A103-06 Preclinical Validation of a Novel Heme-Protein Based Oxygen-Carrier Therapeutic in Shock

Rationale Hemorrhagic shock and respiratory failure are major causes of death. No artificial oxygen carrier based on hemoglobin or hemoproteins has been approved in the US as an alternative to donated blood. A major challenge of free hemoproteins is the vasoconstrictive effects due to the nitric oxide (NO) scavenging. We have engineered a modified Regulator of Carbon Monoxide Metabolism (RcoM) heme-containing protein which shows no vasoconstrictive effects. We aim to validate the oxygen binding, NO dioxygenation rate, stability, and resuscitative effects of RcoM. RcoM has the potential to deliver oxygen as a blood replacement or in refractory hypoxemia in acute respiratory distress syndrome (ARDS). We hypothesize that RcoM is well tolerated and can behave as an artificial oxygen carrier due to ideal oxygen binding rate, low autoxidation rate, slow NO dioxygenation rate, and high thermal/chemical stability. Methods We produced RcoM in microbial expression systems, isolating and purifying the protein with column chromatography. We assessed biochemical features using UV-visible spectroscopy methods to determine the transitions between RcoM oxidation state and ligand binding and dissociation. We carried out competition experiments in which equimolar amounts of RcoM and Hb were exposed to NO to measure NO deoxygenation rate. To assess oxygen carrier capabilities of Rcom, we used a murine model of traumatic, hemorrhagic shock utilizing anesthetized mice with a 2cm abdominal incision and left femoral artery and vein catheterization. Hemorrhage was induced by a 30-minute controlled bleeding to a target blood pressure of 25 to 40 mmHg, shock was maintained for 30 minutes, then infusion by lactated Ringer (LR) solution, 2mM stroma-free Hb, or 2mM RcoM at volume equal to removed blood and monitored for 30 minutes. Mean arterial pressure post resuscitation were compared to assess the tolerance and hemodynamics effects. Results RcoM displays appropriate oxygen binding capacity and has an NO deoxygenation rate ten-fold lower than Hb. In the murine hemorrhagic model, there was no difference for the baseline MAP or in blood removed during the bleeding phase. Post-resuscitation MAP was significantly higher for RcoM (67.0 ± 6.3 mmHg) and Hb (60.4 ± 16.0 mmHg) compared to LR (25.9 ± 21.4 mmHg, p = 0.0029). Conclusion This study demonstrates that resuscitation with RcoM supports the blood pressure in a murine traumatic hemorrhagic shock model, suggesting its potential application as a non-globin artificial oxygen carrier for hemorrhagic shock. The potential of RcoM to augment oxygen delivery in respiratory failure is also being explored. This abstract is funded by: NIH, Department of Defense, Globin Solutions