Current Drug Discovery Technologies - Volume 9, Issue 3, 2012

Blood substitutes, especially hemoglobin based oxygen carriers (HBOC) have been widely studied and reviewed over the past 30 years. The development of HBOCs was highlighted by crosslinking to minimize adverse effects. However, even early attempts at single crosslinking using alpha-alpha crosslinks or diaspirin crosslinking failed clinical trials due to renal morbidity and increased mortality. In fact, preclinical trials may have predicted failure of this first generation product, DCLHb (diaspirin-crosslinked Hb) (HemAssist ®, Baxter). In the 1980's, three small biopharmaceutical companies developed “second generation” HBOCs, the first being Hemosol with their raffinose crosslinked HBOC, hemoglobin- raffimer. The other two development programs modified the HBOC using glutaraldehyde polymerization, in an attempt to further alleviate the toxicities of the “first” generation HBOCs. This paper will review the definitive clinical trials of the two polymerized HBOCs, Biopure's hemoglobin glutamer-250 (bovine) and Northfield's polymerized human Hb, pegylated HBOC and Sangart's peg-conjugated HBOC, with an introductory brief review of Hemosol's hemoglobinraffimer. The paper will then introduce the newest polymerized hemoglobin, zero-linked hemoglobin polymer, which has not yet undergone clinical trials but has undergone some preclinical work that will be discussed in a section on this product. As a new generation HBOC, zero-linked hemoglobin polymer may begin to address the issues presented by the first two generations of HBOCs, and may hold promise as a universally applicable HBOC.

Extracellular soluble hemoglobins (Hbs) have long been studied for their possible use as safe and effective alternatives to blood transfusion. While remarkable progress has been made in the use of cell-free Hb as artificial oxygen carrier, significant problems remain, including susceptibility to oxidative inactivation and propensity to induce vasoconstriction. Hemarina-M101 is a natural giant extracellular hemoglobin (3600 kDa) derived from marine invertebrate (polychaete annelid). Hemarina-M101 is a biopolymer composed of 156 globins and 44 non-globin linker chains and formulated in a product called HEMOXYCarrier®. Prior work has shown Hemarina-M101 to possess unique anti-oxidant activity and a high oxygen affinity. Topload experiment with this product into rats did not revealed any effect on heart rate (HR) and mean arterial pressure (MAP). A pilot study with the hamster dorsal skinfold window chamber model showed absence of microvascular vasoconstriction and no significant impact on mean arterial blood pressure. In vitro nitric oxide (NO) and carbon monoxide (CO) reaction kinetics measurements show that Hemarina-M101 has different binding rates as compared to human Hb. These results revealed for the first time that the presence of this marine hemoglobin appears to have no vasoactivity at the microvascular level in comparison to others hemoglobin based oxygen carriers (HBOCs) developed so far and merits further investigation.

To attenuate hemoglobin's (Hb) intrinsic toxicity, Texas Tech University scientists developed a novel concept of “pharmacologic cross-linking” to formulate an effective oxygen carrier, HemoTech, which consists of purified bovine Hb cross-linked intramolecularly with ATP and intermolecularly with adenosine, and conjugated with reduced glutathione (GSH). In this composition, while ATP prevents Hb dimerization, adenosine permits the formation of homogeneous polymers. ATP also serves as a regulator of blood vessel tone via activation of the P2Y receptor, whereas adenosine counteracts the vasoconstrictive and pro-inflammatory properties of Hb via stimulation of adenosine A2 and A3 receptors. GSH introduces electronegative charge onto the Hb surface that blocks Hb's transglomerular and transendothelial passage. Besides, GSH shields heme from nitric oxide and reactive oxygen species, thus enhancing vasodilation and lowering Hb prooxidative potential. HemoTech underwent favorable initial pre-clinical testing and proof of medical concept, and is under commercial development by HemoBioTech Inc. HemoTech has entered the regulatory process in the US. Several mandated requirements have already been met, including viral/transmissible spongiform encephalopathy (TSE) clearance validation studies and various pre-clinical pharmacological, pharmacokinetic, toxicological, genotoxicity and efficacy tests. These studies provided further evidence that “pharmacologic cross-linking” of the Hb molecule with ATP, adenosine and GSH, is useful for designing a viable Hb-based oxygen carrier.

Many researchers have tested a hemoglobin (Hb) solution as a possible oxygen carrier after discovering that one Hb molecule contains four hemes that bind and release oxygen reversibly, and that blood type antigens are expressed on the surface of red blood cells (RBCs). However, various side effects emerged during the long development of Hbbased oxygen carriers (HBOCs). The physiological significance of the RBC structure is undergoing reconsideration. Fundamentally, excessive native Hb molecules are toxic, but encapsulation can shield this toxic effect. So-called liposomeencapsulated Hb or Hb-vesicles that mimic the cellular structure of RBCs have been developed for clinical applications.

