Defining Fractional Inhibitory Concentration Index Cutoffs for Additive ...

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Jul 16, 2009 - antagonistic combination AMB plus ravuconazole were higher than 1.25. Adequate insight into weak pharmaco- dynamic interactions with in ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2010, p. 602–609 0066-4804/10/$12.00 doi:10.1128/AAC.00999-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 54, No. 2

Defining Fractional Inhibitory Concentration Index Cutoffs for Additive Interactions Based on Self-Drug Additive Combinations, Monte Carlo Simulation Analysis, and In Vitro-In Vivo Correlation Data for Antifungal Drug Combinations against Aspergillus fumigatus䌤 Joseph Meletiadis,1,4* Spyros Pournaras,2 Emmanuel Roilides,3,4 and Thomas J. Walsh4 Laboratory of Clinical Microbiology, Attikon University General Hospital, National and Kapodistrian University, Athens, Greece1; Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece2; Third Department of Pediatrics, Aristotle University, Hippokration Hospital, Thessaloniki, Greece3; and Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland4 Received 16 July 2009/Returned for modification 11 August 2009/Accepted 27 November 2009

The fractional inhibitory concentration (FIC) index range of 0.5 to 4 that is commonly used to define additivity results in no interactions in most combination studies of antifungal agents. These results may differ from those of in vivo studies, where positive and negative interactions may be observed. We reassessed this in vitro FIC index range based on (i) the experimental variation of the checkerboard technique using multiple replicates, (ii) the ability to correctly determine purely additive self-drug and two-drug antagonistic combinations of amphotericin B (AMB) and voriconazole (VRC), (iii) Monte Carlo simulation analysis, and (iv) in vitro-in vivo correlation using experimental models of invasive pulmonary aspergillosis against the same Aspergillus fumigatus isolate based on visual, spectrophotometric, and colorimetric determinations of FICs after 24 and 48 h of incubation. FICs obtained after 24 h of incubation ranged from 0.5 to 1.25 for the self-drug additive combinations of AMB plus AMB and VRC plus VRC and from 2.25 to 4.25 for the antagonistic combination of AMB plus VRC. Monte Carlo simulation analysis showed that self-drug combinations were correctly classified as additive and that the combination of AMB plus VRC was correctly classified as antagonistic for >85% of the simulated FICs when deviation of the 95% confidence interval (CI) of replicate FICs from the additivity range of 1 to 1.25 was used to assess interactions after 24 h. In vitro-in vivo correlation analysis showed that the 95% CIs of the FICs of the in vivo synergistic combination anidulafungin plus VRC determined after 24 h were lower than 1 and the 95% CIs of the FICs of the in vivo antagonistic combination AMB plus ravuconazole were higher than 1.25. Adequate insight into weak pharmacodynamic interactions with in vivo relevance may be obtained by demonstrating that triplicate FICs at 24 h are outside an inclusive additivity range of 1 to 1.25. tance for the development of improved strategies for the management of invasive aspergillosis. Among several in vitro methodologies developed to assess in vitro pharmacodynamic interactions, microdilution broth checkerboard techniques are commonly used to study antifungal combinations. Checkerboard data can be analyzed with different pharmacological mathematical models developed to detect deviations from no-interaction theories and to determine synergistic and antagonistic interactions (8). In vitro antifungal combinations are usually assessed on the basis of the fractional inhibitory concentration (FIC) index, which represents the sum of the FICs of each drug tested, where the FIC is determined for each drug by dividing the MIC of each drug when used in combination by the MIC of each drug when used alone. The FIC index is based on the Loewe additivity zerointeraction theory (4). This theory is based on the hypothesis that a drug cannot interact with itself and therefore the effect of a self-drug combination will always be additive, with an FIC index of 1. An FIC index lower or higher than 1 indicates synergy or antagonism, respectively, because less or more drug would be required in order to produce the same effect as the drugs alone (4). Because of the twofold drug dilution scheme and the 1-di-

Invasive pulmonary aspergillosis is a life-threatening infectious disease, particularly for immunocompromised patients (23). Mortality rates remain high despite the significant progress made in antifungal chemotherapy with the development of new classes of antifungal agents, such as the echinocandins, triazoles, and lipid formulations of amphotericin B (AMB) (10). In search of more-effective chemotherapeutic approaches for treating invasive aspergillosis, combination therapy is an important strategy, as synergistic interactions can potentially increase antifungal efficacy, reduce toxicity, cure faster, prevent the emergence of resistance, and provide broader-spectrum antifungal activity than monotherapy regimens (15). However, combination therapy may also be deleterious in the case of antagonistic interactions, decreasing antifungal efficacy and increasing toxicity (12). Distinguishing synergistic from antagonistic combinations is of major impor-

* Corresponding author. Mailing address: Laboratory of Clinical Microbiology, Attikon University General Hospital, 1 Rimini Str., Haidari, Athens 124 62, Greece. Phone: 30-210-583-1909. Fax: 30210-532-6421. E-mail: [email protected]. 䌤 Published ahead of print on 7 December 2009. 602

