pcth: a novel orally active chelator for the treatment of iron overload disease

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Hemoglobin, 30 (1):93–104, (2006) Copyright © Taylor & Francis Group, LLC ISSN: 0363-0269 print/1532-432X online DOI: 10.1080/03630260500455367 93 LHEM 0363-0269 1532-432X Hemoglobin, Vol. 30, No. 01, December 2005: pp. 0–0 Hemoglobin PROCEEDINGS 15 TH ICOC Taiwan, April 2005 PCTH: A NOVEL ORALLY ACTIVE CHELATOR FOR THE TREATMENT OF IRON OVERLOAD DISEASE PCTH for Iron Overload D. B. Lovejoy et al. David B. Lovejoy, 1 Danuta Kalinowski, 1 Paul V. Bernhardt, 2 and Des R. Richardson 1 1 Iron Metabolism and Chelation Program, Children’s Cancer Institute Australia for Medical Research, Randwick, Sydney, New South Wales, Australia 2 Department of Chemistry, University of Queensland, St. Lucia, Brisbane 4072, Australia Our laboratories have prepared a novel class of iron (Fe) chelators of the 2-pyridylcarboxalde- hyde isonicotinoyl hydrazone (PCIH) class. This article will review the iron chelation efficacy of this series of chelators, both in cell culture and in animal models. Several PCIH analogs were shown to be effective at inducing iron mobilization and preventing iron uptake from the iron-transport pro- tein, transferrin. Moreover, several of these ligands were effective at permeating the mitochondrion and inducing iron release. Studies in mice demonstrated that the PCIH analog, PCTH, was orally active and well tolerated by mice at doses ranging from 50 to 100 mg kg 1 , twice daily (b.d.). A dose-dependent increase in fecal 59 Fe excretion was observed in the PCTH-treated group. This level of iron excretion was similar to that found for the orally effective chelators, pyridoxal isonicotinoyl hydrazone (PIH) and deferiprone (L1). The PCIH group of ligands clearly has the potential for the treatment of β-thalassemia (thal) and Friedreich’s Ataxia (FA). Keywords Desferrioxamine (DFO), Iron (Fe), Iron chelators, PCTH, Pyridoxal isonico- tinoyl hydrazone (PIH) GENERAL INTRODUCTION: IRON AND THE DEVELOPMENT OF -THALASSEMIA AND FRIEDREICH’S ATAXIA Iron (Fe) plays a crucial role in many metabolic processes including heme and DNA synthesis, and as such, iron deficiency results in severe met- Presented at the 15th ICOC, Taichung, Taiwan 22-26 April 2005. Address correspondence to Dr. Des R. Richardson, Iron Metabolism and Chelation Program, Chil- dren’s Cancer Institute Australia for Medical Research, PO Box 81, High Street, Randwick, Sydney, New South Wales, 2031 Australia; Fax: +61-2-9382-0060; E-mail: [email protected] Hemoglobin Downloaded from informahealthcare.com by University of Bath on 11/09/14 For personal use only.

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Page 1: PCTH: A Novel Orally Active Chelator for the Treatment of Iron Overload Disease

Hemoglobin, 30 (1):93–104, (2006)Copyright © Taylor & Francis Group, LLCISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260500455367

93

LHEM0363-02691532-432XHemoglobin, Vol. 30, No. 01, December 2005: pp. 0–0Hemoglobin

PROCEEDINGS 15TH ICOC

Taiwan, April 2005

PCTH: A NOVEL ORALLY ACTIVE CHELATOR FOR THE TREATMENT

OF IRON OVERLOAD DISEASE

PCTH for Iron OverloadD. B. Lovejoy et al.

David B. Lovejoy,1 Danuta Kalinowski,1 Paul V. Bernhardt,2 and

Des R. Richardson1

1Iron Metabolism and Chelation Program, Children’s Cancer Institute Australia for Medical Research, Randwick, Sydney, New South Wales, Australia2Department of Chemistry, University of Queensland, St. Lucia, Brisbane 4072, Australia

