Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 30, 2018
Accepted : May 22, 2018
Published online : June 30, 2018
doi:10.24659/gsr.5.2_104

Review article

Chelation therapy for Alzheimer's disease:
Nanoparticles as new components of this therapy
Nikola Barić
Presika 153, 52220 Labin, Istra, Croatia

Abstract

Objective: Modern treatment of Alzheimer’s disease (AD) with acetylcholinesterase (AChE) inhibitors and N-methyl-Daspartate (NMDA) receptor antagonists, due to only a slightly indicated moderate alleviation of symptoms, as well as theimpossibility of the stagnation and a more significant remission of the disease, or complete recovery, is in constant search for
new medications that would be more effective. Considering that we are becoming increasingly aware of the pathophysiologyof this disease, with emphasis on metal-induced oxidative stress, the accumulation of transitional metals (especially iron),oxidative compounds, free radicals, as well as AGE compounds, the research studies are increasingly directed to the roleof the so-called chelators, i.e. the catchers and cleaners of metal ions. The validity of the opinion about the crucial role oftransitional metals in the induction of oxidative stress and the consequential neurodegenerative damages has been confirmed
by a large concentration of iron, zinc, copper and aluminium in the pathologically affected areas of the brain (parietal andfrontal cortex, hippocampus, amygdala) and by the close relation between the concentration levels and clinical features of thedisease progression.
Methods: With a comprehensive study of the available literature related to AD therapy, relevant data linked to so-calledchelation therapy has been thoroughly analysed, particularly the interesting bonding between chelators and nanoparticles
that enables a better penetration of chelators into the brain and the elimination of the formed complexes by exiting from thebrain through otherwise hardly permeable blood brain barrier (BBB). Thus, this study has in this way not only contributed
to the insight into modern AD therapy, but has also greatly contributed to the knowledge of the complex pathology andpathophysiology of this disease.
Results: Results of the analysis of available studies undoubtedly indicate the crucial role of metal-induced oxidative stressin AD pathophysiology, with a significant role of iron ions. Drawn by electrostatic forces and bound to the β1 chain of theapical amyloid beta (Aβ) monomer that forms the protofilament, the non-toxic ferric iron (Fe 3 + ) is reduced to the toxic formof ferrous iron (Fe 2 + ), which is oxidized in the Fenton reaction through H 2 O 2, forming the extremely toxic hydroxyl radical(*OH) with consequent severe oxidative damage in the surrounding molecular structures. The results of the analysis suggest
the beneficial therapeutic effect of metal chelators that are specially bound to the nanoparticles system.
Discussion: The discussion elaborates the detailed analysis of the comparison of the obtained results with the data from anumber of studies that focus on the same problem area. It is noticed that the author's considerations closely coincide with theopinions of renowned experts. This particularly refers to the crucial role of metal ions, especially iron, in AD pathophysiology,
as well as the importance of iron chelator therapy combined with nanoparticles.
Conclusion: AD is a severe, chronic, neurodegenerative disease with a fluctuating progressive course, and with a lethal end.
The current therapy with AChE inhibitors and NMDA receptor inhibitors does not show satisfying results. The increasingly
frequent therapy with metal chelators, especially iron chelators, combined with nanoparticles that are particles built fromnatural or artificial polymers capable of easily passing through the BBB, has given promising results and requires additionaland more intensive research.

KEY WORDS: Alzheimer's disease, oxidative stress, chelators, nanoparticles, chelation therapy

Contact address: Nikola Barić MD, PhD
Presika 153 52220 Labin, Istra, Croatia
Tel. 385/ 052 /852- 008/
E-mail: nikola.baric@pu.t-com.hr_ 10 4 _Glycative Stress Research 2018; 5 (2): 10 4 - 118(c) Society for Glycative Stress ResearchGlycative Stress Research

Introduction

Following the enormous rise of the worldwide elderly
population (e.g. senectual explosion – relative and absolute
rise of the elderly portio, 65 years and more, in the general
population), there is a concomitant rise in the incidence andprevalence of chronic degenerative diseases, among themespecially neurodegenerative, with a marked position of
Alzheimer's disease (AD). AD is a chronic, progressive andlethal disease, still mostly of unknown polygenetic etiology. It
is closely connected with the aging process and old age. Dueto the typical progressive loss of memory, thought and speech
ability, it presents a very serious disease which enormously
burdens the patient's family, as well as the complete society.
The costs for its prevention, and nowadays not a very
successful treatment, have an exponential trend 1-4).
The yet not adequately explained etiology, and a numberof recent investigations lead to the evident fact that oxidative
stress is the crucial link in AD pathophysiology. This
complex event, according to a number of investigations, isconnected with metal metabolism disregulation, especiallywith the rise of intracerebral concentration of transition
metals, among them especially iron (Fe) and copper (Cu).
Their ions, particularly the nontoxic, redox-inactive ferricion (Fe 3 + ), whose concentration in the pathology affected
brain regions is several times higher than in the nonaffected
regions, have a great affinity toward the beta amyloid(Aβ), an important AD marker. Fe 3 + is by Aβ reduced into
the toxic redox-active Fe 2+ , which enters in the Fenton
reaction, and through the oxidation by H 2 O 2 (hydrogen
peroxide) generates the extremely toxic and destructivehydroxyl radical (*OH). This radical induces the wastingoxidative deteriorations of neuronal lipid membranes (lipid
peroxidation), of protein structures and of DNA molecules.
The Fe 3 + brain interstitial concentration (ISF, interstitial
fluid) and its disorders are now under intensive investigations.
Actually, there are a number of unsolved questions as tothis problem. According to some opinions, it seems that aninverted transferin (Tf) route is possible, which during thisevent attracts Fe 3 + from the subarachnoid space (SAS) in
the superior sagital sinus venous blood flow (passage acrossthe arachnoid granulations). This inverted route is in AD
probably damaged by Aβ sedimentation. Additionally, inAD there is also a decline in Tf (oligodendrocytes) and TfR1
(Transferin receptor protein 1 - vascular endothelium of brain
capillaries) production. On the other hand, it seems that Tf
generated in the brain cannot pass through the arachnoid
layer. It is also possible that in AD there exists an actual
defect of Fe3 + efflux by perivascular lymphatic routes. All
these events certainly require further investigations 1, 3, 5-12).
AD, as a serious disease, is the target of a number oftherapeutic attempts. However, with regard to the poor resultsof all former procedures, among them the actual therapy byacetylcholinesterase (AChE) inhibitors (AChEI), Aricept(donezepil hydrochloride), Exelon (rivastigmine), and Reminyl(galantamine hydrochloride), as well as N-methyl-D-aspartate(NMDA) receptor antagonists (Ebixa, memantine), there isan increased importance of chelation therapy, based on theblockade of the transition metals effect on oxidative stress.
Along with the isolated chelators treatment, the combinationof chelators and the nanoparticle system is becoming moreactualized.
Nanoparticles are small particles, composed of natural
or artificial polymers which, conjugated with chelators, caneasily pass across the blood brain barrier (BBB), and after

