Red blood cell substitutes: fluorocarbon emulsions and haemoglobin solutions B Remy*, G Deby-Dupont* + and M Lamy* + * Department of Anaesthesia and Intensive Care Medicine and 'Centre for Oxygen Research and Development, University of Liege, Liege, Belgium

The problems posed by transfusion of homologous blood have led to the development of substances able to replace the gas transporting properties of blood. Perfluorocarbons (PFCs) emulsions and modified haemoglobin (Hb) solutions have been developed for this goal and are now tested in clinical assays. PFCs are synthetic fluorinated hydrocarbons, capable of dissolving large quantities of oxygen (O^ without binding) at high inspired concentrations of Or and of delivering this O2 to the tissues. They are administered as emulsions containing particles with a diameter of approximately 0.2 urn, capable of entering the microcirculation. They are eliminated unchanged by the lungs within several days. Fluosol-DA* 20% was the first PFC emulsion used in clinical practice. Currently, Oxygent™, a second generation PFC emulsion, is being evaluated in clinical studies. The PFCs are not blood substitutes, but rather a means to ensure tissue oxygenation during extreme haemodilution. Solutions of free Hb do not have the antigenic characteristics of the blood groups, and do not require compatibility testing. They are fully saturated with O2 at ambient FiO2. The Hbs used are derived from either human or bovine sources, or via recombinant DNA technology. In order to maintain satisfactory intravascular half-life and O2 affinity, the Hb molecules are modified by adding internal crosslinks, by polymerization, and/or by encapsulation. After promising animal studies, several of these modified Hb solutions are now being studied in Phase III clinical trials. Among them, diaspirin cross-linked haemoglobin (DCLHb) has been used in cardiac and orthopaedic surgery, and for resuscitation of traffic accident victims. The initial results of multicentre trials are now being analysed. Correspondence to: Dr Bernadette Remy, Department of Anaesthesia and Intensive Care Medicine, University Hospital, B35, Domalne universltaire du Sart Tilman, B-4O00 Liege, Belgium

The transfusion of homologous blood poses numerous problems, including the availability, storage, and transport of the blood itself, the necessity for compatibility testing, the risk of disease transmission, and putative immunosuppressive effects. For these reasons, a considerable research effort has taken place over the past several decades with the goal of finding transfusion alternatives or substances capable of replacing the gas transport properties of blood1. This research has led to the

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development of perfluorocarbon (PFC) emulsions, and of solutions of modified free haemoglobin (Hb); these approaches are totally different but aim at the same goal2-3. These red blood cell substitutes (RBCS) are particularly suited for intensive care and for patients who refuse blood transfusion.

Perfluorocarbon emulsions General characteristics of perfluorocarbons The PFCs are low molecular weight (450-500 Da) linear or cyclic hydrocarbons, occasionally containing oxygen or nitrogen atoms, and in which the hydrogen atoms of the carbon chain have been replaced by fluorine. This hyperfluorination leads to total chemical inertness, and a complete lack of metabolism in vivo. The PFCs are dense transparent liquids with a low surface tension, immiscible in water. They were developed for chemical purpose, but as early as 1966, Clark and Gollan4 brought attention to the capacity of PFC to dissolve gases without covalent binding; they demonstrated that a rat immersed in a solution of perfluorocarbon saturated in oxygen (O2) at atmospheric pressure, breathed normally. The PFC were thus soon considered as RBCS5"7. The transport and liberation of gases by PFCs are based on physical solubility, and the quantity of gas dissolved is linearly related to its partial pressure. The PFCs do not have the O 2 bonding properties of Hb, but act as simple solvents. They are thus only O 2 carriers with a transport capacity that is greater than that of blood under hyperoxic conditions. Because O 2 is transported by PFC without chemical bonding, its unloading, for example at the tissues, is considerably facilitated. Numerous PFCs, with differing structures, have been synthesized and tried as O 2 carriers. They differ by their stability in emulsion and by the physiological response they induce. The most recently synthezised PFCs are linear molecules with 8-10 carbon chains (perfluorooctyl bromide, perfluorooctylethane, perfluorodichlorooctane)5'6. The syntheses of these linear PFCs are carried out using elective processes which have good yields and produce low levels of potentially toxic impurities, consequently reducing the purification steps and the risk of physiological response. As PFCs are chemically and biologically inert, they are not metabolised. They are excreted as vapours by the lungs after passage through the reticuloendothelial system (especially of the liver and spleen). The retention time within the body increases exponentially as a function of the molecular weight, with the exception of PFCs containing a lipophilic extremity (e.g. the bromine atom in perflubron) 6 . 278

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Tissular ischaemia

Acute haemorrhage

Oxygen carriers

Peri operative haemodilution rcardiovascular surgery ^ orthopaedic surgery

Fig. 1 Main potential biomedical applications of perfluorocarbons and modified haemoglobin (Hb) solutions.

Liquid ventilation

•

perfluorocarbon (pure solutions or emulsions)

Organ preservation

Cardiology (myocardial ischemia)

Modified Hb solutions

The main potential biomedical applications of perfluorocarbons as O, carriers3'7'8 are their use in pure form for partial liquid ventilation and intraluminal oxygenation of the intestine, and their use as emulsions in aqueous media as blood substitutes for O 2 transport in ischaemic tissues and in clinical situations of acute haemorrhage or high blood loss surgery (Fig. 1).

Characteristics of perfluorocarbon emulsions Because the PFCs are dense and not hydrosoluble, their use as pure solution in the intravascular space is impossible, but they can be administered as emulsions, containing a dispersion of fine particles, suspended in an isotonic electrolyte solution. In order to produce emulsions which are stable at room temperature, emulsifying agents (surfactants) are necessary. The properties of PFC emulsions depend on both the components of the emulsion, but also on the proportion of the various components and on the sizes of the emulsion particles, which influences the stability of the emulsion, the surface area available for gas exchange, the viscosity, and the intravascular half-life (linked to in vivo toxicity or side effects). The mean size of the particles of the second generation emulsions is approximately 0.2 urn. The surface charge of the particles is also important, because this determines the rapidity of phagocytosis and the interactions with platelets, potentially leading to formation of

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Tfcble 1 Characteristics of an ideal blood substitute Equivalent to natural haemoglobin in terms of O^ and CO2 transport and delivery Maintains arterial blood pressure Similar viscosity to blood Maintains arterial pH Sufficient intravascular persistence Absence of renal toxicity Does not overload reticulo-endothelial system Non-antigenic Does not react chemically with oxygen, activate complement increase white blood cell count react with plasmatic substances or platelets, weak potential to produce methaemoglobin Stable at room temperature Long-term storage possible Moderate cost Easy-to-use Immediate availability

