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  • Heinonen, Pia (2021)
    Oxygen has been used as a medicine since the 18th century and is widely used as prescription and over-the-counter (OTC) medicine. Especially with global COVID-19 pandemic, which started in 2020, the demand for medicinal oxygen has increased significantly and the quality of medicinal oxygen has become increasingly important. Only few studies have been published on the critical process and quality parameters of gases and their impact on product quality. Because oxygen is classified as a medical product, it must be manufactured in accordance with good manufacturing practices regulations (GMP). One part of EU and other GMP guidelines is mandatory annual product quality review (PQR) which must assess the critical quality and process parameters as well as their trends. The aim of the study was to define the critical process and quality parameters for the medicinal oxygen filling process and to analyze the process control, stability, and capability for annual PQR using process data. Process stability and the state of control of processes were assessed using statistical quality and process management tools, such as the Shewhart control diagram and process capability index. Studied process parameters included vacuum level and pressure measured by the filling equipment. Evaluated quality parameters included analysis pressure, O2 and H2O contents. The results of the study showed that the process is not stable and there was a lot of variation between the parameters. Most variation was detected between different cylinder volumes and filling and analysis ramps in all parameters and between different weekdays in H2O content. However, all parameters remained within the specification limits and the Cpk values of all critical parameters were good. By analyzing the data, many variables that can affect the parameters and add variation to the process data can be identified. Based on the results, the necessary measures to improve and optimize the process and quality was identified. In order to stabilize processes and improve performance, the demonstrable variation in process data should be reduced, for example by harmonizing operating methods. According to the study, it was also possible to assess the revalidations required for the process.
  • Krannila, Elina (2012)
    In pharmaceutical industry GMP compliance and quality of operations can be ensured with quality management system (QMS). QMS is an operational system, which consist of multiple different elements depending on the size of the company and nature and complexity of its operations. For the QMS to be functional, documented and defined operations need to be managed and monitored systematically. Conducting internal audits has been considered necessary with regard to QMS, though it has not always been perceived as adding value or seen as an opportunity to utilise more fully. Internal audits are mainly utilized to control compliance to requirements. However, there are possibilities to utilise it more in improving and developing operations, preparation to external audits, quality risk assessment, finding out the best practices, basis for decision making, learning experience as well as the assessment of functionality and effectiveness of the QMS. The aim of this study was to examine the utilisation of internal audits in Orion (Espoo) and find solutions to improve the utilisation of internal audits with QMS. The focus was on how internal audits can monitor and guide QMS and what is required from internal audits for monitoring and guidance of QMS. These aims were approached qualitatively by conducting semi-standardized open-ended interviews. Interviewees (n=9) were selected from both auditor and auditee side and they had their background in quality assurance or production. Data compiled from these interviews was analysed mainly by qualitative methods, using also some quantitative analysis. Monitoring of the QMS can be looked at as the starting point to guide QMS. Valuable information can be gathered with internal audits with regard to QMS. By utilising this information, internal audit process and QMS can be improved and the quality of operations can be ensured. Based on this work internal audits can be utilised to monitor and have the potential to guide QMS under certain conditions. Internal audit topics need to be systematically selected, QMS needs to be monitored and guided based on the internal audit findings, flow and distribution of information needs to be efficient and flexible, and internal audits should be better utilised and managed. Further research is needed on the development and deployment of tools to aid better utilisation of internal audits in the control of QMS. Also ways to measure the effects of internal auditing should be further investigated.
