ALBERT

Description: The safety and efficacy of alternating desferrioxamine and deferiprone for the treatment of iron overload in patients with transfusion-dependent anemias was studied in 60 thalassemia patients regularly treated with desferrioxamine. Patients were randomized to continue desferrioxamine alone (20–60 mg/kg/day, 5–7 days/week) or to alternate desferrioxamine (20–60 mg/kg/day, 2 days/week) with oral deferiprone (25 mg/kg tid, 5 days/week). Both treatment groups were similar for age (19.8 ± 6.1 years for desferrioxamine alone and 18.7 ± 4.8 years for alternate therapy) as was gender distribution and mean standard dose of desferrioxamine at the time of study initiation. Over the following 12 months, all patients were monitored weekly for adverse events and for their white blood cell count. Efficacy of the chelation was evaluated by measurement of the serum ferritin, liver iron concentration (magnetic susceptometry by SQUID), and by Non-Transferrin Bound Iron (NTBI). Compliance was comparable for both arms (96.1 ± 5.0% for alternate therapy vs 95.7 ± 5.7 % for desferrioxamine alone; p=0.7883). There was no significant difference in the proportion of patients with adverse events in the two therapy groups but the chelation regimens were associated with distinct adverse events. The alternate therapy was associated with transient gastrointestinal symptoms, such as vomiting in 5 patients (17%), abdominal pain in 3 patients (10%), or diarrhea in one patient (3%), or transient increase of serum ALT levels in one patient (3%), occurring mainly in the first weeks of therapy and were mild/moderate in severity. Daily infusions of desferrioxamine were associated with abscess at the site of infusion in one patient (3%), and allergic reactions in another patient (3%). Mean serum ALT levels were not significantly different between the two therapies. There were no episodes of agranulocytosis and only one patient, treated with desferrioxamine alone, experienced milder neutropenia. Both therapies resulted in similar decreases of serum ferritin (−349 ± 573 mg/L for the desferrioxamine arm; −248 ± 791 for the alternate arm; p=0.5802), and of liver iron concentrations (−239 ± 474 μg/g wet weight for the desferrioxamine arm; −65 ± 615 μg/g wet weight for the alternate therapy arm; p=0.2263) by the end of the treatment period. No significant changes in NTBI were observed between the two treatment arms (1.10 ± 7.19 μmol/L for the desferrioxamine arm; −0.03 ± 8.13 μmol/L for the alternate arm; p=0.5775). In conclusion, this 12 month study in transfusion-dependent thalassemia demonstrated that the alternating therapy with deferiprone and desferrioxamine is not associated with a significant increase in the incidence of adverse events and that it has comparable efficacy to desferrioxamine alone in controlling iron overload.

Description: Background: The value of understanding deferiprone's comparative pharmacokinetics (PK) in differing indications and human subpopulations derives from its potential versatility in treating, not only transfusional iron overload, but also conditions in which iron mishandling is localized. While some PK on deferiprone in patients with thalassemia has been published, little is known for patients without systemic iron overload, or for those requiring special consideration (e.g., children, patients with hepatic or renal impairment). Reporting of characteristics such as deferiprone's rapid absorption, extensive glucuronidation, and principally urinary excretion has been consistent, but some publications have followed false trails, and lack of IV data and the dearth of PK information in non-iron-overloaded patients have limited the overall picture. Availability of a comprehensive integration of deferiprone human PK would address misconceptions and help in predicting doses for patients with various indications currently being investigated, as well as in special populations. Objective: To provide data on the PK of deferiprone generated from currently unpublished studies, to enable dosing guidance for deferiprone use in conditions beyond adult patients with thalassemia. Methods: Data from PK studies, conducted as part of our development programme with