microthrombi5'6. Two surfactants were proven praaical in the preparation of emulsions: Pluronic F-68 and egg yolk phospholipids. Pluronic F-68 is not acutely toxic, but is no longer used clinically because it is responsible for certain side effects, such as complement activation, which can lead to an inflammatory reaction9. The emulsions are prepared using ultrasonication or high-pressure homogenization, but sonication appears to be partially destructive, liberating free fluoride ions, altering the composition of the emulsion, and increasing the risk of toxicity. The characteristics of the ideal emulsion for use as blood substitute are: the absence of incompatibility and risk of transmission of infectious diseases, a long duration of conservation, an easy access, the absence of metabolism and more particularly non-reactivity with O 2 , no bonding with O2 allowing easy tissue unloading, viscosity and rheological parameters similar to those of blood, permitting the particles to flow through swollen and/or blocked capillaries, where red blood cells might not pass (Table 1). The solubility of O 2 in PFC emulsions is proportional to the partial pressure (Fig. 2), and the O2 transport capacity of these emulsions depends on the PFC concentration. Emulsions containing 45-60% PFC (w/v) appear ideal in terms of O 2 carriage10, but the mechanisms of transport and delivery are entirely different from those of erythrocytes. The oxyhaemoglobin saturation curve for erythrocytes is sigmoid, and a fall in partial pressure from 150 to 50 mmHg leads to unloading of 2 5 % of the bound O2. To obtain similar efficiency when PFC emulsions are used as blood substitutes, an atmosphere enriched in O 2 (Fig. 1) must be used6'11, and 100% O 2 are administered to patients that are included in ongoing Phase II clinical studies with PFC emulsions. The first PFC emulsions were prepared in 1967 using plasma, with large size particles (2-3 ^m), but they were not used in humans. The first generation emulsion authorized by the FDA for injection in humans (for 280

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20-,

Fig. 2 Curves of O2 transport by blood, free natural haemoglobin (Hb), modified haemoglobin and PFC emulsions. DCLHb, diaspirin cross linked Hb; rHb, recombinant Hb.

solution of natural Hb

100

300

500

700 O2 pressure (mm Hg)

percutaneous transluminal coronary angioplasty, PTCA) is Fluosol-DA®, a 20% w/v solution developed by the Green Cross Corporation (Osaka, Japan). This product is a mixture of 70% perfluorodecalin and 30% perfluorotripropylamine, with as the emulsifying agent (3.9% w/v) a mixture of Pluronic F-68, egg-yolk phospholipids and glycerol12. Its use showed that transport and delivery of O2 without major toxic effects was possible, but also presented disadvantages: long tissue retention of one of its components, low concentration of PFC and limited intravascular half-life (both limiting the amount of O2 transported), and unsatisfactory stability. This instability necessitated conditioning as 3 separate solutions with thawing, homogenization, and oxygenation before use; this long procedure was clearly incompatible with the emergency setting. Fluosol also had certain side effects, such as inhibition of white blood cells and complement activation, attributed to the surfactant used to fabricate the emulsion6-9. The main progress of the second generation emulsions is an important increase in the PFC concentration, greatly enhancing the O2-carrying capacity and eliminating the dilution of patient's blood at time of administration. They present a high stability (resistance to heat sterilisation and to storage at +4°C), that is obtained by using a critical amount of egg yolk phospholipids and better emulsification techniques (highpressure homogenization, microfluidization)13. They are formulated 'ready-for-use' in buffered saline with physiological osmolarity, viscosity, and pH values, and have no acute toxicity or major side effects. The small size of the particles (mean diameter 0.2 um, about 1/35 that of an erythrocyte) allows them to become concentrated in the thin layer of plasma between the red blood cells and the vascular wall (near-wall particle excess phenomenon) and to easily maintain perfusion of all the capillaries of the microcirculation during states of local vasoconstriction and ischaemia, when erythrocytes no longer circulate14. Their administration is not associated with haemodynamic effects or with a decrease in cardiac output, and they do not activate complement. British Medical Bulletin 1999;5S (No. 1)

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However, they can produce a dose-dependent flu-like syndrome occurring 4—6 h after infusion. This syndrome includes fever, arterial hypotension, tachycardia, high white blood cell count, and thrombocytopenia. It results from the phagocytosis of emulsion particles by macrophages, with liberation of cytokines and arachidonic acid metabolites. This flu-like syndrome regresses spontaneously in 24 h3. The archetypal second generation emulsion is Oxygent™ (Alliance Pharmaceutical Corp., San Diego, CA, USA), an emulsion based on use of perfluorooctyl bromide, better known by its generic name of perflubron, which is a linear PFC containing 8 carbon atoms. A single terminal bromine atom lends lipophilicity and limits its tissue persistence6. Oxygent™ has a concentration of 60% w/v, and uses egg-yolk phospholipids as emulsifying agents. The particles have a mean diameter of 0.16-0.18 urn. This emulsion dissolves 28 ml O2/100 g at 37°C and 750 mmHg (Fig. 2). It can be stored at refrigerated temperature for up to 2 years. Another second generation product is Oxyfluor™ (HemagenBaxter)3, a 56% w/v emulsion, based around perfluorodichlorooctane (PFDCO, a lipophilic PFC), with egg-yolk phospholipids and safflower oil as surfactants. At equilibrium with 100% O2 at 37°C, it dissolves 17.2% O2 by volume (Fig. 2). It is stable at room temperature for over 1 year. The two emulsions have similar properties of O2 transport and delivery, stability, and viscosity. Third generation emulsions are currently in early preclinical development. They are based on emulsification of a PFC by a phospholipid, but they also contain linear molecules with both hydrocarbon/fluorocarbon properties, which serve to stabilize the emulsion. These mixed-property molecules act as dowels, the hydrocarbon end anchored on one side in the oily chains of the phospholipid film, and the fluorinated end anchored on the other side in the PFC itself. This allows preparation of concentrated emulsions (up to 90% w/v) with a mean particle size of 0.22 u.m, stable for at least 6 months at 40°C (accelerated aging conditions)15. The absence of toxicity of these new emulsions was demonstrated in endothelial cell culture and in animal organ preservation studies16. Potential clinical applications

Because they dissolve large volumes of gas, are highly fluid, and have low surface tension, pure PFC are well suited for use during liquid ventilation to improve oxygenation during acute respiratory distress syndrome. Perflubron (LiquiVent®, Alliance Pharmaceutical Corp.) is an excellent O2 (50 ml/dl) and CO2 (210 ml/dl) transporter for use in total or partial liquid ventilation (PLV or PAGE, perfluorocarbon associated 282

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gas exchange)17-18. It is eliminated by evaporation and is only marginally absorbed through the alveolus. When instilled in the lung, LiquiVent® penetrates into collapsed alveoli, improving oxygenation and increasing pulmonary compliance by reductions of surface tension. Studies have been successful in animal models of respiratory distress syndrome7'19-20, showing improved arterial saturation and CO 2 removal, and usually improved pulmonary mechanics. The first successful uses in humans were reported in the premature newborn and in paediatric patients suffering from severe ARDS21>22. Liquid ventilation brought about an improvement in oxygenation in the first 4 days of the treatment, with improvement in lung compliance. The patients survived, but 2 pneumothoraces potentially attributable to the PFC were reported. PLV, with compensation for losses to evaporation, was also used in the adult with ARDS, and was associated with 50% survival, an improvement in compliance, a fall in physiological shunt, and improved gas exchange23. Two complications potentially attributable to PLV, were reported: one pneumothorax, and one mucus plug. From these preliminary studies, it was concluded that LiquiVent® is safe, that it distributes within the lung under the influence of gravity and that its administration leads to improved gas exchange, compliance, and decreased surface tension19*20'24'25. It would also appear to have a beneficial effect on alveolar macrophages, by reducing their inflammatory response26. Large clinical studies have started using a multicentre protocol. The intraluminal administration of oxygenated PFC has been proposed in situations of ischaemia-reperfusion of the intestinal mucosa (necrotizing enterocolitis, partial mesenteric arterial insufficiency), for rapid delivery of O ? in situ. This intraluminal oxygenation of the intestine has been successfully tested in animal models with preservation of the structure of villi and crypts, and protection of intestinal function27, but no human clinical studies have yet been reported. The clinical use of PFC emulsions are essentially those of perioperative haemodilution, resuscitation from haemorrhagic shock, and those of the treatment of ischaemic problems. The emulsions serve as a temporary vector for O 2 , delaying or avoiding the administration of blood2'6-7'19. The first human experiments using Fluosol were carried out in Japan to treat severe anaemia in surgical patients who refused blood transfusion for religious reasons (401 patients, from 1979 to 1982)28'29. From these studies, it was concluded that the emulsion was safe, had a beneficial effect as a plasma expander, and that it contributed to O 2 delivery, but that the concentration of PFC was too low to obtain a clear beneficial effect. In 1989, Fluosol was approved by the US FDA for use during percutaneous transluminal coronary angioplasty (PTCA), a typical clinical situation uniformly associated with localized myocardial ischaemia30. However, the clinical utility of this intervention remained British Medical Bulletin 1999,55 (No. 1)

