Browsing by department "Farmasian tiedekunta"
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(2017)In vitro liver cell models are important systems to study for example hepatotoxicity, which is one of the major causes for safety-related failures of drug candidates. 2D cell culture-based tests for compound screening are standard procedures in drug discovery, but reliable data for in vivo studies is hard to obtain because cells in a monolayer are in unnatural microenvironment. In turn, cells in 3D culture systems have more natural interactions with other cells and extracellular matrix, and their responses to drugs resemble more in vivo responses. In drug discovery and development, automation of the cell culture processes and compound screening saves time and costs, and improves the consistency and sterility of the procedures. As 3D cell culture systems are becoming more compatible with automation, they are also more promising to be used in drug discovery and development. The aim of the study was to develop and optimize automated processes for preparing 3D cell cultures into 96-well plates. HepG2, a human liver cancer cell line, cultures in nanofibrillar cellulose were prepared into well plates manually or by using automated liquid handling system. To our knowledge, this was the first time that automated processes for cell seeding into NFC were used to prepare 3D cell cultures. Cell seeding steps that could be automated were identified and optimized based on visual analysis of the wells and viability of the cells after seeding. After optimization, manual and automated processes were compared by studying cell viability, morphology and functionality. Alamar blue assay, Live/Dead assay and fluorescence-activated cell sorting were used to study cell viability, and F-actin staining, differential interference contrast microscopy and light microscopy were used to investigate cell morphology. Cell functionality was analyzed by studying albumin secretion. Cells seeded by using automation secreted normal amounts of liver-specific albumin. Cells maintained viability, morphology and functionality for four days after seeding although the results of viability varied. Alamar blue assays showed decreased development of viability although viability of manually seeded cells increased, but in other experiments the results from cultures seeded manually or by using automation were more similar. For example, lower viscosity of nanofibrillar cellulose and longer waiting time of cells at room temperature before automated processes are possible explanations for differences, as well as the natural variability in cell studies. In the future, automated high-throughput screening of compounds could be performed in 3D cell cultures prepared by using automation. That would save time and costs, and increase the correlation between in vitro and in vivo studies.
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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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(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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(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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(2019)Background: The World Health Organization (WHO) outlined in their report published in 2014 that antimicrobial resistance (AMR) is a real public health threat worldwide and the actions against it should be taken. Otherwise, the post-antibiotic era where common community-acquired infections can lead to death, could hypothetically become true. The discovery and development of novel antibiotics (ATBs) against Gram-negative bacteria (GNB)-related infections is difficult due to a dual defence mechanism: the extra protection barrier called the outer membrane and efflux pumps which GNB utilize to protect themselves against external noxious compounds. Efflux pumps are expressed at the basal level in GNB, such as E. coli, but when exposed to sub-inhibitory concentrations of ATBs and the intrinsic extruding capacity is exceeded, GNB start overexpressing these “so-called” multi-drug resistance (MDR) efflux pumps. The most abundant and studied MDR efflux pump in E. coli is a tripartite protein complex AcrAB-TolC which traverse through the bacterial cell envelope and is capable of extruding a broad range of structurally unrelated compounds, thus leading to cross-resistance against several classes of ATBs. It has been suggested that antibacterial activity of existing ATBs could be restored again by inhibiting increased efflux activity through efflux pump inhibitors (EPIs). Objectives: Define the optimal assay conditions and a positive control (EPI) to be used in high throughput screening (HTS) of novel EPIs. The assay consists of one E. coli strain of clinical relevance with high intrinsic efflux activity, one ATB and one EPI, both of them at specific concentrations defined during this study. Methods: The intrinsic efflux activities of seven E. coli strains were studied by Hoechst 33342 (H33342) accumulation assay, both in the absence and presence of five commercially available EPIs. The same assay was used in the dose-response studies in which an optimal concentration of EPIs was identified for further to be utilized in the checkerboard assays. The minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to Clinical and Laboratory Standards Institute. The synergistic effects of ATB and EPI in terms of decreasing the intrinsic MIC value of the ATB were determined in the checkerboard assays partially performed by the Biomek i7 Automated