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. Author manuscript; available in PMC: 2021 Dec 15.
Published in final edited form as: Int J Pharm. 2020 Oct 31;591:120027. doi: 10.1016/j.ijpharm.2020.120027

Characterization of excipient enhanced growth (EEG) tobramycin dry powder aerosol formulations

Amr Hassan 1, Dale Farkas 2, Worth Longest 1,2, Michael Hindle 1
PMCID: PMC8040971  NIHMSID: NIHMS1644157  PMID: 33130220

Abstract

Spray drying can be utilized to produce highly dispersible powder aerosol formulations. However, these formulations are known to be hygroscopic, leading to potential solid-state stability and aerosol performance issues. This study aims to investigate if control of the spray drying particle formation conditions could be employed to improve the solid-state stability and alter the aerosol performance of tobramycin EEG formulations. Eight formulations were prepared, each had the same drug:excipient ratio of 60%w/w tobramycin, 20% w/w l-leucine, 18% w/w mannitol, and 2% w/w poloxamer 188. An experimental design matrix was performed with drying air water content of 1 or 10 g/m3 and spray drying solution l-leucine concentrations of 4.6, 7.6, 15.2 or 23.0 mmol/L. The particle size, morphology and crystallinity of spray dried formulations were characterized together with their dynamic moisture vapor sorption and aerosol performance. Higher crystallization and glass transition %RH were observed for the formulations spray dried using drying air with higher water content indicating more stable characteristics. Initial screening using a handheld dry powder inhaler of the realistic aerosol performance revealed that neither changing l-leucine concentration nor the drying gas water content affect the in-vitro expected lung dose. However, using a novel positive pressure inhaler, formulations produced using spray drying solutions with lower l-leucine concentrations showed better aerosol performance with MMAD around 2 μm and FPF<5μm around 80%.

Keywords: Dry powder aerosol, tobramycin, l-leucine, spray drying, hygroscopic formulation, excipient enhanced growth, solid-state stability, aerosol performance

Graphical Abstract

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1. Introduction

Dry powder inhalers are an effective means of pulmonary drug delivery (Kadota et al., 2020a; Mehta et al., 2020). In most cases, these devices are breath actuated and do not require synchronization of inhalation and actuation by the patient as in case of the pressurized metered dose inhaler (pMDI) (Ivanova et al., 2017). DPIs can also be used to deliver a wide range of active pharmaceutics ingredients (APIs) with doses ranging micrograms (formoterol fumarate; Foradil Aerolizer) to milligrams (tobramycin; Tobi® Podhaler™). These characteristics make them particularly suitable for the delivery of antibiotics to the lungs, which often require high powder doses (Le Brun et al., 2002).

Spray drying is an effective, flexible and scalable technique to produce engineered drug and excipient combination particle formulations for next generation powder aerosol drug delivery applications (Behara et al., 2014; Patil et al., 2019; Son et al., 2013b; Weers et al., 2019). Particle engineering is considered an effective technique to control many formulation characteristics responsible for efficient pulmonary delivery such as size, surface energy, and solid-state stability (Hertel et al., 2020; Kadota et al., 2020b).

