key: cord-0037044-4y1tw6qv
authors: Kobayashi, K.; Horinouchi, H.; Watanabe, M.; Izumi, Y.; Teramura, Y.; Nakagawa, A.; Huang, Y.; Sou, K.; Sakai, H.; Komatsu, T.; Takeoka, S.; Tsuchida, E.
title: Safety and Efficacy of Hemoglobin-Vesicles and Albumin-Hemes
date: 2005
journal: Artificial Oxygen Carrier
DOI: 10.1007/4-431-26651-8_1
sha: 1f4459a9e8d4bdb355a4909fe64d139b6f1ce0a9
doc_id: 37044
cord_uid: 4y1tw6qv

Keio University and Waseda University have worked together on artificial O(2) carrier research for 20 years in close cooperation. Two candidate materials have been selected from the viewpoints of safety, efficacy, and cost performance. One is Hemoglobin-vesicles (HbV) and the other is albumin-heme (rHSA-heme). This chapter summarizes our video presentation that introduced the recent results of our research into HbV and rHSA-heme.

For human beings to survive, it is necessary to continuously deliver oxygen that is needed for the respiration of all tissue cells. Red blood cell, a so-called moving internal-organ, reversibly binds and releases O 2 under physiological conditions. From this point of view, red blood cell substitutes, or O 2 -Infusions, are very important. In order to promote this research, we have emphasized that the establishment of basic science for macromolecular complexes and molecular assemblies is essential. We have systematically studied the Metal Complexes (synthetic heme derivatives) embedded into a hydrophobic cluster, and clarified that the electronic processes of the active sites are controlled by the surrounding molecular environment. Therefore, the reaction activity and its rate constant are observed as cooperative phenomena with the would take considerable time to arrive at a social consensus for its use. We started to produce Hb-vesicles (HbV) using purified Hbs and molecular assembly technologies. In the late 1990's, a mass-production system for recombinant human serum albumin (rHSA) was established and we then prepared albumin-heme hybrids (rHSA-heme) using its non-specific binding ability, which is now considered to be a promising synthetic material.

Based on our effective integration of molecular science and technologies for functional materials developed by Waseda University, and the outstanding evaluation system of safety and efficacy developed by Keio University using animal experiments, we have made strong progress in our research on the O 2 -Infusion Project. During this period, we have received substantial funding support from the Japanese government. In the near future, mass production and clinical tests of O 2 -Infusion will be started by a certain pharmaceutical industry.

Historically, the first attempt of Hb-based O 2 carrier in this area was to simply use stroma-free Hb. However, several problems became apparent, including dissociation into dimers that have a short circulation time, renal toxicity, high oncotic pressure and high O 2 affinity. Since the 1970s, various approaches were developed to overcome these problems [7, 8] . This includes intramolecular crosslinking, polymerization and polymer-conjugation. However, in some cases the significantly different structure in comparison with red blood cells resulted in side effects such as vasoconstriction [9] .

Another idea is to encapsulate Hb with a lipid bilayer membrane to produce HbV that solves all the problems of molecular Hb [10] . Red blood cells have a biconcave structure with a diameter of about 8000 nm. Red blood cells can deform to a parachute-like configuration to pass through narrow capillaries. The possibility of infection and blood-type mismatching, and short shelf life are the main problems. Purified Hb does not contain blood-type antigen and pathogen, thus serves as a safe raw material for HbV.

HbV, with a diameter of 250 nm, do not have deformability but are small enough to penetrate capillaries or constrict vessels that RBC cannot penetrate. The surface of the vesicles is modified with polyethylene glycol (PEG) to ensure homogeneous dispersion when circulated in the blood and a shelf life of two years. The idea of Hb encapsulation with a polymer membrane mimicking the structure of RBC originated from Dr. Chang at McGill University [7] . After that, the encapsulation of Hb within a phospholipid vesicle was studied by Dr. Djordjevici at the University of Illinois in the 1970s [11]. However, it was not so easy to make HbV with a regulated diameter and ade-quate O 2 transport capacity. We made a breakthrough in routinely producing HbV by using fundamental knowledge of macromolecular and supramolecular sciences. Some of the related technologies have already been published in academic journals [12] [13] [14] [15] [16] [17] [18] [19] . Several liters of HbV are routinely prepared in a completely sterile condition. Hb is purified from outdated red blood cells, and concentrated to 40 g/dl. Virus removal is performed using a combination of pasteurization at 60°C and filtration with a virus removal filter. The Hb encapsulation with phospholipids bilayer membrane and size regulation was performed with an extrusion method. The vesicular surface is modified with PEG chains. The suspension of Hb-vesicles is dated at the final stage.

