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  • Salminen, Sanna (2011)
    The background of this study is increase in the ageing population and in medication use. Aged-related changes in pharmacodynamics and pharmacokinetics may change medication response in elderly patients and lead to adverse reactions. For elderly people the risk of being hospitalized due to adverse drug reactions is four times higher than for younger people. Many of these problems could be prevented by avoiding the use of certain drugs in the elderly. Several criteria have been developed to assess medication appropriateness in the elderly. The aim of this study was to develop a new Finnish Medication Risk Assessment (MRA) tool to be used by trained nurses to assess the presence of risks related to use of medicines in outpatients aged 65 years and older. A preliminary tool was developed through a comprehensive literature review of tools to indicate appropriateness and risks of elderly medications, and through expert opinions. The tool was then validated by using three-round Delphi-method. Delphi-method is a qualitative consensus method which is based on group judgement of a subject matter. The first and the second Delphi-rounds measured the tool's suitability and the third Delphi-round measured the importance of the items of the tool in estimating risks related to the use of medications of elderly patients. In this study, 33 expert geriatric panelists were approached of whom 11 physicians, three pharmacists MSc (Pharm.) and four nurses agreed to participate. The results from the Delphi-rounds were evaluated both quantitatively and qualitatively. Through the three-round Delphi-method was developed a MRA -tool that contains 19 items. According to the panelists the items of the tool are either important or moderately important. This indicates that the tool is valid to estimating medication risks in use of medications in this population. Further studies are needed to test the tool among nurses and patients. The MRA -tool was primary developed for estimating risks in medication use, but it could also be used for educational purposes. In the future, it is possible to implement safer and more appropriate pharmaceutical treatment for elderly patients by using this Medication Risk Assessment -tool.
  • Niskanen, Anna (2013)
    Polypharmacy and age-related changes in pharmacodynamics and pharmacocinetics may lead to drug-related problems in elderly patients. Accurate medication reconciliation and medication review on admission may help to control drug-related problems and optimize drug therapy in elderly patients. Several models have been developed to reconciliate and review medications at this point of care. A Finnish model can be developed on the basis of the se models. The aim of this study was to develop a tool for medication reconciliation and medication review on admission for ward pharmacists’ use in the Lahti city hospital. The tool was developed with an action research method in cooperation with the multiprofessional study group. A preliminary tool was developed through doctors’ (n = 2), nurses’ (n = 3) and ward pharmacists’ (n = 2) interviews, a literature review and the expertise of the multiprofessional study group. The preliminary tool was piloted twice in the Lahti city hospital. After the first pilot a view changes were made to the too l by the experiences of the ward pharmacists. Doctors (n = 3) who worked at the study ward during the first pilot were interviewed to find out their views on the medication reconciliation and medication review process so that their views could be taken into consideration in the development of the final version of the tool. After second pilot ward Pharmacists (n = 2), researchers (n = 2) and an expert of geriatrics from the study group took part in a group conversation. Through the group conversation and doctors’ interviews was developed the final version of the tool. The developed tool contains sections for patient’s background information, patient interview, medication reconciliation, drug-related problems, proposed medication changes and doctor’s decisions on the proposed changes. Also instructions of the medication reconciliation and medication review process were developed for ward pharmacists. The developed tool will be used in an intervention study in the Lahti city hospital. In the future a new version of the tool could also be developed to be used in other hospitals in Finland to reconciliate and review medications at the time of hospital admission.
  • Kleme, Jenni (2012)
    The medicines information and counseling given by health care professionals are essential in supporting patients' medication therapy. Given that medication therapies are often associated with medicine-related problems among the elderly, proper knowledge on medicines and their use is especially important for this particular patient group. Benzodiazepines and related drugs are of special concern in the elderly. Despite the current care guidelines, they are commonly used by the elderly, often also regularly and long-term basis. Benzodiazepines and related drugs are associated with multiple potential adverse drug reactions that their elderly users should be aware of. This study aimed at assessing the knowledge on medications, and needs and sources of medicines information on benzodiazepines and related drugs in the elderly. Especially, medicines information related to benefits and adverse drug reactions was studied. Additionally, data on use and subjective experiences of benzodiazepines or related drugs in the elderly were explored. Structured interviews were conducted among patients aged 65 years and using benzodiazepines or related drugs (n = 38) in acute wards (n = 2) of Pori City Hospital in 2004. Elderly patients reported that the package leaflet was the main source of medicines information on benefits and adverse drug reactions relating to medicines they used. The physician was reported as a second source after the package leaflet. More than 50 percent of the elderly (n = 20) had not received information about the benefits or adverse drug reactions of benzodiazepines from their health care providers or relatives. The information received had merely focused on benefits of drug than adverse drug reactions. Most commonly the elderly (61 %, n = 23) knew, that the use of benzodiazepines can cause drug dependence. Least commonly, they were aware that benzodiazepines can cause muscular weakness, depression and falling over. Eight elderly were not aware of any asked adverse drug reactions and nearly two thirds of the patients (63 %, n = 24) knew less than four adverse drug reactions out of eleven. The results indicate that elderly patients are not well aware of the effects of benzodiazepines and related drugs they use. Additionally, they may more often receive information from the package leaflet than health care professionals. Physicians and other health care professionals should pay more attention to counseling elderly patients especially about the benefits and adverse drug reactions of benzodiazepines and related drugs.