There are two FDA approved dry powder inhalers (DPI) that utilize spray dried formulations available now in the market, antibiotic formulation Tobi® Podhaler™ (Geller et al., 2011) and Parkinson’s disease inhalation treatment Inbrija (Paik, 2020). Extensive development efforts were required for the production of these stable pharmaceutical powder aerosol formulations and their remain concerns about the production of amorphous spray dried products and solid-state stability (Li et al., 2016). Amorphous formulations are often characterized as being hygroscopic (King et al., 1982; Otsuka et al., 1993; Yu et al., 2018). Without mitigation, this characteristic may have deleterious effects both on the solid-state stability and aerosol performance of spray dried formulations (Shur et al., 2008). A number of strategies are available to mitigate these effects both at the formulation and packaging levels (Li et al., 2016; Yu et al., 2017a; Zhou et al., 2016). RH can negatively impact powder performance in multiple ways. First, slow diffusion of water vapor through the packaging may lead to decreases in Tg over time, thereby decreasing physical and chemical stability on storage. These processes were effectively mitigated for Tobi® Podhaler™ resulting in a 3 year shelf-live. Second, when the dose is being administered, there may be an instantaneous drop in aerosol performance with increasing RH that is due to capillary bridging. Third, residual powder left in the device may adversely impact the performance of the device if the residual powder is exposed to high RH above its Tg. This was mitigated in Tobi® Podhaler™, by having a 1-week use-life for the device. Finally, it is possible to produce spray dried formulations including a hydrophobic excipient that coats the surface of the particles to protect such formulations against absorption of ambient environmental moisture (Yu et al., 2017b). L-leucine is a hydrophobic excipient that has been used as a dispersion enhancer in spray dried formulations (Boraey et al., 2013; Li et al., 2003; Mangal et al., 2018). It has a low water solubility, and during the spray drying process, it can reach saturation in the droplets earlier than more water soluble components leading to precipitation and formation of an outer layer on the surface of the particle as the drying process continues (Feng et al., 2011). Although there are extensive studies describing the dispersion enhancing effects of the l-leucine surface layer, there is less information about its impact on hygroscopicity and solid-state stability of spray dried materials. Chang et al. studied the effect of l-leucine on the hygroscopicity of spray dried herbal extract (Chang et al., 2014). They observed a decrease in the hygroscopicity of a spray dried material as the percentage of l-leucine increased up to 10 %w/w, and a plateau was observed above this amount. A similar effect was observed when Li et al., investigated the effect of l-leucine on the moisture uptake of a highly hygroscopic spray dried material (Li et al., 2016). Cui et al., were able to prepare a stable formulation of netilmicin through the inclusion of l-leucine. Netilmicin is known for its hygroscopic characteristics, however the optimum formulation with a 30:1 netilmicin: l-leucine ratio showed greater stability when stored at higher temperature and relative humidity conditions compare to the pure spray dried netilmicin (Cui et al., 2018). In another study, Li et al., found that 40 % (w/w) l-leucine in spray dried salbutamol sulfate formulations was sufficient to eliminate the effects of a high humidity environmental exposure on the dispersion behavior (Li et al., 2017). The mechanism of the l-leucine shell formation during the droplet drying process is extensively described in the literature (Longest et al., 2020; Vehring, 2008; Vehring et al., 2007) and depends on the rate of solvent evaporation from the droplet surface, κ, and the diffusion coefficient, D. The evaporation rate, κ, is mainly affected by the drying gas temperature and relative humidity. Increasing the drying gas temperature or decreasing the water vapor content results in increases in the rate of water evaporation from the droplet surface (Vehring et al., 2007). The diffusion coefficient, D, describes the ability of the spray drying solution component to redistribute and move toward the core of the shrinking droplet through molecular motion. These two factors can be combined in one term, the Peclet number Pe, which is defined as the evaporation rate κ divided by 8 times the solute diffusion rate D (Feng et al., 2011). Although l-leucine has a high diffusion rate during the spray drying process, its low solubility allow it to reach saturation at early stages of the drying process and form a layer on the surface of the dry particle (Vehring, 2008). The morphology and the surface composition of spray dried materials will be defined as a result of these factors. For example, the faster the solvent evaporation rate from the surface of a droplet, the greater the surface enrichment of the hydrophobic excipient. Similarly, if the excipient diffusion rate is slower than the receding droplet front then a thin solid layer will form, which in sufficiently large droplets with fast evaporation will lead to buckling and crumpling of the particle surface (Vehring, 2008). For solutes with higher diffusion coefficients, the solubility in the droplet will control when the surface precipitation occurs.

This study aims to investigate the effect of spray drying particle formation conditions (l-leucine concentration and drying gas water vapor content) on the solid-state stability and aerosol performance of tobramycin formulations. We hypothesized by changing l-leucine concentration in the spray drying solution, we can control the amount of l-leucine as well as the thickness of the formed layer on the surface of the dried particles. In addition, by changing the water content of the drying gas, we can control the rate of solvent evaporation from the surface of the sprayed droplet and alter the crystallization kinetics.

In order to investigate the surface enrichment of l-leucine and its ability to improve the stability and aerosol performance of spray dried formulations, tobramycin (TOBI) was selected as the model drug due to its hygroscopic properties (Pilcer et al., 2009) and its applicability of a pediatric inhaled antibiotic formulation. The spray dried powder aerosol formulation is based on the excipient enhanced growth (EEG) concept (Tian et al., 2013; Worth Longest and Hindle, 2011). Micrometer sized combination particles are formulated with a hygroscopic excipient (mannitol) and a dispersion enhancer (l-leucine). We have previously shown that addition of l-leucine to the EEG particles improves their aerosol performance (Son et al., 2013b). The highly dispersible micrometer-sized dry powder aerosols have been shown to have low mouth-throat deposition during inhalation from a DPI (Son et al., 2013a, 2013b); however, in order to maximize pulmonary retention, hygroscopic growth of the particles is beneficial to enable full deposition in the lungs. The challenge is to produce pharmaceutical formulations that are stable under ambient temperature and humidity conditions, but which exhibit hygroscopic growth when inhaled into the humidified airways of the lungs (Longest and Hindle, 2012; Longest and Hindle, 2011) and maintain their aerosol performance.

2. Materials and methods

2.1. Materials

Tobramycin was purchased from Spectrum Chemicals (Gardena, CA), and Pearlitol® PF-Mannitol was donated from Roquette Pharma (Lestrem, France). Poloxamer 188 (Lutrol F68) was donated from BASF Corporation (Florham Park, NJ). L-leucine and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Quali-V hydroxypropyl methylcellulose capsules size 3 were donated from Qualicaps (Whitsett, NC).

