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(2011)Generation of raw materials for dry powder inhalers by different size reduction methods can be expected to influence physical and chemical properties of the powders. This can cause differences in particle size, size distribution, shape, crystalline properties, surface texture and energy. These physical properties of powders influence the behaviour of particles before and after inhalation. Materials with an amorphous surface have different surface energy compared to materials with crystalline surface. This can affect the adhesion and cohesion of particles. Changes in the surface nature of the drug particles results in a change in product performance. By stabilization of the raw materials the amorphous surfaces are converted into crystalline surfaces. The primary aim of the study was to investigate the influence of the surface properties of the inhalation particles on the quality of the product. The quality of the inhalation product is evaluated by measuring the fine particle dose (FPD). FDP is the total dose of particles with aerodynamic diameters smaller than 5,0 µm. The secondary aim of this study was to achieve the target level of the FPD and the stability of the FPD. This study was also used to evaluate the importance of the stabilization of the inhalation powders. The study included manufacturing and analysing drug substance 200 µg/dose inhalation powder batches using non-stabilized or stabilized raw materials. The inhaler formulation consisted of micronized drug substance, lactose <100µm and micronized lactose <10µm. The inhaler device was Easyhaler®. Stabilization of the raw materials was done in different relative humidity, temperature and time. Surface properties of the raw materials were studied by dynamic vapour sorption, scanning electron microscopy and three-point nitrogen adsorption technique. Particle size was studied by laser diffraction particle size analyzer. Aerodynamic particle size distribution from inhalers was measured by new generation impactor. Stabilization of all three raw materials was successful. A clear difference between nonstabilized and stabilized raw materials was achieved for drug substance and lactose <10µm. However for lactose <100µm the difference wasn't as clear as wanted. The surface of the non-stabilized drug substance was more irregular and the particles had more roughness on the surface compared to the stabilized drug substances particles surface. The surface of the stabilized drug particles was more regular and smoother than non-stabilized. Even though a good difference between stabilized and non-stabilized raw materials was achieved, a clear evidence of the effect of the surface properties of the inhalation particles on the quality of the product was not observed. Stabilization of the raw materials didn't lead to a higher FPD. Possible explanations for the unexpected result might be too rough conditions in the stabilization of the drug substance or smaller than wanted difference in the degree of stabilization of the main component of the product <100µm. Despite positive effects on the quality of the product were not seen there appears to be some evidence that stabilized drug substance results in smaller particle size of dry powder inhalers.
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(2023)Drug transporters and metabolizing enzymes have an important role in drug absorption in the small intestine. Food-drug interactions can affect the function of drug transporters and metabolizing enzymes in the small intestine and hence the bioavailability of drugs may change. Certain beverages have clinically relevant interactions with drugs and drinking of them should be avoided during certain drug treatments. However, possible food-drug interactions need more in vitro and in vivo studies, for example in the case of food additives which are used in the food industry increasingly, to investigate their clinical significance as inhibitors. Overall, investigating food-drug interactions is important as they might be as relevant as drug-drug interactions, especially for drugs that pass the gut wall mainly via transporters or have high presystemic metabolism. In this thesis, the inhibitor potential of 23 food additives was studied toward intestinal transporters and CYP enzymes. The food additives included sweeteners, colorants, and antioxidants. Food additives were tested against four efflux transporters with vesicle transporter assays and in OATP2B1 influx transporter with HEK293 uptake assay. The inhibition of CYP enzymes was tested in human intestinal microsomes. Six food additives were identified as possible inhibitors of BCRP, MRP2, OATP2B1, or P-gp. Two food additives were dual inhibitors. IC50 values were determined in dose-response studies for the potential inhibitors. The IC50 values were compared to the maximum expected concentration in the intestinal lumen to evaluate if the in vivo inhibition of intestinal transporters is possible. Only one food additive had a higher IC50 value than the maximum expected concentration. Eight food additives, specifically six antioxidants and two colorants, inhibited CYP-enzyme metabolism by more than 50%. Based on the results of this thesis, further studies could be performed for the identified inhibitors whose daily consumption is higher than the IC50 value. Certain food additives may inhibit CYP enzymes and the microsome assay used in this thesis is valid and could be used to study the metabolism of intestinal drug-metabolizing enzymes. However, the inhibition of transporters and CYP enzymes could be tested in cell lines, for example Caco-2 cells, to have more realistic intestinal test conditions.
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(2024)Congenital heart diseases develop during heart development and encompass structural abnormalities in the heart present at birth, with hypoplastic left heart syndrome (HLHS) representing a rare but life-threatening subtype. HLHS is characterised by the underdevelopment of left-sided heart structures, resulting in a major blood flow obstruction of the heart, impairing systemic circulation. Current knowledge of HLHS aetiology is scarce, which makes the development of effective treatments challenging. Therefore, identifying the disease mechanisms causing HLHS is essential. Notably, HLHS is linked with mutations in the NKX2-5 gene, which encodes for a cardiac transcription factor and has a pivotal role in heart development together with the transcription factor GATA4. This makes these genes intriguing research targets in HLHS. This study aims to enlighten how HLHS patient-derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) differ from those derived from healthy donors in terms of stress response by subjecting hiPSC-CMs to pro-hypertrophic stimuli, namely endothelin-1 (ET-1) and cyclic mechanical stretching. Additionally, the effects of GATA4-targeted compounds on these hypertrophy models were also studied, which included two inhibitors (3i-1262 and 3i-1000) and one activator (3i-0777) of GATA4-NKX2-5 interaction. Differentiation of CMs was performed using a small-molecule induction protocol based on sequential Wnt pathway activation and inhibition. The effects of ET-1 and cyclic mechanical stretching were analysed by High-content analysis for pro-B-type natriuretic peptide (proBNP) expression, and quantitative PCR for hypertrophic gene expression, respectively. Both ET-1 and cyclic mechanical stretching effectively induced hypertrophy in their respective models. This was observed in all cell lines as a higher hypertrophic response of proBNP in ET-1 exposed hiPSC-CMs and upregulation of hypertrophic genes NPPA and NPPB in stretched hiPSC-CMs. GATA4-targeted compounds did not show statistically significant effects on ET-1-induced hypertrophy or stretching-induced hypertrophic gene expression in any cell line, but various trends could be distinguished. As expected, both inhibitor compounds, 3i-1262 and 3i-1000, showed a tendency for antihypertrophic effects since they decreased the percentage of proBNP+ cells in all cell lines. Unexpectedly, the activator compound 3i-0777 also decreased the percentage of proBNP+ cells. We also observed that HLHS-disease cell line HEL 149 seemed to differ from the three other cell lines showing a phenotype that exhibits similar gene expression patterns as seen in heart failure patients. This was mainly observed as a statistically significantly lower basal MYH6 gene expression. However, the limited experimental setup of this study requires further experiments to detect significant differences and draw definitive conclusions regarding the effects of GATA4-targeted compounds on hypertrophic stimuli.
