Combination Antifungal Therapy

23 Pages·2004·0.19 MB·English

ANTIMICROBIALAGENTSANDCHEMOTHERAPY,Mar.2004,p.693(cid:1)715 Vol.48,No.3
0066-4804/04/$08.00(cid:2)0 DOI:10.1128/AAC.48.3.693–715.2004
Copyright©2004,AmericanSocietyforMicrobiology.AllRightsReserved.
MINIREVIEW
Combination Antifungal Therapy
Melissa D. Johnson,1,2* Conan MacDougall,3 Luis Ostrosky-Zeichner,4 John R. Perfect,1 and
John H. Rex4,5
DepartmentsofMedicine,1andPharmacy,3DukeUniversityMedicalCenter,Durham,andCampbellUniversitySchoolof
Pharmacy,BuiesCreek,2NorthCarolina;DivisionofInfectiousDiseases,DepartmentofInternalMedicine,
CenterfortheStudyofEmergingandRe-emergingPathogens,UniversityofTexas—HoustonMedicalSchool,Houston,
Texas;4andAstraZenecaPharmaceuticals,AlderleyHouse,Macclesfield,Cheshire,UnitedKingdom5 D
o
w
n
RATIONALE Greco(79).Inbrief,allapproachestoevaluatingcombinations lo
can be reduced to two elements: (i) a conceptual model for a
Theavailabilityofnewantifungalagentswithnovelmecha- d
predictingtheexpectedresultforacombinationand(ii)aset e
nismsofactionhasstimulatedrenewedinterestincombination
of phrases used to categorize results that are better than ex- d
antifungaltherapies.Inparticular,anddespitethelimitedclin- pected, worse than expected, or as expected. Although many fr
ical data, the high mortality of mold infections and the rela- o
subtle variations are possible, the underlying mathematical m
tively limited efficacy of current agents have produced signifi-
model is based on either the assumption of additive interac- h
cant interest in polyene-, extended-spectrum azole-, and
tions or the assumption of probabilistic (multiplicative) inter- tt
echinocandin-based combinations for these difficult-to-treat p
actions. On the basis of the terminology employed by the :
infections. With the recent publication of the first large ran- //
authorwhofirstcarefullydescribedeachofthesemodels,the a
domized trial of antifungal combination therapy to be con- a
twomodelscanbeusefullyreferredtoastheLoeweadditivity c
ductedintwodecades(166)andtherapidproliferationofnew .
modelandtheBlissindependencemodel(79). a
invitroandinvivodataonantifungalcombinations,wehave s
The terminology used to place results into interpretive cat- m
soughttoreviewtherecentworkandfuturechallengesinthis
egoriesisoftenthesubjectofdebateandconfusion.Grecoet .
area. o
Thefocusofthisreviewisontheefficacyofantifungaldrugs al. (79) have proposed a set of consensus phrases that are rg
in combination with respect to the extent or rate of killing of instructive (Table 1). In this proposal, synergism and antago- o/
nism have clear and intuitive meanings. The phrases used to n
the fungal pathogen, although other potential interactions

describe results that are neither synergistic nor antagonistic J
(such as pharmacokinetic drug interactions) can impact effi- a
cacywhentheseagentsareusedtogether.Thevalueofgiving are,however,somewhattricky.Mathematically,theterm“ad- n
u
ditive”isindeedlogicalfortheLoeweadditivitymodeljustas
twodrugsbecauseeachisseparatelyeffectiveagainstagroup a
of organisms exhibiting a variety of types of resistance is not the term “independent” is logical for the probabilistic Bliss ry
specifically discussed, but this also is an obvious and straight- model. Unfortunately, the term “additive” often conveys an 1
1
imprecisemessageandmaybemisinterpretedasreferringtoa
forwardreasontouseacombinationofagents. ,

positive interaction. Coined terms such as “subadditive” only 2
It cannot be simply assumed that the use of two or more 0
effective drugs with different mechanisms of action will pro- reinforcethiserroneousconception. 1
9
Thissituationhasnoperfectresolution.Possiblealternatives
duceanimprovedoutcomecomparedtotheresultsseenwith
b
a single agent. Combination antifungal therapy could reduce to the term “additive” include the terms “summation” (44), y
antifungal killing and clinical efficacy, increase potential for “no interaction” (141), and “indifferent.” The term “summa- g
u
druginteractionsanddrugtoxicities,andcarryamuchhigher tion”unfortunatelystillcarriesahiddenpositivemessage.Al- e
cost for antifungal drug expenditures without proven clinical though somewhat imprecise, the terms “indifferent” and “no st
benefit (106). Thus, it is important to critically evaluate the interaction” have an inherently conservative emotive nature
roleofcombinationtherapyasnewdatabecomeavailable. and can be used to describe Loewe additivity and Bliss inde-
Conceptual models and terminology. Methods for studying pendence and also to describe results in cases in which the
antifungalcombinationsinvitroandinvivohavedifferedcon- underlying model is not clearly specified. The use of these
siderably over time and among investigators. These tools do terms provides the reader with a constant reminder of the
not differ with respect to their application to combination neutralnatureoftheresult—althoughtherecanbevalueinan
antibacterial or antiviral therapies and have been discussed indifferent (additive) interaction, the biological relevance of
extensively and elegantly in the landmark 1995 review by suchaninteractionisnotalwaysobvious.Forreasonsrelated
mostlytoeaseofexpression(itissimplertospeakofindiffer-
enceandindifferentinteractionsthantospeakofnoninterac-
*Correspondingauthor.Mailingaddress:DepartmentofMedicine, tion and noninteractive interactions), this review uses the
Division of Infectious Diseases, Duke University Medical Center,
phrases synergistic, indifferent, and antagonistic when inter-
NorthCarolina,Box3306DUMC,Room0207EOrangeZone,Duke
HospitalSouthTrentDr.,Durham,NC27710.Phone:[phone]. pretivecategoriesarerequired.
Fax:[phone].E-mail:[email protected]. Withrespecttotheunderlyingmathematicalmodel,Loewe
693
694 MINIREVIEW ANTIMICROB.AGENTSCHEMOTHER.
TABLE 1. Consensusterminologyfordescribingresultsofcombinationtestinga
Terminologyforindicatedconditions
Bothagentsareactive
Category Bothagentsareactive Oneagentis
alone;multiplicative Neitheragentis
alone;additiveeffects activealone;
effectsmodelis activealone
modelispresumed theotherisnot
presumed
Combinationresultisbetterthanexpected Loewesynergism Blisssynergism Synergism Coalism
Combinationresultisasexpected Loeweadditivity Blissindependence Inertism Inertism
Combinationresultisworsethanexpected Loeweantagonism Blissantagonism Antagonism
aTheterminologyshownisthatproposedbyGrecoetal.(79)followingaconsensusconferenceoccurringinSarriselka,Finland.Modelsbasedontheadditive
interactionconceptfirstproposedbyLoeweandMuischnek(111)followtheintuitiveresultinwhichadrugcombinedwithitselfproducesalinearsumofeffects.That
is,1(cid:4)g/mlplus1(cid:4)g/mlgivestheeffectof2(cid:4)g/mlandthisresultisneithersynergisticnorantagonistic.Modelsbasedonthemultiplicativeinteractionconceptfirst
proposedbyBliss(26)followaprobabilisticmodelinwhichthetwoagentstrulyactindependentlyasdeterminedonthebasisoftheirseparateprobabilitiesofeffect.
If1(cid:4)g/mlpermits40%ofthetargetorganismtosurvive,then1(cid:2)1(cid:3)2(cid:4)g/mlshouldpermitonly40%(cid:6)40%(cid:3)16%survival.AsreviewedindetailbyGrecoet
al.(79),bothmodelshavestrengthsandweaknessesbutLoeweadditivity-basedmodelsmoreoftenseemappropriateforcombinationsofantimicrobialagents. D
o
w
n
additivity most often seems appropriate for combinations of both drugs show a minimum drop in MIC of at least two lo
a
antimicrobial agents. This result follows from the detailed dilutionstepsandthus,afourfolddrugconcentrationdrop.As d
comparison of the strengths and weaknesses of the additive anexample,considerdrugsAandB,eachofwhichhasaMIC e
d
andmultiplicativemodelsfoundinthereviewbyGrecoetal. of2(cid:4)g/mlforagivenisolate.Synergywouldonlybedeclared
f
(79).Althoughmanyotherargumentscanbeputforwardand whenbothdrugsincombinationshowedaMICof0.5(cid:4)g/mlor ro
the interested reader is strongly encouraged to study closely less.Mathematically,thiswouldbeFICI(cid:3)(0.5/2)(cid:2)(0.5/2)(cid:3) m
the review by Greco et al., the key argument for us is that 0.25(cid:2)0.25(cid:3)0.5.Importantly,aFICIof0.50000000001does h
t
Loeweadditivitysupportsthethoughtexperimentinwhichan notmeetthedefinitionofsynergy.Forinstance,iftheMICof tp
:
agentcombinedwithitselfisneithersynergisticnorantagonis- onedruginourexampleweretodroptoonly1whenusedin /
/
a
tic. combination, synergy could not occur under these rules no a
Quantitative analyses: the fractional inhibitory concentra- matterhowlowtheotherdrug’sMICincombinationwereto c
.
tion index. Calculation of the fractional inhibitory concentra- become: FICI (cid:3) (1/2) (cid:2) (??/2) (cid:3) 0.5 (cid:2) some value greater as
tion(FIC)index(FICI)bytheuseofthecheckerboardmethod than0.ThiswouldresultinaFICIvalueslightlygreaterthan m
.
haslongbeenthemostcommonlyusedwaytocharacterizethe 0.5andwouldthusbedefinedasindifference. o
r
activity of antimicrobial combinations in the laboratory (66). Conversely, antagonism is declared when at least one drug g
/
TheFICIrepresentsthesumoftheFICsofeachdrugtested, has at least a fourfold increase in MIC. To understand this o
wheretheFICisdeterminedforeachdrugbydividingtheMIC rule,considertheboundaryconditionunderwhichbothdrugs n

