Browsing by Issue Date

Sort by: Order: Results:

Now showing items 1-20 of 166
  • Nordman, Nina (Helsingin yliopisto, 2015)
    Analytical microsystems are attractive in modern bioanalysis where sample amounts often are low and fast analyses are required. Microsystems also provide the prospect of integrating several functional elements on a single platform. The aim of this work was to develop analytical microsystems for fast analysis of bio- and drug molecules. For this, microchips with separation- and injection channels and monolithically integrated electrospray ionization (ESI) emitter were fabricated of epoxy photoresist SU-8 by photolithography and adhesive bonding. For peptide mass fingerprinting and protein sequencing characteristic tryptic peptides were fast and easily separated and detected by microchip capillary electrophoresis (MCE)-ESI/mass spectrometry (MS). Additionally, protein identification based on tandem MS fragmentation data of a single tryptic peptide was achieved. Finally, this rapid (total analysis time below ten minutes) microchip method permitted analysis of human muscle cell lysates. For online coupling of microchip capillary isoelectric focusing (cIEF) to ESI/MS a bilateral sheath flow interface or a two-dimensional separation unit was integrated on-chip. Rapid focusing of peptides by their isoelectric points (pI) was achieved without pretreatment of the SU-8 surface. After focusing the peptides were electrokinetically mobilized toward ESI/MS. The two-dimensional chip design enabled unique separation selectivity for peptides based on both pI values and intrinsic electrophoretic mobilities by multiplex-cIEF-transient-isotachophoresis. Rapid metabolic profiling was demonstrated from urine after intake of tramadol or paracetamol. Both phase I- and II metabolites were separated and detected by MCE-ESI/MS within 35 s. In addition, Michaelis-Menten kinetics was successfully determined for the CYP450-mediated oxidation of bufuralol to 1-hydroxybufuralol. Sample preconcentration (pretreatment) was integrated on-chip by solid-phase extraction (SPE) and liquid-phase microextraction (LPME). For SPE, a monolith zone was firmly anchored at the injection cross of the MCE-ESI/MS microchip by laser induced photopolymerization. The monolith was selective toward hydrophobic and hydrophilic sample molecules and enrichment factors as high as 23-fold was achieved with a loading time as short as 25 s. In addition, LPME was easily downscaled to low volume applications and offered selectivity in the analysis of phase I metabolites compared to SPE. In contrast to previous research in the same field this work offers bioanalysis with several on-chip integrated steps (preconcentration, injection, separation, and analysis) without considerably increasing the short analysis times characteristic of microchip assays.
  • Wang, Chang-Fang (Helsingin yliopisto, 2015)
    Anticancer drugs inhibit the cancer growth by killing the rapidly dividing cancer cells. However, anticancer drugs also kill the dividing healthy cells and cause severe damage to healthy tissues. More specific delivery of the cancer drugs to the cancer tissue can increase the drug delivery efficiency and reduce the drug s side effects. Nanocarriers can increase the solubility of poorly-water soluble anticancer drugs and be modified for targeted drug delivery and theranostic applications. For efficient drug delivery, the drug loading capacity has been one of the key issues for the development of nanoparticle (NP)-based drug delivery systems. The biocompatible and biodegradable porous silicon (PSi) nanomaterial presents high drug loading capacity and tunable surface chemistry which renders it an ideal candidate as a drug delivery carrier. Chemical surface modification, which is one of the approaches to improve the nanomaterials properties, can lead to a stable nanosystem for further drug delivery applications. The main aim of this dissertation was to employ chemical approaches and surface modified PSi nanoparticles (NPs) to improve the drug delivery efficiency for potential cancer therapy applications. Incorporating targeting moieties to the surfaces of the nanocarriers, such as targeting peptides, can increase the nanocarrier s accumulation into the cancer tissue after the intravenous administration. In this thesis, surface modification of amine-terminated PSi NPs was achieved with targeting peptides (RGDS and iRGD) via strain-promoted azide-alkyne cycloaddition click reaction. The functionalization of the PSi NPs with the targeting peptides did not comprise the drug loading capacity, but enhanced the cellular uptake and the drug delivery efficacy of the PSi NPs in vitro. In addition to the targeting NP surface modifications, a multifunctional nanosystem was prepared with simultaneous fluorescence- and radio-labeling, and iRGD surface modification of the carboxylic acid-terminated PSi NPs. Both labelings were accessible for the in vivo biodistribution evaluation in mice by single-photon emission computed tomography and X-ray computed tomography, and ex vivo by immunofluorescence staining, respectively. The iRGD modification enhanced the tumor uptake of the PSi NPs after the intravenous administration. In order to reduce the plasma protein adsorption onto the PSi NPs, five bioactive molecules (peptides and hydrophilic anti-fouling polymers) were used to modify the surface of alkyne-terminated PSi NPs using copper-catalized click chemistry. Dextran 40 kDa modified PSi NPs presented enhanced cellular uptake and the least protein adsorption of all the tested NPs. Furthermore, the chemical conjugation of drug molecules was studied. The targeting peptides were successfully conjugated to antisense interleukin-6 via copper-catalyzed [3+2] azide-alkyne cycloaddition for targeted angiogenic anti-inflammation in cancer. Finally, anticancer drug methotrexate (MTX) was chemically conjugated to the cationic PSi NPs and demonstrated to increase the cellular uptake of MTX with up to 96 h sustained drug release. A hydrophobic anti-angiogenic drug, sorafenib, was also loaded to the MTX-conjugated PSi NPs, and the dissolution rate of this drug was considerably increased. In conclusion, in this thesis different chemical approaches were used to biofunctionalize PSi NPs and to prepare drug-conjugates formulations for potential anti-cancer applications.
