AAC Accepted Manuscript Posted Online 7 December 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.02615-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Clofazimine prevents the regrowth of Mycobacterium abscessus and
2
Mycobacterium avium type strains exposed to amikacin and clarithromycin
3 4
Beatriz E. Ferro1, Joseph Meletiadis2,3, Melanie Wattenberg1, Arjan de Jong1,
5
Dick van Soolingen1,4,5, Johan W. Mouton1,3, Jakko van Ingen1#
6 7
1
8
Nijmegen, The Netherlands; 2Clinical Microbiology Laboratory, Attikon University
9
General Hospital, Medical School, National and Kapodistria University of Athens,
Department of Medical Microbiology, Radboud University Medical Center,
10
Athens, Greece; 3Department of Medical Microbiology and Infectious Diseases,
11
Erasmus Medical Center, Rotterdam, The Netherlands; 4Department of Pulmonary
12
Diseases, Radboud University Medical Center, Nijmegen, The Netherlands;
13
5
14
and the Environment (RIVM), Bilthoven, The Netherlands.
National Tuberculosis Reference Laboratory, National Institute for Public Health
15 16
#
17
[email protected]
Corresponding author. Email: [email protected] /
18 19
Running title: clofazimine prevents regrowth/resistance
20 21
Key words: pharmacodynamics; drug-drug interaction; non-tuberculous
22
mycobacteria, zero-interaction theories, synergy.
23 24 1
25
Abstract
26
Multidrug therapy is a standard practice when treating infections by non-
27
tuberculous mycobacteria (NTM), but few treatment options exist. We conducted
28
this study to define the drug-drug interaction between clofazimine and both
29
amikacin and clarithromycin, and its contribution to NTM treatment. Mycobacterium
30
abscessus and Mycobacterium avium type strains were used. Time-kill assays for
31
clofazimine alone and combined with amikacin or clarithromycin were performed at
32
concentrations 0.25-2×MIC. Pharmacodynamic interactions were assessed by
33
response surface model of Bliss independence (RSBI) and isobolographic analysis
34
of Loewe additivity (ISLA) calculating the percentage of statistically significant Bliss
35
interactions and interaction indices (I), respectively. Monte Carlo simulations with
36
predicted human lung concentrations were used to calculate target attainment
37
rates for combination and monotherapy regimens. Clofazimine alone was
38
bacteriostatic for both NTM. Clofazimine-amikacin was synergistic against M.
39
abscessus (I=0.41, 95%CI 0.29-0.55) and M. avium (I=0.027, 95%CI 0.007-0.048).
40
Based on RSBI analysis, synergistic interactions of 28.4-29.0% and 23.2-56.7%
41
were observed at 1-2×MIC and 0.25-2×MIC for M. abscessus and M. avium,
42
respectively. Clofazimine-clarithromycin was also synergistic against M. abscessus
43
(I=0.53, 95%CI 0.35-0.72) and M. avium (I=0.16, 95%CI 0.04-0.35), RSBI analysis
44
showed 23.5% and 23.3-53.3% at 2×MIC and 0.25-0.5×MIC for M. abscessus and
45
M. avium, respectively. Clofazimine prevented the regrowth observed with
46
amikacin or clarithromycin alone. Target attainment rates of combination regimens
47
were >60% higher than monotherapy regimens for M. abscessus and M. avium.
48
The combination of clofazimine with amikacin or clarithromycin was synergistic in 2
49
vitro. This suggests a potential role of clofazimine in treatment regimens, that
50
warrants further evaluation.
51 52
Introduction
53
The treatment of diseases caused by non-tuberculous mycobacteria (NTM) is a
54
challenge, partly due to the natural resistance of NTM to most antibiotics.
55
Treatment outcomes are generally poor and current treatment recommendations
56
have a very limited evidence base, since very few clinical trials have been
57
performed (1,2).
58
Multidrug therapy is a standard practice when treating mycobacterial infections.
