A Phase Ib open label, randomized, safety study of SANGUINATE™ in patients with sickle cell anemia

A Phase Ib open label, randomized, safety study of SANGUINATE™ in patients with sickle cell anemia B O A S H K N a b c d e a A R A A K S S S C h 1 o ARTICLE IN PRESSJHH 2881; No of Pages 8 rev bras he[.]

w w w r b h h o r g

Brazilian Journal of Hematology and Hemotherapy

Original article

Hemant Misraa, ∗, James Bainbridgea, John Berrymana, Abraham Abuchowskia,

Kenneth Mauricio Galvezb, Luis Fernando Uribec, Angel Luis Hernandezd,

Nestor Rodolfo Sosae

a r t i c l e i n f o

Received22February2016

Accepted22August2016

Availableonlinexxx

Keywords:

Sicklecelldisease

SANGUINATE

Safety

Clinicaltrial

a b s t r a c t

understoodgeneticandbiochemicalpathwayofsicklehemoglobin,currenttherapy contin-uestobelimitedtothesymptomatictreatmentofpain,supplementaloxygen,antibiotics, redbloodcelltransfusionsandhydroxyurea.SANGUINATEisacarbonmonoxidereleasing moleculeandoxygentransferagentunderclinicaldevelopmentforthetreatmentofsickle cellanemiaandcomorbidities

ane-miapatients.TwodoselevelsofSANGUINATEwerecomparedtohydroxyureain24 homozy-gotesforHbSS.Twelvesubjectsreceivedeitheralowdose(160mg/kg)ofSANGUINATEor

15mg/kghydroxyurea.Another12subjectsreceivedeitherahighdose(320mg/kg)of SAN-GUINATEor15mg/kghydroxyurea.TheprimaryendpointwasthesafetyofSANGUINATE

determi-nationoftheplasmapharmacokineticsandassessmentofhematologicmeasurements

tro-ponin Ilevels increased in three patients, one of whom hadan increase in tricuspid regurgitantvelocity;however,noclinicalsignswerenoted.Followinganassessmentofvital signs,tricuspidregurgitantvelocity,electrocardiogram,serumbiochemistry,hematology, urinalysis,andanalysisofreportedadverseevents,SANGUINATEwasfoundtobesafein stablesicklecellanemiapatients

safetyprofileforSANGUINATEinpatientswithsicklecellanemia.Thistrialestablished thesafetyofSANGUINATEatbothdoselevelsandpermitteditsadvancetoPhaseIItrials

©2016Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published

byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/)

E-mailaddress:hmisra@prolongpharma.com(H.Misra)

http://dx.doi.org/10.1016/j.bjhh.2016.08.004

1516-8484/©2016Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction

Treatmentofsicklecellanemia(SCA)isachallengingtaskand

thereisonlyonedrug,hydroxyurea,approvedbytheFoodand

DrugAdministration(FDA) oftheUnitedStates.Painisthe

mostfrequentmanifestationofSCAandisthoughttobea

consequenceofvaso-occlusion.Vaso-occlusivecrisesarethe

primarycauseoffrequenthospitalizationofSCApatientsand

areamajorcauseofdeathinadultSCApatients.1,2Despitethe

well-understoodgeneticand biochemicalpathwayofsickle

hemoglobinandmanydiscoveriesofbiomarkersofsicklecell

pathophysiology,modern-daytherapycontinuestobelimited

to symptomatic treatment of pain, supplemental oxygen,

antibioticstherapy,redbloodcelltransfusions,and

hydrox-yurea.SCAisaninheritedblooddisordercausedbyadefective

hemoglobinprotein.Thepolymerizationofsicklehemoglobin

isassociatedwithmanyabnormaldownstreamprocesses,but

nosinglepathwayhasbeenshowntoplayaprimaryorcritical

roleincomplicationsoccurringinSCApatients.Thiscascade

ofeventsisresponsibleforthedevelopmentofcomorbidities

associatedwithSCAsuchasvaso-occlusion,stroke,legulcers,

andacutechestsyndrome

SANGUINATE(pegylatedbovinecarboxyhemoglobin)

