GTx011, a Novel Agent That Improves Rheological Properties Of Sickle Cell Blood By Increasing Oxygen Affinity For Hemoglobin
Authors: Patel Mira , Oksenberg Donna , Silva Abel , Betz Andreas , Metcalf Brian , Sinha Uma 
Published: October 2019

Abstract
Sickle cell disorder (SCD) is characterized by the presence of non-deformable red blood cells. Literature reports indicate that the T-state structure of hemoglobin (Hb) (highly correlated with the deoxy form) is responsible for the formation of HbS polymers that lead to rigid cells. We hypothesized that the likelihood of polymer formation will be reduced if sufficient HbS remains in the R-state (oxy) conformation. We have designed and synthesized a novel series of compounds that increase the O2 affinity of HbS and improve the rheological properties of SCD blood. In a novel 96-well format oxygen dissociation assay (ODA), compounds including GTx006, GTx007 and GTx011 were all more potent than 5-hydroxy furfural (5HMF), an agent being tested in clinical trials in SCD patients. After two hours of passive deoxygenation, GTx011, at an equimolar concentration to Hb, increases the O2 affinity by six-fold and drastically delays polymerization of HbS. Even at substoichiometric concentrations (GTx011:Hb= 1:3) GTx011 elicits a two-fold improvement in O2 affinity for Hb that translated to 16% more oxy-Hb relative to control (Table 1). We then analyzed the agents in a TCS Hemox analyzer using purified Hb at 25µM. At a GTx011:Hb ratio of 1:3 the oxygen affinity was improved by 15%, while at stoichiometric concentrations, the oxygen affinity was increased by 70% compared to Hb control. These biochemical assays indicated that the GTx agents were altering Hb O2 affinity and should therefore assist in maintaining the oxy conformation of Hb and prevent the formation of polymers.Table 1AssayHb in ODAWashed RBC OECWhole Blood OECViscosityunit(Δoxy state)(%Δp50)(%Δp50)(ΔcP)[Hb]3 µM1 mM1 mM1.5 mM[cmp]1 µM3 µM1 mM3 mM1 mM3 mM1.6 mM8 mM5-HMF<1<1306510470.042.4GTx006210597849711.7>2.5GTx007623697863>802.1>2.5GTx0111656768380>802.5>2.5
To test the agents in a more physiological system, oxygen equilibrium curves (OECs) were measured in washed red blood cells (RBCs) and in whole blood at 20% hematocrit (∼1 mM Hb). In washed RBCs, 5HMF, GTx006, GTx007 and GTx011 at a concentration of 1mM produced partial O2 pressures (p50) of 20, 12, 9 and 7 mm Hg, respectively (control RBCs = 30 mm Hg). To determine the effects of plasma proteins, OECs were measured in whole blood from SCD patients, giving p50s of 27, 18, 11 and 6 mm Hg compared to the control blood p50 of 30 mm Hg (agent concentration of 1 mM). Table 1 shows that 5HMF and GTx006 activities were affected by the presence of plasma proteins but GTx007 and GTx011 activities were not altered.
SCD patients develop anemia due to hemolysis, which partially compensates for an increase in blood viscosity caused by the non-deformable RBCs (ssRBCs). We monitored the effect of our agents on SCD patient blood rheology, ex vivo, to determine if they were capable of decreasing the viscosity of SS blood under hypoxic conditions. We incubated whole blood from SCD patients (30% hematocrit, ∼1.5 mM Hb) with 5HMF, GTx006, GTx007 or GTx011 for 30 mins and then subjected the blood to 2 hours of hypoxia (2.4% O2). Blood viscosity was then measured in a cone-plate viscometer at shear rates ranging from 60 s-1 to 415 s-1. Of the four compounds, GTx011 showed the most pronounced improvement in rheologic measures (see Table 1), changing the viscosity from 6.46 cP (no GTx011) to 4.00 cP (equimolar GTx011). Normoxic SCD blood had a viscosity of 4.28 cP. A similar improvement in blood viscosity under physiologic conditions may be predicted to decrease the residence time of ssRBCs in hypoxic tissue, and allow for a lower level of polymerization in individual red blood cells. In addition, GTx011 has also been shown to delay polymerization and delay sickling. Thus, GTx011 has the potential to elicit a decrease in HbS polymer concentration, reducing the likelihood of forming the rigid cells that cause vaso-occlusion in SCD patients.

Disclosures:
Patel: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Silva:Global Blood Therapeutics: Employment, Equity Ownership. Betz:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Employment, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.