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Hemoglobin-Based Blood Substitutes
CHAPTER
111.8
Hemoglobin-Based Blood Substitutes A L A N S. R U D O L P H
Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington DC 20375, USA
1 Introduction
Transfusion medicine has intrigued man throughout history. In the last century, as the many physiologic roles of blood have been defined, and fractionation methods to separate blood into its cellular and molecular components have progressed, many blood products which dramatically improve human health have been developed. With increasing transfusion practices in the latter part of this century, the increased incidences of disease transmission and the shortages of blood in urban centers and certain areas of the world, have heightened the search for substitutes which mimic various functions of blood. Biomimetic research and development of red blood cell function toward development of a resuscitative artificial oxygen carrying fluid has been one active area of blood substitute research. There is clearly a need for such a product as current estimates of red blood cell usage in the United States are between 12 and 13 million units per year. The efforts to develop such a fluid have focused on using the allosteric oxygen binding properties of the protein hemoglobin or the solublization of oxygen by fluorocarbons. Both of these strategies have been fertile areas of research and have resulted in the development of products which have been, and are now being tested in the clinic. The study of the oxygen delivery properties of these solutions have raised new questions over the appropriate physiologic need and delivery of oxygen both systemically and in the microcirculation. In addition, the development of these products have raised new regulatory challenges as these agencies have struggled with the determination of efficacy of these products in spite of the assumption that a plasma expander that carried oxygen was better than one that did not. Because of the difficulty in clearly demonstrating the efficacy of red blood cell transfusion in the clinic, and the differing clinical practices of red blood cell use, indirect
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demonstrations of efficacy (surrogate end points) through the reduction in the use of allogeneic blood in the clinic have been allowed [1]. There have been numerous proposed uses of a blood substitute. These include elective surgery, emergency surgery and trauma, trauma resuscitation, treatment of anemias, oxygenation of ischemic tissues in stroke, post-angioplasty treatment, peripheral vascular disease, extracorporeal oxygenation in cardiopulmonary bypass, organ preservation solutions, and enhanced oxygen delivery to tumors for irradiation therapies. The ultimate use of any blood substitute will depend on the clinical indication proposed and the physiologic properties of the solution used. The purpose of this paper is to review the recent research and development in the field of hemoglobin-based blood substitutes. The pursuit of an artificial oxygen carrying fluid has been at times full of promise and disappointment, with a history of successes and failures in the clinic. The realities and perceptions of the safety of the blood supply have at times driven research support and clinical development in the field. In spite of this, with new products now entering late phase clinical trials, there is once again hope that artificial oxygen carrying fluids will soon be available.
2 Design considerations in the fabrication of a hemoglobin-based blood substitute
There are a number of important design considerations in the fabrication of a safe and effective red cell substitute. It is important to recognize that the clinical utility of a red blood cell substitute is likely to require administration of a very large therapeutic dose, with grams of protein or fluorocarbon administrated over a short time period. The final design of the system will of course depend on the clinical use of the substitute, but stringent quality control as well as physiologic effects in these dose ranges must be understood. Design considerations include engineering a substitute that is metabolized by the body but has vascular persistence commensurate with desired oxygen delivery characteristics, does not contain histocompatibility antigens and is therefore blood type free resulting in wide patient acceptability, is free of transmittable antigens, can be scaled-up, and has shelf-life properties consistent with the logistics of standard hospital or military use of blood. For hemoglobin based artificial oxygen carrying fluids, there has been considerable progress in achieving these figures of merit. More recently, with the increased concern about the rising costs of health care administration in the US, there has been additional pressures to design a red cell subsitute that is cost competitive and competes favorably with the fully loaded cost of a tested unit of red cells. These design considerations will be used as a basis to evaluate the recent progress of hemoglobin based blood substitutes.
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3 Research issues in the development of hemoglobin-based blood substitutes
Hemoglobin is a tetrameric allosteric protein made up of two a and two/3 subunits. The two a/3 dimeric subunits are loosely bound and dynamically participate in the reversible binding of oxygen by forming the tense (T) and relaxed (R) structures. The cooperativity of oxygen binding and the large oxygen binding capacity of hemoglobin in blood (the normal hemoglobin content of blood in mammals in 15 g/dl) made the choice of using hemoglobin in the design of blood substitutes natural. Attempts as early as 1920 to use hemolysed blood in transfusion demonstrated toxicities and initiated the search for the mechanism of hemoglobin based toxicities. This search was obscured by the difficult task of purifying the molecule. Many of the toxicities observed have since been attributed to trace stromal or other contaminants [2, 3]. Methods to remove stromal contaminants, viral inactivation, and sterile filtration of hemoglobin improved outcomes following its administration. These efforts allowed more definitive studies on the effects of hemoglobin with new important information relevant to the pursuit of a blood substitute still being generated. The three main strategies of hemoglobin-based blood substitutes are seen in Fig. 1.
3.1 Metabolism, biodistribution, and vascular persistence of native and modified hemoglobins The loose binding of the dimeric subunits of native tetrameric hemoglobin
Fig. 1. Three most prominent strategies for hemoglobin-based blood substitutes.