Workstation. The data was analysed by using Microsoft Excel and IBM SPSS Statistics, version 25. Results and discussion: E. coli ATCC 25922 had statistically significantly the highest efflux activity of all wild-type pathogenic and non-pathogenic E. coli strains. However, when H33342 accumulation assay was carried out in conjunction with EPIs, E. coli BAA1161 (uropathogenic strain) had the highest median increase in the intracellular level of H33342. Mefloquine showed to be the most potent of all EPIs at the tested concentrations. However, mefloquine increased the intracellular H33342 accumulation even in efflux-deficient E. coli JW5503 (ΔtolC), thus possible additional modes of action or inhibitory activity towards other efflux pumps might exist. Dose-response studies carried out in ΔtolC E. coli JW5503 suggested that CCCP at 1.25 g/ml and mefloquine at 0.5 g/ml were the optimal concentrations. However, for mefloquine, when tested at 0.5 g/ml, the intracellular level of H33342 was not increased in six remaining E. coli strains. Therefore higher concentrations up to ½ MIC were tested in the checkerboard assays. In the antibacterial susceptibility testing, E. coli BAA1161 was the only strain showing resistance to tetracycline and piperacillin, resulting in MIC ratios (MIC wild-type/MIC mutant) of 512 to 2048. Piperacillin and ofloxacin, which showed a MIC ratio of 4 in two E. coli strains, were chosen to the checkerboard assays in which mefloquine reduced the intrinsic MIC of piperacillin by 16-fold and CCCP by 32-fold in E. coli BAA1161. Conclusions: E. coli BAA1161 was chosen to be used as a model strain in HTS due to the highest median increase in intracellular H33342 accumulation and also for being the only strain with resistance towards the ATBs tested. Mefloquine (16 g/ml) was the EPI of choice for the positive control in HTS because the synergistic effects observed between piperacillin and mefloquine were most probably explained by efflux pump inhibition and not by antibacterial activity of mefloquine itself. Piperacillin (256 g/ml) was selected to be used as an ATB in HTS because it was the only ATB which was potentiated by the tested EPIs.
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(2018)This is a systematic review aiming to investigate the efficacy, effectiveness, and safety of biosimilars in the treatment of inflammatory bowel diseases. Biosimilar drugs used to treat inflammatory bowel diseases include biosimilar infliximab and biosimilar adalimumab. Biosimilar infliximab has been authorized by the European Medicines Agency (EMA) in 2013 and by the US Food and Drug Administration (FDA) in 2016. Biosimilar adalimumab has been authorized by EMA and FDA in 2017 and, at the time the literary search for this systematic review was conducted no studies were found regarding the treatment of adalimumab biosimilar for inflammatory bowel diseases. To acquire marketing authorization for biosimilars, it must be proven that the biosimilar is biologically similar to the original medicinal product. Bioequivalence is demonstrated through physicochemical trials and clinical trials. However, clinical trials do not have to be performed with all of the indications for which the original medical product is registered. After proving bioequivalence with one or more indication it is possible to extrapolate the biosimilar to be used in all of the original medical products indications. This has raised the question of whether biosimilars are really comparable to the originator in indications for which no clinical trials have been conducted. This systematic review was implemented using the Cochrane Handbook for Systematic Reviews and Interventions. Systematic literature searches were made in Cochrane, Medline (Ovid®), PubMed and Scopus databases on 12.05.2017. 14 observational studies, one systematic review and a randomized clinical trial that met the inclusion criteria were included in the systematic review. The quality of the publications was evaluated using the STROBE-, NOS- and CONSORT-checklists and information regarding the efficacy, effectiveness and safety of biosimilars was extracted. CD-patients receiving tumor necrosis factor alpha inhibitors for the first time, the clinical response was achieved in 50.0 % to 97.2 % of patients depending on patient population and the duration of treatment. Similarly, for UC-patients, the clinical response was achieved in 62.2 % to 100.0 %. The clinical remission was achieved among 28.9 % to 84.4 % of CD-patients and among 28.9 % to 84.4 % of UC-patients, depending on patient population and treatment follow-up. After the switch from original infliximab to biosimilar, the proportion of patients in clinical remission during follow-up ranged from 62.3 % to 100.0 % in CD-patients and from 45.5 % to 100.0 % in UC-patients. Clinical remission was sustained throughout the whole follow-up in 70 % to 100 % of CD-patients and 66.7 % to 92.0 % of UC-patients. The incidence of adverse events leading to the discontinuation of drug treatment was between 0.0 % and 25.0 %, and the incidence of all adverse events ranged from 0.0 % to 93.6 % in CD- and UC-patients. Biosimilar infliximab seems to be comparable to the original product regarding the efficacy, effectiveness and safety. This result is supported by the systematic literature review published earlier. Conducting a meta-analysis of the information contained in this systematic literature review could have led to a more final decision considering efficacy, effectiveness and safety of biosimilar-infliximab in the treatment of inflammatory bowel diseases.
