Guest Editorial

New Approaches in Commercial Development of ArtificialOxygen Carriers

The unmet need for an artificial oxygen carrier hasbecome more apparent as problems beset transfusionmedicine. Blood banks are experiencing a criticalblood shortage. The reason for the shortage is anincreasing demand and a serious decline in dona-tions. In the US alone, someone needs a blood trans-fusion every 3 s, and one in three people will requirea transfusion in their lifetime. To meet the currentannual worldwide demand about 100 million units ofblood are needed; 14 million in the US. By 2030,experts anticipate an annual shortfall of 4 millionunits in the US (1).

Blood safety is threatened by transmissible dis-eases, whether endemic, newly emerging, or poten-tial arrivals. Of particular concern are infectiousagents not routinely tested for. Mosquito-borne ill-nesses, avian/swine influenza, tick-borne diseases,retroviruses such as xenotropic murine leukemiavirus-related virus and other pathogens can be spreadby blood transfusions. Increased numbers of newHIV and tuberculosis cases are alarming. Prion dis-eases, especially variant Creutzfeldt–Jakob disease,remain a constant threat as their transmission byblood is probable, and no reliable screening tests ofasymptomatic donors are in place (2–4).

Presently, transfusion medicine is facing anotherdilemma. A meta-analysis of 409 966 blood recipientsrevealed that transfusion of blood stored longer than2 weeks is associated with the risk of death (5).During storage, blood not only loses oxygen deliveryand vasodilatory abilities, but also becomes toxic (6).If the customary blood storage period changes fromthe current 6 weeks to 2 weeks, it will create a cata-strophic shortage of blood. Therefore, a logical andpractical solution to these transfusion medicine prob-lems is a viable artificial oxygen carrier.

Over the last few decades, several concepts inartificial oxygen carriers have been developed,extending from human-, bovine-, swine-, worm-, or

recombinant hemoglobin (Hb)-based products toperfluorocarbons (7). Some developers even contem-plated brewing blood from hematopoietic progeni-tors (8). Many concepts, however, experiencedbiotechnological limitations, which were difficult toovercome. In fact, the recombinant Hb programs,although initially promising, have shown to be tech-nologically more complicated than anticipated andnow are on the decline (7). Similarly, the optimismabout the potential future role for stem cells as adonorless source of blood for transfusion is not yetsubstantiated. So far, it is possible to produce undergood manufacturing practice conditions only a tinyvolume of red blood cells (RBCs) (8). The problem iswith the scale-up process, so this concept at themoment looks surreal.

Because of the obvious biotechnological limita-tions, only a few concepts prevailed and materialized,particularly bovine Hb-based products, which seemto be the most promising approach in the develop-ment of artificial oxygen carriers (7).

The ability of free Hb to sustain life in the absenceof erythrocytes has been recognized since the 1930s(9). Many attempts to create an effective Hb-basedoxygen carrier fell short due to a number of problemsthat were slow to be recognized and difficult toresolve. The proposed formulations in the 1980s and1990s had fundamental limitations related to inad-equate control of Hb intrinsic toxicity (7).

The well-documented, poor clinical performanceof these early products has greatly disappointedinvestors and the medical field (10). The reportedincrease in heart attack and death rates, along withother pathological responses, resulted from an over-simplification of the approach in the rush to market.

From the beginning, it was clear that the develop-ment of a commercially successful artificial oxygencarrier would be a long and arduous process, but noone considered that it would take a few decades torealize that these products, in order to properly func-tion in the human body, must be designed ashemobiotherapeutics with features that alleviate Hbdoi:10.1111/aor.12371

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intrinsic toxicity, and not as only chemically stabi-lized oxygen and carbon dioxide transporters.

Based on the previous failures, the artificial oxygencarrier field has taken a different approach with agoal to create an effective product. Learning fromcostly mistakes of the past, academic and industrialdevelopers have been adding new physicochemical–pharmacologic properties to their products and align-ing them with specific clinical indications.

The patient population targeted for treatment withartificial oxygen carriers is vast and diverse. Most haveserious preexisting conditions, thus they can onlybenefit therapeutically when receiving a productsuited to their health status. For instance, a subjectwith a compromised antioxidant system, who requiresblood transfusion, should get a product that, besidesoxygen transport, must possess antioxidant potential.A patient with systemic inflammation should receive aproduct that will not aggravate it. A person withtraumatic brain injury (TBI) should be transfusedwith an artificial oxygen carrier that has a potential toheal neurons and glial cells. Similarly, for cancerpatients, these products should have ability to blocktumor growth and metastasis and/or facilitate theradiosensitizing effect. Futhermore, these productsshould have a pro-erythropoietic potential, if underconsideration for blood replacement therapy only.The relatively short half-life of a given artificialoxygen carrier of up to 2 days categorizes it as an“oxygen bridge,” where follow-up treatment is stillnecessary, and can be achieved either by the product’sintrinsic erythropoietic effect or transfusion withpacked RBCs. These are only a few strategies beingconsidered in designing an effective artificial oxygencarrier.

