Faculty of Pharmacy

 

Recent Submissions

  • Luhtanen, Päivi (Helsingin yliopisto, 2020)
    The pharmacy operations are strictly regulated in Finland and the operation of a pharmacy business requires a licence. Number of community pharmacies has stayed quite steady for the past 10 years. At the end of the year 2019 there were 817 pharmacies or their subsidiaries in Finland. The number of pharmacies is expected to increase, since 29 new pharmacies has been established since 2016. The inspection of pharmacies is a part of the legal duties of the Finnish Medicines Agency. In Finland, pharmacies are inspected based on a risk assessment, as often as it is necessary to ensure appropriate operations of a pharmacy. During a pharmacy inspection, the focus is on operations that are critical to drug safety and medication safety. The aim of an inspection is to make sure that pharmacy operations comply with the regulations. There are only few studies made on remote inspection of the pharmacies. The Finnish Medicines Agency hasn’t made remote pharmacy inspections before. The aim of this study was to develop, validate and test a questionnaire, which could be used to inspect community pharmacies remotely and to develop and test a process for remote inspections. The study was done in two parts. In the first part, a draft of the questionnaire was developed by studying the regulations and laws regulating the operations of a pharmacy and by using a content analysis. The material for the content analysis was a pre-inspection questionnaire form, answers to the pre-inspection questionnaires and defect lists of the inspection reports of those pharmacies (n=37), which had answered to the pre-inspection questionnaire before pharmacy inspection in 2019. Content of the pre-inspection questionnaire and the answers of pre-inspection questionnaire were compared to the content of the defect lists of inspection reports. The aim of the comparison was to find out how the existing pre-inspection questionnaire could be utilized when developing the questionnaire for the remote pharmacy inspections. In addition, the listed defects of the inspection reports were categorized to explore what were the most common defects observed during pharmacy inspections. In the second part of the study, the content of the developed questionnaire was validated by using a three round modified Delphi survey. Seven experts with good knowledge of the pharmacy inspections were chosen to the Delphi panel. The aim of the Delphi rounds was to achieve full consensus among the experts about the content of the questionnaire. Alongside the Delphi rounds, a process to remote inspect a pharmacy was developed. The remote inspection questionnaire and the process were tested internally in the Finnish Medicines Agency at the end of the second stage of study. The draft of the questionnaire included 15 sections and 164 questions. Based on the comments received during the Delphi rounds, the content of the questionnaire was modified. On the third Delphi round a full consensus of the content of the questionnaire was achieved among the experts. The final questionnaire for the pharmacy remote inspection included 14 sections and 184 questions. The process of the remote pharmacy inspection follows the procedure of an on-site pharmacy inspection. In the internal test, the process of the remote inspection was found to be a good way to inspect pharmacies remotely. The remote inspection process is a new way to inspect pharmacies. With the remote inspection, it is possible to find out the most common defects on the pharmacy operations by using the questionnaire and contact calls. The remote inspection questionnaire and the process need to be further tested to ensure that the process is optimal from the perspective of the authority and the pharmacies.
  • Reunanen, Saku (Helsingin yliopisto, 2020)
    Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic neurons that form the nigrostriatal pathway gradually die. This causes the main motor symptoms of Parkinson’s disease: tremor, rigidity and bradykinesia. While PD affects 1-2% of total population, all currently used medicines are symptomatic, and there is no disease modifying therapy available at present. Although several different animal models for Parkinson’s disease exist, the lack of adequate animal models is often cited as a major obstacle for predicting the clinical success of potential drug candidates. Lewy bodies (LBs) are abnormal aggregates that develop and spread inside nerve cells of human PD patients, their main structural component being α-synuclein. Because α-synuclein is thought to play a major role in the pathology of PD, much research has been focused on it. Different α-synuclein-based animal models of PD exist today, of which the most recent are based on using direct injections of preformed α-synuclein fibrils (PFFs). These new α-synuclein based disease models have helped to understand the disease process in PD better, but cell death in these models takes longer to achieve and is often less pronounced compared to traditional neurotoxin based animal models of PD. The aim of this study was to participate in the development and characterization of a novel mouse model of PD. This new model combines PFF-injections with the commonly used neurotoxin 6-OHDA, which should result in more robust dopamine pathway degeneration than what is seen with the current PFF-based models. The main hypothesis of this study was that the combination of intrastriatal injections of PFFs and a low dose of 6-OHDA would cause gradual spreading of the α-synuclein aggregation pathology in the nigrostriatal dopamine pathway and progressive dopamine neuron loss leading to motor deficits. C57BL/6 mice were stereotactically injected unilaterally with both PFF and 6-OHDA, and their performance was assessed every other week with different behavioral tests until week 12. At the end, brains were collected and optical density of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was measured from striatal sections, and TH and DAT positive cells in the substantia nigra were counted. The amount of Lewy bodies present in the brain slices was also counted from the cortex and substantia nigra areas of the brain. In the histological assays, statistically significant reductions of both TH and DAT were found in the brain sections of the PFF + 6-OHDA combination group and the amount of TH and DAT positive cells were lower in this group compared to the group receiving vehicle treatment only. However, the results of behavioral tests were non-significant, although a non-statistical positive trend in the amphethamine-induced rotations test was observed where mice receiving PFF + 6-OHDA rotated the most. Taken together, combination model that utilizes both PFF and 6-OHDA injections seems like a promising candidate in modelling PD in mice, but much more research and further development of the model is required before this combination model is ready and robust for use in drug development.
