Aerosolized Nanostructured Itraconazole

Recent attention has been focused on new technologies capable of improving the bioavailability of poorly water-soluble drugs and enhancing aerosolized delivery to the lungs. Evaporative precipitation of aqueous solution (EPAS) and spray-freeze into liquid (SFL) are two such technologies utilized to improve the dissolution and bioavailability of poorly water-soluble agents, and can produce nanostructured particles capable of drug delivery to the alveolar space (80,81). The utility of nanostructured itraconazole particles produced by these engineering processes has recently been evaluated in a series of in vitro and in vivo studies.

Manufacture of Nanostructured Itraconazole Particles

Improving the solubility of poorly water soluble drugs is an increasingly important aspect for increased efficacy of treatment regimens. Approximately 40% of investigational active pharmaceutical ingredients (APIs) are estimated to have poor aqueous solubility. The Biopharmaceutics Classification System indicates that class II APIs display poor water solubility with high mucosal permeability (an example of this class of compound is itraconazole). For APIs in this class, solubility and subsequent dissolution rate are rate limiting steps for absorption. Particle size reduction through milling to form micronized particles of API with a high surface area have been shown to improve dissolution and bioavailability (82).

Spray freezing into liquid: Spray freezing into liquid (SFL) is a technique used to formulate APIs to have surface areas from 12 to 83 m2/g, compared to approximately 4 or 5m2/g for unprocessed APIs. Figure 3 indicates the difference in bulk density of an unprocessed itraconazole formulation compared to that of an SFL processed formulation of the same composition. Following processing using the SFL technique there is a marked reduction in bulk density (Fig. 3), which is directly proportional to in increase in surface area, compared to the low bulk density of the raw unprocessed formulation ingredients (Fig. 3).

As there is such an increase in surface area, there is a marked improvement in dissolution rates. During this particle engineering technique (as shown in Fig. 4), an API and stabilizing and/or dissolution enhancing

1

-

(A)

(B)

Figure 3 Powder formulations composed of: 0.75 g Itraconazole, 0.75 g Poloxamer 407, and 0.25 g Polysorbate 80. (A) Processed using the SFL technique; (B) raw unprocessed powder formulation components.

Figure 3 Powder formulations composed of: 0.75 g Itraconazole, 0.75 g Poloxamer 407, and 0.25 g Polysorbate 80. (A) Processed using the SFL technique; (B) raw unprocessed powder formulation components.

component (pharmaceutical excipients selected from a variety of surfactants and polymers) are dissolved in an organic solvent (e.g. acetonitrile). The organic solution is atomized directly into a cryogenic liquid (e.g. liquid nitrogen (83), or CO2 (84)), through an insulated nozzle (a 63-126 ^m inner diameter polyether ether ketone (PEEK) nozzle). During this process the atomized organic solution is instantly frozen (as frozen amorphous microparticles) (83,85-89), and a molecular dispersion is achieved during this rapid freezing, thus preventing phase separation.

The frozen molecularly dispersed microparticles are then placed into a lyophilizer to remove the frozen organic solvent (by sublimation). After the lyophilization process is complete, the resulting dried powder is composed of discrete microparticles where the API is molecularly dispersed with a polymer in a porous matrix (90) (Fig. 5).

Previous studies have indicated that rapid dissolution, of SFL formulations, is due to the amorphous morphology (molecular dispersion), high surface area, and enhanced wetting (related to the high surface area and inclusion of surfactants and/or hydrophilic stabilizing polymers) (91-94).

