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

Prolyl oligopeptidase (PREP) is endopeptidase which cleaves short proline containing peptides. Abnormalities in brain PREP activity has been connected to neurodegenerative diseases. Recently it has been detected that besides its proteolytic activity PREP interacts directly with other proteins which might contribute to generation of neurodegenerative diseases. Further it has been discovered that certain small molecular PREP inhibitors are able to modify these protein-protein interactions (PPIs) and thus have a potential to alleviate the progression of neurodegenerative diseases. This has led to the development of novel second generation PREP ligands which lack the strong inhibitory activity but are potent compounds on modifying the PPIs. Thiazole structure containing PREP modulators has provided most promising class of compounds. It has been detected that these compounds mediate their effects via novel binding site on the enzyme and these effects are not connected to the inhibition of the enzymatic activity. The synthesis of these thiazole containing PREP modulators has proven to be demanding since it have involved a usage of laborious synthesis route and provided low yields. The aim of this research was to examine the synthesis of 2-(2-benzimidazol-1-yl)ethyl)- 4-methyl thiazole containing PREP modulators via previously reported synthesis route. Another aim was to design and develop a synthesis route for 2-(2-(benzimidazol-1- yl)ethyl)-5-bromo-4-methylthiazole, a molecule which serves as valuable intermediate for the lead optimization and generation of second-generation PREP modulators. A synthetic route for 2-(2-(benzimidazol-1-yl)ethyl)-5-bromo-4-methylthiazole was successfully developed. Despite that the total yield of the route remained low. When searching the reasons for the low obtained yield the chemistry behind a thiazole creating cycloaddition reaction and an aromatic halogenation was examined. This led to the discovery of a rare cationic compound which was found to be synthesized from previously undescribed starting materials.

In pharmaceutical sciences the pharmaceutical supply chain is often examined from a quality perspective. As the world is becoming more uncertain due to pandemics and conflicts the societal and political situation where the supply chain operates should be considered. Understanding the big picture helps to consider the cause and effect that lead to medicinal shortages. Effects of these shortages can be seen on every level of the supply chain from the manufacturer to the patient, which is why actors on the supply chain can benefit from understanding the background factors. The aim of the master’s thesis was to examine, whether pharmaceutical field actors could affect realisation of geopolitical risks by preparation and examination that the pharmacotherapy would not be interrupted. Second aim was to bring forward political and societal aspects to pharmaceutical availability which are often side-lined by quality aspects in pharmaceutical context. The study was conducted as a qualitative semi-structured interview between October 2022 and February 2023. Participants (n=11) were recruited via e-mail using representative sampling. Due to recruitment problems, convenience sampling was also used. Questions presented to the interviewees were depending on the group (n=3) they were assigned. Term ”geopolitics” was associated mainly with political and economic factors. Main geopolitical risks for Finland were seen to be small market size and distant location. For Europe, the risks were centralisation of manufacturing (and dependence) to Asia due to economic factors and long disruption-prone supply chain. Transport of pharmaceuticals from Asia to Europe was with sea and air cargo. Inside Europe, transport to Finland was with mainly with lorries utilising ferries. Rail transport was mentioned to be used only on one interview. The transport routes were seen to be staying the same in the future both for Asia-Europe and Europe-Finland. Even though risk management is an important part of functioning of every company, the change in the type of risks requires a new mindset in the pharmaceutical field both from the individual actors as well as international organisations. From risk of strikes and natural disasters we have moved to trade wars, pandemic restrictions, and the strategic acting of industries critical to society. At the same time, the ability/willingness of societies to pay for pharmaceuticals is decreasing, which leads to the manufacturers to find new ways to ensure business.