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controversial6"30, and because technical advances now allow PTCA with autoperfusion catheters which prevent ischaemia, the use of Fluosol is no longer necessary; the product has been withdrawn from the market. The second generation emulsions have been administered to more than 200 volunteers and surgical patients (Phase I studies), with the absence of haemodynamic effects, an increase in cardiac output related to the haemodilution, and no effect on bleeding time, coagulation, and immune function. A PFC dose of 1.35 g/kg could support O 2 delivery despite ongoing blood loss. Transitory side effects have been noted for the highest doses administered (1.8 g PFC/kg), such as a slight and temporary elevation in temperature, and a modest decrease in the platelet count, without bleeding, over the first 2-3 days after administration. Use of second generation emulsions is now considered in the clinical situations of anaemia, trauma, high blood loss surgery, peri-operative haemodilution, ischaemia, and in organ conservation for transplantation. To date, more than one dozen studies have been performed in humans, particularly with Oxygent™, enroling more than 500 subjects in Phases I and II31. Multicentre, Phase II, randomized, controlled, single-blind studies have been completed in about 250 orthopaedic, urological, and gynaecological surgery patients. Several Phase II studies in cardiac surgery with extracorporeal circulation are in their final stages32. Initial results indicate a delaying effect of PFC emulsion administration on blood transfusion, and an absence of toxic effects on haemodynamic, haematological, and biochemical parameters. The use of Oxygent™ seems to be particularly promising during perioperative haemodilution, where the concomitant use of PFC emulsions would allow reductions of the patient's haematocrit below currently accepted thresholds while maintaining or improving tissue oxygenation. Phase HI studies are ongoing in situations requiring protection from tissue ischaemia (e.g. myocardial ischaemia, transient anaemia following high blood-loss surgery, or cerebral protection from gas emboli, for example during open heart surgery), and in situations where allogeneic blood transfusion avoidance is desirable33. The pure PFC or PFC emulsions of the first generation have been used for preservation of many organs (heart, lung, liver, pancreas, kidney), with a good protection of functional activity of the transplant, associated to a better oxygenation reducing free radical damage34. Second generation emulsions have been used for preservation of pulmonary transplants, either as a flush into the pulmonary artery prior to classical cold preservation, or as an autoperfusion by a working heart-lung preparation. In these models, the morphological and functional alterations in the transplant were clearly inferior to those seen when other conservation fluids (EuroCollins, autologous blood, stroma-free haemoglobin) were used, but the viability of the graft did not increase35. Hypothermia for 284

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organ preservation and the shortage of the donor pool of heart-beating cadavers are limiting factors in organ preservation, while warm ischaemic damage hinders attempts to expand the organ donor pool into non-heartbeating cadaver. Oxygent™ supplemented perfusate has already been evaluated, with promising results, for canine kidney salvage postmortem36. Studies are now underway with a concentrated third generation emulsion (stabilized with molecular dowels) for intestine preservation in hypothermia and for organ block preservation in normothermia7'37.

Beneficial and harmful secondary effects Acute toxicity is not seen with the PFC formulations currently in clinical development, but secondary effects have been described during use of pure PFCs and emulsions. With LiquiVent®, the most important side effect is the risk of intravascular passage of the PFC, particularly for severely injured lungs. But animal studies suggest that this passage into the bloodstream is negligible and that the elimination of PFC is effected by evaporation within 24-48 h after stopping the treatment3'19. On the other hand, phagocytosis by alveolar macrophages is seen (demonstrated cytologically), and the possibility of a pathway involving dissolution in lipid, deposition in fat, reuptake by the bloodstream, and ending with elimination by the lung does exist. There does not seem to be direct absorption by the tissues or into bone. The use of PFCs for liquid ventilation is also associated with numerous beneficial side effects (as demonstrated in animal models): positive stimulus for the metabolism of surfactant phospholipids, reduction in intra-alveolar haemorrhage, oedema, and inflammatory infiltration into the lung, and reduction in alveolar debris and intra-pulmonary inflammatory response when compared to gaseous ventilation7'18'23'24'38. These anti-inflammatory properties are similar to those observed for perflubron in in vitro studies: reduction of the production of reactive oxygen species by alveolar macrophages, decrease of the production of cytokines by endotoxin stimulated macrophages, reduced production of H,O 2 and lower chemotactic response of human neutrophils, protection of human alveolar cells in culture during oxidative stress7-39. Finally, a potentially interesting use for LiquiVent® is related to its ability to penetrate and recruit alveoli where it could be used to administer antibiotics, with production of high local concentrations and minimal vascular uptake. This could reduce the various toxicities associated with certain antibiotics. Undesirable and potentially harmful side effects were described for Fluosol, essentially complement and phagocytic cells and adherence of leukocytes, but these effects were principally due to the surfactant used (Pluronic ¥-68), and are no longer seen with second generation emulsions British Medical Bulletin 1999;5S (No. 1)

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using newer surfactants9. A potential limitation to the use of PFC emulsions is the elimination capacity by the reticulo-endothelial system7-8'40. Large doses of emulsion particles could possibly lead to hepatic engorgement and a temporary impairment of immune defense mechanisms, which could be quite dangerous, especially in situations where infection is present or threatened. The small particle size of second generation emulsion considerably decreased this risk. The search for PFCs in various tissues such as lung, liver, spleen, etc. has shown neither high levels of accumulation nor excessive persistence. Until now, there are no reported toxic or side effects that could result from oxidation of the phospholipidic surfactant or from in vivo production of lysophosphatides. Among the beneficial biological effects, are the anti-inflammatory effects of the PFC emulsions. They appear to reduce the erythrocyte aggregation and haemolysis and the platelet activation induced by 'heart assist devices', and to protect the erythrocytes against oxidative haemolysis and lipid peroxidation. They would also inhibit the infiltration of ischaemic muscle by leukocytes and decrease the chemiluminescence produced by neutrophils stimulated by phorbol myristate acetate41.

