How does secondary processing affect the physicochemical properties of inhalable salbutamol sulphate particles? A temporal investigation

Accepted Manuscript Title: How secondary processing affects the physicochemical properties of inhalable salbutamol sulphate particles? A temporal investigation Authors: Joana T. Pintoa, Snezana Radivojeva, Sarah Zellnitza, Eva Roblegga, Amrit Paudela PII: DOI: Reference:

S0378-5173(17)30535-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2017.06.027 IJP 16751

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

7-4-2017 9-6-2017 10-6-2017

Please cite this article as: Pintoa, Joana T., Radivojeva, Snezana, Zellnitza, Sarah, Roblegga, Eva, Paudela, Amrit, How secondary processing affects the physicochemical properties of inhalable salbutamol sulphate particles? A temporal investigation.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.06.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

How secondary processing affects the physicochemical properties of inhalable salbutamol sulphate particles? A temporal investigation

Joana T. Pintoa,b, Snezana Radivojeva, Sarah Zellnitza, Eva Roblegga,b, Amrit Paudela,c*

a

Research Center Pharmaceutical Engineering GmbH, Inffeldgasse 13, 8010 Graz, Austria

b

Institute of Pharmaceutical Sciences, Pharmaceutical Technology and Biopharmacy, University of Graz, Universitätsplatz 1, 8010 Graz, Austria Institute of Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13, 8010 Graz, Austria c

*Corresponding author: Amrit Paudel Research Center Pharmaceutical Engineering GmbH Inffeldgasse 13/II, 8010 Graz, Austria Email: [email protected] Phone: +43 316 873 30912 Fax: +43 316 873 1030912

1

Graphical abstract

Abstract As pulmonary drug delivery is extended from low doses to high doses, physicochemical characteristics of the active pharmaceutical ingredient gain importance in the development of dry powder inhalers. Therefore, the present work aims to understand the impact of distinct engineering techniques on the process induced physicochemical characteristics of salbutamol sulphate particles over time. The particle engineering techniques chosen were jet-milling and spray-drying, two well used processes in the production of predominately crystalline and amorphous inhalable particles, respectively. Fourier transform infrared spectroscopy, modulated differential scanning calorimetry, particle size distribution and tensiometry experiments were used to characterise the engineered powders immediately, 7, 14 and 21 days after production. The rugged spherical amorphous particles (3.75 ± 0.08 µm) obtained via spray-drying showed that they were capable of forming strong agglomerates (5.01 ± 0.22 µm) through “amorphous bridging”. On the other hand, jet-milling produced smaller (2.06 ± 0.08 µm), crystalline, irregular shaped particles with a very large surface area (11.04 ± 0.10 m2/g) that, over time, formed looser particle aggregates of decreasing size (3.76 ± 0.10 µm). Temporal evolution of the properties of spray-dried and jet milled particles showed a notable influence on the efficiency of blending with a model carrier at 0, 7 and 21 days (e.g. relative standard deviation of drug content of 11.3, 7.0 and 21.6%, respectively).

Keywords: dry powder inhaler (DPI), salbutamol sulphate, particle engineering, crystalline, amorphous, physicochemical properties, inter-particle distance, adhesive mixtures. 2

1 Introduction Dry powder inhaler (DPI) products are powder formulations used to deliver active pharmaceutical ingredients (API) via inhalation to the lung. In order to deposit in the lung, API particles are generally required to have an aerodynamic diameter of 1-5 µm. Particulate materials of such size pose a challenge in handling, processing and product performance, due to their poor flowability. Therefore, a common formulation strategy is the use of larger excipient carrier particles – usually sugars such as lactose or mannitol - in order to improve product performance (Pilcer et al., 2012). These dosage forms are called carrier based-DPI platforms and represent the majority of DPI products available on the market. Salbutamol sulphate, a short acting β2-agonist, is prescribed for the treatment of bronchospasm and COPD (Labiris and Dolovich, 2003). The use of salbutamol sulphate in inhalation studies in recent years makes it an ideal model API, as a plethora of physicochemical information can be found in existing data (Brodka-Pfeiffer et al., 2003; Grisedale et al., 2011; Littringer et al., 2013; Zellnitz et al., 2015). This study aims to complement the current knowledge, exploring the impact of secondary processing on physicochemical properties of salbutamol sulphate over time. In order to be formulated into a DPI product, salbutamol sulphate particles must be engineered to the inhalable size (secondary processing). Milling is currently the most common technique to generate particles in 1-5 µm size range and different milling techniques are available (Telko and Hickey, 2005). During milling, a mechanical force is applied to a solid material leading to its particle size reduction. It is reported that such a process can disrupt the crystalline phase, producing random domains of various molecular disorder, in particular, amorphous ones (Shur et al., 2013). Air jet milling is a well-established technique used in the manufacturing of inhalation products. In this case, an air jet stream is used, causing particle acceleration, impact, self-attrition, fracture and consequent size reduction (M. H. Shariare et al., 2011). Alternatively, spray-drying is a one step process also frequently applied in the pharmaceutical industry, where a solution or suspension is converted into a solid powder. In this process, a liquid is atomised into a gas stream, dried at elevated temperatures and the formed solid particles are then separated from the gaseous phase and recovered (Chow et al., 2007). The rapid solvent evaporation during the process enables the dynamic arrest of molecular motions of the API, below the glass transition temperature (Tg), originating an amorphous phase (Paudel et al., 2013). Even though the size of the generated particles is dependent on many factors, spraydrying has proven to be a suitable technique for the production of inhalable sized particles (Chawla et al., 1994; Littringer et al., 2013; Vehring, 2008). 3

