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  • Valve, Kiia (2021)
    Background and objectives: Pharmaceutical services provided by community pharmacies have the potential to improve medication safety and support the implementation of rational pharmacotherapy. The pharmaceutical services are internationally an underused resource to support functioning of social and health care services. The literature review of this Master’s thesis provides an overview of pharmaceutical services, - their funding and remuneration. The primary objective of the empirical study was to create an overview of the development of the pharmaceutical services in Finnish community pharmacies in 2010-2020. The secondary objective was to study differences in the service provision between Finnish provinces. Materials and methods: The study was carried out as a retrospective descriptive survey study annually conducted by the Association of Finnish Pharmacies. Åland was excluded from the provincial review so that individual pharmacies could not be identified. The data was analyzed using Microsoft Excel. The number of pharmacies providing pharmaceutical services annually and the annual number of customers using these services were counted at the national level. At the provincial level, the corresponding data for the prescribing review, medication review, comprehensive medication review and assessment of inhalation technique were analyzed for the years 2017-2020. Results and conclusions: The most common service with the highest number of customers was automated dose dispensing. The second most common service was prescription review. As a whole, the provision of services and the number of customers had increased during the study period in Finnish community pharmacies. Manual dose dispensing was a diminishing service. Differences were found between provinces in the prevalence of services and in the number of customers. It was possible to identify provinces with lower service provision activity, such as Lapland. The service provision prevalence and number of customers varied widely within provinces. The number of customers for a certain service in an individual pharmacy had a large effect on the provincial average, thus, the average number of customers in the provinces does not reflect the provinces' success in implementation of services. Pharmaceutical services, with the exception of the automated dose dispensing, are not well implemented.
  • Korhonen, Juha (2020)
    Traditionally pharmacists’ activities have focused on the manufacturing and compounding of medicinal products. The production of medicines has shifted from pharmacies to industrial mass production since the 1960s, and correspondingly, the production of medicinal products by pharmacists in community pharmacies has declined and is now almost completely absent. It has been concluded that pharmacists are over-educated, and their skills are not fully utilized. Authorities and pharmacy organizations have expressed interest in pharmacists’ extended role, which includes patient care and cognitive services. Professional identity refers to the conscious understanding and awareness of oneself as a professional, which is based on an individual's life history: it is the relationship between the individual, work and profession, and its future. Professional identity has been seen to influence the performance of health workers, in terms of both competence and responsiveness. The purpose of this study is to describe pharmacists’ perceptions of their professional identity and its possible change from the beginning of 1990 to 2019. A systematized review was conducted in October 2019. Altogether 12 publications were selected in the systematized review. All the studies were qualitative. In eight studies, interviews were used as the research method. Two selected studies used a focus group discussion as the research method, and two studies used both interviews and focus group discussions. One study used interviews and focus group discussions as well as survey as research methods. From the basis of this study, no conclusion can be drawn that the professional identity of pharmacists has evolved from a product orientation towards a more patient-centered care provider. Because of the complex and dispersed nature of community pharmacy practices, and the many factors that are involved in them in different contexts, it is difficult to interpret and understand what the core function of a pharmacist is in a community pharmacy. Pharmacists could not describe their activities and their roles explicitly. The researchers concluded that pharmacists had not attained as clear and strong professional identity in their current occupation, as it had been in the past when drugs were still manufactured in the community pharmacy. Community pharmacists balanced between two conflicting roles as pharmacists and business managers. Pharmacists, patients, and politicians alike perceived community pharmacy as a business. This view is further supported by the context, the physical premises of the community pharmacy, which are generally more suitable for retail than patient care. This may represent a challenge for the implementation of pharmacists’ extended role.
  • Niemelä, Akseli (2022)
    Lecithin:cholesterol acyltransferase (LCAT), a key enzyme in maturating high-density lipoprotein (HDL) particles, has been targeted to promote the efficiency of reverse cholesterol transport by small molecular positive allosteric modulators (PAM) of Daiichi Sankyo. For a set of these compounds their Vmax and EC50 values and binding site in the membrane-binding domain (MBD) of LCAT have been determined. Through molecular dynamics (MD) simulations we previously found a metric that qualitatively described which compounds were active, so in this study we aimed to improve it by finding a quantitative metric. This led to the discovery of the Cα distance between CYS50 and ASN65, which correlates with this set’s Vmax values and which can be utilized to predict the Vmax values of novel compounds. Additional simulations were performed to discover whether this metric is changed by a lipid interface present, and to reveal a likely entry pathway PAMs take. As LCAT activation is likely a benign and potentially overlooked effect, we performed a virtual screen of FDA-approved compounds and secondary metabolites associated with LCAT. From secondary metabolites, a key finding was that flavonoids were overwhelmingly associated with LCAT and had a high binding potential to the MBD in docking simulations. The best binding compounds were subjected to MD simulations to discover their Vmax values using the discovered metric. This provided us with a set of compounds, which can be used to validate our in silico model in vitro. Should this model be validated, it can be used in optimising and discovering novel PAMs of LCAT, and it would bring evidence to the benefit of MD in drug discovery processes in general. Furthermore, if our discovered compounds can activate LCAT in vitro, they may be used as precursors for novel PAMs or as therapies by themselves not only for LCAT deficiencies, but perhaps for atherosclerotic cardiovascular diseases as well.
