UNIVERSITY GHENT
FACULTY OF PHARMACEUTICAL SCIENCES
Department of Pharmaceutics
Laboratory of Pharmaceutical Technology
Academic Year 2009-2010
EVALUATION AND CHARACTERISATION OF NANOSTRUCTURE PRESERVATION AFTER BALL
MILLING AND SPRAY DRYING
Liesbet D’HAESE
First Master in Drug Development
Promoter Prof. Dr. C. Vervaet
Commissioners Prof. Dr. S. De Smedt Prof. Dr. T. De Beer
COPYRIGHT
”the author and the promoter give the authorization to consult and to copy
parts of this thesis for personal use only. Any other use is limited by the laws
of copyright, especially concerning the obligation to refer to the source
whenever results from this thesis are cited.”
May 27, 2010
Promoter Author
Prof.Dr. C. Vervaet Liesbet D‟Haese
Acknowledgment
First, I wish to thank my promoter from University Ghent, Prof. Dr. Apr. C.
Vervaet, to make this research internship abroad possible.
As an Erasmus exchange student, I did my research and writing of this master
thesis at the University of Helsinki. Therefore, I want to thank all the members
of the Division of Pharmaceutical Technology for helping me and answering
all my questions while performing the laboratory work. In particular Timo
Laaksonen for guiding me and advising me in the writing process.
Also a great thank to J. Heinämäki for welcoming me the first days.
Last but not least, thanks to my parents for supporting me in this big
adventure. This was one of the most wonderful experiences in my life up to
now and it would have never been possible without them.
TABLE OF CONTENTS
1. INTRODUCTION ..................................................................................... 1
1.1. GENERAL ......................................................................................... 1
1.2. COLLOIDAL SUSPENSIONS AND STABILISERS ........................... 2
1.3. PRODUCTION OF NANOPARTICLES ............................................. 7
1.4. TRANSFORMATION INTO A POWDER ........................................... 8
1.5. THEORITICAL APPROACH OF THE USED TECHNIQUES .......... 12
2. OBJECTIVES ......................................................................................... 15
3. MATERIAL AND METHODS .................................................................. 17
3.1. CHEMICALS .................................................................................... 17
3.2. MILLING PROCEDURE .................................................................. 19
3.3. SPRAY DRYING ............................................................................. 20
3.4. SOLUBILITY .................................................................................... 23
3.5. DISSOLUTION TESTS .................................................................... 23
3.6. DSC ................................................................................................. 26
3.7. XRPD............................................................................................... 26
3.8. TEM ................................................................................................. 27
4. RESULTS AND DISCUSSION ............................................................... 28
4.1. MICROSCOPY ................................................................................ 28
4.1.1. TEM of indomethacin ............................................................. 28
4.1.2. TEM of itraconazole ................................................................ 28
4.2. PHYSICOCHEMICAL CHARACTERIZATION OF THE SPRAY
DRIED POWDERS BY DSC AND XRPD ........................................ 31
4.3. EVALUATION OF SOLUBILITY AND DISSOLUTION RATE .......... 36
4.3.1. solubility and dissolution tests of indomethacin ................ 36
4.3.2. solubility and dissolution tests of itraconazole ................... 39
5. CONCLUSION ........................................................................................ 42
6. REFERENCES ....................................................................................... 44
LIST OF USED ABBREVIATIONS
DLVO theory: Derjaguin, Laundau, Verwey, and Overbeek theory
DSC: Differential Scanning Calorimetry
HPLC: High Performance Liquid Chromatography
Ph. Eur.: European Pharmacopoeia
PVDF: polyvinylidenefluoride
rpm: rotations per minute
SDS: sodiumdodecylsulphate or sodiumlaurylsulphate
TEM: Transmission Electron Microscope
UV: ultraviolet
XRPD: X-Ray Powder Diffraction
Introduction
1
1. INTRODUCTION
1.1. GENERAL
During the last decades, the formulation of drugs as nanoparticles has
become a valuable tool in pharmaceutical technology to obtain a high
dissolution rate, and hence increase (oral) bioavailability. As more and more
possible drug compounds have low solubility and/or dissolution rates, this
formulation approach is very interesting. Since 2000, there has been an
exponential growth of scientific and patent publications and at this moment
there are five products on the market that use this nanoscience strategy, more
specifically nanoparticles. They are all produced by using the top-down
approach (see below). Four of them rely on media milling and one on high-
pressure homogenization (see below). These products are all for oral
administration because this route has important advantages e.g. not painful
like injections and the patient compliance is much better. It is also preferred
from a marketing point of view. Notice that four of the five products are in their
solid state. Beside marketing aspects, physical as well as chemical stability
problems of colloidal suspensions are the major reasons for this. (Van
Eerdenbrugh et al., 2008a).
With this master thesis, we wanted to provide more insight in the
appearance and behaviour of the final product that we obtain after the
process of ball milling and spray drying. Following questions have been made:
How do the particles look like? Can we preserve the nanostructure after spray
drying? Are there any changes in their chemical or physical properties? What
about their crystallinity? Do we need excipients to keep a reasonable
dissolution rate after spray drying?
To study this, we worked with two different drugs: indomethacin (a non-
steroidal anti-inflammatory drug) and itraconazole (a triazole antifungal
agent). What they have in common is their poor solubility in water. We used
them purely as model compounds. The compounds themselves don‟t need
any bioavailability improvements but they are easy to obtain and the increase
Introduction
2
in dissolution rate can already be seen in in vitro experiments. Both products
were processed in a similar way. The same measurements were performed in
order to compare their behaviour and properties after ball milling and spray
drying.
1.2. COLLOIDAL SUSPENSIONS AND STABILISERS
A colloidal suspension consists of two separate phases. One phase
has dimensions remarkably smaller than the other. The suspended phase
(internal phase) is dispersed uniformly throughout the suspending medium
(external phase). The size of the suspended particles ranges between 1 nm
and 1 µm and they have a minimal degree of solubility in this system (Nash,
2007).
The goal of developing a colloidal suspension is to improve
bioavailability of poorly water-soluble drugs after oral administration. This
improvement is caused by a higher dissolution velocity, due to a total surface
area that is typically orders of magnitude larger compared to the coarse
suspension. This phenomenon can be explained by the Noyes-Whitney
equation (1.1).
L
CCsDA
dt
dW )( (1.1)
where:dt
dW: rate of dissolution (g/s)
D : diffusion coefficient (cm2/s)
A: surface area of the solid (cm2)
Cs: solubility or concentration of a saturated solution at
the surface of the solid particle (g/ml)
C: concentration of the solid in the bulk dissolution
medium (g/ml)
L: diffusion layer thickness (cm)
Introduction
3
However, the increase of total surface area has one big disadvantage:
this state is thermodynamically unstable because the interface is bigger (the
Gibbs energy (ΔG) is positive). To minimize the total energy (because a
system always wants to be in its lowest energy state), the nanoparticles tend
to agglomerate. Due to this process, the surface area is minimized and a
thermodynamically stable state is obtained. By adding stabilisers, this process
is slower because they increase the activation energy of the agglomeration
process. Figure 1.1. shows the basic theory of thermodynamics and kinetics.
Thermodynamics defines whether a reaction emerges spontaneously or not.
A reaction will occur when ΔG is negative. However, it doesn‟t explain why a
reaction goes fast or slow. Kinetics can offer the answer here: it depends on
the activation energy (Ea). A reaction goes faster when its activation energy is
lower (http://mcat-review.org/rate-kinetics-equilibrium.php).
FIGURE 1.1. ILLUSTRATION OF THE DIFFERENCE BETWEEN
THERMODYNAMICS AND KINETICS: ΔG = GIBBS ENERGY; Ea =
ACTIVATION ENERGY.
(http://mcat-review.org/rate-kinetics-equilibrium.php)
Because of the amphiphilic character of a stabilizing system, it can act
as a boundary between the hydrophobic product and the hydrophilic medium.
The hydrophobic part is orientated to the particles surface, while the
hydrophilic part is orientated to the medium. It is important to find the right
Introduction
4
type of stabiliser in the right concentration. This is the key to a successful
production. Ionic surfactants can be used to obtain colloidal suspensions. Also
non-ionic surfactants or polymers are possible. They fulfil their function
electrostatically and sterically, respectively (Van Eerdenbrugh et al., 2008a;
Van Eerdenbrugh et al., 2009).
