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  • Wissel, Gloria (Helsingin yliopisto, 2015)
    The main goal of this dissertation is to identify novel modulators acting on ATP Binding Cassette subfamily C member 2 (ABCC2) transporters and α2-adrenoceptors subtypes. With the purpose of identifying novel modulators and their mode of action, a combination of experimental and computational approaches have been used. The first protein presented in this dissertation is the ABCC2 transporter, also known as the multidrug resistance associated protein 2 (MRP2), an efflux transporter expressed in polarized cells where it effluxes a variety of both endogenous and exogenous molecules out of the cell. The most common way to study the interactions between small molecules and ABCC2 transporter is by a vesicle transport assay. Three assays are commercially available, which use different probes to define the ABCC2- transport. With the intent to define the different assays and identify the effect that small molecules have on the ABCC2-transport, a small set of eight compounds and, subsequently a larger library of compounds were tested with the different assays. Additionally, the aim was to identify and characterise novel ABCC2 inhibitors, 16 inhibitors have been identified from the larger library and classification models were built to identify important descriptors that were able to discriminate inhibitors from inactive molecules. Instant structure-activity relationships (SAR) of four scaffolds of ABCC2 modulators are also presented. In addition, some unpublished results are presented, the homology model of ABCC2 and further insights into the SAR of ABCC2 modulators. The other proteins included in this dissertation are the three subtypes of the α2-adrenoceptors, G-protein coupled receptors, involved in the signalling pathway of adrenaline and noradrenaline. A clear subtype characterization/profile of these proteins is not available. Selective molecules could be used in treatment of high blood pressure, in the alleviation of withdrawal symptoms, and as anaesthetic with fewer side effects than the current drugs. To define the affinity of a small set of antagonists and outline the involvement of the first transmembrane helix in ligand binding, a competition binding assay has been used with chimera receptors where the first transmembrane helix has been swapped between the three subtypes. Molecular modelling has been used to explain the different binding affinities to the chimera receptors. Additionally, the aim was to identify novel α2B-adrenoceptor selective compounds, thus a mid-sized library has been screened using a miniaturized binding assay. Hierarchical classification and chemoinformatics analysis has been used to visualize and analyse the screening results.
  • Manevski, Nenad (Helsingin yliopisto, 2013)
    Human UDP-glucuronosyltransferases (UGTs) are important in the metabolic elimination of xenobiotics and endogenous compounds from the body. These enzymes transfer glucuronic acid moiety from the cosubstrate, UDP-glucuronic acid (UDPGA), to nucleophilic groups of small organic molecules, such as hydroxyl, carboxylic, or amino group. The conjugation of these molecules with polar glucuronic acid usually diminishes their pharmacodynamic activity, promotes aqueous solubility and enhances recognition by efflux transporters in the cells, all of which contributes to the efficient metabolic elimination and excretion of the conjugate from the body. Due to these unique properties, UGT enzymes play major roles in drug metabolism and pharmacokinetics. The main goal of this thesis was to investigate the activity and enzyme kinetics of UGTs, as well as the in vitro assay conditions needed to accurately determine the enzyme kinetic parameters. Particular attention focused on the glucuronidation of psilocin, the enhancement of UGT activity by the inclusion of purified bovine serum albumin (BSA), and the enzyme kinetic mechanism of UGT1A9. These goals are especially important in the early phases of preclinical drug development, where in vitro assays serve to explain and predict the glucuronidation of the drug in vivo, both qualitatively and quantitatively. As a starting point, we studied the glucuronidation of psilocin, the hallucinogenic indole alkaloid from mushrooms of the genus Psilocybe, by all the human UGTs of subfamilies 1A, 2A, and 2B. To understand the substrate selectivity of human UGTs, we also studied the glucuronidation of 4-hydroxyindole, a