The Effects of Capsule Fill Weight and Drug/Carrier Blend Ratio on
the Aerosolization of a Model Drug from a Spinhaler
Moawia M. Al-Tabakha* and Adi I. Arida+
* Department of Pharmaceutics, Faculty of Pharmacy and Health Sciences, Ajman
University of Science and Technology Network, P.O. Box 2202, Al-Fujairah, UAE.
Mobile: +971-50-3648300, Tel.: +971-9-2222644, Fax: +971-9-2227644, E-mail:
sphmaa@hotmail.com
+
Faculty of Pharmacy, Philadelphia University, P.O.Box 1, Postal Code 19392,
Jordan.
Tel: +962 2 6300200 Ext. 322, Fax: +962 2 6374440, E-mail: arida@go.com.jo
*Corresponding author
E-mail: sphmaa@hotmail.com
1
ABSTRACT
The purpose of this work was to examine the effect of capsule fill weight and
drug/carrier blend ratio of fluorescein isothiocyanate (FITC)-Dextran used as a
model drug on the aerosolization performance from Spinhaler. Micronised FITCDextran was tumbled with modified -lactose monohydrate in the ratio of 1:25 and
4:22. Factorial design experiments were carried out to test capsule fill weights of 26
and 104 mg for aerosolization using Andersen cascade impactor (ACI). Powders
were characterised in terms of particle size distribution, morphology and thermal
properties. Fine particle fraction based on loaded dose (FPFTotal) was increased
significantly from 14.9 to 23.1% when the blend ratio was increased from 1:25 to
4:22 (fill weight of 26 mg). The Device retention of FIT-Dextran was reduced as the
fill weight and/or blend ratio increased. Mass median aerodynamic diameter
(MMAD) of FITC-Dextran decreased slightly but significantly (from 3.59 to
3.27µm) with the increase in blend ratio for the fill weight 26 mg. Therefore the
increase in the capsule fill weight and/or drug: carrier blend ratio improves the
aerosolization performance. The effect of blend ratio was however greater compared
to fill weight.
Keywords: FITC-Dextran, blend ratio, fill weight, dry powder aerosol, Spinhaler;
Andersen cascade impactor.
2
INTRODUCTION
Respiratory drug delivery, which includes both pulmonary and nasal routes, may
offer certain advantages over other drug delivery systems. The respiratory tract is
void of gastric acid and has a reduced level of degrading enzymes, which are mainly
intracellular, compared to that of the gastrointestinal tract (1, 2). For systemic drugs
absorption is facilitated by a large surface area (50 m2) and an extensive pulmonarycapillary network (3). When considering drugs administered for local action within
the lungs, the oral administration may be limited by unacceptable toxicity to the liver
for example (4); hence topical administration to the lung can be a solution. One of
the advantages of the pulmonary route is the rapid and predictable onset of action
when considering local effects. Peptides and proteins that have poor oral
bioavailability due to inefficient transport across the gastrointestinal epithelium or
high levels of first-pass hepatic clearance can well be delivered through pulmonary
route. Many macromolecular drugs such as polypeptides and proteins have been
developed because of the application of recombinant DNA technology. Recently this
has increased attention to the use of inhalation route for delivery of inhaled proteins
(5). The therapeutic and economic requirements of this route demand the high
efficiency and reproducibility of the delivery system. FITC-Dextran of M. Wt. 4,400
Daltons was used as a model to represent a macromolecular, water-soluble group of
drugs, even though it lacks the secondary, tertiary, or quarternary structures of
proteins; since it belongs to carbohydrate class and not proteins. Many drugs used in
inhalation therapy are water-soluble and hygroscopic (6) which make FITC-Dextran
a suitable choice.
