Development and Characterization of an Amorphous Solid Dispersion of Furosemide in the Form

15 Pages·2017·1.52 MB·English

pharmaceutics
Article
Development and Characterization of
an Amorphous Solid Dispersion of Furosemide
in the Form of a Sublingual Bioadhesive Film to
Enhance Bioavailability
VivianaDeCaro1,* ,AlessiaAjovalasit2,FlaviaMariaSutera3 ,DeniseMurgia1 ,
MariaAntoniettaSabatino2andCleliaDispenza2,4
1 DipartimentodiScienzeeTecnologieBiologicheChimicheeFarmaceutiche(STEBICEF),
UniversitàdegliStudidiPalermo,Palermo,PA90123,Italy;[email protected]
2 Dipartimentodell'InnovazioneIndustrialeeDigitale,IngegneriaChimica,Gestionale,Informatica,
Meccanica,UniversitàdegliStudidiPalermo,Palermo,PA90128,Italy;[email protected](A.A.);
[email protected](M.A.S.);[email protected](C.D.)
3 SiSafLtd,InnovationCentre,NorthernIrelandSciencePark,Queen’sIsland,BelfastBT39DT,UK;
fl[email protected]
4 ConsiglioNazionaledelleRicerche,IstitutodiBiofisica–UOPPalermo,Palermo,PA90146,Italy
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:ThomasRades
Received:26May2017;Accepted:19June2017;Published:24June2017
Abstract: Administeredbyanoralroute,Furosemide(FUR),adiureticusedinseveraledematous
statesandhypertension,presentsbioavailabilityproblems,reportedasaconsequenceofanerratic
gastrointestinalabsorptionduetovariousexistingpolymorphicformsandlowandpH-dependent
solubility. A mucoadhesive sublingual fast-dissolving FUR based film has been developed and
evaluatedinordertooptimizethebioavailabilityofFURbyincreasingsolubilityandguaranteeing
agooddissolutionreproducibility. TheDifferentialScanningCalorimetry(DSC)analysesconfirmed
that the film prepared using the solvent casting method entrapped FUR in the amorphous state.
Asasoliddispersion,FURincreasesitssolubilityupto28.36mg/mL.Drugcontent,thickness,and
weightuniformityoffilmwerealsoevaluated. ThemeasuredYoung’sModulus,yieldstrength,and
relativeelongationofbreakpercentage(EB%)allowedfortheclassificationofthedrug-loadedfilmas
anelastomer. Mucoadhesivestrengthtestsshowedthattheforcetodetachfilmfrommucosagrew
exponentially with increasing contact time up to 7667 N/m2. FUR was quickly discharged from
thefilmfollowingatrendwellfittedwiththeWeibullkineticmodel. Whenappliedonsublingual
mucosa,thenewformulationproducedamassivedrugfluxinthesystemiccompartment. Overall,
theproposedsublingualfilmenhancesdrugsolubilityandabsorption,allowingforthepredictionof
arapidonsetofactionandreproduciblebioavailabilityinitsclinicalapplication.
Keywords: mucoadhesive film; sublingual absorption; amorphous solid dispersion; furosemide
bioavailability;transmucosaldelivery
1. Introduction
Hypertensiveemergenciesandhypertensiveurgenciesarecommonlyencounteredbyawide
varietyofclinicians. Promptrecognition,evaluation,andappropriatetreatmentoftheseconditionsare
crucialtopreventpermanentorgandamage[1].
Furosemide(FUR)isaloopdiureticusedorallyinthetreatmentofedematousstatesassociated
withcardiac,renal,andhepaticfailuresandinthetreatmentofhypertension[2]. Theusualdosage
Pharmaceutics2017,9,22;doi:10.3390/pharmaceutics9030022 www.mdpi.com/journal/pharmaceutics
Pharmaceutics2017,9,22 2of15
is40–120mg/day. Forthetreatmentofmildcasesofedema,dosesaslowas20mgcanbeeffective,
whereasforseverecases,dosesashighas600mg/daymayberequired[3].
Dataonsolubility,oralabsorption,andpermeabilityaresufficientlyexhaustivetoclassifyFUR
intoClassIVoftheBiopharmaceuticsClassificationSystem. Duetothecarboxylandsulfonamidic
groupsinthestructure, withpKaof3.8and9.6, respectively[4], FURshowsaverylowsolubility
in water that increases as a function of pH from 0.01 mg/mL at pH 2 to 1.9 mg/mL at pH 7.4 [5],
determiningahigherabsorptioninthegastricratherthanintestinaltracts. Aerraticbioavailability
ofabout37–51%andlargeinter-andintra-subjectvariabilitiesinrateandtheextentofabsorption
werealsoreported[6]. Absorptionfollowingoraladministrationisinfluencedbythedosageform,
underlyingdiseaseprocesses,andbythepresenceoffood. Theintra-subjectvariabilitywasthoughtto
bemainlydependentontheabsorptionprocess,sincerepeatedintravenous(i.v.) dosesshowedonly
marginalvariability[7].
Thebioavailabilityproblems,reportedasconsequencesofvariableanderraticgastrointestinal
absorption,areprobablyduetothelowandpH-dependentsolubilitytogetherwithvariousexisting
polymorphic forms of FUR. [8]. Indeed, FUR presents different polymorphic forms: four are true
polymorphs (I, II, III, IV), two are solvates (IV-DMSO and V-dioxane) and one is an amorphous
form[9,10]. Itiswellknownthatdifferentpolymorphicformsofanactiveadministeredintheoralor
topicalformcanmodifymanypropertieslikesolubility,stability,color,compressibility,flowability,
and workability and as a consequence can cause differences in bioavailability, toxicological safety,
clinicaleffectiveness,andproductiveefficiency[11].
DifferentapproacheshavebeenproposedtoenhanceFURabsorptionandbioavailabilitysuch
as co-crystallisation [12,13], solid dispersion [14], microemulsification [15], and supramolecular
complexesformation[16].
