Original Contribution
Lipophilic adamantyl- or deferasirox-based conjugates of desferrioxamine B have enhanced neuroprotective capacity: implications for Parkinson disease

https://doi.org/10.1016/j.freeradbiomed.2013.01.027Get rights and content

Abstract

Parkinson disease (PD) is a neurodegenerative disease characterized by death of dopaminergic neurons in the substantia nigra region of the brain. Iron content is also elevated in this region in PD and is implicated in the pathobiology of the disease. Desferrioxamine B (DFOB) is a high-affinity iron chelator and has shown efficacy in animal models of Parkinson disease. The high water solubility of DFOB, however, attenuates its ability to enter the brain. In this study, we have conjugated DFOB to derivatives of adamantane or the clinical iron chelator deferasirox to produce lipophilic compounds designed to increase the bioavailability of DFOB to brain cells. We found that the novel compounds are highly effective in preventing iron-mediated paraquat and hydrogen peroxide toxicity in neuronal-like BE2-M17 dopaminergic cells, primary neurons, and iron-loaded or glutathione-depleted primary astrocytes. The compounds also alleviated paraquat toxicity in BE2-M17 cells that express the PD-causing A30P mutation of α-synuclein. This protection was ∼66-fold more potent than DFOB alone and also more effective than other cell-permeative metal chelators, clioquinol and phenanthroline. These results demonstrate that increasing the bioavailability of DFOB through the conjugation of lipophilic fragments greatly enhances its protective capacity. These novel compounds have potential as therapeutics for the treatment of PD and other conditions of Fe dyshomeostasis.

Highlights

► Lipophilic conjugates greatly enhance the neuroprotective capacity of DFOB. ► They are more protective against iron-mediated paraquat and H2O2 toxicity in neuronal cells. ► They are more protective in iron-loaded, GSH-depleted, or A30P-α-synuclein-expressing cells.

Introduction

Parkinson disease (PD) is a neurodegenerative disease characterized pathologically by the loss of dopaminergic neurons in the substantia nigra (SN) region of the brain and the presence of α-synuclein-containing Lewy bodies [1]. Clinically, PD presents with resting tremor, slowness of initial movement, rigidity, and general postural instability. Additionally, PD also presents with varied nonmotor deficits including sleep deficits and cognitive impairment [2].

A key pathological hallmark of PD is an accumulation of Fe in the SN, the extent of which seems to correlate with disease severity [3], [4]. Iron is essential for normal cell function and is required for many enzymes, including that of dopamine synthesis, tyrosine hydroxylase [5]. However, excess redox-active Fe can catalyze the generation of highly reactive hydroxyl radicals from H2O2, which can damage membranes, proteins, and DNA, causing oxidative stress and cell death [6]. Furthermore, Fe promotes α-synuclein aggregation [7] and has been detected in a redox-active form in Lewy bodies along with α-synuclein [8]. PD is also characterized by the loss of the antioxidant glutathione from the SN [9], [10], [11]. In addition, dopamine is degraded by monoamine oxidases (MAOs) [12] and can autoxidize [13]. Both of these processes generate H2O2, potentially exacerbating the detrimental impact of Fe overload in the PD brain. Together, these factors render the SN highly susceptible to oxidative stress, which is implicated in the loss of SN dopaminergic neurons and the pathogenesis of PD [14], [15].

Exposure to the common pesticide paraquat (PQ) may be a causative event in the pathogenesis of PD. Peripheral/systemic administration of PQ to rodents causes parkinsonism, kills SN dopaminergic neurons, and increases brain reactive oxygen species levels [16], [17]. In humans, recent epidemiological studies indicate a strong association between long-term PQ exposure and PD [18], [19]. PQ is a redox cycling agent that, in the presence of reducing equivalents and oxygen, generates superoxide radicals, which in turn generate H2O2 [20]. This may be particularly relevant in regions of elevated Fe, such as the SN in PD.

In light of the central role of excess Fe in promoting dopaminergic neuron death, a potential therapeutic avenue for PD is to limit excess Fe by application of chelating compounds. One such compound is desferrioxamine B (DFOB), a hexadentate, high-affinity Fe3+ chelator (logβ110=30.5) [21]. DFOB is protective in vivo in several pharmacological animal models of PD [22], [23], [24], [25], [26]. However, in all of these models, DFOB was administered directly into the brain, or peripherally in weanling mice without a fully functional blood–brain barrier (BBB). DFOB is highly water soluble and there is only limited evidence it can cross the BBB or enter cells. It is used clinically for conditions of Fe overload such as β-thalassemia and sickle cell anemia [27], [28], [29]. DFOB has also been evaluated in a single clinical trial for the treatment of Alzheimer disease and elicited a beneficial outcome [30]. Although this effect was ascribed to chelation of aluminium, the potential for effective chelation of brain Fe by DFOB remains a possibility [31]. Novel chelators have been developed with greater capacity to enter cells, and these have shown efficacy in animal models of PD [32], [33], [34], [35], [36]. Hence, Fe chelation therapy remains a potential avenue for the treatment of PD.

