Drug Analysis without Primary Reference Standards : Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material

Drug Analysis without Primary Reference Standards: Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material Primary reference standards for new drugs, metabolites, designer drugs or rare substances may not be obtainable within a reasonable period of time or their availability may also be hindered by extensive administrative requirements. Standards are usually costly and may have a limited shelf life. Finally, many compounds are not available commercially and sometimes not at all. A new approach within forensic and clinical drug analysis involves substance identification based on accurate mass measurement by liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOFMS) and quantification by LC coupled with chemiluminescence nitrogen detection (LC-CLND) possessing equimolar response to nitrogen. Formula-based identification relies on the fact that the accurate mass of an ion from a chemical compound corresponds to the elemental composition of that compound. Single-calibrant nitrogen based quantification is feasible with a nitrogen-specific detector since approximately 90% of drugs contain nitrogen. A method was developed for toxicological drug screening in 1 ml urine samples by LC-TOFMS. A large target database of exact monoisotopic masses was constructed, representing the elemental formulae of reference drugs and their metabolites. Identification was based on matching the sample component s measured parameters with those in the database, including accurate mass and retention time, if available. In addition, an algorithm for isotopic pattern match (SigmaFit) was applied. Differences in ion abundance in urine extracts did not affect the mass accuracy or the SigmaFit values. For routine screening practice, a mass tolerance of 10 ppm and a SigmaFit tolerance of 0.03 were established. Seized street drug samples were analysed instantly by LC-TOFMS and LC-CLND, using a dilute and shoot approach. In the quantitative analysis of amphetamine, heroin and cocaine findings, the mean relative difference between the results of LC-CLND and the reference methods was only 11%. In blood specimens, liquid-liquid extraction recoveries for basic lipophilic drugs were first established and the validity of the generic extraction recovery-corrected single-calibrant LC-CLND was then verified with proficiency test samples. The mean accuracy was 24% and 17% for plasma and whole blood samples, respectively, all results falling within the confidence range of the reference concentrations. Further, metabolic ratios for the opioid drug tramadol were determined in a pharmacogenetic study setting. Extraction recovery estimation, based on model compounds with similar physicochemical characteristics, produced clinically feasible results without reference standards.

Implications of co-twin dependence for twins' social interactions, mental health and alcohol use : A follow-up study of Finnish twins from adolescence to early adulthood

Objective: The aim of the present study was to examine co-twin dependence and its impact on twins' social contacts, leisure-time activities and psycho-emotional well-being. The role of co-twin dependence was also examined as a moderator of genetic and environmental influences on alcohol use in adolescence and in early adulthood. Methods: The present report is based on the Finnish Twin Cohort Study (FinnTwin16), a population-based study of five consecutive birth cohorts of Finnish twins born in the years 1975-1979. Baseline assessments were collected through mailed questionnaires, within two months of the twins' sixteenth birthday yielding replies from 5563 twin individuals. All respondent twins were sent follow-up questionnaires at ages of 17, 18½, and in early adulthood, when twins were 22-27 years old. Measures: The questionnaires included a survey of health habits and attitudes, a symptom checklist and questions about twins' relationships with parents, peers and co-twin. Measures used were twins' self-reports of their own dependence and their co-twin's dependence at age 16, reports of twins' leisure-time activities and social contacts, alcohol use, psychological distress and somatic symptoms both in adolescence and in early adulthood. Results: In the present study 25.6% of twins reported dependence on their co-twin. There were gender and zygosity differences in dependence, females and MZ twins were more likely to report dependence than males and DZ twins. Co-twin dependence can be viewed on one hand as an individual characteristic, but on the other hand as a pattern of dyadic interaction that is mutually regulated and reciprocal. Most of the twins (80.7%) were either concordantly co-twin dependent or concordantly co-twin independent. The associations of co-twin dependence with twins' social interactions and psycho-emotional characteristics were relatively consistent both in adolescence and in early adulthood. Dependence was related to higher contact frequency and a higher proportion of shared leisure-time activities between twin siblings at the baseline and the follow-up. Additionally co-twin dependence was associated with elevated levels of psycho-emotional distress and somatic complaints, especially in adolescence. In the framework of gene-environment interaction, these results suggest that the genetic contribution to individual differences in drinking patterns is dependent on the nature of the pair-wise relationship of twin siblings. Conclusions: The results of this study indicate that co-twin dependence is a genuine feature of the co-twin relationship and shows the importance of studying the impact of various features of co-twin relationships on individual twins' social and psycho-emotional life and well-being. Our study also offers evidence that differences in inter-personal relationships contribute to the effects of genetic propensities.

