Studies have been performed to evaluate the use of micellar electrokinetic chromatography (MEKC), one mode of capillary electrophoresis (CE), as an analytical technique in industrial pharmaceutical analysis. The potential for using chemometrics for the optimisation of MEKC methods has also been studied as well as the possibilities of coupling MEKC with mass spectrometry (MS). Two methods were developed, one for the determination of ibuprofen and codeine and another for pilocarpine, together with their degradation products and impurities in both cases. MEKC was found to be the most suitable mode of CE for the methods. Both methods were optimised by means of experimental design. Valuable information was gathered and optimum conditions were defined which resulted in fast systems with baseline-separated peaks. The ibuprofen-codeine method was validated according to the recommended validation procedures of the International Conference of Harmonisation. The validation was performed on a commercially available tablet formulation to verify the suitability of the method, i.e. for quantification of the two main compounds and to determine the degradation products and impurities in area% of each main peak. The following parameters were determined: selectivity, linearity, accuracy, precision, detection limit, quantitation limit, robustness and range. The results confirm that the method is highly suitable for its intended purpose, i.e. as a routine method for assay and impurity determination. The MEKC method for ibuprofen-codeine was coupled to a mass spectrometer in order to evaluate the potential of partial filling (PF)-MEKC-MS for identification of impurities in pharmaceutical substances and products. The so-called partial-filling technique was used to prevent the non-volatile micelles from entering the MS and was shown to fulfil its purpose of providing detection limits of about 10 pg. The study clearly shows that micellar electrokinetic chromatography is well-suited as an analytical technique in industrial pharmaceutical analysis.

Statistical experimental designs have been used for method development and optimisation of separation. Two reversed phase HPLC methods were optimised. Parameters such as the pH, the amount of tetrabutylammonium (TBA; co-ion) and the gradient slope (acetonitrile) were investigated and optimised for separation of erythromycin A and eight related compounds. In the second method, a statistical experimental design was used, where the amounts of acetonitrile and octane sulphonate (OSA; counter ion) and the buffer concentration were studied, and generation of an α-plot with chromatogram simulations optimised the separation of six analytes. The partial filling technique was used in capillary electrophoresis to introduce the chiral selector Cel7A. The effect of the pH, the ionic strength and the amount of acetonitrile on the separation and the peak shape of R- and S-propranolol were investigated. Microemulsion electrokinetic chromatography (MEEKC) is a technique similar to micellar electrokinetic chromatography (MEKC), except that the microemulsion has a core of tiny droplets of oil inside the micelles. A large number of factors can be varied when using this technique. A screening design using the amounts of sodium dodecyl sulphate (SDS), Brij 35, 1-butanol and 2-propanol, the buffer concentration and the temperature as factors revealed that the amounts of SDS and 2-propanol were the most important factors for migration time and selectivity manipulation of eight different compounds varying in charge and hydrophobicity. SDS and 2-propanol in the MEEKC method were further investigated in a three-level full factorial design analysing 29 different compounds sorted into five different groups. Different optimisation strategies were evaluated such as generating response surface plots of the selectivity/resolution of the most critical pair of peaks, employing chromatographic functions, simplex optimisation in MODDE and 3D resolution maps in DryLab™. Molecular descriptors were fitted in a PLS model to retention data from the three-level full factorial design of the MEEKC system. Two different test sets were used to study the predictive ability of the training set. It was concluded that 86 – 89% of the retention data could be predicted correctly for new molecules (80 – 120% of the experimental values) with different settings of SDS and 2-propanol. Statistical experimental designs and chemometrics are valuable tools for the development and optimisation of analytical methods. The same chemometric strategies can be employed for all types of separation techniques.

This thesis concerns the development and utilisation of techniques for characterisation and transdermal delivery of various systems for pharmaceutical applications. The degree of dissociation of drug molecules and the mobilities of the different species formed are essential factors affecting the rate of drug delivery by iontophoresis. Hence, determination of drug mobility parameters and equilibrium constants are important for the development of iontophoretic systems. With capillary zone electrophoresis using a partial filling technique and methyl-β-cyclodextrin as chiral selector, the enantiomers of orciprenaline were separated. The association constants between the enantiomers of the drug and the selector were also evaluated. Precision conductometry studies were performed for the hydrochloride salts of lidocaine and 5-aminolevulinic acid in aqueous propylene glycol and water as media, respectively. Iontophoresis is a technique for drug delivery where charged molecules are transported into and through skin by application of a weak direct electrical current. The drugs 5-aminolevulinic acid and its methyl ester were used as model compounds and incorporated in two different drug delivery vehicles, a sponge phase and carbopol gel. The bicontinuous structure of the sponge phase, constituted of monoolein and a mixture of propylene glycol and water, makes it interesting for use in iontophoretic delivery, since ions can move more or less freely in the aqueous as well as in the lipid domains. Furthermore, all three components are known for their penetration enhancing abilities. Hydrogels like carbopol gels are interesting media with respect to iontophoretic studies, since devices for iontophoresis often utilize hydrogels as contact interfaces between the skin and the electrodes. The results indicate that the transport achieved iontophoretically using the gel (1 % active substance) was comparable with the passive delivery of clinically used formulations (16 % - 20 % active substance).

