Hemoglobin‐based oxygen carriers: Biochemical, biophysical differences, and safety

Hemoglobin-based oxygen carriers (HBOCs) have a long history of development, yet none are currently licensed for use in the US. Early HBOC studies were interested in the toxicity of infused hemoglobin, followed by the studies of Amberson and colleagues from the University of Maryland.1 As early as 1934, Amberson purified bovine hemoglobin and infused it into cats. In 1949, he described purified human hemoglobin infused into anemic parturients with hemorrhage after childbirth.2 Development continued when the US Army manufactured a tetrameric cross-linked hemoglobin (α-α cross-linked hemoglobin), which later was produced by the Baxter Corporation (Deerfield, IL), as 2,3-diaspirin cross-linked hemoglobin (HemAssist).2 However, it failed in human studies because of decreased cellular perfusion and increased morbidity and mortality. Recombinant HBOC was developed but failed due to bacterial endotoxin concerns (Optro, Somatogen, Boulder, CO).2 During a resurgence of activity in the mid-1980s, manufacturers developed second-generation HBOCs, including HBOC-200 (Oxyglobin; HbO2 Therapeutics, Souderton, PA), approved in the US and the EU for canine anemia; Hemoglobin-glutamer-201 (Hemopure; HbO2 Therapeutics, Souderton, PA) studied in the largest completed clinical trial of an HBOC, approved in South Africa in 2001 and Russia in 2006 and allowed for expanded access use in the US by the FDA.2-4 PolyHeme by Northfield Laboratories (Evanston, IL) and Hemolink by Hemosol, Inc. (Mississauga, Ontario, Canada) were discontinued due to a question of efficacy or to safety concerns.2 ErythroMer (Kalocyte; Baltimore, MD), an encapsulated HBOC, is being developed as a replacement for red blood cells.5 Most recently, Hemarina (Morlaix, FR) has developed HemO2Life, approved in the EU in 2022 for ex-vivo perfusion of kidneys prior to transplantation and orphan designation in the EU for patients undergoing hematopoietic stem cell transplantation.6 Other HBOCs are in various preclinical stages of development. The efficacy of various HBOCs is dependent on biophysical and biochemical characteristics, results of preclinical and clinical studies, and the indication(s) being proposed.2 The challenge for earlier generation HBOCs was safety due to multiple factors (see Table 15, 6, 8-18). The purpose of this Commentary is to define these safety issues related to biophysical and biochemical characteristics. We compare and contrast first, second, third, and new-generation HBOCs based on these characteristics, then with the information learned and correlating to published clinical studies, begin to study how to manage the safety concerns when alternatives are unavailable. First-generation products are referred to as HBOCs that have undergone clinical trials in the US (phase I–III) whereas HBOCs that have yet to undergo clinical trials are referred to as second-generation HBOCs. However, in a recent chapter on classifications of HBOCs, an alternative nomenclature basing the generations on chemical structure has also been proposed.7 Finally, we provide insights into newer generation HBOC development to avoid these issues and derive patient benefit from the efficacies of these products. The manufacturer contended that SAEs in Phase III trials were a result of patient comorbidity, patient management differences, and underdosing the product with ongoing anemia and ischemia and overdosing with CHF. There is no evidence of vasoconstriction in the heart, brain or kidney. This product affects the MAP, SVRI while not affecting LVEDP, coronary output, or coronary perfusion. It is approved for human use in acute anemia in South Africa6, 8 Conjugated: Third Generation While packed red blood cell (pRBC) transfusions remain the mainstay of treatment for patients who are severely anemic, require increased oxygen delivery to tissues, or are acutely bleeding, HBOCs have been in development for decades as an alternative. These products hold potential for scenarios in which pRBCs may not be a feasible option, such as in severe trauma and combat medicine in remote locations (due to limited storage, the requirement for crossmatching, and potential difficulties with cold storage), when there is scarcity in donated blood, and for patients who cannot receive pRBCs, either due to rare blood types/compatibility issues or religious objection. HBOCs, using acellular Hb derived from bovine or human Hb or genetically engineered, have been explored as an option since the 1930s when Amberson used purified human and bovine Hb. HBOCs have been created using different methods, often involving cross-linking, the addition of chemical modifiers, PEGylation, and polymerization (Figure 1).2 Hb within RBCs is found at high concentration which favors stabilization of the Hb tetramer. Additionally, 2, 3-diphosphoglycerate (2,3-DPG), a natural allosteric modifier of Hb adds additional stability to the molecule. When Hb is extracted from RBCs in a dilute solution, it tends to dimerize into α/β dimers, which, if infused, will be cleared rapidly and which could damage kidneys and other organs. Manufacturers treat Hb with chemical reagents that specifically crosslink the dimers and/or polymerize a number of tetramers in a large polymer. This will stabilize Hb in circulation and in some cases improve its function. Despite the variety of chemical approaches used in creating HBOCs, there have been persistent concerns related to serious adverse events throughout their development.2, 8, 19-21 In particular, concerns have arisen relating to cellular oxidative stress, nitric oxide (NO) scavenging, methemoglobinemia, and the inflammatory response seen when heme is lost from Hb.2, 8, 19-21 Various mechanisms have been proposed for the causes of serious adverse events with the use of HBOCs. Among the major biochemical mechanisms that explain HBOC toxicity includes (a) scavenging of endothelial nitic oxide and consequent hemodynamic changes, (b) oversupply of oxygen and autoregulatory responses, and (c) heme-mediated oxidative reactions. These pathways, singularly or collectively, may have contributed to the vascular responses and subsequent injuries.19 Cellular and sub-cellular models using human kidney endothelium, mouse lung epithelium, and alveolar cells have been used to demonstrate the deleterious effects of Hb oxidation and heme loss.21 Free heme is itself a damage-associated molecular pattern (DAMP), which leads to an inflammatory response.21 DAMPs are molecules within cells that are a component of the innate immune response released as danger signals from damaged or dying cells due to trauma or an infection. Heme released from Hb was recently recognized as a DAMP molecule as it triggers a cascade of inflammatory responses.20 Free heme is also a critical mediator of endothelial dysfunction, kidney and lung injury in sepsis, and poor systemic outcomes of hemolytic disorders. It likely also plays a role in poor outcomes from polytrauma.22 Hb undergoes a redox (reduction–oxidation) transition, leading to the formation of higher oxidation intermediates, such as ferryl Hb (HbFe4+), which can attack other biological entities and ultimately self-destruct, leading to heme loss.21 There has been considerable research activity focused in recent years on using haptoglobin (protein scavenger) and hemopexin (heme scavenger).23, 24 However, haptoglobin exhibits weak binding to some HBOCs and the cost of using highly purified scavenger as an additive may have prohibited further development of these important reagents. Newer and current-generation HBOCs have been designed to a certain extent to overcome some of the reported toxicities associated with first generation HBOCs. However, very little is known about possible toxicities, as some of these HBOCs have only recently been produced or reported in the literature.14, 15, 17, 25, 26 Among the most promising intervention strategies designed to control or minimize HBOC toxicity besides haptoglobin and hemopexin, are antioxidants, and reducing agents such as ascorbic acid were used.21 Utilizing the power of genetic engineering, new-generation HBOC prototypes have been engineered with built-in superior oxidative stability, which may pave the way for genetically engineered HBOCs.27 Despite the diverse nature of chemical and genetic modifications during HBOC development, very little attention was given to the impact of these alterations on the efficacy and safety of HBOCs. Recently, a comprehensive biochemical and biophysical characterization was carried out on all HBOCs that have undergone clinical evaluation, allowing for the first-time direct comparison.8 Here, we revisit the documented HBOCs' chemistries and attempt to correlate these unique chemical entities with their published clinical outcomes. This Commentary is divided into the following sections: Introduction (above), Evaluation, Findings, Discussion, and Conclusions. The goal of this work was to synthesize available data to address concerns around the serious adverse events of HBOCs, to aid in the use of pharmacological strategies to address adverse effects. Concretely, we reviewed recent literature on the subject, examining the structure, activity, and biochemical relationships of some discontinued and actively developing HBOCs. We have also analyzed available literature on the serious adverse events of these products. For each of these products, we identified Hb type, molecular weight, SDS-PAGE analysis, p50, n50, catalase activity, autoxidation rate, NO-induced oxidation (kNO), and heme loss kinetics. We then synthesized this data into a table to allow for comparison of these factors. Finally, we interpreted our findings, with the goal of creating a path forward for further innovation and development of HBOCs. In collating the available biochemical data on these HBOC products, various trends were identified (see Table 1). One critical variable common among HBOCs is the different starting materials. Almost all HBOCs began as stroma-free Hb (SFH) or stroma-poor Hb. This means that these Hbs retained some red cell proteins prior to chemical modifications. One particular HBOC, Hemolink, in which all of these contaminants were eliminated, started with almost 99% pure Hb known as HbA0 by using extensive anionic and cationic chromatography; however, the product eventually was discontinued due to adverse effects noted in a number of clinical trials.2, 28 Many HBOCs are derived from human outdated blood prior to chemical modifications with minimal purification steps that remove other red cell proteins to produce SFH or stroma-poor Hb. After treatment with the modifying chemical reagent(s), this results in HBOC solutions that are contaminated with these proteins. Other products derive from bovine blood, and one uses a sea worm hemoglobin, but all require some chemical modification.6 As HBOCs evolved from small, cross-linked molecules with lower molecular weight to larger, polymerized molecules, heme loss kinetics trended lower, with the exception of Sanguinate.8 This is in fitting with the goal of larger HBOCs, which were created with a purpose to reduce protein unfolding, thus decreasing heme exposure and loss.15 Oxygen affinities, as measured by oxygen equilibrium curves (OECs) for HBOCs, range from sigmoidal to non-sigmoidal almost linear, and in some cases with reduced cooperativity (communication among the 4 heme centers). The ideal p50 (oxygen affinity when Hb is half saturated) of HBOCs is still unknown, with earlier cross-linked and/or polymerized products targeting a p50 of approximately 30–35 mmHg, close to the p50 of fresh human blood (27–29 mmHg). Conjugated, encapsulated, and naturally polymerized HBOCs, however, have sought a lower p50, decreasing oxygen offloading to tissues, except in extremely hypoxic conditions by increasing its affinity to Hb. Although some within the HBOC community believe that HBOCs should have higher oxygen affinity (decreased p50) as HBOCs with low oxygen affinity (larger p50) may trigger autoregulatory responses (vasoconstriction) due to the immature offloading of oxygen. Both Hemarina and Sangart (PEGHHb) have lower p50s. One notable exception is the ErythroMer encapsulated product, which is pH responsive, thus allowing for a dynamic p50.5, 25 Cooperativity, (n50; when an oxygen atom binds to one of hemoglobin's four binding sites, the affinity to oxygen of the three remaining available binding sites increases) has not evolved drastically across HBOC products. It varies from 1.0 in Hemolink (O-R-PolyHbA0) to 2.5 in ErythroMer and HEMO2Life.2, 8, 14, 25 In addition to the unique oxygen affinity characteristics of HBOCs discussed above and listed in Table 1, we included some other equally important characteristics that defined these HBOCs further. Collectively, these properties may have some bearing on their safety as well as efficacy. Besides their unique electrophoretic properties, HBOCs differ considerably in their oxidative and redox properties that include autoxidative (spontaneous) oxidation kinetics that describe the trends of iron to oxidize and subsequent oxidative changes. In addition, antioxidative properties are indicated by their own catalase activities, that is, the ability to remove oxidants such as peroxide. Altogether, these reactions are profoundly different from those characteristics of normal unmodified HbA which dissociates into dimers rapidly after transfusion and exhibits non-physiological oxygen binding as well as oxidative behavior. Polyheme (PolyHHb) has increased catalase activity, at 4.01 units/mL, due possibly to the contaminating catalase in SFH. All other products had low catalase activity, between 0 and 0.24 units/mL.8 Cross-linked HBOCs, which were ultimately discontinued, showed lower rates of autoxidation, with rate constants between 0.081 and 0.095 hr−1. All other products, meanwhile, had autoxidation rate constants between 0.19 and 0.26 h−1.8 Most HBOCs had rate constants of NO-induced oxidation between 40 and 45 μM−1 s−1. Recombinant Hb (Optro) showed very high rates of NO-induced oxidation (59.6 μM−1 s−1), which is notable given its discontinuation because of hypertensive effects.8, 14 Polymerized bovine Hb, HBOC-201 (Hemopure) and HBOC-200 (Oxyglobin), showed the least heme loss, with levels of 3.47 and 4.5 h−1, respectively.8 Conversely, the PEGylated Hbs, Hemospan and Sanguinate, showed increased heme loss, with levels of 14.8 and 16.7 h−1, respectively, due possibly to the fact that these HBOCs lack intermolecular crosslinking.8, 19 Because of the proprietary nature of manufactured HBOCs, very little information was made available in the open literature on manufacturing, preclinical, and clinical assessments. It was only recently that most manufacturers were willing to share their products with independent researchers and then only after the termination of their manufacturing programs. This culminated in the publication of a seminal paper comparing side by side all HBOCs that had been clinically tested in humans.8 In that publication, the authors attempted to correlate some of these unique features with their clinical outcome and attempted to draw some general lessons that will enable the design of a safe and effective product and possibly enable the use of earlier products. Based on this publication, clinical trials, and clinical outcomes were added to the biophysical and biochemical characteristics of