Farmasian tiedekunta

 

Recent Submissions

  • Pessi, Jenni (Helsingin yliopisto, 2017)
    This thesis consists of two parts, particle formation and analysis. In the first part, particle formation in microfluidic devices and in devices employing supercritical fluids is investigated, and in the second part, essential issues in analytical methods for determining drug release and solid-state properties are addressed. Microfluidic technology was employed to produce microcapsules for protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water double emulsion droplets with ultrathin shells. All the particles were found to be intact and with a particle size of 23 - 47 µm. The encapsulation efficiency of bovine serum albumin in the microcapsules was 84%. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins. A new, robust, stable, and reproducible method based on expansion of supercritical solutions using carbon dioxide as a solvent was developed to produce nanoparticles. The method, Controlled Expansion of Supercritical Solution (CESS), uses controlled mass transfer, flow, pressure reduction, and particle collection in dry ice. CESS offers control over the crystallization process as the pressure in the system is reduced according to a specific profile. Controlled pressure reduction keeps the particle growth and production process stable. With CESS, we produced piroxicam nanoparticles, 60 mg/h, featuring narrow size distribution (176 ± 53 nm). The Lyophilic Matrix (LM) method was developed for investigating dissolution rates of nanoparticles, powders, and particulate systems. The LM method is based on its ability to discriminate between non-dissolved particles and the dissolved species. In the LM method, the test substance is embedded in a thin lyophilic core-shell matrix. This permits rapid contact with the dissolution medium while inhibiting dispersion of non-dissolved particles without presenting a substantial diffusion barrier. By minimizing method-induced effects on the dissolution profile of nanopowders, the LM method overcomes shortcomings associated with current dissolution tests. Time-gated Raman spectroscopy was applied for solid-state analysis of fluorescent powder mixtures. A setup with a 128 × (2) × 4 CMOS SPAD detector was used for the quantitative analysis of solid-state forms of piroxicam. Time-gating provides an instrumental method for rejecting the fluorescence signal. This study demonstrated that traditional PLS analysis of time-gated Raman spectra resulted in mean RMSE of 4.1%. The time-gated Raman spectroscopy method shows potential for relatively routine quantitative solid-state analysis of photoluminescent pharmaceuticals.
  • Ruokolainen, Miina (Helsingin yliopisto, 2017)
    Redox reactions play an important role in human physiology and pathophysiology. For example, oxidative stress and free radical-mediated oxidation of proteins and lipids are implicated in several diseases such as Alzheimer’s and Parkinson’s disease. Oxidation reactions belong also to the most important phase I metabolism pathways of drugs, which can give rise to pharmacologically active or toxic metabolites. The established methods for in vitro drug metabolism studies, e.g. methods using hepatocytes, human liver microsomes (HLMs), and recombinant enzymes, are relatively time-consuming and expensive. Thus, the potential of several nonenzymatic oxidation methods, such as those based on metalloporphyrins, electrochemistry (EC), and Fenton reaction, have been explored for metabolism studies. However, new methods need to be developed to enable rapid production of drug metabolite standards and since none of the above nonenzymatic methods allow comprehensive prediction of phase I drug metabolism. The titanium dioxide (TiO2) photocatalysis method was developed and applied to evaluate the effect of phosphorylation of tyrosine on the oxidation of (phospho)peptides with the same sequence but different phosphorylation states. The results obtained using ultra-high-performance liquid chromatography – mass spectrometry (UHPLC-MS) show that nonphosphorylated tyrosine was the amino acid most susceptible to hydroxyl radical-initiated oxidation, but oxidation of tyrosine was in most cases inhibited by its phosphorylation. The feasibility of TiO2 photocatalysis for imitation of in vitro phase I HLM metabolism of small drug molecules was studied using UHPLC-MS and compared with the electrochemically assisted Fenton reaction (EC-Fenton) and EC. TiO2 photocatalysis, EC-Fenton, and EC imitated 44%, 31%, and 11%, respectively, of the in vitro phase I HLM metabolites of four model compounds. As TiO2 photocatalysis proved most feasible for the imitation of in vitro phase I HLM metabolism, its feasibility for imitation of in vitro phase I HLM metabolism of five anabolic steroids was also examined. TiO2 photocatalysis was able to imitate over half of the hydroxylation and dehydrogenation metabolites, but its imitation of the metabolites resulting from combinations of these reactions was considerably poorer. To enable even more rapid experiments to study biologically relevant oxidation reactions, TiO2-photocatalysis was simply integrated with desorption electrospray ionization (DESI)-MS by using the same TiO2-coated glass wafer for photocatalytic reactions and DESI-MS analysis. This new method enabled high-throughput investigation of photocatalytic oxidation reactions, as demonstrated using 12 model compounds, and imitation of several drug metabolism reactions of three model compounds studied in more detail. In conclusion, TiO2 photocatalysis proved a feasible method for oxidation of compounds with different polarities. TiO2 photocatalysis cannot predict drug metabolism comprehensively, but offers a potential method for rapid, simple, and inexpensive study of oxidation reactions of biomolecules and imitation of several drug metabolism reactions. Preparative scale synthesis of oxidation products by TiO2 photocatalysis is likely an alternative application of the method, but this remains to be demonstrated.