VOL. 54, 2010

DEFINING FIC INDEX CUTOFFS FOR ADDITIVE INTERACTIONS

lution error of single-drug susceptibility testing methods used for FIC index determination, cutoffs of 0.5 and 4 were suggested for defining additivity/indifference (1, 12, 22). However, the actual variation of the FIC index in checkerboard microdilution methods was not well studied. In addition, most in vitro combination studies resulted in FIC indices within the range of 0.5 to 4 concluding no interaction (additivity/indifference), raising questions about the validity of this arbitrarily chosen FIC range given the absence of in vitro-in vivo correlation studies. Thus, in the present study, we applied the Loewe additivity theory for self-drug combinations of AMB and voriconazole (VRC), two drugs with different modes of action and concentration-effect curves, using a modification of the CLSI M38-A2 broth microdilution technique. We then determined by replication the actual variation of the FIC index for these purely additive self-drug combinations. Because the variation of selfdrug combinations may be different from that of the two-drug combinations, we also studied the variation of the combination of AMB and VRC. The ability of FIC index analysis to correctly determine a purely additive self-drug combination and the two-drug antagonistic combination of AMB and VRC was assessed by Monte Carlo simulation analysis. Finally, we compared the results of two in vivo combination therapy studies using experimental models of invasive pulmonary aspergillosis with in vitro combination studies using FIC index analysis. MATERIALS AND METHODS Isolate and medium. A well-characterized strain of Aspergillus fumigatus (isolate 4215) obtained from a histologically proven fatal case of pulmonary aspergillosis was maintained at ⫺70°C on Sabouraud glucose agar and revived by subculturing at 30°C. A modification of the CLSI M38-A2 method was used in order to prepare the inoculum suspensions (5). An inoculum suspension was prepared from 5- to 7-day-old cultures with a counting chamber in order to obtain two times the final inoculum, which was 2 ⫻ 104 conidia/ml in the medium used throughout the experiments. The nutrient medium was RPMI 1640 medium with L-glutamine and without bicarbonate buffered to pH 7.0 with 0.165 M 3-Nmorpholinopropanesulfonic acid (Cambrex Bio Science Inc., Walkersville, MD). Candida krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality control strains. Susceptibility testing. Susceptibility testing of the isolate was performed according to the CLSI M38-A2 method (6). Twofold serial dilutions of AMB (Apothecon Ben Venue Laboratories, Inc., Bedford, OH) and VRC (Pfizer Pharmaceuticals, New York, NY) were prepared in 50-ml tubes with the medium in order to obtain four times the final concentration, which ranged from 0.015 to 4 mg/liter and from 0.03 to 8 mg/liter, respectively. Fifty microliters of each concentration of the first series of tubes was combined with 50 ␮l of the second series of tubes in 96-well flat-bottom microtitration plates in order to obtain three different 8-by-11 checkerboard designs: AMB plus AMB, VRC plus VRC, and AMB plus VRC. After inoculation with 100 ␮l of 4 ⫻ 104 conidia/ml, the microtitration plates were incubated at 37°C for 48 h and the MICs of the drugs alone and the drug concentrations in isoeffective combinations were determined by the following three methods. All replicate experiments were blindly and independently performed nine times on different days and with individually prepared inocula. (i) Visual (VIS) method. Hyphal growth in each well was assessed visually after 24 and 48 h of incubation with a magnifying reading mirror according to M38-A2 guidelines. The MICs of the drugs alone and the isoeffective combinations were determined as the lowest drug concentrations showing no visual growth (optically clear well). (ii) Spectrophotometric (SPE) method. The hyphal biomass in each well was measured spectrophotometrically at 630 nm after 24 and 48 h of incubation. Percent growth was calculated based on the measured optical density (OD) of each well with the equation 100% ⫻ (ODwell ⫺ ODbackground)/(ODdrug-free well ⫺ ODbackground), where the background was measured in a conidium-free plate (17). The MICs of the drugs alone and of all isoeffective combinations were

603

determined as the lowest drug concentrations showing ⬍10% of the growth of an untreated control. (iii) Colorimetric (COL) method. Fungal metabolism was assessed by the COL method with 2,3-bis{2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium-hydroxide} (XTT) (16). After 48 h of incubation, 50 ␮l of a 0.5-mg/liter concentration of XTT with 125 ␮M menadione (Sigma-Aldrich, St. Louis, MO) was added to each well. The plate was incubated for a further 2 h, after which the color absorbance was measured spectrophotometrically at 450/630 nm. Percent growth was calculated on the basis of color absorbance (A) as 100% ⫻ (Awell ⫺ Abackground)/(Adrug-free well ⫺ Abackground) where the background was measured from a conidium-free plate. The MICs of the drugs alone and of all isoeffective combinations were determined as the lowest drug concentrations showing ⬍10% of the growth of an untreated control. FIC index analysis. For all of the wells of the microtitration plates that corresponded to an MIC, the sum of the FICs (⌺FIC) was calculated for each well with the equation ⌺FIC ⫽ FICA ⫹ FICB ⫽ (CA/MICA) ⫹ (CB/MICB), where MICA and MICB are the MICs of drugs A and B alone, respectively, and CA and CB are the concentrations of the drugs in combination, respectively, in all of the wells corresponding to an MIC (isoeffective combinations). Among all of the ⌺FICs calculated for all isoeffective combinations, we reported the minimum ⌺FIC (⌺FICmin) and the maximum ⌺FIC (⌺FICmax) in order to capture synergistic and antagonistic interactions, respectively (19). We used both ⌺FICmin and ⌺FICmax for all of the data sets in order to get the most information about pharmacodynamic interactions. Monte Carlo simulation analysis. In order to approximate normal distribution, the FICs were transformed to log2 values and the average and standard deviation (SD) were calculated. After passing the D’Agostino-Pearson normality test (GraphPad Prism, San Diego, CA), the mean and SD of log2 ⌺FIC were used in a Monte Carlo analysis in order to simulate 100 experiments performed in triplicate for each combination. For each experiment, the interaction was assessed on the basis of whether the 95% confidence interval (CI) (based on t distribution) of the triplicate log2 ⌺FICs included a cutoff (additive interaction) or not (nonadditive interaction). The number of experiments was recorded where the self-drug combination was not classified as additive and the combination of AMB plus VRC was not classified as the most common interaction (antagonistic) among the 100 simulated experiments. In vitro-in vivo correlation. To find out whether the additivity range determined in the present study correlates with the in vivo outcome, the results of two previously published studies of combination therapy against experimental invasive pulmonary aspergillosis were correlated with the in vitro combination studies of the present study based on FIC index analysis. For that purpose, we chose a synergistic and an antagonistic in vivo combination therapy against experimental aspergillosis using the same A. fumigatus strain used in the present study (variation due to the pathogen is eliminated), the same rabbit model (variation due to the animal model is minimized), and the antifungal agents used in the present study. We previously found that combination therapy against experimental invasive pulmonary aspergillosis with liposomal AMB and ravuconazole was antagonistic (20), whereas combination therapy with VRC and anidulafungin was synergistic (25) based on the analysis of several biomarkers of outcome (survival, pulmonary infarct lesions, lung weight, residual fugal burden, galactomannan levels, and computed tomography scan scores). Thus, in the present study, AMB was combined with ravuconazole and VRC was combined with anidulafungin by the same microdilution checkerboard methodology used in the present study to study self-drug and AMB-plus-VRC combinations as described above. The final drug concentrations were 8 to 0.125 mg/liter for AMB, 2 to 0.004 mg/liter for ravuconazole, 1 to 0.016 mg/liter for VRC, and 0.25 to 0.0005 mg/liter for anidulafungin. Percent growth in each well was calculated based on turbidity after 24 and 48 h, and XTT conversion was measured after 48 h as described above. MICs and ⌺FICs were determined as described above. All experiments were performed in triplicate.