Presentations at the 4th International Symposiumon Artificial Oxygen Carriers held on September 28,2013, in Yokohama, Japan, focused on the mostpromising and innovative concepts in the artificialoxygen carrier field. Many of them are presented inthis Artificial Organs Special Issue.

Encapsulated human or animal Hb designed withhigh or low oxygen affinity reached the next bioengi-neering level and based on preclinical studies, wasshown to be effective in the treatment of variousmedical conditions (11–15). Considering the impactof oxygen on tissue, organ and extremity ischemia,wound healing, tumor growth and metastasis, andeven hearing loss, experiments proved that by con-trolling oxygen delivery, a positive therapeutic effectcan be achieved. It was found that products withdifferent P50 (partial pressure of oxygen at which 50%of Hb is oxygenated) values can stabilize or degradehypoxia inducible factor (HIF)-1 alpha. Induction of

the HIF-1 alpha-regulated erythropoietin gene pro-motes erythropoiesis (16,17). Suppression of theHIF-1 alpha regulated vascular endothelial growthfactor gene blocks angiogenesis in cancer (15). Inwound healing, it is proposed to initially use aproduct with a low P50 to create a slightly hypoxiccondition to stabilize HIF-1 alpha to initiateangiogenic capillary sprouts and later a product witha high P50 to accelerate oxygen-driven collagen syn-thesis (11,12). Similarly, tailoring oxygen deliverycould have a beneficial effect in the treatment ofbrain ischemia (13,14). The scientific bases for thesephenomena were identified (18).

A new concept in liposome-encapsulated humanHb for enhancing circulatory persistence, stability,and immunological neutrality proved promising(19). A strategy of 3,5-bis(2-fluorobenzylidine)-4-piperidone (EF-24) coadministration (20), a com-pound that blocks nuclear factor (NF)-kappa Bactivation that governs induction of 150 inflammatorygenes, may create an opening for artificial oxygencarriers with uncontrolled inflammatory potential.

The treatment of TBI, stroke, and sickle cell disease(SCD) reached another dimension with the use of thenano RBC (nRBC) product, a PEGylated bovine Hbmodified with nitroxides with superoxide dismutase(SOD) mimetic activity. The idea of adding the anti-oxidant property to the Hb molecule resulted in aproduct that showed clinical potential. As found inpreclinical studies, nRBC works through the correc-tion of inadequate blood flow and stabilization ofhemodynamics, thus preventing multiorgan injuries.nRBC reportedly outperformed fresh RBC, openingthe possibility of replacing chronic blood transfusionsto prevent stroke in SCD. The observed immediatevasodilation and blood flow enhancement with a smalltop loading dose of nRBC also allows for the treat-ment of painful vaso-occlusive crisis as well as acutechest syndrome in SCD. Moreover, nRBC is intendedto serve as a bridge and safe alternative to bloodtransfusion in hemorrhagic trauma and TBI, as well asa “golden hour” therapeutic for strokes (21,22).

A strategy of pharmacologic cross-linking that com-prehensively addresses the intrinsic toxic potential ofHb was shown to be extremely effective (17). BovineHb cross-linked intramolecularly with adenosine-5′-triphosphate (ATP) and intermolecularly withadenosine, and decorated with reduced glutathione,resulted in a product with vasodilatory, antioxidant,anti-inflammatory, and erythropoietic potential. Thisnovel modification method incorporates the pharma-cological potential of the cross-linkers. This producthas been extensively tested preclinically and also clini-cally in humans. The performed studies indicate that it

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works as a physiologic oxygen carrier and has efficacyin acute and chronic anemias by providing a tempo-rary oxygen bridge and by preserving HIF-1 alpha innormoxia, which stimulates erythropoiesis (16). Dueto its vasodilatory, antioxidant, and inhibitory effectson NF-kappa B, it is intended for use in ischemicdiseases, particularly in the treatment of thromboticand restenotic events in percutaneous coronary inter-vention patients (17).

