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Drug Resistance Updates 17 (2014) 37–50

Contents lists available at ScienceDirect

Drug Resistance Updates

jo ur nal homepage: www.elsev ier .com/ locate /drup

he role of azoles in the management of azole-resistant aspergillosis:rom the bench to the bedside

eyedmojtaba Seyedmousavia,b, Johan W. Moutona,b, Willem J.G. Melchersa,oger J.M. Brüggemannc, Paul E. Verweij a,∗

Department of Medical Microbiology, Radboudumc, Nijmegen, The NetherlandsDepartment of Medical Microbiology and Infectious Diseases, Erasmus MC, The NetherlandsDepartment of Pharmacy, Radboudumc, Nijmegen, The Netherlands

r t i c l e i n f o

rticle history:eceived 5 December 2013eceived in revised form 21 June 2014ccepted 29 June 2014

eywords:spergillus fumigatus

nvasive aspergillosisoriconazole

a b s t r a c t

Azole resistance is an emerging problem in Aspergillus fumigatus and is associated with a high probabilityof treatment failure. An azole resistance mechanism typically decreases the activity of multiple azolecompounds, depending on the mutation. As alternative treatment options are limited and in some iso-lates the minimum inhibitory concentration (MIC) increases by only a few two-fold dilutions steps, weinvestigated if voriconazole and posaconazole have a role in treating azole-resistant Aspergillus disease.The relation between resistance genotype and phenotype, pharmacokinetic and pharmacodynamic prop-erties, and (pre)clinical treatment efficacy were reviewed. The results were used to estimate the exposureneeded to achieve the pharmacodynamic target for each MIC. For posaconazole adequate exposure can be

osaconazolezole-resistanceanagement

achieved only for wild type isolates as dose escalation does not allow PD target attainment. However, thenew intravenous formulation might result in sufficient exposure to treat isolates with a MIC of 0.5 mg/L.For voriconazole our analysis indicated that the exposure needed to treat infection due to isolates witha MIC of 2 mg/L is feasible and maybe isolates with a MIC of 4 mg/L. However, extreme caution and strictmonitoring of drug levels would be required, as the probability of toxicity will also increase.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Azole resistance is an emerging problem in species of the genusspergillus (Denning and Perlin, 2011; Snelders et al., 2008). Theolyphasic approach to the taxonomic classification of aspergillias resulted in the recognition of new species (Peterson et al.,008). These new or sibling species are difficult to identifysing conventional methods, often requiring molecular techniquesAlcazar-Fuoli et al., 2008; Geiser et al., 2007). Recent epidemiologicesearch indicates that sibling species of Aspergillus may cause inva-ive aspergillosis in susceptible hosts (Balajee et al., 2005; Gerbert al., 1973; Guarro et al., 2002; Hedayati et al., 2007; Jarv et al.,004; Latge, 1999). Many of these species show a susceptibility pro-le that differs from the conventional species, usually with reducedctivity of specific antifungal agents (Alcazar-Fuoli et al., 2008).

In addition to intrinsic resistance within the aspergillus fam-ly (van der Linden et al., 2011a), there are increasing reportsf acquired resistance to azoles (Denning and Perlin, 2011). The

∗ Corresponding author.E-mail address: Paul.Verweij@Radboudumc.nl (P.E. Verweij).

ttp://dx.doi.org/10.1016/j.drup.2014.06.001368-7646/© 2014 Elsevier Ltd. All rights reserved.

majority of reports concern Aspergillus fumigatus (Verweij et al.,2009a), although azole resistance has been reported sporadicallyin other species as well, such as A. flavus (Liu et al., 2012) and A.terreus (Arendrup et al., 2012b).

In A. fumigatus two routes of resistance selection have beenreported; Azole resistance has been reported in patients withchronic cavitating aspergillus diseases that receive long-term azoletherapy (Howard et al., 2009). In these patients the initial infec-tion is caused by an azole-susceptible isolate, but through therapyazole-resistant isolates may be cultured. A second route of resis-tance selection is believed to occur through exposure of A. fumigatusto azole compounds in the environment (Snelders et al., 2008, 2009,2012; Verweij et al., 2009b). Azoles are commonly used for cropprotection or material preservation. Some of the fungicides werefound to have a molecule structure very similar to that of the medi-cal triazoles (Snelders et al., 2012; Verweij et al., 2009b). The fungusis believed to develop mutations that confer resistance to fungi-cides, but due to the molecule similarity with the medical triazoles,

the latter become inactive as well.

A wide range of mutations in A. fumigatus have been describedconferring azole-resistance commonly involving modifications inthe cyp51A-gene, the target of antifungal azoles. Cyp51A mutations

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8 S. Seyedmousavi et al. / Drug R

n A. fumigatus commonly affect the activity of all mold-activentifungal azoles. Specific mutations correspond with varioushenotypes characterized by complete loss of activity of a specificzole, and with decreased activity of others (Verweij et al., 2007).

If a role for the azoles remains in the management ofzole-resistant aspergillosis (Walsh et al., 2008), optimizing drugxposure appears critical to increase the probability of treatmentuccess. In this context, understanding of the pharmacokineticsPK) and pharmacodynamics (PD) and more importantly defininghe pharmacodynamic target of the azole compounds is crucial toncrease the probability of a favorable clinical response (Andes,004). Reduced susceptibility of the fungus for azoles has signifi-ant impact on the ability to achieve the PD-target, and sometimesargets can only be achieved at the cost of increased probabilityf toxicity. Many variables, such as the underlying azole resis-ance mechanism and PK/PD properties of the antifungal agent, aremportant to determine if treatment with an azole remains feasibleAndes et al., 2009). Furthermore, in the absence of extensive clin-cal experience with the treatment of azole-resistant aspergillosis,ata obtained through in vitro susceptibility testing and experimen-al models of infection are needed to design treatment strategies.

We reviewed our current understanding of azole resistance andhe potential role of voriconazole and posaconazole in order touide clinicians to manage patients with azole-resistant aspergillusisease. The results of in vitro and preclinical studies were extrap-lated to humans to provide evidence that may support the usef voriconazole and posaconazole in isolates with attenuated azoleusceptibility.

