Plasma disposition, milk excretion and parasitological efficacy of mebendazole in donkeys naturally infected by Cyathostominae

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ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Research

paper

Plasma

disposition,

milk

excretion

and

parasitological

efﬁcacy

of

mebendazole

in

donkeys

naturally

infected

by

Cyathostominae

Cengiz

Gokbulut

a

_,

_Dilek

_Aksit

b

_,

_Mario

_Santoro

c

_,

_Cristina

_Roncoroni

d

_,

_Ugo

_Mariani

c

_,

Francesco

Buono

e

_,

_Domenico

_Rufrano

f

_,

_Antonio

_Fagiolo

d

_,

_Vincenzo

_Veneziano

e,∗

a_Department_of_{Pharmacology,}_Faculty_of_Medicine,_Balikesir_University,_Balikesir,_Turkey

b_Department_of_Pharmacology_and_Toxicology,_Faculty_of_Veterinary_Medicine,_Balikesir_University,_Balikesir,_Turkey c_Istituto_{Zooproﬁlattico}_Sperimentale_del_Mezzogiorno,_Portici,_Italy

d_Istituto_{Zooproﬁlattico}_Sperimentale_Lazio_e_Toscana,_Rome,_Italy

e_Department_of_Veterinary_Medicine_and_Animal_Productions,_University_of_Naples_Federico_II,_Naples,_Italy f_CREA,_Consiglio_per_la_Ricerca_in_Agricoltura_e_l’analisi_{dell’Economia}_Agraria_Bella,_Potenza,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received8October2015 Receivedinrevisedform 25December2015 Accepted28December2015 Keywords: Donkey Mebendazole Pharmacokinetics Efﬁcacy Milk Cyathostominae

a

b

s

t

r

a

c

t

Mebendazole(MBZ)hasbeenlicensedforuseinhorsesanddonkeys,howevertherearenodata avail-ableintheliteratureregardingitspharmacokineticdispositionandefficacyindonkeys.Thisstudywas designedtodeterminetheplasmadisposition,milkexcretionandanthelminticefficacyofMBZindonkeys naturallyinfectedbyCyathostominae.Theanimalswereallocatedtothreegroups,eachofsixdonkeys. Onegroupwasuntreatedcontrol(C-group)andtheothersweretreatedusingapasteformulationofMBZ administeredperosatthemanufacturer’srecommendedhorsedosageof10mg/kgbodyweight(MBZ1) andatthedoublehorsedosage20mg/kgbodyweight(MBZ2).Bloodandmilksampleswerecollected atvarioustimesbetween1hand120hposttreatmentandanalyzedbyhighperformanceliquid chro-matographywithphotodiodearraydetector.IndividualFECs(FaecalEggCounts)wereperformedoneach animalbeforethetreatment(day-3)andweeklyfromday7untilday56posttreatmentusinga mod-ifiedMcMastertechnique.TheplasmaconcentrationsandsystemicexposureofMBZindonkeyswere relativelylowercomparedwiththeothermethylcarbamatebenzimidazoles.Dose-dependentplasma dispositionsofMBZwereobservedattheincreaseddosage(10mg/kgvs20mg/kg)indonkeys.MBZwas notdetectedinanymilksamplesatadosageof10mg/kg.However,theparentdrugreached0.01␮g/ml peakmilkconcentrationat10.66handAUCmilk/AUCplasmavaluewas0.18±0.02atadosageof20mg/kg

bodyweight.ThisstudyindicatedthatperosadministrationofMBZhasaminimaldispositionrateinto themilkandmaybeusedinlactatingdonkeyswithzeromilk-withdrawalperiod.TheresultsofFECRT forbothMBZdosageswereefﬁcient(>95%efﬁcacy)untilday28.ThistrialdemonstratesthatMBZoral pasteathorsedosage(10mg/kgB.W.)waseffectiveandsafetyforthetreatmentofCyathostominaein donkeys.Therefore,similardosageregimensofMBZcouldbeusedforhorsesanddonkeys.

1. Introduction

Fewdrugshavebeenlicensedforuseindonkeys(Lizarragaetal., 2004).Onlylimitedinformationisavailableonthe pharmacokinet-icsofanthelminticdrugsincludingsomebenzimidazoles(Gokbulut etal.,2006),endectocides(Gokbulutetal.,2005,2011,2013)and tetrahydropyrimidines(Gokbulut etal.,2014), asdonkeyswere oftenneglectedspeciesindomesticanimals.Becauseofthelack

∗ Correspondingauthor.Fax:+390812536280.

E-mailaddresses:vincenzo.veneziano@unina.it,vinvene@unina.it

(V.Veneziano).

ofregistereddrugsfordonkeys,anthelminticslicensedforhorses orruminantsareusedfortreatmentofparasiticinfectionsinthis specieswithsamedosage.Ithasbeenreportedthatdonkeyshavea greatercapacitytometabolizecertaindrugscomparedwithhorses; thus,higherdosageorshorterintervalscouldberequiredfor reach-ingandmaintainingeffectivedrugconcentrations,althoughthere aresomeexceptions(Matthewsetal.,1997;Coakleyetal.,1999; Pecket al.,2002,1997;Lizarragaetal.,2004;Kumetal.,2009; Grosenbaughetal.,2011;Sekkinetal.,2010).

