ALBERT

Description: New treatments directly targeting polymerization of sickle hemoglobin (HbS), the proximate event in the pathophysiology of sickle cell disease (SCD), are needed to address the severe morbidity and early mortality associated with the disease. Voxelotor (GBT440) is a first-in-class oral therapy specifically developed to treat SCD by modulating the affinity of hemoglobin (Hb) for oxygen, thus inhibiting HbS polymerization and downstream adverse effects of hemolytic anemia and vaso-occlusion. GBT440-001 was a phase 1/2 randomized, double-blind, placebo-controlled, single and multiple ascending dose study of voxelotor in adult healthy volunteers and patients with SCD, followed by a single-arm, open-label extension study. This report describes results of voxelotor (500-1000 mg per day) in patients with sickle cell anemia. The study evaluated the safety, tolerability, pharmacokinetic, and pharmacodynamic properties of voxelotor and established proof of concept by improving clinical measures of anemia, hemolysis, and sickling. Thirty-eight patients with SCD received 28 days of voxelotor 500, 700, or 1000 mg per day or placebo; 16 patients received 90 days of voxelotor 700 or 900 mg per day or placebo. Four patients from the 90-day cohort were subsequently enrolled in an extension study and treated with voxelotor 900 mg per day for 6 months. All patients who received multiple doses of voxelotor for ≥28 days experienced hematologic improvements including increased Hb and reduction in hemolysis and percentage of sickled red cells, supporting the potential of voxelotor to serve as a disease-modifying therapy for SCD. Voxelotor was well tolerated with no treatment-related serious adverse events and no evidence of tissue hypoxia. These trials were registered at www.clinicaltrials.gov as #NCT02285088 and #NCT03041909.

Description: Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to formation of hemoglobin S (HbS). A primary and obligatory event in the molecular pathogenesis of SCD is the polymerization of deoxygenated HbS leading to sickling of red blood cells (RBCs). Prolonging the oxygenation of HbS should in principle delay polymerization relative to transit time through microcirculation, thus avoiding sickling. Discovered by Global Blood Therapeutics, GTx011 is a novel orally available small molecule that demonstrates desirable pharmacokinetics in multiple species. In addition, GTx011 binding to HbS produces concentration-dependent left shifts in partial oxygen pressure (p50) of oxygen equilibrium curves, indicating an increase in oxygen affinity. We report here that GTx011 delays in vitro polymerization and prevents sickling. GTx011 was evaluated in an adapted version of the polymerization assay described by Adachi et al. [Blood Cells (1982) 8:213-224]. Purified HbS (pre-incubated with GTx011) in 1.8 M potassium phosphate was passively de-oxygenated, followed by an induction of polymerization via temperature jump from 4°C to 37°C. Polymerization was quantified by measuring turbidity of the HbS solution under continued hypoxia. For sickling experiments, RBCs pre-incubated with GTx011 were subjected to hypoxia (pO2 of ∼30 mmHg) for 0.5 hr and subsequently imaged using a light microscope. In de-sickling experiments, RBCs were first sickled under hypoxia and then treated with GTx011 under continued hypoxia (2 hr) before acquiring images of cells. The percentage of sickled cells in each image was quantified using CellVigene software. GTx011 delayed HbS polymerization in a dose-dependent manner. The delay profile was similar to that of CO-liganded HbA, a well characterized intracellular inhibitor of HbS polymerization. Moreover, GTx011 prevented sickling of RBCs under hypoxia suggesting that GTx011 has the ability to prevent intracellular HbS polymerization. It should be noted that in this experimental system, RBCs were exposed to hypoxia for a relatively longer period of time than typical RBC transit times through microcirculation. Interestingly, GTx011 also reversed sickling of pre-sickled RBCs. The ability of GTx011 to de-sickle RBCs suggests that it may promote transition from the low affinity HbS (T-state HbS) into its high affinity conformer (R-state HbS), a form that is not incorporated into HbS polymers. Thus, by stabilizing the oxy (R) conformation of HbS, GTx011 has the potential to be a therapeutic agent for prevention of HbS polymerization and sickling in SCD patients. Table 1 Assay Polymerization Sickling De-sickling Unit DT cpd -DT HbS (min) (% sickled) (% sickled) [HbS] 50 µM ∼1 mM (20% HCT) ∼1 mM (20% HCT) [Cpd] 25 µM 50 µM 100 µM 1 mM 2 mM 5 mM 1 mM 2 mM 5 mM GTx011 3.9 12.8 22.9 41 33 28 57 34 31 5-HMF 1 1.8 6.3 76 65 50 97 96 89 No Cpd 0 0 0 85 85 85 96 96 96 Table 2 Assay Polymerization Unit DT HbS/HbA -DT HbS (min) [Hb] 50 µM % HbA-CO 20% 30% 40% HbA-CO 2.3 11.8 16.8 Disclosures: Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.

