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The use of catanionic drug-surfactant mixtures was proven to be an efficient novel method of obtaining prolonged drug release from gels. It was shown that various commonly used drug compounds are able to form catanionic mixtures together with oppositely charged surfactants. These mixtures exhibited interesting phase behaviour, where, among other structures, vesicles and large worm-like or branched micelles were found. The size of these aggregates makes them a potential means of prolonging the drug release from gels, as only monomer drugs in equilibrium with larger aggregates were readily able to diffuse through the gel. When the diffusion coefficient for drug release from the formulation based upon a catanionic mixture was compared to that obtained for the drug substance and gel alone, the coefficient was some 10 to 100 times smaller. The effects of changes in the pH and ionic strength on the catanionic aggregates was also investigated, and this method of prolonging the release was found to be quite resilient to variations in both. Although the phase behaviour was somewhat affected, large micelles and vesicles were still readily found. The drug release was significantly prolonged even under physiological conditions, that is, at a pH of 7.4 and an osmolality corresponding to 0.9% NaCl. Surfactants of low irritancy, capric and lauric acid, may successfully be used instead of the more traditional surfactants, such as sodium lauryl sulfate (SDS), and prolonged release can still be obtained with ease. Some attempts to deduce the release mechanism from the proposed systems have also been made using transient current measurements, dielectric spectroscopy, and modelling of the release using the regular solution theory. In these studies, the previous assumptions made concerning the mechanism responsible for the release were confirmed to a large extent. Only small amounts of the drug existed in monomer form, and most seemed to form large catanionic aggregates with the oppositely charged surfactant.

Nasal administration of analgesics for achieving rapid pain relief is currently a topic of great interest. The blood-brain barrier (BBB) restricts access to the central nervous system (CNS) for several central-acting drugs, such as morphine and dihydroergotamine, which results in a substantial effect delay. Evidence for the olfactory transfer of drugs from the nasal cavity to the CNS after nasal administration, bypassing the BBB, is available for both animals and humans. The aims of this thesis were to study the olfactory transfer of morphine to the CNS after nasal administration, and to compare the nasal transport of analgesic drugs across nasal respiratory and olfactory mucosa. In vivo studies in rodents demonstrated that morphine is transferred via olfactory pathways to the olfactory bulbs and the longitudinal fissure of the brain after nasal administration. Further, olfactory transfer of morphine significantly contributed to the early high morphine brain hemisphere concentrations seen after nasal administration to rats. Olfactory transfer was tracked by collecting and analysing brain tissue and blood samples after right-sided nasal administration and comparing the results to the situation after i.v. administration. The olfactory transfer was also visualised by brain autoradiography. In vitro studies indicated that the olfactory mucosa should not be a major barrier to the olfactory transfer of dihydroergotamine or morphine, since transport of these drugs was no more restricted across the olfactory mucosa than across the nasal respiratory mucosa. The in vitro studies were performed using the horizontal Ussing chamber method. This method was further developed to enable comparison of drug transport across nasal respiratory and olfactory mucosa which cannot be achieved in vivo. In conclusion, these analgesic drugs showed potential for olfactory transfer, and access to the CNS by this route should be further investigated in humans, especially for the drugs with central effects that are currently under development for nasal administration.

The aim of this thesis was to identify some functionality related characteristics of pharmaceutically relevant solid materials and to study the effect of their variation on processing behaviour and product performance. For this purpose, particles with different characteristics were prepared under a variety of conditions by crystal agglomeration, wet granulation and spray drying. The effect of particle size distribution on the evolution of the tablet microstructure during and after compression was investigated. The compression behaviour of particles with different nominal strength and degrees of agglomeration was studied and the influence of the surfactant concentration of amorphous particles on the compression behaviour was examined. The response of the powders to compression was described with the help of various techniques characterising the microstructure and tensile strength of the tablets produced. Furthermore, a method suitable for observing drug release from single matrix granules was developed and used to study the effect of granule porosity and compaction pressure on the drug release process. The particle size distribution did not influence the evolution of the tablet porosity or the tensile strength during compression, but it could have an effect on the evolution of the tablet microstructure during short-term storage, depending on the instability mechanism. The compression behaviour of particles prepared by crystal agglomeration and wet granulation was dependent on their degree of agglomeration and their failure strength. For particles with similar solid state properties and compression behaviour, the surface energy appears to have an effect on the bonding strength of adsorption bonds acting at interparticulate junctions. Using the method developed to observe the drug release from single matrix granules, reproducible data was obtained enabling the drug release process to be characterised. Depending on the type of matrix and the compaction pressure, the drug release rate could be enhanced or retarded.

