Measuring Rate and Equilibrium Constants of β‐Cyclodextrin ......is an equilibrium constant which...
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Measuring Rate and Equilibrium Constants of β ‐Cyclodextrin‐Small Molecule Drug Non‐Covalent Interactions with Capillary Electrophoresis By Jennifer Logie Honors thesis submitted to the Faculty of Science Department of Chemistry In partial fulfillment of the requirements of the B. Sc. Degree Supervisor: Maxim Berezovski Department of Chemistry University of Ottawa Ottawa, Canada © April 2011, Jennifer Logie
Transcript of Measuring Rate and Equilibrium Constants of β‐Cyclodextrin ......is an equilibrium constant which...
MeasuringRateandEquilibriumConstantsofβ‐Cyclodextrin‐SmallMoleculeDrug
Non‐CovalentInteractionswithCapillaryElectrophoresis
By
JenniferLogie
HonorsthesissubmittedtotheFacultyofScienceDepartmentofChemistryInpartialfulfillmentoftherequirementsoftheB.Sc.Degree
Supervisor:MaximBerezovski
DepartmentofChemistry
UniversityofOttawa
Ottawa,Canada
©April2011,JenniferLogie
ii
Abstract
Cyclodextrinisacircularoligosaccharidemadeof6‐8glucosemonomers,givingtheideal
conformationtoforminclusioncomplexeswithmanydifferentsmallmolecules.Inthe
pharmaceuticalindustry,itiscommonlyusedasanexcipienttoincreasethesolubilityofsmall
moleculedrugs.Thepurposeofthisresearchwastomeasureequilibriumandrateconstants
betweensmallmoleculesandcyclodextrinsusingtheEquilibriumCapillaryElectrophoresisof
EquilibriumMixtures(ECEEM)method.InECEEM,anequilibriummixtureofsmallmolecule,
cyclodextrinandcomplexisinjectedintoacapillaryandsubjectedtoelectrophoreticseparation.
Therunningbuffercontainsthesameconcentrationofcyclodextrinasintheequilibriummixture.
Bindingparametersofcomplexescanbefoundbyusingthetimepropagationpatternandshapes
ofthepeaksobtainedwhentheconcentrationofcyclodextrinisgraduallyincreased.Inthisstudy,
eightsmallmoleculedrugs:ibuprofen,s‐flurbiprofen,4,4’‐(propane‐1,3‐diyl)dibenzoicacid,
resveratrol,naproxen,diclofenac,folicacidandphenylbutazonewerestudiedfortheir
complexationwithβ‐cyclodextrin.TheECEEMmethodprovedtobeavaluabletechniquefor
determiningthepracticalityofcyclodextrinasadrugdeliverysystemforspecificdrugs.
iii
Acknowledgements
WithoutthehelpandsupportoftheBerezovskilab,thisworkcouldnothavebeencompleted.
Particularlythesupervision,guidanceandinsightofDr.MaximBerezovski;themathskillsofDr.
VictorOkhonin;andfinallytheinstructionandhelpofGlebMironov.
iv
TableofContents
ListofFigures......................................................................................................................................v
StatementofContribution................................................................................................................vi
1.Introduction....................................................................................................................................11.1Rationale.................................................................................................................................................11.2CyclodextrinasaDrugDeliveryAgent....................................................................................................21.3BindingConstantsofCyclodextrin‐SmallMoleculeComplexes..............................................................51.4LiteratureReviewofKDDeterminationMethods...................................................................................61.5PrinciplesofCapillaryElectrophoresis....................................................................................................9
2.ResultsandDiscussion..................................................................................................................132.14,4’(Propane‐1,3Diyl)DibenzoicAcidModel......................................................................................132.2MultiplexApplication............................................................................................................................23
3.References....................................................................................................................................31
4.Experimental..................................................................................................................................334.1ChemicalsandMaterials.......................................................................................................................334.2ExperimentalConditions.......................................................................................................................34
v
ListofFigures
1.StructuresofSmallMoleculeDrugs................................................................................................3
2.β‐cyclodextrinStructureandInclusionComplexation....................................................................4
3.SchematicofECEEMSet‐up..........................................................................................................11
4.LimitofDetectionofPDDAElectropherogram.............................................................................15
5.DegradationofPDDAElectropherogram......................................................................................17
6.PDDAwithvaryingβ‐cyclodextrinconcentrationsElectropherogram.........................................18
7.VelocityofEquilibriumMixtureasaFunctionofβ‐cyclodextrinConcentration..........................20
8.ECEEM‐MSSchematic...................................................................................................................22
9.3‐DAbsorptionPlotsofSmallMolecules......................................................................................24
10.2‐Dand3‐DAbsorptionPlotsofMultiplex.................................................................................25
11.Multiplexwithvaryingβ‐cyclodextrinconcentrationsElectropherogram.................................27
12.MultiplexKDdeterminationGraphs...........................................................................................29
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StatementofContribution
Dr.SergeGorelskydidtheDFTcalculationstodeterminethemodeloftheinclusioncomplex
betweenβ‐cyclodextrinandibuprofen.VictorOkhonincreatedthemathematicalmodelusedto
calculatebindingparametersforthePDDA‐β‐cyclodextrininclusioncomplex.GlebMironovdidall
CE‐MSworkthatisreferencedinSection2.
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1.Introduction
1.1Rationale
Pharmaceuticalcompaniesspendbillionsofdollarseachyearresearchingnewandexcitingdrug
candidates.Gettingthesedrugsintothebodyisanongoingbattleduetotheoften‐lowsolubility
ofthesemolecules.Oftendrugsareformulatedwithexcipients,whichusevariousmethodsto
increasethebioavailabilityofthedrugs(1).Inthisstudy,severalcommondrugsonthemarketwill
beusedtocreateamodelfortheevaluationofdrugdeliverysystems.
Someoftheoldestandmostcommonlyuseddrugsavailablearethenon‐steroidalanti‐
inflammatoryclass(NSAIDS).Althoughthestructuresofthesedrugsvary,theyallserveas
analgesicsandantipyretics,andathighdosesmayhaveanti‐inflammatoryeffects.Theyworkby
non‐specificallyinhibitingtheenzymecyclooxygenase,whichpreventstheproductionof
prostaglandinsandthromboxanesfromarachidonicacid(2).Naproxen,Ibuprofen,andS‐
FlurbiprofenallbelongtotheclassofNSAIDsderivedfrompropionicacid.Diclofenacisanacetic
acidderivative.PhenylbutazoneisanNSAIDderivedfromenolicacidthatisnotcommonlyusedin
humans,howeveritisveryprevalentinveterinarymedicine(2).
