USRE47300E1 - Mitochondrially delivered anti-cancer compounds - Google Patents

Mitochondrially delivered anti-cancer compounds Download PDF

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USRE47300E1
USRE47300E1 US14/955,297 US200914955297A USRE47300E US RE47300 E1 USRE47300 E1 US RE47300E1 US 200914955297 A US200914955297 A US 200914955297A US RE47300 E USRE47300 E US RE47300E
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Stephen John Ralph
Jiri Neuzil
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
    • C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • C07D311/70—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with two hydrocarbon radicals attached in position 2 and elements other than carbon and hydrogen in position 6
    • C07D311/72—3,4-Dihydro derivatives having in position 2 at least one methyl radical and in position 6 one oxygen atom, e.g. tocopherols

Definitions

  • This invention relates, inter alia, to anti-cancer compounds and to methods for treating or preventing cancer.
  • the invention concerns mitochondrially delivered pro-oxidant anti-cancer compounds that generate reactive oxygen species and induce apoptosis of cancerous cells.
  • the list of mitocans currently includes 7 groups, each of them comprising agents with distinct activities, whereby causing mitochondrial destabilisation and the ensuing induction of the intrinsic apoptotic pathway (Neuzil J et al (2007) Mol Pharmacol 71, 1185-1199).
  • Mitocans are proving to be attractive for the treatment of cancer since some of these compounds are potent and selective anti-cancer agents with little effect on normal cells (Neuzil J et al (2007) Mol Pharmacol 71, 1185-119; Ko Y H et al (2004) Biochem Biophys Res Commun 324, 269-275; Bonnet S et al (2007) Cancer Cell 11, 37-51).
  • ⁇ -tocopheryl succinate inducing selective apoptosis of cancer cells
  • ⁇ -TOS ⁇ -tocopheryl succinate
  • 3BP 3-bromopyruvate
  • DCA dichloroacetate
  • PITC ⁇ -phenylethyl isothiocyanate
  • 3BP inhibits hexokinase, an enzyme of the glycolytic pathway predominantly bound to the external face of mitochondria, and also inhibits the mitochondrial enzyme succinate dehydrogenase (SDH), suppressing cellular ATP production and mitochondrial respiration (Ko Y H et al (2004) Biochem Biophys Res Commun 324, 269-275; Xu R H et al (2005) Cancer Res 65, 613-621).
  • SDH succinate dehydrogenase
  • DCA appears to selectively target cancer cells by inhibiting the mitochondrial pyruvate dehydrogenase kinase (Bonnet S et al (2007) Cancer Cell 11, 37-51).
  • PITC selectively kills cancer cells by causing mitochondrial generation of reactive oxygen species (ROS) (Trachootham D et al (2006) Cancer Cell 10, 241-252).
  • ROS reactive oxygen species
  • mitocans include pro-oxidant analogues of vitamin E (Wang X F, Dong L F, Zhao Y, Tomasetti M, Wu K, Neuzil J (2006) Vitamin E analogues as anti-cancer agents: Lessons from studies with ⁇ -tocopheryl succinate. Mol Nutr Food Res 50:675-685).
  • vitamin E analogues epitomized by ⁇ -TOS, as anti-cancer drugs stems from studies with experimentally contrived cancers, such as human xenografts growing in nude mice, where they have been shown to suppress malignancy (reviewed in Neuzil J, Tomasetti M, Mellick A S, Alleva R, Salvatore B A, Birringer M, Fariss M W (2004) Vitamin E analogues: A new class of inducers of apoptosis with selective anti-cancer effects. Curr Cancer Drug Targets 4:355-372).
  • Vitamin E succinate is a potent novel anti-neoplastic agent with high tumor selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L) in vivo.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • vitamin E ⁇ -tocopherol, ⁇ -TOH
  • ⁇ -TOS an esterified, redox-silent and pro-oxidant analogue of vitamin E
  • ⁇ -TOH acts as a potent anti-oxidant in cells
  • ⁇ -TOS acts as a strong cell stressor, causing rapid production of ROS in a range of different cancer cell lines
  • Vitamin E analogues A new class of inducers of apoptosis with selective anti-cancer effects.
  • ⁇ -TOS also has the ability to bind to and inhibit Bcl-2/Bcl-xL (Dong L F, Wang X F, Zhao Y, Tomasetti M, Wu K, Neuzil J (2006) Vitamin E analogues as anti-cancer agents: the role of modulation of apoptosis signalling pathways. Cancer Therapy 4:35-46).
  • Evidence to date suggests that the cancer cell-specific nature of ⁇ -TOS and the lack of toxic effect on normal cells occurs because normal cells are endowed with greater anti-oxidant defenses (Allen R G, Balin A K (2003) Effects of oxygen on the antioxidant responses of normal and transformed cells.
  • Naturally occurring vitamin E consists of a mixture of eight compounds which differ by the methylation patterns of the chromanol ring ( ⁇ -, ⁇ -, ⁇ -, ⁇ -tocopherol) and the number of double bonds of the phytyl side-chain ( ⁇ -, ⁇ -, ⁇ , ⁇ -tocotrienol).
  • ⁇ -, ⁇ -, ⁇ -tocopherol the methylation patterns of the chromanol ring
  • ⁇ -tocopherol the number of double bonds of the phytyl side-chain
  • the vitamin E molecule can be divided into three different domains.
  • the Functional Domain (I) arises from the substitution pattern at position C6 of the chromanol ring. This position determines whether the molecule behaves as redoxactive or redox-silent, since a free hydroxyl group is essential for vitamin E to function as an anti-oxidant.
  • the well documented anti-oxidant properties of the four tocopherol isomers resulted in their application in cancer clinical trials. None of these studies showed a positive outcome concerning the use of free tocopherols in cancer prevention (Pham DQ and Plakogiannis R (2005) Vitamin E supplementation in cardiovascular disease and cancer prevention: Part 1. Ann Pharmacother 39:1870-8).
  • certain chemical modifications at C6 led to ethers (RO—), esters (RCOO—) and amides (RCONH—) that proved to be potent anti-neoplastic agents. See Table II below.
  • the second domain termed the Signaling Domain (II) exhibits some activities that are independent of the anti-oxidant properties of the tocopherols. These properties derive from the methylation pattern of the aromatic ring. For example, ⁇ -tocopherol has been reported to inhibit protein kinase C (PKC) by decreasing diacylglycerol (DAG) levels, while other tocopherols with similar anti-oxidant capabilities (e.g., ⁇ -tocopherol) do not inhibit PKC.
  • PKC protein kinase C
  • DAG diacylglycerol
  • the PKC inhibitory activity of ⁇ -tocopherol is independent of its anti-oxidant capacity (Tasinato A, Boscoboinik D, Bartoli G M, Maroni P and Azzi A (1995) d- ⁇ -Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.
  • Vitamin E prevents diabetes-induced abnormal retinal blood flow via the diacylglycerol-protein kinase C pathway. Am J Physiol 269:E239-246). In some cases, however, the biological activity of the various tocopherols is influenced by structural differences in the Signaling Domain, which do indeed have a profound impact on their anti-oxidant activity against certain species.
