Original ContributionLipophilic adamantyl- or deferasirox-based conjugates of desferrioxamine B have enhanced neuroprotective capacity: implications for Parkinson disease
Graphical abstract
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.
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These authors contributed equally to this work.