CYP2C9 is one of our major drug metabolising enzymes and belongs to the cytochrome P450 (CYP) super family. The aim of this thesis was to gain an understanding of the quantitative structure–activity relationships (QSAR) of CYP2C9 substrates and inhibitors. This information will be useful in predicting drug metabolism and the potential for drug–drug interactions. To achieve this, a well characterised data set of structurally diverse, competitive CYP2C9 inhibitors was identified in our laboratory. Several computational methodologies, many based on GRID molecular interaction fields, were applied or developed in order to handle issues such as compound alignment and bioactive conformer selection. First, a traditional 3D QSAR was carried out in GOLPE, generating a predictive model. In this model the selection of a bioactive conformer and alignment was based on docking in a homology model of CYP2C9. Secondly, we introduced the concept of alignment independent descriptors from ALMOND. These descriptors were used to generate quantitatively and qualitatively predictive models. We subsequently derived conformation independent descriptors from molecular interaction fields calculated in FlexGRID. This enabled the derivation of 3D QSAR models without taking into account the selection of an alignment or a bioactive conformer. A subsequent programming effort enabled the conversion of this model back to 3D aligned pharmacophores. Similar alignment independent descriptors were also used in the development of the software MetaSite® that predicts the site of metabolism for CYP2C9 ligands. Finally, as crystal information on this isoform emerged, the performance of molecular dynamics simulations and homology models and the flexibility of the protein were evaluated using statistical analyses. These modelling efforts have resulted in detailed knowledge of the structural characteristics in ligand interactions with the cytochrome P450 2C9 isoform.

Human Immunodeficiency Virus (HIV) is the causative agent of the pandemic disease Acquired Immune Deficiency Syndrome (AIDS). HIV acts to disrupt the immune system which makes the body susceptible to opportunistic infections. Untreated, AIDS is generally fatal. Twenty years of research by countless scientists around the world has led to the discovery and exploitation of several targets in the replication cycle of HIV. Many lives have been saved, prolonged and improved as a result of this massive effort. One particularly successful target has been the inhibition of HIV protease. In combination with the inhibition of HIV reverse transcriptase, protease inhibitors have helped to reduce viral loads and partially restore the immune system. Unfortunately, viral mutations leading to drug resistance and harmful side-effects of the current medicines have identified the need for new drugs to combat HIV. This study presents computational efforts to understand the interaction of inhibitors to HIV protease. The first part of this study has used molecular modelling and Comparative Molecular Field Analysis (CoMFA) to help explain the structure-active relationship of a novel series of protease inhibitors. The inhibitors are sulfamide derivatives structurally similar to the cyclic urea candidate drug mozenavir (DMP-450). The central ring of the sulfamides twists to adopt a nonsymmetrical binding mode distinct from that of the cyclic ureas. The energetics of this twist has been studied with ab initio calculations to develop improved empirical force field parameters for use in molecular modelling. The second part of this study has focused on an analysis of the association and dissociation kinetics of a broad collection of HIV protease inhibitors. Quantitative models have been derived using CoMFA which relate the dissociation rate back to the chemical structures. Efforts have also been made to improve the models by systematically varying the parameters used to generate them.

Many peptide receptors are of pharmaceutical interest and there is thus a need for new ligands for such receptors. Unfortunately, peptides are not suitable as orally administrated drugs since they are associated with poor absorption, rapid metabolism and low sub-receptor selectivity. One approach that should allow identification of more drug-like ligands is to use the structural information of the endogenous ligand to develop peptidomimetic compounds. The main objective of the work described in this thesis was to convert angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) to small drug-like compounds with retained bioactivity at the AT2 receptor. The study was performed step-wise via incorporation of well-defined secondary structure mimetics and repeated truncation of the peptide. Five scaffolds, comprising a benzene ring as a central element, suitable as a γ-turn or dipeptide mimetics were designed and synthesized. In order to decorate the scaffolds, a method of microwave-assisted alkoxycarbonylation was developed. After incorporation of the scaffolds into Ang II-related peptides or peptide fragments, the affinities for both the AT1 and the AT2 receptor were determined. In the first series of ligands, two tyrosine-related scaffolds were introduced as γ-turn mimetics in Ang II. All five pseudopeptides exhibited good affinities for the AT2 receptor. One compound was chosen for functional studies and was shown to act as an AT2 receptor agonist. After truncation of Ang II it was shown that C-terminal pentapeptide analogs were AT2 receptor selective agonists. A series of pseudopeptides comprising tyrosine-related scaffolds, derived from the pentapeptides, displayed high AT2 receptor affinities. Two compounds had agonistic effect at the AT2 receptor. This study revealed that the N-terminal part was of less importance while a C-terminal Ile residue was a key element for enhanced AT2 receptor affinity. In the final set of compounds, the peptide was truncated to tripeptide C-terminal fragments. After replacing His-Pro by a histidine-related scaffold small drug-like peptidomimetic compounds with nanomolar affinity for the AT2 receptor were identified.

