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Background: Antibiotics have been an important factor in the dramatic decrease of infectious disease mortality in the 20th century. Bacteria are, however, very quick to respond to the changes in their environment because of their short life cycle. Thus, the development of bacterial antibiotic resistance is a natural consequence of the enormous worldwide antibiotic use. The current situation is that the antibiotic resistance develops faster than novel antibiotics are found and developed. The three main resistance strategies of Gram-negative bacteria are: modification of the antibiotic target, enzymatic inactivation of the antibiotic and reduce of the intracellular antibiotic concentration by changing the function of the outer membrane. To decrease the intracellular antibiotic concentration bacteria use efflux pumps. RND efflux pumps are the most important family of efflux pumps regarding antibiotic resistance. They typically function as a part of a tripartite structure which allows the efflux of antibiotics to the extracellular space. Multiple inhibitors have been developed against RND efflux pumps but none has reached the clinical stage of drug development. Objectives: Development and testing of a 384-well plate method for screening efflux pump inhibitors for E. coli (BAA1161) efflux pumps. Methods: Verifying that the absorbance measurement is a sensitive enough method for measuring the bacterial (BAA1161) growth in 384-well plate format. The antibiotic chosen to be used in the screening method was piperacillin and the positive control efflux pump inhibitor was mefloquine. Determining the minimum growth inhibiting concentrations (MICs) of piperacillin and mefloquine in 96- and 384-well plate formats. Verification of the synergistic growth inhibitory effect of piperacillin and mefloquine with the checkerboard method in 96- and 384-well plate formats. Determining the positional effect in the 384-well plate. Determining the highest DMSO concentration without effect on the growth of BAA1161. Screening of 126 natural compounds in 384-well plates to test the developed method. Screening was done in quadruplicates based on the growth inhibitory effect of the natural compounds when combined with piperacillin. Dose-response assay was conducted in combination with and without piperacillin with the compounds that showed growth inhibiting effect during screening. Results and discussion: Absorbance measurement was sensitive enough method for measuring the BAA1161 growth in the 384-well plate. MIC value of mefloquine was 32 μg/ml in both plate formats. Piperacillin’s MIC was 1024 μg/ml in the 96-well plate, but on the 384-well plate there was variation in the MIC. Piperacillin and mefloquine showed synergistic effect on BAA1161 growth inhibition in the checkerboard assays. Positional effect could not be determined, because of the variation in the BAA1161 growth inhibition effect of piperacillin. This randomly occurring phenomenon were piperacillin inhibited BAA1161 growth completely or almost completely with sub-MIC concentration was encountered in all the subsequent experiments in the 384-well plate format. One possible reason for this phenomenon, occuring in the 384-well plate format, could be piperacillin heteroresistance of BAA1161 strain. In the test screen, four compounds, which all included gallic acid ester, showed promising activity. These compounds were: epigallocatechin gallate, hamamelitannin, isopropyl gallate and octyl gallate. In the dose-response assay, hamamelitannin’s and octyl gallate’s effect was synergistic with piperacillin. Conclusions: The developed method can be used to screen novel efflux pump inhibitors. However, to increase the reliability of the method, further optimization is required to eliminate the variability in the effect of piperacillin. When plate format of a method is changed, factors which could affect the functionality of the method in the new format should be carefully assessed. Based on the test screed, gallic acid esters are interesting compounds which combined effects with antibiotics should be studied in the future experiments.

