artificial oxygen carriers as red blood cell substitutes: a selected review and current status

Artificial Organs28(9):813–828, Blackwell Publishing, Inc.© 2004 International Center for Artificial Organs and Transplantation

813

Blackwell Science, LtdOxford, UKAORArtificial Organs0160-564X2004 International Society for Artificial Organs289813828Original ArticleRED CELL SUBSTITUTESH.W. KIM AND A.G. GREENBURG

Received November 2003.Presented in part at the 2003 Joint Congress of the International

Society for Artificial Organs and the American Society for Artifi-cial Organs, held June 18–21, 2003, in Washington, DC, U.S.A.

Address correspondence and reprint request to Dr. Hae WonKim, Department of Surgery, Brown University and The MiriamHospital, 164 Summit Avenue, Providence, RI 02906, U.S.A.E-mail: [email protected]

Artificial Oxygen Carriers as Red Blood Cell Substitutes: A Selected Review and Current Status

Hae Won Kim and A. Gerson Greenburg

Department of Surgery, Brown University Medical School; and The Miriam Hospital, Providence, RI, U.S.A.

Abstract: Two distinct approaches are being explored inred blood cell substitute (RCS) development: hemoglo-bin-based oxygen carriers (HBOCs) and perfluorocar-bon-based oxygen carriers (PFBOCs). HBOCs are basedon intra- and/or intermolecularly “engineered” human oranimal hemoglobins (Hbs), optimized for O2 delivery andlonger intravascular circulation. Some are currently beingevaluated in Phase II/III clinical studies. PFBOCs areaqueous emulsions of perfluorocarbon derivatives thatdissolve relatively large amounts of O2. A PFBOC basedon a 60% (wt/vol) emulsion of perfluorooctyl bromide

has been evaluated in Phase II/III clinical trials. Althoughcurrent PFBOC products generally require patients tobreathe O2 enriched air, they render certain advantagessince they are totally synthetic. This article provides ashort review of the basic principles, approaches, and cur-rent status of RCS development. Results of preclinicaland clinical studies including recent Phase II/III clinicalstudies are discussed. Key Words: Hemoglobin-basedoxygen carrier—Perfluorocarbon-based oxygen carrier—Blood substitute—Hemoglobin—Perfluorocarbon.

The history of “blood substitutes” is closely relatedto that of “blood transfusion” and can be tracedback to early human cultures. Modern scientificattempts to replace human blood started in the early1900s with better understanding of the oxygen trans-port/delivery function of erythrocytes (red bloodcells) and with new recognition that allogeneic bloodtransfusion can only be successful if a donor and arecipient have a matching blood type. Transfusiontherapy was born. The purpose of this article is notto provide a thorough and complete chronologicalreview of the development of artificial oxygen carri-ers as red cell substitutes. Rather, it is intended as abrief background and update on the current status ofartificial oxygen carrier research and clinical devel-opment for a broader audience including those newto the field without much technical knowledge.Substantial efforts exist toward non-red cell substi-

tute applications of artificial oxygen carriers but theyare beyond the scope of this review.

HEMOGLOBIN: AN OXYGEN CARRIER IN THE RED BLOOD CELLS

The complete 3-D structure of Hb was determinedin 1959 by Max Perutz for which he was awardeda Nobel Prize (1). Human hemoglobin (Hb) is a64 kDa tetrameric protein with two a subunits (141amino acids each) and two b-globin subunits (146amino acids each) folded in a compact quaternarystructure (a2b2) (Fig. 1A). Each a or b subunit con-tains one iron-heme group that reversibly binds oneoxygen molecule; when fully saturated with oxygenone Hb molecule carries four oxygen molecules. Intissues, where oxygen is consumed for cellular meta-bolic reactions, changes in local conditions (e.g., pO2,pH, temperature, pCO2) cause Hb to undergo a con-formational change from a high oxygen affinity state(R-state) to lower oxygen affinity state (T-state). ThisR-to-T state transition is facilitated by reversibleelectrostatic binding of 2,3-diphosphoglycerate(DPG), an allosteric effector, between the two b-subunits (b-pocket). This DPG binding decreases Hboxygen affinity facilitating oxygen offloading. While

814 H.W. KIM AND A.G. GREENBURG

Artif Organs, Vol. 28, No. 9, 2004

oxygen (a substrate for aerobic metabolism) is beingunloaded at tissues, CO2 (a cellular metabolic waste)binds to the primary amino groups of globin chains(e.g., N-terminal amino groups and e-amino group oflysine residues). The resulting carbamino-Hb is thentransported to the lungs. The amount of CO2 trans-ported in this way is approximately 20% of the totalCO2 transported in the blood. The remaining CO2 istransported as bicarbonates formed by the action ofthe erythrocytic enzyme carbonic anhydrase. Localconditions in the lungs (e.g., higher PO2, higher pH,and lower temperature) cause Hb to shift back to theR-state and dissociate the DPG. Conversion to R-state favors CO2 release while recombining with O2.The released CO2 is then exhaled. This Hb-mediatedaerobic respiration mechanism is summarized inFig. 2.

RED CELL SUBSTITUTES AND OXYGEN THERAPEUTICS: RATIONALE

AND APPROACHES

When a significant amount of blood is lost due toa traumatic injury or a surgical procedure, circulationthrough the vital organs diminishes causing inade-quate oxygen delivery to tissues. For moderate bloodloss where oxygen delivery is not seriously compro-mised, intravascular blood volume can be restoredwith nonoxygen carrying fluids such as crystalloids(e.g., isotonic saline, Ringer’s lactate) and colloids(e.g., human serum albumin, dextran, hetastarch).However, in severe blood loss, the lost oxygen trans-

port capacity of red blood cells must be restored ororgans cannot sustain their normal functions. Redblood cell transfusion provides additional or replace-ment oxygen carrying capacity improving tissueperfusion and oxygenation. Over the last century,allogeneic donor blood transfusion has evolvedas a lifesaving treatment for many acute anemicconditions.

Although blood transfusion is effective, it is notwithout risks. Allogeneic blood transfusion can causefatal hemolytic reactions, transmit blood-borneinfectious agents, and may compromise overallimmune function. Since the early 1980s when AIDSwas first identified, there has been a general tendencyto avoid allogeneic blood transfusion for fear of HIVand other infectious disease transmission. Allogeneicdonor blood transfusion has some drawbacks: itrequires typing and crossmatching, is limited in sup-ply, has a short storage life, and carries risk of diseasetransmission. It is therefore highly desirable to havean artificial oxygen-carrying fluid that is readily avail-able, free of infectious agents, and can be used inde-pendent of the recipient blood type. Search for a redcell substitute has been ongoing for decades but onlyrecently have a few candidates reached active clinicaltesting. Two distinct approaches, bio-artificial O2

carriers and totally synthetic O2 carriers are beingexplored (Table 1). Bio-artificial O2 carriers are pri-marily hemoglobin-based oxygen carriers (HBOCs)which utilize purified human, animal, or recombinantHbs as raw materials. Synthetic O2 carriers are eitherchemically synthesized metal chelates that mimic

FIG. 1. (A) A 3-D molecular representation of nativetetrameric human hemoglobin (HbA0), a common rawmaterial used in many HBOCs. Hb is a globular proteinconsisting of two a and two b-globin polypeptidechains. Each chain contains an iron-heme prostheticgroup (thick lined) that can reversibly bind one oxygenmolecule. Bovine Hb has a 3-D structure similar to thatof human Hb but there is a substantial difference in theamino acid composition between the two Hbs (74 of thetotal 574 amino acids). (B) A chemical structure and3-D rendering of perfluorooctyl-bromide (perflubron),an active agent used in Oxygent. The molecular lengthof perflubron is roughly 1/5 of tetrameric Hb which hasa diameter of approximately 50 Å.

