Modern Cross-linking Strategies for Synthesizing Acellular Hemoglobin-Based Oxygen Carriers

Abstract

Unmodified cell-free hemoglobin (Hb) is structurally unstable when transfused into the blood stream (1-4). This review examines some of the latest chemical strategies used over the last five years to intra- and inter-molecularly cross-link Hb, thereby stabilizing its quaternary structure. Therefore, this work will address: 1) site-specific chemical modifications of Hb and 2) non-site specific chemical modifications of Hb, including, but not limited to: PolyHeme®, Hemopure®, Oxyglobin® and SOD-Hb. Current strategies for synthesizing PEGylated Hb is outside the scope of this review and will not be discussed herein. For a more thorough review of PEGylated Hb, the reader is directed to the following work:(5, 6).

INTRODUCTION

Cell free hemoglobin (Hb) is the precursor for Hb-based O₂ carrier (HBOC) formulation and / or synthesis. Hb is a 64 kDa tetrameric protein consisting of two noncovalently bound αβ dimers. In the blood stream, Hb dissociates into αβ dimers (7-14). These dimers are small enough to filter out through the kidney tubules resulting in a relatively short half-life of only a few hours (15, 16). The heme portion of the globin chain, which is highly reactive and cytotoxic, can induce renal failure (17, 18). In order to prevent tetramer dissociation and reduce heme-induced toxicity, the quaternary structure of Hb can be stabilized by intramolecular (within individual globin chains or between two adjacent globin chains in a tetramer) and/or intermolecular (between 2 or more Hb tetramers) cross-linking (19-23). This chemical strategy blocks the dissociation of tetrameric Hb into αβ dimers, and thus decreases the associated renal toxicity of cell-free Hb solutions (3, 4, 24-27).

As eluded to earlier, Hb is vulnerable to heme oxidation, which results in several clinical complications including but not limited to renal failure, vasoconstriction and concomitant elevated mean arterial pressure (MAP) above baseline levels (28). In the absence of the protective red blood cell (RBC) membrane or reducing agents (e.g. ascorbic acid, catalase, superoxide dismutase and uric acid), ferrous (Fe²⁺) Hb species will auto-oxidize to the ferric (Fe³⁺) form (methemoglobin [metHb]). The oxidized form of Hb, ferric Hb, cannot bind O₂, thereby lowering the O₂ transporting capacity of Hb, which may result in tissue ischemia (28). In this review, we will cite recent work that attempts to address these clinical problems by chemically modifying Hb.

Despite the volume of work being done in this active research area, many questions remain unanswered regarding HBOC efficacy and safety (28, 29). In this review, we will discuss chemical design strategies to reduce metHb level, tetramer dissociation, heme-induced cytotoxicity and vasoconstriction (17, 18). In light of these challenges that all HBOCs face, this miniature review will discuss recent HBOC design strategies based on 1) site-specific and 2) non site-specific chemical cross-linking.

Site-Specific HBOC Design

Since, HBOCs will be in direct contact with blood components when transfused into a patient, it makes sense to study heme-oxidation in the presence of naturally occurring oxidizing agents in the blood. One common oxidizing agent found in blood is hydrogen peroxide (H₂O₂). In healthy individuals, the plasma concentration of H₂O₂ is sustained at levels ≤0.25 μM. However 1000× more H₂O₂ is present in the plasma when neutrophils travel to damaged tissue sites versus normal tissue (28, 30).

Research at the Food and Drug Administration (Bethesda, MD) demonstrated that highly purified adult human Hb (hHb: purity >99%) exposed to plasma containing hydrogen peroxide (H₂O₂) exhibited altered geometry , and was more prone to oxidation and degradation. The β subunits appear to be especially vulnerable to early oxidative destruction stemming from H₂O₂ attack. Mass spectroscopic and amino acid analysis showed extensive βCys93 and βCys112 oxidation to cysteic acid. Oxidation of βCys93, which resides in the α₁β₂/ α₂β₁ interface, is especially important, since it can lead to the destabilization of the Hb tetramer and promote its dissociation into αβ dimers. Similarly, oxidation of βCys112, which resides in the α₁β₁/ α₂β₂ interface, can destabilize αβ dimers. Other amino acids are also susceptible to oxidation. For example, βMet55 is oxidized to methionine sulfoxide and βTrp15 is oxidized to oxyindolyl and kynureninyl products. These findings lend support for chemically cross-linking adjacent β subunits to prevent tetramer dissociation and blocking susceptible amino acids from oxidation (28).