  • Tallberg, Thomas (Helsingfors universitet, 2017)
    Transactive DNA Response Element Binding Protein 43 (TDP-43) is a RNA binding protein participating in gene expression on a transcriptional level. It is localized in the cell nucleus. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons. In most ALS patients TDP-43 becomes localized into the cytoplasm of neurons and glia cells. The TDP-43 rat ALS model provide insight in ALS disease progression and molecular mechanisms. This animal model has been characterized previously in the literature. Cerebral Dopamine Growth Factor (CDNF) is a neuroprotective and restorative protein in rat animal model of Parkinson's disease. CDNF may have an impact on disease progression in ALS. One of the goals in this work was to recharacterize the TDP-43 rat ALS model and to try repeat published data. The other aim of this work was to treat TDP-43 rats with intraventricular chronic infusion of CDNF, and to compare symptom progression with TDP-43 rats treated with phosphate buffered saline. Behavioral assays were done trice a week and when rats reached endpoint, spinal cords were removed. Motor neuron counting and detection of stress granule formation were investigated in spinal cords with immunohistochemistry. Also, the volume of CDNF diffusion in rat brain after chronic intraventricular CDNF infusion was investigated with immunohistochemistry. In the characterization part, symptom progression was repeated in a similar manner as it has been reported previously. CDNF treatment could not stop the symptom progression nor slow down the progression of symptoms in TDP-43 rats. Motor neuron counting revealed a heavy loss of motor neurons in the lumbal part of the spinal cord in both treatment groups. Diffusion of CDNF was very poor in the rat brain. Higher doses of CDNF and proper administration depth in the brain or route of administration should be reconsidered in the future.
  • Ala-Kurikka, Tommi (Helsingfors universitet, 2013)
    Laminins are a family of heterotrimeric glycoproteins found mainly in basement membranes. They interact with numerous other extracellular matrix components and cell surface receptors, including integrins and α-dystroglycan. Laminins play roles in myriad of functions including tissue morphogenesis, organogenesis, maintenance of tissue integrity and compartmentalization. In central nervous system laminins are involved in every major developmental stage from neural tube closure to synaptogenesis. Laminin expression in central nervous system decreases after maturation but has been found inducible by injury after trauma or disease. Since laminins are known to promote neurite outgrowth and neuronal survival, this has been proposed as a regenerative response to injury. Although the effects of endogenous laminin are clearly inadequate for repair, laminin based compounds could be powerful therapeutic agents. In previous in vivo studies KDI-tripeptide, a neurite outgrowth promoting fragment from γ1-laminin, has proved effective neuroprotective and regeneration promoting compound. Encouraged by these results I set out to test whether KDI would rescue midbrain dopaminergic neurons in unilateral 6-hydroxydopamine-induced rat model of Parkinson's disease. KDI (1-30µg) was injected to the striatum six hours prior to 6-hydroxydopamine. The severity of the lesion was then evaluated by measuring D-amphetamine induced rotation 2, 4 and 6 weeks postlesion and by assessing the number of neurons in substantia nigra pars compacta and optical density of striatum after tyrosine hydroxylase immunostaining at week seven. The only effective KDI dose studied was 3 µg. Compared to control it decreased Damphetamine induced rotational behaviour significantly at week four. KDI, however, failed to save tyrosine hydroxylase positive dopaminergic neurons in substantia nigra pars compacta or their axons in striatum. KDI might be usable in treating Parkinson's disease but it's mode of action doesn't appear to rely on protecting dopaminergic neurons or promoting the branching of their axons. KDI is known to inhibit ionotropic glutamate receptors and could therefore improve motor function by opposing striatal denervation induced overactivity of glutamatergic subthalamic nucleus neurons.
  • Heine, Sari (Helsingfors universitet, 2014)
    Obesity is a significant problem for public health. Obesity develops when systems controlling food intake and consumption are imbalanced. Many different brain areas and transmitters contribute to maintain energy balance. Signals that are secreted proportional to body's fat storage (leptin and insulin) regulate energy balance in a long run. Hormones that are secreted from gastrointestinal tract control food intake in a short run. These hormones are for example cholecystokinin, peptide YY and ghrelin. Drug treatment for obesity is limited because effective drugs are lacking. The only drug to treat obesity in Europe is orlistat but it's effectiveness is modest. The development for new antiobesity drugs has been busy. Problems in drug development have however delayed drugs in the market. The aim of this study was to develop a method with which we could measure how much food zebrafish (Danio rerio) has been eaten and to study how different drugs affect feeding behavior of the zebrafish. The purpose was also to do high throughput screening of antiobesity drug with this method and to study how genes affect feeding. The amount of food that zebrafish ate was able to be measured by utilizing fluorescent rotifers as fish's food. Drugs that are known to affect feeding (fluoxetine and rimonabant) reduced the amount of food zebrafish ate when measurement was done in 6-well plate and with two hours feeding. Sibutramine did not affect food intake, although it has been shown to reduce food intake in zebrafish in another study. The effect of gene knock down was also studied with morpholino oligonucleotides. MANF, th2 or galanin gene knock down did not affect food intake in zebrafish. The conclusion is that the new method is well suited for food intake measurements and drug effectiveness studies. The method can not be used in high throughput screening because results can not be analyzed by a plate reader and the feeding can not be done in 96-well plate.