  • Shahbazi, Mohammad-Ali (2015)
    Porous silicon nanoparticles (PSi NPs) have recently drawn increasing interest for therapeutic applications due to their easily modifiable surface, large pore volumes, high surface area, nontoxic nature, and high biocompatibility. Nevertheless, there is no comprehensive understanding about the role of the surface chemistry of these NPs on the biological interactions and the therapeutic effect of the PSi-based nanosystems. Therefore, extensive attempts are still needed for the development of optimal PSi-based therapeutics. The first step for evaluating the biological activity of the NPs was to investigate the potential toxic effects. Accordingly, the immunotoxicity and hemocompatibility of the PSi NPs with different surface chemistries were assessed at different concentrations on the immune cells and red blood cells, since these are the first biological cells in contact with the NPs after intravenous injection. PSi NPs with positively charged amine functional groups showed higher toxicity compared to negatively charged particles. The toxicity of the negatively charged particles was also highly dependent on the hydrophobic nature of the NPs. Moreover, RBC hemolysis and imaging assay revealed a significant correlation between the PSi NP surface chemistry and hemotoxicity. To further understand the impact of the surface chemistry on the immunological effects of the PSi NPs, the immunostimulatory responses induced by a non-toxic concentration of the PSi NPs were evaluated by measuring the maturation of dendritic cells, T cell proliferation and cytokine secretion. Overall, the results suggested that all the PSi NPs containing higher amounts of nitrogen or oxygen on the outermost surface layer have lower immunostimulatory effects than the PSi NPs with higher amounts of C‒H structures on the NPs surface. Combination cancer therapy by the PSi NPs was then studied by evaluating the synergistic therapeutic effects of the nanosystems. Sorafenib-loaded PSi NPs were biofunctionalized with anti-CD326 monoclonal antibody on their surface. The targeted PSi NPs showed a sustained drug release and increased interactions with the breast cancer cells expressing the CD326 antigen on their surface. These NPs also showed higher antiproliferation effect on the CD326 positive cancer cells compared to the pure drug and sorafenib-loaded PSi NPs, suggesting CD326 as an appropriate receptor for the antibody-mediated drug delivery. In addition, anti-CD326 antibody acted as an immunotherapeutic agent by inducing antibody-dependent cellular cytotoxicity and enhancing the interactions of immune cells with cancer cells for the subsequent phagocytosis and cytokine secretion. Next, the development of a stable PSi NP with low toxicity, high cellular internalization, efficient endosomal escape, and optimal drug release profile was tested by using a layer-by-layer method to covalently conjugate polyethyleneimine and poly(methyl vinyl ether-co-maleic acid) copolymers on the surface of the PSi NPs, forming a zwitterionic nanocomposite. The surface smoothness and hydrophilicity of the polymer functionalized NPs improved considerably the colloidal and plasma stability of the NPs. Moreover, the double layer conjugation sustained the drug release from the PSi NPs and improved the cytotoxicity profile of the drug-loaded PSi NPs. In conclusion, this work showed that the surface modification of the PSi NPs with different chemical groups, antibodies and polymers can affect the toxicological profiles, the cellular interactions and the therapeutic effects of the NPs by modifying the charge, stability, hydrophilicity, the drug release kinetics and targeting properties of the PSi NPs.
  • Palomäki, Jaana (Helsingin yliopisto, 2014)
    Engineered nanomaterials (ENM) are widely used in the industry as their unique characteristics improve competitiveness and potential for innovations in the different sectors of industry: It has been estimated that the market size of nanotechnological innovations will exceed 2 trillion euros in 2015. However, the toxicological hazard and risk assessment of different nanomaterials is far from complete. There is a concern that nanomaterials cause toxic outcomes, and more research efforts are put into hazard assessment of nanomaterials. However, the huge amount of differently modified nanomaterials poses a challenge for toxicologists who will have to develop new, quicker and cheaper, approaches for hazard and risk assessment to guarantee the human health. The human body is protected from foreign material by its immune system. The most common routes for foreign materials to enter the human body are via inhalation, digestion or dermal penetration, and these routes are protected by physical barrier. The physical barrier accompanied with phagocytosing cells are part of the innate immunity: Rapid and non-specific immune system. Phagocytosing cells scavenge foreign material in the body cavities and secrete signaling molecules to further activate other cells of immunity, such as T or B cells, to form a specific immune response. In this thesis, an immunotoxicological approach was used to compare effects of different types of ENM in the phagocytosing cells or in the disease model of atopic dermatitis. Studies performed with metal oxide nanoparticles and carbon nanotubes (CNT) showed that surface treatment or shape of nanomaterial modify their toxic properties in vitro. Mechanistical studies performed with CNT clearly demonstrated that long, rigid carbon nanotubes resemble asbestos fibers in their toxicity whereas other types of CNT showed less toxicity. In addition, the effects of either bulk-sized or nano-sized ZnO particles commonly used in cosmetics were compared in the mouse atopic dermatitis disease model. The results clearly showed that ZnO particles were able to suppress local inflammation, however, there was also an induction of systemic antibody levels. These outcomes were clearly higher if the mice skin had been treated with nano-ZnO shown also to penetrate into the deeper layers on mouse allergic skin. The results suggest that material size, shape and surface modifications are important parameters in their toxicity. Therefore the risk assessment of differently modified nanomaterials shall be performed separately despite the previously performed risk assessment of the bulk-sized form of the same material. These results also underline factors that should be taken into account in the planning of the future studies.
  • Ahonen, Linda (Helsingin yliopisto, 2014)
    Steroids, vitamin D and oxysterols are all part of a group of compounds called lipids, which are generally important in the function of the human body and in many cases the monitoring of these analytes can be of high value. Therefore, the development of a sensitive and universal method, suitable for the simultaneous analysis of these compounds, is of a great importance. In this study, the goal was to develop and test new mass spectrometry based methods for analyzing steroids, vitamin D and oxysterols. First, the feasibility of capillary liquid chromatography-microchip atmospheric pressure photoionization-tandem mass spectrometry was examined for the analysis of anabolic steroids in human urine. The results show that this microchip based method can be feasible for analyzing non-polar and neutral compounds in biological samples. Next, a microfluidic-based liquid chromatography-electrospray ionization-mass spectrometric system (HPLC-Chip/ESI/MS) was compared to a conventional liquid chromatography-electrospray/mass spectrometric system (LC-ESI/MS) for the analysis of steroids. The sensitivity of the HPLC-Chip/MS system was ~50 times higher compared to the conventional LC-MS when injected amounts are compared. The feasibility of the HPLC-Chip/MS system in the analyses of non-derivatized steroids was also tested and the developed method was used in the analysis of mouse plasma samples. An ion mobility mass spectrometry method using a compact traveling wave cell (TWIM-MS) was also developed for the separation of steroid isomers. Three steroid isomer pairs were analyzed in their native form and as their p-toluenesulfonyl isocyanate derivatives. The results show, that the developed TWIM-MS method provides a reliable, fast and repeatable method for separating derivatized steroid isomers. Finally, an ultra-high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometric (UHPLC-APPI-MS/MS) method was developed for the simultaneous analysis of oxysterols and vitamin D related compounds in mouse brain and cell line samples. An UHPLC-APPI-high resolution mass spectrometric method was developed for confirmatory analysis and for the identification of non-targeted oxysterols. Several oxysterols were quantified in the mouse brain and cell line samples. Additionally, 25-hydroxyvitamin D3 was detected in mouse brain samples for the first time. In conclusion, the methods developed in this work are sensitive and selective new methods for the analysis of the selected lipids from biological samples. The methods provide new insights in the analysis of steroids, vitamin D related compounds and oxysterols, and with some additional development some of the presented methods could be implemented e.g. in routine laboratories.