J
of each drug when used in combination by the MIC of each showapreciselytwofoldincreaseinMIC.Continuingwiththe a
drugwhenusedalone.StatedintermsoftheLoeweadditivity same example, this would be the situation when the MICs of n
u
model,theFICImodelassumesthatindifferenceisseenwhen bothagentsincreaseto4andthus,FICI(cid:3)(4/2)(cid:2)(4/2)(cid:3)2 a
thisequationistrue:1(cid:3)(MIC /MIC ) (cid:2)2(cid:3)4.Thisresultremainswithinexperimentalerrorlimits; ry
(cid:2)(MIC /MIdCrugAincombin)a.tiTonomakderutghAisacloonne- a FICI of precisely 4.00000 is defined as indifferent (or addi- 1
drugBincombination drugBalone 1
crete,imagineanorganismforwhichthefluconazoleMICis2 tive), whereas any value greater than 4 is defined as antago- ,

(cid:4)g/ml. If we perform a checkerboard study of fluconazole nistic. The rule that a FICI of (cid:5)4 defines antagonism also 2
0
versusfluconazole,weshouldfindthatthewellwhichreceives handles the situation wherein a small amount of one drug 1
the combination of 1 (cid:4)g/ml (cid:2) 1 (cid:4)g/ml produces an effect dramaticallyincreasestheMICfortheotherdrug.Forexam- 9
b
identical to that of the wells containing 2 (cid:4)g/ml [or FICI (cid:3) ple, a small and ineffective concentration of beta-lactam A y
(1/2)(cid:2)(1/2)(cid:3)0.5(cid:2)0.5(cid:3)1,whichindicatesindifferenceor mightinducetheexpressionofabeta-lactamaseactiveagainst g
u
Loeweadditivity]. beta-lactamBand,thus,causebeta-lactamB’sMICtoincrease e
s
Reproducible variations from an FICI of 1 represent non- fourfoldormore.Inthiscase,theFICIruleforantagonismin t
indifference of at least some magnitude. However, the exper- ourexampleisagainsatisfied:FICI(cid:3)(??/2)(cid:2)(8/2)(cid:3)some
imentalistmustconsiderboththeinherentinaccuracyofMIC valuegreaterthan0(cid:2)4(cid:3)somevaluegreaterthan4.
methodologiesandthequestionofbiologicalrelevance.Thus, Morecomplexcalculationsandmixedinteractions:beyond
ithasbeenproposedthatsynergybedeclaredwhentheFICI the FICI. While the FICI and its variants have long been
is less than or equal to 0.5 and that antagonism be declared employed to depict the characteristics of antimicrobial drug
when the FICI is greater than 4 (66, 141; Instructions to au- combinations,theseapproacheshavehadanumberoflimita-
thors,Antimicrob.AgentsChemother.48:i–xxi,AmericanSo- tions that have been well described by others (62, 106). The
cietyforMicrobiology,2004). biggest challenge is that FICI-based approaches presume a
The logic behind these interpretive categories is worth dis- smooth and linear interaction across all combinations of con-
cussing. They are based on the related assumptions that (i) centrations. In practice, this situation is often not seen; Elio-
testingemploysconcentrationsseparatedbyafactorof2(e.g., poulos and Moellering (66) provide some excellent examples
a sequence similar to 0.25, 0.5, 1, 2, and 4 (cid:4)g/ml) and (ii) ofthecuriousresultsseeninpractice.Inanefforttodevelop
1-dilution-step MIC changes are within experimental error approaches that resolve these difficulties, more sophisticated
ranges. For a result to be synergistic, these rules require that methods have been proposed (as discussed in great detail by
VOL.48,2004 MINIREVIEW 695
Greco et al.) (79). Many of these other methods have only Approaches to analysis of in vivo and clinical studies. Ap-
occasionallybeenemployedinstudiesreportedintheantifun- plyingtheseideastoinvivoandclinicalinvestigationsofcom-
gal literature. The method of Chou and Talalay (44) was re- binationantifungaltherapyisespeciallydifficult,andnostan-
viewedinparticulardetailbyGrecoetal.(79)andisespecially dardsforinterpretationofthesedatahavebeenrecommended
noteworthyforitsfrequentuseinpublicationsonantiviraland todate.Analysisandcomparisonofresultsacrossinvivoand
antineoplastic drug combinations. This method has more re- clinicalstudiesrequirescarefulconsiderationofthenatureof
centlybeenappliedtoworkwithbeta-lactamaseinhibitorsand pathogen, host, host immune status, study design, and study
appears to have broad applications (196). A few antifungal endpoints.Issuesrelatedtoclinicaltrialdesignwithantifungal
studieshaveemployedothermethods.Forinstance,acontour agents have been extensively reviewed elsewhere (25, 167).
and surface plot methodology has been proposed and was Indeed,eachtypeofstudy(invitro,invivo,andclinical)hasits
foundparticularlyusefulincharacterizingthenatureofthree ownsetofstrengthsandweaknesses(Table2).
antifungalagentsincombinationsusingvariousconcentrations A few issues particularly stand out with reference to the
of each agent (74). Similarly, newer and more sophisticated study of antifungal combinations in the clinical setting. First,
methods have been employed in recent years to depict the themethodsusedtoassessantifungaldrugefficacyforhumans D
nature of these complex interactions in animal models of in- are limiting. Whereas other disease states have surrogate o
w
fection. These methods involve the use of response-surface markers,suchasestimatesofviralantigenemia(humanimmu- n
plots which illustrate the Loess fit of the association for a nodeficiency virus [HIV] infection) or sputum CFU (assess- lo
a
particular outcome (such as weight change, fungal tissue bur- mentofearlybactericidalactivityofmycobacterialdrugs),that d
den, or survival) (60, 61, 101). These methods have been fa- canyieldrapid,early,andclinicallyrelevantmeasuresofdrug e
d
cilitated by improvements in the capabilities of technologies efficacy,mostantifungalclinicaltrialsarelimitedtosubjective
f
r
developedfortheperformanceofmorecomplexmathematical efficacy assessments based on clinical outcome. Standardized o
m
and statistical computation. In contrast to older isobologram criteria have been developed for the purposes of enrollment

approaches, the Loess method has the distinct advantage of (8), and the use of blinded efficacy assessment expert panels h
t
allowing visualization of the relationship between agents that canreducebias(25,85,167);however,thesetoolsstilldonot tp
:
havedifferentdose-responsecurves.TheLoessmethodisflex- provide rapid or quantitative means for the evaluation of an- /
/
a
ible and fairly simple to perform using appropriate statistical tifungal drug efficacy. Colony counts are routinely employed a
software, but it requires a fairly high density of data (i.e., with animal models as a means of assessing efficacy, but with c
.
a
intensesampling)andiscomputationallycomplex.Unlikethe the exception of serial studies of cerebrospinal CFU levels in s
resultsseenwithothermodels(suchasnonlinearregression), subjects with cryptococcal meningitis (R. A. Brouwer, A. Ra- m
.
furthermore,theuseoftheLoessmethoddoesnotresultina januwong, W. Chierakul, G. E. Griffin, R. A. Larsen, N. J. o
r
mathematical formula representing a regression function that White,andT.S.Harrison,Abstr.15thCongr.Int.Soc.Hum. g
/
can be easily shared with others. The Loess method has not Anim.Mycol.,abstr.029,2003),studiesofhumansarelimited o
been widely employed to date, but future investigations may toless-invasivemethods.Newertechnologiescanprovidenon- n

