AAC Accepted Manuscript Posted Online 6 April 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.05056-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Title: Ibuprofen potentiates the in vivo antifungal activity of fluconazole against
2
Candida albicans murine infection
3
Running title: In vivo antifungal effect of fluconazole and ibuprofen
4
Sofia Costa de Oliveira 1, 2,*, Isabel M Miranda 1, 2, Ana Silva-Dias 1, 2, Ana P Silva 1, 2,
5
Acácio G Rodrigues 1, 2, 3, Cidália Pina-Vaz1, 2, 3
6 7
1. Department of Microbiology, Faculty of Medicine, University of Porto. Alameda Prof Hernani Monteiro, 4200-319, Porto, Portugal;
8
2. CINTESIS - Center for Health Technology and Services Research, Faculty of
9
Medicine of the University of Porto, Alameda Prof. Hernani Monteiro, 4200
10 11 12 13 14
Porto, Portugal; 3. Burn Unit, Department of Plastic and Reconstructive Surgery, Hospital S. João, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal; 4. Department of Microbiology, Hospital S. João, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal.
15 16
Abstract word count: 75
17
Text word count: 1214
18
*Corresponding author:
19
Sofia Costa-de-Oliveira
20
Department of Microbiology, Faculty of Medicine, Porto University
21
Alameda Professor Hernani Monteiro
22
4200-319 Porto
23
Portugal 1
24
Phone:+351919709956
25
Fax:+351225513603
26
e-mail: [email protected]
27 28 29
30
31
32
33
34
35
36
37
38
39
40
41
2
42
Abstract
43
Candida albicans is the most prevalent cause of fungaemia worldwide. Its ability to
44
develop resistance among patients under azole antifungal therapy is well documented.
45
In a murine model of systemic infection, we show that ibuprofen potentiates
46
fluconazole antifungal activity against a fluconazole resistant strain, reducing
47
drastically fungal burden and morbidity. Therapeutic combination of fluconazole with
48
ibuprofen may constitute a new approach for the management of antifungal
49
therapeutics to reverse resistance conferred by efflux pump overexpression.
50 51 52 53 54 55 56 57 58 59 60 61 62
3
63
Candida invasive infections represent an increasing challenge for clinicians, with an
64
assigned mortality around 40% (1-3). Fluconazole extensive use both for prophylaxis
65
and therapy resulted in the emergence of resistance (4-6).
66
The knowledge of the mechanisms underlying resistance is of crucial importance since
67
it could support approaches to achieve its reversion, leading to the development of
68
new therapeutic strategies. Alterations in C. albicans transcriptome induced by azoles
69
exposure have been extensively reported (5, 7, 8). However, few studies have tackled
70
antifungal resistance reversion.
71
Previously we showed that antifungal resistance conferred by increased efflux activity,
72
due to overexpression of CDR1 and CDR2 genes, in Candida clinical strains could be
73
reversed by ibuprofen (9, 10). This effect is also observed with other drugs, such as
74
pyrazinamide (11) and amphotericin B (12). Ibuprofen is a non-steroidal anti-
75
inflammatory drug used for its antipyretic, analgesic, and anti-inflammatory effects.
76
Herein we aimed to study the in vivo reversion of fluconazole resistance by ibuprofen
77
using a C. albicans systemic model of infection.
78
A C. albicans bloodculture strain (CaS) susceptible to fluconazole, voriconazole and
79
posaconazole was used to induce an azole resistant phenotype. During 60 days, a yeast
80
suspension containing 106 cells in 10 ml of RPMI 1640 medium (Sigma) was daily
81
incubated with fluconazole (Pfizer) at a final concentration of 16 µg/ml (therapeutic
82
serum levels achieved during antifungal therapy) (13, 14). Assessment of the minimal
83
inhibitory concentration (MIC) value to azoles, fluconazole, voriconazole (Pfizer) and
84
posaconazole (Schering-Plough), was determined for the parent strain and the
85
successive fluconazole exposed isolates as well as the susceptibility profile, according
4
86
to the CLSI M27 A3 protocol (15). For posaconazole, strains with MIC ≤0.06 µg/ml were
87
considered susceptible (16). The repeated exposure of C. albicans to fluconazole
88
resulted in the acquisition of an azole cross-resistant phenotype (Table 1), achieved
89
due to overexpression of efflux pumps encoding genes and ERG11 gene (data not
90
shown) displayed by CaR strain.
91
Azole resistant phenotype of CaR strain turned susceptible when MIC values to azoles
92
were re-determined in the presence of 100 µg/ml of ibuprofen, a concentration
93
previously described to impair azole efflux (9, 10). These in vitro results demonstrate
94
that ibuprofen potentiate azoles fungistatic activity.