Haemoglobin solutions Free Hb is not associated with erythrocyte membranes, and thus does not possess the antigenic properties of the blood groups. This fact obviates the necessity for compatibility testing prior to administering these solutions. Solubilised Hb retains its O 2 carrying properties, and is thus normally fully saturated when the subject breathes room air. Furthermore, because of the small size of the Hb molecule compared to the red blood cell, microcirculatory transport of O 2 is presumably more efficient. As early as 1898, Von Stark42 administered a Hb solution to patients in an attempt to treat anaemia, but the chemical instability of these solutions led this line of research to be abandoned. Small quantities of Hb were infused into humans in order to study its clearance by the kidneys; this line of research soon demonstrated signs of renal toxicity43. A systemic and pulmonary vasopressor effect was soon described, which was not due to simple expansion of circulating volume. In 1949, 300 ml of a 6% Hb solution was infused as a last resort to resuscitate a young woman suffering from a severe postpartum haemorrhage unresponsive to infusion of crystalloid, colloid, and homologous blood. This infusion increased the blood pressure and was associated with improved level of consciousness, what suggested that this pressor effect was beneficial44. A moderate increase in peripheral vascular resistance can be beneficial if it contributes to improved perfusion of vital organs, but at the same time, the increases

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of pulmonary and coronary resistances could conceivably have significant undesirable effects in some groups of patients. The toxicity of free Hb solutions was attributed to the presence of lipid and protein contaminants derived from the cell membranes. These substances were nephrotoxic and caused haemolysis. They also activated intravascular coagulation, complement, platelets, and white blood cells, leading to the liberation of inflammatory mediators45. Purification of Hb was improved, beginning in 1970, allowing elimination of certain of the toxic effects of the free molecule, and producing 'stroma free haemoglobin' (SFH)46. Classification of haemoglobins by source Human haemoglobin

Human Hb is obtained from lysis of the erythrocytes contained in expired units of banked blood. It has a greater affinity for O2 than intracellular Hb, because 2,3-diphosphoglycerate (2,3-DPG) is no longer bound to the molecule, reducing the PJ0 from 27 mmHg to 12-14 mmHg (Fig. 2), and thus delivers less O2 to the tissues. Free Hb rapidly leaves the circulation and is eliminated by the kidneys. In the extracellular space, the normal tetramer is split into 2 ctf$ dimers (± 32 kDa), which are excreted in the urine, leading to an osmotic diuresis within one hour of intravenous administration. Extracellular Hb has a high colloid oncotic pressure which limits its concentration in solution to 7 g/ml. Solutions of natural human free Hb have certain undesirable effects, some of which have been attributed to stromal remnants. These include vasomotor effects, activation of the complement, kinin and coagulation systems, nephrotoxicity, interference with macrophage function, antigenic effects, histamine release, and iron deposits. Free Hb is also easily oxidised to methaemoglobin, and must thus be stored in an anaerobic environment. Bovine haemoglobin

The bovine free Hb does not interact with 2,3-DPG and thus has a P50 of approximately 30 mmHg. This value favours O2 delivery to the tissues. Bovine Hb thus appeared to be an interesting potential alternative to the human molecule, especially given its abundant availability and low cost. However, other problems currently limit the use of bovine Hb: the risk of transmission of bovine spongiform encephalopathy, difficulties with purification causing persistence of membrane fragments and consequent possible immune responses and complement activation, and the possible production of antibodies due to infusion of large quantities of bovine proteins. British Medical Bulletin 1999;55 (No. 1)

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Polymerised bovine Hb (HBOC-201, Hemopure®) was subjected to Phase I and II studies in orthopaedic, cardiac, and urological surgery, and was also tested in patients in sickle cell crisis without producing side effects47-48. Another bovine Hb preparation (Biopure Co) has been tested in healthy volunteers. Recombinant haemoglobin

The best characterised recombinant human Hb (rHb 1.1, Somatogen) is obtained by genetic engineering from Escherichia coli whose genome was modified by addition of the genes coding for the globin molecule49. The same Hb has subsequently been produced in yeast and pigs. Using similar technology, a Hb variant has been manufactured (Haemoglobin Presbyterian) which has a higher PJ0 than the normal molecule; this results in improved O2 delivery at the tissue level. To resolve the problem of the affinity of free Hb for O2, a mutant Hb was created, with replacement of one amino acid ({J-asparagine 108 for (5-lysine). The dissociation of Hb into constituent subunits is solved by addition of a covalent bond between the two a chains, at the level of a glycine residue. This Hb has a P50 of 30-33 mmHg (Fig. 2), a plasma half-life 4 times greater than that of free Hb, a storage half-life that is indefinite when frozen, greater than 24 h at 4°C, and 5 h at room temperature. Pilot studies of tolerance were carried out on 24 volunteers with 4 doses (ranging from 0.015 to 0.11 g/kg)50. No renal, hepatic, or pulmonary toxicity was noted. There was no renal excretion, and no significant variation of either systolic or diastolic arterial blood pressure. Fever, higher than 38CC, occurred in 3-8 hours after infusion in 12 subjects, with headache, myalgia, and chills. These symptoms resolved either spontaneously or after ibuprofen (400 or 600 mg). By increasing the purification process, no further episodes of fever were noted. Phase II studies with Optro® are underway in North America for coronary artery bypass surgery. Further studies, for normovolemic haemodilution and in oncology, have been planned. Modified haemoglobin solutions To avoid the inconveniences of free extracellular Hb, and to approach the characteristics of the ideal blood substitute (Table 1), free Hb was modified in order to prolong the intravascular half-life, to slower renal elimination, and to maintain a normal O2 affinity. The following modifications have been used: internal stabilisation of the tetrameric molecule, polymerisation, cross linking of Hb dimers, conjugation with larger molecules, pyridoxylation, and encapsulation within synthetic lipid membranes (Fig. 3). The various solutions of modified Hb currently undergoing Phase I, II and III trials are listed in Table 2. 288

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Haemoglobin solution genetic engineering

from natural SFH (human, bovine) cross-linking : CB-Ot or B-B

Fig. 3 Main ways of preparation of modified haemoglobin solutions

1

1

bacteria

yeaits

1

1

' polymerization ± binding to 1 macromolecules encapsulation i -r 1

natural or modified Hb

macromolecules and polymerization

Cross-linked haemoglobins

The creation of a covalent bond bridging the constituent dimers prevents the rapid renal elimination of the molecule, by avoiding the rapid dissociation seen with the native molecule in the extracellular milieu51. Because these dimers are felt to be responsible for the nephrotoxicity of free Hb, this problem is also resolved by crosslinking. Furthermore, by reacting the Hb with an analogue of 2,3-DPG, the affinity for O 2 can be reduced, even outside the erythrocytes, thus improving tissue O 2 delivery. The cross-linked Hb can be further stabilized by polymerisation. The plasma half-life of these modified Hb varies from 3 h to 30 h, depending on the dose administered, the species of animal, and the degree of polymerisation. They are characterised by a P50 of 30-35 mmHg. One of the easiest ways used for cross-linking is the acetylation of Hb at physiological pH by acetylsalicylic acid (aspirin), or by bis-{3,5 dibromosalicyl)-fumarate (DBBF), the diester of dibromo-acetylsalicylic acid52. The Table 2 Modified haemoglobin solutions undergoing clinical trials Product name (Company)

Type of haemoglobin

Modification

Study phase

HemAssist" (Baxter) PoryHeme* (Northfield) HemoLink"

