REVIEW ARTICLE

BioDrugs 1999 Mar; 11(3): 211-221 1173-8804/99/0003-0211/$05.50/0 © Adis International Limited. All rights reserved.

Potential Clinical Applications of Recombinant Human Haemoglobin in Blood Conservation Bruce J. Leone Mayo Clinic Jacksonville, Jacksonville, Florida, USA

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The Case for Haemoglobin Solution Development: Why Not Red Blood Cell Sources? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Allogeneic Red Blood Cell Transfusion . . . . . . . . . . . . . . . . . . . . 1.2 Autologous Blood Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Autologous Predonation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Acute Normovolaemic Haemodilution . . . . . . . . . . . . . . . . . . . 1.5 Red Cell Salvage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Problems in the Development of Haemoglobin Infusions . . . . . . . . . . . . 3. The Supply of Haemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Recombinant Human Haemoglobin . . . . . . . . . . . . . . . . . . . . . . . 4.1 Modification of Haemoglobin to Produce Functional Human Haemoglobin Somatogen (rHb1.1) . . . . . . . . . . . . . . . . . . . . . . 4.2 Potential Clinical Uses of Recombinant Human Haemoglobin . . . . . . 4.3 Surgical Blood Loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Postoperative Transfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Recombinant Human Haemoglobin as a Prophylactic Oxygen Carrier 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Haemoglobin solutions have been tried as blood substitutes for decades with little success. Problems with early haemoglobin solutions include instability of the haemoglobin tetramer in the plasma, resulting in dissociation of the protein into dimers, as well as excessively high oxygen affinity for clinical oxygen transport capabilities. Newer haemoglobin solutions are currently under development and may prove adequate to transport oxygen while avoiding excessive toxicity. However, these compounds pose other difficulties, including a short plasma halflife and the potential for systemic hypertension due to nitric oxide binding. Several haemoglobin solutions are now undergoing human testing for potential clinical use. Haemoglobin can be harvested from outdated packed red blood cell units and chemically altered for use in clinical scenarios. Likewise, bovine haemoglobin may be obtained and modified for use as a blood substitute. While both these approaches may yield clinically useful oxygen carrying solutions, neither is completely free from infectious risk. The use of recombinant technology

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to produce a genetically altered haemoglobin from Escherichia coli allows avoidance of infectious risks and a functional haemoglobin which does not need further chemical modification. Use of this first generation of recombinant human haemoglobin as a blood substitute may be limited due to its short half-life of <12 hours, but clinical uses such as replacement fluid for acute normovolaemic haemodilution during the perioperative period may allow significant blood savings for patients and the potential to avoid patient exposure to allogeneic transfusions.

The search for an adequate replacement for human red blood cells has spanned well over 3 centuries. Jean Baptiste Denis, physician to Louis XIV, first described the use of lamb’s blood as a transfusion substance in June 1667, with an initially good result. Ultimately, however, a patient died after xenotransfusion, and transfusions were thereafter banned by royal decree.[1] Additional attempts to use haemolysed human blood have also met with similar disastrous outcomes. Among the more notable early clinical trials, Amberson[2] reported the use of a purified human haemoglobin, from haemolysed red blood cells, to resuscitate a young mother suffering life threatening post-partum haemorrhage. While ultimately unsuccessful, the promising early resuscitation of this patient suggested a potential role for haemoglobin as a substitute oxygen carrier for allogeneic blood. However, further research was not fruitful, and until quite recently many have thought the development of a safe and effective ‘artificial blood’ was an unattainable ‘holy grail’ of blood transfusion research. 1. The Case for Haemoglobin Solution Development: Why Not Red Blood Cell Sources? Blood product transfusion usually involves the infusion of red cell concentrates to increase oxygen carrying capacity. Progressive improvements in blood collection and storage have demonstrated that blood from anonymous donors is well tolerated, efficient and cost-effective. However, fear still pervades the general public’s view of allogeneic transfusions, as the possibility of disease transmission, although diminished, is still present (table I).[3] A recent report demonstrates that the volunteer do© Adis International Limited. All rights reserved.