Mebendazole (MBZ, Methyl 5-benzyl-1H-benzimidazole-2yl-carbamate) belongs to the chemical class of benzimidazole methylcarbamatesandhasbeenusedformorethan40yearsin vet-erinaryandhumanmedicinetotreatarangeofinternalparasitic http://dx.doi.org/10.1016/j.vetpar.2015.12.031

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infectionswithbroad-spectrumofactivity.Likeother methylcar-bamatebenzimidazolessuchasalbendazoleandfenbendazole,the poorwatersolubilityofMBZreducesﬂexibilityfordrug formula-tion,allowingitsformulationonlyastablets,paste,orsuspensions perosadministrationinanimalsandthislimitsitsabsorptionand dispositioninthebody(McKellarandScott,1990).Dosage recom-mendationsvaryfrom5mg/kgto50mg/kgdependingbyhostand parasiteinfection.TherecommendeddosageofMBZis5–10mg/kg per osroute in horses (McKellar and Scott, 1990). In addition, 20mg/kgisrecommendedforthetreatmentoflungworm Dicty-ocaulusarnﬁeldiindonkeysandthisshouldbeadministeredfor5 consecutivedays(ClaytonandNeave,1979).

Donkeymilkisgaining popularityin somecountriessuchas Italy,FranceandBelgium(Venezianoetal.,2011)sinceitisthe closestmilktohumanbreastmilkwithhighlactoseratiosandlow fatcontent.Itisidealforfeedinginfantsandinparticularthe redis-coveryofdonkeymilkasafoodsourceforchildrenaffectedbycow milkproteinallergy—CMPA(Iaconoetal.,1992;Vincenzettietal., 2008).Although,MBZhasbeenlicensedforuseinhorsesand don-keys,therearenodataavailableintheliteratureconcerningits pharmacokineticdispositioninanyequinespecies.Therefore,the presentstudywasdesignedtodeterminetheplasmadisposition, milkexcretion,anthelminticefﬁcacyandeggreappearanceperiod (ERP)ofMBZindonkeysfollowingperosadministrationsatthe dosagesof10mg/kgand20mg/kgbodyweight(B.W.).

2. Materialandmethods

2.1. Studyanimals

Eighteen cross-breed female milking donkeys with a mean age (±standard deviation) of 10±4 years, weighting in mean (±standarddeviation)294±49kgwereusedinthepresentstudy. The bodyweight of each animal was estimated 1 day prior to treatment(day-1)usingthenomogramproposedbyTheDonkey Sanctuary(2003).Donkeysweremilkedonceadayand,themilk productionmeasuredbeforeandthroughoutthetrialrangedfrom 1500to1800ml/day.Thestudyanimalsweretaggedfor identiﬁca-tionusinganumberedheadcollar.Duringtheentireexperimental periodthedonkeyswerehousedcommunallyinanoutdoorpen withapermanentandhillypasture;supplementaryfeedingwas givensuchashayandconcentrate(barley,wheatbran,beetpulp, oatmeal,carob).Theanimalshadahistoryofgrazingpasture con-taminated withequine nematode parasites and have not been treated with any anthelmintics during the previous 6 months. Faecal examinations (individual Faecal Egg Counts and pooled coprocoltures)performedbeforethebeginningofthestudy(days -14and-3)showedindividualcounts>500eggspergram(EPG)and highprevalenceofCyathostominaeinallstudieddonkeys.Water wasprovidedadlibitumthroughoutthecourseofthestudy.This investigationwasapprovedbytheAnimalEthicCommitteeof Uni-versityofNaplesFedericoII.

2.2. Experimentalgroups

Onday-3,theexperimentalanimals(n=18)hadanaverageof 1520±522EPG.Theanimalswererankedfromlowesttohighest epgcounts.Based onincreasingepgcounts,replicatesof3 ani-malswereformed.Withineachreplicate,animalswererandomly assignedtotreatment.The18selecteddonkeyswereassigned con-secutivelytothefollowing treatmentgroupsof6animalseach: mebendazolepastehorsetreatedgroup(MBZ1group), mebenda-zolepastedoublehorsedosagetreatedgroup(MBZ2group)and untreatedcontrolgroup(C-group).

Thethree groups weremaintainedtogetherunderthesame conditions. In particular, they co-grazed on the same pasture throughoutthestudyandtherewasnotanychangeofdietduring thelactation.

2.3. Treatmentprocedures

Commercially available equine formulation of mebendazole paste(TelminTM_,_20%,_Janssen,_Italy)_licensed_for_horses_was admin-isteredtodonkeysorallyatthehorsedosage10mg/kgB.W.(MBZ 1)andatthedoublehorsedosage20mg/kgB.W.(MBZ2).Foreach animal,thedosagewascalculatedonthebasisofitsbodyweight. Allgroupsreceivedasingletreatment.

DonkeysoftheC-groupdidnotreceiveanytreatmentbutwere subjectedtothesamehandlingproceduresasthedonkeysofthe treatedgroups,receivingperosaquafontis.

Alltreated animalswereintermittently observedforadverse reactionsfor3h(threetimes)duringthedayoftreatment(day0) andthenweeklyuntiltheendofthetrial.

2.4. Samplingprocedures

Heparinisedbloodsamples(10ml)werecollectedbyjugular venipuncturepriortodrugadministrationand1,2,4,8,12,16,24, 30,36,48,56,72,96,and120hthereafter.Individualmilksamples (15ml)werealsocollectedthroughoutthebloodsamplingperiod, beforedrugadministrationandthereafteratpriortodrug adminis-trationand8,12,24,36,48,72,96and120hposttreatmentinorder tothedeterminationofdrugmilkexcretion.Bloodsampleswere centrifugedat3000rpmfor30minandplasmawastransferredto plastictubes.Alltheplasmaandmilksampleswerestoredat−20◦_C untilestimationofdrugconcentration.

