DTIC ADA539650: In Vivo Reactivation by Oximes of Inhibited Blood, Brain and Peripheral Tissue Cholinesterase Activity Following Exposure to Nerve Agents in Guinea Pigs

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In vivo reactivation by oximes of inhibited blood, brain and peripheral tissue cholinesterase
activity following exposure to nerve agents in guinea pigs 5b. GRANT NUMBER

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6. AUTHOR(S) 5d. PROJECT NUMBER
Shih, T-M., Skovira, JW, O'Donnell, JC, McDonough, JH
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7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT
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US Army Medical Research Institute of Aberdeen Proving Ground, MD
Chemical Defense 21010-5400 USAMRICD-P09-026
ATTN: MCMR-CDR-P
3100 Ricketts Point Road

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US Army Medical Research Institute Aberdeen Proving Ground, MD
of Chemical Defense 21010-5400
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13. SUPPLEMENTARY NOTES
Published in Chemico-Biological Interactions ,187, 207–214, 2010 . This research was supported by the Defense Threat Reduction Agency - Joint Service and
Technology Office, Medical Science and Technology Division.
14. ABSTRACT
See reprint.

15. SUBJECT TERMS
acetylcholinesterase, cholinesterase reactivation, guinea pig, methoxime, nerve agents, oximes, chemical warfare agents, medical
countermeasures
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OF ABSTRACT OF PAGES Tsung-Ming Shih
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Chemico-BiologicalInteractions187 (2010) 207–214
ContentslistsavailableatScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
In vivo reactivation by oximes of inhibited blood, brain and peripheral tissue
(cid:2)
cholinesterase activity following exposure to nerve agents in guinea pigs
Tsung-MingShih∗,JacobW.Skovira,JohnC.O’Donnell,JohnH.McDonough
PharmacologyBranch,ResearchDivision,U.S.ArmyMedicalResearchInstituteofChemicalDefense,AberdeenProvingGround,MD21010-5400,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Thisstudycomparedtheabilityofnineoximes(HI-6,HLö7,MMB-4,TMB-4,carboxime,ICD585,ICD692,
Available online 9 March 2010 ICD3805,and2-PAM)toreactivateinvivocholinesterase(ChE)inblood,brain,andperipheraltissuesin
guineapigsintoxicatedbyoneoffourorganophosphorusnerveagents.Twobis-pyridiniumcompounds
Keywords: withoutanoximegroup,SAD128andICD4157,servedasnon-oximecontrols.Animalswereinjected
Acetylcholinesterase subcutaneouslywith1.0×LD50ofthenerveagentssarin,cyclosarin,VRorVXandtreatedintramuscularly
Cholinesterasereactivation 5minlaterwithoneoftheseoximes.Toxicsignsandlethalityweremonitored;tissueChEactivitieswere
Guineapig
determinedat60minafternerveagent.Someanimalsexposedtosarinorcyclosarin,withorwithoutnon-
Methoxime
oximetreatment,diedwithin60min;however,noanimaltreatedwithanoximedied.ForVRorVX,all
Nerveagents
Oximes animalssurvivedthe60minafterexposure,withorwithoutnon-oximeoroximetherapy.Thefournerve
agentscauseddifferentialdegreesofinhibitioninblood,brainregionsandperipheraltissues.Thetested
oximesexhibiteddifferentialpotencyinreactivatingnerveagent-inhibitedChEinvariousperipheral
tissues,butdidnotaffectChEactivityinthebrainregions.Therewasnodirectrelationbetweenblood
andperipheraltissuesinthereactivatingefficacyofoximetreatments.ChEinhibitedbysarinwasthe
mostsusceptibletooximereactivationwhilecyclosarintheleastsusceptible.Therewasnodifference
intheChEreactivatingpotencybetweenthedimethanesulfonateanddichloridesaltsofHI-6.MMB-4
significantlyreactivatedtheChEinhibitedbythesefournerveagentsinbloodandallthreeperipheral
tissuesoftheguineapig,andamongalltheoximestesteditwasthemosteffectiveinvivoChEreactivator
againstallfournerveagents.
Published by Elsevier Ireland Ltd.
1. Introduction providesadequateprotectionagainstsomenerveagents,suchas
sarin (GB) and VX, it is less effective against other nerve agents,
Theabilityofoximestoreactivatecholinesterase(ChE)activity suchastabun,soman,cyclosarin(GF),andaRussianV-agent(VR)
inhibited by organophosphorus nerve agents is considered to be [2].Forthisreason,numerousstudiesinthelastseveraldecades
themajormechanismoftheirantidotalaction[1].2-PAM(prali- have focused on developing improved oxime reactivators, and
doxime) is a mono-pyridinium oxime approved for use in the several oximes, such as HI-6, HLö7, MMB-4 (methoxime), TMB-
U.S.forthetreatmentofnerveagentexposure.Although2-PAM 4(trimedoxime),carboxime,ICD585,ICD692,andICD3805,have
been found to possess much better in vitro reactivating potency
orinvivoantidotalcapacityagainstnerveagentintoxicationthan
2-PAM [3–7]. Although several of these oximes have been stud-
(cid:2) ied for their ability to reactivate ChE inhibited by single nerve
ResearchwasconductedincompliancewiththeAnimalWelfareActandother
Federalstatutesandregulationsrelatingtoanimalsandexperimentsinvolvingani- agents [8–14], their broad-spectrum capacity to reactivate ChE
malsandadheredtoprinciplesstatedintheGuidefortheCareandUseofLaboratory activityinhibitedbyseveralnerveagentshasnotyetbeenevalu-
Animals,bytheInstituteofLaboratoryAnimalResources,NationalResearchCouncil. atedandcomparedsystematically.Theidealoximeshouldbeable
ThefacilitywherethisresearchwasconductedisfullyaccreditedbytheAssociation
toeffectivelyreactivateChEinhibitedbynerveagentsofdiverse
forAssessmentandAccreditationofLaboratoryAnimalCareInternational(AAALAC).
