Basic Science Offers a Challenge for Developing Hemoglobin Based Oxygen Carriers into Therapeutic Agents

Abstract:

Experimental data, obtained during the course of several years, are connected into a coherent picture, which may help research for the development of HBOCs as therapeutic agents. Oxygen affinity, scavenging of nitric oxide, and yield of production of hemoglobin based oxygen carriers were the areas under consideration.

Keywords::

• Blood substitutes
• oxygen delivery
• facilitate diffusion
• NO scavenging
• vasoconstriction

INTRODUCTION

After the meta analysis reported by Natanson et al. [1], research for therapeutic use of HBOCs is stalled. Adverse clinical events leading to myocardial infarcts, for uncertain causes, produced only speculations. It also proved that FDA directions are inadequate. This is the result of research directed by corporate concerns producing and using scantily characterized fluids, in total disregard of independently obtained experimental data, and controlled only by marketing considerations, producing useless information.

In order to exit from this impasse this writer examined available data, void of corporate and venture capital obligations, so as to produce coherent and objective information directed to the development of HBOCs into therapeutic agents.

AVAILABLE DATA

Oxygen Delivery by Cell-bound and Cell-free Oxygen Carriers

As discussed in previous papers [2,3], in vivo there is a flow of oxygen from lungs to tissues through a gradient that goes from about 120 torr at the lungs to less than 2 torr at the periphery. The flow of oxygen goes through plasma, reaches the capillaries, goes through the parenchyma's tissues and cellular cytosol, and is consumed by mitochondria. The scarce amount of oxygen carried by plasma (10E-5 M) is corrected when, at the capillaries, the flow is replenished by oxygen released from hemoglobin present inside the red cells. In fact, as an oxygen carrier, red cell hemoglobin binds and sequesters oxygen at the lungs, passively releasing oxygen at the capillaries, thus replacing consumed oxygen and satisfying metabolic necessities. After the capillaries in tissues parenchyma, oxygen is retrieved by myoglobin and again released to metabolism at 2 torr or less. This physiologic strategy allows the delivery to metabolism of large amounts of oxygen (about 1 L/min) still at low oxygen tension (near and below 2 torr), avoiding its toxicity.

Oxygen released from red cells hemoglobin must diffuse through the internal viscosity of the cells and their membrane. Then it diffuses through the surrounding plasma, the vascular wall, the interstitial space of parenchyma, cellular membranes and their cytsol, before reaching mitochondria. The scarce solubility of oxygen in watery environments results in a slow rate of diffusion and a significant delay of release kinetics, while consumption by cytochrome oxydase at the mitochondria is very fast, similar to the rate measured in vitro. This rate imbalance is corrected by the “mass action” of a large, overwhelming amount of red cells in plasma with a 40% hematocrit.

Hemoglobin based oxygen carriers (HBOC) add to oxygen delivery a new parameter, which has far-reaching consequences.

Being cell-free, HBOCs are endowed with brownian motions. The translational component of these motions implies that oxygen saturated HBOCs add their diffusion rate to that of free oxygen. The overall result is a virtual increase of oxygen diffusion rate. This phenomenon is the facilitated diffusion first described by Wittemberg [4] using membranes embedded with a variety of oxygen carriers. It adds to the mass action of the hematocrit and interferes with the supply/consumption imbalance mentioned above.

The different efficiency of red cells hemoglobin and HBOCs to oxygen delivery was tested, comparing the competence in vitro of a suspension of red cells with a solution of a cell free HBOC (crosslinked tetrameric Hb). They had identical hemoglobin content (3.0 g/dL), very similar oxygen affinity (P₅₀ near 30 torr), and oxygen binding cooperativity (n near 2.0). The fluids were used for superfusing an intestinal membrane inside a Ussing chamber. The fluids were equilibrated with gas containing 30% oxygen (in air it is 20%). Only the HBOC solution could support the functional transport of amino acids across the membrane, which died almost immediately when superfused with the suspension of red cells. The rate of oxygen consumption by the membrane was matched only by the facilitated diffusion through the media of the oxygen delivered by cell free HBOC.