2.2. Spray dried powder formulations

A series of spray-dried EEG powder formulations were produced with two processing variables under investigation. Firstly, the l-leucine concentration in the spray drying solution was varied across the levels of 4.6, 7.6, 15.2 and 23.0 mmol/L. Previous studies have varied the l-leucine concentration by changing its % composition relative to the drug and other excipients in the final formulation. In this study, the l-leucine concentration and the % solids concentration in the spray drying solution were changed simultaneously in order to maintain a fixed drug : excipient ratio of 60% tobramycin, 20% l-leucine, 18% mannitol, and 2% poloxamer 188 in all the sprayed formulations. This enabled the l-leucine concentration in the sprayed droplet to be varied while maintaining the same composition in the final produced particle. Secondly, the drying gas water content was varied (1g/m3 or 10 g/m3), by controlling the relative humidity of the inlet gas. Poloxamer 188 was employed to enable a stable spray plume during spray drying. A summary of the experimental matrix is shown in Table 1. The EEG powder formulations were produced with a B-90 HP Buchi Nano Spray Dryer (Buchi Labortechnik AG, Switzerland) with a drying inlet temperature of 70°C, spray rate of 8–12 μl/sec and gas flow rate of 120 L/min. A schematic diagram describing the spray drying conditions is shown in Figure 1.

Table 1:

Formulations spray drying conditions, mean (SD) particle size characteristics and solid-state stability parameters.

Formulation Drying gas water content (g/m3) L-leucine mmol/L Spray drying solution solid content (g/100 mL) TOBI Content % Dv50 μm Predicted Diameter μm Span
1 1 4.6 0.3 58 (2) 0.9 (0.0) 0.8 1.46
2 1 7.6 0.5 59 (1) 1.1 (0.0) 0.9 1.49
3 1 15.2 1 59 (3) 1.1 (0.0) 1.1 1.43
4 1 23.0 1.5 57 (3) 1.2 (0.0) 1.3 1.41
5 10 4.6 0.3 58 (1) 0.9 (0.0) 0.8 1.45
6 10 7.6 0.5 60 (3) 1.1 (0.0) 0.9 1.50
7 10 15.2 1 59 (2) 1.1 (0.0) 1.1 1.52
8 10 23.0 1.5 58 (3) 1.2 (0.0) 1.3 1.45
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Figure 1:

Figure 1:

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Schematic diagram of the spray drying process, showing under study parameters as well as the parameter setting.

2.3. Tobramycin content uniformity

The tobramycin content uniformity in the eight formulations was evaluated using a validated liquid chromatography-mass spectrometry (LC-MS) method. 10 mg of TOBI-EEG formulation was dissolved in 100 mL water, and tobramycin was quantitatively analyzed using an LC-MS method. Analysis was performed using Alliance e2695 HPLC (Waters, Milford, MA) coupled with ACQUITY QDA Mass Detector (Waters, Milford, MA). Chromatographic separation was performed using Obelisc R HPLC column (2.1×150mm, 5μm, 100 Å; SIELC Technologies, Inc., Wheeling, IL) and mobile phase consisting 10% acetonitrile and 90% water containing 0.3% v/v formic acid and 0.01% w/v ammonium formate. Isocratic elution was used at a flow rate of 0.3 mL/min and sample injection volume was set at 10 μL. QDa parameters were as follows: capillary voltage, 0.8 kV; cone voltage, 20 V; source temperature, 600 °C. SIR mode was selected to acquire data. Electrospray ionization was used with a positive mode. For tobramycin quantification, single ion monitoring at 468.5 m/z was employed. Empower 4 (Waters, Milford, MA) was used to control LC-QDa system and analyze data obtained. Three replicates were performed to obtain the mean (SD) mass of tobramycin in each mg of TOBI-EEG formulation.

2.4. Particle size characterization

The primary particle volumetric diameter (Dv) of each formulation was determined by laser diffraction using the Sympatec HELOS (submicron R1 lens with 20 mm focal length) equipped with RODOS/M disperser and ASPIROS sample feeder (Sympatec GmbH, Germany). Powder formulations were dispersed at a pressure of 4 bar. The span was calculated and reported for each measurement using the following equation

Span=(Dv90−Dv10)/Dv50. (1)

2.5. Scanning electron microscopy (SEM)

The morphologies of the spray dried formulations were examined using Zeiss EVO 50 XVP (Carl Zeiss AG, Germany) scanning electron microscopy (SEM). Sample powders were spread on a double adhesive carbon tape mounted on an SEM aluminum stub, followed by sputter coating with gold using EMS 550x sputter coater. The images were captured at 10 kV.

