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
British Medical Bulletin 1999;55 (No. 1): 277-298
O The British Council 1999
Intensive care medicine
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 .
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Red blood cell substitutes
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
British Medical Bulletin 1999;55 (No. 1)
279
Intensive care medicine
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
British Medical Bulletin 1999,55 (No. 1)
Red blood cell substitutes
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)
281
Intensive care medicine
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
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British Medical Bulletin 1999;55 (No. 1)
Red blood cell substitutes
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)
283
Intensive care medicine
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
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Red blood cell substitutes
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)
285
Intensive care medicine
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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Red blood cell substitutes
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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Intensive care medicine
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.
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Red blood cell substitutes
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)
British Medical Bulletin 1999;55 (No. 1)
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
289
Intensive care medicine
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
British Medical Bulletin 1999;55 (No. 1)
Red blood cell substitutes
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)
291
Intensive care medicine
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
British Medical Bulletin 1999,55 (No. 1)
Red blood cell substitutes
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
5
6
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)
293
Intensive care medicine
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
British Medical Bulletin 1999,55 (No. 1)
Red blood cell substitutes
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.
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