59
However, the pharmacodynamic interactions among the combined drugs are
60
largely unknown. Understanding these interactions will help to detect synergistic
61
combinations with increased antibacterial killing, which ultimately can result in a
62
better treatment outcome.
63
One of the promising combinations is amikacin and clofazimine, given the key role
64
of amikacin in the treatment of NTM infections (2,3) and the unique characteristics
65
of clofazimine, like its prolonged half-life, preferential accumulation inside
66
macrophages (4) and the recently found bactericidal activity only after two weeks
67
of treatment in the mouse model of tuberculosis (5).
68
Clarithromycin, on the other hand, has substantial in vitro and clinical activity
69
against Mycobacterium avium complex (MAC) and it has been long considered the
70
cornerstone for Mycobacterium abscessus treatment (3).Hence the examination of
71
its interaction with clofazimine is interesting.
3
72
Previous reports showed in vitro synergy between clofazimine and amikacin
73
against both rapidly and slowly growing NTM (1,6). The combination
74
clarithromycin-clofazimine also showed synergy against MAC strains in
75
checkerboard evaluation (7). These checkerboard titrations offer no information on
76
the mechanism of synergistic activity, the exact killing activity of these
77
combinations and its concentration dependence. We therefore investigated the
78
pharmacodynamic interactions between clofazimine and amikacin, and clofazimine
79
and clarithromycin, against two key NTM species, using time-kill assays analyzed
80
with two pharmacodynamic drug interaction models: response surface model of
81
Bliss independence (RSBI) and isobolographic analysis of Loewe additivity (ISLA)
82
(8,9).
83 84
Materials and Methods
85
Bacterial strains, media and antibiotics
86
Mycobacterium abscessus subspecies abscessus CIP 104536 (Collection of
87
Institute Pasteur) and Mycobacterium avium subspecies hominissuis IWGMT49
88
(International Working Group on Mycobacterial Taxonomy) type strains were used.
89
Stocks of each strain were preserved at -80°C in trypticase soy broth with 40%
90
glycerol and were thawed for each assay. Pure powders of amikacin,
91
clarithromycin and clofazimine obtained from Sigma-Aldrich were dissolved in
92
water, methanol and DMSO, respectively.
93 94
Susceptibility testing
4
95
MICs were determined at the beginning and at the end of the experiments,
96
following CLSI recommendations (10) by broth microdilution in cation-adjusted
97
Mueller Hinton broth (CAMHB); commercially available panels were used for
98
amikacin and clarithromycin (RAPMYCO Sensititre®, SLOWMYCO Sensititre®-Trek
99
Diagnostics/Thermo Fisher), while for clofazimine manual broth microdilution was
100
performed in Middlebrook 7H9broth (BD Bioscience). Unexposed M. abscessus
101
type strain was used as a quality control during all MIC determinations.
102 103
Mutation analysis
104
Mutation analysis of rrs and rrl was performed to detect mutations associated to
105
amikacin and clarithromycin resistance, respectively, in mycobacteria exposed to
106
the two antibiotics in the drug-drug interaction experiments. Briefly, mycobacterial
107
suspensions were heat-inactivated at 95°C for 30 min, then DNA was extracted
108
using MagNAPure LC (Roche Life Science). Relevant fragment of rrs and rrl were
109
amplified using previously described primers and PCR conditions (11,12).
110
Sequence analyses were performed to detect mutations at codon 1408 in rrs and
111
2058 and 2059 in rrl.
112 113
Time-kill assays for single drugs
114
The inoculum consisted of mycobacteria in the early logarithmic phase of growth.
115
Individual bottles of 20 ml of Middlebrook 7H9 with oleic acid-bovine albumin-
116
dextrose-catalase growth supplement (BD Bioscience) and 0.05% Tween 80,
117
containing increasing concentrations (from 0.062 to 8 times the MIC for M.
118
abscessus and 0.062 to 32 times MIC for M. avium) of clofazimine were cultured 5
119
with the inoculum ( ~105 – 106 CFU/ml) at 30°C for M. abscessus and 37°C for M.
