Nathan P. Wiederhold, 3,4 * Vincent H. Tam, 1 Jingduan Chi, 1 Randall A. Prince, 1,2 Dimitrios P. Kontoyiannis, 1,2 and Russell E.

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2006, p Vol. 50, No /06/$ doi: /aac Copyright 2006, American Society for Microbiology. All Rights Reserved. Pharmacodynamic Activity of Amphotericin B Deoxycholate Is Associated with Peak Plasma Concentrations in a Neutropenic Murine Model of Invasive Pulmonary Aspergillosis Nathan P. Wiederhold, 3,4 * Vincent H. Tam, 1 Jingduan Chi, 1 Randall A. Prince, 1,2 Dimitrios P. Kontoyiannis, 1,2 and Russell E. Lewis 1,2 The University of Houston College of Pharmacy 1 and The University of Texas M. D. Anderson Cancer Center, 2 Houston, The University of Texas at Austin College of Pharmacy, Austin, 3 and The University of Texas Health Science Center at San Antonio, San Antonio, 4 Texas Received 19 July 2005/Returned for modification 13 September 2005/Accepted 15 November 2005 We conducted a dose fractionation study of neutropenic, corticosteroid-immunosuppressed mice to characterize the pharmacodynamic/pharmacokinetic (PK/PD) parameter most closely associated with amphotericin B (AMB) efficacy in the treatment of invasive pulmonary aspergillosis. Pharmacokinetic parameter estimates were determined by a nonparametric population pharmacokinetic analysis of plasma drug concentrations following single intraperitoneal doses (0.25, 1.0, and 3.0 mg/kg of body weight) of amphotericin B deoxycholate. Three dosage groups (0.5, 0.75, and 1.0 mg/kg) fractionated into three dosing intervals (every 8 h [q8h], q24h, or q72h) were tested to discriminate between the PK/PD parameters (the ratio of maximum concentration of drug in serum [C max ]/ MIC, the ratio of area under the concentration-time curve/mic, and percentage of time above MIC) most closely associated with AMB efficacy over a range of clinically achievable exposures in humans. The efficacy of each regimen was determined by quantitative PCR and survival. Reductions in pulmonary fungal burden and improvements in survival were maximized at the highest peak plasma concentrations in each of the dosage groups. Reductions in pulmonary fungal burden and increased survival were most closely associated with C max /MIC, with maximal activity occurring as the C max /MIC approached 2.4. In our model, C max /MIC is the PK/PD parameter most closely associated with efficacy in the treatment of invasive pulmonary aspergillosis. These data predict that less frequently administered, higher dosages of AMB would optimize efficacy. Amphotericin B (AMB) has been the cornerstone for the treatment of systemic fungal infections for over four decades. Despite recent shifts in the epidemiology of organisms causing these infections in populations at highest risk (4, 12, 17, 21), AMB remains a useful agent due to its broad spectrum and established role in the treatment of endemic and opportunistic mycoses. However, its efficacy is often limited by collateral toxicities in mammalian cells, including infusion-related adverse effects and nephrotoxicity. Reformulation of AMB into lipid carriers has reduced, but not eliminated, these toxicities (24). AMB has long been assumed to be a concentration-dependent fungicidal agent. More specifically, in vitro and in vivo studies have demonstrated concentration-dependent killing versus Candida species (3, 11, 14, 16), suggesting that regimens maximizing peak drug concentrations (i.e., C max /MIC) would be more effective than dosing strategies that maximize overall exposure (area under the concentration-time curve [AUC]/ MIC) or threshold exposures (percentage of time above MIC). Pharmacodynamic observations with Candida species, however, may not be extrapolated to invasive molds such as Aspergillus, for which patterns of antifungal activity may differ based on the unique pathobiology of the fungus. Prior studies of AMB activity in animal models of infection have reported a steep dose-response curve for AMB therapy * Corresponding author. Mailing address: The University of Texas at Austin College of Pharmacy, UTHSCSA, Clinical Pharmacy MSC 6220, 7703 Floyd Curl Dr., San Antonio, TX Phone: (210) Fax: (210) wiederholdn@uthscsa.edu. (2). However, in these studies dosing intervals were not altered. Due to the interrelationship between the pharmacokinetic/pharmacodynamic (PK/PD) components (i.e., the C max / MIC ratio, the AUC/MIC ratio, and the percentage of time above MIC), these study designs are unable to discern which parameter