. Triazole antifungals: mode of action and label indicationor invasive aspergillosis

The antifungal triazoles are synthetic compounds that have1 triazole ring attached to an isobutyl core (e.g., voriconazole,avuconazole, and isavuconazole) or to an asymmetric carbontom with a lipophilic complex mixed functional aromatic chaine.g., itraconazole and posaconazole) (Groll et al., 2003). Triazolesnhibit the synthesis of ergosterol from lanosterol in the fungalell membrane (Groll et al., 2003; Mohr et al., 2008); the tar-et is the cytochrome (CYP)-dependent 14-a-demethylase (CYP51r Erg11p), which catalyses this reaction. Thereby, ergosterol isepleted and methyl-sterols accumulate within the cell mem-rane and lead to either inhibition of fungal cell growth or death,epending on the species and antifungal compound involved. Triaz-les are generally fungistatic, although itraconazole, voriconazole,osaconazole and isavuconazole have been shown to be fungicidalgainst Aspergillus spp such as A. fumigatus, A. flavus, A. niger, A.idulans, A. terreus, A. versocolor and A. sidowii. (Guinea et al., 2008;ohr et al., 2008; Pfaller et al., 2002). The various azoles have dif-

erent affinities for the CYP-dependent 14-a-demethylase, whichn return results in various antifungal activities (Warrilow et al.,010a); and therefore various susceptibilities to Aspergillus spp.our triazole compounds (fluconazole, itraconazole, voriconazole,nd posaconazole) have been clinically licensed and are currentlyn wide use for the prevention and treatment of invasive fun-al infections (EMA, 2012a,b). Fluconazole has a lack of efficacygainst molds such as Aspergillus spp., therefore targeted pro-hylaxis or treatment against aspergillosis cannot be covered byhis agent. Itraconazole is commonly used for the treatment ofhronic and allergic conditions (EMA, 2012a,b). Voriconazole hasroad in vitro activity against Aspergillus spp., is recommended first

hoice treatment of invasive aspergillosis with a label indication indults and children aged 2 and above (EMA, 2012b). In addition,oriconazole is the drug of choice for treatment of central ner-ous system aspergillosis (Schwartz et al., 2005). Posaconazole is

nce Updates 17 (2014) 37–50

licensed only for patients aged 18 years or older (EMA, 2012a); forprophylaxis in patients receiving remission–induction chemother-apy for acute myelogenous leukemia (AML) or myelodysplasticsyndromes (MDS) expected to result in prolonged neutropenia andwho are at high risk of developing invasive fungal infections; forprophylaxis of invasive fungal infections in hematopoietic stem celltransplant (HSCT) recipients who are undergoing high-dose immu-nosuppressive therapy for graft versus host disease and who areat high risk of developing invasive fungal infections; and for sal-vage therapy of invasive aspergillosis in patients with disease thatis refractory to amphotericin B or itraconazole or in patients whoare intolerant of these medicinal products (Cornely et al., 2007;Herbrecht et al., 2002; Ullmann et al., 2007; Walsh et al., 2008).

3. Phenotypic detection of azole resistance and clinicalbreakpoints for Aspergillus spp.

In recent years major advances have been made in the detec-tion of azole resistance in Aspergillus spp. Both the Clinical andLaboratory Standards Institute (CLSI) and European Committeeon Antimicrobial susceptibility Testing-subcommittee on Anti-fungal Susceptibility Testing (EUCAST-AFST) have developed andstandardized phenotypic methods that enable the reliable andreproducible determination of the minimal inhibitory concen-tration (MIC) for conidia-forming molds such as Aspergillus spp.(AFST-EUCAST, 2008; CLSI, 2008). When a collection of fungalstrains is tested, typically a Gaussian distribution of MICs is foundreferred to as the wild type population (Meletiadis et al., 2012).The right side of the distribution, i.e. growth of isolates that isinhibited only by a higher concentration of the drug or any iso-lates/populations to the right side of the wild type distributionmight contain isolates that possess a resistance mechanism. Theseisolates are considered non-wild type (AFST-EUCAST, 2013). Test-ing of large collections of fungi enables the determination of anepidemiological cut-off, which is the concentration of drug thatinhibits 95% of the fungal species. Notably, a clinical breakpoint isneeded to obtain a clinically meaningful interpretation of the MICof individual isolates. A standardized approach is followed, whichincorporates standard dosing recommendations and formulationsof antifungal agents, the Pk/Pd characteristics, information fromexperimental models of infection and results from clinical trials.All this information is analyzed and leads to the clinical break-point, i.e. the classification of the isolate as susceptible to the drugor resistant. There are currently three sets of breakpoints and epi-demiological cut-off values (ECVs) available; The breakpoints waspublished in 2009 by Verweij et al. based on clinical experience andthe available knowledge at that time (Verweij et al., 2009a). Sincethen ECVs have been published by the CLSI (Espinel-Ingroff et al.,2010; Pfaller et al., 2011) and others (Meletiadis et al., 2012) theECOFF by EUCAST-AFT for azole drugs and A. fumigatus (Arendrupet al., 2012a, 2013; Hope et al., 2013; Rodriguez-Tudela et al., 2008).Notably, in all of the above mentioned reports the ECOFF of 1 mg/L isconsidered as breakpoint for voriconazole against A. fumigatus. Thebreakpoints and ECVs are shown in Table 1. However, our recentanalysis on a large collection of A. fumigatus isolates (wild-type andCYP51A-mutants) collected between 2009–2013, indicated that theECOFF for voriconazole and A. fumigatus should be higher and equalto 2 mg/L (van Ingen et al., 2014).

4. Genotypic detection of azole resistance mechanisms inAspergillus spp.

In addition to the phenotypic methods, significant insight hasbeen obtained regarding the underlying genetic mechanisms thatconfer an azole resistant phenotype. In A. fumigatus two distinctbut closely related cyp51 genes were found (cyp51A and cyp51B)

S. Seyedmousavi et al. / Drug Resistance Updates 17 (2014) 37–50 39

Table 1Proposed EUCAST breakpoints (adopted from Rodriguez-Tudela et al., 2008, Arendrup et al., 2012a, Arendrup et al., 2013 and Hope et al., 2013), breakpoints proposed byVerweij et al. (2009a), and CLSI Epidemiological cut-off values (Espinel-Ingroff et al., 2010 and Pfaller et al., 2011) for A.fumigatus and clinically licensed mold-active azoles.

Minimum inhibitory concentration (MIC) mg/L

Azole antifungal Susceptible Intermediate Resistant ECOFF

EUCAST Itraconazole ≤1 – >2 1Voriconazole ≤1 – >2 1 (2)a

Posaconazole ≤0.125 – >0.25 0.25Verweij et al. Itraconazole <2 2 >2 –

Voriconazole <2 2 >2 –Posaconazole <0.5 0.5 >0.5 –

ECVs

Wild-type Non-wild-type

CLSI Itraconazole ≤1 – ≥2 –

tcbwlme(canfpgoeb

Mc2ccwcmbp

Cctcslih

cTnpIbpUp

Voriconazole ≤1

Posaconazole ≤0.25

a Adopted from van Ingen et al. (2014).

hat share 63% sequence identity and encode for two differentyp51 proteins (Mellado et al., 2001; Warrilow et al., 2010b). Azoleinding studies showed that fluconazole has the weakest bindingith cyp51A and cyp51B proteins, which is in keeping with the

ack of activity against A. fumigatus. Furthermore cyp51B showedore tight bindings with azoles compared to cyp51A and is gen-

rally more susceptible to azole compounds compared to cyp51AWarrilow et al., 2010b). Therefore, it has been postulated that theyp51A gene encodes for the major 14-alpha-demethylase enzymectivity required for growth, and the cyp51B gene encodes for alter-ative functions for particular growth conditions or even being

unctionally redundant (Warrilow et al., 2010b). This provides aossible explanation to why mutations in azole-resistant A. fumi-atus isolates are predominantly detected in the cyp51A gene andnly rarely in the cyp51B gene (Diaz-Guerra et al., 2003; Warrilowt al., 2010b). Also in cyp51B no mutations have yet been proven toe correlated to azole resistance (Buied et al., 2013).