  • ⁇ -Tocopherol for example, is a much better scavenger of reactive nitrogen oxide species (e.g., peroxynitrite) than ⁇ -tocopherol.
  • reactive nitrogen oxide species e.g., peroxynitrite
  • the ⁇ -molecule which lacks a methyl group at C5
  • 5-nitro-gamma-tocopherol is elevated in the plasma of subjects with coronary heart disease.
  • the lipophilic side chain of vitamin E isomers distinguishes between tocopherols with saturated isoprenyl units and tocotrienols with unsaturated isoprenyl units.
  • the Hydrophobic Domain (III) determines whether the molecule can bind to lipoproteins and membranes respectively, or be degraded by phase I enzymes (Birringer M, Pfluger P, Kluth D, Austin N and Brigelius-Flohe R (2002) Identities and differences in the metabolism of tocotrienols and tocopherols in HepG2 cells. J Nutr 132:3113-3118; Neuzil J, Massa H (2005) Hepatic processing determines dual activity of vitamin E succinate. Biochem Biophys Res Commun 327:1024-1027).
  • tocopherol derivatives with a modified hydroxyl group have been tested for their pro-apoptotic activity (Table II).
  • the most prominent derivative tested has been ⁇ -TOS (entry 1) bearing a succinylester at position C6 of the chromanol ring. Due to its low pK ⁇ ( ⁇ 6), ⁇ -TOS is fully deprotonated under physiological conditions, leading to a detergent-like molecule which destabilizes mitochondrial membranes and has an effect on complex II.
  • Dicarboxylic esters of tocopherols present the best studied compounds for structure-activity relationship (SAR).
  • Enhancing the stability of these tocopheryl ester derivatives would protect these molecules in vivo, allowing them to stay intact longer, thereby increasing their bioavailability.
  • the isosteric replacement of the esters by amides makes that linkage less prone to enzymatic hydrolysis as well.
  • Several nonspecific esterases exist in the intestinal mucosal cells and in the blood. In contrast, peptidases exhibit a much narrower specificity.
  • pro-drugs with an amino acid in an amide linkage are more stable in the intestine and blood than their corresponding ester analogues (Sugawara M, Huang W, Fei Y-J, Leibach F H, Ganaphthy V and Ganaphthy M E (2000) Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2. J Pharm Sci 89:781-789).
  • ⁇ -tocopherol transfer protein ⁇ -TTP
  • ⁇ -TTP ⁇ -tocopherol transfer protein
  • Tocopherol-associated protein is a ligand-dependent transcriptional activator. Biochem Biophys Res Commun 285:295-299).
  • thrombin-induced PKC activation and endothelin secretion are inhibited by ⁇ -tocopherol but not by ⁇ -tocopherol (Martin-Nizard F, Boullier A, Fruchart, J C and Duriez P (1998) ⁇ -Tocopherol but not ⁇ -tocopherol inhibits thrombin-induced PKC activation and endothelin secretion in endothelial cells. J Cardiovasc Risk 5:339-345).
  • ⁇ -tocopherol causes up-regulation of ⁇ -tropomyosin expression (Aratri E, Spycher S E, Breyer I and Azzi A (1999) Modulation of ⁇ -tropomyosin expression by ⁇ -tocopherol in rat vascular smooth muscle cells. FEBS Lett 447:91-94) and down regulation of LDL scavenger receptors SR-A and CD36, whereas ⁇ -tocopherol is ineffective (Ricciarelli R, Zingg J M and Azzi A (2000) Vitamin E reduces the uptake of oxidized LDL by inhibiting CD36 scavenger receptor expression in cultured aortic smooth muscle cells.
  • the substitution pattern is likely responsible for the rate of side chain degradation because in cell culture, ⁇ - and ⁇ -tocopherol are degraded much faster than ⁇ - or ⁇ -tocopherol (Birringer M, Drogan D and Brigelius-Flohe R (2001) Tocopherols are metabolized in HepG2 cells by side chain ⁇ -oxidation and consecutive ⁇ -oxidation. Free Radic Biol Med 31:226-232).
  • ⁇ -TOS (1) possesses the highest apoptogenic activity tested, followed by ⁇ -TOS (32), ⁇ -TOS (33) and ⁇ -TOS (35) as the least effective (Birringer M, Drogan D and Brigelius-Flohe R (2001) Tocopherols are metabolized in HepG2 cells by side chain ⁇ -oxidation and consecutive ⁇ -oxidation: Free Radic Biol Med 31:226-232). In general, the more highly methylated members of the tocopherol family are the most potent, but this trend is reversed for the tocotrienols (see below).
  • Tocotrienols are efficient anti-cancer agents and their pro-apoptotic property may be related to the inactivation of the Ras family of proteins. Tocotrienols exhibit their pro-apoptotic activity without modifications of the Functional Domain. The hierarchy in the Signaling Domain is also reversed, making ⁇ -tocotrienol (59) the most potent agent in the murine B16-F10 melanoma cell model, followed by ⁇ -(56) and ⁇ -tocotrienol (53) (Table IV; He L, Mo H, Hadisusilo S, Qureshi A A and Elson C E (1997) Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo. J Nutr 127:668-674).
  • Ras farnesylation and RhoA prenylation was inhibited by tocotrienols in A549 cells, a human lung adenocarcinoma cell line containing an activating ras mutation (Yano Y, Satoh H, Fukumoto K, Kumadaki I, Ichikawa T, Yamada K, Hagiwara K and Yano T (2005) Induction of cytotoxicity in human lung adenocarcinoma cells by 6-O-carboxy-propyl- ⁇ -tocotrienol, a redox-silent derivative of ⁇ -tocotrienol. Int J Cancer 115:839-846).
  • ⁇ -Tocopheryl polyethylene glycol succinate (23) has been used as a vehicle for drug delivery systems.
  • This compound was shown to possess anti-cancer activity against human lung carcinoma cells implanted in nude mice.
  • the apoptosis inducing efficacy of the compound was not due to its increased uptake into cells, but rather due to an increased ability to generate reactive oxygen species (Youk H J, Lee E, Choi M K, Lee Y J, Chung J H, Kim S H, Lee C H and Lim S J (2005) Enhanced anticancer efficacy of ⁇ -tocopheryl succinate by conjugation with polyethylene glycol.
  • ⁇ -Tocopheryl phosphate (30) is believed to result from metabolism occurring during tocopherol-associated signaling (Negis Y, Zingg J M, Ogru E, Gianello R, Libinaki R and Azzi A (2005) On the existence of cellular tocopheryl phosphate, its synthesis, degradation and cellular roles: a hypothesis. IUBMB Life 57:23-25).
  • ⁇ -Carboxyethyl hydroxychroman (52), a degradation product of ⁇ -tocopherol often found secreted in the urine, is able to reduce cell proliferation of PC-3 prostate cancer cells by inhibiting cyclin D1 expression (Galli F, Stabile A M, Betti M, Conte C, Pistilli A, Rende M, Floridi A and Azzi A (2004) The effect of ⁇ - and ⁇ -tocopherol and their carboxyethyl hydroxychroman metabolites on prostate cancer cell proliferation. Arch Biochem Biophys 423:97-102).