  • Kontturi, Leena-Stiina (Helsingin yliopisto, 2014)
    Cell therapy is defined as cell transplantation into the patient to treat a certain disease state. Therapies utilizing cells can be divided into two main categories, (1) tissue regeneration or engineering and (2) drug delivery. In tissue engineering, the transplanted cells are used to regenerate the functions of a diseased tissue. In drug delivery, the transplanted cells are used as biological factories that produce therapeutic molecules inside the body. For successful cell therapy applications, cells usually must be combined with biomaterials and bioactive factors to mimic the growth environment in vivo. The properties of these scaffolds are important for outcomes of the treatments, because the local environment determines the functionality of the cells. Thus, research on cell-biomaterial interactions is essential for the progress of cell based therapies. Hydrogels are promising cell therapy materials, because their structure resembles the natural tissue environment; they consist of long polymer chains with high water content and elastic properties, thereby enabling cellular functionality. The aim of this study was to investigate hydrogels for cell therapy applications. Firstly, we encapsulated human retinal pigment epithelial cell line (ARPE-19) genetically engineered to secrete an anti-angiogenic protein (1) into alginate-poly-L-lysine-alginate (APA) microcapsules and (2) into a composite hydrogel of cross-linked collagen and interpenetrating hyaluronic acid (HA). A custom-made cell encapsulation device was designed, built and optimized, and pharmacokinetic/pharmacodynamic (PK/PD) model was developed to investigate the intravitreal drug delivery of the anti-angiogenic protein by the encapsulated cells. Secondly, chondrocytes were encapsulated into the cross-linked collagen/HA hydrogel supplemented with transforming growth factor β1 (TGFβ1). Using the cell encapsulation device, cell microcapsules of symmetrical shape and narrow size distribution were produced. The encapsulated ARPE-19 cells remained viable and functional for at least five months. The cross-linked collagen-HA hydrogel was shown to be a suitable encapsulation matrix for ARPE-19 cells; the cells maintained viability and secreted the anti-angiogenic protein at a constant rate for at least 50 days. Moreover, the hydrogel composition could be modified to adjust the properties of the gel structure without compromising cell viability. This approach is suggested to have potential in the treatment of retinal neovascularization. The developed PK/PD model could be used to predict drug levels and therapeutic responses after intravitreal anti-angiogenic drug delivery. The simulations may augment the design of in vivo experiments. The collagen/HA matrix with TGFβ1 was suitable for chondrocyte encapsulation. The hydrogel supported viability and phenotypic cell stability. This hydrogel is strong, stable and biodegradable, and it can be delivered non-invasively as injection. Overall, it is potentially a useful delivery vehicle of chondrocytes for cartilage tissue engineering. In conclusion, ARPE-19 cells maintain viability in different hydrogels for prolonged periods and secrete the therapeutic transgene product constantly, supporting the suitability of ARPE-19 cells for cell therapy. The cross-linked collagen/HA hydrogel appears to be a potential matrix for cell therapy. It is an injectable system that supports functionality of cells, and it is applicable in drug delivery and tissue engineering.
  • Malinen, Melina (Helsingin yliopisto, 2014)
    New organotypic liver cell cultures are needed to predict the metabolism, excretion, and safety of chemical compounds. Liver cell models are particularly important since the liver largely regulates the ultimate fate of compounds in the body. Approximately 70% of the drugs administered to the body are metabolized or excreted by the liver. Animal models, cell cultures, and cell-free assays are the most common liver models. However, animal models and animal cells do not represent humans due to the interspecies differences in drug metabolizing enzymes and transporters. Instead, the most common cell-free methods, microsomes, are appropriate for drug metabolism studies, but the lack of drug transporters and transcription machinery prevents the complete evaluation of compounds. Primary human hepatocytes are capable of both drug metabolism and drug transport, and are, therefore, considered the gold standard to assess metabolism and toxicity of compounds in vitro. Primary hepatocytes, however, suffer limited availability, high functional variability, and difficulty with maintaining differentiated phenotypes and functions in cell cultures. Therefore, continuous human liver cell lines, such as HepG2 and HepaRG, have been widely used to evaluate drugs and chemicals even though they have defects in their biotransformation functions. The advantages of cell lines are their good availability, easy maintenance, and inducible drug metabolism. Generally, these cells are cultured in a two-dimensional (2D) manner that deviates from the physiological morphology and functions of the hepatocytes. The flattened 2D phenotype leads to reduced polarization and loss of important signaling pathways; this is likely to be a major reason for the failure in the prediction of drug metabolism, pharmacokinetics, and hepatotoxicity. It is believed that for more predictive in vitro models, the liver cells should be maintained in a three-dimensional (3D) microenvironment that allows reconstruction of polarization, and cell-cell and cell-extracellular matrix (ECM) contacts. The 3D cell cultures have been generated by different methods, such as cultures in matrices, scaffolds, bioreactors, and microfluidic platforms. Biomaterial hydrogels have demonstrated great potential for 2D liver cell culturing, but their potential to generate functional 3D liver cell cultures is largely unknown. The main goal of this thesis was to establish improved 3D liver cell cultures with biomaterial hydrogels. Particular attention was focused on the effects of 3D hydrogels on drug metabolism and excretion, cytoarchitecture, and cellular differentiation of HepG2 and HepaRG cell lines. As a starting point, we studied the suitability of wood-derived nanofibrillar cellulose (NFC) hydrogel as a cell culture matrix. NFC hydrogel has not been studied in cell culture before; however, as a novel, defined, animal-free, and abundantly available material, it evoked interest for testing. Herein, the wood-derived NFC was proven to own rheological and structural characters that allow 3D cell culture. Moreover, the NFC was compatible with the HepG2 and HepaRG cells, allowing for the formation of 3D multicellular aggregates with increased apicobasal polarity. When compared to commercial hydrogels, the NFC supported the albumin secretion, an indicator of hepatocellular synthetic function, from HepG2 and HepaRG cells as well or even better. These results demonstrate the potential of wood- derived NFC to function as an ECM analogue, and present the first HepaRG aggregate cultures. Next, the effect of the RAD16-I peptide hydrogel on the HepG2 cell line was investigated in more detail. Immunofluorescence staining and vectorial transport showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of canalicular efflux transporters, multidrug resistance-associated protein 2 (MRP2), and multidrug resistance protein 1 (MDR1), in the spherical HepG2 cell aggregates. The study clearly demonstrated that the peptide hydrogel increases the apicobasal polarity and appearance of bile canaliculi structures in HepG2 cell cultures. The plasticity of HepaRG liver cells was exploited to investigate the impact of 3D NFC and hyaluronan-gelatin (HG) hydrogel cultures on the phenotype of both undifferentiated HepaRG cells (early liver progenitors) and differentiated HepaRG cells (hepatocyte-like cells together with cholangiocyte-like cells). Based on the expression and activity of hepatic markers, drug metabolizing enzymes, and drug transporters, the 3D NFC and HG hydrogels promoted the differentiation of HepaRG liver progenitor cells when compared to the standard 2D technique. Instead, the 3D hydrogel cultures could not really improve the properties of differentiated HepaRG cells. In conclusion, these findings reveal the capability of the NFC, RAD16-I peptide, and HG hydrogels to improve the properties of HepG2 and HepaRG human liver cells. The new spheroid cultures of HepG2 and HepaRG cells may represent added value for pharmacokinetic and toxicity predictions, showing a liver-like cytoarchitecture and demonstrating applicability for drug metabolism and transport studies. Overall, the results deepen our knowledge of the 3D liver cell cultures.
