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  • Kunnari, Mikko (2016)
    Crohn's disease is a type of inflammatory bowel disease. There are no treatment procedures that can cure Crohn's disease, but it is usually controllable with medicinal options. However 70 - 80 % of patients will require surgery and most undergo several during their life, due to weak local potency of drugs and disrupted recovery from surgical treatment. A better method of combined treatment, such as a drug releasing surgical suture, could improve the disease recovery process. One approach would be to coat a surgical suture with nanofibrillar cellulose (NFC) hydrogel containing the active drug ingredient within. NFC is biocompatible, biostable and it can be easily chemically modified. It displays pseudoplastic and thixotropic gel-like behavior in aqueous suspension in addition to high shear thinning properties under low and high shear rates. The shear-thinning behavior is particularly useful in a range of different coating applications. Furthermore, recent studies have shown the potential of NFC in controlled drug release. The aim of this Master's thesis was to investigate the suitability of anionic NFC hydrogel for surgical suture coatings and controlled release applications. The structure of NFC hydrogel was modified with crosslinking cations (Fe3+, Al3+, Ca2+) and alginate. The diffusion studies were performed with two antibiotics, metronidazole and rifaximin together with FITC-dextrans (10 and 250 kDa). The surgical suture was coated with each type of hydrogels (n = 16). Furthermore, the suitability of suture drug formulation for controlled drug release was simulated with STELLA® modeling software. It was shown that the NFC hydrogel structure was stiffened with the use of crosslinking cations; however similar results were not observed with the addition of alginate. Release profiles of model compounds were similar before and after NFC hydrogel crosslinking. At 6 days, 50 - 60 % of 10 kDa dextran (6 µg) was released. For 250 kDa dextran (6 µg) the released amount was 25 - 35 %. During the first 3 days of the diffusion study, all of metronidazole (20 µg) was released. Rifaximin samples were not obtained due to high adsorption to the container surfaces. The release profiles of metronidazole and 10 kDa dextran had linear correlation with square-root diffusion process. 250 kDa dextran followed a near zero-order kinetics after a few hours from the start. The coating was performed successfully with NFC hydrogels except for hydrogels with dextrans or without crosslinking. Metronidazole was predicted to release from the surgical suture almost instantly with STELLA® modeling software. NFC hydrogel shows potential as a matrix for controlled drug release and the coating of surgical sutures. However, the manufacturing method of the NFC hydrogel could be improved with surface modifications of nanofibrils or with the choice of a drug or of its derivatives. With pharmacokinetic simulation models it is possible to predict and estimate different factors which affect drug release from the surgical suture. Furthermore, the simulation models can be used to estimate an effect in the treatment of Crohn's disease.
  • Li, Mingwei (2016)
    Nanofibrillar cellulose (NFC) can form hydrogels with high water content (> 98 %). It has been studied for drug release, and it has been used as a cell culture matrix, due to its similar structure to extracellular matrix (ECM). In addition it has been found that they has no cytotoxicity. Iontophoresis is the application of an electric current over a defined area for the purpose of enhancing permeation across a membrane for ionized drug species. The aim in the experimental work in this Master's thesis is twofold. First, to find out the suitable drug loading concentrations into NFC hydrogels, which can provide a good release profile, a release study with two model drugs, propranolol and ketoprofen, loaded into three types of NFC hydrogels at three different concentrations, was carried out for this purpose. Second, to see if NFC hydrogels are applicable as a drug reservoir in iontophoretic transdermal drug delivery applications, an iontophoresis study was carried out using porcine ear skin model in vitro for human skin with propranolol loaded into NFC hydrogel of type A. In addition, Stella models were used as an aid to find suitable ways to predict the release and permeation behaviour of models drugs in the abovementioned context. The UPLC results from the release study show for both model drugs, the wt. % released had linear correlation with squareroot of time. At 6 hours, more than 70 wt. % propranolol was released from hydrogel reservoir. For ketoprofen, the release varied between 30 - 87 wt. %, where higher initial loading concentrations produced a decrease in the wt. % released from hydrogel. The iontophoresis study did not show a significant difference between the tested current densities (0.50 mA/cm2; 0.25 mA/cm2) produced on the wt. % of drug released. Simulation models could be run with the mathematical equations for diffusion controlled drug release. In conclusion, the NFC hydrogels show potential as drug reservoir for drug release. Additional experimental data using other types of drug reservoirs should be obtained for a better understanding of the suitability of NFC hydrogels as a drug reservoir in iontophoretic transdermal drug delivery.
