- Email: [email protected]
Issues in the development of hemoglobin based oxygen carriers
Seminars in Hematology 56 (2019) 257–261
Contents lists available at ScienceDirect
Seminars in Hematology journal homepage: www.elsevier.com/locate/seminhematol
Review
Issues in the development of hemoglobin based oxygen carriers Timothy N. Estep∗ Chart Biotech Consulting, Erie, CO
a r t i c l e
i n f o
Keywords: HBOCs Development Efficacy Safety Manufacture
a b s t r a c t Hemoglobin based oxygen carriers (HBOCs) have been developed as alternative oxygen transporting formulations for the acute treatment of anemia and ischemia. Efficacy has been demonstrated in a variety of preclinical models and selected human patients; however, a higher overall incidence of mortality and myocardial infarction in those dosed with HBOCs in later stage clinical trials has prevented widespread regulatory approval. Diagnosis of myocardial infarction is confounded by the fact that HBOCs interfere with troponin assays, as well as other clinical chemistry measurements. Analysis of data pertaining to potential toxicity mechanisms suggests that coronary vasoconstriction is an unlikely contributor, but promotion of intravascular thrombosis may occur by several mechanisms. In addition, fluid and anemia management in patients infused with HBOCs has been suboptimal. Elucidation of potential toxicity mechanisms, refinement of use protocols, and definition of improved patient inclusion/exclusion criteria remain active areas of inquiry in understanding the best manner in which to utilize HBOCs. © 2019 Elsevier Inc. All rights reserved.
Introduction The development of technologies enabling the safe, routine transfusion of red blood cells (RBCs) was a landmark achievement of 20th century medicine. However, like all medical interventions, RBC transfusion has limitations, prompting the search for alternative ways in which to augment oxygen transport [1]. The most highly developed alternative is hemoglobin based oxygen carriers (HBOCs) which utilize the inherent ability of this protein to reversibly bind oxygen. Unfortunately, unmodified hemoglobin solutions are not efficacious because they release oxygen poorly in tissues and are rapidly excreted from the circulation [1]. Early preparations were also toxic due to contamination with red cell membrane fragments and endotoxin [1,2]. Subsequent development efforts were focused on improving oxygen transport characteristics, increasing intravascular persistence, and employing more robust purification techniques. Chemical modification resulted in HBOCs with greatly improved oxygen transport functionality and intravascular lifetimes many-fold greater than that of unmodified hemoglobin [1,3]. Improved purification methods essentially eliminated cell membrane and pyrogen contaminants and, after scaleup, enabled the large scale production of high quality HBOC formulations suitable for clinical testing. These formulations inherently possess several desirable properties (Table 1), but, as is often the
∗ Corresponding author. Timothy N. Estep, PhD, Chart Biotech Consulting, LLC, 5623 Highview Drive, Erie, CO 80516, Tel./fax: 303-828-1432. E-mail address: [email protected]
https://doi.org/10.1053/j.seminhematol.2019.11.006 0037-1963/© 2019 Elsevier Inc. All rights reserved.
case in the development of new product categories, resolution of one set of problems revealed additional challenges. The remainder of this communication summarizes the current status and issues of HBOC development. Efficacy Preclinical testing demonstrated that HBOCs are efficacious in the treatment of hemorrhagic shock [4,5]; traumatic brain injury [6]; cardiac arrest [7]; heart [8], brain [9], spinal cord [10], and skeletal muscle ischemia [11]; mitigation of cardiac dysfunction during balloon angioplasty [12]; sickle cell anemia [13]; oxygenation of hypoxic tumors to enhance the efficacy of chemotherapy [14] and radiation [15]; and liver [16] and heart [17] organ preservation (Table 2). Several experiments showed that HBOCs support life after complete or near complete blood replacement in larger animals [18–20]. Clinically, significant reduction in RBC transfusion requirements was observed during surgeries in which HBOCs were infused in lieu of RBCs [21,22]. Compassionate use of HBOCs has reversed ischemia and otherwise stabilized severely anemic patients who refused or were unable to receive blood transfusions [23-25]. These results suggest that HBOCs are able to provide effective oxygen transport in humans and may be useful in the treatment of patients when blood is not available or contraindicated for immunologic reasons. However, results evaluating HBOCs in the resuscitation of human patients from hemorrhagic shock have been disappointing, and scrutiny of clinical results in general has raised concerns with respect to the incidence of serious adverse events.
258
T.N. Estep / Seminars in Hematology 56 (2019) 257–261
Table 1 Advantages of HBOCs. • No typing or crossmatching required • Extended storage stability ◦ Oxygen release characteristics maintained throughout storage ◦ No change in electrolyte composition or solution pH ◦ No particulate accumulation • Enhanced pathogen safety ◦ Validated virus removal and inactivation manufacturing process steps ◦ Breadth of validation requirements enhance safety with respect to new as well as currently known potential contaminants • No anticoagulant in infused formulation • Enhanced ability to perfuse infarcted or ischemic tissues HBOCs = hemoglobin based oxygen carriers. Table 2 Potential HBOC indications. • • • • • • • •
Blood sparing Resuscitation from hemorrhagic shock Resuscitation from cardiogenic shock Salvage of infarcted brain or heart tissue Peripheral vascular perfusion Oxygenation of hypoxic tumors to enhance radiation and chemotherapy Organ preservation Perfusion of heart tissue during balloon angioplasty
HBOC = hemoglobin based oxygen carrier.
Safety A meta-analysis of data from later stage clinical trials as of 2008 implied that HBOC treatment increased the risk of mortality and myocardial infarction (MI) [26]. Interestingly, while controversial [27], several studies that have attempted to define the risks inherently associated with RBC transfusion have also flagged these same adverse events [28-31], with increasing risk associated with longer intervals of red cell storage [32,33]. This in turn leads to the question of whether there may be a related etiology, with the most obvious common factor being hemoglobin. While the concentration of extracellular hemoglobin in RBC units near expiry is far below that of HBOC solutions, evidence suggests that red cells damaged during storage may continue to lyse in vivo after transfusion [34]. Furthermore, unmodified hemoglobin is demonstrably more toxic than modified HBOCs in many test systems [35]. This suggests that similar mechanisms of toxicity may be manifested after the infusion of HBOCs and stored red cells. With respect to putative HBOC associated mortality, the most heavily scrutinized instance is the Phase III US trial of HemAssist, a crosslinked human hemoglobin formulation used to treat patients suffering from traumatic hemorrhagic shock [36]. This study was halted after the enrollment of only 98 total patients because mortality in the treatment arm (24/50) significantly exceeded that of controls (8/50). This in turn resulted in the termination of the development of HemAssist. However, a subsequent case-by-case analysis of patient deaths by several academic investigators revealed that the imbalance was primarily due to the enrollment of sicker patients in the HBOC treatment arm [37]. For example, patients ultimately treated with HemAssist had a higher incidence of prehospital cardiac arrest (19% vs 4%). This analysis ultimately determined that only 2 patient deaths were unexpected, one in each group. A second hypothesis is that the observed mortality difference was due, at least in part, to suboptimal fluid and volume management [38]. Resuscitation studies performed in sheep demonstrated that HemAssist is a surprisingly potent volume expander that can cause exacerbation of hypertension and increased cardiac work when administered in combination with large volumes of crystalloids [38,39]. This volume expansion also limits the degree to which anemia can be corrected, as studies with another HBOC in stable elective surgery patients demonstrated that hemoglobin
concentration after HBOC infusion was lower than that after the infusion of the same amount of hemoglobin as RBCs [22,40]. In this regard it is informative to compare the outcomes of the US HemAssist trauma trial with those from a concomitant European study in a similar patient population [41]. In the latter study, mortality was nearly equally balanced between treatment and control groups. The HemAssist formulation used in these studies was exactly the same, and doses were similar, but in the European protocol patients suffering from cardiac arrest were excluded from enrollment, resuscitation was begun on-scene rather than in the emergency room, and patients were excluded if they had received more than a liter of other fluids prior to HBOC infusion. This suggests that patient inclusion/exclusion criteria and avoiding the concomitant infusion of large volumes of crystalloid can have a significant impact on study outcomes. Suboptimal fluid and anemia management has also been postulated as a contributor to adverse events in clinical studies with other HBOCs [40,42]. Hypotheses that increased mortality in HemAssist infused patients was due to iron-induced infection/sepsis or poorer organ perfusion due to vasoconstriction were not supported by comparison of patient histories, blood pressure differences, or overall indices of organ perfusion between treated and control groups [43,44]. In the only other Phase III clinical trial of an HBOC in traumatic hemorrhage shock, Polyheme, a pyridoxylated, glutaraldehyde polymerized human hemoglobin, was found to support patients without blood transfusion for up to 12 hours postinjury [45]. There was no significant difference