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The Influence of Mechanical Processing of Dry Powder Inhaler Carriers on Drug Aerosolization Performance
PHARMACEUTICAL TECHNOLOGY The Influence of Mechanical Processing of Dry Powder Inhaler Carriers on Drug Aerosolization Performance PAUL M. YOUNG, HAK-KIM CHAN, HERBERT CHIOU, STEPHEN EDGE, TERENCE H.S. TEE, DANIELA TRAINI Advanced Drug Delivery Group, Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia
Received 10 May 2006; revised 18 June 2006; accepted 10 July 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20933
ABSTRACT: The influence of processing on the performance of carrier material used in dry powder inhalers was investigated. a-Lactose monohydrate crystals were processed by ball milling for cumulative time durations and their properties evaluated. As expected, milling reduced the median particle diameter while increasing fine particulate (<10 mm) and amorphous levels. Recrystallization of these partially amorphous samples resulted in a reduction in fines, elimination of amorphous material with little change in median diameter. To study the effects of processing on aerosolization performance, blends of lactose monohydrate with a model drug (nedocromil sodium trihydrate), were evaluated using an in vitro multistage liquid impinger (MSLI) model. In general, milling and storage of the carriers at high humidity (prior to blending) had a significant (ANOVA, p < 0.05) effect on the fine particle fractions (FPF; <6.8 mm). These effects were attributed predominantly to the fines content, showing a strong correlation between increased fines and FPF (R2 ¼ 0.974 and 0.982 for milled and recrystallized samples, respectively). However, this relationship only existed up to 15% fines concentration, after which agglomerate-carrier segregation was observed and FPF decreased significantly. These results suggest that, after processing, high-dose drug formulation performance is dominated by the presence of fines. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1331–1341, 2007
Keywords: milling; surface activation; fines; aerosolization; dry powder; inhalation; recrystallization; amorphous
INTRODUCTION Dry powder inhalers (DPI) are a novel route for drug delivery, with the capability of targeting disease states both locally (in the case of lung diseases such as asthma), and systemically (e.g. in the delivery of proteins and peptides). For effective deposition in the lower airways and deep lung, drug particles with aerodynamic particle sizes of <5 mm are required. However, such systems are highly cohesive due to the high Correspondence to: Paul M. Young (Telephone: þ61 2 9036 7035; Fax: þ61 2 9351 4391; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 1331–1341 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
surface area to mass ratio of the particulates. Cohesive systems pose a problem for the desired deaggregation of particles as uncontrolled agglomeration occurs naturally. Subsequently, such agglomeration may lead to formulation variations and a decrease in DPI efficacy. As a consequence, large inert carrier systems are employed as one method to overcome this problem, where the micron sized drug particles are blended with larger inert material to reduce agglomeration, improve flow and act as a diluent. Ideally, during inhalation, the drug particles are liberated from the carrier to penetrate the lower airways while the carrier impacts on the orthopharynx and is swallowed. As with all these
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systems, it is important to note, however, that the energy supplied by the patient during inhalation needs to be sufficient for liberation and aerosolization of the drug particulates. Currently, lactose is the most popular material approved for use in inhalation as carrier. A naturally occurring disaccharide sugar, lactose is found in milk, and is collected as a by-product of whey produced during the manufacturing of cheese. Lactose monohydrates for use in the pharmaceutical industry, are produced through precipitation from aqueous solution and is available in many particle size grades.1 Furthermore, to obtain inhalation grade lactose monohydrate, the crystals are processed through techniques such as milling and sieving to produce the desired carrier size characteristics.2 However, due to the industrial scale of such processes, inter-batch, and intersupplier variations in the physicochemical properties of the carriers have been observed, leading to variations in the aerosolization performance of the final formulations.2 Such batch-to-batch variation is most likely due to differences in fine particle content, particle size distribution, surface morphology, and amorphous content. Intrinsic carrier particle size has been believed for many years to play an important role in the performance of DPI.3–6 Following a reduction in physical particle diameter, a decrease in mean mass aerodynamic diameter (MMAD) is observed. In general, a decrease in MMAD has been associated with an increase in drug dispersion. However, a recent study by Islam et al.,7 suggested that the volume median diameter of carriers had no significant effect on drug dispersion when studied without the influence of fines content. Earlier studies did not specify the fine particle content, thus with a reduction in particle size, the increasing concentration of fines may have been masking the effect carrier size has on aerosol performance. As fine particles are commonly introduced inadvertently during the comminution process, the influence of fines (e.g. carrier particles with a diameter <15 mm) on DPI performance has been studied extensively. Lucas et al.,8 Zeng et al.,6,9 Louey et al.,10,11 and Islam et al.,7,12 have all demonstrated an improvement in dispersion with the presence of fine particles. More recently, Steckel et al.,13 indicated similar findings, suggested that variation in small quantities of fines (<5% below 15 mm diameter) in different sized sieved fractioned lactose formulations significantly influenced drug aerosolization performance.
Micronized nedocromil sodium trihydrate (NST) was obtained from Sonafi-Aventis (Cheshire, England). Crystalline a-lactose monohydrate (Lactochem1 crystals) was obtained from Borculo Domo (Zwolle, The Netherlands). Water was purified by reverse osmosis (MilliQ, Millipore, Molsheim, France). Analytical grade chloroform
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As discussed, processing methods such as milling may induce variation in surface morphology or result in increased amorphous content. Surface morphology has been demonstrated to directly influence the contact area between drug particle and carrier, leading to variations in interparticulate adhesion. Several studies have reported that variations in contact area, as a result of differing surface structure, could potentially compromise the aerosolization performance of the drug particles.14–16 In addition, the introduction of amorphous material during high energy mechanical processes is associated with a higher surface adhesion energy compared to crystalline surfaces.17–19 As a consequence of raised adhesion energy, poor deaggregation of drug particles is observed.20 No studies have yet been reported in relation to the direct impact of amorphous content on drug dispersion from carriers. However, the presence of amorphous material may cause problems, for example, due to the fusion of particles, resulting in poor dispersion.19–21 Milling is commonly used for processing of powders in the pharmaceutical industry, and in particular for inhalation drugs and carriers. When carriers are milled, various changes to the physical properties are induced. This is important since the reliability of the DPI product mainly depends on batch-to-batch consistency of the lactose monohydrate carrier. However, as mentioned previously, variations between batches do occur, and this study was initiated to investigate the effect of any material changes induced by milling on DPI performance. Further, as part of an ongoing study, the influence of storage at high humidity prior to blending was also investigated. The influence of these processes on DPI aerosolization efficiency was investigated using nedocromil sodium trihydrate as a model drug system, and was correlated with the physical properties of the carrier particles.