Before these latest results were known, cross-linking reagents were engineered to target specific amino acid residues on the Hb molecule (31-33). In 2001, Kluger asked; “Can hemoglobin be chemically altered so that it can effectively deliver oxygen and remain in circulation outside the red cell?”(34). Kluger's group thus began their efforts to selectively introduce cross-links in the cationic amino group regions of the Hb molecule.

In one of their first studies, Kluger's group cross-linked pairs of Hb tetramers with N,N’-5,5’-bis[bis-(3,5-dibromosalicyl)-isophathalyl]- terephthalamide (shown as 1 in Scheme 6) , a tetrafunctional intra-/inter-molecularly capable cross-linking reagent (33, 35). Reagent 1 consisted of two bridge-spanned pairs of reaction sites, which react with the lysyl amino groups (β-82) of hHb to yield the Hb bis-tetramer (shown as 2 in Scheme 6) product with reduced oxygen binding cooperativity (n = 1.3) (35).

Scheme 6.

The bridge-span conformation between adjacent Hb tetramers was shorter in relation to materials with similar internal cross-links (Gourianov & Kluger 2003). Reproduced with permission from the American Chemical Society © 2008.

Gourianov and Kluger expanded on the previous study and then reported on the synthesis of relatively longer bridge-spanned cross-linking reagents with increased rigidity and coupled in pairs. The authors prepared three more tetrafunctional cross-linking reagents. The bridge-span portion of the three reagents connected to Hb are shown as 6, 7 and 8 in Scheme 7 (35). The P₅₀s' (pO₂ at which the HBOC is half-saturated with O₂) of the bis-tetramers synthesized from reagents were 5.0±0.1, 4.2±0.1 and 4.3±0.1 mm Hg respectively, while the bridge-span distance was 14.5, 16.8, and 18.4 Å respectively. In comparison to the shorter bridge-span bis-tetramer shown in Scheme 6, the bis-tetramers shown in Scheme 7 retained greater O₂ binding cooperativity (6, n = 1.8; 7, n = 2.1; 8, n = 2.0). However, the question remained; was the increased O₂ cooperativity due to the inter-tetrameric linkage or protein-protein interactions between adjacent Hb tetramers in solution?

Scheme 7.

The reagent with the longest bridge-span (8) gave the largest yield of Hb bis-tetramer (Gourianov & Kluger 2003). Reproduced with permission from the American Chemical Society © 2008.

In order to answer this question, Gourianov and Kluger extended their report from 2003 and published their findings in 2005 (35, 36). In their subsequent study, they prepared four tetrafunctional reagents (shown as 1, 2, 3 and 4 in Scheme 8). The first two reagents (1 and 2 shown in Scheme 8) consisted of widely positioned acyl groups at 20 and 23 Å, while reagents 3 and 4 contained flexible hydro-carbon manifolds between the reactive ends at 15 and 24 Å, respectively (36). Hb reacted with reagent 1 yielded bis-tetrameric Hb (A) in addition to cross-linked tetrameric Hb (C). Hb reacted with reagent 2 yielded bis-tetrameric Hb (B) in addition to cross-linked Hb (D). Hb species A and B (A, n = 1.4; B, n = 1.2 shown in Scheme 8) displayed minimal O₂ binding cooperativity, while Hb species C and D (C, n = 2.0; D, n = 1.8 shown in Figure 8) exhibited increased O₂ binding cooperativity. Cross-linking reagents 3 and 4 (Scheme 8) possessed flexible but saturated hydrocarbon chains and yielded cross-linked Hb tetramers (E and F) without a second protein moiety (36). Gourianov and Kluger observed that the O₂ binding cooperativity of the tetramers (E, n = 1.7±0.1 and F, n = 1.8±0.1) were similar to tetramers (C and D shown in Scheme 8) suggesting that neither chain conformation nor length of the cross-linker dictated the physical properties of the cross-linked Hb (36). Gourianov and Kluger's results were similar to Eike and Palmer's study, demonstrating that decreased cooperativity (n = 1.2 − 1.5) is usually observed in glutaraldehyde cross-linked and oligomerized Hb (36, 37).

