Red Cell Substitutes Robert M. Winslow Oxygen-carrying plasma expanders (blood substitutes) have been sought for over a century. Development of current products is a result of evolution in the understanding of proteins in general, of hemoglobin in particular, and of how cell-free hemoglobin interacts with the control of local blood flow to ensure adequate tissue oxygenation. Hemoglobinbased products are considered in four “generations” corresponding to major improvements. First-generation products consisted of hemoglobin, freed of red cell membranes (stroma-free hemoglobin [SFH]) , which was renal toxic and vasoactive. Second-generation products were polymerized with aldehyde reagents to reduce or eliminate the renal toxicity, but the products were heterogeneous and still vasoactive. Third-generation products employed more specific intramolecular crosslinking to eliminate polymerization and promote homogeneity, but they also remained vasoactive. Fourth-generation products are based on a new understanding of the way in which microvascular blood flow is controlled and the influence of O2 delivery to vascular walls. After more than a century of research, one of these new solutions should find use as an alternative to red cells for transfusion in certain clinical settings. Semin Hematol 44:51-59 © 2007 Elsevier Inc. All rights reserved.
substitute for blood is a long-sought goal.1 After William Harvey described the circulation of the blood in 1628, Christopher Wren experimented with various solutions, including wine and other substances, without success. Efforts were suspended when death resulted from transfusion of blood itself, until replacement of blood with milk was attempted by Gaillard Thomas and coworkers in an outbreak of cholera in the late 19th century.2 The words of John Brinton3 (1878) predate, in almost startling fashion, some of the claims that have been made by “blood substitute” developers in recent years: “this new operation will, in a few years, have entirely superseded the transfusion of blood, which latter operation is even now being rejected as at once dangerous and unavailing in many parts of the country.” Despite intense research and 128 years, development of an oxygen-carrying plasma expander remains a prized goal in both academia and industry. Many different solutions have been developed and tested to varying degrees in animals and even in humans. Some are perfluorocarbon emulsions, acting simply to increase the amount of non– hemoglobin-bound oxygen. Others are based on hemoglobin, modified in vari-
Sangart, Inc, and Department of Bioengineering, University of California, San Diego, CA. Supported, in part, by NHLBI Grants No. HL-076163 and No. R01 HL62354 and by Sangart, Inc Address correspondence to Robert M. Winslow, MD, Sangart, Inc, 6175 Lusk Blvd, San Diego, CA 92121. E-mail: email@example.com
0037-1963/07/$-see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2006.09.013
ous ways to reduce the toxic effects of hemoglobin and to try to duplicate the oxygen transport properties of red blood cells. Still other products employ encapsulation of hemoglobin in lipid vesicles. This review will deal only with the second group of products, as perfluorocarbon emulsions and lipid vesicles differ in substantial ways from hemoglobin products, and appear to be less promising as general alternatives to blood transfusion. For further information on all types of blood substitute products, the reader is referred to a recent summary of the entire field.4
Stroma-Free Hemoglobin: The First Generation The idea of using hemoglobin, free of red cell membranes (stroma-free hemoglobin [SFH]) was immediately appealing in the early part of the 19th century when it was learned that red cell antigens reside on the cell surface. Von Stark5 treated anemic patients with subcutaneous hemoglobin, but he was not able to prepare a stable solution. Sellards and Minot6 prepared a better solution, made from lysed, washed red cells, and infused from 5 to 33 mL into 33 subjects, who experienced hemoglobinuria. Thirty of the subjects reported no ill effects; the other three reported varying degrees of fever, chills, headache, nausea, flushing and difficulty sleeping. Other small trials were conducted over the following several decades, with similar effects.7–11 51
Figure 1 Reaction of an amine (for example, an ⑀-amino group of a lysine on the surface of hemoglobin) with an aldehyde (for example, glutaraldehyde). The reaction proceeds through a Schiff ’s base intermediate, which is unstable unless reduced to form the final amine adduct.
Perhaps the best described trial of SFH, published by Amberson et al,12 was in a woman with postpartum hemorrhage who had exhausted the supply of crossmatched blood; use of a solution of SFH, while stabilizing her hemodynamics, was associated with oliguria and death from renal failure. Amberson et al noted that hemoglobin solutions appear to cause hypertension and bradycardia, and that such solutions were not yet ready for clinical use. Several studies followed and confirmed that SFH diminishes renal plasma flow and creatinine clearance,13 decreases insulin and para-aminohippurate (PAH) clearance.14 The US Navy sponsored a trial of a solution produced by Merck, Sharp and Dohme, in which 47 patients received infusions, but in 17 blood pressure rose, heart rate fell, and urea clearance was decreased. Rabiner improved the method to produce SFH15 and this solution was employed in a number of small studies. Perhaps the most important was that of Savitsky et al16 in which carefully prepared SFH was infused into eight human volunteers; two reported abdominal pain, seven developed bradycardia and hypertension, and oliguria occurred in all with a fall in creatinine clearance. The authors concluded that vasoconstriction was the cause of the renal problems—in their opinion an effect of hemoglobin itself. Unable to explain these findings, they abandoned SFH as a potential blood substitute.