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has been an important characteristic in the biodistribution and metabolism of hemoglobin-based red cell substitutes. Early attempts to transfuse hemoglobin showed a rapid dissociation of the tetramer and clearance of the dimeric subunits in the kidney, with precipitation in the proximal tubulae resulting in significant nephrotoxicity observed in animal studies [6-8]. This observation was dose dependent as the mechanism of hemoglobin metabolism involves opsonization with serum proteins (haptoglobin, hemopexin, albumin), uptake by the liver and transport to microsomes where heme oxygenase initiates its conversion to bilirubin. With high dose administration, this system is saturated resulting in filtration by the kidney. Early quantitative biodistribution studies with native hemoglobin show the majority in the renal cortex and renal medulla, with significant distribution to the liver, spleen, marrow, and lymph, indicating a considerable extravasation of the protein [9]. Much debate on the effects of hemoglobin has focused on the purification and preparation of the protein as the large dose administrations amplified small impurities present in the solutions. Purification methods introduced by Rabiner et al., 1967 demonstrated that infusion of purified hemoglobin into cynomologous monkeys was not nephrotoxic, while stroma from the same preparation caused renal failure [10, 11]. In spite of this, the clinical trial with purified native tetrameric hemoglobin of Savitsky in 1978 was a major set back for the field as human volunteers showed a fall in urinary output and creatinine clearance following the infusion of small doses of purified hemoglobin [12]. The results of the Savitsky clinical trial led investigators in the early 1980s to explore protein engineering as a means to modify hemoglobin using chemical and recombinant techniques with the purpose of stabiiizing the tetramer and preventing dissociation into dimers with subsequent clearance by the kidneys. Chemical modification to intramolecularly crosslink the dimer subunits of hemoglobin or attach modifiers of oxygen carrying capacity, resulting in a stabilized 64 kDa protein, have been demonstrated by many groups with a variety of cross-linking agents [13-15]. These reactions take advantage of various reactive sites on the surface, internal oxygen binding cavity, and amino terminus of hemoglobin. These agents include aldehydes, pyridoxyl derivatives, trimesic acid, polyethylene glycol, and dextrans. The use of the non-specific aldehydes also results in the formation of intermolecular cross-links resulting in a heterogenous distribution of higher molecular weight polymerized hemoglobin species [16]. Recombinant techniques have also been used to express intramolecularly cross-linked hemoglobin [17]. These modifications result in an increased vascular persistence of hemoglobin and reduced filtration by the kidney, alleviating one of the serious toxicities associated with native hemoglobin infusion [18]. The circulation half-life of the modified hemoglobins is species and dose dependent, but has been reported between 8 and 12 hours, while polymerization extends
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the half-life to 12-16 hours [19]. The biodistribution of chemically stabilized tetrameric hemoglobin may still result in a significant level of extravasated protein which may play a major role in the physiologic effects of hemoglobin. An alternative strategy to shifting the biodistribution and metabolism of native or chemically modified hemoglobins which has recieved more limited attention is the encapsulation of hemoglobin in biodegradable delivery vehicles [20]. The encapsulation of hemoglobin in phospholipid vesicles has been shown to shift the metabolism of hemoglobin to the reticuloendothelial system, specifically sites of phagocytic removal, the liver and spleen [21]. These studies also indicated that there is minimal (< 3%) extravasation of free hemoglobin when encapsulated in 0.2 Izm or larger vesicles.
3.2 Interactions of hemoglobin with cellular and molecular species The interactions of hemoglobin with cellular and molecular species in the vascular compartment also plays a dominant role in the safety of efficacy of hemoglobin-based blood substitutes. Over the last decade, as chemical modifications demonstrated reduced renal handling of hemoglobin, one focus of much work has been on direct effects of hemoglobin in the vascular compartment, with observations of hemoglobin induced vasoactivity manifest as systemic and pulmonary hypertension following the administration of native or chemically modified hemoglobins in animals and humans [22-25], and direct effects on endothelial cells due to the free radical damage from the uptake of heme and iron [26, 27]. The discovery of endothelium-derived relaxation factor as nitric oxide (NO) suggested a possible mechanism for the observed vasoconstrictor response of hemoglobin as hemoglobin is a potent ligand of NO [28]. While there is some debate on changes in flow in vessels and the clinical significance of the hypertension caused by hemoglobin-NO interactions, the extravasation of the chemically modified 64 kDa forms of hemoglobin is thought to result in the observed lack of vasorelaxation and resultant hypertension. The effects of hemoglobin on vascular flow in the microcirculation has been documented as a decrease in the functional capillary density in mesenteric hamster skin fold model [29] while other groups have shown that chemically modified hemoglobin does not alter the flow to various organs in the rat [30]. This loss of functional vasculature as a result of hemoglobin-induced vasoconstriction has also been observed in a high flow tissues such as the vascular plexus of the rabbit eye [31]. Larger molecular forms of hemoglobin, either through polymerization or encapsulation may not extravasate to the same extent. Encapsulation of chemically modified hemoglobin was recently shown to result in significantly less vasoconstrictor activity (60-100 fold) than cell free hemoglobin (chemically
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modified) in an isolated vessel segment model (Fig. 2) [32]. The encapsulation of hemoglobin also has been shown to reduce hemoglobin induced impaired flow in the choroidal plexus of the rabbit eye compared to equivalent concentrations of chemically cross-linked 64 kDa hemoglobin [31]. Preliminary results in our laboratory suggest that the lipid bilayer in liposome encapsulated hemoglobin is also a barrier to heme transfer and may reduce heme oxygenase activity in endothelial cells exposed to encapsulated hemoglobin. The binding of NO by hemoglobin may have consequences for other cell and subcellular elements as well which may have import in surgical settings. Recent reports have examined the effect of hemoglobin on platelet deposition as reduced NO availability increases platelet deposition. In a rat carotid endarterectomy model, influsion of hemoglobin (0.9 g/kg) increased plaletet deposition by 71% [34]. In some surgical procedures, enhanced platelet deposition may be advantageous but this effect warrants further investigation. Hemoglobin binding of NO produced by macrophages as a result of upregulated iNOS activity in bacteremia or endotoxemia models has been another area of active investigation [35, 36]. This activity has resulted in the exploration of chemically modified hemoglobin as an adjunct therapy for sepsis [37]. Finally, there has been recent important evidence that hemoglobin may play a regulatory role in shuttling NO from the lungs to the tissue based on the reactivity of hemoglobin cystein groups and the formation of S-nitrosylthiols [38]. How this proposed activity of hemoglobin may play a role in the development of a hemoglobin based blood substitute remains to be determined. Hemoglobin has been proposed to be a binding protein for endotoxin [39]. Enhanced endotoxin activity in the limulus ameoba lysate assay has been demonstrated (same). The in vivo consequences of such binding has been the subject of some debate. Early reports of hemoglobin exacerbating endotoxemias in animal models were performed with solutions that were suspect for stromal impurities [40, 41]. More recent studies have also demonstrated exacerbated septic consequences in mice that have been infused with pyridoxylated polymerized hemoglobin [42]. Additional considerations in this effect is the nutritional supplement provided by the large dose infusion of the iron containing protein. While encapsulation may limit the exposure of hemoglobin to endotoxin, the liposome surface of liposomal encapsulated hemoglobin has been shown to interact and bind endotoxin as a result of surface adherent hemoglobin [43, 44]. The phagocytosis of liposome encapsulated hemoglobin has also been shown to reduce the inflammatory cytokine response from macrophages exposed to endotoxin [45, 46]. The clinical significance of this effect has not been addressed.