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(2023)In pharmaceutical sciences the pharmaceutical supply chain is often examined from a quality perspective. As the world is becoming more uncertain due to pandemics and conflicts the societal and political situation where the supply chain operates should be considered. Understanding the big picture helps to consider the cause and effect that lead to medicinal shortages. Effects of these shortages can be seen on every level of the supply chain from the manufacturer to the patient, which is why actors on the supply chain can benefit from understanding the background factors. The aim of the master’s thesis was to examine, whether pharmaceutical field actors could affect realisation of geopolitical risks by preparation and examination that the pharmacotherapy would not be interrupted. Second aim was to bring forward political and societal aspects to pharmaceutical availability which are often side-lined by quality aspects in pharmaceutical context. The study was conducted as a qualitative semi-structured interview between October 2022 and February 2023. Participants (n=11) were recruited via e-mail using representative sampling. Due to recruitment problems, convenience sampling was also used. Questions presented to the interviewees were depending on the group (n=3) they were assigned. Term ”geopolitics” was associated mainly with political and economic factors. Main geopolitical risks for Finland were seen to be small market size and distant location. For Europe, the risks were centralisation of manufacturing (and dependence) to Asia due to economic factors and long disruption-prone supply chain. Transport of pharmaceuticals from Asia to Europe was with sea and air cargo. Inside Europe, transport to Finland was with mainly with lorries utilising ferries. Rail transport was mentioned to be used only on one interview. The transport routes were seen to be staying the same in the future both for Asia-Europe and Europe-Finland. Even though risk management is an important part of functioning of every company, the change in the type of risks requires a new mindset in the pharmaceutical field both from the individual actors as well as international organisations. From risk of strikes and natural disasters we have moved to trade wars, pandemic restrictions, and the strategic acting of industries critical to society. At the same time, the ability/willingness of societies to pay for pharmaceuticals is decreasing, which leads to the manufacturers to find new ways to ensure business.
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(2021)Dilated cardiomyopathy is a non-ischemic cardiac disorder predisposing to heart failure, and the characteristics of dilated cardiomyopathy emerge under normal loading conditions. Dilated cardiomyopathy can be consequence of various conditions e.g. genetic mutations, virus infection or toxin exposures. One of the significant causes of familial dilated cardiomyopathy in Finland is mutation S143P in LMNA-gene, coding for A type lamins. Current drug therapy for dilated cardiomyopathy aims to alleviation of symptoms, prevention of complications and progression of the disease, however, efficacy of current therapy is insufficient, and novel therapy strategies are urgently required. Transcription factors are fundamental regulators of gene expression, and GATA4 is a crucial transcription factor both in embryonic and in adult heart and thus an intriguing target for therapeutic manipulation. Compounds targeting GATA4 have shown anti-hypertrophic and cardioprotective effects. Here, effects of two different hypertrophic stimuli, endothelin-1 and mechanical stretch, on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were examined with high-content analysis and quantitative reverse transcription PCR (qRT-PCR), respectively. One hiPSC-CM line was used as a healthy control, whereas the other carried the S143P mutation in LMNA-gene (DCM-CMs). Additionally, effects of GATA4-targeting compound C-2021 on cardiomyocytes were investigated. In summary, according to proBNP staining, DCM-CMs are more hypertrophied at baseline. DCM-CMs seemed to be less susceptible to mechanical stretch-induced enhancement in BNP gene expression. In addition, compound C 2021 may have anti-hypertrophic properties suggesting it to be a potential drug candidate in cardiac diseases. Finally, lamin A seemed to mislocalize to nucleoplasm instead of nuclear lamina in DCM-CMs.
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(2022)Ischemic heart disease (IHD) and subsequent heart failure are caused by irreversible loss of contractile cardiomyocytes due to low oxygen supply to the heart. As the leading cause of death worldwide, IHD raises an urgent need for regenerative therapies that prevent or reverse loss of cardiomyocytes. The fetal mammalian heart grows by cardiomyocyte proliferation and utilizes glycolysis as main energy metabolism pathway, until it is introduced to increased oxygen and fatty acid supply at birth. Subsequently, cardiac energy metabolism shifts from glycolysis to β-oxidation of fatty acids and cardiomyocytes exit the mitotic cell cycle. Due to cessation of proliferation the heart can no longer regenerate after ischemic injury and responds to it by introduction of maladaptive pathological processes leading to heart failure. To gain deeper insight on the roles of cardiac metabolism pathways and hypoxia in cell cycle activation, we evaluated the effects of pharmacological metabolic modulation and oxygen supply on cardiomyocyte phenotype and hypoxia response. Furthermore, we studied the changes in the metabolic genotype of cardiomyocytes under alterations of oxygen supply. We utilized quantitative reverse transcription PCR (qRT-PCR) to evaluate the effects of hypoxia and metabolic maturation on the expression of genes involved in hypoxia signaling and metabolism of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Additionally, we investigated the effects of five metabolism-modulating compounds on cell cycle and phenotype of both metabolically matured and unmatured hiPSC-CMs, by utilizing high content analysis. We observed presence of hypoxia signaling as an increase in vascular endothelial growth factor A (VEGFA) expression following 3-hour hypoxic exposure. High expression of succinate dehydrogenase complex flavoprotein subunit A (SDHA) in hiPSC-CMs, which was downregulated at hypoxia, confirmed occurrence of oxidative metabolism induced by metabolic maturation. Surprisingly, metabolic maturation tended to increase proliferation and decrease stress response signaling of hiPSC-CMs. Introduction of the TCA cycle intermediate succinate decreased proliferation of metabolically unmatured hypoxic hiPSC-CMs by 8.2 %. Finally, inhibition of the mevalonate pathway and ketogenesis caused no alterations in hiPSC-CM phenotype or cell cycle, but introduction of the ketone body β-hydroxybutyrate tended to increase proliferation, supporting current evidence that ketogenesis plays a role in cardiomyocyte cell cycle regulation. Our observations suggest that hypoxic hiPSC-CMs can be useful in investigating gene expression and phenotype. Even so, additional methodologies are needed for in-depth evaluation of metabolic reprogramming and its effects on cardiomyocyte phenotype.