  • Wikman, Essi (2019)
    Streptococcus pneumoniae is a bacterium that causes invasive pneumococcal disease (IPD) such as bacteraemia and meningitis, and pneumonia. The prevalence of pneumococcal diseases is high in infants and in ≥65-year-olds. Also, the incidence of pneumococcal disease is higher in medical risk groups compared to the base population. Pneumococcal diseases can be prevented by vaccinations and since 2010 pneumococcal vaccine PCV10 has been in the national vaccination programme for infants in Finland. The aim for this study is to evaluate the cost-effectiveness of pneumococcal vaccinations in national vaccination programme for the 65-year-olds in medical risk groups (diabetes, chronic coronary artery disease, asthma and COPD). Secondary aim is to examine uncertainty factors that are related to economic evaluations of pneumococcal vaccinations in the elderly. Cost-utility analysis was used as the economic evaluation method. It is a method where health gains are measured by quality-adjusted life years (QALYs). Static multicohort model was chosen for the modelling. Some of the used parameters were acquired from the literature and most of the epidemiology and cost parameters were acquired from research reports and articles published by National Institute for Health and Welfare. Analyses were made for both pneumococcal vaccines that are registered for adults (PCV13 and PPV23) and in 2 different scenarios: Finland’s present situation where PCV10 is in the vaccination programme for infants (scenario A), and hypothetical situation where PCV13 would be in the vaccination programme for infants (scenario B). Based on the analysis, when PCV10 was in the vaccination programme for infants (scenario A), vaccinating 65-year-olds in medical risk groups was cost saving intervention in the health care perspective for both vaccines in chronic coronary artery disease and asthma and COPD risk groups. In diabetes risk group the costs per QALY’s gained were 2 100 € in scenario A. When PCV13 was in the vaccination programme for infants (scenario B), costs per QALY’s gained for PCV13 vaccinations were: diabetes 52 400 €, chronic coronary artery disease 35 900 € and asthma and COPD 22 000 €. The uncertainty of results was tested with deterministic and probabilistic sensitive analysis. In scenario B the results were sensitive for the waning of the PCV13 produced immune protection, the price of the vaccine, the proportion of pneumonia caused by S. pneumoniae, the changes in the pneumococcal disease incidences and the effect that pneumonia has for the health related quality of life. The cost-effectiveness of vaccinating 65-year-olds with pneumococcal vaccines was different depending on the risk group and on which pneumococcal vaccine is in the vaccination programme for infants. In addition, there are several uncertainty factors that have an impact on the results of economic evaluation of pneumococcal vaccinations.
  • Tikkanen, Alli (2019)
    Organic Anion Transporting Polypeptide 2B1 (OATP2B1) is an influx transporter expressed widely throughout the body in tissues such as intestine, liver, brain, placenta and skeletal muscle. Since many clinically used drugs are transported by OATP2B1, changes in the function of the transporter due to genetic polymorphism could lead to altered pharmacokinetics or -dynamics of OATP2B1 substrate drugs. The aim of this Master’s thesis was to create and optimize a cellular uptake assay to study the function of OATP2B1. Furthermore, the aim was to study the effects of six naturally occurring nonsynonymous single nucleotide variants on OATP2B1 transport function in vitro. With site-directed mutagenesis, single nucleotide changes were introduced into the gene coding for OATP2B1. OATP2B1 variants were expressed in human derived HEK293 cell line using baculovirus expression system. A cellular uptake assay with estrone-3-sulfate and a fluorescent probe 4’, 5’-dibromofluorescein (DBF) as substrates was set up and optimized. With the assay, OATP2B1-mediated uptake of variants was compared to the transport activity of OATP2B1 wild type. Amino acid changes Ser486Phe and Cys520Ser impaired OATP2B1 transport function severely. In addition, variant Thr318Ile transported DBF and estrone-3-sulfate less efficiently compared to OATP2B1 wild type, but Arg312Gln, Thr392Ile and Ser532Arg transport function was not affected. A method to study OATP2B1 function was created successfully. According to the results, single amino acid changes in OATP2B1 can impair OATP2B1 function. The results and method can be utilized to understand findings from pharmacogenetic studies in vivo, and to predict consequences of especially rare variants, which can be difficult to detect in small sample populations in clinical studies. However, further studies on the expression level and cellular localization of OATP2B1 variants are needed to fully characterize the impact of the variants studied.
