Aerosolized nanostructured itraconazole as prophylaxis against invasive pulmonary aspergillosis

Journal of Infection (2007) 55, 68e74

www.elsevierhealth.com/journals/jinf

Aerosolized nanostructured itraconazole as prophylaxis against invasive pulmonary aspergillosis Carlos A. Alvarez a,b, Nathan P. Wiederhold a,b,*, Jason T. McConville a, Jay I. Peters c, Laura K. Najvar d, John R. Graybill d, Jacqueline J. Coalson e, Robert L. Talbert a,b, David S. Burgess a,b, Rosie Bocanegra c, Keith P. Johnston f, Robert O. Williams IIIa a

The University of Texas at Austin College of Pharmacy, 1 University Station, A1900, Austin, TX 78712, USA The University of Texas Health Science Center at San Antonio, Pharmacotherapy Education and Research Center, MSC 6220, 7703 Floyd Curl Drive, San Antonio, TX 78299, USA c The University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Pulmonary Diseases/Critical Care Medicine, 7704 Merton Mintor Blvd. 111E, San Antonio, TX 78229, USA d The University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Infectious Diseases, MSC 7881, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA e The University of Texas Health Science Center at San Antonio, Department of Pathology, MSC 7750, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA f The University of Texas at Austin College of Engineering, Department of Chemical Engineering, 1 University Station, C0400, Austin, TX 78712, USA b

Accepted 29 January 2007 Available online 13 March 2007

KEYWORDS Invasive aspergillosis; Prophylaxis; Itraconazole

Summary Objective: Prophylactic strategies against invasive pulmonary aspergillosis are often limited by drug interactions and toxicities. Targeted airway delivery of antifungals to the lungs may avoid these pitfalls. We evaluated the effectiveness of an aerosolized nanostructured formulation of itraconazole produced by spray freezing into liquid (SFL) as prophylaxis against invasive pulmonary aspergillosis caused by A. fumigatus. Methods: Immunocompromised Balb/C mice received either itraconazole by oral gavage (Sporanox Oral Liquid [SOL] 30 mg/kg TID) or by aerosolization (SFL 30 mg/kg via 20 min aerosolizations, or control, BID). Dosing began 2 days prior to pulmonary inoculation with A. fumigatus and continued for 7 days post-inoculation. Changes in lung histopathology were also assessed. In the survival arm, mice were monitored over a 5 day period following discontinuation of therapy and survival was assessed by KaplaneMeier analysis. Results: SFL survival (35%) was greater compared to control (10%; p Z 0.03) and SOL (0%; p Z 0.02). Histopathology demonstrated severe invasive disease involving vessels and small

* Corresponding author. Present address: UTHSCSA, PERC, MSC 6220, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. Tel.: þ1 210 567 8340; fax: þ1 210 567 8328. E-mail address: [email protected] (N.P. Wiederhold). 0163-4453/$30 ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2007.01.014

Aerosolized itraconazole against Aspergillus fumigatus

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airways in control and SOL animals. SFL animals demonstrated colonization with some invasion predominately of large airways. Conclusions: Prophylactic aerosolization of nanostructured SFL significantly improved survival and limited invasive disease of small airways due to A. fumigatus. ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction Invasive aspergillosis remains a leading cause of infectious disease related deaths in patients with hematologic malignancies and those undergoing hematopoietic stem cell transplantation despite advancements in our ability to diagnose and treat infections caused by Aspergillus species.1,2 In a prospective, randomized trial of voriconazole versus amphotericin B deoxycholate as first line therapy for invasive aspergillosis (IA), only 32% of allogeneic stem cell transplant patients had a complete or partial response after 12 weeks of voriconazole therapy.3 Furthermore, therapy is often limited by side effects, such as nephrotoxicity associated with amphotericin B formulations, as well as drug interactions and hepatotoxicity associated with the azoles.4e6 Prophylactic administration of itraconazole (ITZ) has been shown to reduce the occurrence of IA in prospective trials.7,8 However, the usefulness of orally administered ITZ is hampered by low and erratic bioavailability of the oral capsules and side effects associated with the orally administered solution. Gastrointestinal side effects have been reported in up to 25% of recipients of the oral solution in randomized trials and are a major reason for high patient attrition rates.7,8 Recently, attention has been focused on the pulmonary delivery of antifungal agents for the prevention of invasive aspergillosis.9 Targeted delivery to the lungs can achieve high, localized concentrations of antifungals while avoiding systemic toxicities. Spray-freezing into liquid (SFL) is a technology utilized to improve the dissolution and bioavailability of poorly water-soluble drugs, and is capable of producing nanostructured particles for delivery of drugs to the alveolar space.10,11 Our group has recently reported that aerosolized administration of a nanostructured SFL formulation of ITZ is effective in preventing invasive pulmonary aspergillosis (IPA) due to A. flavus.12 The objective of this study was to assess the ability of an aerosolized SFL formulation of ITZ in preventing IPA due to A. fumigatus. An established murine model using pulmonary inoculation was used to simulate the pathogenesis of IPA. The primary endpoint was survival of animals administered SFL ITZ by aerosolization compared to controls and mice administered ITZ by oral gavage. Secondary endpoints included reductions in pulmonary fungal burden and the degree of invasive disease as assessed by histopathology.