Retention Time and Nitric Oxide Scavenging

Since the beginning of HBOCs research scientists were frustrated by the hematuria and systemic vasoconstriction resulting from infusion of purified hemoglobin solutions. Hematuria was responsible for a very short intravascular retention time (half time 20-30 min) and was referred to the ability of tetrameric hemoglobin to dissociate into dimers, not retained by kidney glumeruli. Intra-molecularly crosslinking hemoglobin tetramers eliminated dimer formation, prevented hematuria, and prolonged retention times.

However, MAP increase and vasoconstriction was still observed after infusion of non-dossociable tetrameric HBOCs.

Vasoconstriction was referred to the high affinity of HBOCs for NO, scavenging even nanomolar quantities of NO, the relaxing factor. Consistent with this hypothesis, Urbaitis et al. [6] showed that inhibition of NO synthesis before albumin infusion produced identical MAP increase as infusions of a crosslinked tetrameric hemoglobin (Figure 1).

Figure 1. Comparison of the time course of the MAP increase produced by infusion in rats exchange transfused with solutions of either fumaryly X-linked bovine hemoglobin (a stabilized tetramer) or albumin with L-NAME. Ł) Albumin, Ł) Albumin + L-NAME, Ł) X-linked HB, Ł) L-NAME alone. Adapted from [5].

It was also observed that the vasoconstriction was not evenly distributed over internal organs (Figure 2) [7]. The resulting decrease of blood flow was very pronounced in the intestine, kidneys, and pituitary. The resulting anoxia would produce kidney failure, intestinal and metabolic disorders. Notably, a flow increase was observed for brain and heart. This increase could be referred to the incrased MAP in the absence of a local limited or absent vasoconstriction. The different extent of vasoconstriction in internal organs is the reason why the average MAP increase is never very pronounced.

Figure 2. Effect of infusion of fumaryl X-linked bovine Hb (a stabilized tetramer) on the blood flow of various internal organ in the cat. Notably, heart and brain show an increased blood flow. Adapted from [6].

Asano et al. [8] measured, through a cranial window, the dilation of cerebral arterioles stimulated by the NO producing acetylcholine, in the presence and absence of HBOCs. It was noticed that HBOC infusion, which scavenged NO only in the luminal side, left intact arterioles dilation. Instead the dilation was decreased and even prevented when the HBOC was added through the cranial window onto the abluminal side of the vessels. These data suggested that only when HBOCs extravasate into the muscle layer of the vessels does scavenging of NO result in systemic vasoconstriction.

Extravasation of infused HBOCs occurs through large pores of capillary beds. As shown in Table 1, pores exist with diameters as large as 500 Δ, together with pores as small as 60 Δ [9]. The data in Table 1 and Figure 2 justify that crosslinked tetrames are retained by the tight endothelial junctions of cerebral blood brain barrier and the tight junctions of the heart vascular endothelium, preventing vasoconstriction, while the small pores of glomeruli prevented hematuria. However, they still extravasate from the larger pores in other capillary beds producing systemic vasoconstrictions and MAP increase.

Table 1. Pores in the capillary walls (diameter).

Small pores 80Δ

Glomeruli pores 60-65Δ

Large pores 500-600Δ

Hemoglobin tetramers 60Δ

Hemoglobin dimers 48Δ

Adapted from [9]

Data in the literature indicate that large molecules with average MW near and above 1.0 MDa are needed to prevent extravasation and systemic vasoconstriction. Sakai et al. [10] and Cabrales et al. [11] report that there is an inverse relationship between size of HBOCs and pressor response after infusion. It was null for encapsulated hemoglobins. As shown by Matheson et al. [12], there was no pressor response after infusion of a polymer with average MW near 25 MDa. Failure of extravasation was proven by absence of hemoglobin residues inside the lymph of infused rats. More recently, Yu et al. [13] confirmed those data showing that addition of even very small amounts of tetrameric hemoglobin to Polyheme (Northfield Lab., Evanston, IL) restored a pressor response previously absent.

