scholarly journals Ferrozine-Based Iron Quantification of Iron Overloaded Mice: a Simple, Robust Method for the Assessment of Iron Chelation Therapies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2054-2054
Author(s):  
David T Tran ◽  
Max J Petersen ◽  
Charles O Noble ◽  
Mark E Hayes ◽  
Peter Working ◽  
...  
Keyword(s):  
Nitric Acid ◽  
Iron Chelation ◽  
Iron Removal ◽  
Iron Dextran ◽  
Employment Equity ◽  
Liver Iron ◽  
Iron Quantification ◽  
Tissue Iron ◽  
Nitric Acid Digestion ◽  
Equity Ownership

Abstract Introduction: We describe a low heat nitric acid digestion and colorimetric ferrozine-based iron assay that provides a fast, inexpensive and accurate alternative to high temperature animal tissue processing and inductively coupled plasma mass spectrometry (ICP). This technique is useful for the quantification of iron in iron overloaded animal models and diseases such as β-thalassemia, sickle cell anemia, and myelodysplastic syndromes. We applied it to evaluate iron removal by iron chelation therapies such as liposome-encapsulated deferoxamine (LDFO). Methods: CF-1 mouse liver, spleen, heart, plasma, urine and feces were digested in nitric acid (70%) at 65 °C for 1-2 hours. For large tissues such as the liver, tissues were sectioned at 50 mg fractions (n = 4) to also assess iron homogeneousness. Digested samples were bleached with hydrogen peroxide (30%) and diluted with water before iron quantification by ICP or the ferrozine-based assay. For the ferrozine-based assay, nitric acid was neutralized with ammonium acetate and iron was reduced with ascorbic acid before reaction with ferrozine for the colorimetric assessment of the ferrozine-ferrous iron complex at 550 nm. For iron overloaded models, CF-1 mice (n = 4) were loaded I.V. with iron dextran, iron sucrose, liposome encapsulated iron, or horse ferritin for over a week before sacrifice. Iron removal studies of tissues and excreta from iron overloaded CF-1 (n = 4) started 2 weeks after iron dextran overloading. Animals were dosed with either deferoxamine (DFO) and LDFO by I.V. and Exjade by oral gavage. Urine and feces were collected daily at the start of treatment. Animals were sacrificed 7 days post treatment. Tissues and excrements were digested and measured for iron by the ferrozine-based assay. Results: The ferrozine-based tissue iron quantification assay yields high iron recovery from mouse tissue. CF-1 liver spiked with iron dextran or horse ferritin had an iron recovery of 99±2% and 97±2%, respectively. Liver iron measurements of iron dextran overloaded mouse models resulted in identical iron measurements for the ferrozine-based assay and ICP. For CF-1 mice treated with iron dextran I.V. (0, 100, 300, 600 mg/kg, n = 4), liver iron is highly correlative between the two iron quantification techniques with a slope of 1.03 and R2 of 0.999. In addition, there is a linear iron overloading effect in both the liver (R2 = 0.99) and spleen (R2 = 0.98). CF-1 mice were also iron overloaded with iron sucrose, liposome encapsulated iron, and horse ferritin with dose dependent tissue overload. For all iron overloaded models tested, liver iron and spleen iron were homogenous per tissue when multiple sections were analyzed with an average low 6% difference in iron content. Despite high liver and spleen overloading, none of these iron carriers resulted in statistically significant heart iron overload. Iron dextran overloaded CF-1 mice (100 mg/kg) were treated with LDFO, DFO, and Exjade. LDFO at 100 mg/kg I.V. greatly reduces iron levels in the liver (p = 0.019) and spleen (p = 0.014) compared to non-effective no treatment, free DFO (p = 0.3), and empty liposomes (p = 0.1). Exjade at 30 mg/kg by oral gavage did not result in statistically significant iron removal in the liver or spleen (p < 0.2). Over the first four days, urine and feces were collected daily and also analyzed for iron. Results revealed that iron clearance by LDFO is primarily in the urine (p = 0.022 urine; p = 0.8 feces) while Exjade removed iron appeared in the feces (p = 0.06 feces; p = 0.013 urine). During this short period, drug efficiency in iron excretion (5%) from one dose of the novel LDFO at 100 mg/kg was equivalent to four daily doses of the Exjade at 50 mg/kg/dose. Conclusion: The low heat nitric acid digestion and ferrozine-based tissue iron quantification assay is a simple, precise, highly reproducible tool for the assessment of tissue and excretion iron. The assay enabled the rapid, low cost evaluation of novel iron chelation therapies. We gratefully acknowledge support by NIH SBIR Phase 1 Grant 1R43HD075429-01 and NIH SBIR Phase 2 Grant 2R44HD075429-02. Disclosures Tran: Zoneone Pharma, Inc.: Employment. Petersen:Zoneone Pharma, Inc.: Employment. Noble:Zoneone Pharma, Inc.: Employment, Equity Ownership. Hayes:Zoneone Pharma, Inc.: Employment, Equity Ownership. Working:Zoneone Pharma, Inc.: Consultancy, Equity Ownership. Szoka:Zoneone Pharma, Inc.: Consultancy, Equity Ownership.

scholarly journals Twice Monthly Liposome Encapsulated Deferoxamine (LDFO) Has a High Molar Efficiency in Removing Total Body Iron in an Iron Dextran-Overloaded Mouse Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2322-2322 ◽  
Author(s):  
David T Tran ◽  
Mark E Hayes ◽  
Charles O Noble ◽  
Zhipeng Dai ◽  
Peter K Working ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Iron Overload ◽  
Iron Removal ◽  
Control Group ◽  
Iron Dextran ◽  
Employment Equity ◽  
Transfusion Therapy ◽  
Female Mice ◽  
Equity Ownership