� Our laboratories have prepared a novel class of iron (Fe) chelators of the 2-pyridylcarboxalde-hyde isonicotinoyl hydrazone (PCIH) class. This article will review the iron chelation efficacy of thisseries of chelators, both in cell culture and in animal models. Several PCIH analogs were shown tobe effective at inducing iron mobilization and preventing iron uptake from the iron-transport pro-tein, transferrin. Moreover, several of these ligands were effective at permeating the mitochondrionand inducing iron release. Studies in mice demonstrated that the PCIH analog, PCTH, was orallyactive and well tolerated by mice at doses ranging from 50 to 100 mg kg−1, twice daily (b.d.). Adose-dependent increase in fecal 59Fe excretion was observed in the PCTH-treated group. This levelof iron excretion was similar to that found for the orally effective chelators, pyridoxal isonicotinoylhydrazone (PIH) and deferiprone (L1). The PCIH group of ligands clearly has the potential for thetreatment of β-thalassemia (thal) and Friedreich’s Ataxia (FA).

Keywords Desferrioxamine (DFO), Iron (Fe), Iron chelators, PCTH, Pyridoxal isonico-tinoyl hydrazone (PIH)

GENERAL INTRODUCTION: IRON AND THE DEVELOPMENT

OF �-THALASSEMIA AND FRIEDREICH’S ATAXIA

Iron (Fe) plays a crucial role in many metabolic processes includingheme and DNA synthesis, and as such, iron deficiency results in severe met-

Presented at the 15th ICOC, Taichung, Taiwan 22-26 April 2005.Address correspondence to Dr. Des R. Richardson, Iron Metabolism and Chelation Program, Chil-

dren’s Cancer Institute Australia for Medical Research, PO Box 81, High Street, Randwick, Sydney, NewSouth Wales, 2031 Australia; Fax: +61-2-9382-0060; E-mail: [email protected]

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94 D. B. Lovejoy et al.

abolic disturbances [for reviews see (1,2)]. On the other hand, when iron ispresent in excess of the requirement, it can result in toxicity due to its abil-ity to take part in the generation of free radicals that can damage vital bio-molecules. The most straightforward approach to treat iron overload is theuse of iron-binding drugs, known as iron chelators (1,2). Two significantiron-loading diseases, β-thalassemia (thal) and Friedreich’s Ataxia (FA), arediscussed below.

Pathophysiology of �-Thalassemia

β-Thalassemia is an inherited disorder of hemoglobin (Hb) synthesisthat is common in Mediterranean countries, the Middle East and Asia.Patients suffering from β-thal have a reduction or lack of synthesis of the βchain of Hb, while α chain synthesis remains unimpaired (3–6). The imbal-ance between β and α chains causes the α chains to form unstable aggre-gates, leading to erythrocyte destruction in the bone marrow and spleen(5). This results in ineffective erythropoiesis and, consequently, anemia.The anemia is treated by chronic blood transfusion and this treatment iscomplicated by the increase of iron absorption in these patients, leading toiron overload in vital organs such as the liver and heart (4).

Pathophysiology of Friedreich’s Ataxia

Friedreich’s Ataxia is a severe, inherited, spinocerebellar ataxia with anestimated incidence of one in 30,000 in the European population [forreviews see (7–9)]. The disease primarily affects the nervous system andheart, leading to early confinement in a wheelchair and death. The genedefective in FA, FRDA, encodes a mitochondrial protein known as frataxin(8). A triplet repeat expansion within intron-1 of the FRDA gene results in amarked decrease in frataxin expression. Over the last 5 years, it has becomeclear that this results in mitochondrial iron accumulation, which may gen-erate oxidative stress, and damage critical biological molecules (10). In con-trast to the marked iron overload observed in untreated β-thal major, theiron loading observed in the mitochondrion is less pronounced. Drugs thatreduce oxidative stress have a limited effect on the progression and pathol-ogy of FA, perhaps because they do not remove the accumulated iron (9).

IRON CHELATION THERAPY

Iron overload is treatable by chelation therapy and the most commontreatment utilizes desferrioxamine (DFO) (Figure 1), the only chelatorwidely approved for clinical use (1,2). Desferrioxamine is orally ineffectivebecause it is poorly absorbed across the gastrointestinal tract (11). Therefore,

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PCTH for Iron Overload 95

a cumbersome regimen consisting of subcutaneous infusions 12 hours perday, 5–6 days per week, is required. Additionally, one-third of the patientsreceiving DFO experience pain and swelling at the site of injection, result-ing in low compliance (6). These difficulties associated with administeringDFO led to considerable interest in designing new, orally effective iron che-lators. A number of orally effective chelators have been developed; the bestcharacterized of these being deferiprone (L1) (12). The reader is referredelsewhere for a comprehensive description of these ligands (2,12).