  • Lahti, Aapeli (2022)
    Lääkeaineiden niukkaliukoisuus on yhä enemmän esiintyvä ongelma lääketeollisuudessa. Erityisesti BCS ryhmän II lääkeaineet ovat potentiaalisia liukoisuusominaisuuksia parantaville menetelmille. Tässä työssä näistä menetelmistä keskitytään nanokiteen, ko-kiteen ja ko-amorfisen systeemin muodostukseen ja lääkeaineena käytetään inodmetasiinia (BCS ryhmä II). Kyseisillä menetelmillä on onnistuttu parantamaan indometasiinin liukoisuusominaisuuksia, mutta vertailevia tutkimuksia ei ole aiemmin tehty. Nanokide valmistettiin märkäjauhamalla käyttäen poloksameeri 188 -stabilisaattoria. Ko-kiteen valmistuksessa käytettiin liuottimen haihdutus -menetelmää ja ko-muodostajana sakariinia. Ko-amorfisten systeemien ko-muodostajina käytettiin l-tryptofaania ja sitruunahappoa ja valmistus toteutettiin kuulamyllyllä jauhamalla. Karakterisointimenetelmillä (DLS, DSC ja XRPD) oli mahdollista todentaa nanokiteillä ja ko-kiteillä halutut ominaisuudet (partikkelikoko ja kiderakenne). Ko-amorfinen systeemi ei työssä käytetyllä menetelmällä saavuttanut amorfista rakennetta kummallakaan ko-muodostajalla. Vaikka jauhe osittain muuttui kellertäväksi (viitaten amorfiseen indometasiiniin) olivat XRPD:n ja DSC:n tulokset kiteiselle aineelle tyypillisiä. Nanokiteellä ja ko-kiteellä saavutettiin puhdasta indometasiinia parempi ominaisliukenemisnopeus sekä liukenemisnopeus jauheesta lapamenetelmällä. Systeemien välisessä vertailussa huomattiin, että nanokiteellä oli parempi liukenemisnopeus molemmissa kokeissa. Ero on selkeämmin nähtävissä lapamenetelmässä: pieni partikkelikoko mahdollistaa suuren suhteellisen pinta-alan liukenemista varten. Systeemien fysikaalista stabiilisuutta tutkittiin yhdeksän kuukauden ajan suljetussa muoviastiassa laboratorio-olosuhteissa (huoneenlämpö ja normaali ilmankosteus). Kummassakaan systeemissä ei ollut nähtävissä kiderakenteen muutoksia. Nanokiteillä oli havaittavissa lievää partikkelikoon kasvua, mikä on selitettävissä ennen koetta tehdyn sekoituksen tehottomuudella
  • Saarelainen, Taija (2010)
    Steroid hormones are involved in many physiological functions such as stress response and the maintenance of salt-water balance and pregnancy. Concentrations of steroids in the body fluids are generally very low (below ng/ml). Steroid hormones are metabolically associated and changes in mutual concentration levels of different steroids may signify a disease. Methods that allow the measurement of various steroids simultaneously are of great importance in investigating the role of steroid metabolism for example in formation of cancer. The aim of this work was to develop a sensitive and selective method for simultaneous quantification of 16 steroids in plasma. Nano liguid chromatography-microchip electrospray ionization-tandem mass spectrometry (nanoLC-µESI-MS/MS) was used in order to achieve good sensitivity. C18 enrichment column and separation column, and an electrospray tip were integrated onto the chip that was used in this work. Mass spectrometric parameters were optimized by using a MS calibration and diagnostic chip. It was noticed that the structure of steroids plays an important role on how the compound behave in electrospray ionization. Steroids with 4,5-ene-3-one-structure had much lower limits of detection than steroids without conjugated double bonds (0,075-0,5 ng/ml and 5-25 ng/ml respectively). The chosen sample pretreatment method to extract the steroids from plasma did not work properly, because it was able to extract only a third of the compound's real concentration. Analysis of some compounds was also difficult because of the background noise coming from plasma. The method development was therefore decided to continue with eight steroids that were well detectable and had 4,5-ene-3-one-structure. The limits of detection were 0,075-0,5 ng/ml in biological matrix for these compounds. Eight knock out and seven wild type mouse plasma samples were analyzed using the validated method. The method was able to quantify aldosterone, corticosterone and androstenedione. Developed method did not meet all the aims of this work. Derivatizated compounds, different equipment or totally new method should be used in order to accomplish the aims.
  • Ravela, Reko (2021)
    Objectives To investigate distribution and causes of drug shortages in five selected countries with data from public shortage notification registers. Design Statistical retrospective analysis of national drug shortage registers Data Public shortage notification register data from the first nine months of 2020 in Finland, Sweden, Norway, Spain, and the United States, partly combined with national drug registers. Results Altogether 5132 shortage reports from Finland (n=1522), Sweden (n=890), Norway (n=800), Spain (n=814), and the United States (n=1106) published during the first nine months of 2020 were found in the study. More than half (54%) of the active ingredient level shortages (classified by ATC code) found occurred in only one country, and only 1% occurred in all five countries. On a country level, 19-41% of the shortages were found only in a given country. The distribution of shortages by ATC category and drug form was significantly different between countries, especially between the US and European countries. Injectables were found to have an especially high shortage risk in the US, much less so in European countries. On the other hand, some drug classes were in shortage almost exclusively in Europe but not in the US. Conclusions Although drug shortages are a growing global problem, drug shortages are rarely global, but typically occur only in some countries, while other countries have an uninterrupted supply of the same drug. Drug shortages should be seen not just as a problem of manufacturing disruptions, but as a question of equitable and effective distribution of drug supply on an international level. Price differences and other commercial issues could be factors behind variation of shortages found between countries.Considering the limited and probing nature of the study, further research of shortage register data is certainly warranted. International comparative register study is a meaningful and valid method for further understanding in this field.
  • Tyynismaa, Lotta (2012)
    In Finland first pharmacists started to work on wards in 1980s and 1990s. Ward pharmacy increased mainly in consequence of the lack of nurses. Common tasks were taking care of drug logistics (stock control), dispensing drugs to patient specific doses, preparing and diluting intravenous drugs and providing drug information to ward personnel. During the 2000s, ward pharmacy services have been increasing a lot. New tasks are, e.g., reviewing medications and prescriptions, medication counselling, and taking part in medical rounds. However, the tasks are still rather logistics compared to the United States and the United Kingdom where a pharmacist has an established role in a multiprofessional team. Internationally it has been proved that it is possible to achieve decreased and enhanced quality of care and patient safety with hospital clinical pharmacy services. The aim of this study was to explore the extent and benefits of ward pharmacy services in Finland. An online survey was conducted by sending the invitations to the chiefs of hospital pharmacies (n = 24) and medical dispensaries (n = 94) by using the e-mail register of the University of Helsinki and Satefa (Finnish Association of hospital and health centre pharmacists). Before compiling the questionnaire six theme interviews were conducted to set up the questionnaire. The survey respondents were asked to submit information about development projects and research reports if they had explored the benefits of ward pharmacy services. The response rate was 60 % (n/N = 72/118). A half of the respondents (n = 36) reported having ward pharmacy services in their units. Benefits were explored in 12 units and nine project reports were received. Altogether 157 pharmacists were working in 242 wards at the time of the survey in spring 2011. Most common tasks were providing drug information to ward personnel, drug logistics and dispensing drugs to patient specific doses. Patient oriented tasks were increased, including prescription and medication reviews, taking part on medical rounds and medication counselling gave patient information were reported. The most reported benefits on ward pharmacy services were increased multiprofessional collaboration, saved working time of nurses and physicians, decreased drug costs and decreased number of medication errors and/or enhanced reporting habits and developed functions on wards. Respondents also believed that ward pharmacy services can have positive impact on length of stay, readmission and hospitalisations and mortality during hospitalization, but these benefits were not demonstrated by studies. In the future it would be important to develop the Finnish ward pharmacy services by following the international example and the principles of pharmaceutical care. The help of automation technology and pharmacy technicians should be exploited more in drug logistics. The economical and patient related outcomes of new clinical and patient oriented services should be proved in Finland and the results of the researches and projects made in hospital and health centres should be published more.
