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Browsing by master's degree program "Master's Programme in Pharmacy"

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  • Reunanen, Saku (2020)
    Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic neurons that form the nigrostriatal pathway gradually die. This causes the main motor symptoms of Parkinson’s disease: tremor, rigidity and bradykinesia. While PD affects 1-2% of total population, all currently used medicines are symptomatic, and there is no disease modifying therapy available at present. Although several different animal models for Parkinson’s disease exist, the lack of adequate animal models is often cited as a major obstacle for predicting the clinical success of potential drug candidates. Lewy bodies (LBs) are abnormal aggregates that develop and spread inside nerve cells of human PD patients, their main structural component being α-synuclein. Because α-synuclein is thought to play a major role in the pathology of PD, much research has been focused on it. Different α-synuclein-based animal models of PD exist today, of which the most recent are based on using direct injections of preformed α-synuclein fibrils (PFFs). These new α-synuclein based disease models have helped to understand the disease process in PD better, but cell death in these models takes longer to achieve and is often less pronounced compared to traditional neurotoxin based animal models of PD. The aim of this study was to participate in the development and characterization of a novel mouse model of PD. This new model combines PFF-injections with the commonly used neurotoxin 6-OHDA, which should result in more robust dopamine pathway degeneration than what is seen with the current PFF-based models. The main hypothesis of this study was that the combination of intrastriatal injections of PFFs and a low dose of 6-OHDA would cause gradual spreading of the α-synuclein aggregation pathology in the nigrostriatal dopamine pathway and progressive dopamine neuron loss leading to motor deficits. C57BL/6 mice were stereotactically injected unilaterally with both PFF and 6-OHDA, and their performance was assessed every other week with different behavioral tests until week 12. At the end, brains were collected and optical density of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was measured from striatal sections, and TH and DAT positive cells in the substantia nigra were counted. The amount of Lewy bodies present in the brain slices was also counted from the cortex and substantia nigra areas of the brain. In the histological assays, statistically significant reductions of both TH and DAT were found in the brain sections of the PFF + 6-OHDA combination group and the amount of TH and DAT positive cells were lower in this group compared to the group receiving vehicle treatment only. However, the results of behavioral tests were non-significant, although a non-statistical positive trend in the amphethamine-induced rotations test was observed where mice receiving PFF + 6-OHDA rotated the most. Taken together, combination model that utilizes both PFF and 6-OHDA injections seems like a promising candidate in modelling PD in mice, but much more research and further development of the model is required before this combination model is ready and robust for use in drug development.
  • Anttila, Emmi (2021)
    Mild traumatic brain injury (TBI) is defined as an injury that disrupts the normal functioning of the brain and is the result of external force to the head. It is the most common type of traumatic head injury, and it is common especially in contact sports and within military personnel. Mild TBI typically causes no clear structural changes to the head, but it can induce persistent clinical symptoms, as well as microscopic pathological changes to the brain that may eventually lead to neurodegeneration and increase the risk for several diseases. Mild TBI is a risk factor for several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and chronic traumatic encephalopathy. The primary objective of this study was to develop a repetitive mild TBI mouse model for future research purposes in the field of head trauma and neurodegeneration. The injury was induced as a closed head injury with an electromagnetic impactor. Literature and pilot experiments were used to define the parameters of the impactor required to induce a brain injury of desired severity. The characterization criteria of the mild TBI model considered the criteria used to define human mild TBI, as well as long term effects often reported after repetitive mild TBI: neurodegeneration as tau protein related pathology, neuroinflammation, and memory deficits. The secondary objective of this study was to tentatively test a prolyl oligopeptidase (PREP) inhibitor on the behavioral and histological effects of mild TBI. The functioning of the mild TBI model was studied by histopathological and behavioral assessments. After baseline behavioral assessment and repetitive (1 injury every 24 hours altogether 5 times) mild TBI inductions, the mice were monitored for approximately 3 months, during which several rounds of behavioral tests were performed. Barnes maze and novel object recognition tests were used to assess memory functions, and locomotor activity test was used to assess general locomotor activity. After euthanasia, brain histopathology was performed to study the amount of tau protein and the level of neuroinflammation. Due to the low number of animals in the study, the results are directional and need to be confirmed in subsequent studies. The histopathology showed greater amount of neuroinflammation and tau protein in the brains of injured mice, but statistical evaluations could not be made. Memory functions were slightly worse in the injured mice compared to controls, but significance of the results is unclear. Locomotor activity was not influenced by the mild TBIs. PREP inhibition treatment increased the locomotor activity of the mice, but the significance is unclear. The mild TBI model seems promising and the characterization criteria were partially met. The results of the study need to be verified in subsequent studies with a greater amount of animals. The model developed here can be used to study the involvement of head trauma in neurodegeneration, as well as treatment alternatives to changes caused by mild TBIs. As there currently are no curative treatments to neurodegenerative diseases, research regarding neurodegeneration and its risk factors is highly important.
