Academic year 2013-2014

Goedele CRAYE

First Master of Pharmaceutical Care

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutical Analysis

Laboratory of Process Analytical Technology

UNIVERSITY OF EASTERN FINLAND

SCHOOL OF PHARMACY

Pharmaceutical Technology

CHARACTERISATION OF SIMVASTATIN AND GLIBENCLAMIDE FORMULATIONS

PREPARED BY SPRAY DRYING

Promotor

Prof. Dr. T. De Beer

Co-promotor

Prof. Dr. J. Ketolainen

Supervisor

Dr. R. Laitinen

Commissioners

Prof. Dr. J. Demeester

Prof. Dr. C. Vervaet

Academic year 2013-2014

Goedele CRAYE

First Master of Pharmaceutical Care

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutical Analysis

Laboratory of Process Analytical Technology

UNIVERSITY OF EASTERN FINLAND

SCHOOL OF PHARMACY

Pharmaceutical Technology

CHARACTERISATION OF SIMVASTATIN AND GLIBENCLAMIDE FORMULATIONS

PREPARED BY SPRAY DRYING

Promotor

Prof. Dr. T. De Beer

Co-promotor

Prof. Dr. J. Ketolainen

Supervisor

Dr. R. Laitinen

Commissioners

Prof. Dr. J. Demeester

Prof. Dr. C. Vervaet

COPYRIGHT "The author and the promoters 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."

June 3, 2014

Promoter Author

Prof. Dr. T. De Beer Goedele Craye

SUMMARY

One of the major current challenges in the formulation development is associated

with strategies to enhance the dissolution rate of poorly water soluble drugs. The conversion

of the crystalline material into the amorphous formulation is one method to enhance the

solubility. However, the thermodynamically unstable amorphous formulation is

characterized by a higher risk to recrystallize during manufacturing and storage. A recently

introduced approach to stabilize the amorphous formulation is the co-amorphous drug

formulation concept in which receptor amino acids (AA) from the biological target site of the

drug are used as stabilizers.

The purpose was to use spray drying (SD) as a preparation method for co-amorphous

drug-AA mixtures containing poorly water soluble drugs simvastatin (SVS) and glibenclamide

(GBC). The combination of SVS with lysine (LYS) and GBC with serine (SER) respectively was

based on a recent study in which it was found that both drugs form a co-amorphous mixture

(1:1) with their corresponding amino acid. [33] Since both drugs are poorly water soluble, a

suitable solubilizer was required to perform SD from an aqueous solution. The prepared

samples were characterized with respect to the thermal properties (DSC), the level of

crystallinity (XRPD), intermolecular interactions (FTIR), dissolution and stability properties.

It was possible to perform the spray drying with the use of 5% SLS solution to dissolve

the drug-AA mixtures. A complete amorphous conversion of both drugs and AA was

observed by XRPD. This amorphization resulted in better dissolution properties for only SVS-

5% SLS (SD) and SVS-LYS-5% SLS (CM) over that of the respective crystalline mixtures. Among

all the formulations, SVS-5% SLS (SD) showed the most promising dissolution behaviour.

Interestingly, even though all the prepared spray dried SVS formulations were remaining

partly crystalline with respect to SLS , the changes seen as a function of time in different

storage conditions were small or completely absent. The SVS-LYS-5% SLS (SD inlet 100°C)

mixture was found to be physically stable over at least 9 weeks of storage at 40°C under dry

conditions. The SVS-LYS-5% SLS (CM) was stable over 9 weeks at every condition.

Conclusively, spray drying is a potential preparation method for the amorphization of

drug-amino acid mixtures. Moreover, the amino acids can be considered as promising

stabilizers of the amorphous formulation of poorly water soluble drugs.

SAMMENVATTING

Eén van de actuele uitdagingen in de geneesmiddelenontwikkeling is geassocieerd met

methoden om de oplossingssnelheid van slecht water oplosbare geneesmiddelen te verbeteren.

De omzetting van de kristallijne naar de amorfe geneesmiddelvorm is een mogelijke methode

om de oplosbaarheid te verbeteren. Echter, de thermodynamisch onstabiele amorfe

geneesmiddelvorm is gekenmerkt door een hoger risico op rekristallisatie gedurende de

productie en bewaring. Een recent geïntroduceerd concept om de amorfe vorm te stabiliseren is

het co-amorfe geneesmiddel systeem waarin receptor aminozuren (AZ) gebruikt worden als

stabilisatoren.

Het doel was het sproeidrogen van co-amorfe geneesmiddel-AZ mengsels. De co-amorfe

mengsels omvatten de slecht water oplosbaar geneesmiddelen simvastatine (SVS) en

glibenclamide (GBC) in combinatie met respectievelijk lysine (LYS) en serine (SER). De combinatie

was gebaseerd op een recente studie waarin werd vastgesteld dat beide geneesmiddelen een

amorf mengsel (1:1) vormen met het bijhorende aminozuur. [33] Aangezien beide

geneesmiddelen slecht water oplosbaar zijn, was een oplossende stof vereist om het

sproeidrogen uit te voeren vanuit een waterige oplossing. De bereide formulaties werden

gekarakteriseerd met betrekking tot de thermische eigenschappen (DSC), de mate van

kristalliniteit (XRPD), intermoleculaire interacties (FTIR), dissolutie- en stabiliteitskenmerken.

Het sproeidrogen was mogelijk met behulp van een 5% SLS oplossing om het

geneesmiddel-AZ mengsel op te lossen. De volledige amorfisatie van beide geneesmiddelen en

aminozuren werd aangetoond met XRPD. Deze amorfisatie resulteerde in betere dissolutie-

eigenschappen voor SVS-5% SLS (SD) en SVS-LYS-5% SLS (CM) in vergelijking met de

overeenkomstige kristallijne mengsels. Bovendien bleken de dissolutie eigenschappen van SVS-

5% SLS (SD) het meest belovend. Ondanks de onvolledige reductie van het kristallijn SLS gehalte

in de gesproeidroogde SVS mengsels, was de verandering in functie van de tijd in verschillende

bewaringscondities gering of geheel afwezig. SVS-LYS-5% SLS (CM) bleef stabiel gedurende

tenminste 9 weken bewaring onder iedere conditie. Echter, SVS-LYS-5% SLS (SD inlet 100°C)

bleef stabiel gedurende tenminste 9 weken bewaring bij 40°C onder droge condities.

Kortom, hieruit kan besloten worden dat het sproeidrogen geschikt is voor de bereiding

van amorfe geneesmiddel-aminozuur mengsels. Bovendien worden de aminozuren beschouwd

als veelbelovende stabilisatoren van amorfe geneesmiddel formulaties.

Word of thanks

This thesis would not have been possible without the support of many,

I would like to express my gratitude to all those who made this experience possible.

I would like to thank Prof. Dr. T. De Beer and Prof. Dr. J. Ketolainen for giving me the chance

to participate in this interesting study and for the opportunity to

realise this al in Kuopio, Finland.

I want to express my sincere gratitude to my supervisor Dr. Riikka Laitinen for being

a helpful and critical mentor for me during this thesis.

I appreciate all your time and support, your help and advice,

your optimistic and patient character.

I would like to express a special word of thanks to my parents,

I would not be who I am now without the love, confidence and support they have given me.

I would like to thank Niels,

for your input, constant support, patient and love. Thank you for always being there.

I would like to give special thanks to my sister Ellen,

everything would be so much harder without you on my side.

I also would like to send my warmest thanks to the rest of my family for the support and love.

I want to say thanks to all of my friends, in Belgium and Kuopio,

who stood by me and gave me the support, who did not make me forget to smell the roses.

I especially want to send my warmest thanks to Gaëlle, my person during Erasmus,

with whom I shared good and hard times. Thank you for everything.

Finally, I want to thank the exchange program Erasmus,

which made this rich experience possible.

1. INTRODUCTION ............................................................................................................................... 1

1.1 BACKGROUND ......................................................................................................................... 1

1.2 CHARACTERISTICS OF THE AMORPHOUS STATE ..................................................................... 3

1.2.1 Amorphous versus crystalline structure .......................................................................... 3

1.2.2 The energy of an amorphous system .............................................................................. 4

1.2.3 Physical properties .......................................................................................................... 5

1.3 PREPARATION OF AMORPHOUS MATERIAL ............................................................................ 5

1.3.1 Melting and quench cooling ............................................................................................ 6

1.3.2 Spray drying ..................................................................................................................... 6

1.3.2.1 Spray drying process .................................................................................................... 6

1.3.2.2 Process parameters ..................................................................................................... 8

1.3.2.3 Formulation parameters ................................................................................................. 9

1.3.2.3 The applicability of spray-drying ............................................................................... 11

1.3.3 Freeze-drying ................................................................................................................. 13

1.3.4 Milling ............................................................................................................................ 13

1.4 CHARACTERIZATION OF AMORPHOUS MATERIALS .............................................................. 14

1.4.1 Differential scanning calorimetry (DSC) ........................................................................ 14

1.4.2 X-ray powder diffraction (XRPD) ................................................................................... 15

1.4.3 Infrared spectroscopy (IR) ............................................................................................. 16

1.5 STABILISATION OF THE AMORPHOUS STATE ........................................................................ 17

1.5.1 Solid dispersion ............................................................................................................. 17

1.5.1.1 Definition and different generations ......................................................................... 17

1.5.1.2 Advantages and disadvantages of solid dispersions ................................................. 18

1.5.1.3 Preparation methods................................................................................................. 20

1.5.2 Co-amorphous formulation ........................................................................................... 20

1.5.2.1 Co-amorphous drug-drug formulations .................................................................... 21

1.5.2.2 Co-amorphous drug- amino acid formulations ......................................................... 22

1.6 DESCRIPTION OF THE STUDIED DRUGS ................................................................................. 23

1.6.1 Simvastin (SVS) .............................................................................................................. 23

1.6.2 Glibenclamide (GBC) ...................................................................................................... 23

2. OBJECTIVES OF THE STUDY............................................................................................................ 24

3. MATERIALS AND METHODS .......................................................................................................... 26

3.1 MATERIALS ............................................................................................................................ 26

3.2 METHODS .............................................................................................................................. 26

3.2.1 Solubility test ................................................................................................................. 26

3.2.2 Preparation of the materials ......................................................................................... 27

3.2.2.1 Spray drying process .................................................................................................. 27

3.2.2.2 Cryo-milling process .................................................................................................. 27

3.2.3 Physical characterisation of the materials .................................................................... 28

3.2.3.1 Differential scanning calorimetry (DSC) .................................................................... 28

3.2.3.2 X-ray powder diffraction (XRPD) ............................................................................... 28

3.2.3.3 Fourier-Transform infrared spectroscopy (FTIR) ....................................................... 29

3.2.4 Dissolution test .............................................................................................................. 29

3.2.5 Stability study ................................................................................................................ 29

3.2.6 HPLC analysis ................................................................................................................. 30

4. RESULTS ......................................................................................................................................... 31

4.1 SOLUBILITY TEST .................................................................................................................... 31

4.2 PREPARATION OF AMORPHOUS MATERIAL BY SPRAY DRYING ............................................ 33

4.2.1 Spray drying of glibenclamide (GBC) ............................................................................. 33

4.2.2 Spray drying of simvastatin (SVS) .................................................................................. 34

4.3 PHYSICAL CHARACTERISATION OF THE PREPARED SAMPLES ............................................... 36

4.3.1 Differential scanning calorimetry (DSC) ........................................................................ 36

4.3.2 X-ray powder diffraction (XRPD) ................................................................................... 37

4.3.3 Fourier transform infrared spectroscopy (FTIR) ............................................................ 38

4.4 DISSOLUTION TEST ................................................................................................................ 39

4.5 STABILITY STUDY ................................................................................................................... 41

5. DISCUSSION ................................................................................................................................... 43

5.1 SOLUBILITY TEST .................................................................................................................... 43

5.2 PREPARATION OF AMORPHOUS MATERIAL BY SPRAY DRYING ............................................ 43

5.3 PHYSICAL CHARACTERISATION OF THE PREPARED SAMPLES ............................................... 44

5.4 DISSOLUTION TEST ................................................................................................................ 45

5.5 STABILITY STUDY ................................................................................................................... 46

6. CONCLUSION ................................................................................................................................. 47

7. REFERENCES .................................................................................................................................. 48

LIST OF ABBREVIATIONS

AA Amino acid

ACN Acetonitrile

ANOVA Analysis of variance

API Active Pharmaceutical Ingredients

BCS Biopharmaceutical Classification System

CM Cryo-milling

DSC Differential Scanning Calorimetry

e.g. exampli gratia (for example)

FTIR Fourier-Transform Infrared Spectroscopy

GBC Glibenclamide

HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A

HPLC High-Performance Liquid Chromatography

i.e. Id est (that is)

LYS Lysine

N/A Not applicable or Not Analyzed

N/D Not detected

PM Physical mixture

PVP Polyvinylpyrrolidone

RH Relative Humidity

SD Spray drying

SER Serine

SLS Sodiumlaurylsulfate

SVS Simvastatin

TFA Trifluoroacetic acid

Tg Glass transition temperature

Tm Melting temperature

Tc Crystallization temperature

USP United States Pharmacopeia

XRPD X-ray Powder Diffraction

1

1. INTRODUCTION

1.1 BACKGROUND

The increasing number of active pharmaceutical ingredients (API’s) with more

lipophilic properties and hence poor aqueous solubility arises from the use of combinatorial

chemistry and high throughput screening of potential therapeutic agents in drug discovery.

