Browsing by master's degree program "Proviisorin koulutusohjelma"

Parenteral products are sterile products that are administered as injection, infusion or implantation. Administration of the contaminated parenteral product can cause severe consequences such as sepsis meningitis and even death. Most of the parenteral products used at the hospitals needs to be compounded (e.g. dissolved, diluted) before administration. Whenever possible, compounding should be done in biological safety cabinet using aseptic techniques. According to previous studies errors in aseptic techniques are quite common. Aim of this study was to compare three different environments as compounding area and their effect to the sterility of the compounded parenteral product. Based on the results of this study, changes to the protocols of the hospital could be made. Altogether 220 samples were compounded at two pediatric wards at HUS Helsinki University Hospital. Six volunteers (one pharmacist and five nurses) participated from both wards and each compounded 18 samples in three different environments (patient room, medicine room, biological safety cabinet). The samples were tested for the sterility by membrane filtration within 4 hours or after 24 hours of storage in the refrigerator. The investigator used an observation form to observe the compounding procedures. Environmental monitoring (settle plates) and monitoring of personnel (glove samples) were conducted. Almost all compounded samples (99%, n=213/215) were sterile. There were no significant differences in the contamination rate of the compounded samples between different environments. Five of the collected samples were excluded, because they were contaminated during the sterility test. According to observations, aseptic techniques were well followed. However, disinfection of the septum of the medicine bottle, hand hygiene and cleaning of the compounding area were observed to be deficiently completed. Even though there were lot of variation in the environmental and personnel monitoring the results were quite good. Results from the environmental monitoring were compared to the recommended limits of EU GMP for clean areas. One compounded sample was contaminated with Diezia maris and Corynebacterium mycetoides but the contaminants from the other contaminated sample could not be identified. Aseptic techniques were mainly well followed, however compounding should be done in the biological safety cabinet, since the environmental monitoring results show that the biological safety cabinet was only environment which was within the recommendation limits of the EU GMP for the compounding area of parenteral products. Protocols of the hospital could be changed, since there was no correlation between higher contamination rate of settle plates or compounded samples and not wearing mask and hair cover while compounding in the biological safety cabinet.

The most typical symptoms of dementia include impairment of cognitive brain functions, such as memory and thinking. Most common forms of dementia include Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, which is caused by degeneration of the frontotemporal lobe. Alzheimer's disease is the most common form of dementia, covering about 75% of all the cases. The pathophysiology of Alzheimer's disease includes beta-amyloid plaques and tau proteins, which accumulate in the brain, and which have been linked to damage to nerve pathways and the appearance of the typical symptoms of the disease. The disorder is progressive, but the exact cause remains unknown. However, old age (>65 years), the APOE-4 gene, lifestyle, and some comorbidities, such as cardiovascular diseases, are considered risk factors. Even though extensive research has been conducted, there is currently no curative treatment for Alzheimer's disease. Sleep disorders can be both a symptom of Alzheimer's disease and a risk factor for the onset of the disorder. Therefore, the mechanisms of sleep and circadian rhythm are connected to the pathophysiology of Alzheimer's disease, for example through the glymphatic system that cleans the brain mainly during deep sleep. Many drugs for Alzheimer's disease have a recommended time of administration. The dosing time can be very important issue in terms of the effectiveness of the drug. According to a recent study, sleep and circadian rhythm have not been considered in most studies on new rapid-acting antidepressants. Therefore, we carried out an analogous systematic literature review for Alzheimer's disease and dementia. The aim of this study was to find out whether sleep and circadian rhythm have been considered in the most cited preclinical and clinical drug research articles for Alzheimer's disease and dementia during the last decade (2010–2020). In addition, it was examined which drug groups the studied compounds belonged to, and what was the sex distribution of the test subjects in the studies. The number of subjects was also determined from clinical studies, and the animal species from preclinical studies. The research articles analysed in the study were collected with a systematic literature review of Scopus database. The study found that most studies did not include any consideration of sleep or circadian rhythm. Most of the investigated compounds were small molecules, followed by supplements and herbs, and rest classified as biological drugs. Most of the clinical trials were relatively small studies with less than a hundred subjects or hundreds of subjects. Among the 100 most cited clinical research articles, there were 14 reanalyses and observational studies that were not included in this analysis of subject numbers. In clinical studies, most of the test subjects were usually female, while preclinical studies used commonly male animals. To conduct more open and reliable science in the future, drug research should pay more attention to the subjects’ sleep patterns, the time of drug administration, and reporting on these issues in the articles, which is usually part of the requirements of scientific journals. This could potentially narrow the translational gap between preclinical and clinical research.