  • Liu, Dongfei (Helsingin yliopisto, 2014)
    The co-loading of different therapeutic molecules into a single carrier offers several advantages over individual administration. The aim of this dissertation was to develop a robust platform that would enable precise control over the loading and release of different drug cargos to facilitate combination therapy. Benefiting from numerous attractive features, porous silicon (PSi) materials have emerged as a promising drug delivery system. However, uncontrolled drug release has always been observed on PSi, which can be ascribed to its freely accessible pores. To seal the pores of the PSi materials and to sustain drug release, the drug-loaded PSi particles were embedded into solid lipid nano- and micromatrices. The PSi-encapsulated solid lipid nanocomposites prepared by the traditional emulsion method exhibit superior stability in aqueous solutions and, most importantly, prolonged drug release. Similarly, the microfluidic templated monodisperse PSi solid lipid microcomposites remarkably sustained the release of either hydrophilic or hydrophobic drugs individually. To achieve the stimuli-responsive drug delivery, the drug-loaded PSi particles were assembled into pH-responsive polymeric micro- and nanocomposites. Taking advantage of the high loading degrees of PSi materials for both hydrophilic and hydrophobic therapeutics, multiple drugs with varied physicochemical properties were successfully loaded with ratiometric control. The microfluidic assembled micro- and nanocomposites both exhibited pH-responsive properties and tailored drug release kinetics. In conclusion, PSi-based versatile multicomposites, having the capacity for precisely controlled delivery of simultaneously encapsulated physicochemically distinct cargos, were successfully prepared and characterized. These systems represent a promising platform for future targeted combination therapies.
  • Meinander, Kristian (Helsingin yliopisto, 2014)
    The highly prostate specific serine protease kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen, PSA) is widely used as a biomarker for prostate cancer and it has also been postulated that it may play a part in tumour growth. Especially interestesting is the antiangiogenic effect exerted by proteolytically active KLK3 in cell line models. In order to stimulate the proteolytic activity of KLK3, a series of peptides have been developed by phage display methodology. Even though the peptides are quite potent KLK3 stimulators, they are not directly suitable for in vivo studies or use as drugs. Even though there are many natural and unnatural biologically active peptides, they suffer from rapid clearance via the liver and kidneys and proteolytic degradation of the compounds both in the gastrointestinal tract and other parts of the body. This gives peptides a poor oral bioavailability meaning that they are usually administered as intravenous or intramuscular injections. Several different strategies have been developed in order to access compounds with improved bioavailability including modifications of the peptide structure, development of pseudopeptides and development of small molecular weight peptidomimetics. This thesis concentrates on the further development of the two most potent peptides known to stimulate KLK3, i.e. B-2 and C-4. The main part of the work was concentrated on the replacement of disulphide bridges in the peptides in order to both gain more information on which residues are necessary for obtaining the biological activity and at the same time also gain information on how changes to the geometry of the disulphide bridge affects the activity. A series of different disulphide bridge mimicking building blocks were designed and synthesised with the intention of using them in a protocol for solid-phase synthesis of KLK3 stimulating peptides. Unfortunately, the use of these building blocks in the synthesis of pseudopeptides based on C-4 turned out to be an unsurmountable challenge and the synthesis had to be completed using a different strategy in which the key step was the use of ring-closing metathesis (RCM) for the cyclisation of the partly completed pseudopeptide. Pleasingly, the synthesis of pseudopeptide analogues of the B-2 peptide using the building blocks was more successful. In total three pseudopeptide analogues of C-4 and four of B-2 were synthesised and shown to retain the biological activity of the parent peptides. Based on the information from the synthesised pseudopeptides and a molecular modelling study, a 4-quinolone based peptidomimetic was designed to mimic the C-4 peptide and a synthetic protocol was devised to access this compound. Even though the synthesis of the desired target compound has so far not been successful, the synthetic protocol that was designed has given access to a number of 1,2,8-trisubstituted 4-quinolone derivatives.
  • Magarkar, Aniket (Helsingin yliopisto, 2014)
    Drug delivery is a vital issue in pharmaceutical research; once a drug candidate molecule is identified, it must be delivered to the target area of the body where it can take effect. In addition, non-specific distribution of drug molecules to areas other than the drug target must be decreased to avoid unwanted side effects. To achieve this, nanotechnological drug delivery systems can be used. Nanotechnological drug delivery systems come in a wide variety of forms, including liposomes, dendrimers, nanoparticles, and polymeric micelles. Of these, our research is focused on drug delivery liposomes. Drug delivery liposomes are composed of a membrane that forms a closed spherical sack, with a diameter of approximately 100 nm that can contain drug molecules. The criteria for effectiveness of these drug delivery liposomes (DDLs) are structural stability, its lifetime in the bloodstream, the release rate of the encapsulated content and site specific targeting. Cholesterol is one of the crucial lipid components of the DDL known to increase its stability. They also can have a protective polymer coating such as polyethylene glycol (PEG) that protects the DDL from the body s defense mechanisms. Also the DDL can posses targeting moieties, able to direct the PEGylated liposomes to the specific target. In this study we have investigated surface structure of the DDL and its interactions with elements of the blood stream. While it is difficult to determine an accurate picture of the DDL surface and its interactions with ions and bloodstream proteins with atomistic resolution by experiments alone, computational molecular modelling techniques can provide insights into it. Hence, we have used computational modelling and molecular dynamics simulations to understand the role of each component of the DDL in its structure. The three of the five reported studies in this thesis (I, II, III) are focused on how surface charge plays an important role in the liposome, how it is affected by various components of the DDLs, and how the specific interactions of DDLs and ions present in the blood stream influence it. The chapter IV deals with understanding the properties by systematically varying components such as cholesterol and PEG. Also we have produced the first ever model of the first FDA approved drug delivery liposome (DOXIL ®) at atomistic resolution details. The last study (V) deals with the application of molecular dynamics in targeted drug delivery research. In this study we could identify the reason for failure of specific novel targeting peptide (AETP), which is used to functionalize the DDL, by identifying its interactions with the protective PEG polymer.
