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The aim of this thesis was to synthesize abietic acid derivatives, and to determine their antimicrobial activity. The study was performed in two parts. The first part was carried out at VTT (Technical Research Centre of Finland) and the second part at the Faculty of Pharmacy, University of Helsinki, the Division of Pharmaceutical Chemistry and Technology. Abietic acid as well as its isomers and precursors are important antimicrobial substances among plant kingdom. They are produced in big scale by conifers, which extractives of pitch and wood they are. Abietic acid and its many derivatives have shown to have many bioactivities. Emerging antibiotic resistance of bacteria, viral diseases that are spreading and mutating throughout the world and serious diseases caused by protozoa, cause an ever-growing need to develop new active agents against these pathogens. Abietic acid derivatives are worthy candidates for research into new drug substances. At VTT a synthetic route with abietic acid chloride as intermediate was used (synthetic route 1). It did not work well in the synthesis of esters and yields were low. The synthetic route worked fairly well for amide synthesis. The purifications and analysis of the compounds, two esters and one amide of abietic acid, were left partially unfinished, and none of the products of these reactions were tested for bioactivity. At the Division of Pharmaceutical Chemistry and Technology eight derivatives of abietic acid, from abietic acid and four different amino acids, were obtained via synthetic route 2. Some of these were sent to tests for antibacterial activity. Six synthesized compounds were sent to tests for their bioactivity against bacteria. However, the results were not obtained in time for this Master's thesis, and therefore any conclusions of their structure-activity relationships could not be drawn.

The nucleus accumbens (NAc) is located in the ventral striatum and plays a critical role in drug addiction. NAc receives dopaminergic projections from ventral tegmental area (VTA) which is activated after administration of various abused drugs. Activation of VTA increases the release of dopamine in NAc. Increased dopamine levels induce the release of acetylcholine from striatal cholinergic interneurons. These cholinergic interneurons have been related to the development of addiction and other emotion-related disorders such as depression. Previous studies have shown that a lesion of cholinergic interneurons led to an increase in morphine-induced conditioned place preference in mice. Moreover, an activation of cholinergic interneurons by designer receptors (DREADD) has reduced food consumption motivated by food restriction. The purpose of this study was to investigate whether accumbal cholinergic interneurons mediate alcohol- and morphine-induced conditioned place preference and locomotor activity. The activation of cholinergic interneurons was controlled using DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology. DREADDs are G protein-coupled receptors. Cellular function and activation can be modulated by these receptors. DREADDs are activated by an otherwise inert synthetic ligand, clozapine-N-oxide (CNO). Fluorescent protein, mCherry, is attached to DREADDs so that the expression of receptors in brain tissue can be observed. Cre-spesific adeno-associated viruses (AAV) with DREADD gene were injected bilaterally to the nucleus accumbens of ChATcre mice in stereotactic surgery. The effects of alcohol and morphine were tested with conditioned place preference procedure. Mice were divided to three groups after DREADDs: activating receptor Gq (n = 10), inhibiting receptor Gi (n = 9) and control mC (n = 9). There were both male and female mice in every group. Alcohol did not induce conditioned place preference in any group. The locomotor activity of mice significantly increased after alcohol injection compared to saline injection. However, cholinergic interneurons had no effect on the increased locomotor activity. Morphine-induced conditioned place preference was expressed in every group but there were no significant differences between DREADDs and control group when the first 15 minutes and the whole 30 minutes of the place preference test was analysed. Though, Gq-receptor seemed to decrease the place preference compared to control group when the place preference test was observed in five minute intervals. Morphine also significantly increased the locomotor activity of mice, but there were no differences between the groups. Sex had no influence on the place preference, but female mice were more active than male mice during the alcohol conditioning and the alcohol place preference test. The locomotor activity of female mice also increased more than the activity of male mice after morphine injection. The effect of accumbal cholinergic interneurons on alcohol-induced conditioned place preference remained unclear. Activation of cholinergic interneurons suppressed morphine-induced conditioned place preference compared to control group but not enough so that the effect could be seen during the whole place preference test. The mice were same in the morphine test as in the alcohol test so the mice were conditioned to alcohol before morphine and therefore the results of morphine-induced conditioned place preference are not reliable.

