P1450: voxelotor treatment leads to improved viability of sickle erythrocytes

Topic: 26. Sickle cell disease Background: Sickle cell disease (SCD) is characterized by chronic haemolysis, contributing to vaso-occlusive events and organ damage. Voxelotor, a small molecule reversibly binding to haemoglobin (Hb) and increases its oxygen affinity, decreasing sickle haemoglobin polymerization and sickling, reducing haemolysis. The decrease in erythrocyte sickling might improve erythrocyte deformability and decreased adhesion, while reductions in cell-free haem and labile plasma iron levels might result in decreased inflammation and decreased neutrophil activation, adhesion and reactive oxygen species (ROS) production. Aims: The aim of this study is to assess the effects of voxelotor on erythrocyte deformability and adhesion and neutrophil activation, adhesion and aging and neutrophil ROS production. Methods: SCD patients with severe genotypes (HbSS and HbSβ0-thalassemia) and Hb ≤ 10.5 g/dL were treated with voxelotor 1500 mg daily in a single arm interventional trial in a tertiary teaching hospital. Blood was collected at baseline and after 3 months of treatment. Erythrocyte deformability was assessed by ektacytometry (Lorrca, RR mechatronics, The Netherlands) by measuring elongation index (EI) and point of sickling (PoS). EI max and EI min indicate the highest and lowest erythrocyte deformability upon oxygenation and deoxygenation respectively. PoS is the oxygen tension at which 5% reduction of the EI max is observed. Adhesion of RBCs was measured by flowing whole blood (20x106/ml) over laminin-coated Ibidi slides for 30 minutes with visualization by microscope. Percentage of mean RBC adhesion was normalized to the healthy day control. Antigen expression of neutrophil markers specific for endothelial activation, adhesion and aging were assessed with flow cytometry. ROS production of neutrophils was measured by an Amplex Red Assay (Molecular probes), measuringaH2O2 production under both unstimulated and stimulated conditions with various simulating agents (Figure 1D). Data are presented as medians with interquartile range (IQR). Results: Results of 15 SCD patients (HbSS/HbSβ0-thalassemia (11/4), median age 35 years (IQR 27-46), 5 female) who completed 3 month follow-up are described. Haemoglobin levels increased significantly (from 7.4 (6.9 – 8.1) g/dL to 9.7 (7.9 – 11) g/dL; p=0.002), corresponding to significant decreases in LDH, bilirubin and reticulocyte counts as markers of haemolysis (p<0.05). PoS decreased significantly (from 20.1 (17.5 – 26.1) mmHg to 16.1 (10.3 – 18.1) mmHg, p=0.006; Table 1). EI max and EI min increased (from 0.33 (0.24 – 0.42) to 0.38 (0.28 – 0.48); p=0.029 and from 0.13 (0.07 – 0.26) to 0.21 (0.16 – 0.41); p=0.007 respectively). Erythrocyte adhesion was not different (data not shown). Markers of neutrophil activation, adhesion and aging were generally lower, though the differences were not statistically significant (Figure 1A-C). While neutrophil ROS production under unstimulated conditions increased (p=0.046), it remained unchanged under stimulated conditions (Figure 1D). Summary/Conclusion: Voxelotor significantly improved erythrocyte deformability as suggested by decreased PoS and increased EI max and EI min. However, this did not translate into reduced in vitro adhesion of sickle erythrocytes. While markers of neutrophil adhesion seem to decrease with voxelotor treatment, the differences are not statistically significant, possibly due to the relatively small sample size. Interestingly, neutrophil ROS production under unstimulated condition was significantly higher after treatment than at baseline.Keywords: Sickle cell anemia, Neutrophil, Sickle cell disease, Sickle cell adhesion