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(2019)Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease in which both upper and lower motor neurons degenerate gradually. The disease leads to a total paralysis of almost all skeletal muscles and to death within 3-5 years after onset. At the moment there are two disease modifying medicines available, riluzole and edaravone. Neither is able to cure the disease or even to stop or remarkably slow down its progression. Endoplasmic reticulum (ER) stress has been proposed as one of the pathophysiological mechanisms underlying ALS. During ER stress misfolded of unfolded proteins accumulate in ER lumen. As a defense mechanism, the cell launches unfolded protein response (UPR). UPR response aims to reduce the protein load in ER and restore cell’s normal functions. If the damage is already beyond repair, UPR signal cascades lead to programmed cell death. Neurotrophic factors (NTFs) regulate the growth of nervous tissue and participate in repairing processed. Many of the known NTFs have first seemed promising in the preclinical models of ALS but however failed in clinical trials. Cerebral dopamine neurotrophic factor (CDNF) differs drastically both in structure and function from conventional NTFs. CDNF has seen to relieve ER stress and improve motor behavior in the animal models of Parkinsons’s disease. Recently CDNF entered clinical trials in Parkinson’s patients. Since ER stress is believed to be present not only in ALS but also in Parkinson’s disease and other neurodegenerative diseases, it might have an effect in treating ALS patients. SOD1-G93A is a well-established animal model of ALS in which the animals show typical motor impairments comparable to human disease. In this study we used a novel mouse line obtained from crossing traditional SOD1-G93A model and CDNF knock out models. The study aimed to evaluate the effect of endogenic CDNF loss in survival, onset of symptoms, motor behavioral and spinal motor neuron degeneration in the new line. ER-stress and autophagy marker levels were studied with quantitative polymerase chain reaction (CNDF) and western blotting techniques. Spinal motor neuron loss was examined by anti-choline acetyltransferase antibody (ChAT) stainings. SOD1-G93A CDNF knock out animals were observed to have more severe motor impairments in the early stages of the disease compared to the traditional SOD1-G93A mice. In addition, the degeneration of spinal motor neurons appeared to be more severe in the new line. There were no statistically significant differences in ER stress between the genotypes although a trend of increased ER stress was observed. Endogenous CDNF loss had no effect on the healthy animals. The results suggest that CNDF is a potential treatment for ALS and it might have only little side effect since it does not seen to affect healthy tissue. In medical usage, CDNF might be most effective when administered immediately after disease onset. However, this might be difficult because of the challenges in ALS diagnosis.
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(2018)Drug shortages have become a global issue and reasons for drug shortages are several and multifactorial. Definition of drug shortages is not unambiguous. However, in literature are numerous different suggestions to determine the phenomenon of drug shortages. This study provides more focused information on drug shortages and the reasons behind them. The study was performed in cooperation with Orion Corporation. The aim of this study was to explore the in-depth reasons behind medicine shortages from the perspective of one European pharmaceutical company with special focus on Finland, Germany, the United Kingdom and Sweden. Interviews of the company employees were used to achieve this aim and build a few case studies. Further the aim was to investigate in-depth reasons for drug shortages using data from case studies. Case studies were provided by Orion since this enabled use of unpublished information compare the case studies with relevant legal and regulatory measures in the European pharmaceutical framework which influence drug shortages. Reviewing available data from literature and from EUDRA GMDP database for drug shortages and investigate if the data is detailed enough to understand in-depth reasons for drug shortages. Based on the interview results the most common reasons behind drug shortages in Europe are mainly pharmaceutical market structure 38%. It contains many different factors, such as small stock size, local and foreign manufacturing issues, logistics and distribution issues, changes in demand and regulatory issues. However, the manufacturing (33%) or regulatory (29%) reasons are almost as numerous as pharmaceutical market structure issues. Pharmaceutical market structure issues include most common reasons which are categorized in supply-related and demand-related reasons. According to this study supply-related reasons are more common (73%) than demand-related reasons (27%). Some reasons behind drug shortages overlap and often cause a domino effect, whilst other are unique or stand alone, like reasons resulting from natural disasters. The results of this study seem generalizable because the EUDRA GDMP database shows same results and case studies illustrative same reasons behind drug shortages. This study provides more focused information on drug shortages and the reasons behind them from the perspective of pharmaceutical company and authorities.