  • Viljemaa, Kati (2017)
    The economic burden of adverse events (AEs) is substantial and in direct relation to current increasing drug utilisation. According to previous research, the annual cost of AEs in the U.S. may be as high as 22.9 billion euros. In Europe AEs are considered to contribute to 3.6 percent of hospital admissions, have an impact on 10 percent of inpatients during their hospital admission and are responsible for less than 0.5 percent of inpatient deaths. AEs thus clearly constitute a major clinical issue. Fluoroquinolones have been in clinical use since the 1980s and are globally among the most consumed antimicrobials. Fluoroquinolones are generally well tolerated antimicrobials. The most common AEs are mild and reversible, such as diarrhea, nausea and headaches. Nevertheless, fluoroquinolones are also associated with more serious AEs, including Clostridium difficile associated diarrhea (CDAD), rate-corrected electrocardiographic prolonged QT interval, tendinitis and tendon rupture, dysglycemia, hepatic toxicity, phototoxicity, acute renal failure and serious AEs involving the central nervous system, such as seizures. Health service use and costs specifically associated with fluoroquinolone-related AEs have not been evaluated previously. The theory section of this Marter's thesis considers adverse events and fluoroquinolones. The main principles of conducting a systematic review are also discussed. The empirical section is a systematic review. The aim of this study is to identify health care use and costs associated with ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin and ofloxacin -related AEs. A literature search covering Medline, SCOPUS, Cinahl, Web of Science and Cochrane Library was performed in April 2017. Two independent reviewers systematically extracted the data and assessed the quality of the included studies. All costs were converted to 2016 euro in order to improve comparability. Of the 5,687 references found in the literature search, 19 observational studies, of which 5 were case-controlled, fulfilled the inclusion criteria. Hospitalization was an AE-related health care use outcome in 17 studies. Length of stay associated with AEs varied between <5 - 45 days. The estimated cost of an AE episode ranged between 140 and 18,252 €. CDAD was associated with the longest stays in hospital. However, a mere 10 studies reported AE-related length of stays and only 5 evaluated costs associated with AEs. Although rare, in particular serious fluoroquinolone-related AEs can have substantial economic implications, in addition to imposing potentially devastating health complications for patients. Further measures are required to prevent and reduce health service use and costs associated with fluoroquinolone-related AEs. Equally, better-quality reporting and additional published data on health service use and costs associated with AEs are essential. The strengths of this study are a comprehensive and systematic literature search and transparency of methodologies and reporting. The main weakness is the generalizability of the results.
  • Natri, Ossi (2022)
    Coronary heart disease is a number one killer in westernized countries and the costs from it will continue to grow in the future. It is caused by atherosclerosis, build-up of plaque and chronic inflammation in the arteries of heart, and endogenous lipoproteins have a special role in its development. Among other atheroprotective properties, High density lipoproteins (HDL) have a role in intrinsic mechanism of the reverse cholesterol transport (RCT), of gathering and removing excess cholesterol from peripheral tissues. There have been several HDL raising strategies in the past for the treatment of atherosclerosis, but their success has been modest. Synthetic HDL (sHDL), comprising of various types of phospholipids and proteins or peptides, have been developed to mimic the properties of endogenous HDL. Despite some success in animal studies, failures in clinical studies have turned the focus on the HDL’s interaction with a specific enzyme lecithin:cholesterol acyl transferase (LCAT), responsible for cholesterol esterification, a key step in RCT. ApoA-I, the most abundant protein component of HDL, acts as LCAT cofactor in cholesterol esterification, and many LCAT activating peptides have been developed to mimic the features of apoA-I. The molecular level understanding behind LCAT activation is however still foggy. During enzymatic activation, LCAT goes through conformational changes specific regions, which are generated by interactions with apoA-I or synthetic peptides. These mechanisms have been studied widely with molecular dynamic simulations, in vitro experiments, and imaging. In this study, we investigated 22A (PVLDLFRELLNELLEALKQKLK), apoA-I mimetic peptide known for its as good LCAT activation potency as apoA-I, and four variations of it (21A, 22A-P, 22A-K22Q, and 22A-R7Q), and combined them with phospholipid DPPC to create sHDL nanodiscs by thermal cycling method. We examined the effect of small changes in peptide sequence on LCAT-sHDL binding strength with quartz crystal microbalance with dissipation (QCM-D). The interest was to further test the suitability of thermal cycling method on nanodisc assembly, test the binding strengths against the hypothesis of the role of salt-bridge forming amino acids R7 and K22 in peptide dimerization and its effect on LCAT binding and activation, and to see if QCM could act as a suitable method for the research of sHDL-LCAT interactions. All peptides formed similar sized sHDL particles with diameter of ~10 nm with thermal cycling method. As expected, the LCAT binding tendency of 22A-sHDL was highest, about double compared to four other peptide nanodiscs with almost identical results. The QCM results suggest that binding tendency between LCAT and sHDL is affected by small, one amino acid change in peptide sequence, but it does not necessarily have a big impact on LCAT’s esterification activity, but based on this experiment alone, we cannot make any further conclusions. Electron microscopy revealed exceptional breakdown of 21A-sHDL incubated with LCAT compared to 22A-sHDL. This phenomenon could indicate high lipolytic rate of LCAT but needs further investigation. There were some challenges with the measurement parameters in the beginning, and the variability between parallel measurements with QCM-D was high, which cause a little doubt about the method’s suitability for these kinds of precise measurements. More research for revealing the molecular mechanism behind LCAT activation is needed for the development of more effective treatments.
