scholarly journals Eltrombopag Mobilizes Intracellular Iron Stores at Concentrations Lower Than Those Required with Other Clinically Available Iron Chelators

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1353-1353 ◽  
Author(s):  
Evangelia Vlachodimitropoulou Koumoutsea ◽  
Nichola Cooper ◽  
Bethan Psaila ◽  
Martha Sola-Visner ◽  
John B Porter
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Iron Concentration ◽  
Iron Removal ◽  
Iron Chelators ◽  
Intracellular Iron ◽  
Time Points ◽  
Iron Mobilization ◽  
Cellular Iron

Abstract 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.

A Potential Novel Application of Eltrombopag: A Combination Agent to Enhance Iron Chelation Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3357-3357 ◽  
Author(s):  
Evangelia Vlachodimitropoulou Koumoutsea ◽  
John B Porter ◽  
Nichola Cooper ◽  
Bethan Psaila ◽  
Martha Sola-Visner
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Iron Chelation ◽  
Iron Chelator ◽  
Iron Chelators ◽  
Target Tissue ◽  
Intracellular Iron ◽  
Platelet Counts ◽  
Iron Mobilization ◽  
Cellular Iron

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.

scholarly journals Eltrombopag (ELT) Reverses Iron Mediated Suppression of Insulin Secretion in Pancreatic Cells By Chelating Iron and Decreasing ROS

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1278-1278 ◽  
Author(s):  
Evangelia Vlachodimitropoulou Koumoutsea ◽  
Pimpisid Koonyosying ◽  
John B. Porter ◽  
Nichola Cooper ◽  
Bethan Psaila ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Iron Overload ◽  
B Cell ◽  
Cell Line ◽  
Iron Chelators ◽  
Ros Production ◽  
Iron Loading ◽  
Intracellular Iron ◽  
Pancreatic Cell ◽  
Cellular Iron

Abstract Introduction: Eltrombopag (ELT) is an orally active, nonpeptide, small-molecule thrombopoietin receptor agonist (TPO-R), used to treat chronic immune thrombocytopenic purpura (ITP). We have recently reported its ability to mobilise cellular iron, and act as an iron shuttle when combined with currently licensed chelation therapies (Vlachodimitropoulou et al, Blood 2014, Volume 124, 21). Tissue damage induced by ROS production in iron overload conditions includes endocrine dysfunction including type I diabetes. We have developed a model where iron overload of the pancreatic cell line (RINm5F) inhibits insulin secretion. We investigated the ability of ELT, compared with clinically licensed iron chelators, to reverse ROS production and concomitant suppression of insulin production by iron loading of these cells. Methods: Cell line: RINm5F is a clonal rat pancreatic b cell line (LGC ATCC Sales, UK). These cells secrete insulin following a glucose challenge (Praz et al., 1983, Biochemistry J). Intracellular Iron: Cellular iron loading and mobilisation were measured as a decrease in cellular iron content using the ferrozine assay (Vlachodimitropoulou et al. 2015, British Journal of Haematology). A four-fold increase in intracellular iron compared to control was obtained by serially treating cells with 10% Fetal Bovine Serum (FBS) RPMI media in pancreatic cells over two ten hour periods (Figure 1A). The cells