[1] The drugs have an unsatisfactory therapeutic effect as a consequence of the poor

dissolution rate in the gastro-intestinal tract. These poorly water soluble but well-permeable

drugs with a low bioavailability are called BCS class II drugs. [2]

One of the major current challenges in the formulation development is associated

with strategies to overcome the low bioavailability by improving the dissolution rate of the

BCS class II drugs. [3] The modified Noyes-Whitney equation describes which

physicochemical factors control the dissolution rate:

(1.1)

Where: dC/dt: dissolution rate (mol/s)

A: surface area available for dissolution (cm²)

D: diffusion coefficient of the compound (cm²/s)

Cs: the solubility of the compound in the dissolution medium (mol/cm-3)

C: the concentration of the drug in dissolution medium at time t (mol/cm-3)

Cs-C: concentration gradient

h: the thickness of the diffusion boundary layer (cm)

Considering the Noyes-Whitney equation (1.1), increasing the surface area available

for dissolution (A) may improve the dissolution rate. Reduction of the particle size is one

way to increase the surface area. The dissolution rate also profits from a reduced boundary

layer thickness (h) which can be simply induced by mixing. In fact, increasing the solubility

(Cs) is the key factor to enhance the dissolution rate (dC/dt). [1] Solubility is the amount of

substance that can dissolve in a solvent. When a particular solid compound is dissolved in a

liquid, a solution or homogenous system is obtained. [4]

2

The solubility can be improved by many strategies which are divided into two

categories: physical and chemical modification. Several physical modifications are able to

improve the apparent solubility while the equilibrium solubility can only be enhanced after

chemical modification. The formulation of soluble prodrug and salt formation are classified

under chemical modification. The physical modifications include particle size reduction by

micronization or nanosuspension, (pseudo)polymorphization, complexation with

surfactants/cyclodextrines, the dispersion of a drug in a carrier and the use of the

amorphous form of the drug. It is important to note that an improved apparent solubility

only has an impact on the solubility if it doesn’t decrease immediately. [1]

There are various methods to transform the crystalline drug into the amorphous

equivalent: condensation from the vapour state, quench-cooling, mechanical manipulation

and rapid precipitation of the solution. [5,6] Nevertheless, particular attention should be

paid to the several drawbacks associated with this formulation strategy. More specifically,

the thermodynamically unstable amorphous state has a higher chemical reactivity and

greater risk to recrystallize during manufacturing and storage. [5]

Solid dispersion is one method to stabilise the amorphous form of drugs in order to

profit from the solubility and dissolution rate advantages of the amorphous form. [5] The

term solid dispersion refers to a solid product that contains one or more active hydrophobic

compounds and an inert hydrophilic carrier. [7] In general, hydrophilic polymers are used as

carrier material for the preparation of solid dispersion. However, the use of solid dispersion

offers several drawbacks due to high hygroscopicity of the polymers and the use of a drug

and polymer with a limited miscibility. Another strategy to stabilize the amorphous state is

the recently introduced co-amorphous drug concept in which receptor amino acids from the

biological target site of the drug are used as stabilizers. The concept is based on the

potential interactions between the functional groups of the drug and amino acids resulting

in the stabilization of the amorphous formulation. [8]

3

1.2 CHARACTERISTICS OF THE AMORPHOUS STATE

The transformation of a crystalline drug into its respective amorphous state may

potentially increase the apparent solubility of poorly water soluble drugs. [5] To understand

this, some characteristics of the amorphous state are explained in the following section.

1.2.1 Amorphous versus crystalline structure

Despite the fact that a solid can exist in the well-known crystalline state, it can also

be transformed in the amorphous form. While the crystalline form is characterised by a

regular and well-defined molecular packing, molecules in the amorphous state are

irregularly arranged to one another. Consequently there exists more space between the

molecules resulting in an increased specific volume compared to the crystalline

composition. [6,9]

Secondly, there is a difference between the three dimensional order of the

crystalline and the amorphous form. In contrast to the crystalline form, the amorphous form

is not characterised by a three dimensional long range order. The amorphous state may

consist of a short-range order present over several molecular dimensions. Short-range order

means the distance to the molecules the in closest proximity. [6,9] The structural

differences between crystalline and amorphous material are schematically illustrated in

Figure 1.1. The figure illustrates the short-range order present in both amorphous and

crystalline state on the one hand and the lack of three dimensional long range order in the

amorphous form on the other hand. The molecular arrangement in the amorphous solid

isn’t completely random in comparison to the gas phase. [10]

Fig. 1.1 Schematic representation of the crystalline structure, amorphous structure and

the gas phase. (Modified from Einfalt et al. [10])

4

1.2.2 The energy of an amorphous system

Amorphous drugs are characterised by their high internal energy. Figure 1.2 shows

the enthalpy (H) or specific volume (V) in function of the temperature. The curve for a

crystalline formulation at low temperature shows a small increase in enthalpy and volume

when the temperature is raised. When the temperature is further increased, a discontinuity

appears in the curve at the melting temperature (Tm). At this point, there is a first-order

phase transition to the liquid state. [6]

Vice versa, when the temperature of a liquid is slowly reduced, the liquid will

crystallize at Tm. The observed, reduced volume and enthalpy causes a contraction of the

material. However, if the cooling rate of the liquid is too fast, there is not enough time for

the molecules to form a crystalline structure. The curve follows the equilibrium line of the

liquid state without showing a discontinuity at Tm. The curve will enter the supercooled

liquid region when it continues beyond Tm. The supercooled liquid is a non-equilibrium state

with respect to the crystalline state but it is in equilibrium with the structural changes

caused by the changing temperature. [5,10]

The more the temperature is decreased, the higher the viscosity of the material, the

more the molecular motion is limited. At some point, the movement of the molecules is so

slow that they are kinetically frozen. There appears a change of the slope at the glass

transition temperature (Tg) from where the material enters the glassy state. The glassy state

is a non-equilibrium state, analogous to the supercooled liquid. [5,10]

Figure 1.2 The enthalpy or volume in function of the temperature. (Modified from T.

Einhalt et al. [10])

5

The amorphous state can be seen as a frozen or supercooled liquid which is

characterised by the glass transition temperature (Tg). The Tg is related to the stability of the

amorphous formulation during storage. Molecular motion above this temperature is faster

than the motion at temperatures below the Tg. Consequently, the risk of recrystallization of

the glassy state below the Tg is less compared to the supercooled liquid. [11]

1.2.3 Physical properties

Considering the properties of the amorphous state described above, the amorphous

form possesses higher Gibbs free energy compared to their crystalline counterparts. From

this we see that the molecules in the amorphous form have a greater molecular motion. In

addition, the dissolution of a drug in the amorphous state doesn’t require energy to break

up the lattice structure. Consequently amorphous material has a higher apparent solubility

and an enhanced dissolution rate compared to the crystalline state. [5,7]

Unfortunately, there are also drawbacks due to the high internal energy and

enhanced molecular mobility of the amorphous material. Amorphous material converts

back to the lower energy crystalline state over time, and this can happen during processing,

storage or dissolution. Moreover there is a higher chemical reactivity. Therefore, the use of

amorphous drugs can be limited by the poor inherently physical stability. [10]

1.3 PREPARATION OF AMORPHOUS MATERIAL

Amorphous character of a solid material can be induced by several techniques. The

most common preparation methods are divided into two categories according to the

mechanism behind the amorphization process. The first category contains two major steps.

First, a thermodynamically unstable non-crystalline form (either a melt or solution) is

prepared, starting from the crystalline material. Thereafter, this intermediate is transformed

into its respective thermodynamically unstable amorphous form by quench cooling of the

melt or rapid precipitation from the solution. While the first pathway is an indirect

amorphization process, the second category involves direct conversion of the crystalline

material into its amorphous counterpart (e.g. milling). [10,12] Below the different

preparation methods are described. The spray-drying method (SD) is discussed in more

detail since it was the method used in the experimental part of this thesis.

6

1.3.1 Melting and quench cooling

This relatively simple method consist of three stages and makes use of the molten

material as an intermediate. In the first stage, the drug and the polymer are melted at the

same time. Secondly, the melted mixture is cooled down (e.g. ice bath) with a view to put

back the motion of the molecules. In the case of a rapid cooling, the molecules don’t have

the time to reorganise themselves into the well-defined crystalline structure. This results in

a disorganised structure consisting of kinetically frozen molecules, instead of a crystalline

composition. Finally, the obtained product is pulverized in order to reduce the particle size.

[3,10] Figure 1.2 illustrates the theory behind this method.

The main advantage of the melting method is the fact that this preparation method

doesn’t require a solvent. Nevertheless, the melting method is limited by several

disadvantages. The use of high temperature during the melting step may potentially induce

chemical degradation. There is a risk that the resulting degradation products lower the Tg of

the final amorphous product. This phenomenon is compound dependent. A possible way to

overcome this drawback in some cases is heating with an inert gas. [10] Another method is

to perform the melting stage in such a way that the drug is first suspended in a previously

melted carrier instead of melting both drug and carrier. This adaptation can reduce the

process temperature. Several modifications as hot-stage extrusion, Meltrex or melt

agglomeration were introduced to overcome the drawbacks of the original method. [3]

However, this preparation method is already supplied for a large number of active

pharmaceutical ingredients (API’s) and excipients. [10]

1.3.2 Spray drying

1.3.2.1 Spray drying process

Spray drying is a preparation method used to transform solutions or suspensions into

a solid dry product. [13] This technique is suitable for the preparation of the amorphous

equivalent of a drug compound either alone or in combination with an excipient. [10] The

extremely rapid solvent evaporation during spray drying results in a fast increase in the

viscosity and formation of possibly amorphous particles. The spray drying process consists

of four stages which are illustrated in Figure 1.3.

7

Figure 1.3 Schematic illustration of the spray drying process. (Modified from Ameri et al. [14])

Firstly, there is the atomization of the liquid into a spray of fine droplets by applying

a force. The transportation of the solution or suspension from the container to the nozzle

inlet is possible by using a pump system. The fluid stream is broken in small droplets due to

the interaction with an atomizing gas. [11] The atomizing gas encounters the fluid stream

straight after leaving the nozzle. The atomization provides small droplets resulting in a high

surface area. The increased surface area contributes to an efficient solvent evaporation

which takes place in the second step. [13]

Immediately after atomization, the spray droplets are mixed with the drying gas (i.e.

air or nitrogen) so that the solvent will evaporate. It is important that the drying gas is

conditioned to have the right temperature and humidity with a view to apply his heat and

mass transfer properties. The drying gas is introduced in the drying chamber by an air

dispenser system which ensures an equally gas flow within all the parts of the drying

chamber. The contact between the gas flow and spray droplets is ensured due to the use of

the co-current design. The drying gas inlet and atomizer device are both situated at the top

of the drying chamber. Therefore, the droplets will make contact with the highest

temperature of the drying gas considering the latter has not yet exchanged any of its heat

with the surroundings. [11]

The solvent evaporation inside a droplet is explained with the following mechanism.

One droplet can be seen as a suspension of small particles in a liquid sphere. A rapid

evaporation occurs after the first contact with the drying gas. Thereafter, the surface of the

sphere is covered with a fine layer of dissolved material. The temperature of the particle is

8

low in the beginning but will increase with a rising amount of evaporated solvent. From then

on, the liquid inside the particle starts to evaporate. The faster the liquid is able to diffuse to

the surface of the sphere, the faster the evaporation takes place. [15]

The last step in the spray drying is the separation of the solid particles from the

drying gas stream. The particles are collected with the use of a cyclone separator and/or bag

filtration. The dispersed particles are separated from the continuous gas phase based on the

difference in density between the two phases. The solid particles and gas stream are

exposed to an accelerating flow field. This occurs within a rotating vortex in the cyclone. As

a result of the accelerating flow field, a lag in velocity for the dense particles compared to

the lower density medium, takes place. [11]

The reverse-flow type is the most applied type of cyclone. In this type, a particle-air-

dispersion is introduced tangentially into the top section of the cyclone. The high fluid

velocity creates a vortex which on his turn forces the particles to the wall of the cyclone and

thereafter down to the conical section of the cyclone. The gas stream will reverse at the

bottom of the conical part and get out of the cyclone through the vortex finder.