Evaporative precipitation into aqueous solution: Evaporative precipitation into aqueous solution (EPAS) is another technique whereby an API can be altered to have an increased surface area, and like the SFL technique results in an improvement dissolution rate (90). During this

Organic solution spray plume

Organic solution spray plume

PEEK Tubing

Cryogen

(e.g. Liquid nitrogen)

Lyophilization

PEEK Tubing

Cryogen

(e.g. Liquid nitrogen)

Lyophilization

(F) Powder product

Figure 4 A typical setup for the spray-freezing into liquid technique. (A) Solvent reservoir containing drug with stabilizing excipient(s) co-dissolved in an organic solvent; (B) pump; (C) insulating tubing consisting of PEEK; (D) cryogen (e.g. liquid nitrogen); (E) lyophiliza-tion process to obtain (F) the final dry powder product.

particle engineering technique (as shown in Fig. 6), an API and stabilizing dissolution enhancing component (pharmaceutical excipients selected from a variety of surfactants and polymers) are dissolved in a heated organic solvent (e.g. dichloromethane). The organic solution is atomized directly into a heated aqueous solution bath, which may contain a surfactant, through a crimped tube nozzle (90,95,96). At this stage the volatile solvent rapidly evaporates and the API rapidly precipitates. Depending on the selection of stabilizing excipients, the particle size as determined by crystal growth is inhibited. During this manufacture a colloidal dispersion of crystalline drug is produced. This could be used directly for nebulization or lyophilized (after flash-freezing in a cryogen) to obtain a dry powder product.

Nebulization of Colloidal Itraconazole

Inhalation and deposition of submicron particles (particles below 1 ^m in size) in the lung is a challenge. Submicron particles have a high charge to mass ratio and as a result may easily agglomerate (97) to form large particles that have the potential to impinging in the upper airways of the lung. Conversely, submicron particles that do not agglomerate will essentially posses a very small inertia during inhalation and so will not impinge on the lung wall, they will then be subsequently exhaled. This problem has been

Figure 5 Scanning tunneling electron microscopy micrographs of the SFL (A and B) processed drug (example shown is the poorly water-soluble drug danazol) and the EPAS (C and D) processed danazol. Source: From Ref. 90.

overcome by embedding the submicron particles within carrier materials that are suitably sized to deposit in the deep airways of the lung (98). Another approach is to disperse the submicron material within an aqueous medium and nebulize (95). This nebulization approach effectively removes a processing step, as the lung deposition is controlled by the functionality of the nebulizer (Fig. 7).

In the example of aerosolized itraconazole nanoparticles prepared by either EPAS or SFL technologies (95), comparable emitted doses and respirable fractions were observed when using a micropump nebulizer (Table 1).

Animal Dosing with Nebulized Colloidal Dispersions of Itraconazole

Due to the fact that itraconazole colloidal dispersions could be nebulized within a recognized droplet size range that was consistent with peripheral

Heated solvent reservoir

HPLC pump

Organic solution spray plume

Crimped nozzle

Aqueous solution bath (~80°C)

(E) Flash freezing in cryogen

Powder product

Figure 6 A typical setup for the EPAS technique. (A) Solvent reservoir containing drug with stabilizing excipient(s) co-dissolved in an organic solvent (which may be heated); (B) pump; (C) crimped narrow bore nozzle manufactured from HPLC tubing; (D) aqueous solution bath (this may contain further stabilizing surfactants to prevent aggregation and crystal growth); (E) lyophilization process to obtain (F) the final dry powder product.

lung deposition (99), dosing of a mammalian model was considered possible (100). Previously a small rodent whole body exposure unit had been evaluated for use with an Aeroneb Pro® micro-pump nebulizer (Nektar Therapeutics, formerly Aerogen Inc., Mountain View, CA), using the model drug caffeine (101). This dosing apparatus had been designed to allow up to 14 mice to be housed simultaneously. The apparatus was re-evaluated using a colloidal dispersion of itraconazole which had been prepared using the SFL technique (95). The study indicated that the simultaneous dosing of 10 mice resulted in only a 13% relative standard deviation in itraconazole mouse lung concentration.

Lung Pharmacokinetics of Nebulized Colloidal

Dispersions of Itraconazole

Dosing of the nebulized colloidal dispersions of itraconazole was performed in three groups of mice (14per group) for a 20min nebulization period in the specially designed dosing chamber (indicated above) (95). Interestingly the data revealed that irrespective of the processing technique used to prepare itraconazole (EPAS or SFL, which result in either crystalline or amorphous

1-4 Mrtl droplets containing nanosized (A) itraconazole particles

1-4 Mrtl droplets containing nanosized (A) itraconazole particles

(aqueous droplets 1-4 (jm)

Figure 7 The scheme of nebulization of a colloidal dispersion of a poorly water-soluble drug: (A) A colloidal dispersion of drug is contained within a nebulizer reservoir (e.g., an Aeroneb® Pro micropump nebulizer); (B) the drug is nebulized; and (C) a nebulized spray plume is produced with aqueous droplets between 1 and 4 mm (suitably sized for deep lung deposition); (D) nanoparticles of drug are dispersed within nebulized aqueous droplets.

