Synthesis and Properties of Polymerized, Diaspirin Cross-Linked Hemoglobins

Abstract

During the course of our studies it became clear that there were therapeutic applications for which a polymeric hemoglobin having an extended half-life in circulation would be appropriate. Therefore, a process for the glutaraldehyde-polymerization of diaspirin cross-linked hemoglobin (DCLHb) was developed and used to prepare glutaraldehyde-polymerized DCLHb (GP-DCLHb) in lactated Ringer's solution in sufficient quantities for biological testing. Both isovolemic exchange-transfusion and "top-load" studies (rats; primates and swine, respectively) were completed in which a broad spectrum of physiologic, histopathologic and analytical parameters were monitored and assessed. In general, GP-DCLHb in lactated Ringer's solution was well-tolerated physiologically. When compared to DCLHb, GP-DCLHb offers the advantages of reduced renal clearance of hemoglobin and an extended half-life in circulation. GP-DCLHb has the disadvantages that (1) glutaraldehyde is an ineffective virucidal agent under the conditions of the polymerization reaction and a separate virus inactivation step is required; (2) low-endotoxin (LAL-negative) GP-DCLHb solutions are pyrogenic (rabbits); and (3) unusual deposition of hemoglobin-containing material in the small arterioles of the liver and kidney (rats) was sometimes seen even after a period of time (2 weeks) during which treatment-related organ pathologies are usually resolved, a finding peculiar to GP-DCLHb among the various hemoglobin derivatives we have tested.

αα–Crosslinked Hemoglobin: Was Failure Predicted by Preclinical Testing?

“…The production of αα–Hb has been well documented, both by the US Army [30, 31, 32]and Baxter [33, 34, 35]. Although the same chemical derivative can be produced using bovine [36]or other hemoglobins, the commercial product was made using outdated human blood as raw material.…”

“…Although the oxygen binding of only the Army’s product was described in detail in the public literature [46, 47, 48, 49], nevertheless both products had a P50 and degree of cooperativity similar to that of human blood. A difference between the two, however, that remains problematic, is that the Army’s product was formulated in Ringer’s acetate, while Baxter’s was formulated in Ringer’s lactate [34]. Baxter would raise the question whether large doses of acetate in severely hemorrhaged animals could have accounted for the poor outcome in certain studies carried out at LAIR [unpubl.…”

“…General characteristics of the product were published in descriptions of the process, both by Baxter [33, 34, 37, 44, 53]and LAIR [30, 31, 32, 45, 54]. …”

“…Several preparations of modified αα–Hb were also prepared and studied, including polymerized material [51, 52]and polyethyleneglycol (PEG)–modified αα–Hb [34]. …”

Effects of diaspirin crosslinked hemoglobin infusion in treadmill-exercised swine

“…DCLHb also has a methemoglobin concentration similar to that of swine blood [7]. Infusion of DCLHb has been shown to slightly right-shift the oxygen dissociation curve, thus possibly assisting the delivery of oxygen to the perfused tissue [8]. DCLHb has been shown to have a variety of effects on the cardiovascular system including potentiating the effects of catecholamines and increasing mean arterial blood pressure (MAP) [9][10][11][12].…”