Microvascular and Systemic Impact of Resuscitation with PEGylated Carboxyhemoglobin-Based Oxygen Carrier or Hetastarch in a Rat Model of Transient Hemorrhagic Shock.

BACKGROUND: Hemorrhage is the leading cause of preventable, traumatic death. Currently, prehospital resuscitation fluids provide preload but not oxygen-carrying capacity-a critical blood function that mitigates microvascular ischemia and tissue hypoxia during hemorrhagic shock. Solutions containing polymerized hemoglobin have been associated with vasoactive and hypertensive events. A novel hemoglobin-based oxygen carrier, modified with PEGylation and CO moieties (PEG-COHb), may overcome these limitations. OBJECTIVES: To evaluate the systemic and microcirculatory effects of PEG-COHb as compared with the 6% hetastarch in a rat model of hemorrhagic shock. METHODS: Male Sprague Dawley rats (N = 20) were subjected to severe, controlled, hemorrhagic shock. Animals were randomized to 20% estimated blood-volume resuscitation with either 6% hetastarch or PEG-COHb. Continuous, invasive, cardiovascular measurements, and arterial blood gases were measured. Microcirculatory measurements of interstitial oxygenation (PISFO2) and vasoactivity helped model oxygen delivery in the spinotrapezius muscle using intravital and phosphorescence quenching microscopy. RESULTS: Hemorrhage reduced mean arterial pressure (MAP), arteriolar diameter, and PISFO2, and increased lactate 10-fold in both groups. Resuscitation with both PEG-COHb and hetastarch improved cardiovascular parameters. However, PEG-COHb treatment resulted in higher MAP (P < 0.001), improved PISFO2 (14 [PEG-COHb] vs. 5 [hetastarch] mmHg; P < 0.0001), lower lactate post-resuscitation (P < 0.01), and extended survival from 90 to 142 min (P < 0.001) as compared with the hetastarch group. CONCLUSIONS: PEG-COHb improved MAP PISFO2, lactate, and survival time as compared with 6% hetastarch resuscitation. Importantly, hypertension and vasoactivity were not detected in response to PEG-COHb resuscitation supporting further investigation of this resuscitation strategy.

Alternatives to Transfusion.

OBJECTIVES: We discuss two main categories of blood substitutes: perfluorocarbons and hemoglobin-based oxygen carriers. METHODS: We provide a review of the notable products developed in both categories and include their attributes as well as their setbacks. RESULTS: We contribute a case report tothe growing literature of the successful use of Sanguinate. CONCLUSIONS: We find that artificial oxygen carriers are an attractive field of research because of the practical limitations and the multitude of potential complications that surround human blood transfusions.

Effect of Polyethylene-glycolated Carboxyhemoglobin on Renal Microcirculation in a Rat Model of Hemorrhagic Shock.

