Chronic Iron Overload Research Articles

What Predicts Adrenal Insufficiency in Patients with Thalassemia Major?

Abstract 5299 Background:Thalassemia is one of the most common genetic blood disorders worldwide. With recent improvements in medical therapy, patients with transfusion-dependent thalassemia, i.e., thalassemia major, are living longer. As a result, there is a greater need to address endocrine complications related to chronic iron overload. Adrenal insufficiency (AI), in particular, is important to identify because therapies are available and can be life-saving. Objectives:The objectives of this study are to determine the prevalence of AI in our population of subjects with thalassemia major; to identify risk factors that predict AI in these individuals; and to localize the origin of the AI within the hypothalamic-pituitary-adrenal (HPA) axis. Methods:This is a prospective study of individuals with thalassemia major with an enrollment goal of 30 subjects. All subjects enrolled were initially tested for AI using a glucagon stimulation test. Those found to have AI (stimulated cortisol <18 mcg/dL) subsequently underwent an ovine corticotrophin-releasing hormone (oCRH) stimulation test for confirmatory purposes and to define the physiological basis for the AI. Results:Eleven subjects (8 - 29 years old, 6 female) have been enrolled to date. In our population of patients with TM, the prevalence of AI was 55%. There was no correlation between age, number of years transfused, or ferritin levels and AI. All male patients failed the glucagon stimulation test, whereas 5 of 6 females passed the glucagon stimulation test, p = 0.0024. There was no correlation between 8 AM ACTH levels and 8 AM cortisol levels. There was a significant correlation (p = 0.025) between 8 AM cortisol level and peak cortisol level following glucagon stimulation testing. Of the six subjects with AI, two subjects subsequently failed the oCRH stimulation test (peak cortisol < 21.9 mcg/dL). In these two subjects, peak oCRH ACTH levels were elevated, 144 and 164 pg/mL, respectively, suggesting primary adrenal insufficiency. Conclusions:We conclude that 8 AM cortisol level is a good predictor of adrenal insufficiency in our population, and can potentially be used as a simple screening test for AI with a strong negative predictive value. There appears to be a male predominance of AI in our population. This may indicate a protective role of female sex in this population. Two subjects had classic primary AI with robust ACTH levels in the face of inadequate cortisol production following oCRH testing. Four subjects (all males) who failed the glucagon stimulation test subsequently demonstrated normal ACTH and cortisol response to oCRH, indicating a possible hypothalamic origin to their AI. This dysfunction is likely independent of iron overload and warrants further investigation. Alternatively, these subjects may have impaired sympathetic nervous system function leading to hypoglycemic unawareness. Both outcomes are novel to the field and of medical significance. Disclosures:Geffner:Daiichi- Sankyo: Steering Committee for Clinical Trial; Eli Lilly, Inc.: Research Contract; Endo Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Genentech, Inc: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ipsen: Data Safety Monitoring Board and Research Contract; Novo Nordisk: Research Funding; Pfizer, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Iron Chelation Therapy with Deferasirox Results in Recovery of Hematopoiesis in a Child with Aplastic Anemia

Deferasirox (DFX; Exjade, Novartis Pharma) is a once-daily oral iron chelator that has shown efficacy in the treatment of chronic iron overload [1]. Of note, several reports have been made of not o...

Iron Chelation Therapy for Transfusional Iron Overload: A Swift Evolution

Chronic transfusional iron overload leads to significant morbidity and mortality. While deferoxamine (DFO) is an effective iron chelator with over four decades of experience, it requires tedious subcutaneous infusions that reflect negatively on patient compliance. The novel oral iron chelators deferiprone (L1) and deferasirox (DFRA) opened new horizons for the management of transfusional siderosis. A large body of evidence is now available regarding their efficacy and safety in various populations and settings. Nevertheless, experience with both drugs witnessed some drawbacks, and the search for an ideal and cost-effective iron chelator continues.

