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  • 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.
  • Kampman, Johanna (2016)
    Rheumatoid arthritis (RA) is a chronic autoimmune disease with prevalence around 0.8 % in Finland. Joint inflammation causes pain, tenderness and swelling in joint as well as loss of functional and work capacity. Patients need healthcare resources and medical treatments cause substantial costs to patient and society. Severity of RA can be measured by Disease Activity Score (DAS28) and Health Assessment Questionnaire (HAQ). Previous studies suggest these measures are related to higher direct and indirect cost. The aims of this study are to determine cost of RA in Finland and analyse cost by disease activity and functional disability. Literature review was performed by using some methods from systematic reviews to identify previous studies examining cost of RA by DAS28 and HAQ scores. Secondly in this master thesis was made a quantitative cost analysis which study population was identified from the National register for Biologics in Finland (ROB-FIN) and patient records of the Central Finland Central Hospital. The cost data for direct and productivity costs was received from Finnish national registries. Costs were determined from the followed six months after patients' (N=2285) first routine outpatient visit to specialized healthcare. Distribution of costs was examined by DAS28 and HAQ score based classification. Additionally cost related to RA was determined separately between biologic and non-biologic drug users. Total average costs of the study population were 11 720 € biannually. Direct costs, productivity costs and total costs were higher for patients with higher DAS28 and HAQ scores. Increase in average total costs between best and worst DAS28 and HAQ classifications were 7817 -15 838 € and 8545 - 16 718 €, respectively. In the cost categories changes in both directions were detected between different DAS28 and HAQ score classifications. Drug costs comprised largest part of direct costs (56 %). RA related average total costs increased from best to worst DAS28 and HAQ class for both biologic and non-biologic drug users (p<0,01). Similar study based real life register data is not previous made in Finland. Studies in other countries can`t be directly adapted to Finnish healthcare system, treatment traditions and productivity costs. This study provides information for real-life cots of RA and how they are related to disease activity and functional disability. This information can be used in modelling of cost-effectiveness.
  • Tervahauta, Tuomas (2015)
    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.
  • Kari, Otto K. (2018)
    Nanolääkkeiden pinnalle elimistössä muodostuva biomolekyylikerros eli proteiinikorona vaikuttaa muun muassa jakautumiseen, toksisuuteen ja soluvuorovaikutuksiin. Koronan ominaisuuksien tuntemus jakautumisen eri vaiheissa on siten edellytys tehokkaampien ja turvallisempien nanolääkkeiden kehittämiselle, mutta kehitystyötä on hidastanut soveltuvien menetelmien puute. Turvallisuuden ja tehon ennakoinnin osalta on korostettu leimavapaiden in vitro -menetelmien tarvetta. Tutkielmassa kehitettiin multiparametriseen pintaplasmoniresonanssilaitteistoon ja laskennalliseen mallinnukseen perustuva menetelmä liposomien koronan tiheyden ja paksuuden määrittämiseen. Toisin kuin koronan tutkimiseen yleisesti käytetyt menetelmät, valoon perustuva kajoamaton ja leimavapaa menetelmä ei vaikuta koronan rakenteeseen. Näin voidaan tutkia myös löyhemmin sitoutuneista proteiineista muodostunutta pintakerrosta, mikä vastaa keskeisimpään kirjallisuuskatsauksessa todettuun menetelmäpuutteeseen. Menetelmää sovellettiin neljän biosensorille immobilisoidun liposomiformulaation pinnalle ihmisen seerumissa muodostuvan koronan tutkimiseen. Sen avulla oli mahdollista määrittää ensimmäistä kertaa tiiviin ja löyhän koronan tiheys ja paksuus laimentamattomassa seerumissa. Tulokset tukevat käsitystä ns. erotteluhypoteesin kuvaamasta erillisestä löyhästä proteiinikerroksesta ja avaavat uusia mahdollisuuksia sen biologisen merkityksen arviointiin. Lisäksi voitiin määrittää ensi kerran opsoniinimolekyylien sitoutumiskinetiikka liposomien pinnalle, minkä avulla voidaan arvioida nanolääkkeiden taipumusta poistua verenkierrosta ja aktivoida sisäsyntyinen immuunipuolustus. Menetelmä soveltuu siten liposomien koostumuksen ja pinta-arkkitehtuurin optimointiin prekliinisessä lääkekehitysvaiheessa.
  • Viljamaa, Matleena (2015)
    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.