rep-resentsanovelapproachtotreatingacuteexacerbationsof

SCAbytargetingtheunderlyinginflammationandcausesof

hypoxia.SANGUINATEisacarbonmonoxidereleasing/oxygen

transferagentbeingdevelopedforthetreatmentofanemic

andischemichypoxiathatresultfromcongenitaloracquired

hemoglobinopathies and vasculopathies, such as SCA and

thalassemia,andfromcerebrovascularorperipheralvascular

diseases

As SANGUINATE contains a hemoglobin core, there are

particular concerns regarding potential vasoactivity Trials

performed with previous hemoglobin-based oxygen

carri-ers reported adverse events such as hypertension, cardiac

arrhythmias/conduction disorders, gastrointestinal

symp-toms,abnormalliverfunctiontestsandhemorrhage/anemia.3

The underlying pathophysiological mechanisms for these

cardiac effects are not precisely known but they have

been attributed to the scavenging of nitric oxide.4

SAN-GUINATE,duetothemodificationbypolyethyleneglycoland

the anti-vasoconstrictive activity of carbon monoxide, has

demonstrated no vasoactivity in vivo.5 An ascending dose

studyofthreecohortsofeighthealthyvolunteersfoundno

seriousadverseeventsatdosesof80,120or160mg/kg.6This

PhaseIbtrialrevealednoserioustreatment-relatedadverse

effectsandshoweddose-proportionalpharmacokinetics.As

SCApatientssuffer from inflammationand anemia,which

causeanumberofcomorbidities,anopenlabel,randomized

PhaseIbtrialwasundertakentoensurethesafety of

SAN-GUINATEinclinicallystablepatientswhoarehomozygousfor

SCA

Methods

This open label randomized Phase Ib trial was conducted

in two countries and four medicalcenters in Central and

SouthAmerica.Thesestudieswereconductedincompliance

withtheUSFoodandDrugAdministrationregulations.The

trialwasregisteredasNCT01848925.Protocolsweredesigned togetherwiththeinvestigators.Approvalsweregrantedbythe Ethics CommitteesandRegulatoryAuthorities.Theauthors hadaccesstoprimaryclinicaltrialdata

AdultpatientswithconfirmedSCAwereenrolled.Eligibility criteriaincludedageof18yearsorolder,baselinehemoglobin level>6or<10g/dL,takinghydroxyureaornot,butmusthave been dose stabilized foratleast threemonths and ableto discontinuehydroxyureaforsevendayspriorto randomiza-tion Female patients ofchildbearing age were requiredto useamedicallyacceptableformofcontraceptionduringboth studies and negative serum pregnancy testswere required

atenrollmentandpriortoinfusion.Patientexclusion crite-ria included individuals on a chronic transfusion program (definedasregulartransfusionsevery2–8weeks),acutechest syndrome,seriousinfections,allergiestohydroxyurea,history

ofclinicallysignificantdiseasesandelectrocardiogram(ECG) abnormalities.Moreoverpatientswereexcludediftheyhad had more than sixEmergency Room visits/hospitalizations peryearforSCA-relatedpainevents,renalorliver dysfunc-tion,troponin I>0.31ng/mL,amylaseor lipase>1.1×upper limitofnormal,treatmentwithinvestigationaldrugwithin60 daysorintentiontobeginnewdrugtherapyduringthestudy period

SANGUINATETM (ProlongPharmaceuticals,LLC,South Plain-field, New Jersey) 40mg/mL for intravenous infusion was providedin500mLethylenevinylacetatebloodbagswhich wereshippedinheat-sealedMylar,gasimpermeablefoil over-packbagscontaininganinertgas,whichservedtoprotectthe product.SANGUINATEwasstoredundersecureconditionsina locked,limited-accessrefrigerator,at2–8◦C.Droxia® (hydrox-yurea;BristolMyersSquibb,PrincetonNJ)as100mgcapsules wasobtainedthroughacommercialpharmacyandprovided unalteredaspertheproductlabeling

