Improved aerosolization performance of salbutamol sulfate formulated with lactose crystallized from...

Improved Aerosolization Performance of Salbutamol SulfateFormulated with Lactose Crystallized from Binary Mixturesof Ethanol—Acetone

WASEEM KAIALY,1,2 MARTYN D. TICEHURST,3 JOHN MURPHY,3 ALI NOKHODCHI1

1Chemistry and Drug Delivery Group, Medway School of Pharmacy, University of Kent, Chatham, Kent ME4 4TB, United Kingdom

2Pharmaceutics and Pharmaceutical Technology Department, University of Damascus, Damascus 30621, Syria

3Pfizer Global R & D, Pharmaceutical Sciences, Sandwich, Kent CT13 9NJ, United Kingdom

Received 15 September 2010; revised 25 November 2010; accepted 20 December 2010

Published online 25 January 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22483

ABSTRACT: It has been shown that dry powder inhaler (DPI) formulations typically achievelow fine particle fractions (poor performance). A commonly held theory is that this is due, atleast in part, to low levels of detachment of drug from lactose during aerosolization as a result ofstrong adhesion of drug particles to the carrier surfaces. Therefore, the purpose of the presentstudy is to overcome poor aerosolization performance of DPI formulation by modification oflactose particles. Lactose particles were crystallized by adding solution in water to different ra-tios of binary mixtures of ethanol—acetone. The results showed that modified lactose particleshad exceptional aerosolization performance that makes them superior to commercial lactoseparticles. Morphology assessment showed that crystallized lactose particles were less elon-gated, more irregular in shape, and composed of smaller primary lactose particles comparedwith commercial lactose. Solid-state characterization showed that commercial lactose particleswere "-lactose monohydrate, whereas crystallized lactose particles were a mixture of "-lactosemonohydrate and $-lactose according to the ratio of ethanol—acetone used during crystalliza-tion process. The enhanced performance could be mainly due to rougher surface and/or higheramounts of fines compared with the lactose crystallized from pure ethanol or commercial lactose.© 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:2665–2684,2011Keywords: pulmonary delivery; particle size; crystallization; precipitation; solid state; drypowder inhalers; crystallized lactose; particle shape; aerosolization performance; salbutamolsulfate.

INTRODUCTION

Generally, dry powder inhaler (DPI) formulations con-sist of blends of micronized drug particles in the rangeof 1–5 :m with lactose particles. There are manyphysicochemical properties such as particle size,1 par-ticle shape,2 particle surface texture,3 particle electro-static properties,4 particle hygroscopicity,5 and parti-cle contact area1 that have been shown to have a sig-nificant impact on the drug delivery via DPIs. Thishas attracted researchers in the field of particle en-gineering where many attempts have been made todesign carrier particles with predetermined proper-

Correspondence to: Ali Nokhodchi (Telephone: +44-1634-202947; Fax: +44-1634-883927; E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 100, 2665–2684 (2011)© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

ties that will enhance DPI formulation performance.Many studies were reported about the effect of car-rier size,6–8 carrier surface texture,9–11 and carrierparticle shape12,13 on DPI performance. Typically,carrier particles in drug—carrier formulations areused in the size range of 63–90 :m.14 However, sev-eral studies have shown enhanced DPI performancewhen the carrier size was below 63 :m.15–17 Contra-dictory results have been reported regarding the in-crease of aerosol particle surface smoothness9,16,18,19

or roughness11,18,20,21 on the improvement of DPIperformance. Therefore, an optimum particle sur-face roughness or smoothness was suggested, whichgives a maximal inhalation efficiency.10,22–24 DPIperformance has also been enhanced when theelongation ratio of either carrier particles13,15,16ordrug particles9,25 increased.

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2666 KAIALY ET AL.

From a review of the literature, it can be concludedthat effective methods to improve the performanceof DPI is to modify drug or carrier particles.26–28

As DPI performance is most importantly affected byparticle—particle interactions,29 any changes in drugor carrier particle properties have the potential to al-ter DPI performance. However, it is important to con-sider DPI design as a potential option for improvedperformance of DPI,30 as small variations in the de-sign of inhaler device can have a profound effect onDPI performance.31

The use of binary mixtures of nonsolvent to mod-ify lactose particles for inhalation purpose was firsttime introduced by Kaialy et al.32 In this study, theeffect of using binary mixtures of ethanol—butanol(dipole moment of 1.69 and 1.52, respectively) on lac-tose physicochemical and in vitro deposition proper-ties was investigated. The performance of DPI formu-lation was shown to increase as more butanol wasused to crystallize lactose particles. In the presentstudy, an attempt has been made to crystallize lac-tose particles using two miscible solvents that havea larger difference in dipole moments (ethanol andacetone; dipole moment 1.69 and 2.91, respectively).

The aims of this work were: First, to attempt tomodify the particle morphology of lactose monohy-drate using different ratios of binary mixtures ofethanol—acetone to improve its performance to de-liver salbutamol sulfate (SS) to the lower airwayscompared with the commercial lactose. Second, to ob-serve whether the changes in the physicochemical,and thus aerosolization, properties of the lactose areinfluenced by the relative difference in the dipole mo-ment of solvents; the results obtained in the presentstudy were compared with the previous results ob-tained by Kaialy et al.32 In addition to these two aims,this investigation provides datasets that can be usedto increase the wider understanding about the effectof lactose carrier properties on DPI aerosolization per-formance.

MATERIALS AND METHODS

Lactose (DMV International, Veghel, the Nether-lands) and micronized SS (LB Bohle, Ennigerloh,Germany) were used. Analytical reagent grade of ab-solute ethanol and acetone were obtained from FisherScientific, Loughborough, UK.

Modification of Lactose Crystals

Lactose (30 g) was dissolved in 100 mL of distilled wa-ter to produce a 30% supersaturated lactose solutionunder heating (55◦C) and stirring (400 rpm) condi-tions. When it was prepared, the temperature of thissaturated solution was kept constant and then 5 mLof the solution was added to different combinationsof ethanol—acetone (antisolvents) with constant vol-

ume of 80 mL (80:0, 60:20, 40:40, 20:60, and 0:80)at a constant rate of 5 mL/min. As soon as the lac-tose solution was added, lactose crystals were imme-diately precipitated and the solution was kept undera constant speed of stirring (400 rpm) for 10 min atroom temperature (22◦C). The resulted crystals werecollected by filtration under vacuum and left in thedrying oven for 24 h at 50◦C. The dried crystals werekept in a sealed glass vial until further investigation.

Selection of Lactose Particle Size Fraction

All crystallized lactose powders were sieved to ob-tain particle size in the range of 63–90 :m. This wasachieved by pouring the lactose monohydrate on topof a 90 :m sieve, which was placed on top of a 63 :msieve. The sieves were then shaken for 15 min, and thelactose particles were then collected and transferredto a glass vial and sealed until required for furtherinvestigation. Commercial DMV lactose was treatedin the same manner as a control.

Image Analysis Using Optical Microscopy

A small amount of powder (about 20 mg) was dis-persed on a microscope slide. One hundred particles ineach sample were selected and measured, and the sur-face volume mean diameter, Feret diameters, round-ness (Eq. 1), and elongation ratio (Eq. 2) were cal-culated using image analysis software as describedpreviously.32

Roundness =[

(perimeter)2

(4 × B× area)

](1)

Elongation ratio =(

maximum Feret diameterminimum Feret diameter

)(2)

Scanning Electron Microscope

Electron micrographs of crystallized and original lac-tose samples were obtained using a scanning elec-tron microscope (SEM; Philips XL 20, Eindhoven, theNetherlands) operating at 15 kV. The specimens weremounted on a metal stub with double-sided adhesivetape and coated under vacuum with gold in an argonatmosphere prior to observation. Different magnifica-tions were used to investigate the effect of crystalliza-tion conditions on morphology of lactose particles.

