hsia 2012 a hemoglobin based m

aor_1307 215..228 A Hemoglobin-Based MultifunctionalTherapeutic: Polynitroxylated Pegylated

Hemoglobin

*Carleton Jen Chang Hsia and †Li Ma*SynZyme Technologies LLC, Irvine, CA; and

†Department of Physics, Georgia SouthernUniversity, Statesboro, GA, USA

Abstract: Polynitroxylated pegylated hemoglobin(PNPH) as a multifunctional therapeutic takes advantageof the ability of hemoglobin (Hb) to transport oxygen, theantioxidative stress activities from the redox coupling ofnitroxide and heme iron, and the hypercolloid propertiesof pegylation. The published preclinical data demonstrat-ing that PNPH acts as a neurovascular protective multi-functional therapeutic in an animal model simulatingprehospital resuscitation of traumatic brain injury (TBI)with hemorrhagic shock (HS) are reviewed. Preliminaryresults on the potential utility of PNPH for neurovascularprotection in thrombolytic stroke therapy and for correc-tion of vascular dysfunction through transfusion in sickle-cell disease (SCD) are also discussed. We hypothesize thatwith PNPH, Hb has more than been tamed—it hasbecome a therapeutic and not just a nontoxic extracellularoxygen carrier—and that successful PNPH developmentas a multifunctional therapeutic that protects the neu-rovasculature and reduces oxidative stress may representa paradigm shift in transfusion and critical care medicine,which may meet a number of unmet medical needs result-ing from oxidative stress and inadequate blood flow, suchas HS, TBI, SCD, and stroke. Key Words: Nitroxide—Hemoglobin—Poly(ethylene) glycol—Oxidative stress—Hemoglobin-based oxygen carrier.

CURRENT GENERATIONHEMOGLOBIN-BASED OXYGEN

CARRIERS (HBOCs)

A blood substitute that eliminates the need forrefrigeration and cross-matching, can be manufac-tured in quantity, has a long shelf life, and reducesthe risk of iatrogenic infection is a worthwhile goal.However, hemoglobin-based oxygen carriers(HBOCs) developed during the past half century andtested in humans have done more harm than good byincreasing the risk of death and myocardial infarction(1). This abysmal history, and the resulting negativeviews of HBOCs held by the medical, regulatory, andinvestment communities, means that a paradigm shiftis required. The outcome of the 2008 Food and Drug

doi:10.1111/j.1525-1594.2011.01307.x

Received August 2010; revised May 2011.Address correspondence and reprint requests to Dr. Carleton

Jen Chang Hsia, SynZyme Technologies LLC, 1 Technology Drive,Suite B115, Irvine, CA 92618, USA. E-mail: [email protected]

THOUGHTS AND PROGRESS 215

Artif Organs, Vol. 36, No. 2, 2012

Administration/National Institutes of Health (FDA/NIH) cosponsored workshop on HBOCs was theview that in order to be successful, an HBOC mustdemonstrate a therapeutic index with evidence ofboth safety and efficacy (2).This is based primarily onthe association of increased risk of heart attack anddeath but also, in part, on the lack of therapeuticefficacy of current-generation HBOCs.

The conventional wisdom concerning the causes ofthe adverse effects of experimental HBOCs is thatthe primary mechanism of toxicity is nitric oxide(NO) scavenging at the endothelium, causingvasoconstriction and, paradoxically for a productintended to deliver oxygen, compromising tissue per-fusion and oxygenation (3). After unsuccessful phaseII clinical trials of diaspirin cross-linked hemoglobin(Hb) by Baxter (4), a number of HBOC developerssought to tame Hb by creating Hb derivatives, whicheither reduced NO binding or compensated for NObinding. These strategies included increasing themolecular weight of modified Hb to reduce Hbextravasation into the endothelium, and binding ofNO by (i) glutaraldehyde polymerization of human(5) and bovine Hb (6) by the former NorthfieldLaboratories and Biopure Corp., respectively, and (ii)O-raffinose cross-linking human Hb with residualunpolymerized tetramers by the former HemosolInc. (7). An alternative strategy included geneticallymodifying Hb to reduce NO binding as was done byBaxter (8). These products were tested in advancedclinical trials, but the results were negative (1)