Consequently, the large particles are separated from the vortex while on the contrary the

smaller particles are carried by the gas stream. Afterwards, bag filters are applied to collect

the non separated particles. [11]

1.3.2.2 Process parameters

Immediately after the feed is atomized, the resulting droplets encounter the drying

gas. The temperature of the drying medium at that stage is called inlet temperature. This

process parameter increases the outlet temperature proportionally. Secondly, it has an

enormous influence on the efficiency of the solvent elimination. The more thermal charge

the drying gas obtains, the more solvent can be removed. Subsequently, the final product

shows less sticky properties. The yield also profits from more dry powder due to the fact

they have less tendency to adhere. The air humidity is another process parameter with

influence on the solvent content in the final product. Higher drying gas humidity results in a

final product with a high humidity which on his turn can lead to a decrease in yield because

of the adherence to the glassware. In contrast to the inlet temperature, the humidity of the

inlet air cannot be adapted at any time during the spray drying. [13]

9

The outlet temperature of the drying air is determined by all the other process

parameters. This temperature is measured before entering the cyclone (see Figure 1.3). This

parameter cannot be regulated since it is the resulting temperature of the heat and mass

transfer in the drying cylinder. Furthermore, the outlet temperature plays an important role

in the physical stability of amorphous spray dried particles for the reason that it is normally

higher than the Tg. Beside the physical stability, the outlet temperature also has an impact

on the chemical stability, thermal degradation more specifically. [13]

The fourth parameter is the drying gas flow rate which is defined as the volume of

drying air provided to the spray dry system per unit of time. A higher drying gas flow rate

means more drying energy, resulting in an increased outlet temperature. Moreover, the

residual solvent content is reduced due to the higher drying energy. The cyclone operation

efficiency also benefits from a higher drying gas flow rate. The atomizing air flow also

controls the product properties. First of all, the higher the amount of atomizing gas, the

better the atomization of the liquid into smaller droplets, the smaller the product particles.

Secondly, a higher atomizing air flow means that more cold air has to be heated.

Consequently, the outlet temperature will decrease. [13]

1.3.2.3 Formulation parameters

The first formulation parameter is the feed composition which includes drug,

solvent, polymer and other possible additives. First of all, the selection of an appropriate

solvent is really important. It is possible to achieve a higher yield by using solvents with a

low boiling point. [16] The solid solubility of the components in the solvent in another factor

that should be taken into account. More specifically, the different solubility could result in a

different degree of saturation of the components leading to a heterogeneous instead of

homogeneous amorphous spray dried dispersion. [11] In addition, when an organic solvent

is used, particles with smaller size will be obtained due to the lower surface tension. [16]

If excipients, such as polymers are used, particular attention should be paid to the

drug to polymer ratio. The polymer content in the feed solution has a major influence on

the partial vapour pressure of the solvent. Since polymers molecular interact with the

solvent molecules, the droplet drying will be limited and hence the evaporation rate will be

decreased. [11]

10

The viscosity, caused by high molecular weight polymers, is another solution

property that can be changed by the drug to polymer ratio. The increased viscosity hinders

the solvent transport to the surface during the droplet drying. Beside the drug and

polymers, an additive with a surface activity such as surfactant, may also affect the droplet

drying. After the incorporation of the surfactants at the liquid-air interface, a plasticizing

effect on the surface can be induced, resulting in a possible recrystallization. [11]

The feed concentration has an important impact on the properties of the final

powder. When the solid concentration is rather high, less liquid need to be vaporized which

on his turn results in an increased outlet temperature. Furthermore, higher solid

concentration means that more solid particles are available for particle formation. In view of

the fact that it is easier to separate larger particles, the yield will increase with a higher feed

concentration. [16]

Beside the feed composition and feed concentration, the pump rate (also known as

feed rate) also influences the final product. The spray solution is transported to the nozzle

by the peristaltic pump with this rate. A decrease in outlet temperature arises from a higher

pump rate considering that more solvent has to be evaporated. Consequently, the

difference between inlet and outlet temperature will increase. Furthermore, an increase in

droplet size and hence particle size is observed. [16]

Figure 1.7 summarizes the effects of the most important formulation and process

parameters of the spray drying process on the final product. Regarding to amorphization,

outlet temperature and feed concentration are two most important parameters. An outlet

temperature lower than the boiling point of the solvent results in a final product with a

higher residual solvent level. Consequently, the long-term physical stability will be

decreased. But, this is often solved with the secondary drying of the final product in order to

remove the residual moisture. [11]

11

Figure 1.7 Schematically overview of the influence of increasing process and formulation

parameters on the properties of the final product. (Modified from Buchi Technical

Documents [16])

1.3.2.3 The applicability of spray-drying

The fact that thermolabile components can be easily processed by spray drying is

only one of the several advantages. This one step preparation method is relevant for a wide

range of applications. In addition, process parameters of the spray drying can be easily

controlled when specific final powder properties (e.g. particle size, flow property) are

desired. [26] On the other hand, spray drying requires a lot of energy and the cost of

installation and development of broadly adoptable methods is rather high. [17]

In pharmaceutical technology, spray drying is the preferable method when liquid has

to be removed. Spray drying offers the opportunity to prepare the dry particles with

particular properties. Several parameters may influence the crystalline structure of the

component resulting in material with better plasticity and binding. Because of this, solutions

or suspension containing drug compounds and excipients may be directly tableted after the

spray drying. Direct tableting is advantageous for drug manufacturers from an economical

point of view since no further processing is required when a powder is appropriate for the

direct tableting. [17]

12

The spray drying of active compounds and excipients can also be applied with a view

to improve the aqueous solubility of a drug compound. The increase of surface area

available for the solvent by reducing the particle size is one way to improve the aqueous

solubility. Spray drying is one strategy to create particles with reduced size. In contrast to a

method as milling, the morphology and other particle properties are better under control

with spray drying. It is important to note that the particle properties important for solubility

(e.g. particle size, crystallinity rate) are determined by several process parameters. For this

reason, the reproducibility and scale up of this application may be limited. [17]

Biopharmaceuticals as proteins can be prepared with spray drying. Freeze-drying is

the commonly used method for the production of protein powders for parenteral

administration. Although, this process is not sufficient when fine, flowable powder is

required. Spray drying is a suitable alternative for the manufacturing of parenteral drugs

since this method is able to prepare fine, free flowing powders. Beside the parenteral drugs,

spray drying could also be applied to produce protein powders for administration via the

respiratory tract. Inhalation powders must get deep into the bronchial three. This requires

an appropriate shape and size which can be achieved with the use of spray drying. [17]

Pharmaceutical industry applies the spray dryer for the development of products in a

range from lab to commercial scale. The latter is competent to process an enormous

amount of material per day. Factorial design is a valuable tool for optimizing the process

parameters in such wise that the desired powder properties can be achieved at lager scale.

The evaluation of the four most important parameters (i.e. inlet temperature, feed flow

rate, feed concentration and atomization gas flow rate) in the lab scale development is

required in order to realise a properly scale-up in spray-drying. More specifically, the

influence of these parameters has to be investigated on the following powder properties:

the particle size and the morphology of the final powder, the amount of residual solvent,

the level of crystallinity of the final powder, the yield and stability. In addition, the

optimization of the atomization gas flow and feed rate could also be useful to achieve

powder with uniform particle size on production scale. [16]

13

1.3.3 Freeze-drying

Freeze-drying, also known as lyophilisation, is commonly applied for the drying and

the improvement of the long term storage stability of several pharmaceuticals. The

removed solvent is most often water, but can also be organic. This preparation method

consumes a lot of time and energy. [18]

The freeze-drying process consists of three steps: freezing (crystallization of water),

primary drying (ice sublimation) and secondary drying (desorption of unfrozen water). The

drug-excipient solution is frozen whereby ice crystals of water are formed. The longer the

freezing process continues, the more water is transformed to ice, the higher the

concentration of the drug and the possible excipients the remaining liquid. The high

concentrated suspension will cause on his turn an enhancement in the viscosity. A (partially)

amorphous phase is formed since the freezing process is fast and further crystallization is

prevented by the increased viscosity. At the end of this stage, there is still a small

percentage of unfrozen water in the liquid state. This water is called bound water. [18]

During the primary drying step, the ice sublimes from the frozen product.

Sublimation is the phase transition of a substance directly from the solid to the gas phase,

without passing through the liquid phase. The primary drying step occurs under conditions

where the chamber pressure is below the ice vapour pressure. This can be explained by the

fact that the water doesn’t have to evaporate. Heat needs to be transferred to the system

to provide the heat that is removed by the sublimation of the ice crystals. When the water

vapour is removed from the product, a porous product is formed (i.e. heat of sublimation).

The aim of the secondary drying is the evaporation of the absorbed water from the product

under high temperature and low chamber pressure. This water did not form ice crystals in

the freezing step and consequently was not sublimed in the previous step. [18]

1.3.4 Milling

Milling or grinding, is a process generally used in pharmaceutical industry for particle

size reduction. In addition, milling is also used for the preparation of amorphous material.

The amorphization mechanism behind this preparation method is the mechanically breaking

of the crystalline structure as a result of high-energy ball milling. [10]

14

The mechanical milling performed at cryogenic temperature (well below the glass

transition temperature) is well-known as cryo-milling. During cryo-milling, the cryogenic

conditions are ensured by e.g. immersing the milling chambers after specific time periods in

liquid nitrogen. This method is more efficient for the preparation of amorphous material

due to the fact that recrystallization can be prevented at lower temperatures. In addition,

the low temperature compensates for the released heat, produced by the kinetic energy.

[10]

The temperature of the milling plays an important role in the formulation of

amorphous material. It is shown that amorphization tendency increases when the milling is

performed at milling temperatures below the glass transition temperature. In the case of

temperature above the glass transition temperature, polymorphic changes may occur. [19]

In general, milling can be seen as a green chemistry approach because it doesn’t require any

solvent. For this reason, milling is the preferable method compared to the solution based

preparation methods such as precipitation from the solution. [20] On the other hand, the

milling process consumes a large amount of energy. [10]

1.4 CHARACTERIZATION OF AMORPHOUS MATERIALS

Amorphous materials can be characterised by several analyse techniques. These

methods provide more information about the structure, thermodynamic properties,

changes (e.g. recrystallization) and molecular interactions. [6]

1.4.1 Differential scanning calorimetry (DSC)

Differential scanning calorimetry is the preferable thermo analytical method to

obtain more information about the behaviour of a system as a function of temperature. DSC

serves as a quantitative detection method for processes in which energy is produced

(exothermic) or required (endothermic). In the measurement there are two kinds of

samples: a reference and a test sample. Both samples are heated in such manner that the

difference in temperature between the two samples remains zero (or the temperature

difference when heating both in a similar manner is measured). [1]

15

In the case of an endothermic (energy requiring) process, the system provides

additional energy to the test sample to compensate for the lost in energy. In this way, the

rise in temperature of the test and reference sample is still equivalent. The energy of the

phase transition is then quantified based on the required additional energy. DSC is useful for

the detection of amorphous material since the lack of a melting peak in the DSC could be an

indication of the presence of an amorphous form. [1] Figure 1.4 illustrates the different

processes that can be detected with DSC: glass transition temperature (Tg) of the

amorphous material, crystallization temperature (Tc) at the recrystallization temperature of

the amorphous material and melting temperature (Tm) of the recrystallized material.

Fig. 1.4 The different processes analysed by DSC: endothermic melting peak (Tm),

endothermic glass transition (Tg) and exothermic crystallization (Tc). (Modified from

Gibson et al. [21])

1.4.2 X-ray powder diffraction (XRPD)

The difference in three-dimensional order between amorphous and crystalline

formulation is detected in X-ray powder diffraction (XRPD). A crystalline material is

composed of atoms which are arranged in a periodic pattern i.e. crystal lattice. The crystal

planes in the crystal lattice will constructively diffract the x-rays resulting in a specific

diffraction pattern consisting of well-defined peaks. [6] Since this diffraction pattern is

unique for one component, it is possible to make a distinction between the crystalline

character of the drug and the carrier in one mixture. On the other hand, amorphous

16

material is not characterised by the periodic pattern with long-range order. So, amorphous

material will result in a broad halo peak instead of the well-defined peaks of a crystalline

material. [1] The different between the diffraction pattern of an amorphous and crystalline

material is illustrated in Figure 1.5.

