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  • Hannila, Teija (2013)
    Gene therapy is an experimental technique that involves inserting therapeutic genes into the target cells to treat diseases. Gene transfer can be performed by ex vivo or in vivo method. Ex vivo method means transferring the therapeutic gene in laboratory to the cells that are removed from the patient, after which the cells are returned to the patient. In the in vivo method the gene transfer is performed directly to the target tissue inside the patient's body. Gene therapy clinical trials have been carried out to treat many diseases. The majority of the clinical trials have so far been cancer trials. Nevertheless, the most promising results have been established in treating diseases that arise from mutations in a single gene, i.e. monogenic diseases. Monogenic diseases include e.g. hemophilia and heritable immunodeficiencies. The biggest challenges in the clinical trials so far have been the limited gene transfer efficiency of the currently used gene vectors, the short duration of the transgene expression and the side-effects in viral-mediated gene transfer. Nonviral gene transfer agents have so far been less efficient in vivo than the viral vectors. This is partly due to the interaction between serum components and nonviral vectors. The main purpose of this study was to investigate the effect of serum to the gene transfer efficiency of a nonviral vector polyethyleneimine PEI22K and the combination of PEI22K and cationic liposome Dosper in vitro in the SMC-cells. The potential synergistic increase in the transfection efficiency of PEI22K/Dosper combination was also studied. The secondary goal in this study was to develop an in vitro model which could be used to predict the gene transfer efficiency of gene vectors in vivo. The combination of PEI22K and Dosper resulted in a synergistic increase in the transfection efficiency in serum-free transfection. In the presence of serum the efficiency of PEI22K was higher than the efficiency of PEI22K/Dosper combination. 1-10% serum concentrations did not significantly affect PEI22K`s transfection efficiency, but dramatically decreased the efficiency of PEI22K/Dosper combination. The results suggest that PEI22K is more suitable than PEI22K/Dosper combination for in vivo gene transfer.
  • Sjöberg, Madeleine (2018)
    Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.
  • Sjöberg, Madeleine (2018)
    Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.
  • Pietarinen, Paavo (2012)
    Most xenobiotics are biotransformed by phase I enzymes to a more hydrophilic form in order to get excreted out from the body. In most cases xenobiotics are in lipophilic form when entering body. The most important group in phase I enzymes is cytochrome P450 (CYP) superfamily. Of CYP enzymes probably the most studied is CYP2D6, which is responsible for metabolism of 20-25% of drugs currently on market. Many CYP2D6 substrates belong to therapeutically important drug groups, such as antiarrhytmics, antidepressants, beta-blockers, or neuroleptics. CYP2D6 gene, which encodes the enzyme, exhibits large interindividual variability, which has an effect on the metabolic activity of the enzyme. The frequencies of these genetic variances differ globally on wide scale between and inside populations. Through genotyping it is possible to predict the CYP2D6 metabolic rate, which can be divided into four classes: ultra-rapid metabolizers (UM), extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). The purpose of our study was to examine the frequencies of CYP2D6 genotypes in Finnish population in detail and compare the results to previous studies. Our study population consisted of 857 healthy volunteers whose DNA was extracted. From DNA sample we genotyped 10 different CYP2D6 genetic variants and the copy number of the gene using Applied Biosystems TaqMan genotyping and copy number assays. This study was the largest CYP2D6 genotype frequency study in Finnish population so far. The results supported the findings of a similar study in a Finnish population of smaller scale. Large majority of study subjects were EMs (87.3%) and the second largest group was Ums (7.2%). IMs and PMs were in clear minority (3.0% and 2.5%, respectively). The expected frequencies for UMs (1-2%) are much lower and for PMs higher (~8%) in other North European populations than in Finns. Accordingly, CYP2D6 genetic profile of Finnish population differs from its neighbours, which may be important for the dose requirements, efficacy, and safety for drugs metabolized by CYP2D6.