  • Penttinen, Anna-Maija (Helsingin yliopisto, 2017)
    Parkinson’s disease (PD) is a neurodegenerative disease characterized by intracellular proteinaceous inclusions called Lewy bodies and progressive loss of dopaminergic neurons in the substantia nigra (SN). The first symptoms of PD are non-motor, such as hyposmia and gastrointestinal disturbances, followed by motor symptoms, such as tremor and rigidity. Currently available therapies, medication, surgical procedures and supportive therapies, are symptomatic and do not affect the underlying cause of the disease — the neuronal degeneration. Thus, a new therapy which would restore the dopaminergic phenotype of dying neurons and thus slow down or even halt the progress of the disease is needed. Neurotrophic factors are secretory proteins regulating survival and functioning of the neurons as well as the formation of new neuronal contacts. Neurotrophic factors have shown great potential in animal models of PD, but in clinical trials, the results have been contradictory. One possible explanation for this is poor diffusion and bioavailability of the therapeutic proteins in the target tissue. The aim of this study was to explore the neuroprotective effects of the isoforms of two of the most potent dopamine neurotrophic factors, GDNF (glial cell line-derived neurotrophic factor) and its homolog neurturin (NRTN) in an experimental model of PD, and to characterize a new stable low-dose 6-hydroxydopamine (6-OHDA) rat PD model. In the PD model the degeneration of the nigrostriatal pathway was induced by administrating toxic dopamine analog 6-OHDA into the striatum, where the nerve terminals of the dopaminergic neurons are located. We compared several different administration paradigms to find the optimal parameters to induce a stable lesion model with high success rate. The cell loss induced with low doses (6-9 µg) of 6-OHDA was at similar level as the cell loss induced with higher (20 µg) doses of 6-OHDA. The advantage of using low 6-OHDA doses is the avoidance of non-specific damage, which occurs with higher 6-OHDA doses. Moreover, the low-dose induced lesions have high success rate, reducing the number of animals needed in the experiments and increasing the reliability of the obtained results. The spreading of NRTN in the brain tissue was improved by modifying the extracellular matrix binding sequence of the protein. New NRTN variants were biologically active and were able to initiate signaling via tyrosine kinase Ret (rearranged during transfection). In the neuroprotection assay in rat 6-OHDA model of PD NRTN variant N4 protected the dopaminergic neurons in the SN and fibers in the striatum as well as improved the motor behavior of the animals. In neurorestoration assay, N4 showed a trend in improving the behavioral deficits of the animals. GDNF, on the other hand, was administered to the brain with viral vectors, enabling long-term protein expression in the target tissue. GDNF has been widely studied, but the research has focused on the full-length constitutively secreted α-isoform, whereas the biology of the shorter and activity-dependently secreted β-GDNF has not been studied in vivo before. In the non-lesioned striatum, both isoforms increased striatal dopamine transporter-immunoreactivity. Both isoforms also protected the dopaminergic neurons in SN from 6-OHDA-induced degeneration. The results show that these new and less studied neurotrophic factor isoforms are able to slow down the degeneration of the midbrain dopaminergic neurons. In other words, both NRTN variant N4 and β-GDNF are potential disease-modifying factors for PD.
  • Suvikas-Peltonen, Eeva (Helsingin yliopisto, 2017)
    In hospital care, many medicines require aseptic compounding or preparation before they are administered to patients. The parenteral administration of microbiologically contaminated doses can result in bacteriaemia, other morbidity and even death. The aim os this study was to review the literature for incorrect practices in aseptic drug preparation and administration and recommendations for safer practices in hospitals. The aim was also to develop and content validate an assessment tool for medicine compounding on hospital wards. And at the final stage of this study, the aim was to audit compounding of intravenous medicines on hospital wards by using the developed assessment tool and take microbiological tests for identifying issues posing patient safety risks. A systematic literature search was conducted in PubMed. The first draft of the audittool was based on ISMP “Guidelines for safe preparation of sterile compounds” and a systematic literature search. The tool was then validated by using a two-rounded Delphi-method. A hospital pharmacist conducted external audit based on observation and interviews by using this validated 65-item assessment tool on 20 wards of a secondary care hospital in Finland. In addition microbiological test were collected. Associations between microbial sample results and audit tool results were discussed. In the review, the most reported incorrect practices were multiple use of vials and syringes and lack of overall disinfection during the aseptic preparation and administration. Recommendations to avoid contamination were classified into categories: equipment and medicines; disinfection; working envi-ronment; storing; catheter care; and quality of prepared medicines. The final audittool comprises of 64 items under the following topics: General principles of good compounding practices, Recording and con-firming medicine orders on the wards, Storage of medicines on the wards, Aseptic compounding of intravenous medicines and Quality assurance. The best practices related to logistic practices and quality assurance. Most of the obviated practices related to aseptic practices. All media fill tests were clean but in some of glove samples and settle plate samples were microbial growth. More contamination was found in wards where environmental conditions were inadequate or the use of gloves was incorrect.
