplier and maintained and propagated according to the supplier's instructions in the appropriate medium purchased from the supplier. The experiments reported here used normal cells of three to six passages.

Tumor Growth Suppression 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. After about 8-12 days, the mice were injected intraperitoneally (IP) at the dose and schedule indicated in the figure legend. The drug or carrier was injected at about 2 ml per 25 gm body weight. Drug concentration was less than 1.25 mg / ml (about 3.1 mM). The carrier / solvent consisted of triethanolamine dissolved in water up to 25 mM. The vehicle injected into the false treated animals was always the same as the solvent in which the highest drug dose for the experiment was injected. Mice were monitored daily for physical condition and mortality. Body weight and tumor volume were measured about three times daily before treatment and during and after treatment. Mice were maintained on a 12 hours day / night cycle and free (ad libitum ) and received at institutional guidelines at Stony Brook University animal facility.

Cell death test: For most cell viability measurements, the CellTiter-Glo assay (Promega) was used long enough to not be confounded by early thioctane inhibition of ATP synthesis. (FIG. 9). In a typical experiment, cells were plated in black clear bottom 96-well plates at 5000 cells per well. After 18 to 25 hours, the medium was replaced with a fresh solvent containing thioctane CPI-613 in drug solvent (2.8 mM triethanolamine dissolved in water in serum-containing medium and 0.7 mM in serum free medium) or the same solvent. . The test was performed 24 hours or 48 hours after drug addition, depending on drug dose, according to manufacturer's instructions.

In some cases, the cells were plated at 10,000 cells per cell in 48-well plates, and the medium contained drug solvent (1% EtOH final concentration) after 18-15 hours or a variety of thioctane CPI-045 in the same solvent. It was replaced with fresh embryo containing concentrations. Cells stayed in solvent or drug-containing medium for the remainder of the experiment. Plates were examined 24, 48 and 72 hours after drug addition and cell numbers were estimated as percentage of confluence. Under these conditions, titantan-induced cell death is high in apoptosis at near-limit doses, and cell count estimation is a reliable indicator of death. (FIG. 10) The integrity of the cells remaining at 72 hours was tested by trypan blue exclusion.

Table 2 provides data on the action of thioctans on tumor cells in vitro. The human tumors and primary human cells we investigated for sensitivity to CPI-613 and / or CPI-045 killing are listed. "+" Indicates that the cells will undergo apoptosis or necrosis-like cell death at a dose of about 200-300 μM (in the presence of 10% serum) and about 50 μM in serum-free medium. (FIGS. 8 and 9) "-" indicates that these cells require about 5 times lower drug dose to induce cell death in the media. "nt" refers to a non-test combination. All tumor cell lines were analyzed in the appropriate medium supplemented with 10% serum as in the MDCK normal cells of FIG. 8. In addition, HMEC, SAEC, NHKC primary human cells and SK-BR-3, A549 and H460 tumor cell lines were analyzed in appropriate serum-free medium. Primary cells were contact inhibited and modified cells were present at significant density.

ATP test: Cells were plated in black clear bottom 96-well plates at 5000 cells per well. After 18 to 25 hours, the medium was replaced at drug concentration and for a time interval as indicated by the medium containing drug solvent (triethanolamine) or thioctane (CPI-613 or CPI-157) or lipoic acid. Cell viability and integrity were assessed by recovery after drug discontinuation by trypan blue exclusion. ATP was measured using CellTiter-Glo Luminescence Test (Promega) according to manufacturer's instructions. All measurements were performed in duplicates and were highly consistent. The standard error of the mean reached 0.1 to 2% of the measured value. As a result, error bars are omitted in FIG. The methyl pyruvate medium of FIG. 9 consisted of glucose-free RPMI (Invitrogen) supplemented with 10% dialysis fetal calf serum, 5 mM HEPES (pH 7.4) and 10 mM methylpyruvate (Sigma-Aldrich), comparable to glucose The medium was conventional RPMI (Invitrogen).