Oralmucosaldrugdeliveryisanalternativeandpromisingmethodofsystemicdeliverywhich
offersseveraladvantages[17,18]. Theoralmucosaishighlyvascularized;forthisreason,drugsthat
areabsorbedthroughitdirectlyenterthesystemiccirculation,bypassingthegastrointestinaltractand
first-passmetabolismintheliver.[19].Whensublingualmucosaadministrationischosen,arapidonset
ofactionresultsviaamorecomfortableandconvenientdeliveryroutethantheintravenousroute.[20].
Additionaladvantagesincludeanimprovedbioavailabilityforcertaindrugsandaneasyaccesstothe
absorptionsitessothatthedeliverysystemcanbeappliedandremovedeasily[21,22]. Fastdissolving
oralfilm,inparticularwhenplacedintheoralcavity,rapidlydisintegratesanddissolvestoreleasethe
medicationwithoutchewingandintakeofwater[23,24]. Itgivesquickabsorptionandbioavailability
comparabletointravenousadministration.
Bioadhesive sublingual formulations [25], such as tablets [26], patches, and films, have been
developed using mucoadhesive polymers that can establish a strong adhesive contact with the
mucosa, allowing for an increase in residence time of the delivery system and optimizing drug
bioavailability[19,27]. Thereleasekineticsofagivendrugfromapolymericmatrixcouldbegoverned
predominantlybythepolymermorphologyandexcipientspresentinthesystem[28].
Furthermore,acomparativestudyonpharmacokineticsandpharmacodynamicsofFURafter
administrationofthesametabletsforbothsublingualandoralroutes,usingthei.v. routeascontrol,
hasbeenreported. Theauthorsconcludedthattheinsignificantdifferencesobservedbetweenoraland
sublingualrouteswereduetotheslowdisintegrationoftheoraltabletthatproducesavariabledose
fractionswallowedinthesublingualenvironmentandthatadosageformsuitablyformulatedtobe
appliedonthesublingualmucosawouldimprovethebioavailabilityofFUR[29].
Asaconsequence,theaimofthisworkisthedesignofanadequatenewformulationcapableof
improvingthepharmacokineticsofFURwhenadministeredsublingually.
In this study, a mucoadhesive sublingual fast-dissolving film loaded with amorphous FUR
has been developed. The formulation was designed in order to optimize the bioavailability of
FURbyincreasingsolubilityandguarantyinggooddissolutionprofilereproducibilityandmassive
transmucosalabsorption.
Pharmaceutics2017,9,22 3of15
2. Experimental
2.1. Materials
FUR, Ph. Eur. grade, was purchased from Galeno srl (Carmignano, PO, Italy). Methacrylic
acid-methyl methacrylate copolymer (Eudragit® L-100) was kindly supplied by Rofarma (Milan,
Italy). PolyvinylpyrrolidoneK-90(PVPK-90),Sorbitol,Agar,Triethanolamine(TEA),andNaOHwere
purchased from Sigma-Aldrich (Milano, Italy). Propylene glycol was purchased from Farmalabor
(CanosadiPuglia,Italy).
BufferpH6.8solutionsimulatingsalivawaspreparedusingNaCl,KCl,KSCN,KH PO ,Urea,
2 4
Na SO 10H O, NH Cl, CaCl ·2H O, and NaHCO in distilled water according to Gal et al. [30].
2 4 2 4 2 2 3
Atotalof6mMphosphatebufferedsaline(PBS),Ca2+, andMg2+ freesolution, simulatingpH7.4
plasma, was prepared by dissolving KH PO , anhydrous Na HPO , and NaCl in distilled water.
2 4 2 4
A0.9%salinesolutionwaspreparedbydissolvingNaClindistilledwater.
AllchemicalsandsolventsofanalyticalgradewerepurchasedfromVWRInternational(Leuven,
Belgium) and used without further purification. Porcine mucosae were kindly supplied by the
MunicipalSlaughterhouseofVillabate(Palermo,Italy).
2.2. Methods
2.2.1. PreparationofMucoadhesiveFilm
TEA(450mg)andFUR(600mg)wereaddedtoanaqueoussolution(10mL)ofpropyleneglycol
(150mg),Sorbitol(80mg),andPVP-K90(40mg),andtheresultingsolutionwasstirreduntilcomplete
dissolution. Then,EudragitL100®(1g)andNaOH1M(5mL)wereaddedtothesolutionuntilafluid,
transparent,andhomogeneousviscoussolutionwasformed. Themixturewaspouredintoasilicon
moldhavinganareaof20.25cm2anddriedinanovenat40◦Cfor48h. Thesoformedfilms(plain
patches)werethenlefttoequilibrateatroomtemperatureandhumidityfor24h, checkedforany
imperfectionsorairbubbles,andcutbyabiopsypunchintodisks8.0mmindiameter(area0.5cm2).
Thesampleswerepackedinpolyethylenebags,heat-sealed,andstoredatroomtemperatureinaglass
containertomaintaintheintegrityandelasticityofthefilms.
2.2.2. FilmWeight,Thickness,andDrugLoadUniformity
Five disks from each batch were randomly cut and weighed. The diameter and thickness
were measured with a vernier caliper and an analog micrometer (Mitutoyo Italiana SRL, Milano,
Italy),respectively.
Drugcontentwasestimatedbydissolvingarandomlyselecteddisk,bysonication,intoa200mL
flaskfilledtovolumebydistilledwater. TheamountofFURreleasedfromthefilmwasmeasured
spectrophotometrically(UV/VisShimadzumodel1700instrument,Japan)atλ =331nm,(linearity
max
range0.005–0.1mg/mL,E water=0.158,corr. coeff. 0.999).
1%
Theuniformityofbatcheswasevaluatedbycalculatingtheaveragesandstandarddeviationsfor
alltheconsideredparameters.
2.2.3. DifferentialScanningCalorimetry(DSC)
Differential Calorimetric Analysis was performed on the patches and on their individual
components(sampleweightwasapprox. 8mg)byaPerkinElmerJadecalorimeterwithatemperature
rampfrom30◦Ctoabout250◦Cat10◦C/min,undernitrogenflow.
