Intrapulmonary Posaconazole Penetration at the Infection Site in an Immunosuppressed Murine Model of Invasive Pulmonary Aspergillosis Receiving Oral Prophylactic Regimens Seyedmojtaba Seyedmousavi,a Roger J. M. Brüggemann,b Willem J. G. Melchers,a Paul E. Verweij,a Johan W. Moutona,c Department of Medical Microbiology, Radboudumc, Nijmegen, the Netherlandsa; Department of Pharmacy, Radboudumc, Nijmegen, the Netherlandsb; Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, the Netherlandsc
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nhalation of Aspergillus fumigatus conidia is the most common route of infection in invasive aspergillosis (IA), with the lung being the primary site of infection. Adequate drug penetration to the infection site is therefore crucial for optimal efficacy. Posaconazole is the recommended drug for prophylaxis of Aspergillus diseases in neutropenic patients and stem cell transplantation (SCT) recipients (1–3). Notably, despite relatively low levels of free (unbound) posaconazole exposure in plasma (4–6), there appears to be an adequate protection (adequate prophylaxis) against infection due to Aspergillus fumigatus with an MIC higher than susceptible breakpoints to posaconazole. This is somewhat surprising, compared to some other triazole antifungals in similar animal models and clinical trials of invasive candidiasis (7). This phenomenon could possibly be related to a higher-than-predicted posaconazole penetration at the infection/colonization site, particularly in case of lung infection. Therefore, we set out to explore the posaconazole concentrations and corresponding exposure in pulmonary epithelial lining fluid (ELF) compared to those in blood levels. A clinical A. fumigatus isolate (posaconazole MIC of 0.5 mg/ liter) obtained from a patient with proven IA was used in the experiments. Strain identification was confirmed by sequencebased analysis, as described previously (8). The isolate had been stored in 10% glycerol broth at ⫺80°C. Before performing the experiment, the isolate was cultured once on Sabouraud dextrose agar (SDA) for 5 to 7 days at 35 to 37°C and subcultured twice on 15-cm Takashio slants for 5 to 7 days at 35 to 37°C. The conidia were harvested in 20 ml of sterile phosphate-buffered saline (PBS) and 0.1% Tween 80 (Boom B.V. Meppel, the Netherlands). The conidial suspension was filtered through sterile gauze folded four times to remove any hyphae, and the number of conidia was counted in a hemocytometer. After the inoculum was adjusted to the required concentration, the conidial suspension was stored overnight at 4°C. The in vitro antifungal susceptibility test was performed based on the EUCAST guidelines, using a broth microdilution format (9). A total of 96 outbred CD-1 (Charles River, the Netherlands) female mice, 4 to 5 weeks old, weighing 20 to 22 g, were used. To render the mice neutropenic, cyclophosphamide (150 mg/kg of body weight on day ⫺4 and 100 mg/kg on day ⫺1) was adminis-
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tered. Animals received 4, 8, 16, or 32 mg/kg posaconazole oral solution (Schering-Plough B.V., Boxmeer, the Netherlands) once daily at days ⫺2, ⫺1, and 0 by oral gavage. On day 0, mice were infected with the A. fumigatus isolate through instillation of conidial suspension in the nares. The infection model was confirmed by 0% survival in survival experiments with untreated control animals (n ⫽ 44 mice) (10, 11). Blood and bronchoalveolar lavage (BAL) samples were drawn at 8 predefined time points postinfection (0, 0.5, 1, 2, 4, 8, 12, and 24 h, 3 mice per each time point), as described previously (12). Briefly, the blood samples were drawn through the orbital vein into lithium-heparin-containing tubes and were cooled and centrifuged for approximately 10 min at 1,000 ⫻ g within 30 min of collection. Plasma was aspirated, transferred in two 2-ml plastic tubes, and stored at ⫺80°C. BAL fluid was obtained using a technique described previously (10, 11). After being sacrificed under isoflurane anesthesia followed by cervical dislocation, the mice were secured on a plastic platform. The trachea was exposed by a 1-cm incision on the ventral neck skin for insertion of the cannula and sutured in place. Lungs were instilled 4 times with 0.5 ml of sterile 0.9% saline, with the fluid being immediately aspirated. The aspirates recovered from the instillations were pooled per mouse, placed on ice after each aliquot, and subsequently stored at ⫺80°C. Posaconazole concentrations in plasma and BAL fluid were measured by a validated (for human and mouse matrices) ultraperformance liquid chromatography (UPLC) method with fluorescence detection. Details of the analytical assay are described elsewhere (13). Geometric mean concentrations of total posaconazole in plasma were calculated for each time point (n ⫽ 3 mice). Peak concentrations in plasma (Cmax) were directly observed from the data. Pharmacokinetic parameters were derived using noncom-
Antimicrobial Agents and Chemotherapy
Received 14 January 2014 Returned for modification 9 February 2014 Accepted 17 February 2014 Published ahead of print 24 February 2014 Address correspondence to Johan W. Mouton. [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.00053-14
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Adequate penetration to the infection/colonization site is crucial to attain optimal efficacy of posaconazole against Aspergillus fumigatus diseases. We evaluated posaconazole exposure in pulmonary epithelial lining fluid (ELF) in a murine model of invasive pulmonary aspergillosis. The posaconazole exposure (area under the plasma concentration-time curve from time zero to 24 h postinfusion [AUC0 –24]) in ELF was 20% to 31% of that in plasma for total drug after the third dose, and the relationship between plasma and ELF exposure was linear (r2 ⴝ 0.97, P ⴝ 0.016).