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(2023)Alcohol use disorder (AUD) is a chronic relapsing brain disorder causing a high burden of disease and significant social and economic consequences to both individuals and society. Alcohol addiction, the most severe form of AUD, is characterized by compulsive seeking and use of alcohol, loss of control over limiting alcohol consumption despite negative consequences, emergence of negative emotional states, and long-lasting vulnerability to relapse related to alcohol abstinence. Powerful craving for alcohol and the chronic, relapsing nature of the disease are major problems complicating recovery from alcohol addiction and predicting poor clinical outcome. Relapse to alcohol intake can occur even after an extended period of abstinence in humans, relapse rates being highest during the first three months of alcohol withdrawal. Associative learning is a critical factor in alcohol craving when alcohol consumption is accompanied by conditioned stimulus. Cues associated with alcohol are known to induce craving and alcohol-seeking behavior increasing the risk of relapse, and this craving can be triggered by alcohol itself, alcohol-associated stimulus, or stress. Chronic alcohol exposure has been linked to changes in synaptic plasticity, neurogenesis and cell-signaling. Thus, elucidating the neural mechanisms that underlie alcohol craving and relapse would help to understand the pathology of alcohol addiction and facilitate the development of efficient treatments. In this experiment, the effects of subanesthetic-dose 10 mg/kg ketamine, an NMDAR antagonist and a major inducer of synaptic plasticity, on cue-induced alcohol-seeking behavior after withdrawal were investigated in social context in female mice. Mice were trained to voluntarily drink alcohol, and a novel methodology to study alcohol-seeking behavior after withdrawal allowed to perform the experiment with a minimum of human interference in totally automated social home cage environment. The analyses of behavioral data showed that pairing sweetened alcohol with conditioned stimulus resulted in cue-induced alcohol-seeking behavior, and no differences in alcohol conditioning were observed between treatment groups. However, the behavioral activity in extinction tests after withdrawal showed that alcohol-seeking behavior was not altered by ketamine treatments. In biochemical analyses, the effects of subanesthetic-dose ketamine on ΔFosB and BDNF protein levels in the brain areas important for alcohol addiction were studied. ΔFosB expression levels in the mouse nucleus accumbens were analyzed with western blot and BDNF protein levels in the mouse prefrontal cortex were determined using enzyme-linked immunosorbent assay (ELISA). The results from biochemical analyses showed that levels of ΔFosB and BDNF were unaltered by ketamine treatments. Anyhow, the experiment provided important insights into the interactions of ketamine and alcohol craving and relapse, a topic that has been insufficiently studied in novel preclinical models.
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(2024)Parkinson’s disease (PD) is a progressive neurodegenerative disorder in which dopaminergic neurons in the substantia nigra (SN) degenerate and die. This causes multiple motor symptoms such as rigidity, bradykinesia and tremor and non-motor symptoms such as depression, hallucinations, and cognitive impairment. At the time of the diagnosis, approximately 60% of the dopaminergic cells can already be lost, which underlines the importance of neurorestorative treatments for PD. Currently used treatments are only symptomatic and mostly based on levodopa, which can lose its effectiveness as the disease progresses and additionally cause significant side effects such as dyskinesia. Neurotrophic factors (NTF) such as glial cell-line derived neurotrophic factor (GDNF) and neurturin (NRTN) have been studied in clinical trials with PD patients but have shown only modest effects on motor function. Additionally, they have been administered with invasive techniques such as intraputamenal or intracerebroventricular injections which includes many risks. Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to unconventional NTF’s with unique molecular structure and mode of action. MANF has shown both neuroprotective and neurorestorative properties for nigrostriatal dopamine system in in vivo study in rat model of PD. To enable systemic administration of MANF, the molecule has been modified by retaining only the C-terminal domain to form C-terminal MANF fragment (C-MANF). C-MANF has shown neurorestorative effects when administered intrastriatally in 6-OHDA lesioned rats and when injected subcutaneously (s.c.) in an ALS animal model. The aim of this study was to assess the effect of daily and weekly s.c administered C-MANF in 6-OHDA mouse model of PD. Cylinder and amphetamine-induced rotation tests were performed as behavioural tests and thereafter morphological studies were done by analyzing tyrosine hydroxylase (TH)+ cells in the substantia nigra pars compacta (SNpc) and optical density from TH+ axons in the striatum. Interestingly, weekly C-MANF treatment decreased the number of TH+ cells in SNpc and the density of TH+ fibers in the striatum compared to PBS. However, it decreased ipsilateral rotations and showed some positive effects in the cylinder test. On the other hand, daily C-MANF treatment increased the number of TH+ cells in SNpc and the density of TH+ fibers in the striatum but had a modest effect on ipsilateral rotations and the cylinder test compared to PBS. Although no statistically significant effects were observed in behavioral and morphological studies, s.c administered C-MANF presents a promising treatment option for PD. Particularly, daily administration of C-MANF showed neurorestorative effects in morphological studies; however, further research is required for validation. Additionally, the investigation of higher doses of C-MANF should be considered.
