IV.

KEYNOTE LECTURES (KL)

Articial Cells, Blood Substitutes, and Biotechnology, 36: 187207, 2008
ISSN: 1073-1199 print / 1532-4184 online
DOI: 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, USA
rwinslow@sangart.com
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
187
188 Keynote Lectures
oversupply may lead to oxygen-related toxicity, which may include myocardial
ischemia and infarction.
The evidence for this hypothesis comes from various sources, includ-
ing a consideration of the atmospheric O
2
levels at the time that O
2
delivery
mechanisms evolved, experience with hyperbaric oxygen, articial capillaries,
and the body of research that establishes that tissue oxygen levels are main-
tained over very narrowand lowlevels. Recent data demonstrates that the
response to increased inspired O
2
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-
modied hemoglobin).
The essential elements of the oxygen transport systemin mammals include
red blood cells, low plasma oxygen solubility, barriers to O
2
diffusion, and the
control of microvascular perfusion. Introduction of a cell-free oxygen carrier
disrupts this system, engaging mechanisms that protect against O
2
toxicity,
including vasoconstriction and reduced perfusion. If this happens in an area of
tissue that already has marginal or compromised blood ow, exacerbation of
local hypoxia and infarction could result. The hypothesis predicts that a suc-
cessful cell-free oxygen carrier will limit oxygen release at vascular PO
2
above
that in metabolically active tissues and be most effective at low concentration
where some red blood cell oxygen reserve is still present.
Keynote Lectures 189
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. aggreenburg@biopure.com
Professor Emeritus of Surgery, Brown University, Rhode Island, U.S.A.
Establishing an acceptable safety and efcacy prole for hemoglobin based
oxygen 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 safety
prole. The imputation of causation from numerical counts alone is an in-
complete analysis of a complex situation. Clinical contextualization of the
critical signicant adverse events provides a more complete understanding of
the clinico-pathological processes underlying their emergence. Alternative hy-
potheses for the origin of the events, apart fromthe drug, patient co-morbidities,
situational and disease demands, could include protocol design, failure to pro-
vide mitigation strategies, patient management issues, or inadequate education
of investigators. How clinical contextualization aids the interpretation of safety
prole 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.
190 Keynote Lectures
KL-3 Postinjury Resuscitation with Human Polymerized Hemoglobin:
The USA Multicenter Trial
Steven Gould, M.D., Ph.D.,
President, Northeld, U.S.A.
Professor of Surgery. SAGould@northeldlabs.com
INTRODUCTION: Human polymerized hemoglobin (PolyHeme
R
), a univer-
sally compatible O
2
carrier, may be useful in the early treatment of hemorrhagic
shock when stored blood is not available. This Phase III trial is the rst 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 eld resus-
citation with PolyHeme or crystalloid. PolyHeme patients continued to receive
up to 6 units of PolyHeme during the rst 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
Keynote Lectures 191
patients died; 61 (74%) on Day 1. Major protocol violations occurred in 71
PolyHeme and 53 Control, leaving 590 protocol compliant patients.
PolyHeme Control
(survivors/N) (survivors/N) p-value
Day 1 Protocol Compliant
259/279 (92.8%) 289/311 (92.9%) 1.00
Protocol Violators 57/71 (80.3%) 48/53 (90.6%) 0.14
All Patients 316/350 (90.3%) 337/364 (92.6%) 0.29
Day 30
Protocol Compliant 248/279 (88.9%) 282/311 (90.7%) 0.50
Protocol Violators 55/71 (77.5%) 47/53 (88.7%) 0.15
All Patients 303/350 (86.6%) 329/364 (90.4%) 0.13
CONCLUSIONS: There was no statistically signicant difference in survival
between 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 not
available, thereby addressing a critical unmet medical need.
192 Keynote Lectures
KL-4 Introduction of Development on Transfusion Medicine in China
Chengmin Yang, Lishen Du, Jiaxin Liu
chengminyang2602@163.com
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. This
presentation reviews recent developments and current status of blood transfu-
sion medicine in the elds of Chinese blood groups, government regulations
and laws, clinical blood transfusion, blood transfusion safety, scientic research
in blood transfusion medicine, plasma products and information management
in blood transfusion service. Under the Chinese government regulations and
efforts fromall 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
Keynote Lectures 193
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 eld 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 seientic informations.)
194 Keynote Lectures
KL-5 Process Engineering of Protein-Based Oxygen Carriers
Zhiguo Su, Ph.D. zgsu@home.ipe.ac.cn
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: zgsu@home.ipe.ac.cn
Preparation of protein-based oxygen carriers usually involves three stages in-
cluding protein extraction and purication, protein modication or encapsula-
tion and post-separation of the modied/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 fromany 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, articial cells containing proteins are discussed.
Keynote Lectures 195
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. mintagli@ucsd.edu
P Cabrales, BY Salazar V azquez, A Ch avez 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
rst 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 hemodi-
lution. 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 efcacious 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
196 Keynote Lectures
may even increase release of vasoactive mediators from the endothelium, while
enhancing metabolites removal from the tissue. Present blood substitutes pro-
vide a continuous spectrum of transport properties beyond oxygen carrying
capacity per se, including viscogenic effects and endothelial vasoactive me-
diation leading to enhanced generalized perfusion. Engineering these factors
allows designing for an optimal physiological/resuscitative function while low-
ering oxygen carrier use and its cost.