deferiprone in thalassemia, sickle cell disease, and conditions without systemic iron overload, as well as in children and subjects with impaired renal function or impaired hepatic function are presented. Results: The absolute (oral vs IV) bioavailability of deferiprone was 72% (Studies were conducted with Ferriprox™ 500 mg tablets). Following an oral dose of 1,500 mg (20 mg/kg), the mean maximum serum deferiprone concentration (Cmax) in the fasting state in non-iron-loaded healthy subjects was 20 mcg/mL, and the mean total area under the concentration-time curve (AUC) was 53 mcg·h/mL. Cmax of deferiprone occurs approximately 1 hour after a single dose in fasted subjects, but may be delayed to 2 hours in the fed state. Food decreases the Cmax of Ferriprox tablets by about a third and the AUC by 10%. Steady state is achieved on the first day of dosing and cross-study comparisons indicate dose proportionality. Protein binding of deferiprone in human plasma is ≤20%. Metabolism is predominantly UGT 1A6-mediated conjugation to form a 3-O -glucuronide, which is rapidly cleared by renal excretion (Tmax 2-4 hours in fasting subjects) and lacks iron binding capability. There is no evidence of genetic polymorphism. Most of a dose of deferiprone is rapidly eliminated from plasma, with a t½ of about 2 hours, and is excreted primarily into the urine as the glucuronide. Dose adjustment is not necessary in patients with renal impairment, as confirmed by similar total body clearance to healthy controls. Subjects with mild or moderate hepatic impairment retain sufficient capacity for glucuronidation to also not require dose adjustment. The clearance of deferiprone in children is comparable to that in adults. The pharmacokinetics in patients with Friedreich Ataxia, PKAN and Parkinson's disease, conditions in which deferiprone is currently being evaluated by various investigators, is expected to be comparable to PK in healthy volunteers. Conclusions: Comparative IV and oral dosing of deferiprone reveals that it is extensively and rapidly absorbed from the gut. The PK of deferiprone in patients without systemic iron overload is predicted to be similar to the PK in healthy subjects. Studies in special populations demonstrate that dose adjustment in children or in patients with renal or moderate hepatic impairment is not necessary. Disclosures Spino: ApoPharma Inc.: Employment. Off Label Use: Deferiprone is approved for the treatment of iron overload in thalassemia syndromes. Connelly:ApoPharma Inc.: Employment. Tsang:ApoPharma Inc.: Employment. Fradette:ApoPharma Inc.: Employment. Tricta:ApoPharma Inc.: Employment.

Description: Introduction: The procedures and requirements for the clinical trial application (CTA) to Ethics Committees (ECs) and/or Competent Authorities (CAs) are not fully harmonised, and this is even more evident when non-EU countries are involved. This lack of harmonisation makes more difficult the approach in the case of 'small populations', such as children and patients affected by rare diseases. A phase III efficacy-safety comparative trial (DEEP-2) involving paediatric patients affected by transfusion dependent haemoglobinopathies from seven European and non-European countries (Albania, Cyprus, Greece, Italy, United Kingdom, Egypt, Tunisia) was carried out in the context of a FP7 project (HEALTH-F4-2010-G.A. n. 261483) and included in an agreed Paediatric Investigation Plan. Aims: The aims of this paper are to describe in a complex multi-national/multi-ethnic framework the different provisions and procedures to authorise a paediatric trial in EU/non-EU countries and to evaluate the possible impact of the following key indicators on the timing of ECs approval and CAs authorisation: complexity of the national/local provisions and procedures to authorise a paediatric trial, including the number of ECs and CAs to be addressed; number and type of additional local/national documentation; number of queries from CAs and ECs; geographic setting (EU and non-EU). Methods: The following information was collected from official websites and through a survey addressed to Principal Investigators: The regulatory and legal frameworks in force at the time of the submission of