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Veterinary
Parasitology
j o ur na l h o 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
Plasma
pharmacokinetics,
faecal
excretion
and
efficacy
of
pyrantel
pamoate
paste
and
granule
formulations
following
per
os
administration
in
donkeys
naturally
infected
with
intestinal
strongylidae
Cengiz
Gokbulut
a,∗,
Dilek
Aksit
b,
Giorgio
Smaldone
c,
Ugo
Mariani
d,
Vincenzo
Veneziano
caDepartmentofMedicalPharmacology,FacultyofMedicine,BalikesirUniversity,Balikesir,Turkey
bDepartmentofPharmacologyandToxicology,FacultyofVeterinaryMedicine,BalikesirUniversity,Balikesir,Turkey
cDepartmentofVeterinaryMedicineandAnimalProduction,UniversityofNaplesFedericoII,Naples,Italy
dIstitutoZooprofilatticoSperimentaledelMezzogiorno,BeneventoUnit,Portici,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received21April2014
Receivedinrevisedform17June2014
Accepted19June2014 Keywords: Pharmacokinetics Efficacy Pyrantel Paste Granule Donkey
a
b
s
t
r
a
c
t
Theplasmadisposition,faecalexcretionand efficacyof twoformulationsof pyrantel pamoateindonkeyswereexaminedinacontrolledtrial.Threegroupsofsevendonkeys receivedeitherno medication (control) orpyrantel pasteor granule formulationsat horsedosageof20mg/kgB.W.(equals6.94mg/kgPYRbase)ofbodyweight.Heparinized bloodand faecalsampleswere collectedat varioustimesbetween1 and 144hafter treatment. The samples were analysed byhigh-performance liquid chromatography. Thelastdetectableplasmaconcentration(tmax)ofpasteformulationwas significantly earlier(36.00h)comparedwithgranuleformulation(46.29h).Although,therewasno significantdifferenceon terminalhalflives(t1/2:12.39hvs.14.86h),tmax (14.86hvs. 14.00)andMRT(24.80hvs.25.44h)values;theCmax (0.09g/ml) AUC(2.65gh/ml) valuesofpasteformulationweresignificantlylowerandsmallercomparedwiththoseof granuleformulation(0.21g/mland5.60gh/ml),respectively.Thehighestdryfaecal concentrationswere710.46g/gand537.21g/gandweredeterminedat48hforboth pasteandgranule formulationofPYR indonkeys,respectively.Pre-treatment EPGof 1104,1061and1139wereobservedforthecontrol,PYRpasteandPYRgranulegroups, respectively.Pre-treatmentEPGwerenotsignificantlydifferent(P>0.1)betweengroups. Post-treatmentEPGforbothPYRtreatmentgroupsweresignificantlydifferent(P<0.001) fromthecontrolgroupuntilday35.FollowingtreatmentsthePYRformulationswere efficient(>95%efficacy)untilday28.Inallstudieddonkeys,coproculturesperformedat day—3revealedthepresenceofCyathostomes,S.vulgaris.Faecalculturesperformedon differentdaysfromC-groupconfirmedthepresenceofthesamegenera.Coprocultures fromtreatedanimalsrevealedthepresenceoffewlarvaeofCyathostomes.
©2014ElsevierB.V.Allrightsreserved.
∗ Correspondingauthor.Tel.:+902666121461;
fax:+902666121448.
E-mailaddresses:cengizgokbulut@yahoo.com,cgokbulut@gmail.com
(C.Gokbulut).
1. Introduction
Pyrantel (PYR) is an imidazothiazole derivative, which belongs to the tetrahydropyrimidine group of anthelmintics. PYR is available as tartrateand pamoate http://dx.doi.org/10.1016/j.vetpar.2014.06.026
(syn.embonate)salts.DifferentsaltsofPYRhavedifferent pharmacokinetic properties and consequently different toxicitiestothehost.Thepamoatesaltisalmostinsoluble inwater,poorlyabsorbedfromthegastrointestinaltract, andmostpassesunchangedinthefaeces(Arundel,1983). Reducedsystemicabsorptionofthepamoateform poten-tiallyincreasesavailabilityinthelumenoftheintestine (Bjorn et al., 1996). The tartrate salt of PYR is soluble inwater andabsorbedrapidlyandextensivelyfromthe intestineofmonogastricanimals(Faulkneretal.,1972).