Keynote Lectures 197
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
wzapol@partners.org
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 at-
tenuates the HBOC-induced vasoconstriction. However, NO inhalation at 80
ppm concurrently with HBOC administration oxidizes the plasma Hb impair-
ing 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 decient in
nitric oxide synthase (NOS2) and awake lambs. We infused both tetrameric
hemoglobin and in some studies HBOC-201 (Hemopure). Tetrameric Hb (4
198 Keynote Lectures
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 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 ndings 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 infu-
sion 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.
Keynote Lectures 199
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*,
marketing@lifegen.com
National 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 HBOCs
with low P50 has been prepared. Using porcine blood as the raw material, the
ultra-pure porcine hemoglobin (pHb) was obtained through multiple purica-
tion steps, which was found to have very similar oxygen afnity and mod-
ulating 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 afnity 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).
200 Keynote Lectures
The mean arterial blood pressure (MAP) has no signicant 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 specic serum IgG antibody re-
sponse 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 specic 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.
Keynote Lectures 201
KL-9 Physicochemical Characteristics and Physiological Performances
of Articial Oxygen Carriers (Hb-Vesicles And Albumin-Hemes)
Professor Eishun Tsuchida. Ph.D. eishun@waseda.jp
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 O
2
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 HbVas a molecular assembly, such as stability for storage and in blood circu-
lation, blood compatibility, and prompt degradation in the reticuloendothelial
system (RES). In situ physicochemical characterization claried the narrow
size distribution, the thin lipid bilayer membrane, and conc. Hb solution in-
side the HbV. The HbV suspension shows peculiar viscoelasticity and ligands
binding properties owing to the particle dispersion. Surface modication 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 claried (i) the biodistribution of HbV and the prompt metabolism
and degradation of HbV in RES, (ii) physiological signicance of the cel-
lular structure of HbV in microcirculation, (iii) the efcacy 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
202 Keynote Lectures
carriers have also been developed. Surface modication of albumin-heme by
PEG signicantly 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 articial hemoprotein. Mutation of Ile-142 by
histidine confers the oxygen binding capability to the protoheme. The oxygen-
binding afnity was modulated by the variety of the distal amino acid in the
heme pocket.
Keynote Lectures 203
KL-10 Free Oxygen Flow a New Paradigm
Professor Enrico Bucci, Ph.D. ebucci@umaryland.edu
Professor Emeritus, Dept of Biochemistry and Molecular Biology University
of Maryland Medical School, Baltimore, MD, USA
The classic viewof 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 ow of free oxygen through blood. This is the focus of the
new paradigm. In a steady state where all circulatory parameters are kept con-
stant, blood may be considered a long tubular liquid membrane, surrounded by
vascular walls, interfacing alveolar oxygen and interstitial uid. Consumption
at the periphery drives a ow of oxygen with a gradient from 120 torr in the
lungs alveoli, to 2 torr or less in cells mitochondria drives a ow of free oxy-
gen 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 ow into sustenance by re-
placing 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 uid.
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 car-
riers facilitate oxygen diffusion in plasma, while bathing the endothelial walls.
Therefore they replace consumed oxygen at much faster rate than cell bound
204 Keynote Lectures
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 car-
rier. 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 uids, 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.
Keynote Lectures 205
HL-11 Clinical Application of Cellular Type Articial Oxygen
Carrier-Hemoglobin
Vesicle (Hbv)
Professor K. Kobayashi, M.D., Ph.D., Keio University
K. Kobayashi
1
, H. Horinouchi
1
, M. Watanabe
1
, Y. Izumi
1
, M. Kohno
1
,
T. Ikeda
1
, N. Izawa
1
, Y. Yozu
1
, E. E. Tsuchida
2
and H. Sakai
2
kobayash@sc.itc.keio.ac.jp
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 fromhuman red blood cells are encapsulated in bilayer
liposomes to make cellular type articial oxygen carrier to be used regardless
of patients 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 mod-
ication 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.
206 Keynote Lectures
KL-12 Microcirculation can be a Wonderful Mirror for Blood
Substitutes Performance
Professor Ruijuan Xiu, M.D., Ph.D. xiurj@yahoo.com.cn
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 fulmi-
nant epidemic meningitis among children and acute Cor pulmonale showed
that the microcirculation acts as a Mirror reecting severe spasm and con-
striction of the arterioles and precapillaries in producing local ischemia and
anoxemia, 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 patients
with diminished nail-bed microcirculation, 6 had encephalopathy and one had
hemiplegia. 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 this
patients. 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 from
endotoxin 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 11
American and European AIDS patients and 11 healthy European adults. Severe
Keynote Lectures 207
microvascular dysfunction was demonstrated in the AIDS patients: Normal
spontaneous uctuations in the control capillary blood ow were not present.
The endothelial cells in the capillary wall were damaged signicantly. Perme-
ation of the capillary wall by a heterogeneous mass made the vessel boundaries
indistinct 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 were
found to be suffering from Kaposis 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 signif-
icance of microcirculatory dysfunction in the pathogenesis of major diseases.
The role of O
2
in treatment of ischemic diseases was emphasized, because
many such patients received abundant O
2
via inhalation at the last stage of
their life while the capillary loops were dilated and lled with oxygen saturated
blood. It was found that as bath PO
2
was increased, a progressive constriction
of A
1
, A
2
and A
3
order arterioles in the cremaster muscle of normotensive
and hypertensive rats occurred. Maximum constriction occurred at bath PO
2
of
40-60mmHg. At bath PO
2
beyond 80mmHg, the constricted vessels began to
dilate and irreversible dilation could be caused when bath PO
2
was higher than
90mmHg. It was also found that H
2
O
2
can directly induce the hypertrophic
response of neonatal Wistar rat cardiomyocytes, and H
2
O
2
mediated the reg-
ulation 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.