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(2021)Background: Poor health literacy (HL) is associated to increased hospitalization and decreased seeking for screenings. Shared decision making can increase patient knowledge, decrease anxiety over the care process, improve health outcomes and reduce health care costs. Little is known about factors influencing health literacy and participation in treatment decision making in different population groups. Objectives: To investigate factors predicting HL and participation in the treatment decision making. Methods: A cross-sectional population online survey conducted in Finland in 2019 by Finnish Medicines Agency. Both health literacy and participation in the decision making were assessed by three statements that sum variables were created with score 1-5 (Cronbach’s alpha value 0.584 and 0.810). Age, gender, education, household income and most common chronic diseases were chosen as possible predicting factors. Two-variable Pearson’s chi-squared test was first used to find significant factors followed by logistic regression analysis to take into account several variables. Results: Of all the respondents (n=2104) 76.5% had good HL and 73.4% had willingness to participate in the treatment decision making. In the two-variable test older age (p<0.001), lower education (p<0.001), lower household income (p=0.001), higher number of chronic diseases (p=0.003), having cardiovascular diseases (p=0.003), diabetes (p=0.029) and cancer (p=0.001) predicted poorer health literacy. Male gender (p=0.001), not having chronic diseases (p=0.001), not having a musculoskeletal disorder (p=0.050) or mental health disorders (p<0.001) predicted poorer participation in the treatment decision making. In the logistic regression analysis older age and having cancer predicted poorer health literacy. Male gender and not having mental health disorders predicted less willingness to participate in the decision making. Conclusions: Older age and cancer predicts poorer health literacy and male gender poorer willingness to participate in the decision making. Further research should focus on investigating more in detail the contributing factors to these findings, and how health literacy in elderly and men’s involvement to the decision making could be improved.
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(2016)Chronic myeloid leukemia (CML) is a malignant hematologic disorder, which is fatal without a treatment. Oral tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of CML and transformed the disease to a chronic condition that can be treated at patient's home. The common problem in the treatment of CML is patient's poor adherence to TKIs. The regular, consistent use of TKIs is crucial to keep disease under control. For this reason and to obtain an optimal treatment outcome, adherence to TKIs is extremely important. The aim of the study was to assess reasons for poor adherence to TKI-medications in Finnish CMLpatients, including patient characteristics, treatment related factors, comorbidities and concomitant medications. In addition, patients' experiences, beliefs, knowledge and perception about CML and its treatment were explored and how these could contribute to nonadherent behaviour. This study is part of the larger study, assessing adherence to TKI treatment among Finnish CML population. The data was obtained by using patient questionnaires and semi-structured theme-interview during patient meetings in 2012. Study population consisted of Finnish adult CML patients who had been on TKI -medication (imatinib, nilotinib or dasatinib) for more than six months prior to the study baseline. Patients' adherence was measured using Morisky Medication Adherence 8-Item Scale (MMAS-8) and based on their score, patients were divided into three groups: high, medium and low adherence. Both quantitative and qualitative methods were used in data analysis. Study findings show that 21% (n=18) of the patients were low adherent and 23% (n=20) were high adherent to their treatment. Patient sociodemographic characteristics or experienced adverse drug reactions (ADRs) did not predict adherence, while more concomitant medications and comorbidities were associated with high adherence. However, ADRs had negative effect on the quality of life of several nonadherent patients. All nonadherent patients reported unintentional nonadherence and the most common reason was forgetting. Two-thirds of the patients (n=12) reported intentional nonadherence, which often was a result of experienced ADRs. The knowledge of CML and its treatment was poor among all patients while over half of the nonadherent patients (n=11) thought that they received enough information received. Overall, patients were very satisfied with care provided by the hospitals, physicians and other healthcare professionals. Managing TKI-treatment regimen is challenging for many patients and ADRs can have a negative impact on the quality of life. Healthcare professionals should regularly assess patient adherence and provide information and support for the patients to help them to succeed in medication management. Reasons for poor adherence are complex and have to be identified from each individual patient so that adherence can be improved.