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(2016)Extracellular vesicles are cell-derived vesicles which consist of two lipid layers. Extracellular vesicles involve in intercellular communication, maintaining of homeostase and development of pathophysiological states in human body. Extracellular vesicles are promising biomarkers and drug carriers in future. The aim of this study was to develop a method based on time resolved fluorescence microscopy and autologous extracellular vesicles labelled with environmentally sensitive fluorescent probes for studying the distribution of mitose-inhibitor paclitaxel in prostate cancer cells (PC-3) carried by extracellular vesicles. The efficacy of paclitaxel loaded extracellular vesicles was compared to synthetic liposomes. The two subpopulations of extracellular vesicles, exosome -and microvesicle-enriched, were isolated from the PC-3 cell media by differential ultracentrifugation. The size distribution and particle concentration of extracellular vesicles was determined by nanoparticle tracking analysis. DSPC-Cholesterol liposomes were prepared by reverse-phase evaporation method and the size distribution of the liposomes was determined by dynamic laser diffraction and nanoparticle tracking analysis. Paclitaxel was loaded into the liposomes in hydration phase and into the extracellular vesicles by incubating vesicles and paclitaxel. Unbound paclitaxel was removed from samples by ultracentrifugation. The the dose-dependent sytotoxicity of paclitaxel loaded extracellular vesicles and liposomes was evaluated with Alamar Blue viability assay. The release and distribution of paclitaxel from extracellular vesicles in living PC-3 cells was investigated by confocal microscopy and time-resolved fluorescence microscopy. The exosomes had approximately 50 nm smaller diameter than microvesicles and exosome particle concentrations were significantly higher compared to microvesicles. According to viability assays conducted with wide range of concentrations, paclitaxel loaded in microvesicles were slightly more effective than paclitaxel loaded in exosomes. The time-resolved fluorescence microscopy was useful method for investigating the release and distribution of extracellular vesicle bound paclitaxel, since we succesfully detected changes in Paclitaxel-OregonGreen fluorescence lifetime in different phases of the drug delivery process. With confocal microscopy we detected that paclitaxel loaded extracellular vesicles were already uptaken inside the cells after two hours of incubation and after few hours, paclitaxel was detected in microtubules of PC-3 cells and killed PC-3 cells. Extracellular vesicles may improve the accumulation of paclitaxel into tumor cells thus preventing the side-effects of paclitaxel. Nevertheless, PC-3 cell derived extracellular vesicles have ability to increase the PC-3 cell viability, which limits their potential use as drug carrier due to safety issues. In addition, extracellular vesicles characterization and isolation methods lack standardization and the isolation of exosomes and microvesicles is impossible due to this fact. Extracellular vesicles involvement in physiological and pathophysiological states should be investigated throughoutly and their safety as drug carriers should be examined both in animal and human.
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(2013)γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and it's activity is mediated via metabotropic GABAB - and ionotropic GABAA receptors. Receptors containing αβγ subunit combination are localized in both synaptic and extrasynaptic sites and they mediate fast synaptic phasic activity. αβδ- and α5βγ-containing receptors reside only in extrasynaptic space where they regulate tonic inhibition of the nerve cell. Disruption in tonic inhibition may cause several diseases. Drugs that selectively affect extrasynaptic GABAA receptors are believed to help treatment of diseases like sleep disorder, neuropsychiatry disorders, epilepsy, cognition impairment and recovery from stroke. Drug development of δ- selective GABAA agonists and positive modulators that enhance tonic inhibition as well as α5βγ selective inverse agonists that reduce tonic inhibition and enhance cognition are under investigation right now. Muscimol is a psychoactive molecule which activates all GABAA receptors but has higher affinity to cerebellar granule cell-specific receptor subtypes α6β2γ2 and extra synaptic α6β2δ than to the most common receptor subtype α1β2γ2. However, the binding of [3H]muscimol has produced contradictory results in former studies. Binding to membrane homogenates results to binding levels of the same magnitude in most brain regions, while in receptor autoradiography the high affinity binding is δ-subunit dependent. The affinity of muscimol to GABAAR subtypes has thus far been determined using saturation analysis, i.e. by measuring concentration-dependent binding of [3H]muscimol at equilibrium and by determining KD, the dissociation constant of the ligand. However, this value expresses the affinity of the ligand to the receptor, but does not give any information on the rate of the association and dissociation. In this master's thesis I have investigated association and dissociation rate of [3H]muscimol from recombinant GABAA receptors and from native GABAA receptors present in wild-type (WT) and δ-subunit knock-out (δKO) mice forebrain and cerebellar membranes. We concluded from binding assays that [3H]muscimol dissociates extremely slowly from δ-receptors. This explains further contradictory results: in membrane/filtration assay the washing procedure is generally much faster resulting in only low dissociation from αβγ receptors, while in the long washing procedure of receptor autoradiography only αβδ-binding is retained. The high affinity of muscimol to αβδ receptors is suggested to be due to its extremely slow dissociation from these receptors. Also association of [3H]muscimol to αβδ receptors seems to be slower, but this needs confirming studies. The exceptional binding properties of muscimol make it an interesting leading molecule in development of drugs which act via extrasynaptic GABAA receptors.