Twenty-fouradultSCApatients(HbSS)wererandomized 2:1toreceive,unblinded,eitherasingle2-hintravenous infu-sion of SANGUINATE, or a standard dose of hydroxyurea (HU)with2hatrestingstage.Thefirst12 patientswereto receive either160mg/kgofSANGUINATE(eight patients)or

15mg/kgofhydroxyurea (fourpatients),and thesecond 12 patients were to receive either 320mg/kg of SANGUINATE (eightpatients)or15mg/kgofhydroxyurea(fourpatients).The overallstudydesignispresentedinFigure1

Safetywasevaluatedthroughoutthestudy byassessments overa7-dayperiod.Patientsremainedinthestudycenterfor

aminimumof48hafterthestartofdosingtocompleteaseries

ofsafetyassessmentsincludingbloodandurinelabtests,ECG, andphysicalexamcomparedtopre-dosingbaselinevalues.A 24-hurinecollectionwasperformedasameasureof dehydra-tion.Urinewascollectedover48handcreatinineclearance wastestedtoassesskidneyfunctionevery24h.Vitalsignsas wellasevaluationsofsignsorsymptomsofadverseevents were conductedperiodically byphysicalexam and patient interview

24 Stable SCD patients

All subjects

Randomized 2:1

Visit 1

Screen (Day - 7)

Day of admission (Day 0: includes baseline labs+

treadmill/pain score)

Day after dosing (Day 2: includes spirometry/

treadmill/pain)

Inpatient (Days 3:

includes spirometry /pain)

Outpatient follow-up (Day 5)

Outpatient follow-up (Day 4)

Final visit (Day 7±1)

Day of dosing (Day 1: includes predose echo + troponin + spirometry, and postdose echos + treadmill/pain) Visit 2 (Inpatients)

+ PK blood draws for patients reciving SANGUINATE

Visit 3 Visit 4 Visit 5

SANGUINATE 160 mg/kg (8 patients) Hydroxyurea 15 mg/kg (4 patients)

SANGUINATE 320 mg/kg (8 patients) Hydroxyurea 15 mg/kg (4 patients)

Figure 1 – Overall study design.

Plasma SANGUINATE concentrationswere quantified using

avalidatedhighperformanceliquidchromatography(HPLC)

method Serial blood samples for pharmacokinetic

analy-sis were collected at baseline and at 0.5, 1, 1.5, 2, 4, 6,

8, 12, 24, 36, 48, 72 and 96h from the start of infusion

Non-compartmentalpharmacokineticmethodswereusedto

determinethepharmacokineticparameters,whichincluded

(CmaxandCmin,respectively),timetoreachmaximumplasma

concentration(tmax),theareaundertheplasmaconcentration

mea-surableplasmaconcentration[areaundercurve(AUC)0→],

areaundertheplasmaconcentrationversustimecurvefrom

timezerotoInfinity(AUC0→∞),terminalhalf-life(t1/2)and

theapparenteliminationrateconstant(z).

Results

Overall, the majority of patients were female (15) and of

race ‘other’ (23 reported as Hispanic/Latino, and Black or

mixed/multiracial).Patients in the 160mg/kg SANGUINATE

treatmentgroupwereonaverageslightlyyoungerthanthe

other treatment groups, but this group also included the

oldestpatient inthe trial (54 years) There was no

signif-icant differencein averageheightand weight betweenthe

hydroxyurea-treatedandtheSANGUINATE-treatedpatients

There were no remarkable differences between treatment

groupsinmedicalhistoryandtherewerenounexpected

find-ings(notrelatedtoSCA)foranypatientinthebaselinephysical

examination

Atotalof24stableSCApatientsfrom clinicalcentersin

ColombiaandPanamawereenrolled.Ofthe24patients,22

receivedtheirassignedstudymedicationandcompletedthe

studyasperprotocol.FifteenpatientsreceivedSANGUINATE

and sevenpatientsreceivedhydroxyurea.Two patients dis-continuedbeforereceivingmedications

