Direct hemoglobin S polymerization inhibitor MCP435 completely halts ongoing disease pathophysiology in transgenic sickle cell disease mice

Abstract Background – Direct HbS polymerization inhibitors bind to the surface of Hemoglobin S and block polymerization by disrupting key surface residues involved in intermolecular interactions between HbS molecules in sickle fibers. MCP435, an analogue of clinical stage drug ILX002, is a potent direct polymerization inhibitor that has demonstrated the ability to impede polymerization kinetics in vitro, increasing both polymerization delay time and saturated solubility (Csat) of deoxygenated HbS. However, the degree of pharmacologic inhibition of polymerization required to achieve disease resolution is not known. Although RBCs from healthy individuals with sickle cell trait contain HbS that can polymerize, polymerization kinetics are delayed long enough to avoid RBC damage during the circulatory cycle and prevent all signs and symptoms of disease. Therefore, we hypothesized that long-term administration of MCP435 to Townes mice at 50% occupancy, which results in vitro in polymerization kinetics similar to that of sickle cell trait, would completely halt persistent RBC damage and normalize RBC half-life in circulation, providing mechanistic support that achieving trait-like polymerization kinetics can result in disease resolution. Methods – Humanized homozygous βS/βS Townes mice (female, n=5/group) were randomized to receive either vehicle or 1.0% w/w MCP435 administered ad libitum in food chow for 39 days targeting a 50% Hb occupancy. On Day 30, circulating RBCs were labeled with biotin by injecting tail veins with NHS-biotin. Blood was sampled in a staggered fashion from 2 or 3 mice per group on alternating days for a 9 day period, and biotinylated RBCs quantified by flow cytometry to determine RBC half-life based on a decay model. On Day 39, blood was collected via inferior vena cava for pharmacodynamic and hematologic analyses. Polymerization delay time and saturated solubility (Csat) were assessed ex vivo using a serial dilution of blood lysates (varying HbS concentration), and dynamic sickling performed with incubation under 100% N2 gas for 30 mins. Lastly, we evaluated RBC osmotic fragility ex vivo based on degree of hemolysis following incubation of blood for 30 mins across increasingly hypotonic conditions (10% to 100% H2O). Results – Treatment with MCP435 (1.0% w/w) for 39 days (Hb occupancy 66.4 ± 4.1%) dramatically altered polymerization kinetics ex vivo, significantly increasing delay time and saturated solubility (Csat). Compared with a delay time of < 100 seconds in vehicle mice, delay time was significant increased by at least 12-fold across all HbS dilution levels in blood lysates from MCP435 treated mice. Additionally, HbS solubility in treated blood lysates did not saturate across the entire dilution series up to the maximum HbS concentration of 0.2 g/dL. Comparatively, the Csat was reached at 0.07 ± 0.01 g/dL HbS in the vehicle group. Correspondingly, dynamic sickling also decreased by 49.9 ± 12.0% in the treatment group relative to the vehicle group. As a consequence, RBC half-life in vivo normalized from 1.6 ± 0.2 days to 12.7 ± 2.7 days, an 8-fold increase compared to control (p<0.0001). On Day 31 (Day 1 following biotin labelling), there was a significantly higher fraction of labeled RBCs circulating in MCP435 treated mice compared to controls (89 ± 5.8% vs 46 ± 11%, p=0.015), and this difference was maintained through Day 39 (57 ± 6.5% vs 1.7 ± 0.63%, p<0.0001). RBCs from treated mice also exhibited significantly decreased osmotic fragility. In severe hypotonic conditions (80% H2O), 36 ± 4% of RBCs from treated mice hemolyzed compared to 80 ± 12% of RBCs from the vehicle group (p=0.00011). Consistent with improved RBC survival, other hematologic parameters also normalized, including RBC count (13.4 ± 1.4 vs 6.3 ± 0.15 x 106/mL), Hb (13.4 ± 1.0 vs 6.5 ± 0.2 g/dL), HCT (42.3 ± 2.5% vs 27.6 ± 0.9%), reticulocyte % (10.4 ± 3.5% vs 49.5 ± 3.2%), WBC count (6.7 ± 1.1 vs 16.5 ± 5.2 x 103/mL), and spleen weight to body weight (2.6 ± 0.2% vs 6.6 ± 1.2%). Conclusions – Direct HbS polymerization inhibitors are a potentially transformational new approach for the treatment of SCD. MCP435 achieved functional disease resolution in transgenic SCD mice by preventing polymerization-mediated RBC damage and normalizing RBC survival. This mechanistic proof-of-concept study demonstrated that reproducing sickle trait–like polymerization kinetics with a direct polymerization inhibitor can halt the pathophysiology of SCD.