In Vitro Solubility and Supersaturation Behavior of Supersaturating Drug Delivery Systems

Transcription

1 DOCTORAL T H E SIS In Vitro Solubility and Supersaturation Behavior of Supersaturating Drug Delivery Systems Amani Alhayali Health Science

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3 In Vitro Solubility and Supersaturation Behavior of Supersaturating Drug Delivery Systems A Doctoral Thesis Submitted By Amani Alhayali Division of Medical Sciences Department of Health Sciences Luleå University of Technology May 2018

4 Printed by Luleå University of Technology, Graphic Production 2018 ISSN ISBN (print) ISBN (pdf) Luleå

5 i To my family

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7 Abstract The development of new pharmaceutical products has been challenged by the growing number of poorly water-soluble drugs, which often lead to suboptimal bioavailability. Various approaches, such as the use of amorphous solid dispersions and cocrystals, have been used to improve the solubility, and subsequent bioavailability, of these drug molecules. Supersaturating drug delivery systems (SDDSs) have potential for achieving adequate oral drug bioavailability by increasing the drug solubility and creating a supersaturated state in the gastrointestinal tract. However, there is a need for better understanding of the supersaturation behavior in SDDSs and of the factors affecting supersaturation. The main objective of this thesis was to improve understanding of the supersaturation solubility behavior in SDDSs with a particular focus on rapidly dissolving solid forms (amorphous forms/cocrystals). In the course of the work, a new formulation for ezetimibe using an amorphous solid dispersion was prepared, cocrystals of tadalafil were prepared, and oral films of silodosin were formulated for the first time. These new formulations were thoroughly characterized using a number of solidstate and pharmaceutical characterization techniques. The dissolution and supersaturation behavior of the prepared SDDSs were studied. The effects of various factors on the supersaturation and precipitation characteristics were investigated. These factors included the preparation method, the temperature of the dissolution medium, the type of dissolution biorelevant medium (gastric/intestinal) used, the permeability of the relevant gastrointestinal membranes, the addition of polymers, and the addition of surfactants. The amorphous solid dispersions, cocrystals and oral films that were prepared represent new drug formulations that provide significantly higher dissolution rates and supersaturated solubility than crystalline drug forms. Solid dispersions prepared by the melting method had better supersaturation properties than those prepared by spray drying. The precipitation kinetics of the solid dispersion were faster at 37 C than at 25 C in biorelevant media. Implementation of an absorption tool during in vitro evaluation of supersaturation levels could improve the prediction accuracy of supersaturation and precipitation. A better understanding of the effects of excipients on the supersaturation and precipitation behavior of these types of formulation was obtained in this thesis. The improvement in supersaturation solubility obtained by adding polymers and surfactants was not proportional to the amounts of excipient used. iii

8 This thesis has made notable contributions to the field of pharmaceutical science by advancing our understanding of the supersaturation solubility behavior of the newly prepared SDDSs. Keywords: Poorly soluble drugs; SDDs; Solid dispersions; Cocrystal; Oral films; Supersaturation; Precipitation; Biorelevant media; Dissolution; Absorption. iv

9 List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I II Alhayali A, Tavellin S, Velaga S. (2017) Dissolution and precipitation behavior of ternary solid dispersions of ezetimibe in biorelevant media. Drug Development and Industrial Pharmacy 2; 43(1): Alhayali A, Selo MA, Ehrhardt C, Velaga S. (2018) Investigation of supersaturation and in vitro permeation of the poorly water soluble drug ezetimibe. European Journal of Pharmaceutical Sciences, 117, III Shimpi MR, Alhayali A, Cavanagh KL, Rodríguez-Hornedo N, Velaga S. Cocrystals of tadalafil: Physicochemical characterization, ph-solubility and supersaturation studies. Under revision IV Alhayali A, Vuddanda PR, Velaga S. Silodosin oral films: Development, physico-mechanical properties and in vitro dissolution studies in simulated saliva. Submitted Reprints were made with permission from the respective publishers. Author s contribution to the included papers Papers I, II and IV I was responsible for the planning and execution of the work. I prepared the SDDSs and performed the experimental work including the solid-state characterization and dissolution studies. I analyzed, plotted, and evaluated the results, and wrote the manuscripts. Paper III I participated in planning the study and evaluating the results. I performed the experimental work on ph-related solubility and dissolution. I took part in interpreting the results and writing the manuscript. I was not involved in identifying the cocrytals or predicting the theoretical solubility. v

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11 Conference Contributions i ii Al-Hayali A, Tavelin S, Velaga S. (2014) Dissolution and precipitation behavior of ternary solid dispersions of Ezetimibe in biorelevant media. Poster presentation at: AAPS Annual Meeting and Exposition, San Diego, USA. Alhayali A, Selo MA, Ehrhardt C, Velaga S. (2017) Investigation of supersaturation and permeation of a poorly water soluble drug Ezetimibe. Poster presentation at: 6th FIP Pharmaceutical Sciences World Congress, Stockholm, Sweden. vii

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13 Abbreviations A ANOVA AP API BCS BDDCS BL C C eq C max C s C t D dm/dt DMA DMEM DMF DMSO DS DSC EB EM EZ FaSSGF FaSSIF FBS FDA FeSSIF FTIR GIT h HBSS HPC HPLC HPMC HPMC-AS HPMCP ITZ IUPAC J Surface area Analysis of variance Apical Active pharmaceutical ingredient Biopharmaceutics Classification System Biopharmaceutics Drug Disposition Classification System Basolateral Concentration Equilibrium solubility concentration Maximum concentration Saturation solubility Concentration at time t Diffusion coefficient Dissolution rate Dimethyl acetamide Dulbecco's modified Eagle's medium Dimethyl formamide Dimethyl sulfoxide Degree of supersaturation Differential scanning calorimetry Elongation at break Elastic modulus Ezetimibe Fasted-state simulated gastric fluid Fasted-state simulated intestinal fluid Fetal bovine serum US Food and Drug Administration Fed-state simulated intestinal fluid Fourier transformation infrared spectroscopy Gastrointestinal tract Thickness of the diffusion layer Hank's balanced salt solution Hydroxypropyl cellulose High performance liquid chromatography Hydroxypropyl methylcellulose Hydroxypropyl methylcellulose acetate succinate Hydroxypropyl methylcellulose phthalate Itraconazole International Union of Pure and Applied Chemistry Absorption/flux ix

14 Log P MDSC MOA MQ P PEG pka PTFE PVP PVP K-30 PVPVA PWSDs PXRD SD SDDSs SDS SEDDSs SEM TDF TEER T g TGA T m TPGS TS USP Partition coefficient Modulated differential scanning calorimetry Malonic acid Melt-quenched Drug permeability coefficient Polyethylene glycol Acid dissociation constant Polytetrafluoroethylene Polyvinyl pyrrolidone Polyvinyl pyrrolidone Polyvinyl pyrrolidone-vinyl acetate Poorly water-soluble drugs Powder X-ray diffraction Spray-dried Supersaturating drug delivery systems Sodium dodecyl sulfate Self-emulsifying drug delivery systems Scanning electron microscopy Tadalafil Transepithelial electrical resistance Glass transition temperature Thermo-gravimetric analysis Melting temperature D-ɑ-tocopheryl polyethylene glycol succinate Tensile strength United States Pharmacopeia x

15 Contents 1. Introduction Background Drug bioavailability Biopharmaceutical Classification System Drug solubility, supersaturation and dissolution Formulation strategies for improving the solubility of poorly water-soluble drugs Chemical modification Physical modification Supersaturating drug delivery systems (SDDSs) Spring and parachute theory Precipitation inhibition In vitro investigation of supersaturation Induction of supersaturation in the medium of interest Assessment of drug concentrations in solution as a function of time Factors affecting supersaturation evaluation in vitro Aims of the work Methodology The studied APIs Induction of supersaturation by SDDSs Preparation of ternary solid dispersion (Paper I) Preparation of TDF-MOA Cocrystals (Paper III) Preparation of the casting gel and drug-loaded films (Paper IV) Solid-state characterization Induction of supersaturation by the solvent shift method (Paper II) Supersaturation-precipitation-permeation interplay (without polymer) Supersaturation-precipitation-permeation interplay (with polymer) Preparation of amorphous drug forms (Papers I and III) Solubility and dissolution studies Solubility study (Papers III and IV) Investigation of dissolution and supersaturation solubility (Papers I, III, IV) xi

16 4.6. HPLC quantification methods Statistical analysis Preparation of simulated fluids (Papers I, II and IV) FaSSIF and FaSSGF Simulated saliva Cell culture (Paper II) Results and discussion Preparation of a ternary solid dispersion of ezetimibe (Paper I) In vitro dissolution study Preparation of cocrystals of tadalafil (Paper II) ph-related solubility and Supersaturation index of TDF-MOA cocrystals (Paper III) Dissolution and supersaturation behavior of TDF solid forms Preparation of silodosin oral films (Paper IV) In vitro dissolution in simulated saliva Factors affecting supersaturation behavior (Paper I, II, III) Effects of process, formulation and temperature on supersaturation in FaSSIF (Paper I) Effects of process, formulation and temperature on supersaturation in FaSSGF (Paper I) Effect of implementation of an absorptive compartment on supersaturation behavior (Paper II) Effect of polymer on supersaturation-precipitation-permeation interplay (Paper II) Effect of polymer on the supersaturation behavior of cocrystals (Paper III) Conclusions Future work Acknowledgments References xii

17 1. Introduction Oral drug dosage forms are the most common and the most preferred dosage forms because of the wide range of advantages they provide. Among these are: ease of administration as they can be self-administered by the patient, low production costs compared to other forms, and high patient compliance. Solid oral dosage forms (such as tablets) are associated with greater chemical and physical stability, more accurate doses, and easier handling and manufacturing than other dosage forms (1-4). Drugs in solid formulations that are administered orally need to be in solution (i.e. need to be dissolved) to be absorbed by the gastrointestinal tract (GIT), reach the blood and eventually reach the specific target of action. The extent to which the active pharmaceutical ingredient (API) is available at the site of drug action is defined as the bioavailability (5). The oral bioavailability of a drug can be affected by several factors, such as the drug s aqueous solubility, intestinal permeation and metabolism. Poor aqueous solubility is the most frequent cause of low bioavailability because it is necessary for the drug to be dissolved for absorption to take place after oral administration (1, 2). Since the successful therapeutic effect of a drug depends on its concentration in the systemic circulation, higher doses are needed for poorly water-soluble drugs (PWSDs) to achieve the required plasma concentrations. This is often associated with an increased risk of adverse effects (6). The permeability of the intestinal wall to the drug and its absorption through the wall can also affect both the bioavailability and the solubility of the drug in the intestinal lumen (7). Because the therapeutic activity of the drug is so heavily reliant on its bioavailability, it is important to modify some drug properties to improve the therapeutic activity and reduce the incidence and severity of side effects. Various conventional and advanced approaches have been used by formulation scientists to improve the solubility, and thereby the bioavailability, of PWSDs (8, 9). Many solubility-enhancement technologies are available, including the use of amorphous solid dispersions, lipids, cocrystals, solubilization techniques, surfactants, nanoparticles, cyclodextrins, and others (10, 11). The drug formulations obtained from applying these technologies are called supersaturating drug delivery systems (SDDSs) (10). Supersaturation is an effective oral bioavailability-improving approach that has been gaining in interest over the last decade as the number of poorly water-soluble compounds has increased in drug-discovery pipelines. SDDSs provide intraluminal drug concentrations that are higher than saturation solubility concentrations (i.e. supersaturation solubility concentrations), thus offering potential for improving bioavailability. 1

18 The aim of this thesis was to improve the physical properties of some PWSDs. New drug formulations (using SDDSs) with improved physical properties were prepared, to provide drugs with better aqueous solubility and supersaturated drug concentrations (higher than saturation solubility concentrations) at the GIT. 2

19 2. Background 2.1. Drug bioavailability Drug bioavailability is the key factor to be addressed when developing a formulation for an oral dosage form. For drugs to be therapeutically active at their site of action they must be absorbed through the intestinal barrier and be sufficiently available in the blood to achieve a therapeutic plasma concentration. It is necessary for the drug to be in solution in the gastrointestinal fluid to be well absorbed through the intestinal barrier (Figure 1). Figure 1. Diagram of the relationship between an oral dose of a drug product and its pharmacological effect. Thus, factors such as the solubility, absorption and metabolism of a drug can affect its bioavailability (6, 12). An API that is poorly soluble in the GIT and/or is not well absorbed through the intestinal membrane will reach low plasma concentrations after oral administration. As a result, the bioavailability will be insufficient for the desired pharmacological action. Other factors, such as first-pass metabolism by intestinal cells or liver enzymes (e.g. glucuronidation or oxidation by cytochrome P-450 enzymes, sulfation, etc.) can also limit bioavailability (13) (Figure2). 3

20 Figure 2. Some factors affecting the bioavailability of drugs after oral administration 2.2. Biopharmaceutical Classification System The Biopharmaceutics Classification System (BCS) was developed by Amidon et al. in 1995 (6). It categorizes pharmaceutical drugs into four groups (Class I, II, III and IV drugs, as shown in Figure 3) depending on their solubility and the permeability of the gastrointestinal membrane to the drug. The permeability/flux (J) of a drug through the gastrointestinal membrane is controlled by two factors: the drug permeability coefficient (P) of the membrane and the intraluminal gastrointestinal concentration (C) (Equation 1) (10): J = PC (1) Solubility is a dose-dependent criterion. For the API to be classified as a soluble drug, the maximum oral dose needs to be soluble in 250 ml of aqueous medium in the ph range 1 to 7.5 at 37 C, while permeability is considered to be high if 90% of an oral dose is absorbed across the membranes, according to the US Food and Drug Administration (FDA). The Biopharmaceutics Drug Disposition Classification System (BDDCS) was adapted and modified from the BCS with the focus more on metabolism than on permeability with respect to bioavailability (14, 15). Thus, for PWSDs (BCS classes II and IV), the low intraluminal concentrations of the drug can limit absorption and hence decrease the systemic bioavailability (10). 4

21 Figure 3. The biopharmaceutical classification system Drug solubility, supersaturation and dissolution The solubility of a solute is defined by the International Union of Pure and Applied Chemistry (IUPAC) as the concentration of the solute in a saturated solution, expressed as the proportion of the designated solute in a designated solvent (16). Solubility occurs in a state of dynamic equilibrium and can be changed by changing the solvent environment (such as temperature or ion content) (17, 18). The term apparent solubility describes the solubility at the apparent state of equilibrium between the drug in solution and the drug in an unstable solid state (8, 9). The apparent solubility should not be confused with the equilibrium solubility, which describes the thermodynamic equilibrium between the drug in solution and the most stable solid form of the drug (10). When the concentration of drug molecules in the solvent exceeds the equilibrium solubility, the solution is considered to be supersaturated, and this concentration is called the supersaturation solubility. Dissolution is defined by the IUPAC as the mixing of two phases with the formation of one new homogeneous phase (16). The distinction between solubility and dissolution is important. The solubility (equilibrium solubility or thermodynamic solubility) is a dynamic property representing the maximum quantity of drug or substance that can be dissolved in a given amount of solvent at a given temperature and pressure. It has nothing to do with the time. The dissolution rate is an indication of the speed of dissolution, a kinetic property. 5

22 The US Pharmacopeia (USP) lists the solubility terms shown in Table 1. Drugs with solubility lower than 1 mg/ml are described as very slightly soluble or practically insoluble. In this thesis, these drugs (with solubility lower than 1mg/ml) are referred to as PWSDs (19). Table 1. Definitions of solubility according to the US Pharmacopeia (19) Solubility definition Parts of solvent required for one part of solute Solubility range (mg/ml) Very soluble <1 >1000 Freely soluble From 1 to Soluble From 10 to Sparingly soluble From 30 to Slightly soluble From 100 to Very slightly soluble Practically insoluble From 1000 to > < Formulation strategies for improving the solubility of poorly water-soluble drugs Development of new pharmaceutical products for oral administration has been challenged by the increasing number of poorly water soluble molecules in the pipeline. It has been estimated that about 70% of all new APIs emerging from the discovery pipeline have poor aqueous solubility and more than 40% of marketed drugs are poorly water-soluble (20-22). However, most of these drug molecules permeate well through the intestinal wall despite their poor solubility in gastrointestinal fluids (BCS class II) (6, 22). Enhancement of the dissolution rate of BCS II drugs is a key factor in improving their bioavailability because, for the drugs in this group, the only limitations to improved bioavailability are the dissolution rate and solubility, and slight increases can sometimes make noticeable improvements (2). The solubility of class II drugs can be improved by various formulation strategies that induce intraluminal concentrations higher than saturation solubility concentrations (supersaturation). Commonly used strategies include solid dispersions, self-emulsifying drug delivery systems (SEDDSs), cyclodextrin complexes, cocrystals and nanocrystals. These strategies are based on either chemical or physical modification of the drug molecule. Table 2 provides a list of the most common formulation strategies for improving the solubility of PWSDs; this thesis discusses two formulation strategies: amorphous solid dispersions and cocrystals. 6

23 Table 2. Formulation strategies for improving the solubility of poorly water-soluble drugs (BCS II) Chemical modification Prodrugs Salts Physical modification Cocrystals Particle size reduction Solid dispersion Porous materials Carriers (cyclodextrines, liposomes) Chemical modification Prodrugs The use of prodrugs is a well-known method for improving the solubility of PWSDs. The molecular structure of the API is chemically modified to form an inactive derivative that is swallowed, metabolized by enzymes, and transformed into the parent pharmacologically active form of the drug in the GIT (23) Salts Most drugs are either weak acids or weak bases and the dissolution rates and solubility of these drugs in aqueous media are often improved if the drugs are in the form of a salt. The disadvantages of using the salt form include relatively poor stability and increased incidence of undesirable side effects which may affect patient compliance (24, 25) Physical modification The Nernst-Brunner equation (Equation 2), a modified version of the Noyes- Whitney equation, is the cornerstone for improving the solubility and dissolution rates of BCS II drugs (26). dm dt = AD(C s C t ) h (2) This equation states that the dissolution rate (dm/dt) is directly proportional to the saturation solubility (C s ), the concentration of the drug in the medium at any time (C t ), the available surface area of the dissolving solid (A) and the diffusion coefficient of the compound (D). The thickness of the diffusion layer is expressed as h. Each parameter in the equation is affected by many factors and manipulation of these factors will modify the dissolution rate of the drug, as shown in Figure 4. 7

24 The equation indicates that the dissolution rate can be improved by manipulation of the saturation solubility and the surface area. Most of the physical methods that modify the physical properties of APIs and increase their solubility are based on the Nernst-Brunner equation. These methods include increasing the surface area of the dissolved drug by reducing the drug particle size, increasing the apparent solubility of the drug, and improving the wetting and solubilizing properties of the compound to reduce the thickness of the boundary layer around the drug particles (27) Particle-size reduction One of the oldest methods for improving drug solubility involves reducing the particle size. According to the Noyes-Whitney and Nernst-Brunner equations, a decrease in particle size results in a larger surface area of drug available for solvation and a subsequent increase in the dissolution rate and potential bioavailability of PWSDs (28). Particle size reduction to < 1 μm has been associated with a significant increase in solubility (28, 29). Several methods have been reported for reducing drug particle size to generate nanosized particles; these include milling, spray drying and homogenization (30, 31). The particle size reduction method is considered to be safe as the API is used without any additives or chemical modifications, which implies that there will not be any new adverse effects. Figure 4. Physicochemical factors affecting the dissolution rate according to the Nernst-Brunner equation. 8

25 Amorphous state Solids can be either amorphous or crystalline (4). In the crystalline state the molecules are structured regularly in a lattice formation. Compounds with an irregular molecular arrangement that lacks long range order are referred to as being in the amorphous state. Van der Waals forces, hydrogen bonding and other intermolecular interactions hold the molecules within the crystal lattice, resulting in lower molecular mobility. Because the molecular interactions in the crystalline state are strong, the compound is usually more stable but the dissolution rate is slower. When a crystalline drug is heated, it undergoes melting at temperature T m ; then when the molten drug is slowly cooled, the molecules have sufficient time to move from their current location to a thermodynamically stable point on the crystal lattice, regenerating a crystalline structure (32). If, however, the molten drug is cooled suddenly, it can reach a super-cooled liquid state. If it is cooled further below its T m, the system remains in equilibrium until the glass transition temperature (T g ) is reached, below which it enters a nonequilibrium (rubbery) state and converts into the glassy (amorphous) state of the drug. The glass transition is a second order thermodynamic transition characterized by a step change in the heat capacity which is also associated with changes in other thermodynamic properties such as volume, enthalpy, and entropy (33). The amorphous state of a drug has a higher free energy than the crystalline state and molecules or atoms can convert gradually into the highly ordered crystalline state if they are maintained at a specific temperature for a long time. Amorphous solids have higher dissolution rates than the crystal forms and, when they are dissolved, can result in higher drug concentrations in solution than the saturated solution of the crystalline form (i.e. they can form a supersaturated solution). This phenomenon has been used to increase the solubility of various PWSDs (34-36). The drug in supersaturated solution will eventually regain its equilibrium solubility, as the amorphous form will convert to the stable crystalline form. Therefore, maintaining the PWSD in its amorphous state is one of the great challenges in formulation development Amorphous solid dispersions Pure amorphous drugs are rarely developed alone as pharmaceutical products because their high levels of free energy make them thermodynamically unstable. Therefore, finding ways to stabilize the amorphous form is seen as important. Polymer and surfactant excipients, included to prepare and stabilize the amorphous form, have created tremendous opportunities for the pharmaceutical scientist to address issues relating to the bioavailability of poorly soluble molecules. Solid dispersions in general can be defined as 9

26 molecular mixtures of PWSDs in hydrophilic carriers (20). Solid dispersions are classified into three types (first, second or third generation) according to their development and the carrier/excipient used in the formulation. First generation solid dispersions are prepared using crystalline carriers. Urea and sugars were the first crystalline carriers employed in solid dispersions. The associated improvements in solubility were attributed to faster carrier dissolution, releasing microcrystals or particles of drug. These solid dispersions have the disadvantage that the drug is not released as quickly as from the amorphous form, despite being more stable thermodynamically (37, 38). The second generation of solid dispersions contains amorphous carriers instead of crystalline carriers. The drugs are molecularly dispersed in an irregular form within an amorphous carrier, usually consisting of a polymer, to create the most common type of solid dispersion used to date, amorphous solid dispersions (39). In second generation solid dispersions, the drug is in its supersaturated state because of forced solubilization in the carrier at a molecular level. The dissolution of the polymer dictates the drug release profile (27, 40). Third generation solid dispersions contain a surfactant carrier or a mixture of amorphous polymers and surfactants as carriers with the aim of stabilizing the solid dispersion and achieving the highest degree of bioavailability for PWSDs. Surfactants have surface activity or self-emulsifying properties, and thus help to improve the dissolution properties of PWSDs and potentially improve their bioavailability. Surfactants such as inulin, inutec SP1, compritol 888 ATO, gelucire 44/14 and poloxamer 407 have been used successfully as carriers to increase the bioavailability of various PWSDs (41-45). A combination of surfactants and polymers has also been used to improve dissolution and prevent precipitation of some PWSDs (46, 47). A solid dispersion, therefore, can now be defined as a dispersion of amorphous drug in a polymeric carrier matrix. Selection of a suitable carrier is critical to obtaining a solid dispersion with improved dissolution properties. Drug solubility in the carrier and drug-carrier compatibility are the main factors to be considered when selecting a carrier. Phase separation and drug precipitation can result from inadequate solubility of the drug in the carrier. Drug-carrier incompatibility can cause formulation failure (9, 27, 48). Melting and solvent evaporation methods are mainly used to prepare solid dispersions. Processes that have been used in manufacturing solid dispersions are shown in Figure 5 (20). Polymers The inclusion of polymers in the formulation is one of the most common methods of stabilizing an amorphous API (49). Polymers appear to be the most successful carriers for solid dispersions because of their ability to form amorphous solid dispersions. 10

27 Figure 5. The manufacturing processes used in the preparation of solid dispersions (20). Polymers improve the physical stability of the amorphous compounds by increasing the T g, which delays the crystallization process. Polymer carriers not only stabilize the dispersed amorphous drug but can also increase wettability and prevent drug precipitation in the GIT, as found recently (50). Polymers can be divided into two types: (1) natural product-based polymers which are mainly composed of cellulose derivatives such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose acetate succinate (HPMC-AS), hydroxypropyl methyl cellulose phthalate (HPMCP), ethylcellulose, or starch derivates such as cyclodextrins (40, 51-56); and (2) synthetic polymers such as polyvinyl pyrrolidone (PVP), polyethylene glycols (PEG) and polymethacrylates (39, 57-59). Surfactants The chemical structures of surfactant (surface active agent) molecules have two distinct ends: one end is hydrophilic and the other is hydrophobic. Because of this amphiphilic property, surfactants can be adsorbed onto the surface or interface of a system between two phases, e.g. a water-oil interface. Addition of surfactants to amorphous drug formulations has the potential to reduce nucleation (crystal growth) and crystallization as a result of either alteration of the viscosity of the molecule or changes to the interfacial energy at the amorphous solid interface. However, the ability for surfactants to increase the solubility of PWSDs or to stabilize amorphous APIs in formulation depends on factors such as the chemical structure of the drug and the surfactant, the temperature, and the ph (60, 61). 11

28 Oral films Oral films have received increasing interest in the pharmaceutical industry, especially in recent years. They provide a wide range of potential advantages, including improved compliance, suitability for pediatric and geriatric use, and avoidance of first-pass metabolic effects (62, 63): Compliance: Oral films are easy to administer without chewing or intake of water as the films disintegrate and dissolve rapidly to release the drug when placed in the oral cavity. This is thought to have potential to improve patient compliance. Pediatric and geriatric use: Oral films are useful for special patient groups such as pediatric and geriatric patients because the potential for choking is lessened in patients who have difficulties in swallowing solid oral dosage forms like tablets and capsules. Fast-dissolving oral films provide a solution for these patients and for patients with mental disorders such as mania and schizophrenia, and patients with dysphagia or emesis (63). Metabolism problems: Some drugs are metabolized or degraded in the gastrointestinal environment. This undesirable effect can be avoided by formulating drugs as oral films which will be absorbed directly into the systemic circulation without passing through the GIT, thus avoiding first-pass metabolism in the liver (64, 65). Oral dissolving films are composed of an API dispersed within filmforming polymers and plasticizers. They also contain excipients for sweetening, flavoring, coloring and saliva stimulation. Polymers may be used as drug release platforms. Both natural and synthetic polymers (such as Pullulan, starch, HPMC, polyvinyl alcohol and PVP) can be used in the formulation of oral films in different ratios to obtain effective drug delivery platforms. Modification of the composition of the polymeric matrix can help to achieve desirable formulation properties such as faster disintegration and better mechanical properties (66, 67). Plasticizers can improve the flexibility of the film and reduce brittleness. They reduce the T g of the polymers and enhance the film-forming properties (68). Commonly used plasticizers include PEG, glycerol, diethyl phthalate, triethyl citrate, tributyl citrate, etc (69, 70). Surfactants such as Tweens, sodium lauryl sulphates and Polaxamer 407 play an important role as solubilizing, wetting, and dispersing agents that enable films to disintegrate within seconds, releasing the incorporated API immediately (70, 71). Sweeteners and flavoring agents could be significant in improving palatability and compliance for pediatric and geriatric patients. Sucrose, dextrose and glucose have been used as sweeteners. Excipients such as peppermint oil, cinnamon oil, oil of nutmeg, white vanilla, chocolate, and citrus also provide flavor (72). 12

29 The increase in saliva from the addition of saliva-stimulating agents such as ascorbic acid, citric acid and malic acid can promote the disintegration of oral films (70, 71). Sublingual, fast-dissolving films are oral films that are intended to be placed under the tongue in the mouth. The relatively high vascularity and permeability of the sublingual mucosa and membrane can result in rapid absorption of the formulated drug and instant bioavailability, resulting in a rapid onset of action (73). Fast-disintegrating film formulations can be used to improve the bioavailability of the drugs and to overcome the solubility limitations in aqueous media. Improvements in the solubility and dissolution of the drug are the result of the drug being converted from crystalline to amorphous form and dispersed in the hydrophilic polymer and also as a result of the large exposed surface area of the film. Solvent casting and extrusion methods are the most frequently used methods of preparing oral films Cocrystals Cocrystals are multicomponent solids that contain two or more components in a single homogeneous crystalline system with well-defined stoichiometry. A cocrystal is composed of an API and a coformer. The APIs can be weakly acidic, basic or even neutral compounds while the coformer is a benign nontoxic molecule (such as malonic acid, saccharin, nicotinamide, etc.) or another API. Cocrystal formation has often been guided by hydrogen-bonded assemblies between the neutral molecules of the drug and the cocrystal coformer (74, 75). Over recent decades, many pharmaceutical cocrystals have been prepared and these have received significant interest from the pharmaceutical industry. The most significant application of pharmaceutical cocrystals is improvement in the solubility of PWSDs. The highly soluble coformer, dissolves to a greater extent than the drug, creating supersaturation with respect to cocrystal as a result of nonstoichiometric concentration in solution (75-78). The drug delivery systems obtained as a result of applying these technologies and performing physical modifications to alter the properties of the PWSD are called SDDSs. An amorphous solid dispersion of ezetimibe (EZ; a PWSD) was prepared during the course of the thesis, with the aim of improving its solubility and dissolution. Cocrystals of tadalafil (TDF; a PWSD) were also prepared to improve the dissolution and solubility properties of the powder. In addition, a sublingual silodosin film was prepared to deliver the drug oromucosally. These films also have potential to provide supersaturated concentrations of the drug in the saliva. 13

30 2.5. Supersaturating drug delivery systems (SDDSs) SDDSs are promising tool for improving the oral bioavailability of PWSDs. SDDSs provide drugs with intraluminal concentrations that are higher than the saturation solubility concentrations (i.e. supersaturation). The rationale behind SDDSs is based on Fick s First Law (Equation 1), i.e. the enhanced concentrations at the site of absorption can increase the flux of the formulated drugs across the gastrointestinal membrane. SDDSs are thermodynamically stable or metastable in the formulation. They generate a thermodynamically metastable, supersaturated solution of the drug after dissolution in the aqueous environment of the GIT. The drug is in the supersaturated state when its solubility is higher than its equilibrium solubility in the given medium. The high concentration is achieved by incorporating the drug in a high energy state such as an amorphous phase in amorphous solid dispersions (53) or highly soluble forms such as cocrystals (79) in the SDDS. The drug molecules in the supersaturated state tend to precipitate rapidly as this is a thermodynamically unstable state. Thus, the value of SDDSs in improving solubility and absorption will depend on the extent and duration of maintaining the supersaturated state in the gastrointestinal lumen. Therefore, the challenge is in creating concentrations of the drug that are many times higher than the thermodynamic solubility concentration, and maintaining these high concentrations for an adequate time period for the absorption process to take place. The stabilization and inhibition of precipitation are accomplished by using precipitation inhibitors such as polymers, surfactants, and other excipients that act as stabilizers of supersaturated drugs in the SDDS (80). The generation and maintenance of the metastable supersaturated state are represented by the spring and parachute theory (81) in SDDSs Spring and parachute theory The benefit of supersaturation as a strategy for improving the intestinal absorption and bioavailability of PWSDs depends on two essential steps: the generation (the spring ) and maintenance (the parachute ) of the metastable supersaturated state, as described by Guzman et al. (81) and shown in Figure 6. The spring is formed by using a higher energy form of the PWSD (compared to the crystalline form), which can be achieved using many formulation options (such as the amorphous form or cocrystals, as described previously). Once supersaturation has been induced, precipitation of drug molecules will occur through kinetically or thermodynamically controlled processes. The parachute is the temporary inhibition of precipitation by interfering with nucleation and/or crystal growth, achieved by the use of pharmaceutical 14

