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(2023)Alpha-beta-hydrolase domain containing proteins (ABHD) are involved in lipid metabolism and its regulation in human and animal cells. Approximately 50 of these proteins have been identified and their physiological and pathophysiological functions are still further investigated. ABHD2, ABHD3, ABHD4, ABHD5, ABHD6, ABHD12 and ABHD16 are involved in the metabolism of glycerin esters and phospholipids, in particular lipid mediator 2-arachidonoylglycerol (2-AG) and its metabolites have a significant effect on neuroinflammation via the endocannabinoid system. ABHD12 and ABHD16A were at the center of focus in this thesis as enzymes regulating phosphatidylserine and pro-inflammatory lysophosphatidylserine. In this study, toxicity of five (5) abietane terpene derivatives was tested using mouse E15/16 prenatal cortical neurons, cultured in 96-well plates. There were totally 8 plates cultured in three different batches, 60 cell containing wells per each plate. Wells from each plate were divided into treatment groups of 17, three concentrations of every five compound, control and VEH groups. Those concentrations were 0,1 µM, 1 µM and 10 µM, and for the last two plates 1 µM, 10 µM and 100 µM. Treatment was also separated into three batches like the cell culturing. After treatment, number of living cortical neurons in each treatment groups were counted. For that, cells were treated with immunofluorescent NeuN and DAPI antigens and the fluorescence was imagined with automated microscope. CellProfiler was used to recognize and count the number of living cortical neurons. Immunofluorescent MAP2 antigens were also used but because the shape of MAP2-fluorescing cells, CellProfiler could not recognize them. Intensity of MAP2-fluorescence were measured from those imaged, so the work would not be wasted. One-way ordinary variance analyze ANOVA was carried out for the data to figure out if there were statistically significant results. For compound TAC174, there were several significant results with different concentrations but unfortunately, some results showed signs of toxicity and others improved cell-growth. Some significant results were also found with compounds TAC121, TAC147 and TAD40 showing sings of toxicity, but reliability of those results was questioned. Only one compound, TAC150 was not showing clear toxicity towards mouse cortical neurons, at least with lower concentrations. For conclusion, there were no clear or significant results if these compounds are toxic for cortical neurons. TAC150 showed the least sings of toxicity, therefore it could possibly be considered for further studies in medical field.
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(2017)The aim of this thesis was to synthesize abietic acid derivatives, and to determine their antimicrobial activity. The study was performed in two parts. The first part was carried out at VTT (Technical Research Centre of Finland) and the second part at the Faculty of Pharmacy, University of Helsinki, the Division of Pharmaceutical Chemistry and Technology. Abietic acid as well as its isomers and precursors are important antimicrobial substances among plant kingdom. They are produced in big scale by conifers, which extractives of pitch and wood they are. Abietic acid and its many derivatives have shown to have many bioactivities. Emerging antibiotic resistance of bacteria, viral diseases that are spreading and mutating throughout the world and serious diseases caused by protozoa, cause an ever-growing need to develop new active agents against these pathogens. Abietic acid derivatives are worthy candidates for research into new drug substances. At VTT a synthetic route with abietic acid chloride as intermediate was used (synthetic route 1). It did not work well in the synthesis of esters and yields were low. The synthetic route worked fairly well for amide synthesis. The purifications and analysis of the compounds, two esters and one amide of abietic acid, were left partially unfinished, and none of the products of these reactions were tested for bioactivity. At the Division of Pharmaceutical Chemistry and Technology eight derivatives of abietic acid, from abietic acid and four different amino acids, were obtained via synthetic route 2. Some of these were sent to tests for antibacterial activity. Six synthesized compounds were sent to tests for their bioactivity against bacteria. However, the results were not obtained in time for this Master's thesis, and therefore any conclusions of their structure-activity relationships could not be drawn.
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(2019)Background: Increased use of internet and the development of different services has led into the growth of ecommerce in many sectors and the e-commerce is growing significantly faster than any other economy in Finland. In addition, the search for different health related information from the internet has increased and this has led into increase in the online purchase of different health-related products and services in many countries. However, buying pharmaceutical preparations from online pharmacies have not become that popular in Finland yet, although there are many online pharmacy service providers. Objective: The aim of this study was to explore the acceptance and use of online pharmacies for the purchase of OTC medicines in Finland. The main purpose was to find out what are drivers and barriers to purchase OTC medicines online and which factors could facilitate to overcome customer perceived barriers. Furthermore, the aim was to investigate online purchase behavior for OTC medicines and to find out the insights required to develop more seamless online customer journey. Materials and methods: This study was conducted as a combination of quantitative survey and qualitative interview. The target group of this study was 18-74 year-old-people people living at the Greater Helsinki area. The data was collected with an online survey (n=262), one focus group discussion (n=5) and one-to-one interview (n=3). Participants of both, the survey and interviews, were chosen by convenience sampling and they were drawn in via social media, mainly by Facebook, and in co-operation with a few pharmacies in the Greater Helsinki area. Quantitative analysis of the survey was made by using version 25.0 of the IBM Statistical Program for Social Sciences (SPSS) and data obtained with open questions and interviews was analyzed by using conventional deductive content analysis. Results: In this study sample, 16.5% had bought medicines online. Independence from time and place, convenience and time saving were the biggest drivers to shop OTC medicines online, while the biggest barriers were lack of additional value, high price of the delivery and long delivery time as well as acute nature of the problem. Cheaper price of the medicine was the strongest factor that could get people consider buying online. Results indicate that the online customer journey follows the general five-stage decision making model while purchasing unfamiliar medicines. Internet turned out to be the primary source of information before purchase and self-diagnosis could be made with the help of information found from the internet. In addition, perceptions and experiences of important others and advice from the pharmacist were considered as useful help in the process of self-diagnosis. Conclusions: Barriers for the purchase are currently dominating over the motivating factors. However, the majority of non-buyers would be ready to consider buying medicines online. Currently,the online pharmacies cannot compete with the prices of the medicines, due to the local regulations, but pricing of other pharmacy products is free and those could work as incentive to buy also medicines online. In addition, it would be worth for online pharmacies to invest on developing quick and reasonably priced delivery services and properly working, real-time chat service as well as further increase awareness of their services. Pharmaceutical companies can improve the customer journey by providing the information consumers usually search at their product pages and already at Google-view as a quick links. In addition, online pharmacies should be provided with sufficient information about their products.