  • Halonen, Henna (2013)
    In Finland pharmaceutical policy is a part of health policy. It concerns social decision making on pharmaceutical sector and people and organizations around pharmaceuticals. Pharmaceutical policy 2020 strategy was prepared by the Ministry of Social Affairs and Health, based on a stakeholder (administrative authorities, education and research, industry, medicine wholesalers, patients, pharmacies, professional organizations, public sector) analysis. In Finnish politics this is an assignment of a new kind of evidence-informed policy making (EIPM), which is participative, transparent and democratic. The two main objectives of this study were to evaluate the present state of pharmaceutical policy in Finland and discern the emphasis of pharmaceutical policy by the stakeholders' point of view in the future. The study gave the possibility to a deeper analysis of stakeholders' opinions of the Finnish pharmaceutical policy in the beginning of the year 2010 than it was possible in the official Pharmaceutical policy 2020 strategy document. Besides this, research itself was a way of practicing evidence-based policy. The SWOT-analysis were conducted among key-stakeholders as a main material of this qualitative research (n=19, response rate 59). SWOT analyses included present strengths, weaknesses, opportunities and threats regarding Finnish drug discipline of a stakeholder's point of view. Furthermore, an e-survey (n=10, response rate 31) was conducted in the end of the strategy process in order to enablegive the stakeholders to give feedback of the Pharmaceutical policy 2020 strategy process. The survey also measured that was the consensus among stakeholders reached during the strategy process. It prevailed a very good mutual understanding. Results of the research were clearly readable on the official strategy paper as well as the research could go deeper in the details of stakeholders' words than the official strategy paper. The most important emphases in the results were noticeable: condense the cooperation of stakeholders among the drug discipline to ensure efficient, good quality and patient safe pharmaceutical service and better utilizing of pharmaceutical knowledge among social and public health service. The results of this research can be utilized later as a starting point to measure how Pharmaceutical policy 2020 strategy has came true. With help of this study it's also possible to verify the strategy process and Finnish national medicine policy in the 2020 decade. In Ministry of Social Affairs and Health it is also possible to think, what kind of legislative changes it demands to implement the needed changes among the drug discipline.
  • Oja, Maria (2024)
    Lääkehoitoa toteutetaan erilaisissa toimintaympäristöissä sekä sosiaali- ja terveydenhuollossa, että sen ulkopuolella. Lääkehoitoa toteuttavat pääsääntöisesti sosiaali- ja terveydenhuollon ammattihenkilöt, mutta ympäristön mukaan vaihdellen myös lääkehoidon koulutusta vähän tai ei lainkaan saaneet. Lääkehoidon osaamisen varmistaminen on aina työnantajan vastuulla. Keski-Uudenmaan hyvinvointialueella on ruuhkautuneiden näyttöjen myötä tunnistettu tarvetta keskitetyille näytöille. Tehty pilottitutkimus tuo arvokasta tutkimustietoa näyttötyöpajasta osana lääkehoidon käytännön osaamisen varmistamisen prosessia, sillä aikaisempia tutkimuksia aiheesta ei ole. Tutkimuksen keskeisenä tavoitteena oli tutkia näyttötyöpajan toimivuutta lääkehoidon käytännön osaamisen varmistamisessa, sekä mitä resursseja näyttötyöpajan järjestäminen organisaatiolta vaatii ja mikä olisi optimaalinen näyttötyöpajan osallistujamäärä. Pilottitutkimus toteutettiin järjestämällä näyttötyöpaja, jossa Keusoten yksiköissä työskentelevät nimikesuojatut terveydenhuollon ammattihenkilöt (n=15) osoittivat lääkehoidon käytännön osaamistaan. Näyttötyöpajassa lääkkeiden jakamisen näyttöjä ottivat vastaan farmaseutit (n=3) ja lääkkeiden antamisen näyttöjä sairaanhoitajat (n=2). Aineistona tutkimuksessa toimi näyttötyöpajassa näyttöjä antavien ja vastaanottavien antama palaute, joka kerättiin puolistrukturoidulla palautelomakkeella paikan päällä. Palautekysely lähetettiin myös näyttöjä antaneiden esihenkilöille ja vastaaville sairaanhoitajille sähköisellä Microsoft Forms- lomakkeella. Aineistoa analysoitiin tilastollisesti kuvaavalla tavalla sekä induktiivisella sisällönanalyysillä hyödyntäen Microsoft Excel -ohjelmaa. Näyttötyöpajaan osallistuneet kokivat näyttötyöpajan kehittävän lääkehoidon osaamista. Näyttötyöpajan koettiin nopeuttavan ja helpottavan osaamisen varmistamista sekä. Esihenkilöt ja vastaavat sairaanhoitajat kuvailivat näyttötyöpajan myös vapauttavan näyttöjen järjestämiseen kuluvia resursseja muuhun hoitotyöhön. Sekä osallistujat että näytön vastaanottajat kokivat näyttötyöpajan vertautuvan huonosti aitoon työtilanteeseen. Keskeisiksi näyttötyöpajan kehityskohteiksi nousivat parempi organisointi, tiedonkulun vahvistaminen ja näyttöjen tasalaatuistaminen. Mukauttamalla näyttötyöpajaa enemmän aidon työtilanteen kaltaiseksi voisi näyttötyöpaja tukea osaamisen kehittymistä paremmin, sekä parantaa työyksikön lääkitysturvallisuutta. Näyttötyöpajaa tulisi tutkia ja kehittää lisää, jotta siitä saataisiin sujuva osa osaamisen varmistamisen prosessia. Näyttötyöpaja koettiin pääosin hyödylliseksi ja se koettiin tarpeelliseksi interventioksi helpottamaan ruuhkautunutta lääkehoidon osaamisen varmistamista.
