Micronization of fluticasone successfully reduced the particle size of the bulk material from a mean value (D50
) of 1.6 to 0.2 μm. The solid form of the micronized material was examined by PXRD and TGA/SDTA, and demonstrated no discernable change in the crystal form post the micronization process. (Fig. ). Content uniformity, potency, and homogeneity of the formulations were tested to ensure quality of test material for all studies. In general, nanosuspension performs very well in all the tests. Both nano and regular materials were dosed as suspension in vivo. Control samples (milled vehicle) were very clean with no glass shards observed. The dissolution rate was increased by reducing the particle size and was calculated by the Noye–Whitney equation. Solubility impact was calculated by the Oswald–Freundlich equation (Log(Cs/C∞) = 2αV/(2.303RTρν
) to further characterize the nanosuspension [24
]. For fluticasone, a slight increase of solubility was observed (from 0.7 μM to 1.1 μM) when particle size was reduced to 0.2 μm. Despite the increase of solubility, greater than 99.9% fluticasone still exists as solid crystalline in nanosuspension. Thus, any particles formed from the supernatant during nebulization were ignored. Only aerosol particle from nanoparticle aggregation were considered.
Fluticasone PXRD patterns. Top is the post milling and bottom is the pre-milling API
The first in vivo experiment was designed to compare different dosing techniques and impact of the nanosuspension on lung deposition and systemic exposure. The target was focused on higher and dose depended lung disposition (to enhance local efficacy) with lower systemic exposure (reduce systemic side effect). For this experiment, settings recommended by the manufacturer were used to test both PARI LC and BANG nebulizers. It was found that the PARI LC is more efficient at a higher drug concentration with the lung deposition of 2.4 and 14.9 μg (approximately six folds) for the 1- and 5-mg/kg doses, respectively. The plasma exposures correspond to lung depositions with the evidence of reduced systemic exposures. For the PARI LC nebulizer, plasma concentration increase was observed when dose increased (for the 1- and 5-mg/kg doses: 0.01 and 0.02 μg/mL). However, the degree of increase is much smaller (only two folds) when compared with the lung exposure increase (approximately six folds). The dose-dependent increase in lung exposure along with lower systemic exposure was very much in accordance with the desired outcome. In comparison, the BANG devise lost efficiency at a higher dose with a deposition of 2.0 and 0.3 μg for the 1- and 5-mg/kg doses, respectively. The lack of delivery consistency at higher dose for BANG devise was reflected in plasma level as well. For the 1-mg/kg dose the plasma concentration was determined at 0.01 μg/mL and for the 5-mg/kg dose, plasma level was below the limit of detection. Based on the data, PARI LC nebulizer was chosen for further studies. The PARI LC devise, provided a dose-dependent drug increase in lung disposition and evidence of reduced systemic exposure (compare with the IT dose). Detailed information is illustrated in Figs. and.
Effect of dosing technique on lung deposition
Effect of dosing technique on plasma concentration
Particle size distribution of the aerosol was further investigated by using a ten-stage cascade impactor with PARI LC nebulizer. The obtained data was used to further adjust the nebulizer to maximize the efficiency. Key parameters such as formulation concentration and system flow rates were investigated and found to impact the aerosol particle size and nebulizer efficiency. The system was optimized based on the best-obtained parameters. It was found when the optimized system was used, dose formulation, concentration, and flow rate had minimal effect on the aerosol particle size. The MMAD was obtained by linearly fitting the percent cumulative accumulation at each stage vs particle diameter. The obtained linear equations (y
) were used to calculate the MMAD. In general, the MMAD of our optimized delivery setting was found to be about 3.7 ± 0.3 μm throughout a wide dose range (Table ). The particle size of the nanosuspension formulation used for the studies is 0.2 μm (D50). A MMAD of 3.7 ± 0.3 μm indicates that aerosol particles are indeed aggregates and each aggregate contains from 17 to 20 nanoparticles. The obtained MMAD is well within the respirable range of an aerosol. Furthermore, in this study, the MMAD (<5 μm) of nanosuspension aerosol system is comparable to that of conventional aerosol system prepared from organic propellant [25
], which is clinically proven. Therefore, the delivery efficiency of nanosuspension is believed to be comparable to conventional systems. It is a viable option for pre-clinical drug delivery, and thus can provide a more suitable method for human dose projection.
