ORAL PRESENTATIONS

SESSION I: BLOOD STORAGE, PATHOPHYSIOLOGY OF TRANSFUSION AND OXYGEN-DEPENDENT TUMORS

Chairman: Bonaventura, J.

Duke University and The Bonaventis Research Institute

E-mail: [email protected]

Session Keynotes

This session focuses on the influence of blood storage and transfusion on health and on different unmet medical needs that may be obtained from HBOCs. Most of the presentations focus on transfusion of banked blood or RBCs and the challenges presented by this long-used clinical practice. The belief that transfusion presents problems, irrespective of its well-known life-saving attributes, is now statistically demonstrated. There is rapid progress in identification and “rescue” of in vivo intermediates and functional properties. These changes commence from the moment of exsanguination, at first rapidly and then more slowly over the storage life of blood. Not only have these “defects” been characterized but also we will hear of procedures to restore intermediates and functions lost during storage. Questions concerning the “Transfusion Trigger,” the parameters used before transfusion, will be addressed and discussed. We will also hear that, remarkably, over 90% of extremely low birth weight infants receive RBC transfusions and there are harmful effects, like altered acid-base, glucose and electrolyte balance. One talk this morning will address anaerobic storage of blood and the changes using a proteomic approach. Following this, we will hear about what appears to be an optimal “window” for infusion of RBCs into patients for modulation of tumor oxygenation during cancer therapy and we will learn the answer to the question “Is more {hemoglobin} better?” HBOC researchers will be certainly be interested in hearing about all of these challenges and may also be stimulated to consider how their research might address and ameliorate these problems. Finally, we will gain insight into the past, present, and future of HBOC research from an individual's point of view, an important talk that will generate much discussion.

RED CELL TRANSFUSION IN CLINICAL PRACTICE

Klein, H. G.

Department of Transfusion Medicine, National Institutes of Health, USA

E-mail: [email protected]

Red blood cell (RBC) transfusion is widely used to treat anemia. Although the benefits have never been critically demonstrated, whole blood transfusions proved life-saving, from Blundell's treatment of puerperal hemorrhage (1818) to the management of wounded soldiers in the Great Wars. With prevalence of anemia approaching 95% in intensive care unit (ICU) patients, medical patients currently comprise the largest users of RBC. Some fifty percent of ICU patients, 85% of those in the ICU for greater than one week, receive at least one RBC transfusion; two thirds of ICU transfusions are given for indications other than acute blood loss with “low hemoglobin” the most common indication. Thresholds for RBC transfusion remain controversial. The empiric “10/30 rule,” cited inappropriately and applied too broadly, has biased decades of transfusion practice. Data from clinical studies have contributed more to the understanding of the physiology of anemia than to implementation of clinical guidelines.

Early efforts to curb unnecessary transfusion practices pointed to newly identified infectious disease risks. Advances in blood safety and manufacturing have reduced many of the previous risks associated with allogeneic blood. The introduction of leukoreduced RBC and newer molecular, serologic, and microbial testing call into question past findings of detrimental outcomes associated with transfusion. Controversy over the magnitude of risks of RBC transfusion remains.

With few adequately powered randomized clinical trial assessing outcomes of restrictive versus liberal transfusion strategies, data are needed regarding optimal RBC transfusion thresholds in different clinical settings and optimal quality of RBC. As practitioners await development and validation of accessible, practical, and reliable physiologic markers to guide therapy, the decision to transfuse RBC relies on evaluation of individual patients by skilled clinicians at the bedside who use hemoglobin concentration as no more than a helpful guide.

DYSREGULATED VASOACTIVITY OF THE STORED RBC: RELEVANT MEDIATORS AND THERAPEUTIC IMPLICATIONS

McMahon, T. J.

Duke University Medical Center, Durham, NC, USA

E-mail: [email protected]

Red blood cell (RBC) transfusion in some groups of patients is linked with excess morbidity and mortality, and some risks increase with storage. We present results of investigation into the role of storage-induced loss of key mediators – NO and ATP – whose release from the RBC may be necessary for a normal, recently identified function of the RBC: regulating the flow of blood. Specifically, normal RBCs constrict blood vessels (as does free hemoglobin (Hb)) at higher PO₂ and export vasorelaxing factors when RBC Hb senses lower PO₂ and thus higher metabolic demand. Storage of RBCs impairs this vasoactivity as well as RBC deformability; in vivo, basal, and responsive blood flow are likewise compromised in animal models of stored-RBC transfusion, recapitulating the organ dysfunction documented in transfused, ill humans. SNOs (S-nitrosothiols) and ATP decline during RBC storage, and strategies to restore ATP (e.g., “rejuvenation” of RBCs) or (S)NO show early promise in improving the ability of stored RBCs to improve blood flow in animal models; it is rational to test similar approaches in the context of blood substitute development.

RED BLOOD CELL STORAGE UNDER ANAEROBIC CONDITIONS: A PROTEOMIC INVESTIGATION

Zolla, L., D’Alessandro, A., D’Amici, G.

Università della Tuscia, Viterbo, Italy

E-mail: [email protected]

Red blood cell (RBC) storage is currently strictly regulated by law. Erythrocytes are actually stored at 4°C for as long as 42 days. However, long-stored RBCs are likely to provoke a series of untoward effects after re-infusion, especially in the critically ill; thus the clinical and the academic milieu ought to wonder about the suitability of this blood component after a prolonged storage period.

In order to fill this gap, we thoroughly investigated the membrane proteome of RBCs with high-throughput proteomic tools (2D-IEF-SDS-PAGE, RP-HPLC, ESI-MS/MS – IT QTOF), while samples were collected fortnightly until unit expiry.

As a result, the overall detected spots increased as a function of storage time. Questions arose whether these new spots actually represented new proteins or were produced by fragmentation or aggregation processes. As far as it concerned the former hypothesis, being anucleated, RBCs cannot synthesize new proteins, except for translating the low amounts of mRNA, which are inherited from reticulocyte ancestors. Therefore, these new spots, which appeared at lower and higher molecular masses, were respectively interpreted as fragmentation or aggregation products of yet existing proteins. Indeed, MS/MS analyses confirmed this second hypothesis, since the amino acid sequences of the proteins found under these new (lower/higher molecular weight) spots matched the ones of yet existing proteins at higher/lower molecular weight from fresh RBCs.

Concordingly, it was already known that haemoglobin aggregates also form during storage and bind together to the cytoskeleton and membrane fraction, where they are often removed via the vesiculation process. Nonetheless, haemoglobin aggregates play a key role in RBC storage lesions.

In order to investigate the relative contributions of chemical oxidation and enzyme cleavage to these aggregation or fragmentation processes, the RBC membranes were stored under anaerobic conditions (under helium) in the presence or absence of protease inhibitors. Anaerobic storage drastically reduced the fragmentation events, while no significant differences were observed whether proteases inhibitors were present or not.

Hence, the presence of smearing, mobility shift of intrinsic protein bands, aggregate formation and also protein fragmentation under normal aerobic conditions is likely to be caused by active oxygen species that generate during storage, attack proteins in the cytoskeleton and ultimately impair RBC structure and physiology other than accelerating their removal from the bloodstream of the transfused recipients. Consistently, most of the fragmented proteins of long- aerobically-stored RBCs belonged to the cytoskeletal fraction (prevalently band 4.2, bands 4.1 and 3, spectrin and glyceraldehyde-3-phosphate dehydrogenase – G3PD). Taken together, these data reinforced previous evidences about band 3 being a pivotal target for storage lesions and, as we found out, for the attack by active oxygen species. It is noteworthy that we noticed that the membrane protein G3PD decreased dramatically over time when oxygen was present, but disappeared more slowly when oxygen was removed. The gradual loss of GP3D causes glycolysis to fall off, while glucose metabolism is shifted towards the alternative pentose phosphate (HMS) pathway. It is well known that the energy-less erythrocyte is inevitably lost, since a reduction in the glycolysis rate is disastrous for the RBC metabolism; the blocked production of ATP, DPG and NADH are just a few of the main early consequences.

These data supported the hypothesis that most of the storage-induced lesions could be the result of a cascade of events, primarily induced by oxidative processes. These processes are triggered by a domino effect: being stored in an oxygen-stressed environment for a prolonged period, oxy-hemoglobin is oxidized to methemoglobin and, finally, hemichromes are generated. Oxygen radicals are subsequently released and attack a series of key proteins, among which an eligible target seems to be the cytosolic fraction of band 3. Thus the nearby proteins and enzymes (cytoskeletal proteins – e.g. ankyrin- and G3PD) are compromised. In consequence, neoantigens form from the membrane, RBC physiology and structure are impaired, and glycolysis fades off. Thereon, the no-more functional re-infused RBCs are rapidly removed from the bloodstream or are likely to cause untoward effects in the recipients.

In the absence of oxygen, both G3PD and ankyrin (along with other cytoskeletal proteins) are protected by the reduced likelihood of an oxidative event as well as by being distanced from a major source of oxygen radicals, putatively the oxy-haemoglobin molecule. Moreover, under aerobic conditions oxidation occurred systematically after 10 days of storage, suggestive of a timeline threshold for erythrocyte storage lesions.

Our lab is presently working on the hypothesis that hemichrome, the most dangerous form of haemoglobin, binds preferentially near the G3PD anchoring site of band 3 and represents the main source of OH radicals that are responsible for the main damages to this enzyme.

BLOOD BANKING-INDUCED IMPAIRMENT OF RED BLOOD CELL HEMODYNAMICS

Barshtein, G.¹, Relevy, H.², Koshkaryev, A.¹, Zelig, O.², Manny, N.², Yedgar, S.¹

¹Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel

²Hadassah Hospital Blood Bank, Jerusalem, Israel

E-mail: [email protected]

Background: Blood banking is associated with damage to red blood cells (RBC) that can impair their flow- affecting properties – namely, deformability, aggregability, and adherence to vascular endothelial cells (EC-adherence) – which are important determinants of blood flow. This implies that the transfusion of stored RBC (strbc) with impaired flow-properties might introduce a circulatory risk to recipients.

Objectives: To comprehensively explore the effects of routine cold storage and γ-irradiation on RBC flow-properties.

Methodology: RBC flow properties were monitored as a function of shear stress (τ), using a computerized cell-flow-properties image analyzer, developed in our lab, which enables the direct visual monitoring and subsequent determination of RBC number, shape change, and spatial organization.

Results: RBC aggregability, rigidity and EC-adherence are continuously elevated during cold storage, becoming considerable already at the second week of storage. RBC aggregability was expressed by the formation of larger and stronger than normal aggregates. The reduction of RBC deformability was expressed by a shift in elongation ratio distribution and by a sharp increase (from 1% to 14%) in the portion of undeformable RBC in the cell population. The elevation of strbc/EC adherence correlated with phosphatidylserine (PS) externalization to RBC surface. Blocking surface PS with its specific ligand Annexin-V completely suppressed the elevated adherence, showing that storage-induced PS externalization is responsible for the enhanced RBC/EC adherence. Strbc/EC adhesion was 9-fold higher in recipients’ plasma, compared to that in plasma-free standard buffer.

Γ-Irradiation induced an immediate, marked enhancement in the number of undeformable RBC (from 1% to 40%), but did not affect RBC EC-adherence and aggregability.

Conclusion: RBC flow properties appear to be especially sensitive to storage procedures as they are impaired long before the storage expiration date. Since impaired RBC flow-properties facilitate circulatory disorders, the potential circulatory risk in transfusion of strbc with impaired hemodynamics should be considered.

IMPACT OF HEMOGLOBIN LEVELS ON TUMOR OXYGENATION: THE HIGHER, THE BETTER?

Vaupel, P.

Department of Radiooncology and Radiotherapy, University Medical Center, Mainz, Germany

E-mail: [email protected]

Tumor hypoxia has been linked to tumor progression, the development of acquired treatment resistance, and thus poor patient prognosis. Since anemia is a major factor causing tumor hypoxia, the association between blood hemoglobin concentration (chb) and tumor oxygenation status has been examined.

Published data on the relationship between pretreatment chb values and tumor oxygenation (in terms of median po₂ values, hypoxic fractions) have been summarized. Pretreatment O₂ tension measurements were performed in histologically proven experimental tumors, human breast cancers, squamous cell carcinomas of the head and neck, and cancers of the uterine cervix and of the vulva. In order to allow for a comparison between solid tumors and normal tissues, po₂ measurements were also performed in healthy tissue in anemic and nonanemic patients. Chb was determined at the time of the po₂ measurements.

Based on current information from experimental and clinical studies, there is increasing evidence that anemia is associated with a detrimental tumor oxygenation status. Increasing chb values are correlated with significantly higher po₂ values and lower hypoxic fractions. Maximum tumor oxygenation in squamous cell carcinomas is observed at normal (gender-specific) chb values (approximately 14 g/dl in women and approximately 15 g/dl in men). Above this “optimal” Hb range, the oxygenation status tends to worsen again. In anemic patients, tumor oxygenation is compromised due to a decreased O₂ transport capacity of the blood. At the upper edge of the Hb scale, a substantial increase in the blood's viscous resistance to flow in “chaotic” tumor microvessels is thought to be mainly responsible for the observed restriction of O₂ supply.

Review of relevant clinical data suggests that a maximum oxygenation status in solid tumors is to be expected in the range 12 g/dl < chb < 14 g/dl for women and 13 g/dl < chb < 15 g/dl for men. Considering the “optimal” chb range with regard to tumor oxygenation, the concept of “the higher, the better” is therefore no longer valid. This finding has potentially far-reaching implications in the clinical setting (e.g., inappropriate erythropoietin treatment of nonanemic tumor patients).

CLINICAL AND TRANSLATIONAL ADVANCES IN BLOOD SUBSTITUTES: ONE INVESTIGATOR'S PERSPECTIVE

Jahr, J. S.

Department of Anesthesiology, David Geffen School of Medicine at UCLA

E-mail: [email protected]

The Past: First generation blood substitutes (hbocs) were characterized by small molecules with multiple vasoactive and renal toxicies; alpha-alpha cross-linked hemoglobin and 2,3 diaspirin cross-linked hemoglobin (hemeassist, Baxter) failed clinical trials, but this was predicted based on translational models (Winslow). This prompted multiple biotechnology companies (Hemosol, Biopure, Northfield, and Somatogen) to form and create second generation oxygen therapeutics that eliminated the renal toxity, but are still struggling with the vasoactivity (Greenburg, Jahr, Gould, Weiskopf). My team and I have created a number of translational research platforms to study surrogate markers of end-organ perfusion and oxygenation that could also have predicted the strengths and weaknesses of the second-generation hbocs and avoided some of the negative press and science (Driessen, Cheung, Gunther, Lurie and Jahr).

The Present: These second-generation and a first and third (Hemospan, Sangart) were lumped together in the Natanson report (JAMA), and hurt the entire industry and science. The article accurately pointed out that manufacturers have been reluctant to release product for independent investigation based on the incorrect belief that small, negative findings will hurt the product’s chances for success. In fact, this strategy has backfired on all companies, not just the one with negative results. The current model clearly is not working and financially impossible to sustain either for industry or universities, and preventing the development of potentially useful compounds (HBOC vesicles, large molecules like Oxyvita/large earthworm hbocs, and newer strategies of blocking of nitric-oxide scavenging-Hsia). Current research in our laboratories centers on new pegylated solutions to be presented at an upcoming meeting.

The Future: The only foreseeable future is for a consortium to form of all interested parties and collaborate with validated models evaluating translational models for safety and efficacy, with free transfer of information and sharing materials, with FDA oversight and input, to allow for the current second-generation hbocs to find a clinical niche and for third- and further generations to be evaluated and eventually gain regulatory approval for clinical use. Other industries have done this (aerospace), with success and lack of costly redundancy. It will avoid the negative reports that have plagued the science from press reports, which have had far more deleterious outcomes than the not-perfect results they are reporting. Collaboration and use of validated translational models that reveal identified surrogate markers will allow clinical trials to be executed that will satisfy FDA and other regulatory bodies for approvals needed to fill unmet medical needs and fill small niches in medical care. Hopefully, future oxygen therapeutics and blocking of nitric oxide scavenging will improve clinical outcomes in sepsis, hemorrhagic shock, cardio-pulmonary-neuro resuscitation, stroke, and cardiac or vaso-occlusive disease.

THE NEED OF ALTERNATIVES TO BLOOD TRANSFUSIONS IN NEONATOLOGY

Bacchini, P. L.¹, Romanici, E.¹, Bevilacqua, G.¹, Pioselli, B.², Faggiano, S.², Ronda, L.², Mozzarelli, A.^2,3

¹Neonatal Intensive Care Unit, University Hospital, Parma, Italy

²Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy

³National Institute of Biostructures and Biosystems (INBB), Italy

E-mail: [email protected]

The blood transfusion represents a considerable risk factor for infective illnesses, retinopathy, and chronic lung disease in preterm infants. Blood transfusions are used for anaemia due to group incompatibility, infective events or blood illnesses and, mainly, for anaemia of pre-maturity. Most of the extremely preterm low birth weight infants are transfused once or many times after birth. The use of synthetic erythropoietin has not reduced the complications. The mechanisms leading to retina and lung damages are still not clear. Blood transfusions in newborns, using adult donor red cells, characterized by a higher cell volume and containing haemoglobin with lower oxygen affinity with respect to fetal haemoglobin, cause modifications in capillary flux and oxygen homeostasis, acid-base and glucose disturbances and may introduce toxic molecules resulting from the processing of blood before use. After blood transfusion plasma non-transferrin-bound iron induces tissues oxidative stress.

In the period 01/01/08 – 30/06/09, 39 newborns of gestational age under the 29^th week (weight less than 1000 g) were hospitalized in the Neonatal Intensive Care Unit of Parma and 32 (82%) of them received up to 10 transfusions (an average of 3) with 10–20 cc/kg of packed red cells. Ocular and/or pulmonary complications occurred in 11 (39.3%) of the 28 survived newborns: 4 (14.3%) of them exhibited the chronic lung disease and the retinopathy, 2 of which with retinal detachment, 3 (10.7%) of them only the retinopathy, and the other 4 (14.3%) only the chronic lung disease.

The reduction of the number of transfusions is an unmet clinical need. Therefore, blood substitutes open new perspectives in the field of neonatology. The transfusion of haemoglobin-based oxygen carriers as substitutes of blood would avoid infective risks and could solve the problem of complications. As a first step towards this goal, we are identifying by salivary proteomic analysis the modifications on protein pattern caused by adult blood transfusions in preterm infants and biomarkers of damages.

SESSION II: PHYSIOLOGY AND REGULATION OF OXYGEN AND NITRIC OXIDE

Chairman:

Koehler, R. C.

Department of Anesthesiology and Critical Medicine, Johns Hopkins University, Baltimore, Maryland, USA

E-mail: [email protected]

Session Keynotes

The role of hemoglobin as an O₂ carrier has been expanded to include a role in nitric oxide (NO) signaling. However, hemoglobin outside the red blood cell (RBC) membrane alters the dynamics of both O₂ and NO transport. By facilitating O₂ diffusion between the RBC and endothelium, hemoglobin-based O₂ carriers (HBOC) may improve oxygenation in ischemic tissue but result in excess precapillary loss of O₂ and counteractive vasoconstriction in well-perfused tissue. Nevertheless, regulation of O₂ transport can still be still maintained when blood viscosity and O₂ demand vary. As rbcs become deoxygenated during their systemic arteriovenous transit, ATP released from rbcs may stimulate endothelial NO synthase. Furthermore, NO is generated from nitrosylated hemoglobin and from nitrite reduction by hemoglobin, but it remains unclear if the quantity of generated NO is sufficient to induce vasodilation. Moreover, hbocs in the plasma will scavenge NO released by rbcs unless NO is stored in a protected form, such as on thiols or as N₂O₃. Alternatively, increasing plasma nitrite may stimulate the nitrite reductase activity of plasma-based hemoglobin sufficiently to generate protected NO storage forms and thereby counter the vasoconstrictive actions arising from excess oxygenation and scavenging of free NO. Lastly, oxidation of HBOC heme to the ferric and ferryl state is of concern because of the increased potential to generate reactive oxygen species at the endothelial membrane. Polynitroxylation of hbocs, such as PEG-hemoglobin, confers superoxide dismutase mimetic activity to the protein. Further, by limiting H₂O₂ oxidation to ferryl heme, polynitroxylation also exerts catalase mimetic activity on heme. This approach may not only help deliver O₂ to injured tissue, but do so in a way that limits further oxidative stress. Therefore, several strategies may allow for improved efficiency of O₂ transport selectively to tissues in need without augmenting oxidative stress and without decreasing NO bioavailability.

INFLUENCES OF BOVINE HEMOGLOBIN AND VISCOSITY ON CEREBRAL BLOOD FLOW AND GLUCOSE UTILIZATION

Kuschinsky, W.¹, Waschke, K. F.²

¹Department of Physiology and Pathophysiology, University of Heidelberg, Germany

²Department of Anesthesiology, Alfried Krupp Hospital, Essen, Germany

E-mail: [email protected]

Blood substitution was performed by ultrapurified bovine hemoglobin (UPBHB). The experiments were performed in normal conscious rats after arterial and venous catheters had been inserted during anesthesia. Controlled arterial bleeding was combined with simultaneous replacement of blood by venous infusion of UPBHB until a final hematocrit (hct) <0.03 was reached. 4 hours later radioactive tracers were infused for the measurement of cerebral blood flow ([¹⁴C]-iodoantipyrine) or glucose utilization ([¹⁴C] 2-deoxyglucose). The results showed a mean increase in cerebral blood flow by 82% (local 36 to 126%), and in cerebral glucose utilization by 14% (local 0 to 24%). The relationship between LCGU and LCBF remained tight, although it was reset to higher blood flow levels.

A second series of experiments was performed in a similar way after 30 min of hemorrhage followed by fluid substitution using UPBHB. The results showed a moderate increase in blood flow (+36%) and maintained glucose utilization (+9%) 3 hours after resuscitation. It is concluded that fluid resuscitation by UPBHB can meet the metabolic demands of the brain.

In a third series of experiments the effects of defined changes in blood viscosity on cerebral blood flow were tested. This approach is possible since blood substitutes do not change their viscosity with variations of shear rate. Part of these experiments was performed using crosslinked human hemoglobin solutions (supplied by Enrico Bucci). The results showed the maintenance of blood flow and oxygen delivery to the brain at changing blood viscosities. Thus, changes in blood viscosity are normally compensated for by vascular reactions. When such compensatory mechanisms were exhausted at high viscosity (compensation: dilation) and low oxygen content (compensation: dilation), blood viscosity became relevant, resulting in a reduced cerebral blood flow, which was not sufficient to maintain oxygen delivery to the brain.

EFFECTS OF HEMOGLOBIN-BASED OXYGEN CARRIERS ON OXYGEN TRANSPORT IN THE MICROCIRCULATION

Pittman, R. N.

Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia, USA

E-mail: [email protected]

The supply of oxygen to all cells in an organism is a vital function of the cardiovascular system. Oxygen exchange between the blood and tissue occurs in the microcirculation, most of it across the walls of the arterioles and capillaries. In addition to their role as controllers of blood flow, the arterioles also are a major site for oxygen exchange in tissues with low metabolism and blood flow (e.g., resting skeletal muscle). For tissues with high metabolic rate and blood flow (e.g., contracting skeletal muscle, myocardium, brain), the capillaries become the major site of oxygen exchange. Despite some reports over the last decade that the arteriolar wall (endothelium and/or smooth muscle) consumes a substantial amount of oxygen, recent evidence points to the artifactual nature of these results and they appear to be the result of oxygen consumption by the phosphorescence quenching method used to measure oxygen tension (PO₂). In cases where the oxygen supply is reduced due to a decrease in the oxygen carrying capacity of blood, as occurs from the use of non-oxygen carrying fluids in isovolemic hemodilution and resuscitation following hemorrhage, partial restoration of oxygen carriage using hemoglobin-based oxygen carriers (hbocs) should lead to improvement of tissue oxygenation. However, a side effect of HBOC infusion is often observed in which there is a significant increase in mean arterial pressure (MAP). The most probable mechanism for this hypertensive effect appears to be arteriolar vasoconstriction due to scavenging of NO by the HBOC. This effect has been shown to be related to (1) the reactivity of the HBOC with NO and (2) the physical dimensions of the HBOC. Hemoglobins with reduced NO reactivity have been shown to have a decreased vasoconstrictor effect. The influence of HBOC size might be related to its degree of extravasation from the lumen and/or its ability to interfere with NO production by the endothelium. In terms of oxygen transport, one must consider the question: How does addition of an HBOC affect the oxygen supply to the tissue? Because convective oxygen delivery is the product of blood flow and oxygen content, hbocs with vasoconstrictor action could reduce the improvement in oxygen supply expected from the partial restoration of oxygen content. So, it is important not to cause arteriolar vasoconstriction that adversely affects downstream capillary perfusion. Scavenging of NO by hbocs could also increase the oxygen consumption of mitochondria, since NO acts as a brake on respiration. In the case of a reduced oxygen supply caused by vasoconstriction, this dual action of reduced NO would be expected to exacerbate any existing tissue hypoxia. The P₅₀ of the HBOC also plays a role in the oxygen supply to tissue, since the release of oxygen from the HBOC must occur appropriately in order for adequate tissue oxygenation.