Posaconazole Penetration in ELF Compared to the Blood
partmental analysis with Phoenix, version 6.2 (Pharsight, Inc.). The area under the plasma concentration-time curve from time zero to 24 h postinfusion (AUC0 –24) was determined by use of the log-linear trapezoidal rule. The elimination rate constant was determined by linear regression of the terminal points of the loglinear plasma concentration-time curve. The terminal half-life was defined as ln2 divided by the elimination rate constant. Clearance (CL) was calculated as dose/AUC0 –24. Concentrations of total posaconazole in BAL fluid from three mice per time point were determined as described previously (13). Urea in BAL aspirate and plasma was measured utilizing a modified enzymatic assay (QuantiChrom urea assay kit, DIUR-500; BioAssay Systems) (14, 15). The concentration of posaconazole in ELF was then determined by using the ratio of urea concentration in BAL fluid to that in plasma as described previously (10, 11, 14–19): drug concentrationELF ⫽ drug concentrationBAL ⫻ ureaplasma/ureaBAL. All data analyses were performed using Graph-Pad Prism software, version 5.0, for Windows (GraphPad Software, San Diego, CA). A regression analysis was conducted to determine the linearity between posaconazole concentration in blood and ELF. The goodness of fit was checked by use of the r2 value and visual inspection. Statistical significance was defined as a P value of ⬍0.05 (two-tailed). The Cmax and AUC0 –24 data were log10 transformed to approximate a normal distribution prior to statistical analysis. All animals were alive at the time of sample collection from 0 to 24 h postchallenge, and 192 blood and BAL samples were obtained. The drug concentrations in plasma, including the Cmax of po-
May 2014 Volume 58 Number 5
saconazole, were higher than those in ELF for all dosages (Fig. 1). A significant correlation between mean posaconazole concentrations in plasma and ELF was noted by linear regression analysis (r2 ⫽ 0.61, P ⬍ 0.0001) (Fig. 2a). However, a better correlation was found for AUC0 –24 in plasma and ELF in a linear fashion for the dosages of 4 to 32 mg/kg (r2 ⫽ 0.97, P ⫽ 0.016) (Fig. 2b). The total AUC0 –24 in plasma and ELF is shown in Table 1. The penetration of posaconazole in ELF based on total drug was between 20.21 and 31.39%. At the highest dose (32 mg/kg), the total AUC0 –24/MIC ratio was ⱖ581 in plasma and ⱖ157.56 in ELF for the isolate with an MIC of ⱕ0.5 mg/liter. In the present study, we investigated the intrapulmonary posaconazole penetration in an immunosuppressed murine model of invasive pulmonary aspergillosis receiving oral prophylactic regimens. Our results indicate that posaconazole exposure (AUC0 –24) in ELF was 20% to 31% of that in plasma for total drug, and the relationship between posaconazole and AUC0 –24 in plasma and ELF was significantly linear (r2 ⫽ 0.97). Our findings are comparable to the results of two clinical observations of Conte et al. (20, 21). In the first study, the plasma and intrapulmonary concentrations of posaconazole were evaluated in 25 healthy adult subjects receiving multiple doses (13 or 14) of oral posaconazole suspension 400 mg twice daily with a high-fat meal (20). The AUC0 –12 values in ELF and plasma were 18.3 and 21.9 mg · h/liter, respectively, corresponding to an ELF-to-plasma penetration ratio of 86% of total drug. However, the corresponding exposure in alveolar macrophages (AMs) was appreciably higher (range, 46.2 to 87.7 mg/liter; AUC0 –12 of 715 mg · h/liter). Similarly, in the second study of the above-mentioned group (21),
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FIG 1 Plasma and ELF concentrations of posaconazole following oral administration of 4, 8, 16, and 32 mg/kg to immunosuppressed mice infected with A. fumigatus after the third dose. Each symbol corresponds to the geometric mean plasma levels for three mice.
Seyedmousavi et al.