J
usesimilarmethodsorevendevelopnewermethodstodepict traditionalmethodsfortheestimationoffungalburdenlevels a
thesecomplexandoftenunpredictabledose-responseinterac- inhumantissuesorserum(29,32,45,64,65,70,72,76,83,84, n
u
tionsbetweenmultipleantifungalagents. 86, 108, 109, 110, 112, 131, 132, 134, 177, 211, 220, 221), but a
r
Ultimately, all of these methods attempt to reduce the in- theseassayshavegenerallybeendevelopedwiththeobjective y

teraction to one or more summary terms that capture the ofdiagnosingdiseaseratherthandeterminingdrugefficacy.To 1
1
degreeofdeviationoftheobservedresultsfromtheexpected furthercomplicatematters,eventraditionalmethodsmayfail, ,

2
results.ThemethodofChouandTalalay(44)hastheadvan- asillustratedbystudiesofcaspofunginanditseffectsoncolony
0
tagethatitsplotshowingthefractionaffectedversusthecom- counts of Aspergillus spp. (4, 82, 152). Development and vali- 1
9
bination index provides a simple and visual way to look at a dationofmore-rapidand-reliablemeasuresofantifungaldrug
b
seriesofsummarytermsacrossarangeofpossibledrugcom- efficacy that could be used in the clinical setting would facili- y

bination concentrations. This approach is especially helpful tateclinicaltrialsthatusetheseagents. g
u
whenthestudyshowssynergyundersomeconditionsandan- Beyond this initial hurdle, host factors (such as changes in e
s
tagonismunderothers(38,39).Whensuchasituationoccurs, ongoing immunosuppression, altered physiologic state, and t
itisbothlogicalandplausibletofocusontheresultachieved pharmacokinetic disposition of drugs) greatly impact the per-
withthemaximaltolerablesystemicdrugexposure(37). formance of an antifungal agent in the clinical setting and
No matter how it is defined, the analysis must ultimately cannot always be simulated in studies employing in vitro or
apply some test of biological relevance to the result and pro- animalmodels.Althoughmostoftheseissuesareobvious,the
poseasetofinterpretivecategories.Whileitisnotpossibleto possibilityofapharmacokineticinteractionbetweenthestudy
propose a fixed figure for use in defining interpretive catego- drugs (e.g., studies of the combination of rifampin with an
ries,thehistoricalmodelsuggestedbytheideasdevelopedon azolewouldbelimitedbythefactthatrifampinreducesazole
thebasisoftheFICI(seeabove)seemslogical:synergybegins bloodlevels)isoftenoverlooked.Theinterplaybetweenallof
when the activity of each drug appears to increase at least thesefactorsandthepathogeninthehumancannotnecessar-
fourfold, antagonism begins when the activity of at least one ily be predicted on the basis of laboratory studies, and issues
drug decreases at least fourfold, and indifference lies in be- related to toxicities or lack of efficacy may only be apparent
tween. Although one can debate the point endlessly, these whenstudiedinhumans.
ideas have the virtue of suggesting changes in drug doses or Severalstudieshavesuggestedthatdifferentconcentrations
effectsthatarepotentiallybiologicallyrelevant. ofeachdrugincombinationcanbeassociatedwithresultsthat
696 MINIREVIEW ANTIMICROB.AGENTSCHEMOTHER.
TABLE 2. Technicalandanalyticalissuesassociatedwithdifferentmodelsystemsforstudyingcombinationantifungaltherapy
Characteristic
Category
Invitrostudies Animalmodels Clinicaltrials
Strengths Easilyrepeatedacrossawidevariety Studiescanbedonewithhomogeneous Thisistheanswerthatmatters
ofdrugconcentrations hosts
Easilysubjectedtostatisticaltesting Hostfactorsareintegrated
Easytovarytechnicalfactors Resistantisolatescansometimesbetested
Easytotestmultipleisolates Quantitativeendpoints(tissueburden,
rateofclearance)canbetested
Easytotestisolateswithdefined Arangeofdosesanddosecombinations
typesofresistance canbetested
Weaknesses Relevanceofinvitromethodsnot Infectionmodelsareoftenpoormimicsof Subjectsandinfectingisolates
alwaysclear humandisease areheterogeneous
Hostfactorsareignored Pharmacokineticandtoxicological Noabilitytocontrolnatureof D
behavioroftestdrugs(andtheir infectingisolate o
w
pharmacologicaleffectsoneachother)
n
maynotmimicthatseeninhumans lo
Pharmacokineticfactorsareignored Onlylimitednumbersofisolatescanbe Underlyingdiseasecannotbe a
tested controlled d
Onlylimitednumbersofrepetitionsare Lackofquantitativeendpoints e
d
possible formanydiseases
f
Resistantisolatessometimeshavereduced Limitedabilitytostudya r
o
virulence rangeofdoses m
Veryexpensive
Slow h
t
t
p
:
/
/
a
a
range from synergy to antagonism (19, 157, 161; L. Ostrosky- The approach taken in this review. Recognizing the chal- c
.
a
Zeichner,M.Matar,V.L.Paetznick,J.R.Rodriguez,E.Chen, lengesinherentinthestudyofcombinationantifungaltherapy s
andJ.H.Rex,Abstr.42ndIntersci.Conf.Antimicrob.Agents intheclinicalsetting,itisclearthatinvitroandanimalmodel m
.
Chemother., abstr. M-1816, p. 415, 2002). Studying multiple- investigationscomprisethebulkoftheliteratureonthistopic. o
r
dose combinations in the clinical setting is especially difficult, Therefore,thispaperfocusesonvariousmethodsofstudyand g
/
consideringtheexpenseofclinicaltrialsandthelimitednum- their applications and critically evaluates research findings o
ber of research candidates. However, dose selection of the from these investigations with the goal of providing a frame- n
J
agentsunderstudycancriticallyimpactthetrialresults.Ran- work for future research as well as for translation into the a
domized clinical trials performed to date (24, 166, 213) have clinicalarena. nu
employed standard antifungal doses in combinations that are For the reasons mentioned previously, we will utilize the a
r
consistent with maximally tolerated doses used in mono- terms synergy, indifference, and antagonism to describe the y

