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Polymeric nanocarriers for the treatment of systemic iron overload Hamilton, Jasmine L; Kizhakkedathu, Jayachandran N Mar 24, 2015

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REVIEWPolymeric nanocarriers for1,2(ICveorhenclize ls storcome by evolving to conserve iron [1-3]. Organisms haveacquired highly organized mechanisms of iron acqui-and approximately 1–2 mg of iron is lost daily due tothe sloughing off of epithelial cells, secretions from theHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 DOI 10.1186/s40591-015-0039-1binding proteins which ensure that it is unable to cause2Department of Chemistry, University of British Columbia, 2350 HealthSciences Mall, Vancouver, BC V6T 1Z3, Canadasition, transport and storage; microorganisms use lowmolecular weight (MW) high affinity iron ligands orsiderophores, while more complex life forms like mam-mals use specialized storage and transport proteins [3].skin and gut, and small losses of blood from the gastro-intestinal tract [2,3]. This indicates the conservative na-ture of iron metabolism and recycling. The process ofiron recycling and metabolism is schematically demon-strated in Figure 1.Under normal physiological conditions, iron is com-plexed with proteins like transferrin (Tf) or other iron* Correspondence: jay@pathology.ubc.ca1The Centre for Blood Research, Department of Pathology and LaboratoryMedicine, Vancouver, BC V6T 1Z3, Canadasome unique advantages that these nanomedicines have in treating systemic iron overload as well as their potentialutility in the treatment of other disease states.Keywords: Iron overload, Iron chelation therapy, Iron chelators, Desferrioxamine, Deferiprone, Desferasirox,Nanomaterials, Polymeric chelatorsReviewIronIron is essential for oxygen transport, DNA synthesisand energy metabolism [1]. Thus, it is life sustaining invirtually all living organisms. The usefulness of iron re-sults from its ability to cycle between its ferrous (Fe2+)and ferric (Fe3+) forms in oxidation and reduction reac-tions [1,2]. Although iron is abundant in the earth’scrust, Fe2+ is highly toxic, while Fe3+ is insoluble inaqueous solution at physiological pH, rendering it in-accessible. As a result, obtaining bioavailable iron is acontinual challenge which living organisms have over-Under normal physiological conditions, the humanbody contains 3.5-5 g of iron, with the majority (over70%) existing as hemoglobin [3]. The remainder is foundin myoglobin, intracellular storage iron in the hepato-cytes of the liver, spleen and bone marrow macrophages,and in proteins and enzymes that are involved in cellularrespiration [1-3].Iron metabolism is highly conservative in man, withthe efficient recycling of hemoglobin iron forming themajor component of iron regulation. Intestinal iron up-take also plays a key role in maintaining human ironhomeostasis; 1–2 mg of dietary iron is absorbed dailysystemic iron overloadJasmine L Hamilton1 and Jayachandran N KizhakkedathuAbstractDesferrioxamine (DFO), deferiprone (L1) and desferasiroxtreat secondary iron overload. Although iron chelators haimprovement in available therapy, there is still the need fand drug toxicity. Moreover, all currently approved iron cthe objectives reported for the “ideal” chelator of low MW, iwithout causing toxic side effects, has proven difficult to reamay develop toxicities or become insensitive. In contrast, thhigher MW, polymeric, long circulating iron chelators hachelators toward longer plasma half-lives and reduction into the currently available low MW iron chelators. This article© 2015 Hamilton and Kizhakkedathu; licenseethe Creative Commons Attribution License (htdistribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.Open Accessthe treatment of*L-670) are clinically approved iron chelators used tobeen utilized since the 1960s and there has been muchnew drug candidates due to limited long-term efficacylators are of low molecular weight (MW) (<600 Da) andluding possessing the ability to promote iron excretione in practice. With prolonged iron chelator use, patientsimited research that has been geared towards developinghown promise. The inherent potential of polymeric ironxicity provides optimism and may be a significant additioneviews knowledge pertaining to this theme, highlightsBioMed Central. This is an Open Access article distributed under the terms oftp://creativecommons.org/licenses/by/4.0), which permits unrestricted use,, provided the original work is properly credited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in thisHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 2 of 15free radical production [4,5]. In the plasma, iron is trans-ported by Tf and is unavailable for redox activity. Tf hasa high iron binding capacity which prohibits the accu-mulation of toxic unshielded or non-transferrin boundiron (NTBI). Tf contains 2–3 mg of iron and is hypo sat-urated at ~30% under normal physiological conditions.Tf delivers iron to hepatocytes and specific binding siteson red cell precursors of the bone marrow involved inthe synthesis of hemoglobin. Tf also captures iron re-leased into the plasma from intestinal enterocytes orcells which catabolize senescent RBCs [4,5].Within cells, ferritin is the major storage molecule forreusable iron and accounts for ~27% (1 g) of the totalbody iron in normal individuals [6]. Ferritin has a stor-age capacity of 4500 atoms of iron per ferritin moleculeand iron storage in ferritin ensures that iron is storedwithin cells in a safe redox inactive form. Therefore, fer-ritin reduces the toxicity from free radical generationFigure 1 Iron recycling and distribution in the body. Body iron is primmacrophages of the liver and spleen. Enterocytes obtain iron from the dietRBCs release iron into the circulation where it binds to plasma transferrin, tthe bone marrow and to other sites like hepatocytes of the liver, the mainpathway for iron.while ensuring that iron is also available for mobilizationfor metabolic processes. Ferritin also helps to re-establishnormal redox conditions during oxidative stress by remov-ing ferrous ions and oxygen from the cytoplasm. Underpathological conditions in which ‘iron overload’ occur, ex-cess iron is deposited as insoluble ‘iron cores’ of partiallydegraded ferritin or hemosiderin, primarily in liver, spleen,endocrine organs and myocardium of the heart [1,2].Although electron shuttling is vital in metabolic pro-cesses, under conditions of excess, iron may catalyze harm-ful reactions that generate free radicals which amplify thedevelopment of reactive oxygen species (Figure 2) [7]. Thismay occur via the Haber-Weiss reaction in which hydro-gen peroxide (H2O2) reacts with the superoxide radical(O2) to produce the hydroxyl radical (OH·), the