Development and Characterization of Inhaled Ethanol as a Novel Pharmacological Strategy Currently Evaluated in a Phase II Clinical Trial for Early-Stage SARS-CoV-2 Infection

Abstract

Inhaled administration of ethanol in the early stages of COVID‐19 would favor its location
on the initial replication sites, being able to reduce the progression of the disease and improving its prognosis. Before evaluating the efficacy and safety of this novel therapeutic strategy in humans, its characterization is required. The developed 65° ethanol formulation is stable at room temperature and protected from light for 15 days, maintaining its physicochemical and microbiological properties. Two oxygen flows have been tested for its administration (2 and 3 L/min) using an automated headspace gas chromatographic analysis technique (HS‐GC‐MS), with that of 2 L/min being the most appropriate one, ensuring the inhalation of an ethanol daily dose of 33.6 ± 3.6 mg/min and achieving more stable concentrations during the entire treatment (45 min). Under these conditions
of administration, the formulation has proven to be safe, based on histological studies of the respiratory tracts and lungs of rats. On the other hand, these results are accompanied by the first preclinical molecular imaging study with radiolabeled ethanol administered by this route. The current ethanol formulation has received approval from the Spanish Agency of Medicines and Medical Devices for a phase II clinical trial for early‐stage COVID‐19 patients, which is currently in the recruitment phase (ALCOVID‐19; EudraCT number: 2020‐001760‐29).

Full text

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Pharmaceutics2021,13,342.https://doi.org/10.3390/pharmaceutics13030342www.mdpi.com/journal/pharmaceutics
Article
DevelopmentandCharacterizationofInhaledEthanolasa
NovelPharmacologicalStrategyCurrentlyEvaluatedinaPhase
IIClinicalTrialforEarly‐StageSARS‐CoV‐2Infection
AnaCastro‐Balado
1,2,3,†
,CristinaMondelo‐García
1,2,†
,LetriciaBarbosa‐Pereira
4,†
,IriaVarela‐Rey
1,2,3
,
IgnacioNovo‐Veleiro
5
,NéstorVázquez‐Agra
5
,JoséRamónAntúnez‐López
6
,EnriqueJoséBandín‐Vilar
1,2
,
RaquelSendón‐García
4
,ManuelBusto‐Iglesias
1,2
,AnaRodríguez‐BernaldodeQuirós
4
,
LauraGarcía‐Quintanilla
1,2,3
,MiguelGonzález‐Barcia
1,2
,IreneZarra‐Ferro
1,2
,FranciscoJ.Otero‐Espinar
3
,
DavidRey‐Bretal
7
,JoséRamónLago‐Quinteiro
8
,LuisValdés‐Cuadrado
8
,CarlosRábade‐Castedo
8
,
MaríaCarmendelRío‐Garma
9
,CarlosCrespo‐Diz
10
,OlgaDelgado‐Sánchez
11
,PabloAguiar
7
,
GemaBarbeito‐Castiñeiras
12
,MaríaLuisaPérezdelMolino‐Bernal
12
,RocíoTrastoy‐Pena
12
,
RossanaPassannante
13
,JordiLlop
13,
*,AntonioPose‐Reino
5,
*andAnxoFernández‐Ferreiro
1,2,
*
1
PharmacyDepartment,UniversityClinicalHospitalofSantiagodeCompostela(SERGAS),
15706SantiagodeCompostela,Spain;ana.castro.balado@gmail.com(A.C.‐B.);
crismondelo1@gmail.com(C.M.‐G.);iria.varela.rey@sergas.es(I.V.‐R.);
enrique.jose.bandin.vilar@sergas.es(E.J.B.‐V.);manuel.busto.iglesias@sergas.es(M.B.‐I.);
laura.garcia.quintanilla@sergas.es(L.G.‐Q.);miguel.gonzalez.barcia@sergas.es(M.G.‐B.);
irene.zarra.ferro@sergas.es(I.Z.‐F.)
2
ClinicalPharmacologyGroup,HealthResearchInstituteofSantiagodeCompostela(IDIS),
15706SantiagodeCompostela,Spain
3
DepartmentofPharmacyPharmaceuticalTechnology,FacultyofPharmacy,UniversityofSantiagode
Compostela,15782SantiagodeCompostela,Spain;francisco.otero@usc.es
4
DepartmentofAnalyticalChemistry,NutritionandFoodScience,FacultyofPharmacy,Universityof
SantiagodeCompostela,15782SantiagodeCompostela,Spain;letricia.barbosa.pereira@usc.es(L.B.‐P.);
raquel.sendon@usc.es(R.S.‐G.);ana.rodriguez.bernaldo@usc.es(A.R.‐B.d.Q)
5
InternalMedicineDepartment,UniversityClinicalHospitalofSantiagodeCompostela(SERGAS),
15706SantiagodeCompostela,Spain;ignacio.novo.veleiro@sergas.es(I.N.‐V.);
nestor.vazquez.agra@sergas.es(N.V.‐A.)
6
PathologicalAnatomyDepartment,UniversityClinicalHospitalofSantiagodeCompostela(SERGAS),
15706SantiagodeCompostela,Spain;jose.ramon.antunez.lopez@sergas.es
7
MolecularImageGroup,HealthResearchInstituteofSantiagodeCompostela(IDIS)(IDIS),
15706SantiagodeCompostela,Spain;davidrey.bretal@usc.es(D.R.‐B.);
pablo.aguiar.fernandez@sergas.es(P.A.)
8
PneumologyDepartment,UniversityClinicalHospitalofSantiagodeCompostela(SERGAS),
15706SantiagodeCompostela,Spain;jose.ramon.lago.quinteiro@sergas.es(J.R.L.‐Q.);
luis.valdes.cuadrado@sergas.es(L.V.‐C.);carlos.rabade.castedo@sergas.es(C.R.‐C.)
9
ClinicalAnalyticDepartment,UniversityClinicalHospitalofSantiagodeCompostela(SERGAS),
15706SantiagodeCompostela,Spain;maria.del.carmen.del.rio.garma@sergas.es
10
PharmacyDepartment,UniversityClinicalHospitalofPontevedra(SERGAS),36162Pontevedra,Spain;
carlos.crespo.diz@sergas.es
11
SociedadEspañoladeFarmaciaHospitalaria(SEFH),28001Madrid,Spain;olga.delgado@ssib.es
12
MicrobiologyDepartment,UniversityClinicalHospitalofSantiagodeCompostela(SERGAS),
15706SantiagodeCompostela,Spain;gema.barbeito.castineiras@sergas.es(G.B.‐C.);
maria.luisa.perez.del.molino.bernal@sergas.es(M.L.P.d.M.‐B.);rocio.trastoy.pena@sergas.es(R.T.‐P.)
13
RadiochemistryDepartment,CICbiomaGUNE,ParqueTecnológicodeSanSebastian,
20009SanSebastián,Spain;rpassannante@cicbiomagune.es
*Correspondence:jllop@cicbiomagune.es(J.L.);antonio.pose.reino@sergas.es(A.P.‐R.);
anxordes@gmail.com(A.F.‐F.);Tel.:+34981951423(A.F.‐F.)
†Theauthorscontributedequally.
Abstract:InhaledadministrationofethanolintheearlystagesofCOVID‐19wouldfavoritslocation
ontheinitialreplicationsites,beingabletoreducetheprogressionofthediseaseandimprovingits
prognosis.Beforeevaluatingtheefficacyandsafetyofthisnoveltherapeuticstrategyinhumans,its
Citation:Castro‐Balado,A.;
Mondelo‐García,C.;
Barbosa‐Pereira,L.;Varela‐Rey,I.;
Novo‐Veleiro,I.;Vázquez‐Agra,N.;
Antúnez‐López,J.R.;Bandín‐Vilar,
E.J.;Sendón‐García,R.;
Busto‐Iglesias,M.;etal.
DevelopmentandCharacterization
ofInhaledEthanolasaNovel
PharmacologicalStrategyCurrently
EvaluatedinaPhaseIIClinicalTrial
forEarly‐StageSARS‐CoV‐2
Infection.Pharmaceutics2021,13,342.
https://doi.org/10.3390/
pharmaceutics13030342
AcademicEditor:CarstenEhrhardt
Received:30January2021
Accepted:2March2021
Published:5March2021
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinsti‐
tutionalaffiliations.

Copyright:©2021bytheauthors.Li‐
censeeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsandcon‐
ditionsoftheCreativeCommonsAt‐
tribution(CCBY)license(http://crea‐
tivecommons.org/licenses/by/4.0/).