  • Pessi, Jenni (Helsingfors universitet, 2013)
    Polymer microspheres hold great potential as oral drug delivery system for therapeutic proteins. Microspheres prepared with biocompatible and biodegredable polymers have been extensively studied, since the oral delivery of therapeutic proteins is challenging due to the conditions in the GI-tract. The aims of this research were to apply microfluidics on polymeric microsphere preparation process, to determine what kind of formulations are suitable for this technology, to establish a controlled preparation process that produces advanced particles and to create a template for oral protein drug delivery. With microfluidic fabrication it is possible to gain control over the process and content of each droplet. However, finding suitable formulations for microfluidics is demanding. In this study, biphasic flow was employed to successfully produce double (W/O/W) emulsion droplets with ultra thin shells. Once the process and formulation variables were optimized constant droplet production was achieved. Flow rates used were 500 µl/h in the inner and in the middle phase and 2500 µl/h in the outer phase, respectively. Two formulations were selected for further characterization: 5 % poly(vinyl alcohol) in water in the outer phase, 3 % polycaprolactone in ethyl acetate in the middle phase and either 10 % or 20 % poly(vinyl alcohol) and polyethylenglycol (1:4) in water in the inner phase. All the particles were found to be intact and contain the inner phase, as verified by confocal microscopy. Further, the particles were monodisperse and non-porous, as observed by scanning electron microscopy. Particle size was found to be around 20-40 µm, variation in the particle size within one batch was small and the particles were stable up to 4 weeks. The encapsulation efficiency of the particles was remarkable; as high as 85 % loading of the model compound, bovine serum albumin. Particles released 30 % of their content within 48 hours. In conlusion, developing functional formulations for micfoluidic technology was possible, the microparticles encapsulated the model protein extremely well and all in all microfluidic technology had a lot of potential for droplet manufacturing for pharmaceutical applications.
  • Mikkonen, Heidi (Helsingfors universitet, 2014)
    One way to improve the solubility of a poorly-water-soluble drug is to make amorphous solid dispersion of it with one or several carrier polymers. However, the amorphous solid dispersions are often unstable. Stability and amorphisation of drug substance depend on e.g. the miscibility of the components in dispersion. Moreover, in the early stage of drug development there is available only limited amount of active substance and time to the analyses. In this study, the primary goal was to develop a method combining the preparing (solvent method) and the analyzing (MTDSC, modulated temperature differential scanning calorimetry) methods. In the method developing part, the possible effect of analyzing parameters of MTDSC to the results was also tested. Amorphous solid dispersions were prepared and analyzed with the invented method. The dispersions were made of poorly-watersoluble itraconazole with hydroxypropylmethylcellulose acetate succinate (HPMC-AS) and/or polyvinylpyrrolidone (PVP K30). X-ray powder diffraction (XRPD) and polarized light microscopy (PLM) were also used to make the interpretation of results easier and more reliable. By analyzing the prepared dispersions the differences in the miscibilities of the used polymers with itraconazole were examined and it was also studied how the miscibility affected to the amorphicity of the prepared dispersion. As a secondary goal, it was tested if combining the two polymers would improve the miscibility and amorphicity of the prepared dispersion. In many cases, with the developed method it was possible to make mixed and amorphous solid dispersion with 10-20 % itraconazole concentration. Used small amount of drug was roughly enough to the detection limit of the used analyzing techniques. The analyzing parameters of MTDSC were not seen to affect to the results in this study which makes the use of this method easier. The results of used analyses were in some part contradictory and that is why it is recommended to use several analyzing techniques or methods that combine different kinds of techniques. In the study, it was seen that in the most part of the prepared dispersions there was more HPMC-AS than PVP K30. This was speculated to be caused by the ionic bonds between the basic itraconazole molecules and acidic succinyl groups in HPMC-ASs and also because of more hygroscopic nature of PVP K30 which increases mobility which in turn increases probability of collision of itraconazole molecules. The use of two polymers in the same time was useful especially in the case of 90/10 HPMC-AS/PVP K30 polymer ratio. This was speculated to be caused by different vaporization rates of the used solvents (DCM and methanol) and too slow evaporation phase. To explain and examine this observation more thoroughly, nuclear magnetic resonance (NMR) -measurements were done. When analyzing the prepared dispersions and itraconazole alone, it was observed that with used amorphisation method (solvent method) itraconazole was in a form that differs from the original polymorph. This form of itraconazole was probably some kind of liquid crystal and was examined further by heating the sample and analyzing it by XRPD. Although there are some other studies to support this hypothesis, this interpretation needs some confirmatory analyses with other methods: with high temperature SAXS (small angle X-ray scattering) and NMR.