in 30-day mortality, but the incidence of investigator reported MI was greater in treated (11/349) vs control (3/365) patients (P< .05). With respect to MI more generally, the overall incidence in later stage trials is approximately 3% in HBOC treated patients vs 1% in controls [26,46]. However, this assessment is confounded by the fact that measurement of troponin concentrations, a primary MI diagnostic, may be subject to interference by HBOCs, hemolysis, and HBOC metabolites (see below). Nevertheless, as discussed in more detail elsewhere, insofar as there is a correlation between the investigator reported incidence of MI and the actual incidence, there is a strong correlation between MI, HBOC dose, and HBOC molecular size [46]. Proposed mechanisms for HBOC enhancement of MI risk include coronary vasoconstriction, exacerbation of oxidative stress and inflammation, and inhibition by hemoglobin of the cleavage of the highly thrombotic ultra large von Willebrand factor. The first of these hypotheses derives from the observation that HBOCs frequently increase overall vascular flow resistance due to the consumption of the smooth muscle relaxant nitric oxide [47], However, blood flow measurements in a variety of species, and direct coronary infusion of hemoglobins into a small number of human patients, consistently show that coronary flow is not reduced after HBOC administration even when mean arterial pressure is increased [48-50]. In addition, the increased risk of MI with HBOC molecular size is inconsistent with a coronary vasoconstriction mechanism, since vasoactivity decreases as HBOCs become larger [51]. More likely are intravascular mechanisms by which HBOCs increase the risk of thrombosis through platelet activation, stimulation of procoagulant factors, and/or adverse interactions with dysfunctional endothelium, all of which have been implicated as possibilities in cell culture or preclinical animal studies [46]. A recent analysis of clinical results and preclinical experiments suggests that heme release from oxidized HBOCs and subsequent endothelial and cellular uptake may be a particularly important pathway for tissue damage (unpublished observations; manuscript under review). Several authors have noted that HBOC toxicity frequently manifests in model systems as an exacerbation of preexisting oxidative stress or endothelial dysfunction [52,53]. This explains why, to the author’s knowledge, MI has never been observed in standard preclinical toxicity tests or normal human
T.N. Estep / Seminars in Hematology 56 (2019) 257–261
volunteers. Indeed, when evaluated in model systems of MI or stroke, HBOC administration frequently reduces infarct size due the ability of HBOCs to oxygenate ischemic penumbra [8,9]. However, in these models systems, previously healthy animals were subjected to a singular insult, as compared to human patients who may have multiple risk factors and years of prior exposure to other stressors. Interference with clinical assays It has long been appreciated that HBOCs interfere with spectrophotometric assays due to the intense absorbance of concentrated hemoglobins solutions [54]. More surprising is the interference by HBOCs with immunoassays for troponin. Positive, negative, or minimal biases have been observed, depending on the particular assessment platform utilized [54-56]. Hemolysis may also interfere with troponin determinations, not all of which is ascribable the presence of hemoglobin [57]. Precise, accurate troponin determinations will therefore require assessment of the degree of sample hemolysis, which in itself is compromised by the presence of high concentrations of HBOC. Size exclusion high performance liquid chromatography methods can readily distinguish HBOCs from the unmodified hemoglobin [58], but this type of analysis is not routine in clinical laboratories. A further complication is that bilirubin, a primary heme catabolic product, introduces a strong negative bias into at least some troponin assays, but the degree to which this occurs after HBOC infusion is unknown, in part because bilirubin assays are also subject to strong interference by hemoglobins [59,60]. As a consequence, bilirubin levels in plasma after HBOC infusion are unknown, but likely substantial. The most pressing analytical issue relating to HBOC use is untying the Gordian knot of troponin determinations in the presence of high concentrations of plasma hemoglobin to enable more accurate diagnosis of MI. Manufacture For several contemplated indications HBOCs must be administered at doses equivalent to several units of blood, which equates to several hundred grams of HBOC per patient. This in turn requires substantial manufacturing capacity even to supply material for preclinical and clinical testing. The 10 to 15 million units of RBCs transfused annually in the United States [61] contain approximately 10 0 0 tons of hemoglobin. Thus, if HBOCs are approved for resuscitation or blood sparing indications, an ultimate manufacturing capacity of at least 100 tons per year will likely be required. While substantial, there is precedent in the fact that human albumin is purified from plasma at a scale of several hundred tons per year [62] and hemoglobin is twice as abundant in mammalian blood as albumin. Hemoglobin is also readily recovered from red cells by processes that are efficient and scalable. However, unlike albumin, hemoglobin must be modified to be efficacious, and these modifications must be performed with relatively inexpensive reagents at high yield. This imposes significant constraints on the types of reagents and purification processes that can be economically utilized [63]. In addition, whether human or other mammalian source material is utilized, donors must be carefully scrutinized for the presence of disease, and pathogen inactivation and removal steps incorporated into the manufacturing process. Manufacturing facilities capable of producing several hundred thousand units of HBOCs per year using mammalian red cells as source material have been built by Baxter, Hemosol, and Biopure. Sources of hemoglobin which have been investigated to date include human and bovine blood, human placental blood, transgenic swine, earthworms, and recombinant hemoglobin produced in bacterial or yeast fermentation systems [64]. Late stage clinical testing has been performed with HBOCs derived from human and bovine
259
blood, but recombinant sources may also be viable. Kumar has suggested that at commercial scale transgenic swine could produce unmodified hemoglobin at a cost of $0.40/g, which is less than the acquisition cost of human hemoglobin from red cells [65]. Recombinant hemoglobin produced by fermentation can also be cost effective if multigram yields can be obtained in the fermentation process, a goal which has been achieved for selected HBOCs [66]. One advantage of recombinant production is that targeted mutations can be introduced to improve hemoglobin properties. Relative to unmodified hemoglobin, genetically induced mutations have been utilized to adjust oxygen transport properties, decrease the rate at which hemoglobin inactivates nitric oxide, inhibit autooxidation, reduce the rate of heme loss, and effect subunit crosslinking [66]. Discussion An overarching challenge in the development of HBOCs is the large doses required for indications of interest, presenting both manufacturing and safety challenges. Nevertheless, the fact that several HBOCs have progressed into late stage clinical trials in the US and Europe demonstrates that manufacturing issues can be addressed and these formulations produced in sufficient quantity and quality to satisfy standardized preclinical safety assessments. However, demonstration of safety in human patient populations with more complex risk profiles has proven difficult. It is also unclear to what degree the failure to demonstrate an acceptable therapeutic index in humans is due to inherent properties as opposed to suboptimal use protocols. One fact which has become increasingly clear is that HBOCs are unlike any existing product class, whether it be volume expanders or red cells. Better discerning mechanisms of HBOC toxicity in human patients, developing improved criteria for patient selection, and understanding how to optimally utilize this new class of product are currently primary impediments to HBOC approval and clinical deployment. That said, HBOCs have demonstrated efficacy in both preclinical models and selected human patients. Life has been supported at otherwise lethal hematocrits, hemodynamic stability restored, and indicia of ischemia reversed [1,18,19,20,23-25]. This has led to the conclusion that HBOCs may be useful when RBCs are unavailable or unacceptable [67]. Other demonstrated HBOC advantages include long-term stability, no requirement for blood typing, and crossmatching, the ability to reduce red cell transfusion requirements, and reduced risk of pathogen transmission. [68] With regard to future medical practice, it is interesting to compare and contrast the properties of RBCs with HBOCs. HBOCs maintain their ability to efficiently release oxygen to tissues throughout their storage lifetime [69], whereas RBCs do not [70]. On the other hand, the circulatory half-life of HBOCs is on the order of 12 to 48 hours [68], compared to the multiple week average lifespan of infused red cells [71]. These complementary properties suggest potential synergies in the use of HBOCs and red cells should the former achieve approval for clinical use (Table 3). If patients presenting with blood loss requiring treatment are first admin-
Table 3 Potential synergies between HBOCs and red cell transfusions. • Reduction of need for typing and crossmatching • Sparing of red cells for most needy patients • Leverage excellent oxygen transport characteristics of HBOCs with longer circulatory life of red cells • Additional oxygen transport therapies for those who cannot receive red cell transfusion • Indications for treatment of ischemia for which RBCs are not currently indicated HBOCs = hemoglobin based oxygen carriers; RBCs = red blood cells.