Abstract Introduction: Patients who have β-thalassemia, sickle cell anemia, and myelodysplastic syndromes are sustained by long-term blood transfusion therapy. Transfusion therapy results in iron accumulation initially in the liver, spleen and bone marrow. These are the organs responsible for eliminating apoptotic red blood cells. To prevent iron overload, iron must be removed using iron chelators, administered either orally or infused on a daily basis. One strategy to more effectively deliver chelators to the sites of iron accumulation is to encapsulate them in liposomes. Liposomes also accumulate in the liver, spleen and bone marrow; therefore LDFO targets the chelator directly to the iron stores. We tested the hypothesis that the LDFO approach provides highly efficient (moles chelator administered/moles iron removed) iron chelation. If this hypothesis is supported by the data, lower amounts of chelator could be administered to patients, on a less frequent dosing schedule. This would enable LDFO to be used alone or as part of a combination chelation regime to minimize the need for high daily chelator doses. Methods: We prepared two LDFO formulations composed of (hydrogenated soy phosphatidylcholine, HSPC)/cholesterol (60/40) or (palmitoyloleoylphosphatidylcholine, POPC)/cholesterol (55/45). Animal protocols were approved by the institutional review board. We determined the pharmacokinetics of the encapsulated deferoxamine (DFO) in CF-1 female mice by analyzing serum plasma DFO concentrations by HPLC, after dosing LDFO. CF-1 female mice were overloaded with iron by administering IV, hydrogenated iron dextran 100 mg/kg four times, at three day intervals. Twenty-one days after the last iron dose, mice (n=10) were administered the first dose of the LDFO. The LDFO formulations were administered IV three times at 200 mg/kg at 14-day intervals. A control group (n=10) of non-encapsulated DFO was administered by SC infusion from an implantable minipump at 20 mg/kg/day for a total dose of 280 mg/kg DFO over 14 days. Saline controls were dosed on the same schedule as LDFO. Every 24 h, five mice from each group were alternated from the regular cages into metabolic cages and urine and feces collected. The iron concentration in urine and feces was measured by a modified ferrozine-based spectroscopic assay. At the study end, blood was drawn form the mice and standard blood substances were analyzed as an indicator of organ function. Results: The HSPC/cholesterol and POPC/cholesterol LDFO liposomes had 88 nm and 119 nm diameters and encapsulated 354 and 266 g DFO/mole phospholipid respectively. At 6 and 24 h post IV injection, there is 67.0% and 27.2% ID DFO in plasma for HSPC liposomes and 54.2% and 18.1% ID DFO in plasma for POPC liposomes. In treating iron overloaded mice, IV administered LDFO removed 2.3-2.8 times more iron than deferoxamine mesylate (DFO) given by SC continuous infusion. After LDFO treatment, iron was continuously eliminated for 14 days post dosing. At day 14 LDFO-HSPC and LDFO-POPC cumulatively had a 3.1 and 3.5-fold higher iron elimination in urine compared to SC infused DFO and 1.8 and 1.3-fold higher in feces respectively, corrected for background iron using the saline control group. The second and third dose of LDFO at 14-day intervals showed similar iron elimination patterns to the first dose. The iron removal efficiencies were 68% for LDFO-HSPC, 55% for LDFO-POPC and 24% for Free DFO. The liposome composition shifted the relative iron elimination profiles within the liposome groups. Of the two formulations, LDFO-HSPC produced more fecal iron removal while the LDFO-POPC group gave higher urinary iron removal. The iron elimination curves for both liposome formulations were statistically different than the infused DFO curve (p<0.0001) when analyzed by a regression and covariance analysis, assuming that the slope of the elimination profiles were similar. At study end, liver and renal function markers were normal. Conclusion: In the iron dextran overload mouse model, a single injection of LDFO greatly accelerates iron elimination compared to two weeks of continuous SC infused DFO at similar cumulative dosages. The high molar efficiency of iron removal could lead to an improved treatment that increases chelator coverage and provides substantially better management of iron overload than current regimes in patients suffering from iron overload conditions. Disclosures Hayes: ZoneOne Pharma, Inc: Employment, Equity Ownership. Noble:ZoneOne Pharma, Inc: Employment, Equity Ownership. Dai:ZoneOne Pharma, Inc: Employment. Working:ZoneOne Pharma, Inc: Equity Ownership. Szoka:ZoneOne Pharma, Inc: Consultancy, Equity Ownership.

RNAi-Mediated Inhibition of Tmprss6 Ameliorates Anemia and Secondary Iron Overload in a Mouse Model of β-Thalassemia Intermedia and Decreases Iron Overload in Hfe−/− Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1018-1018
Author(s):  
Paul J Schmidt ◽  
Anoop K Sendamarai ◽  
Ivanka Toudjarska ◽  
Tim Racie ◽  
Jim S Butler ◽  
...  
Keyword(s):  
Mouse Model ◽  
Iron Overload ◽  
Iron Absorption ◽  
Disease Phenotype ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Liver Iron ◽  
Secondary Iron Overload ◽  
Equity Ownership ◽  
Low Levels

