WO2010110887A1

WO2010110887A1 - Fatty acids

Info

Publication number: WO2010110887A1
Application number: PCT/US2010/000879
Authority: WO
Inventors: Robert Shorr; Robert Rodriguez; Paul Bingham; Zuzana Zachar; Lakmal W. Boteju; Patrick P. Zaretski
Original assignee: Robert Shorr; Robert Rodriguez; Paul Bingham; Zuzana Zachar; Boteju Lakmal W; Zaretski Patrick P
Priority date: 2009-03-24
Filing date: 2010-03-24
Publication date: 2010-09-30

Abstract

Organic metabolites are produced in vivo from derivatives of thiol-containing alkyl fatty acids, such as but not limited to lipoic acid. These metabolites are intended to perturb at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, such as lipoate-containing or -utilizing dehydrogenase complexes and/or other enzymes. These metabolites are also intended to influence reactions which generate and/or regulate reactive oxygen and nitrogen species and/or other associated signal transduction pathways and cascades. The target enzymes, receptors, channels, proteins, reactions, pathways, and cascades are found in the organelles of diseased cells of warm-blooded animals, including but not limited to cancer cells. Also produced are structures which are resistant to or prevented from being metabolized into these metabolites, as well as a pharmaceutically-acceptable carrier formed from such metabolites, and methods of use thereof.

Description

Fatty Acids Field of the Invention

This invention relates to therapeutic and diagnostic compounds, and more particularly to organic metabolites of derivatives of thiol-containing alkyl fatty acids, such as but not limited to lipoic acid, which are produced, activated, inactivated, altered by, resistant to, or prevented from modification by in vivo metabolic events.

Background of the Invention US Patent Nos. 6,331,559 and 6,951,887 to Bingham et al; US Patent No. 6,117,902 to Quash et al; and US Patent Application No. 12/105,096 by Bingham et al, all herein incorporated by reference, disclose novel lipoic acid derivatives as potential therapeutic agents useful in the treatment of cancer. These references further disclose pharmaceutical compositions comprising an effective amount of at least one lipoic acid derivative according to its invention along with a pharmaceutically acceptable carrier. Bingham et al. in particular disclose that derivatives of lipoic acid may interfere with energy-generating metabolic events occurring in the mitochondria of cancer cells, more specifically through altering the oxidation-reduction (redox) state within the cell stress by inducing changes in the activity of the pyruvate dehydrogenase (PDH) complex as well as that of similar enzymes. These teachings fail to disclose organic metabolites of the disclosed derivatives that may be produced in vivo. These teachings also fail to disclose derivatives of lipoic acid that would be resistant to or prevented from entering cellular metabolism, or indeed themselves be modified or metabolized, with such metabolism, modification, resistance, or prevention itself potentially being of clinical benefit. More specifically, metabolic events modifying drug structure may occur in any mammalian tissue, such as, for example, heart, kidney, lung, liver, and blood. Both desired and unwanted metabolic events may occur only in the vicinity of particular cell types where the pertinent enzymes are secreted; where environmental conditions such as pH or ionic strength may be altered; or within the cytoplasm, nuclei, and/or organelles (e.g., endosomes, endoplasmic reticulum, and/or mitochondria) of particular cell types. Metabolic events may differ in diseased cells and tissue compared to those occurring in healthy counterparts. Also, while metabolic events occurring distally from the disease site may not be altered from the normal state, these events may also nevertheless influence the effectiveness or activity of any systemically-delivered drug. Finally, metabolic events in vivo may serve to activate inactive compounds, enhance the activity of already-active compounds, or lessen the activity of or even deactivate active compounds.

Metabolic events occurring under conditions of health or disease may therefore be useful for subsequent modifications of compounds, including but not limited to activation of prodrugs (i.e., any compounds that undergo biotransformation before exhibiting pharmacological effects), by healthy or disease-linked enzymes (e.g., without limitation, esterases, proteases, lipases, nucleases, or transferases). Such modifications may also be influenced by alterations in environmental conditions, including but not limited to changes in pH or O₂ concentration, in, at, near, or distal to diseased cells or tissues. Influential events affecting enzymes or conditions may also occur through addition of exogenous enzymes and/or condition-altering agents, such as but not limited to genes, trace elements, transcription factors, and energy-imparting phenomena such as heat, light, and sound of various frequencies. These metabolic events may also occur via enzyme pathways, including but not limited to cytochrome P450, which may or may not be inducible.

Thiol-containing alkyl fatty acids, such as but not limited to lipoic acid, have been suggested to alter the biochemistry of glucose oxidation in cancer cells such as to induce cancer-cell-specific apoptosis or necrosis. To briefly describe current thinking, mitochondria are known to produce both diverse reactive oxygen and nitrogen species (RONS) and over 90% of cellular ATP. The mitochondria of cancer cells are distinct from those of healthy cells. Cancer cells have been suggested to rely almost exclusively on cytoplasmic production of ATP through anaerobic oxidation of glucose. The PDH complex as well as related enzymes that utilize lipoic acid as a cofactor have been linked to alternative biochemical pathways associated with cancer. In part these enzymes take on biochemical functions that help to regulate oxidative stress through RONS levels. It has been suggested that in those cancer cells and tumors where PDH complex levels and/or activity is higher, a less desirable clinical outcome is more likely. This may be due to a greater ability of cells to manage conditions of oxia and hypoxia. For example, shed cells that encounter higher oxygen levels than typically found in a tumor mass are able to survive and form metastatic nodules. (See Steeg PS (2006). Tumor metastasis: mechanistic insights and clinical challenges. Nature Med. 12:895-904, herein incorporated by reference.)