Pharmaceutical molecules can exist in an assortment of solid-state phases – e.g. crystals, salts, cocrystals and amorphous form. Usually, in inhalation products API particles are used in their most stable crystalline form. Research on delivering APIs in their amorphous phase is mostly driven by the development of new oral dosage forms, but has also recently raised the interest of pulmonary drug delivery researchers, in particular in the delivery of biological molecules (Chen et al., 2015). Thus, amorphisation of therapeutic molecules may, on the one hand, represent an attractive drug development approach by improving solubility and bioavailability. On the other hand, it can be an unintended consequence of the manufacturing process and can cause a deleterious effect on product performance and stability (Priemel et al., 2015). Indeed, it is known that engineering techniques can lead to changes in the solid-state of materials such as the production of different crystal phases (Tantry et al., 2007; Vanhoorne et al., 2016) or loss of crystallinity (Della Bella et al., 2016; Hancock and Zografi, 1996). Concerning the impact of different engineering techniques Shur et al., (2013), compared two processes to obtain budesonide in the inhalable size, jet-milling and sono-crystallisation, finding that the micronised material possessed process-induced surface disorder, which was not observed for the sonocrystallised one. These surface disorders revealed to impact aersolisation, as the micronised material showed worse performance than the engineered one, when tested in lactose blends. Early on using salbutamol sulphate Ward and Schultz, 1995 found that jet-milling induced the formation of amorphous domains and hypothesized that this could be deleterious in the effectiveness of inhalation products. Recently Müller et al., (2015) confirmed this former findings, showing that over time storage (45% RH and ambient temperature) led to a decrease in the fine particle fraction (FPF) of salbutamol sulphate blends with lactose monohydrate when partially amorphous particles were used. No difference was found in salbutamol sulphate FPF over time when crystalline particles were investigated. In another study where salbutamol sulphate particles were tested without the presence of carrier particles, Shariare et al., (2011), found that the batch containing a higher percentage of amorphous content showed higher FPF. The authors attributed this to the marked cohesion between API particles and the resulting formation of larger-sized aggregates that could be fluidized. With the increasing interest in the shift from low API doses to high doses in pulmonary drug delivery, drug physicochemical characteristics will, undoubtedly, take on an increasing importance, when final quality attributes of the product are concerned (Hoppentocht et al., 2014). Studies comparing amorphous and crystalline salbutamol sulphate powders are rare and they predominately focus on the impact of environmental conditions on particles’ stability. By controlling the environmental conditions, keeping their influence to a minimum, the authors aim to investigate the possible effect and change of different process-inherited physicochemical 4

properties over time. Therefore, two distinct well used engineering techniques in inhalation, jetmilling and spray-drying, were chosen to produce crystalline and amorphous particles of salbutamol sulphate, respectively. The target specification for the manufactured particles was an inhalable size range between 1-5 µm. To further assess the impact that the distinct particle properties encountered might have in terms of product development, a standard blending procedure with a model carrier, mannitol (a sugar with low propensity to from crystal hydrates, known hemihydrate is formed only when the former is freeze-dried (Alqurshi et al., 2016)), was performed and the quality and homogeneity of the mixture tested at different time points and its respective outcomes were compared.

2 Materials and Methods

2.1 Materials The model API salbutamol sulphate was supplied by Selectchemie (Switzerland, purity 99.5%) and the model carrier material Pearlitol 160® (β-mannitol, purity 99.2%, by Roquette (France). Purified water (TKA Wasseraufbereitunssysteme GmbH, Germany), diiodomethane (99%, Alfa Aesar, USA), ethylene glycol (Emplura®, Merck Millipore, USA), 1- Hexanesulfonic acid sodium salt (≥98%, Sigma-Aldrich, USA), acetic acid (Emprove®, Merck Millipore, USA) and methanol (HPLC grade, Sigma-Aldrich, USA) were also used in the experiments during the present work. 2.2 Particle engineering An aqueous solution of salbutamol sulphate with the solid content of 7.5 % (w/w) was sprayed using an atomising intensity of 0.3 ml/min and an air flow rate of 110 l/min, into the long drying chamber of a B-90 Nano Spray Dryer (Büchi Labortechnick AG, Switzerland). To obtain particles in the inhalable size range, a spray head mesh of 7 µm, an inlet temperature of 120 °C and an outlet temperature of 49°C were used (Littringer et al, 2013). Jet-milling of salbutamol sulphate was performed in a 50 AS spiral jet mill (Hosokawa Alpine, Germany) with compressed air, using an injection pressure of 8.0 bar and a milling pressure of 5.0 bar. 2.3 Powder characterisation The produced powders were characterised immediately, and 7, 14 and 21 days after production. In order to minimise the impact of storage conditions, the engineered samples were stored in closed plastic vials in a low humidity environment (18 ± 2% RH) at room temperature (22 ± 2ºC), controlled using a Thermo-Hygrograph (Opus 10®, Lufft, Germany) (Grisedale et al., 2012). 5

2.3.1 Fourier transform infrared spectroscopy Spectroscopic analysis (n=3) was carried out using an ATR-FTIR (Vertex 70, Bruker, USA). Spectra were collected in between 4000-400 cm-1 range, at a resolution of 2 cm-1 and 64 scans (about 5 minutes per measurement). The analysis was carried out under ambient conditions (22 ± 2ºC, 61 ± 6% RH). Spectral analysis was performed using Opus® software version 6.5 (Bruker, USA) and a spectral environmental compensation algorithm was applied.

2.3.2 Modulated differential scanning calorimetry A sample of 2-4 mg (n=2) was accurately weighed (XP205, Mettler Toledo, USA) and placed into a hermetically sealed aluminium pan. Modulated differential scanning calorimetry analysis (204 F1 Phönix, Netzsch, Germany) was performed using a heating rate of 5 ºC/min from 25 ºC to 230 ºC and a modulation amplitude of ± 0.53 ºC every 40 seconds. Nitrogen was used as a purging gas and the sample chamber flushed at a flow rate of 50 ml/min. The equipment was calibrated using Indium. The results were analysed using Netzsch Proteus Thermal Analysis (Netzsch, Germany).

