Posaconazole Pharmacodynamic Target Determination against Wild-Type and Cyp51 Mutant Isolates of Aspergillus fumigatus in an InVivo Model of Invasive Pulmonary Aspergillosis

Alexander J. Lepak, Karen Marchillo, Jaimie VanHecker, David R. Andes

University of Wisconsin, Madison, Wisconsin, USA

Invasive pulmonary aspergillosis (IPA) is a devastating disease of immunocompromised patients. Pharmacodynamic (PD)examination of antifungal drug therapy in IPA is one strategy that may improve outcomes. The current study explored thePD target of posaconazole in an immunocompromised murine model of IPA against 10 A. fumigatus isolates, including 4Cyp51 wild-type isolates and 6 isolates carrying Cyp51 mutations conferring azole resistance. The posaconazole MIC rangewas 0.25 to 8 mg/liter. Following infection, mice were given 0.156 to 160 mg/kg of body weight of oral posaconazole dailyfor 7 days. Efficacy was assessed by quantitative PCR (qPCR) of lung homogenate and survival. At the start of therapy, micehad 5.59 � 0.19 log10 Aspergillus conidial equivalents (CE)/ml of lung homogenate, which increased to 7.11 � 0.29 log10

CE/ml of lung homogenate in untreated animals. The infection was uniformly lethal prior to the study endpoint in controlmice. A Hill-type dose response function was used to model the relationship between posaconazole free drug area underthe concentration-time curve (AUC)/MIC and qPCR lung burden. The static dose range was 1.09 to 51.9 mg/kg/24 h. Thefree drug AUC/MIC PD target was 1.09 � 0.63 for the group of strains. The 1-log kill free drug AUC/MIC was 2.07 � 1.02.The PD target was not significantly different for the wild-type and mutant organism groups. Mortality mirrored qPCR re-sults, with the greatest improvement in survival noted at the same dosing regimens that produced static or cidal activity.Consideration of human pharmacokinetic data and the current static dose PD target would predict a clinical MIC thresh-old of 0.25 to 0.5 mg/liter.

The incidence of invasive pulmonary aspergillosis (IPA) is in-creasing in parallel with a growing population of immuno-

compromised patients. Recent surveillance data of transplant pa-tients identified this infection as the second most common fungalpathogen in solid organ transplant recipients and the most com-mon pathogen in bone marrow transplantation (1, 2). Despite thedevelopment of new antifungal drugs with enhanced potencyagainst these organisms, morbidity and mortality associated withIPA remain unacceptably high. Numerous factors have beenshown to impact treatment efficacy. One clinical factor under thecontrol of the clinician is the antifungal dosing regimen. Pharma-cokinetic/pharmacodynamic (PK/PD) investigations have beencritical for defining the optimal antimicrobial exposure relative toa measure of in vitro potency, the MIC (3–6).

Posaconazole is the most recently approved advanced-gen-eration triazole with potent anti-Aspergillus activity (7–12).Clinical efficacy has been demonstrated in the prevention andtreatment of IPA (13–15). Furthermore, analysis of posacona-zole concentration monitoring in these scenarios has suggesteda strong clinical concentration-efficacy relationship (15–18).However, definitive determination of the optimal dose and theimpact of variation in in vitro potency (MIC) remain unclear.The recent emergence of Aspergillus fumigatus isolates exhibit-ing reduced triazole susceptibility underscores the potentialimpact of these explorations (19–22). The goals of the currentstudy were to utilize animal model pharmacodynamic ap-proaches to define the optimal posaconazole exposure, discernthe impact of MIC variation associated with emerging resistantA. fumigatus strains, and provide a basis for design of dosingstrategies to successfully treat infections due to these resistantmutants.