  • Seikola, Anniina (2011)
    The Ministry of Social Affairs and Health published a report on development needs of elder care and geriatric pharmacotherapy in 2006. The major concern in this report was related to several challenges in pharmacotherapy of the aged, such as deficiencies in medical knowledge of nurses working with elderly people. One way to improve the medication expertise of those various parties involved in caring elderly people is continuing education (CE). The aim of this study was to explore pharmacotherapy-related training needs of health care professionals involved in the home care services for the elderly in the Social and Health Care Cooperation Region of Lohja, Siuntio, Inkoo and Karjalohja (the LOST Region). This study was started by conducting a survey among nurses working in home care services for the elderly in the LOST Region in 2009 (response rate 47%). To deepen understanding of the key findings of the survey, focus group discussions (FGDs) and face-to-face interviews were conducted among nurses, nursing aids, their managers and physicians (1 FGD among nurses, n=6; 1 FGD among their managers, n=6; and face-to-face interviews with 4 physicians). The survey data were analyzed separately for nurses (n=9), practical nurses (n=53) and home aids (n=9), but results were the same in every group. Of the theoretical training needs, topics related to pharmacokinetics and special characteristics of using medicines in the elderly, effects, adverse effects and interactions of medicines, were most important. In addition, the theoretical training needs covered professional ethics issues, such as accuracy and carefulness of nursing practice. The main training needs related to collaborative practice in pharmacotherapy concerned monitoring medicine user's condition and medication, and dosing medicines (right medicine, dose, strength, dosage) in the right time, and administration routes of medicines. Focus group discussions and face-to-face interviews of the physicians provided a deeper understanding of the results of the survey. One of the main findings of this qualitative part of the study was challenges in cooperation in home care services in the LOST Region. Implementation and monitoring geriatric pharmacotherapy can be improved by improving multiprofessional cooperation and training for nurses and physicians working in home care services. The most important diseases and disorders for which the nurses would like to have shared operational guidelines were diabetes, cardiovascular diseases, pain, memory and psychiatric disorders. Training needs also covered special characteristics of pharmacotherapy for the elderly, and formulations and administration routes of medicines. Finally, a synthesis was made of the results of the survey, focus group discussions and face-to-face interviews. On the basis of the synthesis, a proposal for a multiprofessional training was developed for the LOST Region. The training plan includes topics related to geriatric pharmacotherapy and improving collaborative practices and communication as identified by those involved in different stages of the study.
  • Kallionpää, Roope (2014)
    Estrogens are female sex hormones that have genotoxic and proliferation-enhancing effects in cells. Life-time exposure to estrogens is linked to the risk of several cancers. Estrone is only a weak agonist of estrogen receptor but it serves as a precursor for biosynthesis of 17β-estradiol, 16α-hydroxyestrone and catechol estrogens. While 16α-hydroxyestrone has relatively weak affinity for estrogen receptor, it has prolonged effect due to covalent binding to the receptor. UDP-glucuronosyltransferases (UGTs) are phase II metabolic enzymes that conjugate estrogens with glucuronic acid to render them more watersoluble. Polymorphisms in UGT genes have been linked to excretion of steroids and risk of some cancers. Generally, subfamily UGT1A enzymes conjugate the 3-hydroxyls of estrogens, while the activity of subfamily UGT2B is directed towards 16- and 17-hydroxyls. Previous results on estrone glucuronidation are incomplete and conflicting, while glucuronidation of 16α-hydroxyestrone has not been systematically studied. The aim of this study was to identify UGTs active in the glucuronidation of estrone and 16α-hydroxyestrone and to further examine the glucuronidation kinetics of the active UGTs. Also the effects of bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) and mutations of UGT1A10F90 and UGT1A10F93 on glucuronidation activity were examined. Activity assays were conducted using recombinant enzymes as well as human liver and intestinal microsomes. Resulting glucuronides were analyzed using high performance liquid chromatography and quantified based on their UV absorbance. UGT1A3, UGT1A10 and UGT2A1 showed the highest activity toward estrone glucuronidation, while UGT1A10, UGT2A1 and UGT2B7 were the most efficient UGTs conjugating 16α-hydroxyestrone. UGT1A10 had the highest Vmax in the glucuronidation of both substrates, although it conjugated estrone at a higher rate than 16α-hydroxyestrone. UGT1A10F93 was shown to have a role in the different glucuronidation activities of UGT1A10 toward estrone and 16α-hydroxyestrone. Affinity of 16α-hydroxyestrone was highest for UGT2B7, while UGT2B17 conjugated 16α-hydroxyestrone relatively slowly. The results confirm earlier observations of the preference of UGT2B7 for α-configured hydroxyls while UGT2B17 favors β-configuration. UGT2A1 showed no strict regioselectivity but had a relatively weak affinity for both substrates. DMSO was found to decrease UGT activity. However, its presence is necessary to solubilize lipophilic substrates. DMSO concentration has to be kept constant to produce comparable data for, for example, kinetic studies. BSA was found to alter especially the kinetics of UGT2A1. BSA also seemed to have solubility-enhancing effect.
  • Mattila, Susanna (2012)
    The aim of the stydy was to evaluate how different chemical derivatization methods are suitable for characterization of regional isomers of different glucuronide conjugates. Glucuronidation is one of the phase II metabolic reactions where more water soluble and often inactive substances are produced. Different functional groups may be subjected to glucuronidation. It is important to determine the exact position of glucuronidation, as the isomers may possess different toxicological or pharmacological properties. For example morphine-6-glucuronide is pharmacologically more active than morphine itself. The glucuronide conjugates are commonly detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and/or nuclear magnetic resonance (NMR). MS/MSspectra of native molecule and glucuronidated molecule are usually similar because of an initial loss of 176 Da, i.e. monodehydrated glucuronic acid. This fact often makes it impossible to determine the site of glucuronidation. Samples of NMR-analysis requires larger amounts of sample materials than MS-analysis. Many of those derivatization reagents tested in this study were not reacting as they were supposed to react according to literature. O-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) were forming derivatives as expected and those reagents are very suitable for glucuronide conjugates studies. At the end of the studies the site of the glucuronidation of dopamine- and serotonineglucuronides were evaluated by derivatization with OPA and FMOC. Derivatization with OPA and FMOC successfully gave information about the region of the glucuronide acid in dopamine- and serotoninemolecules. The assumptions supposed to be correct according to NMR-studies presented in literature.