The particle diameter of HbV is regulated to about 250 nm, therefore, the bottle of HbV is turbid, and is a suspension. One vesicle contains about 30,000 Hb molecules, and it does not show oncotic pressure. There is no chemical modification of Hb. Table 1 summarizes the physicochemical characteristics of HbV. O 2 affinity is controllable with an appropriate amount of allosteric effectors, pyridoxal 5-phosphate. Hb concentration is regulated to 10 g/dl, and the weight ratio of Hb to total lipid approaches 2.0 by using an ultra pure and concentrated Hb solution of 40 g/dl, which is covered with a thin lipid bilayer membrane. The surface is modified with 0.3 mol% of PEGlipid. Viscosity, osmolarity, and oncotic pressure are regulated according to the physiological conditions.

HbV can be stored for over two years in a liquid state at room temperature [17] . There is little change in turbidity, diameter, and P 50 . MetHb content decreases due to the presence of reductant inside the HbV, which reduces the 4 K. Kobayashi et al. 

The efficacy of HbV has been confirmed with isovolemic hemodilution and resuscitation from hemorrhagic shock. Some of the results have already been published in academic journals in the fields of emergency medicine and physiology [20] [21] [22] [23] [24] [25] [26] [27] [28] . In this chapter two important facts are described. One is isovolemic hemodilution with 90% blood exchange in a rat model. The other is resuscitation from hemorrhagic shock in a hamster model. To confirm the O 2 transporting ability of HbV, extreme hemodilution was performed with HbV suspended in human serum albumin (HSA) [21, 23] Mean arterial pressure and renal cortical oxygen tension were monitored arterial pressure and renal cortical O 2 tension, and finally all the rats died of anemia. On the other hand, hemodilution with HbV, suspended in HSA sustained both blood pressure and renal cortical O 2 tension, and all the rats survived. These results clearly demonstrate that HbV, has sufficient O 2 transporting capability.

To observe the microcirculatory response to the infusion of Hb products, we used the intravital microscopy equipped with all the units to measure blood flow rates, vascular diameter, O 2 tension, and so on. This system was developed by Professor Intaglietta at the University of California, San Diego. We used the hamster dorsal-skin fold preparation that allows observation of blood vessels from small arteries to capillaries. We evaluated the HbV suspension as a resuscitative fluid for hemorrhagic-shocked hamsters [26] .About 50% of the blood was withdrawn, and the blood pressure was maintained at around 40 mmHg for 1 h. The hamsters either received HbV suspended in HSA (HbV/HSA), HSA alone, or shed blood (Fig. 3) . Immediately after infusion, all the groups showed increases in mean arterial pressure, however, only the albumin infusion resulted in incomplete recovery. The HbV/HSA group showed the same recovery with the shed autologous blood infusion. During the shock period, all the groups showed significant hyperventilation that was evident from the significant increase in arterial O 2 partial. Simultaneously, 6 K. Kobayashi et al. base excess and pH decreased significantly. Immediately after resuscitation, all the groups tended to recover. However, only the HSA group showed sustained hyperventilation. Base excess for the HSA group remained at a significantly lower value one hour after resuscitation. Blood flow decreased significantly in arterioles to 11% of basal value during shock. The HbV/HSA and shed autologous blood groups immediately showed significant increases in blood flow rate after resuscitation, while the albumin group showed the lowest recovery.

We further examined the safety profile of HbV such as cardiovascular responses, pharmacokinetics, influence on reticulo endothelial system (RES), influence on clinical measurements and daily repeated infusion [29] [30] [31] [32] [33] [34] [35] .