10Å

A

B

aa1

a2b2

b1

RED CELL SUBSTITUTES 815


Hb’s reversible O2 binding (2) or artificial fluorinatedorganic fluids that physically dissolve large amountsof O2 (3). Currently, HBOCs and perfluorocarbon-based oxygen carriers (PFBOCs) are the mostpromising red cell substitute candidates and furtherdiscussions will be limited to these two types.

Oxygen therapeutics is the newly designatedbroadly defined term for agents (including artificialoxygen carriers) designed to deliver oxygen tohypoxic tissues and organs. HBOCs and PFBOCs are“oxygen therapeutics” not “blood substitutes” sincethey lack other components of blood such as coagu-lation factors and immune cells.

Hemoglobin-based oxygen carriers (HBOCs)With the understanding of Hb’s unique reversible

oxygen binding property and lack of blood typeantigen, the idea of using purified Hb as a possibleuniversal substitute for red cells has been aroundfor almost a century (4,5). Testing of crudely puri-fied hemoglobin in human subjects was reported asearly as 1916 (6) to study effects on anemia. Butthe first major clinical testing conducted with Hb-saline solution was associated with renal toxicities(7). It was later found that the “crude” Hb prepara-tions contained erythrocyte membrane stromal lip-ids and were often contaminated with bacterialendotoxins; both were considered causes of thenephrotoxicities. Hb solutions prepared “free” ofstromal lipids and endotoxin (stroma-free Hb;SFH) were tested and noted to be without signifi-cant nephrotoxicity (8). Shortly thereafter, twoother problems emerged: SFH was perceived tohave too high an oxygen affinity ( p50 of 10–15 mm Hg vs. 26–28 mm Hg for normal red cell Hb)and too short an intravascular circulation half-time(T1/2 < 1.5 h) to be useful. Acellular free SFH has ahigher oxygen affinity than native intraerythrocyticHb because DPG normally present in the red cellsis lost during purification. The high oxygen affinitywas perceived as detrimental to optimal oxygen off-loading to tissues. Acellular tetrameric Hb (a2b2) insolution readily dissociates into ab dimers that areeasily filtered through the kidneys and excreted inthe urine. The HBOCs in clinical testing today arenot just stroma-free and endotoxin-free Hbs butHbs chemically or genetically “engineered” to pro-duce desirable oxygen offloading characteristics andan extended circulation half-time. HBOCs currentlyin development as red cell substitutes are listed inTable 2 and some key approaches illustrated inFig. 3.

FIG. 2. A simplified diagram of oxygen (O2) and carbon dioxide(CO2) transport by hemoglobin during pulmonary and systemiccirculation. Venous blood arrives in the pulmonary circuit withmostly deoxygenated, 2,3-diphosphoglycerate (DPG) bound,and protonated Hb in a “tense” state (or T-state). Local condi-tions (e.g., higher pO2, higher pH, lower temperature and pCO2)favor Hb to dissociate DPG, release CO2, and shift to the“relaxed” form (or R-state). The R-state Hb has a higher O2

affinity allowing O2 binding to the heme groups. The releasedCO2 is exhaled. When arterial blood circulates through the met-abolically active organs/tissues, local conditions (e.g., lowerpO2, lower pH, higher temperature and pCO2) favor Hb to shiftback to the T-state. This R to T-state transition is facilitated byreversible electrostatic binding of DPG, an allosteric effector, tothe two b-subunits (b-pocket). This DPG binding decreases Hboxygen affinity facilitating oxygen offloading. While oxygen (asubstrate for aerobic metabolism) is being unloaded to tissues,CO2 (a cellular metabolic waste) binds to the primary aminogroups of globin chains (e.g., N-terminal amino groups and e-amino group of lysine residues). The resulting carbamino-deoxyHb compound is then transported back to the lungs viavenous blood. The amount of CO2 transported in this wayamounts to approximately 20% of the total CO2 transported inthe blood. The rest of CO2 is transported as bicarbonatesformed by the action of the erythrocytic enzyme carbonic anhy-drase. Again, the local conditions in the lungs favor Hb to shiftback to the R-state allowing CO2 release while recombiningwith O2. During the process, DPG dissociates from the Hb. Thewhole cycle is repeated as circulation continues.

Hb( H+ )-CO2 + O2

Hb( H+ )-CO2 + O2 HbO2 + CO2 + DPG

Tissues

Lungs

HbO2

Hb(H+)-CO2

HbO2 + CO2 + H++ DPG

– DPG(T-state)

(R-state)

(R-state)

(T-state)

DPG

DPG

DPG

TABLE 1. Key approaches to red blood cell substitute (RCS)

Bio-artificial oxygen carriersModified human or animal hemoglobin-based oxygen carriers

(HBOCs)Stabilized Hb tetramers (intramolecular crosslinking)Polymerized Hbs (intermolecular crosslinking)Conjugated HbsLiposome encapsulate Hbs (Hb vesicles)

Recombinant/transgenic HbsAlbumin-heme hybridsSynthetic oxygen carriers

Synthetic metal chelatesLipid-heme vesiclesHb aquosomesPerfluorocarbon based oxygen carriers (PFBOCs)



Stabilized Hbs (Tetrameric Hbs)One way to alleviate the high oxygen affinity of

SFH is through the use of functional DPG analogsthat specifically attach to the DPG pocket (9). Forexample, the negatively charged pyridoxal-5¢-phosphate (PLP) is a DPG analog that specificallyinteracts with the positively charged amino residuesof the DPG binding site (e.g., Lys82b, His2b,His143b). The reactive aldehyde group of PLP isused to crosslink PLP to the primary amino groupsof the DPG pocket (valines of b1 and b2) via a Schiffreaction which is then reduced forming a covalentbond. Pyridoxylated SFH (PLP-Hb) has a nearnormal oxygen affinity ( p50 = 22–24 mm Hg) butis still dissociable into ab-dimers susceptible to renalexcretion because there is no interdimeric cross-linkage. Using a DPG analog with bifunctional alde-hydes (e.g., 2-nor-2-formyl pyridoxal-5¢-phosphate;NFPLP), low oxygen affinity Hb with an interdimericlinkage between the two primary valines of b1 andb2 subunits (b stabilized Hb) was produced (10). Hbcan also be stabilized using an a-specific crosslinkerbis (3,5-dibromo salicyl)-fumarate (DBBF or dia-spirin) to produce a 99a1–99a2 crosslinked Hb(DCLHb) (11). DCLHb has been shown to have ap50 of 30 mm Hg and substantially longer in-travascular circulation time than unmodified SFH.

Polymerized HbsIntravascular retention times of HBOCs can be

further increased by intermolecular crosslinking(polymerization) of stabilized Hbs using crosslink-ers with bi- or poly-functional groups. For example,PLP-Hb can be polymerized using glutaraldehyde,a bifunctional nonspecific crosslinker, to producepoly (PLP-Hb) with t1/2 of over 30 h following apartial exchange transfusion in adult baboons (12).A low oxygen affinity oligomeric HBOC with desir-able circulation characteristics has been producedusing ring-opened raffinose (o-raffinose), a hexa-functional crosslinker (13,14). With this uniquecrosslinker prior intramolecular stabilization isunnecessary. At 10 g Hb/dL, this o-raffinose HBOCdelivers 4.3 mL O2/dL under the normal arterio-venous oxygen gradient, close to the normal oxygendelivery capacity of blood (Fig. 4). An alternativeapproach to circumvent the high oxygen affinityand limited availability of human SFH is to utilizelow O2 affinity bovine Hb as a starting material(15). Bovine Hb has a naturally low oxygen affinitycompared to human Hb. It is not DPG dependentand can be directly polymerized without priormodification to achieve desired the oxygen affinityand circulation time (16). Potential immunogenicityand transmission of animal-borne disease such as

TABLE 2. HBOCs in development as red blood cell substitutes

Product Hb source Technology Developer Status

DCL-Hb (HemAssist)

Human red cells a-a crosslinked Hb Baxter Healthcare(Deerfield, IL, U.S.A.)