chemically simpler analog of psilocin which lacks the N,N-dimethylaminoethyl side chain. We successfully prevented the oxidative degradation of psilocin, a problem we encountered early in the study, by including an antioxidant (1 mM dithiothreitol) in the assays. Our results showed that psilocin is glucuronidated mainly by UGTs 1A10 and 1A9, whereas the activities of UGTs 1A8, 1A7, and 1A6 were lower. On the other hand, 4-hydroxyindole was glucuronidated mainly by UGT1A6, whereas the activities of UGTs 1A7 1A10 closely correlated with their respective rates of psilocin glucuronidation. To understand in which human tissues psilocin glucuronidation takes place, we studied the expression levels of mRNA for UGTs 1A7 1A10; this work was performed in collaboration with Dr. Michael H. Court of the Tufts University School of Medicine, Boston, Massachusetts. The combined results of the activity and expression studies indicate that although the intestinal enzyme UGT1A10 shows the highest glucuronidation clearance, UGT1A9, an enzyme abundantly expressed in both the liver and kidneys, may be the main contributor to psilocin glucuronidation in vivo. The inclusion of purified albumin is known to significantly enhance glucuronidation rates in vitro. In subsequent studies, we focused our attention on the scope and mechanism of this activity enhancement and investigated albumin effects in a total of 11 human UGTs. Before proceeding with enzyme kinetic assays, we carefully measured the binding of substrates to BSA by either ultrafiltration or rapid equilibrium dialysis. Our results showed that the inclusion of BSA significantly enhances the in vitro glucuronidation activity of almost all the UGTs we tested, either by increasing the apparent substrate affinity (lower Km) or the reaction-limiting velocity (higher Vmax), or both. The nature of albumin effects, however, varied greatly and depended both on the UGT enzyme and the substrate employed. The highest activity increases in the presence of BSA were observed in UGTs 1A7, 1A9, 1A10, 2A1, and 2B7, whereas BSA stimulation was comparatively less pronounced in UGTs 1A1, 1A6, 1A8, 2B4, and 2B15. On the other hand, depending on the substrate used, the addition of BSA to UGTs 1A1, 1A6, and 2B17 sometimes resulted in a lack of any stimulatory effects. Moreover, the activity enhancement by BSA appears independently of the enzyme source used, since both native enzymes in human liver microsomes and recombinant enzymes expressed in Sf9 insect cells yielded similar results. To investigate the mechanism of albumin effects, as well as to elucidate the enzyme kinetic mechanism of human UGTs, we studied bisubstrate enzyme kinetics, the product inhibition, and dead-end inhibition kinetics of UGT1A9. For this purpose, we employed 4-methylumbelliferone as the aglycone substrate and investigated both forward- and reverse-direction UGT-catalyzed reactions. The combined results of our experiments strongly suggest that UGT1A9 follows the compulsory-order ternary-complex mechanism with UDPGA binding first. The addition of BSA quantitatively changes the enzyme kinetic parameters, presumably by removing internal inhibitors that bind to binary (enzyme-UDPGA) or ternary (enzyme-UDPGA-aglycone) complexes, but the underlying compulsory-order ternary-complex mechanism remains unaffected. In addition, based on enzyme kinetic parameters measured in the forward and reverse reaction, we elucidated the thermodynamic equilibrium constant of the overall reaction (Keq = 574), as well as the relative magnitude of the individual rate constants. In summary, the results obtained deepen our current knowledge of UGT enzyme kinetics and set new guidelines for performing in vitro UGT assays. The study of psilocin and 4-hydroxyindole glucuronidation revealed that relatively small structural modifications, such as the loss of the side chain, lead to major changes in UGT substrate selectivity. And provided the substrate binding to BSA is accounted for, the addition of BSA significantly enhances the activities of almost all the UGTs tested and improves the accuracy of the measured enzyme kinetic parameters. These features are especially important for the prediction of UGT activity in vivo. Finally, our results deepen our current understanding of the UGT enzyme kinetic mechanism and conclusively show that UDPGA is the first, and the aglycone substrate is the second binding substrate to form a ternary complex in UGT1A9-catalyzed reactions.