Population is aging. Within aging the morbidity and the use of medicines increase. Polypharmacy and the physiologic changes related to the ageing expose to medication-related problems. This has to be taken into consideration when planning the care of the elderly. Multiprofessional cooperation is seen as a solution to optimize the medicines' use among the aged people. Finnish Medicines Agency (Fimea) has started a network with local multiprofessional health care teams. The aim of the network is to make a national guideline for multiprofessional cooperation and optimizing the medicines' use among the aged people. The objective of the study was to clarify multiprofessional working models to optimize the medicines' use that had been carried out or planned by the teams belonging to the network. The models can work as examples when creating standardized practices to multiprofessional cooperation in Finland. Factors that promote or prevent multiprofessional cooperation and the problems of optimizing the medicines' use were clarified as were the possible solutions to solve them. Factors to strengthen cooperation and its effects were clarified on the basis of experience of the multiprofessional teams. As a material of the study were the interviews (n=15) of health care professionals (n=55) invited to Fimea's multiprofessional network. Fimea had collected the material that consists of group discussions (n=10), pair interviews (n=3) and individual interviews (n=2). The interviews that had been recorded were transcribed and analyzed by using a combine of inductive and deductive content analysis. A theoretical framework in the study was multiprofessional teamwork and networking. According to the interviews, multiprofessional cooperation in optimizing the medicines' use among the aged has been carried out in Finland in both public and private health care. The interviewees think that the most important way to optimize the medicines' use is clear division of tasks and responsibilities. Adding more pharmacists to all over the public health services and fostering the role of the community pharmacies as a part of the health care are seen as solutions. Multiprofessional meetings and education can break barriers between different professionals. The most common problems are the challenges related to economic limitations and to the busy work. There are problems in IT systems and information transfer. At the individual level, the most common problems seem to be in communication and the attitudes. The interviewees' experience is that successful multiprofessional cooperation increases medication safety and improves patients' state. The work of all the professions is faciliatated and burden of the public health service decreases. Lighter medication reviews could be used to find the patients who benefit from the comprehensive medication review. Information transfer and the currency of patients' medication should be secured with functioning IT systems. The results of the study can be utilised when developing multiprofessional practices to optimize the medicines' use. More study is needed to show the profitability of medical reviews, dose dispensing and other services.

Heart failure is a global health issue that can result from various factors, one of which is myocardial infarction. The adult human heart has limited regenerative capacity to cover the loss of cardiomyocytes after myocardial infarction with new cardiomyocytes. The main responses to the loss of cardiomyocytes are fibrotic scar formation and the hypertrophy of remaining cardiomyocytes. Prolonged hypertrophy eventually leads to heart failure. Current treatments for heart failure only relieve the symptoms. Inducing cardiac regeneration could be one possible way to prevent and treat heart failure. Thus, to develop medical treatments that enhance the regenerative capacity, a comprehensive understanding of precise cellular mechanisms behind heart regeneration is crucial. The objective of this study was to establish a high-content analysis method for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) utilizing the Cell Painting assay to identify and categorize morphological alterations induced by various compounds in hiPSC-CMs. To evaluate the morphological impacts, dozens or even hundreds of cell features were measured at the same time. hiPSC-CMs were exposed to two hypertrophy inducers, endothelin-1 and angiotensin II, and to doxorubicin, which is known to be a cardiotoxic compound. In addition, the effects of a GATA4- targeting compound, C-2021, on hiPSC-CMs were examined. C-2021, was expected to have antihypertrophic effect on the cells. Previously used methods, proBNP staining and qPCR, were used to validate the novel method. According to proBNP staining and qPCR, endothelin-1 induced cardiomyocyte hypertrophy greater than angiotensin II. Compound C-2021 did not show statistically significant antihypertrophic properties after hypertrophic stimuli, but some tendency the alleviate the hypertrophy was noticed. Moreover, by utilizing different data processing programs a novel analysis method was developed. With this method, the different treatment groups were clustered based on the morphological alterations caused by compounds exposures. The hiPSC-CMs exposed to endothelin-1, angiotensin II or doxorubicin showed a different morphological profile compared to the control group hiPSC-CMs. Compound C-2021 was also observed to affect cell morphology. However, the data analysis still needs improvements in order to detect which cell features these compounds affect.