higher-than-predicted intrapulmonary penetration of posaconazole was observed in 20 adult lung transplant patients receiving posaconazole suspension 400 mg twice daily with a high-fat meal. The AUC0 –12 values in plasma, ELF, and AMs were 10.99, 11.21, and 530.2 mg · h/liter, respectively. Notably, the lipophilic nature and high intracellular (cell-associated) concentrations of posaconazole might contribute to distribution at the infection/colonization site, in this case ELF (22, 23). Campoli et al. demonstrated that lipophilic characteristics of posaconazole increase the penetration of posaconazole into mammalian host cell membranes, which can mediate its efficacy in prophylactic regimens and likely explains the observed discrepancy between posaconazole low serum level and high potency (23). They exposed intracellular cells to posaconazole and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40- to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. On the other hand, Farowski et al. demonstrated that administration of posaconazole oral solution resulted in significantly increased intracellular concentrations in the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) compared to those in plasma (22). They collected 23 blood samples from 14 patients receiving posaconazole for prophylaxis of fungal infections in order to determine the intracellular concentrations of posaconazole in different compartments of the peripheral blood. These samples were separated by double-discontinuous Ficoll-Hypaque density gradient
TABLE 1 Pharmacokinetic parameters of posaconazole in plasma and ELF following 3 days of once daily oral administration of 4, 8, 16, and 32 mg/kg in immunosuppressed mice
Posaconazole dose (mg/kg) 4 8 16 32
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Cmax (mg/liter)
AUC0–24 mg · h/liter
Plasma
ELF
Plasma
ELF
ELF-plasma Cmax ratio (%)
5.51 8.7 10.92 15.04
1.58 2.83 3.62 5.32
72.69 149.80 198.90 290.50
14.69 42.14 62.23 78.78
28.68 32.53 33.15 35.37
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ELF-plasma AUC0–24 ratio (%) 20.21 28.13 31.29 27.12
centrifugation. The intracellular posaconazole concentrations of PBMCs, PMNs, and red blood cells (RBCs) were determined by liquid chromatography-tandem mass spectrometry. The intracellular concentrations of the PBMCs and PMNs were significantly higher than those of surrounding media (P ⬍ 0.001). The ratios between the intracellular and extracellular concentrations (C/E) were 22.5 ⫾ 21.2, 7.66 ⫾ 6.50, and 0.09 ⫾ 0.05 for the PBMCs, PMNs, and RBCs, respectively. The exposure-response relationships of posaconazole have been defined previously in experimental models of Aspergillus infections (4, 6, 24). Similar to the other triazoles, posaconazole displays exposure-dependent pharmacodynamic characteristics, for which a free AUC0 –24/MIC ratio ranging from 1.67 to 1.78 was the value predictive of success associated with half-maximal efficacy. In general, it is accepted that only the unbound fraction of a drug in serum/plasma is pharmacologically active. Considering the high degree of posaconazole protein binding in plasma (98 to 99%) and negligible bounds in ELF, the results of the current study indicated that effective local concentrations might be achieved even at the lowest dose (4 mg/kg) with a free AUC0 –24/ MIC ratio of 1.76 in plasma and 14.69 in ELF, which is probably high enough to prevent infection with A. fumigatus isolates with an MIC of ⱕ0.5. However, for the isolates with resistant phenotypes and higher MIC to posaconazole (ⱖ16 mg/kg), the obtained free AUC0 –24/MIC in plasma was ⱕ0.18 in plasma and ⱕ4.92 in ELF at the highest dose (32 mg/kg), which indicates the possibility of breakthrough IA. We conclude that penetration of posaconazole in ELF is relatively high, which is consistent with lipophilic characteristics and its increased intracellular permeability. The high intrapulmonary penetration ratio of posaconazole indicates that this agent is an optimal therapeutic choice in prevention of IA due to azole-susceptible and/or azole-resistant A. fumigatus. (Parts of these results were presented at the 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 9 to 12 September 2012, and 6th Trends in Medical Mycology, 11 to 14 October 2013, Copenhagen, Denmark.) ACKNOWLEDGMENTS S.S. and W.J.G.M. have no conflicts of interests. R.J.M.B., P.E.V., and J.W.M. have served as consultants to and have received research grants from Gilead Sciences, Merck, Astellas, and Pfizer.
Antimicrobial Agents and Chemotherapy
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FIG 2 Relationship between posaconazole concentrations (a) and AUC0 –24 (b) in plasma and ELF. The line indicates the linear regression.
Posaconazole Penetration in ELF Compared to the Blood
This study was supported, in part, by an unrestricted research grant from Gilead Sciences.
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REFERENCES
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12.
13.
14. 15.
16. 17.
18. 19. 20.
21.
22.
23.
24.
