Posaconazole Pharmacodynamic Target Determination against WildType and Cyp51 Mutant Isolates of Aspergillus fumigatus in an In

Vivo 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 the
PD target of posaconazole in an immunocompromised murine model of IPA against 10 A. fumigatus isolates, including 4
Cyp51 wild-type isolates and 6 isolates carrying Cyp51 mutations conferring azole resistance. The posaconazole MIC range
was 0.25 to 8 mg/liter. Following infection, mice were given 0.156 to 160 mg/kg of body weight of oral posaconazole daily
for 7 days. Efficacy was assessed by quantitative PCR (qPCR) of lung homogenate and survival. At the start of therapy, mice
had 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 control
mice. A Hill-type dose response function was used to model the relationship between posaconazole free drug area under
the concentration-time curve (AUC)/MIC and qPCR lung burden. The static dose range was 1.09 to 51.9 mg/kg/24 h. The
free 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 results, 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 threshold of 0.25 to 0.5 mg/liter.

he incidence of invasive pulmonary aspergillosis (IPA) is increasing in parallel with a growing population of immunocompromised patients. Recent surveillance data of transplant patients identified this infection as the second most common fungal
pathogen in solid organ transplant recipients and the most common pathogen in bone marrow transplantation (1, 2). Despite the
development of new antifungal drugs with enhanced potency
against these organisms, morbidity and mortality associated with
IPA remain unacceptably high. Numerous factors have been
shown to impact treatment efficacy. One clinical factor under the
control of the clinician is the antifungal dosing regimen. Pharmacokinetic/pharmacodynamic (PK/PD) investigations have been
critical for defining the optimal antimicrobial exposure relative to
a measure of in vitro potency, the MIC (36).
Posaconazole is the most recently approved advanced-generation triazole with potent anti-Aspergillus activity (712).
Clinical efficacy has been demonstrated in the prevention and
treatment of IPA (1315). Furthermore, analysis of posaconazole concentration monitoring in these scenarios has suggested
a strong clinical concentration-efficacy relationship (1518).
However, definitive determination of the optimal dose and the
impact of variation in in vitro potency (MIC) remain unclear.
The recent emergence of Aspergillus fumigatus isolates exhibiting reduced triazole susceptibility underscores the potential
impact of these explorations (1922). The goals of the current
study were to utilize animal model pharmacodynamic approaches to define the optimal posaconazole exposure, discern
the impact of MIC variation associated with emerging resistant
A. fumigatus strains, and provide a basis for design of dosing
strategies to successfully treat infections due to these resistant
mutants.

January 2013 Volume 57 Number 1

MATERIALS AND METHODS

Organisms. Ten Aspergillus fumigatus isolates were chosen, including 9
clinical isolates with and without Cyp51 mutations and one laboratory
isolate with an Fks1 mutation. Organisms were grown and subcultured on
potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI). Only organisms with similar fitness, as determined by growth in lungs (see Table
1) and mortality (see Table 3) in untreated mice over 7 days, were chosen.
Drug. Posaconazole solution for in vivo studies was obtained from the
University of Wisconsin Hospital and Clinics pharmacy. Drug solutions
were prepared on the day of study using sterile saline as the diluent and
vortexed vigorously prior to administration by oral-gastric gavage. Posaconazole powder for in vitro susceptibility was obtained from Merck.
In vitro susceptibility. MICs were determined by broth microdilution
using the CLSI M38-A2 methods (23). MICs were performed in duplicate
three times; the median value is reported in Table 1.
Animals. Six-week-old Swiss/ICR specific-pathogen-free female mice
weighing 23 to 27 g were used for all studies (Harlan Sprague-Dawley,
Indianapolis, IN). Animals were housed in groups of five and allowed
access to food and water ad libitum. Animals were maintained in accordance with the American Association for Accreditation of Laboratory
Care criteria. The Animal Research Committee of the William S. Middleton Memorial VA Hospital and University of WisconsinMadison approved the animal studies.

Received 20 June 2012 Returned for modification 16 August 2012

Accepted 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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Lepak et al.

