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

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.