Health related issues are largely regulated at EU Member State level, whereas areas such as internal market and competition fall mainly under the remit of European Union competence. This creates tension not only between legislation governing health and that concerning internal market but also between national and EU legislation. Here the Court of Justice of the European Union (CJEU) plays a key role in developing case-law through its judgments, defining further interpretation both on European and national level. An example of a sector operating at the interphase between public health and internal market interests is community pharmacy, which was chosen as the focus area of the study. The aim of this master's thesis was, through the case-study example of the regulation of pharmacy establishment, ownership and distribution in EU Member States, to perform a documentary analysis on related CJEU judgments, focusing on statements present in them referring to public health and internal market, discussing potential impacts on the community pharmacy sector as well as relating the outcomes to the broader context of European health policy with reference to existing literature. The study material consisted of publicly available documentation related to four judgments (Case C-531/06, Joined Cases C-171/07 and C-172/07, Joined Cases C-570/07 and C-571/07 and Case C-367/12) that were made between the years 2009 and 2014, the first proceedings initiating in 2006. The prevalence and variety of statements related to public health were found to be much higher in the documents analysed compared to those relating to internal market. The most common argumentation present in the judgments was related to the statement that regulation of ownership of community pharmacies can be justified by public health reasons, deriving from the professionalism inherent to pharmacists as well as ensuring balance between public health and economic interests. This transmits a clear message of the importance of public health and indeed the Court has been perceived as a balancing force to the union's liberalisation agenda. Following this it seems unlikely that the interpretation for national regulation would change in the near future, meaning that Member States should be able to maintain community pharmacy regulation, to the extent that it is implemented in a consistent manner. However, there has been indication of other routes being used to push for the liberalisation agenda and therefore it continues being a part of the debate both at European and national level. The findings of this study support literature suggesting that spillover is taking place in relation to the Court of Justice and health. Furthermore, it has been clearly demonstrated that even though officially the EU has very limited competence (authority) in health, its influence on European health policy is in fact highly significant, taking place to a large extent via routes other than those officially assigned to it in relation to health in particular. Whether this is intentional or unintentional, it does not change the fact that health policy is being influenced. When it happens without explicit intention, the processes lose transparency and are driven by other, potentially competing agendas. Therefore it would be important to assess whether the decision making processes and other processes currently shaping the European healthcare policy are in line with what was originally intended and re-evaluate whether this dynamic is the preferred way to proceed in the future.

Manufacturing execution systems (MES) are computer systems which are used for controlling and automating manufacturing processes. They are increasingly adapted in pharmaceutical industry. Implementation solutions differ, however, and there is no single solution which would be the optimal one for all facilities. Each manufacturing facility has their unique properties and needs which have to be reflected in the implementation. A successful MES project will bring plenty of benefits such as more efficient use of resources and automated data transfer, but the roll out phase might turn to be problematic if the processes of the organization have not been analysed thoroughly enough at decision making. This creates the need for systematic analysis of possible to-be implementation scenarios which is based on the value-drivers of the organization and considers the decision from multiple viewpoints. This study presents a holistic value driver-based framework with a mathematical weighing method to allow for a systematic and scientifically justified decision for identification of the optimal implementation depth of equipment management (EQM) in MES. A Delphi study method was utilized in this study to create the framework. The framework was developed based on literature and brainstorming sessions with experts and validated by means of a Delphi questionnaire round with expert panel consisting of professionals representing the major stakeholders of MES system in a pharmaceutical manufacturing facility. Classical additive weighing method was applied to create a mathematical basis for valuation and comparison of the scenarios. The robustness of mathematical method was tested by means of a sensitivity analysis. A benchmarking survey was done to obtain information on current implementation solutions and decisions leading to current situation. The presented method not only addresses the costs but also takes into account intangible factors. Intangible factors include aspects such as good manufacturing practice (GMP) quality and user acceptance which are not directly transferable into quantitative units but are crucial both for pharmaceutical industry and the success of the implementation project. The framework describes the decision in the form of a value tree with three main branches, namely GMP, cost and process&organization which cover the main viewpoints important for the decision. The presented method also allows the weighing of different factors according to current needs of the facility and decision in question. Hence, the presented framework leads the decision maker through a systematic and comprehensive analysis of different to-be scenarios for EQM implementation. The benchmarking survey identified three major factors of a successful MES implementation, namely effort in design phase, well-defined processes and close discussion with production. The value drivers valued highest by the expert panelists were related to GMP quality. As a use case, the presented framework was applied in a parenterals clinical manufacturing facility to evaluate six different to-be scenarios and based on the results one of them was selected by the management to be implemented. The results from the use case indicate that the framework is a valuable tool in a decision making process, and encourage the further utilization of the framework in future implementation decisions.