Faecalsamplesweretakenfromtherectum, orfromfreshly voided,fromeachstudyanimal,werestoredinarefrigerator(4◦C) andwithin12hindividualfaecaleggcounts(FEC)wereperformed, beforethestartofthetrial(day-3),atdays7,14,21,28,35,42and 56aftertreatment(Nielsenetal.,2010).

2.5. Analyticalprocedures

Astocksolution(100␮g/ml)ofpurestandardofMBZ(Sigma,St. Louis,MO,USA)waspreparedusingacetonitrile(Sigma,St.Louis, MO,USA)asthesolvent.Thiswasdilutedtogive0.01,0.05,0.1, 0.5,1,5␮g/mlstandardsolutionsforplasmaandmilksamplesfor calibrationasstandardcurvesandtoaddtothedrug-freeplasma andmilksamplestodeterminetherecovery.

TheplasmaandmilkconcentrationsofMBZwereanalysedby highperformanceliquidchromatography(HPLC)withphotodiode arraydetectorfollowingliquid–liquidextractionprocedure accord-ingtoapreviouslydescribedmethod(Dawsonetal.,1982).Briefly, drug-freeplasmaormilksamples(1ml)werespikedwith stan-dardstoreachthefollowingfinalconcentrations:0.005,0.01,0.05, 0.1,and0.5␮g/mL.Ammoniumhydroxide(100L,0.1N,pH10)was addedto10ml-groundglasstubescontaining1mlspikedor exper-imentalplasmaormilksamples.Aftermixingfor15s,6mldiethyl etherwasadded.Thetubeswereshakenonaslowrotarymixer for10min.Aftercentrifugationat3000×gfor10min,thediethyl etherlayer(5ml)wastransferredtoathin-walled10ml-conical glass tubeand and concentrated to dryness at 40◦C in a sam-pleconcentrator(Maxi-dryplus,HetoLab.Equipment,Denmark). Thedryresiduewasre-suspendedwith250␮Ldimethyl sulphox-ide(DMSO). Then thetubes were placed in anultrasonic bath andfinally,100␮Lofthis solutionwereinjected intothe chro-matographicsystem.Themobilephaseconsistedofacetonitrileand 0.05Maqueousammoniumphosphatebuffer(50:50,v/v)andwas delivered(1100SeriesQuatPump,Agilent,Waldron,Germany)ata

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Table1

Mean(±SD)plasmaandmilkpharmacokineticparametersofmebendazole(MBZ)followingperosadministrationtodonkeysat10mg/kgbodyweight(n=6)and20mg/kg bodyweight(n=6). Parameters Plasma (10mg/kg) Plasma (20mg/kg) Milk (10mg/kg) Milk (20mg/kg) t1/2␭z(h) 11.97±4.38 13.13±3.85 – 8.40±3.54 tmax(h) 7.33±3.93 8.00±0.00 – 10.66±3.29 Cmax(␮g/ml) 0.04±0.01 0.07±0.01 – 0.01±0.00 AUC0→∞:(␮gh/ml) 0.78±0.35 1.42±0.20 – 0.29±0.04 AUMC0→∞:(␮g.h2_/ml) _19.49_±_7.86 _33.78_±_10.38 _– _7.08_±_0.90 MRT0→∞:(h) 20.34±7.59 23.43±5.55 – 17.63±8.74

AUCmilk/AUCplasma – – – 0.18±0.02

Cmax-milk/Cmax-plasma – – – 0.20±0.03

Cmax:peakplasmaconcentration;tmax:timetoreachpeakplasmaconcentration;AUC0→∞:areaunderthe(zeromoment)curvefromtime0toinﬁnity,AUMC0→∞:area

underthemomentcurvefromtime0toinﬁnity;MRT0→∞:meanresidencetime;t1/2␭z:terminalhalf-life.

flowrateof1ml/min.AnucleosilC18analyticalcolumn(Luna,3␮m, 150mmx4.6mm,Phenomenex,Macclesfield,Cheshire,UK)with nucleosilC18guardcolumn(Phenomenex,Macclesfield,Cheshire, UK)wasusedforanalysisofthemolecules.Photodiodearray detec-tion(1100Series,Agilent,Waldron,Germany)wasatawavelength of254nm.

2.6. Analyticalmethodvalidation

TheanalyticalmethodsusedforMBZinplasmaandmilk sam-pleswerevalidatedpriortothestartof thestudies.Calibration graphforMBZintherange0.005–0.5␮g/mLplasmaandmilkwere preparedusingdrug-freeplasmaandmilksamplesfromdonkeys. Theanalyte wasidentiﬁed withtheretention times of pure referencestandard.Recoveriesofthemoleculeunderstudywere measuredbycomparisonofthepeakareasfromspikedplasmaand milksampleswiththeareasresultingfromdirectinjectionsof stan-dardspreparedinacetonitrile.Theinterandintra-assayprecision oftheextractionandchromatographyprocedureswereevaluated byprocessingreplicatealiquotsofdrug-freedonkeyplasmaand milksamplescontainingknownamountsofthedrugondifferent days.

2.7. Coprologicalexaminations:anthelminticefﬁcacyandegg reappearanceperiod(ERP)

Individualfaecalegg countsweredeterminedusing a modi-ﬁedMcMastertechniquewithadetectionlimitof10EPG,usinga Sheather’ssaturatedsugarsolutionwithaspeciﬁcgravityof1.250 (ReinemeyerandNielsen2013;LesterandMatthews,2014).