MATERIALS AND METHODS Materials
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and n-octane were obtained from Biolab (Victoria, Australia) and Fluka (Germany), respectively. Preparation of Lactose Monohydrate Samples Mechanical treatment of lactose monohydrate samples was achieved by comminution in a small volume ball mill (approximately 1 L) containing 60 ceramic balls (mean diameter of 19.3 0.7 mm). Samples of lactose monohydrate (approximately 100 g) were weighed into the ball mill which was rotated at 42 rev min1 for durations of 10, 20, 30, 40, 50, and 60 min. Each sample was then collected and stored in tightly sealed containers over phosphorus pentoxide prior to sampling or blending. In addition, to crystallize any amorphous material, present in the milled samples, approximately 10 g of each milled lactose monohydrate sample was transferred onto glass Petri-dishes and stored (3 weeks) in tightly sealed containers with a saturated solution of potassium chloride (relative humidity, RH, of 85%). The samples were regularly stirred to ensure moisture penetration into the powder bed. After 3 weeks, each sample was removed and transferred into containers with phosphorous pentoxide (0% RH) for a minimum of 24 h before sampling or blending. Physical Characterization Particle Sizing of Processed Lactose Monohydrate Samples and NST Particle sizing was performed by laser diffraction (Malvern Mastersizer S, Malvern Instruments Ltd., Malvern, UK) using a 300RF lens and automated small volume dispersion sampling unit. Approximately 200 mg of each lactose monohydrate sample or NST was dispersed in about 10 mL of chloroform, and added drop-wise into the sampling unit containing chloroform until an obscuration between 10% and 30% was obtained. Size distributions were based on 2000 sweeps for each sample, with refractive indices of 1.533, 1.358, and 1.444 for lactose monohydrate, NST and chloroform, respectively. Each sample was analysed in triplicate. Scanning Electron Microscopy Visualization of lactose monohydrate surface morphology and the uniformity in blends were investigated using scanning electron microscopy (SEM) (XL30, Philips, Japan) at 10 keV. Each DOI 10.1002/jps
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sample was mounted on a carbon sticky tab and platinum coated (10 nm thickness; Edwards E306A Sputter Coater, UK), prior to analysis. Images were obtained at magnifications of 1000 and 5000. Amorphous Content Quantification of Lactose Monohydrate Organic Dynamic Vapor Sorption (Organic DVS) was used to quantitatively determine the amorphous content in the milled lactose monohydrate samples using a method described elsewhere.22 In simple terms, the technique measures the adsorption of a dispersive molecule (n-octane) into the surface of a sample as a function of partial pressure. Since the relative adsorption of the molecule into amorphous and onto crystalline samples will vary, a calibration curve may be constructed by comparing the relative adsorption in blends of 100% crystalline and 100% amorphous samples. From this, the amorphous content of an unknown sample may be determined. Measurements were conducted using a DVS-1 (Surface Measurement Systems, Alperton, UK), at 258C, using n-octane as the organic probe. Approximately 100 mg of lactose monohydrate was weighed into the sample pan and exposed to a two-step octane partial pressure (p/po) cycle of 0–90%. Equilibrium at each step was determined by a dm/dt of 0.0002% min1. Each milled sample (n ¼ 5), and the amorphous content calculated from the calibration data reported elsewhere.22 In addition, a sample of the 60 min recrystallized sample was analysed to ensure full recrystallization. Drug Content Determination Quantification of NST content uniformity and in vitro deposition was determined by UV spectrophotometry (U-2000 spectrophotometer, Hitachi, Japan) at a wavelength of 376.5 nm. Samples were prepared and diluted appropriately in water. The calibration plot for NST was linear over the range 0.5–50.0 mg mL1 (R2 ¼ 1.00). Lactose monohydrate did not interfere with the analysis at the wavelength used. Dispersion Studies Preparation of Blends The influence of carrier milling on the drug aerosolization efficiency was evaluated using 5% w/w blends of NST. Blends of 1 g were prepared JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 5, MAY 2007
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by geometric hand mixing 50.00 mg NST with 950.00 mg of lactose monohydrate sample in a glass mortar using a spatula. Blend homogeneity was performed by analysing 35.00 1.00 mg samples of each blend (n ¼ 5, due to the relatively small blend size) five times for each powder mixture. In all cases, an acceptable degree of homogeneity was achieved with the mean drug content across all blends being within 100.0 3.0% of the theoretical value and each blend exhibiting a coefficient of variance <5.0%. Approximately 35.00 1.00 mg samples of each blend were manually filled into size 3 hard gelatine capsules (Capsugel1, NSW, Australia) and stored at 45% RH and 258C for 24 h prior to testing. In Vitro Aerosolization Studies
particles with an MMAD < 6.8 mm) as a percentage of the total recovered dose. The total recovered dose (loaded dose) was calculated as the total amount collected from the inhaler, throat, and all stages of the MSLI. Emitted dose was calculated as total recovered from all stages, postdevice. All samples were evaluated in triplicate and were randomized for formulation. Temperature and humidity during the in vitro investigation was 258C and 45% RH, respectively.
RESULTS AND DISCUSSION Particle Sizing of Processed Lactose Monohydrate Samples and NST Particle size analysis of NST gave median diameter of 1.10 mm with 90% particles less than 5.40 mm, suggesting the model drug was suitable for inhalation and DPI studies. The influence of milling time on the particle size distributions of both freshly milled and recrystallized lactose monohydrate samples was investigated. As expected, the milling process resulted in a significant reduction (ANOVA, p < 0.05) in the median particle diameter with respect to time (Fig. 1). Such observations are in agreement with previous investigations.23–25 In general, a decrease in median diameter from 115 1 mm for the untreated lactose monohydrate to 63 1 and 65 1 mm for freshly milled and recrystallized lactose monohydrate samples was observed, respectively. It interesting to note however, that incremental increases in mill time resulted in an
The influence of the carrier humidity conditioning on the aerosolization of NST from the freshly milled and milled-recrystallized lactose monohydrate carrier was investigated using apparatus C, the MSLI (Copley Scientific, Nottingham, UK), according to the method described in the British Pharmacopoeia 2005. Briefly, the apparatus consisted of a USP throat, four stages, each stage containing 20 mL of water, and a filter stage, which, at a flow rate of 60 L min1 produces MMAD cut-off points of 13.0, 6.8, 3.1, and 1.7 mm for stages 1, 2, 3, and 4, respectively. The flow rate through the MSLI was controlled by a GAST rotary vein pump and solenoid valve timer (Copley Scientific). Prior to testing, a 60 L min1 flow rate through the MSLI was set using a calibrated flow meter. The aerosolization performance of each blend was investigated using a CyclohalerTM DPI (Novartis, Surrey, UK). Briefly, a capsule of the formulation was placed into the sample compartment of the CyclohalerTM, inserted into a specially constructed MSLI mouthpiece adapter, primed and tested at 60 L min1 for 4 s. A 3 s delay prior to testing was instigated to allow the pump to settle. Deposited drug fractions were collected from the DPI and MSLI stages using water. In addition, the extracted solution for the filter stage was filtered through a 0.22 mm PVDF filter (Millex GV, Millipore, Billerica, MA) to remove traces of the glass filter. The amount of active contained in each aliquot was determined by UV spectrophotometry using the method described previously. The FPF was defined as the total amount of NST particles deposited in stages 3, 4 and filter (corresponding to
Figure 1. Influence of mill time on the median particle diameter for freshly milled (*) and recrystallized milled (*) lactose monohydrate samples.
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exponential decrease in median diameter (R2 ¼ 0.996). Again, such observations are expected since the probability of individual particles being involved in fracturing processes diminishes as the particles become smaller.25,26 The mean energy required to cause fractures increases with depletion in crystal cleavage planes while the magnitude of local stress from contact with the milling material (in this case, ceramic balls) decreases. In addition, increasing particle aggregation following particle size reduction may occur. As a consequence, energy may be expended in breaking up the aggregates instead of the particles. In such cases, particle size reduction will cease to occur at some practical limit.25 Secondly, as the particles become smaller and more numerous, friction diminishes and the sample may behave as a semi-solid. Larger particles may arch and protect smaller particles from impact, whilst smaller particles coat the grinding medium and cushioning the larger particles from impact. This ‘‘protection’’ may prevent further particle size reduction.27 Representative particle size distributions for the freshly milled and recrystallized lactose monohydrate samples are shown in Figure 2A and B, respectively. From Figure 2A it can be seen that large variations in the size distribution of both freshly milled and recrystallized lactose monohydrate exist, particularly in relation to the small particle fractions. To further investigate this, the variation in percentage particles less than 10 mm (classified as fines for the purpose of this paper) with milling time was studied. Analysis of the fines concentration with respect to mill time indicated a significant (ANOVA, p < 0.05) increase from 4.4% to 18.0% between 0 and 60 min mill time. Again, such observations are expected, since it has been suggested that cleavage planes, commonly found in crystals, fracture into many fine particles. This results in few relatively large particles, a number of fine particles and relatively few particles of intermediate size.27 In comparison, particle size distribution of the recrystallized milled samples suggested a reduced rate of increase in the fines percentage with milling time (4.4–10.1%, between 0 and 60 min mill time). Such observations are most likely due to ‘‘fusion’’ between the mill-induced fines and larger lactose monohydrate particles. Fusion of particles may be achieved by two possible mechanisms, solid–liquid bridge formation and/or particle fusion through amorphous recrystallization. For example, storage of samples at elevated humidDOI 10.1002/jps
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Figure 2. Particle size distributions of (A) freshly milled lactose monohydrate and (B) recrystallized milled samples.
ities may allow water vapor to condense in the capillaries that exist between individual, with increased levels of fine particles resulting in an increased number of capillaries.28,29 Furthermore, highly soluble materials, such as lactose monohydrate, may undergo limited dissolution at interparticulate contact points with subsequent solidification, thus resulting in solid–liquid bridge formation between particles, leading to particulate fusion.30 Similarly, such particle fusion could be facilitated by the presence of amorphous content on particulate surfaces recrystallising at elevated humidity.19,21 Scanning Electron Microscopy Representative SEM images of the 0, 30, and 60 min mill time freshly milled lactose monohydrate samples are shown in Figure 3A–C, respectively. As expected, images were in good JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 5, MAY 2007
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Figure 4. Representative SEM images of (A) 60 min freshly milled lactose monohydrate blend and (B) 60 min recrystallized lactose monohydrate blend.