Atomic Force Microscopy

Atomic force microscopy (AFM) was performed us-ing a Veeco MultiMode atomic force microscopeequipped with an E-type scanner operating via aVeeco Nanoscope IIIa controller (Veeco Instruments,Cambridge, UK). Images were made in tapping modeusing a Veeco RTESP phosphorous-doped silicon tipwith a nominal force constant of 40 nm at a scanning

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 7, JULY 2011 DOI 10.1002/jps

AEROSOLOZATION PERFORMANCE OF SALBUTAMOL SULPHATE CONTAINING CRYSTALLIZED LACCTOSE 2667

rate of 1 Hz and resolution of 256 lines per image.Sufficient particles were imaged at different scales ofscrutiny to produce three representative images persample at scales of 3 :m, 1:m, and 333 nm. Rough-ness analysis was performed using Veeco Nanoscopesoftware (version 6.13; Veeco Instruments) on a 333 ×333 nm region from each image determined to be flatand free of macroscale changes in height such as tran-sitions between overlapping particles or between dif-ferent faces of the same particle lying in differentplanes. Roughness values calculated were both theroot mean square average (Rq) and arithmetic meanaverage (Ra) of the difference in height recorded ateach data point and the mean plane, and presentedas the average of the three measurements for eachsample.

Particle Size Distribution Measurement

Particle size analysis was conducted using a Sym-patec (Clausthal-Zellerfeld, Germany) laser diffrac-tion particle size analyzer. The volume mean diam-eter (VMD) and mean particle size (D10%, D50%, andD90%) were calculated automatically using the soft-ware provided.32 Span value, which is a measure ofthe width of the size distribution, for each sample wasalso calculated using the following equation:

Span =(

D90% − D10%

D50%

)(3)

where D90%, D10%, and D50% are the equivalent volumediameters at 90%, 10%, and 50% cumulative volume,respectively.

Powder Flow Properties

Carr’s index and Hausner ratio were employed to givethe indication of flowability of each tested powder.The powder was filled in a 5-mL measuring cylin-der and after recording the volume (bulk volume), thecylinder was tapped 100 times and the new volumewas recorded (tap volume). Preliminary experimentshowed that 100 taps is enough to reach maximumreduction in the volume of powder bed. Then Carr’sIndex and Hasuner ratio were calculated using thefollowing equations:33,34

CI =[

(tapped density − bulk density)tapped density

]× 100 (4)

Hausner ratio = tap densitybulk density

(5)

Carr’s index values of less than 25% and Hausnerratio values less than 1.25 indicate acceptable flowproperties.

True Density Measurement

The true density of all lactose samples was mea-sured using an ultrapycnometer 1000 (Quantachrom,Boynton Beach, Florida, USA) under helium gas andwas calculated from a mean of three determinations.The input gas pressure was 19 psi and the equilibriumtime was 1 min.

Fourier-Transform Infrared Spectroscopy

Fourier-transform infrared (FTIR) spectra were usedto investigate any changes in crystallized lactosein molecular level during the crystallization or dry-ing processes. These spectra were taken by FTIR(PerkinElmer, Massachusetts, USA) for differentcrystallized lactose samples. The scanning range was450–4000 cm−1. The sample (several milligrams) wasplaced on the middle of the sample stage and force wasapplied by the top of the arm of the sample stage. Af-ter obtaining sharp peaks with reasonable intensity,the spectra acquired were the results of averagingfour scans at 1 cm−1.

Differential Scanning Calorimetry (DSC)

A differential scanning calorimeter (DSC7; MettlerToledo, Switzerland) was used to investigate the solidform of the different lactose samples. The equipmentwas calibrated using indium and zinc. Samples wereheated from 25◦C to 300◦C at a scanning rate of 10 K/min in aluminum pans under nitrogen gas. The melt-ing points and their enthalpies were calculated by thesoftware (Mettler, Switzerland).

Preparation of Powder Formulation

The crystallized sieved lactose in the range of 63–90 :m was blended with SS in a ratio of 67.5:1 (w/w).This blending was carried out using a Turbula R© mixer(Willy A. Bachofen AG, Maschinenfabrik, Basel,Switzerland) at a constant speed of 100 rpm for 30 minto form an ordered mixture. This speed was keptconstant for blending of all formulations. The sam-ples were stored for at least 24 h after the prepara-tion of formulation blends prior to performing depo-sition studies to allow them to physically stabilize orequalize.

The electrostatic properties of the materials shouldalways be considered when processing DPI formula-tions. The crystallized lactose particles experienced alarge number of different manufacturing and han-dling steps. These steps could have an influenceon the electrostatic properties of the powder, poten-tially causing high levels of agglomeration and pow-der sticking on the inhaler walls. However, this wasnot the case in the present study as only very smallamounts of agglomerated powder were observed oninhaler walls during washing processes indicating theefficient aerosolization for all formulation powders.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 7, JULY 2011

2668 KAIALY ET AL.

This was confirmed by emission values as more than94% of drug was recovered from different stages ofmultistage liquid impinger (MSLI), excluding the de-vice and capsule.

Measurement of Dose Uniformity

After blending the drug with carrier, the homogeneityof the drug content in each of the powder formulationswas examined by taking a minimum of five randomlyselected samples from different positions within theblend. Each sample weighing 33 ± 1.5 mg (this wasthe amount of the powder mixture to be introducedinto each capsule) was dissolved in 100 mL of watercontained in a volumetric flask. The amount of theactive drug (SS) in each powder formulation was de-termined using a high-performance liquid chromatog-raphy (HPLC) method, as described elsewhere.32 Thedegree of uniformity (homogeneity) was expressed interms of the percentage coefficient of variation (CV%).

Capsule Filling

Each formulation was filled manually in a hardgelatin capsule (size 3) with 33 ± 1.5 mg such thateach capsule contained 481 ± 22 :g SS, which is inaccordance with nominal dose of Ventolin Rotacaps R©

(GlaxoSmithKline, Victoria, Australia).

Deposition Test of SS

Powder pulmonary deposition profiles of all testeddry powder inhaler formulations were assessed invitro using MSLI equipped with a USP inductionport (Copley Scientific, Nottingham, UK). Prior totesting, 20 mL of distilled water was introducedto the stages 1, 2, 3, and 4 of the MSLI in orderto make the collection surfaces wet. A filter paper(Whatman R©;Maidstone, UK; pore size, <0.45 :m)was introduced in the stage 5 of the impinger. The in-haler (Aerolizer R©, Novartis, Basle, Switzerland) con-tains one capsule and each hard gelatin capsule wasfilled with accurately weighed 33 ± 1.5 mg of the pow-der formulation blend (ratio of drug to carrier was1:67.5). The inhaler containing the capsule was fit-ted in a mouthpiece adaptor and then connected tothe impinger induction port (IP). Then the capsulewas pierced and 10 actuations (10 capsules) of eachpowder were delivered consecutively to the MSLI ata pressure drop of 4 kPa. The suitable operating flowrate for the Aerolizer R© to obtain that pressure was 92L/min and the time that each actuation occurred wasover 2.6 s. The collection procedure of drug from eachstage of MSLI was explained in details elsewhere.32

Several parameters were employed as descriptorsof drug aerolization efficiency. The recovered dose(RD) was the sum of the weights of drug (:g) recov-ered from inhaler device, mouthpiece adaptor, IP, andall stages of the impinger. The emitted dose (ED) wasthe amount of drug emitted from the inhaler device

and collected from the IP and all stages of the im-pinger. The percent total recovery (% recovery) wascalculated as the ratio of the RD to the theoreticaldose (481 ± 22 :g). The percent emission was theratio of the ED to RD.

The impaction loss was calculated as the sum ofdrug amounts collected from the IP and stage 1 of theMSLI expressed as a percentage to the RD. In orderto calculate the fine particle fraction (FPF), a plot wasdrawn for each impaction of the cumulative mass ofpowder less than the stated size of each stage of theimpactor as a percentage to the total ED recoveredfrom the impactor against the log value of the effec-tive cutoff diameter.20,23,15,16,32,35,36 The FPF was cal-culated from that plot as the fraction of powder emit-ted from the inhaler with an aerodynamic diameterof 5 :m or less. Mass median aerodynamic diameter(MMAD) and geometric standard deviation (GSD) cal-culations were described in details elsewhere.15,16,32

RESULTS AND DISCUSSION

Crystallization Procedure

During the crystallization of lactose, it was observedthat lactose crystals were formed immediately dur-ing the addition of lactose solution onto antisolvent(ethanol—acetone). This high speed of nucleationcould be responsible for wide variation in particle sizedistribution of crystallized lactose (unsieved) as it isevident from span values reported in Table 1. Whenthe dried particles were collected, all the sampleswere similar in appearance (off-white powder) and thepercentage yield varied between 74% and 82%. In pre-vious study, when acetone was replaced by butanol,the percentage yield varied between 50%—100%.32

This indicates that the type of solvent plays a ma-jor role in the percentage yield, which could probablybe due to a difference in the solubility of lactose indifferent organic solvents.