most react-ive radical in the body [7]. Although this reaction occurs atminimal levels under normal physiological conditions, itcan be catalyzed by iron, leading to accumulation of freearily located in erythrocytes (>70%) which are efficiently recycled by. Macrophages, which obtain iron from the phagocytosis of senescenthe iron transport protein. Transferrin delivers iron to the erythron ofiron storage site in the body. There is no physiological excretionelehyedctios aHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 3 of 15radicals, which can interact with cellular componentsand disturb metabolic functions [7]. It has been shownthat the increased generation of free radicals can oxidizelipids, proteins, and DNA in major organs with the heartbeing most susceptible. Thus, the disruption of normalcellular redox equilibrium is possible with very smallamounts of misplaced iron, and the magnitude of the bodyiron burden is the most important determinant of the en-suing organ damage.Transfusion associated iron overloadIn contrast to the highly evolved methods of iron acquisi-tion, storage and transport mechanisms, the ability to off-load excess iron remains challenging as there is no knownphysiological pathway to actively excrete iron. This is themajor challenge in disease states like β-thalassemia (β-TM),sickle cell disease (SCD) and myelodysplastic syndromes(MDS) that invariably lead to iron overload [1-3,8,9].Figure 2 Harmful redox cycling of iron. Iron (Fe) can participate in oneharmful free radicals in the presence of oxygen. The hydroxide radical andferrous iron. Ferric iron is in turn reduced by the superoxide radical (O2·-). Rproducing the hydroxyl radical (OH·), which perpetuates free radical produresulting oxidative stress is associated with damage to cellular componentRed blood cell (RBC) transfusions are used to ameliorateanemia in patients with β-TM and MDS and can preventvaso-occlusive events in SCD [8,9]. Patients with these dis-orders develop severe anemia due to ineffective erythropoi-esis and hemolysis, which causes large numbers of marrowerythrocyte precursors to undergo apoptosis before matur-ity into erythrocytes. This leads to severe anemia. In thecase of SCD, there is the added risk of stroke due to thelack of deformability and enhanced stickiness of RBCswhich may cause the obstructive adhesion of sickled cellsto each other and the vasculature [9].Further, ineffective erythropoiesis results in a drasticincrease in plasma iron turnover, with the turnover ofplasma iron occurring at a rate that is 10–15 timesgreater than in patients with normal erythropoiesis [10].As a result, patients can accumulate over 2.5 g of ironannually from this process, which results in a “primary”iron overload state. In addition to this inherent iron ac-cumulation, patients receive red blood cell transfusionsfrequently, which, although highly beneficial in suppress-ing erythropoiesis and anemia, put patients at risk ofdeveloping “secondary” or transfusion associated ironoverload.Each unit of RBC contains approximately 250 mg ofiron [8]. Since humans lack an iron excretion pathway,chronically transfused patients accumulate excess iron ata rate of 0.2-0.4 mg/kg/day if transfused more than twiceper year [8,10]. This excess iron accumulation causes asaturation of the body’s iron regulatory mechanisms anda subsequent disruption of normal iron regulation.As the iron loading from transfusions increase, trans-ferrin in the plasma becomes saturated and NTBI appearsin the serum [11]. This toxic pool of partially ligated ironaccumulates in plasma and is subsequently, and in somecases, preferentially taken up by cells. For example, therate of NTBI uptake by cultured rat heart cells is greaterthan 300 times that of transferrin bound iron [10]. As thisctron oxidation and reduction reactions. This leads to the generation ofdroxide anion (OH−) are produced when hydrogen peroxide reacts withox active or “labile” iron reacts with cellular hydrogen peroxide (H2O2)n, ultimately increasing cellular reactive oxygen species generation. Thend organs [7].toxic pool of NTBI accumulates, an intracellular labileiron pool (LIP) is formed and ultimately facilitates harmfulredox damage to tissues through the formation of the freehydroxyl radical.The excess iron is accumulated primarily in the liver,spleen, endocrine organs and myocardium. The cytosolicLIP mirrors the cellular iron content and its fluctuationsare considered to trigger homeostatic adaptive responses.Once homeostatic mechanisms become saturated, excessiron can ultimately lead to organ dysfunction and death ifleft untreated [8,10,12]. Figure 3 shows some of the poten-tial effects of iron overload on major organs.Iron chelation therapy: treatment of transfusionassociated iron overloadIron chelation therapy is clinically indicated for the treat-ment of transfusion dependent patients with β-TM, SCDand MDS [8,10,13,14]. Iron chelation therapy involves theuse of molecules which can bind iron under physiologicalmsHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 4 of 15Figure 3 Excess labile iron causes damage to the body’s organ systeconditions (iron chelators) to form a non-toxic complex or“chelate” which is subsequently excreted via the feces and/urine, enabling safer body iron levels. Iron chelation ther-apy protects cells against oxidative damage by reducing thepool of reactive iron in the plasma and cytosolic LIP incells. Iron chelation therapy inhibits the lipid peroxidation,protein oxidation and cellular damage that accompaniesiron overload [15,16]. ICT is recommended after receiving10–20 transfusions of erythrocytes in order to prevent se-vere iron loading and damage in major organs [8,10]. Cur-rently, three iron chelators are approved for treatingtransfusion associated iron overload (Figure 4).DesferrioxamineThe most thoroughly characterized iron chelating drug isdesferrioxamine (Desferal®, DFO) which has been thestandard of therapy for over 40 years. Used since the1960s, DFO has demonstrated efficacy in prolonging lifeand improving quality of life for transfusion dependentthalassemic patients (8,13,17). DFO has demonstrated effi-cacy at preventing lipid peroxidation which leads to organdamage, promoting iron excretion, arresting fibrosis, sig-nificantly decreasing deaths by cardiac disease reducinghepatic iron concentrations and extending lifespan in ironoverloaded patients [12-14,16,17].and the development of toxic iron pools in cells and tissues. NTBI in the plasmorganelles, causing peroxidation, DNA damage and protein dysfunction. Thedamaged. These events ultimately lead to organ dysfunction, failure and deat. Chronic transfusion therapy results in the saturation of serum transferrinDFO is a high affinity iron (III) chelator with a log