Fig. 1.5 X-ray powder diffraction pattern for amorphous (bottom) and crystalline (top)

lactose. (Modified from Hancock et al. [6])

1.4.3 Infrared spectroscopy (IR)

Infrared (IR) spectroscopy is a technique used to measure the absorption of IR

radiation after the IR beam passed through the sample. The resulting IR spectrum is a

unique fingerprint representing absorption peaks. The peaks are corresponding to the

frequencies of the bond vibration of the atoms in the material. [22]

IR is the appropriate technique to obtain information about intermolecular

interaction between functional groups of the different components in amorphous and co-

amorphous mixture. It is possible to detect changes in the solid state in the molecular

arrangement. More specifically, interactions such as hydrogen bonding and

interactions can be detected. [23] The conversion of the crystalline to the amorphous form

results in different molecular structure. Therefore, the peaks of amorphous material are

broader compared to the corresponding peaks of the crystalline form. [24] Moreover,

molecular interactions are reflected in peak shifts in the vibrations of the interacting

functional group. [23]

17

1.5 STABILISATION OF THE AMORPHOUS STATE

The amorphous form is characterised by a higher internal energy compared to their

crystalline counterparts. Consequently, the amorphous state could convert back to the

lower energy crystalline state during processing, storage or dissolution. Two approaches to

stabilize the amorphous form are discussed in the following chapter. [10]

1.5.1 Solid dispersion

1.5.1.1 Definition and different generations

Solid dispersion can be defined as a solid product containing one or more active

hydrophobic compounds dispersed in a hydrophilic carrier. [7] Solid dispersions are

classified into three different generations. The first generation solid dispersion contains

crystalline carriers such as urea and sugars. The fast dissolution properties of the carrier

generate the release of fine crystals or particles of the drug. However, the first generation

results in a crystalline solid dispersion, rather than an amorphous formulation. Seeing that

crystalline formulations are more thermodynamically stable in contrast to the amorphous

formulations, the drug release is not comparatively to the release from the amorphous

formulation. To improve the effectiveness of the first generation solid dispersion, a second

generation arises, consisting amorphous carriers. [3]

The second generation is comprised of the irregular dispersion of drug particles

within an amorphous carrier. In general, polymers are used as the amorphous carriers. The

polymer can be fully synthetic (e.g. polyethyleneglycols). There are also natural product-

based polymers principally comprising of cellulose derivates (e.g.

hydroxypropylmethylcellulose) or rather starch derivates. The use of the water soluble

carrier material in the second generation results in a drug particle size reduction, and hence

improved wettability and solubility of the drug. It is important to note that de drug release

profile is influenced by the dissolution properties of the carrier material. [3]

The second generation is divided into three different forms. A system containing a

drug and carrier with a complete miscibility and solubility is called an amorphous solid

solution. The polymers create an amorphous system wherein the crystalline drug particles

can dissolve. The solid solution consists of only one phase due to the homogeneity on the

18

molecular level created by the complete miscibility and solubility. In the case of a drug with

a limited carrier solubility or very high melting point, the system is called amorphous solid

suspension. [3] This formulation is composed of two phases attributable to the lack of a

homogenous molecular structure. The dispersion of the drug particles in the polymer

contribute to the formulation of an amorphous product. A mixture of amorphous solid

solution and amorphous solid suspension is obtained when a drug is both together dissolved

and suspended in the carrier. This heterogeneous structure is characterised by the

properties of the two kinds of solid dispersions. It should be taken into account that the

drug in the amorphous solid dispersion is present in the supersaturated state. Hence, there

is a risk that a phase separation and recrystallization of the drug will occur. It is important to

mention that this has a big implication on the physical stabilization. [3]

The third generation make use of carriers with surface activity or self-emulsifying

properties. The surfactant carrier can be present alone or rather in combination with an

amorphous polymer. There is a growing interest in this generation since they can

accomplish the highest degree of bioavailability for poorly water soluble drugs. [3]

1.5.1.2 Advantages and disadvantages of solid dispersions

The important benefit of solid dispersion is an enhanced physical stability of the

amorphous formulation by the incorporation of the drugs into hydrophilic polymers. The

stability of the amorphous state depends mostly on the molecular mobility of the drug in

view of the fact that drug molecules have to rearrange in order to form the crystalline

structure. When the viscosity is extremely high, below the Tg, there is still some mobility

that can possibly give rise to a change towards the more stable crystalline state. The

characteristic high Tg of the polymer results in a higher Tg of the system in comparison to the

Tg of the pure amorphous drug. Consequently the mobility of the drug in the polymer matrix

is decreased. Specific interactions between the functional groups of the drug and the

polymer can be another explanation for the enhanced physical stability. It is important to

note that the polymer has to be completely miscible with the drug to obtain an effective

stabilisation. [3,25]

19

The enhancement in stability is also attributable to the use of surfactants in the third

generation. This can be explained by the following mechanism: the enhanced wettability of

the drug caused by the decreased interfacial energy barrier between drug and dissolution

medium, result in a better dissolution rate of the drug. Beside the solubility in the medium,

the surfactant provides also a better solubility of the drug in the polymer matrix. [25]

Moreover, the surfactants improve the physical and chemical stability of the drug. The

recrystallization can be prevented by the amphiphilic structure of the surfactant which

increases the miscibility of the drug into the polymer. Secondly, the surfactants can absorb

at the surface of the particles or create micelles to enhance the wettability of the drug. In

this way, the crystallization from the supersaturation state can be prevented. [26]

Furthermore, solid dispersion is a promising strategy to improve the oral

bioavailability of poorly water soluble drugs due to several advantages. Firstly, solid

dispersions create particles with reduced particle size. The drug particles generate a high

surface area gaining an enhanced dissolution rate and hence bioavailability. [3] The

dissolution rate also benefits from the enhanced drug wettability caused by the carrier.

Additionally, the carriers may improve the dissolution more by direct dissolution or co-

solvent effect. Thirdly, it is seen that particles in solid dispersion show a higher degree of

porosity. The presence of porosity is more likely if the solid dispersion is prepared by the

solvent method in which the fast solvent removal causes some holes in the structure.

Moreover, solid dispersion may result in amorphous drugs (see 1.2.3). [3,26]

Despite the fact that solid dispersions have been investigated intensively for a long

time, only a few solid dispersions made it to the market. This can be mainly explained by the

high hygroscopicity of the polymers causing a reduction in the Tg of the glass solution,

occasioned by water. The water can be seen as a plasticiser whereby the molecular mobility

in the glass will increase. Accordingly, there is a major probability that the drug will

recrystallize. Therefore, particular attention should be paid to the temperature and the

moisture of the storage conditions. Another drawback arises from the use of a drug and

polymer with a limited miscibility. A larger amount of polymer is required to ensure

miscibility, resulting in large bulk volumes of the final drug formulation. When they are only

partially miscible, phase separation may occur at some stage (e.g. during storage) which will

lead to recrystallization. [8]

20

1.5.1.3 Preparation methods

In general, every preparation method of amorphous material is suitable for the

preparation of solid dispersion. Although, melting and solvent evaporation are the two

major methods for the reason that they are suitable for large-scale (i.e. industrial). [26] The

general concepts about melting method are already discussed in 1.3.1.

In the solvent evaporation method, both drug and carrier are dissolved in a common,

vaporous solvent such as organic solvents (i.e. methanol, ethanol) or water. The solvent can

be subsequently eliminated by any one of a number of methods e.g. spray, freeze, spray

freeze, oven or vacuum drying, rotary evaporation , heating on a hot plate, supercritical

anti-solvent, fluid-bed coating, ultra-rapid freezing, electrostatic spinning and co-

precipitation. Similar to the melting method, the physical state of the drugs in the final solid

dispersion depends on how fast the solidification occurs. The higher the solidification rate,

the higher the chance that the final product appears in the amorphous state. [3]

The solvent method surmounts some limitations associated with the melting

method. In contrast to the melting method, the drug and polymer don’t have to be exposed

to high temperature when a technique such as freeze drying is applied. This makes it

possible to use polymers with a high melting point (e.g. PVP) or produce solid dispersion of

thermolabile compounds. Another explanation is the fact that organic solvents have a low

evaporation temperature. Nevertheless, the disadvantage of the organic solvent is the

possible toxicity of the final product since it is impossible to remove all the solvent resulting

in residual solvent. Furthermore, the use of organic solvents is associated with

environmental issues. The residual solvent in general can act as a plasticizer by increasing

the Tg. Consequently, the resulting enhanced mobility of the compounds can cause phase

separation. [3,26]

1.5.2 Co-amorphous formulation

Solid dispersion as a stabilisation method of the amorphous form offers several

drawbacks due to the use of polymers (i.e. hygroscopicity and bad miscibility). To overcome

these limitations, the concept of co-amorphous drug systems was introduced as an

alternative way to stabilize amorphous formulations. [5]

21

1.5.2.1 Co-amorphous drug-drug formulations

Binary amorphous drug formulation is another interesting approach to overcome the

limitations of amorphous formulations. Yamamura et al. showed that cimetidine in a binary

system with either naproxen, indomethacin or diflunisal can be converted into its

amorphous counterpart upon precipitation from an ethanol solution. [20] Later, Chieng et

al. (2009) introduced the concept of co-amorphous mixtures in which two pharmacologically

suitable drugs were combined in one co-administration. [24] This new delivery system has

already been applied for several drug combinations. Chieng et al. were able to prepare a

stable amorphous binary mixture of indomethacin and ranitidine, prepared by vibrational

ball milling. [27]

The co-administration offers the opportunity to combine pharmacologically

complementary drugs e.g. naxproxen and indomethacin, both NSAID. [24] The co-

amorphous mixture of simvastatin and glipizide is another pharmacologically relevant

combination to treat metabolic disorders. [28] On the other hand, the co-administration can

reduce the side effects caused by one drug. For example, the combination of cimetidine and

naproxen is reasonable because cimetidine can treat gastro-intestinal disorders induced by

the use of naproxen (NSAID). Furthermore, this therapeutic advantage benefits on his turn

the patient compliance. [29]

Previous studies showed that 1:1 intermolecular interactions are responsible for the

improved physical stability. [29] This can be explained by the several steps before the

recrystallization can occur. First the interactions need to be broken, secondly the molecules

have to rearrange and thereafter they have to meet each other to form the crystal

structure. [24] The interactions between the functional groups of the drugs in the mixture

may also be the primary reason for an improved dissolution rate of the drugs in the co-

amorphous mixture in comparison to the individual drug. [20] Moreover, the drug release

could occur synchronous which can be explained by the rise of a heterodimer consisting

both drugs. The latter was only observed with co-amorphous indomethacin/naproxen

system. [29]

22

1.5.2.2 Co-amorphous drug- amino acid formulations

The binary amorphous drug formulations described above are only applied for two

drugs which make up a pharmacologically relevant pair and are prescribed in similar doses.

In order to make more general use of this promising strategy for a single drug, Löbmann et

al. (2013) introduced the co-amorphous drug-amino acid formulations. In this concept,

amino acids are used to produce highly stable co-amorphous mixture with improved

dissolution properties. Amino acids are part of the daily nutrition which makes them

practically safe. [8,23]

The selection of the amino acids is based on the binding site at the biological target

site of the drug. It is well-known that a pharmacological effect is induced after a strong

interaction between the drug and the receptor amino acids at the active site in the body.

