WO2011046583A1

WO2011046583A1 - Pharmaceutical exploitation of oxidative stress in transformed cells

Info

Publication number: WO2011046583A1
Application number: PCT/US2010/002455
Authority: WO
Inventors: Robert Shorr; Robert Rodriquez; Paul Bingham; Zuzana Zachar
Original assignee: Robert Shorr; Robert Rodriquez; Paul Bingham; Zuzana Zachar
Priority date: 2009-09-11
Filing date: 2010-09-09
Publication date: 2011-04-21

Abstract

Pharmaceutically-acceptable agents, and pharmaceutical compositions thereof, inhibit the reduction of oxidized peroxiredoxin and isoforms thereof, and mutant forms thereof, in the mitochondria and cytoplasm of diseased cells in a warm-blooded animal through interaction with a variety of target enzymes and substrates. These agents upregulate intramitochondrial and intracellular levels of reactive oxygen and nitrogen species and/or the structure, function, activity level, and/or expression level of apoptosis signal-regulating kinase 1. Such upregulation culminates in increased cytochrome c release from the mitochondria, resulting in cell death. These agents and compositions thereof are useful in the treatment, prevention, diagnosis, and/or imaging of diseased cells characterized by cellular hyperproliferation, such as but not limited to cancer.

Description

Pharmaceutical Exploitation of Oxidative Stress in Transformed Cells

Field of the Invention

This invention relates to therapeutic, preventive, diagnostic, and imaging compounds, and more particularly to agents and pharmaceutical compositions therefor which target the thioredoxin-peroxiredoxin system in transformed cells.

Background of the Invention

Free radicals contain one or more unpaired electrons; since all molecules seek to have an equal number of protons and electrons, the unpaired electron spins of these radicals make the radicals highly reactive. Driven by changes in free energy associated with electron or hydrogen transfers, oxidation-reduction (redox) biochemistry is hence fundamental to life both because the ATP supply required by higher organisms for their energy needs depends heavily on such chemistry and because electron-transfer processes such as those involving redox-active species also play a key messenger role in biological systems. Crucial to such activities are reactive oxygen species, including oxygen radicals (e.g., O2"^" and OH^') and nonradical 0₂ derivatives (e.g., H₂0₂), and reactive nitrogen species (e.g., NO^', ONOO^", and N0₂C1), both collectively known as RONS.

RONS are produced continuously by the mitochondria of most cells (0₂ ^'\ H₂0₂, NO^*, ONOO^", and OH^'); cytochrome P450 (0₂ ^" and H₂0₂); macrophages (0₂ ^", H₂0₂, and NO^'); and peroxisomes (H₂0₂). During mitochondrial oxidative metabolism, about 5% of oxygen is converted primarily into 0₂ ^'" with the remainder reduced to water. Given such high reactivity, and especially as RONS have the potential to damage lipids in cell membranes, DNA, and proteins, it is not surprising that there is a cellular antioxidant defense system to manage RONS and their effects. This defense system includes mitochondrial, peroxisomal, and cytoplasmic antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione (GSH) peroxidase, glutaredoxin, peroxiredoxin, and thioredoxin); nonclassic antioxidant enzymes (e.g., hemoxygenase-1); Phase II detoxifying enzymes (e.g., GSH reductase, NQOl, and GSH transferase); and nonenzymatic antioxidants (e.g., lipoic acid, vitamins E and C, GSH, and catechins).

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

The thioredoxin-peroxiredoxin system is a major redox control system in both the cytoplasm and mitochondria of cells. The mitochondrial thioredoxin (Trx) system is known to consist of a 12 kDa redox-active protein called thioredoxin-2 (Trx-2), a homodimeric seleno-protein called thioredoxin reductase-2 (TrxR-2), and nicotinamide adenine dinucleotide phosphate (NADPH). Trx-2 is initially synthesized with an additional N- terminal extension that targets the protein to the mitochondria which is then cleaved to the 12 kDa form. The active site of Trx-2 contains two cysteine residues that undergo reversible oxidation to form a disulfide bond during the transfer of reducing equivalents to a disulfide substrate. The oxidized Trx-2 protein is then recycled to a reduced state through the action of TrxR-2 and NADPH. Through this reversible redox reaction, Trx-2 can regulate the activity of several protein substrates in numerous pathways, including members of the peroxiredoxin (Prx) family that degrade ¾0₂. TrxR is a flavoprotein consisting of homodimers with each subunit containing two active site regions. The first active site is located at the N-terminus of the protein while the second region, containing a selenocysteine component, is located at its C-terminus. The first active site receives electrons from NADPH via the flavin adenine dinucleotide (FAD) molecule and transfers electrons to the selenocysteine redox-active site present at the C- terminus. The selenolate formed in the C-terminal active site region can then act to reduce the required substrate. This region is not only important for the function of TrxR but also represents a target for many therapeutic reagents to specifically inhibit TrxR. While TrxR is the only enzyme in the cell known to reduce Trx, TrxR also reduces low molecular weight disulfide compounds such as lipoic acid and nondisulfide substrates including vitamin K, H₂0₂, various quinones, and selenium-containing compounds. TrxR hence contributes to maintaining the redox state of a cell both through Trx regulation and its own direct action.

