Evaluating the Safety and Efficacy of Hemoglobin-based Blood Substitutes

Principal Investigator: A.I. Alayash, PhD
Office / Division / Lab: OBRR / DH / LBVB

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Overview

Public Health Issue: Hemoglobin-based oxygen carriers (HBOCs) have many potential advantages over human blood, including improved availability, no need to screen for blood type compatibility, and improved long-term storage. Availability of FDA-licensed HBOCs would provide a significant medical advance for the treatment of trauma-associated blood loss in both military and civilian sectors.

Regulatory Contribution: The clinical use of these products has, however, raised a number of concerns, including manufacturing consistency and safety concerns. In addition, HBOCs manufacture results in heterogeneous products (i.e., non-uniform mixtures of modified hemoglobin or Hb) with variable chemical cross-linking, polymerization and surface modifications, making it challenging to characterize this product for consistent potency, efficacy and safety.

Research Approach: The laboratory uses a systematic approach to define the levels of heterogeneity of HBOC products, permitting CBER to evaluate the contribution of the product's component fractions towards the overall stability, oxygen carrying characteristics, safety profile and pharmacokinetics. For example, the risk of tissue damage caused by Hb's ability to initiate oxidative cascades (i.e., autooxidative reactions) can potentially lead to organ failure following HBOC administration. Our in vitro and in vivo studies have developed a number potentially useful protective approaches that can be used in controlling these reactions. We investigate the molecular basis for Hb oxidative side reactions and impact of chemical and/or genetic modifications on integrity and stability of the proteins. We have established 1) with a high degree of accuracy the site(s) of chemical modification on several HBOCs, 2) revealed heterogeneities in size, and non-specificity of modification that were not previously appreciated, 3) defined a number of toxicological pathways, 4) designed protective molecular strategies to suppress or control Hb oxidative side reactions, including the discovery that certain animals can effectively recycle blood levels of the antioxidant vitamin C, 5) correlated Hb and its various oxidative and oxygenation states with the expression of HIF-1a, an "oxygen sensor" and other hypoxia sensitive genes such as erythropoietin (EPO), and heme oxygenase (HO-1) in a model of exchange transfusion and 6) identified CD163 as the macrophage scavenger receptor for small molecular weight Hb species in the absence of haptoglobin, which forms the basis for alternative Hb clearance pathway by human macrophages 7) identified naturally occuring animal Hb (clam Hb, Lucina pectinata)as an oxidatively stable prototype for the desigin of a safe and effective HBOC.

Mission Relevance & Outcomes: The investigation of the structural and functional consequences of HBOC product variability and the impact of these changes on the viability of some HBOCs under normal and extreme physiological conditions has provided important information to improve product characterization and efficacy and reduce product toxicity.

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Publications

Biochim Biophys Acta 2008 Oct;1784(10):1378-81
All hemoglobin-based oxygen carriers are not created equally.
Buehler PW, Alayash AI

Biochim Biophys Acta 2008 Oct;1784(10):1415-20
Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC).
Cooper CE, Silaghi-Dumitrescu R, Rukengwa M, Alayash AI, Buehler PW

Free Radic Biol Med 2008 Oct 15;45(8):1150-8
The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways.
Vallelian F, Pimenova T, Pereira CP, Abraham B, Mikolajczyk MG, Schoedon G, Zenobi R, Alayash AI, Buehler PW, Schaer DJ

Free Radic Biol Med 2008 Sep 1;45(5):659-66
Effects of (-)-epigallocatechin gallate on the redox reactions of human hemoglobin.
Jia Y, Alayash AI

Biochem J 2008 Sep 15;414(3):461-9
Acellular haemoglobin attenuates hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes in haemodiluted rats.
Manalo DJ, Buehler PW, Baek JH, Butt O, D'agnillo F, Alayash AI

Antioxid Redox Signal 2008 Aug;10(8):1449-62
Structural stabilization in tetrameric or polymeric hemoglobin determines its interaction with endogenous antioxidant scavenger pathways.
Buehler PW, Vallelian F, Mikolajczyk MG, Schoedon G, Schweizer T, Alayash AI, Schaer DJ

J Pharmacol Exp Ther 2007 Oct;323(1):49-60
Effects of endogenous ascorbate on oxidation, oxygenation and toxicokinetics of cell-free modified hemoglobin after exchange transfusion in rat and guinea pig.
Buehler PW, D'Agnillo F, Hoffman V, Alayash AI