31 excipients or other components called precipitation inhibitors (10, 82). Precipitation inhibitors are commonly used to maintain supersaturation and inhibit drug precipitation for an extended period of time (83, 84). Figure 6. Schematic illustration of the concentration-time profiles showing the spring and parachute theory of supersaturated drug delivery systems (10) Precipitation inhibition Identification of the optimum precipitation inhibitor is a vital aspect of successful SDDS formulations. Polymers such as cellulose derivatives (e.g. HPC, HPMC), vinyl polymers [e.g. PVP, PVP-vinyl acetate (VA)], and ethylene polymers (e.g. PEG) have been used to stabilize supersaturation (85-89). Precipitation inhibitors used for stabilizing a supersaturated solution act by a variety of mechanisms depending on the properties of the inhibitor, the drug and the medium. The mechanisms usually involve increasing the solubility and/or decreasing nucleation and crystal growth, by increasing the viscosity (which results in reduced molecular mobility, thus decreasing nucleation and crystal growth), adsorption onto the crystal surface (which hinders crystal growth), or changing the level of solvation at the crystal/liquid interface (which slows the incorporation of drug molecules into a crystal lattice) (10). However, some polymers can increase the solubility of drugs (90-93). Surfactants can also delay or hinder precipitation from supersaturated solutions when added at concentrations exceeding their critical micelle concentration. The increase in drug solubility reduces the rate of nucleation and crystal growth. Examples of surfactants are sodium dodecyl sulfate (SDS), D-ɑ-tocopheryl polyethylene glycol succinate (TPGS), and Poloxamers (10). 15

32 2.6. In vitro investigation of supersaturation There are many assays for evaluating supersaturation and precipitation or precipitation inhibition. They differ in their approach to the generation of supersaturation, the techniques used for supersaturation measurement, and the experimental conditions. Evaluation of supersaturation in vitro requires the induction of supersaturation in the medium of interest and then assessment of drug concentrations in solution as a function of time Induction of supersaturation in the medium of interest Supersaturation can be induced and evaluated for either formulated (i.e. SDDSs) or non-formulated drugs Formulated drugs (SDDSs) In a SDDS, supersaturation is induced as an inherent characteristic of the formulation and there is no need for it to be induced as part of the assay. Evaluation of the supersaturation behavior of a SDDS requires an aqueous medium that is relevant for the environment in the GIT (94). One compartment/one phase setups traditionally used to study dissolution are based on USP I or II apparatus. One-compartment and two-compartment ph shift approaches have been used to evaluate the dissolution of formulations that rely on the gastrointestinal ph gradient to induce supersaturation, simulating gastric and intestinal dissolution behavior (95-97). A multi-compartment dissolution setup that includes gastric, intestinal and absorption compartments has also been used to predict supersaturation and precipitation in SDDSs (98) Non-formulated drugs Evaluation of the supersaturation-precipitation potential of non-formulated drugs (APIs) or the precipitation inhibition capacity of an excipient requires induction of supersaturation as a starting step. There are several methods of measuring supersaturated drug concentrations; the most commonly applied are the solvent-shift and ph-shift methods (90). In the solvent-shift method, the PWSD is first dissolved in a solvent that is water miscible and has a significantly higher solubilizing capacity for the drug than the aqueous medium in which supersaturation is to be evaluated [e.g. dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and dimethyl acetamide (DMA)] (99-101). Next, a fraction of this solution is added to the medium under investigation. Finally, supersaturation and/or precipitation can be evaluated. This is a common and simple method of creating supersaturation and is applicable to any PWSD that can be dissolved at significantly higher concentrations in a water miscible solvent (102, 103). 16

33 The ph-shift method is used to evaluate the supersaturation potential of ionizable drugs. The solubility of drugs can increase in polar aqueous solvents as a result of ionization; therefore, any shift in the ph that reduces ionization will decrease the drug solubility and induce a supersaturated state. This method can be considered more biorelevant for weakly basic drugs as a ph shift occurring upon transfer of drugs from the stomach to the small intestine can induce supersaturation in vivo (104, 89) Assessment of drug concentrations in solution as a function of time Assessment of supersaturated concentrations resembles classic concentration assessments during dissolution testing. The measured drug concentrations are combined with the equilibrium solubility of the drug in the test medium (which includes the precipitation inhibitor if one has been added to the drug formulation). Accordingly, the drug concentration can be measured and expressed relative to the equilibrium solubility as the degree of supersaturation (DS), as shown in Equation 3: DS t = C t /C eq (3) Where C t is the drug concentration at time t and C eq is the equilibrium solubility of the drug in the test medium. The saturation extent can be quantified as a measure of the thermodynamic tendency for precipitation (90). DS < 1: subsaturated DS = 1: saturated DS > 1: supersaturated Factors affecting supersaturation evaluation in vitro Formulation-related factors The dissolution characteristics of SDDSs in vitro are mainly affected by the extent of supersaturation maintenance, which is in turn determined by 1) the preparation method and 2) the formulation composition (105). Methods used for preparing solid dispersions, such as quenching of hot melts, spray drying, and film casting, could play a role in stabilizing formulated amorphous drugs in SDDSs. In hot melt quenching, mixtures of the drug and excipient carrier are heated to a molten state, followed by immediate cooling and solidification. This is an effective method of transforming a drug to a critical supersaturated state while avoiding nucleation (50, 106). The supersaturation of a solid dispersion containing itraconazole (ITZ) and low-grade HPMC-AS powder 17

34 (HPMC-AS-LF) by the melting method had better results than using amorphous ITZ, which was physically unstable and recrystallized at high temperature and humidity levels. No physical or chemical instability was observed in the ITZ and HPMC-AS-LF solid dispersion, which was attributed to the temperature- and moisture-activated electrostatic interactions between the drug and their counter ionic polymers (107). Spray-dried dispersions had the single T g of a drug/polymer mixture according to solid-state characterization. The dissolution rate and supersaturation maintenance were increased, which was attributed to the forced aggregation of the polymer into regular micelle structures (108). Some other methods, such as the amorphization strategies, that were used to prepare solid dispersions also had positive results (109). Formulation compositions such as the polymer type and the presence of specific drug:polymer ratios are important for maintaining the stability of the supersaturated state. When the polymer content within the solid dispersion is increased, the DS will be increased significantly (110, 111). According to investigations of the solid dispersions prepared from three polymers (HPMC, PVP, and PEG 6000), formulations incorporating HPMC maintained supersaturation for the longest period (112). The polymer HPMC-AS as precipitation inhibitor had the best effect of 41 types of polymer and surfactant in another study (113). Synergistic enhancement of amorphous stability and dissolution of the drug can be obtained by combining two polymers in ternary solid dispersions (114) or a polymer and a surfactant (115) Assay-related factors The experimental conditions that are chosen for in vitro assays during supersaturation evaluation could affect the outcome of the assay significantly and thus require careful consideration. These conditions include the following: Medium selection Biorelevant dissolution media that simulate fasted and fed conditions in the stomach and small intestine have been developed (116). These provide more accurate predictions of dissolution than simple aqueous buffer solutions. The accuracy of in vitro/in vivo correlations has been improved significantly with the use of biorelevant media (117). The components present in gastrointestinal fluids, including bile salts and phospholipids, could affect the precipitation kinetics. Bevernage et al. have compared the precipitation behavior of PWSDs in simulated and human gastrointestinal fluids. They explored the importance of careful selection of dissolution medium for supersaturation assays and found that simple aqueous buffer solutions should be avoided when studying precipitation kinetics in the intestinal environment as they overestimate the stability of supersaturation. Fasted-state simulated intestinal fluid (FaSSIF) is a reasonable medi- 18

35 um for predicting precipitation behavior in fasted-state human fluids. In contrast, fed-state simulated intestinal fluid (FeSSIF) can significantly underestimate precipitation. Fasted-state simulated gastric fluid (FaSSGF) should be used in place of USP simulated gastric fluid to predict precipitation behavior in the gastric environment as the latter underestimates precipitation (99, 118, 119). Temperature Most of the in vitro supersaturation assays are performed at 25 C or room temperature instead of the more biologically relevant temperature of 37 C. Alonzo et al. investigated the dissolution behavior of amorphous felodipine at 25 C versus 37 C. Amorphous felodipine induced supersaturation during dissolution at 25 C but not at 37 C (120). The altered dissolution behavior of felodipine resulted from temperature-dependent drug crystallization kinetics after contact with the dissolution medium at different temperatures. Implementing an acceptor/absorption compartment Addition of an acceptor compartment to simulate absorption during evaluation of SDDSs is crucial and will improve the predictive power of the assay. Permeation and absorption can affect the dissolution and precipitation kinetics of the PWSD (121). Permeation into a sink compartment may relieve the thermodynamically unstable supersaturated system and act as an alternative for precipitation. As demonstrated by Bevernage et al., precipitation of loviride was significantly reduced in the presence of an absorption compartment in the Caco-2 model when compared to a one-compartment setup without an absorption compartment (122). Different experimental approaches have been used to simulate absorption, including the addition of an immiscible organic layer as an absorptive sink, or the integration of an actual absorption compartment separated from the dissolution medium by a filter/pump combination or a Caco-2 monolayer (121, 123). Hydrodynamics The hydrodynamics of the supersaturation assays will influence the nucleation and crystal growth processes of drug molecules (124, 125). In general, extensive mixing and increased kinetic energy can assist in overcoming the activation obstacle for nuclei formation (10, 126). Very few studies have addressed this aspect of hydrodynamics and its effect on supersaturation and precipitation behavior with respect to PWSDs. Carlert et al. compared the in vitro precipitation of a PWSD (AZD0865) in a stirring model versus a shaking model (95). The precipitation rate was significantly slower in the shaking 19

36 model, indicating the importance of hydrodynamics in supersaturation/precipitation evaluation assays. The mixing that is usually applied during in vitro supersaturation assays is expected to be relatively high compared to in vivo gastrointestinal motility in the fasted state (117, 121). Therefore, it has been hypothesized that in vitro assays will often overestimate precipitation (127), and more research is needed to investigate the nature of in vivo hydrodynamics and its implementation in supersaturation/precipitation models (128). Understanding and applying in vivo hydrodynamics could significantly improve the biorelevance of in vitro supersaturation and precipitation evaluation studies. 20

37 3. Aims of the work The main aim of this thesis was to improve understanding of the supersaturation solubility behavior of SDDSs in vitro. Another aim was to study the effects of various experimental factors on supersaturation and precipitation behavior in vitro. These aims were achieved by formulating and evaluating SDDSs. This was accomplished by the following specific aims: To prepare SDDSs (amorphous solid dispersions, cocrystals, oral films) of the PWSDs EZ, TDF and silodosin and to characterize their physical and mechanical properties (Papers I, III, IV) To investigate the solubility and supersaturation behavior of the formulated products in buffers, FaSSIF and FaSSGF, and simulated saliva (Papers I-IV) To induce supersaturation by two strategies, i.e. using formulated and non-formulated drug systems (Papers I-IV) To investigate the effects of various factors, such as preparation method, medium type, medium temperature (25ºC or 37ºC), and permeation across Caco-2 cell monolayers, on the supersaturation and precipitation behavior of PWSDs (Papers I-II) To investigate the effects of excipients (polymers and surfactants) on the supersaturation and precipitation behavior of SDDSs. The polymers studied were PVP K-30, HPMC and HPMC-AS. The surfactants used were Poloxamer 188 and TPGS (Papers I-IV). 21

38 22

39 4. Methodology A more detailed description of the methodology can be found in the Materials and Methods sections of the individual publications The studied APIs The drugs used in papers I, II, III and IV were EZ (Sigma-Aldrich, Stockholm, Sweden)), TDF (Gift from Yonsei University, South Korea) and silodosin (Ultra Medica, Damascus, Syria). The criteria for selection of drug compounds to be investigated in Papers I-IV were that the drugs: a) should be poorly water soluble, mainly BCS class II compounds; b) should be stable under the selected experimental conditions, especially at the ph used for the solubility and permeability determinations; c) should be used in their confirmed crystalline form and e) should be transported mainly by passive means if included in the permeability study through the Caco-2 cell monolayers Induction of supersaturation by SDDSs Preparation of ternary solid dispersion (Paper I) Melt-quenching method Prior to the preparation of solid dispersions, EZ and PVP K-30 were kept in the oven at 100 C for 24 h to remove any adsorbed water from the solids, and differential scanning calorimetry (DSC) analysis was performed to ensure the crystalline nature of the dried EZ. Crystalline EZ, PVP K-30 and poloxamer 188 in various proportions (see Table 3) were accurately weighed and mixed gently for 1 2 min using a porcelain mortar and pestle. The resulting powder mixtures were placed in a preheated oven at 200 C for 5 8 min. After complete melting, the liquid was immediately put into a freezer at 20 C for 1 2 days. The melt-quenched (MQ) samples were then stored in a desiccator over silica until the day of analysis. Before the analysis, all the formulations were ground and sieved using 125 mm sieves. 23

40 Spray-drying method Crystalline EZ, PVP K-30 and poloxamer 188 in various proportions (see Table 3) were accurately weighed and dissolved (equivalent to 2% w/v of solids) in methanol. The solutions were spray dried using a Buchi mini spray dryer B-29 attached to an inert loop B-295 to trap the residual solvents. The processing conditions were as follows: inlet temperature 80 C, air flow 357 L h 1, aspiration 70 80%, feed-flow rate 3 ml/min and outlet temperature C. Nitrogen gas was used as the drying gas in the closed-loop exper-iment. The collected spray-dried powders were stored in a sealed desiccator over silica until the day of analysis. Table 3. Proportions of the components in the melt-quenched (MQ) and spray-dried (SD) solid dispersions Sample Ezetimibe (%w/w) PVP K-30 (%w/w) Poloxamer188 (%w/w) MQ1 or SD MQ2 or SD MQ3 or SD Preparation of TDF-MOA cocrystals (Paper III) A 7-mL glass vial was charged with 389 mg (1 mmol) of TDF and 104 mg (1 mmol) of malonic acid (MOA). Ethyl acetate 3ml was added to form slurry. The reaction was allowed to stir for a total of 5 hours. The formation of a solid cake indicated product formation. After 5h, the solids were isolated by vacuum filtration and air dried at room temperature to yield 419 mg (85%) of TDF-MOA cocrystals Preparation of the casting gel and drug-loaded films (Paper IV) The casting gel consisted of HPMC or HPMC-AS (65%), glycerol and propylene glycol (7%), and sweetener (2%), with ethanol and water as vehicle, relative to the total weight of the solid base. All weights are w/w ratios. Silodosin was dissolved in ethanol (12-13%) and the remaining excipients were dissolved in water (87-88%). HPMC or HPMC-AS was gradually added to this solution under constant magnetic stirring (800 rpm) at ambient temperature (21± 1 C) until a homogeneous gel was obtained. This casting gel was kept for 6 12 h to remove the air bubbles. Table 4 shows the overall composition of the prepared films. 10g of the gel was cast onto a fluoropolymer-coated polyester sheet using an automated film applicator equipped with a coating knife (Coatmaster 510, Erichsen, Sweden). The silodosin dose of 8 mg was loaded into each 6 cm 2 film by fixing the wet film thickness at 750μm with a casting 24

41 speed of 5 mm/s. The cast films were dried in a convective hot-air oven (Binder, Sweden) at 60 C for min. After drying, the films were carefully peeled off, sealed in plastic (polythene) zip pouches, and stored in a desiccator (23 C/40% RH) until further characterization. Table 4. Formulations of silodosin oral films Formulation code Drug: polymer ratio F1 1:5 (Silodosin:HPMC) F2 1:3 (Silodosin:HPMC) F3* 1:5 (Silodosin:HPMC) F4* 1:3 (Silodosin:HPMC) F5 1:5 (Silodosin:HPMC-AS) F6 1:3 (Silodosin:HPMC-AS) F7* 1:5 (Silodosin:HPMC-AS) F8* 1:3 (Silodosin:HPMC-AS) *Film formulated using TPGS (0.5 % w/w) Solid-state characterization Differential scanning calorimetry (DSC) and modulated differential scanning calorimetry (MDSC) The thermal behavior of the raw materials and SDDSs (Papers I, III and IV) was studied using a DSC Q1000 (TA Instruments, New Castle, DE). The instrument was calibrated before use for temperature and enthalpy using indium. The sample was accurately weighed and placed in a non-hermetic aluminum pan, which was then crimp-sealed. The samples were heated from 25 C to 200 C (EZ), 25 to 320 C (TDF) and 25 to 120 C (silodosin) at a heating rate of 10 C/min under continuous nitrogen purge (50 ml/min). The DSC Q1000 was equipped with a refrigerated cooling system. The data were analyzed using TA analysis software (TA Instruments, New Castle, DE). MDSC was carried out (Paper I) using a Q1000 instrument (TA Instruments, New Castle, DE) for better identification of the T g through the separation of reversible (i.e. T g ) from nonreversible (i.e. enthalpy recovery, fusion) thermal events. Samples (2 5 mg) were accurately weighed and placed in aluminum non-hermetic pans, which were then crimped. These samples were then heated from 25 C to 200 C at 5 C/min with modulation amplitude of ±0.80 C every 60 s Powder X-ray diffraction (PXRD) PXRD patterns were obtained (Papers I, III and IV) using an Empyrean PXRD instrument (PANalytical, Almelo, The Netherlands) equipped with a Pixel3D detector and a monochromatic Cu-Kα radiation X-ray tube ( Å). The tube voltage and amperage were 45 kv and 40 ma, respectively. 25

42 Powdered samples (Papers I and III) were loaded into the oval cavity in the metal sample holder and carefully leveled. The experimental settings were as follows: 2θ ranged from 5 to 40, step size θ. The data were processed using High Score plus Version 3.0 software (PANalytical, Almelo, The Netherlands). The instrument was calibrated using a silicon reference standard. For Paper IV, film samples (3 3 cm 2 films) were placed on a silicone (zero background) plate which was fitted into the sample metal holder. Samples were scanned at a diffraction angle of 2θ between 5 and 40, increasing in step sizes of 0.02º. All patterns were obtained at 25 ± 1 C Thermo-gravimetric analysis (TGA) The moisture content and thermal degradation behavior (Papers I, II and IV) of all the formulation components were investigated using a Thermal Advantage TGAQ5000 (TA Instruments, New Castle, DE) connected to a cooling system. Samples were placed in the platinum pans and the furnace was heated at a rate of 5 C/min. Nitrogen at a flow rate of 50 ml/min was used as a purging gas (balance gas and sample gas: 10 and 90 C min -1, respectively). Universal analysis software was used to analyze the results (TA Instruments, New Castle, DE) Scanning electron microscopy (SEM) The morphology (Papers I and IV) of the samples was examined using a Merlin scanning electron microscope (Zeiss, Oberkochen, Germany) equipped with X-Max 50 mm 2 X-ray detectors (Oxford Instruments, Abingdon, UK). All the samples were coated with tungsten to increase the conductivity of the electron beam. The instrument voltage was 15 kv and the current was 1 na Fourier transformation infrared spectroscopy (FTIR) A Bruker IFS66v/S FTIR spectrometer equipped with a deuterated triglycine sulfate detector was used to obtain the FTIR spectra of the samples in Paper I. Powdered samples were mixed with KBr and IR spectra were obtained in the diffuse reflectant mode (Kubelka Munk). The following experimental settings were used: 64 scans with a resolution of (4 cm -1 ), spectral region cm Raman spectroscopy The Raman spectra for cocrystals prepared in Paper III were recorded on a Chromex Sentinel dispersive Raman unit equipped with a 785nm, 70 mw excitation laser and a TE cooled CCD. Each spectrum is a result of 20 coadded 20-second scans. The unit had continuous automatic calibration using 26

43 an internal standard. The data were collected by Sentinel Soft data acquisition software and processed in GRAMS AI Oral film characterization Mechanical properties The dynamic mechanical strength of the films was tested using a hybrid rheometer in DMA mode (DHR2, TA Instruments, Sweden). Briefly, samples of cast films were cut into rectangular strips of 1 5 cm 2 and 1 cm at each end was held between clamps; thus, the effective testing area was 1 3 cm 2. The upper clamp was then used to stretch the film upwards at a constant linear rate of 0.1 mm/min until the film ruptured. Stress and strain were computed by Trios software. The tensile strength (TS; Equation 4) and the elongation at break (EB; Equation 5) were obtained from the peak stress and the maximum strain, respectively, in the stress vs strain plot. Tensile tests are commonly used to determine the robustness of film preparations. The TS is the maximum force applied to the film sample at the breaking point and the EB is the length of the film during the pulling process. In addition, Young s modulus or the elastic modulus (EM) describes the influence of the strain and its force at this strain on the film area. The EM was obtained from the initial elastic deformation region in the stress vs strain plot (129). Tensile strength (TS) = Peak stress Cross sectional area of the film (4) Elongation at break (EB) = Increase in length at break Initial film length (5) Disintegration time Samples (1 1 cm 2 ) were placed in a Petri dish containing 2 ml of water and shaken at 60 rpm using an orbital shaker water bath at 37 ± 1 C. The disintegration time of the films was evaluated using a modified Petri dish method (130). The time to disintegration or disruption was measured with a stopwatch Induction of supersaturation by the solvent shift method (Paper II) Specific amounts of EZ were dissolved in DMSO and four stock drug solutions were prepared. The solvent shift method was used to induce different degrees of supersaturation (DS10, DS20, DS30 and DS40), based on the equilibrium concentration of EZ in FaSSIF. For example, the concentration of the solution at DS10 was 10 times the equilibrium solubility concentration of EZ.

44 Supersaturation-precipitation-permeation interplay (without polymer) One-compartment setup A one-compartment experimental setup (without Caco-2 cells) was used to investigate the supersaturation and precipitation behavior of EZ in the absence of a Caco-2 cell monolayer. The supersaturation experiments were conducted in 12-well plates (22.1mm diameter). FaSSIF (0.5 ml) was added to each well and supersaturation was induced using the solvent shift method by adding specific volumes of drug/dmso stock solution. The final concentration of DMSO in FaSSIF was <1%. The other experimental conditions were as follows: 60 rpm shaking speed and 37 C temperature. Samples were withdrawn at fixed time points and centrifuged immediately, and the obtained supernatants were diluted and analyzed using high performance liquid chromatography (HPLC) Two-compartment setup Apical compartment (donor compartment) FaSSIF was used as the supersaturation medium in the apical (AP) compartment. The solvent shift method was used to induce DS10, DS20, DS30 and DS40. Basolateral compartment (acceptor compartment). Hank s balanced salt solution (HBSS) was the basis of the transport medium in the basolateral (BL) compartment of the cell monolayer. The HBSS was buffered with HEPES buffer solution 10 mm to a ph of 7.4 and supplemented with 25 mm glucose. TPGS was added to the transport medium in a concentration of 0.2% to provide sink conditions Vectorial transport experiments The transport of EZ across Caco-2 cell monolayers in Transwell inserts was investigated. Samples from the AP side (40 μl) were taken at 0, 5, 15, 30, 45, 60, 90 and 120 min and centrifuged immediately at 17,000 g for 5 10 min. The supernatants were diluted with the mobile phase and analyzed using HPLC to find the concentration of EZ. The amount of EZ in the AP compartment (0.5 ml) was quantified. Samples (200 μl) were withdrawn from the BL side at the same time points and analyzed without dilution. Fresh buffer (200 μl) was added to the BL side at each time point to ensure the maintenance of sink conditions. Each plate set of Transwell membrane inserts was kept in an agitating incubator with an orbital shaker (speed 60 rpm and medium temperature 37 C) and was only taken out for sampling. 28

45 Supersaturation-precipitation-permeation interplay (with polymer) The effect of the polymer on supersaturation and permeation was studied in one-compartment and two-compartment setups with the DS40 sample. PVP K-30 was pre-dissolved in FaSSIF in two concentrations: 0.05 and 0.1% w/v on the AP side of the two-compartment setup and in the one-compartment setup. The DS40 supersaturated solution of EZ was prepared based on the equilibrium solubility of the drug in FaSSIF in the presence of the polymer Preparation of amorphous drug forms (Papers I and III) Crystalline EZ (Paper I) was accurately weighed and placed in a preheated oven. After complete melting, the liquid was immediately put into a freezer at 20 C. The material was then gently ground with a mortar and pestle under controlled water vapor pressure and immediately analyzed by DSC and PXRD. Before the analysis, all the formulations were ground and sieved using 125 mm sieves. Amorphous TDF (Paper III) was prepared by spray drying a 2% w/v solution of TDF in acetone:water (9:1, v/v), as outlined by Wlodarski et al. (131). A Buchi mini spray dryer B-290 (Büchi, Switzerland) was used. An open system was used with nitrogen as the drying and atomizing gas (open, suction mode). The spray drying conditions were as follows: inlet temperature 160 C, pump speed 30 %, and outlet temperature 89 C. The aspirator was operated at 100%. The obtained powder was sieved (500 micron) and analyzed by DSC and PXRD to confirm the amorphous state. The powders were stored at -20 C until further analysis Solubility and dissolution studies Solubility study (Papers III and IV) Cocrystal solubility and determination of eutectic concentrations of TDF and MOA In paper III, the eutectic point between cocrystal and drug was reached by simply adding 200 mg of cocrystal powder (500 microns) to 5 ml of solution at different phs. A 0.1 N HCL solution was used to obtain phs of 1, 2 and 5, an acetate buffer solution was used for phs 3 and 4, and a phosphate buffer solution was used for phs 6 and 7. The solutions were subjected to orbital shaking (60 rpm) for hours at 25 C and the ph at equilibrium was measured. The concentrations of the drug and the coformer at equilibrium at the eutectic point were determined by HPLC. The eutectic point was confirmed by PXRD analysis of the residual solid sample at equilibrium. 29

46 The equilibrium solubility of TDF was measured at a range of phs from 1 to 7; the ph of the solution was adjusted by 1 M HCl or NaOH. An excess amount (approx. 200 mg) of crystalline TDF was added to a 5 ml solution of 0.1 N HCl and slurried in a water bath at 25 C with orbital shaking (60 rpm.) After equilibration for hours, the sample solutions were filtered [using 0.2 µm polytetrafluoroethylene (PTFE) filters] and analyzed by HPLC (Agilent Technologies, Stockholm). The ph at equilibrium was measured and the excess solids were analyzed using PXRD to confirm the solid form at equilibrium Equilibrium solubility determination of silodosin The solubility of silodosin was determined in simulated saliva containing pre-dissolved HPMC or HPMC-AS with or without TPGS in concentrations similar to those used in the film formulations. An excess of drug was added to conical flasks containing 10 ml of saliva and the other polymer-surfactant excipients. The flasks were tightly closed and placed in a shaker water bath at 37 C. After 48 h, the separated aliquots were filtered through a 0.45 μm filter, diluted appropriately and analyzed using HPLC. Each experiment was performed in triplicate (n = 3) and the results were reported as means ± standard deviation Investigation of dissolution and supersaturation solubility (Papers I, III, IV) Dissolution of ternary solid dispersions in simulated intestinal fluids Excess amounts of the solid dispersions of EZ (Paper I) were suspended in 10mL of FaSSIF or FaSSGF in sealed test tubes and placed in a preheated shaking water bath at an orbital shaking speed of 60 rpm. The experiments were performed at 25 C and 37 C. The samples were withdrawn after 5, 15, 30, 45, 60, 75, 90, 120, 180 and 240 min and centrifuged (5 min, 17,000 g), filtered through 0.2 mm PTFE syringe filters to ensure complete separation of the solid phase, and analyzed by HPLC. For comparison, the dissolution properties of crystalline and amorphous EZ in FaSSIF and FaSSGF were also studied. The samples were withdrawn at pre-defined intervals following the above procedure Dissolution of cocrystals in phosphate buffer Non-sink dissolution experiments were conducted to investigate the dissolution and supersaturation behavior of amorphous TDF and TDF-MOA cocrystals (Paper III). Excess amounts (approx. 1 mg/ml) of crystalline TDF, amorphous TDF and an equivalent amount of the cocrystal powder were suspended in a phosphate buffer solution (ph 6.8). The experiments were 30

47 conducted at 25 C with 60 rpm orbital shaking. Aliquot samples were withdrawn at specific time points up to 3 hours, filtered (0.2 µm PTFE filters), diluted and analyzed by HPLC. The dissolution of crystalline TDF, amorphous TDF and the cocrystals was also investigated in the presence of HPMC. HPMC was pre-dissolved in the phosphate buffer solution (0.1% w/v; ph 6.8) and the dissolution experiments were conducted under similar conditions to those above. At the end of the dissolution study, the ph of the solutions was measured and the separated solids were analyzed with PXRD Dissolution of oral films in simulated saliva Non-sink dissolution studies were carried out in simulated saliva at ph 6.8. The films (samples F1-F8; 2 3 cm) were carefully dropped into 10 ml dissolution medium under continuous orbital shaking (60 rpm) at 37 C. Samples of the medium were then withdrawn at different times, filtered through syringe filters (0.45 μm) and analyzed by HPLC. The DS was calculated from the drug concentrations at different times during dissolution of the films (F1-F8) and the drug concentrations at equilibrium. DS calculations for the formulated drug were based on Equation 3 (DS=C t /C eq ). The obtained values of DS for F1-F8 were plotted versus time HPLC quantification methods Solutions were analyzed by reversed phase HPLC (HPLC 1290 series instrument equipped with a quaternary pump and a UV detector; Agilent, Santa Clara, CA). An isocratic method was used. For determination of the concentration of EZ (Papers I and II), a C-18 Eclipse plus 3 mm (4.6mm 100mm) column was used. The mobile phase consisted of 40:60 (v/v) 0.05% sodium 1-heptane sulfonic acid and acetonitrile with a flow rate of 0.5 ml/min. The injection volume was 10 μl and the measurements were carried out at a wavelength of 230 nm. TDF and MOA were quantified using a C-18 Nucleosil column ( mm, 5 microns; Sigma Aldrich, Sweden) for both compounds. For TDF, the mobile phase was 20 mm Na-phosphate buffer (ph 7) and acetonitrile (30:70). Analysis was performed at a flow rate of 0.5 ml/min and a detector wavelength of 260 nm. For MOA, the mobile phase was 25 mm potassium dihydrogen phosphate (ph 2.5) and methanol in a ratio of 93:7. The flow rate and wavelength were 1 ml/min and 210 nm, respectively. Sample separation for silodosin (Paper IV) was performed on an Agilent Eclipse-plus C18 column (5 μm, 250 mm 4.6 mm) with a mobile phase of 25 mm potassium dihydrogen phosphate buffer (ph 7.0) and acetonitrile 40:60 (v/v) at 25 C. The flow rate was 1 ml/min and the determination wavelength was 269 nm. 31

48 4.7. Statistical analysis One-way analysis of variance (ANOVA) with Tukey's post hoc multiple comparisons and the t-test were used to determine statistically significant differences (p < 0.05) in Papers II and IV. All results were expressed as means with standard deviation (n=3). Mechanical properties in Paper IV were tested with n = Preparation of simulated fluids (Papers I, II and IV) FaSSIF and FaSSGF FaSSIF/FeSSIF/FaSSGF powder from biorelevant.com (London, UK) was used to prepare the simulated intestinal fluid. FaSSIF (sodium taurocholate, lecithin, sodium chloride, sodium hydroxide and monobasic sodium phosphate) and FaSSGF (sodium taurocholate, lecithin, sodium chloride and hydrochloric acid) were prepared and the ph adjusted to 6.5 (FaSSIF) and 1.5 (FaSSGF) with NaOH and HCL Simulated saliva Simulated saliva was prepared in the laboratory by dissolving the ingredients in purified water (Table 5) and adjusting the ph to 6.8 with NaOH (132). Table 5. Simulated saliva formulation (132) Ingredients Grams/liter of purified water Sodium chloride (NaCl) Potassium chloride (KCl) Potassium phosphate monobasic (KH2PO4) Urea Sodium sulfate (Na2SO4 10H2O) Ammonium chloride (NH4Cl) Calcium chloride dihydrate (CaCl2 2H2O) Sodium bicarbonate (NaHCO3) Cell culture (Paper II) Caco-2 cells were routinely cultured in 75 cm 2 tissue culture flasks (Greiner Bio One, Frickenhausen, Germany) at 37 C with 5% CO 2 atmosphere in high glucose Dulbecco s modified Eagle s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 1mM sodium pyruvate, 100 U/mL penicillin and 100 μg/ml streptomycin. For the transport studies, the cells were 32