A number of other mitochondrial metabolic events have also been linked to regulation of cellular growth and differentiation, as well as programmed cell death. For instance, RONS may serve as signal transduction molecules in pathways that regulate these functions. Phenotypic, epigenetic, or genotypic changes in enzyme structure, function, and regulation of activity which lead to alterations in oxidative stress levels and/or regulation may underlie pathology and disease. Consequently, such changes may be important targets for the treatment of disease.

The mitochondrial PDH complex plays a central role in the maintenance of glucose homoeostasis in mammals. Carbon flux through the PDH complex is meticulously regulated by elaborate mechanisms including reversible phosphorylation of multiple phosphorylation sites, tissue-specific distribution of dedicated kinases and phosphatases, and long-term hormonal transcriptional controls. Enzyme structure/activity regulation is sensitive to the intramitochondrial redox state and metabolite levels. (See Rigas B and Sun Y (2008). Induction of oxidative stress as a mechanism of action of chemopreventive agents against cancer. Brit. J. Cancer 98:1157-1160, herein incorporated by reference. See also Patel MS and Korotchkina LG (2006). Regulation of the pyruvate dehydrogenase complex. Biochem. Soc. Trans. 34:217-222, herein incorporated by reference.) The ability to modify enzymatic structure, function, and activity as a function of metabolite levels is important to cell survival in the presence of toxic metabolites such as in cancer. (See, e.g., Antoine DJ, Williams DP, and Park BK (2008). Understanding the role of reactive metabolites in drug-induced hepatotoxicity: state of the science. Expert Opin. Drug Metab. Toxicol. 4:1415-1427, herein incorporated by reference.) Changes in oxidative stress may also contribute to differential transcriptional controls of the regulatory components of the PDH complex. Overall, such differences between normal and disease states as regulation by the PDH complex of carbohydrate and amino acid oxidation in response to changes in the concentrations of intramitochondrial metabolites (e.g., pyruvate, acetyl-CoA, and NADH levels); regulation by the PDH complex of the mitochondrial and cellular redox state; and detoxification by the PDH complex of toxic metabolites and redox-affecting signal transduction pathways for growth and differentiation, metastasis, and cell death each present possible new targets for pharmaceutical intervention.

As suggested previously, the Warburg effect is a well-known energy metabolism alteration in tumor cells, which exhibit an increased glycolytic capacity even in the presence of a high O₂ concentration. Warburg originally proposed that the driving force of the enhanced glycolysis in tumor cells was the energy deficiency caused by an irreversible damage of the mitochondrial function in which, similarly to anaerobic muscle, glucose is converted through glycolysis to lactate, which is later secreted. The glycolytic flux in tumor cells is linked to survivability in environments with low O₂ concentrations. (For example, the O₂ concentration is lower than 20 μM in many human tumors.) Anaerobic glycolysis is the main energy pathway in solid tumors (e.g., slow-growing melanomas and mammary adenocarcinoma), and cancer tissue's reliance on anaerobic glycolysis is likely to be associated with increased malignancy. Recent studies suggest that forcing cancer cells into more aerobic metabolism suppresses tumor growth, as the TCA cycle in cancer cells is a variant cycle which depends on glutamine or fatty acids as a primary energy source. The transition to Warburg metabolism therefore obliges shutting down the PDH and related complexes.

Lipoic acid (6,8-dithiooctanoic acid) is a sulfur-containing antioxidant with metal- chelating and anti-glycation capabilities. It is not known whether lipoic acid is produced by cells or is an essential nutrient. Mitochondrial pumps or uptake mechanisms, including binding and transport chaperones, may be important in transporting lipoic acid to mitochondria. Unlike many antioxidants which are active only in either the lipid or the aqueous phase, lipoic acid is active in both lipid and aqueous phases. The anti-glycation capacity of lipoic acid combined with its capacity for hydrophobic binding enables lipoic acid to prevent glycosylation of albumin in the bloodstream. Lipoic acid is the oxidized part of a redox pair, capable of being reduced to dihydrolipoic acid (DHLA). Lipoic acid is readily absorbed from the diet and is rapidly converted to DHLA by NADH or NADPH in most tissues. Additionally, both lipoic acid and DHLA are antioxidants: lipoic acid is active against OH^*, HClO, and O₂, but not against O₂ ^'' or H₂O₂, and DHLA is active against OH^' and HClO, but not against H₂O₂ or O₂. Given the important role of lipoic acid in the regulation of RONS metabolism, then, it may be inferred that derivatives or analogues of lipoic acid would have a similar effect on RONS metabolism.

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

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

Lipoic acid also acts as a cofactor with the PDH complex, and perhaps also the α- KDH and BCAKDH complexes, in detoxifying toxic metabolites. Inhibition or inactivation of the tumor-specific PDH complex and related enzymes that detoxify metabolites may promote autophagic, apoptotic, or necrotic cell death. Indeed, as suggested previously, both lipoic acid and DHLA themselves have been demonstrated to possess potent anticancer effects through the generation of RONS to induce apoptosis in tumor cells. (See, e.g., Wenzel U, Nickel A, and Daniel H (2005). α-lipoic acid induces apoptosis in human colon cancer cells by increasing mitochondrial respiration with a concomitant CV-generation. Apoptosis 10:359-368, herein incorporated by reference.)

It has been discovered that eukaryotes produce organic metabolites (e.g., in mammalian hepatocytes) of the lipoic acid derivatives disclosed by Bingham et al. and Quash et al. , which are not claimed within those teachings. Additionally, it has been discovered that there are structures formed from derivatives of thiol-containing alkyl fatty acids, including but not limited to lipoic acid, which are resistant to or prevented from being metabolized into such organic metabolites. Accordingly, the present invention discloses novel analogs of thiol-containing alkyl fatty acids, such as but not limited to lipoic acid, that can be produced in vivo by metabolic modification of the congeners disclosed in Bingham et al. and Quash et al. These in vivo metabolic events may occur through the action either of endogenous native enzymes associated with such metabolic processes or upon administration of exogenous enzymes and/or condition-altering compounds or agents to propagate the desired metabolic effect. The present invention further discloses novel analogs of thiol-containing alkyl fatty acids, such as but not limited to lipoic acid, that have been modified to serve as prodrugs or so as to minimize metabolic changes in vivo, thereby potentially improving the aqueous solubility, safety, and efficacy of those analogs.