  • Ollikainen, Elisa (2013)
    Protein phosphorylation is an important mechanism in cell signaling. Normally, one third of all proteins in a living organism is phosphorylated every moment. Abnormal amount of phosphorylated proteins is associated with many diseases. Proteins can also be oxidized in a living organism by reactive oxygen species. Usually oxidized proteins are degraded quickly in cells. Increased oxidation and decreased capability to degrade oxidized proteins can cause accumulation of these species in tissues. This has also been associated with several diseases and aging. Titanium dioxide is a widely used catalyst in photocatalytic reactions. When titanium dioxide absorbs UV radiation, hydroxyl radicals and superoxide anions are formed in aqueous solution. These radicals can then react with other compounds and degrade them. The most important factors affecting titanium dioxide photocatalysis are concentration and type of titanium dioxide catalyst, initial concentration of the substrate and pH. The purpose of this work was to investigate the effect of phosphorylation on degradation and oxidation of peptides in titanium dioxide photocatalysis. Since titanium dioxide columns have been used to enrich phosphopeptides, the adsorbtion of phosphorylated and unphoshorylated peptides on titanium dioxide is known to be different. Possibly this has also an effect on reaction products in photocatalysis. Peptides investigated were a non-phosphorylated, a monophosphorylated and a triply phosphorylated insulin receptor peptide. Samples were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). First, the effect of pH on the degradation of the peptides was studied. Acidic conditions decreased the degradation of the non-phosphorylated peptide. Degradation of the phosphorylated peptides was significant in all pH conditions studied. In alkaline conditions all of the investigated peptides were degraded efficiently so these conditions were chosen for further experiments. Degradation of all three peptides was very fast in titanium dioxide photocatalysis. Peptides were degraded significantly already after 0.5 minutes UV exposure. The non-phosphorylated and monophosphorylated peptide produced mainly once (M+O) and doubly (M+2O) oxidized products. Oxidation products of the triply phosphorylated peptide were not detected. Oxidation sites could not be identified since the products were formed in such a small concentrations. Reproducibility was also poor in this study. Further studies are needed to find out, if phosphorylation has effect on oxidation of peptides. In the future, the reaction conditions and the analytical method need to be optimized.
  • Hedström, Anna (2020)
    The ability to regulate release of noradrenaline, dopamine and GABA is one of the most important roles of the nicotinic receptors. The release of neurotransmitters following stimulation of nicotinic receptors is addressed in the thesis, with focus on dopamine and noradrenaline. Release of neurotransmitters, mediated through nicotinic receptors, has been researched using various methods, including brain slices, microdialysis and synaptosomes. Research using synaptosomes have provided valuable information regarding nicotinic receptors and their ability to regulate neurotransmitter release. Research using receptor specific antagonists have provided information regarding the stoichiometry of nicotinic receptor in different regions of the brain. The primary focus in the thesis, was the characterization of [3H]dopamine release following stimulation of nicotinic receptors with varenicline and acetylcholine, using synaptosomes from mouse striatum. Using a-conotoxin-MII, the [3H]dopamine release was divided into a-conotoxin- MII-resistant and -sensitive release. [3H]Dopamine release was mediated through a6b2*- and a4b2*-receptors from striatal synaptosomes. The involvement of other receptors could not be ruled out, but based on these results and results from previous studies, the involvement of other nicotinic receptors is supposedly low.