  • Regina, Sirpa (Helsingin yliopisto, 2017)
    The objective of this doctoral dissertation is to present views, experiences and practices mainly related to the working-age insomniac, insomnia, and the treatment of it, as expressed by pharmacists working in Finnish community pharmacies. The specific aim is to investigate whether pharmacists see the pharmacy as having an important role in the initial stages of treating those who experience insomnia and are seeking to cure it. With regard to the self-treatment of insomnia, the study examines the kind of non-pharmacological advice offered by pharmacists when recommending self-care products and whether or not any underlying causes are being discussed with the customer. A further aim is to investigate what kind of pharmaceutical advice pharmacists provide on hypnotics and the kind of practices they employ in dispensing medicine to individual customers. Pharmacists’ views on working-age people who use hypnotics are also explored. The study was conducted as a qualitative cross-sectional study. The participants were pharmacists working at community pharmacies in mainland Finland (n = 188–277). The data was collected from five online surveys carried out in the spring of 2013. The participants were contacted using the mailing lists of the Finnish Pharmacists’ Association’s member organisations. The surveys could be accessed via a link sent by e-mail. The key questions were open-ended so as to obtain in-depth information on the pharmacists’ views, experiences and practices related to the research topic. The data was analysed mainly by using thematic analysis and both deductive and inductive content analysis. SPSS statistical software was also utilised in the analysis process. According to the study, pharmacists did not consider the pharmacy to be an obvious first choice for providing help to those with insomnia. Determining the cause of insomnia was not considered by all pharmacists to be part of their remit, nor did they necessarily even think it was their job to find out why the customer used hypnotics. Pharmacists did not always automatically provide non-pharmacological advice on insomnia in connection with self-care products, and the treatment instructions they provided differed in both quantity and quality. Even for customers buying hypnotics for the first time, the advice related to the possible side effect, addiction, varied greatly from one pharmacist to the another. Customers whose use of hypnotics exceeded the recommended dose also received very different levels of support when collecting their next batch of hypnotics, since the pharmacists had different ways of dealing with this kind of situation. Pharmacists criticised medical doctors for their treatment practices regarding insomnia. The issue generating the most criticism was deviation from the clinical guidelines for treating insomnia. According to this doctoral dissertation, those with insomnia are far from receiving equal treatment when seeking help at a pharmacy. The quality of service received by customers with sleep problems depends on the pharmacist serving them, on the pharmacist’s competence, and on their motivation to collaborate with the customer. On the other hand, the pharmacists’ positive attitude towards non-pharmacological self-care for insomnia creates opportunities for expanding the range of services provided by pharmacies. More and more frequently, the care pathway for those experiencing insomnia could thus start at the pharmacy. This does, however, require that pharmacists receive further training. Some pharmacists also need to obtain a basic knowledge of insomnia treatment as well as information on doctors’ treatment practices for insomnia.
  • Sjöstedt, Noora (Helsingin yliopisto, 2017)
    Transport proteins embedded in the cell membranes of many organs can affect the absorption, distribution and elimination of numerous drugs. This can lead to the enhanced or restricted uptake or distribution of the drugs, nonlinear pharmacokinetics, transporter-mediated drug-drug interactions (DDIs) and inter-individual variability. Transporters may therefore alter the safety and efficacy of drugs, thus it is important to study drug-transporter interactions in drug development. The breast cancer resistance protein (BCRP, ABCG2) is one of the transporters involved in drug disposition. It belongs to the ATP-binding cassette (ABC) transporter family and uses ATP to expel drugs and other substrates out from cells. BCRP was initially found to cause drug resistance in cancer cell lines, but it is also expressed in healthy tissues such as the intestine, liver and blood-brain barrier, where it is one of the transporters limiting the uptake of many structurally diverse compounds. Despite interest in BCRP and other ABC transporters, it remains poorly understood how they recognize their substrates and which chemical structures are liable to interaction. In this thesis, a vesicle-based in vitro method was used to study the ligand preferences of BCRP. The results were compared to those obtained for the multidrug resistance associated protein 2 (MRP2, ABCC2), which is also implicated in drug transport. The results show that a range of natural compounds and their derivatives are able to inhibit BCRP transport and among these, flavonoids were identified as the most important group for inhibition. Conversely, MRP2 transport was affected by only few of the tested compounds. However, a more similar pattern of inhibition was seen for the two transporters when selected food additives were studied, where several food colourants were identified as inhibitors. In addition, the effect of one assay component (bovine serum albumin, BSA) on the in vitro transport kinetics of BCRP and MRP2 was evaluated. The inclusion of BSA in the vesicle assay lead to moderate changes (up to 2-fold) in transport activity, but the effects on in vitro − in vivo extrapolation are expected to be minor, at least based on the tested compounds. Finally, the vesicle assay was used to study the functionality of selected BCRP variants with polymorphisms in the transmembrane helices and they were found to have significantly impaired transport activity and expression compared to wild type BCRP. In summary, the vesicle-based transport assay was successfully applied to identify and evaluate the effects that BCRP interactions may have on the pharmacokinetics of BCRP substrates.