the capture of metals they can, without any problem, bring
the formed complex out from the brain. Chelation therapy,
combined with the nanoparticle system, today is increasingly
under intensive investigations and is adequately applied in
AD therapy, as well as in other neurodegenerative and evenseveral malignant diseases. The results are very good, andindicate a promising perspective of this therapy 1, 3, 7, 13, 14).
By the method which includes the analysis of accessible
literature involved in AD therapy, this study in detailelaborates a variety of relevant data about the mentioned
therapy, and especially the interesting connection between
chelators and nanoparticles. It is evident that this connection
enables a much better influx of chelators into the brain,
and also a better elimination of generated complexes(metal-chelator-nanoparticle) out from the brain, across theotherwise hardly permeable BBB. In this way the study
contributes, not only to the insight into actual AD therapy,
but also gives a great contribution to the knowledge andunderstanding of the complex pathology and pathophysiologyof this disease ( Fig. 1 ).
The results of the available reviewed studies undoubtedly
indicate the crucial role of oxidative stress, induced by metals,
in AD pathophysiology. The role of iron ions is especiallyemphasized. By electrostatic forces attracted and boundto the β1 chain of the appical Aβ monomer of the forming
protofilament (Aβ monomers fibrillization), nontoxic ferricion (Fe 3 + ) is reduced into toxic ferrous ion (Fe 2+ ), which in
the Fenton reaction (Fe 2+ + H 2 O 2 = Fe 3 + + ¯OH + *OH) is
oxidized by hydrogen peroxide (H 2 O 2), with the formation of
the extremely destructive and toxic hydroxyl radical (*OH).
The final results are the wasting oxidative deteriorationsof the surrounding molecular structures. The results of theanalysis indicate the favourable therapeutic effect of metalchelators, especially conjugated with nanoparticles.
The following two tables ( Tables 1, 2 ) show importantdesirable and undesirable effects of actually utilized chelators,
especially iron chelators. Table 3 shows the effects of metalchelation therapy through the use of nanoparticles.
Fig. 1 shows the schematic presentation of thenanoparticle-chelator conjugate passage, enabled by means of
endocytosis, across the BBB.
It is clearly visible that Fig. 1 is divided into three parts,
A, B, and C. In the upper part (A) on the left side visible is theisolated nanoparticle (green circle) with designated functionalgroups necessary for the conjugation with the chelator (black
points). Nanoparticle surfaces can be coated with Polysorbate
80. It is a nonionic surfactant and emulsifier often usedin foods and cosmetics, and is a water soluble synthetic
compound. Nanoparticles coated with this compound (Tween
80, T - Ω80) can be very easily delivered to the brain. The
surfactant (nanoparticle stabilizer) adsorbs on the surfaceof the nanoparticles and provides steric stabilization tothem. During the brain targeting, the interaction between
T-80 coating and brain micro-vessel endothelial cells isessential. APOE is a fat binding protein, very important for
the formation of lipoproteins. It has a crucial role in the onset
and course of EOAD, it accelerates the delivery of the fullcomplex chelator-nanoparticle across the BBB. Transport
across the BBB is enabled by the low-density lipoprotein(LDL) receptor and the complete complex of endocytosis.
On the left lower part (B), the full complex partially enclosed
in the endosome is visible. Additionally, nanoparticles
have a very thin surrounding interfacial layer. This layer isessential for thermal conductivity. Recently, nuclear magnetic
resonance (NMR) experiments indicate that the thickness_ 10 5 _Chelation Therapy for Alzheimer's Disease

Fig. 1. Schematic presentation of nanoparticle-chelator conjugate entry across the BBB: transcellular pathway.

AD, Alzheimer's disease; BBB, blood brain barrier; APOE gene, protein coding gene, important for making the protein
apolipoprotein E; ApoE, Apolipoprotein E, a class of proteins important in cardiovascular and AD; clathrin; AP-2 complex;
endocytosis; LDL-R, low-density lipoprotein-receptor.

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Table 1. Desirable effects of iron chelators.

High affinity for iron;Some benefits in AD therapy;(L1, Deferiprone), high oral activity, marked lipophilicity;
High BBB penetration ability;

Curcumin: ability for binding toxic metals and formation of tightand inactive complexes; anti-inflammatory property; strong radical
scavenging efficacy; strong neuroprotective role; strong protection

against oxidative stress; it leads to various improvements in the AD

pathology; it can clear amyloid plaques; it easily passes across the BBB;
relatively safe; biologically active polyphenolic compound;clioquinol (iodochlorhydroxyquin) copper and zinc specific chelator;
BBB penetrable; dissolution of β-amyloid plaques; delay of cognitive
decline in treated AD patients; it significantly decreases iron ion induced aggregation of Aβ42;(DFO), desferrioxamine; (EDTA), ethylenediaminetetraacetic acid;(clioquinol), iodochlorhydroxyquin; clinical improvement in AD patients;(DFO) prevents formation of β-pleated sheets of Aβ42 - in the presenceof Al (III) and Fe(III); EDTA prevents this formation only in the presence of Zn(II) and Cu(II);metal chelation can prevent Aβ aggregation; it can also safely dissolve precipitated Aβ peptides;
inhibition of amyloid fibrillization and disintegration of matured

amyloid fibrils; decreasing of amyloidogenic cytoxicity; relatively easy
passage across the BBB (metal-chelator-nanoparticle);

Presentation of the desirable effects of iron chelators. The crucial points are high affinity for iron and high BBB penetration ability. The
table also points out the decrease of amyloid aggregation and inhibition of amyloid fibrillization and disintegration of matured amyloid
fibrils. There are some signs of improved status in AD patients.