Summary of the Invention Consequently, the present invention is directed to organic metabolites formed, following metabolism in eukaryotes in such sites as, without limitation, the mitochondria of liver cells of warm-blooded animals, including humans, from derivatives of thiol-containing alkyl fatty acids, such as but not limited to the lipoic acid derivatives as described in US Patent Nos. 6,331,559 and 6,951,887 to Bingham et al; US Patent No. 6,117,902 to Quash et al.; and US Patent Application No. 12/105,096 by Bingham et al. Such metabolites are intended to influence the structure, function, activity, and/or expression level of at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, including but not limited to dehydrogenases (e.g., without limitation, lipoate- containing or -utilizing enzymes such as the PDH, α-KDH, and/or BCAKDH complexes and aldehyde dehydrogenase) modified in the diseased state. These organic metabolites are also intended to influence reactions associated with RONS generation and regulation and/or other associated signal transduction pathways and cascades. These enzymes, receptors, channels, proteins, reactions, pathways, and cascades may be found in the mitochondria of diseased cells. Such diseased cells may include, but not be limited to, cells characterized by hyperproliferation, such as cancer cells.

The reactions intended to be influenced by the organic metabolites of the present invention may serve to activate inactive compounds, enhance the activity of already-active compounds, or lessen the activity of or even deactivate active compounds. Additionally, the effects of these organic metabolites may be seen in phenotypic, epigenetic, or genotypic alterations. Such modifications may also be influenced by alterations in environmental conditions, including but not limited to changes in pH or O₂ concentration, in, at, near, or distal to diseased cells or tissues. These metabolic events may also occur via enzyme pathways, including but not limited to cytochrome P450, which may or may not be inducible.

Metabolic events producing the organic metabolites of the present invention may occur in specific mammalian cellular organelles, for example endosomes or mitochondria, throughout any mammalian tissue, such as, without limitation, heart, kidney, lung, and liver.

Mitochondrial as well as other metabolic events producing the organic metabolites of the present invention may be specific to a disease wherein diseased cells are characterized by hyperproliferation, such as cancer cells, and may be mediated by enzymes or conditions associated with a specific disease state. Furthermore, metabolic events producing the organic metabolites of the present invention may be required for the activation of prodrugs by disease-linked enzymes or conditions in, at, or near diseased cells or tissues. Disease-linked enzymes include but are not limited to esterases, proteases, lipases, nucleases, or transferases, as well as enzyme pathways, including but not limited to cytochrome P450. Disease-linked conditions include but are not limited to changes in pH or O₂ concentration. Influential events affecting enzymes or conditions may also occur through addition of exogenous enzymes and/or condition-altering agents, such as but not limited to genes, trace elements, transcription factors, and energy-imparting phenomena such as heat, light, and sound of various frequencies as may be introduced in the clinic through far infrared heat therapy, phototherapy, and ultrasound therapy, respectively. The general structure of the organic metabolites of the present invention is:

wherein S may be independently sulfoxidized; wherein Ri and R₂ are independently selected from the group consisting of hydrogen, defined as C_nH_2n, alkenyl defined as C_mH_2m-ι, alkynyl defined as C_mH_2m-3, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; wherein R₃ is alkyl, alkenyl, alkynyl, alkyl sulfide defined as (CH₂)_n-S-, alkoxy defined as (CH₂)_n-0-, alkylamine defined as (CH₂)_n-NH-, -S-S-, -S-O-, -S-NH-, -NH-O-, - NH-NH-, or -O-O-; wherein R₄ is hydrogen or glucuronide; wherein n is 0-10 and m is 1-10; and derivatives, congeners, positional isomers, and salts thereof. It is expressly intended that lipoic acid and DHLA may themselves be the products of the metabolic events described above. Additional specific sulfoxic, glucuronic, and sulfoxic- glucuronic structures are herein disclosed.

Furthermore, there is also provided herein a derivative of a thiol-containing alkyl fatty acid which has the structure:

This structure may in turn be metabolized to one of the above-provided metabolites in vivo.

The present invention also discloses structures formed from derivatives of thiol- containing alkyl fatty acids, such as but not limited to lipoic acid, which are resistant to, or prevented from being metabolized into one or more of the above-defined structures by, such metabolic events as described previously. These structures are formed by the conjugation of derivatives of thiol-containing alkyl fatty acids, such as but not limited to lipoic acid, to natural or synthetic polymers. Non-limiting examples of the constituents of these polymers include carbohydrates, lipids, amino acids, and nucleic acids. This conjugation may occur through an ionic bond, such as but not limited to a salt; a hydrophobic interaction; or through a covalent bond or a covalent reversible or cleavable bond, such as but not limited to an ester linkage. Furthermore, such conjugation may occur either at the carboxyl terminal, at one or both of the sulfur groups, or both. Non-limiting examples of such metabolism-resistant structures are herein disclosed.

It has been observed that the R-isomer of lipoic acid is typically more active then the S-isomer in a variety of cell types. Both isomers separately or as a racemic mix are contemplated for use in the present invention, such that both R- and S-isomers of the organic metabolites of the present invention may be generated and may retain physiological activity. Additionally, where carbohydrates are conjugated to the organic metabolites of the present invention, both D and L isomers of the carbohydrates are contemplated as being within the range of the present invention.

Brief Description of the Drawings FIGURE 1 depicts the kinetics of parent compound metabolic degradation and the formation of two metabolic breakdown products in a murine liver extract.