120
avium, under shaking conditions at 100 rpm (GFL); ventilation through a bacterial
121
filter (FP 30/0.2 Ca/S, Whatman GmbH) was incorporated. A drug-free and
122
inoculum-free bottle with medium served as the growth and sterility control,
123
respectively. At defined time intervals (3, 6, 12, 24, 36, 48, 72, 96 and 120 h for M.
124
abscessus and 12, 24, 36, 48, 72, 96, 120, 144, 168 and 240 h for M. avium), ten
125
μl from undiluted and serially ten-fold diluted in normal saline samples were plated
126
in triplicate on Middlebrook 7H11 agar plates (BD Bioscience). Plates were
127
incubated for 3-5 days at 30°C for M. abscessus, or 7-9 days at 37°C for M. avium
128
and the CFU/ml were calculated (lower limit of detection: 33.3 CFU/ml).
129 130
Time-kill assays for drug-drug interaction
131
Following the same protocol described above, time-kill assays for M. abscessus
132
and M. avium were performed with four concentrations (from 0.25 to 2 times the
133
MIC) of each antibiotic alone and in paired combination with clofazimine. MICs for
134
amikacin, clarithromycin and clofazimine were checked by duplicate at the final
135
sampling time, when colonies were present.
136 137
Pharmacodynamic drug interaction analysis
138
In order to assess the nature and the magnitude of the in vitro interactions between
139
clofazimine and amikacin or clarithromycin against NTM, the data obtained with
140
time-kill assays were analyzed using the response surface analysis of Bliss
141
independence (RSBI) and the isobolographic analysis of Loewe additivity (ISLA) as
142
described previously (8,9). The RSBI was used to describe the kinetics of 6
143
pharmacodynamic interactions at different time points whereas the ISLA was used
144
to describe the full concentration-effect relationships of the drugs alone and in
145
combination at the end of the experiment.
146
For that purpose, the percentage of bacterial load at each drug concentration and
147
combination was calculated by dividing the log10 CFU/ml with the log10 CFU/ml of
148
growth control at each sampling times throughout the experiment. Bliss synergy
149
and antagonism was concluded if the observed bacterial load was statistically
150
significantly (p64 mg/liter vs. 4 mg/liter) in bacteria exposed to clarithromycin
242
alone. Again, this increase was not observed in colonies exposed to the
243
combination of clofazimine-clarithromycin. The A2058G mutation in rrl was found
244
only in colonies exposed to 0.25 and 0.5×MIC clarithromycin alone, not in colonies
245
exposed to any of the clofazimine-clarithromycin combinations.
246 247
Interaction analysis
248
The RSBI analysis of the combination of clofazimine-amikacin against M.
249
abscessus showed independent interactions within the first 12 h which converted
250
to antagonistic interactions at 24-48 h and ultimately to synergistic interaction of
251
29.0% and 28.4% at the end of the experiment for 2 and 1×MIC respectively
252
(Figure 4a). Isobolographic analysis of the full concentration-effect relationship of
253
the same combination showed synergy at the end of the experiment at
254
concentrations ≥ 1×MIC with an I of 0.41, 95%CI 0.29-0.55 (Figure 5a); stasis was
255
achieved at 4 and 8×MIC of amikacin and clofazimine alone; for the combination,
256
stasis was found at 1×MIC of both drugs.
257
The RSBI analysis of the combination clofazimine-clarithromycin versus M.
258
abscessus also showed independence the first 12 h which converted to
259
antagonistic interactions at high concentrations, but ultimately synergistic
260
interaction of 23.5% were observed only at 2×MIC (Figure 4b). Similarly, the ISLA 11
261
of the same combination showed synergistic interaction reaching stasis at 2×MIC
262
when drugs were combined. For each drug alone the endpoint was reached at
263
8×MIC (I=0.53, 95%CI 0.35-0.72 (Figure 5b).