Scheme 8.

The structure of ‘R’ in the cross-linking reagent (1, 2, 3, or 4) leads to the formation of Hb tetramers and/or Hb bis-tetramers (Gourianov & Kluger 2005). Reproduced with permission from the American Chemical Society © 2008.

In 2005, Alagic et al. site-selectively conjugated Hb to superoxide dismutase (38) (shown in Scheme 9) to yield a dual function protein (the P₅₀ of the Hb-SOD conjugate was 6.1 mm Hg as opposed to 5.0 mm Hg for hHb) complex (39). The Hb-SOD conjugate exhibited a 45% decrease in metHb and exhibited greater Hb tetrameric stability compared to hHb. Chang et al. cross-linked Hb with SOD and catalase (40) at least five years prior to Alagic et al.'s study and observed reduced concentrations of O₂ free radicals, free heme and metHb (41).

Scheme 9.

A free thiol group on Hb reacts with the maleimide conjugated superoxide dismutase (38) to yield the Hb-SOD conjugate (Alagic et al. 2005). Reproduced with permission from the American Chemical Society © 2008.

The most recent cross-linking reagent developed by Kluger and Hu utilized hexafunctional aromatic acyl phosphate-carrying amide groups to cross-link Hb (42, 43). Inter- and intramolecular cross-linking was facilitated by two adjacent acyl phosphate groups that reacted with the β globin chains of the tetramer. This approach facilitated an array of reactive groups to connect, at minimum, two Hb tetramers (Scheme 10) (42, 43). This resulted in a cross-linked Hb dendritic array consisting of 2.5 tetramers with an O₂ affinity almost identical to hHb (P₅₀ = 5.0 mm Hg) and a slightly reduced cooperativity (n = 2.0) compared to hHb (n = 2.7) (43).

Scheme 10.

Dendritic Hb consisting of 2.5 tetrameric constituents yielded an oxygen affinity similar to hHb (5.0 torr) (Kluger & Hu 2008). Reproduced with permission from the Royal Society of Chemistry © 2008.

It would be interesting to see in vivo studies on HBOCs emanating from Kluger's lab in the near future.

Non-Site-Specific HBOC Design

The non-site specific ability of glutaraldehyde to cross-link amino acid residues is efficacious for cross-linking proteins such as Hb (3, 4, 19, 44, 45). Glutaraldehyde is a common protein cross-linking agent effective in preventing Hb tetramer dissociation, thereby increasing HBOC retention time without causing the renal toxicity associated with Hb disassociation (46-49). Glutaraldehyde is a five-carbon dialdehyde in equilibrium with the cyclic hemiacetal of its hydrate and is capable of forming oligomers of varying lengths in aqueous solution (44). Because of glutaraldehyde's variable length and the wide variety of reactive species that it can form in aqueous solution, glutaraldehyde is referred to as a non-site-specific cross-linking reagent. Glutaraldehyde is capable of cross-linking Hb at a variety of amino acid residues (19, 50). In particular, glutaraldehyde is capable of reacting with the ε-amino group of lysine and arginine; sulfhydryl group of cysteine; imadazole ring of histidine; and the phenolic ring of tyrosine, among other chemical moieties (19, 51, 52).

The reaction between amino groups on Hb and aldehyde groups on glutaraldehyde yields chemically unstable imine bonds (53, 54). Imine bonds are easily hydrolyzed in aqueous solutions to yield the free glutaraldehyde cross-linker, as well as free Hb (51). Hence, stabilizing the imine bonds present in glutaraldehyde polymerized Hb (PolyHb) dispersions against hydrolysis is important if PolyHbs are to function as clinically useful HBOCs. Sodium borohydride (NaBH₄), a strong reducing agent, was used to reduce the hydrolytically unstable imine bonds of PolyHb into stable amine bonds, however it is possible that NaBH₄ could also reduce other unsaturated functional groups on the Hb tetramer (3). To explore this possibility, Eike and Palmer examined the glutaraldehyde-Hb reaction in closer detail. They studied the effect of NaBH₄ concentration on the oxygen-binding properties (P₅₀, n) and (55) levels of PolyHb dispersions (3, 4).

