Influence of lactose carrier particle size on the aerosol performance of budesonide from a dry powder inhaler

Powder Technology 227 (2012) 74–85

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Influence of lactose carrier particle size on the aerosol performance of budesonide from a dry powder inhaler Waseem Kaialy a, b, c,⁎, Amjad Alhalaweh b, Sitaram P. Velaga b, Ali Nokhodchi a, d,⁎ a

Chemistry and Drug Delivery Group, Medway School of Pharmacy, University of Kent, ME4 4TB, Kent, UK Pharmaceutical engineering group, Department of Health Sciences, Luleå University of Technology, Lulea S-971 87, Sweden Pharmaceutics and Pharmaceutical Technology Department, University of Damascus, Syria d Drug Applied Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran b c

a r t i c l e

i n f o

Available online 10 March 2012 Keywords: Carrier size Density Flowability Roughness Adhesion Homogeneity

a b s t r a c t The purpose of this study was to evaluate the effect of carrier particle size on properties of dry powder and its effect on dry powder inhaler (DPI) performance. Commercial α-lactose-monohydrate, a commonly used carrier in DPI formulations, was carefully sieved to obtain different lactose size fractions, namely Lac A (90–125 μm), Lac B (63–90 μm), Lac C (45–63 μm), Lac D (20–45 μm), and Lac E (b20 μm). The lactose samples were analysed in terms of size, shape, solid state, density, and flowability. Lactose particles were blended with budesonide (b 5 μm) powder to generate five different formulations. These formulations were then evaluated in terms of budesonide–lactose adhesion properties, drug content homogeneity, and in vitro aerosolisation performance. The results demonstrated that lactose samples with smaller particle volume mean diameter have higher amorphous lactose content, higher true density (linear, r2 = 0.9932), higher surface smoothness (linear, r2 = 0.8752), smaller angularity (linear, r2 = 0.921), smaller bulk density, higher porosity (linear, r2 = 0.914), poorer flowability, and higher specific surface area. In general, the smaller the lactose particles the smaller are the budesonide–lactose adhesion properties. Budesonide formulated with smaller lactose particles exhibited smaller aerodynamic diameter and higher amounts of budesonide were delivered to lower stages of the impactor indicating improved DPI aerosolisation performance. However, the use of lactose particles with smaller volume mean diameter had a detrimental effect on budesonide content homogeneity and caused an increase in the amounts of budesonide deposited on oropharyngeal region. Therefore, particle size of the lactose within dry powder inhaler formulations should be selected carefully. Accordingly, higher drug aerosolisation efficiency of lactose particles with smaller size may have to be balanced due to considerations of other disadvantages including poorer flowability, reduced formulation stability, higher potential side effects, and higher dose variability. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Dry powder inhalers (DPIs) are the result of the development of powder and device technologies. In comparison to metered dose inhalers (MDIs), DPIs can be considered, in some cases, more efficient and capable to deliver higher doses of drugs to the lungs [1]. This is because of DPIs are usually activated by inspiratory air flow and thus require little or no coordination between actuation and inhalation (automatic coordination). In drug–carrier DPI formulation development, two different kinds of interaction forces should be taken into account: the cohesive (drug–drug) forces and the adhesive (drug– carrier) forces. Drug–carrier adhesive forces are fundamental

⁎ Corresponding authors at: Chemistry and Drug Delivery Group, Medway School of Pharmacy, University of Kent, ME4 4TB, Kent, UK. Tel.: + 44 1634 202947; fax: + 44 1634 883927. E-mail addresses: [email protected] (W. Kaialy), [email protected] (A. Nokhodchi).

determinants of DPI performance, as excessive drug–carrier adhesive forces limit drug–carrier detachment during aerosolisation leading to poor drug re-dispersion properties [2,3]. For assessing particle adhesion properties in DPI formulations, different techniques could be employed such as colloidal probe technique using Atomic Force Microscope (AFM) [4]. However, AFM characterization is limited to a single or at most only few particles. Indeed, techniques like mechanical and air jet sieving [3,5], centrifugation [6], and impact separation [7], are capable to assess the whole powder interparticulate forces within the adhesive mixtures. Generally, the adhesion of a micrometre-size particle and a solid surface is a result of several physical forces such as Van der Waals forces, electrostatic forces, and capillary forces [8]. The contribution of each one of these forces to the overall particle adhesion is dependent on several factors such as particle surface chemistry, particle contact area, environmental conditions [9], particle mechanical properties [10], particle surface physical properties [3,5,11], solid-state nature, and particle size [12,13].

