To my family

I am my own experiment,I am my own work of art

Madonna

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Bramer, T., Dew, N., Edsman, K. (2006) Catanionic Mixtures

Involving a Drug: A Rather general Concept That Can be Utilized for Prolonged Drug Release from Gels. Journal of Pharmaceutical Science, 4(95):769–780

II Dew, N., Bramer, T., Edsman, K. (2008) Catanionic aggregates formed from lauric and capric acids enable prolonged drug release from gels. Journal of Interface and Colloid Science, 323:386-394

III Dew, N., Edwards, K., Edsman, K. (2009) Gel formation in

systems composed of drug containing catanionic vesicles and oppositely charged hydrophobically modified polymer. Colloids and Surfaces B: Biointerfaces, 70:187-197

IV Dew, N., Edsman, K., Björk, E. Novel Gel Formulations with

Catanionic Aggregates Enable Prolonged Drug Release and Reduced Skin Permeation (2010). Submitted

V Dew, N., Edwards, K., Eriksson, J., Edsman, K., Björk, E. Gel

formulations containing catanionic vesicles composed of alprenolol and SDS: effects of drug release on aggregate structure (2010). Submitted

Reprints were made with permission from the respective publisher.

My contribution to the following papers was as follows:

I – III and V: I was involved in all parts of the research, analysis and writing of the manuscripts, except for the cryo-TEM measurements. IV: I was involved in all parts of the research, analysis and writing of the manuscript.

Additional papers: Bramer, T., Dew, N., Edsman, K. Pharmaceutical Applications for Catanionic Mixtures (2007). Journal of Pharmacy and Pharmacology, 59:1319-1334

Contents

1. Introduction ............................................................................................... 11 1.1 The gel as a pharmaceutical dosage form........................................... 11 1.2 Advantages of gel formulations ......................................................... 12 1.3 Limitations of gel formulations .......................................................... 12

2. Aims of the thesis...................................................................................... 14

3. Catanionic aggregates ............................................................................... 15 3.1 What are catanionic aggregates? ........................................................ 15 3.2 Pharmaceutically interesting aggregates ............................................ 16 3.3 Aggregate - polymer interactions and gel formation .......................... 19 3.4 Topical drug administration ............................................................... 19

3.4.1 Dermal drug administration ........................................................ 20

4. Experimental section ................................................................................. 23 4.1 Materials ............................................................................................. 23 4.2 Phase studies ...................................................................................... 25

4.2.1 Catanionic studies ....................................................................... 25 4.2.2 Vesicle-polymer gel formation studies ....................................... 25

4.3 Gel preparation ................................................................................... 26 4.3.1 Agar gels ..................................................................................... 26 4.3.2 Carbopol gels .............................................................................. 26 4.3.3 Physical gels ............................................................................... 26

4.4 Rheological investigations ................................................................. 26 4.5 Drug release studies ........................................................................... 27 4.6 Skin preparation ................................................................................. 29 4.7 Drug penetration studies ..................................................................... 29 4.8 HPLC analysis .................................................................................... 30 4.9 Skin morphology ................................................................................ 31 4.10 Statistical analysis ............................................................................ 31

5. Results and discussion .............................................................................. 32 5.1 Can drugs be used to form catanionic aggregates? ............................ 32 5.2 Do catanionic aggregates prolong the drug release from gels? .......... 34 5.3 Can the oppositely charged surfactant be less toxic? ......................... 36 5.4 Can catanionic vesicles function as cross-linkers in gels? ................. 39 5.5 Does the gel type affect the drug release? .......................................... 41

5.6 How does drug release affect aggregate structure? ............................ 43 5.7 Could catanionic aggregates in gels be used for topical administration? ......................................................................................... 46

6. Concluding remarks .................................................................................. 49

7. Populärvetenskaplig sammanfattning: Fördröjd läkemedelsfrisättning från geler med katanjoniska aggregat ........................................................... 51

8. Acknowledgements ................................................................................... 53

9. References ................................................................................................. 56

Abbreviations

ANOVA BAC C940 C1342 CAC CI CMC Cryo-TEM CTAB

D DoTAB G´ G´´ HPLC LPC PAA SC s.d. SDS TTAB USP

Analysis of variance Benzalconium chloride Carbopol 940, cross-linked PAA Carbopol 1342, cros-linked PAA with lipophilic modification Critical aggregation concentration Confidence interval Critical micelle concentration Cryogenic transmission electron microscopy Cetyl trimetylammonium bromide The phase angle Apparent diffusion coefficient Dodecyltrimethylammonium bromide The elastic (storage) modulus The viscous (loss) modulus High performance liquid chormatography Lauyl pyridinium chloride Poly(acrylic acid) Stratum corneum Standard deviation Sodium lauryl suphate Trimethyl ammonium bromide United States Pharmacopeia

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1. Introduction

1.1 The gel as a pharmaceutical dosage form Gels are soft, solid-like, or semisolid in nature, and consist of at least two components, one of these being a liquid that is present in a substantial quantity. Although the liquid content is high, on a time scale of seconds, a gel should not flow under the influence of its own weight. Thomas Graham coined the term “gel” in 1864 [1] and it has been in use ever since then. There have been several attempts to define what gels are and, in 1926, Dorothy Jordan Lloyd made the famous statement that “The colloidal condition, the gel, is one which is easier to recognize than to define” [2], probably unaware that her words would be quoted and remain true for a long time to come. There is, as yet, no definition of gels that is applicable to all conditions and includes all gels; therefore, in this thesis a rheological definition is used. The gel characteristics are described using the dynamic mechanical properties, the elastic modulus (G´) and the viscous modulus (G´´). According to the rheological definition the elastic properties are considerably larger than the viscous ones and the former are independent of frequency [3-4].

The term “hydrogel” is widely used and is defined as a gel that is composed of three-dimensional, hydrophilic, polymeric networks and large amounts of water [5-8]. There are, however, other gels that are used for pharmaceutical dosage, such as the xerogels, which mostly contain air, and the organogels, which contain oil. Nevertheless, the most common gel used in pharmaceutical applications is the hydrogel and the terms “gel” and “hydrogel” will be used synonymously henceforth.

Gels consist of three-dimensional networks of cross-linked polymer chains and these polymers can be linked either through covalent bonds, i.e. by chemical cross-linking, or non-covalent bonds, i.e. by physical cross-linking. Covalent cross-linking may be performed prior to or after polymerization. Non-covalent cross-linking occurs after polymerization and may occur as a result of either hydrophobic or electrostatic attractions, or both.

Gel formulations are popular pharmaceutical dosage forms and as a result of which, many administrational routes have been suggested for gels; see, for example, the review by Peppas [6]. As the dosage form is applicable to all topical membranes and as they can be used for both systemical and local

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administration, there are many potential uses for gels: on buccal membranes [9-11], on the skin [12-15], on vaginal mucosa [16-18] and in the nasal cavity [19-21]. Gels can also be used subcutaneously [22-25] and for administration to the stomach [26-29] or colon [30-32]. A gel formulation that offers a prolonged drug release could possibly be used as an alternative to oil based or suspension formulations or implants for subcutaneous use.

1.2 Advantages of gel formulations The contact time of a gel formulation on skin or mucosa is typically much longer than that of an aqueous solution owing to the more favorable adhesive [33-35] and/or rheological [6, 36-38] properties. An extended contact time at the site of administration might increase the absorption of the drug substance, opening up the possibility of: giving a lower dose of the administered drug, using longer dosing intervals, or both. A dosage form with favorable properties might render high patient compliance. There are several circumstances under which a gel might be a suitable dosage form. Compared to ointments and creams the oil content is usually very low in a gel, which makes the cosmetic properties favorable in many places. Despite the fact that the occlusive effects provided by an ointment are abolished when gels are used, there are still many gel formulations that offer sufficient percutaneous absorption to allow a systemic effect to be obtained from the drug substance. In particular, in ocular administration, gel formulations are much more readily tolerated by patients than ointments owing to the more favorable cosmetic properties. Ocular administration is also a typical example of a situation where a prolonged drug release would increase the efficiency of the gel dosage form. An administered dose of a solution of the type of an eyedrop is quickly eliminated due to the nasolacrimal drainage and the bioavailability is reduced additionally as a result of the cornea’s poor drug permeability. By using gel formulations with appropriate rheological properties, the bioavailability in the eye can be increased from a typical 1% [39] by several factors of ten for a number of pharmaceutical substances [40-41]. Gel formulations have also been used to increase the bioavailability of drug substances administered to the nasal cavity [42-44].

1.3 Limitations of gel formulations When applied to mucosa or skin, the mucoadhesive and rheological properties of a gel may increase the residence time on the tissue. The advantages of this can only be utilized if the drug substance is released from the gel throughout the contact time. Given that the gel formulations under

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consideration are mostly comprised of water, the diffusion rate of free, small, molecules in these gels is similar to that in pure water. Gels are therefore quickly emptied of the drug, resulting in no obvious benefits in the form of an extended residence time at the site of application. To prolong the release of drug substances for gel formulations many strategies have been suggested; the drug can be formulated as solid particles in the gel, rendering a suspension [45]; the drug substance may interact with the gel polymer [46] or the drug can be distributed to liposomes [47] or micelles [48], which are incorporated in the gel.

In recent years the possibility of obtaining prolonged drug release from catanionic aggregates, composed of surface-active drug molecules and oppositely charged surfactants and then incorporating these in gels has been explored [49-50].

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2. Aims of the thesis

The aim of this thesis was to explore catanionic aggregates, composed of drug substances and an oppositely charged surfactants, contained in gels as a novel method to achieve a prolonged drug release from gel formulations. The specific goals were:

To investigate how common the formation of catanionic

aggregates was when using drug substances instead of conventional surfactants.