Polypharmacy in older adults is common and there are many things to be corrected in their medication. Medication reviews can be used to identify and address these problems using interprofessional collaboration. Renal insufficiency is common in older adults and its consideration contributes to medication safety. The aim of this study was to investigate the prevalence of renal insufficiency in Lohja home care clients over the age of 65, for whom medication review or comprehensive medication review had been done. The purpose was to investigate from medication review reports how many observations pharmacists made about the drugs that should be avoided or dose reduced. In addition, it was investigated whether the medications of the subjects could be changed during the intervention and whether the plasma creatinine values correlated with the GFR values. The material consisted of the medication review reports of 60 home care clients in the intervention study launched in Lohja year 2015. Medication reviews were done in 2016–2017. Half (n = 30/60) of the subjects had at least one drug for which pharmacist proposed a medication change due to a reduced GFR. Proposals for changes (n=60) were presented 1–7 per subject. The majority of the proposed changes, (52 %, n= 31/60), concerned dose reduction, and 22 % (n=13/60) discontinuation. Other proposals totaled 26 % (n= 16/60). 42 % (n=13/31) of the dose reduction proposals were implemented. Almost all of the drug discontinuation 92% (n=12/13) proposals were implemented. In total, 47 % (n = 28/60) of the proposals were implemented. Nervous system drugs formed the largest group (30 %, n = 18) for which a change was proposed. The second highest number of proposals was for drugs for cardiovascular system (27 %, n=16) and the alimentary tract and metabolism (27 %, n=16). Based on GFR, 93 % (n = 56) of subjects had declined renal function (GFR <90 ml/min). Mild kidney damage (GFR=89–60 ml/min) was the most common; 73 % of men (n=11) and 47 % of women (n=21). In 65 % (n=39) of subjects, plasma creatinine was within or below reference range. Plasma creatinine was above reference value in 25 % (n=15) of subjects. The study confirms that plasma creatinine is not suitable measure of renal insufficiency in the elderly.

Left ventricular noncompaction cardiomyopathy (LVNC) is a unique form of cardiomyopathy, which is believed to arise from arrest in the compaction process during cardiac development. Dysfunctions in cell cycle regulation and increased or decreased proliferation of cardiomyocytes during cardiac development are likely to contribute to the development of LVNC. SCN5A gene encoding the α-subunit of cardiac voltage gated sodium channel Nav1.5 has associated with LVNC- phenotype in a Finnish family. The direct correlation of SCN5A gene mutation and LVNC has not been studied before. There is strong evidence that Nav1.5 channel has an essential role in cardiac development and cardiomyocyte proliferation, therefore perturbed function of the channel might also contribute to the development of LVNC. We used patient-specific human induced pluripotent stem cell -derived cardiomyocytes (hiPSC-CMs), reprogrammed from fibroblasts obtained from LVNC patient carrying SCN5A to study the phenotype of the cells. We utilized immunofluorescent staining in combination with high content analysis (HCA) to investigate the proliferation and Nav1.5 cellular localization. Proliferation potential was assessed at multiple timepoints from three to six weeks. We also investigated the stress response of patient-specific hiPSC-CMs by exposing the cells to mechanical stretch, a hypertrophy inducer, followed by quantitative reverse transcription PCR to study changes in stress biomarker levels. According to our results, the patient-specific hiPSC-CMs have prolonged proliferation compared to control cells as the proliferation peaks towards the last timepoint, whereas in control cells it decreases. Differences were also observed in the hypertrophic gene expression after 24-hour mechanical stretching. An increase in NPPB expression levels caused by stretching was threefold in patient-specific cells to control cells. These results implicate that SCN5A gene has as an important role on cardiomyocyte proliferation. Mutations in SCN5A could correspond to increased proliferation in trabeculations during cardiac development, which might be preventing the compaction process and lead to the development of LVNC. Our results emphasizes that SCN5A has an important role in cardiomyocyte physiology unrelated solely to electrical activity.