sta-bility constant of 30 for the Fe(III) complex and a mo-lecular weight of 560 Da. Due to its hexadentate nature,DFO binds iron in a 1:1 ratio producing a stable com-plex that prevents iron from producing harmful free rad-icals [18]. DFO can access iron by two methods; directlyinteracting with hepatocellular iron and subsequent bil-iary excretion, as well as from the destruction of RBCsin the reticuloendothelial cells (RES), directly or follow-ing its release into plasma as NTBI [8,10]. DFO entersthe liver via active transport and interacts with liver andextracellular iron. This leads to excretion primarily byurine as well as some biliary iron excretion [10]. TheDFO–Fe(III) complex (Figure 4) does not redox-cycleand this reduces the chances of iron redistribution andtoxicity within the body [18].DFO therapy improves lifespan and quality of life[13,14,17]. Borgna-Pignatti et al. showed that mortalityat 20 years of age had fallen significantly after the adventof DFO chelation; diabetes had fallen from 15.5% inthose born between 1970-74 when DFO therapy wasrelatively new, to 0.8% in those born after 1980, whenDFO was more frequently prescribed [17]. This stronglysupports the idea that the age at which transfusiondependent patients begins DFO therapy, as well as ad-herence to therapy may modulate risk of heart diseasea and labile cellular iron (LCI) react with cellular membranes andliver, heart, pancreas and other endocrine organs are most commonlyh if left untreated.rsHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 5 of 15and other complications. Similar studies in the UK haveshown the benefit of iron chelation with DFO in prolong-ing life, significantly reducing the incidence of cardiacdisease, liver failure and other endocrine disorders incompliant patients [13].Despite these advantages, DFO is hardly the “ideal”chelator. Due to its low lipophilicity and high MW (560Da), DFO is not readily absorbed by gastrointestinalFigure 4 The chemical structure of clinically approved iron chelatocells. In addition, it has a very short circulation half-lifeof ~20 minutes in humans and must be subcutaneouslyinfused at doses of 40–60 mg/kg for 8–12 h a day, 5–7days per week. Additionally, DFO at high doses has beenassociated with severe neurotoxicity, causing sensori-neural hearing loss, visual electroretinographic distur-bances, and impaired growth and bone development[19-21]. Thus, the use of DFO has been hindered by itsshortcomings and attempts toward generating more effi-cient iron chelators have continued.DeferiproneDeferiprone (Ferriprox®, Cipla, L1) is the second chelatorto receive approval for the treatment of iron overload. Itwas first reported as a potential orally active iron chela-tor and efficient at in vivo iron removal in 1987 and wassubsequently licensed for use in India in 1994 and Europein 1999 with special conditions [22-24]. Due to questionsregarding safety and chelation efficiency, L1 only receivedfull marketing authorization in Europe in 2002 and by theFDA in 2011.L1 is a bidentate chelator thus, 3 L1 molecules areneeded to chelate one atom of iron [22,23]. As a result,the efficacy of L1 as an iron chelator is highly dependenton the concentration ratio of chelator and iron in the en-vironment. At low L1 to iron concentrations, L1 may bindto iron incompletely. These partially bound forms of ironwith unoccupied coordination sites may accumulate andremain reactive. Furthermore, these partially chelatedforms of iron are able to catalyze the formation of harmfulradicals and other reactive oxygen species [23].In the first study reporting its efficacy, L1 was shown toand their iron (III) complexes.cause iron excretion at a rate proportional to the iron loadof the patients and the dose given in the 4 MDS and 4 β-thalassemia major patients participating in the study [22].Further, the iron excretion levels in urine were found tobe similar to that obtained with therapeutic doses of DFO.Rombos et al. reported that L1 was safe and caused a re-duction in iron overload in Greek thalassemics withoutcausing considerable side effects [24].However, several subsequent studies have shown thatL1 therapy alone may be ineffective in ensuring negativeiron balance in many patients, especially in patients withless severe iron loading [25-28]. Hoffbrand et al. foundno significant reduction in urinary iron excretion in anyof the patients enrolled in the study and no significantchange in the serum ferritin levels of more than half ofthe patients that received L1 treatment for more than 3years [28]. While Cohen et al. found that L1 can reduceand maintain body iron in some but not all patients; L1did not reduce body iron overload to a level below thatachieved by DFO in those patients that had lower base-line iron levels [26]. This demonstrates that the daily L1dose of 75 mg/kg body weight/day induces less iron ex-cretion than the standard daily dose of DFO 50 mg/kgbody weight/day.iron overload [30,31].Hamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 6 of 15Other studies show that L1 reduces serum FTN levelsin some but not all patients and that the effects of pro-longed therapy were not sustained [27]. In addition, al-though L1 can mobilize iron intracellularly and has beenshown to reduce cardiac iron it is unable to promote ad-equate iron removal and prevent death by cardiac diseasein some patients. This was well described in a review byHoffbrand et al. For example, it was reported that 9 out of532 thalassemic patients undergoing consistent L1 treat-ment for 3 years died of heart failure [25]. In addition,Hoffbrand et al. found that out of 51 L1-treated patients,4 out of the 5 patient deaths were caused by cardiac dys-function [28]. This indicates that in some patients withmyocardial iron overload and continuing need for bloodtransfusions, L1 was not reliable at preventing further ironloading [28].One of the major reasons for the limited efficacy of L1in clinical use is its rapid metabolism in the liver. The 3-hydroxyl functional group that is found on the L1 mol-ecule is required for effective iron chelation. However,this is also the site of rapid metabolism by glucoronida-tion in liver cells [18,25,27]. Studies which measured L1recovery in the urine found that over 85% of the L1 dosegiven to patients may be recovered in the urine as theinactive 3-O-glucuronide conjugate [18,25].In addition to the challenging metabolism of L1 de-scribed above, severe side effects of L1 can also be limitingwithout adequate monitoring of patients. Agranulocytosisis considered to be the most serious side effect of L1 use[8,10,26-28]. Milder neutropenia is also common, occur-ring in up to 4.8% of patients in some studies [25]. Thus,it is necessary to carefully monitor blood counts, espe-cially in patients that are given