Darbepoietin-alfa has comparable erythropoietic stimulatory effects to recombinant erythropoietin whilst preserving the bone marrow microenvironment

Abstract

Erythropoiesis stimulating agents are widely used for the treatment of anemia. Recently, we reported erythroid expansion with impaired B lymphopoiesis and loss of trabecular bone in C57BL/6 mice following ten days of treatment with low-dose short acting recombinant human erythropoietin. We have assessed erythropoietin against longer-acting darbepoietin-alfa at a comparable erythroid stimulatory dosage regime. Darbepoietin-alfa and erythropoietin induced similar in vivo erythropoietic expansion. Both agents induced an expansion of the colony-forming unit-erythroid populations. However, unlike erythropoietin, darbepoietin-alfa did not impair bone marrow B lymphopoiesis. Strikingly the bone loss observed with erythropoietin was not apparent following darbepoietin-alfa treatment. This analysis demonstrates that whilst darbepoietin-alfa has similar in vivo erythropoietic potency to erythropoietin, it preserves the bone marrow microenvironment. Thus erythropoietin and darbepoietin-alfa manifest different action showing that erythropoiesis stimulating agents have differential non-erythroid effects dependent on their duration of action.

Figure 1.

Darbepoietin-alfa has similar in vivo potency with unaffected B lymphopoiesis and lower extra-medullary stress erythropoiesis than recombinant human erythropoietin. (A) 9-week old C57Bl/6 mice were treated with once per week high-dose rhEpo or Darbepoietin-α and analyzed 10 days after the first injection (n=9 per treatment; 3 independent experiments of 3 per group). (B) Red blood cell count, (C) hemoglobin and hematocrit in PB 10 days post treatment are shown. (D) Representative FACS plots of erythroid fractions using CD71/Ter-119; (E) quantitation of erythroid differentiation in the bone marrow using CD71 and Ter119; (F–G) Analysis of myeloerythroid progenitor fractions based on CD105/CD150 staining as described by Pronk et al. (H) CFU-E and BFU-E in the bone marrow (n=4 per group). (I) Spleen weight and cellularity (n=13 per group). (J) Erythroid populations in spleen based on CD71/Ter-119 staining (n=9 per group). (K) and CFU-E and BFU-E numbers in the spleen (n=4 per group). Data are represented as mean±SEM. *P<0.05, **P<0.01.

Figure 2.

Largely unaffected B lymphopoiesis with Darbepoietin-α treatment. (A–B) B lymphopoiesis after rhEpo and Darbo treatment analyzed by B220 and IgM expression; (n=9 per group). (C–D) B-cell progenitor populations analyzed by FACS using CD43 and CD19 expression. Representative FACS plots and quantitation of Pre-ProB, Pro-B and Pre-B fractions are indicated. FACS data representative from 3 independent experiments. Data are represented as mean±SEM; (n=9 per group). *P<0.05, **P<0.01.

Figure 3.

Darbepoietin-alfa treatment does not cause bone loss compared to rhEpo. Three-dimensional μCT analysis of the secondary spongiosa of proximal tibia. (A) Quantitation of total bone volume (Bone Volume/Total Volume; %), (B) trabecular number (number of trabecular per mm) and (C) the distance between the trabeculae (trabecular separation; μm) from PBS – Epo- and Darbo-treated mice respectively (n=9 per group; 3 independent experiments of 3 per group). Data represented as mean±SEM. (D–F) Representative images (CTvol) of the trabecular bone within the secondary spongiosa and color-coded quantitative mineralization paraview images of trabecular bone in each treatment category. Red indicates areas of lowest mineral density and blue represents regions of highest mineral density. *P<0.05, **P<0.01.