ThedailyadministrationofPYRtartratetohorsesata continuouslowlevelwashighlyeffectiveagainstcommon equineparasites,includingadultlargestrongyles (Strongy-lusvulgaris,Strongylusedentatus,andTriodontophorusspp.), adult small strongyles (Cyathostomum spp., Cylicocyclus spp.,andCylicostephanusspp.)andadultandfourth-stage Parascarisequorum(Valdezetal.,1995).It wasreported thattherecommendeddoselevelofPYR(13.2mg/kg, body-weight)pamoatepasteformulationwaseffective(95–98%) againstinfectionsofAnoplocephalaperfoliata(FDA,2005). Moreover,arecentinvestigationbyReinemeyeretal.,2010 indicatedthatPYRpamoatepasteformulationwashighly effectiveagainstadult(91.2%)andfourth-stageOxyrusequi larvae(>99%).
Onlylimiteddataareavailableonthe pharmacokine-tics andefficacy ofanthelminticdrugs usedin donkeys becausedonkeysareoftenaneglectedspeciesforstudiesin domesticanimals.Mostofdrugsindifferentclassesusedin horsesandruminantsarecommonlyextrapolatedforuse indonkeyswithoutoptimizationofdosingregimensand determinationofpharmacokineticproperties(Veneziano et al.,2011).Because of thelackof drugs approved for useindonkeys,anthelminticslicensedforusein horses orruminantsareusedatthesamedosagesfortreatment of parasitic infections in donkeys.It hasbeen reported thatdonkeyshaveagreatercapacitytometabolizecertain drugs,comparedwiththecapacityforhorses;thus,higher dosagesorshorterintervalscouldberequiredtomaintain effectivedrugconcentrationsindonkeys(Welfare etal., 1996; Matthewsetal., 1997;Coakley etal., 1999; Peck etal.,2002;Lizarragaetal.,2004;Grosenbaughetal.,2011). Hence,inthepresentstudy,thepharmacokinetic dispo-sition, faecalexcretion andanthelminticefficacyof two differentformulations(pasteandgranule)ofPYRpamoate arereportedindonkeysnaturallyinfectedwithintestinal strongylidaeafteroraladministration.
2. Materialsandmethods 2.1. StudyAnimals
Twenty-onefemalecrossbreeddonkeys(Equusasinus) weighing200–280kgwereusedinthisstudy.The body-weight(BW)ofeachanimalwasestimated1daypriorto treatment(day—1)usingthenomogramproposedbyThe DonkeySanctuary(2003).Theanimalshadameanageof 9.8 (±1.5)years and theyhad a history ofgrazing pas-turecontaminated withequine nematodeparasites and havenotbeentreatedwithanyanthelminticsduringthe previous9months.Faecalexaminations(individualFaecal EggCountsandpooledcoproculture)performedbeforethe
beginningofthestudy(day—3)showedindividualcounts >150eggspergram(EPG)andahighprevalenceof intesti-nalnematodes(Cyathostomes, S.vulgaris) in allstudied donkeys.Thestudyanimalsweretaggedforidentification andhousedcommunallyinanindoorpenuntiltheday0of thetrial.Theanimalswerekeptindoorsandfedhay-based diet.Waterwasprovidedadlibitumthroughoutthecourse ofthestudy.ThisinvestigationwasapprovedbytheAnimal EthicCommitteeofUniversityofNaplesFedericoII. 2.2. Experimentalgroups
Onday—3,21oftheexperimentaldonkeyshadan aver-age of 1101±583 EPG. The animals were ranked from lowest tohighest EPG counts. Based onincreasing EPG counts,replicatesof3animalswereformed.Withineach replicate,animalswererandomlyassignedtotreatment. The21selecteddonkeyswereassignedconsecutivelytothe followingtreatmentgroupsof7animalseach:PYRpaste treatedgroup(PYR-Pgroup),PYR granuletreated group (PYR-G-group),anduntreatedcontrolgroup(C-group). 2.3. Drugadministration
Commercially available equine formulations of PYR paste(StrikePYRpastepamoate38%,Acme,Italy)andPYR pamoategranulate(Strike,PYRpamoate20%,Acme,Italy) licensedforhorseswereadministeredorallytodonkeysat arecommendeddosagerateof20mg/kgB.W.whichequals 6.94mg/kgPYRbase.