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(2018)The nucleus accumbens (NAc) is located in the ventral striatum and plays a critical role in drug addiction. NAc receives dopaminergic projections from ventral tegmental area (VTA) which is activated after administration of various abused drugs. Activation of VTA increases the release of dopamine in NAc. Increased dopamine levels induce the release of acetylcholine from striatal cholinergic interneurons. These cholinergic interneurons have been related to the development of addiction and other emotion-related disorders such as depression. Previous studies have shown that a lesion of cholinergic interneurons led to an increase in morphine-induced conditioned place preference in mice. Moreover, an activation of cholinergic interneurons by designer receptors (DREADD) has reduced food consumption motivated by food restriction. The purpose of this study was to investigate whether accumbal cholinergic interneurons mediate alcohol- and morphine-induced conditioned place preference and locomotor activity. The activation of cholinergic interneurons was controlled using DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology. DREADDs are G protein-coupled receptors. Cellular function and activation can be modulated by these receptors. DREADDs are activated by an otherwise inert synthetic ligand, clozapine-N-oxide (CNO). Fluorescent protein, mCherry, is attached to DREADDs so that the expression of receptors in brain tissue can be observed. Cre-spesific adeno-associated viruses (AAV) with DREADD gene were injected bilaterally to the nucleus accumbens of ChATcre mice in stereotactic surgery. The effects of alcohol and morphine were tested with conditioned place preference procedure. Mice were divided to three groups after DREADDs: activating receptor Gq (n = 10), inhibiting receptor Gi (n = 9) and control mC (n = 9). There were both male and female mice in every group. Alcohol did not induce conditioned place preference in any group. The locomotor activity of mice significantly increased after alcohol injection compared to saline injection. However, cholinergic interneurons had no effect on the increased locomotor activity. Morphine-induced conditioned place preference was expressed in every group but there were no significant differences between DREADDs and control group when the first 15 minutes and the whole 30 minutes of the place preference test was analysed. Though, Gq-receptor seemed to decrease the place preference compared to control group when the place preference test was observed in five minute intervals. Morphine also significantly increased the locomotor activity of mice, but there were no differences between the groups. Sex had no influence on the place preference, but female mice were more active than male mice during the alcohol conditioning and the alcohol place preference test. The locomotor activity of female mice also increased more than the activity of male mice after morphine injection. The effect of accumbal cholinergic interneurons on alcohol-induced conditioned place preference remained unclear. Activation of cholinergic interneurons suppressed morphine-induced conditioned place preference compared to control group but not enough so that the effect could be seen during the whole place preference test. The mice were same in the morphine test as in the alcohol test so the mice were conditioned to alcohol before morphine and therefore the results of morphine-induced conditioned place preference are not reliable.
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(2020)Nucleus accumbens, located in ventral striatum, is an important part of the brain reward system. Accumbens integrates information coming from various brain areas, and it’s important for feeling pleasure, reward learning and reward seeking, including drugs of abuse. Cholinergic interneurons represent a few percent of accumbal cells. Earlier research suggests that accumbal cholinergic activity decreases drug seeking and eating. The aim of this study was to examine the role of cholinergic interneuron activity in alcohol drinking and alcohol related locomotor activity. Cholinergic interneurons (ChI) were manipulated using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), which can be selectively activated with clozapine-n-oxide (CNO). To express DREADDs selectively in ChIs, a cre-dependent viral vector that contained a gene coding for a cell-activating hM3D(Gq)-mCherry (n=9), cell-inhibiting hM4D(Gi)-mCherry (n=9), or control mCherry(n=8), was injected to nucleus accumbens of ChAT-cre- mice. Alcohol drinking was measured using Drinking In the Dark (DID)- model. Three hours after lights-out, the water bottles were replaced with 20% alcohol for two hours, for three days. On the fourth day, mice were injected with CNO or vehicle and alcohol was given for four hours. These cycles were repeated six times. In the locomotor assay, mice were injected with CNO or vehicle, followed by injection of alcohol or saline. Locomotor activity of the mice was observed for 30 minutes. In the DID- assay, the DREADD ligand CNO did not have effects on alcohol drinking within any of the three groups. However, Gi- mice drank more alcohol than Gq-mice even without the presence of CNO. These results are not reliable enough to draw conclusions, as they were confounded by unusually low drinking volumes. In the locomotor assay, CNO alone did not affect locomotion in any group. Together with alcohol, however, CNO decreased locomotion in all three groups, compared to alcohol alone. This is consistent with recent reports suggesting that CNO may have nonspecific behavioral effects.