Characterization of Particle Morphologyof Crystallized Lactose

After sieving, roundness and elongation ratio weremeasured for all crystallized lactose samples and com-pared with commercial lactose (Table 2). Elongationratio, which is the length expressed as a function ofthe width,37 has slightly decreased from 1.64 ± 0.35for commercial lactose particles to the range between1.47 ± 0.24 and 1.55 ± 0.36 for crystallized lactosesamples. Lower elongation ratio for crystallized lac-tose particles indicates that they are less elongated.On the contrary, roundness values have increasedfrom 1.36 ± 0.13 for commercial lactose sample to therange between 1.70 ± 0.36 and 2.09 ± 0.43 for crystal-lized lactose samples. Comparing these results withpreviously published data showed that the presence



Table 1. Unsieved Particle Size Distribution for Commercial Lactose Powder and Different Crystallized Lactose Powders UsingDifferent Volume Fractions of Ethanol—Acetone Expressed as D10%, D50%, D90%, VMD, SV, span, and percent of fine particles 10.50 :mor Less (Values are Mean ± SD, n = 3)

Unsieved Lactose D10% (:m) D50% (:m) D90% (:m) VMD(:m) SV(m2/cm3) 10.50 :m or Less (%) Span

Commercial 16.12 ± 0.61 54.98 ± 0.49 77.54 ± 0.11 51.23 ± 0.59 0.35 ± 0.02 6.92 ± 0.37 1.12 ± 0.02Crystallized:

Ethanol—acetone (80:0)2.01 ± 0.07 9.91 ± 0.26 72.70 ± 0.55 27.01 ± 0.10 1.27 ± 0.00 51.44 ± 0.75 7.13 ± 0.24

Ethanol—acetone (60:20) 8.19 ± 1.01 26.72 ± 0.34 68.87 ± 4.29 33.34 ± 2.33 0.50 ± 0.07 13.79 ± 1.59 2.27 ± 0.09Ethanol—acetone (40:40) 7.87 ± 4.85 27.88 ± 8.16 109.94 ± 14.17 43.48 ± 9.08 0.57 ± 0.24 15.53 ± 9.59 1.70 ± 2.16Ethanol—acetone (20:60) 2.73 ± 0.01 30.96 ± 2.01 126.57 ± 3.27 48.41 ± 1.92 0.80 ± 0.01 29.46 ± 0.47 4.00 ± 0.16Ethanol—acetone (0:80) 2.49 ± 0.10 27.14 ± 0.72 139.76 ± 0.01 53.55±0.08 0.88 ± 0.03 31.82 ± 0.06 5.06 ± 0.13

of acetone in the crystallization medium resulted inmore elongated particles compared with samples ob-tained in the presence of butanol.32

Higher roundness values indicate that crystallizedlactose particles are more irregular in shape and/orrougher in surface.38 Surface VMD for commerciallactose particles was 105.2 ± 1.2 :m, whereas sur-face VMD for crystallized lactose particles was in therange from 76.1 ± 1.5 to 122.9 ± 1.5 :m (Table 2).Generally, the higher the volume of acetone used dur-ing crystallization, the higher is the surface VMD ofthe resulted crystals (Table 2).

The morphology of crystallized lactose was furtherinvestigated by SEM (Figs. 1 and 2). Commercial lac-tose image was similar to typical tomahawk shape re-ported in literature, covered with fines. Crystallizedlactose samples showed different morphologies com-pared with commercial lactose, as it is evident fromFigure 1. It is apparent from SEM images of crystal-lized lactose samples that they comprise of agglom-erated particles. Crystallized lactose particles seemsmaller in size and slightly less elongated, which isin agreement with data obtained by particle size andshape analysis (Table 2). Surface morphology of crys-tallized lactose particles could be described as roughcorrugated surface with fractures. It has been sug-gested that there are favorable interactions betweensolvent and solute on specific surfaces during crystal-lization, resulting in crystal surface conversion fromsmooth to rough in order to reduce surface tension.39

These crystallized lactose particles contain some finelactose particles which could be seen from SEM im-ages and is in good agreement with the percentage of

fine particles less than 10.5 :m reported in Table 3.Comparing different crystallized lactose samples, itwas observed that the composition of binary nonsol-vent has considerably affected the shape of small lac-tose particles that form the larger aggregates. Lactosecrystallized form pure ethanol contained rectangular-shaped subunits (Figs. 1b and 2c). By introducingacetone during crystallization procedure, the resultedsubunits appear to become smaller in size and moreelongated in shape. For lactose particles crystallizedfrom pure acetone, subunits become very smaller insize with different shape (Figs. 1f and 2g). It can beseen that the difference in shape of subunits has af-fected the final shape of their bigger agglomerates.Lactose particles crystallized from higher volumes ofacetone have higher surface roughness (Figs. 1 and 2).This was further quantitatively confirmed by surfaceroughness measurements provided by AFM (Table 4).However, AFM data indicated that lactose particlescrystallized from ethanol—acetone (40:40) have thehighest surface roughness (Table 4).

Particle Size Analysis

Particle size distribution data for unsieved and sieved(63–90 :m) commercial lactose and all crystallizedlactose samples are summarized in Tables 1 and 3.It can be seen that despite all powders were sievedin the same way, crystallized lactose particles weredifferent in particle size distribution when comparedwith commercial lactose sample. It has been reportedthat the differences in particle size of sieved car-rier powders (63–90 :m) is likely to have a con-siderable effect on DPI aerosolization performance.8

Table 2. Roundness, Elongation Ratio, and Volume Specific Surface Area for Commercial Lactose Particles andDifferent Crystallized Lactose Particles Using Different Volume Fractions of Ethanol—Acetone (Values are Mean± SD, n = 100 particles)

Lactose (63–90 :m) Roundness Elongation Ratio Surface Volume Mean Diameter (:m)

Commercial 1.36 ± 0.13 1.64 ± 0.35 105.24 ± 1.17Crystallized: Ethanol—acetone(80:0)

2.09 ± 0.43 1.47 ± 0.24 89.55 ± 1.16

Ethanol—acetone (60:0) 1.91 ± 0.62 1.53 ± 0.39 76.12 ± 1.52Ethanol—acetone (40:40) 2.04 ± 0.71 1.49 ± 0.29 115.67 ± 1.60Ethanol—acetone (20:60) 2.01 ± 0.68 1.55 ± 0.36 108.20 ± 1.65Ethanol—acetone (0:80) 1.70 ± 0.36 1.47 ± 0.29 122.92 ± 1.55


2670 KAIALY ET AL.

Figure 1. SEM of (a) commercial lactose and lactose crystallized by using (b) ethanol—acetone(80:0), (c) ethanol—acetone (60:20), (d) ethanol—acetone (40:40), (e) ethanol—acetone (20:60),and (f) ethanol—acetone (0:80). All samples were taken at ×1000 magnification.

Figure 2. SEM images of (a) salbutamol sulfate (SS), (b) commercial lactose formulation blendand formulation blends containing lactose crystallized by using: (c) ethanol— acetone (80:0),(d) ethanol—acetone (60:20), (e) ethanol—acetone (40:40), (f) ethanol—acetone (20:60), and (g)ethanol—acetone (0:80). All images were taken at ×3000 magnification (some drug particlesare seen on carrier surfaces).