MATERIALS AND METHODS

Organisms. Ten Aspergillus fumigatus isolates were chosen, including 9clinical isolates with and without Cyp51 mutations and one laboratoryisolate with an Fks1 mutation. Organisms were grown and subcultured onpotato dextrose agar (PDA) (Difco Laboratories, Detroit, MI). Only or-ganisms with similar fitness, as determined by growth in lungs (see Table1) and mortality (see Table 3) in untreated mice over 7 days, were chosen.

Drug. Posaconazole solution for in vivo studies was obtained from theUniversity of Wisconsin Hospital and Clinics pharmacy. Drug solutionswere prepared on the day of study using sterile saline as the diluent andvortexed vigorously prior to administration by oral-gastric gavage. Po-saconazole powder for in vitro susceptibility was obtained from Merck.

In vitro susceptibility. MICs were determined by broth microdilutionusing the CLSI M38-A2 methods (23). MICs were performed in duplicatethree times; the median value is reported in Table 1.

Animals. Six-week-old Swiss/ICR specific-pathogen-free female miceweighing 23 to 27 g were used for all studies (Harlan Sprague-Dawley,Indianapolis, IN). Animals were housed in groups of five and allowedaccess to food and water ad libitum. Animals were maintained in accor-dance with the American Association for Accreditation of LaboratoryCare criteria. The Animal Research Committee of the William S. Middle-ton Memorial VA Hospital and University of Wisconsin—Madison ap-proved the animal studies.

Received 20 June 2012 Returned for modification 16 August 2012Accepted 7 November 2012

Published ahead of print 12 November 2012

Address correspondence to David R. Andes, dra@medicine.wisc.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.01279-12

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Infection model. Mice were rendered neutropenic (polymorphonu-clear cells � 100/mm3) by injection of 150 mg/kg of body weight cyclo-phosphamide subcutaneously (s.c.) on days �4, �1, and �3. Priorstudies have shown this to sustain neutropenia for the 7-day experiment(24–26). Additionally, cortisone acetate (250 mg/kg given s.c.) was ad-ministered on day �1 as previously described (25, 27, 28). Throughoutthe 7-day neutropenic period, mice were also given ceftazidime, 50 mg/kg/day s.c., to prevent opportunistic bacterial infection. Uninfected ani-mals given the above immune suppression and antibiotic had 100% sur-vival to study endpoint (data not shown).

Organisms were subcultured on PDA 5 days prior to infection andincubated at 37°C. On the day of infection, the inoculum was prepared byflooding the culture plate with 5 ml of normal saline with 0.05% Tween20. Gentle agitation was applied to release the conidia into the fluid. Theconidial suspension was collected and quantitated by using a hemacytom-eter (Bright-Line; Hausser Scientific, Horsham PA). The suspension wasdiluted to a final concentration of 1 � 107 to 2 � 107 conidia/ml. Viabilitywas confirmed by plating the suspension on PDA and determining CFU.

An aspiration pneumonia model was utilized. Mice were anesthetizedwith a combination of ketamine and xylazine. Fifty microliters of a 1 �107 to 2 � 107 conidial suspension was pipetted into the anterior naresand aspirated into the lungs. The procedure produced invasive aspergil-losis in more than 90% of animals and 100% mortality in untreated in-fected mice by day 3 or 4.

Lung processing and organism quantitation. Processing and quanti-tation of the lung burden were performed as previously described (29, 30).Briefly, at the time of sacrifice for moribund animals or at the end oftherapy (7 days), lungs were aseptically removed and placed in a 2-ouncesterile polyethylene Whirl-Pak bag (Nasco, Fort Atkinson, WI) contain-ing 2 ml of sterile saline. The lungs were manually homogenized usingdirect pressure (31). One milliliter of the primary homogenate was placedin a sterile bead beating tube (Sarstedt, Newton, NC) with 700 �l of 425 to600 �m acid-washed glass beads (Sigma-Aldrich, St. Louis, MO). Theprimary homogenate was bead beaten in a Bio-spec mini bead beater(Bartlesville, OK) for 90 s at 4,200 rpm to yield a secondary homogenate.One hundred microliters of the secondary homogenate was mixed with100 �l of buffer ATL (Qiagen, Valencia, CA) and 20 �l of proteinase K(Qiagen, Valencia, CA) and incubated overnight at 56°C with gentle agi-tation. DNA was then isolated following the DNeasy Blood and Tissueprotocol (Qiagen, Valencia, CA). A final DNA elution step was carried outwith a 100-�l volume. The DNA was stored at �20°C until the day ofquantitative PCR (qPCR).

