Blood Substitutes: How Close to a Solution?

September 3, 2002

The term "blood substitute" is commonly misused when "red cell substitute" is meant. The ideal red cell substitute should deliver oxygen (O2), require no compatibility testing, cause few side effects, have prolonged storage qualities, persist in the circulation, and be available at reasonable cost. While no drug with all of these qualities is on the near horizon, several early generation red cell substitutes are approaching submission for licensure, at least for limited indications.

ABSTRACT: The term "blood substitute" is commonly misused when "red cell substitute" is meant. The ideal red cell substitute should deliver oxygen (O2), require no compatibility testing, cause few side effects, have prolonged storage qualities, persist in the circulation, and be available at reasonable cost. While no drug with all of these qualities is on the near horizon, several early generation red cell substitutes are approaching submission for licensure, at least for limited indications. Hemoglobin-derived red cell substitutes from human bovine and recombinant sources, as well as perfluorochemicals that dissolve O2, are in different stages of development. While each formulation has its own physical characteristics, biologic activities, and adverse reaction profile, all share one characteristic: The physiologic consequences of delivering O2 with small molecules is poorly understood, both accounting for problems seen in the clinical trials and providing therapeutic opportunities for the cancer patient. All the red cell substitutes in phase III trials have a life measured in hours and are unlikely to replace transfusions or drugs that stimulate erythropoiesis for chronic anemia, but they may play a role in cancer surgery, or even in radiation therapy, or in the management of cancer-related vascular occlusive syndromes. [ONCOLOGY 16(Suppl 10):147-151, 2002]

Early transfusion history described the miraculousrecovery of patients suffering life-threatening hemorrhage. However, equally dramaticreports revealed unexpected, unexplained, and occasionally lethal complications.The emergence of HIV as a transfusion-transmitted virus was only the latest suchcomplication and certainly not the last. Little wonder that the medicalestablishment, the public, and, more recently, the biotechnology industry placesuch high hopes on development of safer alternatives to blood. Despite more than40 years of focused research and the recent infusion of hundreds of millions ofventure capital dollars, no credible replacement for blood has yet been approvedfor use in the United States.

Types of Red Cell Substitutes

The term "blood substitute" is a misnomer.So-called blood substitutes in fact replace only one or possibly two functionsof transfused blood. By this definition, several blood substitutes are alreadyin common use: dextran and starch solutions that act as volume expanders;recombinant proteins that replace, among other things, coagulation factors; andeven anticoagulants, such as warfarin and heparin, that are used on occasion tosubstitute for naturally-occurring anticoagulant proteins. For other functionsof blood, those of the platelets and leukocytes, no substitutes are likely toemerge in the near future.

Whereas, the "holy grail" of blood substituteresearch has been to develop a red cells substitute—a small molecule thatdelivers oxygen (O2)efficiently, requires no compatibility testing, can be sterilized, has a longshelf-life at room temperature, reconstitutes easily, persists in thecirculation for days or weeks, and can be provided at a price competitive withthat of human blood. No such substance is on the near horizon.

Candidate red cell substitutes generally fall into threeclasses: perfluorochemicals, hemoglobin-based oxygen carriers, andliposome-encapsulated hemoglobin. Although it is convenient to review thesedrugs as "classes," each formulation should be considered a uniquedrug with its own physical characteristics, biologic activities, and adversereaction profile. General characteristics of these classes are reviewed in Tables 1 and 2.

Perfluorochemical Emulsions

Perfluorochemicals are synthetic, inert, hydrophobicmolecules with an almost unlimited ability to dissolve gases including oxygen.Because these molecules are structurally similar to hydrocarbons, they are notwater-soluble and therefore must be emulsified with surfactants before they aresuitable for intravenous use. This property has complicated their preparationand storage, and the nature of the emulsifier turns out to be as important asthe perfluorochemical itself. The classic early experiments in which a mouse wassubmerged in a beaker of preoxygenated perfluorochemical emulsion and shown tobreathe liquid continues to fascinate medical journalists and catch the publiceye.[1] Similarly, the exchange-transfused "bloodless rat" experimentsseemed to promise a quick transition to a clinically useful oxygen transportdrug.[2] However, early perfluorochemical formulations were impure, persistedfor long periods in the reticuloendothelial system, and proved unsuitable forclinical trials.

Broad application of perfluorochemicals as red cellsubstitutes may be limited by their oxygen-loading and off-loading properties (Figure1). Unlike blood and hemoglobin constructs, perfluorochemicals dissolve O2 in alinear fashion directly related to the partial pressure of O2.In practice this means that these emulsions can carry a great deal of O2,but only if the patient inspires high concentrations of supplemental O2.Furthermore, the compounds may release much of the O2as blood passes through less well-oxygenated environments and long before itreaches the most ischemic tissues. The latter property, the need forrefrigerated storage, and the relatively short circulating half-life have ledsome investigators to postulate that these chemicals will prove most suitablefor hospital use.