49 seeded at a density of 75,000 cells/cm 2 on rat-tail collagen type I-coated Transwell clear inserts (12mm in diameter, pore size 0.4 μm; Corning, VWR, Dublin, Ireland). The cells were allowed to grow for at least 16 days and the medium was exchanged every other day. The transepithelial electrical resistance (TEER) of the cell monolayers was measured using a Millicell ERS-2 epithelial volt-ohm meter equipped with STX-2 electrodes (Millipore, Carrigtwohill, Ireland) and corrected for the background value contributed by the cell culture inserts and medium. To exclude any effects of the sampling process on the cells' integrity and to ensure the tightness of the inter-cellular junctions, the TEER values for all cell monolayers were measured before starting and at the end of each experiment. 33

50 34

51 5. Results and discussion 5.1. Preparation of a ternary solid dispersion of ezetimibe (Paper I) The cholesterol-lowering drug EZ was chosen as the model drug in this study. EZ is a lipophilic molecule with a partition coefficient (logp; octanol/water) of 4.5. It is very weakly acidic (close to neutral) with an acid dissociation constant (pka) of 10.2 (aromatic hydroxyl group) and thus is practically insoluble in water. EZ has been classified as a class II drug; its low dissolution rate in the gastric lumen could potentially lead to dissolution-limited bioavailability ( ). In this study, ternary solid dispersions of EZ were prepared using different proportions of the polymer PVP K-30 and the surfactant poloxamer 188. Two preparation methods were employed: melt quenching and spray drying. Visually glassy materials of pure EZ and the solid dispersions were obtained using the melt-quenching method. The formulations containing EZ, poloxamer and polymer obtained with the spray-drying technique were dry, white powders of EZ and the solid dispersions. Solid-state characterization of the solid dispersions of EZ was carried out using DSC, MDSC, PXRD and FTIR. The amorphous nature of the solid dispersions was confirmed in all the characterization tests. A single T g (no melting peak for EZ) was observed at C for all the solid dispersions when examined by MDSC, suggesting that EZ was molecularly dispersed within the polymer and surfactant to form an amorphous solid solution (Figure 7) (35, 136, 137). SEM images of the solid dispersions showed that the two preparation methods resulted in different particle morphologies for the same formulation, while there were no major differences in particle morphology between the different formulations prepared by the same method. Further, there were no EZ crystals on the surfaces of the particles (Figure 8) In vitro dissolution study (Paper I) It is important to optimize the in vitro experimental conditions in studies of the supersaturation behavior of amorphous solids to ensure that they are biorelevant to the in vivo conditions. This study used biorelevant test media, non-sink conditions and orbital shaking. It has been suggested that non-sink conditions are more biorelevant for evaluating the dissolution behavior of supersaturable formulations where the maximum solution concentrations are determined (138) 35

52 Figure 7. Modulated differential scanning calorimetry reversing heatflow thermograms of (a) melt-quenched (MQ) solid dispersion 1, (b) MQ2 and (c) MQ3; and (d) spray-dried (SD) solid dispersion 1, (e) SD2 and (f) SD3 showing the heat capacity (T g ) in the region C. The effect of hydrodynamics on supersaturation/precipitation is poorly understood. In a recent paper, the precipitation rate was shown to be remarkably slower in the shaking model than in the stirring model, emphasizing the importance of the applied hydrodynamic methodology (95). The factors affecting supersaturation are discussed in more detail later in the thesis. 36

53 Figure 8. Scanning electron microscope photographs of (a) crystalline ezetimibe (EZ), (b) amorphous EZ, and (c) melt-quenched (MQ1) and (d) spraydried (SD1) solid dispersions of EZ Preparation of cocrystals of tadalafil (Paper III) The drug TDF was used as the study drug for preparing cocrystals in this study, with MOA as the coformer. TDF is a phosphodiesterase inhibitor, a pharmaceutically active ingredient marketed under the brand name Cialis and approved by the FDA in 2003 for the treatment of erectile dysfunction. TDF is a white crystalline powder, practically insoluble in water that has been classified as a class II drug by the BCS. It is a neutral drug, with a pka of The TDF logp (octanol:water) is 1.54, which reflects poor drug solubility in water ( ). The pharmaceutical cocrystals of TDF were formed with the coformer MOA, as confirmed by solid-state characterization using Raman spectroscopy, PXRD and DSC. All these techniques unequivocally suggested the formation of TDF-MOA cocrystals. For example, the diffraction pattern for the new form was distinctly different from that of TDF and MOA separately, as shown in Figure 9. 37

54 Figure 9. Powder X-Ray Diffraction patterns for (a) tadalafil (TDF), (b) TDF-MOA cocrystals and (c) malonic acid (MOA). The asterisks denote new diffraction peaks in the cocrystal form in comparison with the individual cocrystal components ph-related solubility and Supersaturation index of TDF- MOA cocrystals (Paper III) Understanding the ph dependence of cocrystal solubility helps to guide the development of formulations for cocrystals as well as helping to determine their supersaturation index and conversion/transformation propensity. TDF- MOA (1:1) cocrystals consist of non-ionic drug, TDF and one diprotic acid MOA, with two pka values (2.83 and 5.69). The TDF-MOA cocrystal solubility dependence on [H + ] is given by Equation 6: S TDF MOA = K sp 1 + K a1,moa [H + ] + K a1,moak a2,moa [H + ] 2 (6) Where K sp is the cocrystal solubility product, and K a1 and K a2 are the ionization constants for MOA. K sp was calculated as the product of the concentrations of the non-ionized constituents according to Equation 7: K sp = 1 + K a1,moa [H + ] [TDF] T [MOA] T + K a1,moa K a2,moa [H + ] 2 (7) Where [TDF] T and [MOA] T are the eutectic concentrations of TDF and MOA, respectively. The TDF-MOA cocrystals converted back into TDF at phs in the range of 1 to 7.4. The solubility was measured at the eutectic point where the solution phase was in equilibrium with both cocrystals and solid drug phases (75). The cocrystal K sp was determined from Equation 7. 38

55 Table 6 summarizes the cocrystal K sp and intrinsic solubility of the drug, S TDF. K sp was used to generate the dependence of the cocrystal solubility on ph. Figure 10a shows that the solubility of the TDF-MOA cocrystals was higher than that of TDF over a wide ph range. For example, even at their lowest solubility, the cocrystals appeared to be over 100 times more soluble than the drug (Figure 10a and Table 6). These trends are consistent with the coformer solubility and ionization properties, in line with the results reported for other cocrystals (75, 143). Table 6. Summary of the tadalafil (TDF)-malonic acid (MOA) cocrystal solubility product (K sp ± standard deviation), the intrinsic solubility of TDF (S TDF ), and the cocrystal:tdf solubility ratio (S CC /S TDF ). The solubility was measured under nonionizing conditions Cocrystal K sp (M 2 ) S TDF (M) S CC /S TDF (ph 1-3) TDF-MOA cocrystal (3.8 ± 1.4 ) (4.6 ± 0.2) The experimental cocrystal solubility could only be measured in the ph range 1 to 3, as the coformer controlled/lowered the ph of the solution. The mathematical model based on K sp and K a values, (Equation 6) predicted an exponential increase in cocrystal solubility with ph, and these theoretical values were in agreement with the experimentally measured solubilities (Figure 10a). Thus, it is possible to predict the dependence of the cocrystal solubility on ph from the Ksp, at ph values where the solubility was not experimentally measured, and to gain insight into the supersaturation behavior of the cocrystals at physiologically relevant phs. It is clear from the shape of the solubility-ph curve that the formation of TDF-MOA cocrystals changed the solubility of TDF to being ph-dependent, demonstrating the potential of cocrystal technology in engineering the solubility behavior of non-ionizing drugs as a function of ph. Cocrystals of saccharin have previously been shown not only to enhance the aqueous solubility of saccharin but also to impart ph sensitivity to cocrystallized non-ionizing drugs like carbamazepine (143) Furthermore, the TDF eutectic concentrations were in agreement with the drug solubility vaues (S TDF ; Figure 10a), suggesting that there was no drug complexation with the coformer in solution. The enhanced solubility of cocrystals has been previously reported for a number of drugs (144, 145). The solubility advantage or supersaturation index (SA), is defined as S cc /S drug, where S cc and S drug are cocrystal and drug solubility, respectively. The SA represents the driving force for drug precipitation and provides a measure of the risk of cocrystal conversions. It also provides guidance for the selection of ph and formulation additives to prevent drug precipitation. Supersaturation index points were calculated from 39

56 experimental cocrystal solubility and eutectic drug concentrations. The theoretical SA was extrapolated according to Equation 8: S TDF MOA S TDF = K sp 1+ K a1,moa H + + K a1,moa K a2,moa H + 2 = S TDF K sp 1+ K a1,moa H + + K a1,moa K a2,moa H + 2 (8) [TDF] T Figure 10b shows the dependence of the SA on ph for TDF-MOA cocrystals. The theoretical and experimentally measured values (in the range ph 1-3) are in good agreement. Interestingly, the SA for TDF-MOA cocrytals increased from approximately 100 at ph 1 (measured) to about 48,000 at ph 6.8 (predicted) (Figure 10b). Figure 10. (a) ph dependence of the solubility of 1:1 tadalafil-malonic acid (TDF- MOA) cocrystals and TDF. Experimentally measured solubilities of the cocrystals and drug are represented by red circles ( ) and blue squares ( ), respectively. [TDF] T concentrations are shown as blue circles ( ). The lines represent the theoretical solubilities of the cocrystals (red dashed line) and drug (blue line). The dependence of the solubility on ph was calculated according to Equation 6 using the K sp value listed in Table 6 and pka values of 2.83 and 5.69 for MOA. The solubility of TDF (S TDF ; line) was determined as the average of the experimental TDF solubility points (blue squares). (b) The cocrystal supersaturation index (cocrystal solubility advantage) as a function of ph for TDF-MOA cocrystals. The symbols represent the calculated cocrystal:tdf solubility ratios from the solubility measurements and the line represents the theoretical dependence on ph (Table 6). The SA values are several orders of magnitude higher than the experimentally measured supersaturation generated by amorphous or cocrystal forms because of the onset of nucleation at a lower supersaturation value. In studying the SA of cocrystals, Good and Rodriguez-Hornedo have shown that the solubility of cocrystals is directly proportional to the solubility of the constituent reactants for a wide range of drugs (including acidic, basic, zwit- 40

57 terionic and neutral drugs) (75). Further, Velaga et al. revealed an increase in the coformer concentration at the eutectic point for carbamazepine-saccharin cocrystals and concluded that the aqueous SA of the cocrystals was the result of a decrease in the solvation energy (log γ) (143) Dissolution and supersaturation behavior of TDF solid forms (Paper III) Figure 11 shows non-sink dissolution profiles for TDF cocrystals, TDF amorphous and crystalline solids in the presence and absence of the precipitation inhibitor HPMC at ph 6.8. In the absence of HPMC, TDF in the form of cocrystals and the amorphous form dissolved faster and reached supersaturation sooner, followed by precipitation. The solubility of the crystalline form of TDF reached a plateau corresponding to the equilibrium solubility of the drug ( M) in phosphate buffer at ph 6.8. The spray dried TDF (amorphous form) and cocrystals dissolved very quickly and reached maximum concentrations (C max ) of M and M, respectively, in less than 5 min. Thereafter, the concentrations decreased because of precipitation or conversion of the cocrystals to TDF, as confirmed by PXRD analysis. The dissolution behavior of TDF-MOA cocrystals resembles that of many other cocrystals (144). The maximum concentration of a drug can be expressed in terms of its supersaturation (C max /S drug ). C max /S drug thus represents the concentration above which precipitation occurs faster than the dissolution of the cocrystals. The cocrystal and amorphous forms generated a supersaturation level of 30 and 10, respectively (Figure 11). 41

58 Figure 11. Dissolution profiles of different solid forms of tadalafil (TDF) in phosphate buffer (ph 6.8±0.4): crystalline TDF (blue), amorphous TDF (green), TDFmalonic acid (MOA) cocrystals (red); and in phosphate buffer containing 0.1% w/v HPMC: amorphous TDF (black) and TDF-MOA cocrystals (purple). Error bars represent standard deviations obtained from triplicate measurements Preparation of silodosin oral films (Paper IV) Silodosin is a selective α 1A adrenoceptor blocker that is a safe, effective treatment for the relief of voiding and storage symptoms in patients with benign prostatic hyperplasia (146). It is a white to pale yellowish powder with a logp (octanol/water) of 2.87, pka1 of 8.53 and pka2 of The bioavailability of oral silodosin capsules is nearly 32% (147). The FDA label states that silodosin is very slightly soluble in water. Oral films of silodosin were successfully prepared with HPMC or HPMC-AS as the formulating polymer, with or without the surfactant TPGS. The shape and morphology of representative film samples were examined using SEM, as shown in Figure 12. The HPMC-based films (F2 and F4), but not the HPMC-AS-based films (F6 and F8), had submicron silodosin particles and tiny pores (Figure 12). This observation was in agreement with the PXRD results. The submicron particles of silodosin may have formed at the point of supersaturation in HPMC during preparation of the casting gel (148). 42

59 Figure 12. Scanning electron microscopy (SEM) micrographs of the formulated films and pure silodosin. The bar represents 100 μm for samples F2, F4, F6, and F8 and 20 μm for pure silodosin. The solid-state characteristics of the films (F1-F8) were investigated using DSC, PXRD and disintegration times. As shown in Figure 13, the melting peak (T m ; C) for the crystalline form was absent from all the DSC thermograms of all the prepared films (F1-F8). This could be the result of molecular dispersion of the drug in the polymer. The fastest disintegration was observed with the HPMC-AS-based film F5 (drug:polymer ratio 1:5) which disintegrated in 15.3±1.2 sec (Table 7). Disintegration was slower for the film based on the HPMC polymer with the same drug:polymer ratio (F1: 35.3±0.6 sec). This effect may have been related to the properties of the polymer, i.e. wettability and surface tension. Thus, HPMC-AS films disintegrated more quickly than HPMC films (149, 150). Addition of the surfactant (TPGS) to the film formulation had an additive effect on the disintegration time (Table 7). These results were in agreement with those of Vuddanda et al., who found an additive effect on speed of disintegration on addition of the surfactant TPGS (151). 43

60 Figure 13. Differential scanning calorimetry of the silodosin film formulations showing (a) HPMC-based films F1-F4 and (b) HPMC-AS-based films F5-F8. The mechanical and tensile properties of the thin films were measured under ambient conditions. The mechanical properties of HPMC films were better than those of HPMC-AS films with respect to stability during patient handling and packing. Table 7. Silodosin oral films: Disintegration time Formulation code Mean disintegration time (seconds)* F1 35.3±0.6 F2 61.0±0.0 F3 65.7±0.6 F4 62.7±1.5 F5 15.3±1.2 F6 33.7±2.5 F7 33.0±1.0 F8 56.7±0.6 *Means ± standard deviations (n 3) In vitro dissolution in simulated saliva (Paper IV) The dissolution studies were conducted in simulated saliva (ph 6.8) under non-sink conditions, mimicking the conditions of the oral cavity. The solubility of pure silodosin in simulated saliva was 0.46 mg/ml. The solubility of silodosin in simulated saliva with additional dissolved HPMC (with and without TPGS) ranged from 0.48 to 0.51 mg/ml at equilibrium after 48 hours, and with additional dissolved HPMC-AS (with and without TPGS) ranged from 0.94 to 1.1 mg/ml. The dissolution results for the films formulated using HPMC and HPMC-AS are shown in Figure 14a and 14b, respectively. In the HPMC-based films, 80% of the drug was released in 10 minutes from F2 (drug:polymer ratio 1:3), while dissolution was poorer for F1 (drug:polymer ratio 1:5), with 80% 44

61 release after 25 minutes. This may be because the increase in polymer concentration led to the formation of a gel-like state which decreased water uptake and retarded drug release, as reported by Singh et al (152). Alhayali et al. have previously reported that, in some cases of solid drug dispersions, the drug concentrations do not change significantly with higher polymer ratios (153). Addition of TPGS also improved the drug release noticeably. Thus, films containing TPGS dissolved faster than TPGS-free films. Other workers have mentioned this effect of the surfactant TPGS in terms of its ability to improve the solubility, dissolution rate and bioavailability of some drugs formulated as oral films (154, 155). In HPMC-AS-based films, about 80% of the drug was released in around 10 minutes (F5 and F6); with no significant differences in dissolution rate between the 1:3 and 1:5 ratios. Addition of TPGS to the HPMC-AS-based films negatively affected the dissolution rate for both ratios. Therefore, this study has demonstrated that HPMC works well as a film-forming polymer when combined with TPGS. Figure 14. Film dissolution of (a) HPMC-based films and (b) HPMC-AS-based films in simulated saliva. In a case study (156), HPMC was the most suitable film-forming material of the excipients tested, providing faster dissolution and easier-to-handle films. Interestingly, the submicron silodosin particles seen in the HPMC films appear not to have affected the faster drug release from these films. Further, it was observed that the dissolution behavior of HPMC-AS (TPGSfree) films was similar to that of HPMC films containing amorphous solid dispersions (Figure 14). These studies suggested that both polymers are capable of increasing and prolonging the supersaturation of the drug and helping to prevent the tendency of the drug to precipitate and recrystallize during dissolution. This could be attributed to the existence of the drug in the amorphous state and it being molecularly dispersed in the polymer matrix. This was also evident from the thermal, solid-state and morphological results. It has been reported that cellulose-derived polymers such as HPMC, HPMC- AS and HPMCP are superior for preparing solid, amorphous dispersions, 45

62 particularly with PWSDs, compared to other polymers. The bulky structure of the polymer network and worse solubility properties (compared to highly water-soluble polymers) facilitate the reduction of drug mobility in the polymer matrix and prevent the drug from recrystallizing, as normally induced by supersaturation during dissolution (157). This consequently improves the product s physical stability and in vitro drug release performance. In general, our dissolution results revealed that either HPMC or HPMC-AS would be useful for preparing films intended for oral cavity absorption, which is more complex than GIT absorption. Figure 15. Degree of supersaturation as a result of formulating the drug silodosin in different oral film formulations containing HPMC (F1-F4) or HPMC-AS (F5- F8). n= 3; means ± standard deviation. The DS values for silodosin formulated as an oral film are presented in Figure 15. The highest DS was obtained for HPMC-based formulations at early time points. F4, in particular, had a DS value of > 1 (supersaturated) before 5 minutes, which was earlier than for the other films formulated using the HPMC polymer (F1-F3), as shown in Figure 15. In the dissolution results, 80% of the drug was released from film formulation F4 during the first 10 minutes (Figure 14). This effect could be attributed to a synergistic effect of the surfactant (TPGS) and the polymer (HPMC), resulting in improved solubility and maintained supersaturation (114, 115, 86). Therefore, film formulation F4 appears to have potential for the development of a silodosin film formulation with improved performance and improved oral sublingual absorption as a result of the high degree of supersaturation solubility (10, 158). 46

63 5.4. Factors affecting supersaturation behavior (Paper I, II, III) Effects of process, formulation and temperature on supersaturation in FaSSIF (Paper I) The supersaturation and precipitation behavior of solid dispersions of EZ was investigated in a one-compartment model under non-sink conditions. Figure 16 shows the dissolution profiles (concentration versus time) for the prepared solid dispersions in FaSSIF. The solubility of crystalline EZ in FaSSIF (ph 6.5) at 25 C and 37 C was 5.9 mg/ml and 10.1 mg/ml, respectively as shown in Figure 16a and b. The apparent solubility (concentration in solution) of EZ from the amorphous form increased and then rapidly decreased at 25 C (Figure 16a and b). In contrast, there was no supersaturated concentration at 37 C for amorphous EZ (Figure 16c and d). This phenomenon of supersaturation and rapid precipitation is typical of amorphous solids and is described as the spring effect (10). Figure 16. Dissolution and precipitation (supersaturation) profiles of ternary solid dispersions of ezetimibe (EZ) in fasted-state simulated intestinal fluid. (a) Meltquenched (MQ1, 2, 3) and (b) spray-dried (SD1, 2, 3) solid dispersions at 25 C; (c) MQ and (d) SD solid dispersions at 37 C. AMP: amorphous EZ; CRY: crystalline EZ. 47

64 For orally administered formulations, the dose-to-solubility ratio has been suggested as an indicator of the precipitation propensity of the drug in the intestinal lumen (6). Drugs with dose-to-solubility (non-ionized) ratios of more than 50:1 have been reported to show a tendency to precipitate in the intestinal environment (159). The dose-to-solubility ratio for EZ is about 1000 at a dose of 10 mg, suggesting that the observed precipitation of stable phases is thermodynamically favored in FaSSIF. Interestingly, the spring effect disappeared (i.e. there was no increase in solubility) as the temperature of the dissolution experiment was increased to 37 C. This may have been because of differences in the relationship between dissolution and the precipitation/crystallization kinetics of crystalline EZ. It is possible that the crystallization of amorphous EZ was faster at 37 C. Similar behavior has been observed with amorphous felodipine and indomethacin in another study (160). Solid dispersions where the drug and polymers are intimately mixed are widely used formulations that can prevent the crystallization of the stable phase and maintain the supersaturation of drugs like EZ. In this study, PVP- K30 and poloxomer 188 were selected from the preliminary studies as the precipitation inhibitor and solubilizer, respectively. PVP has been well established as a crystal growth inhibitor for a range of amorphous drugs (126). In the MQ solid dispersions, concentrations of EZ in solution were maintained at about mg/ml and no precipitation was observed for at least 240 min at 25 C (Figure 16a). Apparently, the presence of the polymer sustained supersaturation, with solution concentrations about 50 times higher than the equilibrium solubility of EZ, possibly by preventing precipitation of the stable phase. However, no differences in the solution concentrations were observed for different drug:polymer ratios in these solid dispersions. Interestingly, in the SD solid dispersions, supersaturation (with solution concentrations about times higher) was followed by desupersaturation at different rates (Figure 16b). SD solid dispersions generated higher initial solution concentrations than those generated by MQ solid dispersions, which could be explained by differences in the particle size/morphology and molecular miscibility. This could also mean that solid dispersions result in a greater driving force for crystallization. The desupersaturation curves also showed that the lower the proportion of polymer in the solid dispersions, the faster the precipitation kinetics will be (Figure 16b). The presence of 10% poloxomer resulted in even faster precipitation of the crystalline phase. The rich drug polymer interactions and changes in viscosity can alter the crystal growth and surface integration kinetics, leading to differences in the crystallization kinetics. Previously published articles have also proposed similar mechanisms to explain the differences in the precipitation kinetics of many amorphous drugs (103). 48

65 In general, solid dispersions prepared by the melting method showed slightly higher initial concentrations and faster precipitation kinetics at 37 C than at 25 C (Figure 16a and c), while solid dispersions prepared by the spraydrying method showed direct precipitation in FaSSIF at 37 C but not at 25 C (Figure 16b and d). The presence of precipitation inhibitors had only a minor effect in maintaining supersaturation at 37 C. These results are perfectly in line with previous observations on the supersaturation and precipitation behavior of other amorphous drugs in the presence of different polymers (161). The results suggest that dissolution experiments performed at 25 C may not be biorelevant and may fail to predict in vivo behavior (162). In the MQ solid dispersions, desupersaturation was dependent on the formulation: a higher polymer/poloxomer content resulted in a slower precipitation rate (Figure 16c). However, in the SD solid dispersions, precipitation was rapid irrespective of the formulation or polymer content (Figure 16d). This suggests that the supersaturation and precipitation behavior of amorphous solids is sensitive to the processing method, possibly because of the history of the material and the presence of nuclei (163) Effects of process, formulation and temperature on supersaturation in FaSSGF (Paper I) Figure 17 shows the dissolution (concentration versus time) profiles for EZ samples in FaSSGF (ph 1.6). The solubility of crystalline EZ in FaSSGF (ph 1.2) at 25 C and 37 C was extremely low: 0.2 mg/ml and 0.1 mg/ml, respectively (Figure 17a and b). This extremely low solubility is thought to be due to the un-ionized state of the drug in the dissolution medium. In contrast to its behavior in FaSSIF, there was no increase in initial solution concentrations or supersaturation for amorphous EZ in FaSSGF. The method of preparation and the temperature had no effect on this behavior. This suggests rapid transformation of the amorphous phase into the crystalline phase, possibly as the result of an extremely high driving force for crystallization. Although some increase in the initial EZ concentrations and supersaturation were observed with the MQ and SD solid dispersions in general, the level and extent of supersaturation were much lower in FaSSGF than in FaSSIF at 25 C and 37 C (Figure 17). Higher polymer/poloxomer content delayed the precipitation kinetics, leading to sustained supersaturation for longer periods as observed in FaSSIF. Although PVP and poloxamer 188 were effective in inhibiting/delaying precipitation from the solid dispersions at 25 C, they were ineffective at 37 C, which is the physiologically relevant temperature. The method of preparation also affected the supersaturation and precipitation kinetics. In general, supersaturation was maintained for relatively longer in biorelevant media at both temperatures when MQ solid dispersions were used 49

66 (Figure 17). It is important to consider these aspects in the design and development of amorphous solid dispersions of poorly soluble drugs. Figure 17. Dissolution and precipitation (supersaturation) profiles of ternary solid dispersions of ezetimibe (EZ) in fasted-state simulated gastric fluid. (a) Melt-quenched (MQ1, 2, 3) and (b) spray-dried (SD1, 2, 3) solid dispersions at 25 ºC; (c) MQ and (d) SD solid dispersions at 37 ºC. AMP: amorphous EZ, CRY: crystalline EZ Effect of implementation of an absorptive compartment on supersaturation behavior (Paper II) The absorption process across the intestinal epithelial barrier could substantially influence the supersaturation and precipitation behavior of a drug. In this study, the effect of absorption across Caco-2 cell monolayers on the supersaturation and precipitation of EZ was investigated in a cell-free, onecompartment setup and compared with results in a two-compartment model. Supersaturation experiments were initially conducted in wells of cell culture plates (one-compartment setup) to gain insight into the supersaturation/precipitation behavior of EZ in the absence of Caco-2 cells (i.e. in a cell-free environment). As illustrated in Figure 18a, the EZ concentration was higher with the DS10 sample than with crystalline EZ, and supersaturation was maintained until the end of the experiment. EZ precipitated with the DS20 and DS30 samples after 15 and 5 min, respectively. Instantaneous precipitation occurred with the DS40 sample in FaSSIF (Figure 18a). In this study, sponta- 50

67 neous nucleation and precipitation of EZ were observed in FaSSIF for all the supersaturated solutions, suggesting a low kinetic barrier to crystallization of the drug. This is typical behavior, because the supersaturated state is thermodynamically unstable and faster precipitation occurs as supersaturation increases, according to the theory of crystal growth (126). In contrast, Bevernage et al. observed no spontaneous precipitation, and nuclei were required to promote precipitation (164). Figure 18. (a) Amount of ezetimibe (EZ) in fasted-state simulated intestinal fluid (FaSSIF) in the one-compartment setup. (b) Amount of EZ in FaSSIF on the apical side of Caco-2 cell monolayers as a function of time, after induction of supersaturation. (c) Cumulative amount of EZ recovered from the basolateral acceptor medium (i.e. HBSS) after vectorial transport across Caco-2 cell monolayers upon induction of supersaturation in the apical compartment. Data represent means ± standard deviations (n=3). Error bars in (c) were too small to show. DS = degree of supersaturation. Figure 18b shows the total amounts of EZ in FaSSIF on the AP side (twocompartment model) without addition of polymer. As depicted, the amount of drug dissolved increased as the DS increased. All DS levels were associated with more stable solutions with lower precipitation tendencies. The reason for this could be that permeation of the drug was an alternative to precipitation for relieving the thermodynamically unstable state that is the predominant feature during supersaturation, and as the concentration (or the 51

68 DS) increased, the possibility of permeation reducing precipitation also increased, as reported by Bevernage et al.. The effect of inducing different degrees of supersaturation on the permeation of EZ across the Caco-2 cell monolayer is shown in Figure 18c. The cumulative amounts of EZ in the transport medium (HBSS) in the BL compartment is plotted as a function of time. As delineated in Figure 18c, the transport of drug to the BL side was delayed for about 45 min with DS10 and 30 min with DS20, DS30 and DS40. This lag-time could be due to the time needed to saturate the monolayer barrier before reaching steady-state flux, as reported by Kanzer et al. (165). The highest amount of EZ transported to the BL compartment after 2 h was 1.28 ± μg from the DS40 sample; ± 2.27 μg was in solution in the AP compartment after 2 h, as shown in Figure 18b. The resulting flux was ± μg/min cm 2 (Figure 19). Although the DS10 sample resulted in the highest amount of EZ dissolved after 2 h in the onecompartment setup (Figure 18a), the highest amount transported after 2 h in the two-compartment setup was from the DS40 sample. The DS40 sample also maintained the highest amount of dissolved drug in the supersaturated state on the AP side of the two-compartment setup (Figure 18a and b). Figure 19. Calculated flux of various ezetimibe (EZ) formulations across Transwell-grown Caco-2 cell monolayers. * Indicates a significant statis-tical difference in the flux between the different polymer concentrations (p < 0.05), and ns indicates that there were no statistically significant differences in flux beween the indicated samples (with varying degrees of supersaturation; DS) (p > 0.05). 52

69 Differences between the DS samples in the amounts of EZ transported to the BL side were less pronounced than differences in the precipitation behavior in the one-compartment setup. However, precipitation behavior in the onecompartment setup was not in accordance with that on the AP side in the two-compartment setup. The different outcomes for the one-compartment and two-compartment setups demonstrate that reliable prediction of supersaturation/precipitation behavior cannot be achieved without including tools to predict absorption in the in vitro setup, as reported by Bevernage et al. (164). As shown in Figure 19, there were no significant differences in flux values between samples with DS10, DS20, DS30 or DS40 for drug transport to the BL side (p > 0.05). However, the EZ fluxes with DS10, DS20, DS30 and DS40 samples differed significantly from the flux with the crystalline EZ sample (p < 0.05). As reported in a previous study (166), the amount of drug transported to the BL side depends on the amount of drug in the supersaturated solution on the AP side. The specific behavior of EZ as it was taken up and metabolized by enterocytes could explain the lack of effect of the various DSs on EZ flux on the apical side of the two-compartment setup, as reported by Oswald et al. (167). Generally, PWSDs that permeate the intestinal wall well should show increased permeation when the concentration of the dissolved drug is increased on the AP side, but some recent reports have shown that solubilized colloidal drugs may permeate poorly (166, 168) Effect of polymer on supersaturation-precipitationpermeation interplay (Paper II) PVP K-30 was selected to assess the effects of polymers on the inhibition of crystallization (i.e. on sustaining supersaturation) and transport (169). In the one-compartment setup with the DS40 sample (Figure 20a), a PVP K-30 concentration of 0.05% w/v resulted in a higher amount of drug in solution but precipitation occurred gradually over 90 min, at which point all the drug had been precipitated. A PVP K-30 concentration of 0.1% w/v resulted in rapid precipitation over the first 30 min; i.e. it did not perform better than 0.05% w/v in keeping the supersaturation state. Precipitation occurred later with both polymer concentrations (0.05% and 0.1% w/v) than in the absence of polymer. In the two-compartment setup, a concentration of 0.05% w/v maintained the supersaturation for 2 h (Figure 20b), but it was difficult to conclude the effect of 0.1% w/v because of system variability. In general, less extensive precipitation was observed in the two-compartment setup, and the amount of EZ dissolved on the AP side was greater in the presence of PVP K-30 (Figure 20b). The polymer PVP K-30 (0.05% w/v) kept the drug in the supersaturated state for 2 h. In the absence of polymer, the drug started to precipitate 53

70 after 15 min. On the other hand, PVP K-30 (0.1% w/v) in the medium on the AP side increased the amount of dissolved drug up to 20 μg after 5 min, which was more than with a concentration of 0.05% w/v and in the absence of polymer (Figure 20b). The effect of PVP K-30 on the supersaturation of EZ in FaSSIF has been demonstrated previously (153). The reason for less extensive precipitation of EZ in the presence of Caco-2 cells may be that permeation offered an alternative to precipitation, as discussed earlier. The same conclusion applies in the absence of polymer. Thus, the in vitro prediction of supersaturation and precipitation is more accurate if it is combined with absorption tools because, in cases like this, the precipitation can be overestimated by the one-compartment experiments compared to the twocompartment experiments. Figure 20. (a) Amount of ezetimibe (EZ) with PVP-K30 in fasted-state simulated intestinal fluid (FaSSIF) in the one-compartment setup. (b) Amount of EZ with PVP- K30 in FaSSIF on the apical side of Caco-2 cell monolayers as a function of time, after nduction of supersaturation to DS40. (c) Cumulative amount of EZ recovered from the basolateral acceptor medum (i.e. HBSS) after vectorial transport across Caco-2 cell monolayers upon induction of supersaturation in the presence of polymer in the apical compartment. Data represent means ± standard deviation (n=3). Error bars in (c) were too small to show. DS=degree of supersaturation. In this study, we also investigated how supersaturation affects the transport of EZ in vitro in a two-compartment setup using Caco2 cells in the presence of PVP K-30. Figure 20c shows the cumulative amounts of EZ on the BL 54