3
Three types of device are recognised for drug delivery to the lungs: pressurized pack
metered-dose inhalers (pMDIs), nebulizers and dry-powder inhalers (DPIs) (7). Of
these systems, DPIs are receiving a resurgence of interest (8). The low resistance unit
dose Spinhaler (0.051 cmH2O1/2/(L/min)) (9) was the first DPI to be introduced to
market by Fisons, which later became part of Rhône-Poulenc Rorer and now RhônePoulenc Rorer is part of Sanofi-Aventis. The minimum airflow required to expel the
powder into the inhaled air from this device was found to be 35 L/min (10). The
emission from this device was found to be dependant on the particle size with the
highest emission occurring for lactose size range (70-100 m) while fine particles <
10 m intensively coated the internal wall of the hard gelatin capsule (11). French et
al. (12) reported that the coarse carrier (PEG 8000) emitted from Spinhaler
exceeded that of the active drug by 20-30%. Lucas et al. (13) considered that the
preferential retention of a drug protein in the device when aerosolized from a blend
with coarse lactose was due to electrostatic and density effects. Podczeck (14)
showed that the adhesion force of drug particles to the capsule walls can be high
resulting in the loss of drug in the device and varies depending on the additives used
with the capsule. Although Spinhaler is known to have poor delivery characteristics
(15), it was used in this work because it allows easier optimisation of powder
formulations as the operation simply relies on loading size 2 capsules containing the
prepared formulations. Modifying the performance of dry powder formulations by
changing the order in which FPL, sieved lactose and the active drug was previously
investigated (16, 17). Such studies revealed that FPL significantly improved the
dispensability of the emitted powder. This effect was attributed to a reduction in the
electrostatic charge of the particles, formation of drug-FPL multiplets and the
reduction of the adhesion force between drug particles and coarse lactose by FPL
4
occupying the strongest binding sites. The highest step-improvement in the aerosol
performance was found at FPL level of 5% (13). On the other hand, there is a lack of
published data regarding the effect of increasing blend ratio of a drug to a carrier and
hence the percentage of drug particles in the mixture. This may be due to the fact that
drugs intended for inhalation in dry powder aerosols are formulated with predetermined dose. Increasing the amount of the model drug in the capsule loaded
formulations may act in similar way to that of FPL. Therefore such study was
conducted and accompanied by a parallel study where the capsule content of FITCDextran was varied by using different fill weights to provide an explanation of the
formulation performance.
MATERIALS AND METHODS
-lactose monohydrate (batch no. 750707) was obtained from Borculo Whey
Products, UK. FITC-Dextran mol wt 4,400 Daltons (lot no. 77H0362) and sorbitan
trioleate (Span 85, lot no. 23H0642) were purchased from Sigma, UK. Chloroform
(batch no. 9896217388) was obtained from Fisher, UK. Gelatin capsules of size 2
(Farillon Limited, UK) was used with Spinhaler from Fisons.
The coarse carrier lactose (75-106 m) was prepared, using a mechanical tap sifter
(Pascall Engineering, England), from -lactose monohydrate in order to remove
particles of other sizes particularly the fine particles. The sieving was carried out as
described in the Pharmacopeial Forum (18) using two sieves, one with an aperture of
75 μm and the other of 106 μm (laboratory test sieves, Endecotts Ltd, England). For
this purpose 25 g of the lactose was loaded to the top sieve (106µm) and sieved for
5
30 minutes. Micronised lactose to be added to the coarse lactose as FPL and
micronised FITC-Dextran were pulverised separately using a fluid jet mill (Gem-T
Air-Impact Mill, Glen Creston Ltd., UK) at the differential pressure set (70-100 psig)
as recommended by the mill company. Before milling, each powder was ground
using a mortar and pestle to reduce any large particles and to aid in achieving a
normal particle size distribution.
Fluorimetric Analysis of FITC-Dextran
Fluorescence measurements were obtained using the LS-5 luminescence
spectrometer (Perkin Elmer, UK). To produce a standard calibration curve, the
fluorescence from three sets of concentrations of FITC-Dextran solution (0.00-0.42
g/ml) in phosphate buffer medium (pH 8.00  0.05) was made. The temperature of
the cuvette in the jacketed slot was maintained at 25 C by circulating water. The
scanned excitation wavelength (ex) and emission wavelength (em) by the LS-5 for
the solution containing FITC-Dextran were found at 492 and 516 nm respectively.
From the equation of the calibration curve, the amount of FITC-Dextran in any
sample was calculated.