  • Laakko, Maria (2017)
    Gene therapy is the therapeutic delivery of nucleic acid sequences into cells, where they can replace a gene that is missing, mutated or poorly expressed. It is a potential treatment to cure e.g. genetic diseases, viral infections and various cancers. The nucleic acid needs to be delivered across the cell membrane and into the nucleus to affect the gene expression. Anionic nucleic acids need a cationic carrier, such as a cationic liposome, to enable their delivery into the cells. The liposomes used in gene delivery usually contain both a cationic lipid to associate with the nucleic acid and a neutral helper lipid to stabilize the structure. The liposome-nucleic acid complex is called a lipoplex. The cationic carrier must include or function as a cell-penetrating enhancer (CPE) to be able to translocate across the cell membrane into the cytosol and to the nucleus. The experimental part of this work was aimed at developing and characterizing an innovative poly-cationic liposomal platform for gene delivery, using a novel synthetic CPE. The CPE used in this study is an oligo-guanidyl derivative (OGD) that had either 4 (OGD4) or 6 (OGD6) cationic charges. Liposomes were surface-engineered with OGD, obtaining a cationic formulation that was then exploited for DNA loading. The study has two main characterization steps: Step 1 was to decorate liposomes with OGD by post insertion using increasing amounts of OGD, and determine the vesicle size and zeta potential by dynamic light scattering (DLS). Step 2 involved DNA loading by post insertion into the cationic liposomes with increasing amounts of DNA. The lipoplex size and zeta potential was determined by DLS, the complexation by electrophoresis, and the thermodynamics of the cationic liposome/DNA association by isothermal titration calorimetry (ITC). The measurements were performed in isotonic buffers (HEPES pH 7.4 and citrate pH 5) and in lower ionic strength TRIS buffer (pH 7.4). The aim of the characterization studies was first to find a liposome composition that includes just enough OGD to obtain a sufficiently high zeta potential and a uniform, sufficiently small size. The optimal formulation contained either 10 % of OGD4 or 5 % of OGD6 of the total lipid amount. The second step was to find the highest stable DNA loading for the lipoplexes. All the characterization studies were performed on OGD4 lipoplexes in TRIS buffer. The optimal OGD4/DNA N/P (nitrogenous/phosphorous) ratio was found to be around 5. Further investigation is needed to determine the best lipoplex composition and manufacturing method using an isotonic buffer. A DNA release study remains to be performed prior to further in vitro and in vivo studies.
  • Tähkäpää, Sanna-Mari (2016)
    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.
  • Suotunen, Pauliina (2020)
    The OATP1B3 belongs to the organic anion transporting polypeptides (OATPs) encoded by the SLCO (solute carrier organic anion) genes which belongs to the SLC (solute carriers) gene superfamily. It is an influx transporter which is primarily expressed on the basolateral membrane of the hepatocytes. It transports many endogenous substrates as well as clinically important drugs such as thyroid hormones and statins into hepatocytes and thus participates in the first step of hepatic metabolism. Single nucleotide polymorphisms (SNPs) of the SLCO1B3 gene can affect the pharmacokinetics and pharmacodynamics of its substrates. The aim of this study was to set up and optimize an in vitro method to study the function and expression of the OATP1B3 transporter and its genetic variants. SNPs 334T> G (Ser112Ala), 699G> A (Met233Ile) and 767G> C (Gly256Ala) and stop codon TAA were introduced into the SLCO1B3 gene by site-directed mutagenesis. Recombinant baculovirus vectors containing the genetic information of OATP1B3 and its variants were used to transiently transfect the HEK293 cells. After optimizing the substrate incubation time and concentration, as well as the viral load and selecting the fluorescent substrate (8-FcA), the uptake assay was used to determine the transport activity of the OATP1B3 variants in HEK293 cells. The transport activity of the artificial WTP variant was also investigated in this study. The transport activities of the Ser112Ala, Met233Ile and Gly256Ala variants did not change significantly from the wild type although the transport activity of the Met233Ile variant appears to be slightly impaired. In turn the WTP variant was unable to transport 8-FcA. Based on this study the function of OATP1B3 variants can be studied using recombinant baculovirus to transiently transfect the HEK293 cells. 8-FcA can be used as a probe substrate in these studies. The results of this study confirm previous knowledge of the functioning of Ser112Ala, Met233Ile and Gly256Ala variants. More studies are needed about the effects of these variants on the transport of OATP1B3 drug substrates. Also studies about the location, cell membrane and total cell expression of the WTP variant are needed to evaluate reliably the reasons of its inactivity.