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(2019)Pharmaceutical companies are required to comply with fair market guidelines and regulations. However, definition of fair market value (FMV) in clinical trial is not unambiguous. In literature are some suggestions how to determine the phenomenon of FMV in clinical trial. Understanding the FMV and how it should be applied into practice when conducting clinical research is challenging. This study provides more focused information on FMV in clinical trials and its determination. FMV should be determined for research-related activities in clinical drug research. FMV of research related activities can be consistent if similar sites are performing similarly conducted studies for similar sponsors. Therapeutic area and geographical location of the trial site can also influence for the FMV. This study was performed in co-operation with Roche. The aim of the study was create a consistent and transparent method to assist in the determination of FMV in medical drug research in relation to the payments paid by the sponsor to the sites. Clinical trial agreements (CTA) and associated agreements were analysed to investigate FMV of research-related activities by study site, study type, therapeutic area and geographical area. Average price and price range of each research-related activity from previous CTAs and associated agreements of Roche Finland was calculated. Based on available data from literature and study results research-related activities and factors affecting to the FMV of clinical trials were discussed to create comprehensive understanding of FMV in clinical drug research. Based on this study average price of the specific research-related activities can be different by therapy area, site, study type and geographical area. All these factors are relevant when assessing FMV of specific research-related activity. Studied therapy area and site seems to have the most important impact when evaluating FMV. For some research-related activities such as national coordinator investigator (NCI) fee price ranges could be very big whereas in other research-related activities such as pharmacy fees prices could be quite similar. Some research-related activities were very study specific which affected evaluation of those activities. CTAs and associated agreements are valid documents to gather information assessing FMV of research-related activities in medical drug research. Average price and price range of the research related activity can be used when assessing FMV in medical drug research. However, price of the specific research-related activity need to be evaluated considering the studied therapy area, site, study type and geographical area.
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(2020)Pharmacogenomic test are laboratory tests that are performed in order to find out what kind of a variable response to a specific drug is most likely for a patient, or in order to determine the background of a deviating adverse drug reaction. Individual drug responses can be caused by a variation in a gene that codes for a protein that is involved in the pharmacokinetics or the pharmacodynamic response of the drug. These genes are called pharmacogenes. Pharmacogenomic tests are most commonly DNA tests that look for the most frequent variations in the pharmacogenes or variations that are associated with variable drug response even if located in the non-coding region of the DNA. Pharmacogenomics is a rapidly emerging branch in scientific research, and the number of known pharmacogenes and available pharmacogenomic tests is constantly growing. Pharmacogenomic tests can be helpful in avoiding ineffective medication, and decrease the probability of severe adverse drug reactions. Several international specialist consortiums, such as Clinical Pharmacogenetics Implementation Consortium (CPIC) and Dutch Pharmacogenetics Working Group (DPWG), have published guidelines regarding dosing of spesific drugs or drug classes when a patient has a certain variation in one of known pharmacogenes. Due tothe amount of knowledge constantly increasing, use of pharmacogenomic tests is believed to expand in the healthcare. The aim of this study was to examine the use of pharmacogenomic test in Finnish healthcare during years 2016 and 2017. A questionnaire was send to Finnish laboratories by email. Purpose of the questionnaire was to collect information of the amount of available and performed pharmacogenomic tests and respondents opinions regarding the future of pharmacogenomic tests. Questionnaire was carried out in co-operation with Ministry of Social Affairs and Health and Division of Pharmaceutical Biosciences in University of Helsinki, using Webropol survey tool. The survey was renewed the following year, without major alterations to its content. Unfortunately, due to the somewhat low response rate of the survey in both 2016 and 2017, the results of the survey cannot be considered to sufficiently represent the collective views of the target group. However, from the giver replies we can rather reliably conclude that pharmacogenomic tests were not used in high volume during years 2016 and 2017. The laboratories reported mainly very small amounts of performed tests. The most abundantly reported test was TPMT gene test, regardless of the year. In spite of the low test volume, respondents were, however, widely unanimous that the use of pharmacogenomic test and their significance in healthcare are very likely to increase in the near future.