0032-5910/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2012.03.006

W. Kaialy et al. / Powder Technology 227 (2012) 74–85

In drug–carrier DPI formulations, the carrier particles form a fundamental component due to low contribution/content of drug within formulation. Therefore, a slight change in carrier physicochemical properties will have considerable effect on the drug aerosolisation behaviour. Size reduction of drug particles, to a certain extent (1–5 μm), enhances the performance of DPI formulation [13,14], however, little attention was given to the effect of carrier particle size as one variable on the DPI performance. In fact, available reports on the effect of carrier particle size on the drug delivery from DPIs are contradictory or inconclusive [15,16]. Some reports demonstrated increased drug respirable fraction in case of smaller carrier particles [17]. However, other reports showed that carrier median diameter has no effect on either aerodynamic diameter [18] or fine particle fraction of drug upon aerosolisation [19]. Conversely, Byron et al. [20] reported that large lactose particles (53–105 μm) outperformed finer lactose particles (b53 μm). Some authors stated that the effect of carrier particle size is influenced by inhaler device type [21], particle surface texture [22], powder micromeritic properties and carrier crystal form [23]. In light of complex interdependencies and lack of consensus in the literature, it is of high scientific and industrial importance to systematically address carrier particle size related properties on the dry powder inhaler performance. The objectives of this study were to provide, for the first time to the best of our knowledge, systemic investigation of the influence of the lactose particle size as one variable on DPI performance from Aerolizer® inhaler device in terms of the drug adhesion tendency toward carrier, drug content uniformity within formulation, and drug in vitro inhalation behaviour. 2. Materials and methods 2.1. Materials Micronized budesonide (BUD) was supplied from IVAX Pharmaceuticals, Ireland. α-Lactose-monohydrate (DMV International, Netherlands) (Pharmatose, 100 M), absolute ethanol (Fisher Scientific, UK), and acetonitrile (Fishser Scientific, UK) were used. 2.2. Sieving As supplied lactose powder was sieved to collect lactose particles in different size fractions. Two different types of sieving were used consequently: mechanical sieving via mechanical shaker (Endecotts Ltd, England) and air depression sieving via air jet sieving machine (Copley Scientific, Nottingham, UK) operating at gas volume flow that generates negative pressure of 4 kPa and rotating nozzle speed of 50 rpm. Lactose bulk powder was poured above 250 μm sieve which has been placed on top of different sieves (Retsch® Gmbh Test Sieve, Germany) with smaller apertures sizes above each other as following: 125 μm, 90 μm, 63 μm, 45 μm, 20 μm, and metal collection plate. The mechanical shaker was tightened closely and then operated for 15 min, after which the amount of powder remained on top of each different sieve (125 μm, 90 μm, 63 μm, 45 μm, or 20 μm sieve) was further subjected to air jet sieving for 15 min. Jet sieving is crucial to remove fine carrier particles adhered to the surface of larger carrier particles which could not be removed by mechanical sieving. After the sieving process were complete, particles with different size fractions: [90–125 μm (Lac A), 63–90 μm (Lac B), 45–63 μm (Lac C), 20–45 μm (Lac D), and b20 μm (Lac E)] were collected and kept in sealed glass vials until used. No significant amounts of lactose powder remained on top of 125 μm sieve following mechanical sieving. 2.3. Particle size measurements Particle size analysis was conducted using a Sympatec (ClausthalZellerfeld, Germany) laser diffraction particle size analyser as described

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in details elsewhere [24]. For budesonide, two different dispersion media were tested (water and water with 1% Tween 20). Each sample was sonicated for 30 s before measurement. 2.4. Image analysis optical microscopy Quantitative particle size and shape analysis were conducted using a computerised morphometric image analysing system (Leica DMLA Microscope; Leica Microsystems Wetzlar GmbH, Wetzlar, Germany; Leica Q Win Standard Analyzing Software). For each lactose sample, a small amount of powder (~20 mg) was homogenously dispersed on a microscope slide to form a thin powder, and minimum 1400 particles were detected randomly from different positions and measured. Particle size and shape were quantified using several parameters including equivalent diameter (EQD, equivalent circle diameter derived from area measurement), aspect ratio (Eq. (1)), angularity (Eq. (2)), and roughness (Eq. (3)) [5]: Aspect ratio ¼

Length Breadth

Angularity ¼

Perimeterconvex Perimeterellipse

Roughness ¼

ð1Þ !2

Perimeter Perimeterconvex

ð2Þ

ð3Þ

where Length is the maximum Feret diameter, Breadth is the minimum Feret diameter (maximum and minimum Feret diameters were calculated from 16 calliper measurements at 6° intervals around the particle), Perimeter is the estimated perimeter of particle with compensation for corners, Perimeterconcex is perimeter of the minimum convex boundary circumscribing the particle, and Perimeterellipse (Eq. (4)) is the perimeter of fictitious equivalent ellipse which has the same area and the aspect ratio of aggregate particle.

Perimeterellipse ¼

sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 þ aspect Ratio2 Perimetercircle 2aspect Ratio

ð4Þ

2.5. Scanning electron microscope (SEM) Electron micrographs of lactose, budesonide (BUD), and BUDlactose formulation samples were obtained using a scanning electron microscope (Philips XL 20, Eindhoven, Netherlands) operating at 15 kV. The specimens were mounted on a metal stub with doublesided adhesive tape and coated under vacuum with gold in an argon atmosphere prior to observation. Different magnifications were used to observe budesonide particles and lactose particle shape and surface topography. 2.6. Density and flowability assessments True density (ρtrue) of all lactose samples (defined as particle mass divided by its volume excluding both open pores and closed pores) was measured using an ultrapycnometer 1000 (Quantachrom, USA) using helium gas at an input gas pressure of 19 psi and an equilibrium time of 1 min. Also, bulk density (ρb), tap density (ρt), and porosity (Eq. (5)) of each lactose powder sample were measured as important descriptors of powder bulk cohesive properties. Carr's index (CI, Eq. (6)) and Hausner ratio (H, Eq. (7)) were measured for all lactose powders as an indication of powder flowability. The method incorporated was described elsewhere [25].

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 porosity ¼

1−

ρb ρtrue

  100

ð5Þ

CI ¼

  ρt −ρb  100 ρb

ð6Þ

H¼

ρt ρb

ð7Þ

2.7. Differential scanning calorimetry (DSC) A differential scanning calorimeter (DSC7, Mettler Toledo, Switzerland) was used to characterise solid state nature of different lactose samples. The equipment was calibrated using indium and zinc. Samples weighing between 4 mg and 5 mg were crimped and sealed nonhermetically in aluminium DSC pans with pin-hole lids. Each sample was heated from 25 °C to 300 °C at a scanning rate of 10 °C/min. A purge gas of nitrogen was passed over the pans with a flow rate of 50 mL/min. Different enthalpies were calculated by the software (Mettler, Switzerland).