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(2015)Health related issues are largely regulated at EU Member State level, whereas areas such as internal market and competition fall mainly under the remit of European Union competence. This creates tension not only between legislation governing health and that concerning internal market but also between national and EU legislation. Here the Court of Justice of the European Union (CJEU) plays a key role in developing case-law through its judgments, defining further interpretation both on European and national level. An example of a sector operating at the interphase between public health and internal market interests is community pharmacy, which was chosen as the focus area of the study. The aim of this master's thesis was, through the case-study example of the regulation of pharmacy establishment, ownership and distribution in EU Member States, to perform a documentary analysis on related CJEU judgments, focusing on statements present in them referring to public health and internal market, discussing potential impacts on the community pharmacy sector as well as relating the outcomes to the broader context of European health policy with reference to existing literature. The study material consisted of publicly available documentation related to four judgments (Case C-531/06, Joined Cases C-171/07 and C-172/07, Joined Cases C-570/07 and C-571/07 and Case C-367/12) that were made between the years 2009 and 2014, the first proceedings initiating in 2006. The prevalence and variety of statements related to public health were found to be much higher in the documents analysed compared to those relating to internal market. The most common argumentation present in the judgments was related to the statement that regulation of ownership of community pharmacies can be justified by public health reasons, deriving from the professionalism inherent to pharmacists as well as ensuring balance between public health and economic interests. This transmits a clear message of the importance of public health and indeed the Court has been perceived as a balancing force to the union's liberalisation agenda. Following this it seems unlikely that the interpretation for national regulation would change in the near future, meaning that Member States should be able to maintain community pharmacy regulation, to the extent that it is implemented in a consistent manner. However, there has been indication of other routes being used to push for the liberalisation agenda and therefore it continues being a part of the debate both at European and national level. The findings of this study support literature suggesting that spillover is taking place in relation to the Court of Justice and health. Furthermore, it has been clearly demonstrated that even though officially the EU has very limited competence (authority) in health, its influence on European health policy is in fact highly significant, taking place to a large extent via routes other than those officially assigned to it in relation to health in particular. Whether this is intentional or unintentional, it does not change the fact that health policy is being influenced. When it happens without explicit intention, the processes lose transparency and are driven by other, potentially competing agendas. Therefore it would be important to assess whether the decision making processes and other processes currently shaping the European healthcare policy are in line with what was originally intended and re-evaluate whether this dynamic is the preferred way to proceed in the future.
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(2019)Anisakiasis is a parasitic disease caused by larval nematodes of the genus Anisakis. Humans become infected by consuming contaminated raw or undercooked seafood products. Most human infections are caused by Anisakis simplex (A. simplex) complex. Currently there is no effective drug for this global emerging disease. Novel active compounds against the nematode are needed for drug development purposes. The research with A. simplex requires the isolation of the larvae from fish, which is time-consuming, unecological and uneconomical. Thus, the utilization of the model nematode Caenorhabditis elegans (C. elegans) in the research of A. simplex is considered in this study. Activities of Tea tree, Java citronella and Ho wood essential oils against C. elegans were studied. Aim of the assays was to examine whether C. elegans could be used as a model for A. simplex. Observed effects on C. elegans were compared to the previously reported effects on A. simplex. Activity of Tea tree and Java citronella essential oils against A. simplex was also examined to confirm previously reported activity. In addition, activity of six coumarins against A. simplex was investigated. The aim of the assays was to discover novel active compounds against the pathogenic nematode. Four coumarins were tested against C. elegans to examine possible comparable effects. Toxicity studies were performed in aquatic medium in a 6 well plate format (A. simplex) and in a 96 well plate format or in 1.5 mL Eppendorf tubes (C. elegans). Tea tree essential oil showed dose dependent activity against C. elegans, producing 100% mortality with the concentration 20 μL/mL after 24 hours exposure. Compared to A. simplex, two to three times higher doses were required to produce same degree of mortality in C. elegans. By contrast, Java citronella and Ho wood essential oils showed no significant activity against C. elegans. The activity of Tea tree and Java citronella essential oils against A. simplex was confirmed. The tested coumarins displayed no significant activity against the nematodes. Due to the contradictory results, further investigation about the suitability of C. elegans as a model for A. simplex is needed. Differences between the effective concentrations are probably caused by the differences in the biology of the nematodes, which result from the phylogenetic distance. Based on current results, the tested coumarins were excluded as potential antinematodal compounds against A. simplex, due to the lack of any significant activity on this model.