Description: Background: Sickle cell disease (SCD) is caused by polymerization of Hemoglobin S, resulting in red blood cell (RBC) sickling, RBC destruction, vaso-occlusive episodes and end-organ damage. No direct anti-polymerization, mechanism-based, preventive therapy is available. GBT440 is a novel small molecule hemoglobin modifier which increases hemoglobin oxygen affinity. In vitro and in vivo studies have shown it to be a potent and direct anti-sickling agent with high specificity for hemoglobin, and that 10-30% hemoglobin modification could be both safe (not compromising oxygen delivery) and effective (preventing HbS polymerization). We hypothesized that a potent antisickling hemoglobin modifier should rapidly interrupt RBC hemolysis, improve anemia and potentially become a safe and effective long-term disease-modifying therapy. We therefore explored safety, pharmacokinetics and pharmacodynamics and potential efficacy in healthy volunteers and SCD patients. Methods: This prospective, randomized, placebo-controlled, double blind, parallel group phase I/II study enrolled healthy volunteers (HV) ages 18-55, and homozygous HbSS SCD patients ages 18-60 with baseline Hb levels ³6 g/dL and ²10 g/dL and without vaso-occlusive crisis or transfusion within 30 days of screening. The study was conducted in two parts: part A tested single ascending doses and part B multiple ascending doses of study drug with 6:2 randomization (GBT440:placebo). Doses administered were: part A, HV cohorts 100mg to 2800 mg, SCD cohort 1000 mg; part B, HV cohorts 300 mg to 900 mg once daily for 15 days, SCD cohort 700 mg once daily for 28 days. The primary endpoint was safety. Secondary endpoints included pharmacokinetics (PK) and pharmacodynamics (PD). Clinical indices of hemolysis were prespecified as endpoints and are described for the SCD multiple dose cohort (700 mg QD for 28 days). Descriptive statistics were used to analyze data. Results: As of July 24 2015, 64 healthy volunteers (HVs) and 16 SCD patients had been enrolled. 54 healthy volunteers had completed the study, 2 were discontinued due to mild-moderate nonserious adverse events (headache, rash) and 8 were in follow-up; 8 SCD patients had completed Part A of the study and 8 were in Part B follow-up. No SCD patients were discontinued; one part B subject had a dose reduction from 700 mg to 400 mg (due to abdominal discomfort). GBT440 was generally well-tolerated, most adverse events were mild, there were no deaths, and there was one serious adverse event (AE) of acute painful crisis in a placebo subject. There were no AEs related to tissue hypoxia. GBT440 showed dose proportional PK, a terminal half-life of 1.5-3 days, high partitioning into RBCs (RBC:Plasma 60-90:1), and a dose dependent increase in hemoglobin oxygen affinity in both HV and SCD patients. In the multiple dose SCD cohort, all patients were evaluable at 28 days (age range 21-52 years; 2 were on hydroxyurea; 3 males/5 females). GBT440-treated patients showed increased hemoglobin, hematocrit and erythrocyte counts with corresponding decreases in LDH, unconjugated bilirubin, reticulocytes and erythropoietin levels with trends evident as early as day 8 compared to no changes in the placebo group (Figure 1 and Table 1). Analysis of peripheral blood smears revealed a marked reduction in sickle cells with GBT440 treatment. Conclusions: Single, oral, daily dosing with GBT440 was well tolerated across a wide dose range and demonstrated dose proportional and predictable PK and PD. GBT440 demonstrated proof of mechanism with a dose-dependent increase in hemoglobin oxygen affinity without causing tissue hypoxia. GBT440 demonstrated proof of concept in SCD patients with rapid reduction in RBC hemolysis and improved oxygen delivery to tissues as evidenced by reduced erythropoietin level, and a marked reduction in circulating sickle cells. These results support further clinical investigation of GBT440 as a potential disease-modifying therapy for SCD. Table 1. Mean change to Day 28 (SEM) GBT440 PLA Hemoglogin (g/dL) 0.8 (0.2) -0.5 (0.4) % Sickle cells in peripheral blood (%) -83 (9) 19 (4) Erythrocyte count (1012/L) 0.5 (0.1) -0.1 (0.2) Reticulocyte count (%) -2.2 (1) 0.8 (1.6) LDH (% change from baseline) -12 (7) -9 (1) Unconjugated bilirubin (% change from baseline) -25 (10) -9.6 (16) Erythropoietin (U/L) -29 (16) 21 (33) GBT = GBT440 treated subject; PLA = placebo subject; SEM = standard error of the mean Figure 1. Figure 1. Disclosures Lehrer-Graiwer: Global Blood Therapeutics: Employment, Equity Ownership. Howard:Pfizer: Consultancy; Novartis: Consultancy, Other: Travel Grant; Aes-Rx: Consultancy. Layton:Agios: Consultancy; Novartis: Consultancy; Glaxo Smith Kline: Consultancy. Mant:Quintiles: Employment, Equity Ownership. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Koller:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Ramos:Global Blood Therapeutics: Employment, Equity Ownership.