Pharmaceutics2021,13,3422of18
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characterizationisrequired.Thedeveloped65°ethanolformulationisstableatroomtemperature
andprotectedfromlightfor15days,maintainingitsphysicochemicalandmicrobiologicalproper‐
ties.Twooxygenflowshavebeentestedforitsadministration(2and3L/min)usinganautomated
headspacegaschromatographicanalysistechnique(HS‐GC‐MS),withthatof2L/minbeingthe
mostappropriateone,ensuringtheinhalationofanethanoldailydoseof33.6±3.6mg/minand
achievingmorestableconcentrationsduringtheentiretreatment(45min).Undertheseconditions
ofadministration,theformulationhasproventobesafe,basedonhistologicalstudiesoftherespir‐
atorytractsandlungsofrats.Ontheotherhand,theseresultsareaccompaniedbythefirstpreclin‐
icalmolecularimagingstudywithradiolabeledethanoladministeredbythisroute.Thecurrenteth‐
anolformulationhasreceivedapprovalfromtheSpanishAgencyofMedicinesandMedicalDevices
foraphaseIIclinicaltrialforearly‐stageCOVID‐19patients,whichiscurrentlyintherecruitment
phase(ALCOVID‐19;EudraCTnumber:2020‐001760‐29).
Keywords:COVID‐19;SARS‐CoV‐2;inhaledethanol;molecularimaging;PET

1.Introduction
Coronaviruses(CoVs)aresingle‐strandedRNAviruseswhichcaninfectanimalsand
humans,causingrespiratory,gastrointestinal,hepaticandneurologicdiseases[1].InDe‐
cember2019,severalhealthauthoritiesreportedpatientswithpneumoniaofanunknown
cause,whichwereepidemiologicallylinkedtoaseafoodmarketinWuhan,China.The
pathogen,anovelcoronavirus(SARS‐CoV‐2),wasidentifiedbylocalhospitalsandthe
infectionwascalledcoronavirusinfectiousdisease2019(COVID‐19)[2,3].InMarch2020,
theWorldHealthOrganization(WHO)classifiedCOVID‐19asapandemic,andinJanu‐
ary2021,ayearafteritseruption,therehavebeenmorethan93millionconfirmedcases
andtwomilliondeaths[4].
SincetheoutbreakoftheCOVID‐19pandemic,ithasbeenseenthatcomplianceof
controlmeasuressuchasphysicaldistancing,theuseofmasks,handcleaning,tracing
contacts,testingofexposedorsymptomaticpersonsandisolationhaverestrictedtrans‐
missions[5].Evenso,theseactionshavenotbeenimplementeduniformly,andtheyhave
notshowntobeenoughtopreventthespreadofSARS‐CoV‐2.
VaccinesareneededtoproducegroupimmunityandreduceCOVID‐19morbidity
andmortality.Inthissense,severalvaccineplatformshavebeenimplicatedintherapid
developmentofcandidatevaccines[6–8],whichallowedthestartingofthevaccination
processworldwideinDecember2020.
AccordingtothepathologicalcharacteristicsofCOVID‐19,especiallyforpatients
withmoderatetosevereCOVID‐19,severaltreatmentstrategieshavebeendeveloped,
including,amongothers,antiviralagents,inflammationinhibitors/antirheumaticdrugs
andlowmolecularweightheparins[9,10].Concerningnewtherapies,itisnecessaryto
highlighttheadministrationofconvalescentplasmawithhighIgGtitersagainstSARS‐
CoV‐2,whichhaverevealedpromisingresults,althoughfurtherstudiesarerequired[11].
Inaddition,regardingantivirals,plitidepsinhasdemonstratedpotentpreclinicalefficacy
againstSARS‐CoV‐2bytargetingthehostproteineEF1A,anditisbeingtestedinaproof
ofconceptstudytoevaluateitssafetyprofile(NCT04382066)[12].Anotherpossiblealter‐
nativeistreatmentwithmonoclonalantibodies,suchasbamlanivimab,whichiscurrently
evaluatedinanexpandedaccessprogram(NCT04603651)[13],orthecocktailofmono‐
clonalantibodiesanti‐spikeSARS‐CoV‐2,whichisbeingtestedin ambulatoryadultand
pediatricpatients(NCT04425629)[14].
Despitealltheabove,ithasbeenseenthat,intheearlystages,antiviraldrugscan
preventtheprogressionofthedisease,whiledexamethasone[15],immunomodulators
andantiviraldrugsseemtoimprovetheclinicaloutcomeofpatientswithsevereCOVID‐

Pharmaceutics2021,13,3423of18
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19[9].However,treatmentremainselusiveinearlystages,inwhichtherearefewstrate‐
giesthatcanbringbenefits;somestrategieshavefailed,andothersareunderevaluation
[11].
Inpandemicsituationslikethis,asignificantnumberofpatientsfindthemselvesin
atherapeuticvacuum,withouteffectivedrugstoaddresstheirtreatment.Inthiscontext,
drugrepositioningisastrategytogenerateadditionalvaluefromanapproveddrug,us‐
ingitforadifferenttherapeuticpurposethanthatforwhichitwasoriginallyintended
[16].Theabsenceofevidence‐basedtreatmentsforCOVID‐19hasledtothestartofalarge
numberofclinicaltrialsinordertoofferpatientsthemosteffectiveandsafesttherapeutic
options.Inthissense,thegeneticcharacteristicsofSARS‐CoVandMERS‐CoVsuggest
thatSARS‐CoV‐2maybesusceptibletodisinfectantssuchasethanol,withagraduation
between62–71°,withprovenactivityagainstenvelopedviruses[17,18].
Inearlystagesofthedisease,activevirusesarelocatedinthethroatandlungs.This
way,theadministrationofviricidalagentsintheinitialreplicationplacecoulddecrease
viremiainthefirststagesofthedisease,andconsequentlyreduceitsprogressionandim‐
provetheprognosisdrastically[19].Inhospitalpharmacydepartments,itiscommonto
elaborateethanolformulationsfortheiruseinnon‐usualroutesofadministration.Ethanol
wasusedincathetersealstopreventbacterialgrowth[20],asaneurolyticintheperipheral
orcentralnervousblockinterminallyillpatients[21],inesophagealvaricosesclerother‐
apy[22],hemorrhagecontrolinhepatocellularcarcinomasurgeries[23],debridementof
thecornealepithelium[24]orevenasanintravenousantidoteinthetreatmentofethylene
glycolormethanolpoisoning[25].
COVID‐19causesaparticularlysevereillnessinolderadults.Thepercentageofhos‐
pitalizedpatientswithinthisagegroupishigh[11],andover95%oftotaldeathcases
occurredinpeopleolderthan60years,withmorethan50%ofalldeathsbeingpeople
aged80yearsorolder[3,26,27].Inthissituation,withnoalternativesamongcommercial
medicines,itisnecessarytodevelopnewtherapeuticapproacheswhichcanbeanade‐
quateoptioninelderlypatients.Inthissense,thelocaladministrationofethanolcouldbe
effectiveagainstaviralenvelopewithnosystemicadverseeffects[28,29].
ThisstudypresentsanovelpharmacologicalstrategyagainstSARS‐CoV‐2within‐
haledethanolanditsgalenic,toxicologicalandpharmacokineticcharacterization.Thecur‐
rentethanolformulationhasreceivedapprovalfromtheSpanishAgencyofMedicines
andMedicalDevicestotestitsefficacy‐safetyinaphaseIIclinicaltrialinelderlypatients
withCOVID‐19,whichiscurrentlyintherecruitmentphase(ALCOVID‐19;EudraCT
number:2020‐001760‐29)[30].
2.MaterialsandMethods
Thisworkincludesaninitialgaleniccharacterizationphase,inwhichthephysio‐
chemicalandmicrobiologicalstabilityoftheformulationinhumidifyingflaskswasde‐
termined,aswellastheconcentrationofvaporizedethanolthroughtwooxygenflows.
Subsequently,atwo‐partpreclinicalphasewascarriedout.Ontheonehand,aninvivo
studytoensurethesafetyoftheethanoladministeredbythisroutewasperformedin
Sprague‐Dawleyrats.Ontheotherhand,evaporatedethanolwasradiolabeled,andits
pharmacokineticsinratswerestudiedusingmolecularimagingtechniqueswithpositron
emissiontomography/computedtomography(PET/TC).Lastly,apreliminaryclinical
studywascarriedoutinsixhealthyvolunteersdividedintotwogroups(1:1),whowere
subjectedtotheadministrationof15minutesofoxygentherapyat2L/minand3L/min
flowthroughaVentimask®(Flexicare,MountainAsh,UK)facemask.Thesedataarede‐
pictedinthesupplementarymaterial,includingFiguresS1andS2.


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2.1.Preparationofthe65°EthanolSolutionandStabilityDeterminationinDisposable
HumidifyingBottles
Avolumeof250mLof65°ethanolsolutionwaspackagedindisposablehumidifier
bottles(INTLCE0482.Ref.3230,generouslydonatedbyOximesaNipponGases,Madrid,
Spain).Toprepare,thestartingpointwas163mLof99.5°ethanolwithPhEurindication
onitslabel(PanReacAppliChem®,Darmstadt,Germany),whichwasmeasuredusinga
graduatedcylinder,andcompletedwithsterilewater(FresniusKabi®,Barcelona,Spain)
upto250mL.Afterhomogenization,itwasnecessarytowaituntilitreachedroomtem‐
peraturetomeasureethanolgraduationwitha60–70°Gay‐Lussacalcoholmeter(Boeco®,
Hamburg,Germany).Sterilizingvacuumfiltrationthrougha0.22‐micronfilterwasper‐
formedinahorizontallaminarflowcabinet.
Inordertodeterminethestabilityofethanolover15daysinadisposablehumidify‐
ingbottle,aninitialethanolgraduationmeasurementwascarriedout,andthenonce
weeklyusingthe60–70°Gay‐Lussacalcoholmeter.Duringthisperiod,theethanolsolu‐
tionwaskeptinafullysealedhumidifierbottleatroomtemperature,andprotectedfrom
light.Paralleltothis,amicrobiologicalstudywascarriedoutbyextractionofthree3mL
samples:onesamplebeforesterilizingfiltrationandanotherafterthisprocessonday0;
andthelastoneafter15daysofstorageatroomtemperatureandprotectedfromlight.
Thesesampleswerecultivatedinathioglycollatebroth(Merck®,Darmstadt,Germany),
Columbiabloodagar(Merck®,Darmstadt,Germany)andSabouraud(Merck®,Darmstadt,
Germany).Allmediumswereincubatedaerobicallyat37°C;thioglycollatefor10days;
andbloodandSabouraudagarplatesfor48hours.Sabouraudagarplatesweresubse‐
quentlyincubatedfor13daysinaerobiosisatroomtemperature.
2.2.DeterminationofEthanolintheAdministeredOxygenFlow
Aflowofoxygenwasmadetopassthroughahumidifiercontainingthe65°ethanol
solution.Theeffectoftwooxygenflowswascompared:2and3L/min.Theseflowscause
theevaporationoftheethanolinthehumidifier.Thisdeterminationofevaporatedethanol
willallowchoosingtheoptimaloxygenflowthatcausestheevaporationwithoutgener‐
atingaerosols,withthisbeingbestsuitedtoitsuselaterintheclinicaltrial.
Theethanolquantificationhasbeencarriedoutbyanautomatedheadspacegaschro‐
matographicmassspectrometryanalysis(HS‐GC‐MS)(Figure1).Itwasperformedusing
aFinniganTraceGCUltrachromatographcoupledwithaFinniganTraceDSQmassde‐
tector,equippedwithaThermoScientificHeadSpace(HS)TriPlusautosampler(Thermo
FisherScientific,SanJosé,CA,USA).Samplingwasperformedin21.5 mLHSglassvials,
whichwerepreviouslysealed.Theseptumwasperforatedwithtwoneedlestoallowa
wayinandawayoutforthegas.Then,oxygenenrichedinethanolgeneratedinthehu‐
midifierwasflushedfor2minineachvialtoreplaceallofitsinternalair,andguarantee
thatthesamplewasrepresentative.Thesampleswerecollectedatseveralintervalsof
time,asfollows:0–2min;2–4min;6–8min;13–15min;28–30min;and43–45min,inorder
todeterminetheconcentrationofethanolat2,4,8,15,30and45minofadministration,
respectively.Allexperimentswereperformedintriplicateforthetwoflowratestested(2
L/minand3L/min).ThevialsweremaintainedandrefrigeratedbeforeHS‐GC‐MSanal‐
ysis.