71 side that were transported over 2 h. Addition of 0.05% w/v PVP K-30 to the AP side resulted in the highest amount of EZ transported to the BL side. Furthermore, the flux of EZ was significantly improved (p < 0.05) with this polymer concentration (0.064 ± 1.5 x10-2 μg/min cm compared to 0.1% w/v polymer and no polymer; Figure 19). PVP K-30 (0.1% w/v) improved the supersaturation solubility of EZ on the apical side in FaSSIF without improving the flux; the total transported amount was 2.97 ± 7.5 x 10-4 μg. In summary, PVP K-30 (0.05% w/v) on the AP side in FaSSIF kept the drug in the supersaturated state for 2 h, improved the flux, and achieved the highest cumulative EZ concentration on the BL side after 2 h (5.54 μg ± 2.6 x10-3 ), as shown in Figure 20b and c. The increase in drug concentration on the donor AP side as a result of solubilizing the excipients may not cause a proportional improvement in the flux in all cases (170). The intrinsic physicochemical properties of the drug and drug-excipient interaction can be expected to affect the outcome. In this study, PVP K-30 improved the flux across the Caco-2 cell barrier (Figure 19). However, some recent studies have shown the opposite effect, reporting that addition of some excipients to the AP side has a negative effect on transport. This was attributed to the fact that increased apparent solubility is associated with a micellar solubilization effect on the excipients which can result in overestimation of the true supersaturation, thus not causing improvement in the permeation rate (166) Effect of polymer on the supersaturation behavior of cocrystals (Paper III) The polymer HPMC was used as a model in this study. In the presence of 0.1% w/v HPMC, cocrystal and amorphous forms achieved much higher concentrations than the crystalline drug form, and the supersaturation was sustained for longer time compared to dissolution in the absence of HPMC (Figure 11). The dissolution of TDF-MOA cocrystals in the presence of 0.1% w/v HPMC resulted in a C max of M, which corresponds to a supersaturation (C max /S drug ) of about 120. The amorphous form was associated with similar concentrations. For both cocrystals and the amorphous phase, supersaturation was maintained much longer (3 hours) in the presence of HPMC; i.e., this suggests slower conversion of the amorphous or cocrystal forms to the drug. HPMC at a concentration of 0.1% w/v provided maximum precipitation inhibition from a solution supersaturated at 29 (171). These findings suggest that cocrystals may increase drug bioavailability under similar conditions. 55

72 56

73 6. Conclusions This thesis has provided a thorough understanding of the in vitro supersaturation solubility behavior of some newly formulated supersaturating SDDSs such as amorphous solid dispersions, cocrystals and oral films. During the course of this thesis: Ternary solid dispersions of EZ containing PVP and poloxamer 188 were successfully prepared using spray-drying and melt-quenching methods. The powders were thoroughly characterized using solid-state tools and were found to be predominantly amorphous. Cocrystals of TDF with MOA were identified and thoroughly characterized. Sublingual oral films of silodosin were prepared successfully for the first time. HPMC-AS polymer-based films showed fully amorphous dispersion as compared to HPMC-based films. It was found that MQ solid dispersions had better supersaturation properties than SD dispersions. The melting method would thus seem to be more promising for enhancing the dissolution of EZ from a ternary solid dispersion. The apparent solubility of EZ solid dispersions was higher in FaSSIF than in FaSSGF and supersaturation was maintained for an adequate time, i.e. for the time required for the absorption process to take place. The precipitation kinetics of EZ in solid dispersions were faster at 37 C than at 25 C in biorelevant media, indicating that dissolution studies currently routinely performed at 25 C may be less relevant. TDF-MOA cocrystal solubility was higher than that of TDF over a wide ph range (1-7) in aqueous media. The dissolution of cocrystals created a supersaturation level that was significantly higher than the saturation point of the crystalline form. The SA of TDF-MOA cocrystals increased by several orders of magnitude with increases in ph. The supersaturated solutions of EZ showed less precipitation tendency in the presence of Caco-2 cells in a two-compartment system and higher precipitation at early time points in a one-compartment chamber. Therefore, the supersaturation studies that are usually conducted in onecompartment chambers or in a test tube could underestimate supersaturation of the drug. Implementation of an absorption tool (such as Caco-2 cell monolayers) in supersaturation evaluation tests in vitro could improve supersaturation and precipitation predictions. 57

74 The effects of polymer and surfactant excipients on the supersaturation and precipitation behavior of SDDs differed. Low concentrations of poloxamer 188 were adequate for obtaining the desired improvements in EZ solubility in ternary solid dispersions. The polymer PVP K-30 improved the supersaturation of EZ at concentrations of 0.05% w/v and resulted in significant improvements in the transport/flux (J). The addition of 0.1% w/v HPMC to the dissolution medium increased supersaturation and slowed down nucleation and/or precipitation kinetics of the drug from formulations of TDF-MOA cocrystals and the amorphous form. The dissolution behavior of films containing HPMC is similar to that of those containing HPMC-AS if the surfactant TPGS 0.5 % w/w is included in the film formulation. 58

75 7. Future work Future work could include: Investigation of other factors possibly affecting supersaturation behavior (such as hydrodynamics and modified biorelevant media) and carrying out online investigation of the precipitation kinetics. Investigation of the supersaturation-precipitation-permeation interplay of EZ in a Caco-2 cell environment for a solid dispersion formulation of the drug. Investigation of the supersaturation behavior of TDF cocrystals in various biorelevant media and aspirated human intestinal fluids. Investigation of the in vivo behavior of the prepared formulations, since only in vitro dissolution tests and cell culture studies were performed in this thesis. Investigation of improvements to the formulation of the silodosin films, since they were prepared for the first time in this thesis. For example, studies of the dissolution of the films in human saliva could be necessary to evaluate their fate in realistic conditions. Permeability investigations are important for evaluating film absorption behavior, and film palatability and crystallization of the drug during storage (stability) also require investigation. 59

76 60

77 8. Acknowledgments Foremost I would like to thank Allah for giving me the power and energy to be here defending my thesis, and for helping me to overcome my weaknesses during the hardest times. The studies in this thesis were carried out at the Department of Health Sciences, Division of Medical Sciences, at Luleå University of Technology. Work was done within the Pharmaceutical and Biomaterial research group. The financial support I received was from the Ministry of Higher Education and Scientific research in Iraq for PhD study. I wish to express my sincere gratitude to: My main supervisor: Professor Sitaram Velaga, for his guidance and support throughout the course of my thesis, and also for his valuable input during meetings and discussions throughout my journey through the research work. My former co-supervisor Staffan Tavelin for his guidance during research work for Papers I and II and for hosting me in his laboratory at Umeå University, Sweden. My new co-supervisor Parameswara Vuddanda for his help and support performing the research work in Paper IV. Prof. Carsten Ehrhardt and his group members for hosting me at their laboratory in the School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland. Prof. Sung-Joo Hwang and his group members for their support during my visit to their laboratory in Yonsei University, South Korea. I would also like to sincerely and gratefully thank my colleagues and friends in the Department of Health Sciences and Division of Medical Sciences for their patience and for being my second work family here in Luleå. Special thanks go to the members of the administration department for helping me to improve my Swedish language mainly; Katarina Virtoft. Thank you Niklas Lehto for your help with the thesis. Many thanks to Katarina Leijon- Sundqvist for encouragement. Many thanks to my former colleague Hassan Ali for his advice and my new colleague Mohammed Elbadawi for support and encouragement. I won t forget that you told me Amani, we are not only 61

78 pharmacists but also engineers when we managed to solve technical problems in the laboratory. Great, heartfelt thanks go to my friend Manar for believing in me: you are really a true friend who had my back at all times. Thanks to all the friends I met in Luleå: Eynas, Asma, Hana, Hiba, Sara, Zahraa we had a really fun time and spent some unforgettable moments laughing and enjoying the winter in the snow here in the north of Sweden. Special thanks to my friends from Egypt: mainly Taisir, Heba, Noha, Basma. You made my time here in Luleå different and gave me constant support to enable me to reach this day. Thanks for your help as my second family during my travel to Dublin, leg surgery and during the days of writing my thesis. I wish to express my deep gratitude to my dearest parents for their encouragement, love and prayers for me throughout my time here. Thank you, you taught me how to be who I want to be, how to forgive, and how to be independent. Gratitude is also extended to my brothers and sisters for all the support they have given to me. My deepest gratitude goes to my husband Hussan for his support and for being so patient during my research, to my beloved son Toto for taking care of me during my illness and making me feel that I am a very important person in his life, and to my son Yaman (the chef) who cooked our food when I was busy with the thesis. You have been the driving force for my carrying out this work. Amani Alhayali June, 2018 Luleå, Sweden 62

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89 DISSERTATIONS FROM THE DEPARTMENT OF HEALTH SCIENCE, LULEÅ UNIVERSITY OF TECHNOLOGY, SWEDEN Doctoral theses Terttu Häggström. Life-story perspective on caring within cultural contexts: experiences of severe illness and of caring. (Nursing) Inger Jacobson. Injuries among female football players. (Physiotherapy) Karl Elling Ellingsen. Lovregulert tvang og refleksiv praksis. (Health Science and Human Services) Annika Näslund. Dynamic ankle-foot orthoses in children with spastic diplegia: interview and experimental studies. (Physiotherapy) Inger Lindberg. Postpartum care in transition: parents and midwives expectations and experience of postpartum care including the use of videoconferencing. (Nursing) Åsa Widman. Det är så mycket som kan spela in en studie av vägar till, genom och från sjukskrivning baserad på intervjuer med långtidssjukskrivna. (Health Science and Human Services) Eija Jumisko. Striving to become familiar with life with traumatic brain injury: experiences of people with traumatic brain injury and their close relatives. (Nursing) Gunilla Isaksson. Det sociala nätverkets betydelse för delaktighet i dagliga aktiviteter: erfarenheter från kvinnor med ryggmärgsskada och deras män. (Health Science and Human Services) Nina Lindelöf. Effects and experiences of high-intensity functional exercise programmes among older people with physical or cognitive impairment. (Physiotherapy) Åsa Engström. A wish to be near: experiences of close relatives within intensive care from the perspective of close relatives, formerly critically ill people and critical care nurses. (Nursing) Catrine Kostenius. Giving voice and space to children in health promotion. (Health Science and Human Services) Anita Melander Wikman. Ageing well: mobile ICT as a tool for empowerment of elderly people in home health care and rehabilitation. (Physiotherapy) Sedigheh Iranmanesh. Caring for dying and meeting death: the views of Iranian and Swedish nurses and student nurses. (Nursing) Birgitta Lindberg. When the baby is premature. Experiences of parenthood and getting support via videoconferencing. (Nursing) Malin Olsson. Meaning of women s experiences of living with multiple sclerosis. (Nursing) Lars Jacobsson. Long-term outcome after traumatic brain injury. Studies of individuals from northern Sweden. (Health Science) Irene Wikman. Fall, perceived fall risk and activity curtailment among older people receiving homehelp services. (Physiotherapy) Christina Harrefors. God vård och användning av digitala hjälpmedel. Föreställningar hos äldre och vårdpersonal. (Nursing) Agneta Larsson. Identifying, describing and promoting health and work ability in a workplace context. (Physiotherapy) Lisbeth Eriksson. Telerehabilitation: Physiotherapy at a distance at home. (Physiotherapy) Amjad Alhalaweh. Pharmaceutical Cocrystals: Formation mechanisms, solubility behaviour and solidstate properties. (Health Science) Katarina Mikaelsson. Fysisk aktivitet, inaktivitet och kapacitet hos gymnasieungdomar. (Physiotherapy) 2012.

90 Carina Nilsson. Information and communication technology as a tool for support in home care. - Experiences of middle-aged people with serious chronic illness and district nurses. (Nursing) Britt-Marie Wällivaara. Contemporary home-based care: encounters, relationships and the use of distance-spanning technology. (Nursing) Stina Rutberg. Striving for control and acceptance to feel well. Experiences of living with migraine and attending physical therapy. (Physiotherapy) Päivi Juuso. Meanings of women's experiences of living with fibromyalgia. (Nursing) Anneli Nyman. Togetherness in Everyday Occupations. How Participation in On-Going Life with Others Enables Change. (Occupational therapy) Caroline Stridsman. Living with chronic obstructive pulmonary disease with focus on fatigue, health and well-being. (Nursing) Ann-Sofie Forslund. A Second Chance at Life: A Study About People Suffering Out-Of-Hospital Cardiac Arrest. (Nursing) Birgitta Nordström. Experiences of standing in standing devices: voices from adults, children and their parents. (Physiotherapy) Malin Mattsson. Patients experiences and patient-reported outcome measures in systemic lupus erythematosus and systemic sclerosis. (Physiotherapy) Eva Lindgren. It s all about survival : Young adults transitions within psychiatric care from the perspective of young adults, relatives, and professionals. (Nursing) Annette Johansson. Implementation of Videoconsultation to Increase Accessibility to Care and Specialist Care in Rural Areas: - Residents, patients and healthcare personnel s views. (Nursing) Cecilia Björklund. Temporal patterns of daily occupations and personal projects relevant for older persons subjective health: a health promotive perspective. (Occupational therapy) Ann-Charlotte Kassberg. Förmåga att använda vardagsteknik efter förvärvad hjärnskada: med fokus mot arbete. (Occupational therapy) Angelica Forsberg. Patients' experiences of undergoing surgery: From vulnerability towards recovery -including a new, altered life. (Nursing) Maria Andersson Marchesoni. Just deal with it Health and social care staff s perspectives on changing work routines by introducing ICT: Perspectives on the process and interpretation of values. (Nursing) Sari-Anne Wiklund-Axelsson. Prerequisites for sustainable life style changes among older persons with obesity and for ICT support. (Physiotherapy) Anna-Karin Lindqvist. Promoting adolescents' physical school. (Physiotherapy) Ulrica Lundström. Everday life while aging with a traumatic spinal cord injury. (Occupational therapy) Sebastian Gabrielsson. A moral endeavour in a demoralizing context: Psychiatric inpatient care from the perspective of professional caregivers. (Nursing) Git-Marie Ejneborn-Looi. Omvårdnad som reflekterande praktik: Att se och använda alternativ till tvång i psykiatrisk vård. (Nursing) Sofi Nordmark. Hindrances and Feasibilities that Affect Discharge Planning: Perspectives Before and After the Development and Testing of ICT Solutions. (Nursing) Marianne Sirkka. Hållbart förbättringsarbete med fokus på arbetsterapi och team Möjligheter och utmaningar. (Occupational therapy) Catharina Nordin. Patientdelaktighet och behandlingseffekter inom multimodal rehabilitering och web-baserat beteendeförändringsprogram för aktivitet. (Physiotherapy) Silje Gustafsson. Self-care for Minor Illness: People's Experiences and Needs. (Nursing) Anna Nygren Zotterman. Encounters in primary healthcare from the perspectives of people with long-term illness, their close relatives and district nurses. (Nursing) 2017.

91 Katarina Leijon Sundqvist. Evaluation of hand skin temperature -Infrared thermography in combination with cold stress tests. (Health Science) Tommy Calner. Persistent musculoskeletal pain: A web-based activity programme for behaviour change, does it work? Expectations and experiences of the physiotherapy treatment process. (Physiotherapy) Licentiate theses Marja Öhman. Living with serious chronic illness from the perspective ofpeople with serious chronic illness, close relatives and district nurses. (Nursing) Kerstin Nyström. Experiences of parenthood and parental support during the child's first year. (Nursing) Eija Jumisko. Being forced to live a different everyday life: the experiences of people with traumatic brain injury and those of their close relatives. (Nursing) Åsa Engström. Close relatives of critically ill persons in intensive and critical care: the experiences of close relatives and critical care nurses. (Nursing) Anita Melander Wikman. Empowerment in living practice: mobile ICT as a tool for empowerment of elderly people in home health care. (Physiotherapy) Carina Nilsson. Using information and communication technology to support people with serious chronic illness living at home. (Nursing) Malin Olsson. Expressions of freedom in everyday life: the meaning of women's experiences of living with multiple sclerosis. (Nursing) Lena Widerlund. Nya perspektiv men inarbetad praxis: en studie av utvecklingsstördas delaktighet och självbestämmande. (Health Science and Human Services) Birgitta Lindberg. Fathers experiences of having an infant born prematurely. (Nursing) Christina Harrefors. Elderly people s perception about care and the use of assistive technology services (ATS). (Nursing) Lisbeth Eriksson. Effects and patients' experiences of interactive video-based physiotherapy at home after shoulder joint replacement. (Physiotherapy) Britt-Marie Wälivaara. Mobile distance-spanning technology in home care. Views and reasoning among persons in need of health care and general practitioners. (Nursing) Anita Lindén. Vardagsteknik. Hinder och möjligheter efter förvärvad hjärnskada. (Health Science) Ann-Louise Lövgren Engström. Användning av vardagsteknik i dagliga aktiviteter - svårigheter och strategier hos personer med förvärvad hjärnskada. (Health Science) Malin Mattsson. Frågeformulär och patientupplevelser vid systemisk lupus erythematosus -en metodstudie och en kvalitativ studie. (Physiotherapy) Catharina Nordin. Patients experiences of patient participation prior to and within multimodal pain rehabilitation. (Physiotherapy) Hamzah Ahmed. Relationship Between Crystal Structure and Mechanical Properties in Cocrystals and Salts of Paracetamol. (Health Science) Johan Kruse. Experiences of Interns and General Practitioners of Communicating with and Writing Referrals to the Radiology Department. (Health Science) For purchase information: Department of Health Science, Luleå University of Technology, S Luleå, Sweden.

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95 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, RESEARCH ARTICLE Dissolution and precipitation behavior of ternary solid dispersions of ezetimibe in biorelevant media Amani Alhayali a, Staffan Tavellin b and Sitaram Velaga a a Department of Health Sciences, Division of Medical Sciences, Luleå University of Technology, Lulea, Sweden; b Departments of Pharmacology and Clinical Neuroscience, Umea University, Umeå, Sweden ABSTRACT The effects of different formulations and processes on inducing and maintaining the supersaturation of ternary solid dispersions of ezetimibe (EZ) in two biorelevant media fasted-state simulated intestinal fluid (FaSSIF) and fasted-state simulated gastric fluid (FaSSGF) at different temperatures (25 C and 37 C) were investigated in this work. Ternary solid dispersions of EZ were prepared by adding polymer PVP-K30 and surfactant poloxamer 188 using melt-quenching and spray-drying methods. The resulting solid dispersions were characterized using scanning electron microscopy, differential scanning calorimetry (DSC), modulated DSC, powder X-ray diffraction and Fourier transformation infrared spectroscopy. The dissolution of all the ternary solid dispersions was tested in vitro under non-sink conditions. All the prepared solid dispersions were amorphous in nature. In FaSSIF at 25 C, the melt-quenched (MQ) solid dispersions of EZ were more soluble than the spray-dried (SD) solid dispersions and supersaturation was maintained. However, at 37 C, rapid and variable precipitation behavior was observed for all the MQ and SD formulations. In FaSSGF, the melting method resulted in better solubility than the spray-drying method at both temperatures. Ternary solid dispersions show potential for improving solubility and supersaturation. However, powder dissolution experiments of these solid dispersions of EZ at 25 C may not predict the supersaturation behavior at physiologically relevant temperatures. ARTICLE HISTORY Received 4 February 2016 Revised 19 June 2016 Accepted 6 July 2016 Published online 25 August 2016 KEYWORDS Poorly soluble drugs; ternary solid dispersions; supersaturation; biorelevant media; dissolution Introduction Supersaturation is becoming an important tool for increasing bioavailability as the number of poorly water soluble compounds increases in drug discovery processes 1. Supersaturation of an orally delivered drug is reached when its intraluminal concentration exceeds its thermodynamic solubility equilibrium concentration 2. The need for designing formulations that can create this high intestinal drug concentration is gaining more attention, because the induction of supersaturation in the intestinal lumen will improve intestinal absorption as well as the therapeutic effect 1. Solid dispersion technology is an interesting option for achieving supersaturated drug concentrations. The drug is dispersed within a polymer at the molecular level to form an amorphous solid dispersion or solid solution 3 5. Methods that have been used to prepare solid dispersions include micronization, melt methods, spray drying and freeze drying, among others 6,7. A wide variety of pharmaceutical excipients (including polymers 4,8 10 and surfactants 10,11 ) have been used to stabilize the drug in the solid, supersaturated state in vivo. In ternary solid dispersions, which have been shown to improve the solubility 12,13 and supersaturation solubility 5 of poorly water soluble and insoluble active pharmaceutical ingredients (APIs), the API is dispersed within a carrier system containing a polymer and a surfactant. The polymer is added to stabilize the system both in the solid state and in solution by preventing precipitation of the drug and phase separation. The surfactant enhances the solubility and dissolution of the API by lowering the surface tension of the drug particles and maintaining supersaturation during dissolution Surfactants can also act as plasticizers that aid and facilitate thermal processing of the polymer and API 15. Ezetimibe (EZ), a cholesterol-lowering drug used in the treatment of atherosclerosis, was chosen as the model drug in this study. It acts by inhibiting the uptake of dietary and biliary cholesterol in the intestine without affecting the uptake of fat-soluble vitamins, triglycerides or bile acids. EZ is a lipophilic molecule with a log P (octanol/water) of 4.5. It is very weakly acidic (close to neutral) with a pka of 10.2 (aromatic hydroxyl group) 16 and thus is practically insoluble in water, with a consequently low dissolution rate in the gastric lumen, potentially leading to dissolution-limited bioavailability 17,18. In fact, EZ has been classified as a class II drug 19. A number of formulations and solid-state strategies have been used to improve its solubility and dissolution, including binary solid dispersions, nanocrystals and cocrystals 16,17,19. In this study, ternary solid dispersions of EZ were prepared using different proportions of the polymer polyvinylpyrrolidone (PVP)-K30 and the surfactant poloxamer 188. Two preparation methods were employed: melt quenching and spray drying. The apparent solubility of EZ from the prepared ternary solid dispersions was studied in two simulated gastrointestinal fluids CONTACT Sitaram Velaga sitram.velaga@ltu.se Department of Health Sciences, Division of Medical Sciences, Luleå University of Technology, Lulea , Sweden Supplemental data for this article can be accessed here. ß 2016 Informa UK Limited, trading as Taylor & Francis Group

96 2 A. ALHAYALI ET AL. [fasting-state simulated intestinal fluid (FaSSIF; ph 6.5) and fastingstate simulated gastric fluid (FaSSGF; ph 1.6)] in terms of supersaturation at two temperatures: 25 C and 37 C. The overall objective of the work was to improve understanding of supersaturation phenomena in ternary solid dispersions of EZ in biorelevant media and to investigate the effects of factors such as the preparation process and the temperature of the dissolution medium on the maintenance of supersaturation. The specific aims of the study were (a) to prepare and characterize ternary solid dispersions of EZ using melt-quenching and spray-drying methods, (b) to investigate the powder dissolution and precipitation behavior (supersaturation) of ternary solid dispersions in vitro in simulated gastric (FaSSGF) and intestinal (FaSSIF) fluids at 25 C and 37 C, and (c) to gain insight into the effects of excipients, preparation processes, dissolution media and temperature on the extent and duration of supersaturation in vitro. Materials and methods Materials Ezetimibe and PVP-K30 were purchased from Sigma-Aldrich (Stockholm, Sweden). Poloxamer 188 was a gift from BASF Chemicals (Ludwigshafen, Germany). Simulated intestinal fluid powders for preparing FaSSIF (sodium taurocholate, lecithin, sodium chloride, sodium hydroxide and monobasic sodium phosphate) and FaSSGF (sodium taurocholate, lecithin, sodium chloride and hydrochloric acid) were purchased from biorelevant.com (London, UK). High performance liquid chromatography (HPLC) grade acetonitrile was purchased from Merck Millipore (Solna, Sweden). All other reagents and solvents were of analytical grade. PTFT syringe filters 0.2 mm were used for all the experiments. Very pure deionized water (Millipore, Solna, Sweden) was used throughout the study. All chemicals and solvents were used without further purification. Preparation of solid dispersions Melt-quenched (MQ) solid dispersions Prior to the preparation of solid dispersions, EZ and PVP-K30 were kept in the oven at 100 C for 24 h to remove any adsorbed water from the solids, and differential scanning calorimetry (DSC) analysis was performed on the dried crystalline EZ to ensure its crystalline nature (data not shown). Crystalline EZ, PVP-K30 and poloxamer 188 in different proportions (see Table 1) were accurately weighed and mixed gently for 1 2 min using a porcelain mortar and pestle. The resulting powder mixtures were placed in a preheated oven (BINDER, Tuttlingen, Germany) at 200 C for 5 8 min. After complete melting, the liquid was immediately put in a freezer at 20 C for 1 2 days. The MQ samples were then stored in a desiccator over silica until the day of analysis. Before the analysis, all the formulations were ground and sieved using 125 mm sieves. Using a similar method, the amorphous form of EZ was prepared for comparison. Spray-dried (SD) solid dispersions Crystalline EZ, PVP-K30 and poloxamer 188 in different proportions were accurately weighed and dissolved (equivalent to 2% w/v of solids) in methanol. The solutions were spray dried using a Buchi mini spray dryer B-290 attached to an inert loop B-295 to trap the residual solvents. The processing conditions were as follows: inlet temperature 80 C, air flow 357 L h 1, aspiration 70 80%, feed-flow rate 3 ml/min and outlet temperature C. Nitrogen gas was used as the drying gas in a closed-loop experiment. The collected SD powders were stored in a sealed desiccator over silica until the day of analysis. Scanning electron microscopy (SEM) The morphology of the particles was examined using a Merlin scanning electron microscope (Zeiss, Oberkochen, Germany) equipped with X-Max 50 mm 2 X-ray detectors (Oxford Instruments, Abingdon, UK). All the samples were coated with tungsten to increase the conductivity of the electron beam. The instrument voltage was 15 kv and the current was 1 na. Solid-state and powder characterization Thermo-gravimetric analysis (TGA) The weight loss and thermal degradation behavior of all the formulation components (EZ, PVP-K30 and poloxamer 188) were investigated using a Thermal Advantage TGAQ5000 (TA Instruments, New Castle, DE) connected to a cooling system. Powdered samples (4 7 mg) were placed in the aluminum pans and the furnace was heated from 20 C to 550 C at a rate of 5 C/ min. Nitrogen at a flow rate of 50 ml/min was used as a purging gas. Universal analysis software was used to analyze the results. Differential scanning calorimetry The thermal behavior of the raw materials and solid dispersions was studied using a DSC Q1000 (TA Instruments, New Castle, DE). The instrument was calibrated before use for temperature and enthalpy using indium. About 1 4 mg of the sample was accurately weighed and placed in a non-hermetic aluminum pan, which was then crimp-sealed. The samples were heated from 25 C to 200 C at a heating rate of 10 C/min under continuous nitrogen purge (50 ml/min). The calorimeter was equipped with a refrigerated cooling system. The data were analyzed using TA analysis software (TA Instruments, New Castle, DE). Modulated differential scanning calorimetry (MDSC) MDSC was performed using a Q1000 instrument (TA Instruments, New Castle, DE) for better identification of the glass transition temperature (Tg) through the separation of reversible (i.e. Tg) from nonreversible (i.e. enthalpy recovery, fusion) thermal events. Samples (2 5 mg) were accurately weighed and placed in aluminum nonhermetic pans, which were then crimped. These samples were then heated from 25 C to 200 Cat5 C/min with modulation amplitude of ±0.80 C every 60 s. Table 1. Proportions of different components in the melt-quenched (MQ) and spray-dried (SD) solid dispersions. Sample ID Ezetimibe (%w/w) PVP-K30 (%w/w) Poloxamer188 (%w/w) MQ1 or SD MQ2 or SD MQ3 or SD Powder X-ray diffraction (PXRD) PXRD patterns for the crystalline and amorphous EZ and ternary solid dispersions were obtained using an Empyrean PXRD instrument (PANalytical, Almelo, The Netherlands) equipped with a Pixel 3D detector and monochromatic Cu Ka radiation. The tube voltage and amperage were 45 kv and 40 ma, respectively. Powdered

97 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 3 samples were loaded into the oval cavity in the metal sample holder and carefully leveled. The experimental settings were as follows: 2h ranged from 5 to 40, step size h. The data were processed using High Score plus Version 3.0 software (PANalytical, Almelo, The Netherlands). Fourier transformation infrared spectroscopy (FTIR) A Bruker IFS66v/S FTIR spectrometer equipped with a deuterated triglycine sulfate detector was used to obtain the FTIR spectra of the samples. Powdered samples were mixed with KBr and IR spectra were obtained in the diffuse reflectant mode (Kubelka Munk). The following experimental settings were used: 64 scans with a resolution of 4 cm 1, spectral region cm 1. In vitro dissolution study Excess amounts of the SD or MQ solid dispersions were suspended in 10 ml of FaSSIF and FaSSGF in sealed test tubes and placed in a preheated shaking water bath at a shaking speed of 60 rpm (orbital shaking). The experiments were performed at 25 C and 37 C. The samples were withdrawn after 5, 15, 30, 45, 60, 75, 90, 120, 180 and 240 min and centrifuged (5 min, 17,000g), then filtered through 0.2 mm PTFE syringe filters to ensure complete separation of the solid phase. The filtrate was diluted adequately with mobile phase and then analyzed by HPLC. For comparison, the dissolution properties of crystalline and amorphous EZ in FaSSIF and FaSSGF were also studied. The samples were withdrawn at pre-defined intervals following the above procedure. All experiments were done in triplicate. High performance liquid chromatography The concentration of EZ was determined in an HPLC 1290 series instrument equipped with a quaternary pump and a UV detector (Agilent, Santa Clara, CA). A C-18 Eclipse plus 3 mm (4.6 mm 100 mm) column was used. The mobile phase consisted of 40:60 (v/v) 0.05% sodium 1-heptane sulfonic acid and acetonitrile. The isocratic method was used, with a flow rate of 0.5 ml/min. The injection volume was 10 ml and the measurements were carried out at a wavelength of 230 nm, in triplicate. Results and discussion Preparation of ternary solid dispersions Melt-quenching and spray-drying methods were used to prepare ternary solid dispersions. Melting and quenching, one of the oldest methods used in the preparation of solid dispersions, relies on mixing the molten drug with a molten or supercooled polymer followed by rapid cooling or solidification 6. With the help of preliminary experiments and prior knowledge, the drug load in the formulations was varied in the range of 5 15% w/w (Table 1). The premixed EZ and excipients were then subjected to melting and quenching. The process temperature of 200 C was based on the melting temperature and thermal decomposition temperatures of the raw materials (see Figure S1). Visually glassy materials of pure EZ and the solid dispersions were obtained. Spray drying is a widely used and efficient method of producing solid dispersions. It is a solvent-based bottom-up method that relies on the rapid evaporation of a solution containing the API and polymers, to yield fine powders 20. In addition to formulation variables, process parameters such as the inlet temperature, Figure 1. Thermo-gravimetric analysis curves of melt-quenched (MQ1) and spraydried (SD1) solid dispersions of EZ showing moisture content. feed rate, flow rate of atomization air, etc. are known to affect the solid-state and physicochemical properties of the resulting solid dispersions 6. In this study, the formulation of EZ, poloxamer and polymer was varied under fixed spray-drying process conditions (Table 1). Dry powders of EZ and solid dispersions were obtained. Figure 1 shows SEM micrographs of crystalline and amorphous EZ and the solid dispersions obtained using the melt-quenching and spray-drying methods. As can be seen, raw EZ is formed as rod/block-shaped crystals. The ground particles of the solid dispersions prepared by melt-quenching were irregular in shape, possibly as a result of micronization of the glassy material, while SD particles were uniform, spherical, porous and aggregated. However, there were no major differences in particle morphology between the different formulations. Further, there were no EZ crystals on the surfaces of the particles (Figure 1). Under fixed process conditions, SD particle formation was not affected by formulation changes. Solid-state characterization of materials Thermal analysis TGA was used to determine weight loss due to degradation of raw materials and the loss of residual solvents/moisture in the processed samples (MQ and SD). Figure S1 (see supplementary figure) shows the TGA weight loss curves for EZ, PVP-K30 and poloxamer 188. It can be seen that EZ, PVP-K30 and poloxamer lost significant weight, attributed to thermal decomposition at 348 C, 401 C and 453 C, respectively. It is important to know the thermal decomposition temperatures in order to avoid the risk of the API or excipients degrading during the preparation of solid dispersions by thermal methods such as melting and quenching and melting followed by extrusion 21. It was invaluable information for the preparation of the solid dispersions in this study. The final product in solvent-based methods like spray drying may contain residual solvents that can compromise the stability of the solid dispersion. TGA weight loss curves for selected solid dispersions are shown in Figure 2. It is clear that the residual solvent levels are ranging between 3.8 and 5.8% in the MQ and SD solid dispersions, suggesting efficient drying of the MQ solid dispersion product. DSC analysis results for crystalline EZ, amorphous EZ, PVP- K30 and poloxamer 188 are shown in Figure 3. In the DSC

98 4 A. ALHAYALI ET AL. Figure 2. Scanning electron microscope photographs of (a) crystalline ezetimibe (EZ), (b) amorphous EZ, and (c) melt-quenched (MQ1) and (d) spray-dried (SD1) solid dispersions of EZ. Figure 3. Differential scanning microscopy thermograms of (a) crystalline ezetimibe (EZ), (b) amorphous EZ, (c) polyvinylpyrrolidone (PVP)-K30 and (d) poloxamer 188. thermograms for EZ, an endothermic peak corresponding to the EZ melting point (Tm ¼ C) was observed. The Tg for PVP-K30 appeared at C and a broad endothermic band was attributed to the sorbed moisture. In fact, the hygroscopic nature of PVP is well known 22. Poloxamer 188 showed an endothermic transition at C which corresponded to the melting point, confirming its crystalline nature. The thermal behavior of these raw materials was in agreement with that in previous reports 18,21. In the DSC thermogram for amorphous EZ, the change in heat capacity at C was followed by an exothermic transition at C and an endotherm at 163 C (Figure 3(b)). These thermal events were attributed to the Tg, recrystallization and melting points typical of an amorphous material. The DSC thermograms of these ternary solid dispersions had overlapping events, which made interpretation of the results difficult. MDSC is a powerful tool that separates the total heat flow signal into reversing and non-reversing parts. The reversing heat flow always contains the Tg while the non-reversing heat flow contains the kinetic transitions, such as crystallization, evaporation, degradation, etc. MDSC was carried out on solid dispersions prepared by melt-quenching and spray-drying methods to separate the Tg from the dehydration peak. Figure 4 shows the MDSC reversing heat flow thermograms for different solid dispersions. The Tg was better resolved and a single Tg (no melting peak for EZ) was observed at C for all solid dispersions. This suggests that EZ was molecularly dispersed within the polymer and surfactant, forming an amorphous solid solution The poloxomer could have increased the solubility of the drug in the polymer, aiding the formation of a solid solution 14,26.