FIGURE 2 illustrates IC₅₀ concentrations for the parent compound and the sulfoxide, glucuronide and sulfoxide-glucuronide metabolites thereof against human H460 non-small cell lung carcinoma (NSCLC), A2780 ovarian tumor cells, and A2780-DX5 ovarian tumor cells upon 48-hour treatment of the same.

FIGURE 3 shows the concentration-response curves for the parent compound and the sulfoxide, glucuronide and sulfoxide-glucuronide metabolites thereof against human H460 non-small cell lung carcinoma (NSCLC), A2780 ovarian tumor cells, and A2780-DX5 ovarian tumor cells upon 48-hour treatment of the same. Detailed Description of the Invention

The present invention comprises organic metabolites formed, following metabolism in eukaryotes in such sites as, without limitation, the mitochondria of liver cells of warmblooded animals, including humans, from derivatives of thiol-containing alkyl fatty acids, such as but not limited to the lipoic acid derivatives as described in US Patent Nos. 6,331,559 and 6,951,887 to Bingham et al.; US Patent No. 6,117,902 to Quash et al; and US Patent Application No. 12/105,096 by Bingham et al. Such metabolites are intended to influence the structure, function, activity, and/or expression level of at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, including but not limited to dehydrogenases (e.g., without limitation, lipoate-containing or - utilizing enzymes such as the PDH, α-KDH, and/or BCAKDH complexes and aldehyde dehydrogenase) modified in the diseased state. These organic metabolites are also intended to influence reactions associated with RONS generation and regulation and/or other associated signal transduction pathways and cascades. These enzymes, receptors, channels, proteins, reactions, pathways, and cascades may be found in the mitochondria of diseased cells. Such diseased cells may include, but not be limited to, cells characterized by hyperproliferation, such as cancer cells.

The reactions intended to be influenced by the organic metabolites of the present invention may serve to activate inactive compounds, enhance the activity of already-active compounds, or lessen the activity of or even deactivate active compounds. Additionally, the effects of these organic metabolites may be seen in phenotypic, epigenetic, or genotypic alterations. Such modifications may also be influenced by alterations in environmental conditions, including but not limited to changes in pH or O₂ concentration, in, at, near, or distal to diseased cells or tissues. These metabolic events may also occur via enzyme pathways, including but not limited to cytochrome P450, which may or may not be inducible. Metabolic events producing the organic metabolites of the present invention may occur in specific mammalian cellular organelles, for example endosomes or mitochondria, throughout any mammalian tissue, such as, without limitation, heart, kidney, lung, and liver. Mitochondrial as well as other metabolic events producing the organic metabolites of the present invention may be specific to a disease wherein diseased cells are characterized by hyperproliferation, such as cancer cells, and may be mediated by enzymes or conditions associated with a specific disease state. Furthermore, metabolic events producing the organic metabolites of the present invention may be required for the activation of pro-drugs by disease-linked enzymes or conditions in, at, or near diseased cells or tissues. Disease-linked enzymes include but are not limited to esterases, proteases, lipases, nucleases, or transferases, as well as enzyme pathways, including but not limited to cytochrome P450. Disease-linked conditions include but are not limited to changes in pH or O₂ concentration. Influential events affecting enzymes or conditions may also occur through addition of exogenous enzymes and/or condition-altering agents, such as but not limited to genes, trace elements, transcription factors, and energy-imparting phenomena such as heat, light, and sound of various frequencies as may be introduced in the clinic through far infrared heat therapy, phototherapy, and ultrasound therapy, respectively.

The general structure of the organic metabolites of the present invention is:

wherein S may be independently sulfoxidized; wherein R₁ and R₂ are independently selected from the group consisting of hydrogen, defined as C_nH_2n, alkenyl defined as C_mH_2m-i, alkynyl defined as C_mH_2m-3, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; wherein R₃ is alkyl, alkenyl, alkynyl, alkyl sulfide defined as (CH₂)_n-S-, alkoxy defined as (CHa)_n-O-, alkylamine defined as (CHa)_n-NH-, -S-S-, -S-O-, -S-NH-, -NH-O-, - NH-NH-, or -O-O-; wherein R₄ is hydrogen or glucuronide; wherein n is 0-10 and m is 1-10; and derivatives, congeners, positional isomers, and salts thereof.

It is expressly intended that lipoic acid and DHLA may themselves be the products of the metabolic events described above. These organic metabolites have but are not limited to the specific sulfoxic, glucuronic, and sulfoxic-glucuronic structures:

(I) This structure may in turn be metabolized to one of the above-provided metabolites in vivo.

The present invention also discloses structures formed from derivatives of thiol- containing alkyl fatty acids, such as but not limited to lipoic acid, which are resistant to, or prevented from being metabolized into one or more of the above-defined structures by, such metabolic events as described previously. These structures are formed by the conjugation of derivatives of thiol-containing alkyl fatty acids, such as but not limited to lipoic acid, to natural or synthetic polymers. Non-limiting examples of the constituents of these polymers include carbohydrates, lipids, amino acids, and nucleic acids. This conjugation may occur through an ionic bond, such as but not limited to a salt; a hydrophobic interaction; or through a covalent bond or a covalent reversible or cleavable bond, such as but not limited to an ester linkage. Furthermore, such conjugation may occur either at the carboxyl terminal, at one or both of the sulfur groups, or both. Non-limiting examples of such metabolism-resistant structures include:

(L)

10

generally wherein R, Ri, and R₂ are independently selected from the group consisting of acyl defined as R₃C(O)-; alkyl defined as C_nH_2n+I ; alkenyl defined as C_mH_2m-i; alkynyl defined as C_mH_2m-3; alkyl sulfide defined as CH₃(CH₂)_n-S-; imidoyl defined as R₃C(=NH)-; hemiacetal defined as R₄CH(OH)-S-; aryl, heteroaryl, cycloalkyl, alkylaryl, or heterocyclyl, any of which can be substituted or unsubstituted; hydroxyl; and hydrogen; wherein R, Ri, and R₂ as defined above can be unsubstituted or substituted; wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; wherein x is 0-16, n is 0-10 and m is 2-10; and derivatives, congeners, positional isomers, and salts thereof.

