Farmasian tiedekunta


Recent Submissions

  • Huovila, Tiina (2017)
    Autophagy is a pathway for cells to degrade intracellular components that are no longer needed or are detrimental for the cells. It is essential for cell homeostasis and survival and has been related to various diseases and pathophysiology. Autophagy is a complex process and there are still several unclear und unknown aspects to it. Regulation of autophagy is essential to prevent unwanted and escess activation, and several pathways and molecules, both stimulatory and inhibitory, are included. Different signaling pathways are sensitive to a variety of environmental clues. Two main autophagy pathways are mTOR-dependent pathway and mTOR-independent pathway. Induction of autophagy in the latter pathway is dependent on the interaction of Bcl-2 and Beclin 1. Prolyl oligopeptidase (PREP) is a peptidase enzyme that has several substrates. PREP-inhibition by KYP-2047 can reduce aggregation of α-synuclein in two ways: by increasing rate of autophagy and by decreasing dimerization. The aim of this study was to find out how PREP affects the interaction between Bcl-2 and Beclin 1 and how this affects autophagy. Based on previous studies, PREP-inhibition seems to increase the amount of Beclin 1 and to affect the phosphorylation of Bcl-2 and Beclin 1, leading to dissociation of the complex. Hypothesis was to see differences in colocalization of Bcl-2 and Beclin 1 in cells treated with different PREP-modifications and for PREP-inhibition to decrease the colocalization. Human embryonic kidney cells 293 (HEK-293) and hPREP knockout cell line created from them by using CRISPR/Cas9-silencing were used in the experiments. Two experiments were performed on regular HEK-cells: inhibitor experiment with KYP-2047 (1 or 10 µM) and overexpression experiment (transfection with either active or inactive hPREP plasmid). After immunofluorescence staining, cells were analysed with confocal microscope and colocation analysis of Bcl-2 and Beclin 1 was performed. The intensity of Beclin 1 in the nuclei was stronger than in other parts of the cell in all samples, which could indicate a stronger activity of its nuclear tasks compared to autophagy. However, the antibody used for immunofluorescence has most likely caused this staining pattern. Based on previous knowledge, it was expected to see differences in colocalization of Bcl-2 and Beclin 1 in cells treated with different PREP-modifications. However, there were no significant differences in colocalization of Beclin 1 and Bcl-2 in any of the experiments but it was nearly 100 percent in all treatments. Since rate of autophagy in cells was not detected, it is impossible to determine, if there were changes in autophagy that were not reflected as changes in colocalization of these two proteins. It is possible that even a small change in colocalization can affect the rate of autophagy or there might be subpopulations where the interaction is interrupted and these changes are so small that they are not detectable with the methods used in this experiment. Both Bcl-2 and Beclin 1 also have functions not related to autophagy, which could be one reason behind the results gained in this study.
  • Kallio, Rosanna (2017)
    A large part of patient safety incidents in health care is related to medicines and medication treatment. Medication safety is an important part of patient safety. In particular, transitions of care endangers continuity of care and patient safety. A poor flow of information between health care units increases a risk of medication errors. An accurate and up-to-date medication list can improve transfer of correct medical information with the patient. In addition to the medication list other organization-level defences are important to patient and medication safety. This study is part of a larger regional development project concerning reformation of service delivery in health and social care. One of the purposes of the project is to integrate health and social services of municipalities in central Uusimaa (Hyvinkää, Mäntsälä, Pornainen, Järvenpää, Nurmijärvi and Tuusula). The aim of this study was to investigate what kinds of medication lists are available in these municipalities and to design a medication list maintained by the patient which is regionally shared. The second aim of the study was to identify defences used by the municipal healthcare systems and to develop a medication management process model for the municipalities. The material consisted of medication lists from the municipalities participating in the study. Twelve (12) lists were selected for analysis. The medication list of Lää was also included in the analysis (N=13). Data of the defences was collected by a questionnaire. In Hyvinkää the material was collected from primary health care and Hyvinkää hospital. Both the medication list and the defence data were systematically reviewed and collected in a Microsoft Excel table. The regional medication list for patients was developed based on the medication list data and the existing literature. The process model for medical treatment was developed based on the defence data. Almost every medication list contained space for personal data (n=11) and basic medication information, such as the name of the drug (n=12), strength (n=12) and dosage (n=13). Regular medication was usually separated from as-needed medication (n=8). Only two of the lists had a column for periodic medication. There were only two lists with columns for over-the-counter medicines and herbal products. Less than half of the lists (n=5) had space for indication. According to the survey, most of the defences listed in the questionnaire were used in all or almost all municipalities. According to the responses, medication lists, for example, are regularly reviewed. Some gaps in defences also emerged. Only in two municipalities high-alert medications were identified. Also only in two municipalities attention was paid to the storage of look-alike medicines. Written instructions for managing medication errors was available only in two municipalities. There are many different medication lists available in health care. A regionally shared medication list maintained by the patient can improve the transfer of the up-to-date medical information with the patient. The list must be comprehensive and easy to use. Both the patient and the healthcare professionals should be responsible for maintaining the accurate medication list. Most of the defences asked in the questionnaire have been taken into account in pharmacotherapy plans. In practice, however, the implementation of defences is unclear. For example patients' medication lists often contain errors even though lists should be reviewed regularly. There should be exact instructions as well as clearly defined roles and responsibilities for medication reconciliation. More attention should also be paid to the use of high-alert and look-alike medicines. Since errors occur despite the defenses, every health care unit should have written instructions in case of medication errors.