therapy. This approach seems logical given limited resources. 1
requiredrangeofinterpretivecategoriesforinvitroinvestiga- 1
Although the possibility exists that greater or lesser benefit tions and to provide the basis for data aggregation and sum- ,
2
may occur with other dose combinations, such information is
marization. This often entails the remapping of the interpre- 0
oflimitedpracticaluse,asfinelycalibratedsystemicexposure 1
tive categories in the original manuscript, with the most 9
adjustmentsareusuallynotpossibleintheclinicalsetting.Itis
common change being the conversion of a large number of b
forthisreasonthatwegenerallybelievethatthemostimpor- y
phrases used to describe indifference into that single term.
tantresultistheresultseenatmaximalsafesystemicexposure g
In analyzing in vitro data, we have followed as closely as u
levels.Statedconversely,wedonotseemuchrelevanceinthe e
possible the rules discussed above respecting the FICI. s
observation that (for example) two drugs are synergistic at t
Analyses of in vivo and clinical data usually require a qual-
15% of their usual dosage but their effect in combination at
itative interpretation of a variety of results; we therefore
maximal doses is no better than monotherapy with the more
focused on objective study endpoints such as time to steril-
potentofthetwocompounds.
ization of tissues or body fluid, survival, or reduction in log
Perhaps the greatest limiting factor for conducting addi-
CFU in infected tissues or fluid, and we comment accord-
tional clinical studies of antifungal drug combinations is the
combination of time and cost (167). Meaningfully powerful ingly on relevant issues of study design and administration.
multicenter studies require several years to complete and re- At times, the terms positive and negative are used to de-
quire enormous financial commitments. Given that combina- scribeinteractionsthatarelessrigorouslycharacterizedbut
tions are being used more frequently in the clinical setting at thattrendinonedirectionoranother.Whenacombination
significant expense without evidence-based clinical support displays a mixed interaction (synergy in some settings, an-
andthatthepotentialforreducedefficacyorincreasedtoxicity tagonisminothers),wenotethecontradictionbutfocuson
exists,itiscriticaltobuildupontheavailableinvitroananimal the interaction most likely to be seen at maximal tolerated
dataanddemonstrateefficacyandsafetyinthesettingofclin- systemic exposures, since this is most clinically relevant in-
icaltrials. teraction (as discussed previously in this paper).
VOL.48,2004 MINIREVIEW 697
MECHANISMSOFINTERACTIONSFORTHE (31,49,182,183,185,186,203).Azolesblockthesynthesisof
ANTIFUNGALAGENTS ergosterol in the fungal cell membrane and may thus render
amphotericin B inactive, since this agent exerts its activity by
Mechanismsofsynergy.Thereareseveralmechanismspro-
bindingtoergosterolinthecellmembrane.(ii)Adsorptionto
posed for antifungal synergy. (i) Inhibition of different stages
thecellsurfacebyoneagentinhibitsbindingofanotheranti-
ofthesamebiochemicalpathwayrepresentsonetypeofinter-
fungal agent to its target site of activity. This mechanism is
action. An example is the combination of terbinafine and
proposedforlipophilicazoles(suchasitraconazoleandketo-
azoles (10, 11, 35, 148), in which both compounds inhibit er-
conazole) which may adsorb to the fungal cell surface and
gosterol biosynthesis and, thus, impair the function of fungal
inhibit binding of amphotericin B to fungal cell membrane
cell membranes. (ii) Increased penetration of an antifungal
sterols(183,185).(iii)Modificationofatargetuponexposure
agent as a result of cell wall or cell membrane antifungal
to an antifungal agent occurs that renders the pathogen less
activity from another agent is possible. This interaction has
susceptibletotheeffectsofotherantifungals.Thismechanism
beenproposedforcombinationsofamphotericinBorflucon-
has been proposed for sequential antagonism observed with
azole with antibacterials such as rifampin (23, 125, 127) and
azoles and amphotericin B, whereby preexposure to an azole D
quinolones (204, 205) and allows these agents to easily pene- compoundcausesreplacementofmembraneergosterolwitha o
tratethefungalcellmembranetoreachtheirtargetoffungal w
methylatedsterolderivativetowhichamphotericinBbindsless n
DNAsynthesis.Suchamechanismmayalsoexplainpotential well(51,67,75).(iv)Otherunknownantagonisticmechanisms lo
synergism between azoles (3, 12, 14, 101, 124, 138, 223) or a
may exist such as that observed for polyenes and flucytosine. d
amphotericin B (126, 156) and flucytosine, in which case the Some have suggested that antagonistic interactions between e
azoleorpolyenedamagesthefungalcellmembrane,enabling d
these agents might be related to changes in fungal cell mem-
f
increaseduptakeofflucytosine.(iii)Atransportinteractionis r
brane function due to the effects of amphotericin B (189); o
proposedforamphotericinB-flucytosine(17,18,20),whereby m
however, additional data are needed to explain this phenom-
amphotericinBactsonthefungalcellmembraneandinhibits enon,becausethesetwodrugsgenerallydisplayaninteraction h
flucytosinetransportacrossthecellmembraneandoutofthe whichtrendstowardssynergy. ttp
yeastcell.Inthisscheme,flucytosineexertsitslethaleffectson :/
/
any surviving fungus when amphotericin B degrades the cell a
FOCUSEDREVIEWOFTHEANTIFUNGALDRUG a
membrane via an oxidative decay, allowing flucytosine to re- c
mainatthesiteofitsactionwithinthecell.(iv)Simultaneous INTERACTIONLITERATURE .a
s
inhibitionofdifferentfungalcelltargets,suchascellwalland The latter portions of this review focus on the available in m
membrane targets, is also possible. This mechanism has been vitroanimalmodelandclinicaldataforthepotentialutilityof .o
suggested for both the apparently synergistic interactions be- antifungalcombinationtherapy.Sincepublisheddataarelim- rg
tweenechinocandins(cellwallactive)andamphotericinB(5, itedforuncommonpathogenssuchasHistoplasmacapsulatum, o/
15, 71; S. Kohno, S. Maesaki, J. Iwakawa, Y. Miyazaki, K. Coccidioides immitis, and Blastomyces dermatitidis and less- n

Nakamura, H. Kakeya, K. Yanagihara, H. Ohno, Y. Higash- common molds, our review focuses on studies of the three Ja
iyama,andT.Tashiro,Abstr.40thIntersci.Conf.Antimicrob. commonfungalpathogens(Cryptococcusneoformans,Candida n
u
Agents Chemother., abstr. 1686, p. 388, 2000; and Ostrosky- spp., and Aspergillus spp.). Analyses respecting these patho- a
Zeichneretal.,Abstr.42ndIntersci.Conf.Antimicrob.Agents gens are more robust and less susceptible to the limitations ry
Chemother., abstr. M-1816, 2002) or azoles (both cell mem- imposed by the availability of only single cases and/or a few 1
1
brane active) (147, 195; Kohno et al., Abstr. 40th Intersci. strains, as occurs with the less common fungi. However, the ,

Conf. Antimicrob. Agents Chemother., abstr. 1686, 2000; limitationsofthisreviewshouldnotsuggestthatcombination 2
0
E. M. O’Shaughnessy, J. Peter, and T. J. Walsh, Abstr. 42nd therapy does not have a place in treatment of less-common 1
9
Intersci.Conf.Antimicrob.AgentsChemother.,abstr.M-856, fungal infections but just that evidence-based data are too
b
p.385,2002;andR.Petraitiene,V.Petraitis,A.Sarafandi,A. limitedatpresenttosupportmeaningfulanalysis. y