Numerical simulations of Kavdia et al. [14] propose that luminal NO scavenging reclaims NO from the abluminal side, depriving it of the muscle layer. It may be argued that when NO diffuses from the endothelium, luminal and abluminal sides are compartments separated by a relative impermeability to gases of vascular walls [2]. The impermeability is also supported by the opposite gradient dynamics produced by oxidation of NO in the luminal side, and by NO reaction with guanidyl cyclase in the abluminal side. It should also be stressed that, even in the absence of HBOCs, in the luminal space NO is very quickly oxidized (scavenged) by plasma oxygen.

It cannot be excluded that other factors besides extravasation contribute to pressor response. The data presented here propose that scavenging of NO from the muscle layer of vascular walls, by extravasating HBOCs, is the main cause of systemic vasoconstriction and pressor response.

Hyperoxygenation

Oxygen toxicity is mostly due to formation of oxygen radicals whose strong oxidative characteristics damage surrounding tissues. They are a natural product of aerobic metabolism, neutralized by reducing enzyme systems including catalase, peroxydase, and SOD. They are also produced by hyperoxygenation, which occurs when oxygen supply exceeds oxygen consumption.

To prevent hyperoxygenation autoregulated vasoconstrictions reduce blood flow. The presence of this autoregulation has been monitored and in some case controlled [7, 15–18].

The vasoconstrictive response to hyperoxygenation implies the presence of sensors sensitive to oxygen tension and rate of oxygen consumption [2]. Initial data emerging from the laboratory of Koehler [12,19] suggest the involvement of the cytochrome P450 system.

There is a difference between the vasonstriction produced by NO scavenging and that produced by hyperoxygenation. The first is systemic and overwhelms regulatory physiology resulting in anoxia and MAP increase. The second is physiologically controlled, local and non-systemic. It may or may not produce MAP increase, depending on the internal distribution of hyperoxygenation and vasoconstriction. Also, it tends to produce normoxia. Formation of oxygen radicals more than vasoconstriction is the toxicity of hyperoxygenation. NO scavenging and hyperoxygenation are two very different toxic events.

Typical data are shown in Figure 3. Infusion of an HBOC, which does not extravasate in the parenchyma lymph, produces a vasoconstriction without MAP increases [12]. The vasoconstriction is due to hyperoxygenation; in fact it persists after inhibiton of NO synthase [19] and is canceled by inhibitors of the cytochrome P450 system.

Figure 3. In rats exchange transfused with ZL-HbBv polymer (average MW 25 MDa) the NO independent vasoconstriction reverts to vasodilation when PVP is added to circulating blood. In the albumin controls, PVP was much less effective, as if vasodilation were oxygen dependent. Adapted from [12].

Page et al. [20] report that the rate of oxygen release by mixtures of red cells and cell-free HBOCs is that of the HBOCs alone. The slower rate of oxygen diffusing from red cells was not detected. This suggests that HBOCs facilitate the diffusion rate also of the oxygen released from concomitant red cells. Thus, infusion of HBOCs in bloods containing red cells would result in a virtual increase in the rate and amount of oxygen offered to metabolism, which may exceed the rate of consumption. Thus, addition of HBOCs, to red cells containing bloods, increases the risk of hyperoxigenation.

Even in the absence of hyperoxygenation, endothelium layers are damaged probably by oxygen radicals originating from HBOCs in plasma “wetting” the vascular walls. Baldwin et al. [21] report the formation, after HBOCs infusion, of venular interendothelial gaps in rats mesentery through which albumin, and presumably HBOCs, extravasate. Dull et al. [22] report formation of gaps in endothelial membranes exposed in vitro to HBOCs.

