Predictive Biomarker Analysis from the GBT021601 Survival Study in Townes Sickle Mice

Introduction: Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene resulting in sickle hemoglobin (HbS). Red blood cell (RBC) deoxygenation leads to HbS polymerization, RBC sickling and subsequent chronic hemolysis, anemia, vascular endothelial cell activation, vaso-occlusion, and ischemia. SCD is associated with extensive morbidity and markedly reduced survival. GBT021601 is an investigational, second-generation HbS polymerization inhibitor that reduces RBC sickling and anemia, and improves both O₂ delivery and consumption in chronically treated Townes Sickle Cell mice that harbor 2 HbS alleles (SS). Objectives of this 2-part study were to (1) characterize the effects of GBT021601 administered ad libitum on compound exposure and pharmacodynamics (PD), (2) examine the impact of long-term (>24 weeks) treatment with GBT021601 on % reticulocytes and overall survival (OS) of Townes SS mice, and (3) assess the utility of early changes in % reticulocytes as a predictive biomarker of survival in Townes SS mice treated with GBT021601.

Methods: In Part 1, 24 male Townes SS mice 8-10 weeks old were split into 3 cohorts (n=8/group) and for 3 weeks were fed standard chow containing 0% (control), 0.05% (low dose), or 0.2% (high dose) of GBT021601. Body weight and food consumption were measured weekly. Mice were bled daily on days 10-21 to determine RBC half-life and on days 10 and 21 for analysis of compound exposure. On day 22 mice were euthanized. Whole blood was collected for analyses of compound exposure, % reticulocytes, Hb levels, RBC numbers, RBC half-life, and RBC deformability. In Part 2, 5-week-old Townes mice (AA and SS genotype; 50% male) were fed either standard chow (AA [n=57] and SS mice [n=54]) or chow containing 0.05% or 0.2% of GBT021601 (SS mice only; n=55 each). Body weight and food consumption were measured weekly. Blood was collected at 1, 14, 31, 43, 55, and 67 weeks of treatment for compound exposure analysis and % reticulocytes. Mice were euthanized when the humane end-point criteria were met or after 72 weeks of treatment, and whole blood was collected for compound exposure and % reticulocyte analyses.

Results: GBT021601 in chow was well tolerated during both study parts; food consumption and body weight were unaffected. Outcomes in Part 1 confirmed the same compound exposure/PD trends as mice orally gavaged with GBT021601 as observed by Dufu et al. (Br J Haematol 2023); Hb levels, RBC numbers, and RBC half-life increased, RBC deformability improved, and % reticulocytes decreased.

Long-term dosing of GBT021601 in the chow of Townes SS mice revealed similar compound exposure and % reticulocytes as our 3-week dosing study (Part 1) and Dufu et al. In Part 2, Townes SS mice fed GBT021601 chow, achieving compound exposures after 1 week of dosing as depicted in Figure 1, had a longer median survival vs control Townes SS mice (60 vs 56 weeks). In control Townes SS mice, % reticulocytes increased during the first 14 weeks of the study and then plateaued, whereas in Townes SS mice fed GBT021601 there was a dose-dependent sustained reduction in % reticulocytes compared with control Townes SS mice. Reductions in % reticulocytes after 1 week of dosing were greater in Townes SS mice achieving a higher compound exposure (Figure 1), a trend sustained after 14 and 31 weeks of treatment. OS increased in Townes SS mice of the same sex achieving a ≤30% reduction in % reticulocytes vs Townes SS mice with % reticulocytes >30% after 1 week of treatment; P=0.00048 (Figure 2). Multivariate survival analysis, including % reticulocytes at 1 week (categorical variable of ≤30% or >30%) and sex as predictors confirmed significant association of >30% reticulocytes with reduced survival (hazard ratio=1.48, P=0.027 using Cox proportional hazard).

Conclusions: Longer-term GBT021601 treatment as part of chow was well tolerated by Townes SS mice, and compound exposure was comparable with studies of a shorter duration of treatment. Early compound exposure was predictive of a long-term survival benefit in Townes SS mice. Early reductions in % reticulocytes were more likely in Townes SS mice with a high GBT021601 exposure and were predictive of a long-term survival benefit. Overall, our findings support the use of GBT021601 to reduce % reticulocytes and improve OS in Townes SS mice and suggest the clinical relevance of early reductions in % reticulocytes as a biomarker to predict long-term survival.