Three different studies adapted the X-ray crystallography ofycobacterium tuberculosis protein to develop an A. fumigatus

yp51A 3-D protein model (Gollapudy et al., 2004; Sheng et al.,004; Xiao et al., 2004). All models show that two ligand entryhannels can be identified in the CYP51A protein. The ligand accesshannels immersed in the endoplasmic reticulum (ER) membraneould allow highly lipophilic sterol substrates as well as azole

ompounds to dock into the channels and restrict access of otheretabolites (Warrilow et al., 2010b). The azole compounds can

ind to the active heme molecule located in the center of the cyp51Arotein and thereby inhibiting its enzyme function.

Different single nucleotide polymorphisms (SNPs) in theyp51A-gene are related to resistance against one or more azoleompounds found in clinical induced azole resistant A. fumiga-us isolates (Table 2). Although several SNPs have been reported,odons 54, 98 and 220 are the most frequently characterized hotpots. According to protein homology modeling, these codons areocated in the opening of one of the ligand access channels, whichs thought to interfere with the entry of azole compounds into theydrophobic access channel (Snelders et al., 2010).

In addition to single point mutations, a combination of genetichanges has been described in azole-resistant A. fumigatus isolates.he duplication of a set of sequences in the promoter region sig-ificantly increases the expression of cyp51A which for one partrovides an explanation for the decrease in azole susceptibility.

mportantly, recombinant experiments showed that only when

oth mutations were introduced the multi azole resistance (MAR)henotype was observed (Mellado et al., 2007; Verweij et al., 2007).p until now, three mechanisms have been described: a 34 baseair tandem repeat (TR) combined with a L98H substitution in the

– ≥2 –– ≥0.5 –

Cyp51A-gene (TR34/L98H) (Klaassen et al., 2010; Mellado et al.,2007; Seyedmousavi et al., 2013b; Snelders et al., 2008), a 53 bpTR without substitutions in the Cyp51A-gene (TR53) (Camps et al.,2012b; Hodiamont et al., 2009), and recently a 46 bp TR with twosubstitutions in the cyp51A-gene (TR46/Y121F/T289A) (Kuiperset al., 2011; van der Linden et al., 2013; Chowdhary et al., 2014).Unlike the point mutations, the resistance mechanisms with a TRappear not have a predictable phenotype for all azole compounds.Isolates with TR53 are associated with a pan-azole-resistant pheno-type and was reported to have caused aspergillus osteomyelitis in apediatric patient in 2006 (Hodiamont et al., 2009). Isolates harbor-ing the TR34/L98H resistance mechanism are all highly resistant toitraconazole and have a MIC of 0.5 mg/L for posaconazole, but theactivity of voriconazole varies, ranging from 1 to >16 mg/L. Likewisein isolates with the TR46/Y121F/T289A, voriconazole is inactive butthe activity of itraconazole may vary ranging from 0.5 to 16 mg/L.Notably, this type of resistance mechanisms has been found in iso-lates that are associated with the environmental route of resistanceselection. A TR in the promoter region has been described in severalazole-resistant plant pathogenic fungi, which adds to the evidencethat selection of this type of resistance mechanism occurs in theenvironment (Chowdhary et al., 2013; Snelders et al., 2009).

Notably, there are several studies indicating that non-Cyp51A-gene mutations might be associated with azole-resistance inAspergillus spp. Bueid et al. reported that Cyp51B overexpressionis a possible azole resistance mechanism in A. fumigatus (Buiedet al., 2012). Other researchers demonstrated that the changes indrug efflux pump of Aspergillus spp. is another potential resistancemechanism. Overexpression of the cdr1B efflux transporter genes(Fraczek et al., 2013), modifications in AfuMDR1 and AfuMDR2genes (da Silva Ferreira et al., 2004), and changes in expression ofAfuMDR3 and AfuMDR4 (da Silva Ferreira et al., 2004; Nascimentoet al., 2003) were linked to high level azole-resistance in A. fumiga-tus. Similarly, Krishan-Natesan et al. showed that overexpressionof ATP-binding cassette and changes in major facilitator super-family class efflux pumps contributed to voriconazole resistancein A. flavus (Natesan et al., 2013). Recently, Camps et al. reporteda novel resistance mechanism, consisting of a mutation in theCCAAT binding transcription factor complex subunit HapE (Campset al., 2012a). The substitution was found in P88L within the exonicregion of HapE gene causing the resistance phenotype. Unlikecyp51A-mediated resistance mechanisms, HapE was associatedwith a fitness cost (Arendrup et al., 2010). Azole resistance in other

species of Aspergillus such as A. flavus (Liu et al., 2012), and A.terreus(Arendrup et al., 2012b) may be also caused by alterations andoverexpression of the azole target 14a-demethylase (Buied et al.,2012).

40 S. Seyedmousavi et al. / Drug Resistance Updates 17 (2014) 37–50

Table 2The minimum inhibitory concentrations (MICs) of clinical A. fumigatus isolates with various Cyp51A-mediated resistance mechanisms conferring azole-resistant phenotypes.

Cyp51A substitution Maximum number of isolates MIC (mg/L) Reference

ITC VRC POS

G54E 6 >8 0.25–0.5 0.25–1 Diaz-Guerra et al. (2003), Howard et al. (2009)G54R 6 >8 0.12–0.5 1 Chen et al. (2005), Howard et al. (2009)G54V 1 >8 0.25–1 ND Garcia-Effron et al. (2008), Howard et al. (2009)G54W 1 >8 0.12–0.5 >8 Garcia-Effron et al. (2008)G138C 10 >8 4 to >8 1 to >8 Howard et al. (2009), Howard et al. (2009)P216L 1 >8 1 1 Howard et al. (2009)M220I 6 >8 1 0.5 Chen et al. (2005)M220K 4 >8 1–2 1 to >8 Howard et al. (2009)M220R 1 >8 2 2 Bueid et al. (2010)M220T 6 >8 0.5–1 0.25–0.5 Howard et al. (2009)M220V 3 >8 1–2 0.5–1 Mellado et al. (2004)M220W 1 ND ND ND Bueid et al. (2010)Y431C 1 >8 4 1 Howard et al. (2009)G432S 1 >8 0.25–0.5 0.25 Alanio et al. (2011)G434C 1 >8 4 1 Howard et al. (2009)G448S 1 >8 >8 0.5–1 Bellete et al. (2010), Howard et al. (2009)TR34/L98H 110 >8 1 to >8 0.25–8 Snelders et al. (2008), Chen et al. (2010),

van der Linden et al. (2011b), Seyedmousaviet al. (2013b)

TR46/Y121F/T289A 29 0.5 to >8 >8 0.25–4 Kuipers et al. (2011), van der Linden et al.(2013)

TR53 1 >8 >8 0.25 Hodiamont et al. (2009)1,1

nsa

5f

toCraif(2v2aAMauiLw

rooaiftopt

F219I 6 >16

P216L 1 >16

Abovementioned studies show that acquired azole resistance isot limited to A. fumigatus, but may be evident in other Aspergilluspp, and that Cyp51A-gene mediated resistance, is only one of prob-bly many mechanisms that confer azole resistance.