2.9.4. Homogeneity test After blending, seven randomly selected samples were taken from different spots of each formulation powder for assay of budesonide content. Each sample weighs 27.5 ± 1 mg (which is equivalent to a unit dose of budesonide: 400 ± 15 μg, in accordance with Budecort400 Rotacaps®) and was dissolved in 50 mL absolute ethanol in a volumetric flask. For each formulation, % uniformity was calculated as the percent amounts of budesonide to the nominal dose, while the degree of budesonide content homogeneity was expressed in terms of percent coefficient of variation (% CV). 2.9.5. Evaluation of drug–carrier adhesion Air jet sieving (air jet sieving machine (Copley Scientific, Nottingham, UK) operating at gas volume flow that generates negative pressure of 4 KPa and nozzle rotating speed of 50 rpm) was employed to assess adhesion forces between budesonide particles and lactose particles [3,5]. For each formulation, quantity of 1 g was placed on top of the 20 μm sieve (Retsch® GmbhTest Sieve, Germany) and four samples (27.5± 1 mg) were removed from different areas of the powder formulation after different functional sieving times (5 s, 15 s, 30 s, 2 min, and 6 min) for quantification of budesonide. For comparison purpose, all amounts of budesonide were expressed as a percentage to average budesonide content determined in each formulation before sieving (Section 2.9.4). All adhesion assessment experiments were conducted in an air-conditioned laboratory (22 °C and 50% RH).

2.8. Powder X-ray diffraction (PXRD) The patterns of all lactoses were collected on a Siemens DIFFRACplus 5000 powder diffractometer with Cu Kα radiation (1.54056 Å). The tube voltage and amperage were set at 40 kV and 40 mA, respectively. The divergence slit and antiscattering slit settings were variable for illumination on the 20 mm area on the sample. Each sample was scanned between 5 and 40° 2θ, with a step size of 0.02° at 2 step/s. The sample stage was spun at 30 rpm. The instrument was pre-calibrated using a silicon standard. 2.9. Characterisation of BUD-lactose DPI formulations 2.9.1. Formulation preparations 3 g of each lactose size fraction powder (Lac A, Lac B, Lac C, Lac D, or Lac E) was blended with budesonide powder at a constant ratio of lactose:budesonide 67.5:1, w/w. This blending was performed in cylindrical aluminium container (6.5 × 8 cm) using a Turbula® mixer (Maschinenfabrik, Basel Switzerland) at a constant speed of 100 rpm for 30 min. Once prepared, all formulations were stored in tightly sealed vials until used. 2.9.2. HPLC quantification of budesonide Budesonide was analysed by HPLC apparatus (Varian prostar, USA). A mixture of acetonitrile:monobasic phosphate buffer (PH = 3.2) (55: 45, v/v) was used as a mobile phase. The absorbance of budesonide was detected with a multiple-wavelength (UV) detector (Varian prostar, USA) at a wavelength of 244 nm. The stationary phase was a Kromasil® C8 column (15 cm × 4.6 mm, Hichrom, UK), the flow rate of mobile phase was 1.1 mL/min, and budesonide retention time was 4.5 min. 2.9.3. BUD agglomeration assessment In order to assess to relative degree of “soft” drug–drug agglomeration within different BUD-Lac formulations, the following approach was applied. Each formulation powder was suspended in distilled water (which dissolves lactose particles but does not dissolve BUD particles) and then subjected to laser diffraction (Sympatec, Clausthal-Zellerfeld, Germany) under stirring conditions (1200 rpm). Particle size data was determined after 5 min, at which lactose particles had completely dissolved and nearly stable particle size values were obtained.

2.9.6. In vitro aerosolisation assessments After blending, each formulation was filled manually into hard gelatine capsules (size 3) with 27.5 ± 1 mg powder. Prior to any investigation, all filled capsules were stored in sealed glass vials for at least 24 h in order to allow any charge-relaxation to occur. Deposition profiles of all formulations were assessed in vitro using Aerolizer® inhaler device (Novartis, Switzerland) and Multi Stage Liquid Impinger (MSLI) equipped with a USP induction port (IP) (Copley Scientific, Nottingham, UK) as adopted from Kaialy et al. [25]. In brief, 20 mL of absolute ethanol was introduced to the stages 1, 2, 3 and 4 of the MSLI. A filter paper (Whatman; pore size, b0.45 μm) was introduced in the stage 5 of the impinger. MSLI was operated at a flow rate of 92 L/min which corresponds to a pressure drop of 4 kPa across the Aerolizer® device (as typical for inspiration by a patient). After ten actuations (ten capsules) for each sample, the initial ethanol on each stage was collected and then all stages of the MSLI were thoroughly washed with more absolute ethanol several times up to 50 mL volumetric flask. Also, the powder remained in capsule shells, deposited on the inhalers with their mouthpiece adapters (I+ M), and deposited on the induction port (IP) were also collected by washing it with absolute ethanol up to 50 mL. Several parameters were employed to quantify budesonide deposition profiles from each formulation including recovered dose (RD), emitted dose (ED), mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), impaction loss (IL), fine particle fraction (FPF), budesonide equivalent aerodynamic diameter (Dae), and lactose equivalent aerodynamic diameter (Dae,lactose) as defined elsewhere [5]. The theoretical aerodynamic diameter (Dae) was calculated from the geometric mean diameter (De) and true density (ρ) using Eq. (8): Dae ¼ De 

rffiffiffi ρ x

ð8Þ

where x is the dynamic shape factor for non-spherical particles which was assumed to be 1. 2.10. Statistical analysis One way analysis of variance (ANOVA) test was applied to compare mean results in this study considering P values less than 0.05

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as indicative of significant difference. When appropriate and when ANOVA indicated significant difference, Tukey's Honestly Significant Difference (HSD) test was performed. 3. Results and discussion 3.1. Particle size analysis Particle size distributions (PSDs) for different lactose size fractions are shown in Fig. 1. All samples showed sigmoidal (log normal) PSDs with different size ranges for each lactose fraction (Fig. 1-a). Lactose samples showed considerably different volume mean diameter (mean particle diameter based on volume, VMD), different fine particle lactose (FPLb 5μm and FPLb 10μm) (Fig. 1-b), different span values, and different theoretical volume specific surface area (SSAv) values for most lactose samples (Fig. 1-c). No substantial amounts of FPLb 5μm and FPLb 10μm were observed in case of Lac A, Lac B, and Lac C (≤1%), however, Lac D contained 4.4 ± 0.1% v/v of FPLb 5μm and 5.2 ± 0.2% of FPLb 10μm, whereas Lac E contains 16.4 ± 2.0% of FPLb 5μm and 36.8 ± 2.4% of FPL10μm (Fig. 1-b). Such observations indicate that it is more difficult to remove fines from smaller lactose particles suggesting that sieving time should be increased with decreasing lactose particle size. Statistically,