  • Somersalo, Petter (2017)
    Cells release different types of phospholipid bilayer-limited vesicles into the extracellular space. These are commonly referred to as extracellular vesicles (EVs). Exosomes (EXOs), ca 50-100 nm in diameter and microvesicles (MVs), ca 100-1000 nm in diameter, having different intracellular origin, are the two main subpopulations of EVs. EVs have been demonstrated to carry a range of proteins and nucleic acids subsequently delivered to recipient cells, making them attractive as drug delivery vehicles. Several mechanisms for the cellular uptake of EVs have been established. When a nanoparticle is introduced into blood plasma, plasma proteins are adsorbed to its surface, forming a protein corona. The formation of the corona is a dynamic process, governed by individual protein concentrations as well as their respective affinities for the surface. Proteins of the corona interact with surrounding cells, thus being able to influence the cellular uptake of the nanoparticle. In the current study, the uptake of PC-3-derived EVs into PC-3 cells was investigated. Moreover, the impact of a human blood plasma-derived protein corona on said uptake was assessed. EVs were isolated from collected PC-3 cell culture medium using differential centrifugation. Experiments were performed separately for MVs (20000xg EV-fraction) and EXOs (110000xg EVfraction). SDS-PAGE analysis revealed adsorption of plasma proteins to EVs, following their exposure to plasma. Prior to uptake experiments DiO-labelled EVs were either incubated or not incubated in plasma. Plasma incubation lasted overnight. PC-3 cells were then treated with either of the two EV-preparations. Following incubation, EV uptake was assessed using confocal microscopy by determining the percentage of positive fluorescent cells in cell cultures. Pre-study plasma incubation resulted in a reduced or unchanged uptake of MVs and in a reduced uptake of EXOs, when compared to their native counterparts. In conclusion, the plasma-derived protein corona was shown not to improve EV uptake. It is worth noting that the current study limits itself to the use of PC-3-derived EVs and PC-3 cells as recipient cells in uptake experiments.
  • Pihlaja, Tea (2017)
    Cytochrome P450 (CYP) enzymes are important catalysers in the first phase of drug metabolism. Roughly two thirds of drugs are oxidized via CYP enzymes, which enable the further modification of drugs, and their excretion. In this thesis, human liver microsomes containing the main hepatic CYP enzymes were immobilized on thiol-ene based micropillar arrays and their stability was evaluated using a CYP2C9 isoenzyme specific luminescent substrate, Luciferin-H. The aim of the study was to develop microfluidic immobilized enzyme reactors (IMERs) for studying enzyme kinetics and drug-drug interactions. For this purpose, the instability issues associated with previously reported CYP-IMERs were carefully addressed. The CYP immobilization protocol used was based on a protocol previously developed in the context of other research projects and relied on biotinylation of human liver microsomes (HLM) with help of fusogenic liposomes. The biotinylated HLMs were then attached to the streptavidin-modified thiol-ene surfaces. The CYP activity was determined by utilizing microfluidics under continuous flow conditions (typically 5 μL/min) in the presence of NADPH. The luminescent metabolite formed by the CYP2C9 enzymes was quantified with a commercial well-plate reader from fractions collected at the microreactor outlet. Half-life was used to compare the differences between enzyme stabilities reached via different immobilization conditions. The effects of flow rate and reaction temperature on the stability of the CYP-IMERs was evaluated together with addition of antioxidative agents and reactive oxygen species (ROS) scavengers. Different functionalization steps as well as storage time and conditions were studied. With Luciferin-H as the model substrate of CYP2C9, the CYP-IMERs showed higher activity and stability at room temperature than at +37 °C. The peak activity could be increased via optimization of the immobilization protocol, though long-term storage diminished the peak activity. The activity of the IMERs typically attenuated within 1-2 hours with little or no improvement achieved via optimization of the immobilization or operation conditions. Only upon addition of the ROS scavengers, the peak activity and stability of the CYP-IMERs could be slightly improved. After functionalization, the IMERs maintained their activity until the time of use when stored in +4 °C for up to 2 weeks, but re-use of IMERs was not possible.