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(2021)Lääketieteen kehittyessä yksilöllisen lääkehoidon tarpeeseen on kiinnitetty enemmän huomiota kuin aikaisemmin ja etenkin lapsille lääkkeiden tarkka annostelu on erityisen tärkeää. Kaupallisilla valmisteilla tarpeeksi pienet annokset eivät usein ole mahdollisia eikä tablettien puolittaminen takaa tarkkaa lääkkeiden annostelua. 3D-tulostamista on ajateltu mahdollisena vaihtoehtona ex tempore -lääkkeiden tuotantoon ja sen mahdollisuuksia on tutkittu laajalti viime vuosien aikana. Tämän tutkimuksen tavoitteena on selvittää, miten ekstruusiomenetelmällä tulostetut varfariinikalvot vertautuvat sairaala-apteekin käyttämiin varfariiniannosjauheisiin, sekä olisiko kyseistä menetelmää mahdollista hyödyntää sairaala-apteekeissa. Tutkimuksessa valmistettiin puolikiinteän aineen ekstruusiolla 0,1 mg:n, 0,5 mg:n ja 2 mg:n varfariinikalvoja, jotka kuivattiin 85 ℃:ssa valmistusprosessin nopeuttamiseksi. Kalvoja verrattiin saman vahvuisiin varfariinia sisältäviin sairaala-apteekin valmistamiin annosjauheisiin. Kalvoissa käytettiin hydroksipropyylimetyyliselluloosaa kalvonmuodostaja-aineena ja glyserolia tuomaan plastisuutta. Annosjauheet koostuivat kaupallisesta 5 mg:n Marevan-valmisteesta ja täyteaineena käytetystä laktoosista. Molemmista lääkevalmisteista mitattiin liukenemisnopeus ja annosyksiköiden yhdenmukaisuus. Molempien valmisteiden toimivuus nenä-mahaletkussa tutkittiin myös, sillä kalvojen on tärkeää soveltua erilaisille potilasryhmille. Kalvot olivat kovia, mikä aiheutti niiden hitaan liukenemisen. Puolikiinteän aineen valmistus ja tulostuksen toteuttaminen tavoitteiden mukaisesti osoittautui oletettua vaikeammaksi. Kalvoissa mitattiin annosjauheita tasaisempi lääkeainepitoisuus. Molempien lääkevalmisteiden kohdalla huomattiin, että kaikki varfariini ei pääse nenä-mahaletkujen läpi. Tärkein huomio oli, että hyvin yksinkertaisella formulaatiolla on mahdollista tuottaa lupaavia lääkevalmisteita. Tämä tutkimus esittelee syitä, joiden vuoksi 3D-tulostusta on hyvä tutkia mahdollisena ex tempore -valmistuksen menetelmänä.
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(2009)Eläinlääkkeiden kehitys kokee nykyaikana huomattavia muutoksia. Perinteisesti 70 % eläinlääkkeistä on ollut tarkoitettu tuotantoeläimille, mutta lemmikkieläinten lääkkeiden kulutus kasvaa jatkuvasti. Transdermaalisen antotavan tärkeitä etuja ovat maksassa tapahtuvan LA:n alkureitin metabolian vähentäminen, noninvasiivisuus ja eläinten omistajan hoitomyöntävyyden parantaminen. Usein kuitenkin eläimille joudutaan käyttämään ihmiselle tarkoitettuja lääkkeitä sopivien eläinlääkkeiden puuttumisen takia Meloksikaami on oksikaamiluokkaan kuuluva, alun perin ihmiskäyttöön kehitetty, tulehduskipulääke jonka indikaatio 1990 luvun lopussa laajennettiin eläinkäyttöön. Tutkimuksen tarkoituksena oli kehittää meloksikaamin preformulaation, jonka in-vitro transdermaalinen virtausnopeus on riittävän korkea meloksikaamin transdermaaliseen annosteluun sialle. Formulointi strategioiksi valittiin keräliuottimien, meloksikaamin liukoisuutta sekä ihon penetraatiota parantavien aineiden käyttö. Formulatioiden perimiaatiokokeita suoritettiin sian ihon preparaattien ja Franz diffusiosolujen avulla, joissa luovuttajakammiossa oli tutkittava formulaatio ja vastaanjottajakammiossa - PB 7,4/saliiniliuos. Sian ihon preparaatti oli valmistettu sian korvan ihosta. Valittujen keräliuottimien ja penetraatiota parantavien aineiden käyttö todettiin tehottomaksi keinoksi nostamaan transdermaalista virtausnopeutta. Tutkimuksen päämäärän saavuttamiseksi päätettiin syntesoida ja kokeilla Enhancer-S:ksi nimitettyä ainetta, joka sekä nostaa meloksikaamin liukoisuutta formulaatiossa että vaikuttaa ihon permeabiliteettiin. Permiaatiokokeiden jälkeen todettiin Enhancer-S apuaineen lisäävän meloksikaamin transdermaalista virtausnopeutta 22 µg/(h·cm2) asti. Todettu meloksikaamin virtausnopeuden taso riittää ylläpitämään 21,5 mg:n vuorokausiannoksen formulaation pinta-alan olevan 41 cm2 (6,4 cm x 6,4 cm).
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(2017)Increasing number of companion animals and market for companion animal drug products will bring about new challenges to the companion animal drug product marketing. Thus animal health companies have to rethink determinants of companion animal drug launch success so that their products could differ from other similar products. The aim of this study was to examine if the dominant approach of the new product launch (product-centered-, traditional sales and marketing- and strategic orientations approach) also more popular than relationship approach in the animal drug product marketing. The approach was chosen because big differences exist between the animal- and the human drug marketing. In addition it was researched how the recent changes of the animal health field has been effected to animal drug product marketing. The study was addressed to animal health marketing experts in Finland, the United Kingdom, Germany, France, Spain and Italy by electronic questionnaire. A previously generated questionnaire was employed. It was used after slight modifications and repiloting. According to the study it was found that there is dominating the same procedures as generally in industries in the launch of veterinary drug products. However, relationship approach, which emphasizes the importance of customer relationships, is quite important in the launch of a new veterinary drug product. Based on the results, relationship approach complements dominant approach in the launch of veterinary drug product. Based on answers given to the open-ended questions, veterinary drug companies consider current chances positive and aim to take advantage of the changes. Based on this study at least the senior management of veterinary drug companies seems to be aware the importance of relationship activities. From the results it can be seen that the relationship activities have not been utilized in the launches as much as it would be potential. Veterinary drug companies lean too much on that the company's products will be succeed with very low relationship activities because veterinary drug market is small and they have much less competitors than the human drug companies has. In addition there is working primarily high experienced people in the veterinary pharmaceutical industry, which has an impact that marketing practices are not updated easily.