TheotherdrugsthatwerestudieddonotbelongtotheNSAIDclass,butwerechosentoshowthe
potentialdiversityofthemethod.FolicAcidisanessentialvitaminfortetrahydrofolateproduction
inthebody,anddietsupplementsareusuallyrecommendedaspartofapre‐natalvitamin
regimen(3).Resveratrolisacommonantioxidantfoundinmanyfoodproducts,andiscurrently
beingtestedforitsvaryinghealthbenefits(4).Finally,4,4’‐(Propane‐1,3‐Diyl)DibenzoicAcid
(PDDA),asmallmoleculewhichdoesnotcurrentlyhaveaclinicaluse,wasselectedduetoits
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structuralpropertieswhichwillbedescribedindetailinSection2.1.Alldrugstructuresarefound
inFigure1.
1.2CyclodextrinasaDrugDeliveryAgent
Theexcipientsusedtoincreasethesolubilityofdrugsvarywidely,inthisstudy,onesuch
excipient,cyclodextrin,willbediscussed(5).Cyclodextrinsarecyclicoligosaccharidesmadeof6‐8
glucosemonomers,withmolecularweightsrangingbetween1000and2000g/mol.Theyforma
toroid‐truncatedcone‐likestructureduetothechairformationoftheglucopyranoseunits(6).
Thehydroxylgroupsareattheexteriorofeitherendofthecone,makingtheexteriorofthe
moleculeveryhydrophilic.Theinteriorofthecyclodextrinisslightlyhydrophobic,makingitan
excellentcavityforhydrophobicsmallmoleculestoenterandformacomplex(6).Theformation
ofthisinclusioncomplexsignificantlyincreasesthewatersolubilityofthedrug,andallowsittobe
transportedthroughthebodytothelipidcellmembraneofthedrugstarget(6).Thestructureof
β‐cyclodextrinandaninclusioncomplexwithibuprofenareshowninFigure2.Thelarge
hydrophiliccyclodextrinmoleculeisnotabletopenetratethroughthehydrophobiccell
membrane,soitisessentialthattheformedinclusioncomplexesareinarapidequilibriumwith
thefreedrug.Thisenablesthefreedrugtogothroughthemembraneandreachitstarget(6).
Differentcyclodextrins(α,βorγ)areuseddependingonthesizeofthesmallmolecule,aseach
cyclodextrinhasadifferentsizedinternalcavity.
Currentlycyclodextrinsareonthemarketforawiderangeofapplications.Thisincludes
householdproducts(suchasFebreeze),cosmetics,andfoods‐wheretheyarenotablyableto
encapsulateflavormolecules.Themostprevalentpharmaceuticalapplicationsareinnasalsprays
andeyedrops,andthereareover30pharmaceuticalproductsonthemarketwhichuse
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cyclodextrinasanexcipient(6).Inthecaseofsmallmoleculedrugswithinthemolecularweight
rangeof100‐400g/mol,beta‐cyclodextrin,whichhassevenglucopyranoseunits,isthebestdrug
deliverymolecule.Thisisduetoaninternalcavitydiameterof5.3Åatthebottomand7.0Åatthe
top(7).Thiscavitydiametermeansthereisgoodmolecularsizeandshapecomplementationto
formastablecomplex(7).ADFTcalculatedmodeldonewithibuprofenshowedthatthe
cyclodextrinholdsthedruginsidethecavitybytwohydrogenbondsbetweenaterminalcarboxyl
groupofibuprofenandtwohydroxylgroupsonthebottomofthecyclicoligosaccharide(7).
1.3BindingConstantsofCyclodextrin‐SmallMoleculeComplexes
Inordertoevaluatethepracticalityofcyclodextrinasadrugdeliverysystemforaparticulardrug,
informationaboutthenatureofthecomplexandthebindingconstantsisnecessary.
Determinationofthedissociationconstant(KD)ofcyclodextrin‐smallmoleculeinclusion
complexesallowsforbettercharacterizationofthecomplex.TheKDisanequilibriumconstant
whichmeasuresthepropensityofacomplextofallapartintosmallercomponents.Forthe
generalreaction
AB A+B
Thedissociationconstantisrepresentedbytheequation:
𝐾! =𝐴 𝐵𝐴𝐵
Itcanalsobeexpressedastheratiooftheon‐rateandtheoff‐rate,whosevaluesgiveevenmore
informationaboutthenatureofthecomplex.
𝐾! =!!""!!"
6
DeterminingtheKDofthenon‐covalentcomplexesbetweencyclodextrinsandsmallmoleculeshas
beenwellstudied,andgenerallyanymethodthatallowstheobservationofchangesin
physiochemicalpropertiesthathappenwithcomplexationcanbeusedtoquantifythedissociation
constant.Althoughthemethodsshouldgiveconsistentresults,oftenthereisalotofdiscrepancy.
Methodsthathavebeenprevalentintheliteraturearesummarizedinthefollowingpages.
1.4LiteratureReviewofKDDeterminationMethods
NuclearMagneticResonance(NMR)hasbeenusedforthecharacterizationofprotein‐ligandnon‐
covalentcomplexestocalculateKD(8),howevernotmuchworkhasbeendonecalculatingthe
bindingconstantsofcyclodextrin‐smallmoleculeinteractions.Theworkthathasbeendone
generallylooksat1Hchemicalshiftchangesofthebeta‐cyclodextrinandcalculatestheKDbased
ontheHildebrand‐Benesimodel(9).Thismodelworksonthebasisthatoneofthereactantsis
presentinexcessamountsoftheother,andyoucanonlytakethatoneintoaccount.Italsoworks
solelyforcomplexeswitha1:1stereochemistry(9).OneotherstudyusingNMRcalculatedtheKD
onthebasisofliganddissociationkinetics.Inthiscase,itmadetheassumptionthattherateof
association,kon,isdiffusionlimitedandequalto1x109M‐1sec‐1.Itthenmonitoredthereaction
byquenchingthereactionatvarioustimepointsandtakinganNMRspectra.Bydoingalineshape
analysisofindividualNMRpeaks,theycalculatedtherateofdissociation,koff,anddeterminedaKD
basedonthisvalue(8).UsingNMRtofindthesebindingparametersmakesalotofassumptions.
Firstly,itisnotalwaysthecasethattheβ‐cyclodextrinisinexcessofthesmallmolecule.Secondly,
inthecaseofcalculatingtheKDonthebasisofliganddissociationkinetics,itislargelyincorrectto
assumetherateofassociationisdiffusionlimited.Finally,youcannotassumethatformed
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complexeswillalwayshave1:1stoichiometry,especiallyathighconcentrations,whenaggregation
ismorelikelytooccur(8).