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Figure1.Diagramoftheoxygenflowsystemandhumidifierwith65°ethanolcoupledtoagas
chromatographymassspectrometry(GC‐MS)system.CreatedwithBioRender.com.
Sampleswereincubatedat45 °Cfor4 min,before0.2 mLofheadspacegaswaswith‐
drawnbytheautosampler,usinga2.5 mLsyringeheatedat50 °C.Thegasvolumewas
injectedintotheinletoftheTraceGCsetat175 °Cinaconstanttemperaturesplitmode,
atasplitratioof50:1andthesplitflowof50mL/min.TheanalyticalcolumnwasaRxi‐
624SilMS,30minlength,withaninternaldiameterof0.25mm(1.4μmfilmthickness)
fromRestek(Pennsylvania,USA).Heliumwasusedascarriergasataflowrateof1.0
mL/mininaconstantflowmode.Theoventemperaturewasinitiallysetat40°Cfor4
min,thenincreasedatarateof50°C/minuntil120°C,andheldat120°Cfor2.5min.The
totalruntimeofeachanalysiswas10min.ThetemperaturesoftheGCtransferlineand
theionsourceoftheMSweresetat220°Cand200°C,respectively.Themassspectrom‐
eterwasoperatedinanelectronimpactionization(EI+)mode,andthechromatograms
wereacquiredinfullscanmodeoverthem/zrangeof20–150,from2to10min,atascan
rateof3.55scans/s.Thechromatographicpeakofethanolwasidentifiedbycomparison
withreferencespectrafromtheNIST2011/Wiley9CombinedMassSpectralLibrary,us‐
ingNISTMassSpectralSearchProgram(version2.0),andconfirmedbycomparisonofits
retentiontime(3.4min),withthatobtainedbyanalysisofauthenticstandardunderthe
sameconditions.Theinstrumentcontrolanddataacquisitionandprocessingwereper‐
formedwithXcalibur2.0.7software(ThermoFisherScientificInc.,Waltham,MA,USA).
Thecalibrationcurveofethanolwaspreparedin21.5 mLHSglassvials,whichwere
previouslysealedwithTeflonseptumcaps,byaddingdifferentvolumesof99.5%(0.5–2
μL)ethanoltoreachtheheadspace(vaporphase)concentrationsintherangeof20–100
mg/L.Theequationanddeterminationcoefficientobtainedfromthreereplicateswerey=
82.285x+9.8808andR
2
=0.9993,respectively.
2.3.PreclinicalStudies
2.3.1.EthanolExposureToxicologicalStudiesandImmunohistochemicalAnalysis
ThesestudieswerecarriedoutonsixfemaleSprague‐Dawleyrats(fourreceiving
inhaledethanolandtwocontrolrats).Theyweresuppliedbytheanimalfacilitiesatthe
UniversityofSantiagodeCompostela,andtheaverageweightwas250±25g.Duringthe
experiments,animalswerekeptinindividualcagesunderacontrolledtemperature(22±
1°C)andhumidity(60±5%)conditions,withday‐nightcyclesregulatedbyartificiallight
(12/12hours)andfedadlibitum.AllanimalexperimentscompliedwiththeARRIVE
guidelines[31]andwerecarriedoutinaccordancewiththeEUDirective2010/63/EUfor
animalexperiments,beingapprovedbytheGalicianNetworkCommitteeforEthicsRe‐
search.Allexperimentalprocedureswereapprovedbytheethicalcommitteeandthelocal
authoritiesbeforeconductingexperimentalwork.
Fortheadministration,65°ethanolwasplacedinadisposablehumidifierandvapor‐
izedbypassinganoxygenflowof2L/min.Thegeneratedethanolvaporswentthrougha

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tubetoaninhalationchamberof20×20×25cm(Bioseb,FL,USA)(Figure2).Forexposure
totheethanolvapours,eachratwasplacedinthechamberfor15minevery8hours(three
timesaday)forfiveconsecutivedays.Followingtheexposuretime,eachratwasremoved
fromtheinhalationchamberandreturnedtoitsindividualcage.

Figure2.Oxygenflowsystemandhumidifierwith65°ethanolcoupledtotheinhalationchamber,
wheretheratsareexposedtoethanolvaporforinhalation.CreatedwithBioRender.com.
Theanimalsweresacrificedbyanintracardiacinjectionof5mLofpotassiumchlo‐
ride(1mEq/mL;B.BraunMedical,S.A,Barcelona,Spain)atday+6oftheinitiationassay.
Upperrespiratorytractswereremovedandindividuallyfixedin10%formalin,dehy‐
drated,paraffinembedded,sectionedinsliceswith4μmthickness,andstainedwithH&E
(haematoxylinandeosin).Thesampleswereblindlyevaluatedbyalungpathologistspe‐
cialistusingamicroscope(Zeiss,Oberkochen,Germany).Sectionswereexaminedusing
lightmicroscopy,anddigitalimageswereacquiredusingLeica
®
software(LeicaMi‐
crosystems,Wetzlar,Alemania).Analyseswereperformedatafinalmagnificationof
×1000.Fivenonoverlappingfieldsofviewpersectionfromtwotothreesections(from
differentregionsofthelung,esophagusandtrachea)peranimalwereanalyzed.
2.3.2.PreclinicalPharmacokinetics
Thepreclinicalpharmacokineticstudieswerecarriedoutwithpositronemissionto‐
mography/computedtomography(PET/TC)methodologyusingradiolabeledethanol.
Theuseofmolecularimaging,particularlyPET/TC,providesanon‐invasiveimaging
techniquethatvisualizesthedistributionofdifferentradiotracersovertimeinanimal
models[32].Inthisstudy,1‐
11
C‐ethanolhasbeensynthetizedinordertoknowthedistri‐
butionofethanoladministeredintotherespiratorytractinratsalongtime,inorderto
assessthetoleranceandsafetyoftheadministrationofvaporizedethanolthreetimesa
dayforfivedaysinSprague‐Dawleyrats.
Synthesisof1‐
11
C‐Ethanol
Thesynthesisof1‐
11
C‐ethanolwascarriedoutusingaTRACERlabFXCProsynthesis
module(GEHealthcare,Chicago,IL,USA).[
11
C]CO
2
wasgeneratedinanIBACyclone
18/9cyclotron(IBARadioPharmaSolutionsHeadquarter,Louvain‐la‐Neuve,Belgium)by
protonirradiation(targetcurrent=22μA,integratedcurrent=2μAh)ofagasN
2
/O
2
mix‐
ture(99/1,startingpressure=20bar)withhighenergy(18MeV)protons.Theradioactive
gaswasfirsttrappedinamolecularsieveovenatroomtemperatureandthenreleasedby
heatingat180°Cundernitrogenflow(20mL/min).Thereleased[
11
C]CO
2
wasbubbledin
areactionvialcontainingCH
3
MgBr(1MsolutioninTHF,250μl,Merck
®
,Darmstadt,Ger‐
many).Aftercompletetrapping,LiAlH
4
(1MsolutioninTHF,500μl,Merck
®
,Darmstadt,
Germany)wasadded,andtheresultingmixturewasstirredat80°Cfor5minutes.The
solventwasthenevaporatedunderheliumflow(5min,60°C;then1min,80°C).There‐