We observed the responses to the infusion of intra-molecularly crosslinked Hb (XLHb) and HbV into conscious hamsters. XLHb (7 nm in diameter) showed a significant increase in hypertension equal to 35 mmHg, and simultaneous vasoconstriction of the resistance artery equal to 75% of the baseline levels [30] (Fig. 4) . On the other hand, HbV at 250 nm, showed minimal change. The small acellular XLHb is homogeneously dispersed in the plasma, and it diffuses through the endothelium layer of the vascular wall and reaches the smooth muscle. Intra-moleculary cross-linked Hb traps nitric oxide (NO) as an endothelium-derived relaxation factor, and induces vasoconstriction, and hypertension. On the other hand, the large HbV stay in the lumen and do not induce vasoconstriction. Several mechanisms are proposed for Hb-induced vasoconstriction. These include NO-binding, excess O 2 supply, reduced shear stress, or the presence of Hb recognition site on the Safety and Efficacy of Hemoglobin-Vesicles and Albumin-Hemes Professor Suematsu at Keio University has revealed the effects of Hb-based O 2 carriers in hepatic microcirculation [29, 32] (Fig. 5) . On the vascular wall of the sinusoid in hepatic microcirculation, there are many pores, called fenestration, with a diameter of about 100 nm. The small Hb molecules with a diameter of only 7 nm extravasate through the fenestrated endothelium and reach the space of Disse. On the other hand, HbV particles, which are larger than the pores, do not extravasate. Heme of extravasated Hb is excessively metabolized by hemeoxygenase-2 in hepatocyte to produce CO and bilirubin. Even though CO acts as a vasorelaxation factor in the liver, the excess amount of Hb rapidly binds CO, resulting in the vasoconstriction and an increase in vascular resistance. On the other hand, HbV (250 nm in diameter) is large enough to remain in the sinusoid, and the vascular resistance is maintained.

So, what is the optimal molecular dimension of Hb-based O 2 carriers? The upper limitation is below the capillary diameter to prevent capillary plugging, and for sterilization by membrane filters (Fig. 6) . On the other hand, smaller sizes exhibit a higher rate of vascular wall permeability with side effects such as hypertension and neurological disturbances. HbV exhibits a very low level of vascular wall permeability. Therefore, the HbV appears to be appropriate from the viewpoint of hemodynamics. We have clarified the influence of HbV on the RES, because the fate of HbV is RES trapping. Circulation persistence was measured by monitoring the concentration of radioisotope-labeled HbV in collaboration with Dr. W.T. Phillips at the University of Texas at San Antonio. The circulation half-life is dose dependent, and when the dose rate was 14 ml/kg, the circulation half-life was 24 h. The circulation time in the case of the human body can be estimated to be twice or three times longer; or about 2 or 3 days at the same dose rate. Gamma camera images of radioisotope-labeled HbV showed the time course of biodistribution. Just after infusion, HbV remains in the blood stream so that the heart and liver that contain a lot of blood showed strong intensity. However, after it is finished playing its role in O 2 transport, a total of 35% of HbV are finally distributed mainly in the liver, spleen and bone marrow.