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span values for different lactose samples were in the following rank order: Lac A b Lac B b Lac C b Lac D b Lac E, whereas SSAv for different lactose samples were in the following rank order: Lac A ≈ Lac B ≈ Lac C > Lac D > Lac E (Fig. 1-c). A higher span of lactose sample is indicative of wider (more heterogonous) size distribution which might lead to higher variability in lung deposition regions upon inhalation. By comparing different lactose samples, logarithmic relationships were obtained when plotting VMD against span or SSAv indicating wider size distribution and higher specific surface area for lactose particles with smaller mean diameter (Fig. 2-a). Similar results were reported previously [26,27]. When wet technique was used to measure particle size distribution of budesonide, it showed multimodal PSD with a median value (d50%) of 3.6 ± 0.6 μm, VMD of 13.6 ± 1.9 μm, and 60% of particles b5 μm (Fig. 3). Such PSD could be considered to be too high especially when SEM visualisation demonstrated that most budesonide particles are less than 2 μm in size (shown later). This could be attributed to high cohesive properties of micronised budesonide leading to the formation of aggregates which could not be deaggregated by water (as a dispersing medium) leading to overestimated particle size. However, when dispersed in water with 1% Tween 20, budesonide showed monomodal PSD with d50% of 1.6 ± 0.0 μm, VMD of 2.0 ± 0.0 μm, and 97% of particles b5 μm (Fig. 3) which is supported by SEM observations (Fig. 6-I), suggesting that this dispersing medium is capable to disperse most budesonide agglomerates into individual particles. Since most budesonide particles were within the size range of 1–5 μm, these particles could be considered as optimal for pulmonary drug delivery. 3.2. Image analysis The image analysis was used to measure size and shape descriptors (e.g., equivalent diameter (EQD), aspect ratio, angularity, and roughness) for all lactose particles which are all shown in Fig. 4-I. EQD of different samples ranged from 119.6 ± 1.1 μm for Lac A to 13.8 ± 0.0 μm for Lac E (Fig. 4-I). Direct linear relationship (r2 = 0.9485) was established between VMD and EQD (Fig. 2-b) indicating that particle size data obtained by image analysis can support the particle size data obtained by laser diffraction. This proves that image analysis with optical microscopy is an effective method for measurement of particle size. All lactose samples showed similar aspect ratio (1.62 ± 0.37, mean ± SD, P > 0.05) indicating that particle elongation of different lactose samples is of no difference, which is important in terms of direct comparison between DPI formulations [28]. Also, this similarity is believed to contribute for good agreement between image analysis and laser diffraction in terms of particle size determination. However, angularity and roughness of different lactose samples varied considerably within the following rank order: Lac A > Lac B > Lac C > Lac D > Lac E (Fig. 4-I). Interestingly, linear correlations were obtained when plotting VMD of different lactose samples against roughness (r 2 = 0.8752) or angularity (r 2 = 0.921) (Fig. 2-b). Such findings demonstrate the “uniform” elongated-shape of all lactose particles regardless of size. However, lactose particle angularity and roughness increase with increasing particle size. In fact, aspect ratio is first order shape descriptor reflects overall particle shape elongation whereas angularity is a second order shape descriptor reflects variations in particle corners [29]. Particle angularity is known to be independent of particle aspect ratio [30]. Variations in angularity between different lactose samples despite their similar aspect ratio could be explained as smaller particles have more rounded edges, however similar degree of elongation.

Fig. 1. Cumulative (% undersize) particle distribution (a), volume mean diameter (█) (VMD), (♦) fine particle lactose less than 5 µm (FPL b 5µm), (●) fine particle lactose less than 10 µm and (FPL b 10µm) (b), (▲) span and (■) volume specific surface area (SSAv) (c) for different lactose size fractions: Lac A (□) (90–125 μm), Lac B (*) (63–90 μm), Lac C (◊) (45–63 μm), Lac D (Δ) (20–45 μm), and Lac E (○) (b20 μm) (mean ± SD, n ≥ 5).

3.3. Scanning electron microscope for different lactoses The use of shape factors obtained by image analyses might not be sufficient to characterise particle morphology, as it is dependent on

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Fig. 2. Relationship between volume mean diameter (VMD), (▲) span, (Δ) volume specific surface area (SSAv), ( true density (ρtrue), (○) bulk density (ρb), (●) tap density (ρt).

particle orientation and contact area with other particles which hinders the accuracy of the technique. Therefore, morphological features of all lactose samples were qualitatively assessed by SEM (Fig. 3-II to -VI). Lac A appeared as coarse particles larger than 100 μm in size (Fig. 4-II), whereas Lac E contains particles with size typically less than 20 μm (Fig. 4-VI). Fine particle lactose (FPLb 5μm and FPLb 10μm) could be visualised in case of Lac D (Fig. 4-V) and Lac E (Fig. 4-VI). In agreement with angularity measurements (Fig. 4-I), larger lactose particles (Lac A, Lac B, and Lac C) showed angular shape with sharp edges and unpolished surfaces. On the other hand, smaller lactose particles (Lac D and Lac E) were less uniform in size and shape having more rounded corners and edges (Fig. 4-VI). In fact, αlactose-monohydrate particles larger than 45 μm behave as brittle particles whereas particles smaller than 45 μm behave as ductile particles [31]. Therefore, larger particles experience larger number of

Fig. 3. Particle size distribution of budesonide measured by laser diffraction after dispersing in water or water/Tween 20 (1%) (mean ± SD, n = 9).