Table 3. Sieved Particle Size (63—90 :m) for Commercial Lactose Powder and Different Crystallized Lactose Powders UsingDifferent Volume Fractions of Ethanol—Acetone Expressed as D10%, D50%, D90%, VMD, SV, Span, and Percent of Fine Particles10.50 :m or Less (Values are Mean ± SD, n = 3)

Sieved Lactose D10% (:m) D50% (:m) D90% (:m) VMD(:m) SV (m2/cm3) <10.50 :m (%) Span

Commercial 58.56 ± 0.36 87.90 ± 0.37 91.07 ± 0.48 91.07 ± 0.34 0.07 ± 0.00 0.00 ± 0.00 0.79 ± 0.33Crystallised:ethanol—acetone (80:0)

57.93 ± 0.59 86.11 ± 0.70 123.19 ± 1.32 89.07 ± 0.77 0.07 ± 0.00 0.00 ± 0.00 0.76 ± 0.00

Ethanol—acetone (60:0) 52.42 ± 0.68 82.76 ± 0.80 121.20 ± 0.52 84.40 ± 0.83 0.13 ± 0.01 1.37 ± 0.14 0.83 ± 0.01Ethanol—acetone (40:40) 17.80 ± 5.60 71.99 ± 11.44 132.02 ± 10.44 72.91 ± 9.87 0.24 ± 0.06 4.61 ± 1.91 1.61 ± 0.28Ethanol—acetone (20:60) 12.92 ± 1.69 63.75 ± 3.22 92.99 ± 3.31 56.47 ± 4.00 0.31 ± 0.03 7.39 ± 1.52 1.26 ± 0.03Ethanol—acetone (0:80) 13.54 ± 0.82 75.95 ± 0.24 114.27 ± 0.37 72.12 ± 0.35 0.27 ± 0.01 7.98 ± 0.52 1.33 ± 0.01

In Tables 1 and 3, VMD and span values were usedto express the particle size distribution of all lactosesamples. The span was used as a measurement ofthe particle size distribution polydispersibility. It canbe seen from Table 3 that for sieved lactose powdersall crystallized lactose samples have lower VMD com-pared with the value reported for commercial lactose.

It is interesting to note that when the VMD wasplotted against percentages of acetone in crystalliza-tion medium, a linear relationship can be establishedwith a high correlation coefficient of 0.982 (from 0 to80 mL acetone) and 0.950 (from 0 to 60 mL acetone)for sieved and unsieved lactose, respectively (Fig. 3).The good linear relationship indicates that the valueof VMD can be predicted when the amount of acetonein the crystallization medium is between 0 and 80 or60 mL.

Particle size distribution (span values) is impor-tant in terms of aerosol quality and efficiency40 asdifferences in aerosol particle sizes lead to differencesin the deposition of particles in different regions ofthe lung. Span value of commercial lactose samplewas low for both unsieved and sieved indicating nar-row size distribution, whereas span values of mostcrystallized lactose samples were higher indicatingwider size distribution (Tables 1 and 3). These highspan values could be explained by poor mixing lead-ing to zones with different levels of supersaturationin the crystallization media during the crystalliza-tion process, which induce heterogeneous growth ofthe crystals resulting in varied particle size distri-bution, and this, in fact, is one of antisolvent crys-tallization technique disadvantages.25 Kaialy et al.32

have reported that if butanol was used in the crystal-

Table 4. Roughness Data Obtained by AFM for DifferentLactose Samples

Rq (nm) Ra (nm)

Commercial lactose 3.3 ± 0.9 2.6 ± 0.7Crystallized using ethanol 2.8 ± 0.4 2.2 ± 0.4Crystallized using ethanol—acetone

(40:40)7.1 ± 4.5 5.8 ± 3.7

Crystallized using acetone 5.3 ± 2.1 4.1 ± 1.3

Rq, root mean square roughness; Ra, arithmetic mean roughness.

lization medium, generally, similar span values couldbe obtained. This could be due to the effect of differ-ent solvent butanol, which produces less elongatedparticles compared with lactose samples crystallizedin the presence of acetone. For unsieved lactose sam-ples, the percentage of fines increased from 6.9 ± 0.4%for commercial lactose sample to 51.4 ± 0.8% for lac-tose crystallized from pure ethanol. For sieved lactosesamples, the percentage of particles less than 10.50:m have significantly increased from 0% for commer-cial lactose to 8.0% for lactose crystallized from pureacetone. It is obvious that for lactose powders crystal-lized from binary mixtures of ethanol—acetone, whenmore acetone was added, more fine lactose particleswas obtained (Tables 1 and 3). This probably could bedue to rougher surface of lactose particles crystallizedfrom higher volumes of acetone compared with sam-ples crystallized from pure ethanol (Table 4), whichmake lactose particles hold higher amounts of finesinside the surface cavities (see also SEM images inFigure 1), which could not be removed by mechani-cal vibrations during sieving. It is known that lactoseis soluble in water, very slightly soluble in ethanol,and insoluble in acetone.41–43 This indicates that thepresence of more acetone results in higher supersat-uration, hence it leads to a quicker precipitation ofthe lactose crystals (heterogeneous nucleation). Thequicker nucleation and precipitation of the lactoseparticles in turn increases the number of dislocationsand defects in the crystal lattice, leading to roughersurfaces and a higher level of disorder (amorphouscontent) in the crystal lattice compared with the sam-ples crystallized in the presence of low acetone vol-ume. This is confirmed by the data reported in Table 5,as lactose samples crystallized in the presence andabsence of acetone showed 13% and 1.4% amorphouscontent, respectively.

During laser particle size analysis, these fines canbe separated and detected by the instrument. On thecontrary, all fine particles were removed by mechan-ical vibration process (sieving) from commercial lac-tose and those crystallized from pure ethanol as theyhave relatively smoother surfaces.

These fine particles are known to be of extremeimportance to affect DPI performance.18 Figure 4


2672 KAIALY ET AL.

Table 5. DSC Enthalpies of Water Dehydration, Amorphous Content Recrystallization, "-Lactose Melting, and $-Lactose Meltingfor Commercial Lactose Sample and Different Crystallized Lactose Samples Using Different Volume Fractions of Ethanol—Acetone(Values are Mean ± SD, n = 3)

Lactose Product

Enthalpy of AmorphousContent Recrystallization

(�Hc) (J/g)Enthalpy of "-LactoseMelting (�H220) (J/g)

Enthalpy of $-Lactosemelting (�H237) (J/g)

AmorphousLactose (%)

Commercial 2.13 ± 0.69 143.87 ± 0.98 0.00 ± 0.00 8.33 ± 4.22Crystallized: ethanol—acetone (80:0) 0.46 ± 0.04 32.07 ± 2.86 100.10 ± 3.62 1.42 ± 0.11Ethanol—acetone (60:0) 1.50 ± 0.52 25.98 ± 7.92 103.45 ± 12.20 4.68 ± 1.64Ethanol—acetone (40:40) 1.93 ± 0.25 19.04 ± 1.11 108.27 ± 2.22 6.02 ± 0.78Ethanol—acetone (20:60) 3.14 ± 0.20 9.53 ± 0.62 111.73 ± 3.67 9.82 ± 0.63Ethanol—acetone (0:80) 4.17 ± 0.09 3.37 ± 0.07 115.74 ± 3.68 13.00 ± 0.28

shows that by comparing crystallized lactose samples,linear relationships could be established between per-centage of fines and acetone volume with r2 value of0.8909 and 0.9162 for sieved and unsieved samples,respectively.

Volume specific surface area for all crystallized lac-tose particles was found to be equal or higher thanvolume specific surface area of commercial lactoseparticles (Tables 1 and 3). It was noted that linearrelationships could be found when Sv was plottedagainst acetone volume for different lactose sampleswith r2 value of 0.947 or 0.8531 for unsieved or sievedlactose samples, respectively (Fig. 5). This shows thatlactose particles crystallized using high volumes ofacetone have high volume specific surface areas.

Higher Sv values (volume specific surface area)of crystallized lactose samples could be due to theirlower size distributions. In addition, the higher Svvalues for crystallized lactose particles could be at-tributed to broader particle size distributions as in-dicated by span values (Tables 1 and 3).44 Linear re-lationships were found between lactose volume spe-cific surface area against both VMD and amountsof fines 10.50 :m or less with r2 of 0.9051 and

0.9729, respectively, (Fig. 6) indicating the strongrelationship between the three parameters. How-ever, in case of corrugated particles, particle sizemeasurement (VMD and Sv) using laser diffrac-tion is less accurate because laser diffraction tech-nique assumes that all particles are spheres withsmooth surfaces.20 Therefore, particle size distri-bution of sieved lactose powders was further ana-lyzed employing particle image analysis for 100 par-ticles (Fig. 7). It can be observed from Figure 7athat the cumulative undersize curves of all crystal-lized lactose powders were shifted toward smaller par-ticle sizes compared with commercial lactose powder.These data are important as it is known that by com-paring two formulations with the same drug content,formulation blend with lower carrier size will havehigher FPF.8,17,18 By observing Figure 7b, it can alsobe noted that most commercial lactose particles fallwithin the particle size range of 91–128 :m that ishigher than the upper sieved fraction value (90 :m).This observation was reported to happen usually forcommercial lactose powders and was attributed to thetomahawk geometry of lactose particles.45 Comparedwith commercial lactose powder, crystallized lactose

Figure 3. Volume mean diameter for different crystallized lactose particles as a function toacetone volume fraction in ethanol—acetone binary mixtures used in crystallization process(error bars represent SD, n = 3).