is critical for efficacy (2). Dose fractionation studies, by varying the dosing interval as well as the dose, allow for further discrimination between these PK/PD parameters and identification of dosing parameters that can be optimized to maximize fungal killing and potentially minimize drug toxicity. In order to characterize the PK/PD parameter most closely associated with reductions in Aspergillus fungal burden and improved survival with AMB, we conducted a dose fractionation study using a murine model of invasive pulmonary aspergillosis (IPA). Sinopulmonary inoculation was used to simulate the pathogenesis of human IPA, and the activities of various AMB dosing strategies were assessed by animal survival and analysis of lung fungal burden using a real-time quantitative PCR (qpcr) assay. (This work was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 30 October to 2 November 2004.) MATERIALS AND METHODS Antifungals. Stock solutions of AMB deoxycholate at 5 mg/ml were prepared by dissolving AMB deoxycholate powder (Fungizone; Bristol-Myers Squibb Co., Princeton, NJ) in sterile water on the day of each experiment. Solutions were further diluted in 5% dextrose water prior to animal dosing. Test isolate. Aspergillus fumigatus (AF 293, the strain used in the A. fumigatus genome sequencing project and kindly provided by David Denning, University of 469

2 470 WIEDERHOLD ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. Maximal a posteriori probability Bayesian prediction of plasma AMB concentrations based on median parameter estimates. Predicted plasma AMB concentrations are based on a three-compartment model. The equation of the best-fit line is shown. Manchester, United Kingdom) was used in the animal infection model. Cultures were grown on potato dextrose agar (Remel, Lenexa, KS) at 37 C for 4 to 6 days. Conidia were isolated by washing agar surfaces with 0.1% Tween 80 (Sigma Chemical Co., St. Louis, MO) in sterile physiological saline (10 ml) and filtered through sterile gauze. Inocula were concentrated by centrifuging conidial suspensions at 10,000 g for 10 min, removing the supernatant, and resuspending conidia in a smaller volume of saline. Conidia were then resuspended to achieve a final inoculum of conidia/ml, as confirmed by hemocytometer counts and serial plating on potato dextrose agar to confirm 95% viability. Susceptibility testing. Susceptibility testing was performed in triplicate according to Clinical and Laboratory Standards Institute (formerly NCCLS) M38-A microdilution methodology (19). Briefly, conidial suspensions of conidia/ml were diluted 1:50 in RPMI growth medium (buffered to ph 7.0 with M 4-morpholinepropanesulfonic acid) and dispensed (100 l) into a microtiter tray containing serial twofold dilutions of AMB. The tray was then incubated for 48 h at 37 C, and the MIC was read at 48 h as the lowest drug concentration that showed complete growth inhibition. Results were verified by use of AMB Etest strips (AB Biodisk North America Inc., Piscataway, NJ). The mean MIC of AMB against AF 293 was 0.25 g/ml by both the microdilution and Etest methods. Mice. Female Swiss Webster mice (20 to 25 g at the time of infection) (Charles River Laboratories) were used for all experiments. Animals were housed (five per cage) in presterilized filter-topped cages in the biohazard isolation suite at the University of Texas M. D. Anderson Cancer Center Animal Care Facilities. Animals had access to sterile food and water ad libitum during the duration of experiments. All experiments were performed in accordance with the highest standards for humane handling, care, and treatment of research animals. This protocol was approved by the University of Texas M. D. Anderson Cancer Center and the University of Houston Institutional Animal Care and Use Committees. Immunosuppression. Cyclosphosphamide (Sigma) was dissolved in sterile saline (15 mg/ml) and injected intraperitoneally (IP) (200 to 250 l) on days 4 and 1 prior to inoculation. This rendered mice neutropenic (absolute neutrophil count, 100/ml) from within 4 days of the first injection to 4 days after the last injection. Cortisone acetate (Sigma) was suspended in sterile saline (65 mg/ml) containing 0.1% Tween 20 (Sigma) and was administered by subcutaneous injection (100 to 150 l) on day 1 before inoculation. Pharmacokinetics. Determinations of single-dose, 24-h plasma AMB concentrations in immunosuppressed, infected mice were made to characterize the pharmacokinetics of AMB. Three IP doses