The potential application of iron chelators for the treatment of neurodegenerative diseases^†

Abstract

Many forms of neurodegenerative disease, for instance Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Hallervorden Spatz syndrome and macular degeneration, are associated with elevated levels of redox active metals in the brain and eye. A logical therapeutic approach therefore, is to remove the toxic levels of these metals, copper and iron in particular, by selective chelation. The increased number of iron-selective chelators now available for clinical use has enhanced interest in this type of therapy. This review summarises the recent developments in the design of chelators for treatment of neurodegenerative disease, identifies some of the essential properties for such molecules and suggests some future strategies.

Graphical Abstract

Many forms of neurodegenerative diseases are associated with elevated levels of transition metals. These metals can be removed by chelation.

1. Introduction

The main agent of risk in most neurodegenerative disorders is age and this may be directly linked to oxidative stress (lipid peroxidation, protein oxidation, DNA and RNA oxidation), which increases in the brain with age and plays a central role in the pathogenic mechanisms of neurodegeneration.

Oxidative stress may be defined as an imbalance between the production of free radicals and the ability of the cell to defend against them through a set of antioxidants and detoxifying enzymes that include superoxide dismutase, catalase and glutathione. When this imbalance occurs, oxidatively modified molecules (lipids, proteins, nucleotides) accumulate in the cellular compartment causing dysfunction.¹ In the case of very sensitive cells such as neurons, the lack of control of defence systems may eventually lead to cell death. Under physiological conditions, free radicals are byproducts of cellular oxygen metabolism, with superoxide (O₂˙⁻), hydroxyl (OH˙) and nitric oxide (NO˙) species being prevalent, while hydrogen peroxide (H₂O₂) and peroxynitrite (ONOO⁻), not radicals themselves, contributing to the cellular redox state and eventually produce radicals through various chemical reactions. Mitochondrial oxidative metabolism phospholipid metabolism, proteolytic pathways and metal ions are potential sources of intracellular free radicals.

The brain is at risk from oxidative damage because of the following specific characteristics:

• high oxygen consumption (20% of the total body basal O₂ consumption);

• critically high levels of both iron and ascorbate

• relatively low levels of antioxidant protective agents

• tendency to accumulate metals with age.

Oxidative damage induced by the redox activity of a target protein, which interacts with free radicals and metal ions, has been found as a typical hallmark in many neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD).

1.1 Oxidative stress and Alzheimer's disease

Oxidative stress is believed to play a major role in the dysfunction and degeneration occurring in AD, as one of the earliest events that takes place in the cytoplasm of vulnerable neurons. A key pathological hallmark of AD is the presence of senile plaques constituted by a highly dense core formed of a mixture of 39–43 residue polypeptides derived from Amyloid Precursor Protein (APP) that accumulate in the cortical interstitium and cerebrovasculature in a characteristic manner. One such peptide, amyloidβ-peptide 1–42 (Aβ1–42) is a minor soluble species but possesses a fibrillogenic activity that renders it central to the pathogenesis and to be particularly toxic to cells in the early stage of the peptide aggregation process.^2,3

There is strong evidence of a relationship between oxidative stress and Aβ cortical deposits and this characteristic is likely to derive mostly from its ability to bind metals and, as a consequence, to mediate redox reactions.^4,5 In fact, Aβ1–42 is a metallo-binding peptide with binding sites for both Cu(ii) and Fe(iii)⁶ (Fig. 1). Metal homeostasis is altered during AD, and as a consequence, metals are reported to accumulate in the neuropil with concentrations that are 3–5-fold increased compared to agematched controls.⁷

Fig. 1

Sequence of the Aβ1–42 peptide, which is a proteolytic fragment of the amyloid precursor protein. The key amino acids for iron(iii) coordination are His 6, Asp 7, Tyr 10 and His 14.