264
For the combination clofazimine-amikacin against M. avium, the RSBI analysis
265
showed independence up to 48 h, then antagonism up to 120 h and synergy at the
266
end of the experiment, of 23.2-56.7% at all concentrations (Figure 4c). Strong
267
synergy was found with the ISLA, (I=0.027, 95%CI 0.007-0.048) since stasis was
268
found at 0.5×MIC of the drugs in combination; the same endpoint was reached at
269
8×MIC and>32×MIC for clofazimine and amikacin alone(Figure 5c). The same
270
profile of interactions was found for the combination clofazimine-clarithromycin;
271
with RSBI analysis independence was observed up to 48 h, then antagonism up to
272
120 h and ultimately synergy. However, the synergistic interactions were found at
273
concentrations 0.25 and 0.5×MIC (53.3% and 23.3%, respectively) (Figure 4d).
274
This was also confirmed with the ISLA (I= 0.16, 95%CI 0.04-0.35) with stasis
275
observed at combinations with 1×MIC of each drug the
278
effect was similar to the effect of clarithromycin alone indicating no interaction as
279
the RSBI analysis showed at the end of the experiment.
280 281
Monte Carlo simulations
282
The percentage of patients that will attain lung concentrations associated with
283
stasis of both M. abscessus and M. avium are shown in Figure 6. For M.
284
abscessus, target attainment rates with clofazimine-amikacin and clofazimine12
285
clarithromycin combinations were higher than monotherapy regimens particularly
286
for isolates with amikacin/clofazimine MIC 16/0.25 and clarithromycin/clofazimine
287
MICs 2/0.25, respectively (>60%difference between combination and monotherapy
288
regimens). For M. avium,>60% difference was found for the clofazimine-amikacin
289
combination for isolates with amikacin/clofazimine MICs 32-128/0.25-1 mg/liter and
290
for the clofazimine-clarithromycin combination for isolates with
291
clarithromycin/clofazimine MICs 32-124/0.5-2 mg/liter. High (>95%) and low (
1
Clofazimine prevents the regrowth of Mycobacterium abscessus and
2
Mycobacterium avium type strains exposed to amikacin and clarithromycin
3 4
Beatriz E. Ferro1, Joseph Meletiadis2,3, Melanie Wattenberg1, Arjan de Jong1,
5
Dick van Soolingen1,4,5, Johan W. Mouton1,3, Jakko van Ingen1#
6 7
1
8
Nijmegen, The Netherlands; 2Clinical Microbiology Laboratory, Attikon University
9
General Hospital, Medical School, National and Kapodistria University of Athens,
Department of Medical Microbiology, Radboud University Medical Center,
10
Athens, Greece; 3Department of Medical Microbiology and Infectious Diseases,
11
Erasmus Medical Center, Rotterdam, The Netherlands; 4Department of Pulmonary
12
Diseases, Radboud University Medical Center, Nijmegen, The Netherlands;
13
5
14
and the Environment (RIVM), Bilthoven, The Netherlands.
National Tuberculosis Reference Laboratory, National Institute for Public Health
15 16
#
17
[email protected]
Corresponding author. Email: [email protected] /
18 19
Running title: clofazimine prevents regrowth/resistance
20 21
Key words: pharmacodynamics; drug-drug interaction; non-tuberculous
22
mycobacteria, zero-interaction theories, synergy.
23 24 1
25
Abstract
26
Multidrug therapy is a standard practice when treating infections by non-
27
tuberculous mycobacteria (NTM), but few treatment options exist. We conducted
28
this study to define the drug-drug interaction between clofazimine and both
29
amikacin and clarithromycin, and its contribution to NTM treatment. Mycobacterium
30
abscessus and Mycobacterium avium type strains were used. Time-kill assays for
31
clofazimine alone and combined with amikacin or clarithromycin were performed at
32
concentrations 0.25-2×MIC. Pharmacodynamic interactions were assessed by
33
response surface model of Bliss independence (RSBI) and isobolographic analysis
34
of Loewe additivity (ISLA) calculating the percentage of statistically significant Bliss
35
interactions and interaction indices (I), respectively. Monte Carlo simulations with
36
predicted human lung concentrations were used to calculate target attainment
37
rates for combination and monotherapy regimens. Clofazimine alone was
38
bacteriostatic for both NTM. Clofazimine-amikacin was synergistic against M.
