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, USA [email protected]
Some clinical trials of early-generation hemoglobin-based oxygen carriers have shown increased incidences of myocardial infarction. This seems a paradoxical observation, since hemoglobin-based solutions have been shown to deliver oxygen to tissues in animal models, leading to the expectation that myocardial oxygenation would be increased. Here, the hypothesis is presented that early-generation, cell-free oxygen carriers oversupply oxygen to tissues and that such oversupply may lead to oxygen-related toxicity, which may include myocardial ischemia and infarction.
The evidence for this hypothesis comes from various sources, including a consideration of the atmospheric O2 levels at the time that O2 delivery mechanisms evolved, experience with hyperbaric oxygen, artificial capillaries, and the body of research that establishes that tissue oxygen levels are maintained over very narrow—and low—levels. Recent data demonstrates that the response to increased inspired O2 is similar to the response to infusion of highly diffusive hemoglobin molecules with high P50 (e.g., □□-crosslinked hemoglobin) in contrast to less diffusive molecules with low P50 (e.g., PEG-modified hemoglobin).
The essential elements of the oxygen transport system in mammals include red blood cells, low plasma oxygen solubility, barriers to O2 diffusion, and the control of microvascular perfusion. Introduction of a cell-free oxygen carrier disrupts this system, engaging mechanisms that protect against O2 toxicity, including vasoconstriction and reduced perfusion. If this happens in an area of tissue that already has marginal or compromised blood flow, exacerbation of local hypoxia and infarction could result. The hypothesis predicts that a successful cell-free oxygen carrier will limit oxygen release at vascular PO2 above that in metabolically active tissues and be most effective at low concentration where some red blood cell oxygen reserve is still present.
KL2 Understanding Causality in HBOC Clinical Trials: The role of Clinical 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 based oxygen carriers, HBOCs, is essential for regulatory agency approval and clinical acceptance. Consideration beyond the counting of “events” typically associated with intent to treat analysis is required to fully understand the “safety profile.” The imputation of causation from numerical counts alone is an incomplete analysis of a complex situation. Clinical contextualization of the critical significant adverse events provides a more complete understanding of the clinico-pathological processes underlying their emergence. Alternative hypotheses for the origin of the events, apart from the drug, patient co-morbidities, situational and disease demands, could include protocol design, failure to provide mitigation strategies, patient management issues, or inadequate education of investigators. How clinical contextualization aids the interpretation of safety profile events will be discussed in the context of a large phase III clinical trial with an appreciation of underlying factors between intent to treat analysis and other 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 (PolyHeme®), a universally compatible O2 carrier, may be useful in the early treatment of hemorrhagic shock 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 blood or the current standard of care. METHODS: Trauma patients with SBP < 90 mmHg were randomized under a waiver of consent to receive initial field resuscitation with PolyHeme or crystalloid. PolyHeme patients continued to receive up to 6 units of PolyHeme during the first 12 hours post injury before receiving blood. Control patients received blood early upon arrival in the ED. RESULTS: The trial enrolled 714 patients at urban Level I trauma centers in 19 states, and involved more than 3500 EMTs and 300 ambulances. Demographics included: 78% males with mean age of 36.3 years in PolyHeme versus 79% males and 37.9 years in Control. Mechanism was blunt injury in 47% PolyHeme and 48% Control. Median transport time was 26 minutes in both groups. Overall, 82 patients died; 61 (74%) on Day 1. Major protocol violations occurred in 71 PolyHeme and 53 Control, leaving 590 protocol compliant patients.
CONCLUSIONS: There was no statistically significant difference in survival between patients receiving PolyHeme without blood for up to 12 hours following injury and those receiving the standard of care including early blood. These data suggest that PolyHeme can be useful when blood is needed but not available, thereby addressing a critical unmet medical need.
KL-4 Introduction of Development on Transfusion Medicine in China
Chengmin Yang, Lishen Du, Jiaxin Liu [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, similar to these advances around world, has made considerable progress. This presentation reviews recent developments and current status of blood transfusion medicine in the fields of Chinese blood groups, government regulations and laws, clinical blood transfusion, blood transfusion safety, scientific research in blood transfusion medicine, plasma products and information management in blood transfusion service. Under the Chinese government regulations and efforts from all levels of blood transfusion research and the service organization as well as the medical stuffs working in blood transfusion medicine, China has made remarkable improvement in blood transfusion safety by enforcing safety policies and quality controls, although there still need more work to be done to reach the same standards as developed countries. This meeting provides us a platform for academic exchange and an opportunity for foreign colleagues to know China. We hope this presentation will stimulate a wide discussion, like a Chinese expression “throws the brick to lead the jade”, regarding advance of transfusion medicine in China. Welcome all the friends in the field to pay greater concern and the support China transfusion medicine. (Thanks very much to prof. Liu wenfang, Gao feng, Tian zhaoshong, Ji yang and “J.of Transfusion Medieine” for that they are friendly providing seientific informations.)