Nanofibrillar cellulose (NFC) can form hydrogels with high water content (> 98 %). It has been studied for drug release, and it has been used as a cell culture matrix, due to its similar structure to extracellular matrix (ECM). In addition it has been found that they has no cytotoxicity. Iontophoresis is the application of an electric current over a defined area for the purpose of enhancing permeation across a membrane for ionized drug species. The aim in the experimental work in this Master's thesis is twofold. First, to find out the suitable drug loading concentrations into NFC hydrogels, which can provide a good release profile, a release study with two model drugs, propranolol and ketoprofen, loaded into three types of NFC hydrogels at three different concentrations, was carried out for this purpose. Second, to see if NFC hydrogels are applicable as a drug reservoir in iontophoretic transdermal drug delivery applications, an iontophoresis study was carried out using porcine ear skin model in vitro for human skin with propranolol loaded into NFC hydrogel of type A. In addition, Stella models were used as an aid to find suitable ways to predict the release and permeation behaviour of models drugs in the abovementioned context. The UPLC results from the release study show for both model drugs, the wt. % released had linear correlation with squareroot of time. At 6 hours, more than 70 wt. % propranolol was released from hydrogel reservoir. For ketoprofen, the release varied between 30 - 87 wt. %, where higher initial loading concentrations produced a decrease in the wt. % released from hydrogel. The iontophoresis study did not show a significant difference between the tested current densities (0.50 mA/cm2; 0.25 mA/cm2) produced on the wt. % of drug released. Simulation models could be run with the mathematical equations for diffusion controlled drug release. In conclusion, the NFC hydrogels show potential as drug reservoir for drug release. Additional experimental data using other types of drug reservoirs should be obtained for a better understanding of the suitability of NFC hydrogels as a drug reservoir in iontophoretic transdermal drug delivery.

Steroid hormones are involved in many physiological functions such as stress response and the maintenance of salt-water balance and pregnancy. Concentrations of steroids in the body fluids are generally very low (below ng/ml). Steroid hormones are metabolically associated and changes in mutual concentration levels of different steroids may signify a disease. Methods that allow the measurement of various steroids simultaneously are of great importance in investigating the role of steroid metabolism for example in formation of cancer. The aim of this work was to develop a sensitive and selective method for simultaneous quantification of 16 steroids in plasma. Nano liguid chromatography-microchip electrospray ionization-tandem mass spectrometry (nanoLC-µESI-MS/MS) was used in order to achieve good sensitivity. C18 enrichment column and separation column, and an electrospray tip were integrated onto the chip that was used in this work. Mass spectrometric parameters were optimized by using a MS calibration and diagnostic chip. It was noticed that the structure of steroids plays an important role on how the compound behave in electrospray ionization. Steroids with 4,5-ene-3-one-structure had much lower limits of detection than steroids without conjugated double bonds (0,075-0,5 ng/ml and 5-25 ng/ml respectively). The chosen sample pretreatment method to extract the steroids from plasma did not work properly, because it was able to extract only a third of the compound's real concentration. Analysis of some compounds was also difficult because of the background noise coming from plasma. The method development was therefore decided to continue with eight steroids that were well detectable and had 4,5-ene-3-one-structure. The limits of detection were 0,075-0,5 ng/ml in biological matrix for these compounds. Eight knock out and seven wild type mouse plasma samples were analyzed using the validated method. The method was able to quantify aldosterone, corticosterone and androstenedione. Developed method did not meet all the aims of this work. Derivatizated compounds, different equipment or totally new method should be used in order to accomplish the aims.

