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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Evaluating the Safety and Efficacy of Hemoglobin-based Blood Substitutes

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


General Overview

The development of a safe and effective blood substitute would greatly improve the emergency treatment of accident victims and wounded soldiers, as well as patients undergoing cardiac surgery, especially when whole blood is in short supply.

One type of artificial blood substitute that scientists have studied extensively is called a "hemoglobin-based oxygen carrier" (HBOC). HBOCs use the natural oxygen-carrying molecule called hemoglobin (Hb) to carry oxygen throughout the body. However, because the Hb used for HBOCs is not inside red blood cells it tends to accumulate to toxic levels in the blood. This cell free Hb can cause high blood pressure; Hb can also escape the blood vessels and damage the kidneys and other organs. Therefore, FDA has not approved any HBOCs for use in the United States, and the regulatory agencies of most other countries also have not approved HBOCs.

Our laboratory is trying to overcome the problem of Hb toxicity in order to enable industry to manufacture safe and effective HBOCs. We are studying how free Hb causes its toxic effects and are developing ways to prevent these effects.

We previously published results of a study in animal models showing that a molecule called haptoglobin found in blood can bind to Hb, preventing it from causing damage. Haptoglobin had this beneficial effect either when it was directly infused into animals or when animals were treated with a drug that increased the body's production of haptoglobin. These findings suggest that it might be possible to design safer HBOCs, since Hb bound to haptoglobin can still carry oxygen and release it to tissues.

Our work is contributing to the regulatory and research efforts of CBER to support development of safe and effective products that improve public health in the US and worldwide.


Scientific Overview

HBOCs have many potential advantages over human blood, including availability compatibility, and long-term storage. However, they also raise a number of concerns, including toxicity. Several major manufacturers recently terminated their clinical trials in the US because of significantly increased adverse events in patients who were infused with currently available investigational HBOC products.

The focus of research of the Biochemistry of Hemoglobin (Hb)-based Substitutes Section is on the structural-functional characterization of modified Hb in relation to its redox (reduction-oxidation) chemistry and toxicity. Specifically, we study the potential contributions of Hb-based reactive intermediates to oxidative and signaling cascades both in vitro and in vivo. We are also investigating several potential molecular interventions for directly or indirectly overcoming Hb toxicity in vitro and in vivo.

The basis of HBOC toxicity is poorly understood; since most research done by industry is proprietary, and there is only minimal exchange of information among investigators occurs. Our major contributions to the field of HBOCs include, 1) defining toxicological pathways that arise from and are driven by the heme prosthetic group of the molecule; 2) designing protective molecular strategies to suppress or control Hb oxidative side reactions; 3) correlating Hb and its various redox and oxygenation states with the expression of hypoxia-inducible factor (HIF-1alpha), an "oxygen sensor," and other hypoxia-sensitive genes in a model of exchange transfusion; and 4) defining the site-specific nature of the interaction between haptoglobin (Hp) and Hb, which is the basis of the Hb clearance pathway in human macrophages. More recently we discovered functions of Hp in controlling blood pressure effects that not only represent a new paradigm for blood-substitutes research but might also have clinical use in the treatment of hemolytic anemias.

Our mission-oriented laboratory research on the safety and efficacy evaluation of HBOCs has been published in major peer-reviewed journals and presented at national and international meetings.


Publications

Antioxid Redox Signal 2013 Jun 10;18(17):2264-73
Haptoglobin Binding Stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine beta145.
Cooper CE, Schaer DJ, Buehler PW, Wilson MT, Reeder BJ, Silkstone G, Svistunenko DA, Bulow L, Alayash AI

PLoS One 2013;8(3):e59841
Haptoglobin Preferentially Binds beta but Not alpha Subunits Cross-Linked Hemoglobin Tetramers with Minimal Effects on Ligand and Redox Reactions.
Jia Y, Wood F, Buehler PW, Alayash AI

Blood 2013 Feb 21;121(8):1276-84
Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins.
Schaer DJ, Buehler PW, Alayash AI, Belcher JD, Vercellotti GM

J Biol Chem 2013 Feb 8;288(6):4288-98
Alpha-hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of alpha subunits of human HbA with hydrogen peroxide.
Mollan TL, Banerjee S, Wu G, Parker Siburt CJ, Tsai AL, Olson JS, Weiss MJ, Crumbliss AL, Alayash AI

Trends Biotechnol 2013 Jan;31(1):2-3
Haptoglobin: the hemoglobin detoxifier in plasma.
Alayash AI, Andersen CB, Moestrup SK, Bülow L

Antioxid Redox Signal 2012 Nov 16 [Epub ahead of print]
Development of Recombinant Hemoglobin-Based Oxygen Carriers.
Varnado CL, Mollan TL, Birukou I, Smith BJ, Henderson DP, Olson JS

Protein Sci 2012 Oct;21(10):1444-55
Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin brigham (alpha(2) beta(2) (Pro100Leu) ).
Mollan TL, Abraham B, Strader MB, Jia Y, Lozier JN, Olson JS, Alayash AI

Free Radic Biol Med 2012 Sep 15;53(6):1317-26
Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions.
Banerjee S, Jia Y, Parker Siburt CJ, Abraham B, Wood F, Bonaventura C, Henkens R, Crumbliss AL, Alayash AI