Additionally, it appears that cell-free Hb deriva-tives tested to date also bring another set of toxici-ties in addition to NO scavenging. These toxicitiesare related to pro-oxidant activity and are notaddressed by the earlier mentioned approaches.They are the result of the combination of oxygenand heme iron in the absence of the controllinginfluence of the antioxidant enzymes in red cells.The result can be the generation of toxic moleculeslike superoxide, hydrogen peroxide, hydroxylradical, oxoferryl porphyrin, etc. (3). Thus, thereduction in oxygen delivery resulting from NOscavenging and vasoconstriction by Hb is furtherexacerbated by oxidative stress through a burst ofsuperoxide generation from heme iron autoxidationin the transfused HBOCs (9). In this view, cell-freeHb without appropriate regulation would actuallyadd to the inflammatory insult of ischemia and rep-erfusion, adding to the underlying pathology in thevery clinical situations where a blood substitutewould be most useful. Polynitroxylation may wellprovide the controlling influence to tame the pro-oxidant activity of Hb (10).

In addition to lipid-encapsulated or “cellular” Hbas done by Waseda University and the Terumo Cor-poration, the most recent strategies to develop cell-free Hbs that address both vasoconstriction and thepro-oxidant activity of cell-free Hb include:

1 Polynitroxylating the HBOC to introduce superox-ide dismutase (SOD) and catalase mimetic activi-ties by Hsia et al. (10–12).

2 Copolymerizing the HBOC with the antioxidantenzymes SOD and catalase to neutralize thenatural pro-oxidant activities of heme iron and toenhance the antioxidant and anti-inflammatoryactivities by D’Agnillo and Chang (13).

3 Pharmacological cross-linking the HBOC withATP, adenosine, and reduced glutathione to dimin-ish intrinsic toxic effects of cell-free Hb by Simoniet al. (14).

4 Limiting the excessive oxygen delivery capacityof the HBOC by modifying the Hb in such a waythat oxygen-binding affinity is increased (P50 isdecreased) by Winslow (15).

5 S-nitrosylating and PEGylating the HBOC toavoid NO scavenging by heme by Nakai (16).

6 Adjunct therapies of NO inhalation by Zapol et al.(17) or sodium nitrite therapy by Petal and Kerbyet al. (18) to attenuate the hypertensive effect ofHBOC.

The ideal HBOC would not only expand circula-tory volume and deliver oxygen but also do so in amanner that treats the oxidative insult of ischemiaand reperfusion. It should perform at least as well asblood, if not better. In fact, the ideal HBOC couldconceivably perform better than stored red cells,given recent evidence that the safety of packed redcells drops off as they are stored for more than aweek or two (19). We propose that polynitroxylatedpegylated hemoglobin (PNPH) with its added cata-lytic antioxidant activity of nitroxides may hold thekey to meeting this challenge.

A NEW GENERATION Hb-BASEDTHERAPEUTIC—PNPH—STRUCTURE

AND DEVELOPMENT

In this review, we describe further refinement of thefirst approach through the creation of PNPH.A sche-matic of PNPH is shown in Fig. 1,and the caption givesstructural details on the molecule.The PNPH has fivestructural and functional components contributing toits unique therapeutic activities: (i) Hb as the proteincenter provides oxygen carrier capability but is car-boxylated to provide thermostability and added anti-inflammatory activity; (ii) the hydrated polyethylene

THOUGHTS AND PROGRESS216


glycol moieties of PNPH provide hypercolloid prop-erties that are important in stabilizing hemodynamicsduring hypotension and hypovolemia; (iii) the nitrox-ide moieties of PNPH not only improve the safetyof cell-free Hb but also provide antioxidant/anti-inflammatory and neuroprotective activities; (iv) thedesirable redox coupling of heme iron with the nitrox-ide is promoted in the stoichiometry; and (v) furtherredox coupling of the nitroxide/heme iron complexwith endogenous plasma antioxidants such as ascor-bate provides additional antioxidant activities (20).These added intravascular and extravascular coupledredox reactions that attenuate vasoconstriction, andthe vascular inflammatory effects of ischemia andreperfusion are the new antioxidative stress therapeu-tic activities of this unique PNPH.Thus, this approachrepresents a paradigm shift in blood substitute devel-opment, away from the traditional focus on oxygendelivery and NO supplementation and toward a newfocus on safely correcting inadequate blood flow andoxidative stress.