99 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 5 Figure 4. Modulated differential scanning calorimetry reversing heat-flow thermograms of (a) melt-quenched (MQ) solid dispersion 1, (b) MQ2 and (c) MQ3; and (d) spray-dried (SD) solid dispersion 1, (e) SD2 and (f) SD3 showing the heat capacity (Tg) in the region ( C). Powder X-ray diffraction (PXRD) Figure 5 shows the PXRD patterns for raw and amorphous EZ and solid dispersions prepared by spray drying. Raw EZ showed several characteristic peaks at 2h angles of 8.2, 9.8, 13.5, 16.3, 19.0, 20.1, 23.6 and 25.5, confirming its crystalline nature. The PXRD pattern for crystalline EZ was similar to a previously published pattern 18. The diffraction peaks completely disappeared in the PXRD pattern of amorphous EZ, resulting in the diffuse hollow pattern distinctive of amorphous materials. All SD solid dispersions (SD1 3) showed diffuse halo PXRD patterns, confirming the amorphous nature of the solid dispersion; i.e. the API was completely dispersed in the polymer and the surfactant matrix 15. Similar PXRD spectra were obtained for solid dispersions prepared by melt-quenching, confirming their amorphous nature (Figure 5). Fourier transformation infrared spectroscopy (FTIR) Diffuse reflectance IR was used to investigate the possible molecular interactions between the drug and polymers in the solid Figure 5. Powder X-ray diffraction patterns of (a) crystalline ezetimibe (EZ), (b) amorphous EZ, and (c) melt-quenched (MQ1) and (d) spray-dried (SD1) solid dispersions of EZ.

100 6 A. ALHAYALI ET AL. Though dilution effects were seen, the IR spectra of the physical mixtures were summations of the individual components, indicating no interactions between the components (Figure 6). In contrast, in the IR spectrum of the MQ solid dispersion MQ2, significant changes were seen: the O H stretching band in EZ at about 3440 cm 1 had disappeared and a C¼O stretch band at 1728 cm 1 had shifted to a lower wave number and broadened. Similarly, changes in the vibrational bands were observed in the IR spectra of the SD dispersions (SD2). These changes in the IR spectra of solid dispersions could be attributed to the formation of strong interactions (H-binding, van der Waals, etc.) between EZ and the PVP. The hydrogen bonding interactions between OH or NH groups on the drug molecules and the carbonyl group on PVP have been cited in many studies previously 31,32. These interactions between the API and the excipients are believed to prevent selfassociation (dimerization) and nucleation of the API phase The mixing effects on the stretching vibration modes of C¼O, C¼C, C N and C F in the range cm 1 are more prominent in MQ solid dispersions than in SD systems, probably related to the level of miscibility between the components. Figure 6. Fourier transformation infrared spectroscopy, showing two regions ( cm 1 and cm 1 ) of (a) crystalline ezetimibe (EZ), (b) amorphous EZ, (c) a physical mixture of EZ, and (d) melt-quenched (MQ2) and (e) spray-dried (SD2) solid dispersions of EZ. dispersions. IR spectroscopy has been extensively used to study interactions between the components of solid dispersions and to aid understanding of the mechanisms behind stabilization and supersaturation behavior 27. Figure 6 depicts the IR spectra for crystalline EZ, amorphous EZ, the physical mixtures of EZ, and selected solid dispersions from melt-quenching and spray-drying methods (MQ2 and SD2 were used as representative solid dispersions in Figure 6). In the IR spectrum of crystalline EZ, a number of characteristic vibrational bands were identified at 3440 cm 1 (O H stretch), 2919 cm 1 (C H stretch), 1728 cm 1 (C¼O stretch), 1603 cm 1 (C¼C stretch), cm 1 (C N stretch), and cm 1 (C F stretch) and cm 1 (ring vibration of paradisubstituted benzene) (Figure 6). The IR spectrum for crystalline EZ was in good agreement with the spectra presented in previous articles 18,28. In the IR spectrum for amorphous EZ, the vibrational bands were broadened in general, and a number of bands had shifted significantly (Figure 6). Clearly, the band corresponding to the O H stretch at 3440 cm 1 was broadened, with a peak at 3321 cm 1, and the C¼O stretching band at 1728 cm 1 had moved to 1723 cm 1. Bathochromic shifts in carbonyl band positions (i.e. lower wave numbers) are indicative of electronic polarization of the double bond as a result of interactions between the lone pair of electrons on the oxygen and hydrogen donor groups. These changes could be attributed to changes in the intermolecular H-bonding interactions between these functional groups in the crystalline and amorphous forms of EZ. Similar observations have been made with many pharmaceutical molecules including indomethacin, celecoxib, furosemide, etc In the IR spectrum of PVP-K30, a broad band was observed in the range cm 1 ; this was assigned to the carbonyl stretching vibrations (data not shown). Poloxamer 188 exhibited characteristic peaks at and 1100 cm 1, corresponding to stretching vibrations of the O H, C H and C O groups (data not shown). In vitro dissolution study It is important to optimize the in vitro experimental conditions in studies of the supersaturation behavior of amorphous solids to ensure that they are biorelevant to the in vivo conditions. This study used biorelevant test media, non-sink conditions and orbital shaking. It has been suggested that non-sink conditions are more biorelevant for evaluating the dissolution behavior of supersaturable formulations where the maximum solution concentration are determined 9. The effect of hydrodynamics on the supersaturation/ precipitation is poorly understood. In a recent paper, the precipitation rate was shown to be remarkably slower in the shaking model than in the stirring model, emphasizing the importance of the applied hydrodynamic methodology 34. Effects of process, formulation and temperature on supersaturation in FaSSIF Figure 7 shows the powder dissolution (concentration versus time) profiles for the prepared solid dispersions in FaSSIF. The solubility of crystalline EZ in FaSSIF (ph 6.5) at 25 C and 37 C was 5.9 mg/ml and 10.1 mg/ml, respectively (Figure 7(a,b)). The apparent solubility (concentration in solution) of EZ from the amorphous form increased and then rapidly decreased at 25 C (Figure 7(a,b)). In contrast, there was no supersaturated concentration at 37 C for amorphous EZ (Figure 7(c,d)). This phenomenon of supersaturation and rapid precipitation is typical of amorphous solids and is described as the spring effect 3. For orally administered formulations, the dose-to-solubility ratio has been suggested as an indicator of the precipitation propensity of the drug in the intestinal lumen 35. Drugs with dose-to-solubility (non-ionized) ratios of more than 50:1 have been reported to show a tendency to precipitate in the intestinal environment 36. Evidently, the doseto-solubility ratio for EZ is about 1000 at a dose of 10 mg, suggesting that the observed precipitation of stable phases is thermodynamically favored in FaSSIF. Interestingly, the spring effect disappeared (i.e. there was no increase in solubility) as the temperature of the dissolution experiment was increased to 37 C. This could be explained by differences in the relationship between dissolution and the precipitation/crystallization kinetics of crystalline EZ. It is possible that the crystallization of amorphous EZ was

101 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 7 Figure 7. Dissolution and precipitation (supersaturation) profiles of ternary solid dispersions of ezetimibe (EZ) in fasted-state simulated intestinal fluid: (a) and (b) meltquenched (MQ1, 2, 3) and spray-dried (SD1, 2, 3) solid dispersions at 25 C; (c) and (d) MQ and SD solid dispersions at 37 C. AMP: amorphous EZ; CRY: crystalline EZ. faster at 37 C. Similar behavior has been observed with amorphous felodipine and indomethacin in another study 37. Solid dispersions where the drug and polymers are intimately mixed are attractive and widely used formulation strategies to prevent the crystallization of the stable phase and maintain supersaturation of drugs like EZ. In this study, PVP-K30 and poloxomer 188 were selected from the preliminary studies as the precipitation inhibitor and solubilizer, respectively. In fact, PVP has been well established as a crystal growth inhibitor for a range of amorphous drugs 38. In the MQ solid dispersions, solution concentrations of EZ were maintained at about mg/ml and no precipitation was observed for at least 240 min at 25 C(Figure 7(a)). Apparently, the presence of the polymer sustained supersaturation, with solution concentrations about 50 times higher than the equilibrium solubility of EZ, possibly by preventing precipitation of the stable phase. However, no differences in the solution concentrations were observed for different polymer/drug ratios in these solid dispersions. Interestingly, in the SD solid dispersions, supersaturation (with solution concentrations about times higher) was followed by desupersaturation at different rates (Figure 7(b)). SD solid dispersions generated higher initial solution concentrations than those generated by MQ solid dispersions, which could be explained by differences in the particle size/morphology and molecular miscibility (Figure 2). This could also mean that solid dispersions result in a greater driving force for crystallization. The desupersaturation curves also show that the lower the polymerto-drug ratio is in the solid dispersions, the faster the precipitation kinetics will be (Figure 7(b)). The presence of 10% poloxomer resulted in even faster precipitation of the crystalline phase. The rich drug polymer interactions (confirmed by FTIR) and changes in viscosity can alter the crystal growth and surface integration kinetics, leading to differences in the crystallization kinetics. Previously published articles have also proposed similar mechanisms to explain the differences in the precipitation kinetics of many amorphous drugs 39. In general, solid dispersions prepared by the melting method showed slightly higher initial concentrations and faster precipitation kinetics at 37 C than at 25 C(Figure 7(a,c)), while solid dispersions prepared by the spray-drying method showed direct precipitation in FaSSIF at 37 C but not at 25 C (Figure 7(b,d)). The presence of precipitation inhibitors had only a minor effect in maintaining supersaturation at 37 C. These results are perfectly in line with previous observations on the supersaturation and precipitation behavior of other amorphous drugs in the presence of different polymers 40. The explanation lies in the increase in the rate of dissolution (supersaturation) and precipitation kinetics at higher temperatures. The results suggest that dissolution experiments performed at 25 C may not be biorelevant and may fail to predict in vivo behavior 41. In the MQ solid dispersions, desupersaturation was dependent on the formulation: higher polymer/ poloxomer content resulted in a slower precipitation rate (Figure 7(c)). However, in the SD solid dispersions, precipitation was rapid irrespective of the formulation or polymer content (Figure 7(d)). This suggests that the supersaturation and precipitation behavior of amorphous solids is sensitive to the processing methods, possibly because of the history of the material and the presence of nuclei 42. Effects of process, formulation and temperature on supersaturation in FaSSGF Figure 8 shows the powder dissolution (concentration versus time) profiles for EZ samples in FaSSGF (ph 1.6). The solubility of crystalline EZ in FaSSGF (ph 1.2) at 25 C and 37 C was extremely low: 0.2 mg/ml and 0.1 mg/ml, respectively (Figure 8(a,b)). This extremely low solubility is thought to be due to the un-ionized state of the drug in the dissolution medium. In contrast to its behavior in FaSSIF, amorphous EZ resulted in no increase in initial solution concentrations or supersaturation in FaSSGF. In fact, the method of preparation and the temperature had no effect on this

102 8 A. ALHAYALI ET AL. Figure 8. Dissolution and precipitation (supersaturation) profiles of ternary solid dispersions of ezetimibe (EZ) in fasted-state simulated gastric fluid: (a) and (b) meltquenched (MQ1, 2, 3) and spray-dried (SD1, 2, 3) solid dispersions at 25 C; (c) and (d) MQ and SD solid dispersions at 37 C. AMP: amorphous EZ, CRY: crystalline EZ. behavior. This suggests rapid transformation of the amorphous phase into the crystalline phase, possibly as the result of an extremely high driving force for crystallization. Although some increase in the initial EZ concentrations and supersaturation were observed with the MQ and SD solid dispersions in general, the level and extent of supersaturation were much lower in FaSSGF than in FaSSIF at 25 C and 37 C (Figure 8). However, as with FaSSIF, higher polymer/poloxomer content delayed the precipitation kinetics, leading to sustained supersaturation for longer periods. The amorphous form of EZ, which is a poorly soluble and very weakly acidic drug, can show increased solubility (supersaturation) in FaSSIF and FaSSGF. Although PVP and poloxamer 188 were effective in inhibiting/delaying precipitation from the solid dispersions at 25 C, they were ineffective at 37 C, which is the physiologically relevant temperature. The method of preparation also affected the supersaturation and precipitation kinetics. In general, supersaturation was maintained for relatively longer in biorelevant media at both temperatures when MQ solid dispersions were used. It is important to consider these aspects in the design and development of amorphous solid dispersions of poorly soluble drugs. MDSC, PXRD and FT-IR showed that although we obtained amorphous solid dispersion formulations, these new systems did not remain stable when they were dissolved in FaSSIF and FaSSGF, especially for SD formulations at 37 C. Conclusions Ternary solid dispersions of EZ containing PVP and poloxamer were successfully prepared by spray-drying and melt-quenching methods. The powders were thoroughly characterized using solid state tools and were found to be predominantly amorphous in nature with Tg values in the range of C. FT-IR indicated the presence of interactions between EZ and the excipients. The results indicate that the melting method would be more promising for enhancing the dissolution of EZ from a ternary solid dispersion. Low concentrations of poloxamer 188 are adequate for obtaining the desired improvements in EZ solubility. The apparent solubility of EZ solid dispersions was higher in FaSSIF than in FaSSGF and supersaturation was maintained for an acceptable time, i.e. for the time required for the absorption process to take place. Further, the precipitation kinetics were faster at 37 C than at 25 C in biorelevant media, indicating that dissolution studies currently routinely performed at 25 C may be less relevant. In general, MQ solid dispersions had better supersaturation properties than SD dispersions. This study nicely demonstrated the effects of the formulation, the process, the temperature of the dissolution medium, and the type of biorelevant medium on the supersaturation and precipitation kinetics of ternary solid dispersions of EZ. Acknowledgements This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Disclosure statement There were no conflicts of interest. References 1. Gao P, Shi Y. Characterization of supersaturatable formulations for improved absorption of poorly soluble drugs. AAPS J 2012;14:

103 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 9 2. Bevernage J, Brouwers J, Clarysse S, et al. Drug supersaturation in simulated and human intestinal fluids representing different nutritional states. J Pharm Sci 2010;99: Brouwers J, Brewster ME, Augustijns P. Supersaturating drug delivery systems: the answer to solubility-limited oral bioavailability? J Pharm Sci 2009;98: Dahan A, Beig A, Ioffe-Dahan V, et al. The twofold advantage of the amorphous form as an oral drug delivery practice for lipophilic compounds: Increased apparent solubility and drug flux through the intestinal membrane. AAPS J 2013;15: Janssens S, Nagels S, De Armas HN, et al. Formulation and characterization of ternary solid dispersions made up of itraconazole and two excipients, TPGS 1000 and PVPVA 64 that were selected based on a supersaturation screening study. Eur J Pharm Biopharm 2008;69: Leuner C, Dressman J. Improving drug solubility for oral delivery using solid dispersions. 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104 10 A. ALHAYALI ET AL. dispersions containing felodipine. Eur J Pharm Biopharm 2008;70: Lindfors L, Forssen S, Westergren J, et al. Nucleation and crystal growth in supersaturated solutions of a model drug. J Colloid Interface Sci 2008;325: Warren DB, Benameur H, Porter CJ, et al. Using polymeric precipitation inhibitors to improve the absorption of poorly water-soluble drugs: a mechanistic basis for utility. J Drug Target 2010;18: Usui F, Maeda K, Kusai A, et al. Inhibitory effects of watersoluble polymers on precipitation of RS Int J Pharm 1997;154: Bevernage J, Brouwers J, Brewster ME, et al. Evaluation of gastrointestinal drug supersaturation and precipitation: strategies and issues. Int J Pharm 2013;453: Greco K, Bogner R. Crystallization of amorphous indomethacin during dissolution: effect of processing and annealing. Mol Pharm 2010;7:

105 SUPPLEMENTARY DATA Figure S1. Supplementary Figure, Thermogravimetric analysis curves of ezetimibe (EZ), poloxamer 188 and polyvinylpyrrolidone (PVP) K-30 showing the degradation temperatures of the main components of the solid dispersion formulations.

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109 Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: Investigation of supersaturation and in vitro permeation of the poorly water soluble drug ezetimibe Amani Alhayali a, Mohammed Ali Selo b,c, Carsten Ehrhardt b, Sitaram Velaga a, a Pharmaceutical Research Group, Department of Health Sciences, Luleå University of Technology, Luleå, Sweden b School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland c Faculty of Pharmacy, University of Kufa, Al-Najaf, Iraq ARTICLE INFO Keywords: Poorly soluble drugs Biorelevant Supersaturation Precipitation Caco-2 cells Absorption ABSTRACT The interplay between supersaturation, precipitation and permeation characteristics of the poorly water-soluble drug ezetimibe (EZ) was investigated. Supersaturation and precipitation characteristics of EZ in the presence of Caco-2 cells were compared to those in a cell-free environment. The effect of the water-soluble polymer polyvinyl pyrrolidone (PVP-K30) on the supersaturation, precipitation and transport of EZ was also investigated and the amount of drug taken up by Caco-2 cells was quantified. A one-compartment setup without Caco-2 cells (i.e. in the wells of cell-culture plates) was used to mimic a non-sink in vitro dissolution chamber. The two-compartment Caco-2 cell monolayer setup (with apical and basolateral compartments) was used to investigate how the absorption of EZ affects supersaturation. EZ in varying degrees of supersaturation (DS; 10, 20, 30 and 40) was introduced into the one-compartment setup or the apical chamber of the two-compartment setup. Samples were collected at specific times to determine supersaturation, precipitation and permeation. At the end of the study, Caco-2 cells were lysed and the intracellular amount of EZ was quantified. In the one-compartment setup, a high DS was associated with rapid precipitation. Supersaturation was maintained for longer time periods and precipitation was lower in the presence of Caco-2 cells. There were no significant differences in the absorption rate of the drug, even at high concentrations on the apical side. Permeability coefficients for all supersaturated solutions (i.e. DS 10 40) were significantly (p < 0.05) different from those when EZ was present in crystalline form. Both concentrations of PVP-K30 (i.e. 0.05% and 0.1% w/v) improved solubility and supersaturation of EZ when added to the apical side, however, the increase in absorption at the higher concentration was not proportional. The amount of intracellular EZ increased with increasing DS in the apical side, until the saturation limit was reached in the cells (i.e. at DS 30 and higher). This study demonstrated that precipitation of EZ could be overestimated when supersaturation was investigated without the implementation of an absorption compartment in vitro, both in the absence and in the presence of polymer. 1. Introduction Development of new pharmaceutical products for oral administration has been challenged by the increasing number of poorly watersoluble molecules in the pipeline. Most of these drug molecules permeate well through the intestinal wall despite their poor solubility in gastrointestinal fluids [i.e. Biopharmaceutical Classification System (BCS) class II compounds] (Amidon et al., 1995; Frank et al., 2014). The solubility and permeation of drugs are key characteristics that determine their oral bioavailability; the poor solubility of class II compounds restricts their absorption and hence their systemic bioavailability. Drugs need to dissolve and be in solution in the intestinal lumen to be available for absorption through the gut wall. Various conventional and advanced approaches have been used by formulation scientists to improve the solubility, and thus the bioavailability, of these drug molecules (Bikiaris, 2011; Singh et al., 2011). Supersaturating drug delivery systems (SDDSs) such as solid dispersions are one approach to improve solubility of orally administered drugs (Alonzo et al., 2011). SDDSs increase the aqueous solubility of poorly soluble drugs by creating a supersaturation state in the gastrointestinal lumen, thus improving bioavailability. A supersaturation state can be created either intentionally, using drug formulation Corresponding author at: Department of Health Sciences, Division of Medical Sciences, Lulea University of Technology, SE Luleå, Sweden. address: sitaram.velaga@ltu.se (S. Velaga). Received 20 November 2017; Received in revised form 30 January 2018; Accepted 31 January 2018

110 A. Alhayali et al. strategies such as SDDSs, or naturally after the passage of weakly basic compounds from the stomach into the small intestine (Brouwers et al., 2009). The drug molecules in a supersaturated state tend to precipitate rapidly, as this is a thermodynamically unstable state. Thus, the improvements to solubility and absorption achieved by SDDSs will depend on the extent of the supersaturation and the duration of maintaining the supersaturation state in the gastrointestinal lumen. The challenge is in creating concentrations of the drug that are many times higher than its thermodynamic solubility concentration, and maintaining these high concentrations for an adequate time period for the absorption process to take place (Alonzo et al., 2011; Hou and Siegel, 2006). The supersaturated state has predominantly been investigated in vitro, as studying supersaturation in vivo is difficult (Gao et al., 2004; Bevernage et al., 2013; Alhayali et al., 2016). Most of these studies have been carried out in one-compartment systems, which neglect the effect of intestinal absorption on supersaturation behaviour. Absorption can affect supersaturation/precipitation of the drug in the intestinal lumen as it can relieve the unstable supersaturated state and prioritise drug permeation over precipitation. Therefore, the prediction power of the dissolution and precipitation kinetics increases when an acceptor absorption compartment is added to the dissolution evaluation setup (Shi et al., 2010; Bevernage et al., 2012). Caco-2 cell monolayers have become the gold standard of intestinal drug disposition studies (Van Breemen and Li, 2005). A two-compartment setup [with apical (AP) and basolateral (BL) sides] was first implemented as a successful predictive tool for measuring the dissolution and absorption of drugs simultaneously by Kataoka et al. (2012, 2003). In these studies, Caco-2 cells were used as a middle layer between the dissolution (donor) and absorption (acceptor) chambers. The system was useful for predicting the oral absorption of poorly water-soluble drugs after administration as solid dosage forms. Later, Bevernage et al. (2012) employed a two-compartment set-up with Caco-2 cell monolayer to investigate the supersaturation behaviour of Loviride to understand the interplay among supersaturation-precipitation-permeation (Bevernage et al., 2012). Frank et al. (2012) has studied the relationship between the true supersaturation and apparent solubility of amorphous solid dispersions of ABT-102. Ezetimibe (EZ) (molecular weight g mole 1 ) is a poorly water-soluble drug and belongs to BCS (class II). EZ is weakly acid compound with pka of 9.75 and log p value of 1.6 (Srivalli and Mishra, 2016). In our previous study, the supersaturation and precipitation behaviour of solid dispersions of EZ was investigated in one-compartment using biorelevant media (Alhayali et al., 2016). Absorption process across the intestinal epithelial barrier could influence considerably the supersaturation and precipitation behaviour. In this study, the effect of absorption (across Caco-2 cell monolayers) on the supersaturation and precipitation of EZ was investigated in two compartments and compared to the supersaturation behaviour in a cell-free, one-compartment setup. Furthermore, the effect of the hydrophilic polymer PVP-K30 (polyvinylpyrrolidone) was also investigated. PVP-K30 a hydrophilic polymer is widely and commonly used as supersaturation inducing-agent and precipitation inhibitor for the formulation of poorly soluble drugs (Patel et al., 2008; Xu et al., 2008; Jahangiri et al., 2016). 2. Materials and methods 2.1. Materials EZ, Dulbecco's modified Eagle's Medium (DMEM), HEPES, Triton X- 100, DMSO, NaOH pellets, KH 2-phosphate and NaCl were purchased from Sigma-Aldrich (Stockholm, Sweden); simulated intestinal fluid (SIF) powder for the preparation of fasted-state SIF (FaSSIF) was purchased from biorelevant.com (Surrey, United Kingdom) and Hanks balanced salt solution (HBSS) was obtained from Gibco (Paisley, United Kingdom). Tocopheryl polyethylene glycol succinate (TPGS) and PVP- K30 were received as free samples from BASF chemicals (Ludwigshafen, Germany). All solvents used for HPLC analysis were of chromatography grade. Pure Millipore water was used Cell culture Caco-2 cells were routinely cultured in 75 cm 2 tissue culture flasks (Greiner Bio One, Frickenhausen, Germany) at 37 C in 5% CO 2 atmosphere in high glucose DMEM supplemented with 20% fetal bovine serum (FBS), 1 mm sodium pyruvate, 100 U/mL penicillin and 100 μg/ ml streptomycin. For transport studies, the cells were seeded at a density of 75,000 cells/cm 2 on rat-tail collagen type I-coated Transwell clear inserts (12 mm in diameter, pore size 0.4 μm; Corning, VWR, Dublin, Ireland). The cells were allowed to grow for at least 16 days and the medium was exchanged every other day (Frank et al., 2014). The transepithelial electrical resistance (TEER) of cell monolayers was measured using a Millicell ERS-2 epithelial volt-ohm meter equipped with STX-2 electrodes (Millipore, Carrigtwohill, Ireland) and corrected for the background value contributed by the cell culture inserts and medium Membrane integrity measurements All sample solutions under study were investigated for toxic effects on the integrity of the cells before starting the transport experiments. After aspiration of the culture medium, insert-grown Caco-2 cell monolayers were washed with pre-warmed HBSS (37 C), then HBSS was added to the AP and BL sides and cells were incubated at 37 C for 1 h. Subsequently, TEER values were measured and used as control values for each sample solution under study. Afterward, the HBSS in the AP compartment of all inserts was replaced with FaSSIF containing different concentrations of EZ or EZ with polymer (the experimental variables under study) and cells were incubated for an additional 2 h. At the end of the incubation period, sample solutions were removed and HBSS was added to both AP and BL sides and TEER values were measured. TEER % was calculated as (TEER at 2 h/teer at time 0) 100. These values were used to select samples for the transport experiments. In addition to these measurements, to exclude any effects of the sampling process on the cells' integrity and to ensure the tightness of the inter-cellular junctions (Yee, 1997), the TEER values for all cell monolayers were measured before starting and at the end of each experiment. Before starting the experiments, DMEM was removed from the AP and BL compartments, the Caco-2 cells were incubated with HBSS (37 C) for 1 to 1.5 h and the TEER values were measured. At the end of the experiment (after 2 h), the experimental medium in the AP and BL compartments was replaced with HBSS and the TEER values were measured Supersaturation-precipitation-permeation interplay (without polymer) One-compartment setup A one-compartment experimental setup (without Caco-2 cells) was used to investigate the supersaturation and precipitation behaviour of EZ in the absence of a Caco-2 cell monolayer. The supersaturation experiments were conducted in 12-wells plates (22.1 mm diameter). FaSSIF (0.5 ml) was added to each well and supersaturation was induced using the solvent shift method starting with a higher concentration of DMSO stock solution. The final concentration of DMSO in FaSSIF was < 1%. Different degrees of supersaturation (DS) (DS 10, DS 20, DS 30 and DS 40) were induced based on the equilibrium concentration of EZ in FaSSIF. For example, the concentration of the solution at DS10 is 10 times the equilibrium solubility of EZ. The other experimental conditions were as follows; 60 rpm shaking speed and 37 C temperature. Samples were withdrawn at fixed time points and centrifuged immediately, and the obtained supernatants were diluted and analysed using HPLC.