  • Katajamäki, Jani (2021)
    Cytochrome P450 (CYP) enzyme inhibition is one of the most common reasons for adverse drug-drug interactions. An especially harmful form of inhibition is time-dependent inhibition (TDI) in which the inhibition potency increases over time and persists even after discontinuation of the drug. Both direct and time-dependent inhibition can be efficiently screened with the so-called cocktail method containing several CYP-selective probe substrates in a single reaction mixture. This method is practical especially in ADME studies of drug development, as it offers lower costs, consumption of fewer reagents and faster implementation in comparison to conventional methods. In addition, the cocktail method can be used to establish new diagnostic CYP inhibitors in vitro. The aim of this Master’s thesis was to participate in the development and optimization of a new cocktail assay method. The method was developed for screening of major drug-metabolizing CYP enzymes in vitro both in a direct and time-dependent manner using pooled human liver microsomes. Based on preliminary testing, included probe substrates were divided into two cocktails to avoid significant inter-substrate interactions: cocktail I containing tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4, and cocktail II containing coumarin/CYP2A6, (S)-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2. First, cocktail incubation conditions were optimized, followed by the determination of probe reaction kinetics, kinetic parameters (Km, Vmax) and inter-substrate interactions with single- or dual-substrate incubations. Finally, suitable probe substrate concentrations and the composition of cocktails was evaluated based on the obtained results. As a result of assay optimization, optimal incubation conditions for yet unoptimized cocktail II were established. In optimized incubation conditions, all probe reactions exhibited saturable Michaelis-Menten kinetics except for tacrine 1-hydroxylation (CYP1A2), which exhibited biphasic kinetics instead (Km1: 7.36, Km2: 517). The selected probe substrate concentrations were all below or near their respective Km values except for (S)-mephenytoin 4’-hydroxylation (40 µM vs. Km of 12.5 µM); however, its concentration could not be reduced in order to maintain sufficient metabolite formation for UHPLC-MS/MS-analysis. Dual-substrate incubation assays demonstrated a need for the reduction of bupropion concentration below 100 µM due to its inhibitory effects on CYP2C8 and CYP3A4. In addition, chlorzoxazone/CYP2E1 and testosterone/CYP3A4 were tested as complementary probe substrates for the cocktails; however, they proved to be unsuitable for both cocktails due to significant interactions (>40% inhibition). Prior to the deployment of the method, some adjustments of probe substrate concentrations are still required in addition to consideration of the suitability of less commonly used CYP3A4 and CYP2E1 probe reactions to improve cocktail coverage. Lastly, validation of the method with known time-dependent model inhibitors should also be conducted. Besides to improvement of the cocktails, new information was generated on inter-cocktail probe-probe interactions and enzyme kinetics of probe reactions, especially for the less-studied astemizole O-demethylation (CYP2J2) and tacrine 1-hydroxylation (CYP1A2). Generated information can be used, for example, in the development of new cocktails.
  • Uoti, Arttu (2021)
    Background and objectives: Cancer is one of the leading causes of death worldwide, and resistance to current treatments demands the continuous development of novel cancer therapies. Cancer immunotherapy aims to induce anticancer immune responses that selectively target cancer cells. Viruses can also be harnessed to elicit tumor-specific immune responses and to improve the response rates of other concomitant cancer therapies. The purpose of this study was to develop a novel viral vector-based cancer vaccine for intratumoral immunotherapy. By using the previously developed PeptiENV cancer vaccine platform, the vector viruses were coated with cell-penetrating peptide (CPP) sequence-containing tumor peptides in an attempt to further drive the immune responses elicited by the vector against cancer cells. The efficacy of the PeptiENV complex as a cancer vaccine was assessed by following its effects on tumor growth and the development of local and systemic antitumor immune responses. Methods: The PeptiENV complex formation was assessed by a surface plasmon resonance (SPR) analysis. Dendritic cell (DC) activation and antigen cross-presentation were studied using the murine JAWS II dendritic cell line. The development of cellular immune responses against tumor antigens was first studied by immunizing mice with the PeptiENV complex. The antitumor efficacy and immunity of intratumoral PeptiENV administration were then studied using the murine melanoma models B16.OVA and B16.F10.9/K1. In addition to intratumoral PeptiENV treatment, some of the B16.F10.9/K1-implanted mice were also treated with an anti-PD-1 immune checkpoint inhibitor (ICI) to study the PeptiENV complex as a biological adjuvant for ICIs. Results: The SPR analysis confirmed that CPP-containing peptides can be stably anchored onto the viral envelope of the viral vector. The in vitro results showed that the PeptiENV complex does not hamper the presentation of antigens at the surface of DCs. Additionally, the viral vector was found to activate DCs seen as a change in the cells’ morphology and surface protein expression. Immunizing mice with the PeptiENV complex induced a robust antigen-specific cytotoxic T cell response. Upon intratumoral administration in vivo, the PeptiENV cancer vaccine was not capable of inducing tumor growth control against B16.OVA melanoma, although it did still elicit robust systemic and local antitumor T cell responses. In the treatment of B16.F10.9/K1 melanoma, however, the PeptiENV complex induced efficient tumor growth control, which resulted in a significant survival benefit. Additionally, co-administration of anti-PD-1 resulted in an additive therapeutic effect. Discussion and conclusions: The present study describes a novel, highly immunogenic viral vector-based cancer vaccine that has the potential to be used as an adjuvant treatment for ICI therapy. Subsequent studies could be conducted to gain a deeper understanding of the immunological mechanisms underlying the antitumor efficacy of the cancer vaccine complex. Moreover, this novel PeptiENV complex could also be further developed as an infectious disease vaccine platform against emerging pandemics. However, the effects of pre-existing antiviral immunity on the efficacy of the cancer vaccine should be explored in future studies.