) roughness, (■) angularity, (□) equivalent diameter (EQD), (×)

fractured faces resulting from inter-particle collisions during powder processing conditions. Morphologically, all lactose particles showed the typical tomahawk habit reported previously for α-lactosemonohydrate [3,5,27]. In fact, tomahawk shape is the shape of lactose crystals grown to maturity, and it was attributed to polar contribution of the crystal lattice during morphology prediction. 3.4. Density, flowability, and porosity characterisation True density (ρtrue), bulk density (ρb), tap density (ρt), Carr's index (CI), Hausner ratio (H), and porosity for different lactose size fractions varied considerably (Table 1). Interestingly, plotting lactose true density against volume mean diameter resulted in strong linear correlation (Fig. 2-c) (r 2 = 0.9932), indicating smaller true density for larger lactose particles. Smaller lactose particles have higher specific surface area and thus have considerably higher percentage of atoms or molecules near the surface (which do not have the equilibrium positions within the solid structure) resulting in differences in particle molecular configuration and molecular arrangements and thus different true density when compared to particles with larger size [5,32]. In DPI systems, particle true density is an important particle physical property as it determines the probability of impaction. In contrast to drug particles (where lower drug true density is likely to enhance DPI performance [33]), mannitol or lactose carrier particles with higher true density produced enhanced DPI performance [25,27]. Plotting lactose volume mean diameter against bulk density or tapped density demonstrated higher bulk density and higher tapped density for lactose powders with larger particles (Fig. 5-d). This could be attributed to increased cohesive forces between particles with smaller sizes. Smaller bulk density and higher porosity for lactose powders containing smaller particles is expected to promote drug-carrier segregation (due to reduced drug-carrier content area and thus reduced drug-carrier interparticulate forces) concurrently with carrier powder consolidation leading to better powder aerosolisation during inhalation, but at the same time poor drug content homogeneity [25].

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Fig. 4. (I) (█) roughness, (○) angularity, (◊) equivalent diameter (EQD) (mean ± SE, n ≥ 1400); and SE micrographs, for different lactose size fractions: (II) Lac A (90–125 μm), (III) Lac B (63–90 μm), (IV) Lac C (45–63 μm), (V) Lac D (20–45 μm), and (VI) Lac E (b20 μm).

Variations in CI and H between lactose powders reflect their considerable different flow characters (Table 1). Higher CI values for lactose powders with smaller VMD could be attributed to powder aggregation arising from their smaller particle diameter and higher fine particle content (FPLb 10μm). Poor flowability of DPI formulation is believed to be disadvantageous in terms of dose metering and processing on industrial scale. Also, poorer powder flowability is, generally, indication of more cohesive powders which proved to be more difficult to fluidize [34].

3.5. Solid state characterisation Thermal traces and XRD patterns of all lactose samples are illustrated in Fig. 5. All samples showed the typical thermal trace and XRD pattern of α-lactose-monohydrate having two distinctive endothermic events at 147.4 ± 0.5 °C (crystal water dehydration) and 217.8 ± 1.2 °C (α-lactose-monohydrate melting), one smaller exothermic event at 174.8 ± 1.6 °C (amorphous lactose crystallisation) [26,27], and XRD diagnostic peaks at 12.5°, 16.4°, and 20.1° [28]. However, the enthalpies of thermal transitions and relative XRD intensities of different lactoses with different particle sizes varied considerably (Fig. 5). It is clear that smaller lactose particles demonstrated higher enthalpies for the crystallisation of amorphous lactose and smaller intensity for characteristic peaks in XRD spectrum (Fig. 5). This indicates that smaller

lactose particles have decreased relative degree of crystallinity since all particles have the same crystal from. 3.6. Budesonide–lactose formulation characterisations 3.6.1. Evaluation of budesonide–lactose formulations with SEM and laser diffraction SEM image of budesonide showed particles with varying degrees of agglomeration (Fig. 6-I) which was confirmed by laser diffraction data (Fig. 7). These particles appeared as crystalline, rough, irregular shape particles which are the typical properties pointed out in previous studies for micronized budesonide [35]. SEM photographs of budesonide–lactose formulation samples showed small budesonide particles (b2 μm) adhered to large lactose particles as individual particles indicating the formation of budesonide–lactose interactive mixtures suitable for DPIs (Fig. 6-II to -VI). With higher magnifications, it was clear that smaller lactose particles (Lac D and Lac E) have fewer protuberances and decreased surface asperities (Fig. 6-V, -VI) compared to larger lactose particles (Lac A and Lac B) that showed fractured surface texture with rounded grains (Fig. 6-II to -IV). This is in agreement with roughness measurements obtained by image analysis (Fig. 4-I). Cavities (macroscopic depressions) on the surfaces of large lactose particles (Lac A and Lac B) are likely to induce mechanical interlocking (entrapment) of budesonide particles and consequently reduced drug-carrier detachment

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Table 1 True density (ρtrue), bulk density (ρb), tap density (ρt), porosity, Carr's index (CI), Hausner ratio (H), and flow character for different lactose size fractions (mean ± SD, n = 7). Lactose product Lac Lac Lac Lac Lac

A (90–125 μm) B (63–90 μm) C (45–63 μm) D (20–45 μm) E (b20 μm)

ρtrue (g/cm3)

ρb (g/cm3)

ρt (g/cm3)

Porosity (%)

CI (%)

H

Flow character

1.49 ± 0.01 1.51 ± 0.00 1.53 ± 0.01 1.55 ± 0.01 1.57 ± 0.01

0.74 ± 0.01 0.69 ± 0.01 0.67 ± 0.01 0.51 ± 0.02 0.28 ± 0.01

0.82 ± 0.01 0.81 ± 0.00 0.80 ± 0.00 0.63 ± 0.01 0.38 ± 0.01

50.0 ± 0.2 54.3 ± 0.4 56.4 ± 0.9 67.1 ± 1.0 82.2 ± 0.9

9.0 ± 0.9 14.8 ± 0.5 16.4 ± 1.1 18.8 ± 1.9 25.3 ± 2.8

1.10 ± 0.01 1.17 ± 0.01 1.20 ± 0.02 1.23 ± 0.03 1.34 ± 0.05

Excellent Good Good Fair Poor

upon inhalation [26]. In case of budesonide–Lac E formulation, fewer drug particles could be observed adhered to flat regions of lactose particle surface (Fig. 6-VI). This might lead to decreased budesonide– lactose adhesion due to decreased direct contact area between budesonide particles and lactose particles. Visual examination of particle morphology in case of budesonide–Lac D and budesonide–Lac E formulations demonstrated that both fine lactose particles (FPLb 5μm) and fine budesonide particles were present (Fig. 6-V, -VI). Also, fine particle agglomerates (FPA: FPL + budesonide) could be observed in case of budesonide–Lac E formulation (Fig. 6-VI). These agglomerates are believed to be formed at the expense of budesonide–lactose ordered interactive mixtures and are likely to deposit on lower airway regions [5].

