In Vivo Efficacy of Anidulafungin and Caspofungin against Candida glabrata and Association with In Vitro Potency in the Presence of Sera

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2007, p Vol. 51, No /07/$ doi: /aac Copyright 2007, American Society for Microbiology. All Rights Reserved. In Vivo Efficacy of Anidulafungin and Caspofungin against Candida glabrata and Association with In Vitro Potency in the Presence of Sera Nathan P. Wiederhold, 1,2 * Laura K. Najvar, 2 Rosie Bocanegra, 2 Destiny Molina, 2 Marcos Olivo, 2 and John R. Graybill 2 College of Pharmacy, The University of Texas at Austin, Austin, Texas, 1 and Department of Medicine/Infectious Diseases, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 2 Received 24 January 2007/Returned for modification 30 January 2007/Accepted 12 February 2007 In vitro studies have demonstrated that anidulafungin has greater potency than caspofungin against Candida glabrata. However, data from in vivo studies demonstrating that it has superior efficacy are lacking. The objective of this study was to compare the activities of anidulafungin and caspofungin against C. glabrata in a murine model of disseminated candidiasis. Two clinical C. glabrata isolates were used, including one with reduced caspofungin susceptibility. MICs were determined by broth microdilution in the presence and absence of sera. For the animal studies, mice were immunosuppressed with 5-fluorouracil one day prior to intravenous inoculation. Treatment with anidulafungin and caspofungin (0, 0.5, 1, 5, and 10 mg/kg of body weight per day) was begun 24 h later and was continued through day 7 postinoculation. The CFU were enumerated from kidney tissue. According to the standard microdilution methodology, anidulafungin had superior in vitro activity. However, this enhanced potency was attenuated by the addition of mouse and human sera. Caspofungin reduced the kidney fungal burden at lower doses compared to that achieved with anidulafungin in mice infected with the isolate with the lower MIC. Against the strain with the elevated caspofungin MIC, both anidulafungin and caspofungin were effective in reducing the kidney fungal burden at the higher doses studied. Despite the greater in vitro activity of anidulafungin in the absence of sera, both echinocandins were similarly effective in reducing the fungal burden in kidney tissue. The superior in vitro activity of anidulafungin did not confer enhanced in vivo efficacy against C. glabrata. Invasive candidiasis caused by Candida glabrata is of increasing clinical concern due to reports of the increasing frequency of cases and the high mortality rates in patients with multiple comorbidities, including elderly patients, immunocompromised patients with cancer, and patients in intensive care units (4, 15, 24, 34, 37). The various susceptibilities of C. glabrata isolates to fluconazole and the reduced response rates to other azole antifungals in breakthrough infections following fluconazole prophylaxis may limit the effectiveness of therapy for infections caused by this species (4, 7, 15, 27, 32, 33, 36). Furthermore, the use of amphotericin B formulations may be limited by infusion-related reactions and nephrotoxicity. Due to the inhibition of a fungal specific target, the beta- (1,3)-glucan synthase enzyme complex, the echinocandins (anidulafungin, caspofungin, and micafungin) avoid the overlapping toxicities and drug interactions with mammalian cells observed with the azoles and the polyenes (10, 35). Broth microdilution studies have demonstrated that each member of the echinocandin class has relatively good activity against Candida isolates, including C. glabrata and other non-c. albicans species (17, 35). Indeed, clinical studies have demonstrated the excellent efficacy of the echinocandins for the treatment of invasive candidiasis (19, 29, 31). * Corresponding author. Mailing address: UTHSCSA, PERC, MSC 6220, 7703 Floyd Curl Drive, San Antonio, TX Phone: (210) Fax: (210) wiederholdn@uthscsa.edu. Published ahead of print on 16 February In vitro studies comparing anidulafungin and caspofungin, including one our group at the University of Texas Health Science Center at San Antonio, have reported that anidulafungin has enhanced potency against C. glabrata (6, 8, 23). These studies, along with case reports describing clinical failures of caspofungin treatment in association with reduced in vitro