  • Kyrö, Minna (2011)
    FTIR spectroscopy (Fourier transform infrared spectroscopy) is a fast method of analysis. The use of interferometers in Fourier devices enables the scanning of the whole infrared frequency region in a couple of seconds. There is no need to elaborate sample preparation when the FTIR spectrometer is equipped with an ATR accessory and the method is therefore easy to use. ATR accessory facilitates the analysis of various sample types. It is possible to measure infrared spectra from samples which are not suitable for traditional sample preparation methods. The data from FTIR spectroscopy is frequently combined with statistical multivariate analysis techniques. In cluster analysis the data from spectra can be grouped based on similarity. In hierarchical cluster analysis the similarity between objects is determined by calculating the distance between them. Principal component analysis reduces the dimensionality of the data and establishes new uncorrelated principal components. These principal components should preserve most of the variation of the original data. The possible applications of FTIR spectroscopy combined with multivariate analysis have been studied a lot. For example in food industry its feasibility in quality control has been evaluated. The method has also been used for the identification of chemical compositions of essential oils and for the detection of chemotypes in oil plants. In this study the use of the method was evaluated in the classification of hog's fennel extracts. FTIR spectra of extracts from different plant parts of hog's fennel were compared with the measured FTIR spectra of standard substances. The typical absorption bands in the FTIR spectra of standard substances were identified. The wave number regions of the intensive absorption bands in the spectra of furanocoumarins were selected for multivariate analyses. Multivariate analyses were also performed in the fingerprint region of IR spectra, including the wave number region 1785-725 cm-1. The aim was to classify extracts according to the habitat and coumarin concentration of the plants. Grouping according to habitat was detected, which could mainly be explained by coumarin concentrations as indicated by analyses of the wave number regions of the selected absorption bands. In these analyses extracts mainly grouped and differed by their total coumarin concentrations. In analyses of the wave number region 1785-725 cm-1 grouping according to habitat was also detected but this could not be explained by coumarin concentrations. These groupings may have been caused by similar concentrations of other compounds in the samples. Analyses using other wave number regions were also performed, but the results from these experiments did not differ from previous results. Multivariate analyses of second-order derivative spectra in the fingerprint region did not reveal any noticeable changes either. In future studies the method could perhaps be further developed by investigating narrower carefully selected wave number regions of second-order derivative spectra.
  • Michallik, Heli (2012)
    Inhibitory GABAergic neurotransmission seems to play a central role in the effects of ethanol on the central nervous system. However, the exact mechanism of ethanol action as well as the role of the GABAA subunits in this mechanism remains unclear. The imidazobenzodiazepine Ro 15-4513 acts as a partial inverse agonist of the GABAA receptors by binding to their benzodiazepine sites which contain a γ2 subunit. In addition, ethanol and Ro 15-4513 seem to bind in a competitive manner and with high affinity to δ subunit-containing extrasynaptic GABAA receptors that mediate tonic inhibition. There exists conflicting evidence about the role of the δ subunit in the mechanism of antialcohol effects of Ro 15-4513. Clinical evidence of the efficacy of γ-hydroxybutyric acid (GHB) in suppressing alcohol withdrawal syndrome has been shown, even though only little preclinical research has been done on this subject. GHB has agonistic effect on the GABAB receptors and on the putative GHB receptors. GHB seems to share a very similar pharmacological profile with ethanol and there is also some proof of their synergistic effects. However, the exact mechanism of ethanol consumption suppressing action of GHB is not exactly known. The aim of this study was to determine the role of the γ2 and δ subunits in the effects of Ro 15-4513 (0, 3 mg/kg) on voluntary ethanol drinking and on the motor coordination suppression by ethanol (1.5 g/kg). In addition the effects of nonselective benzodiazepine flurazepam (0, 6 mg/kg) and GHB (0, 100, 150 mg/kg) on ethanol drinking and the effects of GHB on motor coordination were examined. The rotarod method (∅ 6 cm, fixed speed 6 r.p.m.) was chosen to determine the motor coordination. The drinking-in-the-dark (DID) method was applied to study the drinking effects. In this method the water bottle in the home cage of each mouse was replaced with an ethanol dilution (20 % v/v) for a certain time in the beginning of the dark phase of the light/dark cycle. A knock in mouse line γ2I77-lox with a point mutation in the γ2 subunit gene was used in the experiments. The mutation decreases the affinity of the receptor for certain benzodiazepine structures like that of Ro 15-4513 in the brain. The C57BL/6J mouse line was used as control. Both Ro 15-4513 (3 mg/kg) and GHB (150 mg/kg) significantly reduced ethanol drinking. The GHB dose of 100 mg/kg failed to reach significance probably due to the relatively long drinking time (1 h) used in the experiment in comparison to the short half-life of this drug. Flurazepam (6 mg/kg) significantly enhanced ethanol drinking which as expected was not affected by the mutation of the γ2I77-lox mouse line. Ro 15-4513 (3 mg/kg) failed to reduce the ethanol-induced suppression of motor coordination probably due to a too low dose. The GHB rotarod experiments suggest that the GHB (150 mg/kg) ethanol drinking suppressing effect may have been partly caused by its sedative effects. There was no significant difference between the used mouse lines in the effects of Ro 15-4513. This would suggest that the γ2 subunit does not play a significant role in the effects of Ro 15-4513. However, in order to draw a final conclusion more experiments must be done with the γ2I77-lox as well as with the δ subunit knockout mouse line, which we were unfortunately not able to include in this study as originally planned.