Phase III (trauma)(suspended)

RHb1.1/1.2 (Optro)

E. coli Recombinant human aa fused Hb

Baxter Healthcare(Somatogen < 1998)

Phase II (discontinued)

RHb2.0 E. coli Recombinant Hb Baxter Healthcare PreclinicalHBOC-201

(Hemopure)Bovine red blood

cellsGultaraldehyde

polymerizationBiopure (Cambridge, MA,

U.S.A.)BLA filed (elective

surgery). Approvedfor clinical use in S. Africa

Human POE-Hb(PHP)

Human red cells PEG conjugation Curacyte (Apex)(Munich, Germany)

Phase III (septic shock)

Hb-raffimer (Hemolink)

Human red cells Oligermerization with o-raffinose

Hemosol (Toronto, Canada) Phase III (cardiac surgery) (suspended)

Pyridoxal polyHb(PolyHeme)

Human red cells PLP-Hb polymerized Hb with glutaraldehyde

Northfield Laboratories(Evanston, IL, U.S.A.)

Phase III (trauma) Filed BLA

Hemospan Human red cells Conjugated with maleiimide PEG

Sangart(San Diego, CA, U.S.A.)

Phase II (elective surgery)

HemoZyme Human red cells Polynitroxylated Hb SynZyme(Irvine, CA, U.S.A.)

Preclinical

PolyHb-SOD-CAT Bovine red cells Hb modified with SOD and catalase

McGill University(Montreal, Canada)

Preclinical

PEG-Hb Bovine red cellls PEG conjugated Hb Enzon (Piscataway, NJ, U.S.A.)

Phase Ia (discontinued)

OxyVita Human/bovine red cells

Stabilized Hb with sebacoly diaspirin

IPBL Pharm. (Goshen, NJ) Preclinical

HemoTech Bovine red cells Modified Hb with o-ATP, o-adenosine, and glutathione

HemoBioTech (Amarillo, TX, U.S.A.)

Preclinical



bovine spongiform encephalopathy (BSE) could beof concern with these products.

Conjugated HbsThe intravascular circulation time of a HBOC can

be extended by conjugating Hb with a macromole-cule (17). Human or bovine Hb conjugated withpolyethylene glycol (PEG or sometimes called poly-oxyethylene, POE) appears to protect the moleculefrom renal excretion (18–20). The oxygen affinity ofPEG-Hb can be lowered by first linking to PLP (18).Generally, PEG-Hbs have a larger effective molecu-lar size and exhibit higher viscosity than native Hbat comparable concentration. A bovine Hb conju-gated with PEG was tested in preclinical and PhaseI clinical studies but discontinued (19). Recently,MalPEG-Hb (MP4 or Hemospan), a different typeof PEG modified human Hb has been developed(20). The MP4 is produced by first introducing addi-tional surface thiols with iminothiolane onto the Hbtetramer (which has only two reactive cysteines atthe b93 positions) adding an average of 5 additionalthiols which are then reacted with activated maleiim-ide polyethylene glycol-5000 (PEG-5000). The pro-cess is reported to produce a homogenous productwhich contains 99% pure MalPEG-Hb requiring noadditional purification steps. The conjugation reac-tion of thiolated Hb with PEG is reported to yield 6conjugated thiols with an average molecular weight(MW) of 95 kilodaltons (kDa). This HBOC is a“counterintuitive” HBOC since it has lower Hbconcentration (4 g Hb/dL), higher viscosity (2.5 cP),higher oxygen affinity (pP50 = 6 mm Hg), and highercolloidal oncotic pressure (55 mm Hg) than mostother HBOCs in development (20). This product was

FIG. 4. Oxygen transport characteristics of aHBOC and a PFBOC are graphically comparedwith that of normal whole blood. Normal wholeblood (15 g Hb/dL) can carry approximately 20 mLof O2 (OCC) but delivers only 5 mL O2 per 100 mLblood (ODC) under normal arterial and ven-ous oxygen tensions (paO2 and pvO2 = 100 and40 mm Hg, respectively). Under the same condi-tion, OCC/ODC values of a HBOC (Hb-raffimer,10 g Hb/dL) and a PFBOC (Oxygent, 0 g Hb and60% perflubron emulsion) are 13/4.3 mL O2 and7/1.3 mL O2, respectively. Of note, an ODC of aPFBOC can be achieved to a level equivalent tonormal blood if paO2 of 300 mm Hg or higher ismaintained by inspiring O2-enriched gas (e.g.,FiO2 > 0.6). For simplicity, changes in other rele-vant physiologic O2 transport parameters that maybe affected by these agents are not considered inthis illustration (e.g., total Hb concentration, hemo-dynamic parameters, temperature, pH, pCO2, andshifts in Hb oxygen affinity during arterio-venouscirculation, etc.).

0 100 200 300 400 500 600 7000

5

10

15

20

25

ODC = 5 mL

ODC = 4.3 mL

ODC =1.3 mL

PaO2PvO2

WB (15 g Hb/dL)

Hb_Raffimer (10 g Hb/dL)

PFBOC (60 g PFOB/dL)ODC ~ 5.0 mL

PO2 (mm Hg)

O2

Car

ryin

g C

apac

ity

(mL

O2/

dL

)

ODC ~ 5.0 mL

FIG. 3. Some key approaches of Hb-based oxygen carriers asred blood cell substitutes are illustrated. Stroma-free Hb (SFH)and purified Hb solution prepared from human or bovine redblood cells are the most commonly used raw material. (A, B)Tetrameric stabilization is usually accomplished by intramolecularcrosslinking between the two a or two b subunits using a site-specific bifunctional crosslinker such as di-bromo bis-fumarate(DBBF or diaspirin) and 2-nor-2-formyl pyridoxal-5¢-phosphate(NFPLP) (e.g., NFPLP-Hb, DCLHb, respectively). Similarly, par-tially stabilized Hb can be obtained using a b-pocket specificmonofunctional agent such as pyridoxal-5¢-phosphate (PLP), aDPG analog (e.g., PLP-Hb). (C) Effective molecular weight of Hbcan be increased by conjugating to polyethylene glycol (or poly-oxyethylene) (PEG/POE-2000 or 5000) with or without prior reac-tion with PLP or other DPG analogs (e.g., POE-PLP-Hb, bovinePEG-Hb). (D) Polymerized Hbs of molecular weights significantlygreater than native Hb tetramer (64 kDa) are produced whenHbs, without or with tetrameric prestabilization, are reactedwith polyfunctional crosslinking agents (e.g., glutaraldehyde, o-raffinose, o-adenosine). (E) SFH can also be encapsulated insubmicrometer size liposomes or synthetic membrane vesiclescontaining antioxidant and other enzymes (LEH, Hb nanove-sicles). Please refer to the text for other approaches that arecurrently in development.