Infection model. Mice were rendered neutropenic (polymorphonuclear cells 100/mm3) by injection of 150 mg/kg of body weight cyclophosphamide subcutaneously (s.c.) on days 4, 1, and 3. Prior
studies have shown this to sustain neutropenia for the 7-day experiment
(2426). Additionally, cortisone acetate (250 mg/kg given s.c.) was administered on day 1 as previously described (25, 27, 28). Throughout
the 7-day neutropenic period, mice were also given ceftazidime, 50 mg/
kg/day s.c., to prevent opportunistic bacterial infection. Uninfected animals given the above immune suppression and antibiotic had 100% survival to study endpoint (data not shown).
Organisms were subcultured on PDA 5 days prior to infection and
incubated at 37C. On the day of infection, the inoculum was prepared by
flooding the culture plate with 5 ml of normal saline with 0.05% Tween
20. Gentle agitation was applied to release the conidia into the fluid. The
conidial suspension was collected and quantitated by using a hemacytometer (Bright-Line; Hausser Scientific, Horsham PA). The suspension was
diluted to a final concentration of 1 107 to 2 107 conidia/ml. Viability
was confirmed by plating the suspension on PDA and determining CFU.
An aspiration pneumonia model was utilized. Mice were anesthetized
with a combination of ketamine and xylazine. Fifty microliters of a 1
107 to 2 107 conidial suspension was pipetted into the anterior nares
and aspirated into the lungs. The procedure produced invasive aspergillosis in more than 90% of animals and 100% mortality in untreated infected mice by day 3 or 4.
Lung processing and organism quantitation. Processing and quantitation of the lung burden were performed as previously described (29, 30).
Briefly, at the time of sacrifice for moribund animals or at the end of
therapy (7 days), lungs were aseptically removed and placed in a 2-ounce
sterile polyethylene Whirl-Pak bag (Nasco, Fort Atkinson, WI) containing 2 ml of sterile saline. The lungs were manually homogenized using
direct pressure (31). One milliliter of the primary homogenate was placed
in a sterile bead beating tube (Sarstedt, Newton, NC) with 700 l of 425 to
600 m acid-washed glass beads (Sigma-Aldrich, St. Louis, MO). The
primary 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 with
100 l of buffer ATL (Qiagen, Valencia, CA) and 20 l of proteinase K
(Qiagen, Valencia, CA) and incubated overnight at 56C with gentle agitation. DNA was then isolated following the DNeasy Blood and Tissue
protocol (Qiagen, Valencia, CA). A final DNA elution step was carried out
with a 100-l volume. The DNA was stored at 20C until the day of
quantitative PCR (qPCR).
qPCR plates were prepared on the day of the assay. Standard amounts
of conidia were prepared by hemacytometer counts and were used for
generating standard curves. The results of qPCR are therefore reported as
conidial equivalents (CE) per ml of primary lung homogenate. Samples
were assayed in triplicate using a Bio-Rad CFX96 real-time system (Hercules, CA). A single-copy gene, Fks1, was chosen for quantitation (32).
Primer sequences included the following: forward primer (5=-GCCTGG
TAGTGAAGCTGAGCGT-3=), reverse primer (5=-CGGTGAATGTAGG
CATGTTGTCC-3=), and probe (6-carboxyfluorescein [FAM]-AGCCAGCGGCCCGCAAATG-MGB-3=) (Integrated DNA Technologies,
Coralville, IA). The fks1 mutation (EMFR S678P) was not located in the
primer-probe area of the genome and did not affect the quantitation reaction for that isolate (data not shown). The primer-probe set was validated for all isolates by determining the kinetics and quantitative results
for known quantities of conidia over the dynamic range (102 to 108) (data
not shown). Additionally, conidium-spiked uninfected lung homogenate
was used to test for the presence of PCR inhibitors that may adversely
affect qPCR results (data not shown).
Pharmacokinetics. Murine posaconazole pharmacokinetic data, including the area under the concentration-time curve (AUC) and protein
binding, were derived from our previous study of this animal model (33).
Pharmacodynamic index and magnitude. The AUC/MIC ratio was
used as the PD index for exploration of exposure-response relationships