<p>Homogeneous palladium-catalyzed Heck vinylation of aryl chlorides was investigated under air using Herrmann’s palladacycle and the P(<i>t</i>-Bu)₃-liberating salt [(<i>t</i>-Bu)₃PH]BF₄. Based on the results, controlled microwave heating was utilized to accelerate model Heck reactions with aryl chlorides down to 30 min employing an electron-poor olefin and a mixture of an ionic liquid and 1,4-dioxane as solvent.</p><p>For the first time, a highly regioselective general protocol has been developed for palladium-catalyzed terminal (β-) arylation of acyclic vinyl ethers using inexpensive aryl chlorides as starting materials and the preligand [(<i>t</i>-Bu)₃PH]BF₄ as the key additive. This swift and straightforward protocol exploits non-inert conditions and controlled microwave heating to reduce handling and processing times, and aqueous DMF or environmentally friendly PEG-200 as the reaction medium. Somewhat higher selectivity for the linear β-product was observed in PEG-200. DFT calculations were performed at the B3LYP level of theory for the regioselectivity-determining insertion step in the Heck reaction following the neutral pathway. A series of <i>para</i>-substituted phenylpalladium(II) complexes was investigated in the computational study. The calculations support a ligand-driven selectivity rationale, where the electronic and steric influence of the bulky P(<i>t</i>-Bu)₃ ligand provides improved β-selectivity. The preparative methodology was used to synthesize the β-adrenergic blocking agent Betaxolol.</p><p>Highly stereoselective Pd(0)-catalyzed β-arylation and β-vinylation of a tetra-substituted cyclopentenyl ether have been accomplished using a chiral, pyrrolidine-based and substrate-bound palladium(II)-directing group under neutral reaction conditions. To the best of the author’s knowledge, this P(<i>t</i>-Bu)₃-mediated method represents the first examples of the successful utilization of aryl and vinyl chlorides in asymmetric Heck reactions. The Heck arylation products formed were hydrolyzed and isolated as the corresponding quaternary 2-aryl-2-methyl cyclopentanones in good to moderate two-step yields with excellent stereoselectivity (90-96% ee). Inclusion of vinyl triflates under neutral reaction conditions and one aryl triflate equipped with a strongly electron-withdrawing <i>para</i>-cyano substituent under cationic conditions increased the preparative usefulness of the methodology.</p><p>Furthermore, diastereoselective Heck arylation of both five- and six-membered cyclic vinyl ethers with aryl bromides, using the identical chiral auxiliary and suitable Pd sources, was performed. Arylated products from the tetra-substituted cyclopentenyl ether were also in this case hydrolyzed to the corresponding 2-aryl-2-methyl cyclopentanones with high to excellent enantioselectivity (85-94% ee). Despite low reaction rates and relatively modest yields, arylation reactions with the tri-substituted cyclohexenyl ether were found to be highly diastereoselective (94-98% de).</p><p>Thus, an attractive supplement to direct Pd(0)-catalyzed α-arylation protocols, particularly when the use of organic chlorides, aryl bromides, and milder reaction conditions are of great importance, have been developed.</p>

