vivo adjuvant characterization to in effect in localanesthesia Clonidine complexation hydroxypropyl-beta-cyclodextrin: with Fromphysico-chemical Journal of Pharmaceutical and Biomedical Analysis

(1)

Contents lists available atScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Clonidine complexation with hydroxypropyl-beta-cyclodextrin: From physico-chemical characterization to in vivo adjuvant effect in local anesthesia

Braga M.A.

^a

, Martini M.F.

^b

, Pickholz M.

^b

, Yokaichiya F.

^c,d

, Franco M.K.D.

^c

, Cabec¸ a L.F.

^e

, Guilherme V.A.

^a

, Silva C.M.G.

^a

, Limia C.E.G.

^a

, de Paula E.

^a,∗

aBiochemistryandTissueBiologyDepartment,BiologyInstitute,StateUniversityofCampinas,Campinas,SP,Brazil

bFacultyofPharmacyandBiochemistry,UniversityofBuenosAires,Argentina

cNuclearandEnergyResearchInstitute,IPEN–CNEN/SP,Brazil

dDepartmentofQuantumPhenomenainNovelMaterials,Helmholtz-Zentrum,Berlin,Germany

eTechnologyFederalUniversityofParana,Londrina,PR,Brazil

a r t i c l e i n f o

Articlehistory:

Received5October2015 Receivedinrevisedform 12November2015 Accepted14November2015 Availableonline18November2015

Keywords:

Clonidine Cyclodextrin Drugdelivery Magneticresonance Moleculardynamics

a b s t r a c t

Clonidine(CND),analpha-2-adrenergicagonist,isusedasanadjuvantwithlocalanesthetics.Inthiswork, wedescribethepreparationandcharacterizationofaninclusioncomplexofclonidineinhydroxypropyl- beta-cyclodextrin(HP-␤-CD),asrevealedbyexperimental(UV–visabsorption,SEM,X-raydiffraction, DOSY-andROESY-NMR)andtheoretical(moleculardynamics)approaches.CNDwasfoundtobind toHP-␤-CD (Ka=20M⁻¹)in1:1stoichiometry. X-raydiffractogramsand SEMimagesprovided evi- denceofinclusioncomplexformation,whichwasassociatedwithchangesinthediffractionpatterns ofthepurecompounds.NMRexperimentsrevealedchangesinthechemicalshiftofH3HP-␤-CDhydro- gens(=0.026ppm)thatwerecompatiblewiththeinsertionofCNDinthehydrophobiccavityof thecyclodextrin. Moleculardynamics simulation withthethree CND speciesthat existatpH 7.4 revealedtheformationofintermolecularhydrogenbonds,especiallyfortheneutraliminoformof CND,whichfavoreditsinsertionintheHP-␤-CDcavity.Invitro assaysrevealedthatcomplexation retardeddrugdiffusionwithoutchangingtheintrinsictoxicityofclonidine,whileinvivotestsinrats showedenhancedsensoryblockadeaftertheadministrationof0.15%CND,withtheeffectdecreasing intheorder:CND:HP-␤-CD+bupivacaine>CND+bupivacaine>bupivacaine>CND:HP-␤-CD>clonidine.

Theﬁndingsdemonstratedthesuitabilityofthecomplexforuseasadrugdeliverysystemforclinical useinantinociceptiveprocedures,inassociationwithlocalanesthetics.

1. Introduction

Clonidine (2-[2,6-dichloroaniline]-2-imidazoline, CND) was ﬁrstsynthesizedinthe1960s.Itwasoriginallyusedasnasaldecon- gestant.However,duetoitscapacitytolowerbloodpressure,CND hasbeensuccessfullyemployedinthetreatmentofhypertension forover25years.Nevertheless,otherusesinclinicalpracticewere proposed[1],and CNDhasattractednewinterest inanesthesi- ology,asanadjuvantforgeneralandregionalanesthesiaduring surgeryorinthepostoperativeperiod[2–5].Itiswellknownthat

∗ Correspondingauthorat:Department ofBiochemistryandTissueBiology, InstituteofBiology,StateUniversityofCampinas,RuaMonteiroLobatono255, 13083-862,Campinas,SP,Brazil.Fax:+551935216185.

E-mailaddresses:[email protected],[email protected](E.dePaula).

CNDprolongstheeffectoflocalanesthetics,reducingthedosage requiredfor anesthesia,withminoradverseeffects [6–10].This beneﬁcialactionisduetodifferentmechanisms.Inadditiontoits

␣2-agonistaction (norepinephrine-likein descendinginhibitory pathways),CNDactsanalogouslytoalocalanesthetic,inhibiting andslowingimpulseconductioninCﬁbers[11,12].CNDalsoelic- itsvasoconstriction,mediatedbythepostsynaptic␣2-receptors, whichreducesabsorptionofthelocalanestheticandprolongsits residencetimeattheneuraltissue[6,7].Insummary,theassoci- ationofclonidinewithlocalanestheticssigniﬁcantlyreducesthe timeofonsetofanesthesia,prolongsthedurationandintensityof thesensoryblock,andleadstosedationduetosystemicabsorption [13].

Remkoetal.[14],usingquantumchemicalcalculations,found thatattheCNDequilibriumgeometry,theimidazoleandphenyl ringsofthemoleculearealmostperpendiculartoeachother,and

(2)

istration,isalmostcompletelyeliminatedviaglomerularﬁltration [20].HP-␤-CDhasbeenextensivelystudiedasacarriersystemfor localanesthetics,withadvantagessuchasimprovedsolubilityand clinicalpotency[20–25].

This work proposes the complexation of CND with hydroxypropyl-␤-cyclodextrin, in order to improve its clini- calefﬁcacy in anesthesia. A broad studywas undertaken,with preparation and characterization of the complex, followed by invitroandinvivotests,andmoleculardynamicssimulation,with highlyencouragingresults.

2. Experimental

2.1. Reagentsandchemicals

Clonidinehydrochloridewas donatedby CristáliaInd. Farm.

Ltda.(Itapira,SP,Brazil).Hydroxypropyl-␤-cyclodextrin(Kleptose HP^®) was purchased from Roquette Serv. Tech. Lab. (Lestrem, Cedex,France).D₂OwasobtainedfromSigma.DMEM(Dubelcco’s ModiﬁedEagleMedium)wasacquiredfromNutricell(Campinas, SP,Brazil).Bovinefetalserum,penicillin,andstreptomycinwere obtainedfromCultilab(Campinas,SP,Brazil).Allotherchemicals usedwereofanalyticalgrade.