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3.3 Oxygen delivery The standard transfusion practice of giving patients packed red cells if their hemoglobin drops below 10 g/dl is based on the assumption that an 0 2 reserve is needed and can only be maintained by increasing hemoglobin content. With the blood safety issues of the 1980s these practices have been re-examined in the context of the 'transfusion trigger'. The development of hemoglobin based blood substitutes has also presented the clinician with a new set of issues. The point distribution of the oxygen carrying unit is no longer a 6-7 #m package, but a 64 kDa protein. The transit time of this protein is relatively short (on the order of hours) compared to days for transfused red cells. The optimal oxygen carrying capacity of the protein in the microcirculation is also unknown. In spite of these issues, the most important characteristic of a hemoglobin based blood substitute is the ability of the solution to participate in the cycling of oxygen and carbon dioxide. Removal of hemoglobin from the red blood cell results in a significant decrease in the oxygen carrying capacity (Ps0) of the hemoglobin due to the loss of DPG. In spite of this, there were numerous early studies demonstrating that native bovine hemoglobin when infused into animals delivers oxygen [47,48]. The derivatization of hemoglobin with pyridoxyl phosphates (and other organic phosphates) results in an increase in Ps0, yet there is much debate on what is the optimal oxygen carrying capacity, which may depend on the clinical indication desired. Early demonstrations of efficacy (as defined by oxygen delivery) with purified native hemoglobin preparations also showed that animals could be kept alive after total exchange of red cells with hemoglobin solutions [49, 50]. A few studies with chemically modified or encapsulated hemoglobin have measured direct oxygenation of tissues with oxygen electrodes, or by oxygen-dependent quenching of phosphorescence emitted by metalloporphyrins bound to albumin, and demonstrated oxygen delivery in hemodilution and exchange transfusion models [51, 52]. The encapsulation of hemoglobin does not significantly alter the oxygen binding characteristics of hemoglobin and offers the advantage of cocompartmentalizing modifiers of hemoglobin in order to engineer the desired oxygen carrying capacity. The distribution of oxygen in the microvascular bed has also been a recent focus of attention as the vasoconstrictor properties of hemoglobin have been proposed to limit the oxygen delivery capabilities of these solutions [53]. The alteration in blood viscosity upon red blood cell loss is also thought to be an important determinant in the hemodynamics of oxygen transport in the microvasculature [54].
3.4 Hemoglobin products and clinical trials Over the last 5 years, the clinical development of hemoglobin-based blood
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substitute products has progressed considerably. There are at least 6 companies in phase I - I I I clinical trials (see Table 1). As expected in this stage of development, there are few new reports in the refereed literature on the effects of these solutions on humans. The few reports published appear to suggest that purified, modified hemoglobin is safe at low to moderate doses. Certain side effects of stabilized tetrameric hemoglobin do persist and there is debate on the whether these side effects will limit the utility of these products.
Table 1. Hemoglobin based blood products - - current status Hemoglobin source/ treatment
Product
Company
Human hemoglobin purified from outdated, donated blood, crosslinked 64 kDa protein
HemAssist Baxter In pivotal phase II-III International in Europe and US in cardiac surgery, stroke and acute blood loss
Bovine hemoglobin heterogenous molecular weight
HemoPure BioPure
In Phase II. Phase III studies in elective surgery expected to start in 1997
Human hemoglobin purified from otudated, donated blood; crosslinked and polymerized chemically, heterogenous molecular weight
Hemolink
PhaseI-II. Phase II trials expected to start soon. Indication is orthopedic surgery in patients who have pre-donated blood (much like hemodilution in intent).
Hemososl/ Fresenius
Status
Human hemoglobin PolyHeme Northfield purified from outdated, donated blood; chemically polymerized molecular weight > 64 kDa
In late Phase II (randomized and controlled, but not blinded)in acute blood loss (trauma and urgent surgery). Phase III expected soon.