Figure 3. Representative SEM images of freshly milled lactose monohydrate samples after (A) 0 min, (B) 30 min, and (C) 60 min mill times.
milled and recrystallized mill samples are shown in Figure 4A and B, respectively. From Figure 4A, it can be seen that the samples from the 60 min mill process exhibit discrete fine particulates distributed across the surface of the larger lactose monohydrate particles. In comparison, analysis of the recrystallized 60 min mill time samples (Fig. 4B), suggested many of the fine particulates had become ‘‘fused’’ to the large lactose monohydrate particulate surfaces. Again such observations are in good agreement with particle size analysis discussed previously. Amorphous Content
correlation with the particle size analysis, confirming that increasing the mill time resulted in both a decrease in median particle diameter with a concurrent increase in fines material. Representative high magnification images of the freshly
As previously discussed, the degree of amorphous content present in the milled lactose monohydrate samples was determined using a novel organicDVS technique. As expected, an increase in mill time resulted in an exponential increase in
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Figure 5. Influence of mill time on the degree of amorphous content in freshly milled lactose monohydrate samples. Milled-recrystallized lactose monohydrate samples were completely crystalline (0.00% amorphous content; not shown).
amorphous content (Fig. 5) (R2 ¼ 0.999), correlating with the concurrent decrease in median diameter observed by size analysis. Again, such observations may be expected, since amorphous content was introduced into the sample by surface molecular modification during the milling process.17 As the degree of comminution decreases, it is logical to conclude that the introduction of surface molecular damage would follow suit. In comparison, organic-DVS analysis of the 60 min mill time recrystallized sample suggested a completely crystalline material (0.0% amorphous content). Since the milling process, would most likely induce amorphous domains on the surface of the lactose, differences in interfacial forces between NST and the freshly milled or recrystallized system should exist. The freshly milled sample, under the experimental conditions used here, would be thermodynamically unstable and would have surface amorphous domains with a degree of molecular mobility relative to the environmental conditions (45% RH). Clearly, under such conditions, the force of interaction would be higher and result in a reduced FPF when compared to the re-crystallized lactose system. In Vitro Aerosolization Performance The aerosolization performance of NST from blends of milled and recrystallized lactose monohydrate was studied using a MSLI. Analysis of the deposition data suggested milling resulted in no DOI 10.1002/jps
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Figure 6. Relationship between mill time and fine particle fraction for freshly milled (*) and recrystallized milled (*) lactose monohydrate samples. *R2 relationship for freshly milled samples between 0 and 40 min mill time.
significant difference in either loaded or emitted dose across all mill times and for either freshly milled or recrystallized lactose monohydrate samples (ANOVA, p > 0.05). Mean loaded doses of 1614 79 and 1602 81 mg and emitted doses of 1264 58 and 1274 41 mg were observed for freshly milled and recrystallized samples, respectively. Since no difference in loaded or emitted dose was observed between all samples, the influence of milling and recrystallization of lactose monohydrate carriers on the aerosolization performance could be confidently evaluated. The FPF (MMAD < 6.8 mm) of the loaded dose was used as a measure of DPI performance. The influence of mill time on the FPF of both freshly milled and recrystallized lactose monohydrate samples is shown in Figure 6. In general, the comminution process had a significant influence on aerosolization performance of NST in both freshly milled and recrystallized samples (ANOVA, p < 0.05). Furthermore, with the changes in the physical properties of the carrier induced by ball milling, a linear relationship1 was observed between FPF and milling time for both freshly milled and recrystallized samples (R2 ¼ 0.954 and 0.938 for freshly milled and recrystallized samples, respectively). It is interesting to note however, a deviation from linearity for FPF occurred for the freshly milled samples, after 40 min mill time. In addition, analysis of FPF 1 Linear analysis for freshly milled samples is only applied between 0 and 40 min mill times.
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between freshly milled and recrystallized samples suggested after 10 min mill time, significant differences in FPF over the linear region between 20, 30, and 40 min mill times was observed (ANOVA, p < 0.05). Clearly, the process of milling induces significant variation in aerosolization performance with an apparent linear relationship between mill time and FPF over the range 0–40 and 0–60 min for freshly milled and recrystallized lactose monohydrate samples, respectively. As previously discussed, such observations may be attributed to many physical characteristics including amorphous content, variation in median diameter and an increase in fines. The relationships between such factors are discussed in more detail below. Influence of Median Particle Diameter on In Vitro Performance As discussed earlier, multiple changes in the physical properties of the carrier system were introduced into the system while milling. The most obvious change was the significant reduction in median particle diameter. Since the median particle diameter for both freshly milled and recrystallized samples was similar, analysis of the FPF, with respect to lactose monohydrate median diameter, suggested a poor relationship for both samples. In general, R2 values of 0.894 and 0.823 were observed for freshly milled and recrystallized samples, respectively (Fig. 7). Thus, a relationship between NST aerosolization
efficiency and median particle size was not as evident. A review of the literature revealed conflicting reports concerning the relationship between median particle size and FPF. As previously discussed, many early studies have suggested that reducing the median particle diameter of carriers significantly improves FPF,4,5,31 however, it should be remembered that with any energy induced particle size reduction processes, fine particles are ‘‘introduced’’. Furthermore, no detailed information concerning the fines content was given in these studies. Interestingly, two recent study by Islam et al.,7,12 and Steckel et al.,13 investigating the influence of particle size on DPI performance, reported no significant relationship between FPF and decreasing median particle diameter, which correlates with the data presented here.
Influence of Amorphous Content on In Vitro Performance The presence of meta-stable, that is amorphous, material in the surface of DPI carriers may have implications for product performance. An apparent relationship between amorphous content and FPF for freshly milled samples was observed (R2 ¼ 0.921). However, since recrystallized samples contained no detectable amorphous content there would be no relationship between FPF and amorphous content (e.g. a plot of % amorphous content against FPF in the recrystallized samples, would result in all data sitting at 0%). Furthermore, previous reports have suggested increased amorphous content in DPI systems resulted in decreased aerosolization performance.17,19,21 Clearly such relationships need to be studied further, in isolation of other physical factors.
Influence of Fines Content on In Vitro Performance
Figure 7. Influence of median particle diameter on fine particle fraction of freshly milled (*, R2 ¼ 0.894) and recrystallized milled (*, R2 ¼ 0.823) lactose monohydrate samples.