During a 10-year period, thousands of patients with a widevariety of illnesses received an early perfluorochemical formulation, FluosolDA. This agent was even licensed for use in coronary artery balloon angioplasty.However in controlled clinical trials, patients receiving Fluosol failed to showsubstantial physiologic benefit and were plagued with adverse reactionsattributed by some to complement activation.[3] In any case, production of thisdrug ceased in 1994. An excellent review of this drug and other earlyperfluorochemicals has been published by Keipert.[4]

Assessment of NewlyDeveloped Formulations

Newer perfluorochemical formulations are in phase III trialsand a European multicenter, randomized study of orthopedic surgery patientsreported reduced need for allogeneic blood during profound intraoperativehemodilution.[5] A study of general surgery patients is in progress. Troublingside effects associated with complement activation and cytokine release havebeen attenuated by newer formulations containing smaller emulsion particles.However, a flu-like syndrome (attributed to macro-phage-mediated clearance ofthe emulsion) and a curious sequestration of 15% to 20% of circulating plateletsare commonly observed.

Phase III trials using perfluorochemicals to augment profoundperisurgical hemodilution had been stopped temporarily while an increasedfrequency of stroke in the treatment arm was investigated. It is not clear thatstroke was related to the perfluorochemicals, and these studies have beenrestarted. Despite the several questions that the trials in surgery have raised,the singular ability of these agents to dissolve gases, their ability to besterilized, and their low cost and ease of manufacture continue to makeperfluorochemical emulsions attractive not only as red cell substitutes, butalso as oxygen-carrying therapeutic agents to treat stroke, myocardial ischemia,sickle cell disease, and vascular occlusion, and as sensitizing agents forradiation and chemotherapy for malignancy.[6-10]

Hemoglobin-BasedOxygen Carriers

Encapsulated hemoglobin, nature’s oxygen transport protein,carries 98% of blood oxygen. The well-known sigmoid shape of the oxygendissociation curve describes how hemoglobin binds oxygen rapidly in the lung andreleases it efficiently in the low-oxygen environment of the tissues (Figure1).For these reasons, hemoglobin seemed the logical candidate for a red cellsubstitute. Mammalian evolution undoubtedly encapsulated hemoglobin for areason, however. Outside of the red cell, hemoglobin is vulnerable to oxidativeinactivation, and its chains dissociate into dimers that are cleared by, andtoxic to, the kidney. Inside the red cell membrane, the hemoglobin tetramer isstabilized, provided with organic phosphates that modify oxygen delivery, andprotected from harmful oxidants.

Numerous hemoglobin-based oxygen carriers have been createdduring the last quarter of a century, and several have lately progressed toclinical trials (Table 3). Most hemoglobin-based oxygen carriers are derivedfrom human or bovine blood, washed to remove cellular debris, pasteurized,filtered, and passed over chromatographic columns to ensure purity. The tetrameris then chemically modified by a variety of methods to provide molecules ofdifferent size, molecular weight, oxygen affinity, viscosity, and oncoticactivity.

Recombinant hemoglobin molecules with features not found innature have also been prepared. Red cell substitutes can thus be customized fortheir intended use, providing the characteristics necessary for that use arewell defined. Most candidate hemoglobin-based oxygen carriers have beenengineered to mimic the characteristics of the whole blood oxygen dissociationcurve, and the solutions have frequently been prepared to mimic many of thecharacteristics of blood—albeit at a lower hemoglobin concentration. However,as with perfluorochemical emulsions, the physiology of O2 delivery by small molecules that extravasatebeyond the microcirculation is incompletely understood, and it is possible thatdifferent affinity, viscosity, and oncotic pressure may eventually provesuperior.

Finally, in an ironic recapitulation of red cell evolution,hemoglobin has been encapsulated in liposomes to prolong its intravascularcirculation. However, liposomal hemoglobin has yet to realize the success ofother liposome encapsulated pharmaceuticals, and will not be further discussedin this article.

Indications for Treatment

It has been surprisingly difficult to demonstrate efficacywith the candidate hemoglobin-based oxygen carrier. In a detailed report of asingle patient, Mullon et al described a 21-year-old woman with life-threateningautoimmune hemolytic anemia treated with a polymerized bovine hemoglobinpreparation after severe reactions to repeated incompatible transfusions. Herrapidly falling hematocrit left few options other than oxygen carriers devoid ofred cell membrane antigens.[11] In this situation, proof of drug efficacy isstill indirect, since the lower limits of hemoglobin tolerated by humans havenot been defined.[12] The reversal of signs, symptoms, and electrocardiographicevidence of ischemia in this symptomatic anemic patient are compelling, however,as is the laboratory evidence of reduced end-organ ischemia. In this instance,it seems reasonable to conclude that the 11 hemoglobin-based oxygen carrierinfusions over a 7-day period helped to sustain this patient until herautoimmune process remitted.

