Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers

Advanced Drug Delivery Reviews 40 (2000) 153–169

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Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers c

a

a,b,c ,

Chad R. Haney B.S. , Paul W. Buehler Pharm. D. , Anil Gulati M.D., Ph.D. b

*

a Department of Pharmaceutics and Pharmacodynamics, The University of Illinois at Chicago, Chicago, IL 60612, USA Department of Rehabilitation Medicine and Restorative Medical Sciences, The University of Illinois at Chicago, Chicago, IL 60612, USA c Department of Bioengineering, The University of Illinois at Chicago, Chicago, IL 60612, USA

Received 5 October 1999; accepted 1 November 1999

Abstract The efficacy of blood substitutes, as a whole, has been readily demonstrated, in animals as well as clinical studies. It is well known that stroma free hemoglobin (SF-Hb) is very toxic, due to effects on renal and coagulation functions and vascular tone. Several modifications have been made to the hemoglobin tetramer in an attempt to eliminate its toxicity. Conjugation, cross-linking, polymerization, and recombinant technology have all been used to reduce toxicity, while aiming to optimize the therapeutic value of hemoglobin based blood substitutes. The remaining issue seems to be the hypertensive response seen in many hemoglobin solutions. The cause of the hypertensive response, and hence what chemical modifications are suitable to alleviate it are still under debate.  2000 Elsevier Science B.V. All rights reserved. Keywords: Blood substitutes; Cross-linking; Polymerization; Hypertensive drug effects

1. Introduction When considering the danger of blood-born pathogens, the diminishing donors for blood banks, and other limitations of blood transfusion, the necessity for the development of a blood substitute (oxygen carrier) is clear. Several hemoglobin based oxygen carriers (HBOC) are currently under clinical trials. Numerous reviews have been published on the biological actions and overall development of HBOC’s. Different laboratories have used different *Corresponding author. Tel.: 11-312-996-0826; fax: 11-312996-0098. E-mail address: [email protected] (A. Gulati)

approaches in developing their HBOC. This review will focus on isolation, purification, and properties of modified hemoglobin (Hb). The aim of developing an HBOC is to improve upon the limitations of whole blood by increasing the shelf life and eliminating immune response (no blood typing necessary), while being economical to produce. Additionally, transport of oxygen and carbon dioxide should be similar to that of red blood cells (RBC). Numerous purification procedures and chemical modifications of hemoglobin have been carried out to reduce toxicity and improve the efficacy of HBOC’s. The blood substitutes that are currently in clinical trials are predominantly hemoglobin based. This review will focus on the purifica-

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tion and chemical modifications of hemoglobin in order to develop hemoglobin based blood substitutes.

1.1. Unmodified, partially purified Hb, stroma free Hb ( SFHb) In 1934, Amberson et al. introduced lysed red blood cells into hemorrhaged cats. This crude RBC lysate demonstrated that hemoglobin could be used as an oxygen carrying agent, very effectively [1]. By 1967 it was determined that even trace amounts of stroma lead to anaphylactic shock [2]. Savitsky et al. [3] demonstrated that an unmodified pure Hb solution used as a blood substitute, results in severe toxicity (marked increase in blood pressure, bradycardia, impaired coagulation and renal functions) [3]. Initial preparations of hemoglobin from human red blood cells produced many adverse effects, such as anaphylactic reactions, renal malfunction and vascular coagulation [1,4]. Vascular coagulation was most likely the result of stroma left in the hemoglobin preparation. Anaphylactic reactions we now know are caused by lipopolysaccharides (LPS) bound to the Hb molecule. While kidney malfunction is likely a direct result of Hb toxicity due to tetrameric Hb degradation into dimers, which are able to lodge in the distal tubules of the kidneys and result in renal failure.

2. Purification

2.1. Washing, lysing, and centrifugation Before lysing, RBC’s are typically washed three times with isotonic saline (0.9% NaCl) or isotonic phosphate buffered saline (PBS). Washing with either of these isotonic solutions helps remove plasma proteins, leukocytes, and platelets. RBC’s generally have an anticoagulant added. If heparin is used as an anticoagulant, it must be removed prior to lysing, as it is a polyanionic polysaccharide, with a high affinity for basic proteins like hemoglobin [5]. The most common method of extracting hemoglobin from washed RBC’s is by lysing with cold distilled water. Lysing is typically run over night, at 208C. However, for small volumes (one unit or less), 1 h is sufficient. Keeping the lysing solution cold

helps minimize the auto-oxidation of hemoglobin to methemoglobin (metHb). Freezing alone can be used for lysing; however, it is not as efficient. Hypertonic salt solutions may also be employed, especially in a gradual fashion such that the cell membrane swells, rather than rupturing. Swelling the membrane and allowing the hemoglobin to gradually leak out, while keeping the cell membrane intact, reduces the work necessary to completely separate the hemoglobin from the stroma. In analytical preparations and older publications, toluene or carbon tetrachloride was often used to extract hemoglobin from the cell membrane. Because they are toxic, carcinogenic solvents they are best avoided when preparing hemoglobin as a blood substitute. In addition, toluene can be trapped in the hydrophobic pocket of (Hb) and disrupt spectrophotometric measurements [5]. The lysate is centrifuged for 20 000 3 g for 1 h. Centrifugation sediments the membrane particles, while keeping the hemoglobin in the supernatant. Adding 0.1 M MgCl 2 can help adjust the density of the medium, to enhance the sedimentation of the membrane particles. As an extra precaution, the supernatant should then be filtered with a 0.22 mm filter.

2.2. Dialysis or ultrafiltration Dialysis or ultrafiltration can be used to further remove any cellular debris. Dialysis can be used alone to gradually swell the RBC’s as mentioned earlier and to deoxygenate Hb, which is the preferred state, as will be discussed later. In addition, dialysis is utilized to slowly polymerize Hb with glutaraldehyde. However, strong anion exchange is the most common and a more effective means to complete the isolation of hemoglobin from stroma, enzymes, and hemoglobin variants, most importantly methemoglobin. Although a pH gradient elution will work, a NaCl gradient is more commonly used. Therefore, the supernatant of the lysate needs to be de-salted. De-salting or buffer exchange can be done using ultrafiltration, dialysis, or gel permeation chromatography. Furthermore, the pH of the (Hb) solution needs to be adjusted as well because methemoglobin tends to form rapidly at pH below 7.0. Similarly, the hemoglobin should be concentrated by either dialysis

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or ultrafiltration, as this tends to slow down the auto-oxidation process [6].