FIGURE 1.2. ILLUSTRATION OF THE POTENTIAL ENERGY AS A
FUNCTION OF INTERPARTICLE DISTANCE (CLASSICAL DLVO):
TOTAL POTENTIAL ENERGY (Vtot), ELECTROSTATIC ENERGY (Vel)
AND LIFSHITZ-VAN DER WAALS ENERGY (Vlw) (Van Eerdenbrugh et
al., 2008a).
The Derjaguin, Laundau, Verwey, and Overbeek theory (DLVO theory)
describes the interaction energy between the electric double layers of two
identical charged particles as a function of interparticle distance, illustrated in
figure 1.2. The upper curve shows the electrostatic repulsion, while the lower
curve shows Van der Waals attraction. The sum of those two curves is the
net energy balance. An electrostatic stabiliser influences the electrostatic
curve. Notice that the ζ-potential rather than the Nernst potential rules the
degree of repulsion between identical charged particles, illustrated in figure
1.3. Particles can be stabilised by giving them a maximal ζ-potential. The
energy barrier will be sufficient in suspension, but not when sedimentation
occurs. Then the pressure will cause caking. Adsorption of polymers or non-
ionic surfactants with long chains prevents close approach of the particles
Introduction
5
sterically. When two chains try to come closer, the entropy decreases (partial
loss of conformational freedom), resulting in an increase of Gibbs energy (see
equation 1.2). So, repulsion is preferred from an energetic point of view (Liang
et al., 2007).
ΔG = ΔH – T ΔS (1.2)
Where: ΔG: Gibbs energy (J)
ΔH: enthalpy (J)
T: temperature (K)
ΔS: entropy (J/K)
FIGURE 1.3. DIFFUSE DOUBLE LAYER AND THE ZETAPOTENTIAL
(http://www.malvern.de/LabGer/technology/images/zeta_potential_sch
ematic.png)
Despite of the wide range of stabilizers, only a few are used for
colloidal suspension production. The poloxamers and Tween®80 are the most
used excipients. Also sodiumlaurylsulphate (SDS) as an ionic surfactant is a
common used option. Additionally, we have the lecithins, cellulosics, polyvinyl
alcohol and povidones. You can use them in ratios from 1/3 to 50/1,
representing drug/stabilizer (weight/weight). Their molecular structures can be
seen in figure 1.4. (Van Eerdenbrugh et al., 2008a).
Introduction
6
FIGURE 1.4. MOLECULAR STRUCTURE OF (A) POLOXAMER; (B)
TWEEN®80; (C) SODIUMLAURYLSULPHATE; (D) LECITHINS; (E)
CELLULOSICS; (F) POVIDONES; (G) POLYVINYLALCOHOL.
((A)http://www.medicinescomplete.com/mc/martindale/2009/images/c9003-
11-6.gif;(B)http://ntp.niehs.nih.gov/?objectid=0709A276-0D0E-3EBD-
A3B3CCC2CD707101;(C)http://chemicalland21.com/specialtychem/perchem/
SODIUM%20LAURYL%20SULFATE.htm;(D)http://www.diytrade.com/china/4/
products/489819/Lecithin_Phosphatidylcholine_PC.html;(E)(F)(G)Van
Eerdenbrugh et al., 2009)
Introduction
7
Higher concentrations of stabilisers have, in general, a positive effect
on colloidal suspension production and hence stability. The more hydrophobic
the surface is, the more nanoparticles tend to agglomerate. That is why the
surface hydrophobicity of a particular compound is a decisive factor when
considering the feasibility of obtaining a colloidal suspension (Van
Eerdenbrugh et al., 2009).
1.3. PRODUCTION OF NANOPARTICLES
There are different possibilities to produce nanoparticles. You can
obtain them by building up particles, starting from dissolved molecules that
have to be precipitated (bottom-up approach). They have a great potential to
increase bioavailability but due to some limitations, no products using this
system have been released yet. Some disadvantages of this production
process are: polydispersity, scale-up difficulties, long term process and low
drug loading efficiencies. The most common way is by reducing the particle
size of larger crystals (top-down approach). This includes high pressure
homogenization and media milling. The former technique reduces the particle
size by forcing a suspension through a very thin hole of about 25 µm many
times at high velocity. The latter technique mentioned, grinds the drug
particles and stabiliser using milling media, e.g. glass, zirconium oxide, highly
cross-linked polystyrene resins (Van Eerdenbrugh et al., 2009).
In this study, we used a Planetary micro mill “PULVERISETTE 7
premium line” (Fritsch, Idar-Oberstein, Germany) for wet milling in order to
produce a colloidal suspension. The grinding bowls rotate around their own
axis and around a central axis, though in opposite directions. Consequently,
the grinding material and grinding balls are subject to centrifugal forces which
act alternately in the same and opposite directions. The grinding balls move
along the inner wall of the grinding bowl and hit the opposite wall. Speeds up
to 1100 rotations per minute (rpm) can be reached. Because we can achieve
such high energy values, production of particles below 100 nm are possible
(http://www.fritsch.de/uploads/media/BA_075000_0100_e_02.pdf).
Introduction
8
1.4. TRANSFORMATION INTO A POWDER
As mentioned above, a solid dosage form is preferred over liquid
forms. Patient convenience is one of the reasons. Another reason is the
physical stability: The Ostwald-Freundlich equation (1.3) describes the
process of Ostwald ripening. This is the growth of bigger particles at the
expense of smaller ones. This phenomenon can be understood by a higher
solubility of the smaller particles, so there is a mass transport from high to low
concentration.
212
1 11
303,2
2log
rrRT
MW
S
S (1.3)
Where: S1, S2: solubility of the particles (g/ml) with radius r1 and
r2 (m), respectively
γ: interfacial tension between liquid and solid (N/m)
MW: molecular weight of solid (g/mol)
R: gas constant (J/mol.K)
T: absolute temperature (K)
ρ: density of the solid (g/m3)
Another physical stability issue is sedimentation. Stokes´ law (1.4.) describes
this process.
18
)(2
ss
t
gDV (1.4.)
Where: Vt:sedimentation velocity (m/s)
ρs-ρ :density difference between particle and medium
(kg/m3)
g: gravityconstant (m/s2)
DS: average particle radius (m)
µ: viscosity medium (kg/m.s)
Introduction
9
Agglomeration causes a higher rate of sedimentation. Sedimentation results
in forming a cake at the bottom that is hard to resuspend. It can be explained
by the high interparticular forces in the sediment (Van der Waals attraction at
small distance).
Despite the preference of solid formulations, we have to keep in mind
that the maintenance of the rapid dissolution is the primary goal of our
nanoparticle formulation. Hence, the disintegration of the powder as well as
the redispersion of the nanoparticles cannot be a barrier on the overall
dissolution process (van Eerdenbrugh et al., 2008b).
To transform the colloidal suspensions into a solid state, spray drying
can be used. In the article of Wang et al. (2005), an overview is given of
drying technologies for nanomaterials. They state that spray-drying is often
used because of the low-cost, simplicity, and the ease to scale-up. In our
study, we used the Mini Spray Dryer B-191 (Büchi, Flawil, Switzerland). The
initial step consists of atomizing/spraying suspensions into droplets and their
dispersion into the gas (air). Subsequently, a drying process results in
aggregates of the nanoparticles, from loose flocculates to completely fused
particles (Lee, 2003). They are essentially microparticles, but nanostructured.
The advantage: microparticles are easier to formulate into e.g. tablets,
capsules; while after redispersing in water, the individual nanoparticles are
released and provide an improved therapeutic action (Peltonen et al., 2010).
The article of Cal and Sollohub (2010), gives a clear review about the
spray dryer hardware and process parameters that affect the properties of the
final product. „Trial and Error‟ is the most common way to optimise these
parameters for every single product. The mutual interrelationships among
them can be seen in table 1.1. This tool is very useful when performing the
drying process.
Introduction
10
TABLE 1.1. MUTUAL INTERRELATIONSHIP BETWEEN SPRAY DRY
PARAMETERS (http://www.buchi.com/Mini_Spray_Dryer_B-
290.179.0.html?&no_cache=1&file=308&uid=2283).
Lee (2003) compared particle size distribution of micro- and
nanoparticles before and after spray-drying. In figure 1.5. it can be seen that
nanoparticles show a unimodal peak of 100-500 nm before spray drying, while
microparticles have a bimodal distribution. After spray drying, the
microparticles keep the same distribution. In contrast, the nanoparticles size
distribution has become bimodal and an increase of size can be seen. The
aggregates (microparticles) which have been formed can disintegrate into
nanoparticles after some time. He also noticed that there were no peaks
observed between the main ones during disintegration time. This means that
the aggregates break into primary nanoparticles cooperatively, not one by
one. Due to this possibility of breaking aggregates into nanoparticles, this
nanoformulation will have different dissolution and disintegration
characteristics than a microformulation. Also the dissolution rate will be higher
because primary particles can dissolute before the complete breakup of
aggregates.