RESULTS Table 1 summarizes the results of FIC index analysis for the two self-drug combinations used in the present study. A total of 270 FICs were calculated, 90 for each self-drug combination and 90 for the combination of AMB plus VRC. Self-drug combinations. The self-drug additive combination of AMB plus AMB resulted in a 冱FICmin range of 0.52 to 1.02 (median of 1 for all of the methods) and a 冱FICmax range of 1.13 to 1.5 (median of 1.25 for all of the methods) (Table 1).

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TABLE 1. ¥FICmins and ¥FICmaxs of nine independent replicates for self-drug and two-drug combinations for each method and incubation period Method, time (h), and index

Median (range) with: AMB ⫹ AMB

VRC ⫹ VRC

AMB ⫹ VRC

VIS 24 ¥FICmin 1 (0.63–1) 0.75 (0.63–1) ¥FICmax 1.25 (1.13–1.25)a 1.25 (1.13–1.25)a

1.06 (1.06–1.06) 2.5 (2.25–4.25)a,b

48 ¥FICmin 1 (0.75–1.02) ¥FICmax 1.25 (1.25–1.5)a

1 (0.51–1.01) 1 (0.5–1.03) 1.25 (1.125–2.13)a 2.25 (1.5–2.25)a,b

SPE 24

TABLE 2. Percentages of ¥FICs with 95% CIs overlapping the additivity ranges 1 to 1.25 and 0.75 to 1.25 for the self-drug additive combinations AMB plus AMB and VRC plus VRC and the nonadditive combination AMB plus VRC for 100 simulated checkerboards based on Monte Carlo analysis

Method, time (h), and index

% of values with 95% CIs overlapping indicated additivity range for: AMB ⫹ AMB

VRC ⫹ VRC

AMB ⫹ VRC

1–1.25

0.75–1.25

1–1.25

0.75–1.25

1–1.25

0.75–1.25

90 100

100 100

88 100

100 100

100 5

100 5

95 96

100 96

92 95

100 95

97 5

100 5

90 100

100 100

12a 100

80 100

95 1

100 1

94 100

100 100

78 87

94 87

91 1

97 1

100 96

100 96

88 98

99 98

93 1

100 1

VIS 24 ¥FICmin ¥FICmax 48

¥FICmin 1 (0.63–1) 0.63 (0.5–0.75)a ¥FICmax 1.25 (1.13–1.25)a 1.13 (1.06–1.25)a 48 0.56 (0.5–1)a ¥FICmin 1 (0.52–1) ¥FICmax 1.25 (1.13–1.25)a 2.03 (1.13–2.25)a COL, 48 ¥FICmin ¥FICmax

1 (1–1.02) 1.25 (1.25–1.5)a

0.75 (0.5–1)a 1.25 (1.06–1.5)a

¥FICmin ¥FICmax

1.06 (0.56–1.06) 2.25 (2.25–2.5)a,b 1.03 (0.28–1.06) 2.25 (2.06–2.5)a,b

1.03 (0.53–1.03) 2.25 (2.25–2.5)a,b

a Significantly different than 1 after log2 transformation (one-sample t test, P ⬍ 0.05). b Significantly different from the ¥FICmaxs of the self-drug combinations AMB plus AMB and VRC plus VRC.

SPE 24 ¥FICmin ¥FICmax 48 ¥FICmin ¥FICmax COL, 48 ¥FICmin ¥FICmax

a Using a cutoff of 5% to determine MICs, ¥FICmins were within the additivity range for 85% of the simulations.

