A Novel Non-Covalent Modulator of Hemoglobin Improves Anemia and Reduces Sickling in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is a severe genetic disorder caused by a single point mutation on the β-chain of adult hemoglobin (Hb A), β6 Glu→Val (Hb S). In the deoxygenated state Hb S polymerizes, leading to RBC sickling and precipitating all downstream consequences, including vaso-occlusion (pain crisis), hemolytic anemia, and stroke. Over time, these features cause significant organ damage and eventual organ failure, dramatically impacting both quality of life and expected lifespan.

Numerous small molecules which covalently bind to Hb S have been evaluated clinically, however, the molecules that have demonstrated clinical efficacy all carry a reactive aldehyde group. The reactive aldehyde, a moiety that has the potential to react with any free amine, forms a covalent Schiff base with the N-terminal amine of the α1-Val. At least one member of this class of molecules, Tucaresol, showed a significant safety signal attributed to off-target Schiff base formation.

An early investigation of covalent hemoglobin modification, extracorporeal carbamylation, both improved anemia and decreased the frequency of vaso-occlusive events by 80%, when there was a sufficiently high level of modification (30-50%). These results suggest that a molecule that binds Hb S and stabilizes the oxygenated state can impact both hemolytic anemia and vaso-occlusive crisis, if the molecule can achieve the necessary degree of hemoglobin modification.

PFE-001 is a non-covalent molecule which binds selectively to Hb S and stabilizes the oxygenated state. Biochemical and biophysical studies show that PFE-001 binds specifically to Hb with double digit nanomolar potency and exhibits strong in vivo partitioning into RBCs.

In a two-week multiple dose study using Townes SCD model animals (200 mg/kg, twice daily), PFE-001 significantly improved markers of hemolytic anemia, increased oxygen affinity, and reduced RBC sickling. Following 15 days of treatment blood drawn from PFE-001 treated animals and exposed to intense hypoxic conditions (4% O₂, 4 hr) showed a 37.8% reduction in sickling compared to vehicle treated mice. Oxygen affinity was increased, demonstrated by a 53.7% reduction in p50 and an 84.4% reduction in p20 in the PFE-001 treated group. Hemoglobin levels in mice treated with PFE-001 increased by 42%, a mean increase of 5 g/dL. Hematocrit in the PFE-001 treated group increased to 42%, in contrast to 29% in the vehicle group. Reticulocyte percentages were reduced from 53% in vehicle treated animals to 24% in PFE-001 treated animals.

In addition to the significant impact PFE-001 had on hemolytic anemia, a 10% reduction in sVCAM-1 levels in the PFE-001 treated group indicates a small but statistically significant improvement in vasculopathy following 15 days of treatment. This improvement in vasculopathy suggests that PFE-001 has the potential to address vaso-occlusive crisis in addition to anemia.

In total, the in vitro and in vivo data suggest that PFE-001 is a potent, selective, and effective inhibitor of Hb S polymerization and RBC sickling. PFE-001 can reduce hemolytic anemia, improve vasculopathy, increase oxygen affinity, and reduce RBC sickling under hypoxic conditions. Plans for advancement of PFE-001 to clinical trials are in progress.