were then exposed to iron chelators/ELT, lysed and intracellular iron concentration determined, normalised against protein content. Reactive oxygen species (ROS) estimation: A cell-permeable oxidation-sensitive fluorescent probe 5,6-carboxy-2',7'- dichlorofluorescein diacetate (DCFH-DA); (Molecular Probes, Leiden, Netherlands) was used to measure intracellular ROS. Following iron loading, the cells were pre-incubated with 6 mM H2DCF-DA for 30 minutes at 37°C. Chelators were added and the fluorescence of control and treated cells was read throughout the treatment period in the plate reader (excitation 504 nm, emission 526 nm). Insulin quantification: Following iron loading and chelator treatment, the cells were challenged with Kreb's Ringer Buffer twice, for one hour at a time, containing 2.8mM and 16.7mM glucose (Lu et al. 2010, Toxicology letter). The supernatant was then collected and insulin concentration determined using a standard rat insulin ELISA kit (Life Technologies Limited, UK). Viability: The Sulforhodamide B (SRB) viability assay was used to ensure viability >98% and assess the toxicity on the pancreatic cell line. It is commonly used to measure drug-induced cytotoxicity and is a colorimetric assay dependent on healthy adherent cells. Results: Pancreatic cell iron loading was achieved with serial changes of media containing 10% FBS. This loading method was comparable to treating cells with ferric ammonium citrate (FAC) for 24 hours, which was not adopted as FAC adheres to the extracellular surface and produces bias to our intracellular iron quantification system when using iron chelators (Figure 1A). When cells were then treated with increasing ELT concentrations, a dose-dependent cellular iron removal were demonstrated so that at 10μΜ for 8hours, approximately 40% of total cellular iron was mobilised (Figure 3A). Iron mobilisation by ELT was further enhanced by combination with DFO, DFX or DFP (Figure 3). For example, when 10μΜ DFP is combined with 3μΜ ELT, iron mobilisation increases by a further 17% when compared to DFP treatment alone (Figure 3C). ROS production was also decreased in iron-loaded cells in a concentration-dependent manner by increasing ELT concentrations (Figure 2). These reductions in ROS and cellular iron were associated with restoration of insulin secretion, which was reduced by 2.6 fold following iron loading (Figure 1B). The levels of insulin secretion returned back to higher than baseline levels (better than with DFX 1μΜ) (Figure 1C). Conclusions: This is the first demonstration of a link between cellular iron overload and reduced insulin secretion using pancreatic b-cell line. This is also the first demonstration of improved pancreatic b-cell function, evidenced by restoration of insulin secretion, when iron is chelated and ROS decreased by ELT and other iron chelators. ELT may be useful alone or in combination with other chelators for decreasing iron-mediated ROS induced damage to pancreatic b-cells. Disclosures Porter: Novartis: Consultancy, Honoraria, Research Funding; Bluebird Bio: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Celegene: Consultancy.

