Pegylated Carboxyhemoglobin (SANGUINATE^®) Restores Blood Flow and Tissue Oxygenation in a Novel Rat Model of Acute Vaso-Occlusive Crisis

INTRODUCTION: Sickle Cell Disease (SCD) hemoglobin S (HbS) promotes hemoglobin polymerization and red blood cell (RBC) sickling during hypoxia. PEGylated carboxyhemoglobin bovine (PEG-COHb; SANGUINATE^®) has been previously shown to reverse sickling of SCD RBC in vitro by directed gas transfer to hypoxic RBC. A rat model was used to evaluate the capability of SANGUINATE^® to restore cardiovascular rheology and tissue oxygenation (P_ISFO₂) at sites distal to acute Vaso-Occlusions (VOC).

OBJECTIVES: Evaluate the in vivo capability of PEG-COHb to improve rheology in rats placed in active VOC through transfusion of human SCD RBC while monitoring treatment effects on P_ISFO₂ and systemic functions.

METHODS: Human homozygous (HbSS) whole blood was obtained from consented SCD volunteers. The SCD RBC were isolated by centrifugation, triple-washed and resuspended in lactated Ringer's saline (LRS). Male Sprague Dawley rats were anesthetized, cannulated for fluid transfer, and surgically prepared for microscopic study of the spinotrapezius microcirculation. Isovolemic exchange transfusion of rat blood with suspended SC RBCs (27.5 ± 3.5% total blood volume) was conducted until P_ISFO₂ at the distal portion of the spinotrapezius muscle was < 1 mmHg. VOC was visually confirmed as wide-spread precapillary arteriolar flow-arrest. Animals in VOC were subsequently treated 15 minutes later with PEG-COHb or LRS administered as a bolus of 8.0 ml/kg over 5 minutes. Animals were observed for up to 4 hours post-treatment and then euthanized. Measurements included systemic cardiovascular parameters, survival, blood chemistry, and P_ISFO₂ via phosphorescence quenching microscopy and were obtained across treatment groups and the no treatment [Sham] control group.

RESULTS: Exchange transfusion successfully produced a reduction in P_ISFO₂ in the distal portion of the spinotrapezius muscle for all animals to <1 mmHg presumably through Vaso-Occlusions. VOC was visually apparent as flow arrest was initially observed in capillaries and most small venules and arterioles, and then progressed proximally into sites with larger feed vessels and higher intravascular pressures with subsequent declines in local P_ISFO₂. Intervention with PEG-COHb and LRS both showed a visual improvement in microvascular flow, but yielded differential effects on P_ISFO₂ in all sites (average return to 96 ± 6% and 64 ± 20% of baseline, respectively). PEG-COHb -treated animals survived the entire 4 h observation phase with a final global spinotrapezius P_ISFO₂ level within 10% of baseline values. In contrast, global spinotrapezius P_ISFO₂ declined to 0 mmHg for LRS treated animals by 3 h with rapid decline in mean arterial pressure and death occurring prior to 4 h. Serum lactate levels were monitored to provide an assessment of systemic ischemia from time 0 after exchange transfusion to the last collection point near the end of the experiment. Lactate increased for untreated Sham (34 ± 4.5 to 124 ± 43.0 mg/dl) and LRS groups (48 ± 15.0 to 110 ± 43.0 mg/dl), but remained stable for SANGUINATE^®-treated animals (42 ± 1.5 to 40 ± 4.5 mg/dl). Total microcirculatory collapse occurred in Sham animals by 3h along with a null-reading of P_ISFO₂, which was followed by a rapid decline in mean arterial pressure and death at 3.5 h similar to the LRS group.

SUMMARY: This study was conducted in a novel rat model providing the ability to monitor real-time microcirculatory events and systemic parameters, which is difficult in murine models due to their smaller stature. PEG-COHb treatment promoted VOC resolution as evidenced through improved blood flow and concomitant effects on P_ISFO₂. Importantly, the gas transfer properties of SANGUINATE^® not only acutely resolved VOC, but provided the added benefit of restoring P_ISFO₂ to near baseline values. In contrast, LRS animals showed transient rheology and minimal P_ISFO₂ improvements, but levels declined over time and outcomes were similar to sham controls. Restoring blood flow and P_ISFO₂ are key events to limiting VOC pain and complications and the treatment with PEG-COHb demonstrated the ability to achieve these critical functions suggesting it may provide improvements in treating acute VOC in human SCD individuals.