. Clinical implications of azole resistance in Aspergillusumigatus

There are currently no randomized controlled trials showinghat azole resistance is associated with an increased probabilityf treatment failure compared to infection with wild type isolates.ase series have been published including both patients with azole-esistant chronic aspergillus diseases and azole-resistant invasivespergillosis that show the recovery of an azole-resistant isolates associated with an increased probability of azole treatmentailure compared to infection with an azole-susceptible isolateHamprecht et al., 2012; Hodiamont et al., 2009; Howard et al.,009; Mellado et al., 2013; Rath et al., 2012; Snelders et al., 2008;an der Linden et al., 2009, 2011b, 2013; van Leer-Buter et al.,007; Warris et al., 2002). The characteristics of patients withzole-resistant invasive aspergillosis are shown in Table 3. The.fumigatus isolates were considered resistant if they showed anIC above the breakpoints (Table 1) for any of the mold-active

zoles. The cases and case series listed in Table 3 show overall fail-re to azole therapy. Notably, in one study primary therapy was

nitiated with liposomal amphotericin B in four patients (van derinden et al., 2013). In three of these patients, invasive aspergillosisas diagnosed, and all patients were alive at 12 weeks.

Although the current clinical experience suggests that azoleesistance is associated with treatment failure, it should be rec-gnized that there are numerous factors that impact on treatmentutcome. Patients with underlying malignancy are prone to fail tozole therapy, even if the infection is caused by an azole-susceptiblesolate. Azole exposure might have been insufficient in patientsailing therapy and as most patients may not be culture-positive,

reatment might have been initiated relatively late in the coursef the infection. Therefore, azole-resistant infection might occurredominantly in patients with poor clinical conditions, comparedo wild type isolates. In the absence of robust clinical evidence,

4, 8 0.5, >16 Camps (2012)1 Camps (2012)

experimental models of aspergillus infection can help us to under-stand the implications of MIC elevation on treatment efficacy.

6. Efficacy of voriconazole and posaconazole inexperimental models of azole resistant aspergillosis

Several experimental models have been used to explore PKand PD properties of voriconazole and posaconazole in the settingof azole-resistant aspergillosis. These models are summarized inTable 4 (Howard et al., 2011; Jeans et al., 2012; Lepak et al., 2013b;Mavridou et al., 2010a,b). Using a non-neutropenic murine modelof invasive aspergillosis voriconazole and posaconazole (responsemeasured as survival) showed a clear exposure-dependent relationwith response for both voriconazole-susceptible and voriconazole-resistant strains. For each dose the response was lower in miceinfected with the azole-resistant isolate compared to that of miceinfected with the azole-susceptible isolate. As for voriconazole andposaconazole the dose correlates with exposure, a higher exposureof the azole was required to achieve similar efficacy when harbor-ing azole-resistant strains (Mavridou et al., 2010a,b; Seyedmousaviet al. 2013a).

Jeans et al. developed an in vitro dynamic model of the humanalveolus invasive pulmonary aspergillosis to study the impact ofMIC on exposure–response relationships of voriconazole, againstwild-type and azole-resistant A. fumigatus (Jeans et al., 2012). Theantifungal effect of voriconazole was assessed by measuring lev-els of galactomannan. Galactomannan concentrations began toincrease approximately 16–24 h post-inoculation, and a maximumwas reached approximately after 36 h. The rate of increase andthe maximum galactomannan concentrations were comparable.The isolates with higher MICs required higher area under the con-centration time curves (AUCs) to achieve similar suppression ofgalactomannan compared to the wild-type controls (Jeans et al.,2012).

Howard et al. also used this in vitro model to study the impact

of MIC on PK/PD relationships of posaconazole (Howard et al.,2011). The results were validated using an inhalational murinemodel of invasive pulmonary aspergillosis. Similarly, the admin-istration of posaconazole caused a dose-dependent decline in

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41Table 3Characteristics of patients with azole-resistant Aspergillus fumigatus diseases.

Underlying disease Aspergillus diseasesa Azole susceptibility phenotype Genotype (Mutation incyp51A)

Prophylaxis Treatment Outcome

MIC Method ITC VRC POS

Warris et al. (2002) CGD IPA CLSI >16 4 1 – ITC VRC Failurevan Leer-Buter

et al. (2007)Carcinoma of theoropharynx

IPA CLSI >16 8 0.5 TR34/L98H – VRC Failure

Hodiamont et al.(2009)

CGD Osteomyelitis CLSI >16 16 0.25 – ITC POS, CAS Improvement

van der Lindenet al. (2009)

HIV positive, COPD CNS aspergillosis CLSI >16 8 0.5 TR34/L98H – VRC, L-AmB Clinical failure/died

van der Lindenet al. (2009)

Myocardialinfarction, coloncarcinoma

CNS aspergillosis CLSI >16 16 0.5 TR34/L98H – VRC, CAS Clinical failure/died

van der Lindenet al. (2009)

B celllymphoblasticlymphoma

CNS aspergillosis CLSI >16 16 2 TR34/L98H – VRC, CAS Clinical failure/died

van der Lindenet al. (2011b)

Lung carcinoma Proven pulmonaryaspergillosis

CLSI – 4 – TR34/L98H – VRC Died at 12 weeks

van der Lindenet al. (2011b)

Hematologicmalignancy,Allo-HSCT, GvHD

Proven pulmonaryaspergillosis

CLSI – 8 – TR34/L98H – VRC Died at 12 weeks

van der Lindenet al. (2011b)

Acute myeloidleukemia,Allo-HSCT

Proven pulmonaryaspergillosis

CLSI – 8 – TR34/L98H – VRC Died at 12 weeks

van der Lindenet al. (2011b)

Non-Hodgkinlymphoma,Allo-HSCT, GvHD,lung cavities

Proven pulmonaryaspergillosis

CLSI – 16 – TR34/L98H – VRC Died at 12 weeks

van der Lindenet al. (2011b)

Breast carcinomawith metastasis

Probable pulmonaryaspergillosis

CLSI – 1 – TR34/L98H – VRC Died at 12 weeks

van der Lindenet al. (2011b)