UNRAVELING THE ROLES OF ATP, SNOHB, AND NITRITE IN RED CELL DEPENDENT REGULATION OF NITRIC OXIDE SIGNALING: IMPLICATIONS FOR HBOC THERAPEUTICS

Patel, R. P.¹, Vitturi, D.¹, Rodriguez, C.², Vandromme, M.², George, R. L.², Hutchings, A.², Kerby, J. D.²

¹Department of Pathology and ¹Center for Free Radical Biology,

²Department of Surgery, University of Alabama at Birmingham, Alabama, USA

E-mail: [email protected]

The paradigm of hypoxic sensing by red cells being coupled to increased nitric oxide (NO)-dependent blood flow is well established but the precise mechanism(s) involved remain under investigation. In this presentation we will discuss the three proposed mechanisms, namely S-nitrosohemoglobin dependent vasodilation, nitrite reduction to NO by deoxyhemoglobin, ATP-release and subsequent stimulation of endothelial nitric oxide synthase with a goal that understanding the relative roles of each mechanism will offer novel strategies in the development of hbocs and attenuating their hypertensive effects. In this context, we will discuss data that further develops the concept of NO-dependent signaling being stimulated by a nitrite-reductase activity of deoxyhemoglobin and show data that extrapolates this to hbocs and specifically the attenuation of the latter's hypertensive effects in a murine model of trauma-hemorrhagic shock and resuscitation. Our data support the potential for nitrite as an adjuvant therapy to prevent the hypertensive effects of hbocs and underscore the need to integrate information of how hbocs react with nitrite in the clinical evaluation of these compounds.

INFLAMMATORY RESPONSE TO HEMOGLOBIN INFUSION IS TRIGGERED VIA ACUTE SCAVENGING OF ENDOTHELIAL NITRIC OXIDE

Sovershaev, M., Egorina, E., Yu, B., Bloch, K. D., Zapol, W. M.

Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA, USA

E-mail: [email protected]

Nitric oxide (NO) is a potent anti-inflammatory mediator. The inflammatory and prothrombotic reactions observed in hemolytic conditions have been attributed to scavenging of NO by free intravascular hemoglobin. Therefore, we hypothesized that IV administration of tetrameric Hb or polymerized Hb solution induces an acute inflammatory reaction via endothelial NO scavenging. Awake wild type and NOS3-/- mice were given an IV topload challenge with murine tetrameric Hb, polyheme (Northfield Laboratories), murine met-Hb, met-polyheme®, or murine whole blood, each infused at 1.44 g Hb/kg. In two additional groups, mice breathed 80 ppm NO for 1 hour prior to injection of murine Hb or polyheme. After sacrifice, lungs, livers, and blood were collected at 1, 2, 4, 8, and 24 h after challenge to measure the expression of mrna encoding for IL-6 and TNF and the levels of tissue factor (TF) protein in circulating blood monocytes.

Both murine tetrameric Hb and polyheme challenge led to an increase in levels of IL-6 and TNF mrna levels, which reached a maximum at 2 h in lung and at 24 h in liver tissue from wild type mice; IL6 and TNF levels did not change after similar challenge in NOS3-/- mice. Tetrameric Hb challenge induced expression of TF protein in monocytes from wild-type, but not from NOS3-/- or sgcα1 -/- mice. Challenge with murine met-Hb or met-polyheme did not induce expression of IL-6, TNF or TF. Breathing NO before administration of tetrameric Hb or polyheme completely abolished the induction of IL-6, TNF or TF expression in wild-type mice.

Therefore, IV challenge with either tetrameric murine Hb or polyheme induces an inflammatory reaction via scavenging of endothelial NO. Breathing NO gas before challenge, presumably by enhancing the body's levels of metabolites of NO, can prevent the inflammatory response to extracellular hemoglobin.

PNPH, A NEUROPROTECTANT HBOC: STUDIES OF IN VIVO AND IN VITRO TRAUMATIC BRAIN INJURY

Kochanek, P. M.¹, Wu, X.¹, Ma, L. ², Du, L.¹, Shellington, D.¹, Vagni, V.¹, Clark, R. S. B.¹, Hsia, C. J. C.²

¹Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA

²Synzyme Technologies, Irvine, CA, United States

E-mail: [email protected]

The combination of traumatic brain injury and hemorrhagic shock (TBI+HS) is devastating and is an important challenge in both civilian and military resuscitation. Although Hemoglobin-based Oxygen Carriers (hbocs) are logical candidates for resuscitation in this setting, vasoactivity and neurotoxicity are obvious limiting concerns for clinical translation. Polynitroxylated pegylated hemoglobin (PNPH) is a novel modified bovine Hb that contains 14–15 antioxidant nitroxide moieties along with pegylation that confer putative beneficial properties. We explored the effects of PNPH in three models relevant to TBI resuscitation, namely: 1) experimental TBI in mice followed by 90 min of mild volume-controlled HS and resuscitation (Dennis et al., J Neurotrauma, 2008); 2) TBI in mice followed by 35 min of severe pressure controlled HS and resuscitation; and 3) in vitro studies using rat cortical neurons alone or exposed to an excitotoxic insult (glutamate/glycine). In both in vivo models, PNPH outperformed either Lactated Ringers (LR) or Hextend (HEX) as resuscitation fluids with regard to resuscitation volume required and/or blood pressure achieved. PNPH also exhibited favorable effects on both recovery of brain tissue PO₂ (pbto₂) vs LR, and neuronal death in vulnerable hippocampus vs LR or HEX—as assessed using fluorojade staining. Finally, in neuronal culture, unlike native bovine Hb, PNPH was not toxic across a wide range of concentrations and surprisingly attenuated excitotoxic neuronal death—as assessed using either LDH release or MTT. We conclude that PNPH is a unique, small volume resuscitation solution in experimental TBI+HS that exhibits neuroprotective properties both in vivo and in vitro. Given the possibility of extravasation of hbocs into brain tissue in TBI or polytrauma resuscitation, PNPH or related nitroxylated Hbs may represent very attractive novel small volume oxygen therapeutic agents.

Support: US Army PR054755W81XWH06-01-0247 and NS30318.

APPROACH TO THE THERMODYNAMICS OF OXYGEN TRANSPORT IN VIVO BY NATURAL AND ARTIFICIAL OXYGEN CARRIERS

Bucci, E.

Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, MD, USA

E-mail: [email protected]

Oxygen is a toxic gas, still indispensable to aerobic life. Plasma oxygen concentration is in the range of 10^–5 M, insufficient to sustain metabolism. Oxygen carriers, present in blood, release oxygen into plasma, thereby replacing consumed oxygen and buffering PO₂ near their P₅₀. They are the natural cell-bound carriers, like hemoglobin and myoglobin, and artificial cell-free hemoglobin-based oxygen carriers (HBOC) dissolved in plasma. Cell-bound oxygen delivery is retarded by the slow diffusion of oxygen in plasma and interstitial fluids. The 40% hematocrit of normal blood compensates for the delay, coping with the fast oxygen consumption by mitochondria. Facilitated oxygen diffusion by hbocs corrects for the slow diffusion, making cell-free delivery relatively independent from P₅₀. At all oxygen affinities, hbocs produce hyperoxygenations that are compensated by vasoconstrictions. There is a strict direct correlation between the rate of oxygen replacement and hemoglobin content of blood. The free energy loss of the gradient adds a relevant regulation of tissues oxygenation. Oxygen is retained intravascularly by the limited permeability to gases of vessel walls.

Conflict of interest: Enrico Bucci is a consultant of oxyvita inc. (New Windsor, NY)

Bucci, E, Razynska A. U.S. Patent # (08/733-413). Polymerized Hemoglobins (2000).

SESSION III: HBOCs FROM INDUSTRIAL LABORATORIES AND PERFLUOROCARBONS

Chairman: Weiskopf, R. B.

University of California, San Francisco, CA, USA

E-mail: [email protected]

Session Keynotes

Development of synthetic and semi-synthetic oxygen carriers for use in humans has had a long, tortuous, and at times frustrating course. Two broad classes of compounds have been developed: hemoglobin-based and perfluorocarbons.

Many compounds have demonstrated efficacy in laboratory studies, in a variety of experiments designed to test effective oxygen delivery in the presence of very low concentrations of native hemoglobin. Safety in these pre-clinical studies has been satisfactory generally, although in the case of at least some hemoglobin-based compounds systemic and pulmonary hypertension were noted, likely owing to hemoglobin scavenging of nitric oxide. Administration of some compounds in some species was associated with focal myocardial lesions, elevation of plasma activity of hepatic and pancreatic enzymes, and in some models neurotoxicity. Perfluorochemical administration has been associated with thrombocytopenia.¹ However, the clinical relevance of most of these findings remains unclear.

Design of clinical trials to demonstrate efficacy of both classes of compounds has been challenging. It is not easy to design and conduct ethical trials in humans to show clinically important results of oxygen delivery in states of inadequate oxygenation. Safety concerns have arisen in some of these clinical trials, leading in some cases to termination of a test compound’s further development. In 2008, the U.S. NIH and FDA held a joint workshop to examine these issues for hbocs and to consider the feasibility of, obstacles to, and ethical issues related to future clinical trials of hbocs.

Further development would be advanced, potentially, by further molecular development, as well as novel applications and trial design. This session includes eight presentations describing aspects of oxygen carriers in various stages of development programs. We will hear about a diverse set of compounds with varied potential applications. New compounds are being developed, including one from a marine invertebrate. These approaches are being developed in efforts to limit potential adverse effects, and to prolong intravascular retention, while providing efficacy. Perspectives regarding the balance of benefit and risk vary among clinicians, regulators, and those in industry. These presentations offer the possibility of assisting in re-evaluation of these perspectives and may shed light on how the field may continue to develop rationally.

AN IN-DEPTH EVALUATION OF HBOC-201 SAFETY: SIDE EFFECTS OR TOXICITY?

Greenburg, A. G.

Former VP Medical Affairs, Biopure Corporation, Professor of Surgery, Emeritus, Brown University

E-mail: [email protected]

Established fact: decreased hemoglobin concentration [Hb] is associated with increased mortality and morbidity; this relationship is the driving force of transfusion therapy. Worldwide there are many situations when blood is indicated but neither available nor an option to treat an acutely anemic patient. Periodic problems with the supply and quality of rbcs are recognized. There remains a need for a shelf storable oxygen carrying solution for use when blood is not an option.

While all hbocs are part of a class, the emergence of common adverse events within the class may have diverse origins. The true safety evaluation demands consideration of their manifold dissimilarities.

In clinical trials HBOC-201 and the comparator, prbcs, both demonstrate a correlation between dose and adverse events (AE). Differences in the overall incidence of aes resides in the “side effects”- transient, reversible observations – associated with HBOC-201 use.

The HBOC-201 clinical safety database of 22 studies was subjected to a proper meta-analysis; adjustments for randomization and control solutions were made in a fixed effects model. The cardiovascular SOC had the only statistically significant difference and that only for the organ class, not for any of the individual items.

Attribution of “cardio-toxicity” to HBOC-201 is confounded given differences in solution efficacy [Hb] resulting in “under-treatment” that is then superimposed on preexisting cardiac disease in the presence of significant acute anemia. Statistically, the use of HBOC-201 is not correlated with the serious cardiac adverse events observed in these elective surgery trials. While some of these cardiac cases were noted in 54 cases of HBOC-201 compassionate use, none were attributed to the HBOC by the treating physicians.

When blood is not an option HBOC-201 can be administered safely with the potential for benefit.

CLINICAL APPLICATIONS OF PEGYLATED BOVINE HEMOGLOBIN

Abuchowski, A.

Prolong Pharmaceuticals, Monmouth Junction, NJ, USA

E-mail: [email protected]

Hemoglobin based oxygen carriers have been developed in a variety of iterations, including cross-linked, polymerized, and pegylated forms. A recent review of clinical trials with these products has expressed concern over their toxicity, in particular hypertension and cardiac toxicity. Pegylated hemoglobins appear to have the least toxicity and one form is currently in Phase III clinical trials.

As the use of hbocs has become widespread for investigations in a variety of different applications it has become clear that “one size does not fit all.” Hbocs will need to be specifically formulated for each application. For example, in ischemic diseases like stroke, sickle cell, MI and peripheral ischemia, pegylated hemoglobin in the carbon monoxide form may be ideal.

Physiological concentrations of CO have vasodilatory, antioxidant, anti-inflammatory, anti-apoptotic, and pro-mitochondrial biogenesis properties. In this context, initial release of CO from an HBOC may stabilize and protect the vasculature from subsequent exposure to cell-free oxy-Hb.

In situations involving severe blood loss and hypotension, PEG-Hb formulated in a hypertonic salt solution provides the hyperosmotic/hyperoncotic environment necessary to recover blood pressure and repay oxygen debt.

Data will be presented on both these formulations.

PNPH – A THERAPEUTIC FOR INADEQUATE BLOOD FLOW AND SUPEROXIDE, NITRIC OXIDE DEPENDENT VASCULAR DYSFUNCTIONS

Hsia, C. J. C., Ma, L.

Synzyme Technologies LLC, Irvine, CA, USA

E-mail: [email protected]

Background: The recent meta-analysis concluded that all current generation hemoglobin-based oxygen carriers (hbocs) in advanced clinical trials increased infarction and mortality in the treatment arm (JAMA, 2008 May 21; 299(19): 2304, Natanson, C., et al). The next generation of HBOC needs to have higher therapeutic indexes in order to justify testing in advanced clinical trials.

Objectives: synzyme has developed a polynitroxylated pegylated hemoglobin (PNPH, aka PN-peghb) within which the redox cycling activities of nitroxide and heme iron are coupled to create a hemoglobin-based enzyme mimetic for superoxide dismutase. The high therapeutic index of PNPH is based on its superoxide dismutase, super colloid, endothelium and neuronal protective activities.

Rational: To prove the hypothesis that PNPH is a high-therapeutic-index next generation HBOC, one would have to demonstrate that it can: (1) reduce the aortic stiffness as that is the predictor of all causes of cardiac mortality in hypertensive patients undergoing selective surgery (Hypertension, 2001; 37:1236, S. Laurent, et al.); and (2) reverse oxygen radical induced nitric-oxide-dependent vascular dysfunctions such as those found in sickle cell disease (PNAS, December 18, 2001, vol. 98 no. 26, 15215, M. Aslan, et al.).

Results: PNPH was shown to correct nitric-oxide- dependent vascular dysfunctions induced by superoxide in a transgenic sickle mouse model (Hunter Champion, privileged communication). It also functions as a super colloid neuroprotective therapeutic in a model of traumatic brain injury (TBI) with hemorrhage (J Neurotrauma, 2008 Sep 10, Dennis, A. M. et al.). Low dose PNPH maintained better systemic hemodynamics, brain oxygenation, and reduced neuronal death when compared to the current gold standard of civilian and military pre-hospital resuscitative fluid.

Conclusion: PNPH takes full advantage of both the oxygen and nitric oxide homeostatic properties and also the super colloid properties of peghb. Its novel superoxide dismutase mimetic activity corrects nitric-oxide-dependent vascular dysfunctions, provides neuronal and endothelium protection, and corrects inadequate blood flow for un-met medical needs such as TBI, shock, sickle cell anemia, stroke, and myocardial infarction.

HEMARINA-M101: A NEW GENERATION OF BLOOD SUBSTITUTES COMING FROM SEA

Rousselot, M.¹, Tsai, A. G.², Intaglietta, M.², Zal, F.¹

¹Hemarina SA, France

²University of California, San Diego

E-mail: [email protected]

Extracellular soluble haemoglobins (Hb) have long been studied for their possible use as safe and effective alternatives to blood transfusion. Even if remarkable progress has been made in the use of cell-free Hb as artificial oxygen carrier, significant problems remain, including susceptibility to oxidative inactivation and propensity to induce vasoconstriction.

Hemarina-M101 is natural giant extracellular hemoglobin (3.6-mda) coming from marine invertebrate (polychaete annelid Arenicola marina) and which may have unrestricted future application for human use in situations requiring oxygen delivery to tissues. Hemarina-M101 is a complex of globin and non-globin linker chains, which represent a summit of complexity for oxygen-binding heme proteins. M101's unique properties, including anti-oxydant activity and high oxygen affinity (p50∼7mmhg), are compatible with theoretical and biophysical studies of oxygen transfer in small vessels. Animal studies have revealed no apparent behavioral or physiopathological changes and did not show over allergic response. In vivo experiments realised on rats and hamster showed a lack of hypertension and vasoconstriction and no significant changes in arteriolar and venular hemodynamics. Moreover, its efficacy has been proved on a hypoxic tumours model.

We have not yet determined specific applications for this molecule, but due to its remarkable properties the application field can be large where an oxygen-carrying RBC substitute is needed (anemia, hemorrhagic shock, surgery). However, proof of medical concepts have already been obtained for organ preservation prior transplantation. Indeed, M101 increases considerably the lifetime of cells and quality of cell cultures due to its oxygenation and anti-oxidant properties in comparison with commercial products.

OXYVITA™, NEW THERAPEUTIC OXYGEN DELIVERY PRODUCTS: OVERVIEW INITIAL APPLICATIONS

Harrington, J. P.^{1, 2}, Wollocko, J.², Kostecki, E.², Torres Filho, I.³, Torres, L. N.³, Williams, D.³, Barbee, R. W.³, Reynolds, P. S.², Skaflen, M. D., Ward, K. R.³, Wollocko, H.²

¹SUNY, New Paltz, NY, USA

²OXYVITA Inc., New Windsor, NY, USA

³VCU. School of Medicine, Richmond, VA, USA

E-mail: [email protected]

Oxyvita™-zero-link polymerized hemoglobin is a novel oxygen carrier. The technology was discovered by Professor Enrico Bucci and his team at the University of Maryland and developed to the commercial production scale by scientists at OXYVITA Inc. The product has a well-documented history of in vitro and in vivo studies. These initial studies were performed at independent scientific centers, such as University of Maryland, Johns Hopkins University, University of California (UCLA), Virginia Commonwealth University (VCURES), and State University of New York, New Paltz.

Oxyvita™ is produced through a polymerization reaction of purified hemoglobin using activators that are removed after initial phase of synthesis. This procedure eliminates any chemical linkers remaining in the product, so there are no reaction concerns, such as reversibility, dependency on temperature and pressure, decomposition, and residual toxicity. The strong pseudo-peptide bonds between the hemoglobin molecules within the oxyvita™ polymer, coupled with a thorough physicochemical characterization of the polymer, allow OXYVITA Inc. to have control over the production of several modified hemoglobins. Several of these modified products have already been characterized in vitro and tested successfully in vivo.

The introduction of a powder form of oxyvita™ addresses concerns regarding transport and stability for combat casualty care. The powder form of the product was developed to be dissolved in water or other IV fluids. A special formulation of the powder was created to work as a Small Volume Resuscitation Product (SVRP). SVRP solution/powder can be used at a volume of <1/10th of volume of lost blood to successfully enhance survival for up to 3 hours following extreme hemorrhage (50%-60% blood loss). The poster also presents some of the results of the in vivo studies performed at VCU.

The powder form has the same physicochemical characteristics as the solution from which it is derived. Extensive physicochemical characterization studies were conducted within OXYVITA Inc. and in vitro studies were carried out at SUNY New Paltz. The structural stability and redox studies performed on the oxyvita™ powder when compared to the solution demonstrated similar stability and significant resistance to isothermal urea unfolding. At this time, the known stability of the powder form is 3 years at room temperature, with methb content increase of 0.3% within 3 years. We are currently conducting further studies that will determine stability through 5 years. Both products present dramatic benefits for use as an HBOC in both controlled and remote situations. Further studies are warranted and are being conducted to confirm the utility of this product to enhance oxygen delivery.

COMMERCIAL DEVELOPMENT STATUS OF HEMOTECH, THE HEMOGLOBIN-ATP-ADENOSINE-GLUTATHIONE-BASED BLOOD SUBSTITUTE

Simoni, J.¹, Simoni, G.¹, Moeller, J. F.¹, Feola, M.^1,2

¹Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, Texas, USA

²hemobiotech, Inc., Dallas, Texas, USA

E-mail: [email protected]

To diminish the intrinsic toxic effects of hemoglobin (Hb), Texas Tech University scientists have developed a novel concept of “pharmacologic cross-linking” and formulated an effective oxygen carrier. This novel blood substitute, hemotech, which is under commercial development by hemobiotech and Texas Tech, is composed of purified bovine Hb cross-linked intramolecularly with o-adenosine-5’-triphosphate (ATP) and intermolecularly with o-adenosine, and conjugated with reduced glutathione (GSH). In this pharmaceutical composition, ATP prevents Hb dimerization but adenosine permits the formation of homogeneous Hb polymers. ATP and adenosine counteract the vasoconstrictive properties of Hb. Adenosine also attenuates the pro-inflammatory properties of Hb. GSH introduces a electronegative charge onto the Hb surface that blocks Hb's transglomerular and transendothelial passage, and makes hemotech less visible to phagocytes. In addition, GSH shields heme from nitric oxide (NO) and reactive oxygen species, thus enhancing vasodilation and lowering Hb's pro-oxidant potential. Hemotech does not change the cellular redox equilibrium nor does it activate apoptotic responses. This oxygen carrier prevents NF-kappa B induction and facilitates HIF-1 alpha stabilization and induction of the erythropoietin (EPO) gene under normoxia. Hemotech underwent favorable initial pre-clinical testing, proof of medical concept, and entered the regulatory process in the US. Several mandated requirements have already been met, including viral & prion (TSE) clearance validation studies and various pre-clinical toxicological and efficacy tests. The obtained results provide further evidence that “pharmacologic cross-linking” of the Hb molecule with ATP, adenosine, and GSH is highly effective in designing a viable Hb-based blood substitute.

RECENT ADVANCES OF FLUOROCARBONS AS OXYGEN CARRIERS AND IN NANOMEDICINE

Krafft, M. P.

Systèmes Organisés Fluorés à Finalités Thérapeutiques, Université de Strasbourg, Institut Charles Sadron, Strasbourg Cedex, France

E-mail: [email protected]

Fluorocarbons (fcs) have recently regained interest in nanomedicine, which includes the areas of diagnostics, drug delivery, regenerative medicine, and nanobiotechnology. A first generation of bubble-based contrast agents stabilized by a FC gas has been commercialized for diagnostic ultrasound imaging. Further applications under investigation include targeted microbubbles for molecular imaging, ultrasound-triggered drug and gene delivery, and as clot-breaking agents. Intravascular administration of oxygen in the form of FC-stabilized micron-size bubbles is a new approach in the quest for artificial blood substitutes. FC emulsions (perfluorooctyl bromide, C₈F₁₇Br, PFOB) have recently been used to replace the blood as surrogate oxygen carriers while imaging neuronal populations in vivo using two-photon microscopy. Complete in vivo imaging of neuronal and astrocytic populations with subcellular resolution was achieved, which was impossible in the presence of blood due to the strong absorption and scattering of light by red blood cells. FC gases (PFOB, perfluorohexane, C₆F₁₄, PFH, and perfluorooctyl ethane, C₈F₁₇C₂H₅, PFOE) were shown to facilitate the spreading of dipalmitoylphosphatidylcholine molecules in monolayers at the air/water interface, which qualifies these fcs as effective components for the development of novel synthetic lung surfactant compositions. The development of such therapeutic systems requires deepening our knowledge of the interactions between phospholipids and fcs at various interfaces.