Introduction
11
FIGURE 1.5. (a) NANOPARTICLE SIZE DISTRIBUTION BEFORE/AFTER
SPRAY DRYING AND MEASUREMENTS OF CHANGES FOR 25 HOURS
AFTER FIRST MEASUREMENT OF SPRAY DRIED MATERIAL (0 h); (b)
MICROPARTICLE SIZE DISTRIBUTION BEFORE AND AFTER SPRAY
DRYING (Lee, 2003).
Spray drying often requires the addition of a matrix former to prevent
agglomeration. It ensures the maintenance of the rapid dissolution after the
process. Agglomeration results in a bimodal dissolution profile. It consists of
an initial phase of burst release and subsequently a phase that shows slower
release. The former is due to the individually dispersed nanoparticles, while
the latter can be attributed to agglomerated nanoparticles (Van Eerdenbrugh
et al., 2008c). A negligible drying effect on dissolution was obtained for less
Introduction
12
hydrophobic compounds, whereas compounds with a more hydrophobic
surface were subject to agglomeration. The latter compounds mentioned
need a matrix former. There are several mechanisms for explaining the
agglomeration that occurs while drying the colloidal suspension, e.g. capillary
pressure theory, crystal bridge theory, hydrogen bond theory, chemical bond
theory, etc. There is no sole theory that is generally accepted (Wang et al.,
2005).
With the technique of X-ray microanalysis during electron microscopy,
the degree of dispesion of a drug in different matrix formers can be studied
qualitatively. This technique is only possible for drugs that contain a chemical
element that´s not present in the matrix former, e.g. chloride in itraconazole.
The results in the article of van Eerdenbrugh et al. (2008d) suggest that
dispersion of the drug in the matrix former can influence the dissolution
performance.
1.5. THEORITICAL APPROACH OF THE USED TECHNIQUES
Besides performing the dissolution tests of the spray dried powders, we
also made some measurements to investigate their physical properties. In
following paragraphs, some brief descriptions of the used analytical
techniques are given.
Differential Scanning Calorimetry (DSC) is used to perform a thermo
analysis. It measures changes in chemical or physical properties of a sample.
The sample and the reference material are subjected to a controlled
temperature program, while the difference in energy input (enthalpy) is
measured as function of temperature. The temperature increases linear with
time. The basic principle: when a physical transformation occurs, e.g. a phase
transition, the required heat to maintain the same temperature as the
reference will change. When an exothermic process occurs, which means that
heat is released, the flat base line changes into a positive peak. An
endothermic process, on the other hand, results in a negative peak below the
Introduction
13
base line. This technique helped us to determine changes in crystallization
and melting temperatures after ball milling and spray drying (Dean, 1995;
Pungor, 1995).
Another technique, X-ray powder diffraction (XRPD), identifies the
crystallinity of the powders. By comparing the resulting X-ray diffraction
patterns of the powders, changes in crystallinity can be detected. It was used
to confirm the results of the DSC measurements. The basic principle
underlying this technique: when a focused X-ray beam (wavelength 1 Å)
interacts with a crystal, which can be defined as a solid material consisting of
atoms, ions or molecules that are arranged in a characteristic regular pattern
(Holden & Morrison, 1982), a part of the beam will be diffracted (besides a
transmitted, absorbed, refracted and scattered part). Detection of the intensity
of the diffracted light results in a diffraction pattern, also known as a
diffractogram. This is characteristic for each type of crystal and it depends on
the constituents of the crystal lattice, their arrangement and distance. We can
call it the „fingerprint‟ of a crystal (Suryanarayana & Norton, 1998).
Imaging of the colloidal suspension and the spray dried powders was
done by a Transmission Electron Microscope (TEM) (see figure 1.6.). It uses
a high voltage beam of electrons to illuminate the sample. Because the
wavelength of electrons is much shorter than that of photons, the resolution or
resolving power is much bigger. Resolution defines the smallest distance
between two points that can be distinguished. Where glass lenses focus the
light in an optical microscope, electrostatic and electromagnetic lenses are
used to focus the electron beam in an electron microscope. The beam is
transmitted through the sample. The electrons will partially be scattered out of
the beam. This gives information about the structure after magnifying it by the
objective lenses and projecting on the fluorescent screen (Egerton, 2005).
Introduction
14
FIGURE 1.6. SCHEMATIC ILLUSTRATION OF A TRANSMISSION ELECTRON MICROSCOPE (http://en.wikipedia.org/wiki/Electron_microscope).
Objectives
15
2. OBJECTIVES
The formulation of a drug is a very important part in its development. It
is known that decreasing the particle size, increases dissolution rate and
hence the bioavailability of a drug. Production of nanoparticles can be a very
interesting tool in this context, in particular for products with poor water
solubility. Earlier studies have demonstrated that ball milling can produce
nanoparticles when the right type of stabiliser in the right concentration is
used. However, the obtained product is a colloidal suspension which is not
stable enough to preserve for extended periods of time. This is the main
reason why the current products on the market are in their solid state. Spray
drying is a possible way to transform the colloidal suspension into a powder
after ball milling. This process involves some problems: the nanoparticles
aggregate into microparticles. This might form a barrier in the overall
dissolution process while the primary goal of a nanoparticle formulation is to
improve the dissolution process.
This master thesis wants to provide more insight in the appearance
and behaviour of the powder that we obtain after the process of ball milling
and spray drying. In particular, the preservation of the nanostructure of the
particles was the main objective in this study. When this is possible, the
dissolution improvements obtained after ball milling could also be preserved.
The advantage: microparticles are easier to formulate, while after redispersing
in water, the individual nanoparticles are released and provide an improved
therapeutic action.
First, we prepared the colloidal suspension of indomethacin and
itraconazole by ball milling. These drugs were used as model compounds.
Lutrol®F127 was added to both as a stabiliser before ball milling.
In the second step, the colloidal suspensions were transformed into a
powder by spray drying. Because a matrixformer is needed to keep the
dissolution improvements, we added lactose monohydrate before spray drying
Objectives
16
to a part of the obtained colloidal suspensions. Thus, we obtained 4 different
powders: Indomethacin and Lutrol®F127 (ratio 1/0,25); Indomethacin,
Lutrol®F127 and lactose monohydrate (ratio 1/0,25/10); Itraconazole and
Lutrol®F127 (ratio 1/0,25); Itraconazole, Lutrol®F127 and lactose monohydrate
(ratio 1/0,25/10).
We took pictures of the colloidal suspensions and spray dried powders
with a TEM to observe shape and size of the particles. Then, DSC was used
to perform a thermo analysis. It helped us to determine the changes in
crystallization and melting points after spray drying. To confirm the obtained
results of DSC, we used XRPD to study the crystallinity of the powders.
At this stage, the results for spray dried material with and without
lactose monohydrate as a matrixformer were unclear. This led us to perform
the dissolution tests. They allowed us to observe changes in dissolution rate
before and after spray drying. And also the need for a matrixformer could be
examined.
Material and methods
17
3. MATERIAL AND METHODS
3.1. CHEMICALS
Indomethacin (figure 3.1.) was supplied by Hawkins (Minneapolis,
USA).
Molecular weight: 357,79 g/mol
Melting point: 158°C-162°C
FIGURE 3.1. MOLECULAR STRUCTURE OF INDOMETHACIN
(http://upload.wikimedia.org/wikipedia/commons/thumb/a/a5/Indometha
cin.png/200px-Indomethacin.png).
Itraconazole (figure 3.2.) was supplied by Apotecnia (Murcia, Spain).
Molecular weight: 705,64 g/mol
Melting point: 166 °C
FIGURE 3.2. MOLECULAR STRUCTURE OF ITRACONAZOLE
(http://www.lookchem.com/ITRACONAZOLE/).
Material and methods
18
Poloxamer 407 (=Lutrol® F127) was a gift from Orion (Hollola, Helsinki)
It‟s a polyoxyethylene-polyoxypropylene block polymer. Its general molecular
structure can be seen in figure 3.3.
Molecular weight: between 9840-14600 g/mol
Melting point: 53°C -57°C
FIGURE 3.3. MOLECULAR STRUCTURE OF POLOXAMER 407
WITH a= ca. 98 AND b= ca. 57
(http://www.uspbpep.com/usp28/v28230/usp28nf23s0_m66210.htm).
Lactose monohydrate (figure 3.4.) was supplied by DMV-Fonterra
Excipients (Nörten-Hardenberg, Germany).