KL-5 Process Engineering of Protein-Based Oxygen Carriers
Zhiguo Su, Ph.D. [email protected]
Professor, National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Science, P. O. Box 353, Beijing, 100080, P. R. China E-mail: [email protected]
Preparation of protein-based oxygen carriers usually involves three stages including protein extraction and purification, protein modification or encapsulation and post-separation of the modified/encapsulated product mixture. Because of the possible high dosage in medical applications such as emergency rescue where grams quantity of the protein is infused, the quality requirement for protein-based oxygen carries is very strict. The three stages of the preparation need to be carefully controlled to ensure the end product free from any potential danger or side effects by impurities. The impurities may come from the host where the protein is extracted, from the process of crosslinking, conjugation and encapsulation, from the contamination by the environment, or even from the protein itself after structure change and denaturation. Process engineering is required to design simple, fast, risk-free bioseparation and bioreaction to guarantee an optimal production line that is safe and economical. Strategies for making pegylated proteins, hemoglobin-hemoglobin conjugates, albumin-hemoglobin conjugates, artificial cells containing proteins are discussed.
KL-6 Recovery of Microvascular Perfusion & Oxygen Carrying Capacity: Priorities, Alternatives and Goals in Designing And Using Blood Substitutes
M. Intaglietta, Ph.D., Professor, Department of Bioengineering
University of California, San Diego, CA. USA. [email protected]
P Cabrales, BY Salazar Vázquez, A Chávez 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. The first is in excess, while volume losses are minimally tolerated, therefore plasma volume expansion is the priority in remedying blood losses. Optimal plasma expansion requires sustaining microvascular perfusion and function in hemodilution. Restoring oxygen carrying/delivery capacity results from the product of quantity of oxygen carried and its rate of delivery to the tissue, independently of the relative magnitude of the factors. Thus a blood substitute with modest intrinsic oxygen carrying capacity that via optimal plasma expansion enhances perfusion is as efficacious in delivering oxygen as maximally increasing intrinsic 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, while enhancing metabolites removal from the tissue. Present blood substitutes provide a continuous spectrum of transport properties beyond oxygen carrying capacity per se, including viscogenic effects and endothelial vasoactive mediation leading to enhanced generalized perfusion. Engineering these factors allows designing for an optimal physiological/resuscitative function while lowering oxygen carrier use and its cost.
KL-7 Breathing nitric oxide or intravenous administration of nitrite prevents 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. Zapol [email protected]
Anesthesia Center for Critical Care Research of the Department of Anesthesia and 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 attenuates the HBOC-induced vasoconstriction. However, NO inhalation at 80 ppm concurrently with HBOC administration oxidizes the plasma Hb impairing its ability to carry oxygen. We tested the hypothesis that pretreatment with inhaled NO or intravenously administered nitrite would prevent the systemic vasoconstriction associated with subsequent challenge with murine tetrameric Hb.
Studies were carried out in awake mice, mice congenitally deficient in nitric oxide synthase (NOS2) and awake lambs. We infused both tetrameric hemoglobin and in some studies HBOC-201 (Hemopure). Tetrameric Hb (4 g/dl) prepared from murine whole blood was administered IV (0.012 ml/g BW). Systolic blood pressure was measured in awake mice by tail-cuff. Prior to administration of tetrameric Hb, mice breathed air alone or air supplemented with 80 ppm NO. In a second group of mice PBS (50 μ l, pH 7.4) containing 48 nmol of sodium nitrite or PBS alone was administered via a tail vein and was followed 5 min later by infusion of tetrameric Hb.
Intravenous (IV) infusion of tetrameric Hb induced prolonged systemic vasoconstriction in mice (118 ± 3 to143 ± 7 mmHg). Breathing NO at 80 ppm in air for 15 or 60 min or 200 ppm for 7 min prevented the systemic hypertension induced by subsequent IV administration of murine tetrameric Hb and did not result in conversion of plasma Hb to metHb (2 ± 1% at 15 min and 3 ± 1% at 60 min). IV administration of sodium nitrite 5 min before infusion of murine tetrameric Hb also prevented the development of systemic hypertension. However, 10 min after murine tetrameric Hb infusion, the plasma metHb level increased to 10 ± 2%.