OneofthemostpopularmethodsfordeterminationoftheKDisequilibriumdialysis(11).Like
NMR,thishasbeenwidelystudiedforprotein‐ligandinteractionsandexamplesarelimitedforits
useincyclodextrin‐smallmoleculeinteractions(10).Thismethodreliesontheuseofamembrane
thattheligandcanpassthroughbutthereceptorcannot.Ononesideofthemembranethe
receptorisplaced,whileontheotherthereisaknownconcentrationoftheligand.Thesystemis
thenallowedtoreachequilibrium.Thehighertheaffinitytheligandhasforthereceptor,the
highertheconcentrationofligandwillbebound.Atequilibrium,theconcentrationofthefree
ligandwillbethesameonbothsidesofthemembrane.Knowingtheoriginalconcentrationused,
enoughinformationcanbeobtainedinordertocalculatetheKD.Althoughthismodelisvery
practicalbecauseofitssimplicity,likeNMRitcanbehinderedbytheassumptionsitmakes.The
modelassumesthatallreceptorsareequallyaccessibletoligands,thatreceptorsareeitherfreeor
boundtoaligand,thatbindingdoesnotaltertheligandorreceptor,andthatthebindingis
reversible(11).Despitetheseassumptions,equilibriumdialysisremainsoneofthemostaccurate
methodsforKDdetermination(10).
Phase‐solubilitydiagramshavebeenextensivelyusedforstudyingthebindingwithcyclodextrins
(12).ThismethodwasdevelopedbyHiguchiandConnorsandisbasedonhowcomplexation
affectstheaqueoussolubilityofdrugs(13).Plottingthedrugsolubilityagainsttheconcentration
ofcyclodextrincreatesaphasesolubilitydiagram.Thenoveltyofthismethodcomparedtothose
previouslymentionedisthatitgivesinformationregardingthestoichiometryofthecomplex
throughthelinearityofthecurve(14).Ifitisverylinear,itsuggeststhatthecomplexisfirstorder
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withrespecttoboththedrugandcomplexingagent.Ifthestoichiometryisgreaterthan1:1,it
becomesmoredifficulttocalculateanaccurateKD,butimpliesahigherorderofcomplexation(14).
Themaindownsideofthephase‐solubilitydiagramapproachisthelargequantitiesofcyclodextrin
anddrugrequiredinordertomeasurethesolubility.
Amoreefficientmethodtoevaluatetheinteractionisasurfaceplasmonresonanceassay(15).
Thisassayworksbyimmobilizingligands,inthiscaseβ‐cyclodextrin,onagoldsurfaceand
monitoringthechangesinrefractiveindex.Themagnitudeofthechangeisdirectlyproportional
tothemasschangeatthesurface(15).Monitoringthesignalproducedinrealtimeallowsforthe
calculationofassociationanddissociationrates(usedtocalculatedthedissociationconstant)and
canalsoshowthestoichiometry(16).Thismethodisstillbeingoptimizedandimprovedforuse
withcyclodextrins,particularlyobtainingbettercouplingefficiencyofthecyclodextrintothe
immobilizedsurface,andincreasingthesensitivityoftheassay,asaddingasmallmolecular
weightdrugtoalargemolecularweightcyclodextringivesonlyasmallchangeinsignal(16).
Stopped‐flowisafrequentlyemployedtechniquetostudythekineticsofareaction,particularly
whenlookingatenzymes.Fewstudieshavebeendoneusingstoppedflowtolookatthebinding
ofcyclodextrins,butthiscouldbeduetothenatureoftheequilibrium(17).Cyclodextrin‐small
moleculecomplexesareinarapidequilibrium,andtheratesareveryfast.Themixingdead‐time
andre‐dissociationofthereagentsmeansthattheserapidratesarenotmeasured(7).Thevalues
obtaineddonotrepresentthesystematequilibrium(7).
Electrosprayionizationmassspectrometry(ESI‐MS)isausefultoolforworkingwith
macromoleculesandnon‐covalentcomplexes,asitionizesthemolecules/complexeswithout
fragmentingthem(18).ThegreatestadvantageofESI‐MSisthatitmakesitpossiblytodirectly
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obtainstoichiometricinformation.Byevaluatingthepresenceofcomplex,freedrug,andfree
cyclodextrinusingtheintensitiesoftheirrespectivem/zpeaks,andcalculatingaresponsefactor
tocorrespondtheintensitiestotheconcentrations,theKDcanbecalculated(19,20).Ithaslargely
beenthroughMSthattheaggregationofcyclodextrininsolutionhasbeenverified(18).Although
ESI‐MSelegantlycalculatestheKDbasedontheintensities,thestabilityoftheionizationisahuge
problem,andleadstoalotoferrorintheresult(19).Thebehaviorofthenon‐covalentcomplexes
inthegasphasemaynotrepresentwhatishappeninginsolution(18).Althoughitsusefulnessfor
identificationofcomplexesisnotable,adirectcorrespondencebetweenintensityand
concentrationleavesmuchtobedesired.
AllofthesemethodstodeterminetheKDhavespecificadvantagesanddisadvantages,however
theproblemwithmostofthem,theexceptionbeingMS,isthattheycanonlyquantitatively
evaluatethecomplexationbetweencyclodextrinandasinglesmallmolecule.Thisisaproblemin
thepharmaceuticalindustry,wheremanydrugformulationshavemultipleactiveingredients,and
manydrugsaretakenincombinationdoseswithotherdrugs(21).Aseparationtechniqueis
crucialtothecharacterizationofcomplexesformingbetweencyclodextrinsanddifferentsmall
moleculessimultaneously.
1.5PrinciplesofCapillaryElectrophoresis
Capillaryelectrophoresis(CE)isaseparationtechniquethatseparatesionsbasedontheircharge
andfrictionalforcesaswellastheirhydrodynamicradius.Whenusinganumberofdrugs,their
uniqueabsorptionspectraandelutiontimescanbeusedtoidentifythemindividually.This
separationisdoneunderaninducedelectricfield,whichcausesanelectroosmoticflowofthe
liquidmovingfromthepositiveelectrodetothenegativeelectrode(21).Thisbulkflowofliquid
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causesallspecies(positive,neutralornegative)tomoveinonedirectionduetothesurfacecharge
onsilanolgroupsoftheinteriorcapillarywall.Theelectroosmoticflowisaffectedbyseveral
factorsincludingthepHandtheionicstrengthofthesolution(21).Inthiswork,Equilibrium
CapillaryElectrophoresisofEquilibriumMixtures(ECEEM),atypeofKineticCapillary
Electrophoresis(KCE),willbeappliedinordertostudytheequilibriumbetweenthecyclodextrin
andthechosensmallmoleculedrugs.KCEisageneralconceptusedtodescribeCEseparationof
speciesthatinteractduringelectrophoresis(22).Bychangingtheconditions,specificmethods
undertheumbrellaofKCEhavebeendevelopedtomeasureaffinities(includingbinding
parameters)aswellaspurifyaninteractingmixture(22).