Alcohol use disorder (AUD) is a chronic relapsing brain disorder causing a high burden of disease and significant social and economic consequences to both individuals and society. Alcohol addiction, the most severe form of AUD, is characterized by compulsive seeking and use of alcohol, loss of control over limiting alcohol consumption despite negative consequences, emergence of negative emotional states, and long-lasting vulnerability to relapse related to alcohol abstinence. Powerful craving for alcohol and the chronic, relapsing nature of the disease are major problems complicating recovery from alcohol addiction and predicting poor clinical outcome. Relapse to alcohol intake can occur even after an extended period of abstinence in humans, relapse rates being highest during the first three months of alcohol withdrawal. Associative learning is a critical factor in alcohol craving when alcohol consumption is accompanied by conditioned stimulus. Cues associated with alcohol are known to induce craving and alcohol-seeking behavior increasing the risk of relapse, and this craving can be triggered by alcohol itself, alcohol-associated stimulus, or stress. Chronic alcohol exposure has been linked to changes in synaptic plasticity, neurogenesis and cell-signaling. Thus, elucidating the neural mechanisms that underlie alcohol craving and relapse would help to understand the pathology of alcohol addiction and facilitate the development of efficient treatments. In this experiment, the effects of subanesthetic-dose 10 mg/kg ketamine, an NMDAR antagonist and a major inducer of synaptic plasticity, on cue-induced alcohol-seeking behavior after withdrawal were investigated in social context in female mice. Mice were trained to voluntarily drink alcohol, and a novel methodology to study alcohol-seeking behavior after withdrawal allowed to perform the experiment with a minimum of human interference in totally automated social home cage environment. The analyses of behavioral data showed that pairing sweetened alcohol with conditioned stimulus resulted in cue-induced alcohol-seeking behavior, and no differences in alcohol conditioning were observed between treatment groups. However, the behavioral activity in extinction tests after withdrawal showed that alcohol-seeking behavior was not altered by ketamine treatments. In biochemical analyses, the effects of subanesthetic-dose ketamine on ΔFosB and BDNF protein levels in the brain areas important for alcohol addiction were studied. ΔFosB expression levels in the mouse nucleus accumbens were analyzed with western blot and BDNF protein levels in the mouse prefrontal cortex were determined using enzyme-linked immunosorbent assay (ELISA). The results from biochemical analyses showed that levels of ΔFosB and BDNF were unaltered by ketamine treatments. Anyhow, the experiment provided important insights into the interactions of ketamine and alcohol craving and relapse, a topic that has been insufficiently studied in novel preclinical models.

There is a growing need for antibiotic stewardship since antibiotic resistance is a global and increasing problem. One option would be outpatient parenteral antibiotic therapy (OPAT) which has evolved globally since 1970s. In Helsinki, it has been applied in hospital-at-home units since 2018 with elastomeric antibiotic infusion pumps that enable 24-hour continuous infusion and normal daily life for the patient. The continuous infusion via infusion pumps enables the use of first-line antibiotics whereas with intermittent infusions broad-spectrum antibiotics, that require doses less frequently, are a more likely choice. Thus, antibiotic therapy with elastomeric infusion pumps is likely to enhance antibiotic stewardship. The aim of the study was to analyse if treatment with elastomeric infusion pumps in hospital-at-home unit is cheaper than theoretical hospital stay and to compare the costs. An economic evaluation was performed with the assumption that hospital-at-home care and hospital stay are equal when it comes to the outcomes of the therapy. The economic evaluation was made with cost-minimization analysis. Data were collected manually by nurses in three hospital-at-home units in Helsinki between September 2021 and March 2022. Patients’ age, gender, indication and length of the antibiotic infusion pump therapy, distance from the hospital-at-home unit and problems with the therapy were collected. Cost information were received personally from City of Helsinki and taken from a paper of Finnish Institute for Health and Welfare. The data included 57 patients, of whom one had two treatment periods. The mean age was 60 years. Thirty-two percent of patients were female and 68% were male. The most common indications were bacteremia (n=24) and erysipelas (n=18). A total of 625 hospital bed days were saved, which is 10,8 days per patient on average. Cost savings with elastomeric infusion pump therapy were 89 000–116 000 euros compared to the theoretical treatment in a hospital ward depending on the cost information being used, which is 37–48% of the theoretical hospital stay costs. An economic evaluation was made separately for the treatment of bacteremia. The cost savings were 47 600–150 700 euros or 37–69% of the theoretical costs. Savings in travel costs were 2 300–3 800 euros when elastomeric pump therapy was compared to the conventional hospital-at-home intermittent infusion therapy of 4-6 nurse visits per day per patient. In conclusion, elastomeric infusion pump therapy in hospital-at-home units in Helsinki results in cost savings of 37–48% compared to theoretical hospital stay costs from the perspective of the entity responsible for the costs of the treatment.