The proposed metal coordinating site can in principle bind either iron (Fig. 2A) or copper (Fig. 2B) and provide sites which will readily redox cycle as judged by the relative selectivity of histidine, tyrosine and aspartate for the ion pairs iron(iii)/iron(ii) and copper(ii)/copper(i) (Table 1). The proposed binding site for iron (Fig. 2A and 3) is similar to that of transferrin (2Y, 1D, 1H) except that a second histidine replaces one of the tyrosines (Y, 1D, 2H). This substitution would render the site with a higher affinity for iron(ii) than that of transferrin, thereby facilitating redox cycling. The proposed octahedral site has two sites occupied by water, which by virtue of their lability would render the iron accessible to both reducing and oxidising agents.⁸

Fig. 2

Schematic representation of iron(iii) and copper(ii) bound to the metal coordination site of Aβ1–42. It is proposed that the octahedral field of iron is completed by the coordination of two water molecules.

Table 1

Ligand selectivity of iron, copper and zinc cations

Iron(iii)	O (anionic)
Iron(ii)	O (anionic), N (aliphatic), N (imidazole), S
Copper(ii)	O (anionic), N (aliphatic), N (imidazole), S
Copper(i)	N (imidazole), S
Zinc(ii)	O (anionic), N (aliphatic), N (imidazole), S

Fig. 3

Possible conformation of iron bound to the metal coordination site of Aβ1–42. Light blue, carbon; red, oxygen; dark blue, nitrogen; white, hydrogen; yellow, iron. The structure was energy minimised by HyperChem® 8.0 Amber.

1.2 Oxidative stress and Parkinson's disease

Post-mortem studies in PD brains indicate that a wide range of molecules undergo oxidative damage, including lipids, proteins and DNA.^9,10 In fact, significant neurochemical, physical, histochemical and biochemical evidence confirm the hypothesis that oxidative stress generates the cascade of events, which are responsible of the preferential degeneration of melanised dopaminergic neurons in the substantia nigra pars compacta in PD. Parkinsonian brains possess elevated levels of iron in microglia, astrocytes, oligodendrocytes and dopaminergic neurones of substantia nigra pars compacta.^11–15 Iron can in principle mediate the generation of hydroxyl radicals by interaction with α-synuclein, a 140-amino-acid presynaptic protein that accumulates intracellularly in the form of fibrillar aggregates in neurons and sometimes also in glia cells. The synucleins (α-, β- and γ-) are highly expressed proteins in the nervous tissue. Two missense mutations in the α-synuclein gene (A53T and A30P) are responsible for rare forms of familial PD. The accumulation of fibrillar forms of the protein is one of the decisive events occurring in the pathogenesis of PD.^16–18 The toxicity in part may be generated by oxidative damage, induced by interactions between α-synuclein fibrils and iron.^19,20

1.3 Therapeutic strategies

A wide range of drugs with diverse mechanisms of action has been investigated. Although some of these approaches have demonstrated potential, when studied in cellular and animal models of acute or chronic neurodegeneration, only a few have provided convincing clinical results. Among the relevant therapeutic strategies, it is noteworthy to mention the use of antioxidants, excitotoxicity modulators, the inhibition of the expression of amyloidogenic protein, inhibition of the release of amyloidogenic peptide and the inhibition of Aβ-amyloid aggregation. Oxidative stress, protein aggregation and redox active metal ions are also considered to be promising pharmacological targets. The apparently critical involvement of metals, particularly iron and copper, in both oxidative stress and protein aggregation processes therefore renders chelation therapy a sensible strategy.²¹ The key feature of a suitable chelating agent would be the ability firstly to scavenge the free redox active metal present in excess in the brain and to form a nontoxic metal complex, which is then excreted.

2. Chelation-based therapy

One of the dominant properties of any therapeutic chelator is metal selectivity, typically a high selectivity being required, for instance in the treatment of iron overload associated with β-thalassaemia. In this situation, ligands with a high selectivity for iron over copper and zinc are essential, as chelation therapy is maintained for life. Unfortunately, with the proposed treatment of neurodegenerative diseases by chelation therapy, the identity of the putative toxic metal is not always firmly established. With AD, for instance, iron, copper and zinc have all been associated with the progression of the disease. In contrast, with PD, iron is clearly the major target. Although there are clear guidelines for the design of iron-selective chelating agents,²² this is not the situation with copper.