The self-drug additive combination of VRC plus VRC resulted in a 冱FICmin range of 0.5 to 1.01 (with medians ranging from 0.56 to 1 for all of the methods) and a 冱FICmax range of 1.06 to 1.25 after 24 h and 1.13 to 2.25 after 48 h (with medians ranging from1.13 to 2.03 for all of the methods). The 冱FICmins of the self-drug combination of VRC plus VRC showed significant deviation from 1 by the SPE method. Most of the 冱FICs of self-drug additive combinations ranged from 0.5 to 2. Notably, none of the self-drug additive combinations resulted in an 冱FICmax of 4. Combination of AMB plus VRC. For the antagonistic combination of AMB plus VRC, all of the 冱FICmins were between 0.5 and 1.06, except one (0.28 with the SPE method at 48 h) and all 冱FICmaxs were higher than 1.5 after 48 h and higher than 2.06 after 24 h (Table 1). The 冱FICmins of AMB plus VRC were not significantly different from the 冱FICmins of the self-drug combinations, whereas the 冱FICmaxs of AMB plus VRC were significantly higher than the 冱FICmaxs of the selfdrug combinations (Table 1). These results indicate that a combination with an 冱FICmax of ⱖ1.5 (i.e., ⬎1.25 based on Fig. 1) is most likely to be antagonistic. The median 冱FICmins were very close to 1 for all of the methods, indicating that there were no synergistic interactions. Monte Carlo simulation analysis. Table 2 summarizes the Monte Carlo simulation analysis results. A total of 9,000 experiments (1,500 冱FICmins and 1,500 冱FICmaxs in triplicate) were simulated on the basis of the central estimates and variation calculated from nine replicates. The central estimates of 冱FIC distributions are shown in Table 1, whereas the SDs of

the log2 冱FIC ranged from 0.4 to 0.01 (median, 0.2), with the log2 冱FICmin showing larger SDs than the log2 冱FICmax (0.33 versus 0.13). Based on the 95% CIs of triplicate 冱FICmins and 冱FICmaxs, correct assessment of the additive self-drug combinations of AMB and VRC were found by the VIS and COL methods in ⬎88% and ⬎95% of the simulations using additivity ranges of 1 to 1.25 and 0.75 to 1.25, respectively. For the SPE methods, the corresponding values are ⬎12% for the additivity range of 1 to 1.25 and ⬎80% for the additivity range of 0.75 to 1.25 (the percentage of FICmins within the additivity range of 1 to 1.25 of the SPE method at 24 h increased from 12 to 85% using a 5% instead of a 10% fungal growth cutoff for MIC determination). Of the AMB-plus-VRC combinations, ⬍5% were wrongly classified as additive by all of the methods based on the 冱FICmax, whereas ⬎91% and ⬎97% of the simulated 冱FICmins were within the additivity ranges of 1 to 1.25 and 0.75 to 1.25, respectively (Table 2). Thus, deviation of triplicate 冱FICs from the range of 1 to 1.25 and with even greater certainty from the range of 0.75 to 1.25 can be used to determine pharmacodynamic interaction by all of the methods tested. In order to visualize exactly where in the microdilution checkerboard the additivity ranges lie and which and how many wells are included in these ranges, a photograph of the self-drug combination of AMB plus AMB with the COL method at 48 h is shown in Fig. 1 together with the two MICs,

VOL. 54, 2010

Amphotericin B (mg/l)

DEFINING FIC INDEX CUTOFFS FOR ADDITIVE INTERACTIONS

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Amphotericin B (mg/l)

FIG. 1. Self-drug combination of AMB by the XTT COL method (a classical example of an additive interaction) with an FICmin of 1 and an FICmax of 1.25. Of note, the commonly used additivity range of 0.5 to 4 encompasses a great area of the checkerboard and it contains two series of clear wells and one series of wells with growth, i.e., a stricter criterion for antagonistic interactions than for synergistic interactions. Because of experimental variation, the suggested additivity range encompasses 冱FICs of 1 to 1.25 inclusive. Values inside the wells are the 冱FICs if the wells were clear.

the 冱FICs for each well, and the additivity ranges. Of note, an asymmetry of the 0.5-to-4 additivity range (gray lines) for synergistic (one row of colored wells) and antagonistic (two rows of uncolored wells) interactions exists compared to the 0.5-to-2 additivity range (black line). The additivity range of 1 to 1.25 includes one row of wells on the border between the wells with growth and those without growth. In vitro-in vivo correlation. The results of in vitro-in vivo correlation studies of the combinations of VRC plus anidulafungin and AMB plus ravuconazole are shown in Fig. 2 and summarized in Table 3. Combination therapy with VRC plus anidulafungin was previously found to significantly improve the efficacy of antifungal therapy compared to that obtained with each monotherapy based on several biomarkers of outcome, indicating a synergistic in vivo combination (Fig. 2) (25). The in vitro studies of the combination of VRC plus anidulafungin by all three methods resulted in 冱FICmins lower than 0.5 after 24 h of incubation and lower than 1 after 48 h (Table 3). All 冱FICmaxs ranged from 1 to 1.13, except that obtained for one replicate by the SPE method at 24 h, which was 2.01. However, when a lower cutoff of percent fungal growth was used for MIC determination (⬍5%), the 冱FICmax for that replicate was 1.01. The 95% CI of 冱FICmin was lower than 1 and the 95% CI of 冱FICmax was not higher than 1.25 with all of the methods and incubation periods tested. Combination antifungal therapy with AMB plus ravuconazole was previously found to have reduced efficacy compared to that of each monotherapy based on several biomarkers of outcome, indicating an antagonistic in vivo combination (Fig. 2) (20). In vitro studies of the combination of AMB plus ravuconazole by all three methods resulted in 冱FICmaxs higher than 2 only after 24 h, whereas after 48 h the 冱FICmaxs obtained with all of the methods ranged from 1.06 to 1.5 (Table 3). Notably, none of the 冱FICmaxs was higher than 4. The 95% CI of the 冱FICmin was not lower