Also, these agglomerates suggest that budesonide particles might be less homogeneously distributed throughout budesonide–Lac E formulation in comparison to budesonide in other formulations. BUD–BUD agglomeration assessment suggested smaller degree of BUD–BUD agglomeration in case of smaller carrier particles which appeared as a shift toward smaller size distribution (Fig. 7). Lower degree of drug–drug agglomeration is indicative of better aerosolisation performance upon inhalation. Budesonide particles were distinguished morphologically. However, precise assignment of drug and fine lactose particles is not possible using SEM alone due to possible overlap in size and shape between BUD and fine lactose particles.

Fig. 5. DSC thermal traces and PXRD patterns of different lactose size fractions: Lac A (90–125 μm), Lac B (63–90 μm), Lac C (45–63 μm), Lac D (20–45 μm), and Lac E (b 20 μm). ↓: Crystalline water dehydration endotherm, : amorphous lactose recrystallisation exotherm, :α-lactose melting endotherm.

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Fig. 6. SE micrographs (×1000 and × 3000) for budesonide (BUD) alone and blends containing budesonide formulated with different lactose size fractions: Lac A (90–125 μm), Lac B (63–90 μm), Lac C (45–63 μm), Lac D (20–45 μm), and Lac E (b 20 μm). Arrows refer to budesonide particles and circles refer to fine particle aggregates composed of fine particle lactose and budesonide. VMD: volume mean diameter, CV: coefficient of variation, FPF: fine particle fraction.

3.6.2. Budesonide content uniformity In theory, based on the constant weigh of all doses (400 ± 15 μg), uniformity of budesonide content for all tested formulations should be included within the range of 100 ± 5.5% (mean ± SD, CV = 5.5%). All tested formulations produced similar uniformity (94.6 ± 5.6– 106.0 ± 13.7%, mean ± SD, P > 0.05) which fall within the acceptable range of 100 ± 10% (Table 2). This indicates that the overall blending, sampling, and analysis were satisfactorily accurate. However, %CV of budesonide content varied considerably: BUD-Lac A b BUD-Lac B b BUD-Lac C b BUD-Lac D b BUD-Lac E (Table 2). Inverse linear relationship was obtained between lactose particle VMD and %CV of budesonide content (r 2 = 0.997) (Fig. 10-a) indicating poor homogeneity of budesonide when smaller lactose particles were used in the formulation. This could be attributed to poorer flowability (Table 1) and higher PSD polydispersity (higher span) (Fig. 1-c) for smaller lactose particles, which is likely to lead to enhanced per-location segregation (segregation by size) during formulation blending. Also,

it can be expected that lactose samples with larger size and higher bulk density can be weighed more easily and more reproducibly (than lactose particles with smaller size and smaller bulk density) leading to improved dose metering and thereby reduced variation in budesonide content homogeneity within formulations. Poorer budesonide homogeneity in case of smaller lactose particles is unfavourable since homogenous drug content is essential to achieve uniform metering doses by the patient during inhalation. Nevertheless, formulations containing smaller lactose particles were not subjected to further blending for the comparison purpose of this study. 3.6.3. Budesonide–lactose adhesion assessments The ease of BUD-lactose separation in different formulations was evaluated indirectly by air jet sieving, where particles were subjected to both aspiration (generated by the negative pressure) and airflow (generated by the blow nozzle rotating under the sieve). In this experiment, less amounts of budesonide remained after air jet sieving

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Fig. 7. Particle size distribution of budesonide obtained by laser diffraction from formulations containing different lactose size fractions: Lac A (90–125 μm), Lac B (63–90 μm), Lac C (45–63 μm), Lac D (20–45 μm), and Lac E (b 20 μm) (mean ± SD, n ≥ 3).

indicates weaker adhesion force between budesonide and lactose particles. For BUD-Lac A formulation, amounts of recovered budesonide were similar (P > 0.05) regardless of sieving time (Fig. 8-a) which is indicative of high BUD-lactose adhesion properties. Conversely, in comparison to other formulations, BUD-Lac D formulation generated the lowest amounts of budesonide after all sieving times (Fig. 8-a) suggesting low budesonide–Lac adhesion properties. For BUD-Lac B, BUD-Lac C, and BUD-Lac D formulations, significant decrease in amounts of recovered budesonide was obtained when sieving time increased from 5 s to 120 s (BUD-Lac B), 5 s to 30 s (BUD-Lac C), or 5 s to 15 s (BUD-Lac D) (Fig. 8-a). Fig. 8-b shows that, generally, and after all sieving times, higher amounts of budesonide were collected from formulations containing lactose particles with larger volume mean diameter. These results suggest that BUD-lactose adhesion forces (F) were in the following rank order according to lactose product type: FLac A > FLac B > FLac C > FLac D, indicating that budesonide adhesion to lactose surfaces decreases as lactose particle size decrease. This could be attributed to reduced budesonide–lactose press-on (push-on) forces (collision and friction forces) which act as adhesive forces during mixing [36] in case of smaller lactose particles. Also, based on Zimon's resuspension model [37]; it can be assumed that detachment of small drug particles from large carrier particles occurs laterally on the carrier particle surface (budesonide particles slip along the lactose particle surface till it reaches the edge and falls off). Therefore, the larger the lactose particle size, the longer the distance that the budesonide particles have to slide on lactose surface and thus the greater the drag force which acts as adhesion force. 3.6.4. Deposition study Budesonide mass distribution deposition patterns and aerodynamic diameter as analysed by MSLI from all formulations are