  • Kyynäräinen, Kerttu (Helsingfors universitet, 2013)
    In vivo dissolution of drug molecules depends on the conditions in the gastrointestinal tract, like pH, composition of the fluids and hydrodynamics. Weakly basic compounds dissolve rapidly in low pH in stomach but in intestinal conditions forming of supersaturated solution may occur. This unstable state is the driving force either for rapid absorption from small intestine or for possible precipitation of drug. Difference in precipitation and thus in bioavailability between fasted and fed states can be significant. In this study behaviour of three weakly basic drugs, dipyridamole, ketoconazole and compound A, were studied with aid of two-phase microdissolution method. Three clinically relevant doses were used in the studies. In the study both fasted and fed states were tested as well as the effect of different pH in stomach phase and significance of biorelevant solutions over general buffer solutions. Dissolution of the drugs were examined in the media that simulate gastric fluids (SGF pH 1,2, SGF pH 4,0, FaSSGF pH 1,6 and acetate buffer pH 5,0). When biorelevant simulated intestinal fluid (FaSSIF or FeSSIF) was added to the solution to simulate the drug transfer out of stomach into small intestine, precipitation of different doses were analysed. Also the level of supersaturation compared to solubility results was examined. In addition the effects of various mixing speeds (300 rpm and 150 rpm) and scales (USP I and minidissolution) on precipitation were studied. Concentrations were measured directly from dissolution vessels with fibre optic probes. Re-dissolution of the drugs in small intestine and influence of physical properties on dissolution rate were evaluated with flow through dissolution method. In the fed state simulated microdissolution tests even the high doses of the drugs did not precipitate. Instead, in the tests simulating fasted state the effect of dose was clear and the relative part of dissolved drug were the smaller the higher was the dose. The high doses precipitated fast while the small doses remained much supersaturated. When FaSSGF was used the solutions staid longer in supersaturated state. Higher pH in stomach phase had remarkable impairing effect on the dissolved part of the drug in the small intestine phase and no supersaturated solutions were formed. In the microdissolution smaller mixing speed seemed to cause more precipitation and ranges of the results were larger. Different hydrodynamics in different scale dissolution methods as well caused divergent results. The effect of physical properties of precipitate to re-dissolution could be observed with the flow through dissolution tests. According to this study, two-phased dissolution method fit for few milligrams of API and it can be used to evaluate the precipitation potential of basic drugs in fasted and fed states. Also the effect of higher pH in the stomach on dissolved portion of API can be analysed. The method can be used as a risk assessment method for example when you want to know when the dose raises the risk to precipitation or as a tool to be used in early formulation development. As such dissolution tests can be used to get qualitative data of precipitation phenomenon when comparing basic drug compounds. The dissolution, precipitation and re-dissolution parameters will in future be utilised in pharmacokinetic model to evaluate the effect and significance of these phases on the drug absorption in vivo.
  • Leino, Teppo (Helsingfors universitet, 2013)
    Voltage-gated sodium channels play an essential role in the function of the nervous system as they are responsible for producing action potentials. Abnormal activity of sodium channels is in connection to several diseases such as epilepsy and chronic pain. Voltage-gated sodium channel blockers which are selective towards a specific isoform could provide more efficient and better tolerated drugs to treat these diseases when compared to the drugs used today. Clathrodin is an alkaloid isolated from Caribbean sea sponge Agelas clathrodes. Bioactivity studies have shown that clathrodin changes the function of voltage-gated sodium channels. The aim of this study was to synthesize two kinds of structure analogs of clathrodin and study their structure-activity relationship towards different isoforms of voltage-gated sodium channels. The study is part of the MAREX project (Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications) funded by the European Union. Intention of the project is to find new bioactive compounds in marine organisms. A four-step route was developed for synthesizing 2-aminobenzothiazole analogs. A three-step route was developed as well but the last step seemed to be problematic for some of the compounds. The three-step route provided new compounds as intermediates and some of them were sent to tests for activity. Synthesis of 1H-pyrrole-2-carboxamide analogs of clathrodin failed. 4,5-dihydrooxazole was recognized as a problem as it was formed as a result of a cyclization reaction when bromination was tried on the intermediate. The formed structure was used in synthesizing 2-(1H-pyrrol-2-yl)-4,5-dihydrooxazole analogs of clathrodin. These reactions failed to give any final products which could have been tested for activity. Eight synthesized compounds were sent to tests for activity. Results were received from two of them and they showed no activity towards the voltage-gated sodium channels in 10 µM concentrations. Discussion about structure-activity relationship is not possible based on two compounds only.
  • Svanbäck, Sami (Helsingfors universitet, 2013)
    The dissolution rate is one of the most important physicochemical properties of drug substances. Above all, it demonstrates the energetic interaction between solvent and solute molecules, and is therefore a valuable tool for understanding drug substance properties. Dissolution studies are a widely used method in many areas of the pharmaceutical development process, however, only lately has the value of dissolution testing in drug discovery and early development been assessed. The advantages of dissolution testing over other early screening methods, such as kinetic solubility and in silico screening, lies in the possibility of obtaining solid state dependent quantitative data, from small amounts of drug substances. While the general way of studying drug dissolution has been by the multiparticulate bulk approach, studying the constituent single particles of these systems, could give a deeper understanding of the core factors affecting the dissolution rate of drug substances. The aim of the present study was to develop a static and dynamic method, in which it would be possible to analyze the dissolution process of a single pure drug substance particle, by optical microscopy. Both methods produced practically identical dissolution profiles, for image analysis and UV-spectrophotometric data, from the same systems of a single dissolving particle. The dynamic method developed in the present study is the first flow-through technique, in which it is possible to assess the dissolution of a single freely moving drug particle, by continuous physical analysis. The possibility of using physical analysis instead of chemical analysis poses many advantages. These include reduced materials consumption, reduced experiment times, as well as a reduction in the possible sources of error. Most importantly, the advantage of physical analysis lies in the fact that no prior chemical knowledge about the studied substance is needed. This makes physical analysis an optimal technique for studying new chemical entities. The novel flow-through method succeeded in obtaining the dissolution characteristics and 3D particle morphological data, of a single pure drug substance particle, of sub-milligram initial weight. The theoretical detection limit of 1 pg, poses an intriguing opportunity for further development.