  • Talka, Reeta (Helsingin yliopisto, 2017)
    Tobacco use is the leading cause of preventable death worldwide. Nicotine is the primary addictive component of tobacco, and repeated nicotine exposure often leads to dependence in humans. Nicotine is one of the most commonly co-used substances among polysubstance abuse patients and combined use of nicotine and other drugs of abuse, such as opioids, increases the use of one or both substances. The health consequences associated with polysubstance abuse exceed those of either drug alone. The current pharmacotherapeutic options are ineffective among opioid-substituted patients and the levels of successful smoking cessation are low. At the cellular level, nicotine and opioids have their own molecular mechanisms of action, yet both drugs increase the activity of the reward pathway by increasing dopamine transmission. The purpose of these studies was to investigate the possible effects of different opioid ligands on human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in cell cultures. Our results showed that morphine has a partial agonist effect at α4β2 nAChRs, a very weak antagonist effect at α3* nAChRs and a positive synergistic effect with nicotine on α7 nAChR function. We found that methadone acts as a non-competitive antagonist (NCA) at α4β2 and α3* nAChRs. We also confirmed that methadone is a human α7 nAChR agonist. In the prolonged studies with methadone and morphine, we found that human α3*, α4β2 and α7 nAChRs are differentially regulated by prolonged exposure to methadone and morphine. Buprenorphine was shown to be a weak antagonist at α4β2, α3*, and α7 nAChRs, and codeine had a positive modulatory effect on α4β2 nAChRs and a weak NCA effect on α3* nAChRs. Oxycodone seemed to have a mixed competitive/non-competitive effect on α4β2 nAChRs and a weak NCA effect on α3* nAChRs. Tramadol was shown to be a NCA of α3* nAChRs and a weak NCA of α4β2 nAChRs. Naloxone and naltrexone were mixed competitive/non-competitive antagonists of α4β2 nAChRs, weak NCAs of α3* nAChRs and weak antagonists of α7 nAChRs. Taken together, these studies showed that many opioid ligands have effects on nAChRs that are independent of their agonist or antagonist properties at opioid receptors. These findings suggest that some effects of the nicotine opioid interaction seen in humans can be partially mediated through the receptor-level interplay of these substances. These results, together with earlier findings, highlight the complexity of different nAChRs and the multiplicity of responses to opioid ligands.
  • Helfenstein, Andreas (Helsingin yliopisto, 2017)
    Responding to the aggravating shortage of potent antibiotics, pharmaceutical research is constantly looking for new ideas to combat and contain perilous micro-organisms. As a result of persistent selective pressure, evolution has bestowed a powerful arsenal of antimicrobial survival strategies upon plants, bacteria and fungi. Screening of natural product libraries is an obvious and promising first step in antimicrobial research. We followed that approach by investigating the antimicrobial bioactivity of a set of abietane derivatives. To accommodate for the data that resulted from that screening, we then developed a tool to automate and integrate the data analysis and presentation into the screening workflow. In an attempt to further streamline the process, we evaluated identification techniques for micro-organisms growing in co-cultures based on machine learning. Including the right material in the screening is a key element for a successful campaign. In our last study, we analyzed user-generated online sources to search for potential leads for new anti-infectious drugs. The screening resulted in a potential new compound active against Staphylococcus aureus with moderate cytotoxicity. The abietane derivative showed a minimal inhibitory concentration of 60 µg/ml against S. aureus and 8 µg/ml against multi-resistant S. aureus. Machine learning is used more and more in natural product research, and our study suggested that random forest classifiers and support vector machines are efficient tools to identify micro-organisms from polymicrobial cultures. The proposed models had prediction rates > 90 % in the determination of growth for microbes in polymicrobial cultures. The Internet is an immense and ever-growing resource of information, and we sketched a possible way for how this data can complement the search of antimicrobial plant preparations. In three examples, we showed correlations between mentions of plants and medical terms. Centered on antimicrobial screening, this thesis studies different strategies to enhance the screening process through the help of bioinformatics. We developed, tested and used different tools and methods to support and facilitate library design, detection techniques, and data management. These are only small pieces in the search of new antimicrobial compounds, but they show that there are still many possible approaches to follow.