As previously discussed, a significant increase in fines with increased mill time was observed for both freshly milled and recrystallized lactose monohydrate samples. The relationship between fine lactose monohydrate content and FPF is shown in Figure 8. As with the variation of FPF and mill time FPF (Fig. 6), a linear relationship between FPF and the percentage of fines (<10 mm) present in each sample was observed. However, it is interesting to note that the relationship was more significant with R2 values of 0.974 and 0.982
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being observed for freshly milled and recrystallized lactose monohydrate samples, respectively. Again, as with the variation of FPF with mill time data, linearity for the freshly milled samples only existed up to a certain extent (equivalent to 40 min or 13.9% fines), after which, the FPF decreased. More importantly, when compared to the influence of mill time, the relationship between the fines content of either freshly milled or recrystallized samples and FPF indicated no significant difference with respect to fines content (ANOVA, p < 0.05). In simple terms, samples with less than 15% fines resulted in no significant difference in FPF between samples of milled or recrystallized lactose monohydrate carriers containing similar fines percentage. Such observations correlate with previous investigations. Zeng et al.,6,9 and Islam et al.,7,12 reported a significant reduction in FPF with a reduction in fines content. The FPF was returned to the original level when the fines removed were restored, and further improved with increasing fines content. Various studies reported a trend of improved FPF when fine particles (lactose monohydrate, glucose or PEG 6000) were introduced to coarse carrier mixtures, irrespective of coarse carrier particle size.8,10,11,32 In general, two mechanisms have been proposed to explain such observations: the ‘‘active site theory’’ and ‘‘multiplet/agglomeration’’ theory. In simple terms, the active site theory suggests that the increasing carrier fine concentration
occupies high energy-active sites, thus promoting drug adhesion to occur at relatively lower energy-passive sites.6,8–11,33 Clearly, the result of such an effect would be the easier detachment of drug particles and thus increased FPF. However, although the existences of such sites have been experimentally verified in recent publications,16–19,33 it is suggested that at such high drug loadings (5% w/w) active site effects would be minimal. Alternatively, adhesion and redistribution of ingredients, when fines are present, may produce a mixed agglomerate of drug and fine material (forming multiplets or agglomerates). Such agglomeration may result in improved drug aerosolization, since, due to simple physics, dispersion of larger agglomerates from larger carrier particles will be achieved at lower forces, when compared to typical individual drug carrier systems.10 In the case of such high drug loadings, the authors propose that this mechanism would most likely dominate FPF. Although such general theories are attractive, a deviation from linearity at high fine concentrations (>15% fines) still exists. One of the most likely explanations for the observed decrease in FPF in samples with fines >15% is a mixed agglomerate theory, where at high fines concentrations drug-lactose monohydrate agglomerates fail to adhere to the larger lactose monohydrate carrier particles and become segregated. Such segregation results in a biphasic system in that the typical ‘‘ordered’’ mix of micronized drug/lactose monohydrate becomes a two-component blend: the carrier/drug agglomerate particles and the free agglomerates. Such a blend may result in deviation from an expected agglomerate–carrier relationship. To further investigate the potential for such segregation, the particulate structure of formulations containing different levels of fine lactose monohydrate were investigated. Representative SEM images of formulations containing 18.0% (60 min freshly milled lactose monohydrate) and 10.1% (60 min recrystallized lactose monohydrate) fines are shown in Figure 9A and B, respectively. When comparing samples containing >15% fines (Fig. 9A), large drug-fine agglomerates, that were clearly separated from the coarse carrier, were present in the system. In contrast, with moderate fines content (Fig. 9B), a homogenous blend of mixed drug-fine agglomerates adhering to the coarse carrier surface was depicted. From such observations, it is reasonable to assume, that a critical agglomerate size may exist in drug-fine-
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Figure 8. Relationship between percentage lactose monohydrate fines content (<10 mm), fine particle fraction for freshly milled (*) and recrystallized milled (*) lactose monohydrate samples. *R2 relationship for freshly milled samples where fines are less than 15%.
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markedly reducing the FPF. However, these parameters needed to be studied in isolation (i.e. without the presence of fines) and are suggested for future investigation.
CONCLUSION
Figure 9. Representative SEM images of (A) freshly milled lactose monohydrate blends containing >15% fines and (B) recrystallized lactose monohydrate blends containing <15% fines.
carrier blends where segregation may result in a reduction in aerosolization performance. Indeed, in recent studies similar relationships were observed.10 Finally, although a direct relationship between fine concentration and aerosolization performance was observed (<15% fines), a discrepancy still exists when comparing the regression slopes of freshly milled and recrystallized lactose monohydrate samples (Fig. 8) (slope ¼ 1.56 and 1.08 for freshly milled and recrystallized samples, respectively). It is envisaged that this variation in slope may be due to increased adhesion between the drug particles and amorphous regions. It is also important to note, that in this study, drug particles were blended with the lactose postrecrystallization. It is envisaged that, if blended prior to recrystallization, the drug particles would possibly fuse to the lactose surface as with the fines, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 5, MAY 2007
Industrial processing of carrier materials used in DPI systems induces changes in the physical properties of the particles. Here, a simple ball milling process was used to produce particles which exhibited a reduced particle size, increased levels of fines and amorphous material. In addition, variation in storage conditions of the processed excipient was also shown to induce changes in fines and amorphous content. When used in a high dose DPI system, significant changes in the FPF were observed with increasing milling times. The relationships between milling time, physical property of the carrier and FPF were investigated. In general, the strongest relationship between carrier physical property and FPF was observed when considering the fines content. Such a relationship was independent of storage conditions, with increasing fines (<15%) resulting in a linear increase in FPF. Subsequently, the presence of fines was shown to play the predominating role in influencing DPI performance in this system. Furthermore, increasing fine content above 15% resulted in a deviation from linearity and may be related to changes in overall formulation characteristics. Finally, there appears to be some evidence that the presence of amorphous content may contribute to a decreased FPF. However, like the effect of particle size, further investigation is required to study the effect of these two parameters on dry powder dispersion in isolation, without the masking effect of fines content.
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4. French DL, Edwards DA, Niven RW. 1996. The influence of formulation on emission, deaggregation and deposition of dry powders for inhalation. J Aerosol Sci 27:769–783. 5. Steckel H, Mu¨ller BW. 1997. In vitro evaluation of dry powder inhalers II: influence of carrier particle size and concentration on in vitro deposition. Int J Pharm 154:31–37. 6. Zeng XM, Martin GP, Tee SK, Ghoush AA, Marriott C. 1999. Effects of particle size and adding sequence of fine lactose on the deposition of salbutamol sulphate from a dry powder formulation. Int J Pharm 182:133–144. 7. Islam N, Stewart P, Larson I, Hartley P. 2004. Effect of carrier size on the dispersion of salmeterol xinaofoate from interactive mixtures. J Pharm Sci 93:1030–1038. 8. Lucas P, Anderson K, Stanniforth JN. 1998. Protein deposition from dry powder inhalers: Fine particle multiplets as performance modifiers. Pharm Res 15:562. 9. Zeng XM, Martin GP, Tee SK, Marriott C. 1998. The role of fine particle lactose on the dispersion and deaggregation of salbutamol sulphate in an air stream in vitro. Int J Pharm 176:99–110. 10. Louey MD, Stewart PJ. 2002. Particle interactions involved in aerosol dispersion of ternary interactive mixtures. Pharm Res 19:1524–1531. 11. Louey MD, Razia S, Stewart PJ. 2003. Influence of physico-chemical carrier properties on the in vitro aerosol deposition from interactive mixtures. Int J Pharm 252:87–98. 12. Islam N, Stewart P, Larson I, Hartley P. 2004. Lactose surface modification by decantation: Are drug-fine lactose ratios the key to better dispersion of salmeterol xinafoate from lactose-interactive mixtures? Pharm Res 21:492–499. 13. Steckel H, Markefka P, teWierik H, Kammelar R. 2006. Effect of milling and sieving on functionality of dry powder inhalation products. Int J Pharm 309:51–59. 14. Zeng XM, Martin GP, Marriott C, Pritchard J. 2000. The influence of carrier morphology on drug delivery by dry powder inhalers. Int J Pharm 200: 93–106. 15. Flament MP, Leterme P, Gayot A. 2004. The influence of carrier roughness on adhesion, content uniformity and the in vitro deposition of terbutaline sulphate from dry powder inhalers. Int J Pharm 275:201–209. 16. Young PM, Cocconi D, Colombo P, Bettini R, Price R, Steele DF, Tobyn MJ. 2002. Characterization of a surface modified dry powder inhalation carrier prepared by ‘‘particle smoothing’’. J Pharm Pharmacol 54:1339–1344. 17. Price R, Young PM. 2005. On the physical transformations of processed pharmaceutical solids. Micron 36:519–524.