2.4. Samplingprocedure
Heparinizedbloodsampleswerecollectedbyjugular venipuncturepriortodrugadministrationand1,2,4,8, 12,16,24,30,36,48,56,72,96,120and144hthereafter. Faecalsamples(>10g)werecollectedperrectum through-outtheblood-samplingperiod,beforedrugadministration andthenat4,8,12,16,24,30,36,48,56,72,96and120h inordertodeterminefaecalexcretionofPYRunderstudy. Bloodsampleswerecentrifugedat1825×gfor30minand plasmawastransferredtoplastictubes.Alltheplasmaand faecalsampleswerestoredat−20◦C untilestimationof
drugconcentration. 2.5. Analyticalprocedure
TheparentcompoundofPYRwasanalysedbyhigh per-formanceliquidchromatography(HPLC).Theliquid–liquid phaseextractionprocedureusedforPYRwasadaptedfrom thatdescribedbyMcKellaretal.(1993a).Briefly,1ml drug-freeplasmasampleswerefortifiedwithPYRstandardto reachthefollowingfinal concentrations:0.01, 0.05,0.1, 0.5,1and5g/ml.Morantelcitratewasusedasan inter-nalstandard.Sodiumhydroxide(NaOH)(0.5ml,0.4M)was addedtotubes.Aftervortexfor15s,6mlchloroformwas added.Thetubeswereshakenfor2min.After centrifuga-tionat2000×gfor15min,4mloftheorganicphasewas transferredtotheglasstubeandevaporatedtodrynessat 43(Cinasampleconcentrator—Maxi-dryplus,HetoLab. Equipment,Denmark).Thedry residuewasdissolvedin
300lofthemobilephaseand100lofthissolutionwas injectedintothechromatographysystem.
Faecal material was mixed with a spatula to obtain ahomogeneoussample.Drug-freefaecalsamples(0.5g) werefortifiedwithPYRstandard toreachthefollowing finalconcentrations:1,5,50,100,and300g/g. Moran-telcitratewasusedasaninternalstandard.Acetonitrile (2ml)wasaddedtotubescontaining0.5gfortifiedblank samplesandexperimentalsamples.Aftervortexfor15s, 6ml chloroformwas added.The tubes wereshakenfor 2min.Aftercentrifugationat2000×gfor15min,4mlof theorganicphasewastransferredtoaglasstubeand evap-oratedtodrynessat43(Cinthesampleconcentrator.The dryresiduewasdissolvedin300lor5mlofthemobile phaseand50lofthissolutionwasinjectedintothe chro-matographysystem.BecauseofthephotosensitivityofPYR allpreparativeprocesseswereconductedincovered con-tainers.Todeterminethedryweightofwetfaecalsamples, 1.0gofwetfaecesfromeachsamplewasweighedexactly intoanevaporatingbowlandheatedinanovenat70◦Cfor 10h.Theweightofeachsamplewasdeterminedandthe percentageofeachdrysamplewascalculated.
IntheHPLCsystem,amobilephaseofacetonitrile:water (30:70)with0.6%(v/v)triflouroaceticacid(TFA)pumped (1100 Series QuatPump,Agilent, Waldron, Germany) at flowrate1ml/minwasusedforplasmasamplesand ace-tonitrile:water (15:85) with 0.6% (v/v) TFA pumped at flowrate1.4ml/minwasusedforfaecalsamples.A nucle-osilC18analyticalcolumn(Luna,4m,150mm×4.6mm,
Phenomenex,Macclesfield,Cheshire, UK)withnucleosil C18 guard column(Phenomenex, Macclesfield,Cheshire,
UK)wasusedwithultravioletdetection(1100SeriesPDA detector,Agilent,Waldron,Germany)atawavelengthof 322nm.