Theopinionsorassertionscontainedhereinaretheprivateviewsoftheauthors,and structures.
arenottobeconstruedasreflectingtheviewsoftheDepartmentoftheArmyorthe Theobjectiveofthisstudywasprimarilytocomparetheability
DepartmentofDefense. ofseveraloximes(HI-6,HLö7,MMB-4,TMB-4,carboxime,ICD585,
∗
Corresponding author at: US Army Medical Research Institute of Chemical
ICD692,andICD3805)with2-PAM(seeFig.1)toreactivateblood,
Defense,ATTN:MCMR-CDR-P(Dr.T.-M.Shih),3100RickettsPointRoad,Aberdeen
brain,andperipheraltissueChEinguineapigsintoxicatedbyfour
ProvingGround,MD21010-5400,USA.Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](T.-M.Shih). nerveagents.AsecondobjectivewastocomparetheChEreacti-
0009-2797/$–seefrontmatter.Published by Elsevier Ireland Ltd.
doi:10.1016/j.cbi.2010.03.006
208 T.-M.Shihetal./Chemico-BiologicalInteractions187 (2010) 207–214
Fig.1. Chemicalstructuresofnon-oximes(SAD128andICD4157)andoximes(2-PAM,HLö7[ICD2445],HI-6DMS,HI-6DiCl,ICD585,ICD692,ICD3805,MMB-4[ICD039],
TMB-4,andcarboxime).
vatingpotencyofthetwosaltsofHI-6undertheseconditions.The oxime that is significantly more effective than 2-PAM against a
dichloride(DiCl)saltofHI-6wasstudiedforyears[9,11,12],but spectrumofnerveagents[17,18].
thedimethanesulfonate(DMS)saltofHI-6ismorewatersoluble
andisproposedtobeutilizedinanewHI-6autoinjectorindevel- 2. Materialsandmethods
opment[15].StudieswiththeDMSsaltareneededtoinvestigate
itspharmacodynamicinvivoequivalencywiththeDiClsalt,aswas 2.1. Subjects
reportedforinvitrostudy[16].Twostructurallysimilarcompounds
withoutanoximemoiety(SAD128andICD4157)wereincludedto Male Hartley guinea pigs (Charles River Labs, Kingston, NY;
serveasnegativecontrolsinChEreactivation(Fig.1).Theoverall Crl:(HA) BR COBS) weighing 250–300g served as subjects. They
goalsweretogenerateadatabaseforselectingabroad-spectrum werehousedindividuallyintemperature(21±2◦C)-andhumid-
T.-M.Shihetal./Chemico-BiologicalInteractions187 (2010) 207–214 209
ity (50±10%)-controlled quarters and maintained on a 12-h autoinjectordosestobegiventoa70-kgperson(basedonHI-6DiCl)
light–dark schedule (with lights on at 0600h). Laboratory chow [15].ICD4157andSAD128servedasnegativecontrols.ICD4157is
andtapwaterwerefreelyavailable.Animalswereacclimatedfor1 structurallysimilartoICD585andSAD128isstructurallysimilar
weekpriortoexperimentation. toHI-6.ThedoseofICD4157wasequivalentto58(cid:2)mol/kg,while
thedoseofSAD128usedwas53.6(cid:2)mol/kg,equivalenttoits1/4
2.2. Materials LD dose.
50
Sixtyminutesafterscsalineornerveagentadministration,the
Saline(U.S.P.),AttaneTM(Isoflurane,U.S.P.),andheparinsodium animalsweredeeplyanesthetizedwithisofluraneandeuthanized
werepurchasedfromBraunMedicalInc.(Irvine,CA),Minrad,Inc. by decapitation. Shortly before anesthesia, the severity of toxic
(Bethlehem, PA), and U.S.P., Inc. (Rockville, MD), respectively. signsofeachanimalwasscored.Blood(∼0.5ml)wascollectedinto
2-PAM was purchased from Ayerst Labs, Inc. (New York, NY). a1.0-mlmicrofugetubecontaining50(cid:2)lofheparinsodiumsolu-
HLö7 (1-[[[4-(aminocarbonyl)pyridinio]methoxy] methyl]-2,4- tion(15U/ml).FortheWBsamples,20(cid:2)lofbloodwasdiluted1:25
bis[(hydroxyimino) methyl]pyridinium dimethanesulfonate), in1%Triton-X100solution.FortheRBCsamples,theoriginalblood
MMB-4 (1,1(cid:4)-methylenebis[4-[(hydroxyimino) methyl] pyri- samplewascentrifugedfor5minat14,000rpm,and10(cid:2)lofthe
dinium] dichloride), HI-6 DiCl (1-(((4-(aminocarbonyl)pyridinio) packedRBCwasthendiluted1:50in1%Triton-X100solution.Brain
methoxy)methyl)-2-((hydroxyimino)methyl) regions (brainstem, cerebellum, cortex, hippocampus, midbrain,
pyridinium dichloride), HI-6 DMS, TMB-4 (1,1(cid:4)-trimethyl spinalcord[cervic-thoracicregion]andstriatum)andperipheral
bis-[4-formyl pyridinium chloride] dioxime), carboxime tissue (diaphragm [whole], heart, and skeletal muscle [gastroc-
(1,4-methyl-5-[2(cid:4)-(benzyldimethylammonium)ethyl] nemius])weredissected.Brainsampleswerediluted1:20,while
carbamoylpyridinium-2-aldoxime dichloride), peripheralsampleswerediluted1:5,in1%Triton-X100solution
ICD585 (1-(4-aminocarbonylpyridinio)-3(2- andthenhomogenized.Thehomogenateswerethencentrifuged
hydroxyiminomethylpyridinio)propane dichloride (31,000×gat4◦C;20minforbrainand30minforperipheraltis-
monohydrate), ICD692 (1-[1-(2-hydroxyiminomethyl-3-methyl) sues)andthesupernatantwasdecantedandkeptfrozenat−80◦C
imidazolo]-3-[4-carbamoylpyridinium]-propane dichloride untilChEanalysis.