Pharmaceutics2021,13,3427of18


actorwascooledto40°CandaqueousHCl(4M,1mL)wasimmediatelyadded.Theso‐
lutionwasstirredfor30s,filteredusinga0.2μmfilter,andpurifiedbyhigh‐performance
liquidchromatography(HPLC)usingaMediterraneaSEA18(250×10mm,5μmparticle
size)(TeknokromaAnalítica,Barcelona,Spain)columnasastationaryphaseandul‐
trapurewaterasthemobilephase(flowrate=5mL/min).Thepurifiedproduct(retention
time=7.7min;totalcollectedvolumeofca.2mL)wascollectedinavialanddiluted1:1
withaphysiologicsalinesolution.Theamountofradioactivityofthefinalradiotracerwas
measuredinadosecalibrator(PETDOSEHC,Comecer,CastelBologneseRA,Italy).Ra‐
diochemicalpuritywasdeterminedbyHPLC,usinganAgilent1200SeriesHPLCsystem
(AgilentTechnologies,SantaClara,California,USA)withamultiplewavelengthUVde‐
tector(λ=254nm)andaradiometricdetector(Raytest,Elysia‐raytestGmbH,Strauben‐
hardt,Germany).ATracerExcel120C8(250x4.6mm,5μmparticlesize)(Teknokroma
Analítica,Barcelona,Spain)wasusedasstationaryphaseandpurifiedwaterasmobile
phase(flowrate=1mL/min;retentiontime=3.95min).
LungPETStudies
ThreehealthyfemaleSprague‐Dawleyrats(n=3)weighting350±10gwereusedin
thisstudy.ForPETstudies,anesthesiawasinducedwith5%isofluraneandmaintained
by1.5to2%ofisofluranein100%O2.Thelabelledcompound(ca.7MBq,100μL)was
administeredthroughendotrachealinsufflationsusingthePennCenturyMicroSprayer®
Aerosolizer(FMJ‐250HighPressureSyringeModel,Penn‐Century.Inc.Wyndmoor,USA)
andasmallanimallaryngoscope(Penn‐Century,ModelLS‐2)(Penn‐Century.Inc.Wynd‐
moor,USA)forthecorrectvisualizationoftheepiglottis.Afteradministration,theanimal
wasquicklymovedintothePETcamera,andthePETacquisitionbegan.Thetimegap
betweenadministrationofthedoseandstartofimageacquisitionwas1min.Duringim‐
aging,ratswerekeptnormothermicusingaheatingblanket(HomeothermicBlanketCon‐
trolUnit;Bruker,MA,USA).PETImagingwasperformedusinganeXploreVista‐CTsmall
animalPET‐CTsystem(GEHealthcare,Chicago,IL,USA).Wholebodydynamicimages
wereacquiredinfourbedpositions(20frames:4×5s,4×15s,4×30s,4×60s,4×120s;
totalacquisitiontime=61.33min)inthe400–700keVenergeticwindow.AftereachPET
acquisition,aCTscan(X‐rayenergy:40kV,intensity:140μA)wasperformedforalater
attenuationcorrectionintheimagereconstruction,aswellasfortheunequivocallocali‐
zationoftheradioactivity.Randomandscattercorrectionswerealsoappliedtotherecon‐
structedimage.PET‐CTimagesofthesameanimalwereco‐registeredandanalyzedusing
thePMODimageprocessingtool(PMODTechnologiesLtd,Zürich,Switzerland).Vol‐
umesofinterest(VOIs)wereplacedonmajororgans(lungs,liver,kidneys,heartand
brain),andtime–activitycurves(decaycorrected)wereobtainedascps/cm3ineachorgan.
Curvesweretransformedintorealactivity(Bq/cm3)curvesbyusingacalibrationfactor,
obtainedfrompreviousscansperformedonaphantom(micro‐deluxe,Dataspectrum
Corp.,NC,USA)underthesameexperimentalconditions(isotope,reconstructionalgo‐
rithmandenergeticwindow).
3.Results
3.1.Stabilityofthe65°EthanolPharmaceuticalCompoundingandFlowOxygenEffect
Thegraduationofthehumidifyingbottlewiththehydro‐alcoholicsolutionunder
storageconditions(withoutuse)remainedstablefor15days,withnomicrobiological
growthobservedattheendofsaidperiod.
Theformulationmaintainsoptimalconcentrations(decreaseslessthan5%ofthein‐
itialconcentration)attheendofthethreedailyapplications,withbothflowstested(2and
3L/min).Withtheflowof2L/min,asolutiongraduationof64.1°wasmaintained(98.2%
oftheoriginalconcentrationofthehydro‐alcoholicmixture),whileattheflowof3L/min,
thegraduationobtainedwas63.3°(97.8%).Theethanolcontentoftheinitialsolution,in
thehumidifierbottle,decreased4.63gat2L/minand5.38gat3L/min,after45minof

Pharmaceutics2021,13,3428of18


administration.Thedifferencebetweenflowrateswas0.75gofethanolemittedperday,
higherforaflowof3L/min,whichrepresents16%morethanthetotalamountachieved
withtheflowof2L/min,asshowninFigure3a.
Consideringtheminuteventilationof5L/minforhumanadultsrecommendedfor
short‐termexposure[33],andtheinhalation:exhalation(I:E)ratioof1:2,thetotalamount
ofethanolinhaledafterthethreedailyadministrationsat2L/minand3L/minwas1.51g
and1.79g,respectively(Figure3b).Thedifferenceinthetotalamountoftheinhaledeth‐
anolbetweenflowrateswas0.28ghigherforthehighestflowrateused.

(a)

(b)
Figure3.(a)Thetotalamountofethanolevaporatedfromtheinitialsolution,accordingtoGC‐MSdatadeterminedby
calculationoftheareasunderconcentration‐timecurves(AUC),and(b)theestimatedtotalamountofethanolinhaledby
humanadultstakingintoaccounttherecommendedshort‐termexposurevaluesforinhalation,insedentaryorpassive
activity(b),afterthe45minoftreatmentat2L/minand3L/min.
1.81 2.23
1.53
1.73
1.30
1.42
0.00
1.00
2.00
3.00
4.00
5.00
6.00
2L/min 3L/min
gofethanol
Totalamountofemittedethanol/day
1stadministration 2ndadministration 3rdadministration
0.60 0.74
0.50
0.58
0.41
0.47
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2L/min 3L/min
gofethanol
Totalamountofinhaledethanol/day
1stadministration 2ndadministration 3rdadministration

Pharmaceutics2021,13,3429of18


Kineticparametersoftheethanolvaporizationintheoxygenstream,forbothflow
ratestested,areshowninFigure4.Second‐degreepolynomialequationsweredeveloped
fromtheexperimentaldata,andareasundertheconcentration‐timecurves(AUC)were
calculatedtodeterminetheamountofethanolemittedduringthetreatments.Attheflow
rateof2L/min,theaverageconcentrationsofethanolyielded(51.5±4.5mg/Lofoxygen
(8.7%CV))werehigherthanthatobservedat3L/min(39.9±5.7mg/Lofoxygen(14.4%
CV))duringthe45minoftreatment.Theconcentrationofethanoldecreasedslightlydur‐
ingthetimeforbothflowratestested.Inthefirst15min,theaverageconcentrationsof
ethanoldeterminedataflowrateof2L/minand3L/minwere56.6±1.9(3.4%CV)and
46.6±3.1mg/L(6.6%CV),respectively.Inthesecondperiod(15–30min)oftheexperi‐
ment,theaverageconcentrationswere51.0±1.6(3.2%CV)and38.6±2.0mg/L(5.2%CV),
andfinally,inthelastperiodof15min,theconcentrationsofethanolwere46.5±1.46
(2.9%CV)and34.0±1.0mg/L(3.0%CV),for2L/minand3L/min,respectively.

Figure4.Ethanolconcentration(mg/L)inthevaporphaseofoxygengeneratedinthehumidifier,
duringthe45minutesoftreatment,at2L/minand3L/min.
FollowingthepreviousconsiderationfortheI:Eratio,theaverageconcentrationsof
inhaledethanolatflowratesof2L/minand3L/minwere6.70±0.75mg/L/min(11.2%
CV)and7.99±1.12mg/L/min(14.7%CV),respectively.Theconcentrationswereslightly
higherforthehighestflowrate,sincethedilutionwiththeassistantairwaslower.Specif‐
ically,theinhaledconcentrations(mg/L/min)atthedifferentadministrationswere:7.55±
0.27(0–15min),6.70±0.27(15–30min)and5.58±0.26(30–45min)attheflowrateof2
L/min;and9.37±0.62(0–15min),7.76±0.41(15–30min),6.81±0.19(30–45min)atthe
flowrateof3L/min.
Takingintoaccountthetotalamountofethanolemittedperminuteoftreatment
(mg/min),thevalueswerelowerfortheflowrateof2L/min(100.1±11.5mg/min(11.5%
CV))thanthe3L/min(averageof119.7±17.1mg/min(14.3%CV)),asshowninFigure5.
Thesedifferenceswerehigherinthefirstminutesofethanolenrichedoxygenadministra‐
tion,withtotalamountsofethanolof112.7±4.1(3.7%CV)for2L/minflowrateand139.7
±9.2(6.6%CV)for3L/min,anddecreasedduringthetimeofaround23%and27%,re‐
spectively.Forthesecondandthirdadministrationsat2L/minflowrate,theamountsof
ethanolwere99.7±4.2(4.2%CV)and87.4±4.2(4.2%CV),respectively,whereasataflow
rateof3L/min,thevaluesobservedwere115.8±6.0(5.2%CV)and102.6±3.0(2.9%CV).
y=‐0.0002x2‐0.4307x+59.832
R²=0.9178
y=0.0075x2‐0.7646x+52.023
R²=0.9689
0
10
20
30
40
50
60
70
0 10203040
Ethanol(mg/L)
Time(min)
Evaporatedethanol(mg/L/min)
2L/min
3L/min

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Therefore,basedonpreviousconsiderations,theestimatedfinalaverageamountsof
inhaledethanol(mg/min)were33.6±3.6(10.7%CV)and40.0±5.7(14.2%CV)attheflow
rateof2L/minand3L/min,respectively.Attheflowrateof2L/min,analysisofvariance
(ANOVA)showednosignificantdifferences(p>0.05)amongthedosesofinhaledethanol
duringthethreeadministrations:37.60±2.04(0–15min);33.36±1.34(15–30min);and
29.31±1.32(30–45min).Onthecontrary,significantdifferences(p<0.001)wereobserved
attheflowrateof3L/min:46.55±1.02(0–15min);38.57±2.00(15–30min);and34.04±
1.01(30–45min).