Eike and Palmer (4) obtained similar findings to DeVenuto et al., (56) when varying the NaBH₄ concentration used to quench the polymerization reaction up to a 300:1 (NaBH₄-Hb) molar ratio: i.e. increasing concentrations of NaBH₄ did not deteriorate the oxygen-binding properties of PolyHb dispersions. Eike and Palmer's results (Table 1) show that NaBH₄ does not appear to affect the P₅₀, n, or metHb level when used in amounts up to 300 times the molar concentration of Hb (4). These results can be explained by examining their experimental protocol before the polymerization reaction was quenched with NaBH₄. Prior to quenching the Hb polymerization reaction with NaBH₄, the NaBH₄ quenching solution was degassed for 30 min with nitrogen. Degassing the quenching solution facilitated the transport of hydride ions present in the aqueous phase into hydrogen gas in the gas phase. This phenomenon most likely lowered the initially high concentration of reductant in the quenching solution and thus explains why increasing concentrations of NaBH₄ did not deteriorate the oxygen-binding properties and decrease the metHb level of PolyHb dispersions (3, 4).

Table 1.

Equilibrium oxygen binding properties of PolyHb dispersions synthesized at a 30:1 glutaraldehyde-Hb molar ratio and quenched with various concentrations of NaBH₄ (Eike & Palmer 2004a).

[NaBH4]/[Hb]	P50 (mm Hg)	n	% MetHb
0	13	1.9	17
30	12.1	2.0	15
150	12.5	1.9	19
300	14	2.0	14

In another study by Eike and Palmer (57), various concentrations of glutaraldehyde were used to polymerize bHb and the oxygen affinity was measured at various pHs and chloride ion concentrations. Their results indicated that the oxygen affinity dependence on Bohr H⁺ and Cl⁻ concentration was weakened following Hb polymerization (57). This was similar to other findings, which demonstrated that chemical modification weakens allosteric interactions (48, 58). However, a rise in oxygen affinity was observed with increased cross-link density. This is because of differences in polymerization reaction conditions. It was observed that high cross-link density further weakened the ability of modified Hb to respond to H⁺ and Cl⁻. It was also observed that the Bohr effect was constant at pH 7.0 compared to 7.4 and 8.0 and at high Cl⁻ concentrations. Thus the Bohr effect maintained its dependency on Cl⁻ at pH 7.0 but to a lesser extent at pHs 7.4 and 8.0 (3, 57).

Structurally, the reason for a decrease or increase in P₅₀ with the extent of Hb polymerization can be explained by using the concept of constraining or “freezing” the Hb tetramer into a particular structural state by chemically cross-linking the Hb molecule (59). Perutz et al. showed that Hb in the deoxy conformation (T-state) exhibited a central dyad axis, which was larger than Hb in the oxy conformation (R-state) (60). Hb in either of these two quaternary conformational states exhibits very different and distinct oxygen binding properties. It has been suggested that the presence of physical constraints, which prevents Hb from adopting an R-state conformation, facilitates the synthesis of PolyHbs with low oxygen affinity (59). Conversely, PolyHbs with high oxygen affinity could be explained by a favoring of the quaternary R-state caused by a physical constraint that prevents the widening of the Hb molecule into the T-state (3, 57).

Even though the Bohr effect was suppressed for glutaraldehyde PolyHb dispersions, it was not completely eliminated at physiological pH and Cl⁻ concentration (pH 7.4 and ∼ 100 mM Cl⁻) (61). Although the Bohr effect was not present when the pH was increased from 7.4 to 8.0 for the 20:1 and 30:1 PolyHb dispersions, (Figure 1) it was present when the pH was decreased from 7.4 to 7.0 (a decrease in pH is typical for active muscles via lactic acid buildup). This increase in oxygen affinity is more pronounced in the presence of physiological concentrations of Cl⁻. The fact that Cl⁻ is necessary to observe a decrease in oxygen affinity indicates that Cl⁻ is still necessary for the Bohr effect to be observed in these PolyHb dispersions. Past studies have indicated that ∼ 30% of the Bohr effect is dependent on Cl⁻ (62, 63). Further studies demonstrated that the Bohr effect was abolished with interference of the Cl⁻ binding sites (59). The Val-99α site, for instance, has been identified as a site, if masked, will interfere with both the allostery of Cl⁻ and H⁺ ions (63). In Eike and Palmer's study however, the dependence of the Bohr effect on Cl⁻ binding seems to be partially maintained in PolyHb dispersions (57). The link between the Cl⁻ concentration and the Bohr effect was not completely eliminated even after glutaraldehyde cross-linking. Thus, these Hb polymers were able to maintain some of the oxygen affinity change seen physiologically when varying the pH and Cl⁻ concentration in human blood (3, 57).