  • Somersalo, Petter (2017)
    Cells release different types of phospholipid bilayer-limited vesicles into the extracellular space. These are commonly referred to as extracellular vesicles (EVs). Exosomes (EXOs), ca 50-100 nm in diameter and microvesicles (MVs), ca 100-1000 nm in diameter, having different intracellular origin, are the two main subpopulations of EVs. EVs have been demonstrated to carry a range of proteins and nucleic acids subsequently delivered to recipient cells, making them attractive as drug delivery vehicles. Several mechanisms for the cellular uptake of EVs have been established. When a nanoparticle is introduced into blood plasma, plasma proteins are adsorbed to its surface, forming a protein corona. The formation of the corona is a dynamic process, governed by individual protein concentrations as well as their respective affinities for the surface. Proteins of the corona interact with surrounding cells, thus being able to influence the cellular uptake of the nanoparticle. In the current study, the uptake of PC-3-derived EVs into PC-3 cells was investigated. Moreover, the impact of a human blood plasma-derived protein corona on said uptake was assessed. EVs were isolated from collected PC-3 cell culture medium using differential centrifugation. Experiments were performed separately for MVs (20000xg EV-fraction) and EXOs (110000xg EVfraction). SDS-PAGE analysis revealed adsorption of plasma proteins to EVs, following their exposure to plasma. Prior to uptake experiments DiO-labelled EVs were either incubated or not incubated in plasma. Plasma incubation lasted overnight. PC-3 cells were then treated with either of the two EV-preparations. Following incubation, EV uptake was assessed using confocal microscopy by determining the percentage of positive fluorescent cells in cell cultures. Pre-study plasma incubation resulted in a reduced or unchanged uptake of MVs and in a reduced uptake of EXOs, when compared to their native counterparts. In conclusion, the plasma-derived protein corona was shown not to improve EV uptake. It is worth noting that the current study limits itself to the use of PC-3-derived EVs and PC-3 cells as recipient cells in uptake experiments.
  • Hietala, Tarja (2017)
    Twin screw granulation (TSG) has gained considerable interest as a continuous wet granulation method in the pharmaceutical industry and has been studied the most. However, there is still lack of understanding how continuous granulation affects the material compaction behavior even though it has been noticed in several dry and batch wet granulation studies that the granulation process has an influence on the final tablet strength. Thus, studies on the material compactability and tabletability after continuous wet granulation are relevant for the overall understanding of twin screw granulation process and its effect on material behavior in tableting. Hence, the main objective of this study was to investigate the influence of continuous twin screw granulation on the compactability and tabletability of commonly used excipients. Additionally, the impact of binder on the compaction behavior of materials was examined. Furthermore, the suitability of two "loss in compressibility" models i.e. the Unified Compaction Curve (UCC) model and a porosity model to predict the loss in tablet strength after twin screw granulation and for the materials used was assessed. Earlier, the models have been applied to dry and batch wet granulations only. Full factorial design of three variables (binder type, binder addition method and the number of kneading elements) with two levels was conducted for the ConsiGma1 twin screw granulation of formulations containing microcrystalline cellulose (MCC), mannitol or anhydrous dicalcium phosphate (DCPA) as the main excipient and polyvinylpyrrolidone (PVP) or hydroxypropyl cellulose (HPC) as binder. Magnesium stearate was added as lubricant after granulation prior to tableting. In addition to the full factorial design, granulation with PVP, dry binder addition and four kneading elements was repeated for each main excipient. In total this made 27 experiments. The granules were dried and milled after granulation and all the batches were tableted. Additionally, all the formulations were direct compressed in order to be able to detect the change in compactability and tabletability after granulation. Torque of the granulation was determined as well as bulk density and particle size distribution of the granules. Additionally, the tensile strength and porosity of the tablets were analysed. Tabletability and compactability were determined based on the compaction pressure and the obtained tensile strength and porosity values of the tablets. Furthermore, parameters (PWG, TWG and εWG) describing the loss in compressibility models were calculated. MCC experienced loss in compactability and tabletability after twin screw granulation due to hornification effect. On the other hand, the compaction behavior of mannitol improved due to the formation of porous granules. The compactability of DCPA decreased and the tabletability increased. However, the change was only moderate presumably due to brittle nature of DCPA. Additionally, the binder type had an effect of the compaction behavior of the materials, PVP producing stronger tablets compared with the less hydrophilic HPC. However, the binder addition method played only a small role in modifying the compaction behavior. The UCC model was applicable to MCC as loss in tabletability was detected. Thus, the model can be used to predict tablet tensile strength when MCC is granulated with twin screw granulator. Additionally, the UCC model can be used to design the granulation process to achieve a target tensile strength based on small scale preliminary studies thus reducing the resources needed for case-studies. However, the UCC model was not feasible to mannitol and DCPA because they experienced improvement in tabletability after twin screw granulation. The porosity model was applicable to MCC and DCPA but not to mannitol as it showed improvement in compactability. The porosity model described the loss in compactability of MCC only moderately due to lack of tensile strength data points and the linearity of the tensile strength-porosity relationship. However, the model described well the loss in compactability of DCPA at tablet porosities achieved with compaction pressures used in industry. As a conclusion, the results demonstrate that twin screw granulation can have a significant impact on the final tablet strength and that the compaction behavior of the formulation can change either way depending on the used materials. Furthermore, the small influence of the binder addition method on the tablet strength indicates that the time consuming binder dissolving process step can be excluded from the tablet production chain enabling continuous manufacturing with twin screw granulation.