Human Hb

Internal bond (a,-Oj) (via diaspirin bridge)

Phase III

Human Hb

Pyridoxyiation and polymerisation (via glutaraldehyde) Conjugation

Phases Mb/Ill

(Hemosol) No name (Enzon) Hemopure" (Biopure) Optro" (Somatogen) No name (Apex)

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Bovine Hb Bovine Hb Recombinant Hb (E. col!) Human Hb

(o-raffinose) Conjugation (polyethyleneglycol) Polymerisation (glutaraldehyde) Amino acid substitution and internal bond Pyridoxyiation, polyoxethylene

Phase II Phase 1 Phases IMI Phases Ib/ll Phase 1

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most extensively studied of the stabilised haemoglobins, diaspirin crosslinked haemoglobin (DCLHb) or HemAssist® (Baxter) is prepared by this reaction of natural Hb with DBBF53. DCLHb: a model of cross-linked Hb

The DCLHb solution. DCLHb is prepared from human erythrocytes that have been shown to be negative for viruses (in particular HIV, HBV, and HCV). After washing, the cells are lysed to yield Hb, which is filtered to remove stromal elements. After deoxygenating the Hb, DBBF is added, in order to create a covalent bond (a fumarate bridge) between the 2 a subunits (lysa] 99 - lys^ 99). The product is then pasteurised at 70°C, for viral inactivation, and for the denaturation and precipitation of uncrosslinked Hb and other contaminating proteins54. This is followed by reoxygenation and purification by ion-exchange chromatography. The final product is added with an electrolyte solution and adjusted at physiological pH to produce a sterile and nonpyrogenic solution which has the following composition: DCLHb 10 g/100 ml; pH (37°C) 7.4; Na 146 mM; K 4 mM; Ca 1.15 mM; Mg 0.45 mM; Cl 116 mM; lactate 34 mM, osmolarity 290 mOsm/1; colloid-oncotic pressure (37°C) 44 mmHg. It is frozen at -20°C. At this temperature the DCLHb is stable for one year, with minor oxidation leading to the formation of ± 0.3% methaemoglobin (metHb) per month. The solution can be stored for one month in a refrigerator, and one day at room temperature. DCLHb is non-antigenic and does not require typing and cross-matching55. Because the oxyhaemoglobin dissociation curve is shifted to the right, its affinity for O 2 is reduced (Fig. 2). The intramolecular bridge also affects the transport of C O r DCLHb binds less CO 2 than the native molecule, regardless of the concentration of C O r Only 50% of the binding sites for CO 2 are occupied56. Preclinical studies with DCLHb. Modified haemoglobin solutions have been extensively studied in animal models of haemorrhagic shock57-58 and exchange transfusion, with excellent results. These results include return of heart rate to normal, improved tissue O 2 extraction, return of normal blood flow in most organs, and acceptable oxygenation of peripheral tissues. Overall survival was high, reaching 80% in some studies. DCLHb was more effective than infusion of large volumes of lactated Ringer's solution in re-establishing and maintaining arterial blood pressure and mixed venous O 2 saturations in haemorrhagic shock models. It was as effective as blood, even when the quantities administered reached 50% of those of infused blood. It should be noted, however, that the return to normal venous O 2 values was short-lived. Studies have now been carried out on hundreds of different species (especially the rat, dog, pig, sheep and monkey), without undesirable effects such as antigenicity, complement or white blood cell activation, renal effects, or reticulo-endothelial overload. 290

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No organ toxicity has been noted (more particularly in the kidney, heart and central nervous system). One frequently noted effect is the early and sustained increase in mean arterial pressure, with most often a decreased heart rate. This pressure increase is dose-dependent, but plateaus quickly and is easily controlled using anti-hypertensive agents. This pharmacological property of DCLHb would be mediated by 3 elements of the endogenous vasomotor autoregulatory system: inhibition of NO, stimulation of the production of endothelin, and sensitisation and/or potentialisation of cij- and a2-adrenergic receptor responses to catecholamines59'60. In cases of myocardial ischaemia, DCLHb can improve tissue perfusion because of its low viscosity and the small size of the Hb molecule compared to erythrocytes. This substance could, therefore, constitute a treatment for various ischaemic states. In the animal, DCLHb has proven to be particularly efficacious in supporting cardiac function during coronary angioplasty. Perfusion of DCLHb through the catheter during balloon occlusion has been demonstrated to improve the oxygenation of the myocardium61. In animal models of cerebral ischaemic lesions, isovolemic haemodilution with DCLHb increases cerebral blood flow and oxygenation62 During septic states, tissue O2 delivery is inadequate in relation to demand. Systemic vascular resistance is low, leading to low systolic and diastolic blood pressures. DCLHb attenuates the systemic arterial hypotension induced by injection of endotoxin, without compromising splanchnic or renal perfusion. On the other hand, it significantly worsens the pulmonary arterial hypertension and the arterial hypoxaemia seen in the pig after administration of endotoxin63. Clinical studies with DCLHb

hi a Phase I study of 24 healthy conscious volunteers receiving doses of 25-100 mg/kg, the most frequently noted complication was mild and transitory abdominal discomfort64. At the same time, arterial hypertension and a dose-dependent increase in total creatine phosphokinase (CPK) and iso-LDH5 were seen. A multicentre trial of patients with severe hypovolemic shock consisted of randomisation (within 4 h of the diagnosis of the shock state) to receive either 50 or 100 ml of 10% DCLHb or normal saline solution. This study showed a dose-dependent reduction in mortality, complications, and in the incidence of multiple organ failure, without compromise in renal function65. An investigation of tolerance of DCLHb, using randomisation and a double blind construction was carried out in elective surgery in 82 patients having total hip arthroplasty. Patients received 25-200 mg/kg 10% DCLHb or a control of Ringer's lactate solution prior to induction British Medical Bulletin 1999,55 (No. 1)

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of anaesthesia. This study again showed an immediate increase in arterial blood pressure of 10-20%, peaking at the end of the infusion, and occurring simultaneously with a reduction in heart rate. The vasopressor effect was not dependent on the administered dose and was not associated with increased blood loss. After induction of anaesthesia, arterial pressure decreased in both treatment and control groups, but the 4 groups receiving DCLHb consistently had higher blood pressures and better haemodynamic stability than the control group over the 6 h following the infusion. The volumes of crystalloid and/or colloid administered were comparable in the 2 groups. A randomised, single blind, multicentre phase II study included 70 patients having elective surgery on abdominal aortic aneurysms. These patients were treated with doses of 50, 100, or 200 mg/kg of 10% DCLHb or an equivalent volume of a control infusion of Ringer's lactate. The infusions were started after the induction of anaesthesia, and lasted 15 min. The 2 higher doses of DCLHb significantly increased arterial blood pressure for 6 h following the infusion66. This increase did not cause higher blood loss. A Phase II study in haemodialysis patients showed improved haemodynamic stability with DCLHb, possibly because the molecule did not transfer into the dialysate; renal function in these patients remained stable67. DCLHb was also used in Phase II study in acute ischaemic stroke in man: it increases the mean arterial pressure in correlation with an increase in plasma concentration of 1-endothelin68. DCLHb is the only modified haemoglobin to have reached Phase III studies. In these studies, 750 ml (3 x 250 ml sacs) were administered. We participated in 2 randomised, single blind, human studies, in orthopaedic (n = 24) and cardiac surgery (« = 209; multicentre study). As reported in other studies, we observed an effect of DCLHb infusion on haemodynamic parameters (increase of systolic and diastolic arterial pressure, of systemic vascular resistance with concomitant decrease of heart rate), but these effects plateaued after the first infusion of 250 ml of DCLHb. The main observation of these studies was the effect of DCLHb on the blood saving. In the first postoperative day, 59% of cardiac patients and 92% of orthopaedic patients did not need blood transfusion, and blood savings after 7 days were of 33% and 19% respectively (Fig. 4). No serious side effects were observed. These results are to be published. Phase HI human studies with infusion of more than 1000 ml of DCLHb were planned (in surgery and in trauma patients), but these studies appear to have been stopped. DCLHb also has potential applications in septic shock. In one investigation, 500 ml of DCLHb was administered to patients in septic shock69. An immediate and significant vasopressor effect allowed reductions in the amounts of pressor drugs administered. 292