Description: 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 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.

Description: Background: Sickle cell disease (SCD) is caused by polymerization of Hemoglobin S (HbS), resulting in hemolysis and vaso-occlusion. No therapy achieving pancellular, direct inhibition of HbS polymerization is available. GBT440 is a novel small molecule which increases hemoglobin oxygen affinity and inhibits HbS polymerization and prevents sickling in vitro. This study explored safety, pharmacokinetics (PK) and pharmacodynamics (PD) of GBT440 in healthy volunteers (HV) and subjects with SCD. Methods: This randomized, placebo-controlled, double-blind, phase I/II study enrolled HV and SCD subjects (HbSS and HbSB0). A cohort of 8 subjects with other SCD genotypes (HbSC or HbS/β+thalassemia) is being enrolled. The study was conducted in three parts: Part A, single ascending doses, Part B, multiple ascending doses for 28 days and Part C, 90-day dosing. Longer term dosing (up to 6 months) is being evaluated. The primary endpoint was safety. Secondary endpoints included PK, PD and hematological effects. Results: New and updated results including up to 6 months of dosing and data on other SCD genotypes will be presented at the meeting. As of 26 July 2016, 38 SCD subjects had completed Part B (10 at 500 mg; 12 at 700 mg; 6 at 1000 mg; 10 received placebo [pbo]). An additional 16 SCD subjects have completed or are ongoing in Part C (6 at 700 mg: 6 at 900 mg; 4 on pbo). Enrollment is ongoing for SCD subjects with HbSC or HbS/β+thalassemia. Results are available for SCD subjects enrolled into Part B and Part C (700 mg). The SCD subjects include approximately 60% male, 40% female; 20% were on hydroxyrea (HU); 20% had 2 or more painful crises requiring hospitalization in the prior year; median age was 34 years (range, 20 to 56). There were no drug-related severe or serious adverse events; the majority of AEs were Grade 1 or 2. The most common treatment emergent AEs were headache, back pain and sickle cell crisis. All sickle cell crises on placebo or GBT440 occurred off treatment and were assessed as not related to study drug. PK exposures increased dose-proportionally; mean GBT440 whole blood half-life following multiple doses was approximately 1.5 days and RBC:plasma ratio was 75:1. GBT440-treated subjects demonstrated increased hemoglobin oxygen affinity (p50 shifted to the left towards the normal range), an increase in hemoglobin levels and a sustained decrease in unconjugated bilirubin during the 90 day treatment period (Figure 1, Table 1). Other markers of hemolysis improved concordantly (Table 1). Conclusions: GBT440 was well tolerated with dose-proportional PK and increases in hemoglobin oxygen affinity. GBT440 resulted in marked and sustained reduction in clinical markers of hemolysis and an increase in hemoglobin. These results are consistent with inhibition of HbS polymerization leading to decreased RBC damage, improved RBC lifespan and tissue oxygen delivery, and support further investigation of GBT440 as a potential disease-modifying therapy for SCD. Change from Baseline to Day 90 in Hemoglobin (GBT440 700 mg) Change from Baseline to Day 90 in Hemoglobin (GBT440 700 mg) Figure 1 Figure 1. Disclosures Lehrer-Graiwer: Global Blood Therapeutics: Employment, Equity Ownership. Mant:Quintiles: Employment. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapetics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Tonda:Global Blood Therapetics: Employment, Equity Ownership. Bridges:Global Blood Therapetics: Employment, Equity Ownership. Ramos:Global Blood Therapeutics: Employment, Equity Ownership.

