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Prolyl oligopeptidase (POP, prolyl endopeptidase, EC 3.4.21.26) is a serine-type peptidase (family S9 of clan SC) hydrolyzing peptides shorter than 30 amino acids. POP has been found in various mammalian and bacterial sources and it is widely distributed throughout different organisms. In human and rat, POP enzyme activity has been detected in most tissues, with the highest activity found mostly in the brain. POP has gained scientific interest as being involved in the hydrolyzis of many bioactive peptides connected with learning and memory functions, and also with neurodegenerative disorders. In drug or lesion induced amnesia models and in aged rodents, POP inhibitors have been able to revert memory loss. POP may have a fuction in IP3 signaling and it may be a possible target of mood stabilizing substances. POP may also have a role in protein trafficking, sorting and secretion. The role of POP during ontogeny has not yet been resolved. POP enzyme activity and expression have shown fluctuation during development. Specially high enzyme activities have been measured in the brain during early development. Reduced neuronal proliferation and differentation in presence of POP inhibitor have been reported. Nuclear POP has been observed in proliferating peripheral tissues and in cell cultures at the early stage of development. Also, POP coding mRNA is abundantly expressed during brain ontogeny and the highest levels of expression are associated with proliferative germinal matrices. This observation indicates a special role for POP in the regulation of neurogenesis during development. For the experimental part, the study was undertaken to investigate the expression and distribution of POP protein and enzymatic activity of POP in developing rat brain (from embryonic day 14 to post natal day 7) using immunohistochemistry, POP enzyme activity measurements and western blot-analysis. The aim was also to find in vivo confirmation of the nuclear colocalization of POP during early brain ontogeny. For immunohistochemistry, cryosections from the brains of the fetuses/rats were made and stained using specific antibody for POP and fluorescent markers for POP and nuclei. The enzyme activity assay was based on the fluorescence of 7- amino-4-methylcoumarin (AMC) generated from the fluorogenic substrate succinyl-glycyl-prolyl-7-amino-4-methylcoumarin (Suc-Gly-Pro-AMC) by POP. The amounts of POP protein and the specifity of POP antibody in rat embryos was confirmed by western blot analysis. We observed that enzymatic activity of POP is highest at embryonic day 18 while the protein amounts reach their peak at birth. POP was widely present throughout the developmental stages from embryonic day 14 to parturition day, although the POP-immunoreactivity varied abundantly. At embryonic days 14 and 18 notably amounts of POP was distributed at proliferative germinal zones. Furthermore, POP was located in the nucleus early in the development but is transferred to cytosol before birth. At P0 and P7 the POP-immunoreactivity was also widely observed, but the amount of POP was notably reduced at P7. POP was present in cytosol and in intercellular space, but no nuclear POP was observed. These findings support the idea of POP being involved in specific brain functions, such as neuronal proliferation and differentation. Our results in vivo confirm the previous cell culture results supporting the role of POP in neurogenesis. Moreover, an inconsistency of POP protein amounts and enzymatic activity late in the development suggests a strong regulation of POP activity and a possible non-hydrolytic role at that stage.