Adequate vaccine coverage and vaccine refusal have been prominently featured in the media during the COVID-19 pandemic. The decision-makers have considered adequate vaccine coverage important for maintaining the capacity of healthcare. The purpose of this study is to produce population-based research data about the factors affecting the willingness to take the COVID-19 vaccine. With the help of that information, it is possible to identify the factors that influence individual’s decision-making about whether to take the COVID-19 vaccine. The data of the study is the first Kansalaispulssi-data of the year 2022. Kansalaispulssi is a survey, made by the assignment of the State Council, which is used to research Finnish citizen’s views of the current topics. The research design is cross-sectional. The analysis of the study is done by crosstabulation using the Chi-square test. Age and financial situation of the person were related to the willingness to take the COVID-19 vaccine and the person's view of the role of the vaccine in preventing severe corona disease and getting rid of the COVID-19 pandemic. Age also influenced the person's view of the importance of the vaccine in preventing the COVID-19 disease. Gender, province, or educational level had no effect on a person's vaccine views in this study. Based on the research, Finnish citizens are very pro-vaccine. They also understand well the role of the vaccine in preventing a serious illness caused by the disease.

Digitalization of health care and the corona pandemic have increased availability and use of online services provided by community pharmacies. In Finland, willingness to use online pharmacy services has been studied from population approach. Less is known about the user satisfaction with the core online pharmacy services such as dispensing and medication counseling services. This study aimed to investigate satisfaction with the University Pharmacy’s online services (ya.fi) from customers’ approach. Primarily, customer satisfaction with dispensing and medication counseling services was assessed. In addition, characteristics affecting customer satisfaction were analyzed. The conceptual framework of the study was Andersen's Model of Health Services Use. The data for this study was collected by a cross-sectional survey conducted in August 2020 among University Pharmacy’s online pharmacy customers who had made a purchase during the last three months. The survey instrument consisted mainly of structured Likert-scale questions, which were used to form two sum variables: satisfaction on online dispensing services (3 variables, Cronbach's alpha 0.803) and satisfaction on online counseling services (2 variables, Cronbach's alpha 0.883). Satisfaction on online dispensing services was studied through willingness to recommend and use the services in the future. Satisfaction on online counseling services was studied through a comparison of medication counseling on an online pharmacy and a conventional pharmacy. IBM SPSS (28) -software was used for statistical analysis consisting of bivariate (Kruskal-Wallis and Mann-Whitney U tests) and multivariate (generalized linear model) analyses to identify factors affecting satisfaction with dispensing and medication counseling services. Of 15 172 invitations sent to fill out the survey, 2555 eligible responses were received (16 %). Of the respondents, 92 % had concomitantly used the services of a conventional pharmacy. . The mean of satisfaction on online dispensing services on a scale from 1 to 5 (5 being the most positive option "completely agree") was 4.3 (SD 0.8). Similarly, the mean of satisfaction on online counseling services was 3.7 (SD 0.9). According to the multivariate analyses, significant characteristics affecting satisfaction on online dispensing services were age, form of living (alone/family with children/couple), purchase of prescription or OTC medicine, frequency of internet use and previous visits to a conventional pharmacy. Characteristics affecting satisfaction on online counseling services were education, purchase of prescription medicine, use of chat information service, frequency of internet use and previous visits to a conventional University Pharmacy outlet. The services on ya.fi online pharmacy rated most important by the respondents were services about medicine availabilities (in conventional University Pharmacy outlets and during a shortage) and information about medicines (prices, Kela reimbursements and information about customers' prescriptions). Customers were satisfied with online dispensing and counseling services. Online dispensing services received a higher satisfaction rate than online counseling services. Customers who had used the chat service and purchased a prescription medicine online were more likely to assess online counseling services to be equal or better than in a conventional pharmacy. Active use of internet and purchases of medicines online were factors connected to higher satisfaction with online dispensing services. Most online pharmacy customers had also visited conventional pharmacies. The results from this study can be utilized in the development of online and other pharmacy services.