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(2019)Despite the long history of skin grafting, there is no standardized treatment for split-thickness skin graft donor sites. These sites cause a notable amount of pain and discomfort to the patients and open wounds also introduce a risk for infection. There is an extensive need for treatment options promoting the fastest and least painful healing possible while also being infection-free. The treatment of split-thickness skin graft donor sites is constantly studied and there is plenty of scientific literature available about this topic. In the theory section of this Master’s thesis, the structure of skin, the process of wound healing, skin grafting surgery and wound care products for split-thickness skin graft donor sites are briefly introduced. Additionally, the method of systematic review is described. In the empirical section, a systematic review is performed to compare animal- and non-animal-based wound care products in the treatment of split skin graft donor sites. The methodological quality of the included studies is reviewed. In the literature search, 3552 references were found. In this systematic review a total of 23 articles were included comprising of 21 comparative clinical studies and two previous literature reviews. Of the original studies, 20 reviewed healing, 14 infection and 17 pain of the split-thickness skin graft donor sites. Based on the results of the systematic review, animal-based wound care products might promote healing and reduce pain experienced by patients in the treatment of split-thickness skin graft donor sites when compared with non-animal-based wound care products. The results concerning infection were inconsistent. Generally, the reporting of the clinical original studies was not comprehensive enough for proper evaluation of methodological quality. Some defects, mostly in the blinding of the patients, study personnel and the assessors of outcomes, were also found. Moreover, the studies were heterogeneous in their definitions and measuring of the reported outcomes. Therefore, there is substantial uncertainty in the results of this systematic review. The systematic and transparent way of conducting the literature search, the review of the methodological quality and the reporting of the outcomes can be considered as a strength of this thesis. The main weakness is, that only one person performed the critical steps of this study, which might increase the risk of bias and reduce the repeatability of the study.
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(2019)Literature review part: The enteric nervous system (ENS) often called “the second brain” is considered its own autonomic division that can independently regulate gut function. The ENS is derived from enteric neural crest-derived cells (ENCCs), which colonize the gut during development. Development of the ENS is a complex process, and many signalling pathways are required for a properly functioning ENS, especially GDNF/Gfrα1/RET signalling controlling survival, proliferation, migration, and differentiation of ENCCs. Hirschsprung’s disease (HSCR) is the most common congenital disease affecting gut motility. The prevalence of HSCR is 1:5000, and it is characterized by a complete lack of enteric neurons (aganglionosis) in the distal colon. Due to impaired intestinal motility, infants may have constipation, emesis, abdominal pain or distention, and, in some cases, diarrhea. The most life-threatening symptom is HSCR-associated enterocolitis (HAEC), which occurs in 30-50% of patients. Routine treatment for HSCR is a surgical operation called “pull through” in which the aganglionic segment is removed, and the remaining ganglionic segment is joined to the anus. However, the risk of developing HAEC after successful surgery still exists. Histopathological analysis has revealed that HAEC is accompanied by various changes in the gut epithelium, especially in mucin-producing goblet cells. These changes include hyperplasia of the goblet cells, altered mucin profile, retention of mucin, damaged and disorganized epithelium structure, inflammation, and bacterial adherence to the epithelium. However, a lack of suitable postnatal HSCR mouse models has partially hindered the progress of pinpointing the exact order of these events. A RET mutation found in half of the patients is overwhelmingly the biggest risk factor for HSCR. RET is a receptor on the cell membrane that mediates the effects in GDNF/Gfrα1/RET signaling pathway. of Knock-out mice of Gdnf, Gfra1 and Ret all have intestinal aganglionosis, resembling HSCR. However, to date, no mouse models of HSCR affecting GDNF/Gfrα1/RET signalling exist because pups are born without kidneys and die soon after birth. Experimental part: The GFRa1 hypomorphic mouse line (Gfra1hypo/hypo) created by Dr. Jaan-Olle Andressoo is the first successful model that survives past birth while manipulating GDNF-Gfrα1-RET signalling and phenocopying HSCR. These mice have 70-80% reduction in the expression of Gfrα1 in the developing gut and kidneys, which is sufficient to cause aganglionosis in the distal colon, yet not enough to impair kidney development.These mice are sacrificed between P7-P25 because of welfare problems yet giving a time window for analysis of the development of HAEC. Histological analyses revealed that Gfra1hypo/hypo mice had goblet cell hyperplasia and a shift away from acidic mucin production in the distal colon. Goblet cell hyperplasia was first observed at P10, but the shift in mucin profile already appeared at P5. It is not known what causes goblet cells to change their mucin production, but it seems to be the earliest histopathological change in HAEC preceding goblet cell hyperplasia. qPCR-analysis revealed that Muc2, the main secreted mucin that protects epithelium from invading pathogens, was upregulated at both P5 and P10. mRNA levels of Tnfa were also upregulated at P10. The aforementioned changes were not observed in the duodenum where the ENS had developed normally despite the reduction in Gfra1 expression. This indicates that the changes observed in the colon are likely due to the lack of ENS innervation, rather than a direct effect from GDNF-GFRa1-RET signalling itself. Finally, serum analysis indicated that systemic inflammation did not occur from P10-P16, although one Gfra1hypo/hypo animal had high levels of IL6 and TNFa at P14-16. This indicates that inflammation is not an early stage event and it is preceded by goblet cells related changes. In conclusion, changes in goblet cells seems to be earliest histopathological findings preceeding HAEC.