Genetically engineered human hemoglobin produced in E. coli cross-linked 64 kDa hemodilution
Concluded early Phase II studies, in hemodilution, surgery, and surgical patients who have predonated blood, indications include and acute blood loss
Optro
Somatogen
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In a phase I study of diaspirin cross-linked 64 kDa hemoglobin, a half-life of 3.3 hours at a dose of 100 m g / k g and a dose related increase in lactate dehydrogenase-5-isozyme and serum creatinine kinase was observed in a randomized double blind, cross-over study in 42 healthy adult volunteers [55]. All doses tested were well tolerated but a dose related increase in blood pressure was observed with no evidence of decreased peripheral perfusion at all doses administered. Phase III clinical trials have been initiated with this product. There are other tetrameric hemoglobins stabilized by chemical or recombinant techniques that are also making progress in the clinic with similar side effect profiles. The likely clinical indications for these products will be as adjunct therapies at low to moderate dose application with surrogate efficacy demonstrations that show reduced use of allogeneic blood. Higher molecular weight chemically polymerized human Hb are also making progress in clinical trials specifically aimed at demonstrating efficacy in trauma. Phase I-II clinical trials have been performed and no signficant side effects have been reported. Other products are heterogenous mixtures of cross-linked hemoglobins of varying molecular weight. The larger molecular weight form of hemoglobin is proposed to have much reduced extravasation compared to the 64 kDa form [56]. However, recent data in a random clinical trial on polymerized bovine hemoglobin in patients undergoing pre-operative hemodilution for elective abdominal aortic surgery indicated signficantly increased mean arterial pressure, vascular resistance, and reduced cardiac output, which led the authors to conclude that this preparation impaired oxygen delivery at the dose given (3 ml/kg) [57]. While continuing progress is reported in the popular press, peer-reviewed reports of the efficacy of these products remain to be evaluated. A summary of the status of clinical trials in hemoglobin-based red cell substitutes is seen in Table 1.
4 Future directions in the development of a hemoglobin-based blood substitute
This is an exciting time for the field of blood substitutes as the long awaited promise of a substitute oxygen carrying fluid may soon be realized. These products could dramatically change transfusion practices and offer the clinician new strategies in treating patients that were good candidates for traditional blood therapies. In spite of this progress, the large dose application of hemoglobin-based blood substitutes may not be realized with the first generation products which could limit the clinical utility in unplanned, emergency medicine, especially in military settings where modem hospital practices may not be available to monitor and treat the potential adverse side effects. Research is ongoing into modifications to
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hemoglobin to widen its clinical utility, reduce observed adverse responses and extend its circulation persistence. The encapsulation of hemoglobin has been actively pursued by a number of investigators as this is clearly a more biomimetic strategy for the delivery of hemoglobin. There is wide agreement that encapsulation of hemoglobin offers considerable potential for extending the utility of hemoglobin, increasing the circulation persistence, and shielding hemoglobin from cellular and molecular interactions. Coencapsulation and surface modification also offer the potential to further engineer an oxygen carrying fluid. To date, however, the advantages of the encapsulation of hemoglobin have not outweighed the increased cost of development, which thus have left very few groups actively developing an encapsulated hemoglobin product. One group is the US Navy which actively has pursued the encapsulation of hemoglobin (and the long-term storage of freeze-dried encapsulated hemoglobin) over the last 15 years. Additional strategies for the development of a second generation hemoglobin-based blood substitute include further efforts to protein engineering of the hemoglobin molecule to alter oxygen binding characteristics and decrease reactivity with nitric oxide.
References
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53.
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stroma-free hemoglobin. Surgical Fontrn, 1980, 31, 15-17. Griffiths, E., Cortes, A., Gilbert, N., Stevenson, P., MacDonald, S. and Pepper, D., Haemoglobin-based blood substitutes and sepsis. Lancet, 1995, 345(8943), 158-160. Clif, R. O., Kwasiborski, V. and Rudolph, A. S., A comparative study of the accurate measurement of endotoxin in liposome-encapsulated hemoglobin. Artificial Cells, Blood Substitutes, and Irnmobilization Technology, 1995, 23(3), 331-336. Leslie, S. B., Puvvada, S., Ratna, B. R. and Rudolph, A. S., Encapsulation of hemoglobin in a bicontinuous cubic phase lipid. Biochernica Biophysica Acta, 1996, 1285, 246-254. Langdale, L. A., Maier, R. V., Wilson, L., Pohlman, T. H., Williams, J. G. and Rice, C. L., Liposome encapsulated hemoglobin inhibits tumor necrosis factror release from rabbit alveolar macrophages by a posttranscriptional mechanism. Journal of Leukocyte Biology, 1992, 52, 679-686. Rudolph, A. S., Cliff, R., Kwasiborski, V., Neville, L., Abdullah, F. and Rabinovici, R., Liposome encapsulated hemoglobin modulates lipopolysaccharide-induced tnf-c~ production in mice. Critical Care Medicine, 1997, (in press). Buttle, G. A. H., Kekwick, A. and Schweitzer, A., Blood substitutes in treatment of acute hemorrhage. Lancet, 1940, 2, 507-510. Amberson, W. R., Jennings, J. J. and Rhode, C. M., Clinical experience with hemoglobin-saline solutions. Joztrnal of Applied Physiology, 1949, 1, 469-489. Palani, C. K., DeWoskin, R., Michuda, M., Rosen, A. L. and Moss, G. S., In vivo oxygen transport with a stroma-free hemoglobin solution in primates. Surgical Fonzm, 1974, 25, 199-201. DeVenuto, F., Friedman, H. I. and Mellick, P. W., Massive exchange transfusions with crystalline hemoglobin solution and blood volume. Surgery, 1980, 151, 361-365. Rabinovici, R., Rudolph, A. S., Vernick, J. and Feuerstein, G., A salutary resuscitative fluid: liposome encapsulated hemoglobin/hypertonic saline solution. Journal of Trazzrna, 1993, 35(1), 121-127. Tsai, A. G., Kerger, H. and Intaglietta, M., Microcirculatory consequences of blood substitution with o~c~-hemoglobin. Blood Szzbstitzztes: Physiological Basis of Efficacy, ed. R. M. Winslow, K. D. Vandegriff and M. Intaglietta. Birkhauser, Boston, 1995, 11, 155-174. Tsai, A. G., Kerger, H. and Intaglietta, M., Microvascular oxygen distribution: effects due to free hemoglobin in plasma. Blood Substitutes: New Challenges ed. R. M. Winslow, K. D. Vandegriff and M. Intaglietta. Birkhauser, Boston, 1996, 8, 124-131. Johnson, P. C., Richmond, K., Shonat, R. D., Toth, A., Pal, M., Tischler, M., Tischler, E. and Lynch, R. M., Oxygen delivery regulation: implications for blood substitutes. Blood Substitutes: Physiological Basis of Efficacy, ed. R. M. Winslow, K. D. Vandegriff and M. Intaglietta. Birkhauser, Boston, 1995, 12, 175-186. Pryzebelski, R. J., Daily, E. K., Kisicki, J. C. et al., Phase I study of the safety and pharamacologic effects of diaspirin cross-linked hemoglobin solution. Critical Care Medicine. 1996, 24, 1993-2000.