2.6. Powder X-ray diffraction (XRD)

Powder x-ray diffraction of samples was performed using MiniFlex II Desktop X-ray diffractometer (Rigaku, Japan) to evaluate sample crystallinity. A copper K-α radiation source was used with a scintillation detector at a generation voltage of 30 kV and a current of 15 mA. Data were collected by the 2θ method at a scan speed of 1°/min at the range of 5–80° 2θ.

2.7. Dynamic vapor sorption (DVS)

Dynamic vapor sorption (DVS Adventure, Surface Measurement Systems Ltd., UK) was employed to evaluate the moisture uptake of the spray dried formulations at different % RH values. Approximately 10 mg samples were equilibrated at 0% RH prior to increasing the humidity. This initial equilibration process continued until no change in the sample mass was observed. A mass equilibration criterion of dm/dt = 0.002%/min was chosen for the system to achieve before automatically proceeding to the next step. After the drying step, relative humidity was increased from 0 to 90 % RH at 2% RH/hr, and the sample mass change was recorded automatically. The rate of the mass change of these formulations during both the surface water adsorption and bulk absorption were calculated from the slope of the best fitting tangent of the data points before and after the glass transition inflection. All the DVS experiments were performed at 25 °C.

2.8. In vitro aerosol performance

Initial screening of the aerosol performance was carried out using a previously developed handheld DPI. For these studies, 2 mg of the TOBI-EEG formulation was loaded into size 3 HPMC capsules and placed into a high resistance (0.044 kPa0.5 L−1 min) CC90–3D DPI (Behara et al., 2014), which was built using stereolithography (SLA) in Accura ClearVue (3D Systems, Inc., Rock Hill, SC) (Behara et al., 2014). The aerosol performance of the TOBI-EEG formulation was assessed using a realistic mouth-throat airway model to estimate in vitro both the emitted dose and the fraction of the aerosol reaching the lung. The DPI was sealed in the inlet of the medium-sized VCU mouth-throat and actuated using the medium inhalation profile. The flow profile was characterized by a peak inspiratory flow rate of 65 L/min, total volume inhaled of 2.76 L and average flow rate of 41 L/min (Wei et al., 2017). This profile was generated using the ASL 5000 breathing simulator (Ingmar Medical, PA, USA). Aerosol penetrating the mouth-throat was collected on a respiratory filter positioned at the trachea and estimated as the in vitro total lung dose. Tobramycin deposition on the inhaler, mouth-throat and respiratory filter was collected for quantitative analysis using the validated LC-MC method described previously. The internal surfaces of the MT model were coated with a silicone spray prior to each experiment. Ambient air was used for the actuation of the device (25 °C, 40–50% RH). The realistic in vitro aerosol test setup and as well as inhalation profile used for the DPI actuation are shown in Figure 2. The emitted dose in each run was calculated using the following equation

EmittedDose(%)=(Nominal mass of TOBI loaded in the device−Capsule and device retention)Nominal mass of TOBI loaded in the device×100. (2)

Figure 2:

Figure 2:

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Schematic diagram of the realistic in-vitro aerosol performance test setup using realistic mouth-throat (MT) model, realistic breathing profile, and novel CC90–3D DPI.

Aerosol performance of select formulations were also assessed using a novel positive pressure DPI (Farkas et al., 2020) (Figure 3a), which was developed for pediatric antibiotic aerosol administration. The DPI operates by passing a positive pressure air-jet across an aerosolization chamber and incorporates a 3D rod array to enhance powder deaggregation. The DPI design was created using Autodesk Inventor and exported as .STL files to be prototyped. The DPI files were then prepared using the Object Studio preparation software and were built using an in-house Stratasys Objet24 3D Printer (Stratasys Ltd., Eden Prairie, MN) using VeroWhitePlus material at a 32 μm resolution. Supporting material was cleaned away from the device using a Stratasys water-jet cleaning station and the DPI was allowed to fully dry before use. The volume of air used for the actuation was 750 mL produced at a flow rate around 20 LPM with a nearly square-wave positive-pressure input profile as shown in Figure 3b. The specific airflow rate and profile create a 6 kPa pressure drop across the powder chamber. The device was loaded with 10 mg of formulation, and was connected to the preseparator of the Next Generation Impactor (NGI) operating at 45LPM to entrain the emitted aerosol. The emitted dose and aerodynamic particle size characteristics of the tobramycin aerosol were determined by quantitative assay using the previously described LC-MS method.

Figure 3:

Figure 3:

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Schematic diagram of (a) the positive pressure DPI device used to evaluate the formulations aerosol performance (b) the used compressed air flow profile

2.9. Statistical Analysis

The results in this study are shown as mean values with standard deviation (SD) from at least three measurements. Student’s t-test or one-way ANOVA followed by Tukey’s HSD methods were used to test differences between formulations. A P-value of <0.05 was considered significant.

A multivariance standard least square fitting model was employed to analyze the effect of l-leucine concentration and the drying gas water content on the formulation solid state stability (glass transition RH and crystallization RH). Statistical evaluations were performed using JMP Pro 14 software (SAS Institute Inc., Cary, NC).

