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The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.

For the past thirty years, hemoglobin-based oxygen carriers (HBOCs) have been under development as a red blood cell substitute. Side-effects such as vasoconstriction, oxidative injury, and cardiac toxicity have prevented clinical approval of HBOCs. Recently, high molecular weight (MW) polymerized human hemoglobin (PolyhHb) has shown positive results in rats. Studies have demonstrated that high MW PolyhHb increased O2 delivery, with minimal effects on blood pressure, without vasoconstriction, and devoid of toxicity. In this study, we used guinea pigs to evaluate the efficacy and safety of high MW PolyhHb, since like humans guinea pigs cannot produce endogenous ascorbic acid, which limits the capacity of both species to deal with oxidative stress. Hence, this study evaluated the efficacy and safety of resuscitation from severe hemorrhagic shock with high MW PolyhHb, fresh blood, and blood stored for 2 weeks. Animals were randomly assigned to each experimental group, and hemorrhage was induced by the withdrawal of 40% of the blood volume (BV, estimated as 7.5% of body weight) from the carotid artery catheter. Hypovolemic shock was maintained for 50 min. Resuscitation was implemented by infusing 25% of the animal's BV with the different treatments. Hemodynamics, blood gases, total hemoglobin, and lactate were not different before hemorrhage and during shock between groups. The hematocrit was lower for the PolyhHb group compared to the fresh and stored blood groups after resuscitation. Resuscitation with stored blood had lower blood pressure compared to fresh blood at 2 h. There was no difference in mean arterial pressure between groups at 24 h. Resuscitation with PolyhHb was not different from fresh blood for most parameters. Resuscitation with PolyhHb did not show any remarkable change in liver injury, inflammation, or cardiac damage. Resuscitation with stored blood showed changes in liver function and inflammation, but no kidney injury or systemic inflammation. Resuscitation with stored blood after 24 h displayed sympathetic hyper-activation and signs of cardiac injury. These results suggest that PolyhHb is an effective resuscitation alternative to blood. The decreased toxicities in terms of cardiac injury markers, vital organ function, and inflammation following PolyhHb resuscitation in guinea pigs indicate a favorable safety profile. These results are promising and support future studies with this new generation of PolyhHb as alternative to blood when blood is unavailable.

Amelioration of immune overactivity during sepsis is key to restoring hemodynamics, microvascular blood flow, and tissue oxygenation, and in preventing multi-organ dysfunction syndrome. The systemic inflammatory response syndrome that results from sepsis ultimately leads to degradation of the endothelial glycocalyx and subsequently increased vascular leakage. Current fluid resuscitation techniques only transiently improve outcomes in sepsis, and can cause edema. Nitric oxide (NO) treatment for sepsis has shown promise in the past, but implementation is difficult due to the challenges associated with delivery and the transient nature of NO. To address this, we tested the anti-inflammatory efficacy of sustained delivery of exogenous NO using i.v. infused NO releasing nanoparticles (NO-np). The impact of NO-np on microhemodynamics and immune response in a lipopolysaccharide (LPS) induced endotoxemia mouse model was evaluated. NO-np treatment significantly attenuated the pro-inflammatory response by promoting M2 macrophage repolarization, which reduced the presence of pro-inflammatory cytokines in the serum and slowed vascular extravasation. Combined, this resulted in significantly improved microvascular blood flow and 72-h survival of animals treated with NO-np. The results from this study suggest that sustained supplementation of endogenous NO ameliorates and may prevent the morbidities of acute systemic inflammatory conditions. Given that endothelial dysfunction is a common denominator in many acute inflammatory conditions, it is likely that NO enhancement strategies may be useful for the treatment of sepsis and other acute inflammatory insults that trigger severe systemic pro-inflammatory responses and often result in a cytokine storm, as seen in COVID-19.

