AAC Accepts, published online ahead of print on 18 August 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.03261-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
In vitro Combination Therapy of Isavuconazole against Medically Important
2
Moulds
3 4
Aspasia Katragkou1, Matthew McCarthy1, Joseph Meletiadis2, Vidmantas Petraitis1,
5
Patriss W. Moradi1, Gittel E. Strauss1, Monique M. Fouant3, Laura L. Kovanda3,
6
Ruta Petraitiene1, Emmanuel Roilides4, Thomas J. Walsh1, 5, 6*
7 8
Transplantation-Oncology Infectious Diseases Program, Division of Infectious Diseases,
9
Department of Medicine, Weil Cornell Medical Center of Cornell University, New York,
10
NY, USA1; Clinical Microbiology Laboratory, Attikon University Hospital, Medical
11
School, National and Kapodistrian University of Athens, Athens, Greece2; Astellas
12
Pharma Global Development, Inc., Northbrook, IL3; Infectious Disease Unit, 3rd
13
Department of Pediatrics, Faculty of Medicine, Aristotle University School of Health
14
Sciences, Hippokration Hospital, Thessaloniki, Greece4; Department of Pediatrics, Weill
15
Cornell Medical College of Cornell University, New York, NY, USA5; Department of
16
Microbiology and Immunology, Weill Cornell Medical College of Cornell University,
17
New York, NY, USA6.
18 19 20 21 22 23
Running title: Isavuconazole combination therapy against moulds
24
*Corresponding author:
25
Prof. Thomas J. Walsh
26
Director, Transplantation-Oncology Infectious Diseases Program
27
Professor of Medicine, Pediatrics, and Microbiology & Immunology
28
Weill Cornell Medical Center
29
Henry Schueler Foundation Scholar
30
1300 York Ave., Rm A-421
31
New York, NY 10065
32
TEL: 212-746-6320 (main office)
33
FAX: 212-746-8675
34
[email protected]
35 36 37 38 39 40
Keywords: isavuconazole, moulds, amphotericin B, micafungin, Aspergillus, Mucorales
41
ABSTRACT
42
Whether isavuconazole, an extended-spectrum triazole, possesses synergistic
43
activity in combination therapy with echinocandins or amphotericin B for the treatment
44
of invasive moulds infections has not been studied. Our in vitro combination studies
45
showed that, isavuconazole and micafungin are synergistically active against Aspergillus
46
fumigatus, Aspergillus flavus, and Aspergillus terreus and Cunninghamella bertholettiae.
47
These results suggest that isavuconazole in combination with micafungin may have a role
48
in the treatment of invasive aspergillosis and warrant further investigation.
49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
64
Invasive moulds infections are important causes of morbidity and mortality in
65
immunocompromised hosts. Response to single antifungal agent treatment is often
66
inadequate, as acquired resistance and breakthrough infections have been reported among
67
patients with long-term exposure to even the newest of antifungal agents (1-3).
68
Combination antifungal therapy provides a potential strategy to improve antimicrobial
69
activity and clinical outcomes (4, 5). Isavuconazole is an extended-spectrum antifungal
70
triazole with a favorable pharmacokinetic and safety profile, as well as in vitro activity
71
against a wide variety of fungal pathogens, including non-Aspergillus spp., Fusarium
72
spp., Scedosporium spp. and Mucorales (6, 7). Whether isavuconazole possesses
73
synergistic activity when combined with echinocandins or polyenes is unknown. Herein
74
we examine the combination of isavuconazole with micafungin or amphotericin B
75
deoxycholate against medically important mould isolates.