3. Methods

3.1. Preparationoftheclonidine:HP-ˇ-CDcomplex

Preparationoftheinclusioncomplexwasperformedbymix- ingequimolaramountsofCNDandHP-␤-CDinwater,followed by12hagitation,toachievecompletesolubilization.Thesamples werefreeze-driedandstoredat−20^◦Cforfurtheruse.

3.2. UV-Visabsorptionstudyandstoichiometrydetermination TheinteractionbetweenCNDandHP-␤-CDwasfollowedby UVabsorptionintherange250–310nm.Atitrationapproachwas usedtodeterminethecomplexationstoichiometry[26],withCND spectrarecordedinthepresenceofincreasingHP-␤-CDconcen- trations(CND:HP-␤-CDmolarratiosof1:0,1:1,1:10,1:20,1:30, 1:40,1:50,1:75,and1:100).UsingtheJobplotapproach[27],CND spectrawererecordedfordifferentCND:HP-␤-CDratios,maintain- ingaﬁnalconcentrationofCND+HP-␤-CD=2mM.Allexperiments wereperformedin5mMHepesbuffer,atpH7.4and25^◦C.

TheBenesi–Hildebrandprocedurewasemployedtodistinguish betweenthe1:1and1:2stoichiometries,accordingto[28]:

[CND]

Abs−Abs₀ = 1

[HP−␤−CD]ⁿ (1)

where[CND]and [HP-␤-CD]aretheconcentrationsofCNDand HP-␤-CD,respectively.Abs(Abs₀)representstheCNDabsorbance at271nm,inthepresence(absence)ofHP-␤-CD,and“n”isthe

secondaryelectronemission.

3.4. X-raydiffractionexperiments

PowderdiffractogramsforsamplesofHP-␤-CD,clonidine,the physicalmixture,andtheinclusioncomplexwereobtainedusing aRigakuwideanglegoniometer,equippedwithaCuK_␣radiation source(PhilipsPW1743),operatedat40kVand20mA.Thecrystal analyzerwaspyrolyticgraphite.Ascanrateof1^◦/minwasused, between2=5^◦and60^◦ina–2conﬁguration.

3.5. Nuclearmagneticresonance

NMRanalyseswereperformedwithaVarianInova500MHz (11.75T)instrument,attheBrazilianSynchrotronLightLaboratory (LNBio,Campinas,Brazil).One-andtwo-dimensional¹HNMRspec- trawereacquiredat25^◦C,usingD₂Oassolvent.Samples(10mM) ofCND,HP-␤-CDandthecomplexwerepreparedinD₂OatpH 7.4(adjustedwithNaODandDClsolutions),homogenizedfor6h, andtransferredto5mmtubesforspectrumacquisition.Toavoid anypossibleinteractionwithHP-␤-CD,noexternalstandardswere used[29];instead,theresidualwaterpeak(4.68ppm)wasusedas aninternalreference.

2D-ROESYexperimentswerecarriedouttodeterminenuclear Overhausereffects(NOE),indicatingthespatialproximitybetween CNDand HP-␤-CD hydrogens. Rotating-frame cross-relaxations werecarriedoutusingspin-lockedconditionsandNOEinthetrans- versepositiveplane.Pulsesequencewasemployed,withamixing timeof300ms[30].

DOSY-NMRspectrawererecordedat25^◦C,usingtheDgcteSL (gradient compensated stimulated echo spin lock) HR-DOSY sequence, as described previously [31]. The amplitudes of the pulsedgradientrange were0.12–0.63Tm⁻¹,where anapproxi- mately90–95%decreaseintheresonanceintensitywasachieved atthelargestgradient.Forallexperiments,25differentgradient amplitudeswereused,withanoptimizeddiffusiontimeof0.06s.

Theprocessingprogram(DOSYmacro)wasrunwithdatatrans- formedusingfn=32K.

Fromthemeasureddiffusionconstants(D),thefractionofCND boundtoHP-␤-CD(f)wasdetermined,accordingtoEq.(2):

f=DCND−DCND:HP−␤−CD

D_CND−D_{HP−␤−CD} (2)

allowingforthedeterminationoftheassociationconstant(Ka),as follows[31]:

K_a= f

(1−f)([HP−␤−CD]−f[CND]) (3) 3.6. Moleculardynamicssimulation

Moleculardynamicssimulationsofthedifferentsystemswere performedinordertoshedlightontheinteractionbetweenCND andHP-␤-CDattheatomiclevel.SinceCNDhasapKaof8.0[32],

(3)

Fig.1.Threeformsofclonidine:(A)amine-imidazoline,(B)imino-imidazolineand(C)protonated.

Table1

Schemeofselectedatomsfromtheclonidinemoleculeandtheircorresponding atomiccharge(seeFig.1forassignment).

Amino Imino Protonated

N9 −0.476 −0.633 −0.847

H9 0.460 0.324 0.438

N12 −0.150 −0.584 −0.831

H12 – 0.490 0.446

N7 −0.642 −0.915 −0.879

H7 0.378 – 0.446

C8 −0.384 0.780 1.145

C1 −0.708 0.715 0.240

80%ofthemoleculesarefoundintheprotonatedstateatphys- iologicalpH,while 20% areneutral. In itsprotonatedform,the positivechargeissharedthroughresonancebyallthreenitrogens oftheguanidinegroup.Ontheotherhand,theunchargedCNDform presentstwotautomericisomers:aminoandiminoimidazolidine [33,14],withtheiminoformbeingpredominant[14].Thethree CNDformsareshowninFig.1.Theyhaveabulkyorthochlorine groupthatpreventsthetworingsadoptingacoplanarconforma- tion[14,15].

Inordertocarryoutthesimulations,CND:HP-␤-CDcomplexes with1:1stoichiometrywereconstructedusingeightrepresenta- tiverelativeorientationsforeachoftheforms,surroundedbywater molecules.SimulationswereperformedusingtheGROMACS4.5 softwarepackage[34–37].TheGROMOS-9653a6forceﬁeld[38]

wasusedfortheCNDandHP-␤-CDmolecules.Thehydrogenatoms bondedtocarbonatomsweretreatedastheunitedatomstype.

Waterwasmodeledusingthesimplepointcharge(SPC)model[39].

Equilibriumbonddistances,angles,anddihedralswereobtained fromthegroundstategeometry optimizationofthethree CND forms,usingdensityfunctionaltheory(DFT)withtheB3LYP[40]

functionalandthe6-311G**basisset.Thepartialatomiccharges wereobtainedfroma singlepointHF/6-31G*calculation,using GaussianFrischetal.[41]andSinghandKollman[42]protocols.