2.3. Chromatography Many commercial strong anion exchange preparative columns and media are available, e.g., Waters QMA Acell plus [7], Pharmacia Fine Chemical DEAE Sephadex [8,9], Whatman DE-52 [10]. A 0.05 M Tris–HCl buffer, pH 8.5, is commonly used with a salt gradient of 0 to 0.1 M NaCl. For enhanced separation, pH and salt gradients can be combined, however, a salt gradient alone is usually sufficient. By the use of a salt gradient alone at a pH above 7.4, the production of methemoglobin is reduced, while lowering the pH (6.5 to 7.4) or increasing the ionic strength elutes hemoglobin variants (the main variant being HbA o ). At one point, mixed bed ion-exchange resin (Bio-Rad Bio-Rex RG501-X8) was used to produce an iso-ionic Hb solution, it was later determined that this was an unnecessary step in the process and, therefore abandoned. The advantage of using chromatography is its high selectivity using non-denaturing conditions. Another important consideration for using chromatography is to effectively scale up to commercial volume, unlike some of the other techniques, which make scale up processes difficult [11]. The preparation of stroma and endotoxin free human hemoglobin in adequate quantities for further modification were developed separately by Sheffield, Williams and Winslow [11–13]. Post purification Hb solutions are typically stored in sterile, disposable containers at 2 708C under nitrogen and / or bound with CO to minimize oxidation.

2.4. Pasteurization The need for heat pasteurization is self-evident. Cross-linking and polymerizing hemoglobin stabilizes the protein, such that heating to 74–768C for 90 min or 608C for 10 h will not denature the protein. Hemoglobin that is not cross-linked will denature and precipitate out, making it easy to remove ‘‘unstable’’ Hb. Thus, this increases the safety of the Hb solution. Carbon monoxide (CO) can be utilized at this stage to reduce methemoglobin formation, by displacing oxygen. Bubbling oxygen or air through the CO-Hb solution in direct light removes carbon

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monoxide to return oxy Hb [14]. Bucci et al. flush their Hb product with nitrogen while warming the solution to 408C for 1 h. Contaminants are precipitated out by further raising the temperature to 70– 758C for an additional 3 h after adding sodium hydrosulfite [15].

3. Physical properties

3.1. Reduce O2 affinity 3.1.1. Hill coefficient and P50 The ability of Hb to shift between conformations is reflected by the Hill coefficient (n). A coefficient of 2 or more indicates cooperative oxygen binding between subunits through conformational change, while a coefficient around 1 indicates that Hb is locked in either the T or R conformation, i.e., an absence of co-operative oxygen binding. 2,3-diphosphoglycerate (2,3-DPG) gives Hb, inside the RBC’s, allosteric co-operativity, in vivo. The P50 value is the oxygen half saturation pressure. The P50 of normal human blood is approximately 27 mm Hg, thus the goal of an HBOC is considered to be in this range. This may or may not be the desired situation for HBOC. False P50 values can often be obtained by elevated methemoglobin ( . 10%) in solution. Measurement at 378C will cause a precipitation of methemoglobin ( . 10%) and alter the passage of light though the sample cuvett resulting in false absorbances of oxyHb and a left shifted oxygen saturation curve. 3.1.2. Bovine and recombinant hemoglobin Hb contained within the RBC and stroma free Hb differ greatly in their affinities for oxygen (P50 ) as well as their abilities to release oxygen for use at tissue sites (Hill coefficient). The main reason for these differences is due to the function of 2,3-DPG. Found inside RBC’s, 2,3-DPG stabilizes Hb (by binding to the b1 Lys 82 and the b2 Lys 82) in the deoxy form in the peripheral vasculature, where oxygen concentration is low, and releases from it’s binding site in the lungs, where oxygen binding is required. 2,3-DPG has a specific binding cleft within the tetrameric Hb molecule known as the anion binding site and essentially prevents the conforma-

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tional change between the a1b1 subunits which leads to cooperative O 2 binding. Thus, stroma free Hb as well as some cross-linked Hb bind O 2 more readily and prevent O 2 release. In the RBC, Hb exists in two distinct states or conformations termed T and R. The T state is Hb’s deoxy form while the R state represents the oxy form of Hb. When oxygen binds in the T state there is a conformational change in the Hb molecule to the R state (deoxy to oxy). HBOC’s are created to mimic these values and attempt to provide oxygen in a fashion as close to whole blood as possible. However, because these are acellular formulations, it may be imprudent to try to reach the same values as RBC’s, due mainly to differences in size. Cross-linking agents available attempt to stabilize the Hb molecule in the T state (deoxy form). The use of bovine and recombinant Hb have been evaluated regarding O 2 affinity and dissociation. These options afford other notable benefits such as lack of viral content, consistent supply, and cost effectiveness. Bovine Hb is a logical choice when considering reducing oxygen affinity. It is well known that bovine red cells have low levels of 2,3-DPG, yet have an oxygen affinity similar to that of human Hb [16]. Therefore, acellular bovine Hb would have an oxygen affinity much lower than human Hb. This appears to be true especially in the presence of chloride ions [17]. Chloride ions take the place of 2,3-DPG in bovine RBC’s, providing allosteric cooperativity. Much of the work with bovine Hb centers on macromolecular conjugation to increase plasma retention. Recombinant Hb’s also afford the possibility of lowering oxygen affinity due to the virtually limitless alterations in amino acids, which can be obtained. These modifications (without the aid of chemical cross-linking agents) can have profound effects on the genetically modified Hb’s ability to bind and release oxygen [18,19]. Several groups have also developed methods to produce human Hb by bacterial and animal transgenic models [19,20]. Thus, deriving a pure and safe Hb for chemical modification is used routinely on a large-scale basis.

3.1.3. Pyridoxilation Pyridoxilated Hb’s mimic the binding of 2,3-DPG by cross-linking in the anionic site. Theoretically, this should improve oxygen tissue delivery. How-

ever, many of the pyridoxilated compounds have P50 values well over whole blood (the effect of which is not certain). McGarrity et al., using analytical grade anion exchange chromatography, showed that five species consistently form with pyridoxal 59 phosphate, each of these subspecies having a different oxygen affinity [21]. This heterogeneity could make it difficult to characterize pyridoxylated Hb’s.

3.2. NO binding, scavenging Hemoglobin’s affinity for nitric oxide (NO) is approximately 3000 times greater than for CO [22]. Rohlfs et al. dispute the importance of the effects of NO binding affinity on the hypertensive response seen in HBOC’s. This group examined six various Hb’s (HbA o , aa-Hb, Tm-Hb, b82 Hb, PHP, PEG-Hb and o-Raffinose Hb) and found no correlation between the hypertensive response and rates of reaction with NO. However, they did find an inverse correlation between NO affinity and the hypertensive response [23]. This study evaluated many products that do not readily cross the endothelial layer, therefore these compounds do not bind NO to an extent great enough to elicit a physiologic response (regardless of a high affinity for NO). Their work demonstrates that it is important to consider the complexities of the hypertensive response caused by HBOC’s.