With the necessity of ready permeation of the blood–brain barrier (BBB), the size of useful chelators should probably be limited to less than 300Da, thereby excluding hexadentate ligands and further limiting the potential for the design of selective copper(ii) chelators. As PD is clearly associated with an abnormal distribution of iron in the brain and as most strong iron(iii) chelators also possess an appreciable affinity for copper(ii) (Table 2) an initial search for an iron(iii) chelator would appear to be an appropriate approach.

Table 2

Metal affinity constants for selected ligands (Martell & Smith, 1974–1989)

Ligand	Log cumulative stability constant
	Fe(iii)	Al(iii)	Ga(ii)	Cu(ii)	Zn(ii)	Fe(ii)	pFeIII
DFO (1)	30.6	25.0	27.6	14.1	11.1	7.2	26
EDTA (2)	25.1	16.5	21.0	18.8	16.5	14.3	23.4
N,N-dimethyl-2,3-dihydroxy-benzamide (DMB) (3)	40.2			24.9	13.5	17.5	15
Acetohydroxamic acid (4)	28.3	21.5		7.9	9.6	8.5	13
3-hydroxypyridin-4-one (deferiprone) (5)	37.2	35.8	32.6	21.7	13.5	12.1	20.5b
8-hydroxyquinoline (6)	37.7		40.5	22.9	15.8	22.2	20.6

a

pFe^III = −log[Fe³⁺] when [Fe³⁺]_total = 10⁻⁶ M and [ligand]_total = 10⁻⁵ M at pH 7.4.

b

This value agrees with that recently reported by Nurchi et al.25 but is different to the widely quoted figure of 19.021 which was based on measurements of logβ₃ by Martell & Smith of 35.8. The value of cumulative affinity constant has recently been reported to be higher, namely 36.7²⁵ and 37.1.²⁶ Hexadentate ligands: DFO (1), EDTA (2); bidentate ligands: N,N-dimethyl-2,3-dihydroxy-benzamide (DMB) (3), acetohydroxamic acid (4), deferiprone (5), 8-hydroxyquinoline (6).

Iron(iii)-selective chelators favour oxygen atoms as ligands, notably hydroxamates and catecholates (Table 2). Most tribasic cations, for instance aluminium(iii) and gallium(iii), are not essential for living cells and thus iron(iii) is a practical target for ‘clinical chelator’ design. An additional advantage of high affinity iron(iii) chelators is that, under aerobic conditions, they will chelate iron(ii) and facilitate autoxidation to iron(iii).²³

Ligands can be structurally classified according to the number of donor atoms that each molecule possesses. When a ligand contains two, three or six donor atoms, it is termed bidentate, tridentate or hexadentate (Fig. 4). For biological conditions, the pFe value is a more useful parameter than the conventional stability constant for assessment of the ligand's affinity for the metal.^22,24 For clinically useful iron scavengers, a pFe³⁺ value ⩾ 20 (Table 2) is considered to be essential. Molecular size is also a critical factor, as it influences the penetration of both the wall of the gastrointestinal tract²⁷ and the BBB.²⁸ Lipinski et al.²⁹ suggest a guideline of 500. This molecular-weight limit provides a considerable restriction on the choice of chelator and may effectively exclude hexadentate ligands from consideration.

Fig. 4

Schematic representation of chelate ring formation in iron-ligand complexes.

Fig. 5

General structure of iron(iii) chelators. Hexadentate: desferrioxamine (DFO, 1), ethylenediaminetetraacetic acid (EDTA, 2); bidentate: N,N-demethyl-2-3,-dihydroxybenzamide (DMB, 3), acetohydroxamic acid (4), hydroxypyridinone (5, R₁ = R₂ = CH₃, deferiprone; 5a, R₁ = R₂ = C₂H₅, CP94; 5b, R₁ = nC₄H₉, R₂ = CH₃, CP24), 8-hydroxyquinoline (6), Clioquinol (7), VK20 (8), M30 (9), CP241 (10), CP242 (11).