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(2017)The effect of genes on drug response is studied in the field of pharmacogenetics. Genetic polymorphism occurs in several genes that code drug metabolizing enzymes or drug transporters. A protein coded by a variant gene may be dysfunctional, which can affect the efficiency and safety of the substrate drug individually. The common polymorphisms of the gene ABCG2 coding the efflux transporter BCRP and the gene SLCO1B1 coding the influx transporter OATP1B1 are associated with the interindividual variation in the effectiveness and tolerability of the cholesterol-lowering statins. In this study, the effects of the polymorphisms ABCG2 c.421C>A and SLCO1B1 c.521T>C on rosuvastatin concentration in plasma and the liver were studied with two different pharmacokinetic models. The developed liver model illustrating the enterohepatic circulation of drugs was compared to a commercial Simcyp model. According to the simulations with both models, the effect of the polymorphisms of OATP1B1 and BCRP on the plasma concentration of rosuvastatin is additive. The plasma concentration increases up to fourfold if the same individual has homozygous polymorphic forms of both the OATP1B1 and the BCRP. Based on the modellings, the change of the rosuvastatin concentration in the liver owing to polymorphism does not follow the same pattern as in plasma. In consequence of the polymorphism of the BCRP, the rosuvastatin concentration rises two to three times larger in the liver, which is the site of action of the statins. The polymorphism of the OATP1B1 instead causes the liver concentration to decrease little compared to the wild type. In conclusion, the efflux transporter BCRP seems to have a greater significance on regulating the concentration of rosuvastatin in the liver than the influx transporter OATP1B1. Computer modelling is worth exploiting as a supportive method of other study methods in the pharmacogenetic research, for example when the relative significance of separate transporter proteins is evaluated.
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(2019)Farmasian ammattilaiset ovat lääkealan asiantuntijoita, joilta vaaditaan uudenlaista osaamista muun muassa teknologiakehityksen myötä. Nykypäivän asiantuntijuus edellyttää alakohtaisen eli sisällöllisen osaamisen lisäksi geneerisiä eli yleisiä taitoja ja ammatti-identiteetin muodostumista. Geneerisillä taidoilla tarkoitetaan yleishyödyllisiä taitoja, kuten ongelmanratkaisu- ja kommunikointitaitoja. Ammatti-identiteetillä tarkoitetaan käsitystä omasta työminästä, jonka avulla omaa roolia ja työnkuvaa järkeistetään. Näiden elementtien muodostamaa osaamisen kokonaisuutta kutsutaan kompetenssiksi. Asiantuntijoilta vaadittavan osaamisen muutos on ohjannut yliopistoja vastaamaan paremmin työelämän tarpeisiin. Helsingin yliopistossa toteutettiin Iso Pyörä -koulutusuudistus, jossa koulutusohjelmia uudistettiin komeptenssilähtöisesti. Kaikkiin koulutusohjelmiin ja opintojaksoihin lisättiin osaamistavoitteet, jotka opiskelijoiden tulisi saavuttaa valmistumiseensa mennessä. Osaamistavoitteiden täyttymistä edistää esimerkiksi portfoliotyöskentely, minkä avulla opiskelijat pääsevät hyödyntämään ja kehittämään reflektiotaitojaan. Opiskelijat voivat tuoda opetuksen kehittämiseen aivan uudenlaista näkökulmaa avatessaan käsityksiään esimerkiksi hyvistä opetusmenetelmistä, mitkä ovat auttaneet heitä saavuttamaan laaditut osaamistavoitteet. Toisaalta opiskelijoiden näkökulmasta saadaan tietoa, mikä osaaminen voidaan kokea puutteelliseksi, jolloin opetuksen kehittäminen on mahdollista. Tutkimuksen tavoitteena oli selvittää opiskelijoiden käsityksiä omasta osaamisestaan ja ammatti-identiteetistään sekä millä tasoilla opiskelijat reflektoivat osaamistaan. Tutkimuksessa analysoitiin vuoden 2017 kolmannen vuosikurssin kandiportfolion loppureflektioesseet käyttäen aineistolähtöistä sisällönanalyysimenetelmää. Esseissä opiskelijat reflektoivat osaamistaan suhteessa farmaseutin tutkinnolle asetettuihin osaamistavoitteisiin ja pohtivat omaa ammatti-identiteettiään. Tulosten mukaan opiskelijat saavuttivat monipuolista osaamista