Fig. 8. % Amounts of budesonide (BUD) remained on top of 20 μm sieve after varied functional air jet sieving times (a); and % amounts of budesonide (BUD) remained on top of 20 μm sieve in relation to lactose volume mean diameter (VMD) (b). (mean ± SD, n = 4).

shown in Fig. 9. It can be seen that there is relatively low variations in budesonide mass distribution between formulations containing lactose particles larger than 45 μm (Lac A, Lac B, and Lac C); however, considerably different budesonide deposition patterns were obtained from formulations containing lactose particles smaller than 45 μm (Lac D and Lac E) (Fig. 9). Regardless of formulation, amounts of budesonide remained on capsule shells were 7.5 ± 1% (mean ± SD) (Fig. 9). It is clear that, in comparison to the formulations containing lactose particles larger than 45 μm (Lac A, Lac B, Lac C), formulations containing lactose particles smaller than 45 μm (Lac D and Lac E) deposited higher amounts of budesonide on I + M, IP, and MSLI stage 2, stage 3, stage 4, and filter but considerably smaller amounts on MSLI stage 1 (Fig. 9). All formulations produced linear (r 2 ≥ 0.98) aerodynamic PSD curves of budesonide with different slops (referred to as constant K in this study) (Fig. 9). Constant K in case of BUD-Lac D (26.6) and BUD-Lac E (45.3) formulations were considerably higher

Table 2 Percentage uniformity (UN), coefficient of variation (CV), recovered dose (RD), emitted dose (ED), mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), impaction loss (IL), and fine particle fraction (FPF) of budesonide obtained from formulations containing different lactose size fractions (mean ± SD, n ≥ 3). Lactose product

Lac A (90–125 μm)

Lac B (63–90 μm)

Lac C (45–63 μm)

Lac D (20–45 μm)

Lac E (b 20 μm)

Uniformity (%) CV (%) RD (μg) ED (μg) MMAD (μm) GSD IL (%) FPF (%)

95.1 ± 4.4 4.6 286.2 ± 14.1 250.4 ± 11.6 3.43 ± 0.29 2.17 ± 0.04 77.4 ± 2.9 6.8 ± 1.8

94.6 ± 5.6 5.9 358.8 ± 7.8 315.0 ± 10.5 3.60 ± 0.05 2.08 ± 0.01 77.1 ± 1.1 7.0 ± 0.2

93.1 ± 7.5 8.0 363.0 ± 7.7 316.4 ± 8.4 3.38 ± 0.16 2.15 ± 0.05 74.7 ± 0.8 8.4 ± 0.4

102.3 ± 10.8 10.5 380.6 ± 6.3 324.9 ± 11.3 3.06 ± 0.07 2.25 ± 0.05 58.9 ± 5.4 18.7 ± 3.3

106.0 ± 13.7 13.0 404.2 ± 26.8 349.0 ± 21.3 3.13 ± 0.12 2.27 ± 0.06 40.7 ± 1.9 31.8 ± 1.3

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Fig. 9. MSLI deposition profiles and aerodynamic particle size distribution of budesonide before and after aerosolisation by the Aerolizer® obtained from formulations containing different lactose size fractions: Lac A (90–125 μm), Lac B (63–90 μm), Lac C (45–63 μm), Lac D (20–45 μm), and Lac E (b 20 μm) (mean ± SD, n ≥ 3). (I + M = Inhaler with mouthpiece adaptor, IP = Induction port).

than constant K in case of BUD-Lac A (9.7), BUD-Lac B (10.1), and BUD-Lac C (12.1) formulations (Fig. 9). Such results indicate that formulations containing lactose particles smaller than 45 μm (BUD-Lac D and BUD-Lac E) delivered higher amounts of budesonide to lower stages of the impactor than formulations containing lactose particles larger than 45 μm (BUD-Lac A, BUD-Lac B, and BUD-Lac C). In case of steroids (e.g. budesonide), increased amounts of drug delivered to lower stages of the impactor is not only indicative of enhanced therapeutic efficiency, but also minimised side effects. However, when compared to laser diffraction PSD data, aerodynamic PSD of budesonide aerosolised from all formulations suggested that budesonide particles were not dispersed to individual particles. This could be attributed to high cohesiveness of micronized budesonide particles and/or inadequate dispersing efficiency of the inhaler device. Indeed, all variations in budesonide deposition profiles could be attributed to different lactose size fractions included within each formulation, as the same batch of budesonide, the same budesonide: lactose ratio, and the same blending process were used in preparation of all formulations. In fact, amounts of drug remained in I + M and deposited on throat (IP) reflect the dispersible nature of a DPI formulation. Higher amounts of budesonide deposited on throat generated from formulations containing smaller lactose particles could be attributed to inadequate powder dispersion (or fluidisation) during inhalation resulting from their higher fines content (Fig. 1-b) and poorer flowability (Table 1). Increased amounts of budesonide deposited on I + M and throat (representing oropharyngeal region) are expected to maximise the potential for local side effects (including irritations and infections). It was assumed that powder cohesiveness in case of lactose particles with smaller volume mean diameter could not be totally overcome during inhalation process leading to the formation of aggregates remaining in I + M and/or deposited on throat. The RD and ED of budesonide exhibited a decreased trend with increasing lactose VMD (Fig. 10-b), indicating that smaller lactose particles are expected to deliver higher proportion of budesonide mass to the whole pulmonary region following inhalation. Except for BUD-Lac A formulation, RD of BUD obtained from all formulations was within the acceptable range of recovery: 75–125%. Lower recovery in case of BUD-Lac A indicates that some of drug particles were lost, presumably due to adherence of the drug particles to the wall of glass vials during storage or to the container during blending process. This could be ascribed to the adhesive nature of budesonide which minimise drug losses by indulging in fine carrier particles in case of carrier products with smaller size distributions. Mean diameter characterisation of budesonide particles indicated the following rank order: geometric diameter (De = 1.98 ± 0.01 μm) b