Bidentate and tridentate ligands, by virtue of their much lower molecular weights, are predicted to possess higher absorption efficiencies. The fraction of the absorbed dose for a range of bidentate hydroxypyridin-4-ones (5) has, for instance, been found to fall between 50 and 70%.³⁰ Hydroxypyridinones have also been demonstrated to penetrate the BBB.^31,32

Another important feature for chelation therapy of neurodegeneration is the ability not only to scavenge redox active metal but to remove this excess metal from the brain. One of the successes of deferiprone in the treatment of transfusion—induced iron overloaded thalassaemia patients is its ability to remove iron from the heart.³³ It is able to achieve this by forming a non charged water soluble iron(iii) complex possessing a molecular weight less than 500 and therefore capable of permeating membranes by non facilitated diffusion. This property indicates that hydroxypyridinones may also be able to remove excess iron from the brain (Fig. 6). Indeed studies with hydroxypyridinones in the ferrocene—loaded rat demonstrate the ability of such compounds to remove excess iron from the brain (Fig. 7). It is this ability to remove excess labile iron from cells and mitochondria that has led to the success of deferiprone therapy in patients suffering from Friedreich's ataxia^34,35 and NBIA (Neurodegeneration with brain iron accumulation).³⁶

Fig. 6

Schematic representation of the penetration of deferiprone [LH]o through the BBB. The bidentate ligand scavenges loosely bound iron forming the 3 : 1 complex [inset] which also carries zero net charge. Diffusion through the BBB as the iron complex leads to iron excretion.

Fig. 7

Brain iron in the ferrocene-loaded rat. 3,5,5-trimethylhexanoyl ferrocene is used to load the rat brain. Brain regions: 1, cerebellum; 2, cerebral cortex; 3, hippocampus; 4, brain stem; 5, striatum; 6, substantia nigra. Structure of CP94 given in Fig. 5.

A range of 8-hydroxyquinoline analogues have also been investigated for their potential to treat neurodegeneration. Clioquinol (7) is a small lipophilic molecule which has been demonstrated to have beneficial effects in both AD and PD models^37–40 and in clinical studies.^41,42 8-Hydroxyquinolines, like hydroxypyridinones, form neutral 3 : 1 iron complexes.

Unfortunately, halogenated hydroxy-quinolines possess neurotoxic side effects.^43,44 These side effects may be avoided by the use of nonhalogenated analogues, for instance the brain permeable VK-28 (8) and M30 (9).⁴⁵ A study centred on rats, with 6-OHDA—induced striatal dopaminergic lesions, has shown that, when injected either intraventricularly (1 μg in 5 ml) or intraperitoneally (1 or 5 mg kg⁻¹ day⁻¹ for 10 and 7 days, respectively), VK-28 is able to provide neuroprotection against 6-OHDA at very low doses. Moreover, this study has shown that the mechanism of action of VK-28 is more likely to be related to iron chelation properties than to any direct interference with 6-OHDA, since intranigral or intraventricular 6-OHDA initiates an increase in total iron in the substantia nigra and striatum at the sites of neurodegeneration, in monkeys, rats and mice.⁴⁶

3. Chelator design

The toxicity associated with chelators originates from a number of factors, such as inhibition of metalloenzymes, lack of metal selectivity and redox cycling of complexes (Fig. 8).

Fig. 8

Redox cycling activity of iron complexes. The reducing agent could be vitamin C or reduced coenzymes, for instance NADPH.

3.1 Redox properties of iron complex

If all the coordinating ligands are charged oxygen atoms, as is the case with 3-hydroxypyridinones, then both iron and copper complexes will favour the most oxidised form of the metal ion, namely iron(iii) and copper(ii) (Table 1). This in turn prevents redox cycling, as the corresponding iron(ii) and copper(i) complexes are unstable. This is reflected in the extremely low redox potential of iron(iii) hydroxypyridinone complexes, −610 mV.⁴⁷ In contrast, if some of the coordinating ligands are nitrogen atoms, as is the case with 8-hydroxyquinolines, then redox cycling becomes possible with the concomitant generation of free radicals. This is reflected in the higher affinity of 8-hydroxyquinolines for iron(ii) and copper(i) (Table 2), when compared with those of hydroxypyridinones and the higher redox potential of the iron/hydroxyquinoline complex of −150 mV.⁴⁸