Antimicrobial resistance is a growing problem worldwide. It has been shown that more than 70% of the bacteria that cause nosocomial infections are resistant to at least one antibiotic commonly used to treat them. Two concomitant phenomena that aggravate the diarrheal disease situation, especially in developing countries, are general contamination (spread of pathogens due to unclean water, poor sanitation, and malnutrition) and resistant bacterial strains (the adverse consequences of infections increase as infections prolong). According to the WHO, foodborne diseases (FBDs) were estimated to have caused approximately 91 million people to become ill and 137,000 deaths in Africa in 2010. The number is about a third of the deaths caused by FBD worldwide. Diarrhea caused about 70% of the FBD burden. Bacteria that cause food poisoning include Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococccus aureus and Yersinia enterocolitica, some of which are discussed in more detail in this master’s thesis; antibiotics against which resistance has developed, how bacteria resist antibiotics, and the emergence of resistance in Africa. The antibiotic resistance of bacteria and the mechanisms of resistance against antimicrobial drugs are also discussed shortly. In addition to food poisoning, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus can cause difficult-to-treat infections such as wounds. In addition, this work has first dealt with antimicrobial plant derived compounds in general and their modes of action, and then focused on compounds, fractions and extracts of species of the genera Combretum, Terminalia, Pteleopsis and Anogeissus, as well as their antibacterial effects and uses in traditional medicine. In addition, the antibacterial mechanisms of action of different groups of compounds have been discussed in more detail. This work also deals with the combination studies of some plant extracts, fractions and compounds with antibiotics. Combination studies with antibiotics have generally been studied less than the antibacterial effects alone or the effects of combinations of many plant extracts, as used in African traditional medicine. The experimental part covers, among other things, the preparation and yield determination of crude extracts (water and methanol) as well as the agar diffusion method, the microdilution method, the Time kill tests and the checkerboard method in interaction tests to determine MIC, MBC and FIC values. Due to the Covid 19 pandemic, study results were obtained only by the agar diffusion method against Bacillus cereus. The most antimicrobial extracts were extracts of species of the genus Terminalia.

Parkinson's disease is a neurodegenerative disease where the nigrostriatal dopaminergic cells die gradually causing severe motor symptoms. Current treatment of the disease relieves the symptoms but does not affect the progression of the disease, nor does it have a neuroprotective effect. The most important drug for the treatment of Parkinson's disease is L-dopa, the precursor of dopamine. With long-term use, L-dopa loses its efficacy and patients start to get adverse effects. The most significant adverse effects are abnormal involuntary movements called dyskinesias. In the literature review of this thesis Parkinson's disease and its treatment is briefly described. Review focuses on the description of the brain cholinergic and histaminergic systems and their receptors along with the available studies about cholinergic and histaminergic neurotransmission in Parkinson's disease 6-hydroxydopamine (6-OHDA) rodent model. The experimental part of this thesis consisted of two different set of experiments and in both of these the dopamine neurons were destroyed unilaterally by injecting 6-OHDA into the striatum. The aim of the first experiment was to examine histamine H3-receptor antagonist JNJ-39220675 and α7-nicotinic receptor agonist PHA-543613, and their combination therapy effects on motor function and the concentrations of striatal neurotransmitters in hemiparkinsonian mice. Effects on motor function were studied two and four weeks after the 6-OHDA injection with cylinder test, the D-amphetamine-induced rotations, and the inverted grid test. After behavioral tests, mice were sacrificed and striatal neurotransmitter concentrations were determinated by HPLC. The aim of the second experiment was to examine if nicotine can relieve L-dopa-induced dyskinesias. In this experiment 6-OHDA was injected at two sites into the striatum, which was intended to produce more extensive destruction of dopaminergic neurons than in the first experiment. The extent of the lesion by 6-OHDA was verified before starting chronic L-dopa treatments with cylinder test. One month after the 6-OHDA injection, five mice were sacrificed and their striatum and substantia nigra sections were measured for destruction of dopaminergic neurons by immunohistochemical TH-staining. Chronic L-dopa treatment with benserazide was started 49‚àí63 days after the 6-OHDA injection. At the same time, mice were divided into two groups. Half of them got normal drinking water and half got nicotine water. During the chronic L-dopa treatment, development of dyskinesias was observed once a week by video tracking. The cylinder test was also done once again after starting the L-dopa treatment. In the first experiment, H3-receptor antagonist JNJ-39220675 showed promising results in improving motor function. Mice used the impaired (contralateral) paw more in the cylinder test and rotated less to the ipsilateral side in the D-amphetamine-induced rotation test than control animals two weeks after the 6-OHDA injection. Combination therapy also reduced the ipsilateral rotations but in the cylinder test it had no effect two weeks after 6-OHDA injection. Because the asymmetry in behavioural tests were caused by destroying dopaminergic neurons, balancing of the motor skills can result from decreased levels of dopamine in the intact side or from increased dopamine levels or stronger dopaminergic postsynaptic transmission in the lesion side. The results four weeks after 6-OHDA injection are not reliable because the striatal samples showed that dopamine concentrations in the lesion side were very close to that of the intact side indicating recovery from the lesion. In the second experiment, mice developed dyskinesias which were decreased with nicotine treatment. Mice also used the contralateral side paw less indicative of loss of dopamine neurons. In agreement, TH-immunostaining confirmed significant loss of TH-positive neurons. Based on these findings, the 6-OHDA injection site, the selected drug doses, and the experimental design seem to fit the evaluation of dyskinesias. The occurrence of dyskinesias and nicotine's effect on them was seen strongest in the body movements. Dyskinesias in forelimbs were minor, but the nicotine treatment decreased them also.

