Bruton Tyrosine Kinase Inhibitor Rilzabrutinib Reduces Vaso-Occlusion and Markers of Inflammation and Adhesion in Transgenic Mice with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is characterized by episodic vaso-occlusive crises (VOC), chronic hemolysis, and multiorgan dysfunction. VOC has negative effects on quality of life, is a major cause of hospitalization, and has an elevated risk for death. Oxidative stress, inflammation, enhanced adhesion of activated neutrophils, release of neutrophil traps (NET), hypercoagulability, and endothelial activation are increasingly recognized as playing a primary role in vaso-occlusion, tissue ischemia, and organ damage. Activation of TLR4 and NLRP3 inflammasome have been implicated as key inflammatory pathways in SCD pathophysiology. A growing body of evidence suggests multiple roles for Bruton tyrosine kinase (BTK) as a regulator of the innate inflammatory machinery, the NLRP3 inflammasome, heme and TLR4-mediated inflammasome activation, adhesion molecule expression, and NET formation. Our objective was to evaluate the effect of the BTK inhibitor (BTKi) rilzabrutinib on inflammatory and adhesion molecule expression, and prevention of microvascular stasis in a model of vaso-occlusion in SCD mice.

Methods: HbSSTownes 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 (27^th dose), RA15539667 (14^th 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.

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.

Yes, it is off label. Rilzabrutinib is an investigational therapy being evaluated in autoimmune disease models.