Homogeneous palladium-catalyzed Heck vinylation of aryl chlorides was investigated under air using Herrmann’s palladacycle and the P(t-Bu)3-liberating salt [(t-Bu)3PH]BF4. Based on the results, controlled microwave heating was utilized to accelerate model Heck reactions with aryl chlorides down to 30 min employing an electron-poor olefin and a mixture of an ionic liquid and 1,4-dioxane as solvent. For the first time, a highly regioselective general protocol has been developed for palladium-catalyzed terminal (β-) arylation of acyclic vinyl ethers using inexpensive aryl chlorides as starting materials and the preligand [(t-Bu)3PH]BF4 as the key additive. This swift and straightforward protocol exploits non-inert conditions and controlled microwave heating to reduce handling and processing times, and aqueous DMF or environmentally friendly PEG-200 as the reaction medium. Somewhat higher selectivity for the linear β-product was observed in PEG-200. DFT calculations were performed at the B3LYP level of theory for the regioselectivity-determining insertion step in the Heck reaction following the neutral pathway. A series of para-substituted phenylpalladium(II) complexes was investigated in the computational study. The calculations support a ligand-driven selectivity rationale, where the electronic and steric influence of the bulky P(t-Bu)3 ligand provides improved β-selectivity. The preparative methodology was used to synthesize the β-adrenergic blocking agent Betaxolol. Highly stereoselective Pd(0)-catalyzed β-arylation and β-vinylation of a tetra-substituted cyclopentenyl ether have been accomplished using a chiral, pyrrolidine-based and substrate-bound palladium(II)-directing group under neutral reaction conditions. To the best of the author’s knowledge, this P(t-Bu)3-mediated method represents the first examples of the successful utilization of aryl and vinyl chlorides in asymmetric Heck reactions. The Heck arylation products formed were hydrolyzed and isolated as the corresponding quaternary 2-aryl-2-methyl cyclopentanones in good to moderate two-step yields with excellent stereoselectivity (90-96% ee). Inclusion of vinyl triflates under neutral reaction conditions and one aryl triflate equipped with a strongly electron-withdrawing para-cyano substituent under cationic conditions increased the preparative usefulness of the methodology. Furthermore, diastereoselective Heck arylation of both five- and six-membered cyclic vinyl ethers with aryl bromides, using the identical chiral auxiliary and suitable Pd sources, was performed. Arylated products from the tetra-substituted cyclopentenyl ether were also in this case hydrolyzed to the corresponding 2-aryl-2-methyl cyclopentanones with high to excellent enantioselectivity (85-94% ee). Despite low reaction rates and relatively modest yields, arylation reactions with the tri-substituted cyclohexenyl ether were found to be highly diastereoselective (94-98% de). Thus, an attractive supplement to direct Pd(0)-catalyzed α-arylation protocols, particularly when the use of organic chlorides, aryl bromides, and milder reaction conditions are of great importance, have been developed.

Electron paramagnetic resonance (EPR) dosimetry using the well established dosimeter material alanine is a generally accepted dosimetric method for measurements of high absorbed doses. Alanine EPR dosimetry is however not sensitive enough for high precision measurements of low (&lt; 5 Gy) absorbed doses using reasonably measurement times and small dosimeters. It has therefore not been possible to fully exploit the benefits of EPR dosimetry for applications in radiation therapy. The aim of this thesis was to show that sensitive EPR dosimetry is a competitive method for applications in radiation therapy fulfilling the requirements of measurement precision. Our strategy for reaching this goal was to search for new, more sensitive, EPR dosimeter materials fulfilling the criteria of being tissue equivalent, having a high radical yield and having a narrow EPR spectrum suitable for dosimetry. The best materials were found among formates and dithionates. Doping with small amounts of metal ions and recrystallisation in D2O were tested to further increase the sensitivity. Four promising candidate materials were tested regarding radical stability and dose response and among them lithium formate was chosen for dosimetry in radiation therapy applications. A high precision EPR dosimetry method was developed using lithium formate. The method included the development of a production method for EPR dosimeters with very homogenous shape, mass and composition. A read-out process was developed with maximal measurement precision for reasonably short measurement times. The method also included a dosimeter quality control before actual dose measurements. Measurement accuracy was controlled for every new dosimeter batch. This high precision lithium formate EPR dosimetry method was evaluated for pretreatment verifications of intensity modulated radiation therapy (IMRT) treatment plans. The precision and accuracy was shown to be sufficient (&lt; 5 %) for measurements of doses above 1.5 Gy using one single dosimeter and a measurement time of 15 minutes. The described evaluation is therefore a demonstration of the improved precision at low dose determinations that is available with our sensitive EPR dosimeter materials. While the EPR signal intensity is proportional to absorbed dose, the signal shape is in some cases dependent on the radiation quality. A new method is presented for simultaneous measurements of beam LET (linear energy transfer) and absorbed dose in heavy charged particle beams using potassium dithionate EPR dosimetry. The study shows that when irradiating a dosimeter with 35 MeV carbon ions, the ratio of the signal amplitudes from two radicals in potassium dithionate vary along the track indicating a dependence on linear energy transfer, LET. Potassium dithionate may therefore be a promising EPR dosimeter material for simultaneous measurements of absorbed dose and LET in heavy charged particle radiation fields.

<p>This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, <i>N</i>-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation.</p><p>Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from <i>Phoradendron tomentosum</i>. In addition, the cardiac glycoside digitoxin was isolated from <i>Digitalis purpurea</i>. All these substances expressed cytotoxicity and a neutrophil challenging activity.</p><p>Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC₅₀: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC₅₀ values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies.</p><p>The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC₅₀ 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.</p>