Parkinson´s disease (PD) is the second most common neurodegenerative disease in the world after Alzheimer´s disease. There is still no drug that alters the state of the disease. It has been found that Endoplasmic reticulum (ER) stress is one mechanism in PD. ER stress occurs due to accumulation of unfolded proteins. ER stress triggers Unfolded protein response (UPR) that protects against ER stress by decreasing unfolding of proteins. In the beginning, UPR has protective effect, but in prolonged ER stress UPR triggers apoptotic cell death. There are several key mediators in the UPR pathway. Characterisation of ER stress in PD models may be important for the current and future drug development of PD. If ER stress is a significant factor that affects the disease development, it would be important to find a drug that alters these mechanisms and UPR. This may be a way to halt the disease development. Different animal models of PD, like 6-OHDA (6-hydroxydopamine) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model, have similarities in their mechanisms. It has been found that ER stress occurs both in the brain of PD patients and animal models of PD. That is why studying and further characterisation in animal models is relevant. The aim of this study was to characterize ER stress in 6-OHDA rat model. The expression of some key mediators of the UPR were determined in this study. There were male and female Spraque Dawley rats in this experiment. 6-OHDA or saline was injected intrastriatally in 3 spots by stereotaxic surgery. Two weeks after 6-OHDA lesions, amphetamine-induced rotation test was conducted to the rats. The rats were divided into groups based on lesion size according to the results. For this study, the rats were euthanised at week 2 or week 4 post lesion. The rats were euthanised by carbondioxide, and the death was confirmed by decapitation. The brains were collected and stored in -80°C. Striatum and substantia nigra were collected later. Total RNA was isolated from these samples. Part of the RNA sample was used to conduct cDNA synthesis. Finally, the gene expression of Atf4, Ire1α, Xbp1s, Xbp1t, Grp78 and Chop was measured from these cDNA samples by qPCR (quantitative polymerase chain reaction). The qPCR data describes the expression of exact gene. The data was processed prior to statistical analysis. By statistical analysis, it was possible to compare the expression of these genes between 6-OHDA group and vehicle group. In addition, comparison was made between 6-OHDA treated groups at week 2 and 4. According to the results, only Chop expression had increased in 6-OHDA lesioned rats at week 2 compared to the vehicle group. In other genes there were no statistical differences, unlike in several other studies where the expression was found to be increased. Thus, the characterisation of this model requires further studying, possibly by increasing the sample size and studying later time points as well.

Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic neurons that form the nigrostriatal pathway gradually die. This causes the main motor symptoms of Parkinson’s disease: tremor, rigidity and bradykinesia. While PD affects 1-2% of total population, all currently used medicines are symptomatic, and there is no disease modifying therapy available at present. Although several different animal models for Parkinson’s disease exist, the lack of adequate animal models is often cited as a major obstacle for predicting the clinical success of potential drug candidates. Lewy bodies (LBs) are abnormal aggregates that develop and spread inside nerve cells of human PD patients, their main structural component being α-synuclein. Because α-synuclein is thought to play a major role in the pathology of PD, much research has been focused on it. Different α-synuclein-based animal models of PD exist today, of which the most recent are based on using direct injections of preformed α-synuclein fibrils (PFFs). These new α-synuclein based disease models have helped to understand the disease process in PD better, but cell death in these models takes longer to achieve and is often less pronounced compared to traditional neurotoxin based animal models of PD. The aim of this study was to participate in the development and characterization of a novel mouse model of PD. This new model combines PFF-injections with the commonly used neurotoxin 6-OHDA, which should result in more robust dopamine pathway degeneration than what is seen with the current PFF-based models. The main hypothesis of this study was that the combination of intrastriatal injections of PFFs and a low dose of 6-OHDA would cause gradual spreading of the α-synuclein aggregation pathology in the nigrostriatal dopamine pathway and progressive dopamine neuron loss leading to motor deficits. C57BL/6 mice were stereotactically injected unilaterally with both PFF and 6-OHDA, and their performance was assessed every other week with different behavioral tests until week 12. At the end, brains were collected and optical density of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was measured from striatal sections, and TH and DAT positive cells in the substantia nigra were counted. The amount of Lewy bodies present in the brain slices was also counted from the cortex and substantia nigra areas of the brain. In the histological assays, statistically significant reductions of both TH and DAT were found in the brain sections of the PFF + 6-OHDA combination group and the amount of TH and DAT positive cells were lower in this group compared to the group receiving vehicle treatment only. However, the results of behavioral tests were non-significant, although a non-statistical positive trend in the amphethamine-induced rotations test was observed where mice receiving PFF + 6-OHDA rotated the most. Taken together, combination model that utilizes both PFF and 6-OHDA injections seems like a promising candidate in modelling PD in mice, but much more research and further development of the model is required before this combination model is ready and robust for use in drug development.