Non-Hodgkinlymphoma

Proven pulmonaryand CNS aspergillosis

CLSI – 16 – TR34/L98H – VRC, CAS, AmB Died at 12 weeks

van der Lindenet al. (2011b)

Livertransplantation forhepatic failure aftermethotrexatetreatment forarteritis

Proven pulmonaryand CNS aspergillosis

CLSI – 2 – TR34/L98H – VRC, AmB Died at 12 weeks

van der Lindenet al. (2011b)

Acute myeloidleukemia,Allo-HSCT, GVHD

Proven pulmonaryand CNS aspergillosis

CLSI – 4 – TR34/L98H – VRC, CAS, AmB,POS

Survived at 12 weeks

Howard et al.(2009)

Breast cancer,Pulmonary TB,Celiac disease

CCPA withaspergilloma

EUCAST >8 >8 0.5 H147Y, G448S – VRC Clinical andradiologicalfailure/alive

Howard et al.(2009)

Pulmonary TB withresidual bilateralUL scarring andLUL cavity, Smokeinhalation

CCPA withaspergilloma, CFPA

EUCAST (8isolates)

>8 1 to 4 0.25 to >8 M220K, M220T – ITC Clinical andradiologicalfailure/died

Howard et al.(2009)

COPD, Squamouscell carcinoma withLULsegmentectomy

CCPA withaspergilloma

EUCAST (3isolates)

0.5 to >8 1 to 2 0.125 to >8 M220K, G54E – ITC No improvement/died

Howard et al.(2009)

AML, RULlobectomy,Allo-HSCT, GVHD

Cerebral aspergillosis EUCAST (twoisolates)

0.25 to >8 2 0.06 to 0.125 F46Y, M172V, E427K – ITC Regression of cerebralabscess/IPA withrespiratory failure/died

42

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37–50Table 3 (Continued)

Underlying disease Aspergillus diseasesa Azole susceptibility phenotype Genotype (Mutation incyp51A)

Prophylaxis Treatment Outcome

MIC Method ITC VRC POS

Howard et al.(2009)

COPD, PulmonaryTB, Celiac disease

CCPA withaspergilloma

EUCAST (2isolates)

0.25 to >8 0.5 0.06 to >8 G54R – ITC Clinical failure/died

Howard et al.(2009)

COPD, possiblebronchiectasis

Acute invasivepulmonary infection

EUCAST >8 2 0.25 F46Y, M172V, N248T,D255E, E427K

– ITC No improve-ment/switched toVRC/developedtoxicity/died withoutIPA

Howard et al.(2009)

Bronchiectasis,asthma, AVR,hypermobilitysyndrome,Pulmonary TB

ABPA EUCAST (2isolates)

0.25 to >8 0.5 to 8 0.06 to 2 TR34/L98H – ITC Initialimprovement/thenfailure/alive

Howard et al.(2009)

Pulmonarysarcoidosis

CCPA with bilateralaspergillomas, CFPA

EUCAST (12isolates)

>8 4 to >8 1 to >8 G138 C – ITC Clinical failure/died

Howard et al.(2009)

Bronchiectasis,onychomycosis,�-1-antitrypsindeficiency

Aspergillusbronchitis

EUCAST (2isolates)

>8 4 to 8 0.5 to 1 – – ITC, POS ITC resistancedefined/treated withPOS/alive

Howard et al.(2009)

RUL pneumonia CCPA withaspergilloma

EUCAST (2isolates)

>8 4 0.25 to 0.5 F46Y, M172V, E427 K – ITC No improvement/alive

Howard et al.(2009)

Pulmonary TB, HIVpositive, HAART

CCPA withaspergillomas

EUCAST >8 0.5 1 – ITC No improvement/alive

Howard et al.(2009)

COPD,Bonchiectasis,Pulmonary TB

CCPA withaspergilloma

EUCAST (fourisolates)

0.25 to >8 0.125 to >8 0.03–1 G54R, G54E, G448S – ITC Progression/alive

Kuipers et al.(2011)

Kidney transplant IA EUCAST 2 >16 0.5 TR46/Y121F/T289A – VRC, POS Clinical failure/died

Rath et al. (2012) – Aspergilloma CLSI ≥16 4 0.5 TR34/L98HTR34/L98H

– VRC Clinical failureHamprecht et al.

(2012)AML Proven IA EUCAST >16 2 0.5 TR34/L98H POS L-AmB Improvement/Survived

van der Lindenet al. (2013)

Relapse ALL, HSCT,GvHD

Probable IA CLSI 4 >16 0.25 TR46/Y121F/T289A – VRC, CAS Persistent infection at12 weeks

van der Lindenet al. (2013)

Kidney transplant Proven IA CLSI 2 >16 0.5 TR46/Y121F/T289A – VRC, POS Died at 12 weeks

van der Lindenet al. (2013)

Multiple myeloma,autologous HSCT,relapse

Probable IA CLSI 1 >16 0.25 TR46/Y121F/T289A – VRC, L-AmB Died at 12 weeks

van der Lindenet al. (2013)

�-Thalassemia anddiabetes mellitus

Proven IA CLSI 4 >16 1 TR46/Y121F/T289A – VCZ, L-AMB,CAS

Died at 12 weeks

van der Lindenet al. (2013)

NH B-celllymphoma,allo-SCT

Probable IA CLSI >16 >16 1 TR46/Y121F/T289A – VRC Died at 12 weeks

Vermeulen et al.(2013)

HSCT, GvHD IA CLSI 4 >16 4 TR46/Y121F/T289A Fluconazole L-AmB Clinical failure/died

ITC: itraconazole, VRC: voriconazole, POS: Posaconazole, FLC: Fluconazole, CAS: Caspofungin, AND, Anidulafungin, AmB: Amphotericin B, L-AmB: Liposomal-amphotericin B.ABPA: allergic bronchopulmonary aspergillosis, Allo-HSCT: allogeneic hematopoietic stem cell transplantation, AML: acute myeloid leukemia, AVR: aortic valve replacement, CCPA: chronic cavitary pulmonary aspergillosis, CNS:central nervous system, CFPA: chronic fibrosing pulmonary aspergillosis, CF: cystic fibrosis, CGD: chronic granulomatous disease, COPD: chronic obstructive pulmonary disease, CPA: chronic pulmonary aspergillosis, GvHD:graft-versus-host disease, HAART: highly active antiretroviral therapy, HSCT: hematopoietic stem cell transplantation, IA: invasive aspergillosis, IPA: invasive pulmonary aspergillosis, LUL: left upper lobe, NF: not found, NH:non-Hodgkin, RUL: right upper lobe, TB: tuberculosis, UL: upper lobe.

a According to the classification of EORTC/Mycoses study group (De Pauw et al., 2008).