For this reason it is hypothesized that the functional groups of the receptor amino acids and

drug may also interact in a solid state co-amorphous mixture. These drug–amino acid

interactions may possibly result in a stabilisation of the amorphous drug in the mixture

which on his turn prevents the recrystallization. [8]

Recently, Löbmann et al. extensively studied the preparation of co-amorphous drug

systems with amino acids (AA) as co-amorphous stabilizers. Two poorly water soluble drugs

carbamazepine (CBZ) and indomethacin (IND) were combined with receptor amino acids

from the biological target site of the drug with a view to generate co-amorphous drug-AA

systems. [8,23] Both systems showed a single Tg indicating for the formulation of a

homogenous molecular mixture. Furthermore, the Tg was found to be significantly higher in

comparison with the individual drugs, resulting in an improved stability of the mixtures. The

molecular interactions between CBZ/IND and the amino acids arginine (ARG), phenylalanine

(PHE) and tryptophan (TRYP) were investigated and indentified with the use of FTIR. In was

concluded that at least one AA from the biological target site of the drug was required to

generate specific interactions such as hydrogen bonding. It could be stated that amino acids

can be seen as good co-amorphous drug partners. [23]

23

1.6 DESCRIPTION OF THE STUDIED DRUGS

1.6.1 Simvastin (SVS)

Simvastatin is a cholesterol-lowering agent with an inhibitory effect on HMG-CoA

reductase. HMG-CoA reductase is the catalyst of the conversion of HMG-CoA to

mevalonate. This early and rate-limiting step in the biosynthesis of cholesterol is inhibited

by SVS. Therefore SVS is mainly used to treat hypercholesterolemia. [4] Simvastatin is

classified among the statin drug family and is practically insoluble in water

(solubility at and p - . - g - . This inactive lacton is hydrolyzed after oral

digestion to the analogous β-hydroxyacid form. The β-hydroxyacid form is the active

metabolite with the inhibitory effect on HMG-CoA reductase. [4,30]

Simvastatin is a white to off-white, crystalline and non-hygroscopic powder

characterised by relative low glass transition temperature (29.0 ± 0.6°C) and a melting point

of 139.9 ±0.2°C. [4] The production of a stable amorphous product by SD of a component

with a Tg is a challenge due to the fact that the outlet temperature could rise above the Tg. In

this case, the powder exists in the supercooled rubbery state instead of the glassy state and

is rather sticky instead of free flowing. [9] Simvastatin has a sufficient absorption from the

gastro-intestinal tract. Hence, it is essential to find a strategy to improve the solubility,

dissolution rate and thus the bioavailability of the oral dosage of SVS. [4]

1.6.2 Glibenclamide (GBC)

Glibenclamide is a drug used to treat non-insulin dependent diabetes mellitus

(NIDDM). NIDDM is a chronic metabolic disorder where high glucose blood levels are

observed as a result of insulin deficiency. Glibenclamide, an oral long-acting hypoglycemic

agent, causes a cell membrane depolarization in the pancreatic beta cells by inhibiting the

ATP-sensitive potassium channels. Subsequently, voltage-dependent calcium channels open

as a result of the cell membrane depolarization. This increases the intracellular calcium

concentration in the beta cell, which stimulates on his turn the insulin release. [31] The

antidiabetic drug is a weak acid (pKa = 6.3) and is as good as insoluble in water (38 µmol -

at 37°C) and acid solutions. It has a Tg of 71.9 ± 0.7°C and a melting point at 174.2 ± 0.2° C.

[31,32] Reference is made to Appendix A for the chemical structures of GBC and SVS.

24

2. OBJECTIVES OF THE STUDY

The conversion of crystalline material into the amorphous form is one approach to

enhance the dissolution rate of poorly water soluble drugs. However, the

thermodynamically unstable character of amorphous material could induce recrystallization

during manufacturing and storage. Solid dispersion is one strategy to overcome the poor

physical stability of amorphous material. Several drawbacks arise from the polymers used in

this strategy i.e. the hygroscopicity of the polymer and bad miscibility. [26]

Recently, a promising alternative concept is introduced in which body receptor

amino acids are used as stabilisers resulting in a co-amorphous mixture. However, so far the

co-amorphous formulations have been prepared by quench-cooling and co-milling methods.

There is a clear need for an alternative way to prepare the co-amorphous mixtures, on the

grounds that melting and milling method are not easily up-scalable production methods for

amorphous formulations. [8]

The general aim of this study was to test the spray drying as a production method for

co-amorphous drug-amino acid mixtures containing poorly water soluble drugs simvastatin

(SVS) and glibenclamide (GBC). The combination of SVS and GBC with lysine (LYS) and serine

(SER) respectively, was based on preliminary studies in which it was found that both drugs

form co-amorphous mixture (1:1) with their corresponding amino acid. [33]

Since both drugs are poorly water soluble, an appropriate solubilizer is required in

order to dissolve the drugs to water and to perform the spray drying from an aqueous

solution. Aqueous solution is preferred over organic solutions due to toxicity and

environmental issues associated to organic solvents. In the first place, a solubility test was

performed to find a suitable solubilizer for the individual drugs. The addition of the

solubilizer is the main reason why the concept of solid dispersion became relevant in this

study since the tested solubilizers were polymers and surfactants. The aim was to find an

effective solubilizer in order to minimize the content of the solubilizer in the mixture.

25

After finding a suitable solubilizer, the aim was to prepare co-amorphous drug-amino

acid-solubilizer mixtures followed by characterisation of the final products. The level of

crystallinity in the sample was investigated by x-ray powder diffraction (XRPD). In addition,

Differential scanning calorimetry (DSC) was applied to investigate the thermal properties of

the samples. Fourier-Transform Infrared spectroscopy (FTIR) provided information about

possible interactions between the different components in the mixtures. These techniques

are discussed in more detail in the theoretical part of this thesis (see 1.4). Beside the

physical characterisation, the dissolution and stability properties of the prepared samples

were also investigated. The physical stability of four different co-amorphous drug-amino

acid mixtures was investigated under three different storage conditions: 4°C/0% relative

humidity, 40°C/0% relative humidity and 25°C/60% relative humidity.

26

3. MATERIALS AND METHODS

3.1 MATERIALS

Simvastatin (SVS), glibenclamide (GBC) and the amino acids lysine (LYS) and serine

(SER) were supplied by Hangzou Dayangchem Co., Ltd (Hangzou, China). The solubilizers

polysorbate 20 (Tween 20), polyvinylpyrrolidone K-30 (PVP), and Pluronic F-68 were

obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Sodiumlaurylsulfate

(SLS) and Soluplus were provided by YA-Kemia Oy (Helsinki, Finland) and BASF SE

(Ludwigshafen, Germany) respectively. SVS, LYS and SER were stored at 4°C while GBC and

the solubilizers were stored at room temperature. The compounds were used as received.

KH2PO4 was purchased from Merck KGaA (Darmstadt, Germany) and NaOH from Oy FF-

Chemicals ab. (Haukipudas, Finland). Trifluroacetic acid (TFA) and acetonitrile (ACN) were

provided by Sigma-Aldrich.Co. (St. Louis, MO) and VWR international S.A.S. (Fontenay-Sous

Bois, France), respectively.

3.2 METHODS

3.2.1 Solubility test

The solubility of GBC and SVS in water in combination with 5 different solubilizers

was measured. Two polymers were used (i.e. PVP and Soluplus) and three surfactants (i.e.

Pluronic, SLS and Tween20).

First, 100 mL of 5% (m/v) stock solution was prepared of every solubilizer. Three

dilutions (2%, 1% and 0.5%) were prepared, starting from the 5% stock solution. Thereafter,

an excess of GBC/SVS was added to 10 mL of every solution. This was done in triplicate for

every solution. In total, 63 samples were obtained after preparing also 3 blanco samples (i.e.

10 mL water and an excess of GBC/SVS). The samples were put on a shaker at room

temperature for three days. In the case that the drug powder dissolved, more GBC/SVS was

added. After three days, the solutions were filtrated through a 0.20 µm pore size membrane

filter and analysed with HPLC as described in 3.2.6. The obtained solubilities (µg/mL) with

different concentrations of one solubilizer were compared by performing single-factor

ANOVA analysis (Microsoft Excel, Analysis toolpak). The values were conceded statistical

significant when the p-values were smaller than 0.05 (95% confidence level).

27

The solubility of both drugs in water was also defined with solubilizer and ethanol in

order to investigate the influence of ethanol on the solubility. It is well known that both

drugs show a better solubility in ethanol compared to water. In this way, ethanol could

possibly reduce the amount of solubilizer. The solubility of SVS was determined in water

with 20% ethanol in combination with 1% and 2.5% SLS, 2.5% and 5% Soluplus. The

solubility of GBC was determined in water with 10% ethanol and 2% SLS. The maximum

amount of 20% ethanol was applied since this is the highest concentration that can be safely

used in the spray dryer device used in this study.

3.2.2 Preparation of the materials

3.2.2.1 Spray drying process

The drug:amino acid molar ratio used is this study was 1:1. Thus to prepare a 500 mg

batch, 370.60 mg SVS and 129.40 mg LYS, and 412.29 mg GBC and 87.70 mg SER were

weighed. The selection of solubilizer and the amount needed to dissolve the drug was

based on the solubility study. The solubilizer was dissolved in water and subsequently the

drug and the amino acid was added to the solution. The solution was mixed until the

powders were dissolved. The prepared formulations are shown in Table 4.4.

The amorphous formulations were prepared with a Büchi mini spray dryer (Model B-

191, Büchi Labortechnik AG, Flawil, Switzerland). The spray drying process was performed

under the following conditions: inlet temperature of 120°C and 110°C for GBC and SVS

respectively, outlet temperature in a range of 40-60 °C, aspirator rate of 50 %, pump setting

of 20% and a flow rate of 600 L/h. After the initial runs, the spray drying process of SVS-LYS-

5% SLS mixture with an inlet temperature of 100°C and 150°C was also investigated.

3.2.2.2 Cryo-milling process

For one formulation (see Table 4.4), amorphous SVS-LYS was prepared by cryo-

milling (CM) before mixing with surfactant. The two milling chambers were filled with 500

mg SVS-LYS mixture (molar ratio of 1:1) and two 15 mm stainless steel balls. Milling was

performed at 30 Hz in an oscillatory ball mill (Mixer Mill MM 400, Retch GmbH & Co., Haan,

Germany). The total milling time was 60 minutes. Prior to milling and after every 10

minutes, the milling chambers were immersed in liquid nitrogen for 2 minutes to ensure the

28

cryogenic conditions. When the milling process was finished, the milling chambers were

placed in a desiccator over silica. After they reached the room temperature, the product

was weighed and the correct amount of surfactant was added.

3.3.2.3. Physical mixtures

Physical mixtures (PMs) of the unprocessed starting materials (see Table 4.4) were

prepared by mixing by pestle in a mortar.

3.2.3 Physical characterisation of the materials

3.2.3.1 Differential scanning calorimetry (DSC)

The DSC analysis was performed using Mettler Toledo DSC823e (Schwerzenbach,

Switzerland) attached with a Julabo FT 900 Cooler. The DSC thermograms of the spray dried

samples were obtained under a nitrogen gas flow of 50 mL/min. Aluminium pans (40 µl)

were filled with sample powder (4-8 mg) and heated with rate of 10 K/minute from

temperature -50°C to 220°C. STARe software was used to determine the glass transition

temperatures (Tg, midpoint), the melting temperature (Tm, onset) and possible

recrystallization temperature (Trc, onset) of the samples. They were calculated as the mean

of three independent measurements.

It is important to note that the settings applied for the DSC analysis of SLS were

different (i.e. quench cooling). First, SLS was heated from 25°C above its melting

temperature at 10K/min where it was held for 5 minutes. Afterwards, the melt was

immediately cooled to -50°C where it was held for 15 minutes. Thereafter, it was heated

again from -50°C till the melting point.

3.2.3.2 X-ray powder diffraction (XRPD)

XRPD was performed using Bruker D8 Discover X-ray Diffractometer (Karlsruhe,

Germany) applying Cu Kα radiation (λ= 1.54 Å). The scanning of the samples was carried out

in reflection mode between 5° and 35° 2θ with a step size of 0.011° and scan speed of 0.1°

2θ/s. An acceleration voltage of 40 kV and current of 40 mA were used. The data were

collected by DIFFRAC.V3 program.

29

3.2.3.3 Fourier-Transform infrared spectroscopy (FTIR)

The infrared spectroscopy was performed with a FTIR thermo Nicolet nexus 8700 FT-

IR (Thermo Scientific, Madison, USA). ATR (Attenuated Total Reflectance) Thermo Scientific

OMNIC software was applied to collect the samples. The spectra were recorded in a wave

number range of 550 to 4000 cm-1 with a resolution of 4 cm-1.

3.2.4 Dissolution test

The dissolution test was performed using Distek dissolution system 2100 C (North

Brunswick NJ, USA) with a paddle rotation speed of 50 rpm. The bath-based dissolution

system was equipped with an outer water bath to maintain the constant temperature of

37°C. During one dissolution test, two different formulations were measured in triplicate. A

powder amount equivalent to 20 mg of the drug was place on the bottom of the dissolution

chamber. The chamber was subsequently filled with 500 ml of the preheated dissolution

medium. The dissolution medium was a phosphate buffer with pH 7.2 (conform the United

States Pharmacopeia).

The dissolution profiles were measured over a period of 24 hours. At predetermined

intervals (2, 4, 6, 10, 20, 30, 40, 60, 90, 120, 240, 360, 480 and 1440 minutes) 5 mL aliquots

were taken from the chamber and immediately replaced with the same volume of

phosphate buffer solution (pH 7.2). After filtering the 5 mL samples through a 0.20 µm

membrane filter, the filtrates were analysed by the HPLC as described in 3.2.6. The single-

factor ANOVA analysis was performed with a view to investigate if the dissolution profiles of

two formulations were statistical different (95% confidence level).

3.2.5 Stability study

The formulations selected for stability study were stored at room temperature

(25°C)/60% relative humidity (RH), 4°C/0% RH and 40°C/0% RH. Phosphorous pentoxide was

used to achieve the RH of 0% while the RH of 60% was obtained with a saturated NaBr

solution. Periodically, the samples were analysed with XRPD and FTIR until the onset of

recrystallization was seen.