DEEP-2 in each involved country;The procedures required at local/national level (i.e. number of ECs and CAs to be addressed, parallel or subsequent submission to the CA and the EC, preparation of the CTA form and documents required from CAs and ECs);The timing of ECs approval and CAs authorisation, including number and types of queries, were collected from DEEP-2 Trial Master File. Descriptive analysis, Wilcoxon Rank-Sum test and General Linear Model (GLM) analysis were used to describe results and to analyse significance of the considered indicators. Results: In the EU countries, relevant legislative acts apply and include GCP and specific procedures for paediatric trials, in non-EU countries GCP guidelines apply but have not been implemented in the national laws regulating clinical trials. Moreover, within the 4 EU Member States a different approach was in place, even if under the same rules (i.e. Directive 2001/20/EC as implemented in the national law) with distinctive documents required for the CTA in almost all the EU countries compared with the EC provisions. The CTAs were performed in the period June 2012 - September 2015 in 23 trial sites. The EC approvals and CA authorisations were issued between January 2013 - September 2015. In the EU countries, the authorisation process was completed within 7,3 to 33,8 months (median = 15 months), while in non-EU countries, the authorisation process was completed by 7 months (median = 4 months) (figure 1). In particular, the comparison of the CA time authorisation shows a significant difference between EU and non-EU clusters (p = 0.001); however, if the statistical model is adjusted for the number of EC requests as covariate, the difference is not significant. Thus, it seems that the main factor influencing the time for EC approval is the number of requests for changes/clarifications (mainly on informed consent/assent, study protocol, insurance) (figure 2). Conclusion: Delays in completion of the authorisation phase in many countries seems to be a relevant issue and the timeframes for the authorisation in EU countries are not compliant with the European requirements (60 days for single opinion release and 30 days for its acceptance, as stated in Directive 2001/20/EC). The main reasons for delay is the complexity of the procedures and the requests from the ECs/CAs. In non-EU countries, procedures are different and faster with less requests from ECs and CAs. The upcoming application of a stronger set of rules, CT-Regulation (EU) 536/2014, is expected to harmonise practices in Europe and possibly outside Europe. The final aim of this change should be to assure a good balance between a timely approval and a high-level of children protection. Disclosures Reggiardo: CVBF: Consultancy. Tricta:ApoPharma: Employment.

Description: Although there are 20 yr of clinical experience with deferiprone in treating transfusional iron overload, limited data exist on the safety and efficacy of deferiprone in very young children. Difficulties in swallowing the tablet formulation of deferiprone (Ferriprox®, ApoPharma, Canada), is a limiting factor in the administration of deferiprone in young children. The current study evaluated the tolerability, safety and efficacy of a new liquid formulation of deferiprone (Ferriprox® Oral Solution) in iron-overloaded pediatric patients with transfusion-dependent anemias (≥ 8 transfusions/year). The study also assessed the daily neutrophil count in patients who continued deferiprone therapy during episodes of mild neutropenia. The study was approved by the relevant regulatory authorities and ethics review boards. Informed consent was obtained from the patients’ legal representatives. One-hundred children [91Thal major, 8 HbE, 1 Sickle Cell disease; 46 female and 54 male; 76 Caucasian (Egyptian), 24 Asian (9 Chinese, 13 Indonesian, 2 Malay)] ranging from 1 to 10 yr of age (median 5.0 yr) were enrolled. At enrollment, 51 children were being treated with deferoxamine (mean duration 1.82 ± 1.95 years; range 0.1–7.3 yr), 20 with deferiprone (mean duration 0.5 ± 0.6 yr; range 0. 