Molecular weight: 360,31 g/mol
Melting point: 214°C
FIGURE 3.4. MOLECULAR STRUCTURE OF LACTOSE
(http://www.edinformatics.com/math_science/science_of_cooking/lacto
se.htm).
Material and methods
19
3.2. MILLING PROCEDURE
We used the Premium Line Pulverisette 7 planetary micro mill (Fritsch,
Idar-Oberstein, Germany) to decrease the particle size of both drugs,
indomethacin and itraconazole. The two vessels were prepared by adding
successively 70,0 g zirconium oxide (ZrO2) milling balls with 1 mm diameter
(Fritsch, Idar-Oberstein, Germany); 0,5 g Lutrol® F127 (this is 25 % of the
amount of the drug), 2,0 g drug and 10,0 ml purified distilled water (according
to the European Pharmacopoeia (Ph. Eur.)). After placing the vessels in the
milling machine, we set the following parameters: speed at 1100 rpm, 6 cycles
of 3 minutes milling and 15 minutes pause with reverse option on. Then the
temperature of the vessels was measured as a quality control. A temperature
of approx. 50°C was reached for both drugs. Another 4 cycles of 3 minutes
milling and 15 minutes pause with reverse option and speed at 1100 rpm
were performed.
At least one hour after cooling down, we poured out the milling balls
and the colloidal suspension of the two vessels on a sieve to separate both.
We added 40,0 ml purified distilled water in several steps to wash off the
indomethacin from the milling balls. For the colloidal suspension of
itraconazole, we carried out the same, but now we used 60,0 ml purified
distilled water, because we noticed that we lost too much of the drug by
washing with only 40,0 ml purified distilled water. As mentioned, there is
always some product that keeps sticking on the milling balls by using this
method. Because we didn‟t exactly know what the real concentration of drug
was at that point, we prepared a dilution of the colloidal suspensions to
quantify with high performance liquid chromatography (HPLC).
For the quantification with HPLC we prepared one sample of
indomethacin by diluting our colloidal suspension (approx. 6,66 weight%)
10 000 times in 100 % ethanol (Altia, Rajamäki, Finland) and another sample
in 50%/50% ethanol/purified distilled water. The ethanol was used to ensure
that indomethacin was dissolved. We achieved a solution with a concentration
Material and methods
20
of approx. 6,66 µg/ml. For itraconazole, we diluted our colloidal suspension of
approx. 5 weight% 2000 times in pure methanol (Rathburn, Walkerburn,
Scotland). We achieved a solution with a concentration of approx. 25 µg/ml.
The HPLC measurements were performed by using Agilent 1100
Series (Agilent technologies, Waldbronn, Germany). We used the Luna 3u
C18 (2) 100 A (Phenomenex, AllerOd, Denmark) column as stationary phase
for indomethacin. 0,2% phosphoric acid (H3PO4) pH 2,0 (Riedel de Häen,
Seelze, Germany) and acetonitrile (VWR Prolabo, Briare, France) with ratio
40%/60% was used as mobile phase. For itraconazole, we used the Gemini
NX3u C18 110 A column (Phenomenex, AllerOd, Denmark) 0,1 % trifluoro
acetic acid (CF3COOH) pH 2,0 (Sigma-Aldrich, Helsinki, Finland) and
acetonitrile with ratio 50%/50% was used as mobile phase. Before starting the
measurements, it was important to filter the mobile phase and the samples to
remove possible particles in the solutions. We used Acrodisc LC 13 mm
syringe filter with 0,2 µm PVDF (polyvinylidenefluoride) membrane (Pall,
Portsmouth, UK). First, we performed a prewash with air locked to avoid
bubbles. Secondly, we carried out a pre-treatment of the column with mobile
phase only, to get a basic line. Finally, we inserted the samples. For
indomethacin, 20 µl of each sample was automatically injected in the system
and the flow rate was 1,5 ml/min. Detection occurred with ultraviolet (UV)
spectrophotometry at 320 nm wavelength. For itraconazole, 10 µl was
automatically injected and flow rate was 1,5 ml/min. Detection occurred with
UV at 261 nm.
3.3. SPRAY DRYING
After measuring the concentration of the colloidal suspension with
HPLC (see above), we were able to dilute exactly to a colloidal suspension
that contained 1 weight% of indomethacin. A first sample was prepared by
diluting 25,0 ml of the 5 weight% colloidal suspension of indomethacin to
125,0 ml with purified distilled water. A second sample was prepared in the
same way, but lactose monohydrate as a matrix former was added in an
Material and methods
21
amount according to 10 times the amount of indomethacin, 12,5 g. The
quantification of the colloidal suspension of itraconazole gave us a
concentration of 4,8 wt%. We diluted 35,0 ml of this to 175,0 ml for a first
sample. A second sample was prepared in the same way but now we added
lactose monohydrate, 10 times the amount of itraconazole, 16,8 g.
These 4 samples were used for spray drying, to obtain a dry powder:
- Indomethacin+Lutrol®F127 ratio: 1/0,25
- Indomethacin+Lutrol®F127+Lactose monohydrate ratio: 1/0,25/10
- Itraconazole+Lutrol®F127 ratio: 1/0,25
- Itraconazole+Lutrol®F127+Lactose monohydrate ratio: 1/0,25/10
We used the Mini Spray Dryer B-191 (Büchi, Flawil, Switzerland), see
figure 3.5. The used parameters can be seen in table 3.1. for indomethacin,
and table 3.2. for itraconazole.
TABLE 3.1. SPRAY DRY PARAMETERS FOR INDOMETHACIN SAMPLES
parameter Setting
aspirator 95 %
Pump 15 %
inlet temperature 160°C
outlet temperature ±95°C
air pressure 6,5 bar
air flow 600-800 ml/min
TABLE 3.2. SPRAY DRY PARAMETERS FOR ITRACONAZOLE SAMPLES
parameter Setting
aspirator 95 %
Pump 15 %
inlet temperature 150°C
outlet temperature ±86°C
air pressure 6,5 bar
air flow 600-800 ml/min
Material and methods
22
FIGURE 3.5. SCHEMATIC PRESENTATION OF A SPRAY DRYER:
1.SAMPLE, 2.FEED PUMP, 3.TWO FLUID NOZZLE, 4.FEED CONNECTION,
5.AIR CONNECTION, 6.INLET COOLING WATER, 7.OUTLET COOLING
WATER, 8.HEATER, 9.DRYING CHAMBER, 10.WASTE COLLECTING
VESSEL, 11.TEMPERATURE PROBE OUTLET, 12. CYCLONE: PRODUCT
SEPERATED FROM AIR, 13.PRODUCT COLLECTING VESSEL, 14.
ASPIRATOR EXHAUST GAS TO FILTER, 15.MAIN SWITCH, 16. AIR FLOW
SWITCH, 17.INLET AND OUTLET AIR TEMPERATURE, 18. ASPIRATOR
IN %, 19. PUMP IN %
Material and methods
23
3.4 SOLUBILITY
For the dissolution tests (described in the next chapter), it is important
to work in sink conditions. When performing them, a correlation can be seen
between the in vitro dissolution rates and in vivo observations (Gibaldi and
Feldman, 1967). According to the Ph. Eur., sink conditions are obtained when
the volume of the dissolution medium is at least 3 to 10 times the saturation
volume. Then, the material that is already in solution does not affect the
dissolution rate of the residual material. To know the saturation volume, we
first performed a saturated solubility test of pure indomethacin in the
phosphate buffer with pH 6,8 (see below). The solubility of pure itraconazole
was tested in the medium containing 0,1 M hydrochloric acid and 0,2% SDS
(see below). The tests were performed in duplicate. 50 mg of drug was put in
50 ml medium. The samples were placed on a horizontal-shaker for 24 hours
at 37°C. We filtrated the suspension by a pumping installation through a 0,2
µm hydrophilic polypropylene membrane filter (Pall corporation, Ann Arbor,
Michigan, USA). The obtained solution of indomethacin was measured with
UV spectrophotometry (see chapter 3.5.). HPLC was used to measure the
obtained solution of itraconazole (see chapter 3.2).
3.5. DISSOLUTION TESTS
To comply with the dissolution requirements for solid dosage forms that
have a conventional release and are administered orally, we followed the Ph.