Cytochrome P450 (CYP) enzyme inhibition is one of the most common reasons for adverse drug-drug interactions. An especially harmful form of inhibition is time-dependent inhibition (TDI) in which the inhibition potency increases over time and persists even after discontinuation of the drug. Both direct and time-dependent inhibition can be efficiently screened with the so-called cocktail method containing several CYP-selective probe substrates in a single reaction mixture. This method is practical especially in ADME studies of drug development, as it offers lower costs, consumption of fewer reagents and faster implementation in comparison to conventional methods. In addition, the cocktail method can be used to establish new diagnostic CYP inhibitors in vitro. The aim of this Master’s thesis was to participate in the development and optimization of a new cocktail assay method. The method was developed for screening of major drug-metabolizing CYP enzymes in vitro both in a direct and time-dependent manner using pooled human liver microsomes. Based on preliminary testing, included probe substrates were divided into two cocktails to avoid significant inter-substrate interactions: cocktail I containing tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4, and cocktail II containing coumarin/CYP2A6, (S)-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2. First, cocktail incubation conditions were optimized, followed by the determination of probe reaction kinetics, kinetic parameters (Km, Vmax) and inter-substrate interactions with single- or dual-substrate incubations. Finally, suitable probe substrate concentrations and the composition of cocktails was evaluated based on the obtained results. As a result of assay optimization, optimal incubation conditions for yet unoptimized cocktail II were established. In optimized incubation conditions, all probe reactions exhibited saturable Michaelis-Menten kinetics except for tacrine 1-hydroxylation (CYP1A2), which exhibited biphasic kinetics instead (Km1: 7.36, Km2: 517). The selected probe substrate concentrations were all below or near their respective Km values except for (S)-mephenytoin 4’-hydroxylation (40 µM vs. Km of 12.5 µM); however, its concentration could not be reduced in order to maintain sufficient metabolite formation for UHPLC-MS/MS-analysis. Dual-substrate incubation assays demonstrated a need for the reduction of bupropion concentration below 100 µM due to its inhibitory effects on CYP2C8 and CYP3A4. In addition, chlorzoxazone/CYP2E1 and testosterone/CYP3A4 were tested as complementary probe substrates for the cocktails; however, they proved to be unsuitable for both cocktails due to significant interactions (>40% inhibition). Prior to the deployment of the method, some adjustments of probe substrate concentrations are still required in addition to consideration of the suitability of less commonly used CYP3A4 and CYP2E1 probe reactions to improve cocktail coverage. Lastly, validation of the method with known time-dependent model inhibitors should also be conducted. Besides to improvement of the cocktails, new information was generated on inter-cocktail probe-probe interactions and enzyme kinetics of probe reactions, especially for the less-studied astemizole O-demethylation (CYP2J2) and tacrine 1-hydroxylation (CYP1A2). Generated information can be used, for example, in the development of new cocktails.

The potential of extracellular vesicles (EVs) as diagnostic markers and drug delivery vehicles has been studied increasingly in recent years. One of the challenges in this field has been the isolation of EVs from complex biological fluids such as blood. The methods widely used for the isolation process include for example size exclusion chromatography (SEC) and ultracentrifugation (UC). As these methods use size and density of the particle, the have not been efficient enough in isolating EVs from certain particles such as lipoproteins. Due to the challenges related to these methods, other isolation methods have been sought to improve the efficiency of EV isolation. One of these methods is ion-exchange chromatography (IEC). From the two forms of IEC, anion-exchange chromatography has been studied more in EV isolation due to the negative net charge on EV particles. However, in this study the functionality and efficiency of cation-exchange chromatography (CEC) in EV isolation was studied as very little research has been done on this method. In this study, two CEC-resins were studied to define their applicability in EV isolation. A standard strong cation-exchange chromatographic resin SP Sepharose Fast Flow was compared to a strong tentacle-type resin. In addition to this, we studied the possibility to use a magnesium gradient to separate different forms of lipoproteins from EVs through dextran-sulfite precipitation. Tentacle-type CEC-resin was found to be more efficient in capturing EVs compared to the standard-type resin without magnesium. These EVs could then be eluted from the column with sodium chloride. The use of magnesium gradient allowed the separation of apolipoproteins in the samples. Higher concentrations of magnesium also reduced the number of lipoproteins in the samples altogether but resulted in the loss of EVs as well. These results were promising and showed that cation-exchange chromatography can be used in EV isolation. Tentacle-type resin seemed to be most efficient in removing impurities and capturing EVs. While more research is needed before these findings can be applied to clinical use, these results prove that cation-exchange chromatography can be used in EV isolation as a new, efficient and up scalable method.

Drug metabolism is a series of enzyme catalysed processes that modify foreign compounds into a form that is more easily excreted from the body. Compounds can affect the activity of metabolizing enzymes and this may lead to toxic concentrations of a drug that is metabolized via the enzyme. With prodrugs, on the other hand, the drug might not achieve its biologically active form and therefore the treatment will not be effective. Recognizing and preventing metabolic interactions is important already in the early stages of drug discovery and development. Cytochrome P450 (CYP) enzyme inhibition is one of the major reasons for adverse drug-drug interactions (DDIs). The inhibition can be time-dependent (TDI), which means that the potency of inhibition increases over time. TDI may be reversible or irreversible, latter being more severe as new enzymes need to be produced in the body to restore the enzymatic activity. IC50 shift assay is a method that gives information of new compounds potential to cause TDI. IC50 shift assay does not show whether the TDI is reversible or irreversible, however further studies, e.g. dialysis assay, can be conducted to find it out. If the study compound is irreversibly bound to the enzyme, the enzyme activity should not recover in the dialysis. The aim of this master’s thesis was to develop a dialysis method that could determine the reversibility of the TDI observed in the IC50 shift assay. A dialysis method conducted with microsomes is described in earlier literature. Known inhibitors (both time-dependent and direct) for four CYP isoforms were studied in this work: CYP1A2 (furafylline and fluvoxamine), CYP2C9 (tienilic acid and sulphaphenazole), CYP2D6 (paroxetine and quinidine) and CYP3A4 (verapamil, azamulin and ketoconazole). IC50 shift assays were conducted to each inhibitor before the dialysis experiment. The studied compounds behaved in the dialysis assay mostly as assumed based on the literature. The workflow from IC50 shift assay to dialysis assay worked successfully and the IC50 shift data could be utilized when choosing the test concentrations for dialysis assay. Both the IC50 shift assay and dialysis assay were reproducible and the deviations between replicates and separate studies were relatively low. The method still requires some optimizing, but so far, the results are promising. In the future the dialysis method may be part of in vitro CYP inhibition studies at Orion Pharma.

New drugs against malaria are required, as millions of people are still affected yearly by this deadly disease. The development of drug resistance to current antimalarials is an ongoing process. Membrane-bound pyrophosphatases (mPPases) are potential new drug targets against malaria and other protozoan diseases. mPPases play a crucial role in the survival of the malaria parasite, they couple the energy released from the hydrolysis of pyrophosphate into the transport of protons or ions against an electrochemical gradient. The aim of this study was to identify potential mPPase inhibitors through a docking-based virtual screen of the Tres Cantos Antimalarial Compound Set, which consists of over 13500 malaria-active compounds. The virtual screen against a Thermotoga maritima mPPase protein structure identified a 2,4-diamino-1,6-dihydrotriazine among the top-ranking scaffolds. Four compounds found among the docking results containing this scaffold were synthesised: three with a halophenyl substituent, and one with a hydroxyl substituent. The compounds in their hydrochloride salt forms were synthesised using a three-component method for the synthesis of 2,4-diamino-1,6-dihydrotriazines. The compounds were also freed from the hydrochloride salts into their corresponding molecular forms. The structural characterisation of the compounds, especially the molecular forms, presented challenges. The docking results were also searched to identify compounds containing previously identified mPPase-active substructures. From the docking results, several other interesting compounds were identified in addition to the synthesised compounds. The knowledge and results obtained from this study can be used as openings for potential future docking and synthesis projects in the development of mPPase inhibitors. The activity of the compounds synthesised in the project remains to be evaluated in subsequent investigations.