Modeling Combination Chelation Regimes To Optimize Cellular Iron Removal and Explore Mechanisms Of Enhanced Chelation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2200-2200
Author(s):  
Evangelia Vlachodimitropoulou ◽  
Garbowski Maciej ◽  
John B Porter
Keyword(s):  
Iron Concentration ◽  
Dual Therapy ◽  
Iron Chelator ◽  
Iron Removal ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Synergy Index ◽  
Cellular Iron ◽  
Low Concentrations ◽  
Oral Chelators

Abstract Introduction Monotherapy with clinically available chelators, namely deferoxaime (DFO), deferasirox (DFX) or deferiprone (DFP) is effective but often slow and suboptimal. Combinations of DFO with DFP have been used clinically to enhance cellular iron mobilization but the conditions under which this occurs have not been studied systematically. With the emergence of DFX, the possibility exists to combine this with either DFO or DFP to enhance chelation. We have developed a system to study the optimal concentrations and times of exposure to these chelators, alone or in combination for maximising cellular iron removal. Isobol modeling has been used to determine whether interaction is additive or synergistic. The demonstration of synergy would imply the primary chelator acting as a ‘sink’ for iron chelated and donated to this sink by low concentrations of a secondary ‘shuttle’ chelator as shown in plasma (Evans et al. TransL. Res, 2010). Methods Human hepatocellular carcinoma (HuH-7) cells were chosen as hepatocytes are the major cell of iron storage in iron overload. Iron concentration was determined using the ferRozine (Riemer et al. Anal Biochem. 2004). A threefold increase of intracellular iron compared to control was obtained by serially treating cells with 10% FBS RPMI media. The cells were then exposed to iron chelator then lysed and intracellular iron concentration determined via the ferrozine assay, normalized against protein content. Cell viability was assessed using 0.4% Trypan blue as well as Acridine Orange /Propidium Iodide and was consistently > 98%. Isobolograms were constructed (Tallarida et al, Pharmacol Ther, 2010) as well as a the synergy index (QUOTE 1-1/R) x 100 (%), where R = difference of areas between the line of additivity and the curve of synergy on the isobologram. This index represents how much of the obtained effect exceeds that expected by additivity of two chelators. Results Monotherapy with DFP, DFX or DFO at clinically relevant concentrations of 1 to 30µM iron binding equivalents (IBE), induced both dose and time dependent cellular iron removal. Dual therapy combinations of all 3 chelators enhanced iron removal at 4, 8 and 12 hours. At 4 hours of incubation, whereas 10µM DFO alone had no demonstrable effect on cellular iron removal, addition of DFP at as little as 1µM IBE increased cellular iron removal. Table 1 shows examples of cellular iron removal at specimen chelator concentrations alone or in combination at 8h. The combination of DFX with DFO, DFX with DFP and DFP with DFO all resulted in enhanced cellular iron removal. The combination of DFP and DFX was the most effective. Isobol plot analysis from multiple chelator concentrations demonstrated synergy for all pairs at 4 and 8 hours of exposure. The derived synergy index at 8h indicates that when DFX and DFO are combined, 49% of the chelation effect is due to synergy in this system and 51% in the case of DFP and DFO combination. Most interestingly, the synergistic effect is even greater, in the case of the two oral chelators DFP and DFX when in combination (59%). Figure 1. Conclusion Remarkably low concentrations of a second chelator are required to enhance cellular iron removal by the primary chelator. Isobol analysis shows synergy rather than additivity as the mechanism for enhanced chelation for all 3 combinations, implying a ‘shuttle’ and ‘sink’ effect. Interestingly, the combination of two oral chelators DFP and DFX showed the most marked enhancement of cellular iron removal, without cellular toxicity, suggesting a potentially powerful therapeutic approach, provided this is also well tolerated clinically. The long plasma half life of once daily oral DFX will allow a continuous ‘sink’ for iron shuttled by the shorter acting DFP. Line of Additivity Curve of Synergy below the line Disclosures: Porter: Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Celgene: Consultancy.

All-Trans-Retinoic Acid (ATRA) Causes Extensive Differentiation In the NPM Mutant, Non-APL Leukemic Cell Line OCI-AML3

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3305-3305 ◽  
Author(s):  
Matthew A. Kutny ◽  
Steven J. Collins ◽  
Keith Loeb ◽  
Roland B. Walter ◽  
Soheil Meshinchi
Keyword(s):  
Cell Viability ◽  
Cell Count ◽  
Cell Line ◽  
Cell Lines ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Live Cell ◽  
Leukemic Cells ◽  
Viable Cell Number ◽  
Nuclear Segmentation