  • Lähdeniemi, Veera (2021)
    Drug metabolism is a series of enzyme catalysed processes that modify foreign compounds into a form that is more easily excreted from the body. Compounds can affect the activity of metabolizing enzymes and this may lead to toxic concentrations of a drug that is metabolized via the enzyme. With prodrugs, on the other hand, the drug might not achieve its biologically active form and therefore the treatment will not be effective. Recognizing and preventing metabolic interactions is important already in the early stages of drug discovery and development. Cytochrome P450 (CYP) enzyme inhibition is one of the major reasons for adverse drug-drug interactions (DDIs). The inhibition can be time-dependent (TDI), which means that the potency of inhibition increases over time. TDI may be reversible or irreversible, latter being more severe as new enzymes need to be produced in the body to restore the enzymatic activity. IC50 shift assay is a method that gives information of new compounds potential to cause TDI. IC50 shift assay does not show whether the TDI is reversible or irreversible, however further studies, e.g. dialysis assay, can be conducted to find it out. If the study compound is irreversibly bound to the enzyme, the enzyme activity should not recover in the dialysis. The aim of this master’s thesis was to develop a dialysis method that could determine the reversibility of the TDI observed in the IC50 shift assay. A dialysis method conducted with microsomes is described in earlier literature. Known inhibitors (both time-dependent and direct) for four CYP isoforms were studied in this work: CYP1A2 (furafylline and fluvoxamine), CYP2C9 (tienilic acid and sulphaphenazole), CYP2D6 (paroxetine and quinidine) and CYP3A4 (verapamil, azamulin and ketoconazole). IC50 shift assays were conducted to each inhibitor before the dialysis experiment. The studied compounds behaved in the dialysis assay mostly as assumed based on the literature. The workflow from IC50 shift assay to dialysis assay worked successfully and the IC50 shift data could be utilized when choosing the test concentrations for dialysis assay. Both the IC50 shift assay and dialysis assay were reproducible and the deviations between replicates and separate studies were relatively low. The method still requires some optimizing, but so far, the results are promising. In the future the dialysis method may be part of in vitro CYP inhibition studies at Orion Pharma.
  • Tuominen, Elsi (2021)
    Neurodegenerative diseases and neuronal injury after trauma are common causes of neuronal loss. Adult brain has only a limited regenerative capability to replace the lost neurons caused by several distinct brain diseases. Direct reprogramming of brain resident cells into neurons could provide a promising strategy for efficiently replacing non-functional neurons. To date, the focus has been put largely on astrocyte-to-neuron reprogramming despite the relatively low yield of newly generated neurons reported in vivo. According to our hypothesis oligodendrocytes possess a more diverge transcriptomic profile when compared to neurons and astrocytes thus allowing better cell-specific targeting of reprogramming. Here, we establish the molecular tools for direct neuronal reprogramming of human oligodendrocytes to neurons. We investigate whether the expression of a known neural fate specification factor under selected oligodendrocyte-specific promoters is sufficient to induce oligodendrocyte-to-neuron transformation. Furthermore, we test the established tools in vitro using an immortalized human oligodendrocyte cell line. Our preliminary data shows that the human ERBB3 promoter and a single transcription factor transfected cells express doublecortin (DCX), an early marker of neuronal identity. Only recently, the direct in vitro reprogramming of human oligodendrocyte precursor cells into functional neurons has been reported. The direct reprogramming of oligodendrocytes into neurons provides an exciting alternative of neuronal replacement for astrocyte-to-neuron reprogramming. Overall, the field of direct reprogramming offers interesting possibilities for regenerative medicine providing a method for the production of newly generated disease and patient-specific cells.
  • Pohjavaara, Saana (2021)
    Dilated cardiomyopathy is a non-ischemic cardiac disorder predisposing to heart failure, and the characteristics of dilated cardiomyopathy emerge under normal loading conditions. Dilated cardiomyopathy can be consequence of various conditions e.g. genetic mutations, virus infection or toxin exposures. One of the significant causes of familial dilated cardiomyopathy in Finland is mutation S143P in LMNA-gene, coding for A type lamins. Current drug therapy for dilated cardiomyopathy aims to alleviation of symptoms, prevention of complications and progression of the disease, however, efficacy of current therapy is insufficient, and novel therapy strategies are urgently required. Transcription factors are fundamental regulators of gene expression, and GATA4 is a crucial transcription factor both in embryonic and in adult heart and thus an intriguing target for therapeutic manipulation. Compounds targeting GATA4 have shown anti-hypertrophic and cardioprotective effects. Here, effects of two different hypertrophic stimuli, endothelin-1 and mechanical stretch, on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were examined with high-content analysis and quantitative reverse transcription PCR (qRT-PCR), respectively. One hiPSC-CM line was used as a healthy control, whereas the other carried the S143P mutation in LMNA-gene (DCM-CMs). Additionally, effects of GATA4-targeting compound C-2021 on cardiomyocytes were investigated. In summary, according to proBNP staining, DCM-CMs are more hypertrophied at baseline. DCM-CMs seemed to be less susceptible to mechanical stretch-induced enhancement in BNP gene expression. In addition, compound C 2021 may have anti-hypertrophic properties suggesting it to be a potential drug candidate in cardiac diseases. Finally, lamin A seemed to mislocalize to nucleoplasm instead of nuclear lamina in DCM-CMs.
