Page 5

Red cell substitutes


Figure 4 NO binding and blood pressure in representative examples of hemoglobin-based O2 carriers. Test solutions were infused into rats at the time indicated by the arrow, and mean arterial blood pressure was continuously measured. The blood pressure responses are significantly different for the 3 classes of molecules, and the degree of blood pressure elevation is inversely proportional to the molecular size. The sizes of the molecules are shown at relative scale on the right,57 and the reaction rate constants for NO binding are given on the far right.35

tated diffusion.” Experiments in artificial capillaries confirmed that the release of O2 from cell-free hemoglobin could be controlled by alteration of oxygen affinity (P50) and size of the carrier molecule (Figure 5).38

Figure 5 Exit saturation as a function of residence time (flow rate) in an artificial capillary. Fully oxygenated solutions are injected into a capillary of 50 ␮m diameter and allowed to flow at different rates. The capillary is freely permeable to O2, and N2 is maintained outside the capillary. The effluent solution is collected, and the hemoglobin saturation is measured. The behavior of PEG-Hb is very similar to that of red blood cells, despite the large difference in their oxygen affinities (P50). Both ␣␣Hb and purified Hb release O2 very quickly, again despite a large difference in their P50, indicating molecular size (hence, diffusion) is more important than P50 in this experimental model.38

New Molecules These physiologic experiments defined the requirements for a new generation of oxygen carriers, in which the properties of the molecule were “tuned” to the requirements of the microcirculation to deliver O2 effectively to hypoxic tissue; their essential features were increased oxygen affinity (reduced P50), increased molecular size and homogeneous chemistry. Work by Acharya and coworkers at Albert Einstein University included a description of the conjugation of maleimideactivated polyethylene glycol (PEG) 5,000 molecular weight (MW in daltons) with sulfhydryl groups on the surface of hemoglobin.39 Originally, this technology was described using bifunctional PEG molecules for polymerization. But when the PEG was activated on only one end, the reaction resulted in specific attachments without polymerization, such that about 6 strands of PEG 5,000 MW were attached per molecule. The modification process was streamlined, and a method was established for production of a solution that could be used for research.40 The product, Hemospan (sometimes abbreviated MP4; Sangart, Inc, San Diego, CA), was formulated as 4.2 g/dL of maleimide-activated PEG-5000 hemoglobin conjugate (MalPEG-hemoglobin) in Ringer’s lactate, and the production process was of sufficiently high yield and potentially low cost to be commercially viable if the reagent were safe in animal and human trials. Zero-linked hemoglobin (ZL-HbBv) is polymerized bovine hemoglobin developed by researchers at the University of Maryland.41 Zero-linked hemoglobin is included with the fourth-generation products because coupling between molecules does not use crosslinking agents, such as glutaralde-

Red Cell Substitutes  
Red Cell Substitutes  

seminario en hematología