To investigate if drug-containing catanionic aggregates can be used to prolong the drug release from gels.

To investigate if surfactants with a natural origin, possibly with a lower toxicity than synthetic surfactants, can be used to form catanionic aggregates with drugs.

To investigate if catanionic vesicles composed with a drug can be used as physical cross-linkers and render gel formation using non-cross-linked polymer.

To investigate if the drug release could be prolonged from

physical gels formed from drug containing catanionic aggregates and polymers.

To investigate if drug penetration through biological and synthetic membranes can be reduced and if, or how, these membranes are affected by using gel formulations containing catanionic aggregates.

To study the drug release mechanisms from catanionic aggregates

contained in gels and how the catanionic aggregate structure is affected when the formulations are applied to biological membranes.

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3. Catanionic aggregates

3.1 What are catanionic aggregates? Catanionic aggregates are spontaneously formed when solutions of oppositely charged surfactants are mixed. The driving force for this aggregate formation is the combination of attraction of the opposite charges and the lipophilic interactions between the surfactants. A schematic illustration of how catanionic aggregates are formed is presented in Figure 1. Extensive research has been conducted on aqueous mixtures of classical surfactants bearing opposite charges, which started around sixty years ago [51-55]. The term catanionic mixture was first used by Jokela et al [56] in 1987 and shortly afterwards, Kaler et al further described the formation of catanionic vesicles [57]. Over the years the interest in catanionic aggregates increased and their potential use in different fields has been explored. When it comes to the pharmaceutical arena this is no exception, see for example the review by Bramer et al [58].

Catanionic aggregates have a complex phase behavior, and the formation of micelles and vesicles has been observed in the dilute regions of the phase diagram. A typical tertiary phase diagram of the dilute region for a catanionic surfactant mixture is displayed in Figure 2. Micellar and vesicular regions are generally surrounded by a multi-phase region and separated by precipitation. In each system, the cationic/anionic surfactant ratio and the total surfactant concentration affect which aggregates are formed. The symmetry of the phase diagram was found to depend on the difference in the length of the chains in the tails of the oppositely charged surfactants, see the review by Gradzielski [59]. Prior to the work conducted in this thesis, it was shown that the cationic surfactant can be exchanged for a drug substance, rendering a catanionic aggregate containing a pharmaceutically active ingredient [49-50].

The use of surfactants for pharmaceutical applications is limited, as substances with surfactant properties tend to disrupt cell membranes, cause irritation and, possibly, induce allergic reactions. The most toxic surfactants are the cationic ones and the nonionic ones are the least toxic, the anionic surfactants exhibit a biocompatibility that lies somewhere in between these extremes. The cationic surfactants have also been shown to be allergenic [60]. The Reviews by Sterzel and Drobeck [61-63] thoroughly describe the toxicity issues associated with surfactants. Studies of the cytotoxicity of

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catanionic aggregates composed of conventional surfactants have shown a similar pattern, where the catanionic aggregates have a toxicity somewhere between that of the two oppositely charged components [64].

Figure 1. A schematic illustration of the formation of catanionic aggregates, where (1) vesicles and (2) spherical, elongated and branched micelles might occur.

3.2 Pharmaceutically interesting aggregates Spherical micelles are normally formed upon dissolution of ionic surfactants in water. The formation of micelles is energetically favorable for the system as the area of hydrocarbon exposed to water is reduced upon micelle formation [65]. When using oppositely charged species, the electrostatic interactions also constitute a driving force for aggregate formation. In catanionic mixtures, not only spherical micelles are formed, but also elongated or branched micelles can be found, depending on the total surfactant concentration [66] and the ratio between the two surfactants [67-68]. When the two oppositely charged species do not have the same tail length the micellar growth may be affected, how much depending on how different the two species are. The studies by Raghavan et al [69] and Koehler et al [70] showed that rheology is a useful method of studying micellar growth in catanionic mixtures.

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Figure 2. A typical pseudo-ternary phase diagram of a catanionic mixture, redrawn from Bramer et al [58]. L signifies a micellar/aqueous solution, V- and V+ signify negatively and positively charges vesicles, respectively, and P signifies precipitate.

Conventional vesicles, or liposomes, can be prepared through a variety of methods, e.g. sonication, high-pressure extrusion or thin-film hydration [59]. Kaler noted that the catanionic vesicles were formed spontaneously [57] and, since then, many studies have supported this [59, 71-79]. The concept of vesicles as a thermodynamically equilibrate state is, as yet an unresolved issue; the discussion is almost as colorful as the number of participants. Although this discussion is interesting, there are numerous catanionic mixtures where vesicles are spontaneously formed and show sufficient stability to be applicable in many fields. Within each catanionic system the variations in aggregate size and polydispersity are enormous. These variations depend on the cationic/anionic ratio used, but they are also unique to each system. Additionally, as the vesicles are not manipulated during the production such polydispersity is not unexpected. Catanionic vesicles composed of conventional surfactants span a range of sizes from several m in diameter [74, 80-82] to as small as 20 nm [72, 74-75, 81, 83-84].

Several pharmaceutical applications have been suggested for catanionic aggregates, as recently reviewed [58]. Many of these applications imply an environment of physiological osmolality and pH:s. The ionic strength can be

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expected to affect the catanionic micelles and, indeed it has been shown that the addition of salts alters the critical micelle concentration (CMC) [85] and that the size and shape of the micelles are affected when the electrostatical interactions of the surfactant head groups are screened by salt ions [69, 86-89]. In catanionic systems, the addition of salt may induce phase transitions, as shown by Brasher et al [90]. Physiologically relevant temperatures do not normally pose problems regarding aggregate stability [91-92]. Bramer et al used catanionic vesicles composed of drug substances and oppositely charged surfactants to study the effects of changes in pH and ionic strength [93]. The investigation showed that these aggregates were unaffected by salt concentrations up to double the physiological concentration. As the pH was raised, the drug substances were less charged and it was shown that increases in the pH resulted in smaller micelles. In some cases the elevated pH reduced the presence of vesicles, but the drug release from gels with an adjusted pH and ionic strength could still be prolonged in most cases.

It is not an easy task to elucidate the chain of events that takes place in a gel containing catanionic aggregates during the drug release process. It was proposed early on that the reason for the prolonged drug release from gels containing catanionic aggregates was because of the high distribution of drug substance to large aggregates with low mobility, leaving only a small fraction of monomers free to diffuse out of the gel [50]. With only a small fraction of drug substance available, the driving force for the drug to diffuse out of the gel is also small. Owing to the complex nature of the formulations explored in this thesis, there are many ways to investigate them, but the analysis of the data is not always straightforward.

The drug release mechanism from the catanionic aggregates contained in the gels used in this thesis is of course an area of great interest. Some studies conducted prior to or parallel with this thesis have addressed this topic and, even though it is clear that the drug release is prolonged when catanionic aggregates are employed, it is still not completely clear how these systems function. The apparent diffusion coefficients measured in a study by Brohede et al were very similar to those obtained previously using the modified USP paddle method [94]. Bramer et al applied the regular solution theory to obtain complementary information regarding the drug release mechanism [95]. Even though the theory is simple, it provides a valuable tool to predict the drug release from catanionic aggregates contained in gels. Both these studies supported the initial theory that the aggregates are immobile in the gels because of their size, but a deeper understanding of these systems required further study, as did increasing the applicability of the formulations.

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3.3 Aggregate - polymer interactions and gel formation Polymers can be produced synthetically or be of natural origin, and can be modified to tailor their suitability to different applications. The uses of polymers are numerous and both synthetically and naturally produced polymers are used in pharmaceutical formulations of many kinds. The cross-links of the polymers that form gel networks can be either chemical or physical. The Carbopol gels used in this thesis all have a network of chemically, covalently, cross-linked poly(acrylic acid). Paulson and Edsman showed that cationic drug substances interact with Carbopol gels [48], which Bramer et al also confirmed [49]. Paulsson and Edsman also revealed that cationic drug substances form mixed micelles with hydrophobic modifications on some Carbopol polymers that give rise to a prolonged drug release from these gels [48].

There are examples of when polymers stabilize conventional vesicles and function as rate controllers in drug delivery [96], but there are also examples of when polymer and vesicle interactions lead to instabilities. This can be manifested as faceting of the vesicles, vesicle ruptures or even the formation of discs [97]. Vesicles are able to interact with polymers in several ways, for example, through hydrophobic interactions between the polymer and the hydrophobic parts of the polymer [98-100], be means of electrostatic interactions in charged systems [101-102], or by a combination of both of these [101-102]. Interactions that lead to cross-linking of polymers may result in gel formation.

3.4 Topical drug administration Formulations that are applied to the skin or to mucus membranes are referred to as topical. Drugs administered through the topical route may have both local and systemic effects, depending on where the application is made and on how the formulation is constructed. When a systemic effect is sought, topical administration can offer many advantages over oral or parenteral administration, for example: first pass metabolism is avoided, the risks and inconveniences of parenteral administration are abolished, and large variations in the pH and in gastric emptying are avoided. Oppositely, if a local effect is sought, many adverse effects associated with an oral administration can be avoided when the topical route is used.

