Session: 113. Sickle Cell Disease, Sickle Cell Trait, and Other Hemoglobinopathies, Excluding Thalassemias: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Sickle Cell Disease, Research, Translational Research, Hemoglobinopathies, Immune mechanism, Diseases, Treatment Considerations, Biological Processes
Methods: HbSS Townes SCD mice aged 10-14 weeks (n=12/group) were pretreated with the reversible covalent BTKi rilzabrutinib (40 mg/kg twice daily), the irreversible covalent BTKi RA15539667 (15 mg/kg/day once daily), a P‑selectin blocking monoclonal antibody (mAb, 200 µg IP weekly), a control mAb (200 µg IP weekly), or vehicle control. Control HbAA mice were pretreated with vehicle. All mice were pretreated for 2 weeks prior to hypoxia/reoxygenation (H/R) or hemoglobin (Hb) challenge. After 13 days of dosing, the final doses of rilzabrutinib (27th dose), RA15539667 (14th dose), or vehicle were administered in the morning, mice were weighed, implanted with a dorsal skinfold chamber (DSFC), and challenged with H/R (7% O2, 93% N2 for 1 hour, followed by return to normoxia) or infusion of human HbA (1 µmol heme/kg body weight) via the tail vein. Flowing venules in the DSFC window were selected at baseline and re-examined for stasis (no flow) at 1, 2, 3, and 4 hours after return to normoxia. The percent stasis was determined for each time point and treatment group. Mice were euthanized in CO2 at 4 hours after the last timepoint prior to tissue and blood collection. Complete blood counts were measured manually in fresh blood. Immunoblots measured liver nuclear NF-ĸB phospho(Ser536) and total p65 expression on nuclear extracts from liver (n=3/treatment/ challenge; total n=36). Lung P-selectin, von Willebrand factor, and endothelial cell marker CD31 expression were examined by immunofluorescence staining and expressed relative to CD31 (n=3/treatment/challenge; total n=36). Transcriptomic analysis was performed using Twist capture exome sequencing on liver extracts of the left lobe.
Results: Pretreatment for 14 days with rilzabrutinib or RA15539667 in HbSS mice significantly decreased microvascular stasis at 1, 2, 3, and 4 hours after both H/R or Hb challenge (vs vehicle/control mAb; P<0.01). Rilzabrutinib and RA15539667 significantly decreased spleen weight (P<0.01) and lowered the inflammatory state in HbSS mice as evidenced by the reduction in total white blood cell count (P<0.01). Significant decreases in inflammation were observed in post-challenge tissue samples including decreased activation of NF-κB (P<0.001) in liver and reduced mobilization of P‑selectin and von Willebrand factor by immunofluorescence staining of lung tissue after H/R (P<0.05). In liver of SCD mice from the Hb model, treatment with rilzabrutinib resulted in downregulation of inflammasome-related genes (BTK, caspase-1, NLRP3, IL-1b, IL-18). In both H/R and Hb models, rilzabrutinib treatment led to downregulation of gene expression of markers of inflammation, markers of adhesion (P-selectin, E-selectin/C62L), complement, NETosis, and thrombosis. There were no significant differences among treatment groups compared to vehicle for any red blood cell indices.
Conclusion: Preclinical data provides evidence that treatment with rilzabrutinib ameliorates inflammation through multiple mechanisms of action and prevents microvascular stasis in Townes SCD mice.
Disclosures: Daak: Sanofi: Current Employment, Current equity holder in publicly-traded company. Light: Illexcor Therapeutics: Current equity holder in private company. Chen: University of MN: Current Employment. Storek: Sanofi: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Q32Bio: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Dianthus: Current equity holder in publicly-traded company; Alexion: Patents & Royalties. Ofengeim: Sanofi: Current Employment. Hicks: Sanofi: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Lee: Sanofi: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Vercellotti: Sanofi: Research Funding; Octapharma: Research Funding; CSL Behring: Research Funding; Mitobridge/Astellas: Consultancy, Research Funding; Omeros: Research Funding; Merk: Consultancy. Belcher: Sanofi: Consultancy, Research Funding; Illexcor Therapeutics: Consultancy; Omeros: Research Funding; Octapharma: Consultancy, Research Funding; Mitobridge/Astellas: Consultancy, Research Funding; Hillhurst Biopharmaceuticals: Research Funding; CSL Behring: Research Funding.
OffLabel Disclosure: Yes, it is off label. Rilzabrutinib is an investigational therapy being evaluated in autoimmune disease models.