IRON CHELATORS OF THE 2-PYRIDYLCARBOXALDEHYDE

ISONICOTINOYL HYDRAZONE CLASS

A novel group of chelators, the 2-pyridylcarboxaldehyde isonicoti-noyl hydrazone (PCIH) analogs (Figure 1), have been designed and pat-ented by our laboratories (13–15). These compounds are based on the

FIGURE 1 Chemical structures of the chelators discussed in this review, namely: desferrioxamine(DFO), deferiprone (L1), pyridoxal isonicotinoyl hydrazone (PIH), 2-pyridylcarboxaldehyde isonicoti-noyl hydrazone (PCIH), isonicotinoyl picolinoyl hydrazine (IPH), 2-pyridylcarboxaldehyde benzoylhydrazone (PCBH), 2-pyridylcarboxaldehyde m -bromobenzoyl hydrazone (PCBBH), 2-pyridylcarboxal-dehyde 2-thiophenecarboxyl hydrazone (PCTH), and 2-hydroxy-1-naphthylaldehyde isonicotinoylhydrazone (311).

(CH2)5

N

O HN

H2N

OHO

(CH2)5

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OHO

(CH2)5

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OHO

DFO

N

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OH

Deferriprone (L1)

N

O

HNN

N

PCIH

N

O

HNN

N

PIH

OH

HO

N

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311

OH

O

HNN

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PCBBH

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PCTH

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96 D. B. Lovejoy et al.

parent chelator, pyridoxal isonicotinoyl hydrazone (PIH) (16,17),and are easily synthesized by an economical one-step Schiff base con-densation. The ligand, PIH, was shown to have high iron chelation effi-cacy both in vitro and in vivo, and was orally active in animals and human[for review see (18)]. The PCIH analogs were synthesized due to the factthat PIH was not patented, making its development commerciallyunattractive.

Chemical Characterization of the PCIH Analogs

X-ray crystallography studies demonstrated that the PCIH ligandswere to some extent planar with the 2- and 4-pyridyl rings being slightlytwisted about their respective C–C bonds. This chelator is potentially tri-dentate, binding metal ions through the 2-pyridyl nitrogen atom (N3),carbonyl oxygen (O1) and aldimine nitrogen (N1) (Figure 2) (14).Indeed, PCIH was shown to bind iron as a tridentate ligand and, inter-estingly was shown to be oxidized to the diacylhydrazine, isonicotinoylpicolinoyl hydrazine (IPH) (19), upon forming a complex with iron(Figure 3). In contrast, in the absence of Fe(III), the parent hydrazoneis not oxidized in aerobic aqueous solution (19). To examine whetherthe diacylhydrazine IPH could be responsible for the biological effectsof PCIH, their iron chelation efficacy was compared. In contrast to itsparent hydrazone PCIH, IPH showed little iron chelation activity. Poten-tiometric titrations suggested that this may be because IPH was chargedat physiologic pH, hindering its access across membranes to the intracel-lular iron pools. In contrast, the iron complex of IPH was neutrallycharged, which would permit passage through cell membranes. Thesedata allow a model of iron chelation for this compound to be proposed:the parent aroylhydrazone (PCIH) diffuses through cell membranes tobind Fe and is subsequently oxidized to the IPH-iron complex, whichthen diffuses from the cell (19).

FIGURE 2 X-ray crystal structure of the parent compound of the PCIH class of chelators, namely2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH). The 30% probability ellipsoid is shown[taken from Richardson et al. (14)].

