Abstract
Individuals affected by non-transfusion-dependent thalassemia (NTDT) develop severe ineffective erythropoiesis that causes a number of serious clinical morbidities, such as chronic anemia, splenomegaly and systemic iron overload requiring chelation therapy. In NTDT, low hepcidin levels caused by ineffective erythropoiesis result in increased iron absorption which may in turn exacerbate erythroid cell damage, apoptosis and ineffective erythropoiesis. Minihepcidins (MH) are short peptide mimetics that reproduce the iron restrictive effects of hepcidin. Using a mouse model of NTDT (Hbbth3/+), we previously showed that MH-induced iron restriction significantly reduced erythroid cell damage, leading to reduced ineffective erythropoiesis and improved anemia. Accumulation of tissue iron was significantly diminished in MH treated animals. In clinical settings, use of MH for the treatment of NTDT would likely be concomitant with oral chelation therapy. Therefore we conducted studies in the (Hbbth3/+) mouse to evaluate whether the concurrent use of the oral iron chelator deferiprone (DFP) and MH would alter a) the hematological benefit of MH or b) the tissue iron reduction benefit of DFP. MH may improve the efficacy of DFP by reducing dietary iron absorption, thus increasing net reduction in tissue iron.
Studies were performed in six week old mice using MH M004, which reduces cell surface expression of ferroportin with an EC50of 9.7 nM and causes >80% reduction in serum iron at a dose of 7.5 mg/kg in the rat. M004 (2.65 mg/kg) or vehicle control was administered twice weekly by subcutaneous injection for six weeks. Half of the mice in each group had free access to water containing DFP (1.25 mg/mL).
Treatment with MH alone produced a profile of hematological changes resulting in a significant increase in circulating hemoglobin of 1.6 g/dL. Flow cytometry studies of bone marrow erythroid populations from MH-treated animals demonstrated an increase in the relative proportion of mature erythroid cells, reduced apoptosis and reduced levels of ROS. Parameters of erythrocyte damage (red cell distribution width, red cell morphology, red cell survival time) were improved and an increase in peripheral red cell number was observed. Reticulocyte count and spleen size were both reduced, reflecting improved erythropoietic efficiency. In separate studies in Hbbth3/+ mice that also expressed a hypoxia induced luciferase-reporter gene, treatment with MH resulted in reduction of tissue hypoxia. Mice treated with DFP alone showed no hematologic improvement compared to vehicle control whereas mice treated with MH and DFP together showed similar hematologic benefit as mice treated with MH alone (Hb increase of 2.3 g/dL versus control).
Both DFP and MH administered separately caused a reduction in total liver iron compared to vehicle controls but the different was only statistically significant in MH treated animals. When administered concurrently, the combination of DFP and MH caused a further non-significant reduction compared to either agent alone. Although spleen iron/g wet weight was increased by MH, reflecting the iron-sequestering effect of hepcidin, total spleen iron was not significantly increased because spleen size was also reduced. DFP was equally effective in reducing spleen iron/g wet weight with or without concurrent MH treatment. However total spleen iron was non-significantly reduced (-35%) in DPF-treated mice compared to vehicle controls (1,435 ± 627 µg (mean ± SEM) vs 2,210 ± 213 µg) but a larger, statistically significant reduction in spleen iron was observed in MH + DFP treated mice compared to MH alone (-50%, 1,258 ± 414 vs 2,531 ± 246, p<0.05).
In conclusion, the beneficial hematological effects of MH treatment in this model of NTDT remained even during concurrent treatment with DFP. DFP had no hematological benefits when used as a single agent, reflecting the inability of iron chelators to cause clinically meaningful iron restriction. Reduction in tissue iron by DFP was either unaffected or enhanced by concurrent treatment with MH. The improved effect of DFP on spleen iron burden in the presence of MH may reflect the complementary effects of these agents to increase iron excretion and reduce dietary iron absorption respectively. Enhancement of the clinical efficacy of iron chelators may be an important benefit of MH therapy in addition to the observed increase in circulating hemoglobin.
Disclosures
Casu: Isis Pharmaceuticals, Inc.: Employment; Merganser Biotech LLC: Employment. Nemeth:Merganser Biotech LLC: Stockholders Other. Ganz:Merganser Biotech LLC: Stockholders Other. MacDonald:Merganser Biotech LLC: Employment, Equity Ownership. Rivella:bayer: Consultancy, Research Funding; merganser Biotech LLC: Consultancy, Research Funding, Stock options, Stock options Other; isis Pharmaceuticals, Inc.: Consultancy, Research Funding.
Correcting β-thalassemia by combined therapies that restrict iron and modulate erythropoietin activity
