Evaluation of Purified Poloxamer-188 (vepoloxamer) on Sickle Red Blood Cell (RBC) Membrane Function Properties

Background: Sickle erythrocyte adhesion and membrane fragility contribute to vaso-occlusion and downstream tissue and organ ischemia in sickle cell disease (SCD). Vepoloxamer is an amphipathic triblock copolymer with multi-mechanistic properties believed to improve these pathophysiologic consequences, by sealing damaged cell membranes and inhibiting hydrophobic cellular adhesive interactions. Vepoloxamer reduced both acute vaso-occlusive crisis duration and total opioid analgesic requirements in previous clinical studies. A phase 3 clinical trial of vepoloxamer in acute vaso-occlusive crises is ongoing. Currently, there are no standardized clinical assays to assess membrane properties such as adhesion and fragility that vepoloxamer is believed to target. We evaluated if and to what extent vepoloxamer affected adhesion, thrombosis, and membrane fragility in individual patient samples in our standardized microfluidic flow-based whole blood assays.

Methods: Blood was obtained from pediatric homozygous sickle cell patients at steady state (n=12). Patients were first characterized by measuring the extent of whole bloodadhesion to vascular cell adhesion molecule (VCAM-1) in a standardized microfluidic flow-based assay. Dynamic adhesive interactions (rolling or sliding) of whole blood and isolated leukocytes to P-selectin were assessed by measuring absolute rolling cell number, cell rolling flux (number of rolling cells per total number of flowing cells), and cell rolling velocity at 0.3dyn/cm². Detachment assays were performed by exposing adherent cells to vepoloxamer (10 mg/mL) under physiological flow conditions. Erythrocyte membrane fragility was evaluated based on mechanical stress-induced hemolysis at 3 min (Hem_3min, %).

Results: Vepoloxamer reduced whole blood adhesion to VCAM-1 at 0.1 mg/mL by 18% (p = 0.0015), at 1.0 mg/mL by 69% and at 10.0 mg/mL by 79% (in both cases, p < 0.001), and to HUVECs by >50% 0.1mg/mL (p=0.003, n=10). Vepoloxamer (10mg/mL) did not significantly affect cell-rolling flux in dynamic adhesion assays to P-selectin (p = 0.51 and 0.94, isolated leukocytes and whole blood, respectively). Vepoloxamer reversed established cell adhesion by > 30% in 3 or 10 patient samples tested, although the total average difference in percent detachment did not reach statistical significance (16.3%vs 29.6%, p=0.42, n=10). Finally, vepoloxamer reduced shear-induced hemolysis compared to untreated blood samples (35.2 % vs. 44.6% control, p =0.033).

Conclusion: Vepoloxamer can significantly reduce firm cell adhesion to both VCAM-1 and HUVECS under physiologic flow conditions. Shear-induced hemolysis is also reduced in a statistically significant manner, consistent with the proposed membrane protecting effects of vepoloxamer. Interestingly, P-selectin mediated rolling adhesion was not significantly affected. This may suggest that vepoloxamer may have distinct inhibitory effects to VCAM-1 vs. P-selectin. The current findings suggest that combinatory assessment of the response of adhesive properties and membrane fragility to vepoloxamer treatment may facilitate selection of patients most likely to benefit from vepoloxamer therapy. The microfluidic flow-based platform presented in this study may have the potential to predict and monitor individual patient response to anti-adhesive therapies such as vepoloxamer. There are few reliable biomarkers that objectively predict and assess vaso-occlusive potential, thus further clinical study of this microfluidic flow-based platform are warranted.