SESSION IV: BIOCHEMISTRY AND PHYSIOLOGY OF CHEMICALLY AND GENETICALLY MODIFIED HBOCS (PART I)

Chairman: Kim, H. W.

Brown University, Providence, RI, USA

E-mail: [email protected]

Session Keynotes

Taming acellular hemoglobin-mediated vasoconstriction by chemical modifications or use of vasodilators

In clinical studies, patients treated with test hemoglobin-based oxygen carriers (hbocs) exhibited adverse effects (aes), including hypertension, cardiovascular events and abnormal enzyme levels. There is a concern that Hb's known vasoconstrictive property may be involved in the causality of the aes. Although the mechanism has not been fully elucidated, ferrous Hb scavenging of endogenous NO, a potent endothelium derived vasodilator, appears to play a significant role at least in some HBOC-mediated vasoconstriction in systemic blood vessels. Relatively small particle-sized acellular hbocs may extravasate into the subendothelium where they could readily interrupt NO diffusion to the smooth muscle. However, other mechanisms may also be involved. For example, it has been proposed that some low O₂ affinity hbocs may oversupply O₂ in the cerebral circulation and other vascular beds, triggering autoregulatory hyperoxic vasoconstriction. Thus, current development efforts are directed toward producing higher molecular size hbocs with reduced NO reactivity and/or adjusted O₂ affinity by chemical modifications or genetic engineering of human or animal Hbs. Hbocs based on Hb surface conjugation with polyethylene glycols (pegs) showed reduced vasoactivity. Pegylation also increases solution viscosity, which may promote shear-induced vasodilation. Increasing Hb molecular size by polymerization with glutaraldehyde and other crosslinking agents also reduced but did not eliminate vasoactivity. And yet, an ultra high MW bovine Hb polymerized by the “zero-link” method was reported not to elicit systemic hypertension in animal models. Interestingly, it caused cerebral vasoconstriction. Choice of a chemical modification method and product characteristics ([Hb], MW, O₂/NO affinity, solution properties, etc.) seem to be key factors that influence the final product vasoactivity. In an alternate approach, results of an in-vitro study indicate that certain nitro-vasodilators appear to be effective in attenuating HBOC-mediated vasoconstriction but clinical efficacy of this approach requires further studies.

NEW GENERATION OF PEGYLATED HEMOGLOBIN: EXTENSION ARM FACILITATED PEGYLATION OF αα-FUMARYL HEMOGLOBIN TARGETING THE PEGYLATION EXCLUSIVELY TO AMINO GROUPS

Acharya, S. A.

Albert Einstein College of Medicine, Bronx, NY, USA

E-mail: [email protected]

Though pegylated Hbs are vasoinactive and nonhypertensive, pegylation weakens the interdimeric interactions and increases the O₂ affinity of Hb, thereby limiting their potential as blood substitutes. Extension Arm Facilitated pegylation (EAFP) of Hb, on the other hand, conserves the interdimeric interactions of the parent molecule in pegylated form. Hexapegylated Hb generated by EAFP using iminothiolane and PEG-5K maleimide is also vasoinactive. EAFP of Hb also increases the O₂ affinity, thus limiting its application as blood substitute. Influence of reversible protection of Cys-93(β) as a mixed disulfide during EAFP on the structure and functional properties of pegylated Hbs has now been investigated to develop strategies to generate pegylated Hbs with lower O₂ affinity. Reversible protection of Cys-93(β) lowers the level of pegylation induced perturbation of heme pocket, α1β2 interface, autoxidation, heme loss, and O₂ affinity as compared to the original EAF-pegylatied Hbs. The combination of αα-fumaryl crosslinking and EAF-pegylation of Hb targeted exclusively to Lys residues exhibits a degree of mutual compensatory effect. PEG-shell engineered around Hb as well as pegylation of Cys-93(β) independently increase the O₂ affinity. EAFP-generated PEG-shell is less effective in counteracting the low O₂ affinity inducing influence of αα-fumaryl crosslink compared to the pegylation of Cys-93(β), Asymmetric single site EAF-pegylation of αα-fumaryl Hb (and/or of other intramolecularly crosslinked Hbs) is a novel approach to amplify the shape and flexibility of pegylated Hbs, and O₂ affinity to modulate the in vivo responses. Pattern of pegylation could also be modulated to customize the shape, flexibility, density of the PEG-shell, and O₂ affinity of pegylated Hbs. The simplicity and flexibility of EAFP suggest that it is well suited to design novel pegylated Hbs to optimize the desired chemical, biophysical, and microcirculatory properties of pegylated Hb and paves the way for correlation of the structure of pegylated Hbs with their in vivo properties.

NITRITE REDUCTASE ACTIVITY OF MODIFIED HUMAN HEMOGLOBIN: CORRELATION OF STRUCTURAL AND KINETIC VARIATION

Kluger, R., Lui, F., Chen, S., Foot, J.

Department of Chemistry, University of Toronto, Toronto, Canada

E-mail: [email protected]

The conversion of nitrite to nitric oxide is an important function of red cells that helps maintain blood vessels in their relaxed state. The addition of cross-linked hemoglobin as a red cell substitute (hbocs) leads to vasoconstriction related to scavenging of nitric oxide. While formation of nitric oxide by the reduction of nitrite may counteract the scavenging effect, the reaction must be fast and also must lead to the rapid release of nitric oxide from hemoglobin. Kinetic analysis of nitrite reductase activity is complicated by the multiple changes in spectra of the reacting hemoglobin that are observed during the course of the reaction. We developed a computational algorithm that permits deconvolution of the time-course spectra in order to obtain useful and verifiable kinetic data. We observed the rate of nitrite reduction for a set of cross-linked hemoglobins with various oxygen affinities, with and without added PEG chains (the number of chains per tetramer was varied as well). One result is the observation that the PEG chains enhance the rate of conversion of nitrite to nitric oxide (while producing methb), potentially counteracting the effects of scavenging of nitric oxide but decreasing oxygenation capacity. Recent developments in creating enhanced materials of greater size, without PEG, suggest alternative approaches to providing safe and effective hbocs.

PEGYLATION PROMOTES HEMOGLOBIN TETRAMER DISSOCIATION

Ronda, L.¹, Caccia, D.², Perrella, M.², Bruno, S.¹, Faggiano, S.¹, Pioselli, B.¹, Bettati, S.^1,3, Abbruzzetti, S.⁴, Viappiani, C.⁴, Barocelli, E.⁵, Ballabeni, V.⁵, Bertoni, S.⁵, Mozzarelli, A.^1,3

¹Department of Biochemistry and Molecular Biology, University of Parma, Italy

²Department of Biomedical Sciences and Technologies, University of Milan, and LITA (Interdisciplinary Laboratory of Advanced Technologies), Milan, Italy

³National Institute of Biostructures and Biosystems (INBB), Italy

⁴Department of Physics, NEST CNR-INFM, University of Parma, Italy

⁵Department of Pharmacological and Biological Sciences and Applied Chemistry, University of Parma, Italy

E-mail: [email protected]

Hemoglobin conjugated with polyethylene glycol (PEG) acts as an oxygen carrier free in plasma, substituting red blood cells in supplementing oxygen in hypo-oxygenation pathologies. Given the complexity of oxygen delivery regulation, subtle structural and functional differences in pegylated hemoglobins might be associated with distinct physiological responses and, potentially, to adverse effects. We have compared hemoglobin pegylated under anaerobic conditions, called PEG-Hb^deoxy, with hemoglobin pegylated under aerobic conditions, called PEG-Hb^oxy, a product that mimics Hemospan^®, produced by Sangart. SDS PAGE and MALDI-TOF analyses demonstrated that PEG conjugation yields products characterized by a broad distribution of PEG/hemoglobin ratios. The elution profiles in size-exclusion chromatography indicate that both products exhibit a more homogeneous distribution of molecular weight/hydrodynamic volume under deoxy conditions and at higher concentrations. PEG-Hb^oxy shows high oxygen affinity, low modulation by allosteric effectors, almost no cooperativity, a fast and monophasic CO binding, and a limited dependence of functional properties on concentration, whereas PEG-Hb^deoxy exhibits oxygen binding curves that significantly depend on protein concentration, and a slow CO binding, similar to unmodified hemoglobin. Pegylated CO-hemoglobins, probed by flash photolysis, exhibited a lower amplitude for the geminate rebinding phase with respect to native hemoglobin and a negligible T state bimolecular CO rebinding phase. These findings are explained by an increased dissociation of pegylated hemoglobins into dimers and perturbed T and R states with decreased quaternary transition rates. These features are more pronounced for PEG-Hb^oxy than PEG-Hb^deoxy. The detected heterogeneity might be a source of reported adverse effects when pegylated Hbs are used as blood substitutes. Transfusion experiments in guinea pigs are in progress, evaluating bioclinical parameters as well as proteomic patterns, diagnostic for liver, pancreas and heart damages, aimed to a total body safety assessment.

PHYSIOLOGICAL VS. BIOPHYSICAL RESPONSES OF THE MICROVASCULATURE IN BLOOD VOLUME AND OXYGEN CARRYING CAPACITY RESTORATION

Cabrales, P., Tsai, A. G., Intaglietta, M.

Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

E-mail: [email protected]

Although the initial correction blood losses require plasma expansion, development of oxygen carrying blood substitutes has focused on producing oxygen carrying fluids, under the assumption that plasma expansion is a solved problem. This perception has tended to divert interest in maximizing the effectiveness of plasma expanders as a means for restoring the transport capacity of the circulation, and compensating for decreased oxygen carrying and delivery capacity. Studies on oxygen non-carrying and carrying plasma expansion in the microcirculation are providing new, counterintuitive results with which to improve plasma expansion efficacy, in showing the beneficial role of increasing plasma viscosity and increasing oxygen affinity of the oxygen carrier as a means of reducing carrier use and improving tissue oxygen delivery.

A remaining unaddressed concern is that plasma expansion, particularly in the context of blood substitutes, is designed and used independently of the underlying pathologies that may be present when used. However, it is one of the most common interventions that interacts with the regulation of the blood oxygen supply and inflammation.

Lactated Ringer's solution, dextran 40, and 6% hetastarch used in clinical practice to replace blood losses can induce inflammatory responses causing neutrophil activation and tissue injury. Since plasma expanders are used in conditions with potentially inherent endothelial dysfunction, it may be advantageous to exploit those endowed with the ability to directly deliver antioxidant to the tissue, particularly in the presence of molecular hemoglobin and its latent toxicity. Most patients present some form of endothelial dysfunction due to age or conditions that cause the clinical/surgical intervention leading to the blood loss being treated. Acute or chronic endothelial dysfunction, a precursor, consequence and cause of inflammation, is not a part of the design of plasma expanders or blood substitutes, although it probably accounts for most of the toxicity experienced in the field.

WHOLE-BODY ASSESSMENT OF HBOC-RELATED ADVERSE EFFECTS BY HIGH-RESOLUTION, QUANTITATIVE NANOSPECT/CT TECHNOLOGY

Eke, A.¹, Máthé, D.², Portöroő, I.¹, Mozzarelli, A.^3,4

¹Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Hungary

²Mediso Ltd., Hungary

³Department of Biochemistry and Molecular Biology, University of Parma, Italy

⁴National Institute of Biostructures and Biosystems (INBB), Italy

E-mail: [email protected]

Hemoglobin-based oxygen carriers (hbocs) have dynamically emerged as an alternative to whole blood transfusion under conditions of various medical emergencies. Several strategies have been devised aiming to develop Hb-derivatives that, when free in the plasma, would have overall oxygen carrying properties similar to that of Hb confined within the erythrocytes, among them Euro-PEG-Hb developed by partners of the Eurobloodsubstitutes Consortium. Due to the high affinity of cell-free hemoglobin (Hb) to nitric oxide (NO) – a molecule of central importance in maintaining vascular homeostasis – hbocs can potentially reduce the bioavailability of NO that links up with various pathologies such as systemic hypertension due to vasoconstriction, release of proinflammatory mediators, and a loss of platelet inactivation, conditions that can lead to vascular thrombosis of the heart and other organs thus having lately become center-staged in HBOC-related research. The evolution of these adverse effects in the body is the result of complex processes most likely with organ-specific spatial and temporal dynamics.

Recent advances in the Single Photon Emission Computed Tomography (SPECT) technology offer the means for a non-invasive, whole-body assessment of physiological, pathophysiological, and biochemical processes under in vivo conditions using isotope-labeling techniques.

In exemplary experiments, rat red blood cells (rbcs) were labeled with ^99mtc using stannous pyrophosphate as reducing agent (20 μg Sn (II)/kg bdw, i.v.). ^99mtc-HMPAO (hydroxy-methyl-para-amino-oxime) based labeling of rat platelets was performed using a commercially available Leuco-Scint^® Kit (Medi-Radiopharma Ltd., Érd, Hungary) containing hexaethyl starch as expander and ACD-A as anticoagulant.

Employing a dedicated small animal SPECT scanner (nanospect/CT, Mediso Ltd., Budapest, Hungary) in anesthetized rats, we demonstrate the impact on red blood cell (RBC) and platelet distribution of conditions like NO scavenging by L-NAME (100 mg/kg bdw, i.v.), and blood-to-HBOC exchange.

(Supported by a National Office for Research and Technology Grant 2008ALAP1-01569/2008 given to AE.)

EFFECT OF CELL-FREE HEMOGLOBIN ON THE CEREBRAL CIRCULATION

Koehler, R. C., Cao, S., Zhang, J., Kwansa, H.

Department of Anesthesiology and Critical Medicine, Johns Hopkins University, Baltimore, Maryland, USA

E-mail: [email protected]

Previous work has shown that exchange transfusion with zero-link bovine hemoglobin polymers (ZL-hbbv) does not interfere with NO-dependent dilation of pial arterioles. However, baseline diameter decreased, and the decrease was blocked by inhibitors of O₂-dependent cytochrome P450 synthesis of 20-HETE. Cytochrome P450s can be inhibited by NO and CO. Because Hb can act as a nitrite reductase and possibly generate NO and nitrosthiols, 2 μmol/kg of Na nitrite was infused iv before exchange transfusion, and the pial arteriolar constrictor response was prevented. Exchange transfusion of ZL-hbbv decreased infarct volume induced by middle cerebral artery occlusion (MCAO) but did not produce an increase in cerebral blood flow that might be anticipated from the decrease in hematocrit and viscosity. Pial arterioles were found to initially dilate at the onset of MCAO, but the dilation was not sustained. Exchange transfusion with ZL-hbbv at 35 min of MCAO did not prevent the loss of pial arteriolar dilation. However, infusion of Na nitrite before transfusion preserved pial arteriolar dilation throughout 2 h of MCAO. Polynitroxylation of Hb confers SOD and catalase mimetic activity. Polynitroxylated pegylated bovine hemoglobin (PNPH) was exchange transfused in the carboxy state without nitrite co-administration and was also found to preserve pial arterioles in a dilated state during MCAO. To better delineate the role of CO released from Hb, infarct volume was determined after transfusion of PEG-Hb, PEG-Hb-CO, and PNPH with bound CO at 20 min of 2 h of MCAO in rats. A 10 ml/kg topload transfusion of PEG-Hb, which produces a plasma Hb of only ∼0.5 g/dl (<∼2 g/dl with exchange transfusion), did not significantly decrease infarct volume. However, topload transfusion with PEG-Hb-CO or PNPH with CO decreased infarct volume by 55% and 58%, respectively. Use of PEG-Hb as a CO-releasing molecule may increase its efficacy in rescuing the ischemic brain.

VESSEL TYPE-SPECIFIC HBOC-MEDIATED CONTRACTIONS AND THEIR ATTENUATION BY NITROVASODILATORS

Hai, C.-M., Kim, H. W.

Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI, USA

E-mail: [email protected]

Acellular HBOC-mediated vasoconstriction is an important safety concern. Using isolated blood vessels, we tested the hypothesis that vessel type is an important determinant of HBOC-induced contractions and their attenuation by nitrovasodilators. First, we studied vasoactivities of two different HBOC preparations (HBOC-201 and HBOC-High MW; Biopure) in bovine pulmonary artery, coronary artery, carotid artery, and portal vein, and their attenuation by nitroglycerin, sodium nitroprusside (SNP), and sodium nitrite. Next, we studied the molecular interaction between HBOC-201 and nitrovasodilators in the formation of methemoglobin. Lastly, we studied the molecular mechanism of HBOC-mediated vascular smooth muscle contraction by measuring myosin light chain phosphorylation in HBOC-201-stimulated tissues by 2-D PAGE. We found that both HBOC preparations significantly enhanced contractions of phenylephrine-stimulated pulmonary arteries only at relatively high phenylephrine. In contrast, HBOC-mediated contractions of serotonin-stimulated coronary artery were independent of serotonin. Carotid artery and portal vein did not develop significant contractions in response to either HBOC preparation. HBOC increased the IC₅₀ for nitrovasodilator-induced relaxations; however, the magnitude of increase appeared to be dependent on nitrovasodilator type in the following descending order: SNP > nitroglycerin ∼ sodium nitrite. Furthermore, vessel type-specific sensitivities in HBOC-mediated contractions and nitrovasodilator-induced relaxations exhibited the same descending order: pulmonary artery > coronary artery > carotid artery and portal vein, suggesting that vascular beds that are most sensitive to HBOC are also most responsive to nitrovasodilators. We observed time-dependent and nitrovasodilator-specific methemoglobin formation when HBOC-201 was mixed with nitrovasodilator in vitro. Agonist-induced vascular smooth muscle contractions are typically accompanied by increases in myosin phosphorylation. Unexpectedly, we found that HBOC-mediated contractions of pulmonary arteries were accompanied by small but significant decreases in myosin phosphorylation, suggesting that other molecular mechanisms such as heat shock proteins may be involved in HBOC-mediated vascular smooth muscle contractions.

This study was supported by NMRC grant N00189-07-C-Z086.

THE PH DEPENDENCE OF HEMOGLOBIN AND MYOGLOBIN REDOX COMPROPORTIONATION

(Poster Selected for Oral Presentation)

Holladay, R. S., Nicholls, P., Wilson, M. T., Cooper, C. E.

Department of Biological Sciences, University of Essex, England

E-mail: [email protected]

Ferryl hemoglobin is a strongly oxidising species suggested to be responsible in part for the toxic properties of cell free hemoglobin. Therefore the comproportionation of oxyhemoglobin and ferrylhemoglobin to yield methemoglobin has been considered an important antioxidant mechanism. Given the strong ph effects on ferryl hemoglobin reactivity in general, we studied the ph dependence of this comproportionation reaction. Ferryl and oxy species were mixed in a spectrophotometric split cell, in which the weakly buffered ferryl species was initially at ph 8 and the strongly buffered oxy species at an equal or lower ph; the comproportionation (methemoglobin formation) kinetics was then followed. The kinetic pk values were determined for the two proteins and shown to be close to the previously determined values for the protonation of the ferryl species (pk ∼ 4.7), suggesting that the reactive species is protonated ferryl hemoglobin. This has important implications for haemoglobin-based oxygen carriers (HBOC). At low ph lipid peroxidation by ferryl heme is very rapid. The enhanced ferryl reactivity with oxyhemoglobin under these conditions may play an important role in ferryl toxicity by out-competing lipid peroxidation. Mechanisms to maintain a high oxyhemoglobin: methemoglobin ratio in HBOC may therefore be important for optmising antioxidant defence as well as oxygen delivery.

SESSION IV: BIOCHEMISTRY AND PHYSIOLOGY OF CHEMICALLY AND GENETICALLY MODIFIED HBOCS (PART II)

Chairman: Fronticelli, C.

Johns Hopkins University, Baltimore, MD

E-mail: [email protected]

Session Keynotes

Chemical reactions and molecular biology have been applied to the development of Hemoglobin Based Oxygen Carriers (HBOC). The transformation of the hemoglobin molecule into a HBOC requires a variety of modifications for rendering the acellular hemoglobin molecule physiologically acceptable when infused in circulation. Hemoglobins with different oxygen affinities and molecular sizes have been obtained and tested in vivo using various animal models. A major obstacle to the development of HBOC has been the appearance of peripheral vasoconstriction following infusion. Factors contributing to this negative phenomenon are: 1) NO depletion due to the endothelial extravasation of small size HBOC; 2) increased perivascular PO₂ resulting from the facilitated delivery in plasma of HBOC’s bound O₂.

Approaches for solving the problem of NO depletion have been either to inhibit extravasation by increasing the size of the HBOC molecules, or by introducing in the hemoglobin molecule a decreased NO affinity. Antioxidant enzymes are being coupled to HBOC for preventing autoxidation and the endothelial damage due to the radicals’ formation. The control of perivascular PO₂ and the optimal oxygen affinity of HBOC in different therapeutical applications is being investigated.

In our laboratory we have used recombinant technology to construct two polymeric HBOC with moderate (P50 = 18.0 torr) and high (P50 = 2.0 torr) oxygen affinity and MW of 500 kd and < 1000kd, respectively. In experimental stroke in a mice model, injection of either derivative reduced the infarct volume without inducing MAP increase. This suggests that the efficiency of the HBOC is not strictly correlated to its oxygen affinity. We suggest that polymerized Myoglobin can also act as aviable HBOC.

HEMOGLOBIN-BASED OXYGEN CARRIERS: FROM BLOOD SUBSTITUTES TO PHOTODYNAMIC THERAPY

Olsen, K. W., Boateng, G., Schaffer, J., Vellos, T., Le, M., Scorza, B, Moore, A., Hu, P.

Department of Chemistry, Loyola University Chicago, Chicago, IL, USA

E-mail: [email protected]

Hemoglobin-based oxygen carriers (hbocs) have been proposed as potential blood substitutes, but there are other exciting uses for them, such as photodynamic therapy (PDT) agents. The search for a safer blood substitute has resulted in the identification of properties that are needed to minimize previously encountered side-effects with hbocs. These properties include increased molecular weight to minimize vasoconstriction and incorporation of catalase and superoxide dismutase (SOD) to decrease oxidative damage. Bovine hemoglobin was crosslinked between the β82 Lysines using bis(3,5-dibromosalicyl) fumarate (bb82xlhb). The reactive groups for complementary chemistry were introduced into different proteins using N-γ-maleimidobutyryloxy-succinimide and Traut's reagent, and the proteins were mixed to produce the polymer. We have made high-molecular-weight polymers of Hb alone and of Hb with either or both catalase and SOD. There is a decrease in both the p50 and Hill coefficient for all polymerized species. The polymers containing catalase and/or SOD had smaller autoxidation rates than Hb-only polymers. Hbocs can also be used as the basis for PDT agents. We have modified bb82xlhb with folate and malachite green to make FA-xlhb-MG. Folate targets a wide variety of cancer cells and allows the protein to be brought into the cell with it by endocytosis. The malachite green is a photosensitizer that activates oxygen when excited by light. Since tumors are often hypoxic and PDT depends on oxygen for its toxicity, the Hb in this complex may increase the local oxygen levels near the photosensitizer. The FA-xlhb-MG complex has been tested on SH-SY5Y neuroblastoma and hela cells. Both cell lines took up FA-xlhb-MG in a folate-specific manner. There was no significant cytotoxicity due to these compounds in the absence of light. After exposure to a red light (630 nm) at a 8.0 J/cm² dose, 43% of the SH-SY5Y cells and 56% of the hela cells died.

HYPOXIA, HYPOXIA-INDUCIBLE FACTOR (HIF-1Α), AND HBOCS

Samaja, M.