qPCR plates were prepared on the day of the assay. Standard amountsof conidia were prepared by hemacytometer counts and were used forgenerating standard curves. The results of qPCR are therefore reported asconidial equivalents (CE) per ml of primary lung homogenate. Sampleswere assayed in triplicate using a Bio-Rad CFX96 real-time system (Her-cules, CA). A single-copy gene, Fks1, was chosen for quantitation (32).Primer sequences included the following: forward primer (5=-GCCTGGTAGTGAAGCTGAGCGT-3=), reverse primer (5=-CGGTGAATGTAGGCATGTTGTCC-3=), and probe (6-carboxyfluorescein [FAM]-AGC-CAGCGGCCCGCAAATG-MGB-3=) (Integrated DNA Technologies,Coralville, IA). The fks1 mutation (EMFR S678P) was not located in theprimer-probe area of the genome and did not affect the quantitation re-action for that isolate (data not shown). The primer-probe set was vali-dated for all isolates by determining the kinetics and quantitative resultsfor known quantities of conidia over the dynamic range (102 to 108) (datanot shown). Additionally, conidium-spiked uninfected lung homogenatewas used to test for the presence of PCR inhibitors that may adverselyaffect qPCR results (data not shown).

Pharmacokinetics. Murine posaconazole pharmacokinetic data, in-cluding the area under the concentration-time curve (AUC) and proteinbinding, were derived from our previous study of this animal model (33).

Pharmacodynamic index and magnitude. The AUC/MIC ratio wasused as the PD index for exploration of exposure-response relationships

based upon previous PK/PD investigations (33–35). Both total and free(not protein-bound) concentrations were considered. Neutropenic micewere infected as described above. Treatment consisted of 0.156 to 160mg/kg/24 h of posaconazole administered once daily for 7 days by oralgavage (OG). The doses were selected to vary the effect from maximal tono efficacy. Controls were utilized for each isolate and included a zero-hour and untreated control groups at the end of the study period. Fourmice were used for each group (zero-hour control, no-treatment control,and each dosing regimen).

Data analysis. The qPCR data were modeled according to a Hill-typedose response equation: log10 D � log10 (E/Emax � E)/N � log10 ED50,where D is the drug dose, E is the growth (as measured by qPCR andrepresented as CE/ml of lung homogenate) in untreated control mice,Emax is the maximal effect, N is the slope of the dose-response relation-ship, and ED50 is the dose needed to achieve 50% of the maximal effect.The AUC/MIC for total and free (non-protein-bound) drug was deter-mined for each organism and associated drug exposure. The coefficient ofdetermination (R2) was used to estimate the percent variance in thechange of log10 CE/ml of lung homogenate over the treatment period forthe different dosing regimens that could be attributed to the PD index,AUC/MIC. The dose necessary for net stasis (static dose) and 1-log killand the associated PD targets total and free drug AUC/MIC associatedwith these endpoints were determined. The PD targets were comparedbetween wild-type and Cyp51 mutants by using the t test for normallydistributed data and by using the Mann-Whitney rank sum test for non-normally distributed data.

Survival. Survival to the end of the study period (7 days) was alsorecorded for each group. A laboratory technician not aware of the studydesign or expected results was responsible for determining the time ofsacrifice of moribund animals in accordance with accepted laboratorystandards for the humane treatment of research animals (American As-sociation for Accreditation of Laboratory Care criteria). Survival in dif-ferent groups was compared by t test for normally distributed data and byMann-Whitney rank sum test for non-normally distributed data. Logisticregression was also performed using survival as the outcome using thesoftware program Sigma Stat (Systat Software, Inc., San Jose, CA).