3. Results

3.1. Physical characterization of the spray dried formulations

3.1.1. Primary particle size and scanning electron microscopy

Primary particle size based on laser diffraction is presented in Table 1 as median volume diameter (Dv50) for each formulation with a mean (SD) value across the eight formulations of 1.0±0.2μm. There was a small, statistically significant increase in the particle size with increasing l-leucine concentration due to the increase in the solid content in the spray drying solution. The observed particle size for all the formulations agreed with the predicted size calculated based on the solid content of each spray drying solution. There was no significant change observed in primary particle size when the drying gas water content was changed, while maintaining constant l-leucine concentration.

Scanning electron micrographs (Figure 4) showed that all the formulations have spherical morphology with diameters around 1 μm in agreement with the laser diffraction data. Qualitatively, slightly larger sized particles were observed for formulations sprayed from the higher l-leucine concentration spray drying solution which agreed with the laser diffraction data. Figure 4 reports the scanning electron micrographs of formulations 1, 3, 5, and 7, respectively illustrating the effects of both l-leucine solution concentration (4.6 and 15.2 mmol/L) and drying gas water content (1 and 10 g/m3).

Figure 4:

Figure 4:

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Scanning electron micrographs (a) Formulation 1, (b)Formulation 3, (c) Formulation 5 and (d) Formulation 7

3.1.2. Powder X-ray Diffraction

The solid-state characteristics of the spray dried formulations were evaluated using powder X-ray diffraction (XRD). Figure 5 provides the XRD patterns for formulations 1, 3, 5, and 7 as well as tobramycin, l-leucine and mannitol raw materials. All the raw materials were observed to be crystalline with XRD patterns in agreement with literature data (Lamy et al., 2019; Li et al., 2015; Parlati et al., 2009). Crystalline features of tobramycin and mannitol were absent in the spray dried formulation, which appeared amorphous except for the presence of characteristic crystalline peaks for l-leucine. Peaks at 2θ values 6° and 19.5° were observed corresponding to l-leucine crystalline planes (001) & (−110) respectively. The preferential crystal growth of l-leucine in (−110) plane compared to (004) plane observed in the raw material was attributed to the crystallization of l-leucine on the surface of the formed particle during the drying process (Lamy et al., 2019; Raula et al., 2007). Sou et al. (Sou et al., 2013), attributed this observation to the limited 2 D crystal growth on the surface of the spray dried particles unlike the 3D crystal structure in the raw material. There were no trends observed in the powder formulations XRD patterns corresponding to changes in either l-leucine concentration or drying gas water content.

Figure 5:

Figure 5:

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XRD patterns for Formulations 1, 3, 5, and 7 as well as tobramycin, l-leucine, and mannitol raw materials

3.2. Dynamic water vapor sorption (DVS)

After drying, all spray dried formulations were exposed to relative humidity conditions increasing at a rate of 2% RH/hr. Considering the hygroscopicity of the formulations, prior to the glass transition, the surface water adsorption rate was lower for the formulations generated with the higher drying gas water content of 10 g/m3 across all l-leucine solution concentrations. The glass transition RHs for the formulations, observed as inflections in the adsorption profiles, are shown in Figure 6 and reported as numerical values in Table 2. The formulations produced using the higher drying gas water content (formulations 5–8) had higher glass transition RH’s compared to formulations 1–4 which were produced with the lower drying gas water content. Following the glass transition, the high molecular mobility allows bulk water absorption to take place (Burnett et al., 2004). As shown in Table 2, the bulk water absorption rate was approximately double that of the surface water adsorption rate for all formulations, indicating a faster water uptake due to the high mobility of molecules after the surface water adsorption was complete. The lower hygroscopicity of the powder formulations produced at 10 g/m3 drying gas water content, despite having identical % composition, could been seen when the equilibrium moisture content at 45% RH (selected as below the crystallization RH for all formulations) was evaluated. Samples prepared with the higher drying gas water content (10 g/m3) had equilibrium moisture contents of 7.3 – 7.6 %, compared to 8.2 – 9.0 % for the formulations prepared at 1 g/m3 drying gas water content, indicating their lower hygroscopicity. The improved stability for these samples could also be observed when comparing the first crystallization RH (Figure 6 and Table 2). Formulations 5 – 8 were observed to recrystallize at RH’s of 53 – 55% RH which were significantly higher than values of 49–51% for formulations 1–4. The XRD patterns of the formulations following exposure to humidity were observed to confirm the crystalline feature of the formulations as shown in Figure 7.