A Turn-on Fluorescent Iron Complex and Its Cellular Uptake

In the treatment of chronic iron overload disorders, ligands capable of complexing so-called "labile" (nonprotein bound) Fe are required to enter iron-loaded cells, sequester excess Fe, and then exit the cell (and the body) as an intact Fe complex. Despite the emergence of several ligand families that show high activity in mobilizing intracellular Fe, the mechanism and the locations of these subcellular labile Fe pools are still poorly understood. Our previous studies have unearthed a class of heterocyclic hydrazine-based chelators (e.g., benzoyl picolinoyl hydrazine, H(2)BPH) that show excellent activity at mobilizing Fe from Fe-loaded cells. Herein, we have grafted a fluorescent tag (rhodamine B) onto H(2)BPH to generate a ligand (L(1)) that is nonfluorescent in its uncomplexed form but becomes strongly fluorescent in complex with Fe(III). The free ligand and its 1:2 Fe complex [Fe(III)(L(1))(2)](3+) have both been fully characterized spectroscopically and with X-ray crystallography. Confocal fluorescent microscopy of HeLa cells incubated with [Fe(III)(L(1))(2)](3+) shows that the complex rapidly enters HeLa cells and localizes within endosomes/lysosomes.

Managing unresponsiveness or intolerance to deferasirox therapy: a tale of two doses

Evaluation of: Chang HH, Lu MY, Liao YM et al. Improved efficacy and tolerability of oral deferasirox by twice-daily dosing for patients with transfusion-dependent β-thalassemia. Pediatr. Blood Cancer 56(3), 420–424 (2011).Chronic transfusional iron overload leads to significant morbidity and mortality in patients with β-thalassemia major. The once-daily oral iron chelator, deferasirox, opened new horizons for the management of transfusional siderosis. A large body of evidence is now available regarding its efficacy and safety. Nevertheless, some patients remain unresponsive or intolerant to the adverse events of the drug. Chang et al. evaluated the benefit of twice-daily dosing in this setting. The authors concluded that twice-daily deferasirox improves responsiveness and tolerability. Even though the study included only a small number of patients, it offers promising insights that should be interpreted with caution.

The association between porphyria cutanea tarda and diabetes mellitus: analysis of a long-term follow-up cohort

An association between porphyria cutanea tarda (PCT) and diabetes mellitus (DM) is widely reported, but the pathogenetic link remains unknown. To investigate the natural history of DM in the setting of PCT and to determine which PCT features and risk factors may be associated with the development of DM. This retrospective longitudinal study included 81 Spanish patients with PCT with at least 10 years of strict follow-up. Patients attended our Porphyria Unit for follow-up visits and the data were collected in the period 2004-2008. We classified patients into two groups: patients with glucose metabolism alterations (GMA: DM or impaired fasting glucose), and patients without. PCT features and PCT and DM risk factors were retrieved from clinical charts and compared between groups. We identified 33 patients (41%) with GMA, of whom 27 (82%) developed GMA a long time after the diagnosis of PCT (mean 12·7 years). In bivariate analysis, these patients had significantly higher mean serum ferritin at diagnosis (651 vs. 405 ng mL(-1); P = 0·005), a higher prevalence of persistently elevated serum ferritin (52% vs. 15%; P < 0·001 for trend) and a higher prevalence of family history of DM (48% vs. 19%; P = 0·004). In multivariate analysis, persistently elevated serum ferritin [odds ratio (OR) 10·66, 95% confidence interval (CI) 1·95-58·19; P = 0·006] and family history of DM (OR 4·82, 95% CI 1·34-17·33; P = 0·016) remained significantly associated with the presence of GMA. GMA are highly prevalent in patients with PCT and mostly develop a long time after the diagnosis of PCT. Persistent hyperferritinaemia seems to be a risk biomarker of GMA in patients with PCT, probably in the setting of chronic iron overload and hepatic inflammation. Strict long-term monitoring of glucose metabolism and serum ferritin may be advisable in the routine follow-up of patients with PCT.

Iron Overload Decreases Ca V 1.3-Dependent L-Type Ca 2+ Currents Leading to Bradycardia, Altered Electrical Conduction, and Atrial Fibrillation

