Physiologically, mitochondria perform a variety of key cellular regulatory processes, including ATP production, intracellular Ca2+ regulation, reactive oxygen species (ROS) generation and detoxication, and apoptosis (Tzagoloff (1982) Mitochondria, Plenum Press, New York). Mitochondria use approximately 90% of the consumed O2 for oxidative phosphorylation and ATP synthesis. Thus, the proteins involved in the mitochondrial electron transport chain are probable sites for ROS generation. Intracellular glutathione, glutathione peroxidase, glutathione transferases, catalase, superoxide dismutase, and a variety of other antioxidant defenses keep ROS concentrations in check, which allows cells to function homeostatically thereby preventing oxidative stress (Abid, et al. (2004) J. Biol. Chem. 279:44030-44038; Zhang, et al. (2002) J. Virol. 76:355-363; Li, et al. (2000) Cancer Res. 60:3927-3939; Warner, et al. (1996) Am. J. Physiol. 271:L150-L158; Schiavone & Hassan (1988) J. Biol. Chem. 263:4269-4273). A shift in the balance between ROS generation and destruction to overproduction or decreased detoxication is associated with chronic diseases (Ross, et al. (1997) Am. J. Kidney Dis. 30:489-494).
The etiology of a range of diseases is associated with the generation of excess reactive oxygen species. Steady-state maintenance of ROS/antioxidant ratio is, however, essential for cell signaling. Reactive oxygen species generated in cells include the superoxide anion radical (O2.−), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), hydroxyl radical (OH.), and singlet oxygen (1O2). These ROS are formed as a consequence of endogenous enzymatic and nonenzymatic reactions within the cell and within mitochondria. ROS may also be formed in response to external stimuli and chemicals.
ROS are generated by normal biochemical reactions in the cell. Leakage of electrons from the mitochondrial electron transport chain is a significant source of mitochondrial ROS, particularly superoxide (Boveris & Cadenas (1997) In: Oxygen, Gene Expression, and Cellular Function, Clerch & Massaro, eds., Marcel Dekker, New York, pp. 1-25). Moreover, the TCA cycle enzymes α-ketoglutarate dehydrogenase and the pyruvate dehydrogenase complex also generate superoxide and H2O2 (Starkov, et al. (2004) J. Neurosci. 24:7779-7788). Superoxide is also generated by NADPH oxidase, which is found in phagocytic and nonphagocytic macrophages (Quinn & Gauss (2004) J. Leukoc. Biol. 76:760-781), and by xanthine dehydrogenase/oxidase (Rajagopalan (1997) In: Biotransformation, Guengerich, ed., Elsevier, New York pp. 165-178). Hydrogen peroxide is produced by mitochondrial monoamine oxidase (Cashman (1997) In: Biotransformation supra pp. 69-96) and by the superoxide dismutase (MnSOD and Cu/ZnSOD)-catalyzed dismutation of superoxide (Fridovich (1995) Annu. Rev. Biochem. 64:97-112). In addition, peroxisomal acyl-CoA oxidases also generate hydrogen peroxide (Reubsaet, et al. (1988) Biochim. Biophys. Acta 958:434-442). The myeloperoxidase-catalyzed generation of hypochlorous acid is an important line of defense against invading microorganisms (Winterbourn, et al. (2000) Curr. Opin. Hematol. 7:53-58).
There is no known enzymatic route to detoxify the hydroxyl radical, which may be produced by the Haber-Weiss reaction in the presence of transition metals, particularly iron. Singlet oxygen may be formed by photodynamic processes or from the reaction of hypochlorous acid with hydrogen peroxide.
Reactive nitrogen species have also been implicated in cell damage and death. Nitric oxide synthase catalyzes the synthesis of the radical species nitric oxide (NO.), which may react with superoxide to give peroxynitrite (ONOO−). ROS generation may also be associated with external stimuli. UV and high-energy irradiation, the metabolism of some xenobiotics, air pollutants (O3), and the redox cycling of quinones and nitroaromatics are all associated with ROS generation.
The balance between these sources of ROS depends on the physiologic and pathophysiologic states of the organism, and it is often difficult to pinpoint the source of ROS generation. It is, however, known that ROS exert important regulatory functions (Dröge (2002) Physiol. Rev. 82:47-95). Hence, a basal or tonal concentration of ROS, especially at the level of the mitochondrion, is essential for basic cell signaling processes. In other words, all ROS are not created equal, and compartmentalization and concentration gradients are highly important. Abolishment of all cellular ROS by vigorous use of antioxidants may not be beneficial and, indeed, may prove harmful. The requirement for a basal ROS tone may explain why many antioxidant-based therapies have failed.
Mitochondria are attractive targets for drug-delivery strategies because of their roles in cellular energy metabolism, programmed (apoptotic) cell death, calcium homeostasis, and cell signaling. Moreover, mutations in mitochondrial DNA are associated with a range of human diseases, again making mitochondria attractive targets for mitochondrial gene therapy. Hence, strategies have been developed to target small and large molecules with therapeutic potential to mitochondria (Muratovska, et al. (2001) Adv. Drug Deliv. Rev. 49:189-198; Weissig (2003) Crit. Rev. Ther. Drug Carrier Syst. 20:1-62; Weissig, et al. (2004) Drug Design Rev.-Online 1:15-28).
For example, the high potential gradient across the mitochondrial inner membrane can be exploited to deliver lipophilic cations to mitochondria. Cationic compounds, such as rhodamine 123 and tetraphenylphosphonium (TPP+), have been adopted for mitochondrial membrane potential determinations and a series of cationic antioxidants that preferentially accumulate in mitochondria have been developed (Ross, et al. (2005) Biochemistry (Moscow) 70:222-230). Further, a triphenylphosphonium-based, mitochondria-targeted mixture of ubiquinol (mitoquinol) and ubiquinone (mitoquinone), i.e., MitoQ (Kelso, et al. (2001) J. Biol. Chem. 276:4588-459), as well as MitoVit E (Smith, et al. (1999) Eur. J. Biochem. 263:709-716); MitoPBN (Murphy, et al. (2003) J. Biol. Chem. 278:48534-48545); MitoPeroxidase, a mitochondria-targeted analog of ebselen (Filipovska, (2005) J. Biol. Chem. 280:24113-24126); and glutathione choline ester (MitoGSH) and N-acetyl-L-cysteine choline ester (MitoNAC) have been synthesized for delivery of an antioxidant to mitochondria to selectively prevent mitochondrial oxidative damage.
USPTO Backgrounds
USPTO Backgrounds