Figure 6:

Figure 6:

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Dynamic vapor sorption (DVS) for Formulations 1–8 showing the crystallization and glass transition RH%

Table 2:

Dynamic Vapor Sorption (DVS) Data for spray dried formulations

Formulation Glass Transition RH** Mass Change (% of dry) At 45% RH* Crystallization RH** Mass change (% of dry) at Crystallization point* Mass Change rale (Mass change % of dry/h)
Surface adsorption* Bulk absorption*
1 27% 9.0 49% 9.9 0.25 0.50
2 29% 8.2 51% 9.4 0.25 0.47
3 27% 8.5 50% 9.6 0.24 0.50
4 28% 8.5 50% 9.5 0.27 0.49
5 36% 7.6 53% 8.9 0.23 0.40
6 38% 7.4 54% 9.0 0.20 0.43
7 39% 7.3 55% 9.1 0.23 0.40
8 39% 7.4 55% 9.3 0.23 0.44
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The data from glass transition RH%, Mass change at 45% RH, Crystallization RH%, Mass Change at crystallization RH%, and Mass change rates Surface and bulk were pooled to study the effect of drying gas water content. Pooled T-test were applied to investigating the significant differences between the two drying gas groups.

*

Significantly lower for the 10 g/m3 drying gas group than 1 g/m3 drying gas group

**

Significantly higher for the 10 g/m3 drying gas group than 1 g/m3 drying gas group

Figure 7:

Figure 7:

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Effect of l-leucine concentration on the glass transition, crystallization %RH, and the equilibrium mass at 90% RH. Blue for 1 g/m3 drying gas water vapor content and orange for 10 g/m3

Figure 8 summarizes the effect of drying gas water content and spray drying solution l-leucine concentration on glass transition and crystallization % RH as well as the % mass gain at 90% RH. There was no significant effect of spray drying solution l-leucine concentration on glass transition or crystallization %RH at either drying air water content. However, the equilibrium moisture content at 90% RH was elevated for lowest spray drying solution l-leucine concentration. As described previously, higher glass transition and crystallization % RH were observed at all spray drying solution l-leucine concentrations for the 10 g/m3 drying air water content formulations compared to 1 g/m3 drying air water content.

Figure 8:

Figure 8:

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XRD patterns for formulations 1, 3, 5, and 7 after DVS experiment.

Standard least square regression for a linear model was generated to study the effect of spray drying solution l-leucine concentration and the drying gas water content, as well as their interaction effect on the glass transition RH % and crystallization RH % of the eight formulations. The models show a good fit with R2 0.98 for the glass transition RH % model and 0.94 for the crystallization RH % model. The statistical analysis revealed that the drying gas water content had a significant effect on both crystallization and glass transition RH of the formulations (P = 0.0002 and 0.0017 respectively). Both l-leucine concentration and the first order interaction between the drying gas water content and l-leucine concentration had no significant influences on the solid-state stability of the formulations. These effects can be observed from the leverage graphs shown in Figure 9.

Figure 9:

Figure 9:

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Leverage graphs showing the effect of l-leucine concentration, drying air water content, and the interaction on crystallization and glass transition RH%

3.3. In-vitro evaluation of the spray dried powders

A realistic setup was used to evaluate mouth-throat and estimated in vitro lung deposition of the spray dried formulations aerosolized with the CC90–3D DPI (Figure 2). Four formulations were selected for evaluation, Formulation 1 and 3 sprayed using 1g/m3 water content of the drying air and spray drying solution concentrations of l-leucine of 4.6 and 15.2 mmol/L, respectively, and formulations 5 and 7 sprayed using 10 g/m3 water content of the drying air and spray drying solution concentrations of l-leucine of 4.6 and 15.2 mmol/L, respectively. Neither the drying gas water content nor l-leucine spray drying solution concentration appear to have significant effect on the in vitro estimated lung dose when expressed as a fraction of the 2 mg nominal dose. The mean (SD) estimated lung dose was 48 (6) %, 54 (3) %, 55 (3) % and 53 (6) % for formulations 1, 3, 5 and 7 respectively. There was no statistical difference between the four formulations.

Given the improved stability of formulations generated using the lower water content in the drying gas, these formulations were selected for further aerosol testing using a novel positive pressure DPI. In this experiment, 10 mg of formulation was loaded into the aerosolization chamber of a novel positive-pressure air-jet DPI to aerosolize the powder. The chamber had a geometry consistent with a size 0 capsule. The emitted dose and aerodynamic particle size characteristics of the aerosol were determined and are shown in Table 3. All four formulations produced a high emitted dose between 79 to 82 % of the loaded dose and there was no significant difference between the formulations. The two formulations sprayed using lower l-leucine spray drying concentration (4.6 and 7.6 mmol/L), produced a smaller MMAD (1.8 and 2.0 μm, respectively) compared to 15.2 and 23 mmol/L formulations (MMAD = 2.6 μm for both). Similar trends were observed for other aerosol performance parameters, such as fine particle fractions (FPF) less than 5 μm and 1 μm, respectively. This may be due to lower total solid contents in these spray drying solutions; which affected the primary particle size as shown in Table 1. Formulation 5 produced the highest FPFs and was considered the optimal formulation for further study based on its aerosol performance