DPG analog

a ab b

SFH(5 nm)

AB

E

C D

Cross linker

Heme

(< 1 mm)

* Not to scale



shown to improve microcirculatory blood flow andtissue oxygenation in animal studies (21). Whetherthis product also improves perfusion in the microcir-culation of the critical organs (e.g., brain, heart, lung,liver, kidney, etc.) remains to be seen. The higherviscosity of MP4 could cause increased cardiac work-load, an additional burden to the heart in patientswith hypovolemic shock.

Erythrocytic Hbs are well protected from uncon-trolled oxidation due to a protective cellular environ-ment including antioxidant enzymes. In contrast, freeHbs in solution are more susceptible to oxidant stressresulting in production of harmful radicals such assuperoxide anion (O–*), hydroxyl radical (OH*), orhydrogen peroxide (H2O2). To reduce oxygen radicalmediated damages (reperfusion injuries), a polyHbconjugated with superoxide dismutase and catalase(Hb-SOD-CAT) has been developed (22). Similarly,bovine Hb intramolecularly stabilized with o-ATP,intermolecularly crosslinked with o-adenosine, andsubsequently conjugated with glutathione has alsobeen developed (23).

Hemoglobin vesicles (Hb encapsulated, embedded, and coated vesicles)

In 1957, it was first demonstrated that free Hbcould be encapsulated in a polymer membrane (24).Later, human or animal SFH encapsulated in phos-pholipid vesicles (liposomes) was developed as apotential red cell substitute (25–27). Similarly, totallysynthetic heme imbedded between two lipid bilayers(lipid-heme vesicles) has been developed (28). A sta-ble fat mircrosphere suspension has been achievedby emulsifying triglycerides with lipid-heme as a sur-factant. These lipid-heme products are reported tohave heme concentration and reversible O2 bindingclose to that of normal blood.

More recently, Hb and red cell enzymes (e.g., met-Hb reductase, SOD, CAT, and others) encapsulatedin nanometer size biodegradable polymer (polylac-tide or polyglycolides) vesicles has been developed(24,29). Unlike the lipid vesicles, these nanocapsulescould be prepared to be permeable to glucose andother molecules needed for methemoglobin (metHb)reduction. These Hb nanocapsules could contain Hbconcentration as high as 15 g/dL and maintained nor-mal p50, Bohr, and Hill coefficients (24).

Earlier liposomal or embedded Hbs had two majorproblems: a short intravascular circulation half-life(t1/2 < 2–3 h) and metHb formation (>15% of totalHb) (24,30). The circulation half-life could beimproved significantly through surface modificationusing negative surface charges, sialic acid analogs, orPEG. The circulation t1/2 was extended to more than

24 h. MetHb formation was reduced by incorporat-ing reducing enzymes (e.g., metHb reductase sys-tem). There have been studies in animal models withexchange transfusion and hemorrhagic shock.Results were mostly successful without significantadverse effects reported but complement activationwas shown to occur in rats and pigs (31,32).

Recently, a new type of HBOC called “Hb aquo-somes” has been developed (33). The Hb aquosomeswere prepared by coating Hb molecules on the sugarcoated hydroxyapatite nanoparticles. This productcould load up to 13–14 mg Hb/g of hydroxyapatitecore, had oxygen affinity and cooperativity similar tonative erythrocytic Hb, and was stable for at least30 days. In 50% exchange transfused rats, no unde-sirable changes in physiologic parameters (e.g., bloodpressure, heart rate) were reported.

Hybrid HbsRecently, synthetic porphinatoiron (II) complexes

(e.g., FepivP [Im], FecycP [His]) conjugated to arecombinant human serum albumin (rHSA) havebeen developed (34,35). In this albumin-heme hybridapproach, as many as 8 porphinatoiron (II) complexescould be absorbed to a rHSA molecule. These albu-min-heme hybrids showed reversible oxygen bindingunder physiologic conditions. Of these, rHSA-FecycP(Im) appears to be promising as a red cell substitutesince it has shown to have a p50 similar to nativeerythrocytic Hb and intravascular circulation timegreater than 36 h in anesthetized rats (35,36). It wouldbe of some advantage to use a natural plasma proteinlike HSA as a component of an oxygen carrier solu-tion as it is without undue adverse physiologic effects.

Recombinant/transgenic HbsWith recent advances in recombinant DNA tech-

nologies, native or specifically modified Hbs may beproduced from microorganisms (E. coli, yeast, etc.),transgenic plants, or animals. For example, prestabi-lized recombinant human Hb was produced in E. coliand S. cerevisiae using an expression vector contain-ing two mutant human globin genes, one with a lowoxygen affinity mutant and another tandemly fuseda-globins (37). Recombinant a-a crosslinked humanHb products produced in E. coli did advance toclinical trials but were discontinued due to vasocon-striction and other adverse effects (38). A second-generation product with reduced vasoactivity hasbeen developed and is being tested in preclinicalstudies (39). Human Hb has also been produced intransgenic animals (e.g., mice and pigs). Human aand b globin gene constructs are injected into newlyfertilized mouse or pig eggs and the resultant embryo



developed in a surrogate mother (40,41). Red cellsof transgenic animals thus born contain authentichuman Hb. Harvesting and purification of desiredHb product from these animals is, however, morecomplicated since the red cells contain hybrid Hbs aswell as the animal’s own Hb and human Hbs. Theeffectiveness and economics of these approaches areyet to be revealed.

Perfluorocarbon based oxygen carriers (PFBOCs)An entirely different approach to O2 delivery can

be achieved by using certain organic chemicals withhigh gas solubility. Perfluorinated carbons are gener-ally chemically and biologically inert liquids butdissolve large amounts of gases. The amount of dis-solved oxygen in perfluorocarbons is proportional toambient oxygen tension according to the Henry’sLaw. A mouse submerged in a pure perfluorocabonsolution saturated with oxygen could sustain life foran extended period (42). However, perfluorocarbonsare oil-like fluids that do not mix with water andcannot carry water-soluble salts and metabolic sub-strates. To be useful as an oxygen carrying red cellsubstitute, perfluorocarbons must be dispersed in aplasma like aqueous fluid such as albumin or in phys-iologic electrolyte solution. Current PFBOCs aregenerally stable emulsions of one or more perfluoro-carbons in aqueous media using various emulsifyingagents (surfactants) such as Pluronic-68, egg yolkphospholipids, and triglycerides. In some cases, acolloidal agent (e.g., hydroxyl ethyl starch or HES)is added to balance colloidal osmotic effect. Afterextensive exploratory experiments with various per-fluorochemicals, several perfluorocabon emulsionshave been developed as potential oxygen carryingred cell substitutes and are in various developmentalstages (Table 3).

The first perfluorocarbon emulsion developed asan oxygen carrier was Fluosol-DA (Green Cross

Corp., Osaka, Japan), a 20% (wt/vol) coemulsion ofperfluorodecalin and perfluorotripropylamine withegg yolk phospholipid and Pluronic-68 as emulsifyingagents. Because emulsions contain much less perflu-orochemicals per volume compared with pure liq-uids, the amount of oxygen they could dissolve is alsoless. For examples, breathing ambient air and withthe normal arterial and venous oxygen tensions (100and 40 mm Hg, respectively), Fluosol-DA coulddeliver only 0.4 mL oxygen per 100 mL. To meet themetabolic oxygen demand, the fraction of O2 in theinspired gas (FiO2) patients were required to breathewere 100% oxygen (i.e., FiO2 = 1.0), a situation tobe avoided clinically due to the adverse effects ofelevated oxygen concentration on the lungs (e.g.,oxygen toxicity).