  • Rossi, Vilma (2020)
    Background: Inhaled therapy is the most widely used treatment for asthma and chronic obstructive pulmonary disease (COPD). Inhaled medicinal product has several advantages, including high local drug concentration in the lungs and reduced systemic adverse effects. However, the challenge with inhaled therapy is that many asthma and COPD patients do not know how to use their inhaler properly. Suboptimal inhaler use can lead to poor clinical control. The Association of Finnish Pharmacies has developed inhalation technique assessment service (ITAS) to detect and correct patients’ inhalation technique and to give information regarding the inhaler and inhaled therapy, such as drug storage and oral care. Objective: The aim of the study is to investigate whether asthma and COPD patients’ ability to prepare the Respimat inhaler and the patients’ ability to properly inhale the drug improve after receiving ITAS. The second objective is to find out what patients and pharmacists think about the service and which customer groups benefit the most from the service. Methods: The study design is an uncontrolled pre-post intervention. 33 pharmacies participated in the study. All patients who were buying a prescribed Respimat inhaler, were offered to participate in the study. Patients’ inhalation technique was assessed before (baseline) and immediately after ITAS (follow up 1). In addition, the inhalation technique was assessed the next time the patient came to pharmacy to buy Respimat inhaler (follow-up 2). Questionnaires were used to assess patients’ and pharmacists’ perceptions of ITAS. Results: 228 baseline and follow-up ITAS were performed. The results of follow-up 2 will be published later in a separate article. 14 % of the patients performed all the steps (both inhaler preparation before first inhalation and inhalation process itself) correctly at baseline. After ITAS the number increased to 77 %. At baseline 30 % of the patients had an optimal inhalation technique (all inhalation steps correct) and after ITAS the number increased to 85 %. 70 % of the patients had an acceptable technique (all critical steps correct) before and 93 % after ITAS. Both patients and pharmacists felt that the service was beneficial to the patients when thinking the proper inhaler preparation and proper inhalation technique. Overall patients’ and pharmacists’ satisfaction were high towards ITAS. Our study indicates that patients benefit from ITAS regardless of patient’s age or how long the patient have been using the Respimat inhaler. Conclusions: A pharmacist-led inhalation technique assessment service significantly improves asthma and COPD patients’ inhalation technique with Respimat inhaler. ITAS should be performed regularly as part of the delivery of the inhaled drug to the patient. Further research is needed on the effectiveness of ITAS with other inhalers.
  • Vähä-Mäkilä, Maria (2012)
    The aim of this master`s thesis was to investigate the accuracy of in silico inhalation model to predict pharmacokinetics of orally inhaled products. In literature review special features of the inhalation medication and current statements of medicinal regulatory agencies about bioequivalence (BE) of inhaled products are discussed. The ability of generalized pharmacokinetic BE studies to replace the traditional efficacy studies is a major question in the regulatory agencies. Also the usefulness of published in vitro - in vivo correlations (IVIVC) as an aid for inhaled product development in pharmaceutical industry is considered. Furthermore the most commonly used in silico lung deposition models and their properties are presented. In the experimental part a generic in silico inhalation model was constructed using a proper software and Orion Oyj`s in vitro and in vivo research materials on certain dry powder inhaler (DPI) products. Based on in vitro knowledge the aim of modeling was to predict the pharmacokinetic behavior of a therapeutic drug used in inhaled products. Also the applicability as a tool in clinical study design of inhaled products was estimated. Inhalation model consisted of two separate modeling parts utilizing primary in vitro characterization results of DPI products. Lung deposition of products was predicted with the ARLA (The Aerosol Research Laboratory of Alberta) respiratory deposition calculator available to the public while drugspecific pharmacokinetics was simulated using constructed Stella model (isee systems). ARLA lung deposition model takes into account several factors affecting the final lung dose of medical aerosol. Those include aerosol formulation and the dimensions of the device, as well as breathing conditions and inhalation mode. A rough sensitivity analysis was carried out with ARLA considering the effect of these factors on predicted lung deposition fractions. The predicted plasma concentration profiles, Cmax and AUCt values of the model drug were markedly lower than the experimental values. ARLA deposition model predicted moderately the order of systemic drug exposure obtained with different DPI products. The inhalation model built in the experimental part needs to be refined using more comprehensive and trustable source and reference material. The role of clinical BE studies in the marketing approval of generic inhalation product will be important because currently in silico predictions are still under development.