260
T.N. Estep / Seminars in Hematology 56 (2019) 257–261
istered an HBOC, it could mitigate the need for urgent typing and crossmatching, while providing immediate oxygen transport. HBOCs could also reduce the need for prospective blood typing and crossmatching for certain surgical procedures. This could both reduce laboratory costs and provide more effective care. If blood loss is severe, or ongoing, red cell transfusion would be indicated, leveraging the ability of the cellular formulation to provide longterm oxygen transport capacity after recovering the ability to effectively release oxygen. The more robust storage stability of the HBOCs would also facilitate on-scene, and therefore earlier, resuscitation. In addition, HBOCs will likely be beneficial in indications benefiting from enhanced tissue perfusion for which red cells are not appropriate. Thus, utilization of HBOCs alongside red cell transfusion promises to expand the repertoire of available treatments for hypoxia and ischemia in a way that should both enhance patient care and improve healthcare efficiency. Declaration of Competing Interest The author is a consultant for Omniox, Inc., an early stage biopharmaceutical company developing new medicines for hypoxic diseases. No support was received from Omniox for the production of this manuscript. References [1] Friedman HI, Devenuto F, Kerwin A, Carson K, Bynoe R. Hemoglobin solutions as blood substitutes. J Invest Surg 20 0 0;13:79–94. [2] Feola M, Simoni J, Canizaro PC, Tran R, Raschbaum G, Behal FJ. Toxicity of polymerized hemoglobin solutions. Surg Gynecol Obstet 1988;166:211–22. [3] Reid TJ. Hb-based oxygen carriers: are we there yet? Transfusion 2003;43:280–7. [4] Habler O, Kleen M, Pape A, Meisner F, Kemming G, Mesmer K. Diaspirin-crosslinked hemoglobin reduces mortality of severe hemorrhagic shock in pigs with critical coronary stenosis. Crit Care Med 20 0 0;28:1889–98. [5] Rice J, Philbin N, McGwin G, et al. Bovine polymerized hemoglobin versus Hextend resuscitation in a swine model of severe controlled hemorrhagic shock with delay to definitive care. Shock 2006;26:302–10. [6] King DR, Cohn SM, Proctor KG. Resuscitation with a hemoglobin-based oxygen carrier after traumatic brain injury. J Trauma 2005;59:553–62. [7] Chow MSS, Fan C, Hieu Tran, Zhao H, Zhou L. Effects of diaspirin cross-linked hemoglobin (DCLHb) during and post-CPR in swine. J Pharmacol Exp Ther 2001;297:224–9. [8] George I, Yi GH, Schulman AR, et al. A polymerized bovine hemoglobin oxygen carrier preserves regional myocardial function and reduces infarct size after acute myocardial ischemia. Am J Physiol Heart Circ Physiol 2006;291:H1126–37. [9] Cole DJ, Schell RM, Drummond JC, Przybelski RJ, Marcantonio S. Focal cerebral ischemia in rats: effect of hemodilution with α -α cross-linked hemoglobin on brain injury and edema. Can J Neurol Sci 1993;20:30–6. [10] Bowes MP, Burhop KE, Zivin JA. Diaspirin cross-linked hemoglobin improves neurological outcome following reversible but not irreversible CNS ischemia in rabbits. Stroke 1994;25:2253–7. [11] Horn EP, Standl T, Wilhelm S, et al. Bovine hemoglobin increases skeletal muscule oxygenation during 95% artificial arterial stenosis. Surgery 1997;121:411–18. [12] McKenzie JE, Cost EA, Scandling DM, Ahle NW, Savage RW. Effects of diaspirin crosslinked haemoglobin during coronary angioplasty in the swine. Cardiovasc Res 1994;28:1188–119213. [13] Crawford MW, Shchor T, Engelhardt T, et al. The novel hemoglobin-based oxygen carrier HRC 101 improves survival in murine sickle cell disease. Anesthesiology 2007;107:281–7. [14] Teicher BA, Ara G, Herbst R, Takeuchi H, Keyes S, Northey D. PEG-hemoglobin: effects on tumor oxygenation and response to chemotherapy. In Vivo 1997;11:301–12. [15] Linberg R, Conover CD, Shum KL, Shorr RGL. Increased tissue oxygenation and enhanced radiation sensitivity of solid tumors in rodents following polyethylene glycol conjugated bovine hemoglobin administration. In Vivo 1998;12:167–74. [16] Starnes HF, Tewari A, Flokas K, et al. Effectiveness of a purified human hemoglobin as a blood substitute in the perfused rat liver. Gastroenterology 1991;101:1345–53. [17] Serna DL, Powell LL, Kahwaji C, et al. Cardiac function after eight hour storage by using polyethylene glycol hemoglobin versus crystalloid perfusion. ASAIO J 20 0 0;46:547–52. [18] Vlahakes GJ, Lee R, Jacobs EE, LaRaia PJ, Austen WG. Hemodynamic effects and oxygen transport properties of a new blood substitute in a model of massive blood replacement. J Thorac Cardiovasc Surg 1990;100:379–88.
[19] Gould SA, Sehgal LR, Rosen AL, Sehgal HL, Moss GS. The efficacy of polymerized pyrodoxylated hemoglobin solution as an O2 carrier. Ann Surg 1990;211:394–8. [20] Meisner FG, Kemming GI, Habler OP, et al. Diaspirin crosslinked hemoglobin enables extreme hemodilution beyond the critical hematocrit. Crit Care Med 2001;29:829–38. [21] Schubert A, Przybelski RJ, Eidt JF, et al. Diaspirin-crosslinked hemoglobin reduces blood transfusion in noncardiac surgery: a multicenter, randomized, controlled, double-blinded trial. Anesth Analg 2003;97:323–32. [22] Jahr JS, Mackenzie C, Pearce LB, Pitman A, Greenburg AG. HBOC-201 as an alternative to blood transfusion: efficacy and safety evaluation in a multicenter Phase III trial in elective orthopedic surgery. J Trauma 2008;64:1484–97. [23] Mullon J, Giacoppe G, Clagett C, McCune D, Dillard T. Transfusions of polymerized bovine hemoglobin in a patient with severe autoimune hemolytic anemia. New Eng J Med 20 0 0;342:1638–43. [24] Fitzgerald MC, Chan JY, Ross AW, et al. A synthetic haemoglobin-based oxygen carrier and the reversal of cardiac hypoxia secondary to severe anaemia following trauma. Med J Aust 2011;194:471–3. [25] Marrazzo F, Larson G, Lama TTS, et al. Inhaled nitric oxide prevents systemic and pulmonary vasoconstriction due to hemoglobin-based oxygen carrier infusion: a case report. J Crit Care. Epub 2019;51:213–16 doi.org/10.1016/j.jcrc.2018.04.008. [26] Natanson C, Kern SJ, Lurie P, Banks SM, Wolfe SM. Cell-free hemoglobin-based blood substitutes and risk of myocardial infarction and death. JAMA 2008;299:2304–12. [27] Vostal JG, Buehler PW, Gelderman MP, et al. Proceedings of the Food and Drug Administration’s public workshop on new red blood cell product regulatory science 2016. Transfusion 2018;58:255–66. [28] Koch CG, Li L, Duncan AI, et al. Morbidity and mortality risk associated with red blood cell and blood-component transfusion in isolated coronary artery bypass grafting. Crit Care Med 2006;34:1608–16. [29] Obi AT, Park YJ, Bove P, et al. The association of perioperative transfusion with 30-day morbidity and mortalitiy in patients undergoing major vascular surgery. J Vasc Surg 2015;61:10 0 0–9 e1Epub 2015 Jan 14. doi:10.1016/j.jvs. 2014.10.106. [30] Whitlock EL, Kim H, Auerbach AD. Harms associated with single unit perioperative transfusion: retrospective population based analysis. BMJ 2015;350:h3037 Published online 2015 Jun 12doi: 10.1136/bmj.h3037: 10.1136/bmj.h3037. [31] Osborne Z, Hanson K, Brooke BS, et al. Variation in transfusion practices and the association with perioperative adverse events in patients undergoing open abdominal aortic aneurysm repair and lower extremity arterial bypass in the vascular quality initiative. Ann Vasc Surg 2018;46:1–16. doi:10.1016/j.avsg.2017. 06.154. [32] Koch CG, Li L, Sessler DI, et al. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med 2008;358:1229–01239. [33] Wang D, Sun J, Soloman SB, Klein HG, Natanson C. Transfusion of older stored blood and risks of death: a meta-analysis. Transfusion 2012 June;52(6). doi:10. 1111/j.1537-2995.03466.x. [34] Baek JH, D’Agnillo F, Vallelian F, et al. Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy. J Clin Invest 2012;122:1444–58. [35] Simoni J, Simoni G, Lox CD, Prien SD, Tran R, Shires GT. Expression of adhesion molecules and von Willebrand factor in human coronary artery endothelial cells incubated with differently modified hemoglobin solutions. Artif Cells Blood Substit Immobil Biotechnol 1997;25:211–25. [36] Sloan EP, Koenigsberg M, Gens D, et al. Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock. JAMA 1999;282:1857–64. [37] Sloan EP, Koenigsberg M, Brunett PH, et al. Post Hoc Mortality Analysis of the efficacy trial of diaspirin cross-linked hemoglobin in the treatment of severe traumatic hemorrhagic shock. J Trauma 2002;52:887–95. [38] Vane LA, Funston JS, Kirschner R, et al. Comparison of transfusion with DCLHb or pRBCs for treatment of intraoperative anemia in sheep. J Appl Physiol 2002;92:343–53. [39] Fischer SR, Burnet M, Traber DL, Prough DS, Kramer C. Plasma volume expansion with solutions of hemoglobin, albumin, and Ringer lactate in sheep. Am J Physiol 1999;276:H2194–203. [40] LaMuraglia GM, O’Hara PJ, Baker WH, et al. The reduction of the allogenic transfusion requirement in aortic surgery with a hemoglobin-based solution. J Vasc Surg 20 0 0;31:299–308. [41] Kerner T, Ahlers O, Veit S, et al. DCH-Hb for trauma patients with severe a shock: the European “On-Scene” multicenter study. Intensive Care Med 2003;29:378–85. [42] Northfield laboratories releases summary observations from its elective surgery trial [news release] (2006). Evanston, IL: Northfield Laboratories Inc. Available from from http://phx.corporate-ir.net/phoenix.zhtml?c= 91374&p=irol-newsArticle_print&ID=833808&highlight=. [accessed 16 December 2017]. [43] Sloan EP, Philbin NB, Koenigsberg MD, Gao W. DCLHb Traumatic Hemorrhagic Shock Study Group; European HOST Investigators. The lack of consistent diaspirin cross-linked hemoglobin infusion blood pressure effects in the US and EU traumatic hemorrhagic shock clinical trials. Shock 2010;33:123–33. [44] Sloan EP, Koenigsberg MD, Philbin NB, Gao W. DCLHb Traumatic Hemorrhagic Shock Study Group; European HOST Investigators. J Trauma 2010;68:1158–71.