  • Rissanen, Johanna (2020)
    Lääkevaihto ja sitä täydentävä viitehintajärjestelmä ovat laskeneet lääkekustannuksia Suomessa. Epilepsialääkkeet eivät ole aiemmin kuuluneet lääkevaihdon piiriin, sillä epilepsian hoidossa eri valmisteet eivät välttämättä ole terapeuttisesti tarpeeksi samanarvoisia, ja pienikin muutos hoitotasapainossa voi altistaa epilepsiakohtauksille. Nykyisin epilepsialääkkeitä käytetään kuitenkin usein muihinkin käyttöaiheisiin, kuten psykiatrisiin sairauksiin ja kivun hoitoon. Vuonna 2017 lääkekorvausjärjestelmään tehtiin säästötoimenpiteitä, joiden yhteydessä epilepsialääkkeet sisällytettiin lääkevaihdon piiriin muissa käyttöaiheissa kuin epilepsian hoidossa. Lisäksi otettiin käyttöön poikkeava viitehintaryhmä, joka koski epilepsialääkkeistä pregabaliinia neuropaattisen kivun käyttöaiheessa. Tutkimuksen tavoitteena oli tarkastella epilepsialääkkeiden (pregabaliinin, gabapentiinin, topiramaatin, lamotrigiinin ja valproiinihapon) vaihtamista sekä hintojen kehitystä lääkevaihtoon ja viitehintajärjestelmään sisällyttämisen jälkeen vuoden 2017 alusta vuoden 2019 puoliväliin. Lisäksi tarkasteltiin näiden lääkeaineiden kustannusten, korvausmenojen sekä käyttäjä- ja reseptimäärien kehitystä. Aineistona käytettiin Kansaneläkelaitoksen reseptirekisteriin pohjautuvia tilastoja epilepsialääkkeiden lääkeostoista sekä lääkkeiden hintalautakunnan päätöksiä epilepsialääkkeiden viitehintaryhmistä ja viitehinnoista. Epilepsialääkkeiden vaihtaminen yleistyi tarkastelujakson aikana kaikilla lääkevaihdon piirissä olleilla viidellä lääkeaineella, ja vaihtokieltojen osuus resepteistä laski useimmilla lääkeaineista. Viitehinnat laskivat useimmissa tarkastelluista viitehintaryhmistä, mutta lähes yhtä usein viitehinta ei muuttunut. Viitehinnat laskivat enemmän viitehintaryhmissä, joissa oli useampia vaihtokelpoisia valmisteita. Lääkevaihdon ensimmäisenä vuonna 2017 lääkevaihtoon kuuluvien epilepsialääkkeiden kustannukset ja korvausmenot pääosin laskivat, vaikka lääkkeiden käyttö ei vähentynyt. Lääkevaihdon toisena vuonna kustannukset eivät juuri laskeneet. Pregabaliinin poikkeavan viitehintaryhmän vuoksi vaihtamatta jääneet reseptit aiheuttivat merkittävän osan lääkevaihtoon kuuluvien epilepsialääkkeiden kustannuksista. Pregabaliinille jäi siten todennäköisesti yhä säästöpotentiaalia poikkeavan viitehintaryhmän voimassaolon päätyttyä vuoden 2019 heinäkuussa, mitä on syytä tarkastella jatkotutkimuksissa.
  • Jalonen, Milla (2020)
    There are significant inter-individual differences in the effects of drugs. These differences can be caused by, for example, other diseases, adherence to treatment, or drug-drug interactions. A drug-drug interaction can lead to an increase in the concentration of the active substance in the circulation (pharmacokinetic interactions) or a change in the effect of the drug without changes in plasma concentration (pharmacodynamic interactions). A drug-drug interaction can change the efficacy of a drug or affect the adverse drug reaction profile. The individual’s genetic background, such as diversity in drug-modifying enzymes (polymorphism), also has an effect on the efficacy and the risk for adverse drug reactions of some drugs. A pharmacogenetic test can be used to study how genetic factors affect drug treatments. The aim of this master's thesis was to examine the possibilities of personalized migraine pharmacotherapy from the perspective of pharmacogenomics and drug-drug interactions. Four online drug-drug interaction databases available in Finland were compared. Inxbase is the most widely used interaction database by physicians in Finland and it is also integrated into Finnish pharmacy systems. Other databases used in this study were the international professional database Micromedex as well as Medscape Drug Interaction Checker and Drugs.com Drug Interactions Checker. The latter two are open-access databases available for healthcare professionals and patients. Interaction searches were conducted in the selected databases between acute and prophylactic drugs used for the treatment of migraine (e.g. bisoprolol-sumatriptan). Fourteen acute and 12 prophylactic drugs were selected for this study based on the Current Care Guidelines in Finland (Käypä hoito), and the data were collected in Excel spreadsheets. The first search was completed in December 2019 and the second search in March 2020. In this study, many potential interactions were found between acute and prophylactic drugs used to treat migraine in Finland. For more than half of the drug pairs studied, a potential interaction was found in at least one of the databases. There were significant differences between the interaction databases regarding which interactions the database contains and how the severity of the interactions was classified. Of the interactions found, only 45% were found in all four databases, and each database contained interactions that were not found in the other databases. Even very serious interactions or drug pairs classified as contraindicated were not found to be consistently presented across all four databases. When selecting drug treatment for a migraine patient, potential drug-drug interactions between acute and prophylactic drugs as well as the patient's genetic background should be considered. Individualizing migraine treatment to achieve the best efficacy and to reduce the risk for adverse drug reactions is important because migraine as a disease causes a heavy burden on individuals, healthcare, and society. Pharmacogenetic tests particularly developed to help choosing migraine treatment are not yet available, but tests are available for few other indications in both public and private healthcare. The use of these tests in clinical practice will increase as physicians’ pharmacogenetic knowledge and scientific evidence on pharmacogenetic tests increase. Utilization of pharmacogenetic data requires that test results are stored in electronic health records so that they are available in the future, when changes are made to drug treatment of individuals. More studies are warranted to better understand the clinical impact of pharmacogenomics and drug-drug interactions in migraine care.