04–2 yr), 8 patients with deferasirox (mean duration 0.4 ± 0.5 yr; range 0.1–1.6 yr) and 21 patients were naïve to chelation therapy. Deferiprone therapy was initiated at 50 mg/kg/day, divided in 3 doses, for the first 2 weeks, and then increased to 75 mg/kg/day. The dose was further increased to 100 mg/kg/day for those patients with ferritin 〉 2500 μg/L at baseline. Ninety-five children completed 6 months of therapy. One patient was lost to follow-up, 2 patients voluntarily withdrew consent (1 patient disliked the taste, 1 patient did not comply with weekly visits), and 2 were withdrawn due to adverse events. Therapy with the oral solution of deferiprone was not associated with unexpected adverse reactions. The incidence of gastrointestinal adverse reactions was lower than observed for the tablet formulation in older patients (Table). Oral solution in children ≤ 10 yr old Tablet formulation in children 〉 6 yr old and adults Adverse Reaction (AR) % Patients with AR % Patients with AR Nausea 1% 16% Abdominal Pain 6% 14% Vomiting 6% 12% Arthralgia 4% 11% Neutropenia (0.5 × 109/L ≤ ANC 〈 1.5 × 109/L) 6% 6% Agranulocytosis (0.5 〈 ANC) 2% 1% Five patients experienced single episodes of mild neutropenia [absolute neutrophil count (ANC) 1.5 × 109/L but not less than 1.0 × 109/L], which resolved and did not recur, despite continuous deferiprone use. Another patient experienced 2 transient episodes of mild neutropenia and a third episode that progressed to agranulocytosis (ANC 〈 0.5 × 109/L). Deferiprone was discontinued and the patient was treated with G-CSF. The event resolved (ANC 〉 1.5 × 109/L) within 9 days upon discontinuation of deferiprone. Another patient experienced a single episode of agranulocytosis, which resolved within 9 days upon discontinuation of deferiprone and therapy with G-CSF. During the 6-month therapy, there was a significant decrease in serum ferritin from a mean baseline value of 2532 ± 1463 to 2176 ± 1144 μg/L (p〈 0.0005). The new oral solution of deferiprone was well tolerated and effective in lowering serum ferritin in young children with transfusion dependent anemias and exhibited a safety profile similar or better to that reported for the tablet formulation in older patients. The results also suggest that not all episodes of mild neutropenia progress to agranulocytosis with continued deferiprone therapy, and that further studies are warranted to differentiate those patients from those at risk of developing deferiprone-induced agranulocytosis following neutropenia. This study includes the first report of patients using deferiprone as their first iron chelator.

Description: The identification of a safe, orally active iron chelator is critically important for the prevention of morbidity and early death in patients receiving regular red cell transfusions. Based on our findings in a 1-year multicenter, prospective study of the safety and efficacy of deferiprone in patients with thalassemia major, we have extended the treatment period to 4 years. The mean dose of the chelator was 73 mg/kg per day during 531 patient-years. The rates of agranulocytosis (absolute neutrophil count [ANC] 〈 500 × 109/L) and milder forms of neutropenia (ANC, 500-1500 × 109/L) were 0.2 and 2.8 per 100 patient-years, respectively. Neutropenia occurred significantly more commonly in patients with intact spleens. Gastrointestinal and joint symptoms decreased significantly after the first year of therapy, and led to discontinuation of deferiprone in only one patient in years 2 to 4. The mean alanine aminotransferase (ALT) value of 71 U/L after 4 years of therapy was significantly higher than the baseline value of 61 U/L. Trend analysis showed no increase in the ALT levels or the percentage of patients with ALT levels greater than twice the upper limit of the reference range. Ferritin levels did not change significantly from the values at the time of change from deferoxamine to deferiprone in either the intention-to-treat analysis or in the 84 patients who completed 4 years of therapy. Because of concerns regarding the effectiveness of the studied dose of deferiprone, 47 patients discontinued therapy, whereas 15 patients interrupted therapy because of concerns regarding low iron levels. The results of this study help to define the safety and effectiveness of long-term therapy with deferiprone.