Figure5.Amountofethanol(mg)inthevaporphaseofoxygengeneratedinthehumidifier,dur‐
ingthe45minutesoftreatment,attheflowratesof2L/minand3L/min.
3.2.PreclinicalStudies
3.2.1.EthanolExposureToxicologicalStudiesandImmunohistochemicalAnalysis
Tissueandair‐spacefractions(atelectasis),oedemaandcongestionwereevaluated
intheparaffin‐embeddedsectionsoflungtissuestainedwithhematoxylinandeosin.
Therewerenotypicalpatternsofpulmonarytoxicitiesalongtherespiratorytract,ascan
beseeninFigure6(a.lung;b.andc.tracheaandd.ande.esophagus).

y=‐0.0003x2‐0.8613x+119.66
R²=0.9178
y=0.0226x2‐2.2939x+156.07
R²=0.9689
0
20
40
60
80
100
120
140
160
180
0 5 10 15 20 25 30 35 40 45
Ethanol(mg)
Time(min)
Evaporatedethanol(mg/min)
2L/min
3L/min

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

(a)

(b)(c)

(d)(e)
Figure6.Upperrespiratorytractsfixedin10%formalin,dehydrated,paraffinembedded,sectionedinsliceswith4μm
thicknessandstainedwithH&E:(a)Lung(200×)withdescriptionofabsencedamage;(b,c)Trachea(10×,40×)withde‐
scriptionofabsencedamage;and(d,e)Esophagus(40×,100×)withdescriptionofabsencedamage.
3.2.2.Pharmacokinetics
Synthesisof1‐11C‐Ethanol
Thesynthesisof1‐11C‐ethanolhasbeenpreviouslyreported[34,35].Intheseprevious
works,thefinalpurificationofthelabeledcompoundwasachievedbyfractionaldistilla‐
tion.Inourhands,thissyntheticapproachresultedinthepresenceofradioactiveimpuri‐

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tiesinthefinalsolution.Theundesiredby‐productswereco‐elutedwithmethanol(reten‐
tiontime=3.5min)andisopropanol(retentiontime=4.7min)underouranalyticalHPLC
conditions(Figure7a)andaccountedforca.20%oftotalradioactivity.Takingintoaccount
thatouraimwastoobtainradiochemicallypure1‐11C‐ethanol,andthattheoverallyield
wasnotacriticalaspect,wedecidedtoassayapurificationmethodbasedonHPLC.After
afiltrationsteptoremoveanyeventualprecipitateduetoincompletedissolutionofthe
solidresidueaftertheadditionofhydrochloricacid,agoodseparationofthedifferent
peakscouldbeachievedwhenwaterwasusedasthemobilephase.
Undertheseconditions,approximately600MBqofpure1‐11C‐ethanol(radiochemical
purity>99%;Figure7b)couldbeobtainedinaverageproductiontimeof30min(decay
correctedradiochemicalyieldofaround7%).Thisamountofradioactivity,whichwas
readyforadministrationaftersimpledilutionwithphysiologicsalinesolution,wassuffi‐
cienttotacklesubsequentinvivostudiesinrodents(seebelow).

Figure7.(a)Chromatograms(radioactivitydetector)obtainedafteranalysisofthereactioncrude;
(b)chromatograms(radioactivitydetector)obtainedinqualitycontrolanalysisofthepurified1‐
11C‐ethanol.
LungPETStudies
PETstudieswerecarriedouttodeterminethebiodistributionof1‐11C‐ethanolinrats
afterintratrachealinsufflation.AdministrationwascarriedoutusingthePenn‐Century
MicroSprayer®Aerosolizer,whichisreportedtoprovidearounda20μmdropletsize[36].
Accordingtoourpreviousresults,thisisaveryappropriatesystemforthequantitative
administrationofaerosolsintheratlung[36].Duetotheadministrationprocess,dynamic
imagescouldbestartedaroundoneminuteafteradministration,andhenceinitialdistri‐
butiondata(0.1minafteradministration)waslost.DynamicPETimages(Figure8)show
averyfastlungclearance(t1/2=1.43min),withmostoftheradioactivityalreadycleared
fromthelungsatt=1min.
Thepresenceofradioactivitydelocalizedoverthewholeanimalatearlytimepoints
suggesttranslocationtotheblood,followedbyprogressiveaccumulationintheliver(Fig‐
ure8),suggestingmetabolicoxidationtoacetaldehydebyalcoholdehydrogenaseandcy‐
tochromeP450,whichareextensivelypresentinthisorgan.
BKG1
Reg #1
Reg #2
Reg
#
Reg #4
Cnt
ADHV 1
CPS
1000
123456789100
Time(min)
Radioactivity(arbitraryunits)
[
11
C]methanol
[
11
C]isopropanol
[
11
C]ethanol
a
b

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Figure8.(a)Positronemissiontomography(PET)images(maximumintensityprojections,coronal
views)obtainedatdifferenttimepointsafterintratrachealadministrationof1‐
11
C‐ethanol.Images
havebeencoregisteredwith3D‐renderedcomputedtomography(CT)imagesforanatomicallocal‐
izationoftheradioactivesignal;(b)PETimages(coronalprojections)correspondingtotheseg‐
mentedlungsatdifferenttimespointsafteradministration;imageshavebeencoregisteredwith
representativeCTslices;(c)timeactivitycurvesobtainedafterquantificationofvolumesofinterest
drawnindifferentorgans.Valuesareexpressedasstandarduptakevalues(SUV),mean±standard
errormean,n=3.
4.Discussion
TheemergencyperiodoftheCOVID‐19outbreakhasforcedthescientificcommunity
togenerateevidenceagainsttheclock[37].Despitetherecentdevelopmentofvaccinesto
preventthedisease,newtherapeuticalternativestotreattheestablisheddiseaseshould
bestudied[38,39].AccordingtothepathologicalcharacteristicsofCOVID‐19anddiffer‐
entclinicalstages,especiallyforpatientswithmoderatetoseveredisease,antiviralagents,
inflammationinhibitors/antirheumaticdrugs,lowmolecularweightheparinsandconva‐
lescentplasmawithhighIgGtitersagainstSARS‐CoV‐2havebeenusedandtested[9–
11].Intheearlystagesofthedisease,thetreatmentofCOVID‐19remainselusive.There
arefewstrategiesthatcanbringbenefits,somestrategieshavefailed,andothersareunder
evaluation[11,40–43].
Facedwiththerapeuticgapssuchasthese,cliniciansareforcedtoresorttotherapeu‐
ticalternativessuchasdrugrepositioningandpharmaceuticalcompounding.Thegenetic
characteristicsofSARS‐CoVandMERS‐CoVsuggestthatSARS‐CoV‐2maybesusceptible
todisinfectantswithprovenactivityagainstenvelopedviruses[17,18].Ethanolexertsits

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actionagainstaviralenvelope;alipidbilayertakenfromthehostcellsintheassemblyor
buddingstageoftheviralcycle,causingvirallysiswiththeconsequentreleaseanddeg‐
radationofitscontent[19].Activevirusesinthroatandlungswereisolatedinpatients
withamildconditiononlyuptoday8aftertheonsetofsymptoms,reachingthepeakof
theviralloadbeforeday5.Thisway,theadministrationofviricidalagentsintheplaceof
initialreplicationcoulddecreaseviremiainthefirststagesofthedisease,andconse‐
quentlyreducetheprogressionofthediseaseandimproveitsprognosis[29,44].Forthis
reason,theadministrationofinhaledethanolcouldbepresentedasanewtherapeutic
strategytopreventtheprogressionofCOVID‐19infections[45].Ourhypothesisoftreat‐
mentwithinhaledethanolfocusesonitsuseininstitutionalizedelderlypatients,since
certaintherapiesusedtodatewerecontraindicatedordiscouragedinthisagegroup,
whereCOVID‐19hassevereoutcomeswithahighmortalityrate[46].Priortothestartof
aphaseIIclinicaltrialforearly‐stageCOVID‐19olderadultpatients,alreadyapproved
bytheSpanishAgencyofMedicinesandMedicalDevices(ALCOVID‐19;EudraCTnum‐
ber2020‐001760‐29),galenic,toxicologicalandpharmacokineticcharacterizationofthein‐
haledethanolcompoundedformulationhasbeennecessary.
Afterthevaporizationof65°ethanolfor45minutesinthreeadministrations,thereis
adecreaseinthealcoholcontentofthehydro‐alcoholicmixturecontainedinthedisposa‐
blehumidifierscomparedtotheinitialvalues.Throughbubblingoxygenatdifferent
flowsthroughthe65°ethanolsolution,evaporationofthemixtureisfavored.Thevapor
pressureofethanolismuchhigherthanthevaporpressureofwater,sinceethanolhasa
boilingpoint(78.4°C)considerablylowerthanwater(100°C)[47].Forthisreason,thegas
producedafterbubblingoxygenthroughthe65°ethanolsolutionwillhaveahighereth‐
anolconcentrationthanthestartingmixture.
Consideringtheminuteventilationof5L/minforhumanadultsrecommendedfor
short‐termexposure[33],andtheI:Eratioof1:2,theaverageconcentrationsofinhaled
ethanolinthethreeadministrationsofethanolwere6.70mg/L/minand7.99mg/L/minfor
theflowratesof2L/minand3L/min,respectively,whichcorrespondtothetotalamounts
ofethanolperminuteof33.52mg/minand39.88mg/min.Theseamountswerelowerthan
thoseobservedbyBessonneauandThomas[48]inastudyrelatedtotheexposureafter
handdisinfection,wherethetotalinhaleddoseofethanolrangedbetween150.8and
219.26mg/min.Thetotalamountofalcoholvaporizedduringtheflushingofoxygen
throughtheformulationincreasedwiththeflowrateofoxygen,asobservedbyother
studiesdescribedintheliterature[49].Theconcentrationsofethanolpermin(mg/L/min)
emittedinthehumidifierwerelowerattheflowrateof3L/min(seeFigure4),asthe
higherflowrateofoxygencouldnotreachtheequilibriumwithethanolatthesametime
andcarriedlessethanolvaporperliter(mg/L).Theadministrationduringthetimeof
treatmentwasmorestableattheflowrateof2L/minthan3L/min(seeFigure4).
Thetotalamountofethanolevaporated(mg/min)wasslightlyhigheratthehigher
flowrate(3L/min),astherewasmoreoxygenavailabletocarryethanol(seeFigure5).
Thisphenomenonismanifestedintheresultsobtainedinthisstudy,wheretheconcen‐
trationsofthemixture,afterthethreebubblingcycles,havebeenreducedto98.2%and
97.8%,comparedtotheoriginalconcentrationwith2L/minand3L/minflows,respec‐
tively.Thetotalamountofethanolevaporatedafterthreeadministrationsinthe45min
oftreatmentwas4.64gatthe2L/minflowrate,and5.38gatthe3L/minflowrate(see
Figure3a).
AstudyperformedbyZhang[50]demonstratedthatdepositionofethanolinseveral
localsitesoftherespiratorytractusingahumanupperairwaymodeldependedonfluid
flowsanddiffusionparameters.Lowerflowratesallowedhigherpercentagesofethanol
depositionintherespiratorytractthanhigherflows.Instead,athigherflowrates,thecon‐
centrationgradientneartothewallincreasesalongsidethemasstransfercoefficient,and
consequentlytheabsorptionofethanol.Thisauthorconcludedthatiftheobjectiveisdep‐
ositionofethanolandnotitsabsorption,theuseoflowflowratesshouldberecom‐
mended,duetotheextendedvaporresidencetimesatlowflowrates.Thisisinlinewith