  • Keskimäki, Sanne (2023)
    Plasmidit ovat geneettisiä elementtejä, joita voidaan käyttää esimerkiksi geeninsiirtovektoreina. Transposonit ovat DNA-fragmentteja, joilla on kyky siirtyä genomissa paikasta toiseen. Tutkimuksessa käytettävä transposoni on piggyBac, joka on eristetty tupsumetalliyökkösen (Trichoplusia ni) soluista. Transpositiossa piggyBac tunnistaa ITR-osat (käännetty terminaalinen toistojakso) siirtäen osien välissä olevan DNA:n. Tutkimuksen tavoitteena oli tuottaa kaksi erilaista plasmidia. pAc5.1-piggyBac-plasmidiin sisällytettiin piggyBac ja pMT-In-EGFP-PB-ITR-plasmidiin ITR-osat sekä niiden väliin hygromysiiniresistenssigeeni sekä EGFP-geeni. BTI-Tn-5B1-4-solujen DNA:sta eristettiin piggyBac sekä ITR-osat ja ne siirrettiin plasmidiin pTOPO-piggyBac-R. Tästä plasmidista irrotettiin erilleen piggyBac ja ITR-osat, joista välivaiheiden kautta rakennettiin lopulliset plasmidit. Plasmidit rakennettiin pitkälti pilkkomalla DNA-fragmentteja restriktioentsyymeillä ja yhdistämällä niitä ligaatiolla. Plasmideja tuotettiin suurempia määriä siirtämällä niitä transformaation avulla E.Coli-soluihin lämpöshokkimenetelmällä ja eristämällä tämän jälkeen saadut plasmidit. Tuotettujen plasmidien onnistuminen varmistettiin pilkkomalla ne restriktioentsyymeillä ja tutkimalla DNA-fragmenttien kokoa agaroosigeelielektroforeesilla. Plasmidinäytteet myös sekvensoitiin osittain. Banaanikärpäsen (Drosophila melanogaster) S2-solut transfektoitiin kehitetyillä plasmideilla ja solukonsentraatioita sekä elinkelpoisuutta mitattiin 8 päivän ajan transfektion jälkeen. Tavoitteena oli hyödyntää EGFP-geeniä fluoresenssimittauksiin. Solunäytteisiin lisättiin kokeen aikana hygromysiini, jotta voitiin selvittää, olivatko viljellyt solut saavuttaneet hygromysiiniresistenssin. Tutkimuksen tuloksena plasmidit saatiin kehitettyä, mutta solukokeiden tulokset jäivät epäselviksi. Solunäytteissä ilmeni kasvatuksen aikana kontaminaatioita. Lisäksi EGFP-osia ei voitu luotettavasti mitata käytössä olleella laitteella. Transfektio tulee siis toistaa transposonisysteemin toiminnan tutkimiseksi. Lisäkokeilla voidaan selvittää tarkemmin kehitetyn transposonisysteemin mahdollisuuksia sekä toiminnan yksityiskohtia.
  • Tilli, Irene (2017)
    Melanoma is the most severe case of skin cancer and there is no curative treatment if it has progressed. Despite the recent advances in drug therapy tens of thousands of patients die of melanoma annually. There is still need for new antimelanoma drugs for which marine compounds are a potential source. Halogens are common elements in drug molecules as they enhance their molecular properties. So far most of the halogenated drugs contain fluorine and/or chlorine but the role of bromine and iodine is probably growing in the future due to halogen bonding. Bromotyrosines are originally isolated from Verongiida-order sponges but whether they are truly of bacterial origin is under controversy. All bromotyrosine compounds consist of brominated tyrosine and/or tyramine residues or their derivatives. Purpurealidin I is one of the newest bromotyrosine derivatives extracted from Pseudoceratina purpurea and it has shown activity against melanoma. In this study eight new purpurealidin I derivatives were synthesized following a successful route previously designed. All synthesized derivatives contained the original N-oxime structure which's stereochemistry was determined to be E by X-ray crystallography. Cytotoxicity against A375 melanoma cells was determined for seven compounds synthesized here and for 15 compounds synthesized previously. All seven compounds and one previously synthesized purpurealidin I analog were active with CC50 values between 4,7 and 22,1 µM. The previously synthesized bromotyrosine derivative intermediates and aerophobin-1 analogs were not active. The selectivity of the active compounds was calculated by determining their CC50 value against Hs27 fibroblast cells. None of the compounds showed remarkable selectivity the most selective 2-pyridin containing derivative having four times better selectivity against melanoma. The tyrosine part and N-oxime seem to be important parts to preserve while the tyramine part can be modified more freely and maintain the activity. Still more derivatives need to be synthesized and tested to confirm these observations. More data is also needed considering the selectivity of the compounds.
  • Zwiers, Harry (2021)
    Membraanipyrofosfataasit eli mPPaasit katalysoivat pyrofosfaatin hydrolyysiä kahdeksi ortofosfaattimolekyyliksi vapauttaen samalla energiaa. mPPaasit ovat merkittävässä roolissa useiden patogeenisten alkueläinloisten mahdollisuudessa selvitä ulkoisesta osmoottisesta stressistä ja pH:n vaihteluista, joiden lisäksi mPPaasit vaikuttavat niiden kasvuun ja virulenssiin. mPPaaseja ei toistaiseksi ole löydetty ihmisistä eikä eläimistä, jonka vuoksi ne ovat mielenkiintoisia lääkevaikutuksen kohteita. Tässä työssä syntetisoitiin 9 uutta yhdistettä, joita ei ole aiemmin raportoitu kirjallisuudessa. Johtomolekyyleinä käytettiin aiemmassa tutkimuksessa löydettyjä isoksatsolijohdannaisa, jotka inhiboivat T. maritiman mPPaasia IC50-arvoilla 6‒7 μM (Johansson ym. 2020). Uusiin yhdisteisiin liitettiin formyyliryhmiä useaan eri kohtaan, joiden avulla toivotaan saavan lisää tietoa niiden sitoutumisesta mPPaasiin ja niiden rakenne-aktiivisuussuhteista. Uudet yhdisteet tullaan testaamaan aktiivisuuden varalta in vitro T. maritiman mPPaasissa ja tarvittaessa myös P. falciparumissa. Formyyliryhmät liitettiin isoksatsolirenkaiden 3-, 4- ja 5-asemiin erilaisten substituenttien välityksellä. Isoksatsolirenkaan 3-aseman karboksylaattiryhmään esteröitiin kolme eri bromiformyylifenolia formyyliryhmän paikkaa vaihdellen. Isoksatsolirenkaan 4-asema halogenoitiin mikroaaltoavusteisesti, jonka jälkeen liitettiin formyyliryhmiä sisältäviä heterosyklisiä booriyhdisteitä Suzuki-reaktioilla. Myös isoksatsolirenkaan 5-asemaan sitoutuneeseen fenyylirenkaaseen liitettiin formyyliryhmän sisältävän yhdisteen Suzuki-reaktiolla. Uusien yhdisteiden aktiivisuuskokeiden tulokset julkaistaan myöhemmin. Yhdisteiden rakenne-aktiivisuussuhteisiin tai tehokkuuksiin ei ole tässä työssä vielä mahdollista ottaa kantaa.
  • Saharinen, Janne (2020)
    Prolyloligopeptidase (PREP) and alphasynuclein are linked to various neurological and psychiatric conditions of which the most relevant considering this study is parkinsonism. PREP cleaves small peptides after a proline residue. It has also protein-protein ineractions with alphatubulin, GAP-43 and alphasynuclein. PREP inhibitors have been shown to have an effect to elimination of alphasyuclein via autophagy. Thiazole is a heteroaromatic compound with two heteroatoms (sulphur and nitrogen). Thiazole can be found as a structural component among various active pharmaceutical ingredients with wide array of indications. Synthetic route for thiazole was published in 1887 and a considerable amount of literature regarding the use of thiazole in medicinal and synthetic chemistry has been published. The aim of the study was to extend the the scope of research done in the research group on small-molecular thiazole-based PREP inhibitors. The goal was to develop a synthetic route to access a series of molecules and gain information of the possible biological activities of the produced compounds by determining their IC50-values in vitro, effect on dimerization of alphasynuclein and removal of alphasynuclein via autophagy in a cell culture. Optimization of the synthetic route and search of alternative reactions were among the aims to some extent. During the course of study yields of some steps of the synthesis were improved and some new molecules had biological activity.
  • Ikonen, Jasmina (2016)
    In tablet compression the objective is to obtain a durable tablet. The main deformation mechanism of substance affects how good tablet is obtained. The pharmaceutical powders is often divided into two categories with respect to their principal deformation mechanism: plastic and fragmented. Good tablet formulation requires its components to deform with both of these mechanisms. It is possible to examine in many ways, whether material is plastic or fragmented. These include force-time graphs and indentation methods, as well as different compression equations such Heckel equation. Examination and identification of the deformation mechanisms is important in order to design a formulation which provides the most durable tablet. The aim of experimental work in this study was to test the new compression device and method, and to compare the results of the device shown in the earlier literature results. Comparison with previous research, new in this study was compression rate and without a motor acting compaction system. In this study, there was two compression method developed, dynamic and static. Data from a dynamic method were analysed by time-travel - and force-displacement -curves. Results were parameterized, and on the basis of these parameters the behaviour of various materials was evaluated and compared to the earlier literature. Relaxation study was also performed in this research. The results of these measurements were analysed with the parameterized function fit, after which the results were compared with earlier results presented in the literature. The results of this work in dynamic measurements are cosistent with the research results received earlier. In terms of almost all parameters investigated, substances were divided into two groups in the same way as in the previous literature on the basis of the main deformation mechanism. The results obtained in static measurements, however, were quite inconsistent with previous research. Based on the results it can be stated that the method makes it possible to get consistent results with the literature. However, the method still requires development, and possible error sources and the choice of analytical method should pay special attention.
  • Takala, Anna (2019)
    Medication safety is a part of patient safety, and means safety related to the use of medicines. Medication safety covers the principles and functions of individuals and organizations working in the healthcare sector to ensure the safety of drug treatment and to protects patient from harm. Medication error is any preventable event that may cause or lead to inappropriate medication use or patient harm while the medication is in the control of the healthcare professional, patient or consumer. Medication errors are the leading cause of preventable harm in health care across the world. Therefore, improving medication safety is important from the point of view of the promotion of patient safety. The aim of this study was to gather information about serious medication errors at national level by utilizing data from Valvira to learn from the cases outside the organizations where they occurred. The data of this study consisted of complaints and regulatory statements resolved by Valvira in 2013–2017, in which drug treatment were identified as a main reason and where inappropriateness was found (n=58). Cases were classified with predetermined classification system, and inductive content analysis was used to identify the causes and contributing factors of medication errors. The theoretical framework of the study was the Human Error Theory by James Reason (1990). According its systems-based approach, this study focused on the processes and circumstances of organizations. Of the included 58 cases, medication errors caused patient’s death in 21 cases (36 %) and severe harm in nine cases (16 %). A majority (n=53; 91%) of the errors were estimated to be either definitely or possibly preventable. Most of the patients were older adults (mean age 74 years). The most commonly related drugs in medication errors were enoxaparin (n=7; 6%) and oxycodone (n=7; 6%). The most common therapeutic group causing medication errors was antithrombotic agents (n=17; 13%). Most errors occurred in hospital settings (n=29; 45%) and in elderly care units. Doctors (n=37; 50%) were most often involved in the errors. Most of the medication errors occurred in the prescribing (n=38; 47%), administrating (n=15; 19%) and monitoring stage (n=14; 17%), drug-related problems being most often connected to the drug selection. In severe and fatal cases, there are often several drug related problems identified at different stages of the patient’s drug treatment process. The data of Valvira provide valuable information about medication errors at national level. Qualitative analysis is important especially for learning purposes as it provides better understanding of the causes and contributing factors of medication errors, as well as the complexity of drug treatment processes. Based on this study, it seems that healthcare organizations involved in severe medication error cases have taken into consideration the importance of process development and focused on identifying latent risks in organizational conditions and processes rather than blaming individuals.