  • Aitta-aho, Teemu (2003)
    Epidemiological data suggest an important role of perinatal viral infections in the etiology of schizophrenia. In this thesis the connection between neonatal viral brain infection and its consequences to the development of central nervous system was studied. In schizophrenia the symptoms are divided into three categories as positive, negative and cognitive ones. Positive symptoms refer to hallucinations and delusions, negative symptoms are defined as social withdrawal, apathy and poor motivation and cognitive symptoms include deficits in abstraction and paying attention into subjects. Symptoms suggest that in schizophrenia the received information can not be filtered properly in central nervous system, but comes into patients senses in excess i.e. there are defects in sensorimotor gating. Sensorimotor gating was studied by prepulse inhibition of acoustic startle -phenomenon. Prepulse inhibition refers to the inhibition of the startle reflex by weak prepulse presented before the startling stimulus. In schizophrenic patients prepulse inhibition is decreased and in addition to that psychotomimetic drugs disrupt prepulse inhibition in humans as well as in experimental animals. Sensorimotor gating ability is developed under neuronal development and it can be affected by several neurodevelopmental disturbances. In the present study rats were infected with herpes simplex type 1 virus at neonatal age and later challenged to dopaminergic and glutamatergic systems. Results show controversial data of effects on prepulse inhibition, still some attenuation can be seen. Challenge studies did not show clear and persistent effect either in dopaminergic or glutamatergic tests. Corticosterone, naturally occurring hormone in rats, was administered to rat mothers under gestation until weaning in terms to clarify its effects to neuronal development. Administration was carried out by implanted pellet as well as by drinking water. The latter was found to work out better as it releases corticosterone in pulsatile manner. Corticosterone was administered also in acute test to drug naïve animals. This test showed significant decrease on prepulse inhibition. The same could not be repeated in corticosterone challenge test after perinatal treatments. Nitric oxide synthase inhibitor L-NMMA was administered to neonates under days 5-9 after partus. This was supposed to prevent neonates from neurodevelopmental disturbances affected by virus and corticosterone. Despite various dose levels used, any clear effect could not be seen. In summary, the studies show some effect of treatments on neuronal development and sensorimotor gating measured by prepulse inhibition. In the test groups inspected many treatments showed effect at first, but those effects disappeared at later tests as rats grew up. This might be an outcome of the potential compensatory mechanisms of the central nervous system to counteract harmful neurodevelopmental events.
  • Nordström, Sabina (2010)
    Parkinson's disease is a chronic progressive neurodegenerative disorder, characterized by muscle rigidity, hypokinesia, tremors and bradykinesia. The cause of symptoms in Parkinson's disease is loss of dopaminergic nerve cells in the substantia nigra, which attenuates the nigrostriatal dopaminergic signaltransmission. Oxidative stress, mitochondrial dysfunction, protein misfolding and aggregation, inflammation, excitotoxicity, apoptosis and other routes for cell death, and loss of neurotrophic factors have shown to be mechanisms in the pathogenesis of Parkinson's disease. Microglia might have a double role in the pathogenesis of Parkinson's disease. Microglia stimulated by Į- synuclein does not only produce toxic factors such as certain cytokines and reactive oxygen and nitrogen species, which contribute to the neuronal cell death but also produce anti-inflammatory cytokines and neurotrophic factors, which can be neuroprotective. Deeper knowledge of the mechanisms underlying Parkinson's disease is needed for developing restorative medicines. Three different neurotrophic factor families are known to be important in the research of Parkinson's disease. The GDNF-family consists of glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN). The neurotrophin-family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophins NT3 and NT4/5. The most recently discovered family is the MANF-family, which consists of mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF). In Parkinson's disease the neurotrophic factors could stop, slow or ideally even reverse the neurodegeneration in the dopaminergic system and decrease the functional decline of the neurons. Research has already shown that GDNF has both a neurorestorative and neuroprotective effect in animal models of Parkinson's disease. Clinical trials have however shown controversial results. The challenge with neurotrophic factors can be the administration to the brain through the blood-brain-barrier, sideeffects because of receptor binding in other organs or sites of the body and low diffusionrate. Research of both MANF and CDNF has shown promising neurorestorative and -protective results in vivo. Local diffusion of MANF has been shown to be better than of GDNF. In this Master's thesis research was done on whether MANF and CDNF have a neurorestorative effect on the dopaminergic nerve cells in mixed primary culture in vitro after 6-OHDA exposure. The aim of the study was to receive information about whether MANF and CDNF are as effective as GDNF at repairing celldamages caused by 6-OHDA in vitro in this experimental model. GDNF was used as a posivite control in this study. The results from this study suggest that MANF might have a neurorestorative effect, but this effect is much smaller than with the neurotrophic factor GDNF. The results show no neurorestorative effect with CDNF. Neither the dopamine uptake nor the tyrosine hydroxylase staining showed statistical significance.