Pharmaceutics2021,13,34215of18


ourapproach,inwhichweseekethanoldepositionwithminimalsystemicabsorption,
whichcouldproducedruginteractionsoradversereactions.
Ontheotherhand,itisconvenienttohighlightthesafetyofthecurrentpharmaco‐
logicalstrategyproposed.Preclinicalstudiesinratsandmicethatinhaledethanolprovide
thegreatestevidenceavailabletodateforcharacterizingtherisksofinhalationofethanol
[51,52].Theobservedadverseeffectswereattributedtosystemicandchronicexposureto
ethanol,regardlessoftherouteofadministration[53].Choietal.analyzedtheefficacy
andsafetyoftheadministrationofproteinscarriedinabsoluteethanolinrats.Noallergic
orinflammatoryresponseswereshown,norwasdamagetothealveolarbarrier,orcell
lysisthatcouldindicateacutetoxiceffectsinthelungsorairways[54].Theseresultsare
inlinewithwhatwasobservedinthepresentstudyaftertheadministrationofethanolin
Sprague‐Dawleyrats(females,250g(BW),withminuteventilationof130mL/min)every
8hoursforfivedays,withaninhalationperiodof15min.Bavisetal.describedthatrats
withthesamecharacteristicsasusedinourassayshaveaminuteventilation0.130L/min
[55].Takingthisintoaccount,inourexperimentsat2L/min,theratswereexposedtoa
totalestimatedamountofethanolof301.3mg/day,whichcorrespondstoadoseof1.2
g/kg/day.Atthesameflow,inourstudy,151gwereestimatedasinhaledbyhumanina
day.Consideringahumanbodyweightof60kg,thedoseadministratedwouldcorre‐
spondto0.025g/kg/day.Thus,inthepreclinicalstudy,theexposurelimitswereoveresti‐
matedupto40‐foldhigherthanthoseappliedinthehumanvolunteers.Nosignificant
differenceswereobservedthroughhistologicalstainingbyalungpathologistspecialist
betweentherodentsthatreceivedethanolcomparedtothosethatreceivedcontrol.Lung,
tracheaandesophagussamplesweredescribedasabsentofdamage.
Toourknowledge,nomolecularimagingstudieshavebeenpublishedtodateto
characterizethebiodistributionofradiolabeledethanoladministeredthroughtherespir‐
atorytract.Inordertoobtainafirstorientationofthepossibleresidencetimeofethanol
inthelung,aPET/CTstudyhasbeencarriedoutinrats,thereforebeingthefirstresearch
aboutpulmonarykineticsofethanoladministeredbythisroute.ThePET/CTimagesob‐
tainedaftertheadministrationof1‐11C‐ethanolshowradioactivityatinitialtimesatthe
levelofrespiratorytractandlungs,followedbyafastanddelocalizeddistributionover
thewholeanimal.Thesefindingssuggestatranslocationofethanoltothecirculatorysys‐
tem,followedbyprogressiveaccumulationinitsmainorganofmetabolism,theliver.
TheseresultsareinlinewiththatpreviouslyobservedbyGiffordetal.,inwhich1‐14C‐
ethanolwasadministeredintravenouslyinordertodeterminesitesofconcentrationof
ethanoloritsmetabolites,whichmaycontributetoitstoxicologicalandpharmacokinetic
properties[56].
Atthispoint,anewlineoffutureresearchopenssothatspecializedcenterscantest
itsvirucidalpotential.Nowadays,ourgrouparetestingtheclinicalefficacyandsafetyof
thisnewpharmacologicalstrategyin170early‐stageCOVID‐19institutionalizedelderly
patients.Thisclinicaltrialwillallowustoknowifthestablishedethanolconcentrationis
effective,iftheexposuretimeisadequateandalsothetoxicityprofileofinhaledethanol.
ThismaybepossiblethankstotherecentapprovalofthephaseIIclinicaltrialauthorized
bytheSpanishAgencyofMedicinesandMedicalDevices(ALCOVID‐19;EudraCT:2020‐
001760‐29).
5.Conclusions
Thisresearchworkaimstoshowthedevelopmentandresultsofthegalenic,phar‐
macokineticandtoxicologicalcharacterizationofinhaledethanolasapotentialtherapy
againstSARS‐CoV‐2.Thedeveloped65°ethanolcompoundedformulationremainsstable
atroomtemperatureandprotectedfromlightfor15days.Themostconvenientflowrate
forethanoladministrationis2L/min,ensuringtheinhalationofanethanoldailydoseof
33.6±3.6mg/min,andachievingmorestableconcentrationsduringtheentiretreatment
(45min).Furthermore,ithasalsobeenfoundtoshowsatisfactorypharmacokineticand