The time course of liver uptake was monitored with a confocal fluorescence microscope. Hb-vesicle was stained with a lipid fluorophore. The liver of an anesthetized hamster was exposed and a fluorescence-labeled-HbV was infused intravenously. Due to the motion of respiration, the picture oscillates. However, a static frame can be obtained. The individual particles of HbV cannot be recognized. When the vesicles are accumulated in phagosomes of Kupffer cells, they can be recognized with a strong fluorescence. How is HbV metabolized in macrophages? The transmission electron microscopy (TEM) of the spleen 1 day after infusion of HbV clearly demonstrated the presence of HbV particles in macrophages, where HbV particles that appear as black dots are captured by the phagosomes [34] (Fig. 7) . Red blood cells and HbV contain a lot of ferric iron with a high electron density, so that they show Safety and Efficacy of Hemoglobin-Vesicles and Albumin-Hemes 9 strong contrast in TEM. However, after 7 days, the HbV structure cannot be observed. We confirmed no abnormalities in the tissues and no irreversible damage to the organs or complete metabolism within a week. A Polyclonal anti-human Hb antibody was used as the marker of Hb in the HbV. This antibody does not recognize rat Hb. The red-colored parts indicate the presence of Hb in HbV, and almost disappear after 7 days in both the spleen and liver. This shows that HbV can be metabolized quite promptly. One issue of the Hb-based O 2 carriers is that they have a significant influence on clinical laboratory tests. They remain in the plasma phase in hematocrit capillaries after centrifugation of blood samples, and interfere with the colorimetric and turbidimetric measurements. However, HbV can be simply removed from blood plasma either by ultracentrifugation or centrifugation in the presence of a high-molecular-weight dextran to enhance precipitation. We can obtain a very clear supernatant for accurate analyses [35] . This is one advantage of HbV in comparison with acellular Hb solutions. Accordingly we examined the influence on organ functions by serum clinical laboratory tests after the bolus infusion of HbV at a dose rate of 20 ml/kg. Albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactic dehydrogenase (LDH), which reflect the liver function, moves their values within normal range. Concentrations of bilirubin and ferric ion are maintained at a low level. The concentration of lipids transiently changed. In particular, the cholesterols increased significantly. And phospholipids slightly increased, however, they returned to the original level after 7 days. These results indicate that the membrane components of HbV, once they reappear from RES, are metabolized on the physiological pathway.

We recently tested a daily repeated infusion of HbV in Wistar rats as a safety study. The dose rate was a 10-ml/kg/day infusion for 14 days. All rats well tolerated and survived. Body weight showed a monotonous but slightly depressed increase in comparison with the saline group. However, after 2 weeks there was no significant difference with the saline control group. All the rats seemed very healthy and active. There was no piloerection. As for the hematological parameters, the numbers of white blood cells and platelets did not exhibit a significant difference from the HbV group and the saline control group. Hematocrit showed a slight reduction for the HbV group, probably due to the accumulation of the large amount of HbV in the blood. Histopathological examination one day after the final infusion of HbV showed signifi- 

We have been conducting research on totally synthetic O 2 carriers, or socalled albumin-heme that does not require Hb. Human serum albumin is the most abundant plasma protein in our blood stream, but its crystal structure has not been elucidated for long time. In 1998, Dr. Stephen Curry of the Imperial College of London was the first elucidate the crystal structure of the human serum albumin complexed with seven molecules of myristic acids [36] . He found that the dynamic conformational changes of albumin take place by the binding of fatty acid.

In Japan, recombinant human serum albumin is now manufactured on a large scale by expression in the yeast Pichia pastoris, and it will appear on the market soon [37] . A large-scale plant, which can produce one million vials per year, has been already established. From the viewpoint of clinical application, O 2 -carrying albumin is quite exciting and may be of extreme medical importance. With this background, we have found that synthetic heme derivative is efficiently incorporated into recombinant human serum albumin (rHSA), creating a red-colored rHSA-heme hybrid. This rHSA-heme can reversibly bind and release O 2 molecules under physiological conditions in the same manner as Hb. In other words, our rHSA-heme hybrid is a synthetic O 2 -carrying hemoprotein, and we believe that its saline solution will become a new class of red blood cell substitute. We have already published these chemistry findings and technologies in international journals [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . Figure 8 summarizes the structure of the albumin-heme molecule. The Maximal binding numbers of heme to one albumin are eight, and the magnitude of the binding constants ranged from 10 6 to 10 4 (M -1 ). The isoelectric point of rHSA-heme was found to be 4.8, independent of the binding numbers of heme. This value is exactly the same as that of albumin itself. Furthermore, the viscosity and density did not change after the incorporation of heme molecules, and the obtained solution showed a long shelf life of almost two years at room temperature. The O 2 -binding sites of rHSA-heme are iron-porphyrin, therefore the color of the solution changed in a similar way to Hb. Upon addition of O 2 gas through this solution, the visible absorption pattern immediately changed to that of the O 2 -adduct complex. Moreover, after bub-bling carbon monoxide gas, albumin-heme formed a very stable carbonyl complex. Figure 9 shows the O 2 -binding equilibrium curve of rHSA-heme. The O 2binding affinity, of rHSA-heme is always constant, independent of the number of heme, and the O 2 -binding profile does not show cooperativity. However, the O 2 -transporting efficiency of albumin-heme between the lungs where PaO 2 is 110 Torr and muscle tissue where PtO 2 is 40 Torr increases to 20%, which is similar to 22% efficiency of red blood cells. The O 2 -binding property of albumin-heme can be controlled by changing the chemical structure of heme derivatives incorporated. More recently, we have found that a protoheme derivative is also incorporated into albumin and can bind and release O 2 as well [50] . 