Oneachsamplingday,individualfaecalsampleswereincubated at27◦Cfor7–10daysforlarvalidentification.Onlybeforethestart ofthetrial(days-14 and-3)wereperformedpooled coprocol-tures.Thirdstagelarvaewereidentifiedusingthemorphological keysproposedbyM.A.F.F.(1986).Whenacoprocolturehad100or lessthirdstagelarvae,allwereidentified;whenacoprocolturehad morethan100larvae,only100wereidentified.

TodeterminetheefﬁcacyofMBZagainstintestinalstrongylesat eachfaecalsamplingtime,arithmeticmeanofEPGwascalculated followingtheWAAVPguidelines(Colesetal.,1992,2006).Foreach groups(MBZ1–MBZ2)percentefﬁcacy(%)wascalculatedinterms ofFaecalEggCountReduction(FECR)atthedifferentdaysaccording totheformula:

Efﬁcacy(%)=meanEPGcontrolgroup−meanEPGtreatedgroup meanEPGcontrolgroup ×100

ThecutoffvaluechosenforestablishingefﬁcacywasFECR>90% asreportedbyKaplanandNielsen(2010)forbenzimidazoledrugs. Inaddition,95%conﬁdenceinterval(CI)wereincludedtogivea moreaccurateindicationofthedata(Vidyashankaretal.,2007):

LowerCIof80%wasselectedforclassifyresistanceforMBZ treat-ments.

TheERPwasevaluatedassuggestedbytheAmerican Associ-ation ofEquinePractitioners (AAEP)Parasite ControlGuidelines (Nielsenetal.,2013)anditwasdeﬁnedforbenzimidazolesasthe intervaltime(expressedinweekspost-treatment)whenthe per-centreductioninFECdecreasesbelowacutoffvalueof80%based onthegrouparithmeticmeanFEC.Theexpectedcyathostominegg reappearanceperiodis28–35days(4–5weeks)asindicatedfor benzimidazolesbyNielsenetal.(2013).

2.8. Pharmacokineticsandstatisticalanalysisofdata

Theplasmaconcentrationvstimecurvesobtainedaftereach treatmentinindividualanimals,wereﬁttedwiththeWinNonlin softwareprogram(Version5.2,PharsightCorporation,Mountain View,CA,USA).Thepharmacokineticparametersforeachanimal wereanalysedusingnon-compartmentalmodelanalysisfor per osadministration.Themaximumplasmaconcentration(Cmax)and timetoreachmaximumconcentration(tmax)wereobtainedfrom theplotted concentration-time curveof each drugin each ani-mal.Thetrapezoidalrulewasusedtocalculatetheareaunderthe plasmaconcentrationtimecurve(AUC).Themeanresidencetime (MRT)wascalculatedbythelineartrapezoidalrulewith extrap-olationtoinﬁnity.Similarequationswereusedtodeterminethe pharmacokineticsofMBZinmilk.

The pharmacokineticparameters are reported as mean±SD (standard deviation). Pharmacokinetic parameters lognormally distributedwerestatisticallycomparedwithaone-wayanalysis ofvariance (ANOVA).Allstatisticalanalyseswereperformedby usingMINITABforWindows(release12.1,MinitabInc.,State Col-lege,PA,USA).Meanvalueswereconsideredsigniﬁcantlydifferent atP<0.05.

For comparison of the anthelmintic efﬁcacy of both MBZ dosages,statisticalanalysisofdatawereperformedonarithmetic mean(AM)EPGcountsusingtheparametrict-testtocompare dif-ferencesbetweentreatmentgroupsforsigniﬁcanceattheP<0.01 level.

3. Results

Clinically,noadversereactionwasobservedinanyofthe don-keystreatedwithMBZduringthestudy.Theanalyticalprocedures and HPLC analysis of MBZ were validated. The linear regres-sionlinesforMBZintherangebetween0.005–0.5␮g/mlshowed correlation coefﬁcients of 0.9987. The mean recoveriesof MBZ fromplasmaandmilkwere88.87%±4.37and75.33%±5.32.The detectionlimitoftheanalytical techniquewas0.001_␮g/ml;the quantiﬁcationlimitwas0.005␮g/mlforplasmaandmilksamples. Theinter-andintra-assayprecisions(mean±SD)oftheanalytical

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Table2

Strongyleeggcountsineggspergram(EPG)andpercentagereductionsinfaecaleggcounts(FECR)fordonkeystreatedwithMBZpasteatahorsedosage−10mg/kgB.W. (MBZ1—group)andfordonkeystreatedwithmebendazolepasteatadoublehorsedosage—20mg/kgB.W.(MBZ2-group),comparedwithcontroluntreatedgroup(C-group) ateachtimestudypoints.