Parkinson’s disease is a progressive neurodegenerative disease, in which dopamine neurons are dying in the nigrostriatal dopaminergic pathway. This causes motor symptoms such as slowness of movement, tremor, and rigidity. In addition, various non-motor symptoms appear. All currently used medicines are symptomatic, and there are no disease modifying treatment available for Parkinson’s disease. Several neurotrophic factors have shown promise in animal models of Parkinson’s disease. One of those is cerebral dopamine neurotrophic factor (CDNF) which has been studied in different animal models, including rodents and non-human primates. CDNF is a secreted protein but it is also localized in endoplasmic reticulum (ER). CDNF has two domains, N-terminal and C-terminal, which may have distinct functions. CDNF can be retained in the ER by the ER retention sequence at the end of the C-terminal domain. The C-terminal domain also has an evolutionarily conserved disulfide bridge which is crucial for the biological activity of CDNF. The exact mechanism of CDNF is still unknown. However, it has been shown that CDNF affects the unfolded protein response (UPR) in the presence of ER stress. Neurotrophic factors do not penetrate blood-brain barrier (BBB), for this reason, they need to be injected directly to the brain. Penetration of the BBB is also a problem in the treatment of many other diseases. Various methods for enhancing the BBB penetration of drugs have been studied. For example, permeability of the BBB can be temporarily increased by focused ultrasound combined with microbubbles. Another possibility is the use of a carrier molecule, which can be transported through BBB via specific transport mechanisms. Furthermore, molecule modification offers many applications to achieve enhanced BBB penetration. In view of peripheral administration, a next generation variant of CDNF (ngCDNF) has been developed. The efficacy of this novel variant after intrastriatal injection is equal to that of CDNF in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease. Systemic administration could also enable treatment of non-motor symptoms of Parkinson’s disease. The aim of this experiment was to study the effects of subcutaneously injected ngCDNF on rotation behaviour, and nigrostriatal TH-positive cells in rats with 6-OHDA lesions. 6-OHDA was injected unilaterally to three different sites in the striatum. Two weeks later, the lesion size was estimated, via amphetamine- induced rotation test. ngCDNF, at two dose levels, was injected twice weekly for three weeks. Amphetamine-induced rotation test was assessed every other week, until week 12. At the end, optical density of tyrosine hydroxylase (TH) was measured from sections of the striatum, and TH positive cells in the substantia nigra were counted. In addition, the effect of ngCDNF on anxiety and depression like behaviour, learning, and locomotor activity were studied at three different levels in naïve mice. Behaviour was analyzed by open field test, forced swim test, and fear conditioning test. The ngCDNF did not seem to have clear effect on rats’ behaviour or TH positive cells and fibers compared to the control group, but positive tendency was found in the group with lower dose. The reduced efficacy of ngCDNF,via subcutaneous administration, is likely due to rapid metabolism and insufficient entry of the active form to the brain. In naïve mice, ngCDNF did not reduce anxiety-like behaviour and did not affect locomotor activity after subcutaneous injections. This result supports previous findings, which suggest that the effects of CDNF are specific to the toxin treated cells and CDNF has no effect in naïve animals.