  • Holmström, Anna-Riia (Helsingin yliopisto, 2017)
    Medication errors are one of the most common incidents leading to adverse events in healthcare worldwide. Tackling these major problems requires the implementation of a systems approach to healthcare, stating that risks should be managed proactively by improving the healthcare system. One of the recommended key strategies for learning from medication errors and risk prone processes is the establishment of local and national medication error reporting (MER) systems in healthcare. This study explored national and local MER systems in different countries and what makes them work in learning from medication errors. The study also explored how continuing education in medication safety could be organised for practicing healthcare professionals. The study applied both qualitative and quantitative research methods and utilized various data sources. The study was based on the theory of Human Error and the systems approach to risk management. The study comprised of three phases. Phase I explored the existing MER systems in different countries and their development and implementation. 16 medication safety experts from different countries responded to an online-survey. A national or local MER system existed in 11 of the countries. Blaming for errors, and a lack of time, training and coordination of reporting continue to be the major barriers to reporting. Learning from errors and a non-punitive approach are essential features of a MER system. There is also a need for promoting international networking of medication safety experts and bodies for sharing information and learning from others. Several factors associated with the successful development and implementation of MER systems were also identified. Phase II assessed the inter-rater reliability of medication error classifications in a voluntary Reporting System for Safety Incidents in Health Care Organizations (HaiPro) widely used in Finland. Also medication errors (n=32 592) reported in 2007-2009 and their contributing factors were explored. The inter-rater reliability was found acceptable (κ ≥0.41) in 11 out of 42 (26%) variables (e.g., near miss or actual error) describing the reported medication errors. Thus, the medication errors reaching the acceptable level of inter-rater reliability could be pooled from different healthcare units for the exploration of medication errors at the level of all reporting organisations. The most frequently reported medication errors were: dispensing errors (33%, n=10 906); administration errors (24%, n=7 972); and documentation errors (17%, n=5 641). The most commonly reported contributing factor was deficiencies in communication and course of information related to patients medications. In Phase III educational approaches were developed for introducing medication safety for healthcare professionals as a three-day interdisciplinary course. International higher education experts in pharmacy (n=19) brainstormed four syllabi with teaching and assessment methods. Following this, a combined syllabus was developed. All four syllabi were based on constructive, problem-based learning methods and focused on understanding a systems approach in managing medication safety. Learning linked to learners practice through assignments at the workplace appeared to be the key. The study suggests that MER systems need to be improved in many countries together with their operational environments. Moreover, the operational environments of MER systems must support the functionality of these systems. The key factor for successful MER systems and learning from medication errors is having a systems approach as a theoretical context in all reporting and learning processes throughout the operational environments of MER systems. The current work also suggests that constructive problem-based learning linked to learners practice through assignments is the key when developing a course for continuing education in medication safety for healthcare professionals.
  • Hautala, Jaana (Helsingin yliopisto, 2017)
    Product acceptability and administration of drugs on a free choice basis are key factors determining the success of peroral veterinary medication, and are particularly important when treating chronic diseases of companion animals. Veterinary medicines found unpalatable and repulsive in odour, taste or form will result in refused voluntary intake. Particularly with cats, the issues related to product unacceptability are difficult to overcome, and specific tailor-made peroral dosage forms for cats are currently lacking. Both animal treatment compliance and owner treatment bonding are essential for successful veterinary drug therapy. Product dosing should be simple and easy, and it should be performed without any complications. The present study was undertaken to investigate feline peroral drug therapy and to develop novel feline-specific minitablets with increased palatability supporting the safe, simple, flexible and convenient drug treatment of cats. The acceptability of minitablets was evaluated in a new feline behavioural test setting with domestic pet cats. The minitablets, developed with a focus on target species characteristics, were found to be more acceptable than non-favoured food. However, improvements in minitablet odour and/or taste were required. Feline-specific flavours of non-natural origin together with a model substance having a bitter taste were investigated in the minitablet formulations. For a pharmaceutical industry point of view, synthetic flavours are considered more suitable over natural substances. In the present study, amino acids such as L-leucine and L-methionine, and thiamine hydrochloride, were considered as suitable candidates for feline minitablet formulations. New flavoured polymer coating formulations for feline medication purposes on minitablets were developed. Feline-specific synthetic flavours and their mixtures were incorporated in the aqueous film coatings of the polymethacrylate copolymer of Eudragit® E. The film coatings containing meat flavours of 2-acetylpyridine and 2-acetylthiazole in small concentrations were found the most applicable for minitablet coating and taste-masking purposes. Atomic layer deposition (ALD) was investigated as a novel ultrathin-coating method for pharmaceutical minitablets and for taste masking applications. The ALD thin coating, however, did not provide effective taste masking (with the coating levels studied) for the bitter tasting minitablets composed of heterogeneous excipients. In conclusion, the present results support the more cost-effective product development of palatable feline peroral medication. It is evident that pet and owner compliance as well as drug treatment efficacy and safety can be increased with the feline-specific products introduced here. The present results are also likely to be applicable for other veterinary target species, such as pet dogs. However, minitablets as dosage forms would additionally be suitable for humans.