RESULTSOrganism susceptibility and in vivo fitness. Posaconazole sus-ceptibility testing, genetic mutations where applicable, and therelative fitness in the in vivo murine model of each isolate areshown in Table 1. Cyp51 wild-type MICs ranged from 0.25 to 0.5mg/liter and from 1 to 8 mg/liter in Cyp51 mutants. The organ-isms exhibited similar in vivo fitnesses. At the start of therapy, mice

TABLE 1 In vitro susceptibilities and in vivo fitnesses of select A.fumigatus isolates

A. fumigatusisolate

PosaconazoleMIC(mg/liter)

In vivofitnessa Comment

AF41 0.25 1.53 Wild typeAF293 0.50 1.35 Wild typeDPL EC S 1 0.25 1.98 Wild typeEMFR S678P 0.25 1.61 Fks1 S678P (echinocandin

MIC � 16 mg/liter)F11628 8 1.71 Cyp51 G138CF14403 8 1.5 Cyp51 G54RF16216 2 1.22 Cyp 51 L98H � TRAF72 2 1.88 Cyp51 G54EF14532 1 1.77 Cyp51 M220TF13747 2 1.21 Cyp51 G434Ca Defined as the growth, measured in log10 CE/ml of lung homogenate, of the isolate inuntreated animals.

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had 5.59 � 0.19 log10 CE/ml of lung homogenate, and the infec-tious burden increased to 7.11 � 0.29 log10 CE/ml of lung homog-enate in untreated animals. Each isolate produced 100% mortalityprior to the end of the study in untreated animals (see Table 3).

Pharmacokinetics. Data from our prior PK study of po-saconazole in this mouse model were used for the current study(33). The AUC over the dose range was linear. Thus, for dose levelsthat were not directly measured, the AUC was estimated usinglinear extrapolation or interpolation. The total drug AUC rangewas 1.78 to 800 mg · h/liter over the dose range of 0.156 to 160mg/kg/24 h. Protein binding was 99%.

Dose-response curves. A sigmoid dose-response relationshipwas observed for each isolate, and higher doses were necessary toachieve similar outcomes against organisms with elevated po-saconazole MICs (Fig. 1). A net static outcome was observed withall 10 isolates, a 1-log kill was achieved against 9 isolates (3 of 4wild-type isolates and 6 of 6 mutants), and for 6 strains (3 of 4

wild-type strains and 3 of 6 mutants), a 2-log kill was observed.The dose-response curves were steep, with a 4-fold change in drugexposure producing a 2 to 3 log10 change in antimicrobial effect.

PD index and target. The dose and AUC/MIC needed to pro-duce growth suppression compared to the start of therapy (staticdose) and the regimens associated with a 1-log reduction in or-ganism burden (1-log kill) for each isolate are reported in Table 2.The static dose and 1-log kill dose (when achieved) in Cyp51 wild-type organisms ranged from 1.09 to 2.16 mg/kg/24 h and 2.28 to4.22 mg/kg/24 h, respectively. Comparative values for the Cyp51mutant group were much higher, at 14.5 to 51.9 mg/kg/24 h and22.4 to 150 mg/kg/24 h, respectively. The differences for bothstatic dose and 1-log kill dose (mg/kg) between wild-type andmutant groups were statistically significant (P � 0.01 and 0.04,respectively). The total and free drug AUC/MIC PD targets, how-ever, were comparable among this diverse organism group. Whilethe posaconazole exposure associated with these endpoints

FIG 1 Dose-response curves for each isolate are shown, with solid symbols representing wild-type Cyp51 organisms and open symbols Cyp51 mutants. Micewere given 0.156 mg/kg to 160 mg/kg of posaconazole once daily for 7 days. Each data point is the mean � SD in log10 CE/ml of lung homogenate for four mice.The horizontal dashed line represents the net stasis of burden from the start of therapy. Points above the line represent an increase in burden (i.e., net growth),whereas those below the line represent a decrease in burden.