Transport into and across the cells of the human body is a prerequisite for the pharmacological action of drugs. Passive membrane permeability and active transport mechanisms are major determinants of the intestinal absorption of drugs, as well as of the distribution to target tissues and the subsequent metabolism and excretion from the body. In this thesis, the role of ATP-binding cassette (ABC) transporters and passive permeability on drug absorption and disposition was investigated. Particular emphasis was placed on defining the molecular properties important for these transport mechanisms. The influence of different transport pathways on predictions of intestinal drug absorption was investigated using experimental models of different complexity. Experimental models that include the paracellular pathway gave improved predictions of intestinal drug absorption, especially for incompletely absorbed drugs. Further, the inhibition of the ABC transporters breast cancer resistance protein (BCRP/ABCG2) and multidrug-resistance associated protein 2 (MRP2/ABCC2) was experimentally investigated using structurally diverse datasets that were representative of orally administered drugs. A large number of previously unknown inhibitors were identified among registered drugs, but their clinical relevance for drug-drug interactions and drug-induced toxicity remains to be determined. The majority of the inhibitors affected all three major ABC transporters BCRP, MRP2 and P-glycoprotein (P gp/ABCB1), and these multi-specific inhibitors were found to be enriched in highly lipophilic weak bases. To summarize, the present work has led to an increased knowledge of the molecular features of importance for ABC transporter inhibition and passive membrane permeability. Previously unknown ABC transporter inhibitors were identified and predictive computational models were developed for the different drug transport mechanisms. These could be valuable tools to assist in the prioritization of experimental efforts in early drug discovery.

Aqueous solubility is a key parameter influencing the bioavailability of drugs and drug candidates. In this thesis computational models for the prediction of aqueous drug solubility were explored. High quality experimental solubility data for drugs were generated using a standardised protocol and models were developed using multivariate data analysis tools and calculated molecular descriptors. In addition, structural features associated with either solid-state limited or solvation limited solubility of drugs were identified. Solvation, as represented by the octanol-water partition coefficient (logP), was found to be the dominant factor limiting the solubility of drugs, with solid-state properties being the second most important limiting factor. The relationship between the chemical structure of drugs and the strength of their crystal lattice was studied for a dataset displaying logP-independent solubility. Large, rigid and flat molecules with an extended ring-structure and a large number of conjugated π-bonds were found to be more likely to have their solubility limited by a strong crystal lattice than were small, spherically shaped molecules with flexible side-chains. Finally, the relationship between chemical structure and drug solvation was studied using computer simulated values of the free energy of hydration. Drugs exhibiting poor hydration were found to be large and flexible, to have low polarisability and few hydrogen bond acceptors and donors. The relationship between the structural features of drugs and their aqueous solubility discussed in this thesis provide new rules-of-thumb that could guide decision-making in early drug discovery.