Recently, Oxygent (Alliance Corp. San Diego, CA,U.S.A.), a stable 60% (wt/vol) emulsion of perfluoro-octyl bromide (perflubron) (Fig. 1B) has beendeveloped using egg yolk phospholipid as the soleemulsifying agent (43,44). Under normal arterialand venous oxygen tensions, Oxygent can unloadas much as 1.3 mL oxygen per 100 mL (Fig. 4), aremarkable improvement in oxygen delivery capac-ity (ODC). Yet, the oxygen delivery capacity of Oxy-gent is less than 30% of normal blood (5 mL O2/100 mL blood at 15 g Hb/dL) and may still requireoxygen-enriched air breathing to ensure adequateoxygen delivery. Unlike Fluosol-DA, Oxygent has alonger intravascular half-life (t1/2 = 9 h at 4 kg/Kg inrats) and is excreted from the body in about 4 days(compare to months for some components of Fluo-sol-DA) (45). The longer circulation time and shorterbody residence time of Oxygent are considered moredesirable for most clinical applications. However, insome earlier animal studies with perflubron emul-sion, “flu-like” symptoms with fever were observed(46). The “flu-like” response was secondary to therelease of arachidonic acid metabolites since they

TABLE 3. PFBOCs in development as red blood cell substitutes

Product Developer Perfluoro compounds Status

Fluosol-DA Green Cross Corp. (Osaka, Japan) Perfluorodecalin, Perfluoropropylamine Approved for clinical use for perfusate for angioplasty in 1989. Withdrawn from market in 1994.

Oxygent Alliance Corp. (San Diego, CA, U.S.A.) Perfluorooctyl bromide (perflubron) Phase II/III (suspended)S-9156 Sonus Corp. (Seattle, WA, U.S.A.) Dodecafluoropentane (DDFP) Preclinical. Stabilized

microbubbles for ultra small volume resuscitation

PHER-O2 Sanguine Corp. (Pasadena, CA, U.S.A.) Similar to Fluosol-DA? Preclinical?Perftoran Perftoran (St. Petersburg, Russia) Perfluorodecalin, Perfluoromethyl-

cyclohexylpiperidinApproved by Russian Ministry of

Health for clinical use (1999)Oxycyte PFC Synthetic Blood International

(Kettering, OH, U.S.A.)N/A Preclinical?

Oxyflour HemaGen (St. Louis, MO, U.S.A.) Perfluorodichloro octane Preclinical



were alleviated after administration of corticoster-oids or long half-life cyclooxygenase inhibitors. Suchresponses were related to the perflubron circulationhalf-life that was inversely related with emulsion par-ticle size (47). In anesthetized dogs with acute nor-movolemic hemodilution to 3 g Hb/dL, 60% (wt/vol)perflubron emulsion was effective in maintainingtissue oxygenation (48).

A couple of other PFBOCs based on similar prin-ciples but with different ingredient have been devel-oped and are being tested (Table 4). Of note, S-9156(Sonus Corp, Seattle, WA, U.S.A.), a PFBOC formu-lated as stable microbubbles is reported to dissolve asufficient amount of O2 so that it can be used for ultrasmall volume (1/500 of typical perfluorocarbon dose)resuscitation application (49).

REGULATORY APPROVAL PROCESS FOR RED CELL SUBSTITUTES

In the US, development of a drug, or a biologic inthe case of red cell substitutes, is a long and compli-cated series of scientific and regulatory processes. Asimplified version of the drug/biologic developmentprocess is shown in Table 4. A typical processincludes discovery, preliminary validation studies,preclinical studies, and clinical studies to demon-strate efficacy and safety of a candidate drug or bio-logic product.

PRECLINICAL SAFETY (TOXICITY) STUDIES

Preclinical developmental studies are designed tofulfill two primary objectives. First, they are to pro-duce data that support and validate the effectiveness(efficacy) of a therapeutic candidate for its intendedclinical use. Second, preclinical studies are also con-ducted to test potential toxicity (safety) of a thera-peutic candidate. Toxicity is generally assessed interms of changes in physiologic parameters, bloodand urine chemistries, and organ/tissue function, his-topathology, and other relevant indicators.

Several red cell substitutes have successfully com-pleted preclinical safety studies and moved on toclinical trial phase. Still others are in early preclinicaltest stages designed to obtain preliminary safety andeffectiveness data. A variety of protocols using awide range of in vitro and in vivo models were used.These include cell/tissue cultures, isolated perfusedorgans, and whole animal models including rodents,rabbits, pigs, sheep, and nonhuman primates. Preclin-ical programs generally include tests of major organsystem toxicity (safety) including cardiovascular tox-icity, renal toxicity, and hepatotoxicity. In addition toeffects on immune and reproductive systems, mech-anism of absorption, distribution, metabolism, andelimination (ADME) are also investigated. Reportsof preclinical studies on various candidate red cellsubstitutes are too numerous to be discussed here.The following relates some of the representative pre-clinical studies conducted for a typical oxygen carrier(primarily Hb-raffimer).

Cardiovascular/hemodynamic studiesTransient hypertension is one of the most fre-

quently observed effects of many HBOCs (50–53). In10% topload of conscious and anesthetized rats,Hb-raffimer caused increase in mean arterial bloodpressure (MBP) (54). The hypertensive effect wasgenerally more pronounced in the conscious animals;the MBP increases were 19–20% and 16–28% fornormotensive and spontaneous hypertensive rats,respectively. In pentobarbital/isoflurane anesthetizedrats, a similar dose of Hb-raffimer produced MBPincreases of 16% and 9% for normotensive andhypertensive rats, respectively. Frequently, compen-satory decreases in cardiac output were observed inHb treated animals. In a study of rats with 20%exchange transfusion, Hb-raffimer caused a signifi-cant but relatively moderate increase in mean arte-rial blood pressure (~9%) (55). The systemic vascularresistance was only 30% of that typically elicited byunmodified HbA0. In this model, cardiac output wasnot altered.

TABLE 4. Regulatory process involved in development of red cell substitutes† in the US

1. Discovery of a potential agent2. Preliminary in vitro and in vivo animal testing (efficacy testing)3. Decision to develop a therapeutic product (drug or biologic

product)Comprehensive in vitro and in vivo animal studiesEfficacy studies in relevant animal modelSafety (toxicity) studiesFormulation and dosing development, etc.

4. Investigational New Drug Application (IND)Phase I Clinical studies

Single dose safety studyMultiple-dose safety study, etc.

Phase II Clinical studiesInitial efficacy studiesDose definition studies in patientsPharmacokinetic studies, etc.

Phase III Clinical studies“Pivotal” efficacy studies (randomized, blinded, placebo

controlled, if applicable)Active control studies (new treatment vs. current treatment)Long-term safety studies, etc.

5. New Drug Application (NDA) or Biologic License Application (BLA)

6. Post-market surveillance

† Classified as a biologic product.



The exact cause of the certain HBOC mediatedvasoconstriction/hypertension has not been eluci-dated. Hb scavenging of endothelium-derived NO, apotent vascular relaxation factor, may play a key rolesince nitric oxide (NO) has a high affinity for ferrousHb (56–59). However, endothelin and other mecha-nisms may also contribute to the Hb mediated vaso-activity (60–62).

Repeat dose studiesUnder certain clinical situations, multiple/repeat

administrations of an oxygen therapeutic may benecessary. Multiple-dosing renders a greater poten-tial for toxicity because it may produce higher bloodconcentrations of the material. Overwhelming thenormal clearance/catabolism (liver, spleen, kidneys,etc.) may cause accumulation of potentially toxicbreakdown products.