Pharmacokinetics

ThepharmacokineticsofSANGUINATEweredose-dependent ThemeanbloodlevelsofSANGUINATEforthetwodosegroups are showninFigure2.Forpatients receivingthe160mg/kg dose ofSANGUINATE, the mean peak concentration(Cmax) was2.59mg/mLandthemeanpeakconcentrationwiththe

320mg/kg dosewas 6.46mg/mL Both maximumsoccurred

at2h (completionoftheinfusion).Doublingthedosefrom

160mg/kgto320mg/kgledtoamean150%increaseinpeak bloodlevelofSANGUINATEinthisSCApatientpopulation.Ina previousstudyofhealthysubjectsreceivingasingleinfusion

of160mg/kgSANGUINATE,allofthesixsubjectshadblood levelsbelowthelevelofdetectionat96haftertheinfusion.6

Safety

MoreadverseexperienceswerereportedintheSANGUINATE groupsthan reportedinthe hydroxyureagroups.Ofthe 44 reported adverse events in SANGUINATE-treated patients,

16 were from only two ofthe 15 patients In addition, 46

ofthe51 reportedadverse eventsinvolving pain(including headache).Musculoskeletaland connectivetissue disorder-related adverse events were the mostcommonly reported, with arthralgia accountingfor ten (nineSANGUINATE, one hydroxyurea)ofthe51reports(Table1

Mean increases in systolic and diastolic arterial blood pressures (transient)were expectedfollowingSANGUINATE infusionsduetoitsplasmaexpansionpropertiesinthe blood-stream.Theseincreasesdonotappeartobedose-dependent The mean systolic blood pressure following infusion of

320mg/kg is not appreciably different from the 160mg/kg group (Figure 3 The chart of mean diastolic pressures (Figure4)supportsthesuggestionthattheoncoticeffectmay notbedose-dependent

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

96 84

72 60

48 36

24 12

0

160mg/kg MEAN 320mg/kg MEAN

Figure 2 – Mean plasma concentration of SANGUINATE (mg/mL) by dose.

Manyofthelaboratoryparametersthatwereabnormalat

baselineremainedoutsideofthenormalrangeforthe

dura-tionofthestudy,andformanylaboratoryparameters,there

werenomeaningfuldifferencesbetweentreatmentgroups

Meanvaluesforhemoglobinandhematocritovertimeshow

thattreatmentwithSANGUINATEdoesnotprovidean

appre-ciableincreaseinquantityorconcentrationofhemoglobinin

thesepatients

However,thelevelsofdirect(conjugated)bilirubin(Table2) demonstratedaclearmeandecreaseonthedayofdosing rela-tivetobaselinelevelsforthepatientsreceivingSANGUINATE, which was not seen in the hydroxyurea treatment group Notably,themeanlevelfollowinginfusionof320mg/kg SAN-GUINATE approximates the upperlimit of the laboratory’s normalrange.Thismeantreatmentdifferenceisnot appar-entintheleveloftotalbilirubin,butiscloselymirrored,albeit

50

40

30

20

10

0

-10

-20

Figure 3 – Mean systolic blood pressure.

Table 1 – Summary of adverse events.

Table 2 – Direct Bilirubin and gamma-glutamyl transpeptidase (GGT).

Units Statistic Hydoxyurea Sanguinate(160mg/kg) Sanguinate(320mg/kg)

Baseline result

Visit result

Difference Baseline

RESULT

Visit result

Difference Baseline

result

Visit result Difference

40

30

20

10

0

-10

-20

Figure 4 – Diastolic blood pressure.