  • Myllymäki, Pilvi (2018)
    The majority of potential new chemical entities reaching drug development phase belong to Class II the Biopharmaceutics Classification System (BCS) which complicates formulation of orally administered drugs. Therefore, there is a need to develop methods to increase the solubility and dissolution rate. Transformation of a crystalline drug into its amorphous form can be used to enhance these properties of poorly water-soluble drugs. However, amorphous drugs are thermodynamically unstable and tend to recrystallize back to the crystalline form. Coamorphous forms are a new and promising method to stabilize amorphous form. A relatively new approach is to combine the active drug compound with an amino acid to form a coamorphous system. In this study, co-amorphous systems were prepared from gamma, alpha or amorphous form of indomethacin (IND) and tryptophan (TRP) by ball milling. The solid-state changes during milling were investigated to obtain information about the co-amorphization process. The main objective was to investigate the effects of initial solid state of indomethacin on the formation pathways. In addition, different analytical methods were compared with respect to observed endpoints of the formation process. Raman spectroscopy has not been used in previous studies regarding solid state changes in co-amorphous forms. The presence of fluorescence in amorphous systems may have limited use of the method. A time-gated Raman setup together with X-ray powder diffraction and differential scanning calorimetry (DSC) was used to investigate this kind of potentially fluorescent system. Principal component analysis of spectral data revealed that the three different binary systems had individual and direct pathways towards the same end points during milling. This indicates that the co-amorphous form formed after 60 minutes of ball milling is not dependent on the initial solid-state form of IND. Straight pathways also indicated direct transformation to the coamorphous form. DSC was found to be the most sensitive method to detect changes for the longest period during co-amorphization. Conventional Raman spectroscopy was found to be suitable for investigation of the co-amorphization process. However, time-gated Raman spectroscopy did not show significant advantages compared to conventional Raman data. This study revealed that the most stable form of IND could be used for production of co-amorphous form together with TRP. Raman spectroscopy could potentially be used for investigating coamorphization also as an in-process analytical method.
  • Lindstedt, Hanna (2022)
    Drug-induced liver injury (DILI) is a relatively rare hepatic condition that can be classified as predictable and unpredictable. However, DILI is a primary reason for drug withdrawals, post-marketing warnings, and restrictions of use. DILI is a problem for the drug users but also for the pharmaceutical industry and regulatory bodies. From the perspective of patients' and clinicians', DILI is the major cause of acute liver injury. At present, a major problem predicting DILI in drug discovery is a poor understanding of its mechanisms as well as the complexity of DILI pathogenicity. The main mechanism behind DILI are alterations in bile acid homeostasis, oxidative stress, and mitochondrial dysfunction. More than 50 % of drugs causing DILI are causing mitochondrial impairment. If the normal function of mitochondria is disturbed, the energy production of the cell decreases, and cell function decline leading eventually to the cell death. In this study prediction of mitochondrial toxicity was studied using cryopreserved primary hepatocytes of humans and rats. The aim of the study was to clarify if there are interspecies differences in the prediction of toxicity but also investigate possible differences in the mechanisms behind hepatotoxicity by using three well-known compounds toxic to mitochondria. To determine these differences, total cellular ATP was measured after 2- and 24- hour exposure time to gain information on overall viability and possible adaptive responses. Mitochondrial energy pathways were studied as a real-time monitoring acute exposure of test compounds. Morphology, location, and possible adaptive response of mitochondria were studied using a fluorescent probe and antibody staining combined with high content imaging (HCI). Overall, primary rat hepatocytes were more sensitive to the test compounds than human hepatocytes. Also, there were differences between human hepatocyte batches that may reflect the metabolic differences between hepatocyte donors. Immunolabeling did not bring any additional values compared to the fluorescent probe staining in the study of morphology of mitochondria. Additionally, it was noticed that treatment with paraformaldehyde significantly changed the hepatocyte mitochondria morphology. Overall, more effort is needed to develop image analysis of mitochondria morphology. Finally, studying mitochondrial morphology has proven to be difficult, and this study did not unfortunately reveal any information about the adaptive responses of mitochondria for drug-induced liver injury.
