Browsing by Title

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.

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.

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.

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.

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.

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, e.g.in 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.

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.

Prodrugs are pharmacologically inactive molecules which undergo metabolic bioactivation in vivo to form pharmaceutically active agents. Prodrugs have been designed to improve so called drug-like properties of active parent compounds (APC) i.e. to increase solubility or absorption and to reduce first-pass metabolism etc. In this master's thesis the goal was to establish non-cell-based in vitro methods to study prodrug bioactivation. Four commercially available prodrugs (bambuterol, olmesartan medoxomil (OM), candesartan cilexetil (CC) and famciclovir) were used as test compounds. The prodrugs were incubated in liver and intestinal S9 fractions and blood plasma to study in vitro bioactivation of these prodrugs. Other metabolism of the prodrug and APC (nonproductive metabolism) was studied by comparing incubation with and without cofactors of metabolizing enzymes. Species differences was studied using human, rat and dog matrices. Prodrug concentrations were quantified from the incubation samples using liquid chromatography- tandem mass spectrometry (LC-MSMS) methods developed for this study. Additionally the effect of promoiety on passive permeability was studied with parallel artificial membrane permeability assay (PAMPA). All of the studied prodrugs produced at least low concentrations of APC in one or more incubations. Terbutaline (APC of bambuterol) formation was observed in human plasma and was concentration dependent which is consisted with the literature. Olmesartan and candesartan were formed in S9 fraction in high rate, but not in buffer: indicating enzyme mediated hydrolysis. However, based on literature CC hydrolysis was not expected to occur in intestinal S9 fractions. Penciclovir (APC of famciclovir) was formed only in presence of human or rat liver S9 fraction which was in line with the pre-existing literature. With the method used the nonproductive metabolism could not be estimated. In PAMPA bambuterol, famciclovir and OM had higher permeability than corresponding APCs whereas CC was only more permeable than candesartan in pH 7.4. The in vitro incubation used in this study can be used for screening prodrugs. However both low and high activation rates were observed thus the clinically relevant in vivo APC formation can be achieved with both high and low bioactivation in vitro. Studying the rate of prodrug formation alone estimations about clinically relevant bioactivation rates cannot be concluded. No clear signs of nonproductive could be seen with the prodrugs studied with current method. For the estimation of nonproductive metabolism, metabolite screening studies would need to be developed and conducted parallel to studies prescribed in this master's thesis.

Gene therapy involves the delivery of exogenous DNA into the target cells in order to produce therapeutic protein or to correct a genetic defect. The use of cationic liposomes and polymers as carriers of DNA is based on observations that positively charged carriers bind to anionic DNA protecting its premature degradation and facilitating its cellular uptake in transfection. The modification of carriers and the engineering of DNA are proposed to enable efficient and prolonged protein expression after transfection. Gene therapy is a potential treatment for age related macular degeneration (AMD). The dysfunction of retinal pigment epithelial (RPE) cells is assumed to be a significant factor in the development of AMD. The aim of this Master's thesis was to study non-viral gene delivery to RPE cells and endothelial cells using several carrier/DNA combinations. Carriers in this study were DOTAP/DOPE/PS liposomes, methacrylamide based (PDMAEMA) micelles, and anionic lipid coated DNA complexes (LCDCs). The carriers were complexed with episomal plasmid DNA or minicircles using secreted alkaline phosphatase (SEAP) gene as a marker gene. Adult retinal pigment epithelial (ARPE-19) cells, human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE), human embryonic primary RPE cells and endothelial cells (EaHy 926) were used in transfections. In ARPE-19 cells linear PBuA-PDMAEMA -based complexes reached the transfection efficiency of positive control whereas in human primary RPE cells star-like PBuAPDMAEMA -based complexes were the most efficient. In human primary RPE cells, SEAP secretion lasted at least 18 days when PDMAEMA-based micelles complexed with plasmid or minicircle with cytomegalovirus (CMV) promoter were used. High nitrogen/phosphate (n/p) ratios of polyplexes decreased cell viability. DOTAP/DOPE/PS/DNA lipoplexes transfected EaHy cells with high efficiency. In hESC-RPE, lipoplexes also exceeded the transfection efficiency of the positive control and the marker protein secretion lasted ~20 days. Human elongation factor 1a (EF1a) promoter could not prevent transgene silencing. Gene delivery did not succeed with LCDCs in any transfection. According to the results, PBuA-PDMAEMA-polymers and DOTAP/DOPE/PS-liposomes complexed with episomal plasmid or minicircles are potential gene delivery agents for further studies in AMD. More investigation is needed i.e. to confirm the transfection efficiency of the complexes in non-dividing cells.

Many mental disorders, such as anxiety, mood and substance use disorders, become prevalent in adolescence and continue into young adulthood. Anxiety disorders are among the most common mental disorders in adolescents and approximately 6-13 % of adolescents and young adults suffer from them. Benzodiazepines have been used for the treatment of anxiety and sleep disorders for several years but they are not recommended for young patients due to risk of dependence and abuse. Nevertheless, benzodiazepines are also prescribed to treat mental disorders in children and adolescents under 18 -years of age. There is limited population-based evidence on the use of benzodiazepines among children and adolescents. The aim of this study was to investigate the use of benzodiazepines and benzodiazepine related drugs as anxiolytics and hypnotics in the Finnish population aged 0-25 years during 2006-2014. Data of this study were obtained from the Prescription Register of the Social Insurance Institution of Finland covering reimbursed drug purchases of benzodiazepines (N03AE, N05BA, N05CD, N06CA01) and benzodiazepine related drugs (N05CF) dispensed to 0-25 year olds. Purchases of orally administered dosage forms were included in this study. The majority (97 %) of anxiolytic and hypnotic users in the group of 0-25 year olds were 16-25-year-old adolescents and young adults. Use of anxiolytics and hypnotics among 16-25-year-olds decreased from the prevalence 19.9 / 1 000 to 15.9 / 1 000 inhabitants during the years 2006-2014. There were also decrease in incidence and prevalence of long-term use. The prevalence of long-term use among young adults decreased from 5.5 / 1 000 in 2006 to 3.3 / 1 000 young adults in 2014. The majority of anxiolytic and hypnotic users were females but long-term use was more common in males than in females. There was a decrease in use of almost every studied drug. Oxazepam was the only drug with increased number of users during the study period. Use of benzodiazepines as anxiolytics and hypnotics has decreased among adolescents and young adults since 2008 in Finland. Furthermore, long-term use of these drugs has decreased among young adults. Results indicate that rational drug therapy has been paid more attention in recent years which was reflected in decreased use of benzodiazepines.