In this study, we aimed to increase the bioavailability of DFOB through conjugation to actual or analogues of orally available, lipophilic compounds in clinical use. We recently reported that, relative to DFOB, selected candidates from this DFOB conjugate library were two- to threefold more efficient at mobilizing intracellular Fe from SK-N-MC neuroepithelioma cells and that this activity was closely correlated with lipophilicity [37]. In the current study, we investigated the protective capacity of a subset of the novel DFOB conjugates in PD-relevant in vitro models of oxidative stress. We have directly compared the effectiveness of the novel compounds with DFOB and to other relevant metal-chelating compounds. We also assessed the ability of the compounds to mobilize cell-associated Fe. We found that the novel compounds were more effective at attenuating oxidative stress than other chelating compounds and substantially more effective than DFOB. These results are likely to be due to the increased cell permeativity of the novel compounds over DFOB.

Section snippets

Materials

Acetonitrile (biotech grade), adamantane-1-acetic acid (98%), l-buthionine sulfoximine (BSO), 5-chloro-7-iodo-8-quinolinol (clioquinol), DFOB mesylate (95%), 3,5-dimethyladamantane-1-carboxylic acid (97%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide·HCl (EDC; protein sequence grade), dimethyl formamide (biotech grade), ferric ammonium citrate (FAC), methyl viologen dichloride (PQ), and 1,10-phenanthroline were purchased from Sigma–Aldrich (Castle Hill, NSW, Australia). H2O2 was from

Novel DFOB conjugates as bioavailable iron chelators

The novel compounds were generated by conjugating DFOB with 3,5-dimethyladamantane-1-carboxylic acid (compound 1) or adamantane-1-acetic acid (compound 2) or deferasirox (compound 3; Fig. 1 [37]). Several adamantyl-based compounds are used to treat Parkinson disease (amantadine), Alzheimer disease (memantine), and influenza A (amantadine, rimantadine [43], [44], [45], [46]). These compounds are orally active and are generally well tolerated by patients. Deferasirox is a tridentate Fe chelator

Discussion

DFOB is a high-affinity Fe(III) chelator [21] and is effective at decreasing Fe-mediated oxidative stress, exhibiting efficacy in in vivo models of PD [22], [23], [24], [25], [26]. However, DFOB was effective in these studies only when applied directly into the brain and is much less effective when administered peripherally, highlighting the poor propensity of DFOB to cross the BBB and enter the brain. This is probably due to its short plasma half-life (∼15 min) [56], high water solubility, and

Acknowledgments

Funding from the National Health and Medical Research Council (R.C., A.R.W.), the University of Sydney (R.C., J.L.), and Parkinson’s NSW (R.C., A.R.W.) is gratefully acknowledged. The authors kindly thank Dr. Janetta Culvenor, University of Melbourne, for provision of the α-synuclein antibody.

References (81)

  • T. Jayasena et al.

    Membrane permeability of redox active metal chelators: an important element in reducing hydroxyl radical induced NAD+ depletion in neuronal cells

    Neurosci. Res.

    (2007)
  • C. Wersinger et al.

    Differential cytotoxicity of dopamine and H2O2 in a human neuroblastoma divided cell line transfected with α-synuclein and its familial Parkinson's disease-linked mutants

    (2003)
  • E. Junn et al.

    Human alpha-synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine

    Neurosci. Lett

    (2002)
  • H. Jiang et al.

    Parkinson's disease genetic mutations increase cell susceptibility to stress: mutant alpha-synuclein enhances H2O2- and Sin-1-induced cell death

    Neurobiol. Aging

    (2007)
  • W. Xie et al.

    New insights into the role of mitochondrial dysfunction and protein aggregation in Parkinson's disease

    Biochim. Biophys. Acta

    (2010)
  • S.E. Hamrick et al.

    A role for hypoxia-inducible factor-1alpha in desferoxamine neuroprotection

    Neurosci. Lett.

    (2005)
  • D.W. Lee et al.