DayCgroup MBZ1

group

PvalueFECR(%)Upper–lower95%CIMBZ2

group PvalueFECR(%)Upper–lower95%CI EPG AM EPG Range EPG AM EPG Range EPG AM EPG Range −31347 1000–1800 1680 900–2240 0.1492– – 1533 730–2680 0.5858– 7 857 450–1170 3 0–20 0.000099.6 100–98.4 10 0–40 0.000098.8 99.9–97.1 14 942 500–1340 3 0–20 0.000099.7 100–98.4 7 0–30 0.000099.3 99.9–97.8 21 1185 890–1450 12 0–30 0.0000 99.0 99.7–98.3 12 0–30 0.000099.0 99.7–98.0 28 1528 1110–2030 23 10–50 0.000098.5 99.2–97.5 32 10–80 0.000097.9 99.1–95.2 35 1157 610–2000 175 10–340 0.000784.9 94.3–80.8 128 10–230 0.000488.9 95.9–82.9 42 982 520–1370 378 190–610 0.001561.5 90.7–65.8 305 40–450 0.000868.9 89.4–62.7 49 945 600–1680 570 80–970 0.104036.5 80.9–41.4 472 50–1100 0.054950.1 86.1–32.1 56 738 530–1130 648 180–1170 0.606512.2 78.6–37.5 683 70–1000 0.7509 7.5 75.9–17.5

*_Parametric_t-test_(MBZ-groups_vs_C-group)_values_are_{signiﬁcantly}_different_P_<_0.001_–_arithmetic_means_(AM)_–_Conﬁdence_Intervals_(CI).

techniquepresentedacoefﬁcientofvariationof6.11%±2.22and 4.91%_±3.12forplasmaand5.34%_±2.88and5.84%_±3.65formilk analysis,respectively.

Pharmacokineticparametersof MBZinplasmaand milk fol-lowingperosadministrationatdosages of10and20mg/kgare giveninTable1.Themeanplasmaandmilkconcentrationvstime curvesareshowninFig.1.Dose-dependentplasmadispositionsof MBZwereobservedattheincreaseddosage(10mg/kgvs20mg/kg) indonkeys.MBZreachedthepeakplasmaconcentrations(Cmax: 0.04and0.07␮g/ml)at7.33and8.00hafteratdosagesof10and 20mg/kgbodyweight,respectively.MBZwasnotdetectedinany milksamplesafterperosadministrationatadosageof10mg/kg. However,theparentdrugreached0.01␮g/mlpeakconcentration at10.66hand AUCmilk/AUCplasmavaluewas0.18 ata dosageof 20mg/kgbodyweight.

Regardingtheparasitologicalexaminations,pre-treatmentEPG of1347, 1680and 1533 wereobserved for thecontrol, MBZ 1 andMBZ2groups,respectively.Atthestartofthestudythe pre-treatmentEPGwerenotsignificantlydifferent(P>0.1)between groups.TotalEPGcountsandefficacyvaluesforbothtreatedgroups (MBZ1andMBZ2)comparedwiththecontroluntreatedgroup (C-group)ateachtimestudypointsareshowninTable2.Theresults ofthepost-treatmentEPGforbothMBZtreatmentgroupswere sig-nificantlydifferent(P<0.001)fromthecontrolgroupuntilday28 followingtreatments.TheMBZformulationswereefficient(>95% efficacy)ondays7,14,21untilday28.Thepercentagereductions infaecaleggcountsfortheMBZ1group,comparedtotheC-group, were99.6%onday7;99.7%onday14;99.0%onday21and98.5%on day28.ThepercentagereductionsinfaecaleggcountsfortheMBZ2 group,comparedtotheC-group,were98.8%onday7;99.3%onday 14;99.0%onday21and97.9%onday28.Therewasnosignificant differenceinanthelminticefficacybetweenthetwoMBZdosages. TheERPvaluewere35days(5weeks)forbothtreatedMBZgroups. Inallstudieddonkeys,coproculturesperformedbeforethe treat-mentsrevealedthepresenceofCyathostominae.Faecalcoltures performedondifferentdays from C-group confirmedthe pres-enceofCyathostominae.Coproculturespost-treatmentsrevealed thepresenceoflarvaeofCyathostominaeinbothMBZgroups.

4. Discussion

Thepharmacokineticsandactivityofbenzimidazolesare partic-ularlyinﬂuencedbythephysicochemicalpropertiesandmetabolic pathwaysoftheactivemolecules.Thesecompoundsareonly spar-inglysolubleinwaterandtheirabsorptionandpharmacokinetics areaffectedbytheiraqueoussolubility(Ngumuoetal.,1984).The rapiddissolutionand consequentabsorptionandelimination of

somebenzimidazoles explaintheirrelativelyshort residencein thebody.Thus moresolublecompoundshaveshorterresidence incomparisontolesssolublebenzimidazoles,whichareabsorbed overprolongedperiods(McKellarandScott,1990).Thedifferences inefﬁcacyofthebenzimidazolesagainstparasitesinvivohavebeen attributedlargelytodifferentpharmacokineticproﬁleofeach com-poundwithinthehost(Bogan,1983).

MBZisabroadspectrumanthelminticwhichiseffectiveagainst themajorintestinalparasites ofhorsessuchaslargeandsmall strongyles,Parascarisspp.andOxyurisequi.

