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Keywords:

  • clinical study;
  • Deferiprone;
  • frataxin;
  • Friedreich's ataxia (FRDA)

Abstract

  1. Top of page
  2. Abstract
  3. Rationale for the use of iron chelators in FRDA
  4. Pre-clinical studies
  5. Clinical studies
  6. Conclusions
  7. Conflict of interest
  8. References

Friedreich's ataxia (FRDA) is a neurological disease related to a deficiency of the protein frataxin involved in iron–sulfur (Fe–S) cluster biogenesis. This leads to an increased cellular iron uptake accumulating in mitochondria, and a subsequently disturbed iron homeostasis. The detailed mechanism of iron regulation of frataxin expression is yet unknown. Deferiprone, an iron chelator that may cross the blood–brain barrier, was shown to shuttle iron between subcellular compartments. It could also transfer iron from iron-overloaded cells to extracellular apotransferrin and pre-erythroid cells for heme synthesis. Here, clinical studies on Deferiprone are reviewed in the context of alternative agents such as desferoxamine, with specific regard to its mechanistic and clinical implications.


Abbreviations used
FRDA

Friedreich ataxia

IREs

iron responsive elements

IRP1

iron responsive element binding protein 1

Rationale for the use of iron chelators in FRDA

  1. Top of page
  2. Abstract
  3. Rationale for the use of iron chelators in FRDA
  4. Pre-clinical studies
  5. Clinical studies
  6. Conclusions
  7. Conflict of interest
  8. References

The role of frataxin, the deficient protein in Friedreich's ataxia (FRDA), in iron metabolism is now firmly established. The first clues came from the analysis of yeast mutants with a deleted frataxin homolog gene (Δyfh1), which exhibit mitochondrial iron accumulation and dysregulation of iron uptake (Babcock et al. 1997). Δyfh1 yeast shows the apparent paradox of activating the high-affinity iron uptake system, which is normally induced by cellular iron depletion, while having high intracellular iron content. A closer analysis of Δyfh1 cells reveals that the excess iron content is all concentrated in mitochondria, with concomitant cytosolic iron depletion. In yeast, the high-affinity iron uptake system is regulated at the transcriptional level by the iron sensor and transcription factor Aft1, which is localized in the cytosol. Hence, loss of frataxin results in the induction of iron uptake because of the unbalanced intracellular distribution of the metal, and implies that iron entering the cell is directly channeled into mitochondria, where it accumulates. Experimental evidence indeed supported that mitochondrial iron accumulation may be prevented by diverting the flow of iron coming into the cell into a different vesicular compartment, which may be obtained by over-expressing the vesicular metal transporter Ccc1(Chen and Kaplan 2000). Furthermore, restoring frataxin expression in Δyfh1 yeast, by transfection with a frataxin-expressing plasmid, induces a rapid flow of iron out of mitochondria, re-equilibrating the intracellular iron distribution (Radisky et al. 1999). Important clues to the underlying mechanism came with the discovery that frataxin deficiency leads to a depletion of iron–sulfur (Fe–S) clusters. Fe–S clusters are important cofactors for many proteins with different functions and subcellular localizations, including the Krebs cycle enzyme aconitase and several subunits of respiratory complexes I, II, and III in the mitochondria, enzymes of aminoacid and purine metabolism in the cytosol, and DNA replication and repair factors in the nucleus(Sheftel et al. 2010). Deficiencies of Fe–S dependent enzymes were first observed in endomyocardial biopsies from FRDA patients and in Δyfh1 yeast (Rötig et al. 1997), then in conditional knock-out mice with tissue-specific deletion of the Fxn gene (Puccio et al. 2001; Puccio 2007). Subsequent observations suggested that these deficiencies are the consequence of impaired Fe–S cluster synthesis. An initial clue came by the observation that deletion in yeast of any gene encoding a protein involved in Fe–S cluster biogenesis leads to the same impairment of iron metabolism observed in Δyfh1 cells (Chen et al. 2002). Then, a series of studies, first in yeast, then in mammalian systems, confirmed the role of frataxin as an activator of mitochondrial Fe–S cluster biogenesis (Mühlenhoff et al. 2002; Stehling et al. 2004). The most recent findings revealed that frataxin is part of a multiprotein complex responsible for Fe–S cluster biogenesis in the mitochondrial matrix (Schmucker et al. 2011), in which it acts as an allosteric activator of the cysteine desulfurase enzyme that provides the sulfur moiety for Fe–S cluster assembly (Tsai and Barondeau 2010; Colin et al. 2013). Other postulated functions of frataxin oligomers and multimers in mitochondrial iron storage and detoxification (Vaubel and Isaya 2012) cannot be excluded, but remain controversial, and in any case do not contradict its major role in Fe–S synthesis. Mammalian cells contain Fe–S assembly factors also in the cytosol, but an essential precursor for cytosolic Fe–S synthesis has to be exported from mitochondria, where its synthesis depends on mitochondrial Fe–S cluster biogenesis (Lill et al. 2012; Rouault 2012). Accordingly, frataxin-deficient cells have impaired activities of Fe–S proteins in all cellular compartments (Martelli et al. 2007). This is important to understand the consequences of frataxin deficiency on iron metabolism in mammalian cells, because a key regulator of iron metabolism in higher eukaryotes, iron responsive element binding protein 1 (IRP1), which is localized in the cytosol, is a Fe–S protein. IRP1 and a structurally similar protein, IRP2, regulate iron metabolism in mammalian cells by binding to sequence motifs (iron responsive elements, IREs) that are present in the mRNAs for proteins involved in iron metabolism (Aisen et al. 2001). RNA binding of IRP1 and IRP2 normally occurs when cytosolic iron levels are low. IRP2, which has no Fe–S cluster, undergoes iron-dependent degradation, so its level increases as cytosolic iron decreases, whereas IRP1 functions as a cytosolic aconitase when its Fe-S cluster is fully assembled, and becomes an IRE-binding protein when its Fe-S cluster is partially or fully disassembled. IRE-binding by IRP1 and 2 protects mRNAs of iron import proteins from degradation, like the transferrin receptor, and prevents translation of mRNAs for proteins that need iron for their function, like aconitase, or store iron, like ferritin. A possible scenario when frataxin is deficient, is the activation of IRP1 as IRE-binding protein because of impaired Fe–S cluster biogenesis, resulting in increased cellular iron uptake. IRP1 activation has indeed been detected in frataxin-deficient cells and animal models (Lobmayr et al. 2005). Iron then is funneled into mitochondria where it should be utilized for Fe–S cluster (and heme) synthesis. Up-regulation of mitochondrial iron uptake via mitochondrial iron transporter (Richardson et al. 2010) also occurs when frataxin is deficient, contributing to iron accumulation in these organelles. Overall, these changes can be interpreted as a homeostatic response to restore Fe–S cluster biogenesis when it occurs at low levels, normally the consequence of iron deficiency. However, when frataxin is low, the extra iron imported into mitochondria cannot be efficiently utilized, it is oxidized, and progressively accumulates. Iron oxidation is in part the result of the Fenton reaction between ferrous iron, the normal substrate for Fe–S synthesis, and H2O2, which is produced in excess in mitochondria because of respiratory chain dysfunction, generating the highly toxic hydroxyl radical (OH˙) (Fig. 1).

