One or more 1st generation hemoglobin (Hb) based blood substitute products with adequate safety and efficacy profile may soon be approved for use in surgery. However there are unsettling issues related to nitric oxide (NO) physiology and lack of anti-oxidant properties among these 1st generation products Citation[1-2]. From my perspective of developing a 1st generation and a 2nd generation blood substitute Citation[3-4], I believe that we should continue to strive in the new millennium, for a product that outperforms whole blood, with respect of reduction of mortality and morbidity, in resuscitation scenarios where a blood substitute is critically needed. In these applications, an effective blood substitute needs antioxidant and anti-inflammatory properties to remedy the tissue ischemia, reperfusion and inflammatory injures that are common, for example, in severe hemorrhagic shock and ischemic stroke.
A successful 2nd generation blood substitute should manifest the following properties:
Anti-oxidant activities: The 2nd generation blood substitute needs to address the over-production of both superoxide (O2−) and hydrogen peroxide (H2O2) during reperfusion of ischemic tissues. This requirement has been met by either preparing an Hb co-polymer which incorporates both red cell superoxide dismutase and catalase Citation[[5]], or by the synthesis of a polynitroxylated Hb-based blood substitute with the antioxidant enzyme-mimetic activities Citation[[6]]. The 2nd generation blood substitute needs the capacity to remove the toxic oxylferryl (FeIV=0) intermediate Citation[[7]]. Free and covalently bound nitroxides in polynitroxyl Hb facilitate the return of FeIV=0 to FeIII with the release of oxygen; thus providing a catalase-mimetic activity Citation[8-9]. However, even when resuscitation can be initiated within the 20 minute post-hemorrhagic shock window, free radical injury can still occur from the over-production of peroxynitrite (ONOO−). The 2nd generation blood substitute needs to have the necessary antioxidant activity, to detoxify ONOO−Citation[[10]]. In addition, the formation of the most injurious hydroxyl radical (OH) from H2O2 requires catalysis by transition metal ions. Nitroxides prevent the redox cycling of these metal ions Citation[[11]].
Anti-inflammatory activities: Within the golden hour, free radical mediated injury can be reduced if treatment is initiated within 20 minutes of hemorrhagic shock. As the interval between the injury and treatment increases the shock becomes more refractory as resuscitation with shed blood 45 minutes post shock could not reduce mortality Citation[[12]]. An anti-inflammatory activity, derived in part from anti-oxidant activities, can be demonstrated in a 2nd generation blood substitute by its capability for in vivo inhibition of leukocyte/platelet aggregation and adhesion to microvessels Citation[[13]]. It is possible that a 2nd generation blood substitute with characteristics that include an inhibition of leukocyte/endothelium adhesion and a low P50 will facilitate capillary circulation and thereby reduce the severity of ischemia/reperfusion and inflammatory injuryCitation[[14]].
Vasodilation: A 2nd generation blood substitute can accomplish a beneficial reduction in Hb-associated vasoconstriction by incorporating a recombinant Hb molecule that is designed to reduce extravasation (e.g. recombinant Hb-dimer) and to reduce intrinsic nitrosylation activity (heme reactivity) Citation[[15]]. Limiting Hb-mediated nitrosylation in the vascular space has enabled three 1st generation blood substitutes to advance to phase III clinical trials in controlled surgical patients Citation[[16]]. An alternative method to reduce Hb-associated vasoconstriction is to covalently attach NO analogues to Hb, such as b93 S-nitroso Hb Citation[[17]] and polynitroxyl Hb Citation[[4]]. In a rat hemorrhagic shock model, resuscitation with polynitroxyl Hb compared with ααHb, did not produce pressor response, as a result, improved systemic hemodynamics, regional circulation, base deficit and survival time Citation[[18]].
Oxygen delivery: The 2nd generation blood substitute, because its intrinsic short plasma half life, should complement, instead of replace red cells in the delivery of O2. This is best accomplished with a lower than physiological P50 for oxygen delivery to the hypoxic tissueCitation[[19]]. Dextran Hb with a P50=3∼4 =mmHg was shown be able to meet the resting O2 demand at red cells levels that was as low as 2% hematocrit or lower in the dog Citation[[20]].
A number of 2nd generation blood substitutes have the potential to meet some or most of the above requirements. Their safety, cost and effectiveness to allogeneic blood transfusion will ultimately determine their viability as a 2nd generation blood substitute.
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