Turning to the drawings, FIGURE 1 illustrates the kinetics of parent compound metabolic degradation and the formation of two metabolic breakdown products in a murine liver extract. It is apparent that the sulfoxic metabolic product of a lipoic acid derivative in mouse hepatocytes steadily increases as the lipoic acid derivative is metabolized. However, over this same period, there is virtually no increase in the glucuronic metabolic product of the lipoic acid derivative.

The following non-limiting examples are provided to facilitate understanding of the organic metabolites of the present invention. EXAMPLE 1 Synthesis of Sulfoxide Metabolite

Parent thiol-containing alkyl fatty acid derivative (3.Og, 7.72mmol) in dioxane (35mL) was treated with hydrogen peroxide (3% in water, 3mL, 8mmol). The reaction mixture was stirred at room temperature for 35h. The reaction mixture was diluted with water (7OmL) and ethyl acetate (10OmL), and the layers were separated. The aqueous phase was re-extracted with ethyl acetate (lx70mL). The combined organic phases were washed with water (xl), sat. aq. NaCl (xl) and dried (Na₂SO₄). Evaporation gave the crude product which was purified by using chromatography on silica gel (10Og), with a gradient of dichloromethane to 1.5% methanol in dichloromethane: colorless oil, 1.52g (48.6%); TLC: silica gel, 5% methanol in dichloromethane, Rf = 0.26; MS (ESI (-)ve): 403 (M - I); ¹H

NMR (CDCl₃: δ 8.27 (b, IH), 7.3-7.6 (m, 3H), 7.21-7.3 (m, 7H), 4.03 (dd, J = 10.4Hz, J =

2.4Hz, IH), 3.9385 (d, J = 6Hz, IH), 3.906 (d, J = 6.4Hz, IH), 3.624 (d, 16Hz, 2H), 2.69- 2.68 (m, 2H), 2.4-2.64 (m, 2H), 2.27 (m, 2H), 1.95-2.15 ( m, IH), 1.7-1.95 (m, IH), 1.43-

1.52 (m, 3H), 1.3-1.4 (m, 2H); HPLC: Symmetry (Cl 8) column, 3.5μm, 4.6x75mm, wavelength 205nm, flow rate 1.0 mL/min, run time 35 min., injection delay 5.0 min., mobile phases, A = acetonitrile, B = 0.1% phosphoric acid in water, gradient: 40% A 0-5min., 65%

A 5-10min., 65% A 10-20min., retention time: 7.1min.

EXAMPLE 2

Synthesis of Glucuronide Metabolite

The C-glucuronide of a thiol-containing alkyl fatty acid derivative was synthesized according to the procedure described by Becker et al. (2) D-Glucuronic acid (10.36g, 53.28mmol) in methanol (10OmL) was reacted with tetrabutylammonium hydroxide.30H₂O (42.6g, 53.28mmol). After stirring for one hour, the methanol was evaporated under reduced pressure. The residue was dissolved in pyridine (10OmL). To this solution was added pre- activated parent compound, which was prepared by reacting parent compound (17.2g,

44.24mmol) in pyridine (15OmL) with lj'-carbonyldiimidazole (7.88g, 48.68mmol). After the addition of the pre-activated acid, sodium hydride (lOOmg, cat.) was added. The reaction mixture was stirred for two days at room temperature. The pyridine was evaporated to approximately 10OmL and water (25OmL) was added, with stirring. The solution was cooled in an ice bath and the pH was adjusted to 6 with 1.0N HCl. The aqueous phase was extracted with ethyl acetate (5x10OmL). The combined ethyl acetate extracts were washed with water (IxIOOmL), sat. aq. NaCl (IxIOOmL) and dried (Na₂SO₄). Evaporation gave the crude compound which was purified by column chromatography on silica gel: silica gel (60Og), dichloromethane to 15% methanol in dichloromethane. Evaporation of the eluent in the appropriate fractions that contained the product gave an oil which was triturated with methanol to give a white solid: 5.3g (21%); HPLC, Phenomenex C- 18 column, 100x4.6mm, isocratic 50% acetonitrile in water, lmL/min, detection at 220nm, sensitivity at 0.05 AUFS, gives a single peak with a 1.43min retention time; MS (ESI (-)ve) 563.2 (M - I).

References

Becker B, Barua AB, and Olson JA. 1996. All-trans-retinoyl beta-glucuronide: new procedure for chemical synthesis and its metabolism in vitamin A-deficient rats. Biochem J 314:249-252.

EXAMPLE 3 Analysis of Metabolites

Incubated samples were monitored at 0, 15, 30, 60, 90 and 120 minutes. Human liver

S9 extract (1) containing a thiol-containing alkyl fatty acid derivative and its metabolites were analyzed using LC-MS/MS techniques. (1) Both phase 1 (NADPH) and phase 2 cofactors (UDPGA, PAPS, GSH) were present in the incubation, along with alamethicin and MgCl₂. A reverse-phase C-18 HPLC column (200 x 4.5mm) was used to separate the components in the extract. A mobile phase gradient of acetonitrile 5-95% in water over 30 minutes was used with detection by two MS scanning-techniques (turbospray ESI+ or ESI-). Precursor ion scanning and neutral loss MS-scanning methods were used to detect fragmentation patterns. Applied Biosystems Analyst and Microsoft Excel software with appropriate add-ons were used to identify fragments of each component. Imipramine was used as the control.