S. Seyedmousavi et al. / Drug Resista

Tab

le

4Ph

arm

acod

ynam

ic

ind

ex

(PD

I)

of

vori

con

azol

e

and

pos

acon

azol

e

corr

elat

ed

wit

h

mea

sure

s

of

effi

cacy

in

in

vitr

o

and

pre

clin

ical

mod

els

of

inva

sive

asp

ergi

llos

is.

Vor

icon

azol

e

Posa

con

azol

e

Mav

rid

ou

et

al. (

2010

a)

(mod

ified

toEU

CA

ST

inte

rpre

tati

on)

Jean

s

et

al. (

2012

)

Mav

rid

ou

et

al. (

2010

b)/t

hes

is

How

ard

et

al. (

2011

)

Lep

ak

et

al. (

2013

b)

Mod

el

of

infe

ctio

n

Dis

sem

inat

ed/I

mm

un

ocom

pet

ent/

Mic

e

In

vitr

o

Mod

el

Dis

sem

inat

ed/I

mm

un

ioco

mp

eten

t/M

ice

Pulm

onar

y/N

eutr

open

ic/M

ice

and

in

vitr

o

mod

elPu

lmon

ary/

Neu

trop

enic

/Mic

e

Res

ista

nce

mec

han

ism

s

ofis

olat

es

use

d

Wil

d-t

ype

Wil

d-t

ype

Wil

d-t

ype

Wil

d-t

ype

Wil

d-t

ype

FKS1

(Wil

d-T

ype

susc

epti

bili

ty)

G54

W

G13

8C

G54

W

–

G54

E;

-R

G13

8C

M22

0I

G43

4C

M22

0I

–

G43

4C

M22

0TTR

34/L

98H

L98H

TR34

/L98

H–

TR34

/L98

HEn

dp

oin

t

Surv

ival

Gal

acto

man

nan

ind

exSu

rviv

al

Gal

acto

man

nan

ind

ex/S

urv

ival

qPC

R/S

urv

ival

Tota

l AU

C0-

24/M

ICfo

r

EIin

dic

ated

EI50

:

17.6

(EU

CA

ST)

EI50

:

21.9

6(E

UC

AST

)EI

50:

178

(EU

CA

ST)

EI50

:

167

(EU

CA

ST)

EI50

:

176

(CLS

I)

(Un

bou

nd

VR

C

in

mou

sep

lasm

a:

29.8

7%)

(Un

bou

nd

VR

C

in

hu

man

pla

sma:

42%

)

EI80

:

18.0

(EU

CA

ST)

–

EI80

:

1517

(EU

CA

ST)

EI80

:

309(

EUC

AST

)–

(Un

bou

nd

VR

C

in

in

vitr

om

odel

:

98%

)EI

90:

18.3

(EU

CA

ST)

EI90

:

32.1

(EU

CA

ST)

–

EI90

:

441(

EUC

AST

) –

(Un

bou

nd

POS

in

mou

se:

1.0%

)

EI90

:

55

(CLS

I)

nce Updates 17 (2014) 37–50 43

serum galactomannan concentrations with near-maximal suppres-sion following 20 mg/kg/day. The posaconazole MICs affected theexposure–response relationships, those strains with a higher MIChad higher 50% effective pharmacodynamic index (EI50) (Howardet al., 2011).

In another study, Lepak et al. investigated the pharmacody-namic target of posaconazole in an immunocompromised murinemodel of invasive pulmonary aspergillosis against Cyp51A wild-type isolates and isolates carrying Cyp51A mutations conferringazole resistance (Lepak et al., 2013b). Efficacy was assessed byquantitative PCR (qPCR) of lung homogenate and survival. Mortalitymirrored qPCR results, with the greatest improvement in survivalnoted at the same dosing regimens that produced fungistatic orfungicidal activity. The results demonstrated that more posacona-zole, on a mg/kg basis, was required for efficacy against organismswith reduced in vitro susceptibility.

In conclusion, all models show a clear exposure–response rela-tionship. In most models, the exposures that are required forefficacy are in a similar range and therefore underscore the valueof these models. However, it should be realized that the modelsare designed and optimized to find these relationships, and thepharmacodynamic targets that are derived from the models maytherefore over- or underestimate the ‘true’ target. For instance, theEC50 in survival studies is a reproducible measure for efficacy, butnot necessarily coincides with the same endpoint in humans.

Importantly, the pharmacodynamic endpoint in experimentalmodels of invasive aspergillosis that best predicts the outcome ofpatients with pulmonary infections is not well known and still suf-fers lack of standardization. Using the abovementioned studies theEI50, EI80 and EI90 over 7 or 14 days survival for voriconazole andposaconazole can be estimated (Table 4). In addition, in vitro stud-ies and in vivo models differ concerning the route of infection, theefficacy parameter, the presence or absence neutropenia, level ofprotein binding and other variables.

Both in vitro and in vivo studies have indicated that the ratio ofthe area under the concentration time curve (AUC) at 24 h to theminimum inhibitory concentration (MIC) is the main PK/PD param-eter that best predicts voriconazole and posaconazole efficacy ininvasive aspergillosis. Therefore, for purpose of further discus-sion, we decided to use the effective exposure index at 50% (EI50)AUC0–24/MIC as the value most predictive of treatment, which isgenerally considered in the relationships between the PK/PD eval-uation of antimicrobial agents (Mouton et al., 2005). However,one should consider that higher values such as EI80 or EI90 aremore reliable when translating to the patient setting. Importantly,this higher value was similar to EI50 in voriconazole studies, andnot consistent/achievable in all posaconazole experimental studiesanalyzed in our review. Moreover, the range of PD-target predict-ing therapeutic success using either in vitro or in vivo model was inthe same range in abovementioned voriconazole and posaconazolestudies (Tables 5 and 6).

7. Bridging experimental results to humans: is there a rolefor voriconazole and posaconazole in azole-resistantinvasive aspergillosis?

Based on the estimates of the PD-targets for voriconazole andposaconazole we can now determine if there remains a role forvoriconazole or posaconazole in the management of azole-resistantdisease. The integration of all above information is given in Table 5for voriconazole and Table 6 for posaconazole. The underlying resis-

tance mechanisms are provided for each MIC-value. Based on theestimates of the PD-target, the exposure can be calculated that isneeded to achieve the PD-target for each MIC. The exposure corre-sponds with plasma levels, which are typically higher than those

44

S. Seyedm

ousavi et

al. /

Drug

Resistance

Updates

17 (2014)

37–50

Table 5Probability of attaining therapeutic success of voriconazole against azole-susceptible and azole-resistant Aspergillus diseases according to MIC values (top row), PK/PD target and exposures. Green, exposureusing standard dose; orange: exposure attainable using increased dose; red, required exposure not attainable.

S. Seyedm

ousavi et

al. /

Drug

Resistance

Updates

17 (2014)

37–50

45

Table 6Probability of attaining therapeutic success of posaconazole against azole-susceptible and azole-resistant Aspergillus diseases according to MIC values (top row), PK/PD target and exposures. Green, exposure using standarddose; orange: exposure attainable using increased dose; red, required exposure not attainable.

