C.E.R.A. Acts Differently at the Erythropoietin (EPO) Receptor Compared with Epoetin Beta: UT-7 and CD34+ Cell Stimulation Assays.

Continuous Erythropoietin Receptor Activator (C.E.R.A.), an innovative erythropoietic agent with unique receptor activity, is currently in development to provide correction of anemia and stable maintenance of hemoglobin (Hb) levels at extended administration intervals up to once monthly in patients with all stages of chronic kidney disease (CKD), and is also in development for the treatment of chemotherapy-induced anemia. In vitro studies show that C.E.R.A. has a 45-fold lower affinity for the EPO receptor than epoetin beta, due mainly to a reduced association rate. To further investigate the in vitro activity of C.E.R.A., two cell stimulation studies were undertaken. Study 1 evaluated an assay for the analysis of the molecular mechanism of C.E.R.A.- and epoetin beta-mediated cell activation. UT-7 cells were activated with C.E.R.A. or epoetin beta for 72 h or 96 h, followed by WST (tetrazolium salt) staining and spectrophotometric detection. UT-7 is a human myeloid leukemia cell line expressing the EPO receptor, and has growth dependency on EPO if no other growth factors are present. Results showed that the EC⁵⁰ (concentration giving half maximal stimulation) value was approximately 10-fold higher for C.E.R.A. (range 300–400 pM) than for epoetin beta (30–60 pM). Maximal activation of UT-7 cells was achieved at C.E.R.A. 1000–2000 pM and epoetin beta 100–200 pM, but the maximal stimulation of cells was similar for both agents. Study 2 investigated the effects of C.E.R.A. and epoetin beta on stimulation of the proliferation and differentiation of human CD34+ cells. Human CD34+ stem cells from cord blood and bone marrow were cultivated with C.E.R.A. or epoetin beta for 8–14 days. After labeling, using fluorescence-tagged antibodies to proteins specific for erythroid cells (glycophorin A) and other blood cell types (CD13, CD14, CD16, CD41, CD42b, and CD61), cells were analyzed using three-color flow cytometry with a FACScan instrument (Becton Dickinson, CA). The maximal number of glycophorin A positive cells at plateau phase was used for EC₅₀ calculation. Following stimulation of CD34+ cells, glycophorin A+ cells increased to a similar level with C.E.R.A. and epoetin beta. This stimulation was specific for erythroid precursors since the differentiation of white blood cells and megakaryocytes was not affected by C.E.R.A. or epoetin beta. Notably, mean EC₅₀ values were 43.4-fold higher with C.E.R.A. (2.807 nM) than with epoetin beta (0.076 nM). In conclusion, C.E.R.A. and epoetin beta activate UT-7 cells and induce differentiation and expansion of CD34+ cells. These studies provide further evidence that C.E.R.A. has different EPO receptor binding properties compared with epoetin beta and demonstrate its specificity for the red blood cell line. Preclinical studies have shown that these properties translate into more continuous stimulation of erythropoiesis in vivo compared with epoetin beta and Phase III data indicate that C.E.R.A. achieved and maintained stable Hb levels in all patients with CKD.