  • Wikström, Jonna (Helsingin yliopisto, 2013)
    Cells have multiple functions in the body, including maintenance of the tissue structure and physiological homeostasis. The cells express and secrete proteins and other factors that exert actions in other cells. These principles form the underlying basis for cell therapy and cell transplantations. Transplanted cells can be used to regenerate tissue structures and homeostasis or they can be used as platform for secretion of therapeutic molecules. Biomaterials can be used to augment the cell growth, differentiation and viability in cell therapy. In addition, the biomaterial matrix may help the surgical placement of the cells into the target site. Importantly, the biomaterial may protect the cell from the immunological and inflammatory reactions after transplantation. The immunological protection of the transplanted therapeutic cells is based to selectively permeable artificial membrane. The membrane prevents the passage of high-molecular weight substances such as large antibodies and cytotoxic immune cells, but permits the passage of smaller molecules, like the secreted therapeutic molecules, nutrients, waste products and oxygen. Lately the interest in cell encapsulation and biomaterial cell interactions has increased due to the emerging techniques of cellular engineering and stem cell differentiation. Storage of microencapsulated cells in freeze-dried form would improve the logistics of the cell therapies (e.g. shipment to the hospitals for reconstitution and use). Otherwise, the microencapsulated cells should be kept viable in continuous culture conditions. The goal of this work was to evaluate alginate based microencapsulation of retinal pigment epithelial cell line (ARPE-19) for cell therapy. Cell viability was evaluated with stably expressed secreted alkaline phosphatase (SEAP), live/dead imaging and oxygen consumption. An empirical kinetic model was built based on FITC-dextran release and protein secretion to describe, release and potential accumulation of therapeutic proteins in the cell microcapsules. Primary animal experiments were done to evaluate the protein release and functionality in the cell microcapsules. Alginate based cell microcapsules were frozen and freeze-dried in order to evaluate the possibility for cell microcapsule preservation in dry powder form. In conclusion, ARPE-19 is a potential cell line for long-term cell therapy based on the expression of transgenes. ARPE-19 cells remain vital in the alginate microcapsules, and they are able to express stably transfected transgene over long periods (at least 20 months). The best cell viability was obtained with alginate microcapsules with calcium and barium cross-linking. This method results in adequate pore sizes that allowed secretion of SEAP. The same microcapsules showed biocompatibility after intraperitoneal administration in preliminary animal experiments. Empirical kinetic simulation model was able to predict the possibility of accumulation inside the alginate microcapsules and demonstrated that the accumulation potential depends on the microcapsule structure. Lyophilization of the cell microcapsules showed that the cells were able to retain some viability during freeze-drying and reconstitution when lyoprotectants were used.