  • a commonly observable difference in cancer cell compared to normal cell mitochondria is the greater mitochondrial inner trans-membrane potential ( ⁇ m,i) in cancer cells.
  • ⁇ m,i mitochondrial inner trans-membrane potential
  • the ⁇ m,i is increased to greater negative values ( ⁇ 150 to ⁇ 170 mV, negative inside the matrix) in carcinoma cells (Summerhayes, I. C., Lampidis, T. J., Bernal, S. D., Nadakavukaren, J. J., Nadakavukaren, K. K., Shepherd, E. L. and Chen, L. B. (1982) Unusual retention of rhodamine 123 by mitochondria in muscle and carcinoma cells.
  • acetoin undergoes an ATP dependent reaction, almost doubling the reaction rate to produce citrate in tumour cells (Baggetto, L. G. and Lehninger, A. L. 1987. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes. Biochem Biophys Res Commun. 145:153-159; Baggetto, L. G. and Testa-Parussini, R. 1990. Role of acetoin on the regulation of intermediate metabolism of Ehrlich ascites tumor mitochondria: its contribution to membrane cholesterol enrichment modifying passive proton permeability.
  • the enhanced glycolytic activity due to very high energetic demand increases cytoplasmic levels of lactic acid production in cancer cells.
  • these cells activate plasma membrane proton pumps causing extracellular acidification.
  • the pH of the tumour interstitium is 6.2-6.5, while the pH of normal tissue interstitium is neutral (Gerweck, L. E. 2000.
  • the pH difference between tumor and normal tissue offers a tumor specific target for the treatment of cancer.
  • Tumor pH controls the in vivo efficacy of weak acid and base chemotherapeutics. Mol Cancer Ther. 5:1275-1279).
  • the major type of proton pumps used by cancer cells to maintain their neutral cytosolic pH is the class V ATPase (Sennoune, S. R., Luo, D. and Martinez-Zaguilan, R. 2004. Plasmalemmal vacuolar-type H+-ATPase in cancer biology. Cell Biochem Biophys. 40:185-206).
  • This ATPase has relatively low activity in non-malignant cells, while its activity is increased in cancer cells (Izumi, H., Torigoe, T., Ishiguchi, H., Uramoto, H., Yoshida, Y, Tanabe, M., Ise, T., Murakami, T., Yoshida, T., Nomoto, M.
  • ⁇ TOS mitocan ⁇ -tocopheryl succinate
  • ⁇ TOS mitocan ⁇ -tocopheryl succinate
  • ⁇ TOS a compound with pKa of 5.6
  • ⁇ -Tocopheryl succinate an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability.
  • the vitamin E analogue is a weak acid, of which ⁇ 98% is deprotonated at neutral pH with 10-15-fold higher percentage in the protonated form at the acidic pH of 6.2-6.4 of the tumour interstitium. Since there are no known transporters for compounds like ⁇ -TOS, presumably it crosses the plasma membrane to freely diffuse inside the cells and discharge its apoptogenic activity. Accordingly, the present inventors have found that when the pH of the tissue culture medium was more acidic (pH ⁇ 6.2), it resulted in ⁇ 3-times greater apoptogenic efficacy of ⁇ -TOS against T lymphoma cells compared to media at pH of 7.2.
  • the sixth class of mitocans listed in Table I includes molecules that are delocalized lipophilic cations which accumulate at much greater concentrations in the mitochondrial matrix than in the cytoplasm of cells (Smith, R. A., Porteous, C. M., Gane, A. M. and Murphy, M. P. 2003. Delivery of bioactive molecules to mitochondria in vivo. Proc Natl Acad Sci USA 100:5407-5412). These agents are selectively accumulated in the mitochondrial matrix of cancer cells because of their greater transmembrane potentials across the plasma membrane as well as their more polarized mitochondria with a much greater ⁇ m,i than that in non-malignant cells (Davis, S., Weiss, M. J., Wong, J.
  • the target for the lipophilic cation-based mitocans may be one of the inhibitory binding sites on ATPase (Gledhill, J. R. and Walker, J. E. 2005. Inhibition sites in F1-ATPase from bovine heart mitochondria. Biochem J. 386:591-598).
  • One of the earliest members of this class of compounds to be identified for its anti-cancer activity was rhodamine-123 (Bernal, S. D., Lampidis, T. J., Summerhayes, I. C. and Chen, L. B. 1982. Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro. Science 218:1117-1119; Bernal, S.
  • Rose Bengal works in a similar fashion to rhodamine 123, and Rose Bengal is likewise currently in clinical trials as a therapy for metastatic melanoma and recurrent breast cancer, causing complete remissions in some patients (Provectus PV-10-MM-01, www.ClinicalTrials.gov).
  • the drug F16 is a mechanistically more characterized example of this mitocan class and was shown to increase ROS production, depolarize mitochondria as a weak protonophore and collapse ⁇ m,i leading to mitochondrial permeability transition and selective apoptosis of cancer cells when applied in the micromolar range (Fantin, V. R., Berardi, M. J., Scorrano, L., Korsmeyer, S. J. and Leder, P. (2002) A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth. Cancer Cell 2:29-42). F16 was also reported in the study to inhibit the growth of mammary tumours in mice.
  • a rhodocyanine dye analogue is another example of this type of mitocan that entered phase I clinical trials, although these were terminated due to renal toxicity (Britten, C. D., Rowinsky, E. K., Baker, S. D., Weiss, G. R., Smith, L., Stephenson, J., Rothenberg, M., Smetzer, L., Cramer, J., Collins, W., Von Hoff, D. D. and Eckhardt, S. G. 2000. A phase I and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog MKT 077. Clin Cancer Res. 6:42-49).
  • MPP+ (1-methyl-4-phenylpyridinium) which acts as an inhibitor of mitochondrial respiration by blocking the NADH-ubiquinone oxidoreductase site of complex I. It is likely that MPP+ is also a protonophore (Davey, G. P., Tipton, K. F. and Murphy, M. P. 1992. Uptake and accumulation of 1-methyl-4-phenylpyridinium by rat liver mitochondria measured using an ion-selective electrode. Biochem J.
  • the amphipathic and positively charged ⁇ -helical pro-apoptotic peptide (KLAKLAK) 2 has also been included in this class of mitocans as a delocalized lipophilic cation.
  • the peptide must first be coupled to a targeted delivery system for surface receptor binding and uptake into cancer cells, before it is able to function as a mitocan (Ellerby, H. M., Arap, W., Ellerby, L. M.; Kain, R., Andrusiak, R., Rio, G. D., Krajewski, S., Lombardo, C. R., Rao, R., Ruoslahti, E., Bredesen, D. E. and Pasqualini, R.
  • anti-cancer compounds that have a propensity to accumulate within the mitochondria of cancerous cells and induce the death of those cells.
  • such compounds comprise a pro-oxidant moiety attached to a mitochondrial delivery moiety.
  • the pro-oxidant moiety generates reactive oxygen species within the mitochondria of cancerous cells and induces apoptosis of those cells.