In a rat study of 14-day daily repeat dosing of Hb-raffimer (5–30 mL/kg/day, IV), dose-related slowingof weight gain was observed (63). There were tran-sient pigmentations in the skin, liver, and the kidneys.Similar results were also obtained from a 14-day top-load repeat dosing (1030 mL/kg/day) in a dog study(63). In these studies, aspartate aminotransferase(AST), creatine phophokinase, amylase, and totalbilirubin levels were elevated. Urine was dark andcontained “bilirubin” stained cells. No death wasnoted in either study. All clinical pathologies werereversed over a 14-day recovery period. The repeatdosing subjected the animals to prolonged exposureto Hb-raffimer resulting in unphysiologically highconcentrations in the blood. The cumulative totaldoses were many times those anticipated in clinicaluse of this therapeutic and were tolerated well inthese animal models. The biochemical and histologicfindings were consistent with strained Hb catabolicmechanisms due to abnormally high loads of Hb.These findings suggest that this HBOC is processedthrough the normal Hb catabolic pathway.

ImmunogenicityMost current HBOCs are based on modified Hbs.

Modified human Hbs are not identical chemically tonative human Hbs and could, in theory, triggerimmune responses. In rats with a single 5% topload,Hb-raffimer did not elicit significant immuneresponse (63). However, in a rabbit study with twosubcutaneous immunization doses of 3 mg/kg, Hb-raffimer was found to be a stronger immunogen thanpurified HbA0 (Hemosol, personal communication).Hb-raffimer produced antibodies identical to puri-fied HbA0. In another study with two intramuscularinjections of 13 mg/kg, Hb-raffimer had a different

interaction with antisera compared to unmodifiedhuman Hbs. There was no evidence that novelepitopes were introduced upon chemical crosslinkingof human hemoglobin with oxidized raffinose. Theissue is of particular concern for nonhuman Hb-based oxygen carriers since serious hyperimmune(e.g., anaphylactic shock) responses could result ifrecipients were presensitized (64). Bovine Hb has asubstantially different amino acid sequence (74/574residues) from that of human Hbs but no seriousimmune or allergic response has been reported todate in the bovine Hb preparations tested clinically.

PharmacokineticsAbsorption, distribution, metabolism, and elimi-

nation of most HBOCs have not been well estab-lished. In terms of pharmacokinetics, plasma half-life(t1/2) has shown to be a function of molecular size,concentration, and volume infused. For example, inrats, t1/2 of 3H-Hb-raffimer was 5 h following a 10%topload (6.5 mL/kg) (65). The clearance followedfirst-order kinetics and accumulated in the kidneys,liver, and spleen as assessed by percentage total ini-tial radioactivity. Brain tissue had minimal radio-activity indicting that Hemolink did not cross theblood–brain barrier. With a 10% topload, t1/2 of oli-gomeric (>64 kDa) moieties in plasma were 13.2 h inrats and 5.8 h in mice as measured by size exclusionchromatography (66). The overall t1/2 was 7.8 h forrats and 4.4 h for mice. An optimal t1/2 is difficult todefine and should depend on specific applications.For example, if intended to treat chronic anemia, thet1/2 should be as long as possible but if the intendeduse is “bridging” until red cell availability, t1/2 as shortas one hour may be acceptable.

Renal effectsInitial attempts to use crude Hb solutions as red

cell substitutes were hampered by renal toxicity notunlike that seen in the hemolytic transfusion reac-tions. With modified Hb-derived HBOCs the inci-dence of adverse renal effects appears to be minimalfor all of the modern HBOCs tested to date. Chem-ical modification of Hb that increases the effectivemolecular size appears to prevent glomerular filter-ing and the previously noted adverse renal effects.For example, while unmodified Hb increased renalvascular resistance two fold and glomerular filtrationrate (GFR, an indicator of renal function), by 58%,Hb-raffimer had no deleterious effect on GFR, renalblood blow, or renal vascular resistance (55). Frac-tional GFR and reabsorption increased (67). It hasalso been shown that perfusion with a cell-freeHBOC better preserves or restores renal blood flow



and function following an acute ischemic renal event(67–69).

CoagulationHemodilution up to 50% of blood volume replace-

ment with a HBOC should not significantly impairnormal coagulation mechanisms. Under normal con-ditions, NO constitutively produced in the vascularendothelium and platelets is known to play a role inpreventing undue platelet activation and procoagu-lant reaction cascade. Because Hb has a high affinityfor NO, HBOC could decrease blood NO leveltriggering the hemostatic mechanism. However, intesting to date no unusual coagulopathy has beenreported. Coagulation assays including standardplatelet activation and functional assays, phosphoti-dylserine exposure, microparticle generation, andplatelet activation markers (PAC-1, CD41/62/63)were not significantly different from those of controlsamples treated with Ringer’s lactate (70).

Gastrointestinal effectsIntravenous infusion of some HBOCs caused dys-

phagia in animals and human subjects (71–74). Insome animal models, increased esophageal peristalticvelocity, decreased lower esophageal sphincter relax-ation, increased jejunal tone, and phasic contractionwere observed. These effects were reversed by cal-cium-channel blocker (74,75). NO release from themyenteric neurons of the esophageal smooth muscleis believed to be a primary factor for the relaxationof the lower esophageal sphincter and peristalticlatency in response to swallowing (72). Therefore, Hbinteraction and scavenging of NO, a crucial nonadr-energic noncholinergic (NANC) inhibitory neu-rotransmitter, may be involved in the alteredesophageal motor function. Other mechanismsincluding increased vagal tone could be operative aswell.

CLINICAL TRIALS OF RED CELL SUBSTITUTES (OXYGEN THERAPEUTICS)

The primary purpose of clinical studies is the dem-onstration of safety and efficacy of an oxygen carrierfor a defined clinical indication (76,77). Clear dem-onstration of clinical efficacy is key to success in clin-ical trials. However, meaningful demonstration ofefficacy cannot be achieved without establishing asafety profile of the oxygen carrier (78). In demon-strating efficacy, selecting a clinically meaningfulendpoint is a major issue. A clinical endpoint for anoxygen carrier should depend on clinical indications

(e.g., regional perfusion, acute hemorrhagic shock,peri-surgical applications) and may include improvedsurvival, decreased requirement for transfusion, orlaboratory and clinical indicators that correlatemeaningfully with clinical benefit.

Clinical trials of HBOCs as red blood cell substitutesEarly attempts to use crude Hb solution in human

subjects were not successful. In 1949, Ambersoninfused crude Hb-saline solution to a postpartumanemic patient (7). Following Hb-saline infusion,blood pressure rose significantly but the patientdeveloped renal failure and died. Later, it wasdiscovered that crude Hb contained erythrocyticmembrane stroma which might have caused thenephrotoxicity. Methods were developed to produce“stroma-free” Hb solution (SFH), essentially puri-fied Hb relatively free of red cell membrane stromaand lipids. In 1978, Savitsky infused SFH to normalhuman volunteers (79). All subjects treated with SFHshowed a transient hypertension with concomitantbradycardia. In addition, their renal function wasdepressed (decreased urine output and creatinineclearance) during and after the infusion.

In the early 1980s, modified Hb-based HBOCs thatelicit virtually no renal toxicity were developed.These HBOCs were also designed for increasedintravascular circulation time and reduced oxygenaffinity (higher p50) to facilitate O2 offloading at thetissues. Since then, several HBOCs have been testedin various phases of clinical trials in the US, Europe,and other countries.