Harmful drug effects are common among older medicine users. Potentially harmful drugs for older people have been defined by different criteria. Potentially inappropriate medications (PIMs) defined by Beers criteria have been associated with adverse effects, increased costs, need of hospital care and disabilities. Drugs with anticholinergic properties (DAPs) are associated with anticholinergic side effects, cognitive decline and delirium. Psychotropic drugs have been associated with increased risk of falls and mortality. Concurrent use (≥3) of psychotropic drugs has been considered harmful for older people by the Swedish National Board of Health and Welfare. The aim of this study was to examine the prevalence of potentially harmful drug (PHD) use (DAPs, PIMs, or concurrent use of ≥3 psychotropic drugs) and the accumulation of PHDs in aged people living in nursing homes and assisted living facilities. The objective was also to investigate which patient characteristics are associated with PHD use or accumulation of these drugs. The cross-sectional data was collected in 2011-2012 as a part of a larger study "Reducing inappropriate, anticholinergic and psychotropic drug use among older residents in institutional care". The study population (N=326) consisted of ≥ 65-aged residents living in nursing homes or assisted living facilities in Helsinki (n=227) and Kouvola (n=99). The mean age was 83.5 years, 70.0 % were women and mean Charlson comorbidity index was 2.6. Residents were divided into four groups: aged using 1) DAPs, 2) PIMs defined by Beers criteria, 3) concurrent use of ≥3 psychotropic drugs and 4) no PHDs in use. Both those fulfilling any of these criteria and those fulfilling all the three criteria (accumulation of PHD) are described. Users in these groups were compared to the non-users. A majority of the residents, 78,8 % (95 % CI: 74,4 - 83,3) used ≥1 PHDs: 67,8 % (95 % CI: 62,7- 72,9) used ≥1 DAPs, 32,2 % (95 % CI: 27,1- 37,3) ≥1 PIMs and 32,2 % (95 % CI: 27,1-37,3) used ≥3 psychotropics concurrently. Of the residents, 41 (12.6 %) had a medication treatment that fulfilled all the three criteria of PHD use. These residents used significantly more PHDs than others (average mean 4,8 [range 3-7] vs. 1,6 [range 0-6]). The residents having the PHD accumulation were more often males and used more drugs than others. There were no statistical differences among the other characteristics of these groups. The most common PHDs were mirtazapine (n=66), lorazepam (n=64), oxazepam (n=62), ketiapine (n=58) and stimulant laxatives without opioids (n=58). Use of DAPs was associated with multimorbidity, use of PIMs with weaker health related quality of life, and use of psychotropics with younger age. High number of drugs was associated with all these criteria. Use and accumulation of potentially harmful drugs is common among the aged living in nursing homes and assisted living facilities. New means are needed to optimize drug treatments and to educate professionals taking care of these patients. Special attention should be paid on the use of antipsychotics, benzodiazepines, mirtazapine and stimulant laxatives.

In a pharmacotherapy process prescriber, provider, administrator and evaluator of the effects of medication cooperate in a coordinated way to ensure optimum outcomes of the patient's medications. This requires that all professionals involved in the pharmacotherapy process know their functions and responsibilities in an interprofessional team. No previous studies have explored legislative facilitators and barriers that have impact on the interprofessional pharmacotherapy process even though interprofessional collaboration for assuring safe and effective pharmacotherapy is one of the main objectives of Medicines Policy 2020 in Finland (Ministry of Social Affairs and Health 2011). The aim of this study was to examine how legislation prevents or facilitates the interprofessional pharmacotherapy process. The data consisted acts, decrees, regulations and directives concerning pharmacotherapy process in the healthcare. Changes in the Finnish legislation from 1990 until present were studied, taking also into account the European Union legislation since 1995 when Finland became a member. Research method was qualitative content analysis. A modified form of the causal diagram of the impact of law and legal practices on public health system performance by Burris et al (2012) was used as a theoretical framework. Interprofessional collaboration applicable to pharmacotherapy process has been taken into account in the main enactments. These enactments concern rights and duties of healthcare professional, patient’s rights, resources required in healthcare, communication and information transfer, and pharmacotherapy process. Enactments that complicate the interprofessional pharmacotherapy process relate especially to communicational barriers. Facilitating enactments include guidelines ensuring coordinated clinical practice for effective and safe medication use and improved communication, collaboration and patient-centeredness as well as accessibility of cross-border healthcare. A trend was observed that the legislation enacted on the 21st Century put more emphasis on interprofessional collaboration, development of technology and mobility of citizens. According to this study there are more facilitators than barriers for interprofessional pharmacotherapy process. Recent enactments even emphasize more interprofessional cooperation in health care than did those enacted before the 21st Century. Still, enactments are general: they should be made more detailed to give a better understanding of interprofessional cooperation, also related to the pharmacotherapy process for safe, effective and evidence-based medication use.

