Antiretroviral drugs saquinavir and ritonavir reduce inhibitory concentration values of itraconazole against Histoplasma capsulatum strains in vitro

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Antiretroviral

drugs

saquinavir

and

ritonavir

reduce

inhibitory

concentration

values

of

itraconazole

against

Histoplasma

capsulatum

strains

in

vitro

Raimunda

Sâmia

Nogueira

Brilhante

,

Érica

Pacheco

Caetano

,

Giovanna

Barbosa

Riello

_,

_Glaucia

_Morgana

_de

_Melo

_Guedes

_,

Débora

de

Souza

Collares

Maia

Castelo-Branco

,

Maria

Auxiliadora

Bezerra

Fechine

,

Jonathas

Sales

de

Oliveira

_,

_Zoilo

_Pires

_de

_Camargo

_,

_Jacó

_Ricarte

_Lima

_de

_Mesquita

_,

André

Jalles

Monteiro

_,

_Rossana

_de

_Aguiar

_Cordeiro

,

Marcos

Fábio

Gadelha

Rocha

,

José

Júlio

Costa

Sidrim

a_{SpecializedMedicalMycologyCenter,PostgraduatePrograminMedicalMicrobiology,UniversidadeFederaldoCeará(UFC),Fortaleza,} CE,Brazil

b_{PostgraduatePrograminMedicalSciences,UniversidadeFederaldoCeará(UFC),Fortaleza,CE,Brazil} c_{PostgraduatePrograminVeterinarySciences,UniversidadeEstadualdoCeará(UECE),Fortaleza,CE,Brazil} d_{DepartmentofStatisticsandAppliedMathematics,UniversidadeFederaldoCeará(UFC),Fortaleza,CE,Brazil}

e_{DepartmentofMicrobiology,ImmunologyandParasitology,UniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil} f_{HospitalSãoJosé,Fortaleza,CE,Brazil}

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Articlehistory:

Received25February2015 Accepted25November2015 Availableonline31December2015

Keywords: Histoplasmacapsulatum Saquinavir Ritonavir Itraconazole

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Recent studieshaveshownthatsomedrugsthatarenotroutinelyusedtotreatfungal infectionshaveantifungalactivity,suchasproteaseinhibitorantiretroviraldrugs.Thisstudy investigatedtheinvitrosusceptibilityofHistoplasmacapsulatumvar.capsulatumtosaquinavir andritonavir,anditscombinationwiththeantifungalitraconazole.Thesusceptibilityassay was performedaccordingtoClinical and LaboratoryStandardsInstituteguidelines. All strainswereinhibitedbytheproteaseinhibitorantiretroviraldrugs.Saquinavirshowed min-imuminhibitoryconcentrationsrangingfrom0.125to1␮gmL−1_{forbothphases,and}

riton-avirpresentedminimuminhibitoryconcentrationsrangingfrom0.0312to4␮gmL−1_and

from0.0625to1␮gmL−1_{forfilamentousandyeastphase,respectively.Concerningthe}

anti-fungalitraconazole,theminimuminhibitoryconcentrationvaluesrangedfrom0.0019to 0.125␮gmL−1_{andfrom0.0039to0.0312␮gmL}−1_{forthefilamentousandyeastphase,}

respec-tively.Thecombinationofsaquinavirorritonavirwithitraconazolewassynergisticagainst

H.capsulatum,withasignificantreductionintheminimuminhibitoryconcentrationsofboth drugsagainstthestrains(p<0.05).Thesedatashowanimportantinvitrosynergybetween proteaseinhibitorsanditraconazoleagainstthefungusH.capsulatum.

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

∗ _{Correspondingauthor.}

E-mailaddress:brilhante@ufc.br(R.S.N.Brilhante). http://dx.doi.org/10.1016/j.bjid.2015.11.003

1413-8670/© 2016 Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Histoplasmosisisasystemicinfectioncausedbythe dimor-phic fungusHistoplasmacapsulatum. Itismainly associated withimmunosuppression,especiallyinHIVpatients.1,2 _This disease is characterized by a broad spectrum of clinical manifestations ranging from asymptomatic to dissemi-natedforms.3 _{Histoplasmosisis iswidelydistributed inthe} Americas.4 _{InBrazil,thenumber ofcaseshasincreasedin} severalregions. Someoutbreakshavebeen recordedinthe country,involvingthestatesofRiodeJaneiro,SãoPaulo,Minas Gerais,EspíritoSanto,MatoGrosso,andRioGrandedoSul.4–6 InCearástate,arecentstudyreported254casesof histoplas-mosisinpatientswithHIVintheperiodfrom2006to2010, showingitshighprevalenceinthisregion.7