One of the first HBOCs tested in the clinical trialswas a human Hb crosslinked with di-aspirin (DCLHbor HemAssist; Baxter, Deerfield, IL, U.S.A.).DCLHb has been extensively tested in various pre-clinical and clinical studies. However, in Phase IIIstudies, patients treated with DCLHb had signifi-cantly higher mortality rates than those of controlgroups (80–82). In a study with severe traumatichemorrhagic shock (80), 98 patients (of the total 112enrolled) were treated with 500–1000 mL of eitherDCLHb or saline solution. At 48 h, 20 of the 52patients (38%) infused with DCLHb died while 7 ofthe 46 patients (15%) in control group had died. At28 days, the mortality rates were 46% and 17% forDCLHb and saline control groups, respectively. In astudy of 85 patients with acute ischemic stroke, 25–100 mg/kg DCLHb or saline was administered (81).More serious adverse events and deaths occurred inDCLHb-treated patients than in saline-treatedpatients. In elective surgery patients, DCLHb treat-ment did allow a significant reduction in bloodtransfusion (81,83). However, serious complications



occurred in two aortic surgery patients treated withDCLHb. One patient developed an acute respiratorydistress syndrome and another multi-organ failureresulting in death. In June 1998, upon recommenda-tion of FDA, the study was suspended due to safetyconcerns (83).

A human Hb modified with pyridoxal phosphatefollowed by glutaraldehyde polymerization (Poly-Heme; Northfield Laboratories, Evanston, IL,U.S.A.) has been evaluated in Phase II clinical stud-ies (84). In acute hemorrhage, PolyHeme transfusionis reported to be as effective as allogeneic transfusionin maintaining total Hb concentration with lessallogeneic transfusion (85). Currently, PolyHeme isbeing tested in a pivotal Phase III prehospital traumastudy. The study proposes to demonstrate safety andefficacy of PolyHeme in improving survival whenused to treat severely injured bleeding traumapatients at the scene of injury and during transit tothe hospital. Of note, this study will be conductedwith an informed consent waiver. Due to the natureand extent of injuries, patients eligible for the studywill be unable to provide informed consent. Federalregulations allow clinical research without informedconsent under certain emergency settings. It is antic-ipated that over 700 patients will be enrolled in thisstudy from approximately 20 Level I trauma centersacross the country (Northfield Laboratories pressrelease, May 22/June 12, 2003).

Hb-raffimer (Hemolink, Hemosol, Missisauga,Ontario, Canada), a human Hb modified with multi-functional o-raffinose, has been tested in a series ofclinical trials (86,87). In patients undergoing electivecoronary artery bypass graft surgery (CABG;N = 60), Hb-raffimer used as intraoperative autolo-gous donation (IAD) diluent significantly reducedthe amount of donor allogeneic blood use in thesepatients (87). Patients were randomized to receiveeither Hb-raffimer or 6% hetastarch (control) insequential escalating doses of 250, 500, and 750 mL.In the group of 40 patients given 750 mL, 8 of 18 Hb-raffimer treated patients avoided allogeneic red celltransfusion (44%) while only 4 of 22 control patients(18%) did. However, in early 2003, Hemosol volun-tarily suspended a Phase IIb cardiac surgery study(HLK 213) when it discovered an imbalance in theincidence of certain adverse cardiac effects, a highernumber occurring in the Hemolink treated group(Hemosol Inc. press release, June 11, 2003). Hemosolelected to terminate the study and is currently inves-tigating the causes of the imbalance. Analysis of clin-ical data suggests that diabetes and gender may haveplayed a role. Of note, the Phase IIb study was adifferent model of use.

Hemoglobin glutamer-250 (HBOC-201 or Hemo-pure, Biopure Inc., Cambridge, MA, U.S.A.), abovine Hb polymerized with glutaraldehyde, hasbeen tested as an alternative to red cell transfusionin elective surgical cases (88,89). In 2001, Hemopurewas approved in South Africa for treatment of adultsurgical patients who are acutely anemic and for thepurpose of eliminating, reducing, or delaying theneed for allogeneic red cell transfusion in thesepatients. In October 2002, Biopure filed a biologiclicense application (BLA) to US FDA to marketHemopure in the US for a similar indication in ortho-pedic surgical patients. In August 2003, the FDArequested additional information including clarifica-tion of certain clinical and preclinical data beforemaking its decision to allow marketing the product.To prevent possible contamination with pathogensthat cause BSE and other transmittable diseases, theraw bovine Hb is obtained from a specially managedherd of cattle.

A bovine Hb conjugated with PEG (PEG-Hb;Enzon, Piscataway, NJ, U.S.A.) was tested in Phase Isafety testing in healthy volunteers without seriousadverse effects (19) but no further studies have beenreported. Of note, a human PLP-Hb conjugated withPEG originally developed as a HBOC in Japan (18)is now in a Phase III clinical trial in the US as anantihypotensive agent for distributive shock patientsbased on Hb’s ability to scavenge sepsis-induced NO(90, Curacyte News Release, July 1, 2003).

In addition, MP4 (Hemospan), another PEG-mod-ified human Hb-based HBOC (Sangart Corp., SanDiego, CA, U.S.A.) reported positive results from itsPhase I study with healthy volunteers conducted inSweden in the spring of 2002 (August 2, 2002 Sangartpress release). No adverse effects attributable toHemospan were reported. Of note, subjects treatedwith Hemospan did not experience any hypertensionor gastrointestinal distress. Sangart has now initiateda Ib/II clinical trial in Sweden for Hemospan. It isexpected to enroll 30 patients undergoing electiveorthopedic surgery but no detail regarding the studyprotocol is available.

Two human Hbs produced in E. coli, rHb1.1 andrHb1.2 (Optro, Somatogen, Boulder, CO, U.S.A.),were the first recombinant human Hbs tested in clin-ical trials. Some Phase I/II clinical trials with Optro(5 g Hb/dL) were conducted but discontinued due tohypertensive effect, symptoms suggestive of pyroge-nicity, and other adverse effects (38). The companywas acquired by Baxter Pharmaceutical and a sec-ond-generation product, rHb2.0 with reduced vaso-activity, has been developed and plans for clinicaltestings are being developed (39).



Recent clinical trials of PFBOCs as red cell substitutes

The first perfluorocarbon emulsion clinically testedand approved for clinical use (in 1989, as perfusate forcoronary angioplasty) was Fluosol-DA (Green CrossCorp., Osaka, Japan). It was shown that Fluosol-DAwas effective as a transcatheter perfusate during cor-onary angioplasty surgery (91). However, because oflow oxygen delivery capacity under physiologic con-ditions, cumbersome storage/preparation require-ment, and lack of clear clinical benefit, Fluosol-DAwas withdrawn from the market in 1994 (91–94).

Recently, Oxygent (Alliance Corp., San Diego,CA, U.S.A.), a 60% (wt/vol) perfluorooctyl bromide-based PFBOC with a higher oxygen-dissolving prop-erty and improved emulsion stability, has been testedin clinical trials (95,96). Delayed febrile responses(increase in body temperature by 1–2 degrees) andmoderate thrombocytopenic episodes (<20% reduc-tion in platelet count) were reported to haveoccurred at high dose (1.8 g PFC/dKg) (43,95). Thecause(s) of these adverse effects have not been iden-tified. Because perfluorinated carbons in general arechemically and biologically stable, potential long-term biological effects including absorption, distri-bution, metabolism, and excretion and effects onthe reticuloendothelial system require evaluation.Because current PFBOCs cannot dissolve sufficientO2 under ambient inspired air conditions, an O2-enriched gas is required for patients to achieve ade-quate oxygen delivery. In a recently completed PhaseIII study, acute normovolemic hemodilution withOxygent could provide significant benefit, reductionin allogeneic blood use in patients undergoing elec-tive major noncardiac surgery (96). In this study, FiO2

of the Oxygent group was increased to 1.0 vs. 0.4 forthe controls. At 24 h post-administration, the Oxy-gent group did receive fewer red cell units than thecontrol group but the difference was no longer sig-nificant after postoperative day 3. Only in a subgroupof patients whose blood loss was greater than 20 mL/kg, did the difference remain significant. The inci-dence of adverse events was comparable in bothgroups but more serious adverse events (e.g., cardio-vascular events) occurred in the Oxygent group.Most notably, although statistically not significant,the mortality was twice as high in the Oxygent groupthan in the control (4% vs. 2%). In early 2001, aPhase III cardiac surgery study was terminated earlydue to a significant imbalance in cerebral adverseevents (strokes). Analysis of clinical safety data sug-gests that the adverse events may be linked to overlyaggressive autologous blood harvesting prior to car-diac surgery (95).