3.2 Inhibition of enzyme activity

In general, iron chelators do not directly inhibit haem iron-containing enzymes due to the inaccessibility of porphyrin bound iron to chelating agents. In contrast, in many non-haem iron-containing enzymes, such as lipoxygenase, the aromatic hydroxylase family and ribonucleotide reductase are susceptible to chelator-induced inhibition.⁴⁹ Generally, hydrophobic chelators inhibit lipoxygenases, therefore the introduction of hydrophilic characteristics into a chelator tend to minimise such inhibitory potential,⁵⁰ particularly if their introduction also induces steric interference of the chelation process at the enzyme active site.⁵¹ By careful modification of physicochemical properties, iron chelators can therefore be designed which exert minimal inhibitory influence on many metalloenzymes.^52,53

One series of enzymes which requires careful attention in all studies centred on neurodegeneration are those associated with the synthesis and metabolism of dopamine and 5-hydroxytryptamine, namely tyrosine hydroxylases, tryptophan hydroxylases and catechol-O-methyl transferase. In a study with ferrocene-loaded rats the hydroxypyridinone CP94 (5a) was found to decrease both striatal dopamine and 5-hydroxytryptamine levels.⁵⁴ Significantly the more hydrophilic analogue deferiprone (5) failed to change these levels.⁵⁴ A series of hydroxypyridinones have subsequently been designed which possess a very low ability to inhibit tyrosine hydroxylase⁵⁵ (Table 3).

Table 3

Inhibition of Tyrosine Hydroxylase

Hydroxypyridinone	R	R1	% inhibition of tyrosine hydroxylasea
CP94 (5a)	—	—	43
CP142	H	CH3	18.5
CP132	CH3	CH3	2.5
CP133	CH3	CH2CH(CH3)2	15.5
CP135	CH3	CH2Ph	3.0
CP137	CH2Ph	CH2CH(CH3)2	46.5

a

[chelator] = 10 μM.

3.3 Ability to cross the blood brain barrier

By virtue of their small molecular size and noncharged nature, hydroxypyridinones readily cross the blood brain barrier (BBB),^31,32 there being a clear relationship with the log P_octanol values of N-alkyl derivatives (Fig. 9).³¹ However the introduction of a single hydroxyl function dramatically decreases the ability of the hydroxypyridinone to penetrate the BBB (Fig. 9). There is a practical limit to increasing the log P value in order to enhance BBB permeability due to increasingly efficient first past extraction by the liver, which in turn leads to decreasing oral bioavailability. In an attempt to optimise BBB penetration while not enhancing liver first pass extraction, a range of fluorinated hydroxypyridinones have been synthesised⁵⁶ (Table 4). Brain content was determined by in situ perfusion of guinea pig brains,^57,58 having made allowance for the vascular space by use of ³H-mannitol (Fig. 10). There was no correlation with logP_octanol values, but four were found to cross the BBB more efficiently than deferiprone, the most effective being CP241 2-fluoro-3-hydroxy-1-n-propylpyridin-4-one (10).⁵⁹

Fig. 9

Relationship between blood-brain barrier permeability (Log PS) of a range of hydroxypyridinones with Log P_octanol in adult wistar rats. The vascular perfusion time was 1 min values are expressed as means ± SD (n = 3)³¹ CP20 (deferiprone) (5), R₁ = R₂ = CH₃; CP21, R₁ = CH₃CH₂, R₂ = CH₃; CP94 (5a); CP24 (5b); CP25, R₁ = CH₃(CH₂)₄CH₂, R₂ = CH₃; CP29, R₁ = CH₃(CH₂)₃CH₂, R₂ = CH₃; CP41, R₁ = (CH₂)₃OH, R₂ = CH₃; CP102, R₁ = (CH₂)₂OH, R₂ = Et; CP107, R₁ = (CH₂)₃OH, R₂ = Et.