  • Laurén, Patrick (Helsingfors universitet, 2013)
    Cellulose has already been used as an industrial raw material for over a century. However, during recent years the nanostructural features of the naturally occurring biopolymer have been fully investigated and characterized through different processing methods as nanofibrillar cellulose (NFC). This has led to a rapid development of novel cellulose based nanoscale materials and advancements in the field of composite materials. NFC offers interesting specific properties that differ from many other natural and synthetic polymers, such as self-renewable raw materials, semi-crystalline morphology, broad chemical modification capacity, biocompatibility and biodegradability. Biocompatibility and the biomimetic aspects of NFC have enabled the fabrication of nanoporous membranes and scaffolds that can function as medical devices (e.g. tissue engineering, wound healing, novel active implants). In this study, the properties of plant-derived NFC, as potential injectable drug releasing hydrogel "implants" were investigated. Three different sized candidate molecules were selected (123I-NaI, 123I-β-CIT and 99mTc-HSA, from small to large respectively) and investigated with the use of a small animal SPECT/CT molecular imaging device. Study compounds were mixed with the NFC biomaterial and injected into the pelvic region of mice. Drug release was observed for a period of 24 hours and the results were compared to saline/study compound control injections. In addition, 99mTc labeled NFC hydrogels were prepared for dual label tracing to observe the hydrogel positioning during the SPECT/CT acquisitions. For the smaller compounds (123I-NaI, 123I-β-CIT), no differences were found in the drug release or absorption in between the NFC biomaterial and saline injections. However, a clear difference was found with the large compound (99mTc-HSA). In the NFC hydrogel, the rate of release was slower and the distribution of 99mTc-HSA was more concentrated around the area of injection. In addition, the NFC hydrogel did not migrate from, or disintegrate, at the site of injection, suggesting a robust enough structural integrity to withstand normal movement and activity. In conclusion, the labeling of NFC was found to be a reliable and simple method. NFC hydrogels have the potential use as drug releasing medical devices with larger compounds. NFC matrix did not have any controlled release effect on the studied small molecules. Therefore further studies are required for more specific conclusions.
  • Pulkkinen, Nita (Helsingfors universitet, 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.
  • Jokinen, Nora (Helsingfors universitet, 2013)
    Estimated 180 million people worldwide are infected by hepatitis C virus. It causes liver diseases which are often asymptomatic. Chronic infections can lead to liver cirrhosis, transplantation and hepatocellular carcinoma. Drug development was slow until 1999 when the first cell culture model with autonomously replicating subgenomic HCV replicon was developed. It expresses the viral proteins that are necessary in HCV replication. The current interest in exploring new medicines is concentrated to the essential viral proteins, such as the NS3/4A protease, NS3 helicase, NS5A and NS5B RNA polymerase. HCV belongs to the Hepacivirus genus. Due to its high variability there are at least seven genotypes and several subtypes. Genotype 1 is the most common and the most difficult to treat. The current standard of care continues 24-48 weeks and consists mainly of pegylated interferon alpha and nucleoside analogue ribavirin, both non-specific HCV medicines with severe adverse effects. In 2011 two new direct-acting antivirals, protease inhibitors telaprevir and boceprevir, were approved for the treatment of HCV. A vaccine against HCV has not yet been developed. The aim of this study was to optimize and validate a robust cell-based assay for screening of replication inhibitors against HCV. Genetically modified Huh-7 cells harbor a subgenomic HCV replicon expressing only the viral proteins needed in viral replication. In addition, the replicon encodes a firefly luciferase as a reporter gene. The amount of expressed luciferase is directly correlated with the amount of HCV replication making the replicon system suitable for HTS. The optimized and validated method was used for screening HCV replication inhibitors from a library containing 113 marine-derived substances. Marine environment has been in recent years a very interesting source for finding new drug candidates. This study was part of international MAREX project which aims to discover new active molecules from marine resources. A total of 37 samples (32.7%) exhibited antiviral activity over 50%. A cytotoxicity evaluation in ATP assay was performed with these samples. 10 samples (27.0%) exhibited cytotoxicity below 20%, of which six were synthetic samples and four were extracts. Compounds with high antiviral activity, low cytotoxicity and clear dose-response in further studies should be tested with a cell line expressing the full-length HCV genome. The structural proteins can exhibit some characteristics which inactivate the compound identified as active in the replicon system.
