Prevention of Venular Stasis, Circulatory Collapse and Mortality in Transgenic Sickle Mice Administered MP4CO, a Pegylated Hemoglobin Saturated with Carbon Monoxide

Abstract 850^FN2

Patients with sickle cell disease experience recurrent episodes of painful vaso-occlusion with organ damage due to red cell deformation, hemolysis, activation of endothelium, adhesion of blood cells to each other and to the vessel wall, and ischemia/reperfusion injury. There are few therapeutic options available for patients experiencing a sickle cell crisis. Previous studies in our lab have shown that induction of heme oxygenase-1 or administration of its products, carbon monoxide (CO) and biliverdin can inhibit hypoxia-induced vaso-occlusion in transgenic sickle mice. MP4CO is a 4.3 g/dL solution of human hemoglobin conjugated with polyethylene glycol and saturated with CO. In the current studies, we tested the hypothesis that MP4CO would protect transgenic sickle mice from adverse responses to hypoxia/reoxygenation and intravenous administration of hemin.

Vascular stasis was examined by intravital microscopy following hypoxia/reoxygenation in NY1DD transgenic sickle mice implanted 3–4 days earlier with a dorsal skin fold chamber window (DSFC). Four groups of 6 mice were studied: 1) Control untreated mice; 2) Hemin chloride, 40 μmols/kg i.p. × 3 days; 3) single dose MP4CO, 0.012 mL/g i.v. treated 30min after hypoxia; 4) two doses of MP4CO treated 24h prior to and 30 min after hypoxia. Flowing venules in the DSFC window were randomly selected at baseline. A total of 15–32 subcutaneous venules were selected in each mouse. After baseline selection of venules, the mice were exposed to hypoxia (7% oxygen, 93% nitrogen) for one hour then returned to room air and the same venules were re-examined for stasis at 1h and 4h after hypoxia. Venules without any visible blood flow were counted as static. Percent stasis was calculated by dividing the number of static venules at each time point by the total number of venules selected at baseline. In addition, bioavailability of CO was determined in four NY1DD sickle mice by gas chromatographic measurement of CO in exhaled air 0–3h and 24–27h after treatment with MP4CO. Control, untreated NY1DD mice exhibited 27% and 4% static venules at 1h and 4h, respectively, following hypoxia. This was significantly reduced by the positive control hemin (3%* and 0%*), single dose MP4CO (15%* and 3%), and two doses of MP4CO (0%* and 0%*) at 1h and 4h, respectively (*p<0.05). Exhaled CO increased approximately 6-fold from baseline of 0.8 to 4.63* nmol/hr/g body weight over the 3h following dosing, representing approximately 50% of administered CO.

Based on these data, we evaluated MP4CO in a different transgenic mouse model of sickle cell disease. Intravenous injection of hemin is reported to induce vaso-occlusion more robustly than hypoxia/reoxygenation and to cause mortality in transgenic sickle mice. In this potentially more severe model, we studied the effects of hemin injection (50 μmol/kg, i.v.) in heterozygote Townes-AS mice. These mice were mildly anemic compared to wild type C57 (tHb 9.9 vs 14.0 g/dl) with elevated spleen weights measured at necropsy (269 vs. 87 g). Mice were anesthetized, mechanically ventilated, and catheterized to measure and record arterial pressure continuously. Hemin administration was followed 10 minutes later by 30 minute i.v. infusion of normal saline or MP4CO (0.012 mL/g). In saline treated transgenic sickle mice, hemin induced 100% lethality within 90 minutes, compared with 20% lethality following MP4CO (p<0.05, log rank test). In saline treated mice, hemin administration was followed by a progressive decline in mean arterial pressure, which was absent in MP4CO treated mice (p<0.01). Hemin had no effect on arterial pressure or survival in wild-type C57 mice.

These data, from two different models of transgenic sickle mice, demonstrate that MP4CO reduces the vascular complications resulting from oxidative injury. The data suggest that MP4CO is an effective way to administer therapeutic levels of CO. The dose of MP4CO administered in these mice is known to induce a transient increase in total HbCO saturation to approximately 10% with return to baseline levels within 120 minutes. Taken together, these experiments suggest that MP4CO may provide a novel approach to treating patients with sickle cell crisis. Further experiments will evaluate the dose-response, timing of administration, and relative roles of CO administration vs. induction of heme oxygenase-1 by MP4CO.