Preparation of the powder blends
The carrier system was first prepared by tumbling on a roller (speed of 90 rpm) FPL
(5%) with sieved lactose (75-106 m) for one hour. Powder blends of FITC-Dextran
and the prepared carrier in the ratio of 1:25 and 4:22 were then similarly prepared by
accurately weighing the fractions of powders which were transferred to a small glass
6
vial. The contents of the vial were tumbled on a roller for 1 hr at a speed of 90 rpm.
Loading the capsules with the required weight was made manually using the back of
the 25 ml volumetric flask cap to mount the capsule body and microspatula to
transfer the formulation.
In the European Pharmacopoeia (19), test B is applied to the uniformity of predispensed dose of powders for inhalation. The homogeneity of the mixtures was
evaluated by analysing FITC-Dextran in ten random samples from each blend
(lactose did not interfere with the fluorescence measurements). The coefficient of
variation (% C.V.) was then calculated for each blend to express the degree of
homogeneity. As two different fill weights were used in the current study, the smaller
weight 26 mg was the scale of scrutiny.
Particle Size Distribution and Morphology
Micronised FITC-Dextran, FPL, the sieved lactose (75-106 m) and the modified
sieved lactose containing the 5% w/w of FPL were measured for particle size using
Malvern system 2600 (Malvern Instruments Ltd., UK.) which employs laser
diffraction method. Size analysis of suspended particles was carried out in liquid
made of chloroform plus 0.1% w/v sorbitan trioleate as a wetting agent and using an
appropriate lens. Particle size was expressed in terms of volume diameters of the
median (VMD) and the 10 and 90% fractiles of the size distributions. Additionally,
the volume of the particles below 10 m was calculated. Independent particle size
model was used and the obscuration was adjusted between 0.11 and 0.19. The
suspended particles were measured at least in triplicate.
7
Deposition Profile of FITC-Dextran Using an Andersen Cascade Impactor (ACI)
The storage and testing of the formulation products were undertaken in controlled
temperature and humidity laboratory of 18 C and 35-40 % R.H. respectively. This is
to avoid hygroscopic growth of particles (20). ACI was used to assess depositions
from the different formulations emitted from a Spinhaler at the flow rate of 60
L/min for 4 sec. It consists of 8 impaction plates and a filter stage, starting from stage
0, then 1, 2, 3, 4, 5, 6, 7 and a filter stage, the latter was fitted with glass microfibre
filter paper. The effective cut-off diameters of these stages at 60 L/min are: 6.18,
3.98, 3.23, 2.27, 1.44, 0.76, 0.48 and 0.27 m (21). A pre-separator was fitted on top
of the impactor to prevent particle bouncing and re-entrainment errors and to reduce
overloading of the Andersen stages used (22). The filling of the capsules (size 2) was
made just before aerosolization. The capsule to be tested was introduced to the
Spinhaler. The latter was fitted into a moulded rubber mouthpiece attached to the
throat piece of the pre-separator. After ensuring that the assembly is vertical and
airtight, the flow rate was adjusted at 60 L/min and the pump was then switched off.
The capsule was then pierced by the Spinhaler and the pump was switched on for
the period of 4s to release the dose from the capsule. The masses of FITC-Dextran
deposited on the various sites of the assembly were then washed with phosphate
buffer solution (pH 8.00  0.05) and quantified fluorimetrically. This experiment
was repeated five times for each blend ratio and fill weight. The deposition profile of
FITC-Dextran after aerosolization was expressed in terms of percentage to allow a
comparison between the prepared formulations containing micronised FITC-Dextran
and lactose in the ratio of 1:25 and 4:22 (fill weight of 26 and 104 mg). The data
collected from five separate aerosolization experiments were compared using
8
ANOVA (p<0.05, n=5) for each blend and fill weight, the differing groups being
identified using the least significant difference test. The calculated aerosol
parameters to assess the formulation performance were the device retention which is
calculated by subtracting the emitted FITC-Dextran from the loaded, and the fine
particle fraction (FPF) that is assumed to have a predictive value for the amount of
drug that will reach the lungs in vivo (23) and is calculated as cumulative percentage
under size of stage two (< 3.98 m) based on loaded and emitted dose. The first is an
index of formulation efficiency denoted as FPFTotal, while the second (FPFEmitted) is
an index to the extent of emitted dose dispersion but ignores any dose metering or
device deposition. The mass median aerodynamic diameter (MMAD) is the most
common parameter employed to characterise an airborne particle. The mass
distribution of FITC-Dextran on the various stages of the impactor was converted to
a cumulative percentage under size. The probit values of the cumulative were plotted
as the ordinate versus the log effective cut-off diameters as the abscissa. From a
straight line on points close to the cumulative percentage of 50% (probit = 5), that is
from 20 to 80% (from probit 4.16 to probit 5.84), the MMAD was calculated.