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1. EMA. 2012. European public assessment report (EPAR) for Noxafil, 06/ 09/2012 ed. European Medicines Agency, London, United Kingdom. 2. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens DA, van Burik JA, Wingard JR, Patterson TF. 2008. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin. Infect. Dis. 46:327–360. http://dx.doi.org/10.1086/525258. 3. Herbrecht R, Denning DW, Patterson TF, Bennett JE, Greene RE, Oestmann JW, Kern WV, Marr KA, Ribaud P, Lortholary O, Sylvester R, Rubin RH, Wingard JR, Stark P, Durand C, Caillot D, Thiel E, Chandrasekar PH, Hodges MR, Schlamm HT, Troke PF, de Pauw B. 2002. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N. Engl. J. Med. 347:408 – 415. http://dx.doi.org/10.1056 /NEJMoa020191. 4. Mavridou E, Bruggemann RJ, Melchers WJ, Mouton JW, Verweij PE. 2010. Efficacy of posaconazole against three clinical Aspergillus fumigatus isolates with mutations in the cyp51A gene. Antimicrob. Agents Chemother. 54:860 – 865. http://dx.doi.org/10.1128/AAC.00931-09. 5. Petraitiene R, Petraitis V, Groll AH, Sein T, Piscitelli S, Candelario M, Field-Ridley A, Avila N, Bacher J, Walsh TJ. 2001. Antifungal activity and pharmacokinetics of posaconazole (SCH 56592) in treatment and prevention of experimental invasive pulmonary aspergillosis: correlation with galactomannan antigenemia. Antimicrob. Agents Chemother. 45: 857– 869. http://dx.doi.org/10.1128/AAC.45.3.857-869.2001. 6. Lepak AJ, Marchillo K, Vanhecker J, Andes DR. 2013. Posaconazole pharmacodynamic target determination against wild-type and Cyp51 mutant isolates of Aspergillus fumigatus in an in vivo model of invasive pulmonary aspergillosis. Antimicrob. Agents Chemother. 57:579 –585. http://dx.doi.org/10.1128/AAC.01279-12. 7. Andes D, Marchillo K, Conklin R, Krishna G, Ezzet F, Cacciapuoti A, Loebenberg D. 2004. Pharmacodynamics of a new triazole, posaconazole, in a murine model of disseminated candidiasis. Antimicrob. Agents Chemother. 48:137–142. http://dx.doi.org/10.1128/AAC.48.1.137-142.2004. 8. Snelders E, van der Lee HA, Kuijpers J, Rijs AJ, Varga J, Samson RA, Mellado E, Donders AR, Melchers WJ, Verweij PE. 2008. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med. 5:e219. http://dx.doi.org/10.1371/journal.pmed .0050219. 9. Rodriguez-Tudela JL, Arendrup MC, Arikan S, Barchiesi F, Bille J, Cuenca-Estrella E CEMM, Dannaoui EE, Denning DW, Donnelly JP, Fegeler W, Lass-Flörl C, Moore C, Richardson M, Gaustad P, Schmalreck A, Velegraki AA, Verweij P. 2008. EUCAST definitive document E.DEF 9.1: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds. Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). http://www.eucast.org/fileadmin/src/media/PDFs /4ESCMID_Library/3Publications/EUCAST_Documents/Other _Documents/EUCAST_moulds_DEFINITIVE_document_V_ISO _April_08%20final.pdf. 10. Seyedmousavi S, Brüggemann RJM, Melchers WJG, Mouton JW, Verweij PE. 2013. Posaconazole prophylaxis in experimental azole-resistant
invasive pulmonary aspergillosis, poster M-761. Final Prog. 53rd Intersci. Conf. Antimicrob. Agents Chemother. Seyedmousavi S, Melchers WJG, Verweij PE, Mouton JW. 2012. Intrapulmonary penetration of posaconazole in immunosuppressed mice receiving oral prophylactic regimens, poster M-1741. Final Prog. 52nd Intersci. Conf. Antimicrob. Agents Chemother. Seyedmousavi S, Bruggemann RJM, Melchers WJG, Verweij PE, Mouton JW. 2013. Higher than predicted posaconazole penetration at the infection site in a murine model of invasive pulmonary aspergillosis. Mycoses 56(Suppl S3):151. Verweij-van Wissen CP, Burger DM, Verweij PE, Aarnoutse RE, Bruggemann RJ. 2012. Simultaneous determination of the azoles voriconazole, posaconazole, isavuconazole, itraconazole and its metabolite hydroxyitraconazole in human plasma by reversed phase ultra-performance liquid chromatography with ultraviolet detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 887– 888:79 – 84. http://dx.doi.org/10.1016/j .jchromb.2012.01.015. Kiem S, Schentag JJ. 2008. Interpretation of antibiotic concentration ratios measured in epithelial lining fluid. Antimicrob. Agents Chemother. 52:24 –36. http://dx.doi.org/10.1128/AAC.00133-06. Rodvold KA, George JM, Yoo L. 2011. Penetration of anti-infective agents into pulmonary epithelial lining fluid: focus on antibacterial agents. Clin. Pharmacokinet. 50:637– 664. http://dx.doi.org/10.2165/11594090 -000000000-00000. Summer R, Kotton DN, Sun X, Ma B, Fitzsimmons K, Fine A. 2003. Side population cells and Bcrp1 expression in lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 285:L97–L104. Rennard SI, Basset G, Lecossier D, O’Donnell KM, Pinkston P, Martin PG, Crystal RG. 1986. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J. Appl. Physiol. 60:532–538. Chinard FP. 1992. Quantitative assessment of epithelial lining fluid in the lung. Am. J. Physiol. 263:L617–L618. Von Wichert P, Joseph K, Muller B, Franck WM. 1993. Bronchoalveolar lavage. Quantitation of intraalveolar fluid? Am. Rev. Respir. Dis. 147:148 – 152. http://dx.doi.org/10.1164/ajrccm/147.1.148. Conte JE, Jr, Golden JA, Krishna G, McIver M, Little E, Zurlinden E. 2009. Intrapulmonary pharmacokinetics and pharmacodynamics of posaconazole at steady state in healthy subjects. Antimicrob. Agents Chemother. 53:703–707. http://dx.doi.org/10.1128/AAC.00663-08. Conte JE, Jr, DeVoe C, Little E, Golden JA. 2010. Steady-state intrapulmonary pharmacokinetics and pharmacodynamics of posaconazole in lung transplant recipients. Antimicrob. Agents Chemother. 54:3609 – 3613. http://dx.doi.org/10.1128/AAC.01396-09. Farowski F, Cornely OA, Vehreschild JJ, Hartmann P, Bauer T, Steinbach A, Ruping MJ, Muller C. 2010. Intracellular concentrations of posaconazole in different compartments of peripheral blood. Antimicrob. Agents Chemother. 54:2928 –2931. http://dx.doi.org/10.1128/AAC.01407-09. Campoli P, Al Abdallah Q, Robitaille R, Solis NV, Fielhaber JA, Kristof AS, Laverdiere M, Filler SG, Sheppard DC. 2011. Concentration of antifungal agents within host cell membranes: a new paradigm governing the efficacy of prophylaxis. Antimicrob. Agents Chemother. 55:5732– 5739. http://dx.doi.org/10.1128/AAC.00637-11. Howard SJ, Lestner JM, Sharp A, Gregson L, Goodwin J, Slater J, Majithiya JB, Warn PA, Hope WW. 2011. Pharmacokinetics and pharmacodynamics of posaconazole for invasive pulmonary aspergillosis: clinical implications for antifungal therapy. J. Infect. Dis. 203:1324 –1332. http://dx.doi.org/10.1093/infdis/jir023.