Trx also regulates the activity of various transcription factors, including NF-κΒ and p53, by reducing a key cysteine residue that is required for DNA binding. NF-κΒ controls expression of a number of genes involved in blocking the apoptotic pathway, such as members of the Bcl-2 family (including Bcl-2 itself). By contrast, p53 controls expression of proapoptotic genes, and it has been shown that the Trx system must be intact where p53 induces apoptosis in response to specific stimuli. (See Ueno M, Masutani H, Arai RJ, Yamauchi A, Hirota K, Sakai T, Inamoto T, Yamaoka Y, Yodoi J, and Nikaido T (1999). Thioredoxin-dependent redox regulation of p53-mediated p21 activation. J. Biol. Chem. 274:35809-15, herein incorporated by reference.) Inhibition of Trx via oxidation will therefore not only lead to functional consequences, but also transcriptional regulation within a cell will be altered such that expression of antiapoptotic proteins is reduced.

Besides its function in regulating the DNA binding ability of certain transcription factors, leading to further regulation of both cellular growth and immune function, the main function of Trx is to counteract oxidative stress by scavenging RONS and by regulating other enzymes that help to reduce oxidative stress. While there are other antioxidant systems within a cell, Trx has some specific protein substrates that give it some distinct functional roles for counteracting oxidative stress. Trx specifically reduces other enzymes that function to maintain the redox state of a cell, including members of the Prx family. These proteins regulate levels of H₂0₂ in the cell and thus are important contributors to maintaining redox balance. Trx also reduces GSH peroxidase 3, thus providing a link between these two major antioxidant systems. Trx can also act directly on certain RONS through its action as a potent singlet oxygen quencher and hydroxyl radical scavenger. A functional Trx system is therefore essential to maintain the intracellular redox state and to reduce oxidized proteins; consequently, inhibiting Trx function should lead to an imbalance in the redox state that can ultimately cause cell death.

In addition to its maintenance of the cell's redox state, cytoplasmic Trx directly regulates the apoptotic pathway through binding to apoptosis signal-regulating kinase 1 (ASK1), a MAPKKK enzyme. When reduced, Trx binds to the N-terminal region of ASK1 and inhibits its kinase function; while oxidized (e.g., when RONS are present), Trx is unable to bind to ASK1 , which as a result gains kinase capability and forms part of a larger molecular weight complex with such other compounds as TNF-receptor-associated factors 2 and 6. Apoptosis is then mediated via the JNK and p38 pathways.

The mitochondrial Trx system also regulates apoptotic signalling pathways through an unknown mechanism. While some researchers suggest that, since some inhibitors of TrxR-2 can stimulate an increase in membrane permeability and lead to the release of cytochrome c, mitochondrial Trx-2 affects mitochondrial membrane permeability, mere overexpression of TrxR-2 does not result in any changes to permeability. Other researchers have shown that oxidation of Trx-2 by exposure to H₂0₂ results in increased cell death. (See, e.g., Chen Y, Cai J, and Jones DP (2006). Mitochondrial thioredoxin in regulation of oxidant-induced cell death. FEBS Lett. 580:6596-602, herein incorporated by reference.) Especially as Trx-2 was oxidized more extensively and persistently than its cytoplasmic counterpart, this would suggest that Trx-2 is also an important sensor of oxidative stresses and that it too can regulate the onset and progression of apoptosis. However, while Trx-2 interacts with and inhibits ASK1 in the mitochondria, leading to inhibition of apoptosis, mitochondrial ASK1 has been reported to mediate apoptosis through a JNK-independent pathway that nonetheless still results in cytochrome c release and caspase-3 activation. {See Zhang R, Al-Lamki R, Bai L, Streb JW, Miano JM, Bradley J, and Min W (2004). Thioredoxin-2 inhibits mitochondria- located ASKl -mediated apoptosis in a JNK-independent manner. Circ. Res. 94:1483-91, herein incorporated by reference.) Still, by either the cytoplasmic or mitochondrial pathway, inhibition of Trx results in activation of ASK1 and stimulation of apoptosis.