ThechargesoftheselectedatomsaregiveninTable1.

Thesimulations werecarried out withintheNVT ensemble, usingtheBerendsenthermostat[43].Theelectrostaticinteractions werehandledwiththeSPMEversionoftheEwaldsums[44,45].The settingsfortheSPMEmethodwerearealspacecut-offof1.4nm,a gridspacingof0.12nm,andacubicinterpolation.Inallthesimu- lations,thevanderWaalsinteractionswerecutoffat1.4nm.The wholesystemwascoupledtoacontrolled-temperaturebath,with areferencetemperatureof300K,andtherelaxationconstantwas 0.1ps.Noconstraintswereusedforthebonds.Thetimestepfor theintegrationoftheequationofmotionwas1fs.Thenon-bonded listwasupdatedevery10steps.

3.7. Invitrodialysisexperiments

Drugdiffusionexperimentsexperimentswereconductedusing a two-compartment dialysis system, with a MWCO 1000Da cellulose membrane (Spectrapore) separating the donor (1mL) compartment,containingCNDorCND:HP-␤-CD,fromtheaccep- torcompartment(100mL),containing20mMHEPESbufferatpH 7.4,underconstantstirringat37^◦C.Aliquotswerewithdrawnfrom theacceptorcompartmentatregularintervals,andtheCNDcon- centrationwasdeterminedat271nm.

3.8. Cytotoxicitytests

PerpetualBalb/cmousefibroblasts(3T3cells)wereroutinely grown in DMEM medium containing 10% fetal bovine serum, 100U/mLpenicillin, and 100␮g/mLstreptomycin, at 37^◦C in a humidified incubator with5% CO2. The cells (2×10⁴ per well) wereincubatedin96-wellplatesuntilsemi-confluence,followed by treatmentfor 2hwith CND(freeor complexed withHP-␤- CD) at concentrations from 0 to 10mM. After treatment, the cellmedium wasreplacedby asolution ofMTT (1mg/mL)and thecells wereincubatedfor1hat37^◦C.Subsequently,theMTT solution was removed and 0.1mL of pure ethanol was added todissolvetheformazancrystals.Theformazanabsorbancewas measuredat570nmusing amicroplate reader(ELx800, BioTek InstrumentsInc.,USA).Theresults(mean±SD)wereexpressedas percentagesofthevaluesobtainedforuntreatedcontrols[46].IC50

valuesweredeterminedbynonlinearregressionanalysisusinga sigmoidconcentration-responseequationobtainedforindividual experiments,performedwithOrigin6.0(Microcal^TMSoftwareInc., Northampton,MA).

3.9. Invivoanalgesiatests

The sensory block was measured by threshold animal tail removal due toa thermal stimulus(the tail-ﬂick test)[47].Six groupsofanimals(7maleWistarrats,250–350geach)weretested using20␮L intrathecaladministrationof cyclodextrin(control), 0.25%BVC,0.15%CND,0.15%CND:HP-␤-CD,0.25%BVC+0.15%CND, and0.25%BVC+0.15%CND:HP-␤-CD.Afterinjection,theanimals wereplacedincontainerswiththetailpositionedonaheatsource and thetime for tailremoval wasmeasured, asdescribed previously [47].The protocolswereapproved bytheUniversity of CampinasInstitutionalAnimalCareandUseCommittee(protocol 2708-1/2012),followingtherecommendationsoftheGuideforthe CareandUseofLaboratoryAnimals.

(4)

4.1. ClonidineUV–visabsorptionpropertiesandcomplexation ThehyperchromiceffectobtainedafterCNDcomplexationwas evaluatedbymeasurementsoftheUVabsorptionspectraforsev- eral CND:HP-␤-CD ratios (Fig. 2A and B). The changes in the absorptionpropertieswereusedtoidentifycomplexation[48,49].

TreatmentthespectralshiftsbytheBenesi–Hildebrandapproach [28]permittedtoidentifytheprevalenceof1:1or1:2CND:HP-␤-CD complexationstoichiometry(Fig.2CandD).

InagreementwiththeJobplotanalysis(Fig.2A),thelinearcorre- lation(r²=0.9986)obtainedwiththeBenesi-Hildebrandtreatment (Fig.2D)conﬁrmedthe1:1complexationstoichiometry.Moreover, theratiobetweentheslopeandinterceptofFig.3Calloweddeter- minationofthebindingconstant(Ka)ofCNDtoHP-␤-CD:25M⁻¹. ThesmallKavalueindicatesthattheforcesresponsibleformain- tenanceofthecomplexweresmall[50].

4.2. Scanningelectronmicroscopyanalysis

Fig.3showsSEMimagesofHP-␤-CD,CND,theCND:HP-␤-CD physicalmixture,andtheinclusioncomplex.Toexcludeanypos- sibleinﬂuenceofthefreeze-dryingtreatmentinthemorphology, allsamplesweresubmittedtolyophilization,priortoSEManal- ysis.ThesolidCNDsampleconsistedofhexagonalcrystalsupto 100␮minsize,withtheternarysymmetrythatischaracteristic ofthistypeofcrystal.HP-␤-CDconsistedofamorphousstructures

∼50␮minsize.Thecrystallineandamorphousstructuresofthe purecompounds(CNDandHP-␤-CD)werepreservedinthephys- icalmixture,indicatingthatcomplexationdidnotoccurwhenthe solidhostandguestcompoundsweresimplymixedtogether.The CND:HP-␤-CDcomplexpresentedapoorlydeﬁnedstructurewith noresemblancetotheHP-␤-CDamorphousstructureandcomplete lossofthecrystalstructureofCND.Thesemorphologicalalterations providedevidenceofinclusioncomplexformation.

4.3. X-raydiffraction

X-raydiffraction is a useful tool for the qualitative analysis of complexation, by observation of the ﬁngerprints associated withthesamplecomponents[51].Thediffractogramforclonidine (Fig.4D)clearlyrevealeditscrystallinenature,whileHP-␤-CDwas amorphous(Fig.4A).Thediffractogramforthephysicalmixtureof clonidineandHP-␤-CD(Fig.4C)resembledasuperpositioningof thecrystallineclonidineandtheamorphousHP-␤-CDpatterns.In agreementwiththeSEMresults,thecrystallineclonidinepattern wasnotdetectedintheinclusioncomplexdiffractogram(Fig.4B), wheretheamorphousstructurewasindicativeoftheinsertionof CNDintothecyclodextrinmacrocycliccavity[52].Furthermore,a 67%decreaseintheintensityfortheinclusioncomplex,relativeto thatofpureHP-␤-CD(Fig.4Bvs.4A)wasrecordedinthe2region, revealingareductionintheHP-␤-CDcrystallinity.