Description: Background: Patients with sickle cell disease (SCD) or other rare anemias whose care includes chronic blood transfusions must receive iron chelation to prevent the morbidity of iron overload. Currently, only deferoxamine (DFO) and deferasirox (DFX) are approved chelators in these patient populations. This randomized open-label trial evaluated if the efficacy of deferiprone (DFP) was non-inferior to DFO. DFO was used as the comparator product since DFX was not approved as first-line treatment for SCD at trial initiation. Methods: Participants at 27 sites in 8 countries were randomized in a 2:1 ratio to receive either DFP or DFO for up to 12 months. Those with lower transfusional iron input and/or less severe iron load were prescribed either DFP 25 mg/kg of body weight t.i.d. or DFO 20 mg/kg (children) or 40 mg/kg (adults); those with higher iron input and/or more severe iron load received either DFP 33 mg/kg t.i.d. or DFO up to 40 mg/kg (children) or 50 mg/kg (adults). Dosages could be adjusted over the course of the trial if necessary. Efficacy endpoints were the changes from baseline in liver iron concentration (LIC), cardiac iron, and serum ferritin (SF) at Month 12. The primary endpoint was based on LIC, and for the demonstration of non-inferiority of DFP to DFO, the upper limit of the 95% confidence interval for the difference between treatments had to be no more than 2 mg/g dry weight (dw). All patients had their neutrophil count monitored weekly, whereas other safety assessments and compliance with study therapy were evaluated monthly. Acceptable compliance was defined as taking 80% to 120% of the prescribed dosage. Results: A total of 228 of the targeted 300 patients were dosed with 152 receiving DFP and 76 receiving DFO, to assess non-inferiority. There were no significant differences between the groups in any demographic measures: in each treatment group, 84% of patients had SCD and the remainder had other, rarer forms of transfusion-dependent anemia. Mean age at enrollment was 16.9 years (± 9.6); 53.1% of patients were male; and 77.2% were white, 16.2% black, and 6.6% multi-racial. Over the course of the study, 69% of patients in the DFP group and 79% in the DFO group had acceptable compliance with treatment. Based on the Pocock's α spending function, a more stringent confidence level of 96.01% was applied to the calculation of confidence interval for the evaluation of non-inferiority. For the primary efficacy endpoint, the least squares (LS) mean change in LIC (measured as mg/g dw) was -4.04 for DFP, -4.45 for DFO; the upper limit of the 96.01% confidence interval for the difference was 1.57, thereby demonstrating non-inferiority of DFP to DFO. The upper limit for the subpopulation of patients with SCD also met the non-inferiority criterion. For the secondary endpoints, the change in cardiac iron (measured as ms on MRI T2*, log-transformed) was approximately -0.02 for both; and for SF (measured as μg/L), it was -415 vs. -750 for DFP vs. DFO, respectively. The difference between the groups was not statistically significant for both endpoints. With respect to safety, there was no statistically significant difference between the groups in the overall rate of adverse events (AEs), treatment-related AEs, serious AEs, or withdrawals from the study due to AEs. Agranulocytosis was seen in 1 DFP patient vs. no DFO patients, while events of less severe episodes of neutropenia occurred in 4 vs. 1, respectively. All episodes of agranulocytosis and neutropenia resolved. There was no significant treatment group difference in the rates of any of the serious AEs. Conclusion: The efficacy of DFP for the treatment of iron overload in patients with SCD or other rare anemias is not inferior to that of DFO, as assessed by changes in liver iron concentration. non-inferiority was supported by the endpoints on cardiac iron load and SF. The safety profile of DFP was acceptable and was similar to that previously seen in thalassemia patients, and its use was not associated with unexpected serious adverse events. The results of this study support the use of DFP for the treatment of iron overload in patients with SCD or other rare transfusion-dependent anemias. Note: The authors listed here are presenting these findings on behalf of all investigators who participated in the study. Disclosures Kwiatkowski: Terumo: Research Funding; Imara: Consultancy; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Novartis: Research Funding; Celgene: Consultancy; Apopharma: Research Funding. Fradette:ApoPharma: Employment. Kanter:Sangamo: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Imara: Consultancy; Guidepoint Global: Consultancy; GLG: Consultancy; Cowen: Consultancy; Jeffries: Consultancy; Medscape: Honoraria; Rockpointe: Honoraria; Peerview: Honoraria; SCDAA: Membership on an entity's Board of Directors or advisory committees; NHLBI: Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy; Modus: Consultancy, Honoraria. Tsang:Apotex Inc.: Employment. Stilman:ApoPharma: Employment. Rozova:ApoPharma: Employment. Sinclair:ApoPharma: Employment. Shaw:ApoPharma: Employment. Chan:ApoPharma: Employment. Toiber Temin:ApoPharma: Employment. Lee:ApoPharma: Employment. Spino:ApoPharma: Employment. Tricta:ApoPharma: Employment. OffLabel Disclosure: Deferiprone is an oral iron chelator.