higher doses. Arthralgia,nausea, gastrointestinal symptoms, zinc deficiency andfluctuating liver enzymes have also been reported [25-28].DesferasiroxDesferasirox (Exjade®, ICL-670) is the second orally ac-tive iron chelator and the most recent to become ap-proved for the treatment of transfusion associated ironoverload [29-32]. It has a MW of 373 Da. Although tri-dentate, that is, requiring 2 molecules to bind each ironatom, ICL-670 has been shown to be highly selective foriron without promoting the excretion of other metalslike zinc and copper [29]. Studies in rats and humansdemonstrate that ICL-670 possesses a half-life of 8–16h, which allows ICL-670 plasma levels to be sustained ata therapeutic range for longer than either DFO or L1.Subsequent clinical studies have confirmed the iron che-lation efficacy of ICL-670.ICL-670 has been reported to be significantly more ef-ficient than DFO and L1 at promoting iron excretion. Atequal molar concentrations ICL-670 is reported to befive times more efficient than DFO and ten times moreLike L1, ICL-670 is highly cell permeable. Moreover, itis absorbed by some cells more rapidly than L1 [32]. Theactive molecule is highly lipophilic and cell permeablein vivo and feces is the main route of excretion for ICL-670 and its metabolites. Renal excretion accounts for ap-proximately 8% [30,31]. Unlike L1, which is absorbed butrapidly inactivated through metabolism, ICL670 rapidlyincreases in concentration in the plasma of patients andpersists at detectable levels for several hours.Although the long half-life and iron removal efficacy ofICL-670 allows once-daily dosing and offers significantimprovement in convenience for patients when comparedto DFO and L1, the toxicities reported to accompany pro-longed ICL-670 use should be considered [33-37]. In earlystudies, changes to the renal tubular epithelium were ob-served as side effects of ICL-670 use [33]. Subsequentstudies have confirmed that renal toxicity, hepatic dys-function and thrombocytopenia are the main concerns forpatients undergoing iron chelation therapy with ICL-670.Reports indicate that prolonged use can cause Fanconisyndrome [33-35]. Additionally, a mild, dose-dependentincrease in serum creatinine occurs in some patients.Thus, ICL-670 use requires meticulous monitoring of kid-ney, liver, and hematopoietic function.In a recent report by Kontoghiorghes, the fatalities asso-ciated with ICL-670 use are described to be the highestamong the clinically approved chelators. When comparedto DFO and L1 which have been in use for much longerperiods, the toxicity due to chelation with ICL-670 is high[37]. More importantly, according to this report, ICL-670was listed as the drug associated with the second highestnumber of deaths in 2009. Kontoghiorghes, reported thatthere is a steady increase in the ICL-670 induced deaths inpatients per year and that most are caused in elderly pa-tients with MDS. Although the evidence presented in thisreport was questioned by Riva, the potential seriousness ofICL-670 induced toxicity should not be overlooked [38].Continuous advances toward the development ofimproved iron chelatorsThe shortcomings of DFO, L1 and ICL-670 highlight theneed for improved chelation therapy. Challenges such asthe inefficiency of DFO and necessity for continuoussubcutaneous infusion; the toxicities of L1 and its inabil-effective than L1 [29]. Several studies show a lineardose-dependent increase in the amount of iron excretionby iron overloaded patients and the doses of ICL-670given. ICL-670 was reported to induce iron excretion ina manner that would likely prevent iron accumulation inmost patients requiring standard transfusion therapy fority to adequately control body iron levels with prolongeduse; and the severe toxicities associated with ICL-670,have sustained the interest among researchers to developbetter options for iron chelation.Fe(III) selective chelators are the most fitting for bio-logical applications because they are less likely to depleteother essential metals, which are commonly divalent.However, because the size of a drug influences intestinalabsorption, creating orally active hexadentate chelatorshas proven difficult [39]. Instead, the greatest emphaseshave been placed on generating novel bi and tridentatechelators or modifying the properties of existing ligands.Over the years, several promising agents which vary indenticity, metal selectivity for Fe(III), toxicity, stability ofthe Fe-chelator complex and lipophilicity have been pro-posed and tested in several in vivo models including ro-dents, marmosets, dogs, and primates, with promisingagents progressing to clinical trials [40-43]. Figure 5shows the structures of a few previously reported ironchelators with potential clinical utility.HBEDN, N’-bis (2-hydroxybenzyl) ethylenediamine-N-N’-diace-tic acid (HBED) is a hexadentate, phenolic aminocarboxy-late (MW 388) which has been tested for utility as an ironand toxicity. Initial studies showed that HBED is ineffect-ive at promoting iron excretion when given orally. How-ever, when given subcutaneously or intravenously, HBEDwas more than twice as effective as DFO at mobilizingiron from rats and cebus apella. Importantly, HBED is apowerful antioxidant and was not associated with anymajor toxicity in the models tested. Compared to DFO,HBED showed potential as a therapeutic for treatingtransfusional iron overload with the potential for dosingevery other day. HBED has been used in man but has notbeen further developed for use in treating transfusionaliron overload [40].Pyridoxal isonicotinoyl hydrazone (PIH)Pyridoxal isonicotinoyl hydrazone (PIH) is tridentateiron chelator (MW 287) effective at scavenging and mo-bilizing iron (Figure 5). The potential utility of this classof chelator, as well as its efficacy as anti-proliferativeagents, preventing free-radical mediated injury has beendocumented. [41]. PIH has been shown to chelate bothforms of iron, and like other chelators it caused deple-tion in zinc levels at physiological pH. At neutral pH,the neutral charge of PIH ensures oral absorption andHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 7 of 15chelating agent (Figure 5). HBED has a high affinity andspecificity for Fe(III) and like DFO, renders it virtuallyinert and incapable of forming harmful radicals whichdamage cellular components and organs [40]. HBED hasbeen thoroughly characterized for iron chelation efficiencyFigure 5 The structures of previously reported and potential iron cheallows access to the cytosol, where labile iron can bechelated. PIH showed efficacy at removing iron from ratreticulocytes which contained labile non-heme iron andmobilizing iron from Chang cells. PIH also reduced ironlevels in major organs in mice and was tested in manlators for treating