Table 4

Structure of fluorinated hydroxypyridinones

	R1	R2	R5	R6	logP oct.
Deferiprone	CH3	CH3	H	H	−0.77
CP227	Et	F	H	H	−0.58
CP233	Et	H	F	H	−0.89
CP241	n-Prop	F	H	H	0.07
CP242	n-Prop	H	F	H	−0.46
CP243	iso-Prop	H	F	H	−0.92
CP228	H	CF3	H	H	−0.08
CP246	H	F	CONHMe	H	−1.00
CP221	H	COCF3	H	H	−0.97

Fig. 10

Brain content of fluorinated hydroxypyridinones. Values are mean ± S.E.M where n = 6–12.⁵⁹

3.4 Ability to inhibit prooxidant insults

Hydroxypyridinones have been demonstrated to protect cultured cortical neurones against prooxidant damage induced by iron(iii) NTA, hydrogen peroxide and Aβ1–40.^55,60 Deferiprone is capable of providing protection to these cells at 10 μM (Fig. 11). With the amyloid peptide Aβ1–40, the prooxidant damage almost certainly results from the peptide scavenging iron from the medium and providing a binding site which is capable of facile redox cycling (Fig. 3). These results are summarised for 20 μM deferiprone in Fig. 12 where it is clear that deferiprone confers protection against amyloid b—induced neurotoxicity. A similar finding is observed with the fluoro substituted hydroxypyridinones CP241 (10) and CP242 (11) where both compounds offer protection against 10 μM Aβ1–40 peptide (Fig. 13).⁵⁹ In both cases the neurotoxicity induced by Aβ peptide is completely reversed with chelator concentrations of 30 μM.

Fig. 11

Neurotoxicity studies in primary cortical neurones exposed to human Aβ1–40.⁶⁰ Cytotoxicity was assessed by morphometric analysis of cell viability by HOE 33324 (blue) and/or cell death by propidium iodide (red) using an IN1000 Cell Analyser. Cultures were treated with Aβ1–40 (3 or 20 μM, t = 24 h) (a–c) and/or deferiprone (10, 30 or 100 μM) (d–l).

Fig. 12

Deferiprone (10, 30 or 100 μM, t = 24 h) confers protection against amyloid b-induced neurotoxicity (#p < 0.001 vs. control).⁶⁰ [Aβ] = 20μM. The results shown are the mean ± SEM of three independent experiments in quadruplicate.

Fig. 13

Neurotoxicity studies in primary cortical neurones exposed to human Aβ1–40. Cytotoxicity was assessed by formazan blue production, which monitors the viability of mitochondria. The cells were incubated for 24 h in the presence of Aβ1–40 (10 μM) and various chelators (30 μM). All values are mean ± SEM from three independent experiments, in quadruplicate.

4. Targeting chelators to the brain

As indicated in the previous section, there is a clear limit to the maximum chelatortrans-BBB flux achievable by non facilitated diffusion. To increase this flux above this limit it will be necessary to facilitate chelator transfer. Two approaches have been reported, the use of nanoparticles and the conjugation of chelators to sugars, leading to facilitated transport across the BBB.

4.1 Nanoparticle bound iron chelators

There are a number of nanoparticle approaches available for the transfer of chelators into brain.⁶¹ Polymeric nanoparticles possess high drug loading capacities and have been targeted to the BBB^62,63; nanogels, which consist of net works of cross-linked polymers can also be used to deliver low molecular weight drugs⁶⁴; polymeric nanomicelles possess a core-shell structure, with a hydrophobic cove within a shell of hydrophilic polymers, the core is capable of accommodating up to 25% w/w of drugs⁶⁵; and polymeric nanoliposomes, which are vesicular structures composed of one or more lipid bilayers surrounding an internal aqueous compartment.⁶⁶ These nanoparticles can be targeted to the BBB using a variety of address systems,⁶⁷ including melanotransferrin receptor,⁶⁸ transferrin receptor⁶⁹ and apolipoprotein receptors.⁷⁰