activity, have raised the question of whether other members of this class would maintain their potencies at clinically relevant exposures in the presence of reduced caspofungin susceptibilities (11, 13, 26). However, few in vivo and clinical data from studies that have directly compared the efficacies of different echinocandins against isolates with elevated MICs exist. Therefore, the objective of this study was to compare the in vivo efficacies of anidulafungin and caspofungin against two C. glabrata clinical isolates, including one with reduced caspofungin susceptibility, in a murine model of invasive candidiasis. We hypothesized that anidulafungin would have superior in vivo efficacy, based on preliminary data demonstrating enhanced in vitro potency. MATERIALS AND METHODS Antifungals. Stock solutions were prepared by dissolving drug powders in dimethyl sulfoxide (anidulafungin; Vicuron Pharmaceuticals, King of Prussia, PA) or water (caspofungin; Merck & Co., Inc., Whitehouse Station, NJ). Stock solutions were diluted in RPMI medium buffered to ph 7.0 with M 4-morpholinepropanesulfonic acid or physiologic saline prior to each in vitro and in vivo experiment, respectively. Isolates. Two Candida glabrata clinical isolates (isolates and 05-62) were obtained from the Fungus Testing Laboratory at the University of Texas Health Science Center at San Antonio. The isolates were subcultured twice on Sabouraud dextrose agar before they were tested. Yeast cells were collected and 1616

2 VOL. 51, 2007 IN VIVO EFFICACY OF ANIDULAFUNGIN AND CASPOFUNGIN 1617 TABLE 1. MICs, IC 50 s, and IC 90 s for anidulafungin and caspofungin against C. glabrata isolates and by microdilution methodology in RPMI medium in the presence and absence of mouse and human sera Drug and serum present MIC 2 /MIC 0 ( g/ml) with or without mouse serum MIC 2 /MIC 0 ( g/ml) with or without human serum IC 50 /IC 90 ( g/ml R 2a ) with or without human serum Anidulafungin No serum 0.06/ / / / /0.16 (0.98) 0.31/0.41 (0.95) 5% serum 0.5/0.5 4/4 0.5/0.5 2/2 0.31/0.39 (0.99) 1.7/3.1 (0.97) 50% serum 1/1 8/8 1/2 8/16 Caspofungin No serum 1/2 2/ 32 1/2 2/ /1.6 (0.96) 1.2/ 32 (0.88) 5% serum 1/1 4/ /0.5 2/ /0.33 (0.99) 1.4/ 32 (0.95) 50% serum 2/2 8/ 32 1/1 4/ 32 a R 2 values are for the dose-response curves. washed in sterile saline three times, and the inocula were verified with a hemocytometer. The starting inocula were confirmed by plating serial dilutions and determining the colony counts. Susceptibility testing. Microdilution broth susceptibility testing was performed in duplicate according to the CLSI (formerly NCCLS) M27-A2 method in RPMI growth medium in the presence and absence of 5% and 50% (vol/vol) mouse and human sera (Sigma Aldrich, St. Louis, MO) (21). The MIC 2 was defined as the lowest concentration of anidulafungin or caspofungin that caused a significant decrease in turbidity ( 50%) compared to that of the growth control, and the MIC 0 was defined as the lowest concentration that resulted in no visual growth (an optically clear well) after 24 h of incubation. XTT reduction assay. The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay was performed in triplicate, according to previously reported methods (16), in the presence and absence of 5% (vol/vol) human sera. A standardized inoculum (100 l ofa to CFU/ml) was added to the wells of a 96-well microtiter plate containing serial twofold dilutions of antifungals in RPMI growth medium. The final anidulafungin and caspofungin concentrations ranged from to 32 g/ml. The trays were incubated at 37 C for 22 h, at which time 50 l of the tetrazolium salt XTT and the electron-coupling agent menadione were added to each well for final concentrations of 200 g/ml and 25 M, respectively. After 2 additional hours of incubation at 37 C, the absorbance at 492 nm was read with a microplate spectrophotometer (Syngergy HT; Biotek Instruments, Inc., Winooski, VT). The absorbance readings were converted to percent absorbance, with the absorbance of the growth control wells set at 100% and the absorbance of the medium control wells set at 0%. Animal model. Outbred ICR mice (Harlan Sprague-Dawley) weighing between 25 and 27 g were housed at five mice per cage and had access to food and water ad libitum. One day prior to inoculation the animals were immunosuppressed with intravenous 5-fluorouracil at 150 mg/kg of body weight. On day 0, the animals were