Cellulose powders from various sources were manufactured and characterized to investigate the influence of their crystallinity index, surface area, and pore volume on sorption phenomena and the relevant pharmaceutical functionality. The influence of the cellulose crystallinity index on moisture sorption was important at low and intermediate relative humidities. At high relative humidities, properties such as surface area and pore volume took precedence in governing the moisture sorption process. The theory of physical adsorption of gases onto fractal surfaces was useful for understanding the distribution of water in cellulose and the inner nanoscale structure of cellulose particles. It was found that, as a consequence of swelling, moisture induces a fractal nanopore network in cellulose powders that have a low or intermediate degree of crystallinity. On the other hand, no swelling occurs in highly crystalline cellulose powders and moisture sorption is restricted to the walls of the open pores. No correlation was found between the cellulose crystallinity index and the incorporation and release of nicotine in cellulose mixtures. By loading nicotine in highly porous matrices of the Cladophora sp. algae cellulose, higher stability against oxidative degradation, higher loading capacity, and more steady release into an air-stream was achieved than when commercially available microcrystalline cellulose was loaded. It was also shown that, by manipulating the structure of cellulose, the undesired hydrolysis of acetylsalicylic acid in mixtures with cellulose can be avoided. It was suggested that a broad hysteresis loop between the moisture adsorption and desorption curves of isotherms at low relative humidities could be indicative of an improved compatibility between acetylsalicylic acid and cellulose. In all, this thesis demonstrates how the pharmaceutical functionality of microcrystalline cellulose can be improved via engineering of the structure of native cellulose powders.

The appearance of adverse drug reactions is a common reason for hospitalization in Western countries. Research on underlying biological mechanisms for interindividual variability in drug response aims to better identify patients with exceptional genetic traits, disease conditions or risk of drug-drug interactions and thereby help to prevent adverse drug reactions. Active transport mechanisms are involved in the absorption and disposition of several therapeutic agents. The main objective of this thesis was to investigate factors potentially affecting transport proteins and thus contributing to variability in drug absorption and disposition. Studies of physiological, genetic, environmental, and pathological factors were included. The main focus was the two ATP-binding cassette (ABC) transporters: P-glycoprotein 170 (Pgp) and Breast Cancer Resistance Protein (BCRP). Quantification of transport protein mRNAs along the human intestine indicated that eight of the nine investigated drug transporters were expressed in a region-dependent manner. Effects of drug-drug interactions may therefore vary depending on the site of absorption. The genetic aspect was illustrated by identification of sequence variation in the gene encoding BCRP, the most highly expressed ABC transporter along the human intestine. Drug-drug interactions are important environmental causes of interindividual variability. An evaluation of the effects of Pgp-mediated drug-drug interactions showed that patients receiving Pgp inhibitors had elevated serum concentrations of the Pgp substrate digoxin and that digoxin concentrations were positively correlated with the number of co-administered Pgp inhibitors. The final topic in this thesis was that of drug-disease interactions. BCRP and Pgp were down-regulated during active inflammation in patients with ulcerative colitis. This may contribute to altered concentrations of drug in the intestinal mucosa during periods of inflammation and possibly to changes in drug absorption. To summarize, results of this thesis emphasize the complex background to the interindividual variability of drug transport proteins, where physiological, genetic, environmental and pathological factors all can contribute.

Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems. This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine. The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system. The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an in vitro model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, Yersinia pseudotuberculosis, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.

The use of nano- and micro-particulate carriers as delivery systems for oral vaccines has been under investigation for several decades. Surprisingly little is known of their uptake in the human intestine, despite the fact that substantial improvement is required to achieve adequate immune responses in man after oral administration. In this thesis, various aspects of particle transcytosis across the human intestinal epithelium were studied, in order to identify strategies for improved uptake of nano- and micro-particulate drug delivery systems. The follicle associated epithelium (FAE) overlying Peyer´s patches contains M-cells, which have an increased capacity for uptake of particulate antigens. Therefore, a model of human FAE was developed to study mechanisms of particle uptake and transport. Receptors that could be used for targeting to the FAE had previously not been identified in humans. By use of the model FAE, two new targets were identified on human intestinal FAE; CD9 and β1-integrin. Furthermore, studies of isolated human intestinal tissue showed that an integrin-adherent peptide motif, RGD, could be utilized to achieve selective and improved transport of nanoparticles into human Peyer´s patches. Studies of factors influencing intestinal particle uptake and transcytosis revealed that two cytokines, TNF-α and LTα1/β2, but also one growth factor, TGF-β1, induced uptake of particles in Caco-2 cells and transcytosis of particles in the model FAE. Furthermore, it was shown that an enteric bacterium, Yersinia Pseudotuberculosis, could trigger uptake and transcytosis of particles across model absorptive epithelial cells. In conclusion, this thesis provides a platform for further investigations of particle transcytosis across the human intestinal epithelium. The identification of two new proteins with increased expression in human FAE and a targeting sequence that improves particle uptake into Peyer’s patches, gives new hope for the development of subunit oral vaccines.

