Physiology and pathophysiology of iron in hemoglobin-associated diseases

Iron overload and iron toxicity, whether because of increased absorption or iron loading from repeated transfusions, can be major causes of morbidity and mortality in a number of chronic anemias. Significant advances have been made in our understanding of iron homeostasis over the past decade. At the same time, advances in magnetic resonance imaging have allowed clinicians to monitor and quantify iron concentrations noninvasively in specific organs. Furthermore, effective iron chelators are now available, including preparations that can be taken orally. This has resulted in substantial improvement in mortality and morbidity for patients with severe chronic iron overload. This paper reviews the key points of iron homeostasis and attempts to place clinical observations in patients with transfusional iron overload in context with the current understanding of iron homeostasis in humans.     

Chelation in metal intoxication—Principles and paradigms

The present review provides an update of the general principles for the investigation and use of chelating agents in the treatment of intoxications by metals. The clinical use of the old chelators EDTA (ethylenediamine tetraacetate) and BAL (2,3-dimercaptopropanol) is now limited due to the inconvenience of parenteral administration, their own toxicity and tendency to increase the neurotoxicity of several metals. The hydrophilic dithiol chelators DMSA (meso-2,3-dimercaptosuccinic acid) and DMPS (2,3-dimercapto-propanesulphonate) are less toxic and more efficient than BAL in the clinical treatment of heavy metal poisoning, and available as capsules for oral use. In copper overload, DMSA appears to be a potent antidote, although d-penicillamine is still widely used. In the chelation of iron, the thiols are inefficient, since iron has higher affinity for ligands with nitrogen and oxygen, but the new oral iron antidotes deferiprone and desferasirox have entered into the clinical arena. Comparisons of these agents and deferoxamine infusions are in progress. General principles for research and development of new chelators are briefly outlined in this review.     

Post-transfusional iron overload in the haemoglobinopathies

In this report, we review the recent advances in evaluation and treatment of transfusional iron overload (IO). Results of the French thalassaemia registry are described. According to the disease, thalassaemia major or sickle cell anaemia, mechanisms and toxicity of iron overload, knowledge about IO long-term outcome and chelation treatment results, respective value of IO markers, differ. The recent tools evaluating organ specific IO and the diversification of iron chelator agents make possible to individualize chelation therapy in clinical practice. The severity of IO and the level of transfusional iron intake, the preferential localization of IO (heart/liver) as well as the tolerance and adherence profiles of the patient can now be taken into account. Introduction of cardiac magnetic resonance imaging for the quantification of myocardial iron and use of oral chelators have already been reported as decreasing the cardiac mortality rate related to IO in thalassaemia major patients. Long-term observation of patients under oral chelators will show if morbidity is also improving via a more continuous control of toxic iron and/or a better accessibility to cellular iron pools.     

Partition coefficients of the iron(III) complexes of pyridoxal isonicotinoyl hydrazone and its analogs and the correlation to iron chelation efficacy

Pyridoxal isonicotinoyl hydrazone and its analogs are orally effective Fe(III) chelators which show potential as drugs to treat iron overload disease. The present investigation describes the measurement of the partition coefficient of the apochelator and Fe(III) complex of 20 of these ligands. These measurements have been done to investigate the relationship between lipophilicity and the efficacy of iron chelation in rabbit reticulocytes loaded with non-heme ⁵⁹Fe. The results demonstrate a linear relationship between the partition coefficient (P) of the apochelator and its Fe(III) complex, and a simple equation has been derived relating these two parameters. Experimental data in the literature are in agreement with the equation. The relationship of the partition coefficients of the iron chelators and of their Fe(III) complexes to the effectiveness of the ligands in mobilizing iron in vitro and in vivo is also discussed.     

Iron chelation,quo vadis?

Orally bioavailable chelators for transfusional iron overload have been sought since the introduction of deferoxamine (Desferal) in 1962. Despite tremendous efforts, to date, only deferiprone (Ferriprox) and deferasirox (Exjade) have successfully reached the market, reflecting the difficulty to combine oral activity and safety. Owing to the risk of failure, few new oral chelators can be expected in the future for the treatment of transfusional iron overload. As iron is involved in many disease processes, deferiprone and deferasirox have been proposed to be potentially useful in a variety of indications not characterized by general iron overload. Although it may be possible to obtain clinical benefit from current compounds, more selective chelators tailored to the particular target are needed for successful intervention in these indications.     