TABLE 2 Dose and total and free drug AUC/MIC needed to achieve net stasis and 1-log kill endpoints (when achieved) for each A. fumigatusisolatea

A. fumigatusisolate

Staticdose (mg/kg/24 h)

PosaconazoleMIC (mg/liter) AUCt/MIC fAUC/MIC

1-log killdose (mg/kg/24 h) AUCt/MIC fAUC/MIC

AF41 1.68 0.25 76.41 0.76 4.22 192.58 1.93AF293 1.94 0.50 44.30 0.44 NADPL EC S 1 2.16 0.25 98.50 0.99 3.46 157.59 1.58EMFR S678P 1.09 0.25 49.71 0.50 2.28 103.82 1.04F11628 51.39 8 52.63 0.53 150.33 96.89 0.97F14403 51.93 8 53.05 0.53 125.25 88.95 0.89F16216 46.53 2 195.59 1.96 104.91 330.05 3.30AF72 33.89 2 156.71 1.57 92.76 314.66 3.15F14532 14.51 1 165.42 1.65 22.38 242.65 2.43F13747 46.15 2 194.41 1.94 108.67 334.81 3.35

Mean 25.13 108.67 1.09 68.25 206.89 2.07Median 24.20 87.45 0.87 92.76 192.58 1.93SD 22.83 62.75 0.63 59.50 102.28 1.02a NA, not achieved; AUCt, total drug AUC; fAUC, free drug AUC; SD, standard deviation.

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based upon dose in mg/kg varied nearly 50-fold, expression ofthe exposure as AUC/MIC for the same endpoints varied only4-fold. This supports the relevance of the AUC PD index andeven more so the MIC.

The mean free drug AUC/MIC associated with net stasis was0.67 for the wild-type group and 1.36 for the Cyp51 mutant group.This difference was not statistically significant (P � 0.09). Simi-larly, the 1-log-kill free drug AUC/MIC target was numericallyhigher for Cyp51 mutants (mean of 1.52 for the wild type versus2.35 for the mutant group) but was not statistically significant(P � 0.28). The PD target free drug AUC/MIC for a 1-log kill wasroughly 2-fold larger than the stasis endpoint (2.07 versus 1.09).The free drug AUC/MIC for all organisms was fit to the Hill sig-moid-dose-response model, and the relationship is shown in Fig.2. The free drug AUC/MIC was a robust predictor of the observedoutcome based upon a high coefficient of determination (R2 �0.79).

Survival. Survival to the end study endpoint mirrored theqPCR results, with higher survival rates (50 to 100%) when higherdoses of drug were administered and uniform fatality with very

low concentrations (Table 3). In the wild-type group, the averagesurvival rate was 89.6% in animals administered �2.5 mg/kg/24 hof posaconazole; however, animals that received less than this hadan average survival rate of only 18.8%. As expected, the dosagebreakpoint that correlated with significant differences in the sur-vival rate for the Cyp51 mutants was shifted higher. In this group,the average survival rate was 70.6% in animals administered �10mg/kg/24 h of posaconazole but an average of only 10.9% in ani-mals that received �10 mg/kg/24 h. The differences in survivalusing the above dosing cutoffs were statistically significant by theMann-Whitney rank sum test (P � 0.001). Survival was assessedfor each dosage regimen, and differences in effects for wild-typeversus Cyp51 mutant groups were explored. Large differences insurvival between the two organism groups were seen at the 2.5-mg/kg/24 h dose. At this dose, survival was 100% in the wild-typegroup versus 20.8% in the mutant group (P � 0.01). The free drugAUC at this dose level is approximately 0.3, and the free drugAUC/MIC for wild-type organisms would range from 0.6 to 1.2.This free AUC/MIC value range is similar to that associated with astatic effect based upon qPCR (free AUC/MIC, 0.44 to 0.99). Thefree AUC/MIC for the CYP51 mutant group would be only 0.04 to0.3. These values did not produce appreciable efficacy using theqPCR endpoint. Finally, logistic regression was also performedusing survival as the outcome. There was a statistically significant17% increase in survival associated with every 1-log10 decrease inburden as measured by qPCR. The Hosmer-Lemeshow testshowed a good model fit, and the difference in exposure associatedwith survival and death was statistically significant (P � 0.0001).