InECEEM,aquasi‐equilibriumsystemofthesmallmolecule,cyclodextrin,andcomplexisinjected
intothecapillaryandsubjectedtoelectrophoreticseparation.AschematicofECEEMisshownin
Figure3.Theconcentrationofcyclodextrinismaintainedintherunningbuffer,soitiseffectively
thesamethroughoutthewholesystem(22).UniquetoECEEMarethreemaincharacteristicsfor
rapidequilibriummixtureslikethatofsmallmoleculesandcyclodextrins.(i)Changingthe
concentrationoftheβ‐cyclodextrinwillchangethemigrationtimeoftheequilibriummixture.(ii)
Thefreedrugandthecomplexwillmigratetogetherbecauseoftherapidequilibriumbetween
them.(iii)Finally,thewidthofthepeakfortheequilibriummixtureisdependentonthe
concentrationofβ‐cyclodextrin,therelaxationtime,andtheseparationtime(7).Thisis
fundamentaltostudyingaveryfastinteraction,asitwillnotseparatethecyclodextrinfromthe
drug,butratherseparatedrugswithdifferentKDsfromeachother.Inpreviousworkdonebythe
Berezovskilab,ECEEMwasprovenforthedeterminationofkineticparametersoffastnon‐
covalentinteractions,andaparameterbasedmethodforthequantificationofrateconstants
11
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ofcomplexformationanddissociationwasdeveloped(7).Inthismethod,thebindingconstantsof
theinteractionsbetweenβ‐cyclodextrinandfoursmallmoleculedrugs(ibuprofen,s‐flurbiprofen,
salicylicacidandphenylbutazone)weredetermined.
Furthermore,bycouplingECEEMwithESI‐MS,thebenefitsofboththeseparationtechnique
(ECEEM)andthecomplexidentification(MS)canbecapitalizeduponforabetterunderstandingof
thenon‐covalentbinding.
ThegoalofthisworkwastousethepreviouslyestablishedtechniqueofECEEMtofurtheranalyze
theinteractionsbetweenβ‐cyclodextrinsandsmallmoleculesofgreatercomplexity,aswellas
developamethodfortheanalysisofamultiplexofeightdrugs.Withthisstudy,itisthehopethat
capillaryelectrophoresismaybecomeanimportanttoolfortheevaluationofβ‐cyclodextrinasa
drugdeliveryagentfordrugs.
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2.ResultsandDiscussion
2.1ProofofPrinciple4,4’‐(Propane‐1,3‐Diyl)DibenzoicAcidModel
PreviousworkdonebytheBerezovskigroup(7),showedthedeterminationofbindingconstants
betweenβ‐cyclodextrinandfoursmallmoleculedrugs.Eachofthesesmallmoleculedrugs
structurallyhadthepropensitytobindina1:1stoichiometricratiowithβ‐cyclodextrin,andthey
eitherhadasinglephenylringortwophenylringsincloseproximity.
Inthisresearch,amodelfordeterminingtheKDsofgreaterstoichiometrywasdesired,soasmall
moleculethathadtheabilitytoforma2:1stoichiometricsandwichcomplexwiththeβ‐
cyclodextrinwasselected.Carboxylicacidgroupswerealsopreferred,meaningthedrugwould
haveanegativechargeinsolution.ThisisidealfortheCEmethodalreadyused,asthenegative
drugswillmoveslowerthantheneutralcyclodextrin,andthustheformationofthecomplexwill
causeafastervelocityofthedrugandashortermobilitytime.4,4’‐(Propane‐1,3‐Diyl)Dibenzoic
Acid(PDDA)wasselectedasithadbothoftheseproperties.Itsstructurehadtwophenylrings
withcarboxylicacidgroupsattachedbyathree‐carbonlinker.Becausethissmallmoleculeis
symmetrical,itwillbeeasiertodevelopamodelformultipleKDcalculationsthanitwouldbefora
morecomplicatedstructure.
ThechangeinthemobilitybetweenthefullycomplexedPDDAandthefreeformalsoservesto
maketheanalysiseasy.Thischangeinmobility,ofapproximately3minfora90cmcapillaryin10
mMAmmoniumAcetatebufferseparatedwith26kVfor12min,wasmuchgreaterthanallother
smallmoleculesevaluatedinthisresearch.ThiscanbeattributedtothefactthatPDDAhastwo
negativecharges,soitwillbeslowermovinginthecapillarywheninitsfreeform.ThePDDAis
fullysaturatedatβ‐cyclodextrinconcentrationsofaround2500µM.Inordertousecapillary
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electrophoresistocalculatetheKD,itisessentialthatthedrugsbesaturatedwithinthesoluble
concentrationofβ‐cyclodextrin(upto17,000µM).
Methoddevelopmentofthecapillaryelectrophoresistookalargeamountoftimeinthisresearch.
Initially,thelimitofdetectionofthePDDAneededtobedetermined.Thiswasdoneusinga30cm
capillary,andthemethodpreviouslydevelopedbytheBerezovskilab(7).Thelimitofdetection
wasdeterminedtobe5µMina25mMTris‐Acetatebuffer,asshownintheelectropherogramin
Figure4.Movingforward,aconcentrationofapproximatelysixtimesthelimitofdetection,30µM
ofPDDAwasused,asthisprovidedagoodsignalthatwaseasytoanalyze.Usingthephotodiode
array(PDA)detector,severalwavelengthswerecheckedtofindthewavelengthatwhichthe
maximumintensityofsignalwasseen.Sinceithastwophenylrings,twomainareasofabsorbance
onthespectrawereseen:around200nmand250nm.Themostintensesignalwasseenaround
200nm,soinallfutureseparations,oneofthechannelswassetat200nmforevaluation.
Toensurethecurrentremainedstablethroughouttheseparation,severalparameterswere
changed.Initially,ahighconcentrationofβ‐cyclodextrinof5000µMwaspre‐injectedintothe
capillary,followedbytherunbufferandthenthesampleinjection.Thismethodtookalotoftime,
andwouldhaveaddedalotofextratimewhendoingmultiplerunsforbindingparameter
determination.Instead,rinsingthecapillaryforalongerlengthoftime(approximately3min)with
therunbuffergaveastablecurrentwithouthavingtodoapre‐injection.
