Erythromer (EM), a Nanoscale Bio-Synthetic Artificial Red Cell: Proof of Concept and In Vivo Efficacy Results

BACKGROUND: There is need for an artificial oxygen (O2) carrier for use when: stored blood is unavailable or undesirable. To date, efforts to develop hemoglobin (Hb) based oxygen carriers (HBOCs) have failed, because of design flaws which do not preserve physiologic interactions of Hb with: O2 (they capture O2 in lungs, but do not release O2 effectively to tissue) and nitric oxide (NO) (they trap NO, causing vasoconstriction). EM design surmounts these weaknesses by: encapsulating Hb, controlling O2 capture/release with a novel 2,3-DPG shuttle and attenuating NO uptake through shell properties. METHODS: The EM prototype and its lyophilized form were analyzed: (1) structurally (dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM)), as well as for: (2) payload retention (Drabkin), (3) biocompatibility (ex vivo complement activation), (4) O2 affinity (p50, Hill n, Adair), (5) rheology (cone and plate viscometer in rabbit plasma), (6) NO consumption (chemiluminescence), (7) pharmacokinetic (PK) profile (tracking 99mTc-labeled EM in rats), and (8) in vivo O2 delivery (two rodent models: hemorrhagic shock [rats, instrumented for tissue pO2] and hemodilution [bioluminescent HIF-1α reporter mice]). RESULTS: EM was structurally stable (size: 175±10 nm; polydispersity: 0.26±0.0 by DLS, confirmed by TEM and AFM; zeta potential: 12±2 mV). After 3 months storage, we observed nominal change ( 0.96); which is likely to translate to a t1/2 in humans of ~ 3h. EM NO sequestration varied as a function of shell crosslinking and was below the rate observed for RBCs. In our hemorrhagic shock model in fully instrumented SD Rats (400g), 40% blood volume was removed; animals were then resuscitated with an equal volume of EM (N=6) or normal saline (N=6). EM was suspended at 40 wt/vol%, [Hb]=4mM. EM infusion rapidly stabilized hemodynamics. During the 1st hour, we observed resolution of both lactic acidosis (3.2±1.5 v 8.2±2.1 mM) [for EM and NS, respectively, throughout] and elevated AV O2 difference (24±11 v 67±23%) as well as improved brain pO2 (30.5±1.4 v 17.2±1.3 Torr); p CONCLUSIONS: The ErythroMer prototype has passed rigorous initial ex vivo and in vivo "proof of concept" testing and bench testing, which suggests this design surmounts prior challenges (by HBOCs) in emulating normal RBC physiologic interactions with O2 and NO. In models of major bleeding/anemia, EM reconstitutes normal hemodynamics and O2 delivery, observed at the system, tissue, and cellular level. EM potential for extended ambient dry storage has significant implications for portability and use. Next steps include formulation scaling, detailed study of pharmacokinetics, biodistribution and safety, as well as evaluation in large animal models of hemorrhagic shock. Disclosures Pan:KaloCyte, Inc.: Equity Ownership; Children9s Discovery Institute: Research Funding; National Institutes of Health: Research Funding. Spinella:KaloCyte, Inc.: Equity Ownership; Children9s Discovery Institute: Research Funding; National Institutes of Health: Research Funding. Hare:Children9s Discovery Institute: Research Funding. Lanza:KaloCyte, Inc.: Membership on an entity9s Board of Directors or advisory committees; National Institutes of Health: Research Funding. Doctor:KaloCyte, Inc.: Equity Ownership; Children9s Discovery Institute: Research Funding; National Institutes of Health: Research Funding.