76
A collection of eleven mould isolates was used in this study. Candida
77
parapsilosis (ATCC 22019), C. krusei (ATCC 6258) and A. flavus (ATCC 204304) were
78
used for quality control. Amphotericin B deoxycholate (AMB; Sigma-Aldrich, St. Louis,
79
MO), micafungin (MFG, Astellas Pharma US, Inc., Northbrook, IL) and isavuconazole
80
(ISA; Basilea Pharmaceutica, Basel, Switzerland) were used in this study. The antifungal
81
agents were obtained in lyophilized powder form and were prepared and preserved
82
according to the manufacturer’s instructions. Inoculum preparation and minimum
83
inhibitory concentrations (MICs) were determined according to the reference procedure
84
of the Antifungal Susceptibility testing of the Clinical and Laboratory Standards Institute
85
(CLSI, M38-A2) broth microdilution methodology (8).
86
The in vitro combination testing between ISA and MFG or AMB were studied
87
using a two-dimensional (eight-by-twelve) checkerboard microdilution method in sterile
88
96-well flat-bottom microtitration plates. The choice of the appropriate range of drug
89
concentrations was based on the MICs findings on the individual drug and isolate. ISA
90
and MFG or AMB were added in plate wells as it has been described elsewhere (9, 10).
91
The combined effects of antifungal agents were quantified after incubation for 48 h, by
92
using the metabolic reduction assay XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-
93
tetrazolim-5-carboxanilide) and 25 µM menadione, as the final electron acceptor agent,
94
as described previously (11). Interactions between antifungal agents were analyzed using
95
the Loewe additivity model (12) using the fractional inhibitory concentration (FIC) index
96
and the Bliss independence model (9, 13-17).
97
The results of the FIC index model are summarized in Table 1. For the ISA-MFG
98
combinations, the median FIC indices of all strains ranged from 0.28 to 1.06. These
99
indices for A. fumigatus, A. flavus, A. terreus, C. bertholettiae, L. corymbifera and S.
100
apiospermum indicate synergy. In addition, a mean FIC value of >1.25 for all replicates
101
was not noted in any of the mould isolates tested indicating that no antagonism was
102
found. For the ISA-AMB combinations, the median FIC indices of all strains-replicates,
103
with the exception of A. fumigatus, A. flavus and F. oxysporum, ranged from 1 to 1.18
104
indicating an additive effect. However, a mean FIC value of
1
In vitro Combination Therapy of Isavuconazole against Medically Important
2
Moulds
3 4
Aspasia Katragkou1, Matthew McCarthy1, Joseph Meletiadis2, Vidmantas Petraitis1,
5
Patriss W. Moradi1, Gittel E. Strauss1, Monique M. Fouant3, Laura L. Kovanda3,
6
Ruta Petraitiene1, Emmanuel Roilides4, Thomas J. Walsh1, 5, 6*
7 8
Transplantation-Oncology Infectious Diseases Program, Division of Infectious Diseases,
9
Department of Medicine, Weil Cornell Medical Center of Cornell University, New York,
10
NY, USA1; Clinical Microbiology Laboratory, Attikon University Hospital, Medical
11
School, National and Kapodistrian University of Athens, Athens, Greece2; Astellas
12
Pharma Global Development, Inc., Northbrook, IL3; Infectious Disease Unit, 3rd
13
Department of Pediatrics, Faculty of Medicine, Aristotle University School of Health
14
Sciences, Hippokration Hospital, Thessaloniki, Greece4; Department of Pediatrics, Weill
15
Cornell Medical College of Cornell University, New York, NY, USA5; Department of
16
Microbiology and Immunology, Weill Cornell Medical College of Cornell University,
17
New York, NY, USA6.
18 19 20 21 22 23
Running title: Isavuconazole combination therapy against moulds
24
*Corresponding author:
25
Prof. Thomas J. Walsh
26
Director, Transplantation-Oncology Infectious Diseases Program
27
Professor of Medicine, Pediatrics, and Microbiology & Immunology
28
Weill Cornell Medical Center
29
Henry Schueler Foundation Scholar
30
1300 York Ave., Rm A-421
31
New York, NY 10065
32
TEL: 212-746-6320 (main office)
33
FAX: 212-746-8675
34
[email protected]
35 36 37 38 39 40
Keywords: isavuconazole, moulds, amphotericin B, micafungin, Aspergillus, Mucorales
41
ABSTRACT
42
Whether isavuconazole, an extended-spectrum triazole, possesses synergistic
43
activity in combination therapy with echinocandins or amphotericin B for the treatment
44
of invasive moulds infections has not been studied. Our in vitro combination studies
45
showed that, isavuconazole and micafungin are synergistically active against Aspergillus
46
fumigatus, Aspergillus flavus, and Aspergillus terreus and Cunninghamella bertholettiae.