R.M. Winslow cells, and methods were developed to restrict the hemoglobin conformation to the deoxy (T) state by the use of 2,3-DPG analogs.19 The most popular of these agents was pyridoxal5=-phospate (PLP). Following these two discoveries, several laboratories developed versions of polymerized hemoglobin. Payne20 described the use of the bifunctional aldehyde reagent, glutaraldehyde, which was used for most of the products. Glutaraldehyde technology exploits the reactivity of the ⑀-amino groups of the 42 lysine residues on the surface of the hemoglobin molecule with the aldehyde groups of the crosslinker. The essential problem with this chemistry (Figure 1) is that the reaction proceeds through an unstable Schiff’s base intermediate, which must be subsequently reduced to achieve adequate stability. The stability of this reaction was studied in the US Army laboratory,21,22 which concluded that the reaction products are too heterogeneous to permit adequate characterization (Figure 2). Nevertheless, three products emerged from this approach to hemoglobin modification: PolyHeme (Northfield Laboratories, Evanston, IL), HemoPure (BioPure Corp, Cambridge, MA), and HemoLink (Hemosol Ltd, Mississauga, ON, Canada) (Table 1). PolyHeme is currently completing the first randomized study of an oxygen carrier in the prehospital trauma setting, with results expected to be announced sometime in 2007. HemoLink has been abandoned because of
Polymerized Hemoglobin: The Second Generation The structure of hemoglobin was elucidated in the 1950s and 1960s,17 and the rapid renal elimination of cell-free hemoglobin was considered the result of subunit dissociation. This problem was overcome by the finding that hemoglobin could be chemically modified by reaction with bifunctional crosslinkers such as bis(N-makeimidomethyl) ether (BME).18 The oxygen affinity of such crosslinked hemoglobin was found to be higher (lower P50, the PO2 at which hemoglobin is half saturated with O2) compared to native human red
Figure 2 Analytical anion exchange chromatograms of pyridoxylated hemoglobin reduced with sodium borohydride and polymerized with glutaraldehyde (PLP-HbAo-BH4-GLUT) without reduction. The chromatographic conditions are described in the text. The chromatogram (A) is obtained after dialysis; the separation shown below (B) is that for the same preparation, with the same load, after aging for 4 weeks at 4°C. The counts are shown as a dashed line. Reprinted with permission of John Wiley & Sons, Inc, from Marini et al.21
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Table 1 Products and Status, 2006 Generation “First” “Second”
Technology Red cell lysis Polymerized Hb
Product SFH PolyHeme
Academic Warner Lambert Renal failure GI symptoms Discontinued Northfield ? Phase III trauma Hemosol Cardiac events Discontinued Biopure Vasoconstriction ? Indication US Army Vasoconstriction Discontinued Baxter Death (stroke, trauma) Discontinued Somatogen Vasoconstriction Discontinued Ajinomoto/Apex Generally safe Sepsis trials Enzon Generally safe Discontinued
HemoLink HemoPure Intramolecular crosslink ␣␣-Hb HemAssist rHb1.1 PEG conjugation PHP PEG conjugation PEGhemoglobin PEG conjugation Hemospan Sangart
Phase III elective surgery Preclinical
Abbreviations: SFH, stroma-free hemoglobin; PHP, pyridoxal phosphate hemoglobin polyethyleneglycol; ZL-HbBv, bovine hemoglobin polymerized with Zero-Link technology (see text); Hb, hemoglobin; PEG, polyethylene glycol; GI, gastrointestinal.
