Continuous Erythropoietin Receptor Activator Enhances Iron Metabolism by Hepcidin Down-Regulation and Maintains Stable Hemoglobin Levels by Controlling Maturation Rate of Erythroid-Lineage Cells

Continuous Erythropoietin Receptor Activator (C.E.R.A.) is a chemically synthesized erythropoiesis-stimulating agents (ESA) and differs from epoetin-beta (EPO) through the integration of an amide bond between an amino group of EPO and a specific, linear methoxy polyethylene glycol. Hematopoietic progenitor cells are known to show dynamic changes after ESA administration through erythropoietin signaling. It is also well known that C.E.R.A. increases the number of red blood cells and hemoglobin (Hb) levels and provides stable control of Hb levels in clinical and nonclinical studies, but the effects of C.E.R.A. on the maturation of erythroid-lineage cells and iron metabolism are still unclear. In the present study, we analyzed these effects to extract features of C.E.R.A. against EPO, existing ESA.

C.E.R.A. was administered intravenously into male 8-weeks old C57BL/6 mice at 2 (C2) or 10 (C10) μg/kg in a single injection, and EPO was also administrated intravenously into the same conditioned mice at 0.8 (E0.8) or 2 (E2) μg/kg with 5 day continuous injection. Mice sera and bone marrow cells were collected on 0, 2, 5, 8, 11, and 14 days after first administration. Hematological parameters and serum iron concentrations were analyzed, and mouse serum hepcidin concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. We also analyzed the maturation of erythroid-lineage cells in bone marrow by flow cytometry technique, staining TER-119, transferring receptor (CD71), 7-Amino-Actinomycin D (7-AAD) and annexin V.

While Hb levels reached a maximum level on 5^th day after first administration and then decreased in C2, E0.8 and E2 groups, high Hb levels were maintained after 5^th day in C10 group. In this C10 group, mouse serum hepcidin concentrations were effectively down-regulated until 8^th day after first administration, and serum iron concentrations also dropped to the lowest level on 5^th day and then recovered. Interestingly, relatively-small erythroid-lineage cells were detected according to forward scatter (FSC) data by flow cytometry on 5^th day in C10, which reflects transient iron-insufficient condition. TER-119 and CD71 were expressed on the surface of these small cells, and 7-AAD and annexin V were not. The percent of these TER-119(+) and CD71(+) cells which are at intermediate stage of erythroid-lineage cells maturation was increased and maintained continually in C10 group. Additionally, the percent of TER-119(+) and CD71 (−) cells which are at late stage of erythroid-lineage cells maturation in bone marrow was elevated sustainably and moderately.

These results strongly indicate that C.E.R.A. maintains stable Hb levels by enhancing iron metabolism and controlling maturation rate of erythroid-lineage cells with fewer administrations than EPO. Our previous study showed that EPO treatment mainly down-regulated serum hepcidin concentrations by indirect mechanisms through the involvement of bone marrow cells. Furthermore the present study suggests that C.E.R.A. down-regulates serum hepcidin concentrations more effectively than EPO, and promotes more effective use of iron storage for hematopoiesis. It is revealed that C.E.R.A. controls maturation rate of erythroid-lineage cells in association with enhancement of iron metabolism and provides stable control of Hb levels. It is possible to functional iron-insufficient state undermines the efficacy of C.E.R.A. treatment, but further investigation is needed.

No relevant conflicts of interest to declare.