Co-Amorphous Drug Formulations: Carbamazepine, Mefenamic Acid and Sulfamerazine

CO-AMORPHOUS DRUG FORMULATIONS: CARBAMAZEPINE, MEFENAMIC ACID AND SULFAMERAZINE

Word count: 19.613

Danielle DELOMBAERDE Student number: 01406993

Promotor: Prof. Dr. T. De Beer Supervisor(s): Prof. Dr. Ossi Korhonen, Ph.D. (Pharm.) Katja Pajula

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Pharmaceutical Care

Academic year: 2017-2018

CO-AMORPHOUS DRUG FORMULATIONS: CARBAMAZEPINE, MEFENAMIC ACID AND SULFAMERAZINE

Word count: 19.613

Danielle DELOMBAERDE Student number: 01406993

Promotor: Prof. Dr. T. De Beer Supervisor(s): Prof. Dr. Ossi Korhonen, Ph.D. (Pharm.) Katja Pajula

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Pharmaceutical Care

Academic year: 2017-2018

Deze pagina is niet beschikbaar omdat ze persoonsgegevens bevat. Universiteitsbibliotheek Gent, 2021.

This page is not available because it contains personal information. Ghent University, Library, 2021.

ABSTRACT

The majority of the newly discovered active pharmaceutical ingredients tend to be poorly soluble. Drug dissolution is a very important step in drug absorption and ultimately a therapeutic effect. Different methods have been investigated throughout the years to increase the solubility, and thus potentially the bioavailability. The amorphous state of the drug seems to be the most interesting. It is known to have a higher apparent solubility but unfortunately it lacks physical stability. Low molecular weight compounds such as amino acids can stabilise the amorphous form of the drug.

Calculations were performed to predict the miscibility between three drugs, carbamazepine (CBZ), mefenamic acid (MEF), and sulfamerazine (SMZ) and 20 amino acids. Proline (PRO) was chosen as a stabilising agent for all above-mentioned drugs. This choice was made based on molecular modelling calculations and melting points. Afterwards, both chemical and physical stability were evaluated.

The amorphisation technique that was used throughout this study was melting, followed by quench-cooling. Unfortunately, both mefenamic acid and sulfamerazine degraded upon melting. Proline increased the degradation of our active substances. No ideal method was found for carbamazepine, so this was not further analysed. Mefenamic acid-proline (50:50 molar ratio) and sulfamerazine-proline (20:80 molar ratio) stayed amorphous for respectively six and five days.

After performing the intrinsic dissolution rate tests, there was a difference between the dissolution rates of the co-amorphous forms compared to the pure crystalline drug compounds. Results showed that the dissolution rate increased with a factor three for mefenamic acid-proline and with a factor twenty for sulfamerazine-proline. Nevertheless, the error bars were quite large for amorphous MEF-PRO indicating that the difference in dissolution rate might not be three times higher. SMZ-PRO could possibly lead to a potential new drug formulation with increased dissolution properties when prepared via a different amorphisation method that does not introduce degradation of SMZ.

Different amorphisation methods of these compounds should be performed in the near future to be able to draw more definite conclusions. SAMENVATTING

Het merendeel van de nieuwe geneesmiddelen heeft een lage oplosbaarheid. De oplosbaarheid is een cruciale stap in het absorptieproces. Verschillende methoden zijn de voorbije jaren onderzocht geweest om de oplosbaarheid en dus de biologische beschikbaarheid te verbeteren. Studies toonden aan dat de amorfe vorm van een geneesmiddel mogelijkheden biedt. De amorfe vorm wordt gekenmerkt door een hogere schijnbare oplosbaarheid, maar ook door een lage fysische stabiliteit. Het is bewezen dat moleculen met een laag moleculair gewicht, zoals aminozuren, in staat zijn om op een zodanige manier te interageren met de amorfe vorm waardoor deze stabiel blijft.

Op basis van berekeningen werd de mengbaarheid van drie geneesmiddelen, carbamazepine (CBZ), mefenaminezuur (MEF) en sulfamerazine (SMZ) nagegaan met alle 20 aminozuren. Vervolgens werd proline gekozen als stabilsator voor bovenstaand vermelde geneesmiddelen. De keuze werd gemaakt op basis van calculaties en ook de smeltpunten van de individuele componenten werden in rekening gebracht. Vervolgens werd de chemische en fysische stabiliteit geanalyseerd.

Tijdens deze studie werden de stalen eerst gesmolten en vervolgens snel afgekoeld op een koel metaal oppervlak. Hierdoor werden de stalen amorf. Mefenaminezuur en sulfamerazine braken af onder invloed van warmte. Na toevoeging van proline, werd een toename in afbraak waargenomen. Carbamazepine kon niet geëvalueerd worden omdat geen goede analyse methode werd gevonden. Mefenaminezuur-proline (50:50 molaire ratio) en sulfamerazine-proline (20:80 molaire ratio) bleven amorf gedurende zes en vijf dagen.

Uit de dissolutietesten bleek er een verschil te zijn in oplossnelheid tussen de co- amorfe vorm en het zuiver geneesmiddel. De oplossnelheid was driemaal hoger voor mefenaminezuur-proline en twintig keer hoger voor sulfamerazine-proline. Desondanks, bleken de standaarddeviaties op de grafiek vrij groot te zijn voor MEF-PRO, waardoor er geen duidelijk resultaat kon worden afgeleid. SMZ-PRO zou mogelijks tot een nieuwe formulatie kunnen leiden, maar niet via deze methode aangezien afbraak van het geneesmiddel plaatsvond. Er zouden betere conclusies kunnen getrokken worden uit de resultaten moesten er verschillende technieken om de stalen amorf te maken met elkaar kunnen vergeleken worden. ACKNOWLEDGEMENTS

I would like to thank Prof. Dr. T. De Beer and Ghent University for giving me this opportunity to study abroad at the University of Eastern Finland in Kuopio. It has been a wonderful experience which I will cherish for a lifetime.

I would also like to thank my supervisors, Ossi Korhonen and Katja Pajula. Their guidance throughout these three months was very much appreciated. Despite a few setbacks with some of the equipment, we managed to work through this. They were always there to offer help whenever needed. I could not have imagined any better supervisors.

My family and friends were also of great support throughout the whole experience.

INDEX

1 INTRODUCTION ...... 1

1.1 BACKGROUND ...... 1

1.2 DIFFERENTIATION BETWEEN AMORPHOUS AND CRYSTALLINE ...... 4

1.2.1 Crystalline form ...... 5

1.2.2 Amorphous form ...... 5

1.2.3 Solvate form ...... 5

1.2.4 Differentiation and characterisation ...... 6

1.3 AMORPHISATION METHODS ...... 9

1.3.1 Spray drying ...... 9

1.3.2 Freeze drying ...... 9

1.3.3 Milling ...... 9

1.3.4 Melting followed by quench-cooling ...... 10

1.4 STABILISING THE AMORPHOUS STRUCTURE ...... 10

1.4.1 Polymer-based glass solutions / dispersions ...... 10

1.4.2 Mesoporous silica ...... 11

1.4.3 Co-amorphous drug formulations ...... 11

2 OBJECTIVES ...... 13

3 MATERIALS AND METHODS ...... 15

3.1 MATERIALS ...... 15

3.2 METHODS ...... 16

3.2.1 Solid-state characterisation of the active compounds ...... 16

3.2.1.1 Differential Scanning Calorimetry (DSC) ...... 16

3.2.2 Molecular modelling calculations ...... 17

3.2.2.1 Selecting the conformations for miscibility calculations ...... 17

3.2.2.2 Calculating the Flory-Huggins Interaction Parameter ...... 17 3.2.3 Preparation of the binary mixtures ...... 19

3.2.4 Preparation of the amorphous materials...... 19

3.2.4.1 Melt-quench method ...... 19

3.2.4.2 Linkam heating stage ...... 19

3.2.5 Stability studies ...... 20

3.2.5.1 Chemical stability ...... 20

3.2.5.2 Physical stability ...... 20

3.2.5.3 Polarised Light Microscopy (PLM) ...... 21

3.2.6 High Performance Liquid Chromatography (HPLC) ...... 21

3.2.7 Intrinsic Dissolution Rate Study ...... 22

4 RESULTS AND DISCUSSION ...... 23

4.1 SOLID-STATE CHARACTERISATION OF CBZ, MEF, AND SMZ ...... 23

4.1.1 Carbamazepine (CBZ) ...... 23

4.1.2 Mefenamic acid (MEF) ...... 24

4.1.3 Sulfamerazine (SMZ) ...... 26

4.2 MOLECULAR MODELLING CALCULATIONS AND SELECTION OF ADDITIVES AS STABILISING AGENT ...... 27

4.2.1 Carbamazepine (CBZ) ...... 27

4.2.2 Mefenamic acid (MEF) ...... 30

4.2.3 Sulfamerazine (SMZ) ...... 31

4.3 STABILITY STUDIES...... 34

4.3.1 Chemical stability ...... 34

4.3.1.1 Carbamazepine (CBZ) ...... 34

4.3.1.2 Mefenamic acid (MEF) ...... 34

4.3.1.3 Sulfamerazine (SMZ) ...... 36

4.3.2 Physical stability ...... 38 4.3.2.1 Carbamazepine (CBZ) ...... 38

4.3.2.2 Mefenamic acid (MEF) ...... 38

4.3.2.3 Sulfamerazine (SMZ) ...... 39

4.4 INTRINSIC DISSOLUTION RATE STUDY ...... 40

4.4.1 Mefenamic acid (MEF) ...... 40

4.4.2 Sulfamerazine (SMZ) ...... 42

5 CONCLUSIONS ...... 45

6 REFERENCES ...... 47

LIST OF ABBREVIATIONS

AA Amino Acids

ACN Acetonitril

API Active Pharmaceutical Ingredient

CBZ Carbamazepine

DSC Differential Scanning Calorimetry

GLN Glutamine

GLU Glutamic Acid

HPLC High Performance Liquid Chromatography

IDR Intrinsic Dissolution Rate

MFA Mefenamic Acid

PLM Polarised Light Microscope

PRO Proline

rpm rotations per minute

RSD Relative Standard Deviation

SD Standard Deviation

SMZ Sulfamerazine

TFA Trifluoroacetic acid

1 INTRODUCTION

1.1 BACKGROUND

Many of the newly discovered Active Pharmaceutical Ingredients (API’s) tend to be poorly soluble in water [1]. This matter requires immediate attention to pharmaceutical research and development as most drugs are administered orally. This route is the most convenient way. It has a good cost-effectiveness, patient compliance and does not require a sterile environment during manufacturing [2].

Drug dissolution, in gastric and intestinal fluids, is an essential step for drug absorption in the human body and ultimately a therapeutic effect [3,4]. So, poor dissolution leads to limited absorption resulting in lower plasma concentrations than expected. Often, higher doses are given to achieve the desired plasma concentration which eventually will result in adverse effects. These side-effects have a negative impact on patient compliance [2]. This field creates challenges regarding the pharmaceutical industry to discover new methods to increase the solubility, and thus potentially the bioavailability.

The BCS, Biopharmaceutics Classification System provided by the U.S. Food and Drug Administration, is a system used to classify drugs according to their solubility and permeability across biological membranes. There are four different classes, which are listed in table 1.1. BCS class II and IV are known for their low solubility. This limits the rate and extent of absorption. API’s that are categorised in one of these two classes demand extra attention concerning this subject [4,5].

Table 1.1: The BCS, Biopharmaceutics Classification System, consisting of four different categories which are classified based on solubility and permeability characteristics [5]. This study mainly focusses on class II and class IV compounds. Class Solubility Permeability Bioavailability I High High Very well absorbed II Low High Dissolution rate- limited absorption III High Low Permeability-limited absorption IV Low Low Poorly absorbed

Different approaches have been developed throughout the years to enhance the solubility, and thus the efficacy of poorly soluble drugs. The method, appropriate for the drug, is chosen based on drug properties, the site of absorption and dosage form characteristics [2]. We must take into consideration that each method has its limitations. The techniques can be divided into three main sections: chemical modification, physical modification, and other methods [6].

Chemical modification can be divided into two categories. The first one being the salt form of the drug. This method is frequently used to increase the solubility of acidic and basic drugs. The second option is changing the pH, yet this is only relevant for ionisable drugs [2,6]. Pro-drugs can also be considered to increase the solubility. It allows us to modify the pharmacokinetics, pharmacodynamics, and toxicology of the drug [7].

Particle size reduction, modifications of the crystal habit, polymorphism, drug dispersion in carriers and complexation are all listed under physical modifications [2].

Other methods to improve the solubility are co-crystallisation, co-solvency, solubilising agents, and many more. A more detailed explanation of these methods can be found in ‘Solubility Enhancement Methods – A Promising Technology for Poorly Water Soluble Drugs’ [2].

Out of all the techniques listed above, this study emphasizes amorphisation, a physical modification. One method that can be used to improve the solubility is to convert the drugs to their amorphous state. Amorphous drugs have a greater apparent solubility and dissolution rate than their crystalline opposite. The amorphous structure does not require energy to break the crystal lattice structure. So, there are easily molecular interactions between the solvent molecules and drug molecules leading to a higher apparent solubility. Yet the main disadvantage is the lack of physical stability due to their higher energy form. They are thermodynamically unstable and are likely to recrystallise to the more stable, lower energy state i.e. crystals [1,8,9,10,11].

The amorphous form can increase the apparent solubility of a drug up to a thousand times [12]. It is actually the only method that can resolve the solubility of very poorly soluble drugs. So, amorphisation of the drug seems to be a good solution, but the main concern remains the likeliness to recrystallise due to the physical instability. Besides the physical instability there 2

are multiple factors which influence the stability of amorphous drugs such as environmental conditions, preparation methods and conditions [13,14]. Thus, the main challenge is to guarantee the stability of amorphous drugs over a certain timeframe that is relevant to the pharmaceutical industry, typically a three to five year shelf-life [8]. Fortunately, these can be stabilised by adding excipients [15]. Different approaches have already been investigated over time in other studies such as polymer-based glass solutions and dispersions, mesoporous silica and co-amorphous formulations [3,15]. They will be further discussed in section 1.4. Our study focussed on co-amorphous drug formulations, illustrated in figure 1.3, as a method to improve the dissolution properties of our active compounds.

This study investigated the following drugs: carbamazepine (CBZ), mefenamic acid (MFA) and sulfamerazine (SMZ). CBZ is used for the treatment of epilepsy and trigeminal neuralgia. It is also used off-label for the treatment of bipolar affective disorder. The specific pharmacologic mechanism of the drug can be described as followed, it is able to block sodium ion channels and prevent sustained repetitive neuronal excitation [16,17,18]. Four different anhydrous polymorphs (I, II, III and IV) exist and one dihydrate [19,20]. Form III is the most stable form. Therefore, it is crucial to verify whether the correct polymorph was used in the final formulation [16].