Our findings demonstrate that pretreatment with inhaled NO can prevent the systemic hypertension induced by murine tetrameric Hb. In contrast to concurrent NO inhalation, pretreatment with inhaled NO does not oxidize the plasma Hb. Similarly infusion of nitrite, administered before the IV infusion of HBOCs, can prevent systemic vasoconstriction. Additional studies of NO inhalation before and during the infusion of HBOC-201 in awake lambs will be presented. Pretreatment with inhaled NO or nitrite may enable further clinical development of HBOCs for the treatment of patients with anemia or hemorrhage.
KL-8 Functional and Physiological Characterization of Glutaraldehyde-Polymerized Porcine Hemoglobin
Professor Chao Chen, Ph.D. Vice-President, Northwest University
Baoping Wu, Xiaoli Zhu, Kunping Yan, Ning Dan and Chao Chen*, [email protected]
National Engineering Research Center for Miniaturized detection Systems, Shaanxi Lifegen Co., Ltd. Northwest University
The vasoconstriction and other side effects induced by hemoglobin-based oxygen carriers (HBOCs) have limited the HBOCs application as an oxygen-carrying plasma expander. In this study, a new type of nonvasoactive HBOCs with low P50 has been prepared. Using porcine blood as the raw material, the ultra-pure porcine hemoglobin (pHb) was obtained through multiple purification steps, which was found to have very similar oxygen affinity and modulating mechanism as human hemoglobin comparing with bovine one. With glutaraldehyde as cross-linker, polymerized porcine hemoglobin (pPolyHb) was produced. In addition, various physical and chemical properties, such as molecular weight distribution, oxygen affinity and auto-oxidation tendency of the hemoglobin polymer were analyzed. This pPolyHb has a special property of lower 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 the transfusion of 50% isovolemic exchange in rats and can restore rapidly in the study of anti-hemorrhagic shock function of the expander in rat model. The porcine hemoglobin did not elicit detectable specific serum IgG antibody response in rats, mice and rabbits after repeatedly subcutaneous or intraperitoneal administration of the protein samples in the absence of an adjuvant. Intravenous administration of therapeutic dosage of pPolyHb may induce specific immune tolerance towards this protein, but not affect immune functions in general in the experimental animals. The details of characterization of pPolyHb will be further discussed during the conference.
KL-9 Physicochemical Characteristics and Physiological Performances of Artificial Oxygen Carriers (Hb-Vesicles And Albumin-Hemes)
Professor Eishun Tsuchida. Ph.D. [email protected]
Emeritus 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 side effects of molecular Hbs have become apparent. They imply the physiological importance 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 issues of HbV as a molecular assembly, such as stability for storage and in blood circulation, blood compatibility, and prompt degradation in the reticuloendothelial system (RES). In situ physicochemical characterization clarified the narrow size distribution, the thin lipid bilayer membrane, and conc. Hb solution inside the HbV. The HbV suspension shows peculiar viscoelasticity and ligands binding properties owing to the particle dispersion. Surface modification of HbV with PEG chains ensures the stable dispersion state and the stability for storage over a year at room temperature in a deoxygenated condition. In vivo animal tests clarified (i) the biodistribution of HbV and the prompt metabolism and degradation of HbV in RES, (ii) physiological significance of the cellular structure of HbV in microcirculation, (iii) the efficacy as a transfusion alternative for hemorrhagic shock and extracorporeal cardiopulmonary bypass prime, (iv) tailor-made low P50-HbV for oxygenation of ischemic tissues, and (v) other possible clinical applications to transport oxygen that is essential for respiration of tissue cells, such as a perfusate for organ transplant, and a tissue regeneration. On the other hand, totally synthetic heme albumin-based oxygen carriers have also been developed. Surface modification of albumin-heme by PEG significantly prolonged the circulation lifetime of the heme in the blood stream. Recently, we have found that a natural protoheme IX can also be used as an oxygen binding site of this artificial hemoprotein. Mutation of Ile-142 by histidine confers the oxygen binding capability to the protoheme. The oxygen-binding affinity was modulated by the variety of the distal amino acid in the heme pocket.