47
These results suggest that isavuconazole in combination with micafungin may have a role
48
in the treatment of invasive aspergillosis and warrant further investigation.
49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
64
Invasive moulds infections are important causes of morbidity and mortality in
65
immunocompromised hosts. Response to single antifungal agent treatment is often
66
inadequate, as acquired resistance and breakthrough infections have been reported among
67
patients with long-term exposure to even the newest of antifungal agents (1-3).
68
Combination antifungal therapy provides a potential strategy to improve antimicrobial
69
activity and clinical outcomes (4, 5). Isavuconazole is an extended-spectrum antifungal
70
triazole with a favorable pharmacokinetic and safety profile, as well as in vitro activity
71
against a wide variety of fungal pathogens, including non-Aspergillus spp., Fusarium
72
spp., Scedosporium spp. and Mucorales (6, 7). Whether isavuconazole possesses
73
synergistic activity when combined with echinocandins or polyenes is unknown. Herein
74
we examine the combination of isavuconazole with micafungin or amphotericin B
75
deoxycholate against medically important mould isolates.
76
A collection of eleven mould isolates was used in this study. Candida
77
parapsilosis (ATCC 22019), C. krusei (ATCC 6258) and A. flavus (ATCC 204304) were
78
used for quality control. Amphotericin B deoxycholate (AMB; Sigma-Aldrich, St. Louis,
79
MO), micafungin (MFG, Astellas Pharma US, Inc., Northbrook, IL) and isavuconazole
80
(ISA; Basilea Pharmaceutica, Basel, Switzerland) were used in this study. The antifungal
81
agents were obtained in lyophilized powder form and were prepared and preserved
82
according to the manufacturer’s instructions. Inoculum preparation and minimum
83
inhibitory concentrations (MICs) were determined according to the reference procedure
84
of the Antifungal Susceptibility testing of the Clinical and Laboratory Standards Institute
85
(CLSI, M38-A2) broth microdilution methodology (8).
86
The in vitro combination testing between ISA and MFG or AMB were studied
87
using a two-dimensional (eight-by-twelve) checkerboard microdilution method in sterile
88
96-well flat-bottom microtitration plates. The choice of the appropriate range of drug
89
concentrations was based on the MICs findings on the individual drug and isolate. ISA
90
and MFG or AMB were added in plate wells as it has been described elsewhere (9, 10).
91
The combined effects of antifungal agents were quantified after incubation for 48 h, by
92
using the metabolic reduction assay XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-
93
tetrazolim-5-carboxanilide) and 25 µM menadione, as the final electron acceptor agent,
94
as described previously (11). Interactions between antifungal agents were analyzed using
95
the Loewe additivity model (12) using the fractional inhibitory concentration (FIC) index
96
and the Bliss independence model (9, 13-17).
97
The results of the FIC index model are summarized in Table 1. For the ISA-MFG
98
combinations, the median FIC indices of all strains ranged from 0.28 to 1.06. These
99
indices for A. fumigatus, A. flavus, A. terreus, C. bertholettiae, L. corymbifera and S.
100
apiospermum indicate synergy. In addition, a mean FIC value of >1.25 for all replicates
101
was not noted in any of the mould isolates tested indicating that no antagonism was
102
found. For the ISA-AMB combinations, the median FIC indices of all strains-replicates,
103
with the exception of A. fumigatus, A. flavus and F. oxysporum, ranged from 1 to 1.18
104
indicating an additive effect. However, a mean FIC value of