Figure 4. Percentage of fine particles less than 10.50 :m for different crystallized lactoseparticles as a function to acetone volume fraction in ethanol—acetone binary mixtures used incrystallization process (error bars represent SD, n = 3).

powders had higher contribution of particles in thesize range of 64–91 :m (Figure 7-b). This particle sizerange was reported to be the typical and the preferredcarrier size in DPI formulations.14

Salbutamol sulfate particle size distribution anal-ysis showed a monomodal distribution (Figure notshown) with a VMD of 1.9 ± 0.2 :m, D50% of 1.7 ±0.1 :m, and 90% of the particles less than 3.1 ± 0.3:m, indicating the batch is suitable for respiratorydelivery.

Density and Flowability Assessment

The values of true density, bulk density, and tappeddensity for commercial lactose were 1.546, 0.621, and0.759 g/cm3, respectively (Table 6). The true den-sity of commercial lactose reported in Table 6 is in

good agreement with the data reported in literature(1.545 g/cm3).46Table 6 shows that the true densityof all crystallized lactose is higher than true densityof commercial lactose particles [analysis of variance(ANOVA); p < 0.05].

Unlike true density, all crystallized lactose powdersshowed considerably lower bulk density and tap den-sity than commercial lactose powder (Table 6). Com-parison of bulk and tapped densities of all crystallizedlactose sample showed that higher bulk density andtap density were obtained for lactose powders crystal-lized using higher volumes of acetone. Lower tappeddensity means that crystallized lactose powder bedis more porous and has lower contact points betweenparticles within the powder compared with commer-cial lactose. Carrier powders with lower tap density

Figure 5. Volume specific surface area for different crystallized lactose particles as a functionto acetone volume fraction in ethanol—acetone binary mixtures used in crystallization process(error bars represent SD, n = 3).


2674 KAIALY ET AL.

Figure 6. Relationship between lactose particle volume specific surface area, volume meandiameter, and percent amounts of fines 10.50 :m or less for commercial lactose and differentethanol—acetone crystallized lactose particles (error bars represent SD, n = 3).

were reported to be preferred in DPI powders interms of enhanced aerosolization performance,15,16,47

as they are expected to be easier to disperse uponaerosolization due to lower interparticulate cohesive

forces between particles.48 Lower bulk density andtap density for crystallized lactose powders could beexplained by their smaller and more irregular shapedparticles with rougher surface (Fig. 1, Tables 2 and 4),

Figure 7. . (a) Cumulative particle size and (b) mean particle size distributions for commerciallactose and different ethanol—acetone crystallized lactose powders (n = 100).



Table 6. True Density(ρtrue), Bulk Density(ρbulk), Tapped Density(ρtap), Carr’s Compressibility Index(%), andHausner Ratio (H) for Commercial Lactose Powder and Different Crystallized Lactose Powders Using DifferentVolume Fractions of Ethanol—Acetone (Values are Mean ± SD, n = 3)

Lactose powder ρtrue (g/cm3) ρbulk (g/cm3) ρtap (g/cm3) CI (%) H

Commercial 1.546 ± 0.052 0.621 ± 0.014 0.759 ± 0.002 18.12 ± 1.66 1.22 ± 0.02Crystallized: ethanol—acetone (80:0) 1.554 ± 0.019 0.238 ± 0.003 0.294 ± 0.006 19.02 ± 2.76 1.24 ± 0.04Ethanol—acetone (60:20) 1.589 ± 0.004 0.269 ± 0.008 0.384 ± 0.010 30.00 ± 1.11 1.43 ± 0.02Ethanol—acetone (40:40) 1.610 ± 0.002 0.264 ± 0.003 0.384 ± 0.003 31.11 ± 0.00 1.45 ± 0.00Ethanol—acetone (20:60) 1.601 ± 0.010 0.304 ± 0.002 0.432 ± 0.005 29.63 ± 1.28 1.42 ± 0.03Ethanol—acetone (0:80) 1.605 ± 0.012 0.359 ± 0.002 0.473 ± 0.002 24.07 ± 0.64 1.32 ± 0.01

which leads to the formation of additional void spacebetween particles within the powder.20,49 When com-paring different crystallized lactose samples, higherbulk density and tap density for crystallized lactosepowders prepared using higher volumes of acetonecould be attributed to reduced void space betweenparticles due to their higher content of fine particles(Table 3). This results in lower average contact pointsand consequently higher degree of interparticulateforces.

Carr’s compressibility index (CI) was employed toassess the interparticulate forces within powder sam-ples. CI for commercial lactose powder was 18.1 ±1.7% (fair flow), whereas CI values for crystallizedlactose powders were all higher and ranged between19.0 ± 2.8% and 31.1 ± 0.0% (fair to poor flow). HigherCI values for crystallized lactose powders are indica-tion of their poorer flowability. Poorer flow propertiesfor crystallized lactose powders could be due to thereduced particle size or an increase in Van der Waalsforces (cohesion forces) as a result of high surfacearea for crystallized lactose. In addition, higher in-terparticulate forces for crystallized lactose particlescould be ascribed to their anisometric shape whichpromotes mechanical interlocking between particles(Fig. 1, Table 2). Similar conclusion regarding flowa-bility could be drawn when Hausner ratio was as-sessed (Table 6).

Characterization of the Polymorphic Form

Thermal transitions of commercial lactose and allcrystallized lactose samples were assessed usingDSC. DSC traces of commercial lactose showed dis-tinctive endothermic peak at about 422 K (149◦C),which corresponds to the dehydration of water and thesecond endothermic peak at about 493 K (220◦C) cor-responds to the melting of "-lactose monohydrate12,50

(Fig. 8). Commercial lactose DSC curve also showed athird small exothermic peak at about 446 K (173◦C),which could be attributed to the crystallization ofamorphous lactose51 (Fig. 8). DSC curves of all crys-tallized lactose samples have also shown an endother-mic peak at about 415 K (142◦C) corresponding to thedehydration of crystallization water, the exothermicpeak at about 446 K corresponding to amorphous lac-

tose recrystallization, and the endothermic peak ofpseudopolymorph "-lactose monohydrate melting ap-peared at 493 K (Fig. 8). The DSC traces of all crys-tallized lactose samples showed an extra endother-mic peak at about 510 K (237◦C), which correspondsto the melting of $-lactose.12,50 Enthalpies of all ther-mal transitions were calculated by the software andthey are all listed in Table 5. Figure 8 shows thatfor all crystallized lactose samples, most of the lac-tose crystals have been transformed from "-lactose to$-lactose as a result of the crystallization procedure.Furthermore, it can be seen that by increasing thevolumes of acetone in crystallization medium, the en-thalpy of fusion peak for "-lactose decreases, whereasthe intensity of $-lactose melting peak and enthalpyincreases (Fig. 8, Table 5). Interestingly, plotting "-and $-lactose melting enthalpies against acetone vol-umes used during crystallization resulted in a linearrelationship with r2 values of 0.9943 and 0.9974 for"- and $-lactose, respectively (figure not shown). Itshould be kept in mind that if the polarity or dipolemoment of the solvent changed, different proportionsof "- and $-lactose are formed. This was shown by Ka-ialy et al.32 when they replaced acetone with butanoland different ratios of "- and $-lactose were obtainedcompared with the present study.