  • Häggman, Verner (2019)
    If pharmaceutical quality system fails it causes a hazard to the patient’s health, but also to the manufacturer’s economy. For this reason, the manufacturer’s must make sure their products comply with the quality requirements placed by authorities. To ensure the compliance, the authorities perform inspections at the manufacturing sites. If the site does not comply with the quality requirements, the authority will take necessary measures. The goal of this study was to find what type of quality issues FDA and the authorities within EU have observed while inspecting manufacturing facilities, which of these issues are most common, in which countries the sites companies with issues have been located. The results were assessed from European pharmaceutical company’s point of view. The data for the study was collected from Eudra GMDP database and from FDA Warning letters sent by FDA headquarters from years 2015-2017. Qualitative analysis of content was chosen as the method of analysis. The collected data was classified into main classes and subclasses based on reoccurring topics. The classes were transferred in tables to compare how which of the classes were most common. Most often the facilities with quality issues were located in China and India. The authorities also perform a lot of quality inspections in these countries, but that alone doesn’t explain the large number of quality issues in these countries. The number of sites with quality issues per inspection was also high. Both the authorities of EU countries and FDA had mainly observed similar issues. Often quality issues were related to data integrity. Other common themes were quality management system, cleaning of equipment and facilities and analytical methods. There were also some differences in the observed issues. E.g. FDA had rarely observed issues related to personnel while EU authorities had observed such issues frequently. Quality issues which had led to measures by authorities were often related to larger problems with the quality management or to very basic quality actions. If company doesn’t have well-functioning quality organization, the quality system is often inadequate also in other ways. By comparing their own activities with the issues observed at other companies, it is easier for a company to improve their quality and avoid major quality issuer before they occur.
  • Hahl, Eveliina (2023)
    Introduction: European legislation on orphan medicinal products, Regulation (EC) No. 141/2000 of the European Parliament and of the Council, entered into force in April 2000. Although the prevalence of rare diseases is low according to legislation (less than 5/10,000), 18–30 million people in the European Union (EU) are affected by rare diseases. The introduction of orphan medicine legislation has increased the number of orphan medicines developed but the fairness of the legislation has also raised concern and criticism. The literature review of this Master ́s thesis provides an overview of rare diseases, orphan medicines and EU orphan medicine legislation. The aim of the empirical study was to investigate the evolution of orphan medicine selection during European legislation on orphan medicinal products in 2000–2022. In more detail, aims were to describe the evolution of orphan medicine selection, the approved indications for orphan medicines and the number of orphan medicines approved for children. Methods: The research material was orphan medicines that received a marketing authorisation during the EU orphan drug legislation. This material was collected from the European Commission's Community Register of orphan medicinal products and the European Commission's Community Register of not active orphan medicinal products. Qualitative document analysis was used as the research method, where information on orphan medicines were quantified. Results and conclusions: In the 10-year review of orphan medicine development, the number of new orphan medicine products approved for the market doubled, being 63 products between 2001 and 2010 and 127 products between 2011 and 2020. In the latter 10-year period of the review, the focus of approved indications for orphan medicines shifted slightly from orphan medicines developed for the treatment of cancers (36%) to orphan medicines developed for the treatment of inborn errors of metabolism or immune disorders (43%). In the 10-year reviews, the relative share of orphan medicines approved for children decreased from 55 percent in 2001– 2010 to 40 percent in 2011–2020. Based on the results of the study, the fairness and targeting of the benefits of the orphan medicine legislation should be further investigated. Orphan medicine legislation should encourage the development of medicines for rare diseases for which there is no treatment at all, and for the population most affected, in other words children.
  • Salminen, Veera (2021)
    Continuous monitoring of the safety profile of medicinal products is essential also after marketing authorisation approval to ensure the patient safety. Spontaneous reporting of adverse drug reactions is one of the most important methods to collect post-approval safety data of medicinal products. The advantages of spontaneous reporting system include reaching large population throughout a long period of time for many medicinal products, however, it also has some limitations. One commonly recognized problem of the system in many countries is under-reporting of adverse drug reactions. The national reporting scheme in different countries slightly vary, even between Nordic countries. The main aim of this study was to find out what improvements should be done to the current reporting scheme in Finland so that it would better encourage healthcare professionals to report in relevant situations, which respond to the purpose of the spontaneous reporting system. Physicians (n=20), pharmacists (B.Sc.) (n=78), pharmacists (M.Sc.) (n=21) and nurses (n=13) responded to the anonymous open voluntary online questionnaire. Close-ended questions were analyzed and results summarized in graphs and tables. Statistical analysis was done using chi-squared test. Content analysis was performed for open-ended questions by utilizing both, inductive and deductive approach. In the study, we found some differences in healthcare professionals’ opinions what kind of adverse drug reactions should be reported. Some of the healthcare professionals were also aware that they had not reported all suspected adverse drug reactions that came into their knowledge and several reasons were recognized for this. Seriousness of the reaction was considered the most motivating factor for healthcare professionals to report about suspected adverse drug reactions. The results of this study suggest that in healthcare professionals’ opinion, the most important factors that should be considered to improve reporting in Finland are training for healthcare professionals and simplifying the reporting as much as possible. Some differences were noticed between the occupational groups regarding preferences in the reporting route and especially physicians seemed to prefer formation of the report from the information system as a reporting method more than open web-based reporting form. Mobile application for reporting was not preferred that much among Finnish healthcare professionals. The results of this study support the hypothesis that under-reporting of suspected adverse drug reactions is also present in Finland. The reporting instructions should be clarified, training availability should be considered and reporting should be simplified as much as possible to improve the reporting.
