Deferasirox Research Articles

Abstract LB-045: Iron depletion suppress the stemness markers and functions of cancer stem cells

Abstract Background: Cancer stem cells (CSCs) have been proposed to be responsible for tumor initiating, drug resistance, metastasis, and recurrence. Although CSCs are considered to be important targets therapeutically, clinically effective CSC targeting strategies have not yet been established. Iron plays an important role in critical cellular processes, such as energy production and DNA synthesis. Although adequate iron levels are essential for human health, iron overload is known to cause some types of cancer. CSCs are considered to be involved in the tumor regardless of early stage. Thus we have focused on the relationship between iron and CSCs. We previously reported that the expression of stemness markers was suppressed by iron chelator in miPS and miPS inducible CSC model cells (miPS-LLCcm). Here, we hypothesized that the expression of stemness markers and function were also suppressed by iron chelator in high malignant cancer cells with stemness potential. Method: We used 4 stemness markers expressing cancer cell lines (HSC-2: Oral squamous cancer, TE-4 and OE33: Esophageal cancer, and NT-2: Embryonal cell carcinoma). Deferasirox (DFX), an oral iron chelator, was used. The stemness markers expression including Nanog, Sox2, Oct3/4, Klf-4 and c-Myc was examined by western blot analysis. Self-renewal ability was examined by sphere formation assay. Anti-proliferative effect of DFX was assessed combined with CDDP using HSC-2 cells. Result: DFX suppressed the expression of stemness markers and sphere formation ability in 4 cancer cell lines. In contrast, CDDP could not suppressed them. In HSC-2 cells, combination therapy with CDDP and DFX synergistically suppressed the proliferation with suppression of stemness markers. Conclusion: Our results indicated that iron depletion generally suppressed the stemness markers and functions of high malignant cells same as miPS and miPS-LLCcm. Thus iron depletion can be a novel therapy for CSCs via suppression of CSCs function. Citation Format: Toshiaki Ohara, Yuki Katsura, Kazuhiro Noma, Toru Narusaka, Takuya Kato, Hiroaki Sato, Satoshi Komoto, Yasuko Tomono, Takayuki Ninomiya, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Iron depletion suppress the stemness markers and functions of cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-045.

Deferasirox, an oral iron chelator, with gemcitabine synergistically inhibits pancreatic cancer cell growth in vitro and in vivo.

ObjectivesIron is an essential element for cell proliferation and growth processes. We have reported that deferasirox (DFX), an oral iron chelator, showed antiproliferative activity against pancreatic cancer cells. This study aimed to elucidate the effects of combination of gemcitabine (GEM), standard chemotherapy for pancreatic cancer, and DFX in vitro and in vivo.ResultsGEM+DFX showed antiproliferative activity and induced apoptosis in pancreatic cancer cells in vitro. GEM+DFX suppressed xenograft tumor growth and induced apoptosis without any serious side effects compared with control, GEM, and DFX (average tumor volume: control 697 mm3 vs GEM 372 mm3, p < 0.05; GEM 372 mm3 vs GEM+DFX 234 mm3, p < 0.05). RRM1 and RRM2 protein levels were substantially reduced by DFX in BxPC-3 in vitro.ConclusionGEM+DFX has significant anticancer effects on pancreatic cancer cell through RR activity suppression.MethodsBxPC-3, a human pancreatic cancer cell line, was used in all experiments. Cellular proliferation rate was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay. Apoptosis was evaluated by flow cytometry and by measuring caspase 3/7 activity with luminescence assay. In the tumor xenografts in nude mice models, when five weeks after engraftment, drug administration began (day 0). After treatment for 21 days, the mice were sacrificed and the tumors were excised. Apoptotic cells in xenografts were evaluated by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Protein levels of ribonucleotide reductase (RR) subunit 1 (RRM1) and RR subunit 2 (RRM2) in BxPC-3 cells were assessed by western blot in vitro.

Efficacy and safety of iron chelators in thalassemia and sickle cell disease: a multiple treatment comparison network meta-analysis and trial sequential analysis

ABSTRACTBackground: To compare the efficacy and safety of desferrioxamine (DFO), deferiprone (DFP), deferasirox (DFX) and silymarin in patients with either thalassemia or sickle cell disorder through network meta-analysis.Methods: Electronic databases were searched for appropriate randomized clinical trials comparing iron chelators in patients with iron overload. Random effects model was used to generate direct, indirect and mixed treatment comparison pooled estimates for the following outcomes: serum ferritin, liver iron concentration (LIC), changes in serum ferritin, mortality, urine iron excretion, adverse events, neutropenia, agranulocytosis and number of patients withdrawing the chelating therapy.Results: Thirty-two clinical trials were included in the meta-analysis. DFX/DFO was associated with better serum ferritin levels compared to DFO, DFX, DFO/Silymarin and DFP/DFO. DFX/DFO also lower LIC significantly compared to DFO. DFP/DFO was associated with higher LVEF, low risk of adverse events and reduced end of serum ferritin compared to DFO. Combination of silymarin with either DFP or DFX was observed with reduced end of treatment serum ferritin compared to using either of the drugs alone. DFP was observed with better effects in sickle cell disease. The strength of evidence was very low for most of the comparisons.Conclusion: Relative estimates between the individual iron chelators have been established. However, this evidence should be considered preliminary and may change with the results of future head-to-head clinical trials.