Department of Medicine, Surgery and Dentistry, University of Milan, Italy

E-mail: [email protected]

The assessment of tissue hypoxia might represent a critical step in the comparative evaluation of hbocs. Among the various molecular markers potentially suitable for a semi-quantitative assessment of hypoxia, the expression level of hypoxia-inducible factor (HIF)-1α is a good candidate, because this fast-turnover protein becomes stabilized in the absence of O₂ and accumulates in hypoxic tissues. Recent findings from our and other groups showed that HIF-1α is up-regulated by in vivo hypoxia within minutes, quickly returns to baseline values in the correspondence of reoxygenation, and is expressed differentially in various organs exposed to the same degree of hypoxia. These conjectures point to HIF-1α as a suitable marker of tissue hypoxia during HBOC infusion. However, HIF-1α is not only a simple marker of hypoxia, but also a transcription factor activating/deactivating hundreds of genes, including those related to cell survival, vasodilatation, activation of anaerobic glycolysis and angiogenesis. In this study, we compared HIF-1α induction in rat brain tissue following 50% exchange transfusion with different hbocs, including αα-cross-linked oxyhemoglobin (ααhb) and O₂- or CO-saturated MP4 (MP4OX and MP4CO, respectively) (Sangart Inc.). Preliminary data indicate HIF-1α over-expression in the order MP4OX > MP4CO > ααhb > control. While still under investigation, HIF-1α induction in these experiments may reflect: 1) high Hb-O₂ affinity (or low P50), which limits O₂ release especially in highly metabolizing organs as the brain; 2) NO scavenging by HBOC, which induces vasoconstriction and reduces the O₂ release to tissues; and/or 3) HBOC-induced ROS and inflammation, which increases tissue O₂ demand thereby inducing a further imbalance in the O₂ supply-to-demand ratio, a paradigm of hypoxia. We will discuss the implications of these findings that they may translate into improved tissue adaptation to stress through HIF-1α over-expression.

RECOMBINANT HEMOGLOBINS AS RESUSCITATION FLUIDS IN MOUSE MODELS FOR TRAUMATIC BRAIN INJURY AND HEMORRHAGIC SHOCK

Ho, C.¹, Wu, X.², Ho, N. T.¹, Shellington, D.², Vagni, V.², Simplaceanu, V.¹, Shen, T.-J.¹, Kochanek, P. M.²

¹Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA

²Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA

E-mail: [email protected]

Recombinant hemoglobins (rhbs) hold promise as novel resuscitation agents. Traumatic brain injury (TBI) plus hemorrhagic shock (HS) represent a devastating combination. We have explored the physiological effects of novel recombinant octameric Hbs in our 3-phase mouse model of TBI+HS, modified to have a HS phase of 35 min, a Pre-Hospital Phase of 90 min, and a Hospital Phase of 15 min. After a controlled cortical impact TBI, a brain tissue PO₂ (pbto₂) probe was inserted. HS was induced with blood withdrawal (2.3 ml/100 g) over 15 min. Mean arterial pressure (MAP) was maintained (25–27 mmhg) for HS. At the beginning of the Pre-Hospital Phase, mice were assigned to Lactated Ringers (LR) or rhb groups (n = 6-9/group). Three octameric rhbs have been studied: rhb with normal O₂ affinity [NA-Hb(αN78C)], rhb with high O₂ affinity [HA-Hb(αL29F/αN78C)], and rhb with low O₂ affinity [LA-Hb(αL29W/αN78C)]. After bolus LR or rhb (20 ml/kg), additional LR or rhb (10 ml/ kg/5 min) was given (if MAP< 70 mmhg) through Pre-Hospital Phase. The Hospital Phase commenced with return of shed blood. Pre-Hospital resuscitation fluid volume was > 4-fold higher in LR group (p < 0.01). MAP normalized only in the rhb groups (p < 0.01, vs LR). Total blood Hb in rhb groups was ∼2-3 g/dl higher vs LR (p < 0.05). Lactate was normalized in all groups, lowest in the HA-Hb group (p < 0.05). Pbto₂ was numerically lowest in the HA-Hb group. Octameric rhbs normalized MAP and lactate with much smaller volume than LR after TBI+HS. Differences in Hb oxygen affinity could not account for their vasoactivity, but may impact pbto₂. Current studies are examining the effect of these novel rhbs on neuronal death and brain edema in TBI resuscitation. (Supported by NIH R01GM084614 to CH and US Army PR054755W81XWH06-01-0247 to PMK.)

FUSION HEMOGLOBINS FOR BLOOD SUBSTITUTE PURPOSES

Grey, M., Yanoi, S., Bülow, L.

Dept. of Pure and Applied Biochemistry, Lund University, Lund, Sweden

E-mail: [email protected]

Proximity and organisation between polypeptides are important in many biological systems. Such well-defined closeness can be mimicked in vitro in different ways, including co-immobilization of the proteins to a support material, chemical crosslinking, or in-frame fusion of the corresponding genes. Chemical crosslinking has often been used to prepare Hb conjugates suitable for HBOC applications. However, chemical conjugation, although simple in principle, has several inherent drawbacks: the crosslinks may be introduced at multiple residues and usually both intramolecular as well as intermolecular bonds are formed. Additionally, reactive groups may be introduced leading to the need of including blocking steps in the linking process.

In this presentation we will describe the preparation of Hb fusion proteins using genetic approaches. These Hb chimaeras may facilitate production, purification, and offer protection against harmful ROS due to proximity effects. Hb production in heterologous systems is often difficult due to the instability of, in particular, the α-chain. In order to produce a soluble Hb, the α- and β-chains must hence be produced in equal amounts, otherwise the α-chain will precipitate. Beside utilising the α hemoglobin stabilising protein (AHSP), fusions of either the α- or β-chains have been pursued. In our laboratory we have prepared a diglobin (fhb) by linking the α and β-chains, which ensures equal expression of the α and β-chains. A major advantage of this approach is the use of a single gene allowing its facile transfer to a eukaryotic expression system. Additionally, our fhb can be made secretable from the cells.

When designing hbocs, superoxide dismutase and catalase are two other important protein components. The preparation of Hb fusion proteins involving these enzymes will be described as well as the design of suitable linker regions spanning the protein moities.

ASSESSMENT OF THE OLIGOMERIZATION STATE OF GLOBINS AND CHEMICALLY MODIFIED HEMOGLOBINS BY DYNAMIC LIGHT SCATTERING

(Poster Selected for Oral Presentation)

Bruno, S.¹, Faggiano, S.¹, Ronda, L.¹, Fronticelli, C.²

¹Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy

²Johns Hopkins University, Baltimore, MD, USA

E-mail: [email protected]

The oligomerization state of globins is a crucial piece of information to understand their structure-function relationships and is known to vary significantly within the superfamily. Globins consisting of 1, 2, 4, 12, 24 subunits are known and dissociation equilibria play functional roles. For chemically modified and genetically modified hemoglobins to be potentially used as blood substitutes, the determination of the size is particularly important, as it correlates with the extent of both extravasation and renal filtration, two factors responsible for the toxicity of these products once they are administered to patients. The size and the oligomeric state of different globin-based proteins were assessed by dynamic light scattering (DLS), which measures the hydrodynamic radius (Rh) of molecules based on their diffusion coefficient. The hydrodynamic radius depends on the shape of the molecule; a relationship for Rh vs. molecular weight can be found for similarly shaped proteins. In this work, globins in different oligomeric or polymerization states were studied in order to establish such a relationship. Within the structurally homogeneous group of globin-based proteins, the correlation between hydrodynamic radii and the actual molecular weight proved to be very good, allowing for the discrimination between different oligomerization states even at very low molecular weight. The apparent molecular weight for two pegylated hemoglobins, one obtained under aerobic conditions (called peghboxy) and mimicking the MP4 product from Sangart, and another obtained under anaerobic conditions (called peghbdeoxy) was also determined using the globin-based calibration curve. Consistently with gel filtration chromatography experiments, peghbdeoxy exhibited a higher hydrodynamic radius with respect to peghboxy, hinting at a difference in the tetramer-dimer equilibrium at the concentration we investigated. The temperature-dependent aggregation of hemoglobin and its PEG-decorated derivatives was also determined, showing that pegylation strongly stabilizes hemoglobin.

THE EFFECTS OF SODIUM NITRITE ON VASOACTIVITY ASSOCIATED WITH THE HEMOGLOBIN BASED OXYGEN CARRIER HBOC-201 IN CONTROLLED HEMORRHAGE IN SWINE

(Poster Selected for Oral Presentation)

Scultetus, A. H.^1,2, Arnaud, F.^1,2, Haque, A.¹, Kim, B.¹, Oakada, T.¹, Nigam, S.¹, Moon-Massat, P.¹, Auker, C.¹, Mccarron, R.^1,2, Freilich, D.^1,2

¹Trauma and Resuscitative Medicine Department, Naval Medical Research Center, Silver Spring, MD, USA

²Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA

E-mail: [email protected]

Introduction: Animal studies have shown that HBOC-201 effectively restores blood pressure and oxygenation in pre-hospital trauma resuscitation. However, potential adverse events due to vasoconstriction (most likely due to nitric oxide (NO) scavenging) have hindered regulatory approval of clinical trials. We hypothesized that the NO donor sodium nitrite (nano₂) could reduce vasoconstriction associated with HBOC-201.

Methods: Thirty-six anesthetized Yorkshire pigs were subjected to 55% estimated blood volume (EBV) controlled hemorrhage via carotid artery over 15 minutes. At 15 min (T15), T30 and T45, animals were resuscitated with HBOC-201, HBOC-201+nano₂ [4.5 μmol/kg/infusion (low dose), 10.2 μmol/kg/infusion, or 16.2 μmol/kg/infusion (high dose)], Hextend or Hextend+nano₂ (10.2 μmol/kg/infusion). Each infusion was given concomitantly through separate lines over 10 minutes. At 60 minutes, hospital arrival was simulated and pigs were eligible to receive blood or saline. Pigs were euthanized at 120 minutes. Vasoactivity was assessed via mean arterial pressure (MAP), mean pulmonary artery pressure (MPAP), systemic and pulmonary vascular resistance indices (SVRI and PVRI).

Results: Baseline values were similar among groups. Resuscitation with high dose HBOC-201+nano₂ significantly reduced MAP and MPAP increases during the first and second infusion compared to HBOC-201 and low dose HBOC-201+nano₂. During the third infusion, this effect was lost in the HBOC-201+nano₂ (10.2 μmol/kg) group. HBOC-201+nano₂ (16.2 μmol/kg) was able to significantly reduce MAP and MPAP throughout the pre-hospital phase. SVRI and PVRI were significantly lower with high dose HBOC+nano₂ compared to other groups.

Conclusions: nano₂ reduced HBOC-201 associated vasoactivity in pre-hospital resuscitation in this swine model of controlled hemorrhage in higher doses. Future studies are necessary to optimize dosing and administration route, and to verify this effect in a more clinically relevant model of uncontrolled hemorrhage.

The study was approved by the Naval Medical Research Center/Walter Reed Army Institute of Research Institutional Animal Care and Use Committee.

The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.

MOLECULAR MODELING AND NEUTRON SCATTERING STUDIES OF PEG-HEMOGLOBIN ADDUCTS: IMPLICATIONS FOR THE DESIGN OF NOVEL PEGYLATED HBS AS BLOOD SUBSTITUTES

(Poster Selected for Oral Presentation)

Prabhakaran, M. ¹, Thiayagarajan, P.², Intaglietta, M.³, Li, D.³, Hu, T.⁴, Ananda, K.⁴, Acharya, S. A.⁴

¹American Laboratories, Charlotte, NC, USA

²Argonne National Laboratories, Argonne, IL, USA

³University of California San Diego, San Diego, CA, USA

⁴Albert Einstein College of Medicine, Bronx, NY, USA

E-mail: [email protected]

Pegylated Hb is a new class of vasoinactive blood substitutes. Molecular models of pegylated Hbs generated using extension arm chemistry have been developed. The changes in the accessible surface area of Hb, radius of gyration, and molecular shape have been delineated as a function of pegylation. Pegylation at Cys-93(β), (sites 1, 2) Lys-40(α) (sites 3, 4), Lys-120(β) (sites 5, 6), Lys-61(α) (site 7, 8) and Lys-7(α) (sites 9, 10) have been chosen for building the molecular models. The pegylation induced loss of accessible Hb-surface area decreases significantly when PEG is conjugated through extension arm, reflecting the reduced interactions of PEG-chains with the molecular surface of Hb. The radius of gyration increased significantly until hexapegylation, and then slows down as pegylation proceeds further to octa and decapegylation. The radiuses of gyration of pegylated Hb have been determined experimentally by neutron scattering; these are well correlated with values calculated from molecular models. The molecular shapes of different pegylated Hbs and molecular parameters of these shapes have also been calculated. Results show that hexapegylated Hb is clearly ellipsoidal while control Hb is essentially globular. The shape and flexibility properties of the PEG shell engineered around Hb appears to play a significant role in mechanotranduction mediated NO generation by endothelium on infusion of PEG-Hbs. We speculate that this unique structural aspect of PEG-Hb dictates the efficacy of the designed molecule by facilitating better perfusion as well as tissue oxygenation. The site-specific single site asymmetric pegylation of intramolecularly crosslinked Hbs is now planned as an extension of this structural concept to optimize the ellipsoidal shape properties of PEG-Hb to enhance the mechanotransduction induced NO production.

The studies demonstrate the unique strengths that can be realized when chemical, biophysical, and microcirculatory concepts are synergized to correlate the structure and function of PEG-Hbs as blood substitutes.

THE EFFECT OF ANTIOXIDANT ENZYMES CR PROTECTIVE OSS-LINKED POLYHEMOGLOBIN ON H₂O₂-INDUCED OXIDATIVE DAMAGE TO VASCULAR ENDOTHELIAL CELLS

(Poster Selected for Oral Presentation)

Fan, H. ¹, Yang, Y.¹, Wang, Y.²

¹Department of Blood Engineering, Shanghai Blood Center, China

²Department of Life Science, East China Normal University, China

E-mail: [email protected]

Objective: To investigate the protective effect of antioxidant enzymes cross-linked polyhemoglobin and the cell signal transduction pathway on H₂O₂-induced oxidative damage to vascular endothelial cells.

Methods: Human umbilical vein endothelial cells (HUVEC) were treated with H₂O₂ alone or a combination of H₂O₂ with polyhb and polyhb-SOD-CAT. Introcellular H₂O_2, GSH and Ca²⁺ were measured by the chemical colorimetry. Nuclear translocation of NF-kappa B was detected by the immunohistochemistry method. Gene expression of enos, HO-1 and ET-1 were assessed by RT-PCR. Phosphorylated protein of I kappa B, c-Jun and Erk were analyzed by Western Blotting and BD Phosflow method. Cell death (apoptosis and necrosis) was evaluated by flow cytometry.

Results: Incubation with H₂O₂ alone produced a significant drop in GSH, increases in Ca²⁺ concentration, enos, ET-1 and HO-1 gene expression, I kappa B, c-Jun and Erk protein phosphorylation in HUVEC and induced HUVEC death (apoptosis and necrosis) compared with the control. However, combination of H₂O₂ with polyhb or polyhb-SOD-CAT could reduce GSH dropping, gene expression and protein phosphorylation. Polyhb-SOD-CAT had stronger ability to inhibit the HUVEC death induced by H₂O₂ than polyhb.

Conclusion: polyhb-SOD-CAT can reduce the H₂O₂-induced oxidative damage to vascular endothelial cells by maintaining the intracellular GSH level and inhibiting the activation of NF-kappa B and MAPK cell signal transduction pathway.

HEMOGLOBIN-BASED OXYGEN CARRIER PROTECTS ISOLATED RAT HEART FROM ISCHEMIA/REPERFUSION INJURY BY ATTENUATION OF MYOCARDIAL APOPTOSIS AND RESTORATION OF THE NITROSO-REDOX BALANCE

(Poster Selected for Oral Presentation)

Li, T.¹, Liu, J.¹, Yang, Q.², Zhang, P.¹, Yang, C.-M.³

¹Laboratory of Anesthesiology and Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, China

²Department of Medicinal Chemistry, School of Pharmacy, Chengdu Medical College, Chengdu, China

³Institute of Tianjin Union Biotechnology Development, Tianjin, China

E-mail: [email protected]

Ischemia/reperfusion (I/R) injury mainly caused by oxidative stress plays a major role in cardiac damage. Extent of the I/R injury is also an important factor that determines the function of the transplanted heart. This study first examined whether hemoglobin-based oxygen carriers (hbocs) could protect an isolated rat heart from I/R injury and elucidated the underlying mechanism. Using the Langendorff model, isolated Sprague-Dawley rat hearts were arrested and stored at 4°C for 8 hours, then reperfused for 2 hours. Compared with St. Thomas’ solution (STS) and rat self blood in STS, polymerized placenta hemoglobin (polyphb) in STS greatly improved heart contraction and decreased infarction size. The extent of myocardial apoptosis was also significantly decreased, which was related to reduced inos-derived nitric oxide (NO) production, increased protein ratio of Bcl-2/Bax, and reduced caspase-3 activity and cleavage level. Furthermore, polyphb in STS did not increase malondialdehyde (MDA), peroxynitrite (ONOO¯) and mitochondrial hydrogen peroxide (H₂O₂) formation, but greatly elevated superoxide dismutase (SOD) activity and preserved mitochondrial ATP synthesis, which served to maintain redox homeostasis in I/R heart. In conclusion, our results demonstrated that hbocs protected isolated heart from I/R injury and this protection was associated with attenuation of NO-mediated myocardial apoptosis and restoration of nitroso-redox balance.

SESSION V: BIOCHEMICAL APPROACHES TO MITIGATE HEME-MEDIATED TOXICITY OF HBOCS

Chairman: Alayash, A.

Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA

E-mail: [email protected]

Session Keynotes

Chemically modified or recombinant hemoglobin (Hb)-based oxygen carriers (hbocs) have been developed as volume expanding oxygen therapeutics “blood substitutes” for use in a variety of clinical settings. There is a growing realization that oxidative and nitrosative reactions of acellular Hb, due to the rich free radical chemistry of its heme, can limit the effectiveness and compromise its safety. Additionally, Hb can react with important signaling molecules, like nitric oxide (NO), a vasodilator resulting in untoward hemodynamic imbalances. This session will focus on recent biochemical innovations designed to suppress Hb oxidative and nitrosative side reactions, thus maintaining its oxygen off-loading capabilities. In addition, endogenous antioxidative clearance mechanisms such the role of haptoglobin will be discussed. Animal studies designed specifically to monitor Hb oxidative changes as well as NO intervention strategies will also be discussed.

NO DIOXYGENATION, AUTOOXIDATION, HEMIN LOSS, AND RE-ENGINEERING THE DISTAL POCKET OF RECOMBINANT HB-BASED OXYGEN CARRIERS

Olson, J. S.

Department of Biochemistry & Cell Biology and W. M. Keck Center for Computational Biology, Rice University, Houston, TX, USA

E-mail: [email protected]

The key requirements for a Hb-based oxygen carrier (rhboc) are: (a) moderate O₂ affinity and large dissociation rate constants for efficient transport in capillaries; (b) significantly reduced rates of NO scavenging to prevent hypertensive side-effects; (c) resistance to auto- and chemically-induced oxidation to inhibit oxidative stress; (d) low rates of hemin dissociation to increase shelf-life and decrease in vivo toxicity; and (e) enhanced apoglobin stability for increased expression yields in bacteria. We have examined all five processes, determined the underlying biochemical mechanisms, and then used these principles to develop strategies to engineer the active sites of the α and β subunits to optimize all five properties. O₂ affinity can be adjusted by both allosteric and distal pocket mutations to enhance iron reactivity by alteration of the R to T transition and to adjust the extent of steric hindrance and electrostatic interaction with the bound ligand, respectively. The rate of NO dioxygenation can be decreased by inhibiting the rate of ligand entry and capture using aromatic amino acid substitutions in the interior of the distal pocket. Autooxidation can be slowed by mutations that exclude water from the active site and electrostatically prevent protonation of bound O₂. Hemin loss can be reduced by increasing favorable electrostatic interactions between the heme propionates and the surrounding basic amino acids and preventing protein unfolding and/or dissociation into dimers and monomers. The stability of the globin itself can be increased markedly by mutations based on comparisons of human hba with human hbf and Hbs from deep diving mammals. These approaches are leading to the design of specific, third generation, non-vasoactive rhboc prototypes with increased resistance to oxidation and denaturation, enhanced expression yields, and hopefully reduced production costs for commercial development.

Supported by U.S. NIH Grants RO1 HL047020 and R01 GM035649 and Grant C-0612 from the Robert A Welch Foundation, Houston, Texas, USA.

MODIFYING ELECTRON TRANSFER PATHWAYS IN HEMOGLOBIN

Cooper, C. E., Reeder, B. J., Wilson, M. T.

Department of Biological Sciences, University of Essex, Colchester, England

E-mail: [email protected]

The toxicity of the current generation of Hemoglobin Based Oxygen Carriers (HBOC) has been attributed to three different, not necessarily exclusive, mechanisms. The first claims that the presence of a low oxygen affinity protein in the plasma triggers a vasoconstrictive response; the second that scavenging by oxyhemoglobin of endogenous nitric oxide perturbs intrinsic biochemical and/or physiological signalling pathways. The third mechanism focuses on the “intrinsic toxicity” of haem itself via its oxidation by peroxides:

R + Fe³⁺ + H₂O₂ ⇒ R˙⁺ + Fe⁴⁺= O²- + H₂O

globin + ferric + peroxide ↠ globin + ferryl + water

heme radical haem

Both the protein-bound radical (R˙⁺) and the ferryl iron (Fe⁴⁺ = O²–) are highly oxidising species and can cause tissue damage, for example by initiating lipid peroxidation reactions. We will show that the reactivity of these species is a function of ph and the presence of redox active tyrosine residues. Modifications of these tyrosine residues can therefore have important functional consequences, with possible significance for the next generation of HBOC.

ANIMAL SPECIES AND BIOMARKERS TO EVALUATE EXTRA-CELLULAR HEMOGLOBIN INDUCED OXIDATIVE AND NITROSATIVE PROCESSES

Buehler, P. W., D’Agnillo, F., Butt, O. I., Abraham, B., Alayash, A.

Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, FDA (Food and Drug Administration), Bethesda, MD, USA

E-mail: [email protected]

The toxicity of extra-cellular hemoglobin (Hb) in hemolytic diseases and following hemoglobin based oxygen carrier (HBOC) administration has predominantly focused on undesirable hemodynamic toxicity. Recently, nitric oxide (NO) therapeutic modalities have been explored to “detoxify” extra-cellular Hb/hbocs. However, the impact of extra-cellular Hb/hbocs on oxidative and nitrosative processes in vivo has not been fully established. The in vivo evaluation of these processes in animal species with similar plasma/cellular antioxidant status to humans may provide insight into Hb/HBOC heme iron oxidation, protein oxidative/nitrosative modification, protein stability, and tissue cellular alterations. Evaluation of HBOC plasma oxidative processes in ascorbate (rat) and non-ascorbate (guinea pig) producing species suggests different kinetic profiles of ferric Hb formation in guinea pig compared to rat following transfusion with polymerized bovine Hb. Within the circulation of guinea pigs exposure to plasma ferric Hb was 5-fold greater than in rats. Mass spectrometry data supports increased Hb protein destabilization consistent with plasma Hb oxidation in the guinea pig. Specific sites of oxidation have been identified and may serve as useful and sensitive indices of Hb/HBOC oxidative susceptibility in vivo. Hematoxylin/eosin histopathology revealed minor changes within the heart and kidney, but significant changes in vascular tissue. Evaluation of potential extra-cellular Hb “detoxification” modalities, such as sodium nitrite (nano₂), intended to reduce adverse Hb/HBOC hemodynamic effects enhanced the oxidation of extra-cellular as well as red blood cell Hb at doses required to reduce blood pressure. Moreover, iron deposition and oxidative markers such as 4-hydroxynonenal (4-HNE)-modified protein (marker of lipid peroxidation) were elevated in tissues. These data suggest that in vivo oxidative and nitrosative processes are important factors to consider following Hb/HBOC exposure. However, there is still a need to determine if acute findings are relevant in disease state models and ultimately if biomarkers identified can be applied to pre-clinical safety assessments.

*The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

ENDOGENOUS ANTIOXIDATIVE CLEARANCE PATHWAYS, NOVEL AND NEW CONCEPTS

Schaer, D. J.