Chronic iron overload (CIO) is associated with blood disorders such as thalassemias and hemochromatosis. A major prognostic indicator of survival in patients with CIO is iron-mediated cardiomyopathy characterized by contractile dysfunction and electrical disturbances, including slow heart rate (bradycardia) and heart block. We used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced heart rate (≈20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting that bradycardia originates from altered intrinsic SAN pacemaker function. Indeed, spontaneous action potential frequencies in SAN myocytes with CIO were reduced in association with decreased L-type Ca(2+) current (I(Ca,L)) densities and positive (rightward) voltage shifts in I(Ca,L) activation. Pacemaker current (I(f)) was not affected by CIO. Because I(Ca,L) in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of Ca(V)1.3 channels (in addition to Ca(V)1.2 channels), our data suggest that elevated iron preferentially suppresses Ca(V)1.3 channel function. Consistent with this suggestion, CIO reduced Ca(V)1.3 mRNA levels by ≈40% in atrial tissue (containing SAN) and did not lower heart rate in Ca(V)1.3 knockout mice. CIO also induced PR-interval prolongation, heart block, and atrial fibrillation, conditions also seen in Ca(V)1.3 knockout mice. Our results demonstrate that CIO selectively reduces Ca(V)1.3-mediated I(Ca,L), leading to bradycardia, slowing of electrical conduction, and atrial fibrillation as seen in patients with iron overload.

Oxidative stress and prevention of the adaptive response to chronic iron overload in the brain of young adult rats exposed to a 150 kilohertz electromagnetic field

Iron surcharge may induce an oxidative stress-based decline in several neurological functions. In addition, electromagnetic fields (EMF) of frequencies up to about 100 kHz, emitted by electric/electronic devices, have been suggested to enhance free radical production through an iron dependent pathway. The purpose of this study was therefore to determine a possible relationship between iron status, exposure to EMF, and brain oxidative stress in young adult rats. Samples were micro-dissected from prefrontal cortex, hippocampus, striatum, and cerebellum after chronic saline or iron overload (IO) as well as after chronic sham exposure or exposure to a 150 kHz EMF or after combining EMF exposure with IO. The brain samples were used to monitor oxidative stress-induced lipid peroxidation and activity of the antioxidant enzymes superoxide dismutase and catalase. While IO did not induce any oxidative stress in young adult rats, it stimulated antioxidant defenses in the cerebellum and prefrontal cortex in particular. On the contrary, EMF exposure stimulated lipid peroxidation mainly in the cerebellum, without affecting antioxidant defenses. When EMF was coapplied with IO, lipid peroxidation was further increased as compared to EMF alone while the increase in antioxidant defenses triggered by the sole IO was abolished. These data suggest that EMF exposure may be harmful in young adults by impairing the antioxidant defenses directed at preventing iron-induced oxidative stress.

Iron overload is associated with low anti-müllerian hormone in women with transfusion-dependent β-thalassaemia

To investigate anti-müllerian hormone (AMH) as a best test of ovarian reserve in women with transfusion-dependent β-thalassaemia, and the relationship between AMH and iron overload. A case-control study in a tertiary medical centre. Twenty-nine women with transfusion-dependent β-thalassaemia and 29 healthy controls of a similar age were recruited. Blood sampling, questionnaires and medical record reviews were used. The history of iron overload-related morbidities, haematological phenotypes, serum levels of AMH and ferritin, and hormonal profiles were analysed. The serum levels of AMH, luteinising hormone, and estradiol were lower in women with transfusion-dependent β-thalassaemia than in age-matched normal controls. In women with transfusion-dependent β-thalassaemia, the serum AMH level was significantly inversely related to the ferritin level, but not related to the presence of hypogonadotrophic hypogonadism, diabetes and haematological phenotypes. The serum ferritin level was positively associated with advanced age and the presence of hypogonadotrophic hypogonadism in the study participants. However, the inverse relationship between AMH and ferritin still exists after further adjustment for advanced age in women with transfusion-dependent β-thalassaemia. The present study indicates that the serum AMH levels in women with transfusion-dependent β-thalassaemia are lower when compared with normal healthy women of a similar age, and are significantly negatively correlated with their serum ferritin levels. This implies that ovarian function might be impaired by the chronic iron overload status in women with transfusion-dependent β-thalassaemia.

Effect of Deferasirox on Iron Absorption in a Randomized, Placebo-Controlled, Crossover Study in a Human Model of Acute Supratherapeutic Iron Ingestion