Biochemistry 2007 Sep 18;46(37):10451-10460
The Heme Pocket Geometry of Lucina pectinata Hemoglobin II Restricts Nitric Oxide and Peroxide Entry: Model of Ligand Control for the Design of a Stable Oxygen Carrier
Jesús-Bonilla WD, Jia Y, Alayash AI, López-Garriga J

IUBMB Life 2007 Aug;59(8):498-505
Allosteric effects on oxidative and nitrosative reactions of cell-free hemoglobins.
Bonaventura C, Henkens R, Alayash AI, Crumbliss AL

Antioxid Redox Signal 2007 Jul;9(7):991-9
Gating the radical hemoglobin to macrophages: the anti-inflammatory role of CD163, a scavenger receptor.
Schaer DJ, Alayash AI, Buehler PW

Expert Opin Biol Ther 2007 May;7(5):665-675
First-generation blood substitutes: what have we learned? Biochemical and physiological perspectives.
Alayash AI, D'Agnillo F, Buehler PW

J Biol Chem 2007 Feb 16;282(7):4894-907
Structural basis of peroxide mediated changes in human hemoglobin: A novel oxidative pathway.
Jia Y, Buehler PW, Boykins RA, Venable RM, Alayash AI

Biochem J 2006 Nov 1;399(3):513-24
Ascorbate removes key precursors to oxidative damage by cell free hemoglobin in vitro and in vivo.
Dunne J, Caron A, Menu P, Alayash AI, Buehler PW, Wilson MT, Silaghi-Dumitrescu R, Faivre B, Cooper CE

Anal Chem 2006 Jul 1;78(13):4634-4641
Chemical Characterization of Diaspirin Cross-Linked Hemoglobin Polymerized with Poly(ethylene glycol).
Buehler PW, Boykins RA, Norris S, Alayash AI

Blood 2006 Jan 1;107(1):373-80
CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.
Schaer DJ, Schaer CA, Buehler PW, Boykins RA, Schoedon G, Alayash AI, Schaffner.

Antioxid Redox Signal 2005 Nov-Dec;7(11-12):1755-60
Redox biology of blood revisited: the role of red blood cells in maintaining circulatory reductive capacity.
Buehler PW, Alayash A.

Proteins 2005 Jun 1;59(4):840-55
O-raffinose crosslinked hemoglobin lacks site-specific chemistry in the central cavity: structural and functional consequences of beta93Cys modification.
Boykins RA, Buehler PW, Jia Y, Venable R, Alayash A.

Anal Chem 2005 Jun 1;77(11):3466-3478
Structural and Functional Characterization of Glutaraldehyde-Polymerized Bovine Hemoglobin and Its Isolated Fractions.
Buehler PW, Boykins RA, Jia Y, Norris S, Freedberg DI, Alayash A.

Antioxid Redox Signal 2004 Dec;6(6):941-943
Redox biology of blood.
Alayash A.

Antioxid Redox Signal 2004 Dec;6(6):944-953
Effects of Cell-Free Hemoglobin on Hypoxia-Inducible Factor (HIF-1alpha) and Heme Oxygenase (HO-1) Expressions in Endothelial Cells Subjected to Hypoxia.
Yeh LH, Alayash A.

Antioxid Redox Signal 2004 Dec;6(6):1000-1010
Oxygen sensing in the circulation: "cross talk" between red blood cells and the vasculature.
Buehler PW, Alayash A.

Biochem J 2004 Dec 1;384(Pt 2):367-75
Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation.
Jia Y, Ramasamy S, Wood F, Alayash AI, Rifkind J.

Biochim Biophys Acta 2004 Jun 11;1672(3):164-73
Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran.
Jia Y, Wood F, Menu P, Faivre B, Caron A, Alayash A.

Hypertension 2004 May;43(5):1110-5
Hemodilution With Stoma-Free Hemoglobin at Physiologically Maintained Viscosity Delays the Onset of Vasoconstriction.
Rochon G, Caron A, Toussaint-Hacquard M, Alayash AI, Gentils M, Labrude P, Stoltz JF, Menu .

J Appl Physiol 2004 Mar;96(3):893-903
Differential effects of sodium selenite in reducing tissue damage caused by three hemoglobin-based oxygen carriers.
Baldwin AL, Wiley EB, Alayash A.

Nat Rev Drug Discov 2004 Feb;3(2):152-9
Oxygen therapeutics: Can we tame haemoglobin?
Alayash AI