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Isavuconazole Is Effective for the Treatment of Experimental

Cryptococcal Meningitis
Nathan P. Wiederhold,a Laura Kovanda,b Laura K. Najvar,a,c Rosie Bocanegra,a,c Marcos Olivo,a,c William R. Kirkpatrick,a,c
Thomas F. Pattersona,c
University of Texas Health Science Center at San Antonio, San Antonio, Texas, USAa; Astellas Pharma Global Development, Inc., Northbrook, Illinois, USAb; South Texas
Veterans Health Care System, San Antonio, Texas, USAc

We evaluated the efficacy of isavuconazole against cryptococcal meningitis. Treatment with either oral isavuconazole (120 mg/kg
and 240 mg/kg twice a day [BID]) or fluconazole as the positive control significantly improved survival in mice infected intracra-
nially with either Cryptococcus neoformans USC1597 or H99 and significantly reduced brain fungal burdens for both isolates.
Concentrations of isavuconazole in plasma and brain tissue also demonstrated that the greatest improvements in survival and

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fungal burden were associated with elevated exposures.

I savuconazole is a drug with broad-spectrum antifungal activ-

ity that is approved for the treatment of invasive aspergillosis
and mucormycosis by the U.S. Food and Drug Administration.
TABLE 1 Mean model pharmacokinetic parameters for
isavuconazole
Parametera Mean SD
Although this agent has potent in vitro activity against Crypto- Ka (h⫺1) 12.95 7.699
coccus species (MIC50 and MIC90 values against Cryptococcus Vmax (mol/h) 1.63 1.442
neoformans of ⱕ0.015 and 0.06 ␮g/ml and against Cryptococcus Km (mol) 15.24 6.116
gattii of 0.03 and 0.06 ␮g/ml, respectively, per our experience) V (liters) 1.21 0.701
(1–5), data regarding the in vivo efficacy of isavuconazole Vb (liters) 1.82 1.379
against cryptococcosis are limited. The open-label, multicenter Kcp (h⫺1) 5.98 4.525
VITAL study had only 9 patients with infections caused by Kpc (h⫺1) 7.31 3.894
Kcb (h⫺1) 9.15 3.664
Cryptococcus species, including 3 patients with isolated pulmo-
Kbc (h⫺1) 4.49 2.139
nary disease and 2 with central nervous system [CNS]-only a
Ka, absorption rate constant; Vmax, maximum potential difference; Km, Michaelis-
infection (6). Overall, successful responses to therapy were ob- Menten constant; V, volume of the central compartment; Vb, volume of the brain
served in 6 of the patients who received isavuconazole, while compartment; Kcp and Kpc, rate constants for drug moving to and from the central
therapy failed in 3 patients. Our objective was to assess the in and peripheral compartments; Kcb and Kbc, rate constants for drug flow to and
vivo efficacy of isavuconazole using a murine model of crypto- from the central and brain compartments.
coccal meningitis.
Cryptococcus neoformans clinical isolate USC1597 and isolate
H99 were used in this study (7, 8). Isavuconazole demonstrated zonium sulfate) of 20, 40, 80, 120, and 240 mg/kg by oral gavage
potent in vitro activity, as measured by broth microdilution sus- twice daily. Isavuconazole concentrations were measured using
ceptibility testing according to the CLSI M27-A3 reference stan- an established liquid chromatography-mass spectrometry (LC/
dard (9), with MIC values of ⱕ0.03 ␮g/ml against both isolates. MS) assay (16, 17). Concentrations in plasma and brain tissue
The MICs for fluconazole against these isolates were 1 and 8 ␮g/ were modeled using the nonparametric estimation in Pmetrics
ml, respectively. software (18). Simulations were performed using ADAPT 5
An established murine model of cryptococcal meningoen- from mean model parameters to generate a drug concentra-
cephalitis was used to determine the in vivo effectiveness of tion-time profile and area under the curve (AUC) for each dose
isavuconazole (7, 8, 10, 11). This animal protocol was approved for both plasma and brain tissue. As nonlinear pharmacokinet-
by the Institutional Animal Care and Use Committee at the ics were observed, a 3-compartment Michaelis-Menten model
University of Texas Health Science Center at San Antonio, and fit the concentration data well. Mean model parameters (Table
all animals were maintained in accordance with the guidelines
of the Association for the Assessment and Accreditation of Lab-
oratory Animal Care. Immunocompetent outbred ICR mice Received 27 January 2016 Returned for modification 19 March 2016
(Harlan) were inoculated intracranially with 2,600 to 3,500 Accepted 15 June 2016
CFU/animal as previously described (8), and treatment by oral Accepted manuscript posted online 20 June 2016
gavage began 24 h after inoculation. Similar isavuconazole Citation Wiederhold NP, Kovanda L, Najvar LK, Bocanegra R, Olivo M, Kirkpatrick
WR, Patterson TF. 2016. Isavuconazole is effective for the treatment of
doses have shown efficacy in other animal models of invasive experimental cryptococcal meningitis. Antimicrob Agents Chemother
fungal infections (12–15). Isavuconazole concentrations were 60:5600 –5603. doi:10.1128/AAC.00229-16.
measured in infected mice. Plasma and brain tissue were col- Address correspondence to Nathan P. Wiederhold,
lected after the 11th dose from three mice per group at various wiederholdn@uthscsa.edu.
time points. Groups included isavuconazole equivalent doses Copyright © 2016, American Society for Microbiology. All Rights Reserved.
(1.88 conversion factor between isavuconazole and isavucona-