Medication errors due to infusion pump programming errors are common in neonatal intensive care units and often lead to overdoses of medicines. Medication errors can be prevented with dosing limits set into the smart infusion pump drug library. However, alert fatigue caused by unnecessary alarms has been identified to hinder their use. To ensure the benefit of dosing limits as a defence of intravenous medication process, the aim of this study was to define the dosing limits to the drug library for certain high alert medications, and to pilot them in the neonatal intensive care unit. The study was based on the theoretical framework of preventive medication risk management. Based on the results, the suitability of the dosing limits for the use of the unit and the patient group was assessed. This mixed method study employed register-based research methods. The research data consisted of the infusion rate related medication errors (n=21) reported to the HUS HaiPro-system between January 2018 and December 2019 in the HUS Neonatal Intensive Care Unit. The data was analysed qualitatively and quantitatively to describe the infusion rate related errors, and to identify their mechanisms and contributing factors. Based on the identified mechanisms of the errors, simulated test patient cases were developed. Dosing limits were defined by a multidisciplinary expert group for certain high alert medications, and their suitability for preventing medication errors was investigated by programming infusion pumps according to the test patient cases and analysing the pump alerts. Based on the identified mechanisms of infusion rate-related medication errors (n=21) in the HaiPro-reports, 2-, 5-, and 10-fold infusion rates as well as mixing of infusion rates between medicines were established as test patient cases. As a result of the tests (n=226), the infusion pumps did not alert when programming normal infusion rates (n=32) and 73% (n = 70/96) of the erroneous 2-, 5-, and 10-fold infusion rates were prevented. 10-fold infusion rates were inhibited in all cases (n=32). Interference of infusion rates between medicines was prevented in 24% (n=24/98) of the cases. According to this study, significant infusion rate related medication errors can be prevented in the neonatal intensive care unit with the multidisciplinarily defined dosing limits set in the infusion pump drug library. However, they do not prevent all the infusion rate related medication errors alone, and therefore additional defences are needed. In addition to the neonatal intensive care unit, the method used in this study to define and test the dosing limits may be applied in other pediatric units in the future. By using this method, the suitability of the dosing limits for the use of the unit and the patient group can be ensured before integration of the barrier and thereby promote the benefits of its use. The suitability of the dosing limits set into the infusion pump drug library should be assessed again after implementing the defence into the neonatal intensive care unit.

Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.

Solid materials can exist in two major forms: in crystalline or amorphous form. Amorphous form is defined as no long term order existing in solid structure in molecular scale. Amorphous materials have different physicochemical properties compared crystalline forms of same substance. Amorphous materials doesn't have sharp melting point as crystalline materials. When heated above so called glass transition temperature amorphous materials become rubbery (plasticization) and when cooled below they become glassy (hard and brittle). Amorphous forms can also have different dissolution properties which makes them useful in formulation of poorly soluble drugs. Amorphous forms are less stable compared to crystalline form. That's due amount of free energy stored in it's structures. Amorphous materials can be manufactured in many ways including quench cooling, hot-melt-extrusion, spray drying and lyophilisation (freeze drying). In experimental section effect of grinding method in properties of amorphous indomethacin was studied. Amorphous indomethacin was prepared by quenching of melt in liquid nitrogen. Properties of amorphous indomethacin was studied by x-ray powder diffraction and differential scanning calorimetry. Measurements were performed in different time stamps varied form 0 to 92 days. Measured properties were crystalline content, glass transition temperature, change in heath capacity, heat of crystallization, heat of melting and melting points of crystallized forms. Calorimetry data was recorded only from totally amorphous samples. It can be seen in results that different patches are not comparable statistically but when comparing room temperature ground and liquid nitrogen ground samples to each other differences can be found in every set. Difference is observed in initial time of crystallization (time when crystallinity can be measured first time) and in thermodynamical properties such as change in heat capacity, glass transition temperature and heat of melting. Solid dispersions of indomethacin and xylitol were prepared in 3 different compositions (5%, 10% and 20% xylitol in indomethacin). XRPD and DSC data were measured at different time stamps (aged 1 to 63 days). 5% and 10% dispersions found to be stabile and being amorphous in all time stamps. 20% dispersion was already partly crystallized at 63 days (especially liquid nitrogen ground sample).