  • Subrizi, Astrid (Helsingin yliopisto, 2014)
    Biologics are increasingly used in the treatment of ocular diseases such as age-related macular degeneration (AMD) that cannot be controlled with conventional small molecule drugs. AMD is a multifactorial eye disease that carries significant risk of morbidity and vision loss. In Finland and other western countries, AMD affects one in three people older than 75 years, and until the early 2000s no effective treatment was available for these patients. The marketing approval of anti-VEGF antibodies was a major breakthrough in the management of AMD; indeed these biologics effectively halt choroidal neovascularization and therefore prevent further vision loss in roughly half of the patients with wet AMD. Antibody therapy has been the most successful approach so far, however, other biological therapies such as gene therapy, cell therapy and other therapeutic proteins, may prove beneficial in the treatment of AMD and other vision threatening disorders. This thesis deals with the delivery of biologics, including DNA, cells, proteins and peptides, to the retinal pigment epithelium (RPE), which plays a central role in the development of AMD. Briefly, the main topics and results of this work are presented. New non-viral gene delivery candidates are usually screened for transfection efficiency and toxicity by reading out transgene expression levels relative to a reference formulation after in vitro transfection. The screening protocols, however, can be very different among laboratories, so that comparison of results is often difficult, if not impossible. Our aim was to develop a standardized protocol optimized for the transfection of retinal pigment epithelial cells in vitro. The developed screening protocol provides a relatively simple and reproducible procedure for the pre-selection of potential candidate reagents as non-viral gene delivery systems targeted to the retinal pigment epithelium. The ocular delivery of biologics remains a challenging task due to the barriers of the eye. Short cationic peptides, also known as cell-penetrating peptides (CPPs), have been successfully used as tools to introduce various biologics into cells due to their ability to translocate across the plasma membrane and deliver their cargoes intracellularly. In our work, we have explored the functionality of Tat peptide, one of the most widely studied CPPs. Our results indicate that it is not the sequence of Tat per se that dictates cell uptake, but the cationic charge of the peptide. Moreover no direct penetration was observed; instead all the peptides were endocytosed and, as it is often the case in non-viral gene delivery, ended their journey inside lysosomes. For this reason, we think that the use of Tat peptide for the delivery of biologics to the cytoplasm or nucleus of cells will probably not be very successful. Ocular stem cell therapy holds promise for the reconstruction of the degenerated RPE monolayer in AMD patients; in addition, engineered human RPE constructs may also provide a unique platform for drug discovery and toxicology. We have grown a functional RPE tissue in vitro by using human embryonic stem cells as cell source and the synthetic polymer polyimide as supporting scaffold for the growth and maturation of the cells. The epithelia acquired RPE-like properties, including characteristic RPE phenotype, expression of RPE markers, barrier and phagocytic function. The degeneration of RPE cells in dry AMD is caused by the aggregation of proteins inside RPE cells, and is currently untreatable. We have investigated the cytoprotective properties of heat shock protein 70 kDa (Hsp70) against oxidative damage and the feasibility of rhHsp70 protein therapy as a potential therapeutic approach for dry AMD. This work provides a novel therapeutic option for the treatment of RPE degeneration in AMD.
  • Rosenqvist, Kirsi (Helsingin yliopisto, 2014)
    The effect of systemically and locally administered clodronate on bone quality The aims of this study were to evaluate the effect of clodronate, given systemically or locally, on the quality of bone and to develop a combination product including clodronate and bioactive glass for local administration in the treatment of periodontitis. The beneficial effects of clodronate on bone are known. Clodronate inhibits osteoclasts in bone and reduces bone turnover. It is used in breast cancer patients with non-osseous metastases to reduce the osteolytic complications and bone metastases. It is widely investigated e.g., in healthy women reducing bone loss. The first aim of this thesis was to investigate whether these established effects are similar in women with primary operable breast cancer. Since skeletal bone loss and alveolar bone loss in periodontitis share common mechanisms, the effect of clodronate upon dental application was of interest. Systemic bone conditions impact the periodontium and systemically administered clodronate positively affects the peridontium, but also side effects such as diarrhea, rash and osteonecrosis of the jaw can be problematic. However, clodronate is poorly absorbed from gastrointestinal (GI) tract and the oral bioavailability of clodronate is low. Therefore, systemically administered oral dose of clodronate needs to be high to achieve a therapeutic effect, which in turn leads to increased adverse events. Therefore, the challenge in developing novel drug delivery systems for clodronate is to achieve improved bioavailability and safety. The aim of the second part of this thesis was to develop a new delivery system that would reach the target site in the periodontium, while limiting unwanted side effects and reducing the required dose through local administration. In the first part of this study, the loss of bone mineral density (BMD) was studied in patients with primary operable breast cancer given either clodronate or placebo. Oral clodronate appears to reduce the loss of bone in these patients. In premenopausal patients clodronate significantly reduced the loss of bone after one year and in postmenopausal patients clodronate increased the spinal BMD. Patients receiving clodronate had significantly more incidences of diarrhea than those receiving placebo. This indicates that clodronate is poorly absorbed from GI tract thus causing irritation to the intestine. In addition, the development of bone metastases was compared with patients with primary operable breast cancer given either clodronate or placebo. Clodronate given to these patients was shown to reduce the occurrence of bone metastases. Additionally, there was a significant reduction in mortality. In the second part of the study, a novel combination product of bioactive glass (BAG, SiO2 53%, Na2O 23%, CaO 20% and P2O5 4% (w/w %)) and clodronate was investigated. Firstly, preformulation studies were performed. Clodronate was found to promote the activity of the BAG and a calcium clodronate precipitate formed. Additionally, the bioactivity lasted longer in the combination product than in BAG alone. The optimal ratio for bioactive glass and clodronate and optimal particle size for BAG for local treatment of periodontitis was investigated and selected based on bioactivity of the BAG, safest pH profile of the combination product, as well as highest possible amount of clodronate to achieve the enhanced bioactivity for the BAG. The combination product chosen was 1 g BAG with a particle size of 0.5-0.8 mm and 200 mg of clodronate premoisturized with saline. Finally, the selected combination product was compared to the BAG alone in the treatment of the periodontitis maintenance phase in a pilot study with ten study subjects. Based on the clinical signs and symptoms of inflammation or infection at the site (evaluated by the investigator) and overall satisfaction the combination product is at least as good as bioactive glass alone. Of the two bone remodeling biomarkers (osteoprotegerin and osteocalcin) selected for the study, only osteoprotegerin data indicate that the effect of the novel combination product is beneficial. Osteoprotegerin levels decreased in both combination product and BAG alone treated teeth but less in the combination product treated teeth. However, due to the short time period of investigation as well as the limited number of subjects and treated teeth, the result for bone quality remains only indicative. The positive effect of the combination product on tooth sensitivity may bring additional benefits in comparison to the use of BAG alone in periodontal maintenance therapy.
  • Granqvist, Niko (Helsingin yliopisto, 2014)
    For the last few decades, the expences of pharmaceutical development and drug discovery have been constantly increasing whereas the amount of new pharmaceutical products reaching the market has been diminishing. The drug discovery methods today rely heavily on different screening technologies in the early discovery phase. High-throughput screening is usually the dominant approach along with different computational methods, but these methods lack the ability to monitor the interactions between drugs and cells in real-time. The ability to measure drug-cell interactions and cell responses during drug stimulation in real-time could provide complementary kinetic information to traditional methods already used in drug discovery. This time-resolved information should help to build a better mechanistic understanding of the effect of drug formulation design on the drug release actions, the drug delivery process and the efficacy of the drug, especially when it comes to new biological drugs and nanoparticle formulations. This dissertation addresses challenges in developing functional surfaces and analysis methods based on the surface plasmon resonance technique for pharmaceutical research purposes. The research in this thesis spans from traditional drug-protein interaction studies and preparation of cell model surfaces to interaction studies with living cells. An approach where proteins were immobilized in a hydrogel was used for studying the interaction kinetics between protein kinase C ε and both an activating and an inhibiting single-chain antibody. The affinities determined for the interactions were able to predict the level of activation or inhibition in subsequent cell culture assays. This thesis also presents two types of new analysis methods, i.e. label-enhanced and multi-wavelength surface plasmon resonance (SPR) methods were developed in order to improve the sensitivity of bioassays and accuracy for characterizing ultra-thin films, respectively. The label-enhanced SPR method was shown to improve assay sensitivity up to 100-fold, whereas the multi-wavelength SPR analysis provided the means to characterize organic layers in the range from a few nanometers to hundreds of nanometers, i.e. layer thicknesses of relevance to biological membranes and hydrogels. New surface coating chemistries based on dextran and thiol-PEG were also developed in this thesis in order to enable the preparation of robust biomimetic membranes by vesicle spreading or adsorption. The dextran-based and PEG-based coatings promoted supported lipid bilayer and adsorbed vesicle layer formation, respectively. The new analysis approaches developed in this thesis were further utilized in order to characterize the optical properties of the formed lipid layers on the dextran- and PEG-based coatings. Finally, a new analytical approach for signal processing of the real-time and label-free SPR measurements performed together with living cells is introduced which provides the mean to differentiate between para- and transcellular cell absorption routes of drug molecules. This dissertation contributes to the pharmaceutical research field by introducing new measuring tools, improved in vitro biomimetic models and new approaches for processing of the signal from label-free measurements in order to provide relevant real-time and complementary information to traditional drug development and discovery tools. This will hopefully benefit the pharmaceutical research field and possibly enable a more efficient development of new pharmaceuticals and therapies in the future.