Materials and methods Test isolate Conidia were harvested from Aspergillus fumigatus clinical isolate 293 (AF 293) cultures grown on potato dextrose

agar (Hardy Diagnostics, Santa Maria, CA) by washing and scraping agar surfaces with 0.1% Tween 80 in sterile physiological saline and filtering through sterile glass wool. Conidia were resuspended to achieve a final inoculum of w1  109 conidia/mL, as confirmed by hemocytometer counts and serial plating. The minimum inhibitory concentration of ITZ was 0.25 mg/mL as measured according to CLSI M38-A microdilution methodology.13

Mice Female BALB/c mice (National Cancer Institute, Bethesda, MD), weighing between 18e22 g, were used for all experiments. Animals were housed five per cage and had access to sterile food and water ad libitum. Mice were rendered immunosuppressed by intraperitoneal cyclophosphamide (250 mg/kg) and subcutaneous cortisone acetate (250 mg/kg) two days prior to inoculation. Both cyclophosphamide (200 mg/kg intraperitoneally) and cortisone acetate (250 mg/kg subcutaneously) were readministered on day þ3 following inoculation. 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.14

Itraconazole formulations Two formulations of ITZ were used; commercially available ITZ oral solution (Sporanox Oral Liquid [SOL], Janssen Pharmaceuticals Inc., Titusville, NJ); and amorphous nanostructured SFL ITZ. Amorphous nanostructured ITZ was produced using ITZ powder (Hawkins Chemicals, Minneapolis, MN) and the spray freezing into liquid (SFL) particle engineering process.15 Briefly, an organic feed solution was prepared by dissolving itraconazole (0.1% w/v), polysorbate 80 (0.75% w/v) and poloxamer 407 (0.75% w/v) into acetonitrile, and atomized through a 63 mm poly-ether-ether-ketone nozzle (Upchurch Scientific, Oak Harbor, WA) at 20 mL/min below the surface of liquid nitrogen to produce frozen amorphous particles. Lyophilization yielded the stabilized nanostructured particle aggregates of SFL ITZ. Animals were divided into three groups: SOL administered by oral gavage (30 mg/kg TID; N Z 10 mice per survival and fungal burden arms), aerosolized SFL (30 mg/kg BID; N Z 20 mice per arm), and control (aerosolized sterile distilled water; N Z 20 mice per arm). These doses were chosen based on previous pharmacokinetic studies.16,17 Due to the poor survival rate in mice administered SOL by oral gavage in this study and a prior study only 10 animals were administered this regimen in each the survival burden and fungal burden arms.12 Pulmonary delivery of 30 mg/kg BID of SFL ITZ or sterile distilled water was achieved via

70 a twenty-minute aerosolization with an Aeroneb Pro micropump nebulizer (Aerogen Inc., Moutain View, CA) attached to a modified aerosolization chamber as previously described.18 Airflow within the system was maintained at a rate of 0.1 L/min.