  • Viljemaa, Kati (2017)
    The economic burden of adverse events (AEs) is substantial and in direct relation to current increasing drug utilisation. According to previous research, the annual cost of AEs in the U.S. may be as high as 22.9 billion euros. In Europe AEs are considered to contribute to 3.6 percent of hospital admissions, have an impact on 10 percent of inpatients during their hospital admission and are responsible for less than 0.5 percent of inpatient deaths. AEs thus clearly constitute a major clinical issue. Fluoroquinolones have been in clinical use since the 1980s and are globally among the most consumed antimicrobials. Fluoroquinolones are generally well tolerated antimicrobials. The most common AEs are mild and reversible, such as diarrhea, nausea and headaches. Nevertheless, fluoroquinolones are also associated with more serious AEs, including Clostridium difficile associated diarrhea (CDAD), rate-corrected electrocardiographic prolonged QT interval, tendinitis and tendon rupture, dysglycemia, hepatic toxicity, phototoxicity, acute renal failure and serious AEs involving the central nervous system, such as seizures. Health service use and costs specifically associated with fluoroquinolone-related AEs have not been evaluated previously. The theory section of this Marter's thesis considers adverse events and fluoroquinolones. The main principles of conducting a systematic review are also discussed. The empirical section is a systematic review. The aim of this study is to identify health care use and costs associated with ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin and ofloxacin -related AEs. A literature search covering Medline, SCOPUS, Cinahl, Web of Science and Cochrane Library was performed in April 2017. Two independent reviewers systematically extracted the data and assessed the quality of the included studies. All costs were converted to 2016 euro in order to improve comparability. Of the 5,687 references found in the literature search, 19 observational studies, of which 5 were case-controlled, fulfilled the inclusion criteria. Hospitalization was an AE-related health care use outcome in 17 studies. Length of stay associated with AEs varied between <5 - 45 days. The estimated cost of an AE episode ranged between 140 and 18,252 €. CDAD was associated with the longest stays in hospital. However, a mere 10 studies reported AE-related length of stays and only 5 evaluated costs associated with AEs. Although rare, in particular serious fluoroquinolone-related AEs can have substantial economic implications, in addition to imposing potentially devastating health complications for patients. Further measures are required to prevent and reduce health service use and costs associated with fluoroquinolone-related AEs. Equally, better-quality reporting and additional published data on health service use and costs associated with AEs are essential. The strengths of this study are a comprehensive and systematic literature search and transparency of methodologies and reporting. The main weakness is the generalizability of the results.