hydrate), ICD3805 (3-[4-carbamoyl-1-pyridino]-1-[2,4-bis Processed blood, tissue and brain samples were analyzed for
(hydroxyiminomethyl)-1-pyridino]propane dichloride hydrate), ChEactivityandproteinconcentrationsaccordingtothemethods
ICD4157 (1-[3-[4-aminocarbonylpyridinio] propyl]-2- describedbyShihetal.[20,21],basedonthecolorimetricmethod
methylpyridinium dichloride hydrate), and SAD128 ofEllmanetal.[22]andaBCAproteinassay(PierceBiotechnol-
(1,1(cid:4)-[oxybis(methylene)]bis[(4-tert-butyl)-pyridinium] dichlo- ogy,Inc.).Briefly,inthemodifiedEllmanmethod,7(cid:2)lofsample
ride) were obtained from the depository at the Division of (orenzymestandard),20(cid:2)lofH O,200(cid:2)lofDTNBreagent,and
2
ExperimentalTherapeutics,WalterReedArmyInstituteofResearch 30(cid:2)lofacetylthiocholineiodidesubstratewasaddedtoeachwell
(SilverSpring,MD).Sarin(GB;isopropylmethylphosphonofluori- ofa96wellmicroplate(samplesrunintriplicate),andmeanactiv-
date),cyclosarin(GF;cyclohexylmethylphosphonofluoridate),VX itywasdeterminedbyapplyingBeer–Lambert’sLaw,incorporating
(o-ethylS-(2-(diisopropylamino)ethyl)methylphosphonothioate), theobservedchangeinabsorbance,knownextinctioncoefficientof
and a Russian V-type agent designated VR (o-isobutyl S-(2- theDTNBion,path-lengthcorrection,anddilutionfactor.Allsam-
(diethylamino)ethyl)methylphosphonothioate) were obtained plevalueswerewellwithinthelinearrangeofthestandardcurve.
fromtheUSArmyEdgewoodChemicalBiologicalCenter(Aberdeen Protein concentrations of brain and peripheral tissue samples in
ProvingGround,MD).Nerveagentsweredilutedinice-coldsaline theBCAassayweredeterminedbyinterpolatingfromastandard
prior to subcutaneous (sc) injection. All non-oxime and oxime curve,andappliedtoChEactivityfromthesametissuesamplefor
compoundswerepreparedinsalineforintramuscular(im)injec- finalvaluesin(cid:2)mol/(gproteinmin).Eachtissuesamplewasnor-
tion.Injectionvolumewas0.5ml/kgfortheadministrationofall malizedasapercentageoftheaveragesaline/salinecontrolgroup
nerveagentsandoximecompounds. value for that tissue type. Post-exposure blood enzyme activity
((cid:2)mol/(mlmin)) was normalized as a percentage of each sam-
2.3. Experimentalprocedures ple’scorrespondingbaselineactivitydetermined1–3dayspriorto
study.
One to 3 days prior to the experiment, blood (∼0.5ml) was
drawnusingatoenailclipmethod[19]andwascollectedintoa1.0- 2.4. Dataanalysis
mlmicrofugetubecontaining50(cid:2)lofheparinsodium(15U/ml)
todeterminebaselineChEactivityinwholeblood(WB)andred Statisticalanalysiswasperformedusingaone-wayANOVAto
blood cell (RBC). On the day of the study, groups of guinea pigs compareacrosstissuesforChEactivity,amongtissuesacrossnerve
wereinjectedscwitheithersaline(0.5ml/kg)ora1.0×LD doseof agents,andacrosstreatmentgroupsforeachnerveagent.Apost
50
GB(42.0(cid:2)g/kg),GF(57.0(cid:2)g/kg),VR(11.3(cid:2)g/kg)orVX(8.0(cid:2)g/kg). hocTukeytestwasusedformultiplecomparisons.Statisticalsig-
Five minutes later, when the inhibition of ChE activity by these nificancewasdefinedasp<0.05.Onlycomparisonsbetweeneach
nerveagentsreachedmaximumintheblood[20],saline,SAD128 treatment group in which ChE activity was significantly higher
(20.7mg/kg), ICD 4157 (20.1mg/kg), MMB-4 (19.1mg/kg), car- thantheactivityinthenerveagent/saline-treatedgrouporthe2-
boxime (24.0mg/kg), HLö7 (30.2mg/kg), HI-6 DMS (27.8mg/kg), PAM-treatedgrouparediscussed.Inthisreport,theChEactivity
HI-6DiCl(21.9mg/kg),2-PAM(25.0mg/kg),ICD585(21.8mg/kg), reactivatedbythespecificoximeinanytissueisconsideredtobe
ICD692(22.0mg/kg),ICD3805(23.2mg/kg)orTMB-4(20mg/kg) thatportionoftheactivityinanerveagent/oxime-treatedgroup
wasgivenim.Agroupofanimalsthatreceivedbothscsaline(i.e., thatsignificantlyexceededtheactivityinthenerveagent/saline-
nonerveagent)andimsaline(i.e.,nooxime)injectionsservedas treatedgroup(at60min).