Figure 1.

Oxygen affinity (P₅₀) measured at various Cl⁻ concentrations for unmodified bHb and PolyHb dispersions at pH 7.0 (●), 7.4 (■) and 8.0 (▲) (Eike & Palmer 2004d).

The United States (U.S.) military, commercial, and academic investigators have evaluated other glutaraldehyde-based PolyHb products synthesized from bovine and human Hb. PolyHeme® manufactured by Northfield Laboratories (Evanston, Illinois), is a glutaraldehyde polymerized (P₅₀ = 26−32 mmHg) pyridoxylated hHb (64). Northfield Laboratories presented the results of its Phase III trauma trials at The XI^th International Symposium on Blood Substitutes in Beijing, China (64) . Prior to their presentation, six military-casualty investigators at a U.S. Army laboratory conducted one evaluation of PolyHeme®, which showed adverse performance in Sprague-Dawley rats (65). Northfield's Phase III clinical study showed higher incidents of negative side effects and mortality among accident victims experiencing hemorrhagic shock compared to the control group who received standard saline (crystalloid) solution. Their study included 714 patients across thirty-two Level 1 Trauma care units. Out of 586 accident victims, represented by a “per protocol (PP)” population, (PP; a population excluding 128 patients with treatment regimen violations), Northfield reported that 279 were infused with PolyHeme®, and 31 patients or 11.1% of the PP population expired. Among the remaining 307 patients injected with a standard saline solution, 28 patients or 9.1% of the PP population expired. Both sets of patients who did and did not expire , suffered a combination of negative side effects i.e. pneumonia, myocardial infarction, shock and respiratory failure (64) . Forty percent of the 324 patients treated with PolyHeme® suffered higher incidents of negative side effects compared to 35% of the 322 patients in the control group (64).

Hemopure® (HBOC-201) is a glutaraldehyde-polymerized bovine Hb (bHb) manufactured by the Biopure Corporation (Cambridge, Massachusetts). Hemopure® has a P₅₀ of 36 mm Hg, a circulation half-life of 19 hrs and can be stored at room temp for ≥ 3 yrs (66, 67). Biopure completed Phase III clinical studies of Hemopure® in: 1) adult non-cardiac elective surgery patients in Europe and South Africa and 2) adult elective orthopedic surgery patients in the U.S., Canada, Europe and South Africa (66-70). In all twenty-one clinical trials (including phase II post-cardiopulmonary bypass surgery and phase II aortic aneurysm reconstruction surgery trials conducted in the U.S.), 93% of the 797 adult patients who received Hemopure® had at least one adverse event including an increase (10 to 20 mmHg) in blood pressure (66). Based on Hemopure®'s safety profile from the phase III elective orthopedic surgery trial, the U.S. Food and Drug Administration (FDA) halted Biopure's proposed trauma trial (66). Despite these results, Hemopure® is approved in South Africa for use in acutely anemic adult patients undergoing surgery. A clinical research team in South Africa reported what may be the first report of Hemopure® transfusion into a 23-month-old female with sickle cell anemic disease suffering from cardiac failure. Prior to Hemopure® transfusion, the subject's blood pressure was 70/35 mm Hg. After Hemopure™ transfusion, the patient's blood pressure was 100/55 mm Hg (71). However, O₂ transported from RBCs performs differently versus O₂ transported from HBOCs, which increases oxygenation to tissues experiencing restricted blood flow and may obfuscate clear physiological predictions (72). Ashenden et al. tested 12 human subjects in an exercise trial immediately after 30 g (6 subjects) or 45 g (6 subjects) of Hemopure® infusion against a placebo. The results of the trial indicated that Hemopure® did not increase maximal O₂ uptake (VO_2max). Hemopure® also increased the diastolic (∼8 mm Hg) (p=0.046) and mean (∼7 mm Hg) blood pressure (73).

