The present COVID-19 pandemic due to SARS-CoV-2 novel coronavirus has resulted in high numbers of critically ill patients and deaths. 1 Emerging data on the maternal impact of COVID-19 suggest that the clinical course is similar irrespective of pregnancy. [2][3][4] However, despite these data, our report of two pregnancies with COVID-19-related, rapidly progressive coagulopathy may warrant caution.

SummaryIron chelators are increasingly combined clinically but the optimal conditions for cellular iron mobilization and mechanisms of interaction are unclear. Speciation plots for iron(III) binding of paired combinations of the licensed iron chelators desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) suggest conditions under which chelators can combine as 'shuttle' and 'sink' molecules but this approach does not consider their relative access and interaction with cellular iron pools. To address this issue, a sensitive ferrozine-based detection system for intracellular iron removal from the human hepatocyte cell line (HuH-7) was developed. Antagonism, synergism or additivity with paired chelator combinations was distinguished using mathematical isobologram analysis over clinically relevant chelator concentrations. All combinations showed synergistic iron mobilization at 8 h with clinically achievable concentrations of sink and shuttle chelators. Greatest synergism was achieved by combining DFP with DFX, where about 60% of mobilized iron was attributable to synergistic interaction. These findings predict that the DFX dose required for a halfmaximum effect can be reduced by 3Á8-fold when only 1 lmol/l DFP is added. Mechanisms for the synergy are suggested by consideration of the iron-chelate speciation plots together with the size, charge and lipid solubilities for each chelator. Hydroxypyridinones with low lipid solubilities but otherwise similar properties to DFP were used to interrogate the mechanistic interactions of chelator pairs. These studies confirm that synergistic cellular iron mobilization requires one chelator to have the physicochemical properties to enter cells, chelate intracellular iron and subsequently donate iron to a second 'sink' chelator.

Supplemental digital content is available in the text.

INTRODUCTION Eltrombopag (EP) is an orally bioavailable thrombopoietin receptor agonist developed to increase platelet production in a range of conditions associated with thrombocytopaenia. The finding that EP, in addition to increasing platelet counts in myelodysplastic syndromes (MDS), also slowed progression to acute myeloid leukemia has been linked to antiproliferative effects potentially mediated by chelation of labile intracellular iron pools in leukaemia cell lines (Roth et al, 2012, Blood). However, the ability of EP to progressively decrease total cellular iron and its relative efficacy in this regard compared with clinically available iron chelators such as Desferrioxamine (DFO), Deferiprone (DFP) and Deferasirox (DFX) have not been reported. Using a cell based assay system for total cellular iron we therefore compared cellular iron mobilization with EP to that achieved with clinically established iron chelators. METHODS The permanent cardiomyocyte cell line H9C2 derived from embryonic rat ventricle was chosen to model iron mobilization, as heart failure secondary to iron overload is the most common cause of death among patients with transfusion-dependent anaemias. Iron concentration was determined using the ferrozine assay (Riemer et al. Anal Biochem. 2004). A two fold increase of intracellular iron compared to control was obtained by serially treating cells with 10% FBS DMEM media. The cells were then exposed to iron chelators/EP, lysed, and intracellular iron concentration determined via the ferrozine assay, normalized against protein content. The LDH enzymatic viability assay was used to ensure viability was consistently >98% during experiments, and to assess the toxicity of EP on the cardiomyocyte cell line. RESULTS EP induced both dose and time dependent cellular iron removal from cells at 1, 2, 4 and 8 hours. At 1µM, EP was able to remove 42% of total cellular iron following 8 hours of treatment, and 60.1% and 65.62% at 10µM and 30µM respectively (Figure 1). Figure 2 shows that cell viability is compromised at concentrations of 10µM and 30µM to 96% and 92% respectively with eltrombopag, but not with the commercially used iron chelators, DFO, DFP and DFX. However, at 1µM EP the viability of the monolayer is maintained at >98%. The high effects of iron release noted in figure 1 by EP at 10µM and 30µM could be partially attributed to toxicity of the drug on the monolayer. In table 1 the difference in iron removal between EP and commercially used iron chelators after 8 hours of treatment is shown. Interestingly, with EP at only 1µM, 42.9% of cellular iron was removed, compared to 22.7 %, 34.9% and 19.3% in the case of DFO, DFX and DFP respectively, all at higher concentrations of 30µM iron binding equivalents (IBE). DISCUSSION AND CONCLUSION­­ Remarkably low concentrations of EP (1µM) are required to mobilize cellular iron in our cell system while maintaining cell viability at this concentration. This concentration is achievable clinically (Cmax 2-3 µM two to six hours post administration of 75mg orally) (Neito et al, Haematologica, 2011; Deng et al, Drug Metabolism and Disposition, 2011), and when considered alongside with the long plasma half life and elimination in urine and feces, could render it an effective iron chelator for other indications, particularly if combined with existing iron chelation regimes. It would be of interest to explore how EP performed in an animal iron overloaded/MDS model in isolation or as an adjunct to established chelation therapies. Figure 1: Percentage of intracellular iron removed from cardiomyocytes by increasing concentrations of Eltrombopag at different time points up until 8 hours Figure 1:. Percentage of intracellular iron removed from cardiomyocytes by increasing concentrations of Eltrombopag at different time points up until 8 hours Figure 2: Percentage cytotoxicity of cardiomyocyte monolayer following 8 hours of treatment with Eltrombopag and commercially used iron chelators Figure 2:. Percentage cytotoxicity of cardiomyocyte monolayer following 8 hours of treatment with Eltrombopag and commercially used iron chelators Table 1: Comparison of iron mobilization by Eltrombopag and commercially used iron chelators following 8 hours of treatment. Chelator Iron/protein (nmol/mg) SD % iron removal control 26.78 2.5 - DFO 30μM ibe 20.70 2.2 22.70 DFP 30μM ibe 17.43 1.4 34.89 DFX 30μM ibe 21.61 1.0 19.28 Eltrombopag 1μM 15.53 1.9 42.94 Eltrombopag 10μM 10.70 1.2 60.06 Eltrombopag 30μM 9.21 0.3 65.62 Disclosures No relevant conflicts of interest to declare.