Hpara 7.3193 7.3266 0.0073

aNotmeasuredbecauseofpeaksuperpositioning.

4.4. Nuclearmagneticresonance

Nuclearmagneticresonanceisoneofthemostpowerfultools usedtoobtaininformationaboutinclusioncomplexespresentin solution[53].Table2showschangesinthechemicalshiftsofthe hydrogensofCNDandHP-␤-CD,insolutionandinthepresence ofeachother(CND:HP-␤-CDcomplex),determinedat500MHz.

AssignmentoftheCNDpeakswasinagreementwiththelitera- tureandrevealedmagneticequivalentpeaksforhydrogens10and 11oftheimidazolering(Fig.1),at3.68ppm,aswellasformeta hydrogens3and5(at7.45and7.47ppm)andparahydrogen4(at 7.32ppm)ofthephenylring.Theassignmentofhydrogensbelong- ingtoHP-␤-CD(Fig.5)wasalsoinaccordancewiththeliterature [54–57].

Complexationinducedsubtle (ı<0.05ppm)non-signiﬁcant changesinalloftheCNDhydrogens(Table2).Inthecaseofthe cyclodextrinhydrogens,itwasexpectedthatwhencomplexation occurred,hydrogens3(H3)and5(H5),locatedinthecavityofthe macrocyclicringofthecyclodextrin,wouldbeinﬂuencedbythe inclusionoftheguestmoleculeinthecavity[22,56,58].Inthecase oftheCND:HP-␤-CDcomplex,aslightchange(ı=0.02ppm)was observedfortheH3signal(Table2),whilethechemicalshiftof H5couldnotbesatisfactorilydetermined,duetosuperpositioning withCNDpeaks.

Nuclear overhauser effect (NOE) [30,59,60] experiments (ROESY)wereusedtodiscriminatebetweenthepossiblemodesof encapsulationandprovideinformationontheintermolecularprox- imitybetweenthehydrogensoftheguestmoleculeincludedinthe HP-␤-CDcavityandhydrogensH3andH5[59].NoNOEcross-peaks wereobservedforthearomaticCNDhydrogensandH3,butpeak overlapsinthe3-4ppmregion(affectingimidazolehydrogensof CNDandH5ofHP-␤-CD)hinderedtheanalysis.

The lackof interaction betweenthe aromatic CNDring and theHP-␤-CDcavitycanbeexplainedbythehindrance(stericand polarity)generatedbythebulkychlorineatomssubstitutedinthe orthopositioninthephenylring.Asfoundfortheimidazolering, moleculardynamicssimulations(seebelow)revealedthatonlythe interactionoftheiminoform(representingtheminorityuncharged CNDspeciesatpH7.4)withtheCDcavitywasfavored.

Theuseof diffusion(DOSY) experimentsisanotherinterest- ingNMRapproachabletoprovideinformationabouthost-guest inclusioncomplexformation.Thediffusioncoefﬁcientofasmall moleculesuchasCNDislargeanddecreaseswithcomplexfor- mation,whichenablesDOSYdeterminationoftheguestfraction associatedwiththeCD,togetherwiththebindingconstant(Ka) [61,62].Here,theDOSYspectrumindicatedthatthemobilityof CNDwasonlyslightlydecreasedaftercomplexation(withdiffu- sioncoefﬁcientsof6.20×10⁻¹⁰m²s⁻¹and5.68×10⁻¹⁰m²s⁻¹for freeandcomplexedCND,respectively),showingthatonly14%of

(5)

Fig.2.ClonidineabsorptionpropertiesintheabsenceandpresenceofincreasingHP-␤-CDconcentrations(A)Jobplot;(B)spectraforstoichiometryofcomplexation calculations.(C,D)Benesi–Hildebrandtreatmentstodeterminethe1:1CND:HP-␤-CD(C)or1:2CND:HP-␤-CD(D).

Fig.3. SEMmicrographiesof:(A)HP-␤-CD(1000×);(B)CND(100×);(C)CND:HP-␤-CDphysicalmixture(1000×);(D)CND:HP-␤-CDcomplex(1000×).

Table3

Diffusioncoefﬁcients(D)ofCND,HP-␤-CDandCND:HP-␤-CDcomplex,complex molarfractionandCND:HP-␤-CDassociationconstant(Ka),asdeterminedby¹H NMR.

Compound D(10⁻¹⁰m²s⁻¹) Complexmolarfraction(%) Ka(M⁻¹)

CND 6.20±0.044 – –

HP-␤-CD 2.47±0.011 – –

CND:HP-␤-CD 5.68±0.023 14 20

theCNDinteractedwithHP-␤-CD(Table3).Moreover,thelow valueofthebindingconstant(Ka=20M⁻¹)isinagreementwith thatdeterminedwiththeBenesi–Hildebrandapproach(Fig.2C).

Insummary,theNMRresults(slightchemicalshiftchangesand smallbindingconstant)wereconsistentwiththeformationofa

weaklyassociatedinclusioncomplex,butdidnothelptoidentify thepartoftheCNDmoleculethatwasinsertedintheHP-␤-CDcav- ity.Furtherinsightsintothetopologyofthecomplexweretherefore obtainedusingmoleculardynamicssimulations.

4.5. Moleculardynamicssimulations

Asalreadydiscussed,clonidinecanbefoundinitsneutraland protonatedformsatpH7.4.Inaddition,CNDhastwotautomeric formsforitsneutralstate:aminoandiminospecies,withthelatter beingmorestable[14,15].Inthiswork,theinteractionsofthese threeformswithHP-␤-CDwereinvestigated.Ineachcase,eight representativepairs(CND-HP-␤-CD)wereconstructedinorderto explorethepossiblerelative orientations.Thetwenty-fourpairs

(6)

Fig.4.X-raydiffractogramsfor:HP-␤-CD(A);CND:HP-␤-CDcomplex(B),CND:HP-␤-CDphysicalmixture(C)andCND(D).

