screening of nanocrystalline polymeric formulations and
TRANSCRIPT
Screening of Nanocrystalline
Polymeric Formulations and Their Processibility
Miguel Ângelo Correia de Sousa
Thesis to obtain the Master of Science Degree in
Pharmaceutical Engineering
Supervisors: Prof. Dr. João Fernandes de Abreu Pinto and Prof. Dr. Miguel Ângelo
Joaquim Rodrigues
Examination Committee
Chairperson: Prof. Dr. José Monteiro Cardozo de Menezes
Supervisor: Prof. Dr. João Fernandes de Abreu Pinto
Member of the committee: Prof. Dr. João Pedro Martins de Almeida Lopes
November 2019
i
Preface
The work presented in this thesis was performed at the Research Center Pharmaceutical Engineering
(RCPE) (Graz, Austria) during the period February-July 2019, under the supervision of Dr. Joana Pinto.
The thesis was co-supervised at Faculdade de Farmácia da Universidade de Lisboa by Prof. João F.
Pinto.
ii
Declaration
I declare that this document is an original work of my own authorship and that it fulfills all the
requirements of the Code of Conduct and Good Practices of the Universidade de Lisboa.
iii
Acknowledgments
• A special thanks to Dr. Joana Pinto for receiving me so well at RCPE and for always being there
when it was needed even though she was very busy. And to all the RCPE team at area II, for
all the trainings, DSC and WAXS samples and for always trying to help.
• To Prof. Dr. João F. Pinto for giving me this unique opportunity and despite not being in Austria,
for being present every step of the way.
• To my parents and grandmother who always supported me and cared for me and spent hours
on the phone with me when I was away.
• To my little brother that missed me a lot through all the 6 months that I was abroad.
iv
Resumo
O objetivo da presente tese é o desenvolvimento de formulações nanocristalinas de itraconazol
(ITZ) e a seleção de estratégias de formulação para fabricar nanocristais com as características
desejadas em termos de distribuição de tamanho de partículas e estabilidade. Criando ainda um
procedimento fácil para solucionar o mesmo, a partir de um desenho experimental.
O itraconazol pertence à classe II (baixa solubilidade e alta permeabilidade) do sistema de
classificação biofarmacêutico.
As nanosuspensões são dispersões submicronizadas coloidais em estado líquido de princípios
ativos pouco solúveis, estabilizados por tensioativos, polímeros ou ambos. Para obter uma
nanosuspensão de ITZ foi utilizado um moinho de bolas. A nanosuspensão resultante foi misturada
com 5 diferentes soluções poliméricas de PVP (Polivinilpirrolidona) K30 e K90, PVP/VA (Co-polímero
PVP e acetato de vinil), HPC (Hidroxipropilcelulose) GXF e MF nas proporções de 10, 50 e 90% de
polímero para ITZ. Destas resultaram 15 suspensões que foram secas numa estufa, após terem sido
colocadas sobre superfícies de teflon para a produção de filmes. Estes filmes foram caracterizados por
espectroscopia Raman, calorimetria diferencial de varrimento, dispersão de raios X de ângulo amplo e
a distribuição do tamanho de partículas por difração laser.
A nanosuspensão de itraconazol for fabricada em moinho de bolas, sendo estável a -8ºC
durante 4 meses. A HPC GXF a 90% foi o único polímero capaz de estabilizar o tamanho das partículas
da nanosuspensão após a secagem.
Palavras-chave: Distribuição do tamanho de partícula; HPC; itraconazol; nanosuspensão; PVP.
v
Abstract
The aim of this present thesis is the development of Itraconazole (ITZ) nanocrystalline
formulations and screening of formulation strategies to manufacture nanocrystalline solid products with
the desired characteristics in terms of particle size distribution and stability. Also, to create an easy
procedure to do so by using a design of experiments. Itraconazole belongs to class II (low solubility and
high permeability) of the biopharmaceutical classification system.
Nanosuspensions are liquid colloidal submicron dispersions of poorly soluble active particle
ingredients, stabilized by surfactants, polymers or both. To obtain the nanosuspension of ITZ, a ball mill
was used. The resulting nanosuspension was mixed with 5 different polymeric solutions of: PVP
(Polyvinylpyrrolidone) K30 and K90, PVP/VA (copolymer with PVP and vinyl acetate), HPC
(Hydroxypropylcellulose) GXF and MF in the proportions of 10%, 50% and 90% of polymer to Active
Pharmaceutical Ingredient. These resulted in 15 formulations that were dried in an oven to obtain films.
The films were characterized by Raman Spectroscopy, Differential Scanning Calorimetry, Wide-Angle
X-ray Scattering and Particle Size Distribution (Laser Diffraction).
An itraconazole nanosuspension was manufactured by ball mill and the resulting formulation
was stable at -8 °C for up to 4 months. HPC GXF at 90% concentration produced the only film capable
of stabilizing nanosuspension particle size after drying.
Key-words: HPC; itraconazole; nanosuspension; particle size distribution; PVP.
vi
INDEX
1. Introduction ...................................................................................................................................... 1
1.1. Itraconazole ............................................................................................................................. 1
1.2. Techniques to enhance solubility ............................................................................................ 2
1.3. Nanosuspensions .................................................................................................................... 3
1.3.1. Bottom-up methodologies .................................................................................................... 4
1.3.2. Top-Down methodologies .................................................................................................... 4
1.3.3. Nanosuspension Formulation .............................................................................................. 5
1.3.4. Particle Size Distribution ...................................................................................................... 6
1.3.5. Calorimetric evaluation by Differential Scanning Calorimetry ............................................. 8
1.3.6. Raman Spectroscopy .......................................................................................................... 9
1.3.7. Wide angle X-ray scattering ................................................................................................ 9
1.4. Thesis rational ......................................................................................................................... 9
2. Experimental Part .......................................................................................................................... 11
2.1. Materials ................................................................................................................................ 11
2.2. Manufacturing the nanosuspension of itraconazole by ball milling ....................................... 11
2.3. Screening of the formulation strategies for the production of the dried films ........................ 12
2.3.1. Characterization of the obtained mixtures ......................................................................... 14
2.3.2. Characterization of the obtained films ............................................................................... 14
2.3.2.1. PSD ............................................................................................................................... 14
2.3.2.2. DSC ............................................................................................................................... 14
2.3.2.3. RAMAN .......................................................................................................................... 15
2.3.2.4. WAXS ............................................................................................................................ 15
3. RESULTS ....................................................................................................................................... 16
3.1. Characterization of the nanosuspension. .............................................................................. 16
3.2. Characterization of the films obtained ................................................................................... 18
3.2.1. PSD analysis ........................................................................................................................... 18
3.3.2. Calorimetry (DSC) results ....................................................................................................... 18
3.3.3. Spectra of RAMAN .................................................................................................................. 21
3.3.4. Results from X-ray diffractometry (WAXS) .............................................................................. 23
3.4 Would the amount of ITZ present in the best formulation be enough to treat a patient? ............ 24
4. Discussion ..................................................................................................................................... 25
5. Conclusion ..................................................................................................................................... 28