I
nhalation of Aspergillus fumigatus conidia is the most common route of infection in invasive aspergillosis (IA), with the lung being the primary site of infection. Adequate drug penetration to the infection site is therefore crucial for optimal efficacy. Posaconazole is the recommended drug for prophylaxis of Aspergillus diseases in neutropenic patients and stem cell transplantation (SCT) recipients (1–3). Notably, despite relatively low levels of free (unbound) posaconazole exposure in plasma (4–6), there appears to be an adequate protection (adequate prophylaxis) against infection due to Aspergillus fumigatus with an MIC higher than susceptible breakpoints to posaconazole. This is somewhat surprising, compared to some other triazole antifungals in similar animal models and clinical trials of invasive candidiasis (7). This phenomenon could possibly be related to a higher-than-predicted posaconazole penetration at the infection/colonization site, particularly in case of lung infection. Therefore, we set out to explore the posaconazole concentrations and corresponding exposure in pulmonary epithelial lining fluid (ELF) compared to those in blood levels. A clinical A. fumigatus isolate (posaconazole MIC of 0.5 mg/ liter) obtained from a patient with proven IA was used in the experiments. Strain identification was confirmed by sequencebased analysis, as described previously (8). The isolate had been stored in 10% glycerol broth at ⫺80°C. Before performing the experiment, the isolate was cultured once on Sabouraud dextrose agar (SDA) for 5 to 7 days at 35 to 37°C and subcultured twice on 15-cm Takashio slants for 5 to 7 days at 35 to 37°C. The conidia were harvested in 20 ml of sterile phosphate-buffered saline (PBS) and 0.1% Tween 80 (Boom B.V. Meppel, the Netherlands). The conidial suspension was filtered through sterile gauze folded four times to remove any hyphae, and the number of conidia was counted in a hemocytometer. After the inoculum was adjusted to the required concentration, the conidial suspension was stored overnight at 4°C. The in vitro antifungal susceptibility test was performed based on the EUCAST guidelines, using a broth microdilution format (9). A total of 96 outbred CD-1 (Charles River, the Netherlands) female mice, 4 to 5 weeks old, weighing 20 to 22 g, were used. To render the mice neutropenic, cyclophosphamide (150 mg/kg of body weight on day ⫺4 and 100 mg/kg on day ⫺1) was adminis-
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tered. Animals received 4, 8, 16, or 32 mg/kg posaconazole oral solution (Schering-Plough B.V., Boxmeer, the Netherlands) once daily at days ⫺2, ⫺1, and 0 by oral gavage. On day 0, mice were infected with the A. fumigatus isolate through instillation of conidial suspension in the nares. The infection model was confirmed by 0% survival in survival experiments with untreated control animals (n ⫽ 44 mice) (10, 11). Blood and bronchoalveolar lavage (BAL) samples were drawn at 8 predefined time points postinfection (0, 0.5, 1, 2, 4, 8, 12, and 24 h, 3 mice per each time point), as described previously (12). Briefly, the blood samples were drawn through the orbital vein into lithium-heparin-containing tubes and were cooled and centrifuged for approximately 10 min at 1,000 ⫻ g within 30 min of collection. Plasma was aspirated, transferred in two 2-ml plastic tubes, and stored at ⫺80°C. BAL fluid was obtained using a technique described previously (10, 11). After being sacrificed under isoflurane anesthesia followed by cervical dislocation, the mice were secured on a plastic platform. The trachea was exposed by a 1-cm incision on the ventral neck skin for insertion of the cannula and sutured in place. Lungs were instilled 4 times with 0.5 ml of sterile 0.9% saline, with the fluid being immediately aspirated. The aspirates recovered from the instillations were pooled per mouse, placed on ice after each aliquot, and subsequently stored at ⫺80°C. Posaconazole concentrations in plasma and BAL fluid were measured by a validated (for human and mouse matrices) ultraperformance liquid chromatography (UPLC) method with fluorescence detection. Details of the analytical assay are described elsewhere (13). Geometric mean concentrations of total posaconazole in plasma were calculated for each time point (n ⫽ 3 mice). Peak concentrations in plasma (Cmax) were directly observed from the data. Pharmacokinetic parameters were derived using noncom-
Antimicrobial Agents and Chemotherapy
Received 14 January 2014 Returned for modification 9 February 2014 Accepted 17 February 2014 Published ahead of print 24 February 2014 Address correspondence to Johan W. Mouton. [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.00053-14
p. 2964 –2967
May 2014 Volume 58 Number 5
Downloaded from http://aac.asm.org/ on April 16, 2015 by guest
Adequate penetration to the infection/colonization site is crucial to attain optimal efficacy of posaconazole against Aspergillus fumigatus diseases. We evaluated posaconazole exposure in pulmonary epithelial lining fluid (ELF) in a murine model of invasive pulmonary aspergillosis. The posaconazole exposure (area under the plasma concentration-time curve from time zero to 24 h postinfusion [AUC0 –24]) in ELF was 20% to 31% of that in plasma for total drug after the third dose, and the relationship between plasma and ELF exposure was linear (r2 ⴝ 0.97, P ⴝ 0.016).