_ 10 7 _Chelation Therapy for Alzheimer's Disease

Table 2. Undesirable effects of iron chelators.

Accompanying undesirable chelation of metals in various
tissues leading to serious side effects;
They have a host of adverse effects and substantial obstacles;(DFO), neurotoxicity, poor absorption in the gastrointestinal tract;(DFO), quick degradation after administration, hydrophilic nature;(DFO) induces often weak penetration across the BBB;
(L1, Deferiprone), strong hydrophilicity of the L1-iron complex
induces difficulty in removing the complex across the BBB;
clioquinol (iodochlorhydroxyquin), copper and zinc specific chelator;
association with subacute myelo-optic neuropathy; the second
generation of this chelator is under clinical investigations;
Accompanying undesirable chelation of metals in various tissues leading to serious side effects; They have a host of adverse effects andsubstantial obstacles; (DFO), neurotoxicity, poor absorption in the gastrointestinal tract; (DFO), quick degradation after administration,hydrophilic nature; (DFO) induces often weak penetration across the BBB; (L1, Deferiprone), strong hydrophilicity of the L1-iron complex
induces difficulty in removing the complex across the BBB; clioquinol (iodochlorhydroxyquin), copper and zinc specific chelator; association
with subacute myelo-optic neuropathy; the second generation of this chelator is under clinical investigations;

Table 3. Metal chelation therapy through the use of nanoparticles.

Nanoparticles are composed of natural or artificial polymers, they range in size from10 – 1,000 nm, optimally around or less than 300 nm; particles coated withsurfactants e.g. polysorbate 80 have the optimal ability to cross the BBB;
A number of studies indicate the endocytosis of polysorbate coatednanoparticles as an optimal way for transfer across the BBB; In this case very
important is the absorption of apolipoprotein E ( ApoE ) on the surface of
nanoparticles coated with polysorbate 80; LDL-receptor mediated endocytosis;
Chelator (chelating agent) is a compound which can form several bonds
to a single metal ion (e.g. DFO, Deferiprone or L1, Deferasirox, Curcumin etc.);
Chelators conjugated to nanoparticles have the ability to cross the BBB;This complex by the same route can relatively easily exit from the brain;
A chelate is a chemical compound composed of a metal ion and a chelator(chelating agent);
Nanoparticles have the potential to transfer chelators in and out of the brain;
Chelation therapy is a safer and more effective method of reducing the metal loadin the brain;
Evident attenuation of harmful effects of oxidative damage (oxidative stress);
Reduced drug toxicity;
Improved biodistribution;
Improved therapeutic efficacy;
Evident good effects in AD therapy;
Nanoparticles also have a thin surrounding interfacial layer;
Clearly evident desirable effects are achieved by the combination of chelators and nanoparticles. It is important to emphasize some points:
obvious attenuation of harmful effects of oxidative damage, reduced drug toxicity, improved biodistribution, improved therapeutic efficacy,and evident good effects in the course of therapy.

_ 10 8 _Glycative Stress Researchof the ordered layer is around 1.4 nm (14Å). It is important
to emphasize that the clathrin-mediated endocytosis is theessential internalization pathway for the nanoparticle-chelator
conjugate. Clathrin is a protein and has a fundamental rolein the formation of coated vesicles. AP2-adaptor complex is
crucial in the process of clathrin mediated endocytosis. It is
composed of four components, α-adaptin, β2-adaptin, μ2chain, and σ2-chain. The right lower part ofof Fig. 1 presents
the fully developed endosome, which completely encircles thenanoparticle-LDL complex.
Liu G et al. 1) in their study present the mechanism
by which nanoparticles deliver chelators across the BBB.
They emphasize the great importance of clathrin-mediatedendocytosis and apolipoproteins E and B (ApoE, ApoB),and especially of the nanoparticle mimicry with LDL. Fig. 1
exactly explains the first phase of events. The second phase,the removal of the formed complex, the nanoparticle-chelatormetal, out from the brain, can be explained by the analogous
carrier-mediated transport system, but with the absorbed
Apo-A1. LDL receptors have been found on both luminal andabluminal sides of the BBB endothelial cells.
By NMR technology measurement of silica (SiO 2, silicon
diokside) nanoparticle ( 203 nm Ø) surface functional groups,
Yuan L et al.15) have determined that on a surface area of 1
nm2 there are 4 hydroxyl (-OH), 3,7 amine (-NH2), and 2, 3
carboxyl (-COOH) groups. They emphasise the crucial roleof these groups for binding with chelator adequate structures.
Fig. 2 shows the chemical structure of some chelators
tested in AD.