  • Lázaro Ibáñez, Elisa (2017)
    Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies are a heterogeneous population of membrane particles released by cells to the extracellular space and into biofluids during normal physiological and pathological processes. EVs have been recognized as powerful vehicles for intercellular communication due to their capacity to transfer lipids, proteins, and nucleic acids, thereby influencing the properties and functions of recipient cells. Cells generate EVs with a unique composition based on their characteristics, which has a special relevance in the study of diseases such as cancer. Since specific molecular signatures can be passed on to tumor EVs, they are prime candidates for implementation as cancer biomarkers and in the delivery of therapeutics. Thus, exhaustive research is currently targeted towards elucidating the role of EVs in cell-to-cell communication and their therapeutic and diagnostic use. This thesis aims at broadening our understanding of the applicability and functional relevance of the use of EVs as prostate cancer biomarkers and therapeutic delivery vehicles. First, the practical use of EVs as a source of nucleic acid biomarkers in prostate cancer was assessed by exploring the DNA and RNA content of vesicles. Genomic DNA analysis of apoptotic bodies, microvesicles, and exosomes were performed to detect mutations within the EV cargo. The results were validated in plasma EVs of prostate cancer patients, from which the presence of prostate cancer-relevant genes was identified. Next, the prostate cancer-specific messenger RNA signatures of microvesicles and exosomes were analyzed. Unique nucleic acid signatures distinctive for the cell origin were found in the form of differential levels of mRNA transcripts from EV subpopulations. Overall, the nucleic acid content of EVs provided a new source of diagnostic information that could contribute to early prediction and diagnosis of prostate cancer, especially if combined. The role of EV-mediated intercellular communication was shown by comparing the uptake efficiencies and functional effects of EVs from prostate cancer cells of different metastatic status with non-cancer EVs. Additionally, the ability of EVs to carry and deliver a chemotherapeutic drug, together with their cytotoxic effects on prostate cancer cells were also analyzed. While EV uptake, in general, was an active and continuous process, the internalization rate and the subsequent functional effects of EVs on recipient cells differed based on the vesicle origin. EVs derived from cells of a metastatic source were more efficiently internalized than primary prostate cancer or benign prostate epithelial cell-derived EVs. Similarly, those EVs also induced a more proliferative and migratory phenotype in the recipient cells. Applying prostate cancer EVs in the in vitro delivery of paclitaxel to prostate cancer cells, resulted in an enhanced cytotoxic effect of paclitaxel mediated by EV delivery compared to the free drug. In summary, the results presented in this thesis support the concept that EVs can be utilized in both biomarker discovery and drug delivery fields as multifunctional tools for diagnosis and treatment of diseases such as prostate cancer. The studies presented here will also contribute to set the bases for further functional analysis of the roles of EVs in cell-to-cell communication. This new era of research could lead to faster, non-invasive, and more individualized diagnosis and improved treatments tailored to the specific needs of the patients.
  • Subramanian, Vigneshwari (Helsingin yliopisto, 2016)
    Designing drugs that are selective is crucial in pharmaceutical research to avoid unwanted side effects. To decipher selectivity of drug targets, computational approaches that utilize the sequence and structural information of the protein binding pockets are frequently exploited. In addition to methods that rely only on protein information, quantitative approaches such as proteochemometrics (PCM) use the combination of protein and ligand descriptions to derive quantitative relationships with binding affinity. PCM aims to explain cross-interactions between the different proteins and ligands, hence facilitating our understanding of selectivity. The main goal of this dissertation is to develop and apply field-based PCM to improve the understanding of relevant molecular interactions through visual illustrations. Field-based description that depends on the 3D structural information of proteins enhances visual interpretability of PCM models relative to the frequently used sequence-based descriptors for proteins. In these field-based PCM studies, knowledge-based fields that explain polarity and lipophilicity of the binding pockets and WaterMap-derived fields that elucidate the positions and energetics of water molecules are used together with the various 2D / 3D ligand descriptors to investigate the selectivity profiles of kinases and serine proteases. Field-based PCM is first applied to protein kinases, for which designing selective inhibitors has always been a challenge, owing to their highly similar ATP binding pockets. Our studies show that the method could be successfully applied to pinpoint the regions influencing the binding affinity and selectivity of kinases. As an extension of the initial studies conducted on a set of 50 kinases and 80 inhibitors, field-based PCM was used to build classification models on a large dataset (95 kinases and 1572 inhibitors) to distinguish active from inactive ligands. The prediction of the bioactivities of external test set compounds or kinases with accuracies over 80% (Matthews correlation coefficient, MCC: ~0.50) and area under the ROC curve (AUC) above 0.8 together with the visual inspection of the regions promoting activity demonstrates the ability of field-based PCM to generate both predictive and visually interpretable models. Further, the application of this method to serine proteases provides an overview of the sub-pocket specificities, which is crucial for inhibitor design. Additionally, alignment-independent Zernike descriptors derived from fields were used in PCM models to study the influence of protein superimpositions on field comparisons and subsequent PCM modelling.