18. Buckton G, Darcy P. 1999. Assessment of disorder in crystalline powders—a review of analytical techniques and their application. Int J Pharm 179:141–158. 19. Young PM, Price R. 2004. The influence of humidity on the aerolisation of micronised and SEDS produced salbutamol sulphate. Eur J Pharm Sci 22:235–240. 20. Podczeck F, Newton JM, James MB. 1997. Variations in the adhesion force between a drug and carrier particles as a result of changes in the relative humidity of the air. Int J Pharm 149:151– 160. 21. Ward GH, Schultz RK. 1995. Processed-induced crystallinity changes in albuterol sulphate and its effect on powder physical stability. Pharm Res 12:773–779. 22. Young PM, Chiou H, Tee T, Traini D, Chan HK, Thielmann F, Burnett D. 2007. The use of organic vapor sorption to determine low levels of amorphous content in processed pharmaceutical powders. Drug Dev Ind Pharm 33:91–97. 23. Chen Y, Ding Y, Papadopoulos DG, Ghadiri M. 2004. Energy-based analysis of milling alphalactose monohydrate. J Pharm Sci 93:886–895. 24. Kwan CC, Chen YQ, Ding YL, Papadopoulos DG, Bentham AC, Ghadiri M. 2004. Development of a novel approach towards predicting the milling behaviour of pharmaceutical powders. Eur J Pharm Sci 23:327–336. 25. Berg TGO, Avis LE. 1970. Exploratory experiments on kinetics of comminution. Powder Technol 4:27– 31. 26. Piret EL. 1953. Fundamental aspects of grinding. Chem Eng Prog 49:56–62. 27. Parrott EL. 1974. Milling of pharmaceutical solids. [Review] [95 refs]. J Pharm Sci 63:813–829. 28. Coelho MC, Harnby N. 1978. Moisture bonding in powders. Powder Technol 2:201–205. 29. Schubert H. 1984. Capillary forces: Modelling and application in particulate technology. Powder Technol 37:105–116. 30. Young PM, Price R, Tobyn MJ, Buttrum M, Dey F. 2003. Effect of humidity on aerosolization of micronized drugs. Drug Dev Ind Pharm 29:959– 966. 31. Bell JH, Hartley PS, Cox JS. 1971. Dry powder aerosols. I. A new powder inhalation device. J Pharm Sci 60:1559–1564. 32. Zeng XM, Pandhal KH, Martin GP. 2000. The influence of lactose carrier on the content homogeneity and dispersibility of beclomethasone dipropionate from dry powder aerosols. Int J Pharm 197:41–52. 33. Young PM, Edge S, Traini D, Jones MD, Price R, El-Sabawi D, Urry C, Smith C. 2005. The influence of dose on the performance of dry powder inhalation systems. Int J Pharm 296:26–33.
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Received 10 May 2006; revised 18 June 2006; accepted 10 July 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20933
ABSTRACT: The influence of processing on the performance of carrier material used in dry powder inhalers was investigated. a-Lactose monohydrate crystals were processed by ball milling for cumulative time durations and their properties evaluated. As expected, milling reduced the median particle diameter while increasing fine particulate (<10 mm) and amorphous levels. Recrystallization of these partially amorphous samples resulted in a reduction in fines, elimination of amorphous material with little change in median diameter. To study the effects of processing on aerosolization performance, blends of lactose monohydrate with a model drug (nedocromil sodium trihydrate), were evaluated using an in vitro multistage liquid impinger (MSLI) model. In general, milling and storage of the carriers at high humidity (prior to blending) had a significant (ANOVA, p < 0.05) effect on the fine particle fractions (FPF; <6.8 mm). These effects were attributed predominantly to the fines content, showing a strong correlation between increased fines and FPF (R2 ¼ 0.974 and 0.982 for milled and recrystallized samples, respectively). However, this relationship only existed up to 15% fines concentration, after which agglomerate-carrier segregation was observed and FPF decreased significantly. These results suggest that, after processing, high-dose drug formulation performance is dominated by the presence of fines. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1331–1341, 2007
Keywords: milling; surface activation; fines; aerosolization; dry powder; inhalation; recrystallization; amorphous
INTRODUCTION Dry powder inhalers (DPI) are a novel route for drug delivery, with the capability of targeting disease states both locally (in the case of lung diseases such as asthma), and systemically (e.g. in the delivery of proteins and peptides). For effective deposition in the lower airways and deep lung, drug particles with aerodynamic particle sizes of <5 mm are required. However, such systems are highly cohesive due to the high Correspondence to: Paul M. Young (Telephone: þ61 2 9036 7035; Fax: þ61 2 9351 4391; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 1331–1341 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
surface area to mass ratio of the particulates. Cohesive systems pose a problem for the desired deaggregation of particles as uncontrolled agglomeration occurs naturally. Subsequently, such agglomeration may lead to formulation variations and a decrease in DPI efficacy. As a consequence, large inert carrier systems are employed as one method to overcome this problem, where the micron sized drug particles are blended with larger inert material to reduce agglomeration, improve flow and act as a diluent. Ideally, during inhalation, the drug particles are liberated from the carrier to penetrate the lower airways while the carrier impacts on the orthopharynx and is swallowed. As with all these
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systems, it is important to note, however, that the energy supplied by the patient during inhalation needs to be sufficient for liberation and aerosolization of the drug particulates. Currently, lactose is the most popular material approved for use in inhalation as carrier. A naturally occurring disaccharide sugar, lactose is found in milk, and is collected as a by-product of whey produced during the manufacturing of cheese. Lactose monohydrates for use in the pharmaceutical industry, are produced through precipitation from aqueous solution and is available in many particle size grades.1 Furthermore, to obtain inhalation grade lactose monohydrate, the crystals are processed through techniques such as milling and sieving to produce the desired carrier size characteristics.2 However, due to the industrial scale of such processes, inter-batch, and intersupplier variations in the physicochemical properties of the carriers have been observed, leading to variations in the aerosolization performance of the final formulations.2 Such batch-to-batch variation is most likely due to differences in fine particle content, particle size distribution, surface morphology, and amorphous content. Intrinsic carrier particle size has been believed for many years to play an important role in the performance of DPI.3–6 Following a reduction in physical particle diameter, a decrease in mean mass aerodynamic diameter (MMAD) is observed. In general, a decrease in MMAD has been associated with an increase in drug dispersion. However, a recent study by Islam et al.,7 suggested that the volume median diameter of carriers had no significant effect on drug dispersion when studied without the influence of fines content. Earlier studies did not specify the fine particle content, thus with a reduction in particle size, the increasing concentration of fines may have been masking the effect carrier size has on aerosol performance. As fine particles are commonly introduced inadvertently during the comminution process, the influence of fines (e.g. carrier particles with a diameter <15 mm) on DPI performance has been studied extensively. Lucas et al.,8 Zeng et al.,6,9 Louey et al.,10,11 and Islam et al.,7,12 have all demonstrated an improvement in dispersion with the presence of fine particles. More recently, Steckel et al.,13 indicated similar findings, suggested that variation in small quantities of fines (<5% below 15 mm diameter) in different sized sieved fractioned lactose formulations significantly influenced drug aerosolization performance.