Tolerability and efficacy of deferasirox in patients with transfusional iron overload: results from a German 2-year non-interventional study.

Iron overload (IOL) due to repetitive transfusions of packed red blood cells (pRBC) has a major impact on morbidity and mortality in patients with inherited bone marrow failure syndromes and hemoglobinopathies such as thalassemia and sickle cell disease. However, whether IOL influences the outcome of elderly patients with myeloid malignancies is not yet clear. Moreover, clinical trials have reported high drop-out rates during treatment with the oral iron chelator deferasirox (DFX). Here we report the results of a 2-year prospective observational study that aimed at describing the routine use of DFX in patients with hematological malignancies with regard to safety, efficacy and handling of the drug in a routine setting. A total of 406 patients were included. 58% of the patients were male. Most of the patients had myelodysplastic syndromes (MDS) (68%) and myeloproliferative neoplasms (MPN) (14%). Median time from first transfusion to study enrollment was 1.1 years (0-25.5 years) and most patients were chelation naive (91%) at enrollment. With regard to transfusion burden, most of the patients were moderately or mildly transfusion-dependent with 53% receiving 2-4 and 27% receiving less than 2 units of pRBC per month. Serum ferritin decreased from a mean of 2305 μg/l (± 1449 μg/l) to a mean of 1910 μg/l (± 1529 μg/l) at 24 months. There was no substantial change in transfusion-dependence during the observation period. Dose adjustments were reported in 48% of the patients with dose-escalation strategies being the most frequent reason for dosage increases (49%). The median observation time was 355days (5-1080days). Median duration of exposure to DFX was 322days (2-1078days). Two-hundred and ninety (72%) patients discontinued the trial prematurely after a median time of 235days (1-808days). Death (29%) and adverse events (23%) were the main reasons for discontinuation. Eleven percent of the patients discontinued treatment due to sufficient decrease in serum ferritin. Most frequent adverse events were decrease in creatinine clearance (22%), increase in serum creatinine (18%) and diarrhea (16%). This descriptive trial confirms the efficacy of DFX in decreasing the serum ferritin. Moreover, the high drop-out rates seen in prospective trials are recapitulated in this study, which can be attributed to adverse events in a substantial proportion of patients.

Iron Overload and Chelation Therapy in Hemoglobinopathies

Iron overload (IOL) is highly prevalent among patients with hemoglobinopathies; both transfusion dependent thalassemia (TDT) and non-transfusion dependent thalassemia (NTDT). Whether IOL is secondary to regular transfusions like in TDT, or develops from increased intestinal absorption like in NTDT, it can cause significant morbidity and mortality. In TDT patients, iron accumulation in organ tissues is highly evident, and leads to organ toxicity and dysfunction. IOL in NTDT patients is cumulative with advancing age, and concern with secondary morbidity starts beyond the age of 10 years, as shown by the OPTIMAL CARE study. Several modalities are available for the diagnosis and monitoring of IOL. Serum ferritin (SF) assessment is widely available and heavily relied on in resource-poor countries. Non-invasive iron monitoring using MRI has become the gold standard to diagnose IOL. Three iron chelators are currently available for the treatment of IOL: deferoxamine (DFO) in subcutaneous or intravenous injection, oral deferiprone (DFP) in tablet or solution form, and oral deferasirox (DFX) in dispersible tablet (DT) and film-coated tablet (FCT). Today, the goal of ICT is to maintain safe levels of body iron at all times. Appropriate tailoring ICT with chelator choices and dose adjustment must be implemented in a timely manner. Clinical decision to initiate, adjust and stop ICT is based on SF, MRI-LIC and cardiac T2*. In this article, we review the mechanism of IOL in both TDT and NTDT, the pathophysiology behind it, its complications, and the different ways to assess and quantify it. We will also discuss the different ICT modalities available, and the emergence of novel therapies.

Magnetic resonance imaging during management of patients with transfusion-dependent thalassemia: a single-center experience.

Cardiac and hepatic magnetic resonance imaging evaluation during treatment can tailor physicians' chelation therapy titrations. The aim of the study was to assess the relationship of cardiac and hepatic T2* values with chelation therapy in patients with transfusion-dependent thalassemia (TDT). A total of 106 patients with TDT who were followed up in Istanbul Medical Faculty Thalassemia Center were evaluated for the study. Forty-eight (45%) patients with TDT had more than one consecutive MRI examination. The patients were divided into three subgroups according to the cardiac T2* values as the high-risk group (T2* MRI < 10ms), medium-risk group (T2* MRI 10-20ms), and the low-risk group (T2* MRI > 20ms). The majority of patients used DFX (deferasirox) (79%) and deferiprone (DFP) (17%). Approximately 80% of patients according to cardiac T2* value and 40% of patients according to hepatic T2* value were initially in the low-risk group. Patients with follow-up MRI examinations exhibited significant improvement in liver iron concentration, which correlated with an increase in hepatic T2* values. The decrease of liver iron concentration was prominent in the DFX group (p < 0.01). The serum ferritin level was significantly correlated with liver iron concentrations (rs = 0.65, p < 0.001), hepatic T2* value (rs = -0.62, p < 0.001), but not with cardiac T2* value (rs = -0.20, p = 0.07). Cardiovascular and hepatic MRI is a useful follow-up tool during the assessment of risk groups and chelation therapy of patients with TDT. Consecutive MRI tests showed good monitoring of cardiac and liver iron overload.