The research regarding catanionic surfactant mixtures has increased over time, and includes a growing body of publications regarding the toxicological properties of these systems. In a recent study, Aiello et al investigated the cytotoxicity of catanionic vesicles formed by two conventional surfactants, SDS and CTAB and showed that at low concentrations the effect on cell growth is rather limited [103]. In a study by

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Brito et al the ecological and hemolytic effects of catanionic vesicles constructed with amino acid-based surfactants with low toxicity are described [64]. It was shown that the individual surfactants were more toxic than the mixed aggregates and that several of the new surfactants were more biocompatible than the conventional surfactants. Rosa et al also used amino acid-based compounds instead of cationic surfactants, to form catanionic aggregates [104]. The slightly damaging effect of catanionic aggregates was described as being concentration dependent in a study by Östh et al [105], where excised pig mucosa was employed.

In this thesis, all formulations are model systems and the treatment area of the pharmaceutically active substance is never the main focus of attention. The gels that are studied would most likely be suitable for administration to a mucus membrane, which is also where the greatest benefit from a prolonged release of a drug substance would be obtained. However, in many cases the oppositely charged surfactants are not sufficiently biocompatible to be used on sensitive mucosa, as has been shown with these formulations [105]. Skin is a more resilient tissue that can be used to illustrate the drug penetration process from gel formulations containing catanionic aggregates applied to biological membranes.

3.4.1 Dermal drug administration The skin protects the inner organs against mechanical injury, micro-organisms and loss of fluid. It also regulates body temperature, among its many other tasks [106]. Three layers constitute the skin: the exterior is the epidermis, the middle layer is the dermis and the innermost layer is the subcutis. The top layer of the epidermis is the stratum corneum (SC), which is the principal barrier, and which is, therefore, the barrier of primary interest for the dermal penetration of pharmaceutical products. The stratum corneum is often described with the bricks-and-mortar model [107], where the corneocytes are the bricks, surrounded by lipids, which represent the mortar. A simple sketch of the skin structure is shown in Figure 3.

The use of pig skin as a model for human skin has been well established [108] and the diffusion rate of drugs across the stratum corneum is similar in both species [109]. Artificial alternatives are available in numerous forms, and, notably, silicon membranes have proven to function well as a model of human skin [110-111] even when penetration enhancers are applied [112].

Most drugs penetrate the skin barrier through passive diffusion; therefore an adequate reservoir of the available drug is required. Substances can penetrate the skin through just one or through several routes, these being: intercellular diffusion and transcellular diffusion, through hair follicles or via sweat ducts [113]. The intercellular diffusion route is the most common one, and small lipophilic molecules are most likely to be the quickest to penetrate the skin barrier.

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The degree of permeation of pharmaceutically active ingredients through the skin depends, to a large extent, on the molecular structure and size of the compounds, but also on the other ingredients in the formulation. As lipids surround the corneocytes it has been suggested that lipids in formulations containing conventional liposomes may penetrate the intercellular lipid layers, and thereby modify these lamellae [114]. Since the early 1980’s vesicles have been ‘explored’ as potential transdermal drug carriers [115], and the research conducted in this area has been extensive, see, for example, the reviews listed [116-117]. Like traditional liposomes, catanionic aggregates have also been shown to affect the skin penetration of drug substances [118], however, it is a topic that will be discussed in more detail below.

Figure 3. A schematic presentation of the structure of the skin.

Depending on whether the drug is intended for cutaneous (local) or transdermal (systemic) use, different properties of the formulation are sought. Where a local effect is desired, the aim is to deposit as much as possible of the drug substance at the site of effect. In contrast, when a systemic effect is being sought, as much of the drug as possible should reach the blood stream. To investigate how much of the drug penetrates the skin or a mucosal barrier, the horizontal Ussing chamber setup can be used [105]. Tape stripping is a fast and easy way to quantify the dermal absorption and can be performed both in vivo and in vitro. By analyzing the drug content on each or a group of strips the rate or degree of penetration can be elucidated.

Conventional surfactants, such as SDS, have been shown to increase the systemic absorption of drugs that are similar to the ones used in this thesis, when the skin was pre-treated with SDS [119]. Surfactants of natural origin

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were also classified as permeability enhancers [120-122], but there was no correlation between the degree of skin irritation and the flux enhancement when some of these surfactants were used [120]. Similarly, the upper layers of the SC were unaffected by SDS in another study [123].

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4. Experimental section

4.1 Materials In total nine drug substances were investigated in conjunction with the research conducted for this thesis. Their molecular structures are illustrated in Figure 4 and the oppositely charged surfactants are shown in Figure 5. Additional information regarding the chemicals can be found in the manuscripts.

Figure 4. Molecular structures of the drug substances investigated in the research presented here.

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Figure 5. Molecular structures of the oppositely charged surfactants investigated in this thesis.

Figure 6. Molecular structures of the polymers used in this thesis.

The polymers used were Carbopol 940 and 1342, Agar-agar, SoftCAT SK-MH, UCARE JR-400 and HM(C16-18)-PEG. The molecular structure of the monomer units of these polymers is shown in Figure 6. The Carbopols have a poly(acrylic acid) backbone and are covalently cross-linked synthetic hydrogels [124]. The Carbopol 1342 polymer bears hydrophobic modifications on this backbone. As the pH is raised above six, the Carbopol polymers are charged and swell, forming a highly viscous gel. Agar gels are uncharged and composed of polysaccharide complexes originating from agarocytes of several algae species [125]. These dissolve in water upon heating and, after subsequent cooling, a gelatinous rigid gel is formed.

Three non cross-linked polymers were used to investigate gel formation possibilities when mixed with catanionic vesicles. SoftCAT SK-MH is an N,N-dimethyl-N-dodecylammonium derivate of hydroxyethylcellulose. UCARE JR-400 is an N,N,N-trimethylammonium derivate of hydroxyethylcellulose and HM(C16-18)-PEG is an uncharged poly(ethylene glycol), modified with hydrocarbon chains comprised of C16-18, where the

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hydrophilic section between these modifications is approximately 280 oxyethylene units long.

4.2 Phase studies 4.2.1 Catanionic studies The phase studies were conducted by mixing different proportions of stock solutions of the drug substance and the oppositely charged surfactant to obtain various concentrations (Papers I-III). All stock solutions were prepared using 150 mM sodium chloride solution. The mixed samples were allowed to equilibrate for at least one week before visual inspection, to allow the phase-separated samples to set and to minimize the effects of differences in the preparation procedure. In addition to the visual inspection, some samples were also investigated rheologically and/or with cryo-TEM. Visual inspection has proved to be a highly reliable method of investigation for these types of samples. Precipitates and other phase-separated samples are easily spotted, and changes in the viscosity caused by elongated or branched micelles are fairly distinguishable. Vesicles typically appear to be blue or gray and opaque, and are therefore easily recognized. Rheological studies were performed to determine differences in the viscosity, as described below and cryo-TEM was used to visualize vesicular and micellar structures in both liquid and gels. A detailed description of the cryo-TEM method can be found elsewhere [126]. All samples were kept at ambient temperature for at least two months and then re-inspected visually, to evaluate the storage stability of the mixtures.

4.2.2 Vesicle-polymer gel formation studies When gel formation with different polymers was studied, catanionic vesicle solutions were mixed with polymer solutions (Paper III). The three polymers used had different properties; the HM(C16-18)-PEG was uncharged, the UCARE JR-400 was positively charged and the SoftCAT SK-MH was positively charged bearing lipophilic modifications. The variations were used to investigate which property or properties influence gel formation. Both the polymer and vesicle containing solutions used in this study were double the intended final concentrations, and equal volumes were mixed to obtain these. The samples were mixed with magnetic stirrers for several days prior to their evaluation, as the polymer solutions were highly viscous. Those samples with large amounts of trapped air were centrifuged before the evaluation. All samples that appeared to have resulted in gel formation were investigated rheologically, as described below, and selected samples were investigated using cryo-TEM.

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4.3 Gel preparation 4.3.1 Agar gels All gels were prepared in 150 mM sodium chloride solution. The agar gels were prepared by adding polymer powder corresponding to 1 % of the final weight to either a reference drug solution or a solution containing catanionic aggregates. This dispersion was then heated in a water bath for 20 minutes at 100 °C to ensure complete dissolution of the polymer. When used for the drug release measurements the solution was transferred to the gel containers where it was allowed to form stiff gels prior to the immersion in the receiving media.

4.3.2 Carbopol gels The Carbopol polymer powder was dispersed in 150 mM sodium chloride solution and stirred with magnetic stirrers for at least one hour to allow adequate time for the deaggregation of polymer particles. The pH was raised to approximately 7, using NaOH and the gel was allowed to equilibrate at least overnight before final pH adjustments to 7.4 ± 0.1. When required, sodium chloride solution was added to adjust the final volume so that the desired Carbopol concentration of 2% was obtained. The gel was mixed with an equal volume of reference drug solution or solution with catanionic aggregates prepared at double the desired final concentration to render the desired final concentrations of both drug substance or catanionic aggregates and gel polymer.

4.3.3 Physical gels Polymer powder was dispersed in 150 mM sodium chloride solution and mixed with magnetic stirrers overnight to allow the polymer to dissolve. Polymer solution was prepared at twice the desired final concentration and mixed with equal volumes of solutions containing catanionic vesicles, also prepared at double the intended final concentration. These mixtures were stirred for several days until complete mixing had been obtained.

4.4 Rheological investigations Catanionic aggregates in both solution and gels were subjected to rheological measurements, all performed with a Bohlin VOR Rheometer [127] (Bohlin Reologi, Lund, Sweden). The catanionic aggregate solutions were investigated using viscosity measurements (Paper I). Concentric cylinder systems were used (C8 and C14) and, prior to each measurement,

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the delay time was determined. The gel samples were investigated using dynamic oscillating measurements. Gel samples were transferred to a concentric cylinder (C8 or C14) and centrifuged at 3000 rpm for 3 minutes to remove trapped air and, prior to the measurements being made, silicone oil was applied to the sample surface to avoid evaporation. The measurements were carried out at 20 °C (Papers I, II, IV and V), and from 20 – 37 °C (Paper III). A strain sweep was performed initially on all samples, to find the linear viscoelastic region, where the dynamic oscillation measurements were performed. The elastic (G´) and the viscous (G´´) moduli and the phase angle ( ) were then determined.