3.3. Free radical Chemical modifications not only affect O 2 and NO affinity, but also redox properties. Alayash et al. have shown that there are differences in free radical reaction kinetics between bb cross-linked Hb using fumaryl-monodibromoaspirin (FMDA-Hb) which is similar to bis-3,5-dibromosalicyl fumarate and polymerized / cross-linked with glutaraldehyde. They suggest that glutaraldehyde polymerization has a higher redox potential due to a greater accessibility of the heme pocket compared to aa cross-linked [24]. Motterlini et al. suggests that cross-linking increases the rate at which heme is released [25]. Similar work by Rogers et al. showed that aa cross-linked Hb using bis-(3,5-dibromosalicyl) fumarate (that was also polymerized with glutaraldehyde) had a higher auto-oxidation rate than the aa cross-linked Hb.

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Although the NO binding kinetics are similar between these two modified hemoglobins, the NO induced reduction rates appear to be different [26]. OxyHb readily undergoes spontaneous auto-oxidation to from metHb. Superoxide and hydrogen peroxide (H 2 O 2 ) form from metHb. OxyHb can react with H 2 O 2 to form ferryl-Hb (Fe IV). Ferryl heme is toxic because it can peroxidize lipids and cross-link some proteins. Normally in the RBC, catalase or glutathione peroxidase removes the H 2 O 2 as oxygen and water. Also in the RBC, superoxide is dismuted to oxygen and H 2 O 2 by superoxide dismutase (SOD). Considering that we have been discussing acellular Hb here and that neutrophils can generate sufficient amounts of H 2 O 2 , it is clear that one must be aware of changes in redox properties due to chemical modifications to hemoglobin. Similarly macrophages can be a source of superoxide. Free iron, from heme released from modified Hb, combines with superoxide generated from an immune response leading to the Fenton reaction. The redox based toxicity of Hb is due to the Fenton reaction continuously producing very toxic hydroxyl radicals.

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3.4. Oncotic pressure

Superoxide reduces the ferric iron back to the ferrous form.

The current literature on HBOC’s rarely discusses hemodynamic effects caused by colloid osmotic pressure (COP) properties. COP is a linear function of the number of colloidal particles (up to protein concentration of 10 g / dL). However, polyethylene glycol (PEG) conjugated Hb appears to have a higher apparent COP. This may be due to PEG conjugated Hb having a higher Einstein-Stokes radius compared to a substance of comparable molecular weight, e.g., o-raffinose polymerized Hb [28]. The normal COP of plasma is between 20-25 mm Hg. An increase in COP above normal levels will lead to water leaving the interstitium and entering the vascular space [28,29]. Gould and Moss suggest that Hb solutions at 15 g / dL would increase the COP by 300%, shifting the water balance [30]. One of the advantages of using a colloidal solution in place of a crystalloidal solution, is that edema (which is particularly dangerous in the lungs) can be avoided. Crystalloidal solutions require roughly three times the volume of colloidal solutions to maintain systemic hemodynamics. If the colloidal solution can extravasate, then interstitial edema may be a problem. However, this was not seen in a hemorrhage model, where Hein et al. compared resuscitation with crystalloids to colloids [31].

O ?2 1 Fe 31 → Fe 21 1 O 2

3.5. Viscosity

Not only is it necessary to consider changes in redox potential due to chemical modifications, but storage conditions as well. As mentioned earlier, Hb solutions are stored at temperatures colder than 2408C to slow the rate of metHb formation. Svistunenko et al. demonstrate that not only is the 2408C storage temperature important, but freezing and thawing cycles can increase the rate of superoxide formation in whole blood [27]. It is likely that the same is true for Hb solutions if there is a source for H 2 O 2 . If however the benefits of chemical modifications outweigh the increased redox potential, antioxidants / reducing agents such as ascorbate, uric acid, and deferoxamine (an iron chelator), thiourea and mannitol (hydroxyl radical scavengers) could be coadministered to offset the redox based toxicity.

Endothelial cells try to maintain a constant wall shear stress by increasing the diameter when the flow rate increases. The vasculature dilates in response to increased flow (WSS) and constricts when the flow (WSS) is reduced. This should not be confused with the myogenic response, which is based on stretch receptors that respond to changes in blood pressure. Therefore, when chemically modifying hemoglobin for use as a blood substitute, one must be aware of the changes in viscosity of blood due to the modified Hb solution. If one looks at viscosity alone, then a blood substitute with a viscosity slightly higher than that of plasma would be desirable in that it could reduce vasoconstriction (mechanically) during resuscitation. By comparing aa cross-linked Hb with dextran 70, at two different hematocrit, Tsai et al. have shown that non-oxygen carrying (viscous and

H 2 O 2 1 Fe 21 → Fe 31 1 OH 2 1 OH ?

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colloidal) solutions (dextran 70) may be more effective at a less severe hemorrhage, while an O 2 carrying agent would be necessary at higher degrees of hemorrhage [32]. Their work along with data from Winslow’s group suggest a paradox, where a high P50 delivers too much O 2 and stimulates an autoregulatory response that leads to vasoconstriction [23,32–35]. In practice one must also be concerned with NO binding, preventing renal toxicity, redox potential, O 2 affinity, and possibly other factors that have yet to be identified.

4. Chemical modifications Modification of Hb began with cross-linking of the tetrameric subunits of the molecule with a variety of linking agents. It is important to note that most cross-linking modifications involve the formation of a Schiff base. Increasing the molecular weight of Hb with agents that both cross-link and polymerize soon followed, as other undesirable effects of merely cross-linked Hb surfaced. As we will discuss later, the drive to chemically modify cross-linked and polymerized Hb’s with addition of PEG, dextrans, free radical scavengers and recombinant methods to reduce Hb’s affinity for nitric oxide (NO) leads us into a new era of HBOC’s known as second generation HBOC’s. Cross-linking Hb alters its oxygen binding affinity and subsequently Hb’s ability to deliver oxygen to tissues, therefore, one goal of cross-linking Hb is to optimize oxygen binding and tissue delivery. While oxygen carrying capacity is of debatable importance (essentially depends on the therapeutic indication of the modified Hb), improving vascular retention time and protecting the kidneys from the toxic effects of Hb dimers is essential and the main goal of crosslinking. The Hb tetramer at a MW of 64 kDa cannot be filtered though the glomerulus of the kidney, however, the tetramer readily dissociates into dimers (a1b1) with a MW of 32 kDa. Dimeric break down products of Hb can undergo glomerular filtration and result in renal toxicity via deposition in the distal tubules. Metabolism by the liver and excretion in the bile is a much more desirable mode of elimination.