Kelaher, C. A. Lyman, T. Sein, A. H. Groll, D. Mickiene, J. C. neoformans. (i) In vitro data. Numerous in vitro studies g
u
Bacher, and T. J. Walsh, Abstr. 42nd Intersci. Conf. Antimi- haveattemptedtocharacterizeinteractionsamongantifungal e
crob.AgentsChemother.,abstr.M-857,p.385,2002).(v)Po- agentsdevelopedforthetreatmentofC.neoformansinfections st
tent initial activity of a rapidly fungicidal agent, such as am- (Table 3), and various results among different investigations
photericin B, to reduce fungal burden, which then allows have been obtained. In general, antagonism has been an un-
another agent to subsequently work well as consolidation or commonfindingwithmostdrugcombinationsandconcentra-
clearancetherapyonareducednumberoffungalcells(107),is tions employed in laboratory investigations against C. neofor-
anotherpossiblemechanism.Someoftheseeffectshavebeen mansisolates.
observed with certain fungal pathogens but not with others (a) Flucytosine-amphotericin B combinations. While many
(156),sotheseinteractionsmaydependonanddifferaccord- cliniciansbelievethatthecombinationofamphotericinBand
ing to certain target cell factors and even between different flucytosineisthepreferredstrategyforinductiontherapytreat-
fungalspecies. ment of cryptococcal meningitis in immunosuppressed pa-
Mechanisms of antagonism. Antagonism among antifungal tients,invitrofindingswiththiscombinationhavenotconsis-
agentsmightoccurinoneofseveralways.(i)Directantifungal tentlydemonstratedsynergy(40,74,78,80,92,126,140,189).
action at the same site results in decreased ability of other Positive interactions between amphotericin B and flucytosine
agents to exert their competitive effects on that site or at an against C. neoformans strains have been reported (40, 126).
alteredtarget,asproposedfortheazolesandamphotericinB Investigations that have not confirmed synergy (78, 80, 161)
698 MINIREVIEW ANTIMICROB.AGENTSCHEMOTHER.
TABLE 3. SummaryofkeyfindingsreportedinstudiesofC.neoformanswithcombinationsofclinicallyrelevantantifungalagentsa
Combination Settingsstudied Generalfindings Comments
5FC(cid:2)AmB Invitro(40,63,74,78,80,92,126,140, Indifference(63,74,78,80,140,156, AmBreducesdevelopmentofresistanceto
156,189) 189)synergy(40,126),antagonism 5FC;5FC-resistantstrainshavebeenusedin
with5FC-resistantstrain(80) numerousstudies(80,126,189)andproduce
variousresults
Mice(16,27,60,80,158) Improvedsurvival(16,27,158) Survival(27)orreductionintissueburden
Rabbits(150) Reducedtissueburden(16,150) (150)notnecessarilybetterthanresultswith
AmBalone(60);combinationmoreeffective
thanAMBand5FCaloneagainst5FC-
resistantstrains(158)
Humans(24,42,43,55,103,144,213) Similar(213)orimproved(24,171) Additionof5FCleadstoearliersterilizationof
clinicalsuccessoverall,improved CSF(24);clinicalsuccessratesweresimilar
sterilizationofCSF(24,213) betweenAmB(cid:2)5FCandAmBalone(73vs.
83%)after2weeksoftherapy(213);relapse
hasbeenassociatedwithnouseof5FC
duringinitial2weeks(171)
5FC(cid:2)triazoles Invitro FLC:synergy(138) Synergyin62%of50strainsstudiedforFLU- D
FLC(3,138,140),KTC(31,140),ITC KTC:indifference(31,89) 5FC(138);variousdosesmaybenecessaryto o
(12),PSC(14) ITC,PSC:indifferenceorsynergy achievegreatesteffect;additionof5FC w
(12,14) helpedpreventemergenceof5FC-resistant n
mutants lo
Animalstudies Improved(3,50,61,157),similar Combinationassociatedwithbettersurvival a
FLC:mice(3,60,61,101,139,157)b (14,50,60,212),orworse(89) thanmonotherapyandwasconsistentovera d
KTC:mice(50,78,158)andrabbits survival rangeofdoses(3);effectsmorepronounced e
(150) Reducedtissueburden(50,78,101, atlowerdoses(101),andsingleagentswere d
ITC:mice(157),hamsters(89),and 212) veryeffectiveathigherdosesalone;5FC(cid:2) f
guineapigs(212) KTCrarelyclearedtissuesbetterthaneither ro
PSC:mice(14) agentalone(150);hamsterswith m
combinationdidworsethanwithITCalone
(89);ITC(cid:2)5FCperformedsimilarlytoITC h
(cid:2)AmBandbetterthanITCor5FC t
monotherapyinguineapigs(212);with10 tp
daysoftreatmentofmice,combination :
/
prolongedsurvivalmorethaneitheragent /
a
alonebutnotwhentreatmentwaslimitedto a
5days(157);PSCcombinationnotbetter c
thanmonotherapyintermsofsurvivalbut .
betterthanmonotherapyinreducingfungal a
countsinbraintissue(14) s
m
Humans Goodclinicalsuccess(48,102,193, 63%successrateincryptococcalmeningitis
FLC(48,102,124,193,223) 223) (95%confidenceinterval,48–82%)(102); .o
Increasedsurvival(124) improvedsurvival(32%)versusFLCalone r
(12%)at6monthsinAIDS-associated g
cryptococcalmeningitis(124) /
o
n
AmB(cid:2)triazoles Invitro Indifference(13,78,140) FLC:indifferencein10/15strainstested,
FLC(13,74),KTC(78,140,150,161), indifferencein4/15,andsynergyin1/15with J
a
ITC(13),PSC(13) NCCLSmethods(13);indifferenceamong3
n
strainsusinganinoculumof104CFU/mlon
u
yeastnitrogenbasebrothandresponse a
surfaceplots(74);KTC:noantagonism r
observed(78,140,150);synergyreported y
withonestrainintwostudiesusing 1
nonstandardmethodologies(140,161);ITC: 1
14/15strainsindifferent;1/15synergistic(13); ,
PSC:8/15strainsindifferent;5/15synergistic; 2
2/15indifferentinonestudy(13) 0
Animalmodels FLC/KTC:improvedsurvival FLC:additionofAmBtoFLChaddramatic 1
FLC:mice(2,13)b comparedtoresultswithazole(2, impactonyeastburdeninbraintissueb,but 9
KTC:mice(50,78,158)andrabbits 13,78,158)band/orAmB(2,158) survivalwithAmBwas100%;effectson b
(150) ITC:didnotimproveorworsened survivalweregreatestathighestdosagesof y
ITC:mice(157)andguineapigs(212) survival(157) azole-AMB(2,158);improvedsurvivalat
Reducedtissueburden(2,13,78, lowerdosesofITC(cid:2)AmB,butsurvivalwas g
u
212) worsewhenhigherdoseswereused(157);
e
FLUpreexposuredidnotreducesubsequent s
AmBactivity(13) t
Humans—casereport(47) Casereportofawomanwithmeningitiswho
respondedtothiscombinationafterfailing
AmB
Caspofunginor Invitro(71) Synergy Usedhigherlevelsofcaspofunginthanwould
anidulafungin(cid:2) beusedforhumans
AmB
Caspofunginor Invitro(71,169) Indifference(71,169)orsynergy(71) Onestudyshowedthatechinocandinswereno
anidulafungin(cid:2) betterthanFLCmonotherapy(169);no
FLC antagonism(71,169)
ITR(cid:2)FLC Guineapigs(212) Reducedtissueburden Survivalwas100%inalltreatmentgroups;
improvedsterilizationoftissuescomparedto
FLCbutnotITC
aAmB,amphotericinB;CLT,clotrimazole;FLC,fluconazole;5FC,flucytosine;KTC,ketoconazole;ITC,itraconazole;PSC,posaconazole;RVC,ravuconazole;SPC,
saperconazole;TRB,terbinafine;VRC,voriconazole.
bLarsenetal.,Abstr.5thInt.Conf.CryptococcusCryptococcosis,abstr.P3.1,2002.
VOL.48,2004 MINIREVIEW 699
used lower concentrations of flucytosine and amphotericin B ofamphotericinBandflucytosineinstudiesofmice(16,27,60,
only or used strains with reduced susceptibility to flucytosine, 80,158)andrabbits(150)withflucytosine-susceptiblestainsof
whichmayhaveinfluencedorbiasedtheirabilitytocharacter- C.neoformansindicatethatsurvivalisimprovedandthatsub-
ize the full spectrum of antifungal interactions between these stantialreductionsinlevelsoflogCFUpermilliliteroccurwith
agents. combinationtherapy(16,150).Studiesconductedwithcombi-
(b) Flucytosine-azole combinations. The results of early in- nationtreatmentswhoseresultshaveincludedworse-than-an-
vestigations of flucytosine in combination with older azoles ticipatedeffectsonnegativecultureresults(80)usedsubstan-
such as econazole or miconazole (63) suggested that antago- tiallylowerdosesthanthoseusedbyothers.
nism between these agents occurred. However, more recent (b)Flucytosine-azolecombinations.Incombinationwithtria-
reports of investigations of triazole-flucytosine combinations zoles,flucytosine(whencombinedwithfluconazole)appearsto
most commonly have indicated synergy or indifference (3, 12, have increased effects on survival, fungal tissue burden, and
14, 74, 138, 140, 223). In similarity to the results seen with weightchangesininfectedanimals(3,60,61,101,139).How-
interactionsofpolyenesandflucytosine,ithasbeensuggested ever,relativelyhighdosesofeachagenthavebeenveryeffec-
thattheadditionofatriazole(suchasitraconazole)suppresses tiveasmonotherapy(60,61,101);thus,ithasbeendifficultto D
emergence of flucytosine-resistant mutants (12). Perhaps not show superior or improved outcome. Deleterious effects of ow
surprisingly,thesecombinationshavebeensomewhatlessim- combinationshavebeenuncommon,butinvestigatorsconduct- n
pressive against strains with reduced azole susceptibility and ing one study did observe additive toxicity after 5 days of lo
a
variations among the different triazoles have been reported treatmentwiththecombination(157).Combinationsofflucy- d
(12). e
tosine and itraconazole, ketoconazole, or posaconazole have d
(c)Polyene-azolecombinations.Polyene-azoleinteractionsin resultedinimprovementsinsurvival(50,157)andtissueclear- f
r
the treatment of C. neoformans infections have been studied o
ance (14, 212) in cases of cryptococcal meningitis, despite
m
both with (74, 140) and without (13, 63, 74, 78, 150, 161) the
limited penetration of these azoles into cerebrospinal fluid
use of flucytosine, but in vitro data are relatively scant. In h
(CSF).However,ketoconazole-flucytosinecombinationsrarely t
contrast to the antagonism commonly observed with Candida tp
resulted in better outcomes with regard to tissue clearance
:
spp. for this combination, polyene-azole interactions for C. /
thanketoconazolealoneorevenamphotericinBmonotherapy /a
neoformanshavegenerallyrangedfromindifferenttosynergis- (50,78,150,157).Onegroupstudyinghamsterswithsystemic a
tic.Thismayberelatedtothevarioussterolcompositionsseen c
cryptococcosis(89)reportedworsesurvivalandhigherfungal .a
withC.neoformansisolates(75).Indifferenteffectshavebeen burden in brain tissue after 30 or 60 days of therapy for itra- s
reported most frequently (13, 78, 161), but synergy has also m
conazole-flucytosineincombinationcomparedwiththeresults .
been found for combinations of ketoconazole (78, 161) or o
seenwithitraconazolealone.Fungalburdeninbraintissueof r
posaconazole (13) and amphotericin B. While positive inter- g
infected animals was also worse with the combination than /
actions have been most commonly reported with concurrent o
combinationsofpolyenesandazoles,sequentialexposuretoan withflucytosinealone,buttheuseofflucytosinealonewasalso n
associatedwithpoorsurvival(89). J
azole may reduce subsequent amphotericin B activity. In one a
(c)Polyene-azolecombinations.Experiencewithamphoteri-
study(13)preexposuretofluconazoleappearedtoinhibitsub- n
cinBandtriazolecombinationsindicatesthattheazolecom- u
sequent killing activity of amphotericin B, especially against a
poundislargelythebeneficiary.Itisdifficulttoimproveonthe r
nonreplicating C. neoformans cells. Therefore, the timing of y
antifungalexposuremaybecriticaltointeractionresults. rapid and impressive fungicidal activity of amphotericin B in 1
animal models, and tolerability rather than direct antifungal 1
(d) Echinocandins with other agents. Echinocandins, which ,
activity is the main issue. Improvements in survival (R. A. 2
havenegligibleactivityagainstC.neoformansalone(56,69,99,
0
169),haveexhibitedpositiveinteractionsincombinationwith Larsen,M.Bauer,A.M.Thomas,andJ.R.Graybill,Abstr.5th 1
Int. Conf. Cryptococcus Cryptococcosis, abstr. P3.1, p. 121, 9
amphotericinB(71)orazoles(56,71,169),withsomereports
b
2002) and/or reductions in tissue burden have been reported,
indicating indifference (169) and others indicating possible y
synergy(71).Itisimportantthattheconcentrationsofcaspo- buttheseeffectshaveusuallynotbeensuperiortotheresults g
fungin used have been much higher than could be clinically seen with amphotericin B alone. The results seen in early ue
achievedatcurrentdoseswithhumans.Synergisticinteractions studies with ketoconazole-amphotericin B combinations indi- st
havealsobeenreported(56)forcaspofunginandtacrolimus,a cated that the greatest impact of dual therapy might be on
calcineurin inhibitor. Calcineurin is involved in signaling dur- fungalburdeninthetissuesratherthanintheformofdramatic
ing the stress response and probably regulates 1,3-(cid:7)-D-glucan improvementsinsurvival(50,78,150,158).Theadditionofan
synthase. Although promising conceptually, the effectiveness azole could result in better overall efficacy or possibly enable
of tacrolimus as an antifungal in the clinical setting has been effectivenesswithlowerdoserequirementsforamphotericinB.
limitedbyitsdirectimmunosuppressiveeffects. In one model of murine cryptococcal meningitis (157), worse
(ii)Animalmodelsofcryptococcalinfection.Antifungalin- survival was observed when higher dosages of amphotericin
teractionshavebeenstudiedextensivelyinanimalmodelsofC. B-itraconazole were used in combination; when lower doses
neoformansinfection(includingstudiesofbothmeningitisand were employed, survival was dramatically better among those
systemic disease) (Table 3). A common theme with combina- animalsreceivingdualtherapy.Amongneutropenicandnon-
tiontreatmentshasbeenprolongedsurvivaland/orreductions neutropenicguineapigs(212),increasesinratesoftissuester-
intissueburden,buttheeffectswererarelygreaterthanthose ilization with increases in dosages of concomitant amphoteri-
seenwithamphotericinBmonotherapy. cinB(0.63to2.5mg/kgofbodyweight/day)anditraconazole
(a) Flucytosine-amphotericin B combinations. Combinations were observed relative to the results seen with itraconazole
700 MINIREVIEW ANTIMICROB.AGENTSCHEMOTHER.
alone. This combination also reduced colony counts in brain val,48to82%),withamediantimetoCSFculturenegativity
andmeningealtissuemorethanamphotericinBalone. of23days(whichislongerthanthatobserved,ingeneral,with
(d) Azole-azole combinations. Azole-azole combinations combinations of amphotericin B and flucytosine) (102, 173).
havealsobeenemployedonalimitedbasis.Thecombination However, these rates were substantially better than those re-
of fluconazole and itraconazole in a guinea pig model of sys- ported from other studies conducted with fluconazole (103,
temiccryptococcosisandmeningitis(212)resultedinimprove- 173)oramphotericinB(24,173)alone.Dose-limitingadverse
mentsintissuesterilizationcomparedtotheresultsseenwith effects have been problematic with the combination of flucy-
fluconazolealonebutdidnotaddmuchtoitraconazolemono- tosine and fluconazole and necessitated discontinuation of
therapy at similar doses. Survival in this model for all treat- flucytosinetreatmentin28%ofthestudysubjectsinonetrial
mentgroupswas100%. (102).Thiscombinationwasalsoassociatedwithtoxicitiesina
(iii) Clinical data. (a) Flucytosine-amphotericin B combina- studyofaseriesofnon-HIV-infectedsubjectswithcryptococ-
tions.Asmentionedpreviously,theclassicuseofcombination calmeningitis(P.G.Pappas,J.R.Perfect,andR.A.Larsen,
antifungal therapy in the clinical setting involves the use of Abstr.36thAnn.Meet.Infect.Dis.Soc.Am.,abstr.101,1998).
flucytosine with amphotericin B for the treatment of crypto- Outcomes with itraconazole plus flucytosine in case series D
coccal meningitis. Several clinical trials have been used to (218,219)arecomparableorbetterthanthoseforitraconazole o
w
comparethiscombinationtoamphotericinBmonotherapyand alone, which has erratic oral absorption characteristics and n
have resulted in faster clearance of yeasts from the CSF and great interpatient pharmacokinetic variability (91). A total of lo
a
fewerrelapseswiththeadditionofflucytosinecomparedtothe 22subjectswithdisseminatedcryptococcosisreceiveditracon- d
resultsseenwithamphotericinBtreatmentalone(24,103,171, azole(200to400mg)dailywithorwithoutflucytosine(150to e
d
173, 213), but overall mortality or clinical cure rates with the 200 mg/kg/day) for 6 weeks in an open-label study (219) and
f
r
combinationwerenotconsistentlybetterinanyofthesetrials. experiencedsimilarresultswithrespecttotreatmentsuccess.A o
m
In the first of the studies cited, the addition of flucytosine total of 9 of 12 subjects receiving itraconazole alone experi-