BACKGROUND: Primary resuscitation fluid to treat hemorrhagic shock remains controversial. Use of hydroxyethyl starches raised concerns of acute kidney injury. Polyethylene-glycolated carboxyhemoglobin, which has carbon monoxide-releasing molecules and oxygen-carrying properties, was hypothesized to sustain cortical renal microcirculatory PO2 after hemorrhagic shock and reduce kidney injury. METHODS: Anesthetized and ventilated rats (n = 42) were subjected to pressure-controlled hemorrhagic shock for 1 h. Renal cortical PO2 was measured in exposed kidneys using a phosphorescence quenching method. Rats were randomly assigned to six groups: polyethylene-glycolated carboxyhemoglobin 320 mg · kg, 6% hydroxyethyl starch (130/0.4) in Ringer's acetate, blood retransfusion, diluted blood retransfusion (~4 g · dl), nonresuscitated animals, and time control. Nitric oxide and heme oxygenase 1 levels were determined in plasma. Kidney immunohistochemistry (histologic scores of neutrophil gelatinase-associated lipocalin and tumor necrosis factor-α) and tubular histologic damages analyses were performed. RESULTS: Blood and diluted blood restored renal PO2 to 51 ± 5 mmHg (mean difference, -18; 95% CI, -26 to -11; P < 0.0001) and 47 ± 5 mmHg (mean difference, -23; 95% CI, -31 to -15; P < 0.0001), respectively, compared with 29 ± 8 mmHg for hydroxyethyl starch. No differences between polyethylene-glycolated carboxyhemoglobin and hydroxyethyl starch were observed (33 ± 7 mmHg vs. 29 ± 8 mmHg; mean difference, -5; 95% CI, -12 to 3; P = 0.387), but significantly less volume was administered (4.5 [3.3-6.2] vs. 8.5[7.7-11.4] ml; mean rank difference, 11.98; P = 0.387). Blood and diluted blood increased the plasma bioavailability of nitric oxide compared with hydroxyethyl starch (mean rank difference, -20.97; P = 0.004; and -17.13; P = 0.029, respectively). No changes in heme oxygenase 1 levels were observed. Polyethylene-glycolated carboxyhemoglobin limited tubular histologic damages compared with hydroxyethyl starch (mean rank difference, 60.12; P = 0.0012) with reduced neutrophil gelatinase-associated lipocalin (mean rank difference, 84.43; P < 0.0001) and tumor necrosis factor-α (mean rank difference, 49.67; P = 0.026) histologic scores. CONCLUSIONS: Polyethylene-glycolated carboxyhemoglobin resuscitation did not improve renal PO2 but limited tubular histologic damages and neutrophil gelatinase-associated lipocalin upregulation after hemorrhage compared with hydroxyethyl starch, whereas a lower volume was required to sustain macrocirculation.

Effects of Sanguinate on Systemic and Microcirculatory Variables in a Model of Prolonged Hemorrhagic Shock.

BACKGROUND: Hemorrhage and its complications are the leading cause of preventable death from trauma in young adults, especially in remote locations. To address this, deliverable, shelf-stable resuscitants that provide therapeutic benefits throughout the time course of hemorrhagic shock and the progressive ischemic injury it produces are needed. SANGUINATE is a novel bovine PEGylated carboxyhemoglobin-based oxygen carrier, which has desirable oxygen-carrying and oncotic properties as well as a CO moiety to maintain microvascular perfusion. OBJECTIVES: To compare the crystalloid (Lactated Ringer's Solution; LRS), and the colloid (Hextend) standards of care with SANGUINATE in a post "golden hour" resuscitation model. METHODS: Rats underwent a controlled, stepwise blood withdrawal (45% by volume), were maintained in untreated hemorrhagic shock state for >60 min, resuscitated with a 20% bolus of one of the three test solutions, and observed till demise. Parameters of tissue oxygenation (PISFO2), arteriolar diameters, and mean arterial pressure (MAP) were collected. RESULTS: SANGUINATE-treated animals survived significantly longer than those treated with Hextend and LRS. SANGUINATE also significantly increased tissue PISFO2 2 h after resuscitation, whereas LRS and Hextend did not. SANGUINATE also produced a significantly higher MAP, which was hypotensive compared to baseline, that endured until demise. CONCLUSIONS: Resuscitation with SANGUINATE after prolonged hemorrhagic shock improves survival, MAP, and PISFO2 compared with standard of care plasma expanders. Since the pathologies of hemorrhagic shock and the associated systemic ischemia are progressive, preclinical studies of this nature are essential to determine efficacy of new resuscitants across the range of possible times to treatment.

Bovine Hemoglobin-Based Oxygen Carrier Treatment in a Severely Anemic Jehovah's Witness Patient After Cystoprostatectomy and Nephrectomy: A Case Report.

Patients who are Jehovah's Witnesses are known to the medical community for frequently declining blood products, even at times of life-threatening anemia. Alternatives to red blood cell transfusion are being developed, including hemoglobin (Hb)-based oxygen carriers. We present the case of a 77-year-old male Jehovah's Witness who underwent a cystoprostatectomy and radical nephrectomy with a postoperative Hb nadir of 4.5 g/dL. He received an Hb-based oxygen carrier, PEGylated carboxyhemoglobin bovine (Sanguinate), with gradual improvement in anemia symptoms and eventual discharge to a short-term rehabilitation facility.

Pegylated Bovine Carboxyhemoglobin (SANGUINATE) in a Jehovah's Witness Undergoing Liver Transplant: A Case Report.

