Resuscitation with PEGylated carboxyhemoglobin preserves renal cortical oxygenation and improves skeletal muscle microcirculatory flow during endotoxemia.

PEGylated carboxyhemoglobin (PEGHbCO), which has carbon monoxide-releasing properties and plasma expansion and oxygen-carrying properties, may improve both skeletal microcirculatory flow and renal cortical microcirculatory Po2 (CµPo2) and, subsequently, limit endotoxemia-induced acute kidney injury. Anesthetized, ventilated Wistar albino rats (n = 44) underwent endotoxemic shock. CµPo2 was measured in exposed kidneys using a phosphorescence-quenching method. Rats were randomly assigned to the following five groups: 1) unresuscitated lipopolysaccharide (LPS), 2) LPS + Ringer's acetate (RA), 3) LPS + RA + 0.5 µg·kg·-1min-1 norepinephrine (NE), 4) LPS + RA + 320 mg/kg PEGHbCO, and 5) LPS + RA + PEGHbCO + NE. The total volume was 30 mL/kg in each group. A time control animal group was used. Skeletal muscle microcirculation was assessed by handheld intravital microscopy. Kidney immunohistochemistry and myeloperoxidase-stained leukocytes in glomerular and peritubular areas were analyzed. Endotoxemia-induced histological damage was assessed. Plasma levels of IL-6, heme oxygenase-1, malondialdehyde, and syndecan-1 were assessed by ELISA. CµPo2 was higher in the LPS + RA + PEGHbCO-resuscitated group, at 35 ± 6mmHg compared with 21 ± 12 mmHg for the LPS+RA group [mean difference: -13.53, 95% confidence interval: (-26.35; -0.7156), P = 0.035]. The number of nonflowing, intermittent, or sluggish capillaries was smaller in groups infused with PEGHbCO compared with RA alone (P < 0.05), while the number of normally perfused vessels was greater (P < 0.05). The addition of NE did not further improve CµPo2 or microcirculatory parameters. Endotoxemia-induced kidney immunohistochemistry and histological alterations were not mitigated by PEGHbCO 1 h after resuscitation. Renal leukocyte infiltration and plasma levels of biomarkers were similar across groups. PEGHbCO enhanced CµPo2 while restoring skeletal muscle microcirculatory flow in previously nonflowing capillaries. PEGHbCO should be further evaluated as a resuscitation fluid in mid- to long-term models of sepsis-induced acute kidney injury.

Investigational use of PEGylated carboxyhemoglobin bovine in a Jehovah's Witness with hemorrhagic shock.

BACKGROUND: Jehovah's Witnesses pose a clinical challenge in the setting of critical anemia. Most do not accept transfusions, but some accept hemoglobin-based oxygen carriers on a compassionate-use basis. PEGylated carboxyhemoglobin bovine (PCHB) is an acellular dual-action carbon monoxide (CO)-releasing and oxygen transfer agent currently being investigated in Phase II clinical trials. CASE REPORT: We present the case of a 42-year-old Jehovah's Witness with an acute upper gastrointestinal bleed and hemorrhagic shock who required emergent PCHB for stabilization during lifesaving interventions. After PCHB infusion, the patient's shock and encephalopathy improved with decreased vasopressor requirement. Through gastroenterology and interventional radiology procedures, the patient's bleeding stabilized. While receiving five additional doses of PCHB and other supportive therapies (iron, folate, vitamin B12, darbepoetin alfa), the patient was extubated and weaned off vasopressors. CONCLUSIONS: PCHB was used to stabilize (bridge) a critically ill anemic patient for lifesaving interventions without adverse effects. Additional studies are warranted to explore the drug's safety profile and efficacy in patients declining blood products.

SANGUINATE™ (PEGylated Carboxyhemoglobin Bovine) Improves Cerebral Blood Flow to Vulnerable Brain Regions at Risk of Delayed Cerebral Ischemia After Subarachnoid Hemorrhage.

BACKGROUND: Delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH) has been linked to focal reductions in cerebral blood flow (CBF) and microvascular impairments in oxygen delivery. Effective therapies that restore flow and oxygen transport to vulnerable brain regions are currently lacking. SANGUINATE is a dual-action carbon monoxide-releasing and hemoglobin-based oxygen transfer agent with efficacy in animal models of focal brain ischemia and tolerability in patients with sickle cell disease. METHODS: We performed a safety and proof-of-principle study in 12 SAH patients at risk of DCI across three escalating doses (160, 240, and 320 mg/kg). We used 15O-PET (performed at baseline, after SANGUINATE and at 24 h) to evaluate efficacy for improving CBF and restoring flow-metabolism balance (assessed by oxygen extraction fraction [OEF]) to vulnerable regions (defined as baseline OEF ≥ 0.50). RESULTS: SANGUINATE resulted in a transient rise in mean arterial pressure (116 ± 15-127 ± 13 mm Hg, p = 0.001) that normalized by 24 h and allowed three patients with DCI to be weaned off vasopressors. No adverse events were noted during infusion. Global CBF did not rise (43 ± 8-46 ± 9 ml/100 g/min) although a trend was seen at the highest dose (45 ± 7-51 ± 9, p = 0.044). However, a significant 16% rise in regional CBF associated with reduction in OEF was seen in vulnerable regions, but did not persist at 24 h. CONCLUSIONS: We demonstrated that this novel agent can improve regional CBF and may improve oxygen supply-demand balance. Clinical studies (likely with repeat dosing) are required to evaluate whether this effect can prevent DCI or cerebral infarction.