T.N. Estep / Seminars in Hematology 56 (2019) 257–261 [45] Moore EE, Moore FA, Fabian TC, et al. PolyHeme Study Group. Human polymerized hemoglobin for the treatment of hemorrhagic shock when blood is unavailable; the USA multicenter trial. J Am Coll Surg 2009;208:1–13. [46] Estep TN. Haemoglobin-based oxygen carriers and myocardial infarction. Artif Cells Nanomed Biotechnol 2019;47:593–601. [47] Olson JS, Foley EW, Rogge C, Tsai AL, Doyle MP, Lemon DD. NO scavenging and the hypertensive effect of hemoglobin-based blood substitutes. Free Radic Biol Med 2004;36:685–97. [48] Sharma AC, Rebello S, Gulati A. Regional circulatory and systemic hemodynamic effects of diaspirin cross-linked hemoglobin in the rat. Artif Cells. Blood Substit Immobil Biotechnol 1994;22:593–602. [49] Mongan PD, Moon-Massat PF, Rentko V, Mihok S, Dragovich A, Sharma P. Regional blood flow after serial normovolemic exchange transfusion with HBOC-201 (Hemopure) in anesthetized swine. J Trauma 2009;67:51–60. [50] Meliga E, Vranckx P, Regar E, et al. Proof-of-concept trial to evaluate haemoglobin based oxygen therapeutics in elective percutaneous coronary revascularization. Rationale, protocol design and haemodynamic results. EuroIntervention 2008;4:99–107. [51] Sakai H, Hara H, Yuasa M, et al. Molecular dimensions of Hb-based O2 carriers determine constriction of resistance arteries and hypertension. Am J Physiol Heart Circ Physiol 20 0 0;279:H908–15. [52] Biro GP. Adverse HBOC-endothelial dysfunction synergism: a possible contributor to adverse clinical outcomes. Curr Drug Discov Technol 2012;9:194–203. [53] D’Agnillo F. Redox activity of cell-free hemoglobin: implications for vascular oxidative stress and endothelial dysfunction. In: Kim HW, Greenburg AG, editors. Hemoglobin-based oxygen carriers as red cell substitutes and oxygen therapeutics. New York: Springer; 2013. p. 665–82. [54] Ma Z, Monk TG, Goodnough LT, et al. Effect of hemoglobin- and perflubron-based oxygen carriers on common clinical laboratory tests. Clin Chem 1997;43:1732–7. [55] Björkholom M, Fagrell B, Przybelski R, Winslow N, Young M, Winslow RM. A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethylene glycol. Haematologica 2005;90:505–15. [56] Cameron SJ, Gerhardti G, Engelstad M, Young MA, Norris EJ, Sokoll LJ. Interference in clinical chemistry assays by the hemoglobin-based oxygen carrier. Hemospan. Clin Biochem. 2009;42:221–4. [57] Sodi R, Dam SM, Davison AS, Stott A, Shenkin A. Mechanism of interference by haemolysis in the cardiac troponin T immunoassay. Ann Clin Biochem 2006;43:49–56. [58] Varlet-Marie E, Ashenden M, Lasne F, et al. Detection of hemoglobin-based oxygen carriers in human serum for doping analysis: Confirmation by size-exclusion HPLC. Clin Chem 2004;50:723–31.
261
[59] ver Elst KM, Chapelle JP, Boland P, Demolder JS, Gorus FK. Analytic and clinical evaluation of the Abbott AxSYM cardiac troponin I assay. Am J Clin Pathol 1999;112:745–52. [60] Dasgupta A, Wells A, Biddle DA. Negative interference of bilirubin and hemoglobin in the MEIA troponin I assay but not in the MEIA CK-MB assay. J Clin Lab Anal 2001;15:76–80. [61] Whitaker B, Rajbhandary S, Kleinman S, Harris A, Kamani N. Trends in United States blood collection and transfusion: results from the 2013 AABB blood collection, utilization, and patient blood management survey. Transfusion 2016;56:2173–83. [62] He Y, Ning T, Xie T, et al. Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Natl Acad Sci 2011;108:19078–83. [63] Kim HW, Estep TN. Practical considerations in the development of hemoglobin-based oxygen therapeutics. Curr Drug Discov Technol 2012;9:212–23. [64] Chang TMS. From hemoglobin based oxygen carrier to oxygen therapeutics, blood substitutes, nanomedicine and artificial cells. In: Kim HW, Greenburg AG, editors. Hemoglobin-based oxygen carriers as red cell substitutes and oxygen therapeutics. New York: Springer; 2013. p. 3–26. [65] Kumar R, Manjula BN. Human hemoglobin derived from transgenic swine: a starting material for “blood substitute” formulations. In: Rudolph AS, Rabinovici R, Feuerstein GZ, editors. Red blood cell substitutes basic principles and clinical applications. New York: Marcell Dekker; 1998. p. 309–24. [66] Varnado CL, Mollan TL, Birukou I, Smith BJZ, Henderson DP, Olson JS. Development of recombinant hemoglobin-based oxygen carriers. Antioxid Redox Signal 2013;18:2314–28. [67] Weiskopf RB, Silverman TA. Balancing risks and benefits of hemoglobin-based oxygen carriers. Transfusion 2013;53:2327–33. [68] Dubé GP, Vranckx P, Greenburg AG. HBOC-201: the multi-purpose oxygen therapeutic. EuroIntervention 2008;4:161–5. [69] Nelson D, Azari M, Brown R, et al. Preparation and characterization of diaspirin cross-linked hemoglobin solutions for preclinical studies. In: Chang TMS, editor. Blood substitutes and oxygen carriers. New York: Marcel Dekker; 1993. p. 241–5. [70] Gelderman MP, Yazer MH, Jia Y, Wood F, Alayash AI, Vostal JG. Serial oxygen equilibrium and kinetic measurements during RBC storage. Transfus Med 2010;20:341–5. [71] Luten M, Roerdinkholder-Stoelwinder B, Schaap NP, de Grip WJ, Bos HJ, Bosman GJ. Survival of red blood cells after transfusion: a comparison between red cells concentrates of different storage periods. Transfusion 2008;48:1476–85.