Refractory autoimmune hemolytic anemia illustrates anextremely limited indication for red cell substitutes. Most clinical trials use"blood sparing" or "blood avoidance" during surgery asprimary study end points, and these have been approved by the regulatory agency.For elective surgery, the potential exists to reduce the estimated five millionunits of perioperative red cells transfused annually in the United States.

The short 12- to 48-hour half-life of these small moleculeslimits their blood-sparing utility, however. Mathematical modeling suggests thatbenefits will be confined primarily to nonanemic patients who undergo extremehemodilution and sustain large perioperative blood losses.[13] The potentialapplications include an estimated 600,000 coronary artery bypass graft surgerypatients (in North America), 625,000 orthopedic surgery patients, and 70,000 menundergoing radical prostatectomy yearly. While the short half-life of currentcandidate red cell substitutes all but precludes their use for managing chronicanemia, a "second generation" product with a longer intravascularsurvival might find substantial application for the high percentage of lung orovarian cancer patients estimated to develop chemotherapy-induced anemia.

Universally-compatible, readily-available red cellsubstitutes could revolutionize the treatment of hemorrhagic shock. Six-unithemoglobin-based oxygen carrier infusions have been given in place of red cellsduring acute blood loss in trauma and surgery, and the same investigators haveadministered up to 20 units without apparent toxicity.[14] However, successfulcontrolled trials of therapeutic efficacy have yet to be reported. No one knowshow many trauma patients in shock outside of the hospital would survive solelybecause an oxygen-carrying agent is added to the resuscitation solution. Even incombat settings where hemorrhage is the major cause of death, authoritiesestimate that no more than 20% of military casualties are potentiallysalvageable.[15] Tissue oxygen deprivation is a well-documented factor in thepathophysiology of hemorrhagic shock, but the therapeutic role of oxygencarriers and the point at which transfusion becomes critical are not wellunderstood.

Alternatives to Transfusion

A major impetus for developing safer alternatives to bloodhas faded as transfusion’s risk-benefit calculus has changed for the better inthe United States. Newer treatments now have to compete with a very safe andfamiliar product. The combination of volunteer blood, stringent donoreligibility criteria, and sensitive testing (particularly the addition of directassays for viral nucleic acid) has reduced the risk of transfusion-transmittedhepatitis to fewer than 1 case per 100,000 units transfused, and has virtuallyeliminated the risk of transfusion-associated HIV.[16] Other risks have declinedin parallel.

Unfortunately, more than two-thirds of the world’sdeveloping nations do not have adequate blood.[17] An estimated 13 million blooddonations globally are not tested for HIV and hepatitis viruses, primarily indeveloping countries where 80% of the blood supply comes from paid orreplacement donors, and where the infected population is large. Bloodavailability is scarcely better: One-quarter of maternal deaths frompregnancy-related causes are associated with loss of blood. Operationalproblems, infrastructure costs, and cultural issues pose seeminglyinsurmountable hurdles to building modern blood delivery systems in the sameregions. Inexpensive red cell substitutes, even those that fail to meet therigorous safety standards applied to blood, would allow patients to receivetreatments ordinarily requiring blood transfusion and could improve healthdramatically in many developing countries. Whether such practices, or evenclinical trials of such products, are ethically acceptable remains a subject ofintense debate.[18]

Adverse Reactions

Red cell substitutes still face formidable hurdles. Theapparent lack of serious toxicity should not obscure the cautions that haveslowed their licensure. Small molecules like cell-free hemoglobin deliver oxygenin a fundamentally different way. A great deal has been learned, but thephysiologic consequences and compensatory mechanisms remain incom-
pletely understood. Freely-diffusing red cell substitute-bound oxygen may beresponsible for gastrointestinal irritability, hypertension, and the recentlywell-publicized "excess deaths" in trauma patients that led to thetermination of clinical trials and to the withdrawal of two hemoglobin-basedoxygen carrier formulations from further development.[19]

Most hemoglobin-based oxygen carrier formulations exhibit a"pressor effect," generally attributed to hemoglobin’s ability toscavenge the vasodilator nitric oxide. However, recent experimental evidenceimplicates a second mechanism involving autoregulation of capillary bloodflow.[20] Whether this pressor effect can be put to therapeutic use (in septicshock, for example), or whether it must be eliminated because of the difficultyin controlling the response of different vascular beds remains to be determined.

Costs of Treatments

Economics will also influence the use of red cellsubstitutes. If emotion is set aside, expensive alternatives to red cells shouldbe justified based on their increased safety and efficacy. High cost may notprohibit use in nations like the United States, where zero-risk transfusion hasbecome religion, but expensive alternatives will be totally out of reach for thedeveloping countries that need them the most.