In Finland first pharmacists started to work on wards in 1980s and 1990s. Ward pharmacy increased mainly in consequence of the lack of nurses. Common tasks were taking care of drug logistics (stock control), dispensing drugs to patient specific doses, preparing and diluting intravenous drugs and providing drug information to ward personnel. During the 2000s, ward pharmacy services have been increasing a lot. New tasks are, e.g., reviewing medications and prescriptions, medication counselling, and taking part in medical rounds. However, the tasks are still rather logistics compared to the United States and the United Kingdom where a pharmacist has an established role in a multiprofessional team. Internationally it has been proved that it is possible to achieve decreased and enhanced quality of care and patient safety with hospital clinical pharmacy services. The aim of this study was to explore the extent and benefits of ward pharmacy services in Finland. An online survey was conducted by sending the invitations to the chiefs of hospital pharmacies (n = 24) and medical dispensaries (n = 94) by using the e-mail register of the University of Helsinki and Satefa (Finnish Association of hospital and health centre pharmacists). Before compiling the questionnaire six theme interviews were conducted to set up the questionnaire. The survey respondents were asked to submit information about development projects and research reports if they had explored the benefits of ward pharmacy services. The response rate was 60 % (n/N = 72/118). A half of the respondents (n = 36) reported having ward pharmacy services in their units. Benefits were explored in 12 units and nine project reports were received. Altogether 157 pharmacists were working in 242 wards at the time of the survey in spring 2011. Most common tasks were providing drug information to ward personnel, drug logistics and dispensing drugs to patient specific doses. Patient oriented tasks were increased, including prescription and medication reviews, taking part on medical rounds and medication counselling gave patient information were reported. The most reported benefits on ward pharmacy services were increased multiprofessional collaboration, saved working time of nurses and physicians, decreased drug costs and decreased number of medication errors and/or enhanced reporting habits and developed functions on wards. Respondents also believed that ward pharmacy services can have positive impact on length of stay, readmission and hospitalisations and mortality during hospitalization, but these benefits were not demonstrated by studies. In the future it would be important to develop the Finnish ward pharmacy services by following the international example and the principles of pharmaceutical care. The help of automation technology and pharmacy technicians should be exploited more in drug logistics. The economical and patient related outcomes of new clinical and patient oriented services should be proved in Finland and the results of the researches and projects made in hospital and health centres should be published more.

Lääkehoitoa toteutetaan erilaisissa toimintaympäristöissä sekä sosiaali- ja terveydenhuollossa, että sen ulkopuolella. Lääkehoitoa toteuttavat pääsääntöisesti sosiaali- ja terveydenhuollon ammattihenkilöt, mutta ympäristön mukaan vaihdellen myös lääkehoidon koulutusta vähän tai ei lainkaan saaneet. Lääkehoidon osaamisen varmistaminen on aina työnantajan vastuulla. Keski-Uudenmaan hyvinvointialueella on ruuhkautuneiden näyttöjen myötä tunnistettu tarvetta keskitetyille näytöille. Tehty pilottitutkimus tuo arvokasta tutkimustietoa näyttötyöpajasta osana lääkehoidon käytännön osaamisen varmistamisen prosessia, sillä aikaisempia tutkimuksia aiheesta ei ole. Tutkimuksen keskeisenä tavoitteena oli tutkia näyttötyöpajan toimivuutta lääkehoidon käytännön osaamisen varmistamisessa, sekä mitä resursseja näyttötyöpajan järjestäminen organisaatiolta vaatii ja mikä olisi optimaalinen näyttötyöpajan osallistujamäärä. Pilottitutkimus toteutettiin järjestämällä näyttötyöpaja, jossa Keusoten yksiköissä työskentelevät nimikesuojatut terveydenhuollon ammattihenkilöt (n=15) osoittivat lääkehoidon käytännön osaamistaan. Näyttötyöpajassa lääkkeiden jakamisen näyttöjä ottivat vastaan farmaseutit (n=3) ja lääkkeiden antamisen näyttöjä sairaanhoitajat (n=2). Aineistona tutkimuksessa toimi