2.3.3 Particle size distribution Particle size distribution was evaluated using laser diffraction (HELOS/KR, Sympatec GmbH, Germany). The powder was placed on a vibrating chute (Vibri, Sympatec GmbH, Germany) and dispensed using a dry dispersing system (RODOS, Sympatec GmbH, Germany). A sampling time of 10 seconds (120 seconds real time) was applied and measurement with an R2 lens (0.45-87.5 µm) was triggered, once an optical concentration (Copt) of 0.5% was reached. Based on preliminary test results the primary dispersion pressure was manually adjusted in the range of 0.1-0.5 bar. Measurements were done in triplicate (n=3) at the lowest (0.1 bar) and highest (0.5 bar) pressures. Before each measurement the dispersing system was cleaned using sand (Dv0.5 ≥ 125 µm) and a reference measurement was taken. Particle size cumulative volume distribution (Q3) was calculated and analysed using Windox 5 software (Sympatec GmbH, Germany). Additionally, the mean particle diameter results (Dv0.5) obtained at the lowest and highest pressures were analysed using one-way ANOVA (Appendix A).

2.3.4 Specific surface area and porosity analysis Specific surface area measurements (n=2) were carried out with a TriStar II 3020 gas adsorption system (Micromeritics, USA). Prior to measuring, the samples were degassed under vacuum 6

(VacPrep 061, Micromeritics, USA), for at least 18, hours at room temperature (22 ± 2ºC). A multipoint (7 points) analysis was performed using a nitrogen relative pressure (p/p0) between 0.05-0.20. The Brunauer, Emmmett and Teller (BET) adsorption theory was used to calculate the specific surface areas of the samples. Porosity analysis was performed using the BarrettJoyner-Halenda method (BJH), in which a modification to the Kelvin equation relates the amount of adsorbate (nitrogen) removed during each pressure lowering step to the macro- and mesopore size distribution. In the former, 55 points (in a range 0.01-0.99 p/p0) of the nitrogen adsorption isotherm at 77.350 K were used.

2.3.5 Tensiometry The contact angles of the different powder samples (n=3) were measured by means of the Wilhelmy plate technique, using a Tensiometer K100 (Krüss GmbH, Germany). A rectangular shaped substrate, coated with double-side tape (20x20 mm) was used to fix the powders onto its surface. It was ensured that the tape was uniformly coated with powder and no tape was exposed. The solid sample was hung perpendicular to the liquid surface. Three different liquids were used; two polar (water and ethylene glycol) and one apolar (diiodomethane). Each liquid was placed in a clean glass dish and raised by means of a motorised platform to contact the powder plate. The platform was raised at the speed of 6 mm/min and the immersion distance was 2 mm. The volume of liquid used for the contact-angle measurements was ca. 50 ml. The contact angle was calculated from the measured force by transposing the Wilhelmy equation using Krüss Laboratory Desktop (Krüss GmbH, Germany). Contact angle results were analysed using one-way ANOVA (Appendix A) and were then used to determine the surface energy components by the Good and van Oss equation (Van Oss et al., 1988).

2.4 Adhesive mixtures Adhesive mixtures (n=3) using the engineered APIs (spray-dried and jet-milled) were prepared immediately, and 7 and 21 days after production. For this, 0.2 g of salbutamol sulphate and 9.8 g of mannitol were weighed (API concentration of 2% w/w). Using a stainless steel vessel the API was placed between two even layers of carrier material (sandwich method). The mixtures were then blended in a Turbula blender TC2 (Willy A. Bachofen Maschinenfabrik, Switzerland) for 60 minutes at 62 rpm (Littringer et al, 2013). 2.4.1 Mixing homogeneity Blend homogeneity was evaluated by randomly sampling ten samples of 45.0 ± 0.3 mg from the powder bed. The acquired samples were assayed for the drug content using a validated HPLC 7

method (Faulhammer et al., 2015). The homogeneity of the mixtures was expressed by the relative standard deviation (RSD) of drug content. 2.4.2 Scanning electron microscopy (SEM) Salbutamol sulphate and its blends were examined using a scanning electron microscope (Zeiss Ultra 55, Zeiss, Germany) operating at 5 kV. The particles had been sputtered with gold– palladium prior to analysis. Different magnifications were used to observe the blending homogeneity of the various samples.

3 Results

3.1 Solid-state characterization of the processed salbutamol sulphate

3.1.1 Fourier transform infrared spectroscopy Infrared spectroscopic analysis was carried out in order to understand if any chemical changes were induced during manufacturing. Fourier transform infrared (FTIR) spectra (Figure 1) of the raw material matched the spectra of salbutamol sulphate crystalline phase found in literature (Grisedale et al., 2013). FTIR analysis of the spray-dried material (Figure 1 A) showed an overall broadening and downward shift of peak patterns, particularly in the 3600-3100 cm-1 (Grisedale et al., 2013) and 1320-1000 cm-1 regions. This is characteristic for disordered solid materials, where the broader distribution of bond lengths and energies results in these kind of patterns (Kaushal et al., 2008). Analysis of the milled samples did not show any detectable spectral changes, when compared to the raw material (Figure 1 B). Finally, the peak patterns of both engineered materials remained the same from 0 until 21 days’ time, indicating that no chemical changes occurred over time.