  • such compounds comprise a pro-apoptotic moiety attached to a mitochondrial delivery moiety. The pro-apoptotic moiety induces apoptosis of those cells.
  • a compound for inducing the death of a cancerous cell comprising:
  • a pro-oxidant moiety for (i) generating reactive oxygen species within mitochondria of a cancerous cell and (ii) inducing apoptosis of the cancerous cell;
  • a delivery moiety for delivering the pro-oxidant moiety to the mitochondria of the cancerous cell.
  • a compound for preventing or treating cancer comprising:
  • a pro-oxidant moiety for (i) generating reactive oxygen species within mitochondria of a cancerous cell and (ii) inducing apoptosis of the cancerous cell;
  • a delivery moiety for delivering the pro-oxidant moiety to the mitochondria of the cancerous cell.
  • a method for inducing the death of a cancerous cell comprising the step of administering to a subject a therapeutically effective amount of a compound comprising:
  • a pro-oxidant moiety for (i) generating reactive oxygen species within mitochondria of a cancerous cell and (ii) inducing apoptosis of the cancerous cell;
  • a delivery moiety for delivering the pro-oxidant moiety to the mitochondria of the cancerous cell.
  • a method for preventing or treating cancer comprising the step of administering to a subject a therapeutically effective amount of a compound comprising:
  • a pro-oxidant moiety for (i) generating reactive oxygen species within mitochondria of a cancerous cell and (ii) inducing apoptosis of the cancerous cell;
  • a delivery moiety for delivering the pro-oxidant moiety to the mitochondria of the cancerous cell.
  • a compound in the preparation of a medicament for inducing the death of a cancerous cell comprising:
  • a pro-oxidant moiety for (i) generating reactive oxygen species within mitochondria of a cancerous cell and (ii) inducing apoptosis of the cancerous cell;
  • a delivery moiety for delivering the pro-oxidant moiety to the mitochondria of the cancerous cell.
  • a compound in the preparation of a medicament for the prevention or treatment of cancer comprising:
  • a pro-oxidant moiety for (i) generating reactive oxygen species within mitochondria of a cancerous cell and (ii) inducing apoptosis of the cancerous cell;
  • a delivery moiety for delivering the pro-oxidant moiety to the mitochondria of the cancerous cell.
  • a pharmaceutical or veterinary composition comprising the compound according to the first or second aspect of the present invention, or a physiologically acceptable salt thereof, and a physiologically acceptable carrier.
  • the compound may be in an isolated, purified, substantially purified, synthetic or recombinant form.
  • any suitable type of delivery moiety may be used.
  • the delivery moiety may target the pro-oxidant moiety to the intermembranous space, inner membrane or mitochondrial matrix of a mitochondrion, preferably the delivery moiety delivers the pro-oxidant moiety to the mitochondrial matrix of the cancerous cell.
  • the delivery moiety is a lipophilic cation that selectively accumulates within the mitochondrial matrix of a cancerous cell due to the large membrane potential of the cancerous cell.
  • a particularly preferred lipophilic cation is the triphenylphosphonium cation that is described in the specifications of International Patent Applications No. PCT/NZ98/00173 and PCT/NZ02/00154; and in James A M, Cochemé H M, Smith R A, Murphy M P. (Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools. J Biol. Chem. 2005 Jun. 3; 280(22):21295-312. Epub 2005 Mar. 23.), the entire contents of which are hereby incorporated by cross-reference.
  • the tetraphenylphosphonium cation is another example of a suitable delivery moiety.
  • Suitable delivery moieties potentially include gold-phosphine or gold-carbine complexes as described in the publication by Barnard et al. (Barnard P J, Baker M V, Berners-Price S J, Day D A. Mitochondrial permeability transition induced by dinuclear gold(I)-carbene complexes: potential new antimitochondrial antitumour agents. J. Inorg. Biochem. 2004 October; 98(10):1642-7), the entire contents of which are hereby incorporated by cross-reference
  • pro-oxidant moiety Any suitable type of pro-oxidant moiety may be used and the moiety may generate reactive oxygen species in any suitable way. Examples of preferred pro-oxidant moieties (mitocans) are listed in Table I above.
  • the compound may have more than one pro-oxidant moiety and the moieties may disrupt/target different regions/components of the respiratory chain.
  • the pro-oxidant moiety interacts with mitochondrial complex II. More preferably, the pro-oxidant moiety binds to a ubiquinone-binding site of complex II and can readily displace the natural substrate ubiquinone, ubisemiquinone or ubiquinol (coenzyme Qs) or other quinones or related compounds preferentially interacting with complex II.
  • substrates are specified, for example, in Briere J J, Schlemmer D, Chretien D, Rustin P. (2004) Quinone analogues regulate mitochondrial substrate competitive oxidation. Biochem Biophys Res Commun. April 16; 316(4):1138-42, Tan A K, Ramsay R R, Singer T P, Miyoshi H.
  • Apoptosis may occur solely as a result of the increased levels of reactive oxygen species in the mitochondria of the cancerous cell, or the pro-oxidant moiety, delivery moiety or entire compound may be further pro-apoptotic by way of activating mitochondrial dependent cell death signalling pathways within the cell.
  • the pro-oxidant moiety generates reactive oxygen species by way of binding to complex II and is further pro-apoptotic by way of activating mitochondrial dependent cell death signalling pathways.
  • the compound is cleaved, processed or otherwise metabolised in non-cancerous cells to a harmless form lacking pro-oxidant activity.
  • the pro-oxidant moiety is a pro-oxidant vitamin E analogue.
  • the present inventors have previously found that pro-oxidant vitamin E analogues can bind to complex II and disrupt electron transfer to ubiquinone. This has been described in the specification of International Patent Application No. PCT/AU2007/001371, the entire contents of which are incorporated herein by cross-reference.
  • the inventors have also previously found pro-oxidant vitamin E analogues to be pro-apoptotic.
  • pro-oxidant vitamin E analogues can be processed to harmless anti-oxidant forms in non-cancerous cells.
  • a “pro-oxidant vitamin E analogue” is defined herein as a vitamin E analogue that, when located in mitochondria of a cancerous cell, is redox-silent and is capable of binding to a ubiquinone binding site of complex II and trigger the production of oxygen by-products of metabolism that can cause damage to the cell.
  • An example of a pro-oxidant vitamin E analogue is ⁇ -tocopheryl succinate ( ⁇ -TOS).
  • an “anti-oxidant vitamin E analogue” is a vitamin E analogue that has anti-oxidant (redox) activity when located in mitochondria of a cancerous cell, eg. ⁇ -tocopherol ( ⁇ -TOH). Hence, the biological activities of pro-oxidant vitamin E analogue and anti-oxidant vitamin E analogue are directly opposed.
  • the compound may be used to induce the death of any type of cancerous cell in a subject, eg. lung, liver, kidney, brain, prostate, breast, ovary, lymphoid, skin, eye, colon, gastric, oral squamous, and hematopoietic systems.