Division of Internal Medicine, University Hospital, Zurich, Switzerland

E-mail: [email protected]

Endogenous Hb scavenger systems have evolved to counter systemic Hb toxicity. The primary Hb binding proteins involve the haptoglobins (Hp) and specific scavenger receptor cystein rich domain (SRCR) proteins. The default pathway for Hb clearance in mammalian species involves sequestration of Hb within the high affinity Hb-Hp complex that is subsequently removed from the extracellular environment by the macrophage Hb scavenger receptor CD163. While much research has focused on biochemical properties of Hp and, more recently, on CD163, the true in vivo protective potential of the Hb scavenger system remained largely unknown. Here, three examples will be presented to show how supplementation or modulation of Hb scavenger pathways could alleviate side-effects of extracellular free Hb. 1) Hp protects Hb from oxidative damage such as globin cross-linking, polymerization and amino-acid oxidation when exposed to physiologic oxidants. By preserving structural integrity within the Hb-Hp complex, Hp preserves functionality of the CD163 Hb clearance pathway even under strong oxidative conditions. 2) Intravascular compartmentalization of Hb by Hp mitigates hypertensive responses and oxidative tissue damage associated with large Hb exposures in vivo. 3) Expression and activity of the Hb scavenger receptor CD163 can be pharmacologically modulated in a way to increase Hb clearance and heme detoxification by peripheral blood monocytes. Therefore, supplementation and / or modulation of endogenous Hb scavenger pathways may provide a novel strategy to counter Hb toxicity associated with hemolytic diseases or HBOC administration.

DESIGNING OXYGEN CARRYING BLOOD SUBSTITUTES: WHAT CAN BE LEARNED FROM PLASMA EXPANDERS

Cabrales, P., Intaglietta, M., Tsai, A. G.

Microhemodynamics Laboratory; Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

E-mail: [email protected]

The development of oxygen-carrying plasma expanders is significantly complicated by the ongoing discovery of adverse side-effects. An effective development path to circumvent these problems is to apply the new understanding of plasma expansion physiology to producing a plasma expander that takes us to the limit of what can be attained in restoring tissue perfusion and oxygenation without actually increasing oxygen carrying capacity. Thus the aims should be to restore blood volume, recover cardiovascular and microvascular function and, as the final goal, to provide the necessary oxygen carrying capacity. However, the recovery or maintenance of cardiovascular and microvascular function has its limitations and eventually augmenting oxygen carrying capacity with an oxygen carrier will be needed to prevent anoxic tissue damage. Current designs of oxygen carrying blood substitutes may provide immediate benefits by augmenting oxygen carrying capacity but in the long term will lead to tissue injury, largely due to inability to optimize the recovery of local perfusion. Understanding the underlying changes to blood rheology, which occurs after hemodilution, and the subsequent physiological adjustments will clarify when an oxygen-carrying plasma expander would be beneficial and the potential exposure to toxicity acceptable.

Research supported in part by NIH R01 062354 and HL064395.

THE IMPACT OF RED BLOOD CELL EXCHANGE TRANSFUSION ON CYTOKINE PLASMA LEVELS AND GENE EXPRESSION IN SICKLE CELL DISEASE PATIENTS

Van Hilten, J. A.¹, Kerkhoffs, J.-L.²

¹Dept. of Research & Development, Sanquin Blood Bank SW Region, Leiden, The Netherlands

²Dept. of Hematology, Hemoglobinopathy Unit, Haga Teaching Hospital, The Hague, The Netherlands

E-mail: [email protected]

Introduction: Red cell exchange transfusions (rbcx) are being used to treat and prevent acute as well as chronic complications due to hemolysis and vaso-occlusion in Sickle Cell Disease (SCD) patients. In a preliminary study, we assessed the change in cytokine levels in plasma and gene expression (GE) in whole blood after rbcx.

Methods: Blood samples were collected in 14 SCD patients, as well as 18 healthy controls after informed consent. In all first samples and seven post rbcx samples of the 14 patients, the following cytokines were measured: IL-1β, TNF-α, IL-10 including scd-40L by either ELISA or flowcytometric methods. From six SCD patients blood RNA was stabilized and purified before and after rbcx procedure using 5 units. The quality of RNA on each sample was assessed prior to genome wide screening and analysis of biological correlations.

Results: As compared to the healthy controls, the SCD patients showed non-significant increased plasma levels of IL-1β, TNF-α, IL-10. However scd40l was significant higher in SCD patients (10 ± 6 ng/ml vs 2 ± 0.9 ng/ml; p < 0.01). Rbcx reduced only scd40l with a mean decrease of 36.6% (95% CI 3.8 – 69.4%, p = 0.04). The 12 RNA samples revealed two clusters of normalized GE, which are correlated with gender. A good correlation between GE before and after rbcx in each patient was assessed. Paired analysis looking for > two-fold change showed upregulation of 172 genes, including HLA-DP/Q/R-, HLA-class II–invariant CD74-, TNF-family member lymphotoxin β- and IL-10 genes. However no IL-1 β-, TNF-α-, CD40L- cytokine GE changes were observed. Continuing GE-analyses will reveal further results.

Conclusion: The exchange of Hb-S containing red cells with Hb-A containing red cells using rbcx resulted in a reduced release of an activated platelet cytokine in plasma and an upregulation of pro- and anti-inflammatory cytokine genes.

DIFFERENTIAL INDUCTION OF RENAL HEME OXYGENASE AND FERRITIN IN ASCORBATE AND NON-ASCORBATE-PRODUCING SPECIES TRANSFUSED WITH POLYMERIZED BOVINE HEMOGLOBIN

(Poster Selected for Oral Presentation)

Butt, O. I., Buehler, P. W., Alayash, A., D’Agnillo, F.

Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, FDA (Food and Drug Administration), Bethesda, MD, USA

E-mail: [email protected]

Heme catabolism and iron sequestration systems likely play an important role in mediating the physiological and pathophysiological responses to hemoglobin-based oxygen therapeutics. We previously reported that polymerized bovine hemoglobin (hbg) oxidized more readily in the circulation of guinea pigs, a non-ascorbate (AA)-producing species with a similar plasma and tissue antioxidant profile to humans, than in rats, an AA-producing species. We hypothesized that the increased oxidative susceptibility of hbg as well as species differences in antioxidant defenses could differentially modulate heme catabolic and iron sequestration mechanisms. Using a 50% exchange transfusion (ET) model with hbg, we examined the induction of renal heme oxygenase (HO), H- and L-ferritin expression, non-heme ferrous and ferric iron deposition, and renal AA content in rats and guinea pigs. Hemoglobinuria was evident in both species but hbg was considerably more oxidized in the urine of guinea pigs than in rats. Hbg enhanced HO activity in both species but the induction appeared greater and more sustained in guinea pigs. Rats showed increased H- and L-ferritin expression as early as 4 h post-ET that persisted through 24 h and declined at 72 h. Conversely, guinea pigs failed to show H-ferritin induction and displayed a comparatively delayed increase L-ferritin expression (>24 h). Non-heme iron deposition was greater in rat kidneys consistent with the ferritin response. Notably, both ferric and ferrous iron deposits were detectable in rats while only ferric iron was observed in guinea pigs. Renal AA content increased transiently in rats and paralleled the changes in ferritin and iron deposition while AA levels were unchanged in guinea pigs. These findings highlight potentially important species differences in heme catabolism and iron sequestration processes that may have implications for understanding the human response to hemoglobin therapeutics, especially under conditions of depleted antioxidant status.

*The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

SESSION VI: TOWARDS CELLULAR TYPE OXYGEN CARRIERS

Chairman: Kobayashi, K.

School of Medicine, Keio University, Tokyo, Japan

E-mail: [email protected]

Session Keynotes

Establishment of blood transfusion is one of the major contributions in the field of medicine in the 20th century. However, we are still facing difficult tasks to make transfusion medicine safe and reliable. Looking at newly emerged blood borne infectious diseases, shortage of donor and limitation of storage time, developing artificial blood components will promise the progress of medical science in the 21st century.

Clinical trials of various kinds of cell-free (acellular type) hbocs are underway, especially in North America. Some were suspended and some are now in the final stage. However, the paper published in JAMA in 2008 showed that meta-analysis of cell-free hbocs clinical data showed increased risks of myocardial infarction and death. The US FDA held a public workshop entitled “Safety of Hemoglobin-Based Oxygen Carriers (hbocs)” in late April 2008. Recent results of clinical trials, safety issues of clinical and preclinical studies, remedies to the side-effects such as vasoactivity, and future directions were discussed. However, there was no discussion of cellular-type hbocs.

In Japan, research and development of Hb-vesicle (hbv) and synthetic hemes have been performed by close collaboration of Waseda and Keio for more than 20 years. Hbv is a cellular HBOC and it is expected that toxic effects of cell-free hbocs would be shielded by the cellular structure. Even though we have not come to a clinical stage yet, the preclinical results of our safety and efficacy evaluations of hbv make us confident in advancing to further development of hbv.

This session will focus on the design, function, and potential clinical application of cellular-type hbocs of a new category using nano-biotechnology, which is expected to shield adverse events of cell-free Hbs. Moreover, intrinsic issues of these new materials will be discussed for eventual realization. Presentations from several countries are included.

It has to be emphasized that hbocs are necessary for unmet medical needs, a situation where safe RBC transfusion is not available. Whether cell-free, or cellular hbocs, I hope an oxygen carrier to save lives and alleviate human suffering will appear in the foreseeable future.

STRATEGIES FOR NEW GENERATIONS OF HBOCS

Chang, T. M. S.

Director, Artificial Cells & Organs Research Centre, Departments of Physiology, Medicine & Biomedical Engineering, Faculty of Medicine, Mcgill University, Montreal, Quebec, Canada

E-mail: [email protected]

A recent paper in JAMA shows a major misunderstanding in the historical development and strategies of hbocs. Indeed, the earlier hbocs did have problems but there has been extensive improvement in the safety and efficacy of some first generation HBOC. Thus one cannot combine the historical result of all the hbocs together to make conclusions regarding safety and effectiveness. Surprisingly, the author of the JAMA paper also did not mention that stored donor blood has perhaps more side-effects than the best type of HBOC. Furthermore, there seems to be a misconception that any one HBOC should be safe and effective for all clinical conditions tested. This is despite the fact that, in medical therapy, there is no one therapy that is a “cure all” for all clinical conditions. In order to avoid these types of misunderstandings, we need to define our strategies for hbocs. There are many possible strategies that will be discussed by other groups. In this presentation, we shall discuss our own strategy.

The basic principles of polyhemoglobin (polyhb) and conjugated Hb (Chang, 1964, Science; Chang 1971, Bioph Biochim Com; Chang, 1972, Monograph Artificial Cells) have been independently developed by the industries into polyhb and PEG-Hb, the latter a form of conjugated Hb. Polyhb is useful as a volume expander with good oxygen carrying capacity. PEG-Hb has been suggested to enhance microcirculation, and is a volume expander with low oxygen carrying capacity. In order to show safety and efficacy, they should be tested and used in conditions with no oxidative stress or potentials for ischemia-reperfusion – for example, in stable elective surgery patients with no coronary insufficiency or other ischemic and oxidative stress.

New generations of HBOC include the nanobiotechnological assembly of Hb, catalase (CAT) and superoxide dismutase (SOD) into a soluble polyhb-SOD-CAT complex. (D’Agnillo & Chang, 1998, Nature Biot; Chang, 2008, Bioch Biophyacta). This acts as a volume expander, oxygen carrier, and antioxidant for those conditions with oxidative stress and potentials for ischemia-reperfusion injuries: for instance in severe hemorrhagic shock, coronary insufficiency, stroke, cardioplegia, etc. However, purified SOD and CAT are very costly. To solve this problem we recently extracted hemoglobin, SOD and CAT from the same rbc to prepare polyhb-SOD-CAT (Gu & Chang, 2009, ACBSB 37:69-77). This polyhb-SOD-CAT containing 10800U SOD and 23000U CAT per gram of Hb did not have full protection from oxidants. Addition of more enzymes extracted this way resulted in polyhb-SOD-CAT containing 15060U SOD and 330000U CAT per gram of Hb that has full protection from oxidants. We show that both SOD and CAT have to be included and the correct ratio of SOD and CAT is also very important. In this regard, one has to question the validity of using either SOD or CAT alone to compare with the antioxidative effectiveness of polynitroxyl albumin (Kaul et al., 2005) – furthermore, polynitroxyl albumin is not an oxygen carrier. There is also a PEG-polynitroxyl-Hb that is an antioxidant and plasma expander with minimal oxygen carrying capacity.

The complete artificial rbc (Chang, 1964, Science) has now been developed into nanodimension artificial rbc (nanorbc) with either PEG-lipid membrane or PEG-polylactide membrane. Although the bilayer lipid membrane of PEG-lipid vesicles is similar to that of rbc, rbc is 7 microns in diameter whereas lipid vesicles are around 100-150 nanometers. The smaller the diameter the larger the bilayer lipid membrane to volume relationship, thus the lipid content of 500ml of PEG-lipid vesicles is many times higher than 500 ml of rbc. Thus it should be used in conditions where RES function is not critical. In PEG-polylactide nanorbc we are using a biodegradable membrane that is more costly than lipid. On the other hand, our recent studies on histology and function of livers and spleens in rats after 33% toploading of PEG-PLA-nanorbc show no residual of the membrane in the livers or spleen and no disturbances of their functions (Liu & Chang, 2008, ACBSB 36: 513–524); the same was observed on kidney function and renal histology in rats (Liu & Chang, 2008, 36: 421–430).

PHYSICOCHEMICAL CHARACTERISTICS OF HB-VESICLES AS RED BLOOD CELL SUBSTITUTES

Sakai, H.¹, Sou, K.¹, Horinouchi, H.², Kobayashi, K.², Tsuchida, E.¹

¹Research Institute for Science and Engineering, Waseda University, Tokyo

²Department of Surgery, School of Medicine, Keio University, Tokyo

E-mail: [email protected]

Biconcave rbcs deform to a parachute-like configuration to flow through a narrower capillary. This profile is believed to be effective to stir the intracellular viscous Hb solution to facilitate the gas exchange. On the other hand, physicochemical analyses have revealed that O₂ unloading of Hb is significantly retarded by compartmentalization in RBC. Why has nature selected such an inefficient cellular structure for gas transport? Interestingly, some of the answers to this question have been revised by the research of blood substitutes. They are: (i) retardation and targeting of O₂ unloading at microcirculation to avoid autoregulatory vasoconstriction; (ii) reduction of a high colloidal osmotic pressure, COP, of an Hb solution to zero, to increase blood Hb concentration; (iii) rheology control of blood, a rbcs dispersion, to a non-Newtonian viscous fluid; (iv) prevention of extravasation or excretion through renal glomeruli; (v) preservation of the chemical environment in cells, such as the concentrations of electrolytes and enzymes; (vi) prevention of direct contact of toxic Hbs and endothelial lying; and (vii) modulation of reactions with NO as an endothelium derived relaxation factor, EDRF. These observations reassure the importance of the cellular structure of rbcs to design Hb-based oxygen carriers.

Hb-vesicles (hbv) are artificial oxygen carriers encapsulating concentrated Hb solution (35 g/dl) with a phospholipid bilayer membrane. Concentration of the hbv suspension is extremely high ([Hb] = 10 g/dl, [lipids] = 6 g/dl, volume fraction, ca. 40 vol%) and it has an oxygen carrying capacity that is comparable to that of blood. Hbv is much smaller than RBC (250 vs. 8000 nm), but it recreates the functions of rbcs; (i) the rate of O₂ unloading is slower than Hb solution ; (ii) COP is zero at [Hb] = 10 g/dl, and it has to be co-injected with or suspended in a plasma substitute such as albumin or HES ; (iii) the resulting viscosity of a hbv suspension is adjustable to that of blood; (iv) hbv is finally captured by RES and the components are degraded and excreted. The hbv particle of itself is not eliminated through glomeruli; (v) co-encapsulation of PLP as an allosteric effector to regulate oxygen affinity; (vi) no hemolysis occurs during circulation and the lipid bilayer membrane prevents direct contact of Hb and vasculature; (vii) reaction of NO is retarded to some extent by an intracellular diffusion barrier, and hbv does not induce vasoconstriction.

Hbv has limitations such as the shorter functional half-life (a few days) after intravenous injection. On the other hand, the advantages of hbv are that it is pathogen-free and blood-type-antigen-free; moreover, it can withstand long-term storage of a few years, none of which can be achieved by the RBC transfusion systems. An appropriate clinical indication of hbv will be selected through the ongoing preclinical studies of safety and efficacy by domestic and international collaborations.

This work is supported in part by MHLW and JSPS.

RESUSCITATION WITH HEMOGLOBIN- VESICLE CAN IMPROVE SURVIVAL IN UNCONTROLLED HEMORRHAGE MODEL IN RATS: HEMORRHAGE FROM LARGE VESSEL MODEL

Horinouchi, H.¹, Seishi, Y.¹, Kobayashi, K.¹, Izumi, Y.¹, Kohno, M.¹, Sakai, H.², Tsuchida, E.²

¹Department of Surgery, Keio University School of Medicine

²Advanced Research Institute for Science and Engineering, Waseda University

E-mail: [email protected]

Hemostasis and fluid resuscitation is the rationale of treatment of hemorrhagic shock. On site of injury, however, uncontrolled hemorrhage is often encountered if the bleeding is located at the center of the torso. Standardized resuscitation method has not been established in such cases and swift transportation and surgical hemostasis is essential. Several papers recommended controlled fluid resuscitation improved survival. We made a model of uncontrolled hemorrhage by penetrating the abdominal aorta of the rat and resuscitating with saline, 5% albumin, autologous blood, hbv in 5% albumin solution. Non- resuscitation group was also planned. After shock was introduced, fluid resuscitation was administered (three times volume of saline or isovolume of autologous blood, 5% albumin, hbv/5% albumin) and circulation was observed up to two hours.

Results: Blood loss was 8.0+/–0.9ml in non- resuscitation group, 14.3+/–2.9ml in saline group, 12.6+/–4.1ml in autologous group, 11.8+/–4.0ml in 5% albumin group, and 9.8+/–3.7ml in hbv/5% albumin group. In saline group, despite large volume resuscitation, it failed to maintain blood pressure. In hbv group and in autologous blood group improvement of survival was recognized. Serum lactate significantly increased after 30 minutes of resuscitation in each group but in autologous blood group and in hbv/5% albumin group increase was suppressed compared to the other groups. Oxygen carrying fluid improved survival at 2 hours compared to saline group and albumin group.

Discussion: Large volume of crystalloid solution couldn't improve survival. Autologous blood improved survival and suppressed the increase of serum lactate. Hbv/5% albumin improved survival, suppressed the blood loss and increased serum lactate. Hbv may potentially improve oxygen metabolism in uncontrolled hemorrhage and improve survival.

USING HB-VESICLES IMPROVED OXYGENATION OF CRITICALLY ISCHEMIC SKIN TISSUES

Erni, D.

Department of Plastic Surgery, Inselspital, University of Bern, Switzerland

E-mail: [email protected]

The initial purpose for developing artificial oxygen carriers was to replace blood transfusions in order to avoid their adverse effects, such as immunologic reactions, transmission of infectious diseases, limited availability and restricted storage conditions. With the advent of new generations of artifical oxygen carriers, a shift of paradigm evolved that considers the artificial oxygen carriers as oxygen therapeutics re-distributing oxygen delivery in the favor of tissues in need. This function may find a particular application in tissues rendered hypoxic due to arterial occlusive diseases. In collaboration with the Research Institute of Science and Engineering (RISE) of the Waseda University in Tokyo, we tested the efficacy of liposome encapsulated hemoglobin vesicles (hbv) and their potential in treating critical ischemia in various acute and chronic rodent flap models. Hbvs were shown to improve microcirculatory blood flow, capillary perfusion, tissue oxygenation and oxidative energy metabolism, inflammation and tissue survival, which allowed us to outline the optimal design of hemoglobin vesicles (hbvs) given for the above intention: the hbv should be of a large diameter, and oxygen affinity, colloid osmotic pressure, and viscosity of the hbv solution should be high.

HB COATED NANOPARTICLES

Chauvierre, C., Leclerc, L., Marden, M. C.

Inserm U779, University of Paris XI, France

E-mail: [email protected]

Nanoparticles containing hemoglobin may be an alternative to free Hb or red blood cells. Being intermediate in size, they offer the possibility of microcirculation without excessive viscosity and little probability of escape from the blood vessels. We are developing such a delivery system based on an original redox radical polymerization mechanism of producing core-shell nanoparticles. Different types of cyanoacrylate that form the core and the surface polysaccharide were tested. By variation of these basic building blocks, certain properties of the nanoparticles can be easily modulated.

The nanoparticle size can be varied from 100 nm to 300 nm; the particles are stable at high temperatures, pressures, and after storage for over 3 years. The nanoparticle surface charge varies from −40 to +40 mv depending on the polysaccharide nature used. Hb binding is correlated to the negative value of the Zeta potential. Expecting the Hb to bind within the polysaccharide shell, the composition was modified to increase the mass fraction of this component from about 10 to 50%. An equivalent mass of Hb to polysaccharide was bound in the best cases. Flash photolysis studies of Hb loaded onto (poly-isobutyl- cyanoacrylate / dextran) nanoparticles indicate a normal allosteric behavior was maintained. After Hb binding, the nanoparticles do not show an activation of complement or cytotoxicity. Additional components and linkers are being considered to increase the amount of Hb loaded and to maintain the Hb in an active state.

KINETICS OF CARRYING/RELEASING OXYGEN OF RED BLOOD CELL AND BLOOD SUBSTITUTES IN STORAGE

(Poster Selected for Oral Presentation)

Xiang, W., Li, Y.-J., Li, Y., Wei, G., Ke, L.

Bioengineering College of Chongqing University, Chongqing, China

E-mail: [email protected]

This research analyzes the oxygen-carrying capacity changes of red blood cells in the preservation process from both thermodynamics and kinetics; investigates the effects of saved time and pre-processing operation on erythrocyte's oxygen-carrying function and blood properties.

Objective: To study the dynamic process of red blood cells carrying/releasing oxygen, and construct an evaluation system for erythrocyte oxygen-carrying function. The effects of saved time and pre-processing operation on erythrocyte's oxygen-carrying function and blood properties were analyzed so as to provide a scientific basis and guidance for blood preservation and scientific blood transfusion.

Methods: Observing the changes of oxygen saturation (Sat) and the oxygen partial pressure (PO₂) with time in the red blood cell oxygen dissociation process, and analyzing data by curve fitting; detecting the oxygen carrying function and physical/chemical parameters in different conservation phase of whole blood and suspension RBC.

Results: The curve of oxygen saturation over time (Sat-t) has the typical “S” curve characteristic. Building a more effective oxygen-carrying evaluation system by leading in new oxygen dissociation kinetic parameter T*50 and effective oxygen-carrying volume Q. The effective oxygen-carrying volume decreased with the preservation time, after 1 week, a decrease of 27.5% and 19.2%, respectively. At the end of preservation, the effective oxygen-carrying volume of whole blood was only 39 percent of “new blood” and suspended RBC only retained 51% effective oxygen-carrying volume also. P50 values decreased in the preservation period, at the beginning declining for the first three weeks, but with a significant change in the last week. PH, Na⁺ concentration was declined, K⁺ concentration gradually increased; free Hb increased and reached the initial value of 5∼10 times at the end of preservation. Glucose gradually reduced with the extension of storage time, after saving 35 days, the glucose was close to 0; lactate dehydrogenase increased rapidly over time, and near 3-fold at last. At two weeks it reduced sharply, and then did not change significantly. T*50 decreased in the shelf life. T*50 of suspension RBC reduced sharply at the beginning of two weeks, and then did not change significantly. <whole blood T*50 showed a slow decrease>

Conclusion: Oxygen-carrying function can be described and evaluated systematically by considering thermodynamic parameter P50, oxygen dissociation kinetic parameter T*50, and the effective oxygen-carrying volume Q. Blood storage time and pre-processing operations have an important impact on erythrocyte's oxygen-carrying capacity and blood physical and chemical properties; filtration, centrifugation, and other operations will result in a one-time injury in red blood cells.