Abstract Abstract 1018 β-Thalassemia intermedia (TI), an inherited hemoglobinopathy caused by partial loss of β-globin synthesis, is characterized by anemia, extramedullary hematopoiesis and ineffective erythropoiesis as well as secondary iron overload. Hereditary hemochromatosis (HH) is most frequently caused by mutations in HFE and is marked by excess uptake of dietary iron with concomitant tissue iron overload. In both diseases, increased iron absorption is due to inappropriately low levels of the liver hormone, hepcidin (encoded by Hamp1). The membrane serine protease Matriptase-2 (encoded by Tmprss6) attenuates BMP-mediated Hamp1 induction by cleaving the BMP co-receptor, hemojuvelin. Previously, it has been shown that elevating Hamp1 expression by genetic inactivation of Tmprss6 reduces disease severity in the Hbbth3/+ mouse model of TI and prevents iron overload in Hfe−/− mice. Therefore, a therapeutic approach comprising specific inhibition of Tmprss6 could prove efficacious in TI and HH. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to >80% inhibition of Tmprss6 mRNA in the livers of Hbbth3/+ and Hfe−/− mice with concomitant >2-fold elevation in Hamp1 expression. In the TI model, Tmprss6 silencing leads to ∼30% reductions in serum iron and non-heme liver iron. In Hfe−/− mice, serum iron and non-heme liver iron are similarly reduced, and Perls staining of peri-portal iron is diminished. Remarkably, the partial iron restriction induced by Tmprss6 inhibition in Hbbth3/+ mice leads to dramatic improvements in the hematological aspects of the disease phenotype: the severity of the anemia is decreased as evidenced by an approximately 1 g/dL increase in total hemoglobin and a 50% decrease in circulating erythropoietin levels. As in the human disease, Hbbth3/+ mice exhibit the hallmarks of ineffective erythropoiesis including splenomegaly, decreased erythrocyte survival and marked reticulocytosis. Treatment with LNP formulated Tmprss6 siRNA leads to a dramatic 2–3 fold decrease in spleen size, a 3–4 fold decrease in reticulocyte counts and a >7-day increase in RBC half-life. Histological analysis of spleens from Tmprss6 siRNA treated animals demonstrates restoration of normal splenic architecture, as well as a reduction in the number of Tfr1-positive erythrocyte precursors in the spleen. Furthermore, as evidenced by the near normalization of blood smears, the overall quality of erythropoiesis in treated animals is vastly improved. Taken together, these data demonstrate that RNAi-mediated silencing of liver Tmprss6 elevates Hamp1 expression and reduces iron overload in both TI and HH model mice. More significantly, Tmprss6 siRNA treatment ameliorates all aspects of the disease phenotype in the TI mouse model. These results support the development of an RNAi therapeutic targeting TMPRSS6 for the treatment of TI, HH and potentially other disorders characterized by excess iron absorption due to physiologically inappropriately low levels of hepcidin. Disclosures: Racie: Alnylam Pharmaceuticals: Employment. Butler:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership.

Undertreatment of Patients (Pts) with Myelodysplastic Syndromes (MDS): Analysis of a Large Electronic Medical Records (EMR) Database Cohort of US Pts with MDS

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4768-4768 ◽  
Author(s):  
David P. Steensma ◽  
Bart L Scott ◽  
Xiaomei Ma ◽  
Albert Fliss ◽  
Pavel Kiselev ◽  
...  
Keyword(s):  
Platelet Count ◽  
Iron Chelation ◽  
International Classification Of Diseases ◽  
Initial Therapy ◽  
Employment Equity ◽  
Advisory Committees ◽  
Treatment Groups ◽  
Care Patterns ◽  
The Usa ◽  
Equity Ownership

Abstract Introduction: Little is known about current care patterns for pts with MDS across the US with respect to the use of available therapeutic agents. Using a cohort of 5,162 MDS pts we previously identified from the GE Centricity EMR database (GE Healthcare IT, Princeton, NJ) (Ma X, et al. Blood. 2015;126:abstract 3319), we examined associations between pt characteristics and treatment patterns, including sequence of therapies for pts with MDS. Methods: Pts with data in the EMR from Jan 2006 to end of Feb 2014 were included in this analysis. Pts were grouped by treatment received (erythropoiesis-stimulating agents [ESA], lenalidomide [LEN], hypomethylating agents [HMA; azacitidine or decitabine], and iron chelation therapy [ICT]), either alone, in combination, or as part of a sequence with other therapies. Transfusions were not included in this analysis, as transfusion data were often unrecorded due to transfusions occurring in facilities outside the EMR system. Pt characteristics were evaluated for each treatment group. Results: Of 5,162 pts evaluated, 1,843 (35.7%) received only 1 therapy, 2,079 (40.3%) received ≥ 1 therapy, with only 236 (4.6%) receiving ≥ 2 therapies. Pts who received ≥ 1 treatment of interest are shown in the Figure. Baseline characteristics for treatment groups are shown in the Table. A total of 85 pts were recorded as having deletion 5q by International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) coding; of these, 66 were recorded as receiving ≥ 1 treatment. Pts were in the EMR system for a median of 29 days (date of first entry to date of last entry), with 36%, 26%, and 15% of pts present in the system for > 6 months, 1 year, and 2 years, respectively. The most common initial therapy was ESA (n = 1,508; 72.5% of treated pts). Pts treated with ESA first had a median age of 78.0 years; 1,330 (88%) received ESA exclusively. ESA-only pts were the oldest among the treatment groups (median age 79.0 years), had the highest proportion with comorbidities at baseline (69%), and most commonly had isolated anemia. Only a small proportion of pts treated with ESA first subsequently received LEN (n = 79; 5.2%) or HMA (n = 68; 4.5%) as second therapy; median ages of these patients were 76.0 and 73.5 years, respectively. 682 pts (32.7% of treated pts) received a therapy approved specifically for MDS, i.e. HMA and/or LEN, during their treatment. Pts who received LEN as first treatment (n = 258; 12.4% of treated pts) had a median age of 74.0 years. These pts had a lower median hemoglobin (Hb), lower median absolute neutrophil count (ANC), and similar median platelet count vs pts receiving ESA as first treatment. Most pts who received LEN as their first therapy received it exclusively (244; 94.6% of treated pts); a small number (n = 14) were subsequently treated with HMAs. Pts who received LEN second (n = 99) or third (n = 13) in a sequence of therapies were similar in age (median 76.0 and 74.0 years, respectively) and had similar Hb levels, higher ANCs, and higher platelet counts at baseline than pts who received LEN as first therapy. Most pts (n = 79; 80%) who received LEN as second therapy previously received ESA. Of 252 pts (12.1% of treated pts) who received HMA as first therapy, 228 (90.4%) received HMA only; median age of patients who received HMAs as first therapy was 75.0 years, and median Hb level, median ANC, and median platelet count were lower than in pts who received ESA as first therapy. Another 100 pts and 28 pts received HMA as second and third therapies, respectively; median age was 73.0 years in each group. Pts receiving HMA third had higher median Hb level, ANC, and platelet count than pts who received HMA as first therapy. Only 61 pts (2.9% of treated pts) received ICT as first therapy. Conclusions: Pts diagnosed with MDS in the USA are likely to be undertreated. Consistent with findings from physician surveys (e.g. Sekeres M., et al. J Natl Cancer Inst. 2008;100:1542-51), ESAs are the most commonly used therapies despite the lack of a labeled indication for MDS. ESAs are usually the first therapy chosen by physicians and often the only therapy pts with MDS receive. Use of LEN and HMA, which have been approved for the treatment of MDS for ~10 years, appears low in this EMR. Disclosures Steensma: Genoptix: Consultancy; Celgene: Consultancy; Millenium/Takeda: Consultancy; Ariad: Equity Ownership; Amgen: Consultancy; Janssen: Consultancy. Scott:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ma:Celgene Corporation: Consultancy. Fliss:Celgene Corporation: Employment, Equity Ownership. Kiselev:Celgene Corporation: Employment, Equity Ownership. Swern:Celgene: Employment, Equity Ownership. Sugrue:Celgene Corporation: Employment, Equity Ownership.