Contents lists available at ScienceDirect
Seminars in Hematology journal homepage: www.elsevier.com/locate/seminhematol
Review
Issues in the development of hemoglobin based oxygen carriers Timothy N. Estep∗ Chart Biotech Consulting, Erie, CO
a r t i c l e
i n f o
Keywords: HBOCs Development Efficacy Safety Manufacture
a b s t r a c t Hemoglobin based oxygen carriers (HBOCs) have been developed as alternative oxygen transporting formulations for the acute treatment of anemia and ischemia. Efficacy has been demonstrated in a variety of preclinical models and selected human patients; however, a higher overall incidence of mortality and myocardial infarction in those dosed with HBOCs in later stage clinical trials has prevented widespread regulatory approval. Diagnosis of myocardial infarction is confounded by the fact that HBOCs interfere with troponin assays, as well as other clinical chemistry measurements. Analysis of data pertaining to potential toxicity mechanisms suggests that coronary vasoconstriction is an unlikely contributor, but promotion of intravascular thrombosis may occur by several mechanisms. In addition, fluid and anemia management in patients infused with HBOCs has been suboptimal. Elucidation of potential toxicity mechanisms, refinement of use protocols, and definition of improved patient inclusion/exclusion criteria remain active areas of inquiry in understanding the best manner in which to utilize HBOCs. © 2019 Elsevier Inc. All rights reserved.
Introduction The development of technologies enabling the safe, routine transfusion of red blood cells (RBCs) was a landmark achievement of 20th century medicine. However, like all medical interventions, RBC transfusion has limitations, prompting the search for alternative ways in which to augment oxygen transport [1]. The most highly developed alternative is hemoglobin based oxygen carriers (HBOCs) which utilize the inherent ability of this protein to reversibly bind oxygen. Unfortunately, unmodified hemoglobin solutions are not efficacious because they release oxygen poorly in tissues and are rapidly excreted from the circulation [1]. Early preparations were also toxic due to contamination with red cell membrane fragments and endotoxin [1,2]. Subsequent development efforts were focused on improving oxygen transport characteristics, increasing intravascular persistence, and employing more robust purification techniques. Chemical modification resulted in HBOCs with greatly improved oxygen transport functionality and intravascular lifetimes many-fold greater than that of unmodified hemoglobin [1,3]. Improved purification methods essentially eliminated cell membrane and pyrogen contaminants and, after scaleup, enabled the large scale production of high quality HBOC formulations suitable for clinical testing. These formulations inherently possess several desirable properties (Table 1), but, as is often the
∗ Corresponding author. Timothy N. Estep, PhD, Chart Biotech Consulting, LLC, 5623 Highview Drive, Erie, CO 80516, Tel./fax: 303-828-1432. E-mail address: [email protected]
https://doi.org/10.1053/j.seminhematol.2019.11.006 0037-1963/© 2019 Elsevier Inc. All rights reserved.
case in the development of new product categories, resolution of one set of problems revealed additional challenges. The remainder of this communication summarizes the current status and issues of HBOC development. Efficacy Preclinical testing demonstrated that HBOCs are efficacious in the treatment of hemorrhagic shock [4,5]; traumatic brain injury [6]; cardiac arrest [7]; heart [8], brain [9], spinal cord [10], and skeletal muscle ischemia [11]; mitigation of cardiac dysfunction during balloon angioplasty [12]; sickle cell anemia [13]; oxygenation of hypoxic tumors to enhance the efficacy of chemotherapy [14] and radiation [15]; and liver [16] and heart [17] organ preservation (Table 2). Several experiments showed that HBOCs support life after complete or near complete blood replacement in larger animals [18–20]. Clinically, significant reduction in RBC transfusion requirements was observed during surgeries in which HBOCs were infused in lieu of RBCs [21,22]. Compassionate use of HBOCs has reversed ischemia and otherwise stabilized severely anemic patients who refused or were unable to receive blood transfusions [23-25]. These results suggest that HBOCs are able to provide effective oxygen transport in humans and may be useful in the treatment of patients when blood is not available or contraindicated for immunologic reasons. However, results evaluating HBOCs in the resuscitation of human patients from hemorrhagic shock have been disappointing, and scrutiny of clinical results in general has raised concerns with respect to the incidence of serious adverse events.
258
T.N. Estep / Seminars in Hematology 56 (2019) 257–261
Table 1 Advantages of HBOCs. • No typing or crossmatching required • Extended storage stability ◦ Oxygen release characteristics maintained throughout storage ◦ No change in electrolyte composition or solution pH ◦ No particulate accumulation • Enhanced pathogen safety ◦ Validated virus removal and inactivation manufacturing process steps ◦ Breadth of validation requirements enhance safety with respect to new as well as currently known potential contaminants • No anticoagulant in infused formulation • Enhanced ability to perfuse infarcted or ischemic tissues HBOCs = hemoglobin based oxygen carriers. Table 2 Potential HBOC indications. • • • • • • • •
Blood sparing Resuscitation from hemorrhagic shock Resuscitation from cardiogenic shock Salvage of infarcted brain or heart tissue Peripheral vascular perfusion Oxygenation of hypoxic tumors to enhance radiation and chemotherapy Organ preservation Perfusion of heart tissue during balloon angioplasty
HBOC = hemoglobin based oxygen carrier.
Safety A meta-analysis of data from later stage clinical trials as of 2008 implied that HBOC treatment increased the risk of mortality and myocardial infarction (MI) [26]. Interestingly, while controversial [27], several studies that have attempted to define the risks inherently associated with RBC transfusion have also flagged these same adverse events [28-31], with increasing risk associated with longer intervals of red cell storage [32,33]. This in turn leads to the question of whether there may be a related etiology, with the most obvious common factor being hemoglobin. While the concentration of extracellular hemoglobin in RBC units near expiry is far below that of HBOC solutions, evidence suggests that red cells damaged during storage may continue to lyse in vivo after transfusion [34]. Furthermore, unmodified hemoglobin is demonstrably more toxic than modified HBOCs in many test systems [35]. This suggests that similar mechanisms of toxicity may be manifested after the infusion of HBOCs and stored red cells. With respect to putative HBOC associated mortality, the most heavily scrutinized instance is the Phase III US trial of HemAssist, a crosslinked human hemoglobin formulation used to treat patients suffering from traumatic hemorrhagic shock [36]. This study was halted after the enrollment of only 98 total patients because mortality in the treatment arm (24/50) significantly exceeded that of controls (8/50). This in turn resulted in the termination of the development of HemAssist. However, a subsequent case-by-case analysis of patient deaths by several academic investigators revealed that the imbalance was primarily due to the enrollment of sicker patients in the HBOC treatment arm [37]. For example, patients ultimately treated with HemAssist had a higher incidence of prehospital cardiac arrest (19% vs 4%). This analysis ultimately determined that only 2 patient deaths were unexpected, one in each group. A second hypothesis is that the observed mortality difference was due, at least in part, to suboptimal fluid and volume management [38]. Resuscitation studies performed in sheep demonstrated that HemAssist is a surprisingly potent volume expander that can cause exacerbation of hypertension and increased cardiac work when administered in combination with large volumes of crystalloids [38,39]. This volume expansion also limits the degree to which anemia can be corrected, as studies with another HBOC in stable elective surgery patients demonstrated that hemoglobin
concentration after HBOC infusion was lower than that after the infusion of the same amount of hemoglobin as RBCs [22,40]. In this regard it is informative to compare the outcomes of the US HemAssist trauma trial with those from a concomitant European study in a similar patient population [41]. In the latter study, mortality was nearly equally balanced between treatment and control groups. The HemAssist formulation used in these studies was exactly the same, and doses were similar, but in the European protocol patients suffering from cardiac arrest were excluded from enrollment, resuscitation was begun on-scene rather than in the emergency room, and patients were excluded if they had received more than a liter of other fluids prior to HBOC infusion. This suggests that patient inclusion/exclusion criteria and avoiding the concomitant infusion of large volumes of crystalloid can have a significant impact on study outcomes. Suboptimal fluid and anemia management has also been postulated as a contributor to adverse events in clinical studies with other HBOCs [40,42]. Hypotheses that increased mortality in HemAssist infused patients was due to iron-induced infection/sepsis or poorer organ perfusion due to vasoconstriction were not supported by comparison of patient histories, blood pressure differences, or overall indices of organ perfusion between treated and control groups [43,44]. In the only other Phase III clinical trial of an HBOC in traumatic hemorrhage shock, Polyheme, a pyridoxylated, glutaraldehyde polymerized human hemoglobin, was found to support patients without blood transfusion for up to 12 hours postinjury [45]. There was no significant difference in 30-day mortality, but the incidence of investigator reported