  • Matikainen, Heikki (Helsingfors universitet, 2011)
    Chronic heart failure is a major worldwide health problem. It is a complex and severe syndrome caused by different kinds of cardiovascular diseases. Cardiac hypertrophy is frequently caused by hypertension and can lead to abnormality in heart contraction, activation of many neurohumoral mechanism and heart failure. The most important neurohormonal mechanisms of heart failure are activation of sympathetic nervous system and the renin-angiotensin-aldosterone system, insufficiently contracting left ventricle, cardiac remodeling and myocyte loss owing to apoptosis. Antihypertensive drug treatment is often used to prevent or decelerate progression of cardiac hypertrophy. Activation of the renin-angiotensin-aldosterone system plays a major role in heart failure. During the past decades angiotensin converting enzyme inhibitors (ACEIs) have been used as firstline treatment of heart failure. ACEI treatment has been shown to reduce mortality associated with chronic heart failure and improve prognosis of the disease. Angiotensin receptor blockers (ARBs) were expected to replace ACEIs in the treatment of heart failure but for the present they are only an alternative to ACEIs. Beta-blocking agents which reduce activation of sympathetic nervous system have established themself as the second most important treatment of heart failure. Diuretics are widely used as the treatment of heart failure but only aldosterone antagonists has been shown to improve prognosis of the disease. Also digoxin is still used in the treatment of chronic heart failure. In the future renin inhibitors, neutral endopeptidase inhibitors, vasopressin antagonists and molecules that affect inflammatory cytokines could potentially be capable of improving the prognosis of chronic heart failure patients. The major object in the present study was to investigate development of left ventricular hypertrophy induced by abdominal aorta banding in male Wistar rats and prevention of hypertrophy by calcium sensitizer levosimendan and angiotensin II receptor blocker valsartan. Also functionality of abdominal aorta banding as a rodent model of cardiac hypertrophy and heart failure was estimated. Abdominal aorta was constricted above the right renal arteries. That leads to pressure overload and increase in cardiac load. Heart response to pressure overload by hypertrophy in the form of wall thickening. 64 rats were assigned to different groups, each having eight rats. Three of the groups were treated with levosimendan with different daily doses (0,01 mg/kg; 0,10 mg/kg; 1,00 mg/kg) and three of the groups were treated with valsartan with different daily doses (0,10 mg/kg; 1,00 mg/kg; 10,00 mg/kg) via drinking water for eight weeks after the surgery. Sham-operated group underwent the same surgical procedures without constriction of the aorta. All the groups were compared to abdominal aorta banded group without any medical treatment. Cardiovascular parameters such as isovolumic relaxation time (IVRT), left ventricle end-systolic (ESD) and end-diastolic (EDD) dimensions, ejection fraction (EF), fractional shortening (FS), cardiac output (CO), stroke volume (SV), interventricular septum (IVS) and posterior wall (PW) thickness were measured eight weeks after the surgery by using cardiac ultrasound. In the present study levosimendan slightly improved systolic function of the heart. Improvement of the systolic function was seen in a tendency to improve ejection fraction and fractional shortening in abdominal aorta banded rats compared to abdominal aorta banded rats without medical treatment. Neither levosimendan nor valsartan affected diastolic function of heart. Diastolic function was measured by isovolumic relaxation time. Neither levosimendan nor valsartan had significant effect on development of cardiac hypertrophy. Cardiac hypertrophy was estimated by measuring heart weight-to-body weight ratio (HW/BW), left ventricular wall thicknesses and left ventricular internal dimensions in systole and diastole. The present study indicates that outflow constriction by aortic banding is clearly a model of cardiac hypertrophy but not of heart failure.
  • Hitonen, Heidi (Helsingfors universitet, 2013)
    About 10 % of the patients experience an adverse event during their treatment. About 1 % of the adverse events are serious. Extrapolated from international evidence adverse events cause death of 700 - 1700 patients annually in Finland. Medication errors are the most common single preventable cause of adverse events. According to several studies about half of the adverse events and medication errors are preventable. Therefore medication safety is a central part of the Finnish Patient Safety Strategy. One of the medication safety tools in Finland is unit-based pharmacotherapy plan for which the Ministry of Social Affairs and Health gives instructions in the Safe Pharmacotherapy guide. All social and healthcare units should develop a pharmacotherapy plan which describes the medication processes in the unit. The purpose is to harmonise the principles for the provision of pharmacotherapy, to clarify the division of responsibilities related to its provision, and to define the minimum requirements that must be complied. The aim of the study was to explore how the pharmacotherapy plans were made and to evaluate their implementation and benefits in hospitals, health centres and social care units for the elderly. Pharmacotherapy plans are studied as part of the Finnish Patient Safety Strategy, focusing on medication safety. Head nurses were chosen as target group. The data was collected by phone interviews. Forty (8 %) interviews were received from a random sample that was taken from a register of head nurses held by Tehy ry. The interview was semi-structured theme interview and the data was analysed using content analysis. Most of the studied units (n= 24, 60 %) had started to formulate the pharmacotherapy plan in 2007, soon after the Safe Pharmacotherapy guide was published. Pharmacotherapy plans were drawn up on organisation and/or unit level. There was a lot of variety in the working groups' sizes and professionals taking part in them. More instructions would have been needed. The most common way (n=20, 50 %) to induct the pharmacotherapy plan to the staff was to introduce it at the unit meeting. Most commonly mentioned changes to the operations in the units were related to error reporting (n=15, 38 %), certification procedures for distribution and administration of medicines (n=9, 23 %) and task allocation (n=8, 20 %). There were several benefits of the pharmacotherapy plan of which most common were starting or developing error reporting (n=19, 48 %), clarification and better availability of instructions (n=18, 45 %), better induction of new staff members and substitutes (n=16, 40 %) and standardization of procedures (n=10, 25 %). Allocation of tasks and responsibilities was considered a challenge in developing and using the pharmacotherapy plan. Limited resources caused difficulties in implementing and using the pharmacotherapy plan. The staffs' medication education was kept more up-to date after introduction of the pharmacotherapy plan but the education was also challenging for the units. Pharmacotherapy plans made the units develop their procedures and increased understanding of medication safety. All in all the plans have worked well as part of the Patient Safety Strategy. However the systematic development of the pharmacotherapy and the use of pharmacotherapy plans in social and healthcare units requires more effort.