To support this paradigm shift, we review pub-lished and preliminary data on a PNPH formulationthat is efficacious in oxidative stress models of trau-matic brain injury (TBI) compounded by hemor-rhagic shock (HS), stroke, and sickle-cell disease(SCD).The data presented below support the follow-ing therapeutic functions:

1 PNPH normalizes blood flow, which is at least asimportant as or even more important than itsability to deliver oxygen.The new molecular designof PNPH greatly decreases the natural tendency ofheme iron to autoxidize and generate superoxide,thus preventing vasoconstriction and correctinginadequate blood flow.

2 PNPH has the ability to catalytically reduce oxida-tive stress through the redox coupling of thenitroxide moieties with the heme iron resulting inincreased SOD and catalase mimetic activities,instead of the natural pro-oxidant activities of theheme iron, and thus converts PNPH to a potentneurovascular protectant.

3 PNPH has the ability to further redox couple withplasma ascorbate resulting in augmentation ofSOD and catalase mimetic activities in the form ofa novel peroxidase activity (20).

4 The PNPH structure of multiple (up to 14) nitrox-ides covalently labeled onto the core Hb which isshielded by a hydrated polyethylene glycol shell iswhat uniquely allows it to function as neurovascu-lar protective multifunctional therapeutic in thevascular space.

Recent studies with controlled inhalation ofcarbon monoxide (CO) and infusion of carboxypegylated Hbs (PegHbs) have indicated that COcan exhibit beneficial anti-inflammatory activities(21). In fact, both Sangart Inc. and Prolong Pharma-ceuticals Inc. are now developing their respectivePegHbs as CO formulations. Both companies aretargeting approvals as orphan drugs for SCDtherapy. This confirms the benefits of the CO formu-lation of polynitroxylated dextran-conjugated Hbused in an earlier report (12).

EXPERIMENTAL EVALUATION OF PNPH

The multifunctional therapeutic effects of PNPHprepared from bovine PegHb are indicated by itsefficacy in three indications: (i) TBI + HS; (ii) stroke;and (iii) SCD.

PNPH for TBI + HSA full article has been published (22) which

describes the study of PNPH as an efficacious,novel, antioxidative hypercolloid neuroprotectivesmall volume resuscitative fluid for TBI + HS whencompared with standard therapy in a model simu-lating the prehospital setting. In that article, wepresent data which demonstrate that the pathologyand neurological deficits after TBI can be mini-mized with PNPH as a prehospital treatment, and

FIG. 1. Schematic of PNPH: 8–10 5 kD Peg chains (blue) pro-trude to form an outer shell, which shields the 12–14 nitroxides(yellow) and the hemoglobin (Hb) in the center. PNPH has a P50

of 10–12 mm Hg and has a molecular radius of approximately6.8 nm and a hydrodynamic volume corresponding to that of anoligomerized Hb of 256 kD.



the exacerbation of edema caused by the high-volume resuscitation of current standard care canbe avoided. PNPH requires the least volume torestore and maintain mean arterial blood pressurewhen compared to lactated Ringer’s (LR), standardcivilian therapy, or Hextend (HEX), standard mili-tary therapy, and confers neuroprotection in a rel-evant mouse model of TBI + HS. Mice resuscitatedwith PNPH had fewer Fluoro-Jade C+ identifieddying neurons in Cornu Ammonis 1 versus HEXand LR. PNPH was also shown not only to be non-toxic but also to be neuroprotective against injury(glutamate/glycine exposure and neuronal stretch)in rat primary cortical neuron cultures (P < 0.01).PNPH also maintained cerebral oxygenation betterthan LR and HEX as measured by implanted directoxygen electrodes. In addition, recent data suggest asubstantive attenuation of the development ofintracranial hypertension during resuscitation com-pared to conventional therapy in TBI plus severeHS in mice. PNPH as a therapeutic for TBI repre-sents a major improvement over current HBOCssuggesting that the improvement is due to the com-bined refinements of polynitroxylation and pegyla-tion technologies.

