The Supra Perfusion Agent PEGylated Albumin and Its Tempol Derivative Improve Blood Oxygenation and Ameliorate Inflammatory Response in Sickle Transgenic Mice

Abstract 2117

Sickle cell disease (SCD) is characterized by episodes of vaso-occlusive crisis and chronic inflammation. At present, there is no FDA-approved plasma expander with antioxidant properties for the management of acute vaso-occlusive episodes in SCD. We propose a strategy involving a supra perfusion fluid or a combination of a supra perfusion fluid incorporating an antioxidant that will alleviate oxidative stress and vaso-occlusive complications in SCD. HexaPEGylated albumin (P5K6-Alb or PEG-Alb) was generated by extension arm facilitated (EAF) PEGylation using PEG-5K maleimide. PEG-Alb-T12 was prepared using EAF chemistry with 12 Tempol molecules. Sickle (S+S-Antilles) mice were top-loaded with either 4% PEG-Alb or PEG-Alb-T12 at 10% of mouse blood volume. We used sickle mice and BOLD (Blood Oxygen Level Dependent) MRI imaging of the brain to visualize changes in blood oxygenation. In another series of experiments sickle mice were subjected to 16 hr of hypoxia (8% O₂, 0.5% CO₂, balance N₂) followed by 4 hr of re-oxygenation at ambient air; these mice were top-loaded at 10% of mouse blood volume with either 4% PEG-Alb or PEG-Alb-T12 at the onset of re-oxygenation. Both of these compounds are low viscosity fluids following administration of 4% compound at 10% blood volume. Hemodynamic properties were monitored by videomicroscopy of the cremaster. C57BL mice were used as controls.

We found that S+S-Antilles mice had elevated levels of deoxyhemoglobin (deoxyHb) in brain as previously reported. Although we hypothesized that anti-oxidant Tempol groups would increase the efficacy of PEG-albumin, we found that either PEG-Alb-T12 or PEG-Alb without Tempol reduced deoxyHb levels to those found in normal C57BL mice. In a separate set of experiments, hypoxia-reoxygenation in sickle mice caused a marked 5-fold increase in leukocyte adhesion and 7.2-fold increase in transendothelial leukocyte emigration (each P<0.0001) that was accompanied by a significant decline in red cell velocity, wall shear rate and volumetric flow (P<0.05–0.023) in the venules of the cremaster muscle microcirculation. We found that top-loading either PEG-Alb-T12 or PEG-Alb without Tempol at the onset of reoxygenation ameliorated leukocyte adhesion and emigration, and markedly improved hemodynamic parameters almost to the control values.

In conclusion, in SCD, the presence of deoxyHb increases polymer formation and the risk of vaso-occlusion; both PEG-Alb-T12 and PEG-Alb markedly reduced deoxyHb levels. We have previously demonstrated an inverse correlation between perfusion and deoxyHb levels in these mice (Kennan et al. Mag Res in Med, 2004). These results are consistent on a macroscopic scale with the intravital studies that show increased flow following amelioration of the inflammatory effects of reoxygenation by either PEG-Alb-T12 or PEG-Alb. The observed amelioration of inflammation and increased blood oxygenation may, at least partly, be attributed to the reported tonic release of nitric oxide via increased wall shear stress and mechanotransduction (Tsai et al. 2011), and to oxidant scavenging. Taken together, these studies demonstrate the marked efficacy of both of these agents in reducing inflammation and increasing blood oxygenation and flow, all of which characterize SCD and contribute to its pathology.