Patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Patient

age

does

not

affect

mefloquine

concentrations

in

erythrocytes

and

plasma

during

the

acute

phase

of

falciparum

malaria

José

Luiz

Fernandes

Vieira

,

Larissa

Maria

Guimarães

Borges

,

Michelle

Valéria

Dias

Ferreira

_,

_Juan

_Gonzalo

_Bardarez

_Rivera

_,

Margarete

do

Socorro

Mendonc¸a

Gomes

a_{UniversidadeFederaldoPará(UFPA),InstitutodeCiênciasdaSaúde,Belém,PA,Brazil} b_{LaboratórioCentraldeSaúdePúblicadoAmapá(LACEN-AP),Macapá,AP,Brazil}

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o

Articlehistory: Received10May2016 Accepted13July2016

Availableonline16August2016 Keywords: Malaria Infectiousdisease Parasitology Mefloquine

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Objective:Toevaluatewhetherpatientagehasasignificantimpactonmefloquine concen-trationsintheplasmaanderythrocytesoverthecourseoftreatmentforuncomplicated falciparummalaria.

Methods:A total of20 childrenagedbetween8 and 11years and 20 adultmalesaged between22and 41 yearswith uncomplicated falciparummalaria wereenrolled inthe study.Mefloquinewasadministeredtopatientsinbothagegroupsatadoseof20mgkg−1_. Thesteady-statedrugconcentrationsweremeasuredbyreversed-phasehighperformance liquidchromatography.

Results:Allpatientshadanundetectablemefloquineconcentrationonday0.Inadults,the plasmamefloquineconcentrationsrangedfrom770to2930ngmL−1 _{andtheerythrocyte} concentrationsrangedfrom2000to6030ngmL−1_{.Inchildren,plasmamefloquine} concen-trationsrangedfrom881to3300ngmL−1_{anderythrocyteconcentrationsrangedfrom3000} to4920ngmL−1_{.Therewasnosignificantcorrelationbetweenmefloquineconcentrations} intheplasmaanderythrocytesineitheradultsorchildren.

Conclusion:Inthepresentstudy,weobservednoeffectofpatientageonthesteady-state concentrationsofmefloquineintheplasmaanderythrocytes.Wefoundthatthe meflo-quineconcentrationintheerythrocyteswasapproximately2.8-timeshigherthaninthe plasma.Therewerenosignificantcorrelationsbetweenmefloquineconcentrationsinthe erythrocytesandplasmaforeitheragegroup.

©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:latoxufpa@gmail.com(J.L.Vieira). http://dx.doi.org/10.1016/j.bjid.2016.07.005

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Mefloquineisaquinolinemethanolcompoundwhichis effec-tiveagainsttheasexualbloodstagesofPlasmodiumfalciparum. Itwasfirstintroducedin1984forclinicaluseinThailand. Cur-rentlythedrugisusedforthetreatmentoftheacutephase ofmalaria, eitheraloneorincombinationwithartesunate, oraspreventativemonotherapy forindividuals travelingto endemicareas.1,2

Theexact mechanism of actionof mefloquine remains unknown. However, it probably induces morphological changes in the early ring stages of P. falciparum toinhibit haempolymerization.Administrationofmefloquineresultsin formationofthemefloquine-ferriprotoporphyrinIXcomplex, whichistoxictotheparasite.3,4_{Theconcentrationof} meflo-quinewithinerythrocytesisoneofthemajordeterminants ofitstherapeuticefficacy.Thus,itislikelythatachangein exposuretomefloquinewouldaltertheconcentrationofthe drugwithinredbloodcells,andconsequently,thetreatment outcomes.5,6 _{Studies investigatingthe pharmacokinetics of} mefloquinehavedemonstratedthattheconcentrationofthe drug inblood components isinfluenced by several factors includinggender,bodyweight,diseaseseverity,parasitemia athospitaladmission,drugmalabsorption,vomiting,drug for-mulation,andingestionofcertainfoods.6,7

Itisplausiblethatthepatient’sagemayalsoaffectthe con-centrationofmefloquineinthebloodcomponents.Ahigher rateoftherapeuticfailurehasbeenreportedinchildren com-paredtoadults.Thiswasfoundtobeassociatedwithreduced concentrationsofmefloquineinthebloodasaresultof malab-sorption,diarrheaorvomiting.6,7_{Aschildrenareathigherrisk} forcerebralmalaria,theefficacyofantimalarialdrugsneeds tobecarefullyevaluated.However,thereislimiteddataonthe effectofageontheconcentrationofmefloquinewithin eryth-rocytes. Therefore,the aimofthis study wastodetermine whetherthepatient’sagehasaneffectontheconcentrationof mefloquineintheplasmaanderythrocytesduringtreatment oftheacutephaseofuncomplicatedfalciparummalaria.