OneofthemainproblemswithPDDAwasthataftersampleshadbeenleftforseveralhoursthe
migrationtimechangeddrastically.Althoughthisproblemcouldbesolvedbyusingonlyfresh
samples,thecostofthissmallmoleculewouldhavemadethisimpractical.Tosolvethisproblem,
15
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16
afreshsamplewasrunandthencomparedtosampleskeptovernightatroomtemperature,+4°C
and‐20°C.AsshowninFigure5,Samplesat‐20°Cgaveresultsconsistentwithfreshsamples,soall
PDDAsampleswerefrozenat‐20°Candthenthawedbeforeuse.
WiththeoptimizedmethoddescribedinSection4.2,thePDDAwassubjectedtoelectrophoretic
separationwithincreasingconcentrationsofβ‐cyclodextrinintheequilibriummixture.The
electropherogramshowingtheresultsoftheseexperimentsisshowninFigure6.Themobility
shiftofthepeakforthePDDAinrapidequilibriumwithβ‐cyclodextringraduallydecreasesas
higherconcentrationsareused.Thiscanbeexplainedbythefactthatathigherconcentrations
moreofthePDDAisinthecomplex,whichhasaneutralchargeandwillthusmovefasteralong
thecapillarytowardthenegativeelectrodethanthefree,negativelychargedformofthedrug.
IftheproposedsandwichcomplexbetweenPDDAandβ‐cyclodextrinwasformed,itwouldbe
anticipatedthattherewouldbetwoareasofalargemobilityshiftontheelectropherogram,and
anareainthemiddlewherethepeak’smobilitydoesnotchangeinwhichthedrughadbeen
saturatedasa1:1complex,butnotenoughβ‐cyclodextrinwasaddedtoforma2:1complex.This
wasnotseen,sotocalculatethebindingparametersofthiscomplex,themathematicalmodelfor
1:1stoichiometrydevelopedpreviously(7)wasapplied.
UsingtheshiftinmobilityofthePDDAallowsforthecalculationoftheKD,howevermore
informationaboutthenatureofacomplexrequiresthedeterminationofthekonandkoff.This
mathematicalmodelcalculatesthebindingparametersbasednotjustontheshiftinmobility(and
thusthevelocityoftheequilibriummixture)withthechangeinconcentration,butalsowiththe
shapeofthepeak.Whenthedrugisonlyinoneform(eitherthefreeformorthecomplexed
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Figure5:ECEEMElectropherogramstocheckfordegradationofPDDA(30μM).Comparedfreshsampleatroomtemperature(RT)withsampleskeptatthreedifferenttemperaturecontrols.
18
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Figure6:RepresentativeECEEMelectropherogramsofPDDA(30μM)forvaryingconcentrationsofβ‐cyclodextrin.
19
form),seenatverylowandveryhighconcentrationsofβ‐cyclodextrin,verysymmetricalpeaksare
seen.Howeverwhentheβ‐cyclodextrinconcentrationisaroundthatoftheKD(sothedrugcanbe
foundinbothformssimultaneously),thepeakisasymmetricandwider.Aschematicofthisis
showninFigure3b.Thisreflectsthefactthatwithintheequilibriumcomplexthereisagradientof
thedrug,andthiscausesnonlinearcomplexformation.Essentially,becausethecomplexeddrug
movesfaster,thisgradientofdrugcausesonepartofthepeaktomovefasterthantheotherpart.
Usingthismodel,theKDfortheβ‐cyclodextrin‐PDDAcomplexwascalculatedtobe56.6µMwitha
koffof14.7sec‐1andakonof2.6x105M‐1sec‐1.Theseconstantswerecalculatedbasedondata
collectedfrom20experimentsrepeatedthreetimeseach.Theconcentrationsofβ‐cyclodextrin
usedtocalculatetheKDwerechosenbecausetheywereapproximately0.5‐5timestheKD,
providingthemostpreciseresults.Thevelocityvsβ‐cyclodextrinconcentrationgraphisshownin
figure7.Thedatafitcloselywiththistheoreticalcurve.Unfortunately,thedatacollectedwasnot
consistentwiththetheoreticalpatternforthechangesintheasymmetryandthewidthofthe
peak.Thissuggestedthattherewereperhapscomplexesofgreaterstoichiometricratiosforming,
butthattheycouldnotbedetectedwithcapillaryelectrophoresis,whichgivesnostructural
informationaboutthecomplexesformed.
InformationcalculatedusingtheCEwascomparedtoinformationfoundbyotherlabmembers
workingonthesamecomplexesusingCE‐MStogainabetterinsight.UsingMSasadetectorgives
muchmoreinformationaboutwhatisintheequilibriummixtureasitshowstherespectiveions
fordifferentcomplexesinsolution.Ifthereare2:1complexesforming,itisthoughtthatthey
20
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21
wouldbemoreprevalentathigherconcentrationsofβ‐cyclodextrin.TheideaofanECEEM‐MS
separationofacyclodextrin‐druginteractionwith2:1stoichiometryissummarizedinFigure8.In
fact,usingCE‐MS,wewereabletodeterminethat2:1complexesdoform,butnottheexpected
sandwichcomplex,rathercomplexesbetweenthedrugandadimerofcyclodextrin(Gleb
Mironov,unpublisheddata).Athigherconcentrations,thereismoreβ‐cyclodextrininthe
dimerizedform.KDvaluesforthetwodifferentcomplexeswerecalculatedtobe83.65µMforthe
monomericcomplexand39.08µMforthedimericcomplex(GlebMironov,unpublisheddata).
Interestingly,PDDAhasahigheraffinityforthedimerofβ‐cyclodextrin,whichmaybeattributable
tomorehydrogenbondingwithintheinclusioncavityduetomorealcoholgroupswhentwo
cyclodextrinscombine.ThesevaluesaveragetoaroundtheapparentKD(56.55µM)calculated
usingthecapillaryelectrophoresisdata,verifyingbothmethodsaccuracy.
Insummary,capillaryelectrophoresisworksverywellforcalculatingtheKDandbindingconstants
forcomplexesof1:1stoichiometry.Forcomplexeswithhigherstoichiometry,itwillgiveaKDthat
isrepresentativeoftheweightedaverageforallcomplexesformed,informationthatcanstillbe
usedfordrugdeliverydesigninwhichlowconcentrationsofβ‐cyclodextrinareused(andthus1:1
complexeswillbedominant).Alloftheothercommonlyusedmethods(exceptforMS)for
cyclodextrincomplexbindingcalculationsalsocalculateaweightedaverageKDassuming1:1
stoichiometry,highlightingthatcomplexstoichiometryisabigproblemincyclodextrininclusion
complexcharacterization.CE‐MScanbeusedtocharacterizethebindingofthecomplexingreater
stoichiometricdetail,howevercurrentlynomathematicalmodelhasbeendevelopedtocalculate
otherbindingparametersofthesecomplexessuchaskonandkoff.