Therefore, the increase in retention time will shift the clearance from the kidneys to liver metabolism and clearance by the reticulo endothelial system (RES). Speculatively, this route of elimination could be facilitated more readily by linking a modified (Hb) to albumin. Hb plasma elimination via the RES may be a more desirable mode of elimination from a standpoint of safety, however, the extent to which the RES is involved in the removal of modified Hb from the plasma is unknown. Elimination of Hb and modified derivatives by the RES may also be dependent on overall molecular charge [36]. The more net negativity a molecule possesses the less likely it is to cross the endothelium and be removed from the vascular space or be scavenged by cells of the RES. The use of cross-linking agents, therefore, increases vascular retention time and improves safety over SFHb by preventing renal elimination. There are many possible sites which linking agents can bind on the Hb tetramer [37], however, there are essentially 2 sites where cross-linking occurs. The first is between the b globin chain’s lysine 82 residues resulting in bb cross-linked Hb. The second site occurs between the a globin chains at the lysine 99 residues and results in aa cross-linked Hb. The cross-linking agents most cited in the literature include; salicyl derivatives ((bis-3,5-dibromosalicyl) fumarate, sebecate, succinate and glutarate), benzenecarboxylate derivatives (benzenepentacarboxylate (BPC), benzenetetracarboxylate (BTC)), pyridoxylphosphate derivatives (bis-pyridoxyl tetraphosphate (bis-PL)P4) and 2-nor-2-formylpyridoxal 59-phosphate (NFPLP)) as well as recombinant modifications in Hb amino acids which result in fusion of Hb aa chains. Many other linking agents have been tried; however, they provide little insight into in vivo effects. Agents such as o-raffinose and glutaraldehyde are also effective linking agents, but in addition polymerize Hb molecules. PEG and dextran react with Hb on the exterior of the tetramer to increase MW, while polyoxyethelene (a modified PEG in the form of a-carboxymethyl-v-carboxymethoxylpolyoxyethylene prior to reaction with Hb) acts to polymerize Hb. Many of these modifying agents currently are used in products being tested in various phases of clinical trials. See Tables 1 and 2 below for a summary of chemical modifications and their resulting properties.

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Table 1 A summary of linking sites and linking agents used in developing HBOC’s Modification (agent)

Globin links

AA’s involved

bis-3,5-dibromosalicyl Fumarate bis-3,5-dibromosalicyl Sebecate bis-3,5-dibromosalicyl Succinate bis-3,5-dibromosalicyl Glutarate Benzene (penta / tetra)carboxylate (bis-PL)P4 2-nor-2-formylpyridoxal 59-phosphate Glutaraldehyde o-Raffinose Polyetheleneglycol Polyoxyethylene rHb1.1 rHb0.1 rHb2.0 rHb3.0 rHb4.0 Polynitroxyl Mellitic dianhydride

aa

a1 99 Lys-a2 99 Lys

bb

b1 82 Lys-b2 Lys 82

bb

b1 82 Lys-b2 Lys 82

bb

b1 82 Lys-b2 Lys 82

aa,ab,bb

N/S

bb bb

b1 N ter Val-b2 Lys 82 b1 N ter Val-b Lys 82

N / S (CL), N / S(Polymer) bb (CL), N / S (Polymer) None (external binding) None (Polymerization) aa None aa aa aa N / S Binding (b1,2),aa glob. N / S (CL) possible b1b2

e-(NH 2 ) Lysine Groups b1 82 Lys-b2 82 Lys External e-(NH 2 )Lys External e-(NH 2 )Lys b Asn 108→Lys b Asn 108→Lys a11 Phe / b11 Phe / b8Ile a11 Leu / b11 Phe a11 Phe / b11 Trp e-NH 2 Lys 6aa,10b1,2 e-(NH 2 ) Lysine Groups

AA5amino acid; N / S5non-specific target; CL5cross-link.

4.1. Cross-linking 4.1.1. Salicyl derivatives Salicyl derivatives are linking agents which bind in the 2,3-DPG binding cleft of oxy Hb (b1 82 Lys-b2 82 Lys) and at the a1 Lysine 99-a2 Lysine residues of deoxy Hb [38,39]. The most prominent of the salicyl derivatives used is (bis-3,5-dibromosalicyl) fumarate, which links Hb’s aa subunits at the amino groups of the lysine 99 residues in the presence of tripolyphosphate. The resulting product is Hb with a 4 carbon fumaryl bridge between the aa subunits. The product known as DCLHb (Hemasist  ) possesses a half-life of between 12 and 20 h in humans with a molecular weight of 64 kDa. The Oxygen affinity and Hill coefficient of this compound are 32 mm Hg and 2.6–2.8 respectively [40,41]. Most notably from a pharmacodynamic perspective, regarding DCLHb, is its hypertensive response. The proposed mechanisms of vasoconstriction caused by DCLHb include; NO scavenging,

elevation of plasma endothelin levels, sensitization of adrenergic receptors, and the paradoxical autoregulatory response to high O 2 delivery [23,42– 46]. The vasoconstriction of peripheral vascular beds by DCLHb limit its use as a viable blood substitute. Co-administration with anti-hypertensive agents appears to reduce the hypertensive response exhibited by this compound [47,48]. In addition, polymerization of DCLHb to reduce the hypertensive effect has been attempted. By increasing the MW of DCLHb the likelihood of extravasation across the endothelial layer would theoretically be reduced due to the size of the resulting polymer. The studies performed indicate that polymerized DCLHb’s hypertensive response is reduced in isovolemic states, however, the hypertensive effect is not reduced in hypovolemic states. These findings indicate that in hypovolemic states, such as hemorrhagic shock, damage to the endothelial layer may occur, which could allow even polymeric DCLHb to cross the endothelium, scavenge NO and cause significant

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Table 2 A comparison of properties of HBOC Name

Linking agent

MW [kDa]

P50 [mmHg]

Hill (n)

COP [mmHg]

References

HbA 0 HbBv SFHb NFPLP-Hb (bisPL)P4-Hb PLP-Hb DECHb a DBBF-Hb b DBBF-Hb DCLHb BMAA-PEGHb