secondary iron overload.Hamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 8 of 15but did not promote adequate iron excretion. PIH hasbeen reported to be toxic in cebus monkeys and althoughanalogues of PIH have been created none has been furtherdeveloped for use in treating iron overload in transfusiondependent patients [41].FBS0701FBS0701 (SPD-602) is a novel tridentate chelator of thedesazadesferriothiocin (DADFT) class and has been testedin phase II clinical trials (Figure 5). FBS0701 has a MW of400 (salt form 440), binds iron tightly and has a higheraffinity for Fe(III) than other divalent metals. It canenter cells and has demonstrated efficacy in iron chela-tion and a comparable safety profile to currently ap-proved chelators [42].A one-week, dose escalation, phase Ib study demon-strated its potential clinical utility and efficacy. While aphase II multicenter trial, which dosed patients with 50–375 mg of the FBS0701-salt, showed a statistical signifi-cant reduction in liver iron, confirming the potentialbenefit of this agent to reduce iron burden from transfu-sions. Although adverse events were reported, they didnot appear to be dose related and occurred at lowfrequency. Future studies with larger sample sizes willprovide more information on the potential of this chelatorfor treating transfusion associated iron overload. FBS0701is currently undergoing development and represents apromising agent for future treatment [42].CM1CM1, 1-(N-Acetyl-6-Aminohexyl)-3 Hydroxy-2-Methyl-pyridin-4-One), is an orally active, bidentate L1 analogue,possessing a MW of 256 Da and is currently being devel-oped for the treatment of iron overload (Figure 5). CMIhas shown higher lipophilicity than L1 and can bind bothFe(II) and Fe(III). CM1 is effective at mobilizing cytosoliclabile iron in primary mouse hepatocytes and HepG2 cells,and plasma NTBI. It has been studied in transgenic β-thalassemic mice, and has demonstrated efficacy and lowtoxicity in the liver and peripheral blood of iron over-loaded mice. Importantly, CM1 showed efficacy in pre-venting lipid peroxidation, the underlying cause of cellulardamage. Future studies are needed to determine clinicalutility of this agent [43].Current challenges in iron chelation therapyMonotherapy is inadequate to ensure negative iron balanceAlthough there are three iron chelators used for thetreatment of iron overload, often treatment with any ofthese chelators alone is not sufficient and it is estimatedthat 20% of patients undergoing iron chelation therapywill be inadequately chelated [8]. This is due to factorssuch as poor compliance, inefficacy and toxic side ef-fects. For example, 80% of patients undergoing DFOtherapy will experience reactions at the infusion site; oralDFP therapy alone will ensure a negative iron balance insome but not all patients and DFX may cause kidneyfailure in some patients.Additionally the safety of iron chelators for certain pop-ulations is yet to be clearly defined and remains somewhatcontroversial. This is especially true for pregnant women.Yet, the increasing lifespan of women to childbearing ageand the improvement in therapy over the last few decadeshas demonstrated the increase in the likelihood and casesof pregnancies in transfusion dependent women and theimportance to advance knowledge and treatment optionsfor this vulnerable group of patients.Properties of effective iron chelatorsEffective iron chelation therapy is achieved only if ironchelators can remove equal or greater amount amountsof iron to that accumulated due to transfusion therapy.This requires chelators to be able to reach the targetsites at relevant concentrations. Since there are severaliron pools that develop in iron overload, chelators whichare effective at mobilizing iron from all labile iron poolswould be advantageous. Secondly, the effective protec-tion of the heart by chelation therapy is critical for ironoverloaded patients as heart failure is the leading causeof the death in thalassemia major patients with ironoverload [8,10].An “ideal” chelator should have an ability to bindNTBI over long periods of time in order to ensure ad-equate coverage. A long acting chelator would ultimatelydecrease the amount of iron that is taken up into tissuesand would prevent harmful, iron-catalyzed reactions. Inaddition, iron chelators that are clinically effective musthave high selectivity for Fe(III) in comparison to otherimportant trace metal ions in the body. Table 1 com-pares the features of currently available iron chelatorswith that of “ideal” iron chelator features.Overcoming the challenges in current iron chelationtherapy: the development of new polymeric iron chelatorsAlthough all currently approved chelators are of low MW,previous reports of polymeric iron chelators have demon-strated that high MW chelators can be a viable alternativefor improving the pharmacokinetics and systemic toxicityof small MW chelators (Table 2). The approach to developpolymeric chelators or polymeric nanocarriers for ironchelators has varied from using iron binding dendrimers,hydrogels, the covalent attachment of DFO to a widerange of biocompatible materials and the use of aminoacid amide derivatives. Figure 6 shows the structures ofpolymeric components that have been used to modifyDFO toxicity and systemic circulation. The major motiva-tions for producing polymeric iron chelator is to over-come the challenges of rapid plasma elimination,Table 1 Contrasting features of ideal and currently approved iron chelatorsChelator property Ideal chelator DFO L1 DFXCost Affordable for patients in lowincome countriesModerate Moderate Unaffordable andunavailable for mostRoute ofAdministrationOral i.v injection or s.c infusion Oral OralCirculation t1/2 Long enough to allow once-dailydosing and effective iron removalShort (~20 min) requiresall-day (8-12 h) deliveryModerate; requires at least3 times per day dosingIdeal; 8–16 hours,requiring once-dailydosingTherapeutic index High High at high doses inpatients with high burdenUnpredictable HighToxicity None Neurotoxic, swelling attessAgranulocytosis and mild Reversible kidney failureHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 9 of 15prolonged infusions and widespread DFO toxicity that hashindered the achievement of safe iron levels in many ironoverloaded patients undergoing iron chelation therapyFigure 7. Indeed, several studies have indicated that poly-meric iron chelators possess unique advantages over theirlow MW counterparts.In a 1989 report, Hallaway et al. described the advan-tages associated with attaching DFO covalently to dex-tran and hydroxyethyl starch (HES) [44]. This resultedin a significant increase in the plasma half-life and re-duction in toxicity of DFO with no apparent loss in theiron chelating properties. The increase in