than 1, whereas the 95% CI of the 冱FICmax was higher than 1.25 for the 24-h-based methods. For an analytical method to be valid, a synergistic combination should result in 冱FICmins lower than the 冱FICmins of an antagonistic combination and an antagonistic combination should result in 冱FICmaxs higher than the 冱FICmaxs of a synergistic combination. The 冱FICmins of the synergistic combination of VRC plus anidulafungin were significantly lower than the 冱FICmins of the antagonistic combination of AMB plus ravuconazole, and the 冱FICmaxs of the antagonistic combination of AMB plus ravuconazole were significantly higher than the 冱FICmaxs of the synergistic combination of VRC plus anidulafungin after 24 h, but not after 48 h, of incubation. DISCUSSION Self-drug combinations are convenient models of additivity, and their experimental properties can give valuable insights into the significance of synergistic and antagonistic interactions of dissimilar drugs (2, 11). Therefore, we prepared self-drug combinations, as well as two-drug combinations of antifungal drugs with different modes of action and concentration-effect curves. The results were evaluated by FIC index analysis in order to determine cutoffs for detecting synergy and antagonism. FIC index analysis of the self-drug additive combinations of AMB plus AMB and VRC plus VRC resulted in 冱FICmins ranging from 0.52 to 1.02 and in 冱FICmaxs ranging from 1.13 to 2.31 for all of the methods and incubation periods tested. Notably, the 冱FICmax range was narrower after 24 h (up to 1.25) than after 48 h (up to 2.31). In addition, the 冱FICmaxs of the antagonistic combination of AMB plus VRC were, most of the time, greater than 2 but rarely higher than 4. As shown in Fig. 1, the additivity range of 0.5 to 2 is more symmetrical than the additivity range of 0.5 to 4 that is usually recommended. The additivity range of 0.5 to 4 is broader for antagonism than for synergy since it contains two rows of wells for antagonism

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Voriconazole + Anidulafungin (Synergisc combinaon)

Amphotericin B + Ravuconazole (Antagonisc combinaon) In vitro spectrophotometric 24h

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0.0039 0.0078 0.0156 0.0313 0.0625

0.125

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Amphotericin B (mg/l)

Voriconazole (mg/l)

In vitro spectrophotometric 24h

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0

2%

0%

1%

1%

2%

0%

0%

0%

0%

4%

90%

0%

4%

3%

1%

0%

0%

1%

0%

0%

0%

3%

0%

0%

1%

0%

0%

0%

0%

0%

0%

2%

62%

1

0%

1%

0%

2%

3%

1%

0%

0%

0%

2%

4%

81%

78%

0.5

84% 100%

70% 100%

62%

57% 100% 100% 100%

44%

9% 100%

0.25

100% 100%

95% 100%

97% 100% 100% 100% 100%

84%

7% 100%

0.125 100% 100% 98% 100% 100% 100% 100% 100% 100% 0 100% 100% 100% 100% 100% 100% 100% 100% 100%

31%

1%

60%

41%

0%

69%

0. 0039 0. 0078 0. 0156 0. 0313 0. 0625

3%

2%

2%

2%

2%

2%

2%

0%

2%

90%

18%

10%

12%

11%

13%

8%

2%

3%

1%

88%

0.25

94%

65%

92%

76%

74% 100%

66%

56%

37%

27%

19%

93%

0. 125

0. 25

0. 5

1

2

0

84%

0.125 100% 100% 100% 100% 100% 100% 100% 0.063 100% 100% 100% 100% 100% 95% 96% 0.031 100% 100% 100% 100% 100% 100% 100%

82%

87%

71% 100%

78% 100%

93%

82%

95%

91% 100%

98% 100%

0.016

90% 100% 100% 100% 100% 100% 100%

84%

69%

92% 100%

69% 100% 100% 100% 100% 100%

71%

79%

0.001

0.002

95%

0.0039 0.0078 0.0156 0.0313 0.0625

96% 90% 0.125

93% 0.25

96%

89%

8

0%

0%

0%

0%

0%

0%

0%

0%

0%

0%

0% 100%

4

0%

0%

0%

0%

0%

0%

0%

0%

0%

0%

0%

2

0%

0%

0%

0%

0%

0%

0%

0%

0%

0%

0% 100%

0%

0%

0%

0%

0%

0%

0%

0%

0% 100%

1 0.5 0.25

0%

3%

100% 100% 100% 100% 100% 100% 100% 100% 100%

28%

0%

99%

99%

88% 100% 100% 100% 100% 100% 100%

88%

62%

57%

0% 100%

0.125 100% 100% 100% 100% 100% 100% 100% 0 100% 100% 100% 100% 100% 100% 77%

78%

61%

23%

0%

77%

80%

23%

0%

67%

0

0

0.0039 0.0078 0.0156 0.0313 0.0625

Anidulafungin (mg/l)

0.125

90% 0.25

0.5

1

2

0

Ravuconazole (mg/l)

In vivo experimental aspergillosis

In vivo experimental aspergillosis

60

60 Lung weight (gr)

Lung weight (gr)

67% 77%

0%

0

Amphotericin B (mg/l)

Voriconazole (mg/l)