Clinical-Reported Outcomes Of Deferoxamine Versus Deferasirox In The Treatment Of Homozygous BetaThalassemia

Iron chelation therapy with deferoxamine (DFO),the current standard for the treatment of iron overload in patients with betathalassemia,requires regular subcutaneous or intravenous infusions. This can lead to reduced quality of life and poor adherence,resulting in increased morbidity and mortality in iron-overloaded patients with beta-thalassemia. Deferasirox (DFX) is an orally administered iron chelator that has been approved for use in many countries. The requirement of an effective,well tolerated iron chelator with a less demanding mode of administration has led to the development of deferasirox. The present study was aimed to compare the satisfaction and compliance with deferoxamine versus deferasirox (Exjade®),a novel oral iron chelator in patients with transfusion - dependent beta- thalassemia. A cross-sectional,single-center investigation study was carried out in the Thalassemia Center of Ibn-Atheer Teaching Hospital in Nineveh province,Iraq. One hundred and eight thalassemic patients aged between 2- 20 years old having received multiple blood transfusions and a serum ferritin greater than 1500 ng/ml. Patients were randomised into two groups. Group 1 received deferoxamine at a dose of 20-50mg/kg/day and group 2 received deferasirox at the dose of 10-30 mg/kg/day. Another 56 apparently healthy volunteers were used as a control group. The assessment of chelation was done during the period between November 2013 and February 2014 by measurement of serum ferritin. Satisfaction and compliance was assessed by using a special questionnaire prepared by the researcher. Out of the 108 thalassemic patients enrolled there was no discontinuation in treatment with the two drugs under study. The serum ferritin did not change significantly in any of the chelation groups. In comparison with the patients who were treated with DFO,those receiving DFX reported a significantly higher rate of compliance and satisfaction (P &lt; 0.05). However,no significant difference was observed between the two groups regarding their satisfaction (P &gt; 0.05).Compliance with deferasirox (50 %) was more than that with deferoxamine (20 %). Satisfaction with deferoxamine was significantly lower than deferasirox (p= 0.00).

Iron-chelating agents attenuate NMDA-Induced neuronal injury via reduction of oxidative stress in the rat retina

Excitoneurotoxicity is regarded as one of the mechanisms of the death of retinal ganglion cells induced by retinal central artery occlusion and glaucoma. Oxidative stress is at least in part involved in excitoneurotoxicity. Fenton reaction, which is catalyzed by Fe2+, is known to cause formation of hydroxyl radical, one of reactive oxygen species, suggesting that chelation of iron may be protective against excitoneurotoxicity. In the present study, we histologically evaluated whether zinc-deferoxamine (Zn-DFO) and deferasirox (DFX), common iron-chelating agents, were protective against N-methyl-D-aspartate (NMDA)-induced retinal injury in the rat in vivo. Male Sprague–Dawley rats were subjected to intravitreal NMDA injection (200 nmol/eye). Zn-DFO (1, 3, 10, and 30 mg/kg), Zn (0.1, 0.2 and 0.6 mg/kg) and DFX (20 mg/kg) were intraperitoneally administered. Morphometric evaluations using paraffin-embedded retinal sections, and detection of Fe2+ using SiRhoNox-1, a fluorescent probe of labile Fe2+ in the retinal frozen sections were carried out. Intravitreal NMDA resulted in strong positive signals of SiRhoNox-1 in the ganglion cell layer 24 h after NMDA injection, suggesting that intravitreal NMDA caused Fe2+ accumulation in the retinal ganglion cells. Intravitreal NMDA induced retinal ganglion cell loss 7 days after NMDA injection. Zn-DFO (1, 3, 10, and 30 mg/kg), ZnCl2 (0.2 mg/kg, a corresponding dose of 1 mg/kg Zn-DFO) and DFX (20 mg/kg) prevented the damage of retinal ganglion cells, whereas 0.6 mg/kg ZnCl2, which is a corresponding dose of 3 mg/kg Zn-DFO, did not show any protective effects. Zn-DFO (30 mg/kg) significantly decreased the intensity of the fluorescence of SiRhoNox-1 and the transferrin immunofluorescence 24 h after NMDA injection, the number of TUNEL-positive cells 24 h after NMDA injection, that of 8-OHdG-positive cells, and that of 4-hydroxy-2-nonenal-positive cells 12 and 24 h after NMDA injection. These data suggest that iron-chelating agents protected retinal neurons against excitoneurotoxicity via reduction of iron content and oxidative stress in the rats in vivo. We proposed that treatment with iron-chelating agents would be a new strategy for the retinal diseases caused by excitoneurotoxicity.

