The palladium-catalysed Heck-reaction has been utilised in organic synthesis, where the introduction of aryl groups at the internal, β-carbon of different allylic substrates has been achieved with high regioselectivity. The β-stabilising effect of silicon enhances the regiocontrol in the internal arylation of allyltrimethylsilane, while a coordination between palladium and nitrogen induces very high regioselectivities in the arylation of N,N-dialkylallylamines and the Boc-protected allylamine, producing β-arylated arylethylamines, which are of interest for applications in medicinal chemistry. Phthalimido-protected allylamines are arylated with poor to moderate regioselectivity. Single-mode microwave heating can reduce the reaction times of Heck-, Stille- and radical mediated reactions drastically from approximately 20 hours to a few minutes with, in the majority of cases, retained, high regioselectivity. The use of heavily fluorinated tin reagents, which proved to be unreactive under thermal heating, is shown to be applicable with microwave-heating and the high fluorous content of the products is utilised with the aim of improving and simplifying the work-up procedure.

The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism. Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21. Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme. Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression. The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.

This investigation focused on the mRNA expression of drug metabolising Cytochromes P-450 (CYP) and UDP-glucuronosyltransferases (UGT) and the transport protein P-glycoprotein (Pgp) in the small intestine of humans and rats. The mRNA expression of the investigated genes in the human small intestine (duodenum) varies between individuals giving each one of us personal profile. In general, the most dominant forms are Pgp, CYPs 2C9, 2D6, 3A4, and UGTs 1A1, 1A10, 2B7. However, which of these is the highest expressed one varies between individuals. The correlation in expression between some CYP forms and UGT forms respectively is relatively high, which indicates that they have some regulatory mechanisms in common. It was also shown that the mRNA expression of both CYPs and UGTs may be affected by endogenous and exogenous factors. Sex and ethnic background, affected the mRNA expression of CYP2A6 and 2E1 respectively. Commonly used drugs such as acetylsalicylicacid (ASA) and omeprazole (omep) affect CYP2A6, CYP2E1 (ASA) and CYP3A4, UGT1A4 (omep). The expression of UGT1A4 is also affected by smoking. All these factors are commonly used and can therefore lead to important drug-drug interactions. It was also shown that the human small intestinal CYP mRNA expression pattern differs from that found in the rat. The rat CYP expression is rather constant between the different individuals, and the main rat intestinal forms are CYP1A1, CYP2C, CYP2D6 and CYP3A1. The expression is the same for females and males and no difference can be seen between the different segments of the rat small intestine. As metabolic studies have often been done with rat liver we compared the mRNA expression in the two organs. We found that the mRNA expression of 1A1 was absent in the liver and that the CYP2B1, CYP2Cs, CYP2D1 and Pgp all had a stronger mRNA expression in the small intestine compared to the liver. It is therefore important to realise that results from metabolic studies on liver may not be directly extrapolated to the small intestine. Artemisinin is an orally used drug in multidrug treatment of malaria in Southeast Asia. It has been suggested that artemisinin can induce drug metabolism and therefore be involved in drug-drug interactions. This study shows that artemisinin induces mainly the CYP2B via nuclear receptor CAR.