  • Tilli, Irene (2017)
    Melanoma is the most severe case of skin cancer and there is no curative treatment if it has progressed. Despite the recent advances in drug therapy tens of thousands of patients die of melanoma annually. There is still need for new antimelanoma drugs for which marine compounds are a potential source. Halogens are common elements in drug molecules as they enhance their molecular properties. So far most of the halogenated drugs contain fluorine and/or chlorine but the role of bromine and iodine is probably growing in the future due to halogen bonding. Bromotyrosines are originally isolated from Verongiida-order sponges but whether they are truly of bacterial origin is under controversy. All bromotyrosine compounds consist of brominated tyrosine and/or tyramine residues or their derivatives. Purpurealidin I is one of the newest bromotyrosine derivatives extracted from Pseudoceratina purpurea and it has shown activity against melanoma. In this study eight new purpurealidin I derivatives were synthesized following a successful route previously designed. All synthesized derivatives contained the original N-oxime structure which's stereochemistry was determined to be E by X-ray crystallography. Cytotoxicity against A375 melanoma cells was determined for seven compounds synthesized here and for 15 compounds synthesized previously. All seven compounds and one previously synthesized purpurealidin I analog were active with CC50 values between 4,7 and 22,1 µM. The previously synthesized bromotyrosine derivative intermediates and aerophobin-1 analogs were not active. The selectivity of the active compounds was calculated by determining their CC50 value against Hs27 fibroblast cells. None of the compounds showed remarkable selectivity the most selective 2-pyridin containing derivative having four times better selectivity against melanoma. The tyrosine part and N-oxime seem to be important parts to preserve while the tyramine part can be modified more freely and maintain the activity. Still more derivatives need to be synthesized and tested to confirm these observations. More data is also needed considering the selectivity of the compounds.
  • Jakola, Janne (2017)
    The incidence of wet age-related macular degeneration (AMD) is increasing with ageing. AMD leads to blindness if it is not treated properly. Common treatment is to administrate intravitreal growth factor inhibitors. An ageing population increases the number of patients which overloads the public health services and expands costs. Traditionally, injections have been administered by physicians but because of the limited recourses nurses have been trained to administer injections. In addition, injections can be administered as physician's clinical extra work to alleviate the queue and as an outsourcing service from the private sector. As the resources of the public health service are limited, it is important to evaluate used methods reliably. The target of this research was to investigate the administration costs of intravitreal injections which are administered by physicians, nurses, a physician or a nurse working extra to alleviate the queue or by a private sector. The used method was cost analysis because the effectiveness of the care is the same regardless of the administer of the injection. The source of costs was Ecomed database of HUS, the data of cost accounting and catalogue of billing from the outsourcing service. The costs were examined in perspective of the producer of the service. Based on the cost analysis, the administration costs per injection are following: administered by physicians 51,39 €, nurse-administered 51,19 €, administered by a physician to alleviate the queue 100,46 €, nurse-administered to alleviate the queue 72,87 €, administered by a physician in outsourcing service 276,19 and nurse-administered in outsourcing service 269,85 €. The annual total costs of the producer of the service were 4 563 726 €. By increasing the number of injections administered by a nurse of HUS the need for an outsourcing service can be decreased which may decrease the annual total costs by two million euro. It is important to find cost-effective solutions because the number of patients are increasing. Based on this research it is more profitable to increase the number of injections administered by a nurse of HUS than to train more nurses to administrate injections to alleviate the queue or to work in outsourcing services. The result of this research can be adapted in planning of the public health services.
  • Eteläinen, Tony (2017)
    lcohol addiction is a significant public health problem worldwide, and its treatment is extremely challenging. One major problem in the treatment of alcohol addiction are the later relapses to uncontrollable drinking. Approximately 60-70 % of addicts relapse to drinking within a year from the beginning of the treatment. The current treatment of alcohol addiction is based on a combined psychotheraphy and pharmacological treatments, but even at the best the efficacy remains quite modest. This is why further studies on the underlying mechanisms behind alcohol addiction and development of more effective pharmaceuticals to treat it are an important field of research. Chronic exposure to the rewarding effects of alcohol causes neurochemical adaptations in the brain reward system. These adaptations strive to restrain the recurring rewarding signals caused by alcohol and lead eventually to increased reward thresholds in the reward system. As the reward thresholds increase, the individual