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(2015)Manufacturing execution systems (MES) are computer systems which are used for controlling and automating manufacturing processes. They are increasingly adapted in pharmaceutical industry. Implementation solutions differ, however, and there is no single solution which would be the optimal one for all facilities. Each manufacturing facility has their unique properties and needs which have to be reflected in the implementation. A successful MES project will bring plenty of benefits such as more efficient use of resources and automated data transfer, but the roll out phase might turn to be problematic if the processes of the organization have not been analysed thoroughly enough at decision making. This creates the need for systematic analysis of possible to-be implementation scenarios which is based on the value-drivers of the organization and considers the decision from multiple viewpoints. This study presents a holistic value driver-based framework with a mathematical weighing method to allow for a systematic and scientifically justified decision for identification of the optimal implementation depth of equipment management (EQM) in MES. A Delphi study method was utilized in this study to create the framework. The framework was developed based on literature and brainstorming sessions with experts and validated by means of a Delphi questionnaire round with expert panel consisting of professionals representing the major stakeholders of MES system in a pharmaceutical manufacturing facility. Classical additive weighing method was applied to create a mathematical basis for valuation and comparison of the scenarios. The robustness of mathematical method was tested by means of a sensitivity analysis. A benchmarking survey was done to obtain information on current implementation solutions and decisions leading to current situation. The presented method not only addresses the costs but also takes into account intangible factors. Intangible factors include aspects such as good manufacturing practice (GMP) quality and user acceptance which are not directly transferable into quantitative units but are crucial both for pharmaceutical industry and the success of the implementation project. The framework describes the decision in the form of a value tree with three main branches, namely GMP, cost and process&organization which cover the main viewpoints important for the decision. The presented method also allows the weighing of different factors according to current needs of the facility and decision in question. Hence, the presented framework leads the decision maker through a systematic and comprehensive analysis of different to-be scenarios for EQM implementation. The benchmarking survey identified three major factors of a successful MES implementation, namely effort in design phase, well-defined processes and close discussion with production. The value drivers valued highest by the expert panelists were related to GMP quality. As a use case, the presented framework was applied in a parenterals clinical manufacturing facility to evaluate six different to-be scenarios and based on the results one of them was selected by the management to be implemented. The results from the use case indicate that the framework is a valuable tool in a decision making process, and encourage the further utilization of the framework in future implementation decisions.
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(2023)African medicinal plants have been used to treat symptoms of infection successfully for thousands of years. However, no antimicrobial drugs have been developed from these plants. As antibiotic resistance is increasing rapidly, these traditional African herbal medicines can be an important solution in the fight against antibiotic resistance due to their antimicrobial properties. In this research, various extracts o the leaves of Combretum adenogonium (Combretaceae) and the fruits of Piper cubeba (Piperaceae) and Xylopia aethiopica (Annonaceae) were tested for their growth inhibitory effects against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Extracts were made with methanol, water, hexane, and chloroform. In addition, water and ethyl acetate extracts were separated from an 80 % methanol extract using solvent partition. All the studied plants are used for the treatment of infections and wounds in African traditional medicine. Water was used as extraction solvent since it is commonly used in African folk medicine. Both single solvent technique and sequential extraction were used. The antibacterial effects were screened using agar diffusion and microdilution methods. The interaction between an extract and an antibiotic was measured with a checkerboard method. Time-kill experiments were performed using microdilution and plate count methods. In this study, the chloroform extract of C. adenogonium leaves gave the best inhibitory effect of all studied plants against B. cereus (MIC 78.125 µg/ml). In general, B. cereus was the most susceptible of the selected bacteria against extracts and E. coli was the one with most resistance. Time-kill test showed that the antibacterial efficacy was fairly stable throughout the 24-hour period considered with few exceptions. According to checkerboard results, C. adenogonium chloroform extract and tetracycline appeared to inhibit each other's antibacterial activity against B. cereus. However, only one extract was studied in this study, and it is possible that C. adenogonium contains compounds that would have a potentiating effect on antimicrobials. In general, C. adenogonium extracts were effective against B. cereus. The extracts of P. Cubeba were particularly effective against S. aureus. X aethiopica extracts were equally effective for both B. cereus and S. aureus. Methanol extract X. aethiopica is the only extract studied that gave more than 90% inhibition against P. aeruginosa. Therefore, it could be concluded that X. aethiopica has the broadest activity range of the examined plants.
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(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.