nor screening process may underestimate the actual infectious disease risk factors of donors. [4] 1.1 Allogeneic Red Blood Cell Transfusion

After antigen identification made red blood cell transfusions possible, allogeneic blood became available to treat acute and chronic anaemia. As storage of blood created some dysfunction of oxygen carrying capabilities,[5,6] newer methods of blood preservation were employed, with greater functional red cells the result. At present, the use of adenosinecitrate-dextrose anticoagulant-preservation solutions, along with storage at 4°C, allows effective red cell storage up to 45 days, with viable red cells at functional oxygen carrying capacity within 6 hours of transfusion.[5] Detection of contaminated blood has become a priority for blood collection agencies.[7] Recent reports of transfusion of contaminated blood in Canada and France have resulted in renewed examination of donor and blood product infectious disease screening. Red cell unit screening has resulted in a progressive decrease in the incidence of hepatitis over the last decades (tables II and III). Recent improvements have led to the development of a p24 antigen screening test for detection of HIV-infected blood.[8] This increased screening has been impleTable I. Risk of contracting viral disease from allogeneic transfusions (personal communication, L. Schreiber) Virus

Risk of exposure

Hepatitis B

1 : 60 000

Hepatitis C

1 : 100 000

Human Immunodeficiency

1 : 500 000

Human T Cell Lymphotrophic

1 : 600 000

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Clinical Applications of Recombinant Haemoglobin

Table II. Relative safety of blood transfusions, as evaluated by transfusion-associated hepatitis incidence, over the past four decades (personal communication, L. Stehling) Decade

Incidence of hepatitis-infected red blood cell unit (%)

1960s

33

1970s

5-12

1980s

1.5-4

1990s

0.5

mented despite limited cost effectiveness estimates. The concept of immunomodulation as a result of allogeneic blood transfusions has recently been receiving attention. Immunosuppression is a known consequence of allogeneic transfusions;[9] whether this immune function decrease results in postoperative complications is in question.[10-17] While controversial, many of these reports strongly suggest that immunomodulation resulting from allogeneic blood transfusions may have adverse clinical consequences. Thus allogeneic transfusion may result not only in transmission of disease but also in increased susceptibility to disease. 1.2 Autologous Blood Sources

The use of alternative strategies to avoid allogeneic blood exposure has increased dramatically. Autologous blood can be obtained by predonating red blood cells in the weeks prior to elective surgery, harvesting whole blood perioperatively with acute normovolaemic haemodilution, and scavenging red cells using blood salvage techniques intraoperatively. Effective use of these strategies requires preplanning and equipment; at present, the cost effectiveness of these methods is limited to surgical procedures with high expected blood loss.

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binant eythropoietin to increase the amount of autologous red cells that can be obtained.[18] This approach has been demonstrated to decrease exposure risk to allogeneic transfusions; however, it is very costly and labour intensive. Predonated autologous blood carries a risk similar to that of allogeneic blood, as by far most complications from blood transfusions are due to clerical errors, which autologous predonated blood does not avoid.[19] Moreover, as predonated blood is not screened as is allogeneic blood from anonymous donors, it cannot be returned into the pool of allogeneic banked blood if not used. The consequence of these factors is the extreme inefficiency and financial disadvantage of autologous predonated blood.[20] 1.4 Acute Normovolaemic Haemodilution

As an alternative to autologous predonation, whole blood can be harvested immediately prior to the surgical procedure, either prior to or following the induction of anaesthesia.[21] Haemodilution offers several theoretical advantages, including obviating the necessary delay between blood collection and surgery. In addition, fresh whole blood, with functional clotting factors, can be available immediately following the majority of surgical blood loss.[22] Acute normovolaemic haemodilution has advantages in many situations; however, some disadvantages preclude its more widespread use.[23] Although not as time consuming as predonation, haemodilution does nonetheless involve planning and time during the perioperative period. More importantly, use of haemodilution during surgery may not result in significant red blood cell savings until extreme levels of haemodilution and surgical