Abstract Abstract 3305 The differentiating agent ATRA has been used successfully in the treatment of acute promyelocytic leukemia (APL). By comparison, non-APL AML has not shown similar sensitivity to ATRA induced differentiation. Recent data has suggested that a subset of de novo AML patients with nucleophosmin (NPM1) mutations may benefit from addition of ATRA to conventional therapy. The NPM1 gene has several functions affecting cell cycle proliferation including regulation of ribosome biogenesis and centrosome duplication and it acts as a histone chaperone. Mutation of the NPM1 gene leads to differentiation arrest contributing to AML pathogenesis. We hypothesized that leukemia cells with NPM1 mutations could be induced to undergo differentiation. We tested this hypothesis with the NPM1 mutant AML cell line OCI-AML3 and compared the results to identical assays using the AML cell line HL-60 which has been previously well documented to differentiate in response to ATRA therapy. OCI-AML3 and HL-60 cell lines were treated for 5 days with control media and four ATRA doses including 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell viability was assessed by flow cytometry. Compared to the control condition, OCI-AML3 cells treated with the lowest dose of ATRA (0.2 μM) had a live cell count 21.6% of the control. HL-60 cells treated at even the highest ATRA dose (25 uM) had a live cell count 79.3% of the control. Due to the sensitivity of OCI-AML3 cells to the toxic effects of ATRA, the experiment was repeated with lower doses of ATRA including 0.001 μM, 0.01 μM and 0.1 μM. At the lowest dose of ATRA (0.001 μM), OCI-AML3 cells demonstrated a cell viability of 49% with further decrease to 26% at 0.1 μM dose of ATRA. At similar ATRA doses, cell viability for HL-60 cells was 91% and 85%, respectively (see table 1). Table 1: Cell viability as a percent of control cells after 5 days of treatment at three different doses of ATRA in OCI-AML3 and HL-60 cell lines. Cell Line: ATRA 0.001 μM ATRA 0.01 μM ATRA 0.1 μM OCI-AML3 49% 33% 26% HL-60 91% 91% 85% We subsequently determined the time course of changes in cell growth and the extent of differentiation at each point was determined by morphologic assessment. Both cell lines were treated with ATRA at doses of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM for a total of 4 days. Each day viable cell number was determined. In contrast to the HL-60 cells which had continued growth in lower ATRA doses, OCI-AML3 cells demonstrated exquisite sensitivity to growth arrest at the lowest doses of ATRA. Cell morphology was assessed daily with modified Wright-Giemsa staining of cells. Cells were examined for signs of myeloid differentiation including decrease in nuclear to cytoplasmic (N/C) ratio, nuclear segmentation, and cytoplasmic granules and vacuoles. At the lowest dose of ATRA (0.001 μM), after 4 days of exposure, significant number of OCI-AML3 cells demonstrated morphologic evidence of differentiation. At this ATRA dose and exposure interval, HL-60 cells showed no evidence of differentiation. At an ATRA dose of 1 μM (considered a standard dose used for differentiation of HL-60 cells), the OCI-AML3 cells showed differentiation changes as early as day 2 with nuclear segmentation and decreased N/C ratio while HL-60 cells did not show any change at this time point. After 4 days of ATRA exposure, most OCI-AML3 cells showed segmented nuclei and vacuolated cytoplasm, whereas HL-60 cells showed less distinct signs of differentiation with some cytoplasm granules and cup shaped nuclei. This data suggests that leukemic cells with NPM mutations may be susceptible to the pro-differentiating properties of ATRA. Further substantiation of this data with primary human specimens may ultimately provide the rationale for a novel therapeutic option using ATRA-based differentiation therapy for subsets of non-APL AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Potential of Plant-Based Compounds As Iron Chelators

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3631-3631
Author(s):  
Sarah Lane ◽  
Farzam Viand ◽  
Kayla Bolduc ◽  
Juergen Ehlting ◽  
Patrick B Walter
Keyword(s):  
Iron Overload ◽  
Cell Viability ◽  
Conflicts Of Interest ◽  
Plant Root ◽  
Thuja Plicata ◽  
Iron Chelators ◽  
Total Phenolic ◽  
Root Health ◽  
Phenolic Concentration