Table 3:

Aerosol performance characteristics of optimized formulations aerosolized using a novel positive pressure DPI

Formulation L-leucine mmol/L Predicted Diameter μm Emitted dose (% nominal) MMAD (μm) FPF< 5μm (% of impactor dose) FPF< 1μm (% of impactor dose)
5 4.6 0.8 79 (1) 1.8 (0.0) 80 (3) 18 (1)
6 7.6 0.9 79 (5) 2.0 (0.2) 78 (4) 16 (2)
7 15.2 1.1 82 (3) 2.6 (0.2)* 71 (6) 12 (1)*
8 23.0 1.3 75 (2) 2.6 (0.2)* 75 (5) 9 (2)*
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The Data were analyzed using ANOVA Followed by Tuckey HSD

*

Significantly different compared to formulations 5 and 6

4. Discussion

This study of the effect of spray drying particle formation (dry gas water content) and formulation (l-leucine concentration in the spray drying solution) was performed while keeping constant the mass fractions of the formulation components. This approach allowed comparison of eight formulations, all with the same composition (60%w/w tobramycin, 20% w/w l-leucine, 18% w/w mannitol, and 2% w/w poloxamer 188), ensuring that any differences in the hygroscopicity or aerosol performance of these formulations was derived from the particle formation process rather than the final formulation composition. Previous studies have demonstrated that changing the l-leucine mass fraction within a formulation altered the hygroscopicity of the powder. For example, Mah et al. (Mah et al., 2019) observed a 7.2% decrease in water uptake of a spray dried trehalose formulation when the mass fraction of l-leucine increased from 20% to 60%, which was attributed to l-leucine surface enrichment. However, it should be observed that a decrease in the mass fraction of trehalose, the hygroscopic component in the formulation, could also be responsible for the lower hygroscopicity as the l-leucine fraction is increased.

In this study, we aimed to investigate the stability, hygroscopicity and aerosol performance of tobramycin EEG spray dried formulations using a fixed ratio of formulation components removing the variable of final formulation composition and focusing on particle formation effects. L-leucine is known to form a hydrophobic shell or provide elevated concentrations on the surface of spray dried particles creating an effective protective layer against water uptake (Chang et al., 2014; Cui et al., 2018; Li et al., 2017, 2016). The mechanism of l-leucine surface enrichment during spray drying process is well established in the literature (Longest et al., 2020; Parlati et al., 2009; Tian et al., 2013; Yu et al., 2017b). Due to the low aqueous solubility of l-leucine, it reaches supersaturation in the early stages during the drying process, forming a shell on the surface of the drying droplet. The remaining l-leucine as well as the other formulation components will diffuse towards the core of the droplet. At sufficiently high evaporation rates, usually achieved by using high drying air temperature, a thin l-leucine shell will form early in the drying process prior to complete liquid evaporation and before the droplet has had a chance to significantly decrease in size (Boraey and Vehring, 2014). As the drying process continues, shell buckling or shell collapse may occur leading to the formation of wrinkled particles (Kadota et al., 2020a; Longest et al., 2020; Vehring, 2008; Vehring et al., 2007; Zhou et al., 2014). The formation of such wrinkled particles theoretically leads to a better aerosol performance (Vehring, 2008), although the collapsed or thin l-leucine shell may not be able to actively protect the inner hygroscopic materials against the environmental humidity ingress. In case of a slower evaporation rate, a thicker l-leucine surface shell can be formed. Increasing the thickness of the layer may protect the formed particle against collapsing or buckling (Boraey and Vehring, 2014; Gouaou et al., 2019; Mehanna et al., 2019). In addition, a thicker layer of hydrophobic material could act to reduce moisture uptake into the powder.

As described above, two factors that contribute to the shell formation are the l-leucine concentration in the initial spray drying solution, and the rate of drying of the sprayed droplets inside the spray drying chamber. The drying rate can be controlled by either the drying gas temperature, drying gas composition and flow rate, or the drying gas water content (Longest et al., 2020). In this study, two factors were considered; l-leucine concentration in the spray dried solution at four levels (4.6, 7.6, 15.2, 23 mmol/L) and drying gas water content at two levels (1g/m3, and 10g/m3). A full factorial (2×4) experimental design was created leading to spray drying of eight different formulations, as shown in Table 1. It was hypothesized that increasing the drying gas water content (formulations 5–8), would reduce the rate of evaporation leading to the formation of a thicker l-leucine surface layer. Conversely, a faster evaporation rate would be produced when the water content of the drying gas was reduced (formulations 1–4), and a thinner l-leucine layer was expected. SEM images of all eight formulations revealed spherical surface morphology and no wrinkled particles were observed. This indicated that in all the formulations the l-leucine layer was thick enough to protect the spray dried particles against collapse. The DVS data (Table 2) supported the hypothesis regarding l-leucine shell thickness with the surface absorption rates being lower for the formulations spray dried with the higher drying gas water content. The surface adsorption rates were 0.20–0.23 %/hr for the 10g/m3 drying gas water content compared to 0.24–0.27 %/hr at 1 g/m3. Similarly, the glass transition (36–38%RH) and crystallization (53–55%) RH’s for formulations 5–8 were higher suggesting resistance to moisture penetration of the formed l-leucine layer produced at the lower evaporation rate conditions compared to values of 27–29% and 49–51%, respectively, for formulations 1–4. Finally, a direct observation of hygroscopicity could be obtained by comparing the mass change at a fixed humidity (45%RH). The observed lower equilibrium moisture content of formulations 5–8, compared to formulation 1–4, could be attributed to the changes in l-leucine shell thickness given the identical formulation composition. All these observations support the improved stability of formulations 5–8 which were generated using conditions with a drying gas water content which was used to alter the rate of droplet evaporation and drying. Changing the l-leucine concentration in the spray drying solutions did not produce any significant effects on the hygroscopic properties of the formulations.