Future Directions in Oncology

What does this mean for the oncology patient? The firstgeneration of effective red cell substitutes is nearing licensure. These will benovel agents, not miracle drugs. This first generation has the potential toreduce the need for allogeneic blood in patients who require extensive surgicalprocedures. A second generation might address the issue of chronic anemia duringchemotherapy. We should temper our expectations, however, until we learn moreabout the advantages and limitations of these agents.

Blood transfusion has a long history, and most physicians arerelatively familiar, if not comfortable, with its safety and efficacy profile,particularly the long-term toxicity. In this regard, red cell substitutes are anunknown entity. Red cell substitutes will not replace the need for red cells,but they should reduce or eliminate the need for blood for some patients, andpermit the treatment of others who currently cannot be transfused. If problemsof cost and intravascular half-life can be solved, these drugs could savecountless lives in the developing world.

References:

1. Clark LC, Gollan R: Survival of mammals breathing organicliquids equilibrated with oxygen at atmospheric pressure. Science152:1755-1756, 1966.

2. Geyer RP, Monroe RG, Taylor K: Survival of rats perfusedwith a perfluorocarbon-detergent preparation, in Norman JV, Folkman J, HardisonLE, et al (eds): Organ Perfusion and Preparation, pp 85-95. New York,Appleton-Crofts, 1968.

3. Teicher BA, Schwartz GN, Sotomayor EA, et al: Oxygenationof tumors by a hemoglobin solution. J Cancer Res Clin Oncol 120:85-90,1993.

4. Gould SA, Rosen AL, Sehgal LR, et al: Fluosol-DA as a redcell substitute in acute anemia. N Engl J Med 314:218-221, 1986.

5. Keipert PE: Perfluorochemical emulsions: Futurealternatives to transfusion, in Chang TMS (ed): Principles, Methods, Productsand Clinical Trials, pp 127-156, Zurich, Karger, 1998.

6. Spahn DR, van Brempt R, Theilmeier G, et al: Perflubronemulsion delays blood transfusions in orthopedic surgery. European PerflubronEmulsion Study Group. Anesthesiology 91:1195-1208, 1999.

7. Sakas DE, Whitaker KW, Crowell RM, et al: Perfluorocarbons:Recent developments and implications for neurosurgery, J Neurosurg85:248-254, 1996.

8. Premaratne S, Harada RN, Chun P, et al: Effects ofperfluorocarbon exchange transfusion on reducing myocardial infarct size in aprimate model of ischemia-reperfusion injury: A prospective, randomized study. Surgery117:670-676, 1995.

9. Horn E-P, Standl T, Wilhelm S, et al: Bovine hemoglobinincreases skeletal muscle oxygenation during 95% artificial arterial stenosis.Surgery 121:411-418, 1997.

10. Kaul DK, Liu X, Nagel RL: Ameliorating effects offluorocarbon emulsion on sickle red blood cell-induced obstruction in an ex vivovasaculature. Blood 98:3128-3131, 2001.

11. Mullon J, Giacoppe G, Clagett C, et al: Polymerizedbovine hemoglobin transfusions in a patient with severe autoimmune hemolyticanemia. New Engl J Med 342:1638-1643, 2000.

12. Weiskopf RB, Viele MK, Feiner J, et al: Humancardiovascular and metabolic response to acute isovolemic anemia. JAMA 279:217-221,1998.

13. Brecher ME, Goodnough LT, Monk T: The value ofoxygen-carrying solutions in the operative setting, as determined bymathematical modeling. Transfusion 39:396-402, 1999.

14. Gould SA, Moore EE, Hoyt DB et al: The first randomizedtrial of human polymerized hemoglobin in acute trauma and emergent surgery. JAm Coll Surg 187:113-122, 1998.

15. Pope A, French G, Longnecker DE: Fluid Resuscitation.State of the Science for Treating Combat Casualties and Civilian Injuries,Washington, DC, National Academy Press, 1999.

16. Busch MP: Closing the windows on viral transmission byblood transfusion, in Stramer SL (ed): Blood Safety in the New Millennium,pp 33-54, Bethesda, MD; American Association of Blood Banks, 2001.

17. Safe Blood Starts With Me. World HealthOrganization and International Federation of Red Cross and Red CrescentSocieties publication 12. Geneva, 2000.

18. Varmus H, Satcher D: Ethical complexities of conductingresearch in developing countries. N Engl J Med 337:1003-1005, 1997.

19. Sloan EP, Koenigsberg M, Gens D, et al: Diaspirincross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagicshock. A randomized controlled efficacy trial. JAMA 282:1857-1864, 1999.

20. Winslow RM, Gonzales A, Gonzales ML, et al: Vascular resistance and theefficacy of red cell substitutes in a rat hemorrhage model. J Appl Physiol85:993-1003, 1998.