Oxyglobin® (HBOC-301) is another glutaraldehyde-polymerized bHb manufactured by Biopure for veterinary use (74). HBOC-301 was approved by the FDA and is commercially available for use in treating canine anemia (67). Buehler et al., conducted a comprehensive study on HBOC-301 (PolyHbBv) for various structural modifications (75). Their study utilized un-fractionated samples of bHb and polymerized bHb (PolyHbBv), and four fractionated samples of PolyHbBv. The nonfractionated bHb sample exhibited a P₅₀ and n of (27.2±0.33 mm Hg) and (2.1±0.04), respectively. In contrast, the non-fractionated PolyHbBv sample displayed a P₅₀ and n of (38.4 ±0.5 mm Hg) and (1.4 ±0.02) respectively, while the four fractionated samples of PolyHbBv displayed a mean P₅₀ and n of (35.1±0.4 mm Hg) and (1.4±0.03) respectively (75). These results indicate that the O₂ affinity of fractionated and non-fractionated PolyHbBv was similar, however much lower in comparison to bHb (75). Structural modification did not appear to occur in critical areas of the Hb molecule as a result of glutaraldehyde polymerization. In all samples analyzed, the βMet55 residue positioned at the α₁β₁ interface was not modified, along with the β93Cys residue positioned at the α₁β₂ interface. Overall, the glutaraldehyde PolyHbBv formulation, demonstrated stable oxidative properties and homogeneous functionality (75).

Two years after the in vitro study by Buehler et al., the same group reported on an in vivo evaluation of PolyHbBv showing the comparative effects of a 50% exchange transfusion in an ascorbic acid (antioxidant) producing species (rats) versus a non-ascorbic acid producing species (guinea pigs) (75, 76). Ascorbic acid is a strong reducing agent, so its presence in the blood plasma serves to reduce metHb into functional Hb. MetHb is highly toxic, since it is more structurally unstable versus Hb, and will quickly unfold to release free heme into the blood stream. The authors hypothesized that transfusion of HBOCs in ascorbic acid producing animals should result in less cyto-toxicity versus non-ascorbic acid producing animals. Their results showed that significant portions of PolyHbBv in the guinea pig were oxidatively modified (76). This may be one of the reasons why in vivo studies of potential HBOCs tend to report favorable safety data, because in vivo studies often utilize high ascorbic acid producing species like rats (rats produce 38 μg of ascorbic acid per mg of protein per hour) as opposed to other species (like guinea pigs and humans) that are less capable of endogenous ascorbic acid production (76, 77).

All six PolyHbs (20:1, 30:1, 40:1, PolyHeme®, Hemopure®, Oxyglobin® listed in Table 2) possess relatively low O₂ affinities with high molecular weight distributions. The oxygen affinity of these HBOCs were designed to meet or exceed the P₅₀ of human RBCs (26 mm Hg) in order to properly oxygenate tissues and organs supplied by the vasculature (3, 4).

Table 2.

Physical properties of various glutaraldehyde PolyHbs (Eike 2005).

HBOC	Mw range (kDa)	P50 (mm Hg)	n	% Cross-linked tetramer	% Higher Mw Species
20:1	64−192	20	1.5	49	25
30:1	64−192	13	1.2	24	76
40:1	640−980	13	1.4	<1	100
PolyHeme	64−400	26−32	1.5	<1	NA
Hemopure®	64−500	36−38	1.2	<3	>95
Oxyglobin®	64−500	38	1.3	<5	NA

In 2004, Eike and Palmer developed a sub-class of acellular HBOCs with higher oxygen affinities (P₅₀s < 26 mm Hg) by polymerizing bovine Hb with several oxidized (ring-opened) saccharides (3, 15, 78). The ability of oxidized monosaccharides, disaccharides, trisaccharides and polysaccharides to cross-link and polymerize Hb was assessed. Each saccharide was mixed with NaIO₄ stock solution in a ratio of 2 mol of NaIO₄ for every mole of saccharide multiplied by the number of rings comprising that particular saccharide (3, 78). After overnight reaction, oxidative cleavage of 1,2-diols yielded ring-open dialdehydes. The oxidation reaction resulted in ring-opened saccharides exposing two aldehyde groups on each ring. Oxidation reactions are shown for each saccharide in Schemes 1 through 4 (3, 78).

Scheme 1.

Oxidation of monosaccharides by NaIO₄ to yield ring-opened dialdehydes (Eike 2005).

