revised aac 00792-10 activity of deferasirox in mucorales
TRANSCRIPT
Revised AAC 00792-10
Activity of deferasirox in Mucorales:
Influences of species and exogenous iron
*Russell E. Lewis,1, 2 * Georgios N. Pongas, 1 Nathaniel Albert,1 Ronen Ben-Ami,1
Thomas J. Walsh,3 and Dimitrios P. Kontoyiannis1, 2
1Department of Infectious Diseases, Infection Control, and Employee Health, The
University of Texas M. D. Anderson Cancer Center, 2University of Houston College of
Pharmacy, Houston, TX, 3Transplantation-Oncology Infectious Diseases Program,
Division of Infectious Diseases, Weill Cornell Medical College of Cornell University, New
York, NY
Corresponding author: Dimitrios P. Kontoyiannis, M.D., ScD., F.A.C.P., F.I.D.S.A.,
Department of Infectious Diseases, Infection Control and Employee Health, Unit 1406,
The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,
Houston, Texas 77030, United States of America. Telephone: (713) 792-6237; Fax:
(713)745-6839; E-mail: [email protected]
* Contributed equally
Key worlds: Cunninghamella, Rhizopus, deferasirox
Word count: 926
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00792-10 AAC Accepts, published online ahead of print on 18 October 2010
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ABSTRACT
Differences in deferasirox susceptibility among Mucorales are unknown. Here we show
that Cunnighmamella bertolletiae (4 isolates) and Mucor species (5 isolates) display
higher deferasirox MICs and MFCs compared to Rhizopus species (6 isolates).
Exogenous iron further attenuated deferasirox susceptibility of Mucorales isolates with
low MICs. Vital staining revealed damage to sub-apical compartments in susceptible
strains.
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Mucormycosis is an emerging life-threatening infection in immunocompromised patients
(10, 12), with Rhizopus and Mucor spp. accounting for over 70% culture-confirmed
cases. Previous studies have demonstrated the critical role of iron for the growth and
pathogenesis of Mucorales (1-3, 6). Deferasirox, tridentate iron chelator used for
treatment of iron-overloaded patients, has shown potential as a therapeutic intervention
for mucormycosis (9). Whether deferasirox susceptibility is species-specific among
Mucorales is unknown. For example, although the activity of deferasirox against
Rhizopus oryzae is established in preclinical models (9), its action against the more
lethal, yet uncommon Mucorales species such as Cunninghamella bertholettiae has not
been reported. To that end, we compared MICs and MFCs of deferasirox in Rhizopus
vs Mucor and Cunninghamella spp. using a modification of the standard CLSI M38-A2
method (5).
All experiments were performed in triplicate in RPMI 1640 medium + 2% glucose
at different time points. Clinical isolates of Cunninghamella bertholletiae (4 isolates),
Rhizopus species (6 isolates) and Mucor species (5 isolates) were tested. These strains
were isolated from geographically and temporarily distinct cases of mucormycosis in
immunosuppressed patients and displayed variable susceptibility patterns to
amphotericin B and posaconazole (data not shown). Routine morphological methods
were used for their identification. Sporangiospores were collected from yeast extract
agar glucose plates (YAG, media contains 3.6 µM FeSO4) after 48 hours and
suspended in 0.85% normal saline. Suspensions were adjusted to a standardized
inoculum of 104 sporangiospores/mL in RPMI 1640 with deferasirox concentration range
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of 0.125 – 128 µg/ml with or without 0.125% FeCl3. MICs were deteremined at 24 and
48 hours using the CLSI M38-A2 method (5). MFCs were determined used
standardized methods for filamentous fungi (8). Because permissive growth occurs
during the first few hours of deferasirox exposure, MICs were read as an 80%
(prominent) visual reduction in comparison to growth controls (9). Finally, to assess the
hyphal damage of R. oryzae and C. bertholletiae growing in iron-containing RPMI
medium by using the fluorescent cellular morbidity dye bis-(1,3-dibutylbarbituric acid)
trimethine oxonol (DiBAC) as previously described (4). Briefly, 105 sporangiospores/ mL
were incubated in RPMI 1640 for 18 hrs. Hyphae were then mixed in either RPMI with
amphotericin B (positive control, 2 µg/mL) or RPMI with deferasirox (4xMIC80). After 6 h
of incubation in 37°C, hyphae were washed twice with PBS and re-suspended in DiBAC
(2 µg/mL). Tubes were incubated in the dark with gentle shaking at room temperature.
Samples were then washed twice again with PBS and resuspended for
photomicrography.
Results and discussion: After 48 hrs, the geometric mean (GM) MICs
deferasirox were higher for C. bertholettiae (39.1 µg/ml) and Mucor species (32.2 µg/ml)
than for Rhizopus (12.2 µg/ml) (Table 1). MICs were modestly lower at 24 hours.