  • Knuutila, Kaisa (2010)
    As the development of new active ingredients is becoming more expensive and difficult, the development of new and better dosage forms, like multiparticulate systems, has become more attractive. Multiparticulate systems can be defined as oral dosage forms consisting of small discrete units together providing desired dose. Usually the small units are round pellets approximately 0.5-1.5mm in size. Multiparticulate systems can enhance the properties of already existing active ingredients or reduce the unwanted side effects. The properties can be alternated also by preparing the system as the enteric formulation, which resists the acidic environment of the stomach and releases the active ingredients in the small intestine due to the pH change. The enteric delivery is used mainly to enhance the absorption of acid-labile drugs or to reduce the side-effects of stomach irritating drugs, like iron. Prepared pellets contained iron in sulphate form and they were pelletized with Nica extruder/spheronization system. The pellets contained also ascorbic acid to maintain the iron in its reduced form during iron release and absorption. Microcrystalline cellulose enabled the pellet formation and Eudragit L 30 D-55 was used in the coating to protect the drug containing pellets from the stomach's acidic environment. Some of the pellets were subcoated before the enteric coating. The subcoating contained part of the ascorbic acid and iron sulphate and the film forming water soluble hydroxypropyl methylcellulose, HPMC. Other pellets were coated directly with the enteric coat while 100% of the ascorbic acid and iron sulphate were located in the core pellets. The coating was performed in fluidized bed. The iron concentration was determined with UV-Vis spectrophotometer. Iron itself did not absorb light but with o-phenanthroline it formed orange-red complex which absorbed visible light absorption maximum being at the wavelength of 510 nm. In order to ensure the full absorption the enteric iron products need to release the iron rabidly after they enter the small intestine. Approximately 15-20% (w/w) of enteric coating was needed to fulfill the 5% release limit per hour in dissolution test in 0.1 HCl defined by authorities. The release rate was comparable to a commercially available product. The subcoating did not have a considerable effect to the release rate. The iron precipitated to the pellet surface either during extrusion/spheronization process or drying. 1% Tween 80, 3% Kollidon K-25 and 5 and 10% Eudragit L 30 D-55 solutions were used as pelletizing liquid in order to reduce the precipitation. The 3% Kollidon K-25 produced visually best pellets but some pellets were clued together during the process. The possible retardation in release was not tested.
  • Hannila, Teija (2013)
    Gene therapy is an experimental technique that involves inserting therapeutic genes into the target cells to treat diseases. Gene transfer can be performed by ex vivo or in vivo method. Ex vivo method means transferring the therapeutic gene in laboratory to the cells that are removed from the patient, after which the cells are returned to the patient. In the in vivo method the gene transfer is performed directly to the target tissue inside the patient's body. Gene therapy clinical trials have been carried out to treat many diseases. The majority of the clinical trials have so far been cancer trials. Nevertheless, the most promising results have been established in treating diseases that arise from mutations in a single gene, i.e. monogenic diseases. Monogenic diseases include e.g. hemophilia and heritable immunodeficiencies. The biggest challenges in the clinical trials so far have been the limited gene transfer efficiency of the currently used gene vectors, the short duration of the transgene expression and the side-effects in viral-mediated gene transfer. Nonviral gene transfer agents have so far been less efficient in vivo than the viral vectors. This is partly due to the interaction between serum components and nonviral vectors. The main purpose of this study was to investigate the effect of serum to the gene transfer efficiency of a nonviral vector polyethyleneimine PEI22K and the combination of PEI22K and cationic liposome Dosper in vitro in the SMC-cells. The potential synergistic increase in the transfection efficiency of PEI22K/Dosper combination was also studied. The secondary goal in this study was to develop an in vitro model which could be used to predict the gene transfer efficiency of gene vectors in vivo. The combination of PEI22K and Dosper resulted in a synergistic increase in the transfection efficiency in serum-free transfection. In the presence of serum the efficiency of PEI22K was higher than the efficiency of PEI22K/Dosper combination. 1-10% serum concentrations did not significantly affect PEI22K`s transfection efficiency, but dramatically decreased the efficiency of PEI22K/Dosper combination. The results suggest that PEI22K is more suitable than PEI22K/Dosper combination for in vivo gene transfer.