  • Aaltonen, Timi (2023)
    The obligatory storing of medicines is a vital part of the secure supply of medicines in Finland. Over the past few years, its importance has further increased due to the growing number of medicine shortages. Evaluating the effectiveness of the obligatory storing system is important in order to improve it, but so far research on the matter has been limited. The aim of this study was to investigate how the obligatory storages of pharmaceutical manufacturers and importers are used during medicine shortages in Finland, and to assess the effectiveness of their use in these situations. The material for this study consisted of medicine shortage notifications which had been received by the Finnish, Swedish and Norwegian medicine authorities with a forecasted starting date between January and June 2022. In addition, Finnish exemption permits for lower storage levels from the same time period were investigated. Medicine shortage notifications were grouped based on the obligatory storing status of the medicine. The share of obligatorily stored medicines out of all shortage notifications was the smallest in Finland (10%) when compared to Sweden and Norway. There were no statistically significant differences in any of the countries in the duration of a shortage between obligatorily stored medicines and medicines which are not obligatorily stored. In total, 151 exemption permits had been granted within the time period of the study, 129 (85%) of which did not have a coinciding medicine shortage. This suggests that a patient-affecting shortage had successfully been avoided. The remaining 22 exemption permits were linked to a shortage which started either prior to, or during the validity of the exemption permit. In the Finnish data, 91 notifications concerned obligatorily stored medicines but in 69 (76%) of these cases no exemption permit had been applied for or been granted in relation to the shortage. The results of this study indicate that the obligatory storing of medicines was used to respond to several medicine shortages during the first half of 2022, and in most cases, it seems to have been an effective way to avoid a patient-affecting shortage. However, in some cases the use of an exemption permit was not well-timed, a shortage was experienced despite the releasing of products from the storage, or obligatory storages were not used at all. Based on the results, further research on the practices of obligatory storing and the factors which affect the use of exemption permits is needed to develop the system further and to improve its effectiveness in responding to medicine shortages.
  • Rimpelä, Anna-Kaisa (2014)
    Certain drugs accumulate into pigmented tissues due to their binding to melanin, a macromolecule inside pigmented cells. Melanin can affect the drug's pharmacokinetics by acting as a drug reservoir. Binding can also cause toxic effects by accumulating compounds to pigmented cells. This thesis focuses on ocular melanin. The literature review covers the most common methods used in the study of ocular melanin binding and concentrates on in vitro methods and the analysis and usability of the results in pharmacokinetic modeling. The aim of the experimental part was to study melanin binding of a set of compounds in vitro with melanin isolated from the retinal pigment epithelium (RPE) and choroid of porcine eyes and with primary porcine RPE cells and then construct a pharmacokinetic model of melanin binding with STELLA® software and simulate it with the in vitro results. The compounds chosen for the study; nadolol, timolol, chloroquine, methotrexate, carboxydichlorofluorescein (CDCF) and dexamethasone, are small molecules with diverse physicochemical properties (octanol/water partitioning coefficient (logP), pKa, acid/base status). Some are also efflux substrates. The in vitro binding with melanin was studied at pH 7.4 and in addition at pH 5 for the acidic compounds, since the pH inside melanosomes where melanin is located is acidic. Porcine RPE cells were used to study the amount of uptake and rate of elimination of the set of compounds. The effect of efflux was also evaluated with a general efflux inhibitor probenecid. All the basic compounds bound to melanin in vitro. The acidic compounds did not seem to bind at pH 7.4 but bound at pH 5. Chloroquine, as expected, had the highest binding. In the cell studies, the uptake of chloroquine was significant, at least partly due to melanin binding. The other compounds were taken into the cells to a much smaller extent. The efflux inhibitor did not seem to affect the results. The results of the binding study were used in the models constructed of melanin binding and cellular pharmacokinetics. The constructed model was a very simple one not taking into account many factors affecting cellular pharmacokinetics. The results of both the in vitro studies and the model give a good idea of the importance of melanin binding in ocular drug delivery. The model can be used in the future as a base for more comprehensive models of the effect of melanin binding on ocular pharmacokinetics.