    Inhibition of prolyl hydroxylase protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity: model for the potential involvement of the hypoxia-inducible factor pathway in Parkinson disease

    J. Biol. Chem.

    (2009)
  • K.A. Jellinger

    Neuropathology of sporadic Parkinson's disease: evaluation and changes of concepts

    Mov. Disord

    (2011)
  • I. Ferrer

    Neuropathology and neurochemistry of nonmotor symptoms in Parkinson's disease

    Parkinsons Dis

    (2011)
  • M.E. Gotz et al.

    The relevance of iron in the pathogenesis of Parkinson's disease

    Ann. N. Y. Acad. Sci.

    (2004)
  • J. Sian-Hulsmann et al.

    The relevance of iron in the pathogenesis of Parkinson's disease

    J. Neurochem.

    (2011)
  • D.B. Kell

    Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson's, Huntington's, Alzheimer's, prions, bactericides, chemical toxicology and others as examples

    Arch. Toxicol

    (2010)
  • R.J. Castellani et al.

    Sequestration of iron by Lewy bodies in Parkinson's disease

    Acta Neuropathol.

    (2000)
  • J. Sian et al.

    Alterations in glutathione levels in Parkinson's disease and other neurodegenerative disorders affecting basal ganglia

    Ann. Neurol.

    (1994)
  • R.K. Pearce et al.

    Alterations in the distribution of glutathione in the substantia nigra in Parkinson's disease

    J. Neural Transm.

    (1997)
  • H.L. Martin et al.

    Glutathione—a review on its role and significance in Parkinson's disease

    FASEB J

    (2009)
  • M.B. Spina et al.

    Dopamine turnover and glutathione oxidation: implications for Parkinson disease

    Proc. Natl. Acad. Sci. USA

    (1989)
  • D.G. Graham

    Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones

    Mol. Pharmacol

    (1978)
  • C. Henchcliffe et al.

    Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis

    Nat. Clin. Pract. Neurol.

    (2008)
  • K. Kuter et al.

    Increased reactive oxygen species production in the brain after repeated low-dose pesticide paraquat exposure in rats: a comparison with peripheral tissues

    Neurochem. Res

    (2010)
  • T.P. Brown et al.

    Pesticides and Parkinson’s disease—is there a link?

    (2006)
  • C.M. Tanner et al.

    Rotenone, paraquat, and Parkinson's disease

    Environ. Health Perspect.

    (2011)
  • J.S. Bus et al.

    Paraquat: model for oxidant-initiated toxicity

    Environ. Health Perspect.

    (1984)
  • S. Konetschny-Rapp et al.

    Solution thermodynamics of the ferric complexes of new desferrioxamine siderophores obtained by directed fermentation

    J. Am. Chem. Soc.

    (1992)
  • D. Ben-Shachar et al.

    Role of iron and iron chelation in dopaminergic-induced neurodegeneration: implication for Parkinson's disease

    Ann. Neurol.

    (1992)
  • D. Ben-Shachar et al.

    The iron chelator desferrioxamine (Desferal) retards 6-hydroxydopamine-induced degeneration of nigrostriatal dopamine neurons

    J. Neurochem.

    (1991)
  • M. Santiago et al.

    Neuroprotective effect of the iron chelator desferrioxamine against MPP+ toxicity on striatal dopaminergic terminals

    J. Neurochem.

    (1997)
  • J. Lan et al.

    Desferrioxamine and vitamin E protect against iron and MPTP-induced neurodegeneration in mice

    J. Neural Transm

    (1997)
  • Z. Zhang et al.

    Neuroprotection of desferrioxamine in lipopolysaccharide-induced nigrostriatal dopamine neuron degeneration

    Mol. Med. Report

    (2012)
  • J.L. Kwiatkowski

    Real-world use of iron chelators

    Hematology Am. Soc. Hematol. Educ. Program

    (2011)
  • Cited by (27)

    • Neuroprotection of inositol hexaphosphate and changes of mitochondrion mediated apoptotic pathway and α-synuclein aggregation in 6-OHDA induced Parkinson's disease cell model

      2016, Brain Research
      Citation Excerpt :

      Furthermore, iron interacts with α–synuclein, dopamine and tyrosine hydroxylase which are closely associated with the pathology of PD (Rcom-H׳cheo-Gauthier et al., 2014; Zhang et al., 2014). It is a promising approach for PD to target iron accumulation (Kaur et al., 2003; Liddell et al., 2013). Iron induced DNA damage and cell apoptosis were prevented by IP6 in N27 dopaminergic neurons (Xu et al., 2008).

    • Recent advances in therapeutical applications of the versatile hydroxypyridinone chelators

      2022, Journal of Inclusion Phenomena and Macrocyclic Chemistry
    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text