Peg-Cohb Mediated Carbon Monoxide and Oxygen Transfer to Hypoxic Red Blood Cells Prevent, Slow, and/or Reverse Sickling in Vitro.
Authors: Buontempo Peter , Yglesias Rick , Chen Yuli , Buontempo Catherine , Jubin Ronald G. , Abuchowski Abraham 
Published: October 2019

Abstract
Background: In Sickle Cell disease (SCD), a single amino acid substitution in the beta globin chain converts HbA to sickle genotype HbS. This genetic change promotes HbS polymerization upon deoxygenation that can promote occlusion of small blood vessels that is often associated with increased blood viscosity, and circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-COHb; SANGUINATE) was designed as a novel therapeutic agent to initially release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. Delivery of either CO and/or O2 to hypoxic, sickled red blood cells (RBCs) should return cells to a more normal cell morphology and help re-establish normal blood flow and rheology. PEG-COHb was shown to mediate transfer of either a CO or O2/CO mixture and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying or slowing sickling, which should maintain normal blood flow through hypoxic microvasculature. Unsickling is expected to be expedited by O2 transfer by PEG-COHb. To examine these potential therapeutic effects, current in vitro studies examined the effects of time and dose of PEG-COHb to not only reverse, but also prevent or delay sickling by transferring CO as well as expedite atmospheric O2 transfer to the sickled RBCs.
Methods: Reversal of sickling studies were conducted by deoxygenating RBCs from healthy (control) and SCD volunteers in followed by treatment with either PEG-COHb, fully oxygenated PEG-Hb (PEG-O2Hb) or PEG-BSA for 2 hours. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-COHb and then subjected to hypoxia for 3 hours. Time-dose effects were quantified by area under the curve (AUC) analysis. O2 transfer studies were conducted by treating hypoxic, sickled RBCs to increasing concentrations of PEG-COHb and raising the pO2 from 3.8mm to 40mm. In all studies, the fractions of CO-Hb, O2-Hb and reduced Hb were determined by co-oximetry and sickled RBCs were quantified by imaging flow cytometry of fixed RBC specimens.
Results: PEG-COHb-mediated delivery of either CO or O2 can unsickle hypoxic SCD RBCs. Controls exhibited gas exchange similar to SCD RBCs. Interestingly, sickle reversion time-course studies showed differential kinetics between the CO and O2 capacity to cause unsickling. AUC analysis at 20 minutes demonstrated that both CO and O2 reversed sickling by 41% and 42%, respectively. PEG-O2Hb was able to exert substantial unsickling by 5 minutes, where PEG-COHb showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-COHb prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline (PBS). In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that although, not completly inhibited, sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-COHb increased the rate of unsickling as measured by AUC by 50% and 15% at 4 and 2 mg per mL, respectively. These levels are within the expected therapeutic dosage of SANGUINATE.
Summary: RBCs from patients with SCD undergo a conformational shift upon deoxygenation resulting in HbS polymerization and morphological changes of the RBCs. The occlusive and fragile properties of sickled RBCs are responsible for the development of the numerous comorbidities associated with SCD. It is only when the fraction of oxygenated or carboxylated HbS reaches a sufficient level that reversion to normal cell morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-COHb ability to deliver both O2 and CO to sickled RBC. These data suggested that PEG-COHb is a promising gas transfer agent that has the potential to improve sickle cell morphology by reversing sickling; the underlying pathology of sickle cell disease co-morbidities.

Disclosures
Buontempo: Prolong Pharmaceuticals: Employment. Yglesias:Prolong Pharmaceuticals: Employment. Chen:Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Jubin:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment.