Skip to main content
Login | Suomeksi | På svenska | In English

Browsing by master's degree program "Utbildningsprogrammet för provisorexamen"

Sort by: Order: Results:

  • Ruohonen, Iida (2020)
    Tiivistelmä – Referat – Abstract 15D is a generic, 15-dimensional instrument for measuring health-related quality of life (HRQoL). 15D instrument has been used in multiple studies evaluating the effectiveness of medical interventions in Finland and abroad. 15D-instrument is a self-administered questionnaire traditionally administered in a paper-and-pencil format. With the emergence of novel technologies, electronic modes of delivery of the 15D instrument are becoming increasingly common as methods for data collection. However, there are no previous studies evaluating electronic modes of delivery of the 15D instrument. In previous studies, electronic instruments measuring patient-reported outcomes have shown strong measurement equivalence and high acceptability. The aim of this study is to evaluate acceptability and measurement equivalence of electronic delivery modes of the 15D instrument. A repeated measures, randomized cross-over study was conducted in the Department of Otorhinolaryngology at Helsinki University Hospital during April, May and June of 2019. A total of 160 outpatients who participated in the study were randomized into four groups of 40 patients. Every participant filled two delivery modes of the 15D instrument: a single paper-and-pencil questionnaire and one out of two electronic questionnaires provided in the study. The two electronic delivery modes delivered in the study were a web-based questionnaire and a mobile app-based questionnaire. The order and the mode of the administration varied between the groups. The patients filled the first questionnaire before the doctor’s appointment at the study site. The patients were then asked to fill the second questionnaire after the doctor’s appointment at home within 3 days after the first administration. Information about acceptability concerning different delivery modes were collected using an end-of-study questionnaire. As a part of determining patient acceptability, response rates for different delivery forms and mode preferences were examined. Measurement equivalence was assessed by intra class correlation coefficients (ICCs) and comparison of mean and median for 15D scores and weighted kappa for item scores. Paired observations were also visually analysed with Bland-Altman plots. Subgroup analyses were conducted for identifying differences in observed patient characteristics (age, sex, base-line HRQoL). 86 participants (females 53.5 %; males 46.5 %) aged 18 to 80 (mean ± SD: 48.2 ± 15.7) filled both questionnaires of the 15D instrument resulting in an overall response rate of 54.1%. Response rates for filling both questionnaires were lower in both groups that filled electronic forms at home (41.0 % for mobile app-based and 52.5 % for web-based questionnaires) than in the paper-and-pencil groups (60.0 % and 62.5 %). Overall 74.1 % preferred the electronic delivery mode compared to 16.5 % preferring the paper-and-pencil mode. Statistically significant differences favouring the web-based form over the paper-and-pencil form were observed in the speed of use (p = 0.002) and in the possibility to edit answers (p = 0.018). Similarly, mobile app-based form was favoured over paper-and-pencil group in the possibility to edit answers (p = 0,041). In terms of measurement equivalence for 15D index scores, high association across paper-and-pencil and web-based questionnaires (ICC: 0.910 [Cl 95 % 0.794-0.962] and ICC: 0.935 [Cl 95 % 0.862-0.971]) and high to moderate across paper-and-pencil and mobile app-based questionnaires (ICC: 0.949 [Cl 95 % 0.883-0.978] and ICC: 0.928 [Cl 95 % 0.601-0.980]) were observed. A clinically important and a statistically significant difference in mean 15D scores was identified in the paper-mobile app group. A statistically significant but not clinically important difference in medians was observed in mobile app-paper group with participants returning the second delivery form in time. This study provides strong evidence supporting the use of electronic delivery modes of the 15D instrument regarding measurement equivalence and patient acceptability. However, differences in electronic delivery modes may have an impact on measurement equivalence and representativeness of study participants. In this study, a small sample size and limited data on study participants limit the generalizability of the results. Most effective ways of collecting data electronically concerning all age and patient groups must be identified in future studies. Electronic data collection methods offer many opportunities for utilising HRQoL data. For example, it is important to assess whether HRQoL-instruments can be used as clinical tools in the future.
  • Silén, Heidi (2021)
    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.
  • Kärkkäinen, Krista (2021)
    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.
  • Silén, Jenna (2021)
    The life cycle of Chlamydia pneumoniae is a biphasic developmental cycle, as a obligate intracellular bacterium, it forms various morphological forms, including elementary bodies, reticulate bodies and aberrant bodies belonging to a persistent form. Due to the bacterial life cycle and the fact that chronication of C. pneumoniae infection and formation of persistent infection as well as pathogenesis is a complex problem involving multiple signaling pathways and affecting several different cells, it is useful to seek medication to influence infection from different stages of the bacterial life cycle. There are several different factors that induce persistence and thus models of persistence. Although the detection of aberrant RBs and thus aberrant bodies in C. pneumoniae infected tissues does not provide complete certainty about chronic infection, the bacterium has been linked to chronic health problems such as atherosclerotic cardiovascular disease and asthma. The aim of the study was to develop a persistence model induced by beta-lactam antibiotics, amoxicillin and penicillin G, in A549 cells by monitoring the size, shape, and number of inclusions using the IPA method and the immunofluorescence staining method for infection. In addition, the antibiotic sensitizing effect of three compounds on pulmonary chlamydial infection was studied. This effect was monitored by examining the recovery of persistent infection and by monitoring the protective effect of the compounds on beta-lactam-induced persistence. The work succeeded in finding an infection model that is well suited for studying beta-lactam persistence. Due to treatment recommendations, pulmonary chlamydial infections are practically treated with beta-lactam antibiotics. Based on the methods used, it was found that amoxicillin concentrations of 10 and 25 µg/ml and penicillin G concentrations of 100 U/ml and 250 U/ml were sufficiently effective to transfer bacteria to a state of persistence. It was found that the amoxicillin persistence model is reversible based on the increase in the size of the inclusions, especially at 25 µg/ml and quantitatively at 10 µg/ml. It was concluded that amoxicillin at a concentration of 10 µg/ml is sufficient to induce persistence in a beta-lactam antibiotic-induced persistence model. Further quantitative studies on the persistence model are needed, such as quantitative PCR based on the OmpA gene to determine more accurate dose-response relationships. Glutathione levels should also be monitored in the persistence model.
  • Peltola, Roosa (2020)
    Amyotrophic lateral sclerosis (ALS) is a rare fatal neurodegenerative disease in which both the upper and lower motor neurons degenerate. Pathological features of the disease include misfolded proteins and accumulations in the central nervous system. The molecular mechanisms of the disease include neuroinflammation, glutamate induced excitotoxicity, and endoplasmic reticulum stress (ER-stress). Numerous genetic defects have been identified in the background of ALS, the most common mutations are in the C9ORF72, SOD1, TDP43 and FUS genes. For each gene mutation, it is important to develop a reliable animal model of ALS for studying pathology and testing new therapies. The most common and most recently found gene mutation, the C9ORF72 repeat expansion mutation, does not yet have an established animal disesase model. The molecular mechanisms of the disease include neuroinflammation, glutamate induced excitotoxicity, and endoplasmic reticulum stress (ER- stress). There is no drug treatment to cure or slow ALS, so the need for new drug therapies that affect the course of the disease is significant. Cerebral dopamine neurotrophic factor (CDNF) protects and restores dopamine neurons and controls ER-stress in preclinical models of Parkinson’s disease. CDNF has also been shown to improve motor coordination as well as protect spinal cord neurons from cell destruction in ALS genetic SOD1- G93A mouse and TDP-43M337 animal models. The purpose of this master's thesis study was to characterize the changes related to neurodegeneration and neuroinflammation in the new C9ORF72-500 disease model and study ER stress of the SOD1-93A disease model and the effect of CDNF on ER stress in SOD1-model and on inflammation in C9-model. In the first sub-study, brain sections from C9ORF72 transgenic and wild-type mice at different time points were subjected to six different immunohistological stainings. The results were compared at each time point (30, 70 and 170) between the wild type and the transgenic group. In another sub-study, spinal cord sections from CDNF snd vehicle treated SOD1- G93A mice were subjected to immunofluorescence staining, after which the intensity of their ER stress marker, GRP78, was analyzed using a confocal microscope. GFAP stained brain sections from CDNF and vehicle treated C9ORF72 mice were analyzed using microscope and imaging analyses. The results of the first sub-study showed neuroinflammation at 24 weeks timepoint in the transgenic group compared to wild-type mice. Pathological features of C9-ALS, various protein accumulations, were observed only in the transgenic group, mainly at 24 weeks. No neuronal loss was observed in this study. The obtained results support the previously published research results and support the reliability of the studied disease model. In the second sub-study ER stress levels were higher in SOD1-mice compared to wild-type mice. Single intracerebroventrical CDNF injection reduced ER stress in SOD1-G93A transgenic mice almost to the same level as ER stress in wild-type mice. CDNF treatment also showed a tendency for reducing inflammation in hippocampus and motor cortex of C9ORF72 mice. The results confirm the pathological role of ER stress in ALS and show that CDNF reduces ER stress when administered as early in the disease as possible, when neuronal damage begins to occur but does not yet lead to neuronal destruction. CDNF appears to be a promising drug candidate for the treatment of ALS and should therefore be further investigated.
  • Kainulainen, Saila (2020)
    The solubility of a poorly water-soluble drug can be improved by converting the crystalline drug into an amorphous form. However, the amorphous form is metastable due to the higher energy state and recrystallization may occur during storage and dissolution. The amorphous form can be stabilized by forming an amorphous solid dispersion (ASD), where the drug molecules are dispersed to the solid medium, e.g. hydrophilic polymer. One preparation method for amorphous solid dispersions is spray drying, where a solution containing a drug and polymer is converted into small droplets in a drying chamber, in which the solvent evaporates in a hot gas stream and solid particles are formed. The aim of this study was to investigate whether an ASD of a poorly water-soluble drug can be prepared by spray drying using 20:80 (V/V) ethanol-water mixture as a solvent in a feed solution. Indomethacin (γ-polymorph) was used as a model drug and polyvinylpyrrolidone vinyl acetate (PVPVA) as a polymer. The aim was to find a suitable formulation where the drug is in the amorphous form after spray drying and remains in the amorphous form during storage. The ratios of the drug to polymer in the spray-dried formulations were 1:4, 1:6, 1:8, 1:10, 1:12 and 1:16. The study also examined whether a change in one process parameter, pump feed rate, affects the amorphous nature and stability of the resulting spray-dried solid dispersions. Two different pump feed rates, a higher 30% and a lower 15%, were used in the study. X-ray powder diffraction (XRPD) was used to characterize the solid-state of the spray-dried formulations. XRPD measurements were performed immediately after spray drying and on selected time points during storage. Formulations 1:10 at 30% feed rate, 1:12 at both feed rates and 1:16 at 30% feed rate were amorphous after spray drying. In 1:12 (30%) and 1:16 (30%) formulations indomethacin remained in amorphous form over the study periods (22 and 56 days, respectively). In other formulations, indomethacin was found to be in crystalline α-form immediately after spray drying or recrystallization to the α-form occurred during storage. The interaction between indomethacin and PVPVA was studied by surface plasmon resonance spectroscopy (SPR). The aim of the SPR measurements was to understand the interaction between these substances in the feed solution used in spray drying. PVPVA solutions of various concentrations (1%, 0.5%, 0.1% and 0.01%) were injected to the surface of the gold sensor coated with crystalline γ-indomethacin, and the changes in the SPR signal responses were monitored during the interaction. The same measurements were also performed on a pure gold sensor without indomethacin. An interaction between indomethacin and PVPVA can be observed, and based on the measurements, a polymer layer with a thickness of about 1 nm was formed on the surface of the indomethacin sensor regardless of the concentration of the polymer solution. Thus, even a small amount of polymer in solution is sufficient to cover the indomethacin crystals. This may also occur in the feed solution during spray drying, but further studies with SPR are still needed, especially with amorphous indomethacin. This study showed that an ASD of indomethacin and PVPVA can be successfully prepared by spray drying using an aqueous feed solution. Spray-dried 1:12 and 1:16 formulations at a higher pump feed rate were found to be stable enough for further studies. If the spray-dried material is further formulated into a pharmaceutical product, indomethacin must remain in amorphous form throughout the shelf-life of the product to maintain the improved solubility.
  • Rautiainen, Swarna (2020)
    Endothelial dysfunction is a common characteristic of several diseases including diabetes mellitus, coronary heart disease and stroke. Healthy endothelium ensures vascular homeostasis, regulation of blood flow and the exchange of oxygen and nutrients, as well as immune cell filtration to the surrounding tissues. In many cases, endothelial dysfunction results in ischemia in the surrounding tissues impairing cellular regeneration mechanisms, which can lead to tissue necrosis in the worst case. Therapeutic angiogenesis via stem cell transplantation aims to restore tissue blood flow and thus aid in tissue regeneration and restoration of a functioning tissue. Adipose derived stem/stromal cells (ASC) are a stem cell population with a multilineage differentiation ability. They have been shown to differentiate towards adipogenic, osteogenic, chondrogenic, myogenic and neurogenic lineages among others. Their easy obtainability from liposuction material and abundance in the adipose tissue makes them an especially practical and favorable cell option for stem cell research. In angiogenesis research, ASCs are commonly used in a co-culture with an endothelial cell (EC) type such as human umbilical vein endothelial cell. ASCs secrete extracellular vesicles (EV) that are small membrane bound vesicles with a diameter ranging from 40-1000 nm, and which have the ability to alter the behavior of target cells through their cargo. EV cargo consists of microRNAs, messenger-RNAs and proteins, and the EV cargo of ASCs has been shown to have proangiogenic effects. The aim of this work was to review what is currently known about ASC ability to promote angiogenesis through paracrine secretion and differentiation into endothelial cells or pericytes, interactions between ASCs and endothelial cells in the angiogenesis promoting process and the role of ASC extracellular vesicles in promoting angiogenesis. The methods for this work were database research of related articles using scientific databases and search engines, article categorization and reading, and finally manuscript production. It can be concluded from the current literature that a co-culture environment of ASCs and an endothelial cell type supports the formation of tube-like structures in vitro. Additional insulin like growth factor 1 in culture medium enhances the expression of angiogenesis-related growth factors in both cell types via PI3K/AKT signaling pathway. Further, the activation of platelet derived growth factor receptor β supports ASC ability to promote vascular network formation. On the contrary, the presence of ASC secreted activin A results in the inhibition of vascular network formation. ASCs can differentiate into endothelial cells particularly in three-dimensional culture conditions. In addition, fibroblast growth factor 2 and the activation of the AKT-pathway are crucial for endothelial differentiation. In addition, ASCs have the ability to differentiate into pericytes and assume a stabilizing role on the outside of the microvessels. Concerning ASC derived EVs and their cargo, miR-31, miR-125a and miR-126 have proangiogenic effects in vitro and in vivo. Proangiogenic miRNAs in ASC EV cargo are miR-181b-5p and the let7-family, out of which miR-181b-5p upregulates vascular endothelial growth factor and hypoxia-inducible factor 1α and let7-family influences tube formation ability of ECs. In vivo, ASC derived EVs support fat grafting, enhance wound healing both in healthy and diabetic environment, and provide cardioprotection. Therefore, ASC EVs show potential for therapeutic angiogenesis but currently there is a lack of clinical trials in EV research.
  • Hou, Kathy (2021)
    Antidepressant use among children and adolescents has become more common in many countries. The prevalence of antidepressants is higher for boys but during adolescence girls’ have a higher antidepressant prevalence. In previous studies, the prevalence of selective serotonin re-uptake inhibitors (SSRI) has increased. The aim of this study was to investigate antidepressant use among Finnish children and adolescents aged 1–17 years during 2008–2019. The differences of antidepressant use in different age groups and genders were investigated. Furthermore, the secondary objective was to examine the trends in prevalence and costs of the five most commonly used antidepressant agents. This was a nation-wide register study. The data for this study was from Kelasto which is a statistical database maintained by the Social Insurance Institution of Finland. The extracted data was from 2008–2018 and included each persons’ age, gender, dispensed drug and costs. The data extracted was for 1–17-year-olds who had been dispensed reimbursed antidepressants from community pharmacies. The data was analyzed with Microsoft Office’s Excel program. The results were transferred in to tables and reported as prevalences by age groups, genders, antidepressants and costs. The prevalence of antidepressant use among children and adolescents was 5,0 per 1000 in 2008 and it increased to 10,3 by 2018. In the youngest age group of 1–6-year-olds, antidepressant use decreased. Antidepressant use increased slightly among 7–12-year-olds. Antidepressant use increased the most among 13–17-year-olds. 13–17-year-old girls had the higher antidepressant use prevalence throughout the study. The same group had a 2,4-fold increase in prevalence during the study period which accounted for the biggest increase in the study. The most used group of antidepressants was SSRIs. The total cost for antidepressants among children and adolescents increased by 73,7 % during the study period. The most commonly used antidepressant agents were fluoxetine, sertraline, escitalopram, mirtazapine, and venlafaxine, respectively. Fluoxetine was the most used agent throughout the study. In 2014, sertraline surpassed escitalopram and became the second most used antidepressant agent. Escitalopram and venlafaxine’s cost per user decreased during the study. The cost per user stayed stable for mirtazapine. Fluoxetine and sertraline’s cost per user increased. The Kelasto database does not include data on indications for prescriptions. The prevalence of antidepressants does not necessarily correlate directly to depression among children and adolescents because antidepressants can be used to treat other diseases. More studies need to be conducted on different off-label uses for antidepressants among children and adolescents. This study only investigated the trends on cost for the five most commonly used antidepressants. Further studies on antidepressant costs among children and adolescents are needed. Additionally, it is essential to investigate the reasons for the increase in antidepressant use among children and adolescents.
  • Luhtanen, Päivi (2020)
    The pharmacy operations are strictly regulated in Finland and the operation of a pharmacy business requires a licence. Number of community pharmacies has stayed quite steady for the past 10 years. At the end of the year 2019 there were 817 pharmacies or their subsidiaries in Finland. The number of pharmacies is expected to increase, since 29 new pharmacies has been established since 2016. The inspection of pharmacies is a part of the legal duties of the Finnish Medicines Agency. In Finland, pharmacies are inspected based on a risk assessment, as often as it is necessary to ensure appropriate operations of a pharmacy. During a pharmacy inspection, the focus is on operations that are critical to drug safety and medication safety. The aim of an inspection is to make sure that pharmacy operations comply with the regulations. There are only few studies made on remote inspection of the pharmacies. The Finnish Medicines Agency hasn’t made remote pharmacy inspections before. The aim of this study was to develop, validate and test a questionnaire, which could be used to inspect community pharmacies remotely and to develop and test a process for remote inspections. The study was done in two parts. In the first part, a draft of the questionnaire was developed by studying the regulations and laws regulating the operations of a pharmacy and by using a content analysis. The material for the content analysis was a pre-inspection questionnaire form, answers to the pre-inspection questionnaires and defect lists of the inspection reports of those pharmacies (n=37), which had answered to the pre-inspection questionnaire before pharmacy inspection in 2019. Content of the pre-inspection questionnaire and the answers of pre-inspection questionnaire were compared to the content of the defect lists of inspection reports. The aim of the comparison was to find out how the existing pre-inspection questionnaire could be utilized when developing the questionnaire for the remote pharmacy inspections. In addition, the listed defects of the inspection reports were categorized to explore what were the most common defects observed during pharmacy inspections. In the second part of the study, the content of the developed questionnaire was validated by using a three round modified Delphi survey. Seven experts with good knowledge of the pharmacy inspections were chosen to the Delphi panel. The aim of the Delphi rounds was to achieve full consensus among the experts about the content of the questionnaire. Alongside the Delphi rounds, a process to remote inspect a pharmacy was developed. The remote inspection questionnaire and the process were tested internally in the Finnish Medicines Agency at the end of the second stage of study. The draft of the questionnaire included 15 sections and 164 questions. Based on the comments received during the Delphi rounds, the content of the questionnaire was modified. On the third Delphi round a full consensus of the content of the questionnaire was achieved among the experts. The final questionnaire for the pharmacy remote inspection included 14 sections and 184 questions. The process of the remote pharmacy inspection follows the procedure of an on-site pharmacy inspection. In the internal test, the process of the remote inspection was found to be a good way to inspect pharmacies remotely. The remote inspection process is a new way to inspect pharmacies. With the remote inspection, it is possible to find out the most common defects on the pharmacy operations by using the questionnaire and contact calls. The remote inspection questionnaire and the process need to be further tested to ensure that the process is optimal from the perspective of the authority and the pharmacies.
  • Mäkinen, Emilia (2021)
    Background and objectives: Documenting and processing of dispensing errors at both organizational and national levels is one of the basic preconditions for effective medication risk management. Since the most recently accomplished national register research of dispensing errors in Finland, there have been several changes in the medication dispensing process that advance medication safety. Thus, the previous study does not provide an up-to-date picture of the current situation. The primary objective of this study was to find out the trends in dispensing errors that were reported to the Finnish Pharmacy Association's registry of dispensing errors in 2015–2019. The secondary objective was to identify risk factors expository to dispensing errors in the dispensing process and to review the measures utilized by community pharmacies to prevent dispensing errors. Materials and methods: The retrospective registry study, in which the register of dispensing errors maintained by the Finnish Association of Pharmacists for the period from 1 January 2015 to 31 December 2019, was analyzed. Cases that did not fulfil the definition of a dispensing error (n=829) were removed from the original data (n=17763). In addition, clear errors (n=2130) were corrected in the data and cases (n=499) that were initially insufficiently entered in the register were added. 17433 dispensing errors were included in the study. The data was analyzed using Microsoft Excel. The number, qualities, prescription types, observers, therapeutic harms and contributory factors of the dispensing errors were investigated in the data. The most common groups of medicinal substance, high-alert medications and risk factors in the medication dispensing process were identified in the data. In addition, interventions reported by community pharmacies to prevent dispensing errors were collected from the data. Results: The number of cases reported to the dispensing error register has decreased annually (2015 n=3913, 2016 n=3795, 2017 n=3708, 2018 n=3578, 2019 n=2439). The most common types of dispensing errors are incorrect strength (51 % of all the reported dispensing errors) and incorrect quantity or package size (14 %). Slightly more than a half (51 %) of the reported dispensing errors were noticed by medicine users. The percentage of electronic prescriptions in dispensing errors has increased and is clearly the most common prescription type in dispensing errors (2015: 79 %, 2016: 84 %, 2017: 93 %, 2018: 96 %, 2019: 95 %). The majority of dispensing errors occurred with cardiovascular medicines (29 %) and medicines affecting the nervous system (26 %). 7 % of dispensing errors caused therapeutic harm to the medicine user. As a result of dispensing errors, 21 medicine users were hospitalized. 13 % of dispensing errors occurred with high-alert medications (n=2244). The high-alert medications were involved in one-third (n=7) of dispensing errors that led to hospitalization. Factors related to the employee (25 %), similar packaging (19 %), and similar medicine name (15%) were most commonly considered to be the main contributory factors for the occurrence of the dispensing errors. The risk factors identified in the medicine dispensing process were related to the pharmacy system, the characteristics of the prescription, the storage method of the medicine and the characteristics of the medicine packaging. In the automated dose dispensing process, the risk of dispensing error increased if changes had to be made to the dose dispensing order. The risk factors for automated dose dispensing were related to the pharmacy system and the characteristics of the prescription. The community pharmacies had mentioned taking measures to prevent dispensing errors in one-fifth (21 %) of the reported cases. In addition to developing their own operations, community pharmacies saw cooperation with other healthcare professionals as an important factor in preventing medication errors. In addition, community pharmacies reported exposing properties for dispensing errors of pharmaceutical products and systems to pharmaceutical companies and providers of pharmacy systems and automated dose dispensing. Conclusions: Trends, risk factors of the dispensing process and interventions to prevent dispensing errors can be identified in the dispensing errors reported to the Finnish Association of Pharmacists’ dispensing error registry. The dispensing error register provides valuable information on dispensing errors at the national level, but it is no longer able to fulfil completely the current medication safety needs. In the future, the role of the pharmacy as a promoter of medication safety should be perceived as more comprehensive. In the development of medication safety, special attention should be paid to the risk factors of the dispensing process, the high-alert medications and to new risks arising from the increase of electronic prescriptions and automated dose dispensing. In addition, cooperation between pharmacies and other healthcare professionals and the medication safety culture of pharmacies should be further strengthened.
  • Vilhunen, Noora (2021)
    Pharmacy compounding involves the preparation of customized medications that are not commercially available for individual patients with specialized medical needs. The compounded preparations have many special features, such as the rapid/immediate need for the drug, the preparation of several pharmaceutical dosage forms, and the variation of batch sizes and manufacturing processes. Medicinal products prepared in hospital pharmacies may pose additional risks to patients compared to industrial products. These risks with limited evidence of quality, efficacy and microbiological purity can jeopardize patient safety. The aim of this study was to perform a product specific risk assessment of aseptically processed and terminally sterilized products belonging to the manufacturing range of the hospital pharmacy of Turku University Central Hospital. The study material contained 118 different products. The risk assessment was performed with the help of a risk matrix in which various quality and safety risks have been identified and assessed. The risk points obtained from the different areas of risks were multiplied together to obtain total risk points for each product. The products were qualitatively classified according to the total risk points into low-risk, medium-risk and high-risk products. All total parenteral nutrition (TPN) solutions of the study were classified as high-risk products. TPN solution prepared into a syringe without lipids and TPN solution prepared into an EVA bag without lipids had the highest risk points of the study (6561 points). Most of the eye drops (88 %) and patient controlled analgesia (PCA) pumps (68%) belonged to high-risk category. PCA pump containing morphine, clonidine, bupivacaine, ketamine and saline solution (1944 points) and autologous serum eye drops (1296 points) had the highest risk points of these product types. 60 percent of intraocular injections and half of pain products prepared into syringes were scored as high-risk products. Intravitreal bevacizumab had the highest risk points of intraocular injections (972 points). Medium-risk products were mainly different infusions. Infusions containing defibrotide, oxytocin and onasemnogene abeparvovec had the highest risk points in the medium-risk category. Liquid solutions and patient controlled analgesia (PCA) pumps were the second largest group in this category. All products used in allergy testing, all ointments and all inhalation solutions were in the low-risk category. The risk matrix used in the study can be used to identify high-risk compounded preparations in hospital pharmacies. Risk assessment enables targeting quality assurance more effectively to high-risk products. Risk assessment can be used to manage various risks in pharmaceutical compounding and reduce harm to patients. The results obtained in the study cannot be directly generalized to other hospital pharmacies because the products, manufacturing processes and the amounts of different products prepared vary among hospital pharmacies.
  • Sinisalo, Jade (2021)
    Pharmaceutical contaminants in waste and surface waters have been recognized as an emerging risk to environmental health. Bioaccumulation of pharmaceuticals increases the risk of adverse effects in off-target species, as the chemical concentration within the organism exceeds the concentration of the surrounding environment. An organism’s ability to metabolize foreign organic compounds influences the likelihood of bioaccumulation. Current methods for predicting bioaccumulation in aquatic organisms are labour intensive or too simplistic to cover the variety of chemical and physiological processes involved and may lead to over or underestimations of environmental risk. A promising approach to improve bioaccumulation predictions, without the need of excessive animal testing, is to incorporate in vitro biotransformation data into computational models. The primary aim of this study was to assess whether selected pharmaceuticals (diclofenac, gemfibrozil, haloperidol, levomepromazine, levonorgestrel, sertraline and risperidone), that are well metabolized in humans through key biotransformation pathways, are metabolized by rainbow trout (Oncorhynchus mykiss) liver enzymes under physiologically relevant conditions (11°C, pH 7.8). A secondary aim was to produce fish in vitro intrinsic clearance (CLint, in vitro) data, that could potentially be used as input in computational models to predict bioaccumulation. In vitro biotransformation was studied using a single vial approach according to the Organisation for Economic Co-operation and Development (OECD) Test Guideline 319B: Determination of in vitro intrinsic clearance using rainbow trout liver S9 sub-cellular fraction (RT-S9). Depletion of the test compounds were measured during a 3-hour incubation period. High-performance liquid chromatography with ultraviolet detection (HPLC–UV) was used for qualitative and quantitative analysis of the samples. Levomepromazine, levonorgestrel and sertraline showed significant substrate depletion compared to negative controls while gemfibrozil, haloperidol, and risperidone did not seem to be metabolized. The results for verapamil were inconclusive. Levomepromazine displayed a higher in vitro intrinsic clearance rate (26 ml/h/g liver) than diclofenac (6.2 ml/h/g liver). These results are in accordance with previous studies and support the notion that a direct comparability between fish and human metabolism cannot be assumed, highlighting the need of fish in vitro biotransformation studies. The apparent lack of in vitro metabolism of risperidone, haloperidol, and gemfibrozil combined with their lipophilicity suggest that they are more likely to accumulate within rainbow trout, compared with the compounds that showed depletion during the assays, although repetitions and additional studies are needed to confirm this.
  • Rosqvist, Linn (2021)
    Marine invertebrates are a good and relatively unexplored source of bioactive compounds. These bioactive secondary metabolites can have unique structures and mechanisms of actions, since they are produced by organisms, which means their structures are not limited by the fantasy of chemists. Therefore, bioactive secondary metabolites isolated from marine invertebrates are attractive for drug development. Still, there are challenges regarding bioprospecting marine invertebrates. For example, the amount of material is limited and the environment as well as the biodiversity has to be taken into account when gathering the organisms. The aim of this thesis was to perform the first steps of bioprospecting marine invertebrates; extraction, fractionisation, analysis of bioactivity and identification of bioactive metabolites. The samples used in the experiment, gathered from three different locations, were of the sponge Caulophacus arcticus. The goal was not only to identify one or more bioactive metabolites for eventual further analysis, but also to compare the bioactivity of the samples gathered from different locations. The fractionisation was performed using flash fractionisation, which resulted in eight fractions of each extract. These fractions were tested for anticancer, antibacterial and biofilm inhibiting properties. The bioactivity of the fractions was analysed by performing cell viability assays (MTS assays) on four cell lines, antibacterial growth inhibition assays on five strains of bacteria and biofilm inhibition assays on biofilm of S. epidermidis. The active fractions, the fraction right before and after them and the corresponding fractions of the two other samples were further analysed using UHPLC-HR-MS, in order to identify eventually bioactive compounds and determine the elementary composition of these compounds. The most interesting fractions, from which one or more bioactive compounds were to be identified first, were prioritised based on the bioactivity assays. One compound, which was identified as potentially bioactive with a potentially novel elementary composition, was chosen as a target compound for further analysis. Based on the results, it was also possible to draw the conclusion that there were variations as well as similarities in the bioactivity of samples gathered from different locations. Still, further research is needed to determine if the bioactivity of the same fractions from different samples was caused by the same compounds or not. Even if there are challenges regarding bioprospecting of marine invertebrates, it is still useful to keep studying them in order to find new, bioactive compounds. There is a huge need of new drugs, especially for treating cancer and bacterial infections. Therefore, experiments such as this are relevant also in a bigger perspective. The target compound identified in the experimental part of this thesis might be further analysed in order to determine whether it is bioactive and whether it is profitable to develop it further.
  • Luukkanen, Saana (2021)
    Pharmaceutical costs have been rising globally every year. A significant portion of drug costs is caused by biological drugs, which are often very expensive, yet essential in the treatment of many chronic diseases. Biosimilars are clinically equivalent to biological originator products and are expected to alleviate the increase in drug costs. The biosimilar development process does not need to repeat the complete development process of the originator product, allowing the biosimilar to enter the market at a lower price than the originator after the patent and data protection period for the originator ends. The aim of this study was to find out what impact the market entry of biosimilars has on the prices of the reference products in outpatient care in Finland, and to investigate whether biosimilars create price competition for biological drugs. In addition, the study examined how the prices and market shares of outpatient biosimilars have developed in Finland. The study examined the development of price and market shares for adalimumab, etanercept, insulin glargine, insulin lispro, enoxaparin, filgrastim, pegfilgrastim, somatropin, follitropin alfa, teriparatide and epoetin biosimilars and their reference products. The data for the study was acquired from IQVIA and it covered pharmacy wholesale data between 1.1.2009–31.8.2020 for products under investigation. The weighted average wholesale price and monthly wholesale amounts were determined for each product, and the development of the price and market shares were analyzed. In addition, a linear segmented regression analysis was performed to examine the impacts of market entry of biosimilars on the prices of the reference products. According to the study, the prices of the reference products mainly decreased after the biosimilar entered the market. If the price of the reference product did not fall, it lost its reimbursement under the Health Insurance Act. The market shares of the reference products were marginal when they were no longer reimbursed. The prices of biosimilars did not change as much as the prices of reference products, and for most active substances biosimilar prices remained stable or decreased. The use of biosimilars varies widely between different biologics. The study found that prices of reference products were decreasing mainly as a result of various changes in drug policies. Therefore, biosimilars were not seen to generate genuine price competition between biological products. In many of the drug groups examined, the market shares of biosimilars had future growth potential.
