Oral Presentations

SIII-3 Design of Second-Generation PEGylated Intramolecularly Crosslinked Hbs to Manipulate O₂ Carrying Capacity

Seetharama A Acharya^1,2, Tao, Hu¹, Dongxia Li¹, Fanatao Meng¹, Amy G Tsai³ and Marcos Intaglietta³

¹Departments of Physiology and Biophysics, and Medicine ², Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, and ³Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA; [email protected]

Limitations in formulating of hexaPEGylated uncross-linked Hbs as blood substitutes are (i) very high O₂ affinity independent of the chemistry of conjugation, and (ii) weakening of interdimeric interactions of Hb in a conjugation chemistry specific fashion. The generation of PEGylated intramolecularly cross-linked Hbs overcomes these disadvantages and is helpful to (i) decrease the colloidal osmotic pressure (COP) of PEGylated Hbs; (ii) modulate the O₂ affinity of the PEGylated Hbs to desired levels; (iii) to achieve higher loads of PEGylated Hbs in circulation, if needed, without the danger of kidney filtration. TetraPEGylation of α α -fumaryl Hb, a low O₂ affinity central cavity intramolecularly crosslinked Hb, using extension arm facilitated (EAF) PEGylation and PEGylation protocols based on reductive alkylation, acylation and thiocarbamoylation chemistry have been optimized to generate a spectrum of PEGylated Hbs with O₂ affinities in the range of 12 to 35 mm Hg, at pH 7.4 and with a cooperativity around 2.0. The reversible protection of Cys-93(β) of Hb during the EAF-PEGylation has also been optimized to facilitate the manipulation of the O₂ affinity of the product. PEGylated Hbs are significantly protected from autoxidation in plasma; and unmodified Cys-93(β) in the final formulation of the PEGylated products gives a higher resistance to autoxidation. These new design strategies for development of second-generation PEGylated Hbs are expected to remedy remaining potential toxicities of using acellular Hb and simultaneously provide improved intravascular persistence, and facilitate customizing the PEGylated products for any defined clinical settings.

SVI-1 Evaluation of New Perfluorocarbon Based Oxygen Carriers in a Rat Model of Normovolemic Hemodilution: Effect of Emulsion Viscosity osmolarity and Perfluorocarbon Concentration

AC Silva¹, T Gardeazabal¹, M Cabrera¹, F Vega^1,2, CI Castro¹, E Gutierrez², JC Briceño¹

¹Blood Substitutes Laboratory, Fundacion Cardioinfantil and U. of Los Andes; ²School of Veterinary Medicine, U. of La Salle, Bogota DC, Colombia. [email protected]

The objective of this project was to evaluate the efficacy and safety of new perfluorocarbon based oxygen carriers (PFCOCs) in a rat model of hemodilution. The experiment consisted of a two-exchange normovolemic hemodilution of 40% of volemia in Wistar rats. First exchange was performed with 10%-HES. Second exchange was performed with 80% PFCOC and 20% 10%-HES. FiO₂ was raised to 1.0 after the second exchange. In this study formulations were evaluated according to a factorial experimental design with the following factors and levels: PFC concentration (20 and 30% v/v), viscosity (normal and high) and osmolality (normal and high) with n = 24. A control group (n = 7) was hemodiluted with 10%-HES. Blood gasimetry, hematocrit and hemoglobin content were measured at baseline and 15 min after each exchange. Results: A comparison of oxygen extraction (DavO₂) between the control group and the different emulsions was performed. 30%-PFC emulsions transported significantly more oxygen that the control group (p < 0.01). There was no statistical difference in DavO₂ between the 20%-PFC emulsions and the control group (p > 0.05). The effect of emulsion osmolality and viscosity on DavO₂ was not significant, although the 30%-PFC normoosmolar hiperviscous emulsion performed slightly better. All rats tolerated well the infusion of the emulsions. In conclusion, the new PFCOCs developed appear to be efficacious and safe in the animal model of normovolemic hemodilution. Further work is required to better assess the effect of emulsion viscosity and osmolality on oxygen carrying capacity.

PA-2 Red Cell Hemoglobin Oxygen Affinity Modulates Microvascular Hemodynamics and Perfusion during Acute Anemia

P. Cabrales¹, A.G. Tsai^1,2, M. Intaglietta^1,2

¹La Jolla Bioengineering Institute and ²Department of Bioengineering, University of California, San Diego, La Jolla, CA/USA; [email protected]

Responses to exchange transfusion using red blood cells (RBCs) with modified hemoglobin (Hb) oxygen (O₂) affinity were studied in the hamster window chamber model during acute anemia to determine its role on microvascular perfusion and tissue oxygenation. Allosteric effectors were introduced in the RBCs by electroporation. Inositol hexaphosphate (IHP) and 5-hydroxymethyl-2-furfural (5HMF) were used to decrease and increase Hb-O₂ affinity. In vitro P50s (partial pressure of O₂ at 50% Hb saturation) were modified to 10, 25, 45 and 50 mmHg, normal P50 is 32 mmHg. Allosteric effectors also decreased the Hill coefficient. Anemic condition was induced by two isovolemic hemodilution exchanges using 6% dextran 70 kDa to 18% hematocrit (Hct). Modified RBCs (18% Hct in 5% albumin solution) were infused during a third exchange transfusion. Systemic parameters, microvascular perfusion, capillary perfusion (functional capillary density, FCD) and microvascular pO₂ levels were measured. RBCs with P50 of 45 mmHg increased tissue pO₂ and decreased O₂ delivery (DO₂) and extraction (VO₂) and RBCs with P50 of 60 mmHg reduced FCD, microvascular flow, tissue pO₂, DO₂ and VO₂. Erythrocytes with increased Hb-O₂ affinity maintained hemodynamic conditions, DO₂ and decreased tissue pO₂. This study shows that in an anemic condition, maximal tissue pO₂ does not correspond to maximal DO₂ and VO₂.

Supported by NIH BRP R24-HL64395 and grants R01-HL62354, R01-HL62318 and R01-HL76182.

SV-2 Change of Cytokine Production in Intra-Abdominal Hemorrhage Model-Effect of Hemoglobin Vesicle

Hirohisa Horinouchi, Noaki Aikawa, Mitsutomo Kohno, Yotaro Izumi, Hiromi Sakai, Keitaro So, Teruyuki Komatsu, Eishun Tsuchida and Koichi Kobayashi

Dept. of Surgery, Keio University, School of Medicine, Tokyo, JAPAN, Advanced Research Institute for Sci. & Eng., Waseda University [email protected]

Aim: When intra-abdominal hemorrhage occurs, SIRS(systemic inflammatory response syndrome) is sometimes encountered. The cause of this phenomenon is thought over production of inflammatory cytokines. Safety study was done to verify that intra-abdominal HbV didn't change cytokine production. Method: 30% hemorrhage was induced in male Wister rats (340 g+/–10 g) via right carotid artery. Five minutes after hemorrhage, intra-abdominal administration of either autologous shed blood or Hemoglobin Vesicle (HbV dispersed in saline) solution was done. 30 minutes after hemorrhage, Twice volume of saline was used for resuscitation. After 30, 60, 90, and 180 minutes had passed, 0.5 ml of blood was withdrawn through arterial line and replaced with the same volume of saline. Serum was separated and stored in deep freezer until use. Cytokines (IL-1 alpha, beta, IL-2, -4, -6, -10, GM-CSF, Interferon gamma, TNF-alpha) were simultaneously measured using Bio-Rad Bioplex suspension array system. Results: After hemorrhage, shock was confirmed in each animal (40.6+/–7.2 Torr). Intra-abdominal injection doesn't change MAP and HR. After resuscitation, MAP recovered to the pretreatment level and gradually declined. Cytokine production didn't change significantly. However, in ASB group, increase of TNF alpha was seen while in HbV group, there seemed no response (P = 0.11). Discussion: Intrapritoneal HbV itself doesn't change neither the cours of hemorrhagic shock nor the cytokine production during shock. Shock with organ damage model should be further studied.

