Effect of PSI697, a Small Molecule Inhibitor of P-Selectin, in the Townes Model of Sickle Cell Disease

Chronic vaso-occlusion is a major cause of morbidity and mortality in patients with sickle cell disease (SCD). Persistent vaso-occlusion can damage lungs, liver, kidneys or brain and ultimately lead to end-organ dysfunction. Vaso-occlusive pain crisis is a complex multistep process, initiated by adhesion of fragile sickle erythrocytes and rigid neutrophils to hypoxic and inflamed endothelium. Large multicellular aggregates of blood cells, including platelets and sickled erythrocytes, form on these adherent activated neutrophils in the microcirculation, ultimately causing vascular occlusion and tissue ischemia. Inflammatory mediators, such as P-selectin, play a key role in mediating these heterocellular interactions and attract additional leucocytes to the site of occlusion. P- and E-selectin mediate rolling and tethering of blood cells on the endothelium. Here, we explore cellular and animal models of sickle cell disease to assess PSI-697, an orally active small molecule antagonist of P-selectin. PSI-697 inhibits P-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) with an IC₅₀ of 50-125 µM (Bedard et al, JPET, 2008). Townes SCD mice were used to assess the efficacy for PSI-697 in preventing vaso-occlusion in SCD. Mice were randomized to treatment with vehicle or PSI-697. Animals were treated in a prophylactic setting prior to the surgical preparation. Surgical preparation induces a well described acute inflammatory response in the microcirculation associated with neutrophil adhesion to the endothelium and formation of multicellular aggregates. Alexa-488 labeled Ly-6G neutrophil antibody and Dylight-649 labeled CD42c platelet antibody were injected to quantify adherent neutrophils and neutrophil-platelet aggregates. Cremaster microvasculature was observed by intravital microscopy. Mice treated with 100 mg/kg of P-selectin inhibitor PSI-697 showed a 55% reduction in adherent neutrophils and a 78% decrease in the number of neutrophil-platelet aggregates compared to vehicle treated animals. Neutrophils showed ~7 fold increase in rolling velocity in mice treated with P-selectin inhibitor. Our results demonstrate that prophylactic administration of PSI-697, a small molecule P-selectin antagonist, improved parameters associated with vaso-occlusion in Townes SCD mice.