develops tolerance to the rewarding effects of alcohol, but also craving for the substance and a dysphoric mental state which are highlighted especially during periods of abstinence. It is known that the increase in reward thresholds is an important factor leading to relapses, but the exact nature of the neurochemical adaptations behind it are not known. According to recent studies dynorphin -peptides (DYN) and κ-opioid receptors (KOR) of the endogenous opioid system seem to have an important role in these neurochemical adaptations. It has been shown that chronic alcohol exposure increases the activity of DYN/ KOR -system especially within the nucleus accumbens (NAc), which is an essential structure of the brain reward system. The increased activity of the DYN/ KOR -system in the NAc has been shown to inhibit the development of rewarding signals. Previous studies have shown that inhibiting the increased activity of the DYN/ KOR -system with a selective KOR-antagonist, reduces voluntary alcohol intake and relapse-like alcohol seeking behavior during periods of abstinence, especially in physically addicted animals. In this study we studied the relapse-like alcohol drinking of Long-Evans rats in the alcohol deprivation effect (ADE) model. The effects of selective and long-acting KOR-antagonists, JDTic and nor-BNI, were tested on the ADE-effect which occurs after a period of deprivation. The ADE is defined as a transient increase in alcohol intake after a forced period of abstinence and it has been shown both in rodents with a history of alcohol consumption, and human alcohol addicts. In this study the rats were allowed to consume alcohol (10% ethanol-water solution) voluntarily during 90 minutes for 10 consecutive days after which followed a six days long deprivation period. According to results, both intra-accumbally (15 µg/ 0,3 µl/min/ site) or subcutaneously (10 mg/kg) administered JDTic decreased the ADEeffect significantly compared to vehicle, when administered 24 hours prior the end of the deprivation period. Also intra-accumbally administered nor-BNI (3 µg/0,3 µl/min/site) decreased the ADE-effect significantly compared to vehicle when administered 24 hour prior the end of deprivation. The results are in line with the theory that alcohol induces sensitization of the DYN/ KOR -system within the brain structures involved in reward. In theory it can be speculated that by suppressing the activity of the DYN/ KOR -system, KOR-antagonists can relieve craving for alcohol. This can be seen as a decrease in relapse-like consumption of alcohol. In conclusion, it can be suggested that by suppressing the increased activity of the DYN/ KOR -system induced by chronic alcohol exposure with a selective KOR-antagonist, like JDTic or nor-BNI, it could be possible to reduce the risk of relapse during abstinence and thus improve the efficacy of treatments for alcohol addiction.
  • Nikko, Elina (2017)
    There is a great demand of cultured human hepatocytes for hepatotoxicity studies, drug testing, disease modelling and liver transplantation purposes. The current gold standard, primary human hepatocytes (PHHs), suffer from poor availability and high variability. Furthermore, PHHs are short-lived in in vitro cultures. Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have emerged as potential substitutes for PHHs in in vitro studies. PSCs are widely available, and additionally allow studies of hepatogenesis and open possibilities for personalized medicine. However, obtaining HLCs with mature hepatocyte functions in vitro has turned out to be challenging, and the differentiated cells have remained immature compared to PHHs. In vivo, hepatic differentiation and maturation of PSCs is guided by cues from the environment. Mimicking the 3D cellular environment in vitro has already shown encouraging results, but today, HLCs are still awaiting to fulfil their promise as a new gold standard. The aim of this study was first to select a new working human PSC line for in vitro hepatic differentiation and maturation. Hepatic differentiation and maturation of the selected cell line, embryonic stem cell line ESI-017, was next studied in five different 3D culture conditions (spheroids in suspension culture, four different hydrogels: Matrigel, collagen type I, mixture of Matrigel and collagen type I and alginate) with the aim to find the most favourable culture condition for later studies. For this, the PSCs were first differentiated to definitive endoderm cells and then to hepatoblasts in 2D cultures, on Matrigel- and laminin-521-coated plates, respectively. The PSC-derived hepatoblasts were then transferred for 16 days to the different 3D culture conditions for hepatic maturation. Of the conditions, suspension culture and mixture of Matrigel and collagen type I -hydrogel were estimated most promising and were selected for further studies. Hepatic maturation of the PSC-derived HLCs was estimated by analysing protein and mRNA expression levels of key marker genes, such as CYP3A4, AAT, MRP2, HNF4A, ALB, AFP, CK-8/18 and CK-19 by immunofluorescence staining and qPCR, respectively, and by cell morphology. Based on cell morphology and noticeable level of CYP3A4 expression, suspension culture shows most potential of the studied conditions in hepatic maturation of PSC-derived hepatoblasts. However, given that expression level of many other hepatocyte marker genes in these HLCs remained low compared to PHHs or human fetal liver samples, it is evident that adjustments to protocol and culturing conditions are still needed.