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 Isavuconazole for Cryptococcal Meningitis

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FIG 1 Simulated isavuconazole concentration-time profiles for brain tissue and plasma. Mean model parameters were used to generate drug concentration-time
profiles for brain tissue and plasma: (A) 20 mg/kg; (B) 40 mg/kg; (C) 80 mg/kg; (D) 120 mg/kg; (E) 240 mg/kg. Each dose was administered by oral gavage twice
daily.

1) were used to generate drug concentration-time profiles for centrations in the plasma and brain tissue were achieved
plasma and brain tissue (Fig. 1). The data demonstrated a lin- shortly after the last doses (2.4 and 4.8 ␮g/ml in the plasma and
ear increase in AUC up to the 120 mg/kg dose but became 8.4 and 17.3 ␮g/g in the brain tissue for the 120 mg/kg and 240
nonlinear thereafter (Table 2). This may be of significance as mg/kg dosages, respectively).
the AUC/MIC is the parameter associated with isavuconazole In both the survival and fungal burden arms, the in vivo
efficacy in other animal models of invasive fungal infections efficacy of isavuconazole was evaluated at doses of 120 and 240
(12, 13, 15, 19). The ratio of isavuconazole brain tissue to mg/kg twice daily and the in vivo efficacy of fluconazole at doses
plasma exposure was approximately 1.35 for each dose level. As of 20 and 40 mg/kg twice daily. These doses were chosen based
previously reported, the half-life of isavuconazole in this mu- on the pharmacokinetic results in order to achieve exposures
rine model was short (1.1 h) (19), and the trough levels were similar to that observed in the phase 3 clinical study (20). For
low to undetectable (data not shown). However, elevated con-
the positive control, fluconazole doses were chosen based on
our previous experience with this model (8, 11). In the fungal
TABLE 2 Estimated AUCs for each dose from the pharmacokinetic burden arm, mice were humanely euthanized on day 8 postin-
experiment oculation, and the brains were removed, weighed, and homog-
Dose (mg/kg BIDa) AUCplasma (␮g ⫻ h/ml) AUCbrain (␮g ⫻ h/ml) enized. Dilutions of each homogenate were prepared and
plated on Sabouraud dextrose agar (SDA) for incubation. After
20 7.9868 10.8042
40 16.3214 22.0776 72 h, colonies were counted and fungal burdens (CFU per gram
80 34.065 46.074 of tissue) were determined. In survival studies, mice were
120 53.286 72.064 treated for 10 days and then monitored off therapy until day 30
240 120.13 162.414 postinoculation.
a
BID, twice per day. Survival was significantly improved in mice infected with

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Wiederhold et al.

FIG 2 Survival to day 30 in mice with cryptococcal meningitis due to either USC1597 (A) or H99 (B) and treated with isavuconazole (ISA) or
fluconazole (FLU), both administered twice daily by oral gavage. Survival was plotted by Kaplan-Meier analysis, and differences in median survival and
the percent survival between groups were analyzed by the log-rank test and Fischer’s exact test, respectively. The median survival P value was ⬍0.05
for all groups compared to the control. Against USC1597, percent survival increase significantly (P ⬍ 0.05) for 240 mg/kg ISA and both fluconazole

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doses.

