Hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, recent late stage clinical results of polymerized hemoglobin (PolyHb) solutions (i.e. Hemopure? (OPK Biotech, Cambridge, MA), a glutaraldehyde polymerized bovine Hb; and PolyHeme? (Northfield Laboratories Inc., Evanston, IL), a glutaraldehyde polymerized pyridoxylated human Hb) hamper further development. Both of these commercial products elicit vasoconstriction at the microcirculatory level, and lead to the development of systemic hypertension and oxidative tissue damage. These side-effects are hypothesized to occur either by a nitric oxide (NO) scavenging or oxygen (O2) oversupply mechanism, and are both exacerbated by PolyHb extravasation into the tissue space. In light of these 2 potential mechanisms, it is apparent that PolyHb size will have a profound impact on the extent of vasoconstriction, systemic hypertension and oxidative tissue toxicity. However, commercial PolyHb products are complex mixtures with broad size distributions defined only by the size cutoff of the ultrafiltration membranes used in their manufacture. Furthermore, these mixtures are known to contain up to 1% of individual tetrameric Hb molecules and a significantly higher proportion of lower molecular weight (MW) Hb oligomers (80% with MW < 500 kDa). Hence, the side-effects observed during clinical/pre-clinical trials are attributed to a mixture of low MW Hb polymers with different sizes and points of chemical modification, and not to any one, single PolyHb molecule. This precludes precise characterization of how individual components of these complex PolyHb mixtures interact with the vasculature. An important advance would therefore be the ability to produce molecularly uniform, monodisperse, and high MW PolyHb nanostructures. In this application, we hypothesize that the molecular diameter and topology of recombinant PolyHb (rPolyHb) will regulate vasoactivity and oxidative injury to tissues. To test our hypothesis we propose the following specific aims:
Specific Aim 1 : Use orthogonal split splicing inteins to produce well-defined, monodisperse, high MW rPolyHb nanostructures.
Specific Aim 2 a: Analyze the role of endothelial function on the development of vasoactivity and oxidative tissue injury to rPolyHbs of varying size.
Specific Aim 2 b: Evaluate the pharmacokinetics of rPolyHbs in normal guinea pigs and HFSD guinea pigs.
Specific Aim 3 : Evaluate the ability of rPolyHbs to restore tissue oxygenation and optimize survival in severe blood loss. The proposed work is both significant and innovative, since it seeks to develop safe and efficacious rPolyHbs for use in transfusion medicine. In addition, state-of-the-art biophysical techniques and two unique animal models will be used to understand rPolyHb physiological responses and determine the clinical potential of these novel materials.
The U.S. blood supply is at risk due to the presence of emerging infectious diseases and reduced donation rates. In order to preserve the blood supply and protect the population, this application seeks to develop novel oxygen carrying solutions consisting of monodisperse, structurally well-defined recombinant polymerized hemoglobins that are safe and efficacious by screening these materials using an animal model with similar antioxidant status to humans.