PEGylation and saturation of bovine Hb withcarbon monoxide (CO) gas resulted in anotherpromising product that already completed a PhaseI human clinical trial. This dual CO-releasing andoxygen-transferring agent has anti-inflammatory,antiapoptotic and vasodilatory activities (23).The concept of using CO, which possesses well-documented vasodilatory, cytoprotective, anti-ischemic, and anti-inflammatory properties, resultedin a product that is well tolerated by humans andintended for use in a wide array of medical conditions(23).

With advancements in science, many strategiesto control Hb’s natural vasoconstrictive potentialare now available. Encapsulation, PEGylation,CO-saturation, S-nitrosylation, or incorporation ofATP and adenosine are only a few examples of thestrategies being implemented by artificial oxygencarrier developers (7,11–23). These strategies areevolving, and more concepts are on the way. Thelatest one is prenitrosylation at the alpha hemes tocreate a nonvasoactive oxygen carrier (24). Anotherexample is to create a heme containing transitionmetals other than iron, which participates in the for-mation of reactive oxygen species and has a highscavenging potency toward nitric oxide (NO) (25).To be considered valid, artificial oxygen carriers mustlack the vasopressor effect.

Simplification of manufacturing is an importantelement in commercial development of artificialoxygen carriers. Some developers are purifying Hb tothe single tetrameric molecule free of pyrogens andall pathogens by applying a multistep process includ-ing centrifugation, nanofiltration, heat treatment,liquid and/or solid phase extraction, various chro-matographic techniques, etc. Chemical modificationof Hb includes encapsulation, intramolecular cross-linking, intermolecular modification/polymeriza-tion, conjugation, or surface decoration (7). It hasbeen proposed that an artificial oxygen carrier withbiopolymers with a size between 500 nm and 1 μmand high oxygen affinity can be manufactured by asimple procedure that includes coprecipitation of Hbwith CaCl2 and Na2CO3 to form CaCO3 hybridbioparticles which, after cross-linking and dissolution

of CaCO3, results in pure biopolymer particles. Thisproduct demonstrated biocompatibility and lack ofvasoactive properties (26). New tools available inbiotechnology inevitably contribute to the advance-ment in manufacturing of artificial oxygen carriers.

Organ preservation, especially the heart, is verychallenging and not fully accomplished. The cur-rently used solutions for organ perfusion and preser-vation have no oxygen transport capability. Therelease of oxygen by Hb-based oxygen carriers atlower temperatures is extremely limited. Previousattempts to allosterically correct Hb oxygen deliveryat low temperatures have been unsuccessful. It seemsthat the concept of using Hb from giant tube worms(Riftia pachyptila) resolved the problem. Theseorganisms developed Hb containing 144 globinchains with unique oxygen transport and deliverycapabilities at temperatures ranging from 2 to 30 °C.Besides this, Hb is capable of carrying oxygen in thepresence of sulfide, nitrite, and nitrate. Based onremarkable properties of this Hb, it was proposedthat such an oxygen carrier could have numeroustherapeutic applications wherever oxygen is neces-sary, mainly in hypothermia. The initial studiesproved that this product can be effectively used as anoxygen carrying solution in organ preservation andwound healing (27).

Perfluorocarbons, once prominent in the artificialoxygen carrier field, are less enthusiastically accepted,even though one product is in clinical use. Perfluoro-carbons have many positive effects includingimprovement of microcirculation, transport of NO,clearance of lactates, and the like (28–30), but a ques-tion still remains about their limited ability to be fullyoxygenated and deliver a proper amount of oxygen atnormal partial pressure. Inevitably, advancements inperfluorocarbon chemistry may address this problem.

Other concepts in designing artificial oxygencarriers were not presented during the Yokohamasymposium. Worth noting are new genetically engi-neered Hbs, modified swine Hbs, bovine Hb withincorporated SOD and catalase, and many others,which are at the preclinical development stage (7).

Whereas government agencies are providing onlylimited funding for artificial oxygen carrier R&D,trust and enthusiasm still remain among investorsand within the pharmaceutical industry, driven notonly by the medical impact of delivering this criticallyneeded product, but also the enormous economicpotential of this still undisputed “holy grail” of bio-technology. As the global market for effective artifi-cial oxygen carriers is estimated to be in themultibillion-dollar range, the race continues and thefuture is bright. Some low-tier academic institutions,

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discouraged by not making a quick buck, decided towithdraw from the quest, but this should have noimpact on future commercialization of artificialoxygen carriers.

Jan Simoni, DVM, PhDResearch Professor (ret.)

School of MedicineTexas Tech University Health Sciences Center

andPresident & Chief Scientist

Division of Artificial Oxygen CarriersTexas HemoBioTherapeutics & BioInnovation

CenterLubbock, TX 79424, USA

E-mail: jan.simoni@ttuhsc.edu

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