2.2.4. AttenuatedTotalReflectance(ATR)
ATR-FTIRspectrawererecordedonaFourierTransformInfraredSpectrometer(FTIR)(Spectrum
TwoFTIRspectrometer,PerkinElmer)equippedwithanATRunitplug-and-playwithadiamondcrystal
Pharmaceutics2017,9,22 4of15
forsurfaceanalysis. Spectrawereobtainedbyaccumulationof32scansbetween4000and450cm−1at
4.0cm−1resolutionandrationedtotheappropriatebackgroundspectra.
2.2.5. MechanicalTests
MechanicalpropertiesoffilmswereevaluatedusinganInstronUniversalTestinginstrument
(model3365equippedwith8500digitalcontrol,Instron,Norwood,MA,USA)witha1kgloadcell.
Aspecimenwithdimensionsof10×60×(0.9±0.12)mmwasheldbetweentwogripscoveredby
afeltpadtopreventslippageandpositionedatadistanceof2cm. Atensiletestwasperformedat
arateof100mm/min. Tensilestrengthandelongationatbreakweremeasured. Reporteddataare
averagedontheresultsofaminimumofeightspecimens.
2.2.6. SurfacepHofFilm
Randomlyselectedfilmdiskswereswollenfor2honthesurfaceofanagarplate,preparedby
dissolving2%(wt/vol)agarinwarmedsimulatedsaliva(pH6.8)understirringandthenpouringthe
solutionintoaPetridishuntilitgelledatroomtemperature.
Surface pH was measured using a pH meter (HI 2211 pH/ORP Meter, Hanna Instrument,
Woonsocket,RI,USA)byplacingpHprobeinclosecontactwiththewettedpatchsurface. Thesurface
pHstudywascarriedoutbyselectingthreedisks.
2.2.7. EvaluationoftheSolubilityofFUREntrappedintoFilm
In 1 mL of a buffer solution simulating saliva at pH 6.8, at 25 ± 0.1 ◦C, under stirring, small
amountsoffreeFURwereaddeduntilsaturation. Thesuspensionwaskeptundercontinuousstirring
for1h,andaftertheequilibriumwasreached,theremainingsolidFURwasremovedbycentrifugation
followedbyfiltrationthrougha0.45µmmembranefilter(MerkMillipore,MerckS.p.a.,Vimodrone
(MI),Italy).Theclearsolutionwassuitablydiluted(1:100)andanalyzedbyaUV-Visspectrophotometer
atλ =331nmusingtheappropriateblankandcalibrationcurve(linearityrange0.005–0.1mg/mL,
max
E =0.156,corr. coeff. 0.999).
1%salivapH6.8
ThesameexperimentalprotocolwasusedinordertoevaluatethesolubilityofFURincorporated
intothemucoadhesivefilm. Threedisks,8.0mmindiameter,containing11.5mgofFUReach,were
solubilizedin1mLofbuffertosimulatesalivaaspreviouslydescribed. Thesesuspensionswerethen
sonicatedatseveraltimeintervals,andaftertheequilibriumwasreached,theremainingsolidmaterial
wasremovedbyfiltrationthrougha0.45µmmembranefilter(MerkMillipore,MerckS.p.a.,Vimodrone
(MI),Italy). Theclearsolutionwasthenanalyzed. Theanalyseswereconductedintriplicate.
2.2.8. ExVivoMucoadhesionStrengthMeasurement
To perform the ex vivo mucoadhesive strength evaluation of the prepared film disks, the
modifiedtwo-armedphysicalbalancemethodwasused[31]. Porcinebuccalmucosaexcisedfromjust
slaughteredpigswasusedasmodeltissueandhandledwithoutanypre-treatment. Apieceofmucosa
wasgluedwiththeaidofcyanoacrylateresin(SuperAttakLoctite®,HenkelItaliaSrl,Milan,Italy)on
aglasssupportandkeptinavesselplacedinathermostaticbathat37◦C±1◦C.Thepatchwasfixed
atthelowersideofarubberstopperwithanadhesiveandattachedtothebalancepan. Beforestarting
themeasurements,themucosaltissuewaswettedwith50µLofsimulatedsalivaryfluidandthenthe
patchwasplacedonthetissuessoitjusttouchedthemucosalsurfaceandalightforcewithafingertip
wasappliedfor20s. Atemperatureof37◦C±1◦Cwasmaintainedthroughouttheexperiment. The
measurementsstarted5,10,15,and20minafterapplication,thusallowingfordifferenttimecontacts.
Theadhesivestrengthexpressedasthemass(g)requiredtodetachthefilmfrommucosalsurfacewas
calculatedaccordingtotheequation:
Forceofadhesion(N)=(g×9.81)/1000
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Then,detachmentforceswerecalculatedas:
Detachmentforce(N/m2)=Forceofadhesion(N)/Surfacearea(m2)
Themaximumadhesiveforcewasdeterminedastheaverageofthethreemeasurements(n=3).
2.2.9. SwellingandRadialErosionTests
Aswellingtestwasconductedbyplacingadryfilmdisk(0.5cm2ofarea)onaglasssupportinto
ananalyticalbalanceandweighed. Then,0.1mLofartificialsalivaatpH6.8wereaddedonthedisk
every5minfor50min. Ateverytimeinterval,afterremovaloftheexcesswaterwithafilterpaper,
theweightofthewetdiskwasassessed. Thetestwasperformedononediskofsixdifferentbatches.
Resultswerereportedasmeans±SD(n=6;p<0.05).
Theswellingdegree(SD)wascalculatedusingthefollowingequation:
SD%=(W −W )/W ×100
s d d
whereW correspondstothehydratedweightofthesampleandW representsthesampledryweight.
s d
Furthermore, adiskwasplacedonaglasspositionedongraphpaper. Theexperimentswere
startedbyplacing0.2mLofsimulatedsaliva(pH6.8,37◦C)onthediskandadding0.1mLofsaliva
every5min,forthefirst15min,andthenevery15min. Ateverytimeinterval,aphotographwas
takentoevaluateanychangeinthedisk’smorphology.