Side effect of deferiprone as iron chelator in patient with thalassemia

Background There are currently three available iron chelators: deferoxamine (DFO), deferasirox (DFX) and deferiprone (DFP). In Dr. Cipto Mangunkusumo Hospital and Indonesia, in general, the accessibility of DFP for thalassemia patients has been adequate. Even though its efficacy in removing iron has been proven by countless studies, questions relating to its safety and possible side effects continue to be raised.Objective To assess common side effects of DFP usage by an intensive literature search and compare them to that in a pediatric thalassemia patient, in order to determine if the child’s symptoms in the were potentially caused by DFP.Methods A literature search using MeSH terms was done in PubMed. Full copies of articles that fulfil the inclusion criteria, based on their title, abstract, and subject descriptors, were critically appraised using The Joanna Brigs Institute (JBI) critical appraisal tools.Results A total of 10 research articles from 1998-2013 were deemed applicable to this study including: 2 case reports, 5 prospective cohort studies, 2 retrospective cohort studies, and 1 randomized control trial with a grand total of 1,026 samples.Conclusion Side effects of DFP include: neutropenia, agranulocytosis, increased ALT, gastrointestinal problems, arthralgia or arthropathy, increased appetite or weight, thrombocytopenia, urine discoloration, as well as auditory and visual disturbances. Our case report patient’s symptoms of gum bleeding and haemorrhagic mass are not related to her DFP consumption.

Exploring the binding pattern between pepsin and deferasirox using detailed experimental and computer simulation methods.

Steady-state fluorescence spectroscopy indicated that a ground state complex was formed between deferasirox (DFX) and pepsin. The binding parameters and thermodynamic parameters of pepsin–DFX complex formation suggested the presence of only one high affinity binding site in the binding process of DFX and pepsin and that the binding process was hydrogen bond dominated. According to the MD simulation optimal pepsin–DFX binding model analysis, the binding force between DFX and pepsin was mainly hydrogen bonding, and the hydrophobic interaction was supplemented. Synchronous fluorescence spectroscopy and 3D fluorescence spectroscopy indicated that the binding of DFX to pepsin had minor effect on the protein structure and function. Circular dichroism spectra showed that DFX had no significant effect on the main secondary structure of pepsin. MD analysis also showed that DFX did not affect the looseness of pepsin and the overall secondary structure, but it affected the amino acid residue sequence Leu48-Ala49-Cys50-Ser51-Asp52. Pepsin enzyme activity test showed that the addition of DFX had a slight enhancement effect on the activity of pepsin. Combined with the MD results, DFX bound to pepsin and was closer to the pepsin active site Asp-215, which may affect the electrical environment of Asp-215 residues and enhance the activity of pepsin.

A 3-year study of deferasirox therapy in sickle cell disease patients in Basra, Southern Iraq.

Patients with sickle cell disease (SCD) may require repeated transfusions, which inevitably lead to iron overload (IOL). : This study aims to assess the effectiveness and safety of oral deferasirox (DFX) in patients with SCD and transfusional IOL. A descriptive study has been performed on patients with SCD who have completed at least 3 years on DFX. Height and weight were checked every 3-6 months. The efficacy was assessed based on serum ferritin (SF) levels. The safety was assessed based on adverse events (AEs), alanine aminotransferase (ALT), and serum creatinine (S. Cr) levels. : A total of 102 patients (61 males and 41 females) were recruited. Their mean daily iron intake was 0.13 ± 0.06 mg/kg. SF levels declined significantly from 2434.1 ± 132.9 ng/ml at the start of the study to 1655.8 ± 154.2 ng/ml at the end of the study (P < 0.05), with significant decreases observed after increasing the DFX dose to ≥ 30 mg/kg/day. ALT (12.8 ± 9.9 vs. 12.1 ± 7.1 U/L) and S. Cr (72.4 ± 9.2 vs. 74.1 ± 7.9 mmol/L) levels did not show significant differences from the start to the end of the study (P > 0.05). Thirty-eight patients (37%) developed AEs. The most common were abdominal pain (24.5%), diarrhea (8.0%), and nausea (7.8%). AEs were predominantly transient and mild to moderate in nature. This study has revealed that DFX is a safe, tolerable, and effective drug for reducing IOL in SCD patients, though it is associated with mild and transient adverse events.