image

Figure 1. In healthy individuals, iron is transferred from the cytosol into the mitochondria where iron–sulfur (Fe–S) clusters are formed. In iron deficiency, enhancement of this process counteracts low Fe–S availability and restores iron homeostasis. Fe–S cluster formation requires frataxin as a part of the assembly complex. Frataxin deficiency impairs Fe–S cluster formation in the mitochondria, causing loss of Fe–S clusters in all cellular compartments. Lack of Fe–S cluster leads to iron responsive element binding protein 1 (IRP1) activation and increased cellular iron uptake. Together with enhanced iron transport into mitochondria, this causes iron depletion from the cytosol and accumulation in the mitochondria, where toxic hydroxyl radicals (OH˙) are formed by the Fenton reaction between iron and reactive oxygen species (ROS). Iron chelators with low membrane permeability such as desferoxamine add to cytosolic iron depletion because they cannot access the mitochondria, further boost iron import, and subsequent mitochondrial accumulation, and hence potentially worsen the condition. In frataxin-deficient yeast, over-expression of the Fe-transporter Ccc1 may divert iron into a vesicular compartment, preventing its accumulation in mitochondria. The iron chelator deferiprone can enter mitochondria and redirect iron to other compartments, acting beneficial on the disturbed iron homeostasis. Mfrn = mitoferrin.

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The use of iron chelators in FRDA was first proposed as a mean to eliminate the excess iron accumulating in mitochondria, thus decreasing free radical generation through the Fenton reaction. However, iron chelators that poorly penetrate biological membranes, as desferoxamine, can only do that indirectly, by profoundly depleting iron in the extracellular medium and therefore in the whole cell, a clearly unwanted outcome that could even worsen Fe–S protein deficiencies. To target the iron imbalance in FRDA, an iron chelator should instead be able to reach into mitochondria and redistribute iron to other cellular compartments, without causing overall depletion. Such an iron chelator should therefore be membrane-permeable and have relatively low affinity for iron. Deferiprone, an orally administered, lipid-soluble iron chelator used in several countries as an alternative to desferoxamine to treat iron overload in polytransfused individuals with hemoglobinopathies, can easily cross the blood–brain barrier and cellular membranes. In addition, because of its relatively low affinity for iron, lower than that of transferrin, deferiprone is less likely to cause iron depletion than desferoxamine. These properties made deferiprone an interesting candidate as FRDA therapeutic.

