PEG-HBCO MEDIATED CARBON MONOXIDE AND OXYGEN TRANSFER TO HYPOXIC RED BLOOD CELLS PREVENT, SLOW, AND/OR REVERSE SICKLING IN VITRO.
(Abstract release date: 05/19/16)
EHA Library. Jubin R. 06/09/16; 132953; E1404
Disclosure(s): Ronald G Jubin is an employee of Prolong Pharmaceuticals.
Dr. Ronald Jubin
Contributions
Contributions
Abstract
Abstract: E1404
Type: Eposter Presentation
Background
In Sickle Cell disease (SCD), a mutation promotes HbS polymerization causing abnormal cell morphology that results in occlusion of small blood vessels and increased circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-HbCO; SANGUINATE) is a novel therapeutic agent designed to release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. PEG-HbCO was shown to mediate transfer of either a CO or O2 and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying sickling, which should maintain normal blood flow through hypoxic microvasculature.
Aims
Current in vitro studies examined the effects of time and dose of PEG-HbCO to not only reverse, but also prevent or delay sickling by transferring CO and expedite atmospheric O2 transfer to the sickled RBCs.
Methods
Reversal of sickling were conducted by deoxygenating RBCs from healthy and SCD volunteers followed by treatment with either PEG-HbCO, fully oxygenated PEG-Hb (PEG-HbO2) or PEG-BSA. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-HbCO 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-HbCO 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-HbCO -mediated delivery of either CO or O2 unsickles SCD RBCs. The 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-HbO2 was able to exert substantial unsickling by 5 minutes, where PEG-HbCO showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-HbCO prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline. In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-HbCO 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 PEG-HbCO.
Conclusion
RBCs from patients with SCD undergo morphological changes. It is only when the fraction of oxygenated HbS reaches a sufficient level that reversion to normal morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-HbCO ability to deliver both O2 and CO to sickled RBC. Additionally since ASH 2016, PEG-HbCO was shown to improve rheology and down-regulate inflammation in a hemorrhagic shock model; these pathologies also exist in SCD. These data suggest that PEG-HbCO 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 as well as decrease hypoxia-induced inflammation. SCD patients with hyperhemolysis and acute chest syndrome have been treated with PEG-HbCO under eINDs. Phase 2 trials with PEG-HbCO are underway in SCD vaso-occlusive crisis.
Session topic: E-poster
Keyword(s): Hypoxia-sensing, Sickle cell, Vasoocclusive crisis
Type: Eposter Presentation
Background
In Sickle Cell disease (SCD), a mutation promotes HbS polymerization causing abnormal cell morphology that results in occlusion of small blood vessels and increased circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-HbCO; SANGUINATE) is a novel therapeutic agent designed to release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. PEG-HbCO was shown to mediate transfer of either a CO or O2 and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying sickling, which should maintain normal blood flow through hypoxic microvasculature.
Aims
Current in vitro studies examined the effects of time and dose of PEG-HbCO to not only reverse, but also prevent or delay sickling by transferring CO and expedite atmospheric O2 transfer to the sickled RBCs.
Methods
Reversal of sickling were conducted by deoxygenating RBCs from healthy and SCD volunteers followed by treatment with either PEG-HbCO, fully oxygenated PEG-Hb (PEG-HbO2) or PEG-BSA. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-HbCO 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-HbCO 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-HbCO -mediated delivery of either CO or O2 unsickles SCD RBCs. The 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-HbO2 was able to exert substantial unsickling by 5 minutes, where PEG-HbCO showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-HbCO prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline. In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-HbCO 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 PEG-HbCO.
Conclusion
RBCs from patients with SCD undergo morphological changes. It is only when the fraction of oxygenated HbS reaches a sufficient level that reversion to normal morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-HbCO ability to deliver both O2 and CO to sickled RBC. Additionally since ASH 2016, PEG-HbCO was shown to improve rheology and down-regulate inflammation in a hemorrhagic shock model; these pathologies also exist in SCD. These data suggest that PEG-HbCO 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 as well as decrease hypoxia-induced inflammation. SCD patients with hyperhemolysis and acute chest syndrome have been treated with PEG-HbCO under eINDs. Phase 2 trials with PEG-HbCO are underway in SCD vaso-occlusive crisis.
