A Potential Novel Application of Eltrombopag: A Combination Agent to Enhance Iron Chelation Therapy
Authors: Vlachodimitropoulou Koumoutsea Evangelia , Porter John B , Cooper Nichola , Psaila Bethan , Sola-Visner Martha 
Published: October 2019

Abstract
INTRODUCTION
Eltrombopag (ELT) is an orally available, non-peptide, small-molecule thrombopoietin receptor (TPO-R) agonist approved for the treatment of chronic immune thrombocytopenic purpura (ITP). Additionally ELT appears to bind intracellular iron (Roth et al, 2012, Blood) and our group has previously demonstrated its ability to progressively mobilize iron from cardiomyocytes in vitro. (Vlachodimitropoulou et al, Blood 2014, Volume 124, 21). The ELT concentrations at which iron was mobilized were substantially less (1µM) than with the clinically available iron chelators Desferrioxamine (DFO), Deferiprone (DFP) and Deferasirox (DFX), where 30µM iron binding equivalents (ibe) were required to achieve similar effects (Vlachodimitropoulou et al, 2014. Blood, Volume 124, 21). Importantly , the 1µM effective concentration of ELT for mobilizing cellular iron is nearly twenty-fold less than peak plasma concentrations reported clinically, even with low doses (30mg) of ELT (Gabianski, Journal of Clinical Pharmacology, 2011;51:842-856). At this low dose, increments in platelet counts do not typically exceed 1.2 x the baseline values in healthy volunteers with repeat dosing (Jenkins et al 2007, Blood, 109; 11 ). Hence it is predicted that effective chelating doses of ELT could be given without promoting unacceptable thombocytosis. In principle, still lower concentrations could be used for iron chelation if combined with another iron chelator. Here we explore and compare the concentrations at which effective cellular chelation is achieved with ELT alone or in combination with another chelator.
METHODS
As cardiomyocytes are a target tissue for transfusional iron overload and provide a particular therapeutic challenge once iron has accumulated in them, the cardiomyocyte cell line H9C2, derived from embryonic rat ventricle, was chosen for investigation. As hepatocytes represent the cell type with the largest quantity of iron deposition, a human hepatocarcinoma HuH7 cell line was also evaluated. Cellular iron loading and iron mobilization were measured as a decrease in cellular iron content using the ferrozine assay (Vlachodimitropoulou et al 2015, British Journal of Haematology). The cells loaded with iron using 10% FBS containing media and then exposed to iron chelators/ELT. Cells were then lysed and intracellular iron concentration determined via the ferrozine assay, normalized against protein content. Acridine Orange/Propidium Iodide staining was used to ensure viability was consistently >98% during experiments, and to assess the toxicity of ELT on the cardiomyocyte and hepatocyte cell lines.
RESULTS
Monotherapy with 1µM ELT removed 42% of total cardiomyocyte iron following 8 hours of treatment. This was notably more efficient than in hepatocytes, where only 7% of cellular iron was removed with 1µM ELT monotherapy (Table 1). In Table 1 we can see the difference in iron removal between ELT monotherapy and combination with chelators after 8 hours. The effect in combination with all chelators was substantial. Viability was unaffected by combinations of 1µM ELT with other chelators. The hydrophilic hydroxypridinone iron chelator CP40, which has no iron mobilizing effects when used alone, enhanced iron mobilization by ELT, indicating that ELT can shuttle iron from cells onto a second chelator.
CONCLUSION
Remarkably low concentrations of ELT monotherapy mobilize cellular iron from cardiomyocytes compared with conventional iron chelators. Furthermore, when used at as little as 1μΜ, in combination with standard therapeutic concentrations of DFO, DFP and DFX, the percentage of iron mobilized from cardiomyocytes more than doubled. Experiments with CP40 indicate that ELT acts as a shuttle molecule for chelated iron onto a second 'sink chelator' and that this is the likely mechanism for the enhanced iron mobilization with other iron chelators. While the action of ELT on the TPO-R is highly species-specific and occurs only in humans and primates, we found effective iron mobilization from both rat cardiomyocytes and human hepatocyte cell lines. This is consistent with an iron chelating mechanismdistinct from the TPO-R downstream signaling mechanism of ELT. The concentrations of ELT used to achieve iron mobilization in combination are clinically achievable and are unlikely to increase platelet counts in patients without thrombocytopaenia.

Disclosures
Porter: Celgene: Consultancy; Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.