reduced dosage requirements for amphotericin B and thus encedmarkedimprovement,while8of10receivingthecom- h
t
reduced polyene toxicity (24). Clinical cure or improvement binationexperiencedasimilaroutcome.Therewasonetreat- tp
:
ratesinthisprospective,randomizedmulticentercomparative ment failure in each group. Significant toxicity was not /
/
a
trialconductedwithsubjectswithcryptococcalmeningitis(24) observedduringthisinductionperiod,butlong-termfollowup a
were68%(23of34subjects)forsubjectsreceivingamphoter- datafromthemaintenanceperiodwerenotwelldescribed. c
.
a
icinB(0.3mg/kg/day)plusflucytosinefor6weekscomparedto (c) Triple combinations. Triple combinations of amphoteri- s
47%(15of32)forthosereceiving10weeksofamphotericinB cin B, flucytosine, and triazoles in treatment of cryptococcal m
.
treatment alone (0.4 mg/kg/day). There were significantly meningitis have also been employed, with apparent success o
fewerdeaths(24versus47%[P(cid:8)0.05])andmore-rapidcon- (42, 43; Brouwer et al., Abstr. 15th Congr. Int. Soc. Hum. rg
version of CSF to negative culture results (P (cid:8) 0.001) in the Anim.Mycol.,2003).Recently,treatmentofHIV-infectedpa- o/
combination therapy arm. In a later trial, subjects were ran- tientswiththecombinationofamphotericinBandflucytosine n