Description: Iron toxicity is the main risk factor for morbidity and mortality in patients with transfusion-dependent thalassemia. Current practice is to start chelation therapy only after 10-20 transfusions, or when the serum ferritin (SF) level rises above 1,000 μg/L. This delay is aimed at minimizing the risk of chelation toxicity that was observed with the use of deferoxamine in children with low iron stores. Deferiprone has lower affinity for iron than deferoxamine and data from clinical trials on its use in patients without systemic iron overload indicate a safety profile for its use in those conditions. The current trial was designed to evaluate the safety of the early use of low-dose deferiprone in newly diagnosed pediatric thalassemia and to evaluate if it can postpone iron overload. Sixty-four children recently diagnosed with thalassemia major who had begun receiving blood transfusions every 3-4 weeks to keep pre-transfusion Hb above 10 gm/dl, had not yet started iron chelation therapy and had SF ≥ 400 μg/L or transferrin saturation (TSAT) ≥ 70% or labile plasma iron (LPI) ≥ 0.2 µM were randomized to start deferiprone (DFP) at a dose normally considered to be sub-therapeutic (50 mg/kg/day) or no chelation (NC). Age at 1st transfusion was 8.1 ± 1.7 for DFP-treated and 8.1 ± 1.6 months for NC children. The percentage of patients with LPI ≥ 0.6 µM, SF ≥ 1000 μg/L or TSAT ≥ 70% in each study arm was assessed at 6 months and 12 months. Patients with confirmed SF ≥1000 ng/mL were withdrawn from the study and placed on a standard chelation regimen. Results. Two patients (DFP) were lost to follow after baseline measurements, 1 patient (NC) withdrew consent at baseline, and 10 patients (5 DFP, 5 NC) have yet to complete all follow up visits. All NC patients had been removed from the trial prior to completing 7 months of follow-up 12 due to confirmed SF ≥ 1000 μg/L. Mean ± SD time of follow up was 10.4± 4.9 and 5.9 ± 2.5 months for DFP and NC, respectively. Most common adverse events in patients on DFP versus NC were diarrhoea (19% vs 13%, p= 0.73), vomiting (13% vs 13%, p=1.00), abdominal colic (13% vs 13%), increased liver enzymes (6% vs 3%, p=1.00) and neutropenia (neutrophil count between 1,000-1,500 x 109/L) (6% vs 6%). All adverse events were mild in severity and did not require interruption of DFP use. There were no cases of agranulocytosis or of moderate neutropenia, no arthralgia and no serious infections in DFP-treated patients. Preliminary efficacy results are presented in the table. DFP therapy was associated with a significant reduction in the rate of iron accumulation as measured by SF (P

Description: Introduction: Agranulocytosis/severe neutropenia is an established adverse event during deferiprone (DFP) use. Less is known about milder episodes, which are frequently transient despite continuous deferiprone use. To provide further insight into this topic, we compared the incidence of neutropenia during DFP or deferasirox (DFX) treatment in the randomized Deferiprone Evaluation in Paediatrics (DEEP-2) trial, where blood neutrophil count was regularly monitored in patients randomized to be treated with DFP or DFX. Methods: DEEP-2 was a multicenter, randomized, 12-month, open-label trial comparing DFP vs DFX in pediatric (

Description: Cynomolgus monkeys and rats have been the species of choice for evaluation of general toxicity during the non-clinical development of iron chelators. Each model has its advantages in safety assessment studies, although the relatively poor conservation of iron in rats and other differences in their iron handling compared with primates, including man, have raised questions about their appropriateness. It was reported that in a study in cynomolgus monkeys conducted during the early development of deferiprone, doses greater than 150 mg/kg caused deaths when given orally once daily for c. 20 days of a scheduled 3-month treatment period. We present results from a 12-month oral toxicity study of deferiprone in naive and iron-loaded cynomolgus monkeys, using twice daily dosing and compare them with findings in a similar study in rats. Cynomolgus monkeys (4–6/sex/group) and rats (20/sex/group), iron loaded by intraperitoneal injection of iron