Liver transplantation in practicing Jehovah's Witnesses is challenging because of their religious beliefs preventing them from accepting allogenic blood products. Pegylated bovine carboxyhemoglobin (SANGUINATE) is an oxygen transfer agent, currently under investigation for the treatment of sickle cell disease, which may play a role in these patients by maximizing perioperative oxygen delivery. We report a case involving the use of SANGUINATE in a Jehovah's Witness undergoing liver transplant.

PEGylated carboxyhemoglobin bovine (SANGUINATE) ameliorates myocardial infarction in a rat model.

Artificial oxygen (O2 ) carriers were reported to be protective in ischemia/reperfusion (I/R) in various organs including the heart. In the current study, 20 rats underwent ligation (MI) of the left anterior descending artery, were treated with 10 mL/kg of PEGylated carboxyhemoglobin bovine (SANGUINATE, S+, n = 10) or saline (S-, n = 10) 10 minutes after MI and daily thereafter for 3 days, and were followed by weekly echocardiography for 4 weeks, when they had left ventricular pressure volume relationship (PVR) analyses followed by necropsy. Echocardiography showed an increase in end-systolic dimension rather than end-diastolic dimension, preserved fractional shortening (36 vs. 26%, P < .01), and milder mitral regurgitation in S+ compared with S- rats. PVR revealed a milder increase in end-systolic volume, larger stroke volume (101 vs. 74 µL, P < .005) and cardiac output (33.4 vs. 23.8 mL/min, P = .004) in S+ rats in actual determination and under a wide range of standardized loading conditions 4 weeks after MI. Excised heart showed significantly limited area of MI (8.9 vs. 13.3%, P = .028). The results suggest that SANGUINATE in short-term repeated doses may accelerate weight recovery, preserving the myocardium, mitral competence, and cardiac function after MI. The mechanism of action and optimal treatment for MI remain to be studied.

A phase Ib, open-label, single arm study to assess the safety, pharmacokinetics, and impact on humoral sensitization of SANGUINATE infusion in patients with end-stage renal disease.

The endeavor to study desensitization in kidney transplantation has not been matched by an effort to investigate strategies to prevent sensitization. In this study (NCT02437422), we investigated the safety, impact on sensitization, and pharmacokinetics of SANGUINATE (SG), a hemoglobin-based oxygen carrier, as a potential alternative to packed red blood cells (PRBC) in transplant candidates with end-stage renal disease (ESRD). Ten ESRD subjects meeting inclusion/exclusion (I/E) criteria were planned to receive three weekly infusions of SG (320 mg/kg). The study was stopped after five subjects were enrolled, and their data were analyzed after completing a follow-up period of 90 days. Two subjects had elevated troponin I levels in setting of SG infusion, one of which was interpreted as a non-ST elevation myocardial infarction. All other adverse events were transient. SG pharmacokinetic analysis showed mean (SD) Cmax , Tmax , AUC, and half-life of 4.39 (0.69) mg/mL, 2.42 (0.91) hours, 171.86 (52.35) mg h/mL, and 40.60 (11.96) hours, respectively. None of the subjects developed new anti-HLA antibodies following SG infusion and throughout the study period. In conclusion, SG is a potential alternative to PRBCs in ESRD patients considered for kidney transplantation as it was not associated with humoral sensitization. Larger studies in highly sensitized patients are required to further evaluate for potential safety signals.

SANGUINATE (PEGylated Carboxyhemoglobin Bovine): Mechanism of Action and Clinical Update.