  • Heiskanen, Vilma (2021)
    Binge eating disorder (BED) is the most common eating disorder characterized by compulsive recurrent binge eating episodes with the sense of a lack of control. During a binge eating episode, one eats a larger amount of food, typically high in fat and/or sugar, than would normally be eaten in a discrete period of time. After the episode, negative emotions, such as shame and self-disgust, are present. However, BED does not include compensatory behavior, such as vomiting or excessive exercise. Due to compulsive and uncontrollable eating behavior, it has been suggested that BED represents a food addiction. Eating energy-dense food activates the dopamine, opioid, and endocannabinoid systems in the brain. This elicits the activation of the reward process. Some drugs and medications affect the same neurotransmitter systems, which may produce neuronal alterations in the reward process, leading to an addiction. Several studies have found that cannabidiol (CBD) reduces the self- administration of cocaine, morphine, alcohol, and sucrose in rodents, suggesting an effect on the reward-response. Some of these effects have been shown to be mediated by cannabinoid receptor 2 and TRPV1 receptor. However, the effects of CBD on bingeing behavior have not been studied up to date. The aim of the study was to investigate the effect of CBD on homeostatic feeding and binge eating behavior in C57BL/6 mice. Five separate experiments were conducted. The first experiment investigated the effect of CBD (15, 50, and 150 mg/kg, i.p.) on locomotor activity in a modified open field test over a 2-hour period. In the second test, the effect of CBD (15, 50, and 150 mg/kg, i.p.) on homeostatic feeding was monitored in non-bingeing mice. Next, a limited intermittent access binge eating model without food deprivation or stressors was inducted. Mice had access to laboratory chow ad libitum, but a high energy diet (high in fat, HED) was presented in 24-hour periods every 5-8 days. Then the effect of CBD (15, 50, and 150 mg/kg, i.p.) on HED and chow intake in bingeing mice was investigated. In the fourth experiment, seven days following the administration, the after effect of CBD was studied by monitoring food intake without CBD treatment. Finally, it was investigated whether the effect of CBD can be inhibited by TRPV1 receptor antagonist AMG9810 (1 mg/kg, i.p). In each test, the food intake was monitored at the time point 0,5, 2,5, and 24 h after CBD treatment. Also, water consumption was measured in each experiment. The results revealed that CBD does not affect locomotor activity or homeostatic feeding at a dose of 15, 50, or 150 mg/kg (i.p). However, the results showed that CBD reduces the intake of HED in a dose-dependent manner (15, 50, or 150 mg/kg; i.p.) and, possibly, increases chow intake. No after effect was observed seven days following the administration. Most likely, TRPV1 does not mediate the effect of CBD on HED intake. Furthermore, no significant effects on water intake were observed. In this study, the core aims were to evaluate whether CBD affects homeostatic feeding or binge eating behavior in mice. The results provided a novel insight into the effects of CBD. The findings indicate that the acute systemic administration of CBD reduces HED intake, and possibly, simultaneously increases chow intake, suggesting a balancing effect on feeding in bingeing mice. However, the role of TRPV1 in this effect remains unclear, and further studies are needed.
  • Silmu, Veera (2021)
    Parkinsonin tauti on hitaasti etenevä hermorappeumasairaus, jossa mustatumakkeen dopamiinihermosolut tuhoutuvat. Taudille on tyypillistä dopamiinihermosoluissa esiintyvät Lewyn kappaleet, jotka koostuvat pääasiassa väärin laskostuneesta ja kasautuneesta alfasynukleiiniproteiinista. Myös neuroinflammaation uskotaan olevan osa Parkinsonin taudin patofysiologiaa. Nykyiset lääkkeet vaikuttavat ainoastaan taudin oireisiin, joten tarve uusille lääkkeille on suuri. Pilottikokeen tarkoituksena oli selvittää aiheuttaako adenoassosioidun virus- (AAV) vektorin alfasynukleiinin ja alfasynukleiinifibrillien yhdistelmämalli rotilla liikehäiriöitä ja tyrosiinihydroksylaasi- (TH) positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa sekä saadaanko mallilla aikaan neuroinflammatorinen vaste. Varsinaisen pitkän kokeen tarkoituksena oli selvittää aivojen dopamiinihermokasvutekijän (CDNF) mahdollinen neurorestoratiivinen vaikutus tässä mallissa. Alfasynukleiinin kasautumispatologian tasoa ja CDNF:n neurorestoratiivista vaikutusta selvitettiin käyttäytymiskokeilla sekä mustatumakkeen ja aivojuovion TH-vasta-ainevärjäyksillä. Yhdistelmämallista aiheutuvaa neuroinflammatorista vastetta selvitettiin ionisoidun kalsiumia sitovan adapterimolekyylin 1 (Iba1) ja gliaalisen fibrillaarisen happaman proteiinin (GFAP) vasta-ainevärjäyksillä. Pilottikokeen sylinterikokeessa yhdistelmämalli ei indusoinut liikehäiriötä, mutta pitkän kokeen askel- ja sylinterikokeessa mallin osoitettiin aiheuttavan unilateraalille leesiolle tyypillinen liikehäiriö. Pilottikokeen ja pitkän kokeen TH-vasta-ainevärjäyksissä mallin osoitettiin aiheuttavan TH-positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa. Nämä tulokset osoittavat, että yhdistelmämallilla saadaan aikaan alfasynukleiinin kasautumispatologiaa. Pilottikokeessa osoitettiin myös, että yhdistelmämallilla saadaan aikaan neuroinflammatorinen vaste, mikä osoittaa, että malli soveltuu hyvin uusien lääkkeiden vaikutuksen tutkimiseen Parkinsonin tautiin liittyvässä neuroinflammaatiossa. Pitkän kokeen sylinterikokeessa AAV-CDNF:llä ei ollut vaikutusta mallista aiheutuvaan liikehäiriöön. Sen sijaan askeltestissä kämmenen suunnan mittauksessa AAV-CDNF korjasi liikehäiriötä. AAV-CDNF ei kuitenkaan suojannut TH-positiivisia hermosoluja mustatumakkeessa tai hermopäätteitä aivojuoviossa, minkä perusteella johtopäätöstä CDNF:n neurorestoratiivisesta vaikutuksesta ei voida tehdä.
  • Cavonius, Karin (2021)
    Huntington’s disease (HD) is a rare but devastating neurodegenerative disease, progressively culminating in severe brain atrophy and death. The disease is caused by an inherited mutation resulting in a CAG trinucleotide repeat expansion in the huntingtin gene, leading to the production of a neurotoxic protein, known as mutant huntingtin, with an abnormally long polyglutamine stretch. Even though the genetic background of HD is known, the cellular pathways affected in the disease are complex and not completely understood. Increasing evidence indicates that endoplasmic reticulum (ER) stress – a condition of disturbances in normal ER activity, leading to accumulation and aggregation of misfolded proteins in the ER lumen – is a central factor in the pathogenesis of HD and other neurodegenerative diseases. In the literature review of this thesis, known pathogenic cellular mechanisms of HD and how these cellular mechanisms are connected to ER stress, are discussed. Unpublished data from previous studies in our laboratory have indicated that the ER luminal protein canopy homolog 2 (CNPY2) could play a role in the regulation of neuronal survival, including the viability of mutant huntingtin expressing neurons. The aim of the experimental part of this study was to gain insight into a possible function of CNPY2 in HD, by examining the levels of the protein in neuronal models of HD under various conditions, such as ER stress, and by searching for potential interacting partners of CNPY2 amongst known ER stress regulators. The obtained results show that the levels of CNPY2 are increased in striatal neurons expressing mutant huntingtin, and that the secretion of CNPY2 is increased by these neurons, compared to control neurons expressing normal huntingtin. Further, we show that CNPY2 interacts with the major ER stress regulator binding immunoglobulin protein (BiP) in human neuroblastoma cells treated with the ER stress inducer tunicamycin, and that the intracellular levels of CNPY2 are altered by tunicamycin treatment. Together, these findings indicate that CNPY2 could be involved in the pathogenesis of HD. However, further research on the functions of CNPY2 and its role in ER stress regulation is required to understand the nature of this involvement.