Polymorphism is the ability of a compound to crystallise in at least more than one crystal architecture and have different crystal packing arrangements. Polymorphs have different physicochemical properties, i.e. melting temperature, solubility, dissolution rate and bioavailability [8,16,21]. They can be transformed into one another by heating, applying mechanical stress, or via solvents [21]. The polymorph with the lowest Gibbs free energy, at a certain pressure and temperature, is known to have the greatest stability. Unfortunately, it also has the lowest solubility [22].

MEF is a non-steroidal anti-inflammatory drug. It is used as an antipyretic, analgesic and antirheumatic drug [23]. This compound can exist in three polymorphic structures: MFA I, II, and III. The second form, MFA II, is a meta-stable form with a higher solubility opposed to the two others. Form III is also known to be a meta-stable form. The stability is suggested to be I > II > III at room temperature [24,25].

SMZ is a well-known sulphonamide antibacterial drug. Its structure is derived from sulphanilamide, which contains an aniline group attached to a sulphonamide group. This class of antibiotics is known to complicate the synthesis of bacterial folic acid. It is used to treat urinary tract infections, ear infections, bronchitis, and many more infectious diseases. Sulphonamides are weak acids. They are known to be polar and fairly soluble in water [26]. There are three different polymorphs: SMZ I, II, and III. The second form is the most stable one at room temperature [27].

(a) (b) (c)

Figure 1.2: Carbamazepine (a), mefenamic acid (b) and sulfamerazine (c) [3,24,27].

It is noted that CBZ and MEF belong to BCS class II [3,24]. As previously described in table 1.1, the bioavailability depends on the dissolution rate of these drugs. SMZ is also a poorly water-soluble drug [28]. These products are available on the market. They are widely used amongst the population. So, it would be very interesting to investigate whether it would be possible to increase the dissolution rate and to lower the doses that have to be administered.

1.2 DIFFERENTIATION BETWEEN AMORPHOUS AND CRYSTALLINE

The API’s can exist in different solid forms: crystalline, amorphous, or solvate forms. The individual forms also have an impact on the solubility and dissolution rate [1]. The crystalline form possesses, besides long-range order molecular packaging, short-range molecular order. Whereas amorphous solids might have a short-range molecular order similar to the crystalline form, but lack the long-range order packaging [8,10]. It is important to know which state the solid is in, to optimise the storage conditions and anticipate conversions of one state into another [8].

Figure 1.3: Main perspective of the aim of this study. The blue molecules represent the drug and the red molecules represent the amino acid [15].

1.2.1 Crystalline form

Crystalline materials are characterised by a regular and defined molecular packing. This tends to be the most stable form. Nevertheless, the crystal form is poorly soluble compared to the amorphous state. Figure 1.3 gives us an idea how to visualise the structure. The crystal form is typically characterised by Tm, also known as melting temperature, at which the three- dimensionally ordered crystalline state transforms into a disordered liquid state [8,15,29].

1.2.2 Amorphous form

The physical properties of the amorphous state differ from the crystalline state. The specific ‘free’ volume is greater in amorphous materials due to the random arrangement of the molecules (figure 1.4). The higher free volume results in a faster diffusion rate. Despite the instability, it is known to have a greater solubility (figure 1.3). It has the characteristics of a liquid, but with a much higher viscosity. Amorphous materials are typically characterised by a glass transition temperature, also known as Tg [8,15].

1.2.3 Solvate form

Hydrate forms are the most common solvate form. Water can be incorporated into the crystal lattice which causes the crystalline anhydrates to transform to their hydrate form. This process typically occurs via solution or solution-mediated mechanisms. Freeze- and spray drying are two methods that can lead to hydrate formation due to the exposure to solvents. They can generate both crystalline and amorphous forms. Water, which is present in the environment, can lead to hydrate formation as well [22].

Dehydration however often happens via a solid-solid mechanism, although it also can occur via solution and melt mechanisms. For example, milling generates heat and can cause distortion of the crystal lattice. The dehydration process is a result of a solid-solid mechanism. Dehydration can also occur when dissolving the API in a specific solvent which is removed afterwards. Melting followed by cooling can also result in a transformation of the original state to the amorphous state or anhydrous crystalline state. The final product depends on how strict the dehydration conditions are and on the characteristics of the hydrate [22].

1.2.4 Differentiation and characterisation

A phase-transition, from a solid-state to a liquid state, occurs whenever the crystal form of a drug is heated to its melting temperature (Tm). When the melt is cooled down very quickly, it passes the Tm into a ‘supercooled liquid’ region, meaning there is no time for recrystallisation. The supercooled region is also known as the rubbery state. Molecular motions usually occur in less than 100 s and the viscosity varies from 10-3 and 1012 Pa.s. When the cooling process is continued, the slope changes at a glass transition temperature (Tg). The different phases are illustrated in figure 1.4 [8,15,30].

Figure 1.4: The volume (V) and enthalpy (H) as a function of temperature. Tg = glass transition temperature, and Tm = melting temperature. Image modified from ‘Characteristics and Significance of the Amorphous State in Pharmaceutical Systems’ [8].

The glass transition temperature differs depending on the heating/cooling rate and technique used to measure this temperature. It also depends on the molecular mass, purity, and geometry of the sample. At this temperature the molecular motions change. The molecules have much more molecular mobility in the supercooled liquid state opposed to the amorphous glassy state. Molecules below the Tg are known to be ‘kinetically frozen’. Unfortunately, this glassy state is thermodynamically unstable. The molecules can still move, yet at a much lower rate, i.e. at least more than 100 s. This means the amorphous materials can still crystallise over time into a more thermodynamically stable form. So, amorphous samples must be stored at least at 50 °C below the Tg in order to maintain the glassy state [8,15,30].

The glassy state has two strong features. First, the viscosity is higher than 1012 Pa.s. Second, the enthalpy (H) and volume (V) are also higher compared to the crystal state. Due to the higher internal energy, the amorphous state has improved thermodynamic properties (increased solubility, and potentially bioavailability) and more molecular motions. However, this also results in a greater chemical reactivity which could possibly lead to spontaneous recrystallisation. The purpose is to develop methods that prevent the chemical and physical instability of the amorphous state [8,15].

Because of the different physical properties of the amorphous state, there are many different methods to characterise whether a compound is crystalline, amorphous, or both. X- ray powder diffraction, spectroscopic techniques, diffusion-controlled processes have all been used to characterise amorphous materials. Besides aforementioned methods there are also thermal analytical methods, e.g. DSC (Differential Scanning Calorimetry) [8].

Exothermic Tg Tm

Endothermic

Temperature (°C)

Figure 1.5: DCS thermogram of amorphous sucrose (C12H22O11). Three different events are shown. The first event being the glass transition temperature (Tg), the second one the crystallisation temperature (Tc), and the third one the melting temperature (Tm). Image modified from ‘Characteristics and Significance of the Amorphous State in Pharmaceutical Systems’ [8].

Differential Scanning Calorimetry is a technique that can be used to analyse the thermodynamic properties of a compound. An amorphous material always has a higher heat capacity than its crystalline counterpart. This can be viewed in figure 1.4 [8]. When power- compensated DSC is performed, there are two pans, i.e. one containing the sample and a reference pan which is empty. They are each placed in a separate furnace. Both pans are then subjected to a set temperature programme. The sample requires a different amount of power to maintain the same temperature programme as opposed to the empty pan. A heat flow- temperature (°C) profile can be drawn. A non-isothermal DSC scan is shown in figure 1.5. Crystallisation might occur when the temperature increases above the Tg, or when the melt is cooled. Crystallisation, Tc, is an exothermic process, where the molecules are rearranged into a crystal lattice. Melting however, is an endothermic process which requires energy to separate the crystal lattice [29].

1.3 AMORPHISATION METHODS

There are many different ways to acquire the amorphous state of a drug such as via precipitation from a solution (spray- or freeze drying, adding antisolvents), or mechanical activation (milling), or supercooling of the melt. Melt extrusion can also be used, which is a combination of the techniques listed above. However, the result can differ depending on the preparation method [8,31,32]. The preparation method is chosen based on the physicochemical properties of both drug and excipient [15]. The different methods are briefly described below.

1.3.1 Spray drying

Nano to micron size particles can be created by spray drying. This is a scalable technique for transforming crystalline materials into amorphous end products. It consists of three main steps: atomization, dehydration, and powder collection. First, a solution (drug + AA) is sprayed into a drying room by an atomizer/nozzle. Second, the droplets are dried with heated air. Finally, the components of interest can be achieved by separating the dried material from the drying medium by evaporating the solvent in a cyclone. The thermal stress that is applied when processing can be changed by adjusting the specific parameters. The main advantage of spray drying is the ability to adjust the different parameters [11,33,34].

1.3.2 Freeze drying

Freeze drying, or lyophilization, is a process that can also lead to an amorphous product or partly amorphous solid. It consists of crystallising water and sublimation of the water vapor at reduced pressure resulting in a dry substance. The Tg of the final product depends on the compound and the amount of residual water that is still present. When water is still present, it can lower the Tg [35,36].

1.3.3 Milling

Milling is often an additional process when manufacturing pharmaceutical formulations. The size of the particles of a powder can be reduced by applying mechanical stress. Polymorphic transformation, disorder in the crystal lattice, or amorphisation can also be achieved by milling. Short-range molecular order still exists after the milling process. Unfortunately, heat can be generated during the milling process and thus inducing recrystallisation. Often, the crystal 9

lattice is not entirely separated. Thereby leaving crystal parts behind which eventually can lead to recrystallisation. Cryomilling, or cryogenic grinding, however is performed in liquid nitrogen which is able to inhibit the restoration process [24,33]. Nonetheless, these forms are physically less stable. The explanation lies within the large surface areas created by milling resulting in high energy sites for crystal nucleation and growth [37].

1.3.4 Melting followed by quench-cooling

Quenching is a very simple technique that can be applied to make a compound amorphous. When the melt is cooled down very quickly, there is no sufficient time for the molecules to form a crystal lattice structure. If cooling does not happen fast enough, the product will recrystallise. The molecules are often more randomly distributed as opposed to milling [38]. However, it can only be applied to compounds that do not degrade upon melting [15]. Most crystalline substances are known to have a very high melting temperature, which might lead to degradation making this method inapplicable in many cases [33].

1.4 STABILISING THE AMORPHOUS STRUCTURE

The amorphous structure can be stabilised by adding excipients. Three different possibilities are explained below. However, this study only focussed on the last one, i.e. co- amorphous drug formulations.

1.4.1 Polymer-based glass solutions / dispersions

Polymers can be used as a stabilizer for amorphous drugs. Glass solutions are systems where the drug is completely dissolved to the polymer. Whereas in a glass dispersion the concentration exceeds the solubility to the polymer resulting in a partly dispensed system. Polymer-based glass solutions are used to increase the solubility and dissolution rate of the drug [39,40]. Polymers are known to have a high glass transition temperature (Tg). So, they increase the Tg of the drug in the glass solution resulting in a lower molecular mobility at room temperature [3,41]. The solubility of the drug to the polymer and the intermolecular interactions also contribute to the overall stabilisation process [42]. However, many polymers are likely to be hygroscopic. Water is easily absorbed and acts as a plasticizing agent. This leads back to the initial scenario; a decrease in Tg and an increase in molecular mobility which could

result in recrystallisation of the drug. Another obstacle is the low solubility of the drugs to the polymer. Usually large amounts of polymer are needed which generate large volumes of the final doses [43].

1.4.2 Mesoporous silica

The use of mesoporous silica to improve the solubility of amorphous drugs has been studied for over four decades. These systems are based on the adsorption of amorphous drugs on the surface and pores of silica particles [44]. Porous media are known to have large surface areas and pore volumes besides nanosized pores [45]. Stabilisation is achieved in two different ways. First, the porous media have a high surface free energy because of the large surface area. When drugs adsorb onto the porous materials, a lower Gibbs free energy is reached. The drugs do no longer exist in the crystalline state. They now exist in an amorphous state. This results in a physically stable amorphous system. Second, the specific size of the pores limits crystal nucleation and growth. This could possibly increase the physical stability of the system [46].

The main pitfall of this method is the production method which requires organic solvents for drug loading [47].

1.4.3 Co-amorphous drug formulations

Ultimately, there are the co-amorphous drug formulations, illustrated in figure 1.3, which have gained a lot of interest during these last couple of years. The purpose is to stabilise the API by either using excipients with a low molecular weight (amino acids) or another chemical compound that results in an amorphous binary system which is able to retain its amorphous structure. The low molecular weight of AA allows us to reduce the amount of stabilising agent needed when preparing co-amorphous mixtures. If two drugs can stabilise each other in the amorphous form, they could be merged into one unit when combination therapy is needed [3,15].

The Tg of an amorphous mixture consisting of two compounds can be expressed by equation 1.1 and 1.2, i.e. the Gordon-Taylor equation [8,15]:

푤1푇푔1+퐾푤2푇푔2 푇푔12 = (1.1) 푤1+퐾푤2

With: K: constant

w1 and w2: weight fractions

Tg12: Tg of the amorphous mixture

Tg1 and Tg2: Tg’s of the individual compounds

휌 .푇 퐾 = 1 푔1 (1.2) 휌2.푇푔2

With: ρ1 and ρ2: powder densities of the compounds

Tg1 and Tg2: Tg’s of the individual compounds

The Tg of an amorphous mixture can usually be found in between the two individual Tg’s. An elevated Tg typically results in lower molecular mobility. A lower Tg results in a higher molecular mobility which will result in phase separation and recrystallisation [43]. One Tg will be observed when the system is perfectly miscible in the amorphous form. If not, more than one Tg, or broadening of the glass transition could appear [8]. The use of amino acids as excipients leads to a relatively high Tg. They are also more stable due to the molecular interactions. In co-amorphous blends intermolecular interactions are formed between drug and AA. The short-range order of the drug gets disturbed, so it takes much longer to recrystallise compared to an individual amorphous compound [15].

2 OBJECTIVES

A common problem of the new API’s is that they have very low solubility and bioavailability. The pharmaceutical industry has been investigating many ways to resolve this problem. The most interesting method seems to be amorphisation. When a compound is poorly soluble, it is the only method that could resolve this problem. Amorphisation is known to increase the solubility up to a thousand times. However, the main issue regarding amorphisation is the physical instability of the amorphous form. Luckily, co-amorphous systems have been introduced where molecules with a low molecular weight, i.e. amino acids, or another drug molecule can stabilise the amorphous form of the drug.