  • Bäck, Susanne (Helsingin yliopisto, 2014)
    Neurodegenerative diseases are characterized by progressive loss of distinct neuronal populations. In Parkinson s disease (PD) the most prominent cell loss is seen in the dopamine (DA) neuron population in the substantia nigra pars compacta (SNpc). The resulting decrease in striatal DA levels causes dysregulation of neuronal circuits controlling movement and leads to motor symptoms typical to the disease. As for other neurodegenerative diseases, there are no available treatments that would interfere with the degenerative process in PD. The purpose of this work was therefore to test the therapeutic potential of long-term delivery of the neurotrophic factor (NTF) cerebral dopamine neurotrophic factor (CDNF) in the rat partial 6-hydroxydopamine (6-OHDA) lesion model of PD. When injected unilaterally in the striatum, 6-OHDA causes progressive dose-dependent loss of DA neurons in the SNpc accompanied by asymmetrical motor impairment. The 6-OHDA model used in our NTF studies (2x10 µg 6-OHDA) showed a stable lesion progression with a cell loss at two weeks post-lesion corresponding to that seen in PD at symptom onset. In the 6-OHDA model, the DAergic system is traditionally evaluated using immunodetection methods or measurements of tissue neurotransmitter levels. Imaging methods, such as single-photon emission computed tomography (SPECT), allows in vivo detection of neuronal circuits, and together with the DA transporter (DAT) radioligand 2β-carbomethoxy-3β-(4-[123I]iodophenyl)tropane ([123I]β-CIT), SPECT/CT provided reliable estimations of the DA cell degeneration showing high correlation to immunohistochemical findings. The method is sensitive and selective and provides substantial benefits in pre-clinical research allowing longitudinal studies in living animals. The neuroprotective effect of CDNF was studied by applying the NTF intrastriatally as two-week protein infusion with osmotic pumps, or as gene therapy with a recombinant adeno-associated viral vector in 6-OHDA-lesioned rats. Both CDNF delivery methods normalized the amphetamine-induced rotational asymmetry and provided partial protection of the tyrosine hydroxylase (TH) reactive DAergic cells in the SNpc and DA fibers in the striatum. As for GDNF, there were indications of retrograde transport of CDNF, but contrary to what has been reported for GDNF, CDNF did not affect the intact rat DAergic system. In addition, there were differences between the treatments in the capacity to induce sprouting of TH-reactive fibers. Our results confirm that CDNF can be considered as a potential therapy in PD, and that the neuroprotective mechanism of CDNF differs from that of GDNF.
  • Liu, Peng (Helsingin yliopisto, 2013)
    Poorly soluble drugs are often a challenging problem in drug formulation. Reducing the particle size of the drug to a nano-scale leads to an increased surface area-to-volume ratio, increased dissolution velocity and adhesiveness, and improved in vivo performance of poorly soluble drugs. Wet media milling is one of the most popular techniques to prepare the nanocrystals. The aim of this thesis was to optimize the preparation conditions and characterization methods of nanosuspensions for poorly water-soluble drug compounds, then formulate the nanosuspensions into suitable pharmaceutical dosage forms, and finally test the efficacy in in vivo. Firstly, basic research on the preparation and characterization of nanosuspensions was done using the wet milling technique and poorly water-soluble drugs as model compounds. The various milling parameters, including the milling time, diameter of milling balls, stabilizer type and concentrations, were investigated. The particle size, size distribution and stability of nanosuspensions were considered as the important parameters to evaluate the milling conditions. The drug nanocrystals manufactured at the optimal milling environment exhibited significantly increased dissolution rates, good stability and kept a crystalline state. Secondly, the importance of particle size on dissolution was determined. The conventional dissolution method can effectively discriminate the milled and unmilled samples, but it fails to discriminate the dissolution profiles of nanosuspensions with different particle sizes. A novel condition, i.e. the sample concentration in the dissolution medium is close to the apparent saturation solubility of the drug, were found to discriminate the dissolution profiles of nanosuspensions. Thirdly, nanos-in-micros structure for suitable pharmaceutical formulations, e.g. tablet or pulmonary products was set up. The structure is that drug nanocrystals are buried inside microparticle carriers, in which the mannitol and crystalline L-leucine were as matrix and outer layer formers, respectively. Nanos-in-micros structure avoids problems in further formulation of nanopowders, such as poor flowability and aggregation/sinter, meanwhile functionality of individual nanoparticles in microparticles are remained, such as fast dissolution. Finally, the efficacy of nanocrystal formulations in vivo was tested. Brinzolamide nanosuspensions were prepared in buffers and formulated for ocular administration in order to reduce the intraocular pressure in rats. The nanocrystal formulations exhibited a low cytotoxicity and significant reduction in intraocular pressure compared to the physiologic salt solution and untreated group. In conclusion, the nanosuspensions of poorly water-soluble drug can be easily produced by the wet milling technique when the choice of stabilizers and milling parameters are appropriate. The nanosuspensions were successfully formulated and exhibited a good in vivo performance. More efficient formulations based on drug nanosuspensions should be further researched and developed.