6. Future Work ................................................................................................................................... 29
7. References .................................................................................................................................... 30
Annex ..................................................................................................................................................... 34
Kinetic Viscosity ................................................................................................................................. 34
Polymer Molecular Weights ............................................................................................................... 34
Drying End-point Curves ................................................................................................................... 34
vii
List of abbreviations
API – Active Pharmaceutical Ingredient
BCS – Biopharmaceutical Class System
DSC – Differential Scanning Calorimetry
HPC – Hydroxypropylcellulose
HPH – High Pressure Homogenization
HPMC – Hydroxypropylmethylcellulose
IR – Infrared
ITZ – Itraconazole
IV – Intravenous
LD – Laser Diffraction
LOD – Loss on Drying
NCEs – New Chemical entities
PSD – Particle Size Distribution
PVP – Polyvinylpyrrolidone
PVP/VA – Polyvinylpyrrolidone/Vinyl Acetate
SAXS – Small-Angle X-ray Scattering
WAXS – Wide-Angle X-ray Scattering
XRDP – X-Ray Diffraction Patterns
viii
List of Figures
Figure 1 - Itraconazole Molecule [3]. .................................................................................................. 1
Figure 2 - Schematic diagram of a ball mill [26]. ................................................................................ 5
Figure 3 - Possible outcomes of the process of milling [30]. .............................................................. 6
Figure 4 - HELOS CUVETTE dispersing system [36]. ....................................................................... 7
Figure 5 - Different phases of the construction of the thesis. ........................................................... 10
Figure 6 - Ball mill Retsch PM 100 ................................................................................................... 11
Figure 7 – Schematic representation of the steps taken to prepare the films. ................................. 13
Figure 8 - Capillary viscometer. ........................................................................................................ 14
Figure 9 - DSC of pure itraconazole. ................................................................................................ 19
Figure 10 – Thermograms of nanosuspensions containing 10% HPC GXF + 90%
nanosuspension.(a), HPC GXF 50% + 50% nanosuspension (b), 90% HPC GXF +
10% HPC GXF (c). ....................................................................................................... 20
Figure 11 - Thermograms of nanosuspensions containing 90% PVP K90 + 10%
nanosuspension ........................................................................................................... 21
Figure 12 - Raw ITZ raman spectra. ................................................................................................ 22
Figure 13 - Raman spectra showing a new interaction of PVP K90 10% and HPC GXF 50%
inside the blue circle. ................................................................................................... 22
Figure 14 - Differences between raman spectra of raw HPC GXF, HPC GXF 10% and HPC
GXF 90%. ..................................................................................................................... 22
Figure 15 - WAXS of (a) pure ITZ and (b) nanosuspension. ............................................................ 23
Figure 16 - WAXS of 10% of PVP K30 and 90% of nanosuspensions. ........................................... 23
Figure 17 - Kinetic viscosity of the obtained mixtures. ..................................................................... 34
Figure 18 - Curves of drying curve of mixtures of polymers at 120 ºC when the polymers were
present at 10% (a), 50% (b) and 90% (c) concentrations. ........................................... 35
List of Tables
Table 1 - DoE used to access the better programme for further use in the ball milling. ....................... 12
Table 2 - DoE used to access which polymer better stabilizes the nanosuspension PSD. .................. 13
Table 3 - DoE used to access the better programme for further use in the ball milling and its results. 16
Table 4 - PSD increasing the number of milling cycles using 8g of zirconium oxide beads. ................ 17
Table 5 - PSD increasing the number of milling cycles using 16g of zirconium oxide beads. .............. 17
Table 6 - PSD of the produced batches to validate the nanosuspension manufacturing process. ....... 17
Table 7 - PSD of the obtained films ....................................................................................................... 18
Table 8 - Molecular weight of the chosen polymers. ............................................................................. 34
1
1. Introduction
Nowadays, around 75% of new chemical entities (NCEs) fall under the class II (low solubility,
high permeability) and class IV (low solubility, low permeability) of the biopharmaceutical classification
system (BCS). Over half of this 75% belongs to BCS class II. A class II API will typically exhibit
dissolution rate limited absorption and a class IV API will typically exhibit permeation rate limited
absorption. Itraconazole (ITZ) (fig.1) has a broad antifungal range and a very low water solubility which
makes it a BSC II active pharmaceutical ingredient (API). It is a moderately hydrophobic base with a log
P of 5.66 and a pka of 3.76. Itraconazole's low solubility restricts its bioavailability, ultimately affecting
the efficacy and safety of the final product, which is why it was chosen as a model drug for the purpose
of this thesis. Nanosizing the particle size of the ITZ is a way of enhancing its solubility and therefore its
bioavailability [1][2].
Figure 1 - Itraconazole Molecule [3].
1.1. Itraconazole
Itraconazole (ITZ) is a wide spectrum triazole antifungal agent that can be broadly used as a
treatment to both local and systemic fungal infections. ITZ has a molecular weight (MW) of 705.633
g/mol. This API addresses a broad range of systemic fungal infections, resulting in histoplasmosis,
blastomycosis, cryptococcal meningitis, and aspergillosis. The metabolization of ITZ occurs in the liver
and produces over 30 metabolites. The wide-therapeutic spectrum is due to its main metabolite in vivo,
hydroxyl itraconazole, which has strong antifungal properties. Itraconazole is lipophilic and therefore
more abundant in tissues such as lungs, kidneys, bones, and muscles as well as tissues specific to
fungal infections such as skin, nails and female genital tract [4][5].
In the beginning, ITZ was produced and marketed as capsules, and was later developed as a
hydroxypropyl-b-cyclodextrin solution, this solution is available for intravenous and oral administration.
It has been shown that the cyclodextrin excipient improves bioavailability by 37%. However, due to the
toxicity associated to these cyclodextrin formulations, scientists have been working in the development
of non-cyclodextrin formulations. Nanosuspensions have been a good option to this problem [4][5].
The solubility of ITZ can be defined as the maximum quantity of ITZ that can be dissolved in a
certain quantity of solvent or quantity of solution at a specific temperature. Oral ingestion is the most
convenient and commonly way of taking medication due to its ease of administration, high patient
compliance and cost-effectiveness. Nevertheless, the main challenge in the formulation of oral
2
pharmaceutical forms is their poor bioavailability in comparison to other forms such as intravenous (IV).
The factors that oral bioavailability depend on are aqueous solubility, API permeability, dissolution rate,
first-pass metabolism, pre-systemic metabolism, and susceptibility to efflux mechanisms. However, the
most common reasons for low oral bioavailability are in fact due to poor solubility and low permeability.
ITZ usually needs high doses so that it can achieve the needed therapeutic plasma concentrations after
oral administration. Since the factor that most impacts the bioavailability of a class II like ITZ is its lack
of solubility and not its absorption (class IV), a lot of different techniques have been developed to
improve the solubility of these APIs. The solubility of an API is usually inherently related to its particle
size, as the particle size becomes smaller, the ratio of surface area in comparison to volume increases.
A larger surface area allows a better interaction with the solvent, producing an increase in solubility. The
traditional methods used for particle size reduction rely on mechanical stress to disaggregate the API
such is the case of milling. However, sometimes using this kind of techniques may not be enough to
enhance the solubility to the desired level.