lääkkeiden ja lääkehoitojen näkökulmasta sekä kehittivät geneerisiä taitojaan. Puutteellisesti hallittiin useimmiten kielitaito sekä yrityksen ja yhteiskunnan taloudelliset periaatteet. Opiskelijoiden mukaan farmaseutin ammatti-identiteettiä määrittelevät erityisesti lääkeosaaminen ja terveydenhuolto sekä ammatin arvostaminen. Opiskelijoiden pohtimat valmiudet mukailivat osaamistavoitteita. Opiskelijat osasivat arvioida omaa osaamistaan ja nostaa esille vahvuuksiaan ja heikkouksiaan. Opetussuunnitelmaan on onnistuttu sisällyttämään geneeristen taitojen opetus, sillä opiskelijat kokivat saavuttaneensa näitä taitoja pääasiassa hyvin. Opetusta tulisi kehittää kielitaidon ja liiketalouden kohdalla, sillä nämä koettiin usein puutteellisesti hallituksi. Ammatti-identiteettikäsitykset mukailivat kirjallisuutta, sillä muissa tutkimuksissa on saatu samankaltaisia tuloksia.
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(2014)In early clinical trials of drug development capsule is preferable compared with other solid dosage forms, including tablets, because of its simplicity and blinding capabilities. However a simple capsule formulation is not viable in large-scale production. Usually it's either switched to an economical tablet formulation or to another capsule formulation that can be manufactured on large-scale equipment. Tablets are nearly always formulated for commercialization if they're not technologically impossible to manufacture for a reason which could be bad compression or solubility of a drug. Tablet-pressing process sets more-demanding requirements for pharmaceutical powder properties than encapsulation process, because tablet-press uses larger compression forces and it measures the dose in a different way. The most common problems faced when switching from a capsule dosage form to tablet-pressing process are poor powder flow properties and weak mechanical strength, capping and lamination of tablets. The purpose of this work was to investigate the critical pharmaceutical and technical properties to succeed in switching from a capsule dosage form to a tablet dosage form and tablet-pressing process. The starting point of this work was a simple capsule formulation consisting of carbidopa, directcompressible mannitol and pregelatinized maize starch. The simple formulation was used to build up two mixture designs consisting of very different powder properties to study the critical powder properties and process variables involved in the switch. The capsules were filled on a dosating nozzle capsule-filling machine and the tablets were pressed on a pneumohydraulic tablet press. Weight variability, disintegration time, encapsulation and tabletability were used as the responses of the dosage forms. As a result of the study the automatic capsule-filling machine filled many different types of powders in capsules with low fill-weight variability. The most critical powder properties affecting the capsulefilling process were the particle size, bulk and tapped densities and cohesion of powder. Avicel PH200 improved the tabletability and flowability of the powders, but it also increased the fill-weight variability of the capsules. In this work single powder flow properties described only the flowability of a powder, but they did not determine the performance of a powder in the processes. Therefore, measuring various powder flow properties and correlating them to a manufacturing system is necessary to understand the process. Avicel PH102 was proved to work as an ideal reference material for evaluating the sufficiency of the flow properties of a powder in the tablet-pressing process.
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(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.
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(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.
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Flavonoids act as positive allosteric modulators of lecithin:cholesterol acyltransferase in silico (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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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