  • a subject eg. lung, liver, kidney, brain, prostate, breast, ovary, lymphoid, skin, eye, colon, gastric, oral squamous, and hematopoietic systems.
  • ⁇ -TOS has been previously found by the present inventors to efficiently kill erbB2-low or -high cancer cells.
  • ⁇ -TOS has also been previously found by the inventors to treat mesothelioma. This has been described in the specification of International Patent Application No. PCT/AU2007/001371.
  • ⁇ -TOS has been previously found by the present inventors to induce the death of both normoxic and hypoxic cancerous cells.
  • ⁇ -TOS has the advantage that it may be used to induce the death of both early and late stage tumours in a subject.
  • the subject for treatment may be a human, mammal or animal.
  • the subject is a human or other type of mammal.
  • the compound may be included in the composition as pharmaceutically or veterinarially acceptable derivatives thereof.
  • derivatives of the compound includes salts, coordination complexes with metal ions such as Mn 2+ and Zn 2+ , esters such as in vivo hydrolysable esters, free acids or bases, hydrates, or pro-drugs.
  • Compounds having acidic groups such as phosphates or sulfates can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2-hydroxyethyl)amine.
  • Salts can also be formed between compounds with basic groups, such as amines, with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid.
  • inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid
  • organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid.
  • Compounds having both acidic and basic groups can form internal salts.
  • Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques that will be well known to those of skill in the art.
  • compositions for administration to a human subject will include between about 0.01 and 100 mg of the compound per kg of body weight and more preferably between about 0.1 and 10 mg/kg of body weight.
  • the serum level of the compound is preferably in the vicinity of its IC 50 value, approximately 40-50 ⁇ M.
  • composition may be administered to the subject in any suitable way, including: parenterally, topically, orally, by inhalation spray, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the carrier may comprise any suitable diluent, adjuvant, excipient, buffer, stabiliser, isotonicising agent, preservative or anti-oxidant. It will be appreciated that the carrier should be non-toxic and should not interfere with the efficacy of the compound. The precise nature of the carrier or any other additive to the composition will depend on the route of administration and the type of treatment required. See, for example, Alfonso R. Gennaro. Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000, and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y., the entire contents of which are incorporated herein by reference. Pharmaceutical compositions may be produced, for instance, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Sterile injectable forms of the composition may be aqueous or oleaginous suspension. Such forms will be known to those of skill in the art.
  • the composition may be in the form of a parenterally acceptable aqueous solution which has suitable pH, isotonicity and stability.
  • Orally acceptable dosage forms of the composition include capsules, tablets; pills, powders, liposomes, granules, spheres, dragees, liquids, gels, syrups, slurries, suspensions and the like. Suitable oral forms will be known to those of skill in the art.
  • a tablet can include a solid carrier such as gelatine or an adjuvant or an inert diluent.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, a mineral oil or a synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations will generally contain at least 0.1 wt % of the compound and preferably up to about 25 wt %, depending on its solubility in the given carrier.
  • the composition may be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including cancers of the eye, the skin, or the lower intestinal tract.
  • the composition may be applied in the form of a solution, suspension, emulsion, ointment, cream, lotion, paste, gel, foam, or aerosol. Suitable topical forms will be known to those of skill in the art.
  • the composition may include a delivery vehicle for delivering the compound to a particular organ, tissue or type of cancer, and/or for ensuring that the compound is able to be, for instance, absorbed through the skin or ingested through the gut without loss of biological efficacy.
  • Delivery vehicles may comprise, for example, lipids, polymers, liposomes, emulsions, antibodies and/or proteins. Liposomes are particularly preferred for delivering the compound through the skin to, say, treat mesothelioma.
  • composition may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the compound.
  • sustained-release materials are available and well known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compound for about 1 to 20 weeks.
  • the compound may be in the form of a pro-drug.
  • the pro-drug may have protective groups such that the activity of the compound is not compromised when the composition is taken, say, orally.
  • the pro-drug may deliver the active compound to a particular organ or cell type. Suitable pro-drug forms and protective groups will be known to those of skill in the art.
  • an adduct of ⁇ -TOS is linked to the heptapeptide LTVSPWY, for targeting cancer cells over-expressing the receptor tyrosine kinase, erbB2.
  • a subject may be administered the composition comprising the compound together with one or more other actives to achieve an optimal prophylactic or therapeutic effect.
  • the actives may be, for example, alkylating agents, angiogenesis inhibitors, anti-androgens, anti-estrogens, anti-metabolites, apoptosis agents, aromatase inhibitors, cell cycle controlling agents, cell stressor, cytotoxics, cytoprotectant, hormonals, immunotherapy agents, kinase inhibitors, monoclonal antibodies, platinum agents, a respiratory inhibitor, retinoid, signal transduction inhibitors, taxanes and topoisomerase inhibitors.
  • Particularly preferred agents include glycolytic inhibitors such as 2-deoxyglucose and 3-BP.
  • Mitocans mitochondria targeted anti-cancer drugs as improved therapies and related patents.
  • cancer cells are rendered more sensitive to killing by the combination of ⁇ -TOS and 3-BP (as well as with other drug combinations) compared with either drug used alone.
  • the composition is administered parenterally or topically.
  • the particularly preferred pro-oxidant vitamin E analogue moieties are ⁇ -tocopheryl succinate, ⁇ -tocopheryl maleate, ⁇ -tocopheryl maleyl amide, and 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid ( ⁇ -tocopheryloxyacetic acid).
  • the preferred carrier for the esters ⁇ -tocopheryl succinate, ⁇ -tocopheryl maleate and ⁇ -tocopheryl maleyl amide is a transdermally applicable cream, such as the liposome-based cream “Lipoderm”.
  • the non-hydrolysable ether analogue, ⁇ -tocopheryloxyacetic acid is preferably delivered orally.
  • a compound for inducing the death of a cancerous cell comprising:
  • a pro-apoptotic moiety for inducing apoptosis of a cancerous cell
  • a delivery moiety for delivering the pro-apoptotic moiety to mitochondria of the cancerous cell.
  • a compound for preventing or treating cancer comprising:
  • a pro-apoptotic moiety for inducing apoptosis of a cancerous cell
  • a delivery moiety for delivering the pro-apoptotic moiety to mitochondria of the cancerous cell.
  • a method for inducing the death of a cancerous cell comprising the step of administering to a subject a therapeutically effective amount of a compound comprising:
  • a pro-apoptotic moiety for inducing apoptosis of a cancerous cell
  • a delivery moiety for delivering the pro-apoptotic moiety to mitochondria of the cancerous cell.
  • a method for preventing or treating cancer comprising the step of administering to a subject a therapeutically effective amount of a compound comprising:
  • a pro-apoptotic moiety for inducing apoptosis of a cancerous cell
  • a delivery moiety for delivering the pro-apoptotic moiety to mitochondria of the cancerous cell.
  • a pharmaceutical or veterinary composition comprising the compound according to the eighth or ninth aspect of the present invention, or a physiologically acceptable salt thereof, and a physiologically acceptable carrier.
  • the compound may be in an isolated, purified, substantially purified, synthetic or recombinant form.
  • delivery moiety Any suitable type of delivery moiety may be used.