Pre-clinical studies

  1. Top of page
  2. Abstract
  3. Rationale for the use of iron chelators in FRDA
  4. Pre-clinical studies
  5. Clinical studies
  6. Conclusions
  7. Conflict of interest
  8. References

A study in cell culture systems (cardiomyocytes, macrophages, erythroleukemia cells) that used organelle-targeted fluorescent iron sensors in conjunction with time-lapse fluorescence microscopy showed that deferiprone is indeed able to shuttle iron between subcellular compartments (Sohn et al. 2008). Deferiprone could also mobilize iron from iron-overloaded cells and donate it to extracellular apotransferrin and to pre-erythroid cells for heme synthesis (Sohn et al. 2008). A subsequent study from the same group utilized an inducible shRNA to down-regulate frataxin in cultured cells (Kakhlon et al. 2008). When the shRNA was expressed, these cells developed mitochondrial dysfunction with decreased aconitase activity, mitochondrial membrane potential, ATP production and O2 consumption, and increased vulnerability to apoptosis-inducing agents as staurosporine. Deferiprone, at the concentration of 50 μM, could correct the phenotype triggered by induction of the frataxin shRNA. The authors concluded that the ability of deferiprone to chelate accumulated mitochondrial iron and to make it bioavailable was key for the restoration of cell functions affected by frataxin deficiency. Furthermore, a recent publication reported that deferiprone was one of the compounds able to up-regulate frataxin that could be identified by screening a small chemical library for this property, adding an argument for its use to treat FRDA (Li et al. 2013).

However, these studies have to be considered along other experimental evidence suggesting that iron chelation, even by deferiprone, may be deleterious in FRDA under certain circumstances. A small study indicated that deferiprone at the concentration of 150 μM, but not at 25 μM, decreased aconitase activity in cultured fibroblasts by 70–80%, and at concentrations higher than 50 μM it could also inhibit cell proliferation (Goncalves et al. 2008). Another study demonstrated that frataxin mRNA levels decrease significantly in multiple human cell lines treated with desferoxamine (Li et al. 2008). Such a decrease correlated with low levels of cytosolic iron, as demonstrated by IRP activation, suggesting that frataxin expression is iron regulated. The mechanism of iron regulation of frataxin expression is unknown, however, as there is no IRE in the frataxin mRNA. According to preliminary unpublished results from our laboratory, deferiprone at high concentrations (> 100 μM) can also down-regulate frataxin, contrary to its effect at lower concentration. Taken together, these data suggest that, when deferiprone concentration is relatively low, it does not subtract iron from Fe–S clusters and it can stimulate frataxin expression, possibly by an iron-dependent mechanism activated by a shift of iron from mitochondria into the cytosol. However, when its concentration exceeds about 50 μM in cultured cells, it starts to induce iron depletion rather than redistribution, with deleterious effects on Fe–S enzyme activities and frataxin levels. These laboratory findings provide a possible key of interpretation for the clinical data summarized below.

Clinical studies

  1. Top of page
  2. Abstract
  3. Rationale for the use of iron chelators in FRDA
  4. Pre-clinical studies
  5. Clinical studies
  6. Conclusions
  7. Conflict of interest
  8. References

In a pilot, open-label study, nine adolescent patients with no overt cardiomyopathy were treated with 20–30 mg/kg/day deferiprone for 6 months (Boddaert et al. 2007). This dose needs to be compared with the > 75 mg/Kg/day that is commonly used to treat systemic iron overload. The primary end point was the change in iron content of the dentate nucleus, as estimated by magnetic resonance imaging (MRI) using the H-relaxation rate R2*, previously shown to be increased in subjects with FRDA. This study succeeded in reaching its primary end point by showing a significant decrease in R2*, which occurred in all treated subjects and was proportional to the initial R2* value. The ability of deferiprone to remove excess iron from a central nervous system (CNS) structure was therefore demonstrated. Clinically, particularly the youngest patients exhibited some improvement in ataxia, as indicated by observation of parents/relatives and support staff, and also by a relatively small, but significant (p < 0.05) decrease in the ICARS score (8.5 ± 4.5 points). However, the lack of a placebo group, the small number of subjects, and the short duration of the study impose caution on interpreting these data.