infected intravenously with 0.2 ml of C. glabrata at approximately CFU/mouse. This study was approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio, and all animals were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care (20). Tissue burden studies. Antifungal therapy was begun 24 h after inoculation. Anidulafungin and caspofungin (0, 0.5, 1, 5, and 10 mg/kg per day) were administered by intraperitoneal injection until day 7 postinoculation. The mice were euthanized on day 8, and the kidney tissue was harvested and weighed. The kidneys were homogenized in sterile saline (total volume, 2 ml) with a tissue homogenizer (Polytron dispensing and mixing technology PT 2100; Kinematica, Cincinnati, OH). Serial dilutions were prepared in sterile saline and were plated in duplicate onto Sabouraud dextrose agar. Following 24 h of incubation at 37 C, the colonies were counted and the numbers of CFU per gram of kidney tissue for each animal were calculated. Each study consisted of between 10 and 15 animals per dosing group, and studies were conducted on two separate occasions to ensure reproducibility. Data analysis. Data from the XTT reduction assay were fit to a four-parameter inhibitory sigmoid model (modified Hill equation) by using computer curvefitting software (Prism 4; GraphPad Software, Inc., San Diego, CA) to derive 50% inhibitory concentrations (IC 50 s) and IC 90 s, as well as the steepness of the inhibitory dose-response curve (Hill slope). The goodness of fit for each isolate and drug combination was assessed by determination of the R 2 value and the standard error of the IC 50 value. Differences in fungal burden endpoints (CFU/g) were assessed for significance by using the Kruskal-Wallis test with Dunn s posttest for multiple comparisons. A P value of 0.05 was considered statistically significant for all comparisons. RESULTS Susceptibility testing. The results of susceptibility testing demonstrated that the addition of sera affected the potencies of anidulafungin and caspofungin differently. As shown in Table 1, in the absence of sera, anidulafungin had greater in vitro potency against both C. glabrata isolates by the standard broth microdilution methodology, with MIC 2 values 8 to 16 times lower than those of caspofungin. However, with the addition of either 5% mouse or 5% human serum, the MIC of anidulafungin increased by eightfold against each isolate. This reduction in potency was even greater in the presence of 50% serum (16- to 32-fold increases in the MIC 2 s). In contrast, the in vitro activity of caspofungin was relatively unchanged by the addition of sera to the growth medium. In fact, against isolate , the potency of caspofungin was somewhat enhanced in the presence of 5% human serum, as evident by reductions in the MIC 2 and MIC 0 values from 1 and 2 g/ml, respectively, to 0.25 g/ml. The changes in the MICs observed with the addition of sera are also supported by the XTT viability data (Table 1 and Fig. 1). As shown in Fig. 1A and C, in the absence of sera anidulafungin was more potent than caspofungin against both isolates, with lower IC 50 and IC 90 values. However, the addition of 5% human serum to the growth medium caused the dose-response curves for anidulafungin to shift to the right so that the curves for anidulafungin and caspofungin against each isolate were superimposed (Fig. 1B and D). XTT assays were also performed in the presence of 50% sera. However, this resulted in an inhibition in the reduction of the tetrazolium salt to the formazan derivatives not observed with the addition of 5% sera (data not shown). In vivo tissue burden. Despite the reduced in vitro potency of caspofungin compared to that of anidulafungin in the absence of sera, caspofungin maintained its in vivo potency against both C. glabrata isolates. Against isolate , caspofungin significantly reduced the tissue fungal burden at each dose tested, with the exception of the 5-mg/kg dose