Scheme 4.

Oxidation of a polysaccharide by NaIO₄ to yield ring-opened polyaldehydes (Eike 2005).

Utilization of ring-opened saccharides as Hb cross-linking agents resulted in PolyHb dispersions with even lower P₅₀s compared to the glutaraldehyde PolyHb dispersions shown in Tables 1 & 2. Eike and Palmer's study illustrated that o-methylglucopyranoside (shown in Scheme 1) was the most promising o-saccharide for cross-linking Hb (57). O-methylglucopyranoside exhibited large amounts of cross-linked Hb ranging from 78−89% at pHs 6 and 8 respectively. The high degree of cross-linking may be due to structural differences between methylglucopyranoside and the other monosaccharides used. The extra methyl group in the glucopyranoside ring structure results in a 6-carbon dialdehyde upon oxidation compared to a 4-carbon dialdehyde resulting from the oxidation of glucose and galactose. O-methylglucopyranoside also displayed very low metHb levels compared to most of the other o-saccharide cross-linked polyHb dispersions. Using a 50:1, O-saccharide:Hb molar ratio, o-methyglucopyranoside metHb levels ranged from 10−12%, which was lower than most of the other o-saccharides which averaged ∼51% metHb. The n for o-methylglucopyranoside cross-linked Hb was ∼1.1 and 2.1, while the P₅₀s were 6.1 and 14.6 mm Hg at pHs 6 and 8 respectively (3). Eike and Palmer did not conduct animal studies to examine the effects of PolyHbs with P₅₀s ranging between 6.1 and 14.6 mm Hg (3). However, Patton and Palmer simulated HBOC oxygen offloading to hamster skeletal muscle tissue with varying capillary inlet pO₂s (79). The results of their study showed that high affinity HBOCs are most effective in treating hypoxic anemia. Interestingly, high affinity HBOCs enter the capillary at a higher Hb oxygen saturation and release more oxygen over the pO₂ gradient of the capillary compared to lower affinity HBOCs (79, 80).

Oxidized raffinose (O-raffinose) (Figure 3) is another type of polyaldehyde Hb cross-linking reagent that was used in the synthesis of Hemolink™ (Hemosol Inc, Mississauga, Ontario, Canada) (81). Hemolink™ was developed by the Canadian Military (32). In 2005, Boykins et al. cross-linked highly purified (99%>) deoxygenated hHb with raffinose to form O-R-PolyHbA₀, a polymerized HBOC (82). Intramolecular cross-linking did not predominate the central cavity region, however β₂Lys82-β₁Lys82-β₁Val1 and minor intermolecular cross-linking was discerned outside the central cavity region (82). Amino acid alteration on the β-globin chain, Cys93, and the α-globin chain, Cys104, was verified by the presence of completely oxidized and degraded trisaccharide residues. Out of a total of 5.6 cysteine residues (recovered as cysteic acid) per Hb tetramer, non-site specific chemical modification occurred on four residues: α₁Cys104, α₂Cys104, β₁Cys93 and β₂Cys93 (18, 82). These results suggest the presence of random O-raffinose alterations, which promote the formation of a locked T-state or rigid-body-like protein structure (18, 82). This was in sharp contrast to studies with Oxyglobin®, where no discernable changes occurred in the same regions. Hence, the unstable oxidative properties and skewed functionality of separated Hb species in dispersions of O-R-PolyHbA₀ may be a consequence of the chemical nature of the cross-linking reagent O-raffinose (75).

Dimino and Palmer designed novel high O₂ affinity (low P₅₀) HBOCs by cross-linking relaxed (R)-state bHb with ring-opened 1-o-octyl-β-d-glucopyranoside (1-OGP) and 2-chloroethyl-β-d-fructopyranoside (2-CEFP) (Scheme 5) (52).

Scheme 5.

Periodate oxidation of 1-OGP (A) and 2-CEFP (B) yields dialdehyde-based Hb cross-linking regents (Dimino & Palmer 2007).