Side effect of deferiprone as iron chelator in patient with thalassemia

Background There are currently three available iron chelators: deferoxamine (DFO), deferasirox (DFX) and deferiprone (DFP). In Dr. Cipto Mangunkusumo Hospital and Indonesia, in general, the accessibility of DFP for thalassemia patients has been adequate. Even though its efficacy in removing iron has been proven by countless studies, questions relating to its safety and possible side effects continue to be raised.Objective To assess common side effects of DFP usage by an intensive literature search and compare them to that in a pediatric thalassemia patient, in order to determine if the child’s symptoms in the were potentially caused by DFP.Methods A literature search using MeSH terms was done in PubMed. Full copies of articles that fulfil the inclusion criteria, based on their title, abstract, and subject descriptors, were critically appraised using The Joanna Brigs Institute (JBI) critical appraisal tools.Results A total of 10 research articles from 1998-2013 were deemed applicable to this study including: 2 case reports, 5 prospective cohort studies, 2 retrospective cohort studies, and 1 randomized control trial with a grand total of 1,026 samples.Conclusion Side effects of DFP include: neutropenia, agranulocytosis, increased ALT, gastrointestinal problems, arthralgia or arthropathy, increased appetite or weight, thrombocytopenia, urine discoloration, as well as auditory and visual disturbances. Our case report patient’s symptoms of gum bleeding and haemorrhagic mass are not related to her DFP consumption.

A STABILITY INDICATING MICELLAR LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF DEFERASIROX IN SOLUBILIZED SYSTEM

A simple, isocratic and stability indicating micellar liquid chromatographic (MLC) method containing a surfactant solution above its critical micellar concentration (CMC) was developed and validated for determination of Deferasirox (BTBA) content in active pharmaceutical ingredient and its pharmaceutical formulations. The analysis was performed in on isocratic mode at 400C temperature through Thermo Hypersil ODS 100mmA—4.6mm, 3 µm particle size columns. The UV detection wavelength was set at 250 nm in the mobile phase containing 0.1 M Cetyl trimethyl ammonium bromide (CTAB) buffer (pH – 2.5) and 10% 1-butanol. The method was successfully validated following as per the requirements of ICH guidelines. The proposed method was successfully applied as stability indicating method for determination of BTBA under different stressed conditions. The method allows accurate and reliable determination of BTBA for drug stability assay in pharmaceutical investigations.

Renal Function in Transfusion-Dependent β-Thalassemia Patients: A Decade of Follow-up and Comparison between Chelation Regimes

Introduction: Glomerular and tubular dysfunctions in β-thalassemia major (β-TM) patients (pts) have been attributed to iron overload, chronic anemia and iron-chelation therapy (ICT) toxicity. We studied glomerular and tubular function in β-TM pts treated with 2 different ICT regimes.Patients and methods: We studied 36 β-TM pts (18 females and 18 males). Mean age was 20.92 ± 9.7 (range 5-45) years. Pts were treated at Emek Medical Center under standard protocols for regular blood transfusions and ICT: 26 pts received deferasirox (DFX) for a mean period of 59 months; this was the first ICT for 6 of them; 10 pts were treated with deferoxamine (DFO) or DFO + deferiprone (DFP) (4 and 6 pts, respectively; hereafter DFO+/-DFP group). Clinical data were collected from pts' files.We evaluated serum urea, creatinine (sCr) and electrolytes, and estimated glomerular filtration rate (eGFR) according to the Schwartz formula for pediatric pts and the Chronic Kidney Disease Epidemiology Collaboration formula for adults; fractional excretion of sodium (feNa), fractional excretion of potassium (feK), calcium-to-creatinine ratio (Ca/Cr), uric acid excretion (UAE), and tubular phosphorus reabsorption were calculated. Urinary N-acetyl-β-D-glucosaminidase (uNAG) was also measured as a marker of tubular injury.Results: No significant differences were found in age, gender or mean hemoglobin (Hb) level between the groups. Levels of sCr, K, Na and feNa were in the normal range for all pts, as was eGFR (mean 104.6 ± 19 mL/min per 1.73 m2); in the DFX group, eGFR was slightly lower than in the DFO+/-DFP group (100.9 ± 17.09 vs. 114.0 ± 22.31 mL/min per 1.73 m2; p= 0.0676). Hypercalciuria (Ca/Cr > 0.25) was found in 28% of pts, increased feK (>15%) in 33%, high UAE (>0.56 mg/dL) in 64%, and high levels of TmP /GFR (>5mg/dL) in 25%. There were no differences in these parameters between the DFX and DFO+/-DFP groups.Increased uNAG was found in 9 pts (25%)-30% in the DFX group vs. 10% in the DFO+/-DFP group, and was significantly higher in the DFX group (mean 10.4 ± 6.1 vs. 5.3 ± 2.7 IU/L, p= 0.012). A moderate negative correlation was found between uNAG levels and mean serum ferritin for the prior 10 years (r= -0.35, p= 0.03). In the DFO+/-DFP group, a strong positive correlation was found between uNAG levels and the amount of iron transfused over the prior 10 years (r= 0.7647, p= 0.021); this correlation was not found in DFX pts. A moderate negative correlation was found for urinary Ca/Cr ratio with 10-year mean serum ferritin level and with 10-year amount of transfused iron (r= -0.35, p= 0.34; r= -0.4, p= 0.014, respectively). These correlations were stronger in the DFO+/-DFP group. A positive correlation was found between urinary Ca/Cr ratio and Hb levels, strongest in the DFO+/-DFP group than the DFX group (r= 0.41, p= 0.001, r= 0.7, p= 0.018), respectively.Renal function had been previously evaluated in 20 pts treated with DFO (Smolkin et al, 2008) and those results were compared with the current values. The eGFR significantly declined in pts switched to DFX (mean eGFR in first study 113.5 ± 26 vs. 100.1 ± 17 mL/min per 1.73 m2, p= 0.0093) but not in pts who continued DFO+/-DFP. A significant increase in sCr compared to the previous study was found in pts who switched to DFX (mean 0.51 ± 0.9 vs. 0.67 ± 0.1 mg/dl, p= 0.0008), but not in pts who continued treatment with DFO+/-DFP. The same observation was made regarding urine Ca/Cr (mean 0.08 ± 0.11 vs. 0.176 ± 0.12, p= 0.001).Conclusion: Ahigh prevalence of renal tubular abnormalities was observed in our pediatric and adult β-TM pts, particularly in the DFX group. The marker of tubular injury-uNAG-was negatively associated with mean 10-year serum ferritin, suggesting ICT's involvement in tubular injury. Moreover, uNAG was associated with transfusional iron burden in the DFO+/-DFP group, but not in the DFX group, proposing a mechanism other than iron overload for the pathogenesis of tubular injury in DFX-treated pts. Finally, glomerular function remained within the normal range in all pts; however a significant decline in glomerular function compared to a decade earlier was observed only in the pts currently treated with DFX. Strict follow-up of renal function in β-TM pts, especially children, is warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Wheatgrass Extract (Mugineic Acid): An Inexpensive and Effective Iron Chelator in Patients with Myelodysyplastic Syndromes