SV-3 Resuscitation Effect and Long Term Effect of Hb Vesicle on Organ Function in Beagle Dog

Tatsuhikko Ikeda, Hirohisa Horinouchi, Mitsutomo Kohno, Yotaro Izumi, Masazumi Watanabe, Hiromi Sakai, Keitarou Sou, Eishun Tsuchida, Kouichi Kobayashi

Department of Surgery, Keio University, Tokyo, Japan; Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan [email protected]

Long-term safety study was carried out in 40% hemorrhage shock- resuscitate model in Beagle dog. (Method) Sixteen Beagle dogs were used. After stabilization, 40% of blood volume was bled at a rate of 20 ml/min. We kept MAP below 50mmHg for 1 hour. After 1 hour hemorrhagic shock, either autologous shed blood (ASB) or HbV/5%HSA solution was used for resuscitation. MAP, HR, RR, intracranial tissue hemoglobin saturation was monitored during acute phase. 4 Hours after resuscitation, dogs were extubated and were returned to the cages. One, 3, 7, 14, 28, 56, 84, 168, 360 days after, body weight, CBC, serum chemistry was analyzed. Several dogs were sacrificed on day 28, 168, 360 and histological findings were studied. (Results) Efficacy of HbV for the resuscitation of hemorrhagic shock seemed similar to that of ASB. RBC count had recovered within 7 days after shock. HbV in blood stream decreased its concentration and was not detected in the blood taken on day7. After resuscitation, AST/ALT increased twice to the normal value at day 1 in both HbV and ASB group. This upregulation normalized on day three. There was no death in both HbV and ASB group. All dogs didn't show any abnormal findings during experimental period. Histological exam showed no substantial changes. (Discussion) HbV is thought safe and promising blood substitute in long term study. Early rise of AST/ALT after resuscitation is thought to be a reaction to hypovolemic shock.

SI-3 Efficacy of Liposomal Hemoglobin (Lhb) in Massively Hemorrhaged Cynomolgus Monkeys

T. Ishizuka, H. Goto, Y. Ogata, S. Kaneda, H. Kasukawa

Terumo Corp., Japan; [email protected]

We have developed a liposomal hemoglobin (LHb) preparation as a red blood cells substitute and confirmed the efficacy of LHb in hemorrhaged rats and dogs but not in primates. The purpose of this study was to examine the effects of LHb in massively hemorrhaged cynomolgus monkeys.

Anesthetized animals received an isovolemic exchange transfusion with a colloidal solution (Hespander^TM) until the hemoglobin concentration reached about 3 g/dL. Immediately after that, five animals each were given 20 mL/kg of LHb or saline. Survival time, blood pressure, electrocardiogram (ECG), arterial blood gases and plasma lactate level were monitored during 12 hours.

The survival rates were 4/5 in the LHb group and 2/5 in the Saline group. In the animals that survived, arterial blood pressure was maintained during the observation period at a level similar to the baseline in the LHb group but not in the Saline group. Ischemic changes in ECG and elevation in plasma lactate level were observed at 12 hours in the Saline group but not in the LHb group. No apparent signs of pulmonary dysfunction were observed in either group.

We confirmed the efficacy of LHb as a red blood cells substitute in massively hemorrhaged cynomolgus monkeys. These results suggest that LHb is also useful in primates.

SVII-5 Administration of Hemoglobin Vesicle under Mechanical Ventilation Does Not Affect Lung Function

Izumi Y¹, Yamada T², Ogawa EN², Morisaki H², Sakai H³, Horinouchi H¹, Takeda J², Tsuchida E³, Kobayashi K¹

¹Division of General Thoracic Surgery, and ²Department of Anesthesia, School of Medicine Keio University, Tokyo, Japan. ³Advanced Research Institute for Sci and Eng, Waseda University, Tokyo Japan; [email protected]

Hemoglobin Vesicle (HbV) contains concentrated hemoglobin solution within a phospholipid vesicle. As a resuscitative fluid, it will potentially be administered to patients receiving care in the intensive care unit, which includes mechanical ventilation. HbV did not affect lung function when administered under spontaneous breathing. In this study, we administered HbV when the lung was mechanically ventilated. Rabbits were mechanically ventilated (tidal volume, 30ml/kg), and 30% exchange transfusion was done with HbV or saline. Hemodynamics, and blood gas parameters were monitored for 4 hours. The lung was then resected for histology. Wet to dry ratio was also measured. There was no apparent change in systemic blood pressure in either group. Arterial oxygen tension tended to decrease gradually, but there was no significant difference between groups. Slight lung edema was observed in both groups. There was no significant difference in the wet to dry ration between the groups. The moderate decrease in arterial oxygen tension suggested the presence of ventilation induced lung injury, but there was nothing to suggest that it was aggravated by HbV administration. (Supported by NHLW of Japan)

SI-2 Clinical Evaluation of Hemospan® As an Oxygen-Carrying Plasma Expander in Surgical Patients

PE Keipert, N Winslow, RM Winslow

Sangart Inc., 6175 Lusk Blvd., San Diego, CA; [email protected]

Hemospan® is a hemoglobin-based oxygen carrier (HBOC) consisting of chemically modified human hemoglobin (MalPEG-Hb) in Lactated Ringer's solution. Hemospan is formulated at low Hb concentration (4.3 g/dL), high oxygen affinity (P50 ∼ 5 mmHg) and high colloid osmotic pressure (COP ∼ 50 mmHg), properties that prevent oxygen-induced vasoconstriction while preserving capillary perfusion and oxygen delivery.

Clinical studies with Hemospan in Europe and in the US have enrolled a total of > 300 subjects as of the end of July 2007. The first Phase I study in healthy volunteers did not show any hypertension or gastrointestinal side effects, which have been reported with other first-generation HBOCs. A recently completed dose escalation Phase Ib/II study in orthopedic surgery patients also reported no serious adverse events (SAEs) at Hemospan doses ranging from 200 to 1000 mL. A similar Phase II safety study in prostatectomy patients is currently ongoing in the US, with no safety concerns noted.

A randomized, double-blind Phase II study of Hemospan was completed at 6 Swedish hospitals in 90 patients (ASA Class I-III; age 50 to 89) undergoing hip replacement or fracture surgery with spinal anesthesia. The primary endpoints were the incidence of hypotension and the use of vasopressors. The percentage of patients with hypotensive episodes was significantly lower (48% in the 250-mL dose group, 43% in the 500-mL dose group) compared to 84% in controls (P < 0.025). The incidence of vasopressor use was also reduced in the 250-mL (17%) and 500-mL group (13%) compared to controls (32%). Mean heart rate was less in both treated groups versus controls (P < 0.02), with no differences in PR or QTc intervals (from 24-hour Holter ECG monitoring). Three SAEs were reported, but none was related to treatment.

Hemospan is currently being evaluated in two parallel Phase III studies as an oxygen-carrying plasma expander for surgical patients undergoing primary hip arthroplasty, to prevent and treat the hypotension and functional hypovolemia associated with spinal anesthesia and surgical bleeding. By improving hemodynamic stability during surgery and in the early postoperative period and providing oxygen transport, oncotic pressure and plasma volume expansion, Hemospan treatment may also reduce the incidence of postoperative morbidity. These two randomized, double-blind Phase III studies will enroll a total of 830 patients at approximately 36 sites in six European countries.

PA-5 Effect of Iv Hboc-201 on Coronary Hemodynamics and Function in Patients Undergoing Elective Pci

Hae Won Kim, and the COR-001 Study Group Biopure Corporation

Cambridge, MA 02141, U.S.A; [email protected]

Hemoglobin based oxygen carriers (HBOCs) are potential therapeutic adjuncts to patients with acute coronary syndrome undergoing elective percutaneous coronary intervention (PCI). Feasibility of intravenous (IV) HBOC-201 therapy in this patient population was tested. In a double blinded phase II pilot trial, 45 patients with acute non-ST segment elevation myocardial infarct scheduled for PCI procedure were randomized to receive IV infusion of either a colloid control or 15g or 30 g of HBOC-201. Systemic and coronary hemodynamic parameters were assessed at baseline, post-HBOC infusion and post-PCI. After HBOC-201 administration, systemic arterial blood pressure (SBP), pulmonary capillary wedge pressure and systemic vascular resistance were elevated indicating systemic vasoconstriction. Cardiac output (CO) and mixed venous oxygen saturation (SVO₂) were moderately depressed. However, the elevated SBP could be managed with traditional antihypertensive medications. Despite the systemic effects, coronary hemodynamic and functional parameters (e.g., average peak velocity, coronary blood flow, left ventricular work index) were not altered under resting and adenosine-induced hyperemia indicating that HBOC-201 does not interfere with coronary autoregulatory mechanisms. In addition, there was no angiographic (QCA) evidence of coronary vasoconstriction in major epicardial vessels examined. These results indicate that HBOC-201 could be safely administered to patients undergoing percutaneous coronary revascularization for treatment of an acute ischemic syndrome.

