ILX-002 A Direct HbS Polymerization Inhibitor for the Treatment of Sickle Cell Disease
Authors: Abdulmalik Osheiza , Fleischman Andrew N , Light David R , Balogun Benita , Cyril-Olutayo Mojisola , El-Araby Moustafa , Omar Abdelsattar , Safo Martin K 
Published: June 2024

Presentation Date: 6/8/2024

Presentation Start Time: 6:00 PM

Background

With the exception of gene therapies, approved treatment options for SCD do not adequately control hemolysis or prevent the onset of painful vaso-occlusive crises (VOCs). Individuals with hereditary persistence of fetal hemoglobin and other rare HbS co-inheritance mutations (Hb Stanleyville II; HbSAsn78Lys) are free of SCD symptoms and hemolytic anemia. Increased expression of HbF prevents polymerization in RBCs because the Thr87 residue responsible for key lateral contacts of HbS polymers is replaced by Glu87 in HbF. Similarly, the rare HbSAsn78Lys co-inheritance mutation prevents polymerization by interrupting important polymer-stabilizing contacts on the surface of the HbS αF-helix. ILX-002 is a novel aromatic aldehyde drug candidate that binds to HbS and directly blocks polymerization by disrupting surface residues of the αFhelix. Here we report the discovery of ILX-002, a direct HbS polymerization inhibitor, currently undergoing INDenabling studies.

Methods

150 compounds identified by structure-based rational design were synthesized and screened in vitro in human SS blood. Blood samples were collected from pediatric SCD donors and incubated with test compounds (2mM, 20% Hct), then subjected to 100% N2 gas to completely eliminate oxygen prior to quantification of timedependent RBC sickling by image analysis. RBC partitioning ratio was evaluated based on % hemoglobin modification before and after RBC lysis, and whole blood oxygen equilibrium was assessed with a Hemox analyzer. Promising candidates were screened for suitable pharmacokinetics (PK) and preliminary toxicology in Sprague Dawley rats. Once selected as the lead, a 14-day dose range-finder toxicology study (125, 250, and 500 mg/kg) was conducted for ILX-002. Finally, ILX-002 was administered in food chow (1% or 2% w/w) to humanized homozygous βS/βS Townes mice for 21 days to assess changes in sickling and hemolysis.

Results

Two analogs, ILX-002 and MCP-435, sustained &gt;75% inhibition of sickling for 150 mins in total anoxia (Fig. 1). In contrast, Voxelotor quickly lost potency in the absence of O2. Close interactions of ILX-002 and MCP-435 with key surface residues on the αF-helix by x-ray crystallography confirmed the direct polymer destabilizing mechanism of action. ILX-002 and MCP-435 also demonstrated the highest RBC partitioning ratios of 98% and 99%, respectively, compared to 90% for Voxelotor. Following administration at 100 mg/kg for 14-days in male and female SD rats, a high oral exposure (20,977 µM*h) and blood concentration (1,109 µM) were achieved with ILX-002, compared to 9,297 µM*h and 619 µM, respectively, with MCP-435 and 7,756 µM*h and 485 µM, respectively, with Voxelotor. ILX-002 was selected as the lead due to its favorable PK and long half-life which project to once daily dosing in humans. ILX-002 was extremely well tolerated when administered as escalating doses (125, 250, and 500 mg/kg) for 14-days, with no significant adverse effects observed at any dose level tested up to a blood concentration as high as 3.5 mM. ILX-002 administered for 21 days to Townes mice at about 50% hemoglobin occupancy demonstrated dramatic benefits: complete normalization of hemoglobin levels with profound reductions in reticulocytosis (50% at baseline to &lt; 20%), hemolysis (60% reduction in bilirubin), and inflammation (85% reduction in neutrophil count). ILX-002 inhibited ex vivo sickling by nearly 70% despite inducing a shift in oxygen affinity (ΔP50) of only 20% (compared to a 50% shift in P50 with Voxelotor at 50% occupancy) (Fig. 2).

Conclusions

ILX-002 is a promising oral drug, which may provide durable control of hemolysis and prevent VOCs in SCD patients. ILX-002 profoundly inhibits HbS polymerization, even at low PO2 levels which may occur in some areas of the circulation, with significantly less allosteric effect on oxygen affinity. A low shift in oxygen affinity may allow the drug to safely achieve higher clinical HbS occupancy levels.