size of starchconjugated DFO (S-DFO) resulted in improved plasmahalf-life from 5 min for DFO to 87 min S-DFO in mice.The LD50 in mice increased to 4000 mg/kg for dextran-DFO in comparison to 250 mg/kg for DFO and thereinfusion sideformitieUnsaturated IronBinding CapacityHigh: Long enough to preventdrastic fluctuations in LIPNoneAbility to remove ironfrom heart, liver etc.High Lowwas an absence of pulmonary hypotension when intra-venously administered in dogs. This is a major improve-ment as DFO and its iron complex induced hypotensionin dogs at a dose of 100 mg/kg. Moreover, the blood pres-sure did not return to normal during the 60 minutes of theexperiment. In contrast, neither HES nor dextran causedTable 2 The influence of polymer conjugation on the pharma-DFO Conjugate Pharmacodynamic effectDextran-DFO [44] LD50 increased from 250 mg/kg to 4000 mreduction of pulmonary hypotension in dogStarch-DFO (40SDO2) [47] Reduced retinal toxicity in albino rats,Reduction of pulmonary hypotension in doPEG-Methacrylate-DFO [46] Reduced endothelial cell toxicityHPG-DFO [48] Increased in vivo LD50Conjugated forms of DFO are associated with reduced toxicity both in vitro and in vplasma half-lives than unconjugated DFO [44,46-48].any significant change in blood pressure. Additionally, thehalf-life was more than 10 times greater after conjugation.In 2005 Polomoscanik et al. reported the generation ofa non-toxic formulation of DFO hydroxamic acid basediron chelating hydrogels and evaluated utility to preventiron absorption in the gut [45]. These gels were effectivein preventing gastric iron absorption and did not causeany change in hemoglobin and hematocrit. They alsoconducted a study to determine whether other divalentmetals compete with Fe(II) for binding to the polymericchelator and found that Zn and Cu did compete but thatoverall the binding strength of the polymeric chelatorswas affected only modestly. These agents prevented therise of hematocrit and hemoglobin in treated mice andsuggest that arresting the intestinal uptake of dietaryiron is a viable option for depleting iron levels. The, bone neutropenia are commonModerate HighHigh Highhydrophilic polymeric hydroxamic acid gel was non-toxic and suggests the feasibility of using non-absorbediron binding polymers as oral agents to sequester dietaryiron in the GI tract.In 2009, our group reported the development of well-defined, blood compatible and degradable PEG basedcokinetics and pharmacodynamics of DFOPharmacokinetic effectg/kg,sIncreased circulation half-lifegsIncreased in vivo iron excretion efficiency, increased circulationhalf-life.Excess free iron binding capacity in healthy males–Increased in vivo iron mobilization efficiency in miceIncreased circulation half-life in miceDecreased clearanceivo. Dextran-DFO, HES-DFO and HPG-DFO demonstrated significantly higherHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 10 of 15copolymers conjugated with DFO (P-DFO) for applica-tion in iron chelation therapy [46]. PEG methacrylatewas copolymerized by the RAFT method with a func-tional monomer for the conjugation of DFO to the poly-mer backbone via degradable or non-degradable linkage.The presence of PEG increased the biocompatibility ofthese nanoconjugates. The presence of the hydrolysableester linkages was anticipated to cause slow degradationof the conjugate via the ester linkages between the PEGside chains and copolymer backbone. P-DFO had MWsranging from 27–127 kDa with between 5–26 DFO unitsper polymer chain and demonstrated improved biocom-patibility and toxicity profile as compared to unconju-gated, small MW DFO. Like dextran and HES-DFO,P-DFO had a drastically improved toxicity profile; whileunconjugated DFO exposure in HUVECs resulted indeath at 3 μM, P-DFO was associated with ~90% cellviability up to 700 μM. However, to date P-DFO has notbeen tested for efficacy in vivo.The most significant evidence existing for the potentialclinical utility of polymeric iron chelators was publishedFigure 6 The chemical structure of polymer components used in DFOby Harmatz et al. who tested a DFO-starch conjugate[47]. In this first reported human clinical trial of poly-meric chelators, S-DFO caused clinically significant ironexcretion after single dose infusion of S-DFO. Maximumplasma chelator levels of 6 mM/L were achieved by S-DFO after 4 h intravenous infusion, an order of magni-tude higher than that which occurs with DFO treatment.More importantly, there was also residual iron bindingcapacity present in the plasma of patients for one week,without any observable toxicity [47].Recently, we have achieved the longest half-life in micerecorded to date by conjugating DFO to hyperbranchedpolyglycerols (HPG) [48]. HPG is a class of versatile, bio-compatible, inert, nano polymers that can be synthesizedin a controlled one-step reaction with low polydispersity.Detailed biocompatibility testing of these polymers con-ducted in vitro and in vivo has demonstrated the uniqueadvantages that HPG may have in nanomedicine [49,50].Our group has previously developed HPGs as a syntheticsubstitute for serum albumin that closely mimics thebinding and transport properties of natural albumin andmodification.Hamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 11 of 15is considered to hold advantages over the current clinic-ally used plasma expanders [50]. We have also coatedHPG on red blood cells to mask different antigens to-wards the development of universal blood cells, devel-oped DNA delivery agents, and developed anticoagulantneutralizing agents for use as heparin antidotes [51,52].Due to the biocompatibility, multi functionality and longcirculating nature of high MW HPG, we anticipated thatit would be a promising candidate for the developmentof a new generation of non-toxic macromolecular nano-conjugates for the removal of iron in vivo.HPG based polymeric chelators were developed byconjugating DFO to different MW HPGs with differentDFO density, producing a library of polymeric-DFOconjugates, referred herein as HPG-DFO. HPG-DFOFigure 7 Polymeric iron chelators result in a higher unsaturated ironand are not readily taken up by cells. In contrast, small MW chelators are hfrom the circulation.conjugates varied in properties depending on their MWand DFO density, and the structural features of HPG-DFO were optimized to achieve long plasma circulationtime, high chelation efficiency and low toxicity. All of theHPG-DFO conjugates demonstrated suitable biocompati-bility and the hydrodynamic radius ranged from 4.2 to 7.9nm. The narrow polydispersity of the polymer scaffoldallowed the development of homogeneous conjugates withwell-defined and predictable characteristics in vitro andin vivo. The plasma circulation half-life of DFO was in-creased more than 484-fold (44 h) for