MBZ,fenbendazoleandoxfendazolewereregistered benzimi-dazoleanthelminticsforuseindonkeysinsomecountriesincluding UK and Australia (McKellar and Scott, 1990). The pharmacoki-neticprofileofoxfendazoleandalbendazoleindonkeyshasbeen reportedbefore(Gokbulutetal.,2006).However,the pharmacoki-neticdispositionofMBZisfirstlyreportedinthepresentstudy. OurresultsindicatethattheplasmadispositionofMBZwas differ-entcomparedtothoseofoxfendazoleandalbendazolestudiedin donkeysatthesamedosages(Gokbulutetal.,2006).Ithasbeen reportedthatthepeakplasmaconcentrationsandareasunderthe curveofoxfendazole(Cmax:0.49␮g/ml,AUC:5.17␮g.h2/ml)and albendazole(Cmax:0.08␮g/ml,AUC:0.84␮g.h2/ml)werehigher thanthoseofMBZ(Cmax:0.04␮g/ml,AUC:0.78␮g.h2/ml), respec-tively.Nevertheless,inthepresentstudythet1/2(11.97h)andMRT (20.34h) values of MBZ aremuch longer compared withthose ofoxfendazole(t1/2:4.49h,MRT:10.95h)andalbendazole(t1/2: 6.65h, MRT:9.15h) at thesame dosage (10mg/kg). These dif-ferencesprobablyreflectlowerwatersolubilityandsubsequent lowerabsorptionand/ormoreextensivethefirst-passmetabolism ofMBZcomparedwithoxfendazoleandalbendazole.Similarly,low systemicavailabilityhasbeenobservedwithMBZinhumanand ithasbeensuggestedthat itisa resultoftheverylow levelof absorptionofMBZ fromthegastrointestinaltract(Munst etal., 1980). High intestinalconcentrations couldbe effectiveagainst intestinal nematodes that inhabit thegut lumen, but very low plasmaconcentrationsofMBZsuchasdemonstratedinhorses,may notbeeffectiveagainstmigratinglarvalstagesoflargeandsmall strongyles,andencystedcyathostominlarvae.Ithasbeenindicated thatthebenzimidazolesincludingMBZ(atadosageof8.8mg/kg bodyweight)werehighlyeffectiveagainstadultlargeandsmall strongyles,adultOxyurisequiandthe4th stagelarvaeinhorses (Colglazieretal.,1977).

Theresultsobtainedinthepresentstudyplainlyindicatethat an increase in MBZ dosage is associated with elevation in the plasma level of MBZ in donkeys.Signiﬁcantly higher AUC and Cmax values for MBZwere observedafter perosadministration at 20mg/kg compared to the treatmentat 10mg/kg (Table1).

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0 10 20 30 40 50 60 0.02 0.04 0.06 0.08 0.10

Pla

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a or milk c

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cen

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tion

s (

g/ml)

Time (h)

Plasma (10 mg/kg)

Plasma (20 mg/kg)

Milk (20 mg/kg)

0 10 20 30 40 50 60 0.01 0.1 Pla sm a or milk c on cen tra tion s ( g/ml) Time (h)

Fig.1.Mean(±SD)plasmaandmilkconcentrations(␮g/g)vstimecurvesofmebendazole(MBZ)followingperosadministrationtodonkeysat10mg/kgbodyweight(n=6) and20mg/kgbodyweight(n=6).(Smallergraph:semi-logarithmicplasmaandmilkconcentrationvstimecurvesofMBZ).

The Cmax and AUC values of MBZ increased from 0.04␮g/ml and 0.78␮g.h/mL at a dosage of 10mg/kg to 0.07␮g/ml and 1.42␮g.h/mLatthehigherdosage(20mg/kg),respectively. Con-sequently, the increased dosage regime couldbe a strategy to providehigherplasmaconcentrationandthustoimprovethe effi-cacyagainstthetargetparasitessuchasmigratinglarvalortissue stagesofstrongylesandlungwormsindonkey,althoughthe effi-cacyof suchstrategies shouldbeconfirmedbeforetheycanbe recommended.

There are signiﬁcantdifferences in the pharmacokinetics of benzimidazoleanthelminticsbetweenmonogastricsandruminant species (Marrinerand Bogan, 1981;McKellar et al., 1990).The plasmadispositionofMBZin thepresentstudydiffers substan-tiallyfromthoseobservedinsheepandgoatspreviouslyreported (Galtier etal., 1994;Pandeyand Roy, 1998).The presentstudy indicatesthattheplasmaconcentrationofMBZafterperos admin-istration are relatively lower in donkeyscompared with those observed in sheepand goats. In sheep, Cmax (0.107␮g/ml)and AUCvalues(3.27␮g.h/ml)ofMBZafterperosadministrationata dosageof25mg/kgweregreaterthanthoseobservedfordonkeys inthisstudyafteradosageof20mg/kg.Moreover,PandeyandRoy (1998)reportedthattheplasmaconcentrationofMBZwas main-tainedfor120handthepeakconcentrationofMBZwas5.8␮g/ml afterperosadministrationat adosageof 40mg/kg. Thereason forthelowersystemicexposureindonkeyscomparedwiththat observedinruminantspeciesisprobablyrelatedwithdifferences inthephysiologicaloranatomicproperties betweentheanimal species.Anatomicfeaturesinﬂuencethepassageofdigesta,and thebioavailabilityandpharmacokineticsofanthelminticsmaybe affectedbydifferentguttransittimeinanimalspecies(McKellar andScott,1990).Thebenzimidazoleanthelminticdrugshavelower bioavailabilityinmonogastricanimals(e.g.dogandhorse),which haverelativelyfasterguttransittimethanruminants,sincerapid passageoffoodinthegutcausesadecreaseinthebioavailability ofthedrugs.

BecauseoftherelativelylowsystemicavailabilityofMBZ,the lowlevelofexcretionofMBZinmilk(milktoplasmaratio=0.18 atdobledosage)inassociationwithalowMRLvalueledto

advo-catetheuseofMBZinlactatingdonkeysatadosageof10mg/kg bodyweight.