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PCTH for Iron Overload 97

Studies of Iron Chelation Efficacy In Vitro Using

Cell Culture Models

The iron chelation efficacy of the PCIH analogs was first assessed in theSK-N-MC neuroepithelioma cell line, as its iron metabolism was well charac-terized. Moreover, the effect of other chelators on iron efflux and preven-tion of 59Fe uptake from 59Fe-transferrin (Tf) had been reported previouslyin this cell type (20,21). Some of the PCIH analogs were demonstrated tobe highly efficient at mobilizing 59Fe from cells pre-labeled with Tf and pre-venting 59Fe uptake from this protein in vitro. In fact, at a concentration of50 μM, three PCIH analogs (namely PCTH, PCBBH and PCBH) showedsimilar efficacy as PIH at mobilizing 59Fe from cells and preventing 59Feuptake from 59Fe-Tf (13). Moreover, these latter chelators were effective atmobilizing 59Fe from primary cultures of cardiomyocytes pre-labeled with59Fe-Tf. For example, incubation with control media resulted in the releaseof approximately 18% of total cellular 59Fe and this increased to 30% in thepresence of DFO (Figure 4A). The three most effective PCIH analogs,namely PCTH, PCBH and PCBBH, were significantly more effective thanDFO, resulting in 59Fe release equal to 51–52% of total cellular 59Fe (Figure4A) (22). This was not due to any change in the total amount of cardiomyo-cyte 59Fe (i.e., released 59Fe plus cellular 59Fe), as it remained constantunder all experimental conditions (Figure 4B). Hence, the differential 59Ferelease found in the presence of the chelators could not be explained by

FIGURE 3 X-ray crystal structure of isonicotinoyl picolinoyl hydrazine (IPH) Fe(III) complex. View ofthe [Fe(IPH)(HIPH)] molecule. The 30% probability ellipsoid is shown [taken from Bernhardt et al.(19)].

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98 D. B. Lovejoy et al.

FIGURE 4 (A) The PCIH analogues, PCTH, PCBH and PCBBH, were more effective than DFO atinducing 59Fe efflux from primary rat cardiomyocyte cultures. (B) The total amount of 59Fe (i.e., effluxand cells) is equal under all experimental conditions. For both (A) and (B), primary cultures of rat car-diomyocytes were prelabeled with 59Fe-Tf (0.75 μM) for 18 hours at 37°C, washed, and then reincubatedfor 24 hours at 37°C and 59Fe released from the cells into the medium was then assessed. Results are themean ± SD of three separate experiments. (C) The chelator, PCTH (50 μM), showed similar activity asDFO (50 μM) at decreasing ferritin-59Fe levels of prelabeled cardiomyocytes. Primary cultures of cardi-omyocytes were prelabeled with 59Fe-Tf (0.75 μM) for 18 hours at 37°C. The cells were washed and rein-cubated for 24 hours at 37°C with either control medium or medium containing DFO (50 μM) or PCTH(50 μM). The cells were then lysed and native PAGE 59Fe-autoradiography performed. (D) Densitomet-ric analysis of the results in (C). The results in (C) and (D) are typical from three separate experiments.(E) The chelators, DFO and PCTH, decrease 59Fe uptake into ferritin of cardiomyocytes. Cardiomyo-cytes were incubated with 59Fe-Tf (0.75 μM) for 18 hours at 37°C in the presence of either controlmedium, DFO (50 μM) or PCIH (50 μM). (F) Densitometric analysis of the results in (E). The results in(E) and (F) are a typical from three separate experiments [taken from Wong et al. (22)].

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PCTH for Iron Overload 99

variations in total cellular 59Fe levels. Moreover, PCTH also decreased59Fe-ferritin levels in these iron mobilization studies via either a direct orindirect mechanism (Figures 4C and 4D) (22).

In further studies, the ability of DFO and PCIH to inhibit the uptake of59Fe from 59Fe-Tf into ferritin by cardiomyocytes was assessed (Figures 4Eand 4F) (22). Both DFO and PCTH decreased the intensity of 59Fe-ferritinbands to 2 and 50% of the control, respectively (Figures 4E and 4F). Hence,the efficacy of DFO at preventing 59Fe uptake into ferritin was greater thanthat found for PCTH in cardiomyocytes, which was in contrast to their simi-lar efficacy at directly or indirectly mobilizing 59Fe from this molecule (Fig-ures 4C and 4D). Dialysis experiments demonstrated that PCTH did noteffectively remove 59Fe from 59Fe-Tf, being far less effective than DFO (22).These studies suggested that the chelators were acting at a point distal tothe high affinity iron-binding sites of Tf. These studies using cardiomyo-cytes were important to perform, as cardiac complications caused by irondeposition are a major cause of death in β-thal major patients (4).