Discussion

The etiology of AD

In a number of studies about the etiology of AD, it has
been evidently established that the unusual aggregation ofAβ peptide monomers into highly ordered-β-sheet structures,and consequent amyloid fibrils, are the crucial cause inthese events 16, 17). Without entering the detailed polygenetic
etiology of this disease, it is necessary to emphasize theexistence of its two essential forms: the early form (EOAD,
early onset AD, 5% of all cases) which develops before theage of 65, and the late form (LOAD, late onset AD, 95%of all cases) which develops after the age of 65. They bothprovide practically the same clinical picture 4, 18).
The mutations of the gene complexes: APP (β-amyloid
precursor protein gene, 21q21.3); PSEN1 (presenilin-1 protein
coding gene, 14q24.2); PSEN2 (presenilin-2 protein coding
gene, 1q42.13), and BACE1 (βsecretase-1 protein coding
gene, 11q23.3) lead to EOAD, and mutations of APOE4(apolipoprotein E coding gene, 19q13.32) and ADAM10(ADAM metallopeptidase Domain 10 protein coding generegulator of α-secretase activity-gene, 15q21.3) lead to
LOAD 4, 19).
In general, quite inefficient results of the recent
standard AD therapy (AChE inhibitors: Aricept, donepezilhydrochloride; Exelon, rivastigmine; Reminyl, galantaminehydrochloride; and NMDA receptor antagonists, Ebixa,
memantine) have implemented the necessity of searchingfor new medicaments, and then relatively successful effectsof metal chelators were shown. Unfortunately, a number of
their undesirable and side effects interlope the search formore successful solutions, and these were obtained by theuse of the combination of metal chelators with nanoparticles.
The composite of a chelator with a nanoparticle induces
a relatively easy passage of this complex across the BBB,
the binding of the metal ion, and a very easy efflux ofthe full complex, chelator-nanoparticle-metal, out of thebrain 1, 3, 7, 13, 14, 19, 20).
What are metal chelators and what is the essence of theiractivity? Iron chelator, the chemical compound, by natural or
artificial origin, has a strong affinity for iron ions, and also
shows a certain affinity for other metals, which can lead toundesirable effects. By binding with free iron ions, primarily
with Fe 3 + , and by their drawing out from the formed
amyloid fibrils, the chelator reduces their concentration in theneuropil, with concomitant reduction decline of the Fe 3 + in
Fe 2+ and the generation of harmful hydroxyl radical *OH.
Bandaged by the covalent bond with the chelator, metal ionscan be, through the BBB, thrown out of the brain. Whichcharacteristics must a good chelator have? According to
Budimir A 2), a good chelator must have small molecularweight, small electrical charge, selective affinity for specificions, good bonding ability with metals, good ability for metalbonding in protein aggregates with their dissolution, andfinally small toxicity and minimal side effects. Again, it isimportant to emphasize, which is often confusing, that thechelate is a conjugate of a metal and a chelator (Tables 1, 2).

Metal-chelator-nanoparticles

What are nanoparticles? In the introduction it ismentioned that they are small particles formed from natural
or artificial polymers (a large molecule made up of many
repeated subunits), which bound with chelators can easily
pass through the BBB and adequately bind (chelation) metal
ions with consequent efflux of the formed complex (metalchelator-nanoparticle) also through the BBB out fromthe brain. These nanoparticles are produced by various
companies. Dimensions of these particles fluctuate between
10 - 1,000 nm (100 -10,000Å - the optimal value is around
300 nm [3,000 Å]). If a thin coat, as with polysorbate 80(nonionic surfactant and emulsifier often used in foods
and consmetics-synthetic compound) is added, this enables
nanoparticles to covalently bind with a chelator, and later
also with a metal, and an easy penetration through the BBB 1).
Nanoparticles have a surrounding interfacial layer withfunctional groups responsible for binding with the chelator(carboxylic groups, amine groups). Generally, the thicknessof the ordered layer is approximately around 1-1.4 nm (10 -14
Å). It is an integral part of the nanoparticle, fundamentallyaffecting its properties. It consists of ions and inorganicand organic molecules. Organic molecules are nanoparticle
stabilizers and protectors. Nanoparticles are the bridge
between bulk materials and atomic or molecular structures.
In relation to the bulk material, they have different andusually unexpected properties. On the other hand, chelators
also have functional groups for binding with nanoparticles.
The interfacial layer has nanoscale pores. Through this layer
there is a sustained ionic flux, so it is also important forthermal conductivity ( Fig. 1 ).
Today, there are many methods for nanoparticle
production, but their description surpasses the theme of thepresented study. The reader interested in this problem canfind adequate materials in other literature or in the producer's
information.

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Natural chelators

Without entering the detailed description of a numberof chelators, this study is limited only to the effects of theirthree representatives, green tea polyphenol (epigallocatechin3-gallate, EGCG), curcumin, and desferrioxamine B (DFOB)(Fig. 2-6 ).
Debnath K et al. 14) elaborate in their study how themolecular EGCG binds with β amyloid and during thisprocess induces the inversion of fibrillation kinetics, theremodelling of formerly formed fibrils, and the decrease of
amyloid induced toxicity. Due to the weak chemical stability
and substandard bioavailability, the practical utilization of
EGCG is very difficult. On the other hand, EGCG conjugatedwith nanoparticles (nano EGCG), is even up to 100 times
more effective in amyloid inhibition, disintegration offormed plaques, conjugate stability, and in decreased amyloid
toxicity. The authors also emphasize the much easier crossing
through the BBB. Disintegration of formed fibrils on themolecular level happens through EGCG auto-oxidation,
which then subsequently reacts with free primary amyloidamine groups accompanied with the formation of Schiffbases. The nano-EGCG complex is composed of 25-160 nm(250-1,600 Å) polyaspartic acid-based micelles, connectedwith EGCG by covalent bonds and noncovalent interactions.
The analysis of Fig. 2 clearly shows the explanation ofstrong anti-oxidative EGCG effects. Abundantly present
hydroxyl groups (-OH) on the three phenol rings (A, B,
D), especially on B and D rings, make free radical targets,and give a hydrogen atom to the radicals, neutralize their
unpaired electron, and generate neutral water (H 2 O). The
loss of hydrogen atom induces the generation of the new
free radical on the EGCG backbone. This new radical has atendency for binding with a new incoming hydrogen atomand for the hydroxyl group reparation. After these events,
it is possible that the new oxido/reductive cycle repeats inlimitless manners, with permanent free radicals destruction,
especially the extremely dangerous hydroxyl radicals. Clearlyvisible are the double effects of EGCG: the anti-oxidative
effect and binding with Aβ. The later effect was mentioned
in the upper paragraph 14).
It is also possible that after the EGCG auto-oxidation,
which is induced by molecular oxygen (O 2 ), the produced
reactive quinone complex (quinones = oxidized derivativesof aromatic compounds), through the Schiff base addition,
involving alanine side chain, generates a covalent complex
R2C=RN'. In more detail, as a result of the EGCG autooxidation, one hydron (H +), one EGCG radical (EGCG*), andone superoxide radical anion (O2 * – ) originate. O2 accepts
one electron (e – ) and transforms into O2 * – . Hydron (it hasa very short life), which is very reactive, also accepts one
electron and transforms into the H atom. It is also possiblethat two hydrons form a H 2 molecule. The double bond (=)
between the two nearest C atoms changes its position andbinds to the active oxygen. The carbonyl group is formed.
Consequently, the Schiff base formation and cross link withfree amine group on the adjacent Aβ monomer alanine side
chain is possible.
EGCG binds directly with an unfolded natural Aβ
peptide and inhibits the process of peptide aggregationand fibrillization. The result is the formation of nontoxic