Description: Sickle Cell Disease (SCD) is characterized by hemolytic anemia, vaso-occlusion, and progressive end-organ damage. The underlying mechanism of SCD is the polymerization of sickle hemoglobin (HbS) that occurs when sickle erythrocytes (SS RBCs) are partially deoxygenated in microcirculation, leading to SCD pathophysiologic features. One of the most devastating complications of SCD occurs in the central nervous system (CNS), where overt stroke or repeated silent cerebral infarcts lead to significant physical and neurocognitive consequences. In SCD, the brain's response to insufficient oxygen (O2) delivery is balanced by increased blood flow to preserve O2 supply. However, the systemic endothelial dysfunction in SCD limits the capacity for vascular regulatory and compensatory changes to preserve appropriate tissue oxygenation, especially in tissues with high O2 demand like the brain. Low hemoglobin (Hb) levels and increased cerebral blood flow (CBF) are associated with increased stroke risk, suggesting that anemia-induced tissue hypoxia is an important factor contributing to subsequent morbidity in SCD patients. Voxelotor (GBT440) is a small molecule, HbS polymerization inhibitor being developed by Global Blood Therapeutics (GBT) for the treatment of SCD. By addressing the underlying mechanism of SCD, voxelotor has the potential to be disease-modifying and alleviate the clinical manifestations of SCD. Mechanistically, voxelotor increases Hb-O2 affinity and delays the transition from oxyHb to deoxyHb under hypoxic conditions. This study assessed the impact of a pharmacologically mediated increase in Hb-O2 affinity on brain tissue oxygenation under both normoxic and hypoxic conditions in Townes transgenic sickle mice (SCD mice). Two compounds that increase the Hb-O2 affinity with similar potency, voxelotor and an analog to voxelotor, GBT1118, were considered for the study. The target for Hb occupancy with test compounds was ≥30% based on the therapeutic target occupancies observed with voxelotor in clinical studies. The effects of increased Hb-O2 affinity on brain tissue oxygenation were assessed directly with O2-specific microelectrodes in a cranial window and indirectly with hypoxyprobe staining (pimonidazole) of brain tissue. Unique to this SCD model, the targeted Hb occupancy (≥30%) could not be consistently achieved by voxelotor. In contrast, repeat oral dosing of GBT1118 at 200 mg/kg/day for 2 weeks in SCD mice achieved steady state concentrations of 802 ± 81 µM (mean ± SD; n=5), corresponding to a Hb occupancy of 44 ± 5%. Consequently, GBT1118 decreased the p50 (partial pressure of O2 at which Hb is 50% saturated) values of SCD mouse blood from 39 ± 0.8 mmHg (Vehicle-dosed) to 21 ± 1.6 mmHg (GBT1118-dosed). While we could not achieve the desired Hb occupancy (≥30%) with voxelotor in this model, the Hb occupancy and change in p50 achieved with GBT1118 afforded us the opportunity to ask whether significantly increasing Hb-O2 affinity affects brain O2 tension. Measurements of cortical O2 tension (pO2) showed no difference in pO2 values under normoxia (21% O2) (Figure A), and slightly higher pO2 values under hypoxia (10% O2) (Figure B) for GBT1118-dosed SCD mice compared with vehicle-dosed SCD mice. Collectively across all brain tissues, the GBT1118-induced increase in Hb-O2 affinity reduced tissue hypoxia in SCD mice under hypoxia as measured by pimonidazole staining (Figure C). Together, these results indicate that a pharmacological increase of Hb-O2 affinity does not decrease cortical tissue pO2 in SCD mice and may reduce brain hypoxia under hypoxic conditions. Figure Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Lucas:Global Blood Therapeutics: Research Funding. Muller:Global Blood Therapeutics: Research Funding. Williams:Global Blood Therapeutics: Research Funding. Zhang:Global Blood Therapeutics: Employment, Equity Ownership. Rademacher:Global Blood Therapeutics: Employment, Equity Ownership. Alt:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Research Funding.