Micronized nedocromil sodium trihydrate (NST) was obtained from Sonafi-Aventis (Cheshire, England). Crystalline a-lactose monohydrate (Lactochem1 crystals) was obtained from Borculo Domo (Zwolle, The Netherlands). Water was purified by reverse osmosis (MilliQ, Millipore, Molsheim, France). Analytical grade chloroform
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As discussed, processing methods such as milling may induce variation in surface morphology or result in increased amorphous content. Surface morphology has been demonstrated to directly influence the contact area between drug particle and carrier, leading to variations in interparticulate adhesion. Several studies have reported that variations in contact area, as a result of differing surface structure, could potentially compromise the aerosolization performance of the drug particles.14–16 In addition, the introduction of amorphous material during high energy mechanical processes is associated with a higher surface adhesion energy compared to crystalline surfaces.17–19 As a consequence of raised adhesion energy, poor deaggregation of drug particles is observed.20 No studies have yet been reported in relation to the direct impact of amorphous content on drug dispersion from carriers. However, the presence of amorphous material may cause problems, for example, due to the fusion of particles, resulting in poor dispersion.19–21 Milling is commonly used for processing of powders in the pharmaceutical industry, and in particular for inhalation drugs and carriers. When carriers are milled, various changes to the physical properties are induced. This is important since the reliability of the DPI product mainly depends on batch-to-batch consistency of the lactose monohydrate carrier. However, as mentioned previously, variations between batches do occur, and this study was initiated to investigate the effect of any material changes induced by milling on DPI performance. Further, as part of an ongoing study, the influence of storage at high humidity prior to blending was also investigated. The influence of these processes on DPI aerosolization efficiency was investigated using nedocromil sodium trihydrate as a model drug system, and was correlated with the physical properties of the carrier particles.
MATERIALS AND METHODS Materials
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and n-octane were obtained from Biolab (Victoria, Australia) and Fluka (Germany), respectively. Preparation of Lactose Monohydrate Samples Mechanical treatment of lactose monohydrate samples was achieved by comminution in a small volume ball mill (approximately 1 L) containing 60 ceramic balls (mean diameter of 19.3 0.7 mm). Samples of lactose monohydrate (approximately 100 g) were weighed into the ball mill which was rotated at 42 rev min1 for durations of 10, 20, 30, 40, 50, and 60 min. Each sample was then collected and stored in tightly sealed containers over phosphorus pentoxide prior to sampling or blending. In addition, to crystallize any amorphous material, present in the milled samples, approximately 10 g of each milled lactose monohydrate sample was transferred onto glass Petri-dishes and stored (3 weeks) in tightly sealed containers with a saturated solution of potassium chloride (relative humidity, RH, of 85%). The samples were regularly stirred to ensure moisture penetration into the powder bed. After 3 weeks, each sample was removed and transferred into containers with phosphorous pentoxide (0% RH) for a minimum of 24 h before sampling or blending. Physical Characterization Particle Sizing of Processed Lactose Monohydrate Samples and NST Particle sizing was performed by laser diffraction (Malvern Mastersizer S, Malvern Instruments Ltd., Malvern, UK) using a 300RF lens and automated small volume dispersion sampling unit. Approximately 200 mg of each lactose monohydrate sample or NST was dispersed in about 10 mL of chloroform, and added drop-wise into the sampling unit containing chloroform until an obscuration between 10% and 30% was obtained. Size distributions were based on 2000 sweeps for each sample, with refractive indices of 1.533, 1.358, and 1.444 for lactose monohydrate, NST and chloroform, respectively. Each sample was analysed in triplicate. Scanning Electron Microscopy Visualization of lactose monohydrate surface morphology and the uniformity in blends were investigated using scanning electron microscopy (SEM) (XL30, Philips, Japan) at 10 keV. Each DOI 10.1002/jps
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sample was mounted on a carbon sticky tab and platinum coated (10 nm thickness; Edwards E306A Sputter Coater, UK), prior to analysis. Images were obtained at magnifications of 1000 and 5000. Amorphous Content Quantification of Lactose Monohydrate Organic Dynamic Vapor Sorption (Organic DVS) was used to quantitatively determine the amorphous content in the milled lactose monohydrate samples using a method described elsewhere.22 In simple terms, the technique measures the adsorption of a dispersive molecule (n-octane) into the surface of a sample as a function of partial pressure. Since the relative adsorption of the molecule into amorphous and onto crystalline samples will vary, a calibration curve may be constructed by comparing the relative adsorption in blends of 100% crystalline and 100% amorphous samples. From this, the amorphous content of an unknown sample may be determined. Measurements were conducted using a DVS-1 (Surface Measurement Systems, Alperton, UK), at 258C, using n-octane as the organic probe. Approximately 100 mg of lactose monohydrate was weighed into the sample pan and exposed to a two-step octane partial pressure (p/po) cycle of 0–90%. Equilibrium at each step was determined by a dm/dt of 0.0002% min1. Each milled sample (n ¼ 5), and the amorphous content calculated from the calibration data reported elsewhere.22 In addition, a sample of the 60 min recrystallized sample was analysed to ensure full recrystallization. Drug Content Determination Quantification of NST content uniformity and in vitro deposition was determined by UV spectrophotometry (U-2000 spectrophotometer, Hitachi, Japan) at a wavelength of 376.5 nm. Samples were prepared and diluted appropriately in water. The calibration plot for NST was linear over the range 0.5–50.0 mg mL1 (R2 ¼ 1.00). Lactose monohydrate did not interfere with the analysis at the wavelength used. Dispersion Studies Preparation of Blends The influence of carrier milling on the drug aerosolization efficiency was evaluated using 5% w/w blends of NST. Blends of 1 g were prepared JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 5, MAY 2007
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by geometric hand mixing 50.00 mg NST with 950.00 mg of lactose monohydrate sample in a glass mortar using a spatula. Blend homogeneity was performed by analysing 35.00 1.00 mg samples of each blend (n ¼ 5, due to the relatively small blend size) five times for each powder mixture. In all cases, an acceptable degree of homogeneity was achieved with the mean drug content across all blends being within 100.0 3.0% of the theoretical value and each blend exhibiting a coefficient of variance <5.0%. Approximately 35.00 1.00 mg samples of each blend were manually filled into size 3 hard gelatine capsules (Capsugel1, NSW, Australia) and stored at 45% RH and 258C for 24 h prior to testing. In Vitro Aerosolization Studies
particles with an MMAD < 6.8 mm) as a percentage of the total recovered dose. The total recovered dose (loaded dose) was calculated as the total amount collected from the inhaler, throat, and all stages of the MSLI. Emitted dose was calculated as total recovered from all stages, postdevice. All samples were evaluated in triplicate and were randomized for formulation. Temperature and humidity during the in vitro investigation was 258C and 45% RH, respectively.
RESULTS AND DISCUSSION Particle Sizing of Processed Lactose Monohydrate Samples and NST Particle size analysis of NST gave median diameter of 1.10 mm with 90% particles less than 5.40 mm, suggesting the model drug was suitable for inhalation and DPI studies. The influence of milling time on the particle size distributions of both freshly milled and recrystallized lactose monohydrate samples was investigated. As expected, the milling process resulted in a significant reduction (ANOVA, p < 0.05) in the median particle diameter with respect to time (Fig. 1). Such observations are in agreement with previous investigations.23–25 In general, a decrease in median diameter from 115 1 mm for the untreated lactose monohydrate to 63 1 and 65 1 mm for freshly milled and recrystallized lactose monohydrate samples was observed, respectively. It interesting to note however, that incremental increases in mill time resulted in an
The influence of the carrier humidity conditioning on the aerosolization of NST from the freshly milled and milled-recrystallized lactose monohydrate carrier was investigated using apparatus C, the MSLI (Copley Scientific, Nottingham, UK), according to the method described in the British Pharmacopoeia 2005. Briefly, the apparatus consisted of a USP throat, four stages, each stage containing 20 mL of water, and a filter stage, which, at a flow rate of 60 L min1 produces MMAD cut-off points of 13.0, 6.8, 3.1, and 1.7 mm for stages 1, 2, 3, and 4, respectively. The flow rate through the MSLI was controlled by a GAST rotary vein pump and solenoid valve timer (Copley Scientific). Prior to testing, a 60 L min1 flow rate through the MSLI was set using a calibrated flow meter. The aerosolization performance of each blend was investigated using a CyclohalerTM DPI (Novartis, Surrey, UK). Briefly, a capsule of the formulation was placed into the sample compartment of the CyclohalerTM, inserted into a specially constructed MSLI mouthpiece adapter, primed and tested at 60 L min1 for 4 s. A 3 s delay prior to testing was instigated to allow the pump to settle. Deposited drug fractions were collected from the DPI and MSLI stages using water. In addition, the extracted solution for the filter stage was filtered through a 0.22 mm PVDF filter (Millex GV, Millipore, Billerica, MA) to remove traces of the glass filter. The amount of active contained in each aliquot was determined by UV spectrophotometry using the method described previously. The FPF was defined as the total amount of NST particles deposited in stages 3, 4 and filter (corresponding to
Figure 1. Influence of mill time on the median particle diameter for freshly milled (*) and recrystallized milled (*) lactose monohydrate samples.