Based on these findings, we can say that rHSA-heme can become an entirely synthetic O 2 -carrier, and satisfy the initial clinical requirements for a red blood cell substitute. However, we have another problem to solve before we can use this material as an O 2 -carrier in the circulatory system. This problem is NO scavenging. Of course, it can bind NO, and it may be anticipated that the injection of rHSA-heme also induce hypertensive action. We have evaluated the efficacy and safety of this rHSA-heme solution with animal experiments.

As described before, small Hb molecules extravasate through the vascular endothelium and react with NO, thus inducing vasoconstriction and acute increases in systemic blood pressure. Contrary to the expectations, the observation of the intestinal microcirculation after the infusion of rHSA-heme into an anesthetized rat revealed that the diameters of the venules and arterioles were not deformed at all [51] . Indeed, only a small change in the mean arterial pressure was observed after the administration of the rHSA-heme solution (Fig. 10) . In contrast, the infusion of Hb elicited an acute increase in blood pressure. Why does albumin-heme not induce vasoconstriction or hypertension? The answer probably lies in the negatively charged molecular surface of albumin. One of the unique characteristics of serum albumin is its low permeability through the capillary pore, which is less than 1/100 that for Hb due to the electrostatic repulsion between the albumin surface and the glomerular basement membrane around the endothelial cells.

We are now evaluating the O 2 -transporting ability of this albumin-heme molecule in the circulatory system with further animal experiments [52] . First, we determined the physiological responses to exchange transfusion with rHSA-heme solution into rats after 70% hemodilution and 40% hemorrhage 14 K. Kobayashi et al. More recently, we have synthesized human serum albumin dimer, which can incorporate sixteen hemes in its hydrophobic domain [53] . The human serum rHSA-heme dimer solution dissolves 1.2 times more O 2 compared to that of red blood cells and keeps its colloid osmotic pressure at the same level as the physiological value. a b Fig. 11 . Change of (a) MAP and (b) O 2 -tension in renal cortex during the 70% hemodilution with 5 wt% rHSA and further 40% exchange transfusion with rHSA-heme in anesthetized rats (n = 5). All data are shown as changes from the basal values and expressed as mean ± S.E.

For almost 20 years our group at Keio University in collaboration with Dr. Tsuchida's group at Waseda University have been trying to produce artificial O 2 carriers. To date, we have produced several types of O 2 carriers and evaluated their efficacy and biocompatibility. In this chapter, we have shown what we have done to produce O 2 carriers. Below, we would like to show you the potential applications of artificial O 2 carriers, as well as a glimpse of the vast possibilities that lie ahead.

Unlike vessels in normal tissues, the development of a vasculature in a tumor lacks normal course of angiogenesis and is hence, highly heterogeneous. Consequently, areas of hypoxia are quite common in tumors. In these hypoxic regions, it can be added that tumor cells acquire resistance to treatments such as chemotherapy and radiation. Our rHSA-heme was injected into the responsible artery that supplies circulation to an implanted tumor (Fig. 12) 

One of the most important agenda in transplantation medicine is long-term organ preservation and circumvention of ischemia reperfusion injuries. We 16 K. Kobayashi et al. think that artificial O 2 carriers can be applied as a perfusate for donor tissue in order to overcome these problems. In particular, its O 2 carrying capacity has the potential to significantly extend the preservation period. This will make it easier to transport organs. Also, utilizing the extra time, we may in the future be able to perform additional organ tests for better compatibility, or even perform genetic modifications during this period. We believe that through these applications, the concept of organ preservation can be expanded to organ culture, and furthermore to include the preservation of cells derived from donor tissues.