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(2014)The increasing microbial resistance against conventionally used antibiotics has become a worldwide problem. Plant derived compounds may have different mechanisms of action, which might reduce the resistance problem. The aim of this study was to investigate the antimicrobial activity of three African medicinal plants, Terminalia kaiserana, Terminalia sambesiaca and Combretum psidioides, belonging to the family Combretaceae. All three plant species are used for treatment of bacterial and fungal infections in African traditional medicine, but there is limited, or in the case of Terminalia kaiserana, no research on the chemical composition of these plants. Dried plant material was extracted with methanol and solvent-partition extraction was performed using chloroform, butanol and water. Chemical compositions of the fractions was examined using RP-18 thin layer chromatography. The fractions were screened for antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa using an agar diffusion method. The most effective fraction against both bacteria was the water soluble fraction of the root bark of T. sambesiaca. A microdilution method was used to determine minimum inhibitory concentration (MIC). This method was used for S. aureus, P. aeruginosa and in addition for Mycobacterium smegmatis. The method was modified for M. smegmatis to contain a smaller inoculum size in the beginning of the experiment than for the two other bacteria. The lowest MIC-values against S. aureus were given by the crude extract and water soluble fraction of the stem bark of C. psidioides and by the butanol fraction of the same plant against P. aeruginosa. The results on the antibacterial effects of the screened extracts were notable and significant. The antimicrobial activity against M. smegmatis was not as obvious as for the other tested bacteria but the choloroform fraction of the root bark of T. kaiserana and the butanol fraction of the stem bark of C. psidioides showed promising preliminary results. Separation of fractions and compounds of the root bark of T. kaiserana was performed using Lobar RP-18 column cromatography in order to investigate which fractions or compounds are responsible for the antimicrobial activity. The antimicrobial activity of the fractions was examined using the microdilution method. The most effective fraction against both S. aureus and P. aeruginosa was the fraction 2F8, which containsthe same compounds as the crude extract, but a higher concentration of polar ellagitannins which are probably responsible for the antimicrobial activity of this fraction. Also fraction 2F9 showed antimicrobial activity against P. aeruginosa. This fractions contains ellagic acid derivatives which are probably responsible for the antimicrobial activity. The crude extract of the root bark of T. kaiserana was also fractionated using RP-18 thin layer chromatography, because this method gave better separation compared to column chromatography. Due to limited time the antimicrobial activity of the TLC-fractions will be investigated in the future.
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Aivojen kolinerginen järjestelmä ja histamiinireseptorit Parkinsonin taudin 6-OHDA-jyrsijämallissa (2015)Parkinson's disease is a neurodegenerative disease where the nigrostriatal dopaminergic cells die gradually causing severe motor symptoms. Current treatment of the disease relieves the symptoms but does not affect the progression of the disease, nor does it have a neuroprotective effect. The most important drug for the treatment of Parkinson's disease is L-dopa, the precursor of dopamine. With long-term use, L-dopa loses its efficacy and patients start to get adverse effects. The most significant adverse effects are abnormal involuntary movements called dyskinesias. In the literature review of this thesis Parkinson's disease and its treatment is briefly described. Review focuses on the description of the brain cholinergic and histaminergic systems and their receptors along with the available studies about cholinergic and histaminergic neurotransmission in Parkinson's disease 6-hydroxydopamine (6-OHDA) rodent model. The experimental part of this thesis consisted of two different set of experiments and in both of these the dopamine neurons were destroyed unilaterally by injecting 6-OHDA into the striatum. The aim of the first experiment was to examine histamine H3-receptor antagonist JNJ-39220675 and α7-nicotinic receptor agonist PHA-543613, and their combination therapy effects on motor function and the concentrations of striatal neurotransmitters in hemiparkinsonian mice. Effects on motor function were studied two and four weeks after the 6-OHDA injection with cylinder test, the D-amphetamine-induced rotations, and the inverted grid test. After behavioral tests, mice were sacrificed and striatal neurotransmitter concentrations were determinated by HPLC. The aim of the second experiment was to examine if nicotine can relieve L-dopa-induced dyskinesias. In this experiment 6-OHDA was injected at two sites into the striatum, which was intended to produce more extensive destruction of dopaminergic neurons than in the first experiment. The extent of the lesion by 6-OHDA was verified before starting chronic L-dopa treatments with cylinder test. One month after the 6-OHDA injection, five mice were sacrificed and their striatum and substantia nigra sections were measured for destruction of dopaminergic neurons by immunohistochemical TH-staining. Chronic L-dopa treatment with benserazide was started 49‚àí63 days after the 6-OHDA injection. At the same time, mice were divided into two groups. Half of them got normal drinking water and half got nicotine water. During the chronic L-dopa treatment, development of dyskinesias was observed once a week by video tracking. The cylinder test was also done once again after starting the L-dopa treatment. In the first experiment, H3-receptor antagonist JNJ-39220675 showed promising results in improving motor function. Mice used the impaired (contralateral) paw more in the cylinder test and rotated less to the ipsilateral side in the D-amphetamine-induced rotation test than control animals two weeks after the 6-OHDA injection. Combination therapy also reduced the ipsilateral rotations but in the cylinder test it had no effect two weeks after 6-OHDA injection. Because the asymmetry in behavioural tests were caused by destroying dopaminergic neurons, balancing of the motor skills can result from decreased levels of dopamine in the intact side or from increased dopamine levels or stronger dopaminergic postsynaptic transmission in the lesion side. The results four weeks after 6-OHDA injection are not reliable because the striatal samples showed that dopamine concentrations in the lesion side were very close to that of the intact side indicating recovery from the lesion. In the second experiment, mice developed dyskinesias which were decreased with nicotine treatment. Mice also used the contralateral side paw less indicative of loss of dopamine neurons. In agreement, TH-immunostaining confirmed significant loss of TH-positive neurons. Based on these findings, the 6-OHDA injection site, the selected drug doses, and the experimental design seem to fit the evaluation of dyskinesias. The occurrence of dyskinesias and nicotine's effect on them was seen strongest in the body movements. Dyskinesias in forelimbs were minor, but the nicotine treatment decreased them also.