Indeed, it has also been shown that inhibition of Trx can also generate autophagy and necrosis as a form of tumor cell death. In evaluating whether disrupting autophagy would augment the anticancer activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in imatinib-resistant chronic myelogenous leukemia (CML) cells, it was reported that drugs that disrupted the autophagy pathway dramatically augmented the antineoplastic effects of SAHA in CML cell lines expressing wild-type and imatinib-resistant mutant forms of Bcr-Abl. Disrupting autophagy by chloroquine treatment enhanced SAHA- induced RONS generation, triggering both relocalization and upregulation of the lysosomal protease cathepsin D, which in turn reduced the expression of Trx. {See Carew JS, Nawrocki ST, Kahue CN, Zhang H, Yang C, Chung L, Houghton JA, Huang P, Giles FJ, and Cleveland JL (2007). Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance. Blood 1 10:313-22, herein incorporated by reference.) Additionally, it has been reported that when primary human tracheobronchial epithelial cells and transformed human bronchial epithelial cells were exposed to various concentrations of H₂0₂ and subsequently double-labelled with antibodies specific for Trx and markers of injury or oxidant stress, cells with both mitochondrial injury and oxidative stress produced thioredoxin. However, where the injury is too severe, leading to necrosis, the cell is unable to produce thioredoxin. (See Oslund KL, Miller LA, Usachenko JL, Tyler NK, Wu R, and Hyde DM (2004). Oxidant-injured airway epithelial cells upregulate thioredoxin but do not produce interleukin-8. Am. J Respir. Cell Mol. Biol. 30:597-604, herein incorporated by reference.)

Dependent on the Trx system for its function, Prx is a ubiquitous antioxidant protein that uses specialized cysteine residues to decompose peroxides. Its rapid reactivity towards peroxides as well as its high cellular expression contributes to Prx being a primary antioxidant of peroxides. Additionally, members of the Prx family are emerging as key players in cell signalling events involving fluctuations in peroxide levels. Mammalian cells contain at least six different Prx isoforms sharing similar active sites, with Prx-3 being the dominant mitochondrial isoform along with Prx-5. Prx-3 exists as a dodecameric ring whose compact globular monomeric structure undergoes large conformational changes during oxidation and reduction. Peroxide detoxification proceeds via the reaction of an active site cysteine, with peroxide forming a sulfenic acid. The sulfenic acid then reacts with a resolving cysteine residue in another Prx subunit, resulting in the generation of a disulfide- linked intermolecular oxidized Prx dimer; Trx then reduces the Prx dimer back to its active state. Based on its binary redox state, Prx-3 has been implicated in the regulation of apoptotic signalling by mitochondria. Indeed, it has been observed that Prx-3 is oxidized during receptor-mediated apoptosis, suggesting that both increased levels of mitochondrial H₂0₂ and Prx-3 oxidation mediates the apoptotic process. (See Cox AG, Pullar JM, Hughes G, Ledgerwood EC, and Hampton MB (2008). Oxidation of mitochondrial peroxiredoxin 3 during the initiation of receptor-mediated apoptosis. Free Radio. Biol. Med. 44: 1001-9, herein incorporated by reference.) Consequently, it would be desirable to provide a drug for the treatment of cancer which maintains Prx in its inactive oxidized state, thereby promoting the intramitochondrial and/or intracellular accumulation of RONS and upregulation of the structure, function, activity level, and/or expression level of ASK1, leading to cell death.

High levels of Trx and TrxR have been observed in many different tumor types, both solid and liquid cancers, compared to levels observed in corresponding healthy cells from the same patient. Similarly, Prx has also been reported to be upregulated in breast and lung cancer cells. It has been suggested that Trx may have different functions in the cancer cell depending on the stage of cancer development. At early stages Trx may in fact be beneficial for preventing cancer due to its capability to counteract the oxidative stress caused by many carcinogens. Once a cell has initiated a cancer phenotype then high levels of Trx may assist cancer development due to its growth-promoting and antiapoptotic functions, contributing at later stages of cancer progression to angiogenesis and metastasis. On the other hand, due to the integral role that the Trx system plays in regulating apoptosis and its high expression levels in cancer cells, there is an interest in developing drugs that can target the Trx system. These drugs can either act alone or in combination with another chemotherapeutic drug and may target either Trx or TrxR. Targeting the Trx system will result in modulation of the intracellular redox state, which can favor cells becoming apoptotic due to an accumulation of RONS and alteration of the intracellular redox state which tips the balance of oxidative stress in favor of cell death, and the activation of ASK1 to facilitate downstream effects that lead to stimulation of apoptosis within a cell. Consequently, it would therefore be desirable for the treatment of cancer to provide a drug which maintains Trx in its inactive oxidized state, thereby promoting the intramitochondrial and/or intracellular accumulation of RONS and upregulation of the structure, function, activity level, and/or expression level of ASK1, leading to cell death.