  • Pylkkänen, Sarita (Helsingfors universitet, 2013)
    Harmful drug effects are common among older medicine users. Potentially harmful drugs for older people have been defined by different criteria. Potentially inappropriate medications (PIMs) defined by Beers criteria have been associated with adverse effects, increased costs, need of hospital care and disabilities. Drugs with anticholinergic properties (DAPs) are associated with anticholinergic side effects, cognitive decline and delirium. Psychotropic drugs have been associated with increased risk of falls and mortality. Concurrent use (≥3) of psychotropic drugs has been considered harmful for older people by the Swedish National Board of Health and Welfare. The aim of this study was to examine the prevalence of potentially harmful drug (PHD) use (DAPs, PIMs, or concurrent use of ≥3 psychotropic drugs) and the accumulation of PHDs in aged people living in nursing homes and assisted living facilities. The objective was also to investigate which patient characteristics are associated with PHD use or accumulation of these drugs. The cross-sectional data was collected in 2011-2012 as a part of a larger study "Reducing inappropriate, anticholinergic and psychotropic drug use among older residents in institutional care". The study population (N=326) consisted of ≥ 65-aged residents living in nursing homes or assisted living facilities in Helsinki (n=227) and Kouvola (n=99). The mean age was 83.5 years, 70.0 % were women and mean Charlson comorbidity index was 2.6. Residents were divided into four groups: aged using 1) DAPs, 2) PIMs defined by Beers criteria, 3) concurrent use of ≥3 psychotropic drugs and 4) no PHDs in use. Both those fulfilling any of these criteria and those fulfilling all the three criteria (accumulation of PHD) are described. Users in these groups were compared to the non-users. A majority of the residents, 78,8 % (95 % CI: 74,4 - 83,3) used ≥1 PHDs: 67,8 % (95 % CI: 62,7- 72,9) used ≥1 DAPs, 32,2 % (95 % CI: 27,1- 37,3) ≥1 PIMs and 32,2 % (95 % CI: 27,1-37,3) used ≥3 psychotropics concurrently. Of the residents, 41 (12.6 %) had a medication treatment that fulfilled all the three criteria of PHD use. These residents used significantly more PHDs than others (average mean 4,8 [range 3-7] vs. 1,6 [range 0-6]). The residents having the PHD accumulation were more often males and used more drugs than others. There were no statistical differences among the other characteristics of these groups. The most common PHDs were mirtazapine (n=66), lorazepam (n=64), oxazepam (n=62), ketiapine (n=58) and stimulant laxatives without opioids (n=58). Use of DAPs was associated with multimorbidity, use of PIMs with weaker health related quality of life, and use of psychotropics with younger age. High number of drugs was associated with all these criteria. Use and accumulation of potentially harmful drugs is common among the aged living in nursing homes and assisted living facilities. New means are needed to optimize drug treatments and to educate professionals taking care of these patients. Special attention should be paid on the use of antipsychotics, benzodiazepines, mirtazapine and stimulant laxatives.
  • Renko-Kaski, Riikka (Helsingfors universitet, 2014)
    This study explores the research, characteristics, manufacturing processes, safety and applications of graphene from the perspective of pharmacy and medicine. The study also examines how graphene research and commercialization has developed in the last ten years (2004-2013), with an emphasis on biomedical research globally and separately in Finland. The methods employed are an extensive literature review of scientific publications, and a survey of the biomedical research emphases, geographical distribution, and funding of graphene research based on article and patent databases. Graphene holds considerable potential in pharmaceutical use. Clinical trials can commence as soon as the manufacturing processes develop to produce graphene of sufficient quality. The variety of biomedical uses of graphene is vast: antibacterial products and coatings, gene therapy, tissue technology, sensor and imaging technology, as well as utilization in drug delivery. Graphene can be used to enhance therapeutic effectiveness by creating instruments for targeted and controlled drug delivery. In addition to uses in therapeutics, graphene offers possibilities for diagnostics. The biomedical research and commercialization of graphene have accelerated in the recent years, but research and patenting activity has concentrated in Asia, and especially in China. The research has been university driven and primarily publicly funded. In Finland, graphene research has focused on electrical applications, whereas research in the fields of pharmacy and medicine has been limited. As a so-called high-tech country, Finland could increase research into graphene as an innovative pharmaceutical instrument.