  • Kallio, Arttu (2014)
    Cytochrome P450 (CYP) -enzymes are one of the most important enzymes in the metabolism of xenobiotics. Because many xenobiotics are metabolized with each other by the same CYP-enzymes, it is possible that metabolic interactions will take place. These interactions can be the inhibition or induction of the metabolism of another xenobiotic. The interaction can be harmful e.g. when it causes an accumulation of a toxic metabolite or when it inhibits the metabolism of an active drug substance. The aim of this study was to develop a quantitative method for determining metabolic interactions between drugs and environmental chemicals in human liver microsome (HLM) incubations. HLMs contain high concentrations of CYP-enzymes, enabling the use of CYP-model reactions for observing interactions. The model reactions chosen for this study were O-deethylation of phenacetin (CYP1A2), 7-hydroxylation of coumarin (CYP2A6), 4'-hydroxylation of diclofenac (CYP2C9), 1'-hydroxylation of bufuralol (CYP2D6) and 6β-hydroxylation of testosterone (CYP3A4). Michaelis-Menten constants (Km) and maximal enzymatic activities (Vmax) were determined for each model reaction. The suitability of the model reactions for inhibition studies was assessed with specific inhibitors. The quantitative method was developed for an ultra-high performance liquid chormatograph (UPLC) and for a quadrupole time of flight mass spectrometer (QTOF). Samples were ionized with electrospray ionization (ESI) using positive mode. Device parameters were the same for all the metabolites. The analytical method validation was partly performed according to ICH (International Conference on Harmonisation) guidelines. A sufficient linearity (R2>0,99) and specificity was achieved for the quantitative method. The achieved limits of quantitation (LOQ) were low enough (1-120 nM) for quantitation of the small concentrations of the metabolites formed in the inhibition assays. The measurement reproducibility and the reproducibility and accuracy of the method did not fulfill the acceptance criteria for all the metabolites. Improvement of the results should be tried by e.g. exploring different device parameters. 1'-hydroxydiclofenac was found likely to degrade in the matrix solution because of the acidic conditions, making the reliability of the results poor for this metabolite. The Km value obtained for coumarin differed markedly from literature values, which can be due to a too long incubation time. Therefore, incubation conditions should be optimized for this model reaction in coming studies. The Km values obtained for the model reactions of CYP1A2, CYP2D6 and CYP3A4 were similar to those found in literature. Also the IC50 values were quite well within the range of values reported in literature for the inhibitors of the above mentioned model reactions. The effects of four different polymers, F68, F127, Tetronic 1307 and polyvinyl alcohol (PVA) on the enzyme activities were also studied, at a concentration of 1 mg/ml. In principal, at this concentration the polymers did not cause significant changes in the enzyme activities, although inhibition of the CYP2C9 could have been significant. However, the reliability of CYP2C9 model reaction was found to be poor with the used method. In the future this developed method should be further validated, and the incubation conditions for the model reaction of CYP2A6 should be optimized. After this, the IC50 values for the polymers could be studied to get more reliable information about their potential CYP-inhibition properties.
  • Hossi, Heidi (2016)
    The abuse of drugs is monitored by different authorities and health care. World Anti-Doping Agency (WADA) prohibits the use of doping substances and methods in- and/or out-of-competition. WADA has created strict instructions for Anti-Doping laboratories for analyzing different substances from biological samples. The aim of this study was to develop liquid chromatographic-mass spectrometric (LC-MS/MS) screening analysis for the detection in urine of drugs of abuse. The basis of study was 20 different substances which had different molecular weights, logP and pH values. The purpose was to create the basis of the method where is easy to add new analytes in further studies. Almost all substances chosen in this study were doping substances and the guidelines for the method were created by WADA. The sample pretreatment was pursued to be as generic as possible for plenitude of analytes and easy to perform. The sample pretreatment included two liquid-liquid extraction steps and enzymatic hydrolysis. The LC-MS/MS method worked well for many analytes with some exceptions. Some analytes didn't fit for the sample pretreatment and some didn't give strong enough signal in desired detection level. The gradient of LC-method can be limiting factor when adding new analytes to the method. Especially very lipophilic and polar analytes may cause difficulties. Carry over caused some problems in analyses. As a result it may lead to new sample treatment and LC-MSanalysis for the same batch.
  • Molari, Joonas (2018)
    Currently, there is an undeniable need for more effective treatments of depression. The efficacy of traditional antidepressant drugs becomes apparent after multiple weeks of treatment. New advancements in depression treatments have been made, as glutamatergic NMDA-receptor antagonist ketamine is seen to ameliorate symptoms rapidly, even only hours after drug administration. Understanding ketamine’s mechanism of action as an antidepressant could enable the development of more effective antidepressant drugs. The critical molecular level component in ketamine’s antidepressant effect is considered to be the activation of TrkB tyrosine receptor kinase B, which subsequently leads to the initiation of signaling pathways, which regulate synaptic plasticity. So far, it has not been examined; whether there is a difference in ketamine’s antidepressant effect based on the dosing-time of day. The aim of the present study was to find out if there is a variation between ketamine’s effect on synaptic plasticity and the circadian phase in which the drug is administered. Ketamine’s (200 or 50 mg/kg, i.p.) effects were studied in C57BL/6J–mice during light phase (mouse’s inactive phase) and dark phase (mouse’s active phase) of the day. The phase of the day didn’t affect the activity of TrkB signaling in its related parts (pTrkBTyr816, pGSK3βSer9, p-p70S6KTyr421/Ser424 and p-p44/42MAPKThr202/Tyr204) in prefrontal cortex samples which were analysed in Western blot assay. Ketamine increased dose-dependently the phosphorylation of GSK3βSer9 and p70S6KTyr421/Ser424 as well as decreased p-p44/42MAPKThr202/Tyr204 at 30 minutes after drug administration in both phases of the day. Ketamine (200 mg/kg, i.p.) also lowered the glucose concentration measured from the trunk blood. To examine the effect of hypoglycemia on the activity of TrkB signaling another experiment was conducted. The hypoglycemia induced by insulin detemir (6 IU/kg, i.p.) didn’t affect any measured protein phosphorylation at 60 minutes after drug administration. The results of this study support the notion of ketamine’s rapid and dosedependent induction of neuroplasticity. The possible role of hypoglycemia in ketamine's neuropharmacology should be investigated in future studies.