Spray drying is a method with a high potential in the preparation of protein particles suitable for pulmonary delivery. However, surface induced denaturation of bio-molecules during atomization and subsequent drying can be substantial and it is therefore important to develop new formulation concept for concurrent encapsulation and stabilization of proteins during spray drying. Hence, with an overall objective to increase the knowledge of the formation of particulate systems for systemic administration of proteins by spray drying, the first part of this thesis, systematically investigated the particle formation by droplet size and particle size measurements. It was described how specific properties, such as the solubility and the crystallization propensity of the solute, can affect the product, e.g. the particle size, internal structures, and possibly particle density. A new method using atomic force microscopy (AFM) for the assessment of the effective particle density of individual spray-dried particles was demonstrated. In the second part, two different formulation concepts for encapsulation of protein during spray drying were developed. Both systems used non-ionic polymers for competitive adsorption and displacement of protein from the air/water interface during spray drying. The aqueous two-phase system (ATPS) of polyvinyl alcohol (PVA) and dextran, and the surface-active polymers, hydroxypropyl methylcellulose (HPMC) and triblock co-polymer (poloxamer 188) used for in situ coating, proved efficient in encapsulation of a model protein, bovine serum albumin (BSA). Inclusion of polymeric materials in a carbohydrate matrix also influenced several particle properties, such as the particle shape and the surface morphology, and was caused by changes in the chemical composition of the particle surface and possibly the surface rheology. In addition, powder performance of pharmaceutical relevance, such as dissolution and flowability, were affected.

Most pathogens enter the body via mucosal surfaces. In contrast to parenterally administered vaccination, mucosal vaccination has the advantage of eliciting both a systemic and a local mucosal immune response. An oral biodegradable adjuvant with these features would have great potential. This thesis has focused on the development of a new oral vaccine against diphtheria. Biodegradable polyacryl starch microparticles were used as a mucosal adjuvant. Diphtheria toxin or cross-reacting material of diphtheria toxin (CRM197) was covalently conjugated to the microparticles and fed to mice by oral gavage. Formaldehyde treatment was also studied as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations. All formulations given to mice orally or parenterally, but not intranasally, induced a strong systemic immune response and diphtheria toxin neutralising antibodies. Only formulations administered orally induced a mucosal IgA response as well. The non-toxic recombinant protein CRM197 proved to be a promising antigen candidate in an oral diphtheria vaccine when conjugated to the microparticles. Mild treatment of CRM197 with formaldehyde before conjugation to the starch microparticles potentiated the immunogenicity of the formulation. However, no immune response was detected in healthy volunteers after administration of this vaccine in a phase I trial. The possible reasons for the difference in response between mouse and man are discussed. The use of cDNA expression macro array technology was also evaluated as a tool in vaccine-related research. Tetanus toxoid and aluminium phosphate were used as model parenteral antigen and adjuvant. It was concluded that the antigen modulates the molecular mechanisms of the aluminium phosphate adjuvant to a greater extent than previously recognised.

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders. The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO2) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive. The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein. In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.