It has been demonstrated that the E3 subunit of the pyruvate dehydrogenase (PDH) and a-ketoglutarate dehydrogenase (a-KDH) complexes are each capable of RONS generation upon stimulation by an appropriate agent or under the appropriate cellular conditions. (See Starkov A, Fiskum G, Chinopoulos C, Lorenzo B,l Browne S, Patel M, and Beal M (2004). Mitochondrial a-ketoglutarate dehydrogenase complex generates reactive oxygen species. J. Neurosci. 24:7779-88, passim, herein incorporated by reference.) Given that all three enzymes share the same E3 subunit, while this proposition is unproven, it is likely that, under the appropriate conditions and stimuli, that branched-chain a-ketoacid dehydrogenase (BCAKDH) generates RONS as well. As suggested previously, such increased levels of RONS would lead to an imbalance of the oxidative state of a cell in favor of induction of cell death, an advantageous phenomenon for anticancer therapy. Thus, it would be particularly desirable for the treatment of cancer to provide a drug which increases the intramitochondrial and/or intracellular levels of RONS through interaction with the PDH, a-KDH, and/or BCAKDH complexes of cancer cells, these RONS subsequently acting upon the Trx-Prx system to ultimately induce cancer cell death.

US Patents 6,331,559 and 6,951,887 to Bingham et ah, as well as US Patent Application No. 12/105,096 by Bingham et ah, all herein incorporated by reference, disclose a novel class of lipoic acid derivative therapeutic agents that selectively target and kill both tumor cells and certain other types of diseased cells. These teachings further disclose pharmaceutical compositions, and methods of use thereof, comprising an effective amount of such lipoic acid derivatives along with a pharmaceutically acceptable carrier. However, while these patents describe the structures of and general use for these lipoic acid derivatives, there is no indication in either patent that these derivatives are useful in modulating the structure, function, activity, cellular location or compartmentalization, and/or expression level of the Trx-Prx system, either its individual components or as a whole, to facilitate tumor cell death by the increase of intramitochondrial and/or intracellular levels of RONS and upregulation of the structure, function, activity level and/or expression level of ASK1.

Similarly, US Provisional Patent Application No. 61/136,170 by Bingham, et al, herein incorporated by reference, discloses pharmaceutically-acceptable agents which target and perturb the activity, or regulation thereof, of the altered mitochondrial energy metabolism observed in the modified PDH, a-KDH, and/or BCAKDH complexes associated with most cancers by modification of the oxidation-reduction state therein, such as but not limited to affecting concentrations of metabolic by-products of PDH, a-KDH, and/or BCAKDH complex activity, including RONS. However, there is no indication in this application that these agents are useful in modulating the structure, function, activity, cellular location or compartmentalization, and/or expression level of the Trx-Prx system, through maintained oxidation either of its individual components or as a whole, to facilitate tumor cell death by such increased levels of intramitochondrial and/or intracellular RONS and upregulation of the structure, function, activity level, and/or expression level of ASK1.

As it has been demonstrated that an increase in intramitochondrial and/or intracellular RONS levels in cancer cells and upregulation of the structure, function, activity level, and/or expression level of ASK1 lead to oxidation of Trx and Prx, which consequently induces cancer cell death, it would be beneficial to provide a pharmaceutical agent, and composition therefor, which generates increased intramitochondrial and/or intracellular RONS levels and upregulation of the structure, function, activity level, and/or expression level of ASK1 , thus culminating in tumor cell death, through the maintained oxidation of Trx and/or Prx. Objects of the Invention and Industrial Applicability

Consequently, it is an object of the present invention to provide a pharmaceutically- acceptable agent to be used in diseased cells which causes minimal side effects upon administration.

It is a further object of the present invention to provide a pharmaceutically-acceptable agent to be used in diseased cells which is easily manufactured at the least possible cost and is capable of being stored for the longest possible period.

It is a still further object of the present invention to provide a pharmaceutically- acceptable agent to be used in diseased cells which increases the intramitochondrial and/or intracellular levels of RONS, which in turn culminates in tumor cell death.

It is a still further object of the present invention to provide a pharmaceutically- acceptable agent to be used in diseased cells which upregulates the structure, function, activity level, and/or expression level of ASK1, which in turn culminates in tumor cell death. Summary of the Invention

To achieve the aforementioned aims, the present invention broadly discloses a pharmaceutically-acceptable agent for the treatment of a diseased cell of a warm-blooded animal, and/or prevention, diagnosis, and/or imaging of the disease affecting that cell, which inhibits the reduction of Prx and isoforms thereof, such as but not limited to Prx-1 and Prx-3, and mutant forms thereof. As there are a number of steps in the Trx-Prx system which culminate in the reduction of Prx, inhibition may occur through the maintained oxidation of Prx; through the maintained oxidation of Trx; through the maintained reduction of TrxR; through the inhibition of the NADPH-dependent conversion of glutamate to glutamine; through the inhibition of the NADPH-dependent conversion of glutamate to alpha- ketoglutarate (a-KG); and/or through the related inhibition of the conversion of pyruvate to alanine, which is a reaction necessary for the conversion of glutamate to a-KG. The increased levels of pyruvate within the cell may be due to a downregulation of the structure, function, activity level, and/or expression level of the PDH complex and isoforms thereof, and mutant forms thereof. In turn, the downregulation of the PDH complex may be caused either by an upregulation of the structure, function, activity level, and/or expression level of PDH phosphatase and isoforms thereof, and mutant forms thereof, or by a downregulation of the structure, function, activity level, and/or expression level of PDH kinase and isoforms thereof, and mutant forms thereof. The ultimate effect of the maintained oxidation of Prx is increased intramitochondrial and/or intracellular levels of RONS as well as an upregulation of the structure, function, activity level, and/or expression level of ASK1. Each of these phenomena generates increased levels of cytochrome c, which brings about cell death through apoptosis, necrosis, or autophagy. The agent of the present invention is particularly suited for treatment for diseases characterized by cellular hyperproliferation, such as but not limited to cancer.