Equation 6 was employed to calculate the crys-tallinity of the crystallized lactose particles by em-ploying the heat of recrystallization of amorphous lac-tose from DSC traces.52

% amorphous content = �hc

�Hc× 100 (6)

where �hc is the heat of crystallization of amorphouslactose in J/g. �Hc is the specific heat of crystalliza-tion of amorphous lactose, which can be taken as 32J/g for amorphous lactose,53 and all results are listedin Table 5. Amorphous lactose content for commerciallactose sample was 8.3 ± 4.2%, whereas for crystal-lized lactose samples, amorphous lactose content wasin the range of 1.4 ± 0.1% and 13 ± 0.3% (Table 5). Forcrystallized lactose samples, it was found that whenhigher volumes of acetone were used during crystal-lization, the percentage of amorphous content in thecrystallized samples were high and this relationship


2676 KAIALY ET AL.

Figure 8. DSC thermal curves of (a) commercial lactose sample and lactose crystallized byusing: (b) ethanol—acetone (80:0), (c) ethanol—acetone (60:20), (d) ethanol—acetone (40:40), (e)ethanol—acetone (20:60), and (f) ethanol—acetone (0:80).

was linear between the volume of acetone and per-centage of amorphous in the lactose samples with r2

value of 0.9833 (Figure not shown).In conclusion, DSC results show that crystallized

samples are a combination of anhydrous $-lactose,"-lactose monohydrate, and amorphous forms. How-ever, the contribution of each form was related tothe ratio of ethanol to acetone used in crystallizationmedium.

FTIR Studies

Fourier-transform infrared studies of all samples(Fig. 9) showed broadband at 3600 to 3200 cm−1 cor-responding to the stretching vibration of the hydroxylgroup.54 Other bands of interest include the band at1650 cm−1 that is related to the vibration of the crys-tal water hydroxyl group and the band at 1200 to1070 cm−1 that refers to the asymmetric vibrations ofC—O—C in glucose and galactose. Kirk et al.54 havereported that the band at 920 cm−1 is a specific diag-nostic band for "-lactose anomer. It can be seen fromFigure 9 that this band has appeared in commerciallactose sample and all crystallized lactose samplesindicating the existence of "-anomer. Other "-lactosespecific bands are the free O-H vibrations band fromwater molecules at about 3500 cm−1.54 It can be seenthat this specific peak has appeared in commerciallactose sample, but for crystallized lactose samples it

was slightly shifted to lower wavenumber, 3450 cm−1,(Fig. 9). Kirk et al.54 have reported that the band at950 cm−1 is a specific diagnostic band for $-lactoseanomer. As could be seen from Figure 9, this spe-cific peak does not exist in commercial lactose sample,but it appears in all crystallized lactose samples indi-cating that commercial lactose contains no $-anomer,whereas all crystallized lactose samples contain $-lactose anomer. Peaks at 1260, 900, and 875 cm−1 areindication of amorphous lactose.55 From Figure 9, itcan be observed that these three distinctive peaks ex-ist in commercial lactose sample and all crystallizedlactose samples.

In conclusion, all lactose samples including com-mercial lactose contained some amorphous lactose.Commercial lactose showed "-lactose anomer with-out $-form, whereas all crystallized lactose samplesshowed both "- and $-lactose vibrations. FTIR lactosepseudopolymorphic and polymorphic forms analysiswas in good agreement with the information providedby DSC.

Formulation Assessments

Content Uniformity

Five randomly selected samples of 33 ± 1.5 mg havebeen prepared from formulations containing commer-cial lactose and each crystallized lactose formula-tion blend. Each sample was then dissolved in water



(100 mL volumetric flask) and the amount of SS wasthen analyzed by HPLC described above. The per-centage uniformities was calculated and used to ex-press the blends uniformity. The coefficient of vari-ation (CV%) in SS content was also calculated andused as the homogeneity assessment of the formula-tion blends. All content uniformity assessment dataare given in Table 7. SS content for commercial lac-tose formulation was 474.86 ± 51.55 :g and for crys-tallized lactose formulations, SS content was in therange from 400 ± 17 to 471 ± 40 :g (Table 7). Someof the crystallized lactose formulations blends con-tain lower amounts of SS than commercial lactoseblend (ANOVA; p < 0.05, n = 5). However, SS con-tent in most lactose formulations was within the ac-ceptable range of theoretical dose ±10% (correspond-ing to 433–529 :g). Percent uniformity for crystal-lized lactose formulation blends were in the rangebetween 83.1 ± 3.6% and 98.0 ± 8.3%, which issmaller than percent uniformity of commercial lac-tose formulation (98.7 ± 10.7%; ANOVA; p < 0.05,n = 5; Table 7). Lower average drug content andpercent uniformities for crystallized lactose formula-tions could be attributed to higher amounts of drugseparated from crystallized lactose surfaces duringmixing and storage process, and consequently higheramounts of drug being lost by adhering to contain-ers and glass vials. Nevertheless, the phenomenon ofthe reduced drug content uniformity in crystallizedlactose blends caused by higher drug—carrier segre-gation is considered advantageous in DPI formulationblends aerosolization.

Interestingly, the highest drug recovery(474.9 ± 51.6 :g) and the worst percentage ofuniformity (98.7 ± 10.7%) were obtained for com-mercial formulation blends, which produced thelowest FPF upon aerosolization as shown later. Inorder for a formulation to achieve the pharmacopoeiaguidelines, the formulation blends should not exceeda coefficient of variation (CV) of 5%.

As shown in Table 7, not all crystallized lactose for-mulation passed the homogeneity test. For example,CV% for formulation blends containing lactose crys-tallized from 40:40 or 20:60 ethanol—acetones were8.5% and 7.87%, respectively. In fact, dose variability

delivered by DPI is always challenging. However, de-spite their wider carrier size distributions (Table 3)and poorer flow properties (Table 6), all crystallizedlactose formulations were found to have lower CV%than commercial lactose formulations indicating thebetter drug content homogeneity for these samples. Ithas been shown that a reduction in CV values for thecrystallized lactose samples containing the drug couldbe due to an increase in the roughness of the lactoseparticles.56 The increase in the roughness could causean increase in the adhesion between the drug and thecarrier.

Deposition Profiles of SS

The contents of 10 capsules containing either com-mercial lactose or one of crystallized lactose formu-lations were actuated into a MSLI to perform the invitro deposition tests using Aerolizer R© as describedby pharmacopoeia.57 The effective cutoff diameters ofthe MSLI at flow rate of 92 L/min are as follows: stage1:10.50 :m, stage 2: 5.49 :m, stage 3: 2.50 :m, andstage 4: 1.37 :m. All deposition data parameters arerecorded in Table 8. It can be seen that the formu-lation blends containing crystallized lactose resultedin different deposition profiles of SS when comparedwith commercial lactose formulations under the sameconditions. The RD of SS from all the formulationscontaining crystallized lactose were in the range of(383 ± 25 to 436 ± 69 :g), which is higher thanRD of commercial lactose formulations (317 ± 5 :g)(Table 8). Despite accurate constant weight of all me-tered doses and operating at constant flow rate, dif-ferent RDs and EDs were obtained from crystallizedlactose formulation blends when compared with com-mercial lactose formulation blend. Unlike commer-cial lactose formulations, RD of all crystallized lac-tose formulations fall within the acceptable range of(75%–125%),58 which corresponds to the range from360 to 601 :g. Satisfactory RD for crystallized lactoseformulations indicates that the overall procedures ofmixing, capsule filling, deposition, washing, and drugquantification were good in terms of reproducibility,accuracy, and repeatability. ED for commercial lac-tose formulation was 299 ± 7 :g, whereas ED mea-surements for crystallized lactose formulations were

Table 7. Percent Uniformity, Average Salbutamol Sulfate (SS) Content, and the PercentCoefficient of Variation (CV) in SS for Commercial Lactose Formulations and DifferentCrystallized Lactose Formulations (Values are Mean ± SD, n = 5)

Lactose DPI Formulation Uniformity (%) SS Average Content (:g) CV (%)

Commercial 98.72 ± 10.72 474.86 ± 51.55 10.85Crystallized: ethanol—acetone (80:0) 90.94 ± 1.87 437.42 ± 9.01 2.06Ethanol—acetone (60:0) 97.07 ± 3.43 466.92 ± 16.51 3.53Ethanol—acetone (40:40) 97.96 ± 8.32 471.19 ± 40.04 8.50Ethanol—acetone (20:60) 83.53 ± 6.58 401.80 ± 31.64 7.87Ethanol—acetone (0:80) 83.16 ± 3.55 399.99 ± 17.08 4.27