  • the delivery moiety may be as described above.
  • pro-apoptotic moiety is a pro-oxidant moiety as described above.
  • Examples of preferred pro-oxidant moieties are listed in Table I above as well as in Tables II-IV and FIG. 1 .
  • FIG. 1 Compounds used in the study.
  • the compounds used were ⁇ -TOH, ⁇ -TOS, VE 11 S (hereafter referred to as “VES”), Mito VE 3 S, Mito VE 3 S, Mito VE 7 S, Mito VE 9 S and Mito VE 11 S (hereafter referred to as “MitoVES”), Mito VE 11 AE (hereafter referred to as “Mito VEAE”), Mito VE 11 F [not shown] (hereafter referred to as “MitoVEF”), MitoVE 11 M [not shown] (hereafter referred to as “MitoVEM”), and VES4TPP.
  • ⁇ -TOH and ⁇ -TOS were obtained from Sigma, VES, MitoVES, MitoVEAE, MitoVEF, MitoVEM and VES4TPP were synthesised as described in the section entitled General Materials and Methods.
  • FIG. 2 MitoVES causes apoptosis selectively in malignant cells.
  • FIG. 3 Apoptosis triggered by MitoVES is dependent on ROS and the intrinsic pathway.
  • FIG. 4 MitoVES accumulates in the inner mitochondrial membrane and interferes with the coenzyme Q binding site of complex II.
  • FIG. 5 Molecular modelling of MitoVES binding in Complex II.
  • FIG. 6 MitoVES causes apoptosis in proliferating but not arrested endothelial cells due to accumulation of ROS.
  • EAhy926 cells were seeded in 24-well plate so that they would acquire after an overnight recuperation ⁇ 50% or 100% confluency, while their ⁇ 0 counterparts were seeded at 50% confluency.
  • the proliferating and confluent cells were then exposed to MitoVES at concentrations and for times shown, and assessed for apoptosis level by the Annexin V-binding method (A) and for ROS accumulation using the fluorescent probe DHE (B).
  • the inset in panel A shows the cell cycle distribution in proliferating and confluent EAhy926 cells.
  • Panel C shows ROS accumulation in proliferating and confluent EAhy926 cells using EPR spectroscopy.
  • FIG. 7 MitoVES inhibits angiogenesis in vitro.
  • Wound-healing A-F and tube-forming activity (G, H) were assessed using the EC EAhy926 cells.
  • wound-healing activity the cells were seeded in 35-mm Petri dishes and allowed to reach 100% confluency. The ‘injury’ was then performed, resulting in a denuded gap of 0.4-0.5 mm.
  • Wound-healing activity in control cells and cells supplemented with 1, 5 or 10 ⁇ M MitoVES was assessed on the basis of proliferation and migration of cells into the denuded zone using a light microscope equipped with a grid and a digital camera (A).
  • Panel B shows morphology of the cells in zones of arrested ECs 20 h after the injury for the control culture and from cells exposed to 10 ⁇ M MitoVES.
  • panel C representative images of injured control cells and cells treated with 10 ⁇ M MitoVES at different times are presented.
  • Panel D shows the healing rate for the different conditions derived from the slopes of the individual curves in panel A.
  • panel E the level of apoptosis is shown at 20 h after the injury for control cultures and for cells exposed to MitoVES. EAhy926 cells were seeded at ⁇ 10 5 per well in Matrigel-coated 24-well plates and allowed for 24 h to form tubes at the absence or presence of MitoVES.
  • Panel G shows representative images of the Matrigel cultures of control cells and cells treated for 20 h with 5 ⁇ M MitoVES. At 20 h of treatment, the cells were retrieved from the Matrigel and assessed for apoptosis using the Annexin V binding method (H). Parental EAhy926 cells and their ⁇ 0 counterparts were seeded in Petri dishes at confluency, ‘injured’ as described above, and the wound-healing activity assessed in the absence or presence of 10 ⁇ M MitoVES (MVES) (I; see symbols above panel L).
  • MVES 10 ⁇ M MitoVES
  • the healing rate was estimated from the slopes in panel I and plotted in mmol/h (J). At 20 h, the cells were assessed for the level of apoptosis (K).
  • Panel L documents tube-forming activity in Matrigel of the ⁇ 0 EAhy 926 cells in the absence or presence of 5 ⁇ M MitoVES. Data shown are derived from three independent experiments and are presented as mean values ⁇ S.D., the micrographs are representative images of at least three independent experiments.
  • FIG. 8 MitoVES is a highly effective drug for killing populations of the human breast cancer cell line, MCF7, enriched for cancer stem cells.
  • Adherent MCF7 cells and the corresponding mammosphere cells were cultured and assessed for morphological changes by microscopy (A) and for expression levels of markers as indicators of “sternness” by RT-PCR (B) and flow cytometry (C), including the expression of CD44 and CD24 (D).
  • the adherent MCF7 cells (E) and the MS cells (F) were treated with 5 ⁇ M MitoVES, 50 ⁇ M ⁇ -TOS or 10 ⁇ M parthenolide and assessed for their sensitivity to drug induced cell death.
  • Panel G shows the initial levels of cell death by apoptosis in MS cells exposed to MitoVES and panel H shows the histogram analysis of the MS cells exposed to 5 ⁇ M MitoVES for 3 h and then analysed for annexin V-FITC binding.
  • Adherent MCF7 cells (I) and MS cells (J) were treated for 3 h with ⁇ -TOS (50 ⁇ M) or MitoVES homologues containing different lengths in the aliphatic side chain (5 ⁇ M each, either 11, 7 or 5 carbon chain in length) and the treated cells assessed for production of reactive oxygen species (ROS) by flow cytometry.
  • ROS reactive oxygen species
  • FIG. 9 MitoVES treatment completely blocks progression of breast cancer tumours in transgenic FVB/N c-neu mice that form spontaneous ductal breast carcinomas.
  • the transgenic FVB/N c-neu female mice with spontaneous small breast carcinomas were treated with MitoVES at 3 ⁇ mol per mouse per dose and the tumour volume was assessed by repeated ultrasound imaging, monitoring tumour growth over several weeks.
  • IL-2 intratumoral immunotherapy enhances CD8+ T cells that mediate destruction of tumor cells and tumor-associated vasculature: a novel mechanism for IL-2.
  • J. Immunol. 171, 505150-505163 (2003) human non-malignant mesothelial cells Met-5A, rat ventricular myocyte-like cells HL-1 (Claycomb, W. C. et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc. Natl. Acad. Sci. USA 95, 2979-2984 (2001)) and H9c2, and the human endothelial-like cells EAhy926 (Edgell, C. J., McDonald, C.
  • HL-1 cells maintained in fibronectin/gelatine-coated dishes, were grown in the Claycomb medium supplemented with noradrenalin (Claycomb, W. C. et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc. Natl. Acad. Sci.
  • EAhy926 cells were grown in complete DMEM supplemented with HAT (Edgell, C. J., McDonald, C. C. & Graham, J. B. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl. Acad. Sci. USA 80, 3734-3737 (1983)).