Description: Sickle cell disease is caused by a mutation in the β-globin gene leading to production of hemoglobin S (HbS). HbS polymerizes under hypoxic conditions, leading to changes in cytoplasmic viscosity and formation of rigid, non-deformable sickled red blood cells (RBC). Loss of RBC deformability leads to abnormal blood rheology, diminished oxygen (O2) delivery and contributes to vaso-occlusion. GTx011 is a novel orally bioavailable small molecule that has been shown to inhibit HbS polymerization in vitro by maintaining oxygenated HbS under hypoxic conditions. We investigated the mechanism by which GTx011 modulates O2 affinity. Although it binds to the N-terminal α chain of Hb via a reversible Schiff base, 1D 1H NMR studies indicated that the compound allosterically influenced the intra-dimer interface of Hb (α122His and α103His), and the distal valine surrounding heme pockets for both the α and β chains, suggesting a unique solution phase structure. GTx011’s unique allosteric action upon the heme pocket allows it to improve O2 affinity without sterically blocking the release of O2. This modulation of O2 affinity delays the onset of HbS polymers and therefore we hypothesized will improve the abnormal blood rheology observed in SS blood. We report here the beneficial effect of GTx011 on SSRBC deformability using four independent whole cell systems: 1) the profile of SSRBC during passage through a gel filtration column, 2) the ability and corresponding pressure required for SSRBCs to pass through a polycarbonate filter under hypoxic conditions, 3) the tension required to aspirate SSRBCs into a micropipette under low O2 and 4) changes to blood viscosity during deoxygenation. Under deoxygenating conditions, GTx011 dose dependently enabled RBC deformability required for migration of SSRBCs through a gel filtration column. The pressure required to pass SSRBCs through a polycarbonate filter and the tension required to aspirate SSRBCs into a micropipette under hypoxic conditions were also reduced (Table 1). We have established a novel blood viscosity assay, which employs an enzymatic reaction (ascorbic acid/ascorbic oxidase) for deoxygenation of blood, followed by traditional measurements in cone plate viscometers. This protocol enabled us to reduce O2 levels without the technical limitations of using sodium dithionite, laser induced deoxygenation or passive deoxygenation via N2. Time dependent measures of viscosity, at a fixed shear rate, allowed us to quantify GTx011-induced delay in the onset of polymerization, and the increase in cytoplasmic viscosity. As in polymerization experiments, GTx011 dose dependently delayed the onset of hyperviscosity of SS blood under deoxygenating conditions. HbS modification ranging from 10-30%, was sufficient to achieve an improvement in SS blood hyperviscosity (Table 2) These data suggest that GTx011 is a promising agent that has the potential to improve SSRBC deformability and blood viscosity by inhibiting HbS polymerization, the pathophysiologic hallmark of sickle cell disease. Table 1 Assay Migration Filterability Deformability unit (% deformable cells) (mm Hg) (mDynes/cm2) [HbS] 1 mM 1 mM 1 mM [GTx011] (mM) 0 0.5 1 0 0.5 1 0 0.5 1 Readout 16 42 89 358 162 129 101 32 25 Table 2 Assay Polymerization Viscosity [HbS] 50 μM 1.5 mM GTx011 modified HbS (%) 0 10 30 0 10 30 Delay time (min) 13 18 33 21 26 31 Disclosures Patel: Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Consultancy. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Consultancy, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.