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

and derivatives, congeners, and salts thereof,

wherein R_\ and/or R₂ is aryl or aralkyl;

wherein R₃ and/or R4 is S, Se, O, N, aryl, or a metal;

wherein R is alkyl, alkenyl, or alkynyl, with a chain length of one to eighteen carbons; wherein is alkyl, alkenyl, alkynyl, aryl, -COOH, -OH, -COH, -NH₂OH, -CC1₃, - CF₃, -NH₂, amino acids such as but not limited to glutamate, carbohydrates, nucleic acids, lipids, and multimers thereof;

wherein Ri, R₂, R₅, and/or R may be phosphorylated;

and wherein R_\, R₂, R , and/or ¾ may be so modified as to modulate the binding affinity of the compound to carrier molecules in vivo so as to regulate the amount of circulating time the compound spends in the blood.

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

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

In a further preferred embodiment of the present invention, the agent is combined with a pharmaceutically-acceptable carrier therefor to form a pharmaceutical composition useful for the treatment, prevention, diagnosis, and/or imaging of a disease and/or symptoms thereof.

Brief Description of the Figures

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

FIGURE 1 illustrates the general pathway of the proposed mechanism of action of the agent of the present invention. FIGURE 2 depicts the downstream effects of the upregulated levels of Prx caused by the administration of the agent of the present invention.

FIGURE 3 shows the effects of different agents on the production of mitochondrial H₂0₂ as assessed by monitoring the oxidation of mitochondrial Prx-3.

FIGURE 4 illustrates the effects of different agents on the production of mitochondrial H₂0₂ as assessed by monitoring the oxidation of cytoplasmic Prx-1.

Detailed Description of the Invention

The present invention broadly discloses a pharmaceutically-acceptable agent for the treatment of a diseased cell of a warm-blooded animal, and/or prevention, diagnosis, and/or imaging of the disease affecting that cell, which inhibits the reduction of Prx and isoforms thereof, and mutant forms thereof. While the agent of the present invention is particularly suitable for mitochondrial isoforms of Prx, such as but not limited to Prx-3, it is contemplated that cytoplasmic isoforms would also be amenable to the agent, such as but not limited to Prx-1.

As illustrated in FIGURE 1 , there are a number of steps in the Trx-Prx system which culminate in the reduction of Prx. Inhibition may occur through the maintained oxidation of Prx; through the maintained oxidation of Trx; through the maintained reduction of TrxR; through the inhibition of the NADPH-dependent conversion of glutamate to glutamine by glutamine synthetase; through the inhibition of the NADPH-dependent conversion of glutamate to a-KG by glutamate dehydrogenase; and/or through the related inhibition of the conversion of pyruvate to alanine by alanine aminotransaminase, a reaction necessary for the conversion of glutamate to a-KG.

The increased levels of pyruvate within the cell causing the inhibition of alanine aminotransferase may be due to a downregulation of the structure, function, activity level, and/or expression level of the PDH complex and isoforms thereof, and mutant forms thereof. In turn, the downregulation of the PDH complex may be caused by an upregulation of the structure, function, activity level, and/or expression level of PDH phosphatase and isoforms thereof, such as PDK1, PDK2, PDK3, PDK4, and mutant forms thereof. However, the downregulation of the PDH complex may also be caused by a downregulation of the structure, function, activity level, and/or expression level of PDH kinase and isoforms thereof, such as PDP1 and PDP2, and mutant forms thereof.

As noted in FIGURE 2, the ultimate effect of the maintained oxidation of Prx is increased intramitochondrial and/or intracellular levels of RONS as well as an upregulation of the structure, function, activity level, and/or expression level of ASK1. Each of these phenomena generates increased levels of cytochrome c, which brings about cell death through apoptosis through the peroxidation of cardiolipin, which associates cytochrome c to the inner mitochondrial membrane, and subsequent dissociation of cytochrome c. The rapid release of cytochrome c has also been reported to play a role in the early phase of necrosis. {See Li YZ, Li CJ, Pinto AV, and Pardee AB (1999). Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by beta-lapachone in human carcinoma cells. Mol. Med. 5:232-9, herein incorporated by reference.) Furthermore, it has been reported in murine neuroblastoma cells that autophagy is required for cytochrome c release to effect caspase- dependent apoptosis. {See Castino R, Isidoro C, and Murphy D (2005). Autophagy- dependent cell survival and cell death in an autosomal dominant familial neurohypophyseal diabetes insipidus in vitro model. FASEB J. 19: 1024-6, herein incorporated by reference.) Hence, it is expected that administration of the agent of the present invention to a diseased cell shall result in all three forms of cell death. Where the agent of the present invention modulates the expression level of a target enzyme, this modulation may occur at the transcriptional, translational, or post-translational stage, including epigenetic silencing of the appropriate genes.