Exjade® Reduces Cardiac Iron Burden in Chronically Transfused β-Thalassemia Patients: An MRI T2* Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2781-2781 ◽  
Author(s):  
J. Wood ◽  
A.A. Thompson ◽  
C. Paley ◽  
B. Kang ◽  
P. Giardina ◽  
...  
Keyword(s):  
Ejection Fraction ◽  
Iron Concentration ◽  
Iron Chelation ◽  
Left Ventricular ◽  
Iron Removal ◽  
Left Ventricular Ejection ◽  
Liver Iron ◽  
Ventricular Ejection Fraction ◽  
Cardiac Iron ◽  
The Mean

Abstract Introduction: Despite the routine use of iron chelation therapy, cardiac iron overload results in cardiomyopathy, congestive heart failure and death in approximately 71% of pts with β-thalassemia. Recent MRI studies suggest that the kinetics of cardiac iron uptake and elimination differ from that of liver. Furthermore, different chelators appear to exhibit unique profiles of relative heart and liver iron removal. Deferasirox (DFX; Exjade®) is a once-daily oral iron chelator with demonstrated efficacy in reducing liver iron. In addition, preclinical and single-institution clinical studies have demonstrated cardiac iron removal. This study is a prospective, single-arm multi-institutional trial designed to evaluate the effect of DFX on cardiac iron in pts with β-thalassemia major. Here, we report preliminary results from the first 15 pts who completed 6 months of treatment. Methods: This ongoing study will enroll 30 pts at 4 US centers. DFX is administered at 30–40 mg/kg/day for 18 months. Entry criteria include MRI evidence of cardiac iron (T2* <20 ms) and normal left ventricular ejection fraction (LVEF ≥56%). Serum ferritin is assessed monthly and MRI assessments for liver iron concentration (LIC), cardiac T2* and LVEF are assessed every 6 months. Labile plasma iron (LPI), serum creatinine, biochemical and hematological status are being monitored. Results: At the time of this analysis, 15 of 17 pts had 6 months of evaluation; all were dosed at 30 mg/kg/day. One of the excluded pts was found ineligible (LVEF <56% at baseline) and the other developed cardiac failure prior to 6 months and was switched to continuous DFO (deferoxamine). This pt had markedly elevated cardiac iron (T2*=1.8 ms) at enrollment. All results are reported as mean±SEM (range) unless otherwise stated. Baseline: All 15 evaluable pts (3 male, 12 female; aged 10–43 years) received ≥150 lifetime transfusions. Ferritin was 4927±987 ng/mL (395–10751; n=12). Cardiac T2* was 9.8±1.13 ms (5.0–16.1), LIC was 16.6±4.27 mg/g dw (3.6–62.3) and ejection fraction was 61.2±1.83%. LPI was 0.72±0.28 μmol/L (n=11) and 33% of pts started with abnormal LPI (≥0.5 μmol/L). 6 Month results: At 6 months, the mean decrease in ferritin was 516 ng/mL; 14 of 15 (93%) pts had decreases in hepatic and cardiac iron. The mean reductions in cardiac and hepatic iron were 17.8% (P=0.0136) and 27.0% (P=0.0027), respectively (Figure). There was no change in LVEF by MRI. All patients had normal LPI at 6 months; for pts with abnormal LPI at baseline, the mean LPI dropped from 1.6±0.3 to 0.26±0.1 μmol/L (P=0.003). No pts developed creatinine >upper limit of normal. Four pts had abnormal transaminases on ≥2 occasions but all 4 were abnormal at baseline. Conclusions: The 30 mg/kg/day dose was well tolerated and led to negative cardiac and liver iron balance in 93% of pts. These results are encouraging given this heavily iron-overloaded and heavily transfused population of β-thalassemia pts. Ongoing assessments over 12 and 18 months will elucidate if DFX continues to improve cardiac iron burden and maintain/improve cardiac function in severely iron-overloaded pts. Figure Figure

Initial Liver Iron Predicts Cardiac Chelation Efficacy of Deferasirox (Exjade®) Monotherapy in Chronically Transfused β-Thalassemia (β-Thal) Patients: 18- and 24-Month Data.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4069-4069
Author(s):  
John C. Wood ◽  
Alexis A. Thompson ◽  
Carole Paley ◽  
Tara Glynos ◽  
Barinder Kang ◽  
...  
Keyword(s):  
Iron Overload ◽  
Board Of Directors ◽  
Research Funding ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Employment Equity ◽  
Advisory Committees ◽  
Liver Iron ◽  
Cardiac Iron ◽  
Equity Ownership

