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IV. KEYNOTE LECTURES (KL)

Artificial Cells, Blood Substitutes, and Biotechnology, 36: 187207, 2008ISSN: 1073-1199 print / 1532-4184 onlineDOI: 10.1080/10731190802123855

Keynote Lectures (KL)

KL-1 Oxygen: The Poison is in the Dose

Professor Robert M. Winslow, M.D.President, Sangart, Inc.San Diego, CA. USA

Adjunct Professor of Medicine, UCSD, CA, [email protected]

Some clinical trials of early-generation hemoglobin-based oxygen carriers haveshown increased incidences of myocardial infarction. This seems a paradoxicalobservation, since hemoglobin-based solutions have been shown to deliveroxygen to tissues in animal models, leading to the expectation that myocardialoxygenation would be increased. Here, the hypothesis is presented that early-generation, cell-free oxygen carriers oversupply oxygen to tissues and that such

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188 Keynote Lectures

oversupply may lead to oxygen-related toxicity, which may include myocardialischemia and infarction.

The evidence for this hypothesis comes from various sources, includ-ing a consideration of the atmospheric O2 levels at the time that O2 deliverymechanisms evolved, experience with hyperbaric oxygen, artificial capillaries,and the body of research that establishes that tissue oxygen levels are main-tained over very narrowand lowlevels. Recent data demonstrates that theresponse to increased inspired O2 is similar to the response to infusion ofhighly diffusive hemoglobin molecules with high P50 (e.g., -crosslinkedhemoglobin) in contrast to less diffusive molecules with low P50 (e.g., PEG-modified hemoglobin).

The essential elements of the oxygen transport system in mammals includered blood cells, low plasma oxygen solubility, barriers to O2 diffusion, and thecontrol of microvascular perfusion. Introduction of a cell-free oxygen carrierdisrupts this system, engaging mechanisms that protect against O2 toxicity,including vasoconstriction and reduced perfusion. If this happens in an area oftissue that already has marginal or compromised blood flow, exacerbation oflocal hypoxia and infarction could result. The hypothesis predicts that a suc-cessful cell-free oxygen carrier will limit oxygen release at vascular PO2 abovethat in metabolically active tissues and be most effective at low concentrationwhere some red blood cell oxygen reserve is still present.

Keynote Lectures 189

KL2 Understanding Causality in HBOC Clinical Trials: The role ofClinical Contextualization

Professor A. G. Greenburg, M.D., Ph.D.Vice President, Biopure Corporation, Boston. [email protected]

Professor Emeritus of Surgery, Brown University, Rhode Island, U.S.A.

Establishing an acceptable safety and efficacy profile for hemoglobin basedoxygen carriers, HBOCs, is essential for regulatory agency approval and clini-cal acceptance. Consideration beyond the counting of events typically asso-ciated with intent to treat analysis is required to fully understand the safetyprofile. The imputation of causation from numerical counts alone is an in-complete analysis of a complex situation. Clinical contextualization of thecritical significant adverse events provides a more complete understanding ofthe clinico-pathological processes underlying their emergence. Alternative hy-potheses for the origin of the events, apart from the drug, patient co-morbidities,situational and disease demands, could include protocol design, failure to pro-vide mitigation strategies, patient management issues, or inadequate educationof investigators. How clinical contextualization aids the interpretation of safetyprofile events will be discussed in the context of a large phase III clinical trialwith an appreciation of underlying factors between intent to treat analysis andother approaches.


KL-3 Postinjury Resuscitation with Human Polymerized Hemoglobin:The USA Multicenter Trial

Steven Gould, M.D., Ph.D.,President, Northfield, U.S.A.