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100 |U orthopaedic surgery

„80

0 cardiac surgery

a ™ 60

Fig. 4 Blood sparing effect of 750 ml of DCLHb infused in orthopaedic and cardiac surgery patients (Phase II studies).

I 40 2. 20

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7 days

Liposome encapsulated tetrameric haemoglobin

Encapsulation of natural tetrameric Hb into a synthetic, non-antigenic phospholipid vesicle is an alternative method of administering this substance, which is particularly suited to the intensive care setting70. Encapsulation increases the intravascular half-life, and attenuates the vasoactive effects of free Hb. Hb molecules thus 'packaged' have values of P50 and of the Hill coefficient similar to those of blood. The incorporation of 2,3-DPG into the encapsulated Hb yields a P50 of 30 mmHg. The kinetics of binding and off-loading of O2 is faster for encapsulated Hb than for the erythrocytes71. Initial tolerance studies in the animal of first generation encapsulated Hb revealed side effects such as bradycardia, leukopenia, thrombopenia, increases in the values of transaminases and bilirubin, complement activation, hypertension, and decreases in cardiac output, especially with isovolemic exchange72. A lipid contaminant, lysolecithin, capable of activating complement, was the cause of these effects. The second generation of liposomes contain synthetic phosphatidylcholines and an antagonist of the activation of tissue platelet factor. Third generation liposomes (a lyophilised preparation) are beneficial in the treatment of haemorrhagic shock, where they increase PaO2, improve haemodynamic indices, and survival73. Nonetheless, among their undesirable side effects, it should be noted that these liposomes bind endotoxin, and that lipopolysaccharide (LPS) and encapsulated Hb can exacerbate the manifestations of septic shock. In terms of the elimination of these particles from the circulation, the reticulo-endothelial system of the liver and spleen are the primary areas for this function; some degree of overload of these organs can thus be expected after administration of liposomal Hb. British Medical Bulletin 1999;55 (No. 1)

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Beneficial effects and unsolved problems with Hb solutions

Because modified Hb solutions do not require compatibility testing, have low viscosity, do not pose an infectious risk, and have favourable O2 transport properties, their clinical use would appear to be promising. Studies to date have shown an absence of toxicity and immungenicity74, and only minor side effects, the most consistent of which is a rapid but transitory increase of systemic arterial blood pressure. DCLHb favours tissue perfusion and oxygenation, and could reduce the incidence of ischaemic phenomenon. Nonetheless, the modified Hb solutions do not fulfil the numerous other roles of the blood, including regulatory, metabolic, and defence functions. Further, their plasma half-life is short and their metabolic pathways are poorly characterised. They are thus best seen as an emergency substitute, useful in the short-term; they cannot replace transfusions in certain pathological situations such as chronic anaemia, but can postpone (or even eliminate) the need to transfuse homologous blood. The use of modified Hb solutions also poses a certain number of practical problems. Extracellular Hb can simulate, or mask, post-transfusion haemolysis. The haematocrit value after use of these solutions no longer faithfully reflects O2 transport capacity. SpO2 and mixed venous O2 saturations are still measured correctly, but the corresponding values of PO2 no longer have the same meaning, given the differences in P,o values between intra-erythrocytic and extracellular haemoglobins. The presence of plasmatic Hb can lead to false values from machines that measure concentrations optically, such as that of bilirubin. Similarly, the functioning of machines designed to wash red blood cells, such as blood recovery devices, is disturbed by the presence of plasmatic haemoglobin. An important problem that could arrive with free Hb is linked to the susceptibility of deoxyhaemoglobin to oxidation leading to the production of metHb, which has a peroxidative activity and forms further reactive O2 species75'76. This oxidation of Hb into metHb also easily releases haemin, which rapidly associates with membranes, leading to cytotoxicity. The crosslinking does not decrease the peroxidative activity of Hb. Inside the erythrocyte, enzymes and specific compounds protect the Hb molecule, but these compounds are absent in the solutions of modified Hb. The autoxidation of crosslinked Hb and the release of the haem would be more rapid than with native Hb, so that crosslinked Hb would cause a higher rate of induction of haem oxygenase in endothelial cells77, and contribute indirectly to oxidative stress on the endothelium. The formation of metHb thus, not only lowers the effectiveness of administered modified Hb, but is the source of potentially toxic ferryl Hb, haemin, bilirubin and free iron. Several other risks must be considered: modified Hb would complex endotoxins, at least in vitro, increasing the 294

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biological activity of these compounds, and large doses of free Hb would have a bacterial growth-enhancing effect. Finally, it has to be underlined that little is known about the interactions of the modified Hbs with haptoglobin and about their catabolism (possible toxicity of large amounts of bilirubin?). More studies are clearly needed before accepting that modified Hb solutions are absolutely safe and useful blood substitutes, taking also into account that Hb solutions are only effective for 24 h, with a cost that will probably be higher than that of packed red cell preparations. References 1 2 3 4 5 6 7 8. 9 10 11 12 13 14 15 16 17 18 19