The aim of this study is to stabilise amorphous drug compounds, CBZ, MEF, and SMZ with amino acids resulting in an improved dissolution rate, and potentially bioavailability.

First, molecular modelling calculations were performed to select the most potential stabilising additives out of 20 AA for the amorphous drug compounds. These calculations gave information concerning the miscibility of the drug and amino acid. However, not only calculations were taken into consideration, but also the melting points of the amino acids and drug compounds. The melting points of the drug compounds were determined using the DSC.

Besides looking for substances that stabilise the amorphous drugs, it is also very important that they stay amorphous for a pharmaceutical relevant timeframe, i.e. three to five years. Long-term stability was evaluated visually using a polarised light microscope (PLM). When the samples started to show any signs of recrystallisation, they were defined as unstable. The samples were stored in a desiccator under dry conditions to avoid any environmental influences such as temperature, moist and others.

Out of all different amorphisation methods, this study emphasizes the melt-quench method. However, the melt-quench method can only be applied if there is no thermal degradation of the API upon melting. Chemical stability studies were performed to check whether the heating and/or amino acids caused any degradation of the active substances.

Finally, intrinsic dissolution tests were performed of MEF and SMZ to verify whether the co-amorphous mixtures had better dissolution properties as opposed to the pure crystalline drug compounds.

3 MATERIALS AND METHODS

3.1 MATERIALS For more detailed information about the materials view annex table 6.1.

Carbamazepine (CBZ, M = 236.27 g/mol, China), mefenamic acid (MEF, M = 241.29 g/mol, China) and sulfamerazine (SMZ, M = 264.31 g/mol, Spain), L-Arginine (ARG, M = 174.20 g/mol, China), L-Glutamic Acid (GLU, M = 147.13 g/mol, Brazil), L-Lysine (LYS, M = 146.19 g/mol, Switzerland) and L-Serine (SER, M = 105.09 g/mol, USA) were purchased from Sigma Aldrich. (2S,3R)-(-)–Threonine (THR, M = 119.12 g/mol, China) and (S)-(-)-Proline (PRO, M = 115.13 g/mol, Germany) were purchased from Merck KGaA. Glycine (GLY, M = 75.07 g/mol, Belgium) was purchased from VWR Chemicals. L-Alanine (ALA, M = 89.09 g/mol), L-Asparagine Anhydrous (ASN, M = 132.12 g/mol), L-Aspartic Acid (ASP, M = 133.11 g/mol), L-Cysteine (CYS, M = 121.16 g/mol) and L(+)-Glutamine (GLN, M = 146.14 g/mol, China) were obtained from VWR Life Science.

For HPLC analysis we used trifluoroacetic acid (TFA, M = 114.02 g/mol, USA), that was purchased from Sigma-Aldrich, milli-Q water, acetonitril HPLC grade (ACN, M = 41.05 g/mol, U.K.), that was obtained from Fischer Chemical and methanol (MeOH, M = 32.04 g/mol, Methanol HPLC, J.T. Baker, Deventer, Netherlands).

Milli-Q water (Elga, Purelab ultra, Model ULXXXAM2, SN ULT00002345, Buckinghamshire, UK. Millipore Elix Progard, PR0G00002, Merck Millipore, Billerica, Massachusetts, USA) was used throughout the study [48].

Silica gel (Fisher Scientific, U.K.) was used for the long-term stability study. Nitrogen, komprimerad (N2, M = 28.01 g/mol, Espoo, Finland) was used when preparing the co- amorphous mixture SMZ-PRO [49].

3.2 METHODS

3.2.1 Solid-state characterisation of the active compounds

3.2.1.1 Differential Scanning Calorimetry (DSC)

All drugs were characterised using a DSC (Mettler Toledo DSC 823e, Switzerland). The DSC was connected to a refrigerated cooling accessory (Mettler Toledo, METT – FT900 Julabo, Switzerland) and an autosampler (Mettler Toledo, TSO801RO, Sample Robot, Switzerland) to identify the Tg’s and Tm’s of the different compounds. The operating software of the DSC was STARe (Mettler Toledo, Switzerland). This machine allows us to verify the possible melting points, crystallisation temperatures and the glass transition temperatures of the compounds which were used in this study and project these values in thermograms [50].

Every sample was subjected to a predetermined temperature programme which involved six different steps. In the first segment the sample was kept at 25 °C for one minute. During the second segment the temperature was increased at a rate of 10 °C/min to five degrees above the melting point. At this temperature samples were held for five minutes (segment three). In the fourth segment the sample was cooled of at the same rate as above until -50 °C. At this temperature samples were held for 15 minutes (segment five). In the last segment the temperature was raised to ten degrees above the melting point of the desired compound, again using 10 °C/min [50].

The DSC cell was purged with nitrogen (50 mL/min). Reference standards (i.e., indium, lead, zinc, and highly purified water) were used to calibrate the temperature and the heat flow. A high precision microbalance (Sartorius SE2, Sartorius AG, Germany) was used to weigh the samples. Samples were made of pure CBZ, MEF and SMZ. Three samples were made of each compound in 40 µL aluminium pans (Mettler Toledo, Switzerland). A small hole was punched in the lid of each pan to ensure that the moist could evaporate. First the empty pans were weighed and then the full ones so that the amount of powder, that each pan contained, could be calculated [50].

The glass transition temperatures were determined as midpoint values and melting points as onset values [50].

3.2.2 Molecular modelling calculations

3.2.2.1 Selecting the conformations for miscibility calculations For more detailed information about the settings view annex table 2.1.

To predict the miscibility of the active compounds with amino acid(s) molecular modelling calculations were performed. Material Studio 5.5 (Accelrys Software Inc.) is a programme that allows us to draw the structure of the amino acids and active compounds. Each structure can exist in different conformations. The first step was to find the lowest possible energy conformation, i.e. global minimum energy structure, of each molecule at a fixed temperature [50].

The Quench protocol in Material Studio (Materials Studio 5.5, Forcite Module, Accelrys Software Inc.) was used to determine the different energy conformations. Calculations were fulfilled by COMPASS II force field and force field assigned charges. The first quench was performed at 2000 K. The temperature was set this high to allow free rotation of all bonds. Different temperatures were used for the second quench depending on the drug: CBZ 463 K, MEF acid 503 K, and SMZ 509 K. These temperatures are the melting points of the drugs, which were determined with the DSC (see section 3.2.1.1) [50].

The lowest conformation was determined by selecting the potential energies of all possible different conformations at 2000 K and plotting them in a graph. The conformation with the lowest potential energy was then selected and used in the second quench. The lowest energy conformation was saved for further calculation steps. We only continued working with the results obtained from the second quench, i.e. at the melting points [50].

3.2.2.2 Calculating the Flory-Huggins Interaction Parameter For more detailed information about the settings view annex table 2.2.

The Flory-Huggins model is a well-known and frequently used model. It allows us to understand the thermodynamics behind the mixing and phase-separation in binary systems. The miscibility was calculated between each amino acid (20 AA) and active compound (CBZ, MEF, SMZ). The lowest energy conformation of the drug was calculated with the lowest energy conformation of the amino acid. (Materials Studio 5.5, Blends toolbox, Accelrys Software Inc.)

First the binding energies, and the energy of mixing, Emix, were determined by equation 3.1:

1 ∆퐸 = [푍 (퐸 ) + 푍 (퐸 ) – 푍 (퐸 ) − 푍 (퐸 ) ] (3.1) 푚푖푥 2 푏푠 푏푠 푇 푠푏 푠푏 푇 푏푏 푏푏 푇 푠푠 푠푠 푇

With: Z: coordination number

E: binding energy

s and b: screen and base

χAB, the temperature-dependent Flory-Huggins interaction parameter, was derived from equation 3.2 below:

∆퐸푚푖푥 χ = (3.2) 퐴퐵 푅푇

With: R = gas constant

T = temperature

Last, the Gibbs free energy change of mixing was determined as a function of composition and temperature in equation 3.3:

∆퐺푚푖푥 = 푅푇 (푛퐴 ln ∅퐴 + 푛퐵 ln ∅퐵 + χ퐴퐵푛퐴∅퐵) (3.3)

With: ni: number of moles of components A and B

∅i: volume fractions of components A and B

The miscibility calculations were performed at 293 K for CBZ-AA and at the melting points of MEF and SMZ, respectively 503 K for MEF-AA and 509 K for SMZ-AA. The Flory-Huggins interaction parameter was analysed to evaluate the miscibility of the compounds.

These calculations are described more thoroughly in the following article ‘Phase Separation in Coamorphous Systems: in Silico Prediction and the Experimental Challenge of Detection’ [50].

3.2.3 Preparation of the binary mixtures The molar ratio calculations can be viewed in the annex section 3.

Based on previous calculations and melting points, three amino acids (PRO, GLN, GLU) were selected and the mixtures were investigated more thoroughly in this study.

The binary mixtures containing the drug and selected amino acid(s) were made in different molar ratios. For the first two drugs, CBZ and MEF, a 50:50 molar ratio was made with PRO. A 50:50 molar ratio was also used when making following mixtures: SMZ-CYS, SMZ-LYS, SMZ-SER, SMZ-ASN, SMZ-GLY, SMZ-ALA, SMZ-PRO, SMZ-ARG, SMZ-ASP, SMZ-THR, SMZ-GLN and SMZ-GLU. Afterwards several new combinations were made: 70:30 SMZ-GLN, 85:15 SMZ- GLN, 70:30 SMZ-GLU, 85:15 SMZ-GLU, 70:30 SMZ-PRO, 30:70 SMZ-PRO, 20:80 SMZ-PRO. They were mixed homogeneously, using an agate mortar and pestle, and stored in Eppendorf tubes of 1.5 mL.

3.2.4 Preparation of the amorphous materials

3.2.4.1 Melt-quench method

To make a crystal powder amorphous the melt-quench method was used. This method consists of taking a small amount of the desired powder and placing it on a microscope slide. Then the sample was heated on a normal heating stage (Heidolph Instruments MR Hei- Standard, Schwabach, Germany) to its melting point followed by cooling it down very quickly on a cold metal surface. The microscope slide could also be wrapped in aluminium foil before placing the powder on it, to make it easier to remove the amorphous substance. All materials, microscope slide and spatula, were cleaned beforehand using 70% denaturated ethanol.

3.2.4.2 Linkam heating stage

When using a Linkam heating stage the temperature can be controlled accurately. Overheating might be the case when executing the melt-quench method with a normal heating stage, thus making the Linkam heating stage the preferable choice. A Linkam heating Stage (Linkam Scientific Instruments Ltd., U.K.) allows us to select the heating temperature, the interval time, and the time that this temperature must be hold. The same preparation method applies to the Linkam heating stage. A small amount of powder was placed on thinner 19

microscope slides, that were cleaned beforehand with 70% denaturated ethanol. When the powder started to melt, it was immediately placed on a cold metal surface using a thumb forceps.

3.2.5 Stability studies

3.2.5.1 Chemical stability

Several solutions were made to check whether the active substance degraded upon melting and if the amino acid, used to stabilise the amorphous structure, had any influence.

First, a standard series of pure MEF and pure SMZ was made in 70 % ACN / 30 % H2O with concentrations of 0.1, 10, 20 and 30 µg/mL. These were analysed with HPLC by following the specific settings as described in 3.2.6. A calibration curve was made.

Heated MEF, as made in 3.2.4, was very gently grinded and a certain amount was weighed and dissolved in 70 % ACN / 30 % H2O to eventually have a concentration of 20 µg/mL. This was also analysed with HPLC. The same was done for heated SMZ. A decrease in concentration, which was calculated using the equation of the calibration curve, or extra peaks were interpreted as degradation of the API.

Besides the evaluation of the heated API’s, the crystalline and amorphous binary mixtures of MEF-PRO, SMZ-PRO were also analysed. The preparation methods of the samples are described in 3.2.3 and 3.2.4. A concentration of 20 µg/mL was made by diluting the stock solutions with 70 % ACN / 30 % H2O. A decrease in concentration, which was calculated using the equation of the calibration curve, or extra peaks were interpreted as degradation of the API’s or AA’s.

A separate sample of proline was made to check whether it absorbed UV light at the same wavelength and possibly could interfere with the peak of our compound. The proline sample was dissolved in milli-Q water.

3.2.5.2 Physical stability

Three pure, amorphous CBZ samples and three co-amorphous mixtures CBZ-PRO (50:50) were made on a microscope slide by following the protocol as described under 3.2.4.1. Three

co-amorphous mixtures of MEF-PRO (50:50) and three pure MEF samples were made on a microscope slide using the same quench method. Three co-amorphous mixtures of SMZ-GLN (50:50), SMZ-GLU (50:50), SMZ-PRO (50:50), and SMZ-PRO (20:80) were made on a microscope slide using the same quench method. Three samples of pure SMZ were also made.

Only the amorphous samples were stored in a desiccator under dry conditions (silica gel) at room temperature. If the sample recrystallised immediately after amorphisation, the stability study was ended at that point. To check whether there was any recrystallisation, they were monitored daily with a PLM until initiation of crystallisation occurred (see section 3.2.5.3).

3.2.5.3 Polarised Light Microscopy (PLM)

A polarised light microscope (Nikon LV100D, Japan) gives us a visual representation whether the samples, as made in 3.2.4, were amorphous or crystalline. To verify the physical stability of the amorphous samples, pure drugs, and combination amino acid-drug, the samples were monitored each day until they started to recrystallise [50]. When the sample was made on a microscope slide wrapped up in aluminium foil upper light strays were used. If the sample was made on a microscope slide, the sample could be illuminated from both directions.

3.2.6 High Performance Liquid Chromatography (HPLC)

Chemical degradation of the heated active compounds, the physical binary mixtures (drug + AA) and the amorphous binary mixtures was determined with HPLC (Shimadzu Corporation, Kyoto, Japan) using a SIL-20AC autosampler, LC-20AD pump and an SPD-10AVP UV/VIS detector. An Inertsil ® ODS-3 (4.0 x 150 mm) column (GL Sciences Inc., Fukushima Japan) was used and kept at 40.0 °C in a CT0-10AVP column oven. LC solution software (Shimadzu Corporation, Kyoto, Japan) was the operating software.

The mobile phase of MEF contained 75 % ACN, 25 % milli-Q water and 0.1 % TFA. The mobile phase of SMZ consisted of 70 % ACN, 30 % milli-Q water and 0.1 % TFA. TFA was used as an ion-pairing agent [51]. The flow rate was set to 1.0 mL/min for both drugs. The wavelength, λ, was set to 254 nm for MEF, and 260 nm for SMZ. The injection volume for the MEF samples was 30 µL and 10 µL for SMZ [26]. The analysing time for MEF was programmed to 3.25 minutes and 2.00 minutes for SMZ.