KL-10 Free Oxygen Flow a New Paradigm
Professor Enrico Bucci, Ph.D. [email protected]
Professor 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 hemoglobin carrying 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 the new paradigm. In a steady state where all circulatory parameters are kept constant, blood may be considered a long tubular liquid membrane, surrounded by vascular walls, interfacing alveolar oxygen and interstitial fluid. Consumption at the periphery drives a flow of oxygen with a gradient from 120 torr in the lungs alveoli, to 2 torr or less in cells mitochondria drives a flow of free oxygen through blood. Due to the low water solubility of oxygen, this gradient is too shallow to sustain metabolic consumption. Natural cell-segregated oxygen carriers, hemoglobin and myoglobin, regulate the flow into sustenance by replacing molecule by molecule the oxygen consumed by metabolism. Thus, they buffer the oxygen tension at levels near their P50: near 30 torr at the capillaries by 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 of free oxygen necessary for metabolism, still at low non toxic partial pressure. Due to a low diffusion constant, there is a limited permeability of the arterial walls to oxygen, thereby preserving it for peripheral use. Cell free oxygen carriers 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 on their P50. It depends only on the total oxygen carrying mass of available carrier. Their P50s only regulate the pressure range at which oxygen replacement occurs. The free energy loss that drives the oxygen gradient increases oxygen solubility in body fluids, adding a regulatory parameter to the amount of free oxygen available to metabolism. In the absence of myoglobin, which buffers oxygen tension at 2 torr, tissues like arterial walls and brain are exposed to higher oxygen pressure. This may contribute to adverse clinical events.
HL-11 Clinical Application of Cellular Type Artificial Oxygen Carrier-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]
1)Professor, Division of General Thoracic Surgery, Department of Surgery, School of Medicine, Keio University, Tokyo, Japan 2)Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan
Hemoglobins recovered from human red blood cells are encapsulated in bilayer liposomes to make cellular type artificial oxygen carrier to be used regardless of patient's blood type.
The size of this hemoglobin vesicle (HbV) is about 250 nm.HbV can be stored in a liquid state at room temperature for over two years. This stability is oftained by deoxygenation to prevent metHb formation and surface modification with polyethylene glycol. Oxygen carrying capacity, and safety are examined in small animals and beagles. The results of these experiments show that our HbV may be used in following clinical settings. Hemorrhagic shock perfusate for cardiopulmonary bypass tumor oxygenation to increase sensitivity to chemotherapy and radiotherapy prevention of tissere ischemia.
KL-12 Microcirculation can be a Wonderful Mirror for Blood Substitutes Performance
Professor Ruijuan Xiu, M.D., Ph.D. [email protected]
XI ISBS Executive Vice President Director, Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
Our early clinical and experimental studies on endotoxin shock during fulminant epidemic meningitis among children and acute Cor pulmonale showed that the microcirculation acts as a “Mirror” reflecting severe spasm and constriction of the arterioles and precapillaries in producing local ischemia and anoxemia, which can lead to respiratory failure or coma and even death depending on whether the lung or brain was affected. In 12 cor pulmonale patients with diminished nail-bed microcirculation, 6 had encephalopathy and one had hemiplegia. The capillary density (CD) on the nailfold was a important parameter for evaluation of the degree of blood perfusion in tissue and organs of this patients. Expansion blood volume was one of the key diplomacy to save patient's life. It was found, that the key medicine used to save these patients from endotoxin shock was not vaso-dilator, but microvascular vasomotion regulator.
In 1987–1988, the author studied the vascular perturbations associated with AIDs in San Francisco and Stockholm (Ruslagstull Hospital). Video-microscopy of the nail-fold microcirculation was performed on 11 American and European AIDS patients and 11 healthy European adults. Severe microvascular dysfunction was demonstrated in the AIDS patients: Normal spontaneous fluctuations in the control capillary blood flow were not present. The endothelial cells in the capillary wall were damaged significantly. Permeation of the capillary wall by a heterogeneous mass made the vessel boundaries indistinct and serving to narrow the lumen. A computer image analysis indicated part of the heterogeneous mass was in the endothelial cells cytoplasm. Extravasation of red blood cells was seen in three patients. All patients were found to be suffering from Kaposi's sarcoma.
Based on the above studies, in 1985, a National Key Lab was established in the CAMS. A serial of experments was done in order to investigate the significance of microcirculatory dysfunction in the pathogenesis of major diseases. The role of O2 in treatment of ischemic diseases was emphasized, because many such patients received abundant O2 via inhalation at the last stage of their life while the capillary loops were dilated and filled with oxygen saturated blood. It was found that as bath PO2 was increased, a progressive constriction of A1, A2 and A3 order arterioles in the cremaster muscle of normotensive and hypertensive rats occurred. Maximum constriction occurred at bath PO2 of 40-60mmHg. At bath PO2 beyond 80mmHg, the constricted vessels began to dilate and irreversible dilation could be caused when bath PO2was higher than 90mmHg. It was also found that H2O2 can directly induce the hypertrophic response of neonatal Wistar rat cardiomyocytes, and H2O2 mediated the regulation of cardiomyocyte hypertyophy through different isoforms of PKC. In summary, it is desirable that blood substitutes act as Oxygen Carriers, but it is important to establish the optimal capacity of these Oxygen Carriers.