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

and derivatives, congeners, and salts thereof,

wherein Ri and/or R₂ is aryl or aralkyl;

wherein R₃ and/or R4 is S, Se, O, N, aryl, or a metal;

wherein R₅ is alkyl, alkenyl, or alkynyl, with a chain length from one to eighteen carbons;

wherein Re, is alkyl, alkenyl, alkynyl, aryl, -COOH, -OH, -COH, -NH₂OH, -CC1₃, - CF₃, -NH₂, amino acids such as but not limited to glutamate, carbohydrates, nucleic acids, lipids, and multimers thereof;

wherein Ri, R₂, R₅, and/or R^ may be phosphorylated;

and wherein R_1? R₂, R₅, and/or R$ may be so modified as to modulate the binding affinity of the compound to carrier molecules in vivo so as to regulate the amount of circulating time the compound spends in the blood.

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

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

In a further preferred embodiment of the present invention, the agent is combined with a pharmaceutically-acceptable carrier or excipient therefor to form a pharmaceutical composition useful for the treatment, prevention, diagnosis, and/or imaging of a disease, or symptoms thereof, characterized by cellular hyperproliferation. Examples of pharmaceutically-acceptable carriers are well known in the art and include those conventionally used in pharmaceutical compositions, such as, but not limited to, salts, antioxidants, buffers, chelating agents, flavorants, colorants, preservatives, absorption promoters to enhance bioavailability, antimicrobial agents, and combinations thereof, optionally in combination with other therapeutic ingredients. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically-acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically- and pharmaceutically- acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

The agent of the present invention is particularly suited for treatment for diseases characterized by cellular hyperproliferation. Hence, the pharmaceutical composition of the present invention is expected to be useful in such general cancer types as carcinoma, sarcoma, lymphoma and leukemia, germ cell tumor, and blastoma. Indeed, the agent of the present invention is observed to be selectively and specifically delivered to and taken up by a tumor mass and the transformed cells within, and effectively concentrated within the mitochondria of transformed cells, thereby sparing healthy cells and tissue from the effects of the agent.

More specifically, the pharmaceutical composition of the present invention is expected to be useful in primary or metastatic melanoma, lung cancer, liver cancer, Hodgkin's and non-Hodgkin's lymphoma, uterine cancer, cervical cancer, bladder cancer, kidney cancer, colon cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer, without limitation. Non-limiting examples of other diseases characterized by cellular hyperproliferation amenable to the agent of the present invention include age-related macular degeneration; Crohn's disease; cirrhosis; chronic inflammatory- related disorders; diabetic retinopathy; granulomatosis; immune hyperproliferation associated with organ or tissue transplantation; an immunoproliferative disease or disorder (e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, or systemic lupus erythematosus); vascular hyperproliferation secondary to retinal hypoxia; or vasculitis.

The pharmaceutical composition of the present invention may be administered using any mode of administration both that is medically acceptable and that produces effective levels of the agent without causing clinically unacceptable adverse effects. Although formulations specifically suited for parenteral administration are preferred, the pharmaceutical composition of the present invention can also be formulated for inhalational, oral, topical, transdermal, nasal, ocular, pulmonary, rectal, transmucosal, intravenous, intramuscular, subcutaneous, intraperitoneal, intrathoracic, intrapleural, intrauterine, intratumoral, or infusion methodologies or administration, in the form of aerosols, sprays, powders, gels, lotions, creams, suppositories, ointments, and the like. If such a formulation is desired, other additives well-known in the art may be included to impart the desired consistency and other properties to the formulation. Those skilled in the art will recognize that the particular mode of administering the pharmaceutical composition depends on the particular agent selected; whether the administration is for imaging, diagnosis, and/or staging of a disease, condition, syndrome, or symptoms thereof; the severity of the medical disorder being imaged and/or diagnosed; and the dosage required for imaging and/or diagnostic efficacy. For example, a preferred mode of administering an anticancer agent for the treatment of leukemia would involve intravenous administration, whereas preferred methods for the treatment of colon cancer could involve oral administration of the agent.