39
abscessus (I=0.41, 95%CI 0.29-0.55) and M. avium (I=0.027, 95%CI 0.007-0.048).
40
Based on RSBI analysis, synergistic interactions of 28.4-29.0% and 23.2-56.7%
41
were observed at 1-2×MIC and 0.25-2×MIC for M. abscessus and M. avium,
42
respectively. Clofazimine-clarithromycin was also synergistic against M. abscessus
43
(I=0.53, 95%CI 0.35-0.72) and M. avium (I=0.16, 95%CI 0.04-0.35), RSBI analysis
44
showed 23.5% and 23.3-53.3% at 2×MIC and 0.25-0.5×MIC for M. abscessus and
45
M. avium, respectively. Clofazimine prevented the regrowth observed with
46
amikacin or clarithromycin alone. Target attainment rates of combination regimens
47
were >60% higher than monotherapy regimens for M. abscessus and M. avium.
48
The combination of clofazimine with amikacin or clarithromycin was synergistic in 2
49
vitro. This suggests a potential role of clofazimine in treatment regimens, that
50
warrants further evaluation.
51 52
Introduction
53
The treatment of diseases caused by non-tuberculous mycobacteria (NTM) is a
54
challenge, partly due to the natural resistance of NTM to most antibiotics.
55
Treatment outcomes are generally poor and current treatment recommendations
56
have a very limited evidence base, since very few clinical trials have been
57
performed (1,2).
58
Multidrug therapy is a standard practice when treating mycobacterial infections.
59
However, the pharmacodynamic interactions among the combined drugs are
60
largely unknown. Understanding these interactions will help to detect synergistic
61
combinations with increased antibacterial killing, which ultimately can result in a
62
better treatment outcome.
63
One of the promising combinations is amikacin and clofazimine, given the key role
64
of amikacin in the treatment of NTM infections (2,3) and the unique characteristics
65
of clofazimine, like its prolonged half-life, preferential accumulation inside
66
macrophages (4) and the recently found bactericidal activity only after two weeks
67
of treatment in the mouse model of tuberculosis (5).
68
Clarithromycin, on the other hand, has substantial in vitro and clinical activity
69
against Mycobacterium avium complex (MAC) and it has been long considered the
70
cornerstone for Mycobacterium abscessus treatment (3).Hence the examination of
71
its interaction with clofazimine is interesting.
3
72
Previous reports showed in vitro synergy between clofazimine and amikacin
73
against both rapidly and slowly growing NTM (1,6). The combination
74
clarithromycin-clofazimine also showed synergy against MAC strains in
75
checkerboard evaluation (7). These checkerboard titrations offer no information on
76
the mechanism of synergistic activity, the exact killing activity of these
77
combinations and its concentration dependence. We therefore investigated the
78
pharmacodynamic interactions between clofazimine and amikacin, and clofazimine
79
and clarithromycin, against two key NTM species, using time-kill assays analyzed
80
with two pharmacodynamic drug interaction models: response surface model of
81
Bliss independence (RSBI) and isobolographic analysis of Loewe additivity (ISLA)
82
(8,9).
83 84
Materials and Methods
85
Bacterial strains, media and antibiotics
86
Mycobacterium abscessus subspecies abscessus CIP 104536 (Collection of
87
Institute Pasteur) and Mycobacterium avium subspecies hominissuis IWGMT49
88
(International Working Group on Mycobacterial Taxonomy) type strains were used.