In a separate publication studying TBI with HS,in vivo measurement of hemodynamic response,cerebral perfusion, and intracranial pressure (ICP)demonstrated a beneficial synergistic effect ofbreathing 100% oxygen and PNPH resuscitation,which included reduced ICP (23).

The covalently labeled nitroxides on PNPH alsoappear to have potent antioxidative stress activitiesin the reduced state. Recently published results showthat plasma ascorbate participates in the redoxcycling of the covalently labeled nitroxides on PNPHthrough an unusual redox-catalytic mechanismwhereby reduction of H2O2 is achieved at theexpense of reducing equivalents of ascorbate con-verted into reduced nitroxides (20).

PNPH for strokePNPH was studied as a first treatment for neuro-

protection in ischemic stroke. PNPH reduced infarctvolume by 58%, similar to that published for PegHbat 55% (24,25). Further studies are planned to exploitthe neurovascular protective activities of PNPH toextend the therapeutic window of tissue plasminogenactivator (tPA) treatment.

Several factors contribute to the rationale for theuse of PNPH in stroke. First, PNPH has the ability todeliver O2 to the ischemic tissue through collateralarteries and thereby prolong viability until full reper-fusion can be achieved. By increasing the amount of

O2 carried in the plasma, PNPH with a P50 of10 mm Hg (intermediate between red blood cell[RBC] Hb and ischemic tissue PO2) will facilitate O2

delivery to the endothelial wall.The plasma-based O2

carrier will also deliver O2 to capillaries that are nar-rowed or partially obstructed and poorly perfusedwith RBCs during ischemia. Second, PNPH possessesantioxidant properties by virtue of its SOD, catalase,and peroxidase mimetic activities. These propertiesare likely to stabilize the vasculature during ischemiaand reduce hemorrhagic transformation arising fromdelayed reperfusion. Third, PNPH is synthesized andstored with the heme in the carboxy state.When trans-fused, the CO is released within a matter of minutesand over 97% of the Hb in the RBC and the plasma iscapable of carrying O2 soon after a 10 mL/kg transfu-sion. Thus, PNPH also acts as a CO donor that israpidly converted into an O2 carrier in the circulation.Because CO donors are known to possess vasodila-tory,anti-inflammatory,and anti-apoptotic properties,PNPH may limit reperfusion injury through multiplemechanisms. With decreases in NO production in theendothelium during middle cerebral artery occlusion,collateral blood flow is not maximized and the plateletinhibitory role of endothelial-derived NO is lost. COderived from PNPH can act to increase cGMP andsupplant the loss of NO. Fourth, PNPH has beenobserved to directly protect cultured neurons fromN-Methyl-D-aspartic acid (NMDA). As activation ofNMDA receptors is well known to contribute toischemic injury and to injury from tPA, PNPH mayalso act to directly protect neurons from ischemia andtPA administration in addition to Hb in the case ofhemorrhagic stroke.

These therapeutic mechanisms strongly suggestthat besides neuroprotection, when there is compro-mised blood brain barrier as in TBI and hemorrhagicstroke, PNPH enhances the blood flow in the penum-bra of the ischemic brain and serves as an antioxidantto ameliorate reperfusion injury, thereby leading toinfarct reduction.

PNPH for SCDAgain, several factors contribute to the rationale

for the use of PNPH in SCD. The chronic hemolyticanemia of SCD produces NO scavenging by the cell-free Hb causing NO-dependent vascular dysfunction(26), which can be ameliorated by the antioxidantand NO-sparing effects of PNPH. The cell-free Hb isalso naturally a pro-oxidant and leads to overproduc-tion of superoxide which can also be amelioratedby the multiple antioxidant properties of PNPHincluding its SOD, catalase, and peroxidase mimeticactivities. PNPH, as a therapeutic fluid to not only



augment oxygen delivery but also correct inadequateblood flow while conferring antioxidant andNO-sparing effects, could also represent a paradigmchange in the treatment of vaso-occlusive crisis andacute chest syndrome in SCD. In preliminary collabo-rative PNPH studies, PNPH was shown to correctNO-dependent vascular dysfunctions induced by NOdepletion and overproduction of superoxide in atransgenic sickle mouse model (27). Inhibition ofsickling and acute hemolysis by PNPH coupled withits enhancement of NO bioavailability may representanother paradigm shift in transfusion therapy in SCDpatients.