Treatmentofhistoplasmosis dependson theseverity of infection,clinicalmanifestationsandindividualriskfactors. The therapy indicated for mild to moderate cases is the administration ofazoles, such as itraconazole. Theuse of amphotericinBislimitedtoseverecasesbecauseofitshigh toxicity.Duetotheincreaseofhistoplasmosiscasesinrecent years,particularlyamongHIVpatients,associatedwiththe occurrence of refractory and recurrent infections, there is a needto find newtherapeutic approaches to controlthis mycosis.8,9_{Somestudieshaveshownthatcertaindrugsnot} routinelyusedtotreatfungalinfectionshavesignificant anti-fungalactivity.10_{Amongthese,theantiretroviraldrugshave} demonstratedtheabilitytointerferewiththeviabilityand vir-ulenceoffungalcells.Theproteaseinhibitorsindinavirand ritonavir haveshown in vitro and in vivo inhibitoryeffects againstCandidaalbicans.11Indinaviralsohasshownactivity againstthefungusCryptococcusneoformans,reducingits viru-lenceandmakingitmoresusceptibletothekillingactivityof naturaleffectorcellsoftheimmunesystem.12

Thus,thisstudyaimedtoevaluatetheinvitro susceptibil-ityofH.capsulatumvar.capsulatumtotheantiretroviraldrugs saquinavirandritonavir,aswellastheircombinationwiththe azoleantifungalitraconazole.

Materials

and

methods

Microorganisms

Weusedatotalof20clinicalstrainsofH.capsulatuminthe fil-amentousphaseand10intheyeastphase,isolatedfromthe NortheastandSoutheastregionsofBrazil.Thesamplescame fromtheculturecollectionoftheSpecializedMedical Mycol-ogyCenter,FederalUniversityofCeará,andwerehandledin abiosafetylevel3cabin.

Antifungalagents

Stocksolutionsoftheantiretroviraldrugssaquinavir(Roche HoldingAG,Basel,Switzerland)andritonavir(Abbott Labora-tories,Chicago,USA)andtheantifungalitraconazole(Janssen Pharmaceutica,Beerse,Belgium)werepreparedindimethyl sulfoxide (DMSO). These solutions were stored at −20◦C untiluse. Serialdilutions ofeach antimicrobialagentwere

preparedinRPMI1640(SigmaChemicalCorporation,St.Louis, MO,USA),supplementedwithl_{-glutamine,bufferedatapH} of7.0withMOPS165mmoll−1_{(SigmaChemicalCorporation,} St.Louis,MO,USA).

Preparationoffungalinoculum

Topreparetheinoculum,fungalsuspensionswereprepared insalinefromstockculturesaftersevendaysofincubation, maintainedonBHI(brainheartinfusion)agarandincubated at28◦_{Cforthefilamentousphase.Thecultureswere} main-tainedonBHIagarsupplementedwithsheepbloodat10%and incubated at35◦_{Ctoobtain theyeastphase.Theinoculum} wasadjustedto90–95%bytransmittancespectrophotometry at awavelength of530nm. After reading,the suspensions werediluted1:10inRPMI1640mediumtoobtaininoculums ofapproximately0.5×103to2.5×104cfumL−1.13