The analytical methods used for PYR in plasma and faecalsampleswerevalidatedpriortothestartofthe stud-ies.Theanalytewasidentifiedwiththeretentiontimesof purereferencestandard.Recoveriesofthemoleculeunder studyweremeasuredbycomparisonofthepeakareasof spikedplasmaandfaecalsampleswiththeareasresulting fromdirectinjectionsofstandardspreparedinacetonitrile. Theinter-andintra-assayprecisionoftheextractionand chromatographyprocedureswereevaluatedbyprocessing replicatealiquotsofdrug-freedonkeyplasmaandfaecal samplescontainingknownamountsofthedrugson differ-entdays.
CalibrationgraphforPYRwasprepared(linearrange 0.01–5g/mlinplasmaand1–300g/mlfaecalanalysis). Theslopeofthelinesbetweenpeakareasanddrug concen-trationwasdeterminedbyleastsquareslinearregression andcorrelationcoefficient(r)andcoefficientofvariations (CV) calculated. Linearity wasestablished to determine thePYRconcentration/detectorresponserelationship.The detectionlimitofPYRwasestablishedwithHPLC analy-sisofblankplasmafortifiedwiththestandard,measuring
thebaselinenoiseattheretentiontimeofthepeak.The meanbaselinenoiseatthepeakretentiontime,plusthree standard deviations was defined as the detection limit (LOD).Themeanbaselinenoiseplussixstandarddeviations wasdefinedasthelimitofquantification(LOQ).
2.6. Coprologicalexaminationsandanthelminticefficacy According to generalrecommendations proposed by Nielsenetal.(2010)faecalsamplesweretakenfromthe rectumfromeachstudyanimal,werestoredina refrig-erator(4◦C)andindividualfaecaleggcounts(FEC)were performed,within12hbeforethestartofthetrial(day—3), atdays0,7,14,21,28,35,42and56aftertreatment. Indi-vidualfaecaleggcountsweredeterminedusingamodified McMastertechniquewithadetectionlimitof10EPG,using aSheather’ssaturatedsugarsolutionwithaspecificgravity of1.260(MAFF,1986).
Oneachsamplingday,individualfaecalsampleswere incubatedat27◦Cfor7–10daysforlarvalidentification. Onlybeforethestartofthetrial(day–3)wereperformed pooled coprocultures.Thirdstagelarvaewereidentified usingthemorphologicalkeysproposedbyMAFF(1986). Whenacoproculturehad100orfewerthirdstagelarvae, allwereidentified;whenacoproculturehadmorethan100 larvae,only100wereidentified.
To determine the efficacy of PYR against intestinal strongyles at each faecal samplingtime, thearithmetic meanofEPGwascalculated.FollowingtheAmerican Asso-ciation of Equine Practitioners (AAEP) Parasite Control Guidelines(Nielsenetal.,2013a),foreachanimalspercent efficacy(%)wascalculatedin termsof faecalEggCount Reduction (FECR)at thedifferentdays accordingtothe formula:
FECR(%)= MeanEPG(beforetreatment)−MeanEPG(aftertreatment) MeanEPG(beforetreatment) ×100
2.7. Pharmacokineticsandstatisticalanalysisofdata The plasma concentration vs. time curves obtained after each treatment in individual animals, were fit-tedwiththeWinNonlinsoftware program(Version 5.2, PharsightCorporation,MountainView,CA,USA).The phar-macokineticsparametersforeach animalwereanalysed usingnon-compartmentalmodelanalysis.Themaximum plasma concentration (Cmax) and time to reach
maxi-mumconcentration(tmax)wereobtainedfromtheplotted
concentration-timecurveofeachdrugineachanimal.The trapezoidalrulewasusedtocalculatetheareaunderthe plasmaconcentrationtimecurve(AUC):
AUC0→∞= n
i=1 Ci+Ci−1 2 ×(ti−ti−1)+ Cn zWhereCrepresentstheplasmaconcentration,i-1and iareadjacentdatapointtimes.Theareaunderthefirst
movementcurve(AUMC)wascalculatedusingthe equa-tion: AUMC0→∞= n
i=1 Citi+Ci−1ti−1 2 ×(ti−ti−1)+ Cn 2 z +tnCn zThus,themeanresidencetime(MRT)wascalculatedas: MRT0→∞=AUMC0→∞/AUC0→∞
Terminal half-life (t1/2z) was calculated
as:t1/2z=−ln(2)/z
Wherezrepresentthefirstorderrateconstant associ-atedwiththeterminal(loglinear)portionofthecurve.