1% 16%

40

20

0

0% 0% 2

In vitro colorimetric 48h

6% 14%

0.0005

1

Ravuconazole (mg/l)

0.5

0

0.5

2

0

In vitro colorimetric 48h

0

0.25

4

Anidulafungin (mg/l)

1

0.125

13% 37%

In vitro spectrophotometric 48h

0%

Amphotericin B (mg/l)

Voriconazole (mg/l)

0%

76% 100% 100%

Ravuconazole (mg/l)

In vitro spectrophotometric 48h 0%

92% 100%

0.0039 0.0078 0.0156 0.0313 0.0625

Anidulafungin (mg/l)

1

87%

2

Control

AFG5

AFG10

VRC10X3

VRC+AFG5 VRC+AFG10

40

20

0

Control

LAMB1.5

LAMB3

RAV5

RAV5+LAMB1.5 RAV5+LAMB3

FIG. 2. Schematic representation of the in vitro and in vivo results obtained with the synergistic combination of anidulafungin plus VRC and the antagonistic combination of AMB plus ravuconazole. In vitro checkerboards based on data obtained by the SPE method after 24 and 48 h of incubation and by the COL assay using XTT after 48 h of incubation with A. fumigatus strain 4215 are shown. The numbers in the checkerboards represent growth percentages calculated by dividing the OD of each well with the average OD of the drug-free wells of the last column after subtracting the corresponding background OD. The percentages were normalized to span a range of 0 to 100%. For details about the FIC indices, see Table 3. The lung weights obtained in the experimental rabbit model of invasive pulmonary aspergillosis with A. fumigatus strain 4215 from references 20 and 25 are also shown as a measure of the in vivo fungal burdens for the synergistic combination of anidulafungin plus VRC and the antagonistic combination of AMB plus ravuconazole.

(up to 2 dilutions higher than the MIC) and one row of wells for synergy (down to 1 dilution lower than the MIC) in Fig. 1. The additivity range of 0.5 to 2 is symmetrical because it includes concentrations 1 dilution higher and lower than the FIC⫽1 and it accounts for the 1-dilution error of the MIC in the microdilution susceptibility testing methods. The validity of this additivity range is also supported by the in vitro-in vivo correlation data of the present study. The antagonistic combination therapy of AMB plus ravuconazole against experimental aspergillosis corresponded to an in vitro 冱FICmax range of 2.13 to 3, whereas the in vivo synergistic combination therapy of VRC plus anidulafungin resulted in 冱FICmins of 0.18 to 0.38 after 24 h of incubation. Interestingly, the 冱FICmax fell below 2 to 1.06 to 1.5 and the FICmin increased to 0.63 to 0.75 after 48 h of incubation. It seems that after 48 h of incubation, pharmacodynamic interactions disappear or weaken because of emerging growth at concentrations near the MICs, as shown in Fig. 2. Thus, 24 h of incubation may be preferred for studying antifungal combinations.

Comparing the two self-drug combinations of AMB and VRC, 冱FICmaxs were not significantly different but 冱FICmins of VRC plus VRC were lower than the 冱FICmins of AMB plus AMB, particularly for the SPE method. This may be due to the cutoff used to determine MICs based on the percentage of growth measured spectrophotometrically. Decreasing the cutoff from 10% to 5% resulted in an increase of the 冱FICmin (data not shown). The same was found in Table 3, where the 冱FICmax of the combination of anidulafungin plus VRC was 2.01 with a 10% cutoff for MIC determination and 1.01 with a 5% cutoff for MIC determination. This can be explained by the shallow concentration-effect curve of VRC, which results in checkerboard data with many wells with intermediate growth that otherwise would not be detected in single-drug experiments using twofold dilutions and that cannot be detected visually. For example, an MIC of 2 mg/liter in a twofold dilution scheme may correspond to any concentration between 1 and 2 mg/liter. Combinations of VRC with itself and in general with drugs with shallow concentration-effect curves result in

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DEFINING FIC INDEX CUTOFFS FOR ADDITIVE INTERACTIONS

607

TABLE 3. Correlation between in vitro and in vivo drug combination data In vitro values of 3 replicates (95% CI)

Method, time (h), and index

Anidulafungin ⫹ VRC (synergistic in vivo)b,d

AMB ⫹ ravuconazole (antagonistice in vivo)b,e

VIS, 24 ¥FICmin ¥FICmax

0.18, 0.38, 0.25 (0.10–0.64) 1.01, 1.03, 1.01 (0.99–1.05)

1.01, 0.75, 1.01 (0.60–1.4) 2.13, 2.50, 2.25 (1.86–2.8)

Yes Yes

VIS, 48 ¥FICmin ¥FICmax

0.50, 0.63, 0.75 (0.37–1.02) 1.13, 1.06, 1.13 (1.01–1.20)

0.63, 1.01, 1.01 (0.43–1.7) 1.06, 1.50, 1.50 (0.81–2.2)

No No

SPE, 48 ¥FICmin ¥FICmax

0.38, 0.38, 0.25 (0.18–0.59) 1, 1.02, 2.01c (0.47–3.41)

0.75, 0.75, 1.5 (0.35–2.56) 2.13, 3, 2.5 (1.64–3.86)

Yes Yes

SPE, 48 ¥FICmin ¥FICmax

0.75, 0.63, 0.75 (0.54–0.92) 1.13, 1.06, 1.13 (1.01–1.2)

0.63, 0.63, 1.13 (0.33–1.77) 1.06, 1.5, 1.5 (0.81–2.2)