3.2.7 Intrinsic Dissolution Rate Study

The dissolution test was performed with a Distek Dissolution System 2100C (North Brunswick, NJ). Tablets for the dissolution test were made in stainless steel cylinders using a manual hydraulic press (Pye Unicam) [3]. The mass varied from 25 to 30 mg for MEF. Pure SMZ had about the same mass, however the mass of the physical and amorphous mixture was around 120 to 150 mg (Sartorius GMBH A200S, Gottingen, Germany). All tablets had a surface area of 0.5 cm2. A pressure of two ton was applied to each tablet for 30 seconds. Tablets were always made in triplicate and were then put into a vessel containing 600 mL milli-Q water using a time interval of two minutes. The tablets were stirred at room temperature 23. 2 °퐶 ± 1.6 °퐶 with paddles at 50 rpm.

Tablets were made of pure MEF, the physical mixture MEF-PRO (50:50) and the amorphous mixture of MEF-PRO (50:50). The same method was used for tablets of pure SMZ and the physical mixture SMZ-PRO (20:80). However, the amorphous mixture of SMZ-PRO (20:80) required a different preparation environment, i.e. a nitrogen atmosphere.

Samples of the three tablets were taken as described in the US Pharmacopeia [52]. This was performed at set timepoints for MEF: 15, 30, 45, 60, 120, 180, 240, 300, 1440, 1620, 2880, 3180, and 7200 minutes. The same timepoints were used for SMZ up until 180 minutes. 1000 µL was withdrawn each time and stored in Eppendorf tubes. This was replaced immediately with an equivalent volume of fresh milli-Q water to maintain a constant volume [53]. These samples were then centrifuged for 15 minutes at 13.000 rpm in an Eppendorf Minispin (Eppendorf, Hamburg, Germany). HPLC vials were made using 150 µL of the supernatant of the Eppendorf tubes. The concentration at every time point could be calculated using the equation of the standard curve.

A standard series is required to be able to interpret the dissolution rate. Concentrations of 0.01, 0.25, 0.50 and 0.75 µg/mL were made of MEF in 70 % ACN / 30 % H2O. The same series were made for SMZ with an additional five concentrations of 1.00, 2.00, 3.00, 10.00 and 20.00 µg/mL. These were analysed daily with HPLC (the specific settings are described in section 3.2.6) before analysing the dissolution samples.

4 RESULTS AND DISCUSSION

4.1 SOLID-STATE CHARACTERISATION OF CBZ, MEF, AND SMZ

All melting points (Tm) or glass transition temperatures (Tg) were calculated as an average of three samples. The average mass of the pans is listed above each table. More detailed information can be found in the annex table 1.1.

4.1.1 Carbamazepine (CBZ)

The DSC thermogram of CBZ showed different melting points. The results of each sample are presented in table 4.1. Two melting points were observed at 173.2 ± 0.8 °C and 190.1 ± 0.5 °C. After the first two melting points, two recrystallisation temperatures (Tc) were observed in figure 4.2.

A Tg however, was not detected. Previous articles have mentioned a melting point at 191-192 °C for the exact same compound. We continued working with a melting point of 190 °C throughout the study. This information was achieved from the Material Safety Data Sheet. Most likely the compound exists of two different polymorphs. CBZ is known to have two polymorphs that are enantiotropic. The melting temperatures of the two polymorphs are at 176 and a 189 °C [54]. Data indicates an enantiotropic relationship between polymorph III and I. Solid-solid phase transformation could have occurred during the heating process. The melting points after recrystallisation differ from the first two. Most likely these are not the pure forms anymore. The first thermal event is probably form I and the second event form III, the most stable form. To confirm which polymorphs are present in CBZ, we could perform X-ray powder diffraction (XRPD), Fourier transform infrared spectroscopy (FT-IR) or Raman spectroscopy and compare the results to those previously mentioned in literature [16]. The DSC shows that CBZ is a fast crystallising compound.

Table 4.1: Carbamazepine characterisation. Each DSC pan weighed approximately 3.560 mg. The first and second thermal event represent the melting points of two different polymorphs of carbamazepine. The third and the fourth thermal event represent the melting points after recrystallisation. These are lower compared to the first two, most likely indicating that they are not the pure forms. Thermal events (°C) 1st 2nd 3rd 4th CBZ 1 174.1 190.0 141.7 172.7 CBZ 2 172.5 189.7 142.5 171.9 CBZ 3 173.0 190.7 142.0 173.0 Average ± SD 173.2 ± 0.8 190.1 ± 0.5 142.1 ± 0.4 172.5 ± 0.5

Figure 4.2: DSC thermogram of carbamazepine. Upward peaks represent exothermic events. In between the first and last two thermal events we can observe two recrystallisation peaks. The specific values of the different events are illustrated in table 4.1.

4.1.2 Mefenamic acid (MEF)

The results of each sample are presented in table 4.3. The overall melting point of crystalline mefenamic acid was 229.9 ± 0.1 °C. The second and third melting point after recrystallisation were at 58.8 ± 5.1 and 187.7 ± 3.7 degrees Celsius.

The melting point matches the one previously mentioned in literature [55,56]. The DSC thermogram is shown in figure 4.4. The melting point at 230 °C was used to programme the following molecular modelling calculations. After the single recrystallisation peak, three different thermal events can be observed. It is known that mefenamic acid can exist in three

different polymorphic forms. Further investigation is needed to be able to associate these last thermal events to a specific polymorphic form. Perhaps X-ray powder diffraction or Fourier transform infrared spectroscopy could give us more insight of what the different events actually represent. MEF seems to be a fast crystallising compound.

Table 4.3: Mefenamic acid characterisation. Each DSC pan weighed approximately 2.806 mg. The first thermal event represents the melting point of mefenamic acid which is followed by one recrystallisation peak. When we increased the temperature a second time, three thermal events could be observed. These last two represent two different melting points. Thermal events (°C) 1st 2nd 3rd MEF 1 229.9 55.9 187.8 MEF 2 229.8 55.8 184.0 MEF 3 229.9 64.7 191.4 Average ± SD 229.9 ± 0.1 58.8 ± 5.1 187.7 ± 3.7

Figure 4.4: DSC thermogram of mefenamic acid. Upward peaks represent exothermic events. the specific values of the different events can be viewed in table 4.3.

4.1.3 Sulfamerazine (SMZ)

Sulfamerazine showed one melting point and one Tg, which can be viewed in Figure 4.6. The melting point was at 235.8 ± 0.1 °C and the Tg at 85.7 ± 5.2 °C, both illustrated in table 4.5.

The melting point at 235.8 °C correlates with the literature [57]. This temperature was used in the following section when performing the calculations. No recrystallisation peak can be observed in the DSC thermogram. So, we can assume that SMZ is a slowly crystallising compound. However, the standard deviation of the Tg is quite large. This might be due to an inconsistency when this event was integrated. Once the angle differs only a little bit, this could easily result in a bigger temperature difference. Perhaps when the integration would be performed again, we would have a more accurate glass transition temperature.

Table 4.5: Sulfamerazine characterisation. Each DSC pan weighed approximately 2.821 mg. The first thermal event represents the melting point of sulfamerazine. The second event represents a glass transition temperature. Thermal events (°C) Tm Tg SMZ 1 235.8 80.2 SMZ 2 235.9 86.2 SMZ 3 235.8 90.6 Average ± SD 235.8 ± 0.1 85.7 ± 5.2

Figure 4.6: DSC thermogram of sulfamerazine. Upward peaks represent exothermic events. The specific values of the different thermal events are shown in table 4.5. 26

4.2 MOLECULAR MODELLING CALCULATIONS AND SELECTION OF ADDITIVES AS STABILISING AGENT

4.2.1 Carbamazepine (CBZ)

All Chi parameters, illustrated in table 4.7, are negative at 298 K, i.e. room temperature, except for CBZ-ARG, CBZ-LYS and CBZ-GLN. Which means all other amino acids are miscible with CBZ. The calculations were performed three times for each binary system to achieve a more accurate Chi parameter. However, the differences between these calculations were fairly small, so we decided to run the calculations only once for the two other drugs, i.e. MEF and SMZ.

We decided to choose PRO and GLU as stabilising agents for CBZ. The Chi parameter of these mixtures were respectively -8.3 ± 0.1 and -9.4 ± 0.3. The coordination number of both mixtures is 6, meaning one PRO or GLU molecule is surrounded by six CBZ molecules. This is illustrated in figure 4.8 for CBZ-PRO. When χ has a small or negative value it suggests that at this temperature (room temperature) the two molecules have a favourable interaction. Most likely the mixture will only consist of one single phase at this temperature, resulting in one Tg. If the combination is miscible, a depression of the melting point of the drug can be observed [58]. When they are immiscible (Chi being positive) the amino acid and API will separate into two different phases. So, the amino acid cannot interact with or stabilise the API and no co- amorphous drug formulation can be formed.

However, our decision was not only based on these calculations. We also needed to consider the melting points of both drug and amino acid if we wanted to use melt-quenching as amorphisation method. As previously mentioned, this technique can only be applied to compounds that do not degrade upon melting. The melting points of CBZ are 173.2 and 190.1 °C (see section 4.1.1). So, another condition, besides the miscibility, is that the melting points of the API and excipient have to be quite similar. If the difference is too big, there is a great possibility that the API will overheat and degrade, defeating the purpose of our study. The melting point of proline is 220-222 °C. This information was achieved from Merck. The melting point of glutamic acid is 205 °C (obtained from Material Safety Data Sheet from Sigma-Aldrich). Usually when the mixture (drug-AA) is made, the melting point of the mixture is lower than the pure compounds, reducing the risk of thermal degradation.

We would have a more accurate result of the melting points of all AA if we would have determined them via DSC. The melting points of the AA used throughout this study are either based on literature or on the information that was mentioned by the supplier.

CBZ-SER, CBZ-ASN, CBZ-CYS, CBZ-ALA, CBZ-ILE, CBZ-LEU, all have a Chi parameter even more negative than CBZ-PRO. Meaning they would also be miscible when performing melt- quenching as amorphisation technique. Nevertheless, all melting points of these AA are higher than 230 °C.

The initial purpose of our study was to investigate four mixtures after performing molecular modelling calculations, i.e. two with a positive Chi parameter (CBZ-LYS and CBZ-GLN) and two with a negative Chi parameter (CBZ-PRO and CBZ-GLU). The aim was to verify whether the performed calculations gave a correct prediction of the miscibility. However, due to unfortunate circumstances, further discussed in section 4.3.1.1, the mixtures could not be analysed with HPLC. This was only noted after preparing the samples for the physical stability test of CBZ-PRO. So, no samples were made of CBZ-GLU.

To be sure that the chosen mixtures were completely miscible, we should have analysed the co-amorphous binary mixture of CBZ-PRO and CBZ-GLU via DSC. If one single glass transition temperature (Tg) would appear, we could have concluded that the mixture was homogeneous. Also, if the AA’s were analysed with the DSC, we could have calculated the theoretical Tg using the Gordon-Taylor equation and compared it to the experimental Tg. If the Tg would have been higher than the Tg of the pure drug compound, it would have had a positive influence on the physical stability study [10].

To check whether CBZ degraded upon melting and if PRO had any influence on the possible degradation of CBZ stability studies were performed (see sections 4.3.1.1 and 4.3.2.1).

Table 4.7: Calculating the Chi parameter of all amino acids with carbamazepine at room temperature, i.e. 298 K. Base + screen molecule at 463 K Chi (298 K) Average ± SD CBZ + ARG 1.7 1.6 1.0 1.4 ± 0.4 CBZ + HIS -4.1 -4.3 -3.9 -4.1 ± 0.2 CBZ + LYS 0.3 0.7 0.1 0.4 ± 0.3 CBZ + ASP -5.5 -5.6 -6.0 -5.7 ± 0.3 CBZ + GLU -9.1 -9.6 -9.6 -9.4 ± 0.3 28

CBZ + SER -16.1 -16.3 -15.8 -16.1 ± 0.2 CBZ + THR -2.1 -2.0 -2.0 -2.0 ± 0.1 CBZ + ASN -11.1 -10.3 -11.0 -10.8 ± 0.4 CBZ + GLN 1.7 1.7 2.0 1.8 ± 0.1 CBZ + CYS -8.2 -8.6 -8.4 -8.4 ± 0.2 CBZ + GLY -3.3 -3.1 -3.1 -3.1 ± 0.1 CBZ + PRO -8.3 -8.2 -8.5 -8.3 ± 0.1 CBZ + ALA -11.4 -11.4 -11.4 -11.4 ± 0.0 CBZ + VAL -1.2 -1.4 -1.0 -1.2 ± 0.2 CBZ + ILE -10.9 -11.5 -11.1 -11.1 ± 0.3 CBZ + LEU -10.7 -10.7 -10.6 -10.7 ± 0.1 CBZ + MET -6.7 -7.0 -6.5 -6.7 ± 0.2 CBZ + PHE -3.3 -2.8 -3.2 -3.1 ± 0.3 CBZ + TYR -5.6 -5.4 -5.8 -5.6 ± 0.2 CBZ + TRP -6.8 -5.6 -9.2 -7.19 ± 1.8

Figure 4.8: The central molecule represents proline, which is being surrounded by six carbamazepine molecules. The structures were drawn with Material Studio 5.5 (Accelrys Software Inc.). The blue colour represents nitrogen and the red colour represents oxygen. The coordination number six is an average of Zbs and Zsb, i.e. base-screen cluster and screen-base cluster. They usually differ when the volumes of screen and base molecule are different, which is clearly the case. When the volume of the base (drug) molecule is higher than the volume of the screen (AA) molecule, Zbs is usually higher than Zsb.

4.2.2 Mefenamic acid (MEF)

All Chi parameters for MEF, illustrated in table 4.9, are slightly positive at 503 K (melting point of MEF, see section 4.1.2). Based on these calculations combined with previous characterisation of MEF, PRO was selected as a stabilising agent to form the co-amorphous binary mixture MEF-PRO. MEF-PRO has a Chi parameter of 2.2 at 503 K. We tried making the combination amorphous by using the melt-quench method before finalising our decision. A complete amorphous sample could be achieved of this mixture.