the various products to be able to make more definitive statements regarding products. When critically reviewing the trends, it may make sense to organize the products based on formulation (HBOC Class) and by increasing molecular weight.7 In this case, the smallest molecules, the cross-linked and cross-linked are notable for increased rates of with the being The least may be the largest molecules, and as manufacturers believe that are large to be endothelial ErythroMer may have this with to fresh red blood cells by allowing for p50 based on the pH of the cellular However, some products may be able to overcome this with as the is a result of a in study by the showed that a of and HBOC-201 to the of in a of effects on with earlier generation HBOCs is This is dependent on the of and which is in dependent on cellular Hb the for to is found in the red cell with severe anemia there is reducing of ascorbic and can be to the adverse effects of when above as this with However, in cases the is extremely this may be an and could require pRBCs in the as this be the only way to red cell and it has been documented that early in hemorrhage of Hb above may improve an HBOC with higher Hb such as HBOC-201 may be or which was available as a product, and could be with only of to higher Hb, and which could be The of the identified trends can only be in the of each clinical outcomes. The large product, naturally polymerized and derived from a has recently been studied in and approved in the EU for ex-vivo perfusion of In this its use in the for ex-vivo kidneys has and of Heme loss is a result of HBOCs heme (due to oxidative within the Hb or that can be using haptoglobin or HBOCs have increased rates of autoxidation, to three higher rates in human or bovine Hb within red blood There may be a between scavenging and increased with HBOCs due to of that be by who had with HBOCs showed increased blood due to the of HBOCs, increased However, it is important not to all HBOCs with serious adverse as all HBOCs are not as we have has different effects and be studied For HBOC-201 (Hemopure) has been to not have on in a It is if other products have effects on Additionally, a product no has been to avoid and responses due to a designed While HBOCs have been to pRBCs, the authors that this is not the comparison to It that HBOCs have been by concerns of serious adverse events due to multiple however, with some in their development, we that HBOCs may be in pRBCs are either not available or not an For approved use of HBOC-201 (Hemopure) for expanded access has been documented in almost cases in multiple In it may have been when had either failed or have been or for expanded access have been Despite the long history of HBOC development the of a product licensed in the this an of there was a in the publication rate in about which may have been from a serious adverse The paper by was a in the development of HBOCs. began to address the that all HBOCs in development are different but may have that the that HBOCs be to in the to in referred to as We are that some in the are the that all HBOC studies may have been on hold by the The is not allowed to make or including toxicity or safety concerns of efficacy lack that are not in the we cannot on there are ongoing trials with an HBOC and there have been clinical trials with an HBOC the or data are prior to into clinical studies may with the However, the very are the as for biological prior to a the of if the HBOC is and efficacy. range of studies be studies not specifically be The human to be included in clinical studies will on the proposed and the will also based on the proposed higher potential benefit make higher potential Although some products packed red blood the potential of HBOCs and Hb may make this an to overcome for some products, a more limited All HBOCs are not the and each product to the for to human the outcomes of the and the of HBOC for expanded access one the trials have been not allow a but there are potential to with the of studies control As for the expanded access only approximately have been reported to in the There are other potential including and and the potential for an of treatment and Additionally, the of data by who may only a expanded access in their may be There are multiple HBOC products will to overcome to allow for in the US as indicated in the We are to address potential that may be sought by However, to one when blood is not an option, it will require is in fact a and Additionally, HBOC manufacturers will to an control or no product is as a there be clinical given the history of these products to allow a comparison of of with an HBOC these may be to address as these products from to clinical development and expanded access a not for it provide is limited by the lack of of all of the HBOCs, for and and there are additional products in various stages of preclinical and South clinical trials are to compare and HBOCs. and and and have no of or with this work in All authors that this is not All authors we are the authors and have critically reviewed the All authors this was not or by The authors have no of

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