  • Valkonen, Minna (2013)
    The aim of this work was to investigate the feasibility of titanium dioxide (TiO2) photocatalysis for imitation of phase I metabolism of selected anabolic steroids. The role of the solvent composition and the time of UV exposure in the TiO2 photocatalysis were also investigated. TiO2 photocatalysis has been reported to produce mainly the same phase I reaction types formed in drug metabolism in vitro and in vivo. The selected anabolic steroids were testosterone, methyltestosterone, metandienone, nandrolone and stanozolol. Products from TiO2 photocatalysis were compared to products formed in microsomal incubations (HLM). Comparison was made on the basis of same mass, retention time and similarity of the product ion spectra. The samples were analyzed with ultra performance liquid chromatography (UPLC) and quadrupole time-of-flight mass spectrometry (Q-TOF). Electrospray ionization (ESI) in positive ion mode was used for ionization and product ion scan with two different collision energy was used for collision induced dissociation of the steroids and the reaction products. TiO2 photocatalysis is a simple and fast method. For all the steroids studied, the main reactions observed both in TiO2 photocatalysis and microsomal incubations were dehydrogenation, hydroxylation and combination of these two. Several isomers with same mass and retention time were formed. In addition, dihydroxylation and dihydroxylation+dehydrogenation products of stanozolol were observed both in TiO2 photocatalysis, but these were different isomers in different systems. In most cases the product ion spectra of isomers with same retention time were similar but the weak intensity of some peaks caused uncertainty in the interpretation of spectra. TiO2 photocatalysis might be useful in fast screening of possible drug metabolites. However the feasibility of TiO2 photocatalysis needs to be further studied because the differences in stereochemistry in TiO2 photocatalysis and microsomal incubations. If TiO2 photocatalytic reactions can be scaled up, it might be possible to produce standard compounds for example for doping laboratories.
  • Kokkala, Katja (2010)
    The characteristics of macrolides are discussed in general level in the theoretical part of this Master's thesis. The discussion is focused on the properties of two macrolides in molecular level and their tendency to form tautomeric forms highlighting the structural similarities and differences of these macrolides, which will affect both the mechanisms of action and the metabolism. Attention is also paid to biosynthesis and manufacturing process keeping focus on downstream process, especially the impurities, which arise from the macrolide biosynthesis. Also the principles of argentation chromatography are discussed. In the experimental part of Master's thesis a purification method for one macrolide was developed using argentation chromatography. Conventional chromatographic purifications cannot separate the macrolide from its impurities. The purity of the macrolide after argentation chromatography was 98.6%. Also a new crystallization method was developed, which produces anhydrous form of the macrolide instead of traditional monohydrate form. A method for analysing the macrolide using HPLC was developed. The method was validated according to ICH guidelines. The tautomeric forms and the impurities of the macrolide were analysed using LC/MS. One of these impurities was isolated and analysed with NMR thus confirming its identity. An analysed NMR spectrum of this impurity has not been published according to our best knowledge. A previously unknown impurity was identified based on MS analysis and retention time.