(aqueous droplets 1-4 (jm)

Figure 7 The scheme of nebulization of a colloidal dispersion of a poorly water-soluble drug: (A) A colloidal dispersion of drug is contained within a nebulizer reservoir (e.g., an Aeroneb® Pro micropump nebulizer); (B) the drug is nebulized; and (C) a nebulized spray plume is produced with aqueous droplets between 1 and 4 mm (suitably sized for deep lung deposition); (D) nanoparticles of drug are dispersed within nebulized aqueous droplets.

material respectively) (96), approximately the same level of lung deposition was shown to occur, when the emitted dose from the nebulizer was approximately equivalent. Pharmacokinetic analysis of the EPAS and SFL formulations indicated that the retention time of the drug was similar too, with similar elimination rates. The retention time of the itraconazole in the lung was of key importance in these studies, as it was an indication of how long the drug would remain above the minimum lethal concentration (MLC) for preventing the germination of Aspergillus spp. Data reported by Johnson et al. indicated that the 90 percentile MLC (MLC90) for A. flavus (the concentration at which 90% of A. flavus is terminated), was 0.5 mg/mL of blood serum (102). From this information it was inferred that the MLC90 for lung tissue would be 0.5 mg/g of wet lung tissue weight. The pharmacokinetic profiles obtained for the lung retention time for the

Table 1 Cascade Impactor Data

Formulation

TED (^g/min)

FPF* (%)

MMAD (mm)

GSD

EPAS-ITZ

1743

76

2.70

1.9

SFL-ITZ

1134

85

2.82

1.7

Note: The data was obtained for the nebulization of colloidal dispersions of itraconazole prepared from powders manufactured using either the EPAS or SFL techniques. Abbreviations: FPF, fine particle fraction; GSD, geometric standard deviation; MMAD, mass median aerodynamic diameter; TED, total emitted dose. Source: From Ref. 95.

Note: The data was obtained for the nebulization of colloidal dispersions of itraconazole prepared from powders manufactured using either the EPAS or SFL techniques. Abbreviations: FPF, fine particle fraction; GSD, geometric standard deviation; MMAD, mass median aerodynamic diameter; TED, total emitted dose. Source: From Ref. 95.

nebulized EPAS and SFL colloidal dispersion of itraconazole indicated that the drug remained MLC90 for at least 24 h following a single dose administration (Fig. 8).

In order to obtain lung levels of itraconazole greater than the MLC90 of A. flavus in the lung tissue using conventional dosing regimens (i.e. by oral administration of the drug), it would mean that high and sustained doses of itraconazole would be required. This high and sustained itraconazole dosing regimen needed may be considered higher than that indicated for a typical human dosing regimen (103). The strain and degree of a given fungal infection, treatment may vary. Typically values of 0.5 mg/mL blood serum are considered to be therapeutically important (104). Human doses of orally administered itraconazole vary from 200 to 800 mg itraconazole b.i.d. or t.i.d. (104-110).

Steady State Dosing of Nebulized Itraconazole

In a follow up study for dosing itraconazole to the lung, a repeat single dose study was conducted using the itraconazole prepared using SFL technique, and the steady state dosing kinetics was determined (111). Using the pharmacokinetic data from the single dose study, it was predicted that the steady state lung concentration for nebulized itraconazole (prepared using the SFL technique) would be obtained within four half lives when administered at 12 h intervals (b.i.d.). With this information Vaughn et al. designed a study to dose twelve mice b.i.d. using the nebulized SFL itraconazole, and 12 mice b.i.d. using a conventional orally administered Sporanox® formulation. The study was run for 12 days, and 3 mice were euthanized from each group on days 3, 8, and 12. From the 3 mice euthanized

Time (hours)

Figure 8 Lung deposition in mice for nebulized ITZ formulations (prepared either using the EPAS or SFL techniques) by single dose administration (an equivalent dose exposure of 30mg/kg by aerosolization over a 20-min period). Source: From Ref. 95.