Abstract Introduction: The use of iron-chelators is an important clinical treatment for iron overload diseases such as β-Thalassemia (Thal) and neurodegeneration with brain iron accumulation. Iron overload can impair immune cell, cardiac and neurological function. Iron chelation can alleviate some of this morbidity, but at increasing doses certain chelating agents can have serious side effects. Plant-based treatments may offer an alternative. In plants, Fe is required for photosynthesis and enzyme production but is often limited for uptake from the soil. When Fe is limited, plant roots may produce a range of compounds, including chelators, to assist in solubilizing Fe precipitates. Plant-produced phenolic acids such as p-coumaric acid (Cou), caffeic acid (Caf), or chlorogenic acid (CGA) have shown an affinity for Fe and may play a role in plant iron uptake. Plants adapted to environments where Fe is more difficult to access, such as alkaline soils, could show a higher prevalence of these compounds, along with plants generally abundant in phenolics. In this project, the alkaline tolerant plants Thuja plicata (cedar) and Lavandula x intermedia (lavender), along with the phenolic rich Populus trichocarpa x deltoides(Poplar), were investigated for their potential to produce Fe-chelators in response to low Fe. Methods: Cedar, lavender, and poplar cuttings were clonally propagated and cultivated aeroponically to improve efficiency of root collection. Extracts or exudates from roots grown with or without Fe were isolated for characterization as Fe-chelators. Phenolics from root washings were captured with chromatography and separated by collection into fractions in different solvents. These were evaluated for total phenolic concentration against gallic acid as a standard. An in vitro competition assay was used to characterize Fe-binding ability of root isolates. Isolates were compared to standard chelators DFO and EDTA, and model compounds Cou, Caf, and CGA to determine inhibition of the competition reaction. A bioassay quantified intracellular Fe in monocytic THP-1 cells (to model RE system) grown for 8 weeks with chronic relevant non-transferrin bound iron levels (4-20 μM Fe-citrate, CrFe) and without (Con). Cultures were also investigated for other effects of acute Fe treatment and potential chelators over time. Results: Aeroponic plant cultivation improved root health and growth compared to previous hydroponic methods. Fe-deficient plants produced isolates that were different from Fe-normal plants following an analysis of phenolic fractions. Isolates in isopropanol were found to be 104% more plentiful in Fe-deficient poplars, which may indicate Fe-chelating potential. Between species, lavender had the highest phenolic concentration in root isolates, followed by cedar and poplar. Cedar roots showed an increased composition of phenolics compared to Fe-deficient poplar, supporting the potential for species-specific Fe responses. Analysis of Fe responses between species is ongoing. Competition assays showed that lavender root isolates exhibited 36% greater inhibition than 80 μM EDTA and 46% greater than 100 μM DFO. In direct comparison to DFO at 50 μM, Caf was equivalent, CGA had 30% greater inhibition, and inhibition by Cou was 41% lower. CrFe cells had 104% greater intracellular Fe compared to Con cells. Addition of acute Fe over 24 h significantly increased Fe content of cells grown in both CrFe and Con conditions and altered cell viability. A dose-dependent reduction in Fe levels was seen with increasing CGA in both CrFe and Con cells. Overall, Fe in samples treated with CGA were comparable to those with DFO. The effect of plant root isolates on intracellular Fe and cell viability is ongoing. Conclusion: Plant species from different soil types have altered responses to Fe-deficiency. Lavender and cedar, more tolerant of unfavorable soils, may produce more Fe-chelating phenolics as part of their response to low Fe. This was observed in vitro, as lavender isolates contain chelators that stimulate inhibition of the competition reaction similarly to DFO and EDTA at moderate concentrations. As a model, Caf, CGA and Cou also prove to have Fe-chelating activity comparable to DFO at lower concentrations. After using these plant compounds in bioassays, their successful reduction of intracellular Fe in CrFe THP-1 cells show the promise of plant root isolates to be clinically useful Fe-chelators. Disclosures No relevant conflicts of interest to declare.

scholarly journals Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1497-1503
Author(s):  
JB Porter ◽  
M Gyparaki ◽  
LC Burke ◽  
ER Huehns ◽  
P Sarpong ◽  
...  
Keyword(s):  
Binding Constant ◽  
Therapeutic Potential ◽  
Binding Constants ◽  
Iron Chelator ◽  
Dominant Factor ◽  
Iron Chelators ◽  
Lipid Solubility ◽  
Iron Release ◽  
Intracellular Iron ◽  
Iron Mobilization

A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4- ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2- ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.

scholarly journals Association of Liver Iron Concentration Levels with Myocardial T2* Responses in Transfusion-Dependent Thalassemia Major Patients Treated with Deferasirox and Deferoxamine- Extension of Cordelia Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2155-2155 ◽  
Author(s):  
Dudley J Pennell ◽  
John B Porter ◽  
Antonio Piga ◽  
Jackie Han ◽  
Alexander Vorog ◽  
...  
Keyword(s):  
Research Funding ◽  
Iron Concentration ◽  
Thalassemia Major ◽  
Iron Removal ◽  
Liver Iron Concentration ◽  
Liver Iron ◽  
Time Points ◽  
Cardiac Iron ◽  
Time Point