1.3 Autologous Predonation

Prior to a planned high blood loss surgery, patients may be enrolled in a predonation program. Usually, patients have surgery scheduled 3 weeks in advance and are asked to donate a unit of blood once per week, if possible. Patients receive supplemental iron therapy and, in some instances, recom© Adis International Limited. All rights reserved.

Table III. Relative safety of blood transfusions, as evaluated by transfusion-associated human immunodeficiency virus (HIV) incidence, over the past decade (personal communication, L. Stehling) Year

Incidence of HIV infected red blood cell unit

1985

1%

1996

1 : 450 000 to 600 000

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blood loss are realised.[24,25] Although haemodilution has been shown to be of similar benefit as predonation in small studies,[26] widespread application may depend on large studies showing benefit for acute normovolaemic haemodilution. 1.5 Red Cell Salvage

In particularly high blood loss surgical procedures, blood can be suctioned directly from the surgical field and processed for re-infusion into the patient. The red cell mass suctioned from the field therefore is salvaged, ultimately resulting in lower total oxygen carrying capacity loss for the patient. This approach can result in significant avoidance of allogeneic transfusions, thus decreasing the risks associated with the procedure.[27,28] There are several problems associated with red cell salvage. Firstly, to accomplish red cell salvage, separate equipment must be maintained for use in the operating room. Additionally, personnel must be trained to operate and maintain the system; although frequently this can be done with existing operating room personnel, in particularly difficult cases, the red cell salvage system may require a dedicated operator to function efficiently. The suctioned blood from the surgical field must be free of pathogenic potential; thus use of the salvage system is generally contraindicated in situations in which bacterial or cancer cell contamination may be likely. Indeed, a study suggests that even in uncontaminated wounds, a significant amount of potentially pathogenic material can be found contaminating the reinfused salvaged blood; however, no adverse clinical outcome was noted.[29] Finally, the salvage of the blood via a suction system is subjected to significant shear stress, which may result in red cell damage or lysis. Improper washing of the salvaged blood may result in infusion of free haemoglobin and other products from red cell destruction. 2. Problems in the Development of Haemoglobin Infusions The purification of haemoglobin is a relatively straightforward task. Haemoglobin is the main © Adis International Limited. All rights reserved.

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component of red blood cells, and therefore a ready supply is available from donated blood.[30,31] In addition, haemoglobin has remained a relative constant throughout evolution, resulting in similar structure and function in a variety of species. Although the ferric component of the haeme may be substituted with a copper-based oxygen binding system, the protein structure of haemoglobin has been relatively constant.[32] Difficulties arise, however, with the protein components of the molecule. Because of the complex folding involved in this relatively large (64K dalton (kD) molecular weight) protein tetramer, the oxygen binding and delivery components of haemoglobin are extremely difficult to preserve when isolating haemoglobin from red blood cells. The result of haemolysis of red cells is pure haemoglobin, yet this haemoglobin may be dysfunctional and may be contaminated with small quantities of antigens arising from red and white blood cell membranes which have inadvertently been included.[30] As haemoglobin is ubiquitous in our organ systems, humans have developed an efficient system for the metabolism and recycling of the molecule, in particular the haeme moiety. Free haemoglobin is avidly bound by haptoglobin and transported to the liver, where it undergoes metabolism to bile, and the ferric component can be is salvaged and incorporated in nascent red blood cells. This elegant disposal system takes place within the reticuloendothelial system and thus free haemoglobin is rarely released in large quantities in the bloodstream. Like its sister ferrous compound, myoglobin, haemoglobin is ideally suited to transport oxygen and, when it has reached its utilisable lifespan, these molecules are quickly metabolised and the toxic components eliminated. Problems can arise when excessive haemoglobin enters the bloodstream. While the metabolic processes of the body easily handle quantities (e.g. 100mg) of haemoglobin, large amounts of haemoglobin will overwhelm the ability of haptoglobin to bind haemoglobin and result in unbound free haemoglobin circulating in the plasma phase. This BioDrugs 1999 Mar; 11 (3)