  • Huusko, Karoliina (2013)
    Intracellular drug sequestration is useful to understand when designing new drugs with intracellular targets. The knowledge of the intracellular distribution can also help to understand the side effects and pharmacokinetics of a drug, as well as the lack of response in e.g. some multidrug resistant cancer cells. Intracellular concentrations are also important to know when predicting the role of active transport in the overall transport process when binding site of the transporter is intracellular. The literature review describes the mechanisms causing intracellular drug sequestration along with the consequences of intracellular drug sequestration and methods that are used to study it. Alterations of intracellular distribution of anticancer drugs in multidrug resistant cancer cells are also described as an example of the many factors affecting the distribution pattern of the drugs inside cells. Understanding these mechanisms is valuable when designing strategies to overcome the multidrug resistance. The most commonly applied methods for studying intracellular concentrations of drugs are based on fluorescence microscopy. In experimental work, subcellular fractionation protocol is introduced and applied to determine the concentration of CDCF, clotrimazole and celiprolol in vitro in the plasma membrane and cytoplasm of MDCKII cells. CDFC and celiprolol are substrates of the MDR1 transporter and clotrimazole is an inhibitor. Concentrations in the fractions were measured in wild type cells and in MDR1-transfected cells with and without MDR1 inhibitor verapamil to see if the transporter had an effect on the concentrations. Also the effect of lipophilicity of the drug on partition between plasma membrane and cytoplasm was reviewed. Celiprolol showed a typical behaviour of the MDR1 substrate whereas CDCF and clotrimazole did not. Clotrimazole as a lipophilic compound was accumulated more to the plasma membrane than less lipophilic CDCF and celiprolol. Lipophilicity affected also to the ratio of Km (or Ki)(determined from the concentration in extracellular fluid) and Km (or Ki)(membrane) (determined from the plasma membrane concentration) values, with clotrimazole Ki(membrane) value being larger than respective Ki value, and CDCF and celiprolol Km(membrane) values being smaller than their respective Km values.
  • Mustonen, Merja (2011)
    Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.
  • Sjöstedt, Noora (2011)
    The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.
  • von Schantz, Sofia (2015)
    This study aims to address how easily an individual with no prior inhaler experience can learn to use a dry powder inhaler (DPI) through video education. This is a comparative study of four DPIs (Diskus, Easyhaler, Ellipta and Turbuhaler). Different properties affecting ease of use, patient preference as well as educational videos as a method of providing inhaler instructions were investigated. The study used a triangular methodology. The sample consisted of 31 individuals (24-35 years). All participants were considered inhaler naïve. After watching the video education material for a particular inhaler the participants' demonstrated the use of it. Educational videos for all four inhalers were watched and use of all placebo inhalers was demonstrated in a random order. These demonstrations were videotaped. The demonstrations were thereafter checked against a predefined checklist and all mistakes were recorded. Only 33 % of inhaler demonstrations were completed without the participants making any mistakes that could compromise the efficacy of the inhaled medication in a real-life situation. The frequency of error varied greatly between different types of inhalers. Ellipta proved to be most often used correctly with 55 % demonstrating use without making any mistakes. This was closely followed by Diskus for which 48 % demonstrated correct use. The difference between the average error frequency for Ellipta and Diskus was statistically insignificant. With Easyhaler 19 % percent of participants were able to demonstrate correct use, the corresponding percentage for Turbuhaler was 16 %. When comparing participants' demonstrations for Easyhaler and Turbuhaler, the difference in average error frequency between the devices were not statistically significant. The average frequency of error was lower when using Ellipta in comparison to Easyhaler and Turbuhaler (statistically significant). The same indications were found when comparing average frequency of error for Diskus, to those for Easyhaler and Turbuhaler. Comparing the participants self-reported correct use against the actual numbers it is clear that participants often thought they were using the inhaler correctly when they in fact were not. When asked to rank the inhalers from most preferred to least preferred, Ellipta emerged as a favorite. Turbuhaler received the second highest scores, Diskus the third and Easyhaler was least preferred. However, only the difference between preference scores for Ellipta and Easyhaler was deemed statistically significant. The high frequency of error suggests that even though participants generally considered the inhalers intuitive and easy to use, they would have required more comprehensive inhaler education in order to achieve correct inhaler technique. Further, the results indicate that video demonstrations are not ideal for providing inhaler education for first time inhalers users. The most prominent problem with video education is that it provides no feedback to the user regarding their inhaler technique. This may present real problems as the results of this study show that participants tended to overestimate their own inhaler technique. Patient education plays a central role in asthma care and needs to be given proper attention even though the inhalers might be considered intuitive and easy to operate. Interesting areas for future research include investigating interactive learning videos as a way of improving video education on inhaler technique.