Eur. We used the paddle apparatus Erweka DT-D6 (Erweka, Espoo, Finland)
to measure the dissolution rate of our samples. Known amounts of the
powders were put in a gelatine capsule and a wire was placed around it to
ensure that the capsule would sink. To compare our results, we also
measured the dissolution rate of the colloidal suspension. We took samples of
0,01 ml of the colloidal suspension that we obtained after the ball milling (this
is approx. equal to 2 mg drug). Because this was a very small amount, we first
mixed it with 5 ml of the buffer and then put it in the medium. All samples were
measured in triplicates. The average was calculated and standard deviation
Material and methods
24
was determined. To give a proper summary of the samples we measured, we
refer to table 3.3.
TABLE 3.3. OVERVIEW OF THE SAMPLES MEASURED FOR THE
DISSOLUTION TESTS OF INDOMETHACIN AND ITRACONAZOLE
Samples amount
pure INDa ±5 mg
physical mixtured of INDa and F127b ±2,5 mg
INDa and F127b after spray drying ±2,5 mg
INDa, F127b and LACc after spray drying ±25 mg
colloidal suspension of INDa and F127b after ball milling
0,01 ml
pure ITRe ±3 mg
physical mixtured of ITR and F127b ±3 mg
ITRe and F127b after spray drying ±3 mg
ITRe, F127b and LACc after spray drying ±25 mg
colloidal suspension of ITRe and F127b after ball milling
0,01 ml
aIND: indomethacin
bF127: Lutrol
® F127
cLAC: lactose monohydrate
dphysical mixture: made by mixing IND and F127 (ratio 1/0,25) with pestle and mortar
eITR: itraconazole
For the dissolution tests of indomethacin we used 900,0 ml of a
phosphate buffer with pH 6,8 as a dissolution medium. Sink conditions were
maintained throughout the experiment. The buffer was made according to the
Ph.Eur: First, 8,0 g sodiumhydroxide (myrkkyä II LK, Sweden) was dissolved
to 1,00 L purified distilled water to obtain a concentration of 0,2 M. Then, 54,4
g potassium dihydrogen phosphate (Riedel-de Häen, Seelze, Germany) was
dissolved to 2,00 L purified distilled water which resulted in a concentration of
0,2 M. 500,0 ml of 0,2 M potassium dihydrogen phosphate was placed in a
2000 ml volumetric flask and 224,0 ml 0,2 M sodiumhydroxide was added. We
diluted this to 2000,0 ml with purified distilled water. We checked the pH of
the buffer with a Schott pH-meter (Mainz, Germany).
Material and methods
25
The temperature of the dissolution apparatus was set at 37°C and
paddle speed at 100 rpm. At specified time points, a 5 ml sample was taken
out of the dissolution medium and 5 ml of fresh buffer, also equilibrated at
37°C, was added. Every sample was withdrawn from the same spot between
the surface of the dissolution medium and the top of the rotating blade.
We quantified dissolved indomethacin with a UV spectrophotometer
(Ultrospec III, Pharmacia LKB, Burladingen, Germany) at 318 nm. Buffer was
used as reference. Before measuring, we filtered the samples using Acrodisc
LC 13 mm syringe filter with 0,2 µm PVDF membrane (Pall, Portsmouth, UK).
We generated a calibration curve by making different dilutions of
indomethacin in ethanol (Altia, Rajamäki, Finland) with known concentration.
Ethanol was used as reference. We made the dilutions in duplicate and the
average of the absorbance was taken. The obtained calibration curve can be
seen in figure 3.6. The correlation coefficient (0.9999) confirms that there was
a linear correlation between concentration and absorbance.
FIGURE 3.6. CALIBRATION CURVE OF INDOMETHACIN IN ETHANOL
MEASURED AT 318 NM.
Material and methods
26
For itraconazole, we used 500,0 ml dissolution medium containing 0,1
M hydrochloric acid (Merck kgaa, Darmstadt, Germany) and 0,2% SDS which
was supplied by Sigma-Aldrich (Helsinki, Finland). This was done to ensure
that this poorly soluble drug could dissolve. Sink conditions were obtained.
We took samples of 3 ml at certain time points and 3 ml of fresh medium was
added. The other conditions were the same as the dissolution tests of
indomethacin. We refer to the second paragraph of this chapter.
Quantification of itraconazole was now done by HPLC (Agilent
technologies, Waldbronn, Germany). The method we used is described in the
forth paragraph of chapter 3.2.
3.6. DSC
The operating conditions of DSC (DSC823e, Mettler Toledo GmbH
analytical, Greifensee , Switzerlan) can be consulted in table 3.4. The
powders we measured are the same as those from XRPD. They can be seen
in table 3.5.
TABLE 3.4. PARAMETER SETTINGS OF DSC MEASUREMENTS
Parameter setting
temperature range 25°C-235°C
heating rate 10°C/min
N2 gas flow rate 50 ml/min
3.7. XRPD
XRPD is used to make a comparison of the crystal structure of the
powders before and after the process of ball milling and spray drying. It was
performed by using the θ-θ diffractometer (Bruker AXS D8, Karlsruhe,
Germany). We used symmetrical reflection mode with Cu Kα radiation (1,54
Å) using Göbel Mirror bent gradient multilayer optics. A scintillation counter
measured the scattered intensities. The range of the angle was from 5° to 40°,
with steps of 0,02° and time was 2s/step. Sharp peaks indicate the presence
Material and methods
27
of crystalline powder, while deflected peaks are an indication of amorphous
material. In table 3.5., an overview is given of the powders that we did
measure.
TABEL 3.5. OVERVIEW OF POWDERS MEASURED BY XRPD AND
DSC
pure INDa
physical mixture of INDa and F127
b
INDa and F127
b after spray drying
INDa, F127
b and LAC
c after spray drying
pure ITRe
physical mixtured of ITR
e and F127
b
ITRe and F127
b after spray drying
ITRe, F127
b and LAC
c after spray drying
pure F127b
pure LACc
LACc after spray drying
aIND: indomethacin
bF127: Lutrol
® F127
cLAC: lactose monohydrate
dphysical mixture: made by mixing IND and F127 (ratio 1/0,25) with
pestle and mortar
eITR: itraconazole
3.8. TEM
We used the Transmission electron microscope (FEI Tecnai F12,
Philips Electron Optics, The Netherlands) to study the size and the
appearance of the particles after spray drying. The samples were made by
dispersing some spray dried powder in purified distilled water. A droplet of
suspension was placed on a Formvar Carbon Film on 300 Square Mesh
Copper Grids. The samples were then dried for at least 3 hours.
Results and discussion
28
4. RESULTS AND DISCUSSION
4.1. MICROSCOPY
4.1.1. TEM of indomethacin
Figure 4.1.a and 4.1.b show TEM images of the suspension after ball
milling that contains indomethacin and Lutrol®F127 (ratio 1/0,25). The size of
the particles was around 500 nm. Figure 4.1.c and 4.1.d show TEM images of
the spray dried powder of indomethacin and Lutrol®F127 (ratio 1/0,25).
Nanoparticles can be seen. Figure 4.1.e and 4.1.f are TEM images of the
spray dried powder of indomethacin, Lutrol®F127 and lactose monohydrate
(ratio 1/0,25/10). The difference with the spray dried formulation without
lactose monohydrate is not really clear. We can only say that some
nanoparticles could still be seen. In general, the morphology of the particles is
similar for the colloidal suspension and the spray dried powders. No sharp
surroundings could be seen, only smooth edges. This can be explained by the
surfactant on the surface of the particles.
4.1.2. TEM of itraconazole
Figure 4.2.a and 4.2.b show TEM images of the suspension obtained
after ball milling of itraconazole and lutrol®F127 (ratio 1/0,25). In contrast to
the suspension of indomethacin, the formed nanoparticles looked like
needles. Their size was around 300 nm. In figures 4.2.c and 4.2.d, we can
observe that nanoparticles maintained their size and morphology after the
spray drying of itraconazole and lutrol®F127 (ratio 1/0,25). Figure 4.2.e and
4.2.f show TEM images of spray dried itraconazole, lutrol®F127 and lactose
monohydrate (ratio 1/0,25/10). The cloudiness in the images is due to the
large amount of lactose monohydrate. In general, the morphology of the
particles was similar for the colloidal suspension and the spray dried powders.
Edges are not sharp because of lutrol®F127 on the surface of the particles
and the needle-like shape was maintained after the spray drying process.
Results and discussion
29
FIGURE 4.1. TEM IMAGES OF: (A) and (B) INDOMETHACIN +
LUTROL®F127 AFTER BALL MILLING; (C) and (D) INDOMETHACIN +
LUTROL®F127 AFTER SPRAY DRYING; (E) and (F) INDOMETHACIN +
LUTROL®F127 + LACTOSE MONOHYDRATE AFTER SPRAY DRYING.