494 54.
Tissue Engineering Applied to Specialized Tissues
Gould, S. A. and Moss, G. S., Clinical development of human polymerized hemoglobin as a blood substitute. World Jounzal of Sttrgeo,, 1996, 20(9), 1200-1207. 55. Kasper, S. M., Walter, M., Grune, F., Bischoff, A., Erasmi, H. and Buzello, W., Effects of a hemoglobin-based oxygen carrier (hboc-201) on hemodynamics and oxygen transport in patients undergoing preoperative hemodilution for elective abdominal aortic surgery. Anesthesia and Analgesia, 1996, 83(5), 921-927.
111.8
Hemoglobin-Based Blood Substitutes A L A N S. R U D O L P H
Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington DC 20375, USA
1 Introduction
Transfusion medicine has intrigued man throughout history. In the last century, as the many physiologic roles of blood have been defined, and fractionation methods to separate blood into its cellular and molecular components have progressed, many blood products which dramatically improve human health have been developed. With increasing transfusion practices in the latter part of this century, the increased incidences of disease transmission and the shortages of blood in urban centers and certain areas of the world, have heightened the search for substitutes which mimic various functions of blood. Biomimetic research and development of red blood cell function toward development of a resuscitative artificial oxygen carrying fluid has been one active area of blood substitute research. There is clearly a need for such a product as current estimates of red blood cell usage in the United States are between 12 and 13 million units per year. The efforts to develop such a fluid have focused on using the allosteric oxygen binding properties of the protein hemoglobin or the solublization of oxygen by fluorocarbons. Both of these strategies have been fertile areas of research and have resulted in the development of products which have been, and are now being tested in the clinic. The study of the oxygen delivery properties of these solutions have raised new questions over the appropriate physiologic need and delivery of oxygen both systemically and in the microcirculation. In addition, the development of these products have raised new regulatory challenges as these agencies have struggled with the determination of efficacy of these products in spite of the assumption that a plasma expander that carried oxygen was better than one that did not. Because of the difficulty in clearly demonstrating the efficacy of red blood cell transfusion in the clinic, and the differing clinical practices of red blood cell use, indirect
482
Tissue Engineering Applied to Specialized Tissues
demonstrations of efficacy (surrogate end points) through the reduction in the use of allogeneic blood in the clinic have been allowed [1]. There have been numerous proposed uses of a blood substitute. These include elective surgery, emergency surgery and trauma, trauma resuscitation, treatment of anemias, oxygenation of ischemic tissues in stroke, post-angioplasty treatment, peripheral vascular disease, extracorporeal oxygenation in cardiopulmonary bypass, organ preservation solutions, and enhanced oxygen delivery to tumors for irradiation therapies. The ultimate use of any blood substitute will depend on the clinical indication proposed and the physiologic properties of the solution used. The purpose of this paper is to review the recent research and development in the field of hemoglobin-based blood substitutes. The pursuit of an artificial oxygen carrying fluid has been at times full of promise and disappointment, with a history of successes and failures in the clinic. The realities and perceptions of the safety of the blood supply have at times driven research support and clinical development in the field. In spite of this, with new products now entering late phase clinical trials, there is once again hope that artificial oxygen carrying fluids will soon be available.
2 Design considerations in the fabrication of a hemoglobin-based blood substitute
There are a number of important design considerations in the fabrication of a safe and effective red cell substitute. It is important to recognize that the clinical utility of a red blood cell substitute is likely to require administration of a very large therapeutic dose, with grams of protein or fluorocarbon administrated over a short time period. The final design of the system will of course depend on the clinical use of the substitute, but stringent quality control as well as physiologic effects in these dose ranges must be understood. Design considerations include engineering a substitute that is metabolized by the body but has vascular persistence commensurate with desired oxygen delivery characteristics, does not contain histocompatibility antigens and is therefore blood type free resulting in wide patient acceptability, is free of transmittable antigens, can be scaled-up, and has shelf-life properties consistent with the logistics of standard hospital or military use of blood. For hemoglobin based artificial oxygen carrying fluids, there has been considerable progress in achieving these figures of merit. More recently, with the increased concern about the rising costs of health care administration in the US, there has been additional pressures to design a red cell subsitute that is cost competitive and competes favorably with the fully loaded cost of a tested unit of red cells. These design considerations will be used as a basis to evaluate the recent progress of hemoglobin based blood substitutes.
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3 Research issues in the development of hemoglobin-based blood substitutes
Hemoglobin is a tetrameric allosteric protein made up of two a and two/3 subunits. The two a/3 dimeric subunits are loosely bound and dynamically participate in the reversible binding of oxygen by forming the tense (T) and relaxed (R) structures. The cooperativity of oxygen binding and the large oxygen binding capacity of hemoglobin in blood (the normal hemoglobin content of blood in mammals in 15 g/dl) made the choice of using hemoglobin in the design of blood substitutes natural. Attempts as early as 1920 to use hemolysed blood in transfusion demonstrated toxicities and initiated the search for the mechanism of hemoglobin based toxicities. This search was obscured by the difficult task of purifying the molecule. Many of the toxicities observed have since been attributed to trace stromal or other contaminants [2, 3]. Methods to remove stromal contaminants, viral inactivation, and sterile filtration of hemoglobin improved outcomes following its administration. These efforts allowed more definitive studies on the effects of hemoglobin with new important information relevant to the pursuit of a blood substitute still being generated. The three main strategies of hemoglobin-based blood substitutes are seen in Fig. 1.