Infection model and treatment To simulate pulmonary pathogenesis, mice were placed inside an acrylic chamber, and A. fumigatus conidia were introduced by aerosolizing the conidial suspension with a small particle nebulizer (Hudson Micro Mist, Hudson RCI, Temecula, CA) driven by compressed air.19 A standard exposure time of 1 h was used to allow for complete aerosolization of the condidial suspension. Starting inocula were assessed by CFU enumeration from mice one hour post-inoculation. In the survival arm, each regimen (SOL, SFL, or control) was administered beginning two days prior to inoculation and continuing for a total of 10 days (7 days post-inoculation). Mice were monitored for an additional 5 days following discontinuation of ITZ. Animals that appeared moribund prior to the end of the study were euthanized and death was recorded as occurring the next day. In the pulmonary fungal burden each ITZ regimen was administered as previously described for a total of 10 days. Mice were euthanized on day 8 post-inoculation, lung tissue harvested, and the total lung weights recorded. The survival and pulmonary fungal burden studies were each conducted on two separate occasions.

Pulmonary fungal burden Lungs were homogenized in sterile saline (total volume 2 mL) supplemented with gentamicin and chloramphenicol using a tissue homogenizer (Polytron Dispensing and Mixing Technology PT 2100, Kinematica, Cincinnati, OH). Serial dilutions were prepared in sterile saline and plated in duplicate onto potato dextrose agar. Following 24 h of incubation at 37  C, colonies were enumerated and colonyforming units (CFU) per gram of lung tissue for each animal were calculated. Pulmonary fungal burden was also quantified by real-time quantitative PCR using previously described methods.20,21 Briefly, DNA was extracted from 90 mL of lung homogenate with the use of a commercially available kit (DNeasy Tissue Kit, Qiagen, Valencia, CA) according to the manufacturer’s instructions. DNA samples were analyzed in duplicate with the use of the ABI PRISM 7300 sequence-detection system (Applied Biosystems, Foster City, CA) with primers and dual-labeled fluorescent hybridization probes specific for the A. fumigatus FKS gene (GenBank accession number U79728).22 The threshold cycle (Ct) of each sample was interpolated from a six-point standard curve generated by spiking naı¨ve mouse lungs with known amounts of conidia (102 to 107). An internal standard was amplified in separate reactions to correct for differences in DNA recovery.

Histopathology Histopathological changes in lung tissue of mice that received aerosolized SFL were compared to those SOL

C.A. Alvarez et al. or aerosolized control following 10 days of ITZ administration (N Z 5 per regimen). On day 7 post-inoculation animals were euthanized using halothane and 10% volume/ volume formaldehyde was instilled into the lungs via the trachea. Lungs were then harvested and placed into 10% volume/volume formaldehyde followed by processing and embedding into paraffin wax. Coronal sections of the entire lung were stained with hematoxylin and eosin and viewed by light microscopy. Two blinded investigators, including a pulmonary histopathologist, independently viewed each lung section. The extent of lung damage caused by invasive hyphae was recorded including the number of necrotic foci, and number and size of vascular lesions.

Statistics Survival was plotted by KaplaneMeier analysis, and differences in median survival and percent survival between prophylaxis groups were analyzed by the log-rank test, and chi-square test, respectively. Differences in fungal burden endpoints (CFU/g and CE) were assessed for significance by analysis of variance with Tukey’s post-test for multiple comparisons. A p-value of 0.05 was considered statistically significant for all comparisons.

Results Survival Aerosolized SFL provided a survival benefit as preventive therapy against IPA. As shown in Fig. 1, thirty-five percent of the animals that received aerosolized SFL survived to the pre-determined study endpoint (day þ12 post-inoculation). This survival rate was greater than those animals that received control (10%; p Z 0.03) or orally administered SOL (0%; p Z 0.02). Median survival times were also longer in mice that received aerosolized SFL (7.5 days) compared to those that randomized to control (6.5 days; p Z 0.01) or SOL (5 days; p < 0.001). These data are consistent with the results of a previous study conducted by our group that demonstrated a survival benefit with aerosolized SFL ITZ as preventative therapy against A. flavus. As with the current study, no survival benefit was observed for the orally administered SOL.12

Pulmonary fungal burden Despite improvements in survival no reductions in pulmonary fungal burden were observed for any of the ITZ regimens compared to controls (Fig. 2). Animals randomized to received aerosolized saline had higher lung CFU counts (mean log CFU/gm  SEM; 3.89  0.58) compared to mice that received SOL (3.49  0.07) or aerosolized SFL (3.76  0.51). However, these differences were not significant. The starting inocula assessed one hour post-inoculation were consistent between the different study periods (4.59  0.12). Similarly, when assayed by qPCR lung tissue burden did not differ among controls (log CE mean  SEM; 6.24  0.30), orally administered SOL (4.95  0.45), or aerosolized SFL (5.87  0.34).

