Biological evaluation of diaspirin crosslinked hemoglobin solutions

To be clinically useful, blood substitutes must be efficacious and safe. To ensure the latter, a comprehensive testing plan has recently been recommended by the United States Food and Drug Administration. In response to these recommendations, and as part of a broad program to explore the biocompatibility of potential blood substitutes, we have tested solutions of diaspirin cross-linked hemoglobin (DCLHb) in a variety of in vitro and animal models. Studies directed to the assessment of DCLHb immunogenicity and pharmacokinetics are discussed in this presentation, while the results of other experiments are presented elsewhere in this symposium. Immunogenicity was tested by intravenously infusing five 5 mL/kg doses of DCLHb solution into rhesus monkeys at monthly intervals and evaluating whether this repetitive administration resulted in the development of antibodies or hypersensitivity to this modified hemoglobin. No IgG or IgM antibodies to DCLHb were detected in the sera of primates either during or subsequent to the multiple IV infusions, nor was there evidence of hypersensitivity when these animals were skin-tested with a DCLHb challenge. Thus, DCLHb solutions are not immunogenic when given intravenously to primates. To evaluate the distribution and metabolism of DCLHb, radioactivity-labelled material was synthesized and infused into rats which were subsequently sacrificed at time intervals ranging from 15 minutes to 14 days later. At sacrifice, blood and organs were collected and assayed for radioactivity by liquid scintillation counting and for DCLHb and metabolite content by high performance liquid chromatography. Urine and feces were also collected over various time intervals and similarly analyzed. DCLHb was not degraded during circulation within the bloodstream but was rapidly catabolized in tissues to low molecular weight compounds. These compounds were then excreted through the urine and feces. We therefore conclude that DCLHb is readily metabolized once it is removed from circulation and does not accumulate in tissues.