Pharmaceutics2021,13,34216of18
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
toxicologicalcharacteristicsthroughPET/CTstudiesandhistologicalanalysisofrespira‐
torytractandlungtissueinrats.Clinicalsafetyandefficacyarecurrentlybeingstudied
inaphaseIIclinicaltrial(ALCOVID‐19;EudraCTnumber:2020‐001760‐29)forearly‐stage
COVID‐19‐institutionalizedpatients.
SupplementaryMaterials:Thefollowingareavailableonlineatwww.mdpi.com/1999‐
4923/13/3/342/s1,FigureS1:Therapeuticapproachschemetotheuseofinhaledethanol.,FigureS2:
Physiologicalsafetyparametersmonitoredduringinhalation.
AuthorContributions:Conceptualization,I.N.‐V.,N.V.‐A.,J.R.A.‐L.,F.J.O.‐E.,A.F.‐F.andM.G.‐B.;
methodology,L.B.‐P.,R.S.‐G.,L.G.‐Q.,F.J.O.‐E.,J.R.L.‐Q.,L.V.‐C.,M.C.d.R.‐G.,C.C.‐D.,P.A.,
M.L.P.d.M.‐B.,R.T.‐P.,R.P.andA.R.‐B.d.Q.;formalanalysis,A.C.‐B.,C.M.‐G.,L.B.‐P.,I.V.‐R.,E.J.B.‐
V.,R.S.‐G.,A.R.‐B.d.Q.,L.G.‐Q.D.R.‐B.andP.A.;investigation,A.C.‐B.,C.M.‐G.,I.V.‐R.,J.R.A.‐L.,
D.R.‐B.,E.J.B.‐V.,M.B.‐I.,M.C.d.R.‐G.,I.N.‐V.,N.V.‐A.,A.R.‐B.d.Q.,L.V.‐C.,C.R.‐C.,G.B.‐C.,
M.L.P.M.‐B.,R.T.‐P.,R.P.andJ.L.;resources,I.Z.‐F.,F.J.O.‐E.andO.D.‐S.;datacuration,L.B.‐P.,I.V.‐
R.,I.N.‐V.,N.V.‐A.,M.B.‐I.,D.R.‐B.,C.R.‐C.,M.C.d.R.‐G.,G.B.‐C.andR.P.;writing—originaldraft
preparation,A.C.‐B.,C.M.‐G.,L.B.‐P.,R.S.‐G.,M.B.‐I.,I.Z.‐F.andJ.L.;writing—reviewandediting,
P.A.,M.G.‐B.andL.G.‐Q.;visualization,A.P.‐R.,G.B.‐C.,M.G.‐B.,L.V.‐C.,C.R.‐C.,R.T.‐P.,O.D.‐S.
andJ.R.L.‐Q.;supervision,J.R.A.‐L.,C.C.‐D.,M.L.P.d.M.‐B.,J.L.,A.P.‐R.andA.F.‐F.;projectadmin‐
istration,C.C.‐D.,J.L.,A.P.‐R.andA.F.‐F.;andfundingacquisition,O.D.andI.Z.‐F.,software,E.J.V.‐
V.andJ.R.L.‐Q.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:ThisprojectwaspartiallysupportedbyACIS(SA304C)byProgramaTraslaciona‐
COVID19(CT850A‐4)andFundaciónEspañoladeFarmaciaHospitalaria.Wealsowantto
acknowledgeOximesaNipponGasesandtheInstitutodeInvestigaciónSanitariadeSantiagode
Compostelaforthefungiblematerialgivenforthestudydevelopment.AFF,CMG,EJBVandLGC
aregratefultoISCIIIfortheaidforfinancingtheJR18/00014,CM18/00090,CM20/00135and
CM20/00024personnelcontracts.L.B.‐P.isgratefultotheSpanishMinistryofScience,Innovation
andUniversitiesforherJuandelaCierva—IncorporaciónGrant(AgreementNo.IJCI‐2017‐31665).
PAacknowledgesthe“RamónyCajal”researchfellowship(RYC‐2015/17430).
InstitutionalReviewBoardStatement:Thestudywasconductedaccordingtotheguidelinesofthe
DeclarationofHelsinki,andapprovedbytheInstitutionalReviewBoardofCICbiomaGUNE(pro‐
tocolcodePRO‐AE‐SS059).
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Notapplicable.
Acknowledgments:WeappreciatethesupportoftheSpanishSocietyofHospitalPharmacy(SEFH)
thatwillfacilitatefutureclinicaltrialstodeterminethefeasibilityofthistherapyagainstSARS‐CoV‐
2.Finally,wealsowantedtothankSEFHfortheaward“MejorProyectoI+I”,grantedatthe65th
SEFHVirtualCongress2020.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
References
1. Weiss,S.R.;Leibowitz,J.L.CoronavirusPathogenesis.Adv.VirusRes.2011,81,85–164,doi:10.1016/B978‐0‐12‐385885‐6.00009‐2.
2. Jin,Y.;Yang,H.;Ji,W.;Wu,W.;Chen,S.;Zhang,W.;Duan,G.Virology,Epidemiology,Pathogenesis,andControlofCOVID‐
19.Viruses2020,12,doi:10.3390/v12040372.
3. Rothan,H.A.;Byrareddy,S.N.TheEpidemiologyandPathogenesisofCoronavirusDisease(COVID‐19)Outbreak.J.Autoim‐
mun.2020,109,102433,doi:10.1016/j.jaut.2020.102433.
4. WHO.CoronavirusDisease(COVID‐19)Dashboard.Availableonline:https://covid19.who.int(accessedon16January2021).
5. Baker,M.G.;Kvalsvig,A.;Verrall,A.J.NewZealand’sCOVID‐19EliminationStrategy.Med.J.Aust.2020,
doi:10.5694/mja2.50735.
6. Baden,L.R.;Sahly,H.M.E.;Essink,B.;Kotloff,K.;Frey,S.;Novak,R.;Diemert,D.;Spector,S.A.;Rouphael,N.;Creech,C.B.;et
al.EfficacyandSafetyoftheMRNA‐1273SARS‐CoV‐2Vaccine.N.Engl.J.Med.2020,doi:10.1056/NEJMoa2035389.
7. Polack,F.P.;Thomas,S.J.;Kitchin,N.;Absalon,J.;Gurtman,A.;Lockhart,S.;Perez,J.L.;PérezMarc,G.;Moreira,E.D.;Zerbini,
C.;etal.SafetyandEfficacyoftheBNT162b2MRNACovid‐19Vaccine.N.Engl.J.Med.2020,383,2603–2615,
doi:10.1056/NEJMoa2034577.
8. Knoll,M.D.;Wonodi,C.Oxford‐AstraZenecaCOVID‐19VaccineEfficacy.Lancet2021,397,72–74,doi:10.1016/S0140‐
6736(20)32623‐4.

Pharmaceutics2021,13,34217of18


9. Stasi,C.;Fallani,S.;Voller,F.;Silvestri,C.TreatmentforCOVID‐19:AnOverview.Eur.J.Pharmacol.2020,889,173644,
doi:10.1016/j.ejphar.2020.173644.
10. Li,Y.;Liu,S.;Zhang,S.;Ju,Q.;Zhang,S.;Yang,Y.;Wang,H.CurrentTreatmentApproachesforCOVID‐19andtheClinical
ValueofTransfusion‐RelatedTechnologies.Transfus.Apher.Sci.2020,59,102839,doi:10.1016/j.transci.2020.102839.
11. Libster,R.;PérezMarc,G.;Wappner,D.;Coviello,S.;Bianchi,A.;Braem,V.;Esteban,I.;Caballero,M.T.;Wood,C.;Berrueta,
M.;etal.EarlyHigh‐TiterPlasmaTherapytoPreventSevereCovid‐19inOlderAdults.N.Engl.J.Med.2021,
doi:10.1056/NEJMoa2033700.
12. PharmaMarMulticenter.Randomized,ParallelandProofofConceptStudytoEvaluatetheSafetyProfileofThreeDosesof
PlitidepsininPatientsWithCOVID‐19RequiringHospitalization.2020.Availableonline:clinicaltrials.gov(accessedon25Jan‐
uaary2021).
13. EliLillyandCompany.ExpandedAccessInformationforPhysiciansforBamlanivimab(LY3819253)EmergencyInvestigational
NewDrug(EIND).2021.Availableonline:clinicaltrials.gov(accessedon25February2021).
14. RegeneronPharmaceuticals.AMasterProtocolAssessingtheSafety,Tolerability,andEfficacyofAnti‐Spike(S)SARS‐CoV‐2
MonoclonalAntibodiesfortheTreatmentofAmbulatoryPatientsWithCOVID‐19.2021.Availableonline:clinicaltrials.gov
(accessedon25February2021).
15. Group,T.R.C.DexamethasoneinHospitalizedPatientswithCovid‐19—PreliminaryReport.N.Engl.J.Med.2020,
doi:10.1056/NEJMoa2021436.
16. Andersen,P.I.;Ianevski,A.;Lysvand,H.;Vitkauskiene,A.;Oksenych,V.;Bjørås,M.;Telling,K.;Lutsar,I.;Dumpis,U.;Irie,Y.;
etal.DiscoveryandDevelopmentofSafe‐in‐ManBroad‐SpectrumAntiviralAgents.Int.J.Infect.Dis.2020,93,268–276,
doi:10.1016/j.ijid.2020.02.018.
17. Kampf,G.;Todt,D.;Pfaender,S.;Steinmann,E.PersistenceofCoronavirusesonInanimateSurfacesandTheirInactivationwith
BiocidalAgents.J.Hosp.Infect.2020,104,246–251,doi:10.1016/j.jhin.2020.01.022.
18. LaboratoryBiosafetyGuidanceRelatedtoCoronavirusDisease(COVID‐19).Availableonline:https://www.who.int/publica‐
tions‐detail‐redirect/laboratory‐biosafety‐guidance‐related‐to‐coronavirus‐disease‐(covid‐19)(accessedon21June2020).
19. Kratzel,A.;Todt,D.;V’kovski,P.;Steiner,S.;Gultom,M.;Thao,T.T.N.;Ebert,N.;Holwerda,M.;Steinmann,J.;Niemeyer,D.;
etal.InactivationofSevereAcuteRespiratorySyndromeCoronavirus2byWHO‐RecommendedHandRubFormulationsand
Alcohols.EmergingInfect.Dis.2020,26,doi:10.3201/eid2607.200915.
20. Opilla,M.T.;Kirby,D.F.;Edmond,M.B.UseofEthanolLockTherapytoReducetheIncidenceofCatheter‐RelatedBloodstream
InfectionsinHomeParenteralNutritionPatients.JPENJ.Parenter.EnteralNutr.2007,31,302–305,
doi:10.1177/0148607107031004302.
21. Lipton,S.PainReliefinActivePatientswithCancer:TheEarlyUseofNerveBlocksImprovestheQualityofLife.BMJ1989,
298,37–38,doi:10.1136/bmj.298.6665.37.
22. Sarin,S.K.;Sachdeva,G.K.;Nanda,R.;Vij,J.C.;Anand,B.S.EndoscopicSclerotherapyUsingAbsoluteAlcohol.Gut1985,26,
120–124.
23. Chung,S.C.;Lee,T.W.;Kwok,S.P.;Li,A.K.InjectionofAlcoholtoControlBleedingfromRupturedHepatomas.BMJ1990,301,
421,doi:10.1136/bmj.301.6749.421.
24. Yuksel,E.;Novruzlu,S.;Ozmen,M.C.;Bilgihan,K.AStudyComparingStandardandTransepithelialCollagenCross‐Linking
RiboflavinSolutions:EpithelialFindingsandPainScores.J.Ocul.Pharmacol.Ther.2015,31,296–302,doi:10.1089/jop.2014.0090.
25. Sepúlveda,R.A.;Selamé,E.;Roessler,E.;Tagle,R.;Valdivieso,A.IntoxicaciónPorEtilenglicol,FisiopatologíayEnfrentamiento
Clínico.Rev.Med.Chil.2019,147,1572–1578,doi:10.4067/S0034‐98872019001201572.
26. Statement—OlderPeopleAreatHighestRiskfromCOVID‐19,butAllMustActtoPreventCommunitySpread.Available
online:http://www.euro.who.int/en/health‐topics/health‐emergencies/coronavirus‐covid‐19/statements/statement‐older‐peo‐
ple‐are‐at‐highest‐risk‐from‐covid‐19,‐but‐all‐must‐act‐to‐prevent‐community‐spread(accessedon8June2020).
27. Li,P.;Chen,L.;Liu,Z.;Pan,J.;Zhou,D.;Wang,H.;Gong,H.;Fu,Z.;Song,Q.;Min,Q.;etal.ClinicalFeaturesandShort‐Term
OutcomesofElderlyPatientswithCOVID‐19.Int.J.Infect.Dis.2020,97,245–250,doi:10.1016/j.ijid.2020.05.107.
28. Kampf,G.EfficacyofEthanolagainstVirusesinHandDisinfection.J.Hosp.Infect.2018,98,331–338,
doi:10.1016/j.jhin.2017.08.025.
29. Yan,R.;Zhang,Y.;Li,Y.;Xia,L.;Guo,Y.;Zhou,Q.StructuralBasisfortheRecognitionofSARS‐CoV‐2byFull‐LengthHuman
ACE2.Science2020,367,1444–1448,doi:10.1126/science.abb2762.
30. EnsayoClínicoFaseIIParaEvaluarLaEficaciaySeguridaddeEtanolInhaladoEnElTratamientodeLaCOVID‐19EnEstadio
Inicial.Availableonline:https://reec.aemps.es/reec/estudio/2020‐001760‐29(accessedon9January2021).
31. Kilkenny,C.;Browne,W.;Cuthill,I.C.;Emerson,M.;Altman,D.G.;NC3RsReportingGuidelinesWorkingGroupAnimalRe‐
search:ReportinginVivoExperiments:TheARRIVEGuidelines.Br.J.Pharmacol.2010,160,1577–1579,doi:10.1111/j.1476‐
5381.2010.00872.x.
32. Seoane‐Viaño,I.;Gómez‐Lado,N.;Lázare‐Iglesias,H.;Barreiro‐deAcosta,M.;Silva‐Rodríguez,J.;Luzardo‐Álvarez,A.;He‐
rranz,M.;Otero‐Espinar,F.;Antúnez‐López,J.R.;Lamas,M.J.;etal.LongitudinalPET/CTEvaluationofTNBS‐InducedInflam‐
matoryBowelDiseaseRatModel.Int.J.Pharm.2018,549,335–342,doi:10.1016/j.ijpharm.2018.08.005.
33. U.S.EPA.ExposureFactorsHandbook2011Edition(FinalReport);EPA/600/R‐09/052F;U.S.EnvironmentalProtectionAgency:
Washington,DC,USA,2011.