disordered Aβ oligomers. The EGCG binding to the Aβ
peptide can occur in three ways: through hydrophobic
interactions, through H-bonds, and through the formation of
Schiff bases. The Schiff base formation with the consequentcross-linking of fibrils, prevents the fibril dissociation andthe discharge of the build-up of amyloid monomers. In thisway, the occurence of the new aggregation of free monomers,
their fibrillization, and the generation of additional amountsof harmful hydroxyl radicals is prevented. It is important toemphasize once again that free Aβ-monomers have a strong
intrinsic tendency to aggregate.
Additionally, it is evident that EGCG remodels matureamyloid fibrils and converts them to amorphous protein
aggregates, less toxic to surrounding structures. It seems thatduring this remodelling, the accompanying Fe 3 + chelation
by EGCG has a very important role in this process. Also,
investigations demonstrate that the hydrophobic binding sites
in the amyloid fibril are important for EGCG binding and thementioned remodelling. EGCG amyloid fibril remodelling isdependent on its auto-oxidation. Oxidized EGCG molecules
react with amyloid fibril free amine groups by the formationof Schiff bases and by the cross linking of these fibrils 21-23).
The EGCG binding place with nanoparticles can be
attributed to the hydrogen bond interactions between N
atoms on the nanoparticle surface and H atoms in hydroxyl
groups of EGCG (C-N.....HO-C) ( Fig. 2 ).
EGCG is a polyphenol component obtained from theleaves of the plant Camellia sinensis. Other components aresome polyphenols (epicatechin gallate and epicatechin) andflavanols, three kinds of flavonoids (kaempherol, quercetin,
and the highest quantity of Myricetin). Extracts from theCamellia sinensis leaves were for a long time used in traditional
Indian and Chinese medicine in the treatment of AD ( Fig. 2 ).
In their study, Hedge ML et al. 7) present a detailed analysisof the curcumin natural compound, which, as it is observed,
can signifficantly diminish the intracerebral iron concentrationand slow down the course of AD. It is found that curcuminis the dominant component in the old indian spice tumeric,
together with the two curcuminoids, demethoxycurcumin
and bisdemethoxycurcumin. Recent investigations of thiscompound indicate its ability to strongly bind metal ions, at thesame time forming compact inactive complexes. The strong
anti-inflammatory efficacy of curcumin is also observed. The
analysis of the curcumin molecular structure indicates thatthe enol and phenol hydroxyls have the ability to form metal
complexes. The formation ofthese complexes with Cu andFe, accompanied with the marked cleaning of free radicals
shows curcumin's strong neuroprotective ability. Its significant
decrease of metal toxicity is evident, as well as a high antioxidative efficacy. All facts indicate that curcumin can beeffective in AD therapy; it is also valued for the mentioned
EGCG. The authors of the study emphasize the yet unknown
etiology of AD, and the evident role of metal ions (Cu 2 +, Fe 3+,Zn, and Al) and oxidative stress, as crucial factors in the AD
pathophysiology. They stress again the importance of the brain
accumulation of these ions in a number of neurodegenerative
diseases, especially in AD.

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Fig. 2. Chemical structure of some chelators tested in AD.

Presentation of the chemical structure of some chelators tested in AD. Clearly visible is the structure of desferrioxamine (DFO),
clioquinol (CQ, PBT1), VK-28, EGCG; some facts about DFO and clioquinol are presented in Table 1- 3. AD, Alzheimer's
disease; MW, molecular weight.

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Chelation Therapy for Alzheimer's Disease

Fig. 3. Chemical structures of (A, B) desferrioxamine B (DFOB) and (C) ferrioxamine B: Formation ofhexadentate octahedral complex.
r, radius; Ø, diameter; metal; chelator, nanoparticles; ccb, coordinate covalent bond.

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Fig. 4. Schematic presentation of ferrioxamine molecular structure.

A metal ion is visible in the center of the octahedron; six oxygen atoms comprise a square. O, oxygen; N, nitrogen, H, hydrogen; C, carbon;
small dots, electrons; ferrioxamine is desferrioxamine with conjugated Fe 3+ ion; ccb, coordinate covalent bond.

Fig. 5. A, B, C coordination of the three curcumin ligands to the Ferric ion (Fe 3+).

Curcumin (polyphenol compound (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5 -dione); It can cross the blood-brain-barrier(BBB), strong anti-inflammatory and anti-oxidative effects, can dissolve Amyloid plaques in AD, beneficial effects on the course of AD,
chelation effects binds effectively with redox-active metals like Fe and Cu, formation of curcumin-metal complexes, strong neuroprotector.
Curcumin is found in turmeric, the spice that comes from the root Curcuma longa, a member of the Ginger family, Zingiberaceae. The
curcumin bidentate fashion and octahedral geometry is visible; ccb, coordinated covalent bond; AD, Arzheimer’s disease.

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Fig. 6. Hypothetical schematic presentation of curcumin approach to the amyloid beta (Aβ) protofilament.

The perpendicular view is visible from the protofilament longitudinal axis; Z-axis is not presented; electrons; curcumin is a
polyphenol compound; R, repulsion; A, attraction; N, nitrogen; O, oxygen; C, carbon; H, hydrogen; Fe 3+, ferric ion; F, electrostaticforce; Q, charge; r, distance; (1), hydroxyl group oxidation; (2), Hydrogen attraction-water generation; (3), new H approach;_ 114 _Glycative Stress Research

β-Amyloid accumulation

They also accentuate the connection of AD with
β-amyloid accumulation.The role of Al (aluminium) is stillquite unknown. The mentioned researchers are especially
included in the problem of the two natural metal chelators,
curcumin and epigallocatechin, which, as it is confirmed,
decrease the level of Fe in the brain and suppress the courseof AD.
Bonda DJ et al. 3) also consider that the current AD
therapy with AchE inhibitors is not satisfactory. This therapy
can only weakly mitigate AD symptoms, with the delayof the inevitable decline of important cognitive functions.
They also emphasize the significant rise of costs connectedwith its prevention and therapy. According to them, allthis indicates a strong necessity for a new approach to AD
therapy, which accentuates chelation therapy. Oxidative
stress, induced by a number of undiserable events, is a
crucial factor in the AD etiology. Oxidative damage of
mitochondrial DNA, important proteins and phospholipids,
are significantly greater than in healthy people. Due to thesignificance of transition metals in the induction and strengthof oxidative stress, the importance of metal chelators andchelation therapy is obvious. Among these compounds, theauthors especially emphasize desferrioxamine (DFO), thehexadentate iron chelator, which strongly binds Fe 3 + andmoderately Al, Zn, and Cu. Clinical trials indicate that DFO
slows down the rate of AD progression. However, the great
molecular weight and hydrophylicity, which is typical forhexadentate chelators, disrupts the passage across the BBB.
The authors consider that smaller metal chelators can passthe BBB much more easily, but present signs of toxicity. The
solution to this problem is found in metal chelators and intheir conjugations with nanoparticles. The earlier mentioned
nanoparticles are in fact polymer particles, of magnitude 101,000 nm (100 -10,000Å), optimally around 300 nm (3,000Å).
They are made from natural or artificial polymers. With theability to strongly bind with chelators, they as conjugates caneasily pass across the BBB into the brain, and then out of it.
This crossing through the BBB is enabled by endocytosis( Fig. 1 ). In this way, hydrophylic chelators can also passacross the BBB. Conjugates are significantly less toxic andside effects are rare and weak. They also successfully prevent
Aβ-aggregation, which, as is more and more evident, is theprimary cause for AD toxicity. According to these authors,chelation therapy has a promising future.
Liu G et al. 6) demonstrate that DFO has shown somegood effects applied to AD therapy, but as a lot of otherchelators, has shown many undesirable side effects andimportant obstacles for an undisturbed chelation therapy.
Consequently, they combined DFO with nanoparticles, whichevidently facilitates the treatment and improves the effects.
Among the undesirable effects, it is important to mention thepain in the place of injection, postinjection febrility, hearing
loss and problems with sight, diarrhoea and vomiting,pressure drop and allergic reactions. DFO has been on themarket in USA since 1968.
Fig. 2 shows the chemical structure of some chelators
tested. Electrostatic forces around the central ferric ion(Fe 3 +) form strong octahedral complexes, which, conjugatedwith nanoparticles, relatively easily pass across the BBB. For
the complex formation, DFO needs one of its own molecule(hexadentate chelator), deferasirox (DFX) needs two of its
own molecules (tridentate chelator), and deferiprone (DFP)
needs three of its own molecules (bidentate chelator) (Fig.

3-5). The formation of the octahedral metal complex is
explained in detail by Barić and Cetina 24).
The exact understanding of the chelation process
requires a detailed insight into the metal ion complex
structure and into the electrostatic forces involved inthese processes. It is evident that at the begining there is adominance of attractive electrostatic forces developed in
the electrostatic field between the positive central metal
ion and corresponding negative chelator points. Repulsive
forces between metal d-electrons and negative ligand charges(chelator components) are in this phase evidently small. Dueto the increasing approach of the future octahedron elements,
the ligand field and complex with Fe3+ in the centre acquire
the form of an octahedron, energetically the most favourable
structure (Fig. 3-5 ). In fact, the octahedral complex is theresult of composed electrostatic interactions between thepositive metal centre (here Fe 3 + ) and negative charges ofligand ion pairs. If the ligands are neutral polar molecules,
the metal centre reacts with the ligands negative ends (iondipole interaction – attractive force). Furthermore, ligands
interact with one another in an electrorepulsive manner 24).
Analysis of Fig. 3 and Fig. 4 clearly shows the position
for binding with the nanoparticle, the NH2 group on one
end of DFOB molecule. As an example, the conjugation ispossible with gold nanoparticles (colloid). Data from thenanoparticle producer,"BBI Solutions", serve for the practicaluse of gold nanoparticles which range from 2nm to 250 nm.
Nanoparticles with the diameter of 2 nm have a mass of one
particle 8.08 x 10 –20 g , and with a diameter of 200 nm, amass of 8.08 x 10 –14 g.