PDH and αKDH Enzyme Tests: Tumor cells were grown to 80% confluence on 15 cm plates and treated as indicated by CP-613. Mitochondria were isolated according to the methods of Moreadith and Fiskum. ¹ mitochondria were dissolved in 0.4% lauryl maltoside. 50 μl of mitochondrial lysate was added to 96-well plates. 50 ul of reaction mixture (50 mM Tris, pH 7.5, 2 mM β-NAD +, 225 uMv TPP, 2 mM pyruvate or α-ketoglutarate, 150 μΜ coenzyme A, 2.6 mM cysteine, 1 mM MgCl ₂ ) Added to mitochondrial lysate and the mixture was incubated at 37 ° C. for 45 minutes. At this time, 15 μM lesazurin and 0.5 U / ml diaphorase were added to the mixture and incubated for 5 minutes. NADH production was monitored by measuring fluorescence using an excitation wavelength of 530 nm and an emission wavelength of 590 nm on a microplate reader (Fluorostar). All measurements were performed in duplicates and were highly consistent. The standard error of the mean reached 0.3-4% of the measured value. As a result, error bars are omitted in FIG.

E1α 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 treated with 450 μl Lysis Buffer A [455 μl Zoom 2D Protein Solubilizer 1 (Invitrogen), 2.5 μl 1M Tris Base, 5 μl 100 × Protease Inhibition Cocktail (Complete min, EDTA, Roche); 5 μl 2M DTT] was dissolved in situ. Cell lysates were transferred to 1.5 ml microfuge tubes and sonicated for 15 passages on 50% power on ice. After 10 minutes of incubation at room temperature, 2.5 μl of dimethylacrylamide (DMA, Sigma-Aldrich) was added and the lysate was incubated for 10 minutes. 5 μl of 2M DTT was added to neutralize excess DMA. Lysates were centrifuged at 16,000 × g for 15 minutes.

2-D gels: We used the Zoom Benchtop Proteomics System (Invitrogen) according to the manufacturer's instructions. 30-50 μl of lysate was mixed with 0.8 μl pH 3-10 amphoteric electrolyte, 0.75 μl 2M DTT and reached up to 150 μl using Zoom 2D protein solubilizer 1. 150 μl of sample was loaded into the IPG runner and pH 3-10NL IPG strips were added. The strips were soaked overnight at room temperature. Step protocols were used for isoelectric focusin (250 V, 20 min; 450 V, 15 min; 750 V, 15 min 2000 V, 30 min). Strips were treated for 15 minutes in IX loading buffer and then for 15 minutes in IX loading buffer plus 160 mM iodine acetate. Strips were electrophoresed on NuPAGE 4-12% Bis Tris ZOOM gel (Invitrogen).

Effect of Tioctane on In Vitro Tumors and Primary Cells






Tumor cells







Brain, Glioblastoma U-87 MG + + Brain, Glioblastoma LN-229 + nt Breast, adenocarcinoma SK-Br-3 + + Breast, adenocarcinoma MCF7 nt + Bone, osteosarcoma Saos-2 nt + Cervix, adenocarcinoma HeLa + + Colon Rectal, Adenocarcinoma SW480 nt + Hepatocellular carcinoma Hep G2 + + Kidney, carcinoma A-498 + nt Lung, carcinoma A459 + + Lung, carcinoma A460 + + Muscle, rhabdomyosarcoma RD nt + Ovarian, Carcinoma SKOV-3 + nt Pancreas, adenocarcinoma AsPC-1 + nt Pancreas, adenocarcinoma BxPC-3 + nt Prostate, carcinoma LnCaP nt + Uterus, Sarcoma Mes_SA + + Uterus, Sarcoma, MDR Mes-SA / dx5 + +
Primary cells
Breast epithelial cells HMEC - - Small airway epithelial cells SAEC - nt Keratinocytes NHKC nt -

Western: Proteins were blotted onto PVDF 4.5 μm membranes. PDH El and E2 were detected using mAbs (Invitrogen).

Caspase-3 and PARP Degradation: The degraded caspase-3 was detected on Western blots according to Roy and Nicholson. ² In brief, the mixture of media / cell / apoptotic bodies was centrifuged at 6000 × g after drug or solvent treated cells were scraped. The pellet was dissolved in lysis buffer C (4M urea, 10% glycerol, 2% SDS, 0.003% BPB; 5% 2-mercaptoethanol). 30 μg of total cell lysate protein per well was loaded on a 12% bis-tris gel. Proteins were blotted onto PVDF 4.5 μm membranes. Pro- and active-caspase-3 were detected as anti-caspase-3 mAb (mouse monoclonal [31A1067]; abcam). PARP degradation was detected using monoclonal anti-poly (ADP-ribose) polymerase antibody, clone C-2-10 (Sigma-Aldrich).