1.2. Techniques to enhance solubility
There are physical modifications that can be made to these APIs to improve its solubility namely
techniques like micronization, nanosuspensions, crystal engineering, solid dispersions, cryogenic
techniques or amorphization [6][7]:
- Micronization is a technique used to increase the surface area of the API and in consequence, the
dissolution rate. This micronization can be accomplished by techniques such as jet milling and rotor
squad colloid milling. The downside of this technique is that it is not appropriate for APIs that require
high doses, since it does not change its saturation solubility [7];
- A Solid Dispersion can be described as a solid that consists of at least two different components,
usually a hydrophilic matrix and a hydrophobic API. Until now, at least 3 methods of preparing solid
dispersions have shown their efficacy for commercial production. These methods are: melt-extrusion,
applicable to APIs with not very high melting points, spray drying, useful for drugs soluble in at least one
volatile solvent, and co-precipitation, useful for APIs with high melting point and low solubility in typically
used organic solvents [8][9];
- Cryogenic Techniques to create submicron amorphous particles with high degree of permeability at
very low temperature conditions in order to enhance the dissolution rate of APIs. After cryogenic
processing, spray freeze drying, atmospheric freeze drying, vacuum freeze drying, and lyophilisation
are the typical techniques used to obtain dry powder [10];
- Crystal Engineering techniques are used to control crystallization of APIs, producing powders with
well-defined PSD, crystal form (crystalline or amorphous), surface nature, and surface energy. By
changing some conditions in the crystallization process like adding a different solvent or changing the
stirring process, it is possible to force the crystals to rearrange, these “new” crystals are called
polymorphs. Different polymorphs of an API usually differ in their physicochemical properties such as
solubility, dissolution rate, melting point, and stability. Cocrystals are another form of enhancing
solubility, in this technique two or more molecules are arranged to create a new crystal, usually with
better properties in comparison to the single crystals by which they were formed. These are formed
3
between a molecular or ionic API and a cocrystal former which is solid at room temperature. Sublimation
and ball milling of two or more cocrystal formers are some of the techniques used to obtain cocrystals
[7][11];
- Amorphization is the process used to produce amorphous API particles, this amorphization is
characterized by the disorder in the arrangement of molecules in the solid state. Amorphizing the API
particles usually comes with certain advantages regarding their physical properties such as solubility
and dissolution rate, while maintaining the pharmacological activity. Vitrification, grinding, freeze-drying,
spay drying and rapid precipitation from a solution are some of the techniques used to amorphize an
API [12];
- Nanosuspensions based on particle size reduction techniques in order to improve the solubility of
APIs that are not soluble in both water and oils, preferably with a high Log P (Log P>5) which is the case
of ITZ. There are several ways to get a nanosuspension, such as ball milling or precipitation, but since
this technique was the one chosen for the purpose of this thesis, it will be described in more detail in the
next chapter [13][14].
1.3. Nanosuspensions
Nanosuspensions are characterized as a colloidal submicron dispersions in liquid media of poorly
soluble drugs, stabilized by surfactants, polymers, or both [15]. They can also be defined as a biphasic
system consisting of pure drug particles distributed in an aqueous vehicle with a mean diameter of less
than 1μm of suspended particles [16]. Nanosuspensions have advanced with the developments in
nanotechnology not only to focus on problems of solubility, but also to offer a solution for potential API
compounds that conventional methods could not formulate [15]. Nanosuspensions have the following
advantages [15][17][18][19][20]:
a) Enhance the solubility and bioavailability of the API due to its size reduction;
b) Higher drug loading can be achieved due to the structure and physicochemical properties of the
carrier material;
c) Improved biological performance with reduced toxicity and side effects due to the reduced quantity
of excipients needed to formulate a nanosuspension;
d) Long-term physical stability of the final product due to absence of Ostwald ripening;
e) Little batch-to-batch variation due techniques used like ball milling;
f) Targeted drug delivery by modification of surface properties, the API will be up taken by the
mononuclear phagocytic system to allow regional specific drug delivery. This can be used for
targeting antimycobacterial and antifungal drugs uptake to the macrophages;
g) Ease of manufacture and scale-up depending on the technique used to obtain the nanosuspension.
There are two approaches to get a nanosuspension: “Bottom-up technology” and “Top-down
technology” [16]. The "Top-down" method involves taking bulk materials and fragmenting it, by using
cutting and etching techniques until the appropriate nanosized structures are produced. This process
can be compared to the sculpting of stone, where a wide block is slowly chipped away until the
appropriate form and detail is achieved. The "bottom-up" approach is the direct opposite, it begins with
basic structures, such as small molecules or even atoms and assembles them into a larger structure.
4
This can be compared to a LEGO set where there is the possibility of combining many basic building
blocks in many very different ways to produce structures of the appropriate size and complexity [21][22].
There are several ways to get a nanosuspension inside those two methods (Bottom-up and Top-down).
1.3.1. Bottom-up methodologies
Bottom-up methodologies: Precipitation method - is a method used to prepare nano particles
of poorly soluble APIs. In this technique, the API is dissolved in an organic solvent which is further mixed
with an anti-solvent, in the presence of a surfactant. The quick addition of this anti-solvent leads to a
sudden over-saturation of the substance in the solution and the formation of ultrafine amorphous or
crystalline API. This method involves the creation of a nuclei (nucleation) and the development of
crystals that are mainly temperature dependent. High nucleation speed and low growth rate of crystals
are the key factors to prepare a stable nanosuspension. Some of the advantages of this technique are
that it is a simple and cheap process which produces stable products with low energy requirements, low
equipment costs and it is also easy to scale up. However, some drawbacks are the fact that the growth
of API crystals can only be restricted by the introduction of surfactants, the API must be soluble in at
least one solvent, the final product has a widespread size distribution and there is a possibility of
contamination due to the usage of organic solvents which are toxic [23][24].
1.3.2. Top-Down methodologies
Top-Down methodologies: Ball Milling - Milling is a unit operation where mechanical energy is
applied to physically break down API particles into nanoparticles during the shearing process. A ball mill
(fig.2) comprises a vessel filled with balls, or beads, made of a variety of materials such as ceramic,
agate, silicon nitride, sintered corundum, zirconium oxide, chrome steel, Cr–Ni steel, tungsten carbide
or plastic polyamide. This vessel is filled with the liquid suspension of the API in the presence of surface
stabilizer(s) and is comminuted by milling media at a very high shear speed. This technique does not
need any cooling process, as such, it can be done at a stable temperature. However, the shearing
process can produce a lot of heat that can degrade the sample, therefore there is a possibility of setting
intervals in the process in order to cool the vessel. In the milling process, the particle size is determined
by stress intensity and the number of contact points, as such, size reduction is accomplished due to the
friction and collisions caused by the balls or beads, resulting in nanoparticles. The advantage of media
milling is small batch-to-batch variability. However, the main drawbacks of this technique are the time
consumed by it and the erosion of the balls or beads used in the process may contaminate the sample,
and scaling-up is not an easy process [25][26][27].
5
Figure 2 - Schematic diagram of a ball mill [26].
- High Pressure Homogenization (HPH) - involves 3 steps: Firstly, a liquid suspension of the API and
stabilizer is made, secondly, this suspension is homogenised at a low pressure for pre-milling and then
the final step, now under high pressure for ten to fifteen cycles until a nanosuspension is produced. HPH
is often classified into four groups: (i) Dissocubes (homogenization in aqueous media); (ii) Nanopure
(homogenization in water-free media or water mixtures); (iii) Nanoedge or (iv) Nanojet. Dissocubes - in
this method, the suspension containing the API and stabilizers are forced through a small gap at a high
pressure of up to 1500 bar. It is based on cavitation principle, due to the sudden passage of the
suspension from a cylinder of 3 cm to one with 25 μm. This passage, according to Bernoulli’s law
produces an increase in dynamic pressure, while at the same time reducing the static pressure, which
decreases the boiling point of water to room temperature. As a result, water starts to boil at room
temperature, producing gas bubbles. When the suspension leaves the gap and the pressure returns to
atmospheric level, the gas bubbles implode which is called cavitation. This cavitation forces are high
enough to get the API nanosized. The main advantages of this procedure are that it does not cause
erosion of the processed material, something that does not happen when we’re talking of ball milling.
