Hemoglobin-based Oxygen Carriers
Early attempts in this field used just acellular hemoglobin solutions to theoretically deliver oxygen. However, acellular Hb has three main problems as an oxygen carrier.
Acellular Hb has too high an affinity for oxygen. The p50 for acellular Hb is 10 to 15 mm Hg vs 26 to 28 mm Hg for intact RBC. Therefore, with a decreased p50, the oxygen is held more tightly and is not offloaded easily to the tissues. The higher oxygen affinity of the acellular hemoglobin solution is a result of 2,3-diphosphoglycerate being lost during the purification process. On a related note, without its protective cell membrane, Hb also has too short of an intravascular half-life to be very useful (T1/2 < 1.5 hours).
The tetramers of Hb readily dissociate into α-β dimers when not bound inside an RBC. These dimers are then easily excreted into kidneys, pass through the glomeruli, and subsequently cause significant renal tubular toxicity.[8,9]
Acellular Hb rapidly attaches to nitric oxide (NO). As oxygen binds to Hb, NO binds to a specific cysteine amino acid of Hb. Furthermore, the iron on the hemoglobin molecule is positioned to prevent the bound NO from degradation. When NO is unable to be released to perform its physiologic role of vasodilation, it leads to vasoconstriction and subsequently to the release of proinflammatory mediators, such as interleukin 1, tumor necrosis factor α, and interferon γ, among others. In addition, with decreased NO in the circulation, there is a loss of active platelet inhibition. Thus, when combined, these effects create ideal conditions that can lead to vascular thromboses.
Recent hemoglobin-based oxygen carriers (HBOCs) were developed to solve these problems with infusing acellular Hb. Researchers were able to synthetically modify Hb to address these toxicities. Initially, researchers used two mammalian sources of Hb in the developments of HBOCs: bovine Hb and human Hb from outdated RBC units. Bovine Hb does have key differences from human Hb, including a lower oxygen affinity and a substantially different amino acid sequence (74/574 residues differ). However, somewhat counterintuitively, no clinically serious immune responses have been reported. The use of bovine Hb solves the first problem, high oxygen affinity of acellular human Hb, as listed above.
To avoid the second issue of tetramer dissociation of the hemoglobin with subsequent nephrotoxicity, researchers developed four modifications: (1) stabilization by using an α-chain cross-linker such as diaspirin; (2) polymerization with glutaraldehyde to cross-link tetramers; (3) conjugation, or binding of Hb to a larger molecule, such as polyethylene glycol or dextran; and (4) encapsulation by surrounding the Hb with a lipid membrane derived from natural lecithin or synthetic phospholipids.[3,8]
One of the first HBOCs to be developed was HemAssist, a diaspirin cross-linked Hb produced by Baxter Healthcare in the early 1980s that was modified from outdated, donated human blood. The product ultimately failed clinical trials, and work was discontinued in 1999. Baxter's final clinical trial in the United States was a randomized, controlled, and single-blinded efficacy trial at 18 trauma centers. The trial was designed to enroll approximately 850 patients with presumed or proven hemorrhage with hypoperfusion because of trauma. The patients were randomized to receive within 60 minutes of hospital arrival either up to 1,000 mL HemAssist or normal saline. However, the study was ended prematurely after just 112 patients were enrolled due to an interim data review. Unfortunately, the mortality rate at 48 hours was significantly increased (P = .01) in the HemAssist group (20/52 [38%] patients) vs the control group (7/46 [15%] patients). In addition, the 28-day morbidity rate, evaluated by the multiple organ dysfunction score, was higher in the HemAssist group (P = .03).
PolyHeme, manufactured by Northfield Laboratories, was a first-generation pyridoxylated and polymerized (to glutaraldehyde) Hb also sourced from outdated human blood. Work on this product actually began back in 1969 by the US Army. The half-life was reported at around 24 hours in a healthy volunteer. Trials on the product were initially promising; however, PolyHeme then underwent a phase III multicenter trial in the United States with 714 trauma patients receiving either up to 6 U (50 g Hb/unit) of PolyHeme during the first 12 hours postinjury or crystalloid followed by RBCs in the control group, as clinically indicated. While none of the measured end points reached statistical significance between the two groups, there were notable differences, including higher 30-day mortality in the randomized cohort (13.4% PolyHeme vs 9.6% control), higher multiorgan failure incidence (7.4% PolyHeme vs 5.5% control), and higher overall adverse events (93% PolyHeme vs 88%; P = .04). Based on these and similar results, work on the development of PolyHeme was finally halted in 2009 when the FDA denied approval for its clinical use.
Hemopure (HbOC-201), manufactured by Biopure Corporation, was a purified bovine Hb that had also undergone polymerization with glutaraldehyde. In healthy volunteers, the half-life was found to be extended at 16 to 20 hours. While the product was not approved in the United States because of increased hypercoagulability, it did gain approval for use in South Africa in 2001. Hemopure is still available in the United States through the FDA Expanded Access Program for compassionate use for severe, life-threatening anemia.
Hemospan (Sangart) and Hemolink (Hemosol BioPharma) are two other HBOCs developed around the same timeframe. Hemospan was a human Hb-based product, which was modified by pegylation. Hemolink was also human Hb based but modified with o-raffinose. However, each of these aforementioned HBOCs was evaluated in the meta-analysis described below, which had unfavorable outcomes.
In 2008, a meta-analysis published in the Journal of the American Medical Association assessed the safety of HBOCs in 3,711 patients enrolled in 16 randomized controlled trials involving five different products (HemAssist, Hemopure, Hemolink, PolyHeme, and Hemospan). The trials used HBOCs in a variety of clinical settings, including surgery, trauma care, and stroke patients. The team concluded there was a significant increase in both the risk of death (relative risk [RR], 1.3; 95% confidence interval [CI], 1.05–1.61) and the risk of myocardial infarction (RR, 2.71; 95% CI, 1.67–4.40) compared with control standard treatment groups. Natanson et al estimated there was an alarming 50 patients treated for each treatment-associated myocardial infarction. Furthermore, a subgroup analysis of the trials revealed these risks were not restricted to a particular HBOC or underlying clinical etiology. This study has been widely cited as highlighting the unacceptable increased risk of death associated with these HBOCs.
As noted above, while there are notable differences in the formulations of the products studied in the trials, all of them suffered from the same mechanism of toxicity. Notably, the NO scavenging complication was not addressed by these first-generation products. The hemoglobin molecules rapidly scavenge NO when released into the vasculature. With NO not in the circulation, conditions that are ripe for vascular thromboses are created, including vasoconstriction, loss of platelet inhibition, and increased release of proinflammatory mediators. Therefore, with the information this meta-analysis provided, progress on the development of these products was severely hindered. However, a promising new HBOC has been recently developed.
Am J Clin Pathol. 2020;153(3):287-293. © 2020 American Society for Clinical Pathology