overallcontrols(saline/salinegroup). WedividedtheanalysisofChEreactivationdataintotwocom-
Thedoseof2-PAMwasequivalentto145.0(cid:2)mol/kg,im,which partments:theperipheraltissuesandthebloodcomponents.A“+”
isequivalentto3autoinjectordoses(asinMarkINerveAgentAnti- signwasassignedforeachoftheperipheraltissuesorbloodcom-
doteKit)ina70-kgperson.ThedoseofTMB-4was43(cid:2)mol/kg, ponents,whenanoximetreatmentsignificantlyreactivatednerve
equivalenttoits1/4LD dose.Thedoseofotheroximesstudied agent-inhibited ChE. For example, if an oxime treatment signifi-
50
was 58.0(cid:2)mol/kg, im, which was equivalent to the maximum 3 cantlyreactivatedChEinallthreeperipheraltissuesitscellreceived
210 T.-M.Shihetal./Chemico-BiologicalInteractions187 (2010) 207–214
“+++”maximum,orinthetwocomponentsofblooditscellreceived 3.3. ChEactivityinperipheraltissues(Table1)
“++” maximum. When an oxime treatment did not significantly
reactivateanerveagent-inhibitedChEinanytissue,itwasassigned BasalChEactivitiesinthediaphragm,heartandskeletalmus-
a“−”sign. clewere14.3±0.8,18.2±0.6,and10.5±0.4(cid:2)mol/(gproteinmin),
respectively. There was no significant difference among them.
3. Results Table1summarizedtheChEactivityinperipheraltissuesandblood
componentsfollowingexposuretonerveagentsandsalineor6top
3.1. Signsoftoxicityandlethality oximetreatments.GBandVXproducedinthediaphragmthesame
degreeofChEinhibition(72–74%),whichwassignificantlyhigher
Undertheconditionsofthisstudyallguineapigsexhibitedsigns thanthatinducedbyGF(65%)orVR(52%).Similarly,intheskeletal
ofnerveagentintoxication(salivation,rhinorrhea,tremors,muscle muscle,GBandVXproducedChEinhibition(66–68%)thatwassig-
fasciculations,convulsions)at60minfollowingthe1.0×LD dose nificantlygreaterthanwasproducedbyGF(52%)orbyVR(39%).In
50
ofGB,GF,VRorVX.InthecaseofGBandGFexposure,3of21(14%) theheart,thefournerveagentsproducedChEinhibitionthatwas
and20of27(74%)animals,respectively,thatweretreatedwith significantlydifferentamongthem.TherankorderofChEinhibition
salinediedwithin60min.Oneof9animals(11%)exposedtoGF fromhightolowwasGB>VX>GF>VR.
andtreatedwithSAD128diedduringthisperiod.Oneof8(13%)
and 12 of 19 (63%) animals exposed to GB and GF, respectively, 3.3.1. Diaphragmtissue
andtreatedwithICD4157died.Noanimaldiedwhentreatedwith All oximes studied were capable of significantly reactivat-
anyoxime.InthecaseofVRandVXexposure,allanimalssurvived ing diaphragm ChE activity inhibited by GB. MMB-4 and HLö7
the 60min after exposure with or without oxime or non-oxime showed the highest levels of reactivation, returning ChE activ-
therapy. ityfrom25.7±2.2%to90.1±2.4%and80.1±1.4%ofthecontrol,
respectively. They reactivated GB-inhibited diaphragm ChE to a
3.2. ChEactivityinbrainregions significantlygreaterextentthandid2-PAM(to62.5%ofthecon-
trol). TMB-4 showed the least amount of reactivation with ChE
The striatum (389.3±9.2(cid:2)mol/(gproteinmin)) has the high- activityreachingonly46.4±3.1%ofthecontrol.OnlyMMB-4and
estandthecortex(56.9±1.4(cid:2)mol/(gproteinmin))hasthelowest HLö7 reactivated significantly diaphragm ChE activity inhibited
basal ChE activity among all brain regions. The rank order of byGF,returningChEactivityfrom35.3±3.3%to62.0±5.4%and
basal ChE activity from high to low was striatum>cerebellum> 57.2±1.4%ofthecontrol,respectively.TheyalsoreactivatedGF-
brainstem>spinal cord>midbrain>hippocampus>cortex. Nerve inhibitedChEsignificantlymorethandid2-PAM(to37.3%ofthe
agent-inhibited ChE activities in any of the brain regions were control).Carboxime,HI-6DiCl,MMB-4,and2-PAMweretheonly
notaffectedbytheoximetreatments,withtwoexceptions.Inthe four oximes tested that significantly reactivated diaphragm ChE
cortex,treatmentwithHLö7significantlyelevatedtheChEactiv- inhibited by VR, with ChE activity returning from 48.1±4.1% to
ityinhibitedbyVRfrom19.4±1.8%(mean±SEM)to26.6±2.5% 79.0±3.8%,78.8±2.3%,77.1±6.2%,and69.0±5.0%ofthecontrol,
of control. In the spinal cord, treatment with carboxime signifi- respectively. Following VX exposure, all oximes except ICD 585
cantlyelevatedtheChEactivityinhibitedbyVXfrom46.4±3.2%to reactivatedsignificantlyinhibiteddiaphragmChE.MMB-4showed
55.2±2.4%ofcontrol.Thenon-oximes(SAD128andICD4157)did thegreatestleveland2-PAMshowedtheleastamountofreactiva-
notaffectormodifytheChEactivitiesinhibitedbyanynerveagent tion,withChEactivityreturningsignificantlyfrom27.7±1.2%to
inanybrainregion. 56.0±4.3%and42.6±3.5%ofthecontrol,respectively.