Municipal case management is an activity that assesses various functional capacity indicators, utilizing the elderly’s state of health and coping in everyday life. The goal of case management is to refer clients to suitable services, such as home care or a doctor's visit. The problems related to drug treatments are only superficially reviewed. The involvement of a pharmacist in the assessment of case management would provide an opportunity to address the problems of pharmacotherapy and to provide adequate support for the implementation of pharmacotherapy. In this thesis, a remote service of a pharmacist was piloted for new clients over the age of 65 living at home as part of case management. Pharmacist reviewed medications remotely using medication risk management checklist LOTTA. The study examined the suitability of the LOTTA for medication reviews and the problems associated with medications of the elderly participating in case management. In addition, the suitability of pharmacovigilance assessments as a remote service as part of a comprehensive assessment of functional capacity and coping with everyday life was examined. The research material was collected at the case management unit of the city of Turku. The study involved 50 volunteer Finnish-speaking customers over the age of 65, for whom were assessed for a case management at Turku's case management unit. In addition to the assessment of normal case management, two pharmacists with comprehensive medication review qualifications reviewed medications using the medication risk management checklist LOTTA. Subjects were interviewed by telephone. If the pharmacist estimates that the subject will benefit from a multi-professional comprehensive medication review, the physician and pharmacist collaborated to conduct a review using a videophone application. Subjects background information, responses, observations made by pharmacists, and actions taken by physicians were recorded on an electronic form and analyzed. The mean age of the study participants (n = 50) was 82 years (range 67–98). Of these, 36 were women (72%) and 14 were men (28%). Most subjects were multidrug-treated (average medication 10.3, range 3–28). Each subject had at least one drug can be used with consideration for use in the elderly, as defined in the Fimea Drug 75 + database (Class C). 30% of subjects did not have a medication list and 34% reported lack of regular medication monitoring. 96% of the subjects had experienced a symptom on the LOTTA list that repeatedly interferes with their lives. The most common of these were problems such as constipation (54%). Pharmacists proposed changes for medication for 96 % of subjects. The most common proposed change was a change in the time of dosing (46%). Pharmacists estimated that 14 (28%) subjects would benefit from a multi-professional comprehensive medication review. In these cases, pharmacists made an average of 8.1 proposed changes for the physician, and the physician made an average of 6.9 changes for each subject. The most common challenges in coping with medication were symptom, which may be due to adverse drug reactions, a lack of follow-up to medication, and the absence of a treating physician. The results suggest that medication should be reviewed during the case management. The LOTTA list made it possible to identify and address the pharmacological problems of the elderly. The participation of a pharmacist in the assessment of the need for a multi-professional service remotely was possible, but it must be further developed. More research is needed on the benefits of multi-professional case management with a larger sample size.

Parkinson’s disease is a progressive neurodegenerative disorder which is commonly treated with Levodopa (L-dopa) and Dopa Decarboxylase (DDC)/ Catechol-O-methyltransferase (COMT) inhibitors. The main problem with this treatment is the intestinal conversion of L-dopa to dopamine despite DDC and COMT inhibition which probably occurs by the Tyrosine Decarboxylase (TyrDC) of intestinal bacteria. This study aims to find new inhibitor molecules that would have dual inhibitory effects towards both DDC and TyrDC enzymes. Currently, available DDC inhibitors cannot inhibit the bacterial TyrDC enzyme. A recently found TyrDC inhibitor (S)-α-Fluoromethyltyrosine (AFMT) is not able to inhibit the human DDC enzyme, respectively. The dual inhibition of both decarboxylases could reduce the dosing frequency and side effects related to L-dopa. In addition, the object of this study is to produce the human DDC enzyme by DNA recombinant technique as well as develop and optimize a biochemical DDC inhibition assay to study the effect of selected small molecule compounds towards inhibition of DDC and L-dopa conversion in E. faecalis model by previously developed cell-based assay. The human DDC was successfully produced in a TB medium with a yield of 1.8 mg/mL. The Km value of DDC for L-dopa was found to be 34 μM which indicates a high affinity for L-dopa. In the optimization of the DDC inhibition assay, the sample volume of 80 μL and incubation time of 3 h with detection reagent was found to give the highest fluorometric signal with sufficient robustness. In the initial screening of test compounds, 14 % of the compounds (n=59) were classified as active towards human DDC, while 31 % of the compounds were active towards L-dopa conversion in the E. faecalis model. Of those compounds, five were having dose-dependent dual inhibitory effects, but the IC50 values of them were higher compared to either carbidopa or AFMT. The most effective compounds were 8009-2501 (IC50 37 μM in E. faecalis model and 19 % inhibition at 1000 μM towards DDC enzyme) and 8012-3386 (IC50 248 μM in E. faecalis model and 37 % inhibition at 1000 μM towards DDC enzyme). However, this study confirms the possibility to find dual decarboxylase inhibitors. By optimizing the structures as well as investigating the mechanism of action, selectivity, and structure-activity relationships of the most active compounds, it is possible to find more effective dual inhibitors in the future.