Clinical Applications of Recombinant Haemoglobin

situation arises from massive red cell haemolysis, such as with an ABO incompatible transfusion, or from massive trauma, causing the release of large amounts of myoglobin from its intramuscular compartments. Haemoglobin will rapidly separate into dimeric form from its functional tetrameric state, thus mimicking myoglobin. These dimers may then cause renal tubular necrosis, developing from minute cell membrane impurities contained within these solutions, ultimately resulting in renal failure and the need for permanent dialysis. The infusion of native haemoglobin, even from autologous blood sources, will also result in the dissociation of the haemoglobin tetramer, rapid acute renal tubular necrosis, and lead to chronic renal failure; indeed, this is the mechanism by which deaths occurred during early trials of untreated native haemoglobin solutions.[2] Regulation of the oxygen-haemoglobin dissociation curve can be achieved by alterations in temperature, 2,3-diphosphoglycerate (2,3-DPG), or by pH.[33] Temperature and pH are dependent upon systemic regulatory mechanisms, and thus these factors will not be subject to local regulation at the level of the red blood cell. 2,3-DPG may be regulated, however, by enzyme systems within the red cell itself. Thus 2,3-DPG becomes an important feature in the delivery of oxygen at the tissue level. Conditions which have been shown to alter 2,3DPG content include chronic hypoxia and chronic anaemia, as 2,3-DPG content increases to augment oxygen delivery in the face of decreased tissue oxygen supply. It should be noted, however, that changes in 2,3-DPG represent a chronic adaptation to decreased oxygen supply; acute changes to compensate for hypoxaemia do not appear to be present at the red cell level.[34] 3. The Supply of Haemoglobin Haemoglobin is relatively easily isolated from red cells, as mentioned above. Thus, allogeneic blood may be a source of haemoglobin from which to manufacture haemoglobin-based oxygen carriers (HBOCs). Approximately 10 to 20% of the allogeneic blood collected is discarded for a variety © Adis International Limited. All rights reserved.

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of reasons, the most common being the outdating of blood, that is, storage past the 45-day limit of viability in red cell preservative adenosine-citratedextrose buffered (ACD) blood bags. However, this discarded blood can be harvested for native haemoglobin, and haemoglobin then chemically modified so as to be physiologically active in human plasma without significant dissociation to tetramers. This solution is thus dependent upon the supply of outdated allogeneic blood. Xenotransfusion is also a possible means of supplying haemoglobin. Indeed, Denis’ first attempt at transfusion was a xenotransfusion with lambs’ blood which was successful. More refined modes of xenotransfusion are now available, owing to the unique properties of bovine haemoglobin. The bovine haemoglobin molecule does not require 2,3-DPG to function at physiological oxygen tension; rather, chloride ions modulate the position of the bovine oxyhaemoglobin dissociation curve.[35] Supply of bovine haemoglobin is more readily available than human haemoglobin, owing to the large quantities available as a byproduct of the meat producing industry. While supply and production do not appear to be an issue, zoonoses from bovine sources are however possible. Hand, foot and mouth disease can be traced to bovine sources, and recently the possibility of the transmission of bovine spongiform encephalopathy has raised serious concerns in the meat producing industry about transmission of prion diseases to humans.[36-38] Because the sterilisation process for bovine haemoglobin is not by heat, due to the harmful effects of heat sterilisation on the structure and function of bovine haemoglobin, the possibility exists that complete sterilisation of bovine haemoglobin products may not be possible. Thus the risk of acquiring an infectious agent from bovine haemoglobin is not known at present. 4. Recombinant Human Haemoglobin The production of native haemoglobin involves transcription of DNA to RNA and translation into amino acids, with conformational folding resulting BioDrugs 1999 Mar; 11 (3)

