Rapid Reversal of Red Blood Cell Sickling Promoted By Pegylated Carboxyhemoglobin Bovine Gas Transfer Properties
Authors: Jubin Ronald , Buontempo Peter , Yglesias Ricardo A , Abuchowski Abe , Chen Yuli , Kazo Glenn , Kazo Friedericke 
Published: October 2019

Abstract
Background: Red blood cell (RBC) sickling is the hallmark phenotype of Sickle Cell Disease (SCD) resulting from an inherited hemoglobin (HbS) point mutation genotype. Sickling is a direct consequence of HbS polymerization that occurs as RBC encounter low oxygen environments. Sickled RBCs have increased rigidity restricting their passage through the microvasculature that can lead to a vaso-occlusive crisis (VOC). VOCs are painful, debilitating and can be associated with more severe co morbidities including acute chest syndrome, leg ulcers and stroke. Treatments for VOC are limited and include IV hydration and opioid for pain control. Extensive VOC events often require hospitalization, supplemental oxygen and transfusions. Improved therapies that can be administered early during VOC that directly target the causative RBC sickling events are warranted.
PEGylated carboxyhemoglobin bovine (Sanguinate SG) is currently in clinical development being evaluated for the ability to treat the comorbidities of SCD including VOC. SG carries oxygen (O2) and/or carbon monoxide (CO) in the mammalian vasculature for delivery to tissues to synergistically treat inflammation and vasoconstriction and other comorbidities characterized by tissue hypoxia. Early intervention of VOC with SG treatment could limit the crisis event and reduce pain severity while providing a timely crisis resolution thereby reducing the likelihood of more severe VOC clinically related complications.
Methods: SG treatment effects were evaluated under controlled environmental conditions to determine its capacity for gas exchange with RBCs obtained with IRB consent from healthy and SCD volunteers. Carboxyhemoglobin and oxyhemoglobin levels were monitored to determine dose and time effects as well as the repetitive capacity of SG to facilitate gas transfer processes. RBC treated samples were also analyzed by light microscopy and image capture flow cytometry to visualize and quantify the effects of SG treatment on reversing sickled SCD RBC. A PEGylated bovine serum albumin (PBSA) product was also produced and analyzed in parallel as a control agent.
Results: SG addition to normal oxygenated RBC resulted in CO and O2 exchange between RBC and SG that followed mass balance and reached equilibrium in closed system. Kinetic analysis revealed SG rapidly transferred its CO component to oxygenated RBC with concomitant O2 loading of SG. Using experimentally loaded RBC with CO and SG with O2 produced similar reciprocal gas exchange results. Additionally the primary RBC/ SG reaction products were isolated and cycled demonstrating the ability of SG to continually facilitate gas transfer through multiple exposure events.
Similar studies using SG, oxygenated SG (produced by an RBC exchange reaction) or PBSA control were conducted with SCD RBC. Sickling was induced by incubation of RBC in a hypoxic chamber for 3 hours prior to SG or control treatments. After 2 hours of treatment, cells were fixed by addition of glutaraldehyde. Photomicroscopy showed a marked reduction in the sickled RBC population with both SG or oxygenated SG treatments but not with the PBSA control (Fig. 1). Results from imaging flow cytometry further supported the microscopy findings and revealed a significant quantitative reduction in the percentage of sickled RBC levels.
Summary: RBC rheology and lifespan are negatively impacted by repetitive HbS polymerization/de-polymerization cycling and treatments that reverse RBC sickling during a VOC event could be expected to provide broad clinical benefits through reduction of SCD comorbidities. SG was designed to promote CO and oxygen transfer in concentration dependent manners providing physiological supplementation of O2 transport/delivery in conditions of hemolytic or ischemic anemia.
These ex vivo data demonstrated for the first time that under controlled conditions, a therapeutic agent could be observed serving as an active gas transport agent providing either CO or O2 to sickled RBCs, prompting rapid unsickling. Furthermore, the use of image capture flow cytometry provided a quantitative measurement of sickled RBC fraction decrease. This mechanism may provide a useful biomarker test in future clinical studies to monitor SG treatment effects on SCD patients.
Fig. 1 SG Treatment of Sickled RBC. Hypoxic SCD RBC treated with SG, oxygenated SG or PBSA for 2 hrs prior to fixation and image capture (40X mag). Fig. 1. SG Treatment of Sickled RBC. Hypoxic SCD RBC treated with SG, oxygenated SG or PBSA for 2 hrs prior to fixation and image capture (40X mag).

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