ROS involvement

The analysis of the formation of the chelate composedof the metal ion Fe 3 + and curcumin (chelator) indicates thatin fact there exists a synchronised approach and binding of
three bidentate chelator units (Fig. 5 ). Two units approach thecentral Fe 3 + mutually parallel in the altitude of the equatorial
square plane ofof the complex, and the third unit, locatedin the square equally distant from the first two, approaches
perpendicularly towards the two units.The distance from thecomplex Z-axis is determined by the electrostatic equilibriumin the complex. According to recent literature, two ways ofbinding to Fe 3 + are possible.
Khalil MI et al. 25) in their paper present the exampleof the bidentate coordination of curcumin ligand to the
Fe 3 + ion. According to their opinion, the three curcumin
ligands are coordinated to the Fe 3 + ion in a bidentate fashion
forming the octahedral geometry. In fact, here the proton
discharge from the curcumin enol form occurs (Fig. 5 ).
The speculation about the possibility of the metal complex
formation through the phenolic hydroxyls may be also
correct 7). FTIR (Fourier Transform Infrared Spectroscopy)
studies of metal complexes by Banerjee R 26), indicatethat although the metal chelation can occur through theo-methoxy phenol and the β-diketo group, in most cases
the complexation of curcumin with metal ions involves thediketo group. Although the mechanism of curcumin actionin the therapy of AD is yet insufficiently known, the authoremphasizes its anti-oxidative, anti-inflammatory, anti-cancer,
and neuroprotective characteristics. She also emphasizes the
crucial role of oxidative stress and reactive oxidative species(ROS), which further induce ROS generation and detrimental
neurodegenerative damage. The author also emphasizes thepossibility of good effects of curcumin metal complexes on_ 115 _

Chelation Therapy for Alzheimer's Disease

the course of aggregation and amyloid peptide fibrillization.
The slowing down of the course of aggregation andβ-amyloid fibrillization demand a separate analysis. In thissense, the author of this study (Barić N) presents some of
his own reflections. Attracted by the attractive electrostaticforces (Coulomb's law) of the central metal ion (Fe 3 +),curcumin approaches the octahedral complex ion which isformed between two neighbouring harmonized monomers.
The approach occurs from the side of the Aβ β1 strand in thelevel of the octahedron equatorial square plane. There exists
the attractive electrostatic force between the central metal ion(Fe 3 +) and two lone pairs on the chelator (curcumin) oxygens(electrostatic field). When chelator (curcumin) oxygens havearrived (movement from position A to B) near the octahedral
complex ligands on the equatorial plane-square (N atoms
on the two imidazole rings), a rise in the repulsion between
electrons of O and N occurs, and also a rise in the value ofthe negative charge (q+q) due to the two closely approachednegative charges (O and N). Now we have a rise in theattractive value between the four (4) attractive forces and thecentral metal ion (the anterior part of the complex ion). At
the same time, there is no rise in the attractive forces in theposterior part of the complex. The small movement of theattracted Fe3+ in the anterior direction induces the decline in
the strength of the two posterior and Z-axial attractive forces.
Now there is the possibility of the Fe 3 + movement in theanterior direction toward the curcumin, and its binding with
it. Generally, the repulsive forces are weak. This movement
induces protofilament destabilisation and destruction,
and Fe 3 + bound with the chelator and nanoparticle, areadditionally removed from the brain (Fig. 6 ).
Fig. 6, among other things, shows the hydroxyl radical(*OH) attack on the curcumin phenol ring hydroxyl group(1), its oxidation (hydrogen loss), water formation (2), andfinally the new hydrogen atom approach - reduction (3). The
oxidation/reduction process continues.

β-Amyloid aggregation

In their study, Kochi A et al. 27) declare that AD
progression is connected with the accumulation of protein
aggregates, metal ion dyshomeostasis, and oxidative stress.
All these events are accompanied with metals bound withamyloid β (Aβ), which clearly indicates the connection of AD
pathology with these complexes. Curcumin and its derivatives
show evident anti-amyloidogenic activity. On the other hand,their weak solubility and stability in physiological conditions
lead to their difficult application. The results of this group of
authors indicate that specific curcumin derivates, especially
Gd (gadolinium, at. Number 64) – Cur, have prevailed overthese deffects with the evident decrease of the Cu (II)triggered Aβ aggregation intensity.
Yang F et al. 28) also emphasize the close connection
between the Fe 3 + ions chelation process and Aβ aggregationas central events, both in the pathophysiology, and in AD
therapy. By aggregation from the isolated Aβ monomer,
dimers, olygomers, protofibrils and fibrils can be generated.
Curcumin can also, without any contact with Aβ and withconnected Fe 3 +, bind the free Fe 3 + and stop its binding
with Aβ. The authors present the following curcumincharacteristics: strong binding with Aβ, strong aggregation
inhibition, anti-inflammatory and anti-oxidative effects.
They also emphasize the blockade of Aβ accummulation in
the brain accompanied with the disaggregation of fibrilar
Aβ. The delay of cognitive defficit is evident.