3.1 Metabolism, biodistribution, and vascular persistence of native and modified hemoglobins The loose binding of the dimeric subunits of native tetrameric hemoglobin
Fig. 1. Three most prominent strategies for hemoglobin-based blood substitutes.
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Tissue Engineering Applied to Specialized Tissues
has been an important characteristic in the biodistribution and metabolism of hemoglobin-based red cell substitutes. Early attempts to transfuse hemoglobin showed a rapid dissociation of the tetramer and clearance of the dimeric subunits in the kidney, with precipitation in the proximal tubulae resulting in significant nephrotoxicity observed in animal studies [6-8]. This observation was dose dependent as the mechanism of hemoglobin metabolism involves opsonization with serum proteins (haptoglobin, hemopexin, albumin), uptake by the liver and transport to microsomes where heme oxygenase initiates its conversion to bilirubin. With high dose administration, this system is saturated resulting in filtration by the kidney. Early quantitative biodistribution studies with native hemoglobin show the majority in the renal cortex and renal medulla, with significant distribution to the liver, spleen, marrow, and lymph, indicating a considerable extravasation of the protein [9]. Much debate on the effects of hemoglobin has focused on the purification and preparation of the protein as the large dose administrations amplified small impurities present in the solutions. Purification methods introduced by Rabiner et al., 1967 demonstrated that infusion of purified hemoglobin into cynomologous monkeys was not nephrotoxic, while stroma from the same preparation caused renal failure [10, 11]. In spite of this, the clinical trial with purified native tetrameric hemoglobin of Savitsky in 1978 was a major set back for the field as human volunteers showed a fall in urinary output and creatinine clearance following the infusion of small doses of purified hemoglobin [12]. The results of the Savitsky clinical trial led investigators in the early 1980s to explore protein engineering as a means to modify hemoglobin using chemical and recombinant techniques with the purpose of stabiiizing the tetramer and preventing dissociation into dimers with subsequent clearance by the kidneys. Chemical modification to intramolecularly crosslink the dimer subunits of hemoglobin or attach modifiers of oxygen carrying capacity, resulting in a stabilized 64 kDa protein, have been demonstrated by many groups with a variety of cross-linking agents [13-15]. These reactions take advantage of various reactive sites on the surface, internal oxygen binding cavity, and amino terminus of hemoglobin. These agents include aldehydes, pyridoxyl derivatives, trimesic acid, polyethylene glycol, and dextrans. The use of the non-specific aldehydes also results in the formation of intermolecular cross-links resulting in a heterogenous distribution of higher molecular weight polymerized hemoglobin species [16]. Recombinant techniques have also been used to express intramolecularly cross-linked hemoglobin [17]. These modifications result in an increased vascular persistence of hemoglobin and reduced filtration by the kidney, alleviating one of the serious toxicities associated with native hemoglobin infusion [18]. The circulation half-life of the modified hemoglobins is species and dose dependent, but has been reported between 8 and 12 hours, while polymerization extends
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485
the half-life to 12-16 hours [19]. The biodistribution of chemically stabilized tetrameric hemoglobin may still result in a significant level of extravasated protein which may play a major role in the physiologic effects of hemoglobin. An alternative strategy to shifting the biodistribution and metabolism of native or chemically modified hemoglobins which has recieved more limited attention is the encapsulation of hemoglobin in biodegradable delivery vehicles [20]. The encapsulation of hemoglobin in phospholipid vesicles has been shown to shift the metabolism of hemoglobin to the reticuloendothelial system, specifically sites of phagocytic removal, the liver and spleen [21]. These studies also indicated that there is minimal (< 3%) extravasation of free hemoglobin when encapsulated in 0.2 Izm or larger vesicles.
3.2 Interactions of hemoglobin with cellular and molecular species The interactions of hemoglobin with cellular and molecular species in the vascular compartment also plays a dominant role in the safety of efficacy of hemoglobin-based blood substitutes. Over the last decade, as chemical modifications demonstrated reduced renal handling of hemoglobin, one focus of much work has been on direct effects of hemoglobin in the vascular compartment, with observations of hemoglobin induced vasoactivity manifest as systemic and pulmonary hypertension following the administration of native or chemically modified hemoglobins in animals and humans [22-25], and direct effects on endothelial cells due to the free radical damage from the uptake of heme and iron [26, 27]. The discovery of endothelium-derived relaxation factor as nitric oxide (NO) suggested a possible mechanism for the observed vasoconstrictor response of hemoglobin as hemoglobin is a potent ligand of NO [28]. While there is some debate on changes in flow in vessels and the clinical significance of the hypertension caused by hemoglobin-NO interactions, the extravasation of the chemically modified 64 kDa forms of hemoglobin is thought to result in the observed lack of vasorelaxation and resultant hypertension. The effects of hemoglobin on vascular flow in the microcirculation has been documented as a decrease in the functional capillary density in mesenteric hamster skin fold model [29] while other groups have shown that chemically modified hemoglobin does not alter the flow to various organs in the rat [30]. This loss of functional vasculature as a result of hemoglobin-induced vasoconstriction has also been observed in a high flow tissues such as the vascular plexus of the rabbit eye [31]. Larger molecular forms of hemoglobin, either through polymerization or encapsulation may not extravasate to the same extent. Encapsulation of chemically modified hemoglobin was recently shown to result in significantly less vasoconstrictor activity (60-100 fold) than cell free hemoglobin (chemically
486
Tissue Engineering Applied to Specialized Tissues