Pegylated bovine carboxyhaemoglobin utilisation in a thrombotic thrombocytopenic purpura patient.

OBJECTIVE: To determine if pegylated bovine carboxyhaemoglobin can be utilised in a thrombotic thrombocytopenic purpura (TTP) patient. BACKGROUND: TTP is a condition characterized by thrombotic microangiopathy and has a high mortality rate when left untreated. Therapeutic plasma exchange is well established as the most effective and evidence-based treatment of TTP. METHODS: The ability to administer plasma exchange therapy is limited in Jehovah's Witnesses who decline blood products due to religious beliefs. Pegylated bovine carboxyhaemoglobin is a novel oxygen transfer agent in development for the management of complications of ischaemia due to acute anaemia. RESULTS: Treatment was well tolerated, with grade 1 paresthesia of the right face and arm 1 h after the first infusion of Sanguinate, which spontaneously resolved and did not recur, and grade 1 cardiac troponin elevation after receiving the medication (with peak at 0·079 ng mL-1 ), but further workup with electrocardiogram and echocardiogram was unremarkable. By discharge on day 19, the patient's haemoglobin increased to 8·8 g dL-1 and platelet count to 221 000. CONCLUSIONS: We report the first case of TTP in a Jehovah's Witness that was successfully managed with the use of pegylated bovine carboxyhaemoglobin as an adjunct medication.

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.

Transfusion of hemoglobin-based oxygen carriers in the carboxy state is beneficial during transient focal cerebral ischemia.

Exchange transfusion of large volumes of hemoglobin (Hb)-based oxygen carriers can protect the brain from middle cerebral artery occlusion (MCAO). Hb in the carboxy state (COHb) may provide protection at relatively low volumes by enhancing vasodilation. We determined whether transfusion of rats with 10 ml/kg PEGylated COHb [polyethylene glycol (PEG)-COHb] at 20 min of 2-h MCAO was more effective in reducing infarct volume compared with non-carbon monoxide (CO) PEG-Hb. After PEG-COHb transfusion, whole blood and plasma COHb was <3%, indicating rapid release of CO. PEG-COHb transfusion significantly reduced infarct volume (15 ± 5% of hemisphere; mean ± SE) compared with that in the control group (35 ± 6%), but non-CO PEG-Hb did not (24 ± 5%). Chemically dissimilar COHb polymers were also effective. Induction of MCAO initially produced 34 ± 2% dilation of pial arterioles in the border region that subsided to 10 ± 1% at 2 h. Transfusion of PEG-COHb at 20 min of MCAO maintained pial arterioles in a dilated state (40 ± 5%) at 2 h, whereas transfusion of non-CO PEG-Hb had an intermediate effect (22 ± 3%). When transfusion of PEG-COHb was delayed by 90 min, laser-Doppler flow in the border region increased from 57 ± 9 to 82 ± 13% of preischemic baseline. These data demonstrate that PEG-COHb is more effective than non-CO PEG-Hb at reducing infarct volume, sustaining cerebral vasodilation, and improving collateral perfusion in a model of transient focal cerebral ischemia when given at a relatively low dose (plasma Hb concentration < 1 g/dl). Use of acellular Hb as a CO donor that is rapidly converted to an oxygen carrier in vivo may permit potent protection at low transfusion volumes.

The formation of a crosslinked carboxyhemoglobin membrane at an organic-aqueous interface.

A 30% carboxyhemoglobin solution was encapsulated with a membrane composed of crosslinked hemoglobin as a potential artificial red blood cell by adding a 25 microL droplet of 30% carboxyhemoglobin solution in phosphate buffer to a buffer-saturated toluene solution of a homobifunctional crosslinking agent. Disuccinimidyl glutarate, disuccinimidyl suberate, and terephthaldicarboxaldehyde proved successful in crosslinking hemoglobin at concentrations of 0.05%, 0.08%, and 4.3%, respectively (saturated levels in buffer-saturated toluene). Disuccinimidyl glutarate exhibited maximal crosslinking of 11.0% in approximately 30 minutes. Disuccinimidyl suberate and terephthaldicarboxaldehyde required at least 12 hours to form a viable sphere and crosslinked 39.4% and 37.8% of the hemoglobin in 18 hours. Membrane integrity was assessed by determining the permeability of uncrosslinked hemoglobin through the membrane as a function of time.