4.5 Drug release studies The drug release measurements in Papers I-V were performed using a modified USP paddle method. Custom-made gel containers, shown in Figure 7, were immersed in degassed 150 mM sodium chloride solution. Different volumes were used, depending on the spectrophotometrical detection of the drug substance that was used, ensuring that sink conditions were maintained throughout the experiments. A Pharma Test PTW II USP (Pharma Test Apparatebau, Germany) with six beakers and a peristaltic pump and ismaprene tubing (Ismatec SA, Zürich, Switzerland) was used. The receiving media was continuously pumped through a UV-vis spectrophotometer (Shimadzu UV-1601, Shimadzu, Kyoto, Japan). At regular intervals, the absorbance was measured automatically and stored using IDIS tablet dissolution data-management software (Icalis Data Systems Ltd, United Kingdom).

The gel containers were filled with gel and covered with a mesh-size plastic net to hinder diffusion of the polymer from the container; this was followed by a coarse plastic net to prevent further swelling of the gels. The container was then assembled by fastening the top section with screws. The experiments were performed in triplicates and the absorbance data was used to calculate the apparent Fickian diffusion coefficient, D, for each experiment, using Equation 1.

2/1

02Dt

CQ (1)

where Q is the amount of drug substance released per unit area, C0 is the initial concentration of drug substance in the gel and t is the time to have elapsed since the experiment started. The equation is valid for approximately the first 60% of the release [128-129]. The diffusion coefficients were calculated from data from the first 40 minutes of the experiments, where a lesser amount of drug substance was released. The apparent diffusion

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coefficient measures the rate, at which a substance moves randomly from a region of high concentration to a region of low concentration. This depends on several factors, such as catanionic aggregate deterioration, drug loading within these aggregates and the actual diffusivities of the substances concerned.

Figure 7. The custom-made gel containers used in the modified USP paddle method. The figure has been redrawn from Paper II.

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4.6 Skin preparation Dermatomized pig ear skin was used for the drug penetration studies (Papers IV and V). The ears were obtained from pigs used by another research group. These experiments were approved by an ethical committee (application number C 257/6) and the nature of the performed experiments was not considered to affect the skin. At the end of these experiments the pig was put down and the ears were immediately removed using a scalpel. The skin was then separated from the cartilage and the desired thickness, 0.5 mm, was obtained with a Padgett dermatome (Integra, Plainsboro, NJ, USA). The desired shape of the skin sample was obtained using a circular punch with a 15 mm diameter. Immediately after preparation the skin was frozen at -20 °C until experimental use, no longer than six months.

4.7 Drug penetration studies For the drug penetration studies a horizontal Ussing chamber method was employed (Papers IV and V). The system contains six chambers placed side-by-side on a water-heated block (Horizontal Diffusion Chamber System, Costar, Cambridge, MA, USA). The water temperature was adjusted to maintain the receiving medium in the chambers at 37 °C. Stirring was achieved by placing the equipment on a circular shaker (Unimax, Wernerglas, Sweden); set to 145±1 rpm. In this setup, the area of exposed skin surface is 0.55 cm2. In total, 1.2 mL of 150 mM sodium chloride solution was added to the receiving compartment prior to mounting the skin sample. Skin from a selection of several different pigs was used for each experiment and formulation to obtain a randomized selection of samples. The skin samples were thawed in 150 mM sodium chloride solution at ambient room temperature for 30 minutes and prior to mounting in the horizontal Ussing chambers, the thickness of each skin sample was measured using a digital slide gauge (Schuchart maskin AB, Huskvarna, Sweden). After mounting the skin samples, any air bubbles were removed. The Ussing chambers were covered with parafilm to prevent evaporation, and before the experiment started they were placed on the heat-block for 30 minutes to let the skin samples equilibrate. Catanionic vesicles, composed of tetracaine and SDS or capric acid were used in Paper IV and alprenolol and SDS were used in Paper V. Covalently cross-linked Carbopol gels and physically cross-linked SoftCAT gels were used in both studies. A total of 200 L of each formulation was applied to the donor side of each chamber and 100 L samples were withdrawn from the receiving compartment at different times over a period of up to 24 hours. The samples withdrawn were immediately replaced with fresh sodium chloride solution and diluted with HPLC mobile

30

phase, prior to analysis. The amounts of the drug transferred during the first five hours of the experiment were used to calculate the apparent penetration.

The drug penetration studies were carried out on skin, in Papers IV and V, and silicone membranes, in Paper IV. The conditions were identical regardless of which type of membrane was chosen. The size and shape of the silicone sheets was obtained with the same punch as that taken to prepare the skin samples. Before mounting the silicone sheets in the chambers they were washed with Millipore water and the formulations were applied to the donor compartments after resting on the heatblock for 30 minutes. The potential to adopt silicone sheets as a model for the skin was evaluated with formulations containing the catanionic vesicles composed of tetracaine and SDS in Paper IV.

When the final samples were withdrawn from the Ussing chambers after 24 hours in the skin penetration experiments, the skin samples were either tape-stripped (Paper V) or used for skin morphology studies (Paper IV and V), as explained in Section 4.9. The tape stripping was performed to determine in which part or parts of the skin drug could be found. For the tape stripping, the formulation left on the skin surface was carefully dried off with tissue paper and then 2 cm sections of Scotch Magic tape (3M) were applied to the skin surface. A 100 mL beaker was used as a weight before the tape was separated from the skin samples. The two first tape strips were considered to contain formulation residues and were therefore discarded. The three following ones were considered to comprise the upper stratum corneum and the five last strips the middle of stratum corneum. The tape strips and the remaining skin were extracted separately in 70 % ethanol for at least 24 hours and the drug content was then analyzed using high performance liquid chromatography (HPLC).

4.8 HPLC analysis The tetracaine and alprenolol content of the samples extracted from the receiving side of the skin penetration experiments in Papers IV and V was quantified with the help of HPLC. In addition, the samples from the tape strip extractions in Paper V were analyzed using HPLC. The system selected consisted of a Waters 717 Plus Autosampler, a Shimadzu LC-10AD pump and a Spectra 100 UV detector (Thermo Separation Products). All samples were analyzed on a C-18 Hypersil Gold column, 250 mm x 46 mm (5 m) (Thermo Scientific, UK) and Hypersil Gold 5 m 10 x 4 mm drop-in guard inset (Thermo Scientific, UK). The detector was set to 288 nm for the tetracaine quatification and to 276 nm for the alprenolol quantification. Verapamil and metoprolol were selected as the internal standards for the tetracaine and alprenolol analyses, respectively. The calibration and validation of the methods was performed on spiked samples of known

31

concentrations and three quality control concentrations were used (n=3). Calibration curves were established by linear regression of the chromatographic peak areas and ratio of the peaks (tetracaine/verapamil or alprenolol/metoprolol) as a function of the tetracaine or alprenolol concentration.

4.9 Skin morphology In Papers IV and V, skin samples exposed to every formulation investigated and a set of reference skin samples, which had been exposed to sodium chloride solution or air, were studied. The formulations used contained catanionic aggregates composed of both conventional and natural surfactants, present in both covalently and physically cross-linked gels. After the withdrawal of the final set of samples after 24 hours the skin samples were placed in separate embedding cassettes and submerged overnight in Bouin’s solution. The skin was dehydrated for 24 hours in ethanol, then in a 1:1 mixture of 99.5 % ethanol and infiltration solution and finally in pure infiltration solution (Historesin, Leica Microsystems, Germany). The embedding of the dehydrated skin samples in infiltration solution was carried out using hardeners according to the instructions of the manufacturer. Slices of thickness 2 m were prepared by using a motorized microtome (Leica RM 2156, Leica Microsystems, Germany). The slices were stretched on a Millipore water surface and transferred to a glass slide prior to drying on a heat plate. Borax and toluidine blue were used for staining, and the excess staining fluid was removed with ethanol. Slices from different parts of the skin samples were examined microscopically with an Olympus BX-51 microscope (Olympus, Japan) equipped with a DP 50 digital camera (Olympus, Japan) and Olympus DP-soft (Olympus, Japan) was selected as software. The skin samples were studied at different magnifications and any morphological variations were noted.

4.10 Statistical analysis The drug release studies were performed in triplicate and the mean, standard deviation (s.d.) and the 95% confidence interval (CI) were calculated for the diffusion coefficients for each triplicate. The drug penetration studies were sextuplicated, and the mean and standard deviation were calculated for the apparent penetration. ANOVA and the Bonferroni’s multiple comparisons post hoc test were used. The software utilized was Prism 4 for Windows, by GraphPad Software Inc. (San Diego, CA).

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5. Results and discussion

The results obtained in the research undertaken for this thesis have been extracted from the publications included and are presented in this section. The order of these results is not strictly chronological, but rather, it follows a trail intended to illustrate the relevance of these systems. Prior to the work presented here Paulsson and Edsman discovered the ability of drug substances to form catanionic aggregates with oppositely charged surfactants [50] and Bramer et al explored the phase behavior of some of these mixtures in greater depth, as well as the use of catanionic aggregates for prolonged drug release purposes from gels [49]. In parallel with the work incorporated in this thesis, additional physical-chemical [93] and mechanistic modeling work was also performed [94-95]; however the research presented in this thesis is more focused on the applicability of the catanionic aggregates chosen for incorporation in pharmaceutical gel formulations.