Mitochondrial Ca ⁺² Detection: Cells were sprinkled with 3 × 10 ⁵ on 35 mm glass bottom plates (BD Biosciences), grown overnight, and treated as indicated with drug or solvent. Cells were then loaded with calcium dye Fluo-4, X-Rhod-1 or Rhod-2 (4 μM, Invitrogen) in medium without phenol red 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 fixed exposure time using a FITC filter. Quantification of fluorescence was performed using software supplied by the manufacturer. X-Rhod-1 and Rhod-2 gave similar results (FIG. 13), indicating that these dyes measure mitochondrial Ca + 2 signals. ^3-4

references

1.Moreadith RW and Fiskum G. Isolation of Mitochondria from Ascites Tumor-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. Gerenscser AA and Adam-Vizi V. Selective, high-resolution fluorescence imaging of mitochondrial Ca 2 + concentration. Cell Calcium 30, 311-321 (2001).

4. Gyorgy H, Gyogrgy C, Das S, Garcia-Perez C, Saotome M, Roy SS, and Yi MQ. Mitochondrial calcium signaling and cell death: Approaches for assessing the role of mitochondrial Ca ^{2 +} uptake in apoptosis. Cell Calcium 40, 553-560 (2006).

Example 3

Tioctane  Mitochondria Membrane potential  And Ca Disturbs +2 intake

Substrate effects on tioctane inhibition of ATP synthesis (FIG. 9) suggest that the drug interferes with the TCA cycle in the mitochondrial substrate. If this is the case, we expect that the mitochondrial membrane potential can be compromised above the ¹ lethal threshold dose. Using TMRE, a potential sensitive dye, we observe the expected effect. (FIG. 13) Mitochondrial membrane potential rapidly decreases after initiating drug treatment. The kinetics of membrane potential reduction is very similar to the loss of ATP synthesis in the presence of mitochondrial substrates. (Figure 9)

ATP depletion in mitochondria is known to stimulate homeostatic responses, including uptake of Ca ⁺² released from cytoplasmic stores containing endoplasmic reticulum. In addition, such import of Ca ^{+2 into} the mitochondria is considered to require the mitochondrial membrane potential. Thus, we anticipate that treatment with thioctane above the lethal threshold may result in sustained cellular release of Ca + 2 with transient mitochondrial uptake of iron in terms of progressively damaged membrane potential. By measuring mitochondrial Ca ⁺² and Fluo-4 using X-Rhod-1 and Rhod-2 for the measurement of cytoplasmic Ca + 2, we observe this expected effect. (Figure 13)

This initial mitochondrial Ca + 2 temporarily decreases as the mitochondrial membrane potential decreases over approximately 2 hours, at doses slightly above the CPI- and lethal thresholds (compare FIGS. 9 and 13). This is followed by a second large mitochondrial Ca ⁺² peak that is believed to be associated with the onset of calcium-dependent cell death pathways at 4-6 hours. ³

references:

Garrett R and Grisham CM. Biochemistry. Thomson Brooks / Cole, Southbank, Vic., Australia; Belmont, CA. (2007).

Graier WF, Frieden M, and Malli R. Mitochondria and 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 signaling and cell death: Approaches for assessing the role of mitochondrial Ca ^{2 +} uptake in apoptosis. Cell Calcium 40, 553-560 (2006).

Example 4

Tioctane  Induces various cell death programs

Reduction in mitochondrial energy metabolism is known to correlate with the decision to enter the cell death pathway in some situations, although detailed mechanisms are unsafely understood. At thioctane doses, which exceed the ^1-3 limit, but within about two times this minimum killing dose, all tested cancer cell types undergo morphologically overwhelming apoptosis-like cell death. (FIG. 8) Apoptotic killing was confirmed by conventional Annexin immunostaining and TUNEL DNA end-labelling (results not shown) under these conditions.

At higher drug doses (approximately two times greater than the limit), active thioctane cells lack morphological correlations of apoptosis (as assessed by replating survival and trypan blue or propidium exclusion tests). Induces death, suggesting a necrotic-like pathway (result not shown).