SIII-4 Three-Dimensional (3D) Solution Structure of Maleimide-Poly(ethylene) Conjugated Hemoglobin by Small-angle X-ray Scattering: Implications for Hemospan® as a New Oxygen Therapeutic

KD Vandegriff¹, A Malavalli¹, DA Baker¹, DI Svergun², F Ekström³, C Nilsson³, RM Winslow¹

¹Sangart, Inc., 6175 Lusk Blvd., San Diego, CA USA 92121; ²European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany; ³Swedish Defense Research Agency (FOI), CBRN Defense and Security, Umeå, Sweden; [email protected]

Several general benefits have been attributed to poly(ethylene) glycol (PEG) conjugation to proteins, including increased intravascular retention time. Hemospan® is human hemoglobin conjugated to ∼ 7 linear strands of poly(ethylene) glycol (PEG), ∼ 5-kD each. The design strategy for Hemospan was initiated using poly(ethylene) glycol chemistry to increase hemoglobin macromolecular size without polymerization. Solutions of unmodified hemoglobin (Hb) and PEGylated hemoglobins with either two (P5K2) or ∼ 7 (P5K7, Hemospan) 5-kD PEGs were evaluated using small-angle X-ray scattering (SAXS). The 3D solution structures reveal that PEGylation elongates the dimensions of the hemoglobin molecule: Hb < P5K2 < P5K7, giving maximal molecular dimensions of 7, 11, and 13 nm, respectively. The overall tertiary structure of hemoglobin remains intact upon conjugation to the PEG chains, but its quaternary structure appears compacted, presumably due to the dehydration of the intersubunit interfaces. The major part of the PEG chains protrudes away from hemoglobin, while the rest interacts with the core protein. Based on calculations of the PEG Flory radius, the SAXS structure of Hemospan suggests that the PEG chains exists at the transition point between mushroom and brush conformations. PEGylation introduces a strong intermolecular repulsive effect that increases with the amount of conjugated PEG. These results further define the biological activity of Hemospan as an oxygen therapeutic such that: 1) PEG provides surface shielding of the hemoglobin molecule; 2) hemoglobin tertiary structure is not altered by PEG conjugation; and 3) PEG imparts intermolecular repulsive forces, which may affect the rate or mechanism of Hemospan's intravascular clearance.

SV-4 Application of Hemoglobin Vesicles to Anemia Due to Inflammatory Bowel Disease in a Mouse Model

M Kohno¹, H Horinouchi¹, Y Izumi¹, T Ikeda¹, H Sakai², E Tsuchida², K Kobayashi¹

¹Department of Surgery, Keio University School of Medicine, Tokyo Japan ²Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan; [email protected]

Human inflammatory bowel disease (IBD) is a chronic, relapsing and remitting inflammatory condition of unknown origin that afflicts individuals of both sexes throughout life. The disease is clinically characterized by two phenotypes—ulcerative colitis and Crohn's disease. Anemia affects between 30% and 70% of patients and has great impact on the quality of their lives. They currently receive transfusion of red blood cells for severe anemia. The purpose of this study was to assess the feasibility and safety of infusion of encapsulating concentrated human hemoglobin (Hb vesicles) for treatment of anemia due to IBD. Infusion of Hb vesicles did not exacerbate IBD as determined by a serum biomarker and histologic evaluation compared with saline in the dextran sulfate sodium mouse colitis model. Further, disease activity index for IBD and body weight loss tended to be decreased by infusion of Hb vesicles.

SVI-4 Genetic Engineering of Heme Pocket in Human Serum Albumin: Control of O₂ Binding of Iron Protoporphyrin Ix

T. Komatsu,^1,2 A. Nakagawa,¹ E. Tsuchida¹

¹Reserach Institute for Science & Engineering, Waseda University, Tokyo 169-8555 Japan, ²PRESTO, Japan Science and Technology Agency (JST), Saitama 332-0012 Japan; [email protected]

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O₂ in much the same way as hemoglobin (Hb).¹ We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O₂ reversibly: (i) Ile-142 → His, and (ii) Tyr-161 → Phe or Leu [I142H/Y161F (HF) or I142H/Y161L (HL)].²⁾ Here we report additional new mutations designed to manipulate the architecture of the heme pocket in rHSA–heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme can modulate the O₂ binding equilibrium.³⁾ The HSA(HL/L185N)–heme showed very high O₂ binding affinity (P_1/2^O2: 1 Torr, 22°C), which is 18-fold greater than that of the original double mutant rHSA(HL)–heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O₂ binding primarily by reducing the O₂ dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O₂ binding affinity (P_1/2^O2: 9–10 Torr, 22°C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O₂ transport.

SVII-3 Microcirculation Disorders Under Chronic Venous Insufficiency: Diagnostics and Correction with Taxifolin

VI Kozlov, VP Tihonov, LI Dergatchova, GA Azizov, OA Gurova, VV Baranov

Department of anatomy and Department of laser medicine, People's Friendship University of Russia; Company “Diod”; Company” Centre for Analysis of Substances”, Moscow; Russia [email protected]

The purpose of the present research consisted to investigate the influence bioflavonoid Taxipholin [Dihydroqercetin], possessing antioxydative action, on a skin microcirculation at patients with chronic venous insufficiency [CVI]. 45 patients with CVI [CEAP stage 2 – 4] have been surveyed at treatment of 0.3% gel Taxipholin in comparison with treatment of 2% gel Troxerutine [control group]. Application of gel was made on a leg and foot daily within 3 weeks. Methods: The estimation of skin microcirculation in a leg and foot was made by means of Laser Doppler flowmetry [LDF] [device LAKK, Lazma-Co, Rassia], and also TV-computer capillaroscopy of eponichii on 1 finger foots [Computer capillaroscope, Joint Stock Co, Russia]. A systemic microcirculation estimated by means of biomicroscopy of bulbar conjunctval microvessels. Results: At patients CVI in 100% of cases disorders of microcirculation are revealed. Under biomicroscopy at them increases of permeability of their wall and rheologic frustration were marked both structural changes of microvessels. The degree of insufficiency of microcirculation directly depend on weight of current CVI. The leading pathogenetic mechanism in frustration of microcirculation are: progressive increase of the phenomena of venous stagnation on a microvessel level, local blockade of a capillary blood flow and sharp damage of barrier function of microvessel wall. The most vulnerable is a nutritive part of microvascular network. Frustration of a blood flow at CVI lead to change of myogenic activity of arterioles and precapillaries. At LDF it is expressed in reduction of fluxmotion amplitude, spectral narrowing of oscillations and displacement of their dominant in high-frequency area that is a result of suppression the vasomotion mechanism. Application of gel Taxipholin positively affects dynamics of parameters of a local and systemic microcirculation at patients CVI. After the lead treatment improvement of microcirculation and diminish of stagnation tissue blood flow was observed: average linear speed of erythrocytes in capillaries increased with 320 ± 54 μ m/c up to 358 ± 8,1 μ m/c, the degree of erythrocyte aggregation and adhesions of leukocytes decreased, the parity of arteriolar and venular diameters was normalized, the perivascular zone [on 20 – 25%] decreased. Comparison of gel Taxipholin to gel Troxerutin [placebo] has shown, that at them comparable vasotropic effect. At the same time it has noted been, that reactance of microvessels [on postural test] at patients CVI it becomes authentic above [on 11%] after treatment by gel Taxipholin. Conclusion: Taxipholin possesses vasotropic action at the level of microvessels and owing to antioxydant action positively influences on normalization of microcirculation and decrease in permeability of capillary wall at patients with CVI.

SII-2 Engineering Hemoglobin-Based Oxygen Carriers for Tissue Engineering

Andre F Palmer

Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, USA; [email protected]

A priori knowledge of the dissolved oxygen (O₂) concentration profile within hepatic hollow fiber (HF) bioreactors is important in designing and developing an effective bioartificial liver assist device (BLAD) for eventual clinical use. O₂ transport is limiting within such HF bioreactors due to the poor solubility of O2 in aqueous culturing media, long tortuous O2 diffusion paths within HF membranes, and high hepatocyte O2 consumption rates. We hypothesize that supplementing a hepatic HF bioreactor's circulating media stream with a hemoglobin-based oxygen carrier (HBOC) will improve hepatocyte oxygenation. Our results (experimental and theoretical) indicate HBOC supplementation of the circulating media stream leads to marked improvement in oxygen delivery, and provision of a greater range of oxygen tensions to hepatocytes over the length of the bioreactor. Therefore, our results thus support the use of HBOCs for improving oxygen delivery to hepatocytes maintained within HF bioreactors. In general, the results of this work will be indispensable in improving oxygenation of bioreactors used for tissue engineering.