The PCIH analogs also exhibited low anti-proliferative activity in vitroagainst SK-N-MC cells. This is an important property, as both β-thal and FApatients would require lifelong chelation therapy (9). In terms of the abilityof the chelators to inhibit 3H-thymidine, 3H-leucine and 3H-uridine incor-poration by SK-N-MC cells, these ligands showed similar or less activity thanDFO. For instance, examining 3H-thymidine incorporation after a 20-hourincubation, DFO, PCBBH, PCTH, and PCBH reduced it to 29, 33, 64 and72% of the untreated control, respectively. For comparison, the cytotoxicchelator, 311, reduced 3H-thymidine incorporation to 0.1% of theuntreated control. In cellular proliferation studies over a 90-hour incuba-tion with SK-N-MC, again these three PCIH analogs showed either less orsimilar activity to DFO in terms of inhibiting growth. The PCIH analog thatdemonstrated the highest iron chelation efficacy in vitro with the lowestanti-proliferative activity was PCTH (13). Thus, this chelator constituted ourlead compound that was subsequently screened in vivo (see Studies of IronChelation Efficacy of PCTH In Vivo Using Mice).

The high activity of several of the PCIH analogs encouraged us to assesstheir ability to remove iron from iron-loaded mitochondrial system usingrabbit reticulocytes (23). At present, this is the only well-characterized invitro mammalian model of mitochondrial iron overload (24–26) that isreadily available. This assay provided a preliminary screen to assess thepotential of these agents for treating the mitochondrial iron-loadingobserved in FA. In studies examining the effects of the PCIH analogs on59Fe release from 59Fe-loaded reticulocyte mitochondria, PIH was used as apositive control due to its demonstrated ability to remove 59Fe from thisorganelle (24). As a function of chelator concentration (10–200 μM), thethree most effective compounds at mobilizing mitochondrial 59Fe were

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100 D. B. Lovejoy et al.

PCIH > PIH = PCTH (Figure 5) (23). The high activity of the parent chela-tor, PCIH, was to some extent surprising, as this ligand showed relativelyless iron chelation activity than PCTH when examining the neural cell line,SK-N-MC (13). As expected, DFO due to its high hydrophilicity, showed lit-tle activity at mobilizing mitochondrial iron, being no more effective thancontrol medium at concentrations from 10 to 200 μM (Figure 5) (23). Itcould be suggested that the form of 59Fe in the reticulocyte 59Fe-loadedmitochondrial model may be different from that found in FA patients.Certainly, this is a consideration that requires further investigation.However, the aim of these studies was to determine whether a chelator canactually penetrate the cell membrane and the two mitochondrial mem-branes to bind the accumulated iron. Obviously, this is the first step amongmany to design iron chelators that can target mitochondrial iron pools forthe treatment of FA (9).

Studies of Iron Chelation Efficacy of PCTH In Vivo Using Mice

Considering the high iron chelation efficacy of PCTH in vitro (13,23),studies were critical to determine its activity in vivo using mice. These inves-tigations demonstrated that PCTH was orally active and well tolerated atdoses ranging from 50 to 200 mg kg−1, twice daily (b.d.), for two days. Adose-dependent increase in fecal 59Fe excretion was observed in the PCTH-treated group (Figure 6A) (22)]. This level of iron excretion at 200 mg kg−1

was similar to the same dose of the orally effective chelators, PIH and L1(Figure 6A). Effective iron chelation in the liver by PCTH was shown via itsability to reduce ferritin-59Fe accumulation (Figure 6B) (22).

Mice treated for 3 weeks with PCTH at doses of 50 and 100 mg per kgb.d. showed no overt signs of toxicity as determined by weight loss and arange of biochemical and hematological indices (22). In subchronic ironexcretion studies over 3 weeks, PIH and PCTH at 75 mg per kg b.d. for5 days per week increased fecal 59Fe excretion to 140 and 145% of the vehi-cle control, respectively. This study showed that PCTH was well tolerated at100 mg per kg b.d. and induced considerable iron excretion by the oralroute, suggesting its potential as a candidate to replace subcutaneouslyadministered DFO (22).

CONCLUSIONS

Our studies have demonstrated the iron chelation efficacy of PCTHboth in vitro and in vivo. The effect exerted by PCTH was equivalent to theknown orally-active chelators, PIH and L1, indicating its potential as a sub-stitute for DFO. These promising results encourage acute and chronic stud-ies in iron-overloaded mice that will soon be initiated.