Oxygen Affinity of HBOCs

The optimal oxygen affinity of HBOCs is still debated. The argument goes that low affinity HBOCs would lose oxygen before reaching the capillaries, while high affinity HBOCs would fail to release oxygen at the periphery.

Intramolecularly crosslinked tetrameric human HBOCs have affinities with P₅₀ directly proportional to the length of the crosslinking agent [23]. Using human and bovine hemoglobins, intramolecular crosslinking results in HBOCs with P₅₀ ranging from 12 torr to 100 torr. Polymers have only high oxygen affinities with P₅₀ down to 3-4 torr [12,24]. All tetrameric HBOCs extravasate. Instead polymers with molecular size above 500 KDa do not extravasate [10–12].

Recently, Koehler et al. [25] have demonstrated a relative insensitivity of peripheral oxygen delivery to oxygen affinity, probably resulting from oxygen facilitated diffusion. It even appears that very high oxygen affinity polymers are most efficient in reducing the volume of brain infarcts in mice (Figure 4).

Figure 4. Decrease of infarct volume, produced in mice by occlusion of the middle cerebral artery, after infusion of HBOCs with different P₅₀. DECA is human hemoglobin X-linked with sebacic acid (MW 64 KDa), Polytauro is a recombinant heptameric HB polymer (seven tetramers) enriched with surface SH groups (MW near 500 KDa). (Polytauro)n is a recombinant Hb polymer enriched with surface SH groups (Average MW 1000KDa). Adapted from [24].

The rationale for the high efficiency of high affinity carriers could be that a larger amount of bound oxygen reaches the periphery. Also, it may be noted that high affinity would decrease formation of oxygen radicals. Oxygen radicals are molecules of oxygen with uneven electron shells, which gives them a very strong oxidative activity. In the case of hemoglobin, they result from an irregular share of electrons between heme iron and oxygen when it is released. The probability of error is small; still it is proportional to the number of binding/release events during the dynamics of oxygen binding equilibria. High affinity would displace the dynamics in favor of binding, therefore decreasing the probability of radicals’ formation.

DESIGN CONSIDERATIONS

The main challenges for the synthesis and use of HBOC appear to be their oxygen affinity and toxicity. Yield of synthesis becomes important when large-scale production is anticipated.

Oxygen Affinity

Intramolecular crosslinking of hemoglobin produces a variety of tetrameric HBOCs with different oxygen affinities. Crosslinking is usually obtained positioning a dicarboxylic acid bridging the two beta 82 lysines in the central Hb cavity between the partner beta subunits. As mentioned, the P₅₀ of crosslinked human and bovine hemoglobins is directly proportional to the length of the crosslinking agents. In human hemoglobin it goes from about 12 torr using fumaric acid (making a four carbons bridge) to about 30 torr using sebacic acid (10 carbons bridge in human hemoglobin [23]). In bovine hemoglobin crosslinking with adipic acid produces 2 compounds crosslinked either between the two beta 82 lysines, or between the beta 82 lysine and the alpha 1 valine of the opposite subunits. They have P₅₀ near 60 torr and near 100 torr, respectively [26]. Oxygen binding cooperativity is also proportional to P₅₀, the Hill's index “n” goes from near 1.2 for the highest to near 2.0 for the HBOC with the lower affinities.

Instead, the oxygen affinities of polymeric HBOCs are always high with P₅₀ near 3-5 torr and no oxygen binding cooperativity [12]. The exception is a structured recombinant polymer that includes only 7 tetramers with a P₅₀ near 18 torr and a cooperativity with Hill's “n” near 2.0 [25].

As discussed above, tetrameric crosslinked HBOCs extravasate producing systemic vasoconstrictions while polymers with MW above 500 KDa do not extravasate. It should be stressed that vasoconstrictions due to NO scavenging can be pharmacological corrected [13] by either inhalation of gaseous NO or by IV administration of NO donors.