The essential drawbacks of this technique are the need for pre-treatment to acquire microparticles
before starting the homogenization process and the high costs associated to this technique. Nanopure
- used for suspensions in nonaqueous media, or water mixtures such as PEG (Polyethylene glycol) 400
or PEG 1000, etc. This technique may be done at room temperature, 0 ºC and below, such as -20 ºC
(also known as “deep freeze” homogenization). Nanoedge - is the combination of two different
techniques: Homogenization and Precipitation. Firstly, there is the precipitation of the API and then its
fragmentation via HPH. This fusion of techniques is very helpful since the major downside of the
precipitation method is the crystal growth and lack of long-term stability, and both things can be
surmounted by Nanoedge Technology. Nanojet - is also known as “opposite stream”, utilizes a chamber
where the suspension stream is then divided in two or even more parts that collide between each other.
The reduction of the particle size is obtained due to the high shear forces caused by those collisions at
high pressure [23][24].
1.3.3. Nanosuspension Formulation
While formulating a nanosuspension via ball milling (also known as nanomilling), a stabilizer
(polymeric and/ or surfactant excipient) has a crucial role. In the absence of an adequate stabilizer, an
6
increased particle surface area contributes to the extra Gibbs free energy, which results in unstable
nanosuspension systems which can result in API agglomeration or aggregation (fig.3). The most
important highlight of a stabilizer is wetting the API particles and to prevent particle agglomeration and
compensate the extra free energy of the freshly exposed surfaces by providing a steric or ionic barrier.
The type and amount of stabilizer has an evident effect on the physical stability and in vivo comportment
of the nanosuspension. According to the literature, frequently used stabilizers are poloxamers,
polysorbates, cellulosics, povidones, and lecithins [16][28][29].
Figure 3 - Possible outcomes of the process of milling [30].
Regarding the physical and chemical properties of the API and their impact when formulating a
nanosuspension, there is still a lot of contradiction. A study conducted by George and Gosh show that
an API with high enthalpy of fusion and Log P were the best candidates for a nanosuspension via ball
milling and the selection of an ideal stabilizer was determined by the level of hydrophobicity of the API
itself [31]. A study conducted by Lee et al. suggests that APIs with high MW, low solubility, high melting
point and a surface energy similar to the stabilizer to be used are going to have a better outcome at
getting a nanosuspension with an unimodal particle size distribution (PSD) [32]. On the other hand, a
study made by Bilgili et al. found no correlation between the physicochemical properties of an API and
the production of a stable nanosuspension [33]. Therefore, there is still a lot of studies to be done
regarding this problematic.
When thinking about which polymers to use or not to use, a study conducted by Van
Eerdenbrugh et al. shown that semi-synthetic polymers (hydroxypropylmethylcellulose (HPMC),
hydroxypropylcellulose (HPC), etc.) have less efficacy at stabilizing nanosuspensions in comparison to
linear synthetic ones (povidones like PVP K30 and K90) [34]. However, when combining semi-synthetic
polymers with a surfactant (poloxamers, polysorbates, etc.) there is a possibility of achieving even better
results than just using a linear synthetic polymer. It is not enough to think just about which polymer does
it better, ratios of API:polymer most be considered, these ratios should be in the range between 1:0.05
and 1:0.5 [30].
1.3.4. Particle Size Distribution
Particle Size Distribution (PSD) is measured by laser diffraction (LD) which is the most efficient
light scattering method for particle size analysis covering an extensive range from submicron to
millimetric scale [35]. LD measures the PSD by measuring the angular variation in intensity of light
scattered as a laser beam passes through a dispersed particulate sample and can be mathematically
7
explained by Fraunhofer or Mie's theory. For a single spherical particle, the diffraction pattern reveals a
typical ring structure. The length of radius from the first minimum to the centre depends on the size of
the particle. The scattering of unpolarized laser light by a single spherical particle can be mathematically
described by equation 1:
Eq. 1
where, I(θ) is the total scattered intensity as function of angle θ concerning the forward direction, I0 is
the illuminating intensity, k is the wavenumber 2π/λ, a is the distance from the scatter to the detector,
S1(θ) and S2(θ) are dimensionless, complex functions describing the variation of amplitude in the
perpendicular and the parallel polarized light.
There are two commonly used ways to measure I(θ), the Lorenz-Mie theory and the Fraunhofer
theory. The Lorenz-Mie theory assumes that spherical, isotropic and homogeneous particles and that
all particles can be represented by a common complex refractive index m= n-ik of the material. The
refractive index m must be determined precisely for the calculation, which is especially difficult in practice
for the imaginary element k, and inapplicable for mixtures of components with different refractive indices.
The Fraunhofer theory only contemplates the diffraction at the contour of the particle in the near-forward
direction. There is no need for any previous knowledge regarding the refractive index therefore, I(θ) is
simplified to the following formula with the dimensionless size parameter α=πx/λ. Only Fraunhofer’s
theory is suitable for mixtures of different materials and shapes, since the diffraction scattered light
absorbed by the sample particles is only measured under low angles in forward direction only. This
theory does not necessitate any pre-knowledge of optical properties. The particle size determined by
LD always refers to the equivalent diameter of a sphere sharing the same diffraction pattern [36][37].
The CUVETTE is a wet dispersing system designed for the LD sensor HELOS (fig.4) for
analysing the particle size of suspensions and emulsions that have small sample volumes (less than 50
ml) such is the case of the CUVETTE 50. This technique is non-destructive, which means that the
sample is fully preserved, making this tool a good option for costly samples. This cuvette has an optical
path of over 20 mm, which ensures accurate measurements even at low concentrations.
Figure 4 - HELOS CUVETTE dispersing system [36].
While interpreting the PSD is valuable to know the following definitions: X10 value means that
10% of the PSD is below that diameter; X50 which is the parameter of interest while working with PSD,
8
means that 50% of the PSD is below that diameter; X90 value means that 90% of the PSD is below that
diameter [38]; Span represents the width of the PSD according to equation 2 [39]:
( 𝑋90−𝑋10
𝑋50 ). Eq. 2
1.3.5. Calorimetric evaluation by Differential Scanning Calorimetry
Differential Scanning Calorimetry (DSC) is a fairly simple, non-destructive technique first
developed in the early 1960s. DSC is used mainly to assess the energy of phase transitions and
conformational changes and to measure their dependency on temperature. Over time, technical
improvements have culminated in high sensitivity instruments that also make DSC a very useful method
for studying the thermodynamic properties of various pharmaceutical products. DSC is used to measure
the specific heat capacity of thermally induced events as a function of temperature. The apparent
specific heat (c2) of a solution is calculated according to equation 3:
𝑐2 =𝑐2+1
𝑤2(𝑐−𝑐1) Eq. 3
where, c is the specific heat of the solution, c1 is the specific heat of the solvent, and w2 is the weight
fraction of the solute. DSC measures the excess apparent specific heat (cex), which is the value (c-c1)
in the equation. Expanding the definition of cex (c-c1), the measured heat capacity of the buffer (c1) can
be written as equation 4:
𝑐𝑏 = 𝑚𝑏𝑥𝑐𝑏° Eq. 4
where, mb and Cb° are the mass and the specific heat capacity of the buffer, respectively. Equally, the
heat capacity of the sample solution (c) can be expressed as equation 5:
𝑐𝑠 = 𝑚𝑠𝑥𝑐𝑠° Eq. 5
where, s reffers to the sample. By subtracting these two values, is possible to determine the cex. The
value (mb-ms) may be substituted by the partial specific volume, eliminating mass from the equation, as
new calorimeters use the more precise volume over mass measurements.
The temperature-dependent differential heat flow from the calorimeter is known as a
thermoanalytical curve. The sample’s energy is given by the time integral of the calculated heat flow,
since the scan frequency is constant. The cex is plotted against temperature, revealing the respective
transitions. Integration of cex over the temperature range results in specific calorimetric enthalpy Δhcal.
While integrating is when the issues start arising. For example, during the phase transition, the path of
the baseline is not inherently apparent and may shift after the transition so it needs artificial baselines
or sophisticated software tools [40].