  • Peltonen, Anna (2018)
    Histamine acts as a neurotransmitter in the central and peripheral nervous system and it has a role in various body functions. Histamine neurons spread widely to most of the central nervous system where histamine has an important role in sleep-wake cycles, regulation of appetite, and motor functions. The effects of histamine are mediated mostly by H1-, H2- and H3-receptors in the central nervous system. The synthesis of histamine and the release of histamine from the presynaptic nerve endings are regulated by H3-receptor via negative feedback. H3-receptors are located also on the presynaptic cell membranes of other neurons where they regulate the release of other neurotransmitters. Several animal experiments have shown that H3-receptor-mediated mechanisms have been observed to have an important role in the regulation of the motor functions together with other neurotransmitter systems especially in the basal ganglia area. The histaminergic system is involved in the patophysiology of diseases such as Parkinson’s disease, Tourette’s syndrome and Huntington’s disease where motor performance is impaired. Functional, physiological and genetical changes in the histaminergic system have been observed in patients with these diseases. There are no clinically used histaminergic compounds for the treatment of these diseases, though recently in animal experiments the histaminergic compounds have proved to be promising. The aim of this Master’s thesis study was to examine the effects of histamine deficiency in the brain on the levodopainduced dyskinesias in histidine decarboxylase knock-out mice (HDC KO) (n=9) and wild-type mice (n=12) in a 6-OHDA mouse model of Parkinson’s disease. The mice were injected with a neurotoxic 6-OHDA solution (3 μg) into the right medial forebrain bundle to cause a unilateral dopaminergic lesion. The success of degeneration of dopaminergic neurons were measured by a rotating rod test and amphetamine-induced (2.5 mg/kg) and apomorphineinduced (0.5 mg/kg) rotameter tests. A daily treatment of levodopa and benserazide (4.5 mg/kg, 1.125 mg/kg) was initiated after the behavioural studies for 10 days. On the last day of the treatment the dyskinesias of the mice were filmed for one minute after 20, 40, 60, 80, 100 and 120 minutes after levodopa dose. After the filming, the mice were killed by decapitation and their middle brains were collected for immunohistochemical studies to measure the extent of the dopaminergic lesion. No statistically significant difference was observed between genotypes in levodopa-induced dyskinesias. In previous studies of our study group more severe levodopa-induced dyskinesias were observed in HDC KO mice when the dopaminergic lesion was caused in the striatum in the 6-OHDA mouse model. The degenerated brain area and thereby the extent of the lesion may have importance in observing the difference between levodopa-induced dyskinesias. In this Master’s thesis study the dopaminergic lesions were equally successful with both genotypes. Therefore differently successful lesions between the genotypes can not be the reason why the difference in genotypes in levodopa-induced dyskinesias was not observed. HDC KO mice were observed to have significantly increased ipsilateral rotational behaviour induced by amphetamine in amphetamine-induced rotametry. Previous studies have shown that HDC KO mice have increased dopamine release and high dopamine metabolite levels which might explain the increased rotational behaviour induced by amphetamine in this study. The observations of earlier studies and this Master’s thesis study verify the relation between histaminergic and dopaminergic systems in motor functions.
  • Havia, Mari (2013)
    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channel receptors which are widely distributed in human brain. nAChRs are often expressed pre-synaptically and they modulate the release of other neurotransmitters. nAChRs consist of five subunits: nine different subunits have been identified so far, forming multiple different nAChR subtypes with different pharmacological properties. nAChRs participate extensively in physiological functions and pathophysiological conditions. nAChRs mediate the effects of endogenous agonist, acetylcholine, as well as commonly used substance of abuse, nicotine. Addictive drugs such as nicotine and opioids cause adaptive changes in central nervous system. In addition to binding site of acetylcholine, various allosteric binding sites have been identified in nAChRs. Allosteric ligands are able to modulate the effect of agonist by binding to allosteric binding site. The aim of the experimental part of the pro gradu was to study in vitro interactions of nicotine and three different opioids, codeine, oxycodone and tramadol in SH-SY5Y cells. SH-SY5Y cells express endogenously α3* and α7-nAChRs. Binding assays were performed with radioactive ligand [3H]-epibatidine. Functional interactions of nicotine and the opioids were studied with 86Rb+- efflux assay. Codeine, oxycodone and tramadol exhibited receptor level interactions with nicotine in SH-SY5Y cells. Observed interactions were mediated by nAChRs. The opioids inhibited nAChR activation caused by nicotine without binding to the [3H]-epibatide binding site. Codeine, oxycodone and tramadol appear to affect as weak non-competitive antagonists of nAChRs. These results give further information of nicotine-opioid interactions at receptor level. There are indications that nicotine and opioids also have interactions in vivo, which may be partly explained with these receptor level interactions.