PHARMACOKINETIC PROFILES OF HEMOGLOBIN-VESICLES AS AN ARTIFICIAL OXYGEN CARRIER

(Poster Selected for Oral Presentation)

Taguchi, K.¹, Maruyama, T.¹, Watanabe, H.¹, Sakai, H.², Horinouchi, H.³, Kobayashi, K.³, Tsuchida, E.², Otagiri, M.^1,4

¹Graduate School of Pharmaceutical Sciences, Kumamoto University

²Research Institute for Science and Engineering, Waseda University

³Department of Surgery, School of Medicine, Keio University

⁴Faculty of Pharmaceutical Sciences, Sojo University

E-mail: [email protected]

Hemoglobin-vesicle (hbv) is an artificial oxygen carrier that encapsulates a concentrated Hb solution in lipid vesicles (liposome) and has considerable promise for use in a clinical setting. Despite the large body of pharmacological evidence for hbv, little is known concerning its pharmacokinetic properties. In addition, it is well known that pharmacokinetic properties of liposome change in response to pharmacological conditions. Therefore, in this study, the pharmacokinetic properties of hbv were investigated in mice and rats. Firstly, the pharmacokinetics of hbv components (internal Hb and liposomal lipid) were investigated using ¹²⁵I-hbv and ³H-hbv (internal Hb and liposomal cholesterol were radiolabeled with iodine-125 and tritium-3, respectively). As a result, it was found that hbv were mainly distributed to the liver and spleen, and degraded by mononuclear phagocyte system after circulating in the form of intact hbv. Internal Hb was excreted into the urine and cholesterol into feces via biliary excretion. Second, we examined the pharmacokinetics of ¹²⁵I-hbv during hemorrhagic shock (HS), which was induced by withdrawal of 40% of total blood volume. In the case of rat-induced HS, the half-life of hbv was shorter in HS rats, but it returned to non-HS levels after the second ¹²⁵I-hbv injection. Excretion into urine, the major elimination pathway, did not differ between non-HS and HS rats. Furthermore, the half-life of hbv in humans was estimated to be approximately 3–4 days using an allometric equation. These results suggest that hbv has a reasonable blood retention, metabolic and excretion performance, and could be used as an oxygen carrier.

BACTERIZATION OF TWO STABLE OCTAMERIC RECOMBINANT HEMOGLOBINS αN78C AND βG83C

(Poster Selected for Oral Presentation)

Brillet, T.¹, Vasseur, C.¹, Domingues, E.¹, Ho, C.², Marden, M. C.¹, Baudin-Creuza, V.¹

¹Inserm U779, University of Paris XI, France

²Department of Biological Science, Carnegie Mellon University

E-mail: [email protected]

Studies have shown that adult hemoglobin (hba) tetramer self-dissociates into two dimers in blood, and these dimers are not capable of delivering oxygen to tissues because of their high affinity for oxygen. Several cross-linked hemoglobins have been developed for preventing this dimerization. However, some studies have shown that these Hb tetramers are too small to prevent extravasation, and may disrupt the NO signaling pathway that leads to serious vasomotricity perturbation. Increasing the size of the transporter by polymerization of multiple tetramers is the next logical step for designing a stabilized tetramer in blood. We have shown that an octameric Hb can be produced by engineering the β chain with the G83C mutation (β octamer). This mutation led to the formation of disulfide bounds with another tetramer to form an octameric Hb. We have produced another octamer by making the analogous mutation N78C on the α chain (α octamer).

In this study we compare the properties of the α and the β octamers. Both oxygen affinities and kinetics of recombination of CO after flash photolysis show that these octamers have a behavior comparable with hba. The tetramers within the octamer maintain a good allosteric behavior and respond to allosteric effectors such as IHP. However, the β octamer has a higher affinity for oxygen. We also follow the stability of these octamers in fresh human plasma under air and their possible interaction with haptoglobin, which specifically interacts with the αβ dimers. Both octamers are stable in plasma and do not interact with haptoglobin. Thermal denaturation experiments show that both octamers have a Tm (temperature for 50% of unfolding) of 79°C, 6°C more that hba, probably because of the stabilization induced by the disulfide bonds. The enhanced thermal stability and lack of dimerisation indicate that there are probably two disulfide bonds per octamer.

ENGINEERING POLYMERSOME ENCAPSULATED HEMOGLOBINS

Palmer, A. F.

Department of Chemical and Biomolecular Engineering, The Ohio State University, USA

E-mail: [email protected]

The development of safe and effective artificial blood substitutes is of paramount importance given the limited supply, risk of infections and allergic reactions, and expensive collection and storage costs of donor blood. One design strategy in artificial blood substitute development involves the encapsulation of hemoglobin (Hb) inside the aqueous core of liposomes. In order to improve the intravascular persistence and biocompatibility of liposome encapsulated Hb (LEH), lehs have been surface-conjugated with FDA approved poly(ethylene glycol) (PEG), a biocompatible polymer. However, both the intravascular circulation and biocompatibility of PEG-lehs are restricted by the limited PEG surface coverage that can be achieved (10 mol% of total bilayer lipids), the maximum length of PEG chains that can be incorporated (5000 Da), and the universal membrane thickness of liposomes (3–4 nm). We propose to employ polymersomes or self-assembled vesicles made up of amphiphilic diblock copolymers containing PEG, as the hydrophilic block in order to encapsulate Hb. In this design strategy, 100% pegylation can be achieved, PEG chains with molecular weights ranging from 1.2 to 30 kda can be incorporated, and the membrane thickness can be easily engineered from 3 to 40 nm by simply varying the molecular weight of the hydrophobic block. We hypothesize that the circulatory persistence, hemodynamic properties, biocompatibility and dispersion state of this novel blood substitute will be superior compared to PEG-lehs, while still possessing good oxygen carrying properties.

HB CONTAINING POLYMERIC PARTICLES

Dessy, A.¹, Piras, A. M.¹, Chiellini, F.¹, Chiellini, E.¹, Levantino, M.², Schirò, G.², Cupane, A. ²

¹Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Application (biolab) udr–INSTM Consortium, Reference Center, University of Pisa, Pisa, Italy

²Department of Physical and Astronomical Sciences, University di Palermo, Palermo, Italy

E-mail: [email protected]

Developing alternative strategies to commonly used blood transfusion appears essential because of the scarcity of donors, the short time storage of blood samples, and the presence of blood antigens. In order to obtain nanostructured artificial oxygen carriers, polymeric nanoparticles loaded with human hemoglobin were prepared by using 2-methoxyethanol hemiesters of poly(maleic anhydride alt-butylvinyl ether) 5% grafted with methoxy-PEG2000 (VAM41-PEG). The co-precipitation method was adapted to be performed under controlled atmosphere and temperature. The characterization of these nanostructures revealed a spherical morphology and a diameter distribution of about 140nm, while Drabkin's assay analysis showed a protein loading of 50%. Zeta potential analyses demonstrated the exposure of PEG chains on the surfaces, assigning an antiopsonizing effect and, consequently, a feasible long circulation time in vivo. Preliminary characterization studies evidenced that the protein maintains its secondary structure when loaded inside nanoparticles (Circular Dichroism analysis) and the cooperativity properties of the protein seem to be unchanged (Hill coefficient estimation). In order to minimize methemoglobin formation, the use of both reducing agents, such as ascorbic acid and methylene blue, and chemically modified polymeric matrices was investigated. VAM41-PEG was covalently modified by grafting 30% of the carboxylic moieties with o-methoxyaniline (VAM41-PEG-man). In-vitro quantitative and qualitative evaluation of VAM41-PEG and VAM41-PEG-man on mouse embryo fibroblasts displayed a good cytocompatibility profile for both polymeric matrices. Furthermore, alginate was investigated as alternative biocompatible natural derived material. Hemoglobin loaded beads with 2mm diameter were obtained by ionotropic gelation. UV-VIS spectroscopy and biochemical investigations revealed that hemoglobin maintains its functionality and its quaternary structure when loaded in alginate particles. Ongoing studies confirmed the possibility of reducing beads’ dimension to about 104 μm, preserving a high protein loading (47%).

MODIFIED KONJAC GLUCOMANNAN: A POTENTIAL PLASMA SUBSTITUTE – HEMODYNAMIC AND HEMORHEOLOGICAL PROPERTIES

Li, S., Hu, T., Ma, G., Su, Z.

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, PR China

E-mail: [email protected]

Plasma substitutes (e.g., albumin, gelatin and hydroxyethyl starch) have been used to treat volume deficiency, to stabilize the circulation, and to dilute the blood. However, these colloidal materials have different side-effects such as erythrocyte aggregation and blood bleeding tendency. In this study, konjac glucomannan (KGM), a polysaccharide from the Amorphophallus konjac plant, was carboxylmethylated to render its potential as a new plasma substitute. The hemodynamic and hemorheologic properties of the carboxylmethylated KGM were evaluated in an ameliorative hemorrhagic shock rabbit model. The KGM showed an exceptionally higher colloidal osmotic pressure (COP), indicating its ability for volume expansion, intravascular persistence, and rapid restoration of blood pressure. Thus, less colloid concentration would be effective to increase the mean arterial pressure and to decrease the whole blood viscosity. The negative charge also lowered the erythrocyte aggregation index (EAI), which was a problem for some commercial plasma substitutes.

POSTERS RESENTATIONS

P-01

ATTENUATION OF REDUCTIVE HEXAPEGYLATION INDUCED PERTURBATIONS OF HEME POCKET AND INTERSUBUNIT INTERACTIONS OF HEMOGLOBIN BY αα-FUMARYL INTRAMOLECULAR CROSSLINK

Acharya, S. A.^1,2, Hu, T.¹, Manjula, B. N.^1,2, Simplaceanu, V.³, Ho, N. T.³, Ho, C.³

¹Departments of Physiology & Biophysics and of ²Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

³Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA

E-mail: [email protected]

Although reductive pegylated Hb is vasoinactive, it weakens the interdimeric interactions of Hb and increases its O₂ affinity, thus limiting its application as a blood substitute. Pegylation of αα-fumaryl Hb is considered to generate PEG-Hb conjugates with desired stability and O₂ affinity. Influence of reductive hexapegylation of αα-fumaryl Hb on its structure has been investigated using proton NMR spectroscopy. Influence of hexapegylation of Hb is propagated throughout the molecule: heme environment, α₁β₂ and α₁β₁ interfaces are all perturbed. Αα-fumaryl crossbridge reduces the pegylation induced perturbations of α₁β₂ interface and of heme pocket, but not the α₁β₁ interface. Interestingly, αα-fumaryl Hb exhibits a T-state NMR signature of the α₁β₂ interface in the liganded state, a phenomenon observed in low O₂ affinity mutant Hbs. Hexapegylation of αα-fumaryl Hb reverses this propensity of αα-fumaryl crossbridge to access the T-state conformational space in the liganded state. Consistent with this, hexapegylation increases the O₂ affinity of the crosslinked Hb and brings it close to that of uncrosslinked Hb at ph 7.0 reflecting that αα-fumaryl crosslink reverses the propensity of pegylation to increase the O₂ affinity of Hb. Pegylation wipes out the Bohr effect of crosslinked Hb. It is concluded that PEG-shell induces significant global conformational changes in Hb that induces a higher O₂ affinity. At neutral ph, αα-fumaryl crossbridge completely negates high O₂ affinity inducing potential of pegylation but, on lowering the ph, the propensity of PEG-shell to increase the O₂ affinity is increased and the crosslink is unable to negate the pegylation to induced high O₂ affinity. Implications of the results in designing pegylation strategies that minimize the influence of PEG-shell in increasing the O₂ affinity of Hb without compromising the neutralization of vasoactivity will be discussed.

P-02

A NOVEL CHEMICAL STRATEGY TO OVERCOME DIRECT REDUCTIVE PEGYLATION INDUCED WEAKENING OF INTERDIMERIC INTERACTIONS IN PEG-HB ADDUCTS

Meng, F., Hu, T., Li, D., Prabhakaran, M., Acharya, S. A.

Departments of Physiology and Biophysics, and of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

E-mail: [email protected]

Hexapegylation of Hb by reductive pegylation weakens the interdimeric interactions of the molecule, thus limiting its application as potential blood substitute. The hexapegylation of [Propyl-Val-1(α)]₂-Hb by reductive pegylation generates stable pegylated Hb tetramer. Since Hb with propyl PEG-5K chains on Val-1(α) is essentially a pegylated dimer, it is hypothesized that avoiding the pegylation of Val-1(α) of Hb by prior modification is a unique chemical strategy to generate a pegylated Hb tetramer by reductive pegylation and an alternative to the use of intramolecularly crosslinked Hb. However, products of reductive hexapegylation of Hb dicarboxymethylated either at Val-1(α) or Val-1(β) remained essentially dissociated and did not conserve the stability of the tetramer. Therefore, protecting the Val-1(α) from direct pegylation is not adequate to conserve the stability of Hb after hexapegylation. Preventing the pegylation at the α-amino group of the tetramer as well as increasing stability of the tetramer is required to generate stable pegylated tetramer. Molecular models of di-pegylated Hb with propylpeg chains on Val-1(α) or on Val-1(β) have also been developed. Propylpeg-chain on Val-1(α) exhibits more extensive interactions with protein surface than the one on Val-1(β). The PEG domains of propyl-PEG-5K on Val-1(β) are involved in an interaction, thereby creating a “pseudo outside the central cavity crosslink” effect. Accordingly, we speculate that the stability of the new hexapegylated [Propyl-Va-1(α)]₂-Hb is a cumulative influence of (i) higher interdimeric stability of [Propyl-Val-1(α)]₂Hb; (ii) intramolecular crosslinking like effect afforded by PEG-chains on Val-1(β); and (iii) the absence Val-1(α) pegylation induced destabilization of the tetramer. Engineering higher stability to the αα-end of the Hb molecule through attachment of more hydrophobic alkyl and/or aryl chains on Val-1(α) is a novel approach to generate stable pegylated Hbs using direct pegylation. Impact of this approach to generate PEG-Hbs using low oxygen affinity Hbs by direct pegylation will be discussed.

P-03

INFLUENCE OF EXTENSION ARM FACILITATED PEGYLATION OF HB AND OF αα-FUMARYL HB ON THE THERMAL TRANSITION OF THE PROTEINS

Ananda, K.¹, Li, D.¹, Acharya, S. A. ^1,2

¹Departments of Physiology & Biophysics and of ²Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

E-mail: [email protected]

Extension Arm Facilitated pegylation (EAFP) is an effective approach to generate vasoinactive pegylated Hb with limited perturbation of interdimeric interactions. The extension arm engineered reduces the impact of PEG-shell on the structure of Hb. Influence of extension arm chemistry used in EAFP on the thermal stability of hexapegylated Hb has been investigated. The thermal transition patterns of Hb and pegylated Hbs are fitted by a non two state curve fittings (Table 1). The Tm 1 and Tm 2 for Hb are at 60°C and 70°C. Presumably the first and second transitions correspond to the perturbations of the inter (α₁β₂) and intra (α₁β₁) dimeric interactions. The Tm 1 and Tm 2 for conservatively EAF pegylated Hb are at 57 and 62°C. Αα-fumaryl crosslink sharpens the transition and increases Tm, Tm 1 and Tm 2 being at 75 and 79°C respectively. EAF-pegylated αα-fumaryl Hb exhibits one major transition, Tm 2 at 74°C. Tm 1 and Tm 2 for Hb EAF-hexapegylated nonconservatively are at 70°C and 79°c.αα-fumaryl crossbridge sharpens transition to a major one at 72°C (Tm 2). Direct nonconservatively hexapegylated αα-fumaryl Hb exhibits one major transition at 72°C (Tm 2). Thus, pegylation either by EAF or direct protocol lowers the thermal stability; this is in contrast to the increase that is seen with other proteins. This is presumably a consequence of the quaternary structure of Hb. Nonconservative EAFP affords a higher and sharper transition with uncrosslinked Hb. The lower and broader transition of conservative EAFP with uncrosslinked Hb is presumably a consequence of pegylation of Cys-93(β). Cys-93(β) pegylation influence is masked in crosslinked Hb and accordingly we speculate that if conservative EAFP is targeted exclusively to amino groups in Hb as in nonconservative EAFP, the stability of PEG-adducts will be increased further. Strategy to achieve this has been developed and will be discussed.

Table 1. Thermal Transition Parameters of HexaPEGylated Hbs

	First Transition		Second Transition
Hemoglobin samples	Tm 1 (°C)	ΔH1 (kcal/mol)	Tm 2 (°C)	ΔH2 (kcal/mol)
HbA	60.91 ± 0.6	373 ±21	69.42 ± 0.1	326 ± 17
αα fumaryl Hb	75.06 ± 0.9	155 ± 62	78.35 ± 0.4	126 ± 61
(SP-PEG5K) 6-Hb	56.63 ± 1.9	64 ± 36	61.56 ± 0.8	34 ± 33
(SP-PEG5K) 6-aaHb	55.83 ± 1.5	38 ± 08	73.95 ± 0.1	276 ± 06
(TSC-PEG5K) 6-Hb	69.30 ± 1.3	88 ± 25	79.44 ± 7.0	12 ± 25
(TSC-PEG5K) 6-aaHb	68.05 ± 1.6	47 ± 23	72.47 ± 0.6	35 ± 22
(SPA-PEG5K) 6-aaHb	70.26 ± 0.5	164 ± 37	73.79 ± 0.5	83 ± 37

P-04

HEMATOLOGICAL EFFECT OF SODIUM NITRITE USED TO MODULATE HEMOGLOBIN BASED OXYGEN CARRIER VASOACTIVITY IN HEMORRHAGE MODELS

Arnaud, F.^1,2, Scultetus, A.H.^1,2, Michaud, S.¹, Haque, A.¹, Kim, B.¹, Nigam, S.¹, Moon-Massat, P.¹, Auker, C.¹, Mccarron, R.^1,2, Freilich, D.^1,2

¹Trauma and Resuscitative Medicine Department, Naval Medical Research Center, Silver Spring, MD, USA

²Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA

E-mail: [email protected]

Introduction: The hemoglobin based oxygen carrier HBOC-201 may act as a nitric oxide (NO) scavenger and cause vasoconstriction while administered as a resuscitation fluid to treat hemorrhagic shock. Concomitant infusion of NO donors such as sodium nitrite (nano2) to counteract the NO scavenging effect could have systemic consequences on platelet activity, thrombosis, and hemoglobin oxidation.

Methods: nano2 was infused concurrently with 10 ml/kg HBOC-201 through separate lines in Yorkshire swine with 55% estimated blood volume controlled hemorrhage (at doses 5.4 – 16.2 μmol/kg) or severe liver crush injury (at dose 10.8 μmol/kg). The study compared 3 regimens of resuscitation: HBOC-201 alone, HBOC-201+nano2, and Hextend (HEX)+nano2. Fluids were given for 10 min at 15, 30 and 45 min (pre-hospital) after injury. Thereafter, animals were eligible for blood transfusion (Hb <7 g/dl / MAP< 40 mmhg) and monitored for up to 3 days. Blood samples were collected for complete hematology (CBC), Methemoglobin (methb), thromboelastography (TEG), platelet function (PFA), ADP-aggregation, and coagulation (PT, PTT).

Results: nano2 (10.8 μmol/kg) effectively reduced systemic vasoactivity of HBOC-201 in both hemorrhage models. CBC parameters, which changed due to timely fluid infusions, were unaffected by any of the doses of nano2. There was no reduction in PFA and ADP-aggregation due to nano2 in either injury model. Hemoglobin oxidation (methb) significantly increased linearly to an elevation of 9.5% with 16.2 μmol/kg nano2 infusion in groups treated with HBOC-201; methb elevation (<3%) was less with HEX treatment. Methb elevation continued after the last infusion (45 min) for up to 24 h in HBOC-201 groups.

Conclusion: The concomitant administration of nano2 reduced HBOC-201-induced vasoactivity without affecting platelet function. Significant elevations in methb were observed in animals treated with nano2 and HBOC-201. The results suggest further studies to characterize the effects of NO donors.

Acknowledgments: This work was funded by ONR #604771N.9737.001.A0315.

The study was approved by the Naval Medical Research Center/Walter Reed Army Institute of Research Institutional Animal Care and Use Committee. The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.

P-05

OXIDATIVE STRESS IN GUINEA PIG BRAIN FOLLOWING EXCHANGE TRANSFUSION WITH POLYMERIZED BOVINE HEMOGLOBIN

Butt, O. I., Buehler, P. W., D’Agnillo, F.

Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, FDA (Food and Drug Administration), Bethesda, MD, USA

E-mail: [email protected]

Hemoglobin-based oxygen therapeutics may be useful in limiting or preventing brain ischemia under certain settings. However, besides their oxygen delivery properties, these agents have the potential to trigger oxidative stress. This may be particularly important when plasma reductive defenses are diminished as this could further aggravate blood-brain-barrier (BBB) exposure to hemoglobin-driven oxidative reactions and/or heme degradation products. We previously reported a close relationship between intravascular oxidation of polymerized bovine hemoglobin (Oxyglobin^®, hbg) and reduced plasma ascorbate levels in a 50% exchange transfusion (ET) model in guinea pig, a species that, like humans, can not synthesize ascorbate. In the present study, we used this model to examine the effect of hbg on glial fibrillary acidic protein (GFAP) expression, a marker of BBB injury or activation, heme oxygenase (HO) induction, iron deposition, 8-hydroxy-2’-deoxyguanosine (8-ohdg) and 4-hydroxy-2-nonenal (4-HNE)-modified adducts, markers of oxidative DNA damage and lipid peroxidation, respectively, and cleaved caspase 3 expression, a marker of apoptosis. Hbg produced an increase in GFAP expression as early as 4 hours post-ET that was sustained through 72 hours. Consistent with this BBB activation, hbg increased HO activity and iron deposition, suggesting a possible direct exposure of the brain to hbg and/or its degradation products. Hbg also produced a progressive neuronal accumulation of 8-ohdg and 4-HNE-modified protein adducts as revealed by immunohistochemistry. In addition, hbg-treated guinea pigs showed increased levels of cleaved caspase-3, indicative of enhanced apoptosis. Together, these data suggest that hbg triggers oxidative stress in the guinea pig brain, which, given the similar plasma reductive capacities of guinea pig and humans, could be an important factor to consider in assessing the safety of hemoglobin therapeutics in subjects with diminished antioxidant defenses.

*The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

P-06

P-06 FOLDING AND STABILITY OF MYOGLOBINS AND HEMOGLOBINS

Culbertson, D. S., Olson, J. S.

Department of Biochemistry and Cell Biology and W. M. Keck Center for Computational Biology, Rice University, Houston, TX, USA

E-mail: [email protected]

Understanding hemoglobin assembly is crucial for the development of recombinant hemoglobin as a source for blood substitutes. However, the detailed mechanisms involving subunit assembly and heme binding remain unresolved. Mammalian myoglobins have served as archetype globins for understanding the properties of single domain globins with the 3 on 3 helical fold. After removal of heme, the resultant apo-Mb shows a loss of structure in the proximal F helix and adjacent loops. During denaturation, apo-Mb populates at least one intermediate. In contrast, unfolding of holo-Mb appears to be a simple two-state process with little protein concentration dependence. However, the underlying mechanism is much more complex and the lack of protein concentration dependence implies that heme either interacts with the unfolded polypeptide, self-associates, or both. In addition, the observed steepness of the unfolding curves for holo met-Mb requires that the affinity of hemin for the intermediate and completely unfolded states must be at least 1000-fold weaker than to the native apo-state. As a result, unfolding of holo met-Mb is governed primarily by the affinity of the folded native apo-state for hemin. Hemoglobin unfolding is even more complex due to association of the α and β subunits into dimers and tetramers. Removal of hemin leads to formation of an apo-α₁β₁ dimer and its unfolding appears to involve an intermediate whose stability is dependent on protein concentration. This dependence suggests the formation of a dimer intermediate with partially folded subunits still attached to each other through the α₁β₁ interface. Folding and assembly of holo-Hb is even more complex because there are significant differences in hemin affinity between the α and β subunits, and between tetramers, dimers and monomers.