111 A. Alhayali et al Two-compartment setup Apical compartment (donor compartment). FaSSIF was used as the supersaturation medium in the AP compartment. It was prepared from the original SIF powder using potassium di hydrogen phosphate buffer with the ph adjusted to 6.5 with NaOH. The solvent shift method was used and different DS (DS 10, DS 20, DS 30 and DS 40) were induced as in Section The other experimental details can be found in the Section Basolateral compartment (acceptor compartment). HBSS was the basis of the transport medium in the BL compartment of the cell monolayer. The HBSS was buffered with HEPES 10 mm to a ph of 7.4 and supplemented with 25 mm glucose. TPGS was added to the transport medium in a concentration of 0.2% to provide sink conditions (Hou and Siegel 2006). This concentration of TPGS was chosen following solubility analysis of the drug in the medium and after testing for compatibility with Caco2 cells (data not shown). The other experimental details can be found in the Section Vectorial transport experiments Experiments for the investigation of transport were conducted in the presence of Caco-2 cell monolayers using Transwell inserts. FaSSIF (0.5 ml) was prepared and added to the AP side and different DSs were induced as outlined before. Samples from AP (40 μl) were taken at 0, 5, 15, 30, 45, 60, 90 and 120 min and centrifuged immediately at 17,000 g for 5 10 min. The supernatants were diluted with the mobile phase and analysed using HPLC to find the concentration of EZ. The amount of EZ in the apical compartment (0.5 ml) was quantified. Two-hundred microliter samples were withdrawn from the BL side at the same time points and analysed without dilution. Fresh buffer (200 μl) was added to the BL side at each time point to ensure the maintenance of sink conditions. All experiments were conducted in triplicate. Each plate set of transwell membrane inserts was kept in an agitating incubator with an orbital shaker (speed = 60 rpm and medium temperature = 37 C) and was only taken out for sampling Supersaturation-precipitation-permeation interplay (with polymer) The effect of the polymer on supersaturation and permeation was studied in one-compartment and two compartment setups (as shown in Sections and ) with the DS 40 sample. PVP K-30 was predissolved in FaSSIF in two concentrations: 0.05 and 0.1% w/v in the AP side of the two compartment setup and in the one-compartment setup. The DS 40 supersaturated solution of EZ was prepared as outlined in Section 2.4 and was introduced into the FaSSIF solutions Quantification of intracellular ezetimibe in Caco-2 cells At the end of transport studies, the Caco-2 cell monolayers were washed twice with HBSS and lysed in 200 μl of FaSSIF supplemented with 1% Triton X-100. After 2 3 min, cell monolayers were removed gently from the surface of the insert membranes, diluted with acetonitrile and analysed using HPLC Quantitative analysis of ezetimibe An isocratic high performance liquid chromatography (HPLC) method was used for the determination of EZ concentrations in the collected samples. An Agilent HPLC unit (1290 series) equipped with a quaternary pump and UV detector (Agilent Technologies, Waldbronn, Germany) and a C-18 Eclipse column with a 5 μm (4.6 mm 100 mm) were used. A 40:60 (v/v) mobile phase was prepared from 0.05% w/v sodium 1-heptane sulfonic acid and acetonitrile. A flow rate of 0.5 ml/ min and injection volume of 10 μl were used. The measurements were made at a wavelength of 230 nm. Fig. 1. Changes in normalised transepithelial electrical resistance (TEER) following apical incubation of Caco-2 cell monolayers with various samples. Incubation with HBSS was used as control. Data represent means ± SD (n =4). DS =degree of supersaturation of ezetimibe Statistical analysis One-way ANOVA and a t-test were used to investigate statistically significant (p < 0.05) differences in the data obtained. All experiments were carried out in triplicate (n = 3). 3. Results and discussion 3.1. Integrity of Caco-2 cell monolayers The TEER values were measured to determine the integrity of the cell monolayer on exposure to the different drug solutions under study (Fig. 1). The results were given as percentage of the initial TEER value. All TEER % values were either stable or increased compared to the control TEER value (i.e. 100%). Samples containing PVP-K30 resulted in a decreased values (approximately 80%) but always remained above the threshold of 300 Ω cm 2, which generally is considered to be a hallmark of Caco-2 cell monolayer integrity (Van Breemen and Li, 2005; Frank et al., 2012; Saha and Kou, 2000). The TEER values before and after each experiment were high (at least 1700 Ω cm 2 ) indicating that the cell integrity was kept intact until the end of the experiment Supersaturation-precipitation-permeation interplay (without polymer) Supersaturation experiments were initially conducted in wells of cell culture plates (one-compartment setup) to gain insight into the supersaturation/precipitation behaviour of EZ in the absence of Caco-2 cells (i.e. in a cell-free environment) and to understand whether supersaturation/precipitation experiments conducted in a one-compartment model or in a test tube can predict the behaviour of a drug compound in a two-compartment model. As illustrated in Fig. 2a, the EZ concentration was higher with the DS 10 sample than with crystalline EZ and supersaturation was maintained until the end of the experiment. EZ precipitated with the DS 20 and DS 30 samples after 15 and 5 min, respectively. Instantaneous precipitation occurred with the DS 40 sample in FaSSIF (Fig. 2a). In this study, spontaneous nucleation and precipitation of EZ were observed in FaSSIF in all supersaturated solutions, suggesting a low kinetic barrier to crystallisation of the drug. This is typical behaviour because the supersaturated state is thermodynamically unstable and faster precipitation occurs as supersaturation increases, according to the theory of crystal growth (Lindfors et al., 2008). In contrast, Bevernage et al. (2012) observed no spontaneous precipitation and nuclei were required to promote precipitation (Bevernage et al., 2012).

112 A. Alhayali et al. Fig. 2. Amount of ezetimibe (EZ) in FaSSIF in the one-compartment setup (a). Amount of EZ in FaSSIF on the apical side of Caco-2 cell monolayers as a function of time, after induction of supersaturation (b). Cumulative amount of EZ recovered from the basolateral acceptor medium (i.e. HBSS), after vectorial transport across Caco-2 cell monolayers upon induction of supersaturation in the apical compartment (c). Data represent means ± SD (n = 3). Error bars in (c) are too small to show. Fig. 3. Calculated flux of various ezetimibe (EZ) formulations across Transwell-grown Caco-2 cell monolayers. Data represent means ± SD (n = 3). Symbol (*) represent the significant difference in the flux of the different polymer concentrations (p < 0.05), (ns) indicates data is not significant with respect to the flux of all DSs (p > 0.05). Fig. 2b shows the total amounts of EZ in FaSSIF on the AP side without addition of polymer. As depicted, the amount of drug dissolved was revealed to increase as the DS increased. All DS levels were associated with more stable solutions with lower precipitation tendencies. The reason for this could be that the permeation of the drug was an alternative to precipitation for relieving the thermodynamically unstable state that is the predominant feature during supersaturation, and as the concentration (or the DS) increased, the possibility of permeation reducing precipitation also increased, as reported by Bevernage et al. (2012). The permeation of EZ across the Caco-2 cell barrier at different concentrations was presented as a flux (J) instead of as the apparent permeability (P app), because reliable P app measurements require that the donor concentrations are known and determination of the drug flux across the membranes is independent of the donor concentration. The exact initial concentration of the dissolved drug was not known because of the fast precipitation of poorly water-soluble drugs when supersaturation was induced. This has also been documented for other drugs in previous studies (Kanzer et al., 2010; Hubatsch et al., 2007). The effect of inducing different degrees of supersaturation on the permeation of EZ across the Caco-2 cell monolayer is shown in Fig. 2c. The cumulative amounts of EZ in the transport medium (HBSS) in the BL compartment is plotted as a function of time. As delineated in Fig. 2c, the transport of drug to the BL side was delayed for about 45 min with DS 10 and 30 min with DS 20, 30 and 40. This lag-time could be due to the time needed to saturate the monolayer barrier before reaching steady-state flux, as reported by Kanzer et al. (2010). The highest amount of EZ transported to the BL compartment after 2 h was 1.28 ± 1.1E-04 μg from the DS 40 sample; ± 2.27 μg was in solution in the AP compartment after 2 h, as shown in Fig. 2b. The resulting flux was ± 9.9E-04 μg/min cm 2 (Fig. 3). Although

113 A. Alhayali et al. the DS 10 sample provided the highest amount of EZ dissolved after 2 h in the one-compartment setup (Fig. 2a), the highest amount transported after 2 h in the two-compartment setup was from the DS 40 sample. The DS 40 sample also maintained the highest amount of dissolved drug in the supersaturated state on the AP side of the two-compartment setup (Fig. 2a and b). Differences between the DS samples in the amounts of EZ transported to the BL side were less pronounced than differences in the precipitation behaviour in the one-compartment setup. However, precipitation behaviour in the one-compartment setup was not in accordance with that on the AP side in the two-compartment setup. The different outcomes for the one-compartment and two-compartment setups demonstrate that reliable prediction of supersaturation/precipitation behaviour cannot be achieved without including tools to predict absorption in the in vitro setup, as reported by Bevernage et al. (2012). As shown in Fig. 3, there were no significant differences in flux values between samples with DS 10, 20, 30 or 40 for drug transport to the BL side (p > 0.05). However, the EZ fluxes with DS 10, 20, 30 and 40 samples differed significantly from the flux with the crystalline EZ sample (p < 0.05). As reported in a previous study (Frank et al., 2012), the amount of drug transported to the BL side depends on the amount of drug in the supersaturated solution on the AP side. The specific behaviour of EZ as it was taken up and metabolised by enterocytes could explain the lack of effect of the varying DSs on EZ flux on the apical side of the two-compartment setup, as reported by Oswald et al. (2007). Generally, poorly water soluble drugs that permeate the intestinal wall well should show increased permeation when the concentration of the dissolved drug is increased on the AP side, but some recent reports have shown that solubilised colloidal drugs may permeate poorly (Frank et al., 2012; Fischer et al., 2011) Supersaturation-precipitation-permeation interplay (with polymer) PVP-K30 was selected to assess the effect of polymers on the inhibition of crystallisation (i.e. on sustaining supersaturation) and transport. PVP K-30 is well established as a precipitation inhibitor (Alhayali et al., 2016; Patel and Anderson, 2015). In the one-compartment setup with the DS 40 sample (Fig. 4a), a PVP-K30 concentration of 0.05% w/v resulted in a higher amount of drug in solution but precipitation occurred gradually over 90 min, at which point all the drug had been precipitated. A PVP-K30 concentration of 0.1% w/v resulted in rapid precipitation over the first 30 min but did not perform better than 0.05% w/v on keeping supersaturation state for longer time. Precipitation occurred later with both polymer concentrations (0.05% and 0.1% w/v) than in the absence of polymer. In the two-compartment setup, a concentration of 0.05% w/v maintained the supersaturation for the 2 h (Fig. 4b) while it was difficult to conclude the effect of 0.1% w/v due to system variability. In general, less extensive precipitation was observed in the twocompartment setup, and the amount of EZ dissolved on the AP side was greater in the presence of PVP-K30 (Fig.4b). The polymer PVP-K30 (0.05% w/v) kept the drug in the supersaturated state for 2 h. In the absence of polymer, the drug started to precipitate after 15 min. On the other hand, PVP-K30 (0.1% w/v) in the medium on the AP side increased the amount of dissolved drug up to 20 μg after 5 min, which was more than with a concentration of 0.05% w/v and in the absence of polymer (Fig. 4b). The effect of PVP-K30 on the supersaturation of EZ in FaSSIF has been demonstrated previously (Alhayali et al., 2016). The reason for less extensive precipitation of EZ in the presence of Caco-2 cells may be that permeation offered an alternative to precipitation. The same conclusion applies in the absence of polymer. Thus the in vitro prediction of supersaturation and precipitation is more accurate if it is combined with absorption tools because, in some cases, such as in this study, the precipitation can be overestimated by the one-compartment experiments compared to the two-compartment experiments. In this study, we also investigated how supersaturation affects the transport of EZ in vitro in a two-compartment setup using Caco2 cells in the presence of PVP-K30. Fig. 4c shows the cumulative amounts of EZ on the BL side that were transported over 2 h. Addition of 0.05% w/v PVP-K30 to the AP side resulted in the highest amount of EZ transported to the BL side. Furthermore the flux of EZ was significantly improved (p < 0.05) with this polymer concentration ± 1.5E- 02 μg/min cm as compared to 0.1% w/v polymer and no polymer (Fig. 3). PVP-K30 (0.1% w/v) improved the supersaturation solubility of EZ on the apical side in FaSSIF without improving the flux; the total transported amount was 2.97 ± 7.5E-04 μg. In summary, PVP-K30 (0.05% w/v) on the AP side in FaSSIF kept the drug in the supersaturated state for 2 h, improved the flux, and achieved the highest cumulated EZ concentration on the BL side after 2 h (5.54 μg ± 2.6E-03), as shown in Fig. 4b&c. The increase in drug concentration on the donor AP side as a result of solubilizing the excipients may not cause a proportional improvement in the flux in all cases, as reported by Saha and Kou, The intrinsic physicochemical properties of the drug and drug-excipient interaction can be expected to affect the outcome. In this study, PVP-K30 improved the flux across the Caco-2 cell barrier (Fig. 3) without affecting the amount of EZ taken up by the monolayer, as shown in Fig. 5. However, some recent studies have shown the opposite effect, reporting that addition of some excipient to the AP side has a negative effect on transport. This was attributed to the fact that increased apparent solubility is associated with a micellar solubilisation effect on the excipients which can result in overestimation of the true supersaturation, thus not causing improvement in the permeation rate (Frank et al., 2012) Quantification of intracellular ezetimibe EZ is known to accumulate in the intestinal mucosa (Oswald et al., 2007). To investigate how much of the EZ was taken up by the Caco-2 cells, the cells were lysed and analysed for intracellular EZ using HPLC. Fig. 5 shows the amounts of EZ in each sample solution studied. With DS 10, 7 ± 3.02 μg of EZ was found in the cell monolayer; this was approx. 4.3-fold the amount for the crystalline form. The amounts with DS 20, 30 and 40 were 13 ± 0.99, 32.7 ± 5.80 and 26.8 ± 6.70 μg, approximately 8-, 20- and 16-fold, respectively. These results indicate that the uptake of EZ by the cells increases proportionally with the degree of supersaturation on the AP side, reaching a plateau when the concentration of the drug is 30 times the concentration of the crystalline form at equilibrium, or higher (DS 30 and 40), on the AP donor side. The uptake of the drug by Caco-2 cells from the DS 40 sample in the presence of PVP-K30 (at 0.05 and 0.1% w/v) was approximately and 20-fold the amount quantified for the crystalline form, respectively. These amounts were the same as in the absence of the polymer, indicating that the amount taken up by the cells was not affected by the presence of polymer. Further, the uptake of EZ by Caco2 cells reached saturation for samples with DS 30 and higher, both with and without polymer. In previous work by Saha and Kou (2000), it was reported that the addition of solubilizing excipients to the AP donor side of Caco-2 cells may not only improve the solubility of a poorly soluble drug, but also affect the monolayer drug concentration and the cumulated drug amount, which was in contrast to our findings for EZ. To the best of our knowledge, this is the first study which has quantified the amount of EZ in Caco-2 cells during supersaturation in FaSSIF. 4. Conclusions This study was an attempt to investigate the supersaturation-precipitation-permeation interplay of EZ in a Caco-2 cell environment. The supersaturated solutions of EZ showed higher precipitation earlier at early time points in one compartment chamber while less precipitation tendency had been seen in the presence of Caco-2 cells. The transport of EZ for all degrees of supersaturation was low. Thus, the supersaturation studies that are usually conducted in one compartment chamber or in test tube could underestimate supersaturation, precipitation and

114 A. Alhayali et al. Fig. 4. Amount of ezetimibe (EZ) with PVP-K30 in FaSSIF in the one-compartment setup (a). Amount of EZ with PVP-K30 in FaSSIF on the apical side of Caco-2 cell monolayers as a function of time, after induction of supersaturation to DS 40 (b). Cumulative amount of EZ recovered from the basolateral acceptor medium (i.e. HBSS) after vectorial transport across Caco-2 cell monolayers upon induction of supersaturation in the presence of polymer in the apical compartment (c). Data represent means ± SD (n = 3). Error bars in (c) are too small to show. DS = degree of saturation. mandatory for accurate prediction of bioavailability. Acknowledgments A.A. and M.A.S. are recipients of PhD bursaries from the Iraqi Ministry of Higher Education and Scientific Research (MOHESR); the authors would like to acknowledge their contribution and funding. References Fig. 5. Amount of intracellular ezetimibe (EZ) after transmonolayer transport across Caco-2 cells. Data represent means ± SD (n = 3). transport of the drug. The polymer PVP-K30 improved the supersaturation of EZ at concentrations at 0.05% w/v and resulted in significant improvement in the transport/flux (J). EZ was taken up by the Caco-2 cells, with the amount trapped increasing with increasing amounts of the drug on the AP side. Hence, a more in vivo-like tool is Alhayali, A., Tavellin, S., Velaga, S., Dissolution and precipitation behavior of ternary solid dispersions of ezetimibe in biorelevant media. Drug Dev. Ind. Pharm Alonzo, D.E., Gao, Y., Zhou, D., Mo, H., Zhang, G.G., Taylor, L.S., Dissolution and precipitation behavior of amorphous solid dispersions. J. Pharm. Sci. 100, Amidon, G.L., Lennernäs, H., Shah, V.P., Crison, J.R., A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 12, Bevernage, J., Brouwers, J., Annaert, P., Augustijns, P., Drug precipitation permeation interplay: supersaturation in an absorptive environment. Eur. J. Pharm. Biopharm. 82, Bevernage, J., Brouwers, J., Brewster, M.E., Augustijns, P., Evaluation of gastrointestinal drug supersaturation and precipitation: strategies and issues. Int. J. Pharm. 453, Bikiaris, D.N., Solid dispersions, part I: recent evolutions and future opportunities in manufacturing methods for dissolution rate enhancement of poorly water-soluble drugs. Expert Opin. Drug Deliv. 8, Brouwers, J., Brewster, M.E., Augustijns, P., Supersaturating drug delivery systems: the answer to solubility-limited oral bioavailability? J. Pharm. Sci. 98, Fischer, S.M., Brandl, M., Fricker, G., Effect of the non-ionic surfactant Poloxamer 188 on passive permeability of poorly soluble drugs across Caco-2 cell monolayers. Eur. J. Pharm. Biopharm. 79,

115 A. Alhayali et al. Frank, K.J., Rosenblatt, K.M., Westedt, U., Hölig, P., Rosenberg, J., Mägerlein, M., Fricker, G., Brandl, M., Amorphous solid dispersion enhances permeation of poorly soluble ABT-102: true supersaturation vs. apparent solubility enhancement. Int. J. Pharm. 437, Frank, K.J., Westedt, U., Rosenblatt, K.M., Hölig, P., Rosenberg, J., Mägerlein, M., Fricker, G., Brandl, M., What is the mechanism behind increased permeation rate of a poorly soluble drug from aqueous dispersions of an amorphous solid dispersion? J. Pharm. Sci. 103, Gao, P., Guyton, M.E., Huang, T., Bauer, J.M., Stefanski, K.J., Lu, Q., Enhanced oral bioavailability of a poorly water soluble drug PNU by supersaturatable formulations. Drug Dev. Ind. Pharm. 30, Hou, H., Siegel, R.A., Enhanced permeation of diazepam through artificial membranes from supersaturated solutions. J. Pharm. Sci. 95, Hubatsch, I., Ragnarsson, E.G., Artursson, P., Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2, Jahangiri, A., Barzegar-Jalali, M., Garjani, A., Javadzadeh, Y., Hamishehkar, H., Asadpour-Zeynali, K., Adibkia, K., Evaluation of physicochemical properties and in vivo efficiency of atorvastatin calcium/ezetimibe solid dispersions. Eur. J. Pharm. Sci. 82, Kanzer, J., Tho, I., Flaten, G.E., Mägerlein, M., Hölig, P., Fricker, G., Brandl, M., Invitro permeability screening of melt extrudate formulations containing poorly watersoluble drug compounds using the phospholipid vesicle-based barrier. J. Pharm. Pharmacol. 62, Kataoka, M., Masaoka, Y., Yamazaki, Y., Sakane, T., Sezaki, H., Yamashita, S., In vitro system to evaluate oral absorption of poorly water-soluble drugs: simultaneous analysis on dissolution and permeation of drugs. Pharm. Res. 20, Kataoka, M., Sugano, K., da Costa Mathews, C., Wong, J.W., Jones, K.L., Masaoka, Y., Sakuma, S., Yamashita, S., Application of dissolution/permeation system for evaluation of formulation effect on oral absorption of poorly water-soluble drugs in drug development. Pharm. Res. 29, Lindfors, L., Forssén, S., Westergren, J., Olsson, U., Nucleation and crystal growth in supersaturated solutions of a model drug. J. Colloid Interface Sci. 325, Oswald, S., Koll, C., Siegmund, W., Disposition of the cholesterol absorption inhibitor ezetimibe in mdr1a/b ( / ) mice. J. Pharm. Sci. 96, Patel, D.D., Anderson, B.D., Adsorption of Polyvinylpyrrolidone and its impact on maintenance of aqueous Supersaturation of indomethacin via crystal growth inhibition. J. Pharm. Sci. 104, Patel, R.P., Patel, D.J., Bhimani, D.B., Patel, J.K., Physicochemical characterization and dissolution study of solid dispersions of furosemide with polyethylene glycol 6000 and polyvinylpyrrolidone K30. Dissolut. Technol. 15, Saha, P., Kou, J.H., Effect of solubilizing excipients on permeation of poorly watersoluble compounds across Caco-2 cell monolayers. Eur. J. Pharm. Biopharm. 50, Shi, Y., Gao, P., Gong, Y., Ping, H., Application of a biphasic test for characterization of in vitro drug release of immediate release formulations of celecoxib and its relevance to in vivo absorption. Mol. Pharm. 7, Singh, A., Worku, Z.A., Van den Mooter, G., Oral formulation strategies to improve solubility of poorly water-soluble drugs. Expert Opin. Drug Deliv. 8, Srivalli, K.M.R., Mishra, B., Improved aqueous solubility and antihypercholesterolemic activity of ezetimibe on formulating with hydroxypropyl-βcyclodextrin and hydrophilic auxiliary substances. AAPS PharmSciTech 17, Van Breemen, R.B., Li, Y., Caco-2 cell permeability assays to measure drug absorption. Expert Opin. Drug Deliv. 1, Xu, D.H., Wang, S., Mei, X.T., Luo, X.J., Xu, S.B., Studies on solubility enhancement of curcumin by Polyvinylpyrrolidione K30. Zhong Yao Cai 31, Yee, S., In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man fact or myth. Pharm. Res. 14,

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119 Cocrystal of tadalafil: Physicochemical characterization, ph-solubility and supersaturation studies Manishkumar R. Shimpi 1,2, Amani Alhayali 1, Katie L. Cavanagh 3, Naír Rodríguez- Hornedo 3, Sitaram P. Velaga 1, * 1 Pharmaceutical and Biomaterials group, Department of Health Sciences 2 Chemistry of Interfaces, Luleå University of Technology, SE-97187, Luleå, Sweden. 3 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan , United States Abstract: Tadalafil (TDF), used for the treatment of erectile dysfunction, is a practically water insoluble drug. A cocrystal of TDF with malonic acid (MOA) was identified and characterized using a fleet of solid-state characterization techniques including differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Raman spectroscopy and powder X-ray diffraction (PXRD) and single crystal X-ray diffraction technique. Cocrystal ph-solubility and supersaturation index (SA) (defined as solubility of cocrystal/ solubility of drug) were determined and theoretical models were applied. The dissolution behavior of the cocrystal and amorphous forms in the presence and absence of HPMC was compared. The crystal structure analyses reveals the formation of 1:1 ratio of TDF and MOA in asymmetric unit, wherein TDF molecule shows persistent N H O hydrogen bonding interactions and catemeric chain of MOA forms hydrogen bonds with lactam carbonyl group of TDF. MOA sublimes from the cocrystal lattice, followed by the melting of TDF upon heating the cocrystal in the DSC. TDF-MOA showed higher solubility than TDF over a wide ph range and it was dependent on the ph, with higher solubility at higher ph values. Theoretical models based on Ksp have nicely predicted the cocrystal experimental solubility-ph dependence. Supersaturation index for TDF-MOA cocrystal increased from approximately 100 at ph 1 to about at ph 6.8. In the powder dissolution studies, cocrystal and amorphous forms generated a supersaturation (C max /S drug ) of 30 and 10, respectively and in the presence of 0.1% w/v HPMC, both forms showed a supersaturation of 120 which was maintained for at least 3 hours due to precipitation inhibition. Thus, SA based on equilibrium studies can provide insights on the supersaturation level and the rate of cocrystal conversions in the dissolution studies. 1

120 Key words: Tadalafil, cocrystal, crystal engineering, amorphous, ph-solubility, supersaturation index, dissolution. 1. Introduction Active Pharmaceutical Ingredients (APIs) are often formulated into solid dosage forms due to their favorable properties such as thermodynamic stability, purity, convenience and excellent patient compliance. Different solid forms of an API such as polymorphs, salts, solvates, amorphous form and cocrystals show different physicochemical properties, which can profoundly affect the bioavailability of a resulting dosage form. 1-3 physicochemical properties of a crystalline form (single component or multicomponent) are inherently dependent upon the composition and the packing of molecules in the crystal lattice. 4-6 In this context, crystal engineering 7 is an important tool used as a design strategy to alter the crystalline lattice and improve the properties (e.g. thermodynamic stability, solubility and mechanical etc.) of drug substances Crystal engineering is the understanding of intermolecular interactions in the context of crystal packing and the utilization of such understanding in design of new solids with desired physical and chemical properties. 11 Pharmaceutical cocrystals, often designed based on crystal engineering principles, have been found to be effective in improving physical and technical properties of clinical relevance. 12, 13 In fact, a number of reports has revealed the potential of cocrystals as supersaturating drug delivery systems. 14, 15 For example, indomethacin-saccharin (IND-SAC) cocrystals showed improved solubility and bioavailability of indomethacin as compared to the crystalline form. 16 The cocrystal eutectic point is an isothermal invariant point where the solid cocrystal and drug are in equilibrium with solution and models have been developed to predict cocrystal solubilities in pure solvent from measurement of transition concentrations. 17 The solubility advantage (or supersaturation index) of IND-SAC cocrystals was estimated to be about 65 times over the crystalline form at ph 3 and the increase in cocrystal to drug solubility was related to solvation effects. 16 Furthermore, the aqueous solubility of carbamazepine cocrystals was found to be times greater than the solubility of carbamazepine dihydrate form. 16 Rodriguez-Hornedo et al. have derived mathematical models based on cocrystal dissociation and ionization of the components that explain cocrystal solubility dependence on the ph, pka, cocrystals stoichiometry and coformer concentration. 15, 17, 18 Since solution conditions such as ph complexation determine the cocrystal solubility and stability, cocrystals should be characterized not only by their solubility but also supersaturation behavior. 15 2

121 Tadalafil (TDF, Figure 1) is a phosphodiesterase inhibitor, 19 a pharmaceutical active ingredients, under the brand name Cialis approved by Food and Drug Administration (FDA) in 2003 for the treatment of erectile dysfunction (ED). 20 TDF is a white crystalline powder, practically insoluble in water 21 and consider as a class II drug by Biopharmaceutical classification system. 22 TDF is a neutral drug with pka of TDF logp is 1.54 that reflects poor drug solubility in water. 23 Many approaches have been tested and described in the literature to increase the dissolution rate and bioavailability of TDF. The approaches reported include the use of (1) inclusion complexes, 24 (2) solid dispersion, (3) amorphous hot-melt extrudate, 28 (4) nanosuspension, 29 and (5) crystalline solvates forms. 30 Although Weyna and co-workers have reported four cocrystals of TDF, no dissolution and supersaturation performance was studied. 31 Furthermore, cocrystals of TDF and Malonic acid have not been identified. Malonic acid (MOA, Figure 1), is a pharmaceutically accepted second-smallest aliphatic dicarboxylic acid. The aim of the study was to identify cocrystals of tadalafil-malonic acid (TDF-MOA) cocrystal and to perform comprehensive solid state and structural characterization. Another objective was to determine ph-solubility dependence and provide insights on the solubility advantage (or supersaturation index) offered by the cocrystals. It was also our aim to compare supersaturation behavior of the cocrystal and amorphous forms in the powder dissolution studies in the presence and absence of the precipitation inhibitors like HPMC. Finally, we aim to understand how cocrystal supersaturation index (SA) determined under equilibrium conditions relates to drug supersaturation levels during cocrystal dissolution. O O O O O N H N HO OH H 3 C N O Figure 1. Molecular structures of Tadalafil (TDF) and coformer, malonic acid (MOA). Hydrogen bond donors and acceptors are highlighted in red and blue respectively. 3

122 2. Materials and Methods 2.1 Materials All chemicals and solvents (Purity > 99%) were purchased from Sigma Aldrich or Acros Organics and were used without further purification. HPMC (Pharmacoat grade 606) was obtained from Shin-Etsu chemical company (Japan) and all buffer salts for buffer solution preparation were purchased from Sigma Aldrich (Stockholm, Sweden). 2.2 Methods Screening for cocrystals of TDF Non-stoichiometric slurry crystallization method was used for the screening of TDF cocrystals. Based on the solubilities of TDF and various coformers (including, nicotinamide, tartaric acid, citric acid, adipic acid, glutaric acid, malonic acid, succinic acid, malic acid and vanillin), ethyl acetate and acetonitrile were used as solvents for screening. Excess amount of TDF was added to the saturated solutions of coformers and stirred at room temperature in a tightly caped glass vials. Solids were isolated after h and subjected to PXRD and Raman spectroscopy or DSC Preparation and scale-up of TDF-MOA A 7-mL glass vial was charged with 389 mg (1 mmol) of TDF, 104 mg (1 mmol) of MOA. 3 ml of ethyl acetate was added to form slurry. The cake formation was observed, indicating product formation. To make slurry, 3 ml of ethyl acetate was added in a glass vial. The reaction was allowed to stir for a total of 5 hours, at which time the solids present were isolated by vacuum filtration and air dried at room temperature to yield 419 mg (85%) of TDF-MOA cocrystal. Similar sizes of batches were prepared as per experiment requirements Preparation of single crystals of TDF-MOA cocrystal Approximately mg of TDF-MOA cocrystal was used in the crystallization experiments. For slow evaporation, the cocrystal was dissolved in enough acetonitrile (~2 ml) to produce an under saturated solution at room temperature. The solution was collected by filtration in a 5-mL glass vial and let it stand to evaporate at low temperature (~10-15 o C). Colorless needle-shaped diffraction quality crystals of TDF-MOA (examine under optical microscope) were obtained within 48 hours and isolated from the solution. 4

123 2.2.4 Preparation of amorphous TDF Amorphous TDF was prepared by spray drying of 2% w/v solution of drug in acetonewater (9:1, v/v), following the work by Wlodarski et al. 32 Buchi mini spray dryer B-290 (Büchi, Switzerland) was used. An open system was used with nitrogen as a drying and atomizing gas (open, suction mode). Spray drying process conditions were as follows: inlet temperature 160 C, pump speed 30 %, and outlet temperature 89 C. The aspirator was operated at 100%. The obtained amorphous powder was sieved (500 micron) and analyzed by DSC and PXRD to confirm the amorphous state. The powders were stored at -20 C before further analysis Solid state characterization Raman spectroscopy The Raman spectra were recorded on a Chromex Sentinel dispersive Raman unit equipped with a 785nm, 70 mw excitation laser and a TE cooled CCD. Each spectrum is a result of twenty co-added 20 second scans. The unit has continuous automatic calibration using an internal standard. The data was collected by SentinelSoft data acquisition software and processed in GRAMS AI Powder X-ray diffraction (PXRD) PXRD patterns for the samples were collected using an Empyrean X-ray diffractometer (PANalytical, The Netherlands) equipped with a PIXel3D detector and a monochromatic Cu Kα radiation X-ray tube ( Å). The tube voltage and amperage were set at 45 kv and 40 ma, respectively. Samples were prepared for analysis by pressing a thin layer of the sample onto a metal sample holder. Instrument calibration was performed using a silicon reference standard. Each sample was scanned in the 2θ range of 5 40, increasing at a step size of θ. The data were processed using HighScore Plus Version 3.0 software (PANalytical, The Netherlands) Differential scanning calorimetry (DSC) Thermal analyses of cocrystal samples were performed on a TA instruments DSC Q1000 (V9.9) The sample (1-3 mg) was placed into a non-hermetic aluminum DSC pan, and the weight accurately recorded. The pan was crimped and the contents were heated (10 o C 5

124 min -1 ) under continuously purged dry nitrogen atmosphere (50 ml min -1 ) in the range of o C. The instrument was equipped with a refrigerated cooling system. Indium was used as the calibration standard. The data was collected in duplicate for each sample Thermogravimetric (TG) analysis Thermogravimetric experiments were performed using a Thermal Advantage TGA Q5000 (V3.17) Instruments. Each sample (5-10 mg) was placed in a platinum pan (100 l) and inserted into the TG furnace. The sample was heated with heating rate 10 o C min -1 in the rage of o C, under a nitrogen purge gas (balance gas and sample gas was 10 and 90 o C min -1 respectively) Variable temperature-powder X-ray diffraction (VT-PXRD) High-temperature studies were carried out using an X-ray chamber HTK-10 Anton Paar. The crystalline phase of the sample was determined by powder X-ray diffraction (PXRD) using a PANalytical Empyrean diffractometer equipped with a Cu LFF HR X-ray tube and a PIXcel 3D detector. For the in situ high temperature measurement, the temperature was kept at 25 o C by Anton Paar furnace with the heating rate of 50 o C/min. The diffraction patterns were recorded every 50 C interval. The diffraction patterns were recorded by 0.05 step scanning in the range of 2θ angles from 5 to 30 grad. A platinum slice was used as a sample holder. 2.3 Single-crystal X-ray diffraction The single-crystal X-ray diffraction data of the crystals were collected on a Bruker Nonius Kappa CCD. The data set for compounds were collected at room temperature (293 K). The crystals were quite stable and hence no extraordinary precautions were necessitated for the smooth collection of intensity data. All the data sets were collected using Mo Kα radiation (λ= Å), and crystal structures were solved by direct methods using SIR- 92 and refined by full-matrix least-squares refinement on F2 with anisotropic displacement parameters for non-h atoms using SHELXL Hydrogen atoms were placed on their expected chemical positions using the HFIX command. All the nonhydrogen atoms were refined anisotropically. Structure solution, refinement, and generation of publication materials were performed by using shelxle, V623 software. 33 All packing diagrams are generated using Diamond software. 34 ORTEP diagram were generated using OLEX2 software. 35 6