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(2018)Appetite regulation is a complex process involving regulation of energy homeostasis and the rewarding nature of food. Abnormalities in appetite regulation lead to obesity and eating disorders which are challenging to treat with medicines. Especially obesity is an increasing public health problem and drug development against it is a current subject in research. Hypothalamus is the most important brain area related to appetite regulation. Also, the basal forebrain and the amygdala which are part of the reward system in the brain, contribute to the appetite regulation. There is cholinergic innervation from the basal forebrain to the amygdala and most of the cholinergic activity in the amygdala is originating from the basal forebrain. It is known that the cholinergic system inhibits appetite but there is still no research about impact of cholinergic projections between these two brain areas. Aim of this study was to find out if the cholinergic projections from the basal forebrain to the amygdala effect on appetite regulation. The study included two stereotactic surgery. In the first surgery the mice (n=14) received injections to the basal forebrain that contained genetic materials for DREADDs in AAV. DREADDs appeared in the cholinergic cells of the basal forebrain and emanated within their axons to the amygdala. In the second surgery cannulas were placed to the amygdala. CNO was injected through the cannulas to the amygdala to cause the DREADDs activate or inhibit the cholinergic cells. As a control, mice received vehicle. The feeding experiments were performed in normal conditions or after food restriction and there were either food or sugar pellets available. The pellet dispenser monitored how many pellets mice ate during the six hours after the CNO or vehicle treatment. The success of virus injections was checked after the feeding experiments by antibody dyeing. In any conditions there was no significant differences in the results due to DREADDs and treatment. Because of the small group sizes and dispersion of the results no final conclusions can be made but the additional research about this topic is needed.
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(2018)Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.
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(2019)If pharmaceutical quality system fails it causes a hazard to the patient’s health, but also to the manufacturer’s economy. For this reason, the manufacturer’s must make sure their products comply with the quality requirements placed by authorities. To ensure the compliance, the authorities perform inspections at the manufacturing sites. If the site does not comply with the quality requirements, the authority will take necessary measures. The goal of this study was to find what type of quality issues FDA and the authorities within EU have observed while inspecting manufacturing facilities, which of these issues are most common, in which countries the sites companies with issues have been located. The results were assessed from European pharmaceutical company’s point of view. The data for the study was collected from Eudra GMDP database and from FDA Warning letters sent by FDA headquarters from years 2015-2017. Qualitative analysis of content was chosen as the method of analysis. The collected data was classified into main classes and subclasses based on reoccurring topics. The classes were transferred in tables to compare how which of the classes were most common. Most often the facilities with quality issues were located in China and India. The authorities also perform a lot of quality inspections in these countries, but that alone doesn’t explain the large number of quality issues in these countries. The number of sites with quality issues per inspection was also high. Both the authorities of EU countries and FDA had mainly observed similar issues. Often quality issues were related to data integrity. Other common themes were quality management system, cleaning of equipment and facilities and analytical methods. There were also some differences in the observed issues. E.g. FDA had rarely observed issues related to personnel while EU authorities had observed such issues frequently. Quality issues which had led to measures by authorities were often related to larger problems with the quality management or to very basic quality actions. If company doesn’t have well-functioning quality organization, the quality system is often inadequate also in other ways. By comparing their own activities with the issues observed at other companies, it is easier for a company to improve their quality and avoid major quality issuer before they occur.
