WO2010110887A1 - Acides gras - Google Patents

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WO2010110887A1
WO2010110887A1 PCT/US2010/000879 US2010000879W WO2010110887A1 WO 2010110887 A1 WO2010110887 A1 WO 2010110887A1 US 2010000879 W US2010000879 W US 2010000879W WO 2010110887 A1 WO2010110887 A1 WO 2010110887A1
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metabolite
metabolism
derivative
alkyl
enzyme
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PCT/US2010/000879
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Robert Shorr
Robert Rodriguez
Paul Bingham
Zuzana Zachar
Lakmal W. Boteju
Patrick P. Zaretski
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Robert Shorr
Robert Rodriguez
Paul Bingham
Zuzana Zachar
Boteju Lakmal W
Zaretski Patrick P
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Application filed by Robert Shorr, Robert Rodriguez, Paul Bingham, Zuzana Zachar, Boteju Lakmal W, Zaretski Patrick P filed Critical Robert Shorr
Publication of WO2010110887A1 publication Critical patent/WO2010110887A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • 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.
  • 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.
  • redox oxidation-reduction
  • PDH pyruvate dehydrogenase
  • 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).
  • prodrugs i.e., any compounds that undergo biotransformation before exhibiting pharmacological effects
  • healthy or disease-linked enzymes e.g., without limitation, esterases, proteases, lipases, nucleases, or transferases.
  • modifications may also be influenced by alterations in environmental conditions, including but not limited to changes in pH or O 2 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
  • mitochondria are known to produce both diverse reactive oxygen and nitrogen species (RONS) and over 90% of cellular ATP.
  • RONS reactive oxygen and nitrogen species
  • 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.
  • 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).
  • 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 O 2 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 O 2 concentrations.
  • 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.
  • solid tumors e.g., slow-growing melanomas and mammary adenocarcinoma
  • 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 O 2 , but not against O 2 '' or H 2 O 2 , and DHLA is active against OH ' and HClO, but not against H 2 O 2 or O 2 . 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 ( ⁇ -KDH) and branched chain alpha-keto acid dehydrogenase (BCAKDH) complexes also use lipoic acid as a cofactor.
  • ⁇ -KDH analogous alpha-ketoglutarate dehydrogenase
  • BCAKDH branched chain alpha-keto acid dehydrogenase
  • the lipoyl domain itself is attached by a flexible linker to the E2 core.
  • 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.
  • the lipoate-thioester functionality is translocated into the E2 active site, where a transacylation reaction transfers the acetyl from the "swinging arm" of lipoate to the thiol of coenzyme A.
  • the dihydrolipoate, still bound to a lysine residue of the complex then migrates to the E3 active site, where it undergoes a flavin-mediated oxidation back to its lipoate resting state, producing FADH 2 (and ultimately NADH) and regenerating the lipoate back into a competent acyl acceptor. Should this lipoate species be interrupted, then, there would be no flow of electrons to FADH 2 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 ⁇ - 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). ⁇ -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.)
  • 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.
  • 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.
  • 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, ⁇ -KDH, and/or BCAKDH complexes and aldehyde dehydrogenase) modified in the diseased state.
  • dehydrogenases e.g., without limitation, lipoate- containing or -utilizing enzymes such as the PDH, ⁇ -KDH, and/or BCAKDH complexes and aldehyde dehydrogenase
  • 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.
  • 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 O 2 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 O 2 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.
  • 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:
  • S may be independently sulfoxidized;
  • Ri and R 2 are independently selected from the group consisting of hydrogen, defined as C n H 2n , alkenyl defined as C m H 2m- ⁇ , alkynyl defined as C m H 2m-3 , cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted;
  • R 3 is alkyl, alkenyl, alkynyl, alkyl sulfide defined as (CH 2 ) n -S-, alkoxy defined as (CH 2 ) n -0-, alkylamine defined as (CH 2 ) n -NH-, -S-S-, -S-O-, -S-NH-, -NH-O-, - NH-NH-, or -O-O-; wherein R 4 is hydrogen or glucuronide; wherein n is 0-10 and
  • 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.
  • 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 IC 50 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.
  • NSCLC non-small cell lung carcinoma
  • 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.
  • NSCLC non-small cell lung carcinoma
  • 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, ⁇ -KDH, and/or BCAKDH complexes and aldehyde dehydrogenase) modified in the diseased state.
  • dehydrogenases e.g., without limitation, lipoate-containing or - utilizing enzymes such as the PDH, ⁇ -KDH, and/or BCAKDH complexes and aldehyde dehydrogenase
  • 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.
  • 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 O 2 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.
  • 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 O 2 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:
  • S may be independently sulfoxidized; wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, defined as C n H 2n , alkenyl defined as C m H 2m- i, alkynyl defined as C m H 2m-3 , cycloalkyl, aryl, alkylaryl, heteroaryl, or heterocyclyl, any of which can be substituted or unsubstituted; wherein R 3 is alkyl, alkenyl, alkynyl, alkyl sulfide defined as (CH 2 ) 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 R 4 is hydrogen or glucuronide; wherein n is 0-10 and
  • 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:
  • 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.
  • 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.
  • 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:
  • 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 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.30H 2 O (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,
  • S9 extract (1) containing a thiol-containing alkyl fatty acid derivative and its metabolites were analyzed using LC-MS/MS techniques.
  • phase 1 NADPH
  • phase 2 cofactors UDPGA, PAPS, GSH
  • 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 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:
  • 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% CO 2 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 :
  • compositions of the cell culture medium are outlined in Table 1.
  • 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.
  • 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.
  • the number of viable tumor cells was determined and the concentrations of the test agents that induced 50% of cell growth inhibition (IC 50 ) were derived and compared.
  • the cell- containing medium (20 ⁇ L) was added to 20 ⁇ L of 0.4% Trypan Blue solution, mixed, and 10 ⁇ L 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
  • the number of cells targeted for the study was 4x10 3 per well in 100 ⁇ L 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.
  • test Articles and Vehicle Treatment with Test Articles and Vehicle ⁇
  • 5 ⁇ L 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.
  • 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 ⁇ L 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.
  • IC 50 values for the sulfoxide-glucuronide metabolite against the three tumor cell lines were >700 ⁇ M. Not only were these IC 50 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.
  • 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.

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Abstract

L'invention porte sur des métabolites organiques qui sont produits in vivo à partir de dérivés d'acides gras d'alkyle contenant un thiol, tels que, mais sans y être limité, l'acide lipoïque. Ces métabolites sont destinés à perturber au moins une enzyme ou un complexe d'enzymes, un récepteur, un canal ionique, une protéine de transport ou au moins une sous-unité de chacun de ceux-ci, tels que des complexes déshydrogénases contenant ou utilisant du lipoate et/ou autres enzymes. Ces métabolites sont également destinés à influencer les réactions qui génèrent et/ou régulent des espèces oxygénées et azotées réactives et/ou d'autres voies et cascades de transduction du signal associées. Les enzymes, réacteurs, canaux, protéines, réactions, voies et cascades cibles sont trouvés dans les organelles de cellules malades d'animaux à sang chaud, comprenant mais sans y être limités des cellules cancéreuses. Sont également produites des structures qui sont résistantes à ces métabolites ou empêchées d'être métabolisées en ces métabolites, ainsi qu'un support pharmaceutiquement acceptable formé à partir de tels métabolites. L'invention porte également sur leurs procédés d'utilisation.
PCT/US2010/000879 2009-03-24 2010-03-24 Acides gras WO2010110887A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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