Abstract Background: Beta thalassemia major patients (pts) are at an increased risk of heart failure, due to the deposition of iron in the heart causing myocardial siderosis. Intensive long-term iron chelation therapy (ICT) is required to obtain a normal myocardial T2* (mT2* >20 ms). Previously published studies suggested that cardiac iron removal lags changes in liver iron, and liver iron concentration (LIC) may affect the rate of removal of cardiac iron (Porter et al, ASH 2013). The objective of these analyses was to evaluate the association of the severity of LIC levels with the change in mT2* responses in pts with myocardial siderosis when treated with deferasirox (DFX) and deferoxamine (DFO) for up to 24 months (mo) in the CORDELIA study. Due to the very low pt numbers in the DFO arm, the results for these pts are not presented here. Methods: The study design, inclusion, and exclusion criteria have been reported previously (Pennell et al, Am J Hematol. 2015). Pts were categorized into LIC <7, 7 to <15 and ≥15 mg Fe/g dry weight (here after mg/g) both at baseline (BL) and specific visits, to assess the relation of absolute LIC and changes in LIC overtime, with mT2* and cardiac iron concentration (CIC), respectively. During the study, mT2* (ms), and LIC (mg/g) were measured every 6 mo at the same time point. CIC (mg/g) was analyzed as a post hoc parameter derived from mT2*. The change in mT2* was assessed as geometric mean (Gmean)±coefficient of variation (CV), ratio of the Gmean at specific time points divided by that at BL (Gmean at specific time point/Gmean BL) and both CIC and LIC as mean±SD, unless otherwise specified. Results: Of 197 pts, 160 (81.2%) completed 12 mo of treatment and 146 (74.1%) entered into the extension study whereas 103 pts continued on initially assigned treatment. Pts completing 24 mo of treatment included 65 (87.8%) of 74 pts (mean age 20.1±6.9 years, 59.5% male) on DFX and the results for these pts are presented as follows. Average actual doses (mg/kg/d) were 26.7±8.9, 31.5±7.4, 38.0±2.9 for LIC <7, 7 to <15, ≥15, respectively, during the extension study. The LIC levels for pts categorized by LIC <7, 7 to <15 and ≥15 improved from BL to Mo 24 as follows: 72% decrease (mean absolute change, -15.1±14.1), 66% decrease (-26.6±13.0), and 19% decrease (-10.2±15.7), respectively. For pts with BL LIC <7, 7 to <15, ≥15, mT2* improved from BL to Mo 24 as follows: 43% increase (14.0±18.1 to 21.6±31.1; mean abs change, 7.8±4.0), 50% increase (12.3±34.4 to 19.1±46.4; 8.0±6.0), and 30% increase (11.1±30.8 to 14.5±40.8; 4.1±5.0). The CIC values improved from BL to Mo 24 by 38% (1.8±0.4 to 1.1±0.5), 40% (2.3±0.9 to 1.4±0.7), and 23% (2.6±1.0 to 1.9±1.0), respectively. The mT2* responses for pts categorized according to visit specific LIC levels (LIC <7, 7 to <15, ≥15) from BL to Mo 12 were 22% increase (mean abs change, 3.7±4.3) in LIC <7, 21% increase (2.7±2.0) in LIC 7 to <15, and 7% increase (1.5±3.2) in LIC ≥15. From BL to Mo 24, mT2* increased by 51% (mean abs change, 7.8±5.3), 35% (4.1±2.5), and 11% (2.0±4.4), respectively. The CIC levels improved from BL to Mo 24 by 40% (mean abs change, -1.0±0.8) in LIC <7, 31% (-1.0±0.6) in LIC 7 to <15, and 6% (-0.1±0.8) in LIC ≥15. The change in mT2* (Gmean ratio) at Mo 6, 12, 18 and 24 are shown in the Figure A. The mT2* response was higher in pts who achieved a lower LIC category (LIC <7) at respective time points and this change in mT2* was more apparent at 18 and 24 mo of treatment with DFX. Discussion: Overall, DFX treatment resulted in a substantial decrease in LIC and improved mT2*. These results suggest a greater difference in mT2* improvement and CIC reduction in pts who achieved lower LIC during treatment with DFX. This divergence was progressive with time, being maximal at Mo 24. Thus, a therapeutic response in LIC with DFX may be associated with a greater likelihood of improving mT2*. Pts with high LIC ≥15 may require an effective long-term treatment with higher doses of ICT to have an improvement in mT2*, suggesting that cardiac iron removal is likely to be slow in heavily iron overloaded pts. These results are consistent with the previous report which showed a significant decrease in LIC and increased mT2* responses at Mo 36 in pts who attained lower end-of-year LIC levels when treated with DFX (Porter et al, ASH 2013) and highlight the potential value of monitoring the liver and cardiac responses during ICT. To further understand the kinetics between liver and cardiac iron removal, prospective investigation is warranted. Disclosures Pennell: Novartis: Consultancy, Research Funding; Apotex: Consultancy, Research Funding. Porter:Celgene: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria. Piga:Acceleron: Research Funding; Cerus: Research Funding; Apopharma: Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding; Celgene Corporation: Honoraria. Han:Novartis: Employment. Vorog:Novartis: Employment. Aydinok:Cerus: Research Funding; Sideris: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Inhibition of the Jak/Stat Pathway Downregulates Immunoglobulin Production and Induces Cell Death in Waldenström Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1691-1691
Author(s):  
Stephen M Ansell ◽  
Deanna Grote ◽  
Sherine F. Elsawa ◽  
Mamta Gupta ◽  
Steven C Ziesmer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Thymidine Uptake ◽  
Complete Suppression ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Stat Pathway