  • Takala, Anna (2012)
    Neurosteroids are steroids which are active in the central nervous system. They have many biological and physiological functions in human body. Fluctuations of the neurosteroid concentrations are related to many diseases such as depression, schizophrenia and epilepsy. Neurosteroid levels are measured to understand their role in brain function and human behavior. The aim of the work was to develop a gas chromatographic-atmospheric pressure fotoionization-tandem mass spectrometric (GC-APPI-MS/MS) method for analyzing 19 neurosteroids and their metabolites in urine. Neurosteroids are excreted in urine mainly as conjugates, so they have to be hydrolyzed before analysis. Sample purification is done by liquid-liquid extraction and the analytes are subsequently derivatized to enhance their volatility. Because widely used β-glucuronidase/arylsulfatase-enzyme from Helix pomatia oxidases 3β-hydroxy-5-ene and 3β-hydroxy-5α-reduced steroids, we decided to use β-glucuronidase from Escherichia coli and acid hydrolysis instead of H. pomatia. The quantification of the total neurosteroid concentration in urine was challenging because β-glucuronidase enzyme from E. coli did not hydrolyze glucuronides completely and acid hydrolysis deconjugated also glucuronides in addition to sulfate conjugates. In addition the internal standard d4-allopregnanolone was noticed to be impure and degrade during acid hydrolysis. The limits of detection were reasonably low for the method (2 pg/ml-1 ng/ml). The retention times of the analyte peaks were very repeatable (RSD 0,06-0,11%) and the repeatability of the method was acceptable for all compounds (RSD < 27%). Urine samples from two males and two females were analyzed with the preliminary validated method. We could determine estimated concentrations for dehydroepiandrosterone, dihydrotestosterone, androstenedione, testosterone, estrone, β-estradiol, estriol, 5α-tetrahydrodeoxocorticosterone, cortisone, corticosterone and hydrocortisone. The developed method did not meet all the aims of this work. The method needs further validation and more exact investigation about the effect of the selected hydrolysis method on intact steroids. Also the internal standard should be changed to some other compound, preferably a non-deuterated one.
  • Rinne, Meri (2010)
    Parkison's disease is a progressive neurodegenerative disorder that is characterized by degeneration of dopaminergic neurons in the nigrostriatal dopamine pathway. This is responsible for the major symptoms of Parkinson's disease. The current therapies only treat symptoms without being able to slow down, or reverse, the neurodegenerative process. Therefore current research is directed toward prevention of dopaminergic neuron degeneration. Prolyl oligopeptidase (POP) is a serine peptidase which cleaves small proline-containing peptides. A number of neuropeptides are affected in Parkinson's disease and POP contributes to the degradation of many of these neuropeptides. Reduction in POP activity has observed in Parkinson's disease. However, it is not known if changes in POP activity were a cause or a consequence of Parkinson's disease. POP inhibitors are substrate-like compounds. In our study we used KYP-2047, a novel brain-penetrating POP inhibitor. Administration of KYP-2047 has previously been shown to increase slightly neurotensin levels after a single dose. Neurotensin is an endogenous neuropeptide that has antidopaminergic actions in the brain. A number of neurotensin receptors has been observed to decrease in substantia nigra and striatum after degeneration of nigrostriatal pathway in laboratory animals or Parkinson patients. When given into the brain, neurotensin and neurotensin analogs have decreased rigidity and tremor caused by 6-hydroxydopamine (6-OHDA). The aim of this study was to determine the interactions between POP and neurotensin and their connections with dopamine deficit in a rat model of Parkinson's disease (the turning model of Ungerstedt). POP activity was also studied. In this study two different kinds of lesions were used. Intracerebral injections of 6-OHDA were given either into the MFB (medial forebrain bundle) or striatum. Rotational behaviour was measured five weeks post-lesion. The MFB lesion Wistar rats were given levodopa/carbidopa -suspension with KYP-2047 or entacapone or both of them. The striatum lesion rats were given amphetamine with KYP-2047. Studies were organized in a cross-over manner once a week. Rotational behaviour did not change when a POP inhibitor was given suggesting that neurotensin levels were apparently not much changed. Differences in POP activity assay were not noticed when compared to normal and lesioned cerebral hemisphere. This indicates that there is no POP in long dopaminergic neurons.
  • Hynynen, Marko (2017)
    Smoking poses a significant threat to public health. Major public health benefits could be attained if we could increase the incidence of smoking cessation on the national level. However, smoking cessation is often difficult, therefore different medical means have been devised to make it easier for people to quit. One of these medical means is nicotine replacement therapy(NRT). Since NRT entered the market it has been contested which kind of role counselling should have when people purchase NRT. NRT was deregulated In Finland in 2006. Prior to this thesis there has not been carried out studies that would have tried to investigate where the retailer shops are located after deregulation. The aim of this thesis is to investigate how the deregulation affected to the distribution of NRT sales in different NRT-outlets and pharmacies in 2006-2015. It is also aim to investigate if the new outlets have tangibly increased the coverage of NRT-outlets network in diverse municipalities, more sparsely populated rural municipalities. Finnish Medical Agency‘s (FIMEA) data about NRT-outlets between the years of 2006-2015 was used as source data for this thesis. This data was analysed by comparing the number of NRToutlets and pharmacies in diverse municipalities. The municipalities have been classified regarding the area and population of diverse municipalities, among other parameters. The PDF sheet of FIMEA’s NRT-outlet data was converted into an Excel file, and using that as a basis different diagrams were made. The number of NRT-outlets grew steadily until during the year 2011 there was a slightly bigger increase in the number of NRT-outlets as NRT became available in restaurants too. Afterwards the growth of NRT-outlets diminished. It can be seen from the results that NRT-outlets, more than pharmacies, are concentrated to the crowded municipalities. On the other hand, there has hardly been an increase in the coverage of NRT-outlet network in more sparsely populated rural municipalities since deregulation took place. However, many of the municipalities that have a pharmacy and one or a few other NRT-outlets are located in Southern Finland surrounding bigger city municipalities or in Central Finland.