  • Akhgari, Amir (VTT Technical Research Centre of Finland Ltd, 2015)
    Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in the Middle East and South Asia. It produces a large number of terpenoid indole alkaloids(TIAs), some of which possess important pharmacological properties. This study focused on the establishment of biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities it was necessary first to develop different analytical methods to determine the alkaloid contents and changes in their profiles in transgenic cultures and after various treatments. Targeted and non-targeted analyses from cell andorgan cultures were carried out using gas chromatography-mass spectrometry(GC-MS), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Callus cultures were successfully initiated from five different explants onmodified B5 medium containing phytohormones. The phenotypes of the calli varied, but as was expected the callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems had elevated levels of strictosidine lactam compared to other cultures. Transformation experiments revealed that only leaves but not cotyledons, hypocotyls or stem segments were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. Wild type and gus hairy root clones contained twofold higher amounts of alkaloids than adventitious roots. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS analysis allowed the separation of the most volatile and non-polar alkaloids in a single run. The composition of typical non-polar alkaloids indicated the occurrence of 20 TIAs belonging to nine different groups. The quantities of these alkaloids varied between clones in the order eburenine, vincanine, vallesiachotamine and yohimbine isomer II. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GC-MS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Methyl jasmonate, a well-known elicitor, caused a significant increase in the total alkaloid content of wild type hairy roots as determined by NMR analyses. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased compared to non-elicited cultures. Another studied elicitor, chitosan, did not have any effect on individual alkaloid contents. Transgenic hairy root clones did not exhibit phenotype differences. Multivariate analysis from NMR data showed a clear discrimination between transformed and wild type/gus cultures. This was most probably due to differences in primary metabolites, as the total alkaloid content did not vary between different hairy roots and controls. In general, the production of individual TIAs, analysed by HPLC, was repressed in hairy roots transformed with geraniol synthase(ges) and geraniol 8-oxidase (g8o) genes compared to the wild types. Overexpression of the strictosidine synthase (str) gene resulted in a higher accumulation of serpentine, whereas the production of strictosidine lactam was decreased. There were no significant differences in the contents of other alkaloids compared to the wild type hairy roots. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabled comprehensive investigation of the alkaloids. These data might serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.
  • Suominen, Tina (Helsingin yliopisto, 2015)
    Neurotransmitters and neurosteroids are compounds that regulate the functions of the brain. The neurotransmitters dopamine (DA) and serotonin (5-HT) play a role in several psychological conditions, including schizophrenia, depression and anxiety. DA also has an important role in Parkinson s disease. Neurosteroids are involved in neurodegenerative diseases. In Alzheimer s disease and multiple sclerosis, the levels of neurosteroids are decreased in certain areas of the brain. Neurosteroids differ from classical neurotransmitters in that they are lipid-soluble and can easily cross the blood-brain barrier (BBB). Neurotransmission can be studied in vivo by microdialysis, and as the concentrations of neurotransmitters in the microdialysates are very low, sensitive analytical methods are needed for their analysis. In this work an UPLC-MS/MS method was developed for the determination of 5-HT, DA, their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfonate and glucuronide conjugates. The method was validated and applied for analyzing human brain microdialysis and cerebrospinal fluid (CSF) samples. Intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain. The origin of the determined phase II metabolites in the brain is unknown. Even though sulfonate-conjugated compounds such as dopamine sulfonate (DA-S) and 5-HIAA-S were detected in the human brain, it is unclear whether they were locally formed or transported into the brain through the BBB from peripheral sources. The BBB permeation of DA-S was studied by administration of isotope (13C6)-labelled DA-S, which can be distinguished from endogenous DA-S by mass spectrometry, subcutaneously (s.c.) while brain microdialysis samples were collected and analyzed by UPLC-MS/MS. The fate of 13DA-S in brain was followed by monitoring 13C6-labelled DA-S metabolites and hydrolysis products. The results proved that DA-S permeates through the BBB, and indicated that DA-S finally either permeates through the BBB back to the peripheral circulation or is dissociated or metabolized by unknown mechanisms. While the hydrophilic neurotransmitters DA and 5-HT are well suited for analysis by liquid chromatography coupled to atmospheric pressure ionization, the neurosteroids have more commonly been analyzed by methods based on gas chromatography (GC) coupled to ionization in vacuum. Recently GC has been combined to atmospheric pressure photoionization utilizing heated nebulizer microchips (μAPPI). We now constructed a simpler interface for combining GC to mass spectrometry (MS) using dopant-assisted atmospheric pressure photoionization (APPI), utilizing commercially available hardware. The neurosteroids were analyzed as trimethylsilyl (TMS) derivatives, and the effect of different dopants (chlorobenzene, toluene and anisole) on the ionization and on the sensitivity of the method was investigated. Chlorobenzene was chosen as the best dopant, as the neurosteroid-TMS derivatives formed intense molecular ions with minimal fragmentation, while with toluene and anisole also protonated molecules were observed. The molecular ions of the steroids formed by APPI ionization showed fragmentation patterns in their MS/MS spectra similar to the patterns seen in corresponding spectra obtained by electron impact ionization (EI). Therefore the use of EI libraries could be possible, thus enabling the identification of a wide range of unknown compounds.