No No

COL, 48 ¥FICmin ¥FICmax

0.5, 0.5, 0.51 (0.5–0.51) 1, 1.03, 1.13 (0.91–1.22)

0.63, 0.63, 1.13 (0.33–1.77) 1.25, 1.5, 1.5 (1.09–1.83)

Yes No

Significantly differenta

a Analysis-of-variance P value of ⬍0.05 for a comparison of the ¥FICmin and ¥FICmax of the synergistic combination of anidulafungin plus VRC with the ¥FICmin and ¥FICmax of the antagonistic combination of AMB plus ravuconazole, respectively. b Based on residual fungal burden in the lungs, pulmonary infarct lesions, lung weight, galactomannan index, and survival. c Using a cutoff of 5% growth to determine MICs, the ¥FICmax of the third replicate was 1.01 instead of 2.01, resulting in a 95% CI of 0.99 to 1.06. d Reference 25. e Reference 20.

checkerboard data with more concentrations and therefore levels of growth. The SPE method has greater sensitivity in detecting and differentiating small amounts of fungal growth than the VIS method, which is a more approximate method for fungal growth quantitation. This property of the SPE method could result in more wells with intermediate fungal growth that inevitably would result in reducing the 冱FICmin, particularly for drugs with shallow concentration-effect curves like VRC. This phenomenon could be reduced by analytical tools employing modeling techniques increasing even further the sensitivity of the microdilution checkerboard technique to detect significant pharmacodynamic interactions (21). Other than this difference, the methods did not differ significantly for a certain incubation period. In order to narrow the additivity range and also be able to detect weak interactions, we calculated the FICs of the two self-drug combinations and the combination of AMB plus VRC from replicate experiments. Monte Carlo simulation analysis showed that self-drug interactions were correctly classified as additive on the basis of both 冱FICmin and 冱FICmax more than 88% (⬎78% for the SPE method). The combination of AMB plus VRC was correctly classified as antagonistic because the 95% CI of replicate 冱FICmins and 冱FICmaxs was within the additivity range of 1 to 1.25 more than 91% and ⬍5% of the time, respectively, with all methods. The validity of this analysis also was confirmed in the in vitro-in vivo correlation analysis, where the 95% CI of the 冱FICmins of the synergistic combination of VRC plus anidulafungin was lower than 1 and the 95% CI of the 冱FICmaxs of the antagonistic combination of AMB plus ravuconazole was higher than 1.25 (Table 1). Thus, replication may increase the sensitivity of FIC index analysis in detecting significant pharmacodynamic interactions.

The number of replicates required to detect significant pharmacodynamic interactions depends on the power of the study (usually 80%), the significance level (usually 5%), and the standardized difference, which is the SD of our data and the difference we want to detect. Thus, for an interaction with a mean log2 冱FICmin of ⫺1 (i.e., a 冱FICmin of 0.5) and an SD of 0.3 (i.e., 冱FICmins of 0.4, 0.5, and 0.6) or a mean log2 冱FICmax of 0.84 (i.e., a 冱FICmax of 1.8) and an SD of 0.2 (i.e., 冱FICmins of 1.5, 2, and 2), we need three replicates to detect a significant difference from 1 and 1.25, respectively. The number of replicates can be further reduced or remain the same and accept larger SDs, performing one-sided analyses. There is no need to perform a two-sided analysis, since we are only interested in testing whether the 冱FICmin is lower than 1 in order to capture synergistic interactions and the 冱FICmax is higher than 1.25 in order to capture antagonistic interactions. A previously published study (26) attempted to assess the reproducibility of microdilution checkerboard data for synergistic antibacterial combinations calculating the number of replicates with an FIC index of ⱕ0.5 and those with a higher FIC index and concluding discordant classification (a 7:3 or worse split in FIC distribution) for 25% of the cases, concluding that a final classification should be made whenever there is 80% agreement among the replicates (at least five). However, 0.5 is not a natural cutoff like 1, which derives from Loewe additivity theory. Inevitably, the 0.5 cutoff cuts the FIC distributions at any point resulting in different splits. In addition, the conclusion about 80% agreement is not statistically based. In another study where self-drug and two-drug antibacterial combinations were analyzed, even weak interactions with FICs between 0.5 and 0.99 were proven to be statistically significant (11). As found in the present study and in previously published

608

MELETIADIS ET AL.