Materials

and

methods

Ethicalstatement

The study was approved by the Ethical Committee of Associac¸ão Educacional da Amazônia-SEAMA (079/08). All adultpatients and caretakers or guardians of the children enrolledinthestudy wereinformed aboutthegoals ofthe study,aswell astherisks andbenefits,before giving writ-teninformedconsentpriortoenteringthestudyaccordingto theNationalCommitteeofEthicsinResearch(CONEP;196/96). Thechildrenreceivedverbalinstructionsaboutthestudy,and thecollectionofbloodwasperformedinthepresenceoftheir caretakersorguardians.

Studylocationandpatients

ThestudywascarriedoutattheReferenceCenterforTropical DiseaseinMacapa(Brazil),whichislocatedinanareaoflow malarialtransmissionalongsidetheAmazonRiver.Atotalof

20malechildrenand20maleadultswithslide-confirmedP. falciparuminfectionwereenrolledinthestudy.Theexclusion criteriaweresignsorsymptomsofsevereormixedmalaria (jaundice,renalimpairment,severeanemia,oranalteredlevel ofconsciousness),parasitemiaabove5%,aknown hypersen-sitivity or allergy tomefloquine, individuals that had used antimalarialdrugsinthefour weekspriortoadmission,or individualswithaknownhistoryofpsychiatricdisorders.

Clinicalandlaboratoryassessment

Allpatientsunderwentclinicalandlaboratoryevaluationat admissiononday0(D0)andondays3,7,14,28and42.A com-pletebloodcountwasperformedonD0andday3(D3).Blood samplesforthemeasurementofmefloquinewerecollected onD0andD3.

Mefloquinetreatment

Eachparticipantreceivedmultipleoraldosesof250mg meflo-quinebasetablets (Farmanguinhos;BrazilianHealthOffice) ondays1and2.1,8 _{Thedosewasadjustedfortheweightof} thepatienttoreceiveatotaldoseof20mg/kg.Thedrugwas administered with water30minaftera typicalAmazonian breakfast.Noadditionalfoodordrinkwasprovidedfor2h fol-lowingadministrationofthedrug.Incaseofvomitingwithin 2hofmefloquineadministration,thepatientswerecarefully supervisedbyclinicalstaff.

Bloodsamplecollectionandmeasurementofmefloquine

Venousbloodsamples(4mL)werecollectedfromeachpatient inlithiumheparintubesonD0and 24hafterthe lastdose ofmefloquine(D3).Aftercollection,thesamples(4mL)were immediatelycentrifugedat500×gfor10minat4◦Cforthe separation ofplasma. Acell washbuffer wasadded tothe erythrocytepellet,whichwasthencentrifugedat500×gfor 10min.Thisprocedurewasrepeatedfivetimesforthe separa-tionoferythrocytes.Thesampleswerestoredat−80◦Cuntil analysis.

Areversed-phasehigh performanceliquid chromatogra-phy(HPLC)systemwithultravioletdetection(ProStar;Varian, Walnut, CA)was used forthe analysisofmefloquine after liquid-liquid extractionofthedrug from theblood compo-nentswithmethyltert-butyletheratpH4.0.Theseparation was carried out using a reversed-phasecolumn (ODS C18; 4.6mm×250mm,5␮minternaldiameter;SupelcoInc., Belle-fonte, PA) with a mobile phase composed of acetonitrile: phosphate buffer (pH 2.5) with a ratio of 42:58. Quinidine (2.5␮g/mL)wasusedastheinternalstandard.Thewithin-day andday-to-daycoefficientsofvariationforthelaboratory con-ditionswere6.7%and8.1%,respectively.Theassaywaslinear from150to5000ng/mLandthedetectionlimitwas50ng/mL. Themean recoverywas95%.Thestability ofblankplasma spikedwithmefloquinewas60days.9

Dataanalysis

Thedataaredescribedasthemedianvalueandmeasurement range. TheMann–Whitney Utestwasusedtocompare the

Table1–Baselinecharacteristicsofpatientsincludedinthestudy.