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(2019)Despite the long history of skin grafting, there is no standardized treatment for split-thickness skin graft donor sites. These sites cause a notable amount of pain and discomfort to the patients and open wounds also introduce a risk for infection. There is an extensive need for treatment options promoting the fastest and least painful healing possible while also being infection-free. The treatment of split-thickness skin graft donor sites is constantly studied and there is plenty of scientific literature available about this topic. In the theory section of this Master’s thesis, the structure of skin, the process of wound healing, skin grafting surgery and wound care products for split-thickness skin graft donor sites are briefly introduced. Additionally, the method of systematic review is described. In the empirical section, a systematic review is performed to compare animal- and non-animal-based wound care products in the treatment of split skin graft donor sites. The methodological quality of the included studies is reviewed. In the literature search, 3552 references were found. In this systematic review a total of 23 articles were included comprising of 21 comparative clinical studies and two previous literature reviews. Of the original studies, 20 reviewed healing, 14 infection and 17 pain of the split-thickness skin graft donor sites. Based on the results of the systematic review, animal-based wound care products might promote healing and reduce pain experienced by patients in the treatment of split-thickness skin graft donor sites when compared with non-animal-based wound care products. The results concerning infection were inconsistent. Generally, the reporting of the clinical original studies was not comprehensive enough for proper evaluation of methodological quality. Some defects, mostly in the blinding of the patients, study personnel and the assessors of outcomes, were also found. Moreover, the studies were heterogeneous in their definitions and measuring of the reported outcomes. Therefore, there is substantial uncertainty in the results of this systematic review. The systematic and transparent way of conducting the literature search, the review of the methodological quality and the reporting of the outcomes can be considered as a strength of this thesis. The main weakness is, that only one person performed the critical steps of this study, which might increase the risk of bias and reduce the repeatability of the study.
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(2022)There is a growing need for antibiotic stewardship since antibiotic resistance is a global and increasing problem. One option would be outpatient parenteral antibiotic therapy (OPAT) which has evolved globally since 1970s. In Helsinki, it has been applied in hospital-at-home units since 2018 with elastomeric antibiotic infusion pumps that enable 24-hour continuous infusion and normal daily life for the patient. The continuous infusion via infusion pumps enables the use of first-line antibiotics whereas with intermittent infusions broad-spectrum antibiotics, that require doses less frequently, are a more likely choice. Thus, antibiotic therapy with elastomeric infusion pumps is likely to enhance antibiotic stewardship. The aim of the study was to analyse if treatment with elastomeric infusion pumps in hospital-at-home unit is cheaper than theoretical hospital stay and to compare the costs. An economic evaluation was performed with the assumption that hospital-at-home care and hospital stay are equal when it comes to the outcomes of the therapy. The economic evaluation was made with cost-minimization analysis. Data were collected manually by nurses in three hospital-at-home units in Helsinki between September 2021 and March 2022. Patients’ age, gender, indication and length of the antibiotic infusion pump therapy, distance from the hospital-at-home unit and problems with the therapy were collected. Cost information were received personally from City of Helsinki and taken from a paper of Finnish Institute for Health and Welfare. The data included 57 patients, of whom one had two treatment periods. The mean age was 60 years. Thirty-two percent of patients were female and 68% were male. The most common indications were bacteremia (n=24) and erysipelas (n=18). A total of 625 hospital bed days were saved, which is 10,8 days per patient on average. Cost savings with elastomeric infusion pump therapy were 89 000–116 000 euros compared to the theoretical treatment in a hospital ward depending on the cost information being used, which is 37–48% of the theoretical hospital stay costs. An economic evaluation was made separately for the treatment of bacteremia. The cost savings were 47 600–150 700 euros or 37–69% of the theoretical costs. Savings in travel costs were 2 300–3 800 euros when elastomeric pump therapy was compared to the conventional hospital-at-home intermittent infusion therapy of 4-6 nurse visits per day per patient. In conclusion, elastomeric infusion pump therapy in hospital-at-home units in Helsinki results in cost savings of 37–48% compared to theoretical hospital stay costs from the perspective of the entity responsible for the costs of the treatment.
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(2019)Literature review part: The enteric nervous system (ENS) often called “the second brain” is considered its own autonomic division that can independently regulate gut function. The ENS is derived from enteric neural crest-derived cells (ENCCs), which colonize the gut during development. Development of the ENS is a complex process, and many signalling pathways are required for a properly functioning ENS, especially GDNF/Gfrα1/RET signalling controlling survival, proliferation, migration, and differentiation of ENCCs. Hirschsprung’s disease (HSCR) is the most common congenital disease affecting gut motility. The prevalence of HSCR is 1:5000, and it is characterized by a complete lack of enteric neurons (aganglionosis) in the distal colon. Due to impaired intestinal motility, infants may have constipation, emesis, abdominal pain or distention, and, in some cases, diarrhea. The most life-threatening symptom is HSCR-associated enterocolitis (HAEC), which occurs in 30-50% of patients. Routine treatment for HSCR is a surgical operation called “pull through” in which the aganglionic segment is removed, and the remaining ganglionic segment is joined to the anus. However, the risk of developing HAEC after successful surgery still exists. Histopathological analysis has revealed that HAEC is accompanied by various changes in the gut epithelium, especially in mucin-producing goblet cells. These changes include hyperplasia of the goblet cells, altered mucin profile, retention of mucin, damaged and disorganized epithelium structure, inflammation, and bacterial adherence to the epithelium. However, a lack of suitable postnatal HSCR mouse models has partially hindered the progress of pinpointing the exact order of these events. A RET mutation found in half of the patients is overwhelmingly the biggest risk factor for HSCR. RET is a receptor on the cell membrane that mediates the effects in GDNF/Gfrα1/RET signaling pathway. of Knock-out mice of Gdnf, Gfra1 and Ret all have intestinal aganglionosis, resembling HSCR. However, to date, no mouse models of HSCR affecting GDNF/Gfrα1/RET signalling exist because pups are born without kidneys and die soon after birth. Experimental part: The GFRa1 hypomorphic mouse line (Gfra1hypo/hypo) created by Dr. Jaan-Olle Andressoo is the first successful model that survives past birth while manipulating GDNF-Gfrα1-RET signalling and phenocopying HSCR. These mice have 70-80% reduction in the expression of Gfrα1 in the developing gut and kidneys, which is sufficient to cause aganglionosis in the distal colon, yet not enough to impair kidney development.These mice are sacrificed between P7-P25 because of welfare problems yet giving a time window for analysis of the development of HAEC. Histological analyses revealed that Gfra1hypo/hypo mice had goblet cell hyperplasia and a shift away from acidic mucin production in the distal colon. Goblet cell hyperplasia was first observed at P10, but the shift in mucin profile already appeared at P5. It is not known what causes goblet cells to change their mucin production, but it seems to be the earliest histopathological change in HAEC preceding goblet cell hyperplasia. qPCR-analysis revealed that Muc2, the main secreted mucin that protects epithelium from invading pathogens, was upregulated at both P5 and P10. mRNA levels of Tnfa were also upregulated at P10. The aforementioned changes were not observed in the duodenum where the ENS had developed normally despite the reduction in Gfra1 expression. This indicates that the changes observed in the colon are likely due to the lack of ENS innervation, rather than a direct effect from GDNF-GFRa1-RET signalling itself. Finally, serum analysis indicated that systemic inflammation did not occur from P10-P16, although one Gfra1hypo/hypo animal had high levels of IL6 and TNFa at P14-16. This indicates that inflammation is not an early stage event and it is preceded by goblet cells related changes. In conclusion, changes in goblet cells seems to be earliest histopathological findings preceeding HAEC.