studies, the results of the microdilution checkerboard method for analyzing antifungal combinations is very reproducible, with narrow ranges of FICs, which seldom range from synergistic to antagonistic values (18, 19). Thus, the perception that checkerboard data may be more variable than single-drug susceptibility data may be questioned. For example, there were data sets in the present study in which the interaction pattern inside the checkerboard was shifted to higher concentrations whenever the MIC of a drug was increased, resulting in the same FICs. A cutoff of 1 can also be supported pharmacologically on the basis of the theory of Loewe additivity. An additive combination of two drugs at 0.5 times the MICs should result in the same effect as that obtained at the MIC (e.g., complete growth inhibition). If less drug (e.g., 0.5 times the MIC of drug A and 0.125 times the MIC of drug B) or more drug (e.g., the MIC of drug A and 0.5 times the MIC of drug B) is required to inhibit fungal growth completely, a synergistic and an antagonistic interaction, respectively, should take place with a 冱FICmin of 0.625 and a 冱FICmax of 1.5, respectively. An additivity range of 1 to 1.25 is recommended here because interactions within this 冱FIC range cannot be determined with a twofold dilution checkerboard design. As shown in Fig. 2, 冱FICs of 1 to 1.25 never correspond to the 冱FICmax, which can take values like 1, 1.5, and ⬎2. Figure 2 also shows that there are no 冱FICs between 0.75 and 1, between 1.25 and 1.5, or between 1.5 and 2, etc., because of the twofold dilution scheme. However, one could obtain 冱FICs within those ranges by averaging 冱FICs from replicates or using less-than-twofold dilutions (2). Notably, there is no “indifference” category based on Loewe additivity theory because interactions are classified as either additive or nonadditive (synergistic and antagonistic). The term indifference is used instead of additivity only when one drug is inactive because there is no effect of an inactive drug to be added but only the effect of the active drug (4, 9). Another question that arises from the FIC index analysis is which cutoffs could be used to detect clinically important pharmacodynamic interactions. The general perception is that a strong pharmacodynamic interaction (with a very small 冱FICmin or a very large 冱FICmax) should have a greater impact on clinical outcome than interactions of smaller magnitude. Although there are no data to support this, our in vitro-in vivo correlation study showed that pharmacodynamic interactions of relatively small magnitude (冱FICmins of 0.25 to 0.38 and 冱FICmaxs of 2.13 to 3) had a great impact on the in vivo outcome of experimental pulmonary aspergillosis. Histopathological analysis of lung sections showed that the residual fungal burden in the lungs of rabbits receiving combination therapy with AMB plus ravuconazole (in vitro 冱FICmaxs of 2.13 to 3) is comparable to that in the lungs of untreated control rabbits and is more extensive than that in the lungs of rabbits receiving monotherapy (20). Combination therapy with VRC plus caspofungin or AMB plus caspofungin prolonged survival and reduced fungal burden in in vivo models of experimental aspergillosis, whereas in vitro combination studies by the microdilution checkerboard method resulted in FIC indices ranging from 0.5 to 1 (5, 7, 13, 14, 24, 27). Minor synergy or antagonism may be important in vivo because it can be augmented by pharmacokinetic and other factors, as described previously (3).

ANTIMICROB. AGENTS CHEMOTHER.

A lack of correlation between the in vitro FIC index and the in vivo outcome does not necessarily indicate an inability of the FIC index to detect pharmacodynamic interactions. When two drugs are combined in vitro, different 冱FICs, ranging from the 冱FICmin to the 冱FICmax, would be obtained, depending on the drug concentrations in combinations that produce the same effect as the drugs alone (this effect can be an MIC effect, i.e., 100% growth inhibition or any other effect observed at lower drug concentrations, e.g., 25 or 50% growth inhibition, etc.). Because in vivo drug concentrations fluctuate over time, the derived 冱FICs based on these concentrations also change over time. Thus, a single FIC index is unlike to predict in vivo efficacy. An in vitro synergistic or antagonistic combination may be additive or indifferent in vivo if the administered dosages result in drug concentrations lower or higher that those at which synergy or antagonism was observed. The exact FICbased pharmacokinetic/pharmacodynamic parameter that better predicts in vivo efficacy is unknown, and further research in this direction is required. Although a single isolate was used in the present study, the lack of in vitro-in vivo correlation of combination therapy depends not on particular isolates but on the methodological tools used to correlate FIC index data with the in vivo outcome. Finally, the conclusions of the present study may be applicable to in vitro combination studies with other antifungal agents, as we used representative drugs from different classes of antifungal compounds. In conclusion, microdilution checkerboard data can be used to assess pharmacodynamic interactions based on FIC index analysis. An additivity range of 0.5 to 2 is more symmetrical than a range of 0.5 to 4. Replication can increase the sensitivity of the FIC index analysis in the detection of pharmacodynamic interactions by statistically assessing deviation for an additivity range of 1 to 1.25 (inclusive). FIC index determination at 24 h might be better than longer incubation periods in detecting significant pharmacodynamic interactions. Finally, in vivo experiments and pharmacokinetic/pharmacodynamic analyses of antifungal drug combinations are needed to establish the exact correlation between in vitro pharmacodynamic interactions in antifungal combinations and in vivo outcomes. ACKNOWLEDGMENT This study was supported by the Intramural Research Program of the National Cancer Institute, Bethesda, MD. REFERENCES 1. American Society for Microbiology. 2004. Instructions to authors. Antimicrob. Agents Chemother. 48:i–xxi. 2. Berenbaum, M. C. 1980. Correlations between methods for measurement of synergy. J. Infect. Dis. 142:476–480. 3. Berenbaum, M. C. 1987. Minor synergy and antagonism may be clinically important. J. Antimicrob. Chemother. 19:271–273. 4. Berenbaum, M. C. 1989. What is synergy? Pharmacol. Rev. 41:93–141. 5. Clemons, K. V., M. Espiritu, R. Parmar, and D. A. Stevens. 2005. Comparative efficacies of conventional amphotericin B, liposomal amphotericin B (AmBisome), caspofungin, micafungin, and VRC alone and in combination against experimental murine central nervous system aspergillosis. Antimicrob. Agents Chemother. 49:4867–4875. 6. Clinical and Laboratory Standards Institute. 2002. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. NCCLS document M38-A. Clinical and Laboratory Standards Institute, Wayne, PA. 7. Cuenca-Estrella, M., A. Gomez-Lopez, G. Garcia-Effron, L. Alcazar-Fuoli, E. Mellado, M. J. Buitrago, and J. L. Rodriguez-Tudela. 2005. Combined activity in vitro of caspofungin, amphotericin B, and azole agents against itraconazole-resistant clinical isolates of Aspergillus fumigatus. Antimicrob. Agents Chemother. 49:1232–1235.

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