The melting points of both drug and AA only slightly differ from each other, i.e. 229.9 °C and 220-222 °C. The coordination number is three, illustrated in figure 4.10, meaning one proline molecule is surrounded by three mefenamic acid molecules. However, because Chi is slightly positive this might indicate that phase separation will occur over time, i.e. the mixture is maybe partly miscible. To be sure which combinations are miscible, it might be better to perform the calculations at room temperature seen as this is the temperature where the samples are prepared at. Also, DSC characterisation of the binary mixture MEF-PRO could confirm whether the mixture is homogeneous by observing only one single glass transition temperature (Tg).

Both chemical and physical stability studies were performed to check whether thermal degradation occurred of MEF and if PRO had an influence on the degradation process.

Table 4.9: Calculating the Chi parameter of all amino acids with mefenamic acid at the melting point of mefenamic acid, i.e. 503 K. Base + screen molecule at 503 K Chi (503 K) MEF + ARG 2.0 MEF + HIS 2.0 MEF + LYS 0.7 MEF + ASP 0.5 MEF + GLU 1.4 MEF + SER 0.1 MEF + THR 1.8 MEF + ASN 0.3 MEF + GLN 0.7 MEF + CYS 1.6 MEF + GLY 1.4 MEF + PRO 2.2

MEF + ALA 1.4 MEF + VAL 1.3 MEF + ILE 2.0 MEF + LEU 1.6 MEF + MET 1.3 MEF + PHE 1.7 MEF + TYR 1.5 MEF + TRP 2.5

Figure 4.10: The central molecule also represents proline. This time it is only surrounded by three molecules of mefenamic acid. The structures were drawn with Material Studio 5.5 (Accelrys Software Inc.). The blue colour represents nitrogen and the red colour represents oxygen. The coordination number three is an average of Zbs and Zsb, i.e. base-screen cluster and screen-base cluster. A more detailed description can be found below figure 4.8.

4.2.3 Sulfamerazine (SMZ)

We chose CYS, LYS, SER, ASN and GLY to try and make a co-amorphous mixture grounded on calculations and melting points. The Chi parameters for SMZ are listed in table 4.11. These five amino acids had a Chi parameter that was negative (LYS, SER and CYS) or only slightly positive (ASN and GLY). However, none of the above-mentioned combinations resulted in a co- amorphous system. So, we also tried SMZ-ALA, SMZ-PRO, SMZ-ARG, SMZ-ASP, SMZ-THR, SMZ- GLN, and SMZ-GLU. The prepared samples, after melting and quench-cooling, can be viewed in figure 4.13. Obviously, some decomposition occurred because of the change in colour. A partially amorphous system could be obtained from SMZ-PRO (50:50). SMZ-GLN and SMZ-GLU resulted in a fully amorphous system. Combinations with HIS, VAL, ILE, LEU, MET, PHE, TYR, and TRP were not further analysed because of the major difference in melting points between drug and AA. Most likely degradation of SMZ would occur when analysing these mixtures. We tried

different molar ratios of the SMZ-PRO mixture to investigate which of the two components was inducing the recrystallisation. The 70:30 SMZ-PRO mixture was not amorphous. So, clearly SMZ was the agent inducing recrystallisation at room temperature. Luckily, the 30:70 and 20:80 mixture of SMZ-PRO were amorphous.

The following combinations were further analysed throughout the study: SMZ-PRO (30:70 and 20:80), SMZ-GLN (50:50, 70:30 and 85:15) and SMZ-GLU (50:50, 70:30 and 85:15). Nevertheless, we did not check whether the mixtures were perfectly homogeneous. This could have been done with the DSC. The samples were also only checked visually whether they were amorphous using a PLM. The DSC also could have been used to check whether only a single glass transition temperature (Tg) could be observed.

Unfortunately, our results did not match our predicted calculations. This could be explained by the fact that Chi was determined at the melting point of the drug and not at room temperature. Our calculations do not take the kinetics into consideration that occur at room temperature. Another explanation could be that they might be miscible, but maybe melting and quench-cooling is not the most ideal method because of the decomposition at the melting point.

Both chemical and physical stability test were performed to check whether thermal degradation occurred of SMZ and if PRO, GLN, or GLU had any influence on the possible degradation process.

Table 4.11: Calculating the Chi parameter of all amino acids with sulfamerazine at the melting point of sulfamerazine, i.e. 509 K. Base + screen molecule at 509 K Chi (509 K) SMZ + ARG 0.7 SMZ + HIS 0.8 SMZ + LYS -0.4 SMZ + ASP -0.0 SMZ + GLU 0.6 SMZ + SER -0.3 SMZ + THR 0.6 SMZ + ASN 0.7 SMZ + GLN 0.3 SMZ + CYS -0.1 SMZ + GLY 0.5 SMZ + PRO 0.7 32

SMZ + ALA -0.1 SMZ + VAL -0.1 SMZ + ILE 1.3 SMZ + LEU 1.0 SMZ + MET -0.3 SMZ + PHE 0.7 SMZ + TYR 0.2 SMZ + TRP 1.5

Figure 4.12: Four sulfamerazine molecules are surrounding proline. The structures were drawn with Material Studio 5.5 (Accelrys Software Inc.). The blue colour represents nitrogen, the red colour represents oxygen and the yellow colour represents sulphur. The coordination number four is an average of Zbs and Zsb, i.e. base-screen cluster and screen-base cluster. A more detailed description can be found below figure 4.8.

Figure 4.13: 50:50 molar ratio of sulfamerazine with nine different amino acids. Pure sulfamerazine crystallised immediately after amorphisation. SER-SMZ turned black. In some of the other samples, SMZ-CYS, SMZ-LYS, SMZ-ASN, and SMZ-ARG the powder does not entirely melt even though the samples were heated way above their melting points. SMZ- GLY, SMZ-ALA, and SMZ-THR crystallised instantly after melt-quenching. The dark colour clearly indicates that something is being degraded upon melting. None of these amino acids can stabilise the amorphous form of SMZ, so they were not further investigated in this study.

4.3 STABILITY STUDIES

4.3.1 Chemical stability

4.3.1.1 Carbamazepine (CBZ)

We analysed the same HPLC vial of pure CBZ multiple times. Each analysis gave a different result. We tried several times to optimise the HPLC method, however no good method was found for this compound. Therefore, it was not possible to make a calibration curve and evaluate the thermal degradation. Unfortunately, the crystalline binary mixture and amorphous binary mixture could not be analysed either. A different column perhaps might solve this problem, but this was not available during our study period.

However, research tells us that CBZ can be investigated when using a different column e.g. Discovery ® C18. It has been proven that CBZ can be stabilised by its receptor amino acid TRP when prepared by vibrational ball milling. It was stable for over a period of six months and had improved dissolution properties compared to the crystalline counterpart. Nevertheless, this compound was purchased from Hawkins, Inc. Pharmaceutical Group and could consist of different polymorphs than the compound we purchased [3].

4.3.1.2 Mefenamic acid (MEF)

The retention time of MEF was approximately 2.87 ± 0.00 min. The standard curve had a correlation coefficient R2 of 0.9994 and is illustrated in figure 4.14. The error bars are also displayed, but these are very small, so they cannot be seen.

The chromatogram of heated MEF, obtained by HPLC, showed an extra peak at 1.30 min resulting in a decrease in concentration. The results are listed in table 4.15. Furthermore, the crystalline binary mixture MEF-PRO also showed a slight decrease in concentration (table 4.16). Multiple samples were made of the amorphous binary mixture. They all had an original concentration of 20 µg/mL. When analysing the samples of the amorphous binary mixture of MEF-PRO an extra peak appeared at approximately 3.71 min. This extra peak is responsible for continuous reduction in concentration from 20 µg/mL to 18.26 µg/mL. When we analysed the pure proline sample, we noticed that this amino acid absorbs UV light at the same wavelength

as MEF, i.e. 254 nm. A single peak appeared at 1.33 ± 0.00 min. However, this does not interfere with the peak of MEF.

Standard curve Mefenamic acid 1400000 y = 44006x - 8598.8 1200000 R² = 0.9994

1000000

800000

600000 Peak Peak area

400000

200000

0 0 5 10 15 20 25 30 Concentration (µg/mL)

Figure 4.14: Standard calibration curve of mefenamic acid where the peak area is plotted as a function of the concentration in µg/mL.

Above-mentioned results indicate that MEF partially degrades when using the melt quench method. The small loss of concentration could also be due to the small amount of powder that had to be weighed and/or mistakes when pipetting. So, this is not the most ideal method to make the binary mixture MEF-PRO amorphous, though the degradation was fairly small, i.e. 8.7 %, and tolerated for further analysis. Anyhow, no different amorphisation methods, that use amino acids as a stabilising agent, have been mentioned in literature yet. Perhaps milling or spray/freeze-drying might be a good alternative to avoid the thermal degradation.

Table 4.15: Results of heated mefenamic acid. Theoretical Measured Retention Extra concentration concentration time (min) peak (min) (µg/mL) (µg/mL) 2.81 1.30 2.87 1.30 21.12 20.23 2.86 1.30 2.87 1.30 Average ± SD 2.85 ± 0.03 1.30 ± 0.00

Table 4.16: Results of the crystalline binary mixture mefenamic acid-proline. Theoretical concentration Measured concentration Retention (µg/mL) (µg/mL) time (min) 2.88 20.00 19.22 2.89 2.89 Average ± SD 2.89 ± 0.01

4.3.1.3 Sulfamerazine (SMZ)

The retention time of SMZ was approximately 1.36 ± 0.00 min. The standard curve had a correlation coefficient R2 of 0.9998 and is illustrated in figure 4.17.

Standard curve Sulfamerazine 1200000

y = 37192x - 7568.9 1000000 R² = 0.9998

800000

600000 Peak Peak area 400000

200000

0 0 5 10 15 20 25 30 Concentration (µg/mL)

Figure 4.17: Standard calibration curve of sulfamerazine where the peak area is plotted as a function of the concentration in µg/mL.

The results of heated SMZ can be viewed in table 4.18. The concentration decreased of pure SMZ from 20 µg/mL to 17.81 µg/mL upon heating. The thermal degradation was investigated of the following combinations that are listed in table 4.19. The crystalline physical mixture of the 50:50 SMZ-GLU and 50:50 SMZ-GLN both had a theoretical concentration of 20 µg/mL. The measured concentrations were respectively 21.27 µg/mL and 18.62 µg/mL.

As opposed to the chromatograms of heated SMZ and the crystalline mixtures, which had a single peak at 1.36 min, the amorphous mixtures showed multiple peaks. Two small peaks appeared each time before the peak of SMZ for all molar ratios. Different molar ratios were

analysed to minimise the area of the two peaks and thus, the degradation. When increasing the percentage of SMZ the concentration also became greater. The highest concentrations that could be achieved for SMZ-GLU and SMZ-GLN were very similar at an 85:15 molar ratio, i.e. 17.24 µg/mL and 17.17 µg/mL. The smallest area of the two peaks was achieved with SMZ-PRO 20:80. The crystalline mixture had a concentration of 18.53 µg/mL and the amorphous mixture 17.97 µg/mL. The 30:70 mixture of SMZ-PRO had an even higher concentration, i.e. 18.93 µg/mL, yet the peaks were not as ‘nice’ as the 20:80 ratio. Proline did not absorb UV light at the set wavelength.

So, clearly GLN and GLU degrade the API upon amorphisation. Similar results were achieved for both combinations. The explanation lies in the fact that their structures are almost identical. They only differ in the amino group, – NH2, which is attached to the carboxyl functional group of GLN. Besides the amino acids, the accuracy of pipetting and weighing can also explain the lower concentrations than expected. The melt quench method, as previously mentioned, is not the best method for amorphisation since all amino acids induce degradation of SMZ. Nevertheless, the mixture of SMZ-PRO 20:80 was used for the dissolution study despite a decrease in concentration, i.e. 10.15 %. Maybe when using a different amorphisation method, we could avoid this degradation. We could possibly perform spray-drying, freeze-drying, and milling. Afterwards we could compare the results of all the techniques and choose the one with the highest efficiency.

Table 4.18: Results of heated sulfamerazine. Theoretical concentration Measured concentration Retention (µg/mL) (µg/mL) time (min) 1.36 20.00 17.81 1.35 1.36 Average ± SD 1.35 ± 0.00

Table 4.19: Multiple binary mixtures with varying molar ratios of the following combinations: sulfamerazine-glutamic acid, sulfamerazine-glutamine, and sulfamerazine-proline. SMZ-GLU 50:50 70:30 85:15 SMZ-GLN 50:50 70:30 85:15 SMZ-PRO 30:70 20:80

4.3.2 Physical stability The individual results of each API and stabilising amino acid can be viewed in the annex.

As previously mentioned in the introduction, the storage conditions are usually 50 °C lower than the Tg of the amorphous mixture to maintain the glassy state. Nevertheless, we did not determine the Tg of the amorphous mixtures with the DSC. So, we could not determine the exact temperature the samples had to be stored at to remain amorphous for a pharmaceutical relevant timeframe.

4.3.2.1 Carbamazepine (CBZ) The results of the stability test of CBZ can be viewed in the annex table 5.1.

Two pure CBZ sample stayed amorphous for at least 17 days. Meanwhile the other sample started to crystallise in less than a day. The binary mixture CBZ-PRO stayed amorphous for only two days. However, we are interested in an excipient which stabilises the amorphous form of the active compound. This is not the case, so CBZ was not further analysed.

It has been mentioned in the literature that degradation products can stabilise the amorphous form of the drug [59]. Perhaps this might be the case for carbamazepine. The crystallisation of the pure CBZ sample in less than a day is most likely due to an impurity that was introduced during preparation. Crystallisation is a random process, so it is very important to prevent all influences that could lead to this process.

4.3.2.2 Mefenamic acid (MEF) The results of the stability test of MEF can be viewed in the annex table 5.2.

The three pure MEF samples crystallised immediately after melt quenching. These were not further analysed in this study. The co-amorphous mixture MEF-PRO stayed amorphous up until six days. This is an average that was made of three samples.

Six days nonetheless is not a relevant time period towards the pharmaceutical industry. Perhaps after determining the Tg of the co-amorphous mixture via DSC, we would have been able to optimise the storage conditions. Not only the storage conditions play a significant role in preventing recrystallisation, i.e. molecular mobility. Also, molecular interactions [60,61]

inclusion of impurities [3] and molecular level mixing [62] are crucial. Mixing amino acids with amorphous drugs can also introduce impurities in the amorphous drug.

4.3.2.3 Sulfamerazine (SMZ) The results of the stability test of SMZ can be viewed in the annex table 5.3 and 5.4.

The three pure SMZ samples also crystallised immediately after melt quenching. The outer sides of the 50:50 molar ratio sample of SMZ-PRO crystallised immediately. Although the centre of the sample initially remained amorphous. SMZ-PRO (20:80) stayed amorphous for five days. SMZ-GLU (50:50) started to crystallise after ten days. SMZ-GLN (50:50) stayed amorphous for at least 27 days.