always within the normal range, in the levels of

gamma-glutamyltranspeptidase

Themeanresultsofthechemicalurinalysistestfor

pres-enceofbloodintheurineshowatreatment-specific(though

not apparently dose-specific) difference following infusion

withSANGUINATE.Themeanredbloodcelllevelsseenupon

microscopicanalysisapproximatelymirrorthisfinding,which

may be the result of the increased colloid-osmotic

pres-sureproducedbytheinfusion,causinganincreaseinforced

glomerularfiltration.Asimilarresultwasseenforurinary

pro-tein

TherewerebriefbutsubstantialincreasesintroponinI

lev-elsinthreepatientsreceivingSANGUINATE(320mg/kg)andin

onepatientreceivingHU.Theincreaseswereofshortduration

andnotaccompaniedbyanyclinicallyidentifiedorpatient

reportedadverseexperiences.Oneofthethreepatientswith

elevatedtroponinlevelsalsohadareportedseriousadverse

event due to an increase in tricuspid regurgitant velocity

(TRV) whichwas labeledas asign ofmoderate pulmonary

hypertension;however,associatedsymptomsofpulmonary

hypertensionwerenotpresentafterangiography.Theother

twopatientshadbaselinevaluesnearorabovetheupperlimit

ofthelaboratoryreferencerangefortroponinI(0.028ng/mL)

Thehighestmeasuredlevelwasalsotheshortestlasting:the

serumtroponinIlevelofonepatientwasassayedatnearly

16-timestheupperlimitat1haftertheendofSANGUINATE

infusion.Yet,despitereadingabovethelimitatbaseline,

tro-poninIwasdowntothelowlevel ofnormal(0.007) bythe

timeofthenextmeasurement5hlater,whereitremainedfor

thedurationofthestudy.Becauseoftheshortduration,and

becauseitwasnotaccompaniedbyanyclinically-identifiedor

patient-reportedadverseexperiences,thesewerenotreported

bytheinvestigatorsasserious adverseevents.Onepatient

receivinghydroxyureaalsohadabriefincreaseintroponin

Ilevel(0.045ng/mL) abovetheupperlimitofthelaboratory

referencerangeat72hafterdosing

Discussion

The pathobiology ofSCA is characterizedbyinflammation and oxygen deprivation While hydroxyurea therapy has decreasedthepainfulepisodesofSCA,poorcomplianceand non-response by a significant proportion of SCA patients resultsinthedevelopmentofvaso-occlusivecrisesandother acute comorbidities SANGUINATE is a novel construct of hemoglobin that contains carbon monoxide and pegylated bovine carboxyhemoglobin It has been shown to reduce infarctvolume inanimalmodels7,8 andhas been adminis-tered undermultipleemergency InvestigationalNewDrugs (eINDs).9,10 In vitrostudieshavedemonstratedtheabilityof SANGUINATE to return SCA blood cells to a more normal morphology11 and demonstrated the transfer ofoxygen to hypoxiccells

This first in-patients study assessed the safety-related effectsofSANGUINATEinSCApatients.Sinceonly15patients received SANGUINATEand sevenpatients received hydrox-yurea, the size of the study population was too small to allowacalculationofstatisticalsignificancebetween treat-ment groups It wasobserved that many ofthe laboratory parameters being measured were abnormalat baseline in this population as expected The damage that has devel-opedinthesepatientsfromalifetimeofhemolyticanemia andischemichypoxiameansthattheypresentanextremely diverserangeofpossiblephysiologicalresponsesthatmaybe undetectableusingtheparametersandmethodsofthisstudy Thedecreaseinbilirubinandgamma-glutamyl transpep-tidase maybeduetotheimpactofSANGUINATEupon the redbloodcell.SANGUINATEhasbeenshownto‘unsickle’red bloodcellsinSCApatientsin vitro.12Theimprovementinred bloodcellmorphologyandbloodflowparametersmayreduce hemolysisandtherebyimpactthesebiochemicalparameters This hypothesis remains to beconfirmed in larger clinical studies

tro-ponin I levels seen in some of these patients is also not

clear.ThetransientelevationoftroponinIinthreeof15

sub-jectslastedafewhoursorafewdays,without sequelaeor

repeatedelevations.CurrentAmericanCollegeofCardiology

Foundation(ACCF)/AmericanHeartassociation(AHA)