Aerosolized itraconazole against Aspergillus fumigatus

71 disruption and necrosis observed in the SFL group was mainly superficial and isolated more proximally at branch points of the airways (Fig. 3C) with less vascular congestion and edema compared to the control and SOL groups. Airway disease was also markedly reduced and vascular invasion was observed in only one animal administered aerosolized SFL (Fig. 3D). These histopathology results are consistent with the survival data demonstrating improved survival in the aerosolized SFL group due to reductions in invasive disease and angioinvasion.

Discussion

Figure 1 Survival of A. fumigatus-infected mice that received itraconazole prophylaxis or control. Mice received preventative treatment with aerosolized sterile distilled water (control e solid black line), orally administered Sporonox Oral Liquid (SOL, dashed black line), or aerosolized itraconazole prepared by spray-freeze into liquid (SFL, dashed gray line), were challenged with A. fumigatus, and were followed until day þ12 post-inoculation. Median survival times were significantly longer for SFL compared to control (P < 0.01) and SOL (p < 0.001). No difference in survival was observed between control and SOL groups (p > 0.05).

Histopathology Marked differences in lung histopathology were noted among the different prophylaxis groups. Lungs from animals that received aerosolized control demonstrated the most severe invasive disease of the small airways (Fig. 3A), including epithelial disruption, congestion, necrosis, and angioinvasion. In addition, animals in the control group had the highest number of necrotic foci in the lower airways as well as the largest number of vascular lesions (Fig. 4). Severe invasive disease was also observed in mice administered oral SOL (Fig. 3B). However, the number of necrotic foci and vascular lesions was somewhat reduced in this group compared to controls. In contrast, the epithelial

Preventative strategies against fungal infections are commonly used by many transplant centers in high-risk immunocompromised patients (i.e., hematopoietic stem cell transplant recipients, solid organ transplant recipients, and patients with hematologic malignancies) secondary to the high morbidity and mortality rates associated with invasive aspergillosis. These strategies often include the systemic administration of antifungal agents as prophylaxis. Unfortunately, this practice predisposes patients to the adverse effects of these agents and potential deleterious drug interactions. Aerosolization of antifungal agents may help to avoid or reduce systemic toxicities and drug interactions by limiting exposure to the lungs. Additionally, this strategy may help prevent invasive mycoses that primarily affect the upper and lower respiratory tract due to the high concentrations that can be achieved at the initial sites of infection.23 Aerosolization of amphotericin B has recently gained favor in transplant centers, and studies have demonstrated this strategy to be relatively safe.24,25 One critique of this approach is that the intravenous formulations currently used are not specifically designed for aerosolized administration. Recently, a dry-powder amphotericin B formulation specifically designed for pulmonary delivery was reported to be effective in preventing invasive aspergillosis due to A. fumigatus in a persistently neutropenic rabbit model (A.R. Kugler, T.D. Sweeney, M.A. Eldon, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. LB2-31, 2005). These results agree with previous animal studies demonstrating improvements in survival with aerosolized

Figure 2 Pulmonary fungal burden for mice that received itraconazole prophylaxis or control. Mice received preventative treatment with aerosolized sterile distilled water (control), orally administered Sporonox Oral Liquid (SOL), or aerosolized itraconazole prepared by spray-freeze into liquid (SFL), and challenged with A. fumigatus. A) Fungal burden as measured by colony-forming units (CFU) per gram of lung tissue. B) Fungal burden as measured by real-time quantitative polymerase chain reaction (qPCR) and reported as conidial equivalents (CE). The line for each group represents the mean pulmonary tissue burden.

