ACCEPTED. Yanjun Li 1. M. Hong Nguyen 2,3,4. Harmut Derendorf 1. Shaoji Cheng 2. *Cornelius J. Clancy 2,3

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1 AAC Accepts, published online ahead of print on 21 May 2007 Antimicrob. Agents Chemother. doi: /aac Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Measurement of voriconazole activity against Candida albicans, C. glabrata and C. parapsilosis isolates using time-kill methods validated by high performance liquid chromatography Yanjun Li 1 M. Hong Nguyen 2,3,4 Harmut Derendorf 1 Shaoji Cheng 2 *Cornelius J. Clancy 2,3 1 Department of Pharmaceutics, University of Florida College of Pharmacy, Departments of 2 Medicine and 3 Molecular Genetics and Microbiology, University of Florida College of Medicine, and 4 North Florida/South Georgia Veterans Health System, Gainesville, FL * Corresponding author: Cornelius J. Clancy, MD University of Florida College of Medicine Box JHMHC 1600 SW Archer Rd. Gainesville, FL Ph: Fax: clancyn@medicine.ufl.edu Running title: HPLC validates voriconazole time-kill against Candida Key words: Candida parapsilosis/candida glabrata/candida spp./time-kill/high performance liquid chromatography (HPLC) Word counts: Abstract: 50 Text: 997 1

2 Abstract. We developed an HPLC assay to validate time-kill and post-antifungal effect (PAFE) experiments for voriconazole against C. albicans, C. glabrata and C. parapsilosis isolates. Voriconazole exerted prolonged fungistatic activity but no PAFE at concentrations achievable in human sera. HPLC confirmed that experiments were conducted at the desired steady-state voriconazole concentrations. 2

3 Voriconazole is a triazole agent that inhibits ergosterol synthesis by blocking the action of 14α-demethylase. The drug is fungistatic and exhibits no post-antifungal effect (PAFE) against Candida albicans (1, 3-5, 7, 9). Time-kill and PAFE data are limited against C. glabrata (1, 7), and do not exist against C. parapsilosis isolates. Moreover, standard timekill and PAFE methodologies, although widely used, have not been validated for voriconazole or other antifungals by direct measurements of drug concentrations. In this study, we developed an high performance liquid chromatography (HPLC) assay to validate the results of time-kill and PAFE experiments for voriconazole against C. albicans reference strains (ATCC and SC5314), and C. glabrata and C. parapsilosis bloodstream isolates (2 each). The MICs of all isolates were within the susceptible range, as measured by E-test and microdilution methods (Table 1) (10, 11). For time-kills and PAFEs, colonies from 48-hour cultures on Sabouraud dextrose agar (SDA) were suspended in 9 ml sterile water (2, 7). One microliter of a 0.5 McFarland suspension was added to 10 ml of RPMI 1640 medium with or without voriconazole (0.25-, 1-, 4-, and 16 MIC), and the solution was incubated at 35 C with agitation. The maximal voriconazole concentration in these experiments was 3.04 µg/ml (16 MIC for C. glabrata #1). For time-kills, 100 µl from each solution was serially diluted at desired time points and plated on SDA plates for colony enumeration (0, 2, 4, 8, 12, 24, 36, 48, 60 and 72 hours). For PAFEs, Candida cells were collected after 1 hour of incubation, washed three times, and resuspended in warm RPMI medium (9 ml); colonies were enumerated at desired time periods. Voriconazole exhibited dose-response effects against all 3