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in a functional haemoglobin protein backbone. Addition of the haeme moiety, acting as the oxygen binding ligand at the centre of the protein, with 2 alpha chain proteins and 2 beta chain proteins produces the haemoglobin molecule; encapsulation within the milieu of the red blood cell, and modification of oxyhaemoglobin dissociation by 2,3DPG, produces a system designed to ferry oxygen from areas of high partial pressure (i.e. the lung) to low partial pressure areas (i.e. the peripheral organs). Transcription and translation of proteins for commercial use can be accomplished using recombinant gene technology. Drugs such as insulin can be produced by inserting the appropriate DNA genetic code into the plasmid of a bacteria; typically, Escherichia coli is used as a cell production system for protein synthesis.[32] Thus, with the genetic code for haemoglobin known, production could be accomplished by bacteria, with harvesting of the protein identical to that used at present with other commercially produced drugs. 4.1 Modification of Haemoglobin to Produce Functional Human Haemoglobin Somatogen (rHb1.1)

The production of native haemoglobin would then require the protein product described above to be combined with haeme groups to result in a molecule capable of oxygen binding. This functional molecule would then need to be modified to minimise toxicity and to produce oxygen delivery capabilities. A decrease in toxicity could be accomplished by crosslinking the individual protein chains together by polymerisation or by a linking molecule. Additional modifications would be needed to decrease the molecular oxygen affinity so as to be functional in the bloodstream. Another approach could be to subtly alter the haemoglobin compound itself through genetic engineering. By substitution, addition, or deletion of specific DNA codons, the possibility exists of producing a molecule with properties that do not result in toxicity and do enable effective oxygen delivery (see fig. 1). Human haemoglobin somatogen, a re© Adis International Limited. All rights reserved.

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combinant haemoglobin molecule, contains 2 specific genetic alterations designed to alter the metabolism and the oxygen affinity of native haemoglobin. The first modification is the insertion of a glycine to bind the carboxy and amino termini of the alpha chains of haemoglobin covalently. This results in a ‘di-alpha’ haemoglobin subunit capable of binding 2 beta subunits to form haemoglobin (see fig. 2). The glycine insertion does not alter the conformational structure of the haemoglobin so as to effect function, but does prevent the dissociation of the tetramer into dimers, and thus extends the plasma half-life of the molecule while eliminating renal toxicity. The second modification is the substitution of a lysine for an asparginine at amino acid position 108 of the beta chain subunits (see fig. 3). This modification affects the oxygen affinity of haemoglobulin, which is measured by determining the partial pressure of oxygen at which haemoglobulin is 50% saturated (P50); the lower the P50 value, the higher the affinity of haemoglobulin for oxygen. The haemoglobin produced with these altered

a

b

Fig. 1. Diagram of haemoglobin molecule as transcribed from genetic codons to tetramer. a = The normal transcription and

translation of haemoglobin involves production of a tetrameric protein that is capable of dissociating into dimers outside of the erythrocytic environment. b = Depiction of genetically modified haemoglobin, in which a di-alpha construct has been encoded by the addition of a codon for glycine (white bar), which results in a tetrameric haemoglobin that will not dissociate in the plasma phase.