580 aac.asm.org

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

fumigatus isolates
A. fumigatus
isolate

Posaconazole
MIC
(mg/liter)

In vivo
fitnessa

AF41
AF293
DPL EC S 1
EMFR S678P

0.25
0.50
0.25
0.25

1.53
1.35
1.98
1.61

F11628
F14403
F16216
AF72
F14532
F13747

8
8
2
2
1
2

1.71
1.5
1.22
1.88
1.77
1.21

Comment
Wild type
Wild type
Wild type
Fks1 S678P (echinocandin
MIC 16 mg/liter)
Cyp51 G138C
Cyp51 G54R
Cyp 51 L98H TR
Cyp51 G54E
Cyp51 M220T
Cyp51 G434C

a
Defined as the growth, measured in log10 CE/ml of lung homogenate, of the isolate in
untreated animals.

based upon previous PK/PD investigations (3335). Both total and free
(not protein-bound) concentrations were considered. Neutropenic mice
were infected as described above. Treatment consisted of 0.156 to 160
mg/kg/24 h of posaconazole administered once daily for 7 days by oral
gavage (OG). The doses were selected to vary the effect from maximal to
no efficacy. Controls were utilized for each isolate and included a zerohour and untreated control groups at the end of the study period. Four
mice 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-type
dose 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 and
represented as CE/ml of lung homogenate) in untreated control mice,
Emax is the maximal effect, N is the slope of the dose-response relationship, 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 determined for each organism and associated drug exposure. The coefficient of
determination (R2) was used to estimate the percent variance in the
change of log10 CE/ml of lung homogenate over the treatment period for
the different dosing regimens that could be attributed to the PD index,
AUC/MIC. The dose necessary for net stasis (static dose) and 1-log kill
and the associated PD targets total and free drug AUC/MIC associated
with these endpoints were determined. The PD targets were compared
between wild-type and Cyp51 mutants by using the t test for normally
distributed data and by using the Mann-Whitney rank sum test for nonnormally distributed data.
Survival. Survival to the end of the study period (7 days) was also
recorded for each group. A laboratory technician not aware of the study
design or expected results was responsible for determining the time of
sacrifice of moribund animals in accordance with accepted laboratory
standards for the humane treatment of research animals (American Association for Accreditation of Laboratory Care criteria). Survival in different groups was compared by t test for normally distributed data and by
Mann-Whitney rank sum test for non-normally distributed data. Logistic
regression was also performed using survival as the outcome using the
software program Sigma Stat (Systat Software, Inc., San Jose, CA).

RESULTS

Organism susceptibility and in vivo fitness. Posaconazole susceptibility testing, genetic mutations where applicable, and the
relative fitness in the in vivo murine model of each isolate are
shown in Table 1. Cyp51 wild-type MICs ranged from 0.25 to 0.5
mg/liter and from 1 to 8 mg/liter in Cyp51 mutants. The organisms exhibited similar in vivo fitnesses. At the start of therapy, mice

Antimicrobial Agents and Chemotherapy

Aspergillus Posaconazole Pharmacodynamic Target

FIG 1 Dose-response curves for each isolate are shown, with solid symbols representing wild-type Cyp51 organisms and open symbols Cyp51 mutants. Mice

were 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.