95
The pursuit of knowledge of efflux pumps mechanism in Candida arises from the
96
homology between yeasts and human cells. In eukaryotic neoplasic cells, ATP-
97
dependent drug efflux pumps, such as P-glycoprotein (P-gp) which is encoded by
98
MDR1 gene, are important mediators of resistance, contributing to failure of cancer
99
therapy. In the human kidney, ibuprofen could inhibit methotrexate efflux transporters
100
(17).
A
similar
effect
was
described
for
FK506
(tacrolimus),
a
potent
101
immunossuppressor agent that shows a synergistic effect when combined with
102
antineoplasic agents on tumor cells, decreasing or even suppressing multidrug
103
resistance by competing with cytotoxic drugs for the P-glycoprotein (18-20). Thus, a
104
similar approach could be applied to C. albicans cells.
105
To investigate the potential clinical application between fluconazole and ibuprofen, in
106
vivo experiments were conducted in a murine candidosis model approved by the
107
Directorate-General of Food and Veterinary Medicine (authorization number 6411).
5
108
Female specific-pathogen-free BALB/c mice (age, 6 to 8 weeks; weight, 17 to 20 g;
109
Charles River Laboratories) were injected with 5x105 cells of the CaS or the CaR strain
110
in 0.1ml of sterile saline via the lateral tail vein (21). The fluconazole effective dose
111
that reduced 50% the pathological effects of i.v. C. albicans challenge relative to
112
untreated mice (control) was defined as the 50% effective dose (ED50) (8). Therapy was
113
initiated 3 hours after yeast challenge and was administered daily for a total of 3 days
114
(8, 21). Mice were treated intraperitoneally with fluconazole (8 to 60 mg/kg of body
115
weight/day) (8, 21), with ibuprofen (10 or 20 mg/kg/day) (11, 22) or fluconazole (8 to
116
60 mg/kg of body weight/day) plus ibuprofen (10 or 20 mg/kg/day). Mice weight was
117
daily registered and at day 4 post-infection mice were euthanized and kidneys were
118
aseptically removed. The fungal burden was calculated as the number of colony
119
forming units (CFU) per gram of tissue and isolates were preserved for later MIC
120
determination. For histological studies, kidneys were processed for periodic acid-Schiff
121
(PAS) staining.
122
Mice infected with the CaS strain and treated with 30 mg/kg of body weight/day (ED50)
123
of fluconazole a significant reduction (p8/R
0.06/S
>8/R
0.125/R
314
315
316
317
318
15
1
Title: Ibuprofen potentiates the in vivo antifungal activity of fluconazole against
2
Candida albicans murine infection
3
Running title: In vivo antifungal effect of fluconazole and ibuprofen
4
Sofia Costa de Oliveira 1, 2,*, Isabel M Miranda 1, 2, Ana Silva-Dias 1, 2, Ana P Silva 1, 2,
5
Acácio G Rodrigues 1, 2, 3, Cidália Pina-Vaz1, 2, 3
6 7
1. Department of Microbiology, Faculty of Medicine, University of Porto. Alameda Prof Hernani Monteiro, 4200-319, Porto, Portugal;
8
2. CINTESIS - Center for Health Technology and Services Research, Faculty of
9
Medicine of the University of Porto, Alameda Prof. Hernani Monteiro, 4200
10 11 12 13 14
Porto, Portugal; 3. Burn Unit, Department of Plastic and Reconstructive Surgery, Hospital S. João, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal; 4. Department of Microbiology, Hospital S. João, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal.
15 16
Abstract word count: 75
17
Text word count: 1214
18
*Corresponding author:
19
Sofia Costa-de-Oliveira
20
Department of Microbiology, Faculty of Medicine, Porto University
21
Alameda Professor Hernani Monteiro
22
4200-319 Porto
23
Portugal 1
24
Phone:+351919709956
25
Fax:+351225513603
26
e-mail: [email protected]
27 28 29
30
31
32
33
34
35
36
37
38
39
40
41
2
42
Abstract
43
Candida albicans is the most prevalent cause of fungaemia worldwide. Its ability to
44
develop resistance among patients under azole antifungal therapy is well documented.
45
In a murine model of systemic infection, we show that ibuprofen potentiates
46
fluconazole antifungal activity against a fluconazole resistant strain, reducing
47
drastically fungal burden and morbidity. Therapeutic combination of fluconazole with
48
ibuprofen may constitute a new approach for the management of antifungal
49
therapeutics to reverse resistance conferred by efflux pump overexpression.
50 51 52 53 54 55 56 57 58 59 60 61 62
3
63
Candida invasive infections represent an increasing challenge for clinicians, with an
64
assigned mortality around 40% (1-3). Fluconazole extensive use both for prophylaxis
65
and therapy resulted in the emergence of resistance (4-6).