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(2018)The majority of potential new chemical entities reaching drug development phase belong to Class II the Biopharmaceutics Classification System (BCS) which complicates formulation of orally administered drugs. Therefore, there is a need to develop methods to increase the solubility and dissolution rate. Transformation of a crystalline drug into its amorphous form can be used to enhance these properties of poorly water-soluble drugs. However, amorphous drugs are thermodynamically unstable and tend to recrystallize back to the crystalline form. Coamorphous forms are a new and promising method to stabilize amorphous form. A relatively new approach is to combine the active drug compound with an amino acid to form a coamorphous system. In this study, co-amorphous systems were prepared from gamma, alpha or amorphous form of indomethacin (IND) and tryptophan (TRP) by ball milling. The solid-state changes during milling were investigated to obtain information about the co-amorphization process. The main objective was to investigate the effects of initial solid state of indomethacin on the formation pathways. In addition, different analytical methods were compared with respect to observed endpoints of the formation process. Raman spectroscopy has not been used in previous studies regarding solid state changes in co-amorphous forms. The presence of fluorescence in amorphous systems may have limited use of the method. A time-gated Raman setup together with X-ray powder diffraction and differential scanning calorimetry (DSC) was used to investigate this kind of potentially fluorescent system. Principal component analysis of spectral data revealed that the three different binary systems had individual and direct pathways towards the same end points during milling. This indicates that the co-amorphous form formed after 60 minutes of ball milling is not dependent on the initial solid-state form of IND. Straight pathways also indicated direct transformation to the coamorphous form. DSC was found to be the most sensitive method to detect changes for the longest period during co-amorphization. Conventional Raman spectroscopy was found to be suitable for investigation of the co-amorphization process. However, time-gated Raman spectroscopy did not show significant advantages compared to conventional Raman data. This study revealed that the most stable form of IND could be used for production of co-amorphous form together with TRP. Raman spectroscopy could potentially be used for investigating coamorphization also as an in-process analytical method.
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(2019)Background: Continuous manufacturing has been utilized for decades in many industries since it has many advantages compared to batch manufacturing. Therefore the interest towards continuous processes has arisen also in the pharmaceutical industry. Also, the strict regulations characteristic of pharma industry have started to change more favorable towards continuous manufacturing when the possibilities of continuous processes to produce higher quality products faster and more efficiently, have been proven in many researches. Objectives: Objectives of this thesis were to clarify the effect of the material characteristics on material flowability from continuous feeders and to study how different toolings, like feeding screws, affect the feeding of materials with different characteristics. Based on these results, a possibility to model the feeding results of a material based on only some measured material characteristics was also under investigation. The aim was to develop a clear and systematic procedure which would simplify the determination of the most suitable equipment when starting to feed a new material. Methods: The similarity of flowability of various pharmaceutical powders from continuous feeders was studied. First material characteristics affecting material flowability from a feeder based on literature was determined from 26 pharmaceutical powders. Following this, six materials were chosen to be studied with gravimetric powder feeders using different kinds of research frames. The six materials formed three material pairs, in which two materials had clear similarities in the flowability characteristics. The reason for this was that the flowability from feeders with similar materials could be compared. The feeding of materials was determined investigating the feed rate capacity and accuracy of feed rate of material. Also, the effect of feeder screws and the speed of the screws on the feeding capability of a material was investigated. A model to predict the feeding result based on material characteristics was built using PLSand MLR-methods. Results: The prediction of material feeding was not possible with PLS- and MLR-modeling methods. The feeding of similar materials was wound to be alike. Poor flow characteristics correlated with poor feeding results. PCA- and cluster analysis were found suitable to define the similarity of materials. Conclusions: The success of feeding of pharmaceutical powders is clearly affected by the material flowability properties. The feeding screws and screw speed affect the feeding accuracy, too. The prediction of feeding results of specific material, would need much more data to produce valid and trustworthy models. However, it seems highly possible to be able to build a model with more materials.
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(2018)Medication-related events are a significant cause of in-hospital adverse events. Intravenous medication errors occur more frequently and are more likely to result in serious harm than other medication therapies. Closed loop medication management which seamlessly integrates automated and intelligent systems barriers, is used for reducing medication errors. The aim of this systematic review was to identify what kind of scientific studies exist regarding closed loop medication in intravenous medication therapy and barriers related to it. This study is part of a larger systematic review. The literature search indentified 2292 scientific papers. Of these, only 57 were included in the larger review since most of the references were excluded based on titles, abstracts or full-texts. Of these, 21 studies regarding closed loop medication management in intravenous medication therapy were included in this thesis. The studies conserned intelligent infusion devices, computerized physician order entries, clinical decision support systems, automated workflow management systems reducing compounding errors and bar-code confirmation of drugs and patients. According to this review, closed loop medication management potentially reduces medication errors related to intravenous medication therapy. It seems to be more effective to seamlessly integrate the closed loop medication management barriers to each other and to the medication management process than to implement the barriers separately. It’s important to plan the implementation carefully by a multidisciplinary team. In addition, the latest care guidelines need to be taken in to account. Significant resources must be allocated to training and engaging employees and to systematically maintaining and developing the process to manage the successful implementation of the process. This review provides valuable information for Finnish hospitals implementing the closed loop medication management since the concept is not yet well-known in Finland.