Objectives and Methods of Iron Chelation Therapy

Recent developments in the understanding of the molecular control of iron homeostasis provided novel insights into the mechanisms responsible for normal iron balance. However in chronic anemias associated with iron overload, such mechanisms are no longer sufficient to offer protection from iron toxicity, and iron chelating therapy is the only method available for preventing early death caused mainly by myocardial and hepatic damage. Today, long-term deferoxamine (DFO) therapy is an integral part of the management of thalassemia and other transfusion-dependent anemias, with a major impact on well-being and survival. However, the high cost and rigorous requirements of DFO therapy, and the significant toxicity of deferiprone underline the need for the continued development of new and improved orally effective iron chelators. Within recent years more than one thousand candidate compounds have been screened in animal models. The most outstanding of these compounds include deferiprone (L1); pyridoxal isonicotinoyl hydrazone (PIH) and; bishydroxy- phenyl thiazole. Deferiprone has been used extensively as a substitute for DFO in clinical trials involving hundreds of patients. However, L1 treatment alone fails to achieve a negative iron balance in a substantial proportion of subjects. Deferiprone is less effective than DFO and its potential hepatotoxicity is an issue of current controversy. A new orally effective iron chelator should not necessarily be regarded as one displacing the presently accepted and highly effective parenteral drug DFO. Rather, it could be employed to extend the scope of iron chelating strategies in a manner analogous with the combined use of medications in the management of other conditions such as hypertension or diabetes. Coadministration or alternating use of DFO and a suitable oral chelator may allow a decrease in dosage of both drugs and improve compliance by decreasing the demand on tedious parenteral drug administration. Combined use of DFO and L1 has already been shown to result in successful depletion of iron stores in patients previously failing to respond to single drug therapy, and to lead to improved compliance with treatment. It may also result in a “shuttle effect” between weak intracellular chelators and powerful extracellular chelators or exploit the entero-hepatic cycle to promote fecal iron excretion. All of these innovative ways of chelator usage are now awaiting evaluation in experimental models and in the clinical setting.     

The study of iron mobilisation from transferrin using alpha-ketohydroxy heteroaromatic chelators

A group of heteroaromatic chelators with an alpha-ketohydroxy binding site have been tested for their ability to mobilise iron from transferrin in vitro. When these chelators were mixed with iron-saturated transferrin at physiological pH, biphasic reactions were observed. The alpha-ketohydroxy heteroaromatic chelators were found to cause substantial iron removal compared to other known chelators. These findings suggest that these chelators may have an important role in the study of iron metabolism and a possible clinical use in the treatment of transfusional iron overload in thalassaemia, and other diseases of iron imbalance.     

The study of iron mobilisation from transferrin using α-ketohydroxy heteroaromatic chelators

A group of heteroaromatic chelators with an α-ketohydroxy binding site have been tested for their ability to mobilise iron from transferrin in vitro. When these chelators were mixed with iron-saturated transferrin at physiological pH, biphasic reactions were observed. The α-ketohydroxy heteroaromatic chelators were found to cause substantial iron removal compared to other known chelators. These findings suggest that these chelators may have an important role in the study of iron metabolism and a possible clinical use in the treatment of transfusional iron overload in thalassaemia, and other diseases of iron imbalance.     

In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC₅₀) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development.     