  • Rydman, Saara (2013)
    With advancing age the kidneys undergo anatomical and physiological changes. The most significant physiological changes are decreased renal blood flow and glomerular filtration rate (GFR). GFR is declined approximately in two thirds of the aged. Dosages of drugs eliminated mainly by the kidney should be adjusted carefully in patients with renal insufficiency. Nevertheless, according to earlier studies renal insufficiency among elderly patients is underdiagnosed and renal function is often overlooked when prescribing. Serum creatinine level is used as a screening test for renal dysfunction. However, it is a poor measure among elderly patients. Instead, calculated GFR should be the preferred method in estimating kidney function among this patient group. The aim of this study was to assess if comprehensive medication review can improve the quality of drug therapy among aged nursing home residents with impaired renal function. The data consisted of 153 comprehensive medication reviews (CMRs) conducted by Farenta Oy. CMR case reports were used to assess intervention recommendations made by pharmacists because of renal insufficiency, other kidney-related findings and resulting medication-related interventions. In addition, the prevalence of renal insufficiency, degree of renal insufficiency diagnoses and relationship between serum creatinine and estimated GFR were assessed. Clinical significance of the interventions was not assessed. The mean age of patients was 82,4 years. The estimated GFR was available for 145 patients (94,8%). 86,9% (n=126) had declined renal function (GFR<80 ml/min). Of these, serum creatinine levels were within normal range or under in 73,8% of all patients with renal insufficiency. Physicians had documented 4,8% of renal insufficiency cases in clinical patient files. Pharmacists identified inappropriate drugs due to renal function in 34,9% (n=44) of the patients with renal insufficiency. In total, pharmacists made 71 intervention recommendations. Physicians approved 60,6% of the pharmacists' recommendations as made. At least one intervention recommendation was approved as made in nearly one fourth (23,0%) of the patients with renal insufficiency. The most common drug-related intervention was changing the drug (25,4 %). Cardiovascular drugs accounted for 33,8% of the intervention recommendations, nervous system drugs 19,7 %. According to this study renal insufficiency among aged nursing home residents is common but underdiagnosed. Approximately one third of patients with renal insufficiency are using inappropriate medicines or dosages. These drug-related problems can be indentified and resolved during the comprehensive medication review procedure.
  • Kyynäräinen, Kerttu (2013)
    In vivo dissolution of drug molecules depends on the conditions in the gastrointestinal tract, like pH, composition of the fluids and hydrodynamics. Weakly basic compounds dissolve rapidly in low pH in stomach but in intestinal conditions forming of supersaturated solution may occur. This unstable state is the driving force either for rapid absorption from small intestine or for possible precipitation of drug. Difference in precipitation and thus in bioavailability between fasted and fed states can be significant. In this study behaviour of three weakly basic drugs, dipyridamole, ketoconazole and compound A, were studied with aid of two-phase microdissolution method. Three clinically relevant doses were used in the studies. In the study both fasted and fed states were tested as well as the effect of different pH in stomach phase and significance of biorelevant solutions over general buffer solutions. Dissolution of the drugs were examined in the media that simulate gastric fluids (SGF pH 1,2, SGF pH 4,0, FaSSGF pH 1,6 and acetate buffer pH 5,0). When biorelevant simulated intestinal fluid (FaSSIF or FeSSIF) was added to the solution to simulate the drug transfer out of stomach into small intestine, precipitation of different doses were analysed. Also the level of supersaturation compared to solubility results was examined. In addition the effects of various mixing speeds (300 rpm and 150 rpm) and scales (USP I and minidissolution) on precipitation were studied. Concentrations were measured directly from dissolution vessels with fibre optic probes. Re-dissolution of the drugs in small intestine and influence of physical properties on dissolution rate were evaluated with flow through dissolution method. In the fed state simulated microdissolution tests even the high doses of the drugs did not precipitate. Instead, in the tests simulating fasted state the effect of dose was clear and the relative part of dissolved drug were the smaller the higher was the dose. The high doses precipitated fast while the small doses remained much supersaturated. When FaSSGF was used the solutions staid longer in supersaturated state. Higher pH in stomach phase had remarkable impairing effect on the dissolved part of the drug in the small intestine phase and no supersaturated solutions were formed. In the microdissolution smaller mixing speed seemed to cause more precipitation and ranges of the results were larger. Different hydrodynamics in different scale dissolution methods as well caused divergent results. The effect of physical properties of precipitate to re-dissolution could be observed with the flow through dissolution tests. According to this study, two-phased dissolution method fit for few milligrams of API and it can be used to evaluate the precipitation potential of basic drugs in fasted and fed states. Also the effect of higher pH in the stomach on dissolved portion of API can be analysed. The method can be used as a risk assessment method for example when you want to know when the dose raises the risk to precipitation or as a tool to be used in early formulation development. As such dissolution tests can be used to get qualitative data of precipitation phenomenon when comparing basic drug compounds. The dissolution, precipitation and re-dissolution parameters will in future be utilised in pharmacokinetic model to evaluate the effect and significance of these phases on the drug absorption in vivo.