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(2015)This review focuses on neurotrophic factors, especially CDNF, and Amyotropic lateral sclerosis (ALS). This review finds out which neurotrophic factors have been studied in clinical trials of ALS and what kind of results have been got. Neurotrophic factors are important for development and function of neurons because they prevent apoptosis of neurons. They also play role in differentiation, development and migration of neurons. It is also known that many of the neurotrophic factors have protective and restorative properties. ALS is a rare neurodegenerative disease which causes the destruction of motor neurons and leads to death in three years. The disease degenerate the upper and lower motor neurons. Symptoms are muscle weakness, muscle atrophy, cramps and problems with swallowing. At the moment there is no cure for ALS so it is important to study neurotrophic factors that could prevent the progression of the disease and perhaps to protect or repair destroyed motor neurons. This is why it is important to study potential of CDNF in ALS. The experimental part consists of three different parts. The purpose of the first part study was to determine the distribution of CDNF after intraventricular delivery at different time points. CDNF was labeled with 125I (125I-CDNF). The distribution was determined by gammacounter and autoradiography. To determine the stability of the injected 125-I CDNF we performed SDS-PAGE. The second part studied the diffusion volume of CDNF after intraventricular injection with seven wild type mice. After stereotaxic surgery CDNF-immunohistochemistry staining from coronal sections was done. The last experimental part studied the effect of single intracerebral injection of CDNF on motivation, locomotor activity, anxiety and depression with male and female mice. Light-dark box, open field, rotarod, forced swim test (FST), elevated plus maze and fear conditioning were carried out with male mice. After behavioural tests mice were sacrified for HPLC-analysis. Light-dark box and IntelliCage were carried out with female mice before c-fos staining. Gammacounter and autoradiography shows that 125I-CDNF distributes widely after intracerebroventricular injection. It spread throughout to the brain and also all the way to the spinal cord after one and three hours from injection. After 24 hours 125I-CDNF was cleared so the CDNF signal was very weak. SDS-PAGE showed the stability of radioactive CDNF. CDNF increased locomotor activity and decreased anxiety in male mice. But a statistically significant difference appeared in forced swim test and fear conditioning test. HPLC-analysis supported these results partly. CDNF also increased motivation of female mice in IntelliCage experiment. C-fos staining was observed in CDNF group and PBS group so quantitative analysis should be done from these sections so that reliable conclusions could be done. However, because CDNF distributed to spinal cord and it showed some effect on locomotor activity, motivation and depression it might be potential for ALS disease.
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(2019)Parkinson’s disease is a progressive neurodegenerative disease, in which dopamine neurons are dying in the nigrostriatal dopaminergic pathway. This causes motor symptoms such as slowness of movement, tremor, and rigidity. In addition, various non-motor symptoms appear. All currently used medicines are symptomatic, and there are no disease modifying treatment available for Parkinson’s disease. Several neurotrophic factors have shown promise in animal models of Parkinson’s disease. One of those is cerebral dopamine neurotrophic factor (CDNF) which has been studied in different animal models, including rodents and non-human primates. CDNF is a secreted protein but it is also localized in endoplasmic reticulum (ER). CDNF has two domains, N-terminal and C-terminal, which may have distinct functions. CDNF can be retained in the ER by the ER retention sequence at the end of the C-terminal domain. The C-terminal domain also has an evolutionarily conserved disulfide bridge which is crucial for the biological activity of CDNF. The exact mechanism of CDNF is still unknown. However, it has been shown that CDNF affects the unfolded protein response (UPR) in the presence of ER stress. Neurotrophic factors do not penetrate blood-brain barrier (BBB), for this reason, they need to be injected directly to the brain. Penetration of the BBB is also a problem in the treatment of many other diseases. Various methods for enhancing the BBB penetration of drugs have been studied. For example, permeability of the BBB can be temporarily increased by focused ultrasound combined with microbubbles. Another possibility is the use of a carrier molecule, which can be transported through BBB via specific transport mechanisms. Furthermore, molecule modification offers many applications to achieve enhanced BBB penetration. In view of peripheral administration, a next generation variant of CDNF (ngCDNF) has been developed. The efficacy of this novel variant after intrastriatal injection is equal to that of CDNF in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease. Systemic administration could also enable treatment of non-motor symptoms of Parkinson’s disease. The aim of this experiment was to study the effects of subcutaneously injected ngCDNF on rotation behaviour, and nigrostriatal TH-positive cells in rats with 6-OHDA lesions. 6-OHDA was injected unilaterally to three different sites in the striatum. Two weeks later, the lesion size was estimated, via amphetamine- induced rotation test. ngCDNF, at two dose levels, was injected twice weekly for three weeks. Amphetamine-induced rotation test was assessed every other week, until week 12. At the end, optical density of tyrosine hydroxylase (TH) was measured from sections of the striatum, and TH positive cells in the substantia nigra were counted. In addition, the effect of ngCDNF on anxiety and depression like behaviour, learning, and locomotor activity were studied at three different levels in naïve mice. Behaviour was analyzed by open field test, forced swim test, and fear conditioning test. The ngCDNF did not seem to have clear effect on rats’ behaviour or TH positive cells and fibers compared to the control group, but positive tendency was found in the group with lower dose. The reduced efficacy of ngCDNF,via subcutaneous administration, is likely due to rapid metabolism and insufficient entry of the active form to the brain. In naïve mice, ngCDNF did not reduce anxiety-like behaviour and did not affect locomotor activity after subcutaneous injections. This result supports previous findings, which suggest that the effects of CDNF are specific to the toxin treated cells and CDNF has no effect in naïve animals.