5.1 Can drugs be used to form catanionic aggregates? Prior to the investigations performed in Paper I, formation of catanionic aggregates was observed when sodium dodecyl sulphate (SDS) was mixed with three cationic drug compounds: diphenhydramine, tetracaine and amitriptyline. In Paper I, six additional drug substances were tested for catanionic interactions: lidocaine, ibuprofen, naproxen, alprenolol, propranolol and orphenadine. Five out of these were able to form either catanionic vesicles or high viscosity catanionic micelles, or both, the exception being naproxen. Figure 8 shows cryo-TEM micrographs of vesicles and micelles that are characteristical of the catanionic systems explored in this thesis.

The selection of drug compounds in Paper I was based on simple criteria: the substance should bear a charge, either negative or positive, at physiological pH, have a molecular structure that implied surface activity of the compound and have a toxicity profile that enabled work to be accomplished without complications induced by toxicity issues in the laboratory. The selection of substances was made from the compendium containing the formulae for Swedish pharmaceutical specialties [130]. Both negatively and positively charged drug substances were used in the study along with several oppositely charged surfactants; the results showed that

33

both negatively and positively charged drug compounds have the ability to form catanionic aggregates. The substances used and the aggregates and interactions that were observed are listed in Table 1.

Figure 8. Representative cryo-TEM images of (A) alprenolol/SDS (at a ratio of 4:6 and at 20 mM) vesicles and (B) ibuprofen/TTAB (at a ratio of 4:6 and at 40 mM) micelles. The arrows indicate examples of branches of the micelles and size bar shows 200 nm.

For prolonged drug release purposes, the vesicles or high-viscosity micelles are the most interesting, as these are the largest aggregates. In the systems previously investigated by Bramer et al [49], both of these types of aggregates were formed, and in the systems investigated in Paper I, only two systems showed no type of catanionic interaction. These findings make it obvious that drug substances readily form catanionic aggregates with oppositely charged surfactants.

Based on the simple selection criteria for the drug substances used in Paper I it is likely that many more surface-active drug substances bearing charge would be able to form catanionic aggregates with oppositely charged surfactants. Besides the beneficial use of prolonging the drug release from gels, presented below, it should also be noted that these aggregates might give rise to negative effects in other formulations. Surfactants are not unusual in pharmaceutical formulations and specifically SDS, which was used throughout the work conducted for this thesis, and benzalkonium chloride (BAC), are often used. This poses a risk of inducing undesirable effects such as precipitations or of causing other incompatibilities; there is also a possibility that catanionic aggregate formation might have an impact on the drug release effects and have a negative effect on the formulation properties.

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Table 1. Catanionic interactions and aggregates formed in the systems examined in Paper I. Catanionic interactions were noted with all drug compounds investigated, but one.

5.2 Do catanionic aggregates prolong the drug release from gels? A selection of the mixtures studied in Paper I was examined further with regards to the drug release rate from gels and both micelle and vesicle containing mixtures composed of both cationic and anionic drug substances were tested. These mixtures had proven to be beneficial for obtaining prolonged drug release from gels. The mixtures examined in Paper I confirmed not only that the formation of catanionic aggregates is a general occurrence but also that these aggregates can often be used to prolong the drug release from gels. In Figure 9 the release of orphenadrine from catanionic vesicles and micelles contained in gels is shown. Depending on the ratio of the drug substance to the oppositely charged surfactant, either vesicles or micelles are formed in this system. The results presented in Figure 9 show that both catanionic micelles and vesicles prolong the drug release and that the vesicles render the most prolonged drug release. When comparing vesicle solutions with a variety of total surfactant concentrations, there is no statistically significant difference between their diffusion coefficients. Vesicles prolonged the drug release the most, and the diffusion coefficients were significantly smaller for the vesicles compared to those for

Cation Catanionic interactions

Viscous micelle

formation

Vesicle formation

Anion

Alprenolol SDS

BAC IbuprofenBAC NaproxenBAC SDSDiphenhydramine IbuprofenDiphemhydramine NaproxenLidocaine SDSLPC IbuprofenLPC NaproxenOrphenadrine SDSPropranolol SDSTTAB IbuprofenTTAB Naproxen

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the micelles and reference formulations in the orphenadine/SDS systems investigated. The diffusion coefficients were around 100 times smaller than the reference for vesicles and 10 times smaller for micelles. However, this may not be representative behavior as micelles have been shown to prolong the drug release from gels to the same extent as vesicles in the diphenhydramine/SDS system investigated by Bramer et al [49]. As the orphenadine/SDS and some of the other catanionic aggregates did not mix well with the Carbopol gels, Agar-agar gels were used instead. The drug release rate has been found to be virtually the same from Agar and Carbopol gels when using drug-surfactant systems [49].

Figure 9. The release of orphenadine from 1% Agar-agar gels. This figure has been redrawn from Paper I. The reference gel, containing only orphendaine at 48 mM, is signified by diamonds. The orphenadine/SDS micelles (at a 2:8 ratio at 160 mM) is signified by squares. Orphenadine/SDS vesicles are signified by triangles where the empty triangles show vesicles at 160 mM (at a 4:6 ratio) and the filled triangles show vesicles at 40 mM (at a 3:7 ratio). The error bars show the standard deviation (n=3).

In Paper I, mixtures of lidocaine and SDS, with the same ratio of drug-surfactant, but with different total surfactant concentrations, were examined rheologically. The increase in viscosity was vast, indicating an increased aggregate size. The viscosity of these micelles also rose when the ratio of lidocaine and SDS moved towards a 1:1 relationship, at the same total concentration, which also indicated an increase in aggregate size. However, when the drug release rates from gels containing these different micelles were examined the difference between the diffusion coefficients from all micelle mixtures investigated was small and not statistically significant. This

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indicates that there is a limit to how much the drug release can be prolonged from gels by increasing the size of the micelles.

The drug release studies performed in Paper I included several drug substances and oppositely charged surfactants and, by varying the molar ratios of the components, different aggregate structures were obtained. This shows that, by forming catanionic aggregates with drug substances and oppositely charged surfactants, the drug release from gels can be extensively prolonged, regardless of which type of aggregate was employed.

5.3 Can the oppositely charged surfactant be less toxic? In a pharmaceutical formulation the aim is to minimize all toxicity induced effects and conventional surfactants cannot be used in mucosal formulations. Surfactants of natural origin that were possibly more biocompatible than conventional ones, were investigated in Paper II to see if these could be used to form catanionic aggregates with drug substances. Two carboxylic acids of natural origin were used, lauric and capric acid. These can both be found in coconut oil and mother’s milk [131-133] and lauric acid has been classified as non-harmful and is easily digested in humans [133-135]. A study of capric acid performed in the rat revealed a rather low toxicity on endothelioid cells and heart muscle [136].

The statement from Paper I, that the formation of catanionic aggregates when mixing drug substances with oppositely charged surfactants is a common occurrence, was broadened in Paper II as the surfactants of natural origin proved useful for this purpose. The systems studied in Paper II are listed in Table 2. This phase study was intended to include two additional naturally occurring carboxylic acids: myristic and palmitic acid, however, their solubility in 150 mM sodium chloride solution was insufficient, even though they were heated above the Krafft points. These surfactants were, therefore, excluded from the study. Lauric acid has a Krafft point around 45 ºC and the catanionic aggregates that were formed above this temperature proved to be stable even when the mixtures had reached ambient room temperatures. Capric acid, which has the shortest hydrocarbon tail of the carboxylic acids used in Paper II, is soluble at room temperature and could be used to form catanionic aggregates without heating. Lauric acid has twelve carbons in the tail, but no striking differences in aggregate forming capability can be noted between the two acids, as can be seen from the list presented in Table 2. The most noteworthy difference is that lauric acid more commonly produces vesicle formations, and that these vesicle regions spread over a greater region than in the capric acid systems. Had myristic and palmitic acid been used in the phase study of Paper II, the influence of chain length could have been investigated in greater depth. From the results

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generated with only two surfactants with a slight difference in chain length the effects observed by Raghavan et al [69] could not be confirmed.

Table 2. The catanionic interactions in the systems examined in Paper II.

The drug release study in Paper II was performed with Agar gels, as the mixtures containing lauric acid were not compatible with Carbopol gels. As vesicle formation was more prominent than formation of elongated or branched micelles, the vesicles were employed for the drug release study. The results show that the surfactants of natural origin, capric and lauric acid, can be used just as well as a conventional surfactant such as SDS. In view of the previous results a few interesting comparisons can be made regarding the use of the natural surfactants for prolonged drug release purposes: A mixture of diphenhydramine and lauric acid (at a ratio of 35:65), as was used in

Cation Catanionic interactions

Viscous micelle

formation

Vesicle formation

Anion

Alprenolol Capric acid

Amitriptyline Capric acidAtenolol Capric acidBAC Capric acidDiphenhydramine Capric acidDoTAB Capric acidLidocaine Capric acidOrphenadrine Capric acidPropranolol Capric acidTetracaine Capric acidTTAB Capric acid

Alprenolol Lauric acidAmitriptyline Lauric acidAtenolol Lauric acidBAC Lauric acidDiphenhydramine Lauric acidDoTAB Lauric acidLidocaine Lauric acidOrphenadrine Lauric acidPropranolol Lauric acidTetracaine Lauric acidTTAB Lauric acid

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Paper II resulted in a diffusion coefficient around 10 times smaller than the reference formulation, comprised of only drug substance. This can be compared to a formulation containing SDS instead of lauric acid like the one that was investigated by Bramer et al [49], where the diffusion coefficient was 15 times smaller than that for the reference. An example where the use of lauric acid generates smaller diffusion coefficients than SDS can be found when comparing the results obtained with alprenolol. In Paper II the alprenolol and lauric acid mixture generated a diffusion coefficient around 60 times smaller than the reference formulation, but when SDS was used in a study by Paulsson et al [50] the diffusion coefficient was only 20 times smaller than the reference. As these are conflicting results it is difficult to make any general conclusions regarding which oppositely charged surfactant gives rise to the most prolonged drug release. It must be taken into account that the drug substances do not have typical surfactant structures and, therefore, the catanionic aggregates they produce when mixed with surfactants may not always have similar features, despite being mixed with the same surfactant. Further studies of the aggregate interactions with the various gel polymers used in the above-mentioned studies might explain if these affect the drug release rate from the gels.