a 500 kDa conjugatecompared to that of unconjugated DFO. These conjugateswere also more efficient at mobilizing iron in mice; theiron excretion was significantly higher in mice treatedwith HPG-DFO [48]. Ongoing studies which are aimed atbinding capacity (UIBC). High MW chelators have longer half-livesighly permeable to cells and may be rapidly metabolized and removedHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 12 of 15understanding the circulation behavior of these moleculeswith respect to its MW and DFO density will allow furtheroptimization of toxicity and biodistribution.Apart from the previously described polymeric struc-tures with specific chelators attached to the polymerbackbone, other polymeric chelators have also beengenerated. Winston et al. prepared polymeric chelatorswith hydroxamic acid terminated side chains [53]. Thesepolymeric chelators were composed of amino acid amidederivatives of acrylic and methacrylic acid with the ter-minal carboxyl group converted to the hydroxamic acid.Polymeric chelators demonstrated a high affinity for iron(III) and were able to remove iron from iron overloadedmice when administered via i.p. injection.Zhou et al. reported the synthesis of 3-hydroxypyridin-4-one hexadentate, ligand-containing copolymers by thecopolymerization of 3-hydroxypyridin-4-one hexadentateligand with N,N-dimethylacrylamide (DMAA), and N,N’-ethylene-bis-acrylamide (EBAA) using (NH4)2S2O8 as theinitiator [54]. This class of chelator has demonstratedhigh selectivity and affinity for iron (III), and has poten-tial clinical utility for the treatment of iron overloaddiseases associated with the hyper-absorption of iron(e.g. hemochromatosis).Since iron accumulates as a result of transfusions as wellas dietary absorption, it has been suggested that blockingthe intestinal absorption of iron may also significantly re-duce iron levels in patients. This has been attempted byadministering high affinity, high MW chelators that arenot absorbed by intestinal cells, which bind iron andpromote its removal from the body. Zhou et al. designedhydroxypyridinone-containing polymers which signifi-cantly reduced intestinal iron uptake. In their in vitro in-testinal perfusion study, the accumulated absorbed ironwas significantly reduced compared with the controlgroups in the presence of polymeric iron chelator.Dendrimers have also demonstrated suitability as forthe generation of polymeric iron chelators [55]. Zhouet al. designed novel dendritic iron chelators by termin-ating dendrimers with hexadentate ligands formed fromhydroxypyridinone, hydroxypyranone, and catechol moi-eties and have demonstrated that these novel conjugatescan reduce iron absorption efficiently. This supports theidea that polymeric and dendritic iron chelators may beable to uniquely diminish iron absorption through theintestine and may have clear potential clinical utility dueto their high MW [55].Although the encapsulation of DFO into liposomes hasalso been attempted as a means of improving the thera-peutic index, it has been unsuccessful [56]. This is likelybecause DFO encapsulation does sequesters DFO only ini-tially, however, release profiles may not be ideal due to thehydrophilicity and relatively large molecular weight. An-other reason may be that if untargeted to specific tissues,DFO can still cause toxicity once released into healthy,non-iron overloaded cells. Liposome technology and DFOcan be extremely valuable with a targeted approach to aspecific organ affected by the iron overload. It may also bemuch more useful in treating cancers or tumors that havea high iron requirement but it also has to be targeted toensure a very specific release location. Liposomal encapsu-lation may also be beneficial if liposomes can be tuned torelease DFO slowly. Slow release would be beneficial andwould reduce toxicity associated with high DFO doses.Advantages of polymeric iron chelatorsThere are several advantages associated with the modi-fication of iron chelators with polymeric nanocarriers(Table 2). One of the most important properties of aniron chelator is its circulation half-life as this influencesthe unsaturated iron binding capacity (UIBC) of the chela-tor and ultimately, the rate of NTBI generation and re-moval. As iron in β-TM patients is constantly beingturned over due to the RBC catabolism in macrophages orthe breakdown of ferritin within cells, these pools of ironare redox active and are mainly responsible for the ironloading of plasma and tissues. Thus, in order to achieveeffective iron chelation and the removal of labile iron, 24h chelation coverage is the ideal. The importance of hav-ing a long-circulating chelator and constant coverage hasalso been demonstrated in studies that have shown thatprior to significant changes in cardiac iron in patients, car-diac failure is reversed during continuous administrationof DFO and that NTBI appears within minutes of a chela-tor being cleared from the body [57].Increased half-life is anticipated to have profound effectson compliance to therapy for patients treated with DFO.The current arduous DFO regimen has proven chal-lenging for many patients, especially young children andteenagers, so an optimized version of a long circulatingDFO would be of significant benefit. If polymeric chelatorscan be engineered toward slow, sustained degradation inthe plasma, it is conceivable that patients will requireonce-weekly or bi-weekly injections that will enable suffi-cient iron mobilization. In addition to improving compli-ance, this will likely improve access to DFO for manypatients since less drug will be needed to cause effectiveiron excretion and will reduce toxicities, ultimately allow-ing a paradigm shift in chelation therapy with DFO.Reduction in toxicityThe high MW polymeric DFO conjugates have demon-strated efficacy at clearing excess iron in vivo with re-duced or absent toxic effects [44-48]. The reduction inthe acute toxicity of polymer conjugated DFO as com-pared to the parent drug, allows the administration ofmuch greater amounts of “active ingredient” after poly-mer conjugation. This reduction in toxicity is most likelyHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 13 of 15attributable to a size dependent reduction in cellular up-take as conjugates remain in the vascular space longeronce conjugated to polymers. It is well documented thatlow MW iron chelators can be taken up by many celltypes. DFO has the highest MW among the 3 clinically ap-proved chelators and it has the highest hydrophilicity(with a distribution coefficient of −2) at physiological pH.DFO enters the liver via active transport and can interactwith the LIP and facilitate the iron excretion [10].Although a cell permeable chelator is more likely to haveaccess to labile cellular iron, it is not