Evaluation of the aerosol performance was performed using two novel inhalers using different aerosolization conditions. Screening with a handheld DPI using realistic adult breathing conditions did not show any differences between formulations 1, 3, 5 and 7, as representative formulations produced using varying particle formation conditions. The combination of the inhalation flow conditions and the deaggregation properties of the DPI appeared sufficient to disperse the EEG formulation and produce estimated lung doses of about 50% for each of the formulations. In contrast, using a DPI designed for pediatric applications which is operated with a lower actuation flow rate of 20 L/min, an effect of the l-leucine solution concentration was observed. Formulations 5 and 6 showed significantly lower MMAD and higher FPF<1μm compared to formulations 7 and 8. No statistically significant differences were observed between formulation 5 and 6 or between formulation 7 and 8. In order to further understand the dispersibility of the powders, the formulation particle size was evaluated using Sympatec HELOS using different dispersion pressures as shown in Figure 10. Similar differences were observed when the formulation particle size was measured at low dispersion pressures (0.5 and 1.0 bar). The observation of improved aerosolization properties for formulations formed using spray drying solutions with lower l-leucine concentrations and slower or limited drying rate is interesting and will be subject of further investigation.

Figure 10:

Figure 10:

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Effect of dispersion pressure on the apparent particle size of Formulations 5, 6, 7 and 8 measured using Sympatec HELOS

5. Conclusion

The hygroscopicity of spray dried materials due to their amorphous characteristics can affect both their stability and aerosol performance. The enrichment of a l-leucine hydrophobic layer on the surface of spray dried particles could act as a protective layer against water uptake. The successful formation of this layer was shown to depend on the spray drying conditions, including the rate of the evaporation from the surface of the droplet during the spray drying process and the l-leucine concentration in the spray drying solution. In this study, we investigated the effects of spray drying particle formation conditions (drying air water content and l-leucine concentration in the spray drying solution) on the solid-state stability, hygroscopicity and aerosol performance of tobramycin EEG formulations. It was observed that the formulations sprayed using high water contents drying air had higher glass transition and crystallization RH%, indicating the formation of more stable formulations. Changing l-leucine concentration in the spray drying solution did not significantly affect the stability of these formulations. No effect on the aerosol performance of the formulations produced using different particle formation conditions were observed using simulated adult inhalation from a handheld DPI. However, for the drying gas stabilized formulations, it was shown that improved dispersion was observed for formulations produced using lower l-leucine concentrations in the spray drying solution in a positive-pressure pediatric DPI. Overall, using this new device, a high quality aerosol was observed for the four formulations with emitted dose around 80% of the device loaded mass and MMAD around 2 μm. In conclusion, control of the spray drying particle formation conditions could be employed to improve the solid-state stability of the tobramycin EEG formulation and alter the aerosol performance of tobramycin EEG formulations.

Acknowledgments

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R01HD087339 and by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R01HL139673. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

ABBREVIATIONS

3D

three-dimensional or three-dimensional rod array

API

active pharmaceutics ingredients

DPI

dry powder inhaler

EEG

excipient enhanced growth

FDA

U.S. Food and Drug Administration

FPF

fine particle fraction

HPLC

high-performance liquid chromatography

HPMC

hydroxypropyl methylcellulose

LC-MS

liquid chromatography-mass spectrometry

MMAD

mass median aerodynamic diameter

MT

mouth-throat

NGI

next generation impactor

pMDI

pressurized metered-dose inhaler

R2

coefficient of determination

RH

Relative humidity

SD

standard deviation

SEM

scanning electron microscopy

TOBI

tobramycin

XRD

X-ray diffraction

Footnotes

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Conflict of interest

Virginia Commonwealth University is currently pursuing patent protection of devices and methods described in this study which, if licensed and commercialized, may provide a future financial interest to the authors.

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