Parameter Children(n=20) Adults(n=20)

Age,yearsa _{9(8–11) 31(22–41)}

Weight,kga _{23(19–26) 67(54–78)}

Parasitaemia,microliterb _{2100(900–4500) 2600(800–5100)}

Timeforclearanceofparasites,hoursa _{30.5(24–48) 29.5(24–48)}

Timeforfeverclearance,hoursa _{20(12–30) 18(12–28)}

Whitebloodcellscount,microlitera _{11.300(7.600–13.000) 9300(5.200–12700)}

ErythrocytecountX106_,microlitera _{3.21(3.1–4.1) 4.11(3.9–4.9)}

a _{Dataarepresentedasmedianandranges.}

b _{Dataarepresentedasgeometricalmean.}

Table2–Concentrationsofmefloquinedeterminedintheplasmaanderythrocytesofchildrenandadultswith

uncomplicatedfalciparummalaria.

Agegroups n Mefloquineinplasmaa_(ngmL−1_{) Mefloquineinerythrocytes}b_(ngmL−1_{) Ratioerythrocytetoplasma}b

Children 20 1530(881–3300) 4380(3000–4920) 2.86(1.37–4.97)

Adult 20 1610(770–2930) 4220(2000–6030) 2.72(0.89–3.89)

a _{Dataarepresentedasmedianandranges.}

b _{Ratioofmefloquineconcentrationsintheerythrocytesandplasmaofchildrenandadults.}

concentrationsofmefloquineintheplasmaanderythrocytes forbothage groups.TheSpearman’scorrelationcoefficient wasusedtoestimatethecorrelationbetweenthemefloquine concentrationsintheerythrocytesandplasmaforbothage groups.Allp-valuesweretwo-tailedandp<0.05was consid-eredtobestatisticallysignificant.Allstatisticalanalyseswere performedwithStatisticasoftware(version7.0;StatSoftInc., Tulsa,OK).

Results

The baseline characteristics of all patients are shown in Table1.Thegeometricmeanparasitedensitywassimilarfor bothagegroups.Allpatientscompletedthe42-dayfollow-up period.Therewasnoresurgenceoftheparasiteinthe periph-eralblood,andallpatientsshowedanadequateresponseto treatmentasdemonstratedbycompleteclearanceofparasites fromtheperipheralbloodwithinthreedaysofstarting treat-ment.Themeanparasiteclearancetimeswere30.5h(ranged from24to48h)and29.5h(rangedfrom24to48h)hin chil-drenandadults,respectively.Themeantimeforthefeverto clearwas20h(ranged from12 to30h)inthechildrenand 18h (ranged from 12 to28h)in the adults. Thedrug was welltolerated,asonlytwochildrenexperiencedslight nau-seaanddizzinessandtherewasnoreportofvomitingafter drugintake.

All patientshad no measurable concentrationof meflo-quineonD0.In adults,theconcentration ofmefloquine in theplasmarangedfrom770to2930ngmL−1_{andfrom2000to}

6030ngmL−1_{intheerythrocytes.Theerythrocyte-to-plasma}

concentration ratio ranged from 0.89 to 3.89. In children, theconcentrationofmefloquineintheplasmarangedfrom 881 to3300ngmL−1 _{and from 3000to 4920ngmL}−1 _{in the}

erythrocytes.Theerythrocyte-to-plasmaconcentrationratio ranged from 1.37 to 4.97 in the children. The mefloquine concentrations were similar in the adults and children in both the plasma (U=210; p=0.60) and erythrocyte (U=228;

p=0.94) concentrations. A similar result was found for the erythrocyte-to-plasmaconcentration ratio between the different agegroups(U=192;p=0.34). Themefloquine con-centrations foundinthe erythrocytes and plasmaforboth agegroupsarepresentedinTable2,andthe erythrocyte-to-plasma concentration ratios are presentedin Fig. 1. There werenosignificantcorrelationsbetweentheconcentrationof mefloquineintheerythrocytesandplasmaofadults(s=0.102; p=0.32)orchildren(s=0.215;p=0.21).

Discussion

Thisstudyinvestigatedtheeffectofageontheconcentration ofmefloquineintheplasmaanderythrocytesofpatientswith uncomplicatedfalciparummalariaintheAmazonbasin.Drug

Adults Children 0 1 2 3 4 5 6 7 8 9 10

Ratio erythrocytes to plasma

Fig.1–Ratioofmefloquineconcentrationsinthe

erythrocytesandplasmaofchildrenandadultsonD3after thecommencementoftreatment.Theboxrepresentsthe medianvalueandthefirstandthirdquartiles,andthe whiskersrepresentthemaximumandminimumvalues.

administrationandtheoccurrenceofvomitinganddiarrhea werecarefullymonitoredbytheclinicalresearchstaff.