necessary for all che-lators to have this property. In fact, in some instances itmay be disadvantageous as small chelators which are notspecific for Fe(III) or which have high affinities for Fe(II)may chelate other essential metals, thus exerting unwantedeffects. This is well demonstrated when considering someof the factors underlying the toxicity of low MW iron che-lators. Low MW chelators may remove or displace essen-tial iron or other metals. Iron chelators can interfere withzinc, copper and other micronutrient even though thebinding affinity for these metal ions is relatively small (forinstance, the log cumulative stability constant of DFO-Fe is30.6 versus 11.1 for DFO-Zn 2+) [18]. Zinc deficiency hasbeen reported in patients undergoing DFO and L1 therapy[58]. Additionally, reducing essential iron in the cell can re-sult in reduced cell proliferation by inhibiting intracellularribonucleotide reductase and cell division [59].Polymeric iron chelators: beyond transfusional ironoverloadIron is an essential element for several metabolic pro-cesses and the perturbation of iron recycling and meta-bolism has been shown to be a major factor in severaldiseases. Iron removal has been shown to be a useful ap-proach for the treatment of microbial infectious diseases,reducing the growth rate of some cancer cells and neuro-degenerative diseases [60-62]. Additionally, iron chelationmay be useful in malaria treatment and treatment of theHIV virus [63,64]. Therefore, the ability to modify poly-mers to enhance iron chelation, minimize toxicity andmaximize blood circulation may prove beneficial in de-pleting iron stores in these disease states as well. Poly-meric iron chelators may also be modified to enhancetargeting to specific areas of the body and may thus havepotential clinical utility beyond the treatment of ironoverload.For example, polymeric chelators may be uniquely suitedfor iron depletion at the site of tumors owing to their largesize that can be used to passively target tumors throughtheir compromised endothelial junctions via the enhancedpermeation and retention (EPR) effect [65]. Likewise, theincreased residence time associated with polymeric ironchelators may have utility in treating chemotherapy pa-tients [61]. It has been reported that cancer chemotherapyincreases the levels of NTBI due to toxicity of anticancerdrugs to bone marrow cells. This can reduce the demandfor iron by marrow cells and cause transferrin to becomefully loaded which increases NTBI and may render thehost more susceptible to oxidative damage. While lowMW chelators are prone to enter cells, high MW drugs arealmost exclusively restricted to the vascular and extracellu-lar spaces due to the poor cellular uptake. Thus, polymericchelators are advantageous for such treatments.ConclusionsCurrent iron chelation therapy requires the daily admin-istration of virtually the maximum tolerated doses ofDFO, L1 and ICL-670 in order to ensure that the ratesof transfusional iron loading and iron excretion in trans-fusion dependent patients are well matched. This resultin patients experiencing a wide range of toxicities and inmany cases, the administered doses are still insufficientto mobilize the required amount of iron and producenegative iron balance. As a result, chelators that are lesstoxic and more efficient at iron mobilization would en-sure rapid reduction of labile body iron and prevent thedevelopment or progression of complications associatedwith iron overload. One potentially promising approachto advancing chelation therapy is through the use ofpolymeric chelators.The role of polymers in medical applications has seensubstantial growth over the past three decades. The useof polymers for applications in drug delivery, for artifi-cial organs, medical devices and dentistry is well docu-mented. In this review we have highlighted the potentialof polymeric iron chelators for the removal of toxic ironpools in vivo. Through appropriate design and modifica-tion of biocompatible polymers, several high MW chela-tors have been developed and characterized. Theseconjugates take advantage of the biophysical propertiesof the polymers that can extend plasma circulation andreduce dose dependent toxicity, while retaining excellentchelating properties. In many aspects, polymeric chela-tors are shown to be significantly more effective thansmall MW chelators at in vivo iron removal due to ex-tended plasma circulation times and may represent anew paradigm in treating transfusion associated ironoverload. Although none of the previously designedpolymeric chelators have advanced to the clinic, the suc-cessful phase Ib clinical trials conducted Harmatz et al.suggest the potential for iron removal that exists whenDFO is engineered into a high MW polymer conjugate.Our experience with modifying the properties of lowMW iron chelators like DFO through nanoengineeringwith polymers has grown only moderately from the firstattempt in 1989. It is well established that the undesirableproperties of DFO can be significantly transformed bymodification with nanomaterials, with such improvementsHamilton and Kizhakkedathu Molecular and Cellular Therapies  (2015) 3:3 Page 14 of 15varying based on the properties of the biomaterials used.To date, emphasis has been placed on modifying DFOproperties, although the possibility of engineering othersmall MW iron chelators like L1 and ICL-670 to improvetheir toxicity profile remains uncertain due to their bi andtri-denticity respectively.It is essential that polymer components are safe to use,can be reproduced easily on large scale and are suitablefor transformation into effective pharmaceutical formu-lations that are practical to use clinically. Therefore, fu-ture studies must consider several important questions.It is important to determine whether polymers such asHPG can be further modified to generate more targetedhigh MW chelators. It is also important to determinewhether the high MW chelators have specific degrad-ation routes or are prone to accumulation with chronicuse. Since the MW of drug molecules can play a criticalrole on their cellular and tissue accumulation, determin-ing the toxicity profile of polymers and their likelihoodof reaching target sites in adequate concentrations willbe of great importance.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsJLH conducted a systematic review of the literature, drafted and edited themanuscript. JNK edited the manuscript. Both authors read and approved thefinal manuscript.AcknowledgementsAuthors acknowledge the funding from Canadian Institutes of HealthResearch (CIHR) for funding. JLH acknowledges the Canadian Blood Servicesfor Graduate Fellowship Award and the Center for Blood Research forCollaborative Scholarship. 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