The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease

Abstract

Sickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the β-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Graphical abstract

Figure 1.

Vamifeport induced iron-restricted erythropoiesis with reduced hemoglobin (Hb) concentration in RBCs of HbSS mice. (A) Administration of vamifeport (60 mg/kg) twice daily (BID) for 6 weeks in 6- to 7-week-old HbSS mice resulted in iron-restricted erythropoiesis, as shown by decreased CHCM, mean corpuscular hemoglobin (MCH), MCV, hematocrit, and total Hb without lowering RBC counts. (B) Likewise, reticulocyte counts remained stable with marked reductions in reticulocyte Hb and spleen size. (C) RBC scatterplots revealed a significant increase in the proportion of hypochromic and microcytic RBCs after vamifeport treatment. Results are presented as individual values with mean ± standard deviation (n = 6-8 mice per group). Analysis was performed by using one-way analysis of variance with Dunnett’s multiple comparison of all groups vs the HbSS vehicle group: *P < .05, **P < .01, ***P < .001.

Figure 2.

Vamifeport reduced hemolysis in HbSS mice. Vamifeport significantly reduced plasma markers of hemolysis in HbSS mice, including heme (A), lactate dehydrogenase (LDH) (B), and indirect bilirubin (C). (D) Erythrophagocytosis was reduced in HbSS mice treated with vamifeport, as shown by percentages of Ter119⁺ red pulp macrophages, identified by gating on CD45⁺F4/80⁺ spleen cells. (E) Total iron deposition in the kidneys of HbSS mice was reduced by vamifeport, as measured by inductively coupled plasma optical emission spectrometry. (F) Reduction of total iron deposition in the kidneys of HbSS mice by vamifeport was also illustrated by Perls staining of kidney sections. Results are presented as individual values with mean ± standard deviation (n = 5-8 mice per group). Analysis was performed by using one-way analysis of variance with Dunnett’s multiple comparison of all groups vs the HbSS vehicle group: *P < .05, **P < .01, ***P < .001. Abs, absorbance; BID, twice daily.

Figure 3.

Vamifeport decreased PS exposure and improved mitochondria clearance in mature RBCs of HbSS mice. (A) Vamifeport lowered the exposure of PS on peripheral blood RBCs, as detected by a decreased percentage of Annexin V–positive cells and decreased intensity of Annexin V staining. (B) Although almost all reticulocytes of vehicle- and vamifeport-treated HbSS mice contained mitochondria (left), treatment with vamifeport reduced the occurrence of mitochondria in mature RBCs (right). Results are presented as individual values with mean ± standard deviation (n = 7-10 mice per group). Analysis was performed by using one-way analysis of variance with Dunnett’s multiple comparison of all groups vs the HbSS vehicle group: *P < .05, ***P < .001. BID, twice daily; MFI, mean fluorescent intensity.

Figure 4.

Vamifeport reduced systemic and vascular inflammation, and oxidative stress, in HbSS mice. Vamifeport significantly reduced inflammatory markers in HbSS mice: total leukocyte, neutrophil, and lymphocyte counts (A); plasma levels of the chemokine RANTES (B); expression of the neutrophil chemoattractant Cxcl1, as determined by quantitative polymerase chain reaction in liver (C); sVCAM-1 (D); soluble P-selectin (E); and plasma XO activity (F). Results are presented as individual values with mean ± standard deviation (n = 6-8 mice per group). Analysis was performed by using one-way analysis of variance with Dunnett’s multiple comparison of all groups vs the HbSS vehicle group: *P < .05, **P < .01, ***P < .001. BID, twice daily.

Figure 5.

Vamifeport prevented organ iron loading in the Townes model of SCD (HbSS mice). Mice received water supplemented with the stable iron isotope ⁵⁸Fe to distinguish iron acquired during the treatment period and iron already present in organs. ⁵⁸Fe concentrations in the liver and spleen were measured by using inductively coupled plasma mass spectroscopy. (A) Vamifeport significantly reduced serum iron levels 3 hours postdose. Vamifeport prevented ⁵⁸Fe loading of livers (B) and spleens (C) of HbSS mice. (D) Vamifeport corrected total iron content in HbSS RBCs to the levels of HbAA mice. Results are presented as individual values with mean ± standard deviation (n = 7-10 mice per group). Analysis was performed by using one-way analysis of variance with Dunnett’s multiple comparison of all groups vs the HbSS vehicle group: ***P < .001. BID, twice daily.

Figure 6.

Systemic iron restriction by vamifeport, but not dietary iron restriction, reduced hemolysis and inflammation in HbSS mice. Treatment with vamifeport, but not dietary iron restriction (LID), increased the proportion of circulating hypochromic and microcytic RBCs and reduced CHCM (A) and decreased indirect bilirubin in plasma (B), compared with HbSS mice treated with vehicle. Vamifeport, but not LID, prevented the release of sVCAM-1 into the circulation of HbSS mice (C) and reduced plasma concentrations of RANTES (D). (E) Circulating leukocyte, neutrophil, and lymphocyte numbers were normalized in HbSS mice receiving vamifeport but remained elevated in HbSS mice on LID. (F) Vamifeport, but not LID, decreased serum iron levels. LID, but not vamifeport, efficiently depleted iron from the liver (G), whereas both treatments decreased the expression of liver Hamp (H). Results are presented as individual values with mean ± standard deviation (n = 10-12 mice per group). Analysis was performed by using one-way analysis of variance with Dunnett’s multiple comparison of all groups vs the HbSS vehicle group: *P < .05, **P < .01, ***P < .001. BID, twice daily; QD, once daily.

Figure 7.

Vamifeport improved deformability of RBCs in hypoxia, reduced the adhesion of blood cells to microvasculature, and prevented inflammation-triggered vaso-occlusion in HbSS mice. (A) No change in the point of sickling of RBCs in HbSS mice; measurement of point of sickling is not applicable (n.a.) to HbAA mice. Reduced severity of sickling shown as a decrease in ΔEI (B) and lowered EI at minimal oxygenation (EI_min) (C) with a minor change in the EI at maximal oxygenation (EI_max) (D). (E and F) Scanning electron microscopy analysis revealed RBCs with improved membrane morphology in HbSS mice treated with vamifeport compared with vehicle. Representative intravital microscopy images showing marked blood stasis (vaso-occlusion) in capillaries of vehicle-treated Townes mice (arrows show firmly adherent cells) (G), reduced blood cell adhesion in capillaries of vamifeport-treated HbSS mice (H), the number of adherent blood cells (I), and the percentage of vessels with normal blood flow (J). In average, 12 to 35 vessels per mouse in each group were analyzed. Results are presented as mean ± standard deviation (n = 4-7 mice per group). Analysis was performed by using one-way analysis of variance with Dunnett’s multiple comparison of all groups vs the HbSS vehicle group: *P < .05, **P < .01, ***P < .001. a.u., arbitrary units; BID, twice daily; FU, fluorescence units.