Abstract Abstract 4069 Poster Board III-1004 Introduction Transfused patients with β-thal major are known to experience clinical consequences of cardiac iron overload despite the widespread use of iron chelation therapy. Approximately 71% of patients will suffer cardiomyopathy, congestive heart failure (CHF) and death. Previous trials have confirmed the efficacy of deferasirox (Exjade®) in removing cardiac iron in patients with β-thal major. This ongoing study evaluates the effects of deferasirox on cardiac iron and left ventricular ejection fraction (LVEF) in patients with β-thal major in a prospective, single-arm, multi-center trial using cardiac MRI T2*. All patients have completed 18 months of therapy and we also report preliminary results from 24 months. Methods 28 patients were enrolled at four US centers. Entry criteria included MRI evidence of cardiac iron (T2* <20 ms) and normal LVEF (≥56%). Deferasirox was administered at 30–40 mg/kg/day for 18 months. Following core study completion (18 months), patients could continue treatment for an additional 6 months if their 18-month cardiac T2* was <20 ms and they demonstrated ≥25% improvement in cardiac T2* or LIC from baseline. Serum ferritin (SF) was assessed monthly. Liver iron concentration (LIC), cardiac T2* and LVEF were assessed by MRI every 6 months. Serum creatinine (SCr), biochemical and hematological status were also monitored. All results are reported as mean ± SE (range) unless otherwise stated. Baseline: All 26 evaluable patients (7 M/19 F; aged 10–44 years) received ≥150 lifetime transfusions. SF was 4307 ± 613 ng/mL (312–12,655), cardiac T2* was 9.5 ± 0.8 ms (1.8–16.1), LIC was 20.6 ± 3.15 mg Fe/g dry weight (dw; 3.6–62.3) and LVEF was 61.8 ± 0.8%. Results At the time of analysis, 22 and 9 patients had 18- and 24-month evaluations, respectively. Six patients discontinued the core trial due to patient decision (n=2), adverse events (AEs; n=2) or abnormal lab tests (n=2). Two of these patients died after discontinuing; the first enrolled with markedly elevated baseline cardiac iron (T2* = 1.8 ms) and died secondary to CHF. The second patient withdrew due to an AE and died 2 months later due to sepsis and multi-organ failure. 18-month results: At 18 months, 10/22 patients were on 40 mg/kg/day. The mean improvement in cardiac T2* from baseline in all patients was 2.2 ms (22%; P=0.016), with 13 patients improving, four remaining stable (T2* change <10%) and five worsening. Baseline LIC was a powerful predictor of response (Figure); cardiac T2* in 14 patients with LIC <18.5 mg Fe/g dw improved 2.2% per month, with 13/14 patients showing large improvements and one patient remaining stable. In contrast, in eight patients with LIC >18.5 mg Fe/g dw, mean T2* worsened 1.4% per month (P<0.0001); three patients remained stable and five worsened significantly. Improvements in cardiac iron were correlated with changes in LIC (r2 = 0.27, P=0.013). In general, initial T2* did not predict therapeutic response, although all three patients with T2* <6 ms increased their cardiac iron. LIC decreased 4.1 mg Fe/g dw over the study interval (P=0.003). LVEF remained stable. 24-month results: At 24 months, 7/9 patients were on 40 mg/kg/day. Relative to the 18-month time-point, 8/9 patients (89%) increased their cardiac T2*, with a mean improvement of 2.7% per month. Mean LIC, SF and LVEF were unchanged over the extension. Safety parameters from patients treated with 30–40 mg/kg/day deferasirox (n=25) were in line with previous studies at 20–30 mg/kg/day. Conclusions Deferasirox monotherapy resulted in statistically significant improvements in cardiac and hepatic iron after 18 months. Baseline LIC <18.5 mg Fe/g dw was a strong predictor of favorable response. LVEF remained stable during the study. Patients in the extension (18–24 months) improved their cardiac T2* without further improvements in LIC or SF. Deferasirox monotherapy at 30–40 mg/kg/day provides good cardiac chelation in patients with moderate cardiac and liver iron burdens. More aggressive therapy is warranted for more severe iron overload. Disclosures: Wood: Novartis: Research Funding. Thompson:Novartis: Research Funding. Paley:Novartis Pharmaceuticals: Employment, Equity Ownership. Glynos:Novartis Pharmaceuticals: Employment. Kang:Novartis Pharmaceuticals: Employment, Equity Ownership. Giardina:Novartis: Research Funding, Speakers Bureau. Harmatz:Ferrokin: Membership on an entity's Board of Directors or advisory committees; Apotex: Membership on an entity's Board of Directors or advisory committees. Coates:Hope Pharma: Consultancy, Research Funding; Sangart Pharma: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

The Effect of Supplemental Ascorbate on Defersirox Iron Chelation In the Iron-Loaded Gerbil

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2059-2059
Author(s):  
Maya Otto-Duessel ◽  
Casey Brewer ◽  
Aleya Hyderi ◽  
Jens Lykkesfeldt ◽  
Ignacio Gonzalez-Gomez ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Content ◽  
Iron Concentration ◽  
Iron Chelation ◽  
Iron Dextran ◽  
Iron Loading ◽  
Liver Iron ◽  
Cardiac Iron ◽  
Liver Iron Content ◽  
Wet Weight