Polymerized bHBOCs synthesized with ring-opened 1-OGP exhibited metHb levels ranging from 5% to 10%. For polymerized bHBOCs synthesized with ring-opened 2-CEFP, metHb levels ranged between 2% to 10%. Infusion of HBOCs with high metHb levels (>10%) into animals was demonstrated to increase mortality (83). In contrast, in vivo studies have shown that infusion of acellular Hbs with metHb levels less than (<) 10% did not elicit adverse physiological responses when infused into rats (84). Ring-opened 1-OGP and 2-CEFP are both mild oxidizing agents; therefore, as the concentration of cross-linker increases, the metHb level of the bHBOC product increases. To limit the formation of metHb, the cross-linking/polymerization reaction may be conducted under reduced pO₂ conditions by bubbling N₂ gas through the reaction vessels (note: CO may also be used instead of N₂) (52). As the concentration of ring-opened 1-OGP increased from 0 (1-OGP0) to 30 mM, the P₅₀ decreased from 22 to 7 mm Hg. A similar result was observed for bHBOCs synthesized via polymerization with ring-opened 2-CEFP. As the concentration of ring-opened 2-CEFP was increased from 0 to 30 mM, the P₅₀ decreased from 22 to 8 mm Hg (52). These results indicate that increasing the cross-linker to bHb molar ratio increases the O₂ affinity of the bHBOCs synthesized with ring-opened 1-OGP and 2-CEFP, and suggest that further increases in O₂ affinity could be achieved by further increasing the cross-linker to bHb molar ratio (52). While Dimino and Palmer didn't carry out animal studies on these high O₂ affinity HBOCs, they did develop a mathematical model of oxygen transport in human skeletal muscle tissue to test the oxygen transporting ability of these HBOCs (85). Their simulations with 1-OGP completely replacing hRBCs exhibited the most O₂ delivery to skeletal muscle tissue at a capillary inlet pO₂ of 5 mm Hg, whereas simulations with 2-CEFP (P₅₀ = 15.2 mm Hg) completely replacing hRBCs exhibited the most O₂ delivery to skeletal tissue at inlet pO₂s ranging between 10 and 20 mm Hg (85). This may be because at pO₂s between 4.4 and 15.2 mm Hg, 2-CEFP releases a greater fraction of bound O₂ from the HBOC in the highly sloped region of its O₂-HBOC equilibrium-binding curve compared to the other HBOCs evaluated in their study (85).

Other cross-linked Hbs have been recently reported in the literature. Lu et al. synthesized a conjugate of human serum albumin (HSA) and bHb. The P₅₀ and n of bHb was 24.5 mm Hg and 2.46 and decreased to 18.2 mm Hg and 2.10 for the bHb-HSA conjugate. The O₂ transport efficiency of the Hb-HAS conjugate was 20% in comparison to 28% for RBCs (86).

In another study, Haney et al. isolated Hb from hRBCs and polymerized it with cyclic-diethylenetriaminepentaacetic acid (DTPA) to yield Poly-DTPA-Hb. Analysis of Poly-DTPA-Hb yielded a P₅₀ of 5.1 mm Hg and n of 1.4 (87).

At the Marine Biological Lab (Woods Hole MA), Harrington et al. isolated, purified and analyzed acellular Hb from Lumbricus terrestris (LtHb) and Arenicola marina (ArHb) in order to investigate their physical properties. LtHb and ArHb were found to contain double layered hexameric bilayers with 144 and 156 heme-carrying chains. The subunits are naturally cross-linked into a structurally stable dodecamer. The most exciting feature of LtHb and ArHb is that it seems impervious to oxidation at 37°C with potassium ferricyanide, K₃Fe(CN)₆ (a strong oxidizing agent for converting Hb to metHb) (88).

Recently, Wong and Chang (2007) cross-linked fibrinogen with Hb to form polyhemoglobinfibrinogen (PolyHb-Fg) for potential use as a HBOC with similar clotting properties to whole blood (89). Their in vivo study showed that the blood clotting time was unaffected at 98% exchange transfusion with PolyHb-Fg.

Summary

Natanson et al. recently conducted a thorough meta-analysis on five HBOC products evaluated in sixteen clinical trials (90). This work has resulted in a halt to all clinical trials of HBOCs in the U.S. and underscores the need for more basic research into the origin of HBOC-induced toxicity and efficacy.

Scheme 2.

Oxidation of disaccharides by NaIO₄ to yield ring-opened tetra-aldehydes (Eike 2005).

Scheme 3.

Oxidation of a trisaccharide by NaIO₄ to yield a ring-opened hexa-aldehyde (Eike 2005).