2.2.10. DrugReleaseStudies
Drugreleaseinbuffersolutionsimulatingsalivawasassessedusingtheflowthroughsystem
previouslydescribed[32]. Briefly,thesystemconsistsofacontainerforthebuffersolutionsimulating
saliva(100mL)fromwhichtheliquidisforcedintoaPlexiglassreleasechamber. Theflowrateof
salivawascontrolledbyaperistalticpump(Bio-RadEconoPump,Hercules,CA,USA)andmaintained
constantat0.5mL/min. Inthechamber,thesalivarylayerwettingthesidesofthediskwas0.1mm
thick. Thetemperaturewascontrolledbysubmergingthechamberandthecontainerinathermostatic
bath(37±0.1◦C).Thedrugamountinthesolutioncomingoutfromthereleasechamberwasthen
quantitativelydeterminedbyUVanalysisusingtheappropriateblankandcalibrationcurve. Results
wereaveragedonsixdisksfromsixdifferentbatchesoffilm. Everyexperimentwasconsideredatits
endwhenthecompletedissolutionofthediskoccurredandtheamountofdrugreleasedmatched
the original drug content of the disk. Release data were elaborated using Curve Expert v.1.4 and
Kaleidagraphv.3.5softwareandfittedtotheequationsappliedinreleasestudiesfrommatrixsystems.
Linearornon-linearleastsquaresfittingmethodswereusedtodeterminetheoptimumvaluesforthe
parameterspresentineachequation. Fittingswerevalidatedbyusingχ2. Apvaluelessthan0.05was
consideredtobestatisticallysignificant.
2.2.11. PermeationStudyofFURReleasedfromtheFilmthroughPorcineSublingualMucosa
Mucosal specimens consisted of tissue removed from the ventral surface of the tongue of
freshlyslaughtereddomesticpigs. Specimenswerepreparedasdescribedpreviously[17]. Briefly,
specimens, used within 2 h from animal sacrifice, were dipped for approximately 1 min in saline
solutionpreviouslywarmedto60◦C;theconnectivetissuewasthencarefullypeeledofffromthe
mucosa (slides 150 ± 25 µm thick) to obtain the heat-separated epithelium along with the intact
basal lamina. The thickness was measured using a digital micrometer. Before the beginning of
theexperiments,specimenswereequilibratedinphosphatebufferedsalinepH7.4(PBS)forabout
3 h at room temperature to remove biological matter which could interfere with drug analyses.
TheequilibrationmediumwasreplacedwithfreshPBSevery15min.
Pharmaceutics2017,9,22 6of15
Appropriate sections of mucosa were mounted in vertical jacketed, Franz type diffusion cells
(Permeagear,flatflangejoint,9mmorificediameter,15mLacceptorvolume,SESGmbH—Analysesysteme,
Bechenheim,Germany)usedasatwocompartmentopenmodel. Tissuedisks(12mmdiameter)were
equilibratedfor1hat37±0.1◦CaddingPBSinboththedonorandtheacceptorcompartment.Thisstep
wasfollowedbytheremovalofPBSfromthedonorcompartmentandreplacementwithonepatch
(8mmofdiameter)in0.4mLofsimulatedsalivaappliedtotheapicalsideofthemembrane. Atregular
timeintervals(30min),samples(0.5mL)werewithdrawnfromtheacceptorcompartmentandthe
sample volume was taken out and replaced with fresh fluid. Each experiment was carried out at
37 ± 0.1 ◦C for 6 h and repeated six times using different fractions of the same portion of tissue.
ThepermeatedFURwasdeterminedbyUVanalysisusingtheappropriateblankandcalibrationcurve
(linearityrange0.005–0.1mg/mL,E =0.152,corr. coeff. 0.999). Attestingconcentrations,
1%PBSpH7.4
PBS,artificialsaliva,andformulationcomponentsdonotinterferewithFURquantification.
Drugflux(Js)throughthemucosalmembranewascalculatedatthesteadystateperunitareaby
linearregressionanalysisofpermeationdatafollowingtherelationshipJs=Q/At(mg/cm2 h),where
Qistheamountofthedrugwhichpassesthroughthecelllayersintotheacceptorcompartment,Ais
theactivecross-sectionalareaavailablefordiffusion(0.636cm2),andtisthetimeofexposure(h).
Thepermeabilitycoefficient(Kp)wasthencalculatedaccordingtotheequationKp=Js/Cd(cm/h),
whereCdisthedrugconcentrationinthedonorcompartment(mgcm−3).
The analyses were performed on two disks of six different batches. Results were reported as
means±SD(n=12;p<0.05).
3. ResultsandDiscussion
3.1. FilmFormulation
Becauseoflowcostsandeaseofproduction,oneofthemostcommonapproachestogetdelivery
systemswithsuitabledrugreleaseistoembedthedruginanadequatematrix. Thefilmasmatrix
system is also considered the most suitable in order to obtain a solid dispersion of drugs in their
amorphousform. Inparticular,formulationofabioerodiblemucoadhesivefilminwhichFURwas
homogenouslydispersedhasbeendesignedwiththeobjectiveofimprovingdrugbioavailabilityby
anadequatedeliverythroughoutthemucosa.
Therefore, a readily water-soluble polymer such as Eudragit® L-100 was chosen for the
formulation as the matrixing polymer, together with PVP-K90 for conferring mucoadhesive
properties.Eudragit®L-100(MethacrylicAcidCopolymerTypeAUSP/NF,MethacrylicAcid—Methyl
MethacrylateCopolymer1:1Ph. Eur.) isabiocompatible,mucoadhesiveanionicpolymer. Itssolubility
iscloselyinfluencedbypH,andspecifically,increasesforpHvaluesabove6. Thispolymer,when
dispersedintheaqueousmixturecontainingalltheformulationcomponents,confersonthemixturea
lowviscosity,whichisusefulforpouringandhomogeneouslydistributingitintothemold,without
lossofproduct. Whendried,Eudragit®L-100isabletomaintainFURinitsamorphousformandis
usefulforpreparingdrugloadedfilmsthatensureafastreleaseofthedrug.