unexpected cardiac toxicity in its clinical trials, and the clinical indications for HemoPure have not been announced by the company. Northfield Laboratories pioneered modern clinical trials of oxygen carriers. The company initially reported a series of surgical patients that received relatively large amounts of PolyHeme, showing the product could be used instead of red cells for transfusion.23 A Biological License Application (BLA) was submitted to the US Food and Drug Administration (FDA) in August 2001 based on the results of a trauma trial in 171 patients, but the FDA issued a refusal to file letter, citing difficulties in the design of the clinical protocol. The results of the trial, eventually published in the scientific literature, showed that patients with severe traumatic blood loss were treated in hospital with up to 20 units of PolyHeme (⬃ 50 g of hemoglobin per unit). Nine of 12 patients (75%) with red cell hemoglobin levels less than 1 g/dL survived the traumatic injury compared to a group of historical controls in which only 16% of patients survived with the same hemoglobin level.24 To address the difficulties with the use of historical controls, Northfield Laboratories designed a 720-patient phase III trauma trial, in which patients are randomized at the point of injury to receive either PolyHeme or standard of care (infusion of crystalloid solutions). Upon reaching the hospital, the standard of care arm receives blood as indicated, while the PolyHeme arm receives additional PolyHeme up to 12 hours, and thereafter receives blood if indicated. Northfield Laboratories has a Special Protocol Assessment (SPA) with the FDA that includes the dual primary end points of both superiority and noninferiority compared with blood. The company has not disclosed its power analysis for the study but is planning for a total enrollment of 720 patients. A Data Safety Monitoring Board (DSMB) will review the safety data, and the company’s press releases to date have announced that analysis after enrollment of 60, 120, 250, and 500 patients has not resulted in either adjustment of the power of the
study or breaking the blind to evaluate any imbalance in safety. A controversial aspect of the Northfield Laboratories’ phase III study is its conduct under a waiver of consent, approved by the FDA, for circumstances in which a therapy may be life-saving but the patient may not be conscious or otherwise able to give fully informed consent. The applicability of this waiver has been challenged by ethicists on the basis that since PolyHeme is not FDA-approved, there is insufficient evidence to support the assumption that it is lifesaving. Northfield Laboratories anticipates that the results of the study should be available sometime in the first half of 2007. Assuming PolyHeme is able to achieve at least the noninferiority end point, the company would then submit its BLA to the FDA. Whether the license is issued would depend not only on the results of this phase III study but on approval of Northfield Laboratories’ manufacturing methods and their compliance with Good Manufacturing Practices (GMP) guidelines. Northfield Laboratories has not published data in the peerreviewed literature since 2002, and the scientific development of the product or any follow-on products is unknown. One recent independent publication from a US Army laboratory indicates poor performance of PolyHeme in an animal model of traumatic blood loss.25
Intramolecularly Cross-linked Hemoglobin: The Third Generation The US Army reorganized its Blood Research Division at the Letterman Army Institute of Research (LAIR) in San Francisco in 1985, based on its views, first, that human immunodeficiency virus (HIV) transmission by stored blood was a
54 serious threat to battlefield medical care and, second, that glutaraldehyde-polymerized hemoglobin would become a successful “blood substitute.” LAIR was the main arm of the military research effort in blood and blood-related problems, and its in-house research team had developed its own version of polymerized hemoglobin based on bovine hemoglobin.26 As part of its reorganization, scientists at LAIR undertook a broad evaluation of the entire field of blood substitutes. They concluded, first, that there was no explanation for the vasoconstriction that had been reported consistently over the years with the various types of hemoglobin solutions and, second, that ascribing biological effects to properties of hemoglobin would be extremely difficult with solutions as heterogeneous as those produced using the classical amine-aldehyde reaction chemistry. An alternative solution was a new type of cross-linking reaction, described by Walder et al,27 in which the reagent was bis(3,5-dibromosalicyl)fumarate (DBBF). Because of its dimensions, this molecule could react only between the ⑀-amino groups of ␣1Lys92 and ␣2Lys92, both of which are situated in the interior of the hemoglobin molecule. Thus it was not possible to generate polymers, and uncrosslinked hemoglobin could be easily removed from the reaction mixture. The Army contracted Baxter Healthcare to devise a GMP method for production of this product so that LAIR could undertake studies of the biological effects of cellfree hemoglobin, particularly vasoconstriction. The LAIR group referred to this molecule as ␣␣Hb, and Baxter would later call its version DCLHb, or for clinical use, HemAssist (Baxter, Deerefield, IL). ␣␣-Hb was stable, and its oxygen affinity was close to that of normal human red blood cells. Before the end of the contract period, it became apparent that Baxter intended to develop DCLHb for its own purposes, and LAIR subsequently implemented the production methods for ␣␣Hb at its own in-house pilot plant.28 A substantial body of research was carried out by the LAIR group and its collaborators, culminating in the demonstration that ␣␣Hb was intensely vasoactive: in a pig model of hemorrhagic shock, resuscitation with ␣␣Hb resulted in depressed cardiac output and severely increased vascular resistance (Figure 3).29 These physiologic effects were inferred to be most likely due to scavenging of nitric oxide (NO) by hemoglobin, and this was considered an insurmountable problem. Baxter continued its efforts to commercialize HemAssist, eventually leading to failed clinical trials.30,31 In a review of the development of this product, we concluded that the intense vasoactivity of ␣␣Hb was probably responsible for most, if not all, of its toxic effects.32 Other products that can be classified with ␣␣Hb as thirdgeneration products were also developed: a recombinant molecule, genetically crosslinked between ␣ chains (rHb 1.1 or Optro, Somatogen, Boulder, CO) by Somatogen,33 and by Baxter, a variety of site-directed hemoglobin mutants with altered NO binding at the heme site.34 All of these products produced some degree of vasoconstriction. Research conducted in the 1980s and 1990s established NO as an important local vasodilator. NO is synthesized in endothelial cells and can be scavenged or its synthesis
Figure 3 Hemodynamic response to resuscitation of dehydrated swine after hemorrhage. Animals were hemorrhaged by approximately 50% of estimated blood volume starting at time 0. Infusion of resuscitation solution began at 1 hour after start of the hemorrhage. Infusion of either Ringer’s lactate (RL) or 5% albumin in saline (Albumin) reduced, slightly, vascular resistance, but infusion of either purified hemoglobin (Hb Ao) or ␣␣Hb produced striking vasoconstriction, indicated by the rise in systemic vascular resistance. The net result is that O2 delivery in the animals that received the O2 carriers (cardiac output x arterial O2 content) is not different from those that received RL or Albumin.56
inhibited, resulting in vasoconstriction. It had been known for many years that hemoglobin can avidly bind NO at its heme group—the same site that binds O2. Thus it seemed to most in the field that hemoglobin would always scavenge NO and therefore inevitably produce vasoconstriction. A series of mutants around the heme pocket of hemoglobin appeared to confirm that if NO binding were reduced, vasoactivity would be alleviated.34 However, while NO binding was constant in a variety of different hemoglobin modifications, the degree of vasoactivity could be substantially different (Figure 4).35 This key discovery demanded explanation and led to a new theoretical basis for hemoglobin-induced vasoactivity.