Professor of Surgery. [email protected]

INTRODUCTION: Human polymerized hemoglobin (PolyHemeR), a univer-sally compatible O2 carrier, may be useful in the early treatment of hemorrhagicshock when stored blood is not available. This Phase III trial is the first U.S.A.study to assess survival of patients resuscitated with PolyHeme in lieu of bloodor the current standard of care. METHODS: Trauma patients with SBP


patients died; 61 (74%) on Day 1. Major protocol violations occurred in 71PolyHeme and 53 Control, leaving 590 protocol compliant patients.

PolyHeme Control(survivors/N) (survivors/N) p-value

Day 1 Protocol Compliant259/279 (92.8%) 289/311 (92.9%) 1.00Protocol Violators 57/71 (80.3%) 48/53 (90.6%) 0.14All Patients 316/350 (90.3%) 337/364 (92.6%) 0.29

Day 30Protocol Compliant 248/279 (88.9%) 282/311 (90.7%) 0.50Protocol Violators 55/71 (77.5%) 47/53 (88.7%) 0.15All Patients 303/350 (86.6%) 329/364 (90.4%) 0.13

CONCLUSIONS: There was no statistically significant difference in survivalbetween patients receiving PolyHeme without blood for up to 12 hours fol-lowing injury and those receiving the standard of care including early blood.These data suggest that PolyHeme can be useful when blood is needed but notavailable, thereby addressing a critical unmet medical need.


KL-4 Introduction of Development on Transfusion Medicine in China

Chengmin Yang, Lishen Du, Jiaxin [email protected]

Institute of Transfusion Medicine Chengdu,CAMS & PUMC President, Tianjin Uion Biotechnology Lab. LTD. Tianjin

300457 China

For last decade, the development of blood transfusion medicine in China, sim-ilar to these advances around world, has made considerable progress. Thispresentation reviews recent developments and current status of blood transfu-sion medicine in the fields of Chinese blood groups, government regulationsand laws, clinical blood transfusion, blood transfusion safety, scientific researchin blood transfusion medicine, plasma products and information managementin blood transfusion service. Under the Chinese government regulations andefforts from all levels of blood transfusion research and the service organizationas well as the medical stuffs working in blood transfusion medicine, China hasmade remarkable improvement in blood transfusion safety by enforcing safetypolicies and quality controls, although there still need more work to be doneto reach the same standards as developed countries. This meeting provides us


a platform for academic exchange and an opportunity for foreign colleagues toknow China. We hope this presentation will stimulate a wide discussion, likea Chinese expression throws the brick to lead the jade, regarding advanceof transfusion medicine in China. Welcome all the friends in the field to paygreater concern and the support China transfusion medicine. (Thanks very muchto prof. Liu wenfang, Gao feng, Tian zhaoshong, Ji yang and J.of TransfusionMedieine for that they are friendly providing seientific informations.)


KL-5 Process Engineering of Protein-Based Oxygen Carriers

Zhiguo Su, Ph.D. [email protected], National Key Laboratory of Biochemical Engineering,Institute of Process Engineering, Chinese Academy of Science,

P. O. Box 353, Beijing, 100080, P. R. ChinaE-mail: [email protected]

Preparation of protein-based oxygen carriers usually involves three stages in-cluding protein extraction and purification, protein modification or encapsula-tion and post-separation of the modified/encapsulated product mixture. Becauseof the possible high dosage in medical applications such as emergency rescuewhere grams quantity of the protein is infused, the quality requirement forprotein-based oxygen carries is very strict. The three stages of the preparationneed to be carefully controlled to ensure the end product free from any potentialdanger or side effects by impurities. The impurities may come from the hostwhere the protein is extracted, from the process of crosslinking, conjugationand encapsulation, from the contamination by the environment, or even fromthe protein itself after structure change and denaturation. Process engineeringis required to design simple, fast, risk-free bioseparation and bioreaction toguarantee an optimal production line that is safe and economical. Strategiesfor making pegylated proteins, hemoglobin-hemoglobin conjugates, albumin-hemoglobin conjugates, artificial cells containing proteins are discussed.


