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Antimicrobial Agents and Chemotherapy, April 2006, p. 1552-1554, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1552-1554.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Efficacy of Aerosolized Nanostructured Itraconazole Formulations for Prevention of Invasive Pulmonary Aspergillosis

Barbara J. Hoeben,1,3 David S. Burgess,1,3* Jason T. McConville,1 Laura K. Najvar,3 Robert L. Talbert,1,3 Jay I. Peters,3 Nathan P. Wiederhold,1,3 Bradi L. Frei,1,3 John R. Graybill,3 Rosie Bocanegra,3 Kirk A. Overhoff,1 Prapasri Sinswat,1 Keith P. Johnston,2 and Robert O. Williams III1

The University of Texas at Austin College of Pharmacy,1 The University of Texas at Austin College of Engineering,, Austin,2 The University of Texas Health Science Center at San Antonio, San Antonio,Texas3

Received 21 October 2005/ Returned for modification 18 November 2005/ Accepted 18 January 2006


   ABSTRACT
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Aerosolized evaporative precipitation into aqueous solution and spray freezing into liquid nanostructured formulations of itraconazole as prophylaxis significantly improved survival relative to commercial itraconazole oral solution and the control in a murine model of invasive pulmonary aspergillosis. Aerosolized administration of nanostructured formulations also achieved high lung tissue concentrations while limiting systemic exposure.


   TEXT
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Aerosolized administration of antifungals is gaining favor for the prevention of invasive pulmonary mycoses (4). Aerosolized administration can achieve high, localized lung tissue concentrations while avoiding systemic toxicities. However, the formulations currently used clinically include intravenous preparations that are not specifically designed for aerosolized administration. Evaporative precipitation into aqueous solution (EPAS) and spray freezing into liquid (SFL) are novel technologies utilized to improve the dissolution and bioavailability of poorly water-soluble drugs (8). Both technologies can produce nanostructured particles (<1 micron in diameter) capable of drug delivery to the alveolar space (5).

We hypothesized that aerosolized administration of EPAS and SFL formulations of itraconazole (ITZ) would be an effective prophylaxis strategy against invasive pulmonary aspergillosis. An established murine model was used to simulate the pathogenesis of invasive pulmonary aspergillosis and assess survival following pulmonary inoculation. We also measured steady-state lung tissue and serum ITZ concentrations.

Five-week-old male outbred ICR mice (Harlan Sprague-Dawley, Indianapolis, IN) were rendered immunosuppressed by cortisone acetate administered subcutaneously at a dose of 100 mg/kg of body weight on days –1, 0, +1, and +6 and were inoculated with Aspergillus flavus ATCC MYA-1004 (ITZ MIC, 0.125 µg/ml per CLSI M38-A microdilution methodology) (14) via an inhalation chamber as previously described (13, 16). Animals were divided into four groups: ITZ oral solution (Sporanox oral liquid [SOL]) administered by oral gavage (30 mg/kg three times a day), aerosolized EPAS (30 mg/kg twice a day), aerosolized SFL (30 mg/kg twice a day), and control (aerosolized sterile distilled water). EPAS and SFL formulations were manufactured using pharmaceutical grade ITZ powder (Hawkins, Inc., Minneapolis, MN) (3, 17, 18, 20, 21). For the EPAS formulation, ITZ and poloxamer 407 were dissolved in dichloromethane and the solution was sprayed into a heated aqueous solution containing 4% polysorbate 80, causing rapid evaporation of the dichloromethane and subsequent precipitation of nanostructured crystalline ITZ. For SFL, an organic feed solution was prepared by dissolving ITZ (0.1% wt/vol), polysorbate 80 (0.75% wt/vol), and poloxamer 407 (0.75% wt/vol) into acetonitrile. The organic feed solution was atomized into liquid nitrogen to produce frozen amorphous particles. Lyophilization of the particles yielded stabilized nanostructured particle aggregates. EPAS, SFL, and control were administered via a 20-min aerosolization with an Aeroneb Pro micropump nebulizer (Aerogen, Inc., Mountain View, CA) attached to an aerosolization chamber (12). There is a patent pending for EPAS and SFL ITZ formulations (N. Beck, D. S. Burgess, P. Garcia, I. B. Gillespie, D. A. Hayes, J. E. Hitt, K. P. Johnston, J. McConville, J. Peters, T. L. Rogers, B. D. Scherzer, R. L. Talbert, C. J. Tucker, R. O. Williams III, and T. J. Young., U.S. patent application 200,508,261).