  • Li, Mingchen (2021)
    Parkinson’s disease (PD) is a progressive chronic neurodegenerative disorder, which results in the selective loss of dopaminergic neurons in the substantia nigra (SN). The loss of these neurons results in the dysfunction of the nigrostriatal pathway bringing forth the characteristic motor symptoms seen in PD: postural instability, rigidity, slowness of movement and resting tremors. Non-motor symptoms, such as cognitive deficits, depression and impaired olfaction, typically emerge before motor symptoms. Currently available treatments only provide symptomatic relief with diminishing returns over time and no improvements on the overall outcome of the disease. Neurotrophic factors (NTF) have been of particular interest as a possible curative treatment for PD due to their potential for neuroprotection and neurorestoration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an NTF that has shown promising results in numerous in vitro and in vivo studies of PD. However, therapy with MANF and other NTFs involves surgical intervention for local administration, as NTFs cannot cross the blood-brain barrier (BBB). Therefore, the therapeutic potential of a systemically administered NTF would be tremendous, as it would lead to a significantly more favorable risk-benefit ratio for the patient. The aim of the current investigation is to evaluate the efficacy of a next generation variant of MANF in the 6-hydroxydopamine toxin-induced unilateral lesion rat model of PD. Prior in vivo results suggested that subcutaneously injected MANF variant is able to penetrate the BBB. Amphetamine-induced rotational behavior (AMPH-ROTO) was used to evaluate the severity of the unilateral lesions during the experiment every other week until the end of the experiment at week eight. Animals were divided into treatment groups during week two based on their AMPH-ROTO results. Animals received MANF variant either subcutaneously through an implanted osmotic minipump at two different dosages or as a single dose divided into three separate intrastriatal injections. Tyrosine hydroxylase (TH) immunohistochemical staining was performed on brain sections collected from the striatum and SN for data analysis. In addition to AMPH-ROTO results, the efficacy of treatment was determined via the optical density of TH-positive striatal fibers and the number of TH-positive cells in the SN. Statistically significant differences (defined by p < 0.05 and a non-zero mean difference at a 95 % confidence interval) were observed only in the number of TH-positive cells in the SN favoring intrastriatal MANF variant treatment over both intrastriatal MANF and the vehicle treatment. The main concern regarding the validity of the results was related to the heterogeneous lesion sizes in different treatment groups possibly resulting in unsuccessful randomization due to excessive baseline differences. The inadvertent negative effects of this was further exacerbated by low a priori statistical power, which in the end had likely caused inflated effect sizes. Thus, assessment of the definitions of the used statistical parameters and the limitations of the experimental design suggest that presently, the efficacy of the MANF variant could not be evaluated reliably, in spite of the statistically significant result.
  • Muurman, Tuulikki (2021)
    Background: Poor health literacy (HL) is associated to increased hospitalization and decreased seeking for screenings. Shared decision making can increase patient knowledge, decrease anxiety over the care process, improve health outcomes and reduce health care costs. Little is known about factors influencing health literacy and participation in treatment decision making in different population groups. Objectives: To investigate factors predicting HL and participation in the treatment decision making. Methods: A cross-sectional population online survey conducted in Finland in 2019 by Finnish Medicines Agency. Both health literacy and participation in the decision making were assessed by three statements that sum variables were created with score 1-5 (Cronbach’s alpha value 0.584 and 0.810). Age, gender, education, household income and most common chronic diseases were chosen as possible predicting factors. Two-variable Pearson’s chi-squared test was first used to find significant factors followed by logistic regression analysis to take into account several variables. Results: Of all the respondents (n=2104) 76.5% had good HL and 73.4% had willingness to participate in the treatment decision making. In the two-variable test older age (p<0.001), lower education (p<0.001), lower household income (p=0.001), higher number of chronic diseases (p=0.003), having cardiovascular diseases (p=0.003), diabetes (p=0.029) and cancer (p=0.001) predicted poorer health literacy. Male gender (p=0.001), not having chronic diseases (p=0.001), not having a musculoskeletal disorder (p=0.050) or mental health disorders (p<0.001) predicted poorer participation in the treatment decision making. In the logistic regression analysis older age and having cancer predicted poorer health literacy. Male gender and not having mental health disorders predicted less willingness to participate in the decision making. Conclusions: Older age and cancer predicts poorer health literacy and male gender poorer willingness to participate in the decision making. Further research should focus on investigating more in detail the contributing factors to these findings, and how health literacy in elderly and men’s involvement to the decision making could be improved.
  • Ritamäki, Kaisu (2019)
    Pharmaceutical companies are required to comply with fair market guidelines and regulations. However, definition of fair market value (FMV) in clinical trial is not unambiguous. In literature are some suggestions how to determine the phenomenon of FMV in clinical trial. Understanding the FMV and how it should be applied into practice when conducting clinical research is challenging. This study provides more focused information on FMV in clinical trials and its determination. FMV should be determined for research-related activities in clinical drug research. FMV of research related activities can be consistent if similar sites are performing similarly conducted studies for similar sponsors. Therapeutic area and geographical location of the trial site can also influence for the FMV. This study was performed in co-operation with Roche. The aim of the study was create a consistent and transparent method to assist in the determination of FMV in medical drug research in relation to the payments paid by the sponsor to the sites. Clinical trial agreements (CTA) and associated agreements were analysed to investigate FMV of research-related activities by study site, study type, therapeutic area and geographical area. Average price and price range of each research-related activity from previous CTAs and associated agreements of Roche Finland was calculated. Based on available data from literature and study results research-related activities and factors affecting to the FMV of clinical trials were discussed to create comprehensive understanding of FMV in clinical drug research. Based on this study average price of the specific research-related activities can be different by therapy area, site, study type and geographical area. All these factors are relevant when assessing FMV of specific research-related activity. Studied therapy area and site seems to have the most important impact when evaluating FMV. For some research-related activities such as national coordinator investigator (NCI) fee price ranges could be very big whereas in other research-related activities such as pharmacy fees prices could be quite similar. Some research-related activities were very study specific which affected evaluation of those activities. CTAs and associated agreements are valid documents to gather information assessing FMV of research-related activities in medical drug research. Average price and price range of the research related activity can be used when assessing FMV in medical drug research. However, price of the specific research-related activity need to be evaluated considering the studied therapy area, site, study type and geographical area.