Patient safety is a part of quality and safety of care. Patient safety is defined as freedom for a patient from unnecessary harm or potential harm associated with healthcare. Patient safety covers safety of care, medication safety and safety of devices. Different authorities have promoted patient safety in Finland. The Ministry of Social Affairs and Health set up the Steering Group for the Promotion of Patient Safety for a term extending from 1 November 2006 to 31 October 2009 to coordinate the work for promoting patient safety and to evaluate related development needs at the national level. It has published a national Patient Safety Strategy for the years 2009-2013. Patient safety is also included in the Health Care Act. The National Institute for Welfare and Health (THL) has a Finnish national programme on patient safety: Patient Safety with Skills. Other authorities promoting patient safety in Finland are Finnish Medicines Agency (Fimea) and National Supervisory Authority for Welfare and Health (Valvira). Many studies are related to Patient Safety. In the Seminar of Patient Safety Research 2011 studies were separated to following categories: Patient Safety Culture as a Challenge for Organisations, Medication Safety, Safeguard of Care, Preparation for Patient Safety, Learning of Patient Safety, Control of Patient Documents and Financing of Patient Safety. The aim of this study is to explore Finnish patient safety studies. This study was conducted by using an electronic survey. The survey was sent to members of Finnish Patient Safety Society and a mailing list of Patient Safety Network. The survey was also sent to attendees of the Seminar of Patient Safety Research 2011. Altogether 81 responses were obtained. A patient safety research had been done in 60 per cent of organizations. A patient safety research will be done in 62 per cent of organizations. 10 per cent of the researches were meant for the internal use of the organization but were also published in Finland and abroad. 21 per cent of the researches were published in Finland and abroad. 18 per cent of the researches were published only in Finland and 12 per cent only abroad. 25 per cent of the researches were meant only for the internal use of organization. 14 per cent of the respondents left this question unanswered. A personal grant from a foundation was the most common way of financing for patient safety research. Many different kinds of sponsors were also mentioned. There was co-operation between organizations in 58 per cent of researches. 86 per cent of respondents were interested in a network of patient safety researchers. Using of Reporting System for Safety Incidents in Health Care Organizations (HaiPro) was asked as a detail of this study. HaiPro was used in 65 per cent of organizations. 89 per cent of respondents said that their organization takes advantage of HaiPro but the level of use varied between respondents.

Physiologically based pharmacokinetic modelling (PBPK) can be used to predict pharmacokinetic behaviour of new drug molecules in human. PBPK model represents the body anatomically and physiologically with compartments connected to each other and combines those to drug specific parameters. PBPK modelling can be used to predict the absorption, disposition, and time-concentration profiles of drug molecules. The purpose of the study was to build a PBPK model for new drug molecule under research (compound A) and predict pharmacokinetics in human, to support the selection of dosing interval, formulation, and sampling time points for the first clinical trial. In this work it is described the building of the model in the ”bottom-up”-approach using in vitro parameters in GastroPlusTM-software. The modelling was done also for preclinical species (mouse, rat, dog) comparing the simulations to the observed in vivo data, which gave the confidence to the methods used in the modelling also for human. The model was first built for systemic kinetics and thereafter it was used for predicting pharmacokinetics after oral dosing. Parameters of systemic kinetics were compared also to the predictions from allometric scaling. Based on the preclinical species the most predictive method for the volume of distribution of compound A was the method by Lukacova, which predicted the volume of distribution to be moderate in human (1.7 l/kg). From the in vitro-to-in vivo -extrapolation methods the most predictive method to predict the clearance was the method by Poulin, which predicted low clearance in human (8.1-14.3 l/h). Empirical scaling factors based on the preclinical data were not needed, as the models predicted well the observed in vivo data. Allometric methods predicted the systemic kinetic parameters to be in the similar range. Advanced compartmental absorption transit -model (ACAT) integrated to GastroPlusTM-software predicted the absorption after oral dosing well in the preclinical species (predicted/observed ratio 0.8-1.3 for systemic exposure) despite the low solubility of the compound A. The model predicted the absorption in human to be sensitive to particle size and absorption rate to be clearly affected by the particle size. The feeding status was also predicted to affect on the absorption with larger particle sizes. The gut metabolism was not predicted to limit the oral exposure notably, whereas moderate bioavailability was predicted to be achievable. Compound A could be given in a capsule if the target particle size distribution could be achieved. The built PBPK-model can be used in the future to predict the first clinical doses by comparing the predicted plasma concentrations to in vitro pharmacodynamic parameters and to the plasma concentrations needed for efficacy in the pharmacodynamic models. The model can also be used to predict the drug-drug interactions.