66
The knowledge of the mechanisms underlying resistance is of crucial importance since
67
it could support approaches to achieve its reversion, leading to the development of
68
new therapeutic strategies. Alterations in C. albicans transcriptome induced by azoles
69
exposure have been extensively reported (5, 7, 8). However, few studies have tackled
70
antifungal resistance reversion.
71
Previously we showed that antifungal resistance conferred by increased efflux activity,
72
due to overexpression of CDR1 and CDR2 genes, in Candida clinical strains could be
73
reversed by ibuprofen (9, 10). This effect is also observed with other drugs, such as
74
pyrazinamide (11) and amphotericin B (12). Ibuprofen is a non-steroidal anti-
75
inflammatory drug used for its antipyretic, analgesic, and anti-inflammatory effects.
76
Herein we aimed to study the in vivo reversion of fluconazole resistance by ibuprofen
77
using a C. albicans systemic model of infection.
78
A C. albicans bloodculture strain (CaS) susceptible to fluconazole, voriconazole and
79
posaconazole was used to induce an azole resistant phenotype. During 60 days, a yeast
80
suspension containing 106 cells in 10 ml of RPMI 1640 medium (Sigma) was daily
81
incubated with fluconazole (Pfizer) at a final concentration of 16 µg/ml (therapeutic
82
serum levels achieved during antifungal therapy) (13, 14). Assessment of the minimal
83
inhibitory concentration (MIC) value to azoles, fluconazole, voriconazole (Pfizer) and
84
posaconazole (Schering-Plough), was determined for the parent strain and the
85
successive fluconazole exposed isolates as well as the susceptibility profile, according
4
86
to the CLSI M27 A3 protocol (15). For posaconazole, strains with MIC ≤0.06 µg/ml were
87
considered susceptible (16). The repeated exposure of C. albicans to fluconazole
88
resulted in the acquisition of an azole cross-resistant phenotype (Table 1), achieved
89
due to overexpression of efflux pumps encoding genes and ERG11 gene (data not
90
shown) displayed by CaR strain.
91
Azole resistant phenotype of CaR strain turned susceptible when MIC values to azoles
92
were re-determined in the presence of 100 µg/ml of ibuprofen, a concentration
93
previously described to impair azole efflux (9, 10). These in vitro results demonstrate
94
that ibuprofen potentiate azoles fungistatic activity.
95
The pursuit of knowledge of efflux pumps mechanism in Candida arises from the
96
homology between yeasts and human cells. In eukaryotic neoplasic cells, ATP-
97
dependent drug efflux pumps, such as P-glycoprotein (P-gp) which is encoded by
98
MDR1 gene, are important mediators of resistance, contributing to failure of cancer
99
therapy. In the human kidney, ibuprofen could inhibit methotrexate efflux transporters
100
(17).
A
similar
effect
was
described
for
FK506
(tacrolimus),
a
potent
101
immunossuppressor agent that shows a synergistic effect when combined with
102
antineoplasic agents on tumor cells, decreasing or even suppressing multidrug
103
resistance by competing with cytotoxic drugs for the P-glycoprotein (18-20). Thus, a
104
similar approach could be applied to C. albicans cells.
105
To investigate the potential clinical application between fluconazole and ibuprofen, in
106
vivo experiments were conducted in a murine candidosis model approved by the
107
Directorate-General of Food and Veterinary Medicine (authorization number 6411).
5
108
Female specific-pathogen-free BALB/c mice (age, 6 to 8 weeks; weight, 17 to 20 g;
109
Charles River Laboratories) were injected with 5x105 cells of the CaS or the CaR strain
110
in 0.1ml of sterile saline via the lateral tail vein (21). The fluconazole effective dose
111
that reduced 50% the pathological effects of i.v. C. albicans challenge relative to
112
untreated mice (control) was defined as the 50% effective dose (ED50) (8). Therapy was
113
initiated 3 hours after yeast challenge and was administered daily for a total of 3 days
114
(8, 21). Mice were treated intraperitoneally with fluconazole (8 to 60 mg/kg of body
115
weight/day) (8, 21), with ibuprofen (10 or 20 mg/kg/day) (11, 22) or fluconazole (8 to
116
60 mg/kg of body weight/day) plus ibuprofen (10 or 20 mg/kg/day). Mice weight was
117
daily registered and at day 4 post-infection mice were euthanized and kidneys were
118
aseptically removed. The fungal burden was calculated as the number of colony
119
forming units (CFU) per gram of tissue and isolates were preserved for later MIC
120
determination. For histological studies, kidneys were processed for periodic acid-Schiff
121
(PAS) staining.
122
Mice infected with the CaS strain and treated with 30 mg/kg of body weight/day (ED50)
123
of fluconazole a significant reduction (p8/R
0.06/S
>8/R
0.125/R
314
315
316
317
318
15