Voltage-gated Calcium Channels Provide an Alternate Route for Iron Uptake in Neuronal Cell Cultures

Recent studies suggest that iron enters cardiomyocytes via the L-type voltage-gated calcium channel (VGCC). The neuronal VGCC may also provide iron entry. As with calcium, extraneous iron is associated with the pathology and progression of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. VGCCs, ubiquitously expressed, may be an important route of excessive entry for both iron and calcium, contributing to cell toxicity or death. We evaluated the uptake of ⁴⁵Ca²⁺ and ⁵⁵Fe²⁺ into NGF-treated rat PC12, and murine N-2α cells. Iron not only competed with calcium for entry into these cells, but iron uptake (similar to calcium uptake) was inhibited by nimodipine, a specific L-type VGCC blocker, and enhanced by FPL 64176, an L-VGCC activator, in a dose-dependent manner. Taken together, these data suggest that voltage-gated calcium channels are an alternate route for iron entry into neuronal cells under conditions that promote cellular iron overload toxicity. Special issue dedicated to Dr. Moussa Youdim.     

Iron mobilization from ferritin using alpha-oxohydroxy heteroaromatic chelators.

Several alpha-oxohydroxy heteroaromatic chelators have been shown to mobilize iron from horse spleen ferritin. Although the reactions were slow, taking up to 3 days to reach completion, the amounts of iron mobilized were higher than those reported for other chelators. These results increase the prospects for the clinical use of alpha-oxohydroxy chelators in the treatment of iron overload.     

Iron mobilization using chelation and phlebotomy

Knowledge of the basic mechanisms involved in iron metabolism has increased greatly in recent years, improving our ability to deal with the huge global public health problems of iron deficiency and overload. Several million people worldwide suffer iron overload with serious clinical implications. Iron overload has many different causes, both genetic and environmental. The two most common iron overload disorders are hereditary haemochromatosis and transfusional siderosis, which occurs in thalassaemias and other refractory anaemias. The two most important treatment options for iron overload are phlebotomy and chelation. Phlebotomy is the initial treatment of choice in haemochromatosis, while chelation is a mainstay in the treatment of transfusional siderosis. The classical iron chelator is deferoxamine (Desferal), but due to poor gastrointestinal absorption it has to be administered intravenously or subcutaneously, mostly on a daily basis. Thus, there is an obvious need to find and develop new effective iron chelators for oral use. In later years, particularly two such oral iron chelators have shown promise and have been approved for clinical use, namely deferiprone (Ferriprox) and deferasirox (Exjade). Combined subcutaneous (deferoxamine) and oral (deferiprone) treatment seems to hold particular promise.     

Atrial fibrillation in β-thalassemia patients with a focus on the role of iron-overload and oxidative stress: A review

Cardiac complications including arrhythmia and especially atrial fibrillation (AF) are common causes of death in β-thalassemia patients. The main factor in the etiopathogenesis of these complications is iron overload, which results in increased oxidative stress. Although there is a known association between cardiac complications and iron overload in β-thalassemia patients, there is no comprehensive review on AF and excessive iron with a focus on oxidative stress in these patients. The aim of this article was to review the different aspects of AF in β-thalassemia patients with a focus on the prevention and treatment of AF by using iron chelators and/or anti-oxidants. AF in β-thalassemia patients is more common than in the general population. One of the most important causes of AF is cardiac iron overload and the harmful effects of increased oxidative stress. Iron-induced AF can be reversed by using an intensive iron chelation regimen. Based on a few experimental studies, the combination of iron chelators with some anti-oxidants, including NAC, vitamin C, and acetaminophen, can lead to improved cardiac protection. However, the effect of such combinations on cardiac arrhythmias should be further evaluated with animal and human studies.     

Iron chelators can protect against oxidative stress through ferryl heme reduction

Iron chelators such as desferrioxamine have been shown to ameliorate oxidative damage in vivo. The mechanism of this therapeutic action under non-iron-overload conditions is, however, complex, as desferrioxamine has properties that can impact on oxidative damage independent of its capacity to act as an iron chelator. Desferrioxamine can act as a reducing agent to remove cytotoxic ferryl myoglobin and hemoglobin and has recently been shown to prevent the formation of a highly cytotoxic heme-to-protein cross-linked derivative of myoglobin. In this study we have examined the effects of a wide range of iron chelators, including the clinically used hydroxypyridinone CP20 (deferriprone), on the stability of ferryl myoglobin and on the formation of heme-to-protein cross-linking. We show that all hydroxypyridinones, as well as many other iron chelators, are efficient reducing agents of ferryl myoglobin. These compounds are also effective at preventing the formation of cytotoxic derivatives of myoglobin such as heme-to-protein cross-linking. These results show that the use of iron chelators in vivo may ameliorate oxidative damage under conditions of non-iron overload by at least two mechanisms. The antioxidant effects of chelators in vivo cannot, therefore, be attributed solely to iron chelation.     