22
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Figure8:(A)SchematicrepresentationofECEEM‐MSseparationwithstoichiometryofthecomplexwithincreasedβ‐cyclodextrinconcentrations.(B)ComplexformationwithPDDAaccordingtoMSresults.
23
2.2MultiplexApplication
AfteroptimizingtheCEmethodwithPDDAandshowingitwasaviablemethodofaweighted
averageKDdetermination,themethodwasappliedtoamultiplexofeightdrugs:ibuprofen,(s)‐
flurbiprofen,PDDA,naproxen,folicacid,diclofenac,phenylbutazoneandresveratrol.Thisexploits
thepropertyoftheECEEMmethodtoseparatedrugswithdifferentKDs,andprovidesamore
accuratemodelofamoderndrugformulation(21).
WithaPDAdetector,individualdrugscanbeidentifiedbasedontheiruniqueabsorptionspectra
andshiftinmobility.The3‐Dabsorptionspectrasofeachoftheeightdrugsaresummarizedin
Figure9.Theabsorptionspectrathatwouldbeseenwhenalldrugsarecombinedintoone
formulationisshowninFigure10.Itshouldbenotedthatforabettervisualseparationandless
overlapalongercapillary(90cm)andincreasedseparationtimewasusedforthemultiplex
experiments.Allofthedrugscanbevisualizedat200nm,sothischannelwaschosenforpeak
analysisandcalculations.ThemainproblemwithusingaPDAdetectorasopposedtoaMS
detectoristhatthedrugmustabsorbinauniquerange.Conveniently,themajorityof
pharmaceuticals,suchastheonesusedinthiswork,havearomaticringsinthemthatwillabsorb
athigherwavelengths.Absorbancesunder200nmcangenerallynotbeidentifiedasthe
backgroundfromhydrocarbonsthatmaybecomponentsofthebuffer,includingthecyclodextrin,
istoohigh.
Peakmobilitydatawascollectedfrom17experimentsrepeated4timeseachtoensureaccuracy.
Theelectropherogramswereconsistentwhenalignedusingphenylbutazone,apreviously
determinedinternalstandardfortheseexperiments(7).Phenylbutazonehasanegativecharge
whichisdelocalizedoverfiveatoms,andisverystericallyhindered,meaningthatitcannotforma
24
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26
complexwithcyclodextrin.Ifphenylbutazonewasnotused,itispossibletoalignallrunsusingthe
dipinsignalbytheelectroosmoticflow,howeverifthereisanychangeinthebufferthiscanlead
toinaccurateresults.Overthecourseofthefourrepeats,theammoniumacetatebufferchanged
whichcausedalargechangeinthemobilityshiftforalldrugs.Theuseofaninternalstandard
ensuresthatexperimentsareveryreproducible.Anelectropherogramofoneofthetrialsisshown
inFigure11.Nouniquepeakwasseenforresveratrol.Thisisbecauseresveratrolisaneutral
drug,anddoesnothavethecarboxylategroupsthatgivethenegativechargeoftheotherdrugs.
Becauseofitsneutralcharge,acomplexwithresveratrolwillnotmovefasterwhencomplexed
withcyclodextrin,ascyclodextrinisalsoaneutralmolecule.Resveratrolcomesoutwiththepeak
associatedwiththeelectroosmoticflow,verifiedbyitsabsorptionspectra.
Duetothelargenumberofdrugsused,asimplermethodforKDdeterminationwasappliedthat
reliedonlyontheshiftinmobilityoftheindividualequilibriummixturesandnotthepeak’swidth
andasymmetry.Thiswasdonebecausethereisalotofoverlapinpeaksatdifferent
concentrations,assomecomplexesmovefasterthanothers,anditisimpossibletomeasurethe
peakwidthsaccuratelywhenthisisthecase.AcalculationmethoddevelopedbyKargeretal.(23)
formeasuringthebindingconstantsofpeptideswasappliedtothemultiplexsystem.
𝐾! = 𝛽𝐶𝐷𝑡! − 𝑡!𝑡! − 𝑡!
wheretisthemigrationtimeandthesubscript0isthefreedrug,tisequilibriummixture,andcis
thedrugcomplex.t0wastakenasthemobilitywhentheconcentrationofβ‐cyclodextrinwaszero,
whiletcwastakenasthemobilitywhenthesystemwassaturated.Thiscouldcauseproblemswith
drugsthataredidnotsaturatewithinasolublerangeofcyclodextrin.Thisformulawas
27
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Figure11:RepresentativeECEEMelectropherogramsofmultiplex(15μMofeachdrug)forvaryingconcentrationsofβ‐cyclodextrin.Drugsinthemultiplexare:Phenylbutazone(1),Diclofenac(2),Ibuprofen(3),s‐Flurbiprofen(4),Naproxen(5),FolicAcid(6),PDDA(7)andResveratrol(8).Resveratrolemittedwiththeelectroosmoticflow.
28
appliedtoallmobilityshifts,andtheaverageofthosewithintherangeoftheKD(determinedby
themovementofthepeakontheelectropherogram)wastakentogiveavalueoftheKD.The
calculatedKDsandtherepresentativegraphsofthesixdrugsforKDdeterminationaresummarized
inFigure12.
Phenylbutazoneisknowntonotcomplexwithβ‐cyclodextrin(7),andwasusedastheinternal
standard,sonoKDwascalculated.Resveratrolisaneutraldrug,sousingthismethoditis
impossibletodetermineitscomplexationwithβ‐cyclodextrin,ascomplexationwouldnotincrease
itsvelocity.ResveratrolhighlightsthemainproblemwiththeECEEMmethodforKD
determination.Thistechniqueworksverywellfornegativelychargeddrugs,howeveritdoesnot
workwithneutraldrugs,ascomplexationhasnoaffectonthesedrugsmobility.Forpositively
chargeddrugs,thisparticularmethodofECEEMwouldnotworkaspositivelychargeddrugswould
moveveryquicklytowardsthenegativeelectrode,andtheareaofthemobilityshiftwouldbe
smallanddifficulttoanalyze.Presumably,byreversingtheconditions,anECEEMmethodcouldbe
utilizedforKDdeterminationofpositivelychargeddrug‐cyclodextrininteractions.