Extracorporeal circulation is quite common in cardiac surgery. Improvements are being made in the priming solutions but red cells are often still required to fill the device circuit, particularly in compromised cases and in children [55] . We believe that the use of artificial O 2 carriers in the priming solution can decrease or completely eliminate the need for a transfusion in such cases, and hence reduce the incidence of infection or GVHD.

Tissue ischemia can ensue from impairment of peripheral perfusion due to a variety of diseases such as arteriosclerosis obliterans, diabetes, and Burger's disease. The key event in the progression of ischemic diseases is the inability of red cells to flow through the capillaries, beyond obstruction ulceration and gangrene formation become imminent. We believe that this critical phase can be avoided or delayed by the application of artificial O 2 carriers, which can be designed to flow through these damaged capillaries or collateral circulation [27, 28] .

For patients who present acute lung injury or acute respiratory distress syndrome (ARDS), gas exchange in the lung exhibits severe deterioration and sometimes even the newest mechanical ventilation method fails to establish adequate oxygenation of the blood. In this type of critical case, liquid ventilation using an artificial O 2 carrier can establish optimal oxygenation of the blood and may reproduce the integrity of lung parenchyma [56] . Briefly explained, oxygenated liquid ventilation fluid is administered into the lung through trachea and O 2 molecules are transferred through diseased alveolus by diffusion and oxygenate the blood. Currently, this method is thought to be effective for patients with congenital diaphragmatic herniation. Efficacy for adult acute lung injuries is now under investigation. Perfluorochemicals are the main fluid used for clinical use, however, aqueous artificial O 2 carriers may have the potential to be used for liquid ventilation.

The research field of the red cell substitutes is moving forward very rapidly. Also as you have seen, the paradigm in this field is expanding from red cell substitutes to "O 2 therapeutics". The quality control and the pre-clinical test will be completed on the carriers produced at the pilot plant, after which clinical trials will proceed. We look forward to the day that our research will play an effective role in treating patients.

Macromolecular complexes, dynamic interactions and electronic processes

Macromolecule-metal complexes

Synthesis of amphiphilic porphinatoiron complexes having phosphocholine groups

Liposome-embedded iron-porphyrins as an artificial oxygen carrier

Hemoglobin model-artificial oxygen carrier composed of porphinatoiron complexes

Liposome/heme as a totally synthetic oxygen transporter

Blood substitutes: principles, methods, products, and clinical trials

Oxygen carriers (Blood Substitutes)-raison d'etre, chemistry, and some physiology

Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock

Amsterdam 11. Djordjevich L, Mayoral J, Miller IF, et al (1987) Cardiorespiratory effects of exchange transfusions with synthetic erythrocytes in rats

Preparation conditions of human hemoglobin vesicles covered with lipid membrane

Purufication of concentrated Hb using organic solvent and heat treatment

Layer-controlled hemoglobin vesicles by interaction of hemoglobin with a phospholipid assembly

Virus removal from hemoglobin solution using Planova membrane

Carbonylation of oxyhemoglobin solution using a membrane oxygenator

Poly(ethylene glycol)-modification of the phospholipid vesicles by using the spontaneous incorporation of poly(ethylene glycol)-lipid into the vesicles

Poly(ethyleneglycol)-conjugation and deoxygenation enable long term preservation of hemoglobin vesicles as oxygen carriers

Effective encapsulation of proteins into sizecontrolled phospholipid vesicles using the freeze-thawing and extrusion

Pysiologic responses to exchange transfusion with hemoglobin vesicels as an artificial oxygen carrier in anesthetized rats: changes in mean arterial pressure and renal cortical tissue oxygen tension

Evaluation of the capabilities of a hemoglobin vesicle as an artificial oxygen carrier in a rat exchange transfusion model