Experimental Design
In addition to the ANOVA analysis, factorial design was applied which serves as a
test for the influence of certain factors on one or several responses and if any
interaction occur (24, 25). The experiment shows two factors (i.e. drug to carrier
ratio (factor A) and fill weight (factor B)) on two levels (i.e. low when the blend ratio
is 1:25 or when the fill weight is 26 mg and are given the negative sign - or high
when the blend ratio is 4:22 or when the fill weight is 104 mg and are given the
9
positive sign +). This results in a 22-factorial design. The experiments as shown in
table 1 are listed in standard order as (1), a, b and ab, where (1) denotes the
experiment when all levels are at their lowest while ab denotes the experiment when
the factors A and B are at their highest. The magnitude of any factor can be
calculated by taking the mean when the factor is at highest and subtracting from it
the mean when the factor is at lowest.
RESULTS
Powders Characterisation
The volume percentage of particles < 10 m is considered important for drug
delivery to the lung. Micronised FITC-Dextran revealed that the volume median
diameter (VMD) was 5.30 m with 81.3% of the particles volume below 10 m. The
VMD of micronised lactose was found to be 5.62 m with all particles below 20.70
m. Micronised lactose particles were rounded and have smooth surfaces in
comparison to the micronised FITC-Dextran when examined by an electron
microscope. Sieved lactose fraction (75-106 m) had VMD of 116.39 m which lies
outside the designated size fraction. Sieving of lactose to remove all fine particles
was not completely successful as there were 0.8 % of the particle volumes below
10.0 m as measured by Malvern. The addition of FPL to the coarse lactose fraction
(75-106 m) resulted in lowering the VMD to 112.13 m and an increase in the
fraction <10.0 m to 5.1%. Examination of this powder by an electron microscope
showed the presence of FPL freely dispersed in the powder.
10
Content Uniformity
The blends tested were found to be homogenous and to pass the uniformity of predispensed dose in accordance to European Pharmacopoeia (19) as the coefficient of
variation was < 2.9% (n= 10) for all blends.
Deposition Profile of FITC-Dextran
The deposition profile of FITC-Dextran after aerosolization is given in table 2 and
figure 1. The results indicate that the presence of higher ratios of FITC-Dextran to
lactose improves the emission of FITC-Dextran (p< 0.00007) for the fill weight 26
mg and (p< 0.03) for the fill weight 104 mg. Similarly, increasing the capsule fill
weight and hence the amount of FITC-Dextran resulted in increased emission (p< 8 *
10-7) for the blend ratio 1:25 and (p<0.00001) for the blend ratio 4:22. Therefore the
presence of larger amounts of FITC-Dextran in the capsule is beneficial to the
emission.
When using high blend ratio (4:22) with the fill weight of 26 mg in comparison with
similar FITC-Dextran capsule content using blend ratio of 1:25 and the fill weight of
104 mg, the effect of FITC-Dextran fine particles on the bulk properties of the
aerosolized formulation is obvious. The emitted dose of FITC-Dextran for the first
(50.3%) is significantly lower than the second (60.0%), (p<0.00002) probably
because of flowability issues. This is because fine particles enhance the van der
Waals cohesive forces which operate between neighbouring particles (26). On the
other hand, such difference did not improve performance when considering FPFTotal,
11
while when considering FPFEmitted and MMAD, it is the freely dispersed FITCDextran (hence the higher blend ratio) that significantly improved the performance of
the aerosolized powder.