Survival was determined for different groups of animals on two separate occasions. In the long-term survival study, each ITZ regimen was administered to 10 mice per group beginning 1 day prior to infection and continuing for a total of 12 days (10 days postinoculation). Mice were monitored until day 20 postinoculation. In the acute survival study, each ITZ regimen was administered as previously described to 10 mice per group for a total of 9 days (7 days postinoculation). On day 8 postinoculation, all surviving mice were euthanized. Animals that appeared moribund prior to the end of each arm were euthanized and death was recorded as occurring the next day. Survival was plotted by Kaplan-Meier analysis by using Prism 4 software (GraphPad Software, Inc., San Diego, CA), and differences were analyzed using the log rank test.

ITZ steady-state tissue and serum concentrations were measured in two uninfected mice per time point (0.5, 1, 2, 6, 10, and 24 h) in each formulation group following 8 days of drug administration. Concentrations were separately measured on pooled serum and pooled lung samples using an established high-performance liquid chromatography assay (7). Pharmacokinetic parameters were determined by noncompartmental pharmacokinetic analysis.

Aerosolized EPAS and SFL formulations provided a significant survival benefit compared to that of SOL and the controls (Fig. 1). The SFL formulation had the longest median survival (>20 days), which was significantly greater than the controls (median survival, 5 days; P < 0.005) and SOL (median survival, 4 days; P < 0.001). The EPAS formulation (median survival, 11 days) also demonstrated a survival advantage over SOL (P = 0.04) and a trend toward increased survival compared to that of the controls (P = 0.06). The survival benefits of the EPAS and SFL formulations were reproducible and evident by day 8 postinoculation in both the long-term and acute survival arms (Fig. 1A and B, respectively).


Figure 1
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  		FIG. 1. Survival curves for mice (n = 10 per group per study arm) that received prophylaxis with aerosolized itraconazole prepared by EPAS (•) and SFL ({triangleup}) or orally administered SOL ({square}) formulations of itraconazole and controls ({blacksquare}) and challenged with A. flavus. (A) Long-term survival (day 20). *, SFL P was <0.005 versus controls and 0.001 versus SOL; ¶, EPAS P was 0.06 versus controls and 0.04 versus SOL. (B) Acute survival (day 8). {ddagger}, SFL P was <0.001 versus controls; §, EPAS P was <0.05 versus controls.

 
Serum ITZ concentrations were higher with orally administered SOL (Cmax, 0.99 µg/ml) and remained detectable (>0.05 µg/ml) for 6 h compared to aerosolized EPAS and SFL formulations (Cmax, 0.44 µg/ml), which became undetectable 2 h postadministration. In contrast, peak lung ITZ concentrations for aerosolized EPAS (25.9 µg/g) and SFL (5.3 µg/g) were substantially higher than concentrations achieved with SOL (1.5 µg/g) and remained detectable for greater than 10 h following aerosolized administration of these formulations (Table 1). The tissue concentrations achieved are similar to those previously reported (J. T. McConville, K. A. Overhoff, P. Sinswat, et al., Abstr. 2005 American Association of Pharmaceutical Scientists Annual Meeting, abstr. T3292, 2005).


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  		TABLE 1. Steady-state pulmonary tissue pharmacokinetic parameters of aerosolized EPAS and SFL itraconazole formulations and orally administered SOL

 
Nanostructured formulations of ITZ significantly prolonged survival, achieved higher lung tissue concentrations, and limited systemic exposure compared to orally administered SOL. Prior animal studies have demonstrated the efficacy of aerosolized amphotericin B. Aerosolized administration of amphotericin B deoxycholate and lipid amphotericin formulations resulted in high localized lung tissue concentrations and improvements in survival (2, 6, 19). However, decreased in vitro activity and clinical failures with the use of amphotericin B have been reported for both A. flavus and A. terreus (1, 9, 15, 22).

The prolonged survival and limited systemic exposure with aerosolized delivery of EPAS and SFL ITZ are encouraging. Although oral ITZ prophylaxis in allogeneic hematopoietic stem cell transplant recipients has been shown to reduce the occurrence of invasive aspergillosis and a trend towards reduced fungal-related mortality, this strategy is often limited by gastrointestinal toxicity and drug interactions (10, 11, 23). Further studies are warranted as the clinical application of aerosolized ITZ may help to minimize adverse drug reactions and avoid drug interactions while improving the effectiveness of antifungal prophylaxis.


   ACKNOWLEDGMENTS
 
This work was supported in part by grants from The Dow Chemical Company and the Society of Infectious Diseases Pharmacists.


   FOOTNOTES
 
* Corresponding author. Mailing address: University of Texas Health Science Center at San Antonio, Clinical Pharmacy Programs-MSC 6220, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Phone: (210) 567-8329. Fax: (210) 567-8328. E-mail: burgessd{at}uthscsa.edu. Back


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Antimicrobial Agents and Chemotherapy, April 2006, p. 1552-1554, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1552-1554.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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