  • Böhling, Linda (2021)
    Tablet is the most common pharmaceutical dosage form due to ease of administration, chemical and physical stability, and relatively low manufacturing cost. Direct compression is the preferred method for tablet production. Direct compression formulations typically contain a considerable amount of excipients. Therefore, excipients can have a significant effect on the tableting properties of formulations. More research is needed for better comprehension of the compression behaviour of different materials. The objective of this work was to investigate tableting properties of different excipients and their binary mixtures with two different laboratory scale tableting devices; the Gamlen® D1000 Powder Compaction Analyzer and the FlexiTab®. The excipients used were microcrystalline cellulose (MCC), lactose, mannitol, starch, and dicalcium phosphate (DCP). Different compression pressures were used to survey the compression behaviour of the excipients at a wide pressure range. In addition, potential effects of compression speed, dwell time, and lubrication method were considered. The excipients and their binary mixtures were characterised based on compressibility (solid fraction vs. compression pressure) and tabletability (tensile strength vs. compression pressure). The results obtained with the devices were compared to enhance process understanding. Based on the compressibility curves, it appeared that plastic deformation was the main compression mechanism of MCC and starch and fragmentation the main compression mechanism of lactose, mannitol, and DCP. The tabletability of MCC was excellent, and also the tabletability of mannitol was good. The tabletability of DCP was intermediate, whereas lactose and starch had inferior tabletabilities. In general, the tabletabilities and compressibilities of the binary mixtures were more or less what was expected based on the results of the individual materials. The results obtained with the different speed parameters and lubrication methods were mainly in line with the perceptions of the compression mechanisms of different materials. In overall, the results obtained in the Gamlen and FlexiTab experiments were quite similar. However, tensile strengths appeared generally slightly lower in the FlexiTab experiments. Probable explanations are the higher compression speed of the FlexiTab and differences in hardness measurements. This study indicated that the FlexiTab and Gamlen devices have different benefits. The Gamlen device is clearly very suitable for investigating tableting properties during formulation development, but the FlexiTab device has the advantages of higher compression speed and automatic powder feeding mechanism. Tabletability results were slightly better with the Gamlen, but more experiments are needed for solving the reasons (e.g. compression speed and hardness measurements). More information of the compression behaviour of different materials could be obtained by analyzing punch displacement data and by using different compression equations.
  • Kaugonen, Olga (2017)
    Investigating the role of cell membrane proteins has increased over the last decade, as drugdrug interactions and genetic polymorphisms have been found to cause changes in drug pharmacokinetics and dynamics. In this study the characteristics of the OATP1B1 transporter were reviewed and new in vitro research method to study protein functions was developed. Human Embryonic Kidney cells (HEK) is a human derived mammalian cell-line that is widely used in the study of OATP1B1 transporter. The Sf9 cell line is isolated from Spodoptera frugiperda insect and is one of the standard in vitro tools in a genetic engineering study. In the experimental part of this thesis the goal was to express OATP1B1 transporter in Sf9 and HEK293 cell lines. The wild-type SLCO1B1-gene encoding the OATP1B1 was virulent with baculovirus into the cells by the Bac-to-Bac® Baculovirus Expression System. For expression in the Sf9 cells, the aim of the study was to clone the SLCO1B1-gene into the pFastBac vector. The cloning was not successful in this study although attempts were made for several approaches. The expression of OATP1B1 transporter in HEK293 cells was successful. HEK293 cells expressing OATP1B1 transporter are well suited for the study of the SLCO1B1-gene. The in vitro method developed in this study remains in the research team as a tool to investigate the polymorphisms of the SLCO1B1-gene, the inhibition of the transporter and possible drug interactions.
  • Ryhänen, Tiia (2014)
    Nucleic acid amplification technologies are widely used in the field of molecular diagnostics and biological research for amplifying and detecting specific nucleic acid sequence. Polymerase chain reaction (PCR) is the most established nucleic acid amplification technology providing a fast, sensitive and highly specific method to amplify nucleic acids. But, as a "point-of-care" - diagnostic tool, PCR still remains limiting due to the need to use complex and heavy instrument. This disadvantage of PCR has lead to development of nucleic acid amplification technologies, which are possible to utilize with a simple and cost-effective device. PCR requires thermocycling between three different temperatures, while these isothermal technologies are possible to use in a constant and low temperature. Over ten different isothermal nucleic acid amplification technologies are described in the literature within past two decades. This literature review is focused on six of these existing isothermal technologies. They are briefly described and their potential applications to point-of-care diagnostics are discussed. The experimental part of the thesis focuses on improving the sensitivity, speed and robustness of isothermal nucleic acid amplification, SIBA technology. This was achieved by investigating the optimum configuration of the invasion oligonucleotide (IO) required dissociation of target DNA duplex. The invasion oligonucleotide contains of three functionally district regions; the seeding-, DNA- and a 2'-O- methyl RNA region. The IO can also contain a few non-cognate bases at the 3'end, inverted dt. It was researched how the length of the seeding- and DNA regions, and the ratio of pyrimidine in the seeding region affect to the amplification speed and specificity. SIBA reaction was tested also with IOs where the 2'-O- methyl RNA bases of the IO were partially replaced by 2'-fluoro RNA bases and the inverted dT removed. The results revealed that a higher ratio of pyrimidine in the seeding region speed up the nucleic acid amplification reaction, but too high ratio can also induce non-specific amplification. The optimal length of the seeding region lies somewhere between 3 and 18 bases. The optimal length of the DNA region is around 35 bases. This study also showed that the inverted dT blocker of the IO is unnecessary in SIBA, and by replacing 2'-O- methyl RNA with 2'-fluoro RNA bases is possible to improve the amplification of target template with mutations. Results presented in this thesis have helped to improve existing knowledge of SIBA-assay design. Future work could focus on investigating the impact of the IO's 2'-O- methyl RNA region.