  • Nikko, Elina (2017)
    There is a great demand of cultured human hepatocytes for hepatotoxicity studies, drug testing, disease modelling and liver transplantation purposes. The current gold standard, primary human hepatocytes (PHHs), suffer from poor availability and high variability. Furthermore, PHHs are short-lived in in vitro cultures. Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have emerged as potential substitutes for PHHs in in vitro studies. PSCs are widely available, and additionally allow studies of hepatogenesis and open possibilities for personalized medicine. However, obtaining HLCs with mature hepatocyte functions in vitro has turned out to be challenging, and the differentiated cells have remained immature compared to PHHs. In vivo, hepatic differentiation and maturation of PSCs is guided by cues from the environment. Mimicking the 3D cellular environment in vitro has already shown encouraging results, but today, HLCs are still awaiting to fulfil their promise as a new gold standard. The aim of this study was first to select a new working human PSC line for in vitro hepatic differentiation and maturation. Hepatic differentiation and maturation of the selected cell line, embryonic stem cell line ESI-017, was next studied in five different 3D culture conditions (spheroids in suspension culture, four different hydrogels: Matrigel, collagen type I, mixture of Matrigel and collagen type I and alginate) with the aim to find the most favourable culture condition for later studies. For this, the PSCs were first differentiated to definitive endoderm cells and then to hepatoblasts in 2D cultures, on Matrigel- and laminin-521-coated plates, respectively. The PSC-derived hepatoblasts were then transferred for 16 days to the different 3D culture conditions for hepatic maturation. Of the conditions, suspension culture and mixture of Matrigel and collagen type I -hydrogel were estimated most promising and were selected for further studies. Hepatic maturation of the PSC-derived HLCs was estimated by analysing protein and mRNA expression levels of key marker genes, such as CYP3A4, AAT, MRP2, HNF4A, ALB, AFP, CK-8/18 and CK-19 by immunofluorescence staining and qPCR, respectively, and by cell morphology. Based on cell morphology and noticeable level of CYP3A4 expression, suspension culture shows most potential of the studied conditions in hepatic maturation of PSC-derived hepatoblasts. However, given that expression level of many other hepatocyte marker genes in these HLCs remained low compared to PHHs or human fetal liver samples, it is evident that adjustments to protocol and culturing conditions are still needed.
  • Samushenkova, Anna (2012)
    Numerous scientific studies have revealed the connection between oxidative stress and a wide range of diseases, including cardiovascular, neurodegenerative, inflammatory diseases and cancer. The most probable theory of ageing is based on oxidative stress as well. There exist endogenous and exogenous antioxidants capable of fighting oxidative and nitrosative damage to molecules and tissues of the body. Such compounds may be beneficial in prevention and treatment of different conditions. For example, plant foods contain various amounts of antioxidants. The aim of this thesis is to evaluate the antioxidant-related activities of certain commonly used vegetables (broccoli, brussel sprouts, cauliflower, peas), berries (bilberry, raspberry), herbs (Egyptian basil, oregano, rosemary, thyme) and spices (paprika) and to discuss their role in human health. The sample extracts were tested with four different methods: the determination of total phenols using Folin-Ciocalteu reagent, the DPPH free radical scavenging activity assay, reducing power activity assay and iron (II) chelation. In all assays, with exception of iron (II) chelation, vegetables proved to be less active as a potential source of antioxidants than other samples, while herbs seemed to be the most active samples. Iron chelation potential of samples is approximately the same with exception of paprika (lower than other samples) and bilberry (higher than other samples). The results obtained from different assays are not consistent with each other, and good correlative relationship occurs only between total phenols and iron reduction. On the basis of the results, it can be assumed that herbs and berries may be the main target for the research of pharmaceutically important antioxidants, although in daily diet vegetables and fruits are likely to be the best sources of such compounds. However, the beneficial daily doses of plant foods remain to be considered and further research is needed to provide information on the activity of given samples in vivo.