had 5.59 0.19 log10 CE/ml of lung homogenate, and the infectious burden increased to 7.11 0.29 log10 CE/ml of lung homogenate in untreated animals. Each isolate produced 100% mortality
prior to the end of the study in untreated animals (see Table 3).
Pharmacokinetics. Data from our prior PK study of posaconazole in this mouse model were used for the current study
(33). The AUC over the dose range was linear. Thus, for dose levels
that were not directly measured, the AUC was estimated using
linear extrapolation or interpolation. The total drug AUC range
was 1.78 to 800 mg h/liter over the dose range of 0.156 to 160
mg/kg/24 h. Protein binding was 99%.
Dose-response curves. A sigmoid dose-response relationship
was observed for each isolate, and higher doses were necessary to
achieve similar outcomes against organisms with elevated posaconazole MICs (Fig. 1). A net static outcome was observed with
all 10 isolates, a 1-log kill was achieved against 9 isolates (3 of 4
wild-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 drug
exposure producing a 2 to 3 log10 change in antimicrobial effect.
PD index and target. The dose and AUC/MIC needed to produce growth suppression compared to the start of therapy (static
dose) and the regimens associated with a 1-log reduction in organism burden (1-log kill) for each isolate are reported in Table 2.
The static dose and 1-log kill dose (when achieved) in Cyp51 wildtype organisms ranged from 1.09 to 2.16 mg/kg/24 h and 2.28 to
4.22 mg/kg/24 h, respectively. Comparative values for the Cyp51
mutant group were much higher, at 14.5 to 51.9 mg/kg/24 h and
22.4 to 150 mg/kg/24 h, respectively. The differences for both
static dose and 1-log kill dose (mg/kg) between wild-type and
mutant groups were statistically significant (P 0.01 and 0.04,
respectively). The total and free drug AUC/MIC PD targets, however, were comparable among this diverse organism group. While
the posaconazole exposure associated with these endpoints

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. fumigatus
isolatea
A. fumigatus
isolate

Static
dose (mg/kg/24 h)

Posaconazole
MIC (mg/liter)

AUCt/MIC

fAUC/MIC

1-log kill
dose (mg/kg/24 h)

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

1.68
1.94
2.16
1.09
51.39
51.93
46.53
33.89
14.51
46.15

0.25
0.50
0.25
0.25
8
8
2
2
1
2

76.41
44.30
98.50
49.71
52.63
53.05
195.59
156.71
165.42
194.41

0.76
0.44
0.99
0.50
0.53
0.53
1.96
1.57
1.65
1.94

4.22
NA
3.46
2.28
150.33
125.25
104.91
92.76
22.38
108.67

Mean
Median
SD

25.13
24.20
22.83

108.67
87.45
62.75

1.09
0.87
0.63

68.25
92.76
59.50

AUCt/MIC

fAUC/MIC

192.58

1.93

157.59
103.82
96.89
88.95
330.05
314.66
242.65
334.81

1.58
1.04
0.97
0.89
3.30
3.15
2.43
3.35

206.89
192.58
102.28

2.07
1.93
1.02

NA, not achieved; AUCt, total drug AUC; fAUC, free drug AUC; SD, standard deviation.

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Lepak et al.

FIG 2 The free drug AUC/MIC and microbiological effect are plotted for each
of the 10 A. fumigatus isolates tested. Solid symbols denote wild-type Cyp51
organisms, and open symbols denote Cyp51 mutants. The horizontal dashed
line 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 horizontal
dotted lines represent 1- and 2-log kills, respectively. The coefficient of determination (R2) based on the Hill equation is shown in the upper corner with
associated PD parameters, including Emax, ED50, and slope (N).

based upon dose in mg/kg varied nearly 50-fold, expression of

the exposure as AUC/MIC for the same endpoints varied only
4-fold. This supports the relevance of the AUC PD index and
even more so the MIC.
The mean free drug AUC/MIC associated with net stasis was
0.67 for the wild-type group and 1.36 for the Cyp51 mutant group.
This difference was not statistically significant (P 0.09). Similarly, the 1-log-kill free drug AUC/MIC target was numerically
higher for Cyp51 mutants (mean of 1.52 for the wild type versus
2.35 for the mutant group) but was not statistically significant
(P 0.28). The PD target free drug AUC/MIC for a 1-log kill was
roughly 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 sigmoid-dose-response model, and the relationship is shown in Fig.
2. The free drug AUC/MIC was a robust predictor of the observed
outcome based upon a high coefficient of determination (R2
0.79).
Survival. Survival to the end study endpoint mirrored the
qPCR results, with higher survival rates (50 to 100%) when higher
doses of drug were administered and uniform fatality with very