Abstract Abstract 2059 Introduction: Iron dextran injections are often used to induce iron overload in rodents, for the purposes of assessing iron chelation therapy. In gerbils, we have previously described that deferasirox therapy preferentially clears hepatocellular iron when compared with reticuloendothelial stores. Ascorbate deficiency, which is common in humans with iron overload, produces similar profound disparities between reticuloendothelial and parenchymal iron stores. We postulated that iron-induced ascorbate deficiency might be exaggerating reticuloendothelial iron retention in gerbils receiving deferasirox therapy. This study examined the effect of supplemental ascorbate on spontaneous iron loss and deferasirox chelation efficiency in the iron-dextran loaded gerbil. Methods: 48 female gerbils underwent iron dextran loading at 200 mg/kg/week for 10 weeks. Sixteen animals were sacrificed at 11 weeks to characterize iron loading; eight were on standard rodent chow and eight had chow supplemented with 2250 ppm of ascorbate. 32 additional animals that were not ascorbate supplemented during iron loading transitioned into the chelation phase. Half were subsequently placed on ascorbate supplemented chow and both groups were assigned to receive either deferasirox 100 mg/kg/day five days per week or sham chelation. Animals received iron chelation for twelve weeks. Liver histology was assessed using H & E and Prussian blue stains. Iron loading was ranked and graded on a five-point scale by an experienced pathologist screened to the treatment arm. Iron quantitation in liver and heart was performed by atomic absorption. Results: Table 1 one summarizes the findings. During iron dextran loading, ascorbate supplementation lowered wet weight liver iron concentration but not liver iron content suggesting primarily changes in tissue water content. Spontaneous iron losses were insignificant, regardless of ascorbate therapy. Deferasirox lowered liver iron content 56% (4.7% per week) in animals without ascorbate supplementation and 48.3% (4.0% per week) with ascorbate supplementation (p=NS). Cardiac iron loading, unloading and redistribution were completely unaffected by ascorbate supplementation. Spontaneous iron redistribution was large (1.9% – 2.3% per week). Deferasirox chelation did not lower cardiac iron to a greater degree than spontaneous cardiac iron redistribution. Histologic grading paralleled tissue wet weight iron concentrations. Ascorbate treatment lowered the rank and absolute iron score in liver during iron loading (p=0.003) and there was a trend toward lower iron scoring in sham treated animals (p=0.13). Ascorbate had no effect on histological score or relative compartment distributions of iron in deferasirox chelated animals (p=0.5). Ascorbate supplementation was sufficient to increase total plasma ascorbate levels from 25 ± 12.2 uM to 38.4 ± 11 uM at 10 weeks (p=0.03). In the liver, ascorbate increased from 1203 ± 212 nmol/g of tissue to 1515 ± 194 nmol/g of tissue (p=0.01) with supplementation. No significant change in total ascorbate was observed in the heart. Discussion: We hypothesized that ascorbate supplementation might improve reticuloendothelial iron accessibility to deferasirox by facilitating redox cycling. Although gerbils synthesize their own ascorbate, supplementation was able to raise both serum and hepatic total ascorbate levels. However, increasing ascorbate did not change either the amount or distribution of tissue iron in deferasirox-treated animals. Disclosures: Nick: Novartis: Employment. Wood:Novartis: Research Funding; Ferrokin Biosciences: Consultancy.

R2 and R2* Are Equally Effective In Evaluating Chronic Response To Iron Chelation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3437-3437
Author(s):  
John C. Wood ◽  
Pinggao Zhang ◽  
Hugh Y. Rienhoff ◽  
Walid Abi-Saab ◽  
Ellis J. Neufeld
Keyword(s):  
Clinical Trials ◽  
Los Angeles ◽  
Liver Biopsy ◽  
Dynamic Range ◽  
Iron Concentration ◽  
Iron Chelation ◽  
Phase 2 ◽  
Limits Of Agreement ◽  
Liver Iron ◽  
Equity Ownership

Abstract Introduction Magnetic resonance imaging (MRI) relaxometry, using either R2 or R2* measurements, has generally replaced liver biopsy for estimation of liver iron stores in response to iron chelation therapy, but there have been no longitudinal studies comparing R2 and R2* techniques. We used R2 and R2* liver iron concentration (LICR2 and LICR2*) estimates, transfusional iron burdens, and drug adherence data to calculate iron chelation efficiency in patients undergoing a phase 2 trial of SPD602 (formerly known as FBS0701), a tridentate iron chelator in development. We hypothesized that chelation efficiency estimates derived from LICR2 and LICR2* measurements would be in better agreement than baseline LIC assessments using the two techniques. Methods In the phase 2 clinical trial of SPD602, 51 patients underwent a baseline examination, 39 patients completed 48 weeks, and 26 patients completed 96 weeks. MRI assessment of liver R2 and R2* were performed at baseline, 12, 24, 48, 72, and 96 weeks, analyzed by experienced reference laboratories (Ferriscan R2 by Resonance Health Western Australia; R2* by Children's Hospital Los Angeles, CA), and converted to LIC using established calibrations. Efficiency was calculated according to the following equation: where TII is the transfusional iron intake in mg/kg, ΔLIC is the change in LIC in mg/g dry weight, MWdrug and MWFe are the molecular weights of SPD602 and iron respectively, and drug is the total SPD602 consumed in mg/kg. Results Figure 1 shows a scattergram of LICR2 versus LICR2* at baseline; both axes were log-transformed to normalize the variance. Resulting linear fit had a slope of 0.996, a scaling factor of 0.114 and an r2 of 0.76 (p< 0.0001). 95% limits of agreement were broad, measuring -53.8% to 53.8%. However, chelation efficiency estimates across the two techniques compared more favorably, with r2 values 0.76 and 0.83 when calculated over 0–48 and 49–96 weeks, respectively. Figure 2 demonstrates the LICR2 and LICR2* chelation efficiency estimates in patients completing 96 weeks. The r2 value was 0.89 with excellent 95% limits of agreement [-3.5 to 3.5%]; elimination of two outliers improved the r2 value to 0.95. Discussion Taken together, these data illustrate two important points for individual hematology practitioners. First, while R2 and R2* methods are individually as accurate as liver biopsy in predicting true LIC, large discrepancies between LICR2 and LICR2* can be observed for any given individual as shown by the baseline measurements. These discrepancies do not reflect intrinsic deficiencies of either technique, but arise because R2 and R2* are differentially sensitive to pattern and scale of iron deposition in the liver. Thus LICR2 and LICR2* cannot be used interchangeably in an individual. Secondly, however, longitudinal assessments decrease systematic errors between and within techniques, just as paired statistics often provide greater statistical power than unpaired statistics. As a result, both LICR2 and LICR2* produce chelation efficiency estimates that are internally consistent and physiologically reasonable. The choice of MRI assessment technique for clinical trials and for clinical management depends on many logistical considerations, including cost, the expected dynamic range of iron burden, and the availability of trained, experienced personnel for image quality control and analysis. In summary, the present study demonstrates that well-performed LICR2 and LICR2* measurements yield comparable longitudinal monitoring of chelator effectiveness, suitable for clinical trials and for clinical practice. Disclosures: Wood: Shire: Consultancy, Research Funding; Apopharma: Honoraria, Patents & Royalties; Novartis: Honoraria. Zhang:Shire: Employment. Rienhoff:Shire: Consultancy, Milestone Payments Other. Abi-Saab:Shire: Employment, Equity Ownership, Patents & Royalties; AbbVie: Equity Ownership, Patents & Royalties; Novartis: Equity Ownership, Patents & Royalties; Abbott: Equity Ownership, Patents & Royalties; Pfizer: Equity Ownership, Patents & Royalties. Neufeld:Shire: Consultancy.

