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Antibiotic resistance is a global crisis causing increasing number of infections that cannot be treated with conventional antibiotics. The main reasons for the resistance crisis include overuse and misuse of antibiotics, use in agriculture, and decreased interest of pharmaceutical companies to discover and develop new antibiotics. Apart from mortality, antibiotic resistance causes large economical costs due to longer hospitalizations and more expensive treatments. Bacteria can acquire resistance via multiple pathways. Main path for spread of resistance in bacterial populations is horizontal gene transfer (HGT) in which bacteria receive genetic material that contain resistance genes. Bacteria can acquire new resistance traits via mutations in their genome. The emergence of resistance is a natural feature of bacteria and therefore many bacteria and bacteria can quickly acquire resistance towards novel antibiotics. The resistance properties of potential new antibiotics should be studied already during drug discovery and development. This study determined resistance properties of diazaborine compounds, which have been shown to have antibacterial activity especially against Gram-negative bacteria. The studied properties were mutant prevention concentration (MPC) and spontaneous mutation frequency. MPC measures the antimicrobial compound concentrations in which single-step resistant mutants arise. MPC values can be compared to minimum inhibitory concentration (MIC) values to determine range of mutant selection window (MSW) in which single-step resistant mutants are selectively amplified. Spontaneous mutation frequency is a feature for bacteria in presence of antimicrobials. Spontaneous mutation frequencies demonstrate the proportion of emerged resistant mutants from a bacterial population in each antimicrobial concentration. Four diazaborine compounds were studied with Escherichia coli ATCC25922 and the results were compared with ciprofloxacin. Ciprofloxacin had the lowest MPC at 16xMIC, diazaborine compounds 1, 2 and 3 at 32xMIC and diazaborine compound 4 at 64xMIC. Spontaneous mutation frequency of E. coli was on a normal level with each compound.

Liposomes are nano-sized vesicles in which the aqueous phase is surrounded by lipid-derived bilayer. They are excellent drug vehicles for example in ocular drug delivery because they can, among other things, increase the bioavailability and stability of the drug molecules and reduce their toxicity. Liposomes are known to be safe to use, because they degrade within a certain period of time and they are biocompatible with the cells and tissues of the body. Owing to its structure, the surface of liposomes can also be easily modified and functionalized. Light-activated ICG liposomes allow drug release in a controlled manner at a given time and specific site. Their function is based on a small molecule called indocyanine green (ICG) which, after being exposed to laser light, absorbs light energy and thereby locally elevates the temperature of the lipid bilayer. As a result, the drug inside is released into the surroundings. The blood circulation time of liposomes has often been prolonged by coating the liposomes with polyethylene glycol (PEG). Although PEG is generally regarded as a safe and biocompatible polymer, it has been found to increase immunological reactions and PEG-specific antibodies upon repeated dosing. Conversely, hyaluronic acid (HA), is an endogenous polysaccharide, which is present in abundance for instance in vitreous. Thus, it could serve as a stealth coating material which extends the otherwise short half-life of liposomes. One of the main objectives of this thesis was to find out whether HA could be used to coat liposomes instead of PEG. In order to prepare HA-coated liposomes, one of the lipid bilayer phospholipids, DSPE, had to be first conjugated with HA. For the conjugation, potential synthesis protocols were sought from the literature. Ultimately two different reductive amination-based protocols were tested. Consequently, the protocol in which the conjugation was achieved via the aldehyde group of HA, proved to be working. Thereafter, HA-coated liposomes were prepared by thin film hydration from the newly synthesised conjugate as well as DPPC, DSPC and 18:0 Lyso PC. Calcein was encapsulated in the liposomes. HA-covered liposomes were then compared with uncoated and PEGylated liposomes by examining their phase transition temperatures, ICG absorbances, sizes, polydispersities, and both light and heat-induced drug releases. The aforementioned tests were also conducted when the effects of the HA and ICG doubling were examined and the possibility to manufacture HA liposomes with small size was assessed. HA-liposomes showed similar results as PEG-coated liposomes. In addition, successful extrusion of HA-liposomes through a 30 nm membrane was also demonstrated in the results. Doubling of HA did not significantly affect the results. In contrast, increasing the molar amount of ICG by double caused spontaneous calcein leakage even before any heat or light exposure. Based on these findings, HA could work as a coating material instead of PEG, yet further studies are required for ensuring this conclusion. The other key objective was to evaluate the stability of four different formulations, named as AL, AL18, AL16 and AL14, in storage and biological conditions. Based on the differences in the formulation phospholipid composition, the assumption was that AL would be the most stable of the group and that the stability would decrease so that AL18 and AL16 would be the next most stable and eventually AL14 would be the least stable formulation. As in the previous study, the liposomes were prepared by thin film hydration with calcein being encapsulated inside the liposomes. In the storage stability test, liposomes were stored in HEPES buffer at either 4 °C or at room temperature for one month. In the test conducted in physiological conditions, the liposomes were added either to porcine vitreous or fetal bovine serum (FBS) and the samples were incubated at 37 ºC for five days. Regardless of the experiment, phase transition temperatures as well as light and heat-induced drug releases were initially measured. As the test progressed, calcein release, ICG absorbance, size, and polydispersity were measured at each time point. The initial measurements confirmed the hypothesis about the stability differences of tested formulations. In the storage stability test, all formulations, except AL14, appeared to be stable throughout the study and no apparent differences between the formulations or temperatures were observed. On the other hand, the stability of liposomes stored in biological matrices varied so that the liposomes were more stable in vitreous than in FBS and the stability decreased in both media as expected.

