Title: Five-minute exposure to caspofungin results in prolonged post-antifungal effects and ...

18 Pages·2011·0.56 MB·English

AAC Accepts, published online ahead of print on 2 May 2011
Antimicrob. Agents Chemother. doi:10.1128/AAC.00095-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Title: Five-minute exposure to caspofungin results in prolonged post-antifungal effects and
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3 eliminates the paradoxical growth of Candida albicans
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6 Running Title: 5-minute exposure to caspofungin kills C. albicans
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11 Ryan K. Shields1, M. Hong Nguyen1*, Ellen Press1, Cornelius J. Clancy1,2 lo
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17 Affiliations: (1) University of Pittsburgh, Department of Medicine (2) VA Healthcare System, t
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18 Pittsburgh, Pennsylvania, United States /a
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19 Word Count in Text: 1,489 Abstract: 74 s
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21 Institution: University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213 J
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23 *Corresponding Author: M. Hong Nguyen, Division of Infectious Diseases, Suite S872 Scaife 0
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24 Hall, 3550 Fifth Avenue, Pittsburgh, Pennsylvania 15261, United States of America; telephone: b
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25 412-383-5193; fax: [phone]; e-mail: [email protected] u
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33 Abstract.
34 We studied the impact of brief caspofungin exposures on post-antifungal (PAFEs) and
35 paradoxical effects against five Candida albicans isolates. In time-kill studies, caspofungin at 4x
36 and 16x MIC resulted in significant killing of all isolates. Caspofungin at 8 µg/mL resulted in
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37 lesser killing, and paradoxical effects were evident against 4 isolates. Caspofungin exposures of o
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38 5 to 60 minutes caused prolonged, concentration-dependent killing that approached or exceeded lo
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39 that of time-kill experiments, and eliminated paradoxical growth. e
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45 Caspofungin, an inhibitor of 1,3-β-D-glucan synthesis, demonstrates concentration-dependent
46 fungicidal activity in vitro (6, 9) and in vivo (2, 13) against Candida spp. Our lab and others
47 have demonstrated that caspofungin exerts prolonged post-antifungal effects (PAFEs) against
48 Candida spp., following in vitro exposures of one hour (6, 8, 10, 15). At the same time,
49 caspofungin exerts paradoxical effects in vitro, in which the growth of certain Candida isolates is D
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50 increased at concentrations above the minimum inhibitory concentration (MIC) (19). To date, n
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51 relationships between PAFEs and paradoxical effects have not been investigated. The objective d
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52 of the present study was to evaluate the impact of brief exposures to caspofungin (ranging from 5 f
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53 to 60 minutes) on both PAFEs and paradoxical growth of C. albicans.

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55 Four randomly chosen C. albicans bloodstream isolates collected from the Clinical Microbiology a
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56 Laboratory at the University of Pittsburgh Medical Center and C. albicans SC5314 were .o
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57 included in this study (11). Caspofungin time-kill and PAFE experiments were performed in o
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58 parallel (6, 16-17). In total, four test solutions were used, and consisted of RPMI 1640 alone a
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59 (control) and RPMI 1640 plus caspofungin at concentrations of 4x MIC, 16x MIC and 8 µg/mL. a
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60 We selected 8 µg/mL to simulate peak serum concentrations in vivo (1). After incubation at ,

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61 35°C with agitation for 5, 15, 30, and 60 minutes, the cells were washed with saline three times, 1
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62 then re-suspended in 20 mL of medium (RPMI 1640) and re-incubated at 35°C (PAFE y