Animal model and molecular interactions of Cln5

Neuronal ceroid lipofuscinoses (NCLs) are a family of inherited pediatric neurodegenerative disorders, leading to retinal degeneration, death of selective neuronal populations and accumulation of autofluorscent ceroid-lipopigments. The clinical manifestations are generally similar in all forms. The Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCLFin) is a form of NCL, especially enriched in the Finnish population. The aim of this thesis was to analyse the brain pathology of vLINCLFin utilising the novel Cln5-/- mouse model. Gene expression profiling of the brains of already symptomatic Cln5-/- mice revealed that inflammation, neurodegeneration and defects in myelinization are the major characteristics of the later stages of the disease. Histological characterization of the brain pathology confirmed that the thalamocortical system is affected in Cln5-/- mice, similarly to the other NCL mouse models. However, whereas the brain pathology in all other analyzed NCL mice initiate in the thalamus and spread only months later to the cortex, we observed that the sequence of events is uniquely reversed in Cln5-/- mice; beginning in the cortex and spreading to the thalamus only months later. We could also show that even though neurodegeneration is inititated in the cortex, reactive gliosis and loss of myelin are evident in specific nuclei of the thalamus already in the 1 month old brain. To obtain a deeper insight into the disturbed metabolic pathways, we performed gene expression profiling of presymptomatic mouse brains. We validated these findings with immunohistological analyses, and could show that cytoskeleton and myelin were affected in Cln5-/- mice. Comparison of gene expression profiling results of Cln5-/- and Cln1-/- mice, further highlighted that these two NCL models share a common defective pathway, leading to disturbances in the neuronal growth cone and cytoskeleton. Encouraged by the evidence of this defected pathway, we analyzed the molecular interactions of NCL-proteins and observed that Cln5 and Cln1/Ppt1 proteins interact with each other. Furthermore, we demonstrated that Cln5 and Cln1/Ppt1 share an interaction partner, the F1-ATP synthase, potentially linking both vLINCLFIN and INCL diseases to disturbed lipid metabolism. In addition, Cln5 was shown to interact with other NCL proteins; Cln2, Cln3, Cln6 and Cln8, implicating a central role for Cln5 in the NCL pathophysiology. This study is the first to describe the brain pathology and gene expression changes in the Cln5-/- mouse. Together the findings presented in this thesis represent novel information of the disease processes and the molecular mechanisms behind vLINCLFin and have highlighted that vLINCLFin forms a very important model to analyze the pathophysiology of NCL diseases.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Molecular Interactions Underlying Neuronal Ceroid Lipofuscinoses CLN1 and CLN5

Disorders resulting from degenerative changes in the nervous system are progressive and incurable. Both environmental and inherited factors affect neuron function, and neurodegenerative diseases are often the sum of both factors. The cellular events leading to neuronal death are still mostly unknown. Monogenic diseases can offer a model for studying the mechanisms of neurodegeneration. Neuronal ceroid lipofuscinoses, or NCLs, are a group of monogenic, recessively inherited diseases affecting mostly children. NCLs cause severe and specific loss of neurons in the central nervous system, resulting in the deterioration of motor and mental skills and leading to premature death. In this thesis, the focus has been on two forms of NCL, the infantile NCL (INCL, CLN1) and the Finnish variant of late infantile NCL (vLINCLFin, CLN5). INCL is caused by mutations in the CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. In the Finnish variant of late infantile NCL (vLINCLFin), the CLN5 gene is defective, but the function of the encoded CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases by focusing on the molecular interactions of the defective proteins. In this work, the first interaction partner for PPT1, the mitochondrial F1-ATP synthase, was described. This protein has been linked to HDL metabolism in addition to its well-known role in the mitochondrial energy production. The connection between PPT1 and the F1-ATP synthase was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. The levels of F1-ATP synthase subunits were increased on the surface of Ppt1-deficient neurons when compared to controls. We also detected several changes in lipid metabolism both at the cellular and systemic levels in Ppt1-deficient mice when compared to controls. The interactions between different NCL proteins were also elucidated. We were able to detect novel interactions between CLN5 and other NCL proteins, and to replicate the previously reported interactions. Some of the novel interactions influenced the intracellular trafficking of the proteins. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism introduces a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and to develop strategies for therapies.