30

3.2.6 HPLC analysis

The quantitative determination of the drugs was performed by Gilson HPLC analysis

system with UV-VIS 151 detector (Gilson, USA), 234 auto-injector (Gilson, France), 321 pump

(Gilson, France), system interface module (USA) and Unipoint TM LC system version 3.01

software (USA). A Phenomenex Gemini-NX 5 µm C18 110 Å (250 x 4.6 mm) column and

SecurityGuard precolumn were used.

The drug concentrations were determined using a mobile phase consisting of 70%

acetonitrile (ACN), 30% ultrapure water and 0.1% trifluoroacetic acid (TFA). The flow rate

was 1.2 mL/min, there was an ambient column temperature and the detection wavelength

for GBC and SVS was 222 nm and 238 nm respectively. Standard solutions with

concentrations of 0.5, 1, 5, 10, 25, 50 and 100 µg/mL of the drug were prepared in 70/30

ACN/ultrapure water. It is important to mention that the filtered samples were diluted with

70/30 ACN/ultrapure water if the concentration in the sample was exceeding the

concentration SVS and GBC in the 100 µg/mL standard line solution.

31

4. RESULTS

4.1 SOLUBILITY TEST

The solubilities of GBC in water at different concentrations of the solubilizers are

demonstrated in Table 4.1. In comparison to the GBC concentration in the blanco samples,

the influence of Pluronic and PVP on the solubility was negligible. Moreover the results of

different concentrations of these solubilizers were not statistically significantly different

(p>0.05). When Soluplus/Tween20 was used as a solubilizer, higher concentration levels of

GBC were seen in comparison to Pluronic/PVP. Nevertheless, the defined concentrations

were still really low. The concentration GBC obtained with only 0.5% SLS was found to be

higher than the concentration levels reached with the use of 5% of every other solubilizer.

Thus SLS was the only possible option for the spray-dying studies.

Table 4.1

The average concentration of GBC (µg/mL) in water in combination with different

percentages of solubilizer a

Solubilizer

Soluplusb

Tween 20b

SLSb

Pluronic

PVP

0.5 %

2.53 ± 1.27

2.58 ± 0.88

21.38 ± 1.48

3.11 ± 0.83

1.24 ± 0.20

1%

5.19 ± 0.21 3.56 ± 0.48 51.91 ± 6.94 2.48 ± 0.08 1.06 ± 0.12

2%

8.71 ± 0.10 5.65 ± 0.10 118.19 ± 5.76 2.91 ± 0.57 1.01 ± 0.16

5% 14.32 ± 4.30 11.79 ± 0.38 275.04 ± 14.34 2.21 ± 0.39 1.00 ± 0.10

a

The average concentration of GBC in blanco solution is 0.96 ± 0.16 µg/mL b

The solubilities (µg/mL) with different concentrations of one solublizer were found to be statistical significant (calculated p-values < 0.05)

The concentrations of SVS in water in combination with solubilizer in different

concentrations are shown in Table 4.2. Again, the influence of Pluronic and PVP is negligible

after comparison with the blanco samples. Moreover, it was seen that the concentration

SVS with 5% Soluplus was similar to the concentration SVS with 0.5% Tween20. The same

phenomenon was observed with 5% Tween and 0.5% SLS. Conclusively, the solubility of SVS

in water could reach the highest levels with SLS as solubilizer. The concentration SVS in 0.5%

32

and 5% SLS was approximately 100 times higher than the corresponding concentration GBC

in 0.5% and 5% SLS.

Table 4.2

The average concentration of SVS (µg/mL) in water in combination with different

percentages of solubilizer a

Solubilizer

Soluplusb

Tween 20b

SLSb

Pluronicb

PVPb

0.5 %

27.28 ± 2.49

221.35 ± 33.56

2102.27 ± 570.16

2.40 ± 0.39

2.36 ± 0.24

1%

52.99 ± 5.55 446.87 ± 113.60 5179.33 ± 2051.30 2.37 ± 0.45 1.99 ± 0.22

2%

111.89 ± 6.42 779.35 ± 3.02 13599.06 ± 1350.70 5.39 ± 0.87 3.93 ± 1.10

5% 259.90 ± 33.83 2198.04 ± 640.57 24337.54 ± 863.18 5.57 ± 1.16 4.52 ± 0.83

a

The average concentration of SVS in blanco solution is 1.74 ± 0.19 µg/mL b

The solubilities (µg/mL) with different concentrations of one solublizer were found to be statistical significant

(calculated p-value < 0.05)

In order to analyse the influence of ethanol on the solubility of both drugs, the same

solubility test was done with the addition of ethanol beside the appropriate amount of

solubilizer. It was seen that 2.5% SLS-20% ethanol did not enhance the solubility of SVS

compared to SLS used alone. The concentration SVS with 2.5% Soluplus-20% ethanol was

found to be almost 5 times higher than the concentration of SVS with 2% Soluplus. In the

case of 5% soluplus-20% ethanol, the concentrations were approximately twice the

concentration of SVS 5% Soluplus. Nevertheless, the determined results were still negligible

compared to the best solubilzer (SLS). No enhancement in the solubility of GBC was

observed with 2% SLS-10% ethanol in comparison to the values determined for GBC with 2%

SLS. The exact values are shown in Table 4.3.

In consideration of the defined values discussed above, it was decided to continue

with SLS as solubilizer for SVS and GBC with a view to prepare the aqueous solution required

for the spray drying (see Appendix A for the chemical structure of SLS). Ethanol was not

added to the solution since the concentration of both drugs was not remarkable higher after

the addition of ethanol.

33

Table 4.3

The average concentration of SVS (µg/mL) in water in combination with different

percentages of solubiliser in combination with 20% ethanol

Solubilizer

SLS + 20% ethanol a

Soluplus+ 20% ethanol

1 % 2.5% 5%

5076.75 ± 1767.95

11237.82 ± 2524.58

N/A

N/A

508.61 ± 113.00

581.43 ± 42.48

a The solubilities (µg/mL) with different concentrations of one solublizer were found to be statistical significant

(calculated p-value < 0.05)

4.2 PREPARATION OF AMORPHOUS MATERIAL BY SPRAY DRYING

Table 4.4 summarizes all the prepared formulations in combination with the exact

amount of every component, the calculated weight ratios of the components in the final

product, the conditions of the spray drying and the corresponding yield. The different

components in the PM and the CM mixture were exactly the same as in the spray-dried

mixtures with a view to make a correct comparison. The results are described in the

following chapter.

4.2.1 Spray drying of glibenclamide (GBC)

The first drug-amino acid mixture (GBC-SER 1:1) was spray-dried from 5% SLS

aqueous solution. It is important to mention that not the final product but the spray-drying

solution contains 5% SLS. Considering the results of the solubility test, 5% SLS was needed to

dissolve 275 µg/ml of GBC. As calculated above, to prepare 500 mg GBC-SER 1:1, 412.29 mg

GBC and 87.70 mg SER was weighed. This means that 1500 mL of 5% SLS solution was

needed to dissolve 412.29 mg GBC. Despite the solubility study result, the amount of drug

and SLS was not dissolving properly in 1500 mL of water. Since GBC obtains acid properties,

the solubility should benefit from a basic environment. Therefore, 1M NaOH was added

until a pH of 10 was achieved. After the aqueous solution was obtained, 150 mL of GBC-SER-

5% SLS was spray dried.

34

4.2.2 Spray drying of simvastatin (SVS)

Three drug-amino acid mixtures including SVS-LYS 1:1 spray-dried from 0.5%, 5% SLS

and 5% Tween20 solutions were prepared. To prepare 500 mg SVS-LYS 1:1, 370.60 mg SVS

and 129.40 mg LYS was weighed. The required volume of solubilizer needed to dissolve

370.60 mg SVS was based on the solubility results. The volumes of 0.5% SLS solution, 5% SLS

solution and 5% Tween20 solution were found to be 180 mL, 16 ml and 190 mL respectively.

To be sure that SVS was dissolved properly, 20 mL of 5% SLS solution, 200 ml of 0.5% SLS

solution and 200 mL of 5% Tween solution were used.

When all the components were completely dissolved, the three aqueous solutions

were spray dried for the first time under the following conditions: inlet temperature of

110°C, outlet temperature of 50°C, aspiration rate of 100% and pump rate of 18-20%. The

final powder of SVS-LYS-5% SLS and SVS-LYS-0.5% SLS showed sticky properties and the

yields were rather low. It was observed that no dry powder could be produced starting from

SVS-LYS-5% Tween20 aqueous solution, which was not surprising considering that Tween20

is a viscous liquid.

After the first spray drying procedure of SVS, different conditions were applied to

investigate the effect of processing conditions on the quality, yield and amorphous nature of

the final product. SVS-LYS-5% SLS was prepared by dissolving 500 mg SVS-LYS 1:1 in 16 mL

instead of 20 mL. The decreased volume of 5% SLS solution caused a reduction of 20% of the

amount of SLS. Reference is made to Table 4.4 for the observed spray dry conditions.

Beside the SVS-LYS-5% SLS mixtures prepared with an inlet temperature of 100°C

and 110°C, a third mixture was also spray dried, namely SVS-5% SLS. Taken into account that

the amount of final product with an inlet temperature of 100°C was found to be better than

the procedures in which the inlet temperature of 110°C was used, this preparation was

performed with an inlet temperature of 100°C. Likewise, reference is made to Table 4.4 for

the observed spray dry conditions and exact values.

35

Table 4.4

Summary of the prepared formulations: exact amounts of the components, weight ratios

in the final product, spray drying conditions (Tinlet, Toutlet and pump rate) and the yield

(N/A: not applicable).

Components Spray drying conditions

Exact amount of every component

Weight ratios in final product

TINLET

(°C) TOULTET

(°C)

Pump rate (%)

Yield

GBC-SER-5% SLS (SD)

412.29 mg GBC 87.70 mg LYS 1500 mL 5% SLS

N/A

120

60

20

Low

SVS-LYS-5% SLS (SD 1)

370.6 mg SVS 129.4 mg LYS 20 mL 5% SLS

28.51 % SVS 9.95% LYS 61.54% SLS

110 50 18-20 Low

SVS-LYS-5% SLS (SD 2)

370.6 mg SVS 129.4 mg LYS 16 mL 5% SLS

28.51 % SVS 9.95% LYS 61.54% SLS

150 65 20 Low

SVS-LYS-5% SLS (SD 3)

370.6 mg SVS 129.4 mg LYS 16 mL 5% SLS

28.51 % SVS 9.95% LYS 61.54% SLS

100 45 15 High

SVS-LYS-0.5% SLS (SD)

370.6 mg SVS 129.4 mg LYS 200 mL 0.5% SLS

N/A 110 50 18-20 Low

SVS-LYS- 5% Tween20 (SD)

370.6 mg SVS 129.4 mg LYS 200 mL 5% Tween

N/A 110 50 18-20 N/A

SVS-5% SLS (SD)

370.6 mg SVS 129.4 mg LYS

31.70 % SVS 68.30% SLS

100 45 15 High

SVS-LYS-5% SLS (PM)

370.6 mg SVS 129.4 mg LYS 16 mL 5% SLS

28.51 % SVS 9.95% LYS 61.54% SLS

N/A N/A N/A N/A

SVS-5% SLS (PM) SVS-LYS (CM)+ 5% SLS

370.6 mg SVS 129.4 mg LYS 741.2 mg SVS 258.8 mg LYS 1229.84 mg SLS

31.70 % SVS 68.30% SLS 28.51 % SVS 9.95% LYS 61.54% SLS

N/A

N/A

N/A

N/A

N/A

N/A

N/A

High

36

4.3 PHYSICAL CHARACTERISATION OF THE PREPARED SAMPLES

4.3.1 Differential scanning calorimetry (DSC)

The thermal properties of the prepared samples were investigated using DSC. Table

4.5 provides an overview of the determined melting temperature (Tm), glass transition

temperature (Tg), and recrystallization temperature (Trc) of all the samples.

Table 4.5

Melting temperature (Tm), glass transition temperature (Tg), and recrystallization

temperature (Trc) of the pure material compared to the prepared samples (not analyzed

(N/A), not detected (N/D))

Material Tg (C°) Tm SVS/GBC (C°) Tm SLS (C°) Trc (C°)

SVSa 29.00 ± 0.60 139.90 ± 0.20 N/A N/A

GBCa 71.90 ± 0.70 174.20 ± 0.20 N/A N/A

SLS N/D N/A 103.13 ± 7.20

194.25 ± 0.46

N/A

SVS-LYS-0.5% SLS (SD 110°C) 22.93 ± 6.27 143.74 ± 0.17 97.25 ± 0.44 N/D

SVS-LYS-5% SLS (SD 110°C ) 14.73 ± 2.24 143.70 ± 0.16 84.92 ± 4.78 74.14 ± 13.90

SVS-LYS-5% SLS (SD 100°C) 42.04 ± 24.72 140.60 ± 0.96 98.43 ± 2.16 68.55 ± 9.55

SVS-5% SLS (SD) 11.73 ± 0.59 N/D 95.98 ± 0.57 N/D

GBC-SER-5%SLS (SD) 55.43 ± 1.15 N/D 105.29 ± 2.67

192.93 ± 0.26

N/D

a Reference is made to Riikka L. et al. [33]

Two endothermic melting peaks were observed in the thermogram of pure SLS at

194.25°C and 103.13°C which is in agreement with literature data. [34] Only one

endothermic melting peak of SLS (103.13°C) was observed in all the SD samples of SVS. From

this we concluded that the crystalline character of SLS may have been partially reduced in

the SD SVS samples (see Appendix B). In addition, both peaks (194.25°C 5, 103.13 °C) were

seen in GBC-SER-5% SLS (SD) indicating that the sample probably was mainly crystalline with

respect to SLS.