dextran, were given deferiprone as two equal daily oral doses totalling 0, 75, 150 and 200 (250 after 3 months in monkeys) mg/kg/day; naive monkeys were given 0 and 150 mg/kg/day for 12 months. Measurement of all standard parameters, including toxicokinetics, was included. No mortalities, no adverse clinical signs, and no effects on body weight gain, food intake, cardiovascular function or eye morphology, were observed in either iron-loaded or naive monkeys. Haematological and plasma chemistry parameters were largely unaffected by treatment with deferiprone; intermittent increases in serum activities of some hepatic enzymes were related to iron loading. Similarly, histopathological changes were limited to those associated with iron deposition in tissues. Plasma deferiprone concentrations achieved were significantly in excess of clinical exposures. Both naive and iron-loaded rats were less tolerant of long-term administration of deferiprone. Both divided daily dosing and iron loading may have contributed to the markedly better survival of monkeys in the 12-month study compared with the earlier investigation, and the results of this work confirm the value of the model in the evaluation of the safety of iron chelators.

Description: Transfusion-dependent iron overload, such as occurs in beta-thalassaemia (Cooley’s Anaemia), leads to lethal cardiac toxicity in the second decade of life if not treated by iron chelation, but even with subcutaneous desferrioxamine (DFO) cardiac disease remains a problem, although delayed by 1–2 decades. As we design novel iron chelators, we are testing them in various animal models of iron overload. While assessing outcomes we have observed relatively sparse reports of systematic studies on organs, tissues, cellular, or subcellular iron distribution. Therefore we initiated a series of studies to characterize iron distribution using various approaches. Multiple intraperitoneal injections of iron dextran, 200 mg/kg/week X 4 − 16 weeks, followed by an equilibration period of minimum 1–2 weeks was studied as a means of increasing total body iron load in hundreds of rats under various conditions. Sacrifice varied from 6 weeks to 1 year post iron loading and the concentration of iron in liver, heart, and other tissues, organs, cells and subcellular organelles was examined. Quantitatively, in untreated rats (no chelators), the liver/heart iron ratio was about 10:1, consistent with the accumulation observed in post-mortem studies in humans prior to extensive use of iron chelation. Much less-well described has been the distribution of iron in lymphatic tissues. Our studies revealed that lymph nodes become visibly enlarged. In addition, randomly distributed brown spots appeared in the omentum. Such changes persisted up to one year after iron loading, regardless whether they were treated daily with chelators (DFO or deferiprone) in standard doses for four months. Even after a single intraperitoneal iron-dextran injection of 200 mg/kg, changes were visible. Histopathological analysis (hematoxilin-eosin for general histology and Perl’s Prussian Blue for iron) showed extensive iron accumulation in the omentum, and in the cortical and subcortical regions of the enlarged lymph nodes. Electron microscopy revealed cellular (macrophages) and subcellular (mitochondria) iron localization in the lymph nodes. When iron was administered as iron sucrose (single ip dose), iron accumulation was more extensive in the omentum and in the peritoneal fat in comparison to iron dextran, but the enlargement of the lymph nodes was not observed. Quantitative iron measurement (via validated HPLC method) in the liver and heart after a single iron dextran (N=30, up to 29th day) and iron sucrose dose (N=6 up to 50th day) was in agreement with the histological observations. Iron accumulation in the omentum and lymph nodes after four months of chelation treatment and one year after iron loading indicated the resistance of these unusual iron “pools” to chelation therapy. These studies confirm that different iron formulations may result in different patterns of iron distribution and they also raise questions about the suitability of rats as an animal model for transfusional iron overload in humans.