Fig.5. SchematicrepresentationofanHP-␤-CDmolecule,showingtheassignmentofhydrogensfromtheglucopiranoseunits.Thearrowsindicatehydroxypropylatoms involvedinhydrogenbonds(seetext).

weresimulatedupto100ns.Inordertoobtainaninitialpicture ofthebehaviorofthepairs,calculationwasmadeofthecenter ofmassdistances(d_CMS)betweenCNDandHP-␤-CD.Observation ofthetemporalevolutionofdCMSindicatedthatonlyafewofthe CND-HP-␤-CDpairsremainedassociatedforatleast10nsduring thesimulationrun.Therefore, itwasonlyconsideredthatasso- ciationtookplacewhenthefollowingcriteriawereobeyed:dCMS

<9Å,variationofd_CMS<3Å,andmaintenanceofthecomplexfor

≥10ns.Forthosepairsthatwerefoundtoremainassociatedfor

≥10ns,Fig.6showsthetemporalevolutionofdCMSandtheenergy ofinteraction(E_int,inkJ/mol)betweenCNDandHP-␤-CD,calcu- latedasthesumoftheenergiesassociatedwiththeelectrostatic andvanderWaalsinteractions.

Fortheneutralaminocase,onlytwopairsremainedassociated for∼20ns(Fig.6A).However,theyseparatedandnonewcom- plexeswereformedduringthesimulationrun,conﬁrmingthelow complexationafﬁnityoftheneutralaminoCNDform.

Ontheotherhand,intheimino(alsoneutral)case,fourinitial conﬁgurationsledtocomplexation(Fig.6B).Notably,theblackand redcurvesshowcomplexesthatremainedstableover40ns(black line)andthetotaltime(100ns,redline)ofthesimulationrun.For

thislastcase,aschematicdiagramofthecomplexisgiveninFig.7A, showingtheformationoftwohydrogenbonds,involvingtheimi- dazolegroupofCND(N7andN12)andatomsoutsidethecavity (OHandetheroxygenofthehydroxypropylgroup;seeFigure5)at theouterperimeterofHP-␤-CD.Fig.7Bshowsthetemporalevolu- tionofthehydrogenbondsforthethreenitrogensofagivenimino CND:HP-␤-CDcomplex.

Thefeaturesoftheprotonatedcasewereintermediatebetween theothertwocases(Fig.6).Inallthecaseswherecomplexationwas found,investigationwasmadeoftheinteractions thatprovided stabilization.

Inordertoclarifytheseresults,calculationwasmadeofthe occurrenceofhydrogenbondsbetweeneach ofthenitrogenous groups(NorNH)ofCNDandallthepotentialdonors(D)andaccep- tors(A)ofHP-␤-CD.Thecriteriausedforhydrogenbondformation wasaD-Acut-offdistanceof3.1ÅandanH-D-Aanglecut-offof 30^◦.Itwasconcludedfromthesimulationsthatcomplexforma- tionbetweenCNDandHP-␤-CDdependedontheCNDformand theinitialconditions,asfollows:

(7)

Fig.6.Moleculardynamicsimulations:center-of-mass(dCMS)distanceandinter- actionenergiesasafunctionoftimefortheamino(A),imino(B)andprotonated(C) formsofCND.Justthecomplexesthatremainedassociatedformorethan20nsare shownhere.

-CNDaminoform:hydrogenbondformationwassporadic,even forthetwopairsthatremainedassociatedover20nsofthesim- ulation.

-CNDiminoform:complexstabilizationwasstrongerthanforthe aminoform.Thelonger-lastingcomplex,whichremainedstable overtheentiresimulation,wasessentiallystabilizedbythefor- mationofhydrogenbonds,asillustratedinFig.7,whichshowsthe numberofhydrogenbondsformedasafunctionoftimeforN9H, N12H,andN7.Theaveragehydrogenbondformationwasquite high:0.1forN9H,0.83forN12H,and0.76forN7.Asimilarpattern ofhydrogenbondformationwasdetectedforthecomplexcor- respondingtothegreencurveinFig.6B,despitebeingweaker andless stable(foronly 20ns)thanthecomplexcorrespond- ingtotheredcurve.Thetwootherpairsthatformedcomplexes (withlongerdCMSdistancesandhigherenergies;Fig.6B)were notstabilizedbyhydrogenbonds.

-CNDprotonatedform:thetwocomplexesshowninFig.6Cpre- sentedhydrogenbondstabilizationduringtheﬁrst19and35ns ofthesimulation. Thesecomplexes thereforeremainedstable beyondthelifetimeofthehydrogenbonds,suggestingaslow hydrationprocess.

Fig.8.Histogramsofchlorine–hydrogendistancesfortwoselectedcasescorre- spondingtoamino(inblack)andprotonatedformsofCND.

Theresultsofthesimulationssuggestedthatwhenacomplex wasformed,theimidazoleringwasabletoenterthecyclodextrin cavity(especiallyintheiminocase,asdiscussedbelow),whileentry ofthearomaticringintothecavitywaspreventedbythepresence ofchlorine-containinggroups.

Itisknownthatchlorineatomscanformweakhydrogenbonds oftheO H···Cl Ctype,withatypicaldistanceof2–3Å[63,64], althoughthiskindofinteractionisuncommonandmainlyoccurs inintramolecularsituations[62].Here,theprotonatedandamino CNDformscouldinteractinthis way.Analysiswasmadeofthe distancesbetweenH7(seeFig.1),whichisonlypresentinthese twoforms,andeachchlorineatom.Theresultsshowedthatthe distancetooneofthechlorineswasalwaysintherangedescribed forweakhydrogenbonds.Arepresentativeexampleofeachofthese cases(consideringtheprotonatedandaminoforms)isshownin Fig.8,wheretwowell-deﬁnedpeakscanbeobserved.Thiskindof interactionincreasesthesterichindranceforentryofthemolecule intotheHP-␤-CDcavityandreducestheavailabilityofH3atoms forformationofintermolecularhydrogenbonds.

Inconclusion,themoleculardynamicsresultsshowedthatthe iminoformpresentedthehighestcapacityforassociationwithHP-

␤-CD.However,theprotonatedformistheprevailingspeciesat pH7.4,whichexplainsthelowassociationconstantdetermined experimentally.

Fig.7. (A)SnapshotofaninclusioncomplexbetweentheimineCNDformandHP-␤-CD,stabilizedbyhydrogenbonds;(B)numberofhydrogenbondsasafunctionoftime forthethreenitrogensofagiveniminoCND:HP-␤-CDcomplex.

(8)

Fig.9. DialysisexperimentsforCNDandCND:HP-␤-CDinHepesbuffer,pH7.4,at25^◦C.

Fig.10.Cytotoxicityassaysshowingthesurvivalof3T3cellstreatedfor24hwithCNDorCND:␤-CD(1:1molarratio).Theinsertshowsdetailofthe0–1mMconcentration range.MTTreductiontest;datapresentedaspercentageofcontrol(n=6).