BackgroundHemoglobin (Hb)-based oxygen carriers (HBOCs) have been proposed as alternatives to blood for decades. Previous studies demonstrated that large molecular diameter HBOCs based on polymerized bovine Hb (PolybHb) attenuate Hb side-effects and toxicity. The objective of this study was to test the safety and efficacy of tense state PolybHb after long-term storage.Methods and resultsPolybHb was subjected to diafiltration to remove low molecular weight (< 500 kDa) species and stored for 2 years. PolybHb was studied in parallel with blood, collected from rats and stored leukodepleted under blood bank conditions for 3 weeks. Rats were hemorrhaged and resuscitated to 90% of the blood pressure before the hemorrhage with fresh blood, stored blood, fresh PolybHb, or 2-year-stored PolybHb. Hemorrhagic shock impaired oxygen delivery and cardiac function. Resuscitation restored blood pressure and cardiac function, but stored blood required a significantly larger transfusion volume to recover from shock compared with fresh blood and PolybHb (fresh and stored). Stored blood transfusion elevated markers of organ damage compared with all other groups.ConclusionsThese studies indicate that large molecular diameter PolybHb is as efficacious as fresh blood in restoring cardiac function and confirm the lack of degradation of PolybHb's safety or efficacy during long-term storage.

Acclimatization to hypoxia or high altitude involves physiological adaptation processes, to influence oxygen (O2) transport and utilization. Several natural products, including aromatic aldehydes and isothiocyanates stabilize the R-state of hemoglobin (Hb), increasing Hb-O2 affinity and Hb-O2 saturation. These products are a counter intuitive therapeutic strategy to increase O2 delivery during hypoxia. 5-Hydroxymethylfurfural (5-HMF) is well known Amadori compound formed during the Maillard reaction (the non-enzymatic browning and caramelization of carbohydrate-containing foods after thermal treatment), with well documented effects in Hb-O2 affinity. This study explores the therapeutic potential of 5-HMF on left ventricular (LV) cardiac function (LVCF) during hypoxia. Anesthetized Golden Syrian hamsters received 5-HMF i.v., at 100 mg/kg and were subjected to stepwise increased hypoxia (15, 10, and 5%) every 30 min. LVCF was assessed using a closed chest method with a miniaturized conductance catheter via continuous LV pressure-volume (PV) measurements. Heart hypoxic areas were studied using pimonidazole staining. 5-HMF improved cardiac indices, including stroke volume (SV), cardiac output (CO), ejection fraction (EF), and stroke work (SW) compared to the vehicle group. At 5% O2, SV, CO, EF, and SW were increased by 53, 42, 33, and 51% with 5-HMF relative to vehicle. Heart chronotropic activity was not statistically changed, suggesting that differences in LV-CF during hypoxia by 5-HMF were driven by volume dependent effects. Analysis of coronary blood flow and cardiac muscle metabolism suggest no direct pharmacological effects from 5-HMF, therefore these results can be attributed to 5-HMF-dependent increase in Hb-O2 affinity. These studies establish that naturally occurring aromatic aldehydes, such as 5-HMF, produce modification of hemoglobin oxygen affinity with promising therapeutic potential to increase O2 delivery during hypoxic hypoxia.

Therapeutic agents that increase the Hb affinity for oxygen (O2) could, in theory, lead to decreased O2 release from Hb and impose a hypoxic risk to tissues. In this study, GBT1118, an allosteric modifier of Hb affinity for O2, was used to assess the impact of increasing Hb affinity for O2 on brain tissue oxygenation, blood pressure, heart rate, O2 delivery, and tolerance to hypoxia in Townes transgenic sickle cell disease (SCD) mice. Brain oxygenation and O2 delivery were studied during normoxia and severe hypoxic challenges. Chronic treatment with GBT1118 increased Hb affinity for O2, reducing the PO2 for 50% HbO2 saturation (P50) in SCD mice from 31 mmHg to 18 mmHg. This treatment significantly reduced anemia, increasing hematocrit by 33%, improved cardiac output (CO), and O2 delivery and extraction. Chronically increasing Hb affinity for O2 with GBT1118 preserved cortical O2 tension during normoxia, improved cortical O2 tension during hypoxia, and increased tolerance to severe hypoxia in SCD mice. Independent of hematological changes induced by chronic treatment, a single dose of GBT1118 significantly improved tolerance to hypoxia, highlighting the benefits of increasing Hb affinity for O2 and consequently reducing sickling of RBCs in blood during hypoxia in SCD. [ABSTRACT FROM AUTHOR]