Thecharacteristicsofflexibilityandsoftnessofthefilmshavebeenconferredbytheintroduction
intheformulationofwettingagentsandplasticizers[33];inparticular,SorbitolandPropyleneGlycol
arechosenfortheirabilitytoretainwaterwithinthefilmandfortheirinhibitoryactivityonbacterial
growthespeciallywhentheyareinassociation.
Thequantitativecompositionoffilmshasbeenselectedonthebasisofourpreviousexperiences[34].
Filmswithdifferentmatrixcomponentratioshavebeentestedtoselectthecompositionwiththehighest
FURloadingcapacityandadequatecharacteristicsofmucoadhesionanddrugdischarge.
Mucoadhesive sublingual films of FUR have been prepared by the solvent casting method.
The method is simple, inexpensive, and does not imply the use of organic solvents. The method
hasbeenthemostappropriatetoobtainasoliddispersionofdruginamatrixsystemwithahigh
reproducibilityofresults.
Pharmaceutics2017,9,22 7of15
3.2. FilmWeight,Thickness,andDrugLoadUniformity
Thereproducibilityofthefilmspreparationhasbeenassessedbymeasuringtheaverageweight
(48.50 ± 1.46 mg), thickness (0.750 ± 0.022 mm), drug content (11.53 ± 0.43 mg), and surface pH
Pharmaceutics 2017, 9, 22 7 of 15
(7.2–7.4)ofdisks(area0.5cm2)fromdifferentbatches.Alldataareinaccordancewiththerequirements
oftheItalianPharmacopoeia[FUXIIed]andconfirmhighproductreproducibility.
3.3. Differential Scanning Calorimetry Analysis
3.3. DDiffreuregn-tpiaollySmcaenrn iinngteCraalcotriiomnest rwyeArnea liynsvisestigated using differential scanning calorimetry (DSC).
Thermograms of the single components of the film and their mixtures in the film have been
Drug-polymer interactions were investigated using differential scanning calorimetry (DSC).
recorded. FUR thermograms show a small peak at 219 °C characteristic of the onset of drug melting.
Thermogramsofthesinglecomponentsofthefilmandtheirmixturesinthefilmhavebeenrecorded.
This peak is followed by a sharp exothermic peak at 220 °C attributable to drug decomposition that
FURthermogramsshowasmallpeakat219◦Ccharacteristicoftheonsetofdrugmelting. Thispeakis
takes place upon melting [35]. The thermogram of sorbitol shows a typical sharp melting peak at
followedbyasharpexothermicpeakat220◦Cattributabletodrugdecompositionthattakesplace
about 100 °C, while Eudragit presents two endothermic peaks, at 66 and 220 °C, attributable to
uponmelting[35]. Thethermogramofsorbitolshowsatypicalsharpmeltingpeakatabout100◦C,
unbound water and to the condensation in anhydride, respectively. PVP is an amorphous polymer
while Eudragit presents two endothermic peaks, at 66 and 220 ◦C, attributable to unbound water
and the only feature in its thermogram is associated with the evaporation of absorbed water. The
and to the condensation in anhydride, respectively. PVP is an amorphous polymer and the only
thermogram of the FUR-loaded film (named “Film” in Figure 1) shows a group of endothermic
featureinitsthermogramisassociatedwiththeevaporationofabsorbedwater. Thethermogramof
peaks that can be attributed to the melting of sorbitol domains and a shallow and broad endothermic
theFUR-loadedfilm(named“Film”inFigure1)showsagroupofendothermicpeaksthatcanbe
peak remnant of FUR degradation. This last evidence supports the theory that the drug is embedded
attributedtothemeltingofsorbitoldomainsandashallowandbroadendothermicpeakremnant
within the polymer matrix mostly in the amorphous form. The disappearance of Eudragit peaks,
ofFURdegradation. Thislastevidencesupportsthetheorythatthedrugisembeddedwithinthe
when it is in the form of the film together with the other components, suggests that the film forming
polymermatrixmostlyintheamorphousform. ThedisappearanceofEudragitpeaks,whenitisin
process has removed the water, and condensation reactions are impeded or shifted to higher
theformofthefilmtogetherwiththeothercomponents,suggeststhatthefilmformingprocesshas
temperatures.
removedthewater,andcondensationreactionsareimpededorshiftedtohighertemperatures.
Film
g) PVP K-90
m
W/ Eudragit L-100
weight (ndo up) Sorbitol
w/E FUR
o(
at fl
e
H
50 100 150 200 250
T (°C)
Figure 1. Differential Scanning Calorimetry (DSC) thermograms of the Furosemide (FUR)-loaded
Figure1.DifferentialScanningCalorimetry(DSC)thermogramsoftheFurosemide(FUR)-loadedfilm
film and of all the individual components present in the film.
andofalltheindividualcomponentspresentinthefilm.
3.4. Attenuated Total Reflectance (ATR)
3.4. AttenuatedTotalReflectance(ATR)
The formation of FUR-TEA salt was verified through ATR-FTIR spectroscopy. Spectra of FUR,
TheformationofFUR-TEAsaltwasverifiedthroughATR-FTIRspectroscopy. SpectraofFUR,
FUR-TEA salt, unloaded film, and film loaded with FUR are shown in Figure 2a,b. In the spectra of
FUR-TEA salt, unloaded film, and film loaded with FUR are shown in Figure 2a,b. In the spectra
FUR-TEA and film loaded with FUR, a sharp peak at 1610 cm−1, associated with the carbonyl
of FUR-TEA and film loaded with FUR, a sharp peak at 1610 cm−1, associated with the carbonyl
stretching of the carboxylate anion (–COO−), is present (this peak is absent in the unloaded film)
stretching of the carboxylate anion (–COO−), is present (this peak is absent in the unloaded film)
together with a shift from 1141 cm−1 to 1159 cm−1 due to the stretching of the C–N band, indicating
togetherwithashiftfrom1141cm−1to1159cm−1duetothestretchingoftheC–Nband,indicating
that FUR is entrapped in the film as triethanolamine salt [36].
thatFURisentrappedinthefilmastriethanolaminesalt[36].