Oxygen affinity of HBOCs is a very flexible parameter.

Toxicity Challenge

This chapter is focused only on NO scavenging and hyperoxygenation because experimental data are available only for these two phenomena. Many other toxic events may be anticipated by speculation.

Regarding NO scavenging and systemic vasoconstriction resulting from HBOCs extravasing into the muscle layer of vessels, there is now a concourse of opinion that in order to avoid this problem it is sufficient to synthesize high MW HBOCs molecules [12,10,11]. The high oxygen affinity of polymeric HBOCs does not seem to be a problem.

Hyperoxygenation is a more difficult challenge. It is linked to the facilitated diffusion of oxygen produced by the translational component of HBOCs’ brownian motions. Viscosity inhibits brownian motions, thus inhibiting facilitated diffusion and hyperoxygenation. This may be the rationale behind the observation that adding PVP to infusing fluids, transformed the vasoconstriction produced by a Zero-link polymer into a pronounced vasodilation stimulated by viscosity-reduced blood flow (Figure 3) [12]. Viscosity of blood can be adjusted by plasma expanders, and should be explored as a means to reduce hyperoxygenation.

A promising novel approach is now emerging in the literature: the use of monooxygenated physiologic effectors like carbon monoxide (CO) and nitric oxide (NO) under the form of CO- and S-nitroso-HBOCs.

Vandegriff et al. [30] reported that solutions of carbonmonoxy-MP4 (a PEG decorated HBOC, Sangart, San Diego, CA) were more efficient than oxygenated-MP4 in reducing the volume of cardiac infarcts in rat exposed hearts. A reduction of 20% was obtained with CO-MP4. Very recently, a paper from the laboratory of Koehler [31] reported substantial reduction of mice cerebral infarcts produced by CO-PEG-Hb fluids obtained from Prolong Pharmaceutical (South Plainfield, NJ).

Kawaguchi et al. [32] and Asanuma et al. [33] reported that S-nitroso-PEG-Hb reduces the size of cerebral infarcts in rats, and are cardioprotective against heart ischemia, respectively.

The rationale behind these observations is that infused CO-HBOC are re-oxygenated by the mass action of plasma oxygen partially displacing CO. Thus the infused HBOC transports both CO and O2 to the periphery. SH-NO does not bind to heme and is simply transported to tissues by, and released from, S-nitroso-HBOC. Motterlini [34] produced evidence that CO may mimic NO as a relaxing factor.

Therefore both NO and CO are vasodilating effectors and, very importantly, powerful reducing agents. When released to tissues they will stimulate vasodilation while neutralizing oxygen radicals. These two events may be the cause of the beneficial outcomes reported above.

If this rationale is correct it is a very promising hypothesis.

Yield of Production

Several procedures have been described for the production of intra- and intermolecularly crosslinked HBOCs, purified from residual low MW polymers and non-polymerized hemoglobin.

Most in use is treatment with glutaraldehyde resulting in a distribution of polymers of different size [11,27]. In the experience of this writer, after elimination of low molecular weight species, the yield of non-extravasating polymers (MW>500KDa) is very low. Also, it is not clear how stable is the reduced Shiff base of the intermolecular links. Glutaraldehyde is very toxic. Residual traces in the infusion fluid may become significant when gram quantities of the polymer are infused.

More effective is polymerizing either human or bovine hemoglobin activating with carbodimide the carboxyl residues on the surface of hemoglobin so as to form direct pseudopeptide bonds with the amino groups of a neighbor molecule (Zero-link procedure) [12]. In the laboratory of this writer the final yield, after elimination of low molecular weight species, reaches 40% or more of the starting material. The obtained polymers were very large with MW in the range of 25 MDa.

Polymeric HBOC can also be obtained from recombinant hemoglobin or myoglobin enriched with surface SH groups [25]. The yield of polymerization is near 100%; however, the low yield of recombinant material is a serious challenge for products which, in vivo, should be used in gram quantities. Somatogen (Boulder, CO) developed a large-scale production of recombinant tetrameric HBOCs. The company is now closed.