Anyhow we are interested in a mixture that stays stable for a pharmaceutical relevant time frame. This is not the case for both SMZ-PRO and SMZ-GLU. Maybe we would have been able to optimise the storage temperature after determining the Tg of the co-amorphous binary mixtures via DSC. SMZ-GLN did however stay stable for at least 27 days. This should be investigated over a longer period of time to be able to draw more definite conclusions, but it does show potential.

4.4 INTRINSIC DISSOLUTION RATE STUDY

4.4.1 Mefenamic acid (MEF) The complete dissolution curve (up to five days) can be viewed in the annex table 4.23.

The 50:50 molar ratio of MEF-PRO was further investigated to evaluate the possible intrinsic dissolution advantages. In figure 4.20 A the concentration-time profiles (µg/mL) are shown for pure MEF, the physical mixture MEF-PRO and the co-amorphous mixture MEF-PRO. The dissolution study lasted 5 days. However, only the first two hours gave a fairly linear fit. After two hours MEF started to level off, i.e. saturation.

To convert graph (A) to graph (B) we had to calculate the accessible surface area of the drug in our binary mixture. The mixture contained 101.4 mg MEF and 48.4 mg PRO giving us a total mass of 149.8 mg. The densities of the two compounds, MEF and PRO, were 1.203 g/cm3 and 1.350 g/cm3. The volumes of the individual compounds before compression were respectively, 0.084 cm3 and 0.036 cm3. The total surface area of the tablets was 0.5 cm2 which contained 70% MEF and 30% PRO, i.e. 0.35 cm2 and 0.15 cm2. The accessible surface area of pure MEF was 0.5 cm2. Under the assumption that the surface area remains constant throughout the dissolution study, the release-time profiles could be calculated and are illustrated in figure 4.20 B. The drug concentrations were divided by the accessible area of the drug and then multiplied by 600 mL. The calculations were modified from Löbmann K. 2013 and Allesø M. 2009 [3,60].

The intrinsic dissolution rate of crystalline MEF was 9.3725 µg cm-2 hour-1. The amorphous mixture had a dissolution rate that was almost three times higher than pure MEF. Although, we have to take into consideration that the error bars are considerably large. The dissolution rates of crystalline MEF and the physical mixture were quite similar, though slightly higher for crystalline MEF. Proline seems to inhibit the dissolution process of MEF to a certain extent.

We only checked whether the samples were amorphous visually by using a PLM. Whilst it would have been better if we would have verified if only one Tg was present using the DSC, meaning the mixture was perfectly miscible and homogeneous. The error bars are very large for the amorphous mixture compared to crystalline MEF and the crystalline binary mixture. 40

However, there is no overlap between the error bars. So, there is definitely a difference in dissolution rate. To verify how much faster the amorphous form dissolves, it would be better to perform the dissolution test again for the amorphous mixture. (A) 0.070 Crystalline MEF y = 0.015x + 0.0344 Crystalline binary mixture MEF - PRO R² = 0.8772 0.060 Amorphous binary mixture MEF - PRO

0.050 ) y = 0.0078x + 0.025

µg/mL 0.040 R² = 0.9526

0.030

Concentration( y = 0.0046x + 0.0078 0.020 R² = 0.9741

0.010

0.000 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Time (hours) (B) Crystalline MEF 140 Crystalline binary mixture MEF-PRO Amorphous binary mixture MEF-PRO 120 y = 25.778x + 58.948 R² = 0.8772

100 )

80 µg/cm2

60 y = 9.3725x + 30.036

R² = 0.9526 Release Release ( 40 y = 7.852x + 13.329 R² = 0.9741 20

0 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Time (hours)

Figure 4.20: Intrinsic dissolution profiles of mefenamic acid. Besides the amorphous mixture 41

mefenamic acid-proline, the physical binary mixture and pure compound were also analysed. Assuming the mixture is homogeneous, and the surface area remains constant throughout the study, we can convert the (A) concentration-time profile (µg/mL) into (B) release-time profile (µg/cm2).

4.4.2 Sulfamerazine (SMZ) The complete dissolution curve (up to three hours) can be viewed in the annex table 4.34.

Based on the performed stability studies, the 20:80 molar ratio of SMZ-PRO seemed like it had the most potential to be further investigated on whether it had possible dissolution advantages. In figure 4.21 A the concentration-time profiles (µg/mL) are shown for pure SMZ, the physical mixture SMZ-PRO and the amorphous mixture SMZ-PRO. The dissolution study lasted a total of three hours. After three hours the tablets started to disintegrate. So, the assumption of a constant surface area would have no longer be valid. Only during the first hour there was a good linear fit.

To convert graph (A) to graph (B) we had to calculate the accessible surface area of the drug in our binary mixture. The mixture contained 166.7 mg SMZ and 290.5 mg PRO giving us a total mass of 457.2 mg. The densities of the two compounds, SMZ and PRO, were 1.439 g/cm3 and 1.350 g/cm3. The volumes of the individual compounds before compression were respectively, 0.116 cm3 and 0.215 cm3. The total surface area of the tablets was 0.5 cm2 which contained 35% MEF and 65% PRO, i.e. 0.175 cm2 and 0.325 cm2. The accessible surface area of pure SMZ was 0.5 cm2. Under the assumption that the surface area remains constant throughout the dissolution study, the release-time profiles could be calculated and are illustrated in figure 4.21 B. The calculations were modified from Löbmann K. 2013 and Allesø M. 2009 [3,60].

The intrinsic dissolution rate of crystalline SMZ was 454.93 µg cm-2 hour-1. The amorphous mixture had a dissolution rate that was almost twenty times higher than pure SMZ. The dissolution rate of the physical mixture was 1698.1 µg cm-2 hour-1, i.e. almost four times greater than crystalline SMZ.

The mass of the tablets of the physical and amorphous mixture had to be higher than the pure compound. Seen as the surface area was made up out of 65% of PRO, it caused the tablets to disintegrate practically immediately after starting the dissolution test with tablets of

25 to 30 mg. This was due to the high solubility of PRO in water, i.e. 1500 g/L in H2O at 20 °C. This information was achieved from Merck. Amorphous SMZ-PRO tablets could not be made in a normal atmosphere due to the high hygroscopicity. So, they were made in a nitrogen atmosphere.

The crystalline binary mixture had better dissolution properties compared to the pure crystalline drug. This indicates that the amino acid interacts with the drug in such a way that the drug dissolves slightly quicker. The difference is significant seen as the error bars do not overlap.

Although the dissolution rate appeared to be twenty times higher, degradation still occurred. When analysing the HPLC thermograms the two peaks, as mentioned is 4.3.1.3, were much higher because of the initial lower concentrations. Only the last peak was integrated because we knew this was the peak of SMZ. Also, we did not check whether the mixture was homogeneous, i.e. only one Tg. So, we are not completely sure that the mixture was fully amorphous. Possibly when using a different amorphisation method this could lead to a potential new drug formulation with increased dissolution properties.

(A) Crystalline SMZ 4.000 Crystalline binary mixture SMZ- PRO 3.500 Amorphous binary mixture SMZ- y = 2.5789x + 0.6703 PRO R² = 0.9922 3.000

2.500 µg/mL)

2.000

1.500 Concentration( 1.000 y = 0.4953x + 0.1285 R² = 0.9992 0.500 y = 0.3791x + 0.1101 0.000 R² = 0.9925 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 Time (hours)

(B)

14000 Crystalline SMZ

Crystalline binary mixture SMZ- 12000 PRO Amorphous binary mixture SMZ- y = 8842x + 2298.3 PRO R² = 0.9922

10000 )

8000 µg/cm2

6000 Release Release (

4000 y = 1698.1x + 440.51 R² = 0.9992 2000 y = 454.93x + 132.18 R² = 0.9925 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 Time (hours) Figure 4.21: Intrinsic dissolution profiles of sulfamerazine. Besides the amorphous mixture sulfamerazine-proline, the physical binary mixture and pure compound were also analysed. Assuming the mixture is homogeneous, and the surface area remains constant throughout the study, we can convert the (A) concentration-time profile (µg/mL) into (B) release-time profile (µg/cm2). 44

5 CONCLUSIONS

Carbamazepine was not further analysed in this study because the ideal method for analysis was not found, so thermal analysis could not be performed. A different column perhaps might solve this problem, but this was not available during our study period. Also, the long-term stability of CBZ-PRO (50:50) was shorter compared to pure CBZ and defeating the purpose of our study.

Mefenamic acid was combined with PRO as a stabilising additive in a 50:50 molar ratio. When analysing the thermal degradation with HPLC, a loss in concentration was found of 8.7 % while using melting followed by quench-cooling as amorphisation method. MEF-PRO stayed amorphous for six days. Maybe if the Tg of the mixture was determined via DSC, we would have been able to optimise the storage conditions hopefully resulting in a sample that would stay amorphous for a pharmaceutical relevant timeframe. The amorphous mixture of MEF-PRO seemed to dissolve at a higher rate than crystalline MEF. However, no definite conclusions could be made seen as the error bars are quite large compared to those of the crystalline drug and crystalline binary mixture. Also, we did not define whether the mixture was homogeneous. This could have easily been verified by analysing the amorphous sample with DSC, meanwhile confirming the miscibility calculations. Most likely there is a difference in dissolution rate. The exact rate however could not be determined seen as the amino acid partly induced degradation of the API as well.

Sulfamerazine was also combined with PRO as a stabilising agent, but in a 20:80 molar ratio. Sulfamerazine clearly degraded upon melting. PRO increased the degradation of SMZ. A loss in concentration was found of 10.15 %. SMZ-PRO stayed amorphous for five days. Maybe if the Tg of the mixture was determined via DSC, we would have been able to optimise the storage conditions resulting in a sample that would stay amorphous for a pharmaceutical relevant timeframe. The amorphous mixture of SMZ-PRO clearly had a dissolution rate that was twenty times higher compared to crystalline SMZ. However, degradation of SMZ occurred and we did not verify whether the mixture was homogeneous, so the definite rate could not be determined. Maybe when performing a different amorphisation method this could lead to a potential new formulation with increased dissolution properties.

In conclusion, both drugs degraded upon melting. Perhaps if we could perform multiple amorphisation methods, i.e. ball milling, freeze drying, and spray drying, we could compare the results of these methods and select the one with the best efficiency.

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ANNEX 1. DSC: sample preparation of the three active compounds: CBZ, MEF, and SMZ

Reference mass of the pan: 49.5320 mg

Table 1.1: Calculating the amount of powder in each DSC pan. Empty pan (mg) Full pan (mg) Mass in pan (mg) CBZ 1 49.3169 52.6601 3.3432 CBZ 2 48.9128 53.0261 4.1133 CBZ 3 48.8617 52.0866 3.2249 MEF 1 49.4211 51.7190 2.2979 MEF 2 49.1586 52.6790 3.5204 MEF 3 48.7380 51.3375 2.5995 SMZ 1 50.0764 53.6633 3.5869 SMZ 2 48.8216 51.8695 3.0479 SMZ 3 49.7598 51.5868 1.8270

2. Molecular modelling calculations: BIOVIA Materials studio 2017 R2 1) Draw the structure in 3D atomistic document 2) Forcite tools → calculation → Table 2.1: Settings used to calculate all the possible conformations. Setup Energy Job control Task: Quench Forcefield: COMPASS|| Gateway location: My Quality: Ultra- fine Charges: Forcefield computer assigned Job description: ✓ MORE Quality: ultra-fine automatic Quench every: 1000 steps Run in parallel: 1 of 12 Dynamic options: more MORE cores Dynamics ✓ calculate automatically Ensemble: NVT ✓ list all forcefield types MORE Initial velocities: random Charges Live updates Temperature: 2000 K and Forcefield assigned  update structure 463 K Charge groups ✓ update graphs Time step: 1.0 fs (10-15) ✓ calculate automatically ✓ update textual results Total simulation time: 10.0 Summation method Update every 5.0 ps Electrostatic: Atom based seconds Number of steps: 10 000 Van der Waals: Atom based When job completes Advanced  retain server files Energy deviation: MORE ✓ automatically view 500000000.0 kcal/mol Electrostatic output ✓ include velocities Summation method: ✓ notify on job  include forces Atom based completion Truncation: Cubic spline

Geometry optimization options: Cutoff distance: 18.5 Å more Spline width: 1 Å Algorithm: smart Buffer width 0.5 Å Quality: ultra- fine ✓ energy: 2.0 e-5 kcal/mol ✓ force: 0.001 ✓ displacement 1.0 e-5 kcal/mol/Å Max iterations: 500  keep motion groups rigid

→ select folder by clicking on it twice → Run Selection the conformation with the lowest total potential energy; you can sort the table by selecting the total potential energy column and click on the A Z button. Then to view the lowest conformation you can quickly plot a graph. Select the lowest conformation and copy paste this in a new atomistic document. Do this at a temperature of 2000 K and 463 K.

3) The next step is the miscibility calculation. For this calculation we are going to use the

BLENDS tool. Select calculation. Table 2.2: Settings behind the blends calculation. Setup Energy Job control Task: mixing Forcefield: COMPASS|| Gateway location: My Quality: ultra-fine Charges: Forcefield computer assigned Job description: Blend CBZ + AA Input: Quality: ultra-fine Select CBZ geometry MORE optimisation at 463 K MORE Live updates which fulfils the role of the ✓ calculate automatically  update structure base ✓ list all forcefield types ✓ update graphs Select 1 of the amino acids Charges ✓ update textual results at 463 K which fulfils the Forcefield assigned Update every 5.0 seconds role of the screening Charge groups When job completes molecule ✓ calculate automatically  retain server files Summation method ✓ automatically view output MORE Electrostatic: Atom based ✓ notify on job completion Energy samples: 10 000 Van der Waals: Atom 000 based Energy bin width: 0.02 kcal/mol

Cluster samples: 100 000 Iterations per cluster: 20 MORE ✓ head and tail atoms Electrostatic are non – contact Summation method:  return lowest energy Atom based frames Truncation: None Reference temperature: 298,0 K

→ select the amino acid which you want the screen together with the base (drug, in this case CBZ) and click on ‘run’

THEORY BEHIND THE BLENDS CALCULATION Chi = χ = interaction parameter When chi is negative, the combination is miscible When chi is positive, the combination is less miscible and will likely separate into two phases < - 5: extremely miscible - 2: borderline (phase separation as a function of time)

Base molecule = drug (CBZ, MEF and SMZ) Screen molecule = 20 amino acids

Coordination number = how many molecules can surround the drug compound Interaction energy = total energy - energy carbamazepine alone - energy AA alone

3. Sample preparations and analysis 3.1. Carbamazepine 3.1.1. Making of 20 µg/mL CBZ to determine HPLC method

2.79 mg was weighed and dissolved in a 50 mL volumetric flask.