37

Moreover, it was noticed that the sharp endothermic melting peak of crystalline SVS

at 139.90°C was reduced in the samples SVS-LYS-5% SLS (SD 100°C and SD 110°C) and SVS-

LYS-0.5% SLS (SD 110°C). This was seen as an indication for the partial amorphization of SVS

in these samples. However, a recrystallization peak was observed for SVS-LYS-5% SLS (SD

100°C, SD 110°C) at temperature 68–75°C as a result of crystallization of the amorphous

material during DSC run. This means that SVS may have been fully amorphous after

preparation and part of this recrystallized in DSC (XRPD was needed to confirm this, see

chapter 4.3.2.).

On the contrary, there was no melting peak from SVS (139.90°C) and no

recrystallization peak seen for SVS-5% SLS (SD 100°C). Similarly, the thermogram of GBC-

SER-5% SLS showed no Tm of GBC (174.20°C) and no recrystallization. Consequently, SVS and

GBC may have been completely amorphous in SVS-5% SLS (SD 100°C) and GBC-SER-5% SLS

respectively according to DSC. Furthermore, one single Tg was determined in the

thermograms of SVS spray dried samples. From the thermogram of GBC-SER-5% SLS (SD

110°C) a Tg of 55.43°C was determined, which was lower compared to the Tg of pure GBC

(71.90°C). The Tg of pure serine was unknown.

4.3.2 X-ray powder diffraction (XRPD)

XRPD measurements were performed for the samples prepared by spray drying (SD)

i.e. SVS-LYS-5% SLS (inlet 110°C), SVS-LYS-0.5% SLS, SVS-5% SLS and GBC-SER-5% SLS. This

was done to obtain more information about the level of crystallinity in the samples. The

results can be used to confirm observations obtained by the DSC. In addition, in view of the

fact that diffraction pattern is unique for one component; the XRPD measurements were

compared to the pattern of crystalline SLS. In this way, it was possible to distinct between

crystalline peaks coming from SVS/GBC or SLS. The XRPD patterns of the different samples

are shown Figure 4.1.

It can be concluded that the crystalline peaks from SLS were observed in the same

extent in the XRPD pattern of SVS-LYS-5% SLS (CM) and GBC-SER-5% SLS (SD) indicating

crystalline nature of SLS in these mixtures. It is obvious that the crystalline SLS peaks were

observed in the case of the CM sample for the reason that crystalline SLS was mixed with

amorphous SVS-LYS.

38

0

5 10 15 20 25 30 35

cou

nts

2ɵ

Secondly, it was seen that the crystalline character of SLS was reduced the most in

the SVS-LYS-5% SLS (SD). In comparison, the crystallinity of SLS was less reduced in SVS-LYS-

0.5% SLS (SD) and SVS-5% SLS (SD) since the curves were remaining small crystalline peaks

coming from SLS (6.6°2θ for SVS-LYS-0.5% SLS (SD) and at 18.1, 22.7, 31.5°2θ for SVS-5% SLS

(SD).) Nevertheless, the crystalline nature of SLS was partly preserved in all the SD samples

since the major central peak from SLS was still seen.

The amorphous conversion of both drugs and amino acids was indicated since no

other clearly sharp peaks were seen beside the crystalline peaks from SLS. However, since

crystallinity in GBC-SER-5% SLS was not reduce to as large extent as in other mixtures and

due to preparation difficulties (see 4.2.1) this sample was not characterized further.

Figure 4.1 XRPD pattern of GBC-SER-5% SLS (SD), SVS-LYS-5% SLS (CM), SVS-5% SLS (SD),

SVS-LYS-5% SLS (SD), SVS-LYS-0.5% SLS (SD) versus crystalline SLS.

4.3.3 Fourier transform infrared spectroscopy (FTIR)

FTIR spectra of PM and SD mixtures were compared to detect differences in peak

absorptions of the functional groups between the crystalline and amorphous form. The SD

samples with and without LYS were compared to identify if there were intermolecular

interactions between SVS/SLS and LYS. The spectra were analysed in the regions 1600-1800

cm-1 (C=0 vibration) and 3100-3700 cm-1 (OH-vibration) to detect hydrogen bondings.

GBC-SER-5% SLS (SD)

SVS-LYS-5% SLS (SD)

SVS-5% SLS (SD)

SVS-LYS-5% SLS (CM)

SVS-LYS-0.5% SLS (SD)

SLS

39

0,00

1100,00 1600,00 2100,00 2600,00 3100,00 3600,00

AB

SOR

BA

NC

E (A

U)

wavenumber ( cm^-1)

SVS-5% SLS (PM)

SVS-5% SLS (SD)

SVS-LYS-5%SLS (PM)

SVS-LYS-5%SLS (SD)

SLS (PM)

Crystalline SVS is characterised by hydrogen bondings between the hydroxyl groups

and the esters groups of the molecule. [28] After the SD-process with SLS, a peak shift from

3550 to 3450 cm-1 (OH-region) was seen in combination with a peak broadening. A second

peak shift was seen at the ester C=0 stretch absorption region from 1695 cm-1 to 1720 cm-1.

The shoulder peak at 1180 cm-1 (fenol stretching band of SVS) was not seen in both SD

mixtures. There was one difference observed between the samples with LYS and the

samples without LYS. The FTIR spectra of SVS-LYS-5% SLS (SD) and SVS-LYS-5% SLS (PM)

showed two peaks at 1580 cm-1 and 1645 cm-1 (primary amide group).

Figure 4.2 FTIR spectra of the physical mixture (PM) and spray dried mixture (SD) of SVS-

5% SLS and SVS-LYS-5% SLS

4.4 DISSOLUTION TEST

The samples for the dissolution and stability test were prepared with the 5% SLS

solution based on the results of the physical characterisation (see 4.3). The SVS-LYS-5% SLS

was prepared by CM in addition to the corresponding spray-dried and physical mixture. This

was done to have a fully crystalline reference (i.e. PM), a reference with only amorphous

SVS-LYS (i.e. CM) and mixture were SVS-LYS and SLS are all spray-dried together. This is

important to investigate the differences in dissolution properties. The preparation of SVS-

LYS-5% SLS (CM) was based on the weight ratios of SVS, LYS and SLS in the corresponding

spray dried sample (see Table 4.4). After two times 500 mg SVS-LYS (1:1) was successfully

prepared by CM with a yield of 76%, the correct amount of 1229.84 mg SLS was added.

40

0

0,5

1

1,5

2

2,5

3

0 200 400 600 800 1000 1200 1400 1600

rele

ased

am

ou

nt

of

SVS

(mg)

time (minutes)

SVS-LYS-5% SLS (PM)

SVS-LYS-5% SLS_SD inlet 100 °C

SVS-LYS-5%SLS (CM)

The dissolution test was done for six different formulations in phosphate buffer (pH

7.2 and 37C°). Figure 4.3 shows the dissolution curves of SVS-LYS-5% SLS prepared in three

different ways: spray drying inlet temperature 100°C (SD), cryo-milling of SVS-LYS and

subsequently mixing with SLS (CM) and the physical mixture (PM). The dissolution curve of

SVS-LYS-5% SLS (SD inlet 110°C) is not given in Figure 4.3 due to the lack of significant

difference with SVS-LYS-5% SLS (SD inlet 100°C).

In case of SVS-LYS-5% SLS (SD), the maximum released amount of SVS (1.79 mg) was

already reached after 90 minutes while the maximum released amount of SVS (1.80 mg) for

SVS-LYS-5% SLS (PM) was seen later, after approximately 6 hours. Secondly, the curve of

SVS-LYS-5% SLS (SD) was decreasing after the maximum was reached whereas the curve of

the PM staid constant till the time point of 24 hours. Nevertheless, it can be stated that the

dissolution curve of SVS-LYS-5% SLS (SD) was not significantly different (p>0.05) from SVS-

LYS-5% SLS (PM). On the other hand, the dissolution of SVS-LYS-5% SLS (CM) was faster than

that of SVS-LYS-5% SLS (SD) considering the calculated significant difference between both

samples (p<0.05). In addition this formulation enabled higher concentrations of SVS than

the others. The maximum released amount of SVS (2.46 mg) in the case of SVS-LYS-5% SLS

(CM) was already seen after 30 minutes.

Figure 4.3 Dissolution profiles at pH 7.2 of SVS-LYS-5% SLS prepared by spray drying with

an inlet temperature of 100°C (SD), cryo-milling (CM) and the physical mixture (PM)

showing an enlargement from the region until 120 minutes.

0

1

2

3

0 50 100

rele

ased

am

ou

nt

of

SVS

(mg)

time (minutes)

41

0

1

2

3

0 200 400 600 800 1000 1200 1400 1600

rele

ase

d a

mo

un

t o

f SV

S (m

g)

time (minutes)

SVS-5% SLS (SD INLET 100 )

SVS-5% SLS ( PM)

The dissolution curves of SVS-5% SLS (PM) and SVS-5% SLS (SD inlet 100°C) are shown in

Figure 4.4. For SVS-5% SLS (SD), the maximum released amount of SVS (3.23 mg) was

reached after 90 minutes and was almost twice the maximum released amount of the PM

mixture. The curve SVS-5% SLS (SD) decreased again after the maximum while the curve of

SVS-5% SLS (PM) staid constant. In considering that SVS-5% SLS (SD) was significantly

different from SVS-5% SLS (PM) (p<0.05), it can be stated that SVS-5% SLS was more

promising on the level of dissolution, compared to SVS-5% SLS (PM). It was seen that the

results of SVS-5% SLS (SD) and SVS-LYS-5% SLS (CM) were significant different after one hour

(p<0.05).

Figure 4.4 Dissolution profile (pH 7.2) of SVS-5% SLS mixtures prepared by spray drying

with an inlet temperature of 100°C (SD) and the physical mixture (PM)

4.5 STABILITY STUDY

The physical stability of four formulations was investigated after storage under

different conditions i.e. 4°C/0% RH, 40°C/0% RH and 25°C/60% RH. XRPD and FTIR analyses

were done at regular time intervals (1week/2weeks) to detect the onset of recrystallization.

The stability of the spray-dried samples were compared with SVS-LYS (CM) + 5% SLS. This

was done with a view to investigate if there was any difference in the stability when SLS is

added simply as a crystalline component to the amorphous SVS-LYS or when all these

components are spray-dried together.

0

1

2

3

0 40 80 120

rele

ased

am

ou

nt

of

SVS

(mg)

time (minutes)

42

It can be stated that SVS-LYS-5% SLS (SD inlet 100°C) was found to be stable at least

for 9 weeks at 40°C/0% RH storage conditions. The SVS-LYS-5% SLS (SD inlet 110°C) was

stable for only seven weeks at the same storage conditions. One small peak (6.6°2ɵ, SLS)

was seen after 7 weeks storage at 4°C/0% RH for inlet 100°C while the same peak was seen

for inlet 110°C at week 9. When both samples were stored at 25°C/60% RH, crystalline peaks

were seen at 6.7°2ɵ (SLS) and 9.74°2ɵ (SVS). Both peaks were seen after one week for

sample with inlet 100°C while only one peak (9.4°2ɵ) was seen after week 1 for inlet 110°C.

The onset of the recrystallization was confirmed by the results from the FTIR analyse. The

peak at 1180 cm-1 (fenol stretching band), that disappeared after SD, reappeared in its

original position at week 1 in the FTIR spectrum. Figure 4.5 shows the XRPD patterns and

FTIR spectra of SVS-LYS-5% SLS inlet 100°C after 9 weeks storage under different conditions.

Figure 4.5 XRPD pattern and FTIR spectra of SVS-LYS-5% SLS (SD inlet 100°C) after 9 weeks

of storage under 4°C/0% RH, 40°C/0% RH and 25°C/60% RH conditions compared to time

point zero

Moreover we could state that SVS-LYS-5% SLS (CM) staid stable during at least for 9

weeks of storage at the three different conditions (stability data see Appendix C). The onset

of recrystallization of SVS-5% SLS (SD) was seen after 2 weeks storage at 25°C/60% RH.