125 2.4 Solubility and dissolution studies Solubility of TDF The equilibrium solubility of TDF was measured in a ph range of 1-7 and the ph of the solution was adjusted by 1 M HCl or NaOH. An excess amount (approx. 200 mg) of crystalline TDF was added to 5 ml solution of 0.1 N HCl and slurried in a water bath at 25 C with orbital shaking (60 rpm.) After equilibration for hours, the sample solutions were filtered (using 0.2 μm PTFE filters) and analysed by HPLC (Agilent Technologies, Stockholm). The ph at equilibrium was measured and the excess solids were analysed using PXRD to confirm the solid form in equilibrium Cocrystal solubility and determination of eutectic concentrations of TDF and MOA The eutectic point between cocrystal and drug was reached by simply adding 200 mg of cocrystal powder (500 micron) in a 5 ml of solution at different ph. A 0.1 N HCL solution was used for (ph 1, 2, and ph 5), acetate buffer solution for (ph 3 and 4) and phosphate buffer solution for (ph 6 and 7). The solutions were subjected to orbital shaking (60 rpm) for hours at 25 C and the ph at equilibrium was measured. The concentration of the drug and the coformer at equilibrium at the eutectic point was determined by HPLC. The eutectic point was confirmed by PXRD analysis of the residual solid sample at equilibrium Dissolution experiments Non-sink powder dissolution experiments were conducted to investigate dissolution and supersaturation behaviour of amorphous and cocrystals of TDF. Excess amounts (approx. 1 mg/ml) of the crystalline TDF, amorphous TDF and equivalent amount of the cocrystal powder were suspended in phosphate buffer solution (ph 6.8). Experiments were conducted at 25 C and 60 rpm orbital shaking. Aliquot samples were withdrawn at different time points until 3 hours, filtered (0.2 μm PTFE filters), diluted and analysed by HPLC. Dissolution of crystalline TDF, amorphous TDF and cocrystals were also investigated in the presence of hydroxypropyl methyl cellulose (HPMC). A 0.1% w/v HPMC was predissolved in the phosphate buffer solution (ph 6.8) and the dissolution experiments were conducted under similar condition as before. At the end of dissolution 7

126 study, phs of the solutions were measured and the separated solids were subjected to PXRD analysis HPLC method TDF and MOA were quantified using a quaternary pump HPLC unit (1290 series) 36, 37 equipped with a UV detector (Agilent Technologies, Waldbronn), following methods. A C-18 Nucleosil column ( mm, 5 microns) (Sigma Aldrich, Sweden) was used for both compounds. In the case of TDF, the mobile phase was 20 mm Na phosphate buffer (ph 7) and Acetonitrile (30:70). Analysis was performed at a flow rate of 0.5 ml/min and detector wavelength 260 nm. Injection volume was 10μL. For the analysis of MOA, mobile phase consisted of 25 mm potassium dihydrogen phosphate (ph 2.5) and Methanol in 93:7 ratios. Flow rate, injection volume and the wavelength for UV detection were 1 ml/min, 5 μl and 210 respectively. 3. Result and Discussion 3.1. Screening and identification of TDF-MOA cocrystal A Cambridge Structural Database (CSD; 38 ConQuest version 5.38, May 2017, Filters- only organic molecules, number of hits 8) analysis revealed only six organic molecules of TDF; including TDF 39 (IQUMAI), two solvates 30 (KAJKOY and KAJKUE) and three cocrystals 31 (LASZUC, LATBAL and LATBEP). The crystal structure analysis of TDF reveals that TDF molecules form supramolecular chains utilising hydrogen bond interactions present between the indole group and the lactam carbonyl group of an adjacent TDF molecules. Due to the presence of such hydrogen bond interactions, it is predisposed to form hydrogen bonds with a second molecule (coformer) in the crystal lattice. Weyna et al. have utilized this knowledge in the design of pharmaceutical cocrystals of TDF with methylparaben, propylparaben and hydrocinnamic acid. 31 In all TDF cocrystals, lactam carbonyl group, hydrogen bond acceptor of TDF is linked to the hydrogen bond donor of coformers. Aakeröy et al. 40 and Adsmond et al. 41 engineered ternary cocrystals based on observations of the hydrogen bond donor/acceptor pairings. Therefore, supramolecular synthon history was used for cocrystal screening and included variety of coformers that consist of hydrogen bond donor functionality in this study. Cocrystal screening was conducted using slurry crystallization method, because it is a more efficient and considers thermodynamic phase behaviour of different phases. 8, 12 New pharmaceutical cocrystal of TDF was found with coformer MOA as confirmed by initial solid-state characterization. 8

127 Figure 2. Raman spectra for (a) TDF, (b) MOA and (c) TDF-MOA cocrystal. The arrows and rectangles shows new or change in vibrational frequency in cocrystal form in comparison with cocrystal components. The Raman spectrum of a new solid form was compared with the pure components (Figure 2). The appearance of new vibrational peak at 1736 cm -1 and the disappearance shoulder peak around 1600 cm -1 in cocrystal solid form indicate the formation of a new solid form (Figure 2). There are also additional peaks, as highlighted by arrows and rectangles, suggest the change in intermolecular interactions in the cocrystal. PXRD is a rapid analytical technique used for phase identification of crystalline materials. PXRD patterns for TDF, MOA, and TDF-MOA are shown in Figure 3. The diffraction pattern for the new form was distinctly different from that of TDF and MOA, indicating the formation of cocrystal. Thus, PXRD results corroborates with the Raman spectra confirming the formation of tadalafil-malonic acid (TDF-MOA) cocrystal. Further evidence of new cocrystal formation is presented in the following sections based on solid-state techniques and single crystal X-ray diffraction. 9

128 Figure 3. Powder XRD patterns for (a) TDF, (b) TDF-MOA cocrystal and (c) MOA. The asterisks denote new diffraction peaks in cocrystal form in comparison with cocrystal components. 3.2 Crystal structure description The crystal structure was elucidated using single crystal X-ray diffraction. The obtained data revealed that TDF-MOA crystallizes in the orthorhombic space group P with one molecule each of TDF and MOA in the asymmetric unit as shown in Figure 4. MOA is the coformer possessing carboxylic acid groups capable of forming strong hydrogen bonds with or without proton transfer. The structural analysis found that the carbonyl group of malonic acid has shorter bond length (C24 O7 = 1.22 Å and C25 O6 = 1.19 Å) and C OH group of malonic acid has larger bond length (C24 O8 = 1.33 Å and C25 O5 = 1.34 Å. Thus, malonic acids in the cocrystal exist as a neutral moiety (no proton was transfer occurred), which is evident from the carboxylic acid bond length. 10

129 Figure 4. ORTEP of asymmetric unit in the crystal lattice of TDF-MOA cocrystal. The crystal packing in cocrystal shows that adjacent TDF molecules are hydrogen bonded via N H O=C interactions [N3 H3 O1; N O = Å, N H O = o ] of a neighbouring molecule (the indole group and the lactam carbonyl group) as shown in Figure 5a. The CSD 38 analysis of different TDF crystal forms shows that the adjacent TDF molecule shows persistent N H O hydrogen bonding interactions in all the reported TDF crystal form. 30, 31, 39 The persistent hydrogen bonding motif (bond distance and angle) present between the indole and the lactam carbonyl group an observed in all TDF crystal forms are tabulated in Table 1. Table 1. Characteristics of important hydrogen bonds observed in different TDF crystal forms. Crystal structure with CSD code N-H O=C interactions a N O (Å) H O (Å) N H O ( o ) TDF (IQUMAI) TDF-MPB (LASZUC) TDF-PPB (LATBAL) TDF-HCA (LATBEP) TDF-ACE (KAJKOY) TDF-MEK (KAJKUE) TDF-MOA ( ) a Three columns for each structure represent N O /H O bond length in angstroms and bond angle in degrees respectively. The supramolecular chains of TDF are linked to the MOA molecules through C=O H O hydrogen bonds [C=O2 H8a O8; O O = Å, O H O = o ] as shown in Figure 5b. 11

130 Further, MOA forms the catemeric chains in the crystal lattice along a 2 1 screw axis perpendicular to the b-axis through O H O hydrogen bonds [O5 H6e O7; O O = Å, O- H O = o ] (Figure 5b). TDF molecule forms double layered structures (Figure 5a) through N H O=C interactions and this double layers are separated by catemeric chains of MOA (Figure 5b). In order to check the homogeneity and crystalline phase purity of prepared TDF-MOA cocrystal by reaction crystallization, the experimental and simulated PXRD pattern (generated from the single crystal structure of TDF-MOA using Mercury 42 ) were compared. The experimental PXRD did match the simulated PXRD (standard reference) pattern from the singlecrystal structure (Figure S1) confirm the homogeneity and purity of TDF-MOA crystalline phase. In addition, single endothermic transition observed in DSC curve further attest the single crystalline phase present in the cocrystal (Figure 6a, discussed in details in 3.3 Section). (a) (b) Figure 5. (a) A catamer-like chain via N-H O hydrogen bonds along the b-axis. (b) The catemeric chains are separated by malonic acids zig-zig chain via O-H O hydrogen bonds along the b-axis. To better visualize/understand the potential intermolecular interactions of cocrystal, 1,3-benzodioxole group was removed. 3.3 Thermal behaviour of TDF-MOA cocrystal The thermal behaviour of TDF-MOA cocrystal in relation to the TDF and MOA was carried out using DSC and TGA. The DSC thermogram for TDF, MOA and TDF-MOA cocrystal are presented in Figure 6a. TDF showed a single endothermic transition with 12

131 T onset = o C. MOA shows first endothermic peak at 96.2 o C due to solid phase transition; second endothermic transition at T onset = o C attributed to melting; and a third endothermic peak at o C corresponding to the thermal decomposition. The overlaid DSC and TGA thermograms of MOA are presented in Figure S2. Thermal events observed for MOA are in agreement with the reported thermal behaviour. 43 The DSC thermogram for TDF-MOA cocrystal showed a first endothermic transition at T onset ( Hf) o C 2.0 (139.0 J g ) and second endothermic peak at T onset ( Hf) o C 2.7 (55.71 J g ). The DSC thermogram of the cocrystal was distinctly different from individual components as observed in Figure 6a. Thermal behaviour of the TDF-MOA cocrystal was further investigated by TGA. Interestingly, TGA of TDF-MOA shows first weight loss of around 25% at ~195 o C where the major endothermic event (T onset = o C) occurs in the DSC, which suggests a possible gas released event (Figure 6b). The second major weight loss at >310 o C was possibly due to the degradation of the material (Figure 6b). To get more insights on the first weight loss, TDF-MOA cocrystal was analysed by capillary melting point apparatus. Release of gases from cocrystal powder material was observed in the temperature range o C. Subsequent heating of the powder resulted in a complete melting at ~300 o C. (a) (b) Figure 6. (a) DSC thermograms for tadalafil, TDF; malonic acid (MOA) and TDF-MOA cocrystal. (b) DSC (blue line) and TG (black line) overlaid profile of the TDF-MOA cocrystal. In a separate experiment, the cocrystal powder was heated in a capillary to 210 o C, (i.e. until after the gases are released but essentially before the melting event) and the powder sample was isolated at room temperature and subjected to Raman analysis. As observed in 13

132 Figure S3, the Raman spectra were found to be identical to that of TDF. It was reported that the subsequent heating after the melting of MOA (at around 137 o C) results in endothermic event at T onset = o C, corresponding to the evaporation of acetic acid and carbon dioxide gases as confirmed by IR spectroscopy. 43 Thus, the capillary melting experiments and DSC/TGA data and its analyses suggest the sublimation phenomenon (i.e. solid to gas transformation) of MOA was observed in the cocrystal at 190 o C. We have also collected further evidence of sublimation phenomenon using variable temperature powder X-ray diffraction (VT-PXRD). The characteristic diffraction peaks of the cocrystal were observed until 180 o C (Figure 7) later it convert into crystalline, in line with DSC and TGA observations. Thus, based on the solid-state analysis, we conclude that the TDF-MOA cocrystal undergoes sublimation of MOA from the cocrystal lattice, in the form of gases, followed by the melting of TDF. To the best of our knowledge, this is first example of a cocrystal wherein the coformer sublimates from the crystal lattice leaving behind the drug which subsequently melts. This is another intriguing example where the cocrystallization can impart drastic change in thermal behaviour of the drug or a conformer (i.e. patent compounds). Figure 7. Variable temperature powder X-ray pattern for the TDF-MOA cocrystal. The characteristic peaks of cocrystal and TDF are highlighted using rectangular boxes. 14

133 3.4 TDF-MOA cocrystal solubility and Understanding cocrystal solubility-ph dependence helps to guide formulation development of cocrystals as well as to determine supersaturation index and conversion/transformation propensity of cocrystals. Furthermore, knowledge of how cocrystal solubility-ph is affected by cocrystal components provides tools to engineer cocrystals with customized solubility behaviour. In fact, equations that describe cocrystal solubility in terms of solubility product, cocrystal component ionization constants, and solution ph have been derived for various cocrystals with acidic, basic, amphoteric, and zwitterionic components. 15 TDF-MOA (1:1) cocrystal consists of non-ionic drug, TDF and one diprotic acid MOA, with two pka values 2.83 and While detailed derivations can be found elsewhere, 15 the TDF-MOA cocrystal solubility dependence on is given by, (1) where is the cocrystal solubility product, and are the ionization constants of MOA. was calculated as the product of the concentrations of the nonionized constituents according to the following equation; (2) Where and are eutectic concentrations of TDF and MOA respectively. Table 2. Cocrystal and intrinsic solubility of the drug, S TDF. Cocrystal (M 2 ) S TDF (M) S CC /S TDF (ph 1-3) TDF-MOA cocrystal (3.8 ± 1.4 ) (4.6 ± 0.2) S refers to solubility under nonionizing conditions ± represents standard deviation 15

134 TDF-MOA was found to convert back into TDF at ph and the solubility was measured at eutectic point, where the solution phase is in equilibrium with both cocrystal and drug solid phases. 17 Figure S4 shows presence of cocrystal and drug phases at the eutectic points at different ph. Cocrystal was determined from equation 2. Table 2 summarizes the cocrystal and intrinsic solubility of the drug, S TDF. was used to generate the cocrystal solubility dependence on ph. Figure 8 shows that the TDF- MOA solubility is higher than TDF over a wide ph range. For example, even at its lowest solubility, cocrystal appears to be over 100 times more soluble than the drug (Figure 8 and Table 2). These trends are consistent with coformer solubility and ionization properties, in 16, 17 line with the results reported for other cocrystals. Experimental cocrystal solubilities could only be measured in the ph range (ph 1-3) as the cofomer controlled/lowered the ph of the solution. Mathematical model (eq 1), based on and values, predicts the exponential increase in cocrystal solubility with ph and these theoretical values are in excellent agreement with the experimentally measured solubilities (Figure 8). Thus, it is possible to predict the cocrystal solubility-ph dependence from knowledge of Ksp, at ph values where solubility was not experimentally measured and to gain insight of the supersaturation behavior of cocrystals at physiologically relevant ph. It is clear from the shape of solubility-ph curve that the TDF-MOA cocrystal imparts ph dependent solubility to TDF, demonstrating the potential of cocrystal technology in engineering solubility behavior of nonionizing drugs as a function of ph. Previously, cocrystals of saccharin were shown to not only enhance aqueous solubility but also impart ph sensitivity to nonionizing drugs like carbamazepine. 16 Furthermore, TDF eutectic concentrations are in agreement with drug solubility values (S TDF ) values (Figure 8) suggesting that there is no drug complexation with coformer in solution. 16

135 Figure 8. Solubility-pH dependence of 1:1 TDF-MOA cocrystal and TDF. Experimentally measured cocrystal and drug solubilities are represented by (red ) and (blue ) respectively. concentrations are shown in (blue ). Lines represent theoretical solubilities of cocrystal (red dashed line) and drug (blue line). Solubility-pH dependence was calculated according to eq 1 with value listed in Table 2 and pka values of 2.83 and 5.69 for MOA. S TDF (line) was determined as the average of experimental TDF solubility points (blue squares). 3.5 Supersaturation index (SA) of TDF-MOA cocrystals The enhanced solubility of drug cocrystals has been reported for a number of drugs. 8, 44, 45 The solubility advantage or supersaturation index (SA), defined as Scc/Sdrug, represents the driving force for drug precipitation and provides a metric for the risk of cocrystal conversions. It also provides guidance for the selection of ph and formulation additives to prevent drug precipitation. Recently, Rodriguez-Hornedo el al. have introduced a simple method to tailor cocrystal SA by the rational selection of drug solubilizing agents and ph Supersaturation index points were calculated from experimental cocrystal solubility and eutectic drug concentrations. Theoretical SA is extrapolated according to 17

136 (3) Figure 9 shows the SA dependence on ph for TDF-MOA cocrystal. Evidently, theoretical and experimentally measured values are in good agreement. Interestingly, SA index for TDF-MOA cocrystal increases from approximately 100 at ph 1 to about at ph 6.8 (Figure 9). SA values are orders of magnitude higher than experimentally measured supersaturation generated by amorphous or cocrystal forms due to onset of nucleation at a lower supersaturation value. SA-pH dependence graph provides a means to assess slow and fast cocrystal conversions to less soluble drug. High SA values are expected to lead to fast cocrystal conversions, fast drug nucleation, and low observed supersaturation. Thus, the maximum daily TDF dose of 20 mg (dose number 26, TDF solubility M ( 0.3 μg/ml) and volume of solution 250 ml) is well below the cocrystal solubility under physiological conditions (ph 1-7.4). In studying solubility advantage of cocrystals, Good and Rodriguez-Hornedo have shown that the cocrystal solubility is directly proportional to the solubility of constituent reactants for a wide range of drugs (including acidic, basic, zwitterionic and neutral). 14 Later, Velaga et al have revealed increase in the coformer concentration at the eutectic point for carbamazepine-saccharin cocrystals and concluded that the cocrystal aqueous solubility advantage was a result of decrease in the solvation energy (log γ)

137 Figure 9. Cocrystal supersaturation Index (cocrystal solubility advantage) as a function of ph for TDF-MOA cocrystal. Symbols represent the calculated S cocrystal /S drug values from solubility measurements and line represents theoretical dependence on ph generated from eq 3 with values listed in Table 2 and pka values of 2.83 and 5.69 for MOA. 3.6 Powder dissolution and supersaturation behaviour of TDF solid forms Figure 10 shows non-sink dissolution profiles of TDF cocrystal and amorphous solid in the presence and absence of precipitation inhibitor HPMC at ph 6.8. In the absence of HPMC, cocrystals and amorphous forms generate faster dissolution and supersaturation with respect to TDF, followed by precipitation (Figure 10). Whilst in the presence of 0.1% w/v HPMC, cocrystal and amorphous forms achieved much higher concentrations than the drug and the supersaturation was sustained for 3 hours (Figure 10). TDF crystalline form reached a plateau that corresponds to the equilibrium solubility of the drug M ( 0.36 μg/ml) in phosphate buffer ph 6.8. The spray dried TDF (amorphous form) and cocrystals dissolved very quickly and reached maximum solution concentrations (C max ) of M and M respectively (3.51 and μg/ml respectively), in less than 5 min. Thereafter the concentrations decreased because of precipitation or conversion of cocrystal to TDF as confirmed by PXRD 19

138 analysis (supplementary information). The dissolution behavior of TDF-MOA resembles many other cocrystals in the literature. 44 For example, the ezetimibe L-proline cocrystal showed rapid dissolution and conversion to ezetimibe anhydrate in phosphate buffer (ph 6.5). 8 The maximum concentration is expressed in terms of supersaturation (C max /S drug ), which represents the concentration above which precipitation is faster than the dissolution of the cocrystal. The cocrystal and amorphous forms generated a supersaturation of 30 and 10, respectively (Figure 10). TDF-MOA cocrystal in the presence of 0.1% w/v HPMC showed C max of M ( μg/ml), which corresponds to a supersaturation of about 120. Amorphous form also achieved similar concentrations. For both cocrystal and amorphous phase, the supersaturation was maintained much longer (3 hours) in the presence of HPMC i.e., suggesting slower conversion of amorphous or cocrystal form to drug. HPMC at a concentration of 0.1% w/v was found to provide maximum precipitation inhibition from solution supersaturated at These findings suggest that cocrystals may increase drug bioavailability under similar conditions. C max during cocrystal dissolution reached a supersaturation of about 30 at SA of (Figure 10). At this SA, HPMC was effective in slowing drug nucleation, reaching supersaturation values of 120. Similarly, SA was shown to increase with increasing the ph for cocrystals of ketoconazole, and a faster precipitation of the drug was observed. 49 Therefore, SA appears to be an indicator of the rate of cocrystal conversions. 20

139 Figure 10: Dissolution profiles of different solid forms of TDF in phosphate buffer (ph 6.8±0.4); Crystalline TDF (Blue), Amorphous TDF (Green), TDF-MOA cocrystal (Red) and phosphate buffer containing 0.1% w/v of HPMC; Amorphous TDF (Black) and TDF- MOA cocrystal (Purple) in Error bars represent standard deviations obtained from triplicate measurements. The physical purity of cocrystals and amorphous forms was confirmed by PXRD and DSC (Figure S4). Conclusion Cocrystal of Tadalafil with malonic acid was identified using crystal engineering strategy. TDF-MOA cocrystal material was successfully scaled-up to few grams using solution crystallization method and thoroughly characterized. TDF-MOA cocrystal crystallized in chiral space group P , with one molecule of each TDF and MOA in the asymmetric unit. In the TDF-MOA crystal structure, adjacent TDF molecule shows persistent N H O hydrogen bonding interactions as observed in other TDF crystal forms. The catemeric chain of MOA forms hydrogen bonds with lactam carbonyl group of TDF. Interestingly, 21

140 TDF-MOA cocrystal does not show classical melting point rather MOA sublimes from the cocrystal lattice. In the aqueous media, TDF-MOA cocrystal solubility was higher than TDF over a wide range of ph. Solubility-pH dependence of the cocrystal and SA can be calculated using cocrystal Ksp, pka(s) and drug intrinsic solubility. Theoretical models nicely predicted experimental ph solubility and SA results. Supersaturation index of TDF-MOA cocrystal increased by order of magnitude with the increase in ph, reaching as high as at ph6.8. In powder dissolution studies, cocrystal and amorphous form generated a supersaturation (C max /S drug ) of 30 and 10, respectively. The addition of 0.1% w/v HPMC to dissolution media increased supersaturation to 120 by slowing down nucleation and/or precipitation kinetics of the drug from cocrystal and amorphous form. Therefore, the supersaturation during cocrystal dissolution may not reach SA at such a high SA. Thus, SA calculated from the cocrystal and drug solubility equations represents the driving force for nucleation and provides insights on the cocrystal dissolution-precipitation behavior of cocrystal. Ackowledgements. MRS grateful for the financial support from Kempe foundation for postdoctoral grant and The Knut & Alice Wallenberg foundations, Sweden. Professor Sung-Joo Hwang and his team at Yonsei University, South Korea acknowledged for preliminary screening experiments Crystallographic data and structure refinement parameters of TDF-MOA cocrystal. empirical formula = C 25 H 23 N 3 O 8, formula weight = , orthorhombic, space group = P , a = (4) Å, b = 7.782(3) Å, c = (12) Å, V= (16) Å 3, Z = 4, D calc = g cm -3, T= 293(2) K, μ = mm -1, F(000) = 1032, number of reflection used = 1585, number of parameters = 370, GOF on F 2 = 1.118; R1[I>2σ(I)] =0.0777, wr2= , CCDC number =

141 References 1. Childs, S. L.; Kandi, P.; Lingireddy, S. R. Mol. Pharm. 2013, 8, Kobayashi, Y.; Ito, S.; Itai, S.; Yamamoto, K. Int. J. Pharm. 2000, 2, Jung, M. S.; Kim, J. S.; Kim, M. S.; Alhalaweh, A.; Cho, W.; Hwang, S. J.; Velaga, S. P. J. Pharm. Pharmacol. 2010, 11, Shimpi, M. R.; Velaga, S. P.; Shah, F. U.; Antzutkin, O. N. Cryst. Growth Des. 2017, 4, Srivastava, K.; Shimpi, M. R.; Srivastava, A.; Tandon, P.; Sinha, K.; Velaga, S. P. RSC Adv. 2016, 12, Pandey, J.; Prajapati, P.; Shimpi, M. R.; Tandon, P.; Velaga, S. P.; Srivastava, A.; Sinha, K. RSC Adv. 2016, 78, Desiraju, G. Current Opinion in Solid State & Materials Science 1997, 4, Shimpi, M. R.; Childs, S. L.; Bostroem, D.; Velaga, S. P. CrystEngComm 2014, 38, Ahmed, H.; Shimpi, M. R.; Velaga, S. P. Drug Dev. Ind. Pharm. 2017, 1, Jones, W.; Motherwell, S.; Trask, A. V. MRS Bull 2006, 11, Desiraju, G. R. Crystal Engineering. The Design of Organic Solids; Elsevier: Amsterdam, 1989;. 12. Malamatari, M.; Ross, S. A.; Douroumis, D.; Velaga, S. P. Adv. Drug Delivery Rev. 2017, Bolla, G.; Nangia, A. Chem. Commun. (Cambridge, U. K. ) 2016, 54, Good, D. J.; Rodriguez-Hornedo, N. Cryst. Growth Des. 2009, 5, Bethune, S. J.; Huang, N.; Jayasankar, A.; Rodriguez-Hornedo, N. Cryst. Growth Des. 2009, 9, Alhalaweh, A.; Roy, L.; Rodriguez-Hornedo, N.; Velaga, S. P. Mol. Pharmaceutics 2012, 9, Good, D. J.; Rodriguez-Hornedo, N. Cryst. Growth Des. 2010, 3, Huang, N.; Rodriguez-Hornedo, N. J. Pharm. Sci. 2011, 12, Laties, A. M. Drug Saf. 2009, 1, Gresser, U.; Gleiter, C. H. Eur. J. Med. Res. 2002, 10, El-Badry, M.; Haq, N.; Fetih, G.; Shakeel, F. J. Chem. Eng. Data 2014, 3,

142 22. Benet, L. Z.; Broccatelli, F.; Oprea, T. I. Aaps J. 2011, 4, Krupa, A.; Majda, D.; Mozgawa, W.; Szlek, J.; Jachowicz, R. AAPS PharmSciTech 2017, 4, Badr-Eldin, S. M.; Elkheshen, S. A.; Ghorab, M. M. Eur. J. Pharm. Biopharm. 2008, 3, Christfort, J. F.; Plum, J.; Madsen, C. M.; Nielsen, L. H.; Sandau, M.; Andersen, K.; Mullertz, A.; Rades, T. Mol. Pharmaceutics 2017, Ahead of Print. 26. Choi, J.; Lee, S.; Jang, W. S.; Byeon, J. C.; Park, J. Eur. J. Pharm. Sci. 2017, Choi, J.; Park, J. Eur. J. Pharm. Sci. 2017, Krupa, A.; Cantin, O.; Strach, B.; Wyska, E.; Tabor, Z.; Siepmann, J.; Wrobel, A.; Jachowicz, R. Int. J. Pharm. (Amsterdam, Neth. ) 2017, 1-2, Obeidat, W. M.; Sallam, A. A. AAPS PharmSciTech 2014, 2, Miclaus, M. O.; Kacso, I. E.; Martin, F. A.; David, L.; Pop, M. M.; Filip, C.; Filip, X. J. Pharm. Sci. 2015, 11, Weyna, D. R.; Cheney, M. L.; Shan, N.; Hanna, M.; Wojtas, L.; Zaworotko, M. J. CrystEngComm 2012, 7, Wlodarski, K.; Sawicki, W.; Paluch, K. J.; Tajber, L.; Grembecka, M.; Hawelek, L.; Wojnarowska, Z.; Grzybowska, K.; Talik, E.; Paluch, M. Eur. J. Pharm. Sci. 2014, Hubschle, C. B.; Sheldrick, G. M.; Dittrich, B. J. Appl. Crystallogr. 2011, Pt 6, Crystal-Impact, Brandenburg & Putz, Bonn, Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. J. Appl. Crystallogr. 2009, 2, Prasanna Reddy, B.; Amarnadh Reddy, K.; Reddy, M. S. Res. Pharm. Biotechnol. 2010, 1, Cawthray, G. R. J. Chromatogr. A 2003, 1-2, Allen, F. H. Acta Crystallogr. B 2002, Pt 3 Pt 1, Daugan, A.; Grondin, P.; Ruault, C.; Le Monnier de Gouville, A.; Coste, H.; Linget, J. M.; Kirilovsky, J.; Hyafil, F.; Labaudiniere, R. J. Med. Chem. 2003, 21, Aakeroy, C. B.; Beatty, A. M.; Helfrich, B. A. Angew. Chem., Int. Ed. 2001, 17, Adsmond, D. A.; Sinha, A. S.; Khandavilli, U. B. R.; Maguire, A. R.; Lawrence, S. E. Cryst. Growth Des. 2016, 1,

143 42. Cambridge Crystallographic Data Centre, UK Mercury Caires, F. J.; Lima, L. S.; Carvalho, C. T.; Giagio, R. J.; Ionashiro, M. Thermochim. Acta 2010, 1-2, Babu, N. J.; Nangia, A. Cryst. Growth Des. 2011, 7, Basavoju, S.; Bostroem, D.; Velaga, S. P. Cryst. Growth Des. 2006, 12, Lipert, M. P.; Rodriguez-Hornedo, N. Mol. Pharmaceutics 2015, 10, Maheshwari, C.; Andre, V.; Reddy, S.; Roy, L.; Duarte, T.; Rodriguez-Hornedo, N. CrystEngComm 2012, 14, Kuminek, G.; Rodriguez-Hornedo, N.; Siedler, S.; Rocha, H. V.; Cuffini, S. L.; Cardoso, S. G. Chem. Commun. (Camb) 2016, 34, Chen, Y.; Rodriguez-Hornedo, N. Cryst. Growth Des. 2018, Ahead of Print. 25

144 For Table of Contents Use Only Cocrystal of tadalafil: Physicochemical characterization, ph-solubility and supersaturation studies Manishkumar R. Shimpi 1,2, Amani Alhayali 1, Katie L. Cavanagh 3, Naír Rodríguez- Hornedo 3, Sitaram P. Velaga 1, * 1 Pharmaceutical and Biomaterials group, Department of Health Sciences 2 Chemistry of Interfaces, Luleå University of Technology, SE-97187, Luleå, Sweden. 3 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan , United States An important goal of pharmaceutical solid-state development is to increase drug solubility while maintaining a stable form. Cocrystal of tadalafil with malonic acid was identified using crystal engineering strategy. Interestingly, TDF-MOA cocrystal does not show classical melting point. The studies demonstrate maximum achievable supersaturation is dictated by the aqueous solubility of the cocrystal form of the drug. 26

145 Cocrystal of tadalafil: Physicochemical characterization, ph-solubility and supersaturation studies Manishkumar R. Shimpi 1,2, Amani Alhayali 1, Katie L. Cavanagh 3, Naír Rodríguez- Hornedo 3, Sitaram P. Velaga 1, * 1 Pharmaceutical and Biomaterials group, Department of Health Sciences 2 Chemistry of Interfaces, Luleå University of Technology, SE-97187, Luleå, Sweden. 3 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan , United States. Figure S1. Experimental and simulated (obtained from single crystal data) powder X-ray diffraction data for TDF-MOA cocrystal.

146 Figure S2. DSC and TGA profile for Malonic acid. Figure S3. Raman spectrum for TDF shown in black line. The blue line spectrum is obtained from the powder sample isolated when cocrystal was heated till 210 o C.

147 Figure S4. PXRD of Amorphous-TDF prepared by spray drying method. PXRD of powder materials isolated from solution of ph 1 and 3 was compare with PXRD of TDF and TDF-MOA cocrystal. The arrow indicates peaks corresponding to the cocrystal present in powder materials isolated from solution of ph 1 and 3.