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(2021)Abstract Faculty: Faculty of Pharmacy Degree programme: Master of Science in Pharmacy Study track: Social pharmacy Author: Laura Toivonen Title: Deficiencies and risks related to medication use management in nursing homes identified by Regional State Administrative Agencies during their inspection visits Level: Master´s thesis Month and year: November 2021 Number of pages: 94+7 (appendice) Keywords: Medication safety, medication use process, nursing home, older adult, risk management, guidelines for safe medication practices Supervisor or supervisors: M.Sc. Pharm, PhD student Suvi Hakoinen, University of Helsinki, Keusote; Professor, PhD Marja Airaksinen, University of Helsinki Where deposited: ethesis.helsinki.fi Additional information: Abstract: Nursing home residents are often characterized by older age, multimorbidity and polypharmacy. Medication safety has become an issue as part of client/patient safety in nursing homes in Finland. Still, little is known about medication safety risks and their management in this care context. The aim of this study was to identify deficiencies and risks associated with medication use management in nursing homes using inspection visits by the Regional State Administrative Agencies (AVIs) as a data source. In addition, the aim was to evaluate what issues the AVI-authorities pay attention to in the medication use management during their inspection visits in nursing homes. The data consisted of the latest inspection reports (n = 24) prepared by the Regional State Administrative Agencies (n = 6) on the basis of their nursing home visits (nursing homes for older people). The data were analysed by deductive content analysis methods. In addition to qualitative documentary analysis, quantitative indicators were used to illustrate the frequency of the risks and deficiencies -observed in different phases in the medication use process to identify phases posing risks most commonly. Reason´s system-based risk management theory was applied as a theoretical framework for the study. A total of 372 deficiencies and risks related to medication use process were identified from the inspection reports (n = 24) of Regional State Administrative Agencies. The largest proportion (58,9 %, n = 219) of the deficiencies and risks concerned the management and quality management of the medication use process. Particularly, deficiencies and risks related to lack and competence of personnel were emphasized. Deficiencies and risks were also identified in the self- assessment guidelines for safe medication practices used in the inspected nursing homes. The second highest number of risk observations (26,9 %, n = 100) was reported for ordering, delivery and storage of medicines. In particular, the deficiencies and risks were reported for the storage and warehousing practices. Reported risk observations in other phases of the medicines use process were rare. This study showed that the medication use process in Finnish nursing homes includes deficiencies and risks endangering the safety of the older nursing home residents. In order to manage the risks, both self-assessment and inspection practices by the authorities need development. One way to improve and harmonize both self-assessment and inspection practices could be use of a comprehensive checklist covering of all the relevant issues required for the safe medication practices in nursing homes. Increasing collaboration with pharmacists could also be a way to improve medication safety in nursing homes.
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(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.
