Deferasirox (Exjade®, ICL670): A Journey into Labile Iron Centers of Living Cardiomyocytes.

Introduction and Aims: Iron toxicity that prevails in iron overload is associated with forms of labile cell iron (LCI) that appear in tissues such as heart, endocrine glands and liver. The primary goal of chelation is to reduce LCI appearance by preventing the entry of labile plasma iron into cells and by chelating LCI. The present study was aimed at evaluating the capacity of the novel oral iron chelator deferasirox (a) to access cardiomyocytes and chelate LCI in the cell organelles harboring labile iron and thereby prevent its involvement in reactive oxidant species (ROS) formation and (b) to reduce the cell iron load by extraction of labile and accumulated iron.

Methods: LCI pools were revealed by fluorescence microscopy imaging using novel fluorescent iron sensors addressed to various organelles (cytosol, nucleus, mitochondria and endosomeslysosomes) via specific peptide target sequences. Resident or imported labile iron binds the fluorescence metal sensor and quench its fluorescence. The chelator’s capacity to restore quenched fluorescence in a given cell compartment is indicative of its permeation and chelating potential, which is also assessed with ROS-sensitive probes targeted to cytosol or mitochondria (Glickstein et al 2005, Blood In press). Chelator-mediated extraction of cell iron was assessed in cardiomyocytes from neonatal rats or murine H9C2 cells preincubated with 360μM ⁵⁹Fe-ferric ammonium citrate for 3 h or 24 h (representing respectively radiolabelled LCI=RLCI or stored iron=RSI). Iron extraction was followed as cell iron retention following 0.5–24 h treatment with chelators in culture medium (with 20% fetal calf serum ≡ 1% albumin or with 4% bovine serum albumin).

Results and Discussion: In situ fluorescence LCI tracing studies indicate that LCI chelation by up to 100 μM deferoxamine (DFO) (x10 higher than normally attained in plasma during infusion) demanded incubation times >1 h. Conversely, the relatively smaller and more lipophilic deferasirox at 50–100 μM demonstrably gained access to all LCI pools associated with organelles following 20′–30′ incubation in serum-free medium and 30′–60′ in serum-containing medium (note: deferasirox reaches clinically C_max and trough plasma levels of approximately 100 μM and 20 μM respectively with daily doses of 20 mg/kg). Following 6 h treatment with chelator in serum-containing medium, LCI (measured as RLCI) was 15 and 60% reduced by 20 and 100 μM DFO respectively, and 15 and 34% reduced by 20 and 100 μM deferasirox, respectively. On the other hand, the reduction in mostly stored RSI with 20 and 100 μM chelator was 5 and 45% for DFO and 0 and 20% by deferasirox, respectively.

Conclusions: At drug concentrations equivalent to those attained in plasma with a single daily drug intake, ie 50–100 μM: (a) deferasirox gains relatively fast entry into cardiomyocytes and their intracellular compartments, scavenges LCI and attenuates ROS formation; (b) exit of the (deferasirox)₂-Fe chelates formed from LCI pools (RLCI) by added drug is relatively slower than entry of the free drug, but evident within 1–3 h even at 20 μM drug concentrations (equivalent to trough plasma levels).