  • Sjöberg, Madeleine (2018)
    Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.
  • Sjöberg, Madeleine (2018)
    Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.
  • Pietarinen, Paavo (2012)
    Most xenobiotics are biotransformed by phase I enzymes to a more hydrophilic form in order to get excreted out from the body. In most cases xenobiotics are in lipophilic form when entering body. The most important group in phase I enzymes is cytochrome P450 (CYP) superfamily. Of CYP enzymes probably the most studied is CYP2D6, which is responsible for metabolism of 20-25% of drugs currently on market. Many CYP2D6 substrates belong to therapeutically important drug groups, such as antiarrhytmics, antidepressants, beta-blockers, or neuroleptics. CYP2D6 gene, which encodes the enzyme, exhibits large interindividual variability, which has an effect on the metabolic activity of the enzyme. The frequencies of these genetic variances differ globally on wide scale between and inside populations. Through genotyping it is possible to predict the CYP2D6 metabolic rate, which can be divided into four classes: ultra-rapid metabolizers (UM), extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). The purpose of our study was to examine the frequencies of CYP2D6 genotypes in Finnish population in detail and compare the results to previous studies. Our study population consisted of 857 healthy volunteers whose DNA was extracted. From DNA sample we genotyped 10 different CYP2D6 genetic variants and the copy number of the gene using Applied Biosystems TaqMan genotyping and copy number assays. This study was the largest CYP2D6 genotype frequency study in Finnish population so far. The results supported the findings of a similar study in a Finnish population of smaller scale. Large majority of study subjects were EMs (87.3%) and the second largest group was Ums (7.2%). IMs and PMs were in clear minority (3.0% and 2.5%, respectively). The expected frequencies for UMs (1-2%) are much lower and for PMs higher (~8%) in other North European populations than in Finns. Accordingly, CYP2D6 genetic profile of Finnish population differs from its neighbours, which may be important for the dose requirements, efficacy, and safety for drugs metabolized by CYP2D6.
  • Seikola, Anniina (2011)
    The Ministry of Social Affairs and Health published a report on development needs of elder care and geriatric pharmacotherapy in 2006. The major concern in this report was related to several challenges in pharmacotherapy of the aged, such as deficiencies in medical knowledge of nurses working with elderly people. One way to improve the medication expertise of those various parties involved in caring elderly people is continuing education (CE). The aim of this study was to explore pharmacotherapy-related training needs of health care professionals involved in the home care services for the elderly in the Social and Health Care Cooperation Region of Lohja, Siuntio, Inkoo and Karjalohja (the LOST Region). This study was started by conducting a survey among nurses working in home care services for the elderly in the LOST Region in 2009 (response rate 47%). To deepen understanding of the key findings of the survey, focus group discussions (FGDs) and face-to-face interviews were conducted among nurses, nursing aids, their managers and physicians (1 FGD among nurses, n=6; 1 FGD among their managers, n=6; and face-to-face interviews with 4 physicians). The survey data were analyzed separately for nurses (n=9), practical nurses (n=53) and home aids (n=9), but results were the same in every group. Of the theoretical training needs, topics related to pharmacokinetics and special characteristics of using medicines in the elderly, effects, adverse effects and interactions of medicines, were most important. In addition, the theoretical training needs covered professional ethics issues, such as accuracy and carefulness of nursing practice. The main training needs related to collaborative practice in pharmacotherapy concerned monitoring medicine user's condition and medication, and dosing medicines (right medicine, dose, strength, dosage) in the right time, and administration routes of medicines. Focus group discussions and face-to-face interviews of the physicians provided a deeper understanding of the results of the survey. One of the main findings of this qualitative part of the study was challenges in cooperation in home care services in the LOST Region. Implementation and monitoring geriatric pharmacotherapy can be improved by improving multiprofessional cooperation and training for nurses and physicians working in home care services. The most important diseases and disorders for which the nurses would like to have shared operational guidelines were diabetes, cardiovascular diseases, pain, memory and psychiatric disorders. Training needs also covered special characteristics of pharmacotherapy for the elderly, and formulations and administration routes of medicines. Finally, a synthesis was made of the results of the survey, focus group discussions and face-to-face interviews. On the basis of the synthesis, a proposal for a multiprofessional training was developed for the LOST Region. The training plan includes topics related to geriatric pharmacotherapy and improving collaborative practices and communication as identified by those involved in different stages of the study.