Abstract Abstract 1691 Poster Board I-717 Waldenström macroglobulinemia (WM) is a B-cell malignancy that is characterized by the production of a monoclonal IgM protein and a lymphoplasmacytic infiltrate in the bone marrow. The aberrant production of the monoclonal IgM can result in serum hyperviscosity that can cause significant morbidity in patients with this disease. In previous work, we have shown that IL-6 significantly upregulates IgM secretion by WM cells and that IL-6 secretion is regulated by CCL5 (Rantes). We have also shown that IL-6 mediated IgM secretion in WM requires phosphorylation of Stat1 and Stat3. Because IL-6 induced signaling involves the Jak/Stat pathway, we tested whether the use of a Jak/Stat inhibitor, TG101348, would result in down regulation of CCL5, IL-6 and IgM production and inhibit cell proliferation and viability in WM. First, we determined whether TG101348 could inhibit the production of CCL5 because other Jak inhibitors have been shown to inhibit cytokine production. Using the BCWM.1 cell line as well CD19+ malignant cells from bone marrow specimens from WM patients, we measured CCL5 by ELISA in the culture supernatant 24 hours after treatment with increasing concentrations of the inhibitor. We found that CCL5 secretion was decreased by 50% at a concentration of TG101348 of 250nM and was completely inhibited at 2μM. Next, we measured IL-6 production after treatment with TG101348. We had previously shown that stromal cells are the primary source of IL-6 and therefore used the stromal cell line HS-5 to measure IL-6 by ELISA after treatment with the inhibitor. Our previous work had also shown that IL-6 secretion was mediated by GLI (a member of the Hedgehog pathway) rather than the Jak/Stat pathway. Interestingly, we found that IL-6 secretion was inhibited in a dose dependent fashion but required higher doses for complete suppression (8μM). We then measured IgM production by malignant B-cells 24 hours after treatment with TG101348. Our previous work had shown that IL-6 mediated IgM secretion was dependent on the Jak/Stat pathway. We found that IgM production was inhibited by 50% at 500nM and completely suppressed at 2μM. Finally, we measured the effect of TG101348 on cell proliferation and survival. Using the BCWM.1 cell line, we found that cell proliferation as determined by tritiated thymidine uptake was inhibited in a dose dependent fashion with 50% inhibition at 1μM. Inhibition of cell viability as measured by Annexin V/propidium iodide staining, however, required higher concentrations and cell viability was inhibited with an IC50 of 8μM. These data confirm the role of Jak/Stat signaling in the CCL5-IL-6-IgM axis in WM. We found that TG101348 generally suppressed the signaling and growth of WM cells but that pathways that were known to be Jak/Stat dependent required significantly lower doses to be completely inhibited. These data provide a strong rationale for the use of inhibitors of this pathway, such as TG101348, in the treatment of patients with WM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1497-1503 ◽  
Author(s):  
JB Porter ◽  
M Gyparaki ◽  
LC Burke ◽  
ER Huehns ◽  
P Sarpong ◽  
...  
Keyword(s):  
Binding Constant ◽  
Therapeutic Potential ◽  
Binding Constants ◽  
Iron Chelator ◽  
Dominant Factor ◽  
Iron Chelators ◽  
Lipid Solubility ◽  
Iron Release ◽  
Intracellular Iron ◽  
Iron Mobilization

Abstract A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4- ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2- ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.

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