  • Jäntti, Heli-Noora (2019)
    Farmasian ammattilaiset ovat lääkealan asiantuntijoita, joilta vaaditaan uudenlaista osaamista muun muassa teknologiakehityksen myötä. Nykypäivän asiantuntijuus edellyttää alakohtaisen eli sisällöllisen osaamisen lisäksi geneerisiä eli yleisiä taitoja ja ammatti-identiteetin muodostumista. Geneerisillä taidoilla tarkoitetaan yleishyödyllisiä taitoja, kuten ongelmanratkaisu- ja kommunikointitaitoja. Ammatti-identiteetillä tarkoitetaan käsitystä omasta työminästä, jonka avulla omaa roolia ja työnkuvaa järkeistetään. Näiden elementtien muodostamaa osaamisen kokonaisuutta kutsutaan kompetenssiksi. Asiantuntijoilta vaadittavan osaamisen muutos on ohjannut yliopistoja vastaamaan paremmin työelämän tarpeisiin. Helsingin yliopistossa toteutettiin Iso Pyörä -koulutusuudistus, jossa koulutusohjelmia uudistettiin komeptenssilähtöisesti. Kaikkiin koulutusohjelmiin ja opintojaksoihin lisättiin osaamistavoitteet, jotka opiskelijoiden tulisi saavuttaa valmistumiseensa mennessä. Osaamistavoitteiden täyttymistä edistää esimerkiksi portfoliotyöskentely, minkä avulla opiskelijat pääsevät hyödyntämään ja kehittämään reflektiotaitojaan. Opiskelijat voivat tuoda opetuksen kehittämiseen aivan uudenlaista näkökulmaa avatessaan käsityksiään esimerkiksi hyvistä opetusmenetelmistä, mitkä ovat auttaneet heitä saavuttamaan laaditut osaamistavoitteet. Toisaalta opiskelijoiden näkökulmasta saadaan tietoa, mikä osaaminen voidaan kokea puutteelliseksi, jolloin opetuksen kehittäminen on mahdollista. Tutkimuksen tavoitteena oli selvittää opiskelijoiden käsityksiä omasta osaamisestaan ja ammatti-identiteetistään sekä millä tasoilla opiskelijat reflektoivat osaamistaan. Tutkimuksessa analysoitiin vuoden 2017 kolmannen vuosikurssin kandiportfolion loppureflektioesseet käyttäen aineistolähtöistä sisällönanalyysimenetelmää. Esseissä opiskelijat reflektoivat osaamistaan suhteessa farmaseutin tutkinnolle asetettuihin osaamistavoitteisiin ja pohtivat omaa ammatti-identiteettiään. Tulosten mukaan opiskelijat saavuttivat monipuolista osaamista lääkkeiden ja lääkehoitojen näkökulmasta sekä kehittivät geneerisiä taitojaan. Puutteellisesti hallittiin useimmiten kielitaito sekä yrityksen ja yhteiskunnan taloudelliset periaatteet. Opiskelijoiden mukaan farmaseutin ammatti-identiteettiä määrittelevät erityisesti lääkeosaaminen ja terveydenhuolto sekä ammatin arvostaminen. Opiskelijoiden pohtimat valmiudet mukailivat osaamistavoitteita. Opiskelijat osasivat arvioida omaa osaamistaan ja nostaa esille vahvuuksiaan ja heikkouksiaan. Opetussuunnitelmaan on onnistuttu sisällyttämään geneeristen taitojen opetus, sillä opiskelijat kokivat saavuttaneensa näitä taitoja pääasiassa hyvin. Opetusta tulisi kehittää kielitaidon ja liiketalouden kohdalla, sillä nämä koettiin usein puutteellisesti hallituksi. Ammatti-identiteettikäsitykset mukailivat kirjallisuutta, sillä muissa tutkimuksissa on saatu samankaltaisia tuloksia.
  • Hedström, Anna (2020)
    The ability to regulate release of noradrenaline, dopamine and GABA is one of the most important roles of the nicotinic receptors. The release of neurotransmitters following stimulation of nicotinic receptors is addressed in the thesis, with focus on dopamine and noradrenaline. Release of neurotransmitters, mediated through nicotinic receptors, has been researched using various methods, including brain slices, microdialysis and synaptosomes. Research using synaptosomes have provided valuable information regarding nicotinic receptors and their ability to regulate neurotransmitter release. Research using receptor specific antagonists have provided information regarding the stoichiometry of nicotinic receptor in different regions of the brain. The primary focus in the thesis, was the characterization of [3H]dopamine release following stimulation of nicotinic receptors with varenicline and acetylcholine, using synaptosomes from mouse striatum. Using a-conotoxin-MII, the [3H]dopamine release was divided into a-conotoxin- MII-resistant and -sensitive release. [3H]Dopamine release was mediated through a6b2*- and a4b2*-receptors from striatal synaptosomes. The involvement of other receptors could not be ruled out, but based on these results and results from previous studies, the involvement of other nicotinic receptors is supposedly low.
  • Porru, Anna (2020)
    Medication-related errors have been identified as the single most important risk factor for patient safety across the world. According to previous research, medication errors are common in nursing homes. However, the existing data on medication errors in Finnish nursing homes is scarce, although the challenges and defects in nursing home care services, including drug treatments, are well known. Furthermore, nursing home residents are typically characterized by old age, multimorbidity and polypharmacy. Therefore, they are particularly vulnerable to potential adverse events caused by medication errors. The aim of this study was to investigate the rates and causes of medication errors reported in nursing homes and evaluate their impact on medication safety. Additionally, the proportions of potentially inappropriate medication (PIMs) and high-risk medication involved in the medication errors were determined. The data of the study consisted of 251 medication errors reports that were submitted to the safety incident report system (HaiPro) in nursing homes located in Central Uusimaa healthcare and social welfare joint municipal authority (Keusote) in 2019. Quantitative analysis of the data provided an overview of the medication errors that had occurred in nursing homes and the medicines most commonly involved in them. Content analysis and simplified root cause analysis enabled to study more in-depth the contributing factors of medication errors and potential risks associated with the medication process in nursing homes, as well as the possibilities of preventing similar errors in the future. James Reason's human error theory and in particular its system perspective was applied as a theoretical framework in this study. Medication errors were reported regularly in nursing homes during the follow-up period of the study. The most frequent medication error type was administration error. The majority of these errors were medication omissions, followed by the wrong time of administration and administration to the wrong patient. The most common drug classes causing medication errors were antithrombotics, opioids, antidementia drugs, diuretics, antipsychotics, antidiabetics, and antidepressants. Nearly a quarter of the reported medicines were high-risk medications, most commonly opioids, antithrombotics, or antidiabetic drugs. PIMs accounted for approximately 13% of all medications in the data. Errors were most often caused by unsafe medication practices, communication problems, and deficiencies in the work environment such as excessive workload or time pressure. A significant part of the medication errors were related to transdermal medication patches. The study also showed that the quality of medication error reporting in nursing homes is in part insufficient and should be improved so that the reports can be better used for learning purposes. The results of the study provide valuable additional information on medication errors in Finnish nursing homes and their contributing factors. The information can be used to improve medication safety practices in nursing homes. Safe and uninterrupted medication use process is a goal that should be pursued not only in health care but also in social welfare services such as nursing homes.