Nowadays, targetability studies usually require sample modifications and quite often, examination requires the use of directed light in harmful wavelengths. The surface plasmon resonance (SPR) technique does not need either of those actions. With SPR technology, the targetability of biomolecules can be studied in real-time and without any additional labels. The SPR response is received by measuring the change in surface plasmon resonance conditions due to refractive index changes caused by material interactions in the vicinity of a metal sensor surface. In the present study, the targetability of neonatal Fc receptor (FcRn) was studied by SPR. FcRn-mediated targetability studies were performed against protein A and human colorectal adenocarcinoma (Caco-2) immobilized on SPR sensors. The aim of the study was to confirm the FcRn targetability with bare Fc-fragment and Fc-fragment modified nanoparticles (NPs) designed for oral drug delivery. The NPs consisted of a core porous silicon (PSi) particle, entrapped into a lignin capsule, and finally functionalized with the FcRn-targeting ligand. Results confirmed the binding efficacy of bare Fc-fragment with protein A at pH 6.5, which was the critical pH value for preserving the lignin capsule around the PSi NPs. The cell-based SPR response was significantly higher for FcRn-targeted NPs when compared with non-functionalized NPs. According to these results, FcRn-mediated transcytosis emerges with great potential for oral drug delivery via Fc-functionalized NPs.

There are certain characteristics in children’s medication process, such as weight or body surface area-based drug dosing and off-label use of medications, that expose children to medication errors. Small children especially are prone to physical injuries resulting from medication errors. High-alert medications bear a heightened risk of causing significant, even life-threatening harm to a patient when used in error. The aim of this study was to promote children's medication safety by identifying medication errors and contributing factors to errors associated with the use of high-alert medications in pediatric medication process in a hospital environment. The data of this retrospective register study consisted of voluntary medication error reports (HaiPro) made in the pediatric and adolescent units at Helsinki university hospital (HUS). ISMP's (Institute for Safe Medication Practices) list of high-alert medications in acute care settings was used to limit the data. The data was analyzed by using both quantitative and qualitative methods. The aim of the quantitative analysis was to report the frequencies (n) and proportions (%) of high-alert medications and routes of administration and the aim of the qualitative analysis was to identify the types of medication errors and contributing factors in the data. ISMP’s high-alert medications accounted for approximately one-fifth (19.7%) of all medication error reports made in pediatric and adolescent units in 2018–2020. Twelve medications and intravenous route covered approximately 65.0% of all high-alert medications and routes of administration mentioned in the data. Medication errors were mostly identified in medication administration stage (43.3%) and administration errors were often preceded by prescribing errors. Dosing errors (20.5%) and documenting errors (16.8%) were the most common medication error types in the data. Errors associated with dosing and infusion rate were most often involved in severe medication errors. The most frequently identified contributing factors in the data were associated with the work situation and conditions, documenting and information transfer or medications. More detailed risk analysis considering high-alert medications and the intravenous medication process and targeting preventive barriers to identified risk areas are recommended in pediatric and adolescent units in the future. Barriers should be planned to cover the entire medication process. Among different types of medication errors, multiple dosing errors and errors during the programming of infusion rate require special attention in the future.