J
domizedtoreceiveamphotericinB(0.7mg/kgdaily)plusflucy- for cryptococcal meningitis resulted in reduction of yeast a
tosine (100 mg/kg daily divided in four doses) or placebo for counts in CSF that was faster than that seen with a triple n
u
the initial 2 weeks of therapy followed by randomization to combination with amphotericin B, single-agent therapy with a
r
fluconazoleoritraconazoleconsolidationtherapyfor8weeks. amphotericinB,orthecombinationofflucytosineandflucon- y

Aftertheinitial2weeksoftherapy,60%ofsubjectsreceiving azole(Brouweretal.,Abstr.15thCongr.Int.Soc.Hum.Anim. 1
1
thecombinationofamphotericinBplusflucytosineversus50% Mycol.,2003). ,

2
ofthoserandomizedtoamphotericinBaloneachievedsteril- Published results from a large clinical trial (43) indicated
0
ization of the CSF (P (cid:3) 0.06). No differences were reported that overall treatment success and time to resolution of fever 1
9
between treatment groups with respect to clinical and micro- were better among subjects receiving triple therapy with am-
b
biologicresponsesafterthis2-weekinductionperiod(213),but photericin B, flucytosine, and itraconazole (50 of 50 success- y
theadditionofflucytosinetotheregimenwasassociatedwith fullytreated;feverresolvedin5.9(cid:9)3.7days)thantheresults g
u
fewerrelapses(171). seenwithamphotericinB-flucytosinealone(45of50success- e
(b)Flucytosine-azolecombinations.Incontrasttoexperience fullytreated[P(cid:3)0.03];feverresolvedin8.8(cid:9)5.1days[P(cid:3) st
withamphotericinB,theadditionofflucytosinetofluconazole 0.02]). In this study, 100 subjects with AIDS-associated cryp-
in clinical studies has resulted in less-clear-cut benefits. In an tococcal meningitis were randomized to receive amphotericin
observational study of HIV-infected individuals with crypto- B (0.3 mg/kg of body weight/day) plus flucytosine (150 mg/kg
coccalmeningitis,theadditionofflucytosinetofluconazolefor dividedintodosesadministeredfourtimesdaily)foratotalof
thetreatmentofsubjectswitharangeoflevelsofillnesscon- 6 weeks or amphotericin B and flucytosine in the same doses
sistentlyreducedthefailurerate(222).Inasmall,randomized plus itraconazole (400 mg in 200-mg capsules administered
trial performed in Uganda, the addition of flucytosine to flu- twicedaily)untilnegativecultureresults(meantime,2.4(cid:9)0.6
conazole for the first 2 weeks of induction therapy for the weeks)wereobtained;thisregimenwasfollowedbytreatment
treatment of HIV-infected subjects with cryptococcal menin- with itraconazole alone in the same doses for a total of 6
gitis was associated with increased 6-month survival rates (32 weeks. Secondary prophylaxis for both treatment groups con-
versus 12% [P (cid:3) 0.022]) without a high frequency of serious sisted of the administration of itraconazole capsules (200 mg
toxicities(124).Inanotherstudy(102),theclinicalsuccessrate daily).Fewersubjectsreceivingtripletherapyexperiencedsig-
after 10 weeks of daily treatment with fluconazole (400 mg) nificantlaboratoryadverseevents(21of50versus32of50[P
plus flucytosine (150 mg/kg) was 63% (95% confidence inter- (cid:3)0.045]),whichconsistedmostlyofdecreasedhematocritand
VOL.48,2004 MINIREVIEW 701
metabolicacidosisinthesubjectsreceivingamphotericinBand regimen until further supported by solid comparative clinical
flucytosine. In addition, after 2 weeks of therapy, CSF steril- datademonstratingefficacyaswellassafety.
izationrateswerebetterforthetripletherapygroupthanthe Unlike the results seen with respect to the antagonism ob-
resultsseenwithcontrols.However,thereweremorerelapses servedbetweenpolyenesandazolesinotherfungalorganisms,
amongsubjectsreceivingthetriplecombinationaftertheyhad the combination of amphotericin B and triazoles (when used
beenswitchedtoitraconazolealone.Theresultsofotherstud- concurrently) appears to have positive effects in vitro and in
ies(162,171)havesuggestedthatrelapseratesamongsubjects animal models of cryptococcosis; however, the effects are not
with HIV-associated cryptococcal meningitis are higher with necessarily more positive than the effects of high dosages of
itraconazoleasasecondaryprophylaxisthanwithfluconazole. amphotericin B alone. The addition of amphotericin B to a
ItshouldalsobenotedthatthedoseofamphotericinBusedin triazole could possibly enable reduced dosages of either or
this study was less than that recommended in Infectious Dis- bothagentsandthuspotentiallyreducedrug-associatedtoxic-
easesSocietyofAmericaguidelines(172). ities, which are frequently observed at the high dosages of a
(d)Polyene-azolesequentialtherapy.Sequentialtherapywith polyenerequiredforsuperiorefficacywithhumans.Assequen-
polyenewithorwithoutflucytosinefollowedbyanazole(flu- tial therapy, preexposure with a triazole may reduce subse- D
o
conazoleoritraconazole)hasbeenwellstudiedintheclinical quent activity of amphotericin B in vitro; however, polyene w
setting (171, 213), and it appears that pretreatment with am- preexposure does not appear to reduce subsequent azole ac- n
photericin B with or without flucytosine during the induction tivity, and a sequential approach is supported by large-scale lo
a
phasemightaidthepositiveimpactofsubsequentazoleactiv- clinicaltrialresults(213). d
e
ityduringtheconsolidation,clearance,ormaintenancephase. Combinations of amphotericin B and echinocandins are an d
This strategy is currently used clinically (171, 172). However, interestingareaoftherapeuticstudy,butuntilfurtherdataor f
r
o
relapserateshavebeenhigherwiththeuseofitraconazoleas newechinocandinsareavailablethesecombinationsshouldbe
m
maintenance therapy than the results seen with fluconazole employedonlyintheexperimentalsetting.
h
(171,213).ThisprobablyrelatestothebetterCSFpenetration Three-drugcombinationsareanovelapproachthatappears t
t
p
offluconazoleanditsmorereliablepharmacokineticprofile. to result in excellent clinical cure rates and reduced toxicities
:
/
(iv) Interpretation and recommendations. Judging on the for subjects with cryptococcosis. The results of a few in vitro /a
basis of these data, flucytosine-amphotericin B combinations (74, 140) and animal model (60) studies suggest that this ap- a
c
havenotbeenparticularlyimpressivewithC.neoformansiso- proach might be promising, and the results of small clinical .a
lates in in vitro investigations; however, the results seen with trials(42,43;Brouweretal.,Abstr.15thCongr.Int.Soc.Hum. s
m
animal models of cryptococcosis suggest significant positive Anim. Mycol., 2003) have affirmed this potential. However, .
o
effects of this combination with respect to survival and tissue relapserateshavebeenunacceptablyhigh(43),suggestingthat r
g
clearanceoforganisms.Asdeterminedonthebasisofclinical thedurationofinductiontherapymightneedtobeprolonged /

o
researchexperience,itappearsthattreatmentwithamphoter- when lower doses of these agents are used in combination or n
icin B and flucytosine is the best combination available for that maintenance therapy after induction with three agents J
a
cryptococcal meningitis at the present time; perhaps the dif- needs to be carefully selected to avoid subsequent clinical
n
ferential performance of this combination in vivo relates to failures.Furthermore,itisnotproventhatthesecombinations u
a
host factors that cannot be readily simulated in the test tube. havesuperiorfungicidalactivitycomparedtothecombination r
y
AmphotericinBandflucytosinetreatmentrepresentstheonly ofamphotericinBandflucytosine(Brouweretal.,Abstr.15th 1
combination antifungal therapy regimen recommended as an Congr.Int.Soc.Hum.Anim.Mycol.,2003). 1
,
initial therapy for the treatment of cryptococcal meningitis in Candidaspp.(i)Invitroevidence.Therelationshipsamong 2
0
theguidelinespublishedbytheInfectiousDiseasesSocietyof numerous combinations of antifungal agents have been char- 1
America(172),andweagreewiththeserecommendations. acterizedininvitrostudiesofCandidaspecies(Table4). 9