In Paper IV, the morphology of skin exposed to formulations containing drug only, and drug-containing catanionic aggregates formed with both natural and conventional surfactants was compared to skin exposed to air or saline solution. Additionally, both covalently and physically cross-linked gels were used. The study showed that there were no visible differences between any of the skin samples investigated. It was suggested that the reason for a higher amount of tetracaine to be transferred when the capric acid containing formulations were used than when the SDS containing formulations were used was an effect of a lower monomer concentration in the SDS containing formulations.

Regardless of the conflicting results discussed above, two major conclusions could be drawn from Paper II: that surfactants of natural origin can be used to form catanionic aggregates with drugs and that the drug release from gels can be prolonged to the same extent as when aggregates composed of conventional surfactants are used. From Paper IV it can be concluded that there are no differences in the morphological effect on skin between conventional and natural surfactants and that they are indistinguishable visually from the reference samples.

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5.4 Can catanionic vesicles function as cross-linkers in gels? Formation of cross-links arising from interactions between polymers and catanionic vesicles can lead to gel formation, which was shown in Paper III. Catanionic vesicles composed of the drug substances tetracaine or alprenolol and the oppositely charged surfactant SDS were mixed with three different polymers: one uncharged polymer with lipophilic modifications, one bearing positive charges and one bearing positive charges and lipophilic modifications.

Figure 10. Rheological properties of a 1% SoftCAT SK-MH solution (empty symbols) and upon addition of alprenolol/SDS vesicles (at a 40:60 ratio at 20 mM) (filled symbols). The elastical modulus (G´) is shown with diamonds and the viscous one (G´´) with squares.

Only the polymer bearing positive charges and hydrophobic modifications, called SoftCAT SK-MH, resulted in gel formation when mixed with the catanionic vesicles used in Paper III. Above a critical level of both polymer and vesicles gel formation occurred, which was in accordance with previous studies in similar systems [100]. When catanionic vesicles were added to the polymer solution the rheological properties of the system changed from those of a typical entangled polymer solution to a gel with weak structure; i.e. G´>>G´´ with a slight frequency dependence. The changes in the rheological properties of the polymer solution upon addition of catanionic vesicles composed of alprenolol and SDS is shown in Figure 10. The interactions between polymers and vesicles were durable and produced gels

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with a substantial shelf life, but not too strong to cause associative phase-separations or aggregate ruptures. An illustration of how catanionic vesicles may interact with an oppositely charged polymer bearing lipophilic modifications and how this differs from a covalent gel is shown in Figure 11.

Figure 11. A schematic illustration of (A) how catanionic vesicles interconnect with an oppositely charged polymer bearing lipophilic modifications resulting in gel formation and (B) how this differs from the situation where the vesicles are contained in a covalent gel. Note that the vesicles are a founding part of the interconnections in the polymer network in (A) and that the polymer cross-links in (B) are constituted by the polymer itself.

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Cryo-TEM was conducted to visualize the aggregate structure in the physical gels. As the alprenolol/SDS vesicles were mixed with polymers the vesicle size and the presence of bi-lamellar aggregates decreased. The tetracaine/SDS system was harder to characterize owing to the radiation sensitivity of the samples, but lamellar sheets and vesicles with large openings were observed as polymer was added. Micrographs showing the characteristic structures of both solution and physically cross-linked gel samples can be found in Figure 12.

The study conducted in Paper IV reveals that when catanionic vesicles composed of drug substance and oppositely charged surfactants are mixed with positively charged polymers bearing hydrophobic modifications a gel formation occurs. The rheological properties of these gels have a slight frequency dependence, which shows that the gel strength is not as high as that of to covalently cross-linked gels.

Figure 12. Representative cryo-tem micrographs of catanionic alprenolol/SDS vesicles (at a 4:6 ratio at 20 mM) in solution and mixed with 1% SoftCAT SK-MH polymer solution. The size bar indicates 200 nm.

5.5 Does the gel type affect the drug release? The polymer network that constitutes the gel can be either covalently or physically cross-linked, as mentioned above. The in vitro drug release from the physical gels, composed of catanionic vesicles and oppositely charged polymers bearing hydrophobic modifications, was investigated and compared to that from covalent, Carbopol, gels in Paper III. Carbopol gels with and without lipophilic modifications were used both as reference gels with only the drug and as test formulations with catanionic vesicles. Figure 13 enables a comparison of the fraction of released drug substance over time from the physical and covalent gels investigated in Paper III. Not

42

unexpectedly, the release of drug substance was most prolonged from the Carbopol gels with lipophilic modifications. However, the diffusion coefficients of the physical gels were not significantly different from those obtained using Carbopol gels, even though the physical gels break up as the drug substance is released.

Figure 13. Tetracaine release profiles from physical and covalent gels. Open symbols signify the release from tetracaine/SDS vesicles (at a 35:65 ratio and 40 mM) and when contained in Carbopol 940 signified with squares, Carbopol 1342 illustrated with diamonds and SoftCAT SK-MH illustrated with triangles. The release from reference formulations with only tetracaine 14 mM is shown with filled symbols. The error bars mark the standard deviation (n=3). The figure is redrawn from Paper IV.

As described earlier, the apparent diffusion coefficient was calculated during the first 40 minutes of the drug release process. The differences between the physically and covalently cross-linked gels became more obvious at later times, as is described in more detail in Paper V. The drug release was monitored until it was complete in Paper V, with experiments lasting up to 72 hours compared to the habitual limit of 13 hours in Papers I-IV. In Paper V the gel properties of the samples of physically cross-linked gel removed during the drug release experiment were also investigated rheologically. The amount of gel left in the gel container in the modified USP paddle method decreased as the drug substance was released, which also shows that the vesicles are part of the cross-links that form the gel structure. Further, the gel properties were maintained although the amount of gel decreased as the drug release progressed, as displayed in Figure 14. This also strongly indicates that the vesicles constitute the cross-links of the gels.

0

0,2

0,4

0,6

0,8

1

0 100 200 300 400 500 600 700 800

Fra

cti

on

re

lea

se

d

Time (min)

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Figure 14. Alprenolol release from physical and covalent gels, redrawn from Paper V. The release of alprenolol from alprenolol/SDS vesicles (at a 4:6 ratio at 20 mM) contained in Carbopol 940 is shown with squares, SoftCAT SK-MH 1 % is illustrated with open triangles and 2% polymer concentration is shown with filled triangles. The release from the reference Carbopol 940 gel with only alprenolol 8 mM is shown with diamonds. The error bars show the standard deviation (n=3).

Thus, it can be concluded that drug-containing catanionic aggregates can prolong the drug release from both physically and covalently cross-linked gels. At later times, when large amounts of drug substance have been released, the rate of the drug release from the physical gels is increased compared to the covalently cross-linked gels. In the physical gels catanionic vesicles constitute the cross-links and as the drug substance is released these gels break up. In the covalently cross-linked gels, the structure remains reasonably intact throughout the drug release process. The different properties might be favorable at different scenarios. In a clinical situation, a gel that is broken up as the drug substance is released might offer advantages over a covalently cross-linked gel, as an empty gel would not be left at the site of application when the drug substance has been released.

5.6 How does drug release affect aggregate structure? Previous studies conducted by Bramer et al and Brohede et al supported the initial theory that the aggregates are immobile in the gels because of their size [94-95]. In order to further understand these systems and to increase the applicability of the formulations, the aggregate structure during the drug release process was visualized in Paper V. The effect of drug release and

44

skin application on aggregate structure was visualized using cryo-TEM. Vesicles composed of alprenolol and SDS (at a ratio of 4:6 and 20 mM) contained in both covalently cross-linked Carbopol gels as well as physically cross-linked SoftCAT gels were investigated. The drug release was studied with the USP paddle method until it was complete and gel samples were taken out at various stages of the experiment and visualized using cryo-TEM. Gel samples taken from skin penetration experiments, performed in the horizontal Ussing chambers, were also visualized with cryo-TEM to gain further knowledge regarding the aggregate structure in formulations applied to biological membranes. The drug release profiles showed no nicks or breaks, as is evident from Figure 14, which indicates that no major phase transitions have occurred within the gels during the release process. The differences at later times between the covalent and physical gels also become more clear, even though the diffusion coefficients are not significantly different the fraction of the drug substance released is lower from the covalent gels from 15 hours and beyond this time (p<0.001). The cryo-TEM analysis revealed that vesicles were present in the gels throughout the release process, even when 99% of the drug substance had been released. The structures in the gel samples taken out of the containers in the USP paddle experiment could not be distinguished from the respective reference sample, which indicates that the aggregates are highly stable and possibly immobile. There was neither a difference in the samples taken from the skin surface at any of the time points between one and six hours; nor could they be distinguished from the reference sample. Representative cryo-TEM micrographs are displayed in Figure 15.