  • Säilä, Pasi (2016)
    Oxysterols and vitamin D related compounds are found to be biologically active in brain. They might be involved in different psychiatric and neurodegenerative diseases. These compounds have traditionally been analysed from tissues using somewhat laborious and time-consuming gas chromatograpy and liquid chromatography mass spectrometric methods. To the side of these methods ambient desorption ionization methods have been developed. The advantage of these methods is rapid and easy operation. Usually minimal or no sample pretreatment is required. In addition these methods can be applied to imaging of for example tissues. The aim of this work was to study if it is possible to detect certain oxysterols and vitamin D related compounds from rat brain tissue samples with desorption atmospheric pressure photoionization (DAPPI). The compounds chosen to this study were cholesterol, vitamin D3, 25-hydroxyvitamin D3, 7-dehydrocholesterol, desmosterol and 7-ketocholesterol. DAPPI is especially suitable for efficient ionization of this kind of neutral and non-polar compounds. Detected MS and MSn spectras of the brain tissue samples were compared to those obtained from standard compounds. As a result we could not detect vitamin D3, 25-hydroxyvitamin D3, 7-dehydrocholesterol, desmosterol from rat brain samples with DAPPI. Excluding vitamin D3 it is possible that those other analytes are present at the spectras of brain samples but there is some other compound with same mass which makes the reliable identification of studied compounds impossible. 7-ketocholesterol and cholesterol were the only compunds we detected from brain tissue sections. 7-ketocholesterol can be formed via auto-oxidation in samples containing excess amount of cholesterol. According to this study it is impossible to say if the detected 7-ketocholesterol is formed endogenously or during sample preparation and analysis.
  • Rimmistö, Riikka (2024)
    Tieteen kehittymisestä huolimatta tehokkaiden syöpähoitojen ongelmana on niiden vakavat systeemiset haittavaikutukset, jotka voivat johtaa jopa hoidon keskeyttämiseen. Haittavaikutusten vähentämiseksi lääkehoitoa on pyritty kohdentamaan paremmin syöpäkudokseen esimerkiksi valoaktivoitavien liposomien avulla, jossa liposomiin pakatun valoherkistimen altistaminen valolle aiheuttaa muutoksia liposomin lipidikaksoiskalvoon siten, että lääkeaine pääsee vapautumaan liposomista kohdennetusti säteilyn alueella. Verteporfiini-valoherkistintä on tutkittu paljon hydrofiilisten lääkeaineiden vapauttamisessa liposomeista, mutta lipofiilisten lääkeaineiden vapauttamiseen liittyviä tutkimuksia on julkaistu hyvin vähän. Tämän maisterintutkielman tarkoituksena oli selvittää, voitaisiinko verteporfiinia hyödyntää lipofiilisen, solunsalpaajana tunnetun paklitakselin vapauttamisessa liposomeista valoakivaation avulla. Hypoteesin mukaan myös sytotoksisia vaikutuksia omaavan verteporfiinin yhdistäminen samaan liposomiin paklitakselin kanssa lisäisi valoaktivoitavien liposomien sytotoksisuutta in vitro. Tutkimuksessa arvioitiin valoaktivoidun verteporfiinin aiheuttamien liposomin lipidikaksoiskalvon muutosten suuruutta hydrofiilisen kalseiinin vapautuskokeen avulla, mitä käytettiin liposomiformulaation optimoinnissa. Lisäksi tutkimuksessa selvitettiin valoaktivaation seurauksena liposomeista vapautuneen paklitakselin määrää sekä arvioitiin valoaktivoitavien paklitakseli-verteporfiiniliposomien sytotoksisuutta A549-keuhkosyöpäsolulinjalla. Tutkimuksessa osoitettiin verteporfiinin valoaktivaation lisäävän muutoksia liposomin ja solujen kalvorakenteissa valoaltistuksen keston pidentyessä. Kuitenkaan paklitakselin lisääminen verteporfiiniliposomeihin ei vaikuttanut lisäävän liposomien sytotoksisuutta A549-soluissa valoaktivaatiota käytettäessä, mutta ei myöskään ilman valoaltistusta. Paklitakselin vapautuskoe ei tutkimuksessa käytetyllä menetelmällä onnistunut, joten syytä sille, miksi sytotoksisuuseroa ei havaittu paklitakseli-verteporfiiniliposomien ja pelkkää verteporfiinia sisältävien liposomien välillä, oli vaikea arvioida. Käytetyillä valoaltisteilla ja liposomiformulaatiolla paklitakselin vapautuminen liposomeista valoaktivaation seurauksena oli kuitenkin todennäköisesti erittäin vähäistä.