Multidentate pyridinones inhibit the metabolism of nontransferrin-bound iron by hepatocytes and hepatoma cells.

The therapeutic effect of iron (Fe) chelators on the potentially toxic plasma pool of nontransferrin-bound iron (NTBI), often present in Fe overload diseases and in some cancer patients during chemotherapy, is of considerable interest. In the present investigation, several multidentate pyridinones were synthesized and compared with their bidentate analogue, deferiprone (DFP; L1, orally active) and desferrioxamine (DFO; hexadentate; orally inactive) for their effect on the metabolism of NTBI in the rat hepatocyte and a hepatoma cell line (McArdle 7777, Q7). Hepatoma cells took up much less NTBI than the hepatocytes (< 10%). All the chelators inhibited NTBI uptake (80-98%) much more than they increased mobilization of Fe from cells prelabelled with NTBI (5-20%). The hexadentate pyridinone, N,N,N-tris(3-hydroxy-1-methyl-2(1H)-pyridinone-4-carboxaminoethyl)amine showed comparable activity to DFO and DFP. There was no apparent correlation between Fe status, Fe uptake and chelator activity in hepatocytes, suggesting that NTBI transport is not regulated by cellular Fe levels. The intracellular distribution of iron taken up as NTBI changed in the presence of chelators suggesting that the chelators may act intracellularly as well as at the cell membrane. In conclusion (a) rat hepatocytes have a much greater capacity to take up NTBI than the rat hepatoma cell line (Q7), (b) all chelators bind NTBI much more effectively during the uptake phase than in the mobilization of Fe which has been stored from NTBI and (c) while DFP is the most active chelator, other multidentate pyridinones have potential in the treatment of Fe overload, particularly at lower, more readily clinically available concentrations, and during cancer chemotherapy, by removing plasma NTBI.     

Spike‐in SILAC proteomic approach reveals the vitronectin as an early molecular signature of liver fibrosis in hepatitis C infections with hepatic iron overload

Hepatitis C virus (HCV)‐induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal‐restricted histological distribution of pathological iron deposits has hampered the attempt to perform large‐scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload‐induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike‐in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron‐associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV‐infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV‐infected patients without iron overload.     

Current status in iron chelation in hemoglobinopathies

Although blood transfusions are important for patients with hemoglobinopathies, chronic transfusions inevitably lead to iron overload as humans cannot actively remove excess iron. The cumulative effects of iron overload lead to significant morbidity and mortality, if untreated. Desferrioxamine (DFO) is the reference-standard iron chelator whose safety and efficacy profile has been established through many years of clinical use. DFO side effects are acceptable and manageable however the prolonged subcutaneous infusion regimen of 5-7 days per week is very demanding and results in poor adherence to therapy. Deferiprone (Ferriprox, L1) is a bidentate molecule, orally administrable three-times/day, licensed in Europe and in other regions but in the USA and Canada, for the treatment of iron overload in patients for whom DFO therapy is contraindicated or inadequate. Preliminary evidences suggest that Deferiprone may be more effective than DFO in chelating cardiac iron. The side effects include gastrointestinal symptoms, liver dysfunction, joint pain, neutropenia and agranulocytosis. A weekly assessment of white blood cell counts is recommended because of the risk of agranulocytosis. Deferasirox is a new, convenient, once-daily oral iron chelator that has demonstrated in various clinical trials good efficacy and acceptable safety profile in adult and pediatric patients affected by transfusion-dependent thalassemia major and by different chronic anemias (SCD, BDA, MDS). The long half-life of Deferasirox (16-18 hours) provides sustained 24 hr iron chelation coverage. The efficacy and safety profile have been evaluated in more than 1000 patients in clinical trials allowing FDA registration. Patient satisfaction with Deferasirox was superior than with DFO therapy.     