The results showed that the parent compound was subject to oxidation (+16 amu), and direct glucuronidation (+176 amu), thus generating two metabolites. Only a single oxidation peak was observed during the long chromatographic separation, even though the chemical structure of the parent compound used indicated several potential sites of oxidation. Further analysis of the LC/MS/MS data (e.g., fingerprints, etc.) and bioanalytical data showed that the results were consistent with oxidation occurring on a sulfur (sulfoxide), and not on the alkyl chain or phenyl ring.

Signature peaks for O-glucuronidation include 113, 175 and 193 (in negative ionization mode). All of these peaks were observed. Therefore, these results were consistent with glucuronidation of the terminal carboxyl group of the parent compound. Accordingly, direct glucuronidation was hypothesized to occur on the terminal (carboxyl) OH.

EXAMPLE 4

Anti-tumor Activity of Metabolites Objective:

The objective of this investigation was to assess the in vitro anti-tumor activities of sulfoxide, glucuronide, and sulfoxide-glucuronide metabolites of the thiol-containing alkyl fatty acid derivative CPI-613 against human H460 NSCLC and 2 human ovarian tumor cell lines: A2780 and A2780-DX5 (a doxorubicin-resistant derivative cell line of A2780). The three metabolites were investigated in this study because they are likely to be generated in humans according to an in vitro human hepatocyte study (see Example 1). Materials and methods:

Tumor Cell Lines:

The H460 NSCLC cells were originally obtained from American Type Cell Culture (ATCC). Human ovarian cancer A2780, and human A2780-DX5 ovarian tumor cells were gifts from Dr. Ralph Bernacki (Roswell Park Cancer Institute, Buffalo, NY). All tumor cells were maintained at 37°C in a humidified 5% CO₂ atmosphere in T75 tissue culture flasks containing 25 mL of Roswell Park Memorial Institute (RPMI) 1640, with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. The tumor cells were split at a ratio of 1:10 every 4-5 days by trypsinization and resuspended in fresh medium in a new flask. Cells were harvested for experiments at 70-90% confluency. Cell Culture Medium:

The compositions of the cell culture medium are outlined in Table 1.

Table 1: Compositions of Cell Culture Media Used

Study Design:

The anti-tumor activities of the test articles were assessed by exposing the tumor cells to various concentrations of test articles (or vehicle), or not treated with the test articles or vehicle. The concentration ranges of the test articles evaluated in this study were 0-1 mM.

The duration of treatment of the tumor cells was 48 hours in serum-containing medium.

Subsequent to treatment with the test articles, the number of viable tumor cells was determined and the concentrations of the test agents that induced 50% of cell growth inhibition (IC₅₀) were derived and compared.

Study Procedures:

Cell Seeding for Experiments — To cells grown to 70-90% confluency, medium was removed and the cell monolayers were washed briefly by adding 5 mL of phosphate buffer saline (PBS) followed by aspiration. Trypsin-ethylenediaminetetraacetic acid (EDTA) (4 mL) was added to each flask, and the flask was placed in the tissue culture incubator for five minutes. Serum-containing medium (10 mL) was added to halt the enzymatic reactions, and cells were disaggregated by repeated resuspension with serological pipette. The cell- containing medium (20 μL) was added to 20 μL of 0.4% Trypan Blue solution, mixed, and 10 μL of this cell-containing mixture was placed in a chamber of a hemocytometer. The number of viable cells was determined by counting the number of viable cells (cells that excluded Trypan blue) in the 4 corner squares of the hemocytometer chamber at 10Ox magnification. The volume of cells needed was determined by the following formula: .. . _ ., , , # of cells wanted/mL

Volume of cells needed =

# of cells counted/mL, where # of cells counted/mL = average # of cells on hemocytometer x 2 dilution factor x 10⁴. The number of cells targeted for the study was 4x10³ per well in 100 μL of medium. The actual number of cells were counted and seeded in the wells of a 96-well plate. The cells were then incubated for -24 hours before they were used for testing of anti-tumor activities of the test articles and vehicle.

Treatment with Test Articles and Vehicle ~ On the day of testing, 5 μL of a specific concentration of the test articles (or vehicle) were added to the wells. After exposure to the test articles (or vehicle) for 48 hours, the number of viable cells in the wells was determined (see next section) and the percent of cells relative to no treatment was calculated.

Determination of the Number of Viable Cells by the CellTiter Blue Assay ~ The number of viable cells was determined using the CellTiter Blue Assay in this study. Specifically, reagents were allowed to come to room temperature according to instructions from Promega, Inc. (Madison, WI). CellTiter Blue reagent was added with the 12-channel Eppendorf pipettor, 20 μL per well. The cells were then incubated at 37°C for 1-4 hrs in cell culture incubator. Fluorescence intensity, which is proportional to the quantity of viable cells, was read at 530/590 run.

Calculations of IC₅₀ Values:

Data from fluorescence readings were copied onto EXCEL spreadsheets, and cell growth relative to untreated cells was calculated, using the following equation:

% # cells relative to untreated = (mean luminescence at N/mean fluorescence untreated) x

100% where N = concentration of the test article or vehicle

The calculated values were imported into SigmaPlot v9. A Four-Parameter Logistic Curve of the "mean relative cell growth as a function of the concentrations of the test articles" was generated. The IC50 values were determined from the curves. The R-squared value provided an indication of the degree of fitness of data to the curve.

Results:

Anti-Tumor Activity of Parent Compound: The mean IC50 values for parent compound against H460 NSCLC, A2780 ovarian tumor cells, and A280-DX5 ovarian tumor cells, obtained after 48 hours of treatment, were -200 μM. These mean IC_5O values were consistent with those previously reported.

Anti-Tumor Activity of Sulfoxide Metabolite:

The mean IC50 values for the sulfoxide metabolite could not be determined due to a lack of inhibition of tumor cell growth at concentrations as high as 1,000 μM. These results suggested that the sulfoxide metabolite does not have significant anti-tumor activity. These results are consistent with those previously reported.