Additional information was obtained by applying the principles of factorial design to
the experiments as shown in table 3. The experiments show two factors (i.e. drug to
carrier ratio (factor A) and fill weight (factor B)) on two levels. The fill weight was
shown to be more effective in reducing the percentage of FITC-Dextran retained by
the device compared to the blend ratio (16.4% to 6.7%) as a result of higher
percentage of fines present in the latter formulation. Although both factors acted
synergistically (indicated by the greater FITC-Dextran emission when both factors
were used at their maximum compared to any single factor), there was some degree
of interaction that reduces device emptying (2.6%).
Although the fill weight was more important to percentage emission of FITCDextran compared to blend ratio, this was not reflected in the results of FPFTotal (i.e.
both factors showed a similar effect (6.1 and 6.9% respectively). On the other hand,
the increase in the FITC-Dextran emission by increasing the blend ratio (6.7%)
resulted in similar corresponding increase in the FPFTotal (6.9%). Both factors (blend
ratio and fill weight) acted synergistically, but there was small negative interaction
(1.4%) on the FPFTotal.
When examining FPFEmitted, it was clear that blend ratio is the factor to consider in
order to achieve improvement. The fill weight had a small and negative effect on the
12
FPFEmitted (0.8%). Although the factors acted antagonistically, the effect of blend
ratio was apparent compared to the negative interaction (8.0 to -1.8% respectively).
The results of MMAD indicate similar conclusions to that of FPFEmitted, but the
reduction of MMAD by increasing the blend ratio was as significant as the opposing
increase achieved by increasing the fill weight. The interaction was small (0.06 m)
compared to any individual effect.
It was possible to decrease the retained dose of FITC-Dextran in the device to nearly
half (from 59.0% to 35.9%) by modifying the formulation with a ratio of 1:25 and fill
weight of 26 mg to a ratio of 4:25 and fill weight of 104 mg. The increases of
FPFTotal from 14.9% to 27.8% and FPFEmitted from 36.2% to 43.4% without
significantly affecting the MMAD were also achieved. The deaggregation pattern of
the emitted dose was better when higher ratios of FITC-Dextran to lactose were used
as indicated from the results of fine particle fraction (FPFEmitted) and the MMAD.
DISCUSSION
Powders Characterisation
The measured VMD of sieved lactose fraction (75-106 m) by Malvern showed a
value larger than the designated fraction because the measured particles were not
spherical (27). Fine particles were not completely removed by sieving, because such
particles adhere sufficiently tenaciously to the coarse particles not to be displaced
13
during sieving (28). The presence of the freely dispersed FPL upon mixing with
sieved lactose indicates the quick saturation of the binding sites on the coarse lactose
particle by FPL. A similar observation with lactose fraction (63-90 m) even at a
lower level of added FPL ( 1.5%) was noted by other workers (29).
The larger proportion of fine particles freely dispersed in the powder formulation
with blend ratios of 1:25 and 4:25 are expected to be largely of micronised FITCDextran because the coarse particles are already saturated by the FPL and since
FITC-Dextran can only adhere to the coarse particle directly by replacing FPL
(redistribution of particles) or indirectly by building up particle aggregates. As such,
it is expected that the formulation performance would be attributed to these freely
dispersed particles.
Deposition Profile of FITC-Dextran
The increased emission of FITC-Dextran whether by increasing fill weight and/or
increasing blend ratio indicates that a fraction of FITC-Dextran is adsorbed on the
inner walls of the capsule and the device. When the walls are coated with adsorbed
particles, the percentage retention of the remaining FITC-Dextran by the device
would be expected to decrease allowing an increase in emission. The intensive
coating of the inner wall of a capsule by fine lactose (< 10 m) was previously
reported (11). Such effect can be attributed to a variety of particle-surface
interactions. Particle size, density, electrostatic charge and moisture content of FITCDextran can influence the device retention. Because of FITC-Dextran hygroscopicity
and because a capsule inherently contains 12-15% water, liquid bridging may form
14
resulting in such loss to the device. As FITC-Dextran was micronised by jet milling,
which processes powder by attrition and impaction, the presence of particle charge
would not be surprising and this can contribute to device losses.