  • Järvinen, Päivi (Helsingin yliopisto, 2011)
    The first line medication for mild to moderate Alzheimer s disease (AD) is based on cholinesterase inhibitors which prolong the effect of the neurotransmitter acetylcholine in cholinergic nerve synapses which relieves the symptoms of the disease. Implications of cholinesterases involvement in disease modifying processes has increased interest in this research area. The drug discovery and development process is a long and expensive process that takes on average 13.5 years and costs approximately 0.9 billion US dollars. Drug attritions in the clinical phases are common due to several reasons, e.g., poor bioavailability of compounds leading to low efficacy or toxic effects. Thus, improvements in the early drug discovery process are needed to create highly potent non-toxic compounds with predicted drug-like properties. Nature has been a good source for the discovery of new medicines accounting for around half of the new drugs approved to market during the last three decades. These compounds are direct isolates from the nature, their synthetic derivatives or natural mimics. Synthetic chemistry is an alternative way to produce compounds for drug discovery purposes. Both sources have pros and cons. The screening of new bioactive compounds in vitro is based on assaying compound libraries against targets. Assay set-up has to be adapted and validated for each screen to produce high quality data. Depending on the size of the library, miniaturization and automation are often requirements to reduce solvent and compound amounts and fasten the process. In this contribution, natural extract, natural pure compound and synthetic compound libraries were assessed as sources for new bioactive compounds. The libraries were screened primarily for acetylcholinesterase inhibitory effect and secondarily for butyrylcholinesterase inhibitory effect. To be able to screen the libraries, two assays were evaluated as screening tools and adapted to be compatible with special features of each library. The assays were validated to create high quality data. Cholinesterase inhibitors with various potencies and selectivity were found in natural product and synthetic compound libraries which indicates that the two sources complement each other. It is acknowledged that natural compounds differ structurally from compounds in synthetic compound libraries which further support the view of complementation especially if a high diversity of structures is the criterion for selection of compounds in a library.
  • Kreander, Kari (Helsingin yliopisto, 2006)
    More and more drugs are becoming useless as a result of increasing numbers of drug resistant pathogen bacteria strains. The urge to find new active drugs, pure or modified, has become a critical task to overcome the limitations that older, still in use, drugs have faced. Drug companies and research facilities are screening different sources with different techniques to fill this need. For a successful screening process, optimized high-quality methods are needed. In this study, erythromycin resistant Streptococcus pyogenes strains, mefA, ermB and ermTR, and the Staphylococcus simulans ermC strain of Finnish origin were used to optimize a turbidometric screening assay using a 96-well microplate for detecting new antimicrobials. Optimization was assisted by using quality parameters S/N (signal-to-noise), S/B (signal-to-background) and Z' factor (screening window coefficient) to confirm the reliability and repeatability. The optimized assay was used for screening a small-scale library of natural compounds and their derivatives against the antibiotic resistant strains. The results showed that gallic esters, specially octyl gallate, had potential inhibition effect against tested strains. Lichen acids were also found to be good inhibitors against all tested strains. The search for novel antibacterial agents can be facilitated by virtual screening of compound databases against a known bacterial target. Following this approach, approx. 