  • Meijer, Juri (2012)
    Smoking is one of the major causes for premature deaths worldwide. Tobacco smoke contains nicotine, which activates the nicotinic acetylcholine receptors (nAChR) expressed by the human body. nAChRs are part of the cholinergic system and its endogenous neurotransmitter is acetylcholine. The nAChRs are excitatory and the often regulate the release of other neurotransmitters. Nicotine is one of the most addicting compounds known. The rewarding effects of nicotine are mediated through the activation of the mesolimbic dopamine pathway. The mesolimbic pathway is triggered also by the compounds activating the endogenous opioid system thus mediating the rewarding effects and opioid addiction. The nicotine - opioid interactions have been widely studied. It is observed that majority of opioid abusers and patients receiving opioid replacement therapy are smokers. It has been also detected that nicotine releases endogenous opioid peptides in vivo in the brain regions mediating both addiction and analgesia. In addition, the rewarding effect of nicotine attenuates in opioid receptor knock-out rodents. Furthermore, it has been observed that nicotine's rewarding effects can be reduced with opioid receptor antagonists. In order to prevent smoking's negative effects the use opioid antagonists for smoking cessation has been clinically researched with poor results. Many of the opioids in clinical use have diverse and direct interaction with the nAChRs in vitro. E.g. it has been observed that methadone and morphine have an effect on the function of the nAChRs. This may explain partially the smoking behaviour of replacement therapy patients. Opioids are prescribed mainly for the treatment of moderate to intense pain. Nicotine is too found to be analgesic in vivo but in humans its analgesic effect has been questionable. In the experimental part of thesis binding and functional interactions with human's α4β2-nAChR expressed by SH-EP1-hα4β2 cell line was researched with clinically commonly used opioids codeine, oxycodone and tramadol. Competitive binding was studied using [3H]-epibatidine binding assay and the functional effects were studied using 86Rb+-efflux assay. The results suggest that oxycodone and tramadol act as weak competitive antagonists of α4β2-nAChR in vitro in concentrations that are clinically irrelevant. According to the results, however, codeine acts as positive allosteric modulator of α4β2-nAChR potentiating the effects of nicotine in micromolar concentrations. The effect is similar to galantamine, used in treatment of Alzheimer's disease. The clinical relevance of codeine's potentiating nicotine's effect on the function of α4β2-nAChR cannot be estimated according to the results from these studies. Therefore, in order to confirm the results experiments with codeine need to be done in vivo using e.g. α4- and β2-knock-out mice in order to clarify α4β2-nAChR's role in the analgesic and rewarding effects of codeine. However, the results from the experimental part provide valuable information on the interactions of nicotine and opioids. Results from studies conducted with α4β2-nAChRs have not been published enough to determine the importance of the phenomenon in i.a. drug addiction and analgesia.
  • Savolainen, Mari (2011)
    Neuronal nicotinic receptors are widely expressed throughout the brain and they facilitate fast synaptic neurotransmission. They are also involved in regulation of the release of other neurotransmitters like GABA, dopamine and glutamate. The most common subtypes are alfa4beta2 and alfa7 subunits containing receptors. Neuronal nicotinic receptors are involved in nicotine addiction but also in many neurological diseases like Alzheimer's disease, schizophrenia, depression and attention deficit/hyperactivity disorder. The cholinergic stimulation enhances cognition in vivo and in human. There is not many drugs on the market that act via nicotinic receptors but many drug companies have new nicotinic agonists and antagonist under clinical research. When using nicotinic receptor agonists the problem is desensitization, which occurs in alfa7 nicotinic receptor rapidly after agonist exposure. When desensitized the receptor no longer responds to agonist even if it is there available to bind to receptor. The desensitization may lead to tachyphylaxis and losing of the clinical effect. Conventional agonists, like acetylcholine, bind to the binding site located in the extracellular part on nicotinic receptor subunit. There is also some other binding sites, which are called allosteric binding sites. It has been found out, that allosterically binding ligands, for example PNU-120596, can cause potentiation of agonist induced responses and/or prevent desensitization of receptor. These kinds of agents are called positive allosteric modulators and they are considered to be a new therapeutic option for CNS diseases containing cholinergic deficits. The mechanism of action of positive allosteric modulators is so far unclear. The purpose of my study was to characterize positive allosteric modulators on alfa7 nicotinic receptor. It had been found out earlier in the Millar laboratory that mutation L247T in the transmembrane domain converts positive allosteric modulators to agonists. The aim was to use site-directed mutagenesis to generate mutation in the agonist-binding site of alfa7 and alfa7L247T receptors and see how it effects on the ability of PNU-120596 to act as an agonist on the receptor. Second aim was to generate a mutation in the transmembrane part of the receptor to an assumed binding site of allosteric potentiators' and test how it effects on allosteric potentiator's ability to act as an agonist on the alfa7L247T. Mutated receptors were expressed on oocytes by microinjeting the mRNA into oocyte. The function of receptors was studied with electrophysiology using two-electrode voltageclamp technique. All the mutations were successfully inserted in nicotinic receptor alfa7 and alfa7L247T. Mutation in orthosteric agonist binding site had a very profound effect on wild type alfa7 receptor; it had an effect on either acetylcholine binding or receptor gating. It was not possible to record any proper responses neither with acetylcholine nor with PNU-120596. In the double-mutated receptor alfa7W149M/L247T the W149M mutation had a much greater effect on dose-response curves than it had on PNU-120596 curves compared with alfa7L247T. The transmembrane domain mutation M253L did not have much effect on PNU-120596's ability to act as an agonist to alfa7L247T and either it did not effect on acetylcholine dose-response curves. The results from this study support the previous results that the binding site of positive allosteric modulators is located in the transmembrane domain of the alfa7 receptor. The results are little controversial with the M253L mutation but because the L247T mutation has so profound effect on the function on alfa7 receptor it might be that it masks the other mutation which is located quite close to it. On the other hand it might be so that the amino acid M253 has only effect on the receptor's ability to be potentiated not the allosteric binding.