Figure 15. Representative cryo-TEM micrographs of catanionic 8/12 mM alprenolol/SDS vesicles contained in gel samples at various stages of the drug release process. (A) Prior to the release experiments, (B) after 99% of the drug had been released in the USP paddle experiments, (C) after six hours of application to the skin in an Ussing chamber. The size bar indicates 100 nm.

A rheological investigation was conducted of the samples taken during the USP paddle experiments to gain further knowledge of the physical gels. The amount of gel left in the gel containers decreased as the drug was released,

45

but the rheological properties of the samples collected scarcely changed. This shows that the physical gels are broken down inwards, as the drug is released and the catanionic vesicles and the cross-links are broken up. A comparison of the elastic, G´, and viscous, G´´, moduli of the physical gel formulations investigated is displayed in Figure 16. The findings presented in Paper V support the idea that the physically cross-linked gels are formed when cross-links are created by interconnections of the catanionic vesicles and the polymer, which was suggested in Paper III.

Figure 16. Rheological properties of the physical gels collected at various stages of the drug release experiments. The diamonds show the elastical modulus (G´) and the squares show the viscous modulus (G´´). The SoftCAT SK-MH polymer concentration is indicated by empty symbols for the 1% concentration and by filled symbols for the 2% gels.

There was also an indication in Paper V that the vesicles may be located in compartments between the Carbopol polymers within the gels. The cryo-TEM visualization technique, however does not offer sufficient resolution to enable proper visualization of the polymer and therefore further studies are needed to investigate the actual location of the aggregates within the gel.

To conclude, the data generated in Paper V shows that the vesicles are present in the gels even though most of the drug substance has been released and that they are stable when applied to skin. In addition, this supports the propositions that the catanionic aggregates have very low mobility within the gel network and that the monomers are released from the gels, at the same rate. Some questions regarding the drug release mechanisms and how the aggregates are contained in the gels still remain unanswered. With imaging

46

techniques where the resolution is adequate to visualize the polymer and the catanionic aggregates clearly, further knowledge could be obtained.

Figure 17. Drug release and skin penetration profiles. (A) and (B) illustrate in vitro tetracaine release profiles obtained in the modified USP paddle setup (n=3). (C) and (D) illustrate tetracaine penetration through skin in the horizontal Ussing chamber setup (n=6). Carbopol gels are signified by filled symbols and SoftCAT SK-MH gels by empty ones. Squares represent tetracaine/SDS catanionic vesicles (at a ratio of 35:65 and 40 mM) and triangles signify tetracaine/capric acid catanionic vesicles (at a ratio of 2:8 and 40 mM). Diamonds show reference Carbopol gels with 14 mM tetracaine solution. The error bars reveal the standard deviation.

5.7 Could catanionic aggregates in gels be used for topical administration? As gel formulations are typically used for mucosal or topical administration, the work in Paper IV was focused on determining whether the prolongation of the drug release would also be valid when the catanionic aggregates were used in conjunction with biological tissue. The problem of surfactant toxicity, touched upon previously, limits the choice of tissues that can be used with the systems investigated in this thesis. The skin, being one of the body’s primary defense barriers is resilient and therefore, usually, the diffusion of a drug substance applied on the skin is low, which has the implication that there is virtually no need for a slow release drug formulation

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to be developed for dermal use. However, due to its insensitivity to the surfactants in the catanionic aggregates employed in this thesis skin was used as a model biological membrane. Pig ear skin was used as a model of human skin in Papers IV and V, and silicone sheets were used as a synthetic model of the skin in Paper IV. A prolonged drug release could offer an advantage for formulations intended for mucosal use, where a less frequent dosing interval, or an increased absorption, is sought. These benefits could, possibly, also result in increasing patient compliance.

Catanionic vesicles composed of the drug tetracaine and conventional and natural surfactants, in physically and chemically cross-linked gels were used in Paper IV. Drug release from the formulations was studied with the modified USP paddle method and the skin penetration was investigated with horizontal Ussing chambers. The drug release from all gels containing catanionic aggregates comprised of SDS was slow enough to also prolong the skin permeation, but the vesicles composed with capric acid did not turn out to be as suitable. Figure 17 enables a comparison to be made between the drug release from the gels measured in vitro and the skin permeation ex vivo. It is evident that when the drug release from the formulation is sufficiently prolonged, the drug penetration rate through the skin is also reduced, which can be seen when comparing Figure 17 A and C with B and D. The rheological properties of the physically cross-linked gels composed with capric acid containing aggregates exhibited weak gel properties. The drug release profiles from the USP paddle experiments showed that the drug release from the gels containing these vesicles was not as prolonged as it had been when SDS was used. This indicates that capric acid renders fewer aggregates than SDS. In turn, the presence of fewer aggregates implies that there are fewer cross-links formed, and therefore the resultant gels have a weaker structure. As the molar ratio of surfactant is much larger in the capric acid mixtures than the SDS ones, there are probably also structural differences in the respective catanionic aggregates.

Regardless of whether or not the monomer concentration is higher in the capric acid mixtures than in the SDS mixtures and regardless of whether or not capric acid is more gentle to tissue than SDS, none of the formulations tested in Papers IV or V showed any morphological effects on the skin samples. Nor were they distinguishable from the skin samples exposed to air or saline solution. A selection of micrographs of skin exposed to formulations used in Paper IV is shown in Figure 18. It is possible that capric acid is more gentle to biological membranes and that the monomer concentrations of SDS are too low to induce visible damage, however definitive conclusions cannot be drawn from the research conducted for this thesis. One could anticipate that any damaging effects induced by the surfactants would be more visible on more sensitive tissues.

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Figure 18. Representative micrographs of skin samples exposed to (A) saline, (B) 14 mM tetracaine in Carbopol 940 and (C) tetracaine/SDS (at a 35:65 ratio at a concentration of 40 mM) in 1% Carbopol 940. The size bar indicates 50 m. Note that there are no visible differences in the quality of the skin regardless if it has been exposed to saline, drug or catanionic aggregates composed of drug and oppositely charged surfactant.

If the use of a synthetic material instead of skin was possible, the experiments conducted in Papers IV and V could be simplified. The silicone sheets employed in Paper IV proved to generate apparent penetration that was not statistically significant from the ones obtained when skin was used. Nevertheless, the amount of drug substance found at the donor side of the Ussing chambers after 24 hours was not in the same range. The most likely reason for this is that the silicone is not affected in the same way as the skin by the occlusion and hydration obtained in this setup. The study conducted in Paper IV showed that silicone sheets might be used as a model of skin over a clinically relevant time span. In addition, silicone sheets have previously been shown to function as a model of skin [110-111].

The tape stripping experiments conducted in conjunction with Paper V indicated that around 4 percent of the applied drug substance could be found in the skin after 24 hours and that most of it was located to the deepest layers of the stratum corneum. When a local treatment is sought the aim is to concentrate the drug substance in the skin. In contrast, when a systemic treatment is desired, the goal is to achieve as high drug concentrations in the blood stream as possible. The data generated in Papers IV and V do not provide sufficient evidence to conclude if these systems are more suitable for cutaneous or transdermal administration. The data does suggest, however, that as the amount of drug substance that is transferred from the formulations through the skin or silicone membranes is rather low, the effect of the drug substance applied would most likely only be local. Despite this, with a highly potent drug it would be possible to obtain a systemic effect.

The major conclusion from Papers IV and V is that the prolonged drug release rendered by catanionic aggregates contained in gels offers reduced skin penetration. The effects seen are obtained because the aggregates are stable within the gels even as most of the drug substance is released.

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6. Concluding remarks

Catanionic aggregates composed of a drug substance and an oppositely charged surfactant were investigated as a means of prolonging the drug release from gels. It was revealed that catanionic aggregates offer a promising way to increase the efficiency of pharmaceutical gel formulations. In greater detail, this thesis shows that:

Catanionic aggregates are commonly formed when surfactants are mixed with drug substances and either cationic or anionic drugs can be used provided that they have amphiphilic features. It is likely that many other drug substances than those tested form catanionic aggregates and these could also be used for prolonged drug release purposes. However, as surfactants are used in many pharmaceutical products, formation of catanionic aggregates may also lead to unwanted properties as a result of unforeseen interactions.

Drug release is prolonged considerably when catanionic aggregates are contained in gels. The apparent diffusion coefficient was lowered between 10 and 100 times, depending on the type of aggregate that was being investigated. A prolonged drug release potentially increases the efficiency of the formulation and the patient compliance.

The oppositely charged surfactant can be of natural origin, bearing low irritancy. The aggregates generated with the natural surfactants induced a similar prolongation of the drug release to that induced by aggregates consisting of conventional surfactants. The ability to minimize the toxicity of the excipients in the formulation is promising.

Drug-containing catanionic vesicles form cross-links and result in gels when mixed with certain polymers. The interactions are strong enough to generate gels with an extensive shelf life, but the gel properties are slightly frequency dependent which indicates a somewhat weaker structure than that of the covalently cross-linked gels.

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The drug release from physically cross-linked gels can be prolonged. As the drug substance constitutes the cross-links of a physical gel the gel is broken down when the drug substance is released. A gel that disassociates entirely could be beneficial for clinical use, as empty gel is not left at the site of application when the drug has been released. The apparent diffusion coefficients of drugs contained in physical gels were in the same range as when classical, covalent, gels were used. However, the release is more rapid at later times as the gels break up when the drug is released.