Table1
Cholinesteraseactivityinperipheraltissueandbloodfollowingexposuretonerveagentsandtreatmentsa.
Agent Tissue Saline MMB-4 HLö7 Carboxime HI-6DMS HI-6DiCl 2-PAM
GB Diaphragm 25.7±2.2 90.1±2.4*,# 80.1±1.4*,# 61.4±3.0* 64.9±1.9* 62.1±2.9* 62.5±3.0*
Heart 15.0±1.5 56.2±4.2* 58.7±2.8* 56.0±1.5* 53.7±0.8* 49.4±2.7* 61.7±1.5*
Skeletalmuscle 34.2±3.6 88.7±3.2* 104.8±3.8*,# 52.7±2.1 69.8±7.4* 78.9±4.0* 70.2±2.8*
RBC 8.7±1.9 75.3±4.2*,# 52.1±1.8* 89.8±4.3*,# 83.1±5.0*,# 76.8±2.4*,# 52.5±3.5*
WB 8.8±1.3 83.1±4.6*,# 66.8±3.9* 74.8±1.4* 69.2±4.7* 56.9±3.1* 61.8±4.0*
GF Diaphragm 35.3±3.3 62.0±5.4*,# 57.2±1.4*,# 49.9±2.5 36.6±2.2 44.3±5.7 37.3±3.0
Heart 34.7±2.9 52.3±5.6* 43.5±2.7 48.1±2.2 49.0±3.2 45.6±3.7 41.7±3.4
Skeletalmuscle 48.3±4.1 75.5±4.6*,# 83.7±3.8*,# 50.5±6.1 42.9±4.8 58.5±6.5 55.5±3.4
RBC 5.9±1.1 44.8±4.5*,# 24.1±1.7* 54.3±2.1*,# 55.0±5.1*,# 44.1±4.6*,# 11.0±1.5
WB 16.6±2.7 47.0±2.0*,# 45.1±4.9*,# 44.1±2.7*,# 46.7±4.3*,# 41.8±3.0*,# 20.1±1.0
VR Diaphragm 48.1±4.1 77.1±6.2* 59.4±5.2 79.0±3.8* 63.5±4.7 78.8±2.3* 69.0±5.0*
Heart 54.9±2.0 97.8±6.0*,# 74.6±4.8* 96.9±4.0*,# 82.0±4.8* 69.3±2.7 68.9±2.5
Skeletalmuscle 60.9±4.0 81.1±6.0*,# 63.4±5.2 77.6±4.4 79.7±4.6 95.1±3.0* 92.7±3.3*
RBC 6.6±1.2 81.2±9.8*,# 58.4±3.2*,# 91.9±2.8*,# 45.6±3.3*,# 39.8±2.4*,# 6.6±2.0
WB 30.8±3.6 80.5±2.2*,# 89.1±5.4*,# 89.3±0.8*,# 73.6±9.5*,# 73.2±4.1*,# 50.2±4.7
VX Diaphragm 27.7±1.2 56.0±4.3* 48.0±3.5* 56.2±1.1* 50.3±2.1* 44.3±2.8* 42.6±3.5*
Heart 23.6±1.1 54.6±3.9* 63.0±3.6*,# 62.5±2.1*,# 44.2±1.3* 38.9±1.4* 44.1±3.8*
Skeletalmuscle 32.1±2.4 56.1±3.4*,# 56.7±3.7* 49.1±1.8* 51.9±2.3* 46.3±3.0* 46.6±2.2*
RBC 6.3±1.0 83.9±2.1*,# 84.4±2.6*,# 84.7±3.4*,# 56.8±2.3*,# 44.3±2.4* 37.7±4.7*
WB 26.8±2.1 82.7±1.9*,# 66.4±4.2*,# 78.2±2.1*,# 56.3±1.6* 46.2±1.7* 43.5±3.2*
a EffectsofnerveagentandsalineoroximetreatmentsonperipheraltissueChEactivityintheguineapig.Salineoroximecompoundswasgivenintramuscularly5min
aftera1.0×LD50subcutaneousdoseofGB,GF,VRorVX.Sampleswerecollectedat60minafternerveagent.ChEactivityindiaphragm,heart,andskeletalmusclewas
expressedaspercentofcontrolChEactivityforeachtissue.ChEactivityinRBCandWBwasexpressedaspercentofindividualbaselineactivitythatwasobtained1–3days
priortoexperiment.TotalChEactivitywasexpressedasmean±SEM(%ofcontrolgroup)with6–8animalspergroup.
* Significantlydifferentfromsalinetreatment,p<0.05.
# Significantlydifferentfrom2-PAMtreatment,p<0.05.
T.-M.Shihetal./Chemico-BiologicalInteractions187 (2010) 207–214 211
Thenon-oximes(SAD128andICD4157)didnotmodifytheChE 26.8±2.1%ofcontrolvalue,respectively.BothGBandGFproduced
activitiesinhibitedbyanynerveagentinthediaphragm,withone significantlygreaterChEinhibitionthandidVRandVX.Thelatter
exception.TheChEactivityshowedanelevationfrom25.7±2.2% twonerveagentsshowedasimilardegreeofChEinhibition.
to39.8±5.0%ofthecontrolinanimalsexposedtoGBandtreated The non-oximes (SAD128 and ICD4157) did not significantly
withSAD128. modifytheChEactivitiesinhibitedbyanynerveagentinanyblood
samples.