4 Candida isolates during time-kill experiments (Figure 1; Table 1), as higher concentrations resulted in greater growth inhibition or killing. The range of maximal growth inhibition of isolates at concentrations of 1- and 4 MIC was to 2.78-log and to 2.99-log, respectively, compared to controls (Table 1). At 16 MIC, the range of maximal growth inhibition was to 4.15-log. Voriconazole did not demonstrate PAFEs (data not shown). Voriconazole at 16 MIC was fungicidal against C. parapsilosis #2, reducing the starting inoculum by 2.21-log at 24 hours (fungicidal defined as > 2-log reduction of starting inoculum). Although kills did not achieve fungicidal levels for other isolates, voriconazole at 4- and 16 MIC reduced starting inocula of C. glabrata #2 and C. parapsilosis # 1 (Table 1). Of note, voriconazole was consistently fungistatic at 1- to 16 MIC, and the effect persisted for 72 hours. Indeed, maximal inhibition of the four C. albicans and C. glabrata isolates at 4- and 16 MIC (compared to starting inocula) was not evident until 48 to 72 hours. The two C. parapsilosis isolates, on the other hand, were maximally inhibited by 24 to 36 hours. The time-kill curves of the C. parapsilosis isolates also differed from the other isolates at early time points. The C. parapsilosis isolates at 4- and 16 MIC were inhibited from entering exponential growth, and dose-response effects were clearly evident by 8 hours. The growth of C. albicans and C. glabrata isolates in the presence of voriconazole did not differ from controls during early exponential phase, but dose-response effects became increasingly apparent as exponential growth continued (8 to 24 hours). 4

5 In our HPLC protocol for measuring voriconazole concentrations during time-kill and PAFE experiments, a mm analytic column with mm guard cartridge (Hichrom, Reading, UK) was packed with 5 µm Kromasil at 25 C in an Agilent 1100 Series apparatus (6, 8). Mobile phase acetonitrile-ammonium phosphate buffer (ph 6.0; 0.04 M; 1:1 v:v) was degassed by filtration through a 0.45 µm nylon filter under vacuum; the flow rate was 0.8 ml/min. Voriconazole concentrations were determined from peak areas detected by UV absorption at 255 nm with an 8.2 minute retention time. For each isolate, we tested RPMI medium containing at least one concentration of voriconazole between 1- and 16 MIC. Samples were diluted with 2 volumes of acetonitrile-ammonium phosphate buffer, centrifuged at full speed in a microcentrifuge for 10 minutes, and supernatants (200 µl) applied to the column. The maximum sensitivity was µg/ml, and the method was linear over a range of µg/ml (r ). In each instance, we confirmed that voriconazole concentrations remained constant throughout the duration of time-kill experiments (Figure 2), and the drug was fully removed during PAFE experiments (data not shown). Our findings conclusively demonstrate that voriconazole exerts prolonged fungistatic activity against C. albicans, C. glabrata and C. parapsilosis at concentrations that are achievable in human sera with routine dosing (median average and maximum voriconazole plamsa concentrations in clinical trials are 2.51 and 3.79 µg/ml, respectively) (12). Our findings are potentially relevant clinically, since certain C. parapsilosis isolates exhibit 5

6 diminished susceptibility to echinocandin antifungals and C. glabrata isolates can develop resistance to fluconazole and other antifungal agents. Although voriconazole caused >2-log kill of one C. parapsilosis isolate, further studies will be needed to accurately define the extent to which the drug might be fungicidal against clinical isolates. To our knowledge, this is the first study to verify standard time-kill and PAFE methodologies by directly measuring drug concentrations. We describe a simple and reproducible HPLC method that has a broad, clinically relevant dynamic range and does not require internal standards. The sensitivity of voriconazole measurements within liquid media was greater than that previously reported for human or guinea pig plasma ( and 5 10 µg/ml, respectively) (6, 8). Based on our findings, we can assume that previous studies of azoles that showed fungistatic anti-candidal activity and no PAFEs were conducted under the conditions of steady-state drug concentrations assumed by investigators. This demonstration is crucial as efforts to use pharmacodynamic data to develop optimal antifungal treatment strategies move forward. In particular, HPLC methods will be essential to the design of dynamic in vitro models to assess the pharmacodynamics of voriconazole and other agents prior to the achievement of steady-state conditions. The authors thank Dr. Vipul Kumar for his advice in developing the HPLC assay. Experiments were conducted in Dr. Clancy s laboratory at the North Florida/South Georgia Veterans Health System, Gainesville, FL. This project was supported by the Medical Research Service of the Department of Veterans Affairs. It was conducted as part of the 6

7 University of Florida Mycology Research Unit (NIH PO1 AI to Drs. Nguyen and Clancy). Dr. Clancy has received research support from Pfizer. 7