  • Kallionpää, Roope (2014)
    Estrogens are female sex hormones that have genotoxic and proliferation-enhancing effects in cells. Life-time exposure to estrogens is linked to the risk of several cancers. Estrone is only a weak agonist of estrogen receptor but it serves as a precursor for biosynthesis of 17β-estradiol, 16α-hydroxyestrone and catechol estrogens. While 16α-hydroxyestrone has relatively weak affinity for estrogen receptor, it has prolonged effect due to covalent binding to the receptor. UDP-glucuronosyltransferases (UGTs) are phase II metabolic enzymes that conjugate estrogens with glucuronic acid to render them more watersoluble. Polymorphisms in UGT genes have been linked to excretion of steroids and risk of some cancers. Generally, subfamily UGT1A enzymes conjugate the 3-hydroxyls of estrogens, while the activity of subfamily UGT2B is directed towards 16- and 17-hydroxyls. Previous results on estrone glucuronidation are incomplete and conflicting, while glucuronidation of 16α-hydroxyestrone has not been systematically studied. The aim of this study was to identify UGTs active in the glucuronidation of estrone and 16α-hydroxyestrone and to further examine the glucuronidation kinetics of the active UGTs. Also the effects of bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) and mutations of UGT1A10F90 and UGT1A10F93 on glucuronidation activity were examined. Activity assays were conducted using recombinant enzymes as well as human liver and intestinal microsomes. Resulting glucuronides were analyzed using high performance liquid chromatography and quantified based on their UV absorbance. UGT1A3, UGT1A10 and UGT2A1 showed the highest activity toward estrone glucuronidation, while UGT1A10, UGT2A1 and UGT2B7 were the most efficient UGTs conjugating 16α-hydroxyestrone. UGT1A10 had the highest Vmax in the glucuronidation of both substrates, although it conjugated estrone at a higher rate than 16α-hydroxyestrone. UGT1A10F93 was shown to have a role in the different glucuronidation activities of UGT1A10 toward estrone and 16α-hydroxyestrone. Affinity of 16α-hydroxyestrone was highest for UGT2B7, while UGT2B17 conjugated 16α-hydroxyestrone relatively slowly. The results confirm earlier observations of the preference of UGT2B7 for α-configured hydroxyls while UGT2B17 favors β-configuration. UGT2A1 showed no strict regioselectivity but had a relatively weak affinity for both substrates. DMSO was found to decrease UGT activity. However, its presence is necessary to solubilize lipophilic substrates. DMSO concentration has to be kept constant to produce comparable data for, for example, kinetic studies. BSA was found to alter especially the kinetics of UGT2A1. BSA also seemed to have solubility-enhancing effect.
  • Mattila, Susanna (2012)
    The aim of the stydy was to evaluate how different chemical derivatization methods are suitable for characterization of regional isomers of different glucuronide conjugates. Glucuronidation is one of the phase II metabolic reactions where more water soluble and often inactive substances are produced. Different functional groups may be subjected to glucuronidation. It is important to determine the exact position of glucuronidation, as the isomers may possess different toxicological or pharmacological properties. For example morphine-6-glucuronide is pharmacologically more active than morphine itself. The glucuronide conjugates are commonly detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and/or nuclear magnetic resonance (NMR). MS/MSspectra of native molecule and glucuronidated molecule are usually similar because of an initial loss of 176 Da, i.e. monodehydrated glucuronic acid. This fact often makes it impossible to determine the site of glucuronidation. Samples of NMR-analysis requires larger amounts of sample materials than MS-analysis. Many of those derivatization reagents tested in this study were not reacting as they were supposed to react according to literature. O-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) were forming derivatives as expected and those reagents are very suitable for glucuronide conjugates studies. At the end of the studies the site of the glucuronidation of dopamine- and serotonineglucuronides were evaluated by derivatization with OPA and FMOC. Derivatization with OPA and FMOC successfully gave information about the region of the glucuronide acid in dopamine- and serotoninemolecules. The assumptions supposed to be correct according to NMR-studies presented in literature.
  • Häggman, Verner (2019)
    If pharmaceutical quality system fails it causes a hazard to the patient’s health, but also to the manufacturer’s economy. For this reason, the manufacturer’s must make sure their products comply with the quality requirements placed by authorities. To ensure the compliance, the authorities perform inspections at the manufacturing sites. If the site does not comply with the quality requirements, the authority will take necessary measures. The goal of this study was to find what type of quality issues FDA and the authorities within EU have observed while inspecting manufacturing facilities, which of these issues are most common, in which countries the sites companies with issues have been located. The results were assessed from European pharmaceutical company’s point of view. The data for the study was collected from Eudra GMDP database and from FDA Warning letters sent by FDA headquarters from years 2015-2017. Qualitative analysis of content was chosen as the method of analysis. The collected data was classified into main classes and subclasses based on reoccurring topics. The classes were transferred in tables to compare how which of the classes were most common. Most often the facilities with quality issues were located in China and India. The authorities also perform a lot of quality inspections in these countries, but that alone doesn’t explain the large number of quality issues in these countries. The number of sites with quality issues per inspection was also high. Both the authorities of EU countries and FDA had mainly observed similar issues. Often quality issues were related to data integrity. Other common themes were quality management system, cleaning of equipment and facilities and analytical methods. There were also some differences in the observed issues. E.g. FDA had rarely observed issues related to personnel while EU authorities had observed such issues frequently. Quality issues which had led to measures by authorities were often related to larger problems with the quality management or to very basic quality actions. If company doesn’t have well-functioning quality organization, the quality system is often inadequate also in other ways. By comparing their own activities with the issues observed at other companies, it is easier for a company to improve their quality and avoid major quality issuer before they occur.