Drug penetration rate through the skin can be reduced using catanionic vesicles contained in gels. When the release of drug substance from a gel is prolonged sufficiently, the amount of substance available for diffusion across the skin is reduced. The penetration rate was substantially reduced when catanionic aggregates were used compared to reference gels containing only the drug substance. The catanionic aggregates contain surfactants that are potentially harmful to the skin but no morphological effects were visible, probably owing to the low concentrations of unaggregated surfactant.

Catanionic vesicles were present in gels throughout the drug release process and clinically relevant topical application time. A confinement of drug and surfactant in the gel probably results in local concentrations above the mixed-CAC. In the physical gels, vesicles were also observed when the amount of gel decreased. When combined with the rheological investigations, the results reveal that the vesicles constitute the cross-links of these gels.

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7. Populärvetenskaplig sammanfattning: Fördröjd läkemedelsfrisättning från geler med katanjoniska aggregat

Geler används ofta som läkemedelsbärare vid olika former av behandling och kan användas både för lokal behandling och behandling av hela kroppen. En av de främsta anledningarna till att geler är lämpliga att använda på slemhinnor eller huden är deras förmåga att stanna kvar länge, antingen på grund av deras konsistens eller för att de fäster på huden eller slemhinnan. Att geler stannar kvar längre gör att mer läkemedelssubstans hinner tas upp över den barriär som utgörs av huden eller slemhinnan. Detta kan medföra att patienten inte behöver använda läkemedlet lika ofta, eller att läkemedelsdosen kan minskas. Geler består av ett tredimensionellt nätverk av gelbildare i vatten. Eftersom huvuddelen av innehållet i geler är vatten, ofta närmare 95-99 %, rör sig upplösta läkemedelsmolekyler mycket snabbt genom och ut ur nätverket. En gel töms därför mycket snabbt på läkemedel och en tom gel är inte fördelaktig ur behandlingssynpunkt även om den finns kvar på kroppen länge. Frisättningen från gelen måste därför fördröjas, vilket var målet med den här avhandlingen. Katanjoniska aggregat bildas spontant i vattenlösningar av motsatt laddade ytaktiva ämnen. Ytaktiva ämnen finns i många produkter som vi använder i vardagslivet som tvål, tandkräm och diskmedel. Läkemedel kan ha egenskaper som liknar de vanliga ytaktiva ämnena och därför kan man byta ut ett av dem mot ett läkemedel i de katanjoniska aggregaten. Beroende på hur mycket av de olika ingredienserna man använder får man olika utseende på aggregaten. Eftersom aggregaten består av stora mängder av läkemedel och ytaktivt ämne blir de mycket större än de enskilda byggstenarna som de består av. De största aggregaten kallas vesiklar och ser ut som ballonger, där skalet består av katanjoner. Den ökade storleken gör att läkemedlet fastnar inne i gelnätverket. Det har visat sig att läkemedlet kommer ut 10-100 gånger långsammare ur gelen när det ingår i ett katanjoniskt aggregat. Den här avhandlingen har visat att det är vanligt att läkemedel som har ytaktiva egenskaper bildar katanjoniska aggregat. Den har också visat att

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ytaktiva ämnen med biologiskt ursprung kan användas istället för syntetiska. På så sätt kan antagligen toxiciteten hos de katanjoniska aggregaten minskas och fler användningsområden blir möjliga. I en vanlig gel är polymeren tvärbunden med sina egna komponenter, och strukturen finns kvar även när läkemedlet frisatts ur gelen. Genom att blanda katanjoniska vesiklar och polymerer skapades knutpunkter mellan polymererna genom interaktioner med de katanjoniska vesiklarna. Ett gelnätverk skapades, där läkemedlet var en del av strukturen. I Figur 1 illustreras en jämförelse mellan de olika geltyperna. Frisättningen av läkemedel var ungefär lika långsam från den här typen av geler som från de vanliga gelerna. När läkemedlet frisätts från gelen bryts också gelnätverket ner, och därför blir det ingen tom gel kvar när läkemedlet har försvunnit från gelen. Användningsområden för geler med katanjoner undersöktes också i den här avhandlingen. Det tog längre tid för läkemedlet att spridas genom huden när katanjoniska aggregat användes. Undersökningar visar att vesiklar finns kvar inne i gelerna under hela frisättningsförloppet också då gelerna används på hud. Det fanns inga strukturella skillnader på hud som utsatts för gel med katanjoner eller bara läkemedel jämfört med hud som inte utsatts för någonting. Den här avhandlingen visar att nyttan för gelläkemedel kan ökas med katanjoner. Det är möjligt att använda många olika läkemedel och ytaktiva ämnen, även sådana som inte irriterar kroppen så mycket. Frisättningen av läkemedel från gelerna kan göras så långsam att ett långsammare upptag i kroppen fås. Det visar att katanjoner kan göra att gelläkemedel under lång tid utövar en bättre effekt.

Figur 1. En illustration av hur katanjoniska vesiklar ryms i en vanlig gel där polymeren är sammanlänkad med sina egna komponenter (A) och hur detta skiljer sig från när katanjoniska vesiklar sammanlänkar en polymer (B).

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8. Acknowledgements

I would like to thank the following people: Professors Göran Alderborn and Martin Malmsten, for providing excellent facilities and scientific environment.

Min handledare Katarina Edsman, tack för allt stöd och snabba svar. Tack också för att jag fick komma tillbaka efter ex-jobbet och för trevliga middagar såväl i Uppsala som under resor. Min handledare Erik Björk, för din breda kunskap och skarpa blick och för att du tog över mig. Många roliga konferensresor och olika sportutmaningar har varit en viktig del i min doktorandutbildning. Magnus Bergström och Per Hansson, för att ni svarat på svåra frågor på ett sätt som även jag förstod (ibland). Tobias Bramer, för att du gav mig ett sådant varmt välkomnande och uppmanade mig att komma hem från England. Tack för ditt stöd under våra gemensamma projekt och trevligt sällskap på och utanför institutionen. För trevligt sällskap på USA-resorna och på andra glada tillställningar, som t ex födelsedagsfester. Nelly Fransén, för allt trevligt sällskap och head-bangande samt för den flärdfulla atmosfären i näs-pulver-gel-gruppen. Min rumskompis Jenny Pedersen, för alla trevliga pratstunder, goda middagar och assistans i kniviga situationer. Och för alla tjuvnyp och heliumexperiment som livade upp de tråkiga stunderna. Erik Sjögren, min egen mr President. Det var aldrig tråkigt att vara din vice. Katarina Edwards, min medförfattare, för din skarpa blick, engagemang och kunskap. Robin Grankvist, min ex-jobbare, för att du såg till att min utrustning fungerade innan jag använde den och för de trevliga stunderna på lab.

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Mina nuvarande och tidigare doktorandkollegor på institutionen för att ni gjort det så trevligt att vara på och utanför jobbet. Jag vill också tacka alla andra som jobbar på institutionen för att ni gör det så roligt att gå dit varje dag. Anna-Maria Lundins stipendienämnd, Apotekare CD Carlssons stiftelse, CR Leffmans resestipendium, IF:s stiftelse och Rektors resebidrag från Wallenbergstiftelsen, för generösa bidrag som gjorde det möjligt för mig att presentera min forskning på konferenser på orter av varierande exotisk grad samt att delta i mycket värdefulla kurser. Jag har också ett värdefullt liv utanför institutionen och vill därför tacka följande personer: Ted och Fredrik Dacksten för alla trevliga middagar hemma och på landet, samt bästa platserna på Falsterbo Horse Show varje år. Michael Fant och Tobias Svensson för alla kvällar med film och (o)nyttigheter och för alla gånger jag fått låna er bil. Henrik Fant, tack för din gästfrihet och klarsyn. Rebecca Fransson och Anna Lampa, för att ni är mina blonde bombshells och för att vi tillsammans med Olof Svensson är ”de farliga fyra” som gör det vi är bäst på. Jag vill också tacka Betsy för alla varma åkturer. Kajsa Holmquist, för att du är rationell och snygg, med en underbar humor. Joakim Jansson, för att du lär mig nya saker och alltid fyller på mitt champagneglas. Liv Johannesson, för att du tar med mig ut på springturer och skvallrar med mig, och för att vi dricker vin efteråt. Karin Johansson, för alla trevliga år tillsammans. Hoppas att det blir många fler! Tobias Johansson, för alla underbara resor och äventyr, din humor och sarkasm samt för att du uppskattar det goda i livet på samma sätt som jag. Ulrika Murphy, för att du är så oerhört rolig, glad och omtänksam, och en av de få personer som kan prata lika fort som jag.

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Michael och Edit Murphy för att jag har fått förmånen att nöta på er häst Magnus. Utan ridningen vore livet inte den njutning det är, även om jag fallit av hästen några gånger. Christopher Naffah och Fredrik Svensson för att jag ALLTID får sova på er soffa och sen får pannkakor till frukost. Anna och Åsa Rising för att ni tar hand om mig och mina djur, och för att jag fått vara en del av er familj. Alla mina hundvakter (ni är många), för att ni ställer upp när jag inte hinner. Mest av allt vill jag tacka hela min underbara familj, för all kärlek och ert stöd, i synnerhet: Pelle, min hund, för att du alltid älskar mig mest och ser till att jag åker hem från jobbet i tid. Sunlei, min älskade syster, bästa vän och sugar-mama, and Kevin Redmond, for taking care of me, always welcoming me to your home and showing me the best of times. Mina föräldrar: mamma Elisabet och pappa Francis för att jag har ärvt er hängivenhet, tålamod och nyfikenhet. För att ni alltid har trott på min förmåga. Utan er hade jag inte kunnat genomföra det här. Emil Lundberg, för allt du ger mig. Jag vill vakna med dig varje morgon.

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