Tomasulo P. Transfusion alternatives: impact on blood banking worldwide. In: Winslow RM, Vandegriff KD, Intaglietta M (Eds) Blood substitutes: physiological basis of efficacy. Boston: Birkhauser, 1995; 1-19 Winslow RM. Potential clinical applications for blood substitutes. Biomat Artif Cells Immobil Biotechnol 1992; 20: 205-17 Kaufman RJ. Clinical development of perfluorocarbon-based emulsions as red cell substitutes. In: Winslow RM, Vandegriff KD, Intaglietta M (Eds) Blood substitutes: physiological basis of efficacy. Boston: Birkhauser, 1995: 53-74 Clark LC, Gollan R. Survival of mammals breathing organic liquids equilibrated with oxygen at atmospheric pressure. Science 1966; 152: 1755-6 Riess JG, Le Blanc M. Perfluoro compounds as blood substitutes. Angew Chem Int Edn Engl 1978; 17: 621-34 Riess JG. Hemocompatible fluorocarbon emulsions. In: Sharma CP, Szycker M (Eds) Blood compatible materials and devices. Lancaster: Technomic, 1990; 237-70 Lamy M, Mathy-Hartert M, Deby-Dupont G. Perfluorocarbons as oxygen carriers. In: Vincent JL (Ed) Update in intensive care medicine. Berlin: Springer, 1998; 332-51 Riess JG. The design and development of improved fluorocarbon-based products for use in medicine and biology. Artif Cells Blood Substit Immobil Biotechnol 1994; 22: 215-34 Ingram DA, Forman MB, Murray JJ. Activation of complement by fluosol attributable to the pluronic detergent micelle structure. / Cardiovasc Pharmacol 1993; 22: 456-61 Lanes A, Rico-Lattes I. Micro-emulsions of perfluorinated and semifluorinated compounds. Artif Cells Blood Substit Immobil Biotechnol 1994; 22 (Suppl 4): 1007-18 Faithfull NS. Mechanisms and efficacy of fluorochemical oxygen transport and delivery. Artif Cells Blood Substit Immobil Biotechnol 1994; 22: 181-97 Yokoyama K, Yamanouchi K, Watanabe M et al. Preparation of perfluorodecalin emulsion; an approach to the red cells substitute. Fed Proc 1975; 34: 1478-83 Riess JG, Krafft MP. Elaboration of fluorocarbon emulsions with improved oxygen carrying capabilities. Adv Exp Med Biol 1992; 371: 465-72 Faithfull NS. Oxygen delivery from fluorocarbon emulsions - aspects of convective and diffusive transport. Biomat Artif Cells Immobil Biotechnol. 1992; 20: 797-804 Cornelus C, Krafft MP, Riess J. Improved control over particles sizes and stability of concentrated fluorocarbon emulsions by using mixed fluorocarbon/hydrocarbon molecular dowels. Artif Cells Blood Substit Immobil Biotechnol 1994; 22: 1183-91 Mathy-Hartert M, Krafft MP, Deby C et al. Effects of perfluorocarbon emulsions on cultured human endothelial cells. Artif Cells Blood Substit Immobil Biotechnol 1997; 25: 563-75 Fuhrman BP, Paczan PR, DeFrancisis M. Perfluorocarbon-associated gas exchange. Crit Care Med 1991; 19: 712-22 HLrschl RB, Merz SI, Montoya JP. Development and application of a simplified liquid ventilator. Crit Care Med 1995; 23: 157-63 Faithfull NS. The role of perfluorochemical in surgery and the ICU. In: Vincent J-L (Ed) Yearbook of intensive care and emergency medicine. Berlin: Springer, 1994; 264-75

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20 Overbeck MC, Pranikoff T, Yadao CM, Hirschl RB. Efficacy of perfluorocarbon partial liquid ventilation in a large animal model of acute respiratory failure. Crit Care Med 1996; 24: 1208-14 21 Greenspan JS, Wolfson MR, Rubenstein SD, Shaffer TH. Liquid ventilation of human preterm neonates. / Pediatr 1990; 117: 106-11 22 Gauger PG, Pranikoff T, Schreiner RJ, Moler FW, Hirschl RB. Initial experience with partial liquid ventilation in pediatric patients with the acute respiratory distress syndrome. Crit Care Med 1996; 24: 16-22 23 Hirschl RB, Pranikoff T, Wise C et al. Initial experience with partial liquid ventilation in adult patients with the acute respiratory distress syndrome. JAMA 1996; 275: 383—9 24 Tiituncii AS, Lachmann B. Perfluorocarbons as an alternative respiratory medium. In: Reinhart K, Eyrich K, Sprung C (Eds) Sepsis. Current perspectives in pathophysiology and therapy. Berlin: Springer, 1994; 549-63 25 Eanes R. On the horizon: liquid ventilation. / Obstet Gynecol Neonat Nurs 1995; 24: 119-24 26 Smith TH, Steinhorm DM, Thusu K, Fuhrman P, Dandona P. A liquid perfluorochemical decreases the in vitro production of reactive oxygen species by alveolar macrophages. Crit Care Med 1995; 23: 1533-9 27 O'Donnell KA, Caty MG, Zheng S, Rossman JE, Azizkhan RG. Oxygenated intraluminal perfluorocarbon protects intestinal mucosa from ischemia/reperfusion injury. / Pediatr Surg 1997; 32: 361-5 28 Tremper KK, Friedman AE, Levine EM, Lapin R, Camarillo D. The preoperative treatment of severely anemic patients with a perfluorochemical oxygen-transport fluid, Fluosol-DA. N Engl J Med 1982; 307: 277-83 29 Mitsuno, Ohyanagi H. Present status of clinical studies of Fluosol-DA 20% in Japan. Int Anesthesiol Clin 1985; 23: 169-84 30 Forman MB, Perry JM, Wilson HB et al. Demonstration of myocardial reperfusion injury in humans: results of a pilot study utilizing acute coronary angioplasty with perfluorochemical in anterior myocardial infarction. / Am Coll Cardiol 1991; 18: 911-8 31 Keipert PE. Perfluorocarbon emulsions: future alternatives to transfusion. In: Chang TMS (Ed) Blood substitutes: principles, methods, products and clinical trials. Berlin: Karger, 1998; 127-56 32 Cochran RP, Kunzelman KS, Vocelka CR et al. Perfluorocarbon emulsion in the cardiopulmonary bypass prime reduces neurologic injury. Ann Thorac Surg 1997; 63: 1326-32 33 Spiess BO, Cochran RP. Perfluorocarbon emulsions and cardiopulmonary bypass: a technique for the future. / Cardiothorac Vase Anesth 1996; 10: 83-9 34 Kuroda Y, Morita A, Fujino Y, Tanioka Y, Ku Y, Saitoh Y. Successful extended preservation of ischemically damaged pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) cold storage method. Transplantation 1993; 56: 1087-90 35 Kaplan E, Diehl JT, Peterson MB et al. Extended ex vivo preservation of the heart and lungs. / Thorac Cardtovasc Surg 1990; 100: 687-98 36 Brasile L, DelVecchio P, Rudofsky U, Haisch C, Clarke J. Postmortem organ salvage using an Oxygent™ supplemented perfusate. Artif Cells Blood Substit Immobil Btotechnol 1994; 22: 1469-75 37 Voiglio EJ, Zarif L, Gorry FC et al. Aerobic preservation of organs using a new perflubron/lecithin emulsion stabilized by molecular dowels. / Surg Res 1996; 63: 439-46 38 Steinhorn DM, Leach CL, Fuhrman BP, Holm BA. Partial liquid ventilation enhances surfactant phospholipid production. Crit Care Med 1996; 24: 1252-6 39 Thomassen MJ, Buhrow LT, Wiedemann HP. Perflubron decreases inflammatory cytokine production by human alveolar macrophages. Crit Care Med 1997; 25: 2045-7 40 Flaim SI. Pharmacokinetics and side effects of perfluorocarbon-based blood substitutes. Artif Cells Blood Substit Immobil Biotecbnol 1994; 22: 1043-54 41 Edwards CM, Lowe KC, Rohlke W, Geister U, Reuter P, Meinert H. Effects of a novel perfluorocarbon emulsion on neutrophil chemiluminescence in human whole blood. Artif Cells Blood Substit Immobil Biotechnol 1997; 25: 255-60 42 Von Stark. Die resorbarkeit des haimatins und die bedentungder hemoglobin - preparate. Dtsche Med Wochenschr 1898; 24: 805-8