These data confirm that the titantan CPI-613 inhibitory mitochondrial energy metabolism correlates precisely with the induction of cell death.

It is impressive that the various tumor cells known or estimated to contain inactivation mutations for different and diverse cell suicide pathways ⁴ were all killed at very similar tioctane doses (FIG. 8 and Table 2). This observation suggests that these drugs induce a master signal that may be involved in multiple potentially overlapping terminal cell suicide execution pathways. ⁵

Consistent with this possibility, we find that the Z-VAD-FMK common caspase inhibitor subtly changes the form of cell death in thioctane treated cells, but has no discernible effect on the drug's lethal threshold dose. It became.

To further test the possibility that thioctane-induced cell death can handle multiple terminal execution mechanisms, we tested caspase-3 and PARP-1 degradation to diagnose different cell death pathways. ⁵ We found that both the tioctanes CPI-613 and CPI-045 induce highly variable levels of these two degradation events in other cells. (Figure 14)

Collectively, these results suggest that the titantan may induce a strategic commitment to agnostic killing for the end of the crystal, tactical performance, depending on drug dose and cell type.

references:

Watabe M and Nakaki T. ATP depletion does not account for apoptosis induced by inhibition of mitochondrial electron transport chain in human dopaminergic cells. 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 AlF in caspase-dependent and caspase-independent cell death. Oncogene 23, 2785-2796 (2004).

Example 5

Lipoic Acid Derivative Analog Structure

Non-limiting examples of the various lipoic acid derivative analogs shown below are prepared and disclosed herein.

The foregoing discussion discloses and describes only exemplary embodiments of the invention. Those skilled in the art will readily appreciate from the above discussion and the appended 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. Also, while exemplary embodiments are represented herein, other skilled in the art may recognize other designs or uses of the present invention. Thus, while the invention has been described in connection with exemplary embodiments thereof, it will be apparent to those skilled in the art that many modifications may be made in both design and use, and the application may be modified or modified in any manner It is intended to include change. Therefore, it is manifestly intended that this invention be limited only by the foregoing claims and equivalents thereof.

Claims (53)

Modulation or perturbation of the structure, expression and / or activity of at least one enzyme and / or enzyme complex or subunits thereof, such as modified pyruvate dehydrogenase (PDH) complexes in the mitochondria of diseased cells of warm-blooded animals, including humans Pharmaceutically acceptable modulator. The method of claim 1,
The modulator or disturbance comprises reversible phosphorylation or dephosphorylation. The method of claim 2,
Said reversible phosphorylation or dephosphorylation occurs on the kinase, phosphatase and / or dehydrogenase of an enzyme or enzyme complex or subunit thereof. The method of claim 3, wherein
Said modulator promotes or inhibits kinase activity. The method of claim 4, wherein
Said kinase is selected from the group comprising pyruvate dehydrogenase kinase (PDK) 1, PDK2, PDK3, PDK4 and their respective isoforms. The method of claim 3, wherein
The modulator is characterized in that to promote or inhibit phosphatase activity. The method according to claim 6,
And said phosphatase is selected from the group comprising pyruvate dehydrogenase phosphatase (PDP) 1, PDP2 and their respective isoforms. The method of claim 3, wherein
The modulator is characterized in that to promote or inhibit the dehydrogenase activity. The method of claim 2,
Said reversible phosphorylation or dephosphorylation occurs in said PDH complex. The method of claim 9,
And said modulation occurs on the El subunit of said PDH complex. The method of claim 10,
Said modulator is caused by inactivation of PDP and its isoforms and mutant forms. The method of claim 11,
Inactivation of PDP occurs by inhibition of PDP expression. The method of claim 10,
The modulator is characterized by the activation of PDK and its isoforms and mutant forms. The method of claim 1,
Wherein said diseased cells exhibit sensitivity or insensitivity to treatment using the modulator of claim 1. The method of claim 14,
A conditioner wherein the diseased cells that are not sensitive to the treatment can be induced to express at least one modified enzyme or enzyme complex or subunit thereof so that they are sensitive to the treatment. The method of claim 15,
Regulator is characterized in that the expression is induced by genetic manipulation. 17. The method of claim 16,
And said induction is achieved by transcriptional manipulation. 17. The method of claim 16,
And said induction is accomplished by translational manipulation. 17. The method of claim 16,
Modulator, characterized in that the induction is achieved by post-translational manipulation The method of claim 15,
The modulator is characterized in that the expression is induced by epigenetic manipulation. The method of claim 15,
The modulator is characterized in that expression is induced by phenotypic manipulation. The method of claim 14,
And said diseased cells express at least one modified enzyme or enzyme complex upon treatment with the modulator of claim 1. The method of claim 1,
Said modulator affects the expression level of PDK and its isoforms and mutant forms. The method of claim 1,
Said modulator affects the expression level of PDP and its isoforms and mutant forms. The method of claim 23 or 24,
And said expression level is varied at the level of transcription, translation or post-translation. The method of claim 25,
And said change is epigenetic. The method of claim 9,
Said modulator inhibits the production of toxic metabolites. The method of claim 9,
And said modulator promotes detoxification of toxic metabolites. The method of claim 27 or 28,
And said metabolites are selected from the group consisting of acetaldehyde, peroxide, hydrogen peroxide and hydroxyl radicals. 29. The method of claim 28,
The modulator's effect is characterized by a decrease in acetoin production. The method of claim 9,
And said reversible phosphorylation or dephosphorylation is irreversible. The method of claim 31, wherein
The effect of the phosphorylation or dephosphorylation is a modulator, characterized in that resulting in cell death. 33. The method of claim 32,
The effector is characterized in that apoptosis. 33. The method of claim 32,
The effector is characterized in that the necrosis. The method of claim 1,
A modulator comprising at least one lipoic acid derivative and at least one pharmaceutically acceptable carrier thereof. 36. The method of claim 35 wherein
The lipoic acid derivative is represented by the following formula:

Wherein R ₁ and R ₂ are hydrogen, alkyl C _n H _{2n +1} , alkene C _n H _2n , alkenyl C _n H _{2n -1} , alkane C _n H _2n-2 , alkynyl C _n H _{2n -3} , Alkyl sulfide CH ₃ (CH ₂ ) _n -S-, disulfide alkyl CH ₃ CH _t -SS-. Thiocarbamic ester (CH ₂ ) _n C = NH- and semithioacetal CH ₃ CH (OH) -S-, where n is 1 to 10 and t is 0 to 9; Aliphatic; Acyl defined as R ₃ C (O)-; Heteroaryl; Imidoyl, defined as R ₄ C (═NH)-; Organometallic aryl; Alkyl-organometallic aryl; And semiacetal R ₅ CH (OH) -S-;
R ₁ and R _{2 as} defined above may be unsubstituted or substituted;
R ₃ is selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which may be substituted or unsubstituted;
R ₄ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl and alkylheteroaryl, any of which may be substituted or unsubstituted;
R ₅ is CCl ₃ , CF ₃ or COOH;
And x is 0 to 16;
Its metabolites;
Or a salt thereof. 36. The method of claim 35 wherein
The lipoic acid derivative is represented by the following formula:

Wherein M is a covalent bond-[C (R ₁ ) (R ₂ )] _z -or a metal chelate or other metal complex, wherein the metal is not palladium;
R ₁ and R ₂ is hydrogen, acyl R ₃ C (O) -, alkyl _{C n H 2n +1, C n} H 2n - 1 alkenyl, alkynyl defined as C _n H _2n-3 defined by, aryl , Heteroaryl, alkyl sulfide CH ₃ (CH ₂ ) _n -S-, imidoyl as defined by R ₃ C (= NH)-and hemiacetal as defined by R ₄ CH (OH) -S- Is selected;
R ₁ and R _{2 as} defined above may be unsubstituted or substituted;
R ₃ and R ₄ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl and heterocyclyl, any of which may be substituted or unsubstituted and ;
R ₅ is selected from the group consisting of —CCl ₃ , CF ₃ or COOH;
And x is 0 to 16, z is 0 to 5 and n is 0 to 10;
Its metabolites;
Or a salt thereof. 36. The method of claim 35, wherein the lipoic acid derivative is of the formula:

Wherein R ₁ and R ₂ are hydrogen, alkyl C _n H _{2n +1} , alkene C _n H _2n , alkenyl C _n H _{2n -1} , alkane C _n H _2n-2 , alkynyl C _n H _{2n -3} , Alkyl sulfide CH ₃ (CH ₂ ) _n -S-, disulfide alkyl CH ₃ CH _t -SS-. Thiocarbamic ester (CH ₂ ) _n C═NH— and semithioacetal CH ₃ CH (OH) —S—, where n is 1 to 10 and t is 0 to 9, aliphatic, R ₄ C (O) — Imyl, heteroaryl, imidoyl defined as R ₅ C (= NH)-, organometallic aryl, alkyl-organometallic aryl, semiacetal R ₆ CH (OH) -S-, amino acids, carbohydrates, Independently selected from the group consisting of nucleic acids, lipids and multimers and combinations thereof;
R ₁ and R ₂ may be unsubstituted or substituted;
R ₃ is selected from the group consisting of amino acids, carbohydrates, nucleic acids, lipids and multimers thereof;
R ₄ is independently selected from the group consisting of hydrogen, alkenyl, alkynyl, alkylaryl, heteroaryl, alkylheteroaryl and organometallic aryl, any of which may be substituted or unsubstituted;
R ₅ is selected from the group consisting of hydrogen, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl and alkylheteroaryl, any of which may be substituted or unsubstituted;
R ₆ is CCl ₃ , CF ₃ or COOH;
And x is 0 to 16;
Its metabolites;
Or a salt thereof. 36. The method of claim 35 wherein
The lipoic acid derivative is represented by the following formula:

Wherein M is a covalent bond-[C (R ₁ ) (R ₂ )] _z -or a metal chelate or other metal complex, wherein the metal is not palladium;
R ₁ and R ₂ is hydrogen, acyl R ₄ C (O) -, alkyl _{C n H 2n +1, C m} H 2m - 1 alkenyl, alkynyl defined as C _m H _2m-3 defined by, aryl , Heteroaryl, alkyl sulfide CH ₃ (CH ₂ ) _n -S-, imidoyl as defined by R ₄ C (= NH)-, hemiacetal as defined by R ₆ CH (OH) -S-, amino acids, carbohydrates Are independently selected from the group consisting of nucleotides, nucleic acids, lipids, and multimers and combinations thereof;
R ₁ and R ₂ may be unsubstituted or substituted;
R ₃ is independently selected from the 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 may be substituted or unsubstituted and ;
R ₅ is selected from the group consisting of CCl ₃ , CF ₃ or COOH;
And x is 0 to 16, z is 0 to 5, n is 0 to 10 and m is 2 to 10;
Its metabolites;
Or a salt thereof. The method according to any one of claims 36 to 39, wherein
And said lipoic acid derivative is present only as its (R) -isomer. The method according to any one of claims 36 to 39, wherein
And said lipoic acid derivative is present as a mixture of the (R) -isomer and the (S) -isomer. The method of claim 1,
And said modulator is useful for treating or diagnosing a disease, condition or symptom muscle or symptoms thereof comprising a change in the structure, expression and / or activity of at least one enzyme and / or enzyme complex or subunit thereof. 43. The method of claim 42,
Said at least one enzyme complex is a PDH complex. 43. The method of claim 42,
The disease, condition or syndrome is a modulator further characterized by cell hyperproliferation. 45. The method of claim 44,
And said disease, condition or syndrome is cancer. A patient exhibiting a disease, condition or syndrome comprising at least one enzyme and / or enzyme complex or subunit change in structure, expression and / or activity of the at least one enzyme and / or enzyme complex or subunit thereof. A method of controlling, comprising administering an effective amount of the modulator of claim 1. The method of claim 46,
Said at least one enzyme complex is a PDH complex. The method of claim 46,
The disease, condition or syndrome is a modulator further characterized by cell hyperproliferation. 49. The method of claim 48 wherein
And said disease, condition or syndrome is cancer. A method of diagnosing and predicting treatment benefit in a patient exhibiting symptoms of a disease, condition or syndrome, including changes in the structure, expression and / or activity of at least one enzyme and / or enzyme complex or subunit thereof,
Obtaining a sample of cells from the patient, administering to the cells in vitro an effective amount of the modulator of claim 1 and obtaining results therefrom. 51. The method of claim 50 wherein
Said at least one enzyme complex is a PDH complex. 51. The method of claim 50 wherein
The disease, condition or syndrome is a modulator further characterized by cell hyperproliferation. The method of claim 52, wherein
And said disease, condition or syndrome is cancer.

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