<p>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.</p><p>These modelling efforts have resulted in detailed knowledge of the structural characteristics in ligand interactions with the cytochrome P450 2C9 isoform.</p>

<p>Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).</p><p>Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus <i>Gaeumannomyces graminis</i>. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.</p><p>Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast <i>Pichia pastoris</i> with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.</p><p>Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).</p><p>Mn-lipoxygenase is an <i>R</i>-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other <i>R/S</i> lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.</p><p>The genome of the rice blast fungus, <i>Magnaporthe grisea</i>, contains putative genes of lipoxygenases and LDS. Mycelia of <i>M. grisea</i> were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from <i>G.graminis</i>. </p><p>Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.</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). 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.

The mouse xenobiotic-inducible Cyp2a5 gene is both transcriptionally and posttranscriptionally regulated. One of the most potent Cyp2a5 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. The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the Cyp2a5 expression and the significance of the 71 nt hairpin-loop region for the Cyp2a5 expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in Cyp2a5 expression. 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 Cyp2a5 expression. 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 Cyp2a5. 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. Treating primary mouse hepatocytes with phenobarbital (PB), a Cyp2a5 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 Cyp2a5 proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment. 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 Cyp2a5 promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the Cyp2a5 expression. The presented studies are the first showing involvement of an hnRNP protein in the regulation of a Cyp gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the Cyp gene family.

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.

Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases). Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones. Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases. Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”). Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols. The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.

The period of early life is important for the development of individual brain function and behavior. Human studies have shown altered vulnerability to develop psychopathology and/or excessive drug intake, possibly leading to dependence, as a consequence of early life experiences. In the present thesis, maternal separation (MS), an experimental model for studies of early environmental influences, was used to investigate long-term effects on neurochemistry, voluntary ethanol intake and exploration and risk assessment behavior in rats. Rat pups were assigned to one of three different rearing conditions: daily 15 min (MS15) or 360 min (MS360) of MS and normal animal facility rearing (AFR) during the first three weeks of life. Measurements of adult endogenous opioid peptide levels, opioid- and dopamine receptor density revealed minor MS-induced effects on the opioid system whereas interesting alterations were found in dopamine receptor density. Long-term effects on voluntary ethanol intake showed distinct MS-induced alterations in male Wistar and ethanol-preferring AA (Alko, Alcohol) rats. Female Wistar rats were unaffected, indicating sex differences in the effects of MS on ethanol intake. Male MS15 rats generally had a slower acquisition phase and a low subsequent ethanol intake whereas male MS360 rats had a high ethanol intake. MS15 is therefore suggested to protect against a high voluntary ethanol intake in male rats whereas MS360 may serve as a risk factor. The recently established concentric square field test indicated alterations in risk assessment as well as an increased exploratory drive and somewhat higher risk-taking behavior in adult MS360 rats, while minor effects were seen in MS15 rats. Altogether, these results demonstrate that environmental influences during the period of early life can have long-term effects on neurochemistry and behavior. Of special interest is the finding that MS altered the inherited high ethanol intake in adult ethanol-preferring AA rats.

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.