Clioquinol (7)—encapsulated in poly(butylcyanoacrylate) nanoparticles (PBCA) has been prepared as a vector for the invivo brain imaging of β-amyloid plaques. These preparations cross the BBB with high efficiency.⁷¹ Further more, ¹²⁵I-labelled clioquinol incorporated into nanoparticle has been utilised for invivo biodistribution studies in mice.⁷² PBCA nanoparticles have been used to deliver a range of drugs to the CNS,⁷³ for instance doxorubicin.⁷⁴ The drug is loaded during the initial polymerisation process and the resulting particles are coated with polysorbate 80. Following intravenous administration the surface of the particles become further coated with absorbed plasma proteins, most prominently apolipoprotein E. It has been suggested that these naturally coated particles are mistaken for low-density lipoprotein particles by the cerebral endothelium and internalised by the LDL uptake system.^75,76 Nanoparticles have also been prepared with chelators, desferrioxamine (1) and hydroxypyridinones (5) covalently attached to the outside of the structure (Fig. 14).⁷⁷ An interesting feature of these nanoparticles is that they are reported to have the potential of recrossing the BBB and entering the blood stream. Thus if they bind apolipoprotein A-I in the CSF they will be susceptible to efflux from the brain.⁷⁸

Fig. 14

An illustration of a typical structure of a polymer-based nanoparticle with externally attached chelators. The hydroxypyridinones are covalently attached via amide links to the polymer matrix.

An important limitation to nanoparticle delivery of chelators to the brain is they would have to be administered by a parenteral route, oral presentation would not be possible.

4.2 Facilitated transport of chelator-sugar conjugates

In principle pendant glucose molecules can be attached to drugs in order to enhance their ability to permeate the BBB. Due the heavy requirement of the brain for glucose, the BBB is endowed with a high concentration of the GLUT 1 hexose transporter protein.⁷⁹ This glycosylation strategy has been applied to a range of low molecular weight compounds with mixed success. Thus glycosyl dopamine derivatives offered little advantage over L-dopa or dopamine,⁸⁰ whereas an enkephalin glycopeptides produced strong analagesic effects when administered peripherally⁸¹ and a glucose conjugate of 7-chlorokynurenic acid was found to excert a strong anticonvulsant effect in rodents.⁸² The concept has been extended to chelators, but most of these conjugates have not been directly tested for BBB penetration. Thus glycosylated tetrahydrosalens (12) have been synthesised which possess a high affinity for both copper(ii) and zinc(ii),^83,84 but it remains unclear whether or not they are capable of penetrating the BBB. Feralex (13), a glucose conjugate of a hydroxypyridinone has been studied in a range of in vitro and cell culture studies^85–87 but again no direct demonstration of BBB permeation. A small series of prodrugs containing a 3-glucopyranosyloxy group have also been investigated⁸⁸ and the 4 iodophenyl derivative (14) has been reported to cross the BBB in rat. However the corresponding 3-glucopyranosyloxy derivative of deferiprone (15) failed to cross the guinea pig BBB.⁸⁹

Clearly this approach has potential, as sugar conjugates which behave as a substrate for hexose transporters, will also experience facilitated transport by the duodenum. The addition of the sugar lowers the log P value to the (−3)–(−4) range and thus first pass extraction by the liver should be minimal. A possible scenario for this class of molecule is presented in Fig. 15.

Fig. 15

Schematic representation of iron removal from CSF by a hydroxypyridinone-sugar conjugate. L˙Sug—Hydroxypyridinone-sugar conjugate; L—Hydroxy-pyridinone; FeL₃—3 : 1 Hydroxypyridinone / iron complex. The hydroxypyridinone-sugar conjugate (L˙Sug) is absorbed from the GIT by facilitated diffusion. By virtue of its hydrophilic nature (log P < −3) it is not susceptible to efficient liver first pass extraction and so enters the systemic circulation. By virtue of the high concentration of glucose transporters in the BBB, L˙Sug enters the brain by facilitated transport, where it will be metabolised to the free hydroxypyridinone which can scavenge iron to form the hydrophobic FeL₃ complex, which can permeate the BBB by nonfacilitated diffusion. Once in the systemic circulation FeL₃ will be excreted via the urine.