  • Laustio, Netta (2018)
    During the past few decades, the explosion of discovery in cancer and immunological research has led to the increased understanding of the interactions between the immune system and tumors. These developments have provided vital information about the immune system’s role in cancer development. It is evidenced that the immunity system is capable to distinguish tumor cells from normal tissue by recognizing tumor antigens that are exclusively expressed on tumor cells or are presented in greater amounts on tumor cells than normal cells. Consequently, the immune cells start to attack tumors for protecting the host. The possibility to use the immune system as a weapon against cancer cells leaded to the promising innovation – cancer immunotherapy – which aims to activate the body’s own immune system and its components to mount antitumor immune responses for eliminating cancer cells. The antitumor efficacy and high safety profile of several immunotherapeutic strategies have already been demonstrated thereby resulting in their integration into clinical practice. However, most patients have not benefited from cancer immunotherapy as a single treatment. In this regard, new innovative methods are clearly needed to overcome the obstacles hindering the clinical success of this field. Therapeutic cancer vaccines are emerging as attractive immunotherapies currently being evaluated in both pre-clinical and clinical studies. The purpose of cancer vaccines is to eradicate tumor cells by eliciting antitumor CD8+ T cell responses against the injected tumor antigens. Due to the ability to specifically kill tumor cells and simultaneously trigger immune responses against tumor antigens via direct oncolysis and by encoding transferred tumor antigens, oncolytic viruses are of significant interest for being used as in situ cancer vaccines. Despite these unique properties, several factors such as tumor immunosuppression and immune tolerance to targeted tumor antigens resembling antigens of normal tissues hamper the use of oncolytic vaccines in clinic. Instead of focusing only on CD8+ T cells, it has been suggested that giving more attention to CD4+ T helper cells, which are required for priming and expansion of CD8+ T cell responses, could be the key to improve the efficacy of cancer vaccines. Researchers have also demonstrated that an ongoing antigen-specific CD4+ T cell response can lead to the bystander activation of surrounding T cells with unrelated antigen specificities. Based on this theory, the hypothesis of this study was to employ the pre-existing immunological CD4+ memory against infectious pathogens in generating bystander CD8+ immunity against solid tumors. In this study, mice transplanted with poorly immunogenic B16-OVA tumors were pre-immunized with the chosen vaccine to induce immunological CD4+ memory against an infectious pathogen. Tumors were then treated with already developed cancer vaccine, which was peptide-coated conditionally replicating adenovirus (PeptiCRAd) complex. PeptiCRAd was constructed by electrostatically coating adenovirus with both pathogen-derived and tumor-derived peptide. The intratumorally injected double-coated PeptiCRAd complex was assumed to activate peptide-specific T cells and thus, result in anti-pathogen CD4+ T cell recall responses and the following bystander activation of antitumor CD8+ T cells, which can then mount an effective immune response to destroy cancer cells. The efficacy of this treatment was observed in pre-immunized mice by measuring the growth of injected tumors. The experiment was repeated identically with non-immunized naïve mice to see the difference in the results. The immunological background of this treatment approach was investigated by analyzing mouse tissue samples with standard immunological techniques including ELISA, IFN-γ ELISPOT and flow cytometry. This study showed that long-term immunological memory against the pathogen was successfully accomplished and the strongest inhibition of tumor growth in pre-immunized mice was achieved with double-coated PeptiCRAd, whereas the antitumor efficacy was not seen in naïve mice. Additionally, a new ex vivo method to detect pathogen-specific CD4+ T cells from spleen was developed and the stimulation of cell-mediated immunity by this treatment was supported by finding the highest levels of pathogen-specific CD4+ Th1 cells from mice treated with double-coated PeptiCRAd. Some encouraging results concerning the beneficial immune cell composition of tumors and tumor draining lymph nodes were also obtained from other performed experiments. Though further immunological analyses are required for understanding the precise mechanisms of action behind the treatment, the increased immunogenicity and antitumor efficacy of double-coated PeptiCRAd can still be considered as a consequence of the bystander effect, which can possibly be utilized for developing improved strategies to win the fight against cancer.