  • Kaugonen, Olga (2017)
    Investigating the role of cell membrane proteins has increased over the last decade, as drugdrug interactions and genetic polymorphisms have been found to cause changes in drug pharmacokinetics and dynamics. In this study the characteristics of the OATP1B1 transporter were reviewed and new in vitro research method to study protein functions was developed. Human Embryonic Kidney cells (HEK) is a human derived mammalian cell-line that is widely used in the study of OATP1B1 transporter. The Sf9 cell line is isolated from Spodoptera frugiperda insect and is one of the standard in vitro tools in a genetic engineering study. In the experimental part of this thesis the goal was to express OATP1B1 transporter in Sf9 and HEK293 cell lines. The wild-type SLCO1B1-gene encoding the OATP1B1 was virulent with baculovirus into the cells by the Bac-to-Bac® Baculovirus Expression System. For expression in the Sf9 cells, the aim of the study was to clone the SLCO1B1-gene into the pFastBac vector. The cloning was not successful in this study although attempts were made for several approaches. The expression of OATP1B1 transporter in HEK293 cells was successful. HEK293 cells expressing OATP1B1 transporter are well suited for the study of the SLCO1B1-gene. The in vitro method developed in this study remains in the research team as a tool to investigate the polymorphisms of the SLCO1B1-gene, the inhibition of the transporter and possible drug interactions.
  • Tepponen, Tuomas (2017)
    Multidrug resistance protein 1 (MDR1, p-glycoprotein) belongs to the ATP-binding cassette transporter family and it's encoded by ABCB1/MDR1 gene. It is a protein which transports many different kinds of compounds out of cells, for example from endocytes to the lumen with the use of energy from ATP. MDR1 is there for a restrictive factor for several orally administered drugs. It`s important to have knowledge about MDR1-inhibitors, in order to avoid harmful drug-drug and food-drug interactions that might affect medical treatment. The purpose of this master's thesis was to optimize an in vitro MDR1-vesicle uptake method and use it to screen inhibitors from compound libraries. To optimize the method, the effect of cholesterol loading on ATP-dependent transport of test substrate N-methylquinidine (NMQ) was evaluated, transport kinetics of the vesicles and kinetics of known inhibitors were also tested. With the optimized method, screening was done with a library of 25 food additives and a library of 42 synthetic compounds. The chemical structures of the synthetic compounds were analyzed manually in order to find factors that could explain their ability to inhibit MDR1. Only one inhibitor was found among food additives: curcumin. Other additives didn't increase or decrease the ATP-dependent transport of NMQ. Several inhibitors were found from the library of synthetic compounds, also a couple of compounds were found to increase the active transport of NMQ. Results indicate, that the additives used in this study have low risk to cause MDR1 mediated interactions, if curcumin is excluded. The inhibitory effect of curcumin should be investigated in in vivo-situation, because vesicle-based in vitro-results have tendency to overestimate results. Screening results of the synthetic compounds gives more confirmation to the usefulness of the screening method. The MDR1-inhibition screening method described in this Master`s thesis is valid, and it can be used to screen different compound libraries for MDR1-inhibitors. In the future it could be used to screen different kinds of compounds, which might end up inside humans and cause interactions with drugs.
  • Helminen, Heidi (2017)
    Obesity is considered one of the major public health challenges. One way to control obesity is to regulate appetite. Because brain is the primary regulative unit responsible for food intake, the research in this field has now been allocated especially to the central nervous system. The aim of this thesis was to clarify the role of cholinergic projections from pedunculopontine tegmentum (PPT) to lateral hypothalamus (LH) in food intake. In this study, DREADD-technology (designed receptors exclusively activated by designer drugs) based on chemogenetics was utilized with a gene manipulated mouse strain. For the experimental part of this work the mice were divided in three separate groups: one transducted with an activating DREADD-receptor (hM3Dq), one transducted with an inhibiting DREADD-receptor (hM4Di) and one transducted only with a fluorescent protein called m-Cherry. The last group was also defined as a control group of this study. In addition, the gene which coded m-Cherry fluorescent protein was transducted together with hM3Dq- and hM4Di-receptor genes for the first two groups to be able to examine the receptor expression later. At the baseline level, no differences were observed in food intake between the three study groups. The food intake did not differ between the groups while clozapine-N-oxide (CNO), a selective DREADD-receptor ligand, was administered straight into the LH area (0,03 µg/injection) with or without fasting of the animals. While administrating CNO to the mice intraperitoneally (1 mg/kg), the hM3Dq-group mice were observed to consume more food compared to the hM4Di-group or the control group. This difference was detected while food consumption was examined cumulatively during total four measuring hours. When the animals were fasted before the intraperitoneal administration test, however no differences were found between the study groups regarding food intake. As a conclusion of this study, cholinergic projections from pedunculopontine tegmentum (PPT) to lateral hypothalamus (LH) were not regulating food intake in mice. However, the cholinergic cells in PPT and some of their axons might be involved in the regulation of food intake while the food consumption is studied continuously and long-term. More studies are required to better define the role of the cholinergic projections from pedunculopontine tegmentum (PPT) to lateral hypothalamus (LH) in food intake.