Results and discussion
30
FIGURE 4.2. TEM IMAGES OF: (A) and (B) ITRACONAZOLE +
LUTROL®F127 AFTER BALL MILLING; (C) and (D) ITRACONAZOLE +
LUTROL®F127 AFTER SPRAY DRYING; (E) and (F) ITRACONAZOLE +
LUTROL®F127 + LACTOSE MONOHYDRATE AFTER SPRAY DRYING.
Results and discussion
31
4.2. PHYSICOCHEMICAL CHARACTERIZATION OF THE SPRAY DRIED
POWDERS BY DSC AND XRPD
In figure 4.3. we can see the thermograms of the indomethacin
powders. Figure 4.4. represents the thermograms of the itraconazole
powders. The peaks that are pointing downwards show the melting points of
the materials. The transition from a solid to liquid state requires energy and
thus is an endothermic process. More heat flow is required to maintain the
same temperature increase as compared to the reference material. Normally,
the scale of the thermograms is in mcal/s/g. This means that they are scaled
by the mass of sample used. Here, we scaled the thermograms containing
indomethacin or itraconazole with the mass of indomethacin or itraconazole,
respectively, to get that peak comparable in all the measurements.
exotherm
endotherm
FIGURE 4.3. THERMOGRAM OF, FROM TOP TO BOTTOM:
- PURE LUTROL F127 (GREEN)
- SPRAY DRIED LACTOSE MONOHYDRATE (BROWN)
- PURE LACTOSE MONOHYDRATE (PURPLE)
- SPRAY DRIED INDOMETHACIN + LUTROL F127 + LACTOSE
MONOHYDRATE (LIGHT BLUE)
- SPRAY DRIED INDOMETHACIN + LUTROL F127 (BLACK)
- PHYSICAL MIXTURE OF INDOMETHACIN + LUTROL F127 (PINK)
- PURE INDOMETHACIN (DARK BLUE)
Results and discussion
32
exotherm
endotherm
FIGURE 4.4. THERMOGRAM OF, FROM TOP TO BOTTOM:
- PURE LUTROL F127 (GREEN)
- SPRAY DRIED LACTOSE MONOHYDRATE (BROWN)
- PURE LACTOSE MONOHYDRATE (PURPLE)
- SPRAY DRIED ITRACONAZOLE + LUTROL F127 + LACTOSE
MONOHYDRATE (LIGHT BLUE)
- SPRAY DRIED ITRACONAZOLE + LUTROL F127 (BLACK)
- PHYSICAL MIXTURE OF ITRACONAZOLE + LUTROL F127 (PINK)
- PURE ITRACONAZOLE (DARK BLUE)
In figure 4.3. and 4.4., the endothermic peaks (melting points) of pure
drug (dark blue) and pure stabilizer (green) are slightly shifted to the left when
they are in a physical mixture (pink). The peaks of the spray dried drug and
lutrol F®127 (black) are even more shifted to the left. There are mainly three
reasons for this: (1) a smaller crystalline size might lead to a lower melting
point; (2) impurities in the powder decrease the melting point (as in the case
of the physical mixture); (3) the presence of amorphous material. The peak
area is also smaller compared with pure material. There are two main reasons
for this: (1) a smaller crystalline size might lead to a lower melting energy; (2)
a change to an amorphous state of some of the material after spray drying,
which means that a smaller amount was left in crystalline form. This last
possibility can be rejected when comparing with the diffractogram of spray
dried drug and lutrol F®127 (black) in figure 4.5. and 4.6. They show similar
Results and discussion
33
peaks as their physical mixture, which means that crystallinity was
maintained. Although the peaks are similar to the physical mixture, their
intensity is lower for spray dried itraconazole and lutrol F®127 (black).
Crystalline nanoparticles are difficult to measure with XRPD because of their
small size. Thus, we can conclude that the nanoparticles of itraconazole have
a smaller size than of indomethacin. This explanation can also be supported
by the TEM results of both drugs (see figure 4.1. and 4.2.). The TEM pictures
of itraconazole represent smaller nanoparticles, which are partially maintained
after spray drying. Notice that the spray dried material shows some peak
broadening in the diffractograms which can occur because of smaller particle
sizes. Another possible reason is from stress and strain caused by the milling
process (Van Eerdenbrugh et al., 2008c).
FIGURE 4.5. DIFFRACTOGRAM OF, FROM TOP TO BOTTOM:
- PURE LUTROL F127 (GREEN)
- SPRAY DRIED LACTOSE MONOHYDRATE (BROWN)
- PURE LACTOSE MONOHYDRATE (PURPLE)
- SPRAY DRIED INDOMETHACIN + LUTROL F127 + LACTOSE
MONOHYDRATE (LIGHT BLUE)
- SPRAY DRIED INDOMETHACINE + LUTROL F127 (BLACK)
- PHYSICAL MIXTURE OF INDOMETHACIN + LUTROL F127 (PINK)
- PURE INDOMETHACIN (DARK BLUE)
Results and discussion
34
FIGURE 4.6. DIFFRACTOGRAM OF, FROM TOP TO BOTTOM:
- PURE LUTROL F127 (GREEN)
- SPRAY DRIED LACTOSE MONOHYDRATE (BROWN)
- PURE LACTOSE MONOHYDRATE (PURPLE)
- SPRAY DRIED ITRACONAZOLE + LUTROL F127 + LACTOSE
MONOHYDRATE (LIGHT BLUE)
- SPRAY DRIED ITRACONAZOLE + LUTROL F127 (BLACK)
- PHYSICAL MIXTURE OF ITRACONAZOLE + LUTROL F127 (PINK)
- PURE ITRACONAZOLE (DARK BLUE)
The thermogram of pure lactose monohydrate (purple) can be seen in
figure 4.3. and 4.4. It shows two endothermic peaks: the peak at 149°C
represents the loss of crystalline water. The second one at 218°C represents
the melting point of the crystalline lactose (Gombás et al., 2002). The
thermogram of spray dried lactose monohydrate (brown), on the other hand,
shows an exothermic peak which represents recrystallisation. Spray drying
changes the state of lactose monohydrate from crystalline to amorphous form.
This explains also why the exothermic peak of loss of crystalline water has
disappeared. The same endothermic peak, the melting point of the crystalline
lactose, is also present here, after recrystallisation occurred. The
diffractogram of spray dried lactose monohydrate (brown) in figure 4.5. and
4.6. confirms that most of the material has changed to its amorphous form
when comparing with the diffractogram of pure lactose monohydrate (purple).
Results and discussion
35
The light blue thermograms in figure 4.3. and 4.4., representing spray
dried drug, lutrol®F127 and lactose monohydrate, are different for
indomethacin and itraconazole. For indomethacin, one endothermic peak can
be seen, which is supposed to be the melting point of lactose. It has shifted to
the right. Also the recrystallisation peak has disappeared. It seems that
lactose changes into another crystalline state in presence of indomethacin.
For itraconazole, on the other hand, the same recrystallisation peak of lactose
as pure spray dried lactose monohydrate can be seen. So, also the
endothermic peak, the melting point of lactose, is present. It is slightly shifted
to the left which can be explained by the same reasons as mentioned before.
The bigger area of the endothermic peak is due to the scale of this
thermogram, namely to the mass of drug and not the mass of the whole
sample. The melting peaks of drug and stabilizer have disappeared. This can
be explained by their low mass compared to the mass of lactose in this
sample.
The light blue diffractogram in figure 4.5., representing spray dried
indomethacin, lutrol F®127 and lactose monohydrate, does not show many
peaks. A change to amorphous state of all material can be an explanation for
that. The preservation of the nanoparticle size can also be the reason
because when particles are nanosized, it is difficult to measure their
crystallinity with XRPD. We can reject these statements because the black
curve, representing spray dried indomethacin and lutrol F®127, shows the
same peaks as the physical mixture, which means that the crystalline form
has maintained and that it can be measured. Though, we can conclude that
the lactose monohydrate in this mixture lost its original crystallinity, explaining
the quite flat base line. The other constituents have maintained their
crystalline form, but the amount is so small compared to lactose monohydrate,
that the intensity of the peaks is low. Same conclusions can be made for
spray dried itraconazole and stabiliser in presence of lactose monohydrate.
The only difference here is that the peaks are on the same place as pure
spray dried lactose monohydrate. This is not the case for indomethacin. This
confirms that lactose seems to be in another crystalline state when spray
Results and discussion
36
dried in presence of indomethacin. Itraconazole, on the other hand, does not
influence the behaviour of lactose while spray drying.