Chemistry and Physiology Linked: The Fourth Generation Studies in the hamster microcirculation provided an important perspective on the interaction of cell-free hemoglobin and the control of vascular tone. Prior research indicated that besides NO, other mechanisms control vessel diameter, including O2 itself,36 and a new concept was developed, which was called the “autoregulatory theory” of vascular control.37 According to this idea, the supply of O2 to vascular walls is the predominant determinant of vascular tone, a mechanism that may be separate from, but of greater importance than the NO mechanism. Cell-free hemoglobin oversupplies O2 to vascular walls through a combination of increased concentration of plasma O2 and the diffusion of hemoglobin itself, a process called “facili-
Red cell substitutes
Figure 4 NO binding and blood pressure in representative examples of hemoglobin-based O2 carriers. Test solutions were infused into rats at the time indicated by the arrow, and mean arterial blood pressure was continuously measured. The blood pressure responses are significantly different for the 3 classes of molecules, and the degree of blood pressure elevation is inversely proportional to the molecular size. The sizes of the molecules are shown at relative scale on the right,57 and the reaction rate constants for NO binding are given on the far right.35
tated diffusion.” Experiments in artificial capillaries confirmed that the release of O2 from cell-free hemoglobin could be controlled by alteration of oxygen affinity (P50) and size of the carrier molecule (Figure 5).38
Figure 5 Exit saturation as a function of residence time (flow rate) in an artificial capillary. Fully oxygenated solutions are injected into a capillary of 50 m diameter and allowed to flow at different rates. The capillary is freely permeable to O2, and N2 is maintained outside the capillary. The effluent solution is collected, and the hemoglobin saturation is measured. The behavior of PEG-Hb is very similar to that of red blood cells, despite the large difference in their oxygen affinities (P50). Both ␣␣Hb and purified Hb release O2 very quickly, again despite a large difference in their P50, indicating molecular size (hence, diffusion) is more important than P50 in this experimental model.38
New Molecules These physiologic experiments defined the requirements for a new generation of oxygen carriers, in which the properties of the molecule were “tuned” to the requirements of the microcirculation to deliver O2 effectively to hypoxic tissue; their essential features were increased oxygen affinity (reduced P50), increased molecular size and homogeneous chemistry. Work by Acharya and coworkers at Albert Einstein University included a description of the conjugation of maleimideactivated polyethylene glycol (PEG) 5,000 molecular weight (MW in daltons) with sulfhydryl groups on the surface of hemoglobin.39 Originally, this technology was described using bifunctional PEG molecules for polymerization. But when the PEG was activated on only one end, the reaction resulted in specific attachments without polymerization, such that about 6 strands of PEG 5,000 MW were attached per molecule. The modification process was streamlined, and a method was established for production of a solution that could be used for research.40 The product, Hemospan (sometimes abbreviated MP4; Sangart, Inc, San Diego, CA), was formulated as 4.2 g/dL of maleimide-activated PEG-5000 hemoglobin conjugate (MalPEG-hemoglobin) in Ringer’s lactate, and the production process was of sufficiently high yield and potentially low cost to be commercially viable if the reagent were safe in animal and human trials. Zero-linked hemoglobin (ZL-HbBv) is polymerized bovine hemoglobin developed by researchers at the University of Maryland.41 Zero-linked hemoglobin is included with the fourth-generation products because coupling between molecules does not use crosslinking agents, such as glutaralde-
56 hyde or other bifunctional agents. The key properties of ZLHbBv are its large molecular size and high oxygen affinity (low P50). Animal trials showed a decreased tendency to extravasate.42 Another novel molecule, a recombinant hybrid of human ␣-chains and bovine ␤-chains that is subsequently polymerized using surface sulfhydryl groups, also has high oxygen affinity and improves oxygenation of ischemic brain tissue in the mouse.43 Since less information about these two molecules is currently available, data from studies with Hemospan will illustrate the potential of fourth-generation products.