4.6. Dialysisexperiments

Itisknownthatthediffusionrateofdrugscanbealtereddueto complexation.ThediffusionofCNDcomplexedtoHP-␤-CDthrough polycarbonatemembraneswascomparedtothatofthefreedrug

inHepesbufferatpH7.4,andfollowedupto300min,whenthe curvesreachedequilibrium(Fig.9).Almost90%ofthefreeCND wasreleasedfromthedonorcompartmentafter60min,compared to60%ofthecomplexedCND. Althoughthedifferencewasnot pronounced,whichmayhavereﬂectedthelowafﬁnityofCND:HP-

(9)

Fig.11.SensorialblockagetestinWistarratsafterintrathecaladministrationof0.15%clonidine(freeorHP-␤-CD-complexed),0.25%bupivacaine(BVC)andtheirassociation (BVC+CNDandBVC+CND:HP-␤-CD).MPE%referstothepercent-anesthetizedanimalsasafunctionoftime(n=7).

␤-CD,itwasneverthelesssigniﬁcant(AUCvaluesof25,541.4±21.7 forCNDand24,175.3±38.8forCND:HP-␤-CD,p<0.001),showing thatcomplexationdiminisheddiffusionrateofCNDinvitro.

4.7. Cytotoxicitystudies

Fig.10showsthecellviabilitycurvesfor3T3ﬁbroblastsafter treatmentwithfreeorcomplexedCND.TheIC50valuesdetermined forCNDandCND:HP-␤-CD(3.5and 2.8mM,respectively)were notsigniﬁcantlydifferent(p>0.05,Tukey’stest).HP-␤-CDwasnot foundtobecytotoxic,asshownpreviously[53].Hence,fromthe toxicitypointofview,complexationdidnotchangetheintrinsic effectofclonidine,whichisanimportantconsiderationintermsof itssafedisposal.

4.8. Analgesiatests

Antinociceptivetestsshowedthatintrathecaladministrationof 0.15%(20␮L)CND,whichwasbelowtheconcentrationthatinduces hypotension(100␮g)[7],causedsensoryblockadeofthecaudal nerve of rats during 120min (Fig.11).Complexation with HP-

␤-CDsigniﬁcantlyprolongedthedurationofanesthesiainduced byclonidine (180min,p<0.001,Tukey’s test).Whenassociated with0.25% bupivacaine, this CND concentration prolongedthe anestheticeffectto300min(p<0.01,Tukey’stest),inagreement withpreviousreports[3–9].Finally, thecombinedeffectofthe CND:HP-␤-CDcomplex and bupivacaine resulted in the exten- sionofanesthesiaforaslongas540min(p<0.001,Tukey’stest).

Theseﬁndingsreinforcedthepotentialforclinicalapplicationof theCND:HP-␤-CDcomplexinsurgicalprocedures,inassociation withlocalanesthetics.

5. Conclusions

A CND:HP-␤-CDhost-guest complexwith 1:1stoichiometry wasprepared.XRDandSEManalysesprovidedevidenceofcomplex formation,asshownbythelossofthecrystallinestructuresofthe purecompounds(CNDandHP-␤-CD).NMRexperimentsrevealed that in solution, the constant for binding of CND to HP-␤-CD waslow(20M⁻¹),althoughitwasnotpossibletoobtainmolec- ulardetailsconcerningthetopologyofthecomplex,duetopeak superpositioning.Moleculardynamicssimulationsconﬁrmedthe experimentalresults,withidentiﬁcationoftheformationofstable (thatremainedthroughthesimulationtime)inclusioncomplexes, notablyfortheneutraliminoformofCNDthatwasstabilizedby hydrogenbondingwiththecyclodextrin.

Inrelationtopossibleapplicationsofthecomplexasaphar- maceutical drugdelivery system,dialysisexperiments revealed thatcomplexationslowedtheCNDdiffusionthroughpolycarbon- atemembranesby15–20%,whileinvitrotestsrevealedthatthe

formation of CND:HP-␤-CDdid not alter the toxicity profileof CNDagainstfibroblastcellsinculture.Antinociceptivestudiescon- ductedinvivoconfirmedthatcomplexationincreasedtheadjuvant effectofclonidinewhenusedtogetherwithbupivacaine,alocal anestheticemployed insurgicalprocedures worldwide.Overall, theresultsobtainedinthisworksupportthecombineduseofthe CND:HP-␤-CDdeliverysystemandbupivacaineasanovelapproach toenhanceanesthesiainsurgicalprocedures.

Acknowledgements

FinancialsupportwasprovidedbyFAPESP(grant#2006/00121- 9)andCNPq (fellowshipawardedtoE.P.). Clonidinewaskindly donatedbyCristáliaProd.Quim.Farmac.Ltda.

References

[1]M.P.B.Simonetti,E.A.Valinetti,F.M.C.Ferreira,Clonidine:fromnasal decongestivetopotentanalgesic:hystoricalandpharmacological considerations,Rev.Bras.Anestesiol.47(1997)37–47.

[2]S.Armand,A.Langlade,A.Boutros,K.Lobjoit,C.Monrigal,R.Ramboatiana,A.

Rauss,F.Bonnet,Meta-analysisoftheefﬁcacyofextraduralclonidineto relievepostoperativepain:animpossibletask,Br.J.Anaesth.81(1998) 126–134.

[3]C.J.L.MacCartney,E.Duggan,E.Apatu,Shouldweaddclonidinetolocal anestheticforperipheralnerveblockade?aqualitativesystematicreviewof theliterature,Reg.Anesth.PainMed.32(2007)330–338.

[4]N.Elia,X.Culebras,C.Mazza,E.Schiffer,M.R.Tramèr,D.Phil,Clonidineasan adjuvanttointrathecallocalanestheticsforsurgery:systematicreviewof randomizedtrials,Reg.Anesth.PainMed.33(2008)159–167.

[5]D.M.Pöpping,N.Elia,E.Marret,M.Wenk,M.R.Tramer,Clonidineasan adjuvanttolocalanestheticsforperipheralnerveandplexusblocks:a meta-analysisofrandomizedtrials,Anesthesiology111(2009)406–415.

[6]K.Rhee,K.Kang,J.Kim,Y.Jeon,Intravenousclonidineprolongsbupivacaine spinalanesthesia,ActaAnaesthesiol.Scand.47(2003)1001–1005.

[7]J.F.N.P.Neves,G.A.Monteiro,J.R.Almeida,R.S.Sant’Anna,R.M.Saldanha,E.S.