Pharmaceutics2017,9,22 8of15
Pharmaceutics 2017, 9, 22 8 of 15
(a) (b)
Figure 2. (a) Attenuated Total Reflectance-Fourier Transform Infrared Spectrometer (ATR-FTIR) full
Figure 2. (a) Attenuated Total Reflectance-Fourier Transform Infrared Spectrometer (ATR-FTIR)
spectra of FUR-loaded film, empty film, FUR, and FUR-Triethanolamine (FUR-TEA) salt. (b)
full spectra of FUR-loaded film, empty film, FUR, and FUR-Triethanolamine (FUR-TEA) salt;
(b)MMaaggnnifiificcaatitoionn ofo tfhteh e2020000–01–010000 c0mc−m1 p−o1rptioonrt ioofn thoef stpheecstrpae. ctra.
3.5. Mechanical Tests
3.5. MechanicalTests
Young’s modulus (E), yield strength (YS), and elongation at break (EB) are parameters that
Young’smodulus(E),yieldstrength(YS),andelongationatbreak(EB)areparametersthatprovide
provide an indication of the strength and elasticity of the patch and characterize the mechanical
anindicationofthestrengthandelasticityofthepatchandcharacterizethemechanicalperformance
performance of the film. These parameters are useful to evaluate the manageability of the film,
oftwhheicfihlm h.asT thoe bsee phaanradmleedt ebrys paaretieunstesf uwlitthooeuvt ablrueaatkeinthge. Imt iasn sauggegaebsitleidty thoaftt ha esufiiltmab,lew bhuicchcahl apsattcohb e
hanshdoleudldb hyavpea taie rnetlsatwivietlhyo huitghb rYeaSk ainndg .EIBt [is37s,3u8g]g. eTshtee dmtehaasturaedsu Yitoaubnleg’bs uMccoadlupluastc whassh 7o0u.2ld2 h± a7v.7e a
relaMtiPvae,l tyhhe iygiheldY SstraenndgtEhB w[a3s7 ,53.83] ±. T0.h55e MmPeaas, uanredd thYeo ruenlagt’isveM EoBd%u lwuassw 2a5s9 7± 00..2825%±. 7T.h7eM drPuag,-tlhoaedyeide ld
strefinlmgt hcawn abse 5cl.a3s±sifi0e.d5 5asM aPn ae,laasntodmthere [r3e9l]a. tiveEB%was259±0.85%. Thedrug-loadedfilmcanbe
classifiedasanelastomer[39].
3.6. Surface pH
3.6. SurfacepH
The surface pH of the films ranged from 6.71 to 6.93. Since the detected pH value is compatible
wiTthh ethseu orrfaalc ecapvHityo, fththereefi wlmills nroatn bgee adnfyr okmind6 .o7f1 itrorit6a.t9i3o.nS tion tcheet mheudcoesteacl tteisdsupeH. valueiscompatible
withtheoralcavity,therewillnotbeanykindofirritationtothemucosaltissue.
3.7. Solubility Test
3.7. SolubilityTest
The assessment of solubility in simulated saliva at pH 6.8 showed that FUR dissolves to a
maTxhiemausmse sosmf e3n.1t8o fmsogl/umbLil.i tyWihnesnim tuhlea tFedURsa lwivaasa tinpcHor6p.8orsahtoewd eidn ththaet FmUaRtrdixis sfoillmve sittso asomluabxiilmityu m
of3s.i1g8nimficga/nmtlyL .inWcrheeansethde uFpU tRo 2w8a.3s6i nmcgo/rmpoLr,a atepdprionxtihmeamtealytr nixinfeil-mfolidts hsioglhuebri ltihtyansi gthnei fvicaalnutel yobintacirneeadse d
upftoor 2th8.e3 f6reme gF/UmRL. ,approximatelynine-foldhigherthanthevalueobtainedforthefreeFUR.
3.83..E8.x EVxi vVoivMo MucuoacodahdehseiosinonS tSrternegntghthM Meaeasusurreemmeenntt
ThTehme umcuocaodahdehseisvievep rporpoperetriteiesso offs suubblliinngguuaall fifillmm aarree ooff ggrreeaatt iimmppoortratanncec easa sthtehye yinifnlufleunecnec teheth e
abialibtiylitoyf othf ethdeo dsoasgaegfeo fromrmto tob eber erteatainineedda attt thhee ssiittee ooff aaccttiioonn,, iinn iinnttiimmaatete ccoonntatactc twwitiht hthteh eabasbosroprtpiotnio n
membrane. Ex vivo mucoadhesion strength measurements were conducted with a modified
membrane.Exvivomucoadhesionstrengthmeasurementswereconductedwithamodifiedtwo-armed
two-armed physical balance. It also evaluated the variation in adhesive strength as a function of
physicalbalance. Italsoevaluatedthevariationinadhesivestrengthasafunctionofcontacttime.
contact time.
Theobtainedresults(Table1)areexpressedasforceofadhesionanddetachmentforce,whose
The obtained results (Table 1) are expressed as force of adhesion and detachment force, whose
equationswerepreviouslydescribed.
equations were previously described.
Table1.Forceofadhesionanddetachmentforceofthepatchafterdifferentcontacttimeonporcine
Table 1. Force of adhesion and detachment force of the patch after different contact time on porcine
buccalmucosa(n=3).
buccal mucosa (n = 3).