– – – 2-nor-2-formylpyridoxal 59-phosphate bis-pyridoxal tetraphosphate pyridoxal 59-phosphate bis(3,5 dibromosalicyl) sebecate bis(3,5-dibromosalicyl) fumarate bis(3,5-dibromosalicyl) fumarate bis(3,5-dibromosalicyl) fumarate bis(maleoylglycylamide) polyethlene glycol, bis(3,5-dibromosalicyl) fumarate fumaryl-monodibromoaspirin glutaraldehyde glutaraldehyde, 2-nor-2formylpyridoxal 59-phosphate glutaraldehyde, pyridoxal 59-phosphate glutaraldehyde, bis(3,5dibromosalicyl) fumarate glutaraldehyde, recombinant glutaraldehyde, recombinant glutaraldehyde, recombinant glycolaldehyde, bis(3,5dibromosalicyl) fumarate glycolaldehyde, pyridoxal 59-phosphate methoxypolyoxyethlene glycol methoxypolyoxyethlene glycol, pyridoxal 59-phosphate a-carboxymethyl, v-carboxymethylmethoxypolyoxyethylene, pyridoxal 59-phosphate Recombinant Recombinant Recombinant Recombinant ring opened raffinose trimesoyl tris(methyl phosphate) trimesoyl tris(methyl phosphate)

64 64 64 64 64 64 N /A 64–67 64 64.5 320–640

8–18 17.7 12–24 45–47 31 11–30.5 34 26–33.9 5 32–36 20

2.6–3.0

23.3–45

2.8 2.1–2.2 1.2–2.3 2.2 2–2.45 N /A 2.4 1.7

18–41.9 27 N /A 23.3 23.7 23–35.1 N /A N /A N /A

[26,60,67,83–85] [83,86] [29,30,63,87] [4,60,88,89] [4] [4,6,85,90] [15] [4,23,24,26,35,63,65,83] [24] [91,92] [26,93]

N /A N /A 140–600

35–36.2 12.8 20–22

N /A N /A 1.3

N /A N /A 9–14

[24,83] [24] [60,89]

64–750

12–29.9

1.2–2.2

20–27.75

[4,6,29,30,67,68,79,87,94]

N /A

28–35

2–2.3

N /A

[41]

64–128 64– .1000 128– .1001 N /A

29.9 29.4 35.8 30

1.4 1.5 1.3 1.8–2.2

N /A 11 11 N /A

[79] [79] [79] [84]

32–500

30–34

1.6–1.8

25

[84]

117–123 90–100

10.2 11.3–21

1.4 1.6–1.7

118 27

[23,35,86] [85,94]

97

20.4

1.64

N /A

[23]

64 64 64 64 64–576 65 65

32 4 3.4 5.2 27–52.6 15.4 38.7

2.2 1.8 1.6 1.4 1–1.6 2.04 2.8

42 N /A N /A N /A N /A N /A N /A

[79] [18] [18] [18] [4,23,84] [23] [23]

FMDA-HbBv PolyHbBv PolyNFPLP PolyPLP-Hb GP-DCLHb mono-glxrHb Poly1-glxrHb Poly2-glxrHb PolyDBBF PolyPLP-Hb PEG-HbBv MPOE-PLP-Hb PHP-Hb

rHb1.1 rHb2 rHb3 rHb4 o-raffinose-Hb b82-Hb Tm-Hb

vasoconstriction [49]. The remaining salicyl derivatives are not commonly used; therefore, we will simply mention them in brief. (bis-3,5-dibromosalicyl) sebacate forms a 10 carbon bridge linking the two b chains of oxy Hb at the 2,3-DPG binding cleft [50]. The succinate and glutarate forms

of bis-3,5-dibromosalicylic acid have also been used to link Hb chains with attempts to increase thermal stability and alter oxygen binding [51]. The relatively tight structure of the fumarate cross-linked Hb seems to produce an overall more stable molecule by reducing flexibility. Therefore, of the salicyl deriva-

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tives, (bis-3,5-dibromosalicyl fumarate) is the most common linking agent.

4.1.2. Benzenecarboxylate derivatives The carboxyl groups of benzenepentacarboxylate (BPC) and benzenetetracarboxylate (BTC) can be activated by the use of (1-ethyl-3-(-3-dimethylaminopropyl)-carbodiimide (ECD) to give Oacylurea at each carboxyl functional group. When an amine is present the result is the formation of a covalent amide bond (a pseudo peptide bond) [52]. This activation is necessary to prevent the formation of non-covalent salt bridges. The cross-linking is pH dependent and results in aa, ab and bb combinations of chain links. The resulting reactions yield Hb stabilized in either oxy or deoxy form with P50 values ranging from 5 to 14 mm Hg, depending on the linking conformation [52]. Thus having profound effects on oxygen carrying capacity and Hill coefficients. Most studies evaluate BPC and BTC conjugated to dextran or another type of volume expanding agent [53–55]. The basis is to maintain plasma volume and tissue perfusion and maintain the ability to deliver oxygen to tissues. There are currently no products in clinical trials involving benzenecarboxylate derivatives as cross-linking agents. 4.1.3. Pyridoxal phosphate derivatives The pyridoxal phosphate derivatives cross-link the tetrameric Hb molecule via bb globin chain linkage. The linking agent bis-pyridoxal tetraphosphate (bisPL)P4 contains two phosphate groups linked between two 59-pyridoxal phosphates [16]. The basis for the use of the pyridoxal phosphate derivatives is to mimic the effects of 2,3-DPG. It is important to emphasize that pyridoxal phosphate alone can not accomplish this and must be modified with additional phosphate groups. Reaction with Hb results in a bb linkage between the N terminal Val of the b1 chain and the Lys 82 of the b2 chain. The pyridoxal groups stack in the 2,3-DPG binding cleft, which seems to protect the molecule from phosphate bond hydrolysis [16]. Synthesis of (bis-PL) P4 is performed by pyridoxal modification described by Michelson [56] and Fukui [57]. The reactions are highly light and oxygen sensitive and must be run in a light resistant apparatus. In addition reagents must be treated with nitrogen or argon gas prior to use