Chronic heart failure is a major worldwide health problem. It is a complex and severe syndrome caused by different kinds of cardiovascular diseases. Cardiac hypertrophy is frequently caused by hypertension and can lead to abnormality in heart contraction, activation of many neurohumoral mechanism and heart failure. The most important neurohormonal mechanisms of heart failure are activation of sympathetic nervous system and the renin-angiotensin-aldosterone system, insufficiently contracting left ventricle, cardiac remodeling and myocyte loss owing to apoptosis. Antihypertensive drug treatment is often used to prevent or decelerate progression of cardiac hypertrophy. Activation of the renin-angiotensin-aldosterone system plays a major role in heart failure. During the past decades angiotensin converting enzyme inhibitors (ACEIs) have been used as firstline treatment of heart failure. ACEI treatment has been shown to reduce mortality associated with chronic heart failure and improve prognosis of the disease. Angiotensin receptor blockers (ARBs) were expected to replace ACEIs in the treatment of heart failure but for the present they are only an alternative to ACEIs. Beta-blocking agents which reduce activation of sympathetic nervous system have established themself as the second most important treatment of heart failure. Diuretics are widely used as the treatment of heart failure but only aldosterone antagonists has been shown to improve prognosis of the disease. Also digoxin is still used in the treatment of chronic heart failure. In the future renin inhibitors, neutral endopeptidase inhibitors, vasopressin antagonists and molecules that affect inflammatory cytokines could potentially be capable of improving the prognosis of chronic heart failure patients. The major object in the present study was to investigate development of left ventricular hypertrophy induced by abdominal aorta banding in male Wistar rats and prevention of hypertrophy by calcium sensitizer levosimendan and angiotensin II receptor blocker valsartan. Also functionality of abdominal aorta banding as a rodent model of cardiac hypertrophy and heart failure was estimated. Abdominal aorta was constricted above the right renal arteries. That leads to pressure overload and increase in cardiac load. Heart response to pressure overload by hypertrophy in the form of wall thickening. 64 rats were assigned to different groups, each having eight rats. Three of the groups were treated with levosimendan with different daily doses (0,01 mg/kg; 0,10 mg/kg; 1,00 mg/kg) and three of the groups were treated with valsartan with different daily doses (0,10 mg/kg; 1,00 mg/kg; 10,00 mg/kg) via drinking water for eight weeks after the surgery. Sham-operated group underwent the same surgical procedures without constriction of the aorta. All the groups were compared to abdominal aorta banded group without any medical treatment. Cardiovascular parameters such as isovolumic relaxation time (IVRT), left ventricle end-systolic (ESD) and end-diastolic (EDD) dimensions, ejection fraction (EF), fractional shortening (FS), cardiac output (CO), stroke volume (SV), interventricular septum (IVS) and posterior wall (PW) thickness were measured eight weeks after the surgery by using cardiac ultrasound. In the present study levosimendan slightly improved systolic function of the heart. Improvement of the systolic function was seen in a tendency to improve ejection fraction and fractional shortening in abdominal aorta banded rats compared to abdominal aorta banded rats without medical treatment. Neither levosimendan nor valsartan affected diastolic function of heart. Diastolic function was measured by isovolumic relaxation time. Neither levosimendan nor valsartan had significant effect on development of cardiac hypertrophy. Cardiac hypertrophy was estimated by measuring heart weight-to-body weight ratio (HW/BW), left ventricular wall thicknesses and left ventricular internal dimensions in systole and diastole. The present study indicates that outflow constriction by aortic banding is clearly a model of cardiac hypertrophy but not of heart failure.