First Animal Tests: Does It Work? The first test of efficacy was a simple model of blood replacement and hemorrhage in the rat.44 In this model, a 50% exchange transfusion was first performed with test solution, and the animal was then subjected to a controlled hemorrhage of 60% of the estimated blood volume. The survival of normal (not exchange-transfused) animals is about 50%, so the model is sensitive to the properties of the solutions of interest. Not surprisingly, we found solutions that were vasoactive (␣␣Hb, polymerized hemoglobin) worsened survival in this model, while Hemospan resulted in uniform survival, in spite of the reduced hemoglobin levels in these animals. To exclude the possibility that improved survival was the result of some property of PEG itself (not hemoglobin), we also performed experiments with PEG-modified albumin.45 In these experiments, we continuously exchange-transfused rats with test article until no red cells were detectable; all animals treated with PEG-albumin died, but survival of animals treated with Hemospan proved conclusively that the beneficial effects of Hemospan are due to the presence of hemoglobin and that sufficient O2 could be supplied to sustain life in the absence of red blood cells.
Can O2 Be Released When P50 Is Reduced? The ability of Hemospan to oxygenate tissue even at relatively low concentration was demonstrated in the hamster skin window model.46 Hamsters were first hemodiluted to a hematocrit of 11% with test solution. PO2 was measured inside arterioles and venules, which, combined with the oxygen dissociation curves of hamster red cells and of test O2 carriers, made it possible to calculate the absolute amount of O2 in arterioles and venules, and hence the amount of O2 released across individual capillary beds. These experiments showed that polymerized hemoglobin produced vasoconstriction and release of most of its O2 prior to arriving to capillaries. The opposite was observed with Hemospan: capillary flow increased, and the majority of the O2 was released in capillaries. This phenomenon is called “targeted O2 release” and explains the remarkable efficacy observed in the rat hemorrhage model. Subsequent studies in the hamster model confirmed that Hemospan, like other cell-free hemoglobin O2 carriers, reduces local NO concentration but does not produce vasoconstriction.47
Figure 6 Survival of pigs after severe, uncontrolled hemorrhage by aortic tear. Animals were resuscitated using the solutions indicated and then followed for up to 24 hours.49 Reprinted with permission of Lippincott Williams and Wilkins from Young et al.49
Large Animal Studies To test Hemospan in a model more applicable to intended human use, studies were performed in pigs in collaboration with the Swedish Defense Establishment (FOI). Initial experiments established that Hemospan did not cause increased vascular resistance.48 Massive, uncontrolled hemorrhage from an aortic tear was then studied, also in pigs. Resuscitation was with test articles and compared to standard-of-care, which is volume replacement with crystalloid (Ringer’s lactate) followed by autologous blood. After resuscitation and repair of the aortic tear, animals were maintained for approximately 20 hours to determine untoward late effects. The results (Figure 6) demonstrated that overall survival after resuscitation was better when animals received Hemospan compared to standard of care, and that there was no significant increase in systemic or pulmonary vascular resistance, as had occurred in earlier studies with ␣␣Hb.49
Preclinical Toxicology Further preclinical studies have been performed, including toxicology in rats and monkeys, specific tests in cultured neurons, and in vitro assays of mutagenesis and intracerebral injections in rats, all of which continued to indicate the safety of Hemospan. A critical finding in its development was the absence of subendocardial lesions, as noted in studies with DCLHb.50 Based on this body of research and upon consultation with the US FDA and the Swedish Medical Products Agency, Sangart initiated its program of human clinical trials in 2002.
Phase I—Normal Volunteers Initial clinical studies were performed to determine if Hemospan would be well tolerated by normal human volunteers. In particular, we were interested to observe whether Hemospan would produce hypertension and gastrointestinal pain, both of which had been observed consistently in almost all previous clinical trials with hemoglobin-based products. We
Red cell substitutes
57 induction.52 The incidence of hypotension was reduced in both groups by approximately half (Figure 7), and the incidence of adverse events in the three arms of the study was approximately equal. Some patients who were included in the study underwent repair of acute fractures or revision arthroplasties, and these cases were more complicated than the primary arthroplasties, as expected. A special feature of this study was Holtor monitoring, starting 1 hour before induction of anesthesia and continued for 24 hours. Blinded analysis of these data did not find any safety concerns among the patients treated with Hemospan.
Phase III and Regulatory Approval Strategy Figure 7 Fraction of primary arthroplasty patients without hypotension. In the period following anesthesia induction, the incidence of hypotension, defined as systolic blood pressure ⬍90 mm Hg or 75% of baseline, is slightly less for group B (500 mL Hemospan) but similar for groups A (250 mL Hemospan) and C (Ringer’s lactate). However, during surgery, group C (control) patients continue to experience hypotension such that by the end of surgery 89% have experienced one or more episodes. The incidence of hypotension in both of the treated groups (A and C) remains constant throughout surgery (unpublished data).
infused two cohorts of volunteers, one with 50 mg/kg (about 100 mL) or 100 mg/kg (about 200 mL) of Hemospan, and observed no hypertension and no gastrointestinal (GI) complaints in any patient.51 Haptoglobin disappeared transiently, but there were no abnormalities of liver enzymes, coagulation, and hematologic or urine tests, and no electrocardiographic abnormalities.