In 2005, the Food and Drug Administration approved deferasirox as an oral iron chelating agent for chronic iron overload. To determine usefulness in management of acute iron ingestion, we study the effect of orally administered deferasirox in healthy human adults. A double-blinded, placebo-controlled, randomized, crossover study of 8 healthy human volunteers was conducted. Subjects ingested 5 mg/kg of elemental iron in the form of ferrous sulfate. One hour after iron ingestion, subjects were randomized to receive 20 mg/kg of deferasirox or placebo. Serial iron levels were then obtained. A 2-week washout was used between study arms. The paired t test was used to compare area under time-concentration curves from baseline to both 12- and 24-hour iron levels between groups. Baseline serum iron levels were similar in the 2 groups. Deferasirox significantly reduced serum iron area under concentration-time curves compared with placebo during both 1 to 12 hours and 1 to 24 hours (12 hour=577 μmol-hour/L and 392 μmol-hour/L, 95% confidence interval for the difference 15.8 to 353.0 μmol-hour/L; 24 hour=808 μmol-hour/L and 598 μmol-hour/L, 95% confidence interval for difference 54.4 to 366.7 μmol-hour/L). Orally administered deferasirox significantly reduced serum iron levels when administered 1 hour after iron ingestion during the 12- and 24-hour periods after acute ingestion of 5 mg/kg of elemental iron in healthy human volunteers. Further study is required to determine optimal dosing, but deferasirox may be an important addition to current therapy for acute iron poisoning.

Management of Oral Iron Chelator Deferasirox for Transfusion-Dependent Patients with Hematological Disorders: 2-year Experience at a Single Institution in Japan

Introduction: Deferasirox is an oral iron chelator, approved worldwide for the treatment of chronic iron overload due to transfusion. Deferasirox was permitted two years ago in Japan, but there is little known regarding its efficacy and tolerability in clinical practice. Methods: We conducted a retrospective study of 18 patients with transfusion-dependent anemias treated by deferasirox at our institution. The starting dose was individualized and ranged from 6.4 to 26.3 mg/kg/day. Routine clinical laboratory data were followed, and serum ferritin was assessed every 4 weeks. Results: The mean serum ferritin level of 18 patients at the time of deferasirox induction was 3162 ng/ml. Ten of 18 patients could sustain deferasirox treatment for at least 6 months, at an average maintenance dose of 10.8 mg/kg/day. Serum ferritin reduction was observed in 4 patients, at doses less than 20 mg/kg/day. Eighty-nine percent of the patients had adverse events, and 13 of them in all ultimately discontinued. Myelodysplastic syndrome (MDS) patients showed poor tolerability. Severe infections of grade 3 or more were documented in 6 patients, and 2 of them were fatal. Conclusions: The potential for beneficial iron chelation of deferasirox at less than the recommended 20 mg/kg dose was demonstrated. On the contrary, poor tolerability was documented, with adverse events such as severe infections, especially in MDS patients. Although it was not clearly demonstrated that deferasirox was responsible for impaired immunity, careful watching is required to administrate deferasirox.

Deferasirox for the Treatment of Transfusional Iron Overload In Sickle Cell Anemia: a 2-Yr Prospective Study