9
1.3.6. Raman Spectroscopy
Raman spectroscopy provides valuable structural information about materials. A Raman
spectrum provides a "fingerprint" which is unique to the material. It is a non-destructive chemical
analytical technique that provides information about the structure of the chemical compounds,
polymorphism, crystallinity and molecular interactions. It is based on the interaction of light with the
chemical bonds within a material. Since every atom movement reveals a characteristic location and
intensity, it is possible to get detailed information about its molecular structure.
The Raman spectra has a different origin in comparison to infrared (IR) absorption, in this
technique, an inelastic scattering process is responsible for the appearance of the vibrational bands. In
IR spectroscopy, the radiation is absorbed by available vibrational states (E1, E2, E3, En), it means that
photon energy must obligatorily be coincident with the energy difference between two available states.
So, 𝐸𝑁 − 𝐸1 = ℎ𝜈, where h is the Planck’s constant. However, in Raman, most of the scattered light is
at the same wavelength as the laser source and does not provide useful information (Rayleigh
scattering). Only a very tiny amount of light is scattered at different wavelengths, which depend on the
chemical structure of the analyte, this phenomenon is known as Stokes (or Anti-Stokes) Raman
Scattering. Normally, lasers with a wavelength between 750 and 850 nm are the ones used in this
technique. So, since the nature of the Raman effect is physically different from the IR, the selection rules
are also different, which leads to different spectra. Since this two techniques are complementary, they
are usually combined for a better understanding [41][42].
1.3.7. Wide angle X-ray scattering
X-ray scattering evaluates a broad range of density domains. In general, this covers a whole range of
condensed matter, which includes the nanosystem composition and morphology. It is possible to assess
a full range of sizes by combining wide-angle X-ray scattering (WAXS) and small-angle (SAXS) X-ray
scattering techniques. Approximately, WAXS covers from 2 nm downwards, and SAXS covers from 0.5
nm to 100 nm and perhaps even 1000 nm for a finely tuned instrument. WAXS alone is a powerful
technique for providing information on crystallographic structure and lack of structure, atomic positions
and sizes in a unit cell, to a certain degree, chemical compositions and chemical stoichiometry [43].
1.4. Thesis rational
The objective of this thesis is the development of nanocrystalline formulations of a class II model
drug (ITZ) which lacks solubility in water and screening of formulation strategies to manufacture
nanocrystalline solid products with the desired characteristics in terms of particle size distribution and
stability. Also, to create an easy procedure to do so by a Design of Experiments (DoE) which will be
discussed later.
The approach of this thesis (fig.5) is, first we get the ITZ nanosuspension, and then we add the
polymer solutions of 5 different polymers in 3 different concentrations each, and then are dried in the
drying chamber for the production of films.
10
Figure 5 - Different phases of the construction of the thesis.
The polymers were chosen due to their different characteristics - 2 different PVPs with different
MWs (PVP K30 and PVP K90); 1 PVP/VA as co-polymer and 2 different HPCs with different MWs (HPC
GXF and HPC MF) providing different viscosities to the nanosuspension.
The techniques used for characterization are:
a) Characterization of the ITZ nanosuspension with polymer mixtures: kinetic viscosity.
b) Characterization of the obtained films by: Calorimetry (DSC), crystallography (WAXS), spectroscopy
(RAMAN) and particle size distribution (PSD).
11
2. Experimental Part
2.1. Materials
The ITZ was bought from Shenzhen Nexconn Pharmaceutics Ltd., China. The HPC GXF and
MF were bought from Ashland, USA. PVP/VA, PVP K30 and K90 were bought from Basf, USA. HPMC
E5 was bought from Dupont, USA. Poloxamer 188 was bought from Merck, Germany. The Zirconium
oxide beads were bought from Sigmund Lindner GmbH, Germany.
2.2. Manufacturing the nanosuspension of itraconazole by ball milling
In order to get the itraconazole nanosuspension 1g Itraconazole, 0.4g HPMC and 0.5g
Poloxamer 188 were added to 10 ml of purified water. The nanosuspension was produced via ball milling
(Retsch pm100) (fig.6) in a 250ml agate jar. The absence of the polymer in the formulation prevented
the formation of the nanosuspension.
Figure 6 - Ball mill Retsch PM 100
High pressure homogenization was also considered to produce the nanosuspension, but since
nanocrystals can be abrasive to its disruption unit, this option was discarded [44]. Sonication and the
homogenization (UltraTurrax) were also considered but without success [45][46].
In order to choose the best programme to be used in the ball milling a Design of Experiments
(DoE) was performed (table 1). These experiments were performed with regular agate balls with cycles
of 30 min each at 500 rpm and 10ml of volume. However, using this kind of balls, the particles of the
suspension were never in the range of the nanometers and after some cycles, the sample would
degrade showing a grey colour. Therefore, there was the need to change the agate balls with zirconium
oxide beads. In order to choose the right number of zirconium oxide beads we should use and for how
long, the next 2 experiments were made by increasing the number of cycles. The first experiment was
performed with 8g of beads and a second one with 16g.
12
Table 1 - DoE used to access the better programme for further use in the ball milling.
In blue is shown (tables 4 and 5) when the nanosuspension range is reached, and in orange
(table 5) is shown the programme that was chosen. 4 cycles of 30 min each, which corresponds to 2 h.
After getting a “foamy” nanosuspension another 20ml of water were added, in order to work with
it.
Separating the nanosuspension from the beads was not a hard process, since they are way
heavier than the nanosuspension, however, this separation process unfortunately causes the loss of
some amount of nanosuspension. This nanosuspension was stable up to 4 months.
2.3. Screening of the formulation strategies for the production of the dried
films
The polymers were chosen due to their different characteristics - 2 different PVPs with different
MWs (PVP K30 and PVP K90); 1 PVP/VA as co-polymer and 2 different HPCs with different MWs (HPC
GXF and HPC MF) providing different viscosities to the suspensions.
In order to select the best polymer and polymer concentration the best results could be obtained
a second full factorial DoE (Design of Experiments) was considered with 5 centre points varying from
10 to 90% concentration (Table 2). Results were analysed with the software MODDE from Umetrics.
Time (min) Speed (RPM) Volume (ml)
1 30 500 50
2 5 500 10
3 17.5 300 30
4 30 500 10
5 30 100 10
6 30 500 50
7 5 100 50
8 17.5 300 30
9 5 500 50
10 17.5 300 30
11 5 100 10
13
Table 2 - DoE used to access which polymer better stabilizes the nanosuspension PSD.
Experience Number Run Order Polymer Concentration Polymer
1 9 0.1 PVP K30
2 12 0.9 PVP K30
3 6 0.1 PVP K90
4 2 0.9 PVP K90
5 14 0.1 PVP/VA
6 5 0.9 PVP/VA
7 7 0.1 HPC GXF
8 10 0.9 HPC GXF
9 11 0.1 HPC MF
10 3 0.9 HPC MF
11 15 0.5 HPC MF
12 1 0.5 HPC GXF
13 13 0.5 PVP/VA
14 4 0.5 PVP K30
15 8 0.5 PVP K90
The essential 3 steps to produce the films were (fig.7): 1) all the polymers were diluted in water,
since HPC MF is very viscous, it is very hard to dissolve, and it forms a kind of gel. As such, it had to be
diluted to a point where no apparent viscosity was noticeable. All the other polymer solutions were
diluted with the same amount of water so that there were not any differences between concentrations;
2) all the different polymers were mixed with the ITZ nanosuspension and put into vials in order to get
mixtures with 90%, 50% and 10% of polymer; 3) all the mixtures were put in the drying chamber at 120
ºC and drying end-point curves were made to see if all the films were completely dry.