The impact of nanoparticulate drug carriers, especially polymeric micelles, is growing continuously. However, their drug delivery properties in vivo are difficult to predict. In this thesis, the approach of screening a combinatorial library of nano carriers for their drug delivery properties in a high throughput/high content (HT/HC) manner was tested. The library consisted of self-assembling polymeric micelles, using the amphiphilic polymer DSPE-PEG2000. The physicochemical characterization of micelles was focused on size, shape and stability, tested by various methodologies. The micelles were labeled with the fluorescence dye Alexa568 and the combinatorial character was based on labeling with two different Cell Penetrating Peptides (CPP), RGD and transactivator of transcription (TAT), in three molar ratios each. The cytotoxicity concentration ranges and micelle uptake were tested in the ARPE-19 cell line. Intracellular localization was observed by confocal fluorescence microscopy. Quantitative HCS imaging analysis was performed by image cytometry, whereas only the parameter 'micelles spots per cell' was analyzed exemplarily. The quantitative HCS results were not clear and pointed to insufficient optimization of experimental and analytical parameters. The results suggest that HCS could be a suitable method to analyze a nanoparticulate library for its drug delivery properties, requiring careful optimization of experimental parameters. However, the careful characterization of the micellar library is a critical factor in planning and understanding biological experiments.

The aim of this study was to synthesize antimicrobial and anti-biofilm agents using abietic (AA) and dehydroabietic acids (DHAA). Bacterial biofilms are formed when bacteria cells cluster together within a self-produced extracellular matrix. This lifestyle makes bacteria highly resilient to different environmental stresses and conventional antibiotics when compared to single-cell bacteria. Currently, there are no approved anti-biofilm agents as drugs and only a few number of compounds can selectively target biofilms and eradicate them at low concentrations. Potent drugs targeting them are needed. AA and DHAA are abietane-type diterpenoids found in the resin of conifer trees. Antibacterial effects of resin acids have been widely studied, specifically against methicillin-resistant Staphylococcus aureus strain (MRSA). Through the combination of DHAA with different amino acids, Manner et al. (2015) discovered a new class of hybrid compounds that target both planktonic and biofilm bacteria in Staphylococcus aureus. The study group also discovered two of the most potent abietane-type anti-biofilm agents reported so far in literature. This thesis followed the work of the research group by designing and synthesizing additional AA and DHAA derivatives to target bacterial biofilms. Rings A, B and C of the diterpenoid core were modified and 24 derivatives were successfully synthesized. Amino acids were attached to the compounds either before or after ring modification. Standard structural elucidation techniques were used to confirm the structure of the synthesized compounds.

Antimicrobial resistance (AMR) is a growing global health concern, and the development of new antibacterial agents is crucial to addressing this issue. Commercial antibiotics are not as effective as they used to be to combat infections. Previous studies have demonstrated the promising antimicrobial activity of etrasimod and one of its derivatives, compound 24f, against Gram-positive species. Therefore, as part of this study, we modified the carboxylic acid functional group to produce new derivatives. We synthesized derivatives of etrasimod and 24f, in order to generate a variety of compounds for evaluation of their antimicrobial effectiveness. Furthermore, the study evaluates the compounds' in vitro antibacterial activity and in vivo efficacy using Caenorhabditis elegans worms as an infection model. C. elegans is a widely used model organism in biological research, and it is particularly useful for studying host-pathogen interactions and drug efficacy. In addition, the cytotoxicity on mammalian cells (HeLa) was determined. Compound 18 showed the lowest cytotoxicity level (CC50 = 75.71±14.4 µM) of tested compounds. The antibacterial activity of new etrasimod derivatives was tested against Gram-positive (Staphylococcus aureus, including methicillin-resistant strains (MRSA)) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). The tested compounds showed activity against Gram-positive bacteria but not against any of the Gram-negative strains. Compounds 9 and 18 showed to be the most active compounds, having a minimum inhibitory concentration of 5–6 and 8–10 μM, respectively. Moreover, both compounds showed promising activity in vivo, being able to significantly reduce the bacterial load in infected worms and improve their survival rates in survival experiments. The study provides insights into the development and assessment of potential antibacterial agents, addressing the contemporary challenge of AMR. The study's findings suggest that compounds 9 and 18 could be potential candidates for further development as novel antimicrobial agents.