SVI-2 Haemorheological Characterization of a Perfluorocarbon-Based Oxygen Carrier (Oxygent) in Microcirculatory Scale Flow

JP Peach

University of Vermont, Burlington, VT, USA; [email protected]

Vascular (geometric and topographical) and intravascular (rheological) factors influence the resistance to blood flow. Addition of drugs, such as Oxygent, to the blood may alter the haemorheology and therefore affect the cardiovascular system's ability to adequately perfuse the tissue. In the microcirculatory system, blood exhibits complex rheological properties that are governed, in part, by vessel diameter, haematocrit ratio, temperature and shear rate. The aims of this study were to characterize the rheological properties of Oxygent mixtures and then evaluate its cardiac work load requirements in a physiologically relevant numerical model. To do this, whole blood was diluted with Oxygent or phosphate buffered saline 7.2 (PBS) to produce mixtures with 25, 30, 35 or 40% haematocrit and 60% plasma. A 100-μ m diameter glass-tube viscometer was used to measure the pressure drop over a 32-mm long test section at various wall shear rates (100 – 1,800/sec). Viscosity was then computed and a regression analysis was used to develop equations which predict viscosity from the shear rate for each haematocrit ratio group. To test the effects of temperature on pure Oxygent, its viscosity was determined in the temperature range of 32.2 – 41.1°C with wall shear rates between 17.5 and 2,275/sec. Lower temperatures led to significantly (p < 0.0005) higher viscosity of Oxygent. In an effort to interpret these results in a clinically relevant framework, cardiac workload over a 50-segment vascular network was examined. The pressure drop over the network was used as a measure of cardiac workload. The boundary conditions (inlet pressure, flow rate and haematocrit) were randomly selected for each inlet segment. The pressure drop using an Oxygent-blood mixture was compared to the pressure drop using a PBS-blood mixture having the same boundary conditions. These results will provide a theoretical measure of the cardiac workload required to pump an Oxygent-based blood mixture compared to a PBS- based mixture.

SV-1 Hemoglobin-Vesicles as Artificial Oxygen Carriers: Interactions with Ligand Molecules in the Production Process and in Blood Circulation

H Sakai¹, A Sato¹, K Sou¹, H Horinouchi², K Kobayashi², E Tsuchida¹

¹Research Institute for Sci. & Eng., Waseda University, Tokyo, Japan; ²Dept. of Surgery, School of Medicine, Keio University, Tokyo, Japan; [email protected]

Hb-vesicles (HbV, 250 nm) are artificial O₂ carriers, and the safety and efficacy as a transfusion alternative have been clarified in detail [1–7]. Hb binds not only O₂ but also CO and NO very strongly. In some conditions HbCO dissociates easily. The interactions of HbV with these gaseous ligands are important both in the production process and in blood circulation. HbCO is resistant to heating and it enables pasteurization at 60oC for 10 hrs to guarantee the utmost safety from infection [8]. The purified HbCO is concentrated to ca. 40 g/dL and encapsulated with a lipid bilayer membrane without protein denaturation. The vesicular surface is decorated with PEG. HbCO can be easily converted to HbO₂ by photoirradiation in an aerobic condition. Finally O₂ is completely removed out and the resulting deoxy-state HbV can be stored at room temperature for over 2 years [9]. HbV does not induce vasoconstriction in contrast to molecular Hbs. This difference presumably relates to the reduced uptake of endogenous NO and CO owing to the large dimension and the cellular structure of HbV [3,4]. Recently, we tested intravenous injection of exogenous CO-bound HbV into rats, and found out that CO was released quite promptly in 3 hrs with a cytoprotective effect at reperfusion. This indicates a possibility of a new clinical application of HbV in addition to its use as a transfusion alternative.

PA-4 Accurate and Quantitative Determinatuion of the Structural Profiles of Hemoglobin Vesicle By Means of X-Ray and Light Scattering Techniques

T. Sato¹, H. Sakai¹, K. Sou¹, O. Glatter², E. Tsuchida¹

Research Institute for Science and Engineering, Waseda University, Tokyo, Japan, and ²Institute of Chemistry, University of Graz, Graz, Austria; [email protected]

The Waseda-type Hemoglobin vesicle (HbV), developed as a cellular-type artificial oxygen carrier encapsulating a solution of purified and concentrated human hemoglobin (∼ 35 g/dL) with a surface-modified phospholipid bilayer membrane with poly(ethylene) glycol (PEG), has been structurally characterized by means of the latest approaches of small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS). DLS data on a diluted HbV dispersion evaluated with Optimized Regularization Technique have provided an accurate quantitative estimation of the size and size distribution of HbV; we obtained an averaged hydrodynamic diameter of 238 nm with a narrow size distribution (standard deviation of 20 nm in volume distribution).

A thin layer-cell DLS has for the first time made it possible to observe collective diffusion process of a concentrated (∼10g/dL) HbV dispersion without dilution, overcoming the interference from multiple scattering and absorption caused by the hemoprotein. The result confirms an ergodic (fluid-like) nature and good stability of the HbV dispersion.

The forward SAXS intensity of HbV dispersion represents the feature of a slightly polydisperse sphere having an averaged radius of ∼ 120 nm, whereas the high q-part reflects the internal structure of HbV, e.g., highly concentrated Hb (> 35g/dL) and lipid bilayer. SAXS experiments have also revealed a uni-lamellar structure, the thickness (5.8nm) and internal electron density profile of the vesicle for Hb encapsulation.

SII-4 Hemotech, a Novel Free Hemoglobin-Based Red Cell Substitute with Pharmacological Properties: the Concept and Current Status of Commercial Development

J Simoni, M. Feola, G Simoni, JF Moeller, BT Mittemeyer, AP Bollon

HemoBioTech, Inc., Dallas, Texas, U.S.A. and Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A; [email protected]

The worldwide need for blood substitutes is evident; however, currently tested free hemoglobin (Hb)-based oxygen carriers have toxicity and efficacy problems. These products were developed before the recognition of Hb's intrinsic toxicity; therefore, it is not surprising that the commercial development of several first-generation products has been discontinued. The problems with these blood substitutes revolve around blood vessel constriction and the pro-oxidant and pro-inflammatory properties of heme. To diminish intrinsic toxic effects of Hb, Texas Tech University Health Sciences Center scientists have developed and patented a novel concept of “pharmacologic cross-linking” and formulated an effective free Hb-based blood substitute product. This novel blood substitute, HemoTech, that was licensed to HemoBioTech, Inc. for commercial development, is composed of purified bovine Hb, cross-linked intramolecularly with open ring adenosine 5′-triphosphate (o-ATP) and intermolecularly with open ring adenosine (o-adenosine), and combined with reduced glutathione (GSH). The idea behind the use of o-adenosine was to counteract the vasoconstrictive and pro-inflammatory properties of Hb with the activation of adenosine receptors, which would produce vasodilatation and reduce inflammatory reactions. The concept of conjugation of Hb with GSH was to introduce more electronegative charges onto the surface of Hb, which would block Hb's transglomerular and transendothelial passage, and would make it less visible to phagocytes. In addition, GSH shields heme from reactive oxygen species and nitric oxide. The reaction with o-ATP stabilizes the Hb tetramer, but the reaction with o-adenosine allows the formation of Hb low molecular weight polymers with uniform electronegative charge. HemoTech was subjected to preclinical testing and clinical proof of medical concept. The results of these studies are favorable, indicating that HemoTech has vasodilatory activity and can reduce vasoconstriction that follows hemorrhage, has erythropoietic activity and produces no adverse nephrotoxic, neurotoxic, oxidative, inflammatory or apoptotic reactions. These findings indicate that in order to design a non-toxic and efficacious free Hb-based blood substitute product, pharmacologic cross-linking of the Hb molecule is necessary. Now, HemoTech has entered the regulatory process for commercial development in the U.S. and abroad.