guide-linesdonotdefinethedurationoftroponinelevationneeded

toindicatepathology,anddonotrecommendtheir

diagnos-ticor prognosticuse alone inheart failure.13 Furthermore,

asclarifiedinthe2007JointEuropeanSocietyofCardiology

(ESC)/ACCF/AHA/WorldHeartFederation(WHF)report

“Uni-versalDefinition of MyocardialInfarction”, serial measures

ofcardiactroponinaftertheonsetofclinicalsymptomsare

neededfordiagnosisofcardiacischemia.14Arapidriseand

falloftroponin I does notfit the standard cardiac disease

paradigm, and the returnof troponinI levels tonormal is

indicativeofresponsetotreatmentorspontaneous

improve-ment

Theresponseofonepatient,whoreceived320mg/kg

SAN-GUINATE, ofmulti-day increases in TRV tolevels typically

associatedwithmoderate-to-severepulmonaryhypertension,

substantialincreasesinsystemicbloodpressure,andelevated

troponinItonearly4-foldtheupperlevelofnormal,lasting

severaldays,areresultswhich,albeitparadoxical,were

associ-atedwithnoclinically-identifiedorpatient-reportedadverse

effects.Pulmonaryhypertension, definedbypersistentTRV

valuesabove3.0m/s,hasbeencorrelated withasignificant

increasedriskofmortalityinSCApatients.15Theimpactof

TRVelevationsabove 3.0m/s that lastforonlya fewdays,

however,occurringinSCApatientswithaverageTRVlevels

>2.0m/shasnotbeendetermined.Giventhelackofclinical

adverseeffectsofthepatient,herprofessedwell-being,and

theisolatednatureofthisevent,theimpactofthereported

eventontheunderstandingofriskrelatedtothestudydrug

inSCApatientsisverylimited

InfusionofSANGUINATEatboth160and320mg/kg

gen-erated anexpected increase inarterial pressure dueto its

colloid osmotic properties, which resolved without

appar-ent sequelae Meansystolic and diastolic systemic arterial

pressurewasincreasedinSANGUINATE-treatedpatients

com-pared to hydroxyurea-treated patients, but accompanying

meanincreases inTRVvalueswerenotseen inthis study

TheknowntransienteffectofSANGUINATEonarterial

pres-sure,possiblyincludingpulmonaryarterialpressurethatwas

undetectableinthissmallstudy,isbelievedtobeduesolely

toitsoncoticeffect.Thelackofclinicallymeaningfuladverse

effectsresultingfromthepressureincreasefurthersupports

atransientfluid-volumebasisforthemechanism

(160mg/kg) had a prolonged T1/2 in stable SCA patients

(19.56h) compared to that found in healthy volunteers

(13.75h).7InthePhaseIstudywithhealthyvolunteers,itwas

observedthathaptoglobinlevels weresignificantlyreduced

followingSANGUINATE administration.Becausethe coreof

SANGUINATEishemoglobin,it hasbeenproposed thatthe

clearance mechanismwill bethe same asthe mechanism

used to clear native hemoglobin following hemolysis by

the reticuloendothelial system Because ofthe widespread

hemolysisinSCApatients,thisclearancemechanismwould

beburdenedbythepatient’sextensivecell-freehemoglobin,

thus reducing the rate atwhichthis mechanism canclear SANGUINATE, which would lead to a longer circulatory half-life

Conclusion

Thisisafirstin-patientstudyofpatientswithstableSCAwith either160mg/kgor320mg/kgofSANGUINATE.Whilethere weremoreadverseeventsintheSANGUINATEarm,theywere mild and self-limited Following assessment ofvital signs, echocardiographicmeasuresofTRV,electrocardiogram anal-ysis,laboratorymeasuresofserumbiochemistry,hematology andurinalysis,andreportedadverseevents,noclearevidence

ofclinicallymeaningfulsafetyconcernswereidentified.These resultssupportfurtherdevelopmentofSANGUINATEforthe treatmentofSCAcomorbiditiesandinitiationoffurther clin-icaltrialsdesignedtooptimizedosingandselectappropriate endpointstoassesssafetyandefficacy