of AMB (0.25, 1.0, and 3.0 mg/kg of body weight) were tested, using 18 mice/dose. Plasma samples from three mice were obtained at six time points (0.5, 2, 4, 8, 12, and 24 h) after IP administration of AMB, and concentrations were determined by high-performance liquid chromatography, as previously described (15, 20). The plasma drug concentration profiles of all the animals were analyzed by a population pharmacokinetic analysis, using the Non-Parametric Adaptive Grid (NPAG) program (13). A three-compartment model with first-order absorption from the peritoneum, intercompartmental transfer, and elimination was fit to the observations. The interday variation of the AMB assay was used to formulate the variance structural model. A quadratic relationship was used to describe the relationship between the standard deviation (SD) and AMB concentration (SD c c 2, where c is the concentration of AMB) and was incorporated as the weighting scheme in the search algorithm of the NPAG program. Using the parameter point estimates of the final model, AMB pharmacokinetic profiles resulting from various dosing regimens were simulated with ADAPT II software (Biomedical Simulations Resource, University of Southern California, Los Angeles). The peak concentration was determined from the maximum concentration in the concentration-time profile of the first dose. The AUC over 72 h (AUC 72 ) was derived by integrating serum drug concentration with respect to time from 0 to 72 h. The average daily AMB exposure (AUC 24 ) was derived by dividing the AUC 72 by 3. Time above MIC was determined by comparing various simulated pharmacokinetic profiles over 72 h to MIC. Infection and treatment. To simulate the pulmonary pathogenesis seen in humans, mice ( 10 per dose tested plus controls) were infected via the sinopulmonary route by a previously reported method (9, 15, 23). Briefly, a single droplet (30 l) of a conidial suspension ( conidia/ml), which the mice voluntarily inhaled, was applied to the nares of anesthetized mice via a micropipette; the mice were returned to filter-topped cages once normal breathing resumed. AMB therapy was begun 12 h after inoculation at doses ranging from mg/kg to 3 mg/kg administered IP at one of three dosing intervals (every 8 h [q8h], q24h, or q72h). Mice were observed at least three times daily until day 4 after inoculation. Animals that became moribund before day 4 were euthanized, and death was recorded as occurring 8 h later. Surviving mice were euthanized on day 4 for determination of fungal burden. Pulmonary fungal burden. Pulmonary fungal burden was determined by realtime qpcr by methods previously reported (6, 23). Briefly, DNA samples isolated from homogenized lungs were assayed in duplicate by use of an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA) using primers and a dual-labeled fluorescent hybridization probe specific for the Aspergillus 18S rrna gene (GenBank accession number AB008401). The threshold cycle (C t ) of each sample was interpolated from a seven-point standard curve of C t values prepared by spiking uninfected mouse lungs with known amounts of conidia (10 1 to 10 7 ) from AF 293. An internal standard was amplified in separate reactions to correct for the percent difference in DNA recovery. Results are reported as conidial equivalents (CE) of A. fumigatus DNA. Statistics. Differences in fungal burden endpoints between different dosing intervals for each dose fractionation group were assessed for significance by analysis of variance with Tukey s posttest for multiple comparisons. Survival was plotted by Kaplan-Meier analysis, and differences between dose fractionation groups were analyzed by the log rank test. The relationship of C max /MIC and animal survival during the experimental period was examined by fitting a loglogistic parametric survival time model to all treatment groups at various C max / MIC exposures. A four-parameter nonlinear logistic model (Hill equation) was then fitted to survival functions at various C max /MIC exposures. Analysis was performed with Stata Rel 8 (Stata Corp, College Station, TX) and Prism 4 (GraphPad Software, Inc., San Diego, CA). A P value of 0.05 was considered statistically significant for all comparisons. RESULTS Pharmacokinetics. A three-compartment model satisfactorily described the pharmacokinetics of AMB with the following me- Dose fractionation group TABLE 1. Simulated pharmacokinetic parameters AMB dose (mg/kg) and interval C max ( g/ml) (first dose) AUC 24 ( g h/ml) 0.5 mg/kg q8h q24h q72h mg/kg 0.25 q8h q24h q72h mg/kg 0.33 q8h q24h q48h