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exponential decrease in median diameter (R2 ¼ 0.996). Again, such observations are expected since the probability of individual particles being involved in fracturing processes diminishes as the particles become smaller.25,26 The mean energy required to cause fractures increases with depletion in crystal cleavage planes while the magnitude of local stress from contact with the milling material (in this case, ceramic balls) decreases. In addition, increasing particle aggregation following particle size reduction may occur. As a consequence, energy may be expended in breaking up the aggregates instead of the particles. In such cases, particle size reduction will cease to occur at some practical limit.25 Secondly, as the particles become smaller and more numerous, friction diminishes and the sample may behave as a semi-solid. Larger particles may arch and protect smaller particles from impact, whilst smaller particles coat the grinding medium and cushioning the larger particles from impact. This ‘‘protection’’ may prevent further particle size reduction.27 Representative particle size distributions for the freshly milled and recrystallized lactose monohydrate samples are shown in Figure 2A and B, respectively. From Figure 2A it can be seen that large variations in the size distribution of both freshly milled and recrystallized lactose monohydrate exist, particularly in relation to the small particle fractions. To further investigate this, the variation in percentage particles less than 10 mm (classified as fines for the purpose of this paper) with milling time was studied. Analysis of the fines concentration with respect to mill time indicated a significant (ANOVA, p < 0.05) increase from 4.4% to 18.0% between 0 and 60 min mill time. Again, such observations are expected, since it has been suggested that cleavage planes, commonly found in crystals, fracture into many fine particles. This results in few relatively large particles, a number of fine particles and relatively few particles of intermediate size.27 In comparison, particle size distribution of the recrystallized milled samples suggested a reduced rate of increase in the fines percentage with milling time (4.4–10.1%, between 0 and 60 min mill time). Such observations are most likely due to ‘‘fusion’’ between the mill-induced fines and larger lactose monohydrate particles. Fusion of particles may be achieved by two possible mechanisms, solid–liquid bridge formation and/or particle fusion through amorphous recrystallization. For example, storage of samples at elevated humidDOI 10.1002/jps
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Figure 2. Particle size distributions of (A) freshly milled lactose monohydrate and (B) recrystallized milled samples.
ities may allow water vapor to condense in the capillaries that exist between individual, with increased levels of fine particles resulting in an increased number of capillaries.28,29 Furthermore, highly soluble materials, such as lactose monohydrate, may undergo limited dissolution at interparticulate contact points with subsequent solidification, thus resulting in solid–liquid bridge formation between particles, leading to particulate fusion.30 Similarly, such particle fusion could be facilitated by the presence of amorphous content on particulate surfaces recrystallising at elevated humidity.19,21 Scanning Electron Microscopy Representative SEM images of the 0, 30, and 60 min mill time freshly milled lactose monohydrate samples are shown in Figure 3A–C, respectively. As expected, images were in good JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 5, MAY 2007
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Figure 4. Representative SEM images of (A) 60 min freshly milled lactose monohydrate blend and (B) 60 min recrystallized lactose monohydrate blend.
Figure 3. Representative SEM images of freshly milled lactose monohydrate samples after (A) 0 min, (B) 30 min, and (C) 60 min mill times.
milled and recrystallized mill samples are shown in Figure 4A and B, respectively. From Figure 4A, it can be seen that the samples from the 60 min mill process exhibit discrete fine particulates distributed across the surface of the larger lactose monohydrate particles. In comparison, analysis of the recrystallized 60 min mill time samples (Fig. 4B), suggested many of the fine particulates had become ‘‘fused’’ to the large lactose monohydrate particulate surfaces. Again such observations are in good agreement with particle size analysis discussed previously. Amorphous Content
correlation with the particle size analysis, confirming that increasing the mill time resulted in both a decrease in median particle diameter with a concurrent increase in fines material. Representative high magnification images of the freshly
As previously discussed, the degree of amorphous content present in the milled lactose monohydrate samples was determined using a novel organicDVS technique. As expected, an increase in mill time resulted in an exponential increase in
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Figure 5. Influence of mill time on the degree of amorphous content in freshly milled lactose monohydrate samples. Milled-recrystallized lactose monohydrate samples were completely crystalline (0.00% amorphous content; not shown).
amorphous content (Fig. 5) (R2 ¼ 0.999), correlating with the concurrent decrease in median diameter observed by size analysis. Again, such observations may be expected, since amorphous content was introduced into the sample by surface molecular modification during the milling process.17 As the degree of comminution decreases, it is logical to conclude that the introduction of surface molecular damage would follow suit. In comparison, organic-DVS analysis of the 60 min mill time recrystallized sample suggested a completely crystalline material (0.0% amorphous content). Since the milling process, would most likely induce amorphous domains on the surface of the lactose, differences in interfacial forces between NST and the freshly milled or recrystallized system should exist. The freshly milled sample, under the experimental conditions used here, would be thermodynamically unstable and would have surface amorphous domains with a degree of molecular mobility relative to the environmental conditions (45% RH). Clearly, under such conditions, the force of interaction would be higher and result in a reduced FPF when compared to the re-crystallized lactose system. In Vitro Aerosolization Performance The aerosolization performance of NST from blends of milled and recrystallized lactose monohydrate was studied using a MSLI. Analysis of the deposition data suggested milling resulted in no DOI 10.1002/jps
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Figure 6. Relationship between mill time and fine particle fraction for freshly milled (*) and recrystallized milled (*) lactose monohydrate samples. *R2 relationship for freshly milled samples between 0 and 40 min mill time.
significant difference in either loaded or emitted dose across all mill times and for either freshly milled or recrystallized lactose monohydrate samples (ANOVA, p > 0.05). Mean loaded doses of 1614 79 and 1602 81 mg and emitted doses of 1264 58 and 1274 41 mg were observed for freshly milled and recrystallized samples, respectively. Since no difference in loaded or emitted dose was observed between all samples, the influence of milling and recrystallization of lactose monohydrate carriers on the aerosolization performance could be confidently evaluated. The FPF (MMAD < 6.8 mm) of the loaded dose was used as a measure of DPI performance. The influence of mill time on the FPF of both freshly milled and recrystallized lactose monohydrate samples is shown in Figure 6. In general, the comminution process had a significant influence on aerosolization performance of NST in both freshly milled and recrystallized samples (ANOVA, p < 0.05). Furthermore, with the changes in the physical properties of the carrier induced by ball milling, a linear relationship1 was observed between FPF and milling time for both freshly milled and recrystallized samples (R2 ¼ 0.954 and 0.938 for freshly milled and recrystallized samples, respectively). It is interesting to note however, a deviation from linearity for FPF occurred for the freshly milled samples, after 40 min mill time. In addition, analysis of FPF 1 Linear analysis for freshly milled samples is only applied between 0 and 40 min mill times.