ContactCToimnteac(tm Tiinm)e (min) FFoorrccee ooff AAddhheessiioonn (N(N)) DetachDmeetnatc Fhomrceen (tNF/mor2c) e(N/m2)
5 0.098 1960
5 0.098 1960
10 0.1196 2392
10 0.1196 2392
15 0.2242 4485
15 0.2242 4485
20 20 00..33883344 7667 7667
PPhhaarrmmaacceeuuttiiccss 22001177,, 99,, 2222 99 ooff 1155
Pharmaceutics 2017, 9, 22 9 of 15
The mucoadhesive force grew with increasing contact time of the patch and the hydration of the
latterT. hITneh mpea murctuioccauodlaahdre,hs bievysei vifneo crfroceeracgseir ngegwre twwh eiwt htiitmihn ecin rcecoarnesiatnasgcintc gbo ecntotwanctetaectnitm ttihemeo epf otahft cethhpe aa pntcadht cmahn uadncotdhs teah,h eay nhd yredaxrtpiaootninoeonnf ottifha telh e
ilnactltareetrta.esrIe.n oInpf atphrateir cftuoicrluaclera,srb r, yebqiyun icirnreecdare staions idgnetgtha techhtei tmthieem pceoa cntocthna ctwatcebtre ebt weotbewseeenervnthe tedh p(eF apitgcauhtcrahen 3ad)n. md umcuocsoas,aa,n aenx epxopnoennetniatlial
incinrecaresaesoef othf ethfeo rfcoerscerse qreuqirueidretdo tdoe dtaecthacthh ethpea ptcahtcwh ewreeroeb osebrsveerdve(dF i(gFuigreur3e). 3).
9000
9000
8000
8000
7000
2e (N/m)26m)0007000
Detachment forc 345etachment force (N/000000000456000000000
D
3000
2000
2000
1000
0 5 10 15 20 25
1000
0 5 10 tim1e5 (min) 20 25
time (min)
Figure 3. Exponential trend of detachment force of the patch as a function of contact time on porcine
bFuigFcucigarlue mr3e.u 3Ec.xo Epsxaop.n oennetinatliatrl etnrednodf odfe dtaecthacmhemnetnfot rfcoercoef othf ethpea ptcahtcahs aas fau nfucnticotnioonf ocfo cnotancttactitm tiemoen opno prcoirnceine
bubcucaclcaml umcuoscao.sa.
3.9. Swelling and Radial Erosion Tests
3.9. Swelling and Radial Erosion Tests
3.9. SwellingandRadialErosionTests
The radial erosion test was carried out in order to measure the surface area of the sublingual
cavityT hTwehhreai cdrhaia dilsiea rleo fesfreioocnstiivoteensl tytwe csato svwceaarrser dice adbryroi eutdht ieon ufoitlr mdine rwotrohdemenre atdosi usmsroeeltauhsteuiorsneu rtofhacecc eusaursrre.f aaTcohefe at hrreeaads uioabfl l itenhrgeou ssaiuolbncla itvneigstuyt al
swhhcoaiwcvheitdiys tehwfafhet,ic cathifv teeisrly aecnfof eivncetiritvieadell ybsw ycetohlvleienrfigel dmo fb wtyhh eet hfnieldm fiis ltsmhoal utw toihcocenunor sdc ciiusnsr otshl.ueT tfhiioernsrt a o1dc0ic amulreisnr.o dTsuihoeen trotea stdhtieas hla oebwrsooesrdipottniho antte, st
oaff tsseharoliawvnae,id nt hittheia adlt,is sawkf teuelnrli dnanegr iognfoiettihsa elr afispwlmiedl lteihnraogts iooofcn ct huaerns dfii lnlmots hsthe oafift r wostcec1igu0hrmst iuinnn ttdhilue te hfitero sttot h1tae0l amdbiissnos ordpluuteitoi otnon o,t hfines aaalbbivosaou,rtpt 9ht0ieo n
mdiiosnkf (suFanilgidvuearr,eg to4he)e.s draispki dunerdoesrigooneasn rdaploidss eorofswioenig ahntdu nlotislst hoef wtoetaiglhdti susnotliul ttihoen ,tointaalb doiussto9l0utmioinn, (iFni gaubroeu4t) 9.0
min (Figure 4).

FFiigguurree 44.. TTiimmeelliinnee ffrroomm 00 ttoo 9900 mmiinn ooff tthhee rraaddiiaall sswweelllliinngg ooff tthhee ppaattcchh..
Figure 4. Timeline from 0 to 90 min of the radial swelling of the patch.
The data related to the weight variation showed that the weight of the disk increases in the first
Thedatarelatedtotheweightvariationshowedthattheweightofthediskincreasesinthefirst
25 minT-ihnet edravtaal rdeluaete dto t ot hthe ea wbseoigrphtti voanr ioaft iosnal sivhao waendd ththaet tchoen wseeqiguhetn ot fs twhee ldliinsgk. inAcfrteears etsh aint tthime fei,r st
25min-intervalduetotheabsorptionofsalivaandtheconsequentswelling. Afterthattime,however,
ho2w5e vmeirn, -tihnete wrveailg hdtu de etcor eathsee s adbusoe rtpot itohne odfi sssaolliuvtaio nan odf tthhee dcoonsasgeqe ufeonrmt sawnedl litnhge. rAemftoerv atlh oaft tthime e,
theweightdecreasesduetothedissolutionofthedosageformandtheremovalofthesolutionofthe
solhuotwioenv oefr ,t hthe ed rwuegi g(Fhitg duerecr 5e)a. ses due to the dissolution of the dosage form and the removal of the
drug(Figure5).
solution of the drug (Figure 5).
Pharmaceutics2017,9,22 10of15
Pharmaceutics 2017, 9, 22 10 of 15
Pharmaceutics 2017, 9, 22 10 of 15
200
200
%
% d
sed ease 150
eacr150
eight incrweight in
w 100
100
50
50
0
0 0 10 20 30 40 50 60
0 10 20 30 40 time5 0(min) 60
time (min)
Figure 5. Swelling index measured as percent of weight increased vs. time. Values are presented as
FFiigguurree 55.. SSwweelllliinngg iinnddeexx mmeeaassuurreedd aass ppeerrcceenntt ooff wweeiigghhtt iinnccrreeaasseedd vvss.. ttiimmee.. VVaalluueess aarree pprreesseenntteedd aass
means ± SD (n = 6).
mmeeaannss ±± SSDD ((nn == 66)). .