PA-3 Effect of Hemospan® on Microvessel Diameter and Functional Capillary Density in a Transgenic Mouse Model of Sickle Cell Anemia

AG Tsai ^1,2, P Cabrales¹, MA Young³, RM Winslow^2,3, M Intaglietta²

¹La Jolla Bioengineering Institute, La Jolla; ²Department of Bioengineering, University of California, San Diego, and ³Sangart Inc. San Diego, CA/USA; [email protected]

Targeting O₂ delivery to anoxic regions may reduce the incidence of complete deoxygenation of the RBC and thus be an effective therapy in sickle cell disease. Knockout transgenic mice (n = 6) were treated with an infusion of Hemospan®, a blood substitute that targets oxygen delivery to hypoxic areas (malemide polyethylene glycol-conjugated Hb, Sangart Inc., San Diego, CA) and then subjected to a hypoxic challenge (FiO2 = 0.08). Results were compared to a control group which received saline (n = 5). A 10% blood volume (BV) topload was infused (BV estimated as 6% body weight). Changes of microvessel diameter and functional capillary density (number of capillaries perfused per area of tissue) were studied in vivo using the dorsal skin fold window chamber model. Mean arterial pressure was higher after treatment during normoxia and hypoxia challenge as compared to the control group. Arterioles dilated and venules remained unchanged from baseline during hypoxia in both study groups. FCD was statistically reduced from baseline during hypoxia; however Hemospan® infused animals maintained FCD at a level that was statistically higher than observed with saline infusion; 60 ± 15% and 27 ± 24%, respectively. Thus Hemospan® treatment reduces the severity of hypoxia-mediated decline in FCD. Mechanistically the higher FCD could be due to the higher perfusion pressure in the absence of vasoconstriction. Reduced sickling and inflammatory responses (i.e., lesser leukocyte-adhesion) are likely concomitant mechanisms which support and enhance capillary perfusion.

Supported by NIH BRP Grant HL064395.

SII-3 Design of A Platelet Substitute Utilising Host Fibrinogen to Enhance Haemostatis

GF Walker¹, J.A. Appleby¹, SM Middleton¹, AH Goodall²

¹Haemostatix Ltd, BioCity, Nottingham, UK. ²Cardiovascular Sciences, University of Leicester, UK; [email protected]

A novel platelet substitute is being developed as a safe replacement for platelet transfusion. The product is designed to bind host fibrinogen (Fgn) after injection and interact preferentially with activated platelets at the site of a wound. The Fgn binding peptide Gly-Pro-Arg-Pro (GPRP) was conjugated to human albumin microparticles (MPs). FITC-labelled Fgn binding by the MPs was determined by flow cytometry. To assess MP incorporation into a fibrin clot, GPRP-MPs were added to platelet free plasma with thrombin (0.25 U/mL) and fibrin formation monitored in a Platelet Aggregation Profiler. Impedance aggregometry was used to assess MP interaction with unactivated and ADP-activated platelets in whole blood rendered thrombocytopenic by centrifugation. Bleeding was measured in busulfan-treated thrombocytopenic rabbits dosed with GRPR- (n = 6) or control-MPs (n = 6). GPRP-MPs bound 4 times more FITC-labelled Fgn than control MPs (p < 0.001). After addition of thrombin (0.25 U/mL) and GPRP- or control-MPs to plasma, only GPRP-MPs were incorporated into the fibrin clot. Impedance aggregometry showed that GPRP-MPs in platelet-depleted blood enhanced ADP (3 μ M)-induced aggregation compared with control MPs (Area Under Curve 12.4 ± 2.6 vs 4.0 ± 2.2 U; p < 0.001; n = 6). Without ADP there was little aggregation with either GPRP-MPs or control MPs. (AUC 3.6 ± 2.0 vs 1.9 ± 0.2 U; p = 0.19). In thrombocytopenic rabbits (platelet count 5 x106/mL ± 8; n = 12), blood loss was reduced in rabbits receiving GPRP-MPs compared with control-MPs (9.3 ± 5.5 vs 20.1 ± 7.6 mL; p = 0.02). GPRP-MPs bind Fgn and exhibit haemostatic activity in vitro and in vivo. Fibrinogen-free GPRP-MPs have potential as an effective platelet substitute with significant safety and production advantages.

SV-5 Blood Banking-Induced Impairment of Red Blood Cells Flow Properties

Saul Yedgar¹, Alex Koshkaryev¹, Hannah Relevy², Nogah Manny², Gregory Barshtein¹

¹Department of Biochemistry, Hebrew University Medical School, and ²Blood Bank, Hadassah University Hospital, Jerusalem, Israel; 91120. [email protected]

Background: Blood banking procedures are associated with damage to red blood cells (RBC), which can impair their flow properties, namely their deformability, self-aggregability, and adherence to endothelial cells (EC) of blood vessel walls. Transfusion of such RBC might thus introduce a circulatory risk to recipients. The present study was undertaken to comprehensively explore the effect of cold storage and γ -irradiation on RBC flow properties. Methods: RBC flow properties were monitored prior to and during storage, as well as before and after γ -irradiation, as a function of shear stress, using a computerized cell flow properties analyzer. Results: Routine cold-storage markedly decreased RBC deformability, and strongly elevated their self-aggregability and their adherence to EC, as expressed by the amount of altered RBC and the strength of intercellular interactions. These changes were observed already at the second week of the storage period. The elevation of RBC/EC interaction was well correlated with translocation of phosphatidylserine to the RBC surface. γ -Irradiation induced an immediate, sharp increase in the number of rigid cells, but did not affect RBC adherence and aggregability. Conclusions: RBC hemodynamic behavior appears to be especially sensitive to cold storage, as it is impaired long before the expiration date of blood units, as well as to γ -irradiation. Since impaired RBC flow properties facilitate circulatory disorders, the potential circulatory risk of the practiced blood banking procedure should be taken into account when considering RBC for transfusion.

References: Hovav et al., Transfusion 39: 277-281, 1999; Relevy et al., Transfusion, Published online: Sep 27th, 2007.

SII-1 Peg-Conjugated Hemoglobin (Hemospan®) Liganded with Carbon Monoxide Reduces Myocardial Infarct Size Following Ischemia /Reperfusion in Rats

Mark Young, Jeff Lohman, Ashok Malavalli, Kim Vandegriff, Robert Winslow

Sangart, Inc. San Diego, CA, USA; [email protected]

Carbon monoxide (CO) is reported to confer cytoprotective effects in models of hypoxia, and ischemia. However therapeutic strategies are complicated by the lack of a satisfactory delivery method for CO. Hemospan (MP4) is a polyethylene glycol-conjugated human hemoglobin in development as an oxygen carrying plasma expander. We investigated the effects of Hemospan saturated with CO (CO-MP4) in a rat model of myocardial ischemia and reperfusion, and compared the effects with oxy-MP4 or α α -Hb. Lactated Ringer's (LR) and preconditioning (PC) were employed as negative and positive controls, respectively. Pentobarbital-anesthetized rats were mechanically ventilated (FiO₂ = 0.3) and subjected to 30 min of coronary ligation followed by recovery and 24 hr reperfusion. Treatment (30% blood volume topload) was begun prior to ligation and continued for 24 hr. Non-ischemic area (area at risk, AAR) was distinguished by Evans blue and infarct size (IS) was delineated with triphenyl tetrazolium chloride. Ischemic area was uniform in all animals as AAR did not differ between groups. Infarct size (IS/AAR) was reduced (P < .05) in CO-MP4 animals (37 ± 6%) compared with oxy-MP4 (61 ± 7%), α α -Hb (63 ± 5%), or LR (53 ± 3%). PC elicited the greatest protection (IS/AAR = 13 ± 6%). CO-Hb at the end of ischemia was 5.2± 0.2% of total Hb in CO-MP4 treated rats. Arterial pressure and heart rate did not differ between groups of rats. In separate studies with rats, CO was found to distribute equally between MP4 and RBC-Hb within 20 min, and disappear from the blood within 30 min following cessation of infusion. These data demonstrate that CO is cardioprotective in this model, and that MP4 may be a safe and controlled method of delivery.