Traditional 2D cell cultivating vessels and experimental models cannot often simulate natural chemical and physical environment of different cell types. For example, availability of oxygen, chemical gradients, messaging molecules, fluid pressure, flow and surface topography are factors that may affect significantly in cell differentiation, growth, cellular structure, and metabolism. Modular bioreactors like Quasi-Vivo® -system can be used to simulate these factors. Liposomes are particles of phospholipid bilayer with aqueous space enclosed within. They can be modified in numerous ways, like loading them with hydrophobic and hydrophilic molecules, changing their transition temperature or coating them according to different needs. Doxorubicin is effective and widely used cytostatic agent, but when administered as a free drug it has often severe side-effects, like cardiotoxicity. Goal of this thesis is to determine appropriate manufacturing parameters and verify adequate shelf-life of ICG-Doxorubicin liposomes, that they are applicable for future in vitro experiments. Then survival of HepG2 cell line under flow in Quasi-Vivo®-equipment is determined, after which A549 and HepG2 will be then combined into one two-cell model. Finally, a simple illumination experiment in this cell model with previously made liposomes is conducted, and the effect in whole system is examined. Using protocol presented in this thesis it is possible to produce successfully and repeatedly liposomes with both ICG and doxorubicin encapsulation over 70%. Their shelf-life was at least 14 days when stored in 4°C protected from light. This was determined to be sufficient for in vitro testing. Cultivating A549 and HepG2 cell lines combined in the same system with shared media and fluid flow conditions was successful. Neither of the cell lines show significant difference in viability when compared to static control. When light-activating liposomes are administered to the system and then illuminated, from preliminary results we can see significant difference in drug effect. Both illuminated chambers and off-target chambers connected via Quasi-Vivo® show increased suppression, which shows promise that this in vitro model would be useful for future experiments.

Prolyl oligopeptidase (PREP) is a serine protease which is extensively present in the mammalian system and especially abundant in the brain. Despite the long research history of PREP its physiological function has remained unclear. PREP has been suggested to regulate the functions of many bioactive peptides by hydrolysis and on the other hand to participate in several intracellular processes probably via direct protein-protein interactions. One of the functions suggested for PREP is the regulation of the brain neurotransmitter systems and based on, for instance, the location in the brain PREP has been connected to both excitatory and inhibitory neurotransmitter systems. The literature review of this thesis first describe the brain neurotransmitter systems associated to PREP in general with some examples of diseases related to their malfunctions. In addition the structure of PREP and its location in the brain, both subcellular and cellular levels, and in distinct neurotransmitter systems, are presented, after which the different proposed functions for PREP are reviewed. The aim of the experimental part of this thesis was to investigate the effects of PREP on the brain neurotransmitter concentrations in the mouse nigrostriatal pathway and also to mouse motor behavior. The main research methods were microdialysis, tissue assays and cylinder test. The study was composed of two sections with five week duration each. The first section was performed with wild-type mice expressing naturally PREP and the second section with PREP-knockout (ko) mice and their wild-type littermates. The mice were injected unilaterally above the substantia nigra with adeno associated (AAV1) hPREP viral vector or with AAV1-eGFP (green fluorescent protein) viral vector as a control treatment. The cylinder test was carried out before the injection, and two and four weeks afterwards. Microdialysis was used to study the actions of PREP on the extracellular