  • Nurmi, Riikka (2017)
    Liposomes are spherical nano-sized drug delivery systems which are composed of lipid bilayer. With liposomes drugs can be targeted for example to tumours and targeting can be passive or active. Drug release from liposomes can also be activated by different methods. Light is very promising triggering method, because it enables drug release at specific time and site. This study examined light activated indocyanine green (ICG) liposomes. Drug release from liposomes happens because ICG converts light energy to heat. ICG is clinically approved imaging agent, so ICG liposomes are very promising drug delivery systems even for clinical use. Liposomes were prepared by thin-film hydration method. One aim of the study was to prepare as small ICG-liposomes as possible. The bigger 100 nm liposomes were studied in three different formulations and the purpose was to find differences between those formulations. In formulation A ICG was in PEGs, in formulation B ICG was in lipid bilayer with no PEGs and in formulation C ICG was supposed to be in lipid bilayer although the formulation C included PEGs. In this study, the cell up take of ICG liposomes was studied with pharmacokinetic model and data from in vitro studies was supposed to use in a pharmacokinetic model. In this study, it was possible to prepare 40 nm sized ICG-liposomes. Small liposomes did not release encapsulated calsein as well as bigger 100 nm liposomes. The decreased release from smaller liposomes was probably explained by the results witch pointed out that transition temperature of small liposomes was higher than transition temperature of bigger liposomes. In the future, the lipid composition of the small liposomes need to be reoptimized, that the release would be more effective. This study however proved that small ICG-liposomes can be prepared and the small size lasts even over three months. Three different formulations of 100 nm liposomes were studied and the differences between the properties of the formulations were found. ICG in the lipid bilayer changed properties of the formulation B and the passive release of the calsein and release during the lightning were increased. In formulation C transition temperature was decreased and its storage life was lower than in other formulations. Formulation A was best for the next studies and the phospholipid composition of other formulations need to be optimated that drug release and storage life would be good enough. Intracellular release properties of liposomes were studied with Sytox red probe. Fluorescence of Sytox red increases when it binds with DNA or RNA. With this study, it was proved that liposomes release Sytox red inside the cells and that the lightning time affects to the release. The results weren't useable for pharmacokinetic model, so the model was made based by literature. Pharmacokinetic model can be used in the future studies and different in vitro or in vivo results can be combined with the model.
  • Nenonen, Satu (2017)
    Ankylosing spondylitis is an inflammatory rheumatoid disease, that is typically diagnosed in young adults. The symptoms include inflammatory back pain, rigidity in the lumbar and thoracic spines, and peripheral inflammations. The incidence of ankylosing spondylitis among northern European population ranges from 0.2 to 0.5%. The mortality rate of people with ankylosing spondylitis is about 50% higher than in the average population. First-line treatment for ankylosing spondylitis includes physiotherapy and NSAIDs. TNF inhibitors are used for patients whose symptoms cannot be controlled with first-line treatment. In Finland, there are five TNF inhibitors indicated for ankylosing spondylitis on the market: infliximab, etanercept, adalimumab, golimumab, and sertolizumab pegol. In 2015, the average medication cost for a patient entitled to reimbursement for TNF inhibitors in Finland was over 12 000 €. The cost-effectiveness of TNF inhibitors in the treatment of ankylosing spondylitis compared to conventional care has been extensively studied, but there is less data on the differences between TNF inhibitors. In this thesis, previously published literature on the cost-effectiveness of TNF inhibitors in the treatment of ankylosing spondylitis was reviewed, and a patient-specific simulation model based on data from the National Register for Biologic Treatment in Finland was conducted. The aim of the simulation was to compare the cost-effectiveness of TNF inhibitors (infliximab, etanercept, adalimumab and golimumab) in the treatment of ankylosing spondylitis as the patient's first biological treatment compared to other TNF inhibitors. The simulation was conducted on a lifetime time horizon and incorporated direct health care and medication costs in 2015 euros. As conclusions of the model, all other TNF inhibitors were found dominant over etanercept. The greatest effectiveness was achieved with golimumab, while the costs were lowest with infliximab. The incremental cost-effectiveness ratio of golimumab compared to infliximab was 63 840 €/QALY. In sensitivity analyzes, the model was found to be very sensitive to TNF inhitors' prices. In addition, sensitivity was also observed for the discount rate and time horizon used.