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[16]. The reaction yields approximately 70% crosslinked Hb with (bis-PL)P4 as a linking agent while removing one phosphate (bis-PL)P3 yields only 15%. The (bis-PL)P4 cross-linking reactions have a profoundly higher yield than the diasprin linking agents, which yield cross-linked Hb at about 15%. The advantages of the pyridoxal derivatives as linking agents are (1) synthesis is a two step process and can be completed in three days (including purification), (2) the reaction is very specific unlike the benzenecarboxylates which are pH dependent and give linkage at various amino acids on various globin chains (this is also true of glutaraldehyde), (3) chemical entities can be linked to the pyridoxyl 59-phosphates (this could be a novel way to produce second generation agents), and finally (4) oxygen affinity is reduced and O 2 unloading is improved thus resulting in potentially more efficient O 2 delivery. The O 2 affinity is nearly double that of whole blood at approximately 40 to 45 mm Hg at 378C [16]. The vascular retention of (bis-PL)P4 is also substantially improved over stroma free Hb. In pharmacokinetic studies involving rats given a bolus dose of 200 mg / kg, the half-life is 3 h compared to the same dose of stroma free Hb with a half-life of 1 h [58]. Perhaps most importantly, no a1b1 dimers were detected in the urine of rats treated with the same 200 mg / kg dose after 5 h of collection, while 21% of the total dose of stroma free Hb was detected in the urine after 2 h of collection. Preliminary pharmacodynamic studies in rats indicate that at a dose of 200 mg / kg heart rate and mean arterial pressure remained stable or returned to normal within hours after bolus injection [58]. We should note that hemoglobin based blood substitutes exhibit dose dependent pharmacokinetics, in addition elimination is highly dependent on the type of crosslinking agent used and seems to vary widely between species. 2-Nor-2 formyl pyridoxyl 59-phosphate (NFPLP) is another commonly used pyridoxal phosphate derivative for Hb cross-linking. NFPLP is essentially pyridoxyl 59-phosphate with a formyl group at the pyridoxal rings 2 position. Much like (bis-PL)P4 the Hb b globin chains are linked via b1 N-terminal Val and the b2 Lys 82 residue. The preparation of NFPLP is via the method by Pocker [59] which is again light and O 2 sensitive. The reaction to form

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Hb(NFPLP) yields 65% just slightly less than Hb(bis-PL)P4. The O 2 affinity for Hb(NFPLP) is approximately 40 to 45 mm Hg at 378C. The improvement in vascular retention appears to be very similar to that of Hb(bis-PL)P4 with a half-life of 3 h for Hb(NFPLP) and 1 h for Hb, in one particular study a volume exchange was performed with a 7% solution in a rat model (therefore, the dose was greater than the dose of (bis-PL)P4 Hb in the kinetic evaluation performed by Kiepert et al.) [60]. The same study was performed in rabbits and resulted in a half-life of 7 h for Hb(NFPLP) and 2.5 h for Hb [61]. In rats, the renal excretion of Hb(NFPLP) showed 5% of the dose as Hb in the urine after 5 h as compared 35% in the Hb treated animals indicating that at least in the first 5 h the pyridoxal phosphate derivatives prevent renal excretion and increase vascular retention. This should then lead to metabolism in the liver with excretion in the bile. The same group of researchers established that dimers were not being trapped in the kidneys by radio labeling Hb(NFPLP) with Technecium 99 and quantitating the radioactivity in the kidneys after 24 h of low dose Hb(NFPLP)Tc99 [62]. From the animal pharmacokinetic data which exists on Hb(NFPLP) human kinetic data has been extrapolated to give a half-life of approximately 10 h. There is concern that a 10 h half-life would not be adequate for situations such as hemorrhagic shock. To increase vascular retention of Hb(NFPLP) even further, polymerization with glutaraldehyde has been attempted [60]. Two molecular weight ranges were evaluated in a rat model; 140 to 170 kDa (2–3 Hb linked) and 220 to 380 kDa (4–6 Hb linked). The half-life of the 140 to 170 kDa range was 11.4 h while the 220 to 380 kDa range resulted in a t 1 / 2 of 15.3 h. Thus not increasing the vascular retention much more than the extrapolated human values [60]. Pyridoxal 59-phosphate derivatives are excellent linking agents and are used in 2 products in clinical trials; Northfield Laboratory’s (Polyheme  ), which is a pyridoxylated and glutaraldehyde polymerized HBOC and Apex Corporations pyridoxylated hemoglobin polyethylene (PHP). As mentioned earlier the fact that several modifications can be made to the bis-PL derivatives, it is conceivable that prodrugs may be added to the two pyridoxal groups and lead us to useful second generation HBOC’s.

4.2. Increasing molecular weight The toxicity of hemoglobin is primarily due to dimers and monomers causing renal failure and extravasation of hemoglobin, which binds to NO, causing severe hypertension, as discussed earlier. Again, cross-linking eliminates the renal toxicity but only reduces the extravasation, but does not eliminate it [63–65]. Increasing the molecular weight by polymerization or conjugation reduces the extravasation. Simoni et al. suggest that the net charge of the modified hemoglobin molecule is important in preventing extravasation [36]. Also, polymerization increases the hemoglobin concentration, iso-oncotically. Due to limitations of oncotic activity, the upper limit of stroma free hemoglobin concentration is 7 g / dL [66]. There is a limit to the amount of conjugation or polymerization, i.e. increasing the molecular weight beyond 500 kDa has been shown to be toxic. MW greater than 500 kDa has been shown to activate compliment. Also viscosity is directly proportional to molecular weight. Therefore, there is a limit to the amount of increase in viscosity, before blood flow begins to decrease [4].

4.2.1. Polymerization 4.2.1.1. Glutaraldehyde Glutaraldehyde forms inter- and intra-molecular cross-linked hemoglobin that reduces the renal clearance and extravasation. The site of reaction is thought to be at the e-amino group of the lysine residues and the N-terminal amino groups, where Schiff bases form. It is possible for glutaraldehyde to cross-link not by a Schiff base but by aldol condensation between glutaraldehyde derivatives bound to different hemoglobin molecules [67]. Due to nonspecific binding of glutaraldehyde, it is not known whether there is a preference for any one of the 44 lysine residues or of the four terminal valines. Furthermore, the intramolecular cross-links have not been defined as well, due to glutaraldehyde’s ability to react with the imidazole group of histidine, the sulfhydryl group of cysteine, and the phenolic ring of tyrosine [6,14]. Some methods involve intramolecular cross-links with 2-nor-2-formylpyridoxal 5’-phosphate or diaspirin. Glutaraldehyde polymerization must be preceded