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43 Sellards AW, Minot GR. Injection of hemoglobin in man and its relation to blood destruction with especial reference to the anemias. / Med Res 1916; 34: 469-94 44 Amberson WK, Jennings JJ, Rhode CM. Clinical experience with hemoglobin - saline solutions. / Appl Physiol 1949; 1: 469-89 45 Rabiner SF, O'Brien K, Peskin GW, Friedmand LH. Further studies with stroma-free hemoglobin solution. Ann Surg 1970; 171: 615-22 46 Feola M, Simoni J, Iran R, Canizaro P. Mechanisms of toxicity of hemoglobin solutions. Biomat Artif Cells Artif Organs 1988; 16: 217-26 47 Monk T, Goodnough L, Hughes G, Jacobs E. Evaluation of the safety and tolerance of hemoglobin-based oxygen carrier-201. Anestbesiology 1995; 83: 3A 48 Feola M, Simoni J, Angelillo R et al. Clinical trial of hemoglobin based blood substitute in patients with sickle cell anemia. Surg Gynecol Obstet 1992; 174: 379-86 49 Hoffman SJ, Looker D, Roehrich JM et al. Expression of fully functionnal human hemoglobin in Eschenchia coli. Proc Natl Acad Set USA 1990; 87: 8521-5 50 Shoemaker SA, Gerber MJ, Evans GL et al. Initial clinical experience with a rationally designed genetically engineered recombinant human Hb. Artif Cells Blood Substit Immobil Biotechnol 1994; 22 (Suppl 3): 457-65 51 Yang T, Olsen KW. Thermal stability of Hb cross-linked in the T state by bis (3,5 dibromosalicyl) fumarate. Biocbem Biophys Res Commun 1991; 174: 518-23 52 Walder JA, Zangg RH, Walder RY et al. Diaspirins that cross-link beta chains of hemoglobin: bis (3,5 dibromosalicyl) succinate and bis (3,5 dibromosalicyl) fumarate. Biochemistry 1979; 18:4265-70 53 Przybelski RJ, Daily EK. The pressor/perfusion effect of diaspirin cross-linked hemoglobin (DCLHb). In: Vincent JL (Ed) Yearbook of intensive care and emergency medicine. Berlin: Springer, 1994; 252-63 54 Farmer M, Ebeling A, Marshall T et al. Validation of virus inactivation by heat treatment in the manufacture of diaspirin cross-linked hemoglobin. Biomat Artif Cells Immobil Biotechnol 1992; 20: 429-33 55 Estep TN, Gonder J, Bornstein I et al. Immunogenicity of diaspirin cross-linked human hemoglobin solutions. Biomat Artif Cells Immobil Biotechnol 1992; 20: 603-9 56 Vandegriff KD, Le Tellier YC, Winslow RM. Determination of the rate and equilibrium constants for oxygen and carbon monoxide binding to R-state human hemoglobin cross-linked between the a subunits at lysine 99.) Biol Chem 1991; 266: 17049-59 57 Przybelski RJ, Malcolm DS, Burns DG, Winslow RM. Cross-linked hemoglobin solution as a resuscitative fluid after hemorrhage in the rat. / Lab Clin Med 1991; 117: 143-51 58 Malcolm D, Kissinger D, Garrioch M. Diaspirin cross-linked hemoglobin solution as a resuscitative fluid following severe hemorrhage in the rat. Biomat Artif Cells Immobil Biotechnol 1992; 20: 495-7 59 Schultz SC, Grady B, Cole F et al. A role of endothelin and nitric oxide in the pressor response to diaspirin cross-linked hemoglobin. / Lab Clin Med 1993; 122: 301-8 60 Gulati A, Rebello BS. Diaspirin cross-linked Hb: involvement of adrenergic mechanisms in the pressor effect. Artif Cells Blood Substit Immobil Biotechnol 1994; 22: 603-12 61 McKenzie JE, Cost EA, Scandling DM et al. Effects of diaspirin cross-linked hemoglobin during coronary angioplasty in the swine. Cardiovasc Res 1994; 28: 1188-92 62 Cole DJ, Drummond JC, Patel PM. Effects of viscosity and oxygen content on cerebral blood flow in ischemic and normal rat brain. / Neurol Set 1994; 124: 15-20 63 Aranow JS, Wang H, Zhuang J. Effect of human hemoglobin on systemic and regional hemodynamics in a porcine model of endotoxemic shock. Crit Care Med 1996; 24: 807-14 64 Przybelski RJ, Daily EK, Kisicki JC et al. Phase I study of the safety and pharmacologic effects of diaspirin cross-linked hemoglobin solution. Crit Care Med 1996; 24: 1993-2000 65 Sloan EP, Koenigsberg MD, Bickell WH. The use of diaspirin cross-linked hemoglobin solution in the hospital management of hemorrhagic hypovolemic shock, Acad Emerg Med 1995; 2: Abstract 66 Garrioch M, Larbuisson R, Brichant JF, Lamy M, Daily E, Przybelski R. The hemodynamic effects of diaspirin cross-linked hemoglobin in the operative setting. Crit Care Med 1996; 24 (Suppl 1): A39

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67 Swan S, Halstenson C, Collins A et al. Pharmacologic profile of diaspirin cross-linked hemoglobin in hemodialysis patients. Am ] Kidney Dis 1995, 26: 918-23 68 Saxena R, Wijnhoud AD, Man m't Veld AJ et al. Effect of dispirin cross-linked hemoglobin on endothehn-1 and blood pressure in acute ischemic stroke in man. / Hypertens 1998; 16: 1459-65 69 Reah G, Mallick A, Bodenham AR, Przybelski R. Diaspirin cross-linked hemoglobin improves gastric intramucosal pH in critically ill patients. Int Care Med 1996; 22 (Suppl 3): S441 70 Bcissinger MC, Farmer RL, Gossage JL. Liposome encapsulated Hb as a red cell surrogate. Trans An Soc Actif Intern Organs 1986; 32: 58-63 71 Rudolph AS. Encapsulation of hemoglobin in liposomes. In: Winslow RM, Vandegnff KD, Intaglietta M (Eds) Blood substitutes: physiological basis of efficacy. Boston: Birkhauser, 1995; 90-104 72 Rudolph AS. Transient changes in the mononuclear phagocyte system following administration of the blood substitute, liposome encapsulated hemoglobin. Biomaterials 1994; 15: 796-804 73 Rabinovici R, Rudolph AS, Feuerstein G. A new salutary resuscitative fluid: liposome encapsulated hemoglobin (LEH) - hypertonic saline solution./ Trauma 1993; 35: 121-7 74 Patel MJ, Webb EJ, Shelbourn TE et al. Absence of immunogenicity of diaspirin cross-linked hemoglobin in humans. Blood 1998; 91:710-716. 75 Deby-Dupont G, Pincemail J, Lamy M. Hemoglobin-based red cell substitute: preliminary human studies. In: Vincent JL (Ed) Yearbook of intensive care and emergency medicine. Berlin: Springer, 1994; 264-75 76 Everse J, Hsia N. The toxicities of native and modified hemoglobins. Free Radtc Biol Med 1997; 22: 1075-99 77 Balla J, Nath KA, Balla G, Juckett MB, Jacob HS, Vercellotti GM. Endothelial cell heme oxygenase and ferritin induction in rat lung by hemoglobin in vivo. Am ] Physiol 1995; 268: L321-7

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