3.1.2 Thermal analysis Modulated differential scanning calorimetry (mDSC) thermograms of the spray dried particles are presented in Figure 2. The glass transition regions (Figure 2 A) of the samples at different time points are presented as the overlay of reversing heat flow, as well as the first derivative of reversing heat flow, with respect to the temperature. The latter is presented for the clearer visualisation of Tg distributions. Immediately after production (0 day), spray dried salbutamol sulphate depicted two glass transition events (Tg); the first one at 46.8 ± 4.1 ºC and the second, smaller one, at 69.8 ± 4.6ºC. The derivative signal suggests that the amorphous fraction with 8

lower Tg was the predominant fraction, compared to the fraction with the higher Tg. After 7 days, the first Tg (51.8 ± 0.5 ºC) appeared to be smaller, while the second one (57.2 ± 0.4ºC) was more distinct. After 14 days, both events appeared at slightly higher temperatures and approximately 6°C apart. In addition, and contrary to previous time points, the first Tg seemed more prominent than the second one (derivative signal). After 21 days, both events appeared at the identical region as observed at 14 days. However, the derivative curve suggested that both amorphous fractions representing these two Tgs were present in an almost equivalent extent. Grisedale et al. (2012) were the first authors to report this phenomenon in salbutamol sulphate powders, suggesting that the Tg appearing at higher temperatures was a reflection of the particles’ dryer surface, with respect to the more hydrated bulk. Detection of the hydrated bulk glass transition at 46.8 ± 4.1 ºC (after production) indicated that the material was plasticized (see Appendix A for samples water content). Moreover and based on evidence reported by Grisedale et al., (2012) on the slight decrease of water content, temporal evolution of both glass transition events (increase of both Tg from 7 to 21 days) apparently indicated that water molecules diffuse from within particle’s bulk to its surface, where evaporation rate can, eventually, be accelerated. However, a closer look at the changes observed between 0 and 7 days revealed a significant decrease on the onset temperature of the surface Tg (by 12ºC) in contrast with almost unaltered bulk Tg (46.8 ± 4.1 ºC and 51.8 ± 0.5 ºC, at 0 and 7 days respectively). Thus, “amorphous bridging” is proposed to be triggered by a gradual diffusion of water molecules from within particle’s bulk to its surface and is interpreted as the governing mechanism behind the observed, over time, particle behaviour (see section 4.1). For the spraydried samples it was also possible to observe a non-isothermal crystallisation event (Figure 2 B), further supporting the amorphous nature of these materials. Interestingly, compared to the initial sample (0 days), the crystallisation exotherm first shifted towards higher temperatures and at 21 days towards lower temperatures providing evidence of the temporal evolution of the amorphous structure. Concerning the milled samples (Figure 2 C), a small Tg was observed at all time points, including the starting material (0 days). Evidently, this indicated that traces of the amorphous fraction were generated via milling. Furthermore, the variability on the extent of the detected Tg in the duplicates translated the heterogeneity of aforementioned trace amount of amorphous fraction within the predominant crystalline matrix. This reflects the random nature of mechanical activated sites of amorphous domains generated during milling (M. H. Shariare et al., 2011).

3.2 Micromeritics and surface characterization of the processed salbutamol sulphate 9

3.2.1 Particle size distribution by pressure titration When developing a method for particle size analysis by laser diffraction, one has to ensure the complete de-agglomeration of the particles without inducing fracture of the material. It is recommended, by ISO 13320, to perform pressure titration experiments. During these experiments, the particle size is measured with constantly increasing pressure, in order to determine the pressure that is required to completely de-agglomerate the powder without particle destruction or abrasion. Additionally, the comparison of the changes in the measured particle size distributions, under different dispersion pressures, can provide an indication about strength and degree of de-agglomeration of respirable sized powder aggregates (Jaffari et al., 2013). In the present context, particle size distribution (PSD) characterisation was carried out to evaluate if the PSD and the force necessary to de-agglomerate the powders remained the same through analysis time. Directly after spray-drying (Table 1), at a very low dispersion pressure of 0.1 bar, the mean particle size (Dv0.5) measured was 4.19 ± 0.09 µm. When the pressure was increased to 0.2 and 0.3 bar a marginal decrease in mean particle size was observed (Figure 3 A). The use of 0.4 bar and 0.5 bar (Dv0.5 = 3.74 ± 0.05 µm) led to identical mean particle sizes (Figure 3 A). After 7 days a pressure of 0.1 bar resulted in a mean particle size slightly larger than at 0 days (Dv0.5 = 5.19 ± 0.54 µm). However, when a pressure of 0.2 bar was used a notable decrease in mean particle size was observed (Figure 3 A). Further increase in dispersion pressure (0.3, 0.4 and 0.5 bar) resulted in somewhat smaller mean particle sizes similar to time 0 when the same pressures were applied. Interestingly, after 14 days (Table 1) at 0.1 bar, a marginally lower mean particle size was obtained (Dv0.5= 4.32 ± 0.03 µm) and at a pressure range from 0.2 to 0.5 bar, a similar Dv0.5 to that of 0 and 7 days was found (Figure 3 A). Finally, after 21 days (Table 1), the mean particle size marginally increased again when measured at 0.1 bar (Dv0.5= 5.01 ± 0.22 µm). This time, only when a pressure of 0.4 and 0.5 bar was applied did one a find similar Dv0.5 to the previous time points (Figure 3 A). For the milled materials immediately after production (Table 2) a mean particle size of 6.87 ± 1.03 µm was measured when applying a pressure of 0.1 bar. By solely increasing the pressure to 0.2 bar, it was possible to obtain a considerably smaller mean particle size (Figure 3 B), indicating that the sample probably consisted of very loose aggregates which could be easily dispersed. When further increasing the pressure to 0.3, 0.4 and 0.5 bar, further small decreases in PSD were found (Figure 3 B), possibly due to some particle abrasion caused by such high dispersion pressures. After 7 days (Table 2), a similar mean particle size to time 0 could be observed at 0.1 bar dispersion pressure (Dv0.5 = 6.11 ± 0.52 µm). As already observed for time 0 samples, a pressure of 0.2 bar was enough to decrease the mean particle size by half (Figure 3 10