The pharmacokinetic parameters are reported as mean±SD.Meanpharmacokineticparameterswere statis-ticallycomparedbyone-wayanalysisofvariance(ANOVA). AllstatisticalanalyseswereperformedbyusingMINITAB forWindows(release12.1,MinitabInc.,StateCollege,PA, USA).Meanvalueswereconsideredsignificantlydifferent atP<0.05.
Forcomparisonoftheanthelminticefficacyofboth for-mulations,statisticalanalysisofdata wasperformedon arithmeticmean(AM)EPGcountsusingtheparametric t-testtocomparedifferencesbetweentreatmentgroupsfor significanceattheP<0.01level.
3. Results
The analytical procedures and HPLC analysis of PYR werevalidatedforplasmaandfaecalanalysis.Thelinear rangeswere0.01–5g/mlforplasmaand1–300g/ml fae-calanalysisandshowedcorrelationcoefficientsof0.999 and0.998,respectively.ThemeanrecoveryofPYRfrom plasmaandfaecalsampleswere72.72%and83.56%, respec-tively.Thedetectionlimitoftheanalyticaltechniquewas 0.002g/mland0.25g/ml;thequantificationlimitwas 0.01g/mland1.00g/mlforplasmaandfaecalsamples, respectively. Theinterand intra-assay precisions ofthe analyticalprocedureobtainedafterHPLCanalysisofspiked standardsofPYR(0.01–5g/ml)showedaCVof4.22%and 6.54%forplasmaand6.12%and7.55%forfaecalsamples, respectively.
Mean pharmacokinetic parameters of PYR, adminis-teredorallyaspasteandgranule,are showninTable1, withtheplasmaconcentrationvs.time curves inFig.1. PYRwasdetectedinplasmasamplesbetween2hand36h afterpasteadministrationand1hand48haftergranule administration.Thelastdetectableplasmaconcentration of paste formulation was significantly earlier (36.00h) compared withgranuleformulation(46.29h).Although, therewasnosignificantdifferenceonterminalhalflives (t1/2: 12.39h vs. 14.86h), tmax (14.86hvs. 14.00h) and
MRT(24.80hvs.25.44h)values;theCmax(0.09g/mlvs.
0.21g/ml)andAUCvalues(2.65gh/mlvs.5.60gh/ml) weresignificantlydifferentindonkeysreceivedpaste for-mulation compared with the animals received granule formulation,respectively.Meankineticparametersof dry-faecesconcentrationsafterpasteandgranuleformulations are shown in Table 2 with the dry-faecal concentra-tionvs. time curves(Fig.2).PYRwasdetectedin faecal
Table1
Mean(±SD)pharmacokineticparametersofpyrantel(PYR)pamoatepaste
andgranulefollowingperosadministrationtodonkeysat20mg/kgB.W.
(equals6.94mg/kgPYRbase)bodyweight.
Parameters PYRgroups
Paste Granule t1/2z(h) 12.39±5.35 14.86±5.59 Cmax(g/ml) 0.09±0.02* 0.21±0.07 tmax(h) 14.86±5.52 14.00±9.45 tlast(h) 36.00±0.00* 46.29±4.53 AUClast(gh/ml) 2.65±0.81* 5.60±0.59 AUMClast(gh2/ml) 68.30±36.04* 143.68±64.72 MRTlast(h) 24.80±5.54 25.44±10.68
Cmax:peakplasmaconcentration;tmax:timetoreachpeakplasma con-centration;AUClast:areaunderthe(zeromoment)curvefromtime0to thelastdetectableconcentration,AUMClast:areaunderthemomentcurve fromtime0totlastdetectableconcentration;MRTlast:meanresidence time;t1/2z:terminalhalf-life.