  • Renko, Juho-Matti (2012)
    Review of the literature: The purpose of the review is to go through what is known about mechanisms of actions of different neurotrophic factors (GDNF, neurturin, CDNF and MANF) and how they are transported within the brain. Neurotrophic factors are endogenous and secreted proteins which have a pivotal role in the development and maintenance of neurons. They support the survival of neurons and they can help them to recover from different injuries. Due to these functions neurotrophic factors might be beneficial for the treatment of neurodegenerative disorders like Parkinson's disease. There are a great deal of studies that clearly show the neuroprotective and neurorestrorative function of GDNF and neurturin on dopaminergic neurons. They are also studied in clinical studies with Parkinson's patients but the results have been partly contradictory. The signalling route of GDNF and neurturin via RET tyrosinekinasereceptor is fairly well known but the other mechanisms of action of these factors needs to be studied further. CDNF and MANF constitute a novel, evolutionarily conserved family of neurotrophic factors. They are shown to have neuroprotective and neurorestrorative actions on dopaminergic neurons both in vitro and in vivo in a rodent model of Parkinson's disease. The mechanisms of action of CDNF and MANF are not quite clear at the moment. There are two different domains in their structure both of which are likely to carry different functions. The N-terminal domains of these proteins are close to saposins, lipid and membrane binding proteins, some of which are shown to have neurotrophic and anti-apoptotic effects. The C-terminal domain of MANF, in turn, is structurally close to the SAP-domain of Ku70-protein which binds Bax in the cytoplasm and thus inhibits apoptosis mediated by Bax. CDNF and MANF might protect neurons both via intracellular mechanisms and extracellularly acting like a secreted neurotrophic factor. CDNF and GDNF are transported retrogradially from striatum to substantia nigra. MANF, unlike the others, is transported from striatum to the frontal cortex. MANF and CDNF are shown to have better diffusion properties in the brain parenchyma than GDNF. Experimental part: We studied, by means of microdialysis, the effects of CDNF, MANF and GDNF on the dopaminergic neurotransmission of naive rats within the striatum. Neurotrophic factors (10 µg) and PBS as a negative control were injected into the left striatum in stereotaxic surgery. After this rats recovered one week before the first mircodialysis. The second mircodialysis was performed three weeks after the surgery. The samples were collected from the left striatum of freely moving rats. During the microdialysis neurotransmission was stimulated by replacing the perfusion solution with hypertonic potassium solution and with amphetamine solution. The concentration of dopamine, DOPAC, HVA and 5-HIAA was measured from the dialysate samples. In vivo TH-activity experiment was carried out for three rats in each group. NSD1015 was injected i.p.after which rats were decapitated and their striatums were dissected. The concentration of L-DOPA, dopamine and metabolites on the treated and untreated hemisphere were analyzed from the tissue samples. The amount of L-DOPA in the striatum after NSD1015-treatment indicates how active TH-enzyme is. There were no significant differences in the concentrations of dopamine and metabolites during the baseline. MANF and CDNF increased the release of dopamine from the nerve terminals compared to GDNF and PBS one week after the surgery. Three weeks after the surgery there was still significant increase in the release of dopamine in MANF group compared to GDNF group. Also the dopamine-DOPAC-turnover was increased significantly in MANF group compared to GDNF and PBS groups one week after the surgery. DOPAC/HVA -ratio was significantly smaller in GDNF group than in other groups one week after the surgery. These findings suggest that MANF potentiates dopaminergic neurotransmission most drasticly. The effects of MANF seem to last longer time than the effects of other neurotrophic factors. CDNF seems to increase the release of dopamine from the nerve terminals as well. The potentiation of dopaminergic neurotransmission could be due to increased biosynthesis of dopamine or due to the potentiation of the function of nerve terminals. In the results of the TH-activity experiment there was a trend according to which L-DOPA is synthesized less after the neurotrophic factor treatment that after the PBS treatment. This suggests that neurotrophic factors might decrease the activity of TH-enzyme.