Posaconazole Penetration in ELF Compared to the Blood
partmental analysis with Phoenix, version 6.2 (Pharsight, Inc.). The area under the plasma concentration-time curve from time zero to 24 h postinfusion (AUC0 –24) was determined by use of the log-linear trapezoidal rule. The elimination rate constant was determined by linear regression of the terminal points of the loglinear plasma concentration-time curve. The terminal half-life was defined as ln2 divided by the elimination rate constant. Clearance (CL) was calculated as dose/AUC0 –24. Concentrations of total posaconazole in BAL fluid from three mice per time point were determined as described previously (13). Urea in BAL aspirate and plasma was measured utilizing a modified enzymatic assay (QuantiChrom urea assay kit, DIUR-500; BioAssay Systems) (14, 15). The concentration of posaconazole in ELF was then determined by using the ratio of urea concentration in BAL fluid to that in plasma as described previously (10, 11, 14–19): drug concentrationELF ⫽ drug concentrationBAL ⫻ ureaplasma/ureaBAL. All data analyses were performed using Graph-Pad Prism software, version 5.0, for Windows (GraphPad Software, San Diego, CA). A regression analysis was conducted to determine the linearity between posaconazole concentration in blood and ELF. The goodness of fit was checked by use of the r2 value and visual inspection. Statistical significance was defined as a P value of ⬍0.05 (two-tailed). The Cmax and AUC0 –24 data were log10 transformed to approximate a normal distribution prior to statistical analysis. All animals were alive at the time of sample collection from 0 to 24 h postchallenge, and 192 blood and BAL samples were obtained. The drug concentrations in plasma, including the Cmax of po-
May 2014 Volume 58 Number 5
saconazole, were higher than those in ELF for all dosages (Fig. 1). A significant correlation between mean posaconazole concentrations in plasma and ELF was noted by linear regression analysis (r2 ⫽ 0.61, P ⬍ 0.0001) (Fig. 2a). However, a better correlation was found for AUC0 –24 in plasma and ELF in a linear fashion for the dosages of 4 to 32 mg/kg (r2 ⫽ 0.97, P ⫽ 0.016) (Fig. 2b). The total AUC0 –24 in plasma and ELF is shown in Table 1. The penetration of posaconazole in ELF based on total drug was between 20.21 and 31.39%. At the highest dose (32 mg/kg), the total AUC0 –24/MIC ratio was ⱖ581 in plasma and ⱖ157.56 in ELF for the isolate with an MIC of ⱕ0.5 mg/liter. In the present study, we investigated the intrapulmonary posaconazole penetration in an immunosuppressed murine model of invasive pulmonary aspergillosis receiving oral prophylactic regimens. Our results indicate that posaconazole exposure (AUC0 –24) in ELF was 20% to 31% of that in plasma for total drug, and the relationship between posaconazole and AUC0 –24 in plasma and ELF was significantly linear (r2 ⫽ 0.97). Our findings are comparable to the results of two clinical observations of Conte et al. (20, 21). In the first study, the plasma and intrapulmonary concentrations of posaconazole were evaluated in 25 healthy adult subjects receiving multiple doses (13 or 14) of oral posaconazole suspension 400 mg twice daily with a high-fat meal (20). The AUC0 –12 values in ELF and plasma were 18.3 and 21.9 mg · h/liter, respectively, corresponding to an ELF-to-plasma penetration ratio of 86% of total drug. However, the corresponding exposure in alveolar macrophages (AMs) was appreciably higher (range, 46.2 to 87.7 mg/liter; AUC0 –12 of 715 mg · h/liter). Similarly, in the second study of the above-mentioned group (21),
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FIG 1 Plasma and ELF concentrations of posaconazole following oral administration of 4, 8, 16, and 32 mg/kg to immunosuppressed mice infected with A. fumigatus after the third dose. Each symbol corresponds to the geometric mean plasma levels for three mice.
Seyedmousavi et al.