3.3.2. Hearttissue
AlloximeswerecapableofsignificantlyreactivatingChEactiv- 3.4.1. Redbloodcell(RBC)
ityinhibitedbyGBintheheart.2-PAMshowedthegreatestand Allnineoximesstudiedwerecapableofsignificantlyreactivat-
TMB-4theleastamountofreactivation,withChEreturningfrom ing RBC ChE activity inhibited by GB. ICD692 (90.2% of control),
15.0±1.5% to 61.7±1.5% and 32.9±0.9% of the control, respec- carboxime(89.8%ofcontrol),HI-6DMS(83%ofcontrol),HI-6DiCl
tively.FollowingGFexposure,onlyMMB-4reactivatedsignificantly (77%ofcontrol),ICD585(76%ofcontrol),andMMB-4(75%ofcon-
inhibited heart ChE with activity returning from 34.7±2.9% to trol) were capable of reactivating GB-inhibited RBC ChE activity
52.3±5.6%ofthecontrol.MMB-4,carboxime,HLö7,HI-6DMS,and significantly more than 2-PAM (53% of control). TMB-4 showed
ICD692werecapableofsignificantlyreactivatingheartChEinhib- the least amount of reactivation with enzyme activity returning
itedbyVR.MMB-4andcarboximeexhibitedthehighestlevelsof from 8.7±1.9% to 29.0±1.3% of the control. All oximes except
reactivation following VR exposure, returning ChE activity from 2-PAMandTMB-4significantlyreactivatedRBCChEactivityinhib-
54.9±2.0% to 97.8±6.0% and 96.9±4.0% of the control, respec- itedbyGF.HI-6DMSandcarboximeshowedthehighestlevelsof
tively.TheyreactivatedVR-inhibitedChEsignificantlymorethan reactivation,returningChEactivityfrom5.9±1.1%to55.0±5.1%
did2-PAM(to68.9±2.5%ofthecontrol).HLö7showedtheleast and54.3±2.1%ofthecontrol,respectively.HLö7showedtheleast
significantamountofreactivationinVR-inhibitedheartChE,where amountofreactivationwithChEactivityreturningto24.1±1.7%
activityreturnedto74.6±4.8%ofthecontrolvalue.OnlyICD585 of the control. All oximes except 2-PAM and ICD585 were capa-
was not capable of reactivating heart ChE activities inhibited by bleofsignificantlyreactivatingRBCChEactivityinhibitedbyVR.
VX. HLö7 showed the greatest and HI-6 DiCl the least signifi- CarboximeandMMB-4showedthehighestlevelsofreactivation
cantamountofreactivationwithVX-inhibitedChEreturningfrom withChEreturningfrom6.6±1.2%to91.9±2.8%and81.2±9.8%
23.6±1.1% to 63.0±5.6% and 38.9±1.4% of the control, respec- of the control, respectively. TMB-4 showed the least amount of
tively. HLö7 and carboxime (to 62.5% of the control) reactivated reactivationamongtheseoximeswithChEactivityreachingonly
heartChEsignificantlymorethandid2-PAM(to44.1%ofthecon- 27.5±4.7% of the control. All oximes significantly reactivated
trol). RBC ChE activity inhibited by VX. MMB-4, carboxime, HLö7, and
Thenon-oximes(SAD128andICD4157)didnotmodifytheChE ICD3805showedexcellentlevelsofreactivationwithChEactivity
activitiesinhibitedbyanynerveagentintheheart. returning from 6.3±1.0% to 83.9±2.1%, 84.7±3.4%, 84.4±2.6%,
and 83.9±2.7% of the control, respectively. HI-6 DMS showed
3.3.3. Skeletalmuscletissue the least amount of reactivation, with ChE activity returning to
Alloximesstudied,withtheexceptionofcarboximeandTMB- 56.8±2.3%ofthecontrol.
4, were capable of significantly reactivating skeletal muscle ChE
activityinhibitedbyGB.HLö7showedthegreatestlevelofreacti- 3.4.2. Wholeblood(WB)
vationwithChEactivityreturningfrom34.2±3.6%to104.8±3.8% AlloximesstudiedsignificantlyreactivatedChEactivityinhib-
ofthecontrol.ICD3805andMMB-4alsodisplayedexcellentlev- ited by GB. MMB-4 showed the greatest and TMB-4 showed the
elsofreactivation,withChEreaching89.7±3.6%and88.7±3.2%of leastamountofreactivationwithWBChEactivityreturningfrom
thecontrol,respectively.OnlyMMB-4andHLö7reactivatedsignif- 8.8±1.3%to83.1±4.6%and50.5±2.7%ofthecontrol,respectively.