3 VOL. 50, 2006 AMB PHARMACODYNAMICS IN INVASIVE ASPERGILLOSIS 471 TABLE 2. PK/PD parameters and pulmonary fungal burden in immunosuppressed mice with IPA AMB dose (mg/kg) and interval C max /MIC ratio 24-h AUC/MIC ratio % Time above MIC (over 72 h) Mean CE (SE) Control 6.79 (0.38) q8h (0.39) 0.5 q24h (0.58) 1.5 q72h (0.50) 0.25 q8h (0.48) 0.75 q24h (0.47) 2.25 q72h (0.35) 0.33 q8h (0.59) 1 q24h (0.79) 3 q72h (0.34) dian parameters: absorption rate constant, 3.16 h 1 ; clearance, liters/h; intercompartment (central to peripheral) transfer rate, 28.4 h 1 ; intercompartment (peripheral to central) transfer rate, 40.1 h 1 ; volume of distribution of the central compartment, liters; terminal half-life, 11.2 h. Approximately 98% of the variance could be explained by the maximal a posteriori probability Bayesian prediction of plasma AMB concentrations using the median parameter estimates, as shown in Fig. 1. The AMB steady-state pharmacokinetic profiles resulting from various simulations are presented in Table 1. Pulmonary fungal burden. Based on the pharmacokinetic simulations, fractionated dosing strategies for AMB (0.5, 0.75, and 1.0 mg/kg, each divided into q8h, q24h, and q72h administration intervals) were tested in infected mice. Maximum reductions in Aspergillus CE DNA were observed with the regimen achieving the highest C max /MIC ratio in each of the three dose fractionation groups studied and were significantly lower than in control (untreated) animals (P 0.05) (Table 2 and Fig. 2). Conversely, doses that maximized the percentage of time the concentration exceeded the MIC did not reduce pulmonary fungal burden compared to controls in the 1-mg/kg dose fractionation group. These results suggest that in this murine model of IPA, C max /MIC is the PK/PD parameter most closely related to reductions in fungal burden. Interestingly, no AMB dose resulted in undetectable levels of Aspergillus DNA in the lungs of the animals. This is in contrast to in vitro studies in which rapid and complete fungicidal activity of AMB is frequently observed (16). A threshold C max /MIC (2.4), corresponding to a C max concentration of 0.6 g/ml, was also observed, above which increases in this PK/PD parameter value did not result in further reductions in pulmonary fungal burden. Survival. Similar to the reductions in pulmonary fungal burden, survival was improved with doses that maximized the C max /MIC ratio. In the 0.75-mg/kg dose fractionation group the percentage of animals surviving to the end of the study was significantly higher (93.3%) in animals that received 2.25 mg/kg q72h than in controls (40%, P 0.003). However, survival rates for animals dosed 0.25 mg/kg q8h and 0.75 mg/kg q24h (57.1% and 60%, respectively) did not differ from controls. Similarly, in the 1-mg/kg dose fractionation group survival was significantly improved in animals that received the dose that maximized the C max /MIC ratio (3 mg/kg q72h) compared to controls (86.7% versus 40%, respectively; P 0.02). As shown in Fig. 3, nonlinear logistic regression analysis of the survival fraction versus C max /MIC also demonstrated survival to be optimized as the C max /MIC ratio approached 2.4 (survival fraction, 0.87). According to this model 90% survival would be achieved with a C max /MIC of DISCUSSION Previous in vivo work has demonstrated C max /MIC to be the PK/PD parameter most closely associated with AMB efficacy against Candida species. Using a murine model of invasive candidiasis, Andes and colleagues reported reductions in CFU counts in the kidneys to be most closely correlated with C max / MIC (3). Similarly, we found C max /MIC to be the PK/PD parameter most closely associated with AMB activity in a neutropenic, corticosteroid-immunosuppressed murine model of IPA. Reductions in fungal burden and improvements in survival were most closely associated with peak plasma AMB concentrations, with response reaching a plateau as the C max / MIC ratio approached 2.4. To our knowledge, this is the first study to show C max /MIC to be the PK/PD parameter most FIG. 2. Pulmonary fungal burden per dosing regimen. Mean ( standard error) log Aspergillus qpcr CE are plotted on the y axis. Each bar represents a group of mice receiving different doses of AMB deoxycholate at different dosing intervals. Each group (0.5, 0.75, and 1.0 mg/kg) was fractionated into three different dosing intervals (q8h, q24h, or q72h)., P 0.05 (compared to controls).