Dose Response of Deferoxamine, Deferiprone, and ICL670 Chelation Therapy in a Gerbil Model of Iron Overload.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3621-3621 ◽  
Author(s):  
John C. Wood ◽  
Maya Otto-Duessel ◽  
Michelle Aguilar ◽  
Hanspeter Nick ◽  
Thomas D. Coates ◽  
...  
Keyword(s):  
Dose Response ◽  
Iron Content ◽  
Chelation Therapy ◽  
Iron Chelation ◽  
Iron Loss ◽  
Iron Dextran ◽  
Liver Iron ◽  
Cardiac Iron ◽  
Iron Excretion ◽  
Iron Concentrations

Abstract Introduction: The Mongolian gerbil mimics many of the cardiac functional impairments observed in iron cardiomyopathy, however relatively few chelation studies have been performed in this model. The purpose of this study was to characterize the dose-response of deferoxamine, ICL670, and deferiprone (L1) with respect to liver and cardiac iron chelation in the gerbil Methods: Thirty three adult Mongolian gerbils underwent subcutaneous iron dextran loading with 1500 mg/kg iron dextran divided into three, weekly doses. Chelation began at 4 weeks and continued for 4 weeks. Animals were divided into 9 treatment groups of three animals each(DFO 50, 100, and 200 mg/kg/day (subQ BID), ICL670 25, 50, and 100 mg/kg/day(PO QD), and L1 125, 250, and 500 mg/kg/day(PO TID), 5 days per week). Three control animals were sacrificed at 4 weeks and 8 weeks to estimate sponatenous iron loss. Histology and quantitative iron were performed in all animals. Results: Iron loading yielded liver iron concentrations of 26.6±3.8 mg/g(dry wt) and cardiac iron concentrations of 3.7±0.5 mg/g(dry wt) at 4 weeks (normal < .5 mg/g for both organs). However, organ iron content fell 6.4% in liver and 8.9% in heart per week in animals without chelation therapy, reflecting high spontaneous iron excretion. All three chelators exhibited significant dose-responsiveness for liver iron elimination. However, only ICL670 chelation at 100 mg/kg reduced liver iron content greater than for controls. In fact, animals treated with low dose L1 and DFO had higher iron levels than controls, probably by interfering with spontaneous iron elimination. None of the agents chelated the heart effectively. In fact, 88% of the L1 group, 56% of the ICL670 group and 22% of the DFO group had cardiac iron levels outside the normal range predicted from the 8 wk control animals. Conclusion: Iron chelation in the gerbil model requires doses nearly 3.6 fold greater than in humans to produce discernable iron loss above background iron excretion in short-term studies. Subtherapeutic dosing may actually increase iron levels relative to control animals by decreasing spontaneous iron excretion. Groupwise Iron Concentration and Content HIC(mg/g dry) HIC(mg/g wet) Organ FE(mg) CIC(mg/g dry) CIC(mg/g wet) Organ FE(mg) Control(4wk) 26.6±3.8 7.0±1.4 27.5±2.6 3.74±0.5 0.74±0.1 0.32±0.05 Control(8wk) 23.1±1.1 5.9±0.5 20.5±2.2 2.64±0.19 0.52±0.03 0.20±0.01 DFO 50mg/kg 31.0±3.0 8.2±1.5 28.9±3.4 2.73±0.32 0.56±0.03 0.20±0.02 DFO100mg/kg 25.3±3.3 6.8±1.2 25.0±4.9 3.20±0.46 0.90±0.46 0.33±0.18 DFO200mg/kg 23.5±1.4 5.9±0.4 17.6±2.4 2.77±0.20 0.53±0.07 0.18±0.03 L1 125mg/kg 32.2±1.3 7.7±1.1 23.8±3.4 3.63±0.25 0.79±0.02 0.23±0.02 L1 250mg/kg 29.3±7.4 8.5±2.7 26.7±6.2 3.56±0.85 0.71±0.12 0.21±0.04 L1 500mg/kg 18.5±0.9 5.0±0.6 19.4±1.8 2.68±0.43 0.57±0.08 0.20±0.04 ICL 25mg/kg 24.3±6.3 6.2±1.3 21.5±5.6 3.47±0.09 0.74±0.02 0.25±.02 ICL 50mg/kg 27.6±1.7 6.7±1.1 19.7±4.3 3.22±0.05 0.64±0.14 0.23±0.04 ICL100mg/kg 18.5±3.7 4.1±1.1 13.8±1.8 2.96±0.38 0.59±0.09 0.23±0.04

Iron Chelation Therapy with Deferasirox (Exjade®, ICL670) or Deferoxamine Is Effective in Reducing Iron Overload in Patients with Advanced Fibrosis and Cirrhosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2696-2696 ◽  
Author(s):  
E. Angelucci ◽  
B. Turlin ◽  
D. Canatan ◽  
A. Mangiagli ◽  
V. De Sanctis ◽  
...  
Keyword(s):  
Iron Overload ◽  
Serum Ferritin ◽  
Chelation Therapy ◽  
Iron Concentration ◽  
Iron Chelation ◽  
Iron Chelator ◽  
Advanced Fibrosis ◽  
Liver Iron ◽  
Once Daily ◽  
Tissue Iron