The interaction between increased fill weight and increased blend ratio (i.e. 2.6%) is
opposite to the action of increasing the blend ratio or the fill weight. This is nearly
half the magnitude of the blend ratio’s effect, while it is negligible when compared to
the fill weight effect. Therefore more attention should be given to the fill weight
when higher emission is required. The interaction indicates that the volume occupied
by the powder in the capsule may affect emission.
As the increase in the FPFTotal for increased fill weight or blend ratio (6.1 and 6.9%
respectively) are similar, it indicates that there is a corresponding increase in the
FITC-Dextran deposition in the pre-separator and on the upper stages of the
Andersen impactor resulting from modifying the fill weight which increases
emission. This can be attributed to the effect of the fill weight on modifying the
plates of the Andersen impactor. The more particles deposited on the upper stages
can prevent the succeeding smaller particles from penetrating to the lower stages of
the ACI. This was demonstrated by Graham et al. (30) and Nasr and Allgire (31).
The latter recommended the use of least number of doses and/or fill weight in one
experiment. Therefore a factor can be introduced by the Andersen impactor for high
fill weight.
The small negative effect of increasing the fill weight (0.8%) on the
FPFEmitted, is probably because of the same reason explained before, as the large fill
15
weights would modify the fractionation of the aerosolized powders by Andersen
impactor. and therefore can be used to counteract any negative affect from the fill
weight that might actually be due to Andersen sampling effect.
When considering the results of both FPFEmitted and MMAD, combination of
blend ratio with the fill weight can counteract the negative effect of the latter, while
both were important to increasing device emission and FPFTotal. Hence it is important
to optimise both factors when preparing formulations for inhalation.
CONCLUSION
The results suggest that increasing the fill weight and/or drug: carrier blend ratio
enhances the aerosolization of a model drug FITC-Dextran. The results are explained
on the basis of physical interactions of micronised FITC-Dextran with themselves,
FPL, sieved lactose and capsule and device walls. When greater numbers of
micronised FITC-Dextran particles are available in the studied limits, the emission
and dispersion improved, when flow is also considered. The results highlight the
need to examine different types of permissible fine particles as performance
modifiers for the aerosolized formulations. The interaction of FITC-Dextran particles
with the capsule and device walls needs further investigation.
16
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1677-1681.
20
Table 1.
Factorial design of two factors with two levels experiment (22).
Experiment
Factor A (ratio)
Factor B (fill weight)
Interaction of A and B
(1)
-
-
+
a
+
-
-
b
-
+
-
ab
+
+
+
21
Table 2.
FITC-Dextran deposition (mean  (SD)) after aerosolization using two blend
ratios and two fill weights (n=5).
Formulations (1:25, 26)
(4:22, 26)
(1:25, 104)
(4:22, 104)
Aerosol
parameters
Device (%)
59.0 (2.4)
49.7 (1.2)
40.0 (1.9)
35.9 (2.9)
FPFTotal (%)
14.9 (1.0)
23.1 (0.7)
22.3 (0.8)
27.8 (1.1)
FPFEmitted(%) 36.2 (1.2)
46.0 (1.6)
37.2 (1.0)
43.4 (0.6)
MMAD (µm) 3.59 (0.09)
3.27 (0.07)
3.73 (0.04)
3.53 (0.04)
Abbreviations:
The formulations are represented by the (blend ratio, fill weight). SD: Standard
deviation
22
Table 3.
The effect of the blend ratio and the fill weight and their interaction on the
percentage of FITC-Dextran retained by the device, fine particle fraction
(FPFTotal and FPFEmitted) and mass median aerodynamic diameter (MMAD).
Place of effect
Factor A
(ratio)
Factor B
Interaction
(fill weight) of A and B
Factor magnitude on device retention (%)
- 6.7
-16.4
2.6
Factor magnitude on FPFTotal (%)
6.9
6.1
-1.4
Factor magnitude on FPFEmitted (%)
8.0
-0.8
-1.8
Factor magnitude on MMAD (m)
-0.26
0.20
0.06
23
Figure 1. The effect of varying blend ratio and/or fill weight on the dispersion of
FITC-Dextran formulation.
100%
% FITC-Dextran
80%
60%
40%
20%
0%
1:25, 26
4:22, 26
1:25, 104
4:22, 104
Formulation
FPFT otal
Pre-separator
24
Device