TherapeuticequivalencewasdemonstratedforMBZ1(horse dosage)andMBZ2(doublehorsedosage)basedonFECRtest.In thisstudy,bothMBZdosageswerehighlyeffective(>95%)against cyathosmoninaeindonkeysuntil28daysposttreatment.The cut-offvalues(meanpercentreductioninFEC)forinterpretingresults ofstrongyleFECRTsuggestedintheguidelinesoftheAAEPfor ben-zimidazoles,reportedarangeof95–99%efficacyexpectedinthe absenceofresistanceinhorses.Thecyathostominsparasitizingthe studieddonkeyswereapparentlysusceptibletomebendazole,as confirmedbyFECRvalues.TheEggReappearancePeriod(ERP)of benzimidazoledrugsinthehorsesisnotexpectedtoexceed4–5 weeks(Nielsenetal.,2013);theERPsfoundinourstudyforboth MBZtreatedGroups(horseanddoublehorsedosages)following perosadministrationindonkeysarefairlysimilar(5weeks)tothat notedinhorsesparasitizedbybenzimidazolessusceptiblesmall strongylepopulations.Thetwo dosagesstudiedofMBZ didnot seemtomodifytheanthelminticefficacyindonkeys.

5. Conclusion

Thedonkeyisnotasmallhorseandrequiresspeciesspecific pharmacologicalandparasitologicaltrials.Thepresentstudy indi-catedthatperosadministrationofMBZhasaminimaldisposition rate into themilk and may beused in lactating donkeys with zeromilk-withdrawalperiodathorsedosageof10mg/kgB.W.The resultsofFECRTforbothMBZdosageswereefficient(>95%efficacy) untilday28,demonstratingthatMBZoralpasteathorsedosage waseffectiveandsafetyforthetreatmentofCyathostominaein donkeys.

Inconclusion,similardosageregimensofMBZcouldbeused forhorsesanddonkeysand,thismemberofbenzimidazolecould beusedasanthelminticforstrongylescontrolprogramindonkey farmsfortheproductionofmilkforhumanconsumption.

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Conﬂictofinterest

Noneoftheauthorsofthispaperhasa ﬁnancialorpersonal relationshipwithotherpeopleororganisationsthatcould inap-propriatelyinﬂuenceorbiasthecontentofthepaper.

Acknowledgements

Theauthorsareverygratefultothereviewersforthecomments andsuggestionsthatimprovedthequalityofthismanuscript.This studywaspartiallyfundedbythegrantsfromtheMinistryofHealth oftheItalianRepublic(IZSME14/11RCandIZSLT09/13RC).

References

Bogan,J.A.,1983.Absorptionanddistribution.In:Bogan,J.A.,Lees,P.,Yoxall,A.T. (Eds.),PharmacologicalBasisofLargeAnimalMedicine.BlackwellScientiﬁc Publication,Oxford,pp.9–11.

Clayton,H.M.,Neave,R.M.,1979.EfﬁcacyofmebendazoleagainstDictyocaulus arnﬁeldiinthedonkey.Vet.Rec.104,571–572.

Coakley,M.,Peck,K.,Taylor,T.S.,Matthews,N.S.,Mealey,K.L.,1999.

Pharmacokineticsofﬂunixinmeglumineindonkeysmulesandhorses.Am.J. Vet.Res.60,1441–1444.

Coles,G.C.,Bauer,C.,Borgsteede,F.H.M.,Geerts,S.,Klei,T.R.,Taylor,M.A.,Waller, P.J.,1992.Worldassociationfortheadvancementofveterinaryparasitology (WAAVP)methodsforthedetectionofanthelminticresistanceinnematodes ofveterinaryimportance.Vet.Parasitol.44,35–44.

Coles,G.C.,Jackson,F.,Pomroy,W.E.,Prichard,R.K.,vonSamson-Himmelstjerna, G.,Silvestre,A.,Taylor,M.A.,Vercruysse,J.,2006.Thedetectionofanthelmintic resistanceinnematodesofveterinaryimportance.Vet.Parasitol.136,167–185.

Colglazier,M.L.,Enzie,F.D.,Kates,K.C.,1977.Criticalanthelmintictrialsinponies withfourbenzimidazoles:mebendazole,cambendazole,fenbendazole,and albendazole.J.Parasitol.63,724–727.

Dawson,M.,Allan,R.J.,Watson,T.R.,1982.Thepharmacokineticsand

bioavailabilityofmebendazoleinman:apilotstudyusing[3H]-mebendazole. Br.J.Clin.Pharmaco.14(3),453–455.

Galtier,P.,Coulet,M.,Sutra,J.F.,Biro-Sauveur,B.,Alvinerie,M.,1994.Fasciola hepatica:mebendazoleandthiabendazolepharmacokineticsinsheep.Exp. Parasitol.79,166–176.

Gokbulut,C.,Boyacioglu,M.,Karademir,U.,2005.Plasmapharmacokineticsand faecalexcretionofivermectinanddoramectinfollowingoraladministrationin donkeys.Res.Vet.Sci.79,233–238.

Gokbulut,C.,Akar,F.,McKellar,Q.A.,2006.Plasmadispositionsandfaecal excretionsofoxfendazole,fenbendazoleandalbendazolefollowingoral administrationtodonkeys.Vet.J.172,166–172.

Gokbulut,C.,DiLoria,A.,Günay,N.,Masucci,R.,Veneziano,V.,2011.Plasma disposition,haircoatdepletionandanthelminticefﬁcacyofeprinomectin followingpour-onadministrationindonkeys.Am.J.Vet.Res.72,1639–1645.