equivalent aerodynamic diameter (Dae = 2.22± 0.03 μm)b experimental mass median aerodynamic diameter (MMAD = 3.13 ± 0.12 μm– 3.43± 0.29 μm) (mean ± SD, n = 3) (Table 2). High Dae of budesonide in comparison to De is understandable since budesonide true density is >1 g/cm3 (1.27 ± 0.02 g/cm3, mean± SD). Also, high MMAD of budesonide in comparison to Dae could be attributed to high cohesive properties of budesonide particles in dry powder state (Fig. 6-I). Among, De, Dae, and MMAD, the latter diameter (MMAD) is believed to be the best descriptor of budesonide mean diameter as it is more related to budesonide particles during inhalation. GSD ranged between 2.08 ± 0.01 (for BUD-Lac B formulation) and 2.27 ± 0.06 (for BUD-Lac E formulation) (Table 2), indicating heterodisperse aerodynamic size distribution pattern for budesonide particles generated from all formulations. By comparison, smaller lactose particles produced budesonide particles with smaller MMAD (Fig. 10-c). This indicates lower degree of BUD– BUD agglomeration in case of BUD-lactose formulations containing smaller lactose particles which is supported BUD–BUD agglomeration assessment data obtained by laser diffraction (Fig. 7). This could be attributed to higher surface area available for budesonide in case of larger lactose particles (higher surface loading of BUD to Lac), potentially promoting BUD–BUD agglomeration. In contrast, smaller lactose particles produced budesonide particles with higher GSD (Fig. 10-c) indicating relatively wider aerodynamic size distribution. This could be due to higher degree of PSD polydispersity (higher span) in case of smaller lactose particles (Fig. 2-a). Logarithmic relationships were established demonstrating smaller impaction loss (IL, refers to budesonide particles being adhered to lactose particle surfaces following inhalation), higher fine particle fraction (FPF, refers to the percentage fraction of the drug that is pharmacologically active), and higher constant K of budesonide generated from formulations containing smaller lactose particles (Fig. 10-d, -e, -f). This confirmed improved inhalation behaviour of budesonide aerosolised from smaller lactose particles. This could be due to the fact that larger carrier particles have higher surface rugosities and higher percentage of surface impurities [Fig. 6, 38] which might lead to higher drug–carrier adhesive forces and thus reduced drug–carrier detachment efficiency upon inhalation. Also, it is well known that the presence of fine particles on the carrier surface may decrease the drug–carrier contact area leading to a reduced drug–carrier adhesion forces and hence improved DPI performance [26,27]. Optimal concentration of fine particle lactose (FPL≤ 5 μm) varied between 5% [39] or up to 10–15% [40] which were sufficient to achieve maximum increase in FPF. In this study, higher FPF was still obtained when using carrier powder containing up to 16.4 ± 2.0% fine particles (FPLb 5 μm, BUD-Lac E formulation). However, it should be kept in

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Fig. 10. Relationships between lactose volume mean diameter (VMD), and (●) coefficient of variation (% CV) (a), (○) recovered dose (RD), (◊) emitted dose (ED) (b), (▲) mass median aerodynamic diameter (MMAD), (Δ) geometric standard deviation (GSD) (c), (♦) impaction loss (IL) (d), (■) fine particle fraction (FPF) (e), and (+) constant K (f) of budesonide obtained from formulations containing different lactose size fraction powders (mean ± SD, n ≥ 3).

mind that despite that the use of smaller lactose particles proved enhanced DPI performance; deposition of lactose particles is expected to be influenced at the same time. Generally, for asthma therapy, optimal DPI performance requires maximum amounts of drug and minimum amounts of carrier delivered to the deep lung airway regions. In this study, deposition behaviour of lactose particles was not assessed experimentally. However, comparative indication of lactose aerodynamic behaviour could be obtained through equivalent aerodynamic diameter of different lactose particles (Dae,lactose): 132.0 μm (Lac A), 108.0 μm (Lac B), 80.7 μm (Lac C), 43.4 μm (Lac D), and 16.4 μm (Lac E). These differences between Dae,lactose of different lactose particles are likely to affect deposition patterns of lactose from BUD-lactose formulations during inhalation as lactose particles with smaller Dae,lactose are expected to deposit on lower air way regions. Finally, it should be noted that PSD, solid state, and aerosolisation performance did not show significant changes when different lactose size fractions were stored for at least 6 months in ambient conditions (data not shown). Further systemic stability studies would be required to be taken under elevated temperature and humidity conditions.

4. Conclusion The present study showed the effect of lactose particle size on inhalation behaviour of budesonide from dry powder inhaler. It was clear that variations of particle size within brittle lactose particles has much lesser influence on budesonide deposition profiles in comparison to variations in particle size within ductile lactose particles. The adhesive forces between budesonide particles and lactose particles decrease with decreasing lactose particle size. Generally, the smaller the lactose volume mean diameter, the higher the recovered dose, the higher the emitted dose, the smaller the mass median aerodynamic diameter, and the higher the fine particle fraction of budesonide which are all indicative of enhanced dry powder inhaler performance. However, lactose particles with smaller volume mean diameter generated higher amounts of budesonide on throat which is disadvantageous in terms of increased potential for local side effects. It was also shown that the smaller the lactose volume mean diameters the poorer the budesonide content homogeneity within dry powder inhaler formulation which is detrimental to DPI formulation safety.

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Therefore, this study demonstrated that the beneficial of decreasing lactose particle size in terms of enhanced budesonide aerosolisation efficiency must be balanced with other disadvantageous in terms of poorer flowability, poorer dose uniformity, possible formulation instability, and increased side effects. Clearly, further studies would be warranted to investigate if the influence of carrier particle size or if the “optimal carrier particle size” within dry powder inhaler formulations is dependent on other factors such as drug particle size, drug type, carrier type, or inhalation flow rate. Acknowledgements Waseem Kaialy thanks Mr Ian Slipper (School of Science, University of Greenwich) for taking SEM images. Alhalaweh and Velaga thank the Kempe Foundation (Kempestiftelserna) for an instrumentation grant.

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