SVII-4 Effect and Mechanism of PEG-conjugated Hemoglobin on Hypoxia Microenvironment in HeLa Tumor Model

Min Dai¹, Minghua Yu¹, Jianqun Han¹, Hongwei Li¹, Jian Zhang¹, Qian Liu², Ruijuan Xiu¹

¹Institute of Microcirculation, Chinese Academy of Medical Sciences (CAMS) & Peking Union medical college (PUMC), ²Peking Union Medical College Hospital (PUMCH), Beijing, China; [email protected]

Tumor hypoxia microenvironment is strongly associated with a diminished therapeutic response and malignant progression. So it is then not surprising that hypoxia has been considered an attractive target for the development of novel anti-cancer therapies. A number of artificial oxygen carriers are currently in advanced clinical trials for their ability to replace red blood cells and to ensure adequate tissue oxygenation. However, little has been done to investigate that if intravenous administration of blood substitutes were effective in increasing the oxygenation throughout experimental tumors. To investigate the effect of PEG-conjugated hemoglobin on tumor hypoxia microenvironment, HeLa cells were cultured and injected to the armpit of BALB/c nude mice. PEG-conjugated hemoglobin solution was intravenous administrated twice a week, and then the animals were sacrificed after a month. Hypoxyprobe™ -1 (pimonidazole hydrochloride) was used the detected the Oxygen gradients in tumor tissue. ELISA and immunocytochemistry methods used for the detection of HIF and VEGF protein in blood and tumor tissue separately have been determined. The results showed that hypoxia in tumor tissue were attenuated by PEG-conjugated hemoglobin solution. HIF and VEGF expression were decline compared with the control group. Taken together, our data show that PEG-conjugated hemoglobin suppresses the HIF and VEGF expression due to the increase of tumor tissue oxygenation.

SIV-1 The Study on a Rat Exchange Transfusion Model By Artificial Red Blood Cells (Hemoglobin Vesicle)

Jing Fan, Xueqiao Wang

Tianjin Blood Center Tianjin, P.R. China; [email protected]

Objective: In a rat exchange transfusion model, as an artificial oxygen carrier, the security and validity with the artificial red blood cells (hemoglobin vesicle, HbV) has been verified. Methods: The first, to construct a best rat exchange transfusion model. The second, using the model to transfuse blood with the artificial red blood cells. The finally, to evaluate the capabilities of the artificial red blood cells by some methods (such as: Blood routine test, Blood gas test, Flow cytometry and Pathology), and to compare the therapeutic effect of the different product in the rat acute hemorrhagic shock and resuscitation. Results: The artificial red blood cells (HbV) has been developed to provide the oxygen-carrying ability. The Studies have shown that after approximately 70% of hemoglobin is lost from circulation, then supply the artificial red blood cells suspension, the rats survive rate is 100%. The Pathology results indicate that no significant change in the viscera(heart, liver, kidney, lung). Conclusion:The artificial red blood cells (HbV) are the artificial oxygen carrier with bionics meaning. It can be helpful the acute hemorrhagic shock rats pass the first hour after the severe trauma, that is golden hour. Key words: artificial red blood cells; hemoglobin vesicle; a rat exchange transfusion model

SVII-1 Effect of PEG-Conjugated Hemoglobin Solution with Chemotherapy on characteristics of tumor neovascularization

Jianqun Han¹, Min Dai¹, Minghua Yu¹, Hongwei Li¹, Jian Zhang¹, Qian Liu² Ruijuan Xiu¹

¹ Institute of Microcirculation, Chinese Academy of Medical Sciences (CAMS) & Beijing, China Peking Union Medical College (PUMC), ²Peking Union Medical College Hospital (PUMCH) Beijing, China; [email protected]

It is recognized that hypoxia is a major driving force behind tumor vascularization, metastatic spread and resistance to radiation and chemotherapy. Tortuosity, dilation, sacculation and permeability morphology changes are the notable characteristics of newly formed tumor vessels which conversely worsen tumor tissue oxygen provision. The aim of this study was focused on the tumor microvessels morphology changes when PEG-conjugated hemoglobin combined with cislplatin treatment. Inoculated tumor cells to the submucosa of golden hamster's cheek pouch to establish tumor model. Computer assisted intravital microscopy was used to observe and measure microvessels tortuosity and functional capillary density. At 5 days post-innoculation microvessel tortuosity close to tumor mass was significantly decreased in animals received both cisplatin and PEG-Hb (0.6 g/kg) compared to animals received single agent cisplatin. However, no significant changes was seen in animals received both cisplatin and PEG-Hb (0.3 g/kg) compared to control. Similar tendency was presented in tumor functional capillary density between the two groups. The improvement of newly formed vessel structure was positive corelation with tumor tissue oxygenation. Conclusion: Higher dose of PEG-conjugated hemoglobin solution with chemotherapy treatment is benefit to tumor tissue oxygenation and microvessel normalization. Improved tumor vasculature facilitate oxygen and chemotherapic agents transport to tumor tissue. It is indicated that the evaluated PEG-conjugated hemoglobin may be a potencial adjuvant to chemotherapy in cancer.

SVI-3 Polyoxyethylene-Modified Albumin-Heme Hybrid—Synthesis, Property and Oxygen-Binding Ability

YB Huang¹, T Komatsu², E Tsuchida²

¹State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin Province, P.R.China. ²Advanced Research Institute for Sciences and Technologies, Waseda University, Tokyo, Japan; [email protected]

Recombinant human serum albumin (rHSA) incorporates a synthetic heme, providing an artificial hemoprotein [albumin-heme (rHSA-heme)] which has the potential to bind and release f under physiological conditions (pH 7.3, 37°C) in the same manner as Hb.

In order to improve the circulation persistence in blood stream, we have recently prepared a new albumin-heme hybrid modified by polyoxyethylene (PEG) derivatives, and its structure, property and oxygen-binding ability were carefully investigated. The maleimido-activated or succinimide-activated PEG (Mw. 2 kDa and 5 kDa; M2000, M5000, S2000 and S5000) were used for covering the surface of rHSA-heme by covalently bond.

By controlling the reaction conditions, 4 types of PEG -modified rHSA-heme ([rHSA] 5 wt%) with 6 PEG chains have been synthesized; numbers of the chains were determined by MALDI TOFMS analyses. The PEG (rHSA-heme)s also bind O₂ reversibly dependent of the O₂ partial pressure. The O2-binding affinities (p1/2) were 31∼ 36 Torr and the half times of the oxy-form were around 12 hr (37°C). The viscosity of the S5000 and M5000 modified PEG (rHSA-heme) solutions showed relatively high values, while the viscosity of S2000 and M2000 modified PEG (rHSA-heme) were nearly the same as that of the rHSA-heme solution. The circulation persistence of the heme was also investigated by 20% top-load in rats, and the half lifetime was significantly extended to 15 hr.

The author would like to thank very much to Dr. T. Komatsu and Prof. E. Tsuchida from Advanced Research Institute for Sciences and Technologies, Waseda University, Japan for their great contribution on this oxygen-transfusion project.

SIII-2 Modification of Hemoglobin by Crosslinking with a Band 3 Terminus Derived Peptide as a Blood Substitute

YL Lin, KT Huang

Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan, China; [email protected]

Hemoglobin solution lacking 2,3-diphosphoglycerate (2,3-DPG) is not able release sufficient oxygen in the circulation of human body. In this study, therefore, a peptide composed of 9 amino acids, 7 from N terminus of human erythrocytic Band 3 protein (AcMEELQDD) and cysteine and glutamic acid added sequentially in C terminus is crosslinked to hemoglobin surface serving as a dynamic allosteric effector of hemoglobin for oxygen release. The derivative of polyethylene glycol (PEG), Maleimide-PEG-N-hydroxysuccinimidyl (MAL-PEG-NHS) was used to crosslink hemoglobin with peptide. The crosslinked peptide on hemoglobin is able to modulate the oxygen affinity of hemoglobin, resulting in a higher P₅₀ value of this peptide-modified hemoglobin (peptide-PEG-Hb) in the oxygen dissociation curve as compared to unmodified hemoglobin. Furthermore, succinimidyl propionate-PEG-succinimidyl propionate (SPA-PEG-SPA) was used to crosslink intra- and inter-hemoglobin molecules to prevent the dissociation of hemoglobin tetramers. This resulting hemoglobin polymer exhibited a similar oxygen dissociation behavior to peptide-PEG-Hb. Taken together, we have successfully modified hemoglobin with a conjugated oxygen affinity modulator providing a new aspect of hemoglobin-based blood substitute.