  • Cells deficient in mtDNA were prepared as detailed in Weber, T. et al., (Mitochondria play a central role in apoptosis induced by ⁇ -tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling.
  • VE 11 S (“VES”), MitoVE 3 S, MitoVE 5 S, MitoVE 7 S, MitoVE 9 S, MitoVE 11 S (“MitoVES”), MitoVE 11 AE (“MitoVEAE”), MitoVE 11 F (“MitoVEF”), MitoVE 11 M (“MitoVEM”), and VES4TPP towards cancer cells was assessed on the basis of IC 50 , as detailed in Turanek, J., et al. ((2008). Liposomal formulation of vitamin E analogs as an efficient and selective anti-cancer treatment. Clin. Cancer Res. (submitted)). Apoptosis was assessed using the Annexin V method (Weber, T. et al.
  • Mitochondria play a central role in apoptosis induced by ⁇ -tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 42, 4277-4291 (2003)) and dissipation of the mitochondrial inner transmembrane potential was estimated using the polychromatic probe JC-1 (Molecular Probes) (Weber, T. et al. Mitochondria play a central role in apoptosis induced by ⁇ -tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 42, 4277-4291 (2003)).
  • Cell proliferation was determined, using an ELISA colorimetric kit (Roche) to determine the number of cells in S phase of the cell cycle, based on DNA incorporation of 5-bromo-2-deoxyuridine (BrdUrd) using the manufacturer's protocol.
  • PrdUrd 5-bromo-2-deoxyuridine
  • cells were plated in 24-well plates so that they reached ⁇ 50%, 70%, and 100% confluency after 24-h recuperation. Cells were then harvested and resuspended in buffer containing sodium citrate (1%), Triton X-100 (0.1%), RNase A (0.05 ⁇ g/mL), and propidium iodide at 5 ⁇ g/mL, incubated in the dark for 30 min at 4° C. and analyzed by flow cytometry.
  • the reaction components included NADH, 0.5 mM; succinate, 5 mM; KCN, 10 mM; DCIP, 50 ⁇ M; phenazine methosulphate (PMS), 50 ⁇ M.
  • PMS phenazine methosulphate
  • the quartz cell was then placed into the cavity of the Bruker EMX bench-top spectrometer set at 293 K with the following spectrometer parameters: field sweep 10 mT, microwave power 20 mW, microwave frequency 100 kHz, modulation amplitude, 0.1 mT, sweep time 83.9 s.
  • the detection limit of the stable nitroxide (TEMPO) under identical conditions was ⁇ 50 nM.
  • the cells were seeded in 3.5 mm Petri dishes and allowed to reach complete confluence. Using a sterile yellow pipette tip, the monolayer of the cells was ‘wounded’ by removal of cells, generating a denuded area of 0.4-0.5 mm across. Regrowth of cells (wound healing) in the presence of ⁇ -TOS or ⁇ -TEA was assessed by following the kinetics of narrowing the denuded gap in the microscope equipped with a grid in the eyepiece and healing expressed as the ‘rate of filling the gap’ as the rate of healing in ⁇ m/h.
  • the surface of Matrigel was gently overlayed by a suspension of EAhy926 cells trypsinized from a proliferating culture, so that 200 ⁇ l of the complete cell media including the HAT supplement with 5 ⁇ 10 5 cells was added to each well.
  • the polygonal structures made by a network of EAhy926 capillaries, established.
  • the cells were treated by addition of MitoVES, added to the cell suspension just before it was transferred to the wells or after the tubes were established.
  • Tube-forming activity was estimated by counting the number of complete capillaries interconnecting individual points of the polygonal structures in a selected field in a light microscope. Three fields in the central area of the well were chosen randomly in every well. The number of such capillaries in control cultures was considered 100%.
  • the number of complete capillaries was counted at various times after the onset of the experiment to obtain the kinetics of inhibition of tube-forming activity of EAhy926 cells.
  • the crystal structure of mitochondrial respiratory membrane protein Complex II from porcine heart was obtained from the Brookhaven Protein Databank (code 1ZOY) (Sun F, Huo X, ZhaiY, Wang A, Xu J, Su D, Bartlam M, Rao Z. 2005. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121:1043-1057).
  • the Complex contains four proteins. Three subunits in this Complex, the iron-sulfur protein (Chain B), the large (Chain C) and small (Chain D) trans-membrane proteins are involved in the binding to UbQ.
  • a BLAST search from the NCBI website revealed that the sequence identity between porcine and human Complex II is very high, 97% for the iron-sulfur protein, 90% for the large trans-membrane protein and 94% for the small trans-membrane protein.
  • the protein structure was prepared for docking using AutoDock Tools (Sanner M F (1999) Python: a programming language for software integration and development, J Mol Graphic Mod 17: 57-61) with the heteroatoms being removed first. Polar hydrogens were added to the structure and Kollman United Atom charges were used for the protein atoms.
  • UbQ5 was built from the crystal structure coordinates of the bound UbQ (1ZOY) using InsightII (Accelrys, 2001).
  • MitoVE 11 S was built from the crystal structure MOPHLB01 retrieved from the Cambridge Structural Database (Allen F H (2002) The Cambridge Structural Database: a quarter of a million crystal structures and rising.
  • FIG. 2 shows that mitochondrially targeted redox-silent analog of vitamin E has a considerably higher apoptogenic effect against cancer cells and anti-cancer activity compared to its untargeted ⁇ -TOS counterpart, whilst maintaining its selectivity for cancer cells.
  • MitoVE 11 S Mito- ⁇ -TOS was found to be up to 50-fold more apoptogenic than the prototypic ⁇ -TOS in cancer cells.
  • IC 50 values of ⁇ -TOS, VES4TPP and MitoVE 11 S (labelled “MitoVES”) for different malignant and non-malignant cells are shown in Table V below.
  • the apoptosis assays of FIG. 2 show that MitoVE 11 S causes apoptosis selectively in malignant cells but not the normal equivalent cell types, with the exception of dividing endothelial cells.
  • micrographs of ⁇ -TOS- and MitoVE 11 S-treated Jurkat cells in panel J reveal typical hallmark signs of apoptosis.
  • FIG. 3B shows that MitoVE 11 S treatment of human Jurkat T-lymphoma cells induced apoptosis predominantly via the mitochondrial pathway because a Bax/Bak double knockout cell line proved extremely resistant to MitoVE 11 S and both of these BH3 only proteins are known to be required for mitochondrial outer membrane permeabilization during apoptosis via the intrinsic pathway.
  • MitoVES causes apoptosis in proliferating but not arrested endothelial cells due to accumulation of ROS, thus revealing its potential as a potent anti-angiogenic agent.
  • MitoVE 11 S is an effective inhibitor of angiogenesis and has direct anticancer effects by killing cancer cells via apoptosis.
  • FIG. 7 shows that MitoVES (MitoVE 11 S) inhibits angiogenesis in vitro, thus again affirming its potential as a potent anti-angiogenic agent.
  • MitoVES MitoVE 11 S
  • MitoVE 11 S has a very potent anti-angiogenic activity in targeting and killing proliferating endothelial cells.