Aβ aggregation as the central link in the AD
pathophysiology is also quoted by a number of investigators
14, 16, 20, 24, 26). Finder VH et al. 29), Etienne MA et al. 30), Ahmed
31)
M et al. are also interested in this problem.
In their study, House E et al. 32) present an excellent
example of the importance of Aβ aggregation on thecourse of AD, as well as the effects of metal chelators
DFO and EDTA on these events. Following the courseof Aβ42 fibrillization connected with the addition of Al(III), Fe (III), Zn(II), and Cu(II), during 32 weeks, theauthors have observed the disolution of formed peptide
aggregates in the presence of desferrioxamine (DFO) andethylenediaminetetraacetic acid (EDTA). Either alone, or in
the presence of Al(III) or Fe(III), Aβ has formed aggregates
which have been dissolved with one or the other chelator.
This indicates that chelation of Al(III) and Fe(III) makes aprotective mechanism against the occurence of AD and its
progression.
During the aggregation process, the oblique, nonparallel
approach of two Aβ monomers most probably occurs, withclose contact of a MBD (metal binding domain – primarily
His13-His14) on one, and MetS35 (methionine sulphur 35) on
the other monomer. During these events, an electron "hop"from MetS35 on the incoming monomer to the Fe 3 + boundon the MBD of the fixed monomer is possible. The result
is the reduction of Fe 3 + into Fe 2 +, the Fenton reaction, andgeneration of extremely aggressive and toxic *OH. Afterthe establishment of the parallel congruent relation of theapproaching monomers, the distance between the two redox
centers becomes greater and the oxido/reduction events cometo an end. Fe 3 + remains fixed in the interspace between two
congruent monomers forming the center of the octahedron.
The situation is now mature for the curcumin attack on thislast balanced Fe 3 + position, with its extraction, chelation,and destruction of the formed protofilament, respectively the
fibrile 24).
In their tutorial review, Wanninger S et al. 33) present an
overview of metal complexes containing curcumin and its
derivatives. In detail they elaborate on the synthesis, crucial
characteristics, and application of curcumin metal complexes
and curcumin derivatives as ligands. The metal complex
is built from the central metal ion to which are bound one
or more ligands. The authors are especially interested in
the medical use of these complexes. They emphasize threespheres, which are included in the ivestigation of malignant
diseases, AD, and anti-oxidative/neuroprotective activity. The
study presents a detailed figurative review of the curcumin
molecule and its derivatives as the components of metal
complexes. The authors consider that curcumin, alone orbound with specific metals, in the future will have a growing
importance in the dignostics and therapy of a number of
diseases, especially AD.
Ferrari E et al. 34) consider that the most recent actual
curcuminoids make a new perspective chapter in AD
treatment, and especially emphasize their complexing
ability. Their biological properties can be compared withthe properties of pure curcumin, only their stability ishigher. K 2 T derivatives are obtained by the inclusion of
the -CH 2COOC(CH 3) 3 group onto the central atom of thediketone group. Depending on the solvent, their diketoketoenol tautomerism is retained. The formation of metal
complexes is analysed by NMR and ultra violet (UV)-vis
spectroscopy.
Ferrari E et al. 35) in another study show how
curcuminoids coordinate metal ions through the keto-enol_ 116 _Glycative Stress Researchfunction. They also emphasize that curcumin anti-oxidative
ability is mostly dependent on the phenol group presence,
while the coordination of the keto-enol part does not have amajor influence.
Priya RS et al. 36) in their study explain how the twohydroxyl groups attached to the phenol ring and β-diketo
part of the molecule, by the conjugation system connectedwith rings, are the active centres of curcumin. Two phenol
centres with their - OH groups have anti-oxidative properties,
and the β-diketo part makes the metal chelating center.
This part has a chelation ability for a lot of metal ions withthe possibility of metal complex formation. In this way, theprevention of extremely deteriorative lipid peroxidationis possible. As a strong anti-oxidant, curcumin effectively
declines the formation of amyloid plaques, which is very
important in the development and course of AD. Curcumin
biological activities, which are anti-inflammatory, anticholesteroletic, and anti-Alzheimeric, are the consequenceof its anti-oxidative properties. At the same time curcumin
can act as a metal chelator and as an anti-oxidant. Curcumin
metal complexes also have an anti-oxidative activity and they
do not decrease its biological ability.
Lee WH et al. 37) consider that the brain, due to its
great oxygen consumption, the great concentration ofpolyunsaturated fatty acids, and of redox active transition
metal ions, makes a system very sensitive to oxidative
stress. In the situation of the evident rise of pro-oxidative
ROS compounds, which are present in the case of AD, an
imbalance of the sensitive equilibrium of ROS/defensive
anti-oxidative compounds occurs, with the phenomenon ofdangerous brain structure deterioration. These damages areaccompanied with strong Aβ aggregation, more expressed by
the consequent formation of ROS, and with the decline of the
ACh level. Curcumin is presented as a strong anti-oxidative
compound, able to cleanse brain tissue of free radicals
generated by oxygen, and to stop Aβ plaque formation
and activation of fibril disintegration. Curcumin also has
favourable effects on Parkinson's disease (PD) by decreasing
the concentration of toxic α-synuclein aggregates and ROS
compounds. It is also effective in the treatment of malignant
brain tumors. The authors in detail analyse the chemical
structure of curcumin. By tables they present the fascinating
facts of curcumin therapy in traditional medicine and inrecent applications.
Elmegeed GA et al. 38) based on their investigations,consider that hybrid drugs which simultaneously act
on more targets are much better than monofunctional
drugs in the therapy of the complex AD pathophysiology.
During these investigations this group has made complex
compounds composed of the steroid part and the curcumin

molecule. By adequate synthesis of specific compoundswith the heterocyclic nucleus and the necessary curcumincomponent, they tried to elucidate the effects of these new
AD therapeutics. Their experiments were performed on
albino rat females, and they showed the clearly favourable
effect of specific compounds on the animals diseased by AD.
These experiments revealed elevated values of brain ACh,
GSH (glutathione reduced), paraoxenase, and BCL2 (2B-cell
lymphoma). At the same time, decreased activity of cerebral
AChE and the level of P53 (tumor protein) was found. A
decline of 8-OHG (8-hydroxy guanosine) values was found in
the urine, and a decline of Caspase-3 was found in the blood
serum. The hystological analysis of the brain tissue indicatesa strong rise of ChAT (cholineacetyl transferase) positive
cells. All of these compounds have shown a marked anticholinesterase potential with a strong anti-oxidative and antiapoptotic activity. Future investigations are necessary, as wellas considering the possibility of the use of these compounds
in the therapy of diseased people.
In the end, after this detailed presentation of chelatorsand chelation therapy, it is necessary to emphasize thegreat importance of the daily intake of accessible naturalor synthetic chelators, anti-oxidants and neuroprotectors,
especially for the middle aged and aged population. The
possibility of their purchase on the pharmaceutical andnatural markets is evidently getting better. However, theauthor of this study considers that professional medical
consultation is necessary when deciding about the use of
these compounds. This preventive approach is effective not
only in the treatment of AD, but also in the treatment of theaccelerated pathological aging process.

Conclusion

AD is a wasting chronic neurodegenerative disease,with a fluctuating progressive course and a lethal end. The
current therapy with cholinesterase blockers and NMDA
receptor blockers is not satisfactory. The increasingly
frequent therapy with metal chelators, especially ironchelators which can also be combined with nanoparticles,
formed from natural and artificial polymers and with theability to easily pass across the BBB, gives promising results
and requires further investigations.

Conflict of interest

The authors declare no conflict of interest in this study.

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