  • Hahl, Eveliina (2023)
    Introduction: European legislation on orphan medicinal products, Regulation (EC) No. 141/2000 of the European Parliament and of the Council, entered into force in April 2000. Although the prevalence of rare diseases is low according to legislation (less than 5/10,000), 18–30 million people in the European Union (EU) are affected by rare diseases. The introduction of orphan medicine legislation has increased the number of orphan medicines developed but the fairness of the legislation has also raised concern and criticism. The literature review of this Master ́s thesis provides an overview of rare diseases, orphan medicines and EU orphan medicine legislation. The aim of the empirical study was to investigate the evolution of orphan medicine selection during European legislation on orphan medicinal products in 2000–2022. In more detail, aims were to describe the evolution of orphan medicine selection, the approved indications for orphan medicines and the number of orphan medicines approved for children. Methods: The research material was orphan medicines that received a marketing authorisation during the EU orphan drug legislation. This material was collected from the European Commission's Community Register of orphan medicinal products and the European Commission's Community Register of not active orphan medicinal products. Qualitative document analysis was used as the research method, where information on orphan medicines were quantified. Results and conclusions: In the 10-year review of orphan medicine development, the number of new orphan medicine products approved for the market doubled, being 63 products between 2001 and 2010 and 127 products between 2011 and 2020. In the latter 10-year period of the review, the focus of approved indications for orphan medicines shifted slightly from orphan medicines developed for the treatment of cancers (36%) to orphan medicines developed for the treatment of inborn errors of metabolism or immune disorders (43%). In the 10-year reviews, the relative share of orphan medicines approved for children decreased from 55 percent in 2001– 2010 to 40 percent in 2011–2020. Based on the results of the study, the fairness and targeting of the benefits of the orphan medicine legislation should be further investigated. Orphan medicine legislation should encourage the development of medicines for rare diseases for which there is no treatment at all, and for the population most affected, in other words children.
  • Salminen, Veera (2021)
    Continuous monitoring of the safety profile of medicinal products is essential also after marketing authorisation approval to ensure the patient safety. Spontaneous reporting of adverse drug reactions is one of the most important methods to collect post-approval safety data of medicinal products. The advantages of spontaneous reporting system include reaching large population throughout a long period of time for many medicinal products, however, it also has some limitations. One commonly recognized problem of the system in many countries is under-reporting of adverse drug reactions. The national reporting scheme in different countries slightly vary, even between Nordic countries. The main aim of this study was to find out what improvements should be done to the current reporting scheme in Finland so that it would better encourage healthcare professionals to report in relevant situations, which respond to the purpose of the spontaneous reporting system. Physicians (n=20), pharmacists (B.Sc.) (n=78), pharmacists (M.Sc.) (n=21) and nurses (n=13) responded to the anonymous open voluntary online questionnaire. Close-ended questions were analyzed and results summarized in graphs and tables. Statistical analysis was done using chi-squared test. Content analysis was performed for open-ended questions by utilizing both, inductive and deductive approach. In the study, we found some differences in healthcare professionals’ opinions what kind of adverse drug reactions should be reported. Some of the healthcare professionals were also aware that they had not reported all suspected adverse drug reactions that came into their knowledge and several reasons were recognized for this. Seriousness of the reaction was considered the most motivating factor for healthcare professionals to report about suspected adverse drug reactions. The results of this study suggest that in healthcare professionals’ opinion, the most important factors that should be considered to improve reporting in Finland are training for healthcare professionals and simplifying the reporting as much as possible. Some differences were noticed between the occupational groups regarding preferences in the reporting route and especially physicians seemed to prefer formation of the report from the information system as a reporting method more than open web-based reporting form. Mobile application for reporting was not preferred that much among Finnish healthcare professionals. The results of this study support the hypothesis that under-reporting of suspected adverse drug reactions is also present in Finland. The reporting instructions should be clarified, training availability should be considered and reporting should be simplified as much as possible to improve the reporting.