Rivipansel: A Small Pan-Selectin Antagonist Improves Cerebral Perfusion and Inhibits Leukocyte Adhesion and in Murine Sickle Cell Disease Model

The pathophysiology of sickle cell disease (SCD) is complex and heterogenous. Tissue hypoxia leads to endothelial activation, and formation of multicellular aggregates of red cells, leucocytes and platelets in the microvasculature. This results in a vaso-occlusion further reducing tissue perfusion. Patients with SCD experience intense pain crisis. Some SCD patients also exhibit elevated cerebral blood flow velocities in transcranial Doppler (TCD) and impaired autoregulation. Higher TCD velocities are associated with imminent risk of ischemic stroke and are an indication for transfusion for SCD patients. Similar to human SCD patients, SCD mice also exhibit an increase in cerebral perfusion (Branch et al, Blood, 2015) and irregularly dilated cerebral microvasculature compared to wild-type (WT) mice (Manci et al, Blood, 2006). Selectins play a critical role in mediating initial tethering and adhesion of hematopoietic cells with endothelial cells, and are attractive targets to treat vaso-occlusion. Rivipansel is a potent small molecule pan-selectin inhibitor with a 100 fold greater inhibitory acitivity for E-selectin over P-selectin. Previous studies have demonstrated the effect of Rivipansel (GMI-1070) in chimeric SCD mice using intravital microscopy (IVM) where bone marrow from Berkeley SCD mice was transplanted in wild type (WT) mice (Chang et al, Blood, 2010). Here, we further investigate the effect of Rivipansel on leukocyte rolling in vitro and in vivo in Townes SCD mice with corresponding changes in inflammatory markers. We also extended these studies to explore the effect of Rivipansel in a cerebral perfusion model in Townes SCD mice.

The effect of Rivipansel on cell adhesion in vitro was examined using the microfluidic BioFlux shear flow system. HL-60 cells were continuously flown under physiological conditions over confluent monolayers of Chinese Hamster Ovary cells expressing E-selectin (CHO-E) or P-selectin (CHO-P) treated with Rivipansel, Immunoglobulin G (IgG) control antibody or vehicle. Rivipansel dose-dependently reduced the adhesion of HL-60 to CHO-E monolayers compared to vehicle treated cells, or CHO-P monolayers.

In the Townes SCD mice, Rivipansel was evaluated using IVM of the cremaster. Surgical manipulation and the chronic inflammatory conditions in Townes mice, induce an acute vaso-occlusive condition in the post-capillary venules. Rivipansel or vehicle control were dosed at 5, 10 or 20 mgs/kg intravenously prior to surgical preparation and a second dose was administered 70 minutes later. Labeled neutrophil and platelet specific antibodies were injected to quantify neutrophil-platelet aggregates and neutrophils adhered to the vasculature. Neutrophil rolling velocity increased 2.5 fold and there was a dose dependent increase in rolling neutrophils. Rivipansel, dosed at 20 mg/kg resulted in a 69% decrease in neutrophil adhesion to the endothelium and an 85% decrease in neutrophil platelet aggregates (NPA) attached to the vasculature compared to vehicle treated mice. No effect on was observed in mice dosed a 5 mg/kg. Plasma soluble P and E-selectin levels were significantly reduced in mice dosed at 10 and 20 mg/kg compared to vehicle treated mice.

Additionally, cultured primary brain endothelial cells stimulated with TNF-α demonstrated a 700 fold increase in E-selectin levels as measured by RNAseq. Based on this observation, we studied the effect of Rivipansel on cerebral perfusion studies in a TNFα induced occlusion model in SCD and WT mice where changes in cerebral microcirculation were measured in real time using laser Doppler tissue perfusion. TNFα treated SCD mice demonstrated a marked reduction in tissue perfusion in contrast to WT mice. This was rapidly reversed by intravenous injection of Rivipansel (20 mg/kg). Brains harvested from TNF treated SCD mice showed increased platelet microthrombi compared to WT mice, and treatment with Rivipansel reduced the platelet aggregates seen in the cerebral microvasculature in SCD mice.

In summary, we demonstrated dose dependent decrease in leukocyte adhesion in vitro and in vivo in the Townes mice. In addition administration of Rivipansel is beneficial in improving cerebral perfusion as well improving peripheral microvascular flow in the SCD mice.

All experiments were within guidelines and were reviewed and approved by Pfizer institutional animal care and use committee.