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Figure 3 Histopathology of lungs. Lung histopathology sections were stained with hematoxylin and eosin from mice administered A) control (aerosolized sterile distilled water, 20 magnification), B) Spornonox Oral Liquid by oral gavage (20 magnification), C) aerosolized itraconazole prepared by spray-freeze into liquid (superficial disease at branch point of airway, 10 magnification), D) aerosolized itraconazole prepared by spray-freeze into liquid (20 magnification).

administration of amphotericin B deoxycholate as well as lipid amphotericin B formulations.26e28 However, concerns exist regarding the activity and efficacy of amphotericin B against non-fumigatus Aspergillus species, specifically A. flavus and A. terreus.29e32

Previous studies by our group demonstrated that aerosolized nanostructured formulations of ITZ are effective as prophylaxis and significantly improved survival compared to orally administered SOL and control in a murine model of IPA due to A. flavus.12 In addition, aerosolized SFL achieves

Figure 4 Box plots of the number necrotic foci and vascular lesions on histopathology. Lesions (A. necrotic foci, and B. vascular lesions) observed in lungs of mice that received preventative treatment with aerosolized sterile distilled water (control), orally administered Sporanox Oral Liquid (SOL), or itraconazole prepared by spray-freeze into liquid (SFL), and challenged with A. fumigatus. Boxes represent the 25th and 75th percentiles, and horizontal lines within the boxes represent the median values.

Aerosolized itraconazole against Aspergillus fumigatus high ITZ concentrations localized within the lung tissue.16,17 The results of the current study demonstrate that aerosolized SFL ITZ is also effective as prophylaxis in improving survival in a murine model of IPA due to A. fumigatus. In addition, aerosolized SFL ITZ also markedly limited disease progression and prevented angioinvasion in the lungs compared to oral SOL and control as determined by histopathology. Interestingly, no reduction in fungal burden was observed with either orally administered SOL or aerosolized SFL ITZ. This lack of a reduction in Aspergillus fungal burden following the administration of ITZ was noted in previous work from our group (B.J. Hoeben, D.S. Burgess, L.K. Najvar, R.L. Talbert, J.T. McConville, J.I. Peters, R. Bocanegra, J.R. Graybill, B.L. Jones, N.P. Wiederhold, and R.O. Williams III, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-1005, 2005), and in other studies that evaluated triazoles for invasive aspergillosis.33,34 The survival and histopathology results are encouraging since both drug interactions and adverse effects may limit the effectiveness of orally administered ITZ. Two prospective studies of ITZ prophylaxis in allogeneic HSCT recipients have reported a trend in decreased fungalrelated mortality primarily due to reductions in the proven and probable IA in patients administered oral ITZ compared to patients randomized to receive fluconazole.7,8 However, in each study the effectiveness of prophylaxis was limited by gastrointestinal side effects that occurred in approximately 24% of patients randomized to ITZ and led to high attrition rates. One concern of azole prophylaxis is the potential antagonism of subsequent polyene activity. Several animal studies have demonstrated antagnonism of amphotericin B activity with prior exposure to an azole.35,36 However, the implications of such an interaction remain unknown as antagonism has not been clearly demonstrated clinically.37 The limitations of this study must be considered prior to extrapolation of the results to the clinical setting. No survival benefit was observed with oral administration of SOL despite previous data from our group demonstrated elevated serum concentrations at the dose used in the current study.12,17 One potential explanation is a synergistic toxic interaction between ITZ and cyclophosphamide.38 However, our previous data has demonstrated a similarly poor survival rate in mice administered oral SOL in the absence of cyclophosphamide.12 While not addressed in the current study, other researchers have reported poor survival rates among mice administered the cyclodextrin vehicle in the absence of ITZ.39 In addition, the survival benefit of aerosolized SFL ITZ in this study was modest. However, only a single dose and dosing interval were evaluated. It is unknown if aerosolized SFL ITZ would be more effective with a higher dose, or if further improvements in survival and absence of disease could be achieved with different dosing strategies. In conclusion, aerosolized administration of SFL ITZ was effective as prophylaxis in improving survival in this murine model of IPA due to A. fumigatus. The survival benefit can be explained by the ability of aerosolized SFL ITZ to limit disease progression and angioinvasion, both of which were markedly reduced in comparison to mice that received control or SOL by oral gavage.

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Acknowledgements Financial support provided in part by the San Antonio Area Foundation (to N.P.W.) and The Dow Chemical Company (to R.O.W.).

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