As used herein, "effective amount" refers to the dosage or multiple dosages of the pharmaceutical composition at which the desired effect is achieved. Generally, an effective amount of the pharmaceutical composition may vary with the activity of the specific agent employed; the metabolic stability and length of action of that agent; the species, age, body weight, general health, dietary status, sex and diet of the subject; the mode and time of administration; rate of excretion; drug combination, if any; and extent of presentation and/or severity of the particular condition being treated. The precise dosage can be determined by an artisan of ordinary skill in the art without undue experimentation, in one or several administrations per day, to yield the desired results, and the dosage may be adjusted by the individual practitioner to achieve a desired effect or in the event of any complication. Importantly, when used to image, diagnose, and/or stage cancer, the dosage amount of the agent used should be sufficient to interact solely with tumor cells, leaving normal cells virtually neglected.

The pharmaceutical composition of the present invention can be prepared in any amount desired up to the maximum amount that can be administered safely to a patient. The amount of the pharmaceutical composition may range from less than 0.01 mg/mL to greater than 1000 mg/mL, preferably about 50 mg/mL. Generally, the pharmaceutical composition of the present invention will be delivered in a manner sufficient to administer to the patient an amount effective to deliver the agent to its intended molecular target. The dosage amount may thus range from about 0.3 mg/m to 2000 mg/m², preferably about 60 mg/m². The dosage amount may be administered in a single dose or in the form of individual divided doses, such as from one to four or more times per day. In the event that the response in a subject is insufficient at a certain dose, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent of patient tolerance.

The following non-limiting examples are provided both to facilitate understanding of the process of the present invention and to exemplify the invention and are not to be construed as limiting the invention's scope.

Example 1

Enhanced Oxidation of Prx-3 in H460 Cells

Materials and Equipment Description:

Reagents:

Equipment:

C0₂ incubator, Thermo Forma, series II water-jacketed, class 100

Gyrotory shaker, model G2, New Brunswick

Vortex- Genie, Fisher

Microcentrifuge, Eppendorf, model 5415C

Power source, LKB, 2197

Gel transfer device, model iBlot, (Invitrogen)

Kodak X-Omat 1000A Procedure:

The purpose of this study was to investigate the effect of 6,8-Z>/s-(benzylthio)octanoic acid (CPI-613) on the redox state of mitochondrial Prx-3 in transformed cells. The procedure was adapted from Cox AG, Brown KK, Arner ES, and Hampton MB (2008). The thioredoxin inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation. Biochem. Pharm. 76:1097-109.

H460 cells were seeded at ~1.5e5 cells/plate in 12-well plates (BD) in RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100μg/mL streptomycin (lXPennStrep). 24 hours later, RPMI medium was replaced with glucose-free RPMI supplemented with lOmM MePyr, +10% dFBS, +lXPenn/Strep (pyr/gln RPMI). 16 hours later, medium was replaced with fresh Pyr-gln RPMI containing 300μΜ CPI-613.

Treatment was terminated as follows, all steps done on ice: cells were washed once with PBS, followed by addition of 180μΙ. of NEM-buffer to block reduced cysteine residues. Cells were incubated in NEM buffer for 10 minutes then lysed by adding 45 iL of 5% CHAPS in ddH₂0 for 5 minutes. Lysates were transferred to 1.5mL microfuge tubes and incubated on ice for an additional 5 minutes with occasional vortexing, followed by a 10 minute spin at l l,700x g at 4°C. Supernatants were mixed 1 : 1 with 4x LDS sample buffer (no reducing agent), heated for 8 minutes at 70°C, and electrophoresed under non-reducing conditions on 16% Tris-Glycine gels (Invitrogen). Proteins were transferred to a PVDF membrane using I-Blot (7-minute transfer, 15v). Proteins were detected using polyclonal antibodies (Cell Signaling) to mitochondrial Prx-3 or cytosolic Prx-1.

In this study, the effect of different agents on the production of mitochondrial H₂0₂ was assessed by monitoring the oxidation of mitochondrial Prx-3. Cells were treated with various agents for 15 or 90 minutes and processed as described above. In FIGURE 3, the amount of oxidized Prx-3 reflects the relative amounts of H₂0₂ produced in response to each agent. It is evident that CPI-613 and related compounds induce very rapid oxidation of Prx- 3.

Example 2

Enhanced Oxidation of Prx-1 in H460 Cells

The purpose of this study was to investigate the effect of 6,8-bw-(benzylthio)octanoic acid (CPI-613) on the redox state of cytoplasmic Prx-1 in transformed cells. The study was carried out using the same procedure as in Example 1.

In this study, the effect of different agents on the production of mitochondrial H₂0₂ was assessed by monitoring the oxidation of cytoplasmic Prx-1. Cells were treated with various agents for 15, 90, or 210 minutes and processed as described above. In FIGURE 4, the amount of oxidized Prx-1 reflects the relative amounts of H₂0₂ produced in response to each agent. Notice that oxidation of the cytoplasmic Prx-1 is much slower than that of mitochondrial Prx-3 as seen in FIGURE 3 and probably represents secondary events in response to the original H₂0₂ signal generated in the mitochondrion. Also note the presence of higher order oligomers; these have been hypothesized to be "signolosomes" containing Prx-1 dimers interacting with other proteins.