This thesis presents two new concepts in oral drug administration and the results of evaluation of some relevant formulation factors. Investigation into improving the homogeneity of mixtures for tableting indicated that it may be possible to obtain interactive dry mixtures of micronised drugs containing drug proportions as low as 0.015% w/w. By studying the relationship between disintegration time and tensile strength, it was found that the microstructure surrounding the disintegrant particles may influence the disintegration process. Therefore, avoidance of excipients which are highly deformable or very soluble in water will result in more rapid disintegration. Further, it is possible to increase the bioadhesive properties of a non-bioadhesive carrier material by forming interactive mixtures containing a fine particulate bioadhesive material. The new sublingual tablet concept presented is based on interactive mixtures consisting of a water-soluble carrier covered with fine drug particles and a bioadhesive component. With this approach, it is possible to obtain rapid dissolution in combination with bioadhesive retention of the drug in the oral cavity. Clinical data indicate that this allows rapid sublingual absorption while simultaneously avoiding intestinal absorption. An individualised dose administration system is also presented. This system is based on the use of standardised units (microtablets), each containing a sub-therapeutic amount of the active ingredient. The required dose is fine-tuned by electronically counting out a specific number of these units using an automatic dose dispenser. A patient handling study supported the suggestion that the dosage of some medications can be more easily and safely individualised for each patient with this method than by using traditional methods of mixing different standard tablet strengths or dividing tablets.

Relationships between stresses during the drying process, particle structural and functional properties, and particle engineering by the drying process were addressed in this thesis. In the first part, the importance of the drying phase and the effect of the drying rate on the intragranular porosity of microcrystalline cellulose pellets were investigated. Differences in porosities of dried pellets could be explained by liquid-related differences in densification during convective drying rather than by differences in densification during wet agglomeration. An increased drying rate gave more porous pellets with a lower compression shear strength, and thereby stronger tablets. The next part dealt with modulation of solid-state stability and tabletting behaviour of amorphous lactose by incorporation of different polymers by spray drying. Increased content and molecular weight of poly(vinylpyrrolidone) (PVP) resulted in an increased resistance to crystallisation provoked by heat and moisture. The stabilising effect was even more evident after long-term storage. However, the glass transition temperature was almost unaffected and may, therefore, be questioned as a stability indicator for these types of materials. The presence of the polymers resulted in somewhat less deformable particles. Incorporation of PVP increased the compactability, whilst a surfactant decreased it, which could be shown to be related to differences in particle-particle adhesivity between the different particles. This thesis contributes to increased mechanistic understanding in the area of particle engineering that may lead to better prediction and optimisation of the functionality of pharmaceutical particles, which is of the utmost importance in the development and production of solid dosage forms.

Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of Salmonella enterica serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic Escherichia coli. The immune response elicited using secreted proteins from S. enterica serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant Salmonella atypical fimbriae (SafB/D) complexes, a conserved structure within Salmonella species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.

Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine in vivo. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human jejunum for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2. The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.

The preparation of multiple unit tablets was investigated in this thesis with the intention of gaining a deeper understanding of some of the factors that influence the properties of such tablets. Initially, three different types of pellets (drug, soft and disintegrant pellets) were combined as a model to investigate the ability of the mixture to form disintegrating tablets. The proportions of the different pellets and the type of disintegrant used were factors that independently influenced the tablet properties. Furthermore, the properties of tablets containing drug pellets barrier-coated with an aqueous polymer dispersion were also found to depend on the coating thickness and the compaction pressure. When compacting pellets barrier-coated with a solvent-based polymer solution without incorporating excipient particles in the tablet formulation, a high pellet porosity was advantageous to preserve the original drug release profile, even though highly porous pellets became more densified and deformed than pellets of lower porosity. The influence of the properties of excipient particles on the deformation of the reservoir pellets was also studied and, although the amount of flattening of the pellets was only slightly affected, changes in the pellet shape (irregularity) with alterations in the porosity and size of the excipient particles were more substantial. In contrast, the properties of the excipient particles did not affect the pellet densification. The solvent-based coating used was able to adapt to the changes in volume and shape that the pellets underwent during compaction. The coating structure appears to be changed by compaction and it is proposed that the final structure of the coating is the net effect of two parallel processes, one reducing and one prolonging the transport time of the drug across the coating. Thus, the drug release could be maintained or even prolonged after compaction, despite extensive structural changes of the reservoir pellets.