The results of DSC and XRPD when lactose monohydrate was added
are quite curious. We obtained different thermograms and diffractograms
when another drug was used. In future studies, more research should be
done about how a particular drug and lactose influence each other while spray
drying. At this point, after performing TEM, DSC and XRPD measurements,
we can state that it is possible to partially maintain the nanoparticle structure
after spray drying. And the measurements confirm that the nanoparticle size
of itraconazole is smaller than of indomethacin. The drug keeps its crystallinity
after spray drying which is positive in the context of long-term stability.
Amorphous drugs are quite unpredictable in their behaviour, so a crystalline
form is preferred. The lack of information about the differences between spray
dried material with and without lactose monohydrate, led us to the
performance of some dissolution tests. These tests were essential to give a
final answer to the need of excipients when we want to keep dissolution
improvements after spray drying a colloidal suspension.
4.3. EVALUATION OF SOLUBILITY AND DISSOLUTION RATE
4.3.1. solubility and dissolution tests of indomethacin
The solubility of pure indomethacin in the dissolution medium was 611
µg/ml. This is of course not the solubility of the spray dried material, but it
gives at least an idea of the magnitude of indomethacin‟s solubility. We only
needed the saturation volume to be able to work in sink conditions for the
dissolution tests. By using the known amounts of sample that can be seen in
table 3.3., we were sure that sink conditions were achieved.
By using the calibration curve of indomethacin in ethanol (see figure
3.6.), we obtain the following graphs in figure 4.7. Buffer was set as reference.
We also measured the absorbances of lutrol® F127 and the gelatine capsule
and decreased the measured absorbance value with that amount.
Results and discussion
37
FIGURE 4.7. DISSOLUTION RATES OF, FROM TOP TO BOTTOM:
- BALL MILLED COLLOIDAL SUSPENSION OF
INDOMETHACIN AND LUTROL F127 (GREEN)
- INDOMETHACIN, LUTROL F127 AND LACTOSE
MONOHYDRATE AFTER SPRAY DRYING (LIGHT BLUE)
- INDOMETHACIN AND LUTROL F127 AFTER SPRAY DRYING
(BLACK)
- PHYSICAL MIXTURE OF INDOMETHACIN AND LUTROL F127
(PINK)
- PURE INDOMETHACIN (DARK BLUE)
First, some general comments have to be made. It is impossible to
have a released percentage of more than 100%. Also by taking the standard
deviation into account, we still have a percentage of more than 100% for each
sample that we measured. To be sure that everything was dissolved, we
made the calibration curve of indomethacin in ethanol. The dissolution
samples, on the other hand, were in the phosphate buffer. The polarity of both
media is different and this can influence the absorbance because of a shift of
λmax. An additional reason is the interference with lutrol®F127. The presence
of this component can strengthen the signal. This was confirmed by
performing following experiment: We dissolved a known amount (=2,41 mg) of
the physical mixture of indomethacin and lutrol®F127 (ratio 1/0,25) in ethanol.
Measuring absorbance at 318 nm and ethanol set as reference, resulted in a
Results and discussion
38
dissolved mass percentage >100% (= 136%). To avoid this problem in future
studies, we suggest making a new calibration curve where the same amount
of lutrol®F127 as in the samples is added. Note also that the used amounts of
samples for the dissolution tests were very low. Mistakes in actual weightings
could easily be made. We suggest that future studies are performed with
higher amounts without losing the sink conditions. As the shape of the graph
is more important than the absolute data, this issue does not hinder us from
using the dissolution graphs. Taking into account the purpose of the study, the
graphs are of sufficient quality for comparative purposes.
Before discussing the results, note that we consider the highest
percentage released of a sample as 100%. After recalculation we had
following results: The curve of the colloidal suspension (green), obtained by
ball milling, shows that >90% of the material is dissolved in less than one
minute. The curves of pure indomethacin (dark blue) and the physical mixture
(pink) are similar to each other. It takes around one hour to dissolve
everything. The black curve represents the spray dried material without
lactose monohydrate. It takes less than 15 minutes to dissolve 90% of this
material. For the spray dried material with lactose monohydrate (light blue),
the slope of the beginning of the curve is even steeper. It takes less than 5
minutes to dissolve 90% of the drug.
The curves prove that nanosizing the particles improves dissolution
rate. The high dissolution rate of the colloidal suspension can partially be
maintained after spray drying, due to some remained nanoparticles. By
adding lactose monohydrate as a matrixformer, the high dissolution rate can
be preserved even better. The burst release originates from individual
nanoparticles, while the second phase of slower release is due to the
agglomerated fraction.
Results and discussion
39
4.3.2. solubility and dissolution tests of itraconazole
The solubility of itraconazole, measured with HPLC, was 104 µg/ml.
Sink conditions were reached with the dissolution method used.
FIGURE 4.8. DISSOLUTION RATES OF, FROM TOP TO BOTTOM:
- BALL MILLED COLLOIDAL SUSPENSION OF
ITRACONAZOLE AND LUTROL F127 (GREEN)
- ITRACONAZOLE, LUTROL F127 AND LACTOSE
MONOHYDRATE AFTER SPRAY DRYING (LIGHT BLUE)
- ITRACONAZOLE AND LUTROL F127 AFTER SPRAY
DRYING (BLACK)
- PHYSICAL MIXTURE OF ITRACONAZOLE AND LUTROL
F127 (PINK)
- PURE ITRACONAZOLE (DARK BLUE)
Several problems occurred during the measurements of the dissolution
samples. First of all, the method we used (see material and methods) was not
good. Two separate peaks appeared at the retention time of itraconazole
when the concentration was small. The limit of detection was not reached for
those samples. Unfortunately, there was no other method available in time.
Secondly, we noticed after all our measurements, that the filter we used –
although it is made of a hydrophilic material - captured the drug somehow. We
Results and discussion
40
do not know the reason for this. For better results, we could use a centrifuge
next time to remove possible particles from the samples. Nevertheless, even
with all these problems, we managed to obtain reasonable release curves. Of
course, we can‟t use the absolute data. But this is not really a limitation. For
dissolution tests, it is most important to be able to compare the shape of the
curves of the different formulations.
As we can see in the dissolution graph in figure 4.8., the colloidal
suspension, the physical mixture and the spray dried material without lactose,
only reach around 80% dissolved material while the spray dried material with
lactose reaches 100% dissolved material. The difference is unclear and we
can‟t conclude that for the former ones it is impossible to reach 100%.
Probably, we were not able to integrate the peaks in a sufficiently good way
for those.
As mentioned above, we consider the highest amount that dissolved as
100%. We obtained following results after recalculation: As we can see in
figure 4.8, pure itraconazole (dark blue) has not dissolved in the medium after
two hours. The concentration is 0 at every time point. Probably something
dissolved, but the amount is too low to detect with the HPLC method. The
physical mixture (pink) seems to start dissolving after 10 minutes and in less
than 30 minutes, more than 90% is released. At least we can say that the
stabilizer helps the drug to dissolve. The colloidal suspension (green),
obtained after ball milling, dissolves very fast. The first sample was taken after
1 minute. Already 70% of the drug has dissolved then. All of the drug had
dissolved after 2 minutes. The black curve represents the spray dried material
without lactose as a matrixformer. It had a slower release, which means that
in less then 30 minutes more than 90% of the drug amount had dissolved. On
the other hand, when we added lactose monohydrate before spray drying, the
slope of the beginning of the curve was steeper. We reached 92% dissolved
material after 4 minutes. Everything dissolved within 15 minutes.
Results and discussion
41
For itraconazole, the improvement of dissolution rate of spray dried
material with matrixformer compared to the spray dried material without
matrixformer is higher. This might be caused by its higher hydrophobicity. A
drug with a more hydrophobic surface will have more difficulties to break up
into nanoparticles than a less hydrophobic drug. And hence a matrixformer is
more needed in this case.
The graphs might give a wrong impression because we did not
recalculate our results, considering the highest amount dissolved as 100%. In
table 4.1., the percentage of drug dissolved after 10 minutes is given. Here we
do have considered the largest amount dissolved as 100%. This table makes
it easier to compare the different behaviour of indomethacin and itraconazole
when performing the dissolution test.