Abstract Introduction: Although the direct measurement of iron from a liver biopsy is the reference standard method to determine liver iron concentration (LIC), results are highly unreliable in patients with advanced fibrosis and cirrhosis. As a result, chelation therapy is difficult to monitor in this patient population where effective chelation therapy may be critical. It is therefore important to assess parameters additional to LIC in order to accurately assess body iron in these patients. Aim: To analyze the efficacy of chelation with deferoxamine (DFO) and the investigational once-daily, oral iron chelator deferasirox (DSX) in patients with advanced fibrosis participating in DSX registration studies. Methods: A subgroup of patients from DSX Studies 0107 and 0108 were selected based on a staging result according to the Ischak scale of 5 (incomplete cirrhosis) or 6 (probable or definite cirrhosis), measured either at baseline or after 1 year of chelation therapy. The subgroup of patients with β-thalassemia participating in Study 0107 received DSX (n=26) or DFO (n=30). In Study 0108, the subgroup of patients with β-thalassemia unable to be treated with DFO (n=12) or patients with anemias other than β-thalassemia (n=7) were treated with DSX only. In both studies, patients received chelation therapy according to baseline LIC. Results: In Study 0107, treatment with DSX or DFO led to a decrease in semi-quantitative tissue iron score (TIS) and LIC, which were paralleled by changes in serum ferritin. TIS, LIC and serum ferritin in a subgroup of patients with advanced fibrosis and cirrhosis treated with DSX and DFO (Study 0107) TIS LIC, mg Fe/g dw Serum ferritin, ng/mL DSX (n=26) DFO (n=30) DSX (n=26) DFO (n=30) DSX (n=26) DFO (n=30) *Median (min, max) Baseline* 35.5 (4,39) 34 (10,52) 25.5 (2.4,45.9) 19.5 (3.9,55.1) 4195 (321,12646) 4144 (653,15283) Change from baseline* −2 (−43,20) −2 (−25,16) −9.4 (−42.2,13.1) −3.1 (−24.5,12.4) −1269 (−7082,3609) −951 (−8259,1264 Similarly, in Study 0108, DSX treatment produced a decrease in all 3 parameters in patients with β-thalassemia or rare anemia. TIS, LIC and serum ferritin in a subgroup of β-thalassemia and rare anemia patients with advanced fibrosis and cirrhosis (Study 0108) TIS LIC, mg Fe/g dw Serum ferritin, ng/mL β-thalassemia (n=12) Rare anemia (n=7) β-thalassemia (n=12) Rare anemia (n=7) -thalassemia β (n=12) Rare anemia (n=7) *Median (min, max) Baseline* 35 (4,48) 41 (32,49) 29.4 (3.8,37.4) 26.3 (15,51.3) 4813 (440,11698) 2385 (1553,9099) Change from baseline* 2 (−19,27) −3 (−20,1) −1.6 (−18,9.9) −10 (−13.9,8.8) −986 (−4453,2131) −1322 (−2609,1901) Conclusions: Chelation therapy with DSX or DFO is effective in reducing iron overload in patients with advanced fibrosis and cirrhosis. The trends observed in TIS and LIC were closely mirrored by changes in serum ferritin, highlighting the validity of this method for monitoring chelation therapy in this population.

scholarly journals Intravenous Administration of Liposome Encapsulated DFO Efficiently Removes Iron from the Liver and Spleen of Iron Overloaded Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4815-4815
Author(s):  
David T Tran ◽  
Charles O Noble ◽  
Mark E Hayes ◽  
Francis C Szoka
Keyword(s):  
Iron Overload ◽  
The Body ◽  
Iron Accumulation ◽  
Molar Ratio ◽  
Iron Dextran ◽  
Employment Equity ◽  
Manufacturing Method ◽  
Excess Iron ◽  
Equity Ownership ◽  
Poor Outcomes

Abstract Introduction: Long-term red blood cell transfusions effectively sustains patients who have β-thalassemia, sickle cell anemia, and myelodysplastic syndromes but they also lead to excess iron accumulation in the body. Iron overload is a major cause of morbidity and mortality in transfusion dependent patients. Chelation therapy reverses iron accumulation but marketed chelators have drawbacks such as: long infusions of deferoxamine (DFO, Novartis), large oral tablets with adverse effects (Exjade, Novartis), or twice daily oral dosing (Ferriprox, ApoPharma). These attributes contribute to poor compliance and poor outcomes in iron overload patients. To overcome long infusions and high doses of current therapies we have devised a stable nanoliposome encapsulated DFO (LDFO) for the treatment of iron overload. Methods: LDFO composed of saturated soy phosphatidylcholine and cholesterol (3/2 molar ratio) is manufactured using a proprietary remote loading method that provides high encapsulation of DFO in 90 nm diameter liposomes. For pharmacokinetics and bioavailability studies, DFO and lipid concentrations in CF-1 mice plasma and tissues were analyzed by HPLC utilizing an in-house method. For iron removal efficacy studies, CF-1 mice were overloaded with iron dextran and after 10 days washout were treated with 100 mg/kg LDFO or unencapsulated DFO. Animals were sacrificed 5 days post treatment and tissue iron was measured by a ferrozine based spectroscopic assay. Results: The manufacturing method to prepare LDFO results in a 300 g DFO/mole lipid encapsulation ratio. The formulation has greater than 6 months stability at 4 ºC. LDFO is long circulating and the DFO is bioavailable. At 24 hr post I.V. injection, there is 30% ID DFO in plasma and 10% ID DFO/g in liver whereas unencapsulated DFO is not detectable. Preclinical single dose safety studies in CF-1 mice indicate that LDFO is well tolerated at 300 mg/kg I.V. and 1250 mg/kg I.P. In the iron dextran overload model, LDFO greatly reduces iron levels in the liver and spleen. The absolute efficiency of LDFO is greater than 50% on a mole LDFO injected /mole iron removed from the liver (P<0.0004, n=4) and spleen (P<0.01, n=4). This is corroborated by an elevated iron accumulation in urine and feces from LDFO. Conclusion: LDFO effectively removes iron from the liver and spleen with an overall molar efficiency > 50%. This high efficacy could lead to a dramatically improved treatment that increases compliance and provides substantially better management of iron overload than current treatments in patients suffering from iron overload conditions. Disclosures Tran: ZoneOne Pharma, Inc.: Employment. Noble:ZoneOne Pharma, Inc.: Employment, Equity Ownership. Hayes:ZoneOne Pharma, Inc.: Employment, Equity Ownership. Szoka:ZoneOne Pharma, Inc.: Consultancy, Equity Ownership.

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