Deferasirox was fungistatic against all the tested isolates as viable colonies could be
recovered from test wells. The 48-hour deferasirox GM MFC for C. bertholettiae isolates
(> 256 µg/ml) were substantially higher than Mucor (111.4 µg/ml) and Rhizopus species
(44.7 µg/ml). We then tested the effect of the addition of iron to RPMI medium on the
MIC values of deferasirox in two representative isolates each of Rhizopus and Mucor
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with lower MICs. Testing in the presence of FeCl3 0.125% resulted in significant
increases in deferasirox MICs all of the tested strains (Table 2).
The lack of fungicidal activity observed in our experiments contrasts with the
results reported by Ibrahim et al, who reported that deferasirox was fungicidal at
concentrations < 6.25 µg/mL for R. oryzae (9). However, we believe that the availability
of iron inside the sporangiospores collected from iron containing plates (YAG medium),
as well as trace amounts of iron in the RPMI medium may have attenuated deferasirox
activity when assayed in vitro (7, 9). Importantly, the cultivation techniques used by
Ibrahim et al. resulted in iron-deprived hyphae that are presumably more susceptible to
the iron depleting effects of deferasirox when tested in vitro and in vivo (9). Collectively,
our results, including the attenuation of deferasirox activity by FeCl3, are consistent with
the concept that the fungicidal action of deferasirox is mediated through iron deprivation
(9). It is unknown whether higher serum levels of deferasirox may be required for
antifungal efficacy in hosts with varying degrees of iron overload and free iron.
Pharmacokinetic studies in healthy volunteers have shown that trough deferasirox
trough serum levels range from 3.7-22.4 µg/ml (11) ; a value falls below the average
GM observed for C. bertholettiea in the present study (Table 1).
Additionally, we found high MFCs to deferasirox for C. bertholletiae isolates
compared to Rhizopus and Mucor spp. In view of the greater lethality of C. bertholletiae
in both animal models and human infections (10, 12), it is possible that increased
resistance to iron starvation contributes to the increased virulence of these species in
human and animal hosts.
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Finally, as growing hyphae are likely to require more iron compared to
metabolically inert spores, we evaluated whether the effect of deferasirox is
predominantly seen in the growing hyphal tips versus sub-apical compartments. DiBAC
staining showed increased fluorescence in subapical compartments of C. bertholletiae
and R. oryzae treated with deferasirox or amphotericin B; whereas, little fluorescence
could be detected in hyphae of untreated controls (Figure 1).
In conclusion, our data suggest that deferasirox is less active in vitro against
Cunninghamella compared to Rhizopus and Mucor spp, and multiple factors related to
iron availability during the cultivation and testing, as well as fungal morphotype, can
profoundly influence susceptibility results with this iron chelator. Standardization of test
conditions will be critical for future development and interpretation of in vitro testing of
this novel therapeutic class.
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REFERENCES
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Table 1. Deferasirox Geometric mean inhibitory and fungicidal concentrations for tested
Rhizopus, Cunninghamella, and Mucor spp*.
Mucorales GM MIC 24 hr
(95% CI)
GM MIC 48 hr
(95% CI)
GM MFC 48 hr
(95% CI)
Rhizopus spp.
(n=6)
7.4
(5.2-10.4)
12.2
(8.1-18.5)
44.7
(30.4-65.5)
Cunninghamella spp.
(n=4)
39.7
(17.7-86.4)
39.1
(17.7-86.4)
>256
(ND)
Mucor spp.
(n=5)
6.6
(5.5-7.9)
32.2
(12.2-
111.4
(23.9-256)
GM-geometric mean
* CLSI M 38-A2 method, RPMI 1640 medium
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Table 2. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration
(MFC) of deferasirox against Rhizopus, Mucor, and Cunninghamella
Median MICa µg/mL
Species DEF DEF-FeCl3 AMB
Rhizpous (n=2) 5 >256 2
Mucor (n=2) 5 >256 2
DEF-deferasirox, DEF-FeCl3- deferasirox + 0.125% FeCl3, AMB- amphotericin B
MFC were not determined for DEF-FeCl3 due to visible growth in all test wells
a 48 hour MICs were read as 80% reduction in observable growth. All experiments were
performed in triplicate.
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FIGURE LEGEND
Figure 1. Photomicrographs of untreated and drug-treated Mucorales hyphae
stained with the fluorescent dye DiBAC. Cunninghamella bertholletiae (a-c) and
Rhizopus oryzae (d-f) were prepared from 18 h incubation in RPMI, were
washed and re-incubated in RPMI with amphotericin B (AMB) (2 µg/ml) and
deferasirox (DEF) at 4xMIC80 and stained with DiBAC. Untreated hyphae were
used as negative controls. Bright field microscopy (a1,b1,c1,d1,e1,f1) and
epifluorescence images (a2,b2,c2,d2,e2,f2) are presented at 200x
magnification. The fluorescence of the dark pictures is indicative of hyphal
damage.
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