Phase Ib/II—Dose Escalation Because Hemospan is an oncotic agent, we sought to escalate the dose in a patient population in which volume expansion is routinely performed. Patients who receive spinal anesthesia for elective orthopedic procedures normally experience “functional hypovolemia” due to sympatholysis and thus seemed an ideal population. In Swedish hospitals, these patients routinely receive up to 1 L of crystalloid (most often Ringer’s acetate) prior to or during induction of anesthesia. Patients from this group were thus given from 200 to 1,000 mL of either Hemospan or Ringer’s acetate, in cohorts of six patients (four active and two control per cohort) prior to induction of spinal anesthesia, again with close safety monitoring. The results, which have not yet been submitted for publication, indicated no concerns raised for continued clinical testing of Hemospan.
Phase II—Expanding the Safety Database and Early Efficacy A more extensive 90-patient, multicenter, double-blind phase II study was performed in orthopedic surgery patients who received either 250 mL or 500 mL of Hemospan or an equal volume of Ringer’s lactate prior to spinal anesthesia
There is no established regulatory pathway for cell-free oxygen carriers because none is currently approved for marketing in the United States, Europe, or Asia. In general, the guidelines for the development and approval of biological materials such as Hemospan are given by the International Committee for Harmonization (ICH) and by guidance documents from the US FDA.53 The FDA requires demonstration of both safety and clinical benefit in order for a new product to be approved for marketing in the United States, and much of the motivation for the development of oxygen carriers has derived from the widely held view that red blood cells are unsafe. Thus, a clinical trial in which the incidence of untoward events is shown to be less frequent for a “blood substitute” compared to blood might seem to be an acceptable basis for marketing approval. However, the incidence of unwanted effects from transfusion is now so low54 that thousands of patients would need to be treated in order to demonstrate superiority compared to blood. Equally, it would be nearly impossible to show superior efficacy of a new product compared to blood, since the efficacy of blood itself has never been shown by controlled clinical trials! How, then, can an O2 carrier be shepherded through the regulatory process? One strategy might be to randomly assign intraoperative surgical patients to receive either blood or an O2 carrier once a “transfusion trigger” has been reached. This approach also has been adopted by some companies, but the main difficulty is the absence of a universally accepted transfusion trigger,55 and it is not reasonable to deny some patients blood transfusion when one is needed. Furthermore, the incidence of intraoperative transfusion, especially in elective surgery, is steadily falling in developed countries, and a significant number of cases would need to be enrolled in such a study to treat the 800 to 1,000 patients needed to establish safety. Another approach might be to select patients with known tissue ischemia due to unstable angina pectoris, peripheral limb ischemia, or stroke or patients with sickle cell disease. However, all of these groups of patients have significant underlying pathology, and it would not seem prudent to aggressively study a new product in them until a better understanding of its safety profile is known. For its primary regulatory activity, Sangart has chosen the conservative approach of administering Hemospan both to
58 prevent and treat hypotension in elective orthopedic surgery patients who are given spinal anesthesia. Very often these patients experience periods of functional hypovolemia as the spinal anesthesia causes a redistribution of blood volume; central volume is reduced with consequent reduction in cardiac filling and output, producing hypotension. Particularly the elderly are at risk of coronary, renal, and cerebral ischemia and may suffer significant long-term consequences such as dementia, myocardial infarction, renal failure, or stroke. Since Hemospan has the capacity to preserve capillary perfusion and tissue oxygenation, fewer adverse events related to poor tissue oxygenation compared to controls would be anticipated. The phase II study in this patient population has already shown a significant reduction in protocol-defined hypotensive episodes.
8. 9. 10.
Conclusion Review of the many generations of O2-carrying plasma expanders (“blood substitutes”) that have been developed and tested, and the enormous amount of resources that have been invested in this quest, produces pessimism that this is a solvable problem. However, progression from one class of products to another indicates first, that hemoglobin-based products are capable of delivering O2 to tissue, and that the main obstacle to clinical success has been their propensity to produce vasospasm. Recent research to produce the current generation of hemoglobin-based molecules has been focused on understanding the mechanism of such vasospasm, and the availability of new techniques for direct study of O2 transfer in the microcirculation has permitted the design of molecules, some with very counterintuitive properties. In spite of a history of disappointments, we may be cautiously hopeful that one or more of these new molecules will prove safe enough for clinical use.
Disclosure Robert Winslow is President, Chairman of the Board of Directors and Chief Executive Officer of Sangart, Inc. He receives salary support, stock and stock options from Sangart. The company is actively engaged in developing a product described in this review.
The author recognizes the editorial assistance of Pam Boltz.