higher-than-predicted intrapulmonary penetration of posaconazole was observed in 20 adult lung transplant patients receiving posaconazole suspension 400 mg twice daily with a high-fat meal. The AUC0 –12 values in plasma, ELF, and AMs were 10.99, 11.21, and 530.2 mg · h/liter, respectively. Notably, the lipophilic nature and high intracellular (cell-associated) concentrations of posaconazole might contribute to distribution at the infection/colonization site, in this case ELF (22, 23). Campoli et al. demonstrated that lipophilic characteristics of posaconazole increase the penetration of posaconazole into mammalian host cell membranes, which can mediate its efficacy in prophylactic regimens and likely explains the observed discrepancy between posaconazole low serum level and high potency (23). They exposed intracellular cells to posaconazole and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40- to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. On the other hand, Farowski et al. demonstrated that administration of posaconazole oral solution resulted in significantly increased intracellular concentrations in the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) compared to those in plasma (22). They collected 23 blood samples from 14 patients receiving posaconazole for prophylaxis of fungal infections in order to determine the intracellular concentrations of posaconazole in different compartments of the peripheral blood. These samples were separated by double-discontinuous Ficoll-Hypaque density gradient
TABLE 1 Pharmacokinetic parameters of posaconazole in plasma and ELF following 3 days of once daily oral administration of 4, 8, 16, and 32 mg/kg in immunosuppressed mice
Posaconazole dose (mg/kg) 4 8 16 32
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Cmax (mg/liter)
AUC0–24 mg · h/liter
Plasma
ELF
Plasma
ELF
ELF-plasma Cmax ratio (%)
5.51 8.7 10.92 15.04
1.58 2.83 3.62 5.32
72.69 149.80 198.90 290.50
14.69 42.14 62.23 78.78
28.68 32.53 33.15 35.37
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ELF-plasma AUC0–24 ratio (%) 20.21 28.13 31.29 27.12
centrifugation. The intracellular posaconazole concentrations of PBMCs, PMNs, and red blood cells (RBCs) were determined by liquid chromatography-tandem mass spectrometry. The intracellular concentrations of the PBMCs and PMNs were significantly higher than those of surrounding media (P ⬍ 0.001). The ratios between the intracellular and extracellular concentrations (C/E) were 22.5 ⫾ 21.2, 7.66 ⫾ 6.50, and 0.09 ⫾ 0.05 for the PBMCs, PMNs, and RBCs, respectively. The exposure-response relationships of posaconazole have been defined previously in experimental models of Aspergillus infections (4, 6, 24). Similar to the other triazoles, posaconazole displays exposure-dependent pharmacodynamic characteristics, for which a free AUC0 –24/MIC ratio ranging from 1.67 to 1.78 was the value predictive of success associated with half-maximal efficacy. In general, it is accepted that only the unbound fraction of a drug in serum/plasma is pharmacologically active. Considering the high degree of posaconazole protein binding in plasma (98 to 99%) and negligible bounds in ELF, the results of the current study indicated that effective local concentrations might be achieved even at the lowest dose (4 mg/kg) with a free AUC0 –24/ MIC ratio of 1.76 in plasma and 14.69 in ELF, which is probably high enough to prevent infection with A. fumigatus isolates with an MIC of ⱕ0.5. However, for the isolates with resistant phenotypes and higher MIC to posaconazole (ⱖ16 mg/kg), the obtained free AUC0 –24/MIC in plasma was ⱕ0.18 in plasma and ⱕ4.92 in ELF at the highest dose (32 mg/kg), which indicates the possibility of breakthrough IA. We conclude that penetration of posaconazole in ELF is relatively high, which is consistent with lipophilic characteristics and its increased intracellular permeability. The high intrapulmonary penetration ratio of posaconazole indicates that this agent is an optimal therapeutic choice in prevention of IA due to azole-susceptible and/or azole-resistant A. fumigatus. (Parts of these results were presented at the 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 9 to 12 September 2012, and 6th Trends in Medical Mycology, 11 to 14 October 2013, Copenhagen, Denmark.) ACKNOWLEDGMENTS S.S. and W.J.G.M. have no conflicts of interests. R.J.M.B., P.E.V., and J.W.M. have served as consultants to and have received research grants from Gilead Sciences, Merck, Astellas, and Pfizer.
Antimicrobial Agents and Chemotherapy
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FIG 2 Relationship between posaconazole concentrations (a) and AUC0 –24 (b) in plasma and ELF. The line indicates the linear regression.
Posaconazole Penetration in ELF Compared to the Blood
This study was supported, in part, by an unrestricted research grant from Gilead Sciences.
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REFERENCES
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12.
13.
14. 15.
16. 17.
18. 19. 20.
21.
22.
23.
24.