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(2013)Amphetamine and its derivatives are widely used as medicines but also abused as psychostimulant drugs. The most important action of amphetamine in the central nervous system is to release dopamine to the extracellular space which leads to enhanced dopaminergic neurotransmission. Amphetamine also releases serotonin and norepinephrine by similar mechanisms and it affects indirectly other neurotransmitter systems too. It still remains partly unsolved how amphetamine exactly releases monoamines but it is known to have multiple sites of action. Amphetamine is a substrate for dopamine transporter (DAT) and it acts as a competitive inhibitor of the transporter reducing uptake of dopamine. Amphetamine enters the cell mainly through DAT and partly by diffusing through the cell membrane. The drug induces changes in DAT leading to reverse transport of dopamine from the cytoplasm into the synaptic cleft through DAT. Amphetamine is also substrate for vesicular monoamine transporter 2 (VMAT2) preventing the uptake of dopamine into storage vesicles and promoting its release from the vesicles to cytoplasm. Additionally, amphetamine inhibits monoamine oxidase (MAO), enzyme which degrades monoamines. It also enhances dopamine synthesis and according to recent studies amphetamine augments exocytotic dopamine release. Drug addiction is a chronic disorder related to structural and functional adaptive changes of neurons, called neuronal plasticity. GDNF (glial cell line-derived neurotrophic factor) is one of the many molecules regulating plasticity. It is especially important to the dopaminergic system and some investigations have suggested that it has potential as a protective agent against addiction. The aim of this study was to investigate how the overexpression of endogenic GDNF affects dopaminergic system and how it changes drug responses. A hypermorphic mouse strain (GDNFh), which is overexpressing physiological GDNF, was used. Their wild-type littermates were used as controls. Using brain microdialysis it was measured how the extracellular dopamine concentration changes in striatum and nucleus accumbens (NAcc) after amphetamine stimulation. Amphetamine was administered straight to the brain through the microdialysis probe. Microdialysis was performed on days 1 and 4, and on days 2 and 3 the mice were given amphetamine intraperitoneally. This was done to find out if the response to amphetamine changed after repeated dosing. In addition to these experiments, the biological activity of three small-molecule GDNF mimetics in intact brains was tested by microdialysis. On the first day amphetamine increased striatal dopamine output more in the heterozygous GDNFh mouse than in the wild-type mice. This stronger reaction to amphetamine may be explained by the enhanced activity of DAT in the GDNFh-het mice leading to higher intracellular amphetamine concentration. Also the striatal dopamine levels are increased in the GDNFh-het. On the fourth day no differences were detected between the genotypes. In the NAcc no significant difference was found between the genotypes. Instead in NAcc amphetamine caused a smaller increase in the dopamine output on day 4 than on day 1 in both genotypes suggesting that tolerance was developed. These results confirm that endogenic GDNF has a remarkable role in the regulation of the dopamine system and hence addiction but further investigations are needed to clarify its versatile actions. The small-molecule GDNF mimetics increased striatal dopamine output thus showing biological activity and encouraging to further investigations.