AbstractAbstract 5148 Background:Majority of patients with sickle cell disease receive repeated blood transfusions by adulthood. Because the body has no physiological mechanism to actively excrete excess iron, chelation therapy is important for the management of iron overload and its complications, including iron deposition into the liver, heart and endocrine organs, eventual death. Deferasirox (DFX) is a once-daily, oral iron chelator that is approved as first-line treatment of chronic transfusional iron overload. Its safety, tolerability and efficacy in reducing body iron burden have been demonstrated in patients with β-thalassaemia major and in other chronic transfusion-dependent anaemias, including SCD. Aims and Methods:Objectives of this prospective, non-randomised, phase IV trial were to evaluate the iron overload status, before and after two year-treatment with DFX, using liver iron concentration [LIC, mg/d dry weight (dw)] by magnetic resonance imaging (MRI) hepatic, MRI cardiac (Cardiac T2*, ms), serum ferritin (SF, μ g/L), and to evaluate the safety and tolerability of DFX. Results:A total of 31 patients with SCD and iron overload, defined as the use of ≥ 20 units of RBC units and/or two SF levels ≥ 1000 μ g/L during the 6 months preceding enrollment, received starting dose of 20mg/kg/day of DFX. Efficacy was assessed monthly by measuring change from baseline in SF levels. Safety was evaluated on a monthly basis according to the incidence and type of adverse events and measurement of laboratory parameters, including serum creatinine and liver enzyme levels. Two patients discontinued treatment at 8 and 9 months, due to pregnancy and moving to other city, respectively. One patient died at 18 months due to pulmonary infection and hemorrhagic stroke. DFX was interrupted in 3 patients due to confirmed SF levels <500 μ g/L at 18-month period of treatment and DFX was not reinstated in none of them during the final 6 months of study. Twenty-five patients completed 2-year treatment. Mean ± SD age 26.9 ± 12.5y; 84% female, 90% afrodescendent, 61.3% on regular blood transfusion; median (range) DFX dose over 24 months and DFX exposure were 20 mg/kg/day (15-25) and 90.5 weeks (35.6-98.0), respectively. Mean SF level (μ g/L) did not significantly reduced at 12 months (p=0.052) but significantly dropped at 24 months compared to baseline [from 2344.6 to 1986.3 (p=0.040)]. Mean ± SD LIC significantly dropped at 12 months and at 24 months compared to baseline [from 13.0 ± 5.4 to 10.4 ± 6.3 (p=0.001) and to 9.3 ± 5.7 (p<0.001), respectively]. The proportion of patients with LIC levels (mg/g dw) ≤7.0, >7.0- ≤14.0 and >14.0 from baseline to 24 months by percentage of patients changed from 13.6% to 44.0%, 40.9% to 44.0% and 45.5% to 12.0%, respectively. In all patients, Cardiac T2* was normal (> 20 ms) at baseline, 12 and 24 months of treatment. There was no significant difference between left ventricular ejection fraction values at baseline and after 12 months but this parameter significantly increased at 24 months of treatment compared to baseline [from 62.2 ± 6.0 to 64.6 ± 6.2 (p=0.02)]. The most common drug-related AEs were mild, transient diarrhea (7 pts), headache (7), nausea (5), vomiting (3), skin rash (2), increases in ALT (2), serum creatinine increases that exceeded the ULN (2). No patient experienced progressive increases in serum creatinine or renal failure. Conclusions:Our data confirms that deferasirox is effective in reducing body iron burden in transfused patients with SCD, well tolerated in pediatric and adult patients and with a clinically manageable safety profile. The availability of deferasirox as a once-daily, oral iron chelator would potentially facilitate improved compliance, and thereby reduce morbidity and mortality from iron overload. Disclosures:No relevant conflicts of interest to declare.

A Phase 1B Dose-Escalation Study to Assess the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of FBS0701, a Novel Oral Iron Chelator for the Treatment of Chronic Iron Overload