Figure 7 – Schematic representation of the steps taken to prepare the films.
14
2.3.1. Characterization of the obtained mixtures
The mixtures of polymer in the concentration of 50, 90 and 100% were measured in terms of
kinetic viscosity. The kinetic viscosity was measured with a capillary viscometer with a constant K of
30.55 cm2/s2 of Xylem Analytics, Germany (fig.8).
Figure 8 - Capillary viscometer.
2.3.2. Characterization of the obtained films
The obtained films were characterized by PSD (Particle Size Distribution), DSC (Differential
Scanning Calorimetry), RAMAN and WAXS.
2.3.2.1. PSD
All the obtained films were dissolved in water in order to get rid of the polymer. Without the
polymer dissolved, only the ITZ particles should be measured. The polymer HPC MF could not be
dissolved in water nor in acetone, ethanol, isopropanol, etc. as such, it was excluded, since one of the
main functions of getting nanocrystals is to enhance its dissolution and, its viscosity could detrimentally
impact any further processing.
After dissolving the films, a tiny amount of liquid was taken and put inside the cuvette which was
already filled with water.
Every film was measured 3 times in the equipment HELOS, in a cuvette at 1000 RPM in a range
of 0.45 to 87.5µm using the Fraunhofer mathematical theory.
2.3.2.2. DSC
The DSC instrument used to obtain all the DSC measurements was a Netzsch, DSC 204 F1
Phoenix®. All the DSC measurements performed using a method previously developed in RCPE for
ITZ. The method consisted on analysing each sample using a pierced lid. Program was composed of a
first heating stage from 20ºC to 185ºC with 5ºC/min and a modulation of 5ºC. The temperature was
maintained at 185ºC for 2 min and then lowered to 20ºC again by use of N2 flow. The second heating
15
stage was performed with the same conditions. All samples were analysed in duplicates. The results
were analysed on Netzsch software, area and onset of melting point were calculated on the total curve.
2.3.2.3. RAMAN
Spectra of all the raw materials used to obtain the nanosuspension and the respective films were
taken. The RAMAN spectrometer used was a PerkinElmer RamanStation 400F, all the obtained spectra
were treated using the software SpectraGryph. All the samples were measured in triplicates.
2.3.2.4. WAXS
The WAXS instrument used to obtain all the WAXS measurements was a HECUS S3-MICROcalix®,
the samples were measured in duplicates for 20 min each.
16
3. RESULTS
3.1. Characterization of the nanosuspension
As mentioned in the earlier chapter, the programme in blue (table 3) was chosen for further
usage, because it was the one with the lowest PSD and without any agglomeration. After being chosen,
the exact same program was used but instead of agate balls with zirconium oxide beads (with 8g and
16g) (tables 4 and 5) by adding cycles of 30 min. each. The results show that by doubling the amount
of zirconium oxide beads, it is possible to reach the nanometer range 1h faster, and with way better finer
particles.
Table 3 - DoE used to access the better programme for further use in the ball milling and its results.
Time
(min)
Speed
(RPM)
Volume
(ml)
Degradation Agglomerate X10
(μm)
X50
(μm)
X90
(μm)
1 30 500 50 No no 1.18 5.5 17.37
2 5 500 10 No no 0.9 4.05 12.26
3 17.5 300 30 No yes 1.84 8.5 43.56
4 30 500 10 No no 0.48 1.47 3.6
5 30 100 10 No no 1.39 6.03 17.45
6 30 500 50 No no 0.97 4.32 17.45
7 5 100 50 No no 2.54 11.8 60.74
8 17.5 300 30 No yes 1.6 7.96 26.58
9 5 500 50 No yes 1.91 9.16 48.68
10 17.5 300 30 No yes 1.54 7.78 61.59
11 5 100 10 No no 1.93 8.72 26.99
17
Table 4 - PSD increasing the number of milling cycles using 8g of zirconium oxide beads.
Table 5 - PSD increasing the number of milling cycles using 16g of zirconium oxide beads.
The nanosuspension and its manufacturing process were characterized by their particle size
distribution over time. In order to validate this process another 2 batches were produced by the exact
same procedure (table 6).
Table 6 - PSD of the produced batches to validate the nanosuspension manufacturing process.
Batch: X10 (µm) X50 (µm) X90 (µm)
1 0.33 0.70 1.71
2 0.32 0.69 1.58
3 0.33 0.72 1.72
Cycles X10 (µm) X50 (µm) X90 (µm)
1 0.58 2.19 7.57
2 0.46 1.47 4.13
3 0.40 1.14 3.08
4 0.35 0.87 2.11
5 0.34 0.76 1.80
6 0.33 0.74 1.63
7 0.32 0.68 1.57
Cycles X10 (µm) X50 (µm) X90 (µm)
1 0.44 1.28 3.43
2 0.37 0.95 2.28
3 0.34 0.76 1.89
4 0.33 0.70 1.71
5 0.32 0.66 1.52
6 0.32 0.65 1.53
7 0.31 0.60 1.33
18
The nanosuspension was also analysed by WAXS. The results will be shown in the WAXS
results chapter.
3.2. Characterization of the films obtained
3.2.1. PSD analysis
According to the obtained PSD results (table 7). There are differences in PSD between every
concentration of each polymer used with some tendencies while increasing the concentration of polymer
used, except for PVP/VA. Both PVP K30 and HPC GXF have a lower PSD while increasing the
concentration of polymer, on the other hand, PVP K90 has a higher PSD by doing so, although this
change in PSD for PVP K90 is not that significant. HPC GXF in a 90% (in blue) ratio in relation to the
nanosuspensions was the only one capable of stabilizing the nanocrystals after drying. HPC GXF 90%
also has the lowest span of all. Although PVP K90 was the second-best polymer with no significant
changes in PSD between concentrations, none of the PVPs used could stabilize the nanosuspensions
after drying.
Table 7 - PSD of the obtained films
3.3.2. Calorimetry (DSC) results
The pure ITZ “form I” has an onset peak at 164.3 ºC (fig. 9). Every other film with 10 and 50%
of polymer has an onset peak very near 156.6 ºC (fig.10 (a) and (b)) difference of around 8 ºC. This
melting point may correspond to its “form II” which has a melting point at 155.7 ºC [47].
Polymer
(%)
X10
(µm)
X50
(µm)
X90
(µm)
Span
PVP K30 10 1.35 ± 0.06 9.53 ± 0.68 34.32 ± 0.67 3.45
PVP K30 50 0.69 ± 0.00 5.16 ± 0.02 28.34 ± 0.55 5.35
PVP K30 90 0.44 ±0.00 1.93 ± 0.03 15.58 ± 0.34 7.84
PVP K90 10 0.37 ± 0.00 1.05 ± 0.01 4.46 ± 0.12 3.89
PVP K90 50 0.39 ± 0.00 1.27 ± 0.04 6.05± 0.71 4.45
PVP K90 90 0.44 ± 0.00 1.57 ± 0.05 8.70 ± 0.87 5.26
PVP/VA 10 0.87 ± 0.15 6.75 ± 1.60 26.83 ± 7.83 3.85
PVP/VA 50 1.21 ± 0.05 11.07 ± 0.63 51.62 ± 3.78 4.55
PVP/VA 90 1.09 ±0.03 9.42 ± 0.25 31.54 ± 1.53 3.23
HPC GXF 10 1.58 ± 0.10 13.00 ± 0.69 44.59 ± 9.00 3.30
HPC GXF 50 0.98 ± 0.15 7.66 ± 1.10 19.25 ± 3.24 2.38
HPC GXF 90 0.32 ± 0.00 0.66 ± 0.00 1.55 ± 0.03 1.86
19
All films with 90% of polymer concentration greater affinity for water than the other
concentrations, since every film still showed the presence of water in it (fig.10 (c)), fact already seen in
the drying curves. The onset peak of HPC with 90% concentration is around 144.6 ºC. According to the
study conducted by Zhang and Lee, at around 145 ºC is when the “form III” of ITZ shifts to the “form II”.