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(2019)Flowability of powders is in critical role when manufacturing the most popular dosage forms, tablets and capsules, of pharmaceutical industry. Re-formulation is expensive and time-consuming, so it is important to determine powder flow properties at the initial stage of drug development prior to tabletting and encapsulation processes. There are many different methods, like shear cell, flow through an orifice and bulk and tapped density, to examine powder flowability. Despite the methods, the most reliable means of examining powder flowability is often empirical. In early stages of drug development, it would be good to have faster, more reliable and cheaper methods to examine powder flowability. FT4 Powder Rheometer is a relatively new flowability characterization technique. The aim of this study is to find out whether the library created using the FT4 Powder Rheometer methods makes it possible to characterize the rheological properties of solids in the early stages of drug development. In addition, the aim is to investigate whether FT4 Powder Rheometer methods can predict the success of masses in tableting and encapsulation processes. The information gained from the research can be used in the future, for example, in continuous processes, because flowability plays an important role, especially in the supply of raw materials to the process, which is the most important division of continuous processes. To the library were selected for particle size and shape 15 different types of material. These materials were subjected to five different FT4 Powder Rheometer basic test methods. In addition, the particle size and shape of the materials and the flow through an orifice and the bulk and tapped density were determined to support the results of the powder rheometer. The principal component analysis was used to process the results. As the tablet and capsule masses examined, the masses of a previous study were utilized. Those masses were tableted and encapsulated in that previous study. These tablet and capsule masses contain a variable amount of cohesive drug substance. FT4 Powder Rheometer methods provide more complex information about materials and their behaviour than conventional flowability test methods. From the powder rheometer parameters pressure drop, compressibility and specific energy distinguish the cohesive and the non-cohesive materials, because the cohesive materials with these parameters obtain clearly higher values than non-cohesive materials. Additionally, the cohesion of FT4 Powder Rheometer shear cell test mainly distinguishes highly cohesive materials from other materials. The flow rate index makes it possible to separate the materials to which the change in flow rate particularly affects. Fluidizing materials, due to the air flow, are distinguished by the aeration test. Avicel PH-102 could be used as a rough limit value for well and poorly flowing materials in the created library (excluding the aeration and shear cell test). Stability index -, flow rate index -, specific energy -, pressure drop -, and compressibility-results of the FT4 Powder Rheometer correlated to the proportional proportion of the cohesive drug in the mixture. These parameters could possibly be used to distinguish mixtures containing the cohesive material. Additionally, specific energy, compressibility, pressure drop, basic flowability energy, stability index and flow rate index correlated with the weight variation of the tablets. With these parameters one could possibly assess the tabletability of the mixtures. A much larger library is needed to evaluate and predict the rheological properties of new materials. FT4 Powder Rheometer can possibly be used to predict the tableting success of tablet and capsule masses. This would be interesting to look more extensively, for example as part of a library. Additionally, it would be good to investigate whether the results of powder rheometer correlate to continuous production.
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(2018)Obesity is a health problem linked to Western lifestyle and it is becoming more general. The complexity of regulation of eating makes it difficult to regulate body weight, when multiple neural networks and regions of brain have overlapping functions regarding to energy gain. Sufficient amount of energy is vital for individuals surviving in their living environment. Cholinergic messaging in brain is wide and for example nicotine is known for its appetite reducing effect. Anorexigenic proopiomelanocortin neurons mediate the effect of nicotine. In nucleus accumbens and central nucleus of amygdala extracellular levels of acetylcholine rise during meal, which promotes satiety. Satiety inhibits eating behavior between meals. Amygdala is a part of limbic system and in earlier knowledge it was associated only to regulation of memory, conditioning and fear. Nowadays importance of amygdala in eating behavior research is rising, but most of the studies focus on the effect of cue in regulation of eating. Cholinergic messaging is vigorous in the amygdala and is received from opposite areas of brain between basolateral and central amygdala and therefore this master’s thesis examined the effect of cholinergic messaging in amygdala on regulation of eating behavior. C57BL/6JRcc male mice were stereotaxically implanted with guide cannulas either in the basolateral complex of amygdala (n=10) or central nucleus (n=13). After recovering and habituation to automated pellet dispenser mice were treated with nicotinic and muscarinic receptor agonists and antagonists and eating behavior was recorded for six hours. Nicotine, administered to central and basolateral part of amygdala, lowered the number of pellets mice ate. In central nucleus effect was dose dependent. Mecamylamine had time related effect on eating behavior in basolateral amygdala, but dose dependent response was seen only in cumulative results. Oxotremorine was the only compound which created statistically significant interaction between time and dose. Result was seen in both groups. Scopolamine reduced eating behavior in central nucleus and dose dependency was seen. In basolateral complex scopolamine had time related effect, similar to mecamylamine. The results suggest that amygdala regulates eating behavior even without cue.
