Low‐dose iron chelation as anti‐oxidative therapy in patients with sickle cell disease: A single‐centre pilot study

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The persistent intravascular haemolysis in patients with sickle cell disease (SCD) leads to elevated levels of cell-free haem and iron, resulting in increased production of reactive oxygen species (ROS), which is a key factor in the development of SCD-associated microvascular dysfunction, vaso-occlusion, inflammation and organ damage.1-3 The release of cell-free iron potentiates endothelial activation and subsequent damage by catalysing intracellular ROS production. Oxidative stress also causes erythrocyte membrane damage, with subsequent exposure of phosphatidylserine, and intraluminal exposure of tissue factor, potentially contributing to the hypercoagulable state in SCD.4 Given the profound impact of sustained and unremitting iron release on oxidative stress in SCD, it is imperative to investigate the potential of iron chelation as anti-oxidant therapy in SCD. Iron chelators have been shown to protect various cells, including erythrocytes and endothelial cells, against oxidative toxicity.5, 6 Furthermore, iron depletion has been associated with a substantial reduction in sickling, haemolysis and vaso-occlusive events.7 This is thought to be mediated by a decrease in mean corpuscular haemoglobin concentration (MCHC), which attenuates the polymerization of haemoglobin S (HbS).7 A retrospective study found that SCD patients who used iron chelators to mitigate transfusion-induced iron overload exhibited a decline in oxidative stress compared to their pre-chelation status and patients not using iron chelators.5 This suggests an anti-oxidative effect of iron chelation by scavenging the continuously released cell-free iron.5 Furthermore, we recently demonstrated that scavenging free iron through the iron chelator deferoxamine inhibited neutrophil activation and the release of neutrophil extracellular traps when exposed to sera of SCD patients, underscoring the significance of cell-free iron in inflammation.8, 9 We hypothesized that treatment with the iron chelator deferasirox could reduce oxidative stress in SCD by scavenging the continuously released intravascular cell-free iron (Figure S1). We investigated whether deferasirox treatment reduced erythrocyte sickling, haemolysis, oxidative stress, neutrophil activation, inflammation, endothelial activation and hypercoagulability in SCD patients. Inclusion criteria were homozygous sickle cell disease (HbSS) or sickle-β0 thalassaemia (HbSβ0), age ≥18 years and lactate dehydrogenase >300 U/L. Key exclusion criteria were: iron overload, use of iron chelation, ferritin ≤50 μg/L, estimated glomerular filtration rate <60 mL/min/1.73 m2, blood transfusion ≤16 weeks or vaso-occlusive crisis (VOC) ≤4 weeks preceding study screening. Enrolled patients were treated with deferasirox 360 mg (film-coated Exjade 360 mg tablet) once daily for 6 weeks. Study visits were every 2 weeks during the 6-week treatment period and after a 4-week washout period. Primary end-point was change in point of sickling (PoS), as a marker of erythrocyte sickling, and was measured with the Oxygenscan (Lorrca; RR Mechatronics).10 Secondary end-points were safety and tolerability, elongation index (EI) max and min, haemolysis parameters, iron status, inflammation markers and protein expression, endothelial markers and neutrophil characteristics (See Supplementary methods for detailed description in Data S1). Between July 2021 and September 2022, 15 patients were assessed for eligibility, with 12 patients (median age 32.5 years [interquartile range, IQR 20.5–42.3], 4 females) enrolling in the study (Figure S2). Ten patients (83%) had HbSS and nine (75%) used hydroxyurea. Baseline characteristics are shown in Table S1. Based on patient interviews and pill counts, adherence to the study drug was high and deferasirox was well tolerated, with no serious adverse events. Three adverse events (headache, sore throat and nausea) were documented, all classified as grade 1 and spontaneously resolved within ≤5 days (Table S2). After 6 weeks of deferasirox treatment, the PoS did not change significantly (17.6 mmHg [IQR 14.3–19.6] to 16.8 mmHg [14.2–20.2], respectively, p = 0.58). Similarly, the EImax, EImin and EIdelta did not change (Table 1). There was a significant reduction in reticulocyte counts after treatment as compared to baseline (from 256 × 109/L [IQR 165–284] to 202 × 109/L [117–231], p = 0.012). The decrease in total bilirubin levels did not reach statistical significance (from 45.0 μmol/L [IQR 34.8–59.5] to 38.0 μmol/L [28.3–52.8], p = 0.059; Figure 1; Table S3). The phosphatidylserine exposure on the outer surface of erythrocytes did not change significantly following treatment with deferasirox (from median mean fluorescence intensity [MFI] 20.0 [IQR 13.0–70.0] to 11.0 [8.0–36.0], p = 0.52). In vitro adhesion of erythrocytes to laminin remained unaffected (Table 1). None of the markers of iron status, including ferritin and transferrin saturation, changed significantly during deferasirox treatment. No differences were observed in coagulation and endothelial activation (Table S3). Of the 92 inflammatory proteins, 19 decreased significantly after deferasirox treatment. However, after correcting for multiple testing, the changes did not remain statistically significant (Table S4). Although in vitro neutrophil adhesion increased after treatment, there were no differences in neutrophil phenotype as measured by flow cytometry (Table S5). While markers of haemolysis decreased after 6 weeks of deferasirox treatment, no significant changes were seen in PoS and in vitro erythrocyte adhesion. The reduction in reticulocyte counts and total bilirubin levels could be the result of a decrease in oxidative damage of erythrocytes.11, 12 Previously, it was demonstrated that iron depletion had a positive effect on erythrocyte degradation, which was attributed to a lower MCHC leading to increased haemoglobin solubility and thus reduced polymerization and sickling of erythrocytes.7 In our study, MCHC levels decreased, although this was not statistically significant. Despite a significant decrease in haemolysis, we did not observe an improvement of PoS. This could either suggest that the decrease in haemolysis is mediated by other mechanisms (e.g. reduced erythrocyte membrane damage) than a reduction in HbS sickling or that 6 weeks of deferasirox treatment is perhaps too short to detect decreases in PoS and the other measured parameters. Concomitant use of hydroxyurea in the majority of patients might have limited the decrease in PoS by deferasirox. Our study is limited by the small sample size. In conclusion, treatment with low-dose deferasirox seemed to be safe in patients with SCD. While the short treatment period with low-dose deferasirox seemed to result in decreased haemolysis, no improvements were seen in erythrocyte rheology (PoS). A non-significant reduction in phosphatidylserine expression and inflammatory proteins was observed. Follow-up studies with a larger study population and longer treatment duration are needed to determine the place of low-dose iron chelation as an anti-oxidant treatment in SCD. Study concept and design: EN, AEG, KdH, RvB. Acquisition of data: AEG, LAdL, EN. Experiments: BMB, AEG, LAdL, MV. (Statistical) analysis of data: AEG, LAdL, BMB. Interpretation of data: AEG, EN, BJB, RvB, JH, JCMM. HLM, CGS. Drafting of the manuscript: AEG, BMB, EN. Critical revision of the manuscript for important intellectual content: AEG, BMB, MV, HLM, JCMM, CGS, KdH, JH, RvZ, BJB, RvB, EN. This study was supported by a grant from the Stichting Amsterdam UMC Foundation. The authors declare no conflicts of interest. This study was performed in accordance with the Declaration of Helsinki. Local ethics approval was obtained. All patients gave written informed consent before entering the study. ClinicalTrials.gov identifier: NCT05392101. Data are available upon request. Data S1. Please note: The publisher is not responsible for the content or functionality

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