The concentration of this solution (stock) was:

2.79 푚푔 µ푔 = 55.8 50 푚퐿 푚퐿

For a final concentration of 20 µg/mL we needed:

µ푔 55.8 ∗ 푥 µ퐿 µ푔 푚퐿 = 20 1000 µ퐿 푚퐿

↔ 푥 µ퐿 = 358.42 µ퐿 ≅ 358.4 µ퐿 of the CBZ stock solution.

To have 1 mL in total, in an Eppendorf tube, we still needed to add:

1000 µ퐿 − 358.4 µ퐿 = 641.6 µ퐿 of 70 % ACN / 30 % H2O

400 µL was then pipetted into an HPLC vial for analysis.

3.1.2. Standard calibration curve

2.8396 mg was weighed and dissolved in a 50 mL volumetric flask.

The concentration of this solution (stock) was:

2.8396 푚푔 µ푔 = 56.8 50 푚퐿 푚퐿

Concentrations of 0.1, 10.0, 20.0 and 30.0 µg/mL CBZ were prepared out of this stock solution. A volume of 1 mL was prepared in a 1.5 mL Eppendorf tube. 400 µL of each Eppendorf tube was pipetted in an HPLC vial for analysis.

3.1.3. Preparing 50:50 molar ratios of the binary mixtures, CBZ-AA

Table 3.1: Mass of each compound that was weighed to attain a 50:50 molar ratio. Mass carbamazepine (mg) Mass amino acid (mg) CBZ-PRO 199.8 97.5

3.2. Mefenamic Acid 3.2.1. Making of 20 µg/mL to determine HPLC method

3.4215 mg was weighed and dissolved in a 50 mL volumetric flask.

The concentration of this solution (stock) was:

3.4215 푚푔 µ푔 = 68.4 50 푚퐿 푚퐿

For a final concentration of 20 µg/mL we needed:

µ푔 68.4 ∗ 푥 µ퐿 µ푔 푚퐿 = 20 1000 µ퐿 푚퐿

↔ 푥 µ퐿 = 292.40 µ퐿 ≅ 292.4 µ퐿 of the MEF stock solution.

To have 1 mL in total, in an Eppendorf tube, we still needed to add:

1000 µ퐿 − 292.4 µ퐿 = 707.6 µ퐿 of 70 % ACN / 30 % H2O

400 µL was then pipetted into an HPLC vial for analysis.

3.2.2. Standard calibration curve

The following concentrations were prepared by using the above-mentioned stock solution: 0.1, 10.0, 20.0 and 30.0 µg/mL MEF. A volume of 1 mL was prepared in a 1.5 mL Eppendorf tube. 400 µL of each Eppendorf tube was pipetted in an HPLC vial for analysis.

3.2.3. Preparing 50:50 molar ratios of the binary mixtures, MEF-AA

Table 3.2: Mass of each compound that was weighed to attain a 50:50 molar ratio. Mass mefenamic acid (mg) Mass amino acid (mg) MEF-PRO 100.06 47.70

3.3. Sulfamerazine 3.3.1. Making of 20 µg/mL to determine HPLC method

0.5560 mg was weighed and dissolved in a 20 mL volumetric flask.

The concentration of this solution (stock) was:

0.5560 푚푔 µ푔 = 27.8 20 푚퐿 푚퐿

For a final concentration of 20 µg/mL we needed:

µ푔 27.8 ∗ 푥 µ퐿 µ푔 푚퐿 = 20 1000 µ퐿 푚퐿

↔ 푥 µ퐿 = 719.42 µ퐿 ≅ 719.4 µ퐿 of the SMZ stock solution.

To have 1 mL in total, in an Eppendorf tube, we still needed to add:

1000 µ퐿 − 719.4 µ퐿 = 280.6 µ퐿 of 70 % ACN / 30 % H2O

400 µL was then pipetted into an HPLC vial for analysis.

3.3.2. Standard calibration curve

1.2669 mg was weighed and dissolved in a 20 mL volumetric flask.

The concentration of this solution (stock) was:

1.2669 푚푔 µ푔 = 63.3 20 푚퐿 푚퐿

Concentrations of 0.1, 10.0, 20.0 and 30.0 µg/mL SMZ were made from this stock solution. A volume of 1 mL was prepared in a 1.5 mL Eppendorf tube. 400 µL of each Eppendorf tube was pipetted in an HPLC vial for analysis.

3.3.3. Preparing 50:50 molar ratios of the binary mixtures, SMZ-AA

Table 3.3: Mass of each compound that was weighed to attain a 50:50 molar ratio. Mass sulfamerazine (mg) Mass amino acid (mg) SMZ-CYS 100.1 46.2 SMZ-LYS 83.6 46.3 SMZ-SER 95.6 38.3 SMZ-ASN 95.7 47.7 SMZ-GLY 94.9 26.8 SMZ-ALA 96.9 32.9 SMZ-PRO 93.1 40.7 SMZ-ARG 96.7 63.7 SMZ-ASP 96.1 48.4 SMZ-THR 92.3 41.4 SMZ-GLN 93.4 51.5 SMZ-GLU 89.5 49.8

3.3.4. Preparing 70:30 molar ratios of the binary mixtures, SMZ-AA

Table 3.4: Mass of each compound that was weighed to attain a 70:30 molar ratio. Mass sulfamerazine (mg) Mass amino acid (mg) SMZ-PRO 103.9 19.4 SMZ-GLN 100.1 23.8 SMZ-GLU 100.1 23.8

Making 20 µg/mL of these 70:30 binary mixtures after successful amorphisation

SMZ-PRO

 This was not amorphous, so we stopped analysing this mixture.

SMZ-GLN

100.1 푚푔 0.6453 푚푔 0.6453 푚푔 µ푔 = ↔ = 32.26 123.9 푚푔 0.7987 푚푔 20 푚퐿 푚퐿

So, we needed:

- 620.0 µL of the stock solution

- 380.0 µL of 70 % ACN / 30 % H2O

SMZ-GLU

100.1 푚푔 0.7546 푚푔 0.7546 푚푔 µ푔 = ↔ = 37.73 123.9 푚푔 0.9340 푚푔 20 푚퐿 푚퐿

So, we needed:

- 530.1 µL of the stock solution

- 469.9 µL of 70 % ACN / 30 % H2O

3.3.5. Preparing 85:15 molar ratios of the binary mixtures SMZ-AA

Table 3.5: Mass of each compound that was weighed to attain a 85:15 molar ratio. Mass sulfamerazine (mg) Mass amino acid (mg) SMZ-GLN 100.3 9.9 SMZ-GLU 145.5 14.3

Making 20 µg/mL of these 85:15 binary mixtures after successful amorphisation

SMZ-GLN

100.3 푚푔 0.3032 푚푔 0.3032 푚푔 µ푔 = ↔ = 30.32 110.2 푚푔 0.3331 푚푔 10 푚퐿 푚퐿

So, we needed:

- 659.6 µL of the stock solution

- 340.4 µL of 70 % ACN / 30 % H2O

SMZ-GLU

145.5 푚푔 0.7836 푚푔 0.7836 푚푔 µ푔 = ↔ = 78.36 159.8 푚푔 0.8606 푚푔 10 푚퐿 푚퐿

So, we needed:

- 255.2 µL of the stock solution

- 744.8 µL of 70 % ACN / 30 % H2O

3.3.6. Preparing 30:70 molar ratios of the binary mixtures, SMZ-AA

Table 3.6: Mass of each compound that was weighed to attain a 30:70 molar ratio. Mass sulfamerazine (mg) Mass amino acid (mg) SMZ-PRO 39.2 39.7

Making 20 µg/mL of these 30:70 binary mixtures after successful amorphisation

SMZ-PRO

39.2 푚푔 0.4026 푚푔 0.4026 푚푔 µ푔 = ↔ = 40.26 78.9 푚푔 0.8103 푚푔 10 푚퐿 푚퐿

So, we needed:

- 496.8 µL of the stock solution

- 503.2 µL of 70 % ACN / 30 % H2O

3.3.7. Preparing 20:80 molar ratios of the binary mixtures, SMZ-AA Table 3.7: Mass of each compound that was weighed to attain a 20:80 molar ratio. Mass sulfamerazine (mg) Mass amino acid (mg) SMZ-PRO 54.4 94.8

Making 20 µg/mL of these 20:80 binary mixtures after successful amorphisation

SMZ-PRO

54.4 푚푔 0.5418 푚푔 0.5418 푚푔 µ푔 = ↔ = 54.18 149.2 푚푔 1.4860 푚푔 10 푚퐿 푚퐿

So, we needed:

- 369.1 µL of the stock solution

- 630.9 µL of 70 % ACN / 30 % H2O

4. Intrinsic Dissolution Rate Study 4.1. Mefenamic acid 4.1.1. Standard solutions for pure MEF tablets

A series of standard solutions was analysed each day before analysing the samples of the dissolution test. These standard solutions 0.01, 0.25, 0.50, 0.75 µg/mL were all made by diluting the stock solution with 70 % ACN/ 30 % H2O: 0.5961 푚푔 µ푔 = 11.92 50 푚퐿 푚퐿 Table 4.1: Results of the standard series of MEF that were analysed on day one. Concentration (µg/mL) Area Average Retention time (min) 0.01 307 323 336 322 2.73 2.86 2.85 0.25 10238 10645 11155 10679 2.85 2.85 2.84 0.50 20925 20702 20907 20845 2.85 2.84 2.85 0.75 32212 31900 32903 32338 2.84 2.85 2.85

Standard curve Mefenamic Acid IDR day one 35000

30000 y = 43004x - 188.06 R² = 0.9994 25000

20000

15000 Peak Peak area 10000

5000

0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.2: Standard curve of MEF that was analysed on day one where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.3: Results of the standard series of MEF that were analysed on day two. Concentration (µg/mL) Area Average Retention time (min) 0.01 361 372 360 364 2.83 2.85 2.84 0.25 10984 11370 11068 11141 2.84 2.84 2.84 0.50 21651 21689 21844 21728 2.84 2.84 2.84 0.75 33386 33025 32413 32941 2.84 2.84 2.84

Standard curve Mefenamic Acid IDR day two 35000

30000 y = 43852x - 10.361 R² = 0.9999 25000

20000

15000 Peak Peak area 10000

5000

0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.4: Standard curve of MEF that was analysed on day two where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.5: Results of the standard series of MEF that were analysed on day three. Concentration (µg/mL) Area Average Retention time (min) 0.01 301 373 321 332 2.88 2.85 2.84 0.25 11461 11096 11214 11257 2.84 2.84 2.84 0.50 21796 21724 21452 21657 2.84 2.84 2.84 0.75 33389 33116 33076 33194 2.84 2.84 2.84

Standard curve Mefenamic Acid IDR day three 35000 y = 44122x - 46.126 30000 R² = 0.9996 25000

20000

15000 Peak Peak area 10000

5000

0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.6: Standard curve of MEF that was analysed on day three where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.7: Results of the standard series of MEF that were analysed on day four. Concentration (µg/mL) Area Average Retention time (min) 0.01 479 454 305 413 2.83 2.83 2.84 0.25 12056 12323 12197 12192 2.84 2.84 2.84 0.50 25065 24710 23621 24465 2.84 2.84 2.84 0.75 37346 37377 37334 37352 2.84 2.84 2.84

Standard curve Mefenamic Acid IDR day four 40000 35000 y = 49839x - 208.61 R² = 0.9999 30000 25000 20000

Peak Peak area 15000 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.8: Standard curve of MEF that was analysed on day four where the peak area is plotted as a function of the concentration (µg/mL).

4.1.2. Standard solutions for crystalline MEF-PRO tablets

Table 4.9: Results of the standard series of MEF that were analysed on day one. Concentration (µg/mL) Area Average Retention time (min) 0.01 339 309 334 327 2.85 2.85 2.85 0.25 10800 10680 10838 10773 2.85 2.85 2.85 0.50 21865 21878 21909 21884 2.85 2.85 2.85 0.75 32811 32909 32740 32820 2.85 2.85 2.85

Standard curve Mefenamic Acid + Proline crystalline IDR day one 35000 30000 y = 43964x - 145.55 R² = 1 25000 20000 15000 Peak Peak area 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.10: Standard curve of MEF that was analysed on day one where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.11: Results of the standard series of MEF that were analysed on day two. Concentration (µg/mL) Area Average Retention time (min) 0.01 351 313 319 328 2.80 2.86 2.86 0.25 10056 10075 10030 10054 2.85 2.85 2.85 0.50 20311 20475 20503 20430 2.85 2.85 2.85 0.75 30920 30520 30698 30713 2.85 2.85 2.85

Standard curve Mefenamic Acid + Proline crystalline IDR day two 35000 30000 y = 41107x - 137.13 R² = 1 25000 20000

15000 Peak Peak area 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.12: Standard curve of MEF that was analysed on day two where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.13: Results of the standard series of MEF that were analysed on day three. Concentration (µg/mL) Area Average Retention time (min) 0.01 314 380 320 338 2.80 2.85 2.85 0.25 10220 10262 10325 10269 2.85 2.85 2.85 0.50 20714 20608 20642 20655 2.85 2.85 2.85 0.75 32082 31457 31513 31684 2.85 2.83 2.83

Standard curve Mefenamic Acid + Proline crystalline IDR day 3 35000 30000 y = 42281x - 224.78 R² = 0.9998 25000 20000 15000 Peak Peak area 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.14: Standard curve of MEF that was analysed on day three where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.15: Results of the standard series of MEF that were analysed on day four. Concentration (µg/mL) Area Average Retention time (min) 0.01 374 351 336 354 2.85 2.85 2.85 0.25 10584 10513 10561 10553 2.85 2.85 2.85 0.50 20828 20738 20673 20746 2.85 2.85 2.85 0.75 32032 31494 31683 31736 2.85 2.85 2.85

Standard curve Mefenamic Acid + Proline crystalline IDR day 4 35000 30000 y = 42245x - 100.09 R² = 0.9998 25000 20000

15000 Peak Peak area 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.16: Standard curve of MEF that was analysed on day four where the peak area is plotted as a function of the concentration (µg/mL).