icantlyskeletalmuscleChEactivityinhibitedbyGF,withtheChE MMB-4 reactivated GB-inhibited WB ChE activity significantly
activityreturningfrom48.3±4.1%to75.5±4.6%and83.7±3.8% morethandid2-PAM(to61.8%ofthecontrol).Alloximesexcept2-
of the control, respectively. MMB-4, HI-6 DiCl and 2-PAM were PAM,ICD585,andTMB-4werecapableofsignificantlyreactivating
theonlythreeoximestestedthatsignificantlyreactivatedskele- WBChEactivityinhibitedbyGF.MMB-4showedthegreatestand
talmuscleChEactivityinhibitedbyVR.HI-6DiClshowedgreater ICD692showedtheleastamountofreactivationwithChEactiv-
reactivation than did 2-PAM and MMB-4, returning ChE activity ityreturningfrom16.6±2.7%to47.0±2.0%and36.7±2.4%ofthe
from60.9±4.0%to95.1±3.0%,92.7±3.3%,and81.1±6.0%ofthe control,respectively.Alloximesexcept2-PAMandICD585signifi-
control,respectively.AlloximesexceptICD585significantlyreac- cantlyreactivatedWBChEactivityinhibitedbyVR.Carboximeand
tivatedskeletalmuscleChEactivityinhibitedbyVX.HLö7showed HLö7showedthehighestlevelsofreactivationsuchthatChEactiv-
thegreatestandHI-6DiClshowedtheleastamountofreactiva- ityreturnedfrom30.8±3.6%to89.3±0.8%and89.1±5.4%ofthe
tionwithChEactivityreturningfrom32.1±2.4%to56.7±3.7%and control,respectively.ICD692showedtheleastamountofreactiva-
46.3±3.0%ofthecontrol,respectively.Thenon-oximes(SAD128 tionamongtheseoximeswithChEactivityreturningto63.7±4.7%
andICD4157)didnotmodifytheChEactivitiesinhibitedbyany ofthecontrol.
nerve agent in the skeletal muscle, with one exception. The ChE AlloximessignificantlyreactivatedWBChEactivityinhibitedby
activities showed an elevation from 34.2±3.6% to 57.4±8.2% of VX.MMB-4andcarboximeshowedthehighestlevelsofreactiva-
thecontrolinanimalsexposedtoGBandtreatedwithSAD128. tionwithChEactivityreturningfrom26.8±2.1%to82.7±1.9%and
78.2±2.1%ofthecontrol,respectively.HI-6DiClshowedtheleast
3.4. ChEactivityinblood(Table1) amountofreactivationwithareturninChEactivityto46.2±1.7%
ofthecontrol.
The basal ChE activities in RBC and WB are 2.2±0.06 and
2.5±0.06(cid:2)mol/(mlmin), respectively. The ChE activities in RBC 3.5. Overallassessmentofoximereactivatingcapacity
weremarkedlyinhibited60minfollowingGB,GF,VR,andVXto
8.7±1.9%, 5.9±1.1%, 6.6±1.2%, and 6.3±1.0% of control value, Table2AandBsummarizedthestatisticallysignificantChEreac-
respectively.Thus,allfournerveagentsproducedasimilardegree tivationbyoximetreatmentsintheperipheraltissuesandtheblood
of RBC ChE inhibition. In the WB, however, GB, GF, VR and VX components,respectively.ItisstrikingtonoticethatMMB-4isthe
reducedenzymeactivityto8.8±1.3%,16.6±2.7%,30.8±3.6%,and onlyoximecapableofsignificantlyreactivatingChEinhibitedby
212 T.-M.Shihetal./Chemico-BiologicalInteractions187 (2010) 207–214
Table2
SummaryofthesignificantChEreactivationbyoximetreatmentsinperipheraltissuesandbloodcomponentsfollowingexposuretoGB,GF,VR,andVXa.
A.Peripheraltissues(diaphragm,heart,skeletalmuscle)
MMB-4 HLö7 Carboxime HI-6DMS HI-6DiCl 2-PAM ICD585 ICD692 ICD3805 TMB-4
GB +++ +++ ++ +++ +++ +++ +++ +++ +++ ++
GF +++ ++ − − − − − − − −
VR +++ + ++ + ++ ++ − + − −
VX +++ +++ +++ +++ +++ +++ − +++ +++ +++
B.Blood(redbloodcellsandwholeblood)
MMB-4 HLö7 Carboxime HI-6DMS HI-6DiCl 2-PAM ICD585 ICD692 ICD3805 TMB-4
GB ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
GF ++ ++ ++ ++ ++ − + ++ ++ −
VR ++ ++ ++ ++ ++ − − ++ ++ ++
VX ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
a Foreachoftheperipheraltissues(A)orbloodcomponents(B),anoximetreatmentthatsignificantlyreactivatedanerveagent-inhibitedChEactivitywasassigneda“+”
sign.Whenanoximetreatmentdidnotsignificantlyreactivateanerveagent-inhibitedChEinanytissue,itwasassigneda“−”sign.
allfournerveagentsinallthreeperipheraltissues.HLö7wasalso inbloodandanyperipheraltissuetested,orVR-inhibitedChEin
capableofreactivatingChEinhibitedbythesefournerveagents, bloodandtheheart.Ontheotherhand,althoughtheotherthree
butwithaweakeractiontowardVR-andGF-inhibitedtissueChE ICDcompounds(ICD585,ICD692,andICD3805)were,ingeneral,
activity. The reactivating potency of carboxime, HI-6 DMS, HI-6 abletoreactivateChEinthebloodinhibitedbyallfournerveagents,
DiCl,2-PAMandICD692appearstobeequal.Theyalllackedthe these compounds lacked the capability to do so on GF- and VR-
abilitytoreactivateGF-inhibitedChEintheperipheraltissues.The inhibitedChEinanyperipheraltissues.Overall,ICD585wasnot
leasteffectiveoxime,ICD585wasnotabletoreactivateperiph- abletoreactivateGF-,VR-,andVX-inhibitedChE,butwasableto
eraltissueChEactivityinhibitedbyGF,VRorVX.TherelativeChE onlyreactivateGB-inhibitedChEinallthreeperipheraltissues.