  • Backman, Heidi (2020)
    Theoretical framework: The consolidated pharmaceutical market is becoming increasingly global and the same international pharmaceutical companies operate around the world in different countries, responsible for drug development and production. The high costs of developing novel medicines and the motive for higher profits has led to elevating price level of pharmaceuticals and health care services. Finland and the U.S. offer two extremes at the pharmaceutical market. The pharmaceutical market field in Finland is very structural and rigid, and medicine prices are regulated by law. In the U.S. the prices are based on the laws of supply and demand and the prices differ by different states, retailers and insurance policies. A small-scale longitudal price comparison is also reviewed to showcase the effect of continuously rising medicine prices. Study objective: The idea of this study is to describe and compare pricing mechanisms of pharmaceuticals and price differences between two very different market structures and review how these might affect the cost-effectiveness of national health care spending. These divergences are also mirrored to survey recent global pharmaceutical market problems such as drug shortages, possibly due to less appealing markets of higher price regulation policies. Materials and methods: Price data were collected from national, official, open-source databases. National health care expenditure and comparison to GDP was collected from publications by the OECD. All monetary values have been presented in both currencies (EUR and USD) to present more comparable values. Results: When compared to other OECD-countries the U.S. spent distinctly the largest amount of funds on health care per capita. Finland’s national health care costs were thousand times minor in total spending and less than a half per capita when compared to those of the U.S. With lower expenditure Finland manages to offer access to public, government-funded health insurance program. Meanwhile the prices of prescription medicines in Finland have decreased significantly, the prices for have continuously elevated in the U.S. Conclusions: The outcome of this study is that free markets and a complex supply chain, compared to more regulated markets with more transparency, have higher overall price level in pharmaceuticals and health care services. Free markets and sufficient intellectual property rights are more enticing to pharmaceutical companies. They promote new innovations and developing of much-needed novel therapies to modern health problems, such as AIDS and the global threat of worsening situation of antibiotic resistance. More regulated markets may create problems such as drug shortages and are often considered complex and less appealing market systems due to high level of administrative work but conserve the cost-effectiveness of the use of public funds.
  • Virtanen, Sonja (2020)
    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.
  • Suominen, Laura (2020)
    Background: Alzheimer’s disease (AD) is a worldwide challenge for health care professionals and researchers. Every year, AD causes dementia for millions of patients. No preventive or curative medication is available despite continuous research. Amyloid-beta (Aβ) deposits in brain are one of the main pathological findings in AD. Accumulating Aβ peptides are thought to be the reason behind further disease progression. If the Aβ accumulation could be restricted or Aβ degradation increased their toxic effects would be prevented. Soluble oligomers and protofibrils are the most toxic species of Aβ. Most of the Aβ targeting drugs developed so far have not specifically targeted these toxic species. Neprilysin (NEP) is a major Aβ degrading enzyme that targets mostly the smallest species (monomers and dimers) of Aβ. Another common challenge for protein drugs has been passing the blood-brain barrier (BBB). Different strategies, such as utilising transferrin receptor (TfR) mediated transcytosis, have been studied for drug transport. For example, a rat anti-mouse TfR antibody, 8D3, or its fragments can be used for drug transportation. Objectives: To produce a recombinant protein, sNEP-scFv8D3, combining soluble NEP and single chain variable fragment of 8D3. Testing its ability to degrade different species and isoforms of Aβ in vitro and study in vivo brain uptake. Evaluate whether it is a promising model for future AD treatments. Methods: The recombinant protein was expressed in Expi293 cells and purified with affinity chromatography. The TfR binding was studied with TfR ELISA and enzymatic activity with MCA assay. Aβ ELISA was used for determining the Aβ degradation. Recombinant protein was compared to sNEP. In in vivo studies the brain uptake and blood half-life of radiolabeled sNEP-scFv8D3 of were studied on NLGF mice. Immunohistochemical analyses of brain cryo sections were done to evaluate the co-localisation of Aβ aggregates and sNEP-scFv8D3. Results and discussion: sNEP-scFv8D3 bound to TfR and showed similar enzymatic activity as sNEP. Both sNEP-scFv8D3 and sNEP were able to degrade monomeric Aβ-40 and Aβ-42 but no significant effect was seen on larger aggregates. In mice brain, sNEP-scFv8D3 was detected in same areas as Aβ aggregates. Compared to sNEP, our recombinant protein had better brain uptake. The blood half-life of sNEP-scFv8D3 was approximately 9.5 h and it was cleared fast from the brain. Already 6 h post injection, levels in the brain had dropped more than by half. Further studies are needed to determine whether sNEP-scFv8D3 is effectively transported across the BBB and if it can reduce brain Aβ levels in vivo. Conclusions: In the future, sNEP-scFv8D3 or its improved version could be used at the earliest stages of AD to prevent disease progression. Since sNEP-scFv8D3 degrades only small Aβ aggregates it could be combined with another drug targeting larger oligomers. Together they would decrease the total Aβ deposition in brain.