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(2020)Lääkevaihto ja sitä täydentävä viitehintajärjestelmä ovat laskeneet lääkekustannuksia Suomessa. Epilepsialääkkeet eivät ole aiemmin kuuluneet lääkevaihdon piiriin, sillä epilepsian hoidossa eri valmisteet eivät välttämättä ole terapeuttisesti tarpeeksi samanarvoisia, ja pienikin muutos hoitotasapainossa voi altistaa epilepsiakohtauksille. Nykyisin epilepsialääkkeitä käytetään kuitenkin usein muihinkin käyttöaiheisiin, kuten psykiatrisiin sairauksiin ja kivun hoitoon. Vuonna 2017 lääkekorvausjärjestelmään tehtiin säästötoimenpiteitä, joiden yhteydessä epilepsialääkkeet sisällytettiin lääkevaihdon piiriin muissa käyttöaiheissa kuin epilepsian hoidossa. Lisäksi otettiin käyttöön poikkeava viitehintaryhmä, joka koski epilepsialääkkeistä pregabaliinia neuropaattisen kivun käyttöaiheessa. Tutkimuksen tavoitteena oli tarkastella epilepsialääkkeiden (pregabaliinin, gabapentiinin, topiramaatin, lamotrigiinin ja valproiinihapon) vaihtamista sekä hintojen kehitystä lääkevaihtoon ja viitehintajärjestelmään sisällyttämisen jälkeen vuoden 2017 alusta vuoden 2019 puoliväliin. Lisäksi tarkasteltiin näiden lääkeaineiden kustannusten, korvausmenojen sekä käyttäjä- ja reseptimäärien kehitystä. Aineistona käytettiin Kansaneläkelaitoksen reseptirekisteriin pohjautuvia tilastoja epilepsialääkkeiden lääkeostoista sekä lääkkeiden hintalautakunnan päätöksiä epilepsialääkkeiden viitehintaryhmistä ja viitehinnoista. Epilepsialääkkeiden vaihtaminen yleistyi tarkastelujakson aikana kaikilla lääkevaihdon piirissä olleilla viidellä lääkeaineella, ja vaihtokieltojen osuus resepteistä laski useimmilla lääkeaineista. Viitehinnat laskivat useimmissa tarkastelluista viitehintaryhmistä, mutta lähes yhtä usein viitehinta ei muuttunut. Viitehinnat laskivat enemmän viitehintaryhmissä, joissa oli useampia vaihtokelpoisia valmisteita. Lääkevaihdon ensimmäisenä vuonna 2017 lääkevaihtoon kuuluvien epilepsialääkkeiden kustannukset ja korvausmenot pääosin laskivat, vaikka lääkkeiden käyttö ei vähentynyt. Lääkevaihdon toisena vuonna kustannukset eivät juuri laskeneet. Pregabaliinin poikkeavan viitehintaryhmän vuoksi vaihtamatta jääneet reseptit aiheuttivat merkittävän osan lääkevaihtoon kuuluvien epilepsialääkkeiden kustannuksista. Pregabaliinille jäi siten todennäköisesti yhä säästöpotentiaalia poikkeavan viitehintaryhmän voimassaolon päätyttyä vuoden 2019 heinäkuussa, mitä on syytä tarkastella jatkotutkimuksissa.
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(2020)Erenumab (Aimovig®) is a first-in-class calcitonin gene-related peptide (CGRP) inhibitor approved for the preventive treatment of migraine by the FDA in May 2018 and by European Commission (EC) in July 2018. It is a human monoclonal antibody (mAb) binding to the CGRP receptor, antagonizing the effect of CGRP. The marketing authorization of Aimovig® was based on two phase II and two phase III clinical trials. In all trials, erenumab with doses 70 mg/mL and 140 mg/mL was found to have a significantly superior effect compared to placebo, with a similar safety profile between all groups. These conclusions are mainly in line with studies conducted post marketing authorization. However, questions about the optimal dose, and the frequency and types of adverse events in larger patient populations remain to be studied. A European Public Assessment Report (EPAR) and Summary of Product Characteristics (SmPC) are required by the European Commission for each human medicine with a marketing authorization within the European Union. The SmPC is produced by the applicant and it should contain all relevant information of the medicinal product as distilled during the assessment process. The SmPC can thus be viewed as a kind of summarized version of the EPAR. The aim of this study was to investigate the post-marketing efficacy and safety information of erenumab from three perspectives: 1) the EPAR was compared with recent systematic reviews and meta-analyses assessing the efficacy and safety of erenumab, 2) all existing literature on the efficacy and safety of erenumab on different subgroups of migraine patients was assessed and summarized, and 3) the efficacy and safety information of the EPAR was compared to those of the SmPC, to resolve whether important information is missing. This review found several points regarding the efficacy and safety of erenumab. First, the status of erenumab was further established as a safe and effective treatment for the prevention of migraine. Second, meta-analyses (n=3) with more extensive cohorts compared to those of the EPAR and SmPC, present a further case for the superiority of the 140 mg dose compared to the 70 mg dose. The difference in dose effect is addressed in the EPAR but its assessment may be based on limited information. Third, different subgroups seem to respond differently to erenumab treatment. This aspect should be further investigated by head-to-head studies. Lastly, the safety information of the SmPC seems insufficient due to lack of mention of upper respiratory infections. This adverse event was among the most common in all of the four clinical trials and has since been observed in a real-world study. Based on these findings, neither the EPAR nor the SmPC of erenumab seem to be fully up to date and information related to the dose and upper respiratory infections as a risk should be reconsidered.
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(2010)The objective of the research was to study the effects of different polymers, sugars, and cell handlings on the viabilities of ARPE-19 and ARPE-19-SEAP-2-neo cells after freeze-drying. Also the residual moisture content of the freeze-dried samples and the amount of intracellular sugar after incubation in trehalose medium were measured. The mixtures used in the study contained different polymers (e.g. polyvinylpyrrolidone, alginate, polyethylene glycol) and sugars (sucrose, trehalose and mannitol). Some cells were incubated or heated in trehalose medium before freeze-drying in order to increase the amount of intracellular sugar. The aim of the heating was also to increase the heat shock protein formation in the cells. The samples were mainly freeze-dried in 24 well plates. The same freeze-drying parameters were used in all freeze-drying runs. The freeze-drying cycle lasted 38.5 hours (freezing 2.5 h, primary drying 32 h, and secondary drying 4 h). In the freezing stage the samples were froze to -40 °C and the freezing rate was 1 °C/minute. In the primary drying stage the shelf temperature was mainly -35 °C and the pressure was 150 mTorr. The viabilities of the samples after freeze-drying were determined by measuring the fluorescence after 3 and 6 hours from addition of the Alamar Blue indicator dye. The residual moisture contents were measured by thermogravimeter. According to the results, mixture containing glycerol (9%) and PEG 10 000 (18%) was the best lyoprotectant in the study (70% viability after 3 hours). However, the viability decreased significantly (24% viability) in the measurement after 6 hours. Similar viability decrease was observed among all lyoprotectant mixtures used in the study. Extracellular sugars rarely had positive effect on the viability results. The 12 days incubation in 150 mM trehalose medium before freeze-drying affected positively to the post freeze-drying viability. Shorter incubation time or heating did not induce the same effect. Intracellular sugar measurements revealed, that the amount of intracellular trehalose was multiple after 12 days of incubation in 150 mM trehalose medium compared to the cells that were not incubated. The residual moisture contents of the samples varied between 0-7%. The residual moisture content of the sample containing glycerol 9% and PEG 10 000 18% was 1,5%.
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