low concentrations (Table 3). In the wild-type group, the average

survival rate was 89.6% in animals administered 2.5 mg/kg/24 h
of posaconazole; however, animals that received less than this had
an average survival rate of only 18.8%. As expected, the dosage
breakpoint that correlated with significant differences in the survival rate for the Cyp51 mutants was shifted higher. In this group,
the average survival rate was 70.6% in animals administered 10
mg/kg/24 h of posaconazole but an average of only 10.9% in animals that received 10 mg/kg/24 h. The differences in survival
using the above dosing cutoffs were statistically significant by the
Mann-Whitney rank sum test (P 0.001). Survival was assessed
for each dosage regimen, and differences in effects for wild-type
versus Cyp51 mutant groups were explored. Large differences in
survival between the two organism groups were seen at the 2.5mg/kg/24 h dose. At this dose, survival was 100% in the wild-type
group versus 20.8% in the mutant group (P 0.01). The free drug
AUC at this dose level is approximately 0.3, and the free drug
AUC/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 a
static effect based upon qPCR (free AUC/MIC, 0.44 to 0.99). The
free AUC/MIC for the CYP51 mutant group would be only 0.04 to
0.3. These values did not produce appreciable efficacy using the
qPCR endpoint. Finally, logistic regression was also performed
using survival as the outcome. There was a statistically significant
17% increase in survival associated with every 1-log10 decrease in
burden as measured by qPCR. The Hosmer-Lemeshow test
showed a good model fit, and the difference in exposure associated
with survival and death was statistically significant (P 0.0001).
DISCUSSION

Invasive aspergillosis is a devastating infection for the immunocompromised host (1, 2, 3640). The development of new-generation triazoles, such as posaconazole and voriconazole, has improved the ability to prevent and treat these infections. Despite
this therapeutic advance, up to half of patients continue to succumb to progressive aspergillosis. Numerous factors account for
treatment failure, including persistent host immunodeficiency,
late diagnosis, and inadequate antifungal exposure. Insufficient
drug exposure can be due to an inadequate dose level, pharmacokinetic variability, and more recently the development of Aspergillus species triazole resistance (1922). Determining the optimal
antifungal exposure is requisite for addressing these pharmacologic shortfalls. Animal model pharmacokinetic/pharmacodynamic investigation has proven useful for designing optimal dosing regimens and delineating resistance levels which can be

TABLE 3 Animal survivala for each A. fumigatus isolate to end of study (7 days) for a given posaconazole daily dose
% survival
Posaconazole dose (mg/kg/24 h)
160
40
10
2.5
0.625
0.156
None (untreated controls)
a

AF41
100
75
100
50
0
0

AF293
100
75
100
50
0
0

DPL EC S 1
75
100
100
0
0
0

EMFR S678P

F11628

F14403

F16216

AF72

100
50
100
50
0
0

50
100
100
0
0
0
0

50
75
75
25
0

50
75
25
0
0

50
75
100
25
0
0
0

F14532

F13747

100
100
50
0
25
0

50
100
25
25
25
0
0

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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Antimicrobial Agents and Chemotherapy