B). Pressures of 0.3, 0.4 and 0.5 bar appeared to cause material erosion and further particle size reduction. After 14 days (Table 2), at 0.1 bar, the mean particle size decreased significantly (Dv0.5 = 4.52 ± 0.12 µm) and when increasing the pressure a similar behaviour as for the previous time points was observed (Figure 3 B). Finally, after 21 days (Table 2), at 0.1 bar the mean particle size slightly decreased, once again (Dv0.5 = 3.76 ± 0.10 µm). However, in contrast to other time points, when a pressure of 0.2 bar was applied the PSD only marginally decreased and only when 0.3, 0.4 and 0.5 bar were applied could a similar Dv0.5 be found (Figure 3 B). 3.2.2 Specific surface area and porosity analysis Specific surface area analysis showed (Table 3), a notable decrease in the specific surface area (SSA) (1.377 ± 0.016 m2/g) for the spray-dried powder after 7 days when compared to the initial value after production (1.565 ± 0.029 m2/g). In turn, Barrett-Joyner-Halenda (BJH) porosity results showed an increase in pore size. After 14 days, the SSA increased and the pore size became smaller. Finally, after 21 days, the SSA decreased, but the pore size increased once again. For the milled materials from production until day 7, it was possible to see an increase in SSA and pore size. However, after 14 and 21 days this tendency reverted and a decrease in SSA and pore size was found. The authors are well aware that the limited number of samples used (n=2) is not large enough to evaluate the significance of the obtained results. Notwithstanding, the primary aim of the undertaken characterization was to evaluate the samples’ trends in solid-solid interfacial area over time. 3.2.3 Surface free energy In comparison to other methods such inverse gas chromatography (IGC), the Wilhelmy plate technique has not been so widely applied to evaluate the surface free energy (SFE) of pharmaceutical powder samples. Notwithstanding, Dove et al., (1996) found a good agreement between the data obtained from both the IGC and Wihelmy plate methodologies. In fact, the total free energy values found for the milled salbutamol sulphate at initial time point in the current study (time 0) are similar to the values described by Du et al., (2017) using IGC. However, in the developed study the objective was to identify the different trends in the SFE of the powders over time. Tensiometry measurements (Table 4) of the spray-dried samples revealed that until 7 days after production, the total surface free energy of the solid’s slightly decreased (from 58.63 ± 8.81 mN/m to 40.91 ± 2.92 mN/m) due to the decrease of both the non-polar and polar components. However, after 14 days the total free energy increased again (48.53 ± 8.05 mN/m), particularly, the polar component. After 21 days, this tendency was maintained and the total surface free energy was similar to that found at time 0 (57.68 ± 8.44 mN/m). A similar trend could be 11

observed for the milled material. The solid’s total free energy significantly decreased (from 67.42 ± 5.84 mN/m to 35.00 ± 1.98 mN/m), due to the decrease of both non-polar and polar components 7 days after production. After 14 days, the total free energy increased again (62.48 ± 7.53 mN/m). However, after 21 days, the surface free energy decreased slightly (56.64 ± 4.89 mN/m), once again, mostly due to the polar component.

3.3 Blending

3.3.1 Mixing homogeneity As shown in Table 5, it was not possible to achieve a homogenous mixture (RSD ≤ 5%) for any of the materials at any of the tested time points as indicated by relatively high standard deviations encountered. Concerning the results for the spray-dried powders, it was interesting to note that immediately after production, double the concentration of the targeted salbutamol sulphate content (4% w/w) was found. Regarding the mixing homogeneity, after 7 days, the lowest RSD and the best mixing behaviour was achieved (7% w/w). The RSD of 21.6% (w/w) after 21 days reflected that very heterogeneous blends were produced. Overall inspection of the milled blends results show that very heterogeneous samples were produced with RSD of 35.6% (w/w) and 27.2% (w/w) at both 0 and 7 days, respectively. After 21 days, a more homogenous blend was achieved (13.6% w/w RSD) however these results were still far from that required for a homogenous mixture. 3.3.2 Scanning electron microscopy observations Before being blended with the carrier, API particles were studied using scanning electron microscopy (SEM). Investigation of salbutamol sulphate’s SEM pictures showed that spray-dried spherical particles had a slightly rugged surface (Figure 4). Pictures of time 0 particles of the spray-dried powders revealed that particles were fairly scattered and more distanced from each other (Figure 4 A1 and B1). At 7 and 21 days it was apparent that particles are positioned closer together forming evident particulate clusters (Figure 4 A2-3 and B2-3). Regarding the evaluation of milled salbutamol sulphate’s SEM pictures it showed that jet-milling resulted in very rough irregular particles (Figure 5). Moreover, it was possible to see that at time 0 and 7 days particulate aggregates of variable sizes were present and sparsely scattered throughout the sample field (Figure 5 A1-2 and B1-2). After 21 days, a more homogenous sample of denser particulate aggregates was observed (Figure 5 A3 and B3). Scanning electron microscopy 12

pictures of the spray-dried blends show that at time 0 (Figure 6 A1) a larger amount of API could be observed attached to the carrier particles, compared to day 7 and day 21 (Figure 6 A23). Furthermore, it was possible to observe, after 21 days (Figure 6 A3), that some of the salbutamol sulphate particles “fused” together, also with the carrier. Indeed, the same phenomena had already been observed by the authors in their previous work using amorphous salbutamol sulphate (Faulhammer et al., 2015) and Young et al., (2007) for milled α-lactose monohydrate particles. Concerning the milled blends, SEM images indicated that after 21 days (Figure 6 B3) the carrier was more homogenously covered with API particles compared to earlier time points (Figure 6 B1-2).