Nogueira,F.L.Coutinho,M.M.P.Neves,F.P.Araújo,P.B.Nobrega,Analgesia pós-operatóriaparacesariana.Aadic¸ãodeclonidinaàmorﬁnasubaracnóidea melhoraaqualidadedaanestesia,Rev.Bras.Anestesiol.56(2006)370–376.

[8]I.Tuijl,W.A.Klei,D.B.M.Werff,C.J.Kalkman,Theeffectofadditionof intrathecalclonidinetohyperbaricbupivacaineonpostoperativepainand morphinerequirementsaftercaesariansection:arandomizedcontrolled trial,Brit.J.Anesth.97(2006)365–370.

[9]A.Parameswari,A.M.Dhev,M.Vakamudi,Efﬁcacyofclonidineasanadjuvant tobupivacaineforcaudalanalgesiainchildrenundergoingsub-umbilical surgery,IndianJ.Anaesth.54(2010)458–463.

[10]A.F.A.Braga,J.A.F.Frias,F.S.S.Braga,R.I.C.Pereira,S.M.M.C.Titotto,Spinal anesthesiaforelectiveceasareansection:useofdifferentdosesofhyperbaric bupivacaineassociatedwithmorphineandclonidine,ActaCir.Bras.28(2013) 26–32.

[11]B.Büttner,B.Ott,R.Klose,Effectsofclonidineaddedtomepivacainefor brachialplexusblockade,Reg.Anesthesiol.17(1992)45.

[12]F.J.Singelyn,M.Dangoisse,S.Bartholomée,Addingclonidinetomepivacaine prolongsthedurationofanesthesiaandanalgesiaafteraxillarybrachial plexusblock,Reg.Anesthesiol.17(1992)148–150.

[13]J.C.Crews,Newdevelopmentsinepiduralanesthesiaandanalgesia, Anesthesiol.Clin.NorthAm.18(2000)251–266.

[14]M.Remko,O.A.Walsh,W.G.Richards,Molecularstructureandgas-phase reactivityofclonidineandrilmenidine:twolayeredONIOMcalculations, Phys.Chem.Chem.Phys.3(2001)901–907.

(10)

Franco,A.F.A.Braga,E.dePaula,G.R.Tofoli,L.F.Fraceto,D.R.deAraújo, Sufentanil-2-hydroxypropyl-␤-cyclodextrininclusioncomplexforpain treatment:physicochemical,cytotoxicity,andpharmacologicalevaluation,J.

Pharm.Sci.101(2012)3698–3707.

[22]L.M.A.Pinto,L.F.Fraceto,M.H.A.Santana,T.A.Pertinhez,S.OyamaJunior,E.de Paula,Physico-chemicalcharacterizationofbenzocaine-␤-cyclodextrin inclusioncomplexes,J.Pharm.Biomed.Anal.39(2005)956.

[23]C.M.Moraes,P.Abrami,D.R.deAraújo,A.F.A.Braga,M.G.Issa,H.G.Ferraz,E.

dePaula,L.F.Fraceto,Characterizationof

lidocaine:hydroxypropyl-␤-cyclodextrininclusioncomplex,J.Incl.Phenom.

57(2007)313–316.

[24]C.M.Moraes,P.Abrami,E.dePaula,A.F.A.Braga,L.F.Fraceto,Studyofthe interactionbetweenS(−)bupivacaineand2-hydroxypropyl-␤-cyclodextrin, Int.J.Pharm.331(2007)99–106.

[25]R.A.F.Lima,M.B.deJesus,C.M.S.Cereda,G.R.Tofoli,L.F.Cabec¸a,I.Mazzaro,L.F.

Fraceto,E.dePaula,Improvementoftetracaineantinociceptiveeffectby inclusionincyclodextrins,J.DrugTarget.20(2012)85–96.

[26]N.S.Sosnowska,Fluorometricdeterminationofassociationconstantsofthree estrogenswithcyclodextrins,J.Fluoresc.7(3)(1997)195–200.

[27]A.A.A.Shaﬁ,S.S.Shihry,Fluorescenceenhancementof1-napthol-5-sulfonate byforminginclusioncomplexwith␤-cyclodextrininaqueoussolution, SpectrochimActa.72(2009)533–537.

[28]R.Banerjee,H.Chakraborty,M.Sarkar,Host-guestcomplexationofoxicam NSAIDswith␤-cyclodextrin,Biopolymers75(2004)355–365.

[29]Y.Matsui,S.Tokunaga,Internalreferencecompoundsavailableforthe determinationofbindingconstantsforcyclodextrincomplexesby¹HNMR spectrometry,Bull.Chem.Soc.Jpn.69(9)(1996)2477–2480.

[30]L.F.Cabec¸a,I.M.Figueiredo,E.dePaula,A.J.Marsaioli, Prilocaine-cyclodextrin-liposome:effectofpHvariationsonthe

encapsulationandtopologyofaternarycomplexusing¹HNMR,Magn.Reson.

Chem.49(6)(2011)295–300.

[31]L.M.Arantes,C.Scarelli,A.J.Marsaioli,E.dePaula,S.A.Fernandes, Proparacainecomplexationwith␤-cyclodextrinandp-sulfonicacid calix[6]arene,asevaluatedbyvaried¹H-NMRapproaches,Mag.Res.inChem.

47(2009)757–763.

[32]J.E.Thompson,APracticalGuidetoContemporaryPharmacyPractice,3rded., LippincottWilliamsandWilkins,Baltimore,2009.

[33]T.L.Lemke,D.A.Williams,Foye’sPrinciplesofMedicinalChemistry,6thed., LippincottWilliams&Wilkins,Baltimore,2008.

[34]H.J.C.Berendsen,D.vanderSpoel,R.vanDrunen,GROMACS:a

message-passingparallelmoleculardynamicsimplementation,Comp.Phys.

Commun.91(1–3)(1995)43–56.

[35]E.Lindahl,B.Hess,D.vanderSpoel,GROMACS3.0:apackageformolecular simulationandtrajectoryanalysis,J.Mol.Model.8(2001)306–317.

[36]D.vanDerSpoel,E.Lindahl,B.Hess,G.Groenhof,A.E.Mark,H.J.Berendsen, GROMACS:fast,ﬂexible,andfree,J.Comput.Chem.16(2005)1701–1718.

[37]B.Hess,C.Kutzner,D.vanderSpoel,E.Lindahl,GROMACS4:algorithmsfor highlyefﬁcient,load-balanced,andscalablemolecularsimulation,J.Chem.

TheoryComput.3(2008)435–447.