  • Heinonen, Suvi (2020)
    Diacylglycerol (DAG) is a lipid second messenger, which activates classical and novel protein kinase C (PKC) isozymes at the plasma membrane. Abnormalities in PKC signaling have been linked to several diseases, and defective PKC function connects to multiple pathophysiological components of Alzheimer’s disease. However, aimlessly activating all PKC isozymes with synthetic ligands can be problematic, since the activation of certain isozymes can also promote unwanted processes. There are indications that DAGs with varying degrees of acyl chain saturation may have different and specific PKC activating abilities. Thus, understanding how the structural differences in DAGs relate to their behavior at the lipid bilayer may be beneficial for the development of new, isozyme-specific ligands of PKC. The aim of this master’s thesis was to compare the orientation, positioning and dynamics of two unsaturated DAG molecular species, 1,2-dioleoyl-sn-glycerol (DOG) and 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG) in glycerophospholipid bilayers using conventional molecular dynamics (MD) simulations and umbrella sampling. The glycerophospholipid bilayers were composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) or 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphatidylethanolamine (SDPE), representing the glycerophospholipid environment in the inner leaflet of the plasma membranes in peripheral tissues and in brain tissue, respectively. Both DAG molecular species displayed very dynamic behavior in all systems, with wide distributions of glycerol moiety tilt angles and acyl chain conformations. Multiple occurrences of transbilayer movement (flip-flop) of DAGs was observed during the MD simulations in all systems. In POPE bilayers, SDG was observed to position closer to the aqueous interface compared to DOG, with larger values of solvent accessible surface area (SASA) of the glycerol moiety and the sn-3 hydroxyl group. In SDPE bilayers, no significant difference in this regard was observed between the DAG molecular species. Although potential of mean force (PMF) profiles did not reveal any major differences in the energetically favoured position of the hydroxyl group between the DAG molecular species, the calculations exposed that the dynamics of DOG are affected more by the surrounding lipid environment compared to SDG. Based on these results, it is probable that while the solvent accessibility and overall position of DAGs is affected by the length and degree of saturation of their acyl chains, there are also other mechanisms and processes causing the differing levels of PKC activation yielded by different DAG molecular species.
  • Järvinen, Janina (2021)
    Current treatments for major depressive disorder have notable limitations including the delay achieving the therapeutic effect. Ketamine has been shown to alleviate the symptoms of depression rapidly and promising findings have also been found when using nitrous oxide. However, the mechanisms behind rapid antidepressant effect are not fully discovered. It seems that rapid-acting treatments alter brain energy metabolism, enhance synaptic plasticity, and repair neuronal dysfunction connected to depression. Particularly, the activation of brain derived neurotrophic factor (BDNF) mediated tropomyosin receptor kinase B (TrkB) signaling has been connected to rapid antidepressant effect. Fasting is also known to induce BDNF production and it is thought to activate BDNF-TrkB signaling. In addition, both of these treatments alter the brain energy metabolism. The objective of this study was to find out how fasting and nitrous oxide alone and in combination affect the rapid antidepressant effect and synaptic plasticity related BDNF-TrkB signaling in mice. Another aim of the research was to determine whether the body temperature changes after these treatments as a marker of metabolic rate. The analyzed brain samples of the mice were collected 15 minutes after cessation of nitrous oxide administration. As a result, it was found that the fasting protocol used in this study did not activate the studied BDNF-TrkB signaling. However, after nitrous oxide administration, the studied signaling and markers related to synaptic plasticity were partly activated. The results from the combination of nitrous oxide and fasting were similar compared to nitrous oxide administration only. It is therefore conceivable, that the effects were caused exclusively by nitrous oxide. Furthermore, a fascinating finding related to energy metabolism was that nitrous oxide reduced the body temperature of the mice significantly 15 minutes after cessation of the gas administration. Overall, these results are promising and consistent with previous research indicating that nitrous oxide administration could be related to induced synaptic plasticity and therefore have antidepressant associated effects. Nitrous oxide could be used to understand the mechanisms behind rapid antidepressant effect and it could be a potential option to treat depression in the future. Based on these results, it seems that energy metabolism could be related to rapid antidepressant effect. It also supports the observations that all different rapid-acting treatments alter the brain energy metabolism.