Large size HBOCs are also obtained decorating the surface of hemoglobin with Polyoxyethylene glycol (PEG). The long PEG side chains added on the surface of the molecule confer to tetrameric Hb molecules high viscosity, high oncotic activity, and a large virtual excluded volume [28]. Tests for Hb residuals in the lymph have not been conducted. Still, PEG decorated hemoglobins do not produce hypertension, hence probably do not extravasate. Many papers produced at Albert Einstein Medical College and at UCSD in the laboratories of Drs. Acharya, Intaglietta, and Winslow prove PEG-HBs to be a very adequate HBOC ([29] is a recent publication with references).

Infusion of MP4, the PEG-Hb developed by Sangart (San Diego, CA) produced myocardial infarcts [1]. Whether the PEG derivative included in the fluid used for clinical trials maintained the laboratory characteristics described for MP4 is not known. The transport of a product from laboratory to industry may necessitate new synthesis protocols.

CAVEATS

HBOCs should not be considered blood replacement devices gram per gram to hemoglobin losses. Considering the facilitated diffusion, which involves also the oxygen transported by the red cells, the gram per gram infusion may be an overdose exposing the recipient to hyperoxygenations similar to reperfusion injuries.

A universal HBOC is probably not a real expectation. Different HBOCs will be applied to different clinical settings. The literature proposing better HBOCs than other ones is out of mark. We do not know at present what will be better or worse for human subjects in the field.

Two distinct classes of non-extravasating HBOCs have already been identified. Chemically obtained polymers like Z-link hemoglobins, are characterized by low viscosity, low oncotic activity and high oxygen affinity, and PEG decorated hemoglobins, are characterized by high viscosity, high oncotic activity and moderately high oxygen affinity. In vivo testing to evaluate the different responses of these derivatives would be very informative for the relevance of HBOC rheology to the efficiency and characteristics of peripheral oxygen delivery. It may be added that while the rheology of chemically obtained polymers can be adjusted adding to the fluid visco-oncotic adjuvants, this flexibility is much more limited for PEG-HBOCs.

It is necessary to stress that in vivo testing is conducted on young, healthy animals genetically controlled and that the experimental animals are sacrificed within a few hours or days. This time span may not be long enough for showing side effects. Thus, using these data to project therapeutic use of HBOC may result in unforeseen problems.

Moreover, Yu et al. [33] report that diabetes and dyslipidemia increased the pressor response of a polymeric HBOC infusion. The initial presence of a pressor response in control specimen may have distorted the data. Nevertheless it is possible to infer that damages to vascular endothelial surfaces produced by the disease further dilated their pores creating gaps, which would allow extravasation of additional small-size molecular species. This is where administration of either NO by inhalation or IV administration of nitrites may be useful [13]. It would be interesting to perform similar experiments using a non-extravasating HBOC, void of pressor response.

Inhalation of NO inhibits the vasoconstriction [13] due to NO scavenging, while CO inhalation protects from reperfusion injuries [35]. It may be stressed that both NO and CO are inactivating all heme-containing enzymes including citochrome systems, which interfere with vasoactivity and mitochondrial metabolism.

As a Final Consideration

This writer proposes that we have at hand a powerful means for improving and advancing clinical therapy. We have to learn how and when to administer with HBOCs calibrated amounts of oxygen.

Acknowledgements

This paper is a late effort generated by NIH program project P48517 (EB). A partial support of the Dept of Biochemistry and Molecular Biology of the University of Maryland. Medical School, Baltimore, MD, is also acknowledged. The paper is dedicated to E. Robert Winslow, scientist and dear friend.

Declaration of interest: Enrico Bucci is a consultant of OxyVita Inc (New Windsor, NY). No financial agreements or responsibilities.