DISCUSSION

Invasive aspergillosis is a devastating infection for the immuno-compromised host (1, 2, 36–40). The development of new-gener-ation triazoles, such as posaconazole and voriconazole, has im-proved the ability to prevent and treat these infections. Despitethis therapeutic advance, up to half of patients continue to suc-cumb to progressive aspergillosis. Numerous factors account fortreatment failure, including persistent host immunodeficiency,late diagnosis, and inadequate antifungal exposure. Insufficientdrug exposure can be due to an inadequate dose level, pharmaco-kinetic variability, and more recently the development of Asper-gillus species triazole resistance (19–22). Determining the optimalantifungal exposure is requisite for addressing these pharmaco-logic shortfalls. Animal model pharmacokinetic/pharmacody-namic investigation has proven useful for designing optimal dos-ing regimens and delineating resistance levels which can be

FIG 2 The free drug AUC/MIC and microbiological effect are plotted for eachof the 10 A. fumigatus isolates tested. Solid symbols denote wild-type Cyp51organisms, and open symbols denote Cyp51 mutants. The horizontal dashedline represents the net stasis of infectious burden from the start of therapy.Points above the line represent an increase in burden (i.e., net growth),whereas those below the line represent a decrease in burden. The horizontaldotted lines represent 1- and 2-log kills, respectively. The coefficient of deter-mination (R2) based on the Hill equation is shown in the upper corner withassociated PD parameters, including Emax, ED50, and slope (N).

TABLE 3 Animal survivala for each A. fumigatus isolate to end of study (7 days) for a given posaconazole daily dose

Posaconazole dose (mg/kg/24 h)

% survival

AF41 AF293 DPL EC S 1 EMFR S678P F11628 F14403 F16216 AF72 F14532 F13747

160 50 50 50 50 5040 100 100 75 100 100 75 75 75 100 10010 75 75 100 50 100 75 25 100 100 252.5 100 100 100 100 0 25 0 25 50 250.625 50 50 0 50 0 0 0 0 0 250.156 0 0 0 0 0 0 25 0None (untreated controls) 0 0 0 0 0 0 0 0 0 0a The numbers in the table represent the percentages of animals in the group that survived to the end of the study.

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overcome with dose adjustments (3–6). These approaches havebeen underutilized for filamentous fungal infections.

Results from the current studies demonstrated a strong rela-tionship between dose and effect for a triazole in therapy of inva-sive pulmonary aspergillosis. Furthermore, across the relativelylarge group of organisms included in the present experiments,efficacy was also closely linked to the MIC. More posaconazole, ona mg/kg basis, was required for efficacy against organisms withreduced in vitro susceptibility. The AUC/MIC index was utilizedas the PK/PD index for subsequent exposure-response analysisbased upon the previous in vivo models and clinical results forinvasive candidiasis and aspergillosis (15, 33–35, 41–55). This in-dex provided a useful measure of exposure for modeling the pres-ent data using a sigmoid Emax model.

The primary treatment endpoint chosen for these studies wasquantitative lung PCR. The rationale for this choice was basedupon the relatively large dynamic range between effective and in-effective therapy and reproducibility among biologic replicates.Our experience with this measure was similar to that previouslyreported (56–59). The quantitative burden of Aspergillus fumiga-tus in mouse lungs over the treatment range was closely related toanimal survival over the study period. However, we did find thatsurvival was less sensitive at detecting changes in microbiologicalefficacy. When the survival data were fit to a sigmoid Hill modelexamining the relationship of the free drug AUC/MIC ratio(fAUC/MIC) to survival, the relationship was strong but less thanwith the qPCR endpoint (R2 of 0.63 with survival endpoint, com-pared to 0.79 for qPCR).