4 esista

nid

atamtm(nsbctbd2z2dceet1

cmrfarrevcraarptrffddwrgtaertl

st(2Aib

6 S. Seyedmousavi et al. / Drug R

eeded for treating infection due to wild-type isolates. The feasibil-ty of achieving higher exposure depends on characteristics of therug related to absorption and clearance, but is limited by toxicity.

For voriconazole, a total drug AUC/MIC ratio of 21.96 wasssociated with 50% probability of success (EI50) to suppress galac-omannan concentrations in a dynamic in vitro model of the humanlveolus (Jeans et al., 2012). Using an immunocompetent murineodel of invasive aspergillosis, we observed that achieving a serum

otal AUC0–24/MIC ratio of 17.61 was the PD-target linked to half-aximum antifungal effect predicting therapeutic success (Table 4)

Mavridou et al., 2010b). Although, the calculated pharmacody-amic index (using total drug) is similar for both studies, onehould consider that in vitro simulation of in vivo protein bindingy adding serum proteins in the in vitro models is difficult sinceomplex phenomena may take place (Smith et al., 2010). Givenhe qualitative and quantitative differences between human andovine serum, the unbound fraction of various drugs was markedlyifferent between human and bovine serum (Finlay and Baguley,000; Siopi et al., 2014). Such differences were found for voricona-ole in the serum of human and different animals (Roffey et al.,003). It is generally accepted that only the unbound fraction ofrug is pharmacologically active and therefore when in vitro con-entrations are correlated with in vivo concentrations, in vivo drugxposures should be corrected for the protein binding (Zeitlingert al., 2011). This can alter the PD of voriconazole depending onhe protein-binding differences between 2% fetal bovine serum and00% human serum (Siopi et al., 2014).

Recently, Pascual et al. performed a population pharma-okinetic analysis (NONMEM) on 505 plasma concentrationeasurements involving 55 patients with invasive mycoses who

eceived recommended voriconazole doses in order to describeactors influencing the pharmacokinetic variability, to assessssociations between plasma concentrations and efficacy or neu-otoxicity/hepatotoxicity, and to define intravenous and oral dosesequired for achieving drug exposure with the most appropriatefficacy/toxicity profile (Pascual et al., 2012). A logistic multi-ariate regression analysis revealed the therapeutic target with alinically appropriate efficacy-safety profile, close to that recentlyeported by others (Seyedmousavi et al., 2013e). An independentssociation between voriconazole trough concentrations and prob-bility of response or neurotoxicity was identified for a therapeuticange of 1.5 mg/L (>85% probability of response) to 4.5 mg/L (<15%robability of neurotoxicity). Population-based simulations withhe recommended 200 mg oral or 300 mg intravenous twice-dailyegimens predicted probabilities of 49% and 87%, respectively,or achievement of 1.5 mg/L and of 8% and 37%, respectively,or achievement of 4.5 mg/L. With 300–400 mg twice-daily oraloses and 200–300 mg twice-daily intravenous doses, the pre-icted probabilities of achieving the lower target concentrationere 68–78% for the oral regimen and 70–87% for the intravenous

egimen, and the predicted probabilities of achieving the upper tar-et concentration were 19–29% for the oral regimen and 18–37% forhe intravenous regimen (Pascual et al., 2012). Apparently, patientschieving higher concentrations of voriconazole may show higherxposure and a better response to therapy, but they are at higherisk for toxicity. In contrast, patients achieving lower concentra-ions may have reduced therapeutic response but subsequently aower risk for adverse events.

Whereas the Pascual study is based on trough levels as a mea-ure of exposure (Pascual et al., 2012), because it is much easiero determine than the AUC, all preclinical models are AUC basedJeans et al., 2012; Mavridou et al., 2010b; Seyedmousavi et al.,

013a). However, voriconazole trough levels correlate well withUC as determined in several studies. Estimates of total AUC0–24

n sixty-four healthy subjects showed that standard dose on theasis of 200 mg twice daily oral voriconazole results in a total

nce Updates 17 (2014) 37–50

AUC value of 18–23 mg.h/L (Purkins et al., 2003). Population PKmodeling of voriconazole in 21 healthy volunteers, and 43 patientswith proven or probable invasive aspergillosis, (Hope, 2012) andother PK studies in allogeneic haematopoietic stem cell transplantrecipients (Fig. 1) (Bruggemann et al., 2010a), revealed that thetrough concentration are well correlated with the AUC, and a druglevel of 1 and 4.5 mg/L corresponded with a total AUC0–24 of 43 and151 mg h/L, respectively.

The AUC levels required for efficacy as derived from the troughlevels in the Pascual study (Pascual et al., 2012), correspond wellwith the AUC levels required for efficacy in preclinical models. Thethreshold was consistent with minimum inhibitory concentrationrequired to inhibit the growth of 90% of organisms and epidemio-logical cutoffs of most VRC-susceptible fungal species, as well asclinical reports (Espinel-Ingroff et al., 2010; Pfaller et al., 2011;Verweij et al., 2009a) (Arendrup et al., 2012a; Hope et al., 2013;Rodriguez-Tudela et al., 2008).

Assuming no resistant strains in the Pascual study (Pascual et al.,2012), the ECOFF of voriconazole (1 mg/L) can be used as the uppervalue of the MIC distribution and the denominator in the AUC/MIC.In addition, using EUCAST methodology, Jeans et al. reported thatthe trough concentration/MIC values that achieve optimal efficacywas 1 (Jeans et al., 2012). However, considering both wild-typeand mutant population of A.fumigatus, higher ECOFF is requiredfor voriconazole (2 mg/L) (van Ingen et al., 2014). Given this, theupper value of denominator will be 2 mg/L and the AUC/MIC ratiorequired for optimal treatment is 43. It follows that the AUC/MICratio required for optimal treatment is very close to the pharma-codynamic targets derived from preclinical models (EI50 EUCAST:17.61–21.96) in order to achieve therapeutic success consideringthe differences in voriconazole disposition between human andmouse.

Therefore, it can be expected that isolates with a MIC that isclassified as susceptible can be treated with voriconazole, with aprobability of exposure attainment of over 90% according to pop-ulation pharmacokinetics modeling of Hope et al. using licenseddoses of voriconazole (Hope, 2012; Hope et al., 2013). For isolateswith a voriconazole MIC of 2 mg/L, classified as intermediate sus-ceptibility by Verweij et al. (2009a), the plasma level should exceed1.03 mg/L which is well attainable. Voriconazole MIC of 4 mg/L isclassified as resistant, and in order to achieve the PD-target a higherexposure is needed (≥2.65 mg/L). Higher exposure of voriconazolecan be achieved using dose escalation, but will be associated withincreased probability of toxicity. Clearly if voriconazole would beused in this setting intravenous administration would be requiredas well as close monitoring of plasma levels. For isolates with a MICexceeding 4 mg/L very high plasma levels exceeding 5.30 mg/L areneeded, which are in a range where toxicity can be anticipated.