CONCLUSIONS

A growing body of preclinical evidence suggeststhat PNPH represents a paradigm shift in HBOCdevelopment toward a new focus on correctinginadequate blood flow due to oxidative stress andattenuating ischemia/reperfusion/inflammationinjury. Also, if this holds up in clinical trials, PNPHappears likely to meet the FDA mandate for anHBOC with a meaningful increase in therapeuticindex. The biology of PNPH suggests that it will beof therapeutic value in a wide range of clinical indi-cations; current thinking is that TBI, stroke, andSCD are a reasonable first set of indications toprove the benefits of PNPH. With renewed interestand funding by the NIH and the Department ofDefense for the development of therapeutic Hb,PNPH (and other related high therapeutic indexproducts) could (i) be further studied to betterdefine the contribution to favorable outcome ofeach of the rich array of potential mechanisticeffects of this multifunctional therapeutic; and (ii)be developed to commercialization and given theopportunity to address major healthcare concernsand unmet medical needs in transfusion and criticalcare medicine.

Acknowledgments: The work in TBI + HS in col-laboration with Safar Center at UPMC was sup-ported in part by grant PR054755 W81XWH-06–1-0247 from the US Army.The work in stroke and SCDat Johns Hopkins University Medical Center wasfunded via a Collaborative Agreement betweenSynZyme Technologies LLC and Prolong Pharma-ceuticals Inc.

DISCLOSURE

The authors are shareholders of SynZyme Tech-nologies LLC.

REFERENCES

1. Natanson C, Kern SJ, Lurie P, Banks SM, Wolfe SM. Cell-freehemoglobin-based blood substitutes and risk of myocardialinfarction and death: a meta-analysis. JAMA 2008;299:2304–12.

2. FDA/NIH Workshop. “Hemoglobin based oxygen carriers:current status and future directions,” Bethesda, Maryland,Wednesday, April 29–30, 2008. Available at: http://www.fda.gov/downloads/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConferences/TranscriptsMinutes/UCM051545. Accessed June 29, 2011.

3. Alayash AI. Hemoglobin-based blood substitutes: oxygen car-riers, pressor agents, or oxidants? Nat Biotechnol 1999;17:545–49.

4. Sloan EP. The clinical trials of diaspirin cross-linked hemo-globin (DCLHb) in severe traumatic hemorrhagic shock:the tale of two continents. Intensive Care Med 2003;29:347–9.

5. Gould SA, Moore EE, Hoyt DB, et al. The life-sustainingcapacity of human polymerized hemoglobin when red cellsmight be unavailable. J Am Coll Surg 2002;195:445–52.

6. Jacobs E. Clinical update: hemopure(r)—a room temperaturestable hemoglobin oxygen carrier. Anasthesiol IntensivmedNotfallmed Schmerzther 2001;36(Suppl. 2):S121–2.

7. Hsia JC, Song DL, Er SS, et al. Pharmacokinetic studies in therat on a o-raffinose polymerized human hemoglobin. BiomaterArtif Cells Immobilization Biotechnol 1992;20:587–95.

8. Burhop K. The development of a second-generation, designer,recombinant hemoglobin. Artif Oxygen Carrier 2005;12:127–34.

9. Alayash AI. Oxygen therapeutics: can we tame haemoglobin?Nat Rev Drug Discov 2004;3:152–9.

10. Ma L, Hsia CJC. Taming the hemoglobin: therapeutic andimaging applications of redox coupled nitroxide andhemoglobin. Xth International Symposium on Blood Substi-tutes, Providence, RI, USA, June 12–15, 2005.