  • Siirola, Outi (Helsingfors universitet, 2013)
    There aren't always available suitable authorized drug products for different age and different weight pediatric patients. Hospital pharmacies have to prepare suitable doses and dosage form for these very young patients extemporaneously. In Finland oral powders are usually used in pediatric medication. In previous studies it has been found that part of drug dose sticks to paper of oral powder and the patient doesn't get the entire intended dose. It is suggested that hard capsules may be better dosage form than oral powders, because capsules have smaller area than oral powders, where the powder can stick. The aim of this study was to examine, whether warfarin- and spironolactone capsules prepared by hospital pharmacy meet European Pharmacopeia standards of uniformity of content. Capsules were compounded from commercial tablets and capsulated by Feton-capsulating device. In this study capsules manufactured with automatic capsule filling device attached to analytical balance, oral powders and capsules prepared from pure drug substance were also compared to capsules compounded from tablets. The three month stability of compounded capsules was also examined. In hospital pharmacy many different strengths are compounded from same drug substance, ordered by physician. Ordered strengths can be nearly identical, but whether the small differences in concentration can possibly be prepared in hospital pharmacy is unknown. From both drug substances two strengths with small difference in concentration were prepared and it was studied if statistically significant difference exists. The drug concentrations of preparations were measured by high performance chromatography (HPLC). Aqualab-water activity meter was used to study water activity of samples during the stability testing. Content uniformities of all capsule batches complied with test specified in the European Pharmacopeia.The drug concentrations of capsules were significantly lower than target concentrations. With these drug substances no difference, between the drug concentration of oral powder and capsules, was found. According to this study oral powders can be replaced by capsules. Warfarin and spironolactone capsules remain at least three months, when storaged in room temperature. Warfarin capsules can be prepared accuracy of 0,1 mg and spironolactone capsules accuracy of 0,5 mg.
  • Suutari, Teemu (Helsingfors universitet, 2014)
    Surface plasmon resonance (SPR) is a label free technique to study surface interactions. It is based on photon-plasmon coupling. Laser light is directed through a prism and reflects form a metal surface, often gold. At certain conditions, photons turn into plasmons, which then propagate on the metal surface. The refractive index (RI) of the medium close to the metal surface alters the conditions when plasmons can be generated. By changing the incident angle of the light, photon-plasmon coupling can be matched. Thus, change in the SPR sensogram peak angular position (PAP) indicates change in the RI of the sample. Traditionally, SPR has been used to investigate biomolecule dissociation / association kinetics. Recently, it has gained popularity in living cell sensing. Exosomes are 30-100 nm size lipid bilayer structured vesicles, which are excreted by nearly all cells. They play a role in cell-cell communications. Exosomes carry selected cargo from the cells of origin, including mRNA, miRNA, dsDNA and proteins, and they are directed to specific cells, which internalize them. This initiates responses in the recipient cells. The aim of the study was to harvest exosomes from prostate cancer (LNCaP) cells and use SPR as a novel method to detect exosome internalization by these cells. Adhesion proteins were tested in their efficiency to promote confluent cell monolayer formation on SPR gold substrate sensor surface. Nanoparticle tracking analysis (NTA) showed that exosome purification by ultracentrifugation was successful. It was also found that gold substrate supports confluent LNCaP cell monolayer formation. Adhesion proteins did not shorten the incubation time on gold substrate, but helped the cells remain on the sensor during the SPR experiment. Prostate and platelet exosomes were tested on whether they are internalized by LNCaP cells. Control samples with plain medium and PEI/DNA nanoparticles were used. PEI/DNA particles are nonviral gene delivery vectors, which are known to permeate into cells. The SPR results showed RI increase caused 0.9 ° change in the SPR sensogram with the PEI/DNA sample and no change with the medium sample. Exosomes showed more complex responses, both increasing the PAP approximately 0.1 °. Prostate exosome sensogram returned to baseline after sample rinsing, which did not occur with platelet exosomes. It was concluded that SPR shows a response in cell-exosome interactions, which is most likely because of exosome internalization.
  • Pietiläinen, Johannes (Helsingfors universitet, 2013)
    The aim of this study was to obtain basic knowledge of the applicability of a Büchi Spray dryer B-290 for inhalation particle production and its process parameters effects on particle physicochemical properties. The possibility to anneal the particles where also studied. The greater goal was to provide some information about the solutes' crystallization tendency related to chosen process parameters. Two active pharmaceutical ingredients, salbutamol sulphate and budesonide, where chosen as model substances. Spray drying is a suspended particle processing system which is widely applied and it has been in use from the 1940s. The processed pumpable liquid which contains chosen substances is dispersed into droplets and dried to produce particles that are later collected. Spray dryer is used to process food, biochemical and pharmaceutical substances. In the field of inhalation particle processing, however, it is rather a new technology. This is because of the quality limitations of inhalable particles and the challenges in process optimization. From the many process parameters the concentration of the solid substances, inlet temperature and concentration of organic solvent were chosen as variables for the conducted experiments due to their apparent effects on product quality and especially on solid state. A rudimentary box-annealing system was studied for spray dried substances to verify their solid state transformation tendencies. Salbutamol sulphate was annealed in a box with 65% relative humidity and budesonide in 74 % and 100% relative ethanol activities. Particle size and size distributions were measured with laser diffraction apparatus, crystallinity was analyzed with powder x-ray diffraction and particle morphology was studied with scanning electron microscope. Salbutamol sulphate turned out to be amorphous and budesonide crystalline when spray dried. Both products were within the inhalable size range (1-5µm). Under the current setup the solid state quality of the products was found dependent on the concentration of the solid substances to some extent. Spray dried amorphous salbutamol sulphate was successfully anneaed to a crystalline material and partly crystalline budesonide was annealed to a more crystalline state. Further studies are needed to utilize the full potential spray drying has to offer for inhalation formulating. The kinetics of the annealing procedure and its dependency on the method used still remain largely unexplored.

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