Gokbulut,C.,Naturali,S.,Rufrano,D.,Anastasio,A.,Yalinkilinc,H.S.,Veneziano,V., 2013.Plasmadispositionandmilkexcretionofeprinomectinfollowing pour-onadministrationinlactatingdonkeys.J.Vet.Pharmacol.Ther.36, 302–305.

Gokbulut,C.,Aksit,D.,Smaldone,G.,Mariani,U.,Veneziano,V.,2014.Plasma pharmacokinetics,faecalexcretionandefﬁcacyofpyrantelpamoatepasteand granuleformulationsfollowingperosadministrationindonkeysnaturally infectedwithintestinalstrongylidae.Vet.Parasitol.205,186–192.

Grosenbaugh,D.A.,Reinemeyer,C.R.,Figueiredo,M.D.,2011.Pharmacologyand therapeuticsindonkeys.EquineVet.Educ.23,523–530.

Iacono,G.,Carroccio,A.,Cavataio,F.,Montalto,G.,Soresi,M.,Balsamo,V.,1992.Use ofass’smilkinmultiplefoodallergy.J.Pediatr.Gastroenterol.Nutr.14, 177–181.

Kum,C.,Gokbulut,C.,Sekkin,S.,Boyacioglu,M.,2009.Pharmacokineticsof danoﬂoxacinfollowingintravenousandintramuscularadministrationin donkeys.J.Vet.Pharmacol.Ther.32,105–108.

Kaplan,R.M.,Nielsen,M.K.,2010.Anevidence-basedapproachtoequineparasite control:itain’tthe60sanymore.EquineVet.Educ.22,306–316.

Lester,H.E.,Matthews,J.B.,2014.Faecalwormeggcountanalysisfortargeting anthelmintictreatmentinhorses:pointstoconsider.EquineVet.J.46, 139–145.

Lizarraga,I.,Sumano,H.,Brumbaugh,G.W.,2004.Pharmacologicaland pharmacokineticdifferencesbetweendonkeysandhorses.EquineVet.Educ. 16,102–112.

Marriner,S.E.,Bogan,J.A.,1981.Pharmacokineticsofoxfendazoleinsheep.Am.J. Vet.Res.42,1143–1145.

Matthews,N.,Peck,K.,Taylor,T.,Ray,A.C.,Taylor,T.S.,1997.Pharmacokineticsof phenylbutazoneanditsmetaboliteoxyphenbutazoneinclinicallynormal horsesanddonkeys.Am.J.Vet.Res.58,53–55.

McKellar,Q.A.,Scott,E.W.,1990.Thebenzimidazoleanthelminticagents:areview. J.Vet.Pharmacol.Ther.13,223–247.

Nielsen,M.K.,Vidyashankar,A.N.,Andersen,U.V.,DeLisi,K.,Pilegaard,K.,Kaplan, R.M.,2010.Effectsoffecalcollectionandstoragefactorsonstrongylidegg countsinhorses.Vet.Parasitol.167,55–61.

Nielsen,M.K.,Mittel,L.,Grice,A.,Erskine,M.,Graves,E.,Vaala,W.,Tully,R.C., French,D.D.,Bowman,R.,Kaplan,R.M.,2013.AAEPParasiteControlGuidelines. Am.AssocEquinePract.,Onlineatwww.aaep.org.

Ngumuo,A.J.,Marriner,S.E.,Bogan,J.A.,1984.Thepharmacokineticsof fenbendazoleandoxfendazoleincattle.Vet.Res.Commun.8,187–193.

Pandey,S.N.,Roy,B.K.,1998.Dispositionkineticsofmebendazoleinplasma,milk andruminalﬂuidofgoats.SmallRuminantRes.27,111–117.

Peck,K.,Matthews,N.,Taylor,T.S.,Mealey,K.L.,2002.Pharmacokineticsof sulfamethoxazoleandtrimethoprimindonkeys,mulesandhorses.Am.J.Vet. Res.63,349–353.

Peck,K.,Mealey,K.L.,Matthews,N.S.,Taylor,T.S.,1997.Comparative

pharmacokineticsofcaffeineandthreemetabolitesinclinicallynormalhorses anddonkeys.Am.J.Vet.Res.58,881–884.

Reinemeyer,C.R.,Nielsen,M.K.,2013.Diagnostictechniquesforequineparasitism. In:HandbookofEquineParasiteControl.Wiley-Blackwell,Oxford,UK,pp. 103–127.

Sekkin,S.C.,Gokbulut,C.,Kum,C.,Karademir,U.,2010.Pharmacokineticsof enroﬂoxacinfollowingintravenousandintramuscularadministrationin donkeys.Vet.Rec.3(171),447.

TheDonkeySanctuary,2003.Donkeynomogram.Availableat:drupal.

thedonkeysanctuary.org.uk/ﬁles/donkeys/Nomogram.pdf(accessed25.12.15.). Veneziano,V.,DiLoria,A.,Masucci,R.,DiPalo,R.,Brianti,E.,Gokbulut,C.,2011.

Efﬁcacyofeprinomectinpour-onagainstDictyocaulusarnﬁeldiinfectionin donkeys(Equusasinus).Vet.J.190,414–415.

Vidyashankar,A.N.,Kaplan,R.M.,Chan,S.,2007.Statisticalapproachtomeasure theefﬁcacyofanthelmintictreatmentonhorsefarms.Parasitology134, 2027–2039.

Vincenzetti,S.,Polidori,P.,Mariani,P.,Cammertoni,N.,Fantuz,F.,Vita,A.,2008.