These encouraging results prompted a subsequent 6-month, phase 2, multicentric, randomized placebo-controlled trial testing three doses of deferiprone (20, 40, and 60 mg/Kg/day) versus placebo in FRDA patients aged 7–35, half of whom < 18 years old. In this study, called LA-29, patient inclusion was planned in two stages, with a final target of 80. Patients in the first cohort, up to 52, were randomized to receive either one of the two lower doses of deferiprone (20 patients for each dose) or placebo (12 patients). After completing inclusion of this cohort, and in the absence of any major safety concern, recruitment started of a second cohort of up to 28 patients, to be randomized to receive placebo (eight patients) or the highest dose of deferiprone (20 patients). However, worsening of ataxia was observed in a few patients soon after recruitment of the second cohort was started, leading to the pre-mature termination of this arm of the trial, whereas patients in the first cohort concluded the study and were further offered participation in a 1-year open-label extension, with the drug dosed at 20 or 40 mg/Kg/day. No publication reporting the detailed results of the LA-29 trial has yet appeared, but some preliminary data have been communicated at international meetings. The primary efficacy end point of LA-29, was the change in the score in the International Cooperative Ataxia Rating Scale (ICARS) after 6 months, secondary end points included additional neurological and several cardiac parameters. LA-29 missed its primary end point, there was in fact a worsening of ataxia not only with the highest dose of deferiprone but also with the intermediate 40 mg/Kg/day dose, as indicated by an about 4-point mean increase in ICARS in this group versus stability in the placebo group. Results were inconclusive with the lower dose of 20 mg/Kg/day, with no significant difference between treated and placebo groups, but some parameters showed encouraging trends. Interestingly, a remarkable reduction in cardiac hypertrophy (about 15–20%) was observed in all active drug groups. There were no major safety concerns, in particular no episodes of agranulocytosis, a known idiosyncratic reaction to deferiprone that imposes a strict control of blood cell counts in all subjects taking the drug.

In addition to this randomized controlled trial, a few dozen patients have been treated with deferiprone in open-label studies or on a compassionate basis in several European countries. Only results from an open-label study on 20 children with FRDA, associating deferiprone (20 mg/Kg/day) with the quinone antioxidant idebenone (20 mg/Kg/day) have been published so far (Velasco-Sánchez et al. 2011). They suggest a stabilizing effect in neurologic dysfunctions because of an improvement in the kinetic functions, but a worsening of gait and posture. Heart hypertrophy parameters and iron deposits in dentate nucleus, estimated by MRI, improved significantly.

Conclusions

  1. Top of page
  2. Abstract
  3. Rationale for the use of iron chelators in FRDA
  4. Pre-clinical studies
  5. Clinical studies
  6. Conclusions
  7. Conflict of interest
  8. References

Overall, results of clinical studies in FRDA suggest a beneficial effect of mild iron chelation, as provided by low doses of deferiprone, on cardiac parameters, and confirm the risk of worsening the condition by higher doses of the drug. Further studies are needed to assess if ataxia can be improved by doses of 20 mg/Kg/day or less. Discontinuous, qod or 4–5 qw deferiprone dosing schedules have also been proposed to prevent iron depletion, but these regimens have not yet been tested in a randomized controlled trial.

When considering the data provided by laboratory investigations, these clinical findings are not surprising. In fact, FRDA is more a condition of iron deficiency than of iron overload. Inefficient Fe–S cluster biogenesis leads to consequences and homeostatic responses that resemble those occurring in iron deficiency, including lower activities of iron-depending enzymes, IRP activation, and increased iron uptake. On the one hand, this concept confirms that the approach of iron redistribution with low doses of a membrane-permeable low-affinity chelator as deferiprone has potential therapeutic value in FRDA, on the other hand it indicates that iron deficiency is deleterious for FRDA patients and has to be corrected with iron supplementation, even if this may seem counterintuitive at first sight.

On the safety side, deferiprone carries a significant risk of agranulocytosis, which may occur at any time during treatment, even after a few years. Agranulocytosis is reversible after drug withdrawal, but it must be detected as soon as possible. For this reason, blood counts must be monitored every week during the entire duration of treatment. In the experience of the author, a case of agranulocytosis in FRDA patients who had been taking deferiprone for more than 1 year could be promptly detected and treated thanks to the continuation of such frequent blood count schedule. A progressive decrease in the neutrophil count is commonly observed as well, which might also possibly be prevented or reduced by discontinuous dosing.

The use of deferiprone in FRDA was suggested by the critical evaluation of basic research findings. The following developments are an example of how clinical studies and laboratory investigation can converge in providing a mechanistic interpretation of the effects of a drug and indications for improving its therapeutic potential.

References

  1. Top of page
  2. Abstract
  3. Rationale for the use of iron chelators in FRDA
  4. Pre-clinical studies
  5. Clinical studies
  6. Conclusions
  7. Conflict of interest
  8. References
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