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

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.