11. Krishna MC, Ma L, Samuni A, Trimble CE, Mitchell JB, HsiaCJC. Superoxide dismutase- and catalase-mimetic activity of apolynitroxyl hemoglobin (PNH). Artif Cells Blood SubstitImmobil Biotechnol 1996;24:369.

12. Buehler PW, Haney CR, Gulati A, Ma L, Hsia CJ. Polynitroxylhemoglobin: a pharmacokinetic study of covalently boundnitroxides to hemoglobin platforms. Free Radic Biol Med2004;37:124–35.

13. D’Agnillo F, Chang TM. Polyhemoglobin-superoxidedismutase-catalase as a blood substitute with antioxidantproperties. Nat Biotechnol 1998;16:667–71.

14. Simoni J, Simoni G, Martinez-Zaguilan R, et al. Improvedblood substitute: evaluation of its effects on human endothelialcells. ASAIO J 1998;44:M356–67.

15. Winslow RM. The role of hemoglobin oxygen affinity inoxygen transport at high altitude. Respir Physiol Neurobiol2007;158:121–7.

16. Kawaguchi AT, Nakai K, Fukumoto D, Yamano M, Haida M,Tsukada H. S-nitrosylated pegylated hemoglobin reduces thesize of cerebral infarction in rats. Artif Organs 2009;33:183–8.

17. Yu B, Raher MJ, Volpato GP, Bloch KD, Ichinose F, ZapolWM. Inhaled nitric oxide enables artificial blood transfusionwithout hypertension. Circulation 2008;117:1982–90.

18. Rodriguez C, Vitturi DA, He J, et al. Sodium nitrite therapyattenuates hypertensive effects of HBOC-201 via nitritereduction. Biochem J 2009;422:423–32.

19. Spinella PC, Carroll CL, Staff I, et al. Duration of red bloodcell storage is associated with increased incidence of deep veinthrombosis and in hospital mortality in patients with traumaticinjuries. Crit Care 2009;13:R151.

20. Stoyanovsky DA, Kapralov A, Huang Z, et al. Unusual per-oxidase activity of polynitroxylated pegylated hemoglobin:elimination of H(2)O(2) coupled with intramolecular oxida-



tion of nitroxides. Biochem Biophys Res Commun 2010;399:139–43.

21. Beckman JD, Belcher JD, Vineyard JV, et al. Inhaled carbonmonoxide reduces leukocytosis in a murine model of sickle celldisease. Am J Physiol Heart Circ Physiol 2009;297:H1243–53.

22. Shellington DK, Du L, Wu X, et al. Polynitroxylated pegylatedhemoglobin: a novel neuroprotective hemoglobin for acutevolume-limited fluid resuscitation after combined traumaticbrain injury and hemorrhagic hypotension in mice. Crit CareMed 2011;39:494–505.

23. Blasiole B, Bayir H, Vagni V, et al. Resuscitation of experi-mental traumatic brain injury and hemorrhagic shock in mice:acute effects of polynitroxylated pegylated hemoglobin and100% oxygen. Crit Care Med 2010;38(Suppl.):39. Abstract 145.

24. Koehler RC, Cao S, Zhanf J, Kwansa H. Effect of cell-freehemoglobin on the cerebral circulation. XIIth ISBS meeting inParma, Italy, August 25–27, 2009.

25. Klaus JA, Kibler KK, Abuchowski A, Koehler RC. Early treat-ment of transient focal cerebral ischemia with bovine pegy-lated carboxy hemoglobin transfusion. Artif Cells BloodSubstit Immobil Biotechnol 2010;38:223–9.

26. Hsu LL, Champion HC, Campbell-Lee SA, et al. Hemolysis insickle cell mice causes pulmonary hypertension due to globalimpairment in nitric oxide bioavailability. Blood 2007;109:3088–9.

27. Hsia CJC, Ma L. PNPH—a therapeutic for inadequate bloodflow and superoxide, nitric oxide dependent vasculardysfunctions. XIIth ISBS Meeting in Parma, Italy, August25–27, 2009.aor_1238 220..233



hsia 2012 a hemoglobin based m

Documents