References 1. Winslow R: Historical background, in Winslow R (ed): Blood Substitutes. London, Elsevier, 2006, pp 5-16 2. Thomas T: The intravenous injection of milk as a substitute for the transfusion of blood. NY State J Med 27:449-465, 1878 3. Brinton J: The transfusion of blood and the intravenous injection of milk. Med Record 14:344-347, 1878 4. Winslow RM (ed): Blood Substitutes. London: Elsevier, 2006 5. Von Stark G: Die Resorbierbarkeit des Hämatins und die Bedeutung der Hämoglobinpräparate. Deutsche Med Wochenschr 24:805-808, 1898 6. Sellards A, Minot G: Injection of hemoglobin in man and its relation to
blood destruction, with especial reference to the anemias. J Med Res 34:469-494, 1916 Ottenberg R, Fox C: The rate of removal of hemoglobin from the circulation and its renal threshold in human beings. Am J Physiol 123: 516-525, 1938 O’Shaughnessy L, Mansell H, Slome D: Haemoglobin solution as a blood substitute. Lancet 2:1068-1069, 1939 Fairly N: The fate of extracorpuscular circulating hemoglobin. Br J Med 2:213-217, 1940 Gilligan D, Altschule M, Katersky E: Studies of hemoglobinemia and hemoglobinuria produced in man by intravenous injection of hemoglobin solutions. J Clin Invest 20:177-187, 1941 Cannan R, Redish J: The large scale production of crystalline human hemoglobin: with preliminary observations on the effect of its injection in man, in Mudd S, Thallimer W (eds): Blood Substitutes and Blood Transfusion. Springfield, IL, Thomas, 1942, pp 147-155 Amberson W, Jennings J, Rhode C: Clinical experience with hemoglobin-saline solutions. J Appl Physiol 1:469-489, 1949 Brandt J, Frank R, Lichtman H: The effect of hemoglobin solutions on renal functions in man. Blood 6:1152-1158, 1951 Miller J, McDonald R: The effect of hemoglobin on renal function in the human. J Clin Invest 30:1033-1040, 1951 Rabiner S, Friedman L: The role of intravascular haemoglobins and the reticuloendothelial system in production of a hypercoagulable state. Br J Haematol 14:105, 1968 Savitsky JP, Doczi J, Black J, Arnold JD: A clinical safety trial of stromafree hemoglobin. Clin Pharmacol Ther 23:73-80, 1978 Bolton W, Perutz M: Three dimensional fourier synthesis of horse deoxyhemoglobin at 2 angstrom units resolution. Nature 228:551-552, 1970 Bunn HF, Jandl JH: The renal handling of hemoglobin. J Biol Chem 243:465-475, 1968 Benesch R, Benesch R, Renthal R, Maeda N: Affinity labeling of the polyphosphate binding site of hemoglobin. Biochemistry 11:35763582, 1972 Payne J: Polymerization of proteins with glutaraldehyde. Soluble molecular-weight markers. Biochem J 135:867-873, 1973 Marini M, Moore G, Fishman R, Jesse R, Medina F, Snell S, et al: Reexamination of the polymerization of pyridoxylated hemoglobin with glutaraldehyde. Biopolymers 29:871-882, 1990. Marini M, Moore G, Fishman R, Jesse R, Medina F, Snell S, et al: A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin A0. Biopolymers 28:2071-2083, 1989 Gould S, Moore E, Hoyt D, Burch JM, Haenel JB, Garcia J, et al: The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery. J Am Coll Surg 187:113120, 1998 Carson JL, Noveck H, Berlin JA, Gould SA: Mortality and morbidity in patients with very low postoperative Hb levels who decline blood transfusion. Transfusion 42:812-818, 2002 Handrigan MT, Bentley TB, Oliver JD, Tabaku LS, Burge JR, Atkins JL: Choice of fluid influences outcome in prolonged hypotensive resuscitation after hemorrhage in awake rats. Shock 23:337-343, 2005 DeVenuto F, Zegna A: Preparation and evaluation of pyridoxalatedpolymerised human haemoglobin. J Surg Res 34:205-212, 1983 Walder J, Zaugg R, Walder R, Steele J, Klotz I: Diaspirins that cross-link B chains of hemoglobin: Bis(3,5- dibromo salicyl) succinate and bis(3,5-dibromosalicyl) fumarate. Biochem 18:4265-4270, 1979 Winslow R, Chapman K: Pilot-scale preparation of hemoglobin solutions. Methods Enzymol 231:3-16, 1994 Hess J, Macdonald V, Winslow R: Dehydration and shock: An animal model of hemorrhage and resuscitation of battlefield injury. Artif Cells Blood Substit Immobil Biotechnol 19:499-502, 1992 Sloan EP, Koenigsberg M, Gens D, Cipolle M, Runge J, Mallory MN, et al: Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock. A randomized controlled efficacy trial. JAMA 282:1857-1864, 1999 Saxena R, Wijnhoud AD, Carton H, Hacke W, Kaste M, Przybelski RJ,
Red cell substitutes