Supported by U.S. NIH Grants RO1 HL047020 and R01 GM035649 and Grant C-0612 from the Robert A Welch Foundation, Houston, Texas, USA. DSC is the recipient of a predoctoral fellowship from the NIH T32 HL007812-11 Atherosclerosis and Vascular Biology Research Training Grant program.

P-07

MARKERS OF OXIDATIVE STRESS AND APOPTOSIS FOR PRECLINICAL EVALUATION OF HEMOGLOBIN-BASED OXYGEN THERAPEUTICS

Butt, O. I., Buehler, P. W., Abraham, B., Alayash, A., D’Agnillo, F.

Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, FDA (Food and Drug Administration), Bethesda, MD, USA

E-mail: [email protected]

Hemoglobin-induced oxidative stress and apoptosis may contribute to some of the unresolved toxicities of hemoglobin-based therapeutics. In the present study, we examined sensitive and specific biomarkers of oxidative stress and apoptosis that have greatly improved the detection and characterization of these processes in vivo. Using a 50% exchange transfusion model with polymerized bovine hemoglobin in rodents, we analyzed 4-hydroxynonenal (4-HNE)-modified protein adducts, an index of lipid peroxidation, 8-hydroxy-2-deoxyguanosine (8-ohdg), a marker of oxidative DNA damage, and cleaved caspase 3, a marker of apoptosis thought to be more sensitive than traditional TUNEL detection. Immunohistochemical and western blot analyses revealed enhanced accumulation of 4-HNE modified protein adducts in rat and guinea pig kidneys. Mass spectral analyses identified mitochondrial enzymes of the Krebs cycle as being highly susceptible to 4-HNE modification. Western blot analyses identified similar 4-HNE-modified protein adducts in lungs and hearts of guinea pigs. Nuclear 8-ohdg immunoreactivity was increased in renal proximal tubules and glomeruli in guinea pigs and co-localized with regions expressing high 4-HNE immunoreactivity. Cleaved caspase 3 was detectable by western blot in guinea pig and rat kidneys but was not associated with significant increases in serum creatinine or other common histopathological indices. Cleaved caspase 3 levels were also elevated in guinea pig hearts and lungs. These markers were observed as early as 4 hours post-exchange and generally peaked between 12 – 24 hours, suggesting that early analyses of these processes may be particularly valuable. Clinical trial results with some hemoglobin-based therapeutics have led to suggestions that preclinical testing may not have been sufficiently predictive of safety in humans. The present findings suggest that sensitive and specific markers of oxidative stress and apoptosis may be useful in preclinical studies designed to evaluate the safety of these products.

*The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

P-08

STUDIES OF MECHANISMS FOR ALTERATIONS OF IMMUNOLOGICAL PROPERTIES OF CHEMICALLY MODIFIED HEMOGLOBIN

Yang, M., Yang, X., Yang, P., Xie, Y., Dan, N., Chen, C.

National Engineering Research Center for Miniaturized Detection System, Northwest University, Xi'an, China

Shaanxi Lifegen Co. Ltd., Xi'an, China

E-mail: [email protected]

A common strategy in production of hemoglobin-based oxygen carriers (hbocs) is through glutaradehyde-medicated polymerization of hemoglobin molecules. It has been shown that such modification may alter the ability of hemoglobin (Hb) to stimulate specific immunological reactions in the body. This study investigated the possible underline mechanisms in terms of alterations in antigenicity (immunogenicity) and/or reactiongenicity of the antigen(s) in the resultant proteins. Two different types of hemoglobin polymers with a similar molecular weight distribution (average molecular weight was about 300kd) were made. One was called intramolecular cross-linked Hb, in which chemical cross-linking within tetramers occurred in almost all hemoglobin units of the protein polymers. The other one was called intermolecular cross-linked Hb, where chemical cross-linking within tetramers occurred only in a portion of the hemoglobin units of the protein polymers.

The following observations were made using Western blot assay: 1) mouse antibodies (mab) against bovine Hb (bhb) recognized intermolecularly cross-linked bovine Hb (intercl-bhb), but failed to react with intramolecularly cross-linked bovine Hb (intracl-bhb), indicating that the antigenic epitope(s) of bhb lost functions in intracl-bhb, while it remained in intercl-bhb. 2) mab against intracl-bhb, but not mab against intercl-bhb, did not bind to bhb, suggesting that the intracl-bhb possessed an antigen determinant(s) that does not exist in bhb. 3) mab against intracl-bhb recognized intermolecular cross-linked porcine Hb (intercl-phb); however, mab against intercl-phb did not react with intracl-bhb. Since phb and bhb have very high sequence similarity (large than 95%), this data indicate that the antigenic determinant(s) shared between bhb and phb lost the ability in intercl-phb to stimulate antibody production in mice, but remained related in antigenic reactiongenicity. In summary, this study clearly demonstrated that polymerization of hemoglobin may result in a variety of changes in the immunological properties of the resultant polymers, depending on the nature of hemoglobin used for polymerization and the type of the method to make them. Such changes may include loss of functions of the antigenic determinants existing in the nature hemoglobin, gain of new antigen epitopes and/or loss of immunogenicity, but maintaining antigenic reactiongenicity of the Hb molecules. Based on data of this study, it is reasonable to predict that HBOC, even made from human Hb, may also present immunological risk to humans due to the possibility of introducing new antigenic properties in the resultant modified molecules.

P-09

HBOC-201 IN MANAGEMENT OF SEVERE ANEMIA IN A PATIENT WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Donahue, L. L.¹, Shander, A.², Shapira, I.¹, Kolitz, J.¹, Allen, S.¹, Greenburg, A. G.³

¹North Shore University Hospital and Long Island Jewish Health Systems, Manhasset, NY, USA

²Department of Anesthesiology and Critical Care Medicine, Englewood Hospital and Medical Center, Englewood, NJ, USA

³Biopure Corporation, Cambridge, MA, USA

E-mail: [email protected]

While highly effective, current chemotherapy regimens used in treatment of Acute Lymphoblastic Leukemia (ALL) result in severe myelosuppression with expected nadir blood counts on day 14 of treatment followed by blood count recovery 1–2 weeks later. Standard medical practice for resultant severe/symptomatic anemia is transfusional support with allogeneic red blood cells until recovery of autologous erythropoietic function. This poses problems when blood is not available or not an option.

Here, we report a 36-year-old woman of Jehovah's Witness faith who refused blood, and was newly diagnosed with ALL. Induction chemotherapy initiated concurrent with erythropoiesis stimulation, with baseline hemoglobin of 7.2 g/dl. Hemoglobin dropped to 4.5 g/dl on day 7 and patient became dizzy, tachycardic and hypotensive, unresponsive to numerous fluid boluses. Given the deteriorating symptomatic severe anemia, use of HBOC-201 was considered on compassionate basis. After obtaining an emergency Investigational New Drug from the Food and Drug Administration and an informed consent, patient received a total of 15 units of HBOC-201 over 12 days of treatment, beginning on day 8 after induction. Following each infusion, the patient reported improved vitality and mental acuity and her vital signs stabilized, despite hemoglobin levels between 3–4 g/dl. HBOC-201-related adverse events were limited to methemoglobinemia, which responded to Methylene Blue infusion. By day 20 post-induction, blood counts had recovered and HBOC-201 infusion stopped.

The patient required 6 additional courses of myelosuppressive chemotherapy, during which her hemoglobin level stayed above 7.5 g/dl by concomitant erythropoiesis stimulation/support. Maintenance chemotherapy continued until 24 months after induction chemotherapy and patient still remains in complete remission at time of this report.

This case exemplifies successful management of profound anemia in an ALL patient who refused blood, using HBOC-201 as a bridging treatment to save the patient from the devastating effects of ischemia in severe anemia.

P-10

POSTINJURY RESUSCITATION WITH HUMAN POLYMERIZED HEMOGLOBIN PROLONGS EARLY SURVIVAL: A POST HOC ANALYSIS

Gould, S. A., Hides, G. A., Guthrie, B. J., Omert, L. A.

Northfield Laboratories Inc., Evanston, IL, USA

E-mail: [email protected]

Introduction: Human polymerized hemoglobin (polyheme®) may be useful in the early treatment of hemorrhagic shock when stored blood (rbcs) is not available. In the recent Phase III USA multicenter trial, Day 30 mortality was 13% in the polyheme group versus 10% in the control group (p > 0.05). However, clear analysis comparing polyheme alone to crystalloid alone or to rbcs alone is obfuscated in the aggregate population by the majority of polyheme recipients who also received rbcs. To examine possible early benefit of polyheme, we examined time to death among non-survivors, and performed post hoc subgroup safety analyses.

Methods: Time to death was analyzed for all non-survivors. The following subgroups of all 714 enrolled and treated patients were analyzed: (1) Patients receiving 1U polyheme (n = 112) without rbcs versus controls (n = 147) receiving crystalloid without rbcs (polyvcrys); (2) Patients receiving up to 6U polyheme (n = 152) without rbcs versus controls (n = 218) receiving up to 6U rbcs (polyvrbc).

Results: Time to death was prolonged in the polyheme group during the first 8 hours following injury (figure 1) In the polyvcrys subgroup, Day 1 (2% v 5%) and Day 30 (3% v 5%) mortality were lower in the polyheme group. No patients had MI, and aes/saes were comparable between groups. In the polyvrbc subgroup, polyheme mortality at Day 1 (3% v 5%) and Day 30 (5%) was lower than/identical to control, respectively. Mis were low and identical between groups (1%), and aes/saes were comparable.

Figure 1. Time to death among non-survivors.

Conclusions: polyheme prolongs early survival, likely owing to the benefit of early oxygen-carrying replacement. Further study may help define post-resuscitation treatments to maintain this benefit long-term. The safety of polyheme compared to crystalloid or rbcs alone was comparable. These subgroup analyses are useful when extrapolating the use of polyheme to settings where rbcs are needed, but not available.

P-11

STRUCTURAL INTEGRITY AND REDOX BEHAVIOR OF A ZERO-LINKED POLYMERIC HEMOGLOBIN, OXYVITA™: A NEW GENERATION HEMOGLOBIN-BASED- OXYGEN-CARRIER (HBOC)

Harrington, J. P.^1,2,Wollocko, J.³, Wollocko, H.³

¹State University of New York, New Paltz, NY, USA

²Marine Biological Laboratory, Woods Hole, MA, USA

³OXYVITA Inc., New Windsor, NY, USA

E-mail: [email protected]

Several years ago a novel “zero-link” polymerized hemoglobin was developed to address a fundamental problem of vascular hemoglobin extravasation and its related problems that earlier hbocs exhibited at the clinical level. Modified production methods introduced by OXYVITA, Inc. have led to a new generation HBOC, oxyvita™, having a M.Wt. of 17 mda with a hydrodynamic radius of 210Å and a low osmotic pressure and a viscosity at 6 g/dl similar to human plasma. For safe and effective oxygen binding, transport and delivery, an HBOC must meet essential molecular requirements related to its structural integrity and redox stability. Structural integrity of oxyvita™ was determined using gel permeation chromatography, light scattering, and isothermal urea unfolding studies. Unfolding midpoints (D_1/2) and free energy of unfolding in the absence of denaturant (ΔG°_{u, water}) of different preparations of oxyvita™ (solution and powder forms) were compared to two naturally utilized acellular polymeric hemoglobins (Lumbricus and Arenicola Hbs), which have served as models for elucidating safe and effective HBOC properties. Redox behavior of oxyvita™ was investigated through coupled oxidation and reduction studies (endogenous reducing agents). The results of these studies are consistent with the initial evaluation of oxyvita™ effectiveness as an oxygen delivery system as discussed in our accompanying presentation.

P-12

MANNOSYLATED-RECOMBINANT ALBUMIN AS A NO TRAFFIC PROTEIN FOR THE TREATMENT OF HEPATIC ISCHEMIA/REPERFUSION INJURY

Hirata, K.¹, Ishima, Y.¹, Watanabe, H.¹, Suenaga, A.¹, Maruyama, T.¹, Otagiri, M.^1,2

¹Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan

²Faculty of Pharmaceutical Sciences, Sojo University

E-mail: [email protected]

Hepatic ischemia/reperfusion (I/R) injury is acute hepatic injury caused by liver transplantations. I/R process is known to generate a large amount of reactive oxygen species (ROS). Kupffer cells (kcs), the resident macrophages in the liver, are activated during I/R, and produce ROS, which leads to hepatocyte death. Therefore, inhibition of ROS production by kcs in the initial period of reperfusion will be an effective strategy for inhibiting hepatic I/R injury. Nitric oxide (NO) induces heme-oxygenase 1 (HO-1), which localizes in kcs. Induction of HO-1 is a protective response against oxidative stress. NO is, however, unstable in blood circulation. Therefore, it is difficult to apply it for the treatment of hepatic I/R injury. Mannose receptors (MR) on kcs mediate the endocytosis of glycoproteins possessing mannose or N-acetylglucosamine residues. Therefore, we prepared the mannosylated-recombinant human serum albumins (man-rhsas: D63N, A320T, D494N and triple mutant (D63N/A320T/D494N)) using site-specific mutagenic techniques and Pichia pastoris expression system, and man-rhsas were nitrosylated via Cys34. Pharmacokinetic profiles and biodistribution of ¹¹¹In-man-rhsas were investigated in mice. We also evaluated the hepatic uptake characteristics of man-rhsas at a cellular level using liver endothelial cells (LEC) and kcs. The pharmacological effects of man-rhsas conjugated with NO against hepatic I/R model rat were also examined. As a result, D494N and triple mutant disappeared from the blood circulation and were taken up by the liver rapidly, as compared to wild-type rhsa, D63N, and A320T. Triple mutant was taken up by kcs, but not by lecs, suggesting that the hepatic uptake was selectively mediated by MR on kcs. Finally, triple mutant-NO conjugate exhibited significant inhibitory effects against hepatic I/R injury. These results implicated that man-rhsas is a useful carrier as a NO traffic protein for the treatment of the hepatic I/R injury.

P-13

HB ENCAPSULATION IN VESICLES RETARDS NO- AND CO-BINDINGS AND O₂-RELEASING WHEN PERFUSED THROUGH A GAS- PERMEABLE NARROW TUBE (25 μM)

Okuda, N.¹, Sakai, H.², Atsushi, S.¹,Takeoka, S.¹, Tsuchida, E. ²

¹Graduate School of Advanced Sciences and Engineering, and

²Research Institute for Science and Engineering, Waseda University, Tokyo, Japan

E-mail: [email protected]

Intravenous administration of cell-free Hb based blood substitutes induces vasoconstriction, presumably due to the enhanced reactions with NO and CO as vasorelaxation factors, and the facilitated O₂-releasing that might induce autoregulatory vasoconstriction. In the present study, we examined these gas reactions of cell-free Hbs and cellular Hbs when perfused through gas-permeable artificial narrow tubes.

Stroma-free Hb solution (sfhb), polymerized bovine Hb (polyhb), encapsulated Hb (Hb-vesicles, hbv, 250 nm), and human rbcs at a practical Hb concentration (10 g/dl) were perfused through a fluorinated ethylenepropylene copolymer tube (inner diameter, 25 μm) at a rate of 1 mm/s. Measurement of the reactions was performed using an apparatus that consisted of an inverted microscope and a spectrophotometer, and the level of reactions was determined based on the visible absorption spectrum in the Q band of Hb.

When the tube was immersed in NO or CO atmospheres, both NO- and CO-bindings to deoxygenated sfhb and polyhb were faster than those to hbv and rbcs, and hbv and rbcs were almost identical. The O₂-releasing rates were observed by perfusing the oxygenated fluids while the tube was immersed in a deoxygenated atmosphere. Hbv showed similar O₂-releasing rate with RBC. Polyhb showed a faster reaction than sfhb, due to the lower O₂ affinity of polyhb than sfhb. To identify the physicochemical mechanism of pronounced difference in gas reactions between hbv and sfhb, the diffusion process of the particles in the narrow tubes was simulated using Navier-Stokes equation and Maxwell-Stefen equation. It was clarified that small sfhb diffuses laterally and mixed rapidly. On the other hand, the large dimension hbv does not show such rapid diffusion. This lateral diffusion and the resultant efficient mixing effect will contribute to the fast gas reactions of cell-free Hbs. The purely physicochemical difference in gas reactions would be one factor to determine the absence or the presence of vasoconstrictive effect of Hb-based blood substitutes.

This work is supported in part by MHLW and JSPS.

P-14

MASS SPECTROMETRY BASED LOCALIZATION OF CHEMICALLY INTRODUCED CROSS-LINKS IDENTIFIES STRUCTURAL RELATIONSHIPS: THE HEMOGLOBIN-HAPTOGLOBIN COMPLEX

Pereira, C.P.¹, Linnemayr, C.¹, Beck, M.², Schoedon, G.¹, Wepf, R.³, Schaer, D. J.¹

¹Division of Internal Medicine, University Hospital Zürich, Switzerland

²Institute for Molecular Systems Biology, ETH Zürich, Switzerland

³Electron Microscopy ETH Zürich, EMEZ, Switzerland

E-mail: [email protected]

Detoxification and clearance of extracellular Hb from the circulation occurs through a plasma protein binding pathway in which Hb αβ-dimers bind with an extremely high affinity to haptoglobin (Hp). Depending on specific structural requirements, some hbocs also retain the ability to bind to Hp despite extensive chemical modification. Hp uniquely protects Hb from oxidative impact and alleviates vascular and oxidative toxicity of extracellular Hb. Currently there is no crystal structure of Hp or the Hb-Hp complex available – a fact that might be related to the complex glycosylation pattern of Hp or to the considerable intrinsic structural flexibility within the multimeric Hp molecule. Here we aimed to resolve critical structural features of the Hb-Hp complex by a combined mass-spectrometry and electron microscopy approach. Cross-linking of proteins is a powerful method to investigate protein conformation and protein-protein interactions. A cross-link between two peptides is indicative of the spatial proximity of two linked amino acids at the time of cross-linking. We used an integrative approach to identify and characterize proximity sites between Hb and Hp through chromatographic enrichment and mass spectrometry based targeted sequencing of peptides, which are modified by isotopically coded cross-linkers. In the presented approach we obtained a spatial map of peptide proximity regions within the Hb-Hp complex. Also, the surface accessibility of specific amino acids in either free proteins or within the complex, respectively, could be derived. Together with three-dimensional imaging data obtained by electron microscopy and single particle analysis we are able for the first time to resolve critical structural elements of the Hb-Hp complex. This information will help in understanding mechanisms involved in the powerful protective activities of Hp against systemic extracellular Hb toxicity.

P-15

VASOACTIVITY AND RENAL EXCRETION OF EURO-PEG-HB, A NOVEL HEMOGLOBIN-BASED OXYGEN CARRIER

Portörő, I.¹, Kocsis, L.¹, Hermán, P.¹, Caccia, D.², Perrella, M.², Ronda, L.³, Bruno, S.³, Bettati, S.^3,4, Micalella, C.³, Mozzarelli, A.^3,4, Varga, A.⁵, Vas, M.⁵, Lowe, K. C.⁶, Eke, A.¹

¹Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Hungary

²Department of Biomedical Science and Technology, University of Milan, Italy

³Department of Biochemistry and Molecular Biology, University of Parma, Italy

⁴National Institute of Biostructures and Biosystems (INBB), Italy

⁵Institute of Enzymology, Hungarian Academy of Sciences, Hungary

⁶School of Biology, University of Nottingham, United Kingdom

E-mail: [email protected]

This investigation was aimed at the development of a novel hemoglobin-based oxygen carrier with low vasoactivity and renal excretion properties, both being indicative of low toxicity.

Human hemoglobin was chemically conjugated with seven PEG and seven propionyl groups – which we named Euro-PEG-Hb – under anaerobic conditions. Following different chemical procedures, dissolved in saline at a concentration of 5.8 g%, 7 ml was exchange-transfused into rats of Wistar strain (n = 5) to evaluate vasoactivity and renal loss.

The blood-to-blood substitute exchange (B-to-BS-X) was carried out at a rate of 1 ml/min via an arterial line inserted into the femoral artery. The extent of B-to-BS-X was 52±3 % as determined using the dilution principle, along with that of plasma volume. The level of femoral arterial blood pressure as compared to its control value assessed vasoactivity. Renal excretion was determined from the total amount of Euro-PEG-Hb excreted into collected urine samples divided by the amount transfused using a spectrophotometric assay based on heme's peroxidative activity.

As assessed at 110 minute post-transfusion, Euro-PEG-Hb did not yield sustained hypertension (109 ± 7 mmhg vs. 110 ± 4 in the control and the test period, respectively). Renal excretion of Euro-PEG-Hb by 5–6 hours post-transfusion was 0.4 ± 0.1 % (n = 3); a minimal level when compared to that of human hemoglobin A at 21.4 ± 7 % (n = 5).

Our Euro-PEG-Hb HBOC molecule proved of low toxicity, thus appearing a very promising candidate as a blood substitute.

I. Portörő and L. Ronda contributed equally to the work.

P-16

MICROHEMODYNAMIC EFFECTS OF EURO-PEG-HB (A NOVEL HAEMOGLOBIN-BASED OXYGEN CARRIER) IN THE RAT BRAIN CORTEX

Portörö,I.¹, Kocsis, L.¹, Hermán, P.¹, Caccia, D.², Perrella, M.², Eke, A.¹

¹Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Hungary

²Department of Biomedical Science and Technology, University of Milan, Italy

E-mail: [email protected]

Owing to NO scavenging, a potential adverse effect of hemoglobin-based oxygen carriers (hbocs) is their vasoactivity. Decoration of hemoglobin with polyethylene glycol (PEG) chains is known to ameliorate this effect. Our aim was to characterize the vasoactivity of our novel pegylated HBOC, Euro-PEG-Hb by assessing its impact (vascular resistance, i.e.) on regional hemodynamics in the rat brain cortex.

The experiments were carried out on white laboratory rats of Wistar strain under general anesthesia by urethane (1.3 g/kg bdw i.p.). The general physiological status of the animals was assessed by core (rectal) temperature, systemic arterial blood pressure (SABP), blood gas parameters (ph, po₂, pco₂, BE, Sat), and arterial hematocrit (Hct). Cerebrocortical blood flow velocity (Q) as a key parameter in regional hemodynamics was measured non-invasively within a voxel of 1 mm³ using the laser speckle imaging method.

As a negative reference, hydroxyethyl starch (HES) was used. Blood-to-HBOC/HES exchange was carried out at a rate of 1 ml/min via a femoral arterial line. A total of 7 ml of arterial blood was replaced by 7 ml of HBOC or HES resulting in a marked decrease in Hct (from 39.8 ± 5 % and 39.8 ± 7 % in control, to 22.5 ± 3 % and 23.8 ± 3 % post-exchange for Euro-PEG-Hb with n = 5 and HES with n=6, respectively). The potential vasoactive effect of the test molecule was assessed by the hematocrit corrected regional prenchymal vascular resistance (R_corr) as the mean of microregional values determined within the ROI.

At 110 minutes post-exchange, R_corr returned to control levels in both groups (15.3 ± 3 A.U. and 12.4 ± 5 A.U. vs. 14 ± 2 A.U. and 11.2 ± 6 A.U.).

Euro-PEG-Hb had a favorable influence on the cerebrocortical microregional hemodynamic status in that it increased cerebrocortical microperfusion due to a lowered Hct; otherwise it left the microregional vascular resistance unchanged as did HES, a positively non-vasoactive molecule.

P-17

RED BLOOD CELL TRANSFUSIONS IN PRETERM INFANTS AND THEIR EFFECTS ON ACID-BASE, GLUCOSE, AND ELECTROLYTES STATUS

Pratesi, S.¹, Dani, C.²

¹Neonatal Intensive Care Unit, Careggi University Hospital, Florence, Italy

²Neonatal Intensive Care Unit, University of Florence, Italy

E-mail: [email protected]

More than 90 percent of extremely low-birth-weight infants receive one or more transfusions of red blood cells (rbcs). Harmful adverse effects have been reported after RBC transfusion, such as acid-base, glucose, and electrolyte disturbances. This can occur because many factors in the preparation of irradiated rbcs may lead to a severe burden of hydrogen ions, carbon dioxide, potassium, and glucose. In order to assess if RBC transfusions induce significant changes of plasma acid-base, electrolyte, and glucose status in extremely preterm infants and if these changes might be more marked as the transfusion volume is larger, we designed a retrospective cohort study.