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PCTH for Iron Overload 101

FIGURE 5 Effect of chelators on the mobilization of 59Fe from reticulocytes that have been incubated withthe heme synthesis inhibitor, succinylacetone, to load the mitochondrion with non heme 59Fe. The cellswere labeled with 59Fe-Tf (10 μM; [Fe] = 20 μM) for 60 min. at 37°C in the presence of the heme synthesisinhibitor, succinylacetone (SA; 1 mM). The 59Fe-loaded reticulocytes were then washed and incubated withthe chelators (10–200 μM) in the presence of SA (1 mM) for 60 min. at 37°C. Results are mean±SD (threedeterminations) in a typical experiment of three performed [taken from Richardson et al. (23)].

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102 D. B. Lovejoy et al.

FIGURE 6 (A) The dose-response of orally administered PCTH on fecal 59Fe excretion in mice com-pared to PIH and L1. Mice were administered by gavage for two days: PIH (200 mg per kg b.d.), L1 (200mg per kg b.d.), or PCTH (50–200 mg per kg b.d.). Results are expressed as a percentage of the vehiclecontrol (i.e., 20% propylene glycol in 0.9% NaCl) and are the means of two experiments consisting ofthree mice per group per experiment. (B) Oral administration of PCTH to mice decreases ferritin-59Felevels as determined using native polyacrylamide gel electrophoresis (PAGE) 59Fe-autoradiography.Mice were labeled with 59Fe-lactoferrin as described by Wong et al. (22) and then either the vehicle con-trol or PCTH at doses of 50 or 75 mg per kg b.d. were administered by gavage for two days. Livers wereharvested at the end of the experiment. Lysates were then prepared and subjected to native PAGE 59Fe-autoradiography. The results are typical from three separate experiments [taken from Wong et al. (22)].

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PCTH for Iron Overload 103

ACKNOWLEDGMENTS

The Children’s Cancer Institute Australia for Medical Research is affili-ated with the University of New South Wales and Sydney Children’s Hospi-tal. This project was supported by a National Health Medical ResearchCouncil (NHMRC) fellowship and grant (to DRR) and also an AustralianResearch Council Discovery Grant (DRR and PB).

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17. Ponka P, Borova J, Neuwirt J, Fuchs O, Necas E. A study of intracellular iron metabolism using pyri-doxal isonicotinoyl hydrazone and other synthetic chelating agents. Biochim Biophys Acta 1979;586(2):278–297.

18. Richardson DR, Ponka P. Pyridoxal isonicotinoyl hydrazone and its analogs: potential orally effec-tive iron-chelating agents for the treatment of iron overload disease. J Lab Clin Med 1998;131(4):306–315.

19. Bernhardt PV, Chin P, Richardson DR. Unprecedented oxidation of a biologically active aroylhy-drazone chelator catalysed by iron(III): serendipitous identification of diacylhydrazine ligands withhigh iron chelation efficacy. J Biol Inorg Chem 2001; 6(8):801–809.

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20. Richardson DR, Ponka P. The iron metabolism of the human neuroblastoma cell: lack of relation-ship between the efficacy of iron chelation and the inhibition of DNA synthesis. J Lab Clin Med1994; 124(5):660–671.

21. Richardson DR, Tran EH, Ponka P. The potential of iron chelators of the pyridoxal isonicotinoylhydrazone class as effective antiproliferative agents. Blood 1995; 86(11):4295–4306.

22. Wong CS, Kwok JC, Richardson DR. The novel iron chelator, 2-pyridylcarboxaldehyde isonicoti-noyl hydrazone, is orally active and induces iron excretion in mice. Biochim Biophys Acta 2004;1739(1):70–80.

23. Richardson DR, Mouralian C, Ponka P, Becker E. Development of potential iron chelators for thetreatment of Friedreich’s ataxia: ligands that mobilize mitochondrial iron. Biochim Biophys Acta2001; 1536(2–3):133–140.

24. Ponka P, Wilczynska A, Schulman HM. Iron utilization in rabbit reticulocytes. A study using succi-nylacetone as an inhibitor or heme synthesis. Biochim Biophys Acta 1982; 720(1):96–105.

25. Adams ML, Ostapiuk I, Grasso JA. The effects of inhibition of heme synthesis on the intracellularlocalization of iron in rat reticulocytes. Biochim Biophys Acta 1989; 1012(3):243–253.

26. Richardson DR, Ponka P, Vyoral D. Distribution of iron in reticulocytes after inhibition of hemesynthesis with succinylacetone: examination of the intermediates involved in iron metabolism.Blood 1996; 87(8):3477–3488.

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