concentrations of dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), gamma-aminobutyric acid (GABA) and 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of serotonin (5-HT). In addition to the baseline assay the concentrations were measured after two amphetamine treatments (10 and 30 µM) administered via the microdialysis probe. The probe guide cannulas were inserted to mice striatums 1-2 weeks before the microdialysis measurement. In the end of the experiment the tissue concentrations of DA, DOPAC, HVA, 5-HT and 5-HIAA were measured from striatum and substantia nigra. Both the microdialysis and tissue sample concentrations were quantified with high performance liquid chromatography. In the first study section the PREP enzyme activity was also determined from striatum. Neither the complete deprivation nor over-expression of PREP in the nigrostriatal pathway had clear or consistent effects on the levels of neurotransmitters studied when compared to naturally occurring PREP expression. When comparing the differences between control treated groups of PREP-ko and littermate mice, a greater amphetamine stimulated DA-levels was seen in the former group proposing negative regulatory influence of PREP. In both study sections the tissue assay results were difficult to interpret due to observed responses also with AAV1-eGFP control treatment in comparison with untreated side of the brain. This was seen as a lower DA- and DOPAC-levels in substantia nigra and thus the meaning of the changes caused by PREP treatment is hard to comprehend. The results of the cylinder test may implicate some protective effect of the PREP-ko-genotype against viral vector injections in general. Then again the existence of compensatory mechanisms is possible when using knockout animals and thus the genotype differences are hardly ever unequivocal. The results of this thesis do not suggest outright regulatory effects of PREP on the neurotransmitter functions in the mouse nigrostriatal pathway although the confirmation of this requires further studies, especially in regard to GABAergic and glutamatergic systems. Studies should include a scale of different behavioral tests of motor activity and repeated microdialysis experiment with some defining method changes. The possible function and mechanisms of PREP as a regulator of neurotransmitter intake or release is rationale to study at molecular level with applicable methods.

Spray drying is one way to dry protein medicines and it has many advantages compared to other drying methods, for example it is a fast process. In spray drying high temperature and mechanical stress can inactivate the protein. Disaccharides are generally used as protective agents of protein in spray drying because they have an ability to protect the structure of the protein during drying and storage. Aim of this research was to study the stability of the protein during spray drying and storage by using β-galactosidace as a model protein. Aim was also to characterize the physical properties of trehalose and melibiose and to study how well they protect the protein. Some of the central matters to be examined were the glass transition temperature, crystallinity, water activity, yield of the spray dried powder and protein activity. Especially studying the properties of melibiose in spray drying was important because it has not been used before. The study also included the optimization of the process parameters to be suitable for the product. Trehalose and melibiose transformed to an amorphous form during spray drying. Both XRPD and DSC showed an amorfous form. Trehalose and melibiose proved to be good protective agents for the protein during spray drying and storatge probably because they remained their amorphous structure. β-galactosidase remained activity very well. Optimizing of the process parameters was successful because protein remained its activity and still the powder was quite dry and yield was good. The changes in the structure of the protein were studied with FT-IR but the amount of the protein was too small. Problems caused by the spray drier may have an effect to the results, but on the other hand the spray dryer was made to work optimally.