Background: Patients with red blood cell (RBC) transfusion-dependent myelodysplastic syndromes (MDS) and congenital anemias are at risk of transfusional hemosiderosis and organ injury from reactive iron species. Available iron chelating drugs in the US are either inconvenient for administration (parenteral deferoxamine) or are costly (oral deferasirox or deferiprone - median cost for these drugs exceeds $13,000-$18,000/month, Master S et al Blood 2016; 128:4858). Additionally, available chelators frequently have adverse effects including gastrointestinal (GI) upset or renal insufficiency. Wheatgrass, a food supplement prepared from the cotyledons of the Triticum aestivum Common Wheat plant, is sold either as a freeze-dried powder or as a juice extracted from the fresh leaves of the plant. Recently wheatgrass was found to contain high concentrations of the hexedentate iron chelator mugineic acid, a phytosiderophore that has comparable efficacy to deferasirox (ICL670) in a murine model of iron overload, and which may be a clinically meaningful iron chelator (Das et al J Biochem 2016; 160:163, Mukhopadhyay S et al JCO 2009; 15S:7012). Methods: Twelve patients with transfusion-dependent MDS (n=11) or congenital dyserythropoietic anemia (n=1) self-administered wheatgrass that they either grew themselves in potting soil or in an agricultural setting (n=3), procured from a juice bar (n=7), or obtained in powder form from a nutritional supplement store (n=2). Three patients also had known HFE variants. Seven patients had previously taken deferasirox and 2 patients had also received deferoxamine, but either did not tolerate these drugs or did not achieve adequate chelation. For patients using wheatgrass in the juice form, approximately 30 mL of juice was obtained from the leaves and stems of ~7 day-old wheatgrass. Serum ferritin was monitored over a period of at least 3 months. Serial hepatic T2* MRI was performed in 2 patients, one of whom also continued to take deferasirox. Results: The serum ferritin of patients at baseline was 2370 ng/mL (range, 1553-6089 ng/mL). The median decrement in serum ferritin in patients taking wheatgrass was 834 ng/mL (range of reduction, 251 to 1774 ng/mL). 3 patients reported worsening in GI upset or diarrhea but only 1 patient discontinued wheatgrass due to these effects; other adverse events were not observed. Two patients who grew their own wheatgrass noted mold outbreaks in the culture and switched to wheatgrass obtained from a juice bar. 1 patient noted a reduction in transfusion needs (of uncertain connection to wheatgrass administration), and the hepatic iron concentration measured by MRI decreased in both patients who were assessed radiographically. Conclusion: Mugineic acid in wheatgrass has iron chelating activity in patients with transfusion-associated iron overload. The annual cost of daily wheatgrass supplement from an urban juice bar (~$1500) is approximately 10% of the monthly cost of deferasirox or deferiprone in the US and less than 1% of the annual cost. Formal trials of wheatgrass or of purified mugineic acid in iron-overloaded patients with MDS or congenital anemias are indicated. Disclosures Steensma: H3 Biosciences: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Equity Ownership; Onconova: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy.

Reactive oxygen species levels control NF-κB activation by low dose deferasirox in erythroid progenitors of low risk myelodysplastic syndromes.