Session topic: E-poster
Keyword(s): Hypoxia-sensing, Sickle cell, Vasoocclusive crisis
Abstract: E1404
Type: Eposter Presentation
Background
In Sickle Cell disease (SCD), a mutation promotes HbS polymerization causing abnormal cell morphology that results in occlusion of small blood vessels and increased circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-HbCO; SANGUINATE) is a novel therapeutic agent designed to release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. PEG-HbCO was shown to mediate transfer of either a CO or O2 and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying sickling, which should maintain normal blood flow through hypoxic microvasculature.
Aims
Current in vitro studies examined the effects of time and dose of PEG-HbCO to not only reverse, but also prevent or delay sickling by transferring CO and expedite atmospheric O2 transfer to the sickled RBCs.
Methods
Reversal of sickling were conducted by deoxygenating RBCs from healthy and SCD volunteers followed by treatment with either PEG-HbCO, fully oxygenated PEG-Hb (PEG-HbO2) or PEG-BSA. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-HbCO 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-HbCO 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-HbCO -mediated delivery of either CO or O2 unsickles SCD RBCs. The 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-HbO2 was able to exert substantial unsickling by 5 minutes, where PEG-HbCO showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-HbCO prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline. In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-HbCO 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 PEG-HbCO.
Conclusion
RBCs from patients with SCD undergo morphological changes. It is only when the fraction of oxygenated HbS reaches a sufficient level that reversion to normal morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-HbCO ability to deliver both O2 and CO to sickled RBC. Additionally since ASH 2016, PEG-HbCO was shown to improve rheology and down-regulate inflammation in a hemorrhagic shock model; these pathologies also exist in SCD. These data suggest that PEG-HbCO 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 as well as decrease hypoxia-induced inflammation. SCD patients with hyperhemolysis and acute chest syndrome have been treated with PEG-HbCO under eINDs. Phase 2 trials with PEG-HbCO are underway in SCD vaso-occlusive crisis.
Session topic: E-poster
Keyword(s): Hypoxia-sensing, Sickle cell, Vasoocclusive crisis
Type: Eposter Presentation
Background
In Sickle Cell disease (SCD), a mutation promotes HbS polymerization causing abnormal cell morphology that results in occlusion of small blood vessels and increased circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-HbCO; SANGUINATE) is a novel therapeutic agent designed to release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. PEG-HbCO was shown to mediate transfer of either a CO or O2 and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying sickling, which should maintain normal blood flow through hypoxic microvasculature.
Aims
Current in vitro studies examined the effects of time and dose of PEG-HbCO to not only reverse, but also prevent or delay sickling by transferring CO and expedite atmospheric O2 transfer to the sickled RBCs.
Methods
Reversal of sickling were conducted by deoxygenating RBCs from healthy and SCD volunteers followed by treatment with either PEG-HbCO, fully oxygenated PEG-Hb (PEG-HbO2) or PEG-BSA. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-HbCO 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-HbCO 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-HbCO -mediated delivery of either CO or O2 unsickles SCD RBCs. The 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-HbO2 was able to exert substantial unsickling by 5 minutes, where PEG-HbCO showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-HbCO prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline. In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-HbCO 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 PEG-HbCO.
Conclusion
RBCs from patients with SCD undergo morphological changes. It is only when the fraction of oxygenated HbS reaches a sufficient level that reversion to normal morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-HbCO ability to deliver both O2 and CO to sickled RBC. Additionally since ASH 2016, PEG-HbCO was shown to improve rheology and down-regulate inflammation in a hemorrhagic shock model; these pathologies also exist in SCD. These data suggest that PEG-HbCO 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 as well as decrease hypoxia-induced inflammation. SCD patients with hyperhemolysis and acute chest syndrome have been treated with PEG-HbCO under eINDs. Phase 2 trials with PEG-HbCO are underway in SCD vaso-occlusive crisis.
Session topic: E-poster
Keyword(s): Hypoxia-sensing, Sickle cell, Vasoocclusive crisis
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