modified) in an isolated vessel segment model (Fig. 2) [32]. The encapsulation of hemoglobin also has been shown to reduce hemoglobin induced impaired flow in the choroidal plexus of the rabbit eye compared to equivalent concentrations of chemically cross-linked 64 kDa hemoglobin [31]. Preliminary results in our laboratory suggest that the lipid bilayer in liposome encapsulated hemoglobin is also a barrier to heme transfer and may reduce heme oxygenase activity in endothelial cells exposed to encapsulated hemoglobin. The binding of NO by hemoglobin may have consequences for other cell and subcellular elements as well which may have import in surgical settings. Recent reports have examined the effect of hemoglobin on platelet deposition as reduced NO availability increases platelet deposition. In a rat carotid endarterectomy model, influsion of hemoglobin (0.9 g/kg) increased plaletet deposition by 71% [34]. In some surgical procedures, enhanced platelet deposition may be advantageous but this effect warrants further investigation. Hemoglobin binding of NO produced by macrophages as a result of upregulated iNOS activity in bacteremia or endotoxemia models has been another area of active investigation [35, 36]. This activity has resulted in the exploration of chemically modified hemoglobin as an adjunct therapy for sepsis [37]. Finally, there has been recent important evidence that hemoglobin may play a regulatory role in shuttling NO from the lungs to the tissue based on the reactivity of hemoglobin cystein groups and the formation of S-nitrosylthiols [38]. How this proposed activity of hemoglobin may play a role in the development of a hemoglobin based blood substitute remains to be determined. Hemoglobin has been proposed to be a binding protein for endotoxin [39]. Enhanced endotoxin activity in the limulus ameoba lysate assay has been demonstrated (same). The in vivo consequences of such binding has been the subject of some debate. Early reports of hemoglobin exacerbating endotoxemias in animal models were performed with solutions that were suspect for stromal impurities [40, 41]. More recent studies have also demonstrated exacerbated septic consequences in mice that have been infused with pyridoxylated polymerized hemoglobin [42]. Additional considerations in this effect is the nutritional supplement provided by the large dose infusion of the iron containing protein. While encapsulation may limit the exposure of hemoglobin to endotoxin, the liposome surface of liposomal encapsulated hemoglobin has been shown to interact and bind endotoxin as a result of surface adherent hemoglobin [43, 44]. The phagocytosis of liposome encapsulated hemoglobin has also been shown to reduce the inflammatory cytokine response from macrophages exposed to endotoxin [45, 46]. The clinical significance of this effect has not been addressed.
Hemoglobin-Based Blood Substitutes
487
3.3 Oxygen delivery The standard transfusion practice of giving patients packed red cells if their hemoglobin drops below 10 g/dl is based on the assumption that an 0 2 reserve is needed and can only be maintained by increasing hemoglobin content. With the blood safety issues of the 1980s these practices have been re-examined in the context of the 'transfusion trigger'. The development of hemoglobin based blood substitutes has also presented the clinician with a new set of issues. The point distribution of the oxygen carrying unit is no longer a 6-7 #m package, but a 64 kDa protein. The transit time of this protein is relatively short (on the order of hours) compared to days for transfused red cells. The optimal oxygen carrying capacity of the protein in the microcirculation is also unknown. In spite of these issues, the most important characteristic of a hemoglobin based blood substitute is the ability of the solution to participate in the cycling of oxygen and carbon dioxide. Removal of hemoglobin from the red blood cell results in a significant decrease in the oxygen carrying capacity (Ps0) of the hemoglobin due to the loss of DPG. In spite of this, there were numerous early studies demonstrating that native bovine hemoglobin when infused into animals delivers oxygen [47,48]. The derivatization of hemoglobin with pyridoxyl phosphates (and other organic phosphates) results in an increase in Ps0, yet there is much debate on what is the optimal oxygen carrying capacity, which may depend on the clinical indication desired. Early demonstrations of efficacy (as defined by oxygen delivery) with purified native hemoglobin preparations also showed that animals could be kept alive after total exchange of red cells with hemoglobin solutions [49, 50]. A few studies with chemically modified or encapsulated hemoglobin have measured direct oxygenation of tissues with oxygen electrodes, or by oxygen-dependent quenching of phosphorescence emitted by metalloporphyrins bound to albumin, and demonstrated oxygen delivery in hemodilution and exchange transfusion models [51, 52]. The encapsulation of hemoglobin does not significantly alter the oxygen binding characteristics of hemoglobin and offers the advantage of cocompartmentalizing modifiers of hemoglobin in order to engineer the desired oxygen carrying capacity. The distribution of oxygen in the microvascular bed has also been a recent focus of attention as the vasoconstrictor properties of hemoglobin have been proposed to limit the oxygen delivery capabilities of these solutions [53]. The alteration in blood viscosity upon red blood cell loss is also thought to be an important determinant in the hemodynamics of oxygen transport in the microvasculature [54].
3.4 Hemoglobin products and clinical trials Over the last 5 years, the clinical development of hemoglobin-based blood
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Tissue Engineering Applied to Specialized Tissues
substitute products has progressed considerably. There are at least 6 companies in phase I - I I I clinical trials (see Table 1). As expected in this stage of development, there are few new reports in the refereed literature on the effects of these solutions on humans. The few reports published appear to suggest that purified, modified hemoglobin is safe at low to moderate doses. Certain side effects of stabilized tetrameric hemoglobin do persist and there is debate on the whether these side effects will limit the utility of these products.
Table 1. Hemoglobin based blood products - - current status Hemoglobin source/ treatment
Product
Company
Human hemoglobin purified from outdated, donated blood, crosslinked 64 kDa protein
HemAssist Baxter In pivotal phase II-III International in Europe and US in cardiac surgery, stroke and acute blood loss
Bovine hemoglobin heterogenous molecular weight
HemoPure BioPure
In Phase II. Phase III studies in elective surgery expected to start in 1997
Human hemoglobin purified from otudated, donated blood; crosslinked and polymerized chemically, heterogenous molecular weight
Hemolink
PhaseI-II. Phase II trials expected to start soon. Indication is orthopedic surgery in patients who have pre-donated blood (much like hemodilution in intent).