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149 Paper IV

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151 Silodosin oral films: Development, physico-mechanical properties and in vitro dissolution studies in simulated saliva Amani Alhayali a*, Parameswara Rao Vuddanda a, Sitaram Velaga a Pharmaceutical and Biomaterial Research Group, Division of Medical Sciences, Department of Health Sciences, Luleå University of Technology, Luleå, Sweden * Corresponding: author: Amani Alhayali amani.al-hayali@ltu.se 1

152 Abstract Sublingual film dosage forms for drugs used for fast symptomatic treatment have promise because they allow a rapid onset of action. The aim of this study was to prepare films of silodosin intended for sublingual administration for the symptomatic treatment of benign prostatic hyperplasia in men. Hydroxypropyl methylcellulose (HPMC) or hydroxypropyl methylcellulose acetate succinate (HPMC-AS) were used as film-forming polymers. The effects of the polymers and the surfactant tocopherol polyethylene glycol succinate (TPGS) on the physico-mechanical properties and dissolution behavior of the films in simulated saliva were investigated. The eight silodosin oral films developed (F1 F8) contained 8mg silodosin per 6 cm 2 film and HPMC or HPMC-AS in drug:polymer ratios of 1:5 or 1:3, while four also contained TPGS (0.5 % w/w). The films were characterized using DSC, TGA, SEM, and PXRD and the mechanical properties were investigated by measuring tensile strength, elongation at break and Young s modulus. The mechanical properties of the films were dependent on the ratio of polymer used. The in vitro dissolution and drug release studies indicated that HPMC-AS films disintegrated more quickly than HPMC films. Silodosin was shown to be dispersed within the polymers. Despite silodosin being submicronized in the HPMC films, the dissolution and drug release rate (time for 80% release) from HPMC films was significantly faster than from HPMC-AS films. TPGS increased the drug release rate to a greater extent with HPMC than with HPMC-AS. The degree of saturation of formulation F4 was >1, which shows potential for improving oral absorption of silodosin. Keywords: Silodosin, sublingual oral films, HPMC, HPMC-AS, TPGS, simulated saliva 2

153 1. Introduction Conventional oral dosage forms, such as tablets or capsules, have challenges related to dissolution, absorption and poor bioavailability for some drugs and can also be associated with problems related to patient compliance {{ Singh, Harmanpreet 2015; Singh, Baljeet 2016; Sharma, Rahul 2014 }}. Alternative drug delivery systems such as oromucosal formulations can be used to overcome these drawbacks {{Kalia, Vani 2016 }}. Oromucosal formulations such as oral films have recently been receiving attention from the pharmaceutical industry because of their unique advantages {{Mashru, RC 2005; Slavkova, Marta 2015}}. For instance, oral films are easy to administer, do not require chewing or intake of water, and disintegrate and dissolve rapidly to release the drug when placed in the oral cavity {{De Caro, V 2012; Campisi, Giuseppina 2010 }}. This has the potential to improve patient compliance, mainly for pediatric and geriatric patients but also for others with mental disorders, dysphagia or emesis {{ Koland, M 2010; Mashru, RC 2005 }}. Drugs formulated as oral films intended for sublingual administration will be directly and rapidly absorbed into the systemic circulation without passing through the gastrointestinal tract (GIT), thereby bypassing first-pass metabolism in the liver {{ Preis, Maren 2013; Hanif, Muhammad 2015}}. The relatively extensive vascularity and high permeability of the sublingual mucosa and membranes can facilitate rapid absorption of the formulated drug and instant bioavailability {{Zhang, Hao 2002; Madhav, NV Satheesh 2009; Mashru, RC 2005; Allam, A. 2016}}. Oral films can be prepared by various methods, including solvent casting, hot-melt extrusion, electrospinning, freeze drying and ink-jet printing {{Repka, Michael A 2003; Boateng, Joshua S 2010; El-Setouhy, Doaa Ahmed 2010; Vuddanda 2018}}. The solvent-casting method is appropriate and feasible for manufacturing on an industrial scale. Usually, oral films contain polymers, plasticizers, drug, surfactants and taste-masking agents (sweeteners and flavors) as required. The film-forming ability and water solubility are the main considerations for selecting the polymer. The polymer to plasticizer ratio is also crucial, as this affects the physico-mechanical stability of the final product, and consideration of this attribute is thus required in the design and development process for oral film formulations. Garsuch and Breitkreutz have reported that the cellulose-derived polymer hydroxypropyl methylcellulose (HPMC) forms films well and has better mechanical properties than other tested excipients{{garsuch 2010}}. In another study, Visser et al. reported the optimal physico-mechanical properties of films prepared with combined HPMC and polyol 3

154 plasticizers {{Visser 2015}}. Oral films can be considered as solid dispersions and the systematic determination of the drug s solubility in the film-forming excipients (polymers/surfactants) using the appropriate techniques is an important development step. Cellulose-based polymers such as HPMC and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) help to stabilize the physical structure of the drug, preventing it from recrystallizing, and increasing its supersaturation during dissolution {{Curatolo 2009}}. It could thus be interesting to evaluate the potential of these polymers in the preparation of oral films intended for sublingual drug absorption. The oral cavity has a smaller surface area and less dissolution medium (i.e. saliva) than the GIT and the polymers could provide a vital boost to the dissolution and subsequent absorption of drugs administered as a film {{Patel, Viralkumar F 2011}}. Furthermore, the film-forming properties and potential of HPMC and HPMC-AS in film drug delivery have been extensively investigated. Surfactants are also an important excipient in the preparation of drug-carrying films, particularly for poorly water-soluble drugs. It is well known that surfactants can facilitate wettability and enhance the dissolution rate of poorly water-soluble drugs. For example, Vuddanda et al. have reported that the addition of surfactant enhanced the dissolution of tadalafil nanocrystal-loaded oral films {{Vuddanda 2017}}. Benign prostatic hyperplasia (BPH) is an enlargement of the prostate gland caused by the proliferation of prostatic stromal cells. It s an important cause of lower urinary tract symptoms in men such as frequency, urgency, nocturia, and hesitancy. BPH is a common problem among men after the age of 40 years {{Kapoor, Anil 2012; Rossi, M. 2010}}. Silodosin is a selective α 1A adrenoceptor blocker that is a safe, effective treatment for the relief of both voiding and storage symptoms in patients with BPH {{Chapple, Christopher R 2011}}. Silodosin is a white to pale yellowish-white powder. The partition coefficient [LogP (octanol/water)] of silodosin is 2.87, with dissociation constants pka1 of 8.53 and pka2 of According to the US Food and Drug Administration (FDA) label, silodosin is very slightly soluble in water. The oral bioavailability of silodosin administered as an oral capsule is nearly 32% and the onset of action (maximum urine flow rate) after the first dose occurs in 2-6 hours {{Montorsi 2010}}. Silodosin undergoes extensive metabolism involving glucuronidation in the liver {{Matsubara,Y. 2006; Rossi,M. 2010}}. Because of these issues, an oral film formulation, intended for sublingual administration, could be promising for silodosin. This would facilitate rapid absorption, provide a faster onset 4

155 of action, and have potential for faster relief of symptoms than an oral capsule. In addition, patient compliance could be improved, as some patients find films easier to take than capsules. Also, the sublingual film formulation of silodosin avoids first-pass metabolism in the liver and thus has potential to improve systemic bioavailability. Films can also provide a supersaturated concentration of the drug in the saliva, which can improve the oral mucosal absorption of poorly soluble drugs. To our knowledge this is the first study of the preparation of a sublingual film dosage form for silodosin. The main aim of this study was to prepare oral film formulations of silodosin intended for sublingual administration. The effects of added polymer and surfactant on the physicomechanical and dissolution properties of the films were investigated. The dissolution and supersaturation properties of the films were investigated in small volumes of simulated saliva to realistically mimic the oral cavity. HPMC and HPMC-AS were investigated as filmforming polymer excipients in drug:polymer ratios of 1:3 w/w and 1:5 w/w and tocopherol polyethylene glycol 1000 succinate (vitamin E; TPGS) was included as a surfactant. The silodosin dose in each film was 8 mg (the daily recommended dose of silodosin according to the FDA) and the films were 6 cm 2 (2 cm 3 cm) in area. 2. Materials and methods 2.1. Materials Silodosin was obtained from Ultra Medica (Damascus, Syria). Hydroxypropyl methylcellulose 6cp (Pharmacoat 606) and Hydroxypropyl methylcellulose acetate succinate 3cp (Hypromellose Acetate Succinate NF) grade AS-HF were obtained from Shin-Etsu (Tokyo, Japan). D-α-tocopherol polyethylene glycol 1000 succinate (vitamin E; TPGS) NF grade was received as a gift sample from BASF Chemicals (Ludwigshafen, Germany). Glycerol and acesulfame potassium were purchased from VWR chemicals (Stockholm, Sweden). The water used in all experiments was ultrapure, freshly collected from a Millipore water system (Milli Q, Sweden). The other materials were purchased locally and used as purchased. 5

156 2.2. Methods Drug-polymer miscibility Silodosin and the polymers (HPMC or HPMC-AS) were mixed in three ratios (1:1, 1:3 and 1:5 w/w) to a total weight of 400 mg. The solid dispersions were prepared by the film-casting method. The drug and polymers were dissolved in ethanol:water (1:1 v/v) to a volume of 5 ml. This solution was cast onto a fluoropolymer-coated polyester sheet (Scotchpak release liner 1022, 3M Inc., USA) and dried at 70 C in an oven for 1 hour. The dried samples were then analyzed using differential scanning calorimetry (DSC; TA Instruments Q 1000, USA) to investigate the miscibility of the drug and the polymer Preparation of the casting gel The casting gel consisted of HPMC or HPMC-AS (65%), glycerol and propylene glycol (7%), and sweetener (2%), with ethanol and water as vehicle, relative to the total weight of the solid base. All weights are w/w ratios. Silodosin was dissolved in ethanol (12-13%) and the remaining excipients were dissolved in water (87-88%). HPMC or HPMC-AS was gradually added to this solution under constant magnetic stirring (800 rpm) at ambient temperature (21 ± 1 C) until a homogeneous gel was obtained. This casting gel was kept for 6 12 h to remove the air bubbles. Table 1 shows the overall composition of the prepared films Preparation of drug-loaded films The casting gel (10g) was cast onto a fluoropolymer-coated polyester sheet (Scotchpak release liner 1022, 3 MInc., USA) using an automated film applicator equipped with a coating knife (Coatmaster 510, Erichsen, Sweden). The silodosin dose of 8 mg was loaded into each 6 cm 2 film by fixing the wet film thickness at 750μm with a casting speed of 5 mm/s, estimated from the formula developed by Preis et al, {{ Preis 2012}}. The cast films were dried in a convective hot-air oven (Binder, Sweden) at 60 C for min. After drying, the films were carefully peeled off, sealed in plastic (polythene) zip pouches, and stored in a desiccator (23 C/40% RH) until further characterization. 6

157 Dry film thickness The thickness of the films was measured using a Vernier caliper (Cokraft, Digital caliper, Sweden). The thickness of each film was measured at the four sides and at the middle point. The average and standard deviation were calculated Differential scanning calorimetry (DSC) Thermograms of the drug, the drug/polymer blends and the film samples were recorded using a differential scanning calorimeter (TA Instruments Q 1000, USA) equipped with a refrigerated cooling system. Each sample (1 3 mg) was placed in a standard aluminum pan and sealed. The samples were heated at a rate of 10 C/min from 25 to 120 C under nitrogen purge (50 ml/min). The calorimeter was previously calibrated for temperature and heat capacities using indium and sapphire. The results were analyzed using Universal analysis software (TA instruments, USA) Thermo-gravimetric analysis (TGA) A TGA instrument (TA instruments, USA) was used for thermo-gravimetric analysis and to determine the moisture content of the films. Approximately 5 8 mg of film (small pieces) were placed in a platinum pan and heated from 25 to 150 C at a constant heating rate (10 C/min) under nitrogen flow (50 ml/min). The results were analyzed using Universal analysis software (TA instruments, USA) Powder X-ray diffraction (PXRD) PXRD patterns from pure crystalline silodosin and the film samples were collected using an Empyrean PXRD instrument (PANalytical, Almelo, The Netherlands) equipped with a PIXel3D detector and monochromatic Cu Kα X-Ray radiation (λ = Å). The voltage and current were 45 kv and 40 ma. The samples (3 3 cm 2 films) were placed on a silicone (zero background) plate which was fitted into the metal sample holder. The samples were scanned (diffraction angle 2θ) between 5 and 40, increasing at a step size of All patterns were obtained at 25 ± 1 C. The data were processed using HighScore Plus software (PANalytical, The Netherlands). 7

158 Mechanical properties The dynamic mechanical strength was tested using a hybrid rheometer in DMA mode (DHR2, TA Instruments, Sweden). Briefly, samples of cast films were cut into rectangular strips of 1 5 cm 2 and 1 cm at each end was held between clamps; thus, the effective testing area was 1 3 cm 2. The upper clamp was then used to stretch the film upwards at a constant linear rate of 0.1 mm/min until the film ruptured. Stress and strain were computed by Trios software. The tensile strength (TS) and the elongation at break (EB) were obtained from the peak stress and the maximum strain, respectively, in the stress vs strain plot. Tensile tests are commonly used to determine the robustness of film preparations. The TS is the maximum force applied to the film sample at the breaking point and the EB is the length of the film during the pulling process. In addition, Young s modulus or the elastic modulus (EM) describes the influence of the strain and its force at this strain on the film area. The EM was obtained from the initial elastic deformation region in the stress vs strain plot {{Radebaugh, Galen W 1988 }}. Tensile strength (TS) = Peak stress Cross sectional area of the film (1) Elongation at break (EB) = Increase in length at break Initial film length 100 (2) Scanning electron microscopy (SEM) A Merlin scanning electron microscope (Zeiss, Oberkochen, Germany) equipped with X-Max 50 mm 2 X-ray detectors (Oxford Instruments, Abingdon, UK) was used to examine the morphology of the films. The instrument voltage was 20 kv and the current was 1 na. The selected film samples were coated with tungsten before the examination to increase the conductivity of the electron beam HPLC analytical method The drug content of the films was analyzed in a high performance liquid chromatography (HPLC) system (Agilent systems Inc., USA) with an auto sampler. The sample separation was performed on an Agilent Eclipse-plus C18 column (5 μm, 250 mm 4.6 mm) with a mobile phase of 25 mm potassium-dihydrogen phosphate buffer (ph 7.0) and acetonitrile 40:60 (v/v) at 25 C. The flow rate was 1 ml/min and the determination wavelength was 269 nm {{Runja 2012}}. 8

159 Drug content The films (1 1 cm 2 ) were placed in a volumetric flask containing 10 ml of water and ethanol (1:1 v/v) and kept under magnetic stirring at 100 rpm for 1 h. The obtained solution was filtered through a syringe filter (0.2 μm) and the filtrate was analyzed for drug content using HPLC Disintegration time Samples (1 1 cm 2 ) were placed in a Petri dish containing 2 ml of water and shaken at 60 rpm using an orbital shaker water bath at 37 ± 1 C. The disintegration time of the films was evaluated using a modified Petri dish method {{Garsuch, Verena 2010}}. The time to disintegration or disruption was measured with a stopwatch Solubility studies The solubility of silodosin was determined in simulated saliva containing pre-dissolved HPMC or HPMC-AS with or without TPGS in concentrations similar to those used in the film formulations. The simulated saliva was prepared using compositions mentioned by Hobbs and David {{Hobbs, David 2013}}. An excess of drug was added to conical flasks containing 10 ml of saliva and the other polymer-surfactant excipients. The flasks were tightly closed and placed in a shaker water bath at 37 C. After 48 h, the separated aliquots were filtered through a 0.45 μm filter, diluted appropriately and analyzed using HPLC. Each experiment was performed in triplicate (n = 3) and the results were reported as means ± standard deviation In vitro dissolution in simulated saliva Non-sink dissolution studies were carried out in simulated saliva at ph 6.8. The films (F1-F8; 2 3 cm) were carefully dropped into 10 ml dissolution medium under continuous orbital shaking (60 rpm) at 37 C. Samples of the medium were then withdrawn at different times, filtered through syringe filters (0.45 μm) and analyzed by HPLC. The degree of supersaturation (DS) was calculated from the drug concentrations at different times during dissolution of the films (F1-F8) and the drug concentrations at equilibrium. DS calculations for the formulated drug were based on equation 3: DSt = Ct Ceq (3) 9

160 Where: C t is the drug concentration at time t and C eq is the equilibrium solubility of the drug in the test medium. The obtained values of DS for F1-F8 were plotted versus time Statistical analysis One-way ANOVA and Tukey's post hoc multiple comparisons were used to determine statistically significant differences (p < 0.05). All results were expressed as averages plus standard deviation (n=3). Mechanical properties were investigated using n = Results and Discussion 3.1. Thermal and solid-state properties DSC thermograms of pure and amorphous silodosin showed a sharp endothermic event at ~106 C, confirming its crystalline state. This is in line with results from a previous report by Singh and Aniruddh {{Singh, Aniruddh 2016 }}. To choose the best drug:polymer ratio in the miscible system for forming films, different polymer ratios were investigated (Fig. 1). The melting peak of the crystalline silodosin (~106 C) was absent in the thermograms of all the silodosin:polymer systems under investigation, indicating that all the systems were miscible and formed solid dispersions (Fig. 1). These results confirmed the formation of miscible dispersions with the studied ratios. Thus, the silodosin:polymer ratios 1:3 and 1:5 were chosen for film formulations with either HPMC or HPMC-AS, with or without the surfactant (TPGS) (Table 1). The polymer content was necessary for casting the films and preventing drug recrystallization during storage. The thermal properties of pure silodosin and the formulated films were assessed as shown in Fig. 2. The melting peak of the crystalline form was absent from the DSC thermograms of all the prepared films (F1-F8), which was interpreted as molecular dispersion of the drug in the polymer (Fig. 2). ElMeshad and El Hagrasy have also reported the formation of a uniform dispersion with complete molecular miscibility of different film components in films prepared with HPMC {{ElMeshad and El Hagrasy, 2011}}. A similar finding of solid dispersions of amorphous nifedipine in HPMC-AS was reported by Curatolo et al. {{Curatolo 2009}}. A glass transition temperature (T g ) of 92.9 C for HPMC-based films and C for HPMC- AS-based films was observed (data not shown). The T g values for the films were higher than the temperature in the buccal cavity and also the environmental temperature, which is important for keeping the product stable during storage (from the perspective of the product 10

161 logistics from manufacturing to consumption){{vila, Marta MDC 2014; Garsuch, Verena 2010}}. TGA was performed to determine the moisture content in the prepared films, as shown in Fig. 3. Weight loss was between 0.9 and 1.6 % for the films prepared with the HPMC polymer (F1-F4) and between 1.6 and 1.9 % for the films prepared with the HPMC-AS polymer. These results suggest that the film formulations retained some moisture, possibly as a result of the inherent water sorption properties of both polymers, as suggested by the moisture content in pure HPMC and HPMC-AS (data not shown). A moisture content of about 2% is essential for flexibility of the films and this was found not to affect the physical stability of the solid dispersions, as confirmed by DSC and PXRD analysis (Fig.s 2 and 4). However, it was observed that the films prepared with HPMC-AS, but not the HPMC films, were tacky. The solid state of pure silodosin and the film formulations were analyzed using PXRD (Fig. 4). The PXRD pattern of pure silodosin showed sharp, characteristic peaks at 2θ angles of approximately 10, 11 and 20, illustrating the crystalline nature of the starting material. This is in agreement with the PXRD patterns reported by Singh and Aniruddh {{Singh, Aniruddh 2016}}. These characteristic peaks disappeared and a hollow shape was observed in the PXRD patterns for all the HPMC-AS film formulations (F6-F8), which confirms the formation of a solid dispersion with the drug uniformly dispersed in the polymer. However, in the case of HPMC films (F1-F4), very low intensity diffraction peaks were observed, which suggests that the drug may not have been fully dispersed or may have existed as submicron particles in the polymer matrix Film Morphology As shown in Fig. 5, the surface morphology of the pure silodosin and representative film formulations were characterized using SEM. The SEM micrographs of the pure silodosin show particles with irregular morphology. The HPMC-based films (F2 and F4), but not the HPMC-AS-based films (F6 and F8), had submicron silodosin particles and tiny pores (Fig 5). This observation was in agreement with the PXRD results. The submicron particles of silodosin may have formed at the point of supersaturation in HPMC during preparation of the casting gel. Alonzo et al. reported the formation of submicron particles in HPMC-based amorphous solid dispersions that were related to the degree of supersaturation {{Alonzo, David E 2011}}. This also suggests that more uniform dispersion was obtained in HPMC-AS 11

162 as a result of the higher solubility of silodosin in HPMC-AS than in HPMC {{Tanno, Fumie 2004; Korhonen, Kristiina 2016;}} Mechanical properties The mechanical and tensile properties of the thin films were measured under ambient conditions. The EM, TS and EB of the silodosin films are shown in Table 2. TS was greater in HPMC-based films than in HPMC-AS-based films, while the EB was longer in HPMC-ASbased films. Decreasing the content of the polymers reduced the TS of the films. TS and EM values decreased by 53% and 54%, respectively, in HPMC samples with a drug:polymer ratio of 1:3 compared with a ratio of 1:5. The effect of less polymer was even more profound with HPMC-AS; TS and EM decreased by 73% and 86%, respectively. The EB was also affected by decreasing the proportion of polymer, increasing in HPMC- and HPMC-AS-based films with a drug:polymer ratio of 1:3 by 24% and 67%, respectively, compared with a ratio of 1:5. Interestingly, the addition of TPGS had no significant effect on the EM or TS, whereas a mixed result was seen for the EB. When TPGS was added to the formulations containing the higher proportion of polymer (drug:polymer ratio 1:5), the EB was increased for HMPC films and decreased for HMPC-AS films; however, there was no change in EB when TPGS was added to the films containing the lower proportion of polymer (ratio 1:3). Further studies are needed to determine the cause of this difference. The ability to sustain TS is important for packaging and handling the thin films. The obtained TS values for our HPMC films are similar to those in the literature {{Visser 2015}}. Our TS values are also comparable to those of the commercial products examined by Pries et al. {{Preis, Maren 2014}} with respect to maximum force, displacement and elongation, particularly in comparison with PediaLax and Triaminic Cold & Cough products. Thus, it can be inferred that our formulated batches would be suitable for commercialization. In fact, the elongation properties of the HPMC-AS films considerably exceeded those of the commercial products; hence films made to this formulation would possess superior toughness. Tensile properties are a function of the molecular structure. HPMC-AS has fewer polar substituents, which are known to improve elongation, but also to decrease TS {{Brady, J. 2017}}). In contrast, while TS was higher with HPMC, the films were not as tough, i.e. they were hard and brittle {{Huang, Yuan 2005}}. 12

163 3.4. Film thickness, drug content uniformity The average thickness of the HPMC-based films (F1-F4) ranged from 106.7±0.0 to 116.7±0.0 mm while that of the HPMC-AS-based films (F6-F8) ranged from 86.7±0.0 to 106.7±0.0 mm. Despite the constant monitoring of processing parameters for all film formulations, there were differences in the thicknesses of the two types of film. These differences were attributed to variations in the density of the casting gels for the two polymers as a result of their intrinsic polymer properties. Differences in film thickness between films with the same polymer-based formulations may have resulted from the different solid contents and drug:polymer ratios among the formulations. Evaluation of the drug content in the films showed values ranging from 96 ± 28 to 117±28.9 %, as shown in Table 1. These results indicated uniform distribution of drug in the films, within the acceptable limits for standard oral solid dosage forms, according to the USP {{Liew 2012; Nagy 2010}} Disintegration time: The disintegration times are shown in Table 2. The fastest disintegration was observed with the HPMC-AS-based film F5 (drug:polymer ratio 1:5) which disintegrated in 15.3±1.2 sec. Disintegration was slower for the film based on the HPMC polymer with the same drug:polymer ratio (35.3±0.6 sec). This effect may have been related to the properties of the polymer, i.e. wettability and surface tension. Thus, HPMC-AS films disintegrated more quickly than HPMC films {{Miller-Chou, Beth A 2003; Tanno, Fumie 2004}}. Addition of the surfactant (TPGS) to the film formulation had an additive effect on the disintegration time (Table 2). These results were in agreement to those of Vuddanda et al, who found an additive effect of the surfactant TPGS on speed of disintegration {{Vuddanda 2017}} In vitro dissolution in simulated saliva The solubility of pure silodosin in simulated saliva was 0.46 mg/ml. The solubility of silodosin in simulated saliva with additional dissolved HPMC (with and without TPGS) ranged from 0.48 to 0.51 mg/ml at equilibrium after 48 hours, and with additional dissolved HPMC-AS (with and without TPGS) ranged from 0.94 to 1.1 mg/ml. The dissolution results for the films formulated using HPMC and HPMC-AS are shown in Fig.s 6a and 6b, respectively. In the HPMC-based films, 80% of the drug was released in 10 minutes from F2 (with a drug:polymer ratio of 1:3), while dissolution was poorer for F1 (1:5 drug:polymer ratio), with 80% release after 25 minutes. This may be because the increase in polymer 13

164 concentration led to the formation of a gel-like state which decreased water uptake and retarded drug release, as reported by Singh and Harmanpreet { Singh, Harmanpreet 2017;}}. Alhayali et al. have previously reported that, in some cases of solid drug dispersions, the drug concentrations do not change with different drug:polymer ratios {{Alhayali, Amani 2016}}. Addition of TPGS also improved the drug release noticeably. Thus, films containing TPGS dissolved faster than TPGS-free films. Other workers have mentioned this effect of the surfactant TPGS in terms of its ability to improve the solubility, dissolution rate and bioavailability of some drugs formulated as oral films {{Guo 2013; Vuddanda 2017}}. In HPMC-AS-based films, about 80% of the drug was released in around 10 minutes (F5 and F6), with no significant differences in dissolution rate between the two ratios (1:3 and 1:5). Addition of the surfactant TPGS to the HPMC-AS-based films negatively affected the dissolution rate for both ratios. Therefore, this study has demonstrated that HPMC works well as a film-forming polymer when combined with TPGS. In a case study by Garsuch and Verena {{Garsuch, Verena 2010 }}, HPMC was the most suitable film-forming material of the excipients tested, providing faster dissolution and easier-to-handle films. Interestingly, the submicron particles appear not to have affected the faster drug release from HPMC films. Further, it was observed that the dissolution behavior of HPMC-AS (TPGS-free) films was similar to that of HPMC films containing amorphous solid dispersions (Fig. 6). The dissolution studies were conducted in simulated saliva (ph 6.8) under non-sink conditions, mimicking the conditions of the oral cavity. These studies suggested that both polymers are capable of increasing and prolonging the supersaturation of the drug and helping to prevent the tendency of the drug to precipitate and recrystallize during dissolution. This could be attributed to the existence of the drug in the amorphous state and molecularly dispersed in the polymer matrix. This was also evident from the thermal, solid-state and morphological results. It has been reported that cellulose-derived polymers such as HPMC, HPMC-AS and hypromellose phthalate (HPMCP) are superior for preparing solid, amorphous dispersions, particularly with poorly water-soluble drugs, compared to other polymers. The bulky structure of the polymer network and worse solubility properties (compared to highly water-soluble polymers) facilitate the reduction of drug mobility in the polymer matrix and prevent the drug from recrystallizing as normally induced by supersaturation during dissolution (Li and Taylor, 2018). This consequently improves the product s physical stability and in vitro drug release 14

165 performance. In general, our dissolution results revealed that HPMC and HPMC-AS would be useful for preparing films intended for oral cavity absorption, which is more complex than GIT absorption. The DS values for silodosin formulated as an oral film are presented in Fig. 7. The highest DS was obtained for HPMC-based formulations at early time points. F4, in particular, had a DS value of > 1 (supersaturated) before 5 minutes, which was earlier than the other films formulated using the HPMC polymer (F1-F3), as shown in Fig. 7. In the dissolution results, 80% of the drug was released from film formulation F4 during the first 10 minutes (Fig. 6). This effect could be attributed to the synergistic effect of the surfactant (TPGS) and the polymer (HPMC), resulting in improved solubility and maintained supersaturation {{Kim, M.S.2014; Prasad, Dev 2014; Gao, Ping 2004}}. Therefore, film formulation F4 appears to have potential for the development of a silodosin film formulation with improved performance and improved oral sublingual absorption as a result of the high degree of supersaturation solubility {{Brouwers, J. 2009; Yang, Meiyan 2016}}. 4. Conclusions Oral films of silodosin intended for the sublingual administration route were prepared successfully for the first time. DSC studies confirmed the existence of silodosin in an amorphous form in films formulated with HPMC or HPMC-AS. SEM and PXRD studies revealed the presence of submicron particles of the drug in HPMC-based films, while the drug remained fully amorphous in HPMC-AS films. The mechanical properties of HPMC films were better than those of HPMC-AS films with respect to stability during patient handling and packing. The dissolution behavior of HPMC was similar to that of HPMC-AS when the surfactant TPGS was added (0.5 % w/w) to the HPMC film formulation. TPGS at the tested concentration had no effect on the dissolution of the drug in HPMC-AS-based formulations. Silodosin formulation F4 had a DS >1, which could be promising for improving its oral absorption. Further studies are required to evaluate the dissolution of the film in human saliva, and to investigate the permeability of the oral cavity to the drug and the drug absorption characteristics. Film palatability and crystallization during storage (stability) also require investigation. 15

166 Acknowledgments A. Alhayali is grateful for the financial support from the Iraqi Ministry of Higher Education and Scientific Research (MOHESR) for PhD grant. M. Elbadawi acknowledged for his assistance and advice about mechanical properties section. 16

167 Table1. Silodosin oral films. Summary of the drug:polymer ratios for the eight developed films, and the mean thickness, speed of disintegration and drug content Formulation Drug:polymer Thickness Disintegration time Drug code ratio (mm) (seconds) content (mg) F1 1:5 (Silodosin:HPMC) 106.7± ±0.6 96±28 F2 1:3 (Silodosin:HPMC) 110± ± ±5.8 F3* 1:5 (Silodosin:HPMC) 116.7± ±0.6 96±1.1 F4* 1:3 (Silodosin:HPMC) 113± ± ±26.1 F5 1:5 (Silodosin:HPMC-AS) 106.7± ± ±6.5 F6 1:3 (Silodosin:HPMC-AS) 86.7± ± ±18.4 F7* 1:5 (Silodosin:HPMC-AS) 90± ± ±28.9 F8* 1:3 (Silodosin:HPMC-AS) 86.7± ±0.6 99±19.8 Mean ± Standard deviation (n 3). HPMC = hydroxypropyl methylcellulose; HPMC-AS = hydroxypropyl methylcellulose acetate succinate. *Four films (F3, F4, F7 and F8) also contained the surfactant tocopherol polyethylene glycol succinate. 17

168 Table 2: Mechanical properties of silodosin films Film (F) Young s Modulus (Mpa) Max. Tensile Strength Elongation at Break (%) (Mpa) 1 490± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±4.33 Means ± Standard deviation (n 5) 18

169 Figure 1: Differential scanning calorimetry results for drug-polymer miscibility determination. 19

170 Figure 2: Differential scanning calorimetry results for the silodosin film formulations. (a) HPMC-based films, F1-F4; and (b) HPMC-AS-based films, F5-F8. 20

171 Figure 3: Thermogravimetric analysis results for the formulated silodosin films showing moisture content. 21

172 Figure 4: X-ray diffraction patterns for pure crystalline silodosin and the developed films (F1-F8). 22

173 Figure 5: Scanning electron microscopy micrographs for the formulated films and the pure drug (silodosin). The bar represents 100 μm for F2, F4, F6, and F8, and 20 μm for pure silodosin. 23

174 Figure 6: Film dissolution in simulated saliva for (a) the HPMC-based films and (b) the HPMC-AS-based films. n=3 ± Standard deviation 24

175 Figure 7. Degree of supersaturation as a result of formulating the drug silodosin in film formulations containing HPMC (F1-F4) or HPMC-AS (F5-F8). n= 3± Standard deviation 25

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184 Amani I. Y. Alhayali was born in Mosul, Iraq, on November 15, 1976, to Suhailah Yousif and Ibraheem Y. Alhayali. She graduated from Mosul University, Iraq with a Master degree in Clinical Pharmacy in The dissertation work was carried out at the Department of Health Sciences, Luleå University of Technology with Professor Sitaram Velaga.