  • Hietala, Tarja (2017)
    Twin screw granulation (TSG) has gained considerable interest as a continuous wet granulation method in the pharmaceutical industry and has been studied the most. However, there is still lack of understanding how continuous granulation affects the material compaction behavior even though it has been noticed in several dry and batch wet granulation studies that the granulation process has an influence on the final tablet strength. Thus, studies on the material compactability and tabletability after continuous wet granulation are relevant for the overall understanding of twin screw granulation process and its effect on material behavior in tableting. Hence, the main objective of this study was to investigate the influence of continuous twin screw granulation on the compactability and tabletability of commonly used excipients. Additionally, the impact of binder on the compaction behavior of materials was examined. Furthermore, the suitability of two "loss in compressibility" models i.e. the Unified Compaction Curve (UCC) model and a porosity model to predict the loss in tablet strength after twin screw granulation and for the materials used was assessed. Earlier, the models have been applied to dry and batch wet granulations only. Full factorial design of three variables (binder type, binder addition method and the number of kneading elements) with two levels was conducted for the ConsiGma1 twin screw granulation of formulations containing microcrystalline cellulose (MCC), mannitol or anhydrous dicalcium phosphate (DCPA) as the main excipient and polyvinylpyrrolidone (PVP) or hydroxypropyl cellulose (HPC) as binder. Magnesium stearate was added as lubricant after granulation prior to tableting. In addition to the full factorial design, granulation with PVP, dry binder addition and four kneading elements was repeated for each main excipient. In total this made 27 experiments. The granules were dried and milled after granulation and all the batches were tableted. Additionally, all the formulations were direct compressed in order to be able to detect the change in compactability and tabletability after granulation. Torque of the granulation was determined as well as bulk density and particle size distribution of the granules. Additionally, the tensile strength and porosity of the tablets were analysed. Tabletability and compactability were determined based on the compaction pressure and the obtained tensile strength and porosity values of the tablets. Furthermore, parameters (PWG, TWG and εWG) describing the loss in compressibility models were calculated. MCC experienced loss in compactability and tabletability after twin screw granulation due to hornification effect. On the other hand, the compaction behavior of mannitol improved due to the formation of porous granules. The compactability of DCPA decreased and the tabletability increased. However, the change was only moderate presumably due to brittle nature of DCPA. Additionally, the binder type had an effect of the compaction behavior of the materials, PVP producing stronger tablets compared with the less hydrophilic HPC. However, the binder addition method played only a small role in modifying the compaction behavior. The UCC model was applicable to MCC as loss in tabletability was detected. Thus, the model can be used to predict tablet tensile strength when MCC is granulated with twin screw granulator. Additionally, the UCC model can be used to design the granulation process to achieve a target tensile strength based on small scale preliminary studies thus reducing the resources needed for case-studies. However, the UCC model was not feasible to mannitol and DCPA because they experienced improvement in tabletability after twin screw granulation. The porosity model was applicable to MCC and DCPA but not to mannitol as it showed improvement in compactability. The porosity model described the loss in compactability of MCC only moderately due to lack of tensile strength data points and the linearity of the tensile strength-porosity relationship. However, the model described well the loss in compactability of DCPA at tablet porosities achieved with compaction pressures used in industry. As a conclusion, the results demonstrate that twin screw granulation can have a significant impact on the final tablet strength and that the compaction behavior of the formulation can change either way depending on the used materials. Furthermore, the small influence of the binder addition method on the tablet strength indicates that the time consuming binder dissolving process step can be excluded from the tablet production chain enabling continuous manufacturing with twin screw granulation.