GTx011, a Potent Allosteric Modifier of Hemoglobin Oxygen Affinity, Prevents RBC Sickling in Whole Blood and Prolongs RBC Half-Life in Vivo in a Murine Model of Sickle Cell Disease

Abstract Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life. We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry. In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of <40 mm Hg) evaluated. Moreover, at an O2 tension mimicking typical hypoxic conditions in tissue capillaries (40 mm Hg), 300 µM of GTx011 was sufficient to prevent sickling of human SSRBCs in whole blood (20% Hct). Similarly, ex vivo sickling analysis indicated that, relative to blood from vehicle-treated SS mice, blood from GTx011-treated SS mice showed a pronounced reduction in the number of sickled RBCs under hypoxic conditions with a concurrent reduction in p50. For example, at a pO2 of 10 mm Hg, 19% of SSRBCs in blood from GTx011-treated mice sickled ex vivo compared with 56% in blood from vehicle-treated SS mice. In SS mice chronically dosed with GTx011, a prolongation of the RBC half-life from 2.4 days to 3.8 days was achieved together with a marked decrease in reticulocyte count. This increase in RBC half-life and accompanying reduction in reticulocyte count was observed in mice with GTx011 concentrations in blood that corresponded to >30% calculated Hb target occupancy. Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD. Table SS mice RBC half life Reticulocytes Sickled RBCs Hemoximetry Chronic treatment, PO, BID, 10-12 days (Days) (%) (% at 10 mm Hg) p20 (mm Hg) p50 (mm Hg) Vehicle-treated 2.4 53 56 18 32 GBT440-treated (100mg/kg) 3.8 32 19 4.5 21 Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Zhou:Global Blood Therapeutics: Research Funding. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Archer:Global Blood Therapeutics: Consultancy, Research Funding.