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by pyridoxylation to increase the P50 . However this can increase the concentration of methemoglobin. Cooperativity is often lost as a result of pyridoxylation. There are many methods for polymerizing hemoglobin, each trying to optimize degree of polymerization, methemoglobin levels, P50 , etc. Some of these are conflicting, e.g., increasing the ratio of glutaraldehyde to hemoglobin, reaches a point where it begins to decrease P50 . The method described by DeVenuto and Zegna, for the polymerization of hemoglobin with glutaraldehyde after pyridoxlyation, has been examined and determined to be successful by many groups [68]. Briefly, HbA o is prepared employing the methods mentioned earlier. However, others chose to use stroma free hemoglobin (not ‘‘pure’’ hemoglobin, HbA o ), which leads to a more heterogeneous product. HbA o is pyridoxylated to increase the P50 to 24–29 mm Hg. Excess glutaraldehyde is mixed with hemoglobin, directly or by utilizing dialysis. Glutaraldehyde should be added slowly, to reduce gel formation. The reaction can be monitored by observing changes in COP. The reaction is typically quenched with excess lysine to prevent free aldehyde groups. At this point the polymer is not stable, even when utilizing excess lysine. Kothe et al. have stabilized the polymer by irreversibly reducing the Schiff bases with sodium borohydride [69]. Glutaraldehyde polymerization and cross-linking does not appear to be sensitive to pH or temperature. Bleeker et al. report that glutaraldehyde itself is toxic, even after using sodium borohydride to stop the reaction [70]. They slowly added a 30:1 molar ratio of glutaraldehyde (Sigma, grade I) to deoxyhemoglobin (heterogeneous mixture). The molecular weight range of the polymerized Hb was from 64 kDa to 500 kDa. They thought that the toxic effect maybe due to residual cell membrane. Therefore, they used the same cross-linking and polymerization steps with HbA o and human serum albumin to test glutaraldehyde. Glutaraldehyde cross-linked and polymerized HbA o and albumin showed the same hemorrhagic disorder as seen with the polymerized heterogeneous Hb [71]. Marini et al. found that even using HbA o and reducing with NaCNBH 3 , led to a heterogeneous product. Furthermore, this heterogeneous mixture was shown to be unstable. After four weeks, the

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chromatogram from an analytical grade anion exchange column showed significant changes [6]. Lee et al. examine the effects of a glutaraldehyde polymerized HBOC in an ovine model and found systemic and pulmonary vascular resistance increased 43.9% and 204.2%, respectively, after HBOC infusion. Interestingly, the formation of methemoglobin was significant, accounting for 33.0% of the plasma hemoglobin at 24 h [72]. Based on these difficulties, it will be interesting to see how Northfield Laboratory’s Polyheme  does in their clinical trials.

4.2.1.2. O-raffinose There are two methods using o-raffinose as a cross-linking and polymerizing agent. Carleton Hsia uses non-specific binding, but controlled by time and stoichiometry. The reaction sites are the primary amino groups on the globin chains using the method described by Carleton Hsia. It is stressed that although the polymerization yields a heterogeneous mixture, each Hb monomer has identical, reproducible oxygen affinity using the method by Hsia [14]. Hemosol Corp. employs a patented method for binding o-raffinose to the 2,3-DPG binding site, specifically at lysine-82 on the beta-chains and the terminal valine group from the beta-chain, also controlled by time and stoichiometry. The Hemosol patented method utilizes Tris buffer, avoiding phosphate interactions with the 2,3-DPG binding (reaction) site [73]. There are many advantages to the improvements made by Hemosol on Carleton Hsia’s original method. The remainder of this section will therefore focus on the newer method described by Hemosol. Sodium periodate is used to open up the trisaccharide, raffinose, yielding o-raffinose, a polyaldehyde. Reacting o-raffinose with HbA o yields seven sugar moieties per Hb molecule. The pH of the reaction mixture is controlled to between 5.0 and 7.5. This is the stable pH range of the polyaldehyde, o-raffinose. The progress of the reaction can be monitored by HPLC or FPLC. The reaction yields 40% hemoglobin tetrameric units, less than 5% of dimeric hemoglobin, with the balance being oligomers with molecular weights between 64 and 500 kDa. This is iso-oncotic at 14 g / dL (polyHb / solution), so that it can be used in iso-volumetric

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exchange, without causing blood pressure problems. The cross-linking can be stopped by reduction with borane dimethylamine or similar reagent as discussed previously. Borane dimethylamine does not form hydrogen gas as with sodium borohydride, enhancing the controllability of the method by Hsia. Polymerized and cross-linked o-raffinose Hb has a P50 of 32 mm Hg, and a Hill coefficient 1.6 measured at 378C in PBS buffer, pH 7.2 [73].

4.2.2. Conjugation Dextran can be conjugated to Hb to increase its molecular weight, e.g., benzenecarboxylate derivatives mentioned already. Polyethylene glycol conjugated bovine Hb (Enzon Corp.) is the product of modifying Bovine Hb with 12 succinimidyl carbonate polyethelene glycol molecules, the Hb is not cross-linked and results in a molecular weight of approximately 125 kDa. PEG conjugated bovine Hb increases plasma half life by a factor of 14, from approximately 3 h with bovine Hb to 42 h with PEG modified Hb in a rabbit top load model [74]. Oxygen transport was evaluated comparing PEG-Bovine Hb, autologous blood and Ringer’s lactate in a 30% exchange transfusion and 30% hypovolemic rat shock model. Muscle O 2 tension was evaluated in each situation and findings indicated that recovery to normal tissue oxygenation was 100–130% baseline with PEG-Bovine Hb and 10–30% baseline with Ringer’s lactate in the volume exchange model and 90–130% with PEG-Bovine Hb vs. 80–100% autologous blood in the 30% hypovolemic rat model [75]. Unlike polymerization, conjugation with PEG or dextran, may create a steric hindrance for Hb to interact with the endothelium, negating the debate on whether HBOC’s are hypertensive due to extravasation. Enzon Corp. is currently pursuing PEG-modified bovine Hb as an adjunct for oxygenation during irradiation of hypoxic tumors, which are resistant to radiation therapy [76]. 4.3. Second generation ( HBOC’ s) The term second generation seems a bit premature given the fact that we are all anxiously awaiting the arrival of a so called first generation HBOC to the market place. Perhaps the best definition of a second

generation agent is a Hb modified agent with unique functionality as to set it apart from the compounds in various stages of clinical trials. Current HBOC’s are cross-linked, polymerized and cross-linked, or conjugated. The second generations HBOC’s therefore possess some unique functions other than those listed above. For example Hsia et al., suggest that crosslinked or polymerized metHb can be used to scavenge cyanide. Oral administration of nitrites is avoided, which leaves the RBC’s undisturbed [14].