4 Discussion 4.1 Spray-dried materials Spray-drying of salbutamol sulphate using process parameters previously described in literature (Littringer et al., 2013) resulted in the production of chemically pure (see Annex A) surface rugged amorphous spherical particles with a mean size of 3.7 ± 0.1 µm. The amorphous nature of the produced materials was confirmed, throughout the study (0 to 21 days), by the characteristic broadening, downward shift of the characteristic vibrational bands in FTIR spectral patterns and the glass transition temperature and exothermic events present in mDSC thermograms. Additional analysis, by X-ray scattering at time 0, further showed that the powder was completely X-ray amorphous (results not shown). Furthermore, the existence of two glass transitions indicates the presence of regions of distinct molecular mobility within the particles. As already mentioned in section 3.1.2, the Tg appearing at higher temperatures was a reflection of the dryer surface of the particle with respect to its more hydrated bulk fraction (Grisedale et al., 2012). After 7 days, overall results seem to indicate that particles approach each other, resulting in a slight larger mean particle size. It is hypothesized that this decrease in inter-particle distance led to the disappearance of some of the smaller mesoporosities present at the rugged surface of the single particles and pores of a larger size are formed between neighbouring particles as they come closer together resulting in a transient decrease of the specific surface area. Considering the large surface area of micronised powders and their tendency to form aggregates, these results were not surprising. The tendency of fine powders to form aggregates is driven by the particles urge to near each other in order to lower their surface area. This, in turn, is advantageous in terms of surface energy as energy is released (Podczeck, 1998). Indeed, this was confirmed by the surface free energy results after 7 days. It was also interesting to observe that at this time point, the second glass transition appeared at a lower temperature. An explanation for this phenomenon could be the possible formation of “amorphous bridges” 13

between the particle’s surfaces, as described by Hartman and Palzer for milk powder (Hartmann and Palzer, 2011). Diffusion of water molecules from particle’s bulk to its surface (plasticization), combined with the intrinsically higher molecular motion ability of surface amorphous fraction (compared to bulk), create the ideal environment for “amorphous bridging” to occur (Brian and Yu, 2013). Therefore, when two particles come into contact their surface molecules will diffuse towards each other creating bridges of a more mobile material than the initial, well defined dryer surface crust. After 14 days, particles continue to move closer together resulting in a slight decrease in mean particle size when almost no dispersion pressure (0.1 bar) was applied. At this time point the decrease of inter-particle resulted in pores of a smaller size (decrease of the pore size) that lead to an increase of the specific surface area. On the one hand, the decrease in inter-particle distance is, to a certain point, energetically beneficial. On the other hand, it leads to an increase of contact points between particles and a consequent increase in Lifshitz-van der Waals (non-polar) and dipole-dipole (polar) interactions (Köhler and Schubert, 1991) as shown by the 14 day surface free energy results. Moreover, mDSC results showed that, at this time, the first glass transition event is the more prominent one, indicating a transient phase of rearrangement between the molecules within the particle’s bulk and in the “amorphous bridge”. After 21 days, thickening and lengthening of the “amorphous bridging” leads to the appearance of two identical glass transition events. At this point the former leads to the disappearance of some of the smaller open pores (Hartmann and Palzer, 2011), with a consequent decrease in specific surface area and an increase in pore size. Concerning surface free energy results, a further decrease in particle distance resulted in an increase of the solid’s total free surface energy, mainly due to the polar component, as the non-polar component interactions actually decreased. The thickening and lengthening of the “amorphous bridges” in combination with the increase of the polar component of the SFE seem to have led to an increase in agglomerates strength, as shown by pressure titration. Interestingly, when 0.1 bar is applied PSD analysis showed an increase (although not significant) in mean particle size; which at first may seem contradicting considering the decreasing inter-particle distance observed. However, a possible explanation therefore might be that 0.1 bar was incapable of dispersing the particles compared to previous time points. 4.2 Milled materials Jet-milling of salbutamol sulphate powders generated very irregular shaped rough particles, with a mean particle size of about 2 µm. FTIR patterns are comparable to the pattern of the raw material, indicating that the produced material is predominately crystalline. However, the glass transition events detected by mDSC suggest that some disordered (amorphous) domains might 14

have been produced during milling (Ward and Schultz, 1995). Furthermore, mechanical impaction during milling led to smaller, irregular shaped particles compared to their spray-dried spherical counterparts. Milled particles also present a larger area to volume ratio and consequently a considerably larger specific surface area. The former possibly explaining why higher surface free energy values were found for the crystalline material. Indeed, after 7 days results showed that due to their need to decrease their exposed surface area particles approach each other forming particulate aggregates. However, and in contrast to what was observed for the spray-dried spherical particles, the formation of new slightly larger pores between neighbouring particles resulted, in this case, in a transient increase of the specific surface area at 7 days. It was also apparent that a decrease in inter-particle distance of large surfaced area particles was enough to cause a considerable energy release, resulting in a significant decrease of the solid’s total free surface energy. However, the former had no impact on the aggregate’s strength, as shown by pressure titration. After 14 days, particles came even closer together and a significant decrease in aggregate size was observed via PSD analysis. As previously explained, for the spray-dried powders and also for the jet milled powders, the increased particle contact points give rise to re-established Lishitz-van der Waals (non-polar) and dipole-dipole (polar) interactions. This is reflected by the 14 day surface free energy results. Once again, no correlation could be observed with the pressure titration results, where 0.2 bar, such as at time 0, appears to be enough to break the formed aggregates. After 14 days, as particles continue to approach each other, the newly formed mesoporosities observed after 7 days become slightly smaller and fewer, resulting in a decrease of the specific surface area. After 21 days, SEM and decrease of the mean particle size, pore volume and size indicate further reduction of the interparticle distance. Although, here, contrary to what was observed for the spray-dried powders, the former seems to lead to a release of energy, resulting in a slight lowering of the non-polar and polar components of the surface free energy. Interestingly, and as seen for previous time points, no correlation was found with the pressure titration results, where in turn an increase of aggregates’ strength was observed. The latter, might be due to tribo-electrification, which plays a very important role in particle-particle interactions and cannot be excluded (Naik et al., 2016), or to surface diffusivity of the produced disorder domains which, as seen in the previous section (4.1), can significantly impact particles’ interplay. The authors are currently undertaking further in depth investigation on this topic and the results will be disclosed in a future communication. 4.3 Adhesive mixtures The concept of adhesive mixing describes a practice where fine particles, usually below 10 µm, adhere to the surface of larger ones, forming a homogenous powder bed that is resistant to 15