näyttötyöpajassa näyttöjä antavien ja vastaanottavien antama palaute, joka kerättiin puolistrukturoidulla palautelomakkeella paikan päällä. Palautekysely lähetettiin myös näyttöjä antaneiden esihenkilöille ja vastaaville sairaanhoitajille sähköisellä Microsoft Forms- lomakkeella. Aineistoa analysoitiin tilastollisesti kuvaavalla tavalla sekä induktiivisella sisällönanalyysillä hyödyntäen Microsoft Excel -ohjelmaa. Näyttötyöpajaan osallistuneet kokivat näyttötyöpajan kehittävän lääkehoidon osaamista. Näyttötyöpajan koettiin nopeuttavan ja helpottavan osaamisen varmistamista sekä. Esihenkilöt ja vastaavat sairaanhoitajat kuvailivat näyttötyöpajan myös vapauttavan näyttöjen järjestämiseen kuluvia resursseja muuhun hoitotyöhön. Sekä osallistujat että näytön vastaanottajat kokivat näyttötyöpajan vertautuvan huonosti aitoon työtilanteeseen. Keskeisiksi näyttötyöpajan kehityskohteiksi nousivat parempi organisointi, tiedonkulun vahvistaminen ja näyttöjen tasalaatuistaminen. Mukauttamalla näyttötyöpajaa enemmän aidon työtilanteen kaltaiseksi voisi näyttötyöpaja tukea osaamisen kehittymistä paremmin, sekä parantaa työyksikön lääkitysturvallisuutta. Näyttötyöpajaa tulisi tutkia ja kehittää lisää, jotta siitä saataisiin sujuva osa osaamisen varmistamisen prosessia. Näyttötyöpaja koettiin pääosin hyödylliseksi ja se koettiin tarpeelliseksi interventioksi helpottamaan ruuhkautunutta lääkehoidon osaamisen varmistamista.

Parkinson's disease is a chronic progressive neurodegenerative disorder, characterized by muscle rigidity, hypokinesia, tremors and bradykinesia. The cause of symptoms in Parkinson's disease is loss of dopaminergic nerve cells in the substantia nigra, which attenuates the nigrostriatal dopaminergic signaltransmission. Oxidative stress, mitochondrial dysfunction, protein misfolding and aggregation, inflammation, excitotoxicity, apoptosis and other routes for cell death, and loss of neurotrophic factors have shown to be mechanisms in the pathogenesis of Parkinson's disease. Microglia might have a double role in the pathogenesis of Parkinson's disease. Microglia stimulated by Į- synuclein does not only produce toxic factors such as certain cytokines and reactive oxygen and nitrogen species, which contribute to the neuronal cell death but also produce anti-inflammatory cytokines and neurotrophic factors, which can be neuroprotective. Deeper knowledge of the mechanisms underlying Parkinson's disease is needed for developing restorative medicines. Three different neurotrophic factor families are known to be important in the research of Parkinson's disease. The GDNF-family consists of glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN). The neurotrophin-family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophins NT3 and NT4/5. The most recently discovered family is the MANF-family, which consists of mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF). In Parkinson's disease the neurotrophic factors could stop, slow or ideally even reverse the neurodegeneration in the dopaminergic system and decrease the functional decline of the neurons. Research has already shown that GDNF has both a neurorestorative and neuroprotective effect in animal models of Parkinson's disease. Clinical trials have however shown controversial results. The challenge with neurotrophic factors can be the administration to the brain through the blood-brain-barrier, sideeffects because of receptor binding in other organs or sites of the body and low diffusionrate. Research of both MANF and CDNF has shown promising neurorestorative and -protective results in vivo. Local diffusion of MANF has been shown to be better than of GDNF. In this Master's thesis research was done on whether MANF and CDNF have a neurorestorative effect on the dopaminergic nerve cells in mixed primary culture in vitro after 6-OHDA exposure. The aim of the study was to receive information about whether MANF and CDNF are as effective as GDNF at repairing celldamages caused by 6-OHDA in vitro in this experimental model. GDNF was used as a posivite control in this study. The results from this study suggest that MANF might have a neurorestorative effect, but this effect is much smaller than with the neurotrophic factor GDNF. The results show no neurorestorative effect with CDNF. Neither the dopamine uptake nor the tyrosine hydroxylase staining showed statistical significance.