Adverse outcomes in clinical trials on Hemoglobin Based Oxygen Carriers (HBOCs) appear to have occurred more frequently in HBOC treated than in control treated subjects. The differential may be related to many factors, including study complexity and compliance issues. Adverse outcomes also appear to be related to chronic comorbidities in subjects undergoing elective surgery. Frequently occurring comorbidities in these populations are those related to aging, cardiovascular and metabolic disease (hypertension, atherosclerosis, diabetes, etc.). These are highly prevalent among many population subsets. These conditions have been extensively studied and are characterized by dysfunction of important endothelial vasoregulatory mechanisms, including impaired nitric oxide bioavailability, excessive generation of reactive oxygen species (ROS) and possibly enhanced vasoconstrictor mechanisms. Although less extensively studied, HBOCs have properties that may have an important amplifying effect upon mechanisms operating in endothelial dysfunction, by scavenging nitric oxide, generating further excess of ROS which in turn react with nitric oxide, inhibit nitric oxide synthase and possibly stimulate the release of vasoconstrictors such as endothelin. It is likely that amplification of vasoconstrictor effects is not uniformly operative in all vascular beds, and that some protective autoregulatory mechanisms maintain sufficient blood flow in vital organs as long as sufficient vasodilator reserve is available. When the latter is exhausted in the presence of arterial disease with physical obstructions, blood flow to vital organs may become compromised. This paper suggests avenues of further exploration to elucidate whether the combination of HBOC and endothelial dysfunction is a contributing factor in HBOC related adverse outcomes.

Vasoconstriction is a major adverse effect of HBOCs. The use of a single drug for attenuating HBOC-induced vasoconstriction has been tried with limited success. Since HBOC causes disruptions at multiple levels of organization in the vascular system, a systems approach is helpful to explore avenues to counteract the effects of HBOC at multiple levels by targeting multiple sites in the system. A multi-target approach is especially appropriate for HBOC-induced vasoconstriction, because HBOC disrupts the cascade of amplification by NO-cGMP signaling and protein phosphorylation, ultimately resulting in vasoconstriction. Targeting multiple steps in the cascade may alter the overall gain of amplification, thereby limiting the propagation of disruptive effects through the cascade. As a result, targeting multiple sites may accomplish a relatively high overall efficacy at submaximal drug doses. Identifying targets and doses for developing a multi-target combination HBOC regimen for oxygen therapeutics requires a detailed understanding of the systems biology and phenotypic heterogeneity of the vascular system at multiple layers of organization, which can be accomplished by successive iterations between experimental studies and mathematical modeling at multiple levels of vascular systems and organ systems. Towards this goal, this article addresses the following topics: a) NO-scavenging by HBOC, b) HBOC autoxidation-induced reactive oxygen species generation and endothelial barrier dysfunction, c) NO- cGMP signaling in vascular smooth muscle cells, d) NO and cGMP-dependent regulation of contractile filaments in vascular smooth muscle cells, e) phenotypic heterogeneity of vascular systems, f) systems biology as an approach to developing a multi-target HBOC regimen.

The development of hemoglobin based oxygen therapeutics (HBOCs) requires consideration of a number of factors. While the enabling technology derives from fundamental research on protein biochemistry and biological interactions, translation of these research insights into usable medical therapeutics demands the application of considerable technical expertise and consideration and reconciliation of a myriad of manufacturing, medical, and regulatory requirements. The HBOC development challenge is further exacerbated by the extremely high intravenous doses required for many of the indications contemplated for these products, which in turn implies an extremely high level of purity is required. This communication discusses several of the important product configuration and developmental considerations that impact the translation of fundamental research discoveries on HBOCs into usable medical therapeutics.

Widespread clinical use of acellular hemoglobin (Hb)-based O2 carriers (HBOCs) has been hampered by their ability to elicit both vasoconstriction and systemic hypertension. This is primarily due to the ability of acellular Hb to extravasate through the blood vessel wall and scavenge endothelial-derived nitric oxide (NO). Encapsulation of Hb inside the aqueous core of liposomes retards the rates of NO dioxygenation and O2 release, which should reduce or eliminate the vasoactivity of Hb. Our aim is to determine the extent of systemic and microvascular vasoactive responses (hypertension, vasoconstriction and hypoperfusion) after infusion of vesicle encapsulated Hbs, in which the encapsulated Hb is in either the deoxygenated or carbon monoxide (CO) state (HbV and COHbV, respectively). To investigate this hypothesis, we used the hamster window chamber model subjected to two successive hypervolemic infusions of HbV and COHbV solutions (each infusion represents 10% of the animal's calculated blood volume) at Hb concentrations of either 7 or 10 g/dL. The hypervolemic infusion model used in this study has all the regulatory mechanisms responsible for predicting the vasoconstrictive responses of HBOCs. The results of this study demonstrate the absence of vasoconstrictive and hypertensive responses upon single and multiple infusions of HbV and COHbV solutions. The HbV and COHbV solutions increased the plasma O2 carrying capacity. However, COHbV delivered low therapeutic levels of CO without inducing any microcirculatory disturbances. Significance: Vesicles containing Hb can be used as a new therapeutic agent in transfusion medicine to treat anemia and revert hypoperfusion.