These data indicate that the film absorbs the saliva which is useful for the drug dissolution, but
TThheessee ddaattaa iinnddiiccaattee tthhaatt tthhee fifillmm aabbssoorrbbss tthhee ssaalliivvaa wwhhiicchh iiss uusseeffuull ffoorr tthhee ddrruugg ddiissssoolluuttiioonn,, bbuutt
the swelling of the film is not so great as to cause discomfort for the patient.
tthhee sswweelllliinngg ooff tthhee fifillmm iiss nnoott ssoo ggrreeaatt aass ttoo ccaauussee ddiissccoommffoorrttf foorrt thheep paattiieenntt..
3.10. Drug Release Studies
33..1100.. DDrruugg RReelleeaassee SSttuuddiieess
Usually, drug release from tablets is studied according to the official pharmacopoeias. Current
UUssuuaallllyy,, ddrruugg rreelleeaassee ffrroomm ttaabblleettss iiss ssttuuddiieedd aaccccoorrddiinngg ttoo tthhee oofffifciciiaallp phhaarrmmaaccooppooeeiiaass.. CCuurrrreenntt
test methods require large volumes of dissolution medium and are operated under sink or
tteesstt mmetehthododssre qreuqirueirlaer glearvgoel uvmoelusmofedsi sosof ludtiisosnolmuteidoinu mmaenddiuamre oapnedr aaterde uonpdeerratseindk uorndpseeru sdion-ks inokr
pseudo-sink conditions. These methods do not simulate the conditions prevailing in buccal
cposneduidtioo-nsisn.kT hceosnedmitieotnhso.d sThdeosne omtseimthuodlast edtho encootn dsiitmiounlsatper etvhaei licnognidnitbiouncsc alperenvvairiloinnmg einnt wbuhcecrael
environment where small amounts of liquid exist; non-sink conditions are more appropriate for
semnvailrloanmmoeunntt swohfelriqe usimdaelxl isatm;noounn-tssi nokf cloiqnudiidti oenxsisat;r enmono-rseinakp pcoronpdritiaiotensf oarrde emscorirbei nagppthroepbreihataev ifoorr
describing the behavior of medications. Indeed, for buccal dosage forms, an initial fast release cannot
odfesmcreidbiicnagt itohnes b.eIhnadveioedr ,off omrebduiccacatilodnos.s Iangdeefeodr,m fosr, bauncicnailt diaolsfaagset froerlemass,e acna innnitoiatlb feasmt reealesuasree dcawnnitoht
be measured with the existing official methods, and in vitro dissolution tests should be performed in
tbhee mexeiasstuinrgedo fwfiictiha lthmee ethxiosdtisn,ga nodffiicniavl imtroetdhiosdsos,l uatniodn inte vsittsrosh doiusslodlubetiopne rtfeosrtms sehdoiunlds mbea lplevrofolurmmeeds oinf
small volumes of dissolution medium. For these reasons, release tests were performed using a flow
dsmissaolll uvtoiolunmmese doifu dmis.soFluotriotnh emseedreiuamso.n Fso,rr ethleeassee reteasstosnws, erreelepaseer ftoersmtse wdeuresi pngerfaorflmowed tuhsrionugg ah flcoewll
through cell system able to simulate the sublingual conditions, in particular, the saliva turnover in
styhsrtoeumgha cbelell tsoysstiemmu alabtlee ttho esismuubllainteg uthael csuonbldinitgiounasl ,cionndpiatirotincsu, lainr, ptahreticsualliavra, tthuer nsaolvivear tiunrnthoeveorr ainl
the oral environment [34,40]. This study, assessing the drug release from the dosage form when
ethnev iororanlm eennvtir[o3n4,m40e]n.t T[h34is,4s0t]u. dTyh,ias sssteusdsiyn,g atshseesdsirnugg trheel edarsuegf rroemleatshee fdroomsa gtehef odromsawgeh efnoremx pwosheedn
exposed to small volumes of fresh salivary fluid that is continuously replaced, showed that FUR is
teoxspmosaeldlv tool usmmaesll ovfoflruemshessa olifv farreyshfl usaidlivthaaryt ifslucoidn ttihnauto ius sclyonrteipnluaoceudsl,ys hroepwleadcetdh,a sthFoUwRedis tdhiasct hFaUrgRe dis
discharged from film quickly; in particular, about 80% of FUR was released from the dosage form
fdroismchfialrmgeqdu fircokmly ;fiilnmp aqrutiicckullya;r ,ianb poaurtti8c0u%laor,f aFbUoRutw 8a0s%r eolef aFsUedR fwroams rtheleeadsoesda gferofmor mthew ditohsinag6e0 fmorimn.
within 60 min. Experimental results were reported in Figure 6.
Ewxiptheirnim 6e0n mtailnr. eEsxupltesrwimeerentraelp roerstuedltsin wFeirgeu rreep6o.rted in Figure 6.
100
100
d 80
e
FUR releasedof FUR releas 686000
e of age 40
ntagcent 40
perce per 20
20
0
0 0 20 40 60 80 100
0 20 40 60 tim80e (min) 100
time (min)
Figure 6. Cumulative percent of FUR released from patch in simulated saliva pH 6.8. Values are
FFiigguurree 66.. CCuummuullaattiivvee ppeerrcceenntt ooff FFUURR rreelleeaasseedd ffrroomm ppaattcchh iinn ssiimmuullaatteedd ssaalliivvaa ppHH 66..88.. VVaalluueess aarree
presented as means ± SD (n = 6).
pprreesseenntteedd aass mmeeaannss ±± SSDD ((nn == 66))..