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(2019)The amount of informal caregiving has increased in Finland, with a growing emphasis on the older adults. Although the medication management process in informal caregiving has been studied and is known to have significant risks, research data focusing on the older adults is still limited. The aim of this study was to describe the medication management process of informal carers and care recipients of at least 65 years old. The aim was to identify medication errors and medication risks in the medication management process and to find out how the caregivers manage them. In addition, the study examined the informal carers and care recipients own development proposals to improve medication management process. A total of 21 volunteer informal carers and care recipients living in the Helsinki metropolitan area were recruited to this study. The study was conducted as a qualitative interview survey in the homes of the participants. The interviews were a combination of semi-structured interviews and narrative approach. The material to this study was collected during spring and summer 2018. The study was analysed with abductive content analysis combining both deductive and inductive approach. The aim was to find repetitive elements by encoding and grouping expressions. The results of this study were compared with previous theory and the results were supplemented with a picture of the medication management process and a fishbone diagram was drawn from the risk factors and contributing factors of the medication management process. The families had medication errors in all stages of the medication management process. The most frequent medication errors were found in counselling, medication administration and in medication treatment monitoring. As a contributing factor, the healthcare professionals’ haste and the responsibility of the caregiver recurred in the background of the medication errors. Family caregivers and care recipients wish to have more counselling, more monitoring of medication and better interaction with health care. Carers often felt left alone to take care of another person's medications and felt they lack support from healthcare. By facilitating access to a physician, improving the availability of a physician, increasing the amount of counseling and support provided by healthcare, including pharmacies, could the safety of medication management at home be improved.
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(2018)Liposomes are nanosized drug delivery vesicles composed of phospholipid membranes. They present an attractive drug delivery system due to their bioavailability and flexibility. Liposomes can be prepared by different techniques. They can carry both hydrophobic and hydrophilic molecules and their surface can be modified with targeting molecules. Coating the liposome surface with the PEG derivative makes their pharmacokinetics easier to predict. There are several liposome-based medicinal products already on the market. Triggering of drug delivery systems by different external or internal stimuli allows precise control of drug release. Light-triggered drug release is an attractive alternative due to the easy control and regulation of the stimulus. The problem with light-triggered therapy has previously been the need to use high-energy ultraviolet light that penetrates badly to the tissues and is not safe. In TTA-UC process the low-energy red or green light is converted to high-energy blue light. In this process photosensitive molecules are excited by visible light and after that the energy is transferred from sensitizer to annihilator molecules. Collision of two annihilators leads to the excitation of the other molecule while the other returns back to its general energy state. The excitation breaks up with fluorescence. In this process the highly permeable and safe red light is converted to blue light which has enough energy to induce drug release. The aim of this work was to optimize liposomal preparation method and prepare a pegylated and stabile liposome formulation for TTA-UC process. Hydrophobic light sensitive molecules were loaded into the phospholipid membrane as much as possible. One of the problems in this work was to find proper methods to measure the concentrations of these molecules. The lipid composition for formulation was chosen after thermostability studies. As a quality control, the size, capability to load calcein and phase transition temperature of liposomes were measured. The quality control of light sensitive molecules was operated too. In this work, the formulation for TTA-UC was prepared. In further studies TTA-UC process happened with sufficient efficacy. The formulation was pegylated and stable in physiological conditions and the concentrations of the molecules were high enough. This was the very first time to get TTA-UC to happen in this kind of liposome formulation that may be useful as a drug carrier. Long-term stability studies and further optimization of TTA-UC method are needed in the future. Some drug release studies are important to arrange in the future, too.
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