  • Nurmi, Riikka (2017)
    Liposomes are spherical nano-sized drug delivery systems which are composed of lipid bilayer. With liposomes drugs can be targeted for example to tumours and targeting can be passive or active. Drug release from liposomes can also be activated by different methods. Light is very promising triggering method, because it enables drug release at specific time and site. This study examined light activated indocyanine green (ICG) liposomes. Drug release from liposomes happens because ICG converts light energy to heat. ICG is clinically approved imaging agent, so ICG liposomes are very promising drug delivery systems even for clinical use. Liposomes were prepared by thin-film hydration method. One aim of the study was to prepare as small ICG-liposomes as possible. The bigger 100 nm liposomes were studied in three different formulations and the purpose was to find differences between those formulations. In formulation A ICG was in PEGs, in formulation B ICG was in lipid bilayer with no PEGs and in formulation C ICG was supposed to be in lipid bilayer although the formulation C included PEGs. In this study, the cell up take of ICG liposomes was studied with pharmacokinetic model and data from in vitro studies was supposed to use in a pharmacokinetic model. In this study, it was possible to prepare 40 nm sized ICG-liposomes. Small liposomes did not release encapsulated calsein as well as bigger 100 nm liposomes. The decreased release from smaller liposomes was probably explained by the results witch pointed out that transition temperature of small liposomes was higher than transition temperature of bigger liposomes. In the future, the lipid composition of the small liposomes need to be reoptimized, that the release would be more effective. This study however proved that small ICG-liposomes can be prepared and the small size lasts even over three months. Three different formulations of 100 nm liposomes were studied and the differences between the properties of the formulations were found. ICG in the lipid bilayer changed properties of the formulation B and the passive release of the calsein and release during the lightning were increased. In formulation C transition temperature was decreased and its storage life was lower than in other formulations. Formulation A was best for the next studies and the phospholipid composition of other formulations need to be optimated that drug release and storage life would be good enough. Intracellular release properties of liposomes were studied with Sytox red probe. Fluorescence of Sytox red increases when it binds with DNA or RNA. With this study, it was proved that liposomes release Sytox red inside the cells and that the lightning time affects to the release. The results weren't useable for pharmacokinetic model, so the model was made based by literature. Pharmacokinetic model can be used in the future studies and different in vitro or in vivo results can be combined with the model.
  • Tenhola, Ella (2023)
    Medicine shortages have been an increasing problem in the pharmaceutical industry for several years. While the causes of these shortages have been widely researched, they have been found to be diverse and the root causes are difficult to identify. The pharmaceutical care system has been formed over a long period of time, which has led to a growing problem of shortages for various reasons. This study aims to investigate he views of different pharmaceutical sectors on what reforms to the mandatory reserve supply law could help to prevent and shorten the medicine shortages. The study was conducted through a thematic interview. Abductive analysis and thematic analysis were selected as the method of analysis. The study found that mandatory reserve supply is successful in acting as a buffer against short-term shortages, but that a comprehensive reform of the law would be necessary. According to the study, the reform of the law is linked to a wide range of issues. More flexible processes in regulatory aspects, the profitability of the Finnish market, and hospital procurements are closely related to the mandatory reserve supply law. The reform of the mandatory supply list could bring flexibility to exemptions to maintain lower stock levels by renewing the list of medicines and categorizing them into different groups. The act on public contracts law and the mandatory supply reserve law should be better coordinated to avoid wastage and thus ensure Finland's adequate competitiveness However, it should be noted that ensuring Finland's competitiveness and reducing shortages is not solely the responsibility of the mandatory reserve supply law. It also requires extensive international cooperation and it is also believed that bringing production back to Europe and even to Finland is crucial to shorten medicines shortages.
  • Ylitalo, Merja (2016)
    Ethanol intake and the use of several drugs of abuse lead to the activation of the endogenous opioid system which has an important role in reward and reinforcement. Ethanol can affect also many other neurotransmitter systems, for example the dopaminergic, GABAergic and glutamatergic systems. The ability of opioid antagonists to decrease ethanol intake refers to the important role of the opioidergic system in mediating the reinforcement from ethanol. Important brain areas in the mesolimbic reward system are the ventral tegmental area, nucleus accumbens and ventral pallidum. The ventral pallidum is regarded as the endpoint of the mesolimbic reward system and as the cross point of the motivational circuit and reward circuit. The role of the ventral pallidum and its GABAergic and opioidergic systems in ethanol reinforcement has been proven in many studies. This review goes through the brain areas involved in the reward circuit and ethanol's effects on the neurotransmitter systems connected to the reward system. This review concentrates especially on the opioidergic system and on the role of the ventral pallidum in ethanol reinforcement. The aim of this study was to research the role µ-opioid receptors in the ventral pallidum on ethanol intake using an ethanol-preferring AA (Alko, Alcohol) rat line. The hypothesis of the study was that local inhibition of the ventral pallidum with an excess of µ-opioid receptors effects ethanol intake. We infused µ-opioid receptor gene overexpressing viral vectors (AAV-MOR), control vectors or vehicle into the ventral pallidum of rats. Ethanol drinking of the rats was examined in the limited access paradigm. After the ethanol drinking study rats received injections of an opioid receptor antagonist, naltrexone (0.1 mg/kg and 0.3 mg/kg, s.c) and an opioid receptor agonist, morphine (3 mg/kg, repeatedly, s.c) before the ethanol drinking session to see what effect the drugs have on ethanol drinking. The biological activity of the viral vectors was confirmed with immunohistochemical staining and qPCR. In the ethanol drinking study there were no statistically significant differences between the groups. Naltrexone 0.1 mg/kg dose decreased statistically significantly ethanol drinking only in AAV-MOR group and caused statistically significant difference in ethanol drinking between the AAV-MOR and control vector groups when proportionate to the control. These results suggest that possibly part of to that naltrexone's ethanol intake decreasing effects are mediated via the ventral pallidum. Morphine did not cause statistically significant differences in ethanol drinking between the groups. The results of this study do not exclude the role of the ventral pallidum in controlling ethanol drinking.