SVII-2 Effects of Different Molecular Weight of Hydroxyethyl Starch on Hemorrhagic Shock in Rats

^1,2Li Tao, ²Liu Liang-ming,¹Yang Chengmin

¹Tianjin Concord Biotech Development Research Institute ²State Key Laboratory of Trauma, Burns and Combined Injury, Department 2 Research Institute of Surgery, Daping Hospital, The Third Military Medial University, Chongqing 400042, China [email protected], [email protected]

Objective: To compare the resuscitation effect of 6% different molecular weight hydroxyethyl starch(HES) on hemorrhagic shock in rats. Methods: SD rats were used to make hemorrhagic shock model by 45% hemorrhagic. Effects of different molecular weight HES(HES40, HES130, HES200) on mean arterial blood pressure(MAP), left intraventricular systolic pressure(LVSP), the maximal change rate of left intraventricular pressure± dp/dt_max) of hemorrhagic shock rat were observed. Meanwhile, the artery blood gas, the survival time and 24 hour survival rate were also observed in the present study. Results: Improving hemodynamic parameter effects of our HES on hemorrhagic shock rats were similar to Voluven and Haes. HES130 and HES200 prolonging the survival time of shocked animals was pretty better than HES40, among the three types of molecular weight HES, HES200 had the best effect and equivelant to Voluven and Haes. Conclusion:Three types of molecular weight HES produced by our company have good beneficial effect on hemorrhagic shock. HES200 has a better effect.

Key Words: Hemorrhagic shock; HES; Voluven; Haes

SIII-5 Porphyrin-Histidine System Recognition of CO2 and Other Anions: mimetic of hemoglobin action in physiological conditions

Di Li¹, Wenqian Dong¹, Tianjun Liu²

Chinese Academy of Medical Sciences & Institute of Biomedical Engineering, Peking Union Medical College, Tianjin 300192, China; [email protected]

Hemoglobin plays a crucial role in life, as oxygen carrier in artery and carbon dioxide carrier in vein, with this dual carrier activity, hemoglobin moves the life cycle in animals and human beings. As an important research topic in biomaterials, with the aim to mimetic and substitute natural system, there are a lot of research papers focused on the oxygen carrier system all over the world, such as human and animal hemoglobin modification, natural or synthetic heme-based oxygen carriers, recombinant and transgenic hemoglobin as well as perfluorocarbon based oxygen carriers, etc. All of these reveal that oxygen carrier is very important. However, the action of carbon dioxide has been ignored, and less reports pay attention to the carbon dioxide, which plays an almost the same function as oxygen in life. Since the carbon dioxide accumulation in life will cause disorder of acid-base equilibrium in vivo, and be lethal for the patient suffering of lung illness. Here we design and synthesize some model compounds with porphyrin as platform with histidine as binding point, and try to mimetic the hemoglobin acting on carbon dioxide in physiological conditions and other systems.

In porphyrin chemistry, molecular recognition has been developed rapidly and turned out to be a front topic in recent years. As a favorable host, porphyrin can recognize multifarious ion and bioactive molecule, which could be used in the field of enzyme analogue, phototherapy, molecular probe and so on. Here in this paper the following content was reported : 1. Synthesis of “porphyrin-histidine” system: a series of “porphyrin-histidine” compounds were synthesized with either free amino groups or free imidazole groups or both as binding points. 2. Evaluation of molecular recognition ability for different compounds: The effect of functional groups such as amino or imidazole group as well as ambient environmental effect on molecular recognition was studied. Results showed that molecular recognition ability correlated tightly with the compound structure of host and guest. For the same host, different binding ability to HCO3-, CO32-, H2PO4-, HPO42-, SO42-, SO32- were detected. 3. Comparing the absorbing CO2 ability for different compounds: On the basis of pressure balance, a simple equipment was designed to detect absorption of carbon dioxide. Results showed that free amino group plays a predominant role in carbon dioxide absorption, while imidazole moiety acts less in our system.

Summary: the synthetic “porphyrin-histidine” show the selective recognition ability for different anions, and its binding ability depends on both stereospecific blockade and chemical structure. The absorption of carbon dioxide of these system benefits from the free amino group in the porphyrin-histidine system.

SIII-1 Protection of Isolated Eat Heart by Polyethylene Glycol-Bovine Hemoglobin Solution

X Chang, C Long

Fuwai Hospitai, Beijing, P.R. China; [email protected]

The aim of the study is to investigate the effect of polyethylene glycol-bovine (PEG-bHb), which was used as an oxygen carrier in cardioplegic solution, on the protection of isolated rat hearts. Rat hearts were harvested to transferred to langendorff circuit and went through perfusion- cardioplegia&ischemia -reperfusion. St. Thomas cardioplegic solution was compared by 3 other cardioplegic solutions containing different concentrations of PEG-bHb. The results showed that after reperfusion, cardiac function and histological change were better in PEG-bHb groups, myocardial ATP contant was higher and cardiac troponin I in coronary flow was lower in in PEG-bHb groups. This study demonstrated that PEG-bHb in cardioplegic solutions can provide better myocardial protection during ischemia.

SIV-2 Diffusion Behavior of Hemoglobin-Loaded Nanoparticles with Porous Structure as Oxygen Carriers

Y Sheng, Y Yuan, CS Liu

Engineering Research Center of Biomedical Materials Under Ministry of Education, East China University of Science and Technology, Shanghai, P.R. China; [email protected]

In this study, a series of molecular weight of poly(ethylene glycol)s (PEGs) were used as probes to evaluate the porous structure of hemoglobin-loaded particles (HbP) fabricated by different process conditions and diffusion behaviors of small molecules (such as glucose, ascorbic acid and glutathione) through HbP were investigated. The results show that HbP with higher encapsulation efficiency (80%) and desirable diameter (70-200nm) were produced through an improved double emulsion method; different molecular weight of PEGs, coencapsulated in the internal phase with Hb, successfully detected the porous structure of HbP, especially the pore size which it is difficult to find a popular apparatus to observe directly; meanwhile, distinct diffusion behaviors of small molecules, which was dominated by the pore parameter of HbP, were obtained based on various preparation conditions. It is significant to understand the relationship between porous structure and diffusion behavior so that the preparation of hemoglobin-loaded porous nanoparticles can be controlled, monitored and reproduced with a desired diffusion profile of small molecules.

Many diseases are caused by tissue hypoxia that due to blood circulation obstacles, which is the most serious problem in clinics. Prof. Yang studied many drugs, such as oxygen carrying anti-shock drugs, the treatments drugs of ischemic cardiocerebrovascular diseases and so on in recent years, the reports of preliminary studies on the pharmacodynamics is below: Our preliminary results showed that poly-human hemoglobin solution has capacities of oxygen delivery together with remaining red blood cells, indicating potential clinical applications in improving or curing microcirculation disorder. Experiments of perfusion and preservation of isolated organs demonstrated that our products could significantly protect hearts from ischemia/reperfusion injury and appear improving the function of rat hearts after prolonged storage. We also study blood flow in Golden Hamster's microcirculation. 13 male hamsters were randomly divided into two groups. Control group include 5 hamsters with ACD anticoagulant fresh blood of health people and Experimental group include 7 hamsters with human polymerized hemoglobin The whole exchange transfusion volume was up to 40% and each mouse was completed within 15 minutes. Slow blood flow, stagnation, dyspnea, systemic convulsion and finally asystole appeared immediately in five mice of control group during the period of exchange transfusion. Five mice were all dead within twenty minutes. But only one mouse of the experimental group showed abnormal reactions and was dead after 30 minutes. The remaining six mice's microcirculation kept well and all survived. In short, preliminary study demonstrates that poly-hemoglobin solution has potential clinical application in improving or curing disease of microcirculation disorder and perfusion and preservation of isolated organ.

SIV-5 Reconstruction of Erythrocyte and Its Cellular Mechanical Properties

Xiang Wang, Wei Gao, Li Yang, Wei-yan Peng, Jia-xin Xie

College of Bioengineering, Chongqing University, Chongqing P.R.China; [email protected]

Erythrocyte shape and its biomechanical properties have close relation to its 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 studied. 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. The study implied the physiological function of reconstructed erythrocyte were similar to that of natural erythrocyte.