3 1618 WIEDERHOLD ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. Sigmoidal dose-response curves for anidulafungin (F) and caspofungin (E) against C. glabrata isolates (A and B) and (C and D) in the absence of sera (A and C) and with the addition of 5% human sera (B and D). Symbols represent the means plus or minus standard deviations of experiments performed in triplicate for each drug. Curves were generated by fitting XTT viability data to a four-parameter inhibitory sigmoid model (modified Hill equation). (Fig. 2A), while anidulafungin resulted in no reductions in fungal burden compared to that for the control at doses below 5 mg/kg. However, the differences between anidulafungin and caspofungin at the higher doses studied (5 and 10 mg/kg) were not significant. Both agents were equally effective at reducing the burden of fungal isolate in kidney tissue (Fig. 2B), despite the reduced susceptibility to caspofungin in vitro by standard microdilution methods. DISCUSSION The echinocandins have been shown to be effective for the treatment of invasive candidiasis in clinical trials. In a randomized, double-blind study, a favorable response at the end of the study was noted in 73.4% of patients who received caspofungin but only 61.7% of those randomized to receive amphotericin B deoxycholate (P 0.09) (19). Micafungin has also been shown to have efficacy similar to that of liposomal amphotericin B for the treatment of invasive candidiasis (31), while anidulafungin proved to be superior to fluconazole at the end of therapy and at 2 weeks after the end of therapy (29). More recently, in a headto-head comparison, micafungin and caspofungin were shown to be equally effective in patients with candidemia or invasive candidiasis (3). Whether one member of this class has superior efficacy against infections caused by a particular Candida species remains unknown. Clinical trials to date have not been powered to show such differences; therefore, the response rates according to pathogen have been similar between treatment groups. Our hypothesis that anidulafungin has superior in vivo ac- FIG. 2. Fungal burden as measured by the numbers of CFU per gram of kidney tissue in mice that received the control preparation ( ), anidulafungin (F), or caspofungin (E) at doses of 0.5, 1, 5, and 10 mg/kg as treatment against Candida glabrata infection. (A) Candida glabrata isolate ; (B) Candida glabrata isolate Each group consisted of 24 mice. The line for each group represents the median fungal burden., P 0.05 versus the results for the control.

4 VOL. 51, 2007 IN VIVO EFFICACY OF ANIDULAFUNGIN AND CASPOFUNGIN 1619 tivity was based on the enhanced in vitro potency of this echinocandin against C. glabrata reported in the literature (1, 8, 22, 23, 30). Against C. glabrata, MICs from multicenter surveillance studies for members of this class have ranged from to 2 g/ml, with anidulafungin having increased potency (8, 23). One study of 2,000 bloodstream Candida isolates from two multicenter clinical trials, including 458 C. glabrata isolates, reported lower MIC 50 and MIC 90 values for anidulafugin compared to those for caspofungin for each Candida species, with the exception of C. parapsilosis (23). Similarly, previous work from our group demonstrated that anidulafungin had greater in vitro potency against C. glabrata, as evident by the lower MIC, IC 50, and IC 90 values and as supported by the results of time-kill studies (6). Despite the enhanced in vitro potency of anidulafungin, treatment with anidulafungin did not result in greater reductions in tissue burdens compared to those achieved by treatment with caspofungin. However, the lack of superior in vivo potency of anidulafungin was not due to the inability of this agent to lower colony counts but, rather, was due to the ability of caspofungin to maintain in vivo activity, despite its reduced in vitro potency. In fact, against the more sensitive isolate, caspofungin was more effective than anidulafungin in reducing the tissue burden at the lower doses studied, while both agents were equally effective against the isolate with elevated caspofungin MIC 0 values. The ability of caspofungin to maintain in vivo efficacy, despite its reduced in vitro potency, is not wholly unexpected. Our data are consistent with evidence that suggests that isolates with echinocandin MIC 2 values of 2 g/ml should be regarded as susceptible (28). In a review of the caspofungin clinical trial database, Kartsonis et al. reported that infections caused by Candida isolates with caspofungin MICs of 1 to 2 g/ml responded as well as those with lower MICs (0.25 and 0.5 g/ml) (12). However, it is unclear from this study if infections caused by isolates with higher caspofungin MICs would also respond, since only three isolates with MICs of 4 g/ml were included. The effects of sera on the activities of echinocandins are