The subject of this thesis was to study two intestinal barriers to oral drug bioavailability, drug efflux proteins of the ABC-transporter family, and in particular ABCB1/P-glycoprotein (Pgp), and the drug metabolizing enzyme cytochrome P450 (CYP) 3A4. At the onset of this thesis, similarities between CYP3A4 and Pgp in terms of their tissue distribution and gene regulation, along with overlapping substrate specificities, had generated the hypothesis that CYP3A4 and Pgp may have a complementary function and thus form a coordinated intestinal barrier to drug absorption and gut wall metabolism. In the first part of this thesis, a cell culture model of the intestinal epithelium that expressed both functional Pgp and CYP3A4 was developed. This model was then used to investigate the steroselective drug efflux and metabolism of R/S-verapamil. In summary, the results indicated that the two barriers in the cell culture model were in agreement with those in the human intestine. Both ABC-transporters and CYPs are regulated by drugs that interact with nuclear receptors. However, while the regulation of CYPs is quite well understood, less is known about how repeated drug administration regulates the most abundantly expressed ABC-transporters. Therefore, in the second part of this thesis, the effects of repeated drug administration on the gene regulation of four ABC-transporters and CYP3A4 were studied in intestinal epithelial cell lines in vitro and in the perfused human jejunum in vivo. The in vitro studies revealed that the ABC-transporters are induced by drugs that interact with slightly different sets of nuclear receptors. The in vivo study showed that repeated oral administration of St John’s wort decreased the bioavailability of verapamil, predominantly by induction of intestinal CYP3A4. This part of the thesis provides new information about the regulation of ABC-transporters, shows that the intestinal metabolism is the most significant barrier to oral bioavailability of verapamil and provides evidence for a clinically significant interaction between verapamil and St John’s wort in vivo.

The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems in vitro and after lung administration in vivo were established for the first time. Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells in vitro and after lung administration in vivo. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for in vivo gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI in vitro and in vivo. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for in vivo gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.

New effective experimental techniques in medicinal chemistry and pharmacology have resulted in a vast increase in the number of pharmacologically interesting compounds. However, the number of new drugs undergoing clinical trial has not augmented at the same pace, which in part has been attributed to poor absorption of the compounds. The main objective of this thesis was to investigate whether computer-based models devised from calculated molecular descriptors can be used to predict aqueous drug solubility, an important property influencing the absorption process. For this purpose, both experimental and computational studies were performed. A new small-scale shake flask method for experimental solubility determination of crystalline compounds was devised. This method was used to experimentally determine solubility values used for the computational model development and to investigate the pH-dependent solubility of drugs. In the computer-based studies, rapidly calculated molecular descriptors were used to predict aqueous solubility and the melting point, a solid state characteristic of importance for the solubility. To predict the absorption process, drug permeability across the intestinal epithelium was also modeled. The results show that high quality solubility data of crystalline compounds can be obtained by the small-scale shake flask method in a microtiter plate format. The experimentally determined pH-dependent solubility profiles deviated largely from the profiles predicted by a traditionally used relationship, highlighting the risk of data extrapolation. The in silico solubility models identified the non-polar surface area and partitioned total surface areas as potential new molecular descriptors for solubility. General solubility models of high accuracy were obtained when combining the surface area descriptors with descriptors for electron distribution, connectivity, flexibility and polarity. The used descriptors proved to be related to the solvation of the molecule rather than to solid state properties. The surface area descriptors were also valid for permeability predictions, and the use of the solubility and permeability models in concert resulted in an excellent theoretical absorption classification. To summarize, the experimental and computational models devised in this thesis are improved absorption screening tools applicable to the lead optimization in the drug discovery process.