89
Stocks of each strain were preserved at -80°C in trypticase soy broth with 40%
90
glycerol and were thawed for each assay. Pure powders of amikacin,
91
clarithromycin and clofazimine obtained from Sigma-Aldrich were dissolved in
92
water, methanol and DMSO, respectively.
93 94
Susceptibility testing
4
95
MICs were determined at the beginning and at the end of the experiments,
96
following CLSI recommendations (10) by broth microdilution in cation-adjusted
97
Mueller Hinton broth (CAMHB); commercially available panels were used for
98
amikacin and clarithromycin (RAPMYCO Sensititre®, SLOWMYCO Sensititre®-Trek
99
Diagnostics/Thermo Fisher), while for clofazimine manual broth microdilution was
100
performed in Middlebrook 7H9broth (BD Bioscience). Unexposed M. abscessus
101
type strain was used as a quality control during all MIC determinations.
102 103
Mutation analysis
104
Mutation analysis of rrs and rrl was performed to detect mutations associated to
105
amikacin and clarithromycin resistance, respectively, in mycobacteria exposed to
106
the two antibiotics in the drug-drug interaction experiments. Briefly, mycobacterial
107
suspensions were heat-inactivated at 95°C for 30 min, then DNA was extracted
108
using MagNAPure LC (Roche Life Science). Relevant fragment of rrs and rrl were
109
amplified using previously described primers and PCR conditions (11,12).
110
Sequence analyses were performed to detect mutations at codon 1408 in rrs and
111
2058 and 2059 in rrl.
112 113
Time-kill assays for single drugs
114
The inoculum consisted of mycobacteria in the early logarithmic phase of growth.
115
Individual bottles of 20 ml of Middlebrook 7H9 with oleic acid-bovine albumin-
116
dextrose-catalase growth supplement (BD Bioscience) and 0.05% Tween 80,
117
containing increasing concentrations (from 0.062 to 8 times the MIC for M.
118
abscessus and 0.062 to 32 times MIC for M. avium) of clofazimine were cultured 5
119
with the inoculum ( ~105 – 106 CFU/ml) at 30°C for M. abscessus and 37°C for M.
120
avium, under shaking conditions at 100 rpm (GFL); ventilation through a bacterial
121
filter (FP 30/0.2 Ca/S, Whatman GmbH) was incorporated. A drug-free and
122
inoculum-free bottle with medium served as the growth and sterility control,
123
respectively. At defined time intervals (3, 6, 12, 24, 36, 48, 72, 96 and 120 h for M.
124
abscessus and 12, 24, 36, 48, 72, 96, 120, 144, 168 and 240 h for M. avium), ten
125
μl from undiluted and serially ten-fold diluted in normal saline samples were plated
126
in triplicate on Middlebrook 7H11 agar plates (BD Bioscience). Plates were
127
incubated for 3-5 days at 30°C for M. abscessus, or 7-9 days at 37°C for M. avium
128
and the CFU/ml were calculated (lower limit of detection: 33.3 CFU/ml).
129 130
Time-kill assays for drug-drug interaction
131
Following the same protocol described above, time-kill assays for M. abscessus
132
and M. avium were performed with four concentrations (from 0.25 to 2 times the
133
MIC) of each antibiotic alone and in paired combination with clofazimine. MICs for
134
amikacin, clarithromycin and clofazimine were checked by duplicate at the final
135
sampling time, when colonies were present.
136 137
Pharmacodynamic drug interaction analysis
138
In order to assess the nature and the magnitude of the in vitro interactions between
139
clofazimine and amikacin or clarithromycin against NTM, the data obtained with
140
time-kill assays were analyzed using the response surface analysis of Bliss
141
independence (RSBI) and the isobolographic analysis of Loewe additivity (ISLA) as
142
described previously (8,9). The RSBI was used to describe the kinetics of 6
143
pharmacodynamic interactions at different time points whereas the ISLA was used
144
to describe the full concentration-effect relationships of the drugs alone and in
145
combination at the end of the experiment.