Lipid peroxidation and associated hepatic organelle dysfunction in iron overload

Iron overload can have serious health consequences. Since humans lack an effective means to excrete excess iron, overload can result from an increased absorption of dietary iron or from parenteral administration of iron. When the iron burden exceeds the body's capacity for safe storage, the result is widespread damage to the liver, heart and joints, and the pancreas and other endocrine organs. Clear evidence is now available that iron overload leads to lipid peroxidation in experimental animals, if sufficiently high levels of iron are achieved. In contrast, there is a paucity of data regarding lipid peroxidation in patients with iron overload. Data from experiments using an animal model of dietary iron overload support the concept that iron overload results in an increase in an hepatic cytosolic pool of low molecular weight iron which is catalytically active in stimulating lipid peroxidation. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in homogenates of liver samples from both iron-loaded human subjects and experimental animals. Some current hypotheses focus on the possibility that the demonstrated functional abnormalities in organelles of the iron-loaded liver may play a pathogenic role in hepatocellular injury and eventual fibrosis. The recent demonstration that hepatic fibrosis is produced in animals with long-term dietary iron overload will allow this model to be used to further investigate the relationship between lipid peroxidation and hepatic injury in iron overload.     

Comparison of iron chelator efficacy in iron-overloaded beagle dogs and monkeys (Cebus apella)

Rodents and dogs are frequently used for preclinical toxicologic assessment of candidate iron chelators. Although the iron-clearing profile of a ligand often is known in rodents, and sometimes in primates, such information in dogs is rarely, if ever, available. Because of this, toxicity studies in dogs could be misleading; chelators that may otherwise be suitable for human clinical studies may be abandoned as being unacceptably toxic, simply because, unknown to the investigator, these drugs remove more iron in this species than would have been expected on the basis of iron clearance results in other species. This is a scenario that we encountered during toxicity trials of (S)-beta,beta-dimethyl-4'-hydroxydesazadesmethyldesferrithiocin in dogs. Thus, we developed an iron-overloaded dog model in which it is possible to evaluate iron-clearing efficiencies of potential therapeutic ligands. Seven deferration agents have been screened in this model, and the results were compared with the iron-clearing efficiency of the same ligands in an iron-loaded Cebus apella monkey model. The data suggest that while the iron-clearing efficiencies of most of the drugs were similar between the two species, there can be profound differences. This is consistent with the idea that caution needs to be exercised when carrying out preclinical toxicity evaluations of a chelator in dogs without first measuring the drug's iron-clearing efficiency in this species.     

Synthesis and iron(III)-chelating properties of novel 3-hydroxypyridin-4-one hexadentate ligand-containing copolymers

Iron overload is a critical clinical problem that can be prevented by the use of iron-specific chelating agents. An alternative method of relieving iron overload is to reduce the efficiency of iron absorption from the intestine by administering iron chelators, which can bind iron irreversibly to form nontoxic, kinetically inert complexes that are not absorbed and are therefore excreted in the feces. A series of polymeric chelators with various iron binding capacities were therefore prepared as nonabsorbable iron-selective additives. A novel 3-hydroxypyridin-4-one hexadentate ligand CP254 has been synthesized and incorporated into polymers by copolymerisation with N, N-dimethylacrylamide (DMAA), and N, N'-ethylene-bis-acrylamide (EBAA) using (NH4)2S2O8 as the initiator. The physicochemical properties of CP254 were determined, namely, log K = 33.2 and pFe(3+) = 27.24. The chelating capacity of the CP254-DMAA copolymers was determined at physiological pH. The iron(III) chelation was found to achieve 80% capacity after 1 h and was virtually complete after 5 h, which is much quicker than that of the commercially available chelating resin Chelex100. The chelating copolymers were found to be readily regenerated and reusable. The copolymers possess a high selectivity for iron(III). The conditional affinity (log K') for iron(III) at pH 7.46 was determined to be 26.55, which is not significantly different to that of the hexadentate ligand CP254 (log K' = 26.47). In vitro perfusion studies indicate that the polymeric chelators described in this study can reduce iron absorption from the intestine.