AbstractAbstract 2057 Background:Iron chelators in current use (parenteral deferoxamine, oral deferasirox and deferiprone) are individually efficacious in many patients with transfusion dependent anemias, but each has liabilities relating to safety, ease of administration, patient acceptance, or narrow therapeutic index. The oral chelator FBS0701 is a polyether derivative of the siderophore-related compound, desazadesferrithiocin, a tri-dentate chelator with high affinity binding and selectivity for Fe(III). Animal studies of FBS0701 demonstrated a 4-fold higher no-observable-adverse-effect level in pre-clinical studies compared to deferasirox suggesting a favorable clinical safety profile with respect to nephrotoxicity. Single dose safety and pharmacokinetic studies in both healthy volunteers and iron overloaded patients have established preliminary data to guide further clinical trials. Objectives:To assess multiple-dose pharmacokinetics, safety, and tolerability of FBS0701 in patients with congenital transfusion-dependent anemias. Methods:This was a multicenter, open-label, dose-escalating, multiple dose study of four dose levels (3, 8, 16 and 32 mg/kg/d) of FBS0701. Sixteen patients with documented transfusional iron overload over the age of seventeen were studied. Each dose cohort consisted of 4 patients. After a washout period, patients were dosed daily for seven days. Patients were clinically evaluated on Days 3, 6, 7, 8, 9, 10 and 15 and then weekly thereafter for three weeks. Assessments included physical exam, clinical pathology and ECG. A standard pharmacokinetic protocol of plasma sampling was performed throughout Day 7 and continuing to Day 10. Urine was collected in three intervals over the 24 hour period following the last dose. Each dose level was separated by a minimum of three weeks to assess safety before escalating to the next dose level. Results:FBS0701 was well tolerated in all dose cohorts. There were no serious adverse events related to the drug. With the exception of a change in urine color that was regarded as without clinical significance, adverse events did not show dose-dependency in frequency or severity. Pharmacokinetic parameters derived from plasma and urine drug levels are shown in the table below. There was no evidence of accumulation after 7 days of dosing. Dose- dependent pharmacokinetics were observed as predicted.Parameter*Dose3 mg/kg/day8mg/kg/day16 mg/kg/day32 mg/kg/dayCmax (ng/mL)5,910 ± 2,298 (4)15,000 ± 4,439 (4)38,225 ± 3,947 (4)68, 250 ± 27,519 (4)Tmax (h)1.31 (4)1.18 (4)1.00 (4)1.49 (4)AUC(0-24) (h×ng/mL)19,476 ± 11,327 (4)44,916 ± 30,751 (4)92,261 ± 36,560 (4)157,577 ± 43,484 (4)ëz(h-1)0.0655 ± 0.0606 (4)0.0424 ± 0.0249 (4)0.0421 ± 0.0246 (4)0.0381 ± 0.0091 (2)t ½ (h)16.2 ± 8.32 (4)20.9 ± 11.3 (4)21.3 ± 11.8 (4)18.7 ± 4.48 (2)CL/F (mL/min)162 ± 85.0 (4)225 ± 142 (4)206 ± 78.4 (4)172 ± 60.1 (4)Vz/F (L)185 ± 84.1 (4)311 ± 137 (4)339 ± 166 (4)214 ± 2.94 (2)Ue(0-24)(mg)66.1 ± 24.0 (4)201 ± 68.1 (4)402 ± 203 (4)641 ± 208 (4)Fe(0-24) (%Dose)44.0 ± 16.0 (4)47.4 ± 13.8 (4)39.2 ± 5.86 (4)43.1 ± 15.8 (4)CLr (mL/min)75.2 ± 46.8 (4)105 ± 62.9 (4)83.2 ± 36.5 (4)73.6 ± 37.8 (4)AUC0–24: area under the plasma concentration versus time, zero time point to 24 hours; Cmax: time to maximum observed plasma concentration; tmax: time to maximum plasma concentration; kel: terminal elimination rate constant; t½: half-life calculated by the equation t½ = 0.693/kel; CL/F: the apparent total plasma clearance of drug after oral administration; Vz/F: the apparent volume of distribution during terminal phase after oral administration; Ue: amount of drug excreted into urine; Fe: the fraction of orally administered drug excreted unchanged in urine; CLr: renal clearance Conclusions:FBS0701, a novel tridentate iron chelator, was well tolerated for 7 days at doses up to 32 mg/kg, the high end of the predicted therapeutic dose range. The compound is of interest because in preclinical animal studies there was no evidence of gastrointestinal toxicity and FBS0701 was less able to cause renal toxicity than deferasirox. The pharmacokinetic parameters suggest good dose proportionality and support once-daily dosing that might provide 24 hour protection from non-transferrin bound iron based on the gradual terminal-phase elimination. FBS0701 warrants Phase 2 safety and efficacy studies in transfusional iron overloaded patients. Disclosures:Rienhoff:Ferrokin, Inc.: Employment, Equity Ownership. Viprakasit:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ferrokin, Inc.: Research Funding. Tay:Ferrokin, Inc.: Research Funding. Harmatz:Ferrokin, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Vichinsky:Novartis: Research Funding. Chirnomas:Novartis: Research Funding. Kwiatkowski:Ferrokin, Inc.: Research Funding. Tapper:Ferrokin, Inc.: Employment. Porter:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Neufeld:Novartis, Inc: Research Funding; Ferrokin, Inc: Research Funding.