Biochim Biophys Acta 2012 Sep 20;1820(12):2020-9
Heme binding to human alpha-1 proteinase inhibitor.
Karnaukhova E, Krupnikova SS, Rajabi M, Alayash AI

Nitric Oxide 2012 Jun 30;27(1):32-9
Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: Functional and antioxidative implications.
Roche CJ, Dantsker D, Alayash AI, Friedman JM

FEBS Open Bio 2012 May 24;2:113-8
Effects of carbon monoxide (CO) delivery by a CO donor or hemoglobin on vascular hypoxia inducible factor 1α and mitochondrial respiration.
Reiter CE, Alayash AI

Anal Chem 2012 Feb 7;84(3):1653-60
Slow histidine h/d exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry.
Tran DT, Banerjee S, Alayash AI, Crumbliss AL, Fitzgerald MC

Biochem Biophys Res Commun 2011 Dec 16;416(3-4):421-6
Inactivation of prolyl hydroxylase domain (PHD) protein by epigallocatechin (EGCG) stabilizes hypoxia-inducible factor (HIF-1alpha) and induces hepcidin (Hamp) in rat kidney.
Manalo DJ, Baek JH, Buehler PW, Struble E, Abraham B, Alayash AI

Nitric Oxide 2011 Aug 1;25(2):59-69
Effects of T- and R-state stabilization on deoxyhemoglobin-nitrite reactions and stimulation of nitric oxide signaling.
Cantu-Medellin N, Vitturi DA, Rodriguez C, Murphy S, Dorman S, Shiva S, Zhou Y, Jia Y, Palmer AF, Patel RP

Antioxid Redox Signal 2011 May 1;14(9):1713-28
Blood aging, safety and transfusion: capturing the "radical" menace.
Buehler PW, Karnaukhova E, Gelderman MP, Alayash AI

Tissue Eng Part A 2011 Apr;17(7-8):927-40
Biophysical properties and oxygenation potential of high-molecular-weight glutaraldehyde-polymerized human hemoglobins maintained in the tense and relaxed quaternary States.
Zhang N, Jia Y, Chen G, Cabrales P, Palmer AF

Biochim Biophys Acta 2011 Apr-Jun;1809(4-6):262-8
Induction of hypoxia inducible factor (HIF-1α) in the rat kidneys by iron chelation with the hydroxypyridinone, CP94.
Baek JH, Reiter CE, Manalo DJ, Buehler PW, Hider RC, Alayash AI

Transfusion 2010 Oct;20(5):341-5
Serial oxygen equilibrium and kinetic measurements during RBC storage.
Gelderman MP, Yazer MH, Jia Y, Wood F, Alayash AI, Vostal JG

Biochim Biophys Acta 2010 Oct;1804(10):1988-95
Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite.
Bonaventura C, Henkens R, De Jesus-Bonilla W, Lopez-Garriga J, Jia Y, Alayash AI, Siburt CJ, Crumbliss AL

Clin Lab Med 2010 Jun;30(2):381-9
Setbacks in blood substitutes research and development: a biochemical perspective.
Alayash AI

Biotechnol Bioeng 2010 May 1;106(1):76-85
Functional comparison of hemoglobin purified by different methods and their biophysical implications.
Elmer J, Buehler PW, Jia Y, Wood F, Harris DR, Alayash AI, Palmer AF

Antioxid Redox Signal 2010 Feb;12(2):181-4
Clearance and control mechanisms of hemoglobin from cradle to grave.
Schaer DJ, Alayash AI

Antioxid Redox Signal 2010 Feb;12(2):185-98
Hemoglobin can attenuate hydrogen peroxide-induced oxidative stress by acting as an antioxidative peroxidase.
Widmer CC, Pereira CP, Gehrig P, Vallelian F, Schoedon G, Buehler PW, Schaer DJ

Am J Respir Cell Mol Biol 2010 Feb;42(2):200-9
Mixed S-nitrosylated polymerized bovine hemoglobin species moderate hemodynamic effects in acutely hypoxic rats.
Irwin D, Buehler PW, Alayash AI, Jia Y, Bonventura J, Foreman B, White M, Jacobs R, Piteo B, TissotvanPatot MC, Hamilton KL, Gotshall RW

Biochim Biophys Acta 2009 Aug;1794(8):1234-42
Effects of cross-linking and zero-link polymerization on oxygen transport and redox chemistry of bovine hemoglobin.
Jia Y, Alayash AI

J Clin Invest 2009 Aug;119(8):2271-80
Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs.
Boretti FS, Buehler PW, D'Agnillo F, Kluge K, Glaus T, Butt OI, Jia Y, Goede J, Pereira CP, Maggiorini M, Schoedon G, Alayash AI, Schaer DJ

Blood 2009 Mar 12;113(11):2578-86
Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification.
Buehler PW, Abraham B, Vallelian F, Linnemayr C, Pereira CP, Cipollo JF, Jia Y, Mikolajczyk M, Boretti FS, Schoedon G, Alayash AI, Schaer DJ

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):10451-60
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):665-75
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 A

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 AI

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 AI

Anal Chem 2005 Jun 1;77(11):3466-78
Structural and Functional Characterization of Glutaraldehyde-Polymerized Bovine Hemoglobin and Its Isolated Fractions.
Buehler PW, Boykins RA, Jia Y, Norris S, Freedberg DI, Alayash AI

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

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 AI

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 AI

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 JM

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 AI

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 P

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 AI

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

     
 

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