  • Suo-Yrjö, Ville (2010)
    Nearly all drugs that are abused release dopamine in the nucleus accumbens, the end point structure of mesolimbic dopamine pathway. This why effect of those drugs is closely attached to dopaminergic system. It seems that function of mesolimbic dopamine pathway is necessary for rewarding effects of drugs as cocaine and amphetamine. However rewarding effects of ethanol seems to mediate routes despite of mesolimbic dopamine pathway. This conclusion is a result of several studies that have showed that destruction of synapses of in nucleus accumbens have no effect on ethanol drinking of rats. So it might be that the possible place were effects of ethanol mediate beside of straight stimulation of nucleus accumbens, are GABA- and opioidergic medium spiny neurons that project from nucleus accumbens to ventral pallidum and also from ventral pallidum back to nucleus accumbens. Ventral pallidum is the structure of brain that is thought to be the last common pathway of brain reward circuitry. Ventral pallidum is also found to mediate reinforcement of pleasure of natural rewarders and also drugs that are abused. Meaning of this study was to find how opioidreceptors in ventral pallidum control the drinking of ethanol. The method of study was observe how opioidergic drugs (µ-, δ-, κ-agonist and -antagonist) that are microinjected in brain of AA-rats mediate voluntary ethanol drinking of these animals. Hypothesis of this study was that activation of opioidreceptors in ventral pallidum leads to lower consumption of ethanol and inactivation of these receptors leads to higher consumption of ethanol. Study was performed in National institute for health and welfare, Department of Alcohol, Drugs an Addiction, in research group of Kalervo Kiianmaa. There were used ethanol prefering AA-rats which were microinjected in ventral pallidum with opioidergic drugs. Study involved 72 male rats form generation F99. There were six groups and each had 12 rats. Before the actual trial rats were taught to drink 10 % ethanol/water-solution voluntary. Surgery and placement of canulaes were done with stereotactic device. Bilateral guide canulaes were placed above the ventral pallidum. Each drug was given in three different size doses and also ringer's solution was given as a control. Volume on injections were in each drug and with the ringer's solution 0,3 µl and rate of injection was 0,3 µl/min. Total trial involved four experiment days (three with the drugs and one with the ringer's solution). Injections were given in mixed order. Immediately after injection rats were moved in their homecages and they were given drinking bottle which was filled with ethanol. The consumption of ethanol was observed in time of 10, 20, 30, 50, 70 and 90 minutes. After all experiment's were done rats were decapitated and the places of injections were checked from brain slices that were stained with thionine. Results were analyzed with repeated measures ANOVA and if difference between groups were found the post hoc test were done with Dunnett's test. If the statistical difference were found with repeated measures ANOVA the result were analyzed also in exact time point with ANOVA and Dunnett's test. The only significant result was found with µ-opioidreceptoragonist (DAMGO). It lowered the ethanol consumption significantly. The drop in ethanol consumption was dose dependent and was seen with two highest doses of DAMGO. Clearest difference was seen at the first 50 minutes after rats had started drinking ethanol. The second highest dose of µ-opioidreceptorantagonist (CTOP) had a little tendency to elevate ethanol consumption and was near to be a significant (p=0.08). There were found no effects with other drugs. The main conclusion of this study was that activation of µ-opioidreceptors in the ventral pallidum lowers consumption of ethanol in AA-rats. Inhibition the µ-opioidreceptors had a mild effect of elevating ethanol consumption but this could not be taken as reliable and more studies are needed to be done. δ- and κ-opioidreceptor activation or inhibition had no effect in ethanol consumption in these rats. Conclusions made by these results give support to the theory of role of ventral pallidum as a part of brain reward circuitry. When these results are compared to studies were GABAergic drugs are injected in ventral pallidum and ethanol consumption is observed and also with the knowledge of how these drugs affect the cell's membrane potential, there can be made conclusion that inhibition the activity of ventral pallidum has effects that block pleasure mechanisms that interface with ethanol.
  • Juuti, Hanne (2010)
    The blood-brain barrier protects brain from xenobiotics that are in blood. Different in vivo and in vitro methods have been developed for studying blood brain barrier and those can be found in the literature. There are only few computational models pharmacokinetics of compounds in the brain. In this study permeability factors, which were measured in vitro or in vivo, were collected from literature. Additionally two different pharmacokinetic computer models of blood-brain barrier were described. One of which is called microdialysis model and the other efflux model. Microdialysis model is a very simple two compartmental model, the compartments being the blood and the brain. Five substances were simulated according to the values measured in vivo in rat. The model did not correlate well with the in vivo results, because of the simplicity of the model as the model missed the compartment of brain tissue and the kinetics of transporters. Efflux model has three compartments, blood, blood brain barrier endothelial cells and brain. The model was used to study the impact of the of efflux transporter at the luminal barrier of endothelial cells and passive permeability to the steady-state concentration of a compound in the brain extracellular fluid with theoretical simulations. The relation between free drug concentrations in blood and brain extracellular fluid (Kp,uu) was studied. The impact of Michaelis-Menten kinetics of efflux transporter to the concentration of compound was shown in the results. The efflux model is suitable for theoretical simulations. It is possible to add new active transporters. With theoretical simulations the results from in vitro and in vivo studies can be combined and the different factors can be studied in one simulation.