Lecithin:cholesterol acyltransferase (LCAT), a key enzyme in maturating high-density lipoprotein (HDL) particles, has been targeted to promote the efficiency of reverse cholesterol transport by small molecular positive allosteric modulators (PAM) of Daiichi Sankyo. For a set of these compounds their Vmax and EC50 values and binding site in the membrane-binding domain (MBD) of LCAT have been determined. Through molecular dynamics (MD) simulations we previously found a metric that qualitatively described which compounds were active, so in this study we aimed to improve it by finding a quantitative metric. This led to the discovery of the Cα distance between CYS50 and ASN65, which correlates with this set’s Vmax values and which can be utilized to predict the Vmax values of novel compounds. Additional simulations were performed to discover whether this metric is changed by a lipid interface present, and to reveal a likely entry pathway PAMs take. As LCAT activation is likely a benign and potentially overlooked effect, we performed a virtual screen of FDA-approved compounds and secondary metabolites associated with LCAT. From secondary metabolites, a key finding was that flavonoids were overwhelmingly associated with LCAT and had a high binding potential to the MBD in docking simulations. The best binding compounds were subjected to MD simulations to discover their Vmax values using the discovered metric. This provided us with a set of compounds, which can be used to validate our in silico model in vitro. Should this model be validated, it can be used in optimising and discovering novel PAMs of LCAT, and it would bring evidence to the benefit of MD in drug discovery processes in general. Furthermore, if our discovered compounds can activate LCAT in vitro, they may be used as precursors for novel PAMs or as therapies by themselves not only for LCAT deficiencies, but perhaps for atherosclerotic cardiovascular diseases as well.

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.

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.

P-glycoprotein (ABCB1, MDR1) is an efflux transporter expressed widely through the body, but mainly focused on tissues that have protective or excretive function, such as liver and blood-brain-barrier. Many clinically used drugs from variety of therapeutic groups are substrates of P-glycoprotein, and changes in the function of P-glycoprotein may have impact on the drugs pharmacokinetics and -dynamics. The impact of genetic polymorphism on P-glycoprotein activity have been investigated for several years, but due to contradictory results no consensus has been made. The aim of this Master’s thesis was to investigate the effect of five different P-glycoprotein single nucleotide polymorphisms (SNPs) on transport activity. The study was performed by Spodoptera frugiperda (Sf9) membrane vesicles expressing P-glycoprotein variants. Baculovirus-derived expression system was used to introduce the ABCB1 gene to the cells. Vesicle assay was performed with N-methylquinidine (NMQ), and ATP-dependent transport of P-glycoprotein variants was compared to the reference gene. Amino acid change Cys717Tyr led to no transport activity compared to reference gene, and Arg669Cys associated with higher transport activity of NMQ. Arg588Cys, Ser795Cys and Ile836Val indicated no effect on the transport activity. Other aim for this Master’s thesis was to create a new in-house protocol to study P-glycoprotein polymorphism in vitro. Substrate accumulation assay for Rhodamine-123 in Sf9 cells analysed with flow cytometry was established, as flow cytometry is widely used method in other laboratories to study P-glycoprotein polymorphism. The baseline for flow cytometry assay was created successfully by optimizing substrate concentration and incubation time. According to the results, SNPs can impair P-glycoprotein function. New method to study P-glycoprotein function was created, and this method can be used to further study the effects of genetic polymorphism of P-glycoprotein and to compare the result between studies. The results gained from these in vitro studies can be utilized to understand in vivo pharmacogenetic findings.