The Oral Ferroportin Inhibitor VIT-2763 Improves Erythropoiesis without Interfering with Iron Chelation Therapy in a Mouse Model of β-Thalassemia

Abstract

In β-thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbb^th3/+ mouse model of β-thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbb^th3/+ mouse model of β-thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbb^th3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in β-thalassemia.

Keywords: VIT-2763; chelation; ferroportin inhibitor; ineffective erythropoiesis; iron; thalassemia.

Figure 1

VIT-2763 did not impair the ability of deferasirox (DFX) to reduce iron overload in Hbb^th3/+ mice. (a) The combination of VIT-2763 with DFX resulted in a similar reduction in the liver iron concentration in Hbb^th3/+ mice as DFX treatment alone. (b) Improved kidney iron reduction by the combination of VIT-2763 and DFX. (c) Serum iron levels in mice receiving VIT-2763 alone or in combination with DFX. (d) Transferrin saturation (TSAT) levels in mice treated with VIT-2763 alone or in combination with DFX. Results represent the mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test. * p < 0.05 and *** p < 0.001. # p < 0.05 depicts a comparison to the group treated with the combination of VIT-2763 and DFX. Wild-type (WT) littermates received both vehicles and were included as controls. n = 10–13 mice per group.

Figure 2

The combination of VIT-2763 and DFX ameliorated anemia and ineffective erythropoiesis in Hbb^th3/+ mice. VIT-2763 alone or in combination with DFX significantly increased hemoglobin levels (a), red blood cell (RBC) counts (b), hematocrit (c), and mean corpuscular hemoglobin concentration (MCHC) (d), and significantly reduced mean corpuscular volume (MCV) (e), mean corpuscular hemoglobin (MCH) (f), and reticulocyte counts (g). DFX alone had no effect. Results represent the mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test. * p < 0.05, ** p < 0.01, and *** p < 0.001. Wild-type (WT) littermates received both vehicles and were included as controls. n = 10–13 mice per group.

Figure 3

VIT-2763 alone or in combination with DFX improved erythropoiesis in Hbb^th3/+ mice. (a) Splenomegaly was significantly reduced in Hbb^th3/+ mice treated with VIT-2763 alone or in combination with DFX compared to Hbb^th3/+ mice treated with vehicle or DFX alone. (b) Elevated erythropoietin (EPO) levels were significantly corrected in mice treated with VIT-2763 alone or in combination with DFX. (c–e) VIT-2763 decreased the frequency of polychromatic erythroblasts (c,e populations in gate 3) and increased the proportion of mature erythrocytes (d,e populations in gate 5) in the spleen. Representative dot plots are shown in (e). Results represent the mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test. ** p < 0.01, and *** p < 0.001. Wild-type (WT) littermates received both vehicles and were included as controls. (a,b) n = 10–13 mice per group; (c–e) n = 5–6 mice per group.

Figure 4

VIT-2763 alone or in combination with DFX improved the functional parameters of RBCs in Hbb^th3/+ mice. RBCs were gated as mature RBCs (Ter119^hiCD71^neg) and analyzed for reactive oxygen species (ROS) (CM-H₂DCFDA), apoptosis (annexin V as staining for phosphatidylserine (PS)), and retention of mitochondria (MitoTracker Deep Red). VIT-2763 alone or in combination with DFX reduced the percentage of ROS-positive RBCs (a), lowered PS exposure on peripheral RBCs (b), and improved the elimination of mitochondria in RBCs (c) of Hbb^th3/+ mice compared to Hbb^th3/+ mice treated with the vehicle or DFX alone. Results represent the mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test. * p < 0.05, ** p < 0.01, and *** p < 0.001. Wild-type (WT) littermates received both vehicles and were included as controls. n = 9–13 mice per group.

Figure 5

VIT-2763 alone or in combination with DFX corrected pathologically elevated neutrophil counts in Hbb^th3/+ mice. VIT-2763 alone or in combination with DFX significantly decreased total leukocyte (a), neutrophil (b), and lymphocyte (c) counts, while having no effect on monocytes (d). Results represent the mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test. and *** p < 0.001. Wild-type (WT) littermates received both vehicles and were included as controls. n = 10–13 mice per group.