KL-6 Recovery of Microvascular Perfusion & Oxygen CarryingCapacity: Priorities, Alternatives and Goals in Designing And Using

Blood Substitutes

M. Intaglietta, Ph.D., Professor, Department of BioengineeringUniversity of California, San Diego, CA. USA. [email protected]

P Cabrales, BY Salazar Vazquez, A Chavez Negrete, AG Tsai, M Intaglietta,La Jolla Bioengineering Institute, Faculty of Medicine, Universidad Juarez

del Estado de Durango, DGO, Mexico, Instituto Mexicano del Seguro Social(IMSS), Mexico, D.F., Department of Bioengineering, University of

California, San Diego, CA

Blood losses lower oxygen carrying capacity and circulating blood volume. Thefirst is in excess, while volume losses are minimally tolerated, therefore plasmavolume expansion is the priority in remedying blood losses. Optimal plasmaexpansion requires sustaining microvascular perfusion and function in hemodi-lution. Restoring oxygen carrying/delivery capacity results from the product ofquantity of oxygen carried and its rate of delivery to the tissue, independentlyof the relative magnitude of the factors. Thus a blood substitute with modestintrinsic oxygen carrying capacity that via optimal plasma expansion enhancesperfusion is as efficacious in delivering oxygen as maximally increasing in-trinsic oxygen carrying capacity with marginal plasma expansion properties.Furthermore, enhanced perfusion improves resuscitation, since it restores and


may even increase release of vasoactive mediators from the endothelium, whileenhancing metabolites removal from the tissue. Present blood substitutes pro-vide a continuous spectrum of transport properties beyond oxygen carryingcapacity per se, including viscogenic effects and endothelial vasoactive me-diation leading to enhanced generalized perfusion. Engineering these factorsallows designing for an optimal physiological/resuscitative function while low-ering oxygen carrier use and its cost.


KL-7 Breathing nitric oxide or intravenous administration of nitriteprevents systemic vasoconstriction caused by challenge with murine

tetrameric hemoglobin or HBOC-201 in awake mice and lambs

Professor Warren M. Zapol,Institute of Medicine of the National Academy of Sciences

Binglan Yu, Kenneth D. Bloch, Fumito Ichinose and Warren M. [email protected]

Anesthesia Center for Critical Care Research of the Department of Anesthesiaand Critical Care, Massachusetts General Hospital, Harvard Medical School,

Boston, Massachusetts, USA 02114

One of the major obstacles hindering the clinical development of a cell-free,hemoglobin (Hb)-based oxygen carrier (HBOC) is systemic vasoconstriction.Breathing nitric oxide (NO) increases plasma nitrite concentrations and at-tenuates the HBOC-induced vasoconstriction. However, NO inhalation at 80ppm concurrently with HBOC administration oxidizes the plasma Hb impair-ing its ability to carry oxygen. We tested the hypothesis that pretreatment withinhaled NO or intravenously administered nitrite would prevent the systemicvasoconstriction associated with subsequent challenge with murine tetramericHb.

Studies were carried out in awake mice, mice congenitally deficient innitric oxide synthase (NOS2) and awake lambs. We infused both tetramerichemoglobin and in some studies HBOC-201 (Hemopure). Tetrameric Hb (4


g/dl) prepared from murine whole blood was administered IV (0.012 ml/gBW). Systolic blood pressure was measured in awake mice by tail-cuff. Priorto administration of tetrameric Hb, mice breathed air alone or air supplementedwith 80 ppm NO. In a second group of mice PBS (50 l, pH 7.4) containing48 nmol of sodium nitrite or PBS alone was administered via a tail vein andwas followed 5 min later by infusion of tetrameric Hb.