  • Shevchenko, Anna (Helsingin yliopisto, 2013)
    To efficiently improve physicochemical properties of pharmaceutical solids and facilitate both preclinical development and clinical translation of investigational new drugs, reliable methods for obtaining a diversity of solid-state forms of a given drug are needed along with effective solid-form screening techniques suitable for the early discovery phases. The research described in this thesis is focused on the development of such methods with the aim of tailoring the solubility, hygroscopicity and physical stability of the solid forms of weakly basic drugs. In particular, an effective and fast approach to initial evaluation of the polymorphism and solvatomorphism tendency and physical stability of drug candidates was developed. The introduced experimental procedures can be applied also to new chemical entities, even prior to the final selection of a drug candidate. In selection of the counterion and coformer for a salt or a cocrystal formation, the exact knowledge of the crystal structure of the drugs and lead compounds is of particular importance. We have found the correct crystalline structures of the neat itraconazole and the itraconazole-succinic acid cocrystal. The corrected data enabled us to select the most appropriate coformers for the formation of itraconazole cocrystals and salts. In another study, the possibility to form cocrystals of itraconazole with C2-C10 aliphatic dicarboxylic acids has been addressed. Using a combination of two screening techniques, which are solvent-drop grinding and slow evaporation, we have discovered cocrystals of itraconazole with C2-C7 acids. Most importantly, C7 was identified as having the maximum carbon atom number of the aliphatic chain for successful cocrystallization with itraconazole. These findings have a considerable conceptual and practical value in the field of crystal engineering, putting an additional emphasis on the importance of the weak intermolecular interactions in the crystal structure cohesion. A comparative evaluation of the discovered cocrystals of itraconazole with malonic acid (C3) and two discovered hydrochloric salts of itraconazole (dihydrochloride and trihydrochloride) has been performed. The intrinsic dissolution rate, hygroscopicity, and thermodynamic stability have been determined for these solid-state forms and compared with those of the itraconazole-succinic acid (2:1) cocrystal. The cocrystallization was found as a more suitable mechanism for the dissolution-rate enhancement of poorly water-soluble weak bases than the formation of hydrochloric salts.
  • Talman, Virpi (Helsingin yliopisto, 2013)
    Diacylglycerol (DAG) is a ubiquitous lipid second messenger that transmits signals from cell membrane receptors to intracellular effector proteins containing a specialized DAG recognition motif, the C1 domain. The protein kinase C (PKC) family of serine/threonine kinases is the best-characterized member of DAG effectors, but there are also six other families of proteins with a DAG-responsive C1 domain: (1) the protein kinase D (PKD) family; (2) the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs); (3) the DAG kinases (DGKs); (4) the Munc13 proteins; (5) the chimaerins; and (6) the Ras guanyl-releasing proteins (RasGRPs). DAG-mediated signalling regulates many cellular functions such as cell proliferation, survival, differentiation, apoptosis and motility − processes that are often deregulated in cancer. As key mediators of these processes, several of the DAG effectors are regarded as promising targets for cancer drug development. Furthermore, PKC contributes to neuronal plasticity and inhibits many pathophysiological processes related to Alzheimer s disease. Activation of PKC is therefore considered a potential future therapeutic strategy for the treatment of Alzheimer s disease. The C1 domain thus represents a well-recognized drug target. However, the existing C1 domain ligands are extremely complex in their chemical structure and from the drug development point of view new C1 domain ligands with a feasible synthesis route are needed. The purpose of these studies was to characterize the structure-activity relationships (SAR) of a novel group of C1 domain ligands, derivatives of 5-(hydroxymethyl)isophthalic acid. These compounds have a simple chemical structure and they are easy to synthesize. Furthermore, these studies aimed to investigate the in vitro pharmacology of the isophthalate derivatives especially focusing on their effects on cell proliferation and morphology. The SAR studies revealed the structural elements indispensable for binding to the C1 domain of PKC: the hydroxymethyl group, both of the ester groups and sufficiently hydrophobic ester substituents. Importantly, the SAR model also held true with β2-chimaerin, another C1-domain containing DAG effector. The active isophthalates bound to PKC with low micromolar affinities, and a selected example of these, HMI-1a3, was also shown to bind to the C1 domains of PKD1 and MRCKα at similar concentrations. Several isophthalates had an antiproliferative effect in HeLa human cervical carcinoma cells. HMI-1a3 exhibited the most potent cytotoxic and antiproliferative effect of the derivatives tested. The isophthalates with no affinity to the C1 domain (e.g. NI-15e, the inactive derivative of HMI-1a3) had no effect on HeLa cell viability or proliferation, suggesting a C1 domain-mediated effect. HMI-1a3 also induced a morphological change characterized by cell elongation. It was accompanied with a marked reorganization of actin cytoskeleton: loss of focal adhesions and actin stress fibres. The elongation-inducing effect of HMI-1a3 was inhibited by an MRCK inhibitor, and thus seems to be at least partially mediated by MRCK. However, the mechanism of its antiproliferative effect remains unclear and may be mediated by several of the DAG effectors. In SH-SY5Y neuroblastoma cells, which are widely used as an in vitro model for neuronal differentiation, HMI-1b11 inhibited cell proliferation and supported neurite growth. HMI-1a3 induced SH-SY5Y cell differentiation as well, but unlike HMI-1b11 it also induced cell death. Again, derivatives with poor binding affinity to the C1 domain had no effect. The HMI-1b11-induced response was accompanied by PKC-dependent ERK1/2 phosphorylation and up-regulation of GAP-43, which is known to mediate neuronal differentiation and to contribute to neurite outgrowth. In conclusion, these studies identify derivatives of 5-(hydroxymethyl)isophthalic acid as a promising novel group of C1 domain ligands. The antiproliferative and cytotoxic HMI-1a3 serves as a potential lead molecule for cancer-related drug discovery. HMI-1b11 on the other hand may be useful in drug development related to neurodegenerative diseases because of its non-toxic and differentiation-inducing properties in SH-SY5Y cells. More studies using cell-based and in vivo models are, however, needed to fully assess the potential of isophthalates in drug development.
  • Lindholm, Jesse (Helsingin yliopisto, 2013)
    Major depressive disorder (MDD) affects millions of people every year and produces significant human suffering and economic burden to society. The symptomatology of MDD is heterogeneous and multidimensional, and only two core symptoms, depressed mood and anhedonia, are frequently shared by patients. Consequently, modeling of MDD is challenging, and only depression-related phenomena, not depressed mood itself, can be examined in animals. MDD is commonly treated with antidepressant drugs (or antidepressants, ADs). However, monoamine-based ADs act in a delayed-onset manner and often exhibit only moderate clinical efficacy. Electroconvulsive therapy (ECT) remains the treatment of choice for treatment-resistant depression (TRD) and for cases for which a rapid clinical response is required. Given the practical and ethical limitations of ECT, the development of fast-acting ADs is needed. Importantly, the NMDA receptor antagonist ketamine has been shown to produce rapid and long-lasting AD effects in TRD patients. Changes in the levels and signaling of neurotrophin brain-derived neurotrophic factor (BDNF) have been associated with the etiology of MDD. However, studies of genetically modified mice expressing altered levels of BDNF have not provided a solid link between BDNF deficiency and depression-related behavior. By contrast, emerging evidence indicates that the effects of ADs are mediated by BDNF and its tropomyosin-related kinase B receptor, TrkB. ADs enhance BDNF-TrkB signaling and thereby facilitate neuronal plasticity in the brain. Recent evidence indicates that these changes in plasticity lead to the restoration of juvenile-type plasticity in the adult rodent cortex, which allows environment-driven reorganization of brain networks. Based on these data, the network theory of AD action was formulated. However, it is unclear if this concept can be generalized to diverse neuronal networks. The main aims of this thesis were to investigate the importance of TrkB signaling in the anxiety- and depression-like behavioral phenotype in mice, to examine the role of BDNF-TrkB signaling in the antidepressant-like effects of glutamatergic drugs in mice, to study the network theory of ADs in a mouse fear extinction paradigm and to investigate the behavioral effects of adult fluoxetine treatment in mice exposed to fluoxetine early in life. When examining TrkB signaling-deficient mice (TrkB.T1), we observed that young and aged TrkB.T1 mice exhibited alterations in their exploration and emotional behavior and increased behavioral despair. These findings suggest that altered TrkB signaling leads to depression-like behavior, and thus, TrkB.T1 mice may be used as a genetic model of depression. We next studied selected glutamatergic drugs in behavioral despair models and determined that, similar to their effects in humans, ketamine and the AMPA receptor potentiator LY 451646 produce an antidepressant-like effect in mice. In contrast to classical ADs, these drugs were also effective in BDNF heterozygote knock-out mice. Furthermore, neither of these drugs influenced BDNF protein or Trk-phosphorylation levels in wild-type or BDNF-deficient mice. These data suggest that the antidepressant-like effects of ketamine may be independent of BDNF-TrkB signaling. Disturbances in the serotonergic system during early development may cause permanent behavioral effect in adult animals. In our study, early life exposure to fluoxetine, an AD that enhances serotonergic transmission, led to specific and persistent behavioral changes in adult animals. Intriguingly, adult fluoxetine treatment normalized some of these changes. We therefore examined whether fluoxetine can enable plastic changes in fear circuits in mice in conjunction with an environmental stimulus. We observed that the combination of fear exposure and fluoxetine treatment produced permanent fear extinction in the classical fear conditioning paradigm in mice. Importantly, neither fluoxetine nor extinction alone produced permanent fear erasure. This finding supports the network theory of AD action and clinical observations demonstrating the superiority of the combination of drug administration and psychotherapy for the treatment of post-traumatic stress disorder and depression. In conclusion, these data strengthen the connection between BDNF-TrkB signaling and the antidepressant-like effects of classical ADs and support the network hypothesis of AD action. In addition, these results also suggest that there may be fast-acting AD treatments with a mechanism of action that is independent of BDNF-TrkB signaling.