Crystalline peaks at 6.7°2ɵ (SLS) and at 31.5°2θ were seen. The same peaks were seen when

the sample was stored at 40°C/0% RH after week 4. It could be concluded that 4°C/0% RH

was the best storage condition for SVS-5%SLS (SD) considering that only one peak (6.7°2ɵ

(SLS)) appeared at week six. However, no changes in the FTIR spectra were observed.

43

5. DISCUSSION

5.1 SOLUBILITY TEST

Among the different solubilisers, 5% SLS was able to provide the highest solubility of

GBC with 275.04 ± 14.34 µg/mL. This means that the amount of SLS needed to dissolve GBC

to prepare the aqueous solution for the spray drying was very high compared to the amount

of the drug. In consequence, this formulation has no practical significance. Despite of that,

spray-drying was attempted but the process was not successful (low yield, lowest

amorphization potential according to XRPD). For these reasons this formulation was

discarded from further studies and is not further discussed. The highest solubility of SVS

(24337.54 ± 863.18 µg/mL) was achieved with 5% SLS also.

5.2 PREPARATION OF AMORPHOUS MATERIAL BY SPRAY DRYING

It can be concluded that the preparation of amorphous material with the spray

drying method was possible for aqueous solutions containing SVS and GBC in combination

with SLS (though impractical in the case of GBC). The amorphous character of the final

product is discussed in more detail below. In addition, the spray drying can be seen as a fast

preparation method which is easy to use.

Despite these advantages, two limitations were observed after the spray drying of

GBC-SER-5% SLS, SVS-LYS-0.5% SLS, SVS-LYS-5% SLS and SVS-5% SLS. First off all, it was

noticed that the preparation of the aqueous solutions containing the appropriate amounts

of every component, required more than one day. In consideration of this time-limiting

problem, there is an enormous need for a better solubility of GBC/SVS in water. Secondly,

the obtained yields of the final products were rather low and variable from batch to batch

(see Table 4.4).

In this study, two different inlet temperatures, higher and lower than the original

inlet temperature of SVS were investigated namely 110°C and 100°C. Interestingly, the yield

of inlet temperature 100°C was increased compared to the yield of the SD with inlet

temperature 110°C. Therefore, the inlet temperature of 100°C was also used in the

characterization.

44

5.3 PHYSICAL CHARACTERISATION OF THE PREPARED SAMPLES

The SD of SVS-LYS-5% SLS (inlet 100°C and 110°C) resulted in amorphous SVS

according to XRPD and DSC (see Table 4.5 and Figure 4.1). A recrystallization peak for SVS

was observed, together with the subsequent endothermic melting peak of SVS in the DSC

thermograms. Moreover, the recrystallization peak of SVS-LYS-5% SLS (inlet 100°C) was

higher (18.3 Jg^-1) compared to the peak (8.823 Jg^-1) of SVS-LYS-5% SLS (inlet 110°C). In

consequence, in the case of inlet temperature of 100°C, more energy was required to

recrystallize the sample with indicating for a better amorphization. The totally amorphous

character of SVS was also successfully achieved after the spray drying of the samples SVS-5%

SLS (inlet 100°C) and GBC-SER-5% SLS. This was confirmed by the lack of melting peak of

both drugs in the DSC thermograms (see Table 4.5). Furthermore, no crystalline peaks of the

drug were observed in the XRPD patterns (see Figure 4.1). In conclusion, it can be stated

that the crystallinity in the samples came from SLS.

The successful amorphization of SVS was also confirmed by the FTIR spectra. In

general, the conversion of the crystalline to the amorphous form results in a different

molecular structure. Therefore, the peaks of amorphous form are broader compared to the

corresponding peaks of the crystalline form. Peak shifts may occur due to the amorphous

molecular packing which permits more molecular movement. IR spectra also provide

information about intermolecular interactions between the components in the sample. [24]

The glass transition temperature was decreased for both drugs in comparison with

the Tg of the individual drugs (see Table 4.5). It is true that the stability generally benefits

from a high Tg but it is published that molecular interactions also play an important role in

the stabilization of amorphous form. Moreover, it was observed that the mixture with the

highest Tg is not always the most stable one. [8,23] However, this comparison is not

completely correct since there were no Tg values available for individual LYS and SLS.

Despite the fact that quench cooling of crystalline SLS was performed to determine the Tg,

the value could not be defined. In addition, there was no comparison made to the Tg of the

individual LYS either since no amorphous production of this component was possible with

the methods available. One can only say that it seems that presence of SLS in the mixtures

decreases the Tg.

45

Furthermore, a single Tg was seen in the thermograms of SVS SD samples indicating

for homogenous mixture due to intermolecular interactions between the components. [24]

The intermolecular interactions were confirmed by the FTIR spectra (see Figure 4.2). There

was one difference observed between the FTIR spectra of samples with LYS and the samples

without LYS. The FTIR spectra of SVS-LYS-5% SLS (SD and PM) showed two peaks at 1580 cm-

1 and 1645 cm-1 (primary amide group). However, the fact that the peaks are seen in the FTIR

spectra of the PM and SD mixture doesn’t indicate any interaction. Only when the peaks

observed in the PM spectra are shifted in the SD spectra, it might indicate interactions in the

amorphous mixtures. FTIR spectra confirmed the intermolecular interaction between LYS

and SVS. For this reason, LYS can be seen as a good co-amorphous stabiliser. [23]

5.4 DISSOLUTION TEST

In the case of amorphous material, it was seen that the amount of dissolved SVS was

decreasing after the maximum was reached. This can be explained by the fact that

amorphous material is able to reach the supersaturation state after the introduction into

the dissolution medium. [35,36] Supersaturation refers to the drug concentration which is

temporarily above the equilibrium solubility in the dissolution medium. This was seen for

the formulations in this study on the grounds that the equilibrium solubility of SVS was 1.74

µg/mL (see Figures 4.3 and 4.4). But, once the supersaturation state is created, the

amorphous form has the tendency to convert back to the more stable crystalline form. Thus,

the amount of dissolved drug will decrease till the equilibrium solubility is reached. In this

study it was seen that the supersaturated state prevailed for 8 hours in the case of SVS-LYS-

5% SLS (CM and SD) and SVS-5% SLS (SD). It is the aim to maintain the supersaturation state

as long as possible to benefit from the properties of the amorphous state. [35,36]

If the crystallization occurs very fast, the expected supersaturation is not achieved,

seeing that amorphous material crystallizes faster than it can dissolve in the medium. Since

the dissolution of the crystalline material will be measured, the dissolution profiles of the

amorphous and crystalline material will look similar. [36] In this study, the same level of

supersaturation was observed for SVS-LYS-5% SLS (SD and PM). As there was a solubilizer in

both formulations, it can be stated that according to the results, the supersaturated state

was induced by the presence of SLS. The conversion to the amorphous state was not able to

46

further improve the supersaturation level (see Figure 4.3). In addition, it can was seen that

the supersaturation state was generated for SVS-LYS-5% SLS (CM) and SVS-5% SLS (SD). For

SVS-5% SLS SD mixture, the conversion of the amorphous state was able to further improve

the supersaturation state (see Figure 4.4). The SVS-LYS-5% SLS (CM) and SVS-5% SLS (SD)

samples were significant different (p<0.05) after one hour. From this we see that there is no

difference in the initial dissolution rate and that the SVS-5% SLS (SD) formulation could

produce significantly higher drug concentrations after one hour.

5.5 STABILITY STUDY

It was an interesting observation that even though the formulations were only

partially amorphous initially (remaining SLS crystallinity, see Figure 4.5) the changes

observed as a function of time in different conditions were small or completely absent. It

can be stated that SVS-LYS-5% SLS (SD 100°C) maintained the best physical stability when

stored at 40°C/0% RH since no recrystallization was seen after at least 9 weeks. The SVS-LYS-

5% SLS (CM) batch was stable during at least 9 weeks at every condition. The stability results

can be promising compared to a previous study in which the stability of CM amorphous SVS-

LYS mixture was investigated. In this study, there was recrystallization seen within 12 weeks

of storage at 40°C/0%. [33] The stability of SVS-5% SLS (SD) looked less promising due to the

onset of recrystallization after storage at every condition. In consideration of that, it can be

suggested that LYS plays an important role in the physical stability of SVS. In general, the

onset of recrystallization was seen at an early stage for SD mixtures stored under 25°C/60%

RH. This can be explained by the plasticizing effect due to the high level of moisture. [8]

Some development ideas for the future can be made, based on the results above.

Since both drugs show a better solubility in ethanol, the SD with organic solvent could be a

solution. Nevertheless, the use of organic solvent in SD is related with environmental

problems and possibly toxic organic residual. [26] A more desirable and constantly yield

could be obtained after an extensive investigation of the influence of the different process

parameters on the amount of final powder. At last, it was seen in the XRPD measurements

that the remaining crystallinity in the samples came mostly from SLS. An option that should

be considered with a view to reduce the amount of SLS, is the solubility test of GBC/SVS in

water after the addition of various polymers and SLS in different ratios. [2]

47

6. CONCLUSION

The first aim was to test spray drying as a preparation method for co-amorphous

drug-AA mixtures containing poorly water soluble drugs SVS and GBC and the corresponding

amino acid LYS and SER respectively. It was seen that 5% SLS was able to provide the highest

solubility of GBC/SVS in water. It was possible to perform the SD of SVS-LYS-0.5% SLS

solution, SVS-LYS-5% SLS solution, SVS-5% SLS solution and GBC-SER-5% SLS solution.

Secondly, the prepared samples were characterized with respect to the thermal

properties, the level of crystallinity, intermolecular interactions, dissolution and stability

properties. It can be stated that the crystalline character of SLS was less reduced in the GBC-

SER-5% SLS sample compared to the SD SVS samples. The GBC-SER-5% SLS sample was not

further characterized due to preparation difficulties and remaining crystallinity. However,

the crystalline nature of SLS was still preserved in every SD SVS sample. The amorphization

of the drugs and AA was indicated since no other sharp peaks were seen in XRPD beside the

crystalline peaks from SLS. The amorphization of SD SVS was confirmed by peak broadening

in the FTIR spectra. Lysine can be seen as a good co-amorphous partner because the FTIR

spectra confirmed the intermolecular interactions between LYS and SVS. It can be stated

that the conversion to the amorphous state resulted in better dissolution properties for only

SVS-5% SLS (SD) and SVS-LYS-5% SLS (CM) over that of the respective crystalline mixtures.

Finally, the stability of four samples was investigated under three different storage

conditions. Interestingly, it was seen that even though all the formulations had remaining

SLS crystallinity, the changes observed as a function of time in different storage conditions

were small or completely absent. The SVS-LYS-5% SLS (CM) sample was stable for 9 weeks

storage at 40°C/0% RH and 25°C/60% RH while recrystallization was seen for SVS-LYS-5% SLS

(inlet 100°C, 110°C) already after 1 week. The SVS-LYS-5% SLS (SD 100°C) was stable for 9

weeks at 40°C/0% RH. These results can be promising compared to the result of a previous

study in which the stability of cryo-milled SVS-LYS mixture was investigated. In this study,

there were signs of recrystallization seen for SVS-LYS (CM) within 12 weeks of storage at

40°C/0%. [33] Finally, LYS can be seen as an important and promising stabiliser in the co-

amorphous drug mixture since SVS-5% SLS (SD) showed recrystallization at an early stage,

after storage at every condition.

48

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i

APPENDICES

Appendix A: Chemical structures

Appendix B: DSC thermograms

Appendix C: Stability data

ii

Appendix A: Chemical structures

Table A.1 The chemical structure of simvastatin, glibenclamide, sodiumlaurylsulfate and

lysine

Simvastatin (SVS)

Glibenclamide (GBC)

Sodiumlaurylsulfate (SLS)

Lysine (LYS)

iii

Appendix B: DSC thermograms

Fig. B.1 DSC thermogram of SVS-LYS-5% SLS (SD 110°C) (up) and pure SLS (down)

iv

0,00

550,00 1050,00 1550,00 2050,00 2550,00 3050,00 3550,00 4050,00

AB

SOR

BA

NC

E (A

U)

wavenumber (cm^-1)

Appendix C: Stability data

Figure C.1 XRPD pattern (up) and FTIR spectra (down) of SVS-LYS-5% SLS (CM) after 9

weeks of storage under 4°C/0% RH, 40°C/0% RH and 25°C/60% RH conditions compared

to time point zero

0

5 10 15 20 25 30 35

cou

nts

2θ

Week 0

4°C/0% RH

25°C/60% RH

40°C/0% RH

Week 0

4°C/0% RH

25°C/60% RH

40°C/0% RH