The human tissue kallikreins (KLKs) form a family of 15 closely related serine proteases (KLK1-15). KLK3 is better known as prostate specific antigen (PSA) and it is highly prostate-specific. Kallikreins are attracting increased attention due to their role as biomarkers for screening, diagnosis, and monitoring of various cancers. Although PSA is a very useful marker for prostate cancer in the blood, the expression level of PSA is higher in normal prostatic epithelium than in tumour tissue, and it is further reduced in poorly differentiated tumours. It has been postulated that PSA activators (stimulating compounds) could be beneficial for patients with prostate cancer. The development of peptides as clinically useful drugs is greatly limited by their poor in vivo stability and low bioavailability. As the problems in using peptides as drugs are mainly arising from the peptide backbone, the focus for synthesizing peptide mimicking compounds is on the peptide backbone and how to replace it. The aim of this work was to replace the central disulfide bridge in the most potent PSA activating peptide C-4 by a hydrocarbon linker that had been previously synthesized in the research group. Two strategies for synthesis of the pseudopeptides were applied: a) synthesis of all peptide bonds on solid support by tailoring the reactions for this particular peptide, and b) synthesis of a monocyclic pseudopeptide in solution that could be incorporated directly into the standard protocol of solid phase peptide synthesis. The first strategy (a) proved to be tedious and would have required a lot of optimization to be successful. The cleavage conditions of the orthogonal protecting groups were not directly compatible with synthesis on solid support. The second strategy (b) also proved to be tedious, epimerization at the histidine residue was very prone in the solution phase even with standard peptide coupling reagents. However, the possibility to monitor all steps of the synthesis and to purify intermediate products made this synthetic route more attractive for this type of pseudopetide. The work in this master thesis resulted in a useful strategy to synthesise the desired pseudopeptides.

Parkinson’s disease (PD) is a prevalent neurodegenerative disease characterized by movement disorders, such as bradykinesia, akinesia, and tremor. The degeneration of dopaminergic neurons in the central nervous system (CNS) is the most central aspect of the pathophysiology of PD-related movement disorders. The treatment of PD motor symptoms is based on increasing the diminished dopaminergic signalling in the CNS. This can be achieved by using medications such as dopamine agonists and monoamine oxidase B inhibitors. Levodopa, which acts as a precursor of dopamine in the body, is currently considered the most effective treatment for PD motor symptoms. Unlike dopamine, levodopa can cross the blood-brain barrier. Thus, levodopa must reach the CNS before being metabolized into dopamine to achieve the desired therapeutic effect. Dopa decarboxylase (DDC) inhibitors and catechol-O-methyltransferase inhibitors have been co-administered alongside levodopa to reduce its peripheral metabolism. However, when administered orally, levodopa is also metabolized in the gut by tyrosine decarboxylase, an enzyme produced by gut bacteria. Inhibi tion of bacterial tyrosine decarboxylase (TyrDC) could increase the effectiveness of levodopa treatment and reduce the needed levodopa dosage. The aim of this study was to synthesize and assess the biological activity of novel analogues of previously identified hit compounds which are dual inhibitors of TyrDC and DDC. Our goal was also to gain a deeper understanding of the structure-activity relationships of these compounds. Some of the compounds synthesized in this study were able to inhibit both TyrDC and DDC. Unfortunately, they were also either toxic towar ds human cells, and/or lacked efficacy in a bacterial cell-based assay used to determine the inhibition of levodopa metabolism. However, the data generated in this study can be utilized to design and synthesize new analogs to discover more efficacious and safer TyrDC and DDC dual inhibitors.