  • MitoVE 11 S shows a surprising 5-fold greater potency as an antiangiogenic drug.
  • about 5-10 micromolar MitoVE 11 S compared to about 25-50 micromolar ⁇ -TOS is needed.
  • the ⁇ -TOS level was taken from Lan-Feng Dong, Emma Swettenham, Johanna Eliasson, Xiu-Fang Wang, Mikhal Gold, Yasmine Medunic, Marina Stantic, Pauline Low, Lubomir Prochazka, Paul K.
  • the B1, B9 and B10 cell lines were transformed by stable transfection with GFP-H-Ras using the pEGFP-C3-H-Ras plasmid (Baysal B E, Ferrell R E, Willett-Brozick J E, Lawrence E C, Myssiorek D, Bosch A, van der Mey A, Taschner P E, Rubinstein W S, Myers E N, Richard C W 3rd, Cornelisse C J, Devilee P, Devlin B. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science. 2000 Feb. 4; 287(5454):848-51).
  • the transfected cells were then subjected to clonal selection for those expressing the highest levels of H-Ras-EGFP (see FIG. 4B ).
  • the different ras-transformed cell lines were then exposed to the MitoVES analogue MitoVE 11 S and assessed for apoptosis efficacy, propensity to accumulate ROS and for SDH activity.
  • the predicted position of MitoVE 11 S is shown as the sticks and the translucent grey box is drawn to indicate approximately where the membrane bilayer would exist.
  • the model identifies the position proposed for MitoVE 11 S binding and docking into the Qp ubiquinone site of Complex II with the triphenylphosphonium ion protruding out on the membrane surface sitting in the mitochondrial matrix near the base of the structure of the SDH enzyme head group outside.
  • MitoVES is an Efficient Drug for Killing Breast Cancer Stem Cells
  • cancer stem cells which can be the source for repopulating a tumour. This also highlights the difficulty with treating cancers, because any treatment that kills the bulk of a tumour, but leaves the stem cells alive in the body will fail because the tumour will regrow (Lou and Dean, Oncogene 2007).
  • Cancer stem cells are also highly significant targets because they are commonly resistant to therapy (O'Brien et al. 2008, Li et al., 2008) and are drug resistant, expressing the Multi Drug Resistance (MDR)/ABC transporter glycoproteins on their cell membranes, involved in preventing chemotherapeutic drugs from accumulating in the cancer stem cells.
  • MDR Multi Drug Resistance
  • the cancer stem cells have also been found to be radiation resistant because they have greater DNA damage repair capacity (Neuzil et al., 2007 BBRC; Eyler and Ricj, 2008).
  • Cancer stem cells have been enriched by selective methods including purification based on their propensity to exclude dyes such as Hoechst 33342 providing a “side population” of cells when gated by fluorescence activated cell sorting (Patrawala L et al 2005, Wu and Alman 2008).
  • Another means for enriching for cancer stem cell populations involves the growth in culture of spheroids from tumour cells. This method has been shown to enrich for higher percentages of tumour initiating cells within such cultures (Grimshaw et al., 2008) with many of the properties of cancer stem cells, including radiation resistance (Phillips T M et al., 2007). Based on analyses of gene expression and protein marker expression on the cancer stem cell enriched populations, these cell types have become more characterised.
  • marker genes and proteins expressed by cancer stem cells are increased levels of the Notch/wnt/beta-catenin signalling pathway, ABC transporters, CD133 high, CD44 high and low levels of CD24 surface markers. These cells, based on their marker expression also correlate closely with the more “basal” tumour cell phenotypes isolated from aggressively lethal malignancies and analysed by gene expression profiling (for example, see Sorlie T et al., PNAS, 2001; review in Sotiriou and Pusztai, 2008).
  • this drug is selective and does not significantly affect normal stem cells or other normal cell types.
  • a drug that has been described is parthenolide, a sesquiterpene lactone derived from the Feverfew plant, which has been found to selectively kill leukemic stem cells (Guzman M L et al., 2005).
  • Another similar drug was the compound 4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione (TDZD-8, Guzman, M L et al, 2007).
  • TTZD-8 Guzman, M L et al, 2007
  • the present inventors made the surprising discovery that MitoVES was a highly effective drug for killing populations of the human breast cancer cell line, MCF7, enriched for cancer stem cells by growth as mammospheres (MS) in culture.
  • the results in FIG. 8 show the morphology of the adherent MCF7 cells and the corresponding MS cells, as well as analyses for expression of a number of markers for cancer stem cells. They found that adherent MCF7 cells, although sensitive to killing by ⁇ -TOS, were more so to the drug, MitoVES. However, these cells were not responsive to parthenolide, the agent previously reported to kill cancer stem cells, particularly leukemia stem cells.
  • MS cells derived from MCF7 cells showed low levels of sensitivity to parthenolide and were resistant to ⁇ -TOS.
  • MS cells with their high levels of sternness (indicated by cancer stem cell markers) were surprisingly very sensitive to the drug MitoVES, such that there was >90% apoptosis within 5-6 h after adding the drug to the MS cells.
  • the inventors also found that ⁇ -TOS caused significant production of ROS in adherent MSFT cells but much lower levels in the MS cells.
  • MitoVES caused greater ROS accumulation in adherent MCF7 cells than did ⁇ -TOS, and even higher levels in the MS cells.
  • MitoVES were less efficient than MitoVE 11 S at promoting ROS accumulation, both in the adherent MCF7 cells and in the corresponding MS cultures.
  • MitoVES was a highly effective anti-cancer drug in vivo.
  • the transgenic FVB/N c-neu mice that form spontaneous ductal breast carcinomas due to high level of expression of the oncogene, HER2 were used as a cancer model.
  • the inventors found that MitoVES treatment completely blocked progression of breast cancer tumours arising in these animals ( FIG. 9 ) at concentrations some 10-fold lower than those needed for corresponding activity of ⁇ -TOS. Importantly, no obvious sign of toxicity was observed in any of the treated animals.
  • untargeted mitocans can be modified by addition of a cationic group that presumably anchors them in the luminal leaflet of the inner mitochondrial membrane, thus maximizing their activity.
  • This is epitomized by mitochondrially targeted analogs of vitamin E that show a considerably higher apoptogenic effect against cancer cells including tumor-initiating cells, and anti-cancer activity compared to their untargeted counterparts, whilst maintaining their selectivity for cancer cells.
  • MitoVES (MitoVE 11 S) is surprisingly much greater in potency than ⁇ -TOS, ⁇ up to 50 fold more active on cancer cells in killing them selectively than ⁇ -TOS;
  • MitoVES is more specific in inducing the mitochondrial pathway for cell death than ⁇ -TOS;
  • MitoVES has a very potent anti-angiogenic activity in targeting and killing proliferating endothelial cells, but MitoVES (MitoVES) shows a surprising 5-fold greater potency as an antiangiogenic drug than ⁇ -TOS; and
  • MitoVES is likely to have very broad application in cancer therapy, in view it killing mesothelioma, breast cancer, colon cancer, lymphoma cell lines and cancer stem cells.

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