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63 experiments). For time-kill assays, test solutions were not centrifuged or washed. All tubes were e
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64 sampled at 0, 2, 4, 8, 12, and 24 hours by removing 500µL and enumerating colony forming
65 units (cfu) on Sabouraud-dextrose agar (SDA) plates. All experiments were performed at least
66 twice.
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68 Paradoxical effects were defined as ≥ 1 log (CFU/mL) greater growth in the presence of 8
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69 µg/mL of caspofungin compared to 4x and 16x MIC (19). The killing effect of caspofungin was
70 calculated as the difference between the starting inoculum and the concentrations of isolates at
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71 specific time points. In time-kill experiments, caspofungin at concentrations of 4x- and 16x o
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72 MIC caused reductions in starting inocula of all tested isolates after 24 hours of incubation lo
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73 (Table 1, Figure 1). In contrast, caspofungin at 8 µg/mL (i.e., 64x to 256x MIC values) did not e
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74 reduce the starting inocula of two isolates (1 and 3) (Table 1). For 4 of 5 isolates, paradoxical ro
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75 effects were evident, as caspofungin concentrations at 4x and 16x MIC resulted in greater kills h
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76 than 8 µg/mL. Moreover, for two isolates, caspofungin at 4x MIC caused greater killing than //
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77 16x MIC. Overall, the killing effects of caspofungin at 4x MIC were significantly greater than 8 .
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78 µg/mL (mean log reduction in the starting inocula: 2.25-log vs 0.50-log; p=0.048, Wilcoxon).
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80 For each isolate, brief exposures to caspofungin (5, 15, 30 and 60 minutes) during PAFE n
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81 experiments resulted in concentration-dependent killing (Table 1; Figure 2), which persisted for ry

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82 24 hours. At 4x MIC, killing of four isolates after brief exposures was less than during 2
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83 continuous drug exposure of time-kill experiments. At 16x MIC and 8 µg/mL, however, PAFEs 9
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84 against three and four isolates, respectively, resulted in greater killing than continuous drug g
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85 exposure. In general, maximal PAFEs against each of the isolates were achieved after exposures t
86 as short as 5 or 15 minutes (Table 1). Moreover, removal of caspofungin (8 µg/mL) after 5 to 60
87 minutes abolished paradoxical effects against all four isolates for which they were observed in
88 time-kill experiments. Indeed, 5-minute exposures to caspofungin at 8 µg/mL resulted in
89 significantly greater mean killing effects than continuous exposure to caspofungin at 8 µg/mL
90 (mean log reduction in the starting inocula: 2.48-log .vs 0.30-log;p=0.01, Wilcoxon).
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92 To conclusively demonstrate that the killing effects we observed after brief caspofungin
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93 exposures stemmed from PAFEs rather than as an artifact of the experimental methods, we w
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94 conducted three experiments in parallel: 1) standard time-kills in the presence of continuous a
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95 exposure to caspofungin; 2) standard PAFE experiments, in which caspofungin was washed-out d

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96 after brief exposures; and 3) caspofungin re-exposure experiments, in which the drug was re- m

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97 added to cells at the original concentration after completing the wash-out steps of PAFE tt
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98 experiments. Over 24 hours, kill-curves for time-kill and caspofungin re-exposure experiments a
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99 were similar for caspofungin at 16x MIC and 8ug/mL, whereas maximal kills were observed a
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100 after brief exposures (Figure 3). Paradoxical growth of the four relevant isolates was apparent .o
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101 during both time-kill and re-exposure experiments, but eliminated during PAFE experiments. o
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102 n
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103 In this study, we showed that caspofungin caused prolonged killing of C. albicans isolates in a 5
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104 concentration-dependent manner. Indeed, exposures to caspofungin for as short as 5 or 15 0
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105 minutes resulted in kills over 24 hours that approached or, in some instances, exceeded those b
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106 seen with continuous drug exposure during time-kill experiments. These findings extended u
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107 previous observations from our lab and others that one hour exposure to caspofungin resulted in
108 significant PAFEs (6, 9). At the same time, we showed that brief exposures to caspofungin at 8
109 µg/mL, a typical peak concentration within human serum, eliminated the paradoxical growth of
110 all isolates that manifested the phenomenon during time-kill experiments.
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112 The potent PAFEs stemming from brief caspofungin exposures suggest that the drug rapidly
113 accesses its target and causes sustained inhibition of the β-1,3-D-glucan synthase enzyme
114 complex. Studies of bacterial protein and nucleic acid synthesis inhibitors have demonstrated
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115 that the duration of post-antibiotic effects is determined by the period of time needed for the o
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116 drugs to dissociate from their targets (20-21). As such, the PAFEs in this study are most likely lo
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117 due to ongoing direct interactions between caspofungin and β-1,3-D-glucan synthase; e
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118 alternatively, the drug may be able to intermittently associate with its target from sequestered ro
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119 sites within the plasma membrane or elsewhere in the cell. Regardless of the particular h
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120 mechanisms by which PAFEs are mediated, our data advocate a model in which the maximal //
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121 activity of caspofungin against a given C. albicans isolate is attained at some threshold c.
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122 determined by a combination of the duration of drug exposure, drug concentration and target
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123 affinity. In the face of caspofungin exposure above this threshold, such as resulting from /