reactivatingpotencyoftheseoximecompoundsagainstthefour Thepresentresultsalsoshowedthattheseoximeswereableto
nerveagentswasGB>VX>VR>GF.TheDiClandDMSsaltsofHI-6 reactiveChEinhibitedbyfourdifferentnerveagentsinperipheral
exhibitedsimilarpotencyagainstGB,GF,VRandVX(Table1). tissuestodifferentdegrees(Table2).Therankorderofsusceptibil-
ityforinvivoChEreactivationbyoximetreatmentsinperipheral
4. Discussion tissueswasGB>VX>VR>GF.
It is interesting to note that the capability of an oxime treat-
AswasreportedearlierOPnerveagentsproducedtissuecom- ment to reactivate ChE in the blood and the peripheral tissue
partmentspecificityintheirabilitytoinhibitChEactivity[20];the compartmentswasmarkedlydifferent.Itwasunderstandablethat
oximesstudiedhere(i.e.,2-PAM,HI-6,HLö7,MMB-4,TMB-4,car- theoximecompoundenteredthebloodfirstbeforereactinginthe
boxime,ICD585,ICD692,andICD3805)alsoexhibiteddifferential tissue compartment, and thus, it had a better chance to act on
potencyinreactivatingnerveagent-inhibitedtissueChEactivity. theChEinhibitedintheblood(i.e.,proximityeffect).Therefore,it
When examined carefully the results obtained from the present wasobservedthatmostoftheoximeswerecapableofreactivating
study showed that MMB-4 was the only oxime (among 9 oxime nerveagent-inhibitedChEinthebloodtoagreaterdegreethanin
compoundstested)capableofreactivatingChEinhibitedbyallfour theperipheraltissues.Forexample,withtheexceptionof2-PAM
nerve agents in all three peripheral tissues and the blood com- andTMB-4,alloximestesteddisplayedsignificantChEreactivation
ponents. Additionally, in several cases, ChE activities in animals inthebloodcomponents,whileintheperipheraltissueonlyMMB-
treated with MMB-4 reached control values: in diaphragm and 4andHLö7werecapableofdoingso.Furthermore,themajorityof
skeletalmuscleafterGBandinheartafterVRexposures.HLö7was theoximesstudiedwerecapableofreactivatingChEinhibitedby
secondinitsoverallreactivatingcapacity.InRBCandWBitreac- thefournerveagentsinthebloodcomponentssignificantlybetter
tivatedChEinhibitedbyallfournerveagents.SkeletalmuscleChE thanwas2-PAM.Intheperipheraltissue,however,onlyMMB-4,
activitiesinanimalstreatedwithHLö7afterGBexposurereturned HLö7,orcarboximedisplayedsignificantlybetterChEreactivating
tocontrolvalue.AlthoughHLö7wascapableofreactivatingChEin capacitythandid2-PAM,butonlyinafewtissues.Therewasno
allthreeperipheraltissuesinhibitedbyGBandVX,itlackedthe relationbetweenbloodandperipheraltissuesinthereactivating
ability to reactivate ChE inhibited in the diaphragm and skeletal efficacyofoximetreatments.Therefore,ahigherbloodChEactivity
musclebyVRandChEinhibitedintheheartbyGF.Carboximereac- followingoximetreatmentinnerveagentpoisoningcouldnotbe
tivatedChEinRBCandWBinhibitedbyallfournerveagentsand usedasadirectindicatorofelevatedChEactivityinaperipheral
wascapableofreactivatingVX-inhibitedChEinallthreeandGB- tissue(diaphragm,heart,orskeletalmuscle).
inhibitedChEintwoperipheraltissues.ItreturnedtheChEactivity Itisalsointerestingtopayattentiontoanissuebroughtupby
inthehearttothecontrolvalueafterVRexposure.However,car- a few in vitro studies in recent years [23–25] using RBC ghosts
boximewasnotabletoreactivateGF-inhibitedChEindiaphragm, as AChE sources, which have demonstrated that oxime reactiva-
heart,andskeletalmuscle,andVR-inhibitedChEinskeletalmus- tion of guinea pig AChE in vitro is significantly slower than that
cle.HI-6showedacompletenerveagentpreference.Itwascapable ofhumanormonkeyAChEs,especiallyinthereactivationofGF-,
ofreactivatingGB-,VR-andVX-inhibitedChE,butnotGF-inhibited soman-,orVR-inhibitedenzymebyHI-6andHLö7.However,these
ChE,inbloodortissues.TherewasnodifferenceinChEreactivating in vitro experiments [24,25] were conducted with a pH of 8.0 at
capacitybetweenthetwosaltsofHI-6followingexposuretothe 25◦C, which is not the optimal physiological condition in living
fournerveagentsstudied.Similarresultswerereportedbylabora- animals.Therefore,theseresultsarenoteasilyreconcilablewith
toryinaninvitrostudy[16].2-PAMwasanexcellentreactivator our in vivo guinea pig data. Even the study of Worek et al. [23]
ofChEactivityinhibitedbyGBandVXinblood,andinallthree thatwasperformedatapHof7.4at37◦C,whoseinvitrokinetic
peripheraltissues.ItwasnotabletoreactivateGF-inhibitedChE dataobtainedfromguineapigRBCghostsweremarkedlydifferent
T.-M.Shihetal./Chemico-BiologicalInteractions187 (2010) 207–214 213
fromcurrentinvivoChEreactivationdataobtainedfromguineapig TamiRowland.ThisresearchwassupportedbytheDefenseThreat
RBC.Forexample,invitroreactivationkineticdataindicatedthat Reduction Agency—Joint Service and Technology Office, Medical
GB-inhibitedghostAChEwouldrespondlesstoHLö7,HI-6and2- ScienceandTechnologyDivision.
PAM,inparticulartoHI-6,whiletheinvivoresultsshowedthat
HI-6wasthemosteffectiveRBCAChEreactivatorwhencompared
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