Intravenous (IV) infusion of tetrameric Hb induced prolonged systemicvasoconstriction in mice (118 3 to143 7 mmHg). Breathing NO at 80ppm in air for 15 or 60 min or 200 ppm for 7 min prevented the systemichypertension induced by subsequent IV administration of murine tetramericHb and did not result in conversion of plasma Hb to metHb (2 1% at 15min and 3 1% at 60 min). IV administration of sodium nitrite 5 min beforeinfusion of murine tetrameric Hb also prevented the development of systemichypertension. However, 10 min after murine tetrameric Hb infusion, the plasmametHb level increased to 10 2%.

Our findings demonstrate that pretreatment with inhaled NO can preventthe systemic hypertension induced by murine tetrameric Hb. In contrast toconcurrent NO inhalation, pretreatment with inhaled NO does not oxidizethe plasma Hb. Similarly infusion of nitrite, administered before the IV infu-sion of HBOCs, can prevent systemic vasoconstriction. Additional studies ofNO inhalation before and during the infusion of HBOC-201 in awake lambswill be presented. Pretreatment with inhaled NO or nitrite may enable furtherclinical development of HBOCs for the treatment of patients with anemia orhemorrhage.


KL-8 Functional and Physiological Characterization ofGlutaraldehyde-Polymerized Porcine Hemoglobin

Professor Chao Chen, Ph.D. Vice-President, Northwest UniversityBaoping Wu, Xiaoli Zhu, Kunping Yan, Ning Dan and Chao Chen*,

[email protected] Engineering Research Center for Miniaturized detection Systems,

Shaanxi Lifegen Co., Ltd. Northwest University

The vasoconstriction and other side effects induced by hemoglobin-based oxy-gen carriers (HBOCs) have limited the HBOCs application as an oxygen-carrying plasma expander. In this study, a new type of nonvasoactive HBOCswith low P50 has been prepared. Using porcine blood as the raw material, theultra-pure porcine hemoglobin (pHb) was obtained through multiple purifica-tion steps, which was found to have very similar oxygen affinity and mod-ulating mechanism as human hemoglobin comparing with bovine one. Withglutaraldehyde as cross-linker, polymerized porcine hemoglobin (pPolyHb)was produced. In addition, various physical and chemical properties, such asmolecular weight distribution, oxygen affinity and auto-oxidation tendency ofthe hemoglobin polymer were analyzed. This pPolyHb has a special property oflower P50 and higher average molecular weight than other products reported.Meanwhile, the pPolyHb has long vascular retention time (half-life =18 h).


The mean arterial blood pressure (MAP) has no significant change during thetransfusion of 50% isovolemic exchange in rats and can restore rapidly in thestudy of anti-hemorrhagic shock function of the expander in rat model. Theporcine hemoglobin did not elicit detectable specific serum IgG antibody re-sponse in rats, mice and rabbits after repeatedly subcutaneous or intraperitonealadministration of the protein samples in the absence of an adjuvant. Intravenousadministration of therapeutic dosage of pPolyHb may induce specific immunetolerance towards this protein, but not affect immune functions in general inthe experimental animals. The details of characterization of pPolyHb will befurther discussed during the conference.


KL-9 Physicochemical Characteristics and Physiological Performancesof Artificial Oxygen Carriers (Hb-Vesicles And Albumin-Hemes)

Professor Eishun Tsuchida. Ph.D. [email protected] Professor & Principal Investigator of the Oxygen Infusion Project,