The human cytochrome P450 (CYP) family of monooxygenases is important for metabolism of drugs and endogenous compounds, e.g., vitamin A and D, cholesterol, steroids, fatty acids, and eicosanoids. This thesis describes the tissue distribution, catalytic properties, and possible function of CYP4F8 and CYP4F12. To this respect, methods for immunohistological analysis, and real-time PCR for analysis of their transcripts, were developed. CYP4F8 was originally cloned from human seminal vesicles and proposed to catalyze 19-hydroxylation of prostaglandin H2 (PGH2). This notion could now be supported, as cyclooxygenase-2, CYP4F8, and microsomal prostaglandin E synthase-1 were found to be co-localized in the epithelial linings of seminal vesicles. The three enzymes were also co-localized in the suprabasal layers of epidermis, suggesting a similar function of CYP4F8 in skin. Real-time PCR showed that CYP4F8 mRNA was more than 10-fold increased in psoriatic lesions compared to non-lesional skin. CYP4F8 immunoreactivity was also found in kidney cortex, transitional epithelium, corneal epithelium, and retina. Although transcripts of all three enzymes were detectable in retina, no co-localization was found. Pro inflammatory stimuli were found to increase CYP4F8 mRNA expression in cultured epidermal and corneal keratinocytes. In these tissues CYP4F8 might oxidize fatty acids or other eicosanoids than PGH2. CYP4F12 was originally cloned from the liver and small intestine, and found to oxidize arachidonic acid and two anti-histamines. Immunohistological studies showed that CYP4F12 immunoreactivity was present mainly in the gastrointestinal tract, e.g., stomach, ilium, and colon, but also in placenta. Although CYP4F8 and CYP4F12 have catalytic properties in common, there are important differences. CYP4F12 does not oxidize PGH2, certain eicosanoids, and fatty acids. The prominent expression in the gut suggests that CYP4F12 might be involved in oxidation of drugs.

Ten years ago, the first protease inhibitor targeting the human immunodeficiency virus (HIV) was approved for clinical use. Highly active antiretroviral therapy (HAART), which combined protease and reverse transcriptase inhibitors, quickly became the standard therapy for treating patients infected with HIV and Acquired Immune Deficiency Syndrome (AIDS). Nevertheless, last year the AIDS pandemic reached its highest level ever. Many infected patients, mainly in the developing countries, are still without treatment. Among those patients who receive treatment, an increase in drug resistance and new-infection with drug-resistant strains are seen. To come to terms with these problems, new drugs that are efficient against resistant strains and can be produced at low cost are needed. In this study, we have focused our research efforts on cyclic sulfamides active as HIV-1 protease inhibitors. Distinctive to this compound class, as compared to the inhibitors so far approved for clinical use, was the incorporation of a water mimic that displaces the structural water (W301) observed in the X-ray crystal co-complexes. The first part of the study was aimed at understanding the rationale behind the nonsymmetric binding mode that the inhibitor adopted when bound to the enzyme. Symmetric and nonsymmetric inhibitors were synthesized and the structure-activity relationships and preferable binding modes were rationalized with the help of Comparative Molecular Field Analysis (CoMFA). In the second part of the study, an attempt was made to reduce the size of these inhibitors. As a result, the traditional P1/P1' substituents were removed, while the P2/P2' substituents were elongated in an attempt to reach between the binding sites. The design hypothesis was shown to be successful and inhibitors possessing nanomolar activity were identified.

<p>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.</p><p>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 <i>ab initio</i> calculations to develop improved empirical force field parameters for use in molecular modelling.</p><p>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.</p>

<p>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). </p><p>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. </p><p>The study clearly shows that micellar electrokinetic chromatography is well-suited as an analytical technique in industrial pharmaceutical analysis. </p>

<p>The mouse xenobiotic-inducible <i>Cyp2a5</i> gene is both transcriptionally and posttranscriptionally regulated. One of the most potent <i>Cyp2a5</i> inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA.</p><p>The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the <i>Cyp2a5</i> expression and the significance of the 71 nt hairpin-loop region for the <i>Cyp2a5</i> expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in <i>Cyp2a5</i> expression.</p><p>The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for <i>Cyp2a5</i> expression.</p><p>The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the <i>Cyp2a5</i>. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA.</p><p>Treating primary mouse hepatocytes with phenobarbital (PB), a <i>Cyp2a5</i> transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the <i>Cyp2a5</i> proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment.</p><p>In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and <i>Cyp2a5</i> promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the <i>Cyp2a5</i> expression.</p><p>The presented studies are the first showing involvement of an hnRNP protein in the regulation of a <i>Cyp</i> gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the <i>Cyp</i> gene family.</p>

<p>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.</p><p>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.</p><p>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™.</p><p>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.</p><p>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.</p>

<p>This thesis concerns the development and utilisation of techniques for characterisation and transdermal delivery of various systems for pharmaceutical applications.</p><p>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.</p><p>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).</p>