A recent report suggests that Perftoran, a 10%perfluorocarbon emulsion, was approved by the Rus-sian Ministry of Health for clinical use in 1996 (97).According to the report, Perftoran has been testedin over 2000 patients by the year 2000 for variousclinical conditions including severe anemia, hemor-rhagic/traumatic shock, cerebral ischemia, and car-diac surgery. It claimed the use of Perftoran reducedischemic/hypoxic damage, improved blood rheology/hemodynamics, and decreased edema. However,many details including dosage and nature of anyadverse effects are not readily available.

LESSONS FROM PRECLINICAL AND CLINICAL STUDIES

Most current candidate red cell substitutes wereshown to be generally safe in preclincal and Phase Iclinical safety studies. However, various adverseeffects ranging from mild to severe have emerged inrecent Phase II/III clinical studies. Most adverseeffects were generally mild in nature and reversible.However, some imbalance of serious adverse events(SAE) has occurred in oxygen-carrier treatedpatients in later phase clinical trials. These SAEsoccurred in both HBOC and PFBOC therapeuticsand were not predicted by preclinical and early phaseclinical studies. Because of essential differences ingenetics and physiology between animal models andhuman patients, results from preclinical efficacy andsafety studies do not always translate into efficacyand safety in actual human patients although they aregenerally relatively good predictors of problems. Tobe clinically useful oxygen therapeutics must be freeof serious toxic side-effects. Therefore, a key ques-tion for a preclinical program is: can preclinical stud-ies predict serious adverse effects in the humansubject? Several HBOCs were reported to be safe ina variety of preclinical study models but adverseeffects varying in severity have emerged during clin-ical studies (80, 83, 95; Hemosol News Release June11, 2003). In these cases, results from preclinical ani-mal model studies or Phase I clinical trials failed topredict serious potential adverse effects. Animalsused in preclinical studies are generally healthyand young with vigorous homeostatic mechanisms.For many protocols in preclinical studies, conditionssimulating relevant human pathology such ashemorrhagic shock and cardiac bypass surgery arecreated abruptly in anesthetized animals. Physiologicresponses of such animals may elicit differentresponses from those of humans. In contrast, humanclinical trials are generally conducted in patients withmultiple underlying pathologies including the condi-



tion for which an oxygen therapeutic is indicated.Extrapolation from one to the other is complex. Withapplications of advanced knowledge in molecularbiology and genetics, we may better understand dif-ferences and similarities between human and animalmodels, improving the predictive power of preclini-cal animal studies for potential human adverseresponses.

Of note, both HBOCs and PFBOCs have beenreported to interfere with certain clinical laboratorytests (e.g., serum creatinine, oximetry, coagulationtests) (98,99). When interpreting laboratory testresults of patients following administration of oxygentherapeutics, appropriate corrective measures mustbe considered.

OTHER POTENTIAL INDICATIONS/BENEFITS

Solid hypoxic tumors are often resistant to radia-tion and chemotherapy. Because HBOCs, PFBOCs,and oxygen therapeutics have generally lower viscos-ity than blood, they may provide conditions forimproved perfusion in solid tumors, thereby resultingin higher tumor oxygen tension. Therefore, adminis-tration of oxygen therapeutics prior to radiation/che-motherapy may increase tumor sensitivity to thesetherapeutic modalities. Similarly, red cell substitutesmay be useful in ischemic rescue (e.g., myocardial orcerebral ischemia). In addition, in conjunction witherythropoietin, administration of a HBOC mayaccelerate erythropoiesis. Some of these potentialapplications have previously been discussed (100).

CONCLUSION AND FUTURE

In the US today, blood transfusion is safer thanever. The risk of HIV and hepatitis B transmissionhas been reduced to nearly one in two million (101).However, there persists in the population concernabout the safety of the blood supply. There stillremain risks of infection from blood obtained frommarker-negative “window phase” donors or donorsinfected with rare pathogens or new emerging infec-tious diseases (102). Further, there is continued per-ception of a shortage in the blood supply relative toan increasing demand (103). For these reasons alone,it seems reasonable to seek substitutes for allogeneicdonor blood, primarily the red blood cell component.

Adverse responses thus far revealed in HBOCclinical trials are generally mild in nature but severalincidences of severe adverse events (SAE) did occurwhich resulted in premature termination of clinicaltrials. The SAEs (stroke, ARDS, multi-organ failure,mortality, myocardiac infarction, etc.) appear to be

product-dependent and not generic to oxygen thera-peutics. Generally, HBOCs appear to cause moder-ate changes in cardiovascular and gastrointestinalsystems. The most common adverse response is amild transient hypertension. Other untoward effectsinclude esophageal discomfort, flu-like symptoms,elevated enzymes levels (e.g., bilirubin, amylase,lipase), and jaundice-like discoloration of the skinand eyes. The exact causes of these reactions havenot been elucidated. The hypertension may be, inpart, related to the high affinity of Hb for nitric oxide(NO), an endogenous vasodilator and a neurotrans-mitter. In the vascular system, HBOC could scavengethe endothelium-derived NO causing vascularsmooth muscle to contract. The esophageal spasmobserved with some HBOCs may also have beencaused by Hb interaction with NO. In the loweresophagus, NO is secreted by nonadrenergic noncho-linergic neurons to coordinate rhythmic contractionof circular smooth muscles. Hb interaction with thisneuronal NO might have altered the normal esoph-ageal motility. Exactly how Hb interacts with theesophageal NO is still a mystery that requires furtherelucidations.

There is no direct evidence that a specific propertyof an oxygen therapeutic is responsible for theincreased mortality seen in trauma patients or thehigher incidence of serious adverse events (ARDS,MOF, etc.) in surgical patients. The targeted popula-tions for these studies were seriously ill, acutelyanemic, or bleeding patients with projected highmortality and morbidity. For these types of studies,demonstrating efficacy based on mortality as an end-point would be difficult and require a prohibitivelylarge sample size.

Similarly, SAEs (stroke, thrombocytopenia, etc.)were also observed with PFBOCs but no definitivecausative mechanism identified. Second- or third-generation PFBOCs that could dissolve physiologicamounts of oxygen and are stable in ambient condi-tions are being developed. The advantage of PFB-OCs is their completely synthetic nature allowingheat sterilization. However, the ADME of thesematerials is still largely unknown especially theeffects on the reticuloendothelial system. and shouldbe investigated before any extensive clinical testing.

While clinical trials continue with current candi-date oxygen therapeutics, research efforts continueto search for alternatives, newer formulations, andconcepts to be applied. As the understanding of oxy-gen-delivery physiology and oxygen use grows, sowill the concepts behind the design of oxygen thera-peutics. As the appreciation of the physiology ofshock and resuscitation at many levels grows, the



desire to create specific oxygen therapeutics to meetthe needs of specific situations is expected. As wegain more clinical experience, more effective andsafer oxygen therapeutics will be developed.

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