Posaconazole is currently not licensed for the primary ther-apy of invasive aspergillosis, but may be used for salvage therapy.Similar to the other triazoles, posaconazole displays concentration-dependent with time dependence pharmacodynamic character-istics, for which a total AUC0–24/MIC ratio ranging 167–178 wasthe value predictive of success associated with half-maximal effi-cacy. Estimates of the total AUC0–24 for patients infected withA. fumigatus with a posaconazole MIC of 0.125 mg/L receiving800 mg/day are 13–17 mg.h/L, corresponding to the best responserate (AbuTarif et al., 2010; Ullmann et al., 2006). Our calculation(Table 6) also showed that similar exposure (AUC0–2410.43–11.12and AUC0–2420.87–22.5) are required to achieve optimal responsefor the isolates with MIC 0.64 and 0.125 mg/L, respectively. On theother hand, optimal outcome could be achieved with posacona-

zole plasma concentrations of ∼0.7 mg/L when administered forprophylaxis. However, for purpose of salvage therapy, Walsh et al.showed that an average concentration of 1.25 mg/L was associatedwith a higher probability of a clinical response for patients with

S. Seyedmousavi et al. / Drug Resistance Updates 17 (2014) 37–50 47

ole (leA

ieTdwws∼>

nss(pmpwfcrittvl(araepNdm5bd1pit

Fig. 1. Linear regression analysis between exposure (AUC0–24) of voriconazdopted from Bruggemann et al. (2010a,b).

nvasive aspergillosis receiving posaconazole 800 mg/day (Walsht al., 2007), corresponding to an AUC of approximately 30 mg h/L.herefore, with fixed dosing of 800 mg/day (200 mg four times aay), drug exposures may not be high enough to cover the entireild-type distribution, reliably in persistently neutropenic hostsith invasive aspergillosis. The patients infected with an Aspergillus

train with a MIC of 0.25 mg/L, will need to obtain an AUC0–24 of40–50 mg.h/L, which corresponds with trough concentrations of1.25 mg/L, as shown in Fig. 1 (Bruggemann et al., 2010a,b).

According to available data shown in Table 6, the exposureeeded to treat infection due to isolates that are classified asusceptible can only be achieved with a low probability of expo-ure attainment in isolates with the MIC ranging 0.31–0.125 mg/LArendrup et al., 2012a; Verweij et al., 2009a). Given the currentroblems of increasing the exposure of the drug due to its for-ulation and limited absorption, there appears to be no room for

osaconazole for the treatment of isolates that are not within theild type distribution. However, a new oral tablet and intravenous

ormulation are under development and soon to be brought thelinical practice (Krishna et al., 2012b). The tablet is designed toelease the entire dose of solubilized posaconazole in the smallntestine, maximizing systemic absorption. In an exploratory study,his new solid oral formulation significantly increased exposureo posaconazole relative to the oral suspension in fasting healthyolunteers (Courtney et al., 2004; Krishna et al., 2009, 2012a). Fol-owing single and multiple doses of posaconazole solid oral tablets200 and 400 mg) in healthy subjects, the exposure increased in

dose-related manner. When the dose was increased in a 1:2atio, exposure increased in 1:1.9 and 1:1.8 ratios for days 1nd 14, respectively. On day 1, the dose-normalized posaconazolexposure (AUCtau) was substantially higher than for the oral sus-ension under both fasted and fed conditions (Krishna et al., 2012a).otably, a novel cyclodextrin formulation of posaconazole is underevelopment for intravenous (i.v) use. In a phase 1B study, the phar-acokinetics of two doses of i.v. posaconazole was investigated in

5 patient volunteers (Maertens et al., 2012). The higher protectivelood level of posaconazole was found for the 300 mg given onceaily, for which the average blood concentration at 14 days was

.43 mg/L. The minimum effective concentration was seen in 95% ofatients. Recently, Cornely et al. reported that 300 mg posaconazole

.v. was well tolerated and resulted in higher exposure comparedo the oral suspension (Cornely et al., 2013). A lowest mean Cmin

ft) and posaconazole (right) and plasma trough concentration in humans.

value of 1297 mg/L was achieved for posaconazole i.v 300 mg vs.751 mg/L for posaconazole oral suspension. Although our calcula-tions indicate that a posaconazole exposure of ≥3.33 mg/L wouldbe required to treat infection due to isolates with a posaconazoleMIC of 0.5 mg/L, we believe that this might be achievable using thei.v. formulation. Given that a significant proportion of isolates har-boring an azole resistance mechanism exhibit a posaconazole MICof 0.5 mg/L, this approach requires further investigation in experi-mental models.

8. Concluding remarks

The management of azole-resistant Aspergillus disease remainsa challenge. There are currently no guidelines or recommendationsthat guide clinicians confronted with azole resistance. Furthermore,pre-clinical or clinical evidence that support treatment choices isscarce. Experimental models of infection indicate that liposomal-amphotericin B may be effective (Seyedmousavi et al., 2013c), or acombination of voriconazole or posaconazole with an echinocan-din (Lepak et al., 2013a; Seyedmousavi et al., 2013a,d). In additionto the choice of antifungal regimen other important issues remainsuch as the early detection of azole resistance, especially in culturenegative patients. Also treatment regimens for patients with infec-tion in tissues that are difficult to reach, such as the brain, remainproblematic.

In our current review we explored the role of azole monother-apy in the management of azole-resistant aspergillosis. We believethat only a modest role of voriconazole and posaconazole remains,if any. Clearly, the use of an azole can only be considered in patientsthat fail alternative regimens or are intolerant to polyene therapy.Although it appears that voriconazole can be used to treat infec-tion due to isolates with a MIC of 2 mg/L, in isolates with a MIC of4 mg/L the risk of toxicity is significant. In this setting of dose esca-lation, intravenous administration, extensive monitoring of plasmalevels and close clinical and radiological follow-up is required. Atcurrent there appears to be no role for posaconazole in the treat-ment of isolates with a MIC outside the wild type population. Webelieve that this is due to the difficulty in achieving sufficient expo-

sure. However, we anticipate that adequate drug exposure mightbe achieved with the i.v. formulation that might allow treatmentof infections due to isolates with a MIC of 0.5 mg/L, although thispossibility would need to be explored in experimental models.

4 esista

C

ag

A

v

R

A

A

A

A

A

A

A

A

A

A

A

B

B

B

B

B

B

B

C

C

8 S. Seyedmousavi et al. / Drug R

onflicts of interest

S.S. and W.J.G.M. have no conflict of interests. R.J.M.B., J.W.M.nd P.E.V. have served as consultant to and have received researchrants from Gilead Sciences, Merck, Astellas and Pfizer.

cknowledgement

We thank William Hope for providing details of his work onoriconazole.

eferences

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