MI was greater in treated (11/349) vs control (3/365) patients (P< .05). With respect to MI more generally, the overall incidence in later stage trials is approximately 3% in HBOC treated patients vs 1% in controls [26,46]. However, this assessment is confounded by the fact that measurement of troponin concentrations, a primary MI diagnostic, may be subject to interference by HBOCs, hemolysis, and HBOC metabolites (see below). Nevertheless, as discussed in more detail elsewhere, insofar as there is a correlation between the investigator reported incidence of MI and the actual incidence, there is a strong correlation between MI, HBOC dose, and HBOC molecular size [46]. Proposed mechanisms for HBOC enhancement of MI risk include coronary vasoconstriction, exacerbation of oxidative stress and inflammation, and inhibition by hemoglobin of the cleavage of the highly thrombotic ultra large von Willebrand factor. The first of these hypotheses derives from the observation that HBOCs frequently increase overall vascular flow resistance due to the consumption of the smooth muscle relaxant nitric oxide [47], However, blood flow measurements in a variety of species, and direct coronary infusion of hemoglobins into a small number of human patients, consistently show that coronary flow is not reduced after HBOC administration even when mean arterial pressure is increased [48-50]. In addition, the increased risk of MI with HBOC molecular size is inconsistent with a coronary vasoconstriction mechanism, since vasoactivity decreases as HBOCs become larger [51]. More likely are intravascular mechanisms by which HBOCs increase the risk of thrombosis through platelet activation, stimulation of procoagulant factors, and/or adverse interactions with dysfunctional endothelium, all of which have been implicated as possibilities in cell culture or preclinical animal studies [46]. A recent analysis of clinical results and preclinical experiments suggests that heme release from oxidized HBOCs and subsequent endothelial and cellular uptake may be a particularly important pathway for tissue damage (unpublished observations; manuscript under review). Several authors have noted that HBOC toxicity frequently manifests in model systems as an exacerbation of preexisting oxidative stress or endothelial dysfunction [52,53]. This explains why, to the author’s knowledge, MI has never been observed in standard preclinical toxicity tests or normal human
T.N. Estep / Seminars in Hematology 56 (2019) 257–261
volunteers. Indeed, when evaluated in model systems of MI or stroke, HBOC administration frequently reduces infarct size due the ability of HBOCs to oxygenate ischemic penumbra [8,9]. However, in these models systems, previously healthy animals were subjected to a singular insult, as compared to human patients who may have multiple risk factors and years of prior exposure to other stressors. Interference with clinical assays It has long been appreciated that HBOCs interfere with spectrophotometric assays due to the intense absorbance of concentrated hemoglobins solutions [54]. More surprising is the interference by HBOCs with immunoassays for troponin. Positive, negative, or minimal biases have been observed, depending on the particular assessment platform utilized [54-56]. Hemolysis may also interfere with troponin determinations, not all of which is ascribable the presence of hemoglobin [57]. Precise, accurate troponin determinations will therefore require assessment of the degree of sample hemolysis, which in itself is compromised by the presence of high concentrations of HBOC. Size exclusion high performance liquid chromatography methods can readily distinguish HBOCs from the unmodified hemoglobin [58], but this type of analysis is not routine in clinical laboratories. A further complication is that bilirubin, a primary heme catabolic product, introduces a strong negative bias into at least some troponin assays, but the degree to which this occurs after HBOC infusion is unknown, in part because bilirubin assays are also subject to strong interference by hemoglobins [59,60]. As a consequence, bilirubin levels in plasma after HBOC infusion are unknown, but likely substantial. The most pressing analytical issue relating to HBOC use is untying the Gordian knot of troponin determinations in the presence of high concentrations of plasma hemoglobin to enable more accurate diagnosis of MI. Manufacture For several contemplated indications HBOCs must be administered at doses equivalent to several units of blood, which equates to several hundred grams of HBOC per patient. This in turn requires substantial manufacturing capacity even to supply material for preclinical and clinical testing. The 10 to 15 million units of RBCs transfused annually in the United States [61] contain approximately 10 0 0 tons of hemoglobin. Thus, if HBOCs are approved for resuscitation or blood sparing indications, an ultimate manufacturing capacity of at least 100 tons per year will likely be required. While substantial, there is precedent in the fact that human albumin is purified from plasma at a scale of several hundred tons per year [62] and hemoglobin is twice as abundant in mammalian blood as albumin. Hemoglobin is also readily recovered from red cells by processes that are efficient and scalable. However, unlike albumin, hemoglobin must be modified to be efficacious, and these modifications must be performed with relatively inexpensive reagents at high yield. This imposes significant constraints on the types of reagents and purification processes that can be economically utilized [63]. In addition, whether human or other mammalian source material is utilized, donors must be carefully scrutinized for the presence of disease, and pathogen inactivation and removal steps incorporated into the manufacturing process. Manufacturing facilities capable of producing several hundred thousand units of HBOCs per year using mammalian red cells as source material have been built by Baxter, Hemosol, and Biopure. Sources of hemoglobin which have been investigated to date include human and bovine blood, human placental blood, transgenic swine, earthworms, and recombinant hemoglobin produced in bacterial or yeast fermentation systems [64]. Late stage clinical testing has been performed with HBOCs derived from human and bovine
259
blood, but recombinant sources may also be viable. Kumar has suggested that at commercial scale transgenic swine could produce unmodified hemoglobin at a cost of $0.40/g, which is less than the acquisition cost of human hemoglobin from red cells [65]. Recombinant hemoglobin produced by fermentation can also be cost effective if multigram yields can be obtained in the fermentation process, a goal which has been achieved for selected HBOCs [66]. One advantage of recombinant production is that targeted mutations can be introduced to improve hemoglobin properties. Relative to unmodified hemoglobin, genetically induced mutations have been utilized to adjust oxygen transport properties, decrease the rate at which hemoglobin inactivates nitric oxide, inhibit autooxidation, reduce the rate of heme loss, and effect subunit crosslinking [66]. Discussion An overarching challenge in the development of HBOCs is the large doses required for indications of interest, presenting both manufacturing and safety challenges. Nevertheless, the fact that several HBOCs have progressed into late stage clinical trials in the US and Europe demonstrates that manufacturing issues can be addressed and these formulations produced in sufficient quantity and quality to satisfy standardized preclinical safety assessments. However, demonstration of safety in human patient populations with more complex risk profiles has proven difficult. It is also unclear to what degree the failure to demonstrate an acceptable therapeutic index in humans is due to inherent properties as opposed to suboptimal use protocols. One fact which has become increasingly clear is that HBOCs are unlike any existing product class, whether it be volume expanders or red cells. Better discerning mechanisms of HBOC toxicity in human patients, developing improved criteria for patient selection, and understanding how to optimally utilize this new class of product are currently primary impediments to HBOC approval and clinical deployment. That said, HBOCs have demonstrated efficacy in both preclinical models and selected human patients. Life has been supported at otherwise lethal hematocrits, hemodynamic stability restored, and indicia of ischemia reversed [1,18,19,20,23-25]. This has led to the conclusion that HBOCs may be useful when RBCs are unavailable or unacceptable [67]. Other demonstrated HBOC advantages include long-term stability, no requirement for blood typing, and crossmatching, the ability to reduce red cell transfusion requirements, and reduced risk of pathogen transmission. [68] With regard to future medical practice, it is interesting to compare and contrast the properties of RBCs with HBOCs. HBOCs maintain their ability to efficiently release oxygen to tissues throughout their storage lifetime [69], whereas RBCs do not [70]. On the other hand, the circulatory half-life of HBOCs is on the order of 12 to 48 hours [68], compared to the multiple week average lifespan of infused red cells [71]. These complementary properties suggest potential synergies in the use of HBOCs and red cells should the former achieve approval for clinical use (Table 3). If patients presenting with blood loss requiring treatment are first admin-
Table 3 Potential synergies between HBOCs and red cell transfusions. • Reduction of need for typing and crossmatching • Sparing of red cells for most needy patients • Leverage excellent oxygen transport characteristics of HBOCs with longer circulatory life of red cells • Additional oxygen transport therapies for those who cannot receive red cell transfusion • Indications for treatment of ischemia for which RBCs are not currently indicated HBOCs = hemoglobin based oxygen carriers; RBCs = red blood cells.