segregation (Mangal et al., 2016). Considering the intrinsic differences found in the properties of the API particles originating from different preparation routes, the aim of the present work was not to develop a blending process that allowed the production of a homogenous mixture. Instead it was the objective of the current work to emphasize how distinct process specific particle properties might propagate to blending, as a downstream process. Therefore, a standard blending process using model carrier particles (mannitol) was tested. Consequently, it was not so surprising to see that the mixing process did not yield a homogenous mixture (RSD ≤ 5%) for any of the materials in any of tested time points. However significant differences concerning drug content were found at the different time points and are worthy of discussion. Powder mixing is known to be governed by four main mechanisms: random mixing followed by de-agglomeration and adhesion, and finally redistribution and compression. These steps are mainly driven by the size and strength of particles’ aggregates. Particulate aggregates more strongly bonded together and/or of larger size need more energy to be broken, so longer mixing time or higher speeds may be needed, when compared to their looser bonded, smaller counterparts (Nguyen et al., 2015). Immediately after production, blends of the spray-dried material and mannitol were shown to have double the targeted drug content. A possible hypothesis for this is that directly after production, blending of salbutamol sulphate led to a higher accumulation of charges. Thus, when transferring from the mixing to the storage vessel, the more charged mannitol particles with higher aspect ratio, (Karner et al., 2014) remain stuck to vessel walls. In turn, mannitol particles with salbutamol sulphate attached to their surface will flow better when in contact with the vessel wall, as the spherical API particles will have a higher capacity to absorb charges (Naik et al., 2016). This may result in a higher API to carrier ratio in the storage vessel that initially intended. Nevertheless, further research needs to be conducted regarding this topic. After 7 days, a better mixing homogeneity was achieved. The decrease in surface free energy and possible tribo-electric phenomena at this time point might have beneficially impacted mixing, explaining why a lower RSD was found. After 21 days, the most heterogeneous blend of all the time points was obtained. This was not surprising, if one considers that at this time point strong API particle agglomerates are formed through “amorphous bridging”. SEM pictures of the blend at this time clearly show API particles “fused” to the carrier and to each other, although in the SEM results for the drug alone such phenomenon is not clearly identifiable. Even though tumble blending is a low-energy input process it is possible that at 21 days the energy generated was enough to accelerated “amorphous bridging” kinetics enabling its clear visualisation under SEM (Bridson et al., 2007). Regarding the milled powders, a continual improvement in mixing homogeneity was observed through time. The latter correlates well with overall results, where it was possible to observe that over time particles come closer and closer together forming 16

aggregates of smaller size. Consequently, smaller sized aggregates need less energy to be broken explaining why they were more easily de-aggregated during the mixing process. It is clear that predominately amorphous and crystalline particles affect the resulting mixing homogeneity in contrasting ways. Contrary to the crystalline milled material, where a clear correlation between particulate aggregates size and mixing homogeneity was found, this could not be observed for the amorphous spray-dried material. This indicates that more dynamic aspects are involved for the spray-dried particles, e.g. “amorphous bridging”.

5 Conclusion In the present work, some characteristic differences were found in the physicochemical properties of salbutamol sulphate particles engineered to the inhalable size, using two distinct processing techniques. As expected spray-drying of salbutamol sulphate powders resulted in the production of rugged, spherical amorphous particles and jet-milling in predominately crystalline irregular shaped particles. The time-dependent particle characterization showed that amorphous particles were capable of forming strong agglomerates through “amorphous bridging”. In contrast, the predominately crystalline milled particles with their very large surface area formed looser particle aggregates with decreasing size over time. Further, the resulting difference in the properties demonstrated to have an impact on powder blending at the different time points. Therefore, knowledge of the temporal evolution of particle’s solid-state and surface properties, related to its processing technique, is of utmost importance in order to develop a successful, stable product. In conclusion, this work offers a glance at how API physicochemical properties emerging from (different) particle engineering can impact certain processes like blending in the development of DPI products. In future studies, it will be important to understand when or how particles’ dynamic states through time are stabilized and, if once a homogenous mixture is achieved, the encountered differences can impact the aerosolisation performance of salbutamol sulphate powders. Acknowledgements This work was funded through the Austrian COMET Program by the Austrian Federal Ministry of Transport, Innovation and Technology (BMVIT), the Austrian Federal Ministry of Economy, Family and Youth (BMWFJ) and by the State of Styria (Styrian Funding Agency SFG). The authors would also like to thank Hartmuth Schroettner at FELMI-ZFE - Austrian Centre for Electron Microscopy for his assistance in the scanning electron microscopy measurements. 17

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Figure Captions Figure 1 FTIR mean spectra of salbutamol sulphate raw material and engineered powders by A) spray-drying and B) milling at different time points (0, 7, 14 and 21 days after production). Figure 2 Salbutamol sulphate powders mDSC results through analysis time (mean ± range). A) spray-dried salbutamol sulphate glass transition region, B) spray-dried salbutamol sulphate crystallisation (exothermic) region and C) milled salbutamol sulphate glass transition region. Figure 3 Pressure titration trends observed through analysis time. A) spray-dried powders and B) milled powders. Triplicates were carried out at the lowest (0.1 bar) and highest (0.5 bar) dispersion pressures (mean ± standard deviation). 20

Figure 4 SEM results for the salbutamol sulphate spray-dried. Panel A) 571.6 µm width and B) 114.3 µm width at 1) 0 days, 2) 7 days, 3) 21 days. Figure 5 SEM results for the salbutamol sulphate milled. Panel A) 238.7 µm width and B) 57.16 µm width at 1) 0 days, 2) 7 days, 3) 21 days. Figure 6 SEM results for the salbutamol sulphate blends (114.3 µm width). Panel A) spray-dried powder and B) milled powder at 1) 0 days, 2) 7 days, 3) 21 days (arrows indicate locations where spray-dried salbutamol sulphate particles apparently “fused” to each other and to the carrier).