Susceptibilitytest

The in vitro antifungal activity was determined by the broth microdilution method in accordance with the pro-tocol described in document M27-A3 and standardized by the Clinical Laboratory Standards Institute.14 _{Initially, the} minimum inhibitory concentration (MIC) was determined for each drug. Subsequently, the MIC values were used as the highest concentration to prepare drugs in com-bination. The concentration ranges of the drugs alone were: 0.0039–2␮gmL−1 _{forsaquinavir, 0.0156–8␮gmL}−1 _for ritonavir, and 0.0009–0.5␮gmL−1 _{foritraconazole. The} con-centration ranges of the drugs in combination were: 0.0002–1␮gmL−1_{forsaquinavir,0.00006–4␮gmL}−1_for riton-avirand0.000003–0.0625␮gmL−1_{foritraconazole.Theresults} were determined by visual readings after seven and four daysofincubationat35◦_{Cforstrainsinthefilamentousand} yeastphase,respectively.TheMICsweredefinedasthe low-est concentration ofdrugableto inhibit80% ofthe fungal growth forantiretroviral drugsand itraconazole,as wellas forthecombinationofboth.13_{Druginteractionwasevaluated} bycalculating the fractional inhibitoryconcentrationindex (FICI),whichwasclassifiedassynergistic(FICI≤0.5), indiffer-ent(0.5<FICI<4),orantagonistic(FICI≥4).15TheFICIvalues obtained for each drug combination against H. capsulatum

were comparedthroughWilcoxontest(p<0.05).The analy-sis wascarried out usingIBMSPSS ver. 21.0software (IBM Co.,Armonk,NY,USA).StandardstrainsofCandida parapsilo-sisATCC22019andCandidakruseiATCC6258wereincludedin eachtestasqualitycontrols.

Results

Theproteaseinhibitorssaquinavirandritonavirwere capa-bleofinhibitingthestrainsofH.capsulatum,withMICvalues rangingfrom 0.125to1␮gmL−1 _{forsaquinavirinboth} fila-mentousandyeastphase;andfrom0.0312to4␮gmL−1_and from0.0625to1␮gmL−1_{forritonavirinfilamentousandyeast} phase, respectively (Table 1). Concerning itraconazole, the MICrangedfrom0.0019to0.0625␮gmL−1_{andfrom0.0039to}

Table1–MICsofantiretroviraldrugsanditraconazoleagainststrainsofHistoplasmacapsulatumvar.capsulatumin yeast-likeandmycelialforms.

Strains Saquinavi(␮gmL−1_{) Ritonavir(␮gmL}−1_{) Itraconazole(␮gmL}−1₎

Mycelialform 0.125(2)a _{0.0312(1) 0.0019(2)} 0.25(6) 0.125(1) 0.0078(4) 0.5(8) 0.5(4) 0.0156(6) 1(4) 1(5) 0.0312(6) 2(5) 0.0625(2) 4(4) Yeastform 0.125(3) 0.0625(4) 0.0039(1) 0.25(1) 0.125(3) 0.0078(4) 0.5(4) 0.5(2) 0.0156(2) 1(2) 1(1) 0.0312(3)

a _{Numberoftestedstrains.}

0.0312␮gmL−1 _{forthefilamentousandyeastphase,} respec-tively(Table1).

Synergistic interactions were observed for the combi-nations of saquinavir or ritonavir with itraconazole, and therewasasignificantreductionintheMICofthesedrugs: saquinavir (p=0.0000) and itraconazole (p=0.0000) for fila-mentous and yeast phases; and ritonavir (p=0.0001) and itraconazole (p=0.0003) for filamentous phase and riton-avir(p=0.0297)anditraconazole (p=0.0015) foryeastphase (Table2).Noantagonisticinteractionswereobserved.

Discussion

Inrecentyears,studieshaveshownthatHIVpatientscanhave substantiallylongerlifeexpectancy,dependingonadherence toantiretroviraltreatmentandtheevolutionoftheimmune status.Also,theuseofmoreeffectivetherapydecreasesthe incidenceofopportunisticinfections,including histoplasmo-sis. Even though the use of protease inhibitors, including saquinavir, has been associated with the development of severalsideeffects, recent studieshavedemonstrated that the use of liposome saquinavir formulations significantly decreases the levels of cytotoxicity, besides improving the bioavailabilityofthisdrug.16