146
For that purpose, the percentage of bacterial load at each drug concentration and
147
combination was calculated by dividing the log10 CFU/ml with the log10 CFU/ml of
148
growth control at each sampling times throughout the experiment. Bliss synergy
149
and antagonism was concluded if the observed bacterial load was statistically
150
significantly (p64 mg/liter vs. 4 mg/liter) in bacteria exposed to clarithromycin
242
alone. Again, this increase was not observed in colonies exposed to the
243
combination of clofazimine-clarithromycin. The A2058G mutation in rrl was found
244
only in colonies exposed to 0.25 and 0.5×MIC clarithromycin alone, not in colonies
245
exposed to any of the clofazimine-clarithromycin combinations.
246 247
Interaction analysis
248
The RSBI analysis of the combination of clofazimine-amikacin against M.
249
abscessus showed independent interactions within the first 12 h which converted
250
to antagonistic interactions at 24-48 h and ultimately to synergistic interaction of
251
29.0% and 28.4% at the end of the experiment for 2 and 1×MIC respectively
252
(Figure 4a). Isobolographic analysis of the full concentration-effect relationship of
253
the same combination showed synergy at the end of the experiment at
254
concentrations ≥ 1×MIC with an I of 0.41, 95%CI 0.29-0.55 (Figure 5a); stasis was
255
achieved at 4 and 8×MIC of amikacin and clofazimine alone; for the combination,
256
stasis was found at 1×MIC of both drugs.
257
The RSBI analysis of the combination clofazimine-clarithromycin versus M.
258
abscessus also showed independence the first 12 h which converted to
259
antagonistic interactions at high concentrations, but ultimately synergistic
260
interaction of 23.5% were observed only at 2×MIC (Figure 4b). Similarly, the ISLA 11
261
of the same combination showed synergistic interaction reaching stasis at 2×MIC
262
when drugs were combined. For each drug alone the endpoint was reached at
263
8×MIC (I=0.53, 95%CI 0.35-0.72 (Figure 5b).
264
For the combination clofazimine-amikacin against M. avium, the RSBI analysis
265
showed independence up to 48 h, then antagonism up to 120 h and synergy at the
266
end of the experiment, of 23.2-56.7% at all concentrations (Figure 4c). Strong
267
synergy was found with the ISLA, (I=0.027, 95%CI 0.007-0.048) since stasis was
268
found at 0.5×MIC of the drugs in combination; the same endpoint was reached at
269
8×MIC and>32×MIC for clofazimine and amikacin alone(Figure 5c). The same
270
profile of interactions was found for the combination clofazimine-clarithromycin;
271
with RSBI analysis independence was observed up to 48 h, then antagonism up to
272
120 h and ultimately synergy. However, the synergistic interactions were found at
273
concentrations 0.25 and 0.5×MIC (53.3% and 23.3%, respectively) (Figure 4d).
274
This was also confirmed with the ISLA (I= 0.16, 95%CI 0.04-0.35) with stasis
275
observed at combinations with 1×MIC of each drug the
278
effect was similar to the effect of clarithromycin alone indicating no interaction as
279
the RSBI analysis showed at the end of the experiment.
280 281
Monte Carlo simulations
282
The percentage of patients that will attain lung concentrations associated with
283
stasis of both M. abscessus and M. avium are shown in Figure 6. For M.
284
abscessus, target attainment rates with clofazimine-amikacin and clofazimine12
285
clarithromycin combinations were higher than monotherapy regimens particularly
286
for isolates with amikacin/clofazimine MIC 16/0.25 and clarithromycin/clofazimine
287
MICs 2/0.25, respectively (>60%difference between combination and monotherapy
288
regimens). For M. avium,>60% difference was found for the clofazimine-amikacin
289
combination for isolates with amikacin/clofazimine MICs 32-128/0.25-1 mg/liter and
290
for the clofazimine-clarithromycin combination for isolates with
291
clarithromycin/clofazimine MICs 32-124/0.5-2 mg/liter. High (>95%) and low (