Hemososl/ Fresenius
Status
Human hemoglobin PolyHeme Northfield purified from outdated, donated blood; chemically polymerized molecular weight > 64 kDa
In late Phase II (randomized and controlled, but not blinded)in acute blood loss (trauma and urgent surgery). Phase III expected soon.
Genetically engineered human hemoglobin produced in E. coli cross-linked 64 kDa hemodilution
Concluded early Phase II studies, in hemodilution, surgery, and surgical patients who have predonated blood, indications include and acute blood loss
Optro
Somatogen
Hemoglobin-Based Blood Substitutes
489
In a phase I study of diaspirin cross-linked 64 kDa hemoglobin, a half-life of 3.3 hours at a dose of 100 m g / k g and a dose related increase in lactate dehydrogenase-5-isozyme and serum creatinine kinase was observed in a randomized double blind, cross-over study in 42 healthy adult volunteers [55]. All doses tested were well tolerated but a dose related increase in blood pressure was observed with no evidence of decreased peripheral perfusion at all doses administered. Phase III clinical trials have been initiated with this product. There are other tetrameric hemoglobins stabilized by chemical or recombinant techniques that are also making progress in the clinic with similar side effect profiles. The likely clinical indications for these products will be as adjunct therapies at low to moderate dose application with surrogate efficacy demonstrations that show reduced use of allogeneic blood. Higher molecular weight chemically polymerized human Hb are also making progress in clinical trials specifically aimed at demonstrating efficacy in trauma. Phase I-II clinical trials have been performed and no signficant side effects have been reported. Other products are heterogenous mixtures of cross-linked hemoglobins of varying molecular weight. The larger molecular weight form of hemoglobin is proposed to have much reduced extravasation compared to the 64 kDa form [56]. However, recent data in a random clinical trial on polymerized bovine hemoglobin in patients undergoing pre-operative hemodilution for elective abdominal aortic surgery indicated signficantly increased mean arterial pressure, vascular resistance, and reduced cardiac output, which led the authors to conclude that this preparation impaired oxygen delivery at the dose given (3 ml/kg) [57]. While continuing progress is reported in the popular press, peer-reviewed reports of the efficacy of these products remain to be evaluated. A summary of the status of clinical trials in hemoglobin-based red cell substitutes is seen in Table 1.
4 Future directions in the development of a hemoglobin-based blood substitute
This is an exciting time for the field of blood substitutes as the long awaited promise of a substitute oxygen carrying fluid may soon be realized. These products could dramatically change transfusion practices and offer the clinician new strategies in treating patients that were good candidates for traditional blood therapies. In spite of this progress, the large dose application of hemoglobin-based blood substitutes may not be realized with the first generation products which could limit the clinical utility in unplanned, emergency medicine, especially in military settings where modem hospital practices may not be available to monitor and treat the potential adverse side effects. Research is ongoing into modifications to
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Tissue Engineering Applied to Specialized Tissues
hemoglobin to widen its clinical utility, reduce observed adverse responses and extend its circulation persistence. The encapsulation of hemoglobin has been actively pursued by a number of investigators as this is clearly a more biomimetic strategy for the delivery of hemoglobin. There is wide agreement that encapsulation of hemoglobin offers considerable potential for extending the utility of hemoglobin, increasing the circulation persistence, and shielding hemoglobin from cellular and molecular interactions. Coencapsulation and surface modification also offer the potential to further engineer an oxygen carrying fluid. To date, however, the advantages of the encapsulation of hemoglobin have not outweighed the increased cost of development, which thus have left very few groups actively developing an encapsulated hemoglobin product. One group is the US Navy which actively has pursued the encapsulation of hemoglobin (and the long-term storage of freeze-dried encapsulated hemoglobin) over the last 15 years. Additional strategies for the development of a second generation hemoglobin-based blood substitute include further efforts to protein engineering of the hemoglobin molecule to alter oxygen binding characteristics and decrease reactivity with nitric oxide.
References
1. Fratantoni, J. C., Demonstration of the efficacy of a therapeutic agent. In Blood Substitutes: Physiological Basis of Efficacy, ed. R. M. Winslow, K. D. Vandegriff and M. Intaglietta. Birkhauser, Boston, 1995, pp. 20-24. 2. Rabiner, S. F., Helbert, J. R., Lopas, H. and Friedman, L. H., Evaluation of stroma-free haemoglobin for use as a plasma expander. Journal of Experimental Medicine, 1967, 126, 1127-1142. 3. Feola, M., Simoni, J., Tran, R. and Canizaro, P. C., Mechanisms of toxicity of hemoglobin solutions. Biomaterials, Artificial Cells and Artificial Organs, 1988, 16, 217-226. 4. Baker, S. L. and Dodds, E. C., Obstruction of the renal tubules during the excretion of haemoglobin. British Journal of Experimental Patholog), 1925, 6, 247-260. 5. Ayer, G. D. and Gauld, A. G., Uremia following blood transfusion. Archives of Pathology, 1942, 33, 513-532. 6. Oken, D. E., Arce, M. L. and Wilson, D. R., Glycerol-induced hemoglobinuric acute renal failure in the rat. I. Micropuncture study of the development of oliguria. Journal of Clinical hzoestigations, 1966, 45, 724-735. 7. Anderson, M. N., Mouritzen, C. V. and Gabrielli, E. R., Mechanisms of plasma hemoglobin clearance after acute hemolysis in dogs: Serum haptoglobin levels and selective deposition in liver and kidney. Atznals of Surgery, 1966, 164, 905-912. 8. Birndorf, N. I., Lopas, H. and Robboy, S. J., Disseminated intravascular coagulation and renal failure. Laboratoo' htoestigations, 1971, 25, 314-319.
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