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1. EMA. 2012. European public assessment report (EPAR) for Noxafil, 06/ 09/2012 ed. European Medicines Agency, London, United Kingdom. 2. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens DA, van Burik JA, Wingard JR, Patterson TF. 2008. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin. Infect. Dis. 46:327–360. http://dx.doi.org/10.1086/525258. 3. Herbrecht R, Denning DW, Patterson TF, Bennett JE, Greene RE, Oestmann JW, Kern WV, Marr KA, Ribaud P, Lortholary O, Sylvester R, Rubin RH, Wingard JR, Stark P, Durand C, Caillot D, Thiel E, Chandrasekar PH, Hodges MR, Schlamm HT, Troke PF, de Pauw B. 2002. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N. Engl. J. Med. 347:408 – 415. http://dx.doi.org/10.1056 /NEJMoa020191. 4. Mavridou E, Bruggemann RJ, Melchers WJ, Mouton JW, Verweij PE. 2010. Efficacy of posaconazole against three clinical Aspergillus fumigatus isolates with mutations in the cyp51A gene. Antimicrob. Agents Chemother. 54:860 – 865. http://dx.doi.org/10.1128/AAC.00931-09. 5. Petraitiene R, Petraitis V, Groll AH, Sein T, Piscitelli S, Candelario M, Field-Ridley A, Avila N, Bacher J, Walsh TJ. 2001. Antifungal activity and pharmacokinetics of posaconazole (SCH 56592) in treatment and prevention of experimental invasive pulmonary aspergillosis: correlation with galactomannan antigenemia. Antimicrob. Agents Chemother. 45: 857– 869. http://dx.doi.org/10.1128/AAC.45.3.857-869.2001. 6. Lepak AJ, Marchillo K, Vanhecker J, Andes DR. 2013. Posaconazole pharmacodynamic target determination against wild-type and Cyp51 mutant isolates of Aspergillus fumigatus in an in vivo model of invasive pulmonary aspergillosis. Antimicrob. Agents Chemother. 57:579 –585. http://dx.doi.org/10.1128/AAC.01279-12. 7. Andes D, Marchillo K, Conklin R, Krishna G, Ezzet F, Cacciapuoti A, Loebenberg D. 2004. Pharmacodynamics of a new triazole, posaconazole, in a murine model of disseminated candidiasis. Antimicrob. Agents Chemother. 48:137–142. http://dx.doi.org/10.1128/AAC.48.1.137-142.2004. 8. Snelders E, van der Lee HA, Kuijpers J, Rijs AJ, Varga J, Samson RA, Mellado E, Donders AR, Melchers WJ, Verweij PE. 2008. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med. 5:e219. http://dx.doi.org/10.1371/journal.pmed .0050219. 9. Rodriguez-Tudela JL, Arendrup MC, Arikan S, Barchiesi F, Bille J, Cuenca-Estrella E CEMM, Dannaoui EE, Denning DW, Donnelly JP, Fegeler W, Lass-Flörl C, Moore C, Richardson M, Gaustad P, Schmalreck A, Velegraki AA, Verweij P. 2008. EUCAST definitive document E.DEF 9.1: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds. Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). http://www.eucast.org/fileadmin/src/media/PDFs /4ESCMID_Library/3Publications/EUCAST_Documents/Other _Documents/EUCAST_moulds_DEFINITIVE_document_V_ISO _April_08%20final.pdf. 10. Seyedmousavi S, Brüggemann RJM, Melchers WJG, Mouton JW, Verweij PE. 2013. Posaconazole prophylaxis in experimental azole-resistant
invasive pulmonary aspergillosis, poster M-761. Final Prog. 53rd Intersci. Conf. Antimicrob. Agents Chemother. Seyedmousavi S, Melchers WJG, Verweij PE, Mouton JW. 2012. Intrapulmonary penetration of posaconazole in immunosuppressed mice receiving oral prophylactic regimens, poster M-1741. Final Prog. 52nd Intersci. Conf. Antimicrob. Agents Chemother. Seyedmousavi S, Bruggemann RJM, Melchers WJG, Verweij PE, Mouton JW. 2013. Higher than predicted posaconazole penetration at the infection site in a murine model of invasive pulmonary aspergillosis. Mycoses 56(Suppl S3):151. Verweij-van Wissen CP, Burger DM, Verweij PE, Aarnoutse RE, Bruggemann RJ. 2012. Simultaneous determination of the azoles voriconazole, posaconazole, isavuconazole, itraconazole and its metabolite hydroxyitraconazole in human plasma by reversed phase ultra-performance liquid chromatography with ultraviolet detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 887– 888:79 – 84. http://dx.doi.org/10.1016/j .jchromb.2012.01.015. Kiem S, Schentag JJ. 2008. Interpretation of antibiotic concentration ratios measured in epithelial lining fluid. Antimicrob. Agents Chemother. 52:24 –36. http://dx.doi.org/10.1128/AAC.00133-06. Rodvold KA, George JM, Yoo L. 2011. Penetration of anti-infective agents into pulmonary epithelial lining fluid: focus on antibacterial agents. Clin. Pharmacokinet. 50:637– 664. http://dx.doi.org/10.2165/11594090 -000000000-00000. Summer R, Kotton DN, Sun X, Ma B, Fitzsimmons K, Fine A. 2003. Side population cells and Bcrp1 expression in lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 285:L97–L104. Rennard SI, Basset G, Lecossier D, O’Donnell KM, Pinkston P, Martin PG, Crystal RG. 1986. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J. Appl. Physiol. 60:532–538. Chinard FP. 1992. Quantitative assessment of epithelial lining fluid in the lung. Am. J. Physiol. 263:L617–L618. Von Wichert P, Joseph K, Muller B, Franck WM. 1993. Bronchoalveolar lavage. Quantitation of intraalveolar fluid? Am. Rev. Respir. Dis. 147:148 – 152. http://dx.doi.org/10.1164/ajrccm/147.1.148. Conte JE, Jr, Golden JA, Krishna G, McIver M, Little E, Zurlinden E. 2009. Intrapulmonary pharmacokinetics and pharmacodynamics of posaconazole at steady state in healthy subjects. Antimicrob. Agents Chemother. 53:703–707. http://dx.doi.org/10.1128/AAC.00663-08. Conte JE, Jr, DeVoe C, Little E, Golden JA. 2010. Steady-state intrapulmonary pharmacokinetics and pharmacodynamics of posaconazole in lung transplant recipients. Antimicrob. Agents Chemother. 54:3609 – 3613. http://dx.doi.org/10.1128/AAC.01396-09. Farowski F, Cornely OA, Vehreschild JJ, Hartmann P, Bauer T, Steinbach A, Ruping MJ, Muller C. 2010. Intracellular concentrations of posaconazole in different compartments of peripheral blood. Antimicrob. Agents Chemother. 54:2928 –2931. http://dx.doi.org/10.1128/AAC.01407-09. Campoli P, Al Abdallah Q, Robitaille R, Solis NV, Fielhaber JA, Kristof AS, Laverdiere M, Filler SG, Sheppard DC. 2011. Concentration of antifungal agents within host cell membranes: a new paradigm governing the efficacy of prophylaxis. Antimicrob. Agents Chemother. 55:5732– 5739. http://dx.doi.org/10.1128/AAC.00637-11. Howard SJ, Lestner JM, Sharp A, Gregson L, Goodwin J, Slater J, Majithiya JB, Warn PA, Hope WW. 2011. Pharmacokinetics and pharmacodynamics of posaconazole for invasive pulmonary aspergillosis: clinical implications for antifungal therapy. J. Infect. Dis. 203:1324 –1332. http://dx.doi.org/10.1093/infdis/jir023.