  • Aaltonen, Kalle (2010)
    Rheumatoid arthritis is an inflammatory autoimmune disease with prevalence of 0,8 per cent of Finnish people. Rheumatoid arthritis may lead to immobility and premature death. Treatment of Rheumatoid Arthritis includes disease modifying anti-rheumatic drugs and surgery. TNF-blockers are efficacious new drugs, which halt the progression of joint destruction caused by inflammation. The first TNF-blocker to receive permission of the national agency for medicines was Infliximab in 1999. Since then Infliximab has been followed by Etanercept, Adalimumab, Golimumab and Certolizumab. TNF-blockers have been found to be more efficacious than placebo in both clinical trials and register studies. In addition they are considered to be safe enough for clinical use despite the increased risk for tuberculosis and certain cancers. The number of patients annually treated with TNF-blockers in Finland increased threefold between 2004 and 2008. In 2008 the medication costs per patient were 11 669€ for Etanercept and 13 074€ for adalimumab. Systematic literature review is a study, which searches, identifies and combines individual studies. Usually Systematic reviews include a meta-analysis, which uses statistical methods to combine the results of the studies. Meta-analysis aims for increasing power and generalisibility of the studies and reducing the potential bias in individual studies. In order not to introduce bias by itself the systematic review must be done following the methods approved by the scientific community. In addition the process must be documented in detail. Following a predefined search strategy the systematic literature search found 5308 references. After a process involving the evaluation of the patients, intervention, control, outcomes, study design and the risk of bias 27 studies were selected to be included in the systematic review and meta-analysis. Of the included studies, nine had adalimumab, six had etanercept, five had infliximab, four had golimumab and three certolizumab as intervention. TNF-blocker was used either alone or in combination with methotrexate whereas control was either placebo or methotrexate. Altogether, there were 11 533 patients in the intervention group and 9027 in the control group. The results of the meta-analysis indicate reveal that the patients treated with TNF-blockers are twice as likely to reach a 20 % increase on ACR criteria compared to control patients. The likelihood to reach improvements of 50 and 70 % was 3 and 3.5 times higher, respectively. There were no statistically significant differences in efficacy between individual TNFblockers. Increasing the dosage of a TNF-blocker did not increase efficacy. However, combination of TNF-blocker and methotrexate was superior to monotreatment of TNF-blocker without increasing the likelihood of discontinuation of treatment. There were no statistically significant differences between the efficacy of TNF-blocker monotherapy and methotrexate. Adalimumab, infliximab and certolizumab lead more often to treatment discontinuation compared to etanercept and golimumab, which do not differ from control. This systematic review probably found all studies that investigated the efficacy of TNF-blockers in a randomized controlled trial. Study selection and evaluation were based on widely accepted methods. This study has two weaknesses. Firstly, literature search and study selection and evaluation were done only by a single researcher. Secondly, unpublished studies and study results were not actively obtained outside electronic databases.
  • Pakarinen, Tiina (2014)
    Clinical pharmacy is defined as a service which a pharmacist provides for example to a ward or a medical center. In Finland clinical pharmacy (or ward pharmacy) was started in the 1980s but it hasn't expanded widely until at the end of the 2010s. Need for cost-effectiveness research has been under discussion because of increasing health care costs. This kind of research helps to choose the most effective services. Naturally also clinical pharmacy is under effectiveness consideration. A systematic review was conducted considering the cost-effectiveness research of clinical pharmacy. The aim of this review was to find clinical pharmacy interventions which have been proven costeffective. Literature research found 7 articles. Three of these studied pharmacokinetic patient surveillance and in the rest four articles pharmacist worked as a part of multidisciplinary team. In six studies the cost savings were greater than the costs. The other part of this study was about clinical pharmacy in the hospital district of Helsinki and Uusimaa (HUS). Data was collected from Helsinki University Central Hospital (HUCH) wards which had had clinical pharmacy services during the years 2009-2012. Collected data included clinical pharmacy costs and amount of work gained with those costs, drug consumption, drug waste amounts, amounts of drugs returned to HUS-pharmacy and amounts of HaiPro-reports. Collected data was presented as a time series. The costs of clinical pharmacy had followed the trend of other health care costs during 2009-2012. Wards with clinical pharmacy had somewhat larger amount of drug waste than the other wards. Amounts of drugs returned to the pharmacy were greater in the wards with clinical pharmacy. The amount of HaiPro-reports grew by a factor of 15 from 2009 to 2012. Especially amounts of drug administration errors and errors in writing down prescriptions were greater in clinical pharmacy wards. The data in this thesis describes only a small part of clinical pharmacists' work in the HUCH area. Making final conclusions about the cost-effectiveness of clinical pharmacy isn't possible with this data. The systematic review can give ideas to improve clinical pharmacy in HUS in a more cost-effective direction.