  • Vanhanen, Jenni (2010)
    Histamine is an important neurotransmitter in peripheral as well as in central nervous system. Histaminergic neurons modulate various functions such as sleep-wake cycle, energy metabolism, memory and pain. In addition the brain histaminergic system has been shown to play a role in reinforcement, addiction and addiction related behaviors. After finding the H3 receptor in 1980s it was realised how essential the neuronal histamine is in modulating several central nervous system (CNS) disorders. H3 receptor modulates the synthesis and release of histamine. Furthermore it modulates the release of various other neurotransmitters, such as serotonin, noradrenalin, dopamine, glutamate, γ-aminobutyric acid (GABA) and acethylcoline. The H3 receptor is predominantly expressed in the brain and therefore it is an attractive target for various CNS indications. For more than a decade H3 receptor has gained the interest of many pharmaceutical companies. Several H3 receptor ligands, mainly antagonists or inverse agonists, have been assessed in preclinical as well as in clinical studies. So far there are not enough clinical data on the safety and efficacy of H3 receptor ligands, but there is a strong possibility that H3 receptor antagonists will be used in the treatment of various important disorders, including narcolepsy, schizophrenia and cognitive disorders. Earlier in our research group it was shown that H3 receptor ligands play a major role in ethanol related behaviors. These observations were confirmed in the practical part of this Master's thesis. H3 receptor modulates ethanol stimulation as well as ethanol reinforcement. Both H3 receptor antagonists, ciproxifan and JNJ-10181457 were able to inhibit ethanol-evoked conditioned place preference (CPP). This means they were able to inhibit ethanol reward and reinforcement. Ciproxifan also increased ethanol stimulation. Immepip on the other hand did not alter ethanol-evoked CPP, but it totally inhibited the stimulation of locomotor activity by ethanol. The dopaminergic system regulates both reward and motor functions. The postsynaptic H3 receptors have been shown to be able to heteromerize with both dopamine D1- and D2- receptors in striatum. The formed heteromers modulate dopaminergic neurotransmission in vitro, which may lead to alterations in behavior in vivo. It is therefore possible that the responses we have seen on a behavioral level in this Master's thesis project are due to interactions between histaminergic and dopaminergic systems in striatal areas. The H3 receptor is an interesting target in the drug development of various CNS disorders. The responses seen in this Master's thesis project also indicate that the blockade of H3 receptor inhibit ethanol reward and reinforcement. In conclusion, these findings indicate that H3 receptor antagonists could possibly have therapeutic potential in treating ethanol addiction.
  • Puro, Valtteri (2024)
    Syöpäsairaudet tai niiden hoito aiheuttavat potilaille usein huomattavaa fyysistä ja psyykkistä taakkaa. Merkittävien fysiologisten muutosten lisäksi hoidot johtavat usein voimakkaaseen ahdistuneisuuteen, psyykkisiin liitännäissairauksiin ja heikentyneeseen yleiseen elämänlaatuun. Näiden oireiden kasaantuessa ne heikentävät merkittävästi potilaiden elämänlaatua, arjen toimintakyvyn ja henkisen jaksamisen madaltuessa. Syöpäpotilaiden monet subjektiiviset oireet jäävät kuitenkin usein alihoidetuiksi tai hoitavalta taholta tunnistamattomiksi. Hoitoihin tai sairauteen liittyvien oireiden piiloon jääminen saattaa johtaa madaltuneeseen hoitoon sitoutumiseen, korkeampiin kuolleisuuslukuihin ja on terveydenhuollon tuottajan näkökulmasta tehotonta. Rutiininomainen ja spesifisti kohdennettu oireiden seuranta on tärkeää niiden oikea-aikaista ja tehokasta esilletuontia varten ja se on yhdistetty parantuneeseen hoitovasteeseen syöpäpotilailla. Kliinisten tulosten seurannan tueksi hoidon onnistumista voidaan seurata validoiduilla potilaslähtöisillä mittareilla (vointimittarit, PROM ja potilaskokemusmittarit PREM). Niillä voidaan saada oikein käytettynä riippumatonta ja reaaliaikaista tietoa potilaiden kokemasta terveydentilasta. Potilaskeskeisen (arvoperustaisen) terveydenhuollon kehitystyön edistyessä tutkimustieto hoidon vaikuttavuuden mittaamisesta potilaiden itse raportoimien tulosten avulla perinteisten kliinisten mittareiden lisäksi tulee korostumaan. Syöpähoitoja tarvitsevien määrän kasvu ja uusien, kalliiden pienmolekyylisten, biologisten, ja geeniterapiahoitojen markkinoille tuleminen, tuottavat taloudellisen paineen kestäville ja arkikäytössä vaikuttaville ratkaisumalleille hoidon toteutuksen tutkimukselle. Modernien viestintäjärjestelmien käyttö potilaiden raportoimien tulosten esilletuonnissa ja tallentamisessa voi tarjota mahdollisuuden parantaa vaikuttavuuden mittaamisen toteutusta kliinisessä arjessa. Tutkielman yhteistyöorganisaatio Istekki oy pyrkii kehittämään terveysteknologisia ratkaisuja julkisen terveydenhuollon käyttöön. Kartoittavan katsauksen periaatteiden mukaisesti, tutkimalla järjestelmällisesti olemassa olevaa kirjallisuutta selvitettiin, minkälaisia mittareita erilaisissa syöpähoitoja tarjoavissa ympäristöissä on käytetty. Katsauksen aineisto koostui 20 vertaisarvioidusta alkuperäistutkimuksesta, jotka keskittyivät potilaslähtöisten mittareiden käyttöön onkologisessa ympäristössä. Näistä saatiin tarkasteluun 28 erilaista validoitua potilaslähtöistä mittaria. Digitaalisten alustojen käyttö mittareiden käyttöympäristönä korostui kaikissa tutkimuksissa. Oikein käytettynä potilaslähtöiset mittarit edistivät potilaiden ja hoitohenkilökunnan välistä kommunikaatiota, potilastyytyväisyyttä ja mahdollistivat hoidon vaikutusten reaaliaikaisen seurannan potilasnäkökulmasta. Kuitenkin tekniset, kulttuuriset ja organisatoriset esteet, kuten koulutuksen puute, resurssien niukkuus ja tiedon puute (asenteet), ovat haasteita, jotka hidastavat mittareiden käyttöönottoa kliiniseen arkeen. Vointimittareiden tehokas hyödyntäminen edellyttää kattavaa koulutusta, selkeitä käyttöönotto- ja reagointistrategioita, sekä hyvin saatavissa olevaa teknistä tukea niin potilaille, kuin henkilökunnallekin.