  • Rojalin, Tatu (Helsingin yliopisto, 2016)
    Biophotonics is an emerging area of scientific research that uses light of photons to probe biological specimens, such as tissues, cells and molecules. The field of biophotonics is broad and considerably multidisciplinary. Therefore the prerequisite for understanding biophotonics is the capability to integrate the fundamental knowledge of the physics of light with perspectives of engineering of devices and instruments used to generate, modify, and manipulate light. Also, the fundamentals of biology and medicine are essential particularly comprehension of the biochemical and cellular phenomena that occur in living systems, and how such phenomena can be scaled up to concern the physiology of organisms, for example humans. Biological pathways and processes differ in the healthy and diseased state, and that is why it is essential to develop understanding of pathophysiology and various states of disease such as cancer, neurodegenerative disease or infectious states. Consequently, solid insights into the functions of medical treatments, including biopharmaceutics, are needed. Raman and surface plasmon resonance (SPR) are both light-based technologies enabling label-free measurements with high sensitivity. The primary aim of this Thesis was to tackle the emerging hardships encountered in the fundamental biopharmaceutical research, clinical settings, or in the pharmaceutical industry by introducing applications and data analysis methods based on the cutting edge Raman and SPR technologies. These techniques represent the biophotonic cornerstones as tools for biopharmaceutical applications throughout this Thesis. The scope of this Thesis was essentially broad. First, small drug molecules were investigated with state-of-the-art time-gated Raman technology, showing that the interfering photoluminescent backgrounds can be effectively suppressed thus improving the acquired Raman data significantly. Additionally, EVs were studied with laser tweezers Raman spectroscopy (LTRS) as larger scale analytes and representatives of a highly interesting topic in the current nanomedical field. When Raman data from several different types of single EVs was examined using sophisticated data analysis, distinct subpopulations were observed, and the differences could be related to the biochemical compositions of the vesicle membranes. For the first time, the study showed the importance of measuring single EVs instead of a pool of vesicles. Multi-parametric SPR (MP-SPR) technology was harnessed to develop applications and data analysis methods for small and larger scale analytes. Hence, a new small drug molecule, spin-labeled fluorene (SLF), was investigated in the context of Alzheimer s disease (AD) particularly its potential to interfere with the detrimental amyloid peptide aggregation processes. The developed bio-functional in vitro platform in combination with rigorous data analysis and computational simulations demonstrated the capabilities of SLF when it was employed in various biomimetic aggregation schemes. Moreover, liposomes were examined with the MP-SPR as larger scale nanomedical particles for the purposes of safe and effective nanocarrier development. The administration of a liposomal nanocarrier into the blood circulation was mimicked in the designed bionanophotonic in vitro schemes. Undiluted serum was made to interact with immobilized model liposomes in dynamic flow conditions. The findings revealed that the variation in surface chemistries of the liposomes plays a role when serum essentially immune system components are interacting with the liposomes. In particular, distinct soft and hard protein coronas were observed and characterized during the interactions. Collectively, the results and findings in this Thesis underline the broad potential of biophotonics for biopharmaceutical applications. The technical improvements in instrumentation, and creativity in the application and data analysis development make the future of biophotonics bright.