2678 KAIALY ET AL.

Figure 9. Fourier-transform infrared spectra for (a) commercial lactose sample and differentcrystallized lactose samples using: (b) ethanol—acetone (80:0), (c) ethanol—acetone (60:20),(d) ethanol—acetone (40:40), (e) ethanol—acetone (20:60), and (f) ethanol—acetone (0:80).

higher falling in the range between 360 ± 33 and 418± 68 :g (Table 8). It is known that ED is dependent onpowder flowability and cohesiveness. However, higherED for crystallized lactose formulations, despite theirpoor flowability, indicates the limit effect of powderflowability on ED values when low resistance inhaleris used. Higher RD and ED for crystallized lactose for-mulation blends compared with commercial lactose

formulations indicate that crystallized lactose carrierpowders produced higher amounts of drug deliveredfrom capsules upon inhalation. Therefore, when usingcrystallized lactose formulation blends, higher drugdose is expected to be available to the patient fromthe inhaler device and deposit on the overall airwayregions. Percent recovery was significantly increasedfrom 65.9 ± 1.1% for commercial lactose formulations



Tab

le8.

Rec

over

edD

ose

(RD

),E

mit

ted

Dos

e(E

D),

Per

cen

tR

ecov

ery,

Per

cen

tE

mis

sion

,Im

pact

ion

Los

s,M

ass

Med

ian

Aer

odyn

amic

diam

eter

(MM

AD

),G

eom

etri

cS

tan

dard

Dev

iati

on(G

SD

),an

dF

ine

Par

ticl

eF

ract

ion

(FP

F)

for

For

mu

lati

ons

Con

tain

ing

for

Com

mer

cial

Lac

tose

For

mu

lati

ons

and

Dif

fere

nt

Cry

stal

lize

dL

acto

seF

orm

ula

tion

sw

hen

Act

uat

edfo

rmA

erol

izer

R ©T

hro

ugh

MS

LI

atF

low

Rat

eof

92L

/min

(Mea

n±

S.D

.,n

=3)

Lac

tose

DP

IF

orm

ula

tion

RD

(:g)

ED

(:g)

Rec

over

y(%

)E

mis

sion

(%)

Impa

ctio

nL

oss

(%)

MM

AD

(:m

)G

SD

(:m

)F

PF

(%)

Com

mer

cial

317.

02±

5.14

299.

43±

6.59

65.9

1±

1.07

94.4

5±

0.55

67.7

9±

4.71

3.15

±0.

392.

19±0

.06

19.7

0±1.

89C

ryst

alli

zed:

eth

anol

—ac

eton

e(8

0:0)

436.

20±

68.9

141

7.86

±67

.99

90.6

9±

14.3

295

.75

±0.

5264

.84

±7.

163.

13±

0.09

2.18

±0.

0222

.68

±5.

10E

than

ol—

acet

one

(60:

0)43

0.40

±43

.27

410.

10±

44.4

389

.48

±9.

0095

.24

±1.

5444

.28

±2.

333.

23±

0.31

2.14

±0.

0537

.31

±1.

21E

than

ol—

acet

one

(40:

40)

420.

08±

71.0

339

9.93

±70

.52

87.3

3±

14.7

795

.13

±0.

6637

.97

±5.

823.

44±

2.09

2.09

±0.

0240

.48

±4.

57E

than

ol—

acet

one

(20:

60)

383.

46±

24. 8

636

0.37

±32

.91

79.7

2±

5.17

93.8

7±

2.58

40.8

0±

8.27

3.47

±0.

152.

08±

0.03

38.0

3±

7.17

Eth

anol

—ac

eton

e(0

:80)

413.

45±

58.2

538

9.05

±59

.96

85.9

5±

12.1

193

.99

±1.

4340

.04

±2.

623.

49±

0.07

2.04

±0.

0138

.59

±1.

76

to 90.7 ± 14.3% for the formulation blends containinglactose crystallized from pure ethanol (Table 8). Thisindicates that in case of commercial lactose formu-lations, 34% of the drug is lost probably adhered toglass vial, container walls, or container used duringmixing, or it might be adhered to capsule shells.

Mass median aerodynamic diameter and geomet-ric standard deviationGSD for all formulation blendswere similar (Table 8). However, similarity in drugaerodynamic size distribution obtained for all formu-lation blends (MMAD and GSD) does not mean simi-lar aerosolization efficiency.59

Formulation blends containing crystallized lactoseproduced higher FPF of SS (22.7 ± 5.1% to 40.5 ±4.6%) than commercial lactose formulations (19.7 ±1.9%; ANOVA; p < 0.05; Table 8). This indicates thatemploying crystallized lactose in DPI formulations in-stead of commercial lactose resulted in an increase inthe fraction of SS depositing on the lower airways anddecrease the fraction of SS deposited on the upperstages of the lung. Low FPF for commercial lactoseformulations indicates that SS does not detach wellfrom the carrier surface.

Table 8 also shows that the impaction loss wasfound to decrease significantly (p < 0.05) from 67.8± 4.7% for commercial lactose formulations to 38.0± 5.8% for lactose crystallized from 40:40 ethanol—acetone. This indicated that formulation blends con-taining crystallized lactose should have better pen-etration to the lung compared with commercial for-mulations. This could be the reason for highest FPFobtained for formulation blends containing lactosecrystallized from 40:40 ethanol—acetone. In addi-tion, lower impaction losses and higher FPFs forformulations containing lactose crystallized usingethanol—acetone and pure acetone indicate that SSparticles adhered to corrugated crystallized lactoseparticles can be dispersed about twice as efficientlyas the commercial formulations upon aerosoliza-tion. Comparing the present results with previouslypublished data on crystallization of lactose fromethanol—butanol showed that, generally, lactose crys-tallized in the presence of acetone showed less im-paction loss32. This indicates that the aerosolizationperformance of lactose particles crystallized in thepresence of acetone could be better than lactose par-ticles crystallized in the presence of butanol. Table9 shows that there is no significant difference in theamounts of drug deposited on inhaler & mouthpieceadaptor (I&M; from 4.3 ± 0.5% to 6.1 ± 2.6%) for allformulations. Amounts of drug deposited on IP (whichresembles the human oropharynx) were higher forcrystallized lactose formulations (from 4.4 ± 0.6% to13.0 ± 1.1%) compared with commercial lactose for-mulations (2.5 ± 0.1%) (Table 9). This could prob-ably be due to lower flow properties of crystallizedlactose powders when compared with commercial


2680 KAIALY ET AL.

Table 9. Amounts of Salbutamol Sulfate Deposited on Inhaler& Mouthpiece Adaptor (I&M), Induction Port (IP), and All MSLI StagesExpressed as a Percentage to the Recovered Dose (RD) for Commercial Lactose Formulations and Different Crystallized LactoseFormulations when Actuated Form Aerolizer R© Through MSLI at Flow Rate of 92 L/min (Mean ± S.D., n = 3)

Lactose DPI Formulation I&M IP MSLI 1 MSLI 2 MSLI 3 MSLI 4 MSLI 5 (Filter)

Commercial 5.55 ± 0.55 2.51 ± 0.14 65.28 ± 4.56 6.74 ± 3.17 7.91 ± 1.65 9.47 ± 0.34 2.54 ± 0.32Crystallised: Ethanol—acetone

(80:0)4.25 ± 0.52 4.41 ± 0.56 60.43 ± 7.67 7.26 ± 2.71 9.83 ± 1.72 10.71 ± 2.97 3.10 ± 0.35