PARI LC Nebulizer MADD via Impactor for Nanosuspension on Different Dose Range
Several in vivo experiments were adopted. The first experiment was designed to evaluate if regular suspension is suitable for in vivo aerosol delivery. The regular suspension was produced by using larger particle fluticasone (particle size D50 to D90 1.6–2.9 μm). The particle size range used in this study is within the range used for dry powder inhaler. In general, when larger particles were used for aerosol delivery, the fluticasone level was below LOD in both lung tissue and plasma. This finding was not a surprise. During the inhalation of an aerosolized drug, assuming if all the drug particles travel at a constant velocity, the large particles carry a higher momentum due to its increased mass. The higher momentum makes it difficult for these particles to negotiate the sharp turns in the anatomy of the nasal cavity and the transition to the upper airway. Thus, these larger particles tend to impact the inside of the nasal cavity or the back of the throat [27
]. On the other hand, the smaller particles can change their trajectory with relatively increased ease, and can reach the lower parts of the airways [30
]. Inside the deep lungs, the drug deposition is due to many factors which include Brownian motion, sedimentation due to gravity, and random impaction [31
]. Based on the number of nanoparticles found in each aggregate post nebulization, it is hypothesized that after nebulization, resulting aggregates from regular size particles were too large for inhalation. Due to the larger particle size used (particle size D50 to D90 1.6–2.9 μm), an aggregate with n
> 4 will result in a particle with D50 > 5 μm which is considered too large for inhalation. When particles are larger than 5 μm, majority of the particles are trapped in the nasal cavity and upper airways, and only a small percentage will actually reach the deep lung [33
]. The majority of the fluticasone will then be deposited in the oral/nasal cavity and then swallowed. Because fluticasone is known to have very low oral absorption, low plasma exposure was expected. Based on the observation, it is concluded that regular size fluticasone suspension is not suitable for aerosol delivery. No further studies were conducted with regular size fluticasone.
The robustness of the delivery system was further tested in vivo. In this experiment, the impact of system flow rate on performance was further investigated. The flow rates tested for this study were 0, 2, and 5 L/min to cover the extreme cases, and the dose was set at 1 mg/kg. In general, our system was very robust. Using our delivery system, the lung depositions were not statistically significant, even under the extreme challenge. At a flow rate 0, 2, and 5 L/min, the lung depositions were 2.3, 1.1, and 2.4 μg/g, respectively (Fig. ). Furthermore, the plasma concentration was about 0.01 μg/mL for all the groups (Fig. ). If IT delivery is considered to be 100% on target delivery, the lung exposure via aerosol delivery is approximately 30%. In a clinical setting, the typical fraction delivered to lung is believed to be between 10 and 40% [9
]. The amount of fluticasone deposited in the lung via nanosuspension aerosol delivery falls within the clinical range with low variability. This device provided a much better confidence in delivery in a relevant preclinical model in a range which mimics human exposure. Another highly desired advantage is reflected in the reduced systemic exposure. The systemic exposure from nanosuspension aerosol delivery is approximately 25% of the IT delivery. This low systemic exposure can provide a potential tool to differentiate topical (lung) vs systemic efficacy and side effects. This advantage is particularly important, because a major focus of pulmonary drug development is to improve local exposure/efficacy and minimizing systemic exposure and side effects. The lower systemic exposure observed via nanosuspension aerosol delivery will help researchers further explore the feasibility to differentiating topical (lung) vs systemic efficacy and side effects pre-clinically. Based on the results from various tests, we concluded that the aerosol delivery of fluticasone nanosuspension is very robust and well suited for pre-clinical pulmonary drug delivery. These novel studies demonstrate that combining nano-suspension and aerosol delivery is a valuable tool for pre-clinical pulmonary drug delivery. The major advantages of this delivery system include the absence of a propellant, ease of production, no solubility limit of the compound, and simulation of actual exposure in humans.
Effect of system flow on lung deposition (PARI LC)
Effect of system flow on plasma exposure (PARI LC)