The “form III” is the least stable of them all [47]. HPC GXF at 90% concentration is the only one having
an onset peak below 150 ºC.
Figure 9 - DSC of pure itraconazole.
20
(a)
(b)
(c)
Figure 10 – Thermograms of nanosuspensions containing 10% HPC GXF + 90% nanosuspension.(a), HPC GXF
50% + 50% nanosuspension (b), 90% HPC GXF + 10% HPC GXF (c).
21
Although PVP K90 at a 90% concentration is the second-best formulation in that concentration,
it does not have an onset peak near 145 ºC (fig.11), therefore not showing the presence of the ITZ “form
III”.
Figure 11 - Thermograms of nanosuspensions containing 90% PVP K90 + 10% nanosuspension
3.3.3. Spectra of RAMAN
ITZ has a very specific peak at 1613 cm-1 which corresponds to the C=C of the phenyl group
(fig.12) [48].
Every film shows the same Itraconazole Peak at 1613 cm-1 with no shifts. Every film shows
similar peaks between themselves when they are being compared in the same polymer concentration
but different between different concentrations of same polymer. When the polymer concentration is high
(90%), the polymer peaks are way more noticeable (2800-3200 cm-1) and the ITZ peaks are not even
noticeable except for the biggest one at 1613 cm-1; when it is very low (10%), the ITZ peaks are way
more noticeable (200-1650 cm-1) (fig.14). HPC GXF 50% and PVP K90 10% are the only ones that show
a new interaction shown in the blue circle, although those peaks are practically in the same place as the
raw ITZ, they have a lot more intensity (fig.13). These interactions are only visible (independently of the
intensity) in high concentrations of ITZ (50 and 90%). The Raman intensity is a function of the
polarizability and symmetry and therefore probes the bonding covalence and structure [49]. Both HPC
GXF and PVP K90 have very similar molecular weight which can explain those similar interactions with
a peak at 262, 464 and 641 cm-1. These interactions seem to be detrimental, having in mind that HPC
GXF 90% has way finer particles than HPC GXF 50%, as for PVP K90 10% this interaction does not
seem to be significant since all the concentrations show very similar particle size distributions.
22
Figure 12 - Raw ITZ raman spectra.
Figure 13 - Raman spectra showing a new interaction of PVP K90 10% and HPC GXF 50% inside the blue circle.
Figure 14 - Differences between raman spectra of raw HPC GXF, HPC GXF 10% and HPC GXF 90%.
23
3.3.4. Results from X-ray diffractometry (WAXS)
(a)
(b)
Figure 15 - WAXS of (a) pure ITZ and (b) nanosuspension.
Figure 16 - WAXS of 10% of PVP K30 and 90% of nanosuspensions.
0
2
4
6
8
10
12
14
16
18
17 19 21 23 25 27
Inte
snit
y
2Θ (degree)
0
2
4
6
8
10
12
14
16
18
17 19 21 23 25 27 29
Inte
nsi
ty
2Θ (degree)
0
2
4
6
8
10
12
14
16
18
17 19 21 23 25 27 29
Inte
nsi
ty
2Θ (degrees)
24
WAXS was performed to compare the pure ITZ which is crystalline (fig.15 (a)) with the nanosuspension
(fig.15 (b)) and with one film (fig.16). PVP K30 was chosen randomly and to assess the eventual
conversion of “form I” to “form II”. The data obtained for the film came out inconclusive because of all
the background noise probably due to the amorphous excipients present in both. However, it is possible
that the ITZ is in the amorphous form in the nanosuspension.
3.4 Would the amount of ITZ present in the best formulation be enough to treat a
patient?
To try answering this question, first we have to consider that nowadays, the marketed ITZ in
solid oral pharmaceutical form is marketed with a dose of 100mg (Sporanox among other generics) [50].
The amount of ITZ in the film HPC GXF at 90% concentration can be calculated according to the
following equations:
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝐼𝑇𝑍 𝑖𝑛 𝑡ℎ𝑒 𝑁𝑎𝑛𝑜𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛 =1𝑔 𝐼𝑇𝑍
30 𝑚𝑙 𝐻2𝑂 = 0.0333 𝑔/𝑚𝑙 Eq.6
𝑄𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑜𝑓 𝐼𝑇𝑍 𝑖𝑛 𝑡ℎ𝑒 𝑓𝑖𝑙𝑚 = 0.0333𝑔
𝑚𝑙∗ 0.5 𝑚𝑙 = 16.65𝑚𝑔 Eq.7
The amount of ITZ present in 90% polymer concentration film is 16.65mg. This means that, at
first glance, this dose would be inefficient to treat a patient in need of ITZ.
25
4. Discussion
The nanosuspension using a polymer and a surfactant has proved to be stable. The single use
of a surfactant failed to provide particles in the nanometer range. The effect of using a semi-synthetic
polymer and a surfactant has already been described as one of the best ways of formulating a
nanosuspension. The formulation is not only capable of successfully reducing the particle size but also
of stabilizing the nanosuspension up to 4 month [30].
Ball milling in a wet media has shown to be a capable technique to get a nanosuspension, as
described in the literature [30].
The zirconium oxide beads played a very important role, without them it would not have been
possible to produce a nanosuspension of ITZ by ball milling. The quantity of beads used had also an
important impact in the milling process, by doubling the weight of the beads it was possible to obtain
particle in the nanometer range, 2 cycles faster. A larger quantity of zirconium oxide beads leads to
fewer voids, leading to a smaller PSD [51].
The DoE used for the production of films was successful by showing which polymer was the
best and at what concentration it would better stabilize the ITZ nanosuspension PSD. The outputs also
identified the optimal range of the concentration the polymer for a maximum effect (between 10 and
50% or 50 and 90%, depending on the polymer). This way possible to know in which range of
concentrations the polymer should be further studied.
HPC GXF at 90% concentration had the best results at stabilizing the nanosuspension after
drying, being the only one capable of keeping it in the nanometer range. This fact can be either due to
chemical or physical interactions or maybe both. And of course, the polymer concentration also plays
an important role. HPC GFX at 90% concentration has also the least span of all the obtained films,
meaning that it has the least span of PSD.
Although high viscosity, caused in this case by the addition of a polymer, can be detrimental to
the process of ball milling or HPH affects directly the breakage of particles, when it is added to the final
product, it can better stabilize the nanocrystals [52]. This may be the reason why HPC MF did not
dissolve in water after being mixed with the nanosuspension and duly dried. The high viscosity
proportioned by the HPC GXF was a key factor at stabilizing the PSD of the nanosuspension.
The better stabilization of the nanosuspension size distribution, upon increasing the
concentration of polymer, may be due to the fact that the interaction between the ITZ and the polymer
produce a more compacted form, and therefore have better efficacy at stabilizing it [53].
