Daprodustat Induces Fetal Hemoglobin and Erythropoietic Drive in an in Vitro and In Vivo Model of Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder that results in chronic anemia and painful vaso-occlusive episodes. Chronic anemia increases the risk for development of chronic kidney disease, pulmonary hypertension, and stroke in SCD. The current standard of care therapy, hydroxyurea (HU), inhibits polymerization of abnormal hemoglobin S (HbS) by inducing fetal hemoglobin (HbF) expression, preventing vaso-occlusive complications. However, HU only modestly improves anemia, which can persist due to inadequate erythropoietic (EPO) production. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a class of orally bioavailable drug that stimulate erythropoiesis by inhibiting degradation of HIF1/2α, increasing EPO expression. In addition, the HIF-PHI roxadustat (roxa) has been shown to induce HbF expression in CD34⁺ hematopoietic stem and progenitor cells (HPSCs) in vitro, suggesting multiple therapeutic benefits in SCD. Daprodustat (dapro) is the first FDA-approved HIF-PHI for dialysis-dependent chronic kidney disease. In this study, we sought to evaluate the HbF-inducing and erythropoietic potential of daprodustat in both a CD34⁺ HSPC in vitro model and an in vivo mouse model of SCD.

CD34⁺ HSPCs were isolated from 3 patients with SCD, differentiated in vitro to erythroblasts, and treated with vehicle, 30μM dapro, or 50μM of HU. Flow cytometric analysis on Day 15 (D15) showed that dapro treatment increased % F-cells by 2-fold (p<0.05) compared to vehicle, similar to the effect of roxa on HbF induction (Feng et al., Nature 2022; Sharma et al., ASH abstract 2022). HU treatment increased % F-cells by 3-fold (p<0.001) compared to vehicle.

We then evaluated the effect of dapro in an in vivo SCD model using male 10 wk old C57BL/6J mice transplanted with bone marrow from transgenic SCD Townes mice (Jackson Laboratory). Engrafted mice were treated by oral gavage daily for 3 weeks with either vehicle (10%DMSO;80%PEG;10%saline) (n=4) or 30 mg/kg dapro (n=5). All mice underwent retro-orbital bleeding at baseline (24 hours pretreatment), D10 and D21 for hematological evaluation. Reticulocyte% was also determined by flow cytometry at D21.

At D10, the increase in Hb concentration compared to baseline was higher in the dapro vs. vehicle treated mice (44% vs. 2%, p<0.05). This difference persisted at D21, with an increase in Hb of 50% in the dapro group and a decrease of 30% in vehicle group (p<0.01). Similar trends were observed in Hct and RBC count. Interestingly, at D21, reticulocyte% was lower in the dapro group vs. vehicle group (39% vs. 55%, p<0.01). RBC indices (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC)) decreased earlier in the treatment course and to a greater extent in the dapro vs. vehicle treated mice, but these differences were not significant. Additionally, by D21, there was a significantly greater increase in platelet count in the dapro treated group vs. the vehicle treated group (75% vs. -20%, p<0.001). White blood cell (WBC) count decreased in both groups; the difference was not significant.

Our study demonstrates that dapro has the potential to both induce HbF and erythropoietic drive in preclinical models of SCD. Despite the significant increase in Hb, Hct and RBC count, reticulocyte% was lower in the dapro vs. vehicle treated group at D21, suggesting that dapro may influence Hb through multiple mechanisms, such as decreasing hemolysis through HbF induction or decreasing stress erythropoiesis by correcting anemia. Notably, there was a decrease in Hb, Hct, MCV, MCH, and MCHC in the vehicle group at D21, concerning for development of iron deficiency anemia. There was a similar decrease in MCV, MCH, and MCHC at D21 in the dapro group, in line with the hypothesized vehicle-induced iron deficiency, but dapro treatment may have protected against the development of anemia from iron deficiency. Finally, we saw a decrease in all three cell lines (WBC, RBC, and platelets) in the vehicle group at D21, concerning for bone marrow toxicity from the vehicle, suggesting that the erythropoietic effect of dapro treatment may have been even greater without this confounding effect. In summary, daprodustat proves to be a promising candidate for treatment of anemia in SCD, and the long-term effects of daprodustat on erythropoiesis, HbF induction, and iron metabolism in SCD should be further evaluated.