8 References. 1. Canton E, Peman J, Gobernado M, Viudes A, and Espinel-Ingroff A Synergistic activities of fluconazole and voriconazole with terbinafine against four Candida species determined by checkerboard, time-kill, and Etest methods. Antimicrob Agents Chemother 49: Clancy CJ, Huang H, Cheng S, Derendorf H, and Nguyen MH Characterizing the effects of caspofungin on Candida albicans, Candida parapsilosis, and Candida glabrata isolates by simultaneous time-kill and postantifungal-effect experiments. Antimicrob Agents Chemother 50: Di Bonaventura G, Spedicato I, Picciani C, D'Antonio D, and Piccolomini R In vitro pharmacodynamic characteristics of amphotericin B, caspofungin, fluconazole, and voriconazole against bloodstream isolates of infrequent Candida species from patients with hematologic malignancies. Antimicrob Agents Chemother 48: Ernst EJ, Yodoi K, Roling EE, and Klepser ME Rates and extents of antifungal activities of amphotericin B, flucytosine, fluconazole, and voriconazole against Candida lusitaniae determined by microdilution, Etest, and time-kill methods. Antimicrob Agents Chemother 46: Espinel-Ingroff A In vitro antifungal activities of anidulafungin and 8

9 micafungin, licensed agents and the investigational triazole posaconazole as determined by NCCLS methods for 12,052 fungal isolates: review of the literature. Rev Iberoam Micol 20: Gage R, and Stopher DA A rapid HPLC assay for voriconazole in human plasma. J Pharm Biomed Anal 17: Klepser ME, Malone D, Lewis RE, Ernst EJ, and Pfaller MA Evaluation of voriconazole pharmacodynamics using time-kill methodology. Antimicrob Agents Chemother 44: , MacCallum DM, Whyte JA, and Odds FC Efficacy of caspofungin and voriconazole combinations in experimental aspergillosis. Antimicrob Agents Chemother 49: Manavathu EK, Cutright JL, and Chandrasekar PH Organism-dependent fungicidal activities of azoles. Antimicrob Agents Chemother 42: NCCLS Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard, 2 nd ed. NCCLS document M27-A2. NCCLS, Wayne, PA. 11. Pfaller MA, Diekema DJ, Rex JH, Espinel-Ingroff A, Johnson EM, Andes D, 9

10 Chaturvedi V, Ghannoum MA, Odds FC, Rinaldi MG, Sheehan DJ, Troke P, Walsh TJ, and Warnock DW Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints. J Clin Microbiol 44: Smith J, Safdar N, Knasinski V, Simmons W, Bhavnani SM, Ambrose PG, and Andes D Voriconazole therapeutic drug monitoring. Antimicrob Agents Chemother 50:

11 Table 1. Voriconazole MICs and time-kill data against Candida isolates. Isolate MIC (µg/ml) Maximum log-kill (24 h) 1x MIC 4x MIC 16x MIC C. albicans ATCC Maxiumum log-kill (24-48 h) 1x MIC 4x MIC 16x MIC Maximum log-kill (>48 h) 1x MIC 4x MIC 16x MIC C. albicans SC C. glabrata C. glabrata C. parapsilosis C. parapsilosis Time-kill data are presented as the maximum difference in the growth of the control isolate (grown in the absence of voriconazole) and that of the isolate in the presence of various voriconazole concentrations at 24 hours, 24 to 48 hours, and > 48 hours, respectively. Maximum growth inhibition of each isolate at given concentrations is in bold. MICs as determined by E-test. MICs as determined by the Clinical and Laboratory Standards Institute broth microdilution method (CLSI document M27-A2) were within 2-fold agreement. 11

12 1e+8 Ctrl 0.25*MIC 1*MIC 4*MIC 16*MIC C. albicans ATCC e+8 C. glabrata 1 1e+7 1e+7 N_CFU/mL 1e+6 1e+5 1e Time_hr 1e+8 1e+7 1e+6 1e+5 C. glabrata 2 Fig. 1. Time-kill curves for voriconazole against Candida isolates. Representative curves are presented for each isolate. Experiments were performed in duplicate, without significant differences in results. 1e+6 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 C. parapsilosis 2 1e+4 1e

13 120 C. glabrata 1 %Voriconazole Concentration Time_hr Fig.2. Measurements of voriconazole concentrations in culture media by HPLC. Representative data for 48 hour time-kill experiment against C. glabrata #1 are presented. Concentrations of voriconazole against each Candida isolate were 85% of the starting concentration at all time points. 13