The oxygen carrying capability of hemoglobin vesicles evaluated in rat exchange transfusion models

Surface modification of hemoglobin vesicles with poly(ethyleneglycol) and effects on aggregation, viscosity, and blood flow during 90% exchange transfusion in anesthetized rats

Subcutaneous microvascular responses to hemodilution with a red cell substitute consisting of polyethyleneglycol-modified vesicles encapsulating hemoglobin

Microvascular responses to hemodilution with Hb-vesicles as red cell substitutes: Influences of O 2 affinity

Systemic and Microvascular responses to the hemorrhagic shock and resuscitation with Hb-vesicles

Normovolemic hemodilution with hemoglobin-vesicle solution attenuates hypoxia in ischemic hamster flap tissue

Improved oxygenation in ischemic hamster flap tissue is correlated with increasing hemodilution with Hb vesicles and their O 2 affinity

Distribution of heme oxygenase isoform in rat liver: topographic basis for carbon monoxide-mediated micorvascular relaxation

Molecular dimensions of Hb-based O 2 carriers determine constriction of resistance arteries and hypertension in conscious hamster model

Effects of PEG-modified hemoglobin vesicles on agonist induced platelet aggregation and RANTES release in vitro

Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver

Effects of poly (ethyleneglycol)-modified hemoglobin vesicles on N-formyl-methionyl-leucyl-phenylalanine induced respoonses of polymorphonuclear neutrophils in vitro

Hemoglobin-vesicles as oxygen carriers: influence on phagocytic activity and histopathological changes in reticuloendothelial systems

Pretreatment of serum containing Hbvesicels (oxygen carriers) to avoid their interference in laboratory tests

Crystal structure of human serum albumin complexes with fatty acid reveals an asymmetric distribution of binding sites

Purification and Physicochemical Properties of Recombinant Human Serum Albumin

Transport Albumin: A New Hybridhaemoprotein Incorporating Tetraphenylporphinatoiron(II) Derivative

Properties of oxygen binding by albumintetraphenylporphyrynatoiron(II) derivative complexes

Resonance raman studies of O 2 -binding to orthosubstituted tetraphenyl-and tetranaphthyl-porphinatoiron(II) derivatives with a covalently linked axial imidazole

Physicochemical properties and O 2 -coordination structure of human serum albumin incorporating tetrakis(opivalamido) phenylporphinatoiron(II) serivatives

Human serum albumin incorporating tetrakis(o-pivalamido)phenylporphinatoiron(II) derivative as a totally synthetic O 2 -carrying hemoprotein

Kinetics of CO and O 2 Binding to Human Serum Albumin-heme Hybrid

Reaction of Nitric Oxide with Synthetic Hemoprotein

Photoreduction of autooxidized albuminheme hybrid in saline solution: revival of its O 2 -binding ability

O 2 -Binding properties of double-sided porphyrinatoiron(II)s with polar substituents and their human serum albumin hybrids

Electrochemical studies of albumin-heme hybrid in aqueous media by modified electrode

Effect of heme structure on O 2 -binding properties of human serum albumin-heme hybrids: intramolecular histidine coordination provides a stable O 2 -adduct complex

Preservation stability and in vivo administration of albumin-heme hybrid solution as an entirely synthetic O 2 -carrier

Synthetic dioxygen-carrying hemoprotein: human serum albumin including iron(II) complex of protoporphyrin IX with an axially coordinated histidylglycyl-propionate

Human serum albumin incorporating synthetic heme: red blood cell substitute without hypertension by nitric oxide scavenging

Exchange transfusion of albuminheme as an artificial O 2 -infusion into anesthetized rats: physiological responses, O 2 -delivery and reduction of the oxidized hemin sites by red blood cells

Cross-linked human serum albumin dimers incorporating sixteen (tetraphenylporphinato)iron(II) derivatives: synthesis, characterization, and O 2 -binding property

Oxygenation of hypoxic region in solid tumor by administration of human serum albumin incorporating synthetic hemes

Efficacy of synthetic oxygen-carrying substances

Liquid ventilation using artificial oxygen carrier