MeankineticparametersobtainedfollowingPYRpasteadministration significantlydifferent(*P<0.01)fromthoseobtainedfollowingPYR gran-uleadministration. 0 5 10 15 20 25 30 35 40 45 50 55 60 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Pl asma co nc en trati ons ( μ g/ ml ) Time (h) Paste Granule
Fig. 1.Mean (±SD) plasma concentration (g/g) of pyrantel (PYR) pamoatepasteandgranulefollowingoraladministrationtodonkeysat adoserateof20mg/kgB.W.(equals6.94mg/kgPYRbase)bodyweight.
samplesbetween16hand120h.BothPYRformulations displayedasimilarfaecalexcretionprofilesfollowingoral administration.Thehighestdryfaecalconcentrationswere 710.46g/gand537.21g/gandweredeterminedat48h
Table2
Mean(±SD)faecalkineticparametersofpyrantel(PYR)pamoatepaste andgranulefollowingperosadministrationtodonkeysat20mg/kgB.W. (equals6.94mg/kgPYRbase)bodyweight.
Parameters PYRgroups
Paste Granule tmax(h) 46.00±9.73 47.43±5.86 Cmax(g/g) 839.13±212.98 650.39±264.00 tlast(h) 102.86±11.71 109.71±12.83 AUClast(gh2/g) 29,765.56±4687.66 26,414.17±8184.14 MRTlast(h) 48.43±5.89 53.68±8.18
Cmax:peakfaecalconcentration;tmax:timetoreachpeakfaecal concen-tration;AUClast:areaunderthe(zeromoment)curvefromtime0tothe lastdetectableconcentration;tlast:timetolastdetectablefaecal concen-tration;MRTlast:meanresidencetime;
0 20 40 60 80 100 120 100 200 300 400 500 600 700 800 900 1000 F aecal c on cent rat ions ( µ g/ g) Time (h) Paste Granule
Fig.2. Mean(±SD) dry-faecalconcentration(g/g)ofpyrantel(PYR) pamoatepasteandgranulefollowingoraladministrationtodonkeysat adoserateof20mg/kgB.W.(equals6.94mg/kgPYRbase)bodyweight.
forbothpasteandgranuleformulationofPYRindonkeys, respectively.
Clinically, no adverse reaction was observed in any ofthedonkeys treatedwithPYR duringthestudy. Pre-treatmentEPGof1104,1061and1139wereobservedfor thecontrol,PYR pasteand PYR granulegroups, respec-tively.AtthestartofthestudythepretreatmentEPGwere notsignificantlydifferent(P>0.1)betweengroups.Total EPGcountsandefficacyvaluesforbothtreatedgroups com-paredwiththecontroluntreatedgroup(C-group)ateach timestudypointsareshowninTable3.Theresultsofthe post-treatmentEPGforbothPYRtreatmentgroupswere significantlydifferent(P<0.001) fromthecontrol group untilday35followingtreatments.ThePYRformulations wereefficient(>95%efficacy)ondays7,14,21untilday28. Therewasnosignificantdifferenceinanthelminticefficacy betweenthetwoPYRformulations.
In all studied donkeys, coprocultures performed at day—3revealedthepresenceofCyathostomesandS. vul-garis. Faecal cultures performedon differentdays from C-groupconfirmedthepresenceofthesamegenera. Copro-culturesfromtreatedanimalsrevealedthepresenceoffew larvaeofCyathostomesinbothPYRgroups.