Pharmaceutics2021,13,34218of18


34. Gulyás,B.;Vas,A.;Halldin,C.;Sóvágó,J.;Sandell,J.;Olsson,H.;Fredriksson,A.;Stone‐Elander,S.;Farde,L.CerebralUptake
of(Ethyl‐11C)Vinpocetineand1‐(11C)EthanolinCynomolgousMonkeys:AComparativePreclinicalPETStudy.Nucl.Med.
Biol.2002,29,753–759,doi:10.1016/s0969‐8051(02)00319‐0.
35. DeGrazia,J.A.;Rodden,A.F.;Teresi,J.D.;Busick,D.D.;Walz,D.R.RadioscintigraphicStudiesof11CDistributioninCatsgiven
1‐11C‐Ethanol.J.Nucl.Med.1975,16,73–76.
36. Cossío,U.;Gómez‐Vallejo,V.;Flores,M.;Gañán‐Calvo,B.;Jurado,G.;Llop,J.PreclinicalEvaluationofAerosolAdministration
SystemsUsingPositronEmissionTomography.Eur.J.Pharm.Biopharm.2018,130,59–65,doi:10.1016/j.ejpb.2018.05.037.
37. Castro‐Balado,A.;Varela‐Rey,I.;Bandín‐Vilar,E.J.;Busto‐Iglesias,M.;García‐Quintanilla,L.;Mondelo‐García,C.;Fernández‐
Ferreiro,A.ClinicalResearchinHospitalPharmacyduringtheFightagainstCOVID‐19.Farm.Hosp.2020,44,66–70,
doi:10.7399/fh.11494.
38. Dai,L.;Gao,G.F.ViralTargetsforVaccinesagainstCOVID‐19.Nat.Rev.Immunol.2020,doi:10.1038/s41577‐020‐00480‐0.
39. Fernández‐Ferreiro,A.;González‐Barcia,M.TheHumanBeing,theOnlyAnimalThatNeedsaMastertoLive.OFIL∙ILAPHAR
2020,30,179–182.
40. Cao,B.;Wang,Y.;Wen,D.;Liu,W.;Wang,J.;Fan,G.;Ruan,L.;Song,B.;Cai,Y.;Wei,M.;etal.ATrialofLopinavir–Ritonavir
inAdultsHospitalizedwithSevereCovid‐19.N.Engl.J.Med.2020,2001282,doi:10.1056/NEJMoa2001282.
41. RECOVERYCollaborativeGroup;Horby,P.;Mafham,M.;Linsell,L.;Bell,J.L.;Staplin,N.;Emberson,J.R.;Wiselka,M.;Ustian‐
owski,A.;Elmahi,E.;etal.EffectofHydroxychloroquineinHospitalizedPatientswithCovid‐19.N.Engl.J.Med.2020,383,
2030–2040,doi:10.1056/NEJMoa2022926.
42. Stone,J.H.;Frigault,M.J.;Serling‐Boyd,N.J.;Fernandes,A.D.;Harvey,L.;Foulkes,A.S.;Horick,N.K.;Healy,B.C.;Shah,R.;
Bensaci,A.M.;etal.EfficacyofTocilizumabinPatientsHospitalizedwithCovid‐19.N.Engl.J.Med.2020,383,2333–2344,
doi:10.1056/NEJMoa2028836.
43. Li,L.;Zhang,W.;Hu,Y.;Tong,X.;Zheng,S.;Yang,J.;Kong,Y.;Ren,L.;Wei,Q.;Mei,H.;etal.EffectofConvalescentPlasma
TherapyonTimetoClinicalImprovementinPatientswithSevereandLife‐ThreateningCOVID‐19:ARandomizedClinical
Trial.JAMA2020,324,460–470,doi:10.1001/jama.2020.10044.
44. Talavera,B.;García‐Azorín,D.;Martínez‐Pías,E.;Trigo,J.;Hernández‐Pérez,I.;Valle‐Peñacoba,G.;Simón‐Campo,P.;deLera,
M.;Chavarría‐Miranda,A.;López‐Sanz,C.;etal.AnosmiaIsAssociatedwithLowerIn‐HospitalMortalityinCOVID‐19.J.
Neurol.Sci.2020,419,117163,doi:10.1016/j.jns.2020.117163.
45. Shintake,T.PossibilityofDisinfectionofSARS‐CoV‐2(COVID‐19)inHumanRespiratoryTractbyControlledEthanolVapor
Inhalation.arXiv2020,arXiv:2003.12444.
46. Ly,T.D.A.;Zanini,D.;Laforge,V.;Arlotto,S.;Gentile,S.;Mendizabal,H.;Finaud,M.;Morel,D.;Quenette,O.;Malfuson‐Clot‐
Faybesse,P.;etal.PatternofSARS‐CoV‐2InfectionamongDependantElderlyResidentsLivinginLong‐TermCareFacilities
inMarseille,France,March–June2020.Int.J.Antimicrob.Agents2020,56,106219,doi:10.1016/j.ijantimicag.2020.106219.
47. Durst,H.D.;Gokel,G.W.QuímicaOrgánicaExperimental;EditorialReverté:Barcelona,Spain,1985;ISBN978‐84‐291‐7155‐6.
48. Bessonneau,V.;Thomas,O.AssessmentofExposuretoAlcoholVaporfromAlcohol‐BasedHandRubs.Int.J.Environ.Res.
PublicHealth2012,9,868–879,doi:10.3390/ijerph9030868.
49. Luisada,A.A.;Goldmann,M.A.;Weyl,R.AlcoholVaporbyInhalationintheTreatmentofAcutePulmonaryEdema.Circulation
1952,5,363–369,doi:10.1161/01.cir.5.3.363.
50. Zhang,Z.;Kleinstreuer,C.;Kim,C.S.TransportandUptakeofMTBEandEthanolVaporsinaHumanUpperAirwayModel.
Inhal.Toxicol.2006,18,169–184,doi:10.1080/08958370500434172.
51. Rogers,J.;Wiener,S.G.;Bloom,F.E.Long‐TermEthanolAdministrationMethodsforRats:AdvantagesofInhalationoverIntu‐
bationorLiquidDiets.Behav.NeuralBiol.1979,27,466–486,doi:10.1016/s0163‐1047(79)92061‐2.
52. MacLean,R.R.;Valentine,G.W.;Jatlow,P.I.;Sofuoglu,M.InhalationofAlcoholVapor:MeasurementandImplications.Alcohol.
Clin.Exp.Res.2017,41,238–250,doi:10.1111/acer.13291.
53. Gilpin,N.W.;Richardson,H.N.;Cole,M.;Koob,G.F.VaporInhalationofAlcoholinRats.Curr.Protoc.Neurosci.2008,
doi:10.1002/0471142301.ns0929s44.
54. Choi,W.S.;Murthy,G.G.K.;Edwards,D.A.;Langer,R.;Klibanov,A.M.InhalationDeliveryofProteinsfromEthanolSuspen‐
sions.Proc.Natl.Acad.Sci.USA2001,98,11103–11107,doi:10.1073/pnas.201413798.
55. Bavis,R.W.;Johnson,R.A.;Ording,K.M.;Otis,J.P.;Mitchell,G.S.RespiratoryPlasticityafterPerinatalHypercapniainRats.
Respir.Physiol.Neurobiol.2006,153,78–91,doi:10.1016/j.resp.2005.09.002.
56. Gifford,A.N.;Espaillat,M.P.;Gatley,S.J.BiodistributionofRadiolabeledEthanolinRodents.DrugMetab.Dispos.2008,36,1853–
1858,doi:10.1124/dmd.107.020271.