Aspergillus Posaconazole Pharmacodynamic Target

overcome with dose adjustments (36). These approaches have

been underutilized for filamentous fungal infections.
Results from the current studies demonstrated a strong relationship between dose and effect for a triazole in therapy of invasive pulmonary aspergillosis. Furthermore, across the relatively
large group of organisms included in the present experiments,
efficacy was also closely linked to the MIC. More posaconazole, on
a mg/kg basis, was required for efficacy against organisms with
reduced in vitro susceptibility. The AUC/MIC index was utilized
as the PK/PD index for subsequent exposure-response analysis
based upon the previous in vivo models and clinical results for
invasive candidiasis and aspergillosis (15, 3335, 4155). This index provided a useful measure of exposure for modeling the present data using a sigmoid Emax model.
The primary treatment endpoint chosen for these studies was
quantitative lung PCR. The rationale for this choice was based
upon the relatively large dynamic range between effective and ineffective therapy and reproducibility among biologic replicates.
Our experience with this measure was similar to that previously
reported (5659). The quantitative burden of Aspergillus fumigatus in mouse lungs over the treatment range was closely related to
animal survival over the study period. However, we did find that
survival was less sensitive at detecting changes in microbiological
efficacy. When the survival data were fit to a sigmoid Hill model
examining the relationship of the free drug AUC/MIC ratio
(fAUC/MIC) to survival, the relationship was strong but less than
with the qPCR endpoint (R2 of 0.63 with survival endpoint, compared to 0.79 for qPCR).
It is unclear which qPCR endpoint in this infection model
might correlate with optimal treatment effect in patients. Treatment results from a similar immunocompromised model of invasive candidiasis using an ED50 endpoint have correlated well with
patient survival and clinical success (44, 45, 47, 50, 52, 53, 55). We
report the posaconazole AUC/MIC associated with a net stasis or
inhibitory endpoint, as well as that necessary to produce a further
1-log10 CE/ml reduction in organ burden. While the dose associated with these endpoints varied nearly 50-fold across the group of
Aspergillus isolates, the AUC/MIC varied only 4-fold. The unbound AUC/MIC values associated with the stasis and killing endpoints were 1.09 and 2.07, respectively. We were somewhat surprised at the relatively low values for these estimates compared to
those demonstrated for multiple triazole antifungals in similar
animal models and clinical trials of invasive candidiasis. The
AUC/MIC PD targets identified in the present study are more than
10-fold lower than those for disseminated candidiasis for posaconazole in a similar immunocompromised murine model
(33). The basis for these differences is not evident but clearly is a
fertile area for future mechanistic investigations. Previous PK/PD
investigation with posaconazole and other triazoles in experimental aspergillosis have also explored the question of PD index magnitude (34, 35). We are encouraged to observe congruence in the
PK/PD exposure-response relationships across animal models
and laboratories. The posaconazole AUC/MIC associated with
50% of maximal effect in a similar model with a single A. fumigatus isolate was a free drug ratio of 1.67, compared to the present
study value of 1.76 (34). Similarly, the free drug AUC/MIC needed
to protect 50% of mice from mortality in an acute disseminated
aspergillosis model for three A. fumigatus strains was a value of 3.2
(35).
An additional question probed by the present study includes

January 2013 Volume 57 Number 1

delineating the impact of MIC variation due to the majority of

defined mutations in the gene conferring resistance in the triazole
target. The observations were similar to that described for other
antimicrobial agents, such as quinolones with pneumococci compared to Gram-negative bacilli (60, 61). Principally, MIC is a relatively robust predictor of efficacy for susceptible and resistant
strains across resistance mechanisms.
Experimental PK/PD analyses have been shown to be useful for
predicting clinical outcomes. If one considers posaconazole pharmacokinetics in patients and the MIC distribution for Aspergillus
fumigatus isolates from surveillance studies, one would predict
treatment success for the majority of patients. The kinetics of the
current formulation using a regimen of 200 mg every 6 h given
with a high-fat meal would be expected to produce an AUC exposure 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 clinical
outcome, the highest MIC for which the current posaconazole
formulation and regimen would be predicted to produce a favorable outcome is 0.5 g/ml. For the 1-log-kill endpoint, this value
would shift a single dilution lower (0.25 g/ml). The single clinical
posaconazole experience for which concentration monitoring was
available identified the optimal plasma concentration associated
with clinical efficacy as a value of 1.25 g/ml (15). Unfortunately, organism MICs were not available for additional PK/PD
analysis. Examination of the outcomes of future clinical investigations of the present and new formulations of posaconazole in
treatment of invasive aspergillosis will be important to explore
these experimental PK/PD predictions. In the absence of this clinical evidence, the present studies should be used to guide preliminary susceptibility breakpoint determination.
ACKNOWLEDGMENT
We thank David Perlin for providing Aspergillus isolates DPL EC S 1 and
EMFR S678P.

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