It is unclear which qPCR endpoint in this infection modelmight correlate with optimal treatment effect in patients. Treat-ment results from a similar immunocompromised model of inva-sive candidiasis using an ED50 endpoint have correlated well withpatient survival and clinical success (44, 45, 47, 50, 52, 53, 55). Wereport the posaconazole AUC/MIC associated with a net stasis orinhibitory endpoint, as well as that necessary to produce a further1-log10 CE/ml reduction in organ burden. While the dose associ-ated with these endpoints varied nearly 50-fold across the group ofAspergillus isolates, the AUC/MIC varied only 4-fold. The un-bound AUC/MIC values associated with the stasis and killing end-points were 1.09 and 2.07, respectively. We were somewhat sur-prised at the relatively low values for these estimates compared tothose demonstrated for multiple triazole antifungals in similaranimal models and clinical trials of invasive candidiasis. TheAUC/MIC PD targets identified in the present study are more than10-fold lower than those for disseminated candidiasis for po-saconazole in a similar immunocompromised murine model(33). The basis for these differences is not evident but clearly is afertile area for future mechanistic investigations. Previous PK/PDinvestigation with posaconazole and other triazoles in experimen-tal aspergillosis have also explored the question of PD index mag-nitude (34, 35). We are encouraged to observe congruence in thePK/PD exposure-response relationships across animal modelsand laboratories. The posaconazole AUC/MIC associated with50% of maximal effect in a similar model with a single A. fumiga-tus isolate was a free drug ratio of 1.67, compared to the presentstudy value of 1.76 (34). Similarly, the free drug AUC/MIC neededto protect 50% of mice from mortality in an acute disseminatedaspergillosis model for three A. fumigatus strains was a value of 3.2(35).

An additional question probed by the present study includes

delineating the impact of MIC variation due to the majority ofdefined mutations in the gene conferring resistance in the triazoletarget. The observations were similar to that described for otherantimicrobial agents, such as quinolones with pneumococci com-pared to Gram-negative bacilli (60, 61). Principally, MIC is a rel-atively robust predictor of efficacy for susceptible and resistantstrains across resistance mechanisms.

Experimental PK/PD analyses have been shown to be useful forpredicting clinical outcomes. If one considers posaconazole phar-macokinetics in patients and the MIC distribution for Aspergillusfumigatus isolates from surveillance studies, one would predicttreatment success for the majority of patients. The kinetics of thecurrent formulation using a regimen of 200 mg every 6 h givenwith a high-fat meal would be expected to produce an AUC expo-sure as high as 60 mg · h/liter (i.e., free AUC, 0.6 mg · h/liter) (62).If the stasis endpoint from the present study is relevant for clinicaloutcome, the highest MIC for which the current posaconazoleformulation and regimen would be predicted to produce a favor-able outcome is 0.5 �g/ml. For the 1-log-kill endpoint, this valuewould shift a single dilution lower (0.25 �g/ml). The single clinicalposaconazole experience for which concentration monitoring wasavailable identified the optimal plasma concentration associatedwith clinical efficacy as a value of �1.25 �g/ml (15). Unfortu-nately, organism MICs were not available for additional PK/PDanalysis. Examination of the outcomes of future clinical investiga-tions of the present and new formulations of posaconazole intreatment of invasive aspergillosis will be important to explorethese experimental PK/PD predictions. In the absence of this clin-ical evidence, the present studies should be used to guide prelim-inary susceptibility breakpoint determination.

ACKNOWLEDGMENT

We thank David Perlin for providing Aspergillus isolates DPL EC S 1 andEMFR S678P.

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