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Clinical Applications of Recombinant Haemoglobin

217

Val

-

Val +

Gly

Arg

Arg Tyr Tyr

Fig. 2. Depiction of the amino (+) terminus and carboxyl (–) ter-

minus of alpha haemoglobin chains. The addition of a glycine amino acid results in a covalently-bound di-alpha construct, which stabilises the tetramer while not affecting the conformational folding of the protein or its subsequent ability to bind and transport oxygen.

beta subunits has a P50 of 32mm Hg, meaning slightly less oxygen affinity than native haemoglobin, which has a P50 of 27mm Hg. A mutant variant of native haemoglobin, haemoglobin Presbyterian, possesses this particular substitution in the beta chain subunits.[32] When the di-alpha genetic code is coupled with the beta subunit amino acid substitution, the resulting haemoglobin may be functional and nontoxic without additional chemical modifications. This haemoglobin would be functionally slightly better than the native red cell at offloading oxygen (see fig. 4), as well as needing no specific chemical alterations to avoid haemoglobin associated renal toxicity.[39] Early trials with this recombinant human haemoglobin have been promising. Volunteers tolerated low doses of up to 10g of human haemoglobulin somatogen without significant renal toxicity; however, gastrointestinal motility disorders, particularly of the lower oesophagus, were manifest at low to moderate doses.[40] Higher doses of human haemoglobin somatogen have been utilised in clinical trials in patients undergoing a variety of elective surgery. In one study, Lessen et al.[41] demonstrated that human haemoglobin somatogen doses of up to 100g infused into anaesthetised patients requiring oxygen carrying replenishment were well tolerated without significant toxicity. No gastrointestinal events were noted postoperatively.[41] We examined patients undergoing acute © Adis International Limited. All rights reserved.

normovolaemic haemodilution prior to elective hip arthroplasty. Exchange of 2 units of autologous whole blood with up to 50g of human haemoglobin somatogen were well tolerated in sedated, awake patients immediately prior to a combination of general and regional anaesthesia. No patient suffered any adverse events, with an approximate 10% increase in mean arterial pressure (measured with direct radial arterial cannulation), and the infusions were well tolerated without any clinically significant adverse effects.[42] These results suggest that human haemoglobin somatogen will be a useful blood substitute for use in clinical practice. Human haemoglobin somatogen has a negligible propensity to transmit viral infections commonly associated with blood transfusions, and zoonoses are not a consideration. The manufacturing process used to produce human haemoglobin somatogen is based on a currently used system of recombinant DNA technology, and the manufacturing capability can be expanded to markedly increase supply. The possibilities of recombinant human haemoglobin being a clinically useful blood substitute are present; the key issue will be how to use human haemoglobin somatogen effectively and efficiently in clinical practice. 4.2 Potential Clinical Uses of Recombinant Human Haemoglobin

Early clinical testing of human haemoglobin somatogen has shown that this agent has a plasma half-life of about 12 hours after doses of approximately 1 homologous unit equivalent or 50g of hu-

107

108

109

Normal

Gly

Asn

Val

rHgb 1.1

Gly

Lys

Val

Fig. 3. Depiction of the point mutation engineered in recombinant

human haemoglobin ( rHb1.1; human haemoglobin somatogen) to result in lower oxygen affinity, i.e. a leftward shift in the oxyhaemoglobin dissociation curve. This point mutation is also a naturally occurring mutant variant of native haemoglobin A1; this mutated haemoglobin is also called Haemoglobin Presbyterian.

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Stroma-Free Normal RBC Hb Recombinant Human Hb (rHb1.1) 100

Hb-Oxygen Saturation (%)

90 80 70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

70

80

90

100

PaO2 (mmHg)

Fig. 4. The oxy-haemoglobin dissociation curves of native intra-

erythrocytic haemoglobin (the result of an interaction with 2,3diphosphoglycerate), stroma-free native haemoglobin (without 2,3-DPG interaction), and genetically-engineered recombinant human haemoglobin (rHb1.1; human haemoglobin somatogen).