  • Valkohaapa, Anna-Mari (2014)
    In Finland the elderly residents of long-term care facilities are often prescribed a lot of medications, especially psychotropic drugs. It also happens that a patient or a resident has to be physically or chemically restrained. Chemical restraining can be defined in many ways, for example as using a drug - usually an antipsychotic - to restrict the freedom or movement of a patient and to control his or her behavior. In nursing homes the staff is in a key position when it comes to deciding on the use of chemical restraining or PRN medication. A legislation to guarantee the self-determination of a patient and to define how physical restraining can be used is now being prepared in Finland. Only a few studies on chemical restraining from a nurses' point of view have been made so far. Thus, the aim of this study is to provide more information on the level of knowledge, the attitudes and perceptions of nurses regarding chemical restraining and the effect of those on deciding whether to use chemical restraints or not. Three focus groups with nurses were conducted in Hyvinkää nursing homes (n=13). The groups were recruited both by e-mail and directly from the wards. The focus group discussions were digitally audiotaped and transcribed verbatim. The content of the transcripts was then analyzed using a constant comparative method. According to the study most of the antipsychotics used in long-term care were used daily. However, it is not uncommon for the nurses to be unsure about their knowledge on the use of medicines. It is thus important to help the nursing staff to increase their knowledge and skills in pharmacology. The nurses also wished to get extra training for treating people with dementia. The concept of chemical restraining is quite ambiguous, and the use of chemical restraints is a complex ethical issue because the reasons for and effects of administering it vary depending on the situation. The study shows that the chemical restraining is most often considered justified when it is used to ensure the safety of a patient, relieve anxiety or to keep the working conditions of the staff tolerable. Also a shortage of manpower and a request by the family can influence the decision on using chemical restraints. The lack of proper common guidelines causes confusion and wide variation in the use of chemical restraints. Many interviewees were hoping for more open discussion and cooperation on using chemical restraining. The nurses also mentioned many alternatives to rely on instead of using chemical restraints, such as soothing, comforting and creating a safe feeling for the patients, daily routines and stimulus. One of the key factors for taking to these instead of chemical restraints are the manpower resources in the facilities. Educating the staff can also help them to find more options for chemical restraining and make staff members recognize new or remember forgotten routines for caring for the patients without using psychotropic drugs.
  • Huhtanen, Elina (2023)
    Nonadherence has been a challenge in health care since ancient times, and it is still a major source of poor therapeutic outcomes today. Various theories and theoretical models have been applied to explain adherence. So far no single theory or theoretical model has proved comprehensive, and the effects of various interventions applied have been limited. Theorizing of adherence is still incomplete. The aim of this study was to review the theoretical background of adherence so far, and to find the commonalities in theories and theoretical models, but also to discover possible trends in them. Study material comprised 20 theories and theoretical models. Material was found in reference books and documents with an aspect of adherence. Inclusion criteria involved adult outpatients with chronic diseases, excluding any special groups such as pregnant women. Emphasis was on the relevance of the theory, but comprehensive time span as well as various theory views were also considered. A chronological order was created. A qualitative content analysis was conducted, where phrases describing essential theory contents were categorised into distinctive classes. The incidence of classes and the possible trends of the incidence were analysed. Five classes emerged as commonalities in majority of the theories: Patient factors; sociocultural factors; motivation; phrases that referred to a behavioral view; and cognitive processes. Factors that diminished towards this day were the pivotality of cognitive processes in theory, and the subjective view of the health threat. Several factors were found to increase towards present-day theories and models: phrases that described the individual’s agency, such as self-efficacy or empowerment, concrete expressions of the individual’s abilities, skills and actual opportunity to the desired behavior, phrases that consisted of behavior being a function of its context or environment, and phrases where multifaceted intervention or tailoring the intervention according to the change process phase were considered. The commonalities found in the material reflect its emphasis on psychological behavior theories. The changes reflect the shift in how the patient’s agency is viewed in a patient care relationship. Newer theories also amplify the importance of viewing health behavior change as a series of phases. To achieve patient-centered care, these aspects create an increasing demand to health care professionals’ proficiency in how they encounter their patients. Moreover, there is an important signal for future intervention development in the form of a shift into multifaceted interventions and a system approach. Furthermore, a trend exists from using more general and abstract theories to a more concrete and applied approach. In the future, it is probably more fruitful to pursue knowledge-based and validated models and guidelines that are applicable to practice instead of aiming at developing a comprehensive universal theoretical approach.
  • 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.