not fully understood. In the current study the in vivo efficacies of both echinocandins were consistent with the in vitro activities observed with the addition of sera to the growth medium. Previous studies have reported both positive and negative effects of sera on the activities of members of this class. Using the XTT viability assay, Chiller et al. demonstrated that caspofungin has enhanced inhibitory activity against A. fumigatus in the presence of serum concentrations ranging from 0.05 to 5% (5). Prior heating of the sera did not negate this enhanced activity. Interestingly, in contrast, recent studies have reported a decrease in the activity of echinocandin in the presence of higher concentrations of human sera. Mochizuki et al. reported that the MIC of micafungin against C. albicans increased eightfold in the presence of inactivated serum (18). However, the activity of micafungin remained greater than 50-fold higher than the free concentrations predicted by the protein binding ratio. A recent study comparing each of the three available echinocandins reported a tempering of the activity of each agent with the addition of 50% human sera, resulting in the neutralization of the enhanced potency of micafungin relative to that of caspofungin both in vitro and in vivo (25). In the current study, the addition of 5% and 50% sera negatively modulated the activity of anidulafungin, while the activity of caspofungin was either unchanged or slightly enhanced. Similar to the report by Park et al. (25), our in vitro data were consistent with the results from the animal studies. An alternative explanation for the differences in the in vivo activities of these agents compared to those observed in vitro in the absence of sera could be differences in the pharmacokinetic parameters. While differences in some pharmacokinetic parameters (e.g., transfer rate constants between the kidney tissue, other tissue sites, and the central compartment) for anidulafungin and caspofungin have been observed in mice, both agents persist in the kidneys at concentrations above their respective MIC 90 values for C. glabrata for an extended period following administration of a single dose (9, 14, 17). Furthermore, in this study and in clinical practice, both anidulafungin and caspofungin are administered on a daily basis. Thus, with repeated administration and persistence within the kidney tissue, the overall exposure to both agents is extensive. Prolonged tissue exposure is also observed clinically, as both anidulafungin and caspofungin have long elimination half-lives at steady state (range, 40 to 50 h) that are heavily influenced by tissue disposition and accumulation (Cancidas package insert [Merck & Co., Inc.] and Eraxis package insert [Pfizer, Inc., New York, NY]). While these data suggest that differences in pharmacokinetic parameters between anidulafungin and caspofungin are unlikely to explain the differences in the in vivo activities observed in the current study, the possibility that such differences exist cannot be ruled out. Another limitation of our study is the relative avirulence of C. glabrata in this animal model (2). Whether colony counts within kidney tissue alone are sufficient to gauge in vivo drug activity or whether differences in survival with the use of a more virulent species would also be observed between these agents is unknown. In conclusion, both anidulafungin and caspofungin were effective in reducing the tissue burden of C. glabrata isolates. However, discrepancies between the in vitro activities of each agent and their abilities to reduce the colony counts in vivo were observed. Consistent with the data from the animal studies, the addition of sera negated the enhanced in vitro potency of anidulafungin observed with standard broth microdilution methods and resulted in similar in vitro activities for the two echinocandins. ACKNOWLEDGMENTS This study was sponsored in part by a grant from Vicuron to J.R.G. J.R.G. has received research support from Pfizer, Inc., Schering-Plough Corporation, Merck & Co., and Fujisawa and has served as a consultant and speaker for Merck & Co. and Schering-Plough Corporation. N.P.W. has received research support from and served as a consultant and speaker for Pfizer, Inc. REFERENCES 1. Arevalo, M. P., A. J. Carrillo-Munoz, J. Salgado, D. Cardenes, S. Brio, G. Quindos, and A. 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