TheKDvaluescalculatedforthedrugsDiclofenacandFolicAcidareveryhigh,andhavelarge
errorsassociatedwiththem.Thishighlightstwolimitationsofthismethod.Firstly,veryhigh
concentrationsofβ‐cyclodextrinbegintoinfluencetheelectroosmoticflowbyinterferingwiththe
buffer,whichinturnaffectsthemobilityshiftsoftheequilibriummixturesofdrugsaswellasthe
shapesofthepeaks.Thepeakstendtogetverybroadandshortathighconcentrations,andthus
shouldnotbeusedforKDcalculations.Thesecondlimitationisthesaturationoftheindividual
systems.Evenatveryhighconcentrationsofβ‐cyclodextrin,thepeaksofdiclofenacandfolicacid
continuedmovinganddidnotsaturate.Withoutsaturation(meaningallofthedrugcomplexed
29
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30
withβ‐cyclodextrin),themathematicalequationcannotbeused,asnotcexists.Inthesecases,the
KDforthisinteractionisverylarge,andtheβ‐cyclodextrinisusuallynotarelevantexcipientto
makethedrugmoresoluble.Onthisbasis,thislimitationisnotabigprobleminpracticalsettings‐
inwhichtheuseofcyclodextrinasanexcipientisbeingevaluated.
ItshouldbenotedthattheKDsforPDDA,Ibuprofen,ands‐Flurbiprofenaresmallerthanthose
previouslyreportedinSection2.1(PDDA)andpreviousworkbytheBerezovskigroup(7).Thisis
attributabletothechangeofbuffer.Ammoniumacetatewasusedforthemultiplexexperiments,
sothepHislowerthanwhenTrisAcetatebufferisused,asinSection2.1andpreviouswork.
LowerpHconditionshavebeenshowntoincreasetheaffinityofcyclodextrin‐smallmolecule
interactions(6).
Themainbenefitofthismodelisasapreliminarystudyforwhetherornotcyclodextrinwouldbe
agooddrugdeliveryagentforaspecificdrug.Although,asmentionedinsection2.1,capillary
electrophoresiscannotgiveinformationaboutstoichiometrygreaterthan1:1,itcangiveavalue
fortheapparentKDbetweenasmallmoleculeandcyclodextrin.Fordrugsthatdonotsaturate,or
shiftonlyatveryhighconcentrationsofβ‐cyclodextrin,cyclodextrinislikelyabaddrugdelivery
agent.SmallerKDvaluessuggestthepotentialofthisdrugdeliverysystem,andfurtheranalysis
usingCE‐MScanbeappliedtogiveevenmoreinformationaboutthecomplexation.
31
3.References
1.Heimbach,T.,Fleisher,D.andKaddoumi,A.(2007).Overcomingpooraqueoussolubilityofdrugsfororaldelivery.Biotechnology:PharmaceuticalAspects.5:p.157‐215.
2.Smith,W.,DeWitt,D.,andGaravito,R.(2000).Cyclooxygenases:Structural,cellularandmolecularbiology.AnnualReviewofBiochemistry.69:p.145‐182.
3.Wald,N.(1991).Preventionofneuraltubedefects.Lancet.338:p.121‐137.
4.Baur,J.andSinclair,D.(2006).Therapeuticpotentialofresveratrol:theinvivoevidence.NatureReviewsDrugDiscovery.5:p.493‐506.
5.Kata,M.andSelmeczi,B.(1987).Increasingthesolubilityofdrugsthroughcyclodextrincomplexation.JournalofInclusionPhenomenaandMacrocyclicChemistry.5:p.39‐43
6.Loftsson,T.andBrewster,M.(1996).Pharmaceuticalapplicationsofcyclodextrins.JournalofPharmaceuticalSciences.85:p.1017‐1025.
7.Mironov,G.,Okhonin,V.,Gorelsky,S.,andBerezovski,M.(2011)Revealingequilibriumandrateconstantsofweakandfastnoncovalentinteractions.AnalyticalChem.83:p.2364‐2370.
8.Fielding,L.(2007)NMRmethodsforthedeterminationofprotein‐liganddissociationconstants.ProgressinNuclearMagneticResonanceSpectroscopy.51:p.219‐242
9.Sompornpisut,P.,Deechalao,N.,andVongsvivut,J.(2002)Aninclusioncomplexofb‐cyclodextrin‐L‐phenylalanine:1HNMRandmoleculardockingstudies.ScienceAsia.28:p.263‐270.
10.Ogwu,S.,Alcala,M.,Bhardwaj,R.andBlanchard,J.(1996)Theapplicationofequilibriumdialysistothedeterminationofdrug‐cyclodextrinstabilityconstants.JournalofInclusionPhenomenaandMolecularRecognitioninChemistry.25:p.173‐176
11.HarvardApparatus.(2002).GuidetoEquilibriumDialysis.HarvardBioscience,Inc.p.2‐9.
12.Loftsson,T.,Masson,M.andBrewster,M.(2003)Self‐associationofcyclodextrinsandcyclodextrincomplexes.JournalofPharmaceuticalSciences.93:p.1091‐1097.
13.Marques,H.,Hadgraft,J.andKellaway,I.(1990)StudiesofcyclodextrininclusioncomplexesbyphasesolubilityandDSC.InternationalJournalofPharmaceutics.63:p.259‐266.
14.Kurkov,S.,Ukhatskaya,E.andLoftsson,T.(2011)Drug/cyclodextrin:beyondinclusioncomplexation.JournalofInclusionPhenomenaandMacrocyclicChemistry.69:p.297‐301.
15.Brown,S.,Easton,C.andKelly,J.(2003)Surfaceplasmonresonancetodetermineapparentstabilityconstantsforthebindingofcyclodextrinstosmallimmobilizedguests.JournalofInclusionPhenomenaandMacrocyclicChemistry.46:p.167‐173.
32
16.Kobayashi,H.,Endo,T.,Ogawa,N.,Nagase,H.,Iwata,M.andUeda,H.(2011)Evaluationoftheinteractionbetweenb‐cyclodextrinandpsychotropicdrugsbysurfaceplasmonresonanceassaywithaBiacoresystem.JournalofPharmaceuticalandBiomedicalAnalysis.54:p.258‐263.
17.Zheng,P.,Li,Z.,Tong,L.,andLu,R.(2002)StudyofinclusioncomplexesofcyclodextrinswithOrangeII.JournalofInclusionPhenomenaandMacrocyclicChemistry.43:p.183‐186.