TABLE 4.1. COMPARISON OF PERCENTAGE DRUG DISSOLVED
AFTER 10 MINUTES
Samples amount of IND
a dissolved
(%) after 10 minutes
amount of ITRb
dissolved (%) after 10 minutes
pure drug 55 0
physical mixture of drug and F127 c 54 71
drug and F127c after spray drying 88 73
drug,F127c and LAC
d after spray
drying 99 99
colloidal suspension of drug and F127
c
99 100
a IND: indomethacin
b ITR: itraconazole
c F127: lutrol®F127
d LAC: lactose monohydrate
Conclusion
42
5. CONCLUSION
TEM results showed that we were able to produce colloidal suspensions
of indomethacin and itraconazole by ball milling, using Lutrol®F127 as a
stabilizer (ratio drug/stabiliser was 1/0,25). The size of the nanoparticles was
approximately 500 nm and 300 nm, respectively. The smaller size of
itraconazole nanoparticles is confirmed by XRPD. The peaks show a lower
intensity.
The TEM pictures also show some remained nanoparticles for both
drugs after spray drying. Unfortunately, the difference between the powders
with and without lactose monohydrate is not clear. The big amount of lactose
(ratio drug/stabiliser/lactose 1/0,25/10) results in cloudiness on the pictures.
Performing a thermo analysis with DSC and measuring crystallinity with
XRPD, gave us some more information about phase transitions and changes
in crystallinity of the powders. Linking both results, allows us to conclude that
both drugs stay in their crystalline form after spray drying which is a positive
result, concerning the long-term stability of the material.
Pure lactose monohydrate changed to an amorphous form after spray
drying. This could also be seen in the presence of itraconazole. For
indomethacin, this behaviour cannot be observed. It seems that it is in another
crystalline state. We suggest performing more studies to examine the different
influence of both drugs on the matrixformer while spray drying.
To examine the effect of the processing variables on the dissolution
rate before and after spray drying, we performed dissolution tests. The shape
of both curves of the colloidal suspensions shows that nanosizing particles
strongly improves dissolution rate. This improvement can partially be
maintained after spray drying, due to remained nanoparticles. When adding
lactose monohydrate, the preservation of dissolution rate is even better.
Especially for itraconazole, the difference with and without matrixformer is
Conclusion
43
more obvious. A matrixformer is more needed here because the drug‟s
surface is more hydrophobic. Then, the aggregates have more difficulties to
break up into nanoparticles. The burst release originates from individual
nanoparticles, while the second phase of slower release is due to the
agglomerated fraction.
It would be interesting to do more research in future with different
amounts of stabiliser and matrixformer. Which concentration of stabiliser
allows us to obtain a colloidal suspension and how do both influence the
preservation of the nanoparticle structure and hence the dissolution rate? Also
using other types of stabiliser and matrixformer could be examined. Another
suggestion is to perform the spray drying again with different process
parameters to examine how they influence the powders‟ behaviour and
appearance. The same measurements could be done as in the master thesis,
because they allowed us to make concrete conclusions about our main
objectives.
In conclusion, it was shown that it is possible to partially preserve the
nanoparticle structure of indomethacin and itraconazole after spray drying
their colloidal suspension. This feature ensures that the increased dissolution
rate is partially maintained, and even more when lactose monohydrate is
added.
References
44
6. REFERENCES
Cal, K.; Sollohub, K. (2010). Spray Drying Technique. I: Hardware and
Process Parameters. J. Pharm. Sci., 99, 575-586.
Dean, J. A. (1995). Dean’s Analytical Chemistry Handbook. The McGraw-Hill
Companies, New York, USA, Chapter 15.2.
Egerton, R. F. (2005). Physical Principles of Electron Microscopy. An
Introduction to TEM, SEM and AEM. Springer Science+Business Media, New
York, USA, Chapter 1.4 & Chapter 3.
European Pharmacopoeia 6.7. (2010).
Gibaldi, M.; Feldman, S. (1967). Establishment of sink conditions in
dissolution rate determinations- theoretical considerations and application to
nondisintegrating dosage forms. J. Pharm. Sci., 56, 1238-1242.
Gombás, Á.; Szabó-Révész, P.; Kata, M.; Regdon Jr. G.; Erõs I. (2002).
Quantitative determination of crystallinity of a-lactose monohydrate by DSC. J.
Therm. Anal. Calorim., 68, 503-510.
Holden, A.; Morrison, P. (1982). Crystals and crystal growing. The MIT-Press,
Massachusetts, USA.
Lee, J. (2003). Drug nano-and microparticles processed into solid dosage
forms: physical properties. J. Pharm. Sci., 92, 2057-2068.
Liang, Y.; Hilal, N.; Langston, P.; Starov, V. (2007). Interaction forces between
colloidal particles in liquid: Theory and experiment. Adv. Colloid. Interfac.,
134-135, 151-166.
References
45
Nash, R. A. (2007). Suspensions. In: Encyclopedia of Pharmaceutical
Technology, Swarbrick, J. (Ed.), Informa Healthcare USA, New York, USA,
pp.3597-3610.
Peltonen, L.; Valo, H.; Kolakovic, R.; Laaksonen, T.; Hirvonen, J. (2010).
Electrospraying, spray drying and related techniques for production and
formulation of drug nanoparticles. Expert. Opin. Drug. Del., published ahead-
of-print as doi: 10.1517/17425241003716802.
Pungor, E. (1995). A practical Guide to Instrumental Analysis. Boca Raton,
Florida, USA, 181-191.
Suryanarayana, C.; Norton, M. G. (1998). X-ray Diffraction: A Practical
Approach. Plenum Press, New York, USA.
Van Eerdenbrugh, B.; Van den Mooter, G.; Augustijns, P. (2008a). Top-down
production of drug nanocrystals: Nanosuspension stabilization, miniaturization
and transformation into solid products. Int. J. Pharm., 364, 64-75.
Van Eerdenbrugh, B.; Froyen, L.; Van Humbeeck, J.; Martens, J. A.;
Augustijns, P.; Van den Mooter, G. (2008b). Drying of crystalline drug
nanosuspensions- The importance of surface hydrophobicity on dissolution
behavior upon redispersion. Eur. J. Pharm. Sci., 35, 127-135.
Van Eerdenbrugh, B.; Vercruysse, S.; Martens, J. A.; Vermant, J.; Froyen, L.;
Van Humbeeck, J.; Van den Mooter, G.; Augustijns, P. (2008c).
Microcrystalline cellulose, a useful alternative for sucrose as a matrix former
during freeze-drying of drug nanosuspensions- A case study with itaconazole.
Eur. J. Pharm. Biopharm., 70, 590-596.
References
46
Van Eerdenbrugh, B.; Froyen, L.; Van Humbeeck, J.; Martens, J. A.;
Augustijns, P.; Van den Mooter, G. (2008d). Alternative matrix formers for
nanosuspension solidification: Dissolution performance and X-ray
microanalysis as an evaluation tool for powder dispersion. Eur. J. Pharm. Sci.,
35, 344-353.
Van Eerdenbrugh, B.; Vermant, J.; Martens, J. A.; Froyen, L.; Van Humbeeck,
J.; Augustijns, P.; Van den Mooter, G. (2009). A screening study of surface
stabilization during the production of drug nanocrystals. J. Pharm. Sci., 98,
2091-2103.
Wang, B.; Zhang, W.; Zhang, W. (2005). Progress in drying technology for
nanomaterials. Dry. Technol., 23, 7-32.
Websites:
http://chemicalland21.com/specialtychem/perchem/SODIUM%20LAURYL%20
SULFATE.htm (04-05-2010)
http://en.wikipedia.org/wiki/Electron_microscope (27-04-2010)
http://mcat-review.org/rate-kinetics-equilibrium.php (20-04-2010)
http://ntp.niehs.nih.gov/?objectid=0709A276-0D0E-3EBD-
A3B3CCC2CD707101 (04-05-2010)
http://upload.wikimedia.org/wikipedia/commons/thumb/a/a5/Indomethacin.png
/200px-Indomethacin.png (20-04-2010)
http://www.buchi.com/Mini_Spray_Dryer_B-
290.179.0.html?&no_cache=1&file=308&uid=2283 (04-05-2010)
References
47
http://www.diytrade.com/china/4/products/489819/Lecithin_Phosphatidylcholin
e_PC.html (04-05-2010)
http://www.edinformatics.com/math_science/science_of_cooking/lactose.htm
(20-04-2010)
http://www.fritsch.de/uploads/media/BA_075000_0100_e_02.pdf (20-04-2010)
http://www.lookchem.com/ITRACONAZOLE (20-04-2010)
http://www.malvern.de/LabGer/technology/images/zeta_potential_schematic.p
ng (18-04-2010)
http://www.medicinescomplete.com/mc/martindale/2009/images/c9003-11-
6.gif (04-05-2010)
http://www.uspbpep.com/usp28/v28230/usp28nf23s0_m66210.htm (20-04-
2010)