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between freshly milled and recrystallized samples suggested after 10 min mill time, significant differences in FPF over the linear region between 20, 30, and 40 min mill times was observed (ANOVA, p < 0.05). Clearly, the process of milling induces significant variation in aerosolization performance with an apparent linear relationship between mill time and FPF over the range 0–40 and 0–60 min for freshly milled and recrystallized lactose monohydrate samples, respectively. As previously discussed, such observations may be attributed to many physical characteristics including amorphous content, variation in median diameter and an increase in fines. The relationships between such factors are discussed in more detail below. Influence of Median Particle Diameter on In Vitro Performance As discussed earlier, multiple changes in the physical properties of the carrier system were introduced into the system while milling. The most obvious change was the significant reduction in median particle diameter. Since the median particle diameter for both freshly milled and recrystallized samples was similar, analysis of the FPF, with respect to lactose monohydrate median diameter, suggested a poor relationship for both samples. In general, R2 values of 0.894 and 0.823 were observed for freshly milled and recrystallized samples, respectively (Fig. 7). Thus, a relationship between NST aerosolization
efficiency and median particle size was not as evident. A review of the literature revealed conflicting reports concerning the relationship between median particle size and FPF. As previously discussed, many early studies have suggested that reducing the median particle diameter of carriers significantly improves FPF,4,5,31 however, it should be remembered that with any energy induced particle size reduction processes, fine particles are ‘‘introduced’’. Furthermore, no detailed information concerning the fines content was given in these studies. Interestingly, two recent study by Islam et al.,7,12 and Steckel et al.,13 investigating the influence of particle size on DPI performance, reported no significant relationship between FPF and decreasing median particle diameter, which correlates with the data presented here.
Influence of Amorphous Content on In Vitro Performance The presence of meta-stable, that is amorphous, material in the surface of DPI carriers may have implications for product performance. An apparent relationship between amorphous content and FPF for freshly milled samples was observed (R2 ¼ 0.921). However, since recrystallized samples contained no detectable amorphous content there would be no relationship between FPF and amorphous content (e.g. a plot of % amorphous content against FPF in the recrystallized samples, would result in all data sitting at 0%). Furthermore, previous reports have suggested increased amorphous content in DPI systems resulted in decreased aerosolization performance.17,19,21 Clearly such relationships need to be studied further, in isolation of other physical factors.
Influence of Fines Content on In Vitro Performance
Figure 7. Influence of median particle diameter on fine particle fraction of freshly milled (*, R2 ¼ 0.894) and recrystallized milled (*, R2 ¼ 0.823) lactose monohydrate samples.
As previously discussed, a significant increase in fines with increased mill time was observed for both freshly milled and recrystallized lactose monohydrate samples. The relationship between fine lactose monohydrate content and FPF is shown in Figure 8. As with the variation of FPF and mill time FPF (Fig. 6), a linear relationship between FPF and the percentage of fines (<10 mm) present in each sample was observed. However, it is interesting to note that the relationship was more significant with R2 values of 0.974 and 0.982
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being observed for freshly milled and recrystallized lactose monohydrate samples, respectively. Again, as with the variation of FPF with mill time data, linearity for the freshly milled samples only existed up to a certain extent (equivalent to 40 min or 13.9% fines), after which, the FPF decreased. More importantly, when compared to the influence of mill time, the relationship between the fines content of either freshly milled or recrystallized samples and FPF indicated no significant difference with respect to fines content (ANOVA, p < 0.05). In simple terms, samples with less than 15% fines resulted in no significant difference in FPF between samples of milled or recrystallized lactose monohydrate carriers containing similar fines percentage. Such observations correlate with previous investigations. Zeng et al.,6,9 and Islam et al.,7,12 reported a significant reduction in FPF with a reduction in fines content. The FPF was returned to the original level when the fines removed were restored, and further improved with increasing fines content. Various studies reported a trend of improved FPF when fine particles (lactose monohydrate, glucose or PEG 6000) were introduced to coarse carrier mixtures, irrespective of coarse carrier particle size.8,10,11,32 In general, two mechanisms have been proposed to explain such observations: the ‘‘active site theory’’ and ‘‘multiplet/agglomeration’’ theory. In simple terms, the active site theory suggests that the increasing carrier fine concentration
occupies high energy-active sites, thus promoting drug adhesion to occur at relatively lower energy-passive sites.6,8–11,33 Clearly, the result of such an effect would be the easier detachment of drug particles and thus increased FPF. However, although the existences of such sites have been experimentally verified in recent publications,16–19,33 it is suggested that at such high drug loadings (5% w/w) active site effects would be minimal. Alternatively, adhesion and redistribution of ingredients, when fines are present, may produce a mixed agglomerate of drug and fine material (forming multiplets or agglomerates). Such agglomeration may result in improved drug aerosolization, since, due to simple physics, dispersion of larger agglomerates from larger carrier particles will be achieved at lower forces, when compared to typical individual drug carrier systems.10 In the case of such high drug loadings, the authors propose that this mechanism would most likely dominate FPF. Although such general theories are attractive, a deviation from linearity at high fine concentrations (>15% fines) still exists. One of the most likely explanations for the observed decrease in FPF in samples with fines >15% is a mixed agglomerate theory, where at high fines concentrations drug-lactose monohydrate agglomerates fail to adhere to the larger lactose monohydrate carrier particles and become segregated. Such segregation results in a biphasic system in that the typical ‘‘ordered’’ mix of micronized drug/lactose monohydrate becomes a two-component blend: the carrier/drug agglomerate particles and the free agglomerates. Such a blend may result in deviation from an expected agglomerate–carrier relationship. To further investigate the potential for such segregation, the particulate structure of formulations containing different levels of fine lactose monohydrate were investigated. Representative SEM images of formulations containing 18.0% (60 min freshly milled lactose monohydrate) and 10.1% (60 min recrystallized lactose monohydrate) fines are shown in Figure 9A and B, respectively. When comparing samples containing >15% fines (Fig. 9A), large drug-fine agglomerates, that were clearly separated from the coarse carrier, were present in the system. In contrast, with moderate fines content (Fig. 9B), a homogenous blend of mixed drug-fine agglomerates adhering to the coarse carrier surface was depicted. From such observations, it is reasonable to assume, that a critical agglomerate size may exist in drug-fine-
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Figure 8. Relationship between percentage lactose monohydrate fines content (<10 mm), fine particle fraction for freshly milled (*) and recrystallized milled (*) lactose monohydrate samples. *R2 relationship for freshly milled samples where fines are less than 15%.
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markedly reducing the FPF. However, these parameters needed to be studied in isolation (i.e. without the presence of fines) and are suggested for future investigation.
CONCLUSION
Figure 9. Representative SEM images of (A) freshly milled lactose monohydrate blends containing >15% fines and (B) recrystallized lactose monohydrate blends containing <15% fines.
carrier blends where segregation may result in a reduction in aerosolization performance. Indeed, in recent studies similar relationships were observed.10 Finally, although a direct relationship between fine concentration and aerosolization performance was observed (<15% fines), a discrepancy still exists when comparing the regression slopes of freshly milled and recrystallized lactose monohydrate samples (Fig. 8) (slope ¼ 1.56 and 1.08 for freshly milled and recrystallized samples, respectively). It is envisaged that this variation in slope may be due to increased adhesion between the drug particles and amorphous regions. It is also important to note, that in this study, drug particles were blended with the lactose postrecrystallization. It is envisaged that, if blended prior to recrystallization, the drug particles would possibly fuse to the lactose surface as with the fines, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 5, MAY 2007
Industrial processing of carrier materials used in DPI systems induces changes in the physical properties of the particles. Here, a simple ball milling process was used to produce particles which exhibited a reduced particle size, increased levels of fines and amorphous material. In addition, variation in storage conditions of the processed excipient was also shown to induce changes in fines and amorphous content. When used in a high dose DPI system, significant changes in the FPF were observed with increasing milling times. The relationships between milling time, physical property of the carrier and FPF were investigated. In general, the strongest relationship between carrier physical property and FPF was observed when considering the fines content. Such a relationship was independent of storage conditions, with increasing fines (<15%) resulting in a linear increase in FPF. Subsequently, the presence of fines was shown to play the predominating role in influencing DPI performance in this system. Furthermore, increasing fine content above 15% resulted in a deviation from linearity and may be related to changes in overall formulation characteristics. Finally, there appears to be some evidence that the presence of amorphous content may contribute to a decreased FPF. However, like the effect of particle size, further investigation is required to study the effect of these two parameters on dry powder dispersion in isolation, without the masking effect of fines content.
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