Thisisthefirsttimethatplasmamefloquine concentra-tions have been measured in a riverside population from theBrazilianAmazonbasin.Theconcentrationofmefloquine foundinadultpatientswassimilartothatreportedinprevious studieswhichinvestigatedhealthyCaucasianvolunteersand patientswithuncomplicatedfalciparummalariafrom Thai-landandPeru.10–12 _{Inchildren,themedianconcentrationof} mefloquineintheplasmawassimilartothatobtainedina studyincludingKarenchildren.13_{Furthermore,therewereno} significantdifferencesinmefloquineconcentrationsbetween childrenand adults.Thisisconsistent withprevious stud-ies which investigatedand compared the pharmacokinetic parametersofmefloquineinpatientswithuncomplicated fal-ciparummalariaofdifferentagegroups.Thesestudiesonly foundaneffectofbodyweightondrugclearanceinchildren aged from 6 to 24 months when compared to older chil-dren(agedfrom5to12years),whichweresimilartoadult patients.14–17 _{Inaddition, theconcentrationsofmefloquine} inallpatientswereabove500ngmL−1_{,whichwasassociated}

withhighratesoftreatmentefficacy.15,18

Intheerythrocytes,themefloquineconcentrationswere alsosimilar forboth agegroups. Furthermore, the median concentrationofmefloquineinthe erythrocytes wasabout 2.8-timeshigherthanintheplasmaforbothagegroups.This isconsistent with an in vitrostudy which reported a rela-tivelyconstantdistributionofmefloquineintheerythrocytes and plasma, with a ratio of approximately 2:1, suggest-ing the potential accumulation of mefloquine within the erythrocytes.19 _{Despite the high binding of mefloquine to} plasmaproteins,itpassivelycrossesthemembraneandbinds tothe membranephospholipidsand ferriprotoporphirinIX, formingamefloquine-ferriprotoporphirinIXcomplexwhich istoxicto the Plasmodium.This complex isresponsible for the highdrug concentrationobserved inparasitized eryth-rocytes, which is approximately 3-to 4-times higher than non-parasitizederythrocytes.19–23

Ontheotherhand,thefindingsofthestudywerenot con-sistentwithapreviousstudyonthedistributionofmefloquine in the blood components of patients with uncomplicated falciparummalariainThailand,whichreportedsimilar con-centrationsofmefloquine inthe whole blood and plasma. Furthermore,another study in the same population group whichinvestigatedtheinfluenceofgenderonmefloquine con-centrationsinbloodcomponentsreporteddrugaccumulation intheplasma,withtheplasma-to-erythrocytesratioranging from1.59to3.75.Thereareseveralpossibleexplanationsfor thediscrepanciesbetweenstudies,includingparasitemiaat admission,disease severity,mefloquineformulation,orthe timebetweendrugadministrationandbloodsampling.11,24,10 Therewerenosignificantcorrelationsbetweenthe concen-trationofmefloquineintheplasmaanderythrocytesforeither agegroup.Thismaybeduetotheaffinityofthedrugtoother bloodcomponents, ratherthanthe plasmaorerythrocytes. Thisisinagreementwithtwostudieswhichevaluatedthe distributionofmefloquineinthebloodcomponentsofThai patientswithacuteuncomplicatedfalciparummalariaandin healthyCaucasians.Bothstudiesreportedthatthemefloquine concentrations inblood components was highestin white

bloodcells,followedbyplatelets,plasma,serum,wholeblood, andlowestinerythrocytes.24,10

A potential limitation of the present study isthat only one blood samplewas collected from each patient to esti-matemefloquineconcentrationsinbothbloodcomponents. However,measuringanti-malarialdrugswithalonger half-lifeinthesteadystateappearstoprovidereliabledatawhich can beused toestimate the exposure ofpatients tothese medicines.25

Conclusions

In the present study,we observedno effect ofpatient age onthesteady-stateconcentrationsofmefloquineinplasma and in erythrocytes. In addition, the concentration of the drug in erythrocytes was approximately 2.8-times higher thaninplasma.Finally,therewerenosignificantcorrelations observed betweenthe concentrations ofmefloquinein the erythrocytesandtheplasmaforeitheragegroup.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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