260
T.N. Estep / Seminars in Hematology 56 (2019) 257–261
istered an HBOC, it could mitigate the need for urgent typing and crossmatching, while providing immediate oxygen transport. HBOCs could also reduce the need for prospective blood typing and crossmatching for certain surgical procedures. This could both reduce laboratory costs and provide more effective care. If blood loss is severe, or ongoing, red cell transfusion would be indicated, leveraging the ability of the cellular formulation to provide longterm oxygen transport capacity after recovering the ability to effectively release oxygen. The more robust storage stability of the HBOCs would also facilitate on-scene, and therefore earlier, resuscitation. In addition, HBOCs will likely be beneficial in indications benefiting from enhanced tissue perfusion for which red cells are not appropriate. Thus, utilization of HBOCs alongside red cell transfusion promises to expand the repertoire of available treatments for hypoxia and ischemia in a way that should both enhance patient care and improve healthcare efficiency. Declaration of Competing Interest The author is a consultant for Omniox, Inc., an early stage biopharmaceutical company developing new medicines for hypoxic diseases. No support was received from Omniox for the production of this manuscript. References [1] Friedman HI, Devenuto F, Kerwin A, Carson K, Bynoe R. Hemoglobin solutions as blood substitutes. J Invest Surg 20 0 0;13:79–94. [2] Feola M, Simoni J, Canizaro PC, Tran R, Raschbaum G, Behal FJ. Toxicity of polymerized hemoglobin solutions. Surg Gynecol Obstet 1988;166:211–22. [3] Reid TJ. Hb-based oxygen carriers: are we there yet? Transfusion 2003;43:280–7. [4] Habler O, Kleen M, Pape A, Meisner F, Kemming G, Mesmer K. Diaspirin-crosslinked hemoglobin reduces mortality of severe hemorrhagic shock in pigs with critical coronary stenosis. Crit Care Med 20 0 0;28:1889–98. [5] Rice J, Philbin N, McGwin G, et al. Bovine polymerized hemoglobin versus Hextend resuscitation in a swine model of severe controlled hemorrhagic shock with delay to definitive care. Shock 2006;26:302–10. [6] King DR, Cohn SM, Proctor KG. Resuscitation with a hemoglobin-based oxygen carrier after traumatic brain injury. J Trauma 2005;59:553–62. [7] Chow MSS, Fan C, Hieu Tran, Zhao H, Zhou L. Effects of diaspirin cross-linked hemoglobin (DCLHb) during and post-CPR in swine. J Pharmacol Exp Ther 2001;297:224–9. [8] George I, Yi GH, Schulman AR, et al. A polymerized bovine hemoglobin oxygen carrier preserves regional myocardial function and reduces infarct size after acute myocardial ischemia. Am J Physiol Heart Circ Physiol 2006;291:H1126–37. [9] Cole DJ, Schell RM, Drummond JC, Przybelski RJ, Marcantonio S. Focal cerebral ischemia in rats: effect of hemodilution with α -α cross-linked hemoglobin on brain injury and edema. Can J Neurol Sci 1993;20:30–6. [10] Bowes MP, Burhop KE, Zivin JA. Diaspirin cross-linked hemoglobin improves neurological outcome following reversible but not irreversible CNS ischemia in rabbits. Stroke 1994;25:2253–7. [11] Horn EP, Standl T, Wilhelm S, et al. Bovine hemoglobin increases skeletal muscule oxygenation during 95% artificial arterial stenosis. Surgery 1997;121:411–18. [12] McKenzie JE, Cost EA, Scandling DM, Ahle NW, Savage RW. Effects of diaspirin crosslinked haemoglobin during coronary angioplasty in the swine. Cardiovasc Res 1994;28:1188–119213. [13] Crawford MW, Shchor T, Engelhardt T, et al. The novel hemoglobin-based oxygen carrier HRC 101 improves survival in murine sickle cell disease. Anesthesiology 2007;107:281–7. [14] Teicher BA, Ara G, Herbst R, Takeuchi H, Keyes S, Northey D. PEG-hemoglobin: effects on tumor oxygenation and response to chemotherapy. In Vivo 1997;11:301–12. [15] Linberg R, Conover CD, Shum KL, Shorr RGL. Increased tissue oxygenation and enhanced radiation sensitivity of solid tumors in rodents following polyethylene glycol conjugated bovine hemoglobin administration. In Vivo 1998;12:167–74. [16] Starnes HF, Tewari A, Flokas K, et al. Effectiveness of a purified human hemoglobin as a blood substitute in the perfused rat liver. Gastroenterology 1991;101:1345–53. [17] Serna DL, Powell LL, Kahwaji C, et al. Cardiac function after eight hour storage by using polyethylene glycol hemoglobin versus crystalloid perfusion. ASAIO J 20 0 0;46:547–52. [18] Vlahakes GJ, Lee R, Jacobs EE, LaRaia PJ, Austen WG. Hemodynamic effects and oxygen transport properties of a new blood substitute in a model of massive blood replacement. J Thorac Cardiovasc Surg 1990;100:379–88.
[19] Gould SA, Sehgal LR, Rosen AL, Sehgal HL, Moss GS. The efficacy of polymerized pyrodoxylated hemoglobin solution as an O2 carrier. Ann Surg 1990;211:394–8. [20] Meisner FG, Kemming GI, Habler OP, et al. Diaspirin crosslinked hemoglobin enables extreme hemodilution beyond the critical hematocrit. Crit Care Med 2001;29:829–38. [21] Schubert A, Przybelski RJ, Eidt JF, et al. Diaspirin-crosslinked hemoglobin reduces blood transfusion in noncardiac surgery: a multicenter, randomized, controlled, double-blinded trial. Anesth Analg 2003;97:323–32. [22] Jahr JS, Mackenzie C, Pearce LB, Pitman A, Greenburg AG. HBOC-201 as an alternative to blood transfusion: efficacy and safety evaluation in a multicenter Phase III trial in elective orthopedic surgery. J Trauma 2008;64:1484–97. [23] Mullon J, Giacoppe G, Clagett C, McCune D, Dillard T. Transfusions of polymerized bovine hemoglobin in a patient with severe autoimune hemolytic anemia. New Eng J Med 20 0 0;342:1638–43. [24] Fitzgerald MC, Chan JY, Ross AW, et al. A synthetic haemoglobin-based oxygen carrier and the reversal of cardiac hypoxia secondary to severe anaemia following trauma. Med J Aust 2011;194:471–3. [25] Marrazzo F, Larson G, Lama TTS, et al. Inhaled nitric oxide prevents systemic and pulmonary vasoconstriction due to hemoglobin-based oxygen carrier infusion: a case report. J Crit Care. Epub 2019;51:213–16 doi.org/10.1016/j.jcrc.2018.04.008. [26] Natanson C, Kern SJ, Lurie P, Banks SM, Wolfe SM. Cell-free hemoglobin-based blood substitutes and risk of myocardial infarction and death. JAMA 2008;299:2304–12. [27] Vostal JG, Buehler PW, Gelderman MP, et al. Proceedings of the Food and Drug Administration’s public workshop on new red blood cell product regulatory science 2016. Transfusion 2018;58:255–66. [28] Koch CG, Li L, Duncan AI, et al. Morbidity and mortality risk associated with red blood cell and blood-component transfusion in isolated coronary artery bypass grafting. Crit Care Med 2006;34:1608–16. [29] Obi AT, Park YJ, Bove P, et al. The association of perioperative transfusion with 30-day morbidity and mortalitiy in patients undergoing major vascular surgery. J Vasc Surg 2015;61:10 0 0–9 e1Epub 2015 Jan 14. doi:10.1016/j.jvs. 2014.10.106. [30] Whitlock EL, Kim H, Auerbach AD. Harms associated with single unit perioperative transfusion: retrospective population based analysis. BMJ 2015;350:h3037 Published online 2015 Jun 12doi: 10.1136/bmj.h3037: 10.1136/bmj.h3037. [31] Osborne Z, Hanson K, Brooke BS, et al. Variation in transfusion practices and the association with perioperative adverse events in patients undergoing open abdominal aortic aneurysm repair and lower extremity arterial bypass in the vascular quality initiative. Ann Vasc Surg 2018;46:1–16. doi:10.1016/j.avsg.2017. 06.154. [32] Koch CG, Li L, Sessler DI, et al. Duration of red-cell storage and complications after cardiac surgery. N Engl J Med 2008;358:1229–01239. [33] Wang D, Sun J, Soloman SB, Klein HG, Natanson C. Transfusion of older stored blood and risks of death: a meta-analysis. Transfusion 2012 June;52(6). doi:10. 1111/j.1537-2995.03466.x. [34] Baek JH, D’Agnillo F, Vallelian F, et al. Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy. J Clin Invest 2012;122:1444–58. [35] Simoni J, Simoni G, Lox CD, Prien SD, Tran R, Shires GT. Expression of adhesion molecules and von Willebrand factor in human coronary artery endothelial cells incubated with differently modified hemoglobin solutions. Artif Cells Blood Substit Immobil Biotechnol 1997;25:211–25. [36] Sloan EP, Koenigsberg M, Gens D, et al. Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock. JAMA 1999;282:1857–64. [37] Sloan EP, Koenigsberg M, Brunett PH, et al. Post Hoc Mortality Analysis of the efficacy trial of diaspirin cross-linked hemoglobin in the treatment of severe traumatic hemorrhagic shock. J Trauma 2002;52:887–95. [38] Vane LA, Funston JS, Kirschner R, et al. Comparison of transfusion with DCLHb or pRBCs for treatment of intraoperative anemia in sheep. J Appl Physiol 2002;92:343–53. [39] Fischer SR, Burnet M, Traber DL, Prough DS, Kramer C. Plasma volume expansion with solutions of hemoglobin, albumin, and Ringer lactate in sheep. Am J Physiol 1999;276:H2194–203. [40] LaMuraglia GM, O’Hara PJ, Baker WH, et al. The reduction of the allogenic transfusion requirement in aortic surgery with a hemoglobin-based solution. J Vasc Surg 20 0 0;31:299–308. [41] Kerner T, Ahlers O, Veit S, et al. DCH-Hb for trauma patients with severe a shock: the European “On-Scene” multicenter study. Intensive Care Med 2003;29:378–85. [42] Northfield laboratories releases summary observations from its elective surgery trial [news release] (2006). Evanston, IL: Northfield Laboratories Inc. Available from from http://phx.corporate-ir.net/phoenix.zhtml?c= 91374&p=irol-newsArticle_print&ID=833808&highlight=. [accessed 16 December 2017]. [43] Sloan EP, Philbin NB, Koenigsberg MD, Gao W. DCLHb Traumatic Hemorrhagic Shock Study Group; European HOST Investigators. The lack of consistent diaspirin cross-linked hemoglobin infusion blood pressure effects in the US and EU traumatic hemorrhagic shock clinical trials. Shock 2010;33:123–33. [44] Sloan EP, Koenigsberg MD, Philbin NB, Gao W. DCLHb Traumatic Hemorrhagic Shock Study Group; European HOST Investigators. J Trauma 2010;68:1158–71.