  • Tikkanen, Alli (2019)
    Organic Anion Transporting Polypeptide 2B1 (OATP2B1) is an influx transporter expressed widely throughout the body in tissues such as intestine, liver, brain, placenta and skeletal muscle. Since many clinically used drugs are transported by OATP2B1, changes in the function of the transporter due to genetic polymorphism could lead to altered pharmacokinetics or -dynamics of OATP2B1 substrate drugs. The aim of this Master’s thesis was to create and optimize a cellular uptake assay to study the function of OATP2B1. Furthermore, the aim was to study the effects of six naturally occurring nonsynonymous single nucleotide variants on OATP2B1 transport function in vitro. With site-directed mutagenesis, single nucleotide changes were introduced into the gene coding for OATP2B1. OATP2B1 variants were expressed in human derived HEK293 cell line using baculovirus expression system. A cellular uptake assay with estrone-3-sulfate and a fluorescent probe 4’, 5’-dibromofluorescein (DBF) as substrates was set up and optimized. With the assay, OATP2B1-mediated uptake of variants was compared to the transport activity of OATP2B1 wild type. Amino acid changes Ser486Phe and Cys520Ser impaired OATP2B1 transport function severely. In addition, variant Thr318Ile transported DBF and estrone-3-sulfate less efficiently compared to OATP2B1 wild type, but Arg312Gln, Thr392Ile and Ser532Arg transport function was not affected. A method to study OATP2B1 function was created successfully. According to the results, single amino acid changes in OATP2B1 can impair OATP2B1 function. The results and method can be utilized to understand findings from pharmacogenetic studies in vivo, and to predict consequences of especially rare variants, which can be difficult to detect in small sample populations in clinical studies. However, further studies on the expression level and cellular localization of OATP2B1 variants are needed to fully characterize the impact of the variants studied.
  • Rossi, Vilma (2020)
    Background: Inhaled therapy is the most widely used treatment for asthma and chronic obstructive pulmonary disease (COPD). Inhaled medicinal product has several advantages, including high local drug concentration in the lungs and reduced systemic adverse effects. However, the challenge with inhaled therapy is that many asthma and COPD patients do not know how to use their inhaler properly. Suboptimal inhaler use can lead to poor clinical control. The Association of Finnish Pharmacies has developed inhalation technique assessment service (ITAS) to detect and correct patients’ inhalation technique and to give information regarding the inhaler and inhaled therapy, such as drug storage and oral care. Objective: The aim of the study is to investigate whether asthma and COPD patients’ ability to prepare the Respimat inhaler and the patients’ ability to properly inhale the drug improve after receiving ITAS. The second objective is to find out what patients and pharmacists think about the service and which customer groups benefit the most from the service. Methods: The study design is an uncontrolled pre-post intervention. 33 pharmacies participated in the study. All patients who were buying a prescribed Respimat inhaler, were offered to participate in the study. Patients’ inhalation technique was assessed before (baseline) and immediately after ITAS (follow up 1). In addition, the inhalation technique was assessed the next time the patient came to pharmacy to buy Respimat inhaler (follow-up 2). Questionnaires were used to assess patients’ and pharmacists’ perceptions of ITAS. Results: 228 baseline and follow-up ITAS were performed. The results of follow-up 2 will be published later in a separate article. 14 % of the patients performed all the steps (both inhaler preparation before first inhalation and inhalation process itself) correctly at baseline. After ITAS the number increased to 77 %. At baseline 30 % of the patients had an optimal inhalation technique (all inhalation steps correct) and after ITAS the number increased to 85 %. 70 % of the patients had an acceptable technique (all critical steps correct) before and 93 % after ITAS. Both patients and pharmacists felt that the service was beneficial to the patients when thinking the proper inhaler preparation and proper inhalation technique. Overall patients’ and pharmacists’ satisfaction were high towards ITAS. Our study indicates that patients benefit from ITAS regardless of patient’s age or how long the patient have been using the Respimat inhaler. Conclusions: A pharmacist-led inhalation technique assessment service significantly improves asthma and COPD patients’ inhalation technique with Respimat inhaler. ITAS should be performed regularly as part of the delivery of the inhaled drug to the patient. Further research is needed on the effectiveness of ITAS with other inhalers.
  • Vieraankivi, Marika (2021)
    The ABCG2-protein is an ATP-dependent half transporter. It is found on apical membranes in intestine, liver, kidney, blood-brain barrier and placenta where it regulates absorption, distribution and elimination of many drugs, but also natural compounds and endogenous metabolites. Natural variation found on the ABCG2-gene can alter protein expression and transport activity. The altered function has been linked to pharmacokinetic changes and developing of diseases like gout. Studying natural ABCG2-variants and their effect gathers knowledge not only on their effect on pharmacokinetics but also on the ABCG2- transporters’ mechanism of function. The aim of this study was to combine an activating (I456V or H457R) and an inactivating (Q141K, F431L or T542A) non-synonymous single nucleotide variant in the same gene to study their combined effect on the ABCG2-transporter expression and active transport. Mutations were incorporated into the ABCG2- gene by site directed mutagenesis and the protein was expressed on HEK293-cells. The transport activity for Lucifer-Yellow and estrone sulfate was measured using HEK293-ABCG2-vesicles produced from cell membranes. The protein expression was measured with Western blot and mass spectrometry proteomics. Based on this study, different mutations together can alter each other’s effects, but the combined result is not always equal to the sum of variations. T542A-mutation did not show significant increase on the protein expression on any of the T542A-combinations, even though it has had such an effect in earlier studies. I456V, earlier expressed like wild type ABCG2, seemed to increase protein expression in all combinations. Q141K, F431L and T542A -mutations had lowering not expression dependent effect on the transport activity. F431L-mutation being so dominant that either of the two activating mutations could not restore the active transport in combinations. As seen before, H457R-variant seemed to cause a significant substrate specific activating effect on transport activity also in this study when combined with other mutations. However, H457R had a strong lowering effect on the protein expression and two of the combinations did not produce enough protein for active transport. As seen in this study, the ABCG2-doublemutations can cause altered ABCG2-function and lead to pharmacokinetic changes. These types of in vitro studies are important in studying these less common genetic variants which in lack of study subjects can be hard to study on clinical trials.