USC1597 and treated with isavuconazole (median survival of
28 and ⬎30 days and percent survival of 40% and 70% for the
120 mg/kg and 240 mg/kg dosages, respectively) compared to
the results for placebo (15.5 days and 0% survival) (Fig. 2A).
Similar results were also observed for the positive-control flu-
conazole (⬎30 days and 60% survival for both doses). Against
the H99 strain (Fig. 2B), survival was also increased in mice
treated with isavuconazole (22 and 23 days, respectively) com-
pared to that for placebo (15 days; P ⬍ 0.05). Similar results
were also observed with fluconazole (survival of 21 and 22 days,
respectively). However, percent survivals for isavuconazole
and fluconazole were not significantly different from that for
placebo (overall percent survival of 0 to 10%).
CFU counts were also significantly lower in mice infected
with the USC1597 isolate and treated with isavuconazole
(mean range, 1.6 to 2.87 log10 CFU/g) and fluconazole (2.10 to
2.11 log10 CFU/g) compared to that for placebo (6.58 log10
CFU/g) (Fig. 3A). Brain tissue fungal burdens were also re-
duced in mice infected with C. neoformans H99 and treated with
isavuconazole, but to a lesser degree (3.17 to 5.13 log10 CFU/g
versus 6.87 log10 CFU/g), which is consistent with the reduced
percent survival achieved with isavuconazole against this iso-
late. Similar results were also observed for fluconazole in that
the fungal burden was reduced when this azole was used to treat
mice infected with the H99 isolate, but to a lesser degree than
for mice infected with USC1597.
These results suggest that isavuconazole may have a role in
the treatment of cryptococcal meningitis, as improvements in
survival and reductions in fungal burden were observed in this
experimental model. Improvements in survival and reductions
in fungal burden were also greater with the higher dose, which
achieved higher isavuconazole exposures in the brain and
plasma. These in vivo results are consistent with the in vitro
potency of this agent (2–5, 21). Limitations of this study must
be considered. We did not evaluate isavuconazole in combina-
tion with another agent (amphotericin B or flucytosine), nor
did we assess the use of this agent as consolidation or mainte-
nance therapy in place of fluconazole following induction
treatment as currently recommended in the treatment guide-
lines (22). However, in the VITAL study, 6 of the 9 patients
with cryptococcosis also received isavuconazole as the primary
therapy (6). In addition, this is an immunocompetent model
and thus does not fully mimic the clinical setting where cryp-
tococcal meningitis is observed in immunocompromised indi-
viduals. However, this model has been shown to be beneficial in
evaluating new treatment strategies against cryptococcosis (7,
8, 10, 11). Thus, while our results are encouraging, further
studies are warranted.

FIG 3 Brain tissue fungal burden was assessed on day 8 postinoculation in mice with cryptococcal meningitis due to either USC1597 (A) or H99 (B) and treated
with isavuconazole (ISA) or fluconazole (FLU), and all groups were dosed twice daily by oral gavage. Differences in brain fungal burden (CFU per gram) were
assessed for significance by analysis of variance (ANOVA) with Tukey’s posttest for multiple comparisons. Mean log10 CFU/g P values were ⬍0.0001 for all
groups compared to controls.

5602 aac.asm.org Antimicrobial Agents and Chemotherapy September 2016 Volume 60 Number 9

ACKNOWLEDGMENTS
We thank Arlene Farias for her help with the animal model and Dora
McCarthy for assistance with the in vitro susceptibility testing.
N.P.W. has received research support from Astellas, bioMérieux,
Dow, F2G, Merck, Merz, Revolution Medicines, and Viamet and has
served on advisory boards for Astellas, Merck, Toyama, and Viamet.
T.F.P. has received research grants to the UT Health Science Center at San
Antonio from Astellas, Merck, and Revolution Medicines and has served
as a consultant for Amplyx, Astellas, Cidara, Gilead, Pfizer, Merck, Scyn-
exis, Toyama, Viamet, and Vical. L.K.N. has received travel support from
Viamet Pharmaceuticals, Inc. L.K. is an employee of Astellas. The other
authors declare no conflicts of interest.
FUNDING INFORMATION
This work, including the efforts of Nathan P. Wiederhold and Thomas F.
Patterson, was funded by Astellas Pharma US (Astellas).
Isavuconazonium sulfate and isavuconazole powders were provided by
Basilea.
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