Advanced Research Institute for Sci. & Eng.,Waseda University, Tokyo, Japan

During the long history of development of Hb-based O2 carriers, many sideeffects of molecular Hbs have become apparent. They imply the physiologicalimportance of the cellular structure of RBCs. It is expected that Hb-vesicles(HbV) can solve these side effects. On the other hand, there are intrinsic issuesof HbV as a molecular assembly, such as stability for storage and in blood circu-lation, blood compatibility, and prompt degradation in the reticuloendothelialsystem (RES). In situ physicochemical characterization clarified the narrowsize distribution, the thin lipid bilayer membrane, and conc. Hb solution in-side the HbV. The HbV suspension shows peculiar viscoelasticity and ligandsbinding properties owing to the particle dispersion. Surface modification ofHbV with PEG chains ensures the stable dispersion state and the stability forstorage over a year at room temperature in a deoxygenated condition. In vivoanimal tests clarified (i) the biodistribution of HbV and the prompt metabolismand degradation of HbV in RES, (ii) physiological significance of the cel-lular structure of HbV in microcirculation, (iii) the efficacy as a transfusionalternative for hemorrhagic shock and extracorporeal cardiopulmonary bypassprime, (iv) tailor-made low P50-HbV for oxygenation of ischemic tissues, and(v) other possible clinical applications to transport oxygen that is essential forrespiration of tissue cells, such as a perfusate for organ transplant, and a tissueregeneration. On the other hand, totally synthetic heme albumin-based oxygen


carriers have also been developed. Surface modification of albumin-heme byPEG significantly prolonged the circulation lifetime of the heme in the bloodstream. Recently, we have found that a natural protoheme IX can also be usedas an oxygen binding site of this artificial hemoprotein. Mutation of Ile-142 byhistidine confers the oxygen binding capability to the protoheme. The oxygen-binding affinity was modulated by the variety of the distal amino acid in theheme pocket.


KL-10 Free Oxygen Flow a New Paradigm

Professor Enrico Bucci, Ph.D. [email protected] Emeritus, Dept of Biochemistry and Molecular Biology University

of Maryland Medical School, Baltimore, MD, USA

The classic view of oxygen transport by blood is focused on red cell hemoglobincarrying oxygen from lungs to tissues where it diffuses through the capillaries.It disregards the flow of free oxygen through blood. This is the focus of thenew paradigm. In a steady state where all circulatory parameters are kept con-stant, blood may be considered a long tubular liquid membrane, surrounded byvascular walls, interfacing alveolar oxygen and interstitial fluid. Consumptionat the periphery drives a flow of oxygen with a gradient from 120 torr in thelungs alveoli, to 2 torr or less in cells mitochondria drives a flow of free oxy-gen through blood. Due to the low water solubility of oxygen, this gradient istoo shallow to sustain metabolic consumption. Natural cell-segregated oxygencarriers, hemoglobin and myoglobin, regulate the flow into sustenance by re-placing molecule by molecule the oxygen consumed by metabolism. Thus, theybuffer the oxygen tension at levels near their P50: near 30 torr at the capillariesby the red cells, and near 2 torr by myoglobin in the muscles interstitial fluid.This continuous buffering results in a steady delivery of the large amounts offree oxygen necessary for metabolism, still at low non toxic partial pressure.Due to a low diffusion constant, there is a limited permeability of the arterialwalls to oxygen, thereby preserving it for peripheral use. Cell free oxygen car-riers facilitate oxygen diffusion in plasma, while bathing the endothelial walls.Therefore they replace consumed oxygen at much faster rate than cell bound


carriers. For all carriers, the amount of replaced oxygen does not depend ontheir P50. It depends only on the total oxygen carrying mass of available car-rier. Their P50s only regulate the pressure range at which oxygen replacementoccurs. The free energy loss that drives the oxygen gradient increases oxygensolubility in body fluids, adding a regulatory parameter to the amount of freeoxygen available to metabolism. In the absence of myoglobin, which buffersoxygen tension at 2 torr, tissues like arterial walls and brain are exposed tohigher oxygen pressure. This may contribute to adverse clinical events.


HL-11 Clinical Application of Cellular Type Artificial OxygenCarrier-Hemoglobin

Vesicle (Hbv)

Professor K. Kobayashi, M.D., Ph.D., Keio University

K. Kobayashi1, H. Horinouchi1, M. Watanabe1, Y. Izumi1, M. Kohno1,T. Ikeda1, N. Izawa1, Y. Yozu1, E. E. Tsuchida2 and H. Sakai2

[email protected])Professor, Division of General Thoracic Surgery, Department of Surgery,School of Medicine, Keio University, Tokyo, Japan 2)Advanced ResearchInstitute for Science and Engineering, Waseda University, Tokyo, Japan

Hemoglobins recovered from human red blood cells are encapsulated in bilayerliposomes to make cellular type artificial oxygen carrier to be used regardlessof patients blood type.