Time (hours)

Figure 8 Lung deposition in mice for nebulized ITZ formulations (prepared either using the EPAS or SFL techniques) by single dose administration (an equivalent dose exposure of 30mg/kg by aerosolization over a 20-min period). Source: From Ref. 95.

at each of the time intervals, the lung and serum concentrations could be determined for both pulmonary and orally administered itraconazole. The data showed conclusively that the amount of itraconazole present in the lung at the dose trough level following b.i.d. administration was much greater for the pulmonary administered SFL formulation. Furthermore, the levels obtained were shown to be consistently above the MLC90 for A.flavus. For the orally administered drug this was shown not to be the case. Additionally, information was obtained on the histopathology of the lung. This data indicated that there was no apparent tissue damage as a result of the inhaled SFL itraconazole formulation (112). Further, a qualitative assessment of alveolar macrophage uptake of itraconazole, performed using a bronchoal-veolar lavage (BAL) method, in conjunction with mass spectroscopy, was described (112). The BAL study indicated the presence of itraconazole in isolated alveolar macrophages. Interestingly the studies were able confirm that previously reported incidences of significant side-effects (113,114) were apparent, due to the occurrence of 2 dose related mortalities in the orally administered Sporanox group. The studies concluded that pulmonary dosing with itraconazole could an effective method for delivery of antifungal therapy for the treatment and prophylaxis of invasive fungal infections. Using the nebulized itraconazole formulations high and sustained lung tissue concentrations were achieved via inhalation of an amorphous nanoparticulate ITZ-pulmonary composition while maintaining serum levels which are within the MLC range measured for of A. flavus (102). The lung:serum ratio was significantly greater in pulmonary dosed ITZ and could prove to enhance treatment while reducing side effects, by decreasing the systemic Cmax and maintaining serum levels above the MLC range demonstrated for itracona-zole. By enhancing the local delivery and reducing the potential for side effects of ITZ delivery, this delivery method can greatly improve morbidity and mortality for patients who are at risk of or infected with life-threatening fungal infections.

Nanostructured Itraconazole for the Prevention of Invasive

Pulmonary Aspergillosis

A collaborative study between the University of Texas at Austin and the University of Texas Health Science Center in San Antonio was undertaken using a prophylaxis strategy for the pulmonary administration of itraconazole. Animal models of invasive pulmonary aspergillosis demonstrated that the strategy is effective as prophylaxis compared to the commercially available oral solution (Fig. 9) (115,116). In mice immuno-compromised with high dose cortisone acetate and challenged with A. flavus, both aerosolized EPAS and SFL formulations of itraconazole significantly prolonged and increased survival (up to 60% survival at the end of the study) compared to placebo and mice administered a commercially available

Figure 9 Survival curves for mice that received prophylaxis with aerosolized itraconazole prepared by EPAS (•) and SFL (A), or orally administered Sporanox® Oral Liquid (□) and control group (■). Source: From Ref. 115.

solution of itraconazole by oral gavage (both with a 0% survival level at the termination of the study) (115).

Similar effects of aerosolized SFL itraconazole on survival were observed in a subsequent study in mice immunosuppressed with cortisone acetate and cyclosphosphamide and challenged with the more prevalent and virulent species A.fumigatus (116). In addition, aerosolized SFL itraconazole markedly reduced lung damage as observed by histopathology. In contrast to the extensive damage observed in the lower airways of mice administered placebo or oral itraconazole solution, the epithelial disruption and necrosis observed in the SFL itraconazole group was mainly superficial and isolated more proximally at branch points of the airways with less vascular congestion. Despite these promising data in vitro and in vivo, the clinical utility of nanostructured itraconazole is unknown as these formulations have not been tested in healthy volunteers or patients.

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