4. Discussion
Although,PYRhasbeenusedasananthelminticdrug for almost a half century, there is a paucity of data availablein the literatureon thepharmacokinetics and comparativeefficacyofdifferentformulationsindomestic animalsincludingequidspecies.Therapeuticequivalence was demonstrated for PYR pamoate oral formulations paste and granule based on FECR test. In this study, bothPYR formulationswerehighly effectiveagainstthe intestinalstrongylidaeofdonkeysuntilday28post treat-ment.Thecutoffvalues(meanpercentreductioninFEC) for interpreting results of strongyle FECRT suggested in the guidelines of the AAEP for pyrantel, reported a range of 94–99% effectiveness expected in the absence ofresistanceinhorses.Thecyathostominandstrongylid nematodespresentinthestudieddonkeyswereapparently
susceptibletopyrantelpamoate,asconfirmedbyFECR val-ues.Thepercentagereductionsinfaecaleggcountsforthe PYRpastegroup,comparedtotheC-group,were98.3%on day7;98.5%onday14;98.2%onday21and96.6%onday 28.
Thepercentagereductionsinfaecaleggcountsforthe PYRgranulegroup,comparedtotheC-group,were97.1% onday7;97.3%onday14;96.3%onday21and96.4%onday 28.Thevaluesofefficacyinthepresentstudyperformedon thedonkeyareclosetothosesuggested(92%)byNielsen etal.,2013btoevaluatetheexpectedefficacyofpyrantel againststrongyleparasitesinhorses.TheEgg Reappear-ancePeriod(ERP)ofPYRinthehorsesisnotexpectedto exceed4weeks(ReinemeyerandNielsen,2013);theERPs of PYRpamoate pasteandgranuleformulations follow-ingperosadministrationindonkeysarefairlysimilarto thatnotedinhorseswithPYR-susceptiblepopulationsof nematodesandtreatedatsimilardosages.
Theresultsobtainedinboth treated groupsare con-sistent withthecurrentlyapproved labelclaim for PYR oralformulationsagainstintestinalstrongyleparasitesof horses.ThemodeofadministrationofPYRwouldnotseem toaltertheanthelminticefficacyindonkeys.
Even though, the pharmaceutical formulation of anthelmintic drugs may affect itsbioavailability, which dependsontherateandextentofabsorptionofthedrug fromthegastrointestinaltractintothebloodstream.The pharmacokineticresultsofthepresentstudyobtainedafter perosadministrationofthepasteformulationdiffer sub-stantiallyfromthegranuleformulationofPYRindonkeys atthesamedoserate.Theplasmalevel ofPYRis much lowerfollowingadministrationofpasteformulationthan those observed following granule administration. After oral administration, Cmax (0.09g/ml) was significantly
lower(P<0.01)andAUC(2.65gh/kg)smaller(P<0.01) for PYRpastethanPYRgranule(Cmax: 0.21g/ml,AUC:
5.60gh/kg)givenatsamedoserates(6.94mg/kgB.W.). Itislikelythatthepoorerdissolutionofpasteformulation reducesitsabsorptioncomparedwithgranuleformulation indonkeys.
TheresultsofPYRinthepresentstudydiffer substan-tially fromthosepreviously reportedbyGokbulutet al. (2001)inhorsesatsameformulationandadministration route.Inthepreviousstudy,PYRpastewasadministeredat 13.3mg/kginhorses,whereasinthisstudy,itwas admin-isteredat6.94mg/kgindonkeys.Thelowerconcentrations and shorter MRTof PYRin theplasmaof horses reflect lower bioavailability and shorter persistence compared with donkeys. After correction for dose assuming pro-portionality,Cmax (0.045g/ml), AUC(0.53gh/ml)and
MRT (11.99h) values in horses much lower than those observed in the present study (Cmax: 0.09g/ml, AUC:
2.65gh/mlandMRT:24.80h).Thegastrointestinal pas-sagerateofdigestaisaffectedbyalterationinthequality and quantityofthe feedconsumed and thiscould con-fervariableabsorptiontimeandthereforebioavailability of drugsadministeredorally.It wasindicatedthat vari-ations in the quality (McKellar et al., 1993b; Knox and Steel,1997;OukessouandSouhaili,1998;Gokbulutetal., 2007)andquantity(AliandHennessy,1995;Lifschitzetal., 1997;McKellaretal.,2002;Gokbulutetal.,2010)ofdiet
Table3
Strongyleeggcountsineggspergram(EPG)andpercentagereductionsinfaecaleggcounts(FECR)fordonkeystreatedwithpyrantelpamoatepaste(PYR
P-group)andfordonkeystreatedwithpyrantelpamoategranule(PYRG-group),comparedwithcontroluntreatedgroup(C-group)ateachtimestudy
points.