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124 constant exposure to high drug concentrations, adaptive and compensatory responses may be J
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125 activated that limit further kills and promote growth. By the same token, brief exposure to a u
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126 sufficient concentration of caspofungin may result in a kill equivalent to more prolonged 5
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127 exposures to lesser concentrations. 0
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129 At present, the standard dosing of caspofungin for the treatment of candidiasis is a 70mg loading t
130 dose, followed by 50 mg daily (1). Caspofungin’s concentration-dependent PAFEs that persist
131 for 24 hours suggest that less frequent administration of higher drug doses may be at least as
132 effective as conventional regimens (4). In fact, as predicted from pharmacodynamic (PD)
133 studies (3, 17), once-weekly administration of another echinocandin, micafungin, was shown to
134 be as efficacious as daily administration in a murine model of systemic candidiasis (12). In this
135 regard, it is notable that PD targets for caspofungin are readily achievable in vivo despite less
136 frequent administration due to the tolerability of high doses (5) and the persistence of therapeutic
137 concentrations in tissues (14). In addition to direct killing, caspofungin PAFEs may provide D
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138 indirect benefits in vivo, such as rendering cells more susceptible to phagocytosis and cell wall n
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139 stress, and decreasing adherence to host epithelial cells (16-17). Alternative dosing strategies for d
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140 caspofungin and other echinocandins merit further investigation, including assessments of the f
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141 impact on toxicity, cost and emergence of resistance.

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142 //
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143 The clinical relevance of caspofungin paradoxical effects remains uncertain. We recently a
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144 demonstrated that paradoxical effects were eliminated in 50% human serum, suggesting that they .o
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145 are unlikely to influence the treatment of candidemia (19). Our demonstration that paradoxical o
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146 growth was eliminated during PAFE experiments further argues against a major clinical impact a
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147 unless extremely high caspofungin concentrations are maintained throughout the dosing interval. a
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148 Indeed, in a mouse model of systemic candidiasis, only one of four C. albicans isolates that ,

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149 demonstrated a paradoxical effect in vitro showed a similar pattern in vivo with a higher fungal 1
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150 burden recovered from mice treated with high-dose caspofungin (20 mg/kg) (7). Moreover, the y

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151 apparent paradoxical growth was not reproducible in subsequent experiments (7). Furthermore, e
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152 in two recent clinical trials, there were no differences in responses to echinocandins among
153 patients with candidiasis who received high- or low-dose regimens (150 mg/d vs. 100 mg/d of
154 micafungin, and 150 mg/d vs. 70 mg load, then 50 mg/d of caspofungin) (5, 18). On balance,
155 therefore, the preponderance of evidence suggests that paradoxical effects are not likely to be
156 clinically relevant.
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158 In conclusion, the current study opens the door to future investigations to explore the
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159 mechanisms by which caspofungin and other echinocandins rapidly exert their profound w
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160 antifungal activity against C. albicans. In particular, studies are needed to better understand the loa
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161 interaction between these agents and their target, the enzyme kinetics of β-1,3-glucan synthase, d

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162 and the compensatory responses that are triggered by varying degrees of echinocandin exposure. m

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163 Results from these studies will have important implications for both the treatment of candidiasis tt
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164 and our understanding of Candida cell wall biology. a
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168 Acknowledgements.
169 Experiments were conducted in the laboratories of C.J. Clancy and M.H. Nguyen at the
170 University of Pittsburgh. This project was funded in part by an investigator-initiated research
171 grant from Merck to Dr. Clancy. Dr. Clancy has received research support for unrelated projects
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172 from Pfizer and Astellas. Dr. Nguyen has received research support for other projects from o
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173 Merck, Pfizer and ViraCor-IBT Laboratories. Dr. Shields has received research support for other lo
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174 projects from Merck and Astellas. e
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177 References
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179 1. 2005. CANCIDAS (caspofungin acetate) for injection [prescribing information]. .
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