Iron Overload Accelerates Development of Leukaemia: Evidence From a Mouse Model

AbstractAbstract 122 Introduction:Transfusion-related iron overload is common in MDS. Iron catalyzes, via the Fenton reaction, excess production of reactive oxygen species (ROS), which are known to cause cell senescence and death, promote DNA damage and accelerate carcinogenesis. In addition to the well characterized effects of iron overload on the heart, liver, and endocrine organs, clinical data have suggested iron also causes haematopoietic toxicity in MDS, since iron chelation can lead to dramatic reduction in transfusion requirements and in large registry studies leukaemia-free survival is inversely related to serum ferritin. These observations have led to controversy since they are supported by no mechanistic or animal model data. Hypothesis:We have demonstrated that iron overload increases intracellular ROS (iROS) in early haematopoietic cells in MDS. We hypothesize that iron, via increased iROS, promotes accumulation of DNA damage in MDS HSCs and thus, in the context of the genomic instability of the MDS clone, accelerates progression of MDS to AML. Here we report the results of experiments that establish the biological and mechanistic plausibility of this hypothesis. Results:To establish that the B6D2F1 mouse model, which has been used in studies of cardiac and hepatic iron overload, is also a suitable model of bone marrow iron overload, mice (n=5 per cohort) were given iron dextran (0-150 mg i.p.) and sacrificed 3 days later. Severe weight loss was noted in the iron-loaded animals. Iron deposition was confirmed by Prussian Blue staining in the bone marrow, liver, myocardium, and the red pulp of the spleen.The cardiac effects of this degree of iron overload compromise survival, preventing assessment of longer-term effects of iron on haematopoiesis. We therefore evaluated the effects of lower doses of iron dextran (0, 5, 10, or 20 mg; n=5 per cohort) over 3 months. Increased iROS was seen in lineage negative (lin−) CD45+ bone marrow cells for animals that received 5 mg iron. However, iROS levels decreased progressively from the 10–20 mg treated animals, possibly representing an increase in apoptosis in early haematopoietic cells exposed to the greatest oxidative stress. Consistent with this, we observed increased apoptosis in early erythroid progenitors for the 20 mg iron treated animals (p<0.05).We adapted the chronic iron overload mouse model to evaluate the effect of iron overload on leukaemogenesis. B6D2F1 mice were sublethally irradiated (300 cGy) followed by s.c. injection of 0.5 mg dexamethasone, a protocol which induces a pre-leukaemic state leading, in SJL mice, to AML in 50–75% with a 12 month latency. These mice were then loaded with 0 or 5 mg I.P. iron dextran, n=6 per cohort). Three mice from each cohort were sacrificed and analyzed 3 months after iron loading. Expansion of the splenic white pulp was observed in iron loaded mice and flow cytometric analysis of the bone marrow cells revealed expansion of the lin− CD45+ early haematopoietic population. Furthermore, in one iron loaded mouse we observed a lin− CD45lo population with size and complexity similar to that of haematopoietic progenitors, suggesting blast accumulation. The remaining mice (n=3 per cohort) continued to be observed. One mouse in the iron loaded cohort died eight months after iron loading. Post-mortem examination revealed severe hepatomegaly and splenomegaly, massive splenic and hepatic infiltration by leukaemic blasts, and extensive bone marrow necrosis, fibrosis, and substantial blast accumulation.To establish a plausible mechanism for the promotion of leukaemia development by iron, we tested the ability of iron to cause DNA damage in a haematopoietic cell line. HL60 cells line were treated with ferric ammonium sulfate (10 or 100 μ M) and DNA damage was assessed by flow cytometry for γH2AX, an indicator of DNA double-strand breaks. Elevated γH2AX was observed in HL60 cells 2 hours after iron loading, and sustained DNA damage was noted till the end of the experiment at day 4. Conclusions:Our observations demonstrate that iron is mutagenic in haematopoietic cells and can promote progression of a pre-leukaemic state to frank AML. We postulate that iron is not itself leukaemogenic, but, by causing DNA damage, promotes clonal evolution in MDS. Further evaluation in animal models and in clinical trials is necessary to elucidate the clinical implications of these observations, especially in regard to the deployment of iron chelation therapy. Disclosures:No relevant conflicts of interest to declare.

More pieces to the iron chelation puzzle

More pieces to the iron chelation puzzle

Iron chelation therapy for patients with sickle cell disease and iron overload

A 21-year-old male with sickle cell disease (SCD) presented with severe pallor. He had received a total of 100 red blood cell (RBC) units in his lifetime, had a mean serum ferritin level of 3133 ng/ml, and liver iron concentration (LIC) of 12 mg Fe/g dry weight (dw). He was started on subcutaneous deferoxamine (DFO) infusions at a dose of 56 mg/kg/d, five days a week (equivalent to 40 mg/kg/d, seven days a week) and continued to receive 8–10 RBC units/year as treatment for pain. During the first six months of chelation therapy, his serum ferritin levels fell by around 50% of the pretreatment value, but then started to increase back up to the baseline values. The patient was noncompliant with DFO therapy. He experienced pain at the site of injection, could not sleep and was concerned about carrying a pump and not being accepted by his peers. He dropped out of college and abstained from all social activities. He was referred to a psychologist; however, this failed to improve compliance and he opted to stop DFO therapy altogether.