T.N. Estep / Seminars in Hematology 56 (2019) 257–261 [45] Moore EE, Moore FA, Fabian TC, et al. PolyHeme Study Group. Human polymerized hemoglobin for the treatment of hemorrhagic shock when blood is unavailable; the USA multicenter trial. J Am Coll Surg 2009;208:1–13. [46] Estep TN. Haemoglobin-based oxygen carriers and myocardial infarction. Artif Cells Nanomed Biotechnol 2019;47:593–601. [47] Olson JS, Foley EW, Rogge C, Tsai AL, Doyle MP, Lemon DD. NO scavenging and the hypertensive effect of hemoglobin-based blood substitutes. Free Radic Biol Med 2004;36:685–97. [48] Sharma AC, Rebello S, Gulati A. Regional circulatory and systemic hemodynamic effects of diaspirin cross-linked hemoglobin in the rat. Artif Cells. Blood Substit Immobil Biotechnol 1994;22:593–602. [49] Mongan PD, Moon-Massat PF, Rentko V, Mihok S, Dragovich A, Sharma P. Regional blood flow after serial normovolemic exchange transfusion with HBOC-201 (Hemopure) in anesthetized swine. J Trauma 2009;67:51–60. [50] Meliga E, Vranckx P, Regar E, et al. Proof-of-concept trial to evaluate haemoglobin based oxygen therapeutics in elective percutaneous coronary revascularization. Rationale, protocol design and haemodynamic results. EuroIntervention 2008;4:99–107. [51] Sakai H, Hara H, Yuasa M, et al. Molecular dimensions of Hb-based O2 carriers determine constriction of resistance arteries and hypertension. Am J Physiol Heart Circ Physiol 20 0 0;279:H908–15. [52] Biro GP. Adverse HBOC-endothelial dysfunction synergism: a possible contributor to adverse clinical outcomes. Curr Drug Discov Technol 2012;9:194–203. [53] D’Agnillo F. Redox activity of cell-free hemoglobin: implications for vascular oxidative stress and endothelial dysfunction. In: Kim HW, Greenburg AG, editors. Hemoglobin-based oxygen carriers as red cell substitutes and oxygen therapeutics. New York: Springer; 2013. p. 665–82. [54] Ma Z, Monk TG, Goodnough LT, et al. Effect of hemoglobin- and perflubron-based oxygen carriers on common clinical laboratory tests. Clin Chem 1997;43:1732–7. [55] Björkholom M, Fagrell B, Przybelski R, Winslow N, Young M, Winslow RM. A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethylene glycol. Haematologica 2005;90:505–15. [56] Cameron SJ, Gerhardti G, Engelstad M, Young MA, Norris EJ, Sokoll LJ. Interference in clinical chemistry assays by the hemoglobin-based oxygen carrier. Hemospan. Clin Biochem. 2009;42:221–4. [57] Sodi R, Dam SM, Davison AS, Stott A, Shenkin A. Mechanism of interference by haemolysis in the cardiac troponin T immunoassay. Ann Clin Biochem 2006;43:49–56. [58] Varlet-Marie E, Ashenden M, Lasne F, et al. Detection of hemoglobin-based oxygen carriers in human serum for doping analysis: Confirmation by size-exclusion HPLC. Clin Chem 2004;50:723–31.
261
[59] ver Elst KM, Chapelle JP, Boland P, Demolder JS, Gorus FK. Analytic and clinical evaluation of the Abbott AxSYM cardiac troponin I assay. Am J Clin Pathol 1999;112:745–52. [60] Dasgupta A, Wells A, Biddle DA. Negative interference of bilirubin and hemoglobin in the MEIA troponin I assay but not in the MEIA CK-MB assay. J Clin Lab Anal 2001;15:76–80. [61] Whitaker B, Rajbhandary S, Kleinman S, Harris A, Kamani N. Trends in United States blood collection and transfusion: results from the 2013 AABB blood collection, utilization, and patient blood management survey. Transfusion 2016;56:2173–83. [62] He Y, Ning T, Xie T, et al. Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Natl Acad Sci 2011;108:19078–83. [63] Kim HW, Estep TN. Practical considerations in the development of hemoglobin-based oxygen therapeutics. Curr Drug Discov Technol 2012;9:212–23. [64] Chang TMS. From hemoglobin based oxygen carrier to oxygen therapeutics, blood substitutes, nanomedicine and artificial cells. In: Kim HW, Greenburg AG, editors. Hemoglobin-based oxygen carriers as red cell substitutes and oxygen therapeutics. New York: Springer; 2013. p. 3–26. [65] Kumar R, Manjula BN. Human hemoglobin derived from transgenic swine: a starting material for “blood substitute” formulations. In: Rudolph AS, Rabinovici R, Feuerstein GZ, editors. Red blood cell substitutes basic principles and clinical applications. New York: Marcell Dekker; 1998. p. 309–24. [66] Varnado CL, Mollan TL, Birukou I, Smith BJZ, Henderson DP, Olson JS. Development of recombinant hemoglobin-based oxygen carriers. Antioxid Redox Signal 2013;18:2314–28. [67] Weiskopf RB, Silverman TA. Balancing risks and benefits of hemoglobin-based oxygen carriers. Transfusion 2013;53:2327–33. [68] Dubé GP, Vranckx P, Greenburg AG. HBOC-201: the multi-purpose oxygen therapeutic. EuroIntervention 2008;4:161–5. [69] Nelson D, Azari M, Brown R, et al. Preparation and characterization of diaspirin cross-linked hemoglobin solutions for preclinical studies. In: Chang TMS, editor. Blood substitutes and oxygen carriers. New York: Marcel Dekker; 1993. p. 241–5. [70] Gelderman MP, Yazer MH, Jia Y, Wood F, Alayash AI, Vostal JG. Serial oxygen equilibrium and kinetic measurements during RBC storage. Transfus Med 2010;20:341–5. [71] Luten M, Roerdinkholder-Stoelwinder B, Schaap NP, de Grip WJ, Bos HJ, Bosman GJ. Survival of red blood cells after transfusion: a comparison between red cells concentrates of different storage periods. Transfusion 2008;48:1476–85.