Regarding the ITZ polymorphs, calorimetry has shown that ITZ converts from “form I” to “form
II” which by itself is not enough to explain why any polymer worked better than other, since every film
made of any polymer has shown this form of ITZ. However, after analysing the HPC GXF at 90%
concentration, it is possible that “form III” was present, this “form III” was recently discovered (2016) and
got patented in 2017 [54]. As described in the patent, the “form III” of ITZ has the melting point at 119ºC
26
which unfortunately is absent. However, the authors also mention that at 145 ºC is when there is a shift
from the “form III” to “form II”, which means that if we see this shift is because, the “form III” of ITZ was
in fact present in this film. According to the data obtained by WAXS (fig.15 (b)), the ITZ in the
nanosuspension is amorphous, fact that can be caused by ball milling [54][55]. It is interesting that this
“form III” was obtained, since it is the rarest polymorph of ITZ due to its lack of stability. Another possible
explanation for that shift in the melting point is a chemical interaction between the ITZ and HPC GXF,
however, according to the RAMAN obtained data, there are no interactions.
Unfortunately, the WAXS results regarding the film were inconclusive, maybe a good way of
solving this issue would be utilizing the X-Ray Diffraction Patterns (XRDP) technology instead of WAXS.
In literature it was shown that it is possible to detect the 3 different ITZ polymorphs via XRDP, which in
this case would be a perfect complementary information [47].
Despite the drying curves showing that the drying process is complete (fig.18), there was still
water present, as shown by calorimetry. It is known that a high water content in the final product is
detrimental to the API. This high water content can degrade the API by different mechanisms and
unstabilizes it. However, in this case might be one of the reasons why “form III” appears. It is unfortunate
that with the data taken from the drying curves, it was not possible to get real amount of water that was
lost during the drying process. Because assuming that all the liquid in the nanosuspension and in the
polymeric solutions is water and has a density of approximately 1g/ml, the difference between the mass
at the beginning and the mass at the end of the drying process results in a lot of cases in a higher
amount of water lost than the quantity of water that was really in the vials. The method used to duly
evaluate the amount of water lost after drying would have been Karl-Fischer titration since is way more
accurate than Loss on Drying (LOD), since LOD does not take in account other volatile impurities
besides water [56].
HPC GXF at 50% and PVP K90 at 10% shows a different set of peaks in intensity in the Raman
spectra revealing a chemical interaction between the polymer and the nanosuspension, with this set of
peaks being very similar to the ones present in the raw ITZ. This chemical interaction seems to have a
detrimental effect in the stability of the film affecting its PSD at least for HPC GXF, on the other hand, it
seems to be beneficial for PVP K90 although there is not a very relevant change in its PSD. IR
spectroscopy also would have been a good technique to be used, besides providing a complimentary
information to the data obtained with Raman spectroscopy, in the patent previously described, the
inventors are able to differentiate the 3 different ITZ polymorphs [54].
The physical interactions, starting with viscosity, HPC is way more viscous than the other PVPs
that were used. Although in this case HPC MF is too viscous, HPC GXF seems to have the perfect
viscosity to stabilize the PSD of the nanosuspension after drying. Comparing the MWs of the used
polymers, PVP K90 and HPC GXF are very similar, which is probably why they are the polymers that
better stabilize the nanosuspension PSD after drying. Also, PVP K90 was the second most viscous
polymer after HPC GXF.
27
The HPC MF was a major setback because it formed a gel without allowing dilutions requiring
the use of very low polymer concentrations implying the use of very low amounts of ITZ, for large
quantities of solution, delivering low quantities of ITZ in the films. Bearing in mind that the PSD showed
a tendency to reduce when increasing the viscosity, HPC MF might have shown some promising results.
Since HPC MF reaches a higher viscosity faster than HPC GXF, it is possible that it would have a better
PSD at a lower ratio of Polymer:ITZ in comparison to HPC GXF maybe even at 10% or 50%.
28
5. Conclusion
The objectives presented in this thesis were duly achieved. An ITZ nanosuspensions was
produced, that was mixed with a polymer and then dried, forming films. Only one of the film formulations
(GXF 90%) could stabilize the PSD of the ITZ nanosuspension.
An ITZ nanosuspension was developed for the first time, using the ball milling technique, with a
4 month stability.
I think it is fair to say that the most important factor for better stabilizing the PSD of the ITZ
nanosuspension after drying is the viscosity of the added polymer, since PVP K90 and HPC GXF have
similar MWs.
None of the PVPs used could stabilize the ITZ nanosuspension PSD after drying, with PVP K90
being the best among the other PVPs, including PVP/VA.
HPC GXF in a 90% ratio in relation to the nanosuspension was the only formulation showing
promising results, the PSD of the nanosuspension was maintained after drying, due to its viscosity and
MW characteristics, and due to the polymorph at “form III”. HPC GXF 90% was the only one film showing
the “form III” of ITZ, with this probably being the key factor to obtaining the only favourable PSD along
with the high polymer viscosity. On the other hand, HPC MF failed to promote the formation of a
suspension due to the low solubility in water or to the high viscosity.
It was shown that by following the DoE and the idea behind it presented in this thesis, it is easily
possible to determine the best polymer to stabilize nanocrystals by choosing polymers with different
chain lengths, molecular weights and viscosities. It is also possible to get a notion of what range of
polymer concentrations will give better results regarding PSD and what ranges it can still be further
explored (between 10 and 50% or 50 and 90%).
29
6. Future Work
Although the water content seem to have a positive effect in this exact situation, it still can
degrade the sample, therefore, the final formulation should be further optimized, because even though
the best formulation was 90% of HPC GXF, there are a lot of other ratios between 50% and 90% that
still can be explored and may be promising.
Since 16.65mg of ITZ, at first glance is not enough to treat a patient, maybe considering
increasing the concentration of the polymeric solution of HPC GXF would help increase the ITZ content.
During the experimental part of this thesis, only the stability of the developed nanosuspension
was assessed. After the optimization of the film formulation as previously discussed, the stability of the
obtained film should also be studied.
Using IR and XRDP technologies in order to validate the presence of the “form III” of ITZ.
Further processing should be studied. For example, pulverizing the nanosuspension via spray
coating over mannitol beads with the polymer already impregnated.
30
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34
Annex
Kinetic Viscosity
Figure 17 - Kinetic viscosity of the obtained mixtures.
The polymer HPC MF is way more viscous than the others, factor that will be reflected upon its
dissolution in water, which will be discussed later. Differences between the 3 different concentrations of
each polymer are not very significant. PVP K90 has higher viscosity than the other PVPs which is related
to its bigger chain/MW [52]. The pure nanosuspension and the mixtures of 10% of polymer were not
measured because of the amount of API that was required. Also, HPC GXF is significantly more viscous
in comparison to all the other PVPs.
Polymer Molecular Weights
Table 8 - Molecular weight of the chosen polymers.
Polymer: Molecular Weight (Da)
PVP K30 40000
PVP K90 360000
PVP/VA 45000 - 70000
HPC GXF 370000
HPC MF 850000
Drying End-point Curves
The drying curves were made by taking the respective vials out of the drying chamber and
weighting them, until there was no longer any change in their mass. This way we know that the drying
0123456
(mm
/s2 )
35
process is already over (fig.18). The drying chamber used was a Binder model FD 23 at 120 ºC.
(a)
(b)
(c)
Figure 18 - Curves of drying curve of mixtures of polymers at 120 ºC when the polymers were present at 10% (a),
50% (b) and 90% (c) concentrations.
0 50 100 150 200 250 300 350
Wei
ght
(g)
Time (min)
PVP K30 10% PVP K90 10% PVP/VA 10% HPC GXF 10% HPC MF 10%
10
12
14
16
18
20
22
24
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700
Wei
ght
(g)
Time (min)
PVP K30 90% PVP K90 90% PVP/VA 90% HPC GXF 90% HPC MF 90%
10
12
14
16
18
20
22
24
0 50 100 150 200 250 300 350 400 450 500 550 600
Wei
ght
(g)
Time (min)
PVP K30 50% PVP K90 50% PVP/VA 50% HPC GXF 50% HPC MF 50%