[38]C.Oostenbrink,A.Villa,A.E.Mark,W.F.vanGunsteren,Abiomolecularforce ﬁeldbasedonthefreeenthalpyofhydrationandsolvation:theGROMOS force-ﬁeldparametersets53A5and53A6,J.Comput.Chem.13(2004) 1656–1676.

[39]H.J.C.Berendsen,J.P.M.Postma,W.F.vanGunsteren,J.Hermans,Interaction modelsforwaterinrelationtoproteinhydration,in:B.Pullman(Ed.), IntermolecularForces,Reidel,Dordrecht,1981,pp.331–342.

[40]A.D.Becke,DensityfunctionalthermochemistryIII:theroleofexact Exchange,JChem.Phys.98(1993)5648.

[41]M.J.Frisch,G.W.Trucks,H.B.Schlegel,G.E.Scuseria,M.A.Robb,J.R.

Cheeseman,V.G.Zakrzewski,J.J.A.Montgomery,R.E.Stratmann,J.C.Burant,S.

Dapprich,J.M.Millam,A.D.Daniels,K.N.Kudin,M.C.Strain,O.Farkas,J.

Tomasi,V.Barone,M.Cossi,R.Cammi,B.Mennucci,C.Pomelli,C.Adamo,S.

[46]T.Mosmann,Rapidcolorimetricassayforcellulargrowthandsurvival:

applicationtoproliferationandcytotoxicityassays,J.Immunol.Methods65 (1983)55–63.

[47]S.D.AlSharari,F.I.Carroll,J.M.McIntosh,M.I.Damaj,Theantinociceptive effectsofnicotinicpartialagonistsvareniclineandsazetidine-Ainmurine acuteandtonicpainmodels,J.Pharm.Exp.Ther.342(2012)742–749.

[48]W.Misiuk,M.Zalewska,Spectroscopicinvestigationsontheinclusion interactionbetweenhydroxypropyl-␤-cyclodextrinandbupropion,J.Mol.

Liq.159(2011)220–225.

[49]C.Tablet,I.Matei,M.Hillebrand,Thedeterminationofthestoichiometryof cyclodextrininclusioncomplexesbyspectralmethods:possibilitiesand Limitations,in:StoichiometryandResearch—TheImportanceofQuantityin Biomedicine,AlessioInnocentiEd.,2012.

[50]Y.L.Loukas,V.Vraka,G.Gregoriadis,Drugsincyclodextrinsinliposomes:a novelapproachtothechemicalstabilityofdrugssensitivetohydrolysis,Int.J.

Pharm.162(1998)137–142.

[51]P.Mura,Analyticaltechniquesforcharacterizationofcyclodextrincomplexes insolidstate:areview,J.Pharm.Biomed.Anal.113(2015)226–238.

[52]D.R.deAraújo,S.S.Tsuneda,C.M.S.Cereda,F.D.G.F.Carvalho,P.S.C.Preté,S.A.

Fernandes,F.Yoikaichiya,M.K.K.D.Franco,I.Mazzaro,L.F.Fraceto,A.F.A.

Braga,E.dePaula,Developmentandpharmacologicalevaluationof ropivacaine-2-hydroxypropyl-␤-cyclodextrininclusioncomplex,Eur.J.

Pharm.Sci.33(2008)60–71.

[53]P.Mura,Analyticaltechniquesforcharacterizationofcyclodextrincomplexes inaqueoussolution:areview,J.Pharm.Biomed.Anal.101(2014)238–250.

[54]V.V.A.Castelli,G.Trivieri,I.Zucchelli,L.Brambilla,T.Barbuzzi,C.M.

Castiglioni.Paci,G.Zerbi,M.Zanol,Characterizationofaninclusioncomplex betweencladribineand2-hydroxypropyl-␤-cyclodextrin,J.Pharm.Sci.97 (2008)3897–3906.

[55]A.F.V.B.Soares,L.F.Fraceto,E.R.Maia,I.S.Resck,M.J.Kato,E.S.Gil,A.R.Sousa, L.C.Cunha,K.R.Rezende,Host?guestsystemof4-nerolidylcatecholin 2-hydroxypropyl-␤-cyclodextrin:preparation,characterizationand molecularmodeling,J.Incl.Phenom.Macrocycl.Chem.64(2009)23–35.

[56]E.dePaula,D.R.deAraújo,L.F.Fraceto,Nuclearmagneticresonance spectroscopytoolsforphysicochemicalcharacterizationofcyclodextrin inclusion,in:JieHu(Ed.),Cyclodextrins:ChemistryandPhysics,Res.

Signpost/TransworldRes.Network,2010(Org.)ISBN:978-81-7895-430-1.

[57]J.Y.Tsao,C.P.Wu,H.H.Tsai,K.C.Peng,P.Y.Lin,S.Y.Su,L.D.Chen,F.J.Tsai,Y.

Tsai,Effectofhydroxypropyl-beta-cyclodextrincomplexationontheaqueous solubilitystructure,thermalstability,antioxidantactivity,andtyrosinase inhibitionofpaeonol,J.Incl.Phenom.Macrocycl.Chem.72(2012)405–411.

[58]R.Grillo,N.F.S.Melo,L.F.Fraceto,C.L.Brito,H.G.Trossini,C.M.S.Menezes,E.I.

Ferreira,C.M.Moraes,Physico-chemicalcharacterizationofinclusion complexbetweenhydroxymethylnitrofurazoneand

hydroxypropyl-beta-␣-cyclodextrin,Quim.Nova31(2008)290–295.

[59]H.P.Mo,T.C.Pochapsky,Intermolecularinteractionscharacterizedbynuclear Overhausereffects,Prog.Nucl.Magn.Reson.Spectrosc.30(1997)1–38.

[60]A.Chen,M.Shairo,NuclearOverhausereffectondiffusionmeasurements,J.

Am.Chem.Soc.121(1999)5338–5339.

[61]G.A.Morris,Diffusion-orderedspectroscopy(DOSY),Encycl.Nucl.Magn.

Reson.9(2002)35–44.

[62]K.F.Morris,C.S.Johnson,Diffusion-ordered2-dimensional

nuclear-magnetic-resonancespectroscopy,J.Am.Chem.Soc.114(8)(1992) 3139–3141.

[63]C.B.Aakeröy,T.A.Evans,K.R.Seddon,I.Pálinkó,TheCHClhydrogenbond:

doesitexist?NewJ.Chem.23(1999)145–152.

[64]G.R.Desiraju,T.Steiner,TheWeakHydrogenBondinStructuralChemistry andBiology,vol.9,OxfordUniv.Press,UP,Chichester,1999.