Moreover,ithasbeenobservedthattheproteaseinhibitors canhavebeneficialeffectsonsomefungalinfections,notonly bymodulatingthe immunesystemofthe susceptiblehost, butalsobyexertingdirectactionagainstthepathogen.17 _It

hasalsobeenobservedfrominvitroandinvivostudiesthat proteaseinhibitorsreducethepathogenicityandgrowthofC. albicansprobablybecausethesedrugsactdirectlyonthe pro-ductionoftheenzymeaspartyl-proteinases,whichissecreted bythisorganismintheprocessesofinvasionandcolonization ofhosttissues.18–20_{Additionally,ithasbeenshownthat} riton-avir inhibitstheinvitrohyphalgrowth rateofC.albicans,21 ritonavirandsaquinavirinhibittheadherenceofC.albicans

toendothelialcells,22_{andsaquinavir,ritonavir,andindinavir} attenuatetheinvitroadherenceofC.albicanstoacrylic sub-stances,whichisacommoncomponentoforalappliances.23 Morerecently,itwasobservedthattheproteaseinhibitors saquinavir,darunavir,ritonavir,andindinavirdidnotreduce theinvitrogrowthofC.neoformans,butreducedprotease activ-ityaswellascapsuleproduction,importantvirulencefactors ofC.neoformans.24 _{Althoughthereare somestudieson the} actionofproteaseinhibitorsagainstfungi,noreportsforH. capsulatumhavebeenpublishedsofar.

In the present study, we observed that the protease inhibitorssaquinavirandritonaviraloneinhibitedH. capsu-latum,andsaquinavirandritonavirinteractedsynergistically withitraconazole,asevidencedbytheincreasedinvitro activ-ityoftheantifungaldrug.Theadvantageofthe synergistic effect of the combination of antiretroviral and antifungal drugsaimstoattenuatetoxiceffectscausedbythem, espe-ciallyincasesoflong-termtherapies,suchasinHIV-infected patientspresentingwithhistoplasmosis.2

Casolarietal.25_{foundthatsaquinaviralonedidnotshow} significant antifungalactivity againstthe yeastformsofC.

Table2–EffectsofthecombinationofantiretroviraldrugsanditraconazoleonstrainsofH.capsulatumvar.capsulatumin yeast-likeandmycelialforms.

Strains Drugs MIC–geometricmean (isolateddrugs)

MIC–geometricmean (combineddrugs)

FICIgeometric mean

Results Numberofstrains presentingsynergism Antiretroviral Antifungal Antiretroviral Antifungal

Mycelial form SAQ/ITC 0.4 0.01 0.002 0.0001 0.007 S 20/20 RIT/ITC 1.03 0.01 0.27 0.004 0.5 S 13/20 Yeast form SAQ/ITC 0.45 0.01 0.02 0.001 0.12 S 10/10 RIT/ITC 0.15 0.01 0.01 0.001 0.16 S 10/10

albicansandC.neoformans.However,whencombinedwiththe azoledrugfluconazole,itwasfoundtoinhibitthegrowthof thesefungalspecies,indicatingtheoccurrenceofsynergism betweenthedrugs.Similarly,Palmeiraetal.26_describedthe effectofnelfinavirandsaquinaviragainstthedematious fun-gusFonsecaeapedrosoiandconfirmedthatthesecompounds alone were unable to inhibit the growth of this fungus. However,inhibitiondidoccurwhensub-inhibitorydosesof theseproteaseinhibitorswerecombinedwithamphotericin B.Mikusetal.27_{notedthatritonavirprolongstheclearanceof} voriconazole,althoughtheuseofritonavirwithvoriconazole is contraindicated because the former lowers the plasma levelsofvoriconazole.

Ourdatademonstratedaninvitrosynergisticinteraction betweensaquinavirorritonavirwithitraconazoleagainstthe dimorphicfungusH.capsulatum.Themechanismunderlying theobservedsynergyremainsunknown,butCrommentuyn etal.28_{showedthattheinvivocombinationofantiretroviral} lopinavir/ritonaviranditraconazole interactsthrough phar-macokineticpathways,interferingwiththemetabolismofthe antifungaldrug,whichincreasestheplasmalevelofthe anti-fungalagent,thus,makingitpossibletoreducethedosageof itraconazolewithoutimpairingthetreatment.Basedonour findings,webelievethatsaquinavirorritonavirwith itracona-zoleinteractprobablythroughpharmacodynamicpathways.

Thesedata showan importantinvitrosynergy between saquinavirorritonaviranditraconazoleagainstthefungusH. capsulatum,whichisanimportantpathogenforAIDSpatients.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisresearchwassupportedbyCNPqprocess(303396/2014-8; 552161/2011-0)andCAPES(AE1–0052-000630100/11).

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