Records of infants with gestational age of less than 31 weeks who were transfused with rbcs during the first week of life were reviewed (n = 61). Blood samples were collected before and after transfusions to evaluate hemoglobin level, hematocrit, acid-base, electrolyte, and glucose status. Then infants were stratified into four groups that received a RBC volume of less than 15, 15 to 20, more than 20 to 25, or more than 25 ml per kg. After transfusions, a significant increase of po₂ and decrease of Ca²⁺ and glycemia were observed. Three (4.9%) infants developed hyperkalemia, one (1.6%) had an exacerbation of his hypocalcemia, and another (1.6%) of his hypoglycemia.

RBC transfusions were effective in correcting anemia in our patients and induced a slight increase of ph and po₂ and decrease of Ca²⁺ and glycemia, which were not clinically relevant. A linear direct correlation was observed between potassium intake by RBC transfusions and changes of kalemia in our infants, but there was not an increase of K⁺ plasma level after transfusions. However, caution in programming and performing RBC transfusion in patients at risk of hyperkaliemia seems advisable.

P-18

MAPPING RADICAL MIGRATION THROUGH RESPIRATORY HEMOPROTEINS

Reeder, B. J., Svistunenko, D. A., Cooper, C. E., Wilson, M. T.

Department of Biological Sciences, University of Essex, UK

E-mail: [email protected]

Respiratory hemoproteins such as myoglobin and hemoglobin are capable of generating free radicals in vivo. This redox chemistry of myoglobin and hemoglobin leads to oxidative damage and has been directly linked to the pathogenesis of acute renal failure following rhabdomyolysis and vasospasm flowing subarachnoid hemorrhage. There is evidence that associates infusion of hemoglobin based oxygen carriers (HBOC) with oxidative damage, which may be a leading cause of HBOC toxicity. We have previously reported that manipulation of these radical pathways can modulate the toxicity of these hemoproteins. Understanding the control, or indeed lack of control, of radicals and electron pathways in hemoproteins may lead to the rational design of hemoglobins that have a decreased intrinsic cellular toxicity. We have used myoglobin from the sea hare (Aplysia Fasciata), a protein that has no tyrosine residues but fifteen phenylalanine residues, to add selectively a series of tyrosine residues and thus selectively modulate intra- and inter-molecular radical migration pathways. Using various EPR and spectrophotometric techniques we can follow the radical through the protein and thus determine the fundamental rules that govern free radical movement. The ability of the protein to handle radicals will be compared to the protein's cellular toxicity.

P-19

EFFECT OF LARGE VOLUME RESUSCITATION WITH THE CELLULAR TYPE ARTIFICIAL OXYGEN CARRIER (HB-VESICLE) IN RAT MODEL OF UNCONTROLLED HEMORRHAGIC SHOCK VIA A CAUDAL ARTERY

Seishi, Y.¹, Horinouchi, H.², Sakai, H.³, Tsuchida, E.³, Kobayashi, K.⁴

¹Department of Surgery, Keio University, Japan

²Department of Surgery, Keio University, Japan

³Advanced Research Institute of Science and Engineering, Waseda University, Japan

⁴Department of Surgery, Keio University, Japan

E-mail: [email protected]

Uncontrolled hemorrhage is one of the important factors that threaten survival from major trauma. Fluid resuscitation for uncontrolled hemorrhage has not been standardized. We used Hb-vesicle for the rat model of continuous bleeding from a caudal artery. To induce uncontrolled hemorrhage, an indwelling needle was inserted into the caudal artery of animals. After establishment of shock status, fluid resuscitation was done with 5 materials, Hb-vesicle suspension in 5% albumin solution (hbv-Alb), washed erythrocyte in 5% albumin (wrbc-Alb), 5% albumin (Alb), hydroxyethyl starch (HES), and saline. Animals were observed for a maximum of 2hr after beginning of infusion. In the hbv-Alb group and the wrbc-Alb group, survival was significantly improved and there was no death during the observation period. In both groups, mean arterial pressure drifted above 50mmhg. Blood lactate level in the hbv-Alb group and the wrbc-Alb group did not increase after resuscitation, although that in other groups increased significantly. Hematocrit of the hbv-Alb group, the Alb group, and the HES group showed remarkable decreases, although that of the wrbc-Alb group and the saline group was higher than others. These results indicate that in an uncontrolled hemorrhagic shock model infusion of fluid containing Hb-vesicle is effective for survival. For severe trauma, continuous infusion of hbv-Alb may keep circulating volume and maintain oxygen metabolism in shock organs, even if hemorrhage can not be controlled.

P-20

EFFECT OF LARGE VOLUME RESUSCITATION WITH THE CELLULAR TYPE ARTIFICIAL OXYGEN CARRIER (HB-VESICLE) IN RAT UNCONTROLLED HEMORRHAGIC SHOCK MODEL CAUSED BY BLUNT KIDNEY INJURY

Seishi, Y.¹, Horinouchi, H.², Sakai, H.³, Tsuchida, E.³, Kobayashi, K.⁴

¹Department of Surgery, Keio University, Japan

²Department of Surgery, Keio University, Japan

³Advanced Research Institute of Science and Engineering, Waseda University, Japan

⁴Department of Surgery, Keio University, Japan

E-mail: [email protected]

Uncontrolled hemorrhage is one of the important factors that threaten survival from major trauma. Fluid resuscitation for uncontrolled hemorrhage has not been standardized. We used Hb-vesicle for the rat model of blunt kidney injury. To induce intraperitoneal hemorrhage without laparotomy, the left kidney was injured by percutaneously-inserted wires after hemodilution of hydroxyethyl starch. After establishment of shock status, fluid resuscitation was done with 3 materials, Hb-vesicle suspension in 5% albumin solution (hbv-Alb), washed erythrocyte in 5% albumin (wrbc-Alb), and 5% albumin (Alb). A non-resuscitated group was prepared for comparison. Animals of resuscitation groups underwent intermittent infusion depending on mean arterial pressure (MAP) and were observed for a maximum of 1hr after beginning of infusion. In the hbv-Alb group and the wrbc-Alb group, survival was significantly improved and there was no death during the observation period. In both groups, MAP was maintained at a higher level than the Alb group and the non-resuscitated group. Blood lactate level of all groups increased with time. However, that of the hbv-Alb group drifted lower than the others. Hematocrit of the hbv-Alb group was decreased continuously by infusion, although kept higher than the Alb group. Blood loss of the hbv-Alb group, the wrbc-Alb group, and the non-resuscitated group was less than that of the Alb group. These results indicate that infusion of fluid containing Hb-vesicle is effective for survival. It seems that infusion of hbv-Alb would reduce hemorrhage in blunt injury.

P-21

EFFECTS OF ANTIOXIDANTS ON HUMAN RBC AND ACELLULAR BOVINE HEMOGLOBIN (HB) OXYGEN AFFINITY AND OXIDATION

Simoni, J.¹, Tomison, M. D.¹, Simoni, G.¹, Castillo III, J. X.¹, Moeller, J. F.¹, Campbell, S. J.¹, Wesson, D. E.²

¹Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, USA

²Department of Medicine, Texas A&M College of Medicine, Scott and White Healthcare, Temple, TX, USA

E-mail: [email protected]

Although many antioxidant strategies have been developed, uncontrolled Hb oxidative reactions still hinder the commercial development of blood substitutes. The “radical” nature of Hb is particularly relevant in subjects with compromised antioxidant status. In any viable Hb-based product, the oxidative reactions must be attenuated and proper oxygen-carrying function preserved. Previous work from this laboratory established that the ascorbate (A)-glutathione (GSH)-antioxidant system is essential in the attenuation of pro-oxidant potential of redox-active acellular Hb, and superior to either A or GSH alone (Artificial Organs 2009; 33(2):115–126). In the present study, we investigated this antioxidant system in the presence of uric acid (UA), another important element of the human plasma antioxidant capacity. In this ex vivo model, human RBCs and purified bovine Hb were incubated with clinically acceptable levels of A, GSH, and UA alone, and in combination, and subject to autoxidation or challenged with hydrogen peroxide. Heme oxidation was measured spectrophotometrically using the Ocean Optics USB2000-ISS-UV/VIS System and spectrasuite Software (Version 5.1). This study also focused on the effects of antioxidants on RBC and Hb oxygenation. Oxygen equilibrium curves were obtained using the TCS Scientific Hemox-Analyzer (Model B) and the Hemox Analytical Software (Version 2.00.13), in phosphate buffered saline, ph 7.4, and controlled heme oxidation. These effects were also investigated in the presence of caffeine and its metabolites (CA). The results confirmed that the A-GSH system is extremely effective in preventing Hb pro-oxidant reactions. Surprisingly, UA did not significantly augment these effects. CA showed to be ineffective. The tested antioxidants did not markedly modify RBC oxygen affinity (p50, oxygen pressure at half-saturation), but had various effects on acellular Hb. A and GSH increased p50. UA and CA had no effect. The results of this study could have clinical relevance; however, they warrant further in vivo testing to optimize these findings.

P-22

ACELLULAR HEMOGLOBIN (HB) AS AN ERYTHROPOIESIS-STIMULATING AGENT (ESA)

Simoni, J.¹, Simoni, G.¹, Feola, M.^1,2

¹Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, USA

²hemobiotech, Inc., Dallas, TX, USA

E-mail: [email protected]

A speedy replacement of blood loss with endogenous rbcs seems to be the most attractive future of Hb-based oxygen carriers. Since these products have short circulatory retention time and high autoxidation rates, their erythropoietic potential should be the essential component in blood loss anemia treatment. Although many hypotheses have been proposed for explaining the mechanism behind the erythropoietic effect of Hb, its exact action has not been fully established. Hb is known for its pressor effects, “radical” and “signaling” nature that can be altered by changing its physico-chemical characteristics. Currently, tested blood substitutes express different erythropoietic potential. The regulation of erythropoiesis is a complex process controlled by a highly sensitive feedback system based on oxygen tension and the cellular redox-state that involves oxygen (hypoxia inducible factor (HIF)-1 alpha) and redox (nuclear factor (NF)-kappa B) regulated transcription factors and their target genes. Using in vivo and in vitro models, under hypoxic and normoxic conditions, we investigated the mechanism of erythropoietic action of unmodified bovine Hb and modified with ATP, adenosine, and glutathione (GSH). Results indicate that these Hb solutions have a different impact on HIF-1 stabilization, nuclear translocation, induction of the erythropoietin (EPO) gene, NF-kappa B activation, and production of anti-erythropoietic agents: TNF-alpha and TGF-beta 1. Unmodified Hb suppressed the erythropoiesis by increasing cytoplasmic degradation of HIF-1 and decreasing its binding activity to the EPO gene while activating NF-kappa B. Modified Hb increased the induction of HIF-1 and its binding to the EPO gene and down regulated NF-kappa B, under both oxygen conditions. It seems that adenosine stimulates normoxic induction of HIF-1 and with GSH inhibits the NF-kappa B pathway that is involved in the suppression of erythroid specific genes. Proper chemical modification is required to consider Hb as an ESA.

P-23

INJECTABLE O-15 SYSTEM FOR OXYGEN METABOLISM STUDIES USING HEMOBLOBIN-VESICLE (HBV): AUTOMATIC LABELING AND APPLICATION IN RATS USING PET

Vijay, N. T., Okazawa, H., Kobayashi, M., Mori, T., Kasamatsu, S., Fujibayashi, Y.

Biomedical Imaging Research Center, University of Fukui, Fukui, Japan

E-mail: [email protected]

Artificial red blood cells have emerged as one of the most exciting and promising developments to substitute normal blood, at least for a transient period of time. Evidence from experimental and clinical studies suggests that they are stable enough in vivo and hence can be used as an oxygen carrier for diagnostic purposes. Intravenously injectable ¹⁵O-O₂ preparations could be more efficient in terms of utilization and safety than the established ¹⁵O-O₂ inhalation method for measuring hemodynamic parameters like cerebral blood flow (CBF), oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO₂) in the brain using positron emission tomography (PET). This study examined the possibility of developing an injectable ¹⁵O-O₂ system using hemoglobin vesicles (hbv), a type of artificial red blood cell, and later to measure brain oxygen consumption in rats using O-15 labeled hbv. Though primarily hbv were labeled with O-15 gas using direct bubbling, various other strategies were employed to enhance the labeling efficiency of hbv, including the use of a vacuum pump to eliminate helium fraction from target gas, bubbling combined with vortexing, and addition of L-Cysteine as a reductant to protect hemoglobin molecule from oxidation. L-Cysteine was found to play a significant role in increasing the binding of ¹⁵O-O₂ with hbv. A maximum radioactivity of 204 mbq/ml of hbv was obtained under optimal conditions. CMRO₂ values of 6.8 ± 1.4 ml.min^–1.100g^–1 (n = 5) were obtained in rats using a small animal PET scanner after the injection of ¹⁵O-O₂ labeled hbv. O-15 labeled hbv can be successfully utilized to measure brain oxygen consumption in rats using PET, which indicates that hbv is capable of carrying oxygen in vivo and subsequently in delivering it to cerebral tissue.

P-24

GLUCOCORTICOIDS INDUCE THE CD163 HEMOGLOBIN-SCAVENGER AND RELATED DETOXIFICATION PATHWAYS IN PERIPHERAL BLOOD MONOCYTES

Vallelian, F.¹, Schaer, C. A.¹, Kämpfer, T.¹, Schoedon, G.¹, Schaer, D. J.¹

¹Division of Internal Medicine, University Hospital, Zurich, Switzerland

E-mail: [email protected]

CD163 is the only known receptor-based pathway for clearance of cell-free native hemoglobin (Hb) and for some small molecular sized hbocs from the circulation. Glucocorticoids have been reported to have so far unexplained beneficial effects against Hb related toxicity in non-immune mediated hemolytic anemias. Therefore, we explored potential novel activities of glucocorticoids on monocyte Hb handling. Using LC-MS/MS based analysis of itraq^® labeled peptides and micro-array transcriptome analysis, we found that dexamethasone fundamentally skewed the monocyte phenotype towards enhanced Hb scavenger receptor activity and related heme detoxification pathways. Uptake and breakdown of Hb-Hp complexes by the highly induced CD163 scavenger receptor pathway becomes a primary function of glucocorticoid treated monocytes when exposed to extracellular Hb. Glucocorticoid treatment further modulates Hb regulated expression of numerous genes and protein products involved in anti-oxidative stress responses and iron homeostasis. As a confirmation of our in vitro results, we found that patients who have been treated with high doses of glucocorticoids for various hematologic and inflammatory conditions indeed had a significantly enhanced capacity of monocyte Hb-Hp uptake. Taken together, our data suggests that modulation of the endogenous receptor mechanism for extracellular Hb clearance by glucocorticoids or eventually by other pharmacologic pathways could be employed as a strategy to enhance Hb clearance and to reduce systemic Hb exposure in situations of severe hemolysis or after administration of an HBOC.

P-25

OXIDATIVE STRESS INDUCED BINDING OF HAEMOGLOBIN TO THE RED CELL MEMBRANE: A ROLE FOR NITRITE?

Welbourn, E., Nicholls, P., Wilson, M. T., Cooper, C. E.

Department of Biological Sciences, University of Essex, England

E-mail: [email protected]

Prolonged exercise causes increased haemolysis and modification of red cell membrane proteins, such as spectrin. Haemoglobin binding to red cell membranes is also enhanced. In vitro binding studies showed similar binding when red cell ghosts were treated with purified haemoglobin in the presence of hydrogen peroxide. This suggests oxidative stress triggers haemoglobin binding to the red cell membrane. To investigate this effect further, haemoglobin in isolated red cells was converted to methaemoglobin using sodium nitrite and the amount of binding to membranes measured. Bound haemoglobin increased by 277 ± 15% when measured as % of total protein, and this increased by a further 31 ± 8% when hydrogen peroxide was added after the nitrite. To determine if nitrite was acting solely via oxidising haemoglobin we compared the ability of purified methaemoglobin, made via nitrite or ferricyanide, to induce binding to red cell ghosts following peroxide treatment. Excess oxidant was removed via column chromatography prior to peroxide addition. There was a 71 ± 15% increase in haemoglobin binding to ghosts when nitrite was used to make methaemoglobin rather than ferricyanide. If the nitrite or ferricyanide was not removed from the incubation mixture the increase in binding rose to 171 ± 43%. This suggests that nitrite is altering haemoglobin chemistry in ways other than just oxidising the haem iron, possibly via additional protein modifications.

P-26

MODULATION OF NO BY EPO IMPROVES SURVIVAL OF CRITICALLY ISCHEMIC MUSCULOCUTANEOUS TISSUE

Wettstein, R.¹, Menger, M.², Harder, Y.³

¹Division of Plastic, Reconstructive and Aesthetic Surgery, CHUV University Hospital, Lausanne, Switzerland

²Institute for Clinical & Experimental Surgery, University of Saarland, Homburg/Saar, Germany

³Division of Plastic Reconstructive and Aesthetic Surgery, University Hospitals of Geneva, Switzerland

E-mail: [email protected]

Introduction: Critical soft tissue ischemia after surgery leads to increased morbidity secondary to wound dehiscence and tissue breakdown. With the administration of artificial oxygen carriers, microhemodynamic perfusion and oxygenation could be improved in the jeopardized tissues in the acute experimental setting. The aim of the present study was to test whether systemic administration of erythropoietin (EPO), the primary regulator of erythropoiesis can decrease soft tissue necrosis in a model of sustained acute ischemia and to define the underlying mechanisms.

Materials and Methods: 32 C57BL/6 mice equipped with a dorsal skin fold chamber containing musculocutaneous flap tissue prone to necrosis if untreated were administered either 500 IU or 5000 IU of EPO, saline or L-Name, an unspecific NO synthase antagonist. Capillary perfusion, leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis were determined over a 10-day observation period with intravital microscopy. Inos and enos were immunhistochemically analysed.

Results: A dose-dependent increase in microhemodynamic perfusion was observed with FCD 116 ±32 cm/cm² (EPO 500) vs. 44± 26 cm/cm² (EPO5000) and 9± 7 cm/cm² (saline) (p <0.05). Whereas endothelium-leukocyte interaction and apoptotic cell death was decreased in both EPO groups, EPO 500 significantly decreased tissue necrosis (16±4 EPO 500 vs. 52 ±4% saline, p <0.01). A significant increase in hematocrit occurred only with EPO5000 but not with EPO500. Administration of L-Name completely neutralized the effect of EPO500 on blood rheology and tissue survival.

Conclusion: EPO at the low, non-hematocrit relevant dose led to improved tissue survival. This can be attributed to a vasodilatory effect mediated by NO via upregulation of inos and enos. The increase in hematocrit in the high dose group and L-Name did abrogate the beneficial effect of EPO, reflecting the intricate interplay between blood rheology with NO and hematocrit, factors that have to be considered in the design of oxygen carriers.

P-27

BIORHEOLOGICAL PROPERTIES OF RECONSTRUCTED ERYTHROCYTES AND ITS FUNCTION OF CARRYING-RELEASING OXYGEN

Xiang, W., Wie, G., Li, Y., Weiyan, P., Jiaxin, X., Yaojin, L.

Bioengineering College of Chongqing University, Chongqing, China

E-mail: [email protected]

Erythrocyte shape and biomechanical properties have close relation to their physiological function. In this research the erythrocyte was reconstructed with natural structure protein and lipids based on cellular mechanics and hemorheology concepts. The biomechanical properties of the reconstructed erythrocyte were determined with the micropipette aspiration system. The shapes of reconstructed erythrocyte were obtained with an electron scanning microscope. The oxygen carrying-releasing function was analyzed with the HEMOX analyzer from TCS; the experimental results indicated that the reconstructed erythrocytes were similar to the natural erythrocyte, having biconcave disc shape, good deformability, and carrying-releasing oxygen function.

P-28

ENDOTHELIAL DYSFUNCTION ENHANCES VASOCONSTRICTION DUE TO NITRIC OXIDE SCAVENGING BY A HEMOGLOBIN-BASED OXYGEN CARRIER

Yu, B., Egorina, E., Sovershaev, M., Raher, M. J., Lei, C., Bloch, K. D., Zapol, W. M.

Anesthesia Center for Critical Care Research of the Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

E-mail: [email protected]

Background: At present, there is no FDA-approved hemoglobin(Hb)-based oxygen carrier (HBOC) to substitute for RBC transfusion in the treatment of hemorrhagic shock. Clinical application of hbocs has been stymied by the noxious adverse side-effects of nitric oxide (NO) scavenging. We hypothesized that some of the patients who have an MI or stroke after HBOC administration have an underlying deficiency of endothelial NO production (endothelial dysfunction), which sensitizes them to the NO scavenging effects of HBOC, with enhanced vasospasm after HBOC challenge.

Materials and Methods: polyheme® was obtained from Northfield Laboratories (Evanston, IL). Murine tetrameric Hb was prepared from lysed murine whole blood with a final concentration of 4 g/dl. Hb was given as a top load (16% blood volume) via a tail vein. Systolic blood pressure (SBP) was measured in awake standard diet fed wild type mice (WT, C57BL6), WT mice fed a high fat diet for 4–6 weeks, and diabetic mice (db/db) by tail-cuff in awake mice, and hemodynamics were measured invasively in anesthetized mice.

Results: Intravenous administration of polyheme (1.08 g/kg) did not produce vasoconstriction in normal WT mice, but produced systemic hypertension and vasoconstriction in both high-fat fed and db/db mice. These findings were confirmed using invasive hemodynamic methods at thoracotomy. We observed greater sensitivity to systemic vasoconstriction in db/db as compared to WT mice to infusion of low doses of tetrameric Hb. Pretreatment with inhaled NO (80 ppm, 1 hr) prevented polyheme-induced hypertension in awake db/db mice.

Conclusions: Endothelial dysfunction sensitizes mice to the NO scavenging effects of hemoglobin-based oxygen carriers. Our results suggest that there is a need for testing hbocs in animals with endothelial dysfunction, since db/db and fat fed mice provide a more sensitive test of the adverse vasoconstrictor effects of hbocs seen in patients with vascular disease.

P-29

HIGH VISCOSITY ALGINATE PLASMA EXPANDER MAY INDUCE AGGREGATION OF RED BLOOD CELLS

Zhao, L.^1¼, You, G.^1¼, Liao, F.², Wang, B.¹, Sun, Q.², Xu, H.¹, Han, D.^2*, Zhou, H.^1*

¹Department of Immunohematology, Beijing Institute of Transfusion Medicine, Beijing, China

²National Center for Nanoscience and Technology, Beijing, China

E-mail: [email protected]

Many studies have shown that a high viscosity plasma expander (HVPE) is beneficial in extreme hemodilution as well as in hemorrhagic shock. Sodium alginate (SA) at a comparatively low concentration may induce high plasma viscosity with a low colloid oncotic pressure (COP). Therefore, SA is always mentioned as a good “viscosity modifier” in the study of HVPE. However, some detailed effects of SA on the rheological behaviors of red blood cell (RBC, are yet poorly understood. In the present study, whole blood was diluted using SA solutions to final hematocrits of 10%, 20% and 35%, respectively. Whole blood viscosity (WBV) at different shear rates, plasma viscosity (PV), and rheological behavior of RBC was studied in vitro. The results showed that replacement of plasma with the solution of 0.6% SA in 0.9% nacl (A0.6%) significantly elevated WBV at different hematocrits. Replacement of plasma with A0.6% and the solution of 0.3% SA in 0.9% nacl (A0.3%) at hematocrit of 35% revealed significant increases in WBV and PV compared with the solution of 6% Dextran 70 in 0.9% nacl (D6%). Moreover, at the hematocrit of 35%, A0.6% revealed significant increases in RBC aggregation index compared with D6%. The RBC morphology was consistent with the aggregation index measurement. These results showed that SA may induce RBC aggregation in a dose-dependent manner, indicating that it can be used as an alternative leading to RBC aggregation, and the effect of SA on RBC aggregation may be involved in the regulation of microcirculation.