Anti-Tumor Activity of Glucuronide Metabolite:

The mean IC₅₀ values for the glucuronide metabolite against the three tumor cell lines were >313 μM. These mean IC₅₀ values were significantly higher than those of parent compound, suggesting that the glucuronide metabolite has limited anti-tumor activity. Once again, these results are consistent with those previously reported.

Anti-Tumor Activity of Sulfoxide-Glucuronide Metabolite:

The mean IC₅₀ values for the sulfoxide-glucuronide metabolite against the three tumor cell lines were >700 μM. Not only were these IC₅₀ values significantly higher than those of parent compound in serum-containing medium after 48 hours of treatment, they were actually beyond the concentration range expected to be in the circulation of patients treated with the expected therapeutic doses of parent compound. Therefore, the sulfoxide-glucuronide metabolite is considered not to have any anti-tumor activity. See Table 2 for synopsis of results. Graphic representations of this data are seen in FIGURES 2 and 3.

Table 2: IC50 of Parent Compound and 3 Metabolites Thereof Against Human H460 NSCLC, A2780 Ovarian Tumor, and A2780-DX5 Ovarian Tumor, After 48 Hours Treatment (N=4 in Each Experiment)

IC₅₀ ⁼ concentrations that induced tumor cell growth by 50%; N = sample size; ND = IC₅₀ not determined due to lack of significant inhibition of tumor cell growth at concentration < 700 μM.

Discussion/Conclusion:

The results from this study further showed that the sulfoxide metabolite might be an inactive metabolite. This is because there was no detectable tumor cell growth inhibition induced by this metabolite in all three tumor cell lines. The results from the current study also showed that the glucuronide metabolite of CPI-613 might have limited anti-tumor activity. This is reflected by the significantly higher IC_5O values of glucuronide metabolite against the three tumor cell lines when compared to those of CPI-613. Finally, the results

from this study showed that the sulfoxide-glucuronide metabolite might be an inactive metabolite. This is because not only were the IC₅₀ values of this metabolite significantly higher than those of CPI-613, they were actually beyond the concentration range expected to be in the circulation of patients treated with the expected therapeutic doses of CPI-613.

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. An organic metabolite produced in vivo from at least one derivative of a thiol- containing alkyl fatty acid, this metabolite having the ability to interact with at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, and/or to influence reactions associated with the generation and/or regulation of reactive oxygen and nitrogen species (RONS) and/or other associated signal transduction pathways and cascades.

2. The metabolite of claim 1, wherein the enzymes, receptors, channels, proteins, reactions, pathways, and cascades are found in the organelles of diseased cells of warm- blooded animals.

3. The metabolite of claim 2, wherein the organelles are mitochondria.

4. The metabolite of claim 1, wherein the warm-blooded animals are humans.

5. The metabolite of claim 1, wherein the diseased cells are characterized by hyperproliferation.

6. The metabolite of claim 5, wherein the hyperproliferative cells are cancer cells.

7. The metabolite of claim 1, wherein the thiol-containing alkyl fatty acid is lipoic acid.

8. The metabolite of claim 1 , wherein the derivative of a thiol-containing alkyl fatty acid interacts with a dehydrogenase.

9. The metabolite of claim 8, wherein the dehydrogenase is aldehyde dehydrogenase.

10. The metabolite of claim 8, wherein the dehydrogenase is a lipoate-containing or -utilizing enzyme or enzyme complex.

11. The metabolite of claim 10, wherein the lipoate-containing or -utilizing enzyme or enzyme complex is the pyruvate dehydrogenase (PDH) complex, the alpha- ketoglutarate dehydrogenase (α-KDH) complex, or the branched chain alpha-keto acid dehydrogenase (BCAKDH) complex.

12. The metabolite of claim 1, wherein the metabolite is generated as the R-isomer or S-isomer of the derivative thereof.

13. The metabolite of claim 1, wherein the metabolite is generated as a mixture of the R- isomers and S-isomers of the derivatives thereof.

14. The metabolite of claim 1 wherein the metabolite has the general formula:

wherein S may be independently sulfoxidized; wherein R_\ and R₂ are independently selected from the group consisting of hydrogen, defined as C_nH_2n, alkenyl defined as C_mH_2m-i, alkynyl defined as C_mH_2m-3, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; wherein R₃ is alkyl, alkenyl, alkynyl, alkyl sulfide defined as (CH₂)_n-S-, alkoxy defined as (CH₂)_n-O-, alkylamine defined as (CH₂)_n-NH-, -S-S-, -S-O-, -S-NH-, -NH-O-, - NH-NH-, or -O-O-; wherein R₄ is hydrogen or glucuronide; wherein n is 0-10 and m is 1-10; and derivatives, congeners, positional isomers, and salts thereof.

15. The metabolite of claim 1 , wherein the metabolite is lipoic acid.

16. The metabolite of claim 1, wherein the metabolite is dihydrolipoic acid.

17. The metabolite of claim 1 , wherein the metabolite has the structure:

18. The metabolite of claim 1, wherein the metabolite has the structure:

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

20. The metabolite of claim 1 , wherein the metabolite has the structure:

21. The metabolite of claim 1 , wherein the metabolite has the structure:

22. The metabolite of claim 1 , wherein the metabolite has the structure:

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

24. The metabolite of claim 1 , wherein the metabolite has the structure:

25. A derivative of a thiol-containing alkyl fatty acid having the structure:

26. A metabolism-resistant derivative of a thiol-containing alkyl fatty acid structured to prevent the metabolism of that derivative into the metabolite of claim 1.

27. The metabolism-resistant derivative of claim 26, wherein the derivative is formed from the conjugation of the thiol-containing alkyl fatty acid derivative to natural or synthetic polymers.