  • Sneitz, Nina (Helsingin yliopisto, 2013)
    UDP-glucuronosyltransferases (UGTs) are responsible for the metabolism of many xenobiotics, as well as several endogenous compounds like bilirubin and certain steroids. This thesis involves three major themes: UGT1A1 and Crigler-Najjar syndrome (CN); expression and characterisation of a novel UGT, UGT2A2; and the glucuronidation of various endogenous and synthetic steroids. Fourteen different UGT1A1 mutations were identified in 19 CN patients, four of which were novel. The mutant proteins were expressed in baculovirus infected Sf9 insect cells and their activity towards bilirubin and other substrates was determined. Residual glucuronidation activity and the severity of hyperbilirubinemia were found to correlate well in most cases. In addition, a major advancement to aid the development of gene therapy for CN was achieved by in discovering that ezetimibe glucuronidation correlates with reduction of serum bilirubin in gene therapy treated Gunn rats. This compound might be useful in examining the efficacy of gene therapy in humans in the future. When the novel UGT2A2 was cloned, expressed, and studied alongside UGTs 2A1 and 2A3, results indicated that UGT2A2 is a functional enzyme with broad substrate specificity. UGT2A2 was found to be expressed mainly in nasal mucosa, similar to UGT2A1. In addition, UGT2A1 and UGT2A2 were found to differ largely in their regioselectivity towards estrogens. Lastly, the glucuronidation of estriol, 16-epiestriol, 17-epiestriol, 13-epiestradiol, ent-estradiol, ent-androsterone, and ent-etiocholanolone by different UGT enzymes was studied and analytical methods for analysing these compounds were developed. UGTs 1A10 and 2B7 were found to be the most active UGTs in the glucuronidation of almost every steroid studied. Several patterns in the regio- and stereoselectivity of steroid glucuronidation were observed for the first time. The effects of phenylalanines 93 and 90 on human UGT1A10 on glucuronidation were also studied, exploiting site directed mutagenesis. The results revealed that F93 is involved in the interactions of the enzyme with estrogens and a homology model of UGT1A10 was created based on the results. Knowledge of glucuronidation of endogenous compounds is important, since it may be directly or indirectly linked to clinical conditions, like Crigler-Najjar syndrome, and also because certain drugs that inhibit UGTs, may inhibit the metabolic pathways of endogenous substrates that are metabolised by UGTs. Understanding the glucuronidation of steroids could be used to make better predictions on the role of glucuronidation in the metabolism of newly designed drugs that resemble steroids. Detailed information on steroid glucuronidation may also be used in determining UGT structure via function and the results obtained during this PhD work may also be used in the development of more detailed homology models of other UGT enzymes.
  • Vaikkinen, Anu (Helsingin yliopisto, 2013)
    Direct open air surface sampling/ionization mass spectrometry offers a means to the rapid, sensitive, and reliable analysis of samples in their native states. It can be used for fingerprinting, but also for studying the distribution of compounds on surfaces by mass spectrometry imaging (MSI). The aim of this work was to develop and apply direct open air surface sampling/ionization mass spectrometry methods in the study of neutral and nonpolar compounds, with emphasis on photoionization-based techniques. First, two krypton discharge photoionization lamps were compared in micro atmospheric pressure photoionization and desorption atmospheric pressure photoionization mass spectrometry (DAPPI-MS). The radio frequency (rf) alternating current lamp was shown to have even over an order of magnitude higher ionization efficiency than the direct current (dc) lamp, thus providing better sensitivity. This was possibly because the rf lamp emitted 5 times the number of photons of the dc lamp. Next, a polydimethylsiloxane (PDMS) extraction phase was developed and optimized for DAPPI-MS analyses of aqueous samples to decrease the severe matrix effects often encountered in direct open air surface sampling/ionization mass spectrometry. The method was applied in the analysis of spiked wastewater and urine samples. The direct open air surface sampling/ionization mass spectrometry methods best suited for neutral and nonpolar analytes do not enable analyses at sub-millimeter range, and thus they are not optimal for MSI. A novel laser ablation atmospheric pressure photoionization (LAAPPI) ion source was developed to enable analyses of at ~300 μm resolution. The LAAPPI method was capable of semi-quantitative performance and suited for the analysis of neutral and nonpolar compounds directly from plant and animal tissues. Subsequent work also demonstrated the suitability of LAAPPI for MSI with sage (Salvia officinalis) leaves as tissue model. The final part of the research involved the modification of a laser ablation electrospray ionization (LAESI) source with a heated gas jet to enable the simultaneous study of polar and nonpolar compounds. The heat-assisted LAESI (HA-LAESI) was tested for analytes of different polarities and sizes, and it was found to protonate compounds ranging from selected PAHs to large proteins with more or less equal efficiency. As compared with LAAPPI and LAESI, the HA-LAESI method ionized a wider range of analytes. Biological tissues were successfully fingerprinted, confirming that neutral and nonpolar analytes can be detected in biological matrices, and HA-LAESI was used in MSI. In conclusion, the work presented in this thesis advances the study of neutral and nonpolar compounds through developments in direct open air surface sampling/ionization mass spectrometry. Like other direct open air surface sampling/ionization mass spectrometry methods, however, the described methods are still in their infantry. Additional work is required to establish them as reliable competitors of more traditional techniques.