man haemoglobulin somatogen.[40] Thus, use of a compound such as human haemoglobin somatogen would be for short term oxygen carrying capacity only. Compared with the estimated half-life of allogeneic red blood cells, of the order of weeks to months, it is unlikely that recombinant human haemoglobin would obviate the need for allogeneic red cell transfusions. Moreover, repetitive use of human haemoglobin somatogen could conceivably result in haemosiderosis, as the excessive haemoglobin infused would ultimately be metabolised and the haeme moiety retained to the ultimate detriment of the recipient. However, single doses or limited repeat doses of recombinant human haemoglobin would seem to be a plausible treatment approach. The temporary augmentation of oxygen carrying capacity would be useful in a number of clinical situations, and the ability to temporarily increase oxygen delivery may provide new treatment options in certain situations. 4.3 Surgical Blood Loss

Acute surgical blood loss is most commonly thought of when discussing the need for transfu© Adis International Limited. All rights reserved.

sion to replace oxygen carrying capacity. However, if blood volume status is maintained at pre-bleeding levels, animals tolerate remarkable decreases in haemoglobin levels with no discernible long term effects. In the clinical arena, moderate haemoglobin loss does not appear to result in significant short term morbidity. No data are available regarding minimum postoperative haemoglobin requirements. Several authors have suggested that higher haemoglobin concentrations than are often tolerated intraoperatively may be necessary postoperatively. Using data from Rand,[43] Hint[44] suggested maximal theoretical oxygen delivery was present with a 10 g/dl haemoglobin (30% haematocrit); Messmer[45] has stated that a minimum haematocrit of 30% should be maintained in order to recover from surgery without sequelae. Nelson and colleagues[46] demonstrated an increased incidence of postoperative ischaemia and myocardial infarction in peripheral revascularisation patients with haematocrit values below 29%. Thus oxygen carrying requirements appear to be at the level of 10 g% in the postoperative period in patients with major organ pathology. During the intraoperative period, loss of red cell mass appears to be better tolerated than preoperatively or postoperatively. Laboratory studies have demonstrated tolerance of haemoglobin values to 5 g% in anaesthetised canines with extrinsically applied critical coronary stenoses.[47] However, tolerance to haemoglobin levels is widely variable; although Spahn et al.[48] demonstrated an average haemoglobin of 7.5 g/dl was tolerated in dogs with extrinsic coronary stenosis, the individual animals had a wide variability in the appearance of ischaemia, despite highly controlled pathophysiological conditions. Furthermore, in the presence of multivessel coronary stenoses, tolerance to haemodilution was significantly altered, as higher haemoglobin values were needed to prevent ischaemia in the presence of two vessel extrinsic critical coronary stenoses.[49] Thus patients may tolerate moderate haemodilution during the period of possible surgical BioDrugs 1999 Mar; 11 (3)

Clinical Applications of Recombinant Haemoglobin

blood loss, but require additional oxygen carrying capacity after surgery. Acute normovolaemic haemodilution may be employed during administration of the anaesthetic; aggressive haemodilution, however, would be required to preserve significant red cell mass and potentially avoid transfusions.[25] With availability of human haemoglobin somatogen, significant haemodilution could be employed, with added oxygen carrying capacity supplied by human haemoglobin somatogen.[50] Postoperatively, the patient’s whole blood could be re-infused to restore the oxygen carrying capacity as human haemoglobin somatogen is metabolised from the body. The addition of 3 to 5 g% of oxygen carrying capacity during aggressive haemodilution to haemoglobins of 5 g% native haemoglobin could conceivably save up to 4 units of autologous red cell mass, according to Weiskopf’s calculations.[25] Thus, human haemoglobin somatogen could serve as an important bridge for intraoperative oxygen carrying capacity. As such, acute normovolaemic haemodilution with human haemoglobulin somatogen would represent a cost-effective and efficient means of conserving red cell mass. The ultimate result would be a decrease in the exposure of patients to allogeneic blood transfusions. 4.4 Postoperative Transfusions

A subtle shift in clinical practice may naturally follow from data collected on the tolerance to haemodilution, namely, that transfusion therapy may be effected in the early postoperative period rather than during the intraoperative phase. In this clinical scenario, human haemoglobin somatogen may also play a role. In critically ill patients who have undergone major surgery and are heavily sedated in postoperative intensive therapy units, human haemoglobin somatogen may be infused to augment temporary oxygen carrying capabilities. This could be in lieu of transfusion therapy, and may bridge the patient to a period when effective erythropoiesis increases the oxygen delivery capability, and the patient’s physiological condition improves. © Adis International Limited. All rights reserved.