The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. Furthermore, while exemplary embodiments have been expressed herein, others practiced in the art may be aware of other designs or uses of the present invention. Thus, while the present invention has been described in connection with exemplary embodiments thereof, it will be understood that many modifications in both design and use will be apparent to those of ordinary skill in the art, and this application is intended to cover any adaptations or variations thereof. It is therefore manifestly intended that this invention be limited only by the claims and the equivalents thereof.

Claims

The invention to be claimed is:

1. A pharmaceutically-acceptable agent which inhibits the reduction of peroxidase (Prx) and isoforms thereof, and mutant forms thereof, selectively in a diseased cell of a warm-blooded animal, leading to cell death.

2. The agent of claim 1 , wherein the Prx isoform is Prx-1.

3. The agent of claim 1 , wherein the Prx isoform is Prx-3.

4. The agent of claim 1 , wherein the inhibition is achieved by inhibition of the reduction of thioredoxin (Trx) and isoforms thereof, and mutant forms thereof.

5. The agent of claim 4, wherein the Trx is cytoplasmic.

6. The agent of claim 4, wherein the Trx is mitochondrial.

7. The agent of claim 1 , wherein the inhibition is achieved by inhibition of the oxidation of thioredoxin reductase (TrxR) and isoforms thereof, and mutant forms thereof.

8. The agent of claim 7, wherein the TrxR is cytoplasmic.

9. The agent of claim 7, wherein the TrxR is mitochondrial.

10. The agent of claim 1, wherein the inhibition is achieved by inhibition of the conversion of glutamate to glutamine.

11. The agent of claim 1 , wherein the inhibition is achieved by inhibition of the conversion of glutamate to alpha-ketoglutarate.

12. The agent of claim 11, wherein the inhibition is achieved by inhibition of the conversion of pyruvate to alanine.

13. The agent of claim 12, wherein the inhibition of the conversion of pyruvate to alanine is achieved by upregulation of levels of pyruvate.

14. The agent of claim 13, wherein the upregulation is caused by downregulation of the structure, function, activity level, and/or expression level of the pyruvate dehydrogenase (PDH) complex and isoforms thereof, and mutant forms thereof.

15. The agent of claim 14, wherein the downregulation is caused by upregulation of the structure, function, activity level, and/or expression level of PDH phosphatase and isoforms thereof, and mutant forms thereof.

16. The agent of claim 14, wherein the downregulation is caused by downregulation of the structure, function, activity level, and/or expression level of PDH kinase and isoforms thereof, and mutant forms thereof.

17. The agent of claim 1, wherein the result of the inhibition is the upregulation of levels of reactive oxygen and nitrogen species (RONS) in the mitochondria of the diseased cell.

18. The agent of claim 1 , wherein the result of the inhibition is the upregulation of the structure, function, activity level, or expression level of apoptosis signal-regulating kinase 1 (ASK1).

19. The agent of claim 1, wherein the result of the inhibition is the upregulation of levels of cytochrome c.

20. The agent of claim 1, wherein the form of cell death is autophagy.

21. The agent of claim 1 , wherein the form of cell death is apoptosis.

22. The agent of claim 1, wherein the form of cell death is necrosis.

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

and derivatives, congeners, and salts thereof,

wherein R_\ and/or R₂ is aryl or aralkyl;

wherein R₃ and/or R4 is S, Se, O, N, aryl, or a metal; wherein R₅ is alkyl, alkenyl, or alkynyl, with a chain length of one to eighteen carbons;

wherein is alkyl, alkenyl, alkynyl, aryl, -COOH, -OH, -COH, -NH₂OH, -CCI3, - CF₃, -NH₂, amino acids such as but not limited to glutamate, carbohydrates, nucleic acids, lipids, and multimers thereof;

wherein Ri, R₂, R₅, and/or R^ may be phosphorylated;

and wherein Ri, R₂, R₅, and/or R6 may be so modified as to modulate the binding affinity of the compound to carrier molecules in vivo so as to regulate the amount of circulating time the compound spends in the blood.

24. The agent of claim 23, wherein the agent is present as the R-isomer thereof.

25. The agent of claim 23, wherein the agent is present as a racemic mixture of the R- and S-isomers thereof.

26. The agent of claim 1, wherein the disease is characterized by cellular hyperproliferation.

27. The agent of claim 26, wherein the disease is cancer.

28. A pharmaceutical composition useful for the treatment, prevention, diagnosis, and/or imaging of a disease in a warm-blooded animal comprising the agent of claim 1 and a pharmaceutically-acceptable carrier therefor.

29. The composition of claim 28, wherein the disease is characterized by cellular hyperproliferation.

30. The composition of claim 29, wherein the disease is cancer.