Interactions between transgenic trees and mycorrhizal and pathogenic fungi

The development of biotechnology techniques in plant breeding and the new commercial applications have raised public and scientific concerns about the safety of genetically modified (GM) crops and trees. To find out the feasibility of these new technologies in the breeding of commercially important Finnish hardwood species and to estimate the ecological risks of the produced transgenic plants, the experiments of this study have been conducted as a part of a larger project focusing on the risk assessment of GM-trees. Transgenic Betula pendula and Populus trees were produced via Agrobacterium mediated transformation. Stilbene synthase (STS) gene from pine (Pinus sylvestris) and chitinase gene from sugar beet (Beta vulgaris) were transferred to (hybrid) aspen and birch, respectively, to improve disease resistance against fungal pathogens. To modify lignin biosynthesis, a 4-coumarate:coenzyme A ligase (4CL) gene fragment in antisense orientation was introduced into two birch clones. In in vitro test, one transgenic aspen line expressing pine STS gene showed increased resistance to decay fungus Phellinus tremulae. In the field, chitinase transgenic birch lines were more susceptible to leaf spot (Pyrenopeziza betulicola) than the non-transgenic control clone while the resistance against birch rust (Melampsoridium betulinum) was improved. No changes in the content or composition of lignin were detected in the 4CL antisense birch lines. In order to evaluate the ecological effects of the produced GM trees on non-target organisms, an in vitro mycorrhiza experiment with Paxillus involutus and a decomposition experiment in the field were performed. The expression of a transgenic chitinase did not disturb the establishment of mycorrhizal symbiosis between birch and P. involutus in vitro. 4CL antisense transformed birch lines showed retarded root growth but were able to form normal ectomycorrhizal associations with the mycorrhizal fungus in vitro. 4CL lines also showed normal litter decomposition. Unexpected growth reductions resulting from the gene transformation were observed in chitinase transgenic and 4CL antisense birch lines. These results indicate that genetic engineering can provide a tool in increasing disease resistance in Finnish tree species. More extensive data with several ectomycorrhizal species is needed to evaluate the consequences of transgene expression on beneficial plant-fungus symbioses. The potential pleiotropic effects of the transgene should also be taken into account when considering the safety of transgenic trees.

Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques

Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC ESI-MS. It was discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.

Molecular Interactions Chlorin Assemblies and NMR Separations

Photosynthesis is a chemical process in which the energy of the light quanta is transformed into chemical energy. Chlorophyll (Chl) molecules play a key role in photosynthesis; they function in the antennae systems and in the photosynthetic reaction center where the primary charge separation (CS) takes place. Bio-inspired mimicry of the CS is an essential unit in dye-sensitized solar cells. Aim of this study was to design and develop electron donor-acceptor (EDA) pairs from Chls and fullerenes (C60) or carbon nanotubes (CNT). The supramolecular approach was chosen, as long synthetic sequences required by the covalent approach lead to long reaction schemes and low yields. Here, a π-interaction between soluble CNTs and Chl was used in EDA construction. Also, a beta-face selective two-point bound Chl-C60 EDA was introduced. In addition, the photophysical properties of the supramolecular EDA dyads were analyzed. In organic chemistry, nuclear magnetic resonance (NMR) spectroscopy is the most vital analytical technique in use. Multi-dimensional NMR experiments have enabled a structural analysis of complex natural products and proteins. However, in mixture analysis NMR is still facing difficulties. In many cases overlapping signals can t be resolved even with the help of multi-dimensional experiments. In this work, an NMR tool based on simple host-guest chemistry between analytes and macromolecules was developed. Diffusion ordered NMR spectroscopy (DOSY) measures the mobilities of compounds in an NMR sample. In a liquid state NMR sample, each of the analytes has a characteristic diffusion coefficient, which is proportional to the size of the analyte. With normal DOSY experiment, provided that the diffusion coefficients of the analytes differ enough, individual spectra of analytes can be extracted. When similar sized analytes differ chemically, an additive can be introduced into the sample. Since macromolecules in a liquid state NMR sample can be considered practically stationary, even faint supramolecular interaction can change the diffusion coefficient of the analyte sufficiently for a successful resolution in DOSY. In this thesis, polyvinylpyrrolidone and polyethyleneglycol enhanced DOSY NMR techniques, which enable mixture analysis of similar in size but chemically differing natural products, are introduced.

Quantum Chemical Studies of Intermolecular Interactions

This thesis studies the intermolecular interactions in (i) boron-nitrogen based systems for hydrogen splitting and storage, (ii) endohedral complexes, A@C60, and (iii) aurophilic dimers. We first present an introduction of intermolecular interactions. The theoretical background is then described. The research results are summarized in the following sections. In the boron-nitrogen systems, the electrostatic interaction is found to be the leading contribution, as 'Coulomb Pays for Heitler and London' (CHL). For the endohedral complex, the intermolecular interaction is formulated by a one-center expansion of the Coulomb operator 1/rab. For the aurophilic attraction between two C2v monomers, a London-type formula was derived by fully accounting for the anisotropy and point-group symmetry of the monomers.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.