  • Koivunotko, Elle; Merivaara, Arto; Valkonen, Sami; Chinello, Lisa; Salmaso, Stefano; Korhonen, Ossi (2020)
    Biomimetic native nanofibrillated cellulose (NFC) hydrogel has recently proven its efficacy, safety and diversity at the site of pharmaceutical industry. Yet, properties for the long-term storage in dry condition at room temperature and feasible transportation needs to be developed for NFC hydrogel before it is suitable for freeze-dried biomedical applications. Our aim was to optimize freeze-drying cycle for NFC hydrogel formulation with suitable lyoprotective biomolecules and preserve its properties after freeze-drying process and reconstitution. NFC hydrogel formulations with different combinations of chosen biomolecules were freeze-dried, and physicochemical properties and rheological features were characterized. In addition, morphology of the freeze-dried cakes was studied. The effects of the biomolecules on the water contents in NFC systems were simulated for both of the crystal and amorphous ones. All the results of the characteristics were compared with the non-freeze-dried NFC hydrogel formulations. NFC hydrogel formulation, which had the most optimal preservation properties after freeze-drying and reconstitution, was optimized. We hypothesized that without any chemical modifications native NFC hydrogel can be successfully freeze-dried and subsequently reconstituted with the proper biomolecules only by using biological and natural materials, which are human and xenon-free for the further use in biomedical applications of the native NFC hydrogel.
  • Häkkinen, Aino (2016)
    There is a growing interest towards continuous manufacturing in pharmaceutical industry. When considering solid dosage form manufacturing and continuous wet granulation, twin screw granulation is the most studied technology. In spite of this, process knowledge is not as comprehensive as it is for batch granulation technologies such as high shear granulation. One problem with twin screw granulation seems to be bimodal and broad particle size distribution of granules. Specific causes of bimodal distribution and the ways to possibly gain unimodality either in granulation step or via dry milling are not fully understood. The purpose of this study was to optimize the ConsiGma25 continuous twin screw wet granulation process and study dry milling as a possible way of gaining unimodal particle size distribution in order to optimize particle size for tablet compression purposes. One aim was also to compress tablets from the obtained twin screw granules and evaluate the quality against high shear and direct compressed tablets of same formulation. Central composite circumscribed design was used as a design of experiments for twin screw wet granulation process. Factors (powder feed rate, screw speed, liquid to solid ratio (L/S ratio) and mill screen size) were varied in two levels. In total, 29 experiments were conducted. Tablet compression design was fractional as only 11 experiments from the twin screw granulation design were tableted. High shear granulation was conducted with Diosna P-1/6 using three different L/S ratios. All of these batches were compressed to tablets. Also direct compression was carried out. Formulation used had ibuprofen as active pharmaceutical ingredient, mannitol and MCC as fillers, HPC as binder and croscarmellose sodium as disintegrant. Lubricant, sodium stearyl fumarate, was blended to granules before tablet compression. Torque was measured during granulation in order to evaluate equilibration of the twin screw granulation process. It stabilized quickly and stayed stable after parameter changes and increased as L/S ratio and barrel fill rate increased. Particle size distribution and flowability of granules were analysed and tablets were analysed according to European and United States pharmacopoeias. Also tensile strengths and compactibilities were evaluated. Particle size distributions of unmilled twin screw granules were bimodal and broad. After dry milling, QicPic dynamic image analysis results showed unimodal particle size distributions for all experiments whereas Mastersizer laser diffraction analysis showed more unimodal distributions for experiments milled with smaller mill screen sizes. Mill screen size had the largest effect on particle size and as increased mill screen size increased particle size. Results showed that dry milling was a way to optimize particle size distribution for tablet compression purposes. Also flowability of formulation and process parameters needed to be optimized as granulation parameters had an effect on particle size and manufacturability was enhanced with better flowing formulation compared to previous study. Knowledge of the influence of L/S ratio, screw speed and powder feed rate, on granule size was gained. The effect of these process parameters varied depending whether d10, d50 or d90 was measured and particle size analysis method used. Increased L/S ratio and screw speed increased granule size as increased powder feed rate decreased it. Twin screw tablets showed higher tensile strengths and better compactibility compared to both high shear tablets and direct compressed tablets. Twin screw tablets showed also faster dissolution compared to direct compressed tablets probably due to of lower compression force. Mill screen size had the largest effect on dissolution properties of the twin screw tablets. When larger mill screen size was used, dissolution was slower due to larger particle size. Also increased powder feed rate made dissolution rate slower due to higher fill rate of the barrel. In other tablet properties analysed, no significant differences were seen between different twin screw experiments or between twin screw tablets and direct compressed tablets. All of the twin screw tablets and direct compressed tablets fulfilled the requirements of European and United states pharmacopoeia. As a conclusion, continuous granulation process was successfully optimized and high quality tablets resulted showing especially the effect of dry milling on granule size and shape as well as on tablet properties.