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The infusion of human haemoglobin somatogen may have the added benefit of augmenting haematopoiesis. The human haemoglobin somatogen metabolite would harbour a haeme group in a ‘prepackaged’ form, available for immediate incorporation into newly forming red blood cells. Although the precise mechanisms have not been elucidated, a suggestion of augmented red cell production was seen in the early clinical trial with recombinant human haemoglobin.[42] A haemotapoietic effect would make human haemoglobin somatogen effective as a short term HBOC and an immediate supply of usable iron. If iron overload is a concern then repeat dosing of recombinant human haemoglobin could be based on determination of iron stores, thus enabling repetitive dose administration in the acutely bleeding postoperative patient. Further laboratory and clinical studies are needed in this new area of augmented haematopoiesis to provide additional information as to the potential for human haemoglobin somatogen use. 4.5 Recombinant Human Haemoglobin as a Prophylactic Oxygen Carrier

Recombinant human haemoglobin is roughly equivalent to albumin in molecular size and other physical properties; thus, oxygen delivery through stenosed arteries or via collateral vessels may be enhanced by use of an HBOC. Spahn et al.[48] observed increased coronary blood flow through a fixed coronary stenosis with haemodilution. It is conceivable that a small molecule, such as human haemoglobin somatogen, could pass through a stenosis with much less resistance than a red blood cell. This enhancement of flow could be helpful in a number of clinical situations where decreased blood flow might occur. For example, Cole and colleagues[51-53] have noted a significant decrease in cerebral infarct size in rats treated with α-α crosslinked haemoglobin than control animals. This decrease in infarct size is not apparently due to the hypertensive response seen with this stroma-free haemoglobin compound; these investigators theorised that increased collateral blood flow containBioDrugs 1999 Mar; 11 (3)

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ing the HBOC was responsible for delivery of oxygen to the infarct region, thus lessen the amount of tissue that became ischaemic. Thus mild haemodilution with human haemoglobin somatogen might provide increased peripheral blood flow and collateral blood flow to regions with diminished arterial supply. The viscosity decreasing effect of the haemodilution, coupled with the preservation of oxygen carrying capacity, could find clinical applications in such diverse situations as carotid endarterectomy surgery and sickle cell crisis, both of which may benefit from decreased whole blood viscosity and increased oxygen carrying capacity of the plasma. 5. Conclusion The potential applications for recombinant human haemoglobin have yet to be completely defined. However, current transfusion practices do not eliminate risk from allogeneic transfusions; the use of recombinant haemoglobin would have negligible infectious risk. In its current formulation, human haemoglobin somatogen is covalently cross-linked internally, making chemical additions or modifications unnecessary and extending plasma half-life to hours. Further genetic engineering has obviated chemical alterations to change oxygen affinity; indeed, oxygen affinity is sufficiently low to allow transport and offloading of oxygen under physiological conditions. The current formulation’s use is likely to be limited to perioperative blood replacement owing to the short plasma half-life; potential benefits may also include induction of erythropoiesis as a result of haeme moieties available as a product of human haemoglobin somatogen metabolism. The prospect of subsequent development of better haemoglobins appears excellent, given the genetic technology used in the synthesis of the current human haemoglobin somatogen and the increasing knowledge of oxygen-haemoglobin interactions. References 1. Winslow RM. Hemoglobin-based red cell substitutes. Baltimore (MD): The Johns Hopkins University Press, 1992

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Correspondence and reprints: Dr Bruce J. Leone, Department of Anesthesiology, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA. E-mail: [email protected]

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