200 000 compounds were screened in silico for binding to ErmC' and used for selecting the 49 best-binding, drug-like compounds for in vitro evaluation. As a primary screen, a fluorometric, biochemical assay measuring the inhibition of catechol-O-methyltransferase (COMT), structurally very similar to ErmC', was employed to evaluate the potential activity against ErmC. Out of the selected 49 compounds, two structurally very similar compounds were identified as confirmed hits with reasonable activity (IC50 values of 26 and 73 µM). However, no marked activity was observed in a cell-based assay performed with the Staphylococcus aureus ermC strain. High-performance liquid chromatography (HPLC) was used for microfractionation of natural extracts to overcome limitations of photometric measurement of colored samples that can affect the results of a screening assay. The microfractionation was successfully combined with the 96-well microplate antibacterial assay. The study demonstrated that the use of microfractionation coupled with bioactivity screening is a powerful tool for the identification of active components in natural extracts. To study natural extracts and their safety, a miniaturized Ames test with Salmonella typhimurium TA98 and TA100 strains in a 6-well plate was used. With a miniaturized method, the cost of the test can be decreased, and less time, workspace and amounts of compound are needed than in a normal Ames test. The assay was used to screen mutagenicity and antimutagenicity of rapeseed, pine bark and raspberry extracts and their factions with vinylsyringol, a pure compound from crude rapeseed oil. None of the extracts were shown to be mutagenic. When the metabolic activator (rat liver S9 enzyme) was not added with known positive control (mutagen) and extract, all of the extracts were observed to have antimutagenic properties. The natural extracts were further studied with the Caco-2 model to evaluate their ability to affect the permeability of co-administrated drugs across the cell monolayer. It has been previously shown that some natural extracts can have drug interactions and affect the drug's cellular permeability. Here, the permeability of verapamil, ketoprofen, metoprolol, and paracetamol under the influence of co-administered natural extracts were studied. As a result, none of the extracts had notable effects. In conclusion, it is important to have a proper approach when screening natural products for biological activity. Using the latest technology can be the key for finding new promising drug candidates. Assay validation and miniaturization are good ways to get results quickly and with less money and work. High-quality methods and a thorough investigation that also take safety aspects into account can have a significant effect on the overall success of the screening process.
  • Kauppila, Tiina (Helsingin yliopisto, 2004)
  • Soikkeli, Anne (Helsingin yliopisto, 2012)
    Automation compatibility is a prerequisite for in vitro transport and metabolism assays that are designed for screening large numbers of compounds at the early stages of drug discovery, in parallel with activity optimization. However, automation of more complex assays has also many benefits, even if it does not always increase the throughput of the assay directly. In this thesis work, a new automatable high throughput screening assay was developed for detecting different compound interactions with UGT1A6, one of the human intestinal UGT enzymes. The fluorescence-based assay relies on a robust probe reaction, glucuronidation of 1 naphthol by recombinant human UGT1A6, which yields a highly fluorescent product, 1 naphthylglucuronide. Under the optimized assay conditions, the plate reader-based analysis method was able to detect a given compound interaction with the enzyme, through the probe reaction, in a comparable manner to the (cumbersome) reference HPLC-based method. The new method can analyze the interaction of many different compounds with the UGT1A6 simultaneously. The developed assay was then used to collect data for computational model development. The effects of different compounds on the probe glucuronidation rate were used for a classification model, based on an SVM method, and for 3D-QSAR models, based on CoMFA and CoMSIA analyses. Both the models indicated physicochemical parameters and functional groups that are important for the interaction of different compounds with the UGT1A6. Interactions of two drugs, diclofenac and indomethacin, were studied more thoroughly with the several UGT enzymes, including UGT1A6. The effect of pH on the interaction was examined in both human liver and intestinal microsomes, as well as with many individual recombinant human UGTs. The results indicated that the effects of pH were both enzyme and substrate dependent, suggesting that changes in the enzyme, probably protonation of one or more amino acid side chain, played a major role in the observed effects. Finally, automation of three different types of cell-based assays, Caspase-Glo® 3/7, sulforhodamine B and bidirectional Caco-2 monolayer transport assays, are described in detail. Although, the bidirectional Caco-2 assay was the most challenging for automation, due to the complex assay protocol and the sensitivity of the monolayer to mechanical stress, mainly caused by pipetting, automation improved the utilization potential of the laborious method significantly.
