UC San Diego

Pathophysiological conditions characterized by chronic hemolysis (e.g. genetic and acquired hemolytic diseases) increase circulatory cell free hemoglobin (fHb), free heme and iron. These toxic species elicit a variety of adverse effects, including the scavenging of the potent natural vasodilator nitric oxide (NO) which decreases microcirculatory NO-bioavailability, decreased organ perfusion and renal function, end-organ damage, and increased mortality. Natural clearance mechanisms for fHb, free heme, and iron rely on the scavenger proteins hemopexin (Hpx) and haptoglobin (Hp), respectively. Unfortunately these proteins are depleted during hemolytic states. Thus, treatment of hemolytic conditions would benefit from administration of scavengers of fHb, free heme and iron. A possible functional alternative to Hpx is apohemoglobin (apoHb). ApoHb is produced by removing the heme from Hb and takes advantage of the natural high affinity for heme of the vacant heme-binding pockets in Hb. Hence, apoHb could serve as a novel, practical and scalable alternative to Hx in vivo scavenger for heme.However, apoHb major limitations for in vivo use are its pour stability at physiological temperatures, and short circulatory half-life. Fortunately, Hp can stabilize and extend circulation time of apoHb by forming an anpohemoglobin-haptoglobin (apoHb-Hp) complex. This apoHb-Hp complex binds heme and is more stable at physiological temperature compared to free apoHb. In addition to being able to bind free heme, we have discovered that the apoHb-Hp complex can scavenge free Hb in vivo and in vitro by exchanging Hp bound apoHb αβ dimers for Hb αβ dimers in the circulation. Lastly, to ensure an ease of use for manufacturing conjugation of polyethylene glycol (PEG) to the apoHb (PEG-apoHb) overcomes issues of pour stability and does not diminish the safety and efficacy of the product in physiological conditions.