Ethanol—acetone (60:0) 4.76 ± 1.54 6.77 ± 1.84 37.51 ± 3.97 11.87 ± 2.65 17.33 ± 2.99 17.66 ± 4.19 4.09 ± 0.61Ethanol—acetone (40:40) 4.87 ± 0.66 12.95 ± 1.08 25.02 ± 5.65 15.00 ± 0.92 21.56 ± 2.58 16.68 ± 3.33 3.91 ± 0.33Ethanol—acetone (20:60) 6.13 ± 2.58 9.96 ± 2.72 30.84 ± 5.84 13.76 ± 1.49 20.31 ± 6.11 15.75 ± 3.40 3.25 ± 0.53Ethanol—acetone (0:80) 6.01 ± 1.43 8.80 ± 1.26 31.24 ± 3.73 13.03 ± 1.06 22.92 ± 1.01 15.29 ± 1.52 2.71 ± 0.41

lactose powder as indicated by CI and Hausner val-ues (Table 6). Figure 10 shows that the lower theflow properties of lactose powder incorporated in DPIformulation blend, the higher the amounts of drugdeposited on IP of the MSLI. This shows that flowof carrier particles might have a significant effecton IP of the MSLI. Amounts of drug deposited onMSLI stages 1, 2, 3, and 4 appear to be the most af-fected by using crystallized lactose formulations (Ta-ble 9). Table 9 shows that the amounts of drug de-posited on MSLI stage 1 have significantly decreasedfrom 65.28 ± 4.56% for commercial lactose formula-tions to 25.0—60.4% for crystallized lactose formu-lations. Amounts of drug deposited on lower MSLIstages (2, 3, and 4) have significantly increased forcrystallized lactose formulation blends (Table 9). Inorder to get better understanding of deposition be-havior for different formulation blends and comparetheir deposition profiles, the cumulative amounts ofdrug less that the cutoff diameter of MSLI stages 1,2, 3, and 4 were calculated and shown in Figure 11. Itcan be seen that all crystallized lactose formulationsproduced significantly higher cumulative amounts ofdrug less than 10.50, 5.49, and 2.5 :m compared withcommercial lactose formulations (Fig. 11). However,

Figure 10. Amounts of salbutamol sulfate deposited oninduction port (IP) of the multistage liquid impinger (MSLI)obtained from formulation blends containing lactose pow-ders with different flow properties expressed as Carr’s index(error bars represent SD, n = 3).

no significant difference was observed for cumula-tive amounts of drug less than 1.37 :m for all for-mulation blends, indicating that deposition of SS inthe aerodynamic particles size distribution range be-low 1.37 :m was not influenced by different lactoses(Fig. 11). Generally, similar pattern was observed forthe deposition of SS from DPI formulations whenthe lactose used in the DPI was crystallized fromethanol—butanol rather than ethanol—acetone.32 Itcan also be observed that formulation blends that pro-duced the highest FPF (ethanol—acetone, 40:40) pro-duced the highest amounts of drug less than 10.50and 5.49 :m. However, in case of lactose particlescrystallized from ethanol—butanol, lactose crystal-lized from 20:60 (ethanol—butanol) showed the high-est FPF.32 This indicates that aerosolization perfor-mance of DPI formulations is deeply affected by cu-mulative amounts of drug less than 10.5 and 5.49:m. The enhancement in DPI performance could beascribed to several facts. Indeed, different depositionprofiles for lactose formulation blends were expecteddue to differences in carrier size, shape, polymorphicform, density, and flow properties. It is known thatsurface roughness (rugosity) has a significant influ-ence on DPI performance due to its considerable effect

Figure 11. Cumulative amounts of salbutamol sulfate(SS) less than the stated effective cutoff diameter of mul-tistage liquid impinger stages for formulations containingcommercial lactose and different crystallized lactose pow-ders as a carrier.



Figure 12. Relationship between FPF and roughness data for different lactose formulationblends containing salbutamol sulfate.

on drug—carrier interactive forces.10 Higher carriersurface roughness was reported to give higher FPFdue to lower drug—carrier contact area that leads tolower drug—carrier cohesiveness.11,60 In the presentstudy, the surface roughness of crystallized lactoseparticles was significantly increased compared withcommercial lactose (Fig. 1; Table 4). Therefore, it is ex-pected that DPI formulations containing crystallizedlactose should show different FPF value comparedwith DPI formulations containing commercial lactose.In contrast to the data reported by Heng et al.22 andZeng et al.,13 higher FPF was obtained for DPI formu-lations containing lactose carrier with high rugositydespite using turbulent shear inhaler (Aerolizer R©).This was shown in Figure 12 where carriers withrougher surface produced higher FPF of SS duringinhalation.

Physicochemical analysis of crystallized lactoseparticles showed that these modified lactose particleshave ideal properties to improve aerosolization perfor-mance such as higher amounts of fines61,62 and highersurface area.15,16,63 The lowest FPF was obtained bycommercial lactose formulation that has the highestcarrier size distribution (Fig. 7). In addition, lactosepowders that contains no fine carrier particles (Table3, commercial lactose and crystallized lactose usingpure ethanol) produced the lowest FPF for SS (Table8) indicating the dominating effect of fine carrier par-ticles on DPI aerosolization performance. Similar con-clusion was drawn by other authors emphasizing onthe dominating role of fine carrier particles over meanparticle size of carrier and surface roughness.13,64,65

It can be concluded that the enhanced performanceof crystallized lactose particles is mainly due to theirrougher surface and higher contents of fines.

The proposed mechanism for the increased drugdeposition by addition of fine carrier particles was

the saturation of “active sites”66–69 or hot spots (highadhesion sites)70 on the coarse carrier particles byfine particle additives. When fine particle additives fillthe active sites on the carrier surface that will leaveonly the passive sites (low adhesion sites) availablefor drug adhesion,68,69 leading to low drug—carrieradhesion and consequently increasing drug—carrierdetachment. Also, another mechanism was proposedwhich is “agglomeration theory.”71–75 In this theory,agglomerates were suggested to be formed betweenternary components and drug particles forming mixedagglomerates that are expected to be easier to detachfrom carrier surface upon inhalation than single drugparticles.

Furthermore, crystallized lactose samples werefound to exist in different polymorphic forms. The ef-fect of carrier crystalline form on drug—carrier inter-action in DPI formulations is not much exploited.38

Differences in $-, "-, and amorphous lactose con-tent are expected to affect surface energy of lac-tose particles.76,77 In fact, differences in carrier par-ticle surface energy will affect carrier particle co-hesion forces and drug—carrier adhesion cohesioninteractions.78,79 Our results showed that betteraerosolization performance was achieved for crystal-lized lactose particles that have lower amounts of wa-ter, lower amounts of "-lactose, and higher amountsof $-lactose anomer. These results are different fromwhat has previously been reported when $-lactosepolymorph was stated to be not preferred form tobe used in DPI formulations, whereas "-lactose ispreferred.80 The results reported in Table 5 showthat the contribution of each polymorph—pseudopoly-morph depends on the ratio of ethanol—acetone usedduring the crystallization of lactose. As $-lactosehas a higher true density than "-lactose,81 so thetrue densities of crystallized lactose samples were


2682 KAIALY ET AL.

different according to the percentage of each lactoseform.

The reported true density for each lactose form wasas follows 1.52, 1.545, and 1.59 for amorphous, "-, and$- form.46 In agreement with previous study,15 our re-sults showed that higher true density of carrier parti-cles was associated with higher FPF for formulations.It can be concluded that true density is an importantphysical property that could have remarkable effecton DPI aerosolization.

CONCLUSION

In this study, lactose particles were crystallized usingdifferent fractions of binary mixtures of ethanol andacetone as antisolvents. The crystallized lactose par-ticles had lower VMD, higher amounts of fines, higherspecific surface area, higher true density, lower bulkand tap densities, lower flow properties, lower elon-gation ratios, and higher roundness values comparedwith commercial lactose. The crystallized lactose par-ticles from most ethanol—acetone mixtures showedrougher particle surfaces compared with commerciallactose. It was shown that by changing the volume ra-tio of ethanol—acetone the amount of amorphous, "-,and $-anomer contents will be altered and this canbe used to optimize the aerosolization performanceof lactose in the formulation of SS DPI. All crystal-lized lactose formulation blends showed significantlyhigher FPF and lower impaction loss than commer-cial lactose formulation blends. It was interesting tonote that the highest FPF was obtained for the formu-lation blends containing carrier powder with a poorflowability, indicating that the carrier flowability isnot related to the aerosolization performance.

ACKNOWLEDGMENTS

The authors gratefully thank the University of Dam-ascus for providing PhD scholarship for Waseem Ka-ialy. The authors also appreciate Mr. Ian Slipper,School of Science, University of Greenwich for takingSEM images.

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