Abstract
Introduction
Beta-thalassemia is a inherited disorder characterized by mutations in the beta-globin gene that lead to reduced or absent beta-globin synthesis. As a consequence, in erythroid cells there is a relative excess of alpha globin chains and heme forming toxic hemichromes, which lead to oxidative damage, impaired erythroid cell maturation, premature cell death and anemia. Under these conditions, the anemia and the elevated erythropoietin levels increase the proliferation of the erythroid progenitors, suppressing the expression of the iron master regulator hepcidin. Hepcidin targets the iron exporter ferroportin present on enterocytes and macrophages. High levels of hepcidin limit iron absorption and iron recycling but low levels of hepcidin increase iron absorption. Chronic low levels of hepcidin, as in β-thalassemia, eventually lead to iron overload. The th3/+ mouse model of thalassemia intermedia (Non-Transfusion Dependent Thalassemia or NTDT), reproduces the main features of the disease including anemia, increased erythropoiesis, splenomegaly and iron overload due to low levels of hepcidin. It has been previously demonstrated that modestly increasing hepcidin levels in these mice ameliorates both anemia and iron overload (Gardenghi et al, JCI, 2010).
Methods
We used minihepcidin M004, a modified peptide analog of the 9 N-terminal amino acids of hepcidin, to reproduce the biological effects of hepcidin. In a dose-ranging study, we treated th3/+ animals with daily intraperitoneal (ip) injections of M004 at doses of 6.25-100 µg/day for 2-6 weeks or with subcutaneous (sc) dosing of 52.5 μg twice a week for 6 weeks.
Results
In animals treated with the lower ip doses of M004 (6.25-25 μg/day for 2-4 weeks), mild iron restriction was observed (Tf sat ≥15%). Flow cytometry studies using the markers CD44 and Ter119 demonstrated an increase in the proportion of mature erythroid cells. Consistent with the flow cytometry findings, a 30% increase in peripheral red cells (rbc) was observed associated with a reduction in reticulocyte count (>62%) and a reduction in spleen size (>40%), reflecting improved erythropoietic efficiency. At the 12.5 and 25 µg doses, Hb level increased by 1.8-2.5 g/dL. Longer treatment (6 weeks) with 12.5-25 µg/day led to greater iron restriction and the loss of therapeutic effect at 6 weeks. In the same animals, measurement of total organ iron concentrations demonstrated no changes in the liver and kidney, but a 4-fold iron increase in the spleen. Prussian blue staining confirmed increased sequestration of iron in splenic macrophages and Kupffer cells.
At high ip doses of M004 (100 μg/day for 2 weeks) the mice developed severe iron restriction (Tf sat = 6%) and exacerbation of anemia (Hb reduction of 4.5 g/dL). Accumulation of immature erythroid precursors was observed by flow cytometry studies, reflecting the erythroid maturation block caused by lack of iron for hemoglobin generation.
Subsequent pharmacokinetic studies in rats indicated that plasma levels of M004 increased gradually with sc dosing and were still increasing 24h after dosing, indicating accumulation of drug levels and iron restriction with daily administration. We therefore explored whether less frequent dosing may be preferable to achieve sustained improvement in anemia and normalization of tissue iron. Sc dosing of M004 at 52.5 µg twice weekly was associated with an increase in rbc at 6 weeks to levels observed in WT animals, and an Hb increase of almost 3 g/dL. Reduced spleen weight (-51%) and reticulocyte count (-61%) reflected improved erythropoietic efficiency, further corroborated by flow cytometry studies. Reduction in hemichrome and ROS formation coupled with improvement in rbc morphology and reduced RDW indicated that hematological improvement was likely caused by improved erythroid cell survival as a result of reduced oxidative damage secondary to decreased erythroid iron uptake. Evaluation of total organ iron concentrations showed a reduction of 77% and 54% in the liver and kidney, respectively. Remarkably, the liver iron values were normalized compared to WT animals. Splenic iron increased only ∼1.5 times or 148%. However, when the total weight of the spleen was taken in account, the total splenic iron was reduced (-39%).
Conclusion
Minihepcidin peptides may be beneficial for the treatment of beta thalassemia intermedia by improving anemia and reducing tissue iron burden.
Disclosures:
Casu: Merganser: Research Funding. Goldberg:Merganser: Research Funding. Nemeth:Merganser: Equity Ownership. Ganz:Merganser: Equity Ownership. MacDonald:Merganser : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Rivella:Alexion: Consultancy; Biomarin: Consultancy; Merganser: Equity Ownership, Research Funding; Isis: Consultancy, Research Funding; Bayer: Consultancy; Novartis: Consultancy; Imago: Consultancy.
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