18.Zhang,H.,Zhang,H.,Jin,W.andDing,L.(2006)Determinationofdissociationconstantsofcyclodextrin‐ligandinclusioncomplexesbyelectrosprayionizationmassspectrometry.EuropeanJournalofMassSpectrometry.12:p.291‐299.
19.Gabelica,V.,Galic,N.,Rosu,F.,Houssier,C.andDePauw,E.(2003)Influenceofresponsefactorsondeterminingequilibriumassociationconstantsofnon‐covalentcomplexesbyelectrosprayionizationmassspectrometry.JournalofMassSpectrometry.38:p.491‐501.
20.Mohamed,M.,Wilson,L.,Headley,J.,andPeru,K.(2009)Electrosprayionizationmassspectrometrystudiesofcyclodextrin‐carboxylateioninclusioncomplexes.RapidCommunicationsinMassSpectrometry.23:p.3703‐3712.
21.Neubert,R.andRuttinger,H.(2003)Affinitycapillaryelectrophoresisinpharmaceuticsandbiopharmaceutics.NewYork:MarcelDekker.
22.Krylov,S.(2007)Review:KineticCE:Foundationforhomogeneouskineticaffinitymethods.Electrophoresis.28:p.69‐88.
23.Dunayevskiy,Y.,Lyubarskaya,Y.,Chu,Y.,Vouros,P.andKarger,B.(1998)Simultaneousmeasurementofnineteenbindingconstantsofpeptidestovancomycinusingaffinitycapillaryelectrophoresis‐massspectrometry.JournalofMedicinalChemistry.41:p.1201‐1204.
33
4.Experimental
4.1ChemicalsandMaterials
Chemicalswerepurchasedfromthefollowingcompanies:β‐cyclodextrin(Sigma‐Aldrich,Canada,
EC231‐493‐2),Naproxen(Sigma‐Aldrich,Canada,N8280),4,4’‐Propane‐1,3‐DiylDibenzoicAcid
(SigmaAldrich,Canada,SS499455),Ibuprofen(SigmaAldrich,Canada,I4883),Diclofenac(Sigma
Aldrich,Canada,D6899),Phenylbutazone(SantaCruzBiotechnology,USA,SC‐204843),FolicAcid
(SigmaAldrich,Canada,F7876),(S)‐Flurbiprofen(CaymanChemical,USA,10004207),and
Resveratrol(OntarioHealthResearchInstitute).ForthePDDAmodelexperiments,a25mMTris‐
Acetatebuffer,pH7.80wasusedastheincubationandrunbuffer.Thiswaspreparedbydilutinga
stockbuffer,whichwaspreparedbydissolving12.11gofTris‐base(BioBasicInc,Canada,77‐86‐1)
and2.86mLofaceticacid(BioBasicInc.,Canada,1000)in500mLofddH2O.Multiplex
experimentsweredonein10mMAmmoniumAcetate,pH6.6.Thiswaspreparedfroma100mM
stocksolutionmadebydissolving3.85gofAmmoniumacetate(FisherScientific,Canada,639‐500)
in500mLddH2O.
ForthePDDAmodelingexperiments,theequilibriummixtureofPDDAandcyclodextrinwas
preparedintheincubationbufferwith30uMPDDAand10uMto2500uMofβ‐cyclodextrin.A1
mMstocksolutionofPDDAwasprepareddirectlybydissolving0.0028gofthedrugin10mLof
theincubationbuffer.Allsolutionswerefilteredthrough0.22‐umporesizemembranefilters
(Millipore,Nepean,ON,Canada).ThebaresilicacapillarywaspurchasedfromPolymicro(Pheonix,
AZ,USA)
Forthemultiplexseparationexperiments,theequilibriummixturewaspreparedwith15uMof
eachoftheeightdrugs:PDDA,Ibuprofen,Diclofenac,Naproxen,s‐Flurbiprofen,Phenylbutazone,
34
FolicAcid,andResveratroland10uMto15,000uMβ‐cyclodextrin.Stocksolutionsofalldrugs
(exceptphenylbutazone)werepreparedbydissolvingaweighedamountofthedrugsin10mLof
theincubationbuffer.Inthecaseofphenylbutazone,a10mMstocksolutionwaspreparedby
dissolvingtheweighedamountin95%ethanolandthendilutingintheincubationbuffer.Allof
thesesolutionswerealsofilteredthroughthe0.22umporesizemembranefilters.Thesamebare
silicacapillarywasused.
4.2ExperimentalConditions
ECEEMexperimentswerecarriedoutusingaPA800MDQPharmaceuticalAnalysisCEsystem
(BeckmanCoulter,USA)equippedwithaPDAdetector.
ForthePDDAmodelingexperiments,thefollowingconditionsweremaintained.Thesample
storageandcapillarytemperatureweremaintainedat25°C±0.5°C,anelectricfieldof26kVwith
apositiveelectrodeattheinjectionend,therunbufferwithcyclodextrinintheinletreservoir,and
theincubationbufferintheoutletreservoir.Theelectricfieldappliedcausestheelectroosmotic
flow,abulkflowoftheliquidduetothecationsformingadiffuselayerbeingattractedtowardthe
cathode.Theconcentrationofthecyclodextrinintheequilibriummixtureandtherunbufferwas
thesameforindividualECEEMexperiments.Thecapillarywas29cmlong(20cmtothedetection
window)withaninnerdiameterof50umandanouterdiameterof360um.A5.03mmlongplug
oftheequilibriummixturewasinjectedintothecapillaryfromtheinletendbyapressurepulsefor
20sat1psi.Beforeeachexperimentthecapillarywasrinsedby20psipressurewith0.1MHClfor
3min,0.1MNaOHfor3min,ddH2Ofor3min,25mMTris‐Acetatebufferfor3min,andthe
incubation/runbufferwithcyclodextrinfor1min.Theoutputdatawasabsorbanceintensityinthe
detectionpointasafunctionoftimepassedsincetheapplicationoftheelectricfield.
35
Multiplexexperimentsweredoneunderthesameconditionsexceptwitha89cmlong(80cmto
thedetectionwindow)capillary.A3.35mmplugoftheequilibriummixturewasinjectedintothe
capillaryfromtheinletendbyapressurepulsefor5secat1psi.Beforeeachexperimentthe
capillarywasrinsedby50psipressurewith0.1MHClfor5min,0.1MNaOHfor5min,ddH2Ofor
5min,10mMAmmoniumAcetatebufferfor5minandtheincubationbufferwithcyclodextrinfor
1min.
CE‐MSdatawasobtainedusingaSynapt‐G2‐HD‐MS(Waters,USA)coupledwithaPA800+
PharmaceuticalAnalysisCEsystem(BeckmanCoulter,USA).