4.3.1. Polynitroxyl Hb ( PNH) PNH is an aa cross-linked Hb with (bis-3,5dibromosalicyl) fumarate, where PNH supports 16 nitroxide functional groups covalently bound at various amino acids of the a and b globin chains. The nitroxide functionalities bound to the hemoglobin tetramer are the result of reacting 4(2-BromoAcetamido)-2,2,6,6-tetramethyl piperidine-1-oxyl (BrAcTPO) at a molar ratio of 16 (BrAcTPO) to 1 mole of aa cross-linked Hb. The relative distribution appears to be 6 nitroxides covalently linked to the aa globin and 5 nitroxides on each b chain. The number of nitroxides bound to the Hb tetramer can also be manipulated to a desired pharmacodynamic effect. Initial kinetic data for PNH indicates a halflife of approximately 30 min. This suspiciously short half-life indicates the time by which all nitroxide groups are removed from the cross-linked Hb molecule and does not take into account the half-life of the cross-linked Hb itself. Once removed from the cross-linked Hb the nitroxide groups can continue to exert a pharmacological response. PNH functions as a superoxide dismutase and catalase mimetic, thus removing superoxide (?O 2 ) and the toxic intermediate H 2 O 2 , ultimately converting it to H 2 O and O 2 . PNH’s unique ability to perform the above tasks results in three important pharmacodynamic responses. First, the ability of superoxide to increase the permeability of endothelial tight junctions is removed. Thus, Hb is unable to cross the endothelial layer where it can bind NO and increase vasoconstriction (in addition free nitroxides provide a vasodilatory action). This is most notable in pathological conditions such as hemorrhagic shock, where free radical generation is increased. Therefore, PNH would prevent a leaky endothelial layer by scaveng-

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ing superoxide and reduce vasoconstriction by preventing the passage of PNH followed by nitric oxide scavenging. This provides a potential advantage over polymerized compounds in the lower molecular weight range i.e. 125 kDa that may be able to cross a compromised endothelium resulting from situations such as hemorrhagic shock. PNH prevents neutrophil and platelet interaction with endothelial cells, which results in damage to microvascular circulation. Besides, PNH prevents the formation of ferryl heme (Fe IV), common in ischemic conditions, alleviating the dangers of ferryl heme mentioned previously [77]. Currently, PNH is continuing in preclinical testing with further modifications to the existing compound. A product developed by Chang et al. (PolyHbsuperoxide dismutase-catalase) acts to scavenge superoxide and hydrogen peroxide similar to PNH. PolyHb-SOD-CAT is useful in ischemia and reperfusion injuries again similar to PNH in theoretical mode of action, however, differs markedly in chemistry [78].

4.3.2. Recombinant hemoglobins modified to reduce NO binding rHb1.1 is a purified Hb from E. coli which contains two fused a globin chains and a b Asn-108 →Lys mutation. It is this mutation that appears to reduce the oxygen affinity of the recombinant Hb. rHb1.1 possesses a P50 value of 32 mm Hg that is very similar to human Hb. Other rHb’s possess too high of an oxygen affinity for use as a blood substitute with the exception of rHb1.0 which also possesses the b Asn-108→Lys mutation without the aa globin chain fusing. However, from the standpoint of renal protection rHb 1.1, was a more logical choice to pursue as a viable HBOC [19]. rHb1.1 shows a significant and prolonged (.120 min) hypertensive response when given top load to conscious rats [18]. This is a troublesome consequence of lower MW Hb’s, which as we mentioned earlier is a complex conglomeration of physiological mechanisms including NO scavenging. Recently Somatogen (now Baxter Hb Therapeutics) removed rHb1.1 (Optro  ) from clinical trials. This decision was likely based on the hypertensive response caused by rHb1.1. Baxter has developed several recombi-

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nant Hb’s which modify the ability of Hb to bind NO and as a result reduce the vasoconstrictive effects seen with rHb 1.1, in addition the compounds can be genetically modified to alter oxygen affinity [18]. Doyle et al. have attempted polymerization of rHb1.1 to reduce the hypertensive response [79]. Modification of recombinant Hb to prevent NO binding has resulted in the generation of 3 such Hb’s: rHb2.0, 3.0, and 4.0. All have fused aa globin chains and are based on the rHb1.0 mutant, which does not possess the b chain 108 Lys→Asn mutation. rHb2.0, rHb3.0 and rHb4.0 all have amino acid substitutions in the distal heme pocket. The exchange of amino acids in the heme pockets of the globin chains result in steric hindrance, e.g., Leu (B10), Val (E11) and Leu (G8) have been exchanged for larger amino acid residues Phe and Trp. By exchanging these amino acids the rate constant for NO induced 9 ) is lowered. The k NO 9 for the various oxidation (k NO rHb’s is as follows: rHb1.1 (58), rHb2.0 (24), rHb3.0 (15) and rHb 4.0 (2). The physiological consequence of this appears to be that the lower the rate of NO binding the less the hypertensive response. Over an 80 min time period, rHb1.1 results in a 30% increase in mean arterial pressure (MAP), rHb2.0 results in a increase of 19% MAP, rHb3.0 results in an increase of 15% MAP, and finally rHb4.0 results in a 5% increase in MAP (all doses given at 350 mg / kg of a 5 g / dL Hb solution). rHb4.0 has an a 10 Phe in place of Leu and a b 11 Trp in place of Val. This provides enough hindrance to prevent NO binding, however, seems to prevent oxygen release. The P50 of rHb4.0 is a mere 5.2 mm Hg [18]. Polymerization of rHb’s is another area of interest. By polymerizing and increasing the MW of a Hb based blood substitute, the likelihood of passage across the endothelial layer is reduced. Changing a surface Lys residue to a Cys in Hb results in the ability to create a covalent disulfide bond. With the use of bismaleimidohexane, an agent specific for Cys residues, a 260 kDa polymer can be created. When compared to rHb1.1 the result was a significantly lower MAP with the polymerized rHb [80]. Baxter is currently working on a new rHb, 9 , similar to rHb3.0, but rHb 3011 , which has a low k NO improved P50 near rHb1.1. They have also developed a recombinantly fused dimer of two Hb tetramers [81].

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5. Conclusion Work in the area of Hb based blood substitutes has been extensive with major contributions to understanding this exceedingly complex field. Yet the goal of providing patients with useful products remains years away. The aim of research in Hb solutions should be to provide agents which, are not only safer, more stable, and more readily available than whole blood, but also more efficacious than whole blood and existing therapies where resuscitative fluids are indicated. The past aim of ‘‘blood substitutes’’ to be a mere oxygen carrier is declining and concentration on these compound’s therapeutic abilities is increasing. There are other areas where blood substitutes and hemoglobin solutions are being applied. In fact, some groups have adopted the term, hemoglobin therapeutics [82]. Originally, bis-3,5dibromosalicyl fumarate was used as an anti-sickling agent [39]. Hemoglobin has been conjugated with various molecules to enhance imaging of tissue. Similarly some conjugated hemoglobins have been designed to target tumors. Hemoglobin solutions are being developed to perfuse organs for transplantation. The primary applications of HBOC’S are trauma, preoperative hemodilution, and stroke.

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