Improvements in haemolysis and indicators of erythrocyte survival do not correlate with acute vaso-occlusive crises in patients with sickle cell disease: a phase III randomized, placebo-controlled, double-blind study of the Gardos channel blocker senicapoc (ICA-17043).

Red blood cell (RBC) hydration is regulated in part by the Ca(2+) -activated K(+) efflux (Gardos) channel. Senicapoc selectively blocks potassium efflux through the Gardos channel, reducing RBC dehydration and haemolysis, and increasing haemoglobin levels in sickle cell disease (SCD). This randomized, placebo-controlled trial was designed to determine the safety and clinical efficacy of senicapoc in SCD patients. One hundred and forty-five patients were randomized to receive senicapoc and 144 patients to receive placebo for 52 weeks. Consistent with a previous study, patients in the senicapoc group had significantly increased haematocrit, haemoglobin, and decreased numbers of both dense erythrocytes and reticulocytes when compared to the placebo group. The unblinded Data Monitoring Committee terminated this study early due to a lack of efficacy when it determined that, despite improvements in anaemia and haemolysis, no significant improvement in the rate of sickle cell painful crises was observed in patients treated with senicapoc compared to those on placebo (0·38 vs. 0·31, respectively). Comparisons of the times to first, second and third crises between the senicapoc and placebo groups were not statistically significant. Nausea and urinary tract infections occurred more frequently in the senicapoc group than placebo. Serious adverse events were similar in the two groups.

The effect of fetal hemoglobin on the survival characteristics of sickle cells.

The determinants of sickle red blood cell (RBC) life span have not been well-defined but may include both intrinsic factors (eg, the tendency to sickle) and extrinsic factors (eg, the capacity of the reticuloendothelial system to remove defective RBCs). Fetal hemoglobin (HbF) is heterogeneously distributed among sickle RBCs; F cells contain 20% to 25% HbF, whereas the remainder have no detectable HbF (non-F cells). Autologous sickle RBCs were labeled with biotin and reinfused to determine overall survival, non-F- and F-cell survival, and time-dependent changes in HbF content (%HbF) for the surviving F cells. A total of 10 patients were enrolled, including 2 who were studied before and after the percentage of F cells was increased by treatment with hydroxyurea. As expected, F cells survived longer in all subjects. Non-F-cell survival correlated inversely with the percentage of F cells, with the time for 30% cell survival ranging from 6 days in patients with more than 88% F cells to 16 days in patients with less than 16% F cells. As the biotin-labeled RBCs aged in the circulation, the HbF content of the surviving F-cell population increased by 0.28%/d +/- 0.21%/d, indicating that within the F-cell population those with higher HbF content survived longer.

Phosphatidylserine externalization in sickle red blood cells: associations with cell age, density, and hemoglobin F.

Phosphatidylserine (PS) is normally confined to the cytoplasmic leaflet of the red blood cell (RBC) membrane, but some sickle RBCs expose PS in the outer leaflet (PS+ cells). This study examined the relationships among PS externalization, fetal hemoglobin content, hydration state, and cell age. Sickle RBCs exhibit a wide range of PS externalization. Those with low-level exposure (type 1 PS+) include many young transferrin-receptor-positive (TfR+) cells. This is not specific for sickle cell disease because many nonsickle TfR+ cells are also PS+. RBCs with higher PS exposure (type 2 PS+) appear to be more specific for sickle cell disease. Their formation is most likely sickling dependent because type 2 PS+ dense sickle cells have a lower percentage of fetal hemoglobin (HbF) than PS- cells in the same density fraction (1.7 vs 2.9; n = 8; P <.01). In vivo experiments using biotin-labeled sickle cells showed a sharp decrease in the percentage of circulating, labeled PS+ cells in the first 24 hours after reinfusion. This decrease was confined to type 1 PS+ cells and was thus consistent with the reversal of PS exposure in very young cells. As the labeled cells aged in the circulation, the percentages of type 1 and type 2 PS+ cells increased. These studies indicate that PS externalization in sickle cells may be low level, as observed in many immature cells, or high level, which is associated with dehydration and appears to be more specific for sickle RBCs.

Rehydration of high-density sickle erythrocytes in vitro.

Recent studies have identified older, low-density sickle red blood cells (SSRBCs) that were resistant to dehydration by valinomycin, a K(+) ionophore. These cells, thought to derive from dense SSRBCs that have rehydrated, may represent a terminal cellular phase. To study rehydration, we subjected dense SSRBCs (rho > 1.107 g/cc) to either oxygenated incubation or rapid oxygenated/deoxygenated (oxy/deoxy) cycling (70 seconds per cycle). Light cells (rho < 1.087 g/cc) were generated during both oxy incubation (2.9% +/- 2.1%; n = 42) and oxy/deoxy cycling (5.3% +/- 2.4%; n = 42). The rehydrated cells were K(+)-depleted (K(+) = 20 +/- 14 mmol/kg hemoglobin [Hb]) and Na(+)-loaded (Na(+) = 394 +/- 106 mmol/kg Hb), and had high levels of external phosphatidylserine. In the presence of external calcium, the generation of rehydrated SSRBCs was inhibited during oxy/deoxy cycling, but the percentage with external phosphatidylserine increased. The calcium-mediated inhibition of rehydration was reversed by charybdotoxin, implying that rehydration was delayed in some cells by the Ca(++)-activated K(+) channel. Preincubation of dense SSRBCs with DIDS (4,4'-di-isothiocyanato-2,2'-disulfostilbene) inhibited the generation of light cells during fast oxy/deoxy cycling, but not during oxy incubation. These results suggest that the sickling-induced pathway, previously implicated in SSRBC dehydration, may be involved in the deoxy-dependent component of rehydration for dense, K(+)-depleted cells. Light-cell generation was inhibited by 1 mM bumetanide during both oxy incubation and oxy/deoxy cycling, providing evidence that a bumetanide-sensitive, deoxy-independent pathway, previously described in circulating light SSRBCs, also contributes to the rehydration of high-density SSRBCs.