Day Cgroup PYRPastegroup FECR(%) Pvalue PYRGranulegroup FECR(%) Pvalue
EPGAM EPGRange EPGAM EPGRange EPGAM EPGRange
3 1104 450–1470 1061 160–1770 – 0.8636 1139 170–2440 – 0.9220 7 849 450–1170 14 0–30 98.3 0.0000 24 0–80 97.1 0.0000 14 936 500–1340 14 0–30 98.5 0.0000 26 0–10 97.3 0.0000 21 1036 140–1450 19 0–30 98.2 0.0001 39 0–110 96.3 0.0001 28 1334 170–2030 46 0–110 96.6 0.0002 49 20–80 96.4 0.0002 35 1163 610–2000 213 20–740 81.7 0.0003 124 40–290 89.3 0.0001 42 854 90–1370 520 40–1790 39.1 0.2657 303 70–540 64.5 0.0095 49 823 90–1680 616 10–1950 25.2 0.5055 533 150–1090 35.2 0.1925 56 663 210–1130 737 80–1410 −11.2 0.7371 651 240–1410 1.7 0.9536
*Parametrict-test(PYR-groupsvs.C-group)valuesaresignificantlydifferentP<0.001—arithmeticmeans(AM).
couldaffectthebioavailabilityofanthelminticsin differ-entanimalspecies.Differencesamongthestudiesmaybe associatedwithdiettypeindonkeyscomparedwithhorses. Sinceinthepresentstudy,donkeyswerekeptindoorsand fedhay-baseddiet.Thehorseswereyardedfortheperiod duringandimmediately(4h)afterdrugadministrationand werethenreturned toa grasspaddock(Gokbulutetal., 2001). Grass-based diet probably decreased gut transit timeoffood,thustherapidpassageoffoodinthegutcauses adecreaseinthebioavailabilityofPYRinhorsescompared with those in donkeys in the present study.Moreover, anatomicfeaturesinfluencethepassageofdigesta,andthe bioavailabilityandpharmacokineticsofanthelminticsmay beaffectedbydifferentguttransittimeinanimalspecies (McKellarandScott,1990).
Parasitismcouldhavehadaneffectsincethedonkey usedinthisstudywerenaturallyinfectedwithintestinal parasitesandsuchinfectionsmayhavemodesteffectson absorptionofanthelmintics(McKellaretal.,1991), unfor-tunately the parasitological status of thehorses or the historyofanthelmintictreatmentinthepreviousstudywas unknown.
PYRisnotknowntohaveanysubstantialeffecton fae-calinvertebrates,however,itisapparentthatfaecesfrom treateddonkeyswillhavehighconcentrationsofPYRfor atleast120hforbothformulations.Thisperiodoffaecal excretionofPYRisconsiderablylongerindonkeys com-paredtointhehorses(48h)(Gokbulutetal.,2001). 5. Conclusion
The present study demonstrates relatively limited absorptionofPYR pamoatefollowingperos administra-tionaspasteorgranuleformulationsindonkeys.Although thehigherlevelandlongerpersistenceofPYRinthe gas-trointestinaltractwheretheadultstagesofmostparasitic nematodes reside after both formulations may provide higherandprolongefficacy,lowplasmalevelscouldresult in subtherapeutic plasmaconcentrations. Moreover,the donkeydiffersfromthehorseandrequiresspeciesspecific pharmacological and parasitological studies. Therefore, furtherresearchshouldbecarried outtodeterminethe optimaldosage anddosescheme forantiparasitic com-poundsindonkeys.
Acknowledgement
ThisstudywasfundedbythegrantfromtheMinistryof HealthoftheItalianRepublic(IZSME14/11RC).
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