  • Harju, Elina (2021)
    Extracellular vesicles (EVs) are nano-sized lipid bilayer-delimited particles, released by cells. They take part in intercellular communication by their molecular composition and are part of both physiological and pathophysiological functions. EVs can be extracted from bodily fluids, and they are particularly abundant in blood. The purpose of this thesis was to evaluate the use of Raman spectroscopy in the characterization of EVs. Raman spectroscopy is an analysis method based on the interaction of light and matter, and the inelastic scattering of light, and it is used to get information on the biochemical composition of a substance. Principal component analysis (PCA) was used to investigate if Raman spectroscopy could differentiate two different platelet-derived EV samples, a red blood cell-derived EV-sample and a red blood cell-derived reference material. Evaluation of the characterization also included a stability study of these samples, where it was examined if any temperature dependent changes occurred that could be detected by Raman spectroscopy. Additionally, the applicability of Raman spectroscopy for lipoprotein contamination detection was evaluated by examining if purification of an EV sample decreased the intensity of carotenoid peaks typical for lipoprotein spectra. Raman spectroscopy was able to differentiate all three EV samples and the red blood cell-derived reference material from each other. The most clear differences were found between red blood cell and platelet-derived samples, due to for example the characteristic haemoglobin peaks of red blood cell-derived samples. Differences were also found between the two platelet EV samples, which were thought to implicate difference in protein compositions. The characterization of red blood cell-derived samples proved to be difficult because haemoglobin contained in the samples covered most of the other signal from the samples. Stability studies implicated that during fridge storage the carotenoid peak intensity of platelet-derived EV samples decreases due to the degradation of carotenoids. In the red blood cell-derived samples, no differences assignable to changes in some specific components of the samples were observed. Contamination studies suggested the intensity of the carotenoid peaks may increase due to purification of the sample. This was counter to the assumption and may suggest the carotenoids of the EV samples are not from lipoprotein contamination, but part of the EV composition. In conclusion, Raman spectroscopy proved to be a promising method for characterization and identification of different EV samples.
  • Paavilainen, Nea (2024)
    The eye is well-protected by several anatomical and physiological barriers which also pose significant challenges for ocular drug delivery. Even though ocular pharmacokinetics and the permeability of eye’s important anatomical barriers, such as the cornea and the blood-ocular barriers, have been thoroughly investigated, the significance of active transport in the eye is not completely understood. It is known that several drug transporters are also expressed in ocular tissues, but scientific information on this area is still dispersed and incomplete. The aim of the literature review in this master’s thesis was to compile the current knowledge on the expression and activity of OATP transporters (SLCO; organic anion transporting polypeptides) in cornea and in the blood-ocular barriers. Main principles of ocular pharmacokinetics and common methods for studying transporters are also discussed. The experimental part in this thesis is focused on retinal pigment epithelium (RPE) which is a substructure of the blood-retinal barrier. The transporters of the RPE were studied with three different RPE model systems: human RPE cell line (ARPE-19), fetal primary RPE cells (hfRPE; human fetal retinal pigment epithelium) and ocular tissues of the rabbit. In detail, transporter expression was studied with proteomics from the plasma membrane of isolated rabbit RPE and transporter activity by cellular uptake assays (ARPE-19, hfRPE) in vitro and permeability experiments with rabbit RPE-choroid-sclera ex vivo. As with the literature part, the experimental work was mainly focused on the human and rabbit OATP/Oatp transporters. In this thesis, ten important drug transporters were detected from the rabbit RPE. No significant OATP/Oatp activity was observed either in vitro or ex vivo experiments so these transporters seem not to have a great role in the disposition of their substrates in the studied RPE models. However, signs of other active transport were evident especially in the ARPE-19 cell line, in which significant accumulation of the tested substrate, 4’,5’-dibromofluorescein (DBF), was noted in the presence of several inhibitors. The phenomenon was suspected to result from efflux inhibition, but the responsible transporters could not be unequivocally detected. In conclusion, the findings of this thesis highlight the importance of conducting further research on the transporters of the RPE and choosing a suitable RPE model case-by-case for each study. With the compounds used in this thesis, ARPE-19 and hfRPE cells showed marked differences in efflux activity while the small size and fragile structure of the posterior ocular tissues of the rabbit caused notable difficulties in performing the transporter studies.