  • Karhu, Elina (Helsingin yliopisto, 2016)
    Chlamydia pneumoniae (C. pneumoniae) and Chlamydia trachomatis (C. trachomatis) are among the most common infectious microbes in humans. C. pneumoniae represents approximately 10% of community-acquired pneumonias worldwide. Statistically, everyone becomes infected 2-3 times during their lifetimes. The persistent form of C. pneumoniae is associated with other chronic diseases, such as asthma and chronic obstructive pulmonary disease. C. trachomatis is a sexually transmitted pathogen, which may result in infertility and ectopic pregnancies. Of all the infective agents it is also the one to cause most cases of blindness. The chronic forms of Chlamydiae cannot be cured by antibiotics, and a vaccine against them does not exist. Acute forms can be treated with antibiotics; but, resistance is increasing, given that treatment of Chlamydiae relies on wide-spectrum antibiotics. In this thesis new biologically active extract and compounds from natural sources were found to inhibit the growth of C. pneumoniae and C. trachomatis, the antichlamydial activity against C. pneumoniae being higher with all of the studied compounds and an extract. The antichlamydial effect of six lignans from the berries of Schisandra chinensis (Turczaninov) Baillon and their possible role as lead -compounds in the early drug discovery process were investigated. An aqueous methanol extract from Schisandra chinensis berries and Schisandra lignans showed varying activity on two different clinical isolates of C. pneumoniae, CV-6 and K7. The Schisandra lignans were shown to act at the mid-cycle of the infection. In addition, it was found that the effect is more targeted to the intracellular forms of the bacteria than to the extra cellular forms. Moreover, the results suggest that the antichlamydial mechanism of these lignans is specific to the host cell Chlamydia interaction rather than caused by their antioxidative properties. Schisandra extract and lignans did not affect the growth of three common respiratory tract bacterial pathogens, nor did they inhibit the growth of seven common metabolically active species, suggesting a Chlamydia-selective action of the extract and the lignans. Most of the lignans did not decrease host cell viability at the studied concentrations. As effective and selective growth inhibitors of Chlamydia pneumoniae, non-toxic and most of them not violating the Lipinski s rule of five, these Schisandra lignans present excellent lead candidates for further drug discovery. Schisandra lignans were utilized in constructing a reference set for a novel ligand-based virtual screening process, which proved successful with a hit rate of 1.2%. Six non-toxic lead compounds were found, belonging to new antichlamydial chemotypes. A new chemical space for antichlamydial compounds was defined, for future antichlamydial drug discovery projects.
  • Dimitrow, Sari Maarit (Helsingin yliopisto, 2016)
    Development and Validation of a Drug-Related Problem Risk Assessment Tool for Use by Practical Nurses Working With Community-Dwelling Aged The demand for long-term home health care services for the aged (≥65 years) is growing. Practical nurses (PNs) are those who most often visit the aged using HC services and consequently, are in a key position to monitor the benefits and risks of pharmacotherapy of their clients. The aim of this study was to develop and validate an easy-to-use DRP Risk Assessment Tool (DRP-RAT) for PNs caring for home-dwelling aged ≥65 years focusing on identifying and solving the highest priority DRP risks. The specific aims were: 1) to systematically review articles that describe criteria for assessing inappropriate prescribing in the aged ≥65 years; 2) to describe the development process and content validation of the DRP-RAT; 3) to evaluate the feasibility of the final DRP-RAT among PNs in HC; and 4) to assess the reliability of risk assessments conducted by PNs by using DRP-RAT and to identify the clinically most significant DRPs needing action. Two systematic literature reviews (Study I, year 2010; an unpublished one) and the expertise of the research group were used as a basis for the development of the DRP-RAT. The content of the draft tool was validated by a three-round Delphi survey with a panel of 18 experts in geriatric care and pharmacotherapy (Study II, year 2010). Data for the feasibility study were collected during the training of PNs, working in HC, in the use of the DRP-RAT (Study III, year 2011). The PN-conducted (n=25) DRP risk assessments by DRP-RAT (n=85) and the same clients copied medication lists (n=68), face-to-face discussions and responses to open questions of the returned feedback forms (n=23) were analyzed. In 2013, an experienced geriatrician reviewed HC clients (n=45) medications by using three different reviewing methods on each patient (Study IV). The methods based on: 1) DRP-RAT (n=45) completed by the PNs (n=26) and copied medication lists; 2) health centre s medical records ( gold standard ); and 3) Methods 1 and 2 together. Results of the reviews and contents of the geriatrician s open comments regarding the PNsʼ risk assessments were analyzed. DRPs in the study population identified and reported by the geriatrician were studied from the geriatrician s DRP classifications (n=45) and by a retrospective review of the geriatrician s case reports (n=45). The final DRP-RAT consists of 18 items that assess risks for DRPs in home-dwelling clients. It is divided into four sections: 1) Basic Client Data, 2) Potential Risks for DRPs in Medication Use, 3) Characteristics of the Clientʼs Care and Adherence and, 4) Recommendations for Actions to Resolve DRPs. The DRP-RAT turned out to be feasible among PNs and the PN-completed tool was capable of providing reliable and timely patient information to support physician s clinical decision making. Compared to the gold standard (Method 2), Method 1 resulted in a false negative rating in 7% (95 % CI 1.4 18.3) of the cases (3/45). The geriatrician identified an average of 3.1 potential DRPs per patient. This study indicates that the DRP-RAT, developed and validated in this study, could make it possible to more effectively involve PNs, working in HC, in medication risk management among the home-dwelling aged, and that medication risk management should be focused on the highest priority risks. Actions to facilitate the implementation of the DRP-RAT in the Finnish health care system are needed. Future studies are needed to evaluate the effects of PNsʼ risk assessments using the DRP-RAT on clinical, humanistic and economic outcomes.