Funding

ThisstudywassupportedbyresearchfundingfromProlong Pharmaceuticals

Conflicts of interest

HM,JB,JBandAAareemployeesofProlongPharmaceuticals and InvestigatorsKMG, LFU,ALH andNRS were fundedby ProlongPharmaceuticals

r e f e r e n c e s

1.PerronneV,Roberts-HarewoodM,BachirD,Roudot-Thoraval

F,DelordJM,ThuretI,etal.Patternsofmortalityinsicklecell diseaseinadultsinFranceandEngland.HematolJ

2002;3(1):56–60

2.PlattOS,BrambillaDJ,RosseWF,MilnerPF,CastroO, SteinbergMH,etal.Mortalityinsicklecelldisease.Life expectancyandriskfactorsforearlydeath.NEnglJMed 1994;330(23):1639–44

3.LanzkronS,StrouseJJ,WilsonR,BeachMC,HaywoodC,Park

H,etal.Systematicreview:hydroxyureaforthetreatmentof adultswithsicklecelldisease.AnnInternMed

2008;148(12):939–55

4.SilvermanTA,WeiskopfRB.Hemoglobin-basedoxygen carriers:currentstatusandfuturedirections.Anesthesiology 2009;111(5):946–63

5.CabralesP.Examiningandmitigatingacellularhemoglobin vasoactivity.AntioxidRedoxSignal.2013;18(17):2329–41

6.ZhangJ,CaoS,KwansaH,CrafaD,KiblerKK,KoehlerRC Transfusionofhemoglobin-basedoxygencarriersinthe carboxystateisbeneficialduringtransientfocalcerebral ischemia.JApplPhysiol.2012;113(11):1709–17

7.MisraH,LickliterJ,KazoF,AbuchowskiA.PEGylated carboxyhemoglobinbovine(SANGUINATE):resultsofaPhase

Iclinicaltrial.ArtifOrgans.2014;38(8):702–7

8.AnanthakrishnanR,LiQ,O’SheaKM,QuadriN,WangL, AbuchowskiA,etal.CarbonmonoxideformofPEGylated hemoglobinprotectsmyocardiumagainst

ischemia/reperfusioninjuryindiabeticandnormalmice ArtifCellsNanomedBiotechnol.2013;41(6):428–36

9 KlausJA,KiblerKK,AbuchowskiA,KoehlerRC.Early

treatmentoftransientfocalcerebralischemiawithbovine

PEGylatedcarboxyhemoglobintransfusion.ArtifCellsBlood

SubstitImmobilBiotechnol.2010;38(5):223–9

10.AlaaliY,IocoE,VarelasP,AbdelhakT,MastorodimosV,

MendezM.UseofSanguinateinacutechestsyndrome.J

SickleCellDisHemoglobinopat.2014;1:1

11.ParmarD.AcasestudyofSANGUINATETMinapatientwitha

comorbidityduetoanunderlyinghemoglobinopathy.JSickle

CellDisHemoglobinopat.2014;1:2

12.JubinR,BuontempP,YglesiasRA,AbuchowskiA,ChenY,

KazoG,etal.Rapidreversalofredbloodcellsickling

promotedbyPEGylatedcarboxyhemoglobinbovinegas

transferproperties.Abstract1371.In:56thASHannual

meetingabstractsandprogram.2014

13.MisraH,BuontempoP,BuontempoC,Yglesias,R,ChenY, JubinR,etal.Anti-inflammatoryactivityandrapidreversalof sicklecellmorphologybyPEG-COHbmediatedgastransferin vitro.PresentedinHEMOBRAZIL2015,Braziliancongressof hematology,hemotherapy,andcelltherapy.November20, 2015

14.YancyCW,JessupM,BozkurtB,ButlerJ,CaseyDE,Drazner

MH,etal.2013ACCF/AHAguidelineforthemanagementof heartfailure:areportoftheAmericanCollegeofCardiology Foundation/AmericanHeartAssociationTaskForceon PracticeGuidelines.JAmCollCardiol.2013;62(16):e147–239

15.ThygesenK,AlpertJS,WhiteHD,JointESC/ACCF/AHA/WHF TaskForcefortheRedefinitionofMyocardialInfarction Universaldefinitionofmyocardialinfarction.Circulation 2007;116(22):2634–53