Iron Overload, Cardiac and Other Factors Affecting Pregnancy in Thalassemia Major

The reproductive thalassemic population is growing older and doctors confront the challenge of the thalassemic pregnancy. Pregnancy is characterized by dynamic multiple system changes, resulting in increased basal oxygen consumption, changes in energy substrate use by different organs and increased susceptibility to oxidative stress, while homozygous transfusion-dependent β-thalassemia (β-thal) patients manifest cardiac, hepatic, endocrine, and metabolic disorders attributable to chronic anoxia and iron overload. Pregnant thalassemic patients require significantly larger amount of total blood transfusion during pregnancy and iron overload increases the oxidative stress of pregnancy, while the risk for cardiovascular events, in a high cardiac output state, is augmented and chelation treatment is generally avoided due to the potential teratogenicity. Pregnancy in thalassemia major should be considered high risk, and be cared for by an expert team with special caution and sensitivity.

Mitochondrial dysfunction may explain the cardiomyopathy of chronic iron overload

In patients with hemochromatosis, cardiac dysfunction may appear years after they have reached a state of iron overload. We hypothesized that cumulative iron-catalyzed oxidant damage to mitochondrial DNA (mtDNA) might explain the cardiomyopathy of chronic iron overload. Mice were given repetitive injections of iron dextran for a total of 4 weeks after which the iron-loaded mice had elevated cardiac iron, modest cardiac hypertrophy, and cardiac dysfunction. qPCR amplification of near-full-length (∼ 16 kb) mtDNA revealed > 50% loss of full-length product, whereas amounts of a qPCR product of a nuclear gene (13 kb region of beta globin) were unaffected. Quantitative rtPCR analyses revealed 60–70% loss of mRNA for proteins encoded by mtDNA with no change in mRNA abundance for nuclear-encoded respiratory subunits. These changes coincided with proportionate reductions in complex I and IV activities and decreased respiration of isolated cardiac mitochondria. We conclude that chronic iron overload leads to cumulative iron-mediated damage to mtDNA and impaired synthesis of mitochondrial respiratory chain subunits. The resulting respiratory dysfunction may explain the slow development of cardiomyopathy in chronic iron overload and similar accumulation of damage to mtDNA may also explain the mitochondrial dysfunction observed in slowly progressing diseases such as neurodegenerative disorders.

Dilated Cardiomyopathy in Epidermolysis Bullosa: A Retrospective, Multicenter Study

Dilated cardiomyopathy (DC) has been reported in severe epidermolysis bullosa (EB) subtypes. Poor nutritional status, low carnitine levels, selenium deficiency, chronic iron overload, drugs and viral etiology have been proposed as potential contributors. This was a retrospective, descriptive, multicenter study describing EB patients that developed DC, and determining potential pre-disposing risk factors. Fifteen patients were enrolled in the study; 11 of them were male subjects (73%). Eighty-seven per cent of the participants had dystrophic EB and 13% had junctional EB. Mean age at diagnosis of DC was 12.18 +/- 4.99 years. Chronic anemia was diagnosed in 13 of 15 patients (86.7%). Sixty per cent of patients had prior red blood cell transfusions. At diagnosis, selenium levels were low in 55% of the patients (n = 11) and total carnitine levels were low in 45% of the patients (n = 11). Systolic function was moderately impaired, with a mean shortening fraction of 19.38% (SD = 5.04, n = 8). After a mean follow-up period of 6.3 +/- 4.8 years, six patients were alive without being on any medications (40.0%), two were alive on medications (13.3%) and seven had died (46.7%). Limitations of the study was that it was a retrospective chart review with relatively small sample size. Retrospective chart review, relatively small sample size. This study substantiates the association between DC and EB. Currently, there is no single risk factor identified in EB patients that leads to DC. Further prospective studies are needed.

Acute renal failure and Fanconi syndrome due to deferasirox

Deferasirox is the first oral iron chelator and, as such, is widely used for the treatment of chronic iron overload. However, recent data from large studies confirmed the renal toxicity of deferasirox. We report a case of Fanconi syndrome associated with acute renal failure in a patient receiving deferasirox. In particular, new insights regarding the pathophysiology of the renal disease due to this treatment are discussed. This case highlights the importance of a careful monitoring of kidney function, markers of proximal tubulopathy and ferritinaemia in patients receiving deferasirox.