Flucytosine may be a useful addition to azoles and in this (a)Flucytosine-amphotericinBcombinations.Flucytosinehas b
y
setting could result in improved activity and reduced emer- beenstudiedincombinationwithamphotericinB(21,74,92, g
genceofflucytosine-resistantyeasts.Factorsotherthaninvitro 105,122,140,157,178,191)withmixedresults,dependingon u
e
potentiation, including differential pharmacokinetics, reduc- theisolateandtestconditions.InconcertwithamphotericinB, s
t
tion of selective pressure, and the capability of being used in thepredominantfindinghasbeenthatofsynergy(40,105,133,
lower dosages and therefore of reducing associated drug tox- 156,191);however,indifference(21,74,92,122)hasalsobeen
icity, may be of more importance for these combinations in reported.Insimilaritytotheresultsseenwiththiscombination
vivo.Judgingonthebasisoftheresultsofinvestigationsusing in studies of C. neoformans, it has been suggested that the
animal models, fluconazole-flucytosine combinations appear addition of amphotericin B helps prevent the emergence of
beneficial.Datawithothertriazolesincombinationwithflucy- flucytosine-resistantmutants(156).Theadditionofflucytosine
tosine have been less consistent, and these combinations are intime-killstudies(92),however,didnotappreciablyaffectthe
less likely to be investigated in the clinical setting. Infectious activityagainstC.albicansofbothlowandhighconcentrations
Diseases Society of America guidelines (172) have proposed ofamphotericinB.Inthisstudy,noantagonismwasobserved
fluconazoleplusflucytosineasanalternativeinductiontherapy even with preexposure to flucytosine but other study results
forcryptococcalmeningitis.Sincetherehavebeenonlypoorly have indicated apparent antagonism at higher concentrations
developed comparative outcome studies associated with this (161)orwhenyeastcellswerefirstexposedtoflucytosineand
combinationincasesofcryptococcosisandtoxicitylevelshave thenamphotericinB(122).Dramaticsynergywhenamphoter-
been problematic, it will continue to be used as a secondary icin B and flucytosine were combined in the setting of flucy-
702 MINIREVIEW ANTIMICROB.AGENTSCHEMOTHER.
TABLE 4. SummaryofkeyfindingsreportedinstudiesofCandidaspp.withcombinationsofclinicallyrelevantantifungalagentsa
Combination Settingsstudied Generalfindings Comments
5FC(cid:2)AmB Invitro(21,40,63,74,92,105, Synergy(40,105,133,178,191)or AdditionofAmBhelpspreventemergenceof
122,133,140,156,161,178, indifference(21,74,92,122) 5FCresistance
191)
Mice(157,164,191,209)and Improvedsurvival(164) Mosteffectivecombinationinonestudywhen
rabbits(208) Reducedtissueburden(164,208, comparedwithresultsforAmB-rifampin,
209) 5FC-KTC,andtheseagentsalone(208);
reduceddosagesoftheagentswere
possibleincombinationwhilemaintaining
efficacy(209)
Humanswithinvasivedisease Goodclinicalsuccess AmB(cid:2)5FCclearedculturesfasterthan
(1,36,100,155) fluconazoleinhumanswithperitonitis
(100)
5FC(cid:2)azoles Invitro Noconsensus Extendeddurationofpostantifungaleffect
Econazole(63),miconazole(63, Synergy(129),indifference(19, wasreportedinonestudywithfluconazole- D
191) 105),antagonism(74,140) flucytosine(129),lowconcentrationsof o
CLT(22),KTC(19,20,140), 5FC-KTCappearedantagonisticforC. w
FLC(74,105,129) parapsilosis(19)contoursurfaceplot
n
methodologysuggestednegativeinteraction
betweenfluconazoleandflucytosineovera lo
rangeofconcentrations(74) a
d
e
KTC:mice(158)andrabbits Improvedsurvival(157,158) FLCdosesinrabbitswereequivalentto1,600 d
(208) Reducedtissueburden(115,208) mg/dayinhumans(115);5FC-KTC
ITC:mice(157) appearedtoprolongsurvivalagainstsome fr
FLC:mice(180)andrabbits C.albicansstrainsinamurinemodelmore o
(115) thaneitheragentalone(eveninhigher m
concentrations)butagainstotherstrains
hadnosurvivalbenefitoverasingleagent h
t
(158);effectsmostapparentwith5FC- t
p
resistantC.albicansstrains;inrabbits
:
(115)FLC-AmBcombinationsterilized //
cardiacvegetationsfasterthanFLCbut a
performedsimilarlytoFLCinkidney a
Humans(181) CasereportofsepsisduetoC.albicansthat c
.
wastreatedsuccessfullywith5FCplusFLC a
(181) s
m
AmB(cid:2)azoles Invitro Antagonism Onestudysuggestedindifferenteffectsfor .o
FLC(67,74,105,107,122,154, AmB-FLCagainstC.albicansoverawide r
161,175,185,186,214,216, rangeofconcentrations(74) g
217),bsequential(67,105,107, Slightsynergywithhigherconcentrationsof /
175) KTCandAmB(161);short-termexposure o
Miconazole(31,49,63,154, withmiconazoleresultedinantagonism, n
191)c long-termexposureresultedinpositive J
CLT(22,49) effects(31) a
KTC(31,140,154,161,183, n
198) u
ITC(154,161,184,185) a
r
y
FLC:mice(113,176,199,202) Improved(FLC,PSC,SPC)(113, AmB-FLCeffectsnotasprofoundinaless- 1
andrabbits(115,176) 157,176,202)orsimilarto acutemodelofinfection(202);inrabbits, 1
ITC:mice(157,203) worse(ITC,KTC)(157,203) combinationwasnotbetterthanAMB ,
KTC:mice(158)andrabbits survival aloneinsterilizingcardiacvegetationsand 2
(208) Reducedtissueburden(FLC, kidneys(115).RabbitmodelusedFLC 0
PSC:miced KTC)(115,208)butITC-AmB dosesequivalentto1,600mg/dayin 1
SPC:mice(206) hadpoorerclearanceoftissues humans(115).Inmice,thecombination 9
Sequential:mice(202,203,216) (kidney)(203)withcombination resultedinworsesurvivalandkidney b
fungalburdencomparedtoAmBalone y
(113)againstFLC-susceptibleandlow-level
resistance(MIC,64to125(cid:4)g/ml)strains g
AmB-FLCgavebettersurvivalthanAmBbut u
notFLC(199)andinanotherstudygave e
s
bettersurvivalthanFLCbutnotAmB t
(176).ITC-AmBresultedin100%
mortalityinmice,while90%ofamB-
treatedmicesurvived;inneutropenic
rabbitsAmB-KTCimprovedsterilization
ratesinkidneys(208)relativetoeither
agentalonebutnotasmuchasAmB-5FC
combination;AmB-KTCprolongedsurvival
againstoneC.albicansstrainbutnot2
others(158);combinationsofAmB-KTC
against2C.albicansstrainsweregenerally
notbetterthanAmBaloneinprolonging
survivalininfectedmice(158)
Humanswithcandidemia(166) Goodclinicalsuccess ComparableclinicalcureratestoFLCalone,
fasterbloodstreamsterilizationwiththe
combinationregimen
AmB(cid:2)nystatin Invitro(31) Indifference
Continuedonfollowingpage