5. Conclusion

It is clear that iron- and copper chelators will play an appreciable role in the future treatment of various forms of neurodegeneration. Indeed, with the identification of biomarkers for the various forms of neurodegenerative disease, selective chelators many find a role not only for treatment of the active disease, but also in prophylaxis of disease. The abnormal distribution of these two metals is probably not the primary cause of the various forms of neurodegeneration, but is undoubtedly associated with disease progression. Deferiprone (5) is currently involved in clinical trials for the treatment of Friedreich's ataxia, Parkinson's disease, macular degeneration and Hallervorden-Spatz syndrome. By modifying the structure of this extremely simple molecule, in order to facilitate penetration of the BBB, it is likely that a range of iron-selective chelators will be identified, some of which may find application in neurodegeneration therapy.

List of abbreviations

 
• AD

  Alzheimer's disease

 
• PD

  Parkinson's disease

 
• Aβ1–42

  Amyloid β-peptide 1–42

 
• Aβ1–40

  Amyloid β-peptide 1–40

 
• BBB

  Blood-brain barrier

 
• 6-OHDA

  6-Hydroxydopamine

 
• CSF

  Cerebral spinal fluid

 
• NTA

  Nitrilotriacetic acid

 
• PBCA

  poly(butylcyanoacrylate)

 
• LDL

  Low density lipoprotein

 
• pFe³⁺

  log[Fe³⁺˙6H₂O] at pH 7.4 when [L]_Total = 10⁻⁵ M and [Fe]_Total = 10⁻⁶ M.

Acknowledgements

SR's study was financed by Research Councils UK by the provision of a Dorothy Hodgkin postgraduate award. RCH, YM and XLK acknowledge general support from Apotex, Canada and Vifor, Switzerland. We wish to thank Dr. Francisco Molina-Holgado for the supervision of the neurotoxicity studies.

Professor Bob Hider is emeritus professor of medicinal chemistry at King's College London, where he has worked since 1987. Prior to this he was a lecturer in biological chemistry at Essex University. He has worked with siderophore-based iron uptake processes in microorganisms and the absorption of iron by mammalian cells. His work on membrane structure and transport mechanisms has led to the development of novel oral iron chelators for the treatment of iron overload. As a result, N-alkyl-3-hydroxypyridin-4-ones have been identified as possessing potential for clinical application. Deferiprone is now used worldwide for the treatment of iron overload.

Sourav is a PhD in Pharmaceutical Sciences (King's College London) with an MSc in Drug Discovery (University of London) and a B-Pharm (Bachelor of Pharmacy, Rajiv Gandhi University of Health Sciences, Bangalore, India). His PhD research involved the evaluation of blood-brain barrier permeability and neuroprotective properties of novel iron chelators intended for the prophylaxis of Alzheimer's disease. Through this multidisciplinary work two lead iron chelators possessing neuroprotective properties were identified.

Yong Min Ma was born in 1976 in Zhejiang, P.R. China. He studied chemistry at Zhejiang University, where he obtained his bachelors degree in 1998 and master's degree in 2001 under the direction of Prof. Yong Min Zhang. He then moved to London for PhD study in 2002 in Prof. Hider's group at King's College London. After completion of his PhD in 2005, he continued to work as a postdoctoral fellow in Prof. Hider's group. His research interests are centred on the design of iron chelators for the treatment of iron overload diseases.

Xiaole Kong was originally educated as a computer engineer. After successfully assembling an automatic fluorescent titration system for Professor Hider's lab, he became more interested in scientific research. He commenced a one year training course and then undertook study for a PhD—‘‘Spectrophotometric Determination of Stability Constants of Iron Chelators’’. He was awarded the ‘‘2009 Tadion–Rideal Prize for Molecular Science’’. His specialty is the determination of affinity constants.

Dr Preston is Senior Lecturer in the Institute of Pharmaceutical Science at King's College London. She completed her PhD and post-doctoral training at St Thomas' Hospital Medical School and Brown University, USA and joined King's in 1995. She has been both Vice-Chairman (2004) and Organizing Chairman (2006) of the Gordon Research Conference ‘Barriers of the CNS’ USA and founding member of the International Brain Barriers Society. Research interests are in drug delivery to CNS, cerebrospinal fluid (CSF) dynamics, and brain-barrier function in ageing, stroke, brain tumour challenge using in vivo and BBB cell culture models.

Footnotes

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This article is published as part of a themed issue on Metals in Neurodegenerative Diseases, Guest Edited by David Brown.