28. The metabolism-resistant derivative of claim 27, wherein the polymer is formed from repeating subunits chosen from the group comprising: carbohydrates, lipids, amino acids, and nucleic acids.

29. The metabolism-resistant derivative of claim 28, wherein the carbohydrate is present as the D-isomer thereof.

30. The metabolism-resistant derivative of claim 28, wherein the carbohydrate is present as the L-isomer thereof.

31. The metabolism-resistant derivative of claim 26, wherein conjugation may occur through an ionic bond; a hydrophobic interaction; a covalent bond; or a covalent reversible or cleavable bond.

32. The metabolism-resistant derivative of claim 26, having the structure:

33. The metabolism-resistant derivative of claim 26, having the structure:

34. The metabolism-resistant derivative of claim 26, having the structure:

35. The metabolism-resistant derivative of claim 26, having the structure:

36. The metabolism-resistant derivative of claim 26, having the structure:

wherein R is selected from the group consisting of acyl (defined hereafter as RiC(O)- ); alkyl (defined hereafter as C_nH_2n+O; alkenyl (defined hereafter as C_mH_2m-i); alkynyl (defined hereafter as C_mH_2m-3); alkyl sulfide (defined as CH₃(CH₂)_n-S-); imidoyl (defined hereafter as RiC(=NH)-); hemiacetal (defined hereafter as RiCH(OH)-S-); aryl, heteroaryl, cycloalkyl, alkylaryl, or heterocyclyl, any of which can be substituted or unsubstituted; hydroxyl; and hydrogen; wherein Ri is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; and wherein x is 0-16, n is 0-10 and m is 2-10.

37. The metabolism-resistant derivative of claim 26, having the structure:

(O) wherein Ri and R₂ are independently selected from the group consisting of acyl; alkyl; alkenyl; alkynyl; alkyl sulfide; imidoyl; hemiacetal; aryl, heteroaryl, cycloalkyl, alkylaryl, or heterocyclyl, any of which can be substituted or unsubstituted; hydroxyl; and hydrogen; wherein Ri and R₂ as defined above can be unsubstituted or substituted; wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; and wherein x is 0-16, n is 0-10 and m is 2-10.

38. The metabolism-resistant derivative of claim 26, having the structure:

(P) wherein R is selected from the group consisting of acyl defined as RiC(O)-; alkyl defined as C_nH_{2n+ 1}; alkenyl defined as C_mH_2m-i; alkynyl defined as C_mH_2m.₃; alkyl sulfide defined as CH₃(CH₂)_n-S-; imidoyl defined as RiC(=NH)-; hemiacetal defined as RiCH(OH)-S-; aryl, heteroaryl, cycloalkyl, alkylaryl, or heterocyclyl, any of which can be substituted or unsubstituted; hydroxyl; and hydrogen; wherein Ri is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; and wherein x is 0-16, n is 0-10 and m is 2-10.

39. The metabolism-resistant derivative of claim 26, having the structure:

wherein Ri and R₂ are independently selected from the group consisting of acyl; alkyl; alkenyl; alkynyl; alkyl sulfide; imidoyl; hemiacetal; aryl, heteroaryl, cycloalkyl, alkylaryl, or heterocyclyl, any of which can be substituted or unsubstituted; hydroxyl; and hydrogen; wherein R] and R₂ as defined above can be unsubstituted or substituted; wherein R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; and wherein x is 0-16, n is 0-10 and m is 2-10.

40. A pharmaceutically-acceptable modulator of the structure, function, activity, and/or expression level of at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, such as aldehyde dehydrogenase, the PDH complex, the α-KDH complex, and/or the BCAKDH complex, and/or of the reactions associated with the generation and/or regulation of RONS and/or other associated signal transduction pathways and cascades, these enzymes, receptors, channels, proteins, reactions, pathways, and cascades in the mitochondria of diseased cells of warm-blooded animals, including humans, such modulator comprising at least one derivative of a thiol-containing alkyl fatty acid according to claim 1 and at least one pharmaceutically-acceptable carrier thereof.

41. The modulator of claim 40, wherein the modulator is useful in the treatment and diagnosis of a disease, condition, or syndrome characterized by an alteration of at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, and/or an alteration in reactions associated with the generation or regulation of RONS and/or other associated signal transduction pathways and cascades, these altered enzymes, receptors, channels, proteins, reactions, pathways, and cascades found in the mitochondria of diseased cells of warm-blooded animals.

42. The modulator of claim 41, wherein the disease, condition, or syndrome is further characterized by cellular hyperproliferation.

43. The modulator of claim 42, wherein the disease, condition, or syndrome is cancer.

44. A method of modulating the structure, function, activity, and/or expression level of at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, and/or reactions associated with the generation and/or regulation of RONS and/or other associated signal transduction pathways and cascades, in a patient presenting a disease, condition, or syndrome characterized by an alteration of these enzymes, receptors, channels, proteins, reactions, pathways, and cascades, comprising administration of an effective amount of the modulator of claim 40.

45. The method of claim 44, wherein the disease, condition, or syndrome is further characterized by cellular hyperproliferation.

46. The method of claim 45, wherein the disease, condition, or syndrome is cancer.

47. A method of diagnosing and predicting benefit in a patient presenting symptoms of a disease, condition, or syndrome characterized by an alteration of the structure, function, activity, and/or expression level of at least one enzyme or enzyme complex, receptor, ion channel, transport protein, or at least one subunit of each thereof, and/or reactions associated with the generation and/or regulation of RONS and/or other associated signal transduction pathways and cascades, comprising obtaining a sample of cells from the patient, administering an effective amount of the modulator of claim 40 to the cells in vitro, and obtaining the results therefrom.

48. The method of claim 47, wherein the disease, condition, or syndrome is further characterized by cellular hyperproliferation.

49. The method of claim 48, wherein the disease, condition, or syndrome is cancer.