Anemia is a frequent cytopenia in myelodysplastic syndromes (MDS) and most patients require red blood cell transfusion resulting in iron overload (IO). Deferasirox (DFX) has become the standard treatment of IO in MDS and it displays positive effects on erythropoiesis. In low risk MDS samples, mechanisms improving erythropoiesis after DFX treatment remain unclear. Herein, we addressed this question by using liquid cultures with iron overload of erythroid precursors treated with low dose of DFX (3μM), which corresponds to DFX 5 mg/kg/day, an unusual dose used for iron chelation. We highlight a decreased apoptosis rate and an increased proportion of cycling cells, both leading to higher proliferation rates. The iron chelation properties of low dose DFX failed to activate the Iron Regulatory Proteins and to support iron depletion, but low dose DFX dampers intracellular reactive oxygen species. Furthermore low concentrations of DFX activate the NF-κB pathway in erythroid precursors triggering anti-apoptotic and anti-inflammatory signals. Establishing stable gene silencing of the Thioredoxin (TRX) 1 genes, a NF-κB modulator, showed that fine-tuning of reactive oxygen species (ROS) levels regulates NF-κB. These results justify a clinical trial proposing low dose DFX in MDS patients refractory to erythropoiesis stimulating agents.

Observation on the clinical effectiveness of deferasirox, deferiprone and desferrioxamine in the treatment of iron load in patients with thalassemia major

Objective To explore the clinical effectiveness of deferasirox, deferiprone and desferrioxamine in the treatment of iron overload in patients with thalassemia major. Methods Among the severe thalassemia patients with iron overload being diagnosis, treated and followed up in Guigang City People′s Hospital, fifty cases were tested by MRI T2 and were detected with cardiac and liver iron overload, the patients were selected and randomly divided into the deferoxamine treatment (DFO) group(18 case), deferiprone treatment (DFP) group (22 cases) and deferasirox treatment (DFX) group (10 case), The baseline data of the three groups were statistically analyzed.the serum ferritin (SF) value, the cardiac MRI T2 value and the liver MRI T2 value before and after treatment were measured. Results The patients completed the treatment, after 1 years of treatment, the serum SF value, cardiac and liver MRI T2 values of the patients were improved than those before the treatment (P 0.05); the differences in liver MRI T2 value among the three groups were not statistically significant(F=0.131, P>0.05), There were significant differences in heart iron load MRI T2 values among the three groups ((34.27 ± 6.30) ms vs.(23 ± 2.79) ms vs.(21.8 ± 2.20)ms, F=39.54, P<0.05). Conclusion Deferoxamine, deferiprone and deferasirox can effectively alleviate the iron load in patients with thalassemia major.in terms of cardiac iron overload, the curative effects of deferiprone treatment in patients with thalassemia is superior than desferrioxamine and deferasirox treatment, it is worthy of clinical promotion. Key words: Thalassemia Major; Iron Chelator; Serum Ferritin; MRI T2*Ttechnology

Abstract 925: Tumorigenesis of murine iPS cell is prevented by iron depletion with downregulation of stemness markers

Abstract Background: Iron plays a crucial role in the various metabolic pathways and it is essential for life. However excess of iron is known to cause cancer. There are many reports that iron depletion treatment indicates antitumor effect. Previously, we have confirmed that iron depletion treatment indicates antitumor effect against Cancer stem cell (CSC) model, miPS-LLCcm, which was converted from murine induced pluripotent stem cell (miPS cell) in Okayama university (Chen at el. PLOS ONE 2012), and also suppresses CSC markers. Many tumors contain phenotypically and functionally heterogenous cancer cells due to pluripotency of CSC. Although there are various theories about the origin of CSC, miPS has a pluripotency and regarded as the top of the hierarchy, it can be the candidate of the origin of CSC. miPS cell is known to form teratoma when inoculated into nude mouse because of its undifferentiated status. We hypothesized if we could suppress the stemness and tumorigenicity of miPS cell by iron depletion treatment, it can be applied to the therapy for CSC and undifferentiated status cancer cell. In this study, we investigated the stemness and tumorigenecity of miPS cell with iron depletion treatment. Methods: We used miPS cell(cell name : iPS-MEF-Ng-20D-17) purchased from Riken Cell Bank(Japan). Deferasirox (DFX), an popular commercial available oral iron chelator was used for iron depletion treatment. Western blot analysis was done to examine the expression of the stemness markers including Nanog, Oct3/4, Sox2, Klf4, c-Myc. Subcutaneous model of miPS cell and miPS cell treated with DFX (50μM) were used in vivo study. Tumors were harvested for immunohistochemistry. Results: Stemness markers of miPS cell were suppressed strongly by DFX in western blot analysis. DFX also suppressed the subcutaneous tumor growth. The average tumor volume of the control group was 665.3±430.8mm3 while that of the DFX treated group was 204.3±76.2 mm3(p&amp;lt;0.05) on day 20. Immunohistochemistry of the tumor revealed the suppression of the stemness markers in the DFX treated group compared with the control group. Citation Format: Yuki Katsura, Toshiaki Ohara, Hajime Kashima, Hiroaki Sato, Takuya Kato, Takayuki Ninomiya, Kazuhiro Noma, Yasuko Tomono, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Tumorigenesis of murine iPS cell is prevented by iron depletion with downregulation of stemness markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 925. doi:10.1158/1538-7445.AM2017-925