21

Figure 1

22

Figure 2

23

Figure 3

24

Figure 4

25

Figure 5

26

Figure 6

27

Table 1 Particle size cumulative volume distribution of the spray-dried powders at primary dispersion pressure of 0.1 and 0.5 bar (mean ± standard deviation).

Sample 0.1 bar

Particle size distribution (Q3) Dv0.1 (µm)

Dv0.5 (µm)

Dv0.9 (µm)

0 days

0.67 ± 0.03

4.19 ± 0.09

8.14 ± 0.23

7 days

1.04 ± 0.35

5.19 ± 0.54

10.24 ± 1.94

14 days

0.76 ± 0.01

4.32 ± 0.03

8.16 ± 0.04

21 days

1.08 ± 0.19

5.01 ± 0.22

10.07 ± 0.72

0.5 bar

Dv0.1 (µm)

Dv0.5 (µm)

Dv0.9 (µm)

0 days

0.63 ± 0.02

3.74 ± 0.05

7.61 ± 0.06

7 days

0.58 ± 0.02

3.74 ± 0.04

7.50 ± 0.06

14 days

0.63 ± 0.01

3.75 ± 0.01

7.41 ± 0.07

21 days

0.62 ± 0.02

3.75 ± 0.08

7.55 ± 0.07

28

Table 2 Particle size cumulative volume distribution of the milled powders at primary dispersion pressure of 0.1 and 0.5 bar (mean ± standard deviation).

Sample

Particle size distribution (Q3)

0.1 bar

Dv0.1 (µm)

Dv0.5 (µm)

Dv0.9 (µm)

0 days

0.97 ± 0.08

6.87 ± 1.03

25.40 ± 2.99

7 days

0.92 ± 0.02

6.11 ± 0.52

23.68 ± 0.74

14 days

0.84 ± 0.01

4.52 ± 0.12

18.52 ± 1.33

21 days

0.78 ± 0.03

3.76 ± 0.10

15.39 ± 1.09

0.5 bar

Dv0.1 (µm)

Dv0.5 (µm)

Dv0.9 (µm)

0 days

0.52 ± 0.02

2.16 ± 0.08

6.02 ± 0.39

7 days

0.53 ± 0.02

2.14 ± 0.06

6.12 ± 0.38

14 days

0.53 ± 0.04

2.07 ± 0.04

5.88 ± 0.22

21 days

0.53 ± 0.05

2.06 ± 0.06

6.05 ± 0.25

29

Table 3 Spray-dried and milled powders BET specific surface area and BHJ porosity analysis results (mean ± range).

Sample

BET surface area and BHJ porosity

Spray-dried

Specific surface area (m2/g)

Pore size (nm)

0 days

1.57 ± 0.03

7.69 ± 0.48

7 days

1.38 ± 0.02

9.30 ± 0.31

14 days

1.77 ± 0.01

6.90 ± 0.17

21 days

1.57 ± 0.03

7.93 ± 0.04

Milled

Specific surface area (m2/g)

Pore size (nm)

0 days

11.03 ± 0.20

12.11 ± 0.20

7 days

11.66 ± 0.07

13.80 ± 0.12

14 days

11.25 ± 0.06

12.81 ± 1.64

21 days

11.04 ± 0.10

12.56 ± 0.63

30

Table 4 Spray-dried and milled powders surface free energy calculations results (mean ± standard deviation).

Sample

Surface free energy

Spray-dried

γTOT (mN/m)

γLW (mN/m)

γAB (mN/m)

0 days

58.63 ± 8.81

40.40 ± 2.21

18.23 ± 8.35

7 days

40.91 ± 2.92

34.14 ± 1.22

6.78 ± 3.08

14 days

48.53 ± 8.05

38.24 ± 1.75

10.30 ± 7.49

21 days

57.86 ± 8.44

36.94 ± 1.33

20.91 ± 8.12

Milled

γTOT (mN/m)

γLW (mN/m)

γAB (mN/m)

0 days

67.42 ± 5.84

43.10 ± 1.77

24.32 ± 5.22

7 days

35.00 ± 1.98

31.26 ± 0.93

3.74 ± 1.48

14 days

62.48 ± 7.53

43.10 ± 0.35

19.38 ± 7.51

21 days

56.64 ± 4.89

41.48 ± 1.56

15.16 ± 4.35

γTOT: Solid’s total surface free energy (γLW + γAB) γLW: Solid’s non polar component of the surface free energy γAB: Solid’s polar component of the surface free energy

31

Table 5 Mixing homogeneity results for the spray-dried and milled blends with mannitol (mean ± standard deviation).

Sample

Mixing homogeneity

Spray-dried blends

0 days

7 days

21 days

4.1 ± 0.5

1.8 ± 0.1

1.7 ± 0.4

RSD (%)

11.3

7.0

21.6

Milled blends

0 days

7 days

21 days

1.7 ± 0.6

1.8 ± 0.5

2.3 ± 0.3

35.6

27.2

13.6

Salbutamol sulphate concentration (w/w %)

Salbutamol sulphate concentration (w/w %) RSD (%)

32