Historically, blood substitutes were under development that would provide oxygen carrying capacity as well as fluid replacement for both trauma and surgical indications. Their development was halted by the inability of the products to deliver therapeutic amounts of oxygen targeted to hypoxic tissue as well as from the inherent toxicity of the molecules. This led to the concept of an oxygen therapeutic that would be targeted for indications caused by anemia/ischemia/hypoxia but would not exhibit the toxicity that plagued earlier products. The complex pathophysiology of diseases such as sickle cell and hemorrhagic stroke not only has hypoxia as a pivotal event but also includes inflammation and vasoconstriction that perpetuate the oxygen deprivation. There is a need for an effective therapeutic that addresses the multiple events of inflammation and oxygen deprivation. SANGUINATE acts as a dual mode carbon monoxide (CO) and oxygen delivery therapeutic. SANGUINATE is designed not only to treat hypoxia but also to act on concurrent pathologies such as inflammation and reperfusion injury. This expands the potential therapeutic utility of SANGUINATE beyond anemia into indications such as early brain injury and delayed kidney graft function, where inflammation plays a pivotal pathological role as well as in indications such as sickle cell disease where the inflammation and hypoxia contribute to the development of comorbidities such as vaso-occlusive crisis. Clinical trials in multiple indications are underway.

Use of hyperbaric oxygen therapy and PEGylated carboxyhemoglobin bovine in a Jehovah's Witness with life-threatening anemia following postpartum hemorrhage.

We present a case of a Jehovah's Witness patient who refused blood products, with the exception of albumin and clotting factors, and underwent cesarean section under spinal anesthesia complicated by postpartum hemorrhage. She was fluid resuscitated and treated with multiple uterotonics and internal iliac artery embolization. Because of agitation she required emergency tracheal intubation. Her hemoglobin concentration dropped from a preoperative value of 12mg/dL to 3mg/dL on postoperative day one. She was acidotic, requiring vasopressors for hemodynamic stability and remained ventilated and sedated. She was treated with daily erythropoietin, iron therapy and cyanocobalamin. Because of ongoing hemorrhage, continued acidemia and vasopressor requirements she was co-treated with PEGylated carboxyhemoglobin bovine and hyperbaric oxygen therapy to reverse her oxygen debt. On postoperative day eight her hemoglobin concentration was 7mg/dL, she was hemodynamically stable and vasopressors were discontinued. She was extubated and discharged from the intensive care unit on postoperative day eight. This report highlights the multiple modalities used in treating a severely anemic patient who refused blood, the use of an investigational new drug, the process of obtaining this drug via the United States Food and Drug Administration emergency expanded access regulation for single patient clinical treatment, and ethical dilemmas faced during treatment.

PEGylated carboxyhemoglobin bovine (SANGUINATE): results of a phase I clinical trial.

PEGylated carboxyhemoglobin bovine (SANGUINATE) is a dual action carbon monoxide releasing (CO)/oxygen (O2 ) transfer agent for the treatment of hypoxia. Its components inhibit vasoconstriction, decrease extravasation, limit reactive oxygen species production, enhance blood rheology, and deliver oxygen to the tissues. Animal models of cerebral ischemia, peripheral ischemia, and myocardial ischemia demonstrated SANGUINATE's efficacy in reducing myocardial infarct size, limiting necrosis from cerebral ischemia, and promoting more rapid recovery from hind limb ischemia. In a Phase I trial, three cohorts of eight healthy volunteers received single ascending doses of 80, 120, or 160 mg/kg of SANGUINATE. Two volunteers within each cohort served as a saline control. There were no serious adverse events. Serum haptoglobin decreased, but did not appear to be dose related. The T1/2 was dose dependent and ranged from 7.9 to 13.8 h. In addition to the Phase I trial, SANGUINATE was used under an expanded access emergency Investigational New Drug. SANGUINATE was found to be safe and well tolerated in a Phase I clinical trial, and therefore it will advance into further clinical trials in patients.

Early treatment of transient focal cerebral ischemia with bovine PEGylated carboxy hemoglobin transfusion.

The effect of transfusion of PEGylated hemoglobin (PEG-Hb) was evaluated in anesthetized rats subjected to 2 hours of focal cerebral ischemia and 1 day of reperfusion. PEG-Hb was stored in the carboxy state (PEG-COHb) to reduce autooxidation and increase the shelf life. Transfusion of 10 ml/kg of PEG-COHb at 20 minutes of ischemia did not alter arterial blood pressure or increase red cell flux in the ischemic core. Plasma hemoglobin increased to only 0.6 g/dL, yet infarct volume was markedly decreased and neurological deficits were improved. We conclude that early topload transfusion of PEG-COHb protects the brain from ischemic stroke.