et al: Controlled safety study of a hemoglobin-based oxygen carrier, DCLHb, in acute ischemic stroke. Stroke 30:993-996, 1999 Winslow RM: ␣␣-Crosslinked hemoglobin: Was failure predicted by preclinical testing? Vox Sang 79:1-20, 2000 Looker D, Abbott-Brown D, Cozart P, Durfee S, Hoffman S, Mathews A: A human recombinant haemoglobin designed for use as a blood substitute. Nature 356:258-260, 1992. Doherty DH, Doyle MP, Curry SR, Vali RJ, Fattor TJ, Olson JS, et al: Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin. Nat Biotechnol 16:672-676, 1998 Rohlfs RJ, Bruner E, Chiu A, Gonzales A, Gonzales ML, Magde D, et al: Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide. J Biol Chem 273:12128-12134, 1998 Intaglietta M, Johnson P, Winslow R: Microvascular and tissue oxygen distribution. Cardiovasc Res 32:632-643, 1996 Winslow R., Intaglietta M, Tsai A, Vandegriff K, Wettstein, R: Autoregulation and vasoconstriction: Foundation for a new generation of blood substitutes. Blood 100:210, 2002 (abstr) McCarthy MR, Vandegriff KD, Winslow RM: The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers. Biophys Chem 92: 103-117, 2001 Acharya AS, Manjula BN, Smith P: Hemoglobin crosslinkers. Albert Einstein College of Medicine of Yeshiva University. New York patent (5,585,484), 1-16. 1996 Vandegriff KD, Malavalli A, Wooldridge J, Lohman J, Winslow RM: MP4, a new nonvasoactive PEG-Hb conjugate. Transfusion 43:509516, 2003 Bucci E, Matheson B, Kwansa H, Koehler R. Development of nonextravasating hemoglobin-based oxygen carriers, in Winslow R (ed): Blood Substitutes. London, Elsevier, 2006, pp. 488-497 Matheson B, Razynska A, Kwansa H, Bucci E: Appearance of dissociable and cross-linked hemoglobins in the renal hilar lymph. J Lab Clin Med 135:459-464, 2000 Nemoto M, Mito T, Brinigar W, Fronticelli C, Koehler RC: Salvage of focal cerebral ischemia damage by transfusion of high O2 affinity recombinant polymers in mouse. J Appl Physiol 100:1688-1691, 2006 Winslow RM, Gonzales A, Gonzales M, Magde M, McCarthy M, Rohlfs RJ, et al: Vascular resistance and the efficacy of red cell substitutes in a rat hemorrhage model. J Appl Physiol 85:993-1003, 1998
59 45. Winslow RM, Lohman J, Malavalli A, Vandegriff KD: Comparison of PEG-modified albumin and hemoglobin in extreme hemodilution in the rat. J Appl Physiol 97:1527-1534, 2004 46. Tsai A, Vandegriff K, Intaglietta M, Winslow R: Targeted O2 delivery by cell-free hemoglobin: a new basis for O2 therapeutics. Am J Physiol 285: H1411-H1419, 2003 47. Tsai AG, Cabrales P, Manjula BN, Acharya S, Winslow RM, Intaglietta M: Dissociation of local nitric oxide concentration and vasoconstriction in the presence of cell-free hemoglobin oxygen carriers. Blood 108: 3603-3610, 2006 48. Drobin D, Kjellstrom B, Malm E, Malavalli A, Lohman J, Vandegriff K, et al: Hemodynamic response and oxygen transport in pigs resuscitated with maleimide-polyethylene glycol-modified hemoglobin (MP4). J Appl Physiol 96:1843-1853, 2004 49. Young M, Riddez L, Kjellstrom B, Bursell J, Winslow F, Lohman J, et al: MalPEG-hemoglobin (MP4) improves hemodynamics, acid-base status, and survival after uncontrolled hemorrhage in anesthetized swine. Crit Care Med 33:1794-1804, 2005 50. Burhop KE, Estep TE: Hemoglobin-induced myocardial lesions. Artif Cells Blood Substit Immobil Biotechnol 29:101, 2001 (abstr) 51. Bjorkholm M, Fagrell B, Przybelski R, Winslow N, Young M, Winslow R: A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethylene glycol. Haematologica 90:505-515, 2005 52. Olofsson C, Ahl T, Johansson T, Larsson S, Nellgård P, Ponzer S, et al: A multi-center clinical study of the safety and activity of maleimidepolyethylene glycol hemoglobin (Hemospan) in patients undergoing major orthopedic surgery. Anesthesiology (in press) 53. Silverman, T: Guidance for Industry: Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes. http:// www.fda.gov/cber/gdlns/oxytherbld.htm. Accessed November 2, 2006. 54. Klein H: Transfusion medicine, in Winslow R (ed): Blood Substitutes. London, Elsevier, 2006, pp 17-33 55. Stehling L, Simon T: The red blood cell transfusion trigger. Physiology and clinical studies. Arch Pathol Lab Med 118:429-434, 1994 56. Hess J, Macdonald V, Brinkley W: Systemic and pulmonary hypertension after resuscitation with cell-free hemoglobin. J Appl Physiol 74: 1769-1778, 1993 57. Vandegriff K, McCarthy M, Rohlfs R, Winslow R: Colloid osmotic properties of modified hemoglobins: chemically cross-linked versus polyethylene glycol surface-conjugated. Biophys Chem 69:23-30, 1997