The size of this hemoglobin vesicle (HbV) is about 250 nm.HbV can bestored in a liquid state at room temperature for over two years. This stabilityis oftained by deoxygenation to prevent metHb formation and surface mod-ification with polyethylene glycol. Oxygen carrying capacity, and safety areexamined in small animals and beagles. The results of these experiments showthat our HbV may be used in following clinical settings. Hemorrhagic shockperfusate for cardiopulmonary bypass tumor oxygenation to increase sensitivityto chemotherapy and radiotherapy prevention of tissere ischemia.


KL-12 Microcirculation can be a Wonderful Mirror for BloodSubstitutes Performance

Professor Ruijuan Xiu, M.D., Ph.D. [email protected] ISBS Executive Vice President Director, Institute of Microcirculation,Chinese Academy of Medical Sciences & Peking Union Medical College,

Beijing 100005, ChinaOur early clinical and experimental studies on endotoxin shock during fulmi-nant epidemic meningitis among children and acute Cor pulmonale showedthat the microcirculation acts as a Mirror reflecting severe spasm and con-striction of the arterioles and precapillaries in producing local ischemia andanoxemia, which can lead to respiratory failure or coma and even death de-pending on whether the lung or brain was affected. In 12 cor pulmonale patientswith diminished nail-bed microcirculation, 6 had encephalopathy and one hadhemiplegia. The capillary density (CD) on the nailfold was a important param-eter for evaluation of the degree of blood perfusion in tissue and organs of thispatients. Expansion blood volume was one of the key diplomacy to save pa-tients life. It was found, that the key medicine used to save these patients fromendotoxin shock was not vaso-dilator, but microvascular vasomotion regulator.

In 19871988, the author studied the vascular perturbations associ-ated with AIDs in San Francisco and Stockholm (Ruslagstull Hospital).Video-microscopy of the nail-fold microcirculation was performed on 11American and European AIDS patients and 11 healthy European adults. Severe


microvascular dysfunction was demonstrated in the AIDS patients: Normalspontaneous fluctuations in the control capillary blood flow were not present.The endothelial cells in the capillary wall were damaged significantly. Perme-ation of the capillary wall by a heterogeneous mass made the vessel boundariesindistinct and serving to narrow the lumen. A computer image analysis indi-cated part of the heterogeneous mass was in the endothelial cells cytoplasm.Extravasation of red blood cells was seen in three patients. All patients werefound to be suffering from Kaposis sarcoma.

Based on the above studies, in 1985, a National Key Lab was established inthe CAMS. A serial of experments was done in order to investigate the signif-icance of microcirculatory dysfunction in the pathogenesis of major diseases.The role of O2 in treatment of ischemic diseases was emphasized, becausemany such patients received abundant O2 via inhalation at the last stage oftheir life while the capillary loops were dilated and filled with oxygen saturatedblood. It was found that as bath PO2 was increased, a progressive constrictionof A1, A2 and A3 order arterioles in the cremaster muscle of normotensiveand hypertensive rats occurred. Maximum constriction occurred at bath PO2 of40-60mmHg. At bath PO2 beyond 80mmHg, the constricted vessels began todilate and irreversible dilation could be caused when bath PO2was higher than90mmHg. It was also found that H2O2 can directly induce the hypertrophicresponse of neonatal Wistar rat cardiomyocytes, and H2O2 mediated the reg-ulation of cardiomyocyte hypertyophy through different isoforms of PKC. Insummary, it is desirable that blood substitutes act as Oxygen Carriers, but it isimportant to establish the optimal capacity of these Oxygen Carriers.

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