SIII-6 Effect of Polymerization Reaction on Cation Exchange Chromatography on Molecular Weight Distribution of Polymerized Hemoglobin-Based Oxygen Carriers

X Wang, L Huang, CM Yang, JF Wang

Tianjin University; Tianjin, P.R. China, Institute of Tianjin Uion biotechnology development company, Tianjin, P.R. China, Tianjin Polytechnology University, Tianjin, P.R. China; [email protected], [email protected]

Blood substitutes based on glutaraldehyde cross-linked hemoglobin (PolyHb) are currently being developed for use in human subjects needing blood transfusions. Despite the commercial development of PolyHb dispersions, it seems that the glutaraldehyde molecule is unsuitable cross-linker for a number of reasons: it is a dialdehyde (too reactive), it is too long a molecule (facilitating intertetramer cross-links), and it lacks any intrinsic or functional specificity, leading to uncontrolled polymerization. For these reasons, a new method, polymerization reaction on cation exchange chromatography (PRCEC), is established to provide control of cross-linking (Mw). When Hemoglobin is modified or polymerized by glutaraldehyde, the magnitude of positive charge on the molecule reduces. Because the strength of binding depends on the size of charge carried on molecules in cation exchange chromatography (CEC), hemoglobin or polyHb with low modification or polymerization is enriched. If the polymerization takes place in this asymmetrical system, they have the more reaction odd than hemoglobin (polyHb) with high modification (polymerization). So, polyHb with narrow molecular weight distribution and low average molecular weight may be prepared by this way. The results of this study show that product polymerized by PRCEC (Hb:GDA molar ratio1:12) is polyHb with two tetramers (128kD) to four tetramers (256kD) with the yield of about 53%.

S1-4 Dextran-Hemoglobin

J. Tze-Fei Wong

Department of Biochemistry and Applied Genomics Center, Hong Kong University of Science & Technology, Clear Water Bay, Hong Kong, China [email protected]

Dextran-hemoglobin (DxHb) consists of a covalent conjugate between human hemoglobin and dextran MW20,000. It was the first blood substitute to allow a complete red blood cell replacement in an exchange transfusion model followed by spontaneous recovery under room air (1). It is equally effective in the hemorrhagic shock model (2). Its function as an HBOC is conferred with the following advantages:

• Ease of preparation through coupling between hemoglobin and activated dextran (3).

• Long circulation half life of 2.4 days, or 1.9 days after correction for methemoglobin formation (1).

• Non-entry into either kidneys or lymph (1).

• Utility both as an HBOC and as an oxygen sequestering agent to enhance the efficiency of cancer radiation therapy (4).

1. Tsai, S.P. and Wong, J.T. (1997). Advances in Blood Substitutes, ed. Winslow, R.M. et al, Birkhauser. P. 233–247.

2. Wong, J.T. and Blumenstein, J. (2007). Blood Substitutes, ed. Winslow, R.R. Elsevier. P. 483–487.

3. Xue, H. and Wong, J.T. (1994). Methods Enzymol. 231: 308–322.

4. Hill, R.P., Porter, L.S., Ives, S.A. and Wong, J.T. (1984). Int. J. Radiation Oncology Biol. Phys. 10: 369–373.

PA-1 Inhaled Nitric Oxide Prevents Vasoconstriction after Tetrameric Hemoglobin Infusion

Binglan Yu¹, Michael J. Raher¹, Rong Liu¹, Kenneth D. Bloch^1,2, Fumito Ichinose^1,2, Warren M. Zapol¹

¹Department of Anesthesia and Critical Care, ²Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, U.S.; 02114 [email protected]

Background: Hemoglobin (Hb)-based oxygen carriers (HBOCs) have long been investigated for their potential use as blood substitutes. Clinical development of HBOCs has been limited by the adverse side effect of intense vasoconstriction that is attributed to scavenging of endothelial nitric oxide (NO). The objective of our study was to investigate whether pretreatment with inhaled NO could prevent the systemic hypertension caused by infusion of autologous tetrameric Hb solution in mice. Methods:Tetrameric Hb solution (4 g/dl) was prepared from whole blood of C57BL/6 mice, and administered IV (0.012 ml/g BW) following a period of breathing either air or NO gas (80 ppm). Systolic blood pressure (SBP) was measured non-invasively in awake mice by tail-cuff method, and in anesthetized mice by invasive hemodynamics. Results: After administration of tetrameric Hb, SBP increased from 118 ± 3 to 143 ± 7 mmHg (p < 0.05) in mice breathing air alone. In contrast, when mice were pretreated with inhaled NO (80 ppm, 15 min), subsequent administration of tetrameric Hb while breathing air did not alter SBP (118 ± 3 vs. 120 ± 2 mmHg, p = NS). These findings were confirmed using invasive hemodynamic methods. Breathing NO prior to tetrameric Hb administration did not increase the metHb level in plasma (4 ± 1% at 10 min after administration). However, 80 ppm NO breathing continued for 10 min after tetrameric Hb administration, markedly increased plasma metHb levels (74 ± 10%). Conclusions: Pretreatment of mice with 80 ppm iNO for 15 min prevents the systemic vasoconstrictor effects of subsequent IV tetrameric Hb infusion without causing methemoglobinemia.

SIV-4 Influence of PEG-conjugated Hemoglobin on Tumor Oxygenation and Response to Chemotherapy

Minghua Yu¹, Jianqun Han¹, Min Dai¹, Hongwei Li¹, Jian Zhang¹, Qian Liu², Ruijuan Xiu¹

¹Institute of Microcirculation, Peking Union Medical College (PUMC) & Chinese Academy of Medical Sciences (CAMS), Beijing, China; ²Peking Union Medical College Hospital (PUMCH), Beijing, China [email protected]

The presence of hypoxia in solid tumors has been recognized for more than 50 years. Hypoxic tumors are significantly more malignant, metastatic, radio- and chemoresistant and have a poor prognosis. a number of therapeutic strategies have also been established to overcome tumor hypoxia by improving oxygen supply either by oxygen or carbogen breathing or by increasing the hemoglobin level and oxygen delivery. The use of artificial oxygen carriers represents a new approach to the problem of hypoxia. In the present study, Female athymic BALB/c nude mice bearing the cervical carcinoma were untreated or treated with cisplatin (5 mg/kg i.p.) to determine whether administration of PEG-conjugated hemoglobin (0.3 g/kg i.v. or 0.6 g/kg i.v.) could improve the tumor oxygenation and to determine whether preadministration of PEG-conjugated hemoglobin could enhance the anti-tumor efficacy of cisplatin. Hypoxia marker pimonidazole staining was employed to detect tumor tissue oxygenation status and treatment efficacy was determined by measurements of tumor volume, tumor growth delay and the extent of tumor apoptosis. We found that The application of higher dose (0.6 g/kg) PEG-hemoglobin solution could significantly ameliorate the hypoxic condition in cervical carcinoma xenograft models. Co-administration of PEG-conjugated hemoglobin (0.6 g/kg) with cisplatin produced signifficant tumor growth inhibition and a reduction in tumor vasculature as compared to cisplatin alone, while the bodyweight loss as an indicator of toxicity was more pronounced in the combination treatment groups. Collectively, the evaluated PEG-hemoglobin in this experiment may have positive effects on cisplatin or cisplatin-based chemotherapy, further work still needs to be carried out to minimize its potential toxicities.

SIV-3 Reduction and Suppression of Methemoglobin Loaded In the Polymeric Nanoparticles Used As Blood Substitutes

XL Zhang, YY, CS Liu

Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai; [email protected]

A novel nonenzymatic reduction and suppression route, combination a fast pre-reduction by sodium dithionite (SD) and a sustaining post-reduction by reducing agents present in human plasma, was developed to control the metHb in bovine Hb-loaded nanoparticles (HbP) with porous microstructure to a desirable level. Meanwhile, the influences of the preparation factors were also optimized. In the pre-reduction, the metHb in the raw Hb could be reduced from over 90% to 1.2% using SD combination with gel filtration. In double emulsion preparation, lower emulsion strength, PEGylated polymer and addition of miscible solvent, such as acetonitrile could pronouncedly suppress metHb formation. The resultant metHb level in HbP under the optimal conditions was about 5.6%, which can be further reduced to 1.4% by the reducing agents in plasma with the help of superoxide dismutatse and catalase, named as the sustaining post-reduction. The results achieved are promising for the fabrication of blood substitutes with controlled metHb level, which can fulfill the function of binding/delivering oxygen to tissues in vivo for future trials.