Abstract 2031: Antitumoral synergy of iron chelators and chemotherapies in triple-negative breast cancer cell lines and patient-derived xenograft

Abstract Introduction: Tumor cells present an iron metabolic disorder with high proliferation rate, increased iron storage (ferritin and Labile Iron Pool - LIP) and high sensibility to iron deprivation, which could be a therapeutic target. Anticancer effect of iron chelators deferoxamine (DFO) and deferasirox (DFX) has been revealed in several types of cancers. In breast cancer (BC), the development of new therapeutic approaches is urgently needed for triple negative (TN) subtype which presents poor prognosis and lacks targeting therapy. We investigated the therapeutic potential of iron chelators combined with chemotherapeutic agents in TNBC cell lines and patient-derived xenografts (PDX). Methods: Anticancer effects of iron chelators combined with chemotherapeutic agents (doxorubicin, cisplatin or carboplatin) were evaluated in vitro in 4 TNBC cell lines by MTT assay, annexin V/PI staining and assessment of caspase 3/7 activity. Assessment of LIP, transferrin receptor 1 (TfR1) expression level, Reactive Oxygen Species (ROS) production and mitochondrial membrane potential variations were performed by flow cytometry, and ELISA assay for ferritin level. The activity of DFX alone or combined with doxorubicin/cyclophosphamide (AC) was tested in the HBCx-10 TNBC PDX selected because of its relapse to AC. Iron homeostasis, hypoxia and PI3K pathway were analyzed by immunohistochemistry (IHC) and Western-blot in both cell lines and PDX tumors. In DFX+/-AC treated and untreated tumors, induction of apoptosis was performed by TUNEL assay and a transcriptome analysis is ongoing. Results: Iron chelators acted in synergy effect with three chemotherapies in all cell lines which were tested to inhibit cell proliferation and induce apoptosis. Chelators increased cytotoxicity until 60% compared to chemotherapies alone. In all cell lines, chelators treatment increased TfR1 expression level and decreased LIP and ferritin. Furthermore, down-regulation of PI3K pathway, hypoxia, mitochondrial membrane potential, and the production increasing of ROS were observed. In HBCx-10 PDX, a trend for antitumoral activity of DFX alone was observed (p=0.09) at the end of the experiment (day 81). A significant difference of Relative Tumor Volume (RTV) was observed since day 18 between the AC group and the DFX+AC group (tumor growth inhibition: 37 to 61%, tumor growth delay: 10 to 14 days, 0.005&amp;lt;p&amp;lt;0.04). Same as the observing in vitro, modulations of PI3K pathway and hypoxia are involved in this antitumoral synergy. Except neutropenia (due to chemotherapy), no other hematologic toxicity was observed in both AC and DFX+AC groups. Conclusions: Iron chelators may increase the effectiveness of conventional chemotherapies for TNBC treatments. This antitumoral synergy involves PI3K pathway downregulation, ROS production and decrease mitochondrial membrane potential. Citation Format: Sandrine Tury, Sophie Vacher, Véronique Becette, Franck Assayag, Sophie Chateau-Joubert, Jean-Luc Servely, Elisabetta Marangoni, Ivan Bièche, Céline Callens. Antitumoral synergy of iron chelators and chemotherapies in triple-negative breast cancer cell lines and patient-derived xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2031. doi:10.1158/1538-7445.AM2017-2031

Reactive oxygen species mediated T lymphocyte abnormalities in an iron-overloaded mouse model and iron-overloaded patients with myelodysplastic syndromes.

The adverse effects of iron overload have raised more concerns as a growing number of studies reported its association with immune disorders. This study aimed to investigate alterations in the immune system by iron overload in patients with myelodysplastic syndrome (MDS) and an iron-overloaded mouse model. The peripheral blood from patients was harvested to test the effect of iron overload on the subsets of T lymphocytes, and the level of reactive oxygen species (ROS) was also evaluated. The data showed that iron-overloaded patients had a lower percentage of CD3+ T cells and disrupted T cell subsets, concomitant with higher ROS level in lymphocytes. In order to explore the mechanism, male C57Bl/6 mice were intraperitoneally injected with iron dextran at a dose of 250mg/kg every 3days for 4weeks to establish an iron-overloaded mouse model and the blood of each mouse was collected for the analysis of the T lymphocyte subsets and T cell apoptosis. The results showed that iron overload could reduce the percentage of CD3+ T cells and the ratio of Th1/Th2 and Tc1/Tc2 but increase the percentage of regulatory T (Treg) cells and the ratio of CD4/CD8. We also found that iron overload induced the apoptosis of T lymphocytes and increased its ROS level. Furthermore, these effects could be partially recovered after treating with antioxidant N-acetyl-L-cysteine (NAC) or iron chelator deferasirox (DFX). Taken together, these observations indicated that iron overload could selectively affect peripheral T lymphocytes and induce an impaired cellular immunity by increasing ROS level.