4.1.3. Standard solutions for amorphous MEF-PRO tablets

Table 4.17: Results of the standard series of MEF that were analysed on day one. Concentration (µg/mL) Area Average Retention time (min) 0.01 400 378 334 371 2.80 2.85 2.84 0.25 11361 11007 11335 11234 2.84 2.84 2.84 0.50 22096 21899 22204 22066 2.84 2.84 2.84 0.75 33730 33451 33559 33580 2.84 2.84 2.84

Standard curve Mefenamic Acid + Proline amorphous IDR day one 40000 35000 y = 44720x - 69.121 R² = 0.9999 30000 25000 20000

15000 Peak Peak area 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.18: Standard curve of MEF that was analysed on day one where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.19: Results of the standard series of MEF that were analysed on day two. Concentration (µg/mL) Area Average Retention time (min) 0.01 338 342 385 355 2.78 2.83 2.83 0.25 11244 11228 11007 11160 2.83 2.83 2.83 0.50 21942 22177 22012 22044 2.83 2.83 2.83 0.75 33389 33960 33506 33618 2.83 2.83 2.83

Standard curve Mefenamic Acid + Proline amorphous IDR day two 40000 35000 y = 44808x - 120.92 30000 R² = 0.9999 25000 20000

15000 Peak Peak area 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.20: Standard curve of MEF that was analysed on day two where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.21: Results of the standard series of MEF that were analysed on day three. Concentration (µg/mL) Area Average Retention time (min) 0.01 365 383 390 379 2.83 2.84 2.83 0.25 11607 11673 11494 11591 2.83 2.83 2.83 0.50 22287 22567 22418 22424 2.83 2.83 2.83 0.75 33924 33994 33604 33841 2.84 2.84 2.83

Standard curve Mefenamic Acid + Proline amorphous IDR day three 40000 35000 y = 45023x + 62.779 R² = 0.9998 30000 25000 20000

15000 Peak Peak area 10000 5000 0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Concentration (µg/mL)

Figure 4.22: Standard curve of MEF that was analysed on day three where the peak area is plotted as a function of the concentration (µg/mL).

Intrinsic Dissolution Rate Study of the co-amorphous drug formulation MEF-PRO

0.250

0.200 )

µg/mL 0.150 Crystalline MEF

0.100 y = 0.015x + 0.0344 Crystalline binary

mixture MEF - PRO Concentration( Amorphous binary 0.050 y = 0.0078x + 0.025 mixture MEF - PRO

y = 0.0046x + 0.0078 0.000 0.00 10.00 20.00 30.00 40.00 50.00 Time (hours)

Figure 4.23: Complete intrinsic dissolution curve of mefenamic acid where the concentration (µg/mL) is plotted as a function of time (hours).

4.2. Sulfamerazine 4.2.1. Standard solutions for pure SMZ tablets

A series of standard solutions was analysed each day before analysing the samples of the dissolution test. These standard solutions 0.01, 0.25, 0.50, 0.75, 1.00, 2.00, 3.00, 10.00, 20.00 µg/mL were all made by diluting the stock solution with 70 % ACN/ 30 % H2O: 1.2092 푚푔 µ푔 = 60.46 20 푚퐿 푚퐿 Table 4.24: Results of the standard series of SMZ that were analysed on day one. Concentration (µg/mL) Area Average Retention time (min) 0.01 465 489 542 499 1.37 1.37 1.37 0.25 8293 8480 8380 8384 1.36 1.36 1.36 0.50 17856 17877 17738 17824 1.36 1.36 1.36 0.75 28811 28690 28665 28722 1.36 1.35 1.35 1.00 36752 35552 34819 35708 1.36 1.36 1.36 2.00 74505 74112 75251 74623 1.36 1.36 1.36 3.00 115329 115916 117177 116141 1.36 1.36 1.36 10.00 421880 421959 422457 422099 1.36 1.36 1.36

Standard curve Sulfamerazine IDR day one 450000 400000 y = 42404x - 4812.2 350000 R² = 0.9992 300000 250000 200000

Peak Peak area 150000 100000 50000 0 0.00 2.00 4.00 6.00 8.00 10.00 Concentration (µg/mL)

Figure 4.25: Standard curve of SMZ that was analysed on day one where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.26: Results of the standard series of SMZ that were analysed on day two. Concentration (µg/mL) Area Average Retention time (min) 0.01 244 470 446 387 1.38 1.37 1.37 0.25 8414 8589 8451 8485 1.36 1.36 1.36 0.50 17798 17686 17707 17730 1.36 1.36 1.36 0.75 28899 28493 28888 28760 1.36 1.36 1.36 1.00 35827 35747 35586 35720 1.36 1.36 1.36 2.00 74330 74449 74425 74401 1.36 1.36 1.36 3.00 116037 116900 115846 116261 1.36 1.36 1.36 10.00 422784 422013 421423 422073 1.36 1.36 1.36

Standard curve Sulfamerazine IDR day two 450000 400000 y = 42405x - 4837.1 350000 R² = 0.9992 300000 250000 200000

Peak Peak area 150000 100000 50000 0 0.00 2.00 4.00 6.00 8.00 10.00 Concentration (µg/mL)

Figure 4.27: Standard curve of SMZ that was analysed on day two where the peak area is plotted as a function of the concentration (µg/mL).

Table 4.28: Results of the standard series of SMZ that were analysed on day three. Concentration (µg/mL) Area Average Retention time (min) 0.01 226 377 541 381 1.37 1.37 1.37 0.25 8581 8594 8603 8593 1.36 1.36 1.36 0.50 18219 18096 18194 18170 1.36 1.36 1.36 0.75 29202 29251 29267 29240 1.36 1.36 1.36 1.00 34952 34954 35016 34974 1.36 1.36 1.36 2.00 76483 76357 76412 76417 1.36 1.36 1.36 3.00 118832 119199 118556 118862 1.36 1.36 1.36 10.00 425064 425272 424937 425091 1.36 1.36 1.36 20.00 735677 735712 735809 735733 1.35 1.35 1.36

Standard curve Sulfamerazine IDR day three 800000 y = 37682x + 3780.4 700000 R² = 0.995 600000 500000 400000

Peak Peak area 300000 200000 100000 0 0.00 5.00 10.00 15.00 20.00 Concentration (µg/mL)

Figure 4.29: Standard curve of SMZ that was analysed on day three where the peak area is plotted as a function of the concentration (µg/mL).

4.2.2. Standard solutions for crystalline SMZ-PRO tablets

Table 4.30: Results of the standard series of SMZ that were analysed on day one. Concentration (µg/mL) Area Average Retention time min) 0.01 432 201 200 278 1.38 1.38 1.39 0.25 7454 7835 7798 7696 1.36 1.36 1.36 0.50 16576 16553 16644 16591 1.35 1.36 1.36 0.75 24685 24690 24771 24715 1.35 1.36 1.36 1.00 32946 33072 33101 33040 1.35 1.35 1.35 2.00 71629 71583 71530 71581 1.35 1.35 1.35 3.00 113573 113180 113417 113390 1.35 1.35 1.35 10.00 386621 385657 385028 385769 1.35 1.35 1.35 20.00 786850 786257 785565 786224 1.36 1.35 1.35

Standard curve Sulfamerazine + Proline crystalline IDR day one 900000 800000 y = 39406x - 4315 700000 R² = 0.9999 600000 500000 400000

Peak Peak area 300000 200000 100000 0 0.00 5.00 10.00 15.00 20.00 Concentration (µg/mL)

Figure 4.31: Standard curve of SMZ that was analysed on day one where the peak area is plotted as a function of the concentration (µg/mL).

4.2.3. Standard solutions for amorphous SMZ-PRO tablets

Table 4.32: Results of the standard series of SMZ that were analysed on day one. Concentration (µg/mL) Area Average Retention time (min) 0.01 233 212 195 213 1.38 1.38 1.38 0.25 7709 7953 7932 7865 1.36 1.36 1.36 0.50 16770 16688 16802 16753 1.36 1.36 1.36 0.75 24548 24447 24739 24578 1.36 1.36 1.35 1.00 33276 33269 33230 33258 1.35 1.36 1.35 2.00 72682 72720 72790 72731 1.35 1.35 1.35 3.00 114620 114775 114746 114714 1.35 1.36 1.36 10.00 391602 391679 392250 391844 1.36 1.36 1.36 20.00 800832 800323 800162 800439 1.35 1.36 1.36

Standard curve Sulfamerazine + Proline amorphous IDR day one 900000 800000 y = 40118x - 4713.9 700000 R² = 0.9999 600000 500000 400000

Peak Peak area 300000 200000 100000 0 0.00 5.00 10.00 15.00 20.00 Concentration (µg/mL)

Figure 4.33: Standard curve of SMZ that was analysed on day one where the peak area is plotted as a function of the concentration (µg/mL).

Intrinsic Dissolution Rate Study of the co-amorphous drug formulation SMZ-PRO

5.000

Crystalline SMZ

4.000 )

y = 2.5789x + 0.6703 Crystalline binary µg/mL mixture SMZ - PRO 3.000 R² = 0.9922 Amorphous binary mixture SMZ - PRO

2.000 Concentration( y = 0.4953x + 0.1285 1.000 R² = 0.9992

y = 0.3791x + 0.1101 R² = 0.9925 0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Time (hours) Figure 4.34: Complete intrinsic dissolution curve of sulfamerazine where the concentration (µg/mL) is plotted as a function of time (hours).

5. Stability study 5.1. Physical stability

Table 5.1: Results of the stability test of carbamazepine. Day 1 Day 2 Day 3 Day 4 Day 5 Day 8-11 Day 14 Day 15 Day 16 Day 17 CBZ- Fully Starting to Continued Almost fully crystallised except the border PRO 1 amorphous crystallise crystallisation CBZ- Fully Starting to Continued Almost fully crystallised except the border PRO 2 amorphous crystallise crystallisation Starting CBZ- Fully amorphous Continued light crystallisation, but still amorphous parts to PRO 3 crystallise CBZ 1 Fully amorphous 6 crystals A very throughout 3 new small the rest of Doubling crystals amount of Crystallisation The Continuous the sample of the and crystals in corner has crystals New crystals + growth of the previous CBZ 2 crystallisation in and amount continued (around 5 expanded (12 have ones the corner continuous of growth of – 6) in one crystals) grown growth of crystals the other corner of the other ones the sample crystals CBZ 3 Fully amorphous

Table 5.2: Results of the stability test of mefenamic acid. Day 1 Day 2 Day 3 Day 6 Day 7 Day 8 Day 9 Day 10 Crystallised instantly after using the melt quench method to make it amorphous, so this wasn’t stored in the desiccator and MEF evaluated day by day Small amount of MEF-PRO 1 Fully amorphous crystals + also no longer transparent MEF-PRO 2 Fully amorphous 3 crystals + sample isn’t transparent anymore MEF-PRO 3 Fully amorphous Sample isn’t transparent anymore

Table 5.3: Results of the stability test of sulfamerazine part one. Day 1-6 Day 7 Day 10-12 Day 13-26 Day 27 SMZ Crystallised instantly after melting and quench-cooling, so this was not stored in the desiccator and evaluated daily SMZ- Fully amorphous A few small crystals throughout the sample GLU 1 SMZ- Fully A few small crystals in the upper right corner and the rest of the sample GLU 2 amorphous SMZ- Fully amorphous A few small crystals GLU 3 SMZ- Fully amorphous GLN 1 SMZ- Fully amorphous A few very small crystals GLN 2 SMZ- Fully amorphous GLN 3 SMZ- PRO 1 (50:50) SMZ- PRO 2 The sides crystallised immediately after amorphisation. This is now spreading towards the centre (50:50) SMZ- PRO 3 (50:50)

Table 5.4: Results of the stability test of sulfamerazine part two. Day 1 Day 2 Day 3 Day 4 Day 6 Day 7 Day 8 Day 11 Day 12 SMZ-PRO 1 Fully amorphous Crystallised (20:80) SMZ-PRO 2 Fully amorphous Partly crystallised Crystallisation is spreading (20:80) SMZ-PRO Fully amorphous Very small amount of 3 crystals (20:80)

6. Product information

Table 6.1: Specific information of each product that was used throughout the study. Product Supplier Chemical Abstracts Lot number Service (CAS) (2S,3R)-(-)–Threonine Merck KgaA 72-19-5 S7391112 716 8.16012.0100 Made in China 100 g (S)-(-)-Proline Merck KgaA 147-85-3 S5670219 607 8.16019.0100 – 100 G Made in Germany Acetonitril HPLC grade Fisher Chemical, 75-05-8 1723411 2.5 L U.K. Carbamazepine SIGMA – ALDRICH 298-46-4 MKCD9730 powder Product of China C4024 – 25 g Glycine VWR CHEMICALS 56-40-6 17F084108 101194M – 250 g Made in Belgium L – Alanine VWR LIFE SCIENCE 56-41-7 1357C413 0106 – 100 g L – Arginine SIGMA – ALDRICH 74-79-3 MKBZ9144V W381918 – 1 kg Product of China L – Asparagine VWR LIFE SCIENCE 70-47-3 1276C396 Anhydrous 94341 – 100 g L – Aspartic Acid VWR LIFE SCIENCE 56-84-8 0397C317 0192 – 500 g L – Cysteine VWR LIFE SCIENCE 52-90-4 0277C106 J994 – 100 g L – Glutamic Acid SIGMA – ALDRICH 56-86-0 MKCB2372V W328502 – 1 kg Product of Brazil L – Lysine SIGMA – ALDRICH 56-87-1 BCBW3464 62840 – 100 g Product of Switzerland L – Serine SIGMA – ALDRICH 56-45-1 SLBS8540 S4500 – 100 g Product of USA L(+)-Glutamine VWR LIFE SCIENCE 56-85-9 17l134112 24378.187 – 100 g Made in China Mefenamic Acid SIGMA – ALDRICH 61-68-7 097K0756 M4267 – 50 g Product of China Sulfamerazine, SIGMA – ALDRICH 127-79-7 068K1130 minimum 99.0 % Product of Spain S8876 – 50 g Trifluoroacetic acid SIGMA – ALDRICH 76-05-1 BCBF9404V 99% Product of USA T6608 – 100 mL

Silica gel self-indicating Fischer Chemical 7631-86-9 1339616 SiO2 500 g Granule size 2.5 – 6 mesh Typpi Nitrogen, AGA 7727-37-9 / komprimerad Espoo, Finland 50 L

Master dissertation submitted to the faculty of Pharmaceutical Sciences, performed in collaboration with the Laboratory of Pharmaceutical Technology.

Promotor: Prof. Dr. T. De Beer

Commissioners: Prof. Chris Vervaet and Dr. Pieter-Jan Van Bockstal

The information, conclusions, and points of view in this master dissertation are those of the author and do not necessarily represent the opinion of the promoter or his/her research group.