4 472 WIEDERHOLD ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 3. Four-parameter nonlinear logistic model of survival versus C max /MIC. Dashed lines represent the 95% confidence interval of the regression analysis. Arrows to the y axis and x axis represent the survival fraction (0.87) at a C max /MIC of 2.4. closely associated with AMB efficacy in IPA. Interestingly, the C max /MIC ratio associated with maximum reductions in fungal burden in the current study is lower than that previously reported for disseminated candidiasis (2.4 versus 10, respectively) (1, 3). However, numerous factors may explain the differences in the magnitude of C max /MIC ratios associated with optimal antifungal activity. Killing patterns are unlikely to be identical among different fungal species and different sites of infection. Indeed, in an in vivo study of disseminated and pulmonary candidiasis, higher doses of the deoxycholate formulation of AMB were required to achieve the same reductions in fungal burden in the kidneys versus the lungs, and the differences in potency were reported to correlate with differences in kinetics at the sites of infection (D. R. Andes, N. Safdar, K. Marchillo, and R. Conklin, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-1579, 2003). Despite the reduction in pulmonary fungal burden and increased survival associated with C max /MIC, no regimen was capable of sterilizing the lung tissue. While this is in contrast to in vitro data that demonstrated complete fungicidal activity (16), previous animal models of IPA have shown a similar modest efficacy of AMB in the lungs despite improved survival (15, 18, 22). The reason for incomplete tissue sterilization of infecting organisms by AMB is unknown but may be related to its limited solubility at a neutral ph and virtually unlimited protein binding. Using equilibrium dialysis to measure the unbound concentration in human plasma, Bekersky et al. reported that the percentage of AMB found relative to albumin and 1 -acid glycoprotein increases linearly in a concentrationdependent fashion to a threshold at which the availability of higher unbound free drug concentrations is saturated (5). Surprisingly, the maximum unbound concentration that could be achieved was predicted to be 744 ng/ml. Hence, the poor solubility of AMB coupled with the unusual protein-binding pattern could create a pharmacodynamic ceiling for concentrationdependent activity. Interestingly, the threshold of unbound, presumptively bioactive drug predicted by Bekersky et al. is similar to the resistance breakpoint proposed by investigators studying AMB failures and to the maximally effective peak plasma concentration in our murine model of IPA (0.6 g/ml). Indeed, Collette et al. reported wide variations in AMB bioactivity depending on tissue sites (i.e., liver, lung, brain) (8), and clinical failures have been documented with invasive mycoses despite tissue concentrations of 10- to 100-fold above the MIC (7). As with all animal models, our murine model of IPA has some limitations that must be considered before extrapolating the PK/PD data to human infections. A hyperacute IPA murine model was utilized that may not reflect the more indolent pathobiology of IPA seen in chronically immunocompromised hosts. Also, the pharmacokinetic data, although similar to other reported data from murine models (3, 10, 15), may not reflect the considerable accumulation of AMB in tissues that occurs in humans who receive prolonged administration of this agent. Furthermore, one must be cautious in extrapolating these results to lipid formulations of AMB, as the agents, which differ in their pharmacokinetic parameters, tissue distribution, and potency from the deoxycholate formulation, were not evaluated. Finally, a single isolate of A. fumigatus was evaluated, bringing into question the applicability of these results to A. fumigatus strains or other Aspergillus species with reduced susceptibility to AMB. In summary, these results support plasma C max /MIC as the PK/PD parameter most closely associated with the effectiveness of AMB in IPA. Reductions in fungal burden and improvements in survival were maximized with this parameter. ACKNOWLEDGMENTS We acknowledge financial support from the Society of Infectious Disease Pharmacists (to N.P.W. and R.E.L.), the University of Texas M. D. Anderson Faculty E. N. Cobb Scholar Award Research Endowment (to D.P.K.), and the National Institutes of Health (core grant to the University of Texas M. D. Anderson Cancer Center Animal Care Unit). R.E.L. and D.P.K. receive research support and consultancy fees from Merck & Co., Pfizer, Astellas, Enzon, and Schering-Plough. R.A.P. receives research support from Merck & Co., Bristol-Myers Squibb, Pfizer, Ortho McNeil, and Enzon. V.H.T. receives research support from AstraZeneca. REFERENCES 1. Andes, D Clinical utility of antifungal pharmacokinetics and pharmacodynamics. Curr. Opin. Infect. Dis. 17: Andes, D In vivo pharmacodynamics of antifungal drugs in treatment of candidiasis. Antimicrob. Agents Chemother. 47: Andes, D., T. Stamsted, and R. 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