  • 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.
  • Bimbo, Luis (Helsingin yliopisto, 2012)
    Several of the newly developed drug molecules experience poor biopharmaceutical behavior, which hinders their effective delivery at the proper site of action. Among the several strategies employed in order to overcome this obstacle, mesoporous silicon-based materials have emerged as promising drug carriers due to their ability to improve the dissolution behavior of several poorly water-soluble drugs compounds confined within their pores. In addition to improve the dissolution behavior of the drugs, we report that porous silicon (PSi) nanoparticles have a higher degree of biocompatibility than PSi microparticles in several cell lines studied. In addition, the degradation of the nanoparticles showed its potential to fast clearance in the body. After oral delivery, the PSi particles were also found to transit the intestines without being absorbed. These results constituted the first quantitative analysis of the behavior of orally administered PSi nanoparticles compared with other delivery routes in rats. The self-assemble of a hydrophobin class II (HFBII) protein at the surface of hydrophobic PSi particles endowed the particles with greater biocompatibility in different cell lines, was found to reverse their hydrophobicity and also protected a drug loaded within its pores against premature release at low pH while enabling subsequent drug release as the pH increased. These results highlight the potential of HFBII-coating for PSi-based drug carriers in improving their hydrophilicity, biocompatibility and pH responsiveness in drug delivery applications. In conclusion, mesoporous silicon particles have been shown to be a versatile platform for improving the dissolution behavior of poorly water-soluble drugs with high biocompatibility and easy surface modification. The results of this study also provide information regarding the biofunctionalization of the THCPSi particles with a fungal protein, leading to an improvement in their biocompatibility and endowing them with pH responsive and mucoadhesive properties.
  • Ojala, Tiina (Helsingin yliopisto, 2001)
  • 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.
  • Valo, Hanna (Helsingin yliopisto, 2012)
    Nanotechnology can be used to modify drug delivery by various approaches. Bio-polymer based nanoparticles represent a well-established option to formulate drug delivery systems. New therapeutic compounds are often insoluble or poorly soluble in water, which is a major factor in causing irregular and insufficient absorption, and reduced bioavailability of the drug. Increased dissolution rate can be achieved by decreasing the particle size to nanometer range. Another common reason for the reduced efficacy of the therapeutic compounds is their poor delivery to the desired site of action. Advanced nanoparticle formulations can be used to provide controlled release profiles or they can be combined with ligands for targeted drug delivery. Considering the current needs to produce stable nanoparticle systems for the controlled and actively targeted drug release, the versatile group of biopolymers may offer these functionalities. One topic of this work was to set-up the electrospray apparatus for the production of poly(lactic acid) (PLA) drug nanoparticles. By utilizing electrospray, it was possible to produce spherical drug-loaded PLA-particles with approximately 200 to 800 nm diameters. The main benefits of the electrospray method were to control the particle size as well as the possibility to entrap both hydrophobic and hydrophilic drugs into the polymeric nanoparticles. Second topic of this work was to utilize amphiphilic proteins, hydrophobins, to provide a layer around the drug nanoparticles that can be functionalized by protein engineering techniques. Adsorption of the protein onto the particle surface restricted the particle growth after the nanoparticles were formed, and it also produced a layer around the hydrophobic drug that was possible to functionalize further. Hydrophobin-mediated nanoparticle synthesis was a fast and effective process that was also easy to up-scale. As third topic, nanofibrillar celluloses (NFCs) from various origins were studied as alternative biopolymer carriers for drug nanoparticles. The nanostructured cellulose matrix, an aerogel, prevented the aggregation of the hydrophobin coated nanoparticles during the freeze-drying and storage. Controlled drug release applications could be designed and enabled by utilizing the various modifications of NFC matrices. As a result of this thesis, knowledge about the versatile biopolymer-based materials was provided as a means to construct stable nanoparticle formulations that can offer versatile applications for pharmaceutical nanotechnology.