Lead molecule search is the first part of drug design. This process can be done using computerized docking of ligands into target proteins. Usually this requires expensive software and powerful computer systems specifically made for the process. There are however some programs that are available for free and can be run on home computers. The purpose of this Master's Thesis was to see how these free software can be used for the task of docking and also to create a method or a guideline for such work. Protein kinase C (PKC), a popular target for drug design, was chosen as a target of inhibitor design. PKC is part of a larger family of serine/threonine kinases and formed of 10 isoforms all with different effects on cellular functions. The large amount of related kinases and the similarities in their sequences make finding selective inhibitors a difficult process. Homology models of all PKC isoforms in three known conformations solved by x-ray diffraction (pdb: 1XJD, 2I0E and 3A8W) were created using Modeller. Into these models a set of possible ligands from the free database of molecules ZINC was docked using Autodock Vina utilizing a script created for docking multiple ligands into multiple targets. The dockings resulted in some interesting results. Six molecules were recognized as possible lead molecules for further research. None of these molecules had any patents or previous results of protein kinase inhibition connected to them. The most interesting result was the finding that coluracetam, a nootropic drug of the racetam family, might be a protein kinase inhibitor. Racetams are usually considered drugs that lead to PKC activation. It has been proposed that inhibitors may prolong the lifetime of kinases in the cells leading to increased activity in the long term. In our opinion coluracetam might prove to be a good tool for studying the complex way kinase activity is modulated. The methods and scripts used in this work will be released for free use.

In the United States pharmacists have prescribed medicines and managed patient's drug therapy since the 1970s, and in the United Kingdom pharmacists have been authorization to prescribe medications since 2003. The discussion about the right of Masters of Science in Pharmacy will be renewed prescriptions during the last decade in Finland but few Finnish studies have been published from the subject. In the document Medicines Policy 2020 published by Ministry of Social Affairs and Health states that by prescribing should be used cost-effective modes of operation. The knowledge about pharmacist prescribing benefits and costs, and also prescribing practice in Finland, is needed to evaluate the cost-effectiveness of pharmacists' authorization to prescribe and to support the decision-making concerning pharmacist prescribing. The aim of this master's thesis is to gather all existing knowledge about the economic and other effects of pharmacist prescribing using a systematic literature review method. The aim of theoretical part of this master's thesis is to explain the Finnish prescribing, the participation of pharmacists in drug therapy management in Finland and internationally pharmacist prescribing. The empirical part of this master's thesis is also to assess the quality of the studies of pharmacist prescribing benefits and costs using quality assessment checklists. In addition, this thesis describes the principles of the cost and benefit analyses, economic evaluations and systematic literature reviews. As a result of the literature search were found 1825 references. Based on the inclusion and exclusion criteria, 17 studies were selected to include in the systematic review. Of these studies three were economic assessments, 8 randomized controlled trials and 6 observational studies. The quality of these studies was assessed using four quality assessment checklists. On the basis of a systematic literature review pharmacist prescribing has been studied in the treatment of type 2 diabetes, hypertension, dyslipidemia, anticoagulation, chronic pain, emergency contraception and minor ailments and renewal of long-term medicines. Pharmacists reduced blood pressure by providing follow-up care with prescribing compared with the usual care, but not compared with the case management, which does not include prescribing. In addition, the follow-up care was to improve the treatment results of type II diabetes. The results obtained in the care of dyslipidemia were partly unclear. In the clinic follow-up care with prescribing could be reduced LDL-cholesterol, but not the risk of cardiovascular disease compared with the control group. In the pharmacy follow-up care had no effect on the treatment of patients with LDL-cholesterol compared with the control group. In addition, pharmacist prescribing improved how well patients stayed within INR target range. Pharmacist medication review with pharmacist prescribing achieved in the care of chronic pain patients differed few from the results of pharmacist medication review with feedback for a general practitioner. Pharmacist prescribing could reduce errors in inpatient medication compared with usual care. Much uncertainty is connected to the results of the study. The limited amount of studies, heterogeneity of the studies and methodological quality make the evaluation of real effects more difficult. The included studies of pharmacist prescribing were so heterogeneous. In addition treated disease, assessed benefits and scope of working environment were varied in included studies. Pharmacist prescribing was often studied as part of other care or pharmaceutical service, such as chronic disease management or medication review. The quality assessment of the included studies revealed several sources of bias. The available research information is the insufficient reliable evaluation of economic and other effects of pharmacist prescribing and the need for the further research is big.