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(2024)Alzheimerin taudin riskiä lisää monet tekijät sekä mahdollisesti tietyt lääkkeet. Näiden riskien monitoroinnilla saattaa olla suotuisa vaikutus ennusteeseen. Näin ollen riskiä lisäävät lääkkeet olisi erityisen tärkeää tunnistaa ja lopettaa tai rajoittaa käyttöä korvaamalla muilla lääkeryhmillä tai lääkkeettömillä keinoilla. Pitkäaikaisesti käytössä olleiden lääkkeiden hallittu lopettaminen eli annosten pienentäminen tai lopettaminen vaatii varovaisuutta ja tarkkaa harkintaa, jotta vältytään lopetusoireilta eikä heikennetä elämänlaatua. Tämän Tutkielman tavoitteena oli tutkia, kuinka kognitiivisia haittoja aiheuttavia lääkeaineita voidaan lopettaa hallitusti, jotta vältytään irreversiibeleiltä kognitioon kohdistuvilta haitoilta. Tämä toteutettiin tutkimalla lääkevalmisteiden lakisääteisistä valmisteyhteenvedoista (SmPC), pakkausselosteista ja muista keskeisistä terveydenhuollon ammattilaisille suunnatuista tietolähteistä̈ löytyvää tietoa trisyklisten masennuslääkkeiden ja bentsodiatsepiinien hallitusta lopettamisesta. Tutkimusta varten tarkasteltiin kaikkia Suomessa huhtikuussa 2024 markkinoilla olleita lääkeaineita, joilla on merkittäviä antikolinergisia vaikutuksia. Lisäksi tarkasteltiin tuolloin markkinoilla olleita unilääkkeinä käytettyjä bentsodiatsepiineja. Näistä tutkimukseen sisällytetyt lääkeaineet valittiin sen perusteella, että niiden lopettamisessa tulisi noudattaa varovaisuutta (valittiin trisykliset masennuslääkkeet ja bentsodiatsepiinit). Tutkimus toteutettiin laadullisena sisällönanalyysinä, jossa aineistona käytettiin tutkimukseen valittujen lääkkeiden (amitriptyliini ja nortriptyliini, diatsepaami, oksatsepaami ja loratsepaami) valmisteyhteenvetoja (n = 5), pakkausselosteita (n=5), kansainvälisesti merkittävää hoitosuositusta: 1) American Psychological Association APA: Practice Guideline for the Treatment of Patients with Major Depressive Disorder, Yhdysvallat, 2) National Institute for Health and Care Excellence NICE: Depression in Adults: Treatment and Management, 3) NICE: Medicines Associated with Dependence or Withdrawal Symptoms: Safe Prescribing and Withdrawal Management for Adults, 4) The Maudsley: Deprescribing Guidelines Antidepressants, Benzodiazepines, Gabapentinoids and Z-drugs, Iso-Britannia sekä yhtä kansallista hoitosuositusta (Suomalainen Lääkäriseura Duodecim: Unettomuus Käypä̈ hoito -suositus). Tiedon määrä, sisältö ja laatu vaihtelivat tietolähteiden välillä. Trisyklisten masennuslääkkeiden hallitusta lopettamisesta löytyi selkeästi vähemmän tietoa kuin bentsodiatsepiinien hallitusta lopettamisesta. NICE:n hoitosuositukset täydensivät toisiaan lääkityksen hallitusta lopettamisesta. APA- ja käypä hoito -ohjeistukset sisälsivät tietoa yleisellä tasolla, mutta eivät sisältäneet yksityiskohtia lääkityksen hallitusta lopettamisesta. Muistin ja kognition kannalta huomattiin, kuinka trisyklisten masennuslääkkeiden valmisteyhteenvedoista ja pakkausselosteista puuttui kokonaan tietoa mahdollisista muistiin tai kognitioon kohdistuvista haitoista. Bentsodiatsepiinien mahdollisista muistiin tai kognitioon kohdistuvista haitoista taas löytyi jonkin verran tietoa, mutta tieto oli suhteellisen rajallista ja epätarkkaa.
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(2011)Literature review: There is a need for new disease modifying therapies for Alzheimer disease. In order to develop these, animal models with better Alzheimer disease pathology are required. Old rat models like giving scopolamine or MK-801 or using aged rats don't have many of the characteristics of Alzheimer disease although they diminish cognitive functions in different models. Newer models like transgenic and Aβ-injected or -infused rats have much more analogy to the pathology of Alzheimer disease - at least when Aβ-pathophysiology is concerned. Taupathophysiology however doesn't occur in either of the models. On the other hand Aβ has a bigger role in the pathophysiology of Alzheimer disease so it's more important to have that in both models. These two models seem to be quite similar in modelling the disease. Injecting or infusing Aβ to the brain of the rat may be easier to conduct in practice than to create a transgenic rat line. However in transgenic rats Aβ-pathophysiology is developed intracellularly like in Alzheimer disease instead of giving aggregated Aβ outside of the brain. Still both of the models can be used as well to study new therapies especially affecting Aβ-pathophysiology. Experimental part: The purpose of the study was to validate elevated plus-maze (EPM) as a cognition model with a Trial1/Trial2-protocol (T1/T2) in mice. In this experiment a-five-minute-trial was used and thus different parameters related to cognition were measured. The memory of the mice was tried to be disrupted with time delay (1-18 d) between trials or with muscarinic receptor antagonist scopolamine (0.1-0.8 mg/kg i.p.) 30 minutes pre-T1. These experiments were conducted in C57BL/6J- and ICR:(CD-1)-mice. The only group in the time interval experiment that had a trend of forgetting was the 18 d group of ICR:(CD-1)-mice. Thus 21 d interval was also studied, but clear signs of forgetting couldn't be seen. Scopolamine didn't disrupt memory in ICR:(CD-1)-mice but in C57BL/6J-mice it did significantly with doses 0.2-0.8 mg/kg. Based on this 0.2 mg/kg was selected to be used in further studies in C57BL/6J-mice. In this model the nootropic effects of donepezile (0.3, 0.8 and 1.5 mg/kg s.c.), memantine (5.0 and 10.0 mg/kg s.c.) and an experimental 5-HT6-antagonist SB742457 (1.5 and 6